Analysis of the recognition mechanism of the alternative pathway of complement by monoclonal anti‐factor H antibodies: evidence for multiple interactions between H and surface bound C3b

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Abstract
The ability of the alternative pathway of complement to discriminate targets as either activators or non‐activators is mediated by different binding properties of factor H to surface‐associated C3b molecules. In the present study we have probed the interaction between H and C3b using five anti‐H mAb. The binding sites of the mAb were mapped by Western blotting using both recombinant and trypsin‐generated H fragments. Two mAb bound to CCP1 (90X, 196X), two to CCP5 (MRC OX24, 86X) and one to CCP8‐15a (131X). At a molar ratio 2:1 of 125I‐H:mAb all tested mAb enhanced binding of H to both activator‐ and non‐activator‐bound C3b. At higher concentrations two mAb had an inhibitory effect on H binding to surface‐associated C3b (OX24, 131X). Thus the mAb 131X inhibits H binding to surface‐bound C3b but unlike OX24 it does not bind to the previously described C3b binding site within or near CCP4‐5. These results indicate that there is an additional interaction site on factor H for surface‐bound C3b.