Figure 5.

IgG anti‐αGal activates the classical pathway on bacteria. (a) Complement deposition on bacteria as function of the level of IgG anti‐αGal bound on the bacteria measured by flow cytometry (‘anti‐IgG MFI’). Bacteria, Streptococcus pneumonia serotype 9V (Sp9V) or Escherichia coli O86 (EcO86), were incubated in 1% hypogammaglobulinaemia human serum (HHS) as the complement source supplemented with IgG anti‐αGal at 0, 0·1, 1, 2·5, 5, 10, 15, 20 or 30 mg/l. The data are expressed as the mean and standard deviation of two independent experiments. Curve fittings were performed using a linear regression model for Sp9V (R ² = 0·94) and an exponential model for EcO86 (doubling‐percentage, P = 1·5% (95% CI 1·3%–1·8%), R ² = 0·99). P represents the increase in IgG binding signal associated with doubling of the complement deposition signal. (b) As in the previous panel but experiments were conducted for Sp9V in 1%, 4% or 10% HHS. Curve fitting was performed using linear regression models for 1% HHS (slope 0·00; 95% CI 0·0017–0·0028; R ² = 0·95) and 4% HHS (slope 0·052; 95% CI 0·040–0·064; R ² = 0·95) but in an exponential model for 10% HHS (P = 29% (95% CI 26%–32%), R ² = 0·99). (c) Sp9V, IgG anti‐αGal at 20 mg/l and 10% HHS were incubated at 37° with or without nanobodies: KRA152 (irrelevant specificity), C1qNb75 (inhibits C1q docking to Ig), hC3Nb2 (inhibits C3 cleavage by CP and AP) and hC3Nb1 (inhibits C3 cleavage by AP). Complement deposition on bacteria was detected by flow cytometry. The data are expressed as the mean (bars) of three separate experiments (each represented by a dot) and standard deviation (error bars). (d) As in the previous panel, but for EcO86 in 1% HHS.