Abstract
The H3 subtype of avian influenza virus (AIV) is widespread in avian species and is frequently isolated in surveillance projects; thus, we have developed a more effective diagnostic approach of a monoclonal antibody (mAb)-based sandwich ELISA for the H3 AIV detection. First, we have produced the essential reagent of mAb against AIV H3 strains with the development of an mAb-Mouse immunization with a purified H3-subtype AIV strain and cell fusion to generate hybridoma cells. These cells were screened with hemagglutination inhibition (HI) tests, and optimal cells were subcloned. We chose a hybridoma cell line that steadily secreted a specific H3-subtype AIV mAb, designated 9F12, that belongs to the IgG1 subclass and has a K-type light chain. 9F12 was shown to bind specifically to the H3-subtype AIV antigen by both immunofluorescence assay and Western blot analysis. Finally, a 9F12-based sandwich ELISA was successfully developed and used to specifically test for this antigen. The sandwich ELISA conditions were optimized, and the specificity and sensitivity were validated. The results for clinical sample detection were consistent with viral isolation. Consequently, the 9F12-based sandwich ELISA is a specific, sensitive, robust, rapid and versatile diagnostic tool for H3-subtype AIV and provides a promising strategy for effective influenza virus prevention and control.
Highlights
Influenza A virus, a member of the genus Orthomyxovirus, is classified into different H and N subtypes according to differences in two major antigen genes, hemagglutinin (HA) and neuraminidase (NA)
The monoclonal antibody (mAb) 9F12 reacted with the H3 isolates tested but lacked cross-reactivity with other avian influenza virus (AIV) subtypes, NDV and EDSV, as assessed by the hemagglutination inhibition (HI) analysis (Table 1)
Nine H3 isolates were randomly chosen for identification of the reactivity of the mAb 9F12 by Western blot assay, and the results suggested that the H3 mAb could react with all nine H3 isolates, which showed specific bands at 66 kDa; in contrast, the AIV-H9, NDV and mock reactions had no band (Fig. 1)
Summary
Influenza A virus, a member of the genus Orthomyxovirus, is classified into different H and N subtypes according to differences in two major antigen genes, hemagglutinin (HA) and neuraminidase (NA). The primary reservoirs of influenza A virus are waterfowl and shorebirds (Oshansky et al 2014). The H3N8 equine influenza virus was first isolated from horses in Miami, Florida, in 1963 (Kitchen et al 1963). It is the only subtype affecting equine populations worldwide and has serious implications for public health (Alves Beuttemmuller et al 2016; Sreenivasan et al 2018). An H3N8 influenza virus carrying mammalian adaptation mutations has been isolated from seals (Solorzano et al 2015)
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