Handbook of Experimental Pharmacology

Volume 82

Editorial Board

G.y' R. Born, London P. Cuatrecasas, Research Triangle Park, NC H. Herken, Berlin A. Schwartz, Cincinnati, OH

Radioimmunoassay in Basic and Clinical Pharmacology

Contributors

L. Bartalena· K. Brune' F. Celada . I. Christens son-Nylander G. Ciabattoni . D. Cocchi· M. G. Currie' 1 Dawes' F. Dray E. Ezan . D. M. Geller' 1 Grassi . V. Guardabasso . E. C. Hayes E. Knoll· G. A. Limjuco . 1 Mac1ouf· S. Mamas' S. Mariotti M.L. Michener' P.I Munson' M.G. Murphy' P. Needleman G. Nilsson' F. Nyberg' C.W. Parker' C. Patrono . M. Pazzagli D. S. Pepper' B.A. Peskar . B. M. Peskar . A. Pinchera P. Pradelles . M. Reinke' D. Rodbard . C. Rougeot . D. Schwartz M. Serio . Th. Simmet . S. Spector' L. Terenius . L. E. Underwood lH.Walsh· H.Wisser· H.C.Wong· R.S.Yalow· H.I Zweerink

Editors

C. Patrono and B.A. Peskar

Springer-Verlag Berlin Heidelberg New York London Paris Tokyo

CARLO PATRONO, Professor of Molecular Pharmacology

Institute of Pharmacology Catholic University of the Sacred Heart Largo Francesco Vito, 1 1-00168 Rome

BERNHARD A. PESKAR, Professor of Pharmacology and Toxicology

Institute of Pharmacology and Toxicology Ruhr-University Bochum 1m Lottental, P.O. Box 102148 D-4630 Bochum 1

With 129 Figures

Library of Congress Cataloging-in-Publication Data. Radioimmunoassay in basic and clinical pharmacology. (Handbook of experimental pharmacology; v. 82) Includes index. 1. Radioimmunoassay. 2. Biomolecules - Analysis. 3. Drugs-Analysis. I. Bartalena, L. II. Patrono, Carlo, 1944- . III. Peskar, B. A. (Bernhard A.) IV. Series. [DNLM: 1. Hormones-analysis. 2. Peptides-analysis. 3. Radioimmunoassay. WI HA51L v.82/QW 570 R1294] QP905.H3 vol. 82 615'.1 s 87-9659 [QP519.9.R3] [615'.1901]

ISBN-13: 978-3-642-71811-3 DOl: 10.1007/978-3-642-71809-0

e-ISBN-13: 978-3-642-71809-0

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, re-use of illustrations, recitation, broadcasting, reproduction on microfilms or in other ways, and storage in data banks. Duplication of this publication or parts thereof is only permitted under the provisions of the German Copyright Law of September 9, 1965, in its version ofJune 24, 1985, and a copyright fee must always be paid. Violations fall under the prosecution act of the German Copyright Law.

© Springer-Verlag Berlin Heidelberg 1987

Sofkover reprint of the hardcover I st edition 1987

The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

Product liability: The publisher can give no guarantee for information about drug dosage and application thereof contained in this book. In every individual case the respective user must check its accuracy by consulting other pharmaceutical literature.

2122/3130-543210

List of Contributors

L. BARTALENA, Istituto di Metodologia Clinica e Medicina del Lavoro, Cattedra di Endocrinologia e Medicina Costituzionale, Universita degli Studi di Pisa, Viale del Tirreno, 64, 1-56018 Tirrenia (Pisa)

K. BRUNE, Institut fUr Pharmakologie und Toxikologie, Universitat Erlangen­Niirnberg, Universitatsstr.22, D-8520 Erlangen

F. CELADA, Cattedra di Immunologia dell'Universita degli Studi di Genova, Viale Benedetto XV, 10, 1-16132 Genova

I. CHRISTENSSON-NYLANDER, Department of Pharmacology, University of Uppsala, Box 591, S-75124 Uppsala

G. CIABATTONI, Istituto di Farmacologia, Universita Cattolica del Sacro Cuore, Facolta di Medicina e Chirurgia "Agostino Gemelli", Largo Francesco Vito, 1, 1-00168 Roma

D. COCCHI, Universita degli Studi di Milano, Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, Via Vanvitelli, 32, 1-20129 Milano

M. G. CURRIE, Medical University of South Carolina, Department of Pharma­cology, 171 Ashley Ave., Charleston, SC 29425, USA

J. DAWES, MRCjScottish National Blood Transfusion Service, Blood Compo­nents Assay Group, 2 Forrest Road, Edinburgh EH1 2QW, Great Britain

F. DRAY, U207, INSERM, Institut Pasteur, Unite de Radioimmunologie Analytique, 28, rue du Docteur Roux, F-75724 Paris Cedex 15

E. EZAN, U207, INSERM, Institut Pasteur, Unite de Radioimmunologie Ana­lytique, 28, rue du Docteur Roux, F-75724 Paris Cedex 15

