Methods in Immunology and Immunochemistry - 1st Edition - ISBN: 9780127544014, 9780323148016

Methods in Immunology and Immunochemistry

1st Edition

Editors: Curtis Williams
eBook ISBN: 9780323148016
Imprint: Academic Press
Published Date: 1st January 1968
Page Count: 501
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Description

Methods in Immunology and Immunochemistry, Volume I: Preparation of Antigens and Antibodies is aimed to bring together detailed procedures in the preparation of antigens and antibodies. The text also provides a presentation and discussion of these methods. The book covers topics in immunology such as antigens, its kinds, and the preparation and testing of lipids for immunological study. The production of antiserum; the preparation of immunogens; collecting and handling of serum; and immunization procedures are also explained. The book also discusses the purification of antibodies; methods of labeling antigens and antibodies; and the methods used in the studies of the structure of immunoglobulins. The text is recommended for immunologists who would like to know the different procedures and methods involved in immunology as well as the principles behind it. The book will also serve as a guide for medical staff who prepare products related to immunology.

Table of Contents


Topical Listing of Contents

Contribution to Volume 1

Preface

Contents to Other Volumes

Chapter 1. Antigens

A. Proteins

Crystallization

Chromatographic and Electrophoretic Separations,

Use of Antibodies to Remove Highly Antigenic Impurities

Stabilization of Antigen

B. Bacterial Antigens

1. Disruption of Bacteria and Preparation of Cell Walls

Disruption

Lysis

Grinding

Sonic and Ultrasonic Vibrations

Freeze-Thawing

Shaking

Decompression

High Pressure

Extrusion

Preparation of Whole Cell Walls

Salmonella enteritidis

Mycobacterium tuberculosis

Recovery and Refinement of Cell Walls

2. Cell Wall Antigens from Gram-Negative Bacteria

Trichloroacetic Acid Extraction of the Whole Antigenic Complex

Phenol-Water Extraction of the Lipopolysaccharide

Somatic Degraded Polysaccharide Obtained by Acetic Acid Hydrolysis of the Bacteria

Polysaccharide Obtained by Alkaline Hydrolysis

3. Cell Wall Antigens from Gram-Positive Bacteria

Hemolytic Streptococci

Pneumococci

Other Bacteria

4. Teichoic Acids

Composition

Antigenicity

Preparation of Cell Walls

Extraction

Strain and Species Differences

5. Flagellar Antigens

Isolation of Flagella

Flagellin

Reconstitution of Flagellar Filaments Homogeneity

6. Capsular Polysaccharides

Demonstration

Preparation and Purification

Alkali-Stable Polysaccharides

Alkali-Labile Capsular Polysaccharides

Purification Procedures

Criteria of Purity

7. Extracellular Bacterial Products and Toxins

General Methodology

Culture Conditions

Separation and Sterilization of Culture Supernatant

Concentration

Purification

Tests for Purity

Storing and Stabilizing

Examples

Streptococcal Deoxyribonuclease B

C. Blood Group Antigens

1. Soluble Specific Substances

Extraction

Purification

Chemical Properties

Serological Properties

Testing

2. ABO Antigens from Red Cells

Preparation of Stromata

Ethanol Extraction

Precipitation

Organic Solvent Extraction

Chromatography

Thin-Layer Chromatography

Determination of Serological Activity

D. Viruses

1. Animal Viruses for Use as Antigens

Viral Immunizing Antigens

Viral Serological Antigens

Serological Tests

Concentration Procedures

Purification Procedures: Lipid Solvent Extraction

Protamine Sulfate Precipitation]

