Hybridomas and Cellular Immortality

I. Introduction To Hybridomas.- 1 Somatic Cell Hybrids and Hybridomas.- 1. Somatic Cell Hybrids.- 2. Hybridomas.- References.- II. Antibody Diversity And Lymphocyte Differentiation.- 2 The Nature and Origin of Antibody Diversity.- 1. Introduction.- 2. V-region sequence diversity at the protein level.- 2.1. Nature of the sequence diversity.- 2.2. Origin of the sequence diversity.- 3. Analysis of Ig genes at the DNA level.- 3.1. General considerations, and the mouse X light chain Locus.- 3.2. The mouse ? light chain locus.- 3.3. Generation of VH-region diversity in mouse.- 3.4. Is additional V-region diversity introduced by somatic mutation?.- 4. Conclusions.- References.- 3 Hematopoietic Tumors: Normal or Abnormal Models of Leukocyte Differentiation?.- 1. Introduction.- 2. Cell surface phenotypes.- 2.1. Fc receptors.- 2.2. Thy-1.- 2.3. LEU-3.- 2.4. LY-1/LEU-1.- 2.5. E2.- 2.6. LEU-7.- 3. Discussion.- References.- 4 Dissection of Human Immunoregulatory T Lymphocytes: Implications for Understanding Clinical Disease.- 1. Summary.- 2. Introduction.- 3. Results and Discussion.- 3.1. Differentiation of T lymphocytes.- 3.2. Functions of mature T lymphocyte subsets.- 3.3. Clinical disorders of T lymphocytes.- 3.4. Human T cell malignant diseases.- 3.5. Immunotherapy by slective T cell subset manipulation.- 4. Experimental Procedures.- 4.1. Isolation of lymphoid populations.- 4.2. Production and characterization of monoclonal antibodies.- 4.3. Analysis and separation of lymphocyte populations with fluorescence activated cell sorter.- 4.4. Antigen stimulation of T lymphocytes.- 4.5. Detection of in vitro secretion of IgG.- References.- 5 Immune (?) Interferon and Lymphotoxin Production by T Cell Lines and Hybrids.- 1. Summary.- 2. Introduction.- 3. Results.- 3.1. Growth factor lines.- 3.1.1. Properties.- 3.1.2. Production of LT and IFN.- 3.2. T cell hybrids.- 3.2.1. Analysis.- 3.2.2. LT Production.- 4. Discussion.- 5. Experimental Procedures.- 5.1. T cell lines.- 5.2. T cell hybrids.- 5.3. Lymphotoxin assay.- 5.4. Interferon assay.- References.- III. Monoclonal Antibodies as Probes of Antigen Structure.- 6 Partial Structural Characterization of Human HLA-DR Subsets Using Monoclonal Antibodies.- 1. Summary.- 2. Introduction.- 3. Results and Discussion.- 3.1. Two monoclonal antibodies (L203 and L227) recognize subset of DR antigens.- 3.2. Both subsets of DR antigens are homolgous to murine I-E antigens.- 3.3. The antibody L227 recognizes isolated DR beta chains.- 3.4. Both monoclonal antibodies react with a third population of DR antigens.- 3.5. Amino acid sequence analysis of the L203 and L227 unique populations.- 3.6. The monoclonal antibodies IIIE3 and IVG1 recognize the same subset of DR antigens.- 3.7. III E3 recognizes a subset of L203 and L227 antigens.- 3.8. Amino acid sequence of the III E3/IVG1 antigens.- 4. Conclusions.- 5. Experimental Methods.- 5.1. Cells.- 5.2. Monoclonal antibodies and antisera.- 5.3. Isolation of radiolabeled Dr antigens.- 5.4. Lentil lectin affinity chromatography.- 5.5. Immunoprecipitation.- 5.6. Antibody affinity columns.- 5.7. SDS-Polycrylamide gel electrophoresis.- 5.8. Amino acid sequence determination.- References.- 7 Monoclonal Antibodies for Analysis of HLA Antigens: Further Studies with the W6/32 Antibody.- 1. Introduction.- 2. Results.- 2.1. Interaction of W6/32 antibody with HLA antigen.- 2.1.1. Binding of W6/32 to cells at equilibrium.- 2.1.2. Comparison of the binding of W6/32 IgG and FAB to LB cells at equilibrium.- 2.1.3. Kinetics of binding of W6/32 IgG and Fab to LB cells.- 2.1.4. Binding of W6/32 to soluble HLA antigens.- 2.1.5. Conclusions.- 2.2. Structural studies.- 2.2.1. Amino acid sequencing of W6/32 precipitates.- 2.2.2. Separation of exchange with an excess of non-radioactive "cold"-?-2 microglobulin...- 2.2.3. Separation of preparative SDS-PAGE.- References.- IV. Monoclonal Antibodies in Diagnosis and Therapy.- 8 Monoclonal Antibodies to Hepatitis B Surface Antigen.- 1. Summary.- 2. Introduction.- 3. Results.- 3.1. Fusions and characteristics of monoclonal anti-HBs.- 3.2. Immunoradiometric assay for HBsAg.