Immunoassay: A survey of patents, patent applications and other literature 1980-1991

IMMUNOASSAY

A Survey of Patents, Patent Applications and Other Literature 1980–1991

IMMUNOASSAY A Survey of Patents, Patent Applications and Other Literature 1980–1991 Edited by

JUDITH SIGMOND BERNARD SALOMONS and MARTEN TERPSTRA Technisch Economisch Publiciteitsbureau The Hague, The Netherlands

ELSEVIER APPLIED SCIENCE LONDON AND NEW YORK

ELSEVIER SCIENCE PUBLISHERS LTD Crown House, Linton Road, Barking, Essex IG11 8JU, England This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” WITH 19 TABLES AND 94 ILLUSTRATIONS © 1992 ELSEVIER SCIENCE PUBLISHERS LTD British Library CIP Data Applied For ISBN 0-203-97488-3 Master e-book ISBN

ISBN 1-85166-866-7 (Print Edition) Library of Congress CIP Data Applied For No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Special regulations for readers in the USA This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which photo copies of parts of this publication may be made in the USA. All other copyright questions, including photocopying outside the USA, should be referred to the publisher. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the publisher.

PREFACE

The present book is the result of a comprehensive study of literature (patents, patent applications and other literature, such as journal articles) originating from the United States, Japan and Western Europe and published in a period starting from about January 1980 up to April 1991. More specifically, the authors made a selection of many hundreds of documents on immunoassay procedures and systematized them according to entries listed in the list of contents. Systematizing of documents, particularly patent specifications, is a difficult job in that inventors do not restrict their inventions to a single subject, such as a particular method, but also describe means for effecting the method quite often. Much more difficult is when patent applicants describe inventions directed to procedures which can be carried out by different methods and means. For example, when an inventor discusses an immunoassay procedure to detect, e.g. particular antigens, he or she employs labels which, they state, can be an enzyme, a radio label, a fluorescent or a labelled flocculating substance as well. In these circumstances the authors have referred to those different possibilities instead of repeating the respective reference in other chapters. During their study the authors have discovered that many immunoassays discussed in the literature cover each other or, with other words, much research work is being done while other researchers did the job with the same result before. Therefore, it is of utmost importance that researchers make a comprehensive literature study or have it to be effected by qualified literature searchers first before starting a time and money consuming laboratory work. It is true that many inventors discuss the “prior or state of the art” extensively in the introductory section of the patent disclosure, but it appears that most of them refer to journal articles which, admitted, reveal very attractive scientific approaches to problems to be solved, but it will appear also that patent specifications provide more practical solutions to problems inherent to the procedures discussed.

v

The authors and editor of the present book are aware that the descriptions of the documents referred to (some of them count more than 200 pages of text) do not cover all aspects of the methods and devices covered by the documents, but they have tried to indicate the crux of the respective inventions. More specifically, the authors aimed at providing a review of the state of the art of immunoassay procedures, which may form a foothold to new promising solutions. Regarding the systems of entries to the present book (list of contents) the authors were puzzled about the question whether they had to systematize them according to homogeneous and heterogeneous methods and means, or to proceed with the system as employed herein. It is true that homogeneous procedures have advantages over the heterogeneous ones in that the first are less time consuming while the latter (e.g. in vivo procedures) take more time, sometimes three or more days, but often they are more accurate, particularly for quantitative analysis. It appeared, however, that many documents studied during the literature search have nothing to do with or ignore the kind of procedures involved. Consequently, it was decided to employ a systematization as used in the present book. During the study it was discovered that the Japanese are very active in the field of immunoassay research which became apparent from many patent applications they filed in Japan. Due to the language barrier it is impossible to study original Japanese patent specifications. Fortunately, important Japanese companies and research institutions file their patent applications in the United States, Great Britain, France and Germany and, in addition thereto, the Japanese Patent Office provide English abstracts of Japanese patent applications open to public inspection which have not been filed in other countries. However, quite often these abstracts are too short to get a good insight into the subject matter covered by the respective Japanese document. The authors and editors owe their gratitude to many colleagues who were very helpful in giving advice, in providing copies of literature and in making computer work. In that respect they mention their colleagues and staff members of the Organization of Applied Physical Research (TNO), Rijswijk, the Technical University Delft, the University of Leiden, the European and Dutch Patent Offices, Rijswijk, and the International Patent Research Office, The Hague, all in The Netherlands. Marten Terpstra, editor.

CONTENTS

1. 1.1

Preface

iv

Non-antigen specific Methods, Apparatus and Kits for Immunoassay

1

Enzymatic methods, kits, strips and dipsticks

1

1.1.1

Enzymatic methods

1.1.2

Kits, strips and dipsticks for the enzymatic methods

25

1.1.3

Reagents for enzymatic immunoassay methods

35

1.1.4

Instruments and devices for conducting enzymatic immunoassay

43

1.2

Fluorescence methods, reagents, kits and instruments therefore

1

54

1.2.1

Fluorescence immunoassays

54

1.2.2

Reagents for fluorescence polarization immunoassay

65

1.2.3

Kits and instruments for fluorescence immunoassay

70

1.3

Luminescent, optical and colorimetric immunoassay methods

76

1.3.1

Luminescence

76

1.3.2

Optical and colorimetric methods

87

1.4

Radioimmunoassay

102

1.5

Other methods and means

109

1.6

Reagents

135

Immuno specific Methods and Means

169

2.1

Tumors

169

2.2

Venereal diseases

179

2.

2.2.1

HLTV

179

2.2.2

Gonorrhoea/syphilis

194

vii

2.2.3

Herpes

195

2.3

Hepatitis

196

2.4

Diabetes

203

2.5

Rheuma

207

2.6

Allergies

213

2.7

Heart disease

215

2.8

Pregnancy

221

2.9

Urine

227

2.10

Thyroid

229

2.11

Interferon

232

2.12

Bacteria

235

2.13

Fungi

238

2.14

Testing cattle

243

2.15

Miscellaneous

246

Devices

251

3.1

Apparatus

251

3.2

Strips

258

3.3

Tubes

267

3.4

Miscellaneous

269

Bibliography

271

List of Patentees

273

Register of Keywords

285

3.

viii

1 NON—ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY

Many methods and apparatuses have been proposed the last decade to reliably detect various antigens or haptens in a fluid. The most common methods used up to date are enzymatic immunoassay procedures, radio-immunoassay, fluoroimmunoassay and luminescense methods. Nevertheless, there exist many other procedures which are less known in the art, but which become more and more interesting in connection with their fast determination response and reliability. In the next chapters new developments of the common immunoassay methods and many other recently proposed procedures will be discussed. 1.1 ENZYMATIC METHODS, KITS, STRIPS AND DIPSTICKS 1.1.1 ENZYMATIC METHODS Perlman and Evans (BECTON, DICKINSON AND COMPANY (16.8)) found a method to enhance the sensivity in detecting ligands in biological fluids at very low levels, which method, they say, does not require expensive instrumentation for signal detection. Their method includes use of an enzyme, a metal ion catalyst for an indicator reaction and a blocked modulator for the catalyst. The ligand present in the fluid binds to an antiligand. The resulting bound fraction activates the enzyme to unblock the modulator. The free modulator activates or inhibits the catalyst thereby modulating the rates of an indicator reaction between a substrate and a redox reagent. The presence or absence of the ligand in the fluid is indicated by a signal, such as a colour change or a rate of colour change, consequent to the indicator reaction. The blocked modulator is preferably the free modulator covalently conjugated with a blocking group which can subsequently be removed by the action of the enzyme. Preferred enzymes are hydrolases and preferred blocking groups are

2 IMMUNOASSAY

conjugated to the modulator by chemical linkages which may be cleaved by the hydrolase. The most preferred blocking groups are short peptides. In a preferred embodiment of their method, the enzyme is added to the fluid in an inactive form, and ligand, if present in the fluid, binds to the antiligand whereby the enzyme is activated to cleave a peptide blocking group esterified to the metal binding agent. The liberated metal binding agent complexes and thereby decreases the activity of the catalyst so that catalysis of the indicator reaction, wherein a colored chro-mogen is oxidized to a colorless product, is inhibited. The presence of ligand is indicated by retardation of color disappearance. The most preferred embodiment of the method, however, employs the first component of complement as the enzyme, 8-hydroxyquinoline or benzyl mercaptan as the free modulator and a pentapeptide as the modulator blocking group. The method may be heterogeneous involving a solid phase and a separation step, or it may be carried out by a homogeneous procedure which avoids the separation step. The method makes possible naked eye detection and measurement of the assay signal even though the ligand is present in concentrations as low as 10−12M. This represents signal amplification of the order of 106 and greatly extends the range of ligands which can be detected or determined without expensive or cumbersome equipment. Significant savings in cost and space are thereby achieved, enabling assays to be carried out in small clinical laboratories or even in physician’s offices. Typical of enzymes commonly used in assays are such as redox enzymes, kinases and esterases. An example of the conventional use of enzymes in clinical chemistry assays is in the determination of uric acid, a procedure which has typically employed horseradish peroxidase (HRPO). In that analysis, uric acid is determined using HRPO in conjunction with uricase enzymes. Typical clinical test samples contain uric acid in concentrations less than about 12 milligrams per deciliter (mg/dl), and are contacted in such assays with microbial uricase which converts the uric acid to allantoin and hydrogen peroxide. However, the use of HRPO or other enzymes in such conventional assays is not without problems. For example, the pH values for optimum activity of enzymes such as HRPO and uricase are quite different, and thus neither can be utilized efficiently in such assays. Because uricase is an expensive enzyme, the reaction conditions of such assays ordinarily have been adjusted to facilitate the most efficient use of uricase; such adjustment, in turn, requires concomitant use of a high concentration of HRPO to achieve a rapid kinetic response. In addition, HRPO is known to lose activity in storage, and hence has a limited shelf life. According to Wong and Lee (ABBOTT LABORATORIES (1.3)), a method has been discovered for carrying out diagnostic assays which overcomes the

NON-ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY 3

aforedescribed disadvantages of conventionally-used enzymes. Their method comprises contacting a sample containing an analyte, or a derivative thereof, with a peroxidatively-active material, in the presence of a peroxidase (e.g. microperoxidase) to produce a peroxide reaction product; the reaction product, in the presence of a substance capable of producing a detectable response thereto, can then be determined as a measure of the presence and/or amount of the analyte in the sample. While all of the known procedures take advantage of the amplification of an enzyme to enhance the detectable moieties compared to the target binding pair member moieties, a common problem in such assays is distinguishing such detectable moieties from the many other organic and biological materials present in the phase, particularly liquid phase, wherein the binding reaction and/or the enzymatic reaction are conducted. Diamond and Regina (ALLIED CORPORATION (5.1)), combine the amplification effects of specific binding reactions coupled with enzymatic (or other moderated) reactions with the high sensitivity and low background attainable with gas-phase sensing of detectable moieties. According to their method at the conclusion of a selective binding assay (e.g. immunological or nucleic acid), an enzyme is present in modulated concentration and/or activity to moderate a chemical reaction such as the cleavage of o-nitrophenyl-β-D-galactopyranoside. After a controlled period, the enzymatic product is transferred to the gas phase and concentrated relative to other components in the enzymatic reaction mixture, such as by extraction into ethyl ether, injection into a gas chromatography column and detection by flame ionization or electron capture. Kits for such assays are also disclosed. The immunoassay for biotin, performed by competitively reacting biotin and enzyme labelled biotin with avidin immobilized on a solid support, is described below. Preparation of avidin coated polystyrene test tubes (as described by Parsons, G.H., Jr. in “Methods in Enzymology” (Langone, J.J. and Von Vunakis, H., Ed. 1981, vol. 13, Part B, 224–239, Academic Press, New York). Avidin (5 mg) and bovine-y-globulin (5 mg) were dissolved in 50 mL of 0·05M phosphate buffered saline (PBS) (pH 7·2). To this mixture, 0·1 mL of 25% aqueous glutaraldehyde was added. The resulting solution was incubated at 25°C. for 60 minutes then diluted 10 times with PBS. A 0·4 mL aliquot of this solution was dispensed into each poly styrene tube. The tubes were incubated at 37°C. for 2 hours, and then washed with water (3 times). Preparation of biotinated-β-galactosidase

Biotinated-β-galactosidase was purchased from the Sigma Chemical Co. and used as received.

4 IMMUNOASSAY

Competitive binding reaction: assay for biotin

A phosphate buffered saline solution of bionated-β-galactosidase (1·0× 10−8M in biotin) containing 0·05% Tween 20 was mixed with an equal volume of various biotin solutions ranging in concentration from 1× 10−6 to 1×10 10M. A 0·5 mL aliquot of each of the resulting solutions was dispensed into the avidin coated polystyrene test tubes. The competitive binding reaction was carried out by incubating the tubes at 37°C. for 1·5 hours. The solutions were then discarded and the test tube washed 3 times with water. Subsequently, the quantity of immobilized enzyme was measured via hydrolysis of o-nitrophenyl-β-Dgalactopyra-noside. Each polystyrene tube was charged with 1·0 mL of 0·1M sodium phosphate buffer (pH 7·3), 0·05 mL of 3·36M β-mercaptoethanol and 0·05 mL of 0·03M magnesium chloride. The tubes were incubated at 37°C. for 5 minutes to activate the enzyme, followed by the addition of 0·1 mL of 0·068M onitrobenzyl-β-D-galactopyranoside. The hydrolysis of the substrate proceeded for 1·5 hours at 37°C. Then 1 mL of diethyl ether containing 0·12 mg of nitrobenzene (internal standard) was added and the test tube vigorously shaken for 1 minute. Quantification of o-nitrophenol was effected via gas chromatographic analysis of the ethereal layer utilizing a 6 ft. glass column packed with 1% SP–1240 DA on 100/120 Supelcoport (Supelco Inc.’s trademark) equipped with a flame ionization detector supplied by Hewlett-Packard. The results are tabulated below. Martin and Charles (AMERSHAM INTERNATIONAL PLC (9.3)), have investigated assays which use reagents labelled with the common enzyme horseradish peroxidase (HRP). In general, they got excellent correlation between results obtained using assays with radioactive labels and results obtained using comparable assays with enzyme labels. However, in assays where a body fluid sample is incubated with a reagent carrying an HRP label, they found that a small proportion (about 2%) of samples give rise to quite misleading results. They do not know the cause of these aberrant results. But they believe that they may be due to the presence in the sample of an unidentified substance which inhibits the enzyme action of HRP or inhibits the binding to antibody of an antigen/HRP conjugate. Whether their views as to the cause of the problem are right or wrong, they found a way of overcoming it by a method of performing an assay on a sample of a body fluid including a step of incubating a mixture of the sample with a reagent labelled with an enzyme, wherein the sample may contain an inhibitor for the enzyme. The method comprises the inclusion in the incubation mixture of a correction factor to block the action of any inhibitor present in the sample. Preferably the assay is one which involves an immune reaction between an analyte and its specific binding partner, in which immune reaction the reagent labelled with enzyme also participates. Thus, the analyte may be an antigen or hapten and the specific binding partner its associated antibody; or the analyte

NON-ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY 5

ASSAY FOR BIOTIN

may be an antibody, with an antigen as its specific binding partner. Often the inhibitor is a substance, usually macromolecular, which binds to the enzyme in such a way as to inhibit participation of the labelled reagent in the immune reaction. On other occasions, the inhibitor is an enzyme poison, naturally occurring or as an added preservative, which binds to the enzyme in such a way as to reduce or destroy its enzymic activity. The nature of the assay is not critical. It may for example be an uptake assay, or a competition or immunometric assay for total or for free analyte. The analyte may be a hormone, an enzyme, a biochemical messenger, a steroid, a drug, a drug metabolite, a polypeptide or protein, a catachol-amine, a vitamin, a tumour antigen, a toxin, an alkaloid, a mono, di—or poly-saccharide, or a virus or virus particle. The analyte may itself be antigenic, or may be a small molecule such as a hapten (which can initiate the production of antibodies only when joined to a larger molecule); or may be an antibody. The sample for assay may be taken from any body fluid, such as for example plasma, urine, or serum. We have coined the term “sample correction factor” or SCF for short, which term is occasionally used hereinafter. The correction factor or SCF is a material which blocks the action of any inhibitor for the peroxidase enzyme which may be present in the assay sample. Clearly, the correction factor must be inert to the assay reagents and must not otherwise affect the assay. For example, the correction factor should preferably not itself have enzymatic activity in order to maintain assay precision. One

6 IMMUNOASSAY

suitable material is the apoprotein left after gentle removal of the haem group from HRP. A satisfactory procedure for doing this, involving the use of methylethylketone to separate and dissolve the haem group, has been described (Teale, F.W.J., Biochim. Biophys. Acta, 35, (1959) page 543). It is envisaged that other correction factors might be employed. In particular, it is likely that HRP can be modified in different ways to destroy its enzymic activity without destroying its ability to block the action of the supposed enzyme inhibitor. It is known, that in some cases results obtained by the application of enzyme immuno assays have to be composed (for verification) to radioimmuno assay results. However, in case of some sera (“problem sera”) there are often deviations from the correct values towards much too high results. Research of Harald Haug (BOEHRINGER MANNHEIM GMBH, (24.4)) resulted in eliminating this problem, by proposing a conjugate for enzymeimmuno assays, consisting of an enzyme marker, containing a carbohydrate component (peroxidase) and an immunologically active substance. Prior to or after bonding the marker enzyme with the substance, the marker is oxidised in an aqueous medium with periodine acid or an alkali salt thereof, the oxidation product then being reduced with NaBHy. This procedure yields a marking enzyme derivate, retaining its enzymatic properties unchanged and producing correct values in view of problem sera as well. There are numerous prior art methods for attaching enzyme labels to antigens to form conjugates for use in enzyme immunoassays. In general, these methods employ chemical coupling methods, such as periodate oxidation and glutaraldehyde coupling, or cross-linking agents such as o-phenylene dimaleimide, dihydroxysuccinimide, or soluble dicarbodimides. A serious limitation of chemical coupling methods is the difficulty in achieving consistent and reproducible antigen/enzyme coupling in terms of (a) the site(s) of antigen attachment to the enzyme, (b) the number of antigens attached to each enzyme molecules, and (c) the extent of loss of enzyme activity or change in immunological activity of the antigen following coupling. Because of the difficulty in controlling these variables, the manufacturer of assay kits must carry out quality control tests on each batch of enzyme-labeled antigen that is produced. Even with the quality controls, it is difficult to predict how variations in antigen binding to an enzyme will affect the enzyme on storage. It is therefore an object of Schenk, D.B. (CALIFORNIA BIOTECHNOLOGY INC., (28)) to provide a fused enzyme/peptide protein and methods and systems for producing and using the protein, which overcome the limitations in prior art enzyme immunoassay reagents. His proposal includes a fused protein reagent for use in an enzyme immunoassay. The protein includes an enzymatically active βgalacto-sidase fused, at its C-terminus, to an immunologically active peptide.

NON-ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY 7

The active peptide is preferably derived from an immunologically active region of a peptide hormone, a serum protein, or other suitable polypeptide analyte. The invention further includes a plasmid for use in constructing a fused protein gene. The plasmid contains a complete-sequence β-galacto-sidase gene which terminates at a selected restriction endonuclease site. In producing the fused protein, a nucleotide coding for the immunologically active peptide is inserted in the plasmid at the selected restriction site, and a transfected host which produces the desired fused protein is identified by the presence of βgalactosidase activity and immunospecific reaction with an anti-peptide antibody. The fused protein may be used in a solid-phase enzyme immunoassay based on competitive inhibition between an analyte and the fused protein for binding immunospecifically to a solid support. In another embodiment the fused protein forms part of a homogeneous assay system in which antibody binding to the fused protein modulates the protein’s enzyme activity. The assay research of Maxim, P.E. and Veltri, R.W. (COOPER BIOMEDICAL, INC. (42)) is based upon the discovery that SPA binds preferentially to CIC. This preferential binding affinity allows conditions to be selected wherein the CIC can be bound to SPA and precipitated without preliminary separation of CIC from uncomplexed immunoglobulins. Accordingly, it is possible to eliminate the preliminary separation step used in assays of the prior art and conduct the assay in a single tube with a single precipitation step. This materially simplifies the assay and is therefore a substantial improvement over the known assays for CIC. According to their assay, the serum containing CIC and uncomplexed immunoglobulins is mixed with a solution containing SPA labeled with a suitable label such as an enzyme and incubated for a period of time sufficient to form CICSPA-label complex. The conditions may be chosen so that the CIC-SPA-label complex is formed while no substantial amount of complex between labeled SPA and uncomplexed immunoglobulins is formed. The conditions favourable for this result are a temperature between about 18°C. and about 25°C., preferably about 20°C., an amount of SPA-label in excess of that required for binding all the CIC, and an incubation time sufficient for the completion of complex formation. It has been found that the reaction is essentially complete in about 15 minutes. The CIC-SPA-label complex is then precipitated by addition of a solution, preferably an aqueous solution, of polyethylene glycol, to pro-duce a final concentration of PEG in the analytical solution of between 2·0% and 3·5% by weight. Preferably the concentration of PEG in the analytical solution is about 3·5% by weight. The mixture is then incubated at a temperature between about 4° C. and about 8°C., preferably about 4°C., for a period of time to assure that the precipitation is complete enough to serve for analytical purposes. It has been found that the precipitation is complete enough for quantitative assay after about 3–4 hours.

8 IMMUNOASSAY

Maxim and Veltri proposed a method which comprises: a) contacting the circulating immune complexes in solution in the serum with a staphylococcal protein-A(SPA) linked to a detectable label, whereby a CICprotein-A-label complex is formed; b) selectively precipitating the CIC-SPA-label complex by contacting the complex with polyethylene glycol; c) separating the precipitated CIC-SPA-label complex from the serum; d) measuring the quantity of the label present in the precipitate, and e) comparing the measured quantity of the label with at least one standard prepared by subjecting a solution containing a known amount of CIC or functional equivalent material to the same assay. Steps a) and b) may be conducted simultaneously by contacting the serum with a solution containing labeled SPA and polyethylene glycol, the label being an enzyme label, preferably horseradish peroxidase. The standard proposed is heat aggregated IgG. Enzymatic methods and compositions are provided by DOELLGAST, J. (27 +53), based on the enzyme catalyzed insolubilization of labeled fibrinogen by formation of labeled fibrin in the presence of insolubilized fibrinogen, also subject to enzyme catalyzed fibrin formation, whereby a labeled fibrininsolubilized fibrin complex is formed at the site of the insolubilized fibrinogen. The method can be used for the detection of a wide variety of analytes, being capable of detecting directly blood factors involved in clot formation or inhibition of clot formation, and indirectly, a wide variety of analytes, including haptens, antigens, and receptors, particularly antibodies. Doellgast’s method comprises the improvement that he employs as at least a part of a detection system fibrinogen bound to a substrate and enzyme labeled fibrinogen, where with other than thrombin as the analyte, thrombin is added to the media. The label is, preferably, a fluorescer, and the analyte is a blood clotting factor. A sample suspected of containing at least one blood clotting factor to be assayed as the analyte is combined with prothrombin, labeled fibrinogen and fibrinogen bound to a surface in the presence of any additional blood factors necessary for the formation of thrombrin from prothrombin. The mixture for a sufficient time for fibrin to form and initiate at least partial deposition of the labeled fibrinogen is incubated, and the amount of label bound to the surface or in the supernatant as a measure of the amount of analyte in the sample is detected. Frickey, P.H. et al. (EASTMAN KODAK COMPANY, (54.2)) have developed an immunoassay which does not require a separate wash step to obtain horizontal separation of bound and free ligand. The assay utilizes a dry analytical element which can be used in highly automated analyzers. In these elements,

NON-ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY 9

radial or horizontal separation of bound and free ligand occurs during the spreading of the sample. Therefore, a separate wash step is not required. Minimal sample preparation is required and the assay is complete in as little as three minutes following sample contact with the element, Frickey et al. said. In one embodiment the separation of bound and free ligand is accomplished by slowly contacting the element with the liquid sample. This ensures that the complexing of ligand and receptor occurs during sample spreading. In another embodiment the separation of bound and free ligand is accomplished by using a beaded spreading layer with a porosity such that spreading of the liquid sample occurs slowly enough for complexation to occur during spreading. When an enzyme label is used, the substrate for the enzyme is preferably present in the element e.g. in a reagent layer. Alternatively, the substrate can be added to the element prior to or simultaneously with the liquid sample, or after completion of the binding reaction. It is within the skill of the ordinary worker in clinical chemistry to determine a suitable substrate for a given label. The substrate can be a material which is directly acted upon by the enzyme label, or a material that is involved in a series of reactions which involve enzymatic reaction of the label. If the enzyme label is peroxidase, the substrate is hydrogen peroxide. Using glucose as an example, the substrate is generally present in the reagent layer in an amount of at least about 0·01 moles/m2, and preferably from about 0·01 to about 2·5 moles/m2. A worker skilled in the art would know how to adjust the amount of a particular substrate for the amount of enzyme label used in the assay. It is known that colorometric enzyme-linked-immunosorbent assays are, as a rule, made with three enzymes (e.g. a substrate of alkali phosphatase with pnitrophenyl phosphate, β-galactosidase with o-phenyl-β-D-galactopyrano-side and peroxidases with various colour substrates. Quantitative measurements in each “ELISA” take place by determining the concentration of the coloured reaction product, formed from the enzyme and the first colourless substrate. However, such methods as a rule involve the use of an excessive amount of substrates, which can be 10 × the amount actually operating. This measure is applied to increase the sensitivity and the speed of the test. However, as a consequence, the coloured reaction product has to be quantified by a high-cost, long term photometric analysis. In order to eliminate this drawback, Brynick, D. et al. (FRAUNHOFERGESELLSCHAFT ZUR FOERDERUNG DER ANGEWANDTEN FORSCHUNG, (57A)), developed an immunological enzymatic test with detection by color changes in the visible spectrum with a dyestuff concentration only of the order of magnitude of the substrate turnover. Depending on the concentration of antigens or antibodies, reaction products having different colours are produced, so that the quantitive determination of the antigen or antibody can be effected by comparing

10 IMMUNOASSAY

the coloured reaction product with reference colours. The process involves a dyestuff concentration between 10 and 100 nmole/ml, using as indicator enzyme peroxidase or glucose oxidase or a combined system thereof. As dyestuff 3,3′,5,5′tetramethyl benzidine is proposed. A method of measuring a biological ligand comprises allowing to coexist a biologically active composition comprising an immobilization phase of a particular antibody or a particular ligand and an immobilization phase of a biotinyl enzyme or a biotinyl enzyme inhibitor, a watersoluble conjugate of the ligand or the antibody and the biotinyl enzyme inhibitor or the biotinyl enzyme, and the ligand to be measured in an aqueous solution, and measuring the remaining biotinyl enzyme activity or the biotinyl enzyme inhibitory activity has been developed by Yasushi Kasahara et al. (FUJIREBIO K.K, (60.2)). This method is highly sensitive and specific, and it is suitable as a clinical test for the determination of physiological substances and trace components in humoral fluid. The following is an example of the preparation of a conjugate: 5 mg of avidin was dissolved in 1 ml of 0·1M phosphate buffer solution of pH 6·3, and 100 µl of 2 mg/ml 4-maleimidomethyl cyclohexane-1carboxylic acid succimide ester (CHMS) dioxane solution was added, and allowed to react at room temperature for one hour. The reaction solution was introduced into a column of SEPHADEX G–25 an anion exchange resin containing quaternary ammonium groups, (0·1M phosphate buffer solution–1 mM EDTA Ph 6·5) and gel filtration was carried out. The protein fractions were collected as CHMS induced avidin fractions. Goat anti-human α-fetoprotein (AFP) antibody was purified by using an AFP affinity column, and 10 mgs of the purified IgG was dissolved in 1·0M phosphate buffer solution-lmM EDTA pH 6·0 0·1 ml of a 0·1M 2mercaptoethylamine solution was added to this solution, and the resulting solution was allowed to react at 37°C. for 90 minutes. The reaction solution was passed through a column of SEPHADEX G-25, and the void fractions were collected. The above CHMS induced avidin was added to the void fractions, and the pH of the mixture was adjusted to 6·8. The mixture was allowed to react at 4°C. for 24 hours, and the reaction solution was concentrated by using PEG-20,000. The concentrate was separated by gel filtration using SEPHACRYL S-300 an anion exchange resin, and about 7 mg of the conjugate of anti-AFP IgG-avidin was obtained. The immunoassay according to Kasahara, Yasushi et al. (FUJIREBIO K.K. (60. 2)), is said to be applicable to antigens and antibodies in general, and the antigen and antibody may be selected according to the use to which the biologically active composition is to be put. Among antigens which may be used are haptens and first antibodies in the case of the double antibody method. The antibodies

NON-ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY 11

include the digestion products of an immunoglobulin with protease, such as F(ab ′)2, Fab′ and Fab, and second antibodies as against first antibodies. Moreover, in the case of the double antibody method, any combination of an antigen, a first antibody and a second antibody may be used as a combination of the antigen and the antibody. The following graphs show the IgG µg/ml and the enzyme activity. A method of selective assay of urinary plasminogen activator zymogen (pro-uPA) (commonly known also as pro-urokinase) and urinary plasminogen activator (uPA) (commonly known also as urokinase) in biological fluids as well as in any kind of solution containing them either separately or jointly, has been proposed by Corti, A. et al. (GRUPPO LEPETIT SpA, (65.2)). One feature of this method is in fact that it allows the determination of pro-uPA also in the presence of its enzymatically active derivative u-PA. According to the method u-PA and/or prouPA are selectively immunoadsorbed on the surface of analytical means coated with a monoclonal antibody directed to an epitope which is common to pro-uPA and u-PA and the samples are tested for enzymatic activity with and without activation of the enzymatically inactive zymogen. The accuracy of this assay is due to the fact that the impurities and interferring materials are easily removed from the sample after immunoadsorption of the analyte(s). Plasminogen activators (PA) are serine proteases which convert plasminogen into plasmin, a protease with high specifity for fibrin which is the main constituent of blood clots. At least two immunological distinct types of PAs have been isolated from cultured malignant cell lines: those related to the tissue plasminogen activator (TPA) and those related to the urinary activator (u-PA) (also called urokinase). The u-PA type, originally purified as a two chain protein from urine, has been recently isolated in a proenzyme form (called pro-uPA) for instance, from a human epidermoid carcinoma cell line HEp3 (see Wun T.D., Ossowsky L. and Reich E. (1982) J.Biol.Chem. 257, 7262), from human glyoblastoma cells (see Nielsen L.S. et al. (1982) Biochem 21, 6410) from urine (see Husain S.C. et al. (1983) Arch. Biochem. Biophys 220, 31) and from the human kidney permanent cell line TCL-598 (see Kohno T. et al. (1984)

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Biotechnol 2, 628). A proenzyme immunologically related to u-PA has been identified also from the supernatant of the human epidermoid carcinoma cell line A431. This pro-uPA zymogen is a single chain protein of 50–54000 with no or low amidolytic activity and high fibrin affinity, it is resistant to diisopropylfluorophosphate treatment and inactivation by plasma inhibitors (see Gurewich V. et al. (1984) J.Clin. Invest. 73, 1731) and it is convertible into the two-chain active enzyme (u-PA) by treatment with plasmin. The human u-PA molecule has been isolated in two biologically active forms: a high molecular weight form (MW about 54 000) and a low molecular weight form (MW about 33000). The low molecular weight form (LMW) is derived from the high molecular weight form (HMW) by enzymatic cleavage. The HMW form consists of a 30000 heavy chain (B-chain) and a 20000 light chain (A-chain) linked by a disulfide bond. The LMW form consists of the whole B-chain which contains the active site and a small proteolytically resistant fragment (MW=2427) derived by the A-chain linked by a disulfide bond. The A-chain is proteolytically splitted into a 18000 fragment and the above 2427 fragment. It seems that the LMW u-PA is less active than the HMW u-PA in accelerating clot lysis in vivo (Samama M. et al., Thromb. Haemostas. 40, 578–580, (1979) and Murano G. et al., Blood, 55, 430–436 (1980). Human u-PA is at present mainly obtained from biological sources such as human urine, tissue cultures, particularly normal and tumoral kidney cell cultures, by means of conventional purification techniques. Human u-PA zymogen may play a similar physiological role, once activated to u-PA. Recently it was reported that human u-PA was produced by DNA technoly (see Eur.Pat.Appln.Publ. No. 92182), but Corti A. et al. claim to have improved prior methods by a) causing a test solution to contact with an analytical support, preferably of polystyrene, pvc or polyethylene plate, bearing onto the surface a monoclonal antibody or a conventional antiserum which recognizes a common epitope on u-PA and pro-uPA without either inhibiting the activity of u-PA or inhibiting the enzymatic activation of pro-uPA; b) after rinsing, adding a buffered solution of an enzymatic activator to a series of treated analytical supports while adding only the corresponding buffered solution to another series of treated analytical supports in parallel; c) determining the concentration of the analytes according to a known per se amidolytic assay on a synthetic color developing substrate by reference to a standard curve; the concentration of u-PA being directly determined from the samples which were not treated with the enzymatic activator in the foregoing step, while the concentration of pruPA being determined by difference between the concentration of total analytes measured in the

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samples treated with the enzymatic activator and the samples treated with the buffer only, in the foregoing step. Enzyme binding methods The various prior art immunoassay methods each possess commercial advantages and disadvantages. RIAs are sensitive and easy to set-up but require radioactivity, separation steps and expensive instrumentation. Heterogeneous assays with enzymes or fluorophores eliminate radioactivity and some instrumentation but require separation steps. From a commercial viewpoint it is desirable to eliminate separation steps for several reasons. Separations (1) are labour intensive, (2) are time consuming, (3) require additional equipment, (4) increase variability in results, and (5) preclude high levels of automation. Despite the many commercial advantages of homogeneous immunoassays only three systems, the enzyme-labeled system of Rubenstein et al., US Pat. No. 3,817,837, the substrate-labeled system of Burd et al, 1977, Clin. Chem. 23:1402, and fluorescence polarization (Dandliker et al., 1973, Immunochemistry) have found commercial success. Yet these three assay systems are limited to small (less than 1000) molecular weight analytes and analytes found in concentrations greater than 10–10M. To improve the system Henderson D.R. (MICROGENICS CORPORATION, (108.1)) provides a method and novel compositions for enzyme complementation assays for quantitative analysis of analytes of both high and low molecular weight 150–30000 daltons MW), in high (10–15M) sensitivity. The assays are capable of automation. According to Henderson, polypeptides are produced by recombinant DNA techniques or by chemical polypeptide synthesis techniques. (As used herein the term “polypeptide” is inclusive of peptides and proteins). The polypeptides themselves are enzymatically inactive; however, when reacted together in aqueous medium they associate to form a catalytically active enzyme via a phenomenon known as complementation. β-galac-tosidase is a favored enzyme because it has several substrates, detectable using spectrophotometric and fluorometric methods, has shown utility in previous commercial immunoassays, can be measured at extremely low concentrations and is well characterized genetically. By creating enzymatic activity from insignificant background a high signal-to-noise ratio can be achieved. The novel polypeptides used in the improved assays of the present invention encompass (a) fusion proteins in which analyte is fused to polypeptide, the product of recombinant genes containing sequences coding for analyte and polypeptide; (b) polypeptides genetically engineered for optimal coupling with analytes; (c) polypeptides chemically synthesized for optimal coupling with analytes; and (d) polypeptides genetically

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engineered or chemically synthesized for improved stability to such environmental factors as oxidation, heat, pH, enzymatic degradation and the like. Bolguslaski R.C. et al. (MILES LABORATORIES, INC., (109.2)) provides a homogeneous specific binding assay method and system based on the use of, as labeling substance, a substance which exhibits given reactant activity as a constituent of a predetermined reaction, such substance being referred to as the reactant. The method is based, in part, on the fact that the reaction between a ligand and a specific binding partner thereof to one of which the reactant is coupled alters the activity of the reactant in the predetermined reaction, which reaction thus serves as means for monitoring the specific binding reaction. In view of this basic phenomenon, various manipulative schemes involving various test compositions and devices may be employed in performing the method of the present invention. The preferred fundamental manipulative schemes are the direct binding technique and the competitive binding technique. In the direct binding technique, a liquid medium suspected of containing the ligand to be detected is contacted with a conjugate comprising the reactant coupled to a specific binding partner of the ligand, and thereafter any change in the activity of the reactant is assessed. In the competitive binding technique, the liquid medium is contacted with a specific binding partner of the ligand and with a conjugate comprising the reactant coupled to one or both of the ligand or a specific binding analog thereof, and thereafter any change in the activity of the reactant is assessed. In both techniques, the activity of the reactant is determined by contacting the liquid medium with at least one reagent which forms, with the reactant, the predetermined monitoring reaction. Qualitative determination of the ligand in the liquid medium involves comparing a characteristic, usually the rate, of the resulting reaction to that of the monitoring reaction in a liquid medium devoid of the ligand, any difference therebetween being an indication of a change in activity of the reactant. Quantitative determination of the ligand in the liquid medium involves comparing a characteristic of the resulting reaction to that of the monitoring reaction in liquid media containing known amounts of the ligand. The monitoring reaction preferably is enzyme-catalyzed. Usually, a monitoring reaction is selected which is highly sensitive to the reactant in the conjugate. Luminescent or fluorescent reaction systems are very useful in this regard. Particularly preferred are cyclic reaction systems, especially those in which the reactant is the cycled material. Of the preferred cyclic reaction systems, those which are enzyme-catalyzed are particularly advantageous. The reactant in the conjugate is usually an enzymatic reactant, such as an enzyme substrate or, as is particularly preferred, a coenzyme, and preferably has a molecular weight of less than 9000.

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Anti-enzyme antibody immunoassay Variations on the basic immunoassay technique have been developed to try to overcome some of the problems associated with immunoassays; sensitivity, reliability and cost effectiveness are the major concerns. Although the immunometric assay techniques have been found to be particularly useful in analyzing for antigens and antibodies, there has been difficulty in the past in establishing an optimum level of sensitivity for the assay to be helpful in the detection or monitoring of disease states or maladies in the body. An immunoassay utilizing the inhibition or inactivation of a signal generating molecule to reduce a controlled baseline signal to a lower level of signal corresponding to the amount of analyte present in the sample is subject of research made by Hossom M.G. (MUREX CORPORATION, (113.3)). One embodiment is a heterogeneous enzyme immunoassay, preferably using monoclonal antibodies wherein an insolubilized first antibody binds to an antigen analyte of interest. A second and third antibody, which themselves have been conjugated together, are added so that the second antibody complexes preferentially with the antigen. Then an active enzyme and its specific substrate are added to the complex, simultaneously for example, and enzyme will be bound preferentially to the third antibody which exerts an inhibitory influence on the enzyme. The resultant decrease in enzyme activity is measured and the relative amount of antigen present in the sample is calculated. There are known immunochemical dosage processes, wherein an antibody, directed against a substance to be dosed, is fixed on a matrix, while using dosable labeling enzymes according to prior art methods, the enzyme being covalently bonded to an antibody directed against the substance to be dosed. The research of Guesdon J.L. et al. (INSTITUT PASTEUR, (129.4)) concerns a dosage process for antigens (in the heterogeneous phase), according to which the substance to be dosed is fixed on a matrix through a corresponding antibody, followed by a reaction between a hybride conjugate of an antibody of the substance to be dosed and of an antienzyme, whereafter the enzymatic activity of the matrix is dosed, the matrix having been modified by the foregoing processes. Crosslinked enzymes Covalent methods for coupling single enzyme molecules to single coupling sites on antibodies are already known, e.g. Imagawa et al., J. Appl. Biochem, Volume 4, 400 (1982). It has been appreciated for some time that aggregated antibodyenzyme conjugates can provide enhanced signal generation over that of purely monomeric antibody-enzyme conjugates. Aggregated antibody-enzyme conjugates generally have been prepared using nonspecific coupling chemistries

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such as glutaraldehyde crosslinking. The preparation of these conjugates involves a random crosslinking process in which the antibody or antibodyfragments can be buried deep within an aggregated complex, thus remaining largely inaccessible for antigen binding. In addition, these amorphous macromolecular complexes are difficult to prepare in a reproducible fashion. Immunoassays utilizing such aggregated antibody-enzyme conjugates routinely suffer from very high background blanks due to nonspecific binding, and the ultimate sensitivity achievable in immunometric assays is dramatically reduced. A number of procedures have also been reported that utilize noncovalent chemistry to generate antibody-enzyme conjugates with high enzyme-toantibody ratios. The aforementioned procedures all suffer from the disadvantage that the linkage of antibody to enzyme is not covalent, and, therefore a reversible binding results which is susceptible of unwanted dissociation. Thus, there is a need for a highly reproducible procedure for the preparation of convalently linked polymeric-enzyme/antibody conjugates in which both original enzymatic activity as well as original immunoreactivity are maintained. This need is met by the research of Freytag J.W. et al. (E.I. DU PONT DE NEMOURS AND COMPANY, (135.1)) which, in one aspect, is a process for producing a polymeric-enzyme/antibody conjugate, comprising the sequential steps of: (a) covalently coupling at least two enzyme molecules to produce a prepolymerized enzyme; and (b) coupling covalently the prepolymerized enzyme to an antibody or fragment thereof. In another aspect, the invention is an immunoassay utilizing covalent conjugates of prepolymerized enzyme and antibody fragment. In another aspect, the invention is a covalent conjugate of an antibody and a prepolymerized enzyme. A diagnostic assay for inhibitor of tissue-type and urokinase-type plasminogen activators, and a gene coding for the inhibitor has been developed by Loskutoff D.J. et al. (SCRIPPS CLINIC AND RESEARCH FOUNDATION, (149.2)). It is known that endothelial cells line the luminal surface of the vascular bed and are thought to play an active role in the specific proteolytic breakdown of locally deposited fibrin. The potential of endothelium to initiate and control this process is emphasized by its capacity to synthesize and release plasminogen activators (PAs), including both tissue-type and urokinase-type molecules. Endothelial cells can also produce inhibitors of fibrinolysis. Although these inhibitors probably serve important regulatory roles in controlling the fibrinolytic system of the vascular wall, little is known about their specifity, mode of action, or biochemical nature. The conclusion that these

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inhibitors are actually synthesized by endothelial cells is obscured somewhat by recent report that cultured cells can bind and internalize protease inhibitors from serum-containing culture medium. The possibility of producing relatively unlimited amounts of tissuetype plasminogen activator (t-PA) by recombinant DNA technology as described in British patent application 2,119,804, published November 23, 1983, has generated much interest. The conversion of the relatively inactive molecule into an extremely efficient thrombolytic agent by fibrin itself, suggests that t-PA can exist as an active enzyme only when localized to the fibrin-platelet thrombus itself. Thus, t-PA is considered to be a much more specific thrombolytic agent than urokinase-type plasminogen activator and streptokinase. Recent evidence indicates that there are three immunologically distinct plasminogen activator inhibitors (PAIs). The first is derived primarily from endothelial cells, the second was isolated from placenta, the third is protease nexin. Studies carried out by Loskutoff et al. (149.2) resulted in a diagnostic assay for inhibitor of tissue-type and urokinase-type plasminogen activators, and gene coding for the inhibitor. More in particular Loskutoff et al. developed a biochemical reagent system and methods of preparing and using same, diagnostics utilizing the reagent system, a method for detecting PAI, a substantially pure recombinant proteinaceous molecule that is immunologically similar to human endothelial cell type plasminogen activator inhibitor and has the binding and inhibiting activities of human beta-migrating, endothelial cell plasminogen activator inhibitor, and its gene. The proposed biochemical reagent system comprises (a) a receptor such as an antibody raised in an animal host to endothelial cell plasminogen activator inhibitor; i.e. an anti-plasminogen activator inhibitor, and (b) an indicating means. In one aspect of the assay, the biochemical reagent system is composed of (a) a receptor that can be a polyclonal antibody raised in an animal host and (b) an indicating means. The indicating means and receptor can be a single molecule or can be composed of a plurality of individual molecules. The receptor binds to endothelial cell (beta-migrating) plasminogen activator inhibitor that itself binds to and inhibits tissue-type or urokinase-type plasminogen activators. The indicating means labels the receptor, and in so doing indicates the presence of the inhibitor in a sample to be as-sayed such as serum of patients having thrombotic disease. The receptor of the reagent system of the assay selectively binds to endothelial cell plasminogen activator inhibitor bound to either tissue-type (t-PA) or to urokinase-type (u-PA) plasminogen activators. In another aspect of the assay, a method of forming a polyclonal receptor for use in a biochemical reagent system is contemplated. The method comprises (a) administering to an animal host endothelial cell type plasminogen activator inhibitor (PAI) in an amount sufficient to induce the production of antibodies to

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the inhibitor, the antibodies being a receptor for the inhibitor; (b) collecting antisera containing the antibodies from the immunized host; and (c) recovering the receptor from the antisera. An enzyme immunoassay employing one or more enzyme labelsubstrate pairs which give rise to coloured products and in which absorption by the product of substrate conversion by at least one enzyme label at a first wavelength, at or close to the optimum wavelength for bsorbance was subject of research effected by Philo R.D. (SERONO DIAGNOSTICS PARTNERS, (151.2)). It exceeds the linear range of the detector. The immunoassay includes the steps of measuring the said substrate conversion at a second wavelength at which absorbance by the relevant product is significantly lower than at the said first wavelength and calculating the true absorbance at the said first wavelength by utilizing the result of linear regression analysis of absorbance measurements obtained with product standards at the said first and second wavelengths within the linear range of the detector. The conversion of nitrophenyl-β-D-galactoside (p—and/or o-) to nitrophenol by β-galactosidase is measured at a wavelength at which absorbance by nitrophenol is significantly lower than at the optimum wavelength. The conversion of phenolphthalein monophosphate to phenolphthalein by alkaline phosphatase is measured at a wavelength at which absorbance by phenolphthalein is significantly lower at the optimum wavelength. The conversion of phenolphthalein monophosphate to phenolphthalein is measured at 490nm and the true absorbance at 554nm is calculated utilizing the result of linear regression analysis of absorbance measurements obtained with phenolphthalein standards at 490nm and 544nm within the linear range of the detector. Dual analyte method Blake et al. in Clinical Chemistry (1982) 28, 1469–1473 reported the development of a dual analyte enzyme immunoassay for the two haptenic hormones thyroxine (T4) and triiodothyronine (T3), involving the use of alkaline phosphatase and β-galactosidase as the enzyme labels and phenol-phthalein monophosphate and o-nitrophenyl-β-galactoside (o-NPBG) as the repective substrates. In this assay, firstly unlabelled T3 and T4 compete respectively with T3β-galactosidase conjugate and T4-alkaline phosphatase conjugate for binding to an antibody reagent comprising T3 and T4 specific antibodies. The bound fractions of the two enzyme labels are separated by a second antibody precipitation and, after washing, the precipitate is resuspended in an enzyme substrate solution containing phenolphthalein monophosphate and onitrophenylβ-galactoside. The amounts of two enzyme labels are then determined sequentially in a two-stage incubation protocol, the amount of β-galac-tosidase

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being initially determined by monitoring the absorbance of o-nitrophenol at 420nm and the pH then being raised to determine the amount of alkaline phosphatase by monitoring the absorbance at 540nm. Since the two enzyme reactions are performed sequentially rather than simultaneously this assay is not a true “combo” immunoassay. It was now found, however, that it is in fact feasible to perform true “combo” immunoassays employing two enzyme labels. According to one aspect of the assay of Philo R.D. and Allen G.J. (SERONO DIAGNOSTICS PARTNERS, (151.1)) a dual analyte enzyme immunoassay for assaying two antigens in a single liquid sample is provided, wherein the two immunoreactions are carried out simultaneously and wherein subsequently the two enzyme reactions occur simultaneously. The enzyme labels (and hence indirectly the antigens) can be quantified by direct monitoring of the products of the enzyme-catalysed substrate conversions. Alternatively, the enzyme labels could be quantified by monitoring removal of substrate during the incubation period. Suitable enzyme/substrate pairs are β-galactosidase/nitrophenyl-p-Dgalactosidase (p—and/or o-) and alkaline phosphatase/phenolphthalein monophosphate. The usefulness of macromolecules to be able to distinguish a specific compound in a complex mixture by binding to a specific compound is the basis for the greatly expanding usefulness of specific binding pair assays. Different approaches were employed to reduce background interference or matrix effects on assay results, to improve labeling techniques, to minimize unmodulatable signals, and to expand the range of sensitivity. One group of assays known as “homogeneous” immunoassays relies on the fact that the assays do not require a separation step. Therefore, the assays must be relatively resistant to matrix effects resulting from the physiological fluids, such as blood and urine, which are frequently analyzed. As increasing sensitivity is desired to be able to analyze lower concentrations of a variety of analytes, the matrix or background effects can become extremely troublesome. Therefore, in developing assays of greater sensitivity, it is necessary that they be relatively unaffected by variations in sample composition. Taking these requirements into consideration DiNello R.K. (SYVA COMPANY, (162.1)) provided agglutination assays employing specific binding pairs for causing agglutination of particles. The detection system for agglutination employs a two-enzyme system where the enzymes are related by the product of one being the substrate of the other, where the second enzyme provides a product affording a detectable signal. More particularly the proposed agglutination assay brings together through the intermediacy of the binding of specific binding pair members, particles which differ by having different members of a signal producing system. The signal producing system is characterized by having two enzymes which are related by having the product of one enzyme being the substrate of the other, where the reaction of the second

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enzyme results in a product providing a detectable signal, desirably including a scavenger for the product of the first enzyme. The amount of product providing the detectable signal which is produced is related to the amount of analyte in the media. The composition and ratio of particles can be provided in reagent kits to optimize the assay results. In developing an assay, there are a number of considerations in devising the reagents and protocol. One consideration is the degree of sophistication of the individual performing the assay. There are situations where a relatively untrained individual has to carry out an assay and obtain reasonably quantitative results. In these situations, it is important that the assay be free from interference by materials in the sample, furthermore free of fluctuations with changes in environmental conditions and provide for easy measurement. Also, washings can be a source of error, either because of inadequate washing, leaving nonspecific binding material, or by reversing specific binding. To meet such requirements Zuk R.F. and Litman D.J. (SYVA COMPANY, (162. 2)) provide immunochromatographic methods for detecting an analyte where a quantitative determination may be readily made without special equipment. The analyte is immunochromatographed on a bibulous carrier in the presence or absence of a labeled conjugate where the label is a member of an enzymatic signal producing system, which includes one or more enzymes. After chromatographing the analyte, if the enzyme conjugate was not included in the sample, the chromatograph is contacted with a labeled specific binding pair member which binds to the chromatograph in relation to the distance travelled by the analyte. By providing appropriate reagents, in the case of two enzymes where the substrate of one enzyme is the product of the other enzyme, a final product is produced which provides for a detectable signal, where the distance traveled by the analyte may be defined, which distance is related to the amount of analyte in the sample. Various signal producing techniques are employed in “dip-stick” immunoassay for developing a detectable signal related to the presence or amount of an analyte. One desirable system employs horse radish peroxidase to oxidize a dye precursor to a dye. Ascorbate affects this reaction so that the amount of dye which is produced can vary with the amount of ascorbate present in the assay medium. Since samples, which are assayed are frequently physiological fluids having widely varying amounts of ascorbate, unless the ascorbate interference can be diminished to a satisfactory level, the assay can only provide erratic and uncertain results. It is therefore desirable to find a simple efficient and economic way for reducing ascorbate interference without otherwise interfering with other assay reagents or increasing the complexity of the protocol. To satisfy these demands Tom H. (SYVA COMPANY, (162.3)) provides an immuno-assay method employing a bibulous support to which is attached a

NON-ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY 21

receptor or a ligand (“mip”) and the amount of a “mip”peroxidase conjugate which binds to the “mip” on the support is related to the amount of analyte in a sample, where the peroxidase catalyzes the formation of a dye which binds to the support and ascorbate in the sample interferes with the production of the dye. The support is impregnated with a sufficient amount of periodate to reduce the ascorbate interference to a level which does not interfere with the detection of the analyte. More specifically, an immunoassay is provided for measuring a wide variety of analytes, where the immunoassay employs a bibulous support and horse radish peroxidase and a dye precursor for the production of a detectable signal. The amount of metaperiodate which is impregnated into the support may be widely varied so long as it exceeds a particular minimum. Usually the bibulous support will be saturated with a solution at least about 0·05 M periodate, and preferably at least about 0·1 M periodate. A wide variety of bibulous supports may be employed, particularly cellulosic supports, such as paper. More recently much attention has been focused on assay systems which use labels of a non-radioactive nature, such as chemiluminescent agents, fluorescent agents and enzymes. Such assays are as a rule based on changes in the enzyme’s activity. However, in some cases there are factors, which may decrease the sensitivity of the assays. To eliminate such effect Baldwin T.O. et al. (THE UPJOHN COMPANY, (195)) found an enzyme-linked assay system wherein the enzyme is bacterial luciferase. Bacterial luciferase is a flavin-linked monooxygenase (hydroxylase) which catalyzes the bioluminescent oxidation by O2 of reduced flavin mononucleotide, and a long-chain fatty aldehyde depicted below the yield FMN, the corresponding fatty acid, blue-green light and water. The reaction formula is as follows: The luciferase protein is an αβ dimer with a single active center confined primarily, if not exclusively, to the a subunit. One step method The well-known sandwich enzyme immunoassay system generally includes two methods for the formation of the immunochemical reaction products of the three parties, i.e., an antibody fixed on a carrier, an antigen, and an enzyme-labeled antibody. One of them is a two-step method, comprising a first reaction in which the antibody fixed on the carrier is brought into contact with a liquid containing the antigen to be detected and a second reaction in which the reaction product of the first reaction, after having been separated and washed, is further reacted with the enzyme-labeled antibody. The other is a one-step method comprising one incubation step in which the enzyme-labeled antibody is previously added to the sample solution containing the antigen to be detected. The former two-step

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method is apparently complicated since it involves two incubation steps and two separation and washing steps. On the other hand, the one-step method is easier than the two-step method since it involves one incubation and one separation and washing operations. However, the one-step method requires the operation for adding and blending the labeled antibody, which has been separately prepared. An object of the research of Sakata Y. et al. (WAKO PURE CHEMICAL INDUSTRIES LTD., (199)) was to provide a simpler and more effective method of a sandwich enzyme immunoassay system wherein an immunoreaction product composed of fixed antibody/antigen to be detected/labeled antibody or one composed of fixed antigen/antibody to be detected/labeled anti-immunoglobulinantibody can be formed on an insoluble carrier in one incubation step. It includes a new technique for the two-layer fixation in which an enzyme-labeled antibody layer is coated over the surface of an insoluble carrier, having fixed thereon an antibody or antigen to form a two-layer-fixed constitution. It has been discovered that by using the two-layer-fixed enzyme immunoassay system, an immunoreaction product composed of the fixed antibody/ antigen to be detected/labeled antibody or one composed of the fixed antigen/antibody to be detected/labeled anti-immunoglobulin-antibody can easily be formed on the insoluble carrier only by one operation of incubation with the sample solution containing the antigen or antibody to be detected, while the desired antigen or antibody can be detected by colorimetric determination, in which the insoluble carrier having thereon the immunoreaction product is brought into contact with a solution containing a substrate to the labeling enzyme, which may form an insoluble colored substance (insoluble dye) by reacting with the enzyme. The research provided a method for the detection of an antigen or an antibody by a sandwich enzyme immunoassay which comprises using a combined material of insoluble carrier/antibody/enzyme-labeled antibody or a combined material of insoluble carrier/antigen/enzyme-labeled antiimmunoglobulinantibody which comprises an insoluble carrier having coated thereon a layer of an antibody or antigen specific to the antigen or antibody to be detected and a layer of an enzyme-labeled antibody or enzyme-labeled anti-immunoglobulinantibody specific to the antigen or antibody to be detected, and preferably a combined material of insoluble carrier/antibody/enzyme-labeled antibody or a combined material of insoluble carrier/antigen/enzyme-labeled antiimmunoglobulin-antibody which is formed by chemically or physically binding and fixing an antibody or an antigen specific to the antigen or antibody to be detected on the surface of an insoluble carrier and then coating the surface of the fixed antibody or antigen with an enzyme-labeled antibody or an enzymelabeled anti-immunoglobulin-antibody specific to the antigen or antibody to be detected to form a laminate layer. The known assay methods are unsatisfactory with regard to detection sensitivity and, in assaying samples having low hEGF contents,

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require procedures for the concentration of hEGF and the removal of interfering substances. In partucular the assay methods using radiolabelled compounds are not suited for routine test purposes because of installation problems. This is stated by Hayashi et al. (NIHON CHEMICAL RESEARCH K.K., (116)). The enzyme immunoassay established by them, which uses betagalactosidase-labelled antibodies, encounters no problems in assaying urine samples, which have high hEGF contents, since the detection limit is 0–2 mg/ml. However, other body fluids than urine all have very low hEGF contents, so that they cannot be assayed as they are. Furthermore, this assay method is disadvantageous in that in assaying hEGF in blood samples, the assay is disturbed by galactosidase-like enzymes occurring in the blood. Accordingly, Hayashi et al. studied intensively the problems which may be encountered in the enzyme immunoassay using beta-galactosidaselabelled antibodies and, as a result, found that the hEGF detection sensitivity of the enzyme immunoassay using antibodies coupled to beta-galactosidase largely depends on the kind of the enzyme used for antibody labelling and, surprisingly, further found that the assay sensitivity for hEGF is 100–500 times increased when antibodies coupled to an oxidoreductase such as peroxidase are used. Their results provide an enzyme immunoassay method for epidermal growth factor in a fluid sample, which comprises: reacting immobilized anti-human epidermal growth factor antibody with the fluid sample and then with oxidoreductaselabelled anti-human growth factor antibody, separating the solid phase from the liquid phase, and assaying the enzyme either in the solid phase or in the liquid phase so separated. The gist lies in first causing binding of hEGF in samples to antihEGF antibody coupled to an insoluble carrier in the manner of antigen-antibody reaction, further causing binding of the reaction product to an anti-hEGF antibody-coupled oxidoreductase and assaying the activity of the bound or unbound oxidoreductase to thereby determine the hEGF amounts in the samples. Creatine Kinase MB Three predominant isoenzymes of creatine kinase (CK: E.C. 2.7.3.2) are known: these are dimers consisting of the M and B sub-units. These dimers may comprise two M or two B sub-units, or one M and one B sub-unit. The predominant dimer present in the blood, serum, or plasma of normal individuals is CK-MM isoenzyme, with variable but usually only trace quantities of CK-MB that reflect the normal degradation of skeletal muscle. The CK-BB isoenzyme is not usually present in detectable amounts in serum of normal persons but is present in sig nificant quantities in brain tissue and smooth muscle. Elevations of the BB isoenzyme can occur in pathologic conditions such as metastatic carcinoma of severe burns. The presence of elevated levels of CK-MB

24 IMMUNOASSAY

isoenzyme has been used as a clinically important indication of myocardial infarction in instances where possible sources of significant skeletal muscle damage can be eliminated. Repetitive determinations of CK-MB level in the serum can indicate the time course and severity of infarctions. Differentiation between the CK isoenzymes, therefore, is clinically important and the availability of a rapid, efficient, and highly discriminatory assay for the CK isoenzymes was needed. The immunoinhibition assay of Pankratz (E.I. DU PONT DE NEMOURS AND COMPANY, (135.3)) comprises: a) forming a first reaction mixture by contacting a liquid sample suspected of containing CK-MB isoenzyme with a molar excess of either anti-CK-M or anti-CK-B sub-unit antibody immobilized on a solid phase, the antibody being capable of substantially completely inhibiting the enzymatic activity of the bound sub-unit; b) allowing a reaction to occur whereby complexes are formed between M or B sub-unit-containing isoenzymes and the immobilized antibody; c) separating the solid phase from the reaction mixture; d) forming a second reaction mixture by contacting the solid phase with a soluble anti-CK-M or anti-CK-B sub-unit antibody with the same sub-unit specificity as the immobilized antibody, capable of substantially completely inhibiting the enzymatic activity of the bound sub-unit; and e) determining the enzymatic activity of the uninhibited isoenzyme associated with the solid phase. A process for the immuno-chemical quantitative determination of an immunologically active substance comprising incubating a sample containing the active substance with a binder, which binder comprises a labelled first antibody specific for the substance or an Fab or Fab’ fragment of the antibody under conditions favoring formations of complexes between the substance and the binder is subject matter of research conducted by Baier, Jering and Klose (BOEHRINGER MANNHEIM GmbH, (24.3)). Solid phase bound active substance identical to the substance to be determined, is added, incubating thereby the solid phase bound active substance and the sample under conditions favoring formation of the solid phase bound active substance and uncomplexed binder separating the solid phase from the sample. The sample is contacted with a second antibody in solid phase, the second antibody specifically binding to the binder or the binder-active substance complex, incubating under con ditions favoring formation of complexes between the second antibody and the binder or binder-active substance complex, and removing the solid phase and measuring the amount of labelled binder bound to the solid phase second antibody. The second antibody is specific for complexes

NON-ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY 25

of the binder and active substance but is not cross reactive with the components of the complex. The immunologically active substance is a hapten, and the first antibody is enzymatically labelled with a peroxidase. The first antibody and the second one are Fab or Fab’ fragments. 1.1.2 KITS, STRIPS AND DIPSTICKS FOR THE ENZYMATIC METHODS It will be obvious that for carrying out immunoassay procedures, it is desired to use simple, effective and less time consuming instrumentation. To this end much research has been carried out to develop kits or strips and dipsticks which show the results of the assay in a relatively short period of time. In the following section several proposals will be discussed which are the result of research carried out to develop such kits. Color changes Sudo Y. et al. (FUJI PHOTO FILM CO., LTD., (59.3)) developed a multilayered element for the quantitative analysis of an analyte antigen. The analyte antigen is mixed with an enzyme-labelled antigen, comprising: a waterimpermeable and light-transmissible support; a reagent layer carried by the support and containing a hydrophilic polymer as a binder, and a reaction layer covering the reagent layer and made of a porous matrix. The reaction layer contains an antibody for the antigen and a substrate for the enzyme labelling to the antigen. The antibody is immobilized by a first water-insoluble carrier, and the substrate is immobilized by a second water-insoluble carrier which is different from the first one for immobilizing the antibody. A part of the enzymelabelled antigen is combined with the immobilized antibody to be fixed thereby and the remaining part of the enzyme-labelled antigen reacts with the immobilized substrate to produce an enzymatic reaction product. The reagent layer contains a detection reagent composition for coupling with the enzymatic reaction product to develop a color. The following graph shows the relationship between the optical density of reflected light at 500nm and the concentration of thyroxine (T4) (µg/ml). Incorporating a solid phase into the method of existing assays has made the requisite separation steps easier to perform. For example, by incorporating a solid phase in a fluorescence immunoassay, bound and unbound fluorescent label material may be easily separated without the need for cumbersome double antibody precipitation. This separation is accomplished by merely separating the solid phase containing bound fluorescent label from the liquid phase containing

26 IMMUNOASSAY

unbound fluorescent label. This is of great advantage in automated solid phase fluorescence immunoassays where micro-filtration is used to separate the solid from liquid phase. Moreover, the solid phase may be analyzed for the presence of the fluorescent label. The fluorescence of the residual solid phase located on the microfiltration membrane is analyzed for fluorescence. In contrast, when using enzyme labels the solid phase containing the bound enzyme label must be contacted with an enzyme substrate containing a chromogen and usually a redox reagent. Reaction of the enzyme with the redox reagent causes a color change in the chromogen. Because the chromogen is dissolved in solution, the color appears in the solution rather than on the solid phase. Thus, there is a need for an enzyme assay method in which the color change appears on the solid phase in order to derive benefits generally attributable to radioiodine and especially fluorescence solid phase immunoassays. Thus, there is a need for a solid phase enzyme assay where the color change appears on the solid phase. This would enable enzyme assays to be useful in automated clinical diagnosis where such assays often use a particulate solid phase and in dip-sticks for use in the physician’s office. Halsey J.F. et al. (INCSTAR CORPORATION, (80)) found a solid phase enzyme immunoassay and solid phase nucleic acid hybridization assay wherein a

NON-ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY 27

chromogenic material, capable of changing from a first color state to a second color state in correlation to the amount of enzyme bound to the solid phase, binds to the solid phase. The solid phase is thus analyzed for the second color state. They also designed a dip-stick wherein the reactive membrane is thin and planar with both sides of the membrane exposed for contact with reagents. Still further, they developed a stabilized chromogenic solution wherein the chromo-gen contains a benzidine moiety and is stabilized with a complementary redox reagent. The following figures shows an example of the respective dip-stick. Blocking agents Various compounds have been discovered as useful TBP blocking agents, including tetrachlorothyronine, diphenylhydantoin, salicylate, the various materials disclosed by Hollander (U.S. Pat. No. 3,928,553) and Chopra (U.S. Pat. No. 3,911,096), particularly 8-anilino-l-naphthalenesulfonic acid (ANS), and certain substituted phenylacetic acids, particularly fenclofenac and diclofenac. The structures and general properties of the known TBP blocking agents vary over an extremely wide range. The properties critical to operability as a TBP blocking agent in immunoassays, i.e., the ability to sufficiently dissociate iodothyronines from TBP at concentration levels insufficient to cause significant inhibition of the antibody binding reaction, are generally considered unpredictable from purely structural comparisons, although some theories of TBG blocking have been propounded (Brown and Metheany, J. Phar. Sci. 63: 1214 (1974). Ellis P.B. and Morris D.L. (MILES LABORATORIES, INC., (109.1)) found that 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid (HMS) and salts thereof are particularly advantageous TBP blocking agents for use in iodothyronine immunoassays. The blocking agent compound is included in the immunoassay reaction mixture at a concentration sufficient to release and block the binding of an analytically significant percentage of TBP-complexed iodothyronine, preferably more than 50% and usually more than 70%, while insufficient to interfere significantly with the binding of antibody with iodothyronine. While the precise concentrations of the blocking agent desired for a particular iodothyronine immunoassay will vary according to the iodothyronine under assay and the immunoassay technique followed, as well as other factors, the compound is normally used in concentrations in the reaction mixture of between about 0·1 millimolar (mM) and about 10 mM, preferably greater than about 0·25 mM, and usually less than about 5·0 mM. The HMS blocking agent is added to the assay reaction mixture as the acid or an analytically acceptable salt form thereof, e.g. the sodium, potassium, lithium and ammonium salts.

