ELISA.

To assess the levels of virus-specific antibodies present in immunized mice, ELISAs were performed as previously described (28). Briefly, 96-well plates were coated with lysates from mock- or WT PR8 virus-infected MDCK cells or with PR8 virus HA (200 ng per well; catalog number NR-19240; BEI Resources) or NP (200 ng per well) recombinant proteins. After washing with PBS, coated wells were blocked with PBS containing 1% BSA overnight and then incubated with 1:2 dilutions (starting dilution, 1:100) of mouse serum at 37°C. After 1 h of incubation, the wells of the plates were washed with H₂O and incubated with HRP-conjugated goat anti-mouse IgG for 30 min at 37°C. The reactions were developed with tetramethylbenzidine (TMB) substrate for 10 min at room temperature, quenched with 2 N H₂SO₄, and read at 450 nm.