Rapid Detection of N9 Subtype Avian Influenza Viruses Using an NA-Targeting Lateral Flow Immunoassay

The emergence of highly pathogenic avian influenza viruses (AIVs) poses significant threats to global public health and the poultry economy. Rapid detection methods targeting the hemagglutinin (HA) protein have become increasingly unreliable due to ongoing antigenic drift and genetic mutations. In this study, we screened two monoclonal antibodies (mAbs) against the highly conserved neuraminidase (NA) protein, which were characterized by high affinity and specificity for the N9 subtype. Using the paired mAbs 12A2 and 12F12, an NA-targeting lateral flow immunoassay (NA-LFIA) was developed for the rapid detection of N9 subtype AIVs. The NA-LFIA demonstrated exceptional specificity, exhibiting no cross-reactivity with the N1, N2, or N6 subtypes. The limits of detection (LODs) were established at 10^3.3 TCID₅₀/0.1 mL for the virus and 98 ng/mL for the recombinant NA protein. The test strips exhibited excellent reproductivity for detecting the NA antigen, with intra-assay and inter-assay variations of 5.32% and 6.05%, respectively. Stability testing confirmed a six-month shelf life without any loss of sensitivity. These finds indicate that the NA-LFIA is a robust, rapid point-of-care testing (POCT) tool for the early detection of N9 subtype AIV infection.