EBV transformation and microfusion as the potential source of human monoclonal antisperm antibodies

Peripheral blood lymphocytes were obtained from vasectomized men with high serum titers of antisperm antibodies. An Epstein-Barr virus (EBV) transformation was performed either with B cells or mononuclear leukocytes. The effect of feeder cells (irradiated umbilical cord blood lymphocytes), cyclosporin A, and in vitro stimulation of lymphocytes with sperm extract on EBV transformation was evaluated. Antibody-producing cells were screened for specificity against human sperm by an enzyme-linked immunosorption assay (ELISA) one to six weeks after transformation. Using B cells or leukocyte mononuclear cells, we found that the percentage of wells containing antibody reactive against human sperm was greatest two weeks after transformation (range 3% to 7.5% positive wells). To increase and maintain antibody synthesis by these transformed cells, microfusions were performed in those wells positive for antisperm antibody using the UC 729-6 lymphoblastoid cell partner. Then resultant hybridomas were expanded, subcloned, and preliminarily characterized.