Evaluation of biotinylated cells as a source of antigens for characterization of their molecular profile

Biotinylated lymphoid cells have been suggestedas a useful source of antigen for the immunochemicalcharacterization of their molecular profile. Labellingwith biotin eliminates the problems associated with the useof radioactivity. However, this method has not been widelyused. This reflects: (1) difficulties in optimizing the signal/background ratio because of the lack of a simplemethod to quantify biotinylated proteins in a cell lysate,(2) the loss of reactivity with monoclonal antibody of antigenfollowing biotinylation, because of steric hindrance,and (3) the lack of information about the utility of otherbiotinylated cells as an antigen source. To overcome theselimitations, we developed an ELISA to quantify biotiny-Iated proteins in cell lysates and optimized the signal/backgroundratio. The validity of this approach was confirmedby sodium dodecyl sulfate-polyacrylamide gel electrophoresisof a number of cell surface antigens immunoprecipitatedfrom lymphoid cells by an optimal amount of monoclonalantibody. Furthermore, we showed that biotinylatedmelanoma cells are a useful source of antigen for immunoprecipitationexperiments and that ligation of biotin toantigen does not affect reactivity with monoclonal antibody.Lastly, biotinylated antigens in cell lysates stored at- 80 °C for 6 months maintained their reactivity with monoclonalantibodies. Biotinylated cells thus represent a usefulsource of antigen for characterizing the immunochemicalprofile and analyzing the specificity of antibodies withimmunochemical methods.