The HI titers are expressed as the highest dilution of serum giving complete inhibition of agglutination. Additionally, all HI titer of 1: 20 or greater samples were tested for H5N1 neutralizing antibodies using a traditional neutralization (MN) method. Briefly, positive sera were two fold diluted in serum-free DMEM starting at a dilution of 1:10. Then, 100 TCID50 of virus were added to the serially diluted serum at a 1:1 ratio (V/V), followed by incubation at 37 °C for 1 h. Finally, 0.2 ml of the virus–serum mixtures was transferred to 96-well monolayer plates of MDCK and incubated in 5% CO2 at 37 °C for 72 h. All samples were tested in triplicate. Pathogenic effects were observed every day and cell supernatants were tested by the HA assay to confirm the viral infection. The endpoint for neutralization was expressed as the highest dilution that prevented hemagglutination in all 3 replicates. Both HI titer of 1:20 or greater and MN titer of 1:80 or greater were considered positive. A HPAI H5N1 virus, A/chicken/Guangdong/178/04(H5N1) (Genbank Accession: AY737296), was used for all AIV antibody detection by HI and MN assays. The H5N1 virus was isolated and stored at College of Veterinary Medicine, South China Agricultural University [15]. It belongs to Clade 2.3.2, the most dominant lineage of H5N1 in China since 2009, which has been tested to be suitable for a detection usage [13]. The following antigens were also used for HI assays: H1N1 pdm09 virus