Synopsis
1. Somatic-Cell Mutation Monitoring System Based on Human Hemoglobin Mutants.- 1. Introduction.- 1.1. The Approach.- 1.2. Previous Studies.- 1.3. Requirements of a Red Cell Screening System.- 2. The Hemoglobin Mutants.- 2.1. Hemoglobin Loci.- 2.2. Types of Mutations.- 3. Hemoglobin in Mutation Research: Gametal Mutation Rates.- 3.1. Indirect Estimates.- 3.2. Direct Estimates.- 4. A System for Detecting Somatic Mutations of Hemoglobin.- 4.1. Appropriate Mutants.- 4.2. Immunochemical Detection of Abnormal Hemoglobins in Single Cells.- 4.3. Detection of Rare Mutant Red Cells by Fluorescent Microscopy.- 4.4. Screening for "S Cells" or "C Cells" in Blood of Genetically A/A Subjects.- 4.5. Minimum Frequencies of Somatic Mutations at Globin-Chain Loci.- 4.6. Relationship between Somatic Mutation Frequencies and Gametal Mutation Rates.- 4.7. Relationship between Frequencies of Somatic-Cell Mutants and Compartments at Which Mutations Occur.- 5. Methodological Aspects: Monospecific Anti-Mutant-Hemoglobin Antibodies.- 5.1. Immunizations.- 5.2. Sepharose-Hb.- 5.3. Purification.- 5.4. Red Cell Labeling.- 6. Methodological Aspects: Monoclonal Anti-Globin-Chain Antibodies.- 6.1. Immunizations.- 6.2. Screening.- 6.3. Semiquantitative Assessment of Ab-Hb Binding.- 6.4. Mapping the Sites of Ab-Hb Binding.- 6.5. Possible Recognition of Mutant Hemoglobins in Animals.- References.- 2. Use of Fluorescence-Activated Cell Sorter for Screening Mutant Cells.- 1. Introduction.- 2. Immunologic Identification and Flow Detection of Erythrocytes Containing Amino Acid-Substituted Hemoglobin.- 2.1. Production of Antibodies.- 2.2. Suspension Labeling of Red Cells with Hemoglobin Antibodies.- 2.3. Flow Cytometric Processing.- 2.4. Results Using Hemoglobin S- and C-Specific Antibodies.- 3. Future of the Hemoglobin-Based Assay.- 4. Detection of Erythrocytes with Mutationally Altered Glycophorin A.- 4.1. Background.- 4.2. Gene Expression Loss Variants.- 4.3. Single-Amino Acid-Substitution Variants.- 5. Summary and Conclusions.- References.- 3. Development of a Plaque Assay for the Detection of Red Blood Cells Carrying Abnormal or Mutant Hemoglobins.- 1. Introduction.- 2. Principle of the Method.- 3. Reagents.- 3.1. Anti-Mouse RBC Ghost Sera.- 3.2. Anti-Mouse Hb Antibodies.- 3.3. Indicator Cells. Methods for Coupling Antibodies to Sheep RBC.- 3.4. Complement.- 4. Equipment.- 4.1. Plaque Chambers.- 4.2. Additional Materials.- 5. Procedure for the RBC-Antibody Plaque Assay.- 5.1. Factors Affecting the RBC Plaque Formation.- 5.2. Specificity of the RBC Plaque Assay.- 6. RBC-Protein A Plaque Assay.- 7. Conclusions.- References.- 4. Direct Assay by Autoradiography for 6-Thioguanine-Resistant Lymphocytes in Human Peripheral Blood.- 1. Introduction.- 1.1. Human Mutagenicity Monitoring.- 1.2. 6-Thioguanine-Resistant (TGr) Human Peripheral Blood T Lymphocytes (T-PBLs).- 1.3. Direct Enumeration of TGr T-PBLs by Autoradiography.- 1.4. Phenocopies.- 2. Autoradiographic TGr T-PBL Assay Method.- 2.1. Cell Preparation.- 2.2. Cryopreservation.- 2.3. Cell Culture.- 2.4. Termination, Coverslip Preparation, and Autoradiography.- 2.5. Enumeration of TGr T-PBLs and Calculation of TGr T-PBL Variant Frequency (Vf).- 3. Sample Results.- 3.1. TGr T-PBL VfAssay: Appearance of Slides.- 3.2. TGr T-PBL VfAssay: Sample Data.- 4. Statistical Analysis Methods.- 4.1. Notation and Basic Assumptions.- 4.2. Confidence Intervals for a Single Variant Frequency.- 4.3. Confidence Intervals for Ratios of Variant Frequencies.- 4.4. Sample Size Determinations.- 5. Discussion.- References.- 5. Application of Antibodies to 5-Bromodeoxyuridine for the Detection of Cells of Rare Genotype.- 1. Introduction.- 1.1. Flow Cytometry.- 1.2. The Use of 5-Bromodeoxyuridine for the Detection of Cell Proliferation.- 2. Materials and Methods.- 2.1. Cell Culture.- 2.2. Labeling with BrdUrd.- 2.3. Immunological Methods.- 2.4. Flow Cytometry.- 2.5. Hybridoma Production.- 3. Results.- 3.1. Determination of Antibody Specificity
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