28 IMMUNOASSAY

NON-ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY 29

HMS offers particular advantages as a TBP blocking agent in immunoassays. The compound has been found to be a particularly potent blocking agent. Dissociation of over 50% of TBP-bound iodothyronine in a few minutes is possible using reaction mixture concentrations as low as 1 mM, with concentrations of only 4 mM providing over 90% dissociation. HMS is highly water soluble and has been found to be effective over a fairly broad pH range, giving versatility to the design of test kits. Additionally, HMS will exhibit no substantial inhibitory effect on the catalytic activity of many enzymes at concentrations in which it is an effective TBP blocking agent. By insubstantial inhibitory effect on enzymatic activity is meant that the rate of catalysis is not decreased more than about 70%, more usually less than 50%, and preferably less than 30%. Thus, this compound is further advantageous as a TBP blocking agent in homogeneous immunoassays wherein the label employed is a participant in an enzymatic reaction, e.g. an enzymatic substrate, an enzyme inhibitor, a prosthetic group of an enzyme, a coenzyme, or an enzyme itself, or a fragment thereof. A method for immunoassay of an analyte in a fluid includes contacting the analyte with an antianalyte to give a bound fraction has been developed by O’Connell J.P. (BECTON, DICKINSON AND COMPANY, (16.4)). The bound fraction activates the first component of complement whereby a substrate present in the fluid is modulated to provide a detectable signal. The signal may be detected to establish the presence or absence of the analyte in the fluid, or it may be quantitatively measured to determine the concentration of the analyte in the fluid. Because the enzyme is added to the assay medium in an inactive form and is activated only by antibody bound to antigen, unbound antibody does not activate the enzyme and does not interfere with signal generation induced by bound antibody. Thus, there is no need for separation of bound and unbound fractions. Furthermore, a kit of materials useful in performing the method has been designed. The kit may include an antianalyte, optionally attached to a solid support, and complement or a complement fraction which includes the first component. The kit may also include the standards for the analyte, as, for example, one or more analyte samples of known concentration, or it may include other reagents, such as a substrate or other labeled or unlabeled specific antigens, antibodies or complexes thereof useful in carrying out the assay. It may include solutions, such as saline or buffers. The components of the kit may be supplied in separate containers, as, for example, vials, or two or more of the components may be combined in a single container. Prior art clinical assays are conducted as part of the routine work of medical centers and hospitals. However, conventional methods usually take much time, require a relatively large amount of sample and involve complicated procedures.

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Therefore, it has been desired to develop an assay which requires only a small amount of sample and which gives an accurate result in a short period of time. Nishimura M. et al. (SHIONOGI & CO., LTD, (153.2)) deviced an immunoassay and a kit therefore. The proposed method involving the use, in an enzyme immunoassay, of a detecting support carrying thereon enzyme detecting reagent fixed at a first set of localised predetermined positions in detecting enzyme labels immobilized at a corresponding and second set of fixed localised predetermined positions carried on an immunological reaction support. The immobilization of the labels is the result of an immunoassay reaction. The detecting support being superimposible over the reaction support with the respective sets of positions in contact to permit detection of enzyme labels. Furthermore, a kit for use of the assay method is provided, shown in the next figures. Litai Weng et al. (SYNTEX INC., (161.1)) provide a competitive protein binding assay, where a member of a specific binding pair and a first enzyme are bound to a solid non-porous surface, particularly the wall of a container e.g. microtiter plate. A reagent is provided combining a second enzyme conjugated to a complementary member of a specific binding pair, where the amount of the enzyme conjugate which binds to the solid surface is related to the amount of analyte in the aqueous assay medium. The enzymes are related by the product of one being the substrate of the other. The amount of a detectable product which is formed as a result of turnover by one of the enzymes of a substrate produced by the other enzyme is related to the amount of analyte in the medium.

NON-ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY 31

The ligand analytes are characterized by being monoepitopic or polyepitopic. The polyepitopic ligand analytes will normally be poly(amino acids) e.g. polypeptides and proteins, polysaccharides, nucleic acids, and combinations thereof. Such combinations or assemblages include bacteria, viruses, chromosomes, genes, mitochondria, nuclei, cell membranes, and the like. For the most part, the polyepitopic ligand analytes employed, will have a molecular weight of at least about 5000, more usually at least 10000. In the poly(amino acid) category, the poly(amino acids) of interest will generally be from about 5000 to 5 000 000 molecular weight, more usually from about 20 000 to 1000 000 molecular weight; among the hormones of interest, the molecular weights will usually range from about 5000 to 60 000 molecular weight. Signal Producing System

The signal producing system will have at least three components: The first and second enzymes, where the enzymes are related by the product of the first enzyme being the substrate of the second enzyme, and the substrates of both enzymes. Additional components may be necessary for one or both of the enzyme catalyzed reactions or for interacting or reacting with the product of the second enzyme to provide a detectable signal. The signal producing systems will provide, for the most part, a product which provides for a measurement of electromagnetic radiation, such as a chromophore which absorbs light, or provides light emission, such as fluorescers or chemiluminescers. Solid Surface

The particular mode of conjugation of the mip and the enzyme to the surface is not critical, so long as each retains its properties necessary for the assay. Alternatively, one can coat the solid surface with a poly(amino acid) e.g. polylysine, and allow the poly(amino acid) to act as an intermediate binding agent between the solid surface and a polypeptide ligand or receptor and the enzyme. Kits

As a matter of convenience, the reagents can be provided as kits, where the reagents are in predetermined ratios, so as to substantially optimize the sensitivity of the assay in the range of interest. Normally, the reagents will be provided as powders, particularly lyophilized, where the fewest number of individual formulations will be employed as required by the nature of the reagents and the protocol, to minimize the number of separate measurements and additions by the user. In addition, containers, particularly microtiter plates, will be provided, where the mip and enzyme will be bound to the container wall. Included with the mip-enzyme-conjugate, where the enzyme of the conjugate is

32 IMMUNOASSAY

the second enzyme in the series, may be the substrates and cofactors necessary for the two enzymes. Also included may be various ancillary reagents. It is of interest to be able to simplify presently existing assays by simplifying protocols and maintaining the ease of conducting the assay or enhancing the result. It is particularly important that the assay reagents be provided in predetermined amounts and measurements by the user are avoided. Based on these conditions Zuk R.F. and Leeder S. (SYNTEX (U.S.A.) INC., (161.5)) developed a single step heterogeneous assay involving members of a specific binding pair (“sbp members”) and members of a signal producing system (“sps members”). The signal producing system is capable of producing a detectible signal in relation to the presence or amount of an analyte in a sample suspected of containing the analyte. Exemplary of sps members are enzymes and enzyme substrates, which react with each other to produce a signal. The improvement comprises temporarily delaying the production of the signal without subsequent reagent addition. The delay can be achieved by employing an inhibitor which can be an alternate substrate for the enzyme or a compound which reacts with the product of the enzyme and its substrate in an effective amount. In immunoassay, the immunological reagents comprise immobilized or fixed antibodies or antigens, and labelled antigens or antibodies. The rate of immunological reaction between antigens and antibodies is extremely high, and the binding constant of an antigen-antibody complex is extremely large at a level of from 107 to 1012 l/mol. Therefore, the antigens or antibodies should be analyzed quickly and quantitavely even if their concentration is low. However, there are problems in the reaction rate and the quantitative analysis because of practical problems, such as the difficulty in stirring the immobilized antibody or antigen carrier particles during the reaction, the non-uniformity of the surface area of the carrier particles, or the insufficiency of the removal of the labelled antigens or antibodies which do not form antibody-antigen complexes (“B/F separation”). To overcome such problems Ishida H. et al. (TOYO SODA MANUFACTURING CO., LTD, (172.3)) provide a carrier for a biologically active component for immunoassay of an enzymatic reaction, which comprises: a) a thermoplastic resin bead having an average diameter of from 0·05 to 20 mm, b) from 1 to 25% by weight, based on the bead, of a magnetically responsive powder bonded to the bead, and c) a polymer coated thereon in a thickness of from 2 to 30 µm, the polymer having a number average degree of polymerization of from 20 to 5000 and having functional groups capable of binding, or being activated to bind, the biologically active component.

NON-ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY 33

The carrier is prepared by depositing a magnetically responsive powder on the surface of the thermoplastic resin bead in an amount of from 1 to 25% by weight, based on the bead, and forming a layer of the polymer (c) on the bead in a thickness of from 2 to 30 µm. Uo, Junko et al. (SHIONOGI & CO., LTD., (153.1)) developed a method for assaying an immunologically active substance in a test sample which method comprises: a) preparing an immuno-active membrane of a hydrophobic polymer sheet having a number of hydrophilic spots thereon, onto which a prefixed amount of an immuno-active substance has been immobilized, b) preparing a detecting-membrane having a number of spots which contain a detectable reagent which can be detected by the catalytic effect of an enzyme at positions corresponding to those of the immuno-active membrane, c) making contact the immuno-active membrane with the test sample together with an enzyme-labelled immuno-active substance, simultaneously or successively, whereby reacting the above substances and enzyme-labelled substance competitively or non-competitively, d) adding a substrate to each spot of the immuno-active membrane, e) superimposing the detecting membrane on the immuno-active membrane so that the spots in both membranes correspond to each other, f) detecting the detectable reagent and thereby determining the amount of substance in the test sample. The hydrophilic spots are formed on the hydrophobic polymer sheet by means of a low temperature plasma-treatment method using oxygen, oxygen-containing gas or water vapour, and wherein the hydrophobic polymer, such as polypropylene, preferably is porous. The kit used for carrying out the method comprises an immuno-active membrane, a supporting board and a flame board and a detecting membrane. Serban D. and Rordorf C. (CIBA-GEIGY AG, (37)) found an assay, a purification method of serum amyloid A protein (SAA) and/or serum amyloid Pcomponents (SAP) and a kit therefore. Their method consist in that the proteins are bound to a plastic surface of a carrier or to a carrier bearing nitrated phenyl groups, whereby calcium ions or related bivalent ions have to be present for SAP binding, and that the plastic surface of a suitable carrier, preferably of polystyrene and polyolefins, is incubated with a solution containing SAA and/or SAP the presence of calcium ions or related bivalent ions such as zinc and cupric ions, or with a solution containing SAA without additional ions, and the SAA and SAP bound to the plastic surface is detected and quantified. The proposed test kits contain a carrier having a plastic surface or a carrier coated with a compound bearing nitrophenyl groups, optionally solutions of a

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compound bearing nitrophenyl groups, solutions of a monoclonal or of polyclonal antibodies binding SAA or SAP, and, if the first antibodies are not labelled with an enzyme, solutions of polyclonal, enzyme-conjugated second antibodies binding the first antibodies, enzyme substrates in solid or dissolved form, standard solutions of SAA and/or SAP, buffer solutions and optionally calcium salts or related bivalent salts such as zinc or cupric salts in solid or dissolved form, and optionally pipettes, reaction vessels, calibration curves, colour intensity tables and the like. They preferably contain a carrier coated with keyhole limpet hemocyanin bearing 2,4,6-trinitrophenyl groups. The solutions containing the proteins are subjected to affinity chromatography on solid carriers bearing nitrated phenyl groups. According to the methods for determining antigenes or antibodies in a liquid, proposed by Stocker J. (F. HOFFMANN-LA ROCHE & CO., (74.1)), the particles having the antigenes on its surface, and antibodies are incubated into the liquid. Either the antigenes or the antibodies are of known specifity. The method is characterised in that the antigene/ antibody complex is put into a container having a conically or wedge shaped cavity. At least the cavity of the container is coated with Ig-binding components which are directed against the antibodies. After centrifuging the amount of sediment is determined. The result will be an indication of the presence or absence of the antigene or antibody to be sought. Before and/ or after applying of the antigene/antibody complex into the container, the latter has to be cleaned by washing. As antigenes to be determined, particularly bloodgroup antigenes are subject of the Stocker method. As Ig-binding components Stocker uses anti-Ig as well as protein A. The liquid to be used preferably is a biological liquid or a buffered saline solution. The anti-Ig is purified by affinity chromatography. A method for determining the presence of an analyte in a sample, wherein the analyte being a member of a specific binding pair (“mip”) has been subject of research carried out by Khanna, P. et al. (MICROGENICS CORP., (108.2)). The method employs as reagents fragments of β-galactosidase comprising an N-terminal enzyme donor fragment (“ED”) and a C-terminal enzyme acceptor fragment (“EA”), and wherein the fragments form an active enzyme complex, the enzyme donor being conjugated to a mip which is cross-reactive or complementary to the analyte in binding to its complementary mip; and wherein a mip complementary to the conjugate and/or the analyte is bound to a macromolecule or support. Khanna et al. stated to have improved the method by contacting in an assay medium, the sample, the ED, the EA, the bound mip, and an enzyme substrate, wherein the bound mip and the ED conjugate are complementary and bound ED conjugate is substantially inhibited in forming an enzymatically active complex, and by determining the enzyme activity of the assay medium in comparison to an assay medium having a known amount of analyte.

NON-ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY 35

Hydrophilic spacer molecules in kits are used very often in the immunoassay art. They are coupled to a polymer surface by known type chemical reactions. Finkenaur (ORTHO DIAGNOSTIC SYSTEMS, (122.3)) found specific spacer materials including primary amine terminated poly oxyethylene/oxypropylene compotions, having a chain length of the molecule from about 10 to about 300 Angstroms. Brown et al. (ABBOTT LABORATORIES, (1.12)) provide an analytical device which advantageously can be used in enzyme immunoassays. The material comprises a porous matrix of fibers and a plurality of substantially spherical, solid particles having an average diameter of from about 0·1 to about 5 microns. The particles are retained and immobilized upon the fibers of the matrix. Preferably, the particles have on their surfaces a substance capable of reaction with the analyte in the sample, and the average diameter of the particles is less than the average pore size of the matrix. The device, in a preferred embodiment, comprises a substantially planar layer of the described material. 1.1.3 REAGENTS FOR ENZYMATIC IMMUNOASSAY METHODS Immunoassays usually employ more than one reagent. In most cases, the reagents cannot be combined in a liquid medium prior to running the assay because they contain components that would react on contact with each other. It is desirable to find a method to combine the active materials in liquid form while preventing the reagents from reacting with each other until such time as a means for releasing one or more of the reagents is provided. Generally in immunoassays the reagents are members of a specific binding pair, consisting of ligand and its complementary receptor, one of which is labelled with a member of a signal producing system. Specific binding pair members that are complementary to each other usually react upon contact. Therefore, such reagents are generally stored separately until just prior to the time an assay is conducted. One patented technique for combining interreactive agents in a single reagent is to formulate the reagents dry so that no reactions occur until a liquid sample or diluent is added. Dry reagents, however, impose some restraints on assay

36 IMMUNOASSAY

methods. Achieving a homogeneous blend and avoiding water uptake are matters of concern. Further, premature reaction must be avoided. Dry reagents are expensive and their manufacture and quality control are difficult. For example, it is generally necessary to add the sample and a diluent simultaneously and shake vigorously to assure full dissolution of the powder before the reaction has progressed significantly. Additionally, special processing devices are required. It is, therefore, desirable to develop a new assay method for determining an analyte in a sample wherein two or more specific binding members are combined in a liquid single reagent. Such a reagent avoids the need for dry reagent blending and shaking and does not require simultaneous addition of sample and diluent. A single liquid reagent decreases the time and skill needed to perform an assay. Litchfield et al., “High Sensitive Immunoassays Based on Use of Liposomes without Complement”, Clin Chem 30, (1441–1445 (1984) discuss a liposomebased immunoassay using covalently linked haptencytolysin conjugates to lyse vesicles with entrapped enzymes. US Patent nos. 3,850,578; 4,483,921; and 4, 483,929 disclose immunoreactive liposome reagents in which antigen or antibody is bound to the surface of lipid vesicles. A variety of methods for preparing lipid vesicles are known; see for example, US Patent 4,522,803 disclosing a process for preparing a freeze-dried, liposome mixture. According to Gibbons et al. (SYNTEX (U.S.A.) INC., (161.6)) a composition is provided comprising at least one specific binding pair (sbp) member and its complementary member wherein at least one sbp member is reversibly confined in a material that temporarily renders the confined sbp member incapable of binding with its complementary sbp member. At least one of the sbp members is bound to a member of a signal producing system capable of producing a detectable signal in relation to the amount of analyte in the sample. The confinement is reversed, any remaiming members of the signal producing system are added, and the signal produced in relation to the amount is measured. The binding method may be selected by considering the functional groups of both substances. Such functional groups include, amino groups, carboxyl groups, hydroxyl groups, thiol groups, imidazole groups, phenyl groups, etc. The binding of amino groups, may be carried out by many methods such as the diisocyanate method, the glutaraldehyde method, the difluorobenzene method, and the benzoquinone method, etc. As the method to bind an amino group and a carboxyl group, the peptidebinding method of carboxyl group to succinimido ester, the carbodiimide method, the Woodward reagent method are known. The periodate oxidation method (Nakane method) where a bridge between amino group and sugar chain forms is also utilized. In the case of using a thiol group, for example, a carboxyl group is first converted to a succinimido ester, and this ester group is then allowed to react with cysteine to introduce the thiol group, and both thiol groups are bound by using a thiol-reactive bifunctional cross-linking reagent

NON-ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY 37

such as phenylene-bismaleimide. As the method of utilizing a phenyl group, the diazotization method and the alkylation method are utilized. Other than the above, a suitable method may be selected from the various methods described in “Method in Immunology and Immunochemistry” (C.A. Williams et al., 1976, Aca-demic Press N.Y.) and “Koso Meneki Sokutei-ho” (E. Ishikawa et al., 1978, Igaku-shoin, Japan). The molar ratio of the combination is not limited to 1:1 and suitable ratios can be easily selected. After the binding reaction, the macromolecular enzyme produced is purified by gel filtration, ion-exchange chromatography and affinity chromatography, and lyophilized, if necessary. The antibody against the ligand to be measured (anti-ligand) and the antibody against the enzyme (anti-enzyme) may be produced according to known methods of producing an antibody. Yoshihiro Ashihara et al. (FUJIREBIO K.K., (60.1)) found reagents containing an anti-ligand bound to an anti-enzyme and methods for using them in an immunoassay. Their methods comprise: a) combining in a buffered solution the biological sample; an enzyme or enzyme complex, the enzyme complex comprising an enzyme bound to a first macromolecular compound; a substrate for the enzyme; and an antibody complex, the antibody complex comprising antiligand bound to anti-enzyme and a second macromolecular compound bound to at least one of the antiligand and the anti-enzyme; b) measuring the activity of the enzyme or enzyme complex in the buffered solution; and c) comparing the activity of the enzyme or enzyme complex in the bufferred solution with the enzyme activity of a standard solution containing a known amount of the ligand. The reagents used for carrying out the method consist of an antiligand bound to anti-enzyme and a first macromolecular compound bound to at least one of the anti-ligand and the anti-enzyme. The macromolecular compounds are selected from the group consisting of glucose6-phosphate dehydrogenase, hexokinase, αamylase, malate dehydrogenase, alkaline phosphatase, peroxidase, βgalactosidase, creatine kinase, ribonuclease, and penicillinase. Sugimura M. et al. (DAIKIN INDUSTRIES, LTD. (48)) found a reagent for detecting an antigen-antibody reaction in the form of a latex comprising as its dispersed substance a fluorine-containing polymer up to 142 in refractive index and having a protein adsorbed thereon. The protein is selected from gamma globulin, choriogonadotropin, thymonucleic protein, thyroglobulin, anti-reactive protein antibody, antifibrinogen antibody and anti-gamma globulin antibody. The fluorinecontaining polymer preferably is up to 1·38 in refractive index. The

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fluorine-containing polymer may also be a copolymer of tetrafluoroethylene and at least one of hexafluoropropene and perfluoroalkyl vinyl ether, or a copolymer of vinylidene fluoride and at least one of hexafluoropropene and tetrafluoroethylene. A biologically active composition which comprises an immobilised antigen or antibody phase and an immobilised enzyme, enzyme inhibitor or enzyme activator phase, has been developed by Kasahara Y. et al. (FUJIREBIO K.K., (60. 3)). The composition comprises a single polymer immobilisation phase on which the antigen or antibody phase and enzyme, enzyme inhibitor or enzyme activator phase are immobilised at different locations. The composition may also comprise two polymer materials bonded to each other, one polymer phase having the antigen or antibody immobilised thereon and the other having the enzyme, enzyme inhibitor or enzyme activator immobilised thereon. The polymer materials are in particulate form, with particles of the two polymer materials being mixed together. The antigen or antibody phase and the enzyme, enzyme inhibitor or enzyme activator may also be immobilised on copolymerisable manomers prior to their copolymerisation to form the polymer. A dry analytical element for the determination of creatine kinaseMB comprising an absorbent carrier material, and containing an analytical composition comprising creatine phosphate, and an indicator composition which is capable of providing a detectable change in response to the reaction of creatine phosphate or its reaction product, was found by Findlay J. and Wu A.L. (EASTMAN KODAK COMPANY, (54.3)). The element is characterised by comprising adenosine-5′-diphosphate in an amount of from 1 to 3 millimolar, and a combination of adenylate kinase inhibitors consisting essentially of \ a) P1, P5-di(adenosine-5′)polyphosphate present in an amount of at least 0·3 millimolar, and b) adenosine-5′-monophosphate present in a molar ratio to adenosine5′diphosphate of at least 10:1. The absorbent carrier material is a porous spreading zone carried on a support. The spreading zone contains antibodies for creatine kinase-MM. The element described so far was used for a method for the determination of creatine kinase-MB in an aqueous liquid comprising the steps of A) in the presence of at least one antibody for creatine kinase-MM, contacting a sample of a liquid suspected of containing creatine kinaseMB with an analytical composition comprising creatine phosphate, an indicator composition which provides a detectable change in response to the reaction of creatine phosphate or its reaction product,

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adenosine-5′-diphosphate present in an amount of from 1 to 3 millimolar, and a combination of adenylate kinase inhibitors consisting essentially of: a) P1, P5-di(adenosine-5′)polyphosphate present in an amount of at least 0·3 millimolar, and b) adenosine-5′-monophosphate present in a molar ratio to adenosine5′diphosphate of at least 10:1, and B) determining the detectable change resulting from the presence of creatine-MB. As a result of research carried out by Saxema B.B. (CORNELL RESEARCH FOUNDATION, INC., (44)), a reagent for use in a “sandwich” enzymeimmunoassay comprising a polymer of bioactive substancespecific antibody: enzyme conjugate, wherein the antibody is conjugated to the enzyme by means of a heterobifunctional cross-linking agent. The bioactive substance employed can be a hormone, drug, or any other substance which has biological activity, e.g. LH, hCG, TSH, or FSH. The enzyme employed can be any well-known enzyme marker employed for “sandwich” enzyme-immunoassay such as alkaline phosphatase and horse radish peroxidase. The bioactive substance-specific antibody can be prepared by wellknown means. An example of the heterobifunctional cross-linking agent which can be employed is m-maleimidobenzoyl N-hydroxy-succinimide ester (hereafter “MBS) obtained from Pierce Chemical Co. Monomers of the bioactive substance-specific antibody:enzyme con jugate can be polymerized to form a polymer thereof by means of polymerizing agents such as glutaraldehyde or carbodimide. Samples to be assayed are typically derived from body fluids, for example, blood, urine, serum or spinal fluid. The bioactive substance-specific antibody can be immobilized by well-known means, such as absorption or covalent linkage, on a substrate such as polystyrene tubes, polystyrene beads, glass beads, powder or sticks. Polystyrene beads generally provide a more uniform coating and consequently higher reproducibility as compared to glass beads to which antibody was covalently linked via amino groups (see Fukunaga T., Rathnam P., Landesman R. and Saxena B.B., Obstetrics Gynecology 61:102 (1983)). The use of a solid phase provides the separation of unreacted, excess reagents or undesirable interfering proteins in the body fluid sample and avoids their interference during the next step of the reaction. Also, in the assay of the present invention, the body fluid sample first reacts with the antibody on the solid phase and is then removed,

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thus there is no interference of urine during the step, whereas in the liquid radioimmunoassay system, the body fluid is present throughout the incubation period. The use of immunoglobulin purified from the antisera eliminates the absorption of other serum proteins to the solid phase, thus enhancing the sensitivity, specificity and speed of binding. The particular assay employed for measuring the concentration of the enzyme will, of course, depend upon the enzyme employed. For example, a colorimetric assay for determining the concentration of alkaline phosphatase is disclosed in Fukunaga T., Rathnam P., Landesman R., and Saxena B.B., Obstetrics Gynecology 61:1–2 (1983). In an immunoassay method for the determination of (i) a bacterial polypeptide capable of binding to the Fc portion of an immunoglobulin and/or (ii) the high affinity homologous antibody to the polypeptide Berglund L. and Inganäs, M.W. (PHARMACIA AB, (132.2)) made an improvement by using an antibody preparation directed against the polypeptide and having antibody activity under conditions such that the immunoglobulin potentially binding to the polypeptide will substantially not bind to the polypeptide, the immune reaction between the antibody preparation and the polypeptide being carried out under such conditions. The polypeptide is selected from the group consisting of Protein A and Protein G, so that Protein A or its homologous antibody respective Protein G or its homologous antibody will be determined. The immune reaction is carried out at a pH below 4·0, preferably below about 3·5. The method can be carried out using a competitive immunoassay method or a heterogeneous immunoassay method. A system has been devised which uses a cyclic process to amplify the antigenantibody reaction. In this system, alkaline phosphate catalyses the dephosphorylation of nicotinamide adenine dinucleotide phosphate (NADP), producing nicotinamide adenine dinucleotide (NAD) which acts as a coenzyme for alcohol dehydrogenase in the presence of an excess of the substrate ethanol to produce NADH; the reduced and oxidized nucleotides are catalytically cycled back and forth by diaphorase whereby the second substrate paraiodonitrotetrazolium violet forms a formazan dye in amplified amounts. Thus, the sensitivity of the system can be increased by a factor of 250, and as little as 10–18 moles of alkaline phosphatase has been detected in this system. Nilsson et al. (SOCKERBOLAGET AB, (156)), however, have found that although a good sensitivity and reproducibility is obtained by this amplification method when proteins are analysed, the sensitivity is not sufficient for analysing the presence of pathogenic organisms, e.g. bacteria, viruses or fungi, or components thereof in, for instance, pathophysiological samples. Their method of detecting the presence of a cell, virus or circulating body component or antibody thereto in a sample, which comprises:

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a) contacting the sample with a reagent bound to a solid support, which reagent is capable of binding a cell, virus or circulating body component or an antibody thereto; b) contacting the reagent bound to a solid support which in step a) was contacted with the sample with the same or another reagent as that used in step a), which reagent is coupled to a macromolecular waterinsoluble carrier to which a substance is multivalently bound, the substance being capable of initiating a reaction the product of which is detectable, or which reagent is coupled to a substance multivalently bound to a macromolecular waterinsoluble carrier, the substance being capable of initiating a reaction the product of which is detectable, and c) the reagent bound to the solid support which was contacted with the sample in step a) and with the reagent coupled to the macromolecular waterinsoluble carrier or substance in step b), is reacted with a compound which is a precursor of a detectable reaction product or which is capable of initiating a reaction cascade the end product of which is detectable, to form a detectable reaction product, whereby the presence of any cell, virus or circulating body component or antibody thereto which has become bound to the reagent steps a) and b) is detected. Homogeneous immunoassay employing double ligand binding conjugates comprising an anti-idiotype binding partner was subject of research by Li (ORTHO DIAGNOSTIC SYSTEMS INC., (122.1)). His reagent system comprises: an insolubilized ligang binding partner; a double binding partner conjugate comprising an anti-idiotype binding partner, capable of blocking the ligand from binding with the insolubilized ligand binding partner, coupled to a second binding partner; and an insolubilized label for which the second binding part is specific. The label is selected from the group of labels having a characteristic spectral emission consisting of fluorescent molecules, phosphorescent molecules, and chemiluminescent molecules and wherein binding of the second binding partner to the label results in a detectable change of the labels’s characteristic spectral emission. The label is an enzyme whose activity upon a substrate to produce a detectable product is altered upon binding of the second binding partner to the enzyme. Kasper (BOEHRINGER MANNHEIM GMBH, (24.8)), developed a reagent for determining a reaction partner of an immunological reaction. It has a substance R3 bound to a solid phase, at least one unmarked receptor R2 bindable to R3 and to another reaction partner, and a marked specific receptor R1, and physically

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separated therefrom, a further amount of the substrate R3 bound to a solid phase, an unmarked receptor R′2 which does not react with the other reaction partner of R2 and a further amount of a marked specific receptor R1· The receptor R2 and R′2, respectively, are bound through an anti-lg-antibody to the solid phase. Both receptors R2 and R′2 are haptenized and are bound to the solid phase through an antibody which is directed against the hapten. Note: Though the above reagent has been described for enzymatic markings, it is also applicable to radio, fluorescence—and colorometric markings. To protect an enzyme against inactivation on preparation, by reverse phase evaporation in the presence of organic solvent, and on storage as an aqueous suspension, Kung and Canova-Davis (KUNG, V.T. and CANOVA-DAVIS, (94)), prepared a liposome assay reagent for determination of an analyte in a homogeneous immunoassay. The reagent includes a suspension of oligolamellar lipid vesicles containing encapsulated glucose-6-phosphate dehydrogenase (G6PD), at a specific activity of between about 1–10 units mu-mole vesicle vesicle lipid, and glucose6-phosphate (G6P) at a concentration of at least about 5 mM. The next figure shows a graphic representation of turbidity against time for several antigen/antibody reaction. It symbolizes research conducted by Jefferis and Steensgaard (THE UNIVERSITY OF BIRMINGHAM, (180)), to precipitate antigen/anti-bodies using monoclonal antibodies, the monoclonal antibodies being selected so as to be specific to two distinct antigenic binding sites (L or C 2 or C 3) on a protein (IgG) in a sample under test. The proportions of sub-populations of immunoglobulins (IgG kappa, IgG lambda) in a sample is determined by reacting the sample with a combination of antibodies (II and IV) both of which are specific to the heavy chains (H) of both sub-populations (IgG kappa, IgG lambda) and reacting the sample with an antibody combination (I and II) specific

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to the heavy chain (H) and to an antigenic determinant expressed by only one (IgG kappa) of the sub-populations. Both being linked by disulphide bonds D. A crystalisable fragment Fc of both heavy chains H comprises C gamma 2 and C gamma 3 regions of equal lengths. The fragment Fc may be separated from the remainder of the molecule by enzyme action, and this fragment Fc may be subjected to a further enzyme action to leave a residual fragment pFc′ whose extent is effectively that of the C gamma 3 region. Each chain in the Fab fragment has a variable region V and a constant region C. 1.1.4 INSTRUMENTS AND DEVICES FOR CONDUCTING ENZYMATIC IMMUNOASSA Y It is known to conduct binding assays on a strip of material provided with a plurality of reagent zones, in which a developing solution forms a solvent front which passes along the strip by capillary action picking up and facilitating reaction between a sample and assay reagents located at the reagent zones (see for example British patent specification nr. 1,589,234). A feature of such strips is the existence of a test location at which, under certain conditions determined by the assay protocol and the sample composition, a labelled reagent becomes immobilised, giving an indication of the assay result. In early assays, the labelled

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reagent was a binding partner or analogue of the analyte to be measured, labelled with a radioactive isotope. Such assays require instrumentation to detect the level of radioactive label and may present health risk problems. A solution to this has been the use of enzyme labels which produce a characteristic signal (such as a colorimetric signal) with an appropriate substrate. A significant problem in the design of such so-called “dipstick” enzymelabelled binding assays is the application of the appropriate enzyme substrate in order to produce a detectable signal. The signal may be developed by adding substrate to the appropriate position on the reagent strip after allowing the assay to proceed to completion. Alternatively, the appropriate part of the strip may be removed and chemically analysed. All of these represent steps which would be at least inconvenient, if not impossible for home use of the assay. Baker T.S. et al. (BOOTS—CELLTECH DIAGNOSTICS LTD., (26)) developed a device for performing an enzyme-labelled binding assay which comprises an absorbent material and a developing solution, wherein the absorbent material is provided with a plurality of reagent zones including an indicator reagent zone, and is capable of transporting the developing solution by capillary action sequentially through each reagent zone, and wherein the indicator reagent zone includes a reagent capable, directly or indirectly, of immobilising an enzyme-labelled reagent in an amount dependent upon the assay result, characterised in that the developing solution includes a signal producingsubstrate for the enzyme. The substrate moves slower through the absorbent material than the enzyme-labelled reagent or any compound of the enzymelabelled reagent formed in the assay. The absorbent material is suitably in the form of an elongate strip provided with transverse reagent zones. The device is useful for performing immunoassays including immunometric assays and dual analyte assays. Prior devices for the detection of an antigen or hapten employ an antibody attached to a solid support which is contacted sequentially with static volumes of test liquid, conjugate and substrate/indicator solutions. A period of incubation must be observed for each of these contacting steps as the reactions involved are diffusion controlled. That is, the reactions take place only at the surface of the solid support and sufficient time must elapse to enable enough antigen to migrate to the reaction vessel wall to give a positive test result. Increasing the surface area to volume ratio by carrying out these reactions in a tube having a small bore reduces incubation periods to about ten minutes each. A device developed by Chandler H.M. (ALLELIX INC., (4.1)) employs a method whereby a continuous flow of the various reactive solutions are passed through a tube having the antibody affixed thereto so that the principles of affinity capture and concentration are utilized. The reaction at the tube wall is forced to completion quickly by continuously bathing the solid reactant with a solution having a constant concentration of the co-reactant. This also means that

NON-ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY 45

the sensitivity of the assay is increased since a much lower concentration of coreactant in the test sample can be detected by this flow. The next drawing shows a perspective view of the Chandler device. A great number of medical tests or assays are now available, but often the physician or patient must wait hours or even days before such test results can be made available. Samples for testing are often sent to a testing laboratory where skilled technicians employ specially prepared reagents and expensive equipment to obtain the desired data. Assays conducted in this manner are, therefore, expensive as well as slow. In non-medical applications, test results are often needed quickly for agricultural applications, such as testing for pesticide residues, or for industrial applications, such as testing for the presence of harmful chemicals in the workplace or in products. Anderson H.B. (ALLELIX INC., (4.2)) developed a device comprising a disk having a thin flexible membrane applied to one side thereof, the membrane defining a conduit and a plurality of reagent reservoirs. The reservoirs are isolated from one another or from the conduit by frangible seals. An assay tube having an assay reagent bonded to the inner wall thereof is attached to the exit end of the conduit. An injector is provided for injecting a test sample into the conduit for flowing thereof through the assay tube. Means are also provided for forcing the contents of each reservoir through the conduit and assay tube in the desired order by causing the rupture of each frangible seal sequentially. The drawing shows an exploded view of the device. An apparatus for performing automated heterogeneous immunoassay for the detection or determination of antigenic or haptenic substances or antibodies in a plurality of samples has been designed by Chandler H. (COMMONWEALTH SERUM LABORATORIES COMMISSION, (41)). It comprises (a) a plurality of capillary tubes (12), each of the capillary tubes having antibodies or antigenic or haptenic substances attached to the internal surface thereof; (b) a motor for passing each of the capillary tubes, in sequence, to a plurality of operation stations; (c) means at a first operation station (I) for admitting individual samples, in sequence, to each of the capillary tubes as it is passed to the first operation station; (d) means at one or more subsequent operation stations (II), (III), for admitting immunoassay reagent to each of the capillary tubes as it is passed to the subsequent operation station(s); and (e) means at a final operation station (IV), (V), detecting or determining the result of the immunoassay in each of the capillary tubes as it is passed to the final operation station. The apparatus designed by Valkirs G.E. et al. (HYBRITECH INC, (75.1)) comprises, as a first member, a membrane or filter to which is bound antibody, preferably a monoclonal antibody against the target antigen, or which is capable of separating from the sample being analyzed cells or cellular debris with which the antigen being assayed is associated to thereby fix the antigen to the porous membrane. The apparatus further comprises, as a second member, an absorbent

46 IMMUNOASSAY

NON-ANTIGEN SPECIFIC METHODS, APPARATUS AND KITS FOR IMMUNOASSAY 47

member having capillary path-ways therethrough generally transverse to its upper and lower surfaces. The second member is in capillary communication with the porous first member and is selected to have a capillary pore size so as to induce flow of liquid through the first member without the use of external means when the hydrostatic pressure of the sample and subsequent addends used in the assay are not sufficient to induce flow through the first member. The second member may also provide support for the first member. A cylindrical container (10) is provided having an upper opening (12) defined by sidewall (14). The container may be made of glass or a suitable plastic material. As in the figure, container (10) also has a lower opening (16), in which

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is inserted a removable plug (18), to permit insertion of the porous member (20), a circular membrane or filter disc, and an optional member (21), which rest on cylindrical absorbent member (22), which is also inserted through opening (16). A portion of container (10) is constricted to provide an integral funnel to direct sample onto the member (20) and to assure that effective washing of sample and other addends onto the member (20) is accomplished. See figure below. The next figure is a cross section of a device which acts as a protein support modified into pig skin gelatin which adheres to a plastic film. The film is adapted to reversably immobilise a biologically active substance. The device has been developed by Boitieux J.L. and Thomas D. (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE (INSERM), (82.1)). The gelatin comprises at its surface accessable locations constituted by ligands which are adapted to reversably combine with an antibody or a biologically active substance. The support onto which antibodies are fixed may form a membrane having am enzymatic activity and which can be combined with an electro-chemical catcher. Sample solids removal Biochemical assays, and in particular, immunoassay and so-called “DNA probes”, have been carried out in very many different formats. A format which is very popular within the industry is to carry out immunoassay reactions in a well on a test plate, so as to bind to the wall of the well a labelled substance, in an amount which depends upon the amount of a test substance originally present in a sample.

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The usual method of detection in such assays is by spectrophotometric detection of a coloured reaction product, fluorescence, or, more recently, by electrochemical measurements, as disclosed, for example, in International Patent Application No. WO86/03837. Such formats can give very good quantitative results, but generally require complex apparatus for implementation and are not convenient to use for single test samples. In recent times, a number of proposals have been made to carry out biochemical assays, and in particular immunoassays, in an absorbent matrix material, within which a specific binding species, such as an antibody, is localised. An example of such a system is illustrated in US-PS 4,615,983. The nature of the surface of the materials employed in these types of matrixes enables a wide range of non-specific binding reactions to take place in addition to the desired specific binding reactions. For the same reasons, however, it is difficult to wash excess immunological reagents from the matrix, after the specific binding reaction has taken place. Stanley and Johannsson (IQ (BIO)LIMITED, (86A)) discovered that a device can be constructed in which a matrix for holding a ligand, usually the sample, in contact with a reaction surface may be combined with a filter, to enable solids present in the sample to be removed, and prevent them from coming into contact with the test surface. The apparatus proposed by Stanley and Johannsson consists of a reaction surface adapted to bind a first biochemical ligand, a liquid absorbent member adjacent the reaction surface adapted to absorb washing solution applied to the reaction surface, a filter matrix adapted to contain a

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second biochemical ligand, capable of being specifically bound with the first biochemical ligand, and to retain the second ligand in contact with the reaction surface to overlie the reaction surface in close contact therewith. The filter matrix is removeable to facilitate washing of the reaction surface. An enzyme labelled substance is used which is conjugated to a biochemical ligand. The next figure is an exploded view of Stanley’s apparatus. The absorbent matrix was Whatman 541 Chromatography Paper, which is an inert cellulose matrix, to which conjugates of alkaline phosphatase do not bind non-specifically. A top layer 3 of the device in cludes a hole 4 defining a target area 5 of the absorbent filter matrix 2. A glass fibre filter 6 covers hole 4, and a label 7 is provided for indicia, to indicate the subject of the tests. Non-absorbent reaction surface 1 is provided with a coating of an antibody, shown schematically as 10. Coating of the polystyrene sheet with the antibody is carried out by any conventional method, for example as disclosed in Eu-PA 132 948. After being coated with the antibody, surface 1 is treated with bovine serum albumin (BSA) and other agents to serve as blocking agents for reducing non-specific binding. In an embodiment of the apparatus for determining levels of progesterone in a sample, for example for pregnancy testing, the antibody 10 may be antiprogesterone, and a conjugate of progesterone with alkaline phosphatase is provided in matrix layer 2. When a sample to be analysed is introduced onto the target area 5 of filter matrix 2, the conjugate is dissolved, and binds specifically with antibody 10, in competition with the progesterone in the sample. Filter 6 reduces the amount of

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fatty and similar materials reaching matrix 2, and filter matrix 2, in turn, reduces the amount of these substances reaching surface 1. In the next figure a measurement vessel is shown, which is used for a method for carrying out immunoassay by using enzyme labels developed by Harjunmaa H. and Luotola J. (LAB-SYSTEMS OY, (96.2)). The inside face is covered with an antigen 1 and which also acts as the reaction vessel. Into the measurement vessel, the sample has been administered, which includes the antibody 2 to be determined and, moreover, the corresponding antibody 3 labelled with an enzyme. In the immunological reaction, labelled antibody and unlabelled antibody have adhered to the antigen

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present on the bottom and walls of the measurement vessel by means of an immunological bond in the proportion in which they are present in the reaction solution. The enzymatic immunoassay for estimating the concentration of antigen or antibody, developed by Higo Y. and Kamada S. (TOYO SODA MANUFACTURING CO, LTD., (172.2)), bringing an antigen-antibody complex, which is labelled by an enzyme and is present heterogeneously in a solution, into contact with a substrate where pH of the solution is so adjusted as to be suitable for the enzyme to be activated and also for measuring the fluorescence of the substrate, measuring the time-variation of fluorescent intensity of the substrate produced by the enzyme reaction and estimating the concentration of the antigen or the antibody from the slope of the substantially straight linear portion, as estimated from at least two points, on a characteristic curve representing variation of the fluorescence intensity. The curve is shown in the figure. while the test apparatus used to carry out the assay is shown in the figure.

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An immunoassay for determining qualitatively or quantitatively the presence of an analyte in a sample by means of specific binding, wherein the sample is contacted with a moveable solid phase carrier material, e.g. magnetic particles (620, see figure) on which is immobilised a first binding reagent having specifity for the analyte and with a labelled reagent which can participate in either a “sandwich” or a “competition” reaction with the first reagent in the presence of the analyte and following an incubation period sufficient to allow the reaction to take place is the result of research by Davis P.J. et al. (UNILEVER N.V., (175. 1)). The carrier material is moved within the assay medium (618) to a location adjacent a signal sensing means (602), the label generating a signal, such as chemiluminescent light, continuously in the assay medium and the magnitude of the signal generated in the vicinity of the sensing means being used as a measure of the extent to which the binding reaction has occurred. Preferably the assay medium incorporates a masking or quenching agent which suppresses signal generated in regions of the assay medium remote from the signal sensing means. The label used may be glucose oxidase, the assay medium contains glucose and a chemiluminescent 2,3-dihydro-l,4-phthalazinedione such as luminol, the consumer reagent is catalase enzyme, the reagent associated with the second population of localisable particles is horseradish peroxidase, and the assay medium optionally also an enhancer such as incorporates paraiodophenol.

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1.2 FLUORESCENCE METHODS, REAGENTS, KITS AND INSTRUMENTS THEREFORE 1.2.1 FLUORESCENCE IMMUNOASSAYS Skilled persons in the art on immunoassay procedures know that every assay method has its advantages and its drawbacks. Fluoroimmunoassay (FIA) provides direct detection of the label and is readily adaptable to homogeneous assay procedures. However, known homogeneous FIA methods using organic fluorochromes, such as fluorescein or rhodamine derivatives, have not achieved the high sensitivity of RIA or EIA, largely because of light scattering by impurities suspended in the assay medium and by background fluorescence emission from other fluorescent materials present in the assay medium. The present chapter has to do with various improvements of the FIA. Cell analysis Methods for detecting cells by utilizing the immunological competence of blood cells such as lymphocyte and erythrocyte or tissue cells have begun to be applied and investigated very lately. Lymphocytes include T cell (T lymphocyte) and B cell (B lymphocyte). Both of them are originated from a common marrow stem cell, and they function in cooperation with each other and cause immunoreaction in cooperation with macrophage, monocyte, multinuclear leukocyte, etc. In various immunological diseases, measurement of the abnormality in quantity of T cell playing an important role in cell-mediated immunity and B cell producing humoral antibody is said to be useful for diagnosis or grasping the pathology of the diseased. As a first prior art showing a method for quantitating cells in blood, there is known Nippon Rinsho (Japanese Clinic), Vol. 40, Special Fall Number, p. 985 (1982). In this publication methods for quantitating T cells and B cells are described. In this first prior art, T cells are quantitated by utilizing their property of forming a rosette with sheep erythrocyte (SRBC) in a test tube. Alternatively, T cells are dyed by the membrane fluorescent antibody technique by use of an antibody reactive specifically to T cell (anti-T cell antibody). B cells are quantitated by utilizing their property of forming a rosette with mouse erythrocyte (MRBC) in a test tube. Alternatively, since T cell has immunoglobulin on the membrane surface, fluorescence-labelled anti-human immunoglobulin serum is applied and positive cells are observed under a fluorescence microscope (the membrane fluorescent antibody technique).

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In the above mentioned methods utilizing rosette formation reaction, much labour and a long time are required for quantitating T cells or B cells because of, for example, troublesome manual operations, a long time required for rosette formation reaction, and need for microscopic examination using a counting chamber. Furthermore, the membrane fluorescent antibody technique is disadvantageous in that the detection sensitivity is low because the number of molecules of a label compound (a fluorescent substance) which can be attached to one molecule of antibody is limited. A second prior art is disclosed in US-PS 4,510,244. This prior art that an antigen having fluorescent particles bound thereto is combined with hybridoma cells, followed by introducing a liquid containing the resulting immune complex into a cell sorter to isolate individual fluorescencelabelled cells from unlabelled other cells. Even by this method, cells to be analyzed cannot be detected with sufficient sensitivity. Imai K. and Nomura Y. (HITACHI, LTD., (70.3)) found a method for cell analysis which makes it possible to detect specific cells selectively with high sensitivity. In accordance thereto, cells to be analyzed are combined with an antibody cognate therewith carrying micro-capsules as a label, after which microcapsules bound to the cells to be analyzed are detected, whereby the labelled cells to be analyzed are measured. The label substance used is micro-capsules containing a substance having optical characteristics. The method is characterized in that in the detection, the cells to be analyzed are detected without breakage of the micro-capsules. In a preferred embodiment, plural types of cells to be analyzed can be detected for the same sample by using plural kinds of micro-capsules. In this case, different kinds of micro-capsules have antibodies cognate with different types of cells to be analyzed attached thereto and contain different substances having optical characteristics. According to Imai and Nomura, a substance having optical characteristics which enables optical detection or a precursor thereof is encapsulated in microcapsules, and the micro-capsules are combined with cells to be analyzed by utilizing the immunological competence of the cells, whereby discrimination of the cells is facilitated. Since a large amount of substance having optical characteristics can be encapsulated in micro-capsules, light emission or colour production due to the optical substance and the like is increased when the microcapsules are combined with cells, so that discrimination of cells to be analyzed from the surroundings (the background) is facilitated. As the micro-capsule, a lipid membrane such as liposome and the like can be suitably used but the micro-capsule is not limited thereto. Any micro-capsule may be used so long as it can contain a sufficient amount of a substance having optical characteristics or a precursor thereof. As the optical substance as indicator at the time of detection which is encapsulated in the micro-capsules,

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there can be used fluorescent substances, dyes, chelate complex compounds, precursors thereof, etc. The fluorescent substances are preferably water-soluble ones such as fluorescein isothiocyanate isomer (FITC), carboxyfluorescein, phycoerythrin (PE), tetramethylrhodamine (TRITC), rhodamine, eosin (2′,4′,5′, 7′-tetrabro-mofluorescein) and acridine orange (3,6-bis-dimethylaminoacridine). As dyes and chelate complex coumpounds, there can be preferably used watersoluble dyes and water-soluble chelating agents such as complex of nitrosoPSAP and FE2+, eriochrom black T, 2-(2-thiazolyazo)-4(methyl)-5-sulfomethylaminobenzoic acid (TAMSMB), tetramethylrhodamine isothiocyanate (RITC), rhodamine B, oxamine red, acid orange I, xylenol orange, methylxylenol blue, methylthymol blue, 5-Br-PAPS, 5-Br-PASS, dimethyl-sulfonazo-III, chromazurol B, nitro-PAPS and basophenanthroline. As the precursors, pH indicators can be preferably used. In this case, colour production, whereby cells to be analyzed can be detected, can be caused by changing the pH value of a suspension of the combined product of cells to be analyzed and microcapsules, without breaking the microcapsules. However, certain fluorescent compounds cease to emit fluorescence owing to selfquenching in some cases when their concentration is high. Therefore, in encapsulating the such compound in the microcapsules, its concentration is properly selected. The identification and suppression of human T cell classes and subclasses has previously been accomplished by the use of spontaneous antiantibodies or selective antisera for human T cells obtained by immunizing animals with human T cells, bleeding the animals to obtain serum, and adsorbing the antiserum with (for example) autologous but not allogeneic B cells to remove antibodies with unwanted reactivities. The preparation of these antisera is extremely difficult, particularly in the adsorption and purification steps. Even the adsorbed and purified antisera contain many impurities in addition to the desired antibody, for several reasons. Kung P.C. (ORTHO PHARMACEUTICAL CORPORATION, (123.1)) discovered a novel hybridoma which is capable of producing novel monoclonal antibody against an antigen found on essentially all normal human peripheral T cells. The antibody so produced is monospecific for a single determinant on normal human T cells and contains essentially no other anti-human immuneglobulin, in contrast to prior art antisera (which are inherently contaminated with antibody reactive to numerous human antigens) and to prior art monoclonal antibodies (which are not monospecific for a human T cell antigen). Moreover, this hybridoma can be cultured to produce antibody without the necessity of immunizing and killing animals, followed by the tedious adsorption and purification steps necessary to obtain even the impure anti-sera of the prior art. The hybrid is formed by fusing splenocytes from immunized Balb/cJ mice with P3X63Ag8Ul myeloma cells.

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The figure shows the fluorescence intensity of T cells, B cells, null cells and macrophages. More recently, methods have been developed wherein the complexes formed between specific binding protein and bindable substance are visualized by labelling the complexes directly or indirectly with colloidal metal particles, particularly gold particles. Depending on the circumstances, these particles can be detected e.g. by direct visual examination, by microscopic or spectrofotometric techniques. A description of the “sol particle immuno-assay” (SPIA) technique and of specific applications and improvements thereof will be found e.g. in US-PS 4,446,238. Use has also been made of a latex agglutination method as described e.g. in US-PS 3,857,931 which has certain advantages over the haemagglutination method. Indeed, the red blood cell carriers are themselves antigenic and often cause specific agglutination which interferes with the desired antigen-antibody reaction and renders the efficiency of the determination to a large extent dependent on the nature and composition of the material under analysis. The synthetic polymer latexes employed are devoid of this disadvantage. However, their sensitivity is in general insufficient. The method of de Jaeger N.C.J. et al. (JANSSEN PHARMACEUTICA N.V., (87)) differs from the previous methods essentially by the fact that as a marker to detect or determine the complex formed between a specific binding protein and the corresponding bindable substances there are used latex particles which can be detected visually due to their capability to absorb light in the visual spectrum, or to emit light after irradiation. The former detection is based on colour, the latter on fluorescence. Preferably the marker consists of coloured or colourable latex particles and the determination is based on the colour characteristics of the latex particles as such or, in the instance that colourable latex particles are used, on the colour characteristics of the coloured particles derived therefrom. The colour, respectively fluorescence signal is, if necessary after development, easily detected and optionally quantified.

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Microbead quenching Zuk R.F. and Litman D.J. (SYNTEX (U.S.A.) INC., (161.4)) provided a specific binding pair assay involving a fluorescent bead which is conjugated a member of a specific binding pair and, preferably, also a label, normally a catalyst, which is a component of a signal producing system, which system also includes the fluorescent bead. A second conjugate involves a catalyst, normally an enzyme, and a member of the specific binding pair, so that the amount of catalyst which binds to the fluorescent bead through the intermediacy of the specific binding of the specific binding pair members is related to the amount of analyte in the assay medium. Upon becoming bound to the particle, the catalyst, itself or in conjunction with the label conjugated to the particle, produces a product which results in quenching of the fluorescent bead in proportion to the amount of second conjugate catalyst bound to the bead. Unbound catalyst does not produce a product that causes quenching. Thus, the residual fluorescence will be related to the amount of analyte in the medium. The assay reagents include particles which are small inert beads, which are highly fluorescent due to the presence of a plurality of fluorescent chromophoric groups. A member of a specific binding pair and preferably, also a component of a signal producing system, are conjugated to the fluorescent particle. Also included as an assay reagent is a catalyst, normally an enzyme, conjugated to a specific binding pair member, either directly or indirectly. When the catalyst binds to the fluorescent particle through the intermediacy of binding of specific binding pair members, the catalyst, by itself or in combination with the label conjugated to the particle, produces a product which binds to the particle resulting in the quenching of the fluorescent beads. The reduction in fluorescence of the particles can be related to the amount of analyte in the assay medium by employing standards having known amounts of analyte. The analyte will be a member of a specific binding pair consisting of ligand and its homologous receptor. The solid phase particles or beads will be bound, directly or indirectly, covalently or non-covalently, to one of the members of the specific binding pair. There is an exception where a specific type of receptor to a specific ligand is the analyte. Three specific binding components are required, viz. receptor, antireceptor or ligand for the receptor, which may be bound to the particle, and ligand for the receptor or antireceptor respectively. Thus the receptor as an analyte allows for a number of alternative conjugates. In addition, a catalytic member of the signal producing system will be bound or become bound to the reciprocal member of the specific binding pair. By appropriate choice of specific binding pair conjugates, the amount of signal producing member bound to the particle can be related to the amount of analyte in the assay medium.

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According to Yen-Ping Liu and Ullman E.F. (SYNTEX (U.S.A.) INC., (161.2)) methods of measuring analytes have been developed employing functionalized fluorescent particles and functionalized energy absorbent particles capable of quenching a substantial portion of the fluorescent signal, when in close proximity to the fluorescent particle due to specific non-covalent binding. Particularly, members of binding pairs are bound to fluorescent particles and absorbent particles to provide reagents for the determination of an analyte, which is also a member of a specific binding pair. When the functionalized particles are brought together in an assay medium in the presence of analyte, the number of energy absorbent particles brought into close juxtaposition to the fluorescent particles is related to the amount of analyte in the medium. The presence of the energy absorbent particle adjacent the fluorescent particle results in a substantial diminution of the fluorescence from the fluorescent particle, so that the observed fluorescent signal is greater changed in relation to small changes in the amount of analyte in the medium. The quencher particle may be charcoal and the light emitting particle is an addition polymer fluorescent particle. The method of Halfman C.J. (UNIVERSITY OF HEALTH SCIENCE/ THE CHICAGO MEDICAL SCHOOL, (184)) makes use of a prepared amount of the same analyte labelled with a suitable fluorescent dye. A measured amount of the sample together with a predetermined amount of the labelled analyte is placed in an aqueous solution to which is added a predetermined amount of antibody. A surfactant is also added to the solution in an amount greater than the threshold amount necessary to cause the surfactant to form micelles in the solution. Fluorescent emission from the solution is then measured. In one embodiment the surfactant preferentially quenches the emission from free labelled analyte to effect a homogeneous response. Measurement of the intensity of the fluorescence of the solution accordingly provides a measure of the fraction of labelled analyte which has been bound by the antibody in the solution, as may be inferred from Eq. (1). Not every fluorescent dye is suitable for use with every surfactant. It is necessary that the surfactant-dye combination be selected so that the surfactant is operative to effect differernt fluorescent intensities between bound and free labelled analyte. Such differential effects may generally be expected to occur when micelles are operative to sequester the free from the bound labelled analytes and maintain them in different environments. Flow cytometer measurement—Laser beam A method developed by Saunders G.C. (UNITED STATES DEPARTMENT OF ENERGY, (178)) relates generally to measuring binding assays and more particularly to measuring binding assays carried out with different size particles

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wherein the binding assay sample is run through a flow cytometer without separating the sample from the marking agent. Flow cytometry methods have been developed over time to a point where a single file stream of cells or particles passes through a laser beam. This laser beam can excite any fluorescent chemicals present in or on the particles. The emission of the fluorescent chemicals when struck by the laser and also such other properties as light scatter due to the particles passing through the laser beam are measured by detectors. This technique combined with deflection plates as described in a 1973 article (A New Multiparameter Separator for Microscopic Particles and Biological Cells, Steinkamp, et al., Review of Scientific Instrumentation, Vol. 44, No. 9, Sept. 1973, p. 1301) explained how the fluorescence of the subject cells or particles could be used as a criterion to separate them. The article also described the use of uniform plastic microspheres to evaluate the combined methods. The method proposed by Saunders comprises determining the amount of a binding reactant present in a sample by providing particles with a coating of binder and also a known quantity of smaller particles with a coating of binder reactant, the binding reactant being the same as the binding reactant present in the sample, the smaller particles also containing a fluorescent chemical; then combining the particles with the sample and allowing the binding reaction to occur for a set length of time; followed by combining the smaller particles with the mixture of the particles and the sample produced in the previous step and allowing the binding reactions to proceed to equilibrium; and then simultaneously measuring the fluorescence and light scatter of the combined mixture of sample, particles, and smaller particles as the combined mixture passes through a flow cytometer equipped with a laser which causes the fluorescent chemical in the smaller particles to fluoresce, and finally comparing the number and strength of the fluorescent events caused by the particles having a light scatter measurement within a predetermined range against similar number and strength of events caused by particles reacted with a sample of known binder reactant percentage. Fluorescence Polarization Fluorescence polarization techniques have been only applied with reasonable success to the measurement of analytes of relatively low molecular weight. Since the tracer employed must generally resemble the analyte in order to compete effectively for antibody receptor sites, the tracer itself, in such instances, will be relatively large and will tend to retain the polarization of plane polarized light. This approach of doing a competitive binding immunoassay using a fluoresceinlabelled compound as the tracer generally works well because of the substantial difference in polarization observed when the tracer is free versus

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when it is bound by specific antibody. A polarization unit change from 0·02 free) 0·20 (bound) can be considered typical of many sun tracers. A few examples of fluorescence polarization immunoassays applied to quantitation of higher molecular weight (greater than about 10000 daltons) substances can be found. An example demonstrating such assays is of H. Maeda, Clin. Chem. 24:2139 (1978). Bennett L.G. amd Chiapetta, E.G. (ABBOTT LABORATORIES, (1.10)) offers an advance in the art of detection of the presence of amount of CRP (C-Reactive Protein) in a biological sample. They encompass a fluorescence polarization assay and reagents useful in the assay for CRP. In particular their method provides the advantages of the fluorescence polarization techniques previously set forth, for the measurement of CRP levels and encompasses a method for determining CRPs in a sample comprising: a) intermixing with the sample a fluorescent tracer having a ligand analog to CRP; and wherein the ligand analog has a maximum of one common epitope with the ligand so as to be specifically recognizable by a common antibody or other receptor binding site; and an antibody capable of specifically recognizing the ligand and the tracer, whereby a tracer-antibody complex is formed; and b) determining the amount of tracer-antibody complex formed in step a) by fluorescence polarization technique, as a measure of the concentration of the ligand in the sample. A problem encountered in the use of fluorescent polarization immunoassay techniques to determine analytes in serum or plasma samples is background fluorescence present in varying degrees in the samples. Icteric serum or plasma can contribute a significant error to the desired polarization measurement. A major fluorescent component of icteric serum or plasma is albumin-bound bilirubin. Bilirubin is the final product of heme catabolism and in normal individuals is present in serum at less than 1 mg/dl. In various disease states affecting the liver, bilirubin is markedly elevated, reaching 10–20 mg/dl in some cases. Neonates often attain high levels in the 10–20 mg/dl range due to poor liver function immediately post-partum. Bilirubin is relatively nonfluorescent when it is in aqueous solution, but becomes highly fluorescent if bound to albumin (Chen, Arch. Biochem. Biophys. 160, 106–112) and bilirubin-albumin binding is very tight (Gray, et al., J. Biol. Chem. 253, 4370–4377). Therefore, serum or plasma samples with elevated bilirubin levels will exhibit an elevated fluorescence due to the presence of the bilirubin-albumin complex. In order to avoid erroneous results due to background fluorescence, a blank reading is usually taken on a serum or plasma sample in the TDx Analyzer prior to performance of an assay, which is then subtracted from the final assay reading

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to arrive at a corrected value. However, background subtraction may be ineffective to adequately compensate for background fluorescence in some elevated bilirubin samples, and degredation of the bilirubin-albumin complex during the course of the assay can result in inaccurate compensation for background fluorescence in such samples. Bennet L.G. (ABBOTT LABORATORIES, (1.15)) found that problems with prior art systems are overcome by conducting fluorescence polarization immunoassays for analytes in serum or plasma samples in the presence of dioctyl sodium sulfosuccinate. The dioctyl sodium sulfosuccinate (“DSS”) is employed as an anionic surfactant in fluorescence polarization immunoassays to reduce background resulting from bilirubin-serum albumin complex in serum samples. It has been unexpectantly found that dioctyl sodium sulfonate is highly effective for this purpose at relatively low concentrations with little or no degredation effect on antibody employed in the assay, thereby permitting the use of flurescence polarization techniques to determine the concentration of analytes. DSS is the compound sulfobutanedioic acid 1,4-bis(2-ethylhexyl)ester sodium salt having the structural formula:

In addition to the sodium salt, other salts, such as the potassium salt, calcium salt, lithium salt, magnesium salt and other equivalent salts are included as well. Chiapetta E.G. and Kucera R.J. (ABBOTT LABORATORIES, (1.1)) proposed a fluorescence polarization immunoassay wherein the immunoassay is conducted in the presence of from about 0–001 to about 1·0 percent (weight/volume) of dioctyl sodium sulfosuccinate. The proposed method can be carried out at pH values between 3 and 12 and at temperatures between 15 and 40°C. As reagents the following are proposed. Stabilizing Media Reagent Composition for Calibrators/Controls

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Pre-treatment Reagent Composition

The pH is adjusted with 6 N HCl to ph 8·0.

