Immunology in Plant Sciences

Plant Hormone Immunoassays Based on Monoclonal and Polyclonal Antibodies.- 1 Introduction.- 2 Synthesis of Immunogens.- 2.1 Synthesis of ABA Conjugates.- 2.1.1 Procedure 1: Synthesis of ABA-(C1)-BSA Conjugates (Modified from Weiler 1980).- 2.1.2 Procedure 2: Synthesis of ABA-C4?-BSA Conjugates (According to Weiler 1980).- 2.2 Synthesis of Indole-3-Acetic Acid. Conjugates.- 2.2.1 Procedure 3: Synthesis of IAA-BSA Conjugates Via the Mixed Anhydride.- 2.3 Synthesis of Gibberellin Conjugates.- 3 Radio- and Enzyme-Labeling of Plant Hormones.- 3.1 Radiolabeled Immunotracers.- 3.2 Enzyme-Labeled Plant Hormones.- 3.2.1 Procedure 4: Synthesis of Alkaline Phosphatase-Labeled IAA (Modified from Weiler et al. 1981).- 3.2.2 Procedure 5: Coupling of ABA-C4? -Hydrazones to Alkaline Phosphatase.- 4 Immunological Procedures.- 4.1 Immunization of Rabbits.- 4.2 Immunization of Mice.- 5 Antiserum Processing.- 6 Production of Monoclonal Antibodies.- 6.1 Generation of Hybridomas.- 6.2 Monoclonal Antibody Production and Characterization.- 7 Radioimmunoassay Procedures.- 7.1 Procedure 6: Radioimmunoassay of Plant Hormones.- 8 Enzyme Immunoassays Based on Monoclonal Antibodies.- 8.1 Procedure 7: Enzyme Immunoassay of Plant Hormones Based on Mouse Monoclonal Antibodies.- 9 Plant Immunoanalysis.- 9.1 General Remarks.- 9.2 Validation of Immunoassays.- 9.3 Processing of Plant Material.- 9.3.1 Procedure 8.- 9.3.2 Procedure 9.- References.- Radioimmunoassay and Gas Chromatography/Mass Spectrometry for Cytokinin Determination.- 1 Introduction.- 2 Theoretical Considerations for Analytical Methods.- 3 Radioimmunoassay.- 3.1 General Principles.- 3.2 Cytokinin Antisera.- 3.3 Cytokinin Determination in the Primary Extract.- 3.4 Cytokinin Determination After Preliminary Purification of the Primary Extract.- 3.5 Methods.- 3.5.1 General Isolation Protocol.- 3.5.2 Synthesis of Cytokinin Protein Conjugates.- 3.5.3 Immunization.- 3.5.4 Synthesis of 3H-Cytokinin Riboside-Dialcohols.- 3.5.5 RIA Protocol.- 4 Gas Chromatography/Mass Spectrometry.- 4.1 General Principles.- 4.2 Identification of Cytokinins.- 4.3 Quantitation of Cytokinins.- 4.4 Methods.- 4.4.1 General Isolation Procedure and GC/MS.- 4.4.2 Synthesis of Deuterium-Labeled Cytokinins.- 4.4.3 Practical Approach.- 5 Concluding Remarks.- References.- Immunodetection of Phytochrome: Immunocytochemistry, Immunoblotting, and Immunoquantitation.- 1 Introduction.- 1.1 Antibody Specificity.- 1.2 Immunodetection Methods.- 2 Immunocytochemistry.- 2.1 Rationale.- 2.2 Method.- 2.3 Controls.- 2.4 Evaluation of Method.- 3 Immunoblotting.- 3.1 Rationale.- 3.2 Method.- 3.3 Controls.- 3.4 Evaluation of Method.- 4 Immunoquantitation.- 4.1 Rationale.- 4.2 Method.- 4.3 Controls.- 4.4 Evaluation of Method.