Characterization of high-affinity antifluorescyl IgM antibodies

High-affinity IgM rabbit antibodies were elicited using the fluorescein hapten system. Purity and identification of IgM and IgG anti-fluorescyl antibodies was determined by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis. Ultracentrifugation studies verified that liganded antibodies were of a high mol. wt (901 kd). Comparative analyses of IgM and IgG anti-fluorescyl antibody active sites substantiated the observation that purified IgM preparations, similar to their IgG counterparts, possessed high-affinity antibody active sites. Dissociation rate data confirmed that some IgM molecules within the purified antibody populations possessed association constants at 10 11 M −1, comparable with values of 10 11 M −1 or greater obtained for some anti-fluorescein IgG populations studied in this laboratory. A diffusion-controlled association rate of 4.6 × 10 8 M −1 sec −1 (Levison et al., 1971) was assumed in dissociation rate calculations. This is the first report of purified IgM antibodies possessing exceptionally high affinities.