The above formulation has been found to stabilize C-Revitive protein for 30 days at 45°C. The pH is adjusted to 8·0 with 6N HCl. This pretreatment composition has been found to be effective in eliminating bilirubin interference at bilirubin concentrations of 20 mg/dl while using a CRP assay sample volume of 8·0 microliters. Heavy Antigens Kramer P.B. (ORTHO DIAGNOSTIC SYSTEMS INC., (122.2)) also found that the sensitivity of the polarization assay for a ligand rapidly drops off with increasing molecular weight of the ligand. He proposed a method which entails the competition between the large molecular weight ligand in the aqueous sample for the binding site on an antiligand receptor with a user supplied reagent comprising fluorescently labelled, binding site simulator means. In the most preferred embodiment, the binding site simulator means will comprise a peptide. The ideal peptide is produced in accordance with its ability to immunologically simulate the antigenic determinant

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or binding site present on the large molecular weight ligand to which the antiligand binds. Thus, presence of the sample ligand results in binding of the antiligand thereto thereby reducing the amount of anti-ligand available to combine with the fluorescently labelled peptide. Free, fluorescently labelled peptide exhibits fluorescence depolarization while fluorescently labelled peptide bound to anti-ligand exhibits detectably less fluorescence depolarization. The fluorescence polarization immunoassay of Imai K. and Nomura Y. (HITACHI LTD, (70.2)) comprises mixing microcapsules, a sample containing an antibody or antigen, and a complement. The microcapsules contain a fluid with a specific viscosity containing a fluorescent substance and being labelled with an antigen or antibody. An immunoreaction under the condition where the viscosity of the fluid in the microcapsules is different from that of a solution outside the microcapsules is caused so that microcapsules are lysed by activated complement. The reaction mixture is irradiated with exciting light. Polarization fluorescence is measured due to the reaction mixture, and thereby the concentration of a target substance in the aforesaid sample is determined. According to Imai and Nomura the fluidity or viscosity of the fluid in the microcapsules is different from that of the liquid outside the microcapsules. A viscosity modifier is used for increasing the viscosity inside or outside the microcapsules. The viscosity modifies is preferably a water-soluble organic compound having a viscosity higher than that of water, and there can be used, for example, polyvinyl alcohol, glycerin, sucrose, etc. The reagent is prepared so that one of the viscosities inside and outside the microcapsules is higher than another. Absorbing material Fluoroimmunoassay (FIA) provides direct detection of the label and is readily adaptable to homogeneous assay procedures. However, known homogeneous FIA methods using organic fluorochromes, such as fluorescein or rhodamine derivatives, have not achieved the high sensitivity of radio-immunoassay (RIA) or enzymeimmunoassay (EIA), largely because of light scattering by impurities suspended in the assay medium and by background fluorescence emission from other fluorescent materials present in the assay medium. Scattering is particularly troublesome with fluorochromes having a short (50 nm or less) Stoke’s shift (the differ ence between the wavelength of the absorption and emission). For example, the Stoke’s shift of fluorescein isothiocyanate in only 20–30 nm. Background fluorescence is particularly troublesome when the assay medium is serum. The sensitivity of an assay in serum may be reduced up to one hundred fold compared to an identical assay in buffer. The development of time-resolved fluoroimmunoassay (TR-FIA) has contributed to overcoming these problems. In this procedure, a fluorochrome

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label with a relatively long fluorescence emission decay time is excited with a pulse or light, and fluorescence emission from the label is measured after a preselected delay. Background emission of short decay time (generally less than 10 ns) essentially ceases during the delay and thereby does not interfere with measurement of the specific emission from the label. TR-FIA is most effective when the fluorescent label has a decay time of 100–1000 ns and a long Stoke’s shift (100 nm or greater). Wagner D.B. and Baffi R.A. (BECTON, DICKINSON AND COMPANY, (16. 3)) developed a method for separation-free solid phase immunoassay of an analyte including contacting an anti-analyte attached to the surface of a solid support with the analyte, a light absorbing material and a fluorescent tracer for the analyte. The resulting mixture is incubated. The method includes applying excitation light to the mixture and time resolved measurement of fluorescence emission from the tracer. All excitation light and fluorescence emission are absorbed by the light absorbing material except that absorbed and emitted by the tracer bound to the anti-analyte whereby the only fluorescence emission detected is from the bound tracer. Since free tracer in the fluid phase of the assay medium does not emit fluorescence, separation of the bound and free fractions is unnecessary. 1.2.2. REAGENTS FOR FLUORESCENCE POLARIZATION IMMUNOASSAY Wang C.J. and Stroupe S.D. (ABBOTT LABORATORIES, (1.2)) found a method for determining ligands in a sample comprising intermixing with the sample a biologically acceptable salt of a tracer of the formula:

wherein R is a ligand-analog having a single reactive primary or secondary amino group which is attached to the carbonyl carbon of the carboxyfluorescein wherein the ligand-analog has at least one common epitope with the ligand so as to be specifically recognizable by a common antibody, and an antibody capable of specifically recognizing the ligand and the tracer, and then determining the

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amount of tracer antibody conjugate by fluorescence polarization techniques as a measure of the concentration of the ligand in the sample. Flecainide acetate, or N-(2-piperidylmethyl)-2,5-bis (2,2,2-trifluoroethoxy) benzamide acetate, is a drug used to treat ventricular arrhytmias and tachycardia. Although flecainide acetate has been proven to be effective clinically, it can cause undesirable side effects if optimal dosage levels are exceeded. Therapeutic blood flecainide levels have been found to range from 0·2 to 1·0 microgram per milliliter, with 0·5 microgram per milliliter considered the mean effective concentration. Monitoring of serum flecainide levels combined with clinical data can provide the physician with useful information to aid in adjusting patient dosage, achieving optimal therapeutic effects while avoiding useless subtherapeutic or harmful toxic dosage levels. In the past, patient serum or plasma flecainide levels have typically been measured by high performance liquid chromatography (HPLC) or gas chromatography (GC). Although these methods are reasonably specific for detecting drug levels, they are not without drawbacks. They involve extensive sample preparation, time-consuming instrument set-up and a need for highly trained personnel. To eliminate such drawbacks Heiman D.F. (ABBOTT LABORATORIES, (1. 16)) developed a fluorescence polarization immunoassay for flecainide. Furthermore he found tracers for use in the assay, and methods for making such tracers. A first aspect of his idea relates to the discovery of tracers having specific structures. The tracers and their precursors can both be represented by a structural formula, wherein: Q is hydrogen, hydroxyl or a leaving group, or fluorescein or a fluorescein derivative; Z is NH, C=O, SO2 or C=NH, and R is a linking group including up to 10 heteroatoms and having a total of from 0 to 20 carbon atoms arranged in a straight or branched chain and containing up to two ring structures. Fino F.J. and Shipchandler M.T. (ABBOTT LABORATORIES, (1.7)) proposed a method for the production of 4′-aminomethylfluorescein derivatives as tracers in fluorescence polarization immunoassays as disclosed in US patent 4,510,251 to Kirkemo and Shipchandler. The method disclosed therein for the preparation of 4′-aminomethylfluorescein is not without drawbacks, however, as it has sub

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sequently been learned by Shipchandler that alkylation occurs as an unavoidable side reaction, thereby diminishing the yield of 4′-aminomethylfluorescein. According to Fino and Shipchandler a method is provided, whereby 4′aminomethylfluorescein can be prepared without concomitant alkylation and thus with greatly improved yield. In addition, a novel class of 4′aminomethylfluorescein derivatives, viz., 4′-Nalkylaminomethylfluorescein compounds, has been discovered. These fluorescein derivatives is said to possess greater sensitivity, and hence utility, than 4′-aminomethylfluorescein in certain specific instances. A first aspect of their research relates to the discovery of a class of 4′Nalkylaminomethylfluorescein derivatives having the structural formula:

and acid addition salts thereof, wherein R1 is C1 to C8 alkyl; R3 is hydrogen, alkyl, halo, amino or carboxyl; R4 is hydrogen, alkyl, halo, amino or carboxyl, and R5 is hydrogen, alkyl, halo, amino or carboxyl. These 4′-N-alkylaminomethylfluorescein derivatives possess particularly useful properties for certain fluorescence polarization immunoassays, wherein they are utilized as fluorescent tracers having the formula:

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and the acid addition salts thereof, wherein R1, R3, R4 and R5 are as defined above, and R2 is a ligand-analog having at least one common epitope with a ligand so as to be specifically recognizable by a common antibody. According to another proposal of Fino and Shipchandler (ABBOTT LABORATORIES, (1.14)) for the production of 4′-aminomethylfluorescein, the compound has the structural formula:

and acid addition salts thereof; wherein Rl, is hydrogen or a ligand-analog having at least one common epitope with a ligand so as to be specifically recognizable by a common antibody; and R2, R2 and R4 each may be hydrogen, alkyl, halo, amino or carboxyl. The process where these compounds may be prepared comprises reacting an acetamidomethylfluorescein or 4′-haloacetamidomethylfluorescein derivate having the following structural formula:

wherein R, R′ and R″ each may be hydrogen or halo; and R2, R3 and R4 each may be hydrogen, alkyl, halo, amino or carboxyl; in the presence of a nonhydroxylic ether and a strong acid and recovering the 4′aminomethylfluorescein thus produced. Asymmetrical fluorescein derivatives without functionalities for attaching to other molecules are subject of many literature places, such as in Chemical Abstracts, e.g. CA 74, 45540v and CA 53, 9573g. Symmetrical fluorescence derivatives having alkyl or oxisubstituents are disclosed in e.g. US patent 4,318, 846. Khanna P. and Colvin W. (SYNTEX (U.S.A.) ING., (161.3)) developed fluorescent compounds, which are analogs of fluorescein, being particularly 1,8unsubstituted-9-substituted-6-hydroxy-3H-xanthen-3-ones, having one aliphatic

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substituent at any of the remaining positions, where the aliphatic substituent is separated from the annular carbon atom by from 0 to 1 oxygen atom. Normally present will be a functionality, particularly a non-oxocarbonyl functionality, for conjugation to a member of an immunological pair (MIP), referred to as a ligand and receptor. The fluorescent precursors will have at least about 14 carbon atoms, and usually not more than about 40 carbon atoms. Preferably there is at least one, usually two chlorine atoms at other than the 1,8-positions and there may be as many as 7 chlorine atoms. In addition to chlorine, the only other heteroatoms are fluorine, bromine, and iodine, chalcogen, particularly oxygen and sulfur, and nitrogen, there being at least 4 heteroatoms and not more than about 16 heteroatoms and preferably not more than about 12 heteroatoms. Coupling agents: tracers According to the outcome of research by Allen and Thompson (BECTON DICKINSON AND COMPANY, (16.2)), a process is provided for preparing coupling agents, intermediate and coupled compounds. A useful intermediate in preparing coupled compounds has the following formula:

wherein Y is a divalent aromatic hydrocarbon radical; R is an organic radical having at least one active hydrogen substituent group (in particular, an amine, thiol or hydroxyl substituent group); and A is selected from the group consisting of —NO2; NH2; —COOH;

wherein R″ is alkyl. In accordance with another aspect of the present invention, there is provided coupled compounds having the following structural formula:

wherein Z is selected from the group consisting of

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B is an organic radical; R is an organic radical having at least one active hydrogen substituent group wherein R is coupled through the active substituent group (preferably an amine, thiol, or hydroxyl substituent group); and Y is a divalent aromatic hydrocarbon radical. Another tracer proposed by Wagner (BECTON DICKINSON AND COMPANY, (16.6)), comprises a vesicle derivatized with a ligand, a portion of such vesicle wall being formed from an amphiphilic chelating agent having complexed therewith detectable metal atoms which are comprised of metal atoms being fluorescent when complexed with an activating agent. The metal atoms are a rare earth metal. The amphiphilic chelating agent comprises from 5 to 60 mole percent of the materials forming the vesicle. 1.2.3 KITS AND INSTRUMENTS FOR FLUORESCENCE IMMUNOASSA Y A multilayer analytical element for fluorometric assay of an analyte contained in an aqueous sample liquid, the assay comprising a stage of subjecting the analyte, a predetermined amount of a fluorescent-labelled analyte substance or analogue thereof, and a predetermined amount of a protein specifically bindable to the analyte to competitive binding reaction, a state of substantially separating the resulting bound fluorescent-labelled complex (B) of the protein (including the resulting bound fluorescent-labelled complex (B) of the protein and the analyte, as well as the bound fluorescent-labelled complex (B) of the protein and the analyte substance or analogue thereof) from the free fluorescent-labelled analyte substance or analogue thereof (F), and a stage of determining the fluorescence strength of the labelled complex (B), has been subject of research of Nagatomo S. (FUJI PHOTO FILM CO., LTD., (59.2)).

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Certain improved apparatus in which the required procedures are all automated have been proposed so as to replace the above-mentioned conventional method. These automated apparatus are able to provide high reproducibility without such high skills and are convenient for measuring large numbers of sample liquids. Nevertheless, these have serious disadvantages such as high expense. In order to obviate the disadvantages of such automated apparatus, a multilayer analytical elements in which reaction reagents are in advance incorporated have been proposed as a simple analytical means where supply of the reagent solutions is substantially not required, whereby no trained skills are required. These multilayer analytical elements are advantageous in that they are inexpensive as compared with the automated apparatus. However, although various improvements have been proposed with respect to the multilayer analytical elements utilizing chemical reactions or enzymatic reactions, it is still difficult to assay trace components or components having high structural specifity. Based on the above consideration Nagatomo et al. developed: (a) a porous fibrous or non-fibrous spreading sheet containing a predetermined amount of the fluorescent-labelled analyte substance or analogue thereof: (b) a porous sharing sheet enabling the substantial separation of the bound fluorescent-labelled complex (B) of the protein (including the resulting bound fluorescent-labelled complex (B) of the protein and the analyte, as well as the bound fluorescent-labelled complex (B) of the protein and the analyte substance or analogue thereof) from the free fluorescent-labelled analyte substance or analogue thereof (F); and (c) a reaction sheet containing a predetermined amount of the protein specifically bindable to the analyte fixed therein; being under laminated structure in this order. The figure below shows the assembly of the kit proposed, wherein 101 is the porous spreading sheet, 111 is the intermediate porous sharing sheet and 121 is the reaction sheet. Prior assays involving immunoadsorption and immobilization have several shortcomings. Firstly, the reprocibility of the immobilization is often inconsistent and affects the precision of the assay adversely. The adsorption may also affect the stability of the immune complexes adversely and cause artificially low immonofluorescence intensity readings. Elution inefficiency and/or

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interference from spurious eluted materials may also cause artificially low fluorescence intensity readings. In contrast to the known prior art competitive immunofluorescence assays, the assay of Kameda N. (PARAGON DIAGNOSTICS, (128)) does not involve solid phase readings or readings of supernatants or eluates that may contain interfering materials. Instead the Kameda assay involves precipitating the immune complex with a nonfluorescent, nonlight scattering immunoprecipitant, dissolving the resulting immunoprecipitate with a solvent that does not add background fluorescence or light scatter, and reading the solution. A waveguide coated with single-stranded probe nucleic acids carrying an internally reflected wave signal is contacted with an analyte solution containing denatured test DNA or RNA and fluorescent marker dye, has been developed by Sutherland R.M. et al. Analyte nucleic acid with sequences homologous to that of the probe polynucleotide will hybridize therewith with concomitant binding of the fluorescent dye to the resultant duplex structures. Fluorescence resulting from the interaction of the wave signal at the waveguide/analyte interface with the signal generating centers created within the space probed by the evanescent component of the wave signal is detected and provides useful information on the sequences homologous to that of the probe nucleic acids. The figure schematically shows an embodiment of the respective measuring system of Sutherland et al. In general, in conventional fluorescence detection apparatus, a light source for producing a continuous light is used in the projector system, while, in the receiving and measuring system, fluorescence is received and converted into an electric signal by a photosensor such as a photodiode or a photomultiplier tube, and the electric signal is DC-amplified and then used to measure the intensity of the fluorescence.

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In such a fluorescence detection system, however, consideration must be given to error factors such as incidence of an external disturbance light into the photosensor, a dark current in the photosensor, an offset of the amplier. The projector system and the photosensor must have a tightly closed structure for perfectly preventing the incidence of an external disturbance light. Further, a high sensitivity performance is required for the measurement of a very minute amount of a product of an ammunoreaction. Such a very minute amount is characteristic of the immunoreaction. Therefore, a number of difficulties are experienced in the construction of this type of fluorescence detection apparatuses. In view of the above-mentioned problems, an object of Hayashi H. (TOYO SODA MANUFACTURING CO., LTD, (172.1)) was to provide a fluorescence detection apparatus suitable for determining minute amounts of a matter involved with a high sensitivity for the purpose of assaying an immunoreaction. Another object was to design a durable, simple and inexpensive apparatus suitable for efficiently detecting or measuring fluorescence intensities involved in many samples. The proposed fluorescence detection apparatus for immunoassays comprises an optical system (2, 3, 4) for irradiating a sample (5) containing a fluorescent substance obtained by an immunoreaction with a light emitted from a fluorescent discharge tube (2); a photosensor (8) receiving fluorescence emitted from the sample (5) for converting the received fluorescence into an electric signal, a power source (1) controlled by a clock pulse generator (1A) to intermittently perform the irradiation with a light emitted from the discharge tube (2) at a predetermined frequency, and a phase sensitive detector (10) for detecting the electric signal in synchronism with the frequency of intermittent irradiation to detect an intensity of the fluorescence by way of the electric signal.

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The figure below shows a graph of pulse responses to lamp voltage, exciting light and fluorescent light. The current method for immunofluorescent (IF) test involves a subjective evaluation of the end point (titer) which is dependent, inter alia, upon the observer’s expertise, experience and judgement. This subjectivity is further complicated by the rapid fading of the fluorescent reaction product (FRP) under the test conditions routinely employed. Thus, as the art is presently known, the outcome of an IF test becomes a function of time and judgement. In fact, there being no better or objective method for IF assay, researchers have generally conceded that rapid fading of fluorophores would have to be tolerated if IF is opted as the procedure of choice. See McKay et al., Immunology 43, 591–602 (1981). Various techniques have been used to protect the sample from fading. These are summarized in the next table. Of these, a more practical and feasible technique appears to be the use of chemical additives in the mounting medium to protect the fluorophore from the effects of the excitation light. Protection from fading would make exposure of the specimen to the exciting light less critical. This would allow ease in the localization of the fluorescent specimens and permit more accurate discrimination between weakly positive and negative results, which is difficult if the sample is rapidly fading. Certain tests, such as determination of the type of Herpes present, require finding any positive cells that may be present on the entire slide. This searching procedure may take several minutes and must be done during excitation to recognize the presence of the positives. If fading is rapid, positives may be missed. Protection from fading in these cases is critical. Keeping the above considerations in mind Picciolo G.L. and Kaplan D.S. (US as represented by the DEPARTMENT OF HEALTH AND HUMAN SERVICES,

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Methods for the Reduction of Fading

(179.1)) developed a device and a process for quantitation of end point in the formation of fluorescent reaction product in microfluorophotometry. The process comprises: a) incorporating a protective agent in a suitable mounting medium in an amount sufficient to reduce fading of fluorescent reaction product less than 25% of initial fluorescent intensity; b) calibrating photometer used in the microscopy with a stable emitter; and c) recording the intensity of fluorescence of the fluorescent reaction product by means for measuring light intensity. Furthermore, a device was developed for calibration of the photometer and a kit comprising separate containers for suitable mounting medium, buffer, suitable immunofluorescent reagents, fading retardant means, a photometer calibrating device and the like and optional instructions. The figure schematically shows the arrangement of the device of Picciolo and Kaplan.

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1.3. LUMINESCENT, OPTICAL AND COLORIMETRIC IMMUNOASSA Y METHODS 1.3.1 LUMINESCENCE Luminescent immunoassays have the capability to address most of the drawbacks inherent to the RIA, ELISA and FIA but are, in general, described to be less sensitive than RIA and ELISA. However, while this may be true for the serum-based homogeneous assays, where the Rayleigh and Raman scattering caused by the high protein content in the solution and the intrinsic fluorescence

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from certain amino acids residue reduces the signal-to-background ratio, it is not necessarily true for the heterogeneous assays. One promising heterogeneous technique involves the use of a solid support (e.g., paper disk, plastic dip stick, walls of the test tube and glass or plastic beads) to which an antibody or antigen is absorbed or covalently bonded. This immunoreactive solid surface is then exposed to serum and labelled ligand. The latter react immunologically with the antigen-antibody complex on the solid surface. After a brief wash to remove unreacted serum and unbound labelled ligand, the signal from the antigenantibody complex on the solid phase is measured by, e.g., a fluorometer or a simple light box. There exists continuing interest in further improving the sensitivity of heterogeneous luminescent immunoassays. To this end Khalil H.M. and Pourfarzaneh M.T. (WHITTAKER CORPORATION, (201)) developed a method for detecting the presence of subnanomolar quantities of an analyte of interest in a sample wherein the sample is exposed to a reactant having an affinity for the analyte, or a complex containing the analyte, to bind with the reactant. The reactant comprises microspheric particles labelled with a reporter substance capable of being detected either alone, in complex, or both, and the microspheres further labelled with a substance, also having an affinity for the analyte or a complex containing the analyte. The detection of the presence of the analyte is made by visually determining the association with the analyte-reactant complexes, with the aid of a fluorometer of a light box. The graph shows a comparison of visual and fluorometric readings of CMV G assay. Luminescent substrate preparation Luminescent labels are attractive alternatives for use in specific binding assays for a variety of reasons. Luminescence is broadly defined as the production of visible light by atoms that have been excited by the energy produced in a chemical reaction, usually without an associated production of heat. Chemical energy excites electrons in the light-emitting molecules to higher energy states, from which electrons eventually fall to lower energy states with the emission of quanta of energy in the form of visible light. One of the most important families of chemiluminescent molecules are the phthalylhydrazides. The most familiar member of this family is luminol, or 5amino-2,3-dihydro-1,4-phthalazinedione, which has a gross chemical composition of C8H7N3O2 and a double ring structure with a melting point of about 320°C. While, however, the luminol reaction offers important potential benefits in the measurement of the presence and amount of a reaction component, for many potential applications the intensity of the emitted light is too low. Further, the

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light emitted from commercial luminol exhibits an early flash of light within the first few seconds of the initiation of the reaction, followed by a progressive and rapid decrease in light emission over time. The integrated light intensity during any fixed period of time is therefore likely to be different from that measured over any other equal period of time. This variability may result in irreproducibility between tests. Having the above considerations in mind, Higgins K.W. et al. (MAST IMMUNO-SYSTEMS, INC., (103)) prepared a luminescent substrate having a concentration of catalytic inhibitors of less than about 100 ppm. The preparation is obtained by heating commercial grade luminol in a basic solution, crystallizing the luminol and separating the luminol crystals from the boiled solution. The heating, crystallization and separation steps are preferably repeated sequentially at least four times, with the starting material for each sequence after the first being the luminol preparation produced in the previous sequence. The luminol preparation has an enhanced pattern of activity, in that light output is substantially constant over a period of at least about one hour, with the intensity of light emitted by the preparation being at least about ten times that of commercially available luminol. Because of these enhanced characteristics, the

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luminol preparation is particularly adapted for use as a tag in specific binding assays where the concentration of analyte to be detected is low. Mathis G. and Davin T. (COMMISSARIAT A L’ENERGIE ATOMIQUE, (39. 2)) also have tried to eliminate the drawback of the ELISA, RIA and, particularly, the FIA by proposing a homogeneous process for the detection and/or determination by luminescence of an analyte in a medium susceptible of containing it by evidencing the product obtained by the reaction between the analyte and a corresponding receiver. The process comprises: 1) the addition to the medium of a first reactant comprised of an analyte receiver; 2) the addition of a second reactant comprised of at least one of the elements of the product obtained from the reaction of the analyte with at least one of its receivers, one of the two reactants being coupled with a luminescent compound and the other reactant comprising a heavy atom or patterns containing a heavy atom; 3) the incubation of the resulting medium either after the addition of each reactant or after the addition of both reactants; 4) excitation of the resulting medium; and 5) measuring, in balance or kinetic conditions, the signal emitted by the luminescent compound, the signal being modulated by the heavy atom effect. Luminescence suppressing agent As is known in the art of luminescence-immuno-tests that they consist of an antigen or antibody which is marked with e.g. phthalhydrazides being adapted to luminescence properties, preferably a substrate bound antibody or antigen, and an oxidizing reagent which suppresses the luminescence action, Gadow A. (HENNING BERLIN GmbH CHEMIE und PHARMAWERK, (68.3) prepared an oxidizing reagent which is a preprepared, e.g. a bacterostaticum stabilized solution of catalase and the initiator of a pre-prepared alkaline peroxidase solution being at least 20 minutes old. 4–20 × 106 units of catalase from bovine lever, having a molecular weight of about 240000, are solved in 1 liter 0·15 mol/1 NaCl and 0·1% by weight sodiumazide. One unit of catalase is, per definition, the amount of enzyme converting 1 µmol hydrogenperoxide at a pH value of 7·0 and at a temperature of 25°C in one minute. The final concentration of catalase units/1 to be selected depends upon the condition or state of the reagent which is responsible for the suppression of the chemiluminescence signal of phthalhydrazides and their derivatives, and has accordingly to be adapted or optimized, respectively.

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In accordance with a method developed by Jolley M.E. (PANDEX LABORATORIES, INC., (127)) a solid phase immunoassay is provided for the quantitation of antigen, hapten or antibody analyte in a liquid sample. The solid phase immunoassay incorporates a luminescent label such as a fluorescent label, a phosphorescent label of an atomic fluorescent label. This solid phase immunoassay utilizes (i) a plurality of water insoluble particles of about 10 microns or less in size, or (ii) cells, to which an immunoreactant is attached. The analyte or an analyte containing reaction product is reacted with or in competition with or for the immunoreactant while the particles or cells are in a substantially suspended state. The particles or cells which have, or which through subsequent reaction will have, a luminescent label attached thereto are concentrated by microfiltration to a volume substantially less than the volume of the liquid sample which initially contained the analyte. The luminescence of substantially all of the luminescent label attached to the concentrated particles or cells is measured. Also in accordance with the method of Jolley, a solid phase immunoassay is provided for the quantitation of analyte occurring on or attached to cells or other particulate material contained in a liquid sample. Sidki A.M. and Smith D.S. (INTERNATIONALE OCTROOI MAATSCHAPPIJ “OCTROPA” B.V., (86)) propose a process of carrying out a specific binding assay, for the qualitative detection or quantitative or semi-quantitative determination of an analyte which forms a component of a specific binding reaction, using a labelled conjugate form of a derivative or analogue of one of the relevant specific binding partners, the labelled form being capable of emitting delayed luminescence upon photo-excitation. The labelled conjugate form of one of the relevant specific binding partners comprises a derivative or analogue of the specific binding partner with a derivative of a luminescent substance which shows a delayed light emission upon photo-excitation, the delayed light emission of which is subject to quenching by molecular oxygen. Further the process is characterized in that the assay is carried out by the use of a time-resolved photometric method in the presence of an effective amount of a quenchinhibiting substance able to prevent quenching by molecular oxygen. The figure below shows antibody dilution curves, showing quenching of phosphorescence (one form of delayed luminescence) and delayed fluorescence (another form of delayed luminescence) of erythrosinlabelled primidone by antibodies to primidone. The horizontal axis gives values of final dilution of the antibodies. The vertical axis gives observed luminescence values as a percent of the value obtained without immunoglobulins present. Curve A shows phosphorescence obtained in the presence of nonspecific sheep immunoglobulins (control). Curve B shows delayed fluorescence, and curve C shows phosphorescence, in each case as obtained in presence of antibodies to primidone in the indicated dilution.

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Chemiluminescent reactions A special type of chemiluminescence is bioluminescence, which is defined as the emission of light by living organisms, due to an energyyielding chemical reaction in which a specific biochemical substance, called luciferin, undergoes oxidation, catalyzed by a specific enzyme called luciferase. Using lantern extracts from hundreds of thousands of fireflies, scientists at John Hopkins University determined the chemical structure of firefly luciferin to be C13H12N2O3S2. It can now be synthesized. The reaction of luciferyl adenylate with oxygen is postulated to give a fourmembered-ring alphaperoxylactone intermediate and to release adenosinemonophosphate (AMP). This breaks down in the energy-yielding step to give carbon dioxide and a lightemitting excited molecule. This loses its energy as a photon, in the yellow region of the spectrum in this case. A chemiluminescence is provided by Dattagupta N. and Clemens A.H. (MOLECULAR DIAGNOSTICS, INC., (112)) comprising the contacting of a chemi-luminescence precursor, an oxidant, an enzyme and a nitrogen compound selected from the group consisting of ammonia and a water-soluble organic amine. The reaction of such process can be used in detection of nucleic acid hybrids, antibodies, antigens and peroxidase enzymes and in producing light. Another chemiluminescence process comprises the contacting of a chemiluminescence precursor, an oxidant, an enzyme, a chemilumines-cence enhancer and a nitrogen compound selected from the group consisting of ammonia and water-soluble organic amines. The reaction of such process can be used in

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detection of nucleic acid hybrids, antibodies, antigens and peroxidase enzymes and in producing light. A nucleic acid probe capable of participating in a chemiluminescent reaction comprising a defined nucleic acid sequence, the sequence being linked to any one of a) a chemiluminescence precursor, b) a chemiluminescence enhancer, and c) an enzyme, the remaining two of a), b) and c) not linked to the sequence being in amixture of the linked sequence. A method for determining a particularsingle stranded polynucleotide sequence in a test medium, comprisingthe steps of: (a) combining the test medium with a polynucleotide probe having a base sequence substantially complementary to the sequence to be determined, (b) labelling either the resulting hybrids or probe which has not hybridized with the sequence to be determined with one of the participants in an enhanced chemiluminescent reaction involving a chemilumi-nescent precursor, an enzyme, an oxidant, and a chemiluminescence enhancer, (c) initiating such chemiluminescent reaction with the labelled hybrid or probe, and (d) detecting the resulting light emission. Luminescent assays making use of peroxidase catalysed oxidation of a 2,3dihydro-1,4-phthalazinedione (DPD) can be of several major types, the commonest of which are those wherein horseradish peroxidase is conjugated to a ligand in order to label it and a luminescent reaction is used to detect or quantitate the label. The research of Barnard G.J. et al. (NATIONAL RESEARCH DEVELOPMENT CORP., (114a)) relates exclusively to assays wherein a chemiluminescent compound is used directly to label ligands such as proteins, hormones, haptens, steroids, nucleic acids, metabolites, antigens and/or antibodies. The chemiluminescent DPD such as luminol or isoluminol is normally conjugated to a ligand. Chemiluminescence can be detected by adding peroxidase and an oxidant to the reacted conjugate. Barnard et al. found that certain saturated heterocyclic amines enhance the chemiluminescent oxidation of a DPD. These amines have the characteristic that they lose an electron easily to form positive ions. They are either tertiary amines or secondary amines in which the hydrogen atom attached to the nitrogen is well shielded or sterically hindered. More precisely, the amines of interest are saturated bicyclic compounds having a nitrogen atom at one or both bridgehead

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positions and piperidine ring compounds having four lower (C1−4) alkyl groups at the 2—and 6—positions (the carbon atoms adjacent to the nitrogen). The assay provides a luminescent or luminometric assay which comprises carrying out a chemiluminescent reaction between a catalyst having an accessible heme group, preferably a peroxidase enzyme, most preferably microperoxidase, an oxidant, preferably hydrogen peroxide, and a chemilumines-cent DPD conjugated to a ligand which is a molecular residue relatable to a substance to be assayed, and detecting or measuring the chemilumi-nescence produced, characterised in that the reaction is carried out in the presence of such an enhancer. It includes also a kit for use in the assay comprising the chemiluminescent DPD conjugate, peroxidase enzyme and amine enhancer. The amine enhancers can contain ring substituents, or, in the case of the already substituted piperidines, further ring substituents, preferably lower (C14) alkyl, hydroxy, chloro or bromo. The currently preferred enhancer amines for use in the assay are: (1)

(2)

(3)

(4)

A test kit for hapten chemiluminescent immunoassay consisting of as a merchantile unit (A) an antibody specific to a hapten, (B) a chemiluminescent labelled hapten conjugate including a) a plurality of groups displaying chemiluminescence, b) a linkage group, and

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c) a plurality of haptens, the linkage groups b) being a chainlike polymer having repeating functional units and having bound thereto the plurality of groups displaying chemiluminescence a) and the plurality of haptens c), and directions for carrying out the hapten luminescence immunoassay, was found by Gadow A. (HENNING BERLIN GMBH CHEMIEUND PHARMAWERK, (68.1)). The antibody (A) was prepared using as the immunogen a hapten bound to a linkage group other than linkage group b). The linkage group b) is a peptide, a glycoprotein, a glycolipid or a carbohydrate. The molar ratio of the linkage group b) to the group a) capable of chemiluminescence to the hapten c) is at least 1:10:10. Optical interference detection The method of NICOLI D.F. and ELINGS V.B., (115) is directed to a measurement technique for homogeneous immunoassays whereby the presence of analyte is detected quantitatively using optical interference and specifically a Bragg scattering peak. Nicoli and Elings found an apparatus and method for the optical detection of a binding reaction between a ligand and an antiligand, including a pattern formed by a spatial array of microscopic dimensions of antiligand material, ligand material interacting with the antiligand material to produce a binding reaction between the ligand and the anti-ligand in the pattern, a source of optical radiation including energy at at least one wavelength directed to the pattern at a particular incidence angle to produce scattering of the energy from the pattern in accordance with the binding reaction and with a strong scattering intensity at one or more Bragg scattering angles, and at least one optical detector located relative to the pattern and aligned with a Bragg scattering angle to detect the strong scattering intensity at the Bragg scattering angle to produce a signal representative of the binding reaction. Recently, certain antigen-antibody complexes or conjugates associated closely with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and certain glomerular nephritis have been found. Accordingly, it has currently become more significant to determine an antigen, antibody, or antigenantibody complex or conjugate in body fluids to aid in the diagnosis or remedy of physiological or pathological states or conditions in both human beings and animals. There are methods known for quantitatively determining an antigen or antibody to be determined in a sample solution by spectrophotometrically or electrically measuring the results of an agglutination reaction using the same materials as used in the slide-type method.

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The afore-mentioned spectrophotometric method, however, encounters difficulties in providing an accurate quantitative determination of the antigen or antibody. In this method, the absorbancy or turbidity of a reaction mixture is measured while the reaction is still proceeding therein so that the absorbancy or turbidity thereof will tend to vary to a great extent with the lapse of time because the reaction proceeds even after the preselected period of reaction. Mochida E. et al. (MOCHIDA SEIYAKU K.K., (110a)) found a method for the quantification of an antigen, antibody, or antigen-antibody complex in a sample solution involving measuring the results of an immunochemical agglutination reaction or an agglutination inhibition reaction by spectrophotometry, wherein after the initiation of the agglutination between the antigen, antibody, or antigenantibody complex to be determined and sensitized carrier particles to which a substance specifically bindable to the antigen, antibody, or antigen-antibody complex is bound, a fixing compound is added to fix aggregates formed by the agglutination. A measuring reagent kit or pack includes all the components for use in carrying out the above method. The figure is a diagram comparing the measurement method of Mochida et al. with the conventional method with respect to the measurement accuracy. Shaffar M.R. (ABBOTT LABORATORIES, (1.17)) discovered a method for determining the presence or amount of a ligand in a sample suspected of containing the ligand. The method comprises (a) combining to form an assay solotion: (i) the sample, (ii) an effective amount of a luminescent reagent; and (iii) an effective amount of a reagent system which in the presence of the ligand

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to be determined is capable of providing a change in the transmittive properties of the assay solution within a wavelength band that overlaps the emission wavelength of light emitted by the luminescent reagent; (b) activating the luminescent reagent; and (c) measuring the intensity of light emitted by the assay solution as a measure of the concentration of the ligand in the sample. A wide variety of luminescent reagents may be employed. For example, chemiluminescent, phosphorescent or bioluminescent reagents can be used. The choice of the luminescent reagent will depend upon the particular ligand to be determinede and the chromogenic reagent system employed. Representative of the class of luminescent reagent that may be employed include, for example: anthracenes, triphenyl-benzenes, diphenylnaphthalenes, perylenes, chrysenes, oxalates, luminols, lophines, lucigenins, luciferins, acridiniums, transazodicarboxylates, pyrogallols and coelen-terate chromophores. These luminescent reagents can be triggered to produce light by using organic compounds such as hydrogen peroxide, potassium ferricyanide, potassium permanganate, metal ions and the like, or by biological catalysts such as luciferase or peroxidase, or by physical means such as changes in temperature, pH or radioactive emissions, e.g. x-rays.

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1.3.2 OPTICAL AND COLORIMETRIC METHODS Optical methods As a method of quantifying complement in a serum, so-called 50% hemolysis method (CH 50) developed by Meyer is known. In this method, sheep red blood cells sensitized with optimum amount of hemolysin (anti-sheep red blood cell antibody) is reacted with a serum to be tested at 37°C for 60 minutes. On doing this, the sensitized sheep red blood cells are lysed by the action of complement in the test serum, so that the complement can be quantified by determining the degree of hemolysis. However, the hemolytic reaction can only be halted by cooling the reaction mixture in an ice bath. Further, in this method, the degree of hemolysis is determined by centrifuging the reaction mixture and measuring the absorbance at 541nm of the supernatant obtained by the centrifugation. Thus, the procedure is time-consuming and further the test results are not always accurate. Accordingly, the object of Rokugawa K. and Tamayama Y. (TOSHIBA K.K., (171) is to provide an immunoassay by which antigen or antibody can be quantified accurately in a very short time. In the immunoassay of Rokugawa and Tamayama, microcapsules which can be lysed by complement activity, which contain an optically determinable substance, on whose surfaces an antibody or an antigen specific to an antigen or an antibody to be quantified is bound are provided. The microcapsules are mixed with a test sample containing the antigen or antibody to be quantified and with complement. By so doing, an antigen-antibody complex is formed between the antibody or antigen bound on the surfaces of the microcapsules and the corresponding anti-gen or antibody in the test sample. The complement is activated by the antigen-antibody complex, so that at least some of the microcapsules are lysed by the complement activity. As a result, the optically determinable substance contained in the microcapsules is released from the microcapsules to the reaction mixture. Thereafter an optical measurement is conducted at different wavelengths for the reaction mixture which is still suspending the intact microcapsules. Agglutination assay A variety of proposals have been made to automate agglutination as-says, thereby enabling an automated light measurement to serve in place of visual observation to determine the extent of clumping. An example includes US-Patent 4,205,954 to Babson (1980)n and various patents to Sawai, et al. Application of such agglutination assays to a centrifugal field (e.g., in a microcentrifugal analyzer) is disclosed in US-Patent 4,202,665 to Wenz, et al.

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In other forms of immunoassays, the target binding member is assayed by reaction either successively or concurrently with an immobilized binding pair member and a labelled reagent binding pair member. In the competition mode, for example, when one is analyzing for a hapten or antigen, the analyte hapten or antigen competes with labelled hapten or antigen for a limiting number of sites on immobilized antibody. By washing away unbound binding members, the label remaining on the immobilized antibody is an inverse function on the analyte concentration. In the inhibition mode, analyte hapten or antigen reacts with immobilized antibody before labelled hapten or antigen is introduced. In the sandwich mode, the target antigen or antibody forms a bridge between an immobilized antibody or antigen, respectively, and a labelled antibody or antiantibody, respectively. By washing away unbound labelled moiety, the label remaining is a positive function of the concentration of target antigen or antibody. In certain cases, particles have been used as a convenient form of solid substrate for the immobilization of the antibody or antigen in such tests. See USPatents 4,332,783 to Pernice et al. (sandwich mode); 4,481,298 to Cone, Jr. (sandwich employing two antibodies). In some cases washing the solid phase is a way of separating unbound binding members (and especially unbound labelled reagent binding member) from the solid phase. Centrifugation could be used in such washing. In comparing the two types of assays described above, it should be apparent that they have the disadvantage of requiring sufficient multifunctionality of the antigen or antibody to cause agglutination (clump-ing) rather than mere attachment. Lentrichia B.B. et al. (ALLIED CORPORATION, (5.2)) enable a variety of measurements, especially made in the centrifugal field such as in a microcentrifugal analyzer, in which the detecting step can be performed by simple optical density measurement or light scatter measurement. Accordingly, a method has been provided for the determination of a target binding pair member in a biological sample which comprises the steps: (a) reacting in a liquid phase in a chamber in a centrifugal field: (i) the sample to be analyzed, (ii) a plurality of first particles bearing on their surface a binding pair member complementary to target binding member, and (iii) a plurality of second particles bearing on their surface a binding pair member competitive with the target binding pair member for binding sites on the complementary member; the specific gravity of the first and second particles being sufficiently different with respect to the centrifugal field and the specific gravity and viscosity of the

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liquid phase to cause detectably different rates of migration in the centrifugal field for first particles, for second particles and for first particles bound to second particles; (b) detecting the concentration of particles at a radial locus in the chamber after a specified time of reaction and centrifugation; and (c) correlating the concentration of particles over time with concentration measured when samples of known target binding member concentration are reacted in the centrifugal field with the plurality of first particles and plurality of second particles. An article entitled “Detection of Antibody-Antigen Reactions at a glass-liquid Interface as a Novel Optical Immunoassay Concept”, (1984) R.M. Sutherland et al. (Proceedings of 2nd Optical Fibre Conference (Stuttgart 1984) page 75), describe optic waveguide apparatus wherein an antibody species is covalently immobilized onto the surface of a planar or fibre-optic waveguide. The reaction of immobilized antibody with antigen in sample solution is detected using the evanescent wave component of a light beam, totally internally reflected many times within the waveguide. The evanescent wave has a characteristic penetration depth of a fraction of a wavelength into the aqueous phase, thus optically interacting with substances bound to or very close to the interface and only minimally with the bulk solution. Although the tight confinement of power in the evanescent wave has many advantages, the use of conventional waveguides can create launching difficulties because of the small waveguide size. Stewart W.J. (PLESSEY OVERSEAS LTD., (134)) proposes an opticwaveguide biosensor including an optic-waveguide (1) (see figure) provided with a coating (7) sensitized to a specific assay species, and input and output coupling members (3, 5). Light signal response is enhanced by incorporating a partially reflecting, partially transmitting, low refractive index medium (15) between each coupling member (3, 5) and the waveguide (1). The thickness of this medium (15) is chosen so that light may be coupled by frustated total internal reflection and so that the medium (15) serves as a resonant mirror. It may be in the form of a single layer formed of e.g. magnesium fluoride or alumina material. Alternatively, it may be of multilayer structure dielectric material. Almost all current tests using agglutination/flocculation particles coated with antigen rely on differences in settling patterns of particles in the presence or absence of antibodies. These tests depend for their accuracy and sensitivity upon the training and experience of the person reading the test. Significant variations occur due to the subjectivity of different individuals.

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The microtiter-surface-flocculation assay of Nath N. (AMERICAN NATIONAL RED CROSS, (8)) eliminates subjectivity being machinereadable without sacrificing the sensitivity. The following figures show various symbols used in figures which show the method of Mr. Nath and the spectrometer therefore. Haemoglobin antigen research A nephelometer can be used to analyse samples of whole blood without the need to remove red blood cells or haemoglobin. According to Bradwell A.R. and Deverill I. (ALTA DIAGNOSTICS MACHINES LTD., (6)) a method of quantifying, in a whole blood sample in which the red cells are lysed, a component is provided, which will react with a reagent to form an antigen-antibody complex, comprising mixing the sample with the reagent to obtain the complex, exposing the sample to a source of radiation and measuring the intensity of radiation scattered through a given angle by the complex. In a particular embodiment the intensity of the scattered radiation is measured at intervals, to determine the rate of formation of the antigen-antibody complex. Preferably, the wavelength of the radiation is selected such that it is a wavelength at which the intensity of the radiation scattered through the angle by the antigen/antibody complex is high and the absorption of the radiation by haemoglobin and other proteins is low. It is particularly preferred that the intensity of the scattered radiation is at a local maximum and the absorption of the radiation is at a local minimum. Typical wavelengths of suitable radiation are 460–530 nm, more preferably 460–510 nm. The red cells are lysed such that they fragment into particles of a size which does not scatter light of these wavelengths and so reduces interference. Because the methods described in the previous aspects do not require centrifuging or an ultraviolet light source and its attendant power supply, it is

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Symbols used

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possible to construct the apparatus for carrying out these methods such that it is portable and relies on an internal power supply. The figure is a spectrophotometer scan of a cuvette containing saline, polyethylene glycol, zaponin/KCN and whole blood (line A); the same cuvette two minutes after the addition of anti IgG antiserum (line B); and the interference filter employed in the device. It is known that the properties of a species in a liquid sample may be detected by applying to an optically active part of the surface of a substrate a thin film of a material capable of binding the species to be assayed and thence applying the sample onto the thin film and observing the change in the optical properties of the surface as the species within the sample bonds to the applied thin film. The “optical active” surface may be a grating. A beam of suitably polarised light incident on the grating will give rise to diffraction or reflection properties which are dictated by the manner in which the sample reacts with the applied thin film of material. The angle of incidence of the beam may be a right angle, or may be variable, resulting in a reflectivity dip at a particular angle. Finlan M.F. (AMERSHAM INTERNATIONAL plc, (9.2)) sought to improve the sensitivity and versatility of this technique by providing a method and apparatus for assaying a species in a biological sample fluid. The apparatus comprises an SAW device (1) with a slab (2) of piezoelectric material on the

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upper surface (5) of which is formed an input transducer (3) and an output transducer (4). A source of RF energy is applied to the input transducer to generate a surface acoustic wave. Applied to the surface (5) is a thin layer (8) of a material capable of binding a species to be assayed. The sample (13) to be tested is applied to the top of the layer (8). A collimated light beam (1) from a source (9) is applied to the thin film from underneath the slab (2) and is collected by a photodetector (12). When the slab (2) is energised, the vibration sets up an effective diffraction grating which is coupled to the thin film and acts to diffract the light beam (10) applied to it. The energy in the diffracted beam, as measured by the photodetector (12), is indicative of the progress and result of the reaction between the layer (8) and the sample. A common test for pregnancy involves coating small polystyrene latex spheres with the hormone, human chorionic gonadotropin (HCG). When a woman becomes pregnant, the level of HCG in the urine increases significantly. This is an indirect test in which a quantity (as de termined by titer by an established procedure) of antibodies to HCG is added to a sample of female urine and is allowed to incubate for from about 5 to about 10 minutes therein. Next, HCGcovered latex spheres are mixed with the urine and the mix is allowed to incubate for from about 5 to about 10 minutes. If agglutination of the spheres takes place, the urine does not contain HCG to the level establishing a pregnant condition; if the spheres remain in single suspension, HCG was present beyond that level. These tests can be generalized to detect any antigen or antibody. The fact of agglutination preferably should be visible to the ordinary observer. According to Gisever I. and Keese C.R. (GENERAL ELECTRIC COMPANY, (61.2)), a large number of small droplets of a first liquid are dispersed in a second liquid in the nature of an emulsion. The second liquid is an aqueous medium and the first liquid is relatively immiscible with the second liquid. The resulting liquid droplets receive a coat-ing of a specific protein (e.g., a coating of

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a particular antibody, that will interact specifically with some select protein (e.g. a select antigen). The initial protein coating can be provided in the aqueous medium used to prepare the emulsion or can be added after the emulsion has been prepared. Preferably the concentration of this protein is known. A contact period between the protein material and the liquid droplets of less than one hour is usually sufficient, when protein concentrations upwards of 10 micrograms/ml. are employed. In those instance in which the desired protein coating does not adhere to the liquid droplets unaided, the necessary attachment should be obtained chemically by introducing a small amount of a fluorinated polar compound (e.g., pentafluorobenzoyl chloride) to the first (i.e., the droplet) liquid. Having coated the liquid droplets with the requisite specific protein, the emulsion is then gently centrifuged segregate the droplets from the bulk of the aqueous medium. The supernatant aqueous medium is then removed (e.g., by decanting). Next, the coated droplets are washed at least once with an aqueous solution of a non-specific protein (about 100 micrograms of the non-specific protein per milliliter of 0·15 molar sodium chloride solution). The protein-coated droplets are then re-suspended in dilute (i.e., about 0·15 molar NaCl) saline solution at pH 7·5. It may be necessary to use a buffer, such as 0·01 molar trihydroxymethylaminomethane. These protein-coated liquid droplets present in a concentration of from about 106 to about 1010 droplets/cc are now suitable for contact with a liquid sample to be tested for the presence or absence of the specific protein. The liquid sample is normally a body fluid, such as blood or urine. Wyatt P.J. (THE CENTER FOR IMMUNOLOGICAL STUDIES, (31)) found a method for the detection and quantitation of immunological substances, which of (a) affixing to a surface a reactant material known to interact and to bind to, or to have a relatively high affinity for, the immunological substance to be detected, the substance being present in at least one medium; (b) impinging the affixed surface with a source of coherent radiation; (c) measuring a pattern of radiation scattered from the affixed surface; (d) reacting the affixed surface with at least one medium containing the immunological substance; (e) impinging the reacted affixed surface with substantially the same coherent radiation at substantially the same aspect and orientation utilized to perform step (b); (f) measuring a pattern of radiation scattered from the reacted affixed surface; and (g) comparing the patterns of radiation derived from steps (b) and (f) to determine any differentiations therebetween. The scattering of coherent light from a surface therefore results in the formation of speckled patterns which may be observed by any conventional means, such as the naked eye or projected on to a screen. The degree of speckle is preferably quantitatively measured by suitable measurement means such as a photometric detector or the like. Moreover, detection means by which the

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relative change of speckle pattern may be observed and regular low frequency features detected on a background of relatively higher frequency speckle, such as by spatial filtering, may be utilized to compare the foregoing measured patterns and thereby determine any differentiations therebetween. An embodiment of the Wyatt method is shown in the figure below. Colorimetrical measurement Although colorimetrical measurement and indication are widely accepted in laboratory work nowadays and thus in immunoassay procedures also, it became apparent from the present literature analysis that many researchers in the immunoassay field based their investigations on colorimetric measurements employed for purposes others than immunoassay. The next paragraph is devoted to some typical examples of colori-metric procedures which particularly are adapted to immunoassay. Other procedures and devices will be discussed in chapters following hereafter. A typical example of an immunoassay with chromatographic medium and enzyme labelled reagent is by Edwards J.C. et al. (AMERSHAM INTERNA TIONAL plc., (9.1)). A suspension of antibody complex was formed by mixing suitable amounts of sheep anti-thyroxine serum (gamma globulin fraction) and donkey anti-sheep gamma globulin serum in phosphate buffer and incubating overnight at room temperature. The following reagents were incubated together in a series of test tubes for 2 hours at room temperature. 100 µl of a standard solution of thyroxine in human serum. 200 µl of thyroxine conjugated to beta-galactosidase and diluted in phosphate buffer. 400 µl of the suspension of second antibody complex described above. A 50 µl volume of the reaction mixture from each test tube was then slowly applied to the centre of a glass fibre disk (Whatman GF/D).

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A 400 µl volume of a 20 mM solution of ONPG (orthonitrophenyl galactoside) in phosphate buffer containing 1 mg bovine serum albumin/ ml, 9 mg NaCl/ml and 10 mM magnesium chloride was then slowly applied to the centre of each filter disk. The disks were left for 5 minutes at room temperature before visual observation of the intensity of the yellow spot at the centre of the disk. Hossom M.G. (MUREX CORPORATION, (113.4)) provides a method for colorimetrically quantifying the relative amount of a component as a part of a total sample within a single test format. The method for the semi-quantitation of a component of an analyte of interest, present in a specimen, where the analyte is composed of at least two components, consists of (a) immobilizing an antibody directed against the component on a solid support material in a delimited area defining a reaction zone; (b) contacting with the immobilized antibody a specimen containing the analyte such that substantially all of the component binds to the antibody while the specimen is retained on or within the reaction zone; (c) reading the reaction zone to determine the relative amount of analyte present; (d) washing the reaction zone to remove substantially all material not bound to the antibody; (e) reading the reaction zone to determine the relative amount of component remaining bound to the antibody; and (f) calculating from the readings the ratio of the component to the analyte. Conventional enzyme immunoassays suffer from the problem that it is difficult to have high sensitivity together with a wide assay range. The problem is caused by the limited dynamic range of the signal detection system. Considering for example a 2-site enzyme immunoassay, for high sensitivity a marked increase in signal is required for a unit increase in analyte concentration, but at high analyte concentrations the signal can be greater than the dynamic range of the measurement system. If the signal intensity at high analyte concentrations is reduced to the dynamic range of the detection instrument, then the signal change for a unit increase at low analyte concentrations will be reduced and assay sensitivity decreased. The problem is particularly acute for enzyme immunoassays utilising spectro-photometric endpoint detection. Philo R.D. (SERONO DIAGNOSTICS PARTNERS, (151.2)) has devised a means of carrying out enzyme immunoassays, employing enzyme labels capable of converting a substrate to a coloured product whereby this problem is overcome. Accordingly he provided an enzyme immunoassay employing one or more enzyme label-substrate pairs which give rise to coloured products and in which absorption by the product of substrate conversion by at least one enzyme label at a first wavelength, at or close to the optimum wavelength for absorbance, exceeds the linear range of the detector, which immunoassay includes the steps of measuring the substrate conversion at a second wavelength at which absorbance by the relevant product is significantly lower than at the first wavelength and calculating the true absorbance at the first wavelength by

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utilizing the result of linear regression analysis of absorbance measurements obtained with product standards at the first and second wavelengths within the linear range of the detector. Selection between the two chosen wavelengths for a particular enzymesubstrate pair thereby enabling the requirement for high sensitivity or wide assay range to be met may conveniently be achieved automatically with appropriate instrumentation. Ranby and Bergsdorf (CYTRX BIOPOOL LTD., (46.1 and 46.2)), developed two solid phase procedures for the quantification or qualification of antigens during which they seek to overcome the difficulties with falsely high or falsely positive measurement results. In a first embodiment of the methods of Ranby and Bergdorf (46.1), the purpose is achieved by—exposing two aliquots, preferably of the same size, of the sample containing the antigen that is to be determined to two solid phases with identical anti-antigen-antibody-coated surfaces, in which one exposure occurs in the presence of antibodies present in the aliquot with the same specifity as the antibodies with which the solid phases are coated, and the other exposure preferably occurs in the presence of antibodies present in the aliquot with nonspecifity for the antigen in question, but otherwise essentially the same as the antibodies present in the aliquot in exposure 1, and the antigen is quantified by means of the difference between the bonded antigen in exposure 1 and the bonded antigen in exposure 2. The second embodiment (46.2) comprises, with reference to the next figure, the steps of coating a first solid phase (18) and a second solid phase (22) with an antigen-specific antibody, adding a first dye to the first solid phase (18), exposing the first solid phase (18) to a standard solution with a known amount of the antigen, exposing the second solid phase (22) to a solution with an unknown amount of antigen, and determining the amount of antigen bound to the first solid phase (18) and to the second solid phase (22). In another embodiment, a second dye with a color different from the first dye can be added to the second solid phase (22). Niskanen et al. (ORLON CORPORATION LTD, (121.2)) developed a method and apparatus for interpreting the results of agglutination tests, in which the apparatus includes a light indicator with which it is possible to observe the change, resulting from an agglutination reaction, in the amount of light reaching the light indicator via the route taken by external illumination, not generated by the apparatus itself, to the light indicator; and in the agglutination reaction which is carried out in the aforesaid route of light reaching the apparatus from an external source, not generated by the apparatus, a carrier is used which is coated with a biologically interesting material which can be identified by an agglutination reaction.

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The carrier used in the agglutination reaction is a latex particle or a particle prepared from some other organic material an animal red blood cell, a particle made of inorganic material, a microbe or a liposome. The apparatus may include more than one photodiode or phototransistor. The light entering the light indicator 5 (see the figure) can be filtered with the light filter 6. The amount Øl of light entering the light indicator 5 is proportional to the difference between the amount Øt of light entering the drop 1 from the environment and the amount Øs of light leaving the drop after scattering, absorption, reflection and refraction. The amount of light scattered in the drop 1 is proportional to the size and the number of particles within the drop. The light indicator can therefore be used to observe changes occurring in the size and the number of particles in the drop. The light indicator 5 and the signal processing circuit 7 are protected by the plastic covering 8.

