Immunological Methods

List of Contributors
Preface
Abbreviations List
1 The Quality of Antibodies and Cellular Receptors
I. Introduction
II. Simple Equilibria
III. Competitive Equilibria
References
2 The Isolation and Characterization of Immunoglobulins, Antibodies, and Their Constituent Polypeptide Chains
I. Introduction
II. Fractionation with Neutral Salts at High Concentration
III. Purification of Ig’s
IV. Fractionation by Gel Filtration Chromatography
V. Electrophoretic Separation on a Solid Supporting Medium
VI. Isolation of Antibody by Affinity Chromatography on Sepharose Immunoadsorbents
VII. Immunoadsorbents with Insolubilized Glutaraldehyde-Treated Proteins
VIII. Separation of Polypeptide Chains
IX. Use of Protein A from Staphylococcus aureus as an Immunoadsorbent for the Isolation of Ig’s
References
3 Peptide Mapping at the Nanomole Level
I. Objective
II. Principle of the Method
III. Materials
IV. Procedure
V. Critical Appraisal
VI. An Example of the Application of the Method to Antigenic Variants of Influenza-a Virus Hemagglutinin
References
4 Electrophoresis of Proteins in Polyacrylamide Slab Gels
I. Introduction
II. Procedures for Polyacrylamide Gel Electrophoresis
III. Conclusion
References
5 Resolution of Immunoglobulin Patterns by Analytical Isoelectric Focusing
I. Introduction
II. Principle of the Method
III. Materials
IV. Procedures
V. Application, Sensitivity, and Reproducibility of IEF
References
6 Isolation of Monoclonal Antibody by Preparative Isoelectric Focusing in Horizontal Layers of Sephadex G-75
I. Introduction
II. Principle of the Method
III. Material
IV. Procedure
V. Applications
VI. Limitations
VII. Degree of Purification and Sensitivity
VIII. Reproducibility
References
7 Isotachophoresis of Immunoglobulins
I. Introduction
II. Procedure
III. Discussion
References
8 The Chemical Modification of Proteins, Haptens, and Solid Supports
I. Introduction
II. Theoretical Background
III. Experimentation
Suggested Reading
References
9 Reagents for Immunofluorescence and Their Use for Studying Lymphoid Cell Products
I. Introduction
II. Reagents for Immunofluorescence
III. Staining Procedures
IV. General Comments
Suggested Reading
References
10 Radiolabeling and Immunoprecipitation of Cell-Surface Macromolecules
I. Introduction
II. Labeling Procedures
III. Lysis of Labeled Cells
IV. Specific Purification of Labeled Cell-Surface
Components
References
11 Haptenation of Viable Biological Carriers
I. Introduction
II. Preparation of ONS Esters
III. Haptenation of Carriers
IV. CML Culture Conditions
V. Observations on CML Responses to Haptenated
Lymphocytes
References
12 Production and Assay of Murine Anti-Allotype Antisera
I. Production of Anti-Allotype Serum
II. Quantification of Anti-Allotype Serum
III. Applications
References
13 Preparation of Mouse Antisera against Histocompatibility Antigens
I. Objective
II. Principle of the Method
III. Materials and Procedure
IV. Controls
V. Critical Appraisal
References
14 Technique of HLA Typing by Complement-Dependent Lympholysis
I. Introduction
II. Principles of the Test
III. Details of the Test
IV. Family Studies
V. Some Comments on the Cytotoxicity Test
VI. Technique for Detecting B-Cell Antigens of the HLA System
References
15 The MLR Test in the Mouse
I. The Conventional Primary MLR
II. In Vitro Secondary MLR
III. Critical Comments
References
16 A Sensitive Method for the Separation of Rosette-Forming Cells
I. Objective
II. Principle of the Method
III. Formation of Rosettes
IV. Cell Fractionation
V. Recovery, Depletion, and Enrichment
VI. Applications, Sensitivity, and Limitations
VII. Conclusion
References
17 The Use of Protein A Rosettes to Detect Cell-Surface Antigens
I. Introduction
II. Principle of the Method
III. Materials
IV. Procedures
V. Controls
VI. Critical Aspects
VII. Applications
References
18 Hapten-Gelatin Gels Used as Adsorbents for Separation of Hapten-Specific B Lymphocytes
I. Principle
II. Description of the Technique
III. Applications
IV. Limitations
References
19 Assay for Plaque-Forming Cells
I. Objective
II. Principle of the Method
III. Material
IV. Preparation of Cell Suspensions
V. Plaquing Procedures
VI. Calculations
VII. Critical Factors
References
20 Plaquing and Recovery of Individual Antibody-Producing Cells
I. Objective
II. Materials
III. Procedure
References
21 Assay for Specific Alloantigen-Binding T Cells Activated in the Mixed Lymphocyte Reaction
I. Objective
II. Principle of the Method
III. Cells and Alloantisera
IV. Detection of T-Cell Markers and Stimulator Antigens on Responder Blasts
V. Assay for Alloantigen-Binding Cells
VI. Critical Appraisal: Applications and Limitations
VII. Conclusion
References
22 Assay for Antigen-Specific T-Cell Proliferation in Mice
I. Objective
II. Principle of the Method
III. Materials
IV. Procedure
V. Critical Appraisal
References
Note Added in Proof
23 Antigen-Specific Helper T-Cell Factor and Its Acceptor
I. Introduction
II. Principle of the Method
III. Materials
IV. Procedure
V. Calculations
VI. Critical Appraisal
References
24 In Vitro Immunization of Dissociated Murine Spleen Cells
I. Objective
II. Principle of the Method
III. Materials
IV. Procedure
V. Critical Appraisal
References
25 Induction of a Secondary Antibody Response In Vitro with Rabbit Peripheral Blood Lymphocytes
I. Objective
II. Principle of the Method
III. Materials
IV. Procedure
V. Comments
References
26 Induction of Immune Responses with Clonal Dominance at High Antibody Levels
I. Introduction
II. Principle of the Method
III. Vaccine Preparation
IV. Immunization
V. Serial Transfer of Limited Spleen Cell Numbers
VI. Critical Appraisal
References
27 Limiting Dilution Analysis
I. Objective
II. Principle of the Method
III. Materials
IV. Methods
V. Limitations and Sensitivity
Suggested Reading
References
28 Establishment and Maintenance of Murine Lymphoid Cell Lines in Culture
I. Objective
II. Principle of the Method
III. Materials
IV. Procedure
V. Critical Appraisal
Suggested Reading
References
29 Clonal Growth of Cells in Semisolid or Viscous Medium
I. Introduction
II. Materials
III. Procedure
IV. Applications
References
30 Preparation of Sendai Virus for Cell Fusion
I. Growth of Virus
II. Titration of Virus
III. Concentration of Virus
IV. Inactivation of Virus
V. Assay of Infectivity
References
31 Fusion of Lymphocytes
I. Objective
II. Principle of the Method
III. Material
IV. Procedure
V. Critical Appraisal
References
32 Soft Agar Cloning of Lymphoid Tumor Lines: Detection of Hybrid Clones with Anti-SRBC Activity
I. Objective and Principle of the Method
II. Material
III. Procedure
IV. Critical Appraisal
References
33 Isotope Laboratory
I. Introduction
II. Materials
III. Special Procedures
IV. Radiation and Contamination Surveillance
Suggested Reading
References
34 Analysis of Immunological Data
I. Introduction
II. Worked Examples
References
Subject Index