TECHNICAL REPORTS SERIES No. 233 Laboratory Training Manual on Radioimmunoassay in Animal Reproduction JOINT FAO/IAEA DIVISION OF ISOTOPE AND RADIATION APPLICATIONS OF ATOMIC ENERGY FOR FOOD AND AGRICULTURAL DEVELOPMENT INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1984 LABORATORY TRAINING MANUAL ON RADIOIMMUNOASSAY IN ANIMAL REPRODUCTION TECHNICAL REPORTS SERIES No.233 LABORATORY TRAINING MANUAL ON RADIOIMMUNOASSAY IN ANIMAL REPRODUCTION A JOINT UNDERTAKING BY THE FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS AND THE INTERNATIONAL ATOMIC ENERGY AGENCY INTERNATIONAL ATOMIC ENERGY AGENCY VIENNA, 1984 LABORATORY TRAINING MANUAL ON RADIOIMMUNOASSAY IN ANIMAL REPRODUCTION IAEA, VIENNA, 1984 STI/DOC/10/233 ISBN 92-0-115084-9 © IAEA, 1984 Permission to reproduce or translate the information contained in this publication may be obtained by writing to the International Atomic Energy Agency, Wagramerstrasse 5, P.O. Box 100, A-1400 Vienna, Austria. Printed by the IAEA in Austria January 1984 FOREWORD Since the development of the radioligand assay some twenty years ago the whole field of endocrinology in both humans and animals has been revolutionized. The ability to measure the extremely low quantities of hormones that exist in blood and tissues has increased our knowledge of the reproductive function in domestic animals to an enormous extent, and is now coming to be used at the farm level. Reproduction must always be regarded as one of the major limiting factors in animal production and many of the modern methods for improving reproduc- tion rely heavily on the ability to measure hormone levels in blood and milk. This has produced a world-wide demand for laboratory facilities to carry out hormone assays and the need for specialist training to allow these assays to be undertaken. The need to measure nanogram and picogram quantities and the use of radionuclides requires a good deal of skill and care and this Manual has been prepared to aid training and provide the sort of information that rarely appears in scientific papers. It represents a further step in the Joint FAO/IAEA Division's series of Laboratory Training Manuals, and has been designed to aid training programmes of the type carried out during the Joint FAO/IAEA Interregional Training Course on Radioimmunoassay and its Application in Research on Animal Reproduction at Cornell University in July 1982. Many of the laboratory exercises described in this Manual are based on those conducted during the course. This Manual has been produced on similar lines to the Joint FAO/IAEA Division's Laboratory Training Manual on the Use of Nuclear Techniques in Animal Parasitology (Technical Reports Series No. 219). Indeed, Part I and the Basic Exercises in Part V are largely intact, but sections on the biology of reproduction and a general introduction to the radioimmunoassay have been incorporated. The Applied Exercises have been replaced by a series of exercises describing all aspects of the radioimmunoassay of steroid and protein hormones involved in the regulation of reproduction. The difficulties, particularly in developing countries, of measuring the soft beta emissions from 3H-labelled materials have been borne in mind, and emphasis has been placed on methods based on radioiodinated steroids (125 I) so that relatively simple gamma counting procedures may be used. For these techniques, and for much helpful advice, FAO and the IAEA are grateful to Dr. W.M. Hunter and Dr. J.E.T. Corrie of the MRC Immunoassay Team, Edinburgh, UK, and to Dr. B.A. Morris of the University of Surrey, UK. FAO and the IAEA would also like to thank the scientists who contributed to the success of the course at Cornell University, particularly Drs T.J. Reimers and F.W. Lengemann, and those responsible for preparing the Manual: Dr. W.R. Carr of the ARC Animal Breeding Research Organisation, Roslin, UK; Dr. J.D. Dargie of the Animal Production and Health Section, Joint FAO/IAEA Division; Dr. L.-E. Edqvist of the Department of Clinical Chemistry of the Swedish University of Agricultural Sciences, Uppsala, Sweden; Dr. T.J. Reimers of the Diagnostic Laboratory, New York State College of Veterinary Medicine, Cornell University, Ithaca, USA; and Dr. H.A. Robertson of the Animal Research Centre, Agriculture Canada, Ottawa, Canada. CONTENTS Some basic symbols and units frequently used in this Manual 1 PART I. GENERAL INTRODUCTION TO EXPERIMENTAL WORK WITH ISOTOPES AND RADIATION 3 I— 1. Properties of radionuclides and radiations 3 I-1.1. Atomic model. Radioactivity 3 1—1.2. The equation of Einstein 7 1—1.3. Radioactive decay law. Specific activity 8 I— 1.4. The energy of radiations 12 1-1.5. Interaction of radiation with matter 14 1-1.5.1. Alpha particles 1-1.5.2. Beta particles 1-1.5.3. Gamma