ELISA for FPenvM766 Antibody Detection

The ELISA was essentially performed as described previously [74 (link)], using lysed Vero cells previously infected with 5 PFU FPenvM766 as the plate-bound antigen. Briefly, 96-well maxisorp microtitre plates (Nunc, Naperville, IL, USA) were coated with Vero lysates in PBS^- (5 ×10⁴ cells/well) in 0.05 M carbonate–bicarbonate buffer, pH 9.6, and incubated overnight at 4°C. The sera from all of the animals, drawn at T0 and at different times p.i. (T1, T2), were then added at 1:20 dilution, and the binding was revealed using goat anti-mouse HRP-conjugated serum (1:2000 dilution; Dako) and tetramethylbenzidine substrate (Sigma). The absorbance for each well was measured at 450 nm with a microplate reader (550; Bio-Rad Laboratories, Hercules, CA, USA).