Webster R G, Brown L E, Laver W G
Virology. 1984 May;135(1):30-42. doi: 10.1016/0042-6822(84)90114-4.
Competitive radioimmunoassays using monoclonal antibodies established that the neuraminidase of A/RI/5+/57 (H2N2) influenza can be divided into four overlapping antigenic regions. Antigenic regions 1 and 4 are sufficiently far apart so that there was no competition between antibodies for these sites. Region 1 is conserved in neuraminidases from N2 viruses over a 10-year period, while the other regions changed antigenically during this time. The antibodies belonging to groups 2 and 3 completely inhibited catalytic activity on fetuin substrate, whereas antibodies in groups 1 and 4 inhibited weakly or not at all. Antigenic region 2 can be further divided into four overlapping areas (2a, 2b, 2c, and 2d) based on the reactivity patterns of monoclonal antibodies with antigenic variants, chemically modified neuraminidase, and the ability of the antibodies to inhibit enzyme activity of different molecular weight substrates. Previous studies [R. G. Webster, V. S. Hinshaw , and W. G. Laver (1982) Virology 117, 93-104; D. C. Jackson and R. G. Webster (1982) Virology 123, 69-77] characterized only region 2 of the neuraminidase molecule. Each of the monoclonal antibodies inhibited virus release from MDCK cells when incorporated in an agar overlay, and some antibodies in each group inhibited hemagglutination by intact virus, but only antibodies in group 2 neutralized virus in embryonated eggs and permitted selection of antigenic variants. The results indicate that antibodies to some antigenic sites on the neuraminidase may inhibit virus release more efficiently than others, depending on their relation to the enzyme active center. None of the monoclonal antibodies inhibited the hemolytic activity of viruses possessing N2. Based on antigenic mapping and biological properties of the monoclonal antibodies, a topographical map of the neuraminidase can be constructed. It is proposed that antigenic regions 1 and 4 are spacially separated and, based on their failure to inhibit biological activity, may be located on the bottom surface of the molecule; region 3 may be on the top surface of the molecule but at some distance from the catalytic center. Antigenic region 2 probably encompasses most of the top surface of the molecule; region 2d being closest to the enzyme center, with subregions 2a and 2b adjacent to it on the top surface. Chemical treatment of the neuraminidase with trinitrobenzenesulfonic acid (TNBS) causes modification of the 2b region, confirming the antigenic mapping results.
利用单克隆抗体进行的竞争性放射免疫分析表明，A/RI/5+/57（H2N2）流感病毒的神经氨酸酶可分为四个重叠的抗原区域。抗原区域1和4相距足够远，以至于针对这些位点的抗体之间不存在竞争。区域1在10年期间N2病毒的神经氨酸酶中是保守的，而其他区域在此期间发生了抗原性变化。属于第2组和第3组的抗体完全抑制了对胎球蛋白底物的催化活性，而第1组和第4组的抗体抑制作用较弱或根本没有抑制作用。基于单克隆抗体与抗原变体、化学修饰的神经氨酸酶的反应模式以及抗体抑制不同分子量底物酶活性的能力，抗原区域2可进一步分为四个重叠区域（2a、2b、2c和2d）。先前的研究[R.G.韦伯斯特、V.S.欣肖和W.G.拉弗（1982年），《病毒学》117卷，93 - 104页；D.C.杰克逊和R.G.韦伯斯特（1982年），《病毒学》123卷，69 - 77页]仅对神经氨酸酶分子的区域2进行了表征。当掺入琼脂覆盖物中时，每种单克隆抗体都抑制了病毒从MDCK细胞的释放，并且每组中的一些抗体抑制了完整病毒的血凝作用，但只有第2组中的抗体在鸡胚中中和了病毒并允许选择抗原变体。结果表明，针对神经氨酸酶上某些抗原位点的抗体可能比其他抗体更有效地抑制病毒释放，这取决于它们与酶活性中心的关系。没有一种单克隆抗体抑制具有N2的病毒的溶血活性。基于单克隆抗体的抗原图谱和生物学特性，可以构建神经氨酸酶的拓扑图。有人提出抗原区域1和4在空间上是分开的，并且基于它们未能抑制生物活性，可能位于分子的底面；区域3可能位于分子的顶面，但与催化中心有一定距离。抗原区域2可能涵盖分子的大部分顶面；区域2d最靠近酶中心，子区域2a和2b在顶面上与之相邻。用三硝基苯磺酸（TNBS）对神经氨酸酶进行化学处理会导致2b区域的修饰，证实了抗原图谱结果。
