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Antigen-Antibody Reactions In Vivo - 1st Edition - ISBN: 9780127544052, 9781483220604

Antigen-Antibody Reactions In Vivo

1st Edition

Methods in Immunology and Immunochemistry, Vol. 5

Editors: Curtis A. Williams Merrill W. Chase
eBook ISBN: 9781483220604
Paperback ISBN: 9781483207179
Hardcover ISBN: 9780127544052
Imprint: Academic Press
Published Date: 28th December 1976
Page Count: 586
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Methods in Immunology and Immunochemistry, Volume V: Antigen-Antibody Reactions In Vivo deals primarily with immune phenomena in tissues or in cell preparations. This book covers a variety of topics, including anaphylaxis, tolerance, immune suppression with chemical agents, radiation effects, antibody synthesis in vitro, immunological methods, and applied electron microscopy. Organized into 10 chapters, this volume begins with an overview of systemic anaphylaxis investigations in other more resistant species. This text then presents the analysis of mechanisms involved in the pathogenesis of the Arthus phenomenon, which shed light on the understanding of other lesions of hypersensitivity. Other chapters consider the effects of antigen–antibody interaction on connective tissue. This book discusses as well the degree and duration of acquired tolerance. The final chapter deals with the application of electron microscopy in the elucidation of the mechanisms of immune reactions. This book is a valuable resource for immunologists, students, and research workers.

Table of Contents

Contributors to Volume


Contents of Other Volumes

Chapter 19. Anaphylaxis

A. Systemic Anaphylaxis

Active Anaphylaxis: Antigens

Procedures with Guinea Pigs







Passive Anaphylaxis: Principles

Procedures with Various Species

B. Local Anaphylaxis

Active Cutaneous Anaphylaxis

Passive Cutaneous Anaphylaxis

General Procedures

Special Procedures for PCA in Guinea Pigs

Procedures for Mice, Rats, Rabbits

Semiquantitative Passive Cutaneous Anaphylaxis

Prausnitz-Küstner Skin Tests in Man for Reagins (IgE Antibody)

Passive Transfer of Human Reagins into the Monkey

C. Anaphylaxis in Isolated Tissues and Cells

1. Anaphylaxis in Isolated Tissues

2. Anaphylactic Responses with Isolated Lung Slices

3. Anaphylactic Release of Histamine from Rat Mast Cells

4. Responses of Isolated Leukocytes and Platelets in Reaginic Allergy

D. Assay of Pharmacologically Active Materials

1. Assay of Histamine

2. Assay of Serotonin

3. Biological Assay of Slow Reacting Substances—SRS-A, Bradykinin, Prostaglandins,

4. Immunoassay for Bradykinin and Kininogen

Chapter 20. The Arthus and Related Reactions

A. Cutaneous Arthus Reactions

Active, Direct Passive, Reversed Passive Arthus Reactions

Local Passive Arthus Reactions

Reactions Induced by Soluble Complexes

Tissues Employed in Arthus Studies

Elicitation Requirement for Precipitating Antibody and for Complement Fixation

B. Methods of Modifying Arthus Reactions

Elimination of Polymorphonuclear Leukocytes

Depletion of Serum Complement

Preparation of Cobra Venom Factor



C. Arthus Phenomenon in Disease Processes

Serum Sickness Arteritis

Acute Nephrotoxic Nephritis

Cutaneous Reaction to Anti-Kidney Basement Membrane Antibody

Chapter 21. Analysis of Antigen-Antibody Reactions in Cornea


Local Antigen-Antibody Reactions in the Cornea

Wessely Phenomenon

Anaphylactic Keratitis

Local Intracorneal

Injections in Rabbits with Both Antigen and Antibody

Chapter 22. Immunological Tolerance

A. Introduction

B. Tolerance to Heterologous Serum Proteins and Related Substances

1. Induction in Neonatal Animals, 188. Rabbits

2. Induction of Tolerance in Normal Adult Animals

3. Induction of Tolerance in Irradiated Normal Animals

C. Tolerance to Homologous and Heterologous Erythrocytes




D. Induction of Tolerance to Chemical Haptens

Induction by Feeding

By Injection

E. Induction of Tolerance in Vitro


Polymerized Flagellin


Chapter 23. Immune Suppression and Induction of Tolerance with Chemical Agents

A. Introduction

B. Biochemical and Pharmacological Properties of the Agent



5-Fluoro-2-Deoxyuridine (FUDR)


