Optimization of an ovine antibody-secreting cell assay for detection of antigen-specific immunoglobulin production in peripheral blood leukocytes.

Enumeration of antibody-secreting cells in peripheral blood by enzyme-linked immunospot (ELISPOT) has been used in human studies to detect antigen-specific antibody production at mucosal tissue sites. An alternative assay for detecting and quantitating antigen-specific antibody responses involves culturing circulating peripheral blood antibody-secreting cells and quantitating specific antibody production in culture supernatant by ELISA. In the present study, antigen-specific peripheral blood lymphocytes were isolated from subcutaneously immunized sheep and the parameters for maximizing in vitro antibody production by in vivo-induced antibody-secreting cells optimized for this species. Maximum antibody-secreting cell responses were observed in peripheral blood collected four days after antigen challenge. The addition of lipopolysaccharide and antisheep immunoglobulin had no effect on in vitro antibody secretion by blood antibody-secreting cells, while the effects of pokeweed mitogen were highly variable. However, the combination of anti-Ig and recombinant ovine interleukin-6 to peripheral blood lymphocyte cultures was found to markedly and consistently enhance specific antibody production. In unstimulated cultures, the optimal peripheral blood lymphocyte concentration for generating the greatest antibody responses was 5.0 x 107 cells per mL, but in cultures stimulated with recombinant ovine interleukin-6/antisheep immunoglobulin, the optimal cell concentration was lowered to approximately 1.0 x 107 cells per mL. In vitro, peak immunoglobulin production was usually achieved by day one in unstimulated cultures. In recombinant ovine interleukin-6/antisheep immunoglobulin-stimulated cultures, antibody levels were similar to unstimulated cultures by day one, however, the levels continued to rise during incubation to reach a maximum between days four and five of incubation. This optimized antibody-secreting cell culture assay is amenable for increasing the sensitivity and reducing the cell numbers required for quantitating antigen-specific antibody induction in large-scale immunization trials in sheep and other large animal species.