Immunological Methods

Immunological Methods, Volume III provides information pertinent to the fundamental aspects of immunological methods. This book presents recombinant DNA technology as applied to immunology.

Organized into 25 chapters, this volume begins with an overview of the major histocompatibility complex. This text then examines the use of monoclonal antibodies in the identification of lymphocyte membrane antigens and explains the use of monoclonal antibodies in histocompatibility typing. Other chapters consider the methods in two-dimensional electrophoretic analysis of proteins and another to lymphokines that support B cells. This book discusses as well the production and expansion of cloned B and T cell lines and hybridomas. The final chapter deals with the sophisticated methodology used with particular animal species, namely, birds, sheep, and amphibians to probe specific immunological questions.

This book is a valuable resource for immunologists and investigators with extensive experience in using immunological methods. Protein chemists and biologists will also find this book useful.

Contenu

Contributors
Preface
Contents of Previous Volumes
Abbreviations List
1 Methods in Molecular Immunology: Chromosomal Walking in the Major Histocompatibility Complex
I. Introduction
II. Materials
III. Construction of Cosmid Libraries
IV. Screening of Cosmid Libraries
V. Characterization of Cosmid Clones
VI. Appraisal
References
2 DNA-Mediated Transformation of Lymphoid Cells
I. Introduction
II. Dominant Selectable Markers
III. Techniques of Stable Gene Transfer
IV. Other Techniques of Stable Gene Transfer
References
3 Cloning of cDNAs with Vectors Permitting Expression in Host Cells
I. Introduction
II. General Strategies and Their Problems
III. Experimental Procedures
IV. Some Comments
References
4 Construction of Vectors for Immunoglobulin Reverse Genetics
I. Introduction
II. General Strategy
III. Material and Stock Solutions
IV. Experimental Procedures
V. Examples of Constructions
References
5 Strategies of Oligonucleotide Synthesis
I. Introduction
II. Strategies
III. Experimental Section
IV. Reagents and Solvents That Must Be Purified or Prepared
References
6 High-Performance Liquid Chromatography of Biologically Active Proteins in the Nanogram (Picomole) Range
I. Introduction
II. Materials and Methods
III. Separation by Size: Gel Permeation
IV. Separation by Charge: Ion Exchange and Hydroxyapatite
V. Separation by Hydrophobicity: Reverse Phase
VI. Quantitation and Limits of Detection
VII. Protein Purification
References
7 The HPLC of Immunoglobulins
I. Introduction
II. HPLC: Theory, Practice, and Equipment
III. Immunoglobulins on HPLC
IV. Discussion
References
8 Preparation of Liposomes Carrying Lymphocyte Membrane Proteins
I. Introduction
II. Isolation of Membrane Proteins
III. Preparation of Liposomes
IV. Applications
References
9 Detection of Glycolipid Antigens with Monoclonal Antibodies
I. Introduction
II. Preliminary Tests
III. Lipid Extraction
IV. Solid-Phase RIA
V. Hapten Inhibition and Hapten Binding
VI. Thin-Layer Chromatography
VII. Critical Appraisal
References
10 Mapping of Novel B Lymphocyte Differentiation Antigens
I. Introduction
II. Definition of B Cell Differentiation Antigens by Negative Selection Assay
III. Two-Dimensional Gel Analysis of B Cell Differentiation Antigens
IV. Appendix
References
11 Use of Large-Scale Two-Dimensional ISODALT Gel Electrophoresis System in Immunology
I. Objective
II. Materials
III. Methods
IV. Critical Appraisal
References
12 Tissue Typing Using Biosynthetically Labeled Monoclonal Antibodies
I. Introduction
II. Biosynthetic Labeling
III. Purification
IV. Activity and Stability
V. Binding Assay
VI. Applications and Limitations
References
13 Assays for Lymphokines Supporting B Cell Growth
I. Introduction
II. Materials
III. Preactivation Conditions
IV. Recultivation Conditions
V. Measurement and Proliferation
References
14 Establishment of Cellular Microenvironments for in Vitro B Cell Progenitor Differentiation
I. Introduction
II. Principle of the Method
III. Materials
IV. Procedure
V. Comments
References
15 Development of B Cell Progenitors in Semisolid Agar Cultures
I. Introduction
II. Experimental Methods
III. Summary of Results
References
16 Isolation of Human Alloreactive T Helper Clones and Their Use as Polyclonal Activators of B Cells
I. Objective and Principle of the Method
II. Materials
III. Procedure
IV. Critical Appraisal
References
17 Cytolytic T Cell Clones and Hybridomas
I. Introduction
II. Materials
III. Methods
IV. Application
References
18 Conditions for the Establishment in Vitro of Interleukin 3-Dependent Murine B Cell Precursor Lines
I. Introduction
II. Materials
III. WEHI-3 Supernatants
IV. Assay for IL-3 Activity
V. Preparation of Cells
VI. Establishment of IL-3-Dependent B Cell Precursor Lines
VII. Cloning IL-3-Dependent B Cell Precursors
VIII. Characterizing the Cell Lines
IX. Concluding Remarks
References
19 Assays for the Detection of Rheumatoid Factor-Producing Cells
I. Introduction
II. Hemolysis Assay
III. Plaque Assay
IV. Radioimmunoassay
References
20 Fluorescence Cell Sorter Techniques in Immunology
I. Introduction
II. Description of a Cell Sorter
III. Preparation of Reagents
IV. Sample Preparation
V. Cell Separation
VI. Data Acquisition
VII. Single-Parameter Data Analysis and Interpretation
VIII. Examples
21 Tables for Evaluating Limiting Dilution Experiments
I. Introduction
II. Percentage of Single Clones
III. Confidence Limits
IV. Test for Independence in 2 x 2 Contingency Tables
References
22 Development of an Avian Lymphoid Chimera
I. Introduction
II. Principle of the Method
III. Details of the Procedure
IV. Evaluation of Chimeras
V. Comments
References
23 Avian Embryos in Immunology
I. Introduction
II. Materials
III. Methods
References
24 Sheep as an Experimental Model for Immunology: Immunological Techniques in Vitro and In Vivo
I. Introduction
II. Maintenance of Experimental Sheep
III. In Vivo Procedures
IV. In Vitro Procedures
References
25 Methods Used to Study the Immune System of Xenopus (Amphibia, Anura)
I. Introduction: The Immune System of Xenopus
II. Strains and Species Available
III. Operating on Xenopus
IV. Preparing Lymphocytes for Cell Culture
V. Making Reagents
VI. Analyzing the Immune System
VII. Functional Assays Analyzing Immune Response
VIII. Somatic Cell Hybrid Between Frog Lymphocytes and Mouse Myeloma Cells
IX. Cell Markers, Ploidy Markers
X. Appendix
References
Index