IN-VITRO IMMUNIZATION - GENERATION OF NEUTRALIZING MONOCLONAL-ANTIBODIES TO HUMAN INTERLEUKIN-10

The potential of the in vitro immunization technique to evoke an immune response against an immunomodulatory protein was evaluated using, as antigen, human interleukin-10 (IL-10), a novel cytokine with pleiotropic effects on human and murine lymphocytes and macrophages. After pre-priming the support cells for 48 h and subsequent 3-day stimulation of splenocytes from a non-immune BALB/c mouse with recombinant human IL-10 (rhIL-10; 2 mu g/ml), significant stimulation of splenocytes was observed. 7 days after fusion with the non-secreting myeloma line X63/Ag8.653, IL-10-specific antibodies were detected by ELISA and dot blot in more than 70% of the hybridoma supernatants. After limiting dilution of the hybridoma cells showing IL-10-neutralizing activity in a bioassay using murine MC/9 mast cells, the isotype of the monoclonal antibodies (mAbs) obtained was 20% IgM, 16% IgG and 6% IgA. All other antibodies elicited IgM as well as IgG isotypes. The neutralizing activity of the specific mAbs tested was dose-dependent. Our results show that in vitro immunization can be employed successfully to generate functional mAbs to immunomodulatory proteins, even if these exhibit cross-species activity.