Different C5 inhibitors reveal residual TP activity on foreign activating cells. (A) CP-mediated lysis of shRBCs. Hemolysin-treated shRBCs were mixed with 5% serum in the presence of specific inhibitors. Released hemoglobin was measured as a marker of hemolysis (the average of 3 independent assays with standard deviation [SD] is shown). (B) AP-mediated lysis of rabbit erythrocytes. Rabbit erythrocytes were incubated in 25% human serum in presence of inhibitors or controls (average of 3 independent assays with SD is shown). (C) Different combinations of C5 inhibitors protect rRBCs. As in panel B, but eculizumab and another commercial anti-C5 antibody (clone: 046-10.2.2) were used at concentrations that substantially exceed the typical C5 concentration of the serum dilution in this assay (average of 3 independent assays with SD is shown). (D-E) TP activity of serum from PNH patients on eculizumab. rRBCs were incubated in 25% human serum derived from 2 PNH patients (#A and #B) on eculizumab in absence or presence of additional complement inhibitors as indicated (average of 2 independent assays with SD is shown). (F) Determination of free C5 levels through sandwich enzyme-linked immunosorbent assay using eculizumab as capture antibody and a polyclonal anti-C5 as a detection antibody. Eculizumab deposited on the microtiter plate captures free C5 from NHS, but not when NHS has been premixed with 0.5 µM eculizumab. No free C5 was captured from patient serum or patient serum that had been diluted 1:1 (1-in-2) with NHS (average of 3 independent assays with SD is shown; the NHS/eculizumab mixture [negative control] was only assayed twice). Ecu, eculizumab; PAS-Cov., PASylated Coversin; PBS, phosphate-buffered saline; PBSE, PBS supplemented with EDTA (to 5 mM).