Immunological Methods - 1st Edition - ISBN: 9780124427501, 9781483269993

Immunological Methods

1st Edition

Editors: Ivan Lefkovits Benvenuto Pernis
eBook ISBN: 9781483269993
Imprint: Academic Press
Published Date: 28th March 1979
Page Count: 592
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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

Details

No. of pages:
592
Language:
English
Copyright:
© Academic Press 1979
Published:
Imprint:
Academic Press
eBook ISBN:
9781483269993

About the Editor

Ivan Lefkovits

Affiliations and Expertise

Basel Institute for Immunology, Basel, Switzerland

Benvenuto Pernis