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.

List of Contributors

Preface


Contents of Volume I


Abbreviations List


1 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


References


2 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


References


3 Two-Dimensional Gel Electrophoresis


I. Introduction


II. One-Dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gels (SDS-PAGE)


III. Two-Dimensional Gel Techniques


References


4 Measurements of Antibodies Specific for DNA


I. Introduction


II. Antigen Preparations


III. Anti-DNA Assays


IV. Critical Appraisal


References


5 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


References


6 Methods in Surface Physics for Immunology


I. Introduction


II. Review of Surface Physics


III. Methods of Measurement


IV. Thin Layer Immunoassay Techniques


V. Conclusions


References


7 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


References


8 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


References


9 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


References


10 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


References


11 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


References


12 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


References


13 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


References


14 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


References


15 Long-Term Culture and Cloning of Specific Helper Cells


I. Objective and Principle of the Method


II. Materials


III. Procedure


IV. Critical Appraisal


Reference


16 The Cloning of Alloreactive Cells


I. Objective


II. Principle


III. Materials


IV. Procedures


V. Critical Appraisal


References


17 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


References


18 Enzyme Immunoassay for the Detection of Hybridoma Products


I. Introduction


II. Materials


III. Enzyme Immunoassay


IV. Conclusion


References


Index