D. M. GELLER, Washington University School of Medicine, The Edward Ma­linckrodt Department of Pharmacology, Box 8103, 660 South Euclid Ave., St. Louis, MO 63110, USA

J. GRASSI, Section de Pharmacologie et d'lmmunologie, Laboratoire d' Etudes Radioimmunologiques, CEA, Departement de Biologie CEN-Saclay, F-91191 Gif sur Yvette Cedex

V. GUARDABASSO, Istituto di Ricerche Farmacologiche "Mario Negri", Via Eritrea, 62, 1-20157 Milano

VI List of Contributors

E. C. HAYES, Immunology Research, Merck Sharp & Dohme Research Labora­tories, P.O. Box 2000, Rahway, NJ 07065-0900, USA

E. KNOLL, Abteilung fUr Klinische Chemie, Robert-Bosch-Krankenhaus, Auer­bachstr.ll0, D-7000 Stuttgart 50

G. A. LIMJUCO, Immunology Research, Merck Sharp & Dohme Research Labo­ratories, P.O. Box 2000, Rahway, NJ 07065-0900, USA

J. MACLOUF, U 150, INSERM - LA334 CNRS, H6pital Lariboisiere, 6 rue Guy Patin, F-75475 Paris Cedex 10

S. MAMAS, U207, INSERM, Institut Pasteur, Unite de Radioimmunologie Ana­lytique, 28, rue du Docteur Roux, F-75724 Paris Cedex 15

S. MARIOTTI, Istituto di Metodologia Clinica e Medicina del Lavoro, Cattedra di Endocrinologia e Medicina Costituzionale, Universita degli Studi di Pisa, Viale del Tirreno, 64, 1-56018 Tirrenia (Pisa)

M. L. MICHENER, Washington University School of Medicine, The Edward Malinckrodt Department of Pharmacology, Box 8103, 660 South Euclid Ave., St. Louis, MO 63110, USA

P. J. MUNSON, Laboratory of Theoretical and Physical Biology, National Insti­tute of Child Health and Human Development, National Institutes of Health, Building 10, Room 8C312, Bethesda, MD 20892, USA

M. G. MURPHY, Department of Pediatrics, Division of Endocrinology, The U ni­versity of North Carolina at Chapel Hill, Clinical Sciences Building 229H, Chapel Hill, NC 27514, USA

P. NEEDLEMAN, Washington University School of Medicine, The Edward Ma­linckrodt Department of Pharmacology, Box 8103, 660 South Euclid Ave., St. Louis, MO 63110, USA

G. NILSSON, Department of Physiology, Faculty of Veterinary Medicine, The Swedish University of Agricultural Sciences, P.O. Box 7045, S-75007 Uppsala 7

F. NYBERG, Department of Pharmacology, University of Uppsala, Box 591, S-75124 Uppsala

C. W. PARKER, Department of Internal Medicine, Division of Allergy and Im­munology, Washington University School of Medicine, Box 8122, 660 South Euclid Ave., St. Louis, MO 63110, USA

C. PATRONO, Istituto di Farmacologia, Universita Cattolica del Sacro Cuore, Facolta di Medicina e Chirurgia "Agostino Gemelli", Largo Francesco Vito, 1, 1-00168 Roma

M. PAZZAGLI, Dipartimento di Fisiopatologia Clinica, Unita di Endocrinologia, Universita degli Studi di Firenze, Viale Morgagni, 85, 1-50135 Firenze

D. S. PEPPER, Scottish National Blood Transfusion Service, Headquarters Unit Laboratory, 2 Forrest Road, Edinburgh EH1 2QN, Scotland, Great Britain

List of Contributors VII

B. A. PESKAR, Institut fUr Pharmakologie und Toxikologie, Ruhr-Universitat Bochum, 1m Lottental, D-4630 Bochum 1

B. M. PESKAR, Abteilung fUr Experimentelle Klinische Medizin, Ruhr-Universitat Bochum, 1m Lottental, D-4630 Bochum 1

A. PINCHERA, Istituto di Metodologia Clinica e Medicina del Lavoro, Cattedra di Endocrinologia e Medicina Costituzionale, Universita degli Studi di Pisa, Viale del Tirreno, 64, 1-56018 Tirrenia (Pisa)

P. PRADELLES, Section de Pharmacologie et d'lmmunologie, Laboratoire d'Etudes Radioimmunologiques CEA, Departement de Biologie CEN-Saclay, F-91191 Gif sur Yvette Cedex

M. REINKE, Institut fUr Pharmakologie und Toxikologie, Universitat Erlangen­Niirnberg, Universitatsstr.22, D-8520 Erlangen

D. RODBARD, Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 8C312, Bethesda, MD 20892, USA

C. ROUGEOT, U207, INSERM, Institut Pasteur, Unite de Radioimmunologie Analytique, 28, rue du Docteur Roux, F-75724 Paris Cedex 15

D. SCHWARTZ, Washington University School of Medicine, The Edward Malinckrodt Department of Pharmacology, Box 8103, 660 South Euclid Ave., St. Louis, MO 63110, USA

