Immunological Methods

Immunological Methods a compendium of basic research techniques being used in one of the largest immunology research institutes, the Basel Institute for Immunology, with particular emphasis given to new methodology. The procedures have been described by individuals judged to be highly expert in their specialties. In many instances the methods developed or adapted to unique uses by the contributors have not previously been described in detail. The book contains 34 chapters covering techniques for detection, isolation, and purification of antibodies (including dansylation, two-dimensional chromatography, isoelectric focusing, polyacrylamide gel electrophoresis, and isotachophoresis); measurement of equilibrium constants (equilibrium dialysis, filtration, and sedimentation); and isotope and fluorescent labeling and detection of cell-surface components. Techniques such as isotope laboratory maintenance; chemical modification of proteins, haptens, and solid supports, and haptenation of viable biological carriers; production of antisera against allotypes and histocompatibility antigens and production of antibody with clonai dominance; histocompatibility and MLR testing; and cell separation by haptenated gels and by velocity sedimentation of rosette-forming cells are also discussed. Other chapters cover detection of antibody-secreting and alloantigen-binding cells; immune responses in vitro and their analysis by limiting dilution; production of T-cell factors; hybridoma production by cell fusion; maintenance of cell lines and cloning in semisolid media; and the mathematical analysis of immunological data.

Table of Contents:
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