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.