M. SERIO, Dipartimento di Fisiopatologia Clinica, Unita di Endocrinologia, Universita degli Studi di Firenze, Viale Morgagni, 85, 1-50135 Firenze

TH. SIMMET, Institut fUr Pharmakologie und Toxikologie, Ruhr-Universitat Bochum, 1m Lottental, D-4630 Bochum 1

S. SPECTOR, Department of Neuroscience, Roche Institute of Molecular Biology, Roche Research Center, Building 102, Nutley, NJ 07110, USA

L. TERENIUS, Department of Pharmacology, University of Uppsala, Box 591, S-75124 Uppsala

L. E. UNDERWOOD, Department of Pediatrics, School of Medicine, Division of Endocrinology, The University of North Carolina at Chapel Hill, Clinical Sciences Building 229H, Chapel Hill, NC 27514, USA

J. H. WALSH, Center for Ulcer Research and Education, Veterans Administration Wadsworth, Wilshire and Sawtelle Bvds., Building 115, Room 115, Los Angeles, CA 90073, USA

H. WISSER, Abteilung fUr Klinische Chemie, Robert-Bosch-Krankenhaus, Auer­bachstr.ll0, D-7000 Stuttgart 50

VIII List of Contributors

H. C. WONG, Department of Medicine, University of California, Los Angeles, CA 90024, USA

R. S. YALOW, Solomon A. Berson Research Laboratory, Veterans Administration Medical Center, 130, West Kingsbridge Road, Bronx, NY 10468, USA

H. J. ZWEERINK, Immunology Research, Merck Sharp & Dohme Research Labo­ratories, P.O. Box 2000, Rahway, NJ 07065-0900, USA

Preface

Thirty years have elapsed since the first description by S. A. BERSON and R. S. Y ALOW of the basic principles of radioimmunoassay (RIA). During this period of time, RIA methodology has been instrumental to the growth of many areas of biomedical research, including endocrinology, oncology, hematology, and pharmacology. It has done so by providing a relatively simple universal tool allowing, for the first time, the detection of endogenous mediators that are present in body fluids at concentrations as low as 10- 12_10- 10 M. The fundamental nature of this discovery and the wide-ranging fall-out of basic and clinical knowledge derived from its application have been acknowledged by the many honors tributed to its pioneers, including the Nobel Prize awarded to Dr. Y ALOW

10 years ago. Although several excellent books have been published during the past decades

covering various aspects of RIA methodology, we felt the need, as pharmacologists, for a comprehensive discussion of the methodological and conceptual issues related to the main classes of mediators of drug action and to drugs themselves. Thus, we gladly accepted the challenge provided by the invitation to edit a volume of the Handbook of Experimental Pharmacology on Radioimmunoassay in Basic and Clinical Pharmacology. We tried to balance the emphasis placed on more general aspects of the RIA methodology and that on specific mediators. A potentially endless list of substances of pharmacological interest was necessarily limited by considerations of space and general interest, although we did make some last-minute adjustments in order to include very recent and exciting developments.

A number of introductory chapters provide the reader unfamiliar with RIA with all the basic information concerning the production and characterization of antibodies, labeling techniques, statistical aspects, and validation criteria. Moreover, each of these chapters provides the experienced reader with further insight into problems related to the development and validation of RIA for newly discovered mediator(s). In the following chapters, the emphasis is placed on the technical details relevant to each class of compounds and on specific aspects of their application to basic and/or clinical pharmacological studies. New develop­ments in this area such as monoclonal antibodies and nonradioactive labeling techniques are also given adequate coverage.

We hope this book will represent a valuable working instrument in the hands of those investigators interested in measuring changes in the synthesis and metabolism of a variety of endogenous mediators, as well as in the kinetics of

x Preface

drugs, as related to experimental or clinical models of disease and to pharma­cological intervention.

We are indebted to Mrs. P. TIERNEY, Mrs. A. ZAMPINI, and Ms. K. BUSCHEY for invaluable help in the handling and editing of the manuscripts, to Mrs. D. M. WALKER for providing all the necessary support and advice from the publisher, and to all the authors for generously sharing with us the effort and time that this project required.

Rome Bochum

C. PATRONO B.A. PESKAR

Contents

CHAPTER 1

Radioimmunoassay: Historical Aspects and General Considerations R. S. Y ALOW. With 1 Figure . . .

