ROLE OF LOCAL ANTIBODY DENSITY EFFECTS ON IMMUNOSORBENT EFFICIENCY

This study evaluates the effect of immobilized antibody density on the performance of an immunosorbent. In contrast to previous studies that emphasize the correlation of high volume averaged antibody density wi th immunosorbent performance, we have studied the effects of locally h igh antibody density and spatial distribution on the antigen binding e fficiency under conditions of dynamic loading and elution. The distrib ution of an anti-human Protein C monoclonal antibody immobilized on 3M Emphaze AB1 Biosupport Medium was evaluated. The distribution of immo bilized antibody was controlled by a two-step sequence of permeation a nd reaction. Labeled antibody was visualized by immunofluorescence. Co nditions of low pH, low temperature, and the presence of a competitor nucleophile sufficiently depressed the Thiele modulus for coupling to enable permeation of the antibody. The adsorption of the permeated ant ibody was enhanced by the presence of 0.75 M Na2SO4, and then the pH w as raised to achieve rapid covalent coupling. Bead-averaged antibody d ensities of 1-11 mg/ml of hydrogel support were studied. Immunosorbent s containing more evenly distributed antibody gave a two- to three-fol d greater antigen binding efficiency than those with locally high anti body densities. No appreciable changes in mass transfer characteristic s were observed using breakthrough analysis for immunosorbents with di stributed versus locally high antibody density.