Methods of Hybridoma Formation

ISBN-13: 9781461291794 / Angielski / Miękka / 2011 / 504 str.

Arie H. Bartal; Yashar Hirshaut

Opis

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.

Spis treści

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.- 7.1. Advantages.- 7.2. Disadvantages.- 7.3. Future Prospects.- References.- Intrasplenic Immunization for the Production of Monoclonal Antibodies.- 1. Introduction.- 2. Materials and Equipment.- 2.1. Preparation of Alum-Precipitated Immunogen.- 3. Method.- 3.1. Surgical Intervention.- 4. Technical Comments.- 5. Discussion.- References.- Use of Heteromyelomas in the Enhancement of Human Monoclonal Antibody Production.- 1. Introduction.- 2. Principles and Perspectives of Heteromyeloma.- 3. Antigen-Primed Human Fusion Partners.- 4. Technical Aspects.- 4.1. Preparation of Human Fusion Partners.- 4.2. Fusion Procedure.- 4.3. Cloning Procedure.- 4.4. Mainteinance of Heteromyeloma and Hybridoma Cell Lines.- References.- Human Hybridomas: Fusion Partners.- 1. Introduction.- 2. Selection of a Fusion Partner.- 3. Morphology of Plasmacytoma and Lymphoblastoid Cells.- 4. Hybridomas Generated With Human Plasmacytomas.- 5. Hybridomas Generated With Lymphoblastoid Cell Lines.- 6. Lymphoblastoid vs Plasmacytoma Cells as Fusion Partners.- 7. Development of a Better Fusion Partner.- 7.1. Double Drug-Resistant Cell Lines.- 7.2. Non-lg-Secreting Partners.- 7.3. Hybrid Fusion Partners.- 8. Large-Scale Production of Human Mabs.- 8.1. Ascites Production.- 8.2. Serum-Free Culture.- 9. Advantages of Human Monoclonal Antibodies.- 10. Limitations and Future Developments.- References.- Antibody-Secreting Human-Human Hybridomas: Technical Progress, Generation Obstacles, and Applications.- 1. Introduction.- 2. Malignant Fusion Partners.- 3. Immunization Procedures.- 4. Production of Human Mabs by DNA Recombinant Technology.- 5. Some Areas of Application of Human Mabs.- 5.1. In Vivo Application in Nonneoplastic Diseases.- 6. HMabs in Cancer Biology.- 6.1. HMabs and Oncogenes.- 6.2. The Antiidiotype Approach.- 6.3. Antigenic Modulation.- 6.4. Intratumoral Phenotypic Diversity and Metastatic Activity.- 7. Conclusions.- References.- Interspecific Hybridomas.- 1. Introduction.- 2. Reasons for Preparing Interspecific Hybridomas.- 3. Applications of Interspecific Hybridomas.- 3.1. Interrodent Hybridomas.- 3.2. Human-Mouse Hybridomas.- 3.3. Rabbit-Mouse Hybridomas.- 3.4. Other Interspecific Hybridomas.- 4. Species-Specific Chromosome Loss in Interspecific Hybridomas.- 5. Approaches to Minimizing the Problem of Chromosome Loss in Interspecific Hybridomas.- 6. Summary and Opportunities for Future Studies.- References.- Rat-Rat Hybridoma Formation and Rat Monoclonal Antibodies.- 1. Introduction.- 2. Rat Immunoglobulins.- 2.1. Physicochemical Properties.- 2.2. Biological Properties.- 2.3. Allotypes of Rat Immunoglobulins.- 3. LOU/C Ileocecal Immunocytomas.- 3.1. Origin of the Louvain Rat Strain.- 3.2. LOU/C Ileocecal Malignant Immunocytomas.- 4. Genetic Studies on the Susceptibility of Rats to Immunocytomas.- 5. Transplantation of LOU/C Rat Immunocytomas.- 6. Storage of LOU/C Immunocytoma Cells.- 7. In Vitro Culture.- 8. Biosynthesis of Myeloma Immunoglobulins.- 9. Production of Monoclonal Immunoglobulins.- 10. Rat Hybridomas.- 11. Fusion Cell Lines.- 12. Immune Lymphocytes—Rat Strains.- 13. Immunization Protocol.- 14. Preparation of the Fusion Line Cells.- 15. Media.- 16. Feeder Layer.- 17. Fusion.- 18. Screening Assays.- 19. Cloning.- 20. Freezing and Thawing.- 21. Production of Rat Monoclonal Antibodies.