Fig. 2

Neuraminidase induces C3 activation in vitro. a Scheme showing the activation, deactivation, and degradation of the complement factor C3, based on [44, 45]. The anti-C3 antibody used for the immunoblot detects C3 α chain (113 kDa) before activation, and the iC3b α’ chain fragment (62.5 kDa) generated after activation [46]. b Blood serum andcerebrospinal fluid (CSF) samples were used as the source of factor C3. They were incubated with NA at 37 °C for 1 h (NA/37 °C), and then analyzed by immunoblot with anti-C3. Controls included: incubation in saline (NaCl) without NA, both at 37 °C (NaCl/37 °C) and at 4 °C (NaCl/4 °C); a positive control of complement activation with zymosan (Zym/37 °C). Complement activation correlates with fading of the C3 α band and concurrent appearance of iC3b α’ band. As expected, activation occurred in the positive controls (Zym/37 °C) but not in the negative controls (NA/4 °C), both in serum and CSF samples. NA was also able to activate complement (NA/37 °C), although not as much as zymosan, as the appearance of the iC3b α’ band is delayed. A partial activation was detected in the negative control NaCl/37 °C, which can be ascribed to auto-activation of complement when it is maintained for 1 h at 37 °C. The specificity of the anti-C3 antibody was proved by omitting the primary antibody during the immunoblot procedure (1st Ab bypass)