- 4. Discussion.- 5. Experimental Procedures.- 5.1. Production of monoclonal antibodies to HBsAg.- 5.2. Characterization of monoclonal antibodies to HBsAg.- 5.3. Tissue culture.- 5.4. Assay procedure.- References.- 9 Wandering Around the Cell Surface: Monoclonal Antibodies Against Human Neuroblastoma and Leukemia Cell Surface Antigens.- 1. Summary.- 2. Introduction.- 3. Results.- 3.1. An anti-neuroblatoma antibody.- 3.2. The neuroblastoma determinant is also detectable on leukemia cells.- 3.3. Using a combination of monoclonal antibodies to detect neuroblastoma cells in marrow.- 3.4. Other monoclonal antibodies against neuroblastomas or leukemias.- 3.5. "Operationally tumor specific" antibodies.- 3.6. In vitro immunization.- 3.7. Detection of antibodies inhibiting leukemia cell proliferation.- 3.8. Isolation of cell surface molecules using monoclonal antibodies.- 3.9. Monoclonal antibodies can be used to isolate minor components of the cell surface.- 3.10. Concluding summary.- 4. Discussion.- 5. Experimental Procedures.- 5.1. Production and characterization of monoclonal antibodies.- 5.2. In vitro immunization.- 5.3. Detection of monoclonal antibodies inhibiting Reh cell proliferation.- 5.4. Affinity chromatography of neuroblastoma antigens.- References.- 10 Monoclonal Antibodies to Human Melanoma-Associated Antigen p97.- 1. Summary.- 2. Introduction.- 3. Results.- 3.1. Monoclonal antibodies to p97.- 3.2. Tissue distribution of p97.- 3.3. Structural studies of p97.- 3.4. Relationship of p97 to other tumor-associated antigens.- 4. Discussion.- 4.1. Screening hybridomas.- 4.2. Antigen specificity.- 4.3. Comparison of tumor-associated antigens.- 4.4. Nature, function, and practical application of tumor-associated antigens defined by monoclonal antibodies.- References.- 11 A Biochemical and Biosynthetic Analysis of Human Melanoma-Associated Antigens with Monoclonal Antibodies.- 1. Summary.- 2. Introduction.- 3. Results.- 4. Discussion.- 5. Materials and Methods.- 5.1. Cells.- 5.2. Monoclonal antibodies.- 5.3. Serological assays.- 5.4. Biosynthetic labelling procedures.- 5.5. Indirect immunoprecipitation/SDS-PAGE.- 5.6. Cell fractionation procedure.- 5.7. Materials.- References.- 12 Monoclonal Antibodies to a Tumor Specific Antigen on Rat Mammary Carcinoma Sp4 and their use in Drug Delivery Systems.- 1. Introduction.- 2. Results and Discussion.- 2.1. Monoclonal antibody specifying tumor specific antigen associated with rat mammary carcinoma Sp4.- 2.2. Anti-tumor activities of Sp4/A4 monoclonal antibody.- 2.3. In vivo localization of Sp4/A4 monoclonal antibody.- 2.4. Anti-tumor activity of Sp4/A4 antibody-adriamycin conjugates.- 3. Conclusions.- 4. Materials and Methods.- 4.1. Tumors.- 4.2. Mammary carcinoma Sp4 monoclonal antibody (Sp4/A4).- 4.3. Treatment of Sp4 with Sp4/A4 monoclonal antibody.- 4.4 Complement-dependent cytotoxicity test.- 4.5. Antibody-dependent cell-mediated cytotoxicity.- 4.6. Radiolabelling of Sp4/A4 monoclonal antibody for in vivo localization.- 4.7. Antitumor activity of Sp4/A4 monoclonal antibody conjugated to adriamycin.- References.- 13 Monoclonal Antibody to a Tumor Specific Epitope of Murine Lymphoma Cells: Use in Characterization of Antigen and in Immunotherapy.- 1. Summary.- 2. Introduction.- 3. Results and Discussion.- 3.1. Production of monoclonal antibody.- 3.2. Reactivity of MAb 124-40 with C6XL lymphoma cells.- 3.3. Lack of reactivity of MAb 124-40 with other normal or malignant lymphoid cells.- 3.4. Characterization of Ag 124-40.- 3.5. Expression of similar cell surface component by normal T cells and thymocytes.- 3.6. Immunotherapy with MAb 124-40 and pokeweed antiviral protein conjugate.- 4. Conclusions.- 5. Experimental Procedures.- 5.1. Mice.- 5.2. Cell Lines.- 5.3. Hybridoma construction.- 5.4. Antibody binding assay.- 5.5. Flow microfluorometry.- 5.6. Radioiodination.- 5.7. Immunoprecipitation.- 5.8. Polyacrylamide gel electrophoresis.- References.- V. New and Relevant Methodology.- 14 Human Hybridomas.- 1. Summary.- 2. Introduction.- 3. Results.- 4. Discussion.- 5. Experimental Methods.- 5.1. Selection of somatic cell hybrids.