Reactions of Antibodies with Soluble Antigens
Methods in Immunology and Immunochemistry, Vol. 3
- 1st Edition - September 20, 2014
- Editors: Curtis A. Williams, Merrill W. Chase
- Language: English
- Paperback ISBN:9 7 8 - 1 - 4 8 3 2 - 0 7 1 5 - 5
- eBook ISBN:9 7 8 - 1 - 4 8 3 2 - 2 0 5 8 - 1
Methods in Immunology and Immunochemistry, Volume III: Reactions of Antibodies with Soluble Antigens provides information pertinent to antigen–antibody and hapten–antibody… Read more
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.
Contributors to Volume III
Preface
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
Applications
Preparations of Antigenic Fragments and Characterization
Quantitative Inhibition Assay
C. Ammonium Sulfate Precipitation of Soluble Antigenantibody Complexes
1. Determination of Antibody-Binding Capacity
Principle
Procedure
Analysis of Data
2. Estimates of Antibody Based on Antigen-Binding Capacity
Modified Antigen-Binding Capacity Technique
Verification of Estimates
Specificity
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
Diffusion-Controlled
Interactions
Activation Energy for Dissociation of Antibody-Hapten Complexes
B. Equilibrium Dialysis
Principle
Cells and Membranes for Equilibrium Dialysis
Manipulations and Auxiliary Equipment
Experimental Conditions: Equilibration Period, Membrane Adsorption, Antibody Concentration, Haptens with Low Association Constants
Calculations
Heterogeneity of Antibody Molecules
Average Association Constants
Sips Distribution Function
Limitations
Microtechnique
C. Quenching of Antibody Fluorescence by Haptens and Antigens: A Method for Determining Antibody-Ligand Affinity
Principles
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
Apparatus
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
Introduction
Applications: Relative Binding Capacity
Confirmation of Antibodies Formed to Natural Substances
Test for Affinity
Labeling of Antibody Sites
Reagents
Quantitative Hapten Inhibition
Relative Association Constants
Heterogeneity Constant
Heterogeneity Index
F. Thermodynamic and Kinetic Investigation of the Antigenantibody Reaction by Fluorescence Labeling Techniques
Survey
Absorption, Fluorescence and Phosphorescence
Polarization of Fluorescence
Molar Fluorescence
Thermodynamic Parameters
Kinetic Parameters
Basic Instrumentation
Standards
Performance Tests
Available
Polarometers
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
Introduction
Reagents
Radioiodinated Insulin
Specimens for Analysis
Buffers
Immunodiffusion and Immunoelectrophoresis
Reaction with Anti-Globulin Sera and Radiolabeled Antigen
Staining
Autoradiography
Quantitative Aspects
Applications
Author Index
Subject Index
- Edition: 1
- Published: September 20, 2014
- Language: English
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