FIGURE 14.

HS-coated ELISA (A) and effect of FH on C3bB(Ni²⁺) C3 proconvertase and C3bBb(Ni²⁺) C3 convertase formation in the presence of HS (B) or SA (C) detected by microplate/WB assays. A, microtiter plates were coated with 3 or 30 μg/ml HS in PBS, in the presence or in the absence of 3 μg/ml C3b. Immobilized HS was detected with a monoclonal mouse anti-HS antibody (1:100) followed by HRP-conjugated goat anti-mouse antibody (1:2000). Values are given as the OD averages with standard deviation (n = 3 each). B and C, C3bB(Ni²⁺) and C3bBb(Ni²⁺) complexes were obtained by incubating C3b and HS-coated (B) or SA-coated (C) (3 or 30 μg/ml) wells with FB (1000 ng/ml), FD (5 ng/ml), and NiCl₂ (2 mm) at 37 °C for 30 min, in the presence or in the absence of FH (2640 ng/ml). The amount of C3bB or C3bBb formed was calculated as the intensity of the B (93 kDa) or Bb (60 kDa) bands, respectively, and reported in the bottom graphs as pixel²·10⁶. FH band (150 kDa) could be visualized in the WB. Results of a representative microplate/WB experiment of n = 3 are shown.