Bentonite Adsorption

Fluorocarbon Treatment

Concentration-Purification Procedures

Adsorption and Elution from Electrolytes

Precipitation Methods

Adsorption

Liquid Partition Systems

Density Gradient Centrifugation

2. Plant Viruses for Use an Antigens

Methods: Differential Centrifugation

Zonal Density Gradient Centrifugation

Zonal Density Gradient Electrophoresis

Celite Filtration

Granulated Agar Gel Column

Tests of Purity and Intactness: Analytical Centrifugation

Electron Microscopy

Immunological Methods

3. Bacterial Viruses

Preparation of T2 Phage

Preparation of f2 Phage

Preparation of the Components of T-Even Phages

E. Conjugated and Synthetic Antigens

1. Coupling of Diazonium Compounds to Proteins

General Procedure

Preparation of Diazonium Salt

Coupling Procedure

Notes

2. Hapten Conjugation of Proteins Thiolated with S-Acetylmercaptosuccinic Anhydride

Preparation of Thiolated Proteins

Isolation

Coupling Reaction

3. Immunogenic Dinitrophenyl and Trinitrophenyl Proteins

Principle

Crystallization of 2,4-Dinitrobenzenesulfonic Acid

Dinitrophenylation Reaction

Characterization of DNP-Proteins

Trinitrophenyl-Proteins: Preparation and Characterization

4. Conjugation of Penicillin and Its Derivatives

Penicilloyl Conjugates

Polylysine Conjugates from Penicillenic Acid

from Penicillin

Protein Conjugates

Polypenicoyl Conjugates

Penicillenate Conjugates

Preparation of Thiolated Protein

Protein Conjugation

Penicillamine-Protein Conjugates

General: Purification, Recovery, Stability

Analysis

Red Blood Cell Conjugates

5. Steroid-Protein Conjugates to Elicit Antihormonal Antibodies

Steroid Haptens Prepared with (O-Carboxymethyl) hydroxylamine: Testosterone

Pregnenolone

Conversion of the Oxime to Methyl Ester

Conversion to Progesterone Oxime

Preparation of Progesterone Oxime Methyl Ester

Estrone

Deoxycorticosterone Hemisuccinate

Preparation of Estrone 17-Albumin Conjugate

6. Protein-Protein Conjugation

Diisocyanates

Ferritin-Globulin

Insulin-Albumin

Protein-Erythrocytes

Carbodiimides

Bradykinin-Albumin

Angiotensin-Albumin

Cytochrome c-Globulin

Dihalogenated Dinitrobenzenes

Globulin-Erythrocytes

Bifunctional Organic Mercurials: Complexing of Thiolated Antigens

Phosphine Oxide Reagent

Thiosuccinylation of Proteins

Thiolated Proteins Crosslinked by Phosphine Oxide Reaction

Ethylene Maleic Anhydride Copolymer

Protein-Protein Conjugation

Diazonium Compounds

Ferritin-Globulin

Protein-Erythrocytes

N-Ethyl Benzisoxazolium Fluoroborate

Angiotensin-Polylysine

7. Synthetic Polypeptides as Antigens

Linear Polypeptide Antigens

Preparation of Glutamic Acid-LysineTyrosine Copolymer

Analysis

Dissolution of the Copolymer and Storage of the Antigen

Multichain Polypeptide Antigens

Preparation of Polylysine Polymerized with Polyalanine and Copolymerized with Anhydrides of Carboxyglutamate and Carboxytyrosine

Analysis

Polypeptidyl Proteins

Preparation of Polytyrosyl Gelatin

Preparation of Polylysyl Albumin

Analysis

Immune Response

8. Immunogens with Nucleoside and Nucleic Acid Specificity

Nucleic Acids and Oligonucleotides Complexed with Methylated Bovine Serum Albumin

Methylation Procedure

Complexing Immunization

Methods of Analysis

Specificity of Antibodies

Applications: to Melted DNA

to Synthetic Polydeoxyribonucleotide

to Oligodeoxyribonucleotides

to Transfer-RNA

to Synthetic Polyribonucleotides

Purine- and Pyrimidine-Protein Conjugates

Purine-6-oyl-Proteins

Acetyluracil-Proteins

Proteins Coupled with Ribosides and Ribotides

Nucleoside-5'-oyl Conjugates

Nucleoside Conjugates of Synthetic Polypeptides

Preparation of Uridine-5'-Carboxylic Acid

Uridine Conjugation with Multichain Polyalanine

F. Lipids in Immunological Studies

Lipid Immunogens of Tissues

Particulate Fraction

Soluble Lipids

Immunization

Lipid Serological Antigens

Total Lipid Extracts,

Cytolipin H

Glycosphingolipids

Cardiolipin

Forssman Substance

Partially Purified Lipid Fractions

Complement Fixation Tests with Lipids

Absorption Studies with Antiserum

Lipid Purification

Chapter 2. Production of Antiserum

A. Immunogen Preparation

Soluble Antigens

Colloidal Carriers as Adsorbents: Type Cγ Alumina

Simple Alumina

Aluminum Phosphate

Blood Charcoal

Nascent Alumina

Calcium Alginate

Polyaerylamide Gel

Special Carriers: Bentonite

Methylated Bovine Serum Albumin

Freund's Adjuvant

Water-in-Oil Emulsions

Alternative Adjuvant

Compositions

B. Immunization Procedures

1. Nonhuman Mammals: Principles of Immunization

Rabbits

Horses, Goats, Sheep

Guinea Pigs

Mice

Other Animals

2. Human Subjects

Antitoxin Production

Antipolysaccharide Antibodies

Miscellaneous Human Antibodies

3. Chickens

Protein Antigens

Haptens

Characteristics of Antibody Response

Purification of Chicken Immunoglobulins

Physicochemical Properties (γG-, γM-Immunoglobulins)