A. Historical Aspects. . . . . . B. Principle, Practices, and Pitfalls

I. Principle II. Practices

III. Pitfalls . C. Conclusions References. .

CHAPTER 2

Basic Principles of Antigen-Antibody Interaction

1

1 1 2 2 4 5 5

F. CELADA. With 4 Figures . . . . . 7

A. Introduction to the Immune System 7 B. Antigens. . . . . . . . . . . . 10

I. Chemical Nature . . . . . . 10 II. Antigenic Determinants (Epitopes) . 11

III. Haptens and Carriers . . . . . . 11 IV. Size of Determinants. . . . . . . 11 V. Sequential and Conformational Epitopes 11

C. Antibodies . . . . . . . . . . . . . 12 I. The Ab Combining Site (Paratope) 13

D. The Immune Interaction . 14 I. The Forces Involved. 14

II. Paratope-Epitope Fit. 15 III. Specificity . . . . . 16 IV. Affinity . . . . . . 16

E. Effects of Ab-Ag Interaction 19 I. Free Ag Binds Ig Receptor 19

II. Free Ag Binds Free Ab 19 III. Free Ab Binds Cell-Associated Ag . 20

F. Immune Interaction as a Signal: Role of Conformational Changes 20 I. Antibody. 20

II. Antigen 21 References. . . . 21

XII Contents

CHAPTER 3

Production of Antisera by Conventional Techniques G. CIABATTONI. With 7 Figures. . . . . . . . . . . . . . . . . . . 23

A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 23 B. Substances Which Are Able to Evoke Immunogenic Responses Per Se 24 C. Special Problems with Small Pep tides . . . . . . . . . . 28 D. Haptens Covalently Coupled to Protein Carriers. . . . . . 29

I. Preparation of Derivatives Containing Reactive Groups 29 II. Covalent Coupling to Protein Carriers . . . . . 34

E. Immune Response to Hapten-Protein Conjugates . . 40 I. Effect of Method and Duration of Immunization 40

II. Role of Adjuvants in Antibody Production . . . 43 III. Effect of Carrier Protein on Characteristics of Antisera . 44 IV. Effect of Hapten: Protein Molar Ratio on Characteristics of

Antisera . . . . . . . . . . . . . . . . . . . . . . . 45 V. Effect of Site of Hapten Linkage to Protein on Characteristics of

Antisera . . . . . . . 46 F. Characterization of Antisera 48

I. Titer. . . 48 II. Affinity . 50

III. Sensitivity 53 IV. Specificity 54

References. . . . 61

CHAPTER 4

Production of Monoclonal Antibodies for Radioimmunoassays K. BRUNE and M. REINKE. With 1 Figure . . . . . . . .

A. Introduction . . . . . . . . . . . . . . . . . . . I. Rationale for the Production of Monoclonal Antibodies

II. Applications of Monoclonal Antibodies III. Problems.

B. Techniques. . I. Materials.

II. Methods . C. Results and Outlook. References. . . . . . .

CHAPTER 5

Radioiodination and Other Labeling Techniques J. GRASSI, J. MAcLOuF, and P. PRADELLES. With 5 Figures

A. Labeling for Immunologic Assay: Introduction B. Radioiodination. . . . . . . . . . . . . . . . .

69

69 69 70 70 71 71 73 79 83

91

91 92

Contents

I. General Considerations II. Chemistry . . . . . .

C. Nonisotopic Labeling . . . I. General Considerations

II. Labeling with Enzymes. III. Labeling with Fluorescent Compounds IV. Labeling with Luminescent Compounds V. Other Labeling Methods

D. Conclusions References. . . . . . . . . .

CHAPTER 6

Strategies for Developing Specific and Sensitive Hapten Radioimmunoassays

XIII

92 103 111 111 113 121 124 126 128 130

E. EZAN, S. MAMAS, C. ROUGEOT, and F. DRAY. With 23 Figures 143

A. Introduction . . . . . . . . . 143 B. Hapten-Carrier Conjugation . . . 143

I. Haptens with Amino Groups . 145 II. Haptens with Carboxyl Groups 147

III. Haptens with Other Functional Groups 149 C. Strategy for Increasing Specificity . . . . . 152

I. General Considerations: From Hapten Size to Epitope Size 152 II. Steroids . . . . . . 152

III. Other Small Haptens. . . . . 157 IV. Peptides . . . . . . . . . . 158 V. Transformation of Immunogen 161

D. Immunoassay Sensitization. . . . 163 I. Increase in Tracer Specific Activity 164

II. Modification of Reagent Concentrations and Assay Procedures 169 E. Validation . . . . . . . . 171

I. Nonantigenic Materials . . . . . . . . . . . . 172 II. Antigen-like Materials . . . . . . . . . . . . . 172

III. Example of Validation: Peptide Radioimmunoassay 173 References. . . . . . . . . . . . . . . . . . . . . . 175

CHAPTER 7

How to Improve the Sensitivity of a Radioimmunoassay G. CIABATTONI. With 2 Figures.

A. Introduction . . . . . . . . . B. Basic Considerations. . . . . . C. The Influence of Labeled Tracer. D. The Influence of Antiserum Dilution. E. The Role of Incubation Volume. F. Temperature and pH Effects . . . .

