Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies
Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies
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DOI：
10.1128/cvi.00644-12
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发表时间：
2013-04-01
影响因子：
--
通讯作者：
Tortorella, Domenico
中科院分区：
文献类型：
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作者：
Gardner, Thomas J.;Bolovan-Fritts, Cynthia;Tortorella, Domenico
人巨细胞病毒（CMV）感染可引起强烈的体液免疫应答和抗CMV抗体产生。病毒感染的诊断可以通过使用各种血清学测定进行;然而，针对CMV的血清抗体的定量可能不能准确测量患者控制病毒感染的能力。表达绿色荧光蛋白（GFP）融合蛋白的CMV菌株可用作体外评估CMV感染特性的筛选工具。在这项研究中，我们采用了CMV病毒株，AD 169，异位表达黄色荧光蛋白（YFP）融合到立即早期2（IE 2）蛋白产物（AD 169（IE 2-YFP）），以定量CMV感染人细胞。我们创建了一个高通量的基于细胞的检测，需要最少量的材料，并提供了一个平台，用于快速分析病毒感染的初始阶段，包括病毒附着，融合和立即早期病毒基因表达。利用AD 169（IE 2-YFP）细胞感染系统开发了针对病毒表面糖蛋白gH的单克隆抗体中和试验。将高通量测定扩展至测量来自CMV阳性受试者的血清的中和能力。这些发现描述了一种灵敏和特异的测定方法，用于定量在限制CMV传播和传播中起作用的关键免疫反应。总的来说，我们已经证明，一个强大的高通量感染检测可以分析CMV生命周期的早期步骤，并量化生物试剂的效力，以减轻病毒感染。
Infection by human cytomegalovirus (CMV) elicits a strong humoral immune response and robust anti-CMV antibody production. Diagnosis of virus infection can be carried out by using a variety of serological assays; however, quantification of serum antibodies against CMV may not present an accurate measure of a patient's ability to control a virus infection. CMV strains that express green fluorescent protein (GFP) fusion proteins can be used as screening tools for evaluating characteristics of CMV infection in vitro. In this study, we employed a CMV virus strain, AD169, that ectopically expresses a yellow fluorescent protein (YFP) fused to the immediate-early 2 (IE2) protein product (AD169(IE2-YFP)) to quantify a CMV infection in human cells. We created a high-throughput cell-based assay that requires minimal amounts of material and provides a platform for rapid analysis of the initial phase of virus infection, including virus attachment, fusion, and immediate-early viral gene expression. The AD169(IE2-YFP) cell infection system was utilized to develop a neutralization assay with a monoclonal antibody against the viral surface glycoprotein gH. The high-throughput assay was extended to measure the neutralization capacity of serum from CMV-positive subjects. These findings describe a sensitive and specific assay for the quantification of a key immunological response that plays a role in limiting CMV dissemination and transmission. Collectively, we have demonstrated that a robust high-throughput infection assay can analyze the early steps of the CMV life cycle and quantify the potency of biological reagents to attenuate a virus infection.