FIG. 9.

Exogenous C1 reconstitutes hemolytic activity and C3 activation for CP-treated NHS. (A) NHS was incubated alone, with 6.3 μg CP, or with 1 μg CVF for 1 h at 37°C. Heat-inactivated NHS (HI NHS) was used as an additional control. After the incubation, 2 μg of C1 or 10 μg BSA was added to the indicated samples. Sensitized RBCs were then added to all samples, and hemolysis was determined. (B) NHS was incubated alone, with 9 μg CP, or with 1 μg CVF for 1 h at 37°C. After the incubation, 3 μg C1 was added to the indicated samples, followed by the addition of 25 μl of zymosan to all samples for 10 min at 37°C. Samples were washed and incubated with 25 mM methylamine for 1 h at 37°C to remove bound C3 fragments, and the supernatant was collected. A C3 ELISA was performed on the samples, using a polyclonal antibody to C3, and a standard curve was utilized to determine the value of C3. For both experiments, data are the means for four independent experiments. Error bars denote SEM.