181

181 182 184 186 187 188

XIV Contents

G. Disequilibrium Conditions . . . . H. Other Factors Affecting Sensitivity References. . . . . . . . . . . . .

CHAPTER 8

Statistical Aspects of Radioimmunoassay

189 190 191

D. RODBARD, V. GUARDABASSO, and P. J. MUNSON. With 8 Figures 193

A. Introduction . . . . . . . . . 193 B. The Logit-Log Method . . . . 194 C. Is the Logit-Log Method Failing? 194 D. The Four-Parameter Logistic . . 196 E. Examples of Problems . . . . . 198

I. Evaluating Goodness of Fit. 199 II. Pooling of Information Over Assays 202

F. Strategy to Deal with Failure of Logistic Models 203 I. Does it Matter? . . . . . 203

II. Choice of Other Methods. 204 III. A "Universal" Approach. 209

G. Concluding Remarks 210 References. . . . . . . . . . . 210

CHAPTER 9

Validation Criteria for Radioimmunoassay C. PATRONO. With 3 Figures. . . . . . . . . . . . . . . . . . . . 213 A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 213 B. Conditions Necessary, but not Sufficient for Establishing the Validity

of RIA Measurements . . . . . . . . . . . . . . . . . . . . . 214 C. Appropriate Biological Behaviour of the Measured Immunoreactivity 215 D. Limited Cross-Reactions of Structurally Related Substances. 216 E. Use of Multiple Antisera. . . . . . . . . . . . . . . . . .. 218 F. Identical Chromatographic Behaviour of Standards and Unknowns 219 G. Comparison with an Independent Method of Analysis . . . . .. 221 H. Are we Measuring the Right Compound, in the Right Compartment? 222 J. How to Evaluate an RIA Kit Critically? 223

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

CHAPTER 10

Measurement of Opioid Peptides in Biologic Fluids by Radioimmunoassay F. NYBERG, I. CHRISTENSSON-NYLANDER, and L. TERENIUS. With 1 Figure. 227

A. Introduction . . . . . . . . . . . . . . . . . B. General Aspects. . . . . . . . . . . . . . . .

I. Distribution and Processing of Opioid Pep tides II. Characterization of Opioid Receptors . . . .

227 228 228 230

Contents XV

C. Methodological Aspects . . . . . . 231 I. Sampling Protocol. . . . . . . 231

II. Sample Workup (Preseparation) . 231 III. Radioimmunoassay of Opioid Peptides from Individual Systems 232

D. Functional Aspects . . . . . . . . . 239 I. Opioid Pep tides in Biologic Fluids . 239

E. Clinical Aspects. . . . . . . . . . . 241 I. Pain. . . . . . . . . . . . . . 241

II. Stress, Physical Exercise, and Shock 241 III. Narcotic Dependence . . . . . . 242 IV. Psychiatry . . . . . . . . . . . 242 V. Neuroendocrinology and Endocrine Tumors 243

F. Concluding Remarks 244 References. . . . . . . . . . . . . . . . . . . 245

CHAPTER 11

Radioimmunoassay of Pituitary and Hypothalamic Hormones D. COCCHI . . . . . . . . . . . . 255

A. Introduction . . . . . . . . . . 255 B. General Principles of Determination 255

1. The Antibody. . . . . 255 II. The Labeled Antigen. . . . . 256

III. Reference Preparations. . . . 256 IV. Separation of Bound from Free Labeled Hormone. 257 V. Future Development of RIAs . . . . . . . . 257

C. Radioimmunoassay of Anterior Pituitary Hormones 258 I. Prolactin. . . . . . . 259

II. Growth Hormone. . . . . . . . . . . . . 261 III. Glycoprotein Hormones . . . . . . . . . . 264 IV. Adrenocorticotropin, Endorphins, and Related Peptides 267

D. Radioimmunoassay of Hypothalamic Hypophysiotropic Hormones 270 I. Premises . . . . . . . . . . . . . . . . . . . 270

II. General Principles of Determination . . . . . . . 272 III. Features Peculiar to the Determination of a Single

Hypophysiotropic Hormone . . . . . . . . . . 274 IV. Significance of Hypophysiotropic Peptides in Biologic Fluids 278

References. . . . . . . . . . . . . . . . . . . . . . . . .. 281

CHAPTER 12

Radioimmunoassay of Nonpituitary Peptide Hormones G. NILSSON. With 7 Figures . . . . . . . . . . . . . . . . . 291

A. Hormones Involved in Regulation of Carbohydrate Metabolism 291 I. Insulin. . . . . . 291

II. C-Peptide of Insulin 298 III. Glucagon . . . . 301

XVI

B. Hormones Regulating Calcium Homeostasis I. Physiology of Calcium Homeostasis

II. Parathyroid Hormone III. Calcitonin

References. . .

CHAPTER 13

Radioimmunoassay of Gastrointestinal Polypeptides J.H. WALSH and H.C. WONG. With 18 Figures.