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1.4 RADIOIMMUNOASSAY From the present study it became apparent that the last five years the development of radioimmunoassay comes a little behind other immunoassay methods discussed herein. Nevertheless there have been found a number of developments on radio-procedures worthwhile to be discussed. Many radioimmunoassay test methods and devices have been developed since the pioneering work of Ekins in 1960 and Yalow and Berson in 1960. The standard method of conducting radioimmunoassays, as described by these pioneering researchers, is based upon a theory of competitive binding. Such a test method for a particular antigen of interest, utilizing competitive binding principals, requires an antibody which is specific to the antigen and a quantity of radiolabelled antigen. Liquids containing the unknown antigen and the radiolabelled antigen are reacted with the antibody, and both the unknown antigen molecules and the radiolabelled antigen molecules compete for the binding sites of the antibody molecules. The competitive immunoreaction is conducted until near-equilibrium conditions have been achieved between the bound and unbound reactants. Thereafter, the antibody bound antigen is separated from the liquid reactants by any of several means. Following the separation, the radioactivity of the antibody bound antigen is determined. Further tests are thereafter conducted utilizing a plurality of liquids containing differing known concentrations of the antigen and after equilibrium conditions have been achieved in each of the immunoreactions, the antibody with the bound radioactive antigen from each of such reactions is examined for its radioactivity. Thereafter, a calibration curve is created which correlates the radioactivity from the immunoreactions utilizing known samples with the concentration of antigen in the known samples. Finally, using the calibration curve and the radioactivity of the unknown antigen immunoreaction the concentration of antigen in the unknown sample is determined. The object of research carried out by Tsay and Shah (INTERNATIONAL IMMUNOASSAY LABORATORIES, INC. (84)) provide a radioimmunoassay test method which does not require near-equilibrium conditions in its immunoreactions, such that it provides rapid results. The test method of Tsay and Shah for the quantitative radioimmunoassay of an analyte of interest in endogenous liquid sera utilizes an immunoreagent that is specific to said analyte of interest and which is immobilized on a on a substrate, and an exogenous liquid containing radiolabelled analyte of interest. The endogenous and exogenous liquids are combined with the immobilized immunoreagent in a reaction vessel and an immunoreaction is permitted to occur. The immunoreaction is halted at a time when the rate of change of the quantity of radiolabelled analyte of interest being bound with said immunoragent

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is proportional to the concentration of the analyte of interest in the endogenous liquid. Thereafter, the radioactivity of the immobilized immunoreagent with its bound analyte is determined. Substantially identical test procedures are then performed utilizing liquids containing a zero concentration and other known concentrations of the analyte of interest in place of said endogenous liquid and a calibration curve is created from the results obtained for the known concentration liquids. The test results for the endogenous liquid are then compared with the test results for the known concentration liquids through the use of the calibration curve, and a quantitative determination of the concentration of analyte of interest in the endogenous liquid is completed. The following figure shows a calibration curve of the type utilized in the Tsay test method. Kallikrein determination The determination of kallikrein in human urine by radioimmunoassay for the diagnosis of various deseases including hypertension as well as to a kit to be used for said determination will be discussed now. Kallikrein is an enzyme which acts upon kininogen in plasma, and liberates kinin which is a physiologically active peptide. The biological actions of kallikrein are developed through the liberated kinin. These actions include, for example, vasodilation, blood pressure lowering, smooth muscle contraction, capillary vessel permeability raising, catecholamine secretion raising, and prostaglandin biosynthesis acceleration. It is known that kallikrein is excreted not only in human urine but in urines of many animals. The kallikrein in urine is produced by the biosynthesis in the kidney, and participates particularly in the control of renal blood flow and blood pressure. Thus, kallikrein plays an important role in maintaining the constancy of a living

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body, showing a broad range of physiological activities. Accordingly, the decrease of biosynthesis or secretion of kallikrein gives rise to serious diseases. It is known, for instance, that the amount of kallikrein excreted in the urine of a patient suffering from essential or nephrogenous hypertension is distinctly low compared with that of a normal person, and that the amount of kallikrein excreted in urine is large in case of primary aldosteronism of Bartter’s syndrome and is small in case of renal insufficiency. Thus, in treating various diseases including hypertension, it is highly useful in the diagnosis of the diseases to determine quantitatively the kallikrein excreted in urine. Known methods of quantitative determination of kallikrein include a method in which the lowering of whole body blood pressure of a dog administered with the specimen intravenously is used as an index of the amount of kallikrein (Frey, E.K., Arch. Lin. Chir., 142, 663 (1926)) and a method in which the hydrolytic activity of kallikrein on various synthetic substrates such as methyl ester of tosylarginine, ethyl ester of benzoylarginine, propy-phenylalanyl-arginine methylcoumarinamide, and D-valyl-leucyl-arginine para-nitroanilide is estimated. These methods are not only complicated in their procedure but poor in the specificity of the determination. In recent years immumoassays, particularly radioimmunoassay, utilizing the specificity of antigen-antibody reaction have been developed, and it has been reported that kallikrein can be accurately determined by said assay. As is apparent from these reports by the application of radioimmunoassay an accurate determination of trace amount of kallikrein excreted in urine has become possible, and the correlation between various diseases and the amounts of kallikrein excreted in urine has come to be investigated in detail. In the methods disclosed in these reports, however, the reaction of kallikrein labelled with a radioactive substance and kallikrein in the specimen (urine) which act as antigens with the anti-kallikrein antibody acting as an antibody is carried out in liquid phase, so that the separation of the antigen labelled with a radioactive substance which has formed a complex (B-body) with the antibody from the labelled antigen remaining free (F-body) is troublesome. In the prior methods mentioned above, B/F separation is carried out by the double antibody method or polyethylene-glycol precipitation method. In these methods of separation, however, incubation or centrifugation is necessary for separation, so that the time required for measurement is lenghtened and the operations are complicated. For a solution see Moriya on page 235. Peptide and protein oligomers In the sandwich immunoassay of Pestka (HOFFMANN-LA ROCHE INC. (73)) a monoclonal antibody which recognizes a single, select epitope on the target

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peptide or protein is coupled to a solid support in a manner known per se. Suitable solid supports for this purpose include, for example, plastic microtiter plates, such as most preferably polyvinylchloride plates. The supported monoclonal antibody when contacted with the test solution containing the target protein will bind to all materials which contain the select epitope and thus will bind to monomeric, dimeric, trimeric and higher oligomeric forms of the target peptide or protein, if present. In the second phase of this assay procedure, the monoclonal antibody coupled to the solid support is probed with the same monoclonal antibody which has been labelled with a detectable radiolabel. Any target monomeric peptide or protein which has been bound to the monoclonal antibody coupled to the solid phase will not have the epitopic binding site available since that site is already occupied. However, if the target peptide or protein is present in oligomeric forms then there are one or more free sites available to which the labelled monoclonal antibody can bind. Thus, the immunoassay will detect selectively any dimers or higher oligomers. Since the larger oligomers have more sites available than the smaller ones, more label should bind to the higher oligomers. In principle the number of sites available to the labelled antibody in an oligomer of n subunits is equal to n-1. In practice, however, it can be expected to be somewhat less because some of the oligomers may bind to the first antibody through more than one site. Target peptides or proteins whose oligomeric forms can be selectively detected by means of the Pestka immunoassay include the interferons, such as leukocyte, fibroblast and immune interferons, hybrid leukocyte interferons, growth hormone, insulin, lymphokines such as interleukin-1 and interleukin-2, serum albumin, somatostatin, chorionic gonadotropins, enzymes such as urokinase and the like. The aforesaid peptides and proteins can comprise human or other mammalian sequences such as ovine, porcine, murine, equine, feline, canine, bovine and the like. Such peptides or proteins can be naturally derived, synthetically or produced by recombinant DNA technology. They can be glycosylated or nonglycosylated. Solid-phase support The support proposed by Kerschensteiner (KERSCHENSTEINER (90)) comprises a test tube liner having one closed end that is preferably rounded in shape. The liner has applied to its surfaces a coupling solution comprising a fixative to render the liner water-insoluble, a bioactive protein, and a bifunctional coupling agent to bind the bioactive protein to the liner surfaces. Test tube liners may be prepared from proteinaceous polymeric materials, polysaccharides, polyesters and polyamides, with the first-named class of materials being preferred. Fixatives may be selected from alkali metal and

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alkaline earth metal sulfates, bicarbonates and chlorides; lower alkanols, certain polyols such as polyethylene glycol, and mixtures of these. Bifunctional coupling agents may include certain aldehydes, such as α,β-unsaturated aldehydes, dialdehydes, and mixtures. The work of Kerschensteiner also includes a method for the preparation of the solid-phase support which comprises providing the above described test tube liner; contacting the liner with a sufficient quantity of the above-described coupling solution to render the liner water-insoluble, and to covalently bond to the surfaces of the liner the desired bioactive material; and thereafter separating the treated liner from the coupling solution, whereupon it may be dried for storage or prior immediate use. More particularly, the solid-phase immunoassay supports may be produced by the treatment of a preformed soft gelatin capsule half with a coupling solution comprising a fixative, which may be an alkali metal salt, a bifunctional coupling agent and a bioactive protein which selectively reacts with the substance which is the subject of the immunoassay; processes for their production; and methods of use therefor. T4 measurement Iodothyronine (T4) secreted from the thyroid, and the measurement of serum T4 concentration has become the test commonly employed as an initial procedure in the diagnosis of states of altered thyroid function, such as hyperthyroidism or hypothyroidism. In addition, it is well known that several conditions other than thyroid disease may cause abnormal serum levels of T4. Among these are pregnancy, estrogenic or androgenic steriods, oral contraceptives, hydantoins and salicylates, stress, hyper—and hypoproteinemia, and conditions (hereditary or acquired) which cause alterations in serum levels of thyroid binding globulin (TBG) the major serum T4 transport system. The method, revealed in by Siebert et al. (BECTON, DICKINSON AND COMPANY (16.1)) for the immunoassay of iodothyronine (T4) uses a combination of particular monoclonal antibodies, which are produced by two new and separate hybridoma cell lines. Combinations of the monoclonal antibodies from the two cell lines are used in an immunoassay for T4 of high accuracy over the range T4 concentrations encountered in serum samples. The major problem with the homogeneous enzyme assays is that the sensitivity of radioassays is better than the sensitivity of enzyme assays. The development of homogeneous radioassay would be a step forward because a number of ligands of interest have very low concentration in biological fluids. It also should be noted that none of the homogeneous assays is particularly well suited for in vivo ligand detection.

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Accordingly, an object of Burton and Hoop (BURTON (27A)) was to produce a homogeneous radioassay having excellent sensitivity and reproducibility. Another object was to provide an apparatus for performing a homogeneous radioassay. A process and apparatus for radioassay of ligand in solution which eliminates the separation step required in conventional techniques. In the next figure a chamber (10) is provided containing a quenching solution (22), a plurality of ligand molecules (LM) and a plurality of receptor molecules (R). One of the pluralities forms a free species labelled with a beta particle emitter (LM*) while the other is immobilized on a solid support (16), e.g. the chamber wall or a microbead, within the chamber (10). Ligand (L) introduced with the sample competes with ligand molecules (LM) already in the chamber for receptor sites on the receptor molecules (R) and the free species is allowed to diffuse about the chamber (10). A beta particle detector (14) in communication with the chamber (10) at a fixed position detects only those beta particles omitted from within the quenching distance of the quenching solution (22). The quenching properties of the solution (22) are used in place of the conventional separation step. The process and apparatus are easily adapted for continuous monitoring of ligand level and are particularly well suited for use in radioimmunoassay. The apparatus can be miniaturized allowing implantation in an animal body and in vivo monitoring of ligand level in bodily fluids.

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Iodine marked pyréthrinoîdes Demout, Touyer and Mouren (ROUSSEL-UCLAF (142)) developed radioactive derivatives marked by iodine according to the general formula:

wherein: X1= a halogen or a trifluoromethyl X2=a halogen R=the rest of an aminaled acid R-NH2 or the rest of a derivative of the formula, this rest including an iodome acceptor marked by iodine125 or iodine131 isotopes. Solid phase radioassays were first described by CATT and his coworkers in 1967. See Nature 213 (1967) pp. 825–827. Conventional radioassays, however, are non-homogeneous and require, as described before, separation steps. On a consequence, homogeneous procedures were used such as the homogeneous enzymatic and fluorescence methods which do not require separately steps. BURTON and HOOP (27A) developed a homogeneous radioassay process, which comprises: (a) providing a chamber and an associated sensor for generating s signal representative of the number of beta particles emitted by radioactive atoms incident thereon, said chamber containing: (i) a plurality of receptor molecules capable of coupling with the ligand, and (ii) a plurality of ligand molecules capable of coupling with the receptor molecules, at least one of the pluralities of receptor molecules and ligand molecules being a species immobilized on a solid support within the chamber, the others of the pluralities of receptor molecules and ligand molecules comprising free species labeled with radioactive atoms which emit beta particles; (iii) a chamber medium; (iv) a fixing agent for fixing the position of the sensor with respect to the suport; (b) introducing a solution containing the sample into the chamber, the solution and the chamber medium comprising a quenching solution adapted to

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quench substantially all beta particles emitted by the radioactive atoms before the particles travel a predetermined distance D; (c) allowing ligand in the sample and ligand molecules to compete for sites of attachment on the receptor molecules and allowing the free species within the chamber to diffuse about the solution; and (d) detecting the signal generated by the sensor while the solution and the free species remain within the chamber and comparing the signal with a base value to detect the presence of ligand in the sample. As stated herebefore, heterogeneous radioassays have the drawback that they require separating steps and near-equilibrium conditions which commonly takes much time in carrying out the procedure. 1.5 OTHER METHODS AND MEANS Magnetic means The separation of the solid phase from the reaction solution always includes washing of the solid phase, which at present, as a rule, requires manual operations. If small polymer particles are used, like in the method of the present invention, these operations include centrifuging or magnetic deposition. The object of Luotola et al. (LABSYSTEMS OY (96.1)) was to provide a simple manual method for the determination of antibodies or antigens, their method is also suitable for use with such antibodies or antigens as are placed on the surface of cells or other particles of organic origin. Saxholm (SAXHOLM (146)) also uses magnetic means for testing reactions. In order to compare the test results of bacterial sensitivity to various antibiotics, or the other biologically active substances, it is essential that uniform and good contact between the article containing the biologically active substance and the substrate is obtained. It is also important to achieve as uniformly adjusted contact as possible from one sensitivity test to the next in order to compare the results and establish a basis for reliability. Thus, the object of Saxholm was to provide an article containing an active constituent suitable for testing various reactions i.e. changes in growth and/or other reactions with an agent in a substrate by diffusion of the active constituent in the article into said substrate, or by diffusion of the agent contained in the substrate into the article. According to Saxholm a biologically active substance is incorporated in a unitary body with a magnetically responsive material for carrying out diffusion testing. These may be, microbiological, immunological, serological and other biochemical examinations. The body is applied against a

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substrate or medium by application of an external magnetic field and a reaction is produced at the site of the body and is measured by means of a reader. In order to insure deposit of the body on the substrate a predetermined location and corresponding reading of the reaction legion at such location, the support for the substrate and the dispenser and rear are provided with suitable releasable coupling and orienting devices such that the dispenser and reader can be respectively engaged and oriented on the support in predetermined secured positions. The next figure shows an embodiment of the apparatus used. On the other hand, Davis et al. (UNILEVER PLC (175.1)) developed nonseparation particle-based immunoassay systems wherein one step is required (homogeneous immunoassay). More particularly, the immunoassay of Davis concerns the qualitative or quantitative determination of the presence of an analyte in a sample by means of specific binding, wherein the sample is contacted with a movable solid phase carrier material, e.g. magnetic particles (620) on which is immobilised a first binding reagent having specificity for the analyte and with a labelled reagent which can participate in either a “sandwich” or a “competition” reaction with the first reagent in the presence of the analyte and following an incubation period sufficient to allow the reaction to take place. The carrier material is moved within the assay medium (618) to a location adjacent a signal sensing means (602), the label generating a signal, such as chemiluminescent light, continuously in the assay medium and the magnitude of the signal generated in the vicinity of the sensing means being used as a measure of the extent to which the binding reaction has occurred. Preferably the assay medium incorporates a masking or quenching agent which suppresses signal generated in regions of the assay medium remote from the signal sensing means. See the following figure. One problem with the presently available methods of magnetic separation is that the particle formed of magnetic material which have been in use must be

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coated, or treated extensively to provide a binding or attaching site for molecular attachment. Magnetic material, unless treated, does not provide sufficient, stable attachment or binding sites for biological or biochemical material. As a result, magnetic separation techniques have been hampered either by the need for extensive pretreatment of the magnetic material or have yielded poor results when there has been no pretreatment. Recently, magnetotactic bacteria have been discovered in nature, and have been found to be amenable to cultivation in the laboratory. See, e.g., Frankel, Blakemore & Wolfe, Science, v. 203, pp. 1355–56 (1979). Schwartz and Blakemore (BIO-MAGNETECH CORPORATION (18)) found magnetic material, e.g. magnetic bacteria or magnetic particles contained therein, which are treated to render them receptive to binding or attachment to the substance sought to be detected or removed. Following binding or attachment to the treated magnetic material, the medium is subjected to a magnetic field, which results in removal of the magnetic material and the substance bound or attached thereto. Biological magnetic material may be derived from lysed magnetic bacteria, such as Aquaspirillum magnetotacticum or the whole magnetic bacteria may be used. Lysed magnetic bacteria yield magnetosomes, which consist of particles of magnetite (i.e., Fe3O4) which are surrounded by a sheath or membrane, either as individual particles or in chains of particles. Whole magnetic bacteria may also be used, as the magnetic properties of these bacteria result from the magnetic particles or magnetosomal units which are a part of the bacteria. Both the magnetosomes and the whole bacteria possess biological membranes capable of interaction with and attachment of foreign molecules, such as antigens, antibodies, and chemically reactive groups. In order to enhance the ability of these membranes to form stable attachments to foreign molecules, the magnetosomes and whole bacteria may be treated with bifunctional cross linking

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reagents. Typical examples of these bifunctional cross linking reagents include cyanogen bromide, with the structure or glutaraldehyde, which has the structure

According to Chagnon et al. (ADVANCED MAGNETICS INC.) (2A) magnetic particles covered by silanes are used, the mean size of the particles being 0·03 µm. Magnetic gel Institut Pasteur found a magnetic gel 14 years ago (see French patent 2,334,106) which can be used for immunoenzymatic determinations and which consists of an acrylamide/agarose gel containing magnetic particles and coupled to a protein, such as an antigen or an antibody, via a coupling agent, such as glutaraldehyde, this type of gel coupled to an antigen or an antibody being used for the immunoenzymatic determination of the corresponding antibody or antigen present in a biological liquid, this gel being incubated with the biological liquid, then washed several times and separated off with the aid of a magnetic field, and then incubated with antibodies or antigens labelled with an enzyme commonly used in immunoenzymatic determination, such as glucose oxidase or peroxidase, and the enzymatic activity of the gel being measured by reading the optical density. Dodin et al. (INSTITUT PASTEUR (129.2)) used such a gel in a method comprising the following steps: (a) a biological medium supposedly contaminated by a pathogenic germ possessing a specific antigenic determinant is brought into contact with particles of the magnetic gel, (b) the particles of magnetic gel are then removed by magnetic means and (c) are inoculated onto a nutrient agar medium, (d) the agar medium is incubated to develop culture colonies of the pathogenic germs; (e) areas containing serum having antibodies specific to the pathogenic germ to be detected are formed in the agar, (f) the germs are lysed by means of lysing agents, (g) the lysate is incubated with the serum for a time enabling the serological antibody-antigen reaction to take place by immunodiffusion on the agar, and

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(h) the quantity of germs contained/in the biological medium is read directly, by virtue of the antigen-antibody reaction on the agar. Electrophoresis 1. An immunoassay method for measuring a concentration of an anti-gen contained in a sample by fixing said antigen with an immobilized antibody according to antigen-antibody reaction, and developed by Tokinaga et al. (HITACHI, LTD. (70.1)), comprises (a) a step of immobilizing an antibody over substantially the whole surface of a supporting matrix for electrophoresis; (b) a step of moving the antigen in the sample to be measured by electrophoresis with direct current to fix the antigen by the antigen-antibody reaction onto the immobilized antibody; (c) a step of moving a labelled antibody to said fixed antigen by direct current electrophoresis for reaction with said antigen, and (d) a step of measuring an enzyme activity, luminescence, or fluorescence of said labelled antibody to measure the concentration of the antigen in the sample. 2. The antibody-immobilized matrix is a membrane, 3. preferably a porous cellulose acetate membrane, or 4. a polyacrylamide gel membrane. 5. A potential gradient is applied in a vertical direction to the surface of the antibody-immobilized matrix membrane and the antigen and the labelled antibody are subject to the electrophoresis in the vertical direction. The figure substantially shows the apparatus used by Davis et al.

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Use of microcapsules and globulins Polymers Solid phase reactions Kakimi et al. (FUJI PHOTO FILM CO., LTD. (59.1)) have found that by using microcapsules obtained by introducing into the wall surfaces thereof one or more functional groups an antigen or antibody can be bound to the microcapsules via a cross linking agent in which one group is chemically bound to an antigen or antibody and the other group is bound to the microcapsule wall. As a result, the antigen or antibody is strongly bound to the microcapsules by chemical reaction so that a reagent having excellent storability over long period of time results. The thus obtained microcapsule reagent is highly stable, especially in that it can withstand freeze-dyring which is a representative of storage for long periods of time. The term “microcapsule” refers to a microcapsule comprising a wall material having encapsulated therein an oily substance as the core. The term “functional group” refers to a group which has a site for binding an antigen or antibody via a cross linking agent. Typical examples of functional groups which are introduced into and chemically bound to the wall surface of a microcapsule include an amino group, a carboxy group, a hydroxy group, a mercapto group, etc. Introduction of functional groups into the wall surface of the microcapsule in accordance with Kakimi et al. can be carried out either using compounds containing functional groups as a wall substance of the microcapsule, or, after preparation of a microcapsule, by subjecting the surface thereof to a chemical treatment as will be later explained. Chemical treatments include a method for converting a precursor as a wall substance into a substance having the desired functional groups through a chemical reaction, a method for reacting a compound having functional groups with the wall surface of a microcapsule and a method for binding a compound containing functional groups to the wall surface of a microcapsule using a crosslinking agent. Any compound(s) can be employed as the wall substance(s) for the microcapsule employed without any particular limitation so long as they are capable of chemically bonding to an antigen or antibody without inactivating an antigen or antibody and are capable of encapsulation. The method of Marshall (MUREX CORPORATION (113.2)) comprises a relation between finely divided particulate solid material capable of forming a stable suspension in a liquid medium and an inert porous filter matrix means of sufficient pore size. The method comprises: (a) combining a specimen, a first binding component, insoluble particles, and second binding component labelled with a signal

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generating material in a solid phase retention and separation apparatus having a sufficient pore size such that said particles are trapped within said filter yet permitting rapid passage of fluid therethrough in such a manner that an immunological reaction occurs if analyte is present in the specimen, resulting in the formation of an immunocomplex of insolubilized first binding component: analyte: second labelled binding component on or within said filter means; (b) separating bound from unbound material; and (c) determining the presence and/or amount of signal produced which is correlative with the amount of analyte present in the solution. According to Kronvall (PHARMACIA AKTIEBOLAGET (132.1)) antibodies are specifically fixed to a polypeptide which is derived from microorganisms and which can bind the Fc-part of the antibodies. The polypeptide is bound to the surface of the minute particles. A particularly suitable example of polypeptide (I) in accordance with the above is the so called protein A from Staphylococcus aureus or fragments thereof, said fragments being of a polypeptide nature and being capable of binding the Fc-part in the antibodies. Another example is polypeptide from Staphylococcus epidermidis. Polypeptides from some strains of Streptococcus pyogenes (e.g. group A and C) may also be mentioned as polypeptides capable of reacting with the Fc-part of certain immunoglobulins. In this connection polypeptide (I) (e.g. protein A can be bound to the surface of the bacteria bodies which produce the polypeptide, thereby affording a particularly simple procedure of producing reagent material for the method. Polypeptides bonding antibodies to the surface of small particles which are insoluble in a liquid in which an immunoreaction is effected, is well known in the art. Kronvall (PHARMACIE A.B. (132.1)) also uses such a bonding method but fixes the antibodies to a polypeptide from microorganisms. Kronvall aims at a very high density of antibodies on the surface of the particle, the antibodies having their antigen binding parts (Fab-parts) extending outwardly from the particle, so that an effective binding with the antigen in question can take place. In this way, the agglutimation obtained can be readily observed and indicated. The polypeptide capable of binding the Fc-part in the antibodies is preferably derived from killed staphylococcae. According to an immunoassay for simultaneously quantifying a plurality of different kinds of antigens or antibodies as provided, comprising the steps of: (1) providing a plurality of microcapsules having on their outer surface an antibody or antigen specific to one kind of antigen or antibody of the plurality of kinds of antigens or antibodies to be quantified, the microcapsules being capable of being lysed by complement activity, each kind of the plurality of kinds of microcapsules containing within the microcapsule a different quantifiable subsance which does not interact with

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another quantifiable substance contained in another kind of microcapsule, and is selective to the anti-gen or antibody to be quantified; (2) mixing the plurality of kinds of the microcapsules, a complement, and a test sample which may contain at least one of the kinds of antigens or antibodies to be tested for; and (3) quantifying the quantifiable substances released from the microcapsules simultaneously upon lysis by the complement activity, and wherein said microcapsules are formed of cells capable of containing said quantitatively determinable substance within the capsule defined by the cell memebrane. The microcapsules consist of sheep red blood cell ghosts. The immunoassay according to claim 3, and the quantifiable substance is a water-soluble macromolecule and the macromolecule is quantified by means of a molecular sieve. Crosslinking For effecting an immunoresponse Kakimi (FUJI PHOTO FILM CO. (59.1)) also uses microcapsules. A wall is employed having an oily substance encapsulated therein as a core and an antigen or antibody bound to a functional group of the wall surface of the microcapsules via a cross linking agent, wherein the avarage particle size of the microcapsule is in the range of from 0·1 to 30 µm and the microcapsules have a variable specific gravity ranging from about 0·80 to about 1·20. The functional group being selected from the group consisting of an amino group, a carboxy group, a hydroxy group and a mercapto group. There is an extensive literature relating to conjugates of biological significance which are deliberately designed to combine the activities of two previously independent substances. The most prominent example is enzyme linked immunoassay (ELISA), where an enzyme is conjugated to an antibody to serve as a label. Other applications include immunotoxins wherein a toxin is linked with an antibody to effect the specific delivery of the toxin to a target tissue, delivery of therapeutic agents by linking these agents to antibodies, and reagents for detection or localization of particular tissues by targeting specific tissue receptors with a heteromolecule containing a binding portion and labe. A review of such conjugates in tumor therapy appeared in Immunological Reviews (1982) 62:1–216. A number of approaches to formation of the desired linkages have been employed. Perhaps the most popular approach utilizes the commercially available reagent N-succinimidyl-3-(20pyridyldithio)propi-onate (SPDP). SPDP offers the possibility of disulfide bridge between itself and one moiety and an amide between itself and the other. Thus, for example, two proteins can be linked wherein one contains a free amino group with which to form the amide, and

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another contains a sulfhydryl to form the disulfide. Alternatively, each of two proteins with side chain amino groups can be derivatized with SPDP to provide the needed sulfhydryl functionality for a disulfide bridge between them. Still another cross-linking agent has been prepared by Nitecki and Moreland (CETUS CORPORATION (34.2)). It deals with bifunctional chemicals useful for linking aldehyde containing moieties to moieties containing functionalities which are or can be reactive with sulfhydryl having the formula: wherein L is a leaving group selected from—H or—S — Ar, wherein Ar represents optionally substituted phenyl or pyridyl;

wherein Y is alkylene or oxaalkylene and Z is alkylene or a polypeptide residue briding the N-terminal amino group and C-terminal carboxy group thereof; the SPACER is oxa-alkylene or oxa-alkylene substituted with hydroxyl and specifically includes residues having formulas selected from wherein each n2 is independently 2–4 and a) –(CH2)n2—O—(CH2)n2—O—(CH2)n2 b) –CH2—CH—CH2)—O—(CH2)n3—O—CH2—CH—CH2 OH “OH wherein N3 is 2–6. Others are hydrazides, semicarbazides, and thiosemicarbazides of wmercaptocarboxylic acids which lack SPACER diamine. Insoluble microparticles as binding agens for immunoassay is also subject of a method developed by Marshall (MUREX CORPORATION (113.2)). A separation filter is used having a sufficient pore size such that the particles are trapped within the filter yet permitting rapid passage of fluid therethrough in such a manner that an immunological reaction occurs if analyte is present in the specimen, resulting in the formation of an immunocomplex of the insolubilized

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first binding component, analyte, second labeled binding component on or within the filter menas. Immobilizing immunochemicals on a carrier in the so called agglomeration processes is widespread in the art of immunoassay. Hosaka et al. (TORAY INDUSTRIES (170.1)) also use such a method but they covalently bind the immunochemicals by covalent bonding caused by the reaction between an amino group of the immunochemicals and an epoxy group on the particle surface. In case there is the possibility that the epoxy group will not totally be consumed but remain active in its reaction with the immunochemicals, hydrophilic proteins which do not interfere with the objective immunological tests, such as serum albumin and gelatin, may be reacted with the remaining epoxy group, whereby the epoxy group is made no longer reactive. In this case, such hydrophilic proteins may be mixed and reacted together with the immunochemicals to be immobilized, or the immunochemicals may be reacted alone in advance and thereafter the hydrophilic proteins such as serum albumin and gelatin may be substituted by amino acids such as glycine and alanine. Polysterene latex Lurhuma (LURHUMA (98 + 38)) used synthetic polystyrene latex as an immunically inert basis for binding antibodies. The latex particles are initially subjected to a chemical treatment with glutar aldehyde, preferably in a phosphate buffer at a pH of 7·2. A reaction is followed by one or more washing stages in the same buffer, then by a separation step preferably a centrifugation at about 15000 revolutions per minute during 10 minutes. After centrifuging the precipited matter is removed. Polystyrene particles are used most widely as non-organism originating carrier particles. Polystyrene is a stable synthetic polymer; the quality can be controlled. Because polystyrene is hydrophobic and has a property of adsorbing various proteins, immobilization of an antigen or antibody on polystyrene usually is carried out by physical adsorption. When an antigen or antibody is immobilized by physical adsorption, an equilibrium may occur between the immobilized antigen (or antibody) and a free antigen (or antibody) and result in a competitive reaction which takes place between the antigen (or antibody) immobilized on particles and the free antigen (or antibody) toward a corresponding antibody (or antigen) which is an objective substance of the measurement. This competitive reaction works to inhibit agglutination. As a result, there occur insufficient sensitivity stability in many instances. Moreover, substances difficult to be physically adsorbed to polystyrene cannot be immobilized by this method. Because of these problems, the practical application of polystyrene particles is limited as compared with red corpuscles as a carrier.

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According to Hosaka and Murao (TORAY INDUSTRIES, INCORPORATED (170.1)) the immunoparticles are prepared by immobilizing, by covalent bonding, immuno-chemicals containing an amino group onto fine particles having an average diameter of 0·03 to 10 µm which fine particles comprise a polymer having the repeating unit of glycidyl acrylate and/or glycidyl methacrylate and which fine particles do not substantially have on the surface thereof a hydrophobic component other than the above unit. Tsutsui et al. (MITSUBISHI CHEMICAL INDUSTRIES LIMITED (110)) have made various investigations with the intention of eliminating the abovementioned antigen-antibody reactions and found that in the assay of an antigenantibody reaction, if the reaction is carried out with a sample which has been treated with a polyanion, both the recovery of the substance to be detected and the assay accuracy are improved to provide more accurate assay values of the antigen or antibody. The polyanions that can be used are those substances comprising natural or synthetic polymers such as polysaccharides or polystyrene having therein plural anions such as sulfonyl anions or carboxyl anions, said materials being soluble in the reaction medium used in the antigen-antibody reaction. Specific examples of these polyanions include dextran sulfate, heparin, polystyrene sulfonic acid, cellulose phthalate acetate, hyaluronic acid, chondroitin sulfate and the like. According to the method, a sample containing the antigen or antibody to be assayed is treated with the polyanion and then subjected to the antigen-antibody reaction with the corresponding antibody or anti-gen in the reaction medium. The treatment with the polyanion may be carried out (i) by carrying out the antigen-antibody reaction in the medium in which the polyanion has been included or (ii) by treating the sample with a solid or liquid phase containing the polyanion prior to the antigen-antibody reaction (in the latter case, the antigen— or antibody-containing sample thus treated may be subjected to the antigenantibody reaction after the polyanion has been removed therefrom, but usually it is subjected to the reaction as it carries the polyanion. It is noticed that latex agglutination is used in several other methods discussed in the present analysis as will be apparent from e.g. chapter of Section II. T3 and T4 detection by thyroglobuline The competitive T3 or T4 method of Ferrua and Moulin (COMMISSARIAT A L’ENERGIE ATOMIQUE (39.1)) consists of the competition of T3 and/or T4 with thyroglobuline for the antibody anti-T3 and/or anti-T4 at limited quantity and thereafter to determine either the quantity of thyroglobuline fixed to the antibodies anti-T3 and/or anti-T4, or the quantity of thyroglobuline non-fixed to the antibodies anti-T3 or anti-T4. Generally the thyroglobuline is immobilised in a solid support. The antibodies may be labelled by the enzyme, fluorescent, luminescent label methods or by indirect methods by other labelled antibodies.

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Cross-linked heteroantibodies Cross-linked heteroantibodies can cause normal autologous cells of the immune system to destroy any unwanted cell for which an antibody is available. Treatment or control of tumors, viral infected cells, fungi, bacteria, parasites and the like is made possible through the use of the heteroantibody complex. According to Segal and Perez (THE GOVERNMENT OF THE UNITED STATES OF AMERICA (179.2)) 1. cross-linked antibody heteroaggregates bind one antibody of the heteroaggregate specifically with a receptor entity on a cytotoxic cell and a second antibody of the heteroaggregate binds specifically with a surface entity on a cell different from and to be lysed by the cytotoxic cell. 2. the cytotoxic cell is selected from the group consisting of antibodydependent cytolytic cell and cytotoxic T lymphocytes. 3. the antibodies are cross-linked by avidin coupled to one antibody and biotin coupled to the second antibody. Polymer coating A method by Findlay and Wu (EASTMAN KODAK COMPANY (54.1)) for preparing a blush polymer coating composition containing an immunologically reactive species comprises milling the species and the other materials used in the composition to uniformly disperse the species therein. This coating composition can be used to prepare analytical elements for determining analytes (e.g. creating kinase-MB) whereby the effects of potential interferents are immunochemically removed. By milling the immunologically reactive species, e.g. antisera, into the coating composition, the resulting element has improved stability resulting in improved keeping in high humidity environments. Rauterberg (RAUTERBERG (139.2)) discovered a method whereby substances can be immobilised on synthetic solid phases in a more simple manner. To this end he treated the solid phases with softening agents or additives containing groups which can be activated or converted by bi—or multivalent reactants. Berglund and Inganäs (PHARMACIA AB (132.2)) designed a method and means for the immunoassay determination of (I) a bacterial polypeptide capable of binding to the Fc portion of an immunoglobulin and/or (II) the high affinity antibody to said polypeptide. The characteristic feature of the method resides in using an antibody directed against the polypeptide and having antibody activity under conditions such that the immunoglobulin potentially binding to the polypeptide will substantially not bind to the polypeptide, and carrying out the immune reaction between the antibody preparation and the corresponding polypeptide epitope under such conditions.

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D-Alanyl-D-Alanine dipeptides The method developed by Corti et al. (GRUPPO LEPETIT S.P.A. (65.1)) has been designed for determining a glycopeptidic antibiotic of the vancomycin class or a derivative or aglycon thereof. The method includes: a) causing a test solution to contact a surface supporting a D-AlanylD-Alanine carboxy terminal oligopeptide conjugated with a suitable macromolecular carrier capable of adsorbing onto said surface; b) after rinsing, adding antibodies specifically directed against the substance to be detected (analyte); c) revealing said antibodies bound to the antigen on the stationary phase by means of a species-specific anti-IgG-antibody directed to ward these bound antibodies (anti-antibody antiserum) coupled to a detectable marker. The D-Alanyl-D-Alanine carboxy terminal oligopeptide may be a tri-, tetra-, penta-, hexa-, or heptapeptide wherein the carboxy terminal dipeptide is represented by D-Alanyl-D-Alanine. The preferred DAlanyl-D-Alanine carboxy terminal oligopeptide of the invention is the tripeptide Epsilon-aminocaproyl-DAlanyl-D-Alanine (hereinafter: EpsilonAca-D-Ala-D-Ala). Dougherty et al. (RESEARCH CORPORATION (140.2)) discovered a method by which haptens may be detected in a highly sensitive test by a sandwich assay. This assay depends on the use of a class of antibodies having heretofore unknown properties, not the least of which is the unique ability to be successfully utilized in a hapten sandwich assay. These antibodies are further characterized as not simply having specificity for a particular hapten or small molecule, but also as being able to specifi cally recognize a portion of the molecule, or a particular functional group thereon. An additional feature of the invention is the method by which these unusual antibodies are obtained a particular method of “in vitro” immunization, using an unconjugated hapten as immunogen, is, at present, the only known method by which a hybridoma capable of producing such highly specific antibodies can be produced. These unusual monoclonal antibodies in a sandwich assay for haptens have been shown to be able to detect the presence of extremely small amounts, as low as parts per trillion, of a hapten in a test sample. Furthermore, the technique has been found to be applicable to testing in both aqueous and non-aqueous media. The monoclonal antibodies of the present invention are, of course, also useful in any type of immunoassay in which monoclonal antibodies are typically employed. In one embodiment, the present invention relates to monoclonal antibodies utilizable in a hapten sandwich assay. In a second embodiment, the hybridomas capable of producing recoverable quantities of these monoclonal antibodies is encompassed; it also relates to the

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method by which these hybridomas are produced. This method comprises immunizing B-cells in a culture medium with an unconjugated hapten or hapten mimic, in the presence of an effective amount of mitogen, and fusing, under cell fusion conditions, the immunized B-cells with an immortal cell line. A further aspect is an improvement in immunoassay techniques for the detection of an analyte comprising contacting at least on antibody capable of recognizing the analyte or a portion thereof with a sample suspected of containing the analyte, the improvement comprising employing as the antibody a monoclonal antibody. By “analyte” is meant either an antigen or hapten. In a more particular and preferred application, the assay is a hapten sandwich assay which comprises contacting a fluid sample suspected of containing the hapten with a first and a second antibody capable of participating in a sandwich assay, said antibodies capable of recognizing a portion of the hapten molecule and one of the antibodies being linked to a reporter molecule; allowing time sufficient for an antibody-hapten-antibody complex to form; and determining the presence of the hapten by detection of the reporter molecule. In a more specific aspect of the aforementioned assay, the fluid medium which constitutes the hapten sample may be non-aqueous; e.g., air or an organic solvent extract of soil, plant or animal tissue. Lipp and Buck (MODERN DIAGNOSTICS, INC. (111.1)) feature the detection of antinuclear antibodies in a biological sample by adhering a plurality of eukaryotic nuclei to a solid support, e.g., a 96-well ELISA plate; contacting the sample with the support under conditions sufficient to allow antinuclear antibodies in the sample to form immune complexes with nuclear antigens in the cell nuclei; and detecting the immune complexes as an indication of the presence of antinuclear antibodies in the sample. Preferably, underlying the nuclei on the solid support is a coating, e.g., of nuclear antigens, which is substantially unreactive with antibodies to non-nuclear antigens, and which, like the nuclei, serves to bind antinuclear antibodies in the sample. It is particularly advantageous to use two different nuclear antigencontaining coatings, e.g., one prepared by rupturing the same nuclei as are used in intact form, and the second from a different source the antigen of this second source. A heterogeneous binding assay in which a liquid component and a granular particulate solid phase are incubated together for a predetermined period of time, is deemed to be improved by Baker et al. (BAKER ET AL. (12.1)). The density of the liquid component is maintained substantially equal to the density of the granular particulate solid phase by the addition of a density modifying medium having a density greater than the density of the granular particulate solid phase and wherein the density of the liquid component is reduced upon expiry of the predetermined period of time to allow the granular particulate solid phase to separate under the influence of gravity by diluting the

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liquid component and density modifying medium mixture with a liquid of lower density than the mixture. The granular particulate solid phase comprises an alkyl dextran, crosslinked with N,R′-methylene bisacrylamide and density modifying medium comprise a colloidal suspension of silica particles coated with polyvinylpyrrolidine. The method developed by Hosada et al. (TEIJ LIMITED (167.2)) utilizes antigen-antibody reaction in a solution comprises allowing to exist a protein of 16000 to 50000 in average molecular weight and 1·0 to 5·0 in isoelectric point or a mixture containing the protein as antigen-antibody reaction-adjusting agent in the immunoreaction solution, and adjusting final concentration of the antigenantibody reaction-adjusting agent in the immunoreaction solution to 0·02 to 0·9 wt %. The reagent kit for the measurement contains as part of its consistuents the antigen-antibody reaction-adjusting agent in such amount that its final concentration in the immunoreaction solution becomes 0·02 to 0·9 wt %. Polyanions Tsutsui et al. (MITSUBISHI CHEMICAL INDUSTRIES (110)) developed a method which is characterised in that a sample containing antigens or antibodies to be assayed is treated with a polyanion which is soluble in the reaction medium and thus treated sample is used for the antigen-antibody reaction. The buffer solution includes glycine buffers, phosphoric acid buffers, citric acid buffers, barbital buffers, borate buffers, Tris[tris(hydroxymethyl) aminomethane]-hydrochloric acid buffers, Tris-malate buffers, ammonia buffers and the like. The stabilizers includes, for example, amino acids, polypeptides, proteins and the like which do not participate in the intended immunological reaction and they are usually present at concentrations of 0·001% to 1%, preferably 0·05% to 0·6%. Preferred examples of the preservatives include sodium azide and merthiolate. Preferred examples of the chelating agents include ethylenediaminetetraacetic acid, nitrilotriacetic acid, cyclohexanediaminetetraacetic acid and the like. As the surfactant, nonionic surfactants are generally preferred. The method of making an aminoabsorbant proposed by Bosmans et al. (DR. L. WILLEMS INSTITUUT, LIMBURG UNIVERSITY BELGIUM (211)) comprises the following steps: the insoluble porous polymer, activated, non-activated or coupled by the antigen or antibody is mixed together with the porous base matrix in a pulverulent state to form a dry mixture, the dry mixture is subjected to an agglomeration treatment to form agglomerates, the agglomerates thus formed are subjected to a crushing treatment to form crushed agglomerates. The crushed agglomerates are compressed together to form a compressed tablet and the tablet is subjected to a rolling or laminating process to obtain a film or a sheet.

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A cross-linked antibody heteroaggregate, wherein one antibody of the heteroaggregate binds specifically with a receptor entity on a cytotoxic cell and a second antobody of the heteroaggregate binds specifically with a surface entity on a cell different from and to be lysed by the cytotoxic cell has been found by Segal (THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES (179.2)). The cytotoxic cell is selected from the group consisting of antibody-dependent cytolytic cell and cytotoxic T lymphocytes. The antibodies are cross-linked by avidin coupled to one antibody and biotin coupled to the second antibody. The method developed by Findlay and Wu (EASTMAN KODAK COMPANY (54.1)) has to do with the preparation of a blush polymer coating composition containing an immunologically reative species. The method comprises milling the species and the other materials used in the composition to uniformly disperse the species therein. This coating composition can be used to prepare analytical elements for determining analytes (e.g. creating kinase-MB) whereby the effects of potential interferents are immunochemically removed. By milling the immunologically reactive species, e.g. antisera, into the coating composition, the resulting element has improved stability resulting in improved keeping in high humidity environments. Berglund and Inganäs (PHARMACIA AB (132.2)) have discovered a method for the immunoassay determination of (1) a bacterial polypeptide capable of binding to the Fc protion of an immunoglobulin and/or (11) the high affinity antibody to said polypeptide. The characteristic feature of the method resides inusing an antibody directed against the polypeptide and having antibody activity under conditions such that the immunoglobulin potentially binding to the polypeptide will substantially not bind to the polypeptide, and carrying out the immune reaction between the antibody preparation and the corresponding polypeptide epitope under such conditions. An assay for determining a substance capable of binding to a DAlanyl-DAlanine dipeptide or a D-Alanyl-D-Alanine carboxy terminal oligopeptide is subject matter of European Patent 221 282 to Corti et al. (GRUPPO LEPETIT S.P.A. (65.1). More particularly, the method includes: (a) causing a test solution to contact a surface supporting a D-AlanylD-Alanine carboxy terminal oligopeptide conjugated with a suitable macromolecular carrier capable of adsorbing onto said surface; (b) after rinsing, adding antibodies specifically directed against the substance to be detected (analyte); (c) revealing the antibodies bound to the antigen on the stationary phase by menas of a species-specific anti-IgG-antibody directed toward these bound antibodies (anti-antibody antiserum) coupled to a detectable marker. The D-

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Alanyl-D-Alanine carboxy terminal oligopeptide may be a tri-, tetra-, pentahexa-, or heptapeptide wherein the carboxy terminal dipeptide is represented by D-AlanylD-Alanine. The preferred D-Alanyl-D-Alanine carboxy terminal oligopeptide is the tripeptide Epsilon-aminocaproxyl-D-Alanyl-DAlanine. Hapten Sandwich Assay Attempts to conduct a sandwich assay for hapten detection have been largely unsuccessful, primarily because of the small size of the molecules. The hapten molecule is typically “swamped” by the large size of the IgG or IgM antibody molecule, thereby substantially preventing the binding of the second antibody necessary to form the sandwich. Thus, although the sandwich assay is extremely useful in the detection of polyvalent antigen molecules, it has not yet been effectively applied to smaller, functionally monovalent hapten molecules. The failure of application here is due both to the small size of the molecule to be detected and to the inherent nature of the available antibodies. As an alternative to sandwich assays, hapten immunoassays of the competitive binding type are known; however, they are typicaly less sensitive than is desirable; they also require the use of hapten mimic, i.e. a small molecule of similar size, symmetry and conformation to the hapten itself which, in the case of a highly toxic hapten may also be toxic, thus increasing the inherent danger of the test procedure. There has not heretofore been a truly satisfactory method for safe and highly sensitive hapten assay. Doughety et al. (RESEARCH CORPORATION (140.2) have discovered a method by which haptens may be detected in a highly sensitive test by a sandwich assay. The following example is representative to their method; this example demonstrates the procedure of conducting a hapten sandwich assay in a nonaqueous medium: Into a pasteur pipette containing a glass wool plug is placed about 0·5 gm of dirt suspected of containing TCDD’s. The dirt is extracted by passing 1·0 ml of hexane through the pipette. 50 µl of the hexane extract is then applied to the antibody produced by HB 9049 (this antibody being capable of recognizing the dichlorobenzene portion of the 2,3,7,8-TDCC molecule), the antibody having been attached to a glass fiber filter, and wet with N1N1-dimethylformamide. Incubation is permitted for a period of about one minute and the excess reagent removed. 50 µl of the same antibody to which horseradish perioxidase has been conjugated, is thus added, incubated for one minute, and the excess reagents again draw off. The enzyme substrate, hydrogen peroxide with 4aminoantipyrene is then added as two 50 µl drops. After a two minute development period, the pink color of the oxidized 4-aminoantipyrene is visible if 2,3,7,8-TCDD is present. Samples known to contain 10 ppt of TCDD are

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consistently positive, while samples containing only 1 ppt of TCDD give fiant indications of the reporter pink color in about 50% of the tests. See European patent 242 589 (140.2) to Dougherty. The immunoassay method of Hosoda et al. (TEIJIN LTD (167.2)) utilises an antigen-antibody reaction in a solution and comprises allowing to exist a protein of 16000 to 50 000 in average molecular weight and 1·0 to 5·0 in isoelectric point or a mixture containing the protein as antigen-antibody reaction-adjusting agent in the immororeaction solution, and adjusting final concentration of the antigen-antibody reactionadjusting agent in the immunoreaction solution to 0·02 to 0·9 wt %. The reagent kit for the measurement contains as part of its constituents the antigen-antibody reaction-adusting agent in such amount that its final concentration in the immunoreaction solution becomes 0·02 to 0·9 wt %. Methods have been proposed to efficiently bind amyloid proteins to plastic surfaces. One of those methods have been described by the Swiss scientists Serban and Rordorf (CIBA-GEIGY A.G. (37)). Following most forms of tissue injury, infection or inflammation, the concentrations of a number of plasma proteins increase, and then return to normal again as healing or recovery occurs. This process is known as the acute phase response. In individuals with chronic inflammation, high levels of some acute phase proteins may persist. Examples of acute phase reactants are C-reactive protein (CRP) and serum amyloid A protein (SAA). SAA is an α1 globulin consisting of a single polypeptide chain of molecular weight between 11 500 and 14000 Dalton. Its plasma concentration, which is normally around 1µg/ml or below in healthy individuals, increases substantially within few days following tissue injury or exposure to inflammatory stimuli. Related to the acute phase reactant CRP is serum amyloid P-component (SAP), a 9·5S α1 glycoprotein of 235′000 Dalton molecular weight. Whereas mouse SAP concentration substantially increases in inflammation, human SAP is not a major acute phase reactant, although its level can be moderately increased in some chronic inflammatory diseases and malignancies [M.B. Pepys, Clinics in Immunology and Allergy, Vol. 1, pp. 77– 101 (1981)]. The method developed by Serban and Rordorf relates to an immunological analysis for serum amyloid A protein (SAA) and serum amyloid P-component (SAP). The method is based on the efficient binding of SAP to a plastic surface and to carriers bearing nitrated phenyl groups in the presence of calcium and related bivalent ions, and of SAA to a plastic surface and to carriers bearing nitrated phenyl groups with or without calcium ions. The method of immunological analysis allows for rapid and reliable screening of serum samples with a high sensitivity. The need of preactivating hydropholic plastic surfaces for the immobilization of organo-chemical and biologic materials is recognised in the immunoassay art.

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Gadow (HENNING BERLIN GmbH (68.2)) found a method which comprises contacting the plastic surface with a solution or suspension of a polypeptide to form a coating on the plastic surface. The peptide comprises a hydrophobic amino acid capable of being adsorbed by the plastic surface and an amino acid having a side chain capable of activation and coupling with the organo-chemical and biologic materials, and washing the thus coated surface. The biological material is an antibody or an antigen, while the polypeptide is a phenylalanine-lysine copolymer. One of the most widely used polynucleotide hybridization assay procudures is known as the Southern blot filter hybridization method or simply, the Southern procedure (Southern, E., J. Mol. Biol., 98, 503, 1975). The Southern procedure is used to identify target DNA or RNA sequences. The procedure is generally carried out by subjecting sample RNA or DNA isolated from an organism, potentially carrying the target sequence of interest, to restriction endonuclease digestion to form DNA fragments. The sample DNA fragments are then electrophoresed on a gel such as agarose or polyacrylamide to sort the sample fragments by length. Each group of fragments can be tested for the presence of the target sequence. The DNA is denatured inside the gel to enable transfer to nitrocellulose sheets. The gel containing the sample DNA fragments is placed in contact (blotted) with nitrocellulose filter sheets or diazotized paper to which the DNA fragments transfer and become bound or immobilized. The nitrocellulose sheet containing the sample DNA fragments is then heated to approximately 85° C to immobilize the DNA. The nitrocellulose sheet is then treated with a solution containing a denatured (single-stranded) radio-labeled DNA probe. The radiolabeled probe includes a strand of DNA having a base sequence complementary to the target sequence and having a radioactive moiety which can be detected. Morrison (AMOCO CORP. (10)) found a method for assaying a sample for target polynucleotide strands. His method comprises contacting a sample with reagent under binding conditions wherein the reagent includes a first polynucleotide probe and a second polynucleotide probe. The first and second probes are capable of assuming a position wherein the probes are bound to each other and at least one of the probes is capable of assuming a second position wherein the probe is bound to the target polynucleotide strand. The first and second probes include a first label moiety positioned on one of the probes and a second label moiety positioned on the opposite probe. The first and the second label moieties are capable of interacting when the first and second probes are bound to each other to produce a signal capable of detection characteristic of the reagent strands being in one of the two positions. The sample contacted with the reagent is monitored for the signal, the presence of which is related to the presence of target polynucleotide strands in the sample. The present method allows a polynucleotide sample to be assayed without the need for immobilization steps and without radioactivie labeling techniques. Preferably, at

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least one label moiety is located at the 3′-terminus of one of the probes and the second label moiety is located at the 5′-terminus of the opposite probe. Immobilisation As described herefore, for many immunoassays it is important to immobilize proteins e.g. on plastic surfaces. Lentfer (MALLINCKRODT DIAGNOSTICS (Germany) GmbH (101)) found a procedure for the immobilization of proteins on polystyrene surface which includes a pre-treatment of the polystyrene surface with a bis-diazonium compound of the general formula I where

Rl stands for a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group or a nitro group and where R2 stands for a hydrogen atom, a halogen atom or an alkyl group and where X stands for an anion and the subsequent adsorption of the protein on the surface pretreated. Liposomes Liposomes, as known in the art, are spherical shells formed when mixtures phospholipids with or without cholesterol are dispersed in aqueous solutions, and are made up of one or several concentive phospholipid bilayers within which other molecules can be incorporated, and they simulate many permeability properties of membranes and are used for the administration of certain drugs. Guo et al. (COOPER-LIPOTECH (43.2)) developed an assay system including liposomes having surface-bound, analyte-related binding molecules and entrapped reporter molecules, and a cellulose acetate surface reagent having surface molecules adapted to bind the liposomes to the reagent in proportion to the amount of analyte present. The reagent is prepared, by treating a cellulose acetate substrate with a base, to form a hydrophilic, hydroxyl group shell on the substrate surface, reacting the modified substrate with a bifunctional reagent in an organic solvent, and coupling the surface molecules to the activated substrate in an aqueous solution.

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Heterogeneous assay polymerisation A polymerizing method for the determination of the presence of more than one analyte in a sample, proposed by Nowinski and Hoffman (GENETIC SYSTEMS CORP. (63.1) comprises (a) combining the fluid sample with an addition monomer/analyte conjugate for each analyte being determined to form a fluid sample mixture; (b) combining said mixture with reporter/reactant conjugates specific for bonding to each analyte thought to be contained in the fluid sample mixture under conditions favorable for the formation of the reporter-labeled analyte complexes and reporter-labeled monomer/ analyte complexes, each reporter/ analyte conjugate providing a detectably diflferent signal from every other reporter present in the fluid sample mixture. (c) separating the reporter-labeled monomer/analyte-conjugate complexes by initiating addition polymerization in said mixture; and (d) detecting the incorporation of each reporter into each polymerized complex as a measure of the analytes present in the sample. The monomer comprises a polymerizable, ethylenically unsaturated organic monomer selected from the group consisting of:

where R1 is a hydrogen or lower alkyl having from 1 to 8 carbon atoms, R2 is selectred from the group consisting of

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and R3 and R4 are selected from the group consisting of H and compounds which will provide a reactive ethylenically unsaturated group. Inhibin ROBERTSON et al (20, 136, 188, 197) developed an immunoassay for the estimation of inhibin in an inhibin-containing sample, which comprises the step of using an antibody directed against inhibin. Preferably, the antibody is contained in an antiserum, raised by injecting an animal with an antigen selected from the group consisting of naturally-occurring or recombinant inhibin, or subunits, fragments or derivatives thereof. However, the indicated methods have various drawbacks which can be eliminated according to M.A. VODIAN and co-searchers (160) by their invention, relating to a serum pretreatment method employed in conjunction with immunoassays for enhancing the detection of analyte in serum samples has been discovered by 160. Many analytes are highly antigenic. If the serum donor has produced antibody against an analyte, the formation of antibody complexes with the analyte can interfere with an immunoassay for the detection of serum analyte. The method comprises: step (1): dissociating the analyte from the serum antibody and denaturing the serum antibody by contacting the serum sample with an activated chaotrope at acid pH without the application of elevated heat, the chaotrope being of the type which is activated at room temperature by acid pH and which is de-activated by neutral pH, then step (2): de-activating the chaotrope by neutralizing the pH of the serum sample, and then step (3): performing an immunoassay on the serum sample for assaying the dissociated analyte in the presence of the denatured serum antibody, the immunoassay being of the type which is operable in the presence of de-activated chaotrope. The following additional steps: step (a): prior to step (1), loading the serum sample into a treatment well, and step (b): after step (2) and prior to step (3), transferring the serum sample from the treatment well to a reaction vessel. Immunoactive components immobilized in a porous material Immunoabsorbent usable in immunologe determinations based on a porous matrix of polytetrafluoroethylene wherein there is covalently bound an organic,

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hydrophilic or hydrophobic, water-insoluble polymer, to which are covalently associated antigenes and/or antibodies in well determined amounts and, if there are various antigenes and/or antibodies, in well determined proportions, has been developed by BOSMANS et al (186). The method for the preparation of such an immunosorbant comprises the following steps: the insoluble porous polymer, activated, non-activated or coupled by the antigen or antibody is mixed together with the porous base matrix in a pulverulent state to form a dry mixture, the dry mixture is subjected to an agglomeration treatment to form agglomerates, the agglomerates thus formed are subjected to a crushing treatment to form crushed agglomerates, the crushed agglomerates are compressed together to form a compressed tablet and the tablet is subjected to a rolling or laminating process to obtain a film or a sheet. An immunoassay of a fluid sample wherein the analyte is a hapten and the reaction environment is monitored turbidimetrically, the assay being based upon a hapten modulated precipitin reaction involving the competitive immunochemical interaction of a hapten and hapten mimic with an antibody in the presence of a precipitin reaction enhancer, has been found by LUCAS et al. Their assay comprises controlling the rate of the precipitin reaction by adding to the fluid sample an enhancer modulating effective amount of a mixture consisting essentially of physiological and buffering salts, the relative concentration of salts to enhancer effectively optimizing the kinetics of the precipitin reaction so as to produce an essentially linear change in optical density of the fluid sample over the dynamic analytical range of the hapten. The enhancer is selected from the group consisting of polyethylene glycol and Dextran, while the mixture of physiological and buffer salts consists essentially of about 150 mM sodium chloride and at least about 100 mM buffering salts. According to LOOR et al. at least two antigens in a sample may be detected using an immunometric dual sandwich assay containing an effective amount of at least one monoclonal antibody against each anti-gen, which antibodies are separately conjugated with the same or different signal moieties as labels, and an effective amount of at least one unlabeled monoclonal antibody against each antigen which unlabeled antibodies are immobilized on a single support. Preferably the antibodies are all products of different cell lines and the antigens are prostatic acid phosphatase and prostate antigen. Aalberse et al. (J. Imm. Methods 87 : 51–57 (1986) describes the use of hapten-modified antigens instead of solid phase coupled antigens in a radioallergosorbent test-type assay. In such assay a patient sample suspected of containing specific allergen IgE is reacted with trinitrobenzene sulfonic acid (TNP) modified specific allergen for two hours then further reacted overnight with a solid phase coupled anti-TNP to form IgE-allergen-TNP-anti-TNP complex. The solid phase is washed and again reacted overnight with 125–1-antiIgE antibody, rewashed and counted for the presence of 125–1 isotope which is

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directly porportional to the concentration of allergen specific IgE in the patient sample. In this approach the authors claim to have gained the benefit of liquid phase kinetics in their first reaction but fail to subtantiate the extent of TNP labeling of their allergens. Directly labeling allergens with haptens such as TNP poses the problem of missing certain vital allergenic components that might not be labeled during such a process and thus will not be quantified in said process. To avoid the problems described above EL SHAMI et and colleagues found a method for measuring the level of circulating antigen (AG1), antibody (AB1) or hapten (H) in a liquid sample, which comprises forming in a liquid phase reaction a soluble complex wherein the antigen (AG1), antibody (AB1) or hapten (H) is linked through, respectively a specific antibody (Ab), antigen (Ag) or antihapten (Anti-H), to a matrix which is soluble in the liquid phase and carries a ligand (X), subsequently forming an insolubilised complex comprising a solid support linked to the ligand (X) of the soluble complex through an anti-ligand (Y); the insolubilised complex carrying a label (Z) linked to the antigen (AG1) through an anti-antigen (Anti-Ag1) to the antibody (Ab1) through an antiantibody (Anti-Ab12) or to the hapten (H), washing the insolubilised complex, and checking the washed insolubilised complex for the presence of the label (Z) to provide a measure of the level of said antigen (Ag1), antibody (Ab1) or hapten (H) in the sample. The next schematic figure shows the detection of antigens or antibodies: The method for the determination of a biological substance in a sample developed by LEE/CANAVAGGIO (97), characterized in that: (a) a ligand having a fixing affinity for said biological substance is immobilized on a solid phase;

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(b) the sample is incubated in the presence of said solid phase on which the ligand is immobilized; (c) the solid phase is washed after incubation; and (d) the presence of said biological substance, fixed to the solid phase via the ligand, is revealed by a property inherent to said biological substance. Solid-phase hybridization assay A nuleic acid hybridization method for detecting a particular plynucleotide sequence present in a virus or cell contained in a test sample such as a biological fluid has been developed by Carrico (MILES LABORATORIES INC (109.4). The test sample is treated to release nucleic acids from the virus or cell of interest in single stranded form and to immobilize such nucleic acids on a solid substrate such as a filter membrane. Release of nucleic acids can be accomplished with alkali, lytic enzymes, detergents, and the like. The immobilized nucleic acids are contacted with a probe to form DNA-RNA or RNA-RNA hybrids which are then detectable by binding of an anti-hybrid antibody reagent, preferably labeled. The method does not require the use of labeled probe and provides quantitative results due to the efficiency and stability of the immobilization of sample nucleic acids. An immunoassay for the detection of an immunoglobulin in a sample utilizing one or more ligand binding partners specific thereto, at least one of the ligand binding partners having a detectable label attached for determining when a reaction between the immunoglobulin and ligand binding partner has taken place, is subject of an assay method of H. GRAHAM. Improvement with respect to prior methods is, he says, that it comprises providing an immunoassay control to be performed at the time and as part of the immunoassay wherein the control confirms the procedural steps of the assay including the performance of sample addition to the ligand binding partners and the performance of requisite wash steps. The control portion of the readout and the test portion of the readout together combine to form a discernible pattern, wherein such pattern is a “plus” and wherein the control portion is one leg of the “plus” while the test portion is the other leg of the “plus”. The method developed by Rabbani (ENZO BIOCHEM INC (55)) involves generating the signal in a specific, indicative viscous, or thick medium. The specific medium comprises a network, a solution, and a signal precursor. The indicative medium also comprises a network, a solution, and a signalling moiety, just as the viscous medium. The thick medium also comprises a specific component, a solution, and a signalling moiety. The network is suspended or dissolved in the solution. Signal localization results because the network reduces convection in the specific or indicative medium or because the viscosity reduces diffusion in the viscous or thick medium.

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Gamma interferon (y IFN) detecting test An in vitro method of detecting a cell-mediated immune response to a specific antigen in a human or animal, of WOOD and CORNER (40), comprises the steps of: (i) incubating a whole blood sample from the human or animal with the specific antigen, and (ii) detecting the presence of gamma interferon (y IFN) released by sensitized lymphocytes in the whole blood sample to indicate a cellmediated immune response to the specific antigen. A diagnostic kit is also disclosed. Membrane for immunosensor The research of TANIGUCHI et al. relates to a potential-causing membrane for use in an immunosensor, which comprises an electrically-conductive film being prepared by electrolytic polymerization of an electrolytically polymerizable monomer, having at least one functional group capable of binding the antigen or antibody through the functional group to the film. Examples of the monomer include 3-bromo pyrrole, 3,4-dichlorothiophene, 3-bromofuran, o-chlorophenol and mbromo-aniline. Examples of the monomer having those functional groups include halogenated monomers such as 3-bromopyrrole, 3chloropyrrole, 3bromothiophene, etc. Monomers substituted with COOH groups or CHO groups such as pyrrole-3-carboxylic acid, pyrrole-N-carboxylic acid, etc. Quite often it is desirable to determine in a test sample a bindable substance in excess of the predetermined amount and the quantitative measurement of any such excess. LIBERTI (76) found a method to carry out the determination which is comprised by (a) providing an array of complementary binding substance having a predetermined binding capacity for the bindable substance; (b) contacting the array of complementary binding substance with: (i) the test sample, for a time sufficient for any bindable substance present in the test sample to bind the complementary binding substance, and (ii) labeled bindable substance in a carrier medium, which also may be the test sample, the amount of the labeled bindable substance being sufficient, when added to the predetermined amount, to substantially fill the binding capacity of the complementary binding substance, the array of complementary binding substance and the labeled binding substance

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being in contact for a time sufficient for the labeled bindable substance to bind to the complementary binding substance, and (c) determining the absence or significant presence of unbound labeled bindable substance to differentiate whether or not the bindable substance is present in the test sample in excess of the predetermined amount. 1.6 REAGENTS Immunoassays usually employ more than one reagent. In most cases, the reagents cannot be combined in a liquid medium prior to running the assay because they contain components that would react on contact with each other. It is desirable to find a method to combine the active materials in liquid form while preventing the reagents from reacting with each other until such time as a means for releasing one or more of the reagents is provided. Generally in immunoassays the reagents are members of a specific binding pair, consisting of ligand and its complementary receptor, one of which is labelled with a member of a signal producing system. Specific binding pair members that are complementary to each other usually react upon contact. Therefore, such reagents are generally stored separately until just prior to the time an assay is conducted. One patented technique for combining interreactive agents in a single reagent is to formulate the reagents dry so that no reactions occur until a liquid sample or diluent is added. Dry reagents, however, impose some restraints on assay methods. Achieving a homogeneous blend and avoiding water uptake are matters of concern. Further, premature reaction must be avoided. Dry reagents are expensive and their manufacture and quality control are difficult. For example, it is generally necessary to add the sample and a diluent simultaneously and shake vigorously to assure full dissolution of the powder before the reaction has progressed significantly. Additionally, special processing devices are required. It is, therefore, desirable to develop a new assay method for determining an analyte in a sample wherein two or more specific binding members are combined in a liquid single reagent. Such a reagent avoids the need for dry reagent blending and shaking and does not require simultaneous addition of sample and diluent. A single liquid reagent decreases the time and skill needed to perform an assay. Findlay and Wu (EASTMAN KODAK COMPANY (54.3)) found an analytical composition and element and an immunochemical method using them, which are useful for the determination of creatine kinaseMB in biological fluids. Specific amounts of a P1, P5-di(adenosine-5′) polyphosphate, adenosine-5′monophosphate (AMP) and adenosine-5′-diphosphate (ADP) are used to

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significantly reduce interference by adenylate kinase, especially the liver isoenzyme, in the determination. DNA-bonds Protein and dimide derivatives In an immunoassay carried out by Rauterberg (RAUTERBERG (139.1)) macromolecules of the protein type are used, such as casein, gelatin or human serum albumin, which are covalently bound to native doublestrand or singlestrand DNA. To induce the covalent bind between the macromolecule and DNA carbodiimide derivatives are employed. As solid phase Rauterberg uses artificial resins, glass or cellulose e.g., in the form of beads. Polymer hydrogel According to Bosley et al. (UNILEVER NV (174.2)) a solid substrate is provided to which is fixed a gel, especially e.g. a hydrogel polymer, which either carries immunological sensitisation or is physically associated with some other material conferring microchemical analytical specificity, e.g. an electroactive membrane and/or chemically selective electrode. Such a gel in a layer carried within a capillary-fill cell can be used for official immunoassay. Serum amyloids A and P It is known that following most forms of tissue injury, infection or inflammation, the concentrations of a number of plasma proteins increase, and then return to normal again as healing or recovery occurs. This process is known as the acute phase response. In individuals with chronic inflammation, high levels of some acute phase proteins may persist. The usual test for measuring changes in acute phase and related proteins until recently has been the erythrocyte sedimentation rate. This test is cheap and easily performed, but, as an indirect method, not very accurate and reproducible. Serban and Rordorf (CIBA GEIGY AG (37)) found that serum amyloid Pcomponent (SAP) binds to a plastic surface in the presence of calcium ions and related bivalent ions such as zinc and cupric ions and, more efficiently, to organic or inorganic carriers bearing nitrated phenyl groups in the presence of calcium ions or related bivalent ions. Further they found that serum amyloid A protein (SAA) binds to a plastic surface and, more efficiently, to organic or inorganic carriers bearing nitrated phenyl groups, optionally without or in the presence of calcium, zinc or related bivalent ions. This preferential binding of SAA and SAP to a plastic surface and to a carrier bearing nitrated phenyl groups, also in the presence of a large excess of irrelevant protein, is the basis of their method of immunological analysis for SAA and SAP.

