Determination of membrane antigens by a covalent crosslinking method with monoclonal antibodies.
Monoclonal antibodies that recognize cell surface proteins may serve as very useful tools for the study of the biological functions of membrane proteins. However, solubilization of the antigens with detergents may lead to major conformational changes of the protein, making their determination with monoclonal antibodies by immune blot or ordinary immunoprecipitation methods difficult. This is especially evident when the monoclonal antibodies recognize tertiary structures of the proteins in the membrane. We have generated two monoclonal antibodies which are specific for the cell surface antigens of multidrug-resistant human cell lines. However, the antigens of both monoclonal antibodies were difficult to detect by either immune blot or ordinary immunoprecipitation methods. We used a cleavable crosslinking reagent dithiobis(succinimidyl propionate) to covalently link the monoclonal antibody with its antigenic determinant in the membrane of intact cells. By this method, we were able to detect the antigens for these two monoclonal antibodies following solubilization, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This method should have wide applicability in determination of membrane antigens recognized by monoclonal antibodies when immune blot or ordinary immunoprecipitation methods are not successful.
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000302:isolation & purification; D000954:Antigens, Surface / Q000032:analysis*; D002460:Cell Line; D011232:Chemical Precipitation; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007938:Leukemia / Q000276:immunology; D012967:Sodium Dodecyl Sulfate; D012995:Solubility; D013388:Succinimides
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000954:Antigens, Surface; D003432:Cross-Linking Reagents; D013388:Succinimides; D012967:Sodium Dodecyl Sulfate; C011240:dithiobis(succinimidylpropionate)