Evaluation of Commercial Fluorescein Isothiocyanates Used in Fluorescent Antibody Studies

Commercial preparations of fluorescein isothiocyanate (FITC) for immunofluorescence applications were obtained from 12 sources and examined for purity by quantitative infrared spectrophotometry and by labeling efficiency for bovine serum albumin (BSA). Quantitative photometric measurements were made of nonspecific staining (NSS) produced by conjugates prepared from the dyes. The purity of FITC from different sources was highly variable. The risk of NSS appears to increase as the purity of the dye decreases. In immunofluorescence applications it is desirable to use the purest FITC available in order to obtain conjugates with minimum NSS. It is recommended that 70% FITC, as determined by BSA labeling efficiency, be accepted as the minimum purity for immunofluorescence applications.