Studies on Fluorescent Antibody Staining II. Inhibition by Sub-Optimally Conjugated Antibody Globulins.

Abstract

This is a study of methods of preparing fluorescent antibody reagents that have maximal sensitivity and maximal immunological specificity. Essentially, the goal is to attach an optimal number of fluorescein isothiocyanate (FITC) radicals to antibody globulins. Attaching too many FITC groupings alters the antibody globulins and creates nonspecific staining. Attaching too few fluorescent groupings inhibits the staining brightness, and thereby the sensitivity of the fluorescent antibody technique. Optimal sensitivity and specificity have been obtained by the following procedure. Antibody globulins are obtained by ammonium sulfate precipitation and DEAE-cellulose chromatography. The globulins are conjugated with FITC at 8-10 mg FITC/g of protein. Unreacted FITC is removed by passage through a Sephadex column. The fluorescent antibody is then fractionated by gradient elution on DEAE -cellulose to separate optimally coupled globulin from under- and over- coupled antibody globulin.