Development and validation of a recombinant-H5 hemagglutinin-based competitive ELISA for serodiagnosis of avian influenza A subtype H5 antibodies
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Hemagglutinin (HA) protein is a major antigen presenter within avian influenza viruses, which in turn triggers a substantial immunogenic response within the infected host. This study set out to exhibit a successfully designed, developed, optimized, and validated a highly sensitive and effective competitive ELISA based on recombinant-HA proteins as antigens from two unique strains of AIV H5 that is capable of detecting a wide range of strains of North American and Eurasia lineages, including clade 184.108.40.206 groups B and C viruses. An important reason behind the development of this assay was to achieve the ability to overcome a glaring drawbacks of needing access to high biocontainment facilities in order to perform the golden standard HI assay internationally recognized as the best characterization assay for AIV within aviary samples. This competitive ELISA is able to perform under a low biocontainment environment due to the stability, non-infectious nature of the reagents and the lack of need for live virus. This assay has the capability to be deployed worldwide to facilities that would not be able to perform HI assays and have the ability to effectively detect AIV-H5 antibodies within samples. An unforeseen obstacle within the HI assay was the necessity to have a homologous or calibrated virus for the detection of new and emerging strains that was discovered while testing this assay, while our cELISA did not have the same hindrance. The cELISA’s were based on recombinantly expressed AIV-H5 HA full-length protein from 2 strains: A/Canadagoose/Oregon/AH0012452/2015 and A/Teal/Germany/Wv632/2005. Both cELISA showed high sensitivity and specificity, with low variation and no cross-reactivity to other viruses following calibration and optimization of the assays. This study shows the rec-H5 cELISA was able to perform with great confidence, equally or even outperforming the HI assay results based on our statistical analysis.
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