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In vitro immunization: generation of neutralizing monoclonal antibodies to human interleukin-10
1Laboratory of Infectious Diseases, University Children's Hospital, Zurich, Switzerland.
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 micrograms/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.
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 micrograms/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.