Basic Exercises in Immunochemistry: A Laboratory Manual

ISBN-13: 9783540094531 / Angielski / Miękka / 1979 / 314 str.

A. Nowotny

Spis treści

1 Isolations and Preparations.- Exercise No. 1 Fractionation of Serum Proteins by Ammonium Sulfate.- Exercise No. 2 Isolation of Immunoglobulins by Gel Filtration on Sephadex G 200 Column.- Exercise No. 3 Isolation of IgG by DEAE Cellulose.- A Column Chromatography.- B Batch-Type Operation.- Exercise No. 4 Dissociation of Macroglobulins With 2-Mercaptoethanol.- A.- B.- Exercise No. 5 Enzymatic Cleavage of IgG..- Exercise No. 6 Chromatographic Separation of the IgG Fragments.- Exercise No. 7 Separation and Isolation of H and L Chains of IgG.- Exercise No. 8 Affinity Chromatography and Its Application to Antibody Isolation.- Exercise No. 9 Cellulose Acetate Paper Electrophoresis of Human Serum.- Exercise No. 10 Disc Electrophoresis in Polyacrylamide Gel.- A Separation of Serum Proteins.- B SDS Gel Electrophoresis of Erythrocyte Membrane Proteins.- c Molecular Weight Determination by SDS Gel Electrophoresis.- Exercise No. 11 Separation of Human Hemoglobins by Electro-focusing.- Exercise No. 12 Conjugation of Antibodies With Fluorescein and Rhodamine.- A Fluorescein.- B Rodamine.- Exercise No. 13 Enzymatic Labeling of Proteins With 125I.- A.- B.- Exercise No. 14 Preparation of Peroxidase-Labeled Antibody.- Exercise No. 15 Preparation of Soluble Dinitrophenyl Proteins.- Exercise No. 16 Preparation of Dinitrophenyl- and Trinitrophenyl-Labeled Erythrocytes.- A.- B.- Exercise No. 17 Isolation of Cell Membranes From Ascites Tumors.- Exercise No. 18 Preparation and Use of Synthetic Liposomes.- Exercise No. 19 Isolation of Murine Transplantation Antigen Preparation.- Exercise No. 20 Isolation of Bacterial H-Antigens.- Exercise No. 21 Extraction of Bacterial 0 Antigens (Endotoxins or Lipopolysaccharides).- A Trichloroacetic Acid Method.- B Phenol-Water Procedure.- c Chloroform-Methanol Extraction of Endotoxic Glycolipid (EGL) From Rough Mutants.- Exercise No. 22 Preparation of 14C-Labeled Endotoxin and Endotoxic Glycolipid (EGL).- Exercise No. 23 Isolation of Bacterial Capsular Polysaccharides, K Antigens.- Exercise No. 24 Extraction of Teichoic Acid.- Exercise No. 25 Extraction of Pneumococcus Type XIV Polysaccharide.- Exercise No. 26 Isolation of Phytohemagglutinin.- Exercise No. 27 A and B Blood Group Antigen Preparations From Erythrocytes.- A Solubilization With Dilute NaOH.- B The Use of Detergent.- Exercise No. 28 Isolation of NN Blood Group Antigen From Human Erythrocytes.- Exercise No. 29 Isolation of Forssman Hapten From Erythrocytes.- Exercise No. 30 Isolation of Ragweed Pollen Allergens.- Exercise No. 31 Ultrasonic Disruption of Bacteria and Density-Gradient Centrifugation of the Products.- Exercise No. 32 Vacuum Distillation and Freeze Drying.- 2 Structural Studies.- 2.1 2.1.1 Qualitative Methods, Hydrolytic Procedures.- Exercise No. 33 Determination of Optimal Hydrolytic Conditions.- A Carbohydrate Liberation.- B Liberation of Amino Acids and Amino Sugars.- Exercise No. 34 Hydrolysis of Lipopolysaccharide With Acid.- Exercise No. 35 Partial Hydrolysis of Lipopolysaccharide With Ion Exchanger.- A Discontinuous Method.- B Continuous System.- 2.1.2 Separation and Isolation of the Split Products.- Exercise No. 36 The Methods of Analytic and Preparative Paper Chromatography.- A Preliminary Runs.- B Preparative Procedure.- Exercise No. 37 Separation and Isolation of Peptides or Oligosaccharides by High-Voltage Paper Electrophoresis.- Exercise No. 38 Demonstration of Ion Exchange Column Chromatography of Amino Acids.- Exercise No. 39 Gas-Liquid Chromatography for Fatty Acid Separation,.- Exercise No. 40 Carbohydrate Analysis by Gas-Liquid Chromatography.- Exercise No. 41 Gas-Liquid Chromatographic Analysis of Amino Acids.- Exercise No. 42 Demonstration of Thin-Layer Chromatography.- 2.1.3 Some Other Qualitative Methods for Structural Studies.- Exercise No. 43 Qualitative Analyses of the Constituents of an Unknown Natural Product.- Exercise No. 44 Identification of N-Terminal and C-Terminal Amino Acids.- A N-Terminus.- B C-Terminus 1.- C Paper Chromatography of the Amino Acids and Their Derivatives.- Exercise No. 45 Staining of Gels for Carbohydrates.- Exercise No. 46 Methylation of Carbohydrates.