BIOTINYLATION - A NONRADIOACTIVE METHOD FOR THE IDENTIFICATION OF CELL-SURFACE ANTIGENS IN IMMUNOPRECIPITATES

A nonradioactive method was employed to detect different cell membrane antigens on human polymorphonuclear granulocytes, monocytes and plate lets. We compared the reactivity of one monoclonal antibody, N1III10, assumed to be Fc gamma RII-specific by functional assays, with other w ell-characterized monoclonal antibodies and human sera. Intact cells w ere incubated with biotin N-hydroxysulfosuccinimide ester which prefer entially reacts with lysine residues in polypeptides. Biotin-labeled c ells were lysed and the antigen was isolated from the cell lysate by i mmunoprecipitation with the antibody bound to Protein A-Sepharose. The precipitates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and vis ualized by a streptavidin-alkaline phosphatase system with a suitable substrate. Using this biotin-labeling system we could show that N1III1 0 detects a 40 kDa antigen on monocytes and platelets, comparable to t hat expected of Fc gamma RII monoclonal antibodies.