- 5 Concluding Remarks.- References.- Radioimmunoassay for a Soybean Phytoalexin.- 1 Isolation of Glyceollin I.- 2 Synthesis of Glyceollin I-BSA Conjugate.- 2.1 Preparation of p-Aminohippuric Acid Substituted BSA.- 2.2 Coupling of Glyceollin I to p-Aminohippuric Acid Substituted BSA.- 3 Generation of Antisera.- 4 Preparation of the [125I]-Glyceollin I Tracer.- 5 Radioimmunoassay for Glyceollin I.- 6 Specificity of the Radioimmunoassay.- 7 Application of the Radioimmunoassay for Glyceollin I to the Quantitation of Phytoalexins in Infected Soybean Tissue.- References.- The Measurement of Low-Molecular-Weight, Non-Immunogenic Compounds by Immunoassay.- 1 Introduction.- 1.1 The Analytical Role of the Immunoassay.- 1.2 Types of Immunoassay.- 1.3 Automation.- 2 Development of an Immunoassay.- 2.1 Synthesis of Immunogen.- 2.1.1 Methods of Conjugation.- 2.1.1.1 Carboxylic Acid-Directed Reagents.- 2.1.1.2 Alcohol-Directed Reagents.- 2.1.1.3 Phenolic-Directed Reagents.- 2.1.1.4 N-in-Aromatic-Ring-Directed Reagents.- 2.1.1.5 Amine-Direct Reagents.- 2.1.1.6 Ketone-Directed Reagents.- 2.1.1.7 Sugar-Directed Reagents.- 2.1.2 Bridge Conjugates.- 2.1.2.1 The 6-Amino-N-Hexanoic Acid Method.- 2.1.2.2 Alcohol-Directed Reagents.- 2.1.2.3 The 4-Aminohippuric Acid Method.- 2.1.3 Choice of Protein.- 2.2 Production of Antiserum.- 2.2.1 Immunisation Procedure.- 2.3 Synthesis of Labelled Tracers.- 2.3.1 Radiolabelled Tracers.- 2.3.1.1 Aromatic Ring Substitution.- 2.3.1.2 Incorporation at the Point of Conjugation.- 2.3.1.3 Reduction with Sodium [3H]-Borohydride.- 2.3.1.4 [125I]-Incorporation.- 2.3.2 Enzyme-Linked Tracers.- 2.4 ELISA Procedure.- 2.4.1 Microtitration Plates.- 2.4.2 Buffers.- 2.4.3 Initial Titre Measurement.- 2.4.4 Coating Concentration.- 2.4.5 Standard Curve.- 2.4.6 Serum Dilution.- 2.4.7 Second Antibody Dilution.- 2.4.8 Second Antibody Development.- 2.4.8.1 Alkaline Phosphatase.- 2.4.8.2 Peroxidase.- 2.4.8.3 ?-Galactosidase.- 2.4.9 Assay Validation.- 2.4.10 Future Developments.- 2.4.10.1 Fluorescent Enzyme Assays.- 2.4.10.2 The Biotin-Streptavidin System.- 2.4.10.3 The Enzymatic Radioimmunoassay.- 2.4.10.4 Chemiluminescent-Labelled Antibodies.- 2.4.11 Speed of Assay.- 3 Immmunoassays.- 3.1 Alkaloids.- 3.1.1 Nicotiana Alkaloids.- 3.1.1.1 Nicotine.- 3.1.1.2 Cotinine.- 3.1.2 Indole Alkaloids of Catharanthus roseus.- 3.1.2.1 Vincristine and Vinblastine.- 3.1.2.2 Vindoline.- 3.1.2.3 Ajmalicine and Serpentine.- 3.1.3 Tropane Alkaloids.- 3.1.4 Quinoline Alkaloids.- 3.1.4.1 Quinine.- 3.1.4.2 Quinidine.- 3.1.5 Papaver Alkaloids.- 3.1.5.1 Morphine and Codeine.- 3.1.5.2 The Benzylisoquinoline Pathway.- 3.2 Terpenoids.- 3.2.1 Litnonin.- 3.2.2 Bruceantin.- 3.2.3 Quassin.- 3.2.4 Iridoid Glucosides.- 3.3 Flavonoids.- 3.3.1 Naringin.- 3.4 Anthrones.- 3.4.1 Sennosides.