FIG 6.

Sera from CspZ-YA- but not CspZ-vaccinated mice blocked FH binding to CspZ. Sera from five C3H/HeN mice inoculated with CspZ, CspZ-YA, or PBS (negative control) were obtained at 42 dpii. (a) Schematic diagram showing the experimental setup. GST-CspZ was added to a microtiter plate coated with anti-GST, in order to orient CspZ with the FH-binding site exposed. Wells were treated with PBS or the indicated dilution rates of mouse sera and then incubated with human FH. The levels of bound FH were quantified using sheep anti-human FH and goat anti-sheep HRP IgG as primary and secondary antibodies, respectively. The work was performed in three independent experiments; within each experiment, samples were run in triplicate. (b) Data are expressed as the percent FH binding, derived by normalizing the levels of bound FH from mouse serum-treated wells to that in PBS-treated wells. Data shown are the mean ± SEM of the percent FH binding from three replicates in one representative experiment. The dilution rate of the sera to inhibit 50% of FH bound by CspZ was obtained from curve-fitting and extrapolation of data from panel A (50% inhibitory dilution rate, 1:16,072 ± 1,595).