Plates were incubated at 37 • C for 3-4 h or 4 • C overnight (preliminary studies showed that there was no difference in the result). After incubation, plates were rinsed 4 times with PBS/Tween 20 and incubated for 1 h at room temperature with the previously collected sera. Briefly, 2-fold serial dilutions of each serum sample were prepared in a PBS solution containing 0.2 to 0.5% BSA. Hyperimmune serum from previously infected birds with the LPAI subtype H9N2 or commercial anti-M2e antibody were used as positive controls; and sera from healthy, unvaccinated birds were used as a negative control. After appropriate washes, peroxidaseconjugated goat anti-chicken IgY (Sigma Aldrich,) was prepared in a 1 : 10,000 dilution in PBS and was added to each well and plates were incubated for an additional hour at room temperature. After subsequent rinsing, the plates were developed using 3,3 ,5,5 tetramethylbenzidine (TMB) peroxidase substrate (Thermo Fisher Scientific Inc., Rockford, IL) followed by a room temperature incubation period of 15 to 30 min. The absorbance was read in a SpectraMax 250 microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm.