Monoclonal Antibodies: Principles and Practice
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Table of contents
- Contentsv
- Preface to First Editionx
- Preface to Second Editionxii
- Preface to Third Editionxiii
- Georges Köhler (1946–1995)xvi
- List of Abbreviationsxvii
- Chapter 1. Introduction1
- Chapter 2. The Antibody Response7
- 2.1 Early History7
- 2.2 Structure of Antibodies14
- 2.3 A Typical Antibody Response16
- 2.4 What Distinguishes Self from Nonself?18
- 2.5 Effector Functions of Antibodies20
- 2.6 Tumour Immunity20
- 2.7 The Cost of the Immune System: Autoimmunity and Transplant Rejection21
- Chapter 3. Cellular Basis of the Immune System26
- 3.1 The Clonal Selection Theory26
- 3.2 Lymphocyte Development--General Aspects28
- 3.3 B Cells30
- 3.4 T Cells38
- 3.5 Summary--Cellular Mechanisms in Antibody Production43
- Chapter 4. Nature of Antigens50
- 4.1 What Types of Substances are Antigenic?51
- 4.2 Proteins that are Very Different from Self52
- 4.3 Proteins that are Very Similar to Self: Immune Response Genes52
- 4.4 Proteins that are Identical to Self: Can Self Tolerance be Broken?55
- 4.5 Denatured (Unfolded) Proteins56
- 4.6 Recognition of Short Peptides by T and B Lymphocytes58
- 4.7 Antibodies Against Short Peptides Sometimes Recognize the Native Protein59
- 4.8 Carbohydrates and Lipids61
- 4.9 Haptens: Small Molecules, Such as Drugs, Hormones and Synthetic Compounds62
- 4.10 Adjuvants63
- 4.11 Summary65
- Chapter 5. Antibody Structure and Function72
- 5.1 Structure of Antibodies72
- 5.2 Proteolytic Fragmentation of Immunoglobulins78
- 5.3 Properties of the Individual Immunoglobulin Classes79
- 5.4 Biosynthesis and Assembly of Immunoglobulins89
- 5.5 Serum Electrophoresis91
- Chapter 6. Genetics of Antibodies101
- 6.1 Immunoglobulin Allotypes102
- 6.2 Antibody Genes104
- 6.3 Sequential Rearrangement and Expression of Antibody Genes in the Developing B Cell108
- 6.4 RNA Processing109
- 6.5 Generation of Antibody Diversity110
- 6.6 Genetics of Immunoglobulin Class Switches110
- 6.7 Affinity Maturation and Somatic Mutation in B Cells111
- 6.8 Immunoglobulin Genes and B Cell Neoplasia112
- Chapter 7. Introduction to Monoclonal Antibodies116
- 7. I Myeloma (Plasmacytoma)117
- 7.2 Monoclonal Antibodies of Predefined Specificity Produced by Hybridomas122
- 7.3 Differences Between Conventional and Monoclonal Serology129
- Chapter 8. Production of Monoclonal Antibodies141
- 8.1 Choice of Normal Lymphocyte Donor141
- 8.2 Immunization Protocol142
- 8.3 Choice of Myeloma Cell Line for Fusion146
- 8.4 Equipment Required for Fusion and Growth of Hybridomas149
- 8.5 Preparation of Spleen Cells150
- 8.6 Preparation of Myeloma Cells151
- 8.7 Preparation of HAT and HT Medium153
- 8.8 Fusion Protocol: Important Variables153
- 8.9 Early Growth158
- 8.10 Screening Assays160
- 8.11 Cloning175
- 8.12 Large-scale Cultures176
- 8.13 Storage of Hybrids in Liquid Nitrogen177
- 8.14 Growing of Hybridomas in Animals179
- 8.15 Mix-ups of Cells179
- 8.16 Mycoplasma Contamination180
- Chapter 9. Purification, Fragmentation and Isotopic Labelling of Monoclonal Antibodies192
- 9.1 Determination of Antibody Class193
- 9.2 Methods Used in Antibody Purification196
- 9.3 Purification of Monoclonal IgG209
- 9.4 Purification of IgM214
- 9.5 Fragmentation of Monoclonal Antibodies215
- 9.6 Radiolabelling of Monoclonal Antibodies224
- 9.7 Conditions for Stability and Storage of Monoclonal Antibodies227
- Chapter 10. Analysis of Antigens Recognized by Monoclonal Antibodies234
- 10.1 Cellular Distribution of Antigens Detected by Monoclonal Antibodies234
- 10.2 Determination of the Biochemical Nature of the Antigen235
- 10.3 The Biotin–Avidin System238
