Methods of Hybridoma Formation

Laymen often consider modern laboratory research to be based on an endless array of sophisticated technologies whose complex capabilities are as important to the outcome of any project as the inventiveness and creativity of the scientists who employ them. Scientists at times may share this point of view until they are con­ fronted by unexpected findings that demand new approaches, and they discover that yesterday's "sophisticated tools" are today's "blunt instruments." This experience provides a more sobering view of the current state of our scientific methods. It also serves as an impetus for the further development of technology that prepares us for the next stage of advance. Immunologists were confronted by such a technological crises in the late 1970s when they finally were forced to admit that poly­ clonal antibodies, although quite sensitive reagents, were not spe­ cific enough to answer many of the questions then confronting virologists and tumor biologists. The answer to the need for specific­ ity came with the development of monoclonal antibody technology. In the last ten years there have been considerable advances in monoclonal antibody techniques. Today these reagents are much more versatile than they were initially and can be applied to a broad range of problems. Still, most workers who are using these anti­ bodies are convinced that their potential is far from exhausted, and that at least in some fields we are currently in the early stages of learning how to use them properly.

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Current Methodologies in Hybridoma Formation.- 1. Introduction.- 2. Homokaryons, Heterokaryons, Synkaryons.- 3. Selection of Hybridomas.- 4. The Art of Making Hybridomas.- 4.1. Hybridoma Planning.- 5. Phases in Hybridoma Formation.- 5.1. Preparatory Phase.- 5.2. Prefusion Phase.- 5.3. Cell Fusion Phase.- 5.4. Early Postfusion Phase.- 5.5. Late Postfusion Phase.- 5.6. Long-Term Hybridoma Cultivation and Preservation.- 5.7. Utilization of Hybridoma-Derived Antibodies.- 6. Difficulties in Hybridoma Formation.- 6.1. Contamination.- 6.2. Equipment Failure.- 6.3. Methodological Difficulties.- 6.4. Basic Biological Problems.- 6.5. Technical Skill.- 6.6. Miscellaneous Factors.- 7. Conclusions.- References.- Retroviruses and Hybridoma Formation: Bystanders or Active Participants?.- 1. Introduction.- 2. Retroviruses, Definition, and Characterization.- 3. Morphology of Retroviruses.- 3.1. A-Type Particles.- 3.2. B-Type Particles.- 3.3. C-Type Particles.- 3.4. D-Type Particles.- 4. Retroviruses in Animals and Cell Lines.- 5. Retroviruses in Hybridomas.- 6. Hybridoma Retroviruses: Bystanders or Active Participants.- 7. Hazards Associated With Hybridoma Retroviruses.- 8. Toward Obtaining Purified Virus-Free Monoclonal Antibodies.- 9. Recommendations for Monoclonal Antibody Preparations.- References.- Mechanisms of Cell Fusion and Selection in the Generation of Hybridomas.- 1. Introduction.- 2. Historical Review.- 3. Fusion Techniques.- 3.1. PEG.- 3.2. Electrofusion.- 4. Hybrid Selection Techniques.- 4.1. HAT.- 4.2. Taggart Hybridoma Technology (AAT Selection).- 4.3. Irreversible Biochemical Inhibitors.- 4.4. Toxin-Antitoxin Selection.- 4.5. Cell Sorting.- 5. Future Directions and Conclusions.- References.- Chemically Mediated Cell Fusion.- 1. Introduction.- 2. Mechanism of Polyethylene Glycol-Mediated Cell Fusion.- 2.1. Fusogens and Hybridogens.- 2.2. Hydrophilic Character of Hybridogens.- 3. Relationship of Hybridogens to Cryoprotective Agents, Genetic Transformation Agents, and Inducers of Erythroleukemia Cell Differentiation.- 4. Mechanism of PEG-Mediated Cell Fusion.- 5. Hybridoma Formation by PED-Mediated Cell Fusion.- 5.1. Preparation of PEG.- 5.2. Cell Fusion Procedure.- 5.3. Biochemical Selection for Hybridomas.- References.- Electrofusion of Cells.- 1. Introduction.- 2. Electric Field Conditions.- 3. Electrode Material and Fusion Media.- 4. Enzyme Pretreatment.- 5. Breakdown and Postbreakdown Conditions.