Figure 5.
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.)

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.)

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