- 10.4 Radioiodination of Soluble Protein Antigens242
- 10.5 Radiolabelling of Soluble Proteins with Tritium by Reductive Methylation250
- 10.6 Radiolabelling of Cellular Proteins251
- 10.7 Solubilization of Membrane Proteins257
- 10.8 Isolation of Radiolabelled Antigens by Immunoprecipitation267
- 10.9 Electrophoretic Methods for Analysis of Protein Antigens273
- 10.10 Western Blots294
- 10.11 Topographical Analysis of Proteins by Monoclonal and Polyclonal Antibodies302
- 10.12 Use of Monoclonal Antibodies as Probes for Protein Conformation303
- 10.13 Use of Antibodies in the Production and Identification of Cloned DNA Sequences304
- Chapter 11. Affinity Chromatography327
- 11.1 Choice of Affinity Matrix and Coupling Reaction327
- 11.2 Coupling of Antibodies and Other Proteins to Activated Gels333
- 11.3 Optimization of Antibody Activity of Immunoadsorbents335
- 11.4 Use of Antibody Affinity Columns336
- 11.5 Elution of Antigen from Immunoadsorbents340
- 11.6 Storage of Affinity Columns345
- 11.7 Preparation of Protein for Amino Acid Sequencing346
- Chapter 12. Immunofluorescence352
- 12.1 Principles of Immunofluorescence354
- 12.2 The Fluorescence Microscope355
- 12.3 Choice of Fluorochromes358
- 12.4 Choice of Filters for Fluorescence Microscopy364
- 12.5 Direct and Indirect Immunofluorescence365
- 12.6 The Fluorescence-activated Cell Sorter (FACS)366
- 12.7 Multicolour Fluorescence368
- 12.8 Conjugation of Antibodies with Fluorochromes370
- 12.9 The Biotin–Avidin System381
- 12.10 Fluorescent Substrates for Alkaline Phosphatase384
- 12.11 Staining Cells with Fluorescent Antibodies384
- 12.12 Nonspecific Fluorescence389
- 12.13 Lack of Staining392
- Chapter 13. Immunohistology400
- 13.1 Effects of Fixation on Antigen Recognition by Antibodies---The Balance Between Morphology and A400
- 13.2 Tissue Preparation and Fixation401
- 13.3 Automated Processing for Paraffin Wax Embedding405
- 13.4 Polyester Wax Sections405
- 13.5 Frozen Sections407
- 13.6 Antibody Staining Strategy410
- 13.7 Processlng of Slides for Antibody Staining412
- 13.8 Enzyme Staining416
- 13.9 Postfixation of Slides After Antibody Staining420
- 13.10 Counterstaining420
- 13.11 Mounting with Cover Slips420
- 13.12 Immunofluorescence on Tissue Sections421
- Chapter 14. Construction, Screening and Expression of Recombinant Antibodies424
- 14.1 The Bacteriophage Display Library System424
- 14.2 The Choice of Fv, scFv or Fab Fragments425
- 14.3 Single-chain Fv (scFv) Antibodies426
- 14.4 Libraries of Antibody Genes from Naive and Immunized Sources428
- 14.5 Bacteriophage Display Libraries429
- 14.6 Experimental Details431
- 14.7 Methods for Producing Libraries435
- 14.8 Vectors for Expression of Recombinant Fab Antibody437
- 14.9 Isolation of Antibody-displaying Phage by Panning440
- 14.10 Increasing Library Complexity through Recombination„Production of Fab Fragments of Antibodie442
- 14.11 Affinity Maturation443
- 14.12 In vivo Mutation and Rational Selection449
- 14.13 Increasing the Complexity of Phage-displayed Recombinant Antibody Libraries453
- 14.14 Expression Systems453
- 14.15 Affinity Purification of Antibody Fragments from E. coli Supernatant457
- 14.16 Discussion457
- Chapter 15. Generation of Conventional Antibodies465
- 15.1 Strategies for the Preparation of Highly Specific Polyclonal Antibodies466
- 15.2 Preparative SDS-PAGE468
- 15.3 Immunization471
- 15.4 Purification and Fragmentation of Rabbit, Sheep and Goat IgG473
- 15.5 Specific Antibodies from Nonspecific Antisera475
- Glossary480
- Index485
Monoclonal Antibodies now have applications in virtually all areas of biology and medicine, and much of the world's biotechnology industry has its foundations in the exploitation of this technology.