A. Introduction . . . . . . . B. Gastrin ........ .

I. Physiologic Relevance II. Chemical Composition

III. Characteristics of Antibodies and Labeled Peptides IV. Other Published Assays. . . .

C. Cholecystokinin. . . . . . . . . . I. Published Radioimmunoassays .

D. Secretin . . . . . . . . . I. Chemistry. . . . . .

II. Antibody Production . III. Radioiodination . . . IV. Radioimmunoassay Procedure. V. Characterization of Antibodies.

VI. Sample Preparation for Radioimmunoassay VII. Other Published Radioimmunoassays .

E. Somatostatin . . . . . . . I. Antibody Production. . . . . .

II. Radioiodination . . . . . . . . III. Sampling and Treatment of Serum IV. Serum Extraction . . . . . . V. Radioimmunoassay Procedure. .

VI. Detection Limit . . . . . . . . VII. Other Published Radioimmunoassays .

F. Motilin ........ . I. Chemistry. . . . . .

II. Antibody Production . III. Radioiodination . . . IV. Radioimmunoassay Procedure. V. Characterization of Antibodies.

VI. Other Published Radioimmunoassays . G. Gastric Inhibitory Peptide . . . .

I. Antibody Production. . . . II. Characterization of Antisera.

III. Radioiodination . . . . . . IV. Radioimmunoassay Procedure. V. Other Published Radioimmunoassays .

Contents

305 305 305 308 310

315

315 316 316 316 317 317 317 318 318 319 319 320 321 322 322 323 324 324 324 325 325 325 326 326 327 328 328 328 330 331 331 331 331 332 332 333 334

Contents

H. Neurotensin . . . . . . . I. Chemistry. . . . . .

II. Preparation of Antigen III. Immunization of Animals. IV. Radioiodination . . . . . V. Radioimmunoassay Procedure.

VI. Characterization of the Antibody. VII. Stability of Neurotensin. . . . .

VIII. Other Published Radioimmunoassays . J. Vasoactive Intestinal Polypeptide

I. Chemistry. . . . . . II. Antibody Production. . .

III. Radioiodination . . . . . IV. Radioimmunoassay Procedure . V. Characterization of Antibodies.

VI. Sample Preparation for Radioimmunoassay VII. Other Published Radioimmunoassays .

K. Pancreatic Polypeptide and Peptide YY I. Pancreatic Polypeptide

II. Peptide YY References. . . . . . . . . .

CHAPTER 14

Radioimmunoassay of Atrial Peptide Blood and Tissue Levels M. L. MICHENER, D. SCHWARTZ, M. G. CURRIE, D. M. GELLER, and

XVII

334 334 335 335 335 337 338 339 339 340 340 340 341 342 343 344 344 345 345 345 346

P. NEEDLEMAN. With 2 Figures . . . . . . . . . . 351

A. Introduction . . . . . . . . . . . . . . . . 351 B. Development of Atriopeptin Radioimmunoassay 352

I. Immunization. . . . . 352 II. Iodination of Peptides . 353

III. Titering and Sensitivity. 353 C. Assay of Tissues and Plasma 354

I. Atrial Extracts . . . . 354 II. Plasma Immunoreactivity. 354

III. Brain Atriopeptin . . . . 357 D. Processing of the Atriopeptin Prohormone 358 E. Summary 359 References. . . . . . . . . . . . . . . . 359

CHAPTER 15

Immunochemical Methods for Adrenal and Gonadal Steroids M. PAZZAGLI and M. SERIO. With 8 Figures

A. Chemistry and Nomenclature. . . B. Immunoassay Methods for Steroids . .

363

363 364

XVIII Contents

I. Basic Principles . . . . . . . . . . 364 II. Synthesis of the Immunogen Derivative 365

III. The Radioactive Tracer. . . . . . . 366 IV. Sample Preparation Before Immunoassay 367 V. Bound/Free Separation Systems . . 368

VI. Monoclonal Antibodies to Steroids . 368 VII. Quality Assessment. . . . . . . . 368

VIII. Alternative Immunoassay Methods . 369 C. The Adrenal Cortex. . . . . . . . . . 373

I. Physiology . . . . . . . . . . . 373 II. Control Mechanisms for the Release of Cortisol 376

III. Control Mechanisms for the Release of Aldosterone 378 IV. Biosynthesis of Adrenal Steroids . . . . . . . 378 V. Metabolism of Adrenal Steroids . . . . . . . 378

VI. Immunochemical Methods for Adrenal Steroids 379 D. The Testis . . . . . . . . . . . . . . . . . . . 384

I. Physiology . . . . . . . . . . . . . . . . 384 II. Transport of Testosterone in Blood and Its Action at the

Target Level. . . . . . . . . . . . . . 385 III. Immunochemical Methods for Androgens . 386

E. The Ovary . . . . . . . . . . . . . . . . . 388 I. Physiology . . . . . . . . . . . . . . 388

II. Immunochemical Methods for Ovarian Steroids 388 References. . . . . . . . . . . . . . . . . . . . . 393

CHAPTER 16

Radioimmunoassay of Thyroid Hormones L. BARTALENA, S. MARIOTTI, and A. PINCHERA. With 1 Figure . 401