- 22. Determination of Antibody Isotype.- 23. Purification of Rat Monoclonal Antibodies.- 24. Euglobulin Property.- 25. Stability.- 26. Major Strategies.- 27. Purification of Rat Monoclonal Antibodies From In Vitro Culture Supernatant.- 28. Purification of Rat Monoclonal Antibodies From Ascitic Fluid or Serum by Kappa Allotype Immunoaffinity Chromatography.- 29. Purification of Rat Monoclonal Antibodies by Using Anti-Isotype Immunoaffinity Chromatography.- 30. Purification of Rat Monoclonal Antibodies by Protein A Affinity Chromatography.- 31. Conventional Techniques of Rat Monoclonal Antibody Purification.- 32. IgM.- 33. IgD.- 34. IgE.- 35. IgA.- 36. IgG1.- 37. IgG2a.- 38. lgG2b.- 39. IgG2c.- 40. Fragmentation of Rat Monoclonal Antibodies.- 41. Sensitivity to Reduction.- 42. Enzymatic Proteolysis.- 43. Conclusions.- References.- Generation of Mutant Monoclonal Antibodies.- 1. Introduction.- 2. Modifications in Hybridoma Technology.- 3. Mutant Monoclonal Antibodies.- 4. Class and Subclass Switch Variants.- 5. Structural Mutants With Changes in Effector Functions and Antigen Binding.- 6. Conclusion.- References.- Ranking the Affinities of Monoclonal Antibodies.- 1. Introduction.- 2. Theoretical Aspects.- 2.1. Definition of Affinity.- 2.2. Methods of Measuring Affinity Constants.- 2.3. Calculating K from Binding Curves.- 3. Practical Applications.- 3.1. Suitability of Antigen.- 3.2. The Need for Measuring the Concentration of Monoclonal Immunoglobulin.- 3.3. Final Choice of Monoclonal Antibody.- References.- Intrasplenic Inoculation: A Method for Rescuing Hybridoma Clones.- 1. Introduction.- 2. Technical Procedure and Remarks.- 2.1. Materials.- 2.2. Intrasplenic Inoculation Procedures.- 3. Hybridoma Cell Inoculum.- 4. Harvesting Ascitic Fluid.- 5. Discussion.- References.- Cryopreservation in Hybridoma Production.- 1. Introduction.- 2. Materials and Methods.- 2.1. Growth of Cells.- 2.2. Fusion Protocol.- 3. Results.- 3.1. Strategy for the Production of Hybridomas.- 3.2. Cryopreservation and Thawing Procedures.- 4. Discussion.- References.- Early and Late Freezing of Hybridoma Clones.- 1. Introduction.- 2. Freezing Cells.- 3. Methods for Freezing Hybridoma Clones.- 4. Conclusions.- References.- Flow Cytometry in Hybridoma Technology.- 1. Introduction.- References.- Regulatory Issues Surrounding Therapeutic Use of Monoclonal Antibodies: Points To Consider in the Manufacture of Injectable Products Intended for Human Use.- 1. Introduction.- 2. Development and Characterization of Hybridoma Cell Lines.- 3. Quality Control Tests.- 3.1. Special Considerations Regarding Viral Contamination.- 3.2. Special Considerations Regarding Nucleic Acid Contamination.- 4. Preclinical Animal Testing.- 5. Preclinical Laboratory Testing.- 6. Special Considerations for Monoclonal Antibodies of Human Origin.- 7. Summary and Conclusion.- References.- Human-Mouse Hybridoma Formation.- 1. Introduction.- 2. Somatic Cell Hybridization of Immunoglobulin-Producing Lines.- 3. Production and Characterization of Human-Mouse Hybrids.- 3.1. Production of Hybrids.- 3.2. Stability of Human-Mouse Hybrids.- 4. Expression of Human Enzymes.- 5. Karyotype of Human-Mouse Hybrids.- 6. Stability of Immunoglobulin Secretion.- 7. Membrane Transport by Human-Mouse Hybrids.- 8. Applications of Human-Mouse Hybridoma Technology.- 8.1. Human Genetic Analysis and Gene Mapping.- 8.2. Production of Specific Human Monoclonal Antibodies.- 8.3. Other Biological Research Applications of Human-Mouse Hybrids.- 8.4. Human-Mouse Monoclonals in Cancer.- 9. Conclusions.- References.