96-well ELISA plates (Immulon) were coated with 50uL of 1 μg/ml recombinant N1 or N2 protein (BEI Resources NR-19234 and NR-43784) and incubated overnight at 4°C. Plates were blocked for 1 h at room temperature with 150 μL PBS with 0.01% Tween-20, 3% normal goat serum and 3% milk powder. Ferret sera was heat inactivated at 56°C for 30 minutes. Two-fold serial dilutions were performed in a blocking buffer and incubated on the ELISA plate for 2 h at room temperature. After washing with PBS-0.01% Tween-20, plates were incubated with peroxidase-conjugated goat anti-ferret IgG (Abcam ab112770). SureBlue TMB peroxidase substrate (KPL) was added to each well and the reaction stopped with 250 mM HCl. Absorbance was read at 450 nm.

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