Rapid human metapneumovirus microneutralization assay based on green fluorescent protein expression
Rapid human metapneumovirus microneutralization assay based on green fluorescent protein expression
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DOI：
10.1016/j.jviromet.2005.05.005
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发表时间：
2005-09-01
影响因子：
3.1
通讯作者：
Buchholz, UJ
中科院分区：
文献类型：
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作者：
Biacchesi, S;Skiadopoulos, MH;Buchholz, UJ
我们描述了一种简单和快速的微量中和试验的基础上的重组人偏肺病毒（HMPV）表达增强型绿色荧光蛋白（rHMPV-GFP）。将试验血清稀释液与固定量的rHMPV-GFP孵育并接种到Vero细胞上，通过活细胞的荧光显微镜观察未中和的rHMPV-GFP的生长。孵育3天后可测定初步滴度。GFP表达足以在孵育4-5天后通过自动扫描仪读取，这也提供了永久记录。相比之下，传统的血清中和试验需要更长的孵育时间加上固定和染色或免疫染色的额外步骤。基于rHMPV-GFP的滴度可以通过Reed和Muench的50%感染性终点方法测定[Reed，L. J.，Muench，H.，1938.一个简单的方法估计百分之五十的终点。Am. J. Hyg. 27，493-497]，或通过自动扫描和非线性回归以确定GFP荧光的50%终点。后一种方法的灵敏度要高出两到三倍。该测定还允许自动化和放大，使其适用于广泛的HMPV血清流行病学研究和需要大规模血清学的实验，例如疫苗研究。(c)2005 Elsevier B. V.保留所有权利。
We describe a simple and expedited microneutralization assay for human metapneumovirus virus (HMPV) based on a recombinant HMPV expressing the enhanced green fluorescent protein (rHMPV-GFP). Test serum dilutions were incubated with fixed amounts of rHMPV-GFP and inoculated onto Vero cells, and the growth of non-neutralized rHMPV-GFP was visualized by fluorescent microscopy of living cells. A preliminary titer could be determined following 3 days of incubation. GFP expression was sufficient to be read by an automated scanner after 4-5 days of incubation, which also provided a permanent record. In comparison, the conventional serum neutralization assay requires a longer incubation time plus the additional steps of fixation and staining or immunostaining. rHMPV-GFP-based titers could be determined by the 50% infectivity endpoint method of Reed and Muench [Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoint. Am. J. Hyg. 27, 493-497], or by automated scanning and non-linear regression to determine the 50% endpoint of GFP fluorescence. The latter method was two- to three-fold more sensitive. This assay also permits automation and up-scaling, making it suitable for broad HMPV seroepidemiology studies and experiments that require large scale serology, such as vaccine studies. (c) 2005 Elsevier B.V. All rights reserved.