- 6. Viability of Electrofused Hybrids.- 7. Selection of Antibody-Secreting Lymphocytes.- 8. DNA Transfection.- 9. Conclusion.- Appendix: Murine Hybridoma Electrofusion Protocol for the GCA-Electrofusion System.- References.- Enhancement of Hybridoma Formation.- 1. Historical Introduction.- 2. Modes of Hybridoma Enhancement.- 2.1. Enhancement at the Prefusion Phase.- 2.2. Enhancement at the Cell Fusion Phase.- 2.3. Enhancement at the Early Postfusion Phase.- 2.4. Enhancement at the Late Postfusion Phase and in the Long-Term Cultivation of Hybridoma.- References.- Modification of HAT Medium and Hybridoma Formation.- 1. Introduction.- 2. Experimental Design.- 3. Addition of Dexamethasone to HAT Medium (HAT-DEX).- 4. Dexamethasone Dose Is Critical.- 5. Timing of Dexamethasone Addition Is Critical.- 6. Stability of Dexamethasone-Treated Hybridoma Clones.- 7. Dexamethasone and Hybridoma Formation.- 8. Addition of Insulin to HAT Medium (HIAT).- 9. HIAT and Mouse-Mouse Hybridoma Formation.- 10. HIAT in Human-Mouse Hybridoma Formation.- 11. HIAT in Human-Human Hybridoma Formation.- 12. Interferon and Hybridoma Formation (HAT-INF).- 13. Summary.- References.- Culture Methods for the Selection and Isolation of Stable Antibody-Producing Murine Hybridomas.- 1. Introduction.- 2. The Nature and Origins of Hybrid Cell Chromosome Instability.- 3. Chromosome Instability Reduces the Yield of Antibody-Producing Hybridomas.- 4. Methods for Isolation of Stable Antibody-Producing Hybridomas.- 4.1. Syntenic Culture Selection of the Mouse Heavy Chain Immunoglobulin Locus.- 4.2 Limiting Dilution Cloning of Hybridomas.- 5. Conclusions.- References.- Selection of Growth Factors and Myelomas To Enhance Monoclonal Antibody-Producing Hybridoma Formation.- 1. Introduction.- 2. Selection of Murine Myelomas for Fusion Partners.- 2.1. MOPC 21 (P3-X67Ag8).- 2.2. Sp2/O (Sp2/O-Ag14).- 2.3. NS-1 (P3/NS1/1Ag4-1).- 2.4. 653 (P3/X63-Ag8.653).- 2.5. FO.- 2.6. S194/5.XX0.BU-1.- 2.7. FOX-NY.- 3. Growth Factors.- 4. Feeder Cells.- 4.1. Peritoneal Macrophages.- 4.2. Thymocytes and Splenocytes.- 4.3. Cell Lines.- 5. Soluble Growth Supplements.- 5.1. Human Endothelial Cell Growth Supplement (HECS).- 5.2. Bovine Endothelial Cell Growth Supplement (ECGS).- 6. Myeloma-Conditioned Medium.- 7. Medium Nutrient Supplements.- 7.1. Growth in Low Molecular Weight Serum Protein Media.- 8. B-Cell Growth Factors.- 9. Technology Notes.- References.- Proliferation and Immune Secretion of B-Cell Hybridomas.- 1. Introduction.- 2. Stimulation of B-Cell Lymphocytes in Different Phases of the Cell Cycle.- 3. Hybridization and Limiting Dilution.- 4. Cell Cycle of Hybridomas.- 5. Growth Promoters.- 5.1. Dextran Sulfate - A Stimulator of Resting B-Cells.- 5.2. Cell-Cell Interaction.- 5.3. Lipopolysaccharide - A Stimulator of Activated B-Cells.- 5.4. Macrophage Supernatant.- 5.5. Human Endothelial Culture Supernatant and Human Umbilical Cord Serum.- 6. Comparison of Normal B-Cells to Tumor B-Cells.- 7. Proliferation and Differentiation of Hybridomas.- 8. Conclusion.- References.- Statistical Assessment of Hybridoma Monoclonality After Subcloning by the Limiting Dilution Technique.- 1. Introduction.- 2. Statistical Assessment.- References.- In Vitro Immunization for the Generation of Hybridomas Using Serum-Free Medium.- 1. Introduction.- 2. Growth of Lymphoid Cells and Hybridomas in Serum-Free Media.- 3. Theoretical and Practical Aspects of In Vitro Immunization.- 4. Methods.- 4.1. Preparation of Serum-Free Medium.- 4.2. In Vitro Immunization Protocol Using Serum-Free Medium.- 4.3. Fusion Protocol.- 5. Comparison of Serum-Containing vs Serum-Free Media for Hybridoma Formation.- 6. Rate of Growth of Nascent Hybridomas in Serum-Containing vs Serum-Free Medium.- 6.1. Cloning in Serum-Free Medium.- 6.2. Antibody Subtype Identification.- 6.3. Immunosorbent Assay for Levels of Monoclonal Antibody.- 6.4. Purification of Monoclonal Antibodies From Serum-Free Medium.- 6.5. Cryopreservation in Serum-Free Medium.- 7. Conclusions.-…