- 5.2. Production of human immunoglobulin (Ig) chain.- 5.3. Isozyme analysis.- 5.4. Chromosome analysis.- References.- 15 Procedures for In Vitro Immunization and Monoclonal Antibody Production.- 1. Summary.- 2. Introduction.- 3. Results.- 4. Discussion.- 5. Methods.- 5.1. Culture conditions.- 5.2. Immunization in culture.- 5.3. Hybridization.- 5.4. Enzyme linked immunosorbent assay (ELISA).- 5.5. HPLC-ELISA.- 5.6. Immunogold preparation.- 5.7. Immunogold staining.- 5.8. Immunogold separation.- References.- 16 Liposome-facilitated In Vitro Induction of Primary Cell-Mediated Immunity to Human Cancer Antigens: Potential Adjunct to Hybridoma Technology.- 1. Summary.- 2. Introduction.- 2.1. In vitro immunization.- 2.2. cell-free immunizations.- 2.3. Liposome-mediated immunity.- 3. Results.- 3.1. Summary of studies.- 3.2. In vitro xenoimmunization to human tumor antigens.- 3.3. In vitro alloimmunization to human tumor antigens.- 4. Discussion and Conclusions.- 5. Experimental Procedures.- 5.1. Preparation of tissue culture cells.- 5.2. Preparation of tumor cell membranes.- 5.3. Preparation of liposome-membrane antigens.- 5.4. Quantitation of protein antigens.- 5.5. Quantitation of liposomes.- 5.6. In vitro immunization.- 5.6.1. Generation of cytotoxic activity with liposomes.- 5.6.2. Inhibition of cytotoxicity.- 5.6.3. Cell-mediated cytotoxicity assay.- 5.6.4. Elicitation of blastogenic response.- References.- 17 Comparison of Three Immunoassays for Screening Anti-Hepatitis B Hybridomas.- 1. Summary.- 2. Introduction.- 3. Results and Discussion.- 4. Experimental Procedures.- 4.1. Antisera.- 4.2. Antibody-enzyme conjugation.- 4.3. Immunoassays.- References.- VI. Poster Abstracts.- 1. T-Lymptocyte Subsets of Rhesus Macaque as Determined by Monoclonal Antibodies: T?, T?, T-NULL, and Histamine Receptor Bearing T Lymphocytes.- 2. Production of Human Immunoglobulin Secreting Clones Without the Use of H.A.T. Sensitive Fusion Partners.- 3. Species Distribution of Antigenic Sites on Mouse LDH-C4 Determined by Monoclonal Antibodies.- 4. A New Non-Immunoglobulin Producing Human Myeloma Cell line for Production of Hybridomas.- 5. Monoclonal Antibodies to Human Prostate and Bladder Tumor Associated Antigens.- 6. Antibody Purification by Recycling Isoelectric Focusing.- 7. Monoclonal Antibodies Against Human Ejaculated Sperm-Application in Antibody Mediated Infertility.- 8. Analysis of Peripheral Blood and Salivary Gland Lymphocytes in Sjogren's Syndrome.- 9. A Monoclonal Antibody Which Identifies a Subpopulation of Human B Lymphocytes and Monocytes.- 10. In Vivo Localization of Tumor Associated Antigens with Radioactively Labeled Monoclonal Antibodies.- 11. Characteristics of Monoclonal Antibodies to Herpes Simplex Type 2.- 12. Type Specific Monoclonal Antibodies to Equine Infectious Anemia Virus: Development and Use in the Characterization of Canine and Feline Derived Viruses.- 13. Biochemical and Biological Analysis of Venezuelan Equine Encephalomyelitis Viruses (VEE) Using Monoclonal Antibodies.- 14. Monoclonal Antibodies Specific for Herpes Simplex Virus Type 1 Mediate Antiviral Effects In Vitro and In Vivo.- 15. Monoclonal Antibodies Against Antigens of Amastigotes and Epimastigotes of Trypanosma Cruzi.- 16. Differential Effects of Four Monoclonal Antibodies to Human ?2-Microglobulin on In Vitro Lymphocyte Functions.- 17. Selective Inhibition of Functional Sites on Cell Bound Human C3b By Monoclonal Antibodies.- 18. Generation of Monoclonal Antibodies Reacting With Human Normal and Neoplastic Mammary Duct Epithelial Cells.- 19. Resolution of Human Liver Mao A and B Using A Monoclonal Antibody Immunoaffinity Column.- 20. Isolation of Antibodies that are Specific for Human Carboxypeptidase B But Not for the Zymogen Procarboxypeptidase B by Affinity Chromatography.- 21. Dexamethasone Effects on Hybridoma Formation.- 22. Class Antigen in the Rat: Evidence for More Than One Using Monoclonal Antibodies.- 23. An Immunohistochemical Method for Large Scale Specificity Screening of Monoclonal Antibodies.- 24. Monoclonal Antibodies to Chicken Hemopoietic-Lymphoid Cellular Differentiation Antigens.- Author Index.