IgG and complement bind to nHZ in FG-independent manner but do not elicit the full ROS, TNF, and MCP-1 response in the absence of native FG. (A) IgG and complement binding to HZ during opsonization. HZ obtained from the supernatants of either plasma- (nHZ, lane 1), serum- (HZ SERUM, lane 4), or Albumax- (HZ ALBUMAX, lane 5) supplemented cultures of P falciparum or nHZ digested with plasmin (HZ PLASMIN, lane 2) or denatured by heating at 100°C for 5 minutes (HZ 100°C, lane 3) were opsonized with freshly drawn serum for 30 minutes at 37°C. One representative blot of 3 with similar results is shown. Binding of FG, the opsonins IgG (IgG heavy chain, IgG light chain), and complement (C3β-chain) to the different HZ types was analyzed by Western blotting of extracted and 10% SDS-PAGE separated proteins with specific anti-FG γ-chain, anti-IgG, and anti-C3c Abs. Proteins from 50nmoles, 10nmoles, and 10nmoles HZ (in terms of heme content) were analyzed for FG, both IgG chains, and complement, respectively. (B) FG is essential for full ROS, TNF, and MCP-1 release. IgG and complement alone are no substitutes for FG. Full responses as seen with opsonized FG-containing HZ are indicated with ‘+’ and significantly lower or no responses with ‘-’ or ‘- -’ (see Figure 4 for numeric data on which this qualitative summary is based); n.d. indicates not determined.