Applied Research Note: Development and validation of a highly specific polyclonal antibody targeting neuraminidase of novel H3N8 avian influenza virus

Avian influenza viruses (AIV) of the H3N8 subtype pose a significant threat to both the poultry industry and public health. This study aimed to develop and validate a highly specific polyclonal antibody targeting the neuraminidase (NA) protein from a novel H3N8 AIV, which exhibits tri-basic hemagglutinin cleavage sites and shares genetic proximity to recent human isolates. The NA gene of H3N8 AIV was cloned and introduced into E. coli BL21 and Rosetta competent cells to induce the recombinant protein expression. Optimization procedures, including IPTG concentration, time, and temperature, were implemented to enhance protein expression efficiency. Polyclonal antibodies were generated and validated through western blotting, indirect immunofluorescence assay (IFA), and indirect ELISA. As a result, the pET-32a-NA (N8) vector was successfully constructed. The expression of recombinant NA protein with a size of approximately 70 KDa was obtained and then optimized with a final IPTG concentration of 0.6 mM, at 27°C for 14 h. Western blotting and IFA analysis demonstrated that the prepared polyclonal antibody effectively and specifically bound to NA(N8) protein. The titer of the polyclonal antibodies reached 1:409600 by indirect ELISA. These results indicate the potential of these antibodies for the development of detection assays and biological studies required for H3N8 AIVs.