The generation of human hybridomas using B lymphocytes and the K6H6/B5 heteromyeloma (mouse x human) requires that the lymphocytes be in active division. Because most lymphocytes in the normal B-cell repertoire are in a resting state, polyclonal B-cell activators (PBA) are generally used before fusion. The two PDAs most commonly used are EBV and pokeweed mitogen (PWM). The infection of lymphocytes with EBV results in blast formation, Ig secretion and, ultimately, immortalization of the cells. PWM induces blastogenesis resulting in the increase of mitotically active B lymphocytes. The objective of this study was to compare the efficacy of the fusion of PBLs after either EBV transformation or stimulation with PWM. PBLs from 3 different patients immunized with a cancer vaccine were separated by gradient centrifugation, and the resultant lymphocytes were treated with EBV or PWM. EBV transformation was accomplished by exposing the lymphocytes to the supernatant of the B95-8 marmoset lymphoblastoid cell line. After overnight incubation, cells were washed and kept in culture until fully transformed. PWM stimulation was achieved by incubation of lymphocytes in the presence of PWM (10 μg/ml) for 48 h. EBV transformed or PWM stimulated PBL were fused with K6H6/B5 cells using poly(ethylene glycol) (1500) at a 1:1 ratio. The cells were then placed at 5 × 104 cells per well in RPMI-1640 media (+15% FBS, 1% hybridoma cloning factor, ORIGEN®) followed by HAT/ouabain selection. Hybridomas producing antibodies reactive with the specific antigen (radioimmunoassay) were cloned by limiting dilution. Fusions after EBV transformation showed growth in 99100% of the plated wells (hybrid frequency of 400/107 lymphocytes), with 90-100% of the hybrids producing Igs, and these were mainly IgM (%IgG/%IgM: 25.3/99, 0.7/100, 0/99). The cloning efficiency was 78-96%, with 16-65% of the clones remaining Ig producers. Fusions after PWM stimulation exhibited growth in 67-97% of the plated wells (hybrid frequency of 256-388/107 lymphocytes) with 71-80% of the hybrids producing Igs from both classes (% IgG/%IgM: 50/38, 64/55, 87/72). The cloning efficiency was 64-74% with 8-50% of the clones remaining Ig producers. Although it is possible to generate stable hybridomas from both EBV and PWM stimulatory systems, the EBV transformed lymphocytes gave higher hybrid frequency and better cloning efficiency with a larger number of hybrids remaining Ig producers as compared to the PWM stimulated lymphocytes.
|Original language||English (US)|
|Journal||Human Antibodies and Hybridomas|
|State||Published - 1996|
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