Immunoassay (eBook)

Front Cover 1
Immunoassay 4
Copyright Page 5
Contents 8
Contributors 28
Preface 32
CHAPTER 1. PAST, PRESENT AND FUTURE OF IMMUNOASSAYS 34
CHAPTER 2. IMMUNE FUNCTION AND ANTIBODY STRUCTURE 38
1. Cellular and Molecular Basis of the Humoral Immune Response 38
2. Antibody Structure 44
3. Antigen Binding 51
4. Antibody Structure and Immunologic Diversity 53
5. Conclusion 54
CHAPTER 3. THEORY OF IMMUNOASSAYS 58
1. Binding Theory 58
2. Equilibria Involving Many Ligands and a Heterogeneous Population of Binding Sites 61
3. Principles of Saturation Analysis 62
4. Kinetics of Immunoassay 67
5. The Theory of Two-Site Immunoassays 71
6. Graphical Presentation of Immunoassay Data 73
7. Curve Fitting of Immunoassay Data 75
8. Detection Limit of Immunoassays 80
References 82
CHAPTER 4. DATA INTERPRETATION AND QUALITY CONTROL 84
1. Introduction 84
2. Assay Validation 86
3. Determining Immunoassay Sensitivity 90
4. Tests of Similarity (Parallelism) 95
5. Alternative Methods for Defining Cross Reactivity 115
6. Quality Control 116
7. Summary 125
References 125
CHAPTER 5. PRODUCTION AND PURIFICATION OF ANTIBODIES 128
1. Introduction 129
2. Polyclonal versus Monoclonal Antibodies 129
3. Choice of Animal 130
4. Immunization Procedures 134
5. Obtaining and Processing Blood and Lymphocytes 137
6. Monoclonal Antibody Production 139
7. Antibody Purification 145
References 148
CHAPTER 6. ANTIBODY ENGINEERING 150
1. Introduction 150
2. Cloning Antibody Variable Region Genes 151
3. Expression of Recombinant Antibodies 157
4. Antibody Structure-Function Studies 162
5. Therapeutic Antibodies 170
6. Designer Antibodies 183
7. Future Directions 188
References 190
CHAPTER 7. INTERFERENCES IN IMMUNOASSAYS 198
1. Introduction 198
2. Detecting Interferences 199
3. Cross-Reactivity and Heterogeneous Analytes 201
4. Reducing Cross-Reactive Interferences 204
5. Heterophile and Anti-Animal Antibodies 207
6. Recognizing and Reducing Interference from Endogenous Antibodies 215
7. Interferences Due to Masking of Antigens 218
8. Interference with the Indicator Mechanism 219
9. Matrix Effects 219
10. Interference Problems in the Future 220
References 220
CHAPTER 8. LABELING OF ANTIBODIES AND ANTIGENS 224
1. Introduction 224
2. Requirements for Quality of Labeled Reactants 225
3. Chemical Reactions for Conjugation 226
4. Reagents for Conjugation 227
5. Conjugation of Antibody IgG to Enzymes 227
6. Conjugation of Antibody Fragments to Enzymes 229
7. Conjugation of Antigens, Avidin, and Streptavidin to Enzymes 234
8. Conjugation of Small Molecules to Antibodies and Antigens 235
References 237
CHAPTER 9. SOLID PHASES IN IMMUNOASSAY 238
1. Interfacial Immunochemistry 238
2. Diversity of Solid Phases 246
3. Immobilization Procedures 249
4. SPI in the Future 255
References 256
CHAPTER 10. IMMUNOASSAY CONFIGURATIONS 260
1. Introduction 260
2. Competitive Immunoassays 261
3. Noncompetitive Immunoassays 262
4. Indirect Detection of the Immunocomplexes 266
References 269
CHAPTER 11. THE AVIDIN-BIOTIN SYSTEM 270
1. Introduction 270
2. Components of the System 271
3. Amplified Detection Systems 285
4. Improvement of Capture Systems 288
5. Auxiliary Enhancement Systems 288
6. Selected Protocols 292
References 298
CHAPTER 12. RADIOIMMUNOASSAY 302
1. Radioactive Isotopes 303
2. Counting of Radioactive Isotopes 303
3. Characteristics of a Tracer 306
4. Preparation of Tracers 306
5. Iodinated Tracers 306
6. Iodination Methods 306
7. Iodination Damage 311
8. Comparison of Labeled and Unlabeled Ligand 312
9. Purification of Iodinated Tracer 312
10. Chemical Evaluation of Tracer 313
11. Immunological Evaluation of Tracer 315
12. Variations on the Use of Radiolabeled Tracers 316
Reference 317
CHAPTER 13. ENZYME IMMUNOASSAY 320
1. Introduction 320
