Methods of Protein and Nucleic Acid Research: 2 Immunoelectrophoresis Application of Radioisotopes

ISBN-13: 9783642874901 / Angielski / Miękka / 2012 / 206 str.

L. a. Osterman

Opis

A consideration of the different types of immunoelectrophoresis is the natural con- clusion to the methods of electrophoretic fractionation described in the preceding book for proteins and nucleic acids. Immunoprecipitation is the essential com- ponent process of immunoelectrophoresis, and is in itself a highly specific method of purifying proteins. It can also be used for their detection after conventional elec- trophoresis or isoelectrofocusing. It is therefore worthwhile considering the mecha- nism and specific features of immunoprecipitation before considering im- munoelectrophoresis itself. Some of these features, in particular, the ambiguity of the immune response and the polymorphism ofimmune reactions, cannot be under- stood without abrief but not superficial knowledge of the mechanism of generating immunity, the structure and function of the immunoglobulins, and the nature of antigen-antibody interactions. A knowledge of these topics is necessary for an understanding of the radioimmune methods dealt with in the second Part of the book.In the majority of cases biochemists and molecular biologists are inadequately acquainted with contemporary immunochemistry, which is currently undergoing rapid development. All this justifies the inclusion of a detailed introduction to immunochemistry as the first chapter. Chapter 2 is devoted to adescription of the use of antisera in the immunological assay of components separated by conventional electrophoresis and IEF. The remaining three Chapters analyze the different forms of immuno- electrophoresis, usually carried out in agarose gels.

