Reactions of Antibodies with Soluble Antigens - 1st Edition - ISBN: 9780127544038, 9781483220581

Reactions of Antibodies with Soluble Antigens

1st Edition

Methods in Immunology and Immunochemistry, Vol. 3

Editors: Curtis A. Williams Merrill W. Chase
eBook ISBN: 9781483220581
Imprint: Academic Press
Published Date: 1st January 1971
Page Count: 536
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Methods in Immunology and Immunochemistry, Volume III: Reactions of Antibodies with Soluble Antigens provides information pertinent to antigen–antibody and hapten–antibody reactions in vitro, in free solution and in gels. This book presents the development of research in immunology and immunochemistry.

Organized into three chapters, this volume begins with an overview of protein–antiprotein reactions. This text then discusses the inhibitory activity of protein fragments, which suggested that antigenic combining sites of proteins were limited regions of the whole antigen molecule. Other chapters consider the measurement of inhibitory activity, which is still the principal assay to characterize antigenic sites of proteins. This book discusses as well the immunological techniques prior to the development of gel-diffusion methods. The final chapter deals with fluorescence labeling techniques that provide powerful approaches for exploring the thermodynamic and kinetic parameters of antigen–antibody interactions.

This book is a valuable resource for mathematicians and immunologists.

Table of Contents

Contributors to Volume III


Contents of Other Volumes

Chapter 13. Precipitation Reactions

A. The Quantitative Precipitation Reaction

Theoretical Considerations

Precipitin Reaction and Factors Influencing Values for Antibody Nitrogen

Flocculation Reaction

Quantitative Reactions with Chicken Antibody

Preliminary Qualitative and Semi-Quantitative Tests

Quantitative Analysis for Antibody, 40; Methods for Analyzing Precipitates

Analysis of Supernatants

Applications: Antibody-Antigen Ratios

Quantitation of Antigens in Mixtures

Detection of Heterogeneity

Co-Precipitating (Non-Precipitating) Antibody

Structural Relationships among Macromolecules

Macromolecules Altered by Chemical Treatments

B. Inhibition of Precipitation Reactions


Preparations of Antigenic Fragments and Characterization

Quantitative Inhibition Assay

C. Ammonium Sulfate Precipitation of Soluble Antigenantibody Complexes

1. Determination of Antibody-Binding Capacity



Analysis of Data

2. Estimates of Antibody Based on Antigen-Binding Capacity

Modified Antigen-Binding Capacity Technique

Verification of Estimates


D. Turbidimetric Analysis of Precipitation Reactions

Comparison of Native Antigens

Altered Antigens

Comparisons of Antisera

E. Estimation of Antigen Concentration in Complex Mixtures by Optical Density of Turbid Suspensions

Chapter 14. Precipitation Analysis by Diffusion in Gels

A. Introduction

1. Characteristics of Antigen-Antibody Reactions in Gels

Choice of Method

Sources of Error

2. Diffusion Theory for Reactions in Gels

Solutions of the Diffusion Equation

One-Dimensional Diffusion

Two-Dimensional Diffusion

B. Qualitative Analysis of Antigen-Antibody Reactions in Gels

1. Simple Diffusion in Tubes

2. Double Diffusion in Tubes

3. Double Diffusion in Plates

4. Simple and Double Diffusion in Cells

5. Double Diffusion in Cellulose Acetate Membrane (CAM)

C. Quantitative Determinations by Precipitation Analysis in Gels

1. Diffusion Coefficient of Antigens by Simple Diffusion in Tubes

2. Diffusion Coefficients by Double Diffusion in Gels

3. Diffusion Coefficients in Agar Plates

4. Determining the Effective Size of Antigens and Antibodies

5. Concentration of Antigen and Antibody in Relative Units on Diffusion Plates

6. Antigen Titration by Simple Radial Immunodiffusion in Plates