- 3.5 Steroids.- 3.5.1 Digitalis Cardenolides.- 3.5.1.1 Digoxin.- 3.5.1.2 Digitoxin.- 3.5.2 Solanum Steroidal Glycoalkaloids.- 3.5.2.1 Solanidine-Derived Glycoalkaloids.- 3.5.2.2 Solasodine-Derived Glycoalkaloids.- 3.6 Miscellaneous.- 3.6.1 Physiologically Active Metabolites.- 3.6.1.1 Colchicine.- 3.6.1.2 Chloramphenicol.- 3.6.1.3 Paraquat.- 3.6.2 Contamination of Food and Feedstuffs.- 3.6.2.1 Aflatoxin B1.- 3.6.2.2 Sterigmatocystin.- 3.6.2.3 Ochratoxin A.- Appendix: Sources of Materials and Equipment for ELISA.- References.- Radioimmunoassay and Western Blot Analysis of Acyl Carrier Protein Isoforms in Plants.- 1 Introduction.- 2 Production of ACP Antibody.- 2.1 Purification of Spinach ACP Isoforms.- 2.2 Immunization Procedure.- 3 Radioimmunoassay.- 3.1 Preparation of Radiolabeled ACP.- 3.2 Radioimmunoassay Procedure.- 3.3 Applications.- 3.3.1 ACP Levels During Soybean Seed Development.- 3 3 2 Immuno-Cross-Reactivity Among ACPs.- 4 Immunological Analysis of ACP Isoforms.- 4.1 Immunorelationship of Spinach ACP Isoforms.- 4.2 Western Blot Analysis of ACP Isoforms in Plant Tissue.- 4.2.1 Preparation of Plant Tissue Extracts.- 4.2.2 Western Blot Analysis.- 5 Conclusion.- References.- Immunofluorescent Labelling of Enzymes.- 1 Introduction.- 2 General Methodology.- 2.1 Staining Sequences.- 2.2 Antibody Preparation.- 2.3 Specimen Preparation.- 2.4 Fluorescence Microscopy.- 2.5 Controls.- 3 Examples for Localization of Enzymes in Plant Tissue by Immunofluorescent Labeling.- 3.1 In Situ Immunofluorescent Labeling of Ribulosebisphosphate Carboxylase in Leaves of C3 and C4 Plants.- 3.2 In Situ Immunofluorescent Labeling of a ?-Glucosidase for Coniferin and of UDP-Glucose: Coniferyl Alcohol Glucosyltransferase.- 3.2.1 Localization of ?-Glucosidase.- 3.2.2 Localization of Glucosyltransferase.- References.- Quantitative Immunochemistry of Plant Phosphoenolpyruvate Carboxylases.- 1 Purification of PEPC - Preparation of Immune Sera.- 1.1 Extraction of PEPC.- 1.2 Purification of PEPC.- 1.3 Preparation of Antisera.- 1.4 Purification of Antisera.- 1.4.1 Fractionation by Ion-Exchange Chromatography.- 1.4.2 Affinity Chromatography Utilizing Protein A Immobilized on a Support.- 1.4.3 Affinity Chromatography Utilizing Enzyme Immobilized on an Activated Support.- 2 Quantitative Immunoprecipitation in Gels.- 3 Immunotitration Coupled to Enzyme Activity.- 3.1 Measurement of PEPC Residual Activity in the Supernatant..- 3.2 Measurement of PEPC Activity in the Immunoprecipitates.- 3.3 Applications.- 3.3.1 PEPC During Establishment of CAM and C4-Type Photosynthesis.- 3 3 2 Immunotitration of PEPCs in Relation to Plant Photosynthethic Types.- 3.3.3 Miscellaneous.- 4 Quantitative Immunoprecipitation in Extracts.- 4.1 HPLC Techniques.- 4.2 SDS-PAGE Techniques.