Monoclonal Antibodies (eBook)

Front Cover 1
Monoclonal Antibodies: Principles and Practice 4
Copyright Page 5
Contents 6
Preface to First Edition 11
Preface to Second Edition 13
Preface to Third Edition 14
Georges Köhler (1946–1995) 17
List of Abbreviations 18
Chapter 1. Introduction 20
Chapter 2. The Antibody Response 26
2.1 Early History 26
2.2 Structure of Antibodies 33
2.3 A Typical Antibody Response 35
2.4 What Distinguishes Self from Nonself? 37
2.5 Effector Functions of Antibodies 39
2.6 Tumour Immunity 39
2.7 The Cost of the Immune System: Autoimmunity and Transplant Rejection 40
Chapter 3. Cellular Basis of the Immune System 45
3.1 The Clonal Selection Theory 45
3.2 Lymphocyte Development--General Aspects 47
3.3 B Cells 49
3.4 T Cells 57
3.5 Summary--Cellular Mechanisms in Antibody Production 62
Chapter 4. Nature of Antigens 69
4.1 What Types of Substances are Antigenic? 70
4.2 Proteins that are Very Different from Self 71
4.3 Proteins that are Very Similar to Self: Immune Response Genes 71
4.4 Proteins that are Identical to Self: Can Self Tolerance be Broken? 74
4.5 Denatured (Unfolded) Proteins 75
4.6 Recognition of Short Peptides by T and B Lymphocytes 77
4.7 Antibodies Against Short Peptides Sometimes Recognize the Native Protein 78
4.8 Carbohydrates and Lipids 80
4.9 Haptens: Small Molecules, Such as Drugs, Hormones and Synthetic Compounds 81
4.10 Adjuvants 82
4.11 Summary 84
Chapter 5. Antibody Structure and Function 91
5.1 Structure of Antibodies 91
5.2 Proteolytic Fragmentation of Immunoglobulins 97
5.3 Properties of the Individual Immunoglobulin Classes 98
5.4 Biosynthesis and Assembly of Immunoglobulins 108
5.5 Serum Electrophoresis 110
Chapter 6. Genetics of Antibodies 120
6.1 Immunoglobulin Allotypes 121
6.2 Antibody Genes 123
6.3 Sequential Rearrangement and Expression of Antibody Genes in the Developing B Cell 127
6.4 RNA Processing 128
6.5 Generation of Antibody Diversity 129
6.6 Genetics of Immunoglobulin Class Switches 129
6.7 Affinity Maturation and Somatic Mutation in B Cells 130
6.8 Immunoglobulin Genes and B Cell Neoplasia 131
Chapter 7. Introduction to Monoclonal Antibodies 135
7. I Myeloma (Plasmacytoma) 136
7.2 Monoclonal Antibodies of Predefined Specificity Produced by Hybridomas 141
7.3 Differences Between Conventional and Monoclonal Serology 148
Chapter 8. Production of Monoclonal Antibodies 160
8.1 Choice of Normal Lymphocyte Donor 160
8.2 Immunization Protocol 161
8.3 Choice of Myeloma Cell Line for Fusion 165
8.4 Equipment Required for Fusion and Growth of Hybridomas 168
8.5 Preparation of Spleen Cells 169
8.6 Preparation of Myeloma Cells 170
8.7 Preparation of HAT and HT Medium 172
8.8 Fusion Protocol: Important Variables 172
8.9 Early Growth 177
8.10 Screening Assays 179
8.11 Cloning 194
8.12 Large-scale Cultures 195
8.13 Storage of Hybrids in Liquid Nitrogen 196
8.14 Growing of Hybridomas in Animals 198
8.15 Mix-ups of Cells 198
8.16 Mycoplasma Contamination 199
Chapter 9. Purification, Fragmentation and Isotopic Labelling of Monoclonal Antibodies 211
9.1 Determination of Antibody Class 212
9.2 Methods Used in Antibody Purification 215
9.3 Purification of Monoclonal IgG 228
9.4 Purification of IgM 233
9.5 Fragmentation of Monoclonal Antibodies 234
9.6 Radiolabelling of Monoclonal Antibodies 243
9.7 Conditions for Stability and Storage of Monoclonal Antibodies 246
Chapter 10. Analysis of Antigens Recognized by Monoclonal Antibodies 253
10.1 Cellular Distribution of Antigens Detected by Monoclonal Antibodies 253
10.2 Determination of the Biochemical Nature of the Antigen 254
