title: The use of Bcl-2 over-expression to stabilize hybridomas specific to the HERG potassium channel.
abstract: We encountered a high degree of clonal hybridoma loss in the course of generating antibodies specific for the hERG potassium channel. A protein that is crucial for controlling heart rhythm, is abundant in parts of the brain and is abnormally expressed in some tumors. Intracellular domains of the protein were used for immunogens and generated adequate antibody responses in mice. Subsequent hybridomas created using Ag8 myeloma fusion partner yielded clones that secreted specific antibody but none could be successfully maintained in culture. A variety of mechanisms, including polyploidy inherent to hybridoma development or production of cytotoxic antibodies, may be responsible for eventual loss of cell viability by mechanisms that may include apoptosis. When spleen cells were fused to the NSO myeloma cell line that stably over-expresses the anti-apoptotic protein Bcl-2, hybridoma clones were generated that remained viable in culture with high level of hERG-specific antibody production. When the parental NSO cell line not over-expressing Bcl-2 was used, no stable hybridomas were produced. Antibodies secreted by NSO-Bcl-2 hybridomas were specific for hERG and performed well in immunoblot, immunoprecipitation and immunofluorescence assays. This work demonstrates a feasible option when faced with antigens that seem to be associated with clonal instability in the process of generating monoclonal antibodies.
authors: Department of Molecular Pharmacology, Wilf Family Cardiovascular Research Institute, Albert Einstein College of Medicine, Bronx, NY 10461, United States.
authors: Jakub
authors: J
authors: Sroubek
authors: Yamini
authors: Y
authors: Krishnan
authors: Jordan
authors: J
authors: Chinai
authors: Susan
authors: S
authors: Buhl
authors: Matthew D
authors: MD
authors: Scharff
authors: Thomas V
authors: TV
authors: McDonald
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D017209:Apoptosis / Q000235:genetics / Q000276:immunology; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics / Q000276:immunology; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D006825:Hybridomas / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009101:Multiple Myeloma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D013154:Spleen / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D051017:Apoptosis Regulatory Proteins; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D019253:Proto-Oncogene Proteins c-bcl-2