A. Introduction . . . . . . 401 B. lodometric Techniques. . 403

I. PBI . . 403 II. BEl. . . . . . . . 403

III. T41c. . . . . . . . 404 C. Radioligand Assays of Total T 4 (TT 4) and Total T 3 (TT 3) . 404

I. TT 4 Assays. . . . . . . . . . . . . . . . . . . . 404 II. TT 3 Assays. . . . . . . . . . . . . . . . . . . . 406

III. Factors Affecting the Diagnostic Accuracy of Total Thyroid Hormone Determination . . . . . . . . . . . . . . .. . 408

D. Free Thyroid Hormone Assays . . . . . . . . . . . . . .. . 412 I. Indirect Methods for the Estimation of Free Thyroid Hormone . 413

II. Direct Methods for Free Thyroid Hormone Determination . 416 III. Factors Affecting the Diagnostic Accuracy of Free Thyroid

Hormone Measurements . . . . . . . . . . . . . " . 420 E. Radioimmunoassay of Other lodothyronines, lodotyrosines, and

Products of Thyroid Hormone Degradation. . . . . . .. . 423

Contents

I. rT3 RIA ........ . II. RIA of Diiodothyronines. .

III. RIA of Monoiodothyronines IV. RIA of Tetraiodothyroacetic Acid (TETRAC) and

Triiodothyroacetic Acid (TRIAC). . V. RIA of Iodotyrosines (MIT and DIT)

References. . . . . . . . . . . . . . . .

CHAPTER 17

Radioimmunoassay of Catecholamines

XIX

423 424 424

424 424 425

E. KNOLL and H. WISSER. With 6 Figures . . . . . . . . . . . 433

A. Introduction . . . . . . . . . . . . . . . . . . . . . . 433 B. Radioimmunoassay of Catecholamines and Their Metabolites 434

I. Antibodies to Catecholamines . . . . . . . 434 II. Antibodies to Metabolites of Catecholamines 438

C. Summary 445 References. . . . . . . . . . . . . . . . . . . 445

CHAPTER 18

Radioimmunoassay of Prostaglandins and Other Cyclooxygenase Products of Arachidonate Metabolism B. A. PESKAR, TH. SIMMET, and B. M. PESKAR. With 2 Figures. . . .. 449

A. Introduction . . . . . . . . . . . . . . . . . . . . . . .. 449 B. Biosynthesis and Metabolism of Prostaglandins and Thromboxanes 449 C. Development of Radioimmunoassays for Prostaglandins and

Thromboxanes . . . . . . . . . 452 I. Preparation of Immunogens . 452

II. Immunization . . . . . . . 453 III. Labeled Ligands. . . . . . 453 IV. Separation of Antibody-Bound and Free Fractions of Ligands 455

D. Validation of Radioimmunoassays for Cyclooxygenase Products of Arachidonic Acid Metabolism . . . . . . . . . . . . . .. 455

E. Factors that Affect the Validity of Prostaglandin and Thromboxane Radioimmunoassay Results. . . . . . . . 456

I. Extraction and Purification Procedures . . . . . . . .. 456 II. The Blank Problem . . . . . . . . . . . . . . . " 458

III. Problems Associated with the Determination of Tissue and Plasma Concentrations of Prostanoids . . . . . . . .. 458

F. Radioimmunoassay for Various Prostaglandins and Thromboxanes 459 I. Prostaglandin F 2a . . . . . . . . . . 459

II. 15-Keto-13,14-dihydro-prostaglandin F 2" 459 III. Prostaglandin E2 . . . . . . . . . . . 460 IV. 15-Keto-13,14-dihydro-prostaglandin E2 . 460 V. Prostaglandin E1 . . . . . . . . . . . 461

xx Contents

VI. Prostaglandin D2 . . . . . . . . . . . . . . . . .. . 462 VII. Indirect Determination of Prostaglandin Endoperoxides and

Thromboxane A2 . . . . . . . . . . . . . . . 462 VIII. Thromboxane B2 and 11-Dehydro-thromboxane B2 . 463

IX. 15-Keto-13,14-dihydro-thromboxane B2 . . . . . . 464 X. 6-Keto-prostaglandin F la . . . . . . . . . . . . 464

XI. Metabolites of Prostaglandin 12 and 6-Keto-prostaglandin F la 465 XII. Main Urinary Metabolites of Prostaglandin F 2a and

Prostaglandin E2 . . . . . . . . . . . . . . . . . 466 G. Comparison of Radioimmunoassay with Other Methods for

Quantitative Determination of Cyclooxygenase Products of Arachidonic Acid Metabolism. . . . . . . . . . . . . . . . . . 467

H. Radioimmunoassay of Cyclooxygenase Products of Arachidonic Acid Metabolism in Basic and Clinical Pharmacology . 469

J. Concluding Remarks 470 References. . . . . . . . . . . . . . . . . . . 471

CHAPTER 19

Radioimmunoassay of Leukotrienes and Other Lipoxygenase Products of Arachidonate H. J. ZWEERINK, G. A. LIMJUCO, and E. C. HAYES. With 6 Figures 481

