A ternary complex composed of CCP1-3, C4b, and PfRh4. (A) ELISA of ternary complex. Microtiter plates were coated with C4b (1 μg/well). After incubation with rPfRh4, CCP1-3, or both as indicated, bound rPfRh4 was detected using anti-PfRh4 monoclonal antibody 10C9 (black bars) that was raised against rPfRh4. Monoclonal antibody 2E8 (white bars) was raised to the C-terminal end of PfRh4 and recognizes native PfRh4 but not the region encompassed by rPfRh4. The inset shows the reactivity of these monoclonal antibodies in an ELISA using microtiter plates coated with rPfRh4. Data are the mean ± SD for 3 independent experiments. (B) Immunoprecipitation of ternary complex. Purified C4b, CCP1-3, or rPfRh4 were incubated together as indicated (at 0.02 mg/mL). Western blots were performed after immunoprecipitation with anti-PfRh4 10C9 (lanes 1-3) or 2E8 (lane 4) monoclonal antibody, respectively. Immunoprecipitated material was probed with a rabbit polyclonal anti-PfRh4 antibody, a goat polyclonal anti–human C4 antibody and a monoclonal anti-CR1 antibody 1B4, respectively. PfRh4 and C4b samples were run under reducing conditions whereas CCP1-3 was under nonreducing conditions. Arrowheads highlight specific protein bands. Western blot results are representative of 2 or 3 independent experiments. (C) A ternary complex composed of CCP1-3, C4b, and rPfRh4 as inferred from SPR. Measurement by SPR of CCP1-3 binding to C4b immobilized on a CM5 sensorchip in absence (blue lines) and presence (black lines) of a 2-fold molar excess of rPfRh4. CCP1-3 concentrations were 4μM, 2μM, 1μM, 0.1μM, and 0.05μM; single measurements were made at 4μM and 1μM while all others were duplicate measurements. (D) Plots of responses versus concentration of CCP1-3 (either alone, blue, or mixed with rPfRh4, red) from the sensorgrams in panel C are shown, with extrapolations to R_max values. The much higher responses obtained when rPfRh4 is coinjected with CCP1-3 are consistent with formation of ternary complexes rather than binary complexes. (E) The percentage of maximal binding (estimated from extrapolated R_max values in panel D) versus concentration of CCP1-3 (either alone, blue, or mixed with rPfRh4, red). The near identical-slope of both binding curves indicates that the affinity of CCP1-3 for C4b, while it cannot be quantified because of subsaturation concentrations of CCP1-3, is not radically altered by rPfRh4 and is consistent with a ternary complex in which C4b and rPfRh4 occupy distinct sites on CCP1-3.