Fc Site-Specific Labeling of Immunoglobulins with Calf Intestinal Alkaline Phosphatase

Abstract

A strategy is described for the site-specific conjugation of alkaline phosphatase to the Fc region of immunoglobulins. Mild oxidation of immunoglobulins by sodium periodate followed by reductive amination with excess cystamine and sodium cyanoborohydride covalently attaches cystamine specifically to the oligosaccharide chains of the antibody's Fc region. The disulfide bonds of the conjugated cystamine moieties are then reduced to free thiols with a low concentration of dithiothreitol. The thiol-modified antibodies (typically containing three to four thiols per Fc region) are then brought into contact with maleimide-functionalized alkaline phosphatase to form antibody—alkaline phosphatase conjugates. The Fc site-specific conjugation methodology was optimized and successfully applied to a number of different monoclonal and polyclonal antibodies. The conjugates prepared show assay performances superior to those prepared by more conventional means. Evidence obtained indicates that this superior conjugate performance results from total retention of the antibody's antigen binding activity following the Fc site-specific conjugation procedure. © 1994, American Chemical Society. All rights reserved.