A new panel of NS1 antibodies for easy detection and titration of influenza A virus.

The non-structural protein NS1 of the influenza A virus is a good target for the development of diagnostic assays. In this study, three NS1 monoclonal antibodies (mAbs) were generated by using recombinant NS1 protein of H5N1 virus and found to bind both the native and denatured forms of NS1. Two of the mAbs, 6A4 and 2H6, bind NS1 of three different strains of influenza A virus, namely H1N1, H3N2, and H5N1. Epitope mapping revealed that residues 42-53 of H5N1 NS1 are essential for the interaction with both mAbs. Between the three strains, there is only one amino acid difference in this domain, which is consistent with the observed cross-reactivities. On the other hand, mAb 1G1 binds to residues 206-215 of H5N1 NS1 and does not bind NS1 of H1N1 or H3N2. Furthermore, all three mAbs detected NS1 proteins expressed in virus infected MDCK cells and indirect immunofluorescence staining with mAbs 6A4 and 2H6 provided an alternative method for viral titer determination. Quantifying the numbers of fluorescent foci units yielded viral titers for three different isolates of H5N1 virus that are highly comparable to that obtained by observing cytopathic effect induced by virus infection. Importantly, this alternative method yields results at 1 day post-infection while the conventional method using cytopathic effect yields results at 3 days post-infection. The results showed that this new panel of NS1 antibodies can detect NS1 protein expressed during viral infection and can be used for fast and easy titration of influenza A virus. J. Med. Virol. 82:467-475, 2010. (c) 2010 Wiley-Liss, Inc.