An Introduction to Radioimmunoassay and Related Techniques

List of AbbreviationsChapter 1. The Background to Radioimmunoassay 1.1. Introduction 1.2. Terminology 1.3. Early Development of Radioimmunoassay 1.4. Basic Principles of Binding Assays 1.5. Binder Dilution Curves and Standard Curves 1.6. Methods for Plotting the Standard Curve 1.7. The Measurement of K Value 1.8. A Model System for Binding Assays 1.9. An Application of the Model SystemChapter 2. Requirements for Binding Assays - Purified Ligand 2.1. Requirements for a Binding Assay 2.2. The Need for Purified Ligand 2.3. Availability of Pure Ligand 2.3.1. Large Protein Hormones 2.3.2. Carcino-Fetal Antigens 2.3.3. Steroid Hormones, Drugs and other Small Molecules 2.3.4. Small Peptide Hormones 2.4. Dissimilarity between Purified Ligand and Endogenous Ligand 2.5. Standards 2.5.1. Characteristics of a Standard 2.5.2. Preparation and Calibration of an International Standard 2.5.3. Practical Use of Standards 2.6 Storage of Materials Used in Binding AssaysChapter 3. Requirements for Binding Assays - Tracer Ligand Using Radioisotopic Labels 3.1. Radioactive Isotopes 3.2. Counting of Radioactive Isotopes 3.3. Choice of Counter 3.4. Some Practical Aspects of Isotope Counting 3.5. Essential Characteristics of a Tracer 3.6. Preparation of Tracers 3.7. Iodinated Tracers 3.7.1. Iodination Methods 3.7.2. Choice of Iodination Procedure 3.7.3. Practical Aspects of Chloramine T Iodination 3.7.4. Iodination Damage 3.7.5. Purification of Iodinated Tracer 3.7.6. Chemical Assessment of Tracer 3.7.7. Immunological Assessment of Tracer 3.8. Variations on the Use of Radio-labeled Tracers 3.8.1. Universal Tracers 3.8.2. Autoradiographic Radioimmunoassay 3.8.3. Internal Sample Attenuators 3.8.4. Liposome Immune AssayChapter 4. Requirements for Binding Assays - Tracer Ligand using non-Isotopic Labels 4.1. Particle Labels 4.2. Enzyme Labels (Enzymoimmunoassay, El A) 4.2.1. Heterogeneous Enzymoimmunoassays 4.2.2. Homogeneous Enzymoimmunoassays 4.3. Fluorescent Labels (Fluoroimmunoassay, FIA) 4.3.1. The Nature and Measurement of Fluorescence 4.3.2. Types of Fluoroimmunoassay 4.4. Bacteriophage Labels 4.5. Luminescent Labels 4.6. Advantages of non-Isotopic Labels 4.7. Conclusions - The Place of non-Isotopic Binding AssaysChapter 5. Requirements for Binding Assays - The Binder 5.1. Antibodies and the Immune Response 5.1.1. Chemistry of Antibodies 5.1.2. Chemistry of Antigens 5.1.3. Cellular Basis of the Immune Response 5.1.4. Physiology of the Immune Response 5.2. Preparation of Antisera for use in Radioimmunoassay (RIA) 5.2.1. Production of Antibodies 5.2.2. The Nature and Dose of the Immunogen 5.2.3. Preparation of Haptens as Immunogens 5.2.4. The Use of Adjuvant 5.2.5. The Animal Species 5.2.6. The Route of Immunization 5.2.7. The Timing of Injections and Collection of Antisera 5.2.8. Selection of Antisera for Use in a Radioimmunoassay 5.2.9. Storage of Antisera 5.3. Monoclonal Antibodies 5.4. Cell Receptors 5.5. Circulating Binding Proteins 5.6. Radioassay for the Detection of Endogenous Antibodies, Circulating Binding Proteins and ReceptorsChapter 6. Requirements for Binding Assays - Separation of Bound and Free Ligand 6.1. Efficiency of Separation Procedures 6.2. Practicality of Separation Procedures 6.3. Methods for the Separation of Bound and Free Ligand 6.3.1. Electrophoresis 6.3.2. Gel Filtration 6.3.3. Adsorption Methods 6.3.4. Fractional Precipitation 6.3.5. 