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Short time bleaching of fluorescein isothiocyanate. A possible parameter for the specific binding of conjugates in immunofluorescence

K Schauenstein, G Bock and G Wick

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.

Volume 28, Issue 9, pp. 1029-1031, 09/01/1980
Copyright © 1980 by The Histochemical Society


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