H3n2 ha or h3n2 na

Sera with serial dilutions were added to 96-well plates coated with 5 μg ml⁻¹ NP₅-BSA (for detecting high-affinity anti-NP) or NP₂₅-BSA (for detecting both high- and low-affinity anti-NP) obtained from Biosearch Technologies and incubated at room temperature for 2 h, followed by incubation with HRP-conjugated secondary antibodies against mouse IgM, IgG1, IgG2a, IgG2b, and IgG3 (Southern Biotechnology). The plates were developed with TMB peroxidase substrate kit (Bio-Rad Laboratories, Hercules, CA) and optical densities at 450 nm were measured. A mixture of sera from wild type mice immunized with NP-KLH was used to establish standard curves in each plate and antibody levels were shown as relative titers. Influenza A H3N2 specific antibodies in the sera or BAL fluid were measured similarly as above except that 96-well plates were coated with 2 μg ml⁻¹ H3N2 HA or H3N2 NA (Sino Biological) and HRP-conjugated secondary antibodies against mouse IgG and IgA (Southern Biotechnology) were used.