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Latex A reagent for detecting an antigen-antibody reaction in the form of a latex comprising as its dispersed substance a fluorine-containing polymer up to 142 in refractive index and having a protein adsorbed thereon, has been developed by Sugimura et al. (DAIKIN INDUSTRIES, LIMITED (48)) Polypeptide coating A process for preactivating a hydrophobic plastic surface for the immo-bilization of organo-chemical and biologic materials devised by Gadow and Wood (HENNING BERLIN GMBH (68.2)) comprises contacting the plastic surface with a solution or suspension of a polypeptide to form a coating on the plastic surface, the polypeptide comprises a hydrophobic amino acid capable of being adsorbed by the plastic surface and an amino acid having a side chain capable of activation and coupling with the organo-chemical and biologic materials, and washing the thus coated surface. The polypeptide, preferably, is a phenylalanine-lysine copolymer. The hydrophobic amine may also be valine or leucine. NA-hybridization Nucleic acid hybridization assays have great potential in the diagnosis and detection of disease. Further potential exists in agriculture and food processing where nucleic acid hybridization assays may be used to detect plant pathogenesis or toxicant producing bacteria. One of the most widely used polynucleotide hybridization assay procedures is known as the Southern blot filter hybridization method is used to identify target DNA or RNA sequences. The hybridization process is very specific. The labelled probe will not combine with sample DNA if the two DNA entities do not share substantial complementary base pair organization. Hybridization can take from 3 to 48 hours, depending on given conditions. The use of nucleic acid hybridization assays has also been hampered in part to rather long exposure times to visualize bands on X-ray film. A typical Southern procedure may require one to seven days for exposure. The method for assaying a sample for target polynucleotide strands of Morrison (AMOCO CORPORATION (10)) includes contacting a sample with reagent under binding conditions wherein the reagent includes a first polynucleotide probe and a second polynucleotide probe. The first and second probes are capable of assuming a position wherein the probes are bound to each other and at least one of the probes is capable of assuming a second position

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wherein the probe is bound to the target polynucleotide strand. The first and second probes include a first label moiety positioned on one of the probes and a second label moiety positioned on the opposite probe. The first and second label moieties are capable of interacting when the first and second probes are bound to each other to produce a signal capable of detection characteristic of the reagent being in one of the two positions. The sample contacted with the reagent is monitored for the signal, the presence of which is related to the presence of target polynucleotide strands in the sample. The present method allows a polynucleotide sample to be assayed without the need for immobilization steps and without radioactive labelling techniques. Preferably, at least one label moiety is located at the 3′-terminus of one of the probes and the second label moiety is located at the 5′-terminus of the opposite probe. A plurality of label moieties can be used for each probe, preferably twoone at each termini. For example, a first label moiety may be associated with the first probe at a 3′-position and a second label moiety associated with the 5′position. A second probe having a similar label moiety organization, a first label moiety in the 3′-position, and a second label moiety in the 5′-position, will hybridize to the first probe such that the first and second label moieties of opposite probes are in close proximity and can interact. An embodiment of the method includes the additional steps of preparing probes by splicing polynucleotide segments having base sequences substantially identical to the target sequences into amplification means to form multiple copies of the reagent polynucleotide segments. Preferably, the amplification means include a high copy number plasmid or phage which, when incorporated into bacteria, is reproduced. Carbohydrates and proteins A method of detecting the presence of a cell, virus or circulating body component or an antibody thereto in a sample, found by Nilsson et al. (SOCKERBOLAGET AB (formerly known as SVENSKA SOCKERFABRIKS AB (156)) comprising: a) contacting the sample with a reagent bound to a solid support, which reagent is capable of binding a cell, virus or circulating body component or an antibody thereto, b) contacting the reagent bound to the solid support which in step a) was contacted with the sample with the same or another reagent as that used in step a), which reagent is coupled to a macromolecular water-insoluble carrier to which a substance is multivalently bound, the substance being capable of initiating a reaction the product of which is detectable, or which reagent is coupled to a substance multivalently bound to a macromolecular

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water-insoluble carrier, the substance being capable of initiating a reaction the product of which is detectable, and c) the reagent bound to the solid support which was contacted with the sample in step a) and with the reagent coupled to the macromolecular waterinsoluble carrier or substance in step b), is reacted with a compound which is a precursor of a detectable reaction product or which is capable of initiating a reaction cascade the end product of which is detectable, to form a detectable reaction product, whereby the presence of any cell, virus of circulating body component or antibody thereto which has become bound to the reagent in steps a) and b) is detected. Suitable reagents may be selected from a carbohydrate or carbohydrate derivative, a polypeptide, a protein or another cell or virus surface component or a circulating body component, an antibody to a carbohydrate or carbohydrate derivative, a polypeptide, a protein or another cell or virus surface component or a circulating body component, a metal conjugate or a single-stranded oligonucleotide. When the reagent is a carbohydrate or carbohydrate derivative, it is often a receptor for a cell, virus or circulating body component, such as a receptor for a bacterial membrane protein mediating bacterial adhesion to surfaces such as epithelial tissue which contains such receptors. In chemical terms, the carbohydrate or carbohydrate derivative may be a polysaccharide, glycolipid, neo-glycolipid, glycoprotein, or neoglycoprotein. The term “neo” —indicates that the compound is prepared synthetically to correspond to a naturally occurring compound by coupling the carbohydrate moiety to the lipid/protein moiety. Human C3 and C3a complement systems Nilsson and Svensson (NILSSON AND SVENSSON (117.1)) developed a reagent for the determination of a substance, C3a, which is released upon activation of the mammalian complement system (= C). In particular human C3 and human C3a are concerned. The complement system consists of about 20 components which have to react in a well-defined reaction sequence (= complement activation) in order that destruction of e.g. invading microorganisms can take place in the final stage. The complement system is considered to be a fundamental element of the mammal’s defense against infections caused by bacteria and by viruses. The components discovered first were called “complement factors” and were named C1, C2, … C9. Activation may be induced by a large number of substances such as e.g. immune complexes, aggregated immunoglobulin, protein A, bacterial polysaccharides etc, and can be correlated to inter alia some autoimmune diseases and inflammatory processes. It has been suggested before that the

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fragments released on activation should be utilized as markers to indicate that activation has taken place. Thus, a number of immunochemical methods have been developed for measuring C3a, C4a and C5a (3, 4, 16). The Nilsson and Svensson reagent and determination method are characterized in that in its reaction with C3a it is substantially not inhibited by C3 so that it can be employed for C3a assays, for example in plasma samples, in the presence of native C3. Liposomes A reagent comprising liposomes that are composed of (1) at least one phospholipid or one glycolipid, (2) a marker material enclosed in the liposomes, and (3) an antigen, an antibody, or a part of the antibody immobilized on the liposome surface through a cross-linking agent. This reagent is characterized in that functional groups remaining on the liposome surface after immobilization of the antigen, the antibody, or a part of the antibody on said liposome, are blocked. With these reagents samples, such as a serum that contains a material to be detected, can be analyzed precisely without dilution. A liposome assay reagent for determination of an analyte in a homogeneous immunoassay discovered by Kung and Canova-Davis (KUNG AND CANOVADAVIS (94)) includes a suspension of oligolamellar lipids containing encapsulated glucose-6-phosphate dehydrogenase (G6PD), at a specific activity of between about 1–10 units/µmole lipid, and glucose-6-phosphate (G6P) at a concentration of at least about 5 mM. The encapsulated G6P protects the enzyme against inactivation on preparation, by reverse phase evaporation in the presence of organic solvent, and on storage as an aqueous suspension. Alkylene oxide chains According to Nagaoka et al. (TORAY INDUSTRIES (170.2)) immobilization of a physiologically active material while keeping the function of the active material on a high level takes place by immobilizing the active material into a carrier through an alkylene oxide chain. Single liquid sbp’s Immunoassays usually employ more than one reagent. In most cases, the reagents cannot be combined in a liquid medium prior to running the assay because they contain components that would react on contact with each other. It is desirable to find a method to combine the active materials in liquid form while preventing the reagents from reacting with each other until such time as a means for releasing one or more of the reagents is provided. Generally in immunoassays

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the reagents are members of a specific binding pair, consisting of ligand and its complementary receptor, one of which is labelled with a member of a signal producing system. Specific binding pair members that are complementary to each other usually react upon contact. Therefore, such reagents are generally stored separately until just prior to the time an assay is conducted. Gibbons et al. (SYNTEX (U.S.A.) INC. (161.6)) found a method of combining specific binding reagents, in a single liquid medium in a manner which temporarily delays reaction between the reagents. The method involves encapsulating one reagent as a means for rendering the reagent temporarily nonreactive with the other reagents followed by specific release of the entrapped material at a prescribed time. The encapsulated reagent and the other reagent or reagents are present in a single liquid medium. The sample suspected of containing the analyte is combined with a composition that includes in a single liquid medium at least (1) one sbp member reversibly confined in a material that temporarily renders the confined sbp member incapable of binding with its complementary sbp member and (2) the complementary sbp member. At least one of the sbp members is bound to a member of a signal producing system. Other members of the signal producing system may also be present in the single liquid medium. In some cases, these additional members of the signal producing system are encapsulated with the sbp member. Alternatively, additional members of the signal producing system may be in a separate medium. The reagent system developed by Li (ORTHO DIAGNOSTIC SYSTEMS INC. (122.1)) comprises: 1) an insolubilized ligand binding partner; 2) a double binding partner conjugate comprising an anti-idiotype binding partner, capable of blocking said ligand from binding with the insolubilized ligand binding partner, coupled to a second binding partner; and 3) an insolubilized label for which the second binding partner is specific. The label may be a fluorescent, phosphorescent, chemiluminescent label or an enzyme, such as a phosphatase. The substrate used by Li is p-nitrophenyl phosphate while the detectable product is p-nitrophenol. Tracer composition Allen and Thompson (BECTON DICKINSON AND COMPANY (16.2)) employ bifunctional aromatic compounds as rigid coupling compounds, paranitrophenylisocyanate of the structural formula:

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wherein Y is a divalent aromatic hydrocarbon radical; R is an organic radical having at least one active hydrogen substituent group (in particular, an amine, thiol or hydroxyl substituent group); and A is selected from the group consisting of —NO2; NH2; 3 —COOH; it may be employed for coupling thyroxine to a fluorescent dye to form a tracer. Metal marked vesicle The tracer employed by Wagner and Baffi (BECTON DICKINSON AND COMPANY (16.6)) comprises a vesicle derivatized with a ligand, a portion of such vesicle wall being formed from an amphiphilic chelating agent having complexed therewith detectable metal atoms. The metal atoms are rare earth metals which are fluorescent when complexed with an activating agent, such as a beta-diketone and a Lewis base. Protein A binding The ability of protein A to bind the Fc portion of immunoglobulin molecules is widely used as a basis for separation of IgG’s from other proteins by affinity chromatography. In such separations, protein A is generally immobilized by cross-linking to a solid phase support such as agarose, and the sample is applied as a solution in a buffer which disfavors the binding of other proteins in the mixture. The immunoglobulins are then recovered from the solid phase by elution using buffers of altered composition. The separation achieved, however, is less than complete. Juarez-Salinas and Ott (BIO-RAD LABORATORIES (19)) discovered that the binding affinity of protein A for IgG’s in general is substantially increased in the presence of a salt concentration of 0.5 moles per liter or higher. The effect is achievable by either equilibrating the protein A with a salt solution of the selected concentration, dissolving the immunoglobulin mixture in such a solution, or both. The unusually strong binding which results is particularly useful in separating these immunoglobulins from other proteins, such as those normally present in an ascites fluid. The method comprises contacting the molecules with protein A in the presence of an aqueous solution of an inert inorganic salt at a concentration of at least about 0·5 M, such that when the pH of said aqueous solution is less than about 8·0 said concentration is equal to or greater than about 1 M and when said concentration is less than about 1 M said pH is equal to or greater than about 8·0.

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The salt is selected from the group consisting of ammonium, alkali metal and alkaline earth metal halides and sulfates, while the protein A is cross-linked with agarose. Polystyrene binding A procedure developed by Lentfer (MALLINCKRODT DIAGNOSTICA (GERMANY) GMBH (101)) for the immobilization of proteins on polystyrene surfaces includes a pre-treatment of the polystyrene surface with a bis-diazonium compound of the general formula I where

R1 stands for a hydrogen atom, a halogen atom, an alkylgroup, an alkoxy group or a nitro group and where R2 stands for a hydrogen atom, a halogen atom or an alkyl group and where X stands for an anion and the subsequent adsorption of the protein on the surface pretreated. Non-analyte containing matrix It was very difficult to provide a non-analyte containing matrix for the standard process of defining physiological analytes. In the prior art an adulteration of the calibration curves steepness had also to be taken into consideration. Kasper (BOEHRINGER MANNHEIM GMBH (24.8)) provided a process and a reagent for determining a reaction partner in an immunologic reaction. In accordance with this method the reaction partner to be determined is brought into contact with a marked, specific receptor R1 and with at least one unmarked receptor R2, whereby one of the unmarked receptors R2 is bonded to a substance R3. In order to establish the sample standard value, the unmarked receptor R2 (bonded by the receptor, fixed to the solid phase) is then substituted by another unmarked receptor R′2, which does not react with the reaction partner of R2, which has to be determined. Hydrazine Although there are a number of heterobifunctional linking moieties in the art (referred to by one commercial supplier of these as “double agents”), there is no specific linker class available which provides the capacity for reaction with an aldehyde at one of its termini and reaction to form a disulfide at the other. Such a

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class of reagents is particularly useful in binding glycoproteins to proteins which have or which can be provided with sulfhydryl groups. Cellulose acetate liposome binding An object of Guo et al. (COOPER-LIPOTECH (43.2)) was to develop a surface reagent which gives specific-to-non specific liposome binding ratios which they deem several times higher than those achievable using prior art surface reagents. Their reagent comprises a cellulose acetate substrate whose surface has been treated to replace surface acetate groups with hydroxyl groups, and an array of surface molecules attached to the substrate through the hydroxyl groups. Preferably, the reagent is base-hydrolyzed under conditions which result in a hydrophilic surface shell of hydroxyl groups that protects the material from dissolution by organic solvents capable of dissolving cellulose acetate, and the surface molecules are attached to the substrate through bifunctional linking groups which are attached to the support in the presence of an organic solvent. The immunoassay system of Guo et al. includes the surfaces reagent and liposomes containing surface-bound, analyte-related binding molecules and entrapped reporter molecules. The liposomes are adapted to bind to the surface reagent by direct binding to the reagent surface molecules, in a competitiveinhibition assay, or by binding to a bifunctional analyte in a sandwich-type assay. Reporter groups in the liposome reagent are preferably surface-bound and/ or encapsulated enzymes. Monomer conjugates The advantages and drawbacks of both heterogeneous and homogeneous procedures for immunoassay are well known in the art and have been described in pregoing chapters of the present literature study. There is a need for an immunoassay method which is sensitive to submicromolar concentrations of analyte; which has fast reaction kinetics; and which minimizes the number of manipulations necessary to achieve a result. According to Nowinski and Hoffman (GENETIC SYSTEMS CORPORATION (63.1)) an immunoassay method is provided comprising: a) combining said fluid sample with an addition monomer/analyte conjugate for each analyte being determined to form a fluid sample mixture; b) combining said mixture with reporter/reactant conjugates specific for bonding to each analyte thought to be contained in said fluid sample mixture under conditions favorable for the formation of the reporterlabelled analyte complexes and reporter-labelled monomer/analyte complexes, each reporter/

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analyte conjugate providing a detectably different signal from every other reporter present in said fluid sample mixture; c) separating said reporter-labelled monomer/analyte-conjugate complexes by initiating addition polymerization in said mixture; and d) detecting the incorporation of each reporter into each said polymerized complex as a measure of the analytes present in the sample, wherein the reporters are selected from the group consisting of radioisotopes, enzymes, enzyme inhibitors, enzyme cofactors, luminescent materials, and chromophores. The monomer used is a polymerizable, ethylenically unsaturated organic monomer selected from the group consisting of:

wherein R1 is a hydrogen or lower alkyl having from 1 to 8 carbon atoms, R2 is selected from the group consisting of:

and R3 and R4 are selected from the group consisting of H and compounds which will provide a reactive ethylenically unsaturated group. The graph shows the absorbance time versus the monomer concentration.

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Reagents for protein production by cloned DNA segment Keene (DUKE UNIVERSITY (53A)) provided a method for producing a protein antigen which is reactive with an autoantibody associated with an autoimmune disease, such as lupus erythematosus (SLE) and capable of producing such antigens in large amounts and without requiring donation of large amounts of material from a host. This method comprises: introducing genetic information from a cDNA gene library obtained from a human host into plural recipient cells, wherein the gene library is obtained from a host that expresses a La protein antigen reactive with the autoantibody, thereby producing transformed cells; selecting a producer cell from the transformed cells which contains a gene coding for the La protein antigen and which expresses the anti-gen by detecting a binding reaction between an autoantibody obtained from a second and different host and protein antigen expressed by the producer cell, thereby identifying a cloned DNA segment which can be utilized in the production of the protein antigen.

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Aldosterone Among various steroid hormones, determination of aldosterone has been considered most difficult and conventional immunoassay is not satisfactory for its determination. In general, the adequacy of immunoassay depends much on aptitude of antihapten antiserum employed therein, characteristics of which vary the structure of immunogen. For immunogen, compounds having functional groups in a free state as many as possible are considered to be desirable. As the hapten of ALD, there are known its 21-hemisuccinate, 3(Ocarboxymethyl)oxime, 18,21-bishemisuccinate, etc; however, all of them are at least partly blocked in their functional groups and hence are not satisfactory. Considering the above situation, Kono et al. (SHIONOGI & CO., LTD. (153. 1)) have attempted to provide haptens of ALD which have all the functional groups of ALD in a free state and succeeded in providing such haptens. Oligosaccharide It is known that when antibodies bind to proteinaceous antigens such as glycoproteins and proteoglycans, the epitope to which the antibody is directed may be a sequence of amino acids on the protein or a sequence of sugar units on one of the glyco group oligosaccharide chains. Investigation of the antigenicity of the oligosaccharides is of significant biochemical and medical interest. Oligosaccharides, when isolated as such from immunogenic glycoproteins and proteoglycans, do not bind to their known antibodies in vitro; for example, their simple solutions do not bind to antibodies. The probable reason for the absence of antibody-binding ability is that epitopes are only recognised by antibodies when they are presented to the antibodies in specific orientation and concentration. According to Feizi and Tang (RESEARCH CORPORATION LIMITED (140. 3)) there is provided a biochemical reagent comprising an antigenic conjugate between an oligosaccharide and an immobilising carrier, said oligosaccharide being bound in spaced relationship to the carrier by means of an interposed nonantigenic spacer molecule whereby the oligosaccharide is presented in antigenically active steric configuration for binding on encountering its antibody. The term “antibody” is used as including all carbohydrate-binding moieties. Feizi and Tang also provide a method for the screening of antibodies which comprises contacting a solution containing an antibody with the immobilised antigenically active conjugate aforesaid and thereafter detecting the occurrence (or non-occurrence) of binding. The spacer molecule is preferably a lipid molecule and, more preferably, a glyceride or phosphatide which possesses at least two hydrophobic polyalkylene chains. The combination of the oligosaccharide and the lipid is referred to as a “neoglycolipid”.

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The spacer molecule may be a long chain fatty acid of from 5 to 50, preferably 5 to 30 and more preferably 10 to 25, carbon atom length, an example being stearylamine and similar amino derivatives of lauric, myristic and palmitic acids. Simple amino hydrocarbons may also be employed. The amino group is necessary for chemical bonding to the oligosaccharide to form the antigenic conjugate. Neoglycolipids may be formed by reacting an oligosaccharide having at least one aldehyde group with an amine having one or more hydrophobic groups. Iodine marked pyrethrinoids Demoute et al. (ROUSSEL-UCLAF(142)) found radioactive derivatives on the basis of iodine having the general formula:

wherein X1 is a halogen (chlorus or bromium) or a trifluoromethyl, X2 is a halogen and R represents the rest of an amine acid R-NH2 or the rest of a derivative of the latter, the rest comprising an acceptor group of iodine and being marked by iodine125 or 131. Heparin-albumin conjugate Van Dijk and Bloembergen (RIJKSUNIVERSITEIT UTRECHT (140A) developed a vessel and a microtiter plate with vessel-shaped recesses. They contact human blood serum with a heparin-albumin conjugate to a solid support consisting of polyvinyl chloride. Polytetrafluoroethylene porous matrix For immobilizing immunoactive components Bosmans, Martens and Raus (DR. L WILLEMS INSTITUUT; LIMBURG UNIVERSITEIT CENTRUM (186)) used an immunosorbent based on a porous matrix of polytetrafluoroethylene, wherein there is covalently bound an organic or inorganic, hydrophilic or hydrophobic, water-insoluble polymer, to which are covalently associated antigenes and/or antibodies in determined amounts and, if there are various antigenes and or antibodies, in determined proportions. The method for the preparation of such an immunosorbent comprises the following steps: the insoluble porous polymer, activated, non-activated or coupled by the antigen or antibody is mixed together

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with the porous base matrix in a pulverulent state to form a dry mixture, the dry mixture is subjected to an agglomeration treatment to form agglomerates, the agglomerates thus formed are subjected to a crushing treatment to form crushed agglomerates, the crushed agglomerates are compressed together to form a compressed tablet and the tablet is subjected to a rolling or laminating process to obtain a film or a sheet. In an immunoassay developed by Lucas et al. (COULTER ELECTRONICS (45.2)) the rate of the precipitation reaction is controlled by adding to a fluid sample an enhancer modulating effective amount of a mixture consisting essentially of physiological and buffering salts, the relative concentration of salts to enhancer effectively optimizing the kinetics of the precipitin reaction so as to produce an essentially linear change in optical density of the fluid sample over the dynamic analytical range of a hapten. The enhancer preferably is selected from the group consisting of polyethylene glycol and Dextran and the mixture of physiological and buffer salts consists essentially of about 150 mM sodium chloride and at least about 100 mM buffering salts. Carbohydrate CA 19–9 determinant It has been shown that the 19–9 antibody reacts with a carbohydrate antigenic determinant which has been identified as a sialytated lacto-N-fucopentaose II, an oligosaccharide which shares structural features with Lewis blood group substances. The antigen of a tumor-specific monoclonal antibody is a ganglioside containing sialylated lacto-N-fucopentaose II, Fed. Proc. 41, 898, (1982). In early studies using a competition radioimmunoassay, the 19–9 antibody was shown to have high sensitivity in identifying patients with gastrointestinal adenocarcinomas and to have high specific antigens to colorectal carcinoma in sera of patients are detected by monoclonal antibodies, Cancer Res. 40, 3602– 3609, (1982). The CA 19–9 epitope has also been identified on a glycolipid extracted from SW1116 cells and from meconium, as well as on a glycoprotein from SW1116 cells. See Magnani et al., A Monoclonal Antibody Defined Antigen of colon Carcinoma, Science 212, 55, (1981). In an embodiment of the method of DelVillano and Liu (CENTOCOR, INC. (32.2)) a forward sandwich immunoassay is employed to detect the CA 19–9 antigen. In this assay, a patient sample containing the antigen is initially incubated with a solid-phase immunosorbent containing immobilized 19–9 antibody. Incubation is continued for a sufficient period of time to allow antigen in the patient sample to bind to immobilized antibody on the solid-phase immunoadsorbent. After this first incubation, the solidphase immunoadsorbent is separated from the incubation mixture and washed.

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Latex-agglutination Latex-agglutination procedures are widely used in immunoassays. In German patent application, 3 002 973 for example, a latex-agglutination reaction in the presence of a resin—or formamide derivative is described. That method has been developed to avoid or reduce faulty diagnoses by unspecific agglutination reactions in urine and sera. Toth (BEHRINGWERKE AKTIENGESELLSCHAFT (17)) found that the above disadvantages can be avoided when the latex reaction is carried out in the presence of a compound having the general formula

wherein X=0 or NH and n=2–9. According to Collet-Cassart et al. (INTERNATIONAL INSTITUTE OF CELLULAR AND MOLECULAR PATHOLOGY (85)) in a particle agglutination assay for an antigen (Ag), there is included in the mixture a limited amount of a substance which binds univalently with a proportion of the Ag present, that Ag which is so bound being unable then to cause agglutination of the particles. In this way, unusually large concentrations of Ag can be assayed in that a proportion of the Ag is bound to the univalent substance and the particle agglutination assay is in effect conducted on the smaller amount of Ag still remaining free in solution. Examples of suitable compounds are: butyrolacton, valerolacton, caprolacton, pyrrolidon, valerolactam and caprolactam. Advantageously, butyrolactam (pyrrolidon) has excellent dissolution properties. Liberti (IMMUNICON CORPORATION (76)) found a method for determining whether or not one of a pair of substances having mutual specific binding affinity, and consisting of a bindable substance and a complementary binding substance, is present in a test sample in excess of a predetermined amount. The method comprises: a.) providing an array of complementary binding substance having a predetermined binding capacity for said bindable substance; b.) contacting said array of complementary binding substance with: (i) said test sample, for a time sufficient for any bindable substance present in said test sample to bind to said complementary binding substance; and (ii) labelled bindable substance in a carrier medium, the amount of said labelled bindable substance being sufficient, when added to said predetermined amount, to substantially fill the binding capacity of said

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complementary binding substance, said array of complementary binding substance and said labelled bindable substance being in contact for a time sufficient for said labelled bindable substance to bind to said complementary binding substance; and c.) determining the absence or significant presence of unbound labelled bindable substance to differentiate whether or not said bindable substance is present in said test sample in excess of said predetermined amount. Idiotypic conjunction binding It is known that variations on the basic immunoassay technique have been developed to try to overcome some of the problems associated with immunoassays: sensitivity, reliability and cost effectiveness being major concerns. Although immunometric assay techniques have been found to be particularly useful in analyzing for antigens and antibodies, there has been difficulty in the past in establishing an optimum level of sensitivity for the assay to be helpful in the detection or monitoring of disease states or maladies in the body. Hossom (MUREX CORPORATION (113.1)) developed a method for detection or quantitation of an analyte of interest suspected of being in a specimen comprising (a) contacting a first antibody with a sample suspected of containing the analyte in such a manner that said first antibody reacts with any of the analyte present in the specimen to form a first complex; (b) contacting with any of the first complex a second antibody which recognizes and binds preferentially to the juncture of the idiotypic determinant of the first antibody and the antigenic determinant of the analyte of the first complex to form a second complex; and (c) determining the presence or amount of the second complex as an indication of the presence or amount of the analyte of interest in the specimen. Adsorption selectivity The Raji cell assay depends upon the presence of cell surface receptors for immune complexes, and these receptors only appear at precise times in the growth of the culture. Moreover, complement fractions or antibodies directed against them are often employed in radioimmuno or enzyme-linked immunosorbant assays. Unfortunately, both complement and antibody used to immobilize the complement are labile proteins. This instability leads to loss of sensitivity, specificity and reproducibility. Neither Raji cell-nor com-plement-based assays can generally detect all classes and subclasses of antibodies in the immune complex; they are restricted to IgM, IgG1, IgG2, and IgG3. Raji cell—and other complement-based methods also can generally only detect complexes with a

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molecular mass greater than 1 000 000 Daltons. These two criteria are the most limiting for Raji cell and complement-based assay systems. Roper (ROPER (23)) found immunologically non-specific peptide linked amino acids, which have a particular affinity for attaching immune complexes. As described herein, the immunologically non-specific peptide linked amino acids and similar modified peptide linked amino acids are employed to directly and selectively affix immune complexes from serum or other body fluids. The immunologically non-specific peptide linked amino acids which can be employed advantageously in the practice of the present invention including oligopeptides, polypeptides, and proteins as well as modified or substituted oligopeptides, polypeptides and proteins. Preferably, polypeptides and proteins that are glycosylated or modified with functionally equivalent substituents, such as thiosugars, hydroxy or thioamino acids, hydroxy or thiolipids, or chemically related or similar substances, have been found to be capable of directly bonding with immune complexes. More preferably, glycosylated proteins are utilized and certain globulin fractions such as immunologically non-specific gammaglobulins are bound to be particularly effective in practice. Adding detergents Adding a detergent in such an amount to the incubation medium of the immune reaction between the soluble partner or partners and the partner adsorbed on the solid carrier that its concentration is from 0·0001 to 0·01% (w/v) has been proposed by Baier (BOEHRINGER MANNHEIM GMBH (24.2)). The detergent used by him is a polyoxyethylenesorbitan monolaureate, a polyoxyethylenesorbitan monooleate, an alkylpolyether-alcohol mixture, a polyoxyethylene lauryl ether, poly oxyethylene octylphenol ether, a polyethyleneoxide-alkyl ether adduct, a C16 or C18 fatty alcohol with 10 oxyethylene units or sodium dodecyl sulphate. Surface active agents The process developed by Schmitt et al. (BOEHRINGER MANNHEIM GMBH (24.5)) includes the use of a surface active agent having an HLB value of more than 20. Protein coated liquid droplets Giaever and Keese (GENERAL ELECTRIC COMPANY (61.1)) developed a diagnostic method for determining a select protein in a liquid sample comprising the steps of:

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(a) preparing an emulsion in which a first liquid is dispersed as a large number of small protein-coated liquid droplets in a second liquid, the second liquid being an aqueous medium, and the first liquid being relatively immiscible with the second liquid, and the protein coating the droplets include molecules of a protein having the property of interacting specifically with the select protein; (b) contacting the protein-coated droplets in the emulsion with a solution containing proteinaceous material and (c) determining the presence or absence of agglutination of the proteincoated droplets. The diameters of the droplets are from 0–1 to about 0.5 micrometers. The second liquid is a fluorocarbon or a silicone oil. Dual sandwich assay It is often desirable to detect more than one antigen in a fluid simultaneously due to small sample volume, low reagent cost and short overall assay time. M. Kuriyama et al., JNCI, 68, 99–105 (1982) describe the advantage of using a combination test of tissue-specific markers to detect the presence of two proteins of human prostate-specific origin, namely, prostatic acid phosphatase and prostate antigen. Kuriyama et al., however, did not detect the two antigens by a simultaneous method but rather combined the results of the separate measurements of each anti-gen. Loor et al. (CETUS CORPORATION (34.1)) developed a method which comprises contacting the sample with at least two labelled monoclonal antibodies, each being directed against a different antigen in the sample and each being separately conjugated with the same or different signal moieties as labels, and with at least two immobilized monoclonal antibodies, each being directed against a different antigen in the sample and each being immobilized on the same support. In a preferred embodiment the antigenic materials are prostatic acid phosphatase and prostate antigen. In a second aspect the method represents a direct immunometric assay for simultaneously detecting the presence of at least two antigens in a sample which comprises the steps of: (a) incubating the sample with at least two immobilized monoclonal antibodies, each being directed against a different antigen in the sample and each being immobilized on the same support;

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(b) incubating the incubation product of step (a) with at least two labelled monoclonal antibodies, each being directed against a different antigen in the sample; (c) detecting the amount of labelled antibodies associated with the incubation product of step (b) or the amount of unassociated labelled antibodies; and (d) determining the amount of antigens in the sample by relating the measured amounts of labelled antibodies from step (c) with a control or with samples containing known amounts of the antigens. A third aspect of the method for detecting comprise the steps of: (a) forming a complex of a labelled monoclonal antibody against one antigen in the sample, the antigen, and a monoclonal antibody against the same antigen immobilized on a support to which is also immobilized at least one other monoclonal antibody against a different antigen in the sample which antibody is complexed to that antigen, which antigen is in turn complexed to a monoclonal antibody against that antigen; and (b) measuring either the amount of labelled antibodies to the complex or the amount of unbound labelled antibodies to detect the presence of the antigens in the sample. The complex may be formed by a single-step or double-step incubation of the reagents. Antigen monoclonal antibody precipitate In 1975 Kohler and Milstein (Nature, 256, pp 495–497) reported a method of producing monoclonal antibodies directed against a single antigenic determinant. The advantages of monoclonal antibodies are known in the art. However, since monoclonal antibodies are specific to a singel determinant, or interaction with antigen they can only form soluble linear complexes, rather than cross-linked complexes which might be insoluble (precipitating). Jefferis and Steensgaard (THE UNIVERSITY OF BIRMINGHAM (180)) found a method to obtain an antigen/antibody precipitate using monoclonal antibodies (samples I or II or II or IV (see next graph)). Auto-antibody versus DNA The determination of auto-antibodies against double-strand-DNA is one of the most reliable parameters in diagnostic procedures.

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To prove auto-antibodies against double-strand-DNS is important with regard to differential diagnosis, in limiting SLE from other autoantibody illnesses (collagenoses). To improve prior art methods, Rauterberg (INSTITUT FÜR IMMUNPATHOLOGIE RUPRECHT-KARLS-UNIVERSITÄT (139.1 + 81)) developed an immunoassay against DNA, including the activation of a solid phase, activation taking place on a macromolecule, ensuring a covalent binding of DNA. The proposed method is mainly of importance in the diagnosis of Lupus erythematosus. Inhibiting non-specific immunoreaction Sakai and Hirata (DAIICHI PURE CHEMICALS CO., LTD. (47.1)) designed an immunoassay for a target antigen in a sample also containing materials capable of inducing non-specific immunoreactions, wherein a specific immunoreaction proceeds between the target antigen and the corresponding antibody, their method comprises adding to the sample ultrafine particles having an average particle size of 0·2 microns or smaller, which particles contain a substance not participating in the immunoreaction but being capable of inducing reactions with the materials in the sample and absorbing the materials into the particles, to thereby inhibit non-specific immunoreactions between the materials and the corresponding antibody. The ultrafine particles have an average particle size of about 0·01– 0·1 µm, and the substance which is capable of reacting with the materials is a substance having stabilizing effects as a protective colloid, such as serum albumin. Recently, Aalberse et al. (J. Imm. Methods 87 :51–57 (1986)) describe the use of hapten-modified antigens instead of solid phase coupled antigens in a radioallergosorbent test-type assay. In such assay a patient sample suspected of containing specific allergen IgE is reacted with trinitrobenzene sulfonic acid (TNP) modified specific allergen for two hours then further reacted overnight

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with a solid phase coupled anti-TNP to form IgE-allergen-TNP-anti-TNP complex. The solid phase is washed and again reacted overnight with 125-I-IgE antibody, rewashed and counted for the presence of 125–1 isotope which is directly proportional to the concentration of allergen specific IgE in the patient sample. In this approach the authors claim to have gained the benefit of liquid phase kinetics in their first reaction but fail to substantiate the extent of TNP labelling of their allergens. Directly labelling allergens with haptens such as TNP poses the problem of missing certain vital allergenic components that might not be labelled during such a process and thus will not be quantified in said process. Also, the authors failed to show enhanced reaction timing (two days) as compared to the method described in U.S. Patent No. 3,720,760. El Shami et al. (DIAGNOSTIC PRODUCTS CORPORATION (50)) have tried to circumvent the problems associated with the traditional method of allergen testing and the modifications thereof. They effected the measurement of circulating antigens or antibodies in biological fluids using an approach making use of a specific antigen or antibody chemically attached to a soluble matrix or backbone which is subsequently labelled with a given ligand. After an initial liquid phase reaction with a patient sample, the immunocomplex formed between the patient antigen or antibody and an anti-antigen or anti-antibody labelled with a signal producing probe is immobilized in situ on a solid support containing an anti-ligand directed at the ligand attached to the liquid matrix. This approach of attaching antigens or antibodies to a liquid soluble matrix which is subsequently labelled with a given ligand serves at least two distinct purposes: (a) it increases the potential number of immunocomplexes that could be immobilized on a solid support through an anti-ligand since only few ligands need to be attached to the liquid matrix to effect complete immobilization of the entire immunocomplex. Direct labelling of antigens or antibodies with a given ligand without the use of a liquid matrix as described herein would limit the number of immunocomplexes to be immobilized on the solid support by an anti-ligand since the chances are that only a few antigens or antibodies would be labelled with a given ligand and that would require the use of elaborate affinity chromatographic techniques to separate the ligand labelled antigens or antibodies from the unlabelled antigens or antibodies. (b) the benefits of using liquid phase kinetics in the first reaction are obvious since this facilities the formation of the immmunocomplex between the antigens or antibodies attached to the liquid matrix and the signal producing labelled anti-antigen or anti-antibody.

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If liquid phase kinetics are not sought, the teachings of El Shami et al. could be used to prepare efficient solid phase matrices by attaching antigens or antibodies to a liquid matrix and labelling the matrix with a given ligand then pre-reacting the matrix with a solid phase support containing an anti-ligand. Double solid phase method Previous techniques have attempted to reduce nonspecific absorption (or bonding) to a minimum. For example, the surface of the solid phase has been thoroughly “blocked” to make it incapable of absorbing additional components. In addition, the trapped antibodies have e.g. been enzymatically split and the antigen-bonding fraction ((Fab)2-fragment) has been isolated and immobilized on the solid phase. However, total success has not been achieved with these techniques. Moreover, it should be noted that difficulties in using the ELISA technique have recently been noted to an increasing extent; see, for example, Gaffney et al., 1984, Thromb. Haemostas, 52 (1): 96–97, Boscato, et al., 1986, Clin. Chem., 32(8): 1491–1495. To avoid these difficulties Ranby and Bergsdorf (CYTRX BIOPOOL LTD. (46)) developed a method comprising the steps of coating a first solid phase and a second solid phase with an antigen-specific antibody. The first solid phase is then exposed to a solution containing the same antigen-specific antibody used to coat the first and second solid phases and the antigen. The second solid phase is exposed to a solution containing a non-specific antibody and the antigen. The amount of antigen bound to the first solid phase and to the second solid phase is then determined by means well known to those of ordinary skill in the art. To quantify the amount of antigen in the sample, the amount of antigen bound to the first solid phase is subtracted from the amount of antigen bound to the second solid phase. Zahradnik (ZAHRADNIK (215)) also uses two binding partners, of which the first is bound to biotin or biotin-binding protein, while the second partner to the substance to be analysed is detectably labelled. The biotin-binding protein or the biotin is bound to a carrier. The object of Baier et al. (BOEHRINGER MANNHEIM GMBH (24.3)) was to remove serum components which are not of interest in the case of the determination or which can interfere, to be able to wash out the solid phase without loss of sensitivity and to achieve an increase of sensitivity. To this end he used a method comprising incubating a sample containing an active substance with a binder, the binder comprises a labelled first antibody specific for the substance or an Fab or Fab’ fragment of the antibody under conditions favoring formations of complexes between the substance and the binder, adding solid phase bound active substance identical to the substance to be determined, incubating the solid phase bound active substance and the sample under

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conditions favoring formation of the solid phase bound active substance and uncomplexed binder separating the solid phase from the sample, contacting the sample with a second antibody in solid phase, the second antibody specifically binding to the binder or the binder-active substance complex, incubating under conditions favoring formation of complexes between the second antibody and the binder or binder-active substance complex, removing the solid phase and measuring the amount of labelled binder bound to the solid phase second antibody. According to Yasoshima et al. (KONISHIROKU PHOTO INDUSTRY CO., LTD. (91.1)) the amount of unreacted labelled antigen and the amount of binding complex of the labelled antigen and the antibody of a first and second detecting layers are separately measured to determine the amount of antigen in a fluid sample. The blocking layer may comprise a porous medium. As has been stated in the introduction of the present literature study, many researches and assigned companies propose methods for conducting an immunoassay which, in fact, have been curved by patents or patent applications filed by others. Many examples could be mentioned, such as a method by Lee and Canavaggio (LEE AND CANAVAGGIO (97)) comprising: (a) a ligand having a fixing affinity for said biological substance is immobilized on a solid phase; (b) the sample is incubated in the presence of said solid phase on which the ligand is immobilized; (c) the solid phase is washed after incubation; and (d) the presence of said biological substance, fixed to the solid phase via the ligand, is revealed by a property inherent to said biological substance, or that by Horikawa et al. (KANEBO. LTD. (89)) comprising the steps of binding a specimen antigen or antibody and a labelled antibody or labelled anti-gen to an antibody or antigen immobilized on fibroin, determining the amount of the specimen antigen or antibody using the labelled material, and dipping the system in a buffer solution of about 2 to 4 in pH to dissociate the bound antigens or antibodies for repeated use of the immobilized antibody or antigen. Solid phase hybridization According to Carrico (MILES LABORATORIES, INC. (109.4)) an NA hybridization method is provided to improve conventional techniques which involve chemical modification of either the probe nucleic acid or sample nucleic acids for the purpose of labelling and detection. The necessity of chemically modifying nucleic acids severely limits the practical use of the technique since it requires the large-scale preparation of labelled probes involving complicated and expensive synthetic and purification procedures or the in situ synthesis of labelled sample nucleic acids by the analyst. In particular, the resulting labelled polynucleotide must retain the ability to hybridize efficiently with its complementary sample or probe sequence. Such a requirement severaly limits

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the availability of useful synthetic approaches to label modification of polynucleotides intended for use in hybridization assays. The early hybridization techniques involved the use of radioactive labels such as H3, P32, andI125. In order to eliminate the pregoing drawbacks Carrico developed a method for detecting a particular polynucleotide sequence present in a virus or cell contained in a test sample such as biological fluid. The test sample is treated to release nucleic acids from the virus or cell of interest in single stranded form and to immobilize such nucleic acids on a solid substrate such as a filter membrane. Release of nucleic acids can be accomplished with alkali, lytic enzymes, detergents, and the like. The immobilized nucleic acids are contacted with a probe to form DNA RNA or RNA RNA hybrids which are then detectable by binding of an anti-hybrid antibody reagent, preferably labelled. The method does not require the use of labelled probe and provides quantitative results due to the efficiency and stability of the immobilization of sample nucleic acids. Ig immunoassays with an integrated control procedure Much research labor is directed to eliminate the drawbacks of weak optical spots in assay results, as will be apparent from the pregoing paragraphs of the present study. The object of Graham (ORTHO DIAGNOSTIC SYSTEMS INC. (122.4)) was to provide methods useful for colloidal gold immunoassays and other immunoassays for improving the identification of a positive result. In accordance with the principles and aspects of his methods, there are provided control procedures which form an integral part of the performance of an immunoassay such that the proper performance of the procedure coupled with active immunoassay ingredients in conjunction with a positive sample is necessary in order to achieve an observable positive reaction. Further, to improve the detectability of such a positive result, the control and sample results are ideally combined in a pattern to assist the operator in discriminating a positive result from a negative or no result condition. The invention is especially useful for ensuring that sample has been properly added and washed in immunoglobulin assays. In an embodiment a “plus” pattern is employed wherein one leg of the plus results from successful control while the other leg of the plus results from the presence of the ligand or ligand binding partner has to be detected in the sample. Failure of the reagents or the operator to perform the assay in a correct manner results in a failure of the control portion or zone of the readout and hence a “plus” readout does not appear and a false positive is avoided. Similarly, if the assay components are active and the assay is performed properly, a “minus” readout will result in the absence of ligand or ligand binding partner in the sample. Only if the reagent materials are active, the assay is performed properly,

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and the ligand or ligand binding partner is present in the sample, will a “plus” obtain due to ‘labelling’ of both the control and test zones. The immunoassay of Graham comprises the steps of: a) providing a first zone having a ligand for which the immunoglobulin is specific and a second zone having a binding substance capable of reacting with a component in the sample that is ubiquitously present in all human fluid samples undergoing similar immunoassay testing; b) contacting the first and second zones with the human fluid sample; c) removing unbound materials from the first and second zones; d) contacting the first and second zones with a labelling reagent comprising labelled anti-human IgG or IgM capable of reacting with both sample immunoglobulins and the sample component; e) removing unreacted materials from the first and second zones; and f) observing the first and second zones for the presence of label wherein the presence of label in both of the first and second zones is necessary in order to obtain a positive result. Rabbani (ENZO BIOCHEM, INC. (55)) found a method for detecting an analyte moiety by means of a localized signal. The method involves generating the signal in a specific, indicative, viscous, or thick medium. The specific medium comprises a network, a solution, and a signal precursor. The indicative medium comprises a network of polymers in the form of beads, a solution, and a signalling moiety. The network is suspended or dissolved in the solution. Signal localization results because the network reduces convection in the specific or indicative medium or because the viscosity reduces diffusion in the viscous or thick medium. According to Nilsson et al. (NILSSON ET AL. (117.2)) a method is suggested by which a great deal of work in the hitherto used methodologies is avoided and the structures involved in the binding between two interacting macromolecules may be identified relatively and efficiently distinguished from other structures located outside the actual contact surfaces. In this method antibodies are produced which are capable of reacting with a specific part of a contact surface of either of two macromolecules interacting to form a complex, the method comprising a) separately producing polyclonal antisera containing antibodies against the respective macromolecules, c) contacting the immunoglobulin fractions of the antisera with each other, such that complementary antibodies may react to form antibody complexes, c) separating the pairs of complexed antibodies obtained from antibodies which have not reacted with each other, and

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d) dissociating the antibody complexes and separately recovering at least one antibody component of the antibody complexes. The antibodies obtained may be used for identifying and recovering antigens or fragments thereof from a mixture or as substitute antigens. Wood and Corner (COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION (40)) developed a rapid and simple in vitro assay for detecting cell-mediated immune responses in humans and animals. This method comprises: (i) incubating a whole blood sample from the human or animal with the specific antigen; and (ii) detecting the presence of gamma interferon (µIFN) released by sensitized lymphocytes in the whole blood sample to indicate a cellmediated immune response to the specific antigen. Preparation of mouse monoclonal antibodies Mapping of cell-surface antigens is of great importance in medicine. Histocompatibility antigen mapping reveals which donors of organs, or tissues such as blood, are most likely to be rejected or accepted by needful recipients of the same species. Tissue cells of donor and recipient must be well matched with respect to the different sets of cell surface antigens specific to the tissue to be transferred or transplanted. Matching helps to avoid foreign tissue rejection by the immune system, which functions to remove invasive cells i.e. those cells which do not originate from the selected recipient, and are often depicted as “non-self”. Such mapping is also of medical value in autoimmune disease, in which the organism rejects its own cells as if foreign, by immune reaction of auto antibodies against cells which are “self”, i.e. they do originate from the selected recipient, but are not recognized as self. Rheumatic fever, Rheumatoid arthritis, Lupus Erythematosis are some examples of such disorders. Antibodies (proteins) have the ability to combine with and recognize other molecules (antigens). Monoclonal antibodies are not different from other antibodies except that they are completely uniform in their properties and recognize only one antigen or a portion of an antigen known as a determinant. A method for immunologically distinguishing epidermal keratinocyte cells and subsets thereof from other epidermal cells is subject matter of research of Safai et al. (SLOAN-KETTERING INSTITUTE (154.1)). The method includes: (a) contacting said cells with monoclonal antibody selected from the group consisting of Gpsk 1 (HB 8218), Gpsk 2 (HB 8281), Gpsk 3 (HB 8282),

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Gpsk 4 (HB 8283) and Gpsk 5 (HB 8284) immunologically binding keratinocyte antigen wherein the monoclonal antibody is produced by a hybridoma cell line formed by fusing a myeloma derived cell line with splenocytes derived from a mammal immunized with guinea pig epidermal cells; and (b) detecting immunological binding between keratinocyte antigens and said antibody, wherein Gpsk-1 and Gpsk-5 bind epidermal basement membrane keratinocyte antigen, Gpsk 2 binds epidermal basal and suprabasal layer keratinocyte antigen, Gpsk 3 and 4 bind epidermal spinous and overlaying layer keratinocyte antigen, and Gpsk 5 bind epidermal spinous, granular and horny cell layer keratinocyte antigen. The use of monoclonal antibodies or reagents so as to produce sufficiently pure labelled antibody has also been recognized by Forrest et al. (SERONO DIAGNOSTICS LIMITED (150)). Their process employs at one monoclonal antibody reagent and comprises incubating of mixture of: (a) the liquid sample, (b) labelled antibodies to the antigen under assay, (c) a reagent comprising antibodies to the antigen under assay conjugated with a reagent Z, the reagent Z being a hapten or antigenic substance, and, (d) an antibody reagent raised to reagent Z which selectively interacts with reagent Z by non-covalent bonding, but which is not directly bindable to either component (a) or component (b) the said reagent (d) being bound to a solid phase support and at least said components (b) and (c) comprising monoclonal antibodies; separating the solid fraction from the liquid fraction, determining the amount of label in one of the said fractions and, therefrom the amount of antigen present in the sample. The reagent Z may be fluorescein isothiocyanate, but also may be an enzyme or a radioisotope. Substituted aldosterones in radiation and enzyme i.a. As the hapten of aldosterone, there are known its 21-hemisuccinate, 3-(Ocarboxmethyl)oxime, 18,21-bishemisuccinate, etc.; however, all of them are at least partly blocked in their functional groups and hence are not satisfactory. Considering the above situation, Kono et al. (SHIONOGI & CO. LTD (153.1)) have attempted to provide haptens of aldosterone which have all the functional groups of aldosterone in a free state and now succeeded in providing such haptens. Accordingly, their main object is to provide substituted aldosterones available as haptens of aldosterones, and another object is to provide a process for preparation of the substituted aldosterones.

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The substituted aldosterones are represented by the formula:

wherein either one of R1 and R2 is hydrogen and the other is —S(CH2)mCOR3 or —OCO(CH)nCOR3, provided that when R1 is hydrogen, R2 is—S(CH2)mCOR3 or —OCO(CH2)nCOR3 and when R2 is hydrogen, R1 is —S(CH2)mCOR3; m being an integer from 1 to 3, n being an integer from 1 to 5 and R3 being hydroxyl, lower alkoxy or a residue of tyramine, tyrosine lower alkyl ester, histamine, histidine, 7-aminoheptanoyltyrosine lower alkyl ester or β-D-galactosidase as optionally iodinated (particularly radioiodinated). Specific examples of —S (CH2)mCOR3 are carboxymethylthio, carboxylethylthio, carboxypropylthio, etc. and specific examples of —OCO(CH2)nCOR3 are hemimalonyloxy, hemisuccinyloxy, hemiglutaryloxy, hemiadipoyloxy, hemipimeloyloxy, etc. Of these substituted aldosterones (I), the compounds wherein R3 is a hydroxyl group may be combined with proteins such as bovine serum albumin. Immunization of rabbits with the resulting products as antigens gives anti aldosterone antiserum. They may be also combined with enxymes for labelling such as horseradish peroxidase. The substituted aldosterones (I) wherein R3 is other than hydroxyl may be labelled with radioiodine such as 125I or 131I according to a conventional Chloramine-T or enzymatic method to give a labelled product for RIA. An example of suitable substituted aldosterones (I) is N-(p-hydro xyphenethyl)-2(11β,18—epoxy-18a,21-dihydroxy-3,20-dioxo-4-pregnen4-ylthio(acetamide. The substituted aldosterones (I) can be producted, for instance, by subjecting a compound of the formula:

wherein R1 and R2 are each as defined above and X is a glycol protective group to elimination of the protective group.

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A biochemical reagent prepared by Feizi and Tang (RESEARCH CORPORATION LIMITED (140.3)) comprises an oligosaccharide, preferably one which has been liberated from an immunogenic glycoprotein or proteoglycan, which is immobilised on a carrier via an intermediate spacer molecule such as a lipid. The lipid molecule should preferably have at least two long lipid tails so that the oligosaccharide is held in spaced relationship to the carrier where it exhibits antibody-binding ability which is almost indistinguishable from that of the original glycoprotein or proteoglycan. The reagent has its application in biochemical treating of oligosaccharides and systems which bind to them. The preparation steps are shown in the following reaction scheme: Latex-agglutination reactant From experiences it appeared that it has been difficult to pretreat immunoassay liquids with fibres from a cation exchanging resin of the carbonacid type.

It has been proposed to carry out a latex-agglutination reaction in the pressure of a resin or formamide derivative; see e.g. German patent 3002973. Such a reaction reduces the above problems, but it does not obviate them. Surprizingly Toth (BEHRINGWERKE A.G. (17)) found that the technical problems can be released when the reaction takes place in the presence of a compound of the general formula:

wherein X=O or NH, and n=2–9

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Suitable compounds are: butyrolactone, valerolactone, caprolactone, pyrrolidine, valerolactam and caprolactam. According to Collet-Cassart et al. (INTERNATIONAL INSTITUTE OF CELLULAR AND MOLECULAR PATHOLOGY (85)) in particle agglutination assay for an antigen, there is included in the mixture a limited amount of a substance which binds univalently with a proportion of the Ag present, that Ag which is so bound being unable then to cause agglutination of the particles. In this way, unusually large concentrations of Ag can be assayed in that a proportion of the Ag is bound to the univalent substance and the particle agglutination assay is in effect conducted on the smaller amount of Ag still remaining free in solution. A method by Liberti (IMMUNICON CORPORATION (76)) determines whether or not one of a pair of substances having mutual specific binding affinity, and consisting of a bindable substance and a complementary binding substance, is present in a test sample in excess of a predetermined amount. This method comprises: a) providing an array of complementary binding substance having a predetermined binding capacity for said bindable substance; b) contacting said array of complementary binding substance with: (i) said test sample, for a time sufficient for any bindable substance present in said test sample to bind to said complementary binding substance; and (ii) labelled bindable substance in a carrier medium, the amount of said labelled bindable substance being sufficient, when added to said predetermined amount, to substantially fill the binding capacity of said complementary binding substance, said array of complementary binding substance and said labelled bindable substance being in contact for a time sufficient for said labelled bindable substance to bind to said complementary binding substance; and c) determining the absence or significant presence of unbound labelled bindable substance to differentiate whether or not said bindable substance is present in said test sample in excess of said predetermined amount. Idiotypic binding assay A method for detection or quantitation of an analyte of interest suspected of being in a specimen has been developed by Hossom, M.G. (MUREX CORPORATION (113.1)). It comprises (a) contacting a first antibody with a sample suspected of containing the analyte in such a manner that said first antibody reacts with any of the analyte present in the specimen to form a first

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complex; (b) contacting with any of the first complex a second antibody which recognizes and binds preferentially to the juncture of the idiotypic determinant of the first antibody and the antigenic determinant of the analyte of the first complex to form a second complex; and (c) determining the presence or amount of the second complex as an indication of the presence or amount of the analyte of interest in the specimen. Glycocylated polypeptides Roper, M.D. (BIOSTAR MEDICAL PRODUCTS INC. (23)) found that certain immunologically non-specific peptide linked amino acids are very useful for attaching immune complexes. The immunologically non-specific peptide linked amino acids which can be employed advantageously in the practice of the present invention include oligopeptides, polypeptides, and proteins as well as modified or substituted oligopeptides and proteins that are glycosylated or modified with functionally equivalent substituents, such as thiosugars, hydroxy or thioamino acids, hydroxy or thiolipids, or chemically related or similar substances, have been found to be capable of directly bonding with immune complexes. More preferably, glycosylated proteins are utilized (hereinafter glycoproteins) and most preferably, certain globulin fractions such as immunologically non-specific gammaglobulins are particularly effective. Whether the immune complexes comprise any single class or subclass of antibody or whether the fluid containing the immune complexes derives from corporeal or extracorporeal origin, does not appear to be limiting to the utility of the respective reagents, as physico-chemical alterations to the antibodies, after binding to antigens render them selectively susceptible to immobilization. Preparing an emulsion in which a first liquid is dispersed as a large number of small protein-coated liquid droplets in a second liquid, such as an aqueous medium, and wherein the first liquid being relatively immiscible with the second liquid, and the protein coating the droplets including molecules of a protein having the property of interacting specifically with the select protein, in subject of research by Glaever and Keese (GENERAL ELECTRIC COMPANY, National Foundation for Cancer Research Inc. (61.1)). The protein-coated droplets in the emulsion are contacted with a solution containing proteinaceous material, and then the presence or absence of agglutination of the protein-coated droplets are determined. Preferably the second liquid is a fluorcarbon or silicone oil. To reduce interferences from a serum or plasma in a heterogeneous immunoassay, Baier, M. (BOEHRINGER MANNHEIM GmbH) (24.2) proposes to add a detergent to the incubation medium of the reaction in an amount of 0·0001% to 0·01%. The detergent used is a polyoxyethylenesorbitan

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monolaureate, a polyoxyethylene-sorbitan monooleate, an alkylpolyether-alcohol mixture, a polyoxyethylene lauryl ether, polyoxyethylene octylphenol ether, a polyethyleneoxide-alkyl ether adduct, a C16 or C18 fatty alcohol with 10 oxyethylene units or sodium dodecyl sulphate. A problem inherent to many immunoassay tests is the separation of non-specific influences in a test sample from the specific ones. One of the solutions encountered has been proposed by Sakai and Hirata (DAIICHI PURE CHEMICALS CO LTD.) (47.1) comprises adding to said sample ultrafine particles having an average particle size of 0·2 microns or smaller, which particles contain a substance not participating in the immunoreaction but being capable of inducing reactions with the materials in the sample and absorbing the materials into the particles, to thereby inhibit non-specific immunoreactions between the materials and the corresponding antibody. The substance which is capable of reactin with the materials is a substance having stabilizing effects as a protective colloid, preferably serum albumins. Horikowa et al. (KANEBO, LTD.) (89) found a method of immunoassay which comprises the steps of binding a specimen antigen or antibody and a labelled antibody or labelled antigen to an antibody or anti-gen immobilized on fibroin, determining the amount of the specimen antigen or antibody using the labelled material, and dipping the system in a buffer solution of about 2 to 4 in pH to dissociate the bound antigens or antibodies for repeated use of the immobilized antibody or antigen. The method of Nilsson et al. (117.2) is based upon the concept that in order to identify the specific domains or surfaces of a pair of interacting macromolecules, e.g. protein-cell receptor, which are directly involved in the binding, one utilizes the fact that antisera against the two macromolecus each contain certain antibodies directed against the binding domain of the respective macromolecule, which two groups of antibodies therefore should, on one hand, exhibit antiidiotype specificity to each other (i.e. have complementary binding structures) and, on the other hand, immunologically correspond to the binding domain of the respective macromolecule, and that one selects these binding-specific antibodies by mixing the two antibody preparations such that an isolatable complex is formed between these anti-idiotype antibodies, and subsequently dissociating the complexes into individual antibodies. In such manner antibodies specifically directed against a binding domain, which is not known in any detail, of a macromolecule may readily be produced, and the antibodies may then be utilized for isolating the antigen fragment in question. The methods of NILSSON et al. for the production of antibodies capable of reacting with a specific part of a contact surface of either of two macromolucules interacting to form a complex, said method comprising

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a) separately producing polyclonal antisera containing antibodies against the respective macromolecules, b) contacting the immunoglobulin fractions of the antisera with each other such that complementary antibodies may react to form antibody complexes, c) separating the pairs of complexed antibodies obtained from nonreacted antibodies, and d) dissociating said antibody complexes and separately recovering at least one antibody component of the antibody complex. Antibodies to skin cells The findings of Safai et al. (SLOAN-KETTERING INSTITUTE) (154.1) provide a background to potential dermatological applications of mono clonal antibodies to human epidermis. The hybridoma cell lines producing the monoclonal antibody bind to and subset normal guinea pig keratinocyte cell antigen from the four different epidermal cell layers, wherein the monoclonal antibodies are selected from the group consisting of Gpsk-1 (HB 8218), Gpsk-2 (HB 8281), Gpsk-3 (HB 8282), Gpsk-4 (HB 8283) and Gpsk-5 (HB 8284) and wherein (1) Gpsk-1 and Gpsk-5 bind epidermal basement membrane keratinocyte antigen, (2) Gpsk-2 binds epidermal basal and suprabasal layer keratinocyte antigen, (3) Gpsk-3 and Gpsk-4 bind epidermal spinous and overlaying layer keratinocyte antigen, and (4) Gpsk-5 binds epidermal spinous, granular and horny cell layer keratinocyte antigen. Forrest et al. (SERONO DIAGNOSTICS LIMITED) (150) developed a method of immunoassay of an antigen in a liquid sample wherein a complex is formed between antigen contained in the said sample and two or more antibody reagents, and the said complex is bound to a solid support by non-covalent bonding; and the amount of complex becoming bound to the support is determined; the process employing at least one monoclonal antibody reagent. Labelling methods including radio-active, fluorimetric and enzyme labelling may be used to effect determination of the binding of the complex to the solid support. The solid support may take the form of particles, beads, wall-coatings on the reaction vessel or an insert of large surface area. The method is particularly applicable to the assay of TSH, CEA, HCG, alphafeto protein, immunoglobulins, viruses, allergens, bacteria, toxins, drugs and vitamins. Use of monoclonal reagents is said to improve the specificity of the process, and also decreases non-specific binding.

2 IMMUNO SPECIFIC METHODS AND MEANS

2.1 TUMORS Immunological tests for diagonizing tumors were based upon a coagula-tion test for immunocomplex (IC). Coagulating I & C diminishes the specificity of these tests. Ishii et al. (OTSUKA PHARMACEUTICAL CO., LTD. (124)) seek to overcome this drawback by testing for com-plement-binding ICs wherein the consistuent antigens are tumor associated antigens (tumor markers) such as BFP (basic fetoprotein), AFP (α-fetoprotein) TAG (tumor associated glycolinkage). Such an IC as bound to an insolubilized anti-complement, can be specifically bound to a labeled substance or such an IC alone can be specifically insolubilized and labeled. On human B-cells two antigens have been described: B-LAST-1 and BB-1. Freedman et al. (DANA-FARBER CANCER INSTITUTE, INC. (49)) describe an antigenic protein on the surface of activated human Bcells, having an apparent molecular weight of approximately 75 000 daltons under reducing conditions and 67 000 daltons under non-reducing conditions of the IgM isotype. It is reactive with less than 1% of unstimulated human B-cells, with less than 6% of stimulated or resting T-cells and monocytes, and unreactive with neoplasms of non-B-cell origin, or neoplasms of B-cell origin corresponding to stages of differentiation other than the mid-stage of Bcell differentiation. The monoclonal antibody can be produced with several kinds of cell cultures, amongst whom a hybridoma cell line having the identifying characteristics of ATCC No. HB 8716. It can be used in the conventional immunological assay techniques. For developing a monoclonal antibody exhaustive screening and fusing of hybridoma clones is necessary. Kortright and Hofheinz (COULTER CORPORATION (45.1)) identified two cell surface markers associated with the onset of adenocarcinoma and squamous cell carcinoma.

