| Avian influenza (AI) is one of fatal diseases caused by influenza A virus (AIV) that is classified in the family of Orthomyxoviridae. And it is a considerable zoonoses pathogeny that not only infects poultry, but also infects mammalian animals including human. A long-term surveillance of AIV antibody in natural host waterfowl is necessary for prevention and control of the disease and has a significance in ecology and public health.A prokaryotic expression of HA1 protein of AIV H5N1 subtype was performed using the recombinant plasmid pETHA1-H5 constructed previously. The results showed that the insoluble HA1 protein was successfully expressed in the Escherichia coli BL21(DE3) transformed with the plasmid pETHA1-H5 after IPTG induction. The yield of the expressed HA1 protein purified by Ni2+-NTA column was about 22.75 mg/L in bacterial culture. Western-blot analysis revealed that the recombinant HA1 protein has specific reactive ability with the positive sera of AIV H5N1 subtype from various poultry including chicken, duck and goose, while no cross-reactivity between the recombinant HA1 protein and antibodies against other susceptive poultry viruses was found in Dot-ELISA. The results illuminated that the recombinant HA1 protein with well bioactivity can be used as a detection antigen for H5-antibody of AIV.An H5 subtype-specific indiredt anzyme-linked immunosorbent assay (iELISA) was developed using the purified recombinant HA1 protein as a diagnosis antigen, and HRP-labeled rabbit anti-goose immunoglobulin G (IgG) conjugate as second. The working conditions of iELISA were optimized by serial optimizing tests. The high binding ninety-six-well microtiter plates were coated with HA1 antigen as 2μg/ml overnight at 4℃after incubation at 37℃for 1h, and optimal dilutions for goose serum and secondary antibody were 1:100 and 1:1000 respectively. The optimal coating buffer, blocking solution and dilution solution were 0.05 M Tirs-HCl (pH8.5), 0.05 M phosphate-buffered saline (PBS) containing 5% skimmed milk and PBST containing 0.1%BSA, respectively. The reaction conditions for serum samples and the conjugate were both of 45 min at 37℃, and the exposure time for TMB was about 10 min at room temperature. Using hemagglutinin inhibition (HI) test as a reference, receiver-operating characteristic analysis (ROC) of the iELISA assay was performed by detecting AIV H5- antibodies of 417 goose serum samples. The negative–positive cutoff was defined as 0.25, and its diagnostic sensitivity (DSN) and specificity (DSP) were 91.15% and 91.1% respectively. The HA1-iELISA assay showed a high specificity for H5 antibody with cross-reactivity for antibodies against other poultry viruses. The CV of inter-assay and intra-assay were less than 12% indicating the better stability and reproducibility. The coincidence of HA1-iELISA with HI,VN and AIV-ELISA tests were about 90% as 100 goose serum samples were tested, indicated the high diagnostic sensitivity and specificity of HA1-iELISA.HA1-iELISA has potential application in detecting H5-antibody of AIV of goose. Its components were tested for developing HA1-iELISA kit with high quality and long-term validity. The results showed that the HA1-coated plates maintained well activity at a low temperature after vacuum dehydration. The conservation time of each working solution were prolonged significantly by adding of protective and stabilizing agents. The assembled HA1-iELISA kit could be stored for at least 6 months without loss of reactivity at 4℃. The rapid and specific HA1-iELISA assay was provided as an available technique for diagnosis and serological of H5 subtype of AIV, and a good fundament for the further commercial development of the kit. |
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