'Double'Antibody Methods 6.3.6. Solid-Phase Systems 6.3.7. Conclusions - The Choice of a Separation Procedure 6.4. Immunoradiometric Techniques (IRMA) 6.4.1. Advantages of the Immunoradiometric AssayChapter 7. Requirements for Binding Assays - Extraction of Ligand from Biological Fluids, and Collection and Storage of Samples 7.1. General Aspects of Extraction Procedures 7.2. Extraction using Particulars Adsorbents 7.3. Extraction with Organic Solvents 7.4. Dissociation Procedures 7.4.1. Conversion of Conjugated Steroids to the Unconjugated Forms 7.4.2. Dissociation of Ligand from Endogenous Binding Proteins 7.4.3. Other uses of Dissociating Agents 7.5. Measurement of 'Free' Hormones 7.6. Conclusions — The Elimination of Extraction Procedures 7.7. Sample Collection for RadioimmunoassayChapter 8. Requirements for Binding Assays - Calculation of Results 8.1. Calculation of Results by Manual Extrapolation 8.2. Data Transformation of the Standard Curve 8.3. The Logit Transformation 8.4. Identification of Outliers 8.5. Estimation of Confidence Limits to the Result of an Unknown 8.6. Electronic Aids to Calculation of ResultsChapter 9. Characteristics of Binding Assays - Sensitivity 9.1. Definition of Sensitivity 9.2. Methods of Increasing the Sensitivity of a Binding Assay 9.2.1. Reducing the Amount of Tracer 9.2.2. Improving the Quality of Tracer 9.2.3. Reducing the Amount of Binder 9.2.4. Increasing the Incubation Time 9.2.5. Reducing the Incubation Time — Disequilibrium Assays 9.2.6. Order cf Addition of Reagents 9.2.7. Purification of Binder 9.2.8. Increasing the Sample Volume 9.2.9. Temperature of Incubation 9.2.10. Increasing the Number of Replicates 9.2.11. Extraction and Concentration 9.2.12. Immunometric Assays 9.3. Methods of Decreasing the Sensitivity of an Assay - Increasing the Antibody Concentration 9.4. Targeting of Binding Assays - The Importance of Ranges 9.5. Optimization of an Assay by Theoretical Analysis 9.6. ConclusionsChapter 10. Characteristics of Binding Assays - Specificity 10.1. Definition of Specificity 10.2. Specific non-Specificity 10.2.1. The Basis of Specific non-Specificity 10.2.2. Assessment of Specific non-Specificity 10.2.3. Relevance of Specificity to Physiological and Clinical Studies 10.2.4. Methods for Improving Specificity 10.3. Non-Specific non-Specificity 10.3.1. Presence of Materials which Interfere with the Binder-Ligand Reaction 10.3.2. Variations of Blank Values in Samples 10.3.3. Destruction or Sequestration of Binder, Tracer or Ligand 10.3.4. The Detection and Elimination of non-Specific non-SpecificityChapter 11. Characteristics of Binding Assays - Precision 11.1. Definitions 11.2. Factors Affecting Precision 11.2.1. Errors in the Reagents and Design of the Assay 11.2.2. Errors in the Technical Operation of the Assay 11.3. Quality Control to Monitor the Precision of a Binding Assay 11.3.1. Result Statistics 11.3.2. Standard Curve Statistics 11.3.3. Replicate Statistics 11.4. Practical Use of a Quality-Control Scheme 11.5. External Quality-Control Schemes 11.6. Summary - Optimizing the Precision of a Binding AssayChapter 12. Characteristics of Binding Assays - Relation to Other Types of Assay 12.1. Definitions 12.2. Receptor Assays 12.3. Assays Using Circulating Binding Proteins 12.4. Immunoassays for Hormones 12.5. Immunoassays for non-Hormonal Compounds 12.6. ConclusionsChapter 13. Automation of Binding Assays 13.1. Automation of Binding Assays 13.2. Identification and Dispensing of the Sample 13.3. Addition of Reagents 13.4. Incubation 13.5. Separation of Bound and Free Ligand 13.6. Counting of Radioactivity 13.7. Calculation of Results 13.8. Batch Processing 13.9. ConclusionsChapter 14. Organization of Assay Services 14.1. Who Should Perform Radioimmunoassay 14.2. Organization of an Assay Laboratory 14.3. Organization of Assay ServicesAppendices Appendix I. Manufacturers and Suppliers of Equipment Appendix II. Suppliers of Special Reagents and Chemicals Appendix III. Suppliers of General Reagents and Materials Appendix IV. Manufacturers of Reagent Kits for Radioimmunoassay and Related Techniques Appendix V. Safety Precautions in the Handling of Radioactive Isotopes Appendix VI. Commonly Used Procedures for the Attachment of Ligands to Carrier Molecules or Solid-Phase MaterialsReferencesSubject Index