- 5 Immunoaffinity Chromatography.- 6 Concluding Remarks.- References.- Immunochemical Methods for Higher Plant Nitrate Reductase.- 1 Introduction.- 2 Nitrate Reductase Purification.- 2.1 Seedling Growth Conditions.- 2.2 Extraction.- 2.3 Affinity Chromatography.- 2.4 Assays.- 3 Production of Nitrate Reductase Antiserum.- 3.1 Preparation of Nitrate Reductase.- 3.2 Immunization.- 3.3 Serum Collection and Isolation.- 4 Characterization of Nitrate Reductase Antiserum.- 4.1 Antiserum Titration.- 4.2 NR Antiserum Monospecificity.- 4 2 1 Immunodiffusion.- 4.2.2 Crossed Immunoelectrophoresis.- 5 Applications.- 5.1 Quantification of Nitrate Reductase Antigen.- 5.1.1 Protection of Nitrate Reductase Inactivation.- 5.1.2 Rocket Immunoelectrophoresis.- 5.1.3 Other Methods.- 5.2 Western Blot.- 5.2.1 Native Polyacrylamide Gel Electrophoresis.- 5.2.2 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE).- 5.2.3 Electrophoretic Transfer of Proteins from Polyacrylamide Gels to Nitrocellulose Sheets.- 5 2 4 Immunodetection of Nitrate Reductase.- 5.3 Immunopurifcation of Nitrate Reductase.- 5 3 1 Immuneline Electrophoresis.- 5.3.2 Antibody Column.- 6 Summary.- References.- Immunological-Cytochenlical Localization of Cell Products in Plant Tissue Culture.- 1 Introduction.- 2 The Laticifer.- 2.1 Laticifer Morphology and Differentiation in the Asclepiadaceae.- 2.2 Methods of Preparing Anti-Latex Antibodies.- 2.3 Section Preparation.- 2.4 Laticifer Identification in Zygotic Material.- 3 The Tissue Culture System for the Asclepiadaceae.- 3.1 Culture Techniques.- 3.2 Embryoid Differentiation and Laticifer Identification.- 4 Future Uses of Immunological-Cytochemical Techniques in Tissue Culture.- 4.1 Embryogenesis and Plant Hormones.- 4.2 Embryogenesis and Embryogenic Protein.- 4.3 Cell Sorting.- References.- Measurement of Oat Globulin by Radioimmunoassay.- 1 Introduction.- 1.1 Theory of the RIA.- 1.2 Oat Globulin.- 2 Requirements for the Radioimmunoassay.- 2.1 Preparation of Pure Antigen.- 2.2 Preparation of Radiolabeled Antigen.- 2.3 Antibody Production.- 2.4 Serum and IgG Preparation.- 2.5 Preparation of Oat Seed Extracts.- 3 Designing the RIA.- 3.1 Selecting the Amount of Radiolabeled Antigen.- 3.2 Titration of the Antibody.- 3.3 Precipitation of the Antigen/Antibody Complexes.- 3.4 Conducting the RIA.- 3.5 Preparing the Standard Curve.- 3.6 Data Treatment.- 4 Summary.- References.- Immunocytocheniistry of Chloroplast Antigens.- 1 Introduction.- 2 Light Microscopy.- 2.1 Procedures for Indirect Immunofluorescence.- 3 Electron Microscopy.- 3.1 The Peroxidase Anti-Peroxidase (PAP) Methods (Post-Embedding).- 3.2 Peroxidase Anti-Peroxidase Procedure.- 3.3 Colloidal Gold.- 3.4 Post-Embedding Labeling with Colloidal Gold on Lowicryl-Embedded Tissue.- 4 Conclusions.- References.