Preparation and functional properties of polyclonal and monoclonal antibodies to murine MD-1

Abstract

Rabbits, rats and hamsters were immunized with KLH-coupled synthetic peptide sequences of the murine MD-1 molecule. Serum from immunized animals bound in Western gels to a 25 KDa protein extracted from LPS stimulated mouse spleen cells, as did a rat hybridoma (SH1.2.47) prepared from peptide-immunized rats. CHO cells transfected with a plasmid cDNA construct encoding murine MD-1, the target antigen for the antibodies in question, were also stained (in FACS) by the same antibodies. Patching and capping of the antigen(s) detected by any one of these sera abolished binding of all antibodies in subsequent FACS analysis, consistent with the hypothesis that they all detected the same antigen. In a final study to assess the possible involvement of MD-1 in regulation of cell activation for cytokine production following allostimulation, we found that all of the antibodies inhibited IL-2 and IFNγ production, while enhancing IL-4 and IL-10 production, in mixed leukocyte reactions (MLR) in vitro.