Figure 2

Complement cascade activation assays. (A) Heat-aggregated IgG 1% normal human serum for 1 hour at 31-41°C demonstrated that C5a generation differed significantly as a function of temperature (ANOVA P < 0.01). There was a 2.5 fold increase in C5a generation at 31°C vs. 37°C (*t-test P =0.05). Data are mean ± SEM for 4 independent experiments. C3/C4-analysis samples were generated by incubating 1% C8-deficient serum (to prevent hemolysis) with 0.25 mL EA for one hour at 31-41°C. (B) Total C3-fragment opsonization did not differ significantly as a function of temperature (ANOVA P = 0.5). (C) Cell membrane bound iC3b differed significantly as a function of temperature (ANOVA P < 0.01), with a 2-fold increase at 31°C vs. 37°C (t-test P = 0.04). Data are mean ± SEM for 4 independent experiments. (D) A representative Western blot analysis of membrane-bound C3-fragments confirmed that iC3b was the predominant form. (E) C4-fragment opsonization differed significantly as a function of temperature (ANOVA P = 0.04). There was an almost 2-fold increase in C4 generation at 31°C vs. 37°C (*t-test P = 0.05). Data are mean ± SEM for 4 independent experiments. (F) A representative Western blot analysis demonstrated a mixture of C4b, iC4b and C4d fragments.