Development of a High-Throughput Respiratory Syncytial Virus Fluorescent Focus-Based Microneutralization Assay.
Development of a High-Throughput Respiratory Syncytial Virus Fluorescent Focus-Based Microneutralization Assay.
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DOI：
10.1128/cvi.00225-17
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发表时间：
2017-12
期刊：
影响因子：
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通讯作者：
Esser MT
中科院分区：
文献类型：
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作者：
Shambaugh C;Azshirvani S;Yu L;Pache J;Lambert SL;Zuo F;Esser MT
呼吸道合胞病毒（RSV）特异性中和抗体是抵抗RSV感染的主要保护机制，免疫预防性单克隆抗体帕利珠单抗在预防早产儿RSV相关下呼吸道感染中的疗效证明了这一点。因此，RSV中和试验已成为临床前动物模型、流行病学研究和临床试验中评估血清抗体中和活性的关键功能方法。在这项研究中，我们鉴定了一种24小时，基于荧光焦点的微量中和（RSVA FFA-MN）方法，该方法不需要更换培养基或感染前或感染后处理，以检测表达绿色荧光蛋白的RSV毒株A2（RSVA-GFP）感染的细胞，使用高容量成像系统进行自动图像采集和焦点计数。RSVA FFA-MN方法显示出灵敏度，检测限（LOD）和定量限（LOQ）为1：10或3.32 log 2;在4.27至9.65 log 2 50%抑制浓度（IC 50）范围内呈线性;并且具有精密度，试验内和试验间变异系数<21%。该精密度允许选择统计学合理的3倍血清应答升高临界值标准。通过在每次试验中纳入合并人血清样品作为阳性对照（PC），证明了该方法的重复性和耐用性。在两个实验室之间进行的3年检测中，该PC生成的数据在98.7%的时间内（n = 1，720）落在平均值的2.5个标准差内。这种高通量和可靠的RSV微量中和试验已被证明可用于检测来自临床前候选疫苗评价研究、流行病学研究以及儿科和成人疫苗临床试验的血清。
Neutralizing antibodies specific for respiratory syncytial virus (RSV) represent a major protective mechanism against RSV infection, as demonstrated by the efficacy of the immune-prophylactic monoclonal antibody palivizumab in preventing RSV-associated lower respiratory tract infections in premature infants. Accordingly, the RSV neutralization assay has become a key functional method to assess the neutralizing activity of serum antibodies in preclinical animal models, epidemiology studies, and clinical trials. In this study, we qualified a 24-h, fluorescent focus-based microneutralization (RSVA FFA-MN) method that requires no medium exchange or pre- or postinfection processing to detect green fluorescent protein-expressing RSV strain A2 (RSVA-GFP)-infected cells, using a high-content imaging system for automated image acquisition and focus enumeration. The RSVA FFA-MN method was shown to be sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 1:10, or 3.32 log2; linear over a range of 4.27 to 9.65 log2 50% inhibitory concentration (IC50); and precise, with intra- and interassay coefficients of variation of <21%. This precision allowed the choice of a statistically justified 3-fold-rise seroresponse cutoff criterion. The repeatability and robustness of this method were demonstrated by including a pooled human serum sample in every assay as a positive control (PC). Over 3 years of testing between two laboratories, this PC generated data falling within 2.5 standard deviations of the mean 98.7% of the time (n = 1,720). This high-throughput and reliable RSV microneutralization assay has proven useful for testing sera from preclinical vaccine candidate evaluation studies, epidemiology studies, and both pediatric and adult vaccine clinical trials.