Conjugation of fluorescein isothiocyanate to antibodies. I. Experiments on the conditions of conjugation.

Abstract

In the present study experiments concerning the conjugation of fluorescein isothiocyanate (FITC) to antibodies, which were previously performed by Goldstein, Slizys and Chase (1961), McKinney, Spillane and Pearce (1964) and Wood, Thompson and Goldstein (1965) are compiled, reproduced, extended and discussed. The results of the experiments indicate that if a relatively pure IgG, obtained by DEAE Sephadex chromatography, is conjugated with FITC of high quality, a maximal molecular fluorescein/protein (F/P) ratio is reached in a short time if reaction temperature, pH and protein concentration are high. Maximal labelling was obtained in 30–60 minutes at room temperature, pH 9.5 and an initial protein concentration of 25 mg/ml. The separation of optimally labelled antibodies from under- and over-labelled proteins may be achieved by gradient DEAE Sephadex chromatography. Electrophoretically distinct IgG molecules proved to have about the same affinity for FITC. A correlation between the activity of antibodies in fluorescent and precipitation techniques was found.