Conjugation of fluorescein isothiocyanate to antibodies. II. A reproducible method

Abstract

A reproducible method for conjugation of fluorescein isothiocyanate to antibodies, based on previous experiments, is described in detail. The negative electrical charge of antibodies was found to increase by conjugation. This negative charge may cause the non-specific fluorescence of acidophilic tissue components if overlabelled antibodies are used. Even non-specific nuclear fluorescence was observed when the substrate was previously washed in phosphate buffer. This was probably caused by acid extraction. Underlabelled antibodies showed a weak specific fluorescence. Antibodies with molecular fluorescein/protein ratios between 1 and 4 or `optical density ratios' (E₄₉₅¹:E₂₈₀¹) between 0.3 and 0.95 were found to be optimal. However, even antibodies conjugated to an optimal degree may demonstrate non-specific fluorescence if used in high concentrations. Restricted preservation testing of reconstituted lyophilized conjugates revealed a negative effect of exposure to daylight at room temperature.