Avian influenza (AI) is a serious infectious diseas e caused by negative sense single strand RNA viruse s 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 sequ ence of an Iranian strain of H5N1 virus from clade 2.2, (A/chicken/Ira n/53-3/2008), was obtained from Genbank. The nucleo tide sequence data was codon optimized for insect cells. Using pIEx-3 vect or a molecular construct, encoding a secretion sign al peptide of adipokinetic hormone (AKH) and 6xHis-Tag coding sequence, upstream of the cloning site for expressing fusion recomb inant HA protein with N-terminal tags, were designed and ordered for synt hesis 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 supernata nt of Sf9-cell cultures. Purified rH5 was analyzed using SDS-PAGE, followed by western blot assay. Using the rH5 antigen, an EL ISA 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 anti gen concentration, serum dilution and conjugate ant ibody concentration. Optimum condition for antigen concentration, conjug ate antibody dilution and serum sample dilution wer e 100 ng/ml, 1:1000 and 1:500 dilution, respectively. Based on the optic density observed, an ELISA titer (ET) predictionequation was derived from a positive/negative (P/N) ratio standard curve. The c orrelation coefficient of the results in intra- and inter-assayay of the test was statistic lly significant (P<0.05). The test was specific with no cross reaction with the sera contain ing high titers against Newcastle disease (ND), infectious bronchitis (IB), infectious bursal disease (IBD), infectious laryngotracheitis (ILT) and avian influenza virus H9N2 The H5-ELISA developed was val idated for the detection of H5 antibody in a limite d number of available sera, but it has to be validated against a large nu mbers of known H5 positive sera. The assay has pote ntial to be employed as aserological tool for the detection of antibody agai nst H5 viruses in poultry and avian populations, wh ich may be the host for several influenza virus subtypes.

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