Serum antibodies to H5N1 AIV NP were detected using an indirect ELISA method as described by [24] , which used prokaryotically-expressed NP as the antigen. The testing steps as follows, after washing of the plates, 50 ml of the test serum mixed in the test wells with 50 ml of antigen diluted 1:10 in ELISA buffer. After incubation at 37uC for 1 h, 100 ml horseradish peroxidase conjugate, diluted 1:1000 in ELISA buffer, was added to each well and plates were further incubated at 37uC for 1 h. After two washing steps, 100 ml of TMB substrate was added and incubated at room temperature for 10 min. The reaction was stopped by adding 100 ml H 2 SO 4 2 M. The extinctions were measured at 490 nm with a micro ELISA reader (Bio-Rad). 