Collaborative Anti-Viral Research Group, Institute of Molecular and Cell Biology, Singapore, Singapore.
J Med Virol. 2010 Mar;82(3):467-75. doi: 10.1002/jmv.21709.
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.
甲型流感病毒的非结构蛋白 NS1 是开发诊断检测的理想靶标。本研究利用 H5N1 病毒的重组 NS1 蛋白生成了 3 种 NS1 单克隆抗体（mAb），发现它们可与天然和变性的 NS1 结合。2 种 mAb（6A4 和 2H6）可与 H1N1、H3N2 和 H5N1 3 种不同株系的流感 A 病毒的 NS1 结合。表位作图显示 H5N1 NS1 的 42-53 位残基对与这 2 种 mAb 的相互作用是必需的。在这 3 个株系中，该结构域仅有一个氨基酸的差异，这与观察到的交叉反应性一致。另一方面，mAb 1G1 与 H5N1 NS1 的 206-215 位残基结合，而不与 H1N1 或 H3N2 的 NS1 结合。此外，3 种 mAb 均能检测到感染病毒的 MDCK 细胞中表达的 NS1 蛋白，用 mAb 6A4 和 2H6 进行间接免疫荧光染色为病毒滴度测定提供了另一种方法。对 3 种不同 H5N1 病毒分离株进行荧光焦点单位计数，得到的病毒滴度与观察病毒感染引起的细胞病变效应得到的结果高度可比。重要的是，该替代方法可在感染后 1 天得出结果，而使用细胞病变效应的传统方法则在感染后 3 天得出结果。结果表明，该新的 NS1 抗体组能够检测病毒感染期间表达的 NS1 蛋白，可用于快速、简便地测定流感 A 病毒。J. Med. Virol. 82:467-475, 2010. (c) 2010 Wiley-Liss, Inc.