2. Enzymes as Labeling Substances 321
3. Conjugation Procedures 323
4. Determining Enzyme Activity 327
5. The Variety of Enzyme Immunoassays 332
6. Prospects 337
References 338
CHAPTER 14. FLUORESCENCE IMMUNOASSAYS 342
1. Introduction 342
2. Principles of Fluorescence Spectroscopy 343
3. Fluorescence Immunoassay Instrumentation 347
4. Fluorescent Labels 347
5. Homogeneous Fluorescence Immunoassays 349
6. Heterogeneous Fluorescence Immunoassays 355
References 366
CHAPTER 15. CHEMILUMINESCENCE IMMUNOASSAY 370
1. Introduction 370
2. Chemiluminescent Labels and Labeling 372
3. Detection of Labels 378
4. Types of Chemiluminescent Immunoassay 378
5. Chemiluminescent Detection of Enzyme Labels 380
6. Automated Chemiluminescent Immunoassay Systems 384
References 384
CHAPTER 16. BIOLUMINESCENCE IMMUNOASSAYS 388
1. Introduction 388
2. Principles of Bioluminescent Light Emission 389
3. Bioluminescence Immunoassay Instrumentation 390
4. Enzyme Labels and Bioluminogenic Substrates 390
5. Bioluminescence Enzyme Immunoassays 390
6. Other Bioluminescence Immunoassay Systems 392
7. Availability of Bioluminescence Immunoassays 393
References 393
CHAPTER 17. NEPHELOMETRIC AND TURBIDIMETRIC IMMUNOASSAY 396
1. Introduction 396
2. Historical Perspective 397
3. Characteristics of the Immunoprecipitin Reaction 399
4. Principles of Light Scattering 401
5. Definition and Principles of Nephelometry and Turbidimetry 403
6. Instrumentation 414
References 418
CHAPTER 18. SIMULTANEOUS MULTIANALYTE IMMUNOASSAYS 422
1. Introduction 422
2. Labels and Immunoassay Formats 423
3. Labels 424
4. Discrete Test Zones 427
5. Test Combinations 432
6. Conclusions 432
References 434
CHAPTER 19. NONCOMPETITIVE IMMUNOASSAY FOR SMALL MOLECULES 438
1. Introduction 438
2. Anti-idiotypic Antibodies as Novel Probes in Immunodiagnostics 439
3. Idiometric Assays for Estradiol 445
4. Idiometric Assays for Progesterone 449
5. Conclusions 452
References 453
CHAPTER 20. FREE HORMONE MEASUREMENTS 456
1. Introduction 457
2. Overview of Thyroid, Steroid, and Secosteroid Hormones 459
3. Transport of Thyroid, Steroid, and Secosteroid Hormones in the Blood 461
4. The Theory of Free Hormones and Alternative Concepts 471
5. History of Free Hormone Assays 476
6. Classification of Free Hormone Assays 476
7. Principles of Free Hormone Assays 484
8. Total Hormone/Total Hormone-Binding Globulin Ratio 493
9. Tests Based on the Measurement of "Thyroid Hormone Binding" 494
10. Calculated Free Hormone Concentrations 496
11. Non-Sex Hormone-Binding Globulin-Bound Testosterone (NSB-Te) 497
12. Assay Parameters of Free Hormone Assays 497
13. Specimens for Free Hormone Assays 502
14. Interferences with Free Hormone Measurements 503
15. Selected Issues Regarding Interpretation of Free Hormone Results 507
16. Analytical Goals for Free Hormone Measurements 508
17. Quality Control of Free Hormone Assays 508
18. Conclusions and Perspectives of Free Hormone Measurements 509
References 509
CHAPTER 21. AUTOMATION OF IMMUNOASSAYS 516
1. Introduction 516
2. Automation of Immunoassay 517
3. Automated Immunoassay Systems 521
4. Performance of Immunoassay Systems 524
5. Future Trends 534
References 535
CHAPTER 22. THIN-FILM IMMUNOASSAYS 538
1. Introduction 538
2. Basic Features of Dry Reagent Assays 540
3. Examples of Thin-Film Immunoassays 551
4. Conclusion 566
References 566
CHAPTER 23. IMMUNOBLOTTING TECHNIQUES 570
1. Introduction 570
2. Principles 571
3. The Basic Protocol 572
4. Detection Methods 576
5. Specialized Uses and Applications 582
6. Conclusions 585
Reference 585
CHAPTER 24. DEVELOPMENT OF IN-HOUSE IMMUNOLOGICAL ASSAYS 588
1. Introduction 588
2. Development of an Immunoassay Method 589
3. A Practical Example--An ELISA for p53 596
4. Conclusions 600
References 600
INDEX 602