C. Collection and Handling of Serum

Examination of Trial Bleedings

Collection and Separation of Serum

Preservation and Storage

Sterile Filtration

Sterility Testing

D. Animal Handling

Rabbits

Handling

Injections

Gastric Intubation

Bleedings

Restraint

Terminal Bleeding

Guinea Pigs

Handling

Restraint

Injections

Bleedings

Anesthetization

Mice: Maintenance

Injections

Intranasal Inoculation

Bleedings

Hamsters: Orbital Venous Plexus Bleeding

Rats

Handling

Intravenous Injection

Bleedings

Horse, Sheep, Goats

Primates

Avian Species: Restraint

Injection

Bleedings

Terminal Bleeding

Anesthesia

Euthanasia and Disposal

Common Physiological Data on Laboratory Vertebrates

Legislation Regarding Animals

Chapter 3. Purification of Antibody

A. Preparation of Immunoglobulin

1. Nomenclature

Classes

Proteolytic Fragments

Intraclass Heterogeneity

Antigenic Differences Among Heavy Chains

Gm and Inv Factors in Human Immunoglobulins

Allotypes in Nonhuman Species



2. Immunoglobulin Concentrates

Ethanol Fractionation

Neutral Salt Fractionation

Use of Complexing Precipitants

3. Chromatographic Separation

DEAE-Cellulose Chromatography: Preparation of γG-Immunoglobulin

Fractionation of Serum

γG

γA

γM

γD

Quantitation of γ-G Immunoglobulin

Species Differences

Technical Considerations: Variability among DEAE-Celluloses

Overloading with Protein

Euglobulin Recovery

Special Cases: Myeloma Proteins and Waldenström Macroglobulins,

Bence-Jones Proteins

Purification of Normal γ-A and γ-M Immunoglobulins

Carboxymethyl (CM)-Cellulose Chromatography

4. Electrophoretic Separation on Solid Supporting Media

5. Separation by Electrophoresis-Convection

6. Ultracentrifugal Separation of γG-and γM-Immunoglobulins

B. Preparation of Specific Antibody

1. Antipolysaccharide Antibody Purified from Specific Complexes

Extraction with 15% Salt

Digestion of Specific Complexes with Enzymes

Elution of Antidextran from Sephadex (Dextran) Complexes

2. Antibody Purified from Erythrocyte-Antibody Complexes

Recovery of Antibody and Dissociation Efficiency

Effect of Complement

Antibody Recovery from Erythrocytes by Heat

by Ether

Antibody Recovery from Red Cell Stroma

Cold Agglutinins

Rh Antibodies

Kell

Anti-A Dissociated from A Substance-Polyleucine Complexes

Leukocyte Agglutinins

Mouse Antibodies

Dog Antibodies

3. Antiprotein Antibodies Isolated by the Thiolated Antigen Method

4. Antibody Dissociated from Complexes with Carbon Dioxide in SaltFree Medium

5. Anti-Hapten Antibodies: Anti-2,4-Dinitrophenyl (DNP) and Anti-2,4,6Trinitrophenyl (TNP, Picryl-)

Purification Principles

Methods

Anti-DNP Isolated with TNP-Albumin Antigen

Anti-DNP Antibodies of Low or High Affinity Isolated by DNP-Glycine

Purification of Specific AntiTNP Antibodies

6. Anti-Carbohydrate Hapten Antibody

Specific Precipitation

Antibody Solubilization

Removal of Antigens

Removal of Lactose

Yields of Antibody

Purity and Homogeneity

7. Immunoadsorbents: Cellulose Derivatives

Technical Considerations

Principles

Adsorption of Antibody

Elution of Antibody

Analytical Procedures

Aminoaryl-Celluloses: Preparation of p-Aminobenzylcellulose

Coupling to Proteins

Preparation of Benzidine-Carboxymethylcellulose and Conjugation to Proteins

Preparation of m-Aminobenzyloxymethylcellulose,

Coupling to Proteins

Aminoethylcellulose Coupled to 6-Methylpurine

Carboxymethylcellulose Coupled to Proteins and to Hapten Derivatives by Carbodiimide

Phenolic Derivatives of Cellulose

Chapter 4. Labeling of Antigens and Antibodies

A. Radioisotopic Labeling

1. Internal (Biosynthetic) Labeling of Proteins (C14, S35,H3)

2. External Labeling of Proteins (I131, I125, I130)

3. External Labeling of Proteins (S36-Sulfanilic Acid by Diazotization-Coupling)

4. Paired-Label Technique for Identifying Peptides from the Combining Sites of Antibodies

5. Labeling of Erythrocytes with Chromium-51

B. Ferritin-Labeled Antigens and Antibodies

Purification of Ferritin

Preparation of Antibody and Antigen

Preservation of Coupling Agent

Conjugation Procedures

Purification of Ferritin-Antibody

Evaluation of Conjugates

Applications

C. Fluorescent Labels

Chapter 5. Studies of Immunoglobulin Structure

A. Immunoglobulin: Conventions, Terminology, Models

B. Immunoglobulin Polypeptide Chains

Disulfide Bond Cleavage

Dissociation and Fractionation of Polypeptide Chains

Starch Gel Electrophoresis

Ion Exchange Chromatography

Gel Filtration

Reconstitution of Immunoglobulins: from Reduced Alkylated Chains

from Reduced Chains with Formation of Interchain Disulfide Bonds

from Half-Molecules

C. Enzymatic Hydrolysis: Isolation of Fragments

Papain Hydrolysis,

Use of Insoluble Papain

Pepsin Hydrolysis

5 S Fragments

Their Reduction and R e oxidation

Reduction of Fab Fragments, Recovery of Fd Fragment

Treatment with Other Enzymes

D. Application of Structural Methodology

Author Index

Subject Index

Details

No. of pages:
501
Language:
English
Copyright:
© Academic Press 1967
Published:
Imprint:
Academic Press
eBook ISBN:
9780323148016

About the Editor

Curtis Williams

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