7. Determination of Relative Antigen and Antibody Concentration in Tubes

8. Determination of Antigen and Antibody Concentrations in Absolute Weight Units

D. Immunoelectrophoretic Analysis (IEA)

1. Introduction

2. IEA in Agar Gels

3. IEA on Cellulose Acetate Membranes (CAM)

4. IEA by Transfer from Hydrolyzed Starch Gel

5. Two-Dimensional (Laurell) Immunoelectrophoresis for Estimation of Antigens in Relative Units

E. Characterization of Precipitates In Gels

1. Color Reactions for Identifying Antigens in Gel Precipitates, with Index

2. Photography of Precipitates in Gels

3. Interferometry for Analysis of Antigen-Antibody Reactions in Gels

4. Autoradiography of Precipitates in Gels

F. Preparation of Materials for Diffusion in Gels

1. Selection and Preparation of Gels

2. The Preparation and Properties of Agarose

G. Special Techniques with Diffusion in Gels

1. Single Antigen-Antibody Systems Prepared from Mixed Reactions in Gelatin

2. Particulate Antigens for Reactions in Agar Gel

Chapter 15. Hapten Reactions and Kinetics of Interaction with Antibodies

A. Equilibrium and Kinetic Studies of Antibody-Hapten Reactions

Univalent Haptens for Study of Interactions with Antibody

Equations for Binding Data

Heterogeneity of Binding Constants

Equilibrium Dialysis and Rate Constants

Kinetics Concepts



Activation Energy for Dissociation of Antibody-Hapten Complexes

B. Equilibrium Dialysis


Cells and Membranes for Equilibrium Dialysis

Manipulations and Auxiliary Equipment

Experimental Conditions: Equilibration Period, Membrane Adsorption, Antibody Concentration, Haptens with Low Association Constants


Heterogeneity of Antibody Molecules

Average Association Constants

Sips Distribution Function



C. Quenching of Antibody Fluorescence by Haptens and Antigens: A Method for Determining Antibody-Ligand Affinity


Tryptophan Fluorescence

Ligands Effective in Quenching

Requirement for Purified Antibody

Affinity Constant Procedure

Temperature Control

Calculations and Plotting

Evaluations of Maximal Quenching (Qmax)

Precision, Accuracy, and Range

Applicability to Mono-DNP Antigens

Comparison with Equilibrium Dialysis

Computer Program for Analysis of Fluorescence Quenching of Antibodies by Ligands

D. Determination of the Kinetics of Antibody-Hapten Reactions with the Temperature-Jump Method

Theoretical Considerations


Reaction Cell

Optical System

Choice of Dye-Hapten

Salt-Precipitation of Antibody Globulin

Choice of pH and Electrolyte

Experimental Procedure

Antibody-Hapten Systems Studied

E. Hapten Inhibition Of Reactions Of Antibody With Conjugated Haptens


Applications: Relative Binding Capacity

Confirmation of Antibodies Formed to Natural Substances

Test for Affinity

Labeling of Antibody Sites


Quantitative Hapten Inhibition

Relative Association Constants

Heterogeneity Constant

Heterogeneity Index

F. Thermodynamic and Kinetic Investigation of the Antigenantibody Reaction by Fluorescence Labeling Techniques


Absorption, Fluorescence and Phosphorescence

Polarization of Fluorescence

Molar Fluorescence

Thermodynamic Parameters

Kinetic Parameters

Basic Instrumentation


Performance Tests



Labeled Components

Method of Titration

Representative Equilibrium Results

Sensitivity and Accuracy

Basic Assumptions

FORTRAN Computer Program WD

Appendix I Identification of Multiple Antibody Components by Radioimmunoelectrophoresis and Radioimmunodiffusion



Radioiodinated Insulin

Specimens for Analysis


Immunodiffusion and Immunoelectrophoresis

Reaction with Anti-Globulin Sera and Radiolabeled Antigen



Quantitative Aspects


Author Index

Subject Index


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© Academic Press 1971
Academic Press
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About the Editor

Curtis A. Williams

Merrill W. Chase

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