Summary: Avian influenza (AI) is a serious infectious disease caused by negative sense single strand RNA viruses that belong to the genus influenza virus in the family Orthomyxoviridae. In the present study, we developed an indirect enzyme-linked immunosorbent assay (ELISA) employing a recombinant H5 antigen expressed in sf9 insect cells. The HA nucleotide sequence of an Iranian strain of H5N1 virus from clade 2.2, (A/chicken/Iran/53-3/2008), was obtained from Genbank. The nucleotide sequence data was codon optimized for insect cells. Using pIEx-3 vector a molecular construct, encoding a secretion signal peptide of adipokinetic hormone (AKH) and 6xHis-Tag coding sequence, upstream of the cloning site for expressing fusion recombinant HA protein with Nterminal tags, were designed and ordered for synthesis by GenScript, USA. The recombinant construct was used to transfect Sf9 insect cells in a commercial serum-free medium. The H5-His protein was purified from the supernatant of Sf9-cell cultures. Purified rH5 was analyzed using SDS-PAGE, followed by western blot assay. Using the rH5 antigen, an ELISA assay was developed. The H5-ELISA was compared with the hemagglutination inhibition (HI) test for the assessment of the specificity of the test. The H5-ELISA condition was optimized for antigen concentration, serum dilution and conjugate antibody concentration. Optimum condition for antigen concentration, conjugate antibody dilution and serum sample dilution were 100 ng/ml, 1:1000 and 1:500 dilution, respectively. Based on the optic density observed, an ELISA titer (ET) prediction equation was derived from a positive/negative (P/N) ratio standard curve. The correlation coefficient of the results in intra- and inter-assayay of the test was statistically significant (P<0.05). The test was specific with no cross reaction with the sera containing high titers against Newcastle disease (ND), infectious bronchitis (IB), infectious bursal disease (IBD), infectious laryngotracheitis (ILT) and avian influenza virus H9N2The H5-ELISA developed was validated for the detection of H5 antibody in a limited number of available sera, but it has to be validated against a large numbers of known H5 positive sera. The assay has potential to be employed as a serological tool for the detection of antibody against H5 viruses in poultry and avian populations, which may be the host for several influenza virus subtypes.

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