Methods in Immunology and Immunochemistry

Topical Listing of ContentsContribution to Volume 1PrefaceContents to Other VolumesChapter 1. AntigensA. Proteins Crystallization Chromatographic and Electrophoretic Separations, Use of Antibodies to Remove Highly Antigenic Impurities Stabilization of AntigenB. 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 BC. 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 ActivityD. 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 PhagesE. 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 PolyalanineF. 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 PurificationChapter 2. Production of AntiserumA. 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 CompositionsB. 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 TestingD. 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 AnimalsChapter 3. Purification of AntibodyA. 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-ImmunoglobulinsB. 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 CelluloseChapter 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 ApplicationsC. Fluorescent LabelsChapter 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 MethodologyAuthor IndexSubject Index