Actinomycin D

C. Immunosuppressive Properties of the Agents

1. 6-Mercaptopurine

2. Cyclophosphamide

3. Methotrexate

4. 5-Fluoro-2-deoxyuridine (FUDR)

5. Actinomycin D

Chapter 24. Methods of Applications of Radiation in Immunological Research

A. Radiation Measurements

Characteristics of the X-Ray Beam


Roentgen Units

Importance of Rate of Exposure

Problems in Determining Absorbed Dose

Electronic Equilibrium

B. Representative Effects of Radiation

1. In Vitro Radiation Effects on Serum

2. Total Body Irradiation

3. Low Level Protracted Semicontinuous Radiation

4. Partial Body Radiation: Selective Shielding

C. Concluding Remarks


Depression or Enhancement of Antibody Synthesis by X-Ray Irradiation

Chimeras Produced by Irradiation

Late Effects of Radiation

Chapter 25. Phagocytosis

A. Phagocytosis

1. Preparation of Phagocytic Cells

2. Media and Cell Counting

3. Direct Evaluation of Ingestion

4. Evaluation of Phagocytosis by Measuring Phagocytic Metabolism

B. Clearance in Vivo


Antigen-Antibody Complexes

Bacterial Clearance

Clearance of Carbon from the Blood Stream

Effect of RES Stimulation

Clearance of Bacteria from Serous Cavities

C. Elimination Of 51Cr-Labeled Erythrocytes


Studies of Normal Erythrocyte Lifespan

Assessment of Anti-Erythrocyte Antibodies

Patterns of Elimination

Labeling and Sampling Methods


Chromium Dose Calculation

Blood Collections

Elimination Rates

Immediate Elimination by Natural Antibodies or by RES

Immune Clearance

Onset of Antibody Formation, Slow Immune Clearance, Calculations

Sensitivity of the 51Cr-Elimination Method

Chapter 26. Antibody Synthesis in Vitro

A. Tissue Culture Methods for Antibody Production in Vitro

1. Introduction

2. Technique of Lymph Node Cultures

3. Culture of Spleen Cells for Primary Response

4. Measurement of Synthesized Antibody

B. Feeder Cell Techniques For Antibody Formation in Vitro

General Considerations

Immunization by Diphtheria Toxoid in Rats in Rabbits

Stimulation of Immune Rat Lymph Node Fragments by Diffusible Materials Produced by Nurse Cells in a Parabiotic Chamber

Stimulation of Immune Rabbit Lymph Node Fragments by Rabbit Thymus Preparations in Miniature Parabiotic Chamber, or by Thymosin

C. Single-Cell Techniques in Antibody Production


Micromanipulation Equipment

Advance Preparation of the Cover Slip in the Micromanipulation Chamber

Cell Depot Droplets for Selection of Single Cells

Handling of Mammalian Cells

Isolation and Washing Procedures

Single Cell Isolation

Special Bicarbonate-Free Nutrient Medium

Detection of Antibody to Bacterial Antigen Synthesized by Single Cells

Bacterial Adherence to Single Cells

Immobilization of Motile Bacteria

Observations on Bacterial H and O Agglutination

Quantitation of Antibody in Microdroplets by Serial Dilution

Determination of Mercaptoethanol Sensitivity of Antibody

Handling of Plaque-Forming Cells (PFC)

