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

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 surfaceassociated 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 (13IX). At a molar ratio 2:1 of 12si-ll :mAb all tested mAb enhanced binding of H to both activatorand 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.

Exceptional Resistance of Human H2 Glioblastoma Cells to Complement-Mediated Killing by Expression and Utilization of Factor H and Factor H-Like Protein 1

“…Because factor H has multiple binding sites for C3b (34,50), a polyclonal Ab or a combination of antifactor H mAb is needed for its complete neutralization. Using a combination of two anti-factor H mAbs (131X and OX24) together with the YTH53.1 mAb in a cytotoxicity assay lysis of H2 cells increased 5-fold.…”

“…For neutralization experiments, we used two Abs. The 131X mAb binds to factor H, but not to FHL-1, and inhibits partly binding of factor H to C3b (34). The OX24 mAb binds to SCR 5 of both factor H and FHL-1 and inhibits partly binding of factor H/FHL-1 to C3b.…”

“…By immunoblotting with the 196X mAb, which detects both factor H and FHL-1 by binding to the most N-terminal SCR of both molecules (34), H2 cells were shown to produce both the 150-kDa factor H and the 42-kDa FHL-1 protein (Fig. 5B).…”

Each of the Three Binding Sites on Complement Factor H Interacts with a Distinct Site on C3b

“…Francisco, CA) was used as the secondary antibody, respectively. After three washing steps, the bound secondary antibody was detected by adding the standard alkaline phosphatase substrate (38).…”

“…The other two binding sites have not yet been well characterized. Results obtained by using either several anti-fH mAbs (38) or deletion mutants lacking [5][6][7][8][9][10] SCR domains (39) showed that there is more than one binding site on fH for C3b. One of the two new sites was localized to and the other was suggested to lie within .…”