- 2.2 Quantitative Analytical Determinations.- Exercise No. 47 Dry Weight and Inorganic Ash Determinations.- A Total Dry Weight.- B Inorganic Ash.- Exercise No. 48 Microdetermination of Nitrogen.- Exercise No. 49 Microdetermination of Phosphorus.- Exercise No. 50 Protein Determination by the Biuret Method.- Exercise No. 51 Determination of Primary Amino Compounds With Ninhydrin.- Exercise No. 52 Carbohydrate Determination by Phenol-Sulfuric Acid.- Exercise No. 53 Ultramicrodetermination of Reducing Carbohydrates.- Exercise No. 54 Enzymatic Determination of Carbohydrates.- A Glucose.- B Galactose.- Exercise No. 55 Determination of Amino Sugars.- Exercise No. 56 Determination of Heptoses and Pentoses.- A Heptose 1.- B Pentose.- Exercise No. 57 Determination of Sialic Acid and 2-Keto-3-Deoxyoctonic Acid Derivatives.- A Sialic Acid.- B 2-Keto-3-Deoxyoctonic Acid.- Exercise No. 58 Chemical and Biologic Analysis of Compounds Separated by Preparative Thin-Layer Chromatography.- A Chemical Analyses.- B Measurements of Biologic Activities.- Exercise No. 59 Oxydation of Carbohydrates With Periodate. Quantitative Spectrophotometric Determination.- Exercise No. 60 Measurements of the Products of Periodate Oxydation.- A Formaldehyde Determination.- B Formic Acid Determination.- Exercise No. 61 Reduction of Carbohydrates With NaBH4.- A Determination of the Degree of Polymerization.- B Identification of the Reducing Terminal Carbohydrate.- c Investigation of the Glycosidic Linkages by Periodate and Borohydrate Treatment (Smith Degradation).- Exercise No. 62 Quantitative Determination of Free Hydroxyl Groups.- Exercise No. 63 Lipid Determination With the Hydroxylamine Method.- Exercise No. 64 Qualitative and Quantitative Analysis of Carboxylic Acids.- 3 Immunologic and Other Biologic Assays.- 3.1 Antibody Production.- Exercise No. 65 Immunization and Adjuvant Effect.- A Soluble Immunogen.- B Particulate Immunogen.- Exercise No. 66 Demonstration of Antibody Production at Cellular Level (Immunoplaque Method).- 3.2 3.2.1 Antigen-Antibody Interactions, Methods of Agglutination.- Exercise No. 67 Bacterial Agglutination.- Exercise No. 68 Hemagglutination and Its Inhibition.- Exercise No. 69 Passive Hemagglutination and Its Inhibition.- A Neter’s Method.- B Boyden’s Procedure.- C Carbodiimide Coupling Method.- D Inhibition of Passive Hemagglutination.- Exercise No. 70 Charcoal Agglutination.- 3.2.2 Precipitation Methods.- Exercise No. 71 Double Gel Diffusion/Ouchterlony Method.- Exercise No. 72 Immunoelectrophoresis.- Exercise No. 73 Quantitative Immunoelectrophoresis/Rocket Method.- Exercise No. 74 Crossed Immunoelectrophoresis.- Exercise No. 75 Counter Immunoelectrophoresis.- Exercise No. 76 Semiquantitative Micro-Precipitin Assays.- A Microtitration in Gel.- B Determination of Optimal Antigen/Antibody Ratio by Gel Diffusion.- Exercise No. 77 Quantitative Precipitation.- Exercise No. 78 Quantitative Radial Immunodiffusion.- Exercise No. 79 Radioimmunoassays for Cyclic AMP and Cyclic GMP Determinations.- A Cyclic AMP.- B Cyclic GMP.- Exercise No. 80 Solid-Phase Radioimmunoassay.- 3.2.3 Other Reactions of Antibodies and Immunocytes.- Exercise No. 81 Complement Fixation.- A Preparation of Reagents.- B Hemolysin Titration.- c Titration of the Complement.- D Qualitative Complement Fixation (Determination of the Proper Antigen Dose for Quantitative Complement Fixation Test).- E Anticomplementary Controls.- F Quantitative Complement Fixation.- Exercise No. 82 Total and Viable Cell Counts.- Exercise No. 83 Direct and Indirect Staining of Bacteria With Fluorescein-Labeled Antibodies.- Exercise No. 84 Unlabeled Antibody-Enzyme Method to Localize Antigens.- Exercise No. 85 Isolation of Human Lymphocytes by Sedimentation.- Exercise No. 86 Isolation of Adhering Leukocytes.- A.- B.- Exercise No. 87 Rosette-Forming Cell Assay.- Exercise No. 88 Passive Cutaneous Anaphylaxis.- Exercise No. 89 Lymphoblast Assay.- Exercise No. 90 T Cell-Mediated Cytotoxicity of Influenza Virus-Infected Cells.- 3.2.4 Miscellaneous Biologic Reactions.- Exercise No. 91 The Local Shwartzman Phenomenon.- Exercise No. 92 Measurement of the Activity of the Reticuloendothelial System.- Exercise No. 93 Enhancement of Nonspecific Resistance to Infection.- Exercise No. 94 Determination of Toxicity.- Exercise No. 95 Measurement of Pyrogenicity.