Development of Antigen-Capture ELISA and Blocking ELISA for Detecting the H5-Subtype Avian Influenza Viruses

The purpose of this study is to use monoclonal antibodies (mAbs) against a H5N2 avian influenza virus (AIV) HA1 domain to develop an antigen-capture enzyme-linked-immunosorbent assay (AC-ELISA) and a blocking enzyme-linked-immunosorbent assay (B-ELISA) for early detection of H5 subtype AIV and anti-H5 AIV antibodies, respectively. Besides, we also evaluated the sensitivity and the specificity of a H5-subtype AIV B-ELISA developed in our laboratory by coating with whole virus as the antigen (called Virus-B-ELISA) and improved the virus coated step of the Virus-B-ELISA to save time. These mAbs were used as the capture antibodies and detector antibodies for the detection of H5 AIV by AC-ELISA. These mAbs were also labeled with horseradish peroxidase to become the tracers for the detection of H5 antibodies in chicken serum by B-ELISA. The result showed that the AC-ELISA only detected the H5 subtype Eurasia lineage strain and did not cross react with other lineage AIV (cut-off value=0.1). The detection limit was as little as 4.2×104/0.1 mL EID50. As to the B-ELISA coated with recombinant HA1 protein (rHA1) as the antigen (called rHA1-B-ELISA), the serum polyclonal antibodies induced by H5 AIV couldn’t effective binding with rHA1. The measurements of the H5-positive or negative serum are not significantly different. In Virus-B-ELISA, the sensitivity based on the hemagglutination inhibition test was 95.76％ (113/118) and the specificity was 90.78％ (266/293). The Kappa value between original Virus-B-ELISA and improved Virus-B-ELISA was 0.9539 meant an almost perfect consistency. So the improved Virus-B-ELISA can detect the H5 subtype AIV antibodies more rapidly than the original one. The AC-ELISA and the Virus-B-ELISA are useful to detect H5 subtype Eurasia lineage AIV antigen and H5 subtype AIV antibody, respectively.