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 platelets. We compared the reactivity of one monoclonal antibody, N1III10, assumed to be Fc gamma RII-specific by functional assays, with other well-characterized monoclonal antibodies and human sera. Intact cells were incubated with biotin N-hydroxysulfosuccinimide ester which preferentially reacts with lysine residues in polypeptides. Biotin-labeled cells were lysed and the antigen was isolated from the cell lysate by immunoprecipitation with the antibody bound to Protein A-Sepharose. The precipitates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and visualized by a streptavidin-alkaline phosphatase system with a suitable substrate. Using this biotin-labeling system we could show that N1III10 detects a 40 kDa antigen on monocytes and platelets, comparable to that expected of Fc gamma RII monoclonal antibodies.
Flesch
B
B
Institute of Transfusion Medicine, University of Kiel, Germany.
Ntambi
E
E
Neppert
J
J
D000469:Alkaline Phosphatase; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001710:Biotin / Q000737:chemistry*; D001792:Blood Platelets / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006098:Granulocytes / Q000276:immunology; D015395:Histocompatibility Antigens Class I / Q000032:analysis*; D006801:Humans; D007962:Leukocytes / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009000:Monocytes / Q000276:immunology; D011233:Precipitin Tests / Q000379:methods; D017452:Receptors, IgG / Q000032:analysis*
D000911:Antibodies, Monoclonal; D015395:Histocompatibility Antigens Class I; D017452:Receptors, IgG; D001710:Biotin; D000469:Alkaline Phosphatase