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The first has a molecular weight of approximately 490 kd, and is expressed in the cell membrane. The second form is found in both the cytoplasm and membrane of carcinoma cells and has a molecular weight of approximately 438 kd (KC-4). The KC-4 monoclonal antibody is particularly useful in its application to binding with the antigenic determinants on and in carcinoma cells which occur in the specific diseases of adenocarcinoma and squamous cell carcinoma regardless of the human organ of origin. Preferably, tissues to be sampled are frozen, because it reduces the error margin in selectivity to 3%. A sample hybrid cell lines capable of producing monoclonal antibodies specific for the KC-4 antigen are on deposit with the American Type Culture Collection, and are assigned the Nos. 8709 (IgG3) and HB 8710 (IgM). There has been identified a monoclonal antibody, KC-4 Monoclonal antibody, which is specific for a unique epitope, the KC-4 antigen, of a human carcinoma tumor cell. Critical for detection is the binding without hindrance of the KC-4 Monoclonal antibody with its epitope. Kortright (COULTER CORPORATION (45.2)) found that the aforementioned binding is hindred by sialic residues on the KC-4 antigen. Treating samples to be assayed, with neurominidase at room temperature increases, according to the author, the ability to detect human carcinoma tumors in a pathology sample by at least a factor of two. In tumors nuclear and nucleolar antigens were found not present in non-tumor tissues. Busch (ONCOS, LTD. (120)) developed a monoclonal antibody to a tumor nucleolar antigen, harvested from mouse ascites. Serum levels of galactosyltransferase isoenzyme II (GT-II) have been found to correlate well with the presence of cancer. Murine monoclonal antibodies to GT lacked in specificity. Brandt and Uemura (KONISHIROKU PHOTO INDUSTRY CO. LTD. (91.3)) developed a murine monoclonal antibody specific for the GTII produced by a hybridoma line. Certain RNA tumor viruses and human tumor cells contain genomic sequences, termed oncogenes, that encode specific proteins which have been shown to be responsible for the induction of various human and animal cancers. Albanese et al. (ONCOGENE SCIENCE, INC. (119)) discovered that normally intracellular, oncogene-encoded p21 proteins may be detected in biological fluids such as serum and that this can be utilized to diagnose or monitor neoplastic conditions or states. The detection method is based on immunological principles. In the figure below, a standard curve of the RIA for RAS p21 protein in the solution-phase is depicted. The presence of the p53 phosphoprotein in the nucleus of cells may be indicative of transformation of normal cells to a tumorigenic state.

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Immunofluorescence and—precipitation methods for p53 exist. Miller and Doyle (ABBOTT LABORATORIES (1.8)) developed an immunohistochemical p53 assay method which permits the detection of p53 antigen in human cells, and in tissue specimens. Cells are fixed on a glass slide and incubated with anti-p53 antibodies which react with the p53 antigen in the specimen. The p53 antigen-anti-body complex is then localized by addition of anti-Ig which is either labeled directly or indirectly with an enzyme. Next, a chromogen sub strate solution for the enzyme is added to the slide and color is developed. The slide is then examined under a

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microscope for specifically stained cells which indicate the presence of p53 antigen. The authors claim an improvement in sensitivity and simplicity in comparison to conventional methods. Immunization with tumor cell preparations (generally intact irradiated cell suspensions or mechanically disrupted lysates of cells) has variable efficacies. Furthermore, the means of monitoring the immune response of individual patients is not available for tailoring the immunization dose and the immunization schedule for optimal clinical outcome. In addition, it is often the case that autologous tumor cell preparations are not practical because of a lack of adequate tumor from the patient to be treated. Fareed et al. (INTERNATIONAL GENETIC ENGINEERING, INC. (83)) developed a method for detecting regression-associated antibodies (RAAbs). Using reducing SDS polyacrylamide gel electrophoresis regression-associated antigens (RAAs) with a molecular mass from about 19 to about 23 kd or from about 38 to about 45 kd or within the range from about 65 to about 71 kd. Preferably these regression associated antigens having the above molecular masses do not bind to cationic exchange resins, such as DEAE Sephacel, at 50 mM Na3PO4 (PH7) or to heparin agarose resins at 200 mM Na3PO4 (PH 7). In the figure below immunoblots are depicted of serum dilutions for samples from two immunized patients (Pt. 1, Pt. 2) and an immunized rabbit tested against the particulate fraction (lanes numbered 1) and conditioned medium (lanes numbered 2) from A375 cells. Pt. 1 received intralymphatic (I.L.) immunotherapy with intact, irradiated tumor cells, Pt. 2 received subcutaneous (S.C.) immunization, and rabbit 209 was immunized subcutaneously with the particulate fraction of A375 (ING-A) cells. For determining a leucocyte subset requires expensive instrumentation and specific monoclonal antibodies. Some techniques require lysing of the red blood cells. Kortright and Hofheinz (COULTER CORPORATION (45.3)) provide a method to remove erythrocytes without lysing. A hydrid cell line, American Type Culture Collection No. CRL 8994, producing a K-16 monoclonal antibody specific to an epitope of glycophorin A exposed on the surface of the membrane of a human red blood cell was developed. The K-16 monoclonal antibody were coated on magnetic non porous microspheres. After mixing with blood sample at a microsphere-to-cell ratio of 14:1 the beads were magnetically removed. After one minute, a first supernatant was decanted and after a second period of one minute a second supernatant was decanted; the percentage of erythrocyte deple tion and the percentage of leukocytes remaining in both of the two consecutive supernatants were determined. Measurements indicated that from 7 samples, the average depletion

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of erythrocytes amounted to 99·97 + 0·02 %. Bogoch (BOGOCH (25)) isolated two proteins from brain tumor cells: Astrocytin and Malignin, having molecular weights of about 8 kd, respectively 10 kd. Both can be used in immunoassays. The determination of carcinoembryonic antigen (CEA) lacks accuracy and sensitivity. Giorgio et al. developed a new preparatory method. A sample is diluted from 0·2 ml to 0·5 ml with an acidic buffer, pH 6·3. It is mixed with 35 mg to 80 mg of silica, average primary particle size is from about 7–16 nanometer, and a specific surface area from 130 to 180 m2/g. Thereafter the prepared sample is subject to the appropriate assay for CEA, of which the type is not critical. The presented preparation is more economic than prior art ones. A comparison of Standard CEA Assay, and determination to the presented method is given in the table below. Because of drawbacks in prior art CEA assays subclinical tumors of the colon could not be detected. Schoemaker et al. (CENTOCOR, INC. (32.3)) developed an assay which is based upon the use of two monoclonal antibodies against the high molecular weight CEA: the 1116NS-3d (3d6) and the COL-1 antibody. The 3d6 is employed to form a solid phase absorbent; the other, COL-1, is employed as a labeled antibody. A comparison of CEA measurement according the 3d6/COLl method, and the ABBOTT method is shown in the figure below.

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*(CPM)=Counts Per Minute ** % Bo=Percentage of binding of CEA to antibody at zero antigen concentration.

Few monoclonal antibodies are relatively specific for human pulmonary adenocarcinoma. Yoshida et al. (KYOWA HAKKO KOGYO CO., LTD. (95.1)) found that the monoclonal antibody ALC-186, produced by hybridomas between spleen cells of an antibody-producing mouse immunized with human pulmonary adenocarcinoma tissue membrane components, and mouse myeloma cells has a particularly good reactivity with pulmonary adenocarcinoma. The specificity of the assay based on ALC-186 is illustrated in the scatterchart depicted below. The ALC-186 assay is suitable for diagnosis and for indicating the stage of pulmonary adenocarcinoma. The serodiagnostic systems for the anti-human lung adenocarcinoma monoclonal antibody, ALC-186 and the anti-human stomach adenocarcinoma monoclonal antibody, AMC-382 are of great clinical significance. The rate of detection of these methods are, however, respectively 48% and 61·5%. Yoshida et al. (KYOWA HAKKO KOGYO CO., LTD. (95.2)) pertain to have improved these methods. A mixture of anti-human lung adenocarcinoma monoclonal antibody ALC-186 and anti-human stomach adenocarcinoma monoclonal antibody AMC-382, allowing a human serum sample to react with the first antibody to bind adenocarcinoma antigens. Hereafter said mixture in an enzyme-labeled is allowed to react with the adenocarcinoma antigens. By measuring the enzyme activity the quantity is determined. Similarities between the mouse mammary tumor virus (MMTV) and human mammary tumor virus (HMTV) have been noted. Although improvements in immunoassays for human breast cancer have provided increased sensitivity or specificity, or both, it has not been possible

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until now to obtain a high positive staining rate without having an associated high rate of false positives. Keydar (TEL AVIV UNIVERSITY; TEVA PHARMACEUTICAL INDUSTRIES, LTD. (190)) intents to improve the detection of HMTV. A monoclonal antibody was developed from the T47D clone-10 breast cancer line (HMTV). Several procedures for detection and characterization of ovarian cancer with monoclonal antibodies do exist. However, the lack of sufficient tumor specificity is still a problem. Zurawski and Haisma (CENTOCOR, INC. (32.5)) overcame the drawbacks in his produce based on the cell surface antigen (designated CA-TL3) associated with primary and metastatic human ovarian carcinoma of the serous, mucinous, endometrioid and clear cell types. It is expressed by primary and metastatic ovarian tumor cells of these histological types. This antigen is further characterized by its specific reactivity with the OV-TL3 monoclonal antibody.

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The OV-TL3 has two antigenic determinants with molecular weights of 20 and 40 kd. It is only slightly sensitive to proteolytic digestion. Using immunoscintigraphy, it can be used for tumor imaging. Also it is applicable in the standard immuno-techniques. To improve the detection of malignant colon cells, Chester (CHESTER (36)) developed a radioimmunoassay for soluble circulating immunocomplex (CIC). The antibodies are produced from rabbits immunized with byproducts of human colon cell culture. A remarkable step in the procedure is the dissociating of CIC with 0·2M glycine/HCL pH 2·8. The labeled antibody is combined with the antigen component of the immunocomplex to produce a new labeled immunocomplex. The newly formed immunocomplex is precipitated with PEG 6000. The specificity of the test is demonstrated in the following table. Baldwin (XOMA CORPORATION (212)) used I131-labeled monoclonal antibody to diagnose with precision the presence and position of a cancer deposit particularly in the colorectal region.

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1 Each 2 All

X represents the average of a patient’s serum sample that was assayed in duplicate. values below 15 were considered normal.

3

Hybridoma cell line XMMCO-791 was used to fabricate the antibody. After labeling, it is administered to the patient. Thereafter these is screened for radioactivity using a gamma camera. An example of the resulting image, is depicted below being the anterior radionuclide image of the pelvis showing localisation of the antibody. Markers which are identified with the neoplastic transformation of one type of cell are found to be present on cells of other normal tissues. Conversely, it is also found that markers associated with particular types of tumors in a number of

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individual patients will not be universally associated with that type of tumor on all patients. Singhal (IMRE CORPORATION (78)) wishes to provide assays which may be employed with both known and presently undiscovered tumor markers. It comprises three essential steps. First, the immune complexes within the patient’s serum or plasma sample are enriched relative to other serum components, typically by immunoadsorption. After the initial enrichment, the immune complexes are separated from the enriched fraction, typically by immobilization on a solid-phase receptor specific for the immune complexes. it has been found that the enrichment step followed by the separation of the immune complexes is critical in detecting specific immune complexes which are present at very low levels, typically on the order of less than 1 µg/ml in serum. After separation, the immune complexes may be detected by conventional immunological techniques, conveniently employing labeled receptors specific for the tumor marker. Ishii and Akazawa (OTSUKA PHARMACEUTICAL CO., LTD. (0100914)) designate compounds from the group consisting of basic fetoprotein (BFP), αfetoprotein (AFP), tumor associated glycolinkage (TAG), and carcinoembryonic antigen (CEA) as target tumor associated antigen for determining agent in immunoassays. CA-19–9 antibody It has been shown that the 19–9 antibody reacts with a carbohydrate antigeneic determinant which has been identified as a sialylated lacto-Nfucopentaose II, an oligosaccharide which shares structural features with Lewis blood group substances. See MAGNANI et al. The antigen of a tumor-specific monoclonal antibody is a ganglioside containing sialylated lacto-N-fucopentaose II, Fed. Proc. 41, 898, (1982). In early studies using a competition radioimmunoassay, the 19–9 antibody was shown to have high sentitivity in identifying patients with gastrointestinal adenocarcinomas and to have high specificity for normal individuals. See Koprowski et al. Specific antigen in serum of patients with colon carcinoma, Science, 212, 53, (1981); and, Herlyn and cosearchers. Specific antigens to colorectal carcinoma in sera of patients are detected by monoclonal antibodies, Cancer Res. 40, 3602–3609, (1982). The CA 19–9 epitope has also been identified on a glycolipid extracted from SW1116 cells and from meconium, as well as on a glycoprotein from SW1116 cells. See Magnani et al. A Monoclonal Antibody Defined Antigen of colon Carcinoma, Science 212, 55 (1981). In a embodyment of an assay developed by DelVILLANO and Line (32.2), a forward sandwich immunoassay is employed to detect the CA 19–9 antigen. In this assay, a patient sample containing the antigen is initially inclubated with a

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solid-phase immunoadsorbent containing immobilized 19–9 antibody. Incubation is continued for a sufficient period of time to allow antigen in the patient sample to bind to immobilized antibody on the solid-phase immunoadsorbent. After this first incubation, the solid-phase immunoadsorbent is separated from the incubation mixture and washed. The primary antigen-antibody reaction is carried out under acidic conditions. 2.2 VENEREAL DISEASES 2.2.1 HLTV The ability to purify the major charged species, pl 6·8, of human interleukin-I (IL-I) has allowed the amino acid sequence to be elucidated. The animo acid sequence corresponds exactly with the sequence deduced from a cloned cDNA for this species of human interleukin. The knowledge of the sequence of IL-I has allowed the synthesis of various portions of the secreted form of IL-I. This in turn has permitted the production of monospecific antibodies which react with synthesized peptide regions of the human IL-I species pl 6·8 molecule and the intact IL-I molecule. Schmidt et al (MERCK & CO. INC. (107.3)) discovered that several distinct peptide regions of the secreted form of purified human interleukin-I species pl 6·8 have been found to exhibit characteristics associated with highly immunogenic protein moieties and are used to produce specific antipeptide antibodies. The antibodies raised against the individual peptides are specific for the peptide used for their production and for IL-I, pl 6·8. The individual antibodies bind to both the precursor of IL-I, pl 6·8 and the mature or extracellular IL-I, pl 6·8. The interleukin-2 (IL-2) and its receptor — (IL-2R) per se, are well known entities. The measurement of IL-2 receptors (IL-2R) was limited to the assessment of cell-associated IL-2R by a variety of immunological techniques, involving several monoclonal antibodies against the human IL-2R. The observation of a soluble or released form of this molecule has heretofore not been reported. Therefore, this soluble molecule has never been measured in a body fluid. With regard to cell-associated IL-2R, the quantitative measurement of IL-2R has been accomplished by binding of the monoclonal antibody followed by cellular analysis with an expensive fluorescence-activated cell sorter (FACS) or similar apparatus. Nelson et al. (THE UNITED STATES OF AMERICA as represented by the Secretary of U.S. Department of Commerce (177.2)) developed a sandwich ELISA assay for IL-2R, based upon an alkaline phosphatase conjugated

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monoclonal antibody (7G7/B6) directly, instead of the FITC-conjugated 7G7/B6 and alkaline phosphatase conjugated rabbit anti-FITC. Measurements are read with a simple spectrophotometer. The authors describe the test as rapid, sensitive, and inexpensive. An example of measurements results for serum IL-2R receptors in healthy people and patients with malignant diseases is given in the figure below. Large-scale production of recombinant IL-2 (rIL-2), thus permitting investigation of its effects. The bioactivity of IL-2 has traditionally been measured using a bioas say method [Biochemical Biophysical Research Communications, 109, 363— (1982)], in which the detection limit for blood IL-2 concentration is 1 to 10 µg/m2. However, as IL-2 exhibits bioactivities in a very small amount, a method of

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measuring it at an accuracy higher than that of the bioassay method, is urgently desired. Moriya et al. (TAKEDA CHEMICAL INDUSTRIES, LTD. (164)) developed for IL-2 measurement an enzyme immunoassay (EIA) method, which uses antiIL-2 antibodies obtained by immunizing an animal with rIL-2. The method gives a higher sensitivity than the bioassay method. Kung et al. (T CELL SCIENCES, INC. (163)) used IL2R—and CD8measurements in the diagnosis and therapy of diseases. The measurement of such molecules can be valuable in monitoring the effect of atherapeutic treatment on a subject, detecting and/or staging a disease in a subject, and in differential diagnosis of a physiological condition in a subject. These measurements can also aid in predicting therapeutic outcome and in evaluating and monitoring the immune status of patients. In specific embodiments, measurements of serum or plasma interleukin2 receptor levels can be made, to detect or stage leukemia or lymphoma. In other embodiments, IL2R levels, or CD8 levels, can be used to differentially diagnose renal allograft rejection, as distinguished from Cyclosporing A nephrotoxicity. In another embodiment, CD8 levels can be measured to differentially diagnose rheumatoid arthritis, as distinct from other joint diseases. In particular embodiments, measurements of the soluble T cell surface molecules can be accomplished by sandwich enzyme immunoassays. The measurements are illustrated in a case of renal allograft rejection, and a comparison of CD8-levels in several diseases, amongst other hepatitis and an AIDS related complex (see the figures below).

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Gottlieb (IMREG, INC. (79)) pertains to measure in AIDS or ARC infected humans and mammals, the capacity of the immune system in order to determine its potient of being restored to a higher level of activity. Hereto the response of IL-2 production to mitogens is measured. The subject’s proliferative response, namely the uptake of labeled thymine, is regarded as a measure for capacity of the immune system to respond to antigen. Lindner and Wechter (THE TEXAS A&M UNIVERSITY SYSTEM (192)) provide a serum isolated from the serum of marmoset monkeys (Callithrix jachus) afflicted with or recovering from marmoset wasting disease. It is applicable in the standard immunological techniques. Paul (ABBOTT LABORATORIES (1.11)) developed a quick screening procedure for detecting individuals infected with HLTV-III virus who are antiHLTV-III negative, and therefore nor indicated as HLTVIII positive by currently available antibody screening tests. It comprises: 1) coating a solid support with antibody to HLTV-III; 2) contacting the coated solid support with a biological sample; 3) contacting the solid support with antibody to HLTV-III from a different animal species than that utilized in step I; 4) contacting the solid support with an antibody specific for the antibody of step 3, and conjugated to a label such as an enzyme, radioisotope or fluorescent label; and

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TABLE 1 HTLV-III Antigen and Antibody Enzyme Immunoassay Results of Serum Samples

5) detecting the label as a measure of the presence of HLTV-III viral antigen in the sample. Some test results are depicted in the table below.

Burger and Goldstein (EPITOPE, INC. (56)) developed novel monoclonal antibodies against an antigen produced as a result of the infection, with the hybrid continuous cell lines 3D8, 3G12, and 1E2.

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The following table illustrates that clones 3D8, 1E2 and 3G12 detected HLTVIII antigen expression on H9 cells only when the H9 cells had been infected. Infection with either cell free HLTV-III virus or HLTV-III positive T4 cells was equally effective; the isotype-control monoclonal antibody had no effect. Saxinger and Gallo (UNITED STATES OF AMERICA represented by the United States Department of Commerce (177.1)) claim to have developed an ELISA technique which is more specific, and accurate than known techniques. It is particularly suited to the detection of human Tcell leukemia-lymhoma virus type III (HLTV-III). HLTV-III antigens are more purified disrupted, and coated in the wells of microtiter plates. Test sera is tested and confirmed by a confirmatory neutralization screening which includes an additional 2-hour incubation period before the test sample was incubated with the antigen-coated wells. During this extra 2 hours of incubation, the wells are exposed to unlabeled sheep antibody (heteroantiserum) to HLTV-III which reacts with and saturates HLTV antigen sites on the well, thus preventing the test serum from attaching to the well in the subsequent step. As a control in the test, adjacent wells are exposed to normal sheep serum during the additional incubation period. The preferred dilution of sheep antiserum is 1:2 at a titer of 10000 or more. Sheep antiserum showing reactivity with proteins from phytohemagglutinin (PHA) — stimulated human lymphocyte preparations coated on microtiter plate wells are absorbed with PHA lymphocyte preparations until the reactivity is removed. The sheep antiserum used in this process requires absorption with one volume of cell equivalents per three volumes of serum to reach the end point. A suppression of the absorbance by greater than fifty percent in the sample exposed to the unlabeled sheep antiserum to HLTV-III, relative to a standard normal sheep serum, is considered a positive confirmatory result for the presence of antibody to HLTV-III. Saxinger and Gallo (UNITED STATES OF AMERICA represented by the United States Department of Commerce (177.4) obtained an additional improvement by adding guanidine salts as GuHCL in a molarity of 4–8 molar. The test sensitivity is 3–10 times more than the current commercial screening tests. In the table below a comparison of Sensitivities of HLTV-III Antibody Detection by Indirect ELISA, competitive ELISA and Western Blot Procedure is presented. For detecting an antibody to human immunodefiency virus (HIV) Ikeda and Mizukoshi (FUJIREBIO KABUSHIKI KAISHA (60.4)) pro pose a method which is based upon the use of a passive particle agglutination comprises carrier particles sensitized with HIV antigenic component.

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TABLE 2 Comparison of Sensitivities of HTLV-III Antibody Detection by Indirect ELISA, competitive ELISA and Western Blot Procedure

Preferably gelatin particles are used because they show little nonspecific reaction, and it is easily modified to add properties necessary for the use of the particles. The sensitization of the antigenic component of HIV may be carried out by known methods for sensitizing an antigen on carrier particles. It is preferably carried out under slightly acidic conditions such as pH 5 to 7. The sensitized carrier particles are usually lyophilized, and supplied with a reconstituting solution, and a diluent. To optimize conditions for agglutination the pH of the diluent is preferably 7 to 9. The measurement of the antibody to an antibody to HIV may be carried out by following the conventional method of utilizing indirect passive hemagglutination.

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Cosand et al. (GENETIC SYSTEMS CORPORATION (63.2)) disclose DNA sequences comprising a portion of the envelope (env) region of the LAV genome, coding for a protein which is immunologically reactive with antibodies to LAV. A recombinant plasmid capable of replication in bacterial host cells is also disclosed. The amino acid sequence of an (env) encoded protein is stated. Montagnier et al. (INSTITUT PASTEUR and CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (129.5)) elucidated two glycoproteins encoded by the env gene of the AIDS-virus. It concerns in particular the glycoproteins, gp150 and gp135, having a molecular weight in the range of 150–135 kd. More particularly, they resemble the already known glycoprotein gp110 in that they can be detected by labeling the virus with a high concentration of 35S-cysteine. The novel glycoproteins are capable of forming complexes with concanavaline A, which then can ce dissociated in the presence of Omethyl-alpha-D-mannopyranoside. They are also capable of forming complexes with “Lentyl-Lectines”. Montagnier et al. (INSTITUT PASTEUR and THE UNITED STATES OF AMERICA as represented by the Department of Health and Human Services (179.3 and 129.3)) isolated retroviruses associated with Acquired Immune Deficiency Syndrome (AIDS), including Lymphadenopathy Associated Virus (LAV), from the sera of patients afflicted with Lymphadenopathy Syndrome (LAS) or AIDS. LAV is a Human Immunodeficiency Virus (HIV). Viral extract, structural proteins and other fractions of the retrovirus immunologically recognize the sera of such patients. Immunological reaction is used to detect antibodies that specifically bind to antigenic sites of the retrovirus in samples of body fluids from patients with AIDS or risk of AIDS. Cells inoculated with LAVl developed after 15 days reverse transcriptase activity. No such activity was present in the control. That the new virus isolate was a retrovirus was further indicated by its density in the above sucrose gradient, which was 1–16, and by its labelling with [3H] uridine (see figure). A fast sedimenting RNA appears to be associated with the LAVl virus. Berman et al. (AKZO N.V. (3.4)) describe three principal genes of the HLTVIII virus. I. gag Gene Products Positioned at the 5′ end of the genome, the gag open reading frame sequence encodes a 55K Mr—(molecular mass) precursor protein. This precursor is processed into three mature proteins. Amino acids 1 through 132 correspond to a 17K virion protein and is probably involved in morphogenesis. The next mature protein is the major capsid structural protein, p24. The remainder of the gas poly-protein (p15), a highly basic protein, is probably involved in nucleic acid binding.

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II. pol Gene Products The predicted amino acid sequence of the second long open reading frame from HLTV-III shows homology and similarity with other pol genes of other retroviruses. This 3044 nucleotides long open reading frame has the potential to code for a 1015 amino acid precursor protein for the reverse transcriptase present in HLTV-III virion. In addition to the 5′ reverse transcriptase information, there exists 3′ genetic information probably involved in endonuclease and integrase functions necessary for viral replication. III. env Gene Products The env-lor region of HLTV-III encodes a precursor protein about 863 amino acids long. This primary gene product, Mr 160K, can be detected in infected cells. This precursor is cleaved into two products, a hydrophilic exterior protein (gp120) that is glycosylated and a hydrophobic transmembrane segment. The virion gp41 corresponds to the transmembrane protein. There is a high degree of conservation of amino acid sequence at the transmembrane protein processing site. The regions outside of this are less well conserved and show considerable difference at the nucleotide and amino acid sequence level when different viral isolates are compared. Chang and Ghrayeb (CENTOCOR (32.4)) developed a diagnosing assay for HLTV-III based on three recombinant HLTV-III core proteins expressed by cloned DNA segments of the gag region of the HLTV-III genome. Immunoreactive, chimeric HLTV-III core proteins and methods of producing these proteins are also described. Crowl et al. (HOFFMANN-LA ROCHE & CO. (74.2)) identified recombinant envelope proteins of HLTV-III. A composition using the homogeneous envelope AIDS protein overcomes the nonspecificity of the prior tests or assays. Yet another aspect of this invention is a diagnostic method for detecting and/or determining the presence of the antigen in human blood. The amino acid sequence of the AIDS envelope protein is disclosed, as well as a simplified restriction site map of the 3.15 Kb EcoRI-Xhol segment of the HLTV-III genome which contains the env coding region (cross-hatched arrow). Montagnier et al. (INSTITUT PASTEUR (129.7)) describe a retrovirus capable to provoke a disease with the symptoms of AIDS, designated by HIV-2, of which samples have been deposited at ECACC under the numbers 87.01.1001 and 87. 01.1002 and at NCIB under the numbers 12.398 and 12.399. The virus multiplies itself in human lymfocytes called LAV-type II. In vitro the virus has a tropism for human T4 lymfocytes. Core proteins of UTLV had molecular weights of respectively: 12, 16, and 26 kd (p12, p16, and p26).

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Membrane glycoproteins had molecular weights of 36, 42–45, 130–140 kd (gp36, gp42 and gp140). Immunologically the glycoproteins of HLTV-II are close related to those of HLTV-III. These different antigens are applicable to the diagnosis of the disease, particularly by contact with a serum of the patient on whom the diagnosis is to be carried out. Also related immunogenic compositions containing more particularly the glycoprotein sp140 are disclosed. Nucleo-tidic sequences useable particularly as hybridization probes derived from the RNA of HIV-2 are described. Chang et al. (CHANG ET AL. (35)) provide an immunochemical assay for detecting antibodies against HLTV-III employing a HLTV-III antigenic polypeptide produced by recombinant DNA methodology. The HLTV-III antigen, designated HLTV-III polypeptide 121, contains 83 amino acids and is probably derived from a viral envelope protein. Immunochemical assays utilizing the peptide are highly sensitive, specific and reproducible. The assays are an alternative to assays based on the whole virus which are equivalent, if not superior, in performance. The assays can be used to screen blood or other bodily fluids for presence of HLTV-III and to diagnose AIDS. Gene segments from the env-lor regio of the HLTV-III genome, were expressed in E.coli as peptides. HLTV-III polypeptide 121 is strongly reactive with AIDS patient sera. The peptide produced and purified as a fusion protein on a large scale. Solid phase immunoassays employing this recombinant peptide as an immunoabsorbent can reliably and reproducibly detect antibodies in sera of patients with HLTV-III infection. In two representative serum panels, the assay detected the presence of antibodies in 120 of 121 sera from patients with AIDS or AIDS-related complex (ARC), and only in 1 of 92 normal controls. Based upon HLTV-III polypeptide 121 as immunoreactive agent, sensitive and specific immunoassays for HLTV-III infection have been developed. A comparison between solid phase immunoabsorbents using recombinant HLTV-III antigen, polypeptide 121, and inactivated, disrupted HLTV-III as antigen. The research of Kennedy (SOUTHWEST FOUNDATION FOR BIOMEDICAL RESEARCH (157)) is based upon the assumption that the critical epitopes involved in the induction of protective virus neutralizing antibody are associated with the two viral envelope glycoprotein subunits, gp 120 and gp 41. They developed a synthetic peptide, the amino acid sequence of which is sufficiently homologous to the amino acid sequence of the gp 41 and gp 120 subunits of the gp 160 envelope glycoprotein of HLTV-III, LAV or ARC to produce an immunogenic response in a patient and which has a hydrophilic region therein. It may be used in diagnostic immunoassays for AIDS and ARC. Thorn et al. (CAMBRIDGE BIOSCIENCE CORPORATION (29)) identified specific peptide fragments or constructs thereof which are particularly

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immunoreactive to anti-HLTV-III antibodies. By modifying these diagnostic peptides, the degree of immunoreactivity is signifantly increased. Then high levels of expression of these diagnostic peptides by genetic engineering methods were achieved. Also diagnostic assays for the detection of antibodies to HLTV-III in samples suspected of containing antibodies against the HLTV-III virus have been developed. Cosand (GENETIC SYSTEMS CORPORATION (63.4)) pertains the discovery of novel peptides mimicking proteins encoded in gag and env regions of the HLTV-III virus. The polypeptides can be used as reagents in the determination of exposure of a human host to the virus. Of particular interest is the use of polypeptides in screening blood products. Wang et al. (UNITED BIOMEDICAL INC. (176)) propose the use of a peptide with an amino acid sequence, corresponding to a segment of the envelope protein, p41, for identification of HLTV-III. The use of a synthetic analogue for HLTV-III detection is suggested in the form of an ELISA-method. Pert et al. (THE UNITED STATES OF AMERICA represented by The Secretary The United States Department of Commerce (177.3)) developed short peptides of the formula:

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Ra-Ser-Thr-Thr-Thr-Asn-Tyr-Rb (I) where Ra represents an amino terminal residue Ala—or D-Ala and Rb represents a carboxy terminal residue—Thr or—Thr amide or a derivative thereof with an additional Cys—residue at one or both of the amino and carboxy terminals, or a peptide of formula (II): R1-R2-R3-R4-R5 (II) where R1 is an amino terminal residue Thr-, Ser-, Asn-, Leu-, Ile-, Arg-, or Glu R2 is Thr, Ser or Asp R3 is Thr, Ser, Asn, Arg, Gln, Lys or Trp R4 is Tyr and R5 is a carboxy terminal amino group or a derivative thereof with a corresponding D—amino acid as the amino terminal residue, and/or a corresponding amide derivative at the carboxy terminal residue and/or additionally a Cys-residue at one or both of the amino and carboxy terminals, or a physiologically acceptable salt thereof. Such peptides bind to T4 receptors are useful in preventing viral infectivity by viruses which bind to the T4 receptors. These peptides are believed to act as competitive blocking agents. The figure below displays the inhibition of HLTV-III virus infectivity by several peptides. Essex et al. (PRESIDENT AND FELLOWS OF HARVARD COLLEGE (67)) detected in HTLV-III infected cells a protein with a molecular weight of 27 kd, coded for by ORF position 3′ to the env-gene of HLTV-III. They also developed a scheme of methods to use which according to them, is to be preferred in testing for the various HLTV-III antibodies by the methods indicated in the following table: Bedarida (TECHNOGENETICS S.P.A. (166)) pertains to have developed a HLTV-III diagnosing method with a relatively high specificity for IgM antibodies. The novelty of the procedure is the hot elution of the antigens of the antibodies caught by means of the same relevant antigens, and the subsequent fixation of the purified antibodies to a second layer of antigens, before the addition of the respective conjugate. Dawson (ABBOTT LABORATORIES (1.13)) uses for the detection of antibodies to HTLV-III in biological samples labeled, recombinant p24, and p41 protein expressed in E. coli. The proteins are bound to a solid support. Rosen et al. (ORTHO PHARMACEUTICAL CORPORATION (123.2)) describe synthetic peptides useful for detection of antibodies to HLTV-III virus. Also their diagnostic and therapeutic compositions and methods of use are described.

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*Abbreviations: CIF, cytoplasmic immunofluorescence; CV, concentrated virus; E, ELISA; IC, infected cell; ICV, immunoprecipitation with concentrated virus; IPIC, immunoprecipitation with infected cell homogenate; WBIC, Western Blotting with infected cell homogenate; WBCV, Western blotting with concrated virus.

Burger and Goldstein (EPITOPE, INC. (56)) claim to have developed a novel method for detecting HLTV-III virus. The antigen may be bound to a lymphocyte, or to a macrophage, and is selected from the group of antigens characterized as molecules of approximately 24, 28, 39, 41, 55, 65, 49, and 52 kilodaltons, respectively. Folks et al. (UNITED STATES OF AMERICA, represented by The United States Department of Commerce (177.5)) discovered that Leu-3-cells surviving infection with the AIDS retrovirus can be induced with IUdR to express infectious virus. A cellular clone (8E5), isolated by limiting dilution of a mass culture of survivor cells, was found to contain a single, integrated, defective provirus that was constitutively expressed. Treatment with IUdR (100 µg/ml) for 24 hours of 8E5 cells failed to induce infectious virus, cocultivation with Leu-3+ cells generated the characteristic syncytia associated with acute AIDS retrovirus infection. The single integrated copy of proviral DNA directs the synthesis of all major viral structural proteins except p64 and p34 as monitored by immunoblotting.

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Gallatin and Clark (FRED HUTCHINSON CANCER RESEARCH CENTER (58.2)) identified in CD4+ T cells two distinct populations by using fluorescence: Hermes-lhi and Hermes-llo subpopulations. The Hermes-lhi are preferentially lost in the course of HlV-infection. The ratio between the first and second populations of cells is determined and compared to a control or patient-specific standard to provide an indication of the onset and course of acquired immunodeficiency syndrome in the patient. The figure below is a set of three histograms of one-color flow cytometric analyses of the distribution of Hermes-1 on normal macaque lymphocytes in blood (PBL), thoracic duct lymph (TDL), and mesenteric lymph node (MNL), showing that Hermes-lhi cells predominate over Hermes-llo cells in blood but that the reverse relationship occurs in lymph. Pockl and Haushofer (WALDHEIM PHARMAZEUTIKA GESELLSCHAFT m.b.H. (200)) developed an assay for HLTV-III antibodies. The sample to be tested is contacted with HLTV-III antigens. These are produced by T-cells, transformed with HLTV-III, and are fixed to a solid support. All antibodies which did not react with the HLTV-III antigens are removed. The measurements are according a standard fluorescence immunoassay. In certain conditions, leukemic T cells express large numbers of IL-2 receptors constitutively. This may happen in HLTV-I associated Adult T cell Leukemia, malignant B lymphocytes of hairy cell leukemia and allografts. Waldmann (THE UNITED STATES OF AMERICA as represented by The Secretary, U.S. Department of Commerce (177.2)) provides a method of eliminating disease-associated Tac-positive cells. Anti-Tac monoclonal antibodies are produced by using hybridoma technology. The monoclonal antibodies are conjugated to ricin A chain using a thiol-containing crosslinker, N-succinimidyl-3-(2-pyridyld-ithio) propionate. Conjugates are adjusted to 1 mg/ml with reduced and alkylated human IgG and stored at−20°C. The addition of carrier protein assures stability of the

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conjugates, and the alkylation prevents disulfide toxin exchange between specific antibody and carrier protein. The addition of anti-Tac antibody coupled to the A chain of the toxin (ricin) effectively inhibited protein synthesis and led to cell death of an HLTVI-associated, Tac-positive ATL cell line, HUT102-B2. The inhibitory action of anti-Tac conjugated with ricin A could be abolished by the addition of excess unlabeled anti-Tac or IL-2. The use of other cytotoreic conjugates is possible. The results of anti-Tac therapy of a patient with adult T cell Leukemia is depicted below. The patient was treated with form infusions (solidbars). Jacob and Williams (MUREX MEDICAL RESEARCH LIMITED (114)) envisage for the testing for antibodies to the AIDS related viruses in a liquid sample, a filter comprising a zone of a filter material covered with antigens to said virus. After passage of the specimen, and washing as necessary, the products can then be visualised or otherwise tested in conventional manner. Polypeptide antibodies BANNWARTH et al (74.3) found polypeptides eliciting antibodies against AIDS virus, they say, and therefor, are useful in the diagnosis and thereby of AIDS. The polypeptide involved has an amino acid sequence represented by the following formula

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MRGSEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLI CTTA VPWNASWSNKLLEQIWNNMTWMEWDREINNYTGSVDLQPSLDSC (ENV(80))I as well as fragments and fusion polypeptides thereof eliciting the antibodies. The next figure schematically shows the progress of the BANNWARTH et al. method. 2.2.2 GONORRHOEA/SYPHILIS Guinet and Perrollet (INSTITUT PASTEUR DE LYON ET DU SUD EST (130)) prepared lectine bonding factors of Neisseria gonorrhoeae, the proposed method including the preparation, from a Neisseria gonorrhoeae strain, a parietal antigen extract, followed by solubilisation, the obtained extract being set into contact with a saccharated, immobilised receptor, the said lectine factors, fixed on the receptor, being finally separated therefrom. The proposed antigens (lectine factors) can be used in diagnosing gonorrhoeea, and as component of a preventive farmaceutical drug. Two main categories of serologic tests for syphilis are available: tests for reaginic antibody and tests for treponemal antibody. Reaginic tests use

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cardiolipin as antigen, they are sensitive, but lack a high degree of specificity. The treponemal tests use treponemal antigens and, because they involve a demanding procedure, and used principally as confirmatory tests. Yabusaki (ADVANCED POLYMER SYSTEMS, INC. (2)) developed a test for syphilis-associated antibodies, which should overcome the drawbacks of prior art methods. It comprises a stable aqueous suspension of cardiolipin antigen ionically coupled to latex particles via a polypeptide bridge. This reagent is used in a flocculation or agglutination type assay that includes the following steps: (1) incubating a test sample suspected of containing syphilis-associated antibodies with the antigen reagent under conditions that permit reaction between any such antibody in the sample and the cardiolipin antigen component of the reagent, and (2) determining whether an agglomerate or flocculant has formed. 2.2.3 HERPES Immunofluorescence is routinely employed in testing human serum for the presence of anti-treponemal antibodies associated with syphilis. However it does not allow for quick screening of a number of serum samples since each sample must be individually studied under a fluorescent microscope. Coates and Binder (AMERICAN HOECHST CORPORATION (7)) developed a single assay method that can be used to screen quickly test samples for the presence of anti-treponemal antibody and then confirm the presence of detected anti-treponemal antibody. The unique feature of the method of the present invention resides in tagging the complex of anti-treponemal antibody and specific antigen with antihuman immunoglobin which has been tagged with both an enzyme label and a fluorescent label. It is this dual labeling that enables the assay method to be used for both detection and confirmation of the presence of antitreponemal antibodies. Fluorochrome conjugated antisera are well known in the art. Enzymes that are particularly preferred as labeling agents include, for example, horeseradish peroxidase, alkaline phosphatase, glucose oxidase, lactoperoxidase and βgalactosidase. Assays for Cytomegalovirus (CMV) are reliable in model experimental systems, but not equally reliable on clinical samples. In the latter, probably, molecules of beta2microglobulin (beta2m) become associated with the outer envelope of CMV and mask the antigenic determinants that are present on the surface of CMV. Griffiths et al. (THE ROYAL FREE HOSPITAL SCHOOL OF MEDICINE (143)) developed two methods avoiding the errors introduced in prior art methods by the inhibitory action of the beta2m.

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The first approach is based upon the removal of beta2m by an enzyme contained in urine which is activated at a pH=5·5 at 35° to 40°C. Thereafter consistent results are obtainable equal to model experiments. Secondly, a sample to be assayed for virus with a reagent that reacts with beta2m and which will form an anti-beta2m/beta2m/virus conjugate and subsequently assaying for virus. Presently known assays for the detection of antibodies to varicellazoster virus (VZV) consist of methods for adhering virally infected cells or lysates of virally infected cells to a solid support, e.g., fluorescent antibody to membrane antigen, (FAMA), immune adherence hemagglutination (IAHA), enzyme-linked immunosorbent assay (ELISA), followed by contacting these with the biological fluid and then a second antibody conjugated to an enzyme or a fluorescent probe. Neff et al. (MERCK & CO. INC. (107.2)) provide an improved assay capable of detecting subgroups of antibodies specific to purified gA, gB and gC VZV glycoproteins. Another object is to provide a preparative method and diagnostic use for these purified glycoproteins and their controls as detecting antigens. Antibodies in mammalian biological fluids to each of the three major VZV glycoprotein groups, gA, gB and gC, can be detected, for example, by isolating each viral glycoprotein from VZV-infected cells in vitro, adhering these to a plastic surface and contacting with a biological fluid containing the suspected antibodies. Subsequent treatment with a conjugate consisting of, for example, an antibody-enzyme complex (with specificity for either one, two or all three glycoprotein groups), followed by contact with an enzyme substrate and subsequent determination of the quantity of reacted enzyme substrate, i.e., the ELISA assay, provides a relative measure of the antibody(ies) present directed against VZV glycoproteins. Gallo et al. (THE UNITED STATES OF AMERICA as represented by the Secretary, U.S. Department of Commerce (177.6)) describe a new DNA virus, designated Human B Lymphotropic Virus (HBLV), isolated from the blood leukocytes of patients with lymphoproliferative disorders. The virus belongs morphologically to the Herpes family of viruses. HBLV is associated with some malignancies in AIDS and nonAIDS patients, but is distinctly different than human T-cell Lymphotropic Virus Type III (HLTV-III). A comparison of HBLV and Herpes Simplex Virus morphology is depicted in the table below: 2.3 HEPATITIS Prior art methods for detecting hepatitus B core antigen (HBcAg) involved the use of immune adherence hemagglutination assay (IAHA) or cemplement fixation (CF) assay. A disadvantage of such assays is that the end-point

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Comparison of HBLV and Herpes Simplex Virus Morphology

determination requires interpretation of observed results and therefore is necessarily subjective to some degree. These assays are not sufficiently sensitive for routine use to detect the presence of HBcAg in biological fluids, expecially sera or plasma, in order to prevent transmission of hepatitis B disease by transfusion and also to diagnose the presence of this disease in a person. McAleer and Miller (MERCK & CO., INC. (107.1)) developed an assay for HBcAg. Its applicability is restricted to plasma with an IAHA titer of hepatitis B core antibody (HBcAb) of preferably 1:20–000, or higher. HBcAb in a biological fluid is adsorbed on a surface which is then coated with BSA. The coated surface is then incubated first in the sample and then in the presence of radiolabelled HBcAb. Several synthetic polypeptides have been described purporting a protective efficacy against hepatitis B virus. Sparrow et al. (BAYLOR COLLEGE OF MEDICINE TEXAS MEDICAL CENTER (14)) provided a cyclic polypeptide having a disulfide bond in a hydrophilic region of the peptide. Also provided is a composition for eliciting production of antibody to hepatitis B surface antigen and method of neutralizing the infectivity of hepatitis type B virus. A cyclic polypeptide is prepared having a disulfide bond in a hydrophilic region, namely residues 117–137 or 122–137 and an amino acid sequence unique from that associated with the native 25000 molecular weight polypeptide derived from hepatitis B surface antigen. In the figure below the results of a competitive binding assay of an anti-a monoclonal antibody (A-12) with purified HBsAg (µ) and with the cyclic peptide 1, (o). (Micro wells coated with HRsAg/ade were incubated with a mixture of antibody and the respective antigen. The residual antibody activity bound to the HBsAg attached to the micro wells was detected by the addition of a 125I-labeled goat anti-mouse IgG reagent. Martin and Kung (COOPER-LIPOTECH, INC (43.1)) claim to have improved a red blood cell agglutination test for the determination of hepatitis B surface antigens.

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Analyte is added to agglutinatable particles coated with antianalyte molecules to produce particle agglutination. The extent of agglutination is enhanced by mixing the particles and analyte with an analyte-binding reagent composed of lipid bodies. The reagent bodies act by promoting multiple analyte bridge connections between individual bridged particles and a reagent body. As analyte lipid vesicles are used. Dyes or fluorescent molecules may be attached to the vesicle surfaces. The lipids may include a sterol, with glycolipid or phosphohrid. Halbert and Anken (DIAMEDIX CORPORATION (51)) pertains to have developed a sensitive direct immunoassay system for the detection of hepatitis B antigens in body fluids. The antibodies which react with an antigen or antigens and which are bonded to an insoluble member, are incubated with a test sample. During this first period of incubation a portion of an antigen present in the test sample will combine with the antibody immobilized on the insoluble member. The antibody bonded member, to which antigen is attached, is then washed and incubated with an enzyme tagged antibody reagent. During the second incubation, the tagged antibody reacts with antigen fixed to the antibody member in the first incubation. Thus, an immobilized “sandwich” is formed of an insoluble member—antibodyantigen-enzyme tagged antibody. After the second incubation, the member is washed again to remove unreacted enzyme antibody reagent. The member is then exposed to a substrate which is converted by the enzyme to produce an end product. The tagged antibody reagent will be fixed in the second incubation only if antigen was present in the sample. The amount of enzyme tagged antibody fixed is proportional to the amount of antigen or antigens present in the test sample

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up to the maximum capacity of the test. The concentration of the end product is determined by a spectrophotometer. Persons with high risk of contracting HBV infection are increasingly vaccinated against HBV infection. Vaccines against HBV, however, are very expensive. Prior to vaccination a prescreening of immunoglobulins giving protection against HBV infection is generally carried out and persons already having immunity against HBV are selected. However, persons selected to be immune, occasionally may not possess full protection against subsequent HBV infections. Kuypers and Matthyssen (AKZO, N.V. (3.3)) claim to have developed a novel method for the determination of protecting anti-HBV immunoglobulins is based on the use of two different HBsAg reagents: one insolubilized and having the antigenic type AX and one labelled and having the antigenic type AY, wherein A represents the epitope or epitopes which are common to all HBsAg serotypes, wherein X and Y both represent combinations of the epitopes which are not common to all HBsAg serotypes, and where the combinations X and Y have no epitopes in common. Kuypers and Wolters (AKZO N.V. (3.2)) developed a determination method for the HBcAg. A sample is incubated with carrier-bound immunoglobulins directed against HBsAg, the carrier-bound and liquid phases are separated from one another, the carrier-bound phase is incubated with an agent which breaks the HBsAg coat and releases HBcAg and, simultaneously or subsequently, the solution obtained is incubated with immunoglobulins directed against HBcAg, after which any binding of HBcAg with immunoglobulins directed against it is determined, giving a qualitative and/or quantitative measure of HBcAg in the sample. The possible binding of HBcAg to immunoglobulins directed against it can be detected by any conventional immunoassay. Suitable agents for breaking the coat of HBsAg are detergens like certain sodium dodecyl compounds, and polyoxyethylene derivatives. Nonionic detergents are preferred.

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The effectivity of the described is illustrated in the table below. Related to its significance, screening methods for HBV need continuously to be improved in sensitivity, and costefficiency. Prior art methods for achieving the required sensitivity—the detection threshold must be below 1 nanogram per milliliter of serum—are all of the radioimmunological type. They require specialistic handling, and are costly. Duheille et al. (SANOFI (145)) developed a nephelometric assay. When applied to the quantification of antigen/antibody reactions, it is then called immunonephelometry. It has been employed mainly for the specific determination of proteins in serum by measuring the intensity of the light scattered by the insoluble immune complexes formed between each of these proteins and a corresponding immunoserum. Pursuing their studies on the formation of the nephelometric signal as a function of the size, number and characteristics of the dispersing particles, the great advantage of new microparticulate supports, which behave as indicators and amplifiers of the nephelometric signal were discovered. It concerns polyfunctional hydrophilic spherical microparticles of uniform size which contain from 5 to 95% of units originating from the copolymerization of an aldehyde of the formula

R1 being an alkyl radical, from 5 to 90% of units originating from the copolymerization of an acrylic acid ester of the formula

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in which R2 is H or alkyl and R3 is a hydroxyalkyl group, less than 15% of units originating from an acrylic acid derivative chosen from amongst

R4, R5 and R6 being alkyl radicals, and from 0·1 to 10% of units originating from a crosslinking product. With this method levels of antigen HBsa concentration in a range extending from about 0·15 ng/ml up to almost 80 ng/ml were detected. See the figure below, in which the scattered light intensity has been plotted on the ordinate (in arbitrary units) and the antigen HBsa concentration has been plotted on the abscissa in nanograms per milliliter.

HC-virus Research by Houghton et al. (CHIRON CORPORATION (36A)) provides a family of cDNA sequences derived from hepatitis C virus (HCV). These sequences encode antigens which react immunologically with antibodies present in individuals with non-A non-B hepatitis (NANBH), but which generally are absent from individuals infected with hepatitis A virus (HAV) or hepatitis B virus (HBV), and also are absent from control individuals. A comparison of these cDNA sequences with the sequences in Genebank, and with the sequences of hepatitis delta virus (HDV) and HBV shows a lack of substantial homology. A comparison of the sequences of amino acids encoded in the cDNA with the sequences of Flaviviruses indicates that HCV is a Flavivirus or Flavi-like virus. The HCV cDNA sequences are useful for the design of polynucleotide probes, and for the synthesis of polypeptides which may be used in immunoassays. Both the polynucleotide probes and the polypeptides may be useful for the diagnosis of HCV-induced NANBH, and for screening blood bank specimens and donors

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for HCV infection. In addition, these cDNA sequences may be useful for the synthesis of immunogenic polypeptides which may be used in vaccines for the treatment, prophylactic and/or therapeutic, of HCV infection. Polypeptides encoded within the cDNA sequences may also be used to raise antibodies against HCV antigens, and for the purification of antibodies directed against HCV antigens. These antibodies may be useful in immunoassays for delecting HCV antigens associated with NANBH in individuals, and in blood bank donations. Moreover, these antibodies may be used for treatment of NANBH in individuals. The reagents provided in the invention also enable the isolation of NANBH agent(s), and the propagation of these agent(s) in tissue culture systems. Moreover, they provide reagents which are useful for screening for antiviral agents for HCV, particularly in tissue culture or animal model systems. It is known that the hepatitis virus “non-A-non-B” cause grave cases of liver cirrhosis, hepatocellular carcinoma, etc. and consequently the identification of Flavi virus and the detection of an antigen-antibody system, related therewith are of great importance. The reagents and diagnostic methods developed up till now essentially concern the detection of antigens, related to a “non-A-non-B” hepatitis virus, transmitted during blood transfusions. Pillot (INSTITUT PASTEUR (129.6)) discloses an anti-NANBH (anti-hepatitis non-A-non-B) antibody, composed of IgM’s, isolated from the serum of monkeys, artificially infected by feces extracts of patients, suffering from epidemic hepatitis “NANB”. IgG-fractions are marked with β-galactosidase, and the preferred substrate is orthonitrophenyl-β-D-galactopyranoside. Also enzymatically marked IgG’s are provided to be used together with IgM anti NANB for the detection of epidemical hepatitis virus NANB. The IgG are derived from sera of patients convalescing from epidemical hepatitis virus NANB. These IgG are isolated on a DEAETrisacryl column. Reagents for the detection of epidemical hepatitis virus NANB containing IgM’s, fixed on a solid support. This reagent is applicated in a diagnosis method for epidemical hepatitis virus NANB. HD-virus The hepatitis D virus is thought to be associated with, but not part of the hepatitis B virus. It is hypothesized that the delta agent is dependent on hepatitis B virus for replication. Liver derived delta antigen is in short supply and, therefore, few laboratories have access to suitable reagents for monitoring delta infection. A new source of delta antigen as a test reagent is, therefore, urgently required.

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Shattock and Morgan (SHATTOCK AND MORGAN (152)) derive hepatitis D antigens from serum of human beings, chimpansees, or of squirrel (sciuridae) family. The serum is treated with condensates of ethylene oxide with fatty alcohols, or with an antibody-antigen complex dissociating agent as potassium thiocyanate. The dissociating agent is preferably used simultaneously with the surfactant. The blood derived delta antigen is used as a diagnostic agent in the detection and determination of different classes of antibodies to hepatitis D virus (delta agent) by ELISA and/or RIA. 2.4 DIABETES In 1974 the relationship between diabetes and the major histocompatibility complex in man, called HLA was discovered. Most associations between HLA and disease susceptibility have involved the D region of HLA. Human la antigens map to the HLA-D region. These appear to be encoded by at least three distinct loci, DR, DQ and DP, each with its distinct alpha and beta chains. Each HLA-DR alloantigenic specificity (DRl-wl4) represents a sero-logically defined reaction pattern between well-characterized antisera and major histocompatibility complex (MHC) class II molecules. Each specificity corresponds to a particular epitope recognized by an alloantiserum which is present on HLA-D-encoded cell surface molecules. If specific haplotypes account for HLA associations with disease, then the serologic HLA specificity alone is not a sufficently specific marker to optimally predict disease risk. Restriction endonuclease digestion of genomic DNA may be used to identify specific nucleotide sequences, the presence or absence of which can function as markers for individual genes or gene segments. Fragments which hybridize to a single probe can sometimes be assigned to specific genes. When such a fragment is variably expressed in different individuals, it is referred to as “polymorphic” and can potentially relate to functional differences. Investigators have previously reported a large number of restriction endonuclease fragment polymorphisms (RFLP) which exhibit some correlation with diabetes, hybridizing to DQB probes. (Cohen-Haguenauer et al, PNAS 82:3335–3339, 1985; Owerbach et al., Nature 303:815–817, 1983.) Heretofore, noticeably absent from the art has been the clinical ability to identify individuals who are at increased risk of diabetes prior to the onset of symptoms. The ability to identify individuals at increased risk of diabetes could have several important applications.

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Research by Nepom et al. (GENETIC SYSTEMS CORPORATION (63.3)) discloses methods for identifying individuals at increased risk of diabetes. Essentially it involves the steps of (a) incubating a first mono clonal antibody (MAb) reactive with DQw3·2 and DQw3·1 with a portion of a cell collection; (b) incubating a second monoclonal antibody reactive withDQw3·1with a separate portion of the cell collection; (c) detecting the presence of immunocomplexes formed between the first MAb and the cells, and the second MAb and the cells; and (d) determining from the results of the step of detection whether haplotypes associated with susceptibility to diabetes, such as the DQw3·2 haplotype, are present. The first and second monoclonal antibodies may be labeled. In a preferred embodiment of this method, the first MAb is P100·1 and the second MAb is A10 or GS 159·2, positive reactivity with P100·1 and negative reactivity with A10 or GS 159·2 indicated the presence of a haplotype associated with susceptibility to diabetes. Diabetic control necessitates the monitoring of the levels of glucosylated H6 (primarily HbAlc). Prior art methods have many drawbacks. Dean (MILES LABORATORIES, INC. (109.3)) claims to have overcome these by developing an immunoassay method and reagent system for determining nonenzymatically glucosylated proteins and protein fragments in a biological fluid based on the specific binding of such proteins and fragments with anti (Amadori-rearranged glucose) e.g. “Amadori-rearranged glucose” refers to the 1amino-1-deoxy-D-fructosyl residue, and its conformers, which is the rearranged form taken by glucose upon reaction with amino groups in proteins. Such residue has the formula:

As used herein is intended to mean antibodies, and fragments and aggregates thereof which selectively bind to the Amadori-rearranged glucose residue, formula (A), irrespective of the structure of the haptenic residues on the immunogen used to stimulate production of such antibodies. The antibodies are raised against an immunogen comprising an immunogenic carrier material bearing 1-deoxy-l-fructosyl residues or conformers of such residues. Measurement of nonenzymatically glucosylated proteins and fragments thereof provides a useful index of blood glucose levels. Heparansulphat-Proteoglycane occurs in basal membranes and acts therein as an ion-selective filter. In pathological situations, like Diabetes mellitus a decrease of this proteoglycane in the basal membrane could be observed. Such decrease is assumed to be related to diabetic complications, like nephropathy. The verifications of this decrease are, as a rule, based on tissue extraction, but it

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would be advantageous, to determine such change through a serum analysis as well. Timpl et al. (MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN E.V. (104)) found that by their method it is possible to isolate heparansulphate-proteoglycane of low density from basal membrane containing tissue. By the application of this antigen and the corresponding high-specific antibody an immunologic determination of high sensitivity could be established. The proposed method permits the determination of small amounts (ng/ml) of heparansulphate-proteoglycane of low density in body fluids (blood serum). Measurements of the blood levels of neg-proteins of diabetic patients have been shown to correlate well with physicians’ ratings of monitoring tests. Glycosylated hemoglobin Alc is the most abundant of a group of four glycosylated derivatives designated collectively as HbA1. Measurements of HbA1 therefore reflect the average blood glucose values of the preceding weeks. Measurements of plasma neg-albumin levels may consequently reflect a more rapid assessment of patient compliance. These parameters are a clinically valuable adjunct to urinary and blood glucose determinations, especially when patient compliance is poor, or when strict diabetic control is essential. Many of the tests to determine negprotein samples in blood are specifically designed for the analysis of HbAlc. In addition they are tedious sensitive, require highly specialized instruments, laborintensive, etc. There is therefore a need for simple, accurate and precise routine assay that is generally applicable for the measurement of neg-forms of hemoglobin and albumin or any other clinically important protein. Hammond (FARMOS-YHTYMÄ OY (57)) developed a method based upon an immunoassay of neg-protein analyte comprises contacting a sample containing said analyte with a phenyl-boronic acid, bound to a solid phase, contacting said solid phase with a labelled antibody to the said analyte, separating the solid phase with label bound thereto and then measuring the bound or the unbound label. The new method may be used to determine the relative amounts of proteins, e.g. hemoglobin and albumin, that are present in blood samples in a nonenzymatically glycosylated (neg) form (a parameter which has been advocated as a convenient indicator for the diagnosis and management of diabetes mellitus. From standard curves as the on in the figure below the concentrations can be extrapolated. The validity of the measurement in biological samples of amylases, in diagnosing pancreatic disease is limited, because of those amylases produced by e.g. salivary glands. (With amylase is human alpha amylase indicated.) Bruns and Benjamin (THE UNIVERSITY OF VIRGINIA ALUMNI PATENTS FOUNDATION (193)) developed a method to measure pancreatic amylases in the presence of salivary, and other related, amylases.

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It was achieved by providing a monoclonal antibody reactive with human salivary but not pancreatic amylase. The hybridoma is the fusion product of spleen donor cells from A/J mice, H-2a haplotype, immunized with the purified human amylase, with SP2/0 myeloma cells H-2d haplotype. The antibody is attached to it an insoluble matrix such as latex or an immunochemical stain. First the total amylase concentration is measured. A potion of the sample is reacted with the bound antibody. After centrifuging, the pancreatic amylase concentration is measured in the supernatant. Subtracting the last concentration determination of pancreatic amylase from the first concentration determination of total amylase results in the determination of the concentration of salivarytype amylase.

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The figure below depicts the antibody reacting with the salivary type amylase but not the pancreatic type amylase. In solid phase immunoassays usually it is the antibody which is bound to a solid support. Krauth (KRAUTH (93)) developed an immunoassay wherein supported hapten or antigen is used to separate “bound” and “free” fractions. An increase in sensitivity is claimed. The method may be employed in assays for a wide variety of antigens and haptens. Although the present invention is applicable to an assay which does not provide for regeneration of the supported antigen or hapten, the invention has particular applicability to an assay wherein the supported antigen or hapten is regenerated, and is particularly suitable for use in an automated assay. In such an automated assay, the supported antigen or hapten is provided in a flow-through chamber, with the automated apparatus being, for example, of a type disclosed in U.S. Pat. No. 4009005. After the separation, an eluting liquid is passed through the chamber to elute the antibody from the supported antigen, whereby the supported antigen in the chamber may be reused in an assay. 2.5 RHEUMA Diseases of chondrocytes are usually accompanied by auto-immune reactions. To this groups of disease belong for example chronical polyarthritis, osteoarthrose. In spite of intensive research no reliable diagnosis method or methods for monitoring of the disease were available. Research of Von der Mark and Mollenhauer (MAX-PLANCK-GESELLSCHAFT ZUR FÖRDER-UNG DER WISSENSCHAFTEN E.V. (104.2)) intends to meet these demands with a method based upon the specific membrane protein of chondrocytes. The validity of this concept was proven in a test whereby rabbits were subcutaneously injected with chondrocyte-membranes or the individual membrane proteins. As a result of this immunisation they developed degenerative joint damage. Partly the inflammation reactions in the knee-joint of the hindlegs were so explicit that outwards open wounds did develop. Reactions of these proportions have not been described in the literature on the production of immunoreactions against chondrocyte components. Chondrocyte-specific membrane proteins which display an immune reaction with autoantibodies can be obtained by homogenizing cartilage tissue, isolating the membrane vesicles from the homogenizate, and separating and extracting said proteins from the vesicles. The chondrocyte-specific membrane proteins are suitable for diagnosis and monitoring autoimmune reactions against chondrocytes.

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Table 1 Comparison of the reactive antigens of osteoarthritis and chronical polyarthritis.