A. Introduction . . . . . . . . . . . . . . . . . . . 481 B. Biosynthesis and Metabolism of Lipoxygenase Products 482 C. Biologic Activities of Lipoxygenase Products . . . . . 483 D. Biologic Assays to Measure Lipoxygenase Products . . 484 E. Physicochemical Separation and Identification of Lipoxygenase Products 485 F. Radioimmunoassays for Lipoxygenase Products. 485

I. Introduction . . . . . . . . . . . . 485 II. Radioimmunoassays to Measure LTC4 . 486

III. Radioimmunoassays to Measure LTD4 . 491 IV. Radioimmunoassays to Measure LTB4 . 492 V. Radioimmunoassays to Measure 12-HETE 493

VI. Radioimmunoassays to Measure 15-HETE 494 G. Sample Preparation for Radioimmunoassays . 494 H. Combination of Radioimmunoassays with Physicochemical Separation

Methods. . 495 J. Conclusions 496

References. . . 497

CHAPTER 20

Radioimmunoassay of Cyclic Nucleotides c. W. PARKER. With 6 Figures . .

A. Introduction . . . . . . . . B. Preparation of the Immunogen

501

501 502

Contents XXI

C. Immunization. . . . . . . . . . . . . . . . . . . . . . 504 D. Preparation of the Radioindicator Molecule. . . . . . . . . 504 E. Evaluation of Antisera and Routine Immunoassay Procedures. 507

1. Double-Antibody Procedure . . 509 II. Ammonium Sulfate Precipitation 509

III. Charcoal. . . . . . . . . 509 F. Tissue Extraction . . . . . . . . . 510 G. Derivatization of Tissue Samples . . 510 H. Pitfalls in Immunoassay Measurements. 511 J. Validation of the Immunoassay . . . . 512

K. Special Applications of the Immunoassays 513 References. . . . . . . . . . . . . . . . 514

CHAPTER 21

Radioimmunoassay of Platelet Proteins D. S. PEPPER. With 1 Figure . . . .

A. Introduction . . . . . . . . . B. Sample Collection and Processing C. Assay of p-Thromboglobulin . . D. Assay of Platelet Factor 4 . . . E. Assay of Other Platelet Proteins. F. Applications of Platelet-Specific Protein Radioimmunoassays G. Discussion References. . .

CHAPTER 22

A Competitive Binding Assay for Heparin, Heparan Sulphates and Other Sulphated Polymers

517

517 520 524 526 528 529 533 535

J. DAWES and D. S. PEPPER. With 8 Figures 543

A. Introduction . . . . . . . . . . . 543 B. Radiolabelling of Glycosaminoglycans 544

I. Derivatisation. . . . . 544 II. Iodination . . . . . . . . . . 545

C. Synthesis of Binding Reagent . . . . 546 D. Assay for Therapeutic Heparins and Heparinoids 546

1. Method . 546 II. Sensitivity . . . . . . . . 547

III. Specificity . . . . . . . . 548 IV. Studies in Human Volunteers 549

E. Assay for Endogenous Heparan Sulphate. 552 I. Method . 552

II. Sensitivity . . . . . . . . . . . 554 III. Specificity . . . . . . . . . . . 554 IV. Results of Rat and Human Studies 554

XXII Contents

F. Other Applications 557 557 558

G. Summary References. . . . . .

CHAPTER 23

Radioimmunoassay of the SomatomedinsfInsulin-like Growth Factors L. E. UNDERWOOD and M. G. MURPHY. With 6 Figures . . . . . . . . 561

A. Introduction and Nomenclature of the Somatomedins . . . . . . . 561 I. Methods for Measuring Somatomedins Before the Development of

Radioimmunoassays. . . . . . . . . . . . 562 B. Radioimmunoassay for Sm-CjIGF-I . . . . . . . 563

I. Antisera Production, Sensitivity, and Specificity 563 II. Influence of Binding Proteins . . . . . . . . 563

III. Clinical Utility of Measurements of Sm-CjIGF-I in Plasma 567 IV. Heterologous Radioimmunoassays for Sm-CjIGF-I . . . 570 V. Measurement of Sm-CjIGF-I in Tissues and Media Extracts 571

C. Radioimmunoassay for IGF-II 571 References. . . . . . . . . . . . . . . . . . . . . . . . .. 572

CHAPTER 24

Radioimmunoassay of Drugs and Neurotransmitters S. SPECTOR. With 3 Figures . . . . . . . . . . 575

A. Introduction . . . . . . . . . . . . . . . . . . . . . 575 B. Methods for Coupling Neurotransmitters to Carrier Proteins 576

I. Acetylcholine 576 II. Serotonin. . . . . . . . . . . . . . . 578

III. Melatonin. . . . . . . . . . . . . . . 579 C. Methods for Coupling Drugs to Carrier Proteins 580

I. Barbiturates. 580 II. Curare . . . . 580

III. Clonidine. . . 581 IV. Phenothiazines. 582 V. Benzodiazepines 584

VI. Butyrophenones 585 VII. Atropine 586

VIII. Opiates . 586 References. . 588

Subject Index 591