FIGURE 3.

iC3b deposition on isogenic Cap+ and Cap− meningococci. The five isogenic Cap+ and Cap− pairs of N. meningitidis were incubated with C2-depleted human serum for either 10 or 30 min and bacteria-bound iC3b was analyzed by Western blotting with anti-iC3b mAb G-3E. The position of the α₁′ (68 kDa) fragment of C3 is indicated in the control lane that contains purified iC3b (see text for an explanation of the α₁′ “doublet”). Both blots (10 min (left) and 30 min (right) incubations) were exposed to the alkaline phosphatase substrate for the same duration. “α₁′ + L” and “α₁′ + O” indicate the positions of adducts of LOS or opacity-associated protein covalently linked to the α₁′ iC3b fragment, respectively (36, 46, 47). The asterisks in the “10-min blot” indicate high-molecular mass complexes that contain iC3b in the lanes containing Cap+ W and Y strains. Proteins on the membrane migrating below ∼40 kDa were stained with Coomassie Blue and served as a loading control. Cap+ and Cap− derivatives of strain W171 were incubated with heat-inactivated C2-depleted serum (lanes marked W + HIS) and served as controls to validate the specificity of covalently bound C3 fragments to bacteria.