The Third Edition of this well established book meets the needs of both newcomers to the area and experienced researchers, by providing an integrated treatment of both the production and application of monoclonal antibodies. As in previous editions, detailed and critical accounts of the theory, production, purification, fragmentation, storage and radiolabelling of monoclonal antibodies are given, along with descriptions of their use in antigen characterization, affinity chromatography and immunofluorescence. The present volume has been comprehensively updated to cover recent rapid advances, particularly with respect to the applications of molecular biology, the use of antibodies in closing and heterologous expression of genes, immunohistology and phage display libraries. Since the previous edition, there has been a growing trend towards the replacement of procedures using radioactive isotopes, and the current edition incorporates these newer technologies.
The text is oriented towards problems solving, and makes it easy to adapt each procedure to individual needs. Extensive cross-referencing, a glossary and a comprehensive index make this book an essential reference.
This book will be vital both for laboratories already producing or using monoclonal antibodies, and for workers in many disciplines who are contemplating their use.
*
* Provides an integrated treatment of both the production and application of monoclonals in cell biology, biochemistry, and immunology.
* Gives detailed and critical accounts of the theory, production, purification, storage, and relabelling of monoclonals, and their use in antigen characterization, affinity chromorography, and immunofluroscence.
* Comprehensively updated to cover the rapid advances that have occurred since the publication of the Second Edition.
The Third Edition of this well established book meets the needs of both newcomers to the area and experienced researchers, by providing an integrated treatment of both the production and application of monoclonal antibodies. As in previous editions, detailed and critical accounts of the theory, production, purification, fragmentation, storage and radiolabelling of monoclonal antibodies are given, along with descriptions of their use in antigen characterization, affinity chromatography and immunofluorescence. The present volume has been comprehensively updated to cover recent rapid advances, particularly with respect to the applications of molecular biology, the use of antibodies in closing and heterologous expression of genes, immunohistology and phage display libraries. Since the previous edition, there has been a growing trend towards the replacement of procedures using radioactive isotopes, and the current edition incorporates these newer technologies.
The text is oriented towards problems solving, and makes it easy to adapt each procedure to individual needs. Extensive cross-referencing, a glossary and a comprehensive index make this book an essential reference.
This book will be vital both for laboratories already producing or using monoclonal antibodies, and for workers in many disciplines who are contemplating their use.
*
* Provides an integrated treatment of both the production and application of monoclonals in cell biology, biochemistry, and immunology.
* Gives detailed and critical accounts of the theory, production, purification, storage, and relabelling of monoclonals, and their use in antigen characterization, affinity chromorography, and immunofluroscence.
* Comprehensively updated to cover the rapid advances that have occurred since the publication of the Second Edition.