2.12 IFN-γ detection
Peripheral blood mononuclear cells (PBMC) were obtained from cattle as described before (Romera et al., 2014). PBMC were cultured with 5 μg/ml iFMDV for 5 days. Supernatants were analyzed using ELISA as described previously (V. Quattrocchi et al., 2014). Briefly, plates were coated with a mAb against IFN-γ (kindly donated by Dr. L. Babiuk). Samples and recombinant IFN-γ standard (Serotec, UK) were added and IFN-γ was detected using rabbit polyclonal anti-IFN-γ Abs. After incubation, biotinylated goat anti-rabbit IgG antibody was added, followed by HRP-conjugated streptavidin (KPL, USA). Plates were washed, incubated with (OPD)-H2O2 and read at 492 nm. IFN-γ concentration was calculated from interpolation of data in a standard curve.