Convertase composition and fluid-phase convertase induced activation of the terminal pathway. (A) Binding of C3 or C3b onto immobilized C3b. A total of 2600 RU of biotinylated C3b (at thioester moiety) was immobilized onto a streptavidin SAP sensor chip and C3 (0.6 µM) or C3b (0.6 µM) were probed for binding. (One typical assay of 2 independent assays is shown.) (B) Binding of C3 or C3b to on chip assembled convertases. 4600 RUs of C3b were immobilized onto a CMD sensor chip. Then a mixture of factor B and factor D were injected (from 20 to 200 seconds) to facilitate the formation of C3bBb convertases before evaluating the binding (at 260-440 seconds) of C3b or C3 to C3bBb. At 740 seconds, the convertases were decayed by injection the decay accelerator CR1(1-3) at 1 µM. (One typical assay of 2 independent assays is shown.) (C-D) Effects of soluble complement components and fluid phase C3bBb convertases on nonopsonized and C3b opsonized rRBC. (C) Washed and nonopsonized and (D) highly dense C3b-opsonized rRBC were mixed with physiological concentrations of a C5-C9 mix and either PBS (red) or a separately prepared fluid-phase convertase C3bBb stored in EDTA buffer (green). The separately prepared soluble C3bBb was assembled by incubating soluble C3b with factor B and factor D in PBS supplemented with 5 mM MgCl₂ for 2 minutes before stopping de novo convertase formation by supplementing EDTA to 10 mM. Incubation of cells in water (dark gray) served as reference and was set to 100% lysis; cells incubated in PBS are shown in orange. Released hemoglobin was measured as a marker of hemolysis. The average of 3 independent assays with SD is shown. An ordinary 1-way ANOVA using Tukey's multiple comparisons test was applied. (E-F) Effects of soluble complement components and fluid phase C3bBb convertases on antibody sensitized shRBC that either were or were not opsonized with C4b; otherwise as in panels C and D. ns, not significant.