and X-ray photons 1-2. Radiation detection and assay of radioactivity 19 1-2.1. Autoradiography 21 1-2.2. Ionization detectors 22 1-2.2.1. Electroscope 1-2.2.2. Gas-filled detectors with collector/cathode voltage bias 1-2.2.3. Ionization chamber 1—2.2.4. Proportional counter 1-2.2.5. Geiger-Müller (GM) counter 1-2.3. Solid scintillation counting 29 1-2.4. Liquid scintillation counting 32 1-2.5. Semiconductor radiation detectors 34 1-2.6. Inverse-square-law effect 36 1-2.7. Counting efficiency (counting yield) 37 1-2.8. Counting statistics (natural uncertainty) 39 1-3. Radiation protection 42 1—3.1. Basic considerations and units 42 1—3.2. Protection of personnel 45 1-3.2.1. External exposure 1-3.2.2. Internal exposure 1-3.3. Control of contamination 49 1—3.3.1. Decontamination 1-3.3.2. Special laboratory design features 1-3.4. Waste disposal 54 1-3.4.1. Disposal of radioactive animals References to Part I 54 Bibliography to Part I 55 Working notes to Part I 56 PART II. MENTAL EXERCISES 61 Working notes to Part II 62 PART III. INTRODUCTION TO PRACTICAL WORK 65 III— 1. Principles 65 III—2. Basic considerations 65 III—2.1. Chemical effects 66 III—2.2. Radiochemical purity 66 III—2.3. Isotope effects 67 III—2.4. Radiation effects 67 III—2.5. Exchange reactions 68 III—2.6. Radionuclide preparation and delivery of dose 68 III—2.7. Procedures with animals 69 III-2.8. Handling of samples 69 III—3. Reproductive endocrinology in mammals 71 III—3.1. Hormones 71 III—3.2. Puberty 73 III—3.3. Oestrous cycle 74 III—3.4. Control of the corpus luteum 74 III—3.5. Early pregnancy 75 III—3.6. Pregnancy and parturition 75 III—3.7. Applications of radioimmunoassay in assessing reproductive efficiency 77 III—3.8. Hormones during pregnancy in cattle 78 III—3.9. Hormones during pregnancy in sheep 80 III—3.10. Hormones during pregnancy in pigs 82 III—3.11. Hormones during pregnancy in mares 82 III—3.12. General comments 83 References to Part III 84 PART IV. GENERAL INTRODUCTION TO RADIOIMMUNOASSAY 85 IV—1. Principles of radioligand assays 85 IV-2. Radioimmunoassay 89 IV—2.1. Production of antibodies 89 IV-2.2. Production of labelled antigens 90 IV-2.3. Separation of antibody-bound from free hormone 93 IV—2.3.1. Chemical precipitation IV-2.3.2. Absorption with coated charcoal IV-2.3.3. Absorption with Protein A IV-2.3.4. Second antibody preparation IV—2.3.5. Solid phase separation IV—2.4. Optimizing the conditions for an assay 97 IV—2.5. Validation of RIA 100 IV—2.5.1. Specificity IV-2.5.2. Sensitivity IV-2.5.3. Accuracy IV-2.5.4. Precision IV-2.6. Quality control in RIA 107 IV-2.6.1. Internal quality control schemes References to Part IV 111 PART V. LABORATORY EXERCISES 113 SECTION A. BASIC EXERCISES 113 Exercise 1. The plateau and operating voltage of a GM counter 113 Exercise 2. The resolving time of a GM counter 115 Exercise 3. Counting and sampling statistics 118 Exercise 4. External absorption of beta particles 119 Exercise 5. Self-absorption and self-scattering of beta particles 121 Exercise 6. Solid integral scintillation counting 125 Exercise 7. Solid differential scintillation counting 127 Exercise 8. Estimating the efficiency of a gamma counter 129 Exercise 9. Rapid radioactive decay 130 Exercise 10. Inverse-square law and attenuation of gamma rays 133 Exercise 11. Liquid scintillation counting: determination of optimum counter settings 135 Exercise 12. Preparation of samples for liquid scintillation counting 137 Exercise 13. Quench correction 137 Exercise 14. Reducing the effects of quenching 140 Exercise 15. Cerenkov counting in a liquid scintillation counter 143 SECTION B. APPLIED EXERCISES 145 Exercise 16. Sampling and handling of assay data 145 16.1. Collection and storage of samples for radioimmunoassay 145 16.2. Construction of standard curves 146 Exercise 17. Preparation and titration of antisera for progesterone assays 147 17.1. Immunization of sheep, goats, horses or donkeys against progesterone 148 17.2. Titration of anti-progesterone serum and anti-rabbit gammaglobulin 149 Exercise 18. Radioiodination of sex steroids 154 18.1. Iodination procedure for progesterone 157 18.2. Iodination procedure for testosterone 160 Exercise 19. Radioimmunoassay of progesterone in serum and milk 160 19.1. Preparation of samples 160 19.2. Double-antibody separation with 12SI-progesterone 163 19.3. Radioimmunoassay procedure for extracted plasma or serum 166 19.4. Radioimmunoassay procedure for cow milk 169 19.5. Radioimmunoassay using 12SI-progesterone and antibody-coated tubes 169 19.6. Procedure using charcoal-dextran separation and [3H] progesterone 173 19.7. Titration of anti-progesterone serum using charcoal- dextran separation and [3H] progesterone 176 Exercise 20. Radioimmunoassay of testosterone in serum or plasma 178 20.1.