Spis treści

I Immunoelectrophoresis.- 1 Fundamentals of Immunochemistry.- 1.1 The Immune System of Higher Organisms.- 1.1.1 Blood Components.- 1.1.2 Phagocytosis, Complement, Cytointoxication.- 1.1.3 Leucocytes.- 1.1.4 Antibodies.- 1.1.5 The Lymphatic System and the Spleen.- 1.1.6 Decay of the Immune Response.- 1.1.7 Kinetics of Immunization.- 1.1.8 Generation of the Immunological Arsenal.- 1.2 Structure of the Immunoglobulins.- 1.2.1 General Description of Structure. Classes of Immunoglobulins.- 1.2.2 Antigen-binding Sites.- 1.2.3 Ambiguity of the Immune Response.- 1.2.4 Synthesis and Assembly of Immunoglobulin Molecules.- 1.3 Myeloma and Hybridoma Cells.- 1.4 Preparation of Immune Serum and Purification of Antibodies.- 1.4.1 Immunization of the Rabbit.- 1.4.2 Preparation of Antiserum.- 1.4.3 Purification of Immunoglobulin G (IgG).- 1.4.4 The Use of Immunosorbents for Purification of Antibodies.- 1.5 Production of Monoclonal Antibodies Using Hybridoma.- 1.6 Commercially Available Antibodies.- 1.7 Detection and Estimation of Antibody Concentration.- 1.7.1 Ring Test.- 1.7.2 Determination of the Ratio of Equivalence.- 1.7.3 Hemagglutination and Hemolysis.- 1.7.4 Complement Binding Reaction.- 1.7.5 Ouchterlony’s Method.- 2 The Use of Immune Methods to Detect Protein Zones Following Conventional Electrophoresis and Electrofocusing.- 2.1 Immunochemical Detection of Antigens in Gels.- 2.2 Preparation of Immune Replicas.- 2.2.1 Agarose Replica.- 2.2.2 Diazopaper Replica.- 2.2.3 Nitrocellulose Replica.- 3 Immunoelectrophoresis According to Grabar and Williams.- 3.1 Running the Experiment.- 4 Laurell Rocket Immunoelectrophoresis.- 4.1 Principle of the Method.- 4.2 Running the Experiment. Description of the Underlying Processes.- 5 Crossed Immunoelectrophoresis.- 5.1 Method of Clarke and Freeman.- 5.2 Tandem Crossed Immunoelectrophoresis.- 5.3 Methods of Fractionation in the First Dimension.- 5.4 Technique of Overlaying Agarose Gel with PAAG.- 5.5 The Use of an Intermediate Gel.- 5.6 Crossed Immunoaffinity Electrophoresis.- 5.7 Fused Rocket Immunoelectrophoresis.- II The Use of Radioisotopes.- 1 Isotopes, Scintillators, and Scintillation Counters.- 1.1 Radioactive Isotopes.- 1.1.1 Excess of Neutrons.- 1.1.2 Shortage of Neutrons.- 1.1.3 Rate of Radioactive Decay.- 1.1.4 Total Radioactivity of the Sample.- 1.1.5 Specific Activity (SA).- 1.1.6 Carrier-free Radioisotopes.- 1.2 Scintillators.- 1.2.1 Liquid Scintillators.- 1.2.2 The Spectrum of Light Pulse Intensities.- 1.2.3 Counting Efficiency.- 1.2.4 Quenching.- 1.2.5 ?-Radiation Detection. Solid Scintillators.- 1.3 Liquid Scintillation Counters. Principle of Operation.- 1.3.1 Photomultiplier.- 1.3.2 Amplifiers (Linear and Logarithmic).- 1.3.3 Logarithmic Voltage Amplifier.- 1.3.4 Pulse-Height Discriminators. “Thresholds”.- 1.3.5 Background Noise.- 1.3.6 Coincidence Circuit.- 1.3.7 Scalers.- 1.3.8 Accuracy of Counting.- 1.3.9 Chemiluminescence.- 1.3.10 Photoluminescence.- 1.3.11 Counting Vials.- 1.3.12 Cerenkov Counting.- 1.4 Adjustment (Tuning) of Liquid Scintillation Counters.- 1.4.1 Adjustment of One Channel for Single Isotope Counting.- 1.4.2 Adjustment of Two Channels for Dual Isotope Counting.- 1.4.3 Quench Correction for Single Isotope Counting.- 1.4.4 Quench Correction for Dual Isotope Counting.- 1.4.5 Adjustment of Two Channels for Dual Isotope Counting (3H and 14C) in a Counter with a Linear Amplifier.- 1.4.6 Dual Label Counting of 3H and 125I in a Liquid Scintillation Counter.- 2 Preparation and Counting of Labelled Samples.- 2.1 Samples as Aqueous Solutions.- 2.2 Dioxane-based Scintillators.- 2.3 Toluene- and Xylene-based Scintillators.- 2.4 Solubilization of Biological Specimens.- 2.5 Counting on Filters.- 2.6 Counting Efficiency. The Recovery of Radioactivity.- 2.7 Counting of Powders in Suspension.- 2.8 Counting After TLC and Gel Electrophoresis.- 2.9 Digestion of PAAG with 30% H2O2.- 2.10 Solubilization of PAAG without H2O2.- 2.11 Elution of Proteins and Nucleic Acids from Gels and Counting of the Eluates.- 2.12 Autoradiography.- 2.12.1 Drying of PAAG Slabs.- 2.13 Indirect Autoradiography.- 2.14 Fluorography.- 2.14.1 Fluorography of Plates After TLC.- 2.14.2 Fluorography of PAAG Slabs.- 2.15 Increase in X-ray Film Sensitivity by Prior Exposure to Light.- 2.16 Quantitative Counting.- 2.17 Counting of Dual Label in PAAG.- 2.18 Scanning Plates with Endwindow Counters (Berthold Company).- 2.18.1 Scanning Instrument.- 2.18.2 Berthold LB 2832 Linear Analyzer.- 2.18.3 Photography of Radioactive Spots on Plates Using a Spark Chamber (Berthold LB 292).- 2.19 Detection of Radioactivity in a Flowing Liquid.- 3 Labelling of the Proteins.- 3.1 Iodination with Chloramine T.- 3.2 Iodination in the Presence of H2O2 and Lactoperoxidase.- 3.3 Addition of Special Iodinated Reagents.- 3.4 Iodination with Na125I in the Presence of ICl.- 3.5 Labelling with 14C and 3H Using Formaldehyde and Borohydride.- 3.6 Enzymatic Methods.- 4 Labelling of Nucleic Acids.- 4.1 Chemical Reactions.- 4.1.1 Methylation with Labelled Dimethylsulfate (DMS).- 4.1.2 Incorporation of 3H from Tritiated Water in the Presence of Sodium Metabisulfite (Na2S2O5).- 4.1.3 Slow Isotope Exchange of Hydrogen.- 4.1.4 Labelling the 3?-Terminal Ribonucleotide: The Method of Randerath.- 4.1.5 Complexing Denatured DNA with Labelled N-hydroxymethylene Aminoacid.- 4.1.6 Iodination of Nucleic Acids.- 4.2 Enzymatic Reactions.- 4.2.1 T4-Polynucleotide Kinase.- 4.2.2 Terminal Deoxynucleotide Transferase.- 4.2.3 T4 RNA-Ligase.- 4.2.4 DNA-Polymerase I from E. coli (Kornberg Enzyme).- 5 Labelling of Proteins and Nucleic Acids in Vivo.- 5.1 Higher Animals.- 5.2 Cell Cultures from Higher Organisms.- 5.3 Microorganisms.- 6 Determination of Concentrations by Isotope Dilution.- 7 Radioimmune Methods.- 7.1 Immunoprecipitation and Adsorption from Solution.- 7.1.1 The System of Two Antibodies.- 7.1.2 The Use of Staphylococcus aureus and of Sepharose-linked Protein A.- 7.1.3 Radioactively Labelled Antibodies.- 7.1.4 Radioactive Protein A.- 7.1.5 Immunoprecipitation System with Avidin-Biotin Complex.- 7.2 Competitive Radioimmunoassay (RIA).- 7.2.1 RIA with Precipitation of the Immune Complexes.- 7.2.2 RIA with Filtration.- 7.2.3 Solid-phase RIA.- 7.3 Enzymic Immune Methods.- Some Additional Remarks on the Use of Radioactive Isotopes.- References to Part I.- References to Part II.