Monoclonal Antibodies - Principles and Practice

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Monoclonal Antibodies: Principles and Practice

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Contents

Preface to First Edition

Preface to Second Edition

Preface to Third Edition

Georges Köhler (1946–1995)

List of Abbreviations

Chapter 1. Introduction

Chapter 2. The Antibody Response

2.1 Early History

2.2 Structure of Antibodies

2.3 A Typical Antibody Response

2.4 What Distinguishes Self from Nonself?

2.5 Effector Functions of Antibodies

2.6 Tumour Immunity

2.7 The Cost of the Immune System: Autoimmunity and Transplant Rejection

Chapter 3. Cellular Basis of the Immune System

3.1 The Clonal Selection Theory

3.2 Lymphocyte Development--General Aspects

3.3 B Cells

3.4 T Cells

3.5 Summary--Cellular Mechanisms in Antibody Production

Chapter 4. Nature of Antigens

4.1 What Types of Substances are Antigenic?

4.2 Proteins that are Very Different from Self

4.3 Proteins that are Very Similar to Self: Immune Response Genes

4.4 Proteins that are Identical to Self: Can Self Tolerance be Broken?

4.5 Denatured (Unfolded) Proteins

4.6 Recognition of Short Peptides by T and B Lymphocytes

4.7 Antibodies Against Short Peptides Sometimes Recognize the Native Protein

4.8 Carbohydrates and Lipids

4.9 Haptens: Small Molecules, Such as Drugs, Hormones and Synthetic Compounds

4.10 Adjuvants

4.11 Summary

Chapter 5. Antibody Structure and Function

5.1 Structure of Antibodies

5.2 Proteolytic Fragmentation of Immunoglobulins

5.3 Properties of the Individual Immunoglobulin Classes

5.4 Biosynthesis and Assembly of Immunoglobulins

5.5 Serum Electrophoresis

Chapter 6. Genetics of Antibodies

6.1 Immunoglobulin Allotypes

6.2 Antibody Genes

6.3 Sequential Rearrangement and Expression of Antibody Genes in the Developing B Cell

6.4 RNA Processing

6.5 Generation of Antibody Diversity

6.6 Genetics of Immunoglobulin Class Switches

6.7 Affinity Maturation and Somatic Mutation in B Cells

6.8 Immunoglobulin Genes and B Cell Neoplasia

Chapter 7. Introduction to Monoclonal Antibodies

7. I Myeloma (Plasmacytoma)

7.2 Monoclonal Antibodies of Predefined Specificity Produced by Hybridomas

7.3 Differences Between Conventional and Monoclonal Serology

Chapter 8. Production of Monoclonal Antibodies

8.1 Choice of Normal Lymphocyte Donor

8.2 Immunization Protocol

8.3 Choice of Myeloma Cell Line for Fusion

8.4 Equipment Required for Fusion and Growth of Hybridomas

8.5 Preparation of Spleen Cells

8.6 Preparation of Myeloma Cells

8.7 Preparation of HAT and HT Medium

8.8 Fusion Protocol: Important Variables

8.9 Early Growth

8.10 Screening Assays

8.11 Cloning

8.12 Large-scale Cultures

8.13 Storage of Hybrids in Liquid Nitrogen

8.14 Growing of Hybridomas in Animals

8.15 Mix-ups of Cells

8.16 Mycoplasma Contamination

Chapter 9. Purification, Fragmentation and Isotopic Labelling of Monoclonal Antibodies

