- Volume
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v. 2 ISBN 9780124427020
Description
Immunological Methods, Volume II compiles procedures that are appropriate for studies in immunology. This book discusses the determination of equilibrium binding parameters of monoclonal antibodies specific for cell surface antigens; two-dimensional gel electrophoresis; and measurements of antibodies specific for DNA. The methods in surface physics for immunology; HLA-DR typing by complement-dependent B lymphocyte lysis; and protein A plaque assay for the detection of immunoglobulin-secreting cells are also elaborated. This text also covers the in vitro production and testing of antigen-induced mediators of helper T-cell function; limiting dilution analysis of precursors of cytotoxic T lymphocytes; and induction of antibody formation in mouse bone marrow. Other topics include the long-term culture and cloning of specific helper T cells; cloning of alloreactive T cells; and enzyme immunoassay for the detection of hybridoma products. This publication is valuable to immunologists and medical practitioners researching on immunological methods.
Table of Contents
List of ContributorsPrefaceContents of Volume IAbbreviations List1 Determination of Equilibrium Binding Parameters of Monoclonal Antibodies Specific for Cell Surface Antigens I. Introduction II. Theoretical Background III. Technical Approach IV. Examples V. Critical Appraisal VI. Appendix References2 Biochemical Characterization of Cell Surface Antigens Using Monoclonal Antibodies I. Introduction II. Production of Antibodies III. Labeling Procedures for Cell Surface Antigens IV. Preparation of Cell Membranes V. Analysis of Cell Surface Antigens VI. Conclusion References3 Two-Dimensional Gel Electrophoresis I. Introduction II. One-Dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gels (SDS-PAGE) III. Two-Dimensional Gel Techniques References4 Measurements of Antibodies Specific for DNA I. Introduction II. Antigen Preparations III. Anti-DNA Assays IV. Critical Appraisal References5 High-Pressure Liquid Chromatography of Proteins and Peptides I. Introduction II. General Description of HPLC III. Components of an HPLC System IV. Examples of HPLC Applications References6 Methods in Surface Physics for Immunology I. Introduction II. Review of Surface Physics III. Methods of Measurement IV. Thin Layer Immunoassay Techniques V. Conclusions References7 Hapten-Modified Antibodies Specific for Cell Surface Antigens as a Tool in Cellular Immunology I. Introduction II. Hapten Sandwich Labeling III. Preparation of Hapten Sandwich Labeling Reagents IV. Selected Examples of the Application of Hapten Sandwich Labeling Reagents V. Critical Appraisal References8 HLA-DR Typing by Complement-Dependent B Lymphocyte Lysis I. Introduction II. Principle of the Test III. Details of the Test IV. Modifications of the Test V. Critical Appraisal VI. Equipment, Supplies, and Solutions References9 The Protein A Plaque Assay for the Detection of Immunoglobulin-Secreting Cells I. Introduction II. Reagents III. Cell Suspensions IV. Plaquing Procedure V. Counting of Plaque-Forming Cells VI. General Comments VII. Appendix References10 In Vitro Production and Testing of Antigen-Induced Mediators of Helper T-Cell Function I. Introduction II. Materials III. Use of the Marbrook Culture System for Inducing Antigen-Specific Helper Supernatants IV. Testing the Activity of Antigen-Specific Factors V. Plaque Assay VI. Summary References11 A Helper Cell Assay of Cellular Antigens and Its Applications to Hapten-Specific ? Cells I. Introduction II. Principles of the Method and Its Modifications III. Materials IV. Procedures V. Comments References12 Limiting Dilution Analysis of Precursors of Cytotoxic ? Lymphocytes I. Principle of the Method II. Materials III. Methods IV. Experimental Design V. Analysis of the Results VI. Developments of the Limiting Dilution Assay VII. Limitations of the Assay References13 Induction of an Antibody Response in Cultures of Human Peripheral Blood Lymphocytes I. Objective II. Principle of the Method III. Materials IV. Procedure V. Comments References14 Induction of Antibody Formation in Mouse Bone Marrow I. Introduction II. Collection of Mouse Bone Marrow Cells III. Distribution of Antibody-Forming Cells Over Various Bone Marrow Compartments IV. Calculation of the Antibody-Forming Cell Activity of the Total Bone Marrow V. Kinetics of the Response VI. Critical Factors References15 Long-Term Culture and Cloning of Specific Helper ? Cells I. Objective and Principle of the Method II. Materials III. Procedure IV. Critical Appraisal Reference16 The Cloning of Alloreactive ? Cells I. Objective II. Principle III. Materials IV. Procedures V. Critical Appraisal References17 The Technique of Hybridoma Production I. Introduction II. Prefusion Steps III. Preparation of Medium Constituents, Media, and Cell Suspensions IV. Cell Fusion with Polyethylene Glycol V. Products of Fusion VI. Freezing and Thawing of Hybridoma Cells References18 Enzyme Immunoassay for the Detection of Hybridoma Products I. Introduction II. Materials III. Enzyme Immunoassay IV. Conclusion ReferencesIndex
- Volume
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[v. 1] ISBN 9780124427501
Description
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
by "Nielsen BookData"