Production of IgG-producing hybridomas by in vitro stimulation of murine spleen cells.

An in vitro stimulation protocol has been established which allows production of IgG-secreting murine hybridomas. This procedure has been examined using jack bean urease and human luteinizing hormone as antigens. Parameters which have been optimized include selection of media and serum supplements, thymocyte-conditioned media, antigen dosage, length of stimulation and the effect of medium changes during stimulation and additions of polyclonal mitogen. Murine spleen cells (1 X 10(8) in 10 ml) were incubated with varying doses of jack bean urease and human luteinizing hormone in a six-well plate in supplemented DMEM with 5% normal rabbit serum and 10% thymocyte-conditioned media. Following 5 and/or 8 days stimulation, the spleen cells were fused with SP2/0 cells and plated in 96-well plates. Stable hybridomas were obtained for both antigens from over 25% of the wells identified in initial screening for specific antibody production. All monoclonal antibodies obtained in the LH stimulation experiments, with one exception, were of the IgM isotype. A large number of IgG-producing hybridomas were isolated following prolonged (8 day) stimulation with high concentrations of urease, during which time the medium remained unchanged. Addition of polyclonal mitogen (E. coli lipopolysaccharide) at 10 micrograms/ml markedly increased the production of hybridomas secreting anti-urease, but most were of IgM class.