In vitro stimulated lymphocytes as a source of human hybridomas.

Stimulation of peripheral blood lymphocyte (PBL) cultures from tetanus toxoid (TT)-immunized donor with Epstein-Barr virus (EBV) yielded cells with much higher frequencies of hybrid formation (36 X 10(-7) compared to unstimulated PBL or cells cultured with pokeweed mitogen or TT antigen. The proportion of hybridomas (approximately 1%) producing anti-TT antibody was similar in EBV- and TT-stimulated cultures. A marked increase in immunoglobulin secretion was observed after hybridization and preselection of EBV subcultures for high anti-TT production prior to fusion resulted in a fivefold increase in TT-specific hybridomas (p less than 0.001). Most (20/21) specific hybrids produced IgM anti-TT, whereas one (1/21) produced IgG anti-TT, possibly due to the immature stage of differentiation in EBV-stimulated parental cells. The ability to choose an antigen, immunize a human subject and expand the rare antigen-specific B cells from PBL, in vitro, with EBV, prior to fusion, should yield an increasing spectrum of human monoclonal antibodies for diagnostic, therapeutic or basic studies.