A rapid immune plaque assay for the detection of Hendra and Nipah viruses and anti-virus antibodies
A rapid immune plaque assay for the detection of Hendra and Nipah viruses and anti-virus antibodies
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DOI：
10.1016/s0166-0934(01)00377-9
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发表时间：
2002-01-01
影响因子：
3.1
通讯作者：
Eaton, BT
中科院分区：
文献类型：
--
作者：
Crameri, G;Wang, LF;Eaton, BT
已开发出快速免疫斑块分析方法，以量化生物危害4级制剂Hendra和Nipah病毒，并检测对这两种病毒的中和抗体。这些方法依赖于这两种病毒在24小时内在Vero细胞的单层中迅速产生大的合胞体的事实，以及Hendra病毒磷酸蛋白(P)的单特异性抗血清在Hendra和Nipah病毒感染的细胞甲醇固定后检测到Hendra和Nipah病毒诱导的合胞体。P蛋白是病毒核糖核蛋白核心的组成部分，也是病毒依赖RNA的RNA聚合酶的组成部分，在受感染的细胞中大量合成。在免疫斑块分析中，抗P抗体由碱性磷酸酶连接的第二抗体和Western印迹底物5-溴-4-氯-3-吲哚基磷酸和对硝基蓝四氮唑定位。对快速免疫空斑试验的改进也用于检测来自马来西亚的一组猪场血清中的尼帕病毒抗体，结果表明免疫空斑试验与传统的血清中和试验具有很好的一致性。在甲醇固定后，平板可保存长达7个月，并可用于免疫斑块分析，以补充酶联免疫吸附试验筛选血清中的尼帕病毒抗体。目前，所有酶联免疫吸附试验阳性的血清都要接受确证血清中和试验。通过识别在酶联免疫吸附试验中产生假阳性的血清，使用免疫空斑分析可以减少在生物危害级别4的条件下需要对尼帕病毒抗体进行验证性中和试验的血清数量。(C)2002 Elsevier Science B.V.保留所有权利。
Rapid immune plaque assays have been developed to quantify biohazard level 4 agents Hendra and Nipah viruses and detect neutralising antibodies to both viruses. The methods rely on the fact that both viruses rapidly generate large syncytia in monolayers of Vero cells within 24 h and that monospecific antiserum to the Hendra virus phosphoprotein (P) detects that protein in both Hendra and Nipah viruse-induced syncytia after methanol fixation of virus-infected cells. The P protein is a constituent of the ribonucleoprotein core of the viruses and a component of the viral RNA-dependent RNA polymerase and is made in significant amounts in infected cells. In the immune plaque assay, anti-P antibody is localised by an alkaline phosphatase-linked second antibody and the Western blot substrates 5-bromo-4-chloro-3-indolyl phosphate and p-nitro blue tetrazolium. A modification of the rapid immune plaque assay was also used to detect antibodies to Nipah virus in a panel of porcine field sera from Malaysia and the results showed good agreement between the immune plaque assay and a traditional serum neutralisation test. After methanol fixation, plates can be stored for up to 7 months and may be used in the immune plaque assay to complement the enzyme-linked immunosorbent assay screening of sera for antibodies to Nipah virus. At present, all enzyme-linked immunosorbent assay positive sera are subject to confirmatory serum neutralisation tests. Use of the immune plaque assay may reduce the number of sera requiring confirmatory neutralisation testing for Nipah virus antibodies under biohazard level 4 conditions by identifying those that generate false positive in the enzyme-linked immunosorbent assay. (C) 2002 Elsevier Science B.V. All rights reserved.