LAUSR

&NA; The non‐structural protein 1 (NS1) of influenza A virus (IAV) is a multifunctional protein that antagonizes host antiviral responses, modulating virus pathogenesis. As such, it serves as a good target for research and diagnostic assay development. In this study, we have generated a novel monoclonal antibody (mAb) 19H9 and epitope mapping revealed that two residues, P85 and Y89, of NS1 are essential for interacting with this mAb. Furthermore, residues P85 and Y89 are found to be highly conserved across different IAV subtypes, namely seasonal H1N1 and H3N2, as well as the highly pathogenic H5N1 and H5N6 avian strains. Indeed, mAb 19H9 exhibits broad cross‐reactivity with IAV strains of different subtypes. The binding of mAb 19H9 to residue Y89 was further confirmed by the abrogation of interaction between NS1 and p85&bgr;. Additionally, mAb 19H9 also detected NS1 proteins expressed in IAV‐infected cells, showing NS1 intracellular localization in the cytoplasm and nucleolus. To our knowledge, mAb 19H9 is the first murine mAb to bind at the juxtaposition between the N‐terminal RNA‐binding domain and C‐terminal effector domain of NS1. It could serve as a useful research tool for studying the conformational plasticity and dynamic changes in NS1.