Factors Affecting the Degree of Nonspecific Staining Given by Fluorescein Isothiocyanate Labeled Globulins

Abstract

Experimental data showed that nonspecific staining (NSS) as measured by the tissue culture cell technique increased in a linear fashion as the fluorescein to protein ratios (F/P) increased. Similar results were found at levels of conjugated protein ranging from 0.5 to 2.5%. When the same data were plotted without regard to protein concentration, it was evident that NSS was related directly to the fluorescein isothiocyanate (FITC) content of the conjugates. The F/P ratios were misleading when used to judge the NSS characteristics of conjugates since reagents having the same F/P ratios may differ widely in FITC concentration. It was shown that the NSS values given by conjugates prepared with some impure dyes were considerably higher than those of conjugates labeled with pure dyes. Globulins labeled by the dialysis method for short periods of time with the purer dye preparations yielded conjugates which gave markedly reduced NSS but whose specific staining titers were comparable to those of reagents prepared by routine methods. If conjugates of sufficiently high specific titer can be obtained, dilution is the simplest and most effective way of reducing or eliminating NSS as measured in these studies. Isomer I and II of FITC vary in respect to the nonspecific staining given by their respective conjugates depending upon the filter system employed.