Liquid Monolayer Technique and Inversion of Micromanipulation System

Carboxymethyl Cellulose Plaque Technique

Serial Transfers of PFC

Micromanipulation and Autoradiography of Cells Labeled in Vitro

Chromosomal Analysis of Antibody-Forming Cells Arrested in Metaphase

Deoxycholate Method of Rupturing Cells

Microadaptations of the Ford Technique

Microradioiodination of PFC-Secretions

Electrophoresis on Cellulose Acetate


Microdensitometric Analysis

D. Plaque Techniques for Recognizing Individual Antibodyforming Cells

1. Standard Techniques

2. Indirect Techniques

3. The Thin Layer Technique

4. Modified Techniques

5. Hemolytic Plaques to Detect Synthesis of Antibodies to Diverse Antigens

6. Plating Error and Efficiency of Plating

E. Rosette-Plaque Methods for Detection Of Allotype and Hapten Receptors In Plaque-Forming Cells


Lymphocyte Receptor for Sheep Erythrocytes vs. Direct Plaque Technique

Expression of Allotype Heterozygosity

Detection of Individual Hapten Binding and Anti-Hapten Secreting Cells

Preparation of Lymph Node Cells, Types of Target Cells, Rosette Plaque Assay

Rosette Formation vs. Plaquing: Lymphocytes from NIP-Immunized Rabbits Passively Sensitized by Anti-Rabbit Allotype Antibody

Chapter 27. Immunohistological Methods

A. Preparation of Tissue for Study With Labeled Antibody and Light-Microscopy

1. Introduction

2. Theory and Application

3. Preparative Methods

B. Fluorescent Antibody as Specific Cytochemical Reagents

1. Preparation of Fluorescein-Coupled Globulins

2. Tissue Preparation for Immunofluorescence Staining

3. "Staining" with Labeled Antibody

4. Controls on Specificity of Fluorochrome Staining

5. Microscopic Equipment for Observing Fluorescence

6. Photography of Preparations Stained with Fluorescent Antibody

7. Tetramethylrhodamine Isothiocyanate (TRITC) Conjugates

8. Visualization of Antigens by Fluorochrome Reagents of Contrasting Color

9. Staining with FITC-Coupled and Ferritin-Coupled Univalent Antibody Fragments ("Hybrid Antibodies")

C. Fluorescent Proteins for the Determination of Cell Death


Preparation of FITC-Labeled Bovine Albumin

Detection of Dead Cells: in Organ Cultures

In Tissues in Vivo

Fixation and Sectioning of Tissues

Differentiation of Phagocytosed FIBSA and Uptake by Dead Cells

D. Staining of Histones in Nuclei of Lymphoid Cells

Applicability: Lysine-Rich Histones in Smears, Frozen Cryostat Sections of Mammals, Aves, Plants, Squash Preparations, Electropherograms of Isolated Histones on Cellulose Acetate


Formalin Fixation, Ammoniacal Silver, Formalin Developer

Cytophotometry for Relative Density of Stain

Selective Staining of: Normal Lymphoid Cells vs. Antigen-Stimulated Animals,

Nuclear Histones of Cancer Cells

Comments: Noninterference by Prior or Subsequent Feulgen Staining

Characterization of Cell Types by Altered Prestaining with Formalin

Staining of Arginine-Rich Histones by Fast Green after DNA-Extraction

Chapter 28. Application of Electron Microscopy to Problems in Immunology

A. Introduction

Study of Cells during Antibody Synthesis, Distribution of Antigens, Cell-Specific Antigenic Markers. Cytopathology, Antibody as Cytologic Stain, Requirement for Electron-Dense Antibody Molecules

Complexing of Antibody with Ferritin, with Enzymes, with Viruses

B. Immune Labeling for Electron Microscopy

1. Ferritin-Conjugated Antibody

2. Hybrid Antibody Markers for Electron Microscopy

3. Enzyme-Labeled Antibody Markers for Electron Microscopy

C. Electron Microscopy of Immunoglobulins and Immune Complexes

General Considerations for Visualizing Soluble Proteins by Negative Contrast,

Support Films: Carbon Covered Nitrocellulose Layers or Adhesive-Glue Grids,

Negative-Staining Heavy-Metal Salts

Properties of Protein Molecules Capable of Visualization

Staining Procedures

Sequential Drop Method Allowing Reactions on Grids before Staining

One-Step Direct Staining

Carbon Film Transfer Method from Mica

Immunological Applications, 502: Rabbit IgG Linked by Bivalent Hapten for Structures of IgG, Fc, Fab

Complement Fixation by Antigen-Antibody Tetramers

Human IgA Dimer

IgM Ultrastructure

Clq Ultrastructure

C3 Component of Complement

Author Index

Subject Index


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© Academic Press 1976
28th December 1976
Academic Press
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About the Editors

Curtis A. Williams

Merrill W. Chase

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