Short time bleaching of fluorescein isothiocyanate. A possible parameter for the specific binding of conjugates in immunofluorescence.
The fluorescence kinetics of fluorescein isothiocyanate (FITC)-antibody conjugates during short (0.01 sec) laser excitations were analyzed for possible information about the immunological specificity of binding to antigenic substrates in direct immunofluorescence assays with antigen-coated Sephadex beads. First results of these investigations suggest marked differences in the bleaching characteristics of specifically and nonspecifically bound FITC conjugates: FITC-anti-bovine serum albumin (BSA) and FITC-anti-rabbit immunoglobulin (Ig) were found to fade significantly more slowly when bound to the respective homologous antigen (anti-BSA to BSA, and anti-rabbit Ig to rabbit Ig) as compared to nonspecifically adherent anti-BSA to rabbit Ig, and anti-rabbit Ig to BSA. Possible implications of these data for discrimination of nonspecific staining in immunofluorescence are discussed.
D000941:Antigens / Q000032:analysis*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D012710:Serum Albumin, Bovine; D013861:Thiocyanates
D000941:Antigens; D005452:Fluoresceins; D013861:Thiocyanates; D012710:Serum Albumin, Bovine; D016650:Fluorescein-5-isothiocyanate