9.1 Determination of Antibody Class

9.2 Methods Used in Antibody Purification

9.3 Purification of Monoclonal IgG

9.4 Purification of IgM

9.5 Fragmentation of Monoclonal Antibodies

9.6 Radiolabelling of Monoclonal Antibodies

9.7 Conditions for Stability and Storage of Monoclonal Antibodies

Chapter 10. Analysis of Antigens Recognized by Monoclonal Antibodies

10.1 Cellular Distribution of Antigens Detected by Monoclonal Antibodies

10.2 Determination of the Biochemical Nature of the Antigen

10.3 The Biotin–Avidin System

10.4 Radioiodination of Soluble Protein Antigens

10.5 Radiolabelling of Soluble Proteins with Tritium by Reductive Methylation

10.6 Radiolabelling of Cellular Proteins

10.7 Solubilization of Membrane Proteins

10.8 Isolation of Radiolabelled Antigens by Immunoprecipitation

10.9 Electrophoretic Methods for Analysis of Protein Antigens

10.10 Western Blots

10.11 Topographical Analysis of Proteins by Monoclonal and Polyclonal Antibodies

10.12 Use of Monoclonal Antibodies as Probes for Protein Conformation

10.13 Use of Antibodies in the Production and Identification of Cloned DNA Sequences

Chapter 11. Affinity Chromatography

11.1 Choice of Affinity Matrix and Coupling Reaction

11.2 Coupling of Antibodies and Other Proteins to Activated Gels

11.3 Optimization of Antibody Activity of Immunoadsorbents

11.4 Use of Antibody Affinity Columns

11.5 Elution of Antigen from Immunoadsorbents

11.6 Storage of Affinity Columns

11.7 Preparation of Protein for Amino Acid Sequencing

Chapter 12. Immunofluorescence

12.1 Principles of Immunofluorescence

12.2 The Fluorescence Microscope

12.3 Choice of Fluorochromes

12.4 Choice of Filters for Fluorescence Microscopy

12.5 Direct and Indirect Immunofluorescence

12.6 The Fluorescence-activated Cell Sorter (FACS)

12.7 Multicolour Fluorescence

12.8 Conjugation of Antibodies with Fluorochromes

12.9 The Biotin–Avidin System

12.10 Fluorescent Substrates for Alkaline Phosphatase

12.11 Staining Cells with Fluorescent Antibodies

12.12 Nonspecific Fluorescence

12.13 Lack of Staining

Chapter 13. Immunohistology

13.1 Effects of Fixation on Antigen Recognition by Antibodies---The Balance Between Morphology and Antigenicity

13.2 Tissue Preparation and Fixation

13.3 Automated Processing for Paraffin Wax Embedding

13.4 Polyester Wax Sections

13.5 Frozen Sections

13.6 Antibody Staining Strategy

13.7 Processlng of Slides for Antibody Staining

13.8 Enzyme Staining

13.9 Postfixation of Slides After Antibody Staining

13.10 Counterstaining

13.11 Mounting with Cover Slips

13.12 Immunofluorescence on Tissue Sections

Chapter 14. Construction, Screening and Expression of Recombinant Antibodies

14.1 The Bacteriophage Display Library System

14.2 The Choice of Fv, scFv or Fab Fragments

14.3 Single-chain Fv (scFv) Antibodies

14.4 Libraries of Antibody Genes from Naive and Immunized Sources

14.5 Bacteriophage Display Libraries

14.6 Experimental Details

14.7 Methods for Producing Libraries

14.8 Vectors for Expression of Recombinant Fab Antibody

14.9 Isolation of Antibody-displaying Phage by Panning

14.10 Increasing Library Complexity through Recombination—Production of Fab Fragments of Antibodies in Bacteriophage

14.11 Affinity Maturation

14.12 In vivo Mutation and Rational Selection

14.13 Increasing the Complexity of Phage-displayed Recombinant Antibody Libraries

14.14 Expression Systems

14.15 Affinity Purification of Antibody Fragments from E. coli Supernatant

14.16 Discussion

Chapter 15. Generation of Conventional Antibodies

15.1 Strategies for the Preparation of Highly Specific Polyclonal Antibodies

15.2 Preparative SDS-PAGE

15.3 Immunization

15.4 Purification and Fragmentation of Rabbit, Sheep and Goat IgG

15.5 Specific Antibodies from Nonspecific Antisera

Glossary

Index