The chondrocytes specific membrane proteins are characterized by molecular weights (Mw) of 155 000, 116 000, 78 000, 76 000, 66 000, 42 000, 38 000, 30 000 bzw. 28 000. The characterization with antibodies of the reactive antigens of osteoarthritis and chronic polyarthritis is given in the table below. Test result with immunodot, ELISA, and inhibitions-ELISA were consistent. Qualitatively the authors claim the dominance of the cellmembranes as target of the auto-immunoreactions, in the contrary to components researched by others, which are regarded as being of lesser importance. The proven immunoreactivity of mammal membrane components of the chondrocyte with human sera is of significant commercial importance of the test system. Patients suffering from systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and mixed connective tissue disease (MCTD) tend to present the same clinical symptoms during the initial stages. A single reliable serological technique capable of distinguishing among different rheumatic autoimmune diseases was lacking. Research by Jacob et al. (RESEARCH CORPORATION (140.1)) provided a procedure by which the test can be prepared and performed at low cost and easily performed in a clinical laboratory. Anti-RNA polymerase I antibodies were detected in 100% of patients with SLE and were undetectable in 100% of normal patients. Anti-RNA polymerase antibodies were also detected in 100% of MCTD patients and in 78% of RA patients. RNA polymerase I is a complex enzyme, that is to say, the enzyme is composed of eight different polypeptides designated as: Sl(Mr = 190000), S2

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(Mr=120000), S3(Mr=65 000), S4(Mr =42000), S5(Mr = 25000), S6(Mr=21 000), S7(Mr=19000) and S8(Mr=18000). Sera from patients with SLE contained immunoglobulins directed against the S3 subunit of RNA polymerase I, as well as antibodies to the S2 or S5 subunits, RA patient’s sera contained antibodies only to S3 while MCTD patients sera contained antibody to S4 in addition to antibody to the S3 an S5 subunits. The identification of specific reaction patterns of the antibodies with the individual subunits of the RNA polymerase I is indicative of a particular class of rheumatological disease. There is strong evidence to suggest that there is a particular B-cell alloantigen, the occurrence of which is significantly associated with patients who develop rheumatic fever, Patarroyo, Manuel E. et al, Streptococcal Diseases and the Immune Response, Academic Press, 1980, page 369). The occurrence of this alloantigen in unaffected, apparently normal individuals, and its inheritance in unaffected family members of patients provides evidence of the genetic nature of the alloantigen. Rheumatic fever attacks in susceptible individuals are sequelae of streptococcal sore throats. In the present state of the art for preventing rheumatic fever attacks, documented sore throats as evidenced by positive cultures and rise in streptococcal antibody are treated with penicillin for ten days either by mouth or by injection. The reason for this procedure is that failure to heal the streptococcal infection during this period of time may result in rheumatic fever in 3½ of the age group (5 to 18 years) at greatest risk. If the genetic marker present at birth were available from B-cells, a small amount of heparinized cord blood obtained from each newborn could be tested for the presence of the marker. Positive tests would identify the child as rheumatic fever susceptible individual (about 19′ of the population). Zabriskie and Buskirk (THE ROCKEFELLER UNIVERSITY (189)) describe a method that is capable of producing novel monoclonal antibodies which can be employed to test for individuals at risk of rheumatic fever. One of these hybridomas has successfully identified a genetic marker on approximately 95% of documented rheumatic fever patients. The monoclonal antibody is derived from hybridoma cell line D8/17 which reacts specifically with the complementary rheumatic fever associated antigen on human B-lymphocytes, but not with known HLA antigens of the A, B, C, DR-1 through DRW8 loci on human B-lymphocytes. In order to improve existing tests for rheumatic diseases Vorlaender et al. (HEYL CHEMISCH-PHARMAZEUTISCHE FABRIK (69)) propose a method based upon the presence of auto-antibodies against natural body collagens (C.Steffen et al., Klin. Wschr. 51, 222–229 (1973); C. Steffen, Z. Rheumatol. 37, 131–147 (1978)) in sera and synovial bodyfluids. An immunological test kit is proposed containing:

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a) a carrier upon which the collagen is immobilised; b) an anti-antibody reagent marked with enzyme; c) a substrate, which is capable of enzymatic katalysed forming of dye. The kit might contain several kinds of collagen, type I to VI. As carrier amongst other, diazobenzyloxymethyl, diazophenylthioether, or nitrocellulose might be used. The test based upon an antibody (rheumatoid factor) to the Fc fraction of IgG is not satisfactory because it has been found to give unacceptably large numbers of false positives or negatives, and it does not assess the response to therapy or predict activation or reactivation of the disease process. Hurwitz et al. (HURWITZ ET AL. (72)) discovered a rheumatoid arthritis protein (RHP) characteristically present in detectable amounts in the sera of RA patients but is not detectable in sera from normal individuals or in sera from patients with other arthritides. This protein is not RF nor any of the known acute phase reactants. RHP can be isolated and used to prepare polyclonal and monoclonal antibodies which can be used to detect RA and to follow the course of treatment of the disease. This protein can be recognized and distinguished from other proteins by the following characteristic properties: 1. Isolectric pH range of 5·1 to 5·3. 2. Precipitated from human serum in 0·02 molar acetate buffer at pH 5·5 (the euglobulin fraction). 3. Soluble in 0·026 molar ethylene glycol tetraacetic acid (EGTA) at pH 7·5. 4. Present in euglobulin fraction of human sera. 5. Molecular weight of about 135 000 as detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS.PAGE). 6. Enlarges the size of the Clq precipitin ring in radial immunodiffusion (RID). 7. Inhibits the hemolytic activity of Clq. 8. Inhibits the binding of Clq to fibronectin. 9. Over 90% by weight of the total molecular weight is accounted for by the following amino acids.

10. Nonreactive with antibodies to human IgG, IgA and IgM. The production of RHP antibody in rabbit is done according to wellknown procedures. To produce a much greater concentration of less pure antibody, selected hybridoma may be injected into mice, preferably syngenic or semi-syngenic mice.

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One of the most important medical uses for the RHP of this invention is for the production of antibodies to RHP. These, in turn can be employed to detect RHP in currently afflicted individuals, individuals in remission, or individuals at risk of the occurrence of the disease. An antibody composition used in any test designed to determine the presence of RHP must contain sufficient antibody to react with the RHP, which for this purpose may be considered an antigen, to produce a detectable product. Such diagnostically effective amounts of antibody will vary appreciably. In an advanced stage of atherosclerotic disease atherosclerotic plaque antigen can be released into the blood system where it interacts with anti-plaque antibody to form complexes. Calenoff (VASOCOR (196)) developed a method to determine the presence of atherosclerotic plaque antigen in patient serum as an indication of the presence of advanced atherosclerosis, comprising the contacting the serum with antiplaque antibody for a time sufficient to permit binding of the atherosclerotic plaque antigen with the anti-plaque antibody, and determining the atherosclerotic plaque antigen binding, if any, with the anti-plaque antibody. The anti-plaque antibody can be bound to an insoluble support, contacted with serum to permit atherosclerotic plaque antigen conjugation with the anti-plaque antibody, and the amount of resultant atherosclerotic plaque antigen bound to the insoluble support can be determined. Alternatively, a precise amount of atherosclerotic plaque antigen can be bound to the insoluble support, and a mixture of a predetermined excess amount of labeled anti-plaque antibody and serum can be contacted therewith. The labeled anti-plaque antibody bound to the insoluble support or remaining in the mixture can be determined. Also, a precise amount of anti-plaque antibody can be bound to the insoluble support, and a mixture of a predetermined amount of labeled atherosclerotic plaque antigen and serum contacted therewith. The labeled atherosclerotic plaque antigen bound to the insoluble support or remaining in the solution can then be determined.

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Accurate measurements of the apo-A-lipoproteins I and II may assist in predicting an individual’s prognosis for atherosclerosis, specifically for coronary atherosclerosis disease. The two most frequently used techniques are ultracentrifugation and electrophoresis. However, these techniques are not easily carried out in an ordinary clinical laboratory. Curtis and Edgington (SCRIPPS CLINIC AND RESEARCH FOUNDATION (149)) developed the present invention contemplates monoclonal antibodies that immunologically binding with apolipopro-tein A, but are free from immunoreaction with and binding to apolipoproteins B, C, D and E. Monoclonal anti-apolipoprotein A receptors were formed as described herein from murine (mouse) splenocytes fused with murine myeloma cells. The polyclonal anti-apolipoprotein A antibodies described were formed from rabbits. The hybridomas that produce the monoclonal anti-apo-A-I and anti-apo-A-II receptors of this invention were given the following designations for reference purposes and were deposited with the American Type Culture Collection (ATCC), Rockville, Maryland on Mar. 5, 1985 under the following ATCC accession numbers. The monoclonal receptor molecules of the present invention are particularly useful in methods for assaying the presence and amount of an apolipoprotein A with assays as RIA, ELISA and FIA. Assays with the apo-A-I, apo-A-II monoclonal antibodies, and combinations thereof confirm that all HDL particles express at least one of the three apolipoprotein epitopes defined by antibodies A-II-1, A-I-4, and A-I-7; A-II-1, AI-4, and A-I-9; or A-II-1, A-I-7, and A-I-9, and thus establish limits on the degree of heterogeneity. The figure below illustrates the maximum binding capacity of mouse ascites fluids containing human apo-A-I—and apo-A-II-specific monoclonal receptors (antibodies). The upper portion of the figure shows binding of 125I-HDL. The lower portion of the figure is a graph showing data of binding of 125I-apo-A-I or 125I-apo-A-II. The fluid-phase RIAs were incubated for 18 hours at 4 degrees C. and contained 125I-HDL, 125I-apo-A-I, or 125I-apo-A-II at final concentrations of 66·7, 33·3, and 33·3 nanograms per milliliter (ng/ml), respectively. The coefficient of variation for all data points was less than 10 percent.

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There are prior art methods for separating circulating immune com-plexes (antigen/antibody) from body fluids in order to eliminate their influence on potential deseases such as rheumatic deseases. Circulating immune complexes depositing on the bloodvessel’s walls way induce, amoung others, vasculitis. Such methods, however, ensure no selective separation of the plasma components and consequently require the addition of large amounts of foreign plasma or human blood. Thus, the aim of SCHLÖSSLER et al (185) was to provide a novel adsorbant for the selective separation of circulating immune complexes from body fluids by the biospecific bonding thereof to protein Clq, which is bonded to poly(hydroxymethylmethacrylate) copolymer, or cellulose or silicondioxide containing materials as a solid phase, immobilised by a bridgebond. The immuno complexes bonded in this way are removed as the solid phase, which then is regenerated by non-denutarated media. 2.6 ALLERGIES Worm parasitoses cause an in increase in the circulating specific immunoglobulins, such as IgE, IgG4 IgM. All prior art tests have their drawbacks. This includes the immunological diagnosis methods. In a comparison, the best method for diagnosing hydatidosis, affected carriers still had a negative immunological response. Leynadier and Luce (INSTITUT PASTEUR (129.1)) developed a diagnosis reagent for parasitoses and allergies, and notably for worm parasitoses, which caused an increase in the circulating specific immunoglobulins fixed on the basophils, such as the IgE’s in particular. The reagent enables extremely reliable diagnosis to be obtained and is relatively simple in use, whilst the techniques which enable it to be obtained ensure sufficient concentrations of basophils in the reagent for the results obtained in the presence of antigen, in the course of the diagnosis, to be statistically interpretable with full safety. A suspension of specific IgE’s carrying basophil granulocytes from a blood sample taken from a human or animal patient who is being examined for parasitosis or allergy, which contains from 300 to 1000 basophils per mm3. By centrifugation a layer containing essentially leucocytes is collected. It is mixed with a buffer non-destructive relative to basophil cells, such as Hepes buffer for example, said homogenized mixture being then deposited on a liquid of density 1·079–1·085 whose osmolarity is comprised between 280 and 320 milliosmoles, to give rise, for centrifugation after a suitable period and at a suitable speed, to a ring which is constituted by a suspension enriched in basophils and which overlies the density liquid, which ring is taken off, then washed, preferably by means of the same buffer as above, to be deposited in different wells of a diagnosis read-out slide or dish.

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The diagnosis reagent has the following composition: Hepes buffer, molar solution: 4 to 25 ml KCl 10%: 0·932 ml CaCl2 10%: 0·550 ml MgCl2 10%: 0·510 ml NaCl 9%: 850 to 900 ml Water q.s.p.: 1000 ml pH: 7·4–7·6 The composition can be adapted for a diagnosis. Selective metachromatic dyes, or rapid fixing reagents may be added. Counting the number of basophil granulocytes may be done by filling various wells on a slide or disc with the reagent and various antigen concentrations. The main limitations of the commonly employed procedures for allergological diagnosis: A —by the possibility of falsely positive results which are estimated to be about 25% both for the skin tests and for the quantitative determination of the specific IgE; B —by the possibility of falsely negative results which are estimated to be of the order of 20% both for the skin tests and for the quantitative determination of the specific IgE; C —by the difficulties of performing and of standardizing the provoking or challenging tests and by the possibility of aspecific responses of said tests. Marcucci (MARCUCCI (102)) designed an allergological test for detecting an allergic condition, said test consisting in contacting the mucous membrane direct with the allergen or the anti-IgE antibody linked to a solid phase, so that a rapid in situ incubation is obtained of the allergen or of the anti-IgE antibody with the mucous membrane antibodies, and in the successive in vitro determination of the specific or total IgE through radioimmunological or immunoenzymatic procedures. This testing procedure is carried out by employing a plastic material application device comprising one or more receptacles or housing in which some supports are inserted, such supports bearing allergens or anti-IgE antibodies linked to the supports themselves. The testing procedure can also be employed for performing a particular test of specific challenging. 1: Receptacle. 2,3: Supports bearing allergens or anti-IgE antibodies. The primary known method of testing the antigen/antibody reaction of a suspected allergen is known as “cytotoseic testing”. It is a subjective test, depending on the accuracy of the technician, limited to microscope readings. Pasula (PASULA (131)) developed an apparatus for the objective determination of the degree of reaction between a suspected allergen and the

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white blood cells of a patient. Its object is also to obtain reactions of thousands of white blood cells. From a list blood sample the number of white blood cells is counted. A second sample is mixed with the suspected allergen. After counting the number of white blood cells of the mixture both counts are com-pared for a determine thereby if the suspected allergen has caused an allergic reaction. Coupled to the output of the counter, an analyzer may generate a size distribution of the white blood cells. This does require however that the red blood cells have been removed beforehand. 2.7 HEART DISEASE Methods for biochemical diagnosis of myocardial infarction have been successively developed by assaying the activities of the enzymes flowing out into the blood from cardiac cells by ischemic cardiac disorder. Among them, activities of lactate dehydrogenase, creatine phosphokinase, etc, can be assayed with relative ease. However, these biochemical diagnostic methods involve problems such as (1) a problem with respect to specificity of the target enzyme, (2) uncertainty of the correlation between the amount of the enzyme flowing out into the blood from the cardiac muscle at the infarcted portion and the extent of myocardial infarction, and (3) loss of the correlation thereof with the amount of infarcted cardiac muscle during the use of a thrombolytic agent. A cardiac myosin molecule is a structural protein with a molecular weight of about 500000, and has a subunit structure comprising two heavy chains with a molecular weight of 200 000, two light chains I (LCI) with a molecular weight of 27 000 and two light chains II (LCII) with a molecular weight of 20 000. Cardiac myosin light chain or its fragment is considered to reflect directly the degradation process of cardiac cells by ischemia since it is released into the

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blood with the accompaniment of destruction of cell membranes due to ischemic cardiac disorder. In immunological assay of cardiac myosin light chain, the method of using monoclonal antibody specific to cardiac myosin light chain as an antibody reagent has advantages, as compared to the method using antiserum, such as that (1) the antibody has high specificity to cardiac myosin light chain and little crossreaction with skeletal myosin light chain and that (2) the antibody with high specificity can be supplied in a large amount and continuously, and therefore is more suitable for practical application of the diagnosis of myocardial infarction by assay of cardiac myosin light chain. Yazaki (YAMASA SHOYU KABUSHIKI KAISHA (213)) proposes to solve the problems with the assays in establishing a method of immunological assay of myosin light chain as a diagnostic method of myocardial infarction or skeletal muscle disease. This method comprises using as an antibody reagent a monoclonal antibody having specificity to the myosin light chain which recognizes the common antigenic determinant of the myosin light chain in the human serum and the myosin light chain of at least one of xenogeneic animals and using as an antigen reagent the myosin light chain of a xenogeneic animal to which said monoclonal antibody can be bound. The figure below shows the standard curves obtained with the presented method. In vivo immunodetection of thrombi and fibrin deposits remains an important clinical problem. In humans, the detection and localization of deep vein thrombi and coronary artery thrombi are two clinically important problems. Blood clots when thrombin cleaves two pairs of small peptides from fibrinogen to yield fibrin monomers. Anti-fibrin serum cross-reacts strongly with fibrinogen, and that only one instance is known where a fibrin-specific serum was produced. Polyclonal antibodies to fibrin-fibrinogen, to fibrinogen alone or to degradation products of both polypeptides have been developed for use in the detection of venous thrombosis in humans. The antibodies had a substantial degree of fibrin-fibrinogen cross-reactivity. A different approach for the detection of deepvein thrombosis involved by radioiodinating fibrinogen. This method, however, was found to be inferior to the labeling of platelets with 111In, for the localization of thrombi in deep-vein thrombosis. Recent methods have been developed, including methods based on monoclonal antibodies specific against fibrin. Nevertheless a need continues to exist for highly specific antifibrin monoclonal antibodies and for synthetic epitopic peptides capable of raising such nonfibrinogen-cross-reacting antibodies. The antibodies would be useful for the in vivo detection of thrombi.

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Matsueda (THE GENERAL HOSPITAL CORPORATION (62)) proposes a novel method of screening for fibrin-specific antibodies and to fibrin-clot specific monoclonal antibodies screened by this method. The method of screening for the fibrin-specific antibodies uses completely cross-linked fibrin for the selection of antibodies that preferentially bind to clotted fibrin. They are secreted from hybridoma cell lines capable of secreting monoclonal antibodies against fibrin, without fibrinogen cross-reactivity. The use of the cross-linked fibrin clot provides fibrin-clot specific monoclonal antibodies that have increased binding to in vitro and in vivo thrombi. Generally, in the method of screening, the whole purified clot is immobilized to a solid support. The hybridoma culture medium, containing the fibrin-specific monoclonal antibodies, is contacted with the immobilized clot. The fibrinspecific monoclonal antibodies may then be detected and screened by radioimmunoassay or by enzyme-linked immunosorbent assay (ELISA). The fibrin-clot specific antibodies screened by this method are useful for the in vitro and in vivo detection of thrombi and fibrin deposits in humans and animals.

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These monoclonal antibodies may also be used in complexes with thrombolytic agents. The figure below shows the in vivo localization of a cross-linked fibrinclot screened monoclonal antibody 5D7 to experimental rabbit thrombi. The compound N-(2-piperidylmethyl)-2,5-bis(2,2,2-trifluoroethoxy) benzamide acetate (generic name: flecainide acetate) is a useful antiarrhythmic. It is desirable to have reliable and efficient methods to monitor for drugs in order to assure that therapeutic dosages are maintained while avoiding dosages which could cause undesirable side effects. Banitt et al. (RIKER LABORATORIES (141)) developed novel compounds, which are derivatives of flecainide and which are useful as precursors for the synthesis of conjugates with proteins, enzymes and other molecules such as fluorescent compounds. The compounds are flecainide derivatives, amongst other of the formula:

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wherein, among others:

while each R1 is independently alkylene of 2–6 carbon atoms, any R1 group further defined as containing at least 2 carbon atoms which separate both i) a piperidyl nitrogen atom and the X moiety closest to the piperidyl nitrogen, and ii) if m is 2, the two X moieties; and R2 is alkylene of 1–12 carbon atoms with the provisos that if m is 0 and U is—OH or—NHR11, then R2 has at least 2 carbon atoms separating U and the piperidyl nitrogen; if m is 1 or 2 and U is—OH or -NHR11 then R2 has at least 2 carbon atoms separating U and the X moiety closest to U; and if m is 2, then R2 contains no more than about 6 carbon atoms. With suitable carriers, the proposed compounds can be used to provide antigenic materials for the raising of antibodies in sheep. The procedures are, preferably, used in conjunction with a polarization analyzer. Procainamide is an effective and widely prescribed antiarrhythmic drug. It has narrow therapeutic index and can produce serious toxic side effects. The relationship between the dosage of this drug which is administered and the resulting plasma concentration of procainamide has been found to be quite variable among patients. Therefore, monitoring the procainamide level in patient serum or plasma is recommended for safe and effective therapy. HPLC methods are lengthy. EIA and SLFIA are affected by other components of the sample. Heiman (ABBOTT LABORATORIES (1.4)) proposes a fluorescence polarization assay for procainamide; to tracers, immunogens and antibodies for use in the assay; and to methods for making the tracers, immunogens and antibodies.

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The tracers and the immunogens can be represented by the structural formula shown in the figure below,

wherein, amongst other possibilities: Q is a poly(amino acid), a poly(amino acid) derivative, fluorescein, or a fluorescein derivative; n is 0, 1 or 2; R is a linking group including up to 5 heteroatoms when Q is poly(amino acid) or a poly(amino acid) derivative or other immunologically active carrier and up to 8 heteroatoms when Q is fluorescein or a fluorescein derivative, and having a total of from 0 to 18 carbon atoms and heteroatoms; R′ is an alkyl radical having from 1 to 3 carbon atoms; Y is OH, NH2, CH3, F, Cl, Br, CF3 or H when Q is fluorescein or a fluorescein derivative, and Y is H, OH, CH3, F or Cl when Q is a poly(amino acid) or a poly (amino acid) derivative; and Z is H or F when Q is fluorescein or a fluorescein derivative, and is H when Q is a poly(amino acid) or a poly(amino acid) derivative or other immunologically active carrier, and Z is attached to any of the carbon atoms of the benzene ring other than those attached to Y or the carboxamide group. When Q is a poly(amino acid) or a derivative thereof, the compound can be used as an immunogen. When Q is a fluorescein or a derivative thereof, the compound can be used as a tracer. Available RIA for digoxin produce inconsistencies in results. Although FPIA have several advantages over RIA, they do remain susceptible to the protein concentration of the sample. Precipatation of the proteins before FPIA results in a discrepancy in the digoxin concentration of about 10%. Wang (ABBOTT LABORATORIES (1.9)) claims to have improved the FPIA for digoxin by the use of novel precipitation reagent which also functions simultaneously to extract the analyte. The novel reagent is a 3·5% 5-sulfosalicylic acid in an aqueous solution including from about 40% to about 60% of a straight or branch chained organic alcohol having from 1 to 4 carbon atoms.

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2.8 PREGNANCY Current inhibin bioassay systems are time consuming, expensive, have limited sensitivity and precision and are of limited practicability in their application to large sample numbers. By Robertson (BIOTECHNOLOGY AUSTRALIA Ltd (188)) a more convenient inhibin assay has been developed relying on monoclonal and polyclonal antibodies against inhibin. Most preferably the antibody is capable of neutralizing inhibin bioactivity. The antibody is contained in an antiserum raised with an antigen selected from the group consisting of naturally-occurring or recombinant inhibin, or sub-units, fragments or derivatives thereof. Particularly preferred antigens include preparations containing inhibin, purified bovine 58kD inhibin, purified bovine 31kD inhibin, human inhibin, or human or bovine inhibin or fragments thereof produced using recombinant DNA technology. Suitable animals include mammals such as mice, rabbits, horses, donkeys, dogs, sheep, and goats, and birds such as chickens. The assays are intended for inhibin measurement in follicular fluid or serum from various species (including humans). Preferably the assay is a radioimmunoassay or an enzyme-liked immunosorbent assay (ELISA), or a fluorescence-based immunoassay. The immunoassay is further characterized by the step of using labelled 58kD or 31kD inhibin as tracer. More preferably said tracer is labelled with 125iodine (125I) with an enzyme, or with a fluorescent marker. Preparation and purification of 125I-labelled inhibin tracer comprises the steps of iodination of inhibin using a Chloramine T procedure and purification of 125Iinhibin by an affinity fractionation step, using Natrex Red A. The purification procedure additionally comprises a gel filtration step. An example of measurements with anti-31kD inhibin is given in the figure below. The figure shows inhibin, FSH, LH, oestradiol and progesterone concentrations in the sera of normal women during the menstrual cycle, as-sayed using anti-31kD inhibin. Human chorionic gonadotropin (HCG) is a placental glycoprotein hormone composed of two non-identical alpha and beta subunits. The 145 amino acid beta subunit (beta HCG) of HCG has over 80% homology with the beta subunit (beta LH) of LH. The single major structural difference between the beta subunits of these two hormones is the presence in beta HCG of an extra COOH-terminal extension (residues 115–145). Because of these extensive chemical homologies between HCG an LH, specific immunological detection of HCG is problematic.

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Bellet (THE GENERAL HOSPITAL CORPORATION (62.2)) developed a highly sensitive and specific monoclonal-immuno-radiometric assay (M-IRMA) for HCG, using monoclonal antibodies (Mabs) directed against a 37-amino acid synthetic polypeptide analogous to the carboxyl terminus (CTP) of beta-HCG. Accordingly, in one embodiment, a method is described for the determination of human chorionic gonadotropin in a sample, which comprises: (a) contacting said sample with a first capture monoclonal antibody and a second capture monoclonal antibody which are bound to a carrier, wherein said first and second capture antibodies are epitopically specific for distinct epitopes of the carboxy terminal region of the beta-subunit of human chorionic gonadotropin; (b) incubating the components of step (a) for a period of time and under conditions sufficient to form an immune complex between said human chorionic gonadotropin, said first capture monoclonal antibody, said second capture monoclonal antibody and said carrier; (c) adding to said carrier of step (b), a detectably labeled indicator monoclonal antibody, wherein said indicator monoclonal antibody is epitopically specific for the alpha-subunit of human chorionic gonadotropin; (d) determining the detectably labeled indicator monoclonal antibody in said carrier or in liquid phase. The advantage of the present invention, however, lies in the discovery that utilizing as capture antibodies two monoclonal antibodies of distinct epitopic specificity for the COOH terminal region of the Beta-subunit of HCG results in a surprising increase in assay sensitivity that would not be expected given the affinities of the individual capture antibodies for HCG. The antibodies are derived from the cell lines FB08 and FB09. As radiolabelled indicator antibody a monoclonal anti-HCG antibody was derived from cell line H13 that recognizes an epitope on the alpha subunit. This monoclonal-immunoradiometric assay (M-IRMA) exhibited a very high sensitivity for HCG and was capable of detecting less than 50 pg/ml. Moreover,

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this assay was specific for HCG. No cross reactivity was observed with human LH at a concentration of 1 000 ng/ml. The difference with a prior art test is illuminated in the figure below. The top figure shows a serial study of two patients using a commercial immunoradiometric assay (RIA-GNOST HCG) based on a monoclonal anti-beta HCG antibody (top) and an assay (M-IRMA 9-12-13) based on anti-peptide antibodies (bottom). Patient 1: testicular tumor. Patient 2: gestational trophoblastic tumor. Lower limit of sensitivity of each assay is indicated by shaded area. The bottom figure shows HCG levels as determined by anti-peptide based MIRMA in the sera of 10 patients that had undetectable HCG levels by commercial immunoradiometric assay (CM-IRMA) except in two instances (Patient A: HCG greater than 50 ng/ml; patient B HCG = 2·5 ng/ml). Patient A, B, C, D, F, H, I and J were followed for a gestational trophoblastic tumor and patient E was followed for an ovarian tumor.

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A great many antigens and antibodies are currently detectable by immunoassay methods in which the first member of the specific binding pair is detected by its reaction with the second member to form immune complexes. Some such assays employ a conjugate of the second member of the pair with an enzyme (e.g., horseradish peroxidase, or “HRP”) which typically acts on a substrate (e.g., peroxide) to cause a colour change in a colour reagent (e.g., tetramethylbenzidine, or “TMB”, in an acidic medium). Such conjugates generally are stabilized, prior to use, in buffers containing sulphate and/or phosphate. One commonly detected antigen is human chorionic gonadotropin (“HCG”), a hormone which becomes present in the blood and urine in appreciable amounts during pregnancy. Since the alpha chain of HCG is the same as that of several other fertility hormones, it is the beta chain which is detected, using anti-beta HCG antibodies. Buck and Gibson (MODERN DIAGNOSTICS, INC. (111.2)) claim improvement in the above-described type of immunoassays. One such improvement is the use of TMB at an alkaline pH, i.e., between 7·0 and 9·5, which results in increased colour reagent stability, superior colour development, and the ability to stain solid substrates (e.g., polystyrene reaction tubes) to provide a permanent record of a positive reaction. Another improvement consists of the conjugation of one member of the specific binding pair with an enzyme such as HRP using a gMBS-SATA linkage; this linkage providing enhanced enzyme stability and specificity of reactants. Yet another improvement is the use of coated supports (generally, tubes) in which the coating is a mixture of polyclonal and monoclonal antibodies; preferably, there are three or more different monoclonal antibodies, and the protein polyclonal:monoclonal ratio is preferably about 7:1. The improvements of the invention permit the screening of human serum or urine for the presence of β-HCG in systems featuring speed, simplicity, sensitivity, and accuracy. Because the detection system is stable over long periods of time, it can be sold over the counter as a pregnancy detection kit for home use, or it can be used in a clinical laboratory or doctor’s office. There has long been a need for a simple but reliable technique to indicate the fertile period of the menstrual cycle (i.e. the period in which viable sperm and a viable ovum may be present together in the reproductive tract of the female. Baker and Coulson (BAKER AND COULSON (12.2)) developed an assay for determining the relative concentrations of two antigenic solutes in a sample. The assay involves a first antibody to the first antigenic solute, a second antibody to the second antigenic solute and a ligand molecule having substituents to which the first and second antibodies may bind. The second antibody is immobilized upon a solid phase support and the other components are in solution. The components are mixed with a sample containing the antigenic solutes causing

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competition between the ligand molecule and each of the antigenic solutes for binding to the first and second antibodies. In the situation where there is a relatively higher concentration of the first antigenic so lute to the second antigenic solute in the sample, a significant number of the ligand molecules become attached to the solid phase via the second antibody yet are capable of binding to a labelled antibody having the same specificity as the first antibody. The presence of labelled antibody bound to the solid phase support is indicative of the above mentioned situation. The assay is used for determining the fertile period of the menstrual cycle by monitoring the relative concentration of oestrone-3-glucuronide and preganediol-3-glucuronide in urine. A reagent kit for use in performing the assay and method is described. A ligand molecule is described which comprises two, different antigenic radicals bound together through a bridging support molecule in which the bridging support molecule is a divalent radical derived from an organic compound having two reactive functional groups. Pregnancy diagnostic tests have used both biological assay methods employing laboratory test animals and immunological test procedures. In recent years, such biological tests have been supplanted by immunological or immunochemical tests offering many advantages. Results are usually obtained in shorter periods of time than with the older methods of diagnosis, and costs and other problems incurred in maintaining laboratory animals are avoided. In addition, immunological tests demonstrate greater specificity and sensitivity than bioassay techniques. However the tests may give inaccurate results due to false positives traceable to impurities, excessive amounts of antibody coating, and too large or too small substrate particles. One of the most frequent sources of error is related to the skills of the person interpreting the test. Olson et al. (MELOY LABORATORIES (106)) provide a test method for quickly and accurately determining the presence or absence of antigenic or enzymatic material in a body fluid which yields a color change when the specimen is positive. As indication for pregnancy the presence of human chorionic gonadotropin (HCG) in a woman’s urine is detected. A test utensil is provided having a receptacle, e.g., a well or indentation therein in which two reagent spots are adhered to the surface thereof. The first of the two reagent spots comprises a carrier or substrate material of synthetic or natural derivation, having a dye bound thereto. The dyed substrate comprises particles which are coated or sensitized with an antibody or antiserum which is the specific complement to the antigenic material which is being analyzed, the antigen and its antibody complement forming a specific antigen-antibody pair. Within the well, and in close proximity to the first reagent spot is a second reagent spot formed from a dyed reagent. The second reagent spot may be

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formed from a dyed substrate such as a serum protein or inert molecule, or formed from dyed carrier particles coated in such a way that an antigen-antibody reaction does not result. The specific dyed second reagent spot cited as part of the HCG test example will be dyed particles. Both reagent spots are provided in a dry condition. Bacteria particles which ultimately form the agglutinating agent are dyed blue with Acid Blue 15. The bacteria particles which carry the normal animal serum are dyed yellow using Auramine 0 (C.I. No. 41000). The blue dyed particles are sensitized with the IgG fraction of a rabbit anti-HCG serum. Normal rabbit serum which may also be produced according to conventional methods is used to coat the yellow dyed bacteria particles. The test is performed by adding several drops of conventionally filtered urine specimen to the receiving portion of the test device 10 or 20 containing the spots. If the device 10 illustrated in Fig. 00 is employed, an inert and sterile stirring, rod is used to agitate the specimen. It is to be understood that the instant invention is in no way limited to such application but extends to a wide range of immunological analyses, as aforementioned. The production of estriol by the fetus and placenta forms the basis of one of the most commonly used fetoplacental monitoring tests. Assessment of the levels of estriol in serum and urine may be useful to assess fetal well-being. In general, competitive binding immunoassays have provided a preferable alternative to chemical methods such as gas chromatography and high pressure liquid chromatography. Fluorescence polarization immunoassay techniques are well known in the art; however, to date the use of such techniques for the determination of total estriol levels has not been described. Vanderbilt et al. (ABBOTT LABORATORIES (1.6)) improved the fluorescence polarization assay for total estriol. Both the tracers and immunogens can be represented by the structural formula shown in the figure below, wherein R1 is H or R-Z-Q; R2 and R3 are H when R1 is RZ-Q and are taken together as R-Z-Q when R1 and R4 are H; R4 is R-ZQ. R2 and R3 are H and is H when either R1 or R2 taken together with R3 is R-Z-Q. Q is a poly (amino acid), a poly (amino acid) derivative or another immunologically active carrier, or fluorescein or a fluorescein derivative; Z is CO, NH, CH2NH or CS when Q is fluorescein or a fluorescein derivative and is N, NH, SO2, PO2, PSO or a glucuronide moiety.

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2.9 URINE Up to 75% of tests for urinary tract infection performed on symptomatic patients produce a negative result. This leads to considerable uncertainty in evaluating the test results, since a negative finding can indicate either (a) no infection or (b) undetected infection due to the test limitations. Mckenzie (UNIVERSITY OF DUNDEE (183+105)) proposed a method wherein to the urine sample from a patient a mixture of antigens is added, prepared from an organism which is known to occur in urinary tract infection by killing said organism without substantial reduction of the antigen content of the organism, said organism being obtained independently of said patients’s urine, and detecting the presence or absence of a combination of antibody from said urine sample with antigen from said mixture. Alternatively a labelled reagent is added which is known to combine with antibody which combines with said antigens, washing excess of said reagent from said mixture and thereafter detecting the presence or absence of said label on the mixture. The increase in effectivity in comparison to prior art tests, is indicated in the figure below. IgG antibody to mixed coliform antigen in urine samples from symptomatic patients and from asymptomatic controls. The broken line represents the mean +2 st’d. deviations of the control results. Values for all symptomatic groups were significantly higher than for control values (pSCHNAPER AND AUNE (148)) developed a screening for immune deficiency in a nephrotic patient comprising determining the presence or absence of the lymphokine, soluble immune response suppressor (SIRS), in a urine sample of said patient. In the recent years immunoassays, particularly radioimmunoassay, ulilizing the specificity of antigen-antibody reaction have been developed, and it has been reported that kallikrein can be accurately determined by such an assay. See chapter 1.4. As is apparant from these reports by the application of radioimmunoassay an accurate determination of trace amount of kallikrein excreted in urine has become possible, and the correlation between various diseases and the amounts of kallikrein excreted in urine has come to be investigated in detail. In the methods disclosed in these reports, however, the reaction of kallikrein labeled with a radioactive substance and kallikrein in the specimen (urine) which act as antigens with the anti-kallikrein antibody acting as an antibody is carried out in liquid phase, so that the separation of the antigen is labeled with a radioactive substance which has formed a complex (B-body) with the antibody

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from the labeled antigen remaining fee (F-body) is troublesome. In the prior methods mentioned above, B/F separation is carried out by the double antibody method of polyethylene-glycol precipitation method. In these methods of separation, however, incubation or centrifugation is necessary for separation, so that the time required for measurement is lengthened and the operations are complicated. To avoid the above problems MORIYA and Co-workers (64) developed a method of determining kallikrein in urine in which an antihuman urine kallikrein antibody, human urine kallikrein labelled with a radioactive substance and an urine specimen containing kallikrein undergo an antigen-antibody reaction, and the radioactivity of the resultant reaction complex is measured, free kallikrein in the urine specimen and free kallikrein labelled with a radioactive substance in the reaction mixture can be easily separated from the antigen-antibody immobilized to a vessel and carrying-out the reaction in the said vessel. For this purpose a kit is provided which comprises a combination of a vessel having said anti-human urine kallikrein antibody immobilized therein, human urine kallikrein labelled with a radioactive substance, standard human urine kallikrein and a buffer solution to be used for reaction.

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Numerous studies have been carried out to detect choriocarcinoma in pregnant women. To eliminate drawbacks of prior methods (complex ity, low sensivity, etc) CANFIELD et al (182) developed a method for detecting chorionic sonadotropin in urine. Their procedure is based on the determination of the presence of soluble desialylated glycoproteins in biological fluids, and comprises contacting a sample of the biological fluid with a suitable amount of an appopriate lectin capable of selectively binging to the desialylated glycoprotein to produce a complex. The resulting complex is separately recovered from the biological fluid. The recovered complex is contacted under appropriate conditions with at least one detectable antibody directed to an antigenic determinant on the desialylated glycoprotein and capable of selectively binding to glycoprotein present in the complex. The presence of antibodies so bound is detected and, thereby the presence of desialylated glycoprotein in the biological fluid determined. See also chapter 2.8 on pregnancy. 2.10 THYROID When glycoprotein hormones like thyroid stimulating hormone (TSH) are measured by the conventional EIA and RIA using polyclonal problems concerning the specificity and reproducibility may arise. The sandwich method of the conventional RIA or EIA is very troublesome and requires highly skilled technique. The “one step method” is simple and convenient, but is inferior in sensitivity and hence is less reliable. The one step method using a combination of an a subunit specific monoclonal antibody has a high specificity. However it has still a drawback in that inhibition by other glycoprotein hormones can not be completely avoided. Nakashima et al. (JURIDICAL FOUNDATION THE CHEMOSEROTHERAPEUTIC RESEARCH INSTITUTE (88)) developed an improved method for the measurement of TSH in blood or body liquid without inhibition by the other glycoprotein hormones present. Further, an improved immunoassay of TSH by a sandwich method is using (3 subunit specific monoclonal antibodies recognizing different epitopes as the solid phase antibody and as the labelled antibody, respectively. The figure below illustrates the effectiveness of the new procedure in eliminating the effect of HCG on the measurement of TSH in a serum containing HCG collected from a pregnant woman by a one step EIA of the present invention and a reference prior art method. Agglutination assays, including those for the determination of triiodo-Lthyronine (T3) and thyroxine (T4) have suffered from nonspecific protein interference. Invariably, it has been necessary to perform preliminary procedures to overcome this source of interference.

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Pig. (A) RIOR

Siegel and Marx (TECHNICON INSTRUMENTS CORPORATION (165)) propose assays in which the effects of non-specific protein interference are avoided or overcome without the need for pretreatment steps. As such, it is now possible to provide a homogeneous immunoassay format which is particularly suitable for use in automated analysis systems. These advantages are achieved by the specific binding enzyme-resistant ligand assay test material, which material comprises (a) a solid phase incorporated with one partner of a specific binding pair comprising said ligand or a binding analog thereof and a specific binding protein therefor; (b) conjugate comprising the other partner of said specific binding pair incorporated with a substance which protects the specific binding protein of said pair from enzyme inactivation when bound with its partner; and (c) an active protein-inactivating enzyme. The invention further provides a specific binding method of assaying for an enzyme-resistant ligand in sample, which method consists essentially of the step of: (i) combining said sample in a reaction mixture with (a) a solid phase incorporated with one partner of specific binding pair comprising said ligand or a binding analog thereof and a specific binding protein therefor; (b) a conjugate comprising the other partner of said specific binding pair incorporated with a substance which protects the specific binding protein of said part from enzyme inactivation when bound with its partner and (c) an active protein-inactivating enzyme, and (d) detecting any resultant binding in said same reaction mixture.

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Fig. (B) (88.1)

The binding is of the nature antigen-antibody. Substances which can be used for this purpose include large molecules which sterically protect the binding protein from enzyme attack when bound. Exemplary of such large molecules are high molecular weight polymers like dextran or Ficoll. The specific binding protein, e.g., antibody, is present in the reaction mixture in a concentration of about 0·04 percent (w/v) to about 0·07 percent (w/v). Likewise, the protein inactivating enzyme is present in a concentration of not more than about 4·0 mg/ml, and preferably from about 2·0 to about 4·0 mg/ml. Iodothyronine (T4) Iodothyronine (T4) secreted from the thyroid, and the measurement of serum T4 concentration has become the test commonly employed as an initial procedure in the diagnosis of states of altered thyroid functions, such as hyperthyroidism or hypothyroidism. In addition, it is well known that several conditions other than thyroid disease may cause abnormal serum levels of T4. Among these are pregnancy, estrogenic and androgenic steroids, oral contraceptives, hydantoins and salicylates, stress, hyper—and hypoproteinemia, and conditions (hereditary

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or acquired) which cause alterations in serum levels of thyroid binding globulin (TBG) the major serum T4 transport system. SIEBERT et al. (16.) found a method for the radioimmunoassay of thyroxine. It comprises: (a) affixing to a solid phase particular mouse monoclonal antibodies produced by hybridoma call lines designated as ATCC HB 8499 and ATCC HB 8500, the antibodies being specific for thyroxine and having a cross reactivity to triiodothyronine of no more than 0·12%; (b) reacting the monoclonal antibodies with a composition comprising thyroxine and a predetermined amount of radiolabeled thyroxine to cause binding therebetween; (c) separating the solid phase from unreacted thyroxine and radiolabeled thyroxine; (d) determining the amount of radiolabeled thyroxine bound on the solid phase; (e) repeating steps (a) to (d) with a plurality of other compositions comprising predetermined and different amounts of thyroxine; (f) repeating steps (a) to (d) with a composition comprising an unknown amount of thyroxine; and (g) determining the unknown amount of thyroxine by ascertaining which of the solid phases reacted with a predetermined amount of thyroxine has the same amount of bound radiolabeled thyroxine as the solid phase reacted with said unknown amount of thyroxine. The solid phase may be a polymer such as polypropylene, p.v.c., polysterene, latex and polyacrylamide. The method in accordance with claim 1 wherein from 0·0025% to 0·05% of the antibodies produced by the hybridoma designated ATCC HB 8499 and from 0·005% to 0·025% of antibodies produced by the hybridoma designated ATCC HB 8500 are attached to the solid phase. 2.11 INTERFERON Interferons (IFNs) are measured by determining the extent of cell destruction by a virus. However the cell death, and its cause, can not be determined directly. Other techniques are based on quantitation of various other viral functions. These are applicable only with limited cell-virus combinations. Revel and Wallach (YEDA RESEARCH AND DEVELOPMENT COMPANY LTD. (214)) developed an assay that is based on a simple solid-phase immunoassay for viral proteins such as those of Vesicular Stomatitus Virus (VSV), a virus which is widely used to probe for the antiviral activity of IFN. Its proteins bind effectively to polyvinyl-chlo ride (PVC)-immunoassay plates.

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A virus infected cell culture (10 pfu VSV) is mixed, in microwells, with serial dilutions of the sample to be assayed and a sample of known IFN concentration. After incubation the virus is lysed with a 0·25% sodium desoxycholate solution pH 9·6. The amount of VSV-protein is quantitated by means of RIA or ELISA. The decrease of VSV-protein in IFN treated cultures is correlated with inhibition of formation of infectious virions. It is obtained at IFN concentrations lower than those that reduce all killing by the virus, therefore more sensitive than cytopathetic assays. It is also applicable to cells which do not exhibit a virus cytopathetic effect. The enhanced sensitivity is demonstrated in the figure below. The figure illustrates the quantitative estimation of the relationship between the effect of IFN on viral cytopathy and its effect on viral yield. MDBK (a) and FS11 (b) cells were treated with various concentrations of IFN-β for 20 hrs, infected with VSV and further incubated for 20 hrs until cytopathic effect had developed in those cells not treated with IFN. The yield of viral protein in the culture and the decrease of neutral red dye uptake by the cell, as an indication of cell death, were then determined in parallel. Viral protein yield (■) is expressed as a percentage of its level in the absence of IFN, and the decrease in neutral red uptake (O) as a percentage of the dye taken up by non-infected culture. Generally, assays for human interferon-gamma (huIFN-gamma) are based on the ability of interferons to inhibit lysis of cultured human fibroblasts infected by viruses. However, they are variable and imprecise because the fibroblasts and viruses used are different, time-consuming, and subjective with regards to result interpretation. Moreover, the as-says do not distinguish between the different types of interferons, IFNalpha, IFN-beta and IFN-gamma. Chang et al. (CENTOCOR, INC. (32.1)) developed immunochemical assays for biologically active huIFN-gamma and methods for immunopurification of huIFNgamma. The assays and immunopurification techniques employ monoclonal antihuIFN-gamma antibodies which react specifically with the active form of huIFNgamma. The antibodies do not react with the inactive form of huIFN-gamma.

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Additionally the antibodies are interferon-type specific, that is, they react with interferongamma but not the alpha and beta types of interferon. Another significant attribute of the antibodies is that they react with both natural huIFNgamma and recombinant huIFN-gamma. The test is said to be capable of detecting 0·02 ng huIFN-gamma/ml, and to take 5–6 hours. The method is based on two monoclonal antibodies, specific for the active form of huIFN-gamma: B1 and B3. The effects of the B3 antibody indicate that the macrophage activation factor is identical to huIFN-gamma. The epitope for the B1 antibody is not associated with the active site. The specificity for the active form, which is demonstrated in the table below, may be used for affinity purification of the active molecule. Presumably the IFNgamma subspecies with molecular weight 20·000, 25·000 and 45·000 are bound by the B1-antibody affinity column. Recovery of 81% of the huIFN-gamma activity is possible. A method for the assay of oligomeric forms of a peptide or proteins has been developed by PESTKA (73.1). It comprises: a monoclonal antibody which recognizes a single, select epitope on the target peptide or protein is coupled to a solid support in a manner known per se. Suitable solid supports for this purpose include, for example, plastic microtiter plates, such as most preferably polyvinylchloride plates. The supported monoclonal antibody when contacted with the test solution containing the target protein will bind to all materials which con tain the select epitope and this will bind to monomeric, dimeric, trimeric and higher oligomeric forms of the target peptide or protein, if present. In the second phase of the assay procedure the monoclonal antibody coupled to the solid support is probed with the same monoclonal antibody which has been labeled with a detectable radio label. Any target monomeric peptide or protein which has been bound to the monoclonal antibody coupled to the solid phase will not have the epitopic binding site available since that site is already occupied. However, if the target peptide or protein is present in oligomeric forms then there are one or more free sites available to which the labeled monoclonal antibody can bind. Thus, the immunoassay will detect selectivily any dimers or

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higher oligomers. Since the larger oligomers have more sites available than the smaller ones, more label should bind to the higher oligomers. In principle the number of sites avaibable to the labeled antibody in an oligomer of n subunits is equal to n-1. In practice, however, it can be expected to be somewhat less because some of the oligomers may bind to the first antibody through more than one site. Target peptides or proteins whose oligomeric forms can be selectively detected by means of the PESTKA immunoassay include the interferons, such as leukocyte, fibroblast and immune interferons, hybrid leukocyte interferons, growth hormone, insulin, lymphokines such as interleukin-1 and interleukin-2, serum albumin, somatostatin, chorionic gonadotropins, enzymes such as urokinase and the like. The aforesaid peptides and proteins can comprise human or other mammalian sequences such as ovine, porcine, murine, equine, feline, canine, bovine and the like. Such peptides or proteins can be naturally derived, synthetic or produced by recombinant DNA technology. They can be glycosylated or nonglycosylated. 2.12 BACTERIA The isolation or concentration of bacterial species, in particular bacteria of the genus of Mycobacterium, from sputum specimens is problematic. Traditional methods for the detection, isolation and identification of mycobacteria include the acid-fast staining procedures, culture techniques and biochemical confirmatory procedures. Techniques used to concentrate mycobacteria from sputum have also included, first, treatment with DTT (to liquify) and NaOH (to kill off nonmycobacteria and to release mycobacteria from associated white blood cells), followed by centrifuging for about 15 minutes at 3800 X g and neutralization of the solution. Recovery of pelleted mycobacteria is inefficient, and this method is not ideally adapted to a sensitive assay procedure because recovery is not quantitative. Furthermore, the NaOH reagent used can kill the mycobacteria if left unneutralized too long in the specimen. Other concentration techniques have proven to be largely ineffective, and are not widely used. Kacian (GEN-PROBE INCORPORATED (57B)) claims to have developed improved methods for liquifying mucoid, and concentrating a bacterial species from a biological specimen wherein bacteria are associated with white blood cells. For liquification dithiothreitol (DTT) is used for reduction of disulfide bonds, and deoxyribonuclease I (DNAse I), purified from bovine pancreas, for DNA digestion. Liquification may be by either sequential or simultaneous exposure.

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DTT is used in a 0·01 M solution in 0·9% sterile saline. From DNAse I about 1500 Kunitz units/ml sample are used, in 0·85% (w/v) NaCl, 5mM NgCl2, 5mM CaCl2 (0·02% sodium aside preservative). For further processing it is crucial that the white blood cells are kept cellularly intact: the Mg2+ and Ca+ promote digestion while simultaneously promoting white blood cell stability. After liquification the sputum is centrifuged for 3 minutes in a clinical centrifuge. The DNAse I is preferred because it remains active in the presence of the lysing agent a 16% sodium deoxycholate solution in water. The lysing agent must lyse the white blood cells and be releasing associated bacteria while at the same time preserving the released bacteria as intact cells. But it should at least not inhibit the activity of one DNAse, which is additionally added during lysing. The bacteria should not be lysed before inactivation of the DNAses and/or RNAses because of possible interference with the nucleic acid hybridization procedure. Sodium dodecyl sulfate is such an inactivating agent, and in addition solubilizes proteins and other cell components. The lysing tubes are capped, and sonicated for 15 minutes. The lysed concentrated bacteria sample may be subjected to a nucleic acid hybridization assay in the presence of diisobutylsulfosuccinate, which speeds up the hybridization process and inhibit any activity of residual DNAse. Alternately the bacterial species may be assayed following concentration and lysing of the white blood cells. If the selected bacterial species is to be cultured, the concentrated specimen may be treated so as to kill undesired species while preserving the selected species. In the case of mycobacteria, it may be achieved by treating the concentrated specimen with dilute sodium hydroxide. Alternately, it appears that treatment with sodium dodecyl sulfate will selectively kill many other bacterial species. In tests to identify the Lancefield group of streptococci enzymes like lysozyme are used for extracting the specific group antigens. Either they are insufficiently effective, or they cause non-specific clumping of the latex particles. Webster (OXOID LIMITED (125)) found that the problems of the prior art enzymes do not arise with the use of achromopeptidase to expose the streptococcal antigen sites. It releases group D antigens from streptcocci cells, more correctly, even in the presence of group G antigens. The achromopeptidase is used in a solution of 0·01 M Tris-HCl, pH 8·0. The mixture with the streptococcus to be identified may be incubated at 4°C overnight or alternatively for 10 minutes at 37°C-56°C. A comparison of latex agglutination tests with different enzymes: OA: Oxoid reagent using achromopeptidase (obtained from Takeda Chemical Industries Ltd. Japan) made up in 0·01M Tris-HCl pH 8·0. WSP: “STREPTEX” (Wellcome Diagnostics) which uses pronase

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OSP: Oxoid reagent using SIGMA PROTEASE (pronase from Sigma Chemicals Co., USA) made up in 0·1M phosphate buffered saline pH 7·4. OSL. Oxoid reagent using SIGMA LYSOZYME (lysozyme from Sigma Chemicals Co., USA) made up in 0.1M phosphate buffered saline, pH 7·4. OBP: Oxoid reagent using B.D.H. Pronase made up in 0·1M phosphate buffered saline pH 7·4. The results are shown below, wherein Because of the high frequency of group A Streptococci as etiological agents of human disease, simple and fast methods of diagnosis are being sought. It would be advantageous to have assay procedures for ligands extracted from cells or cell fragments which permit simultaneously carrying out the extraction of the ligand from the sample and reaction of the ligan with at least two antiligands therefor to form a detectable reaction product. Accordingly the method developed by Founds and Plasio (VXR, INC. (198)) provides a method for performing an assay of a ligand extracted from a sample of cells or cell fragments, the method comprising: (1) simultaneously carrying out the extraction of the ligand from the sample and reaction of the ligand with at least two anti-ligands therefor to form a detectable reaction product: and (2) detecting the reaction product. In some embodiments the procedure further provides solid phase ligand/antiligand assays wherein at least one anti-ligand is immobilized in or on a solid support. The graph illustrates the results obtained in immunoassay to determine the optimum pH for simultaneous extraction and immunochemical binding of group A streptococcal antigen. Optimum pH is 4·4. Most of the assays for recognition of enterotoxin-producing bacteria require time-consuming steps, expensive manipulations and/or laboratory animals. The most promising introduced recently is the Bilken test but it also requires four days. Finkelstein and Zhengshi (THE CURATORS OF THE UNIVERSITY OF MISSOURI (187)) developed a test which can be performed on colonies of bacteria from a primary isolation culture plate without subcultering. Antibodies to be the heat-labile enterotoxin (H-LT) is bound under proper conditions of pH and ionic strength to the latex carrier. The amount of antibody used to sensitize the carrier has to be determined experimentally. (a) 3·2 mg of protein per ml. The isolation to be tested is preferably pretreated with polymysein to release the H-LT. During the actual test the specimen is incubated with the sensitized carrier particles under such ionic and pH conditions that any H-LT is detectable by a visible macroscopic insoluble complex.

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++++ denotes a strong positive reaction ++ denotes a positive reaction + denotes a weak positive reaction − denotes no reaction

2.13 FUNGI Many chlamydial infections go untreated because of the limitations connected with the diagnostic techniques. A major problem resides in the culturing of the slowgrowing of chlamydial cultures in living cells, and the contamination thereof by extraneous, non-chlamydial proteins.

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Makela et al. (ORION CORPORATION, LTD. (121.1)) prepared the Relipopolysaccharide of Re-lipopolysaccharide (Re-LPS) mutants of gram-negative bacteria. Antisera prepared by the immunization of rabbits with Re-mutant strains (S. typhirmurium and S. minnesota) and Re-LPS-porin complex contain high-titer antibodies as against chlamydial glycolipid. The preparation can be used in all serological or immunological methods, in which antibodies to group-specific antigen of chlamydia need to be demonstrated. Antiserum to group specific chlamydial antigen can be produced directly by immunization with a Re-mutant of a gramnegative bacterium, without isolation of the Re-lipopolysaccharide. Maybroda et al. (DNA PLANT TECHNOLOGY OPERATING CO. and KOPPERS AGRI-RESEARCH COMPANY (92.1+52)) developed several monoclonal antibodies for the detection of Pythiaceae infection of plants. Also a hybridoma, with the identifying characteristics of ATCC 8878 or 8750, producing a monoclonal antibody to an antigen from at least one strain of a member of the family Pythiaceae, is disclosed.

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Titration of antibody for latex sensitization

*One heptose residue of the LPS core component is present in the mutant in addition to the lipid A KDO-oligosaccharide.

Staphylococcus aureus as etiological agent of bovine mastitis is commercially identified by culturing the organism and classifying it by conventional taxonomic procedures. Norcross and Obdebeeck used aureus strain Wood 46 to produce a staphylococcal alpha hemolysin, which they crudely purified by the method of Coulter. This preparation was then used as an ELISA reagent. The ELISA procedure of Adams and McGuire (PROSCIENCES, INC. (137A)) uses highly purified antigens with a molecular weight range of 18000 to 26000 daltons. The significance of their use as antigens is that virtually all S. aureus infected cows have antibodies in their milk which bind these antigens;

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such antibodies are lacking in the milk of uninfected cows. The antigen preparation does not contain alpha or beta hemolytic activity or significant quantities of polysaccharide. The most relevant advantages in comparison with bacterial culture tests are the reduction in time consumption and its relatively low costs. If one animal becomes befallen with bovine leukemia virus (BLV), the whole herd is threatened. The methods for the detection of BLV differ greatly in sensitivity. And a method for prophylaxis is necessary. Guillemain and Portail (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (82.3)) claim to provide a detection method based on a monoclonal antibody which recognizes an antigenic site of a BLV protein which is known to have no antigenic correspondence with internal proteins of retroviruses of other mammals. As prophylaxis the authors propose to fix at the bottom of well ELISA plates with a monoclonal antibody directed against single site (epitope) of major protein (p24) of BLV. Quantification of milk progesterone levels has been performed by radioimmunoassay (RIA), a technique which requires expensive equipment and handling of hazardous radioactive materials. Another tech nique is the enzyme immunoassay (EIA) wherein an enzyme bound to progesterone competes with the progesterone contained in the sample to be analyzed. The procedures, however, do not allow use of the test, usually dairymen at the location of the cow, with testresults available in a short period, e.g. less than 15 to 20 minutes. Babu et al. (PITMAN-MOORE, INC. (133)) provide a practical assay procedure for progesterone in mammalian body fluids such as milk or plasma. Proposed is to bind monoclonal antibodies to rods, in combination with a dye. Pregnancy in cows should be able to detect by differences in colour. Heartworm disease (dirofilariasis) infected animals may show no outward evidence of disease. Infection with Dirofilaria immitis is commonly demonstrated in an intensive test on microfilariae, larval forms of the parasite. A significant proportion of infected dogs lack microfilariae. Mosier (MALLINCKRODT, INC. (100)) developed a method for rapid separation of D. immitis immune complexes. It involves the steps of (a) lowering the pH of the sample to below 3.0 to effect the separation of circulating parasite antigens of Dirofilaria immitis from antibodies therefor in the sample; (b) heating the sample to a temperature within the range of approximately 56°C. to 90°C. for a sufficient period of time to denature the separated antibodies; and (c) increasing the pH of the resulting sample to within the range of approximately 7 to 8 to produce a sample which may be assayed for the presence of circulating

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parasite antigens of Dirofilaria immitis without interference from the separated antibodies. The virus neutralisation test of Calnek and Jenick for the measurement of the level of antibody to avian encephalomyelitis (AEV) is timeconsuming and not entirely reliable. The embryo-susceptibility test of Taylor and Schelling is unacceptably slow. Smart (THE STATE OF VICTORIA (159)) discovered the suitability of goat antiserum as a mammalian anti-avian encephalomyelitis antiserum comprising the immunoglobulin fraction of mammalian blood serum containing antibodies to avian encephalomyelitis which antiserum is substantially free of anti-chicken immunoglobulin. The antiserum is used in a ELISA assay for the detection of avian encephalomyelitis. Current inoculum for immunoprophylaxis is against Anaplasma marginale or A. centrale may result in disease, and also transmit other hemoparasites (Babesia, Theileria, Trypanosoma) and viruses (bovine leukemia virus) to the host. In nursing calves it can cause neonatal isoerythrolysis. Barbet et al. (WASHINGTON STATE UNIVERSITY RESEARCH FOUNDATION (194)) isolated substantially pure antigenic surface pro teins of Anaplasma marginale have been identified, and are capable of inducing immune responses in ruminants which neutralizes virulent Anaplasma marginale. The antigenic surface proteins have one or more components having a molecular weight of about 105 000 daltons, 86 000 daltons, 61 000 daltons, 36 000 daltons, 31 000 daltons, or 15 000 daltons, and can be purified by an immunoaffinity chromatography process comprising the steps of disrupting Anaplasma marginale initial bodies by treatment with a detergent, passing the disrupted initial bodies over a chromatography column comprising an insoluble matrix coupled to monoclonal antibodies against a determinant on said antigenic surface protein to selectivity bind said antigenic surface protein to said monoclonal antibodies and recovering the bound substantially pure antigenic surface protein from said insoluble matrix. The antigens have further utility in diagnostic tests for anaplasmosis. They can be synthesized by polypeptide procedures or by genetic engineering. Diagnosing of fish having symptoms of “Hitra disease” by means of tissue analysis, takes from 2 to 7 days. Jørgensen and Hjelmeland (APOTHEKERNES LABORATORIUM A.S. (11)) developed a method which takes about 1 to 2 hours, whereby the protein VS-Pl is detected immunologically. Also a test kit according to the invention may be used, comprising a solid matrix coated with an antibody having affinity/specificity to VSPl.

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2.14 TESTING CATTLE From the experience it appeared that radioimmunoassay and fluorescent immunoassay procedures are not suitable for in-field use and consequently cannot satisfy the needs of cattle breeders. This is partly due to the time needed for individually performing tests for each of many hundreds of animals in a large scale breeding operation. In US Patent 4 399 229 to KELTON a method is described which requires less test time by binding antibodies to protein A, which is associated with the cell wall of the bacterium staphilococens aureus. Protein A binds to the Fc portion of almost all antibodies and, since it has four Fc binding regions per protein A molecule, the protein A both orients the antibody molecules and concentrates the number of antibody molecules in a given volume. From the above considerations Isied et al. (BIOMETALLICS INC. (22)) developed a method of binding an antibody with protein A cells (34) which includes a sequence of incubation and dilution steps to produce a preselected amount and concentration of antibody with a prese lected distribution of antibody among the protein A cells. In addition, a method is provided for preparing an antibody entrapped porous matrix (22) and apparatus (20) which includes a specific porous matrix (22) with a preselected position of antibody bound bacterium cells (34) therein along with means for drawing fluids through the porous medium (40) and means for facilitating the deposition of fluids onto the surface of the porous matrix. The apparatus (20) and method is useful for testing for the level of progesterone in animal body fluids, such as milk, plasma, serum, whole blood and saliva. The method is schematically shown in the next figure. Testing pulmonary surface AKIMO et al. (167.1) developed a method for assaying human pulmonary surface active substance and a reagent test therefor. They used a first monoclonal antibody which recognizes apoprotein of the human pulmonary surface active substance and a second labeled monoclonal antibody which recognizes the apoprotein but binds to an antigen site different from that to which the first monoclonal antibody binds. Assaying complement system fragments (195.2) The complement system of humans and other mammals involves more than 20 components which participate in an orderly sequence of reac tions resulting in complement activation. Numerous studies indicate that the complement system

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is a fundamental element of normal host defense mechanisms. As a consequence, complement activation is commonly associated with a variety of patholgical states such as certain maligancies, myocardial infarction, systemic lupus erythematosis, and adult respiratory distress syndrome. Complement activation can occur by either of two primary modes known as the “classical” pathway and the “alternative” pathway, respectively. Activation via the classical pathway is usually associated with an immunologic stimulus whereas activation via the alternative pathway is most commonly associated with non-immunologic stimuli. Regardless of the initiating stimulus both pathways converge, followed by the conversion of the C3 component of complement into its C3a and C3b fragments. This cleavage of C3 into its subcomponents is considered to be one of the significant events signalling activation of the alternate complement cascade. Following the conversion of C3, a C5 convertase enzyme complex is formed. This enzyme cleaves the C5 component to yield the fragments C5a and C5b. Complement activation by the classical pathway mechanism is uniquely characterized by the fact that this route leads to the conversion of the C4 component to its fragments C4a and C4b. For this purpose the invention proposes the use of buffered acrinol, incubation, recovery from the supernatant and labeled complement/anti-body incubation. A suitable kit comprises three containers, respectively containing buffered acrinol, labeled complement fraction and antibody. The acrinol is applied by 2-ethoxy-6,9diaminoacridine or the lactate monoydrade salt thereof.

