Determination of intrinsic association constant of mouse monoclonal IgM antibody to human hepatitis B surface antigen

The ultracentrifugation technique utilizing Airfuge was employed to determine the intrinsic association constant of mouse monoclonal IgM antibodies to human hepatitis B surface antigen (HBsAg). The IgM antibody was reduced to Fab fragments by tryptic, digestion in the presence of mercaptoethanol, followed by two passages over a Sephacryl S-200 column. The centrifugation conditions were optimized to sediment Fab-HBsAg complexes, leaving the unbound Fab in the supernatant. The analyses of the binding data by Scatchard and Sips methods yielded good agreement, and the intrinsic association constants ranged from 0.13 to 5.3 × 10 7 (1/mol) for three IgM antibodies. The heterogeneity indices deduced from the Sips analysis were 1.0. The present protocol would allow determination of the intrinsic association constant for the binding of a multivalent antibody to a comparatively large antigen bearing multiple combining sites.