Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: Sensitive and high-throughput analysis utilizing secreted alkaline phosphatase
Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: Sensitive and high-throughput analysis utilizing secreted alkaline phosphatase
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DOI：
10.1016/j.jviromet.2011.11.003
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发表时间：
2012-01-01
影响因子：
3.1
通讯作者：
Wang, Lin-Fa
中科院分区：
文献类型：
--
作者：
Kaku, Yoshihiro;Noguchi, Akira;Wang, Lin-Fa
尼帕病毒（NiV），副粘病毒科，亨氏病毒属，与密切相关的亨德拉病毒（Hey）沿着被归类为生物安全级（BSL）4病原体。开发了一种新的血清中和试验，用于在BSL 2条件下使用表达分泌型碱性磷酸酶（SEAP）并用NiV F/G蛋白假型化的重组水泡性口炎病毒（VSV）（VSV-NiV-SEAP）测量NiV中和抗体。该新测定的独特特征是能够通过使用普通ELISA板读数器测量上清液中的SEAP活性来获得中和滴度。这相对于表达绿色荧光蛋白或荧光素酶的第一代NiV-假型具有显著的优势，后者需要昂贵且特定的测量设备。使用来自不同物种的NiV和Hey特异性血清组，VSV-NiV-SEAP测定证明中和抗体状态（阳性/阴性）与通过常规活NiV试验获得的一致，并且给出比后者更高的抗体滴度。此外，当在一个稀释度下筛选66个果蝠血清时，VSV-NiV-SEAP测定产生与活NiV测试相同的结果，并且仅需要非常少量（2 μ l）的血清。结果表明，这种新的VSV-NiV-SEAP检测方法是安全的，可用于使用ELISA读板器高通量筛选血清，并且具有高灵敏度和特异性。(C)2011 Elsevier B. V.保留所有权利。
Nipah virus (NiV), Paramyxoviridae, Henipavirus, is classified as a biosafety level (BSL) 4 pathogen, along with the closely related Hendra virus (Hey). A novel serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing secreted alkaline phosphatase (SEAP) and pseudotyped with NiV F/G proteins (VSV-NiV-SEAP). A unique characteristic of this novel assay is the ability to obtain neutralization titers by measuring SEAP activity in supernatant using a common ELISA plate reader. This confers a remarkable advantage over the first generation of NiV-pseudotypes expressing green fluorescent protein or luciferase, which require expensive and specific measuring equipment. Using panels of NiV- and Hey-specific sera from various species, the VSV-NiV-SEAP assay demonstrated neutralizing antibody status (positive/negative) consistent with that obtained by conventional live NiV test, and gave higher antibody titers than the latter. Additionally, when screening sixty-six fruit bat sera at one dilution, the VSV-NiV-SEAP assay produced identical results to the live NiV test and only required a very small amount (2 mu l) of sera. The results suggest that this novel VSV-NiV-SEAP assay is safe, useful for high-throughput screening of sera using an ELISA plate reader, and has high sensitivity and specificity. (C) 2011 Elsevier B.V. All rights reserved.