An indirect sandwich ELISA for the detection of avian influenza H5 subtype viruses using anti-hemagglutinin protein monoclonal antibody

Abstract

A sandwich ELISA test using AIV H5 subtype specific monoclonal antibody (clone 2H4) to an epitope of hemagglutinin protein has been developed. The monoclonal antibody was used to capture the antigen from clinical samples (swabs and tissues). Captured antigens from clinical samples were detected using polyclonal sera, purified AIV H5N1 particles were titrated in the sandwich ELISA and the limit of detection was determined to be approximately 1.0 ng of influenza viral protein in virus preparations. Fifteen AIV strains of H1-H15 subtypes and some other pathogens were tested by this system, and the test is specific to H5 subtype viruses as it failed to detect other AIV subtype viruses and other pathogens. Varieties of clinical samples originating from laboratory experiments (n=382) and from fields (n=288) were employed to test the efficacy of DAS-ELISA test. The test compared very well with the traditional method for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs. In comparison to virus isolation the sensitivity and specificity of sandwich ELISA were found to be 98.6% and 97.6% respectively. In addition, the DAS-ELISA was used to test samples of experimentally infected birds and clinical samples obtained from central China in 2005. The assay proved to be sensitive and specific for the rapid detection of AIV H5 subtype virus form the tissues and swabs in infected animals.