Factors Affecting the Rate of Reaction of Fluorescein Isothiocyanate with Serum Proteins

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Abstract
Summary: Many of the commercial fluorescein isothiocyanate (FITC) products are of a low degree of purity. Their use as reference standards for spectrophotometric measurements in determining fluorescein:protein ratios of labeled globulin is shown to lead to very appreciable error. The extent of reaction of FITC products with rabbit γ-globulin under standardized conditions showed very close correlation with the purity of the isothiocyanate products as determined by infrared measurements. Rabbit and bovine α-globulin were found to react considerably more rapidly than horse α-globulin with FITC. In all species studied, the albumin fraction reacted much more rapidly than the γ-globulin fraction. A systematic study of the effects of temperature, concentration and pH on the rate and extent of reaction of FITC with normal rabbit γ-globulin indicated that the rate can be greatly accelerated by modifying the reaction conditions. At the reaction temperature of 25°C, pH 9.5, protein concentration 2.5% and buffer salt concentration of 0.05 M, conjugation of FITC with rabbit γ-globulin was essentially completed in 30 min. With these reaction conditions, the fluorescein: protein ratio of the conjugate was very reproducible and could be controlled by the initial amount of dye added. The reaction of FITC with immune rabbit γ-globulin at 25°C and pH 9.5 gave results which were comparable to those obtained with normal rabbit γ-globulin. Antibody properties, as determined by staining titer, were not adversely affected.

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