Figure 1.
Effect of anti-FH.07 on in vitro AP complement activation. (A) AP-mediated C3b deposition on immobilized zymosan incubated with 10% (vol/vol) NHS in the presence of anti-FH.07 (binding CCP18) or anti-FH.09 (binding CCP6), as determined by ELISA. C3b deposition without addition of mAbs was set to 100%. (B) As in panel A, but with immobilized LPS instead of zymosan. (C) IC50 of intact or Fab′ fragments of anti-FH.07 in the AP-mediated C3b deposition assay on immobilized LPS. (D) Fluid phase regulation by FH was assessed with a fluid phase cofactor activity assay. Purified C3b was incubated with FH and FI, with addition of the indicated mAbs (anti-FH.11 binds CCP1-4). Cleavage of the α′-chain of C3b was visualized with SDS-PAGE run under reducing conditions, stained with PageBlue. Lanes were run on the same gel but were noncontiguous. (E) C3b/c concentration determined by ELISA after incubation of NHS with the indicated mAbs to assess fluid phase activation of C3. (F-G) Representative SPR sensograms (n = 2) of the binding of full-length FH (F) or a fragment of FH comprised of domains 18 to 20 (G) to immobilized anti-FH.07. For full-length FH, a twofold diluting concentration range starting at 200 nM was probed. For FH18-20, a twofold diluting concentration range starting at 150 nM was probed. Injections were done in random order. The first 200 seconds of the 600-second dissociation phase are shown. Red lines depict the fits of a 1:1 association model. Data in panels A-C,E are presented as mean of n = 3 with standard deviation. ****P < .0001, 1-way ANOVA. ns, not significant; RT, room temperature.