Rapid fluorescein and protein assay method for fluorescent-antibody conjugates

A method is presented for the rapid determination of the fluorescein content, protein content, and fluorescein-to-protein ratio for immune globulin conjugates with fluorescein isothiocyanate as the fluor. This method is based on the absorbance of the fluorescent antibody at those wavelengths primarily associated with the fluorescein and gamma-globulin fractions, and permits these materials to be determined by a single nondestructive analytical procedure. A small sample of the fluorescent antibody, in many cases 0.1 ml or less, is adequate for the above determinations. A nomograph is presented which allows simultaneous determination of the materials from the observed absorbance at the two wavelengths. The method is sufficiently accurate for most applications of the fluorescent-antibody techniques. Although this procedure has been developed primarily for fluorescent-antibody conjugates prepared from rabbit gamma-globulin, it can be used directly for antibodies prepared from other animals provided the gamma-globulin is relatively free from albumin.