SPR analysis of C5 binding to C3b. (A) Binding of C5 and mini-FH to C3b immobilized on a carboxymethyldextran-coated sensor chip by amine coupling (800 RUs). Mini-FH at a concentration of 140 nM or C5 at 100 nM was applied to the C3b surface (reference-subtracted responses are shown throughout). (B) C5 binding to “physiologically,” convertase-immobilized C3b. C3 convertases were assembled onto the amine coupled C3b sensor chip followed by flow of C3, which becomes activated in situ and binds covalently. According to this procedure, another 900 RUs of C3b were immobilized in a physiological manner via the convertase onto the 800 RUs of amine-coupled C3b. Mini-FH was assayed at 1.5 µM in 2 consecutive injections (2 overlaid dark blue curves), followed by C5 at 1.1, 0.6, 0.3, and 0.1 µM (black curves) and then another injection of mini-FH at 1.5 µM (cyan). The lower amount of RUs achieved for the third injection of mini-FH indicates that the C3b surface was not completely regenerated after being exposed to higher C5 concentrations. (C) As in panel B, but analytes were injected in the following sequence: mini-FH at 140 nM, C5 at 110 nM mixed with both eculizumab at 1.7 µM and coversin at 1.7 µM (C5/eculizumab /coversin), C5/eculizumab (110 nM:1.7 µM), C5/coversin (110 nM:1.7 µM), and C5 alone at 110 nM. The C5 inhibitors themselves did not bind to the sensor chip (not depicted). Ecu, eculizumab.