Figure 3

Percent inhibition of C2 activity by IgGs purified from sera of immunized mice. Inhibition assays were used to assess the ability of immune IgGs to inhibit C2 activity in-vitro. IgGs were purified by affinity chromatography on Protein A-Sepharose, from serum collected 2 weeks post-last booster. The assays were performed at three different concentrations of IgG i.e. 250, 125 and 62.5 μg/ml (final amount of 6.25, 3.125, and 1.56 μg per well respectively) and with 3.3 ng C2 per well. The C2-IgG mixtures were incubated overnight at 4°C. Residual C2 activity was measured after activation with 8 mM DTT. The fluorogenic substrate used was 20 μM Z-Phe-Arg-AMC and the fluorescence recorded (excitation 360 nm and emission 460 nm). The percent inhibition of C2 activity was determined from the slopes of the linear plots of residual C2 activity.