B-Cell Hybridoma Production by Avidin-Biotin Mediated Electrofusion
Kohler and Milstein’s method for the production of cell lines secreting specific antibodies directed to an antigen of interest has provided major advancements in the biomedical sciences and is widely used. The method is based on the random fusion of two cell types in the presence of polyethyleneglycol (PEG),1,2 although viruses were used in earlier experiments.3 Antibodies to a wide variety of antigens have been reported. Spleen cells from hyperimmunized mice are fused to azaguanine-resistant myeloma cells (which lack the enzyme hypoxanthine guanine ribosyltransferase, and therefore die in the presence of aminopterin). Hybrids are selected in a medium containing aminopterin supplemented with hypoxanthine and thymidine (HAT). Unfused myeloma cells are killed by the aminopterin, and unfused spleen cells die off after about 1 to 2 weeks in culture. These and all chemically induced fusions are random and may produce several hundred hybrid colonies, which have to be screened for the secretion of the desired antibodies. Usually several experiments have to be performed to obtain monoclonal antibodies of the required specificity and antigen binding affinity.
KeywordsSpleen Cell Myeloma Cell Cell Fusion Keyhole Limpet Hemocyanin Fluorescent Microsphere
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