10.3 The Biotin–Avidin System 257
10.4 Radioiodination of Soluble Protein Antigens 261
10.5 Radiolabelling of Soluble Proteins with Tritium by Reductive Methylation 269
10.6 Radiolabelling of Cellular Proteins 270
10.7 Solubilization of Membrane Proteins 276
10.8 Isolation of Radiolabelled Antigens by Immunoprecipitation 286
10.9 Electrophoretic Methods for Analysis of Protein Antigens 292
10.10 Western Blots 313
10.11 Topographical Analysis of Proteins by Monoclonal and Polyclonal Antibodies 321
10.12 Use of Monoclonal Antibodies as Probes for Protein Conformation 322
10.13 Use of Antibodies in the Production and Identification of Cloned DNA Sequences 323
Chapter 11. Affinity Chromatography 346
11.1 Choice of Affinity Matrix and Coupling Reaction 346
11.2 Coupling of Antibodies and Other Proteins to Activated Gels 352
11.3 Optimization of Antibody Activity of Immunoadsorbents 354
11.4 Use of Antibody Affinity Columns 355
11.5 Elution of Antigen from Immunoadsorbents 359
11.6 Storage of Affinity Columns 364
11.7 Preparation of Protein for Amino Acid Sequencing 365
Chapter 12. Immunofluorescence 371
12.1 Principles of Immunofluorescence 373
12.2 The Fluorescence Microscope 374
12.3 Choice of Fluorochromes 377
12.4 Choice of Filters for Fluorescence Microscopy 383
12.5 Direct and Indirect Immunofluorescence 384
12.6 The Fluorescence-activated Cell Sorter (FACS) 385
12.7 Multicolour Fluorescence 387
12.8 Conjugation of Antibodies with Fluorochromes 389
12.9 The Biotin–Avidin System 400
12.10 Fluorescent Substrates for Alkaline Phosphatase 403
12.11 Staining Cells with Fluorescent Antibodies 403
12.12 Nonspecific Fluorescence 408
12.13 Lack of Staining 411
Chapter 13. Immunohistology 419
13.1 Effects of Fixation on Antigen Recognition by Antibodies---The Balance Between Morphology and Antigenicity 419
13.2 Tissue Preparation and Fixation 420
13.3 Automated Processing for Paraffin Wax Embedding 424
13.4 Polyester Wax Sections 424
13.5 Frozen Sections 426
13.6 Antibody Staining Strategy 429
13.7 Processlng of Slides for Antibody Staining 431
13.8 Enzyme Staining 435
13.9 Postfixation of Slides After Antibody Staining 439
13.10 Counterstaining 439
13.11 Mounting with Cover Slips 439
13.12 Immunofluorescence on Tissue Sections 440
Chapter 14. Construction, Screening and Expression of Recombinant Antibodies 443
14.1 The Bacteriophage Display Library System 443
14.2 The Choice of Fv, scFv or Fab Fragments 444
14.3 Single-chain Fv (scFv) Antibodies 445
14.4 Libraries of Antibody Genes from Naive and Immunized Sources 447
14.5 Bacteriophage Display Libraries 448
14.6 Experimental Details 450
14.7 Methods for Producing Libraries 454
14.8 Vectors for Expression of Recombinant Fab Antibody 456
14.9 Isolation of Antibody-displaying Phage by Panning 459
14.10 Increasing Library Complexity through Recombination—Production of Fab Fragments of Antibodies in Bacteriophage 461
14.11 Affinity Maturation 462
14.12 In vivo Mutation and Rational Selection 468
14.13 Increasing the Complexity of Phage-displayed Recombinant Antibody Libraries 472
14.14 Expression Systems 472
14.15 Affinity Purification of Antibody Fragments from E. coli Supernatant 476
14.16 Discussion 476
Chapter 15. Generation of Conventional Antibodies 484
15.1 Strategies for the Preparation of Highly Specific Polyclonal Antibodies 485
15.2 Preparative SDS-PAGE 487
15.3 Immunization 490
15.4 Purification and Fragmentation of Rabbit, Sheep and Goat IgG 492
15.5 Specific Antibodies from Nonspecific Antisera 494
Glossary 499
Index 504