Conformational activation of C5. (A) Presence of C5 on highly dense C3b opsonized rRBCs. Nonopsonized cells (control, green) and C3b opsonized cells (dark blue) were mixed with C5-C7 and stained with a fluorescein isothiocyanate (FITC)-conjugated anti-human C3c mAb as a measure for C3b opsonization (first panel), a biotinylated anti-human C5a Ab (second panel) or simultaneously with both antibodies (third panel). C5 detection was achieved by fluorescence-labeled eculizumab as well (fourth panel). Fluorescence signals were measured by flow cytometry. (One typical assay of 3 independent assays is shown.) (B) Only rRBCs with the highest C3b deposition lysed after allowing lysis. C3b-opsonized cells from panel A carrying C5-7 complexes were exposed to C8 and C9 to enable whole assembly of MAC. After allowing lysis, the remaining cells were stained again with the antibodies (cyan). For better comparison, C3b-opsonized cells (dark blue) from before lysis are shown again. (One typical assay of 3 independent assays is shown.) (C) C5-9 binding to C3b-coated SPR sensor chip. A total of 2000 RUs of biotinylated C3b was immobilized on an SAP sensor chip followed by consecutive injections of buffer, C6-C9, C9-neoAb (anti-C5b-9 mAb), control Ab (anti-6x-His-tag mAb), C5, C5-C9, C9-neoAb, and control Ab. All complement proteins had a concentration of 0.375 µM, the antibodies were injected at 0.05 mg/mL. An overlay of the C5 binding curve (red) and the C5-C9 binding curve (green) was done to point out differences between binding signals. (One typical assay of 2 independent assays is shown.)