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It has been demonstrated that sera from patients with connective tissue disorders contain antibodies to many nuclear constituents. For example, circulating antibodies that react with native DNA have been found to be highly specific to patients suffering from systemic lupus erythematosus. The appearance and exacerbation of the nephritis attendant in SLE appears to be realted to the formation and depositions of antibody/DNA and other immune complexes in the kidneys and particularly the renal glomeruli. Although the exact reasons for the production of these antibodies and the reasons for their particular specificity to SLE are as yet unknown, the detection and measurements of these autoantibodies has become increasingly important in the clinical diagnosis and evaluation of this disease. A precise method for measuring various small amounts of anti-DNA antibody has been developed using 14C-labeled DNA. See T. PINCUS et al, “Measurement of Serum DNA-Binding Activity In Systemic Lupus Erythematosus”, New England Journal of Medicine, 281, 701–705 (1969). This method appears to be the most definitive test for SLE presently available. It is reported to be positive in 75 percent of SLE pa tients studied, whereas other leading tests were positive in only about 25–64 percent of SLE cases. Moreover, this anti/DNA antobody test has shown greater specificity for SLE than other diagnostic tests used, such as immunodiffusion, complement-fixation, and precipitation. However, this test is a test for antibodies that react with DNA although DNA may not be the authentic antigen. Furthermore, this method is not applicable to autoimmune disorders in which the antigen is a protein. Protein antigens are associated with most forms of SLE as well as other types of autoimmune diseases. Such protein antigens are useful in diagnostic and treatment methods as a result of the ability to react specifically with autoantibodies associated with an autoimmune disease. However, isolation and purification of these antigens have heretofore not been fruitful because of the scarcity and instability of antigenic material and because of the complexity of the separation techniques. KEENE (53A) found a method for producing La protein antigen which is reactive with an autoantibody associated with systemic lupus erythematosus in a host, which comprises: —introducing genetic information from a gene library obtained from a first host into plural recipient cells, wherein said gene library is obtained from a host that expresses a La protein antigen reactive with said autoantibody, thereby producing transformed cells; —selecting a producer cell from the transformed cells which contains a gene coding for the La protein antigen and which expresses the antigen by detecting a binding reaction between an autoantibody obtained from a second and different host and protein antigen expresses by the producer cell, thereby identifying a

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cloned DNA segment which can be utilized in the production of the protein antigen. The shown gene library is a DNA gene library. 2.15 MISCELLANEOUS Assays for myasthenia gravis depend on an acetylcholine (hereinafter “AChR”) receptor protein complex. The preferred AChR source is amputated human legtissue which is limited in supply. AChR from nonhuman sources is less suitable. Lindstrom (THE SALK INSTITUTE FOR BIOLOGICAL STUDIES (144)) found that the AChR receptor protein can be isolated in large quantities from the human medullablastoma derived cell line TE671. For practical purposes in immunoassays the AChR from TE671 equals the quality of AChR from human leg muscle tissue. The author claims that the fraction of the autoantibodies directed to the main immunogenic region of the AChR can be determined by deter mining the fraction which are inhibited from binding to AChR upon incubating the AChR bound to the wells with serum combined with an excess of a monoclonal antibody to the main immunogenic region of the AChR. The discovery makes practicable solidphase immunoassays for serum antiacetylcholine receptor protein autoantibodies; accordingly the invention provides solid supports with the TE671 line-derived acetylcholine receptor protein affixed

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thereto for use as substrates in such solid-phase immunoassays and kits for carrying out such immunoassays with such substrates. Monitoring of the ethosuximide level in the blood of epileptic patients is recommended for safe and effective therapy. Liquid and gas chromatography methods require extraction procedures. Enzyme immunoassay and substratelinked fluorescence immunoassay have drawbacks because of the instability of the reagents, and influencing of the enzyme reaction by corresponding enzyme inhibitors or catalysts in the biological mixture. Heiman et al. (ABBOTT LABORATORIES (1.5)) developed a fluorescence polarization assay for ethosuximide, and the various components needed for preparing and carrying out such an assay, and the methods of making these components. A general structural formula for the tracers and the immunogens of the present invention as well as the classes of reactants used in preparing them, is disclosed below. Wherein the composition of a radical group depends on the composi

tion of the other radical groups. The assay is conducted by measuring the degree of polarization of plane polarized light that has been passed through a sample containing antiserum and tracer. Activity of the serum thymic factor (FTS) in blood is detected with the rosette inhibition assay. This reflects however thymic development and function. A specific method for quantitation of FTS is needed to define the physiological and pathological characteristics of FTS, especially “in vivo”. Erickson et al. (SLOANKETTERING INSTITUTE FOR CANCER (154.2)) disclose four radioimmunoassays (RIA) for the quantitation of FTS. Preferred compounds comprise the FTS decapeptide analogues of the following formula:

wherein X and T are both hydrogen. Each assay employs an antibody specific for FTS, the monoclonal antibody or the antibody from the antiserum of a host animal; synthetic FTS or FTS analogue as the hormone standard; and a radiolabeled FTS analogue as the tracer.

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Using rabbit antiserum RIA III and synthetic porcine FTS, a plasma FTS concentration of 44 pg/ml was detected. Deich et al. (PRAXIS BIOLOGICS (135A)) prepared peptides and proteins related to an epitope comprising an outer membrane protein of Haemophilus influenzae by using novel purification methods and by recombinant DNA and chemical synthetic techniques. The author describes the nucleotide sequences encoding PBOMP-1 and PBOMP-2 related peptides and proteins. The peptides, proteins and viruses both “live” and “inactivated” are used as immunogens in vaccine formulations to protect against H. influenzae infections. The peptides and proteins are also used as reagents in immunoassays as well as to prepare immunoglobulins for passive immunization. Use of the nucleotide sequences encoding the PBOMP related peptides and proteins in hybridization assays is also described. The qualitative and quantitative measurement of the cholesterol epoxides in biological fluids and tissues due to their relatively low concentration has been a difficult, costly and time-consuming task. The used procedures do not lend themselves readily to routine, quick, precise and economical clinical analyses in medical practice. Schaffner (SCHAFFNER (147)) provides a clinical diagnostic method for qualitatively and quantitatively measuring the presence of cholesterol epoxide. The method utilizes a specific cholesterol epoxide reaction to produce a novel cholesterol epoxide adduct molecule, structurally highly different from that of cholesterol, cholesterol epoxide, and other related steroid molecules normally present in biological fluids and tissues. In a preferred embodiment, the enzyme, S-glutathione transferase, of the mammalian liver soluble supernatant fraction is employed to convert cholesterol 5α,6α-epoxide to the S-glutathione conjugate, 3β,5α-di-hydroxycholestan-6β-yl-S-glutathione. This conjugate as a hapten is linked through stable covalent bonding to a protein carrier, such as bovine serum albumin, to produce an immunogen suitable to initiate an immune response. The resultant antibodies are sensitive and specific to the cholesterol epoxideglutathione adduct product rather than to cholesterol epoxide itself. One or more of these antibodies may be selected for use in an immunoassay for the adduct. The resultant antibodies, either polyclonal or monoclonal, are thus used to provide a method for determining the presence or concentration of cholesterol epoxide in a sample of fluid. The sample will first be contacted with a hapten (preferably glutathione) in the presence of a hapten-linking agent (preferably, Sglutathione transferase) to form a ringopened 3,5(6)-transdiaxialdihydroxycholestane-6(5)-yl-hapten adduct. The adduct may be detected or assayed by immunoassay procedures using the prepared antibodies. Normally plant diseases are not diagnosed until in a late stage of the disease the symptoms become visible.

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Grothaus and Stocker (DNA PLANT TECHNOLOGY OPERATING CO. and KOPPERS AGRI-RESEARCH COMPANY (92.2+52)) give a general outline for an antigen-antibody assay. The monoclonal antibodies are made via hybridoma technology, in which hybridoma are formed by the fusion of short-lived antibody producing cells and long-lived myeloma cells to produce long-lived antibody synthesizing cell lines. Each hybrid cell line produces a unique and characteristic antibody that has the ability to bind, with a very high degree of specificity, to a single type of antigen. The bonding of immunoglobulin antibodies to a fixed substrate in an assay has the disadvantage of several time-consuming washing steps in a blood assay. Stocker (F. HOFFMANN-LA ROCHE & CO. (74.1)) proposes to bind the forenamed antibodies directly to the bottom of the centrifuge tubes, thus eliminating the washing and increasing of the sensitivity of the test.

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3. DEVICES

Though many devices, sandwichsystems, dipsticks and other utilities for immunoassay procedures have been referred to in the foregoing chapters, it is deemed worthwhile to describe a number of devices which were eye-catching during the study. Therefore, with chapter 3 it is not intended to cover a complete review of all the devices and systems encountered, but only refers to interesting apparatus since about 1980. 3.1 APPARATUS A dipstick is developed by Wang (PROFILE DIAGNOSTIC SCIENCES INC. (137)) for the detection of viruses. It consists of an extended solid phase having conjugated thereon antiviral antibody (Abv) to form immuno-complexes with antigens characteristic of the viruses to be detected. Next the dipstick is contacted with microspheres having conjugated thereto the Abv to bind the microspheres to the immunocomplexes. The presence of microspheres on the dipstick is proof of the presence of viruses in the sample. It is pertained that when the microspheres consist of magnetic material the presence may be detected electro-magnetically. Using dyed or fluorescent microspheres, detection is visual. It is not intended for labelling/detection with enzymes. Donneux et al. (HYBRITECH INCORPORATED (75.2)) proposes a dispenser for beads which are used as support in immunological assays. Shaped like a gun the trigger is equipped with two dents allowing only one bead at a time to enter the barrel, which can be used for a precis deposition of the bead wherever the user wishes it to be placed. Ebersole et al. (E.I. DU PONT DE NEMOURS AND COMPANY (135.2)) developed a device for the rapid detection and (or) purification of analytes in a sample. The system makes use of a fluid receptacle containing a capture reagent and a manifold having two ports. A sample containing an analyte disposed within the fluid receptacle is subjected to a capture reagent and processed by inserting

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the open end of the recep tacle in one port. Vacuum, gas, reagents, and wash fluids are then applied to the second port to effect rapid interaction between the reagent and analyte, efficient washing of the reagent and detection and/or collection of the analyte. The manifold may be adapted for the simultaneous placement of a plurality of receptables. Test results show good culture and antigen detection. Shinefeld and Murtagh (ANGENICS INC. (10A)) discovered a simple apparatus for agglutination tests. It consists of a capillary tube connecting a reagent reservoir, and a reservoir containing a sorbent, which will maintain a flow of reagent medium (16) through the capillary tube. Applied to an indirect assay for penicillin in milk at 5 ng/ml it compares favorably to the commercially available SPOT test. Aside from needing no rocking or other handlings the results of the new assay were stable for over one hour. The apparatus is depicted in the figure below: Nerbe and Wnne (NERBE UND WÖNNE (114A)) found a reactor for blood diagnosis reactions. Its specific feature is the incorporation of 0·9 weight procent of glyceringlycol in the reactorwall which acts as a catalyst. A further additive is 1%. of siliciumpowder. The main component of the reactor material is preferably polystyrol or polypropylene. Maggio et al. (SYNBIOTICS CORPORATION (160)) developed a device for detecting specific analytes in solids and semisolids, as fecal material or sludge. It includes a homogenization vessel and an insert positioned within the vessel for providing an incubation chamber for solid phase immunological reagents. An essential feature is a screen incorporated into the insert which passes extracted components, including analyte, from the homogenate into the incubation chamber while screening out unextracted components. Homogenization alone can either enhance or diminish the sensitivity and accuracy of a solid phase immunoassay, depending upon the characteristics of the particular sample being analyzed. Kemeny (KEMENY (89A)) has designed a washing device consisting of a washing liquid supply chamber in the shape of a perspex tank, which communicates with shallow conical recesses, providing seatings for synthetic rubber orings 11 which act as seals.

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The passageways 7 are arranged in the base plate in an evenly spaced 8 by 12 array, similar to the arrangement of wells in a conventional microtitre plate. The receiving chamber 2 is similar to the top. The end overlaps of the top plate 16 are provided with locating pegs 17. By applying vacuum to the receiving chamber, washing liquid is passing through the discs. Thus they also can be dried. See the figure below. For enhancing the sensitivity and the shortening of the test duration in RAST or PRIST tests Kemeny advises to reduce the volume of liquid that contains the labelled indicator up to the saturation volume of the assaydisc. Also, the liquid saturation volume of the carrier is preferably reduced to 5 1 or less. As carrier cellulose paper discs are recommended. Sakuma (OLYMPUS OPTICAL CO., LTD. (118)) seeks to improve the detection and judging of agglutination patterns with a de vice having a plurality of inclined bottom surface portions, inclination angles of these portions being increased in a step-wise manner. See the following figure. The particle patterns thus formed on respective bottom surface portions are separately detected to produce agglutination and non-agglutination signals. The Antigen-antibody

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reaction is judged in accordance with the agglutination and non-agglutination signals. Incorporated in an automated apparatus, the pattern signals thus extracted are successively supplied to a comparator and are compared with a threshold value given by a reference D.C. voltage supply source. The comparator produces output signals representing agglutination or non-agglutination. These output signals may be logic “1” or “0”signal. DeLaage et al. (LYONNAISE DES EAUX and IMMUNOTECH (99 + 77)) designed a system for the continuous measurement of organic compounds in a circulating fluid. Continuously a small volume is diverted through a cartridge containing an immunoabsorbent fixing the appropriate antibody in equilibrium with a corresponding antigenic tracer. Variations in tracer concentrations are measured thereby allowing the determination of the organic compound concentration. Kaspar (BOEHRINGER MANNHEIM GMBH (24.7)) provides an apparatus applicable in a centrifugal analytic automate extending the use of sandwich assays to assays normally afflicted by the “High Dose” Hook-Effect. A significant change after phase-separation, of the ratio: change divided by measurement value per time-unit is indicative of the occurrence of the HookEffect. In a normal liquid immunoassay the ligand and the receptor have to move towards each other by diffusion. Van Eijk and IJsselmuiden (THE DUTCH MINISTER OF WELFARE, HEALTH AND CULTURE (158)) developed a device wherein the liquid to be analyzed is made to pass through the carrier, to which an antibody or antigen is bound, with a velocity of 0·1–50 ml/cm2/min. To prevent non specific binding about 0·50% by volume of Tween 20 is added. The carrier consists of a plate with holes in which the liquid is applied. Alric and Renaud (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS) (33)) pertains to have found an improved ELISAapparatus. Macromolecular complexes on a nitro cellulose leaf are detected by incubating the leaf under a plate with several longitudinal canals filled with a reagent. After

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the reaction the canals are rinsed and the plate replaced by another one with canals perpendicular to the first mentioned canals. The canals of the second plate can be filled with another reagent. Eventually another similar plate can be employed for a third reagent. The canals have a width of one to four mm, and open over their whole length. Likewise, the canals can be filled in the same step with different reagents. Guigan (GUIGAN (66)) proposed a method of performing biological analysis of a liquid sample using a one-piece plastic container, said container being compartmented to provide a storage chamber for the sample, a calibrated cell, and a plurality of storage chambers for various liquids such as a conjugate liquid, a substrate liquid, and a blocking liquid. A succession of centrifuging operations serve to successively pass the sample and said liquids into a reaction vat containing a bead which is initially covered in antigens or antibodies. Means are also provided for rinsing the bead. Harris and Stone (BECKMAN INSTRUMENTS, INC. (15)) propose to overcome the limitations of heterogeneous immunoassays with an apparatus applicable in an automated instrument. A reaction capsule having a hydrophobic membrane, which may be repeated by wetted, is used. A pressure differential across the membrane causes liquid flow therethrough to be initiated and the hydrophobic state is then achieved by flowing gas through the membrane. The system includes a turntable supporting a plurality of reaction capsules and eccentric means for agitating the turnable and capsules. The turntable is rotated to position the capsules at various processing stations, including sample introduction (86), reagent introduction, wash, substrate introduction and read stations. A single cylinder two-inlet valve may be used, one inlet connected to liquid and a second inlet connected to a gas source, to provide both liquid and gas flow through the membrane. Chandler (ALLELIX INC. (4.3)) proposes for performing several immunoassays simultaneously. It consists of a plurality of tubes containing assay reagents, a sample tube, and conduit and valve means from the various tubes to the assay tube. It is supposed to be inserted in a programmable apparatus which controls the flows of the various reagents through the assay tube by means of plungers acting on pistons located in each reagent tube. Magnetic means The attraction of particles from a liquid dispension by magnetical means is not novel in the art. A typical example is the method and device developed by Luotola et al. (LAB-SYSTEMS OY (96.1)). The method is particularly designed to be carried out in immunoassays wherein to a solution containing the antibody to be determined, magnetic. particles (2) coated with the corresponding antigen as well as tracer particles (3) coated with the corresponding antigen are added.

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After the immunological reaction the magnetic particles and the tracer particles adhering to them by the intermediate of the antibody are separated from the reaction solution, and the radiation emitted by the separated particles is measured. The magnetic particles are separated from the reaction solution by pushing a magnetic piece (5) into the solution and by pulling it out of the solution after the magnetic particles have adhered to it, whereupon the radiation emitted by the separated particles is measured. The next figure shows a schematic view of the device used by Luotola. SAXHOLM (146) has designed an apparatus for testing reactions, wherein a biologically active substance is incorporated in a unitary body with a magnetically responsive material for carrying out diffusion testing. These may be, microbiological, immunological, serological and other biochemical examinations. The body is applied against a substrate or medium by application of an external magnetic field and a reaction region is produced at the site of the body and is measured by means of a reader. In order to ensure deposit of the body on the substrate a predetermined location and corresponding reading of the reaction region at such location, the support for the substrate and the dispenser and rear are provided with suitable releasable coupling and orienting devices such that the dispenser and reader can be respectively engaged and oriented on the support in predetermined secured positions. The next figure shows the apparatus proposed by SAXHOLM.

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Davis et al. (UNILEVER NV(175.1)) also uses magnetic particles on which is immobilised a first binding reagent having specifity for the analyte and with a labelled reagent which can participate in either a sandwich or a competition reaction with the first reagent in the presence of the analyte and following an incubation period sufficient to allow the reaction to take place. The carrier material is moved within the assay medium (618) to a location adjacent a signal sensing means (602), the label generating a signal, such as chemiluminescent light, continuously in the assay medium and the magnitude of the signal generated in the vicinity of the sensing means being used as a measure of the extent to which the binding reaction has occured. Preferably the assay medium incorporates a masking or quenching agent which suppresses signal generated in regions of the assay medium remote from the signal sensing means. The device used by DAVIS is shown in the next figure. In accordance with another embodiment of the immuno-assay by DAVIS (175. 2) the sample is contacted with a movable solid phase carrier material, e.g. particles (2) on which is immobilised a first binding reagent having specificity for the analyte and with a labelled reagent which can participate in either a “sandwich” or a “competition” reaction with the first reagent in the presence of the analyte and, following an incubation period sufficient to allow the reaction to take place, the carrier material is moved within the assay medium (20) to a location adjacent a signal sensing means (19), in the immediate vicinity of which a signal influencing agent, e.g. a substrate, is released into the assay medium and cooperates with the label in the generation of a signal, such as chemiluminescent light, the magnitude of the signal generated in the vicinity of the sensing means being used as a measure of the extent to which the binding reaction has occurred. Preferably the assay medium incorporates a masking or quenching agent which

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suppresses any signal generated in regions of the assay medium remote from the signal sensing means. The next figure schematically shows the device used. Bosley et al. (UNILEVER NV (174.2)) designed an apparatus for carrying out a microchemical test, comprising a solid substrate having a surface which carries a polymer hydrogel formed in situ thereon and covalently bonded thereto, for example with the gel in contact with a metal conferring microchemical analytical specificity on the apparatus, e.g. an immunological reactant or an electrode. For example such a gel in a layer carried within a capillary-fill cell can be used for optical immunoassay. 3.2 STRIPS For immunoassays Koyama (KONISHIROKU PHOTO INDUSTRY CO., LTD. (91.2)) proposes a novel strip which functions according to the principle of the sandwich assay. Between two supportlayers several layers are positioned containing antigen, labelled antigen, Diffusion rate control layers, and a readily rupturable vessel with developer are also present. A cross section is depicted in the figure below. It is as well suited for RIA, ELISA or fluorescence techniques. Incubation with the sample to be measured takes 24 hours or more.

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A comparison with a prior art test is given in the table below. By Campbell et al. (BECTON DICKINSON AND COMPANY (16.5)) a strip was developed which uses fluorescent labels but can be visually determined without instrumentation, and further tracer treatment. As support nitrocellulose is used. Dyed liposomes are used as tracers, they are bound to the support or to the analyte. The binder is applied to the test area as a spot having a diameter of from 3 to 5 mm. The concentration of the binder placed in the defined test area will vary depending upon the assay to be performed; however, their binder is generally present in a concentration of, most generally, at least 10 g/cm2. Only distinct areas are treated with binder. The remainder of the test strip includes suitable blocking agent to prevent nonspecific binding. The test areas may be provided with antibodies to the analyte having a different affinity for the analyte. The assay protocol and procedure is designed in a manner such that the concentration and/or affinity of the antibody positioned in test areas are coordinated with the other assay parameters, such as tracer concentration, so that when the amount of analyte in the sample is below normal (low), there will be a visual readout in certain test areas. Those who correspond with an analyte concentration below a given value will be coloured. Those who stand for higher values will be colourless. See for an example the scheme below.

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Bernstein (NEW HORIZONS DIAGNOSTICS CORPORATION (114B)) developed a practical device for solid phase immunoassays. It consists of a swab to collect the sample, and a compartmentalized tube or box containing reagents, and a capture membrane (18) wherein a ligand receptor reaction can be examined. A test for group A streptococci resulted in a distinctive reaction within 5 minutes. Rapkin et al. (SMITHKLINE DIAGNOSTICS, INC. (155)) proposes a simple device for in situ whole blood separation and analysis. It consists of a carbohydrate layer covered with a reagent material both packed in a sandwich design between two layers of plastic. An opening in the laminated device is provided whereby the blood can directly contact the carbohydrate porous material. Christian (THE REGENTS OF THE UNIVERSITY OF CALIFOR\ NIA (181)) proposes a multiple assay card consisting of a three-layer laminate construction in which various recessed channels and chambers are interconnected with protruding flexible chambers and channels to provide a completely selfcontained analytical system adapted for treating and analyzing a sample utilizing a multiple assay rod. The configurations of the various channels and chambers are arranged so that a wide variety of analyses involving numerous steps can be accomplished using the card. The card is activated and analysis and/or treatment carried out by passing a roller bar or other pressure device over the top of the card to force solutions and reagents through the various card channels. In addition, the movement of the pressure bar over the card closes and opens various channels within the card to provide controlled and programmable transfer of solutions during treatment of the microassay rod. An assay rod having

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fluorescent marker layers is provided for use in analysis methods utilizing fluorescently marked detection substances. Hewett (HEWETT (60B)) discovered a method for detection of an immunoreactive substance which has capillary action as the motive force for movement of an aqueous assay medium where first and second specific binding pair members are bound to the surface. Complex formation with the first specific binding pair member inhibits complex formation with the second binding pair member. The distance traveled by the homologous specific binding pair member is indicative of the presence of an analyte. The homologous second binding pair member provides for a detectable signal. For determining blood cell type with binding to the red blood cell, antibodies to the red blood cell type antigen are employed and the formation of a relatively well defined front in a zone containing the antibody to the blood cell type antigen is indicative of the blood cell type. Giaever and Harrigan (GENERAL ELECTRIC COMPANY (61.3)) proposes to overcome faint images in immunological characterization due to non-specific sticking protein by using a device consisting of several layers of biological particles. The surface preferably being metallic, with the biological particles disposed within at least one first part of the area having the capability for interacting with other biological particles specific thereto and at least one second part of the area in which the biological particles do not have the aforementioned capability. Typically the first part of the overall area is shaped in a predetermined pattern to facilitate recognition of a positive test upon exposure to a liquid sample containing biological particles specific to those disposed within the first part of the area. Locally the upper surface is inactivated by inadiation. Exposing the top surface to a solution containing antibodies specific to the antigen, the non-irradiated antigen pattern will pickup the specific antibody while the deactivated (radiated) antigen will not. If the protein to be detected is absent from the serum, the predetermined patterns remain invisible. By Quash (INSTITUT NATIONAL DE LA SANT ET DE LA RECHERCHE MEDICALE (82.2)) a strip was developed wherein the solid support carries lateral chains of hydrazine derivatives and/or amino acids containing a—SH group for the binding of the antigen or antibody. An easily manageable strip is developed by Lefkovits (CELLDYNAMICS AG (30)). Various antigens are adsorbed on a sheet of waterinsoluble, adsorptive material, each sheet is cut into parallel tracks, the latter are individually affixed next to one another on a support foil and the track support foil is cut into parallel strips, across the longitudinal direction of the tracks. The test strips obtained are used for immunoassays, by being allowed to incubate with the biological liquid to be examined and subsequently with the solution of a conjugate of a

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complementary antibody and covalent-bonded enzyme, radioisotope, fluorescent agent or dye. Preferred are sheets of nitrocellulose with a pore size from 0·01 to 10 m. A test strip is 8 cm long, 3 mm wide and contains with a distance of 1 mm between them, 10 fields of 3 × 3 mm. The upper part serves for handling and encoding. To enhance the sensitivity of immunoassays Baier et al. (BOEHRINGER MANNHEIM GMBH (24.6)) developed a reagent paper. It consists of a fleece of cellulose/synthetic fibre in a weight ratio of 1 to 10, to 90/99, with a thickness of 0·1–1·5 mm and an open-pore structure. As synthetic fibre a.o. polyamide or polyester fibres may be used. The paper may be activated by NaIO4-treatment. The antibodies are before fixing to the support treated with an acid buffer at pH 2 to 4. A fleece of 6×6 mm may bind about 40 g antibody. It is pertained that during the test no desorption occurs, and shorter reaction times should be possible due to the high binding capacity. The preparation process is suited for a continuous production of the fleece. There is a prior art method, including two incubations for the sensitive determination of polyvalent antigens (peptides, proteins) with the use of two antibodies which are directed against different antigen determinant is known as the 2-site immunoradiometric or immunoenzymometric assay. However, this process suffers from some disadvantages: in the first incubation step, one reactant is present in solid phase and the other in solution. The velocity constant of the reaction is thus smaller than it would be were both reaction components. Moreover, the binding of a bindable antibody specific for the antigen on to a solid phase requires relatively large amounts of the first antibody. Finally, the obtaining of two antibodies of different specificity against the same antigen is, as a rule, laborious and frequently only possible to a limited extent. To avoid such disadvantages P.TANSWELL et al. (24.1) have found a process for the determination of a polyvalent antigen by incubation with three different receptors, the first and third of which are present in liquid phase and are capable of binding with the antigen and the second receptor is present in solid phase and is capable of binding with the first receptor. The third receptor carries a marking and does not cross-react with the first and second receptors, separation of the solid phase from the liquid phase and measurement of the marking in one of the phases, wherein the antigen is simultaneously incubated with all three receptors or with the first and second receptor, whereafter the phases are separated. The solid phase is possibly incubated with the third receptor and the phases are again separated. As receptors are used either specifically bindable complete antibodies, antibody fragments or conjugates of antibodies or antibody fragments with haptens. As first receptor,

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there is preferably used a complete antibody or an antibody covalently bound with a hapten. Many researchers in the field of immunoassay aim to the development of tests avoiding the need of effecting many operations. It is true that many clinical laboratory tests require much time and many operations which may include mistakes and faulty results, particularly when laborants have to do with qualitative tests which almost all require heterogeneous test methods. It is therefor that many researchers direct their interest to the development of solid state methods using strips or kits which, however, results in quantitative analysises only. In that such methods quite often are sufficient to immediately determine the presence of antibody representative to some desease, it will be obvious that in the literature many examples of strips and kits have been disclosed which in one aspect or the other may contribute to a quick and reliable quantitative test result. It was discovered that the patent literature studied during the present analysis cover various methods or processes for carrying out homogeneous assays and, simultaneousely herewith, disclose the means used, such as strips, sandwiches and so on. The present chapter only deals with the composition of such elements only. In the next figure a perspective vies is shown which schematically shows an embodiment of an element for quantitative analysis of an immunoreactive analyte, which element has been developed by KONDO (959.4). The element of immunoassay is denoted generally by reference numeral 10 and has a strip or web-like shape. The strip 10 comprises a support 13, and an antibody immobilizing matrix 11 and a labelled antigen retaining matrix 12 juxtaposed on the support 13. The antibody immobilizing matrix 11 contains a carrier for immobilizing an antibody for the analyte antigen so that the antibody can readily contacts with the components in the aqueous sample solution but the antibody is never released or dissolved in the aqueous sample solution. The matrix 11 provides a field or domain in which a competitive antigen-antibody binding reactions between three members of an antigen, a labelled antigen and a common antibody therefor take place, and has a function of retaining the antigen-antibody complex (B) and the labelled antigen-antibody complex (B) so that they are not dissolved in water. This

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leads to the result that both of these complexes are present only in the matrix 11 with the unreacted free labelled antigen (F) is present in water, whereby B/F separation is effected without any particular operations or steps purposely incorporated. See the following figure. Any known carriers may be used, as long as they contain no ingredients harmful to antigen-antibody reaction or the harmful ingredients or matters are not released even if such matters might be contained. Exemplary materials for the carrier include granulated, globular and plateshaped plastics, glass, fibers and gelled materials. On the other hand, examples of the material for the matrix which contains the carrier for the immunoreactive substance (i.e. the antibody in this instance) are micro-porous sheet-shapped materials, such as filter paper made of natural or synthetic polymer fibers, non-woven fiber cloth, micro-porous sheet dispersed therein with micro particles, polymer films having binder surfaces, and membrane filters. The carrier may be laminated with or mixed internally in the matrix. Alternatively, the antibody may be immobilized in the matrix per se so that the matrix is utilized as the carrier. In such as case, it is preferable to use a material having a large specific surface and selected from the materials including filter paper made of natural or synthetic polymer fibers, non-woven fiber cloth, microporous sheet including micro particle, polymer films having binder surfaces, and membrane filters.

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Testing for drugs An assay system and apparatus for detecting ligands in solution, typically drugs, such as morphine in body fluids e.g. urine, and semi-quantitively determining them by comparison with a reference, has been subject of research QUIDEL AND LEE (138). Haptens, upon being injected as simple chemicals, do not give rise to antibodies. However, antibodies can be raised against haptens when they are conjugated to antigenic carriers prior to using them for immunization. Some ligands of interest are drugs including opiates suchs as morphine and heroin, meperidine and methadone. Other drugs of interest include the amphetamines, narceine, epinephrine, ephedrine and L-Dopa. Their system comprises a reference solid support area comprising a first component of a reference coupling pair which is capable of coupling with a second component of a reference coupling pair. The second component of the reference coupling pair is labeled with a moiety which can be detected and measured. This second component is referred to as the reference conjugate. A test solid support area is also included in the apparatus, the test area comprising a receptor of an immunochemically binding pair. The other component of the immunochemically binding pair comprises the ligand to be determined, i.e. the test ligand. The receptor is capable of immunochemically binding the test ligand and an immunochemically identical ligand labeled with same moiety used to label the reference conjugate. In a preferred form, the reference coupling pair comprises avidin and biotin, and the immunochemically binding pair comprises a pair selected from the group of pairs consisting of (a) an antigen and an antibody to said antigen, (b) a hapten and an antibody to said hapten, and (c) a protein and an antibody to the protein. An example for carrying out the QUIDEL AND LEE method is as follows. I. Reagents: (i) Enzyme conjugate cocktail: contains 40 nM ALP-morphine conjugate and 7 nM ALP-biotin conjugate (the reference conjugate) in conjugate buffer. (ii) Dipstick: the reference pad (3/16″ × 1/8″) is attached to one end of a plastic handle (3/16″ × 2½″) and the indicator pad (3/16″ × 1/8″) is placed next to it. The reference pad contains avidin immobilized on paper. The indicator pad contains rabbit anti-morphine immobilized on paper. (iii) Developer Solution: 5 mM 5-Bromo-4-chrolo-3-indolyl phos phate in 0·3 M AMP buffer at pH 10·15 with 0·02% NaN3. II. Assay procedure:

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(i) Pipette 400% 1 of sample into a 10 × 50 mm glass tube. (ii) Add 100% 1 of enzyme conjugate cocktail. Vortex. (iii) Place dipstick in solution and incubate for 10 minutes at room temperature. (iv) Remove dipstick. Wash for 20 seconds under tap water. (v) Place dipstick in developer solution and incubate for 10 minutes at room temperature. (vi) Remove dipstick and blot on a clean paper towel. (vii Blue color is quantitated by reading the pads on a MacBeth reflectance ) photometer. The next figure shows the responses of both the indicator and the reference pad to samples containing different concentrations of morphine. The quantity K/S plotted on the Y-axis is related to reflectance R by the equation k/S =(1-R)2/2R, and is linearly proportional to the amount of the blue dye deposited on the pads. Multizone, particularly multilayer or sandwich analytical elements are well known in the art of immunoassay, which inherently perform the required separation step so that no additional manipulations are needed after application of the liquid test medium. Examples thereof have been given in e.g. chapter 1.1.2 of the present study. GREENQUIST (109.5 and 6) discoverd a specific binding assay in a multizone, or multilayer, test device which concentrates the detectable reaction product of the interaction between a chemical label group and an interactive detection reagent therefor without interfering with the specific binding reactions involved in the assay. Its principal advantages resides in the use of a labeled reagent which provides a detectable signal in the form of a detectable reaction product as a result of the

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interaction of the labeled reagent with an interactive detection reagent and which can be rendered immobilized in the detection zone. No separately migratable detectable product is generated as with prior art devices and immobilization and concentration of the detectable reaction product results from highly specific chemical interactions. The test device comprises, in fluid flow contact, (1) a reagent zone incorporated with the immmobilized reagent which will be an immobilized form of the analyte or a binding analog thereof, or an immobilized form of a binding partner of the analyte, depending on the immunoassay scheme used, and (2) a detection zone incorporated with an immobilized form of an interactive detection reagent for the labeled reagent. The labeled reagent is a form of a binding partner of the analyte, or a form of the analyte or a binding analog thereof, which is labeled with a chemical group having a detectable chemical property which generates a detectable product upon interacting with the immobilized interactive detection reagent in the detection zone. The immobilized reagent in the reagent zone and the labeled reagent are selected to comprise specific binding partners which will bind to one another dependent upon the amount of analyte present. When the labeled reagent is a labeled form of the analyte or an analog thereof, the immobilized reagent will be an immobilized form of a binding partner for the analyte, and the analyte and labeled reagent will compete for binding to the immobilized reagent. When the labeled reagent is a labeled form of a binding partner for the analyte, the immobilized reagent will be an immobilized form of the analyte or an analog thereof, and the labeled reagent that does not become bound to analyte will become immobilized by binding to the immobilized reagent. Whether labeled analyte or labeled binding partners are involved, a portion of the labeled reagent will remain or become unbound to the immobilized reagent dependent upon the amount of analyte present. 3.3 TUBES In RIA-assays frequently a second antibody directed against the complex of the first antibody and the antigen is used. Trouyez (BIOMERIEUX (21)) proposes to bind the second antibody to the bottom of tubes of which 48 are mounted together in a container. Sapatino and Johnson (BECTON, DICKINSON AND COMPANY (16.7)) developed a multi-well screening system useful for the screening of hybrid cell cultures such as hybridomas. A carrier tray is equipped with a plurality of posts, each of them adapted to bind an immunogenic substance. The tray is positioned on a multi-well assembly wherein each well is aligned with a post. The device may also be useful in immunoassays.

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In a round-bottomed test tube non-agglutinated erythrocytes manifest itself as a more or less thick ring. Scheepens et al. (AKZO N.V. (3.1)) propose to improve the readability of immunoassays by using test tubes having a bottom in the shape of an inverted prism or truncated prism, such that the lower edge of the test tube forms a strip or line having a length which is at least equal to one-fourth of the distance between 2 opposite walls of the test tube or at least equal to one-fourth of the diameter of the test tube. For detecting immunoreactive components Hibino et al. (DAIICHI PURE CHEMICALS CO., LTD. (47.2)) developed a simple technique. It consists of a transparent capillary tube packed with labelled immunoreagents and a solid support. If necessary, the capillary tube may additionally be packed with the solid matrix holding the substrate, chro-mogenic agent, inhibitor and the like. A tube may contain several markers. The uptake of the sample may take from about 1 minute to about 9 minutes. Clagett (ULTRA DIAGNOSTIC CORPORATION (173)) proposes a device including one or more reaction chambers. Each reaction chamber is adapted to receive and retain a volume of test fluid in fluid communication with nonoverlapping first and second reaction surfaces. To the first reaction surface is immobilized an analyte binding partner that is in turn saturated with analyte conjugate: analyte component conjugated to one or more components, termed ligand/marker, that serve ligand and marker function. The analyte conjugate has a higher disassociation constant with reference to the immobilized analyte binding partner than does the analyte to be assayed. To the second reaction surface is immobilized the ligand binding partner. A test fluid sample is introduced into the reaction chamber and retained therein to permit two reactions. In a first reaction between analyte and analyte binding partner at the first reaction surface, analyte proportionately displaces analyte conjugate into the test fluid sample. In a second reaction the displaced analyte conjugate becomes sequestered at the second reaction surface by bonding with immobilized ligand/ marker binding partner. Thereafter the marker activity of sequestered analyte analog is measured, the measured activity being a function of the analyte concentration that is referable to standards and controls. According to Davis and Porter (UNILEVER NV (174.1)) specific binding assays can be carried out more efficient by mixing the sample, the specific binding partner, and the labelled form of the binding partner in a single reaction liquid. Competitive interference of the binding partner and its labelled form is prevented by the use of a slow-release form of the latter, such as a sucrose glaze. The binding partner may be bound to a solid support in the shape of posts of which a plurality is arranged on a plate. The plate can be lowered upon a tray with tubes corresponding with the posts.

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3.4 MISCELLANEOUS Bensinger (FRED HUTCHINSON CANCER RESEARCH CENTER (58)) improved the binding of Staphylococcus Protein A (SPA) by binding covalently to a support by first coating the silica support with an alkylamine and then coupling SPA to the alkylamine by means of a carbodiimide. It may be applicated in the removal of immunoreactive reagents like circulating immune complexes in the plasma of tumor hosts as immunoabsorbent. In the continuous treatment of blood from a patient the immunoabsorbent material may comprise one to three milligrams of the microbial ligand for each gram of support, and about 20–40 ml of the plasma component may be passed through the immunoadsorbent material per minute. By Place (BATTELLE MEMORIAL INSTITUTE (13)) particles were developed whose volume, and hence their density, depends on pressure and temperature. After rising such particles as solid support in an immunoassay separation from a liquid can be facilitated by changing the density to one higher than the density of the liquid. To prevent self-agglutination Dorsett (THE UNIVERSITY OF TENNESSEE RESEARCH CORP. (191.1)) recomments to treat the antigen which is bound on a solid particle, with a detergent like sodium dodecyl sulfate. It is employed to reduce the weight of the antigen which preferably does not exceed 150.000, and used in such an amount and time that antigenic characteristics are not disrupted. Treatment of the antigen prior to binding to support is preferred. Degen et al. (PALL CORPORATION (126)) developed a biologically active membrane. It consists of hydrophylic, microsporous polyamide. After treatment with an activating agent, like trichloro-S-triazine, and reacting with an acceptor molecule it can be used for recovery of ligands or affinity chromatography. As acceptor a wide variety of biological active compounds can be used. Huang and Ho (HUANG AND HO (71+191.2)) developed a membrane lytic immunoassay. In one embodiment of this assay, an antigen is first covalently coupled with lipids and this antigen-lipid complex is mixed with a hexagonal phase forming lipid to form bilayer liposome vesicles additionally containing a self-quenching fluorescent dye. When this antigen-containing liposome is brought into contact with a solid surface coated with antibody molecules, binding occurs between the antigen and the antibody, disrupting the liposome and releasing the dye. To assay a biological fluid for free antigen the fluid is first contacted with the solid surface-antibody complex, to saturate the bound antibody. Binding by the liposomes is thereby inhibited, leading to reduced dye release. Comparing dye release against a standardized curve of dye release

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versus known antigen concentrations allows for rapid determination of the unknown antigen concentration in the biological fluid.

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LIST OF PATENTEES

Page 1)

Abbott Laboratories

2) 2A) 3)

Advanced Polymer Systems, Inc. Advanced Magnetics Inc. Akzo N.V.

4)

Allexis Inc.

5)

Allied Corporation

1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17)

1) 2) 3) 4) 1) 2) 3) 1) 2)

US 4,751 190 US 4,668,640 EP 0 195 320 EP 0 199 042 EP 0 199 963 EP 0200 960 EP 0 201 751 EP 0204 922 EP 0206 014 EP 0210 410 EP 0 216 191 EP 0 217 403 EP 0226 903 EP 0 232 736 EP 0240 021 EP 0242 847 US 4,743,561 PCT/WO 87/03692 EP 0 125 995 US 4,628,036 US 4,701,410 US 4,701,421 EP 0 227 169 US 4,665 034 US 4,690,801 EP 0 241 611 US 4,629,689 EP 0 201 755

64 67 3 226 254 233 68 177 227 63 188 36 197 70 64 68 88 201 115 276 205 205 192 46 46 263 3 91

274 LIST OF PATENTEES

6) 7) 8) 9)

Alto Diagnostics Machines Ltd. American Hoechst Corporation American National Red Cross Amersham International plc.

10) 10A) 11) 12)

Amoco Corporation Angenics Inc. Apothekernes Laboratorium A.S. Baker, T.S. et al.

13) 14)

Battelle Memorial Institute Baylor College of Medicine Texas Medical Center Beckman Instruments, Inc. Becton, Dickinson & Company

15) 16)

17) 18) 19) 20) 21) 22) 23) 24)

Behringwerke Aktiengesellschaft Bio-Magnetech Corporation Bio-Rad Laboratories, Inc. Bio-Technology Australia Proprietary Ltd. Biomérieux, S.A. Biometallics Inc. Biostar Medical Products, Inc. Boehringer Mannheim GmbH

1) 2) 3)

1) 2)

EP 0 223 427 US 4,645,737 PCT/WO 86/07463 US 4,666,863 EP 0246 846 PCT/WO 86/07462 EP 0 232 967 PCT/WO 88/01374 PCT/WO 87/04252 US 4,656,143 EP 0086 095

97 201 92 101 98 4 131, 141 260 250 126 231

EP 0 224 439 EP 0 082 789

277 203

PCT/WO 86/06004 1) US 4,636,478 2) US 4,670,406 3) US 4,680,275 4) US 4,693,970 5) US 4,703,017 6) US 4,707,453 7) EP 0 087 899 8) EP 0 245 981 EP 0 117 436 US 4,677,067 US 4,704,366 PCT/WO 87/05702

263 109 71, 145 67 30 268 72, 146 276 1 154, 169 114 146 133

EP 0 078 734 PCT/WO 87/018 11 PCT/WO 86/07 152 1) US 4,624,930 2) US 4,639,425 3) US 4,670,383 4) EP 0 209 155 5) EP 0 215 457 6) EP 0 226 182 7) EP 0 227 921

276 250 156, 170 271 156, 171 25, 162 6 156 270 262

LIST OF PATENTEES 275

8) 25) 26) 27) 27A) 28) 29) 30) 31) 32)

Bogoch, S. Boots-Celltech Diagnostics Ltd. Bowman Gray School of Medicine, The Burton, J, et al. California Biotechnology Cambridge Bioscience Corporation Celldynamics AG Center for Immimological Studies, The Centocor, Inc.

33)

Centre Nationale de la Recherche

34)

Cetus Corporation

35) 36) 36A) 37) 38) 39)

Chang, T. W. Chester, S. J. Chiron Corp. Ciba-Geigy AG Cliniques Universitaires de Kinshasa Commissariat a l’Energie Atomique

40)

42) 43)

Commonwealth Scientific and Industrial Research Organisation Commonwealth Serum Laboratories Commission Cooper Biomedical, Inc. Cooper-Lipotech, Inc.

44) 45)

Cornell Research Foundation, Inc. Coulter Corporation

46)

Cytrx Biopool Ltd.

47)

Dalichi Pure Chemicals Co., Ltd

41)

1) 2) 3) 4) 5) 1) 2)

1) 2)

1) 2) 1) 2) 3) 1) 2) 1)

EP 0 243 655 US 4,624,932 PCT/WO 87/02774 PCT/WO 86/06489 PCT/WO 88/04429 PCT/WO 86/06742 EP 0 233 045 PCT/WO 87/03965 US 4,62 1,063 US 4,666,865 EP 0 114818 EP 0 225 709 PCT/WO 87/05399 PCT/WO 89/01629 EP 0 211 777

43, 147 25 45 8 109, 111 7 195 270 100 241 153, 185 179 193 182 263

US 4,690,890 EP 0 240 200 EP 0 199 438 US 4,687, 734 EP 0 318 216 EP 0 206 302 EP 0 238 396 EP 0 246 152 PCT/WO 87/00927 PCT/WO 87/05400

157 120 194 182 207 34, 129, 140 121 123 81 137, 165

PCT/WO 83/01119

48

US 4,617, 262 US 4,636,479 PCT/WO 87/02778 US 4,693, 969 US 4,708, 930 PCT/WO 87/01392 PCT/WO 87/03096 PCT/WO 87/06346 PCT/WO 89/00292 US 4,680, 274

7 204 132, 148 40 175 153, 176 178 102, 161 102, 161 159, 171

276 LIST OF PATENTEES

2) 48) 49) 50) 51) 52) 53) 53A) 54)

Daikin Industries, Ltd. Dana-Farber Cancer Institute, Inc. Diagnostic Products Corporation Diamedix Corporation DNA Plant Technology Operating Co. Doellgast, G. J. Duke University Eastman Kodak Company

55) 56) 57) 57A)

Enzo Biochem, Inc. Epitope, Inc. Farmos-Yhtymä Oy Fraunhofer-Gesellschaft zur Förderung der Angewandten Forschung Fred Hutchinson Cancer Research Center

58)

59)

Fuji Photo Film Co., Ltd

60)

Fujirebio K.K.

60A) Gen Probe Incorporated 60B) Gene Labs Inc. 61) General Electric Company

62)

General Hospital Corporation, The

63)

Genetic Systems Corporation

64)

Green Cross Corporation, The

US 4,690, 907 EP 0 199 367 US 4,692, 405 EP 0 245 926 US 4,642, 285 EP 0 222 998 PCT/WO 86/06489 US 4,751, 181 1) US 4,703, 002 2) US 4,670, 381 3) EP 0 200 541 EP 0 212 670 EP 0 248 534 EP 0 227 240 PCT/WO 88/04328

276 39, 141 175 160 204 248, 256 8 150, 253 123, 127 9 39, 139 137, 164 190, 197 211 10

1)

US 4,614,513

277

2)

PCT/WO 89/03532

198

1) 2) 3) 1) 2) 3) 4)

US 4,650,769 EP 0 097 952 EP 0 236 768 US 4,621,048 US 4,649,105 EP 0 094 777 EP 0 233 061 PCT/WO 88/07539 PCT/WO 88/03650 US 4,619,904 US 4,634,681 US 4,672,024 PCT/WO 87/06263 PCT/WO 88/03174 US 4,711,840 EP 0 201 716 PCT/WO 86/07464 EP 0 085 402

117, 119 73 26 38 10,11 39 190 243 269 157, 171 99 269 224 229 132, 149 192 210 235

1) 2) 3) 1) 2) 1) 2) 3)

LIST OF PATENTEES 277

65)

Gruppo Lepetit S.p.A.

66) 67)

Guigan, J. Harvard College, President and Fellows of Henning Berlin GmbH Chemie—& Pharmawerk

68)

69)

1) 2)

EP 0 221 282 124, 128 EP 0 248 144 11 US 4,673,653 263 PCT/WO 87/02988 197

1) US 4,645,646 2) US 4,657,873 3) EP 0 236 997 EP 0 199 261

70)

Heyl Chemisch-pharmazeutische Fabrik GmbH & Co. KG> Hitachi, Ltd.

71) 72) 73) 74)

Huang, L. and Ho, R.J.Y. Hurwitz, C. et al. Hoffman-La Roche, Inc. Hoffman-La Roche & Co. AG., F.

75)

Hybritech Inc.

76) 77)

Immunicon Corporation Immunotech S.A.; Lyonnaise des Eaux S.A. Imre Corporation US 4,711, 839 Imreg, Inc. EP 0 238 851 Incstar Corporation EP 0 231 010 Institut für Immunpathologie EP 0 205 643 RuprechtKarls-Universität; Rauterberg, E.W. Institut National de la Santée et de 1) EP 0 214 053 la Recherche Médicale (Inserm) 2) EP 0 230 166 3) PCT/WO 88/01745 International Genetic Engineering, Inc. EP 0 237 252 International Immunoassay Laboratories, US 4,618,485 Inc. International Institute of Cellular and EP 0 101 228 Molecular Pathology

78) 79) 80) 81)

82)

83) 84) 85)

86 130, 141 82 216

1) US 4,628,035 116 2) EP 0 222 341 66 3) EP 0 241 042 57 US 4,708,933 278 US 4,645,745 216 US 4,623,621 107 1) EP 0 058 780 35, 257 2) EP 0 199 301 193 3) EP 0 219 106 200 1) US 4,632,901 49 2) EP 0 209 994 259 PCT/WO 86/06170 138, 154, 169 EP 0 210 107 262 184 188 28 159

50 270 248 178 105 154, 169

278 LIST OF PATENTEES

86)

Internationale Octrooi Maatschappij ‘Octropa’ b.v. 86A) IQ/Bio Ltd. 87) Janssen Pharmaceutica N.V. 88) Juridical foundation, The Chemo-Sero Therapeutic Research Institute 89) Kanebo, Ltd 89A) Keminy, D.M. 90) Kerstensteiner, D. 91) Konishiroku Photo Industry Co., Ltd

92) 93) 94) 95)

Koppers Agri-Research Company; DNA Plant Technology Operating Co. Krauth, G.H. Kung, V.T. and Canova-Davis, E. Kyowa Hakko Kogyo Co., Ltd.

96)

Lab-Systems Oy

97) 98)

Lee, H. and Canavaggio, M.E. Lurhuma, Z.; Cliniques Universitaires de Kinshasa Lyonnaise des Eaux S.A.; Immunotech S.A. Mallinckrodt, Inc. Mallinckrodt Diagnostica (Germany) GmbH Marcucci, F. Mast Immunosystems, Inc. Max-Plank-Gesellschaft zur Förderung der Wissenschaften e.v.

99) 100) 101) 102) 103) 104) 105) 106) 107)

McKenzie, H; University of Dundee Meloy Laboratories, Inc Merck & Co. Inc.

108)

Microgenics Corporation

US 4,637,985

82

PCT/WO 88/04428 52 EP 0 227 173 59 EP 0 212 522 236

1) 2) 3) 1) 2)

1) 2) 1) 2)

PCT/WO 87/02780 PCT/WO 87/02780 US 4,623,629 US 4,613,567 US 4,615,983 US 0 226 888 EP 0 222 998 EP 0 246 643 US 4,666,866 US 4,622,294 EP 0 218 257 EP 0 242 727 PCT/WO 86/06493 PCT/WO 87/01810 PCT/WO 86/05591 EP 0 238 396

162, 172 260 108 162 267 176 248 256 213 43, 144 181 182 112, 264 53 136, 162 121

EP 0 210 107

262

US 4,656,251 US 4,654,299

249 131, 147

PCT/WO 87/02465 221 EP 0 214 399 81 1) EP 0 198 259 211 2) PCT/WO 89/00164 214

1) 2) 3) 1)

EP 0 233 048 US 4,639,419 US 4,617,260 EP 0 211 756 EP 0 218 531 US 4,708,929

234 232 203 202 185 14

LIST OF PATENTEES 279

109)

Miles Laboratories, Inc.

110) Mitsubishi Chemical Industries, Ltd. 110A) Mochida Seiyaku K.K. 111) Modern Diagnostics, Inc. 112) 113)

Molecular Diagnostics, Inc. Murex Corporation

114) Murex Medical Research Ltd. 114A) National Research & Development Corp. 114B) Nerbe 115) Nicoli, D.F. and Elings, V.B. 116) Nihon Chemical Research K.K. 117) Nilsson, U.R.

2) 1) 2) 3) 4) 5) 6)

1) 2) 1) 2) 3) 4)

PCT/WO 89/02597 US 4,622,293 US 4,629,688 US 4,629,692 EP 0 209 702 EP 0 212 599 EP 0 212 603 US 4,672,045 US 4,690,908 EP 0 206 779 EP 0 210 863 EP 0 210 449 PCT/WO 87/02779 PCT/WO 87/03690 PCT/WO 87/06006 PCT/WO 87/06345 PCT/WO 87/03374 PCT/WO 88/01746

35 28 14 210 137, 163 274 274 122, 126 88 125 231 84 155, 170 117, 121 15 101 200 85, 260 268 86 24 143 165, 172 261 176 176 247 103 42, 145 65 36 163 58 197 175 244 278

118) 119) 120) 121)

Olympus Optical Co., Ltd. Oncogene Science, Inc. Oncos, Ltd. Orion Corporation, Ltd.

122)

Ortho Diagnostic Systems Inc.

123)

Ortho Pharmaceutical Corporation

124) 125)

Otsuka Pharmaceutical Co., Ltd. Oxoid Limited

PCT/WO 88/03648 US 4,647,544 EP 0228 8 10 EP 0 241 443 EP 0 241 444 US 4,66 1,460 EP 0 214 520 EP 0 226 468 US 4,652,518 EP 0 218 188 US 4,66 1,444 US 4,681,859 EP 0 228 225 EP 0 249 418 US 4,657,760 PCT/WO 87/06005 EP 0 100 914 US 4,692,417

126)

Pall Corporation

US 4,693, 985

1) 2)

1) 2) 1) 2) 3) 4) 1) 2)

280 LIST OF PATENTEES

127) 128) 129)

Pandex Laboratories, Inc. Paragon Diagnostics Pasteur, Institut

130) 131) 132)

Pasteur de Lyon et du Sud-Est, Inst. Pasula, M.J. Pharmacia Aktiebolaget

133) 134) 135)

Pitman-Moore, Inc. Plessey Overseas Ltd. Pont de Némours and Company, E.I. du

1) 2) 3) 4) 5) 6) 7)

1) 2)

1) 2) 3)

135A) 136) 137) 137A) 138) 139)

Praxis Biologics Inc. Prince Henry’s Hospital Profile Diagnostic Sciences Inc. Proscience Inc. Quidel Rauterberg, E.W.

140)

Research Corporation

140A) Rijksuniversiteit Utrecht 141) Riker Laboratories, Inc. 142) Roussel-Uclaf 143) Royal Free Hospital School of Medicine, The 144) Salk Institute for Biological Studies 145) Sanofi 146) Saxholm, R. 147) Schaffner, C.P. 148) Schnaper, H.W.

1) 2) 1) 2) 3)

US 4,652, 533 PCT/WO 83/04102 US 4,640, 897 US 4,677, 055 US 4,708, 818 EP 0 122 833 EP 0 206 842 EP 0 242 300 PCT/WO 87/04459 EP 0 214 057 US 4,614,722 US 4,711, 841 EP 0 213 093 EP 0 223 349 EP 0 205 236 US 4,657,853

82 74 220 115 192 16 192 208 193 201 221 118 41, 124, 128 249 92 17

EP 0 198 413 EP 0 227 440 PCT/WO 88/04932 PCT/WO 87/05702 US 4,663,277 PCT/WO 88/04325 EP 0 203 238 EP 0 205 643 EP 0 214 330 EP 0 107 861 EP 0 242 589 PCT/WO 87/02777 PCT/WO 88/03649 PCT/WO 87/06578 EP 0 235 000 PCT/WO 86/05593

259 25 255 133 259 248 273 139, 159 123 215 124, 129 151, 168 152 224 110, 152 202

PCT/WO 87/04251 253 US 4,690,906 206 US 4,657,868 112, 264 US 4,639,420 256 US 4,665,021 235

LIST OF PATENTEES 281

149)

Scripps Clinic and Research Foundation

150)

Serono Diagnostics Limited

151) 152) 153) 154) 155) 156) 157) 158) 159) 160) 161)

162)

163) 164) 165) 166) 167) 168) 169) 170)

1)

US 4,677,057

218

2)

PCT/WO 88/01273 US 4,659,678

17 166, 173

Serono Diagnostics Partners 1) (A Massachusetts Limited Partnership) 2) Shattock, A.G. and Morgan, B.M. Shionogi & Co., Ltd. 1) 2) Sloan-Kettering Institute 1) 2) Smithkline Diagnostics, Inc. Sockerbolaget AB Southwest Foundation for Biomedical Research Staat der Nederlanden, Minister van Welzijn, Volksgezondheid & Cultuur State of Victoria, The Synbiotics Corporation Syntex (U.S.A.), Inc. 1) 2) 3) 4) 5) 6) Syva Company 1) 2) 3) T Cell Sciences, Inc. Takeda Chemical Industries, Ltd. Technicon Instruments Corporation Technogenetics S.p.A. Teijin Limited 1) 2) Teva Pharmaceutical Industries, Ltd. Tel Aviv University Tokyo Shibaura Denki K.K. Toray Industries, Inc. 1)

EP 0 238 353 EP 0249 357 US 4,619,896 US 4,686, 179 EP 0200 507 US 4,649, 115 US 4,634,682 EP 0 198 628 EP 0241 140 PCT/WO 87/02775

19 18, 102 209 34, 151, 167 31 166, 173 255 268 42, 142 194

EP 0 233 385

263

PCT/WO 86/06382 US 4,703,001 US 4,629,690 US 4,650,770 US 4,652,531 US 4,654,300 EP 0202 081 EP 0 243 001 US 4,663, 278 EP 0 100 619 EP 0 103 958 PCT/WO 87/05912 EP 0210 839 US 4,650,951 PCT/WO 87/03375 PCT/WO 86/07154 PCT/WO 89/01624 US 4,707,438

249 134, 260 31 61 71 60 33 37, 145 20 21 21 187 187 238 197 251 126, 129

US 4,623,618 US 4,656,144

121, 122

282 LIST OF PATENTEES

2) 171) 172)

Toshiba K.K. Toyo Soda Manufacturing Co., Ltd.

173)

Ultra Diagnostic Corporation

174)

Unilever N.V.

175)

Unilever PLC

176) 177)

United Biomedical Inc. United States of America as represented by the Secr. US Department of Commerce

178) 179)

United States Department of Energy US as represented by the Department of Health and Human Services

180) 181)

University of Birmingham, The University of California, The Regents of University of Columbia in the City of New York University, Duke (see 53A) University of Dundee University of Health Sciences/The Chicago Medical School Universität Humboldt, zu Berlin Universiteit Centrum, Limburg Dr. L. Willems Instituut University of Missouri University, Monash University, The Rockefeller University, Tel Aviv; Teva Pharamaceutical Industries, Ltd.

182)

183) 184) 185) 186) 187) 188) 189) 190)

1) 2) 3)

PCT/WO 87/06007 EP 0 132 556 EP 0212 455 EP 0216 177 EP 0 234 083 EP 0201 339

144 89 75 54 33 276

1) EP 0 111 762 277 2) EP 0 226 470 140, 266 1) EP 0 247 796 55, 113, 265 2) PCT/WO 88/01744 265 EP 0 214 709 195 1) EP 0 202 890 190 2) PCT/WO 89/01340 199 3) EP 0 249 394 196 4) PCT/WO 87/05394 190 5) PCT/WO 87/06258 197 6) PCT/WO 88/01387 202 US 4,665,020 62 1) US 4,622,291 77 2) US 4,676,980 123, 127 3) US 4,708, 818 192 US 4,618,589 43, 159 US 4,673,657 269 PCT/WO 87/00289

236

US 4,751,181 EP 0 233 048 US 4,640,898

150, 253 234 61

EP 0 222 146 PCT/WO 86/06491

219 134, 152

US 4,677,080 245 PCT/WO 87/05702 133, 228 PCT/WO 86/06171 216 US 4,707,438 182

LIST OF PATENTEES 283

191)

University of Tennessee Research Corp.

1)

US 4,695,537

278

2) University System, The Texas A&M University of Virginia Alumni Patents Foundation 194) University Research Foundation, Washington State 195) Upjohn Company, The 196) Vasocor

PCT/WO 87/04795 US 4,692,403 PCT/WO 87/00526

278 188 212

EP 0 196 290

249

US 4,614,712 EP 0 248 630

22 218

197) 198) 199) 200) 201) 202)

PCT/WO 87/05702 EP 0 217 583 EP 0 231 830 EP 0 213 106 EP 0 201 211 PCT/WO 86/06491

133 244 23 198 79 127

US 4,708,862 EP 0 205 177 US 4,622,292 EP 0 201 079

183 222 239 161

192) 193)

Vincent’s Institut of Medical Research VXR, Inc. Wako Pure Chemical Industries, Ltd. Waldheim Pharmazeutika Gesellschaft mbH Whittaker Corporation Willems Institut, Dr. L.; Limburg Universiteit Centrum 203) Xoma Corporation 204) Yamasa Shoyu K.K. 205) Yeda Research & Development Co., Ltd. 206) Zahnradnik, R.

284

REGISTER OF KEYWORDS

Absorbing material, 63 Agglutination assay, 87 Aldosterone, 145 Aldosterones, 162 Alkylene oxide chains, 140 Anti-enzyme antibody, 14

Glycocylated polypeptides, 165 Haemoglobin antigen research, 94 Hapten Sandwich Assay, 124 Heavy Antigens, 62 Heparin-albumin conjugate, 147 Human C3 and C3a, 143

Biotin, 3 Biotinated-β-galactosidase, 3 Blocking agents, 26

Idiotypic binding assay, 165 Idiotypic conjunction binding, 150 Immobilisation, 127 Inhibin, 129 Integrated control, 158 Iodine marked pyréthrinoîdes, 107 Iodine marked pyrethrinoids, 147

CA-19-9 antibody, 177 Carbohydrate CA 19-9 determinant, 148 Carbohydrates, 138 Cell analysis, 53 Chemiluminescent reactions, 80 Creatine Kinase MB, 23 Crosslinked enzymes, 15 Cross-linked heteroantibodies, 119 Crosslinking, 115

Kallikrein, 103 Latex, 136 Latex-agglutination, 149, 163 Liposomes, 128, 140 Luminescent substrate preparation, 76 Luminescence suppressing agent, 78

D-Alanyl-D-Alanine dipeptides, 120 DNA-bonds, 135 Double solid phase, 156 Dual analyte method, 18

Magnetic gel, 111 Magnetic means, 255 Microbead quenching, 57 Microcapsules, 113 Monomer conjugates, 143 Mouse monoclonal antibodies, 160

Electrophoresis, 112 Enzyme binding, 12 Flow cytometer measurement—Laser beam, 59 Fluorescence Polarization, 59

NA-hybridization, 137

Gamma interferon, 133 Globulins, 113

Oligosaccharide, 147 One step method, 21 285

286 REGISTER OF KEYWORDS

Optical interference detection, 83 Polymer coating, 119 Polymer hydrogel, 136 Polypeptide antibodies, 193 Polypeptide coating, 137 Polysterene latex, 117 Polystyrene binding, 142 Polytetrafluoroethylene porous matrix, 147 Protein A binding, 141 Protein coated droplets, 152 Serum amyloids A and P, 136 Single liquid sbp’s, 140 Skin cells, 168 Solid phase hybridization, 158 Solid-phase support, 105 T4 measurement, 106 T3 and T4 detection by thyroglobuline, 119 Tracer composition, 141