Identification and Characterization of Single-Chain Antibodies that Specifically Bind GI Noroviruses
Identification and Characterization of Single-Chain Antibodies that Specifically Bind GI Noroviruses
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DOI：
10.1371/journal.pone.0170162
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发表时间：
2017-01-17
期刊：
影响因子：
3.7
通讯作者：
Palzkill, Timothy
中科院分区：
文献类型：
--
作者：
Hurwitz, Amy M.;Huang, Wanzhi;Palzkill, Timothy
诺如病毒感染通常会导致急性胃肠炎的爆发并迅速传播，导致诊断前的许多健康和经济挑战。因此，快速和可靠的诊断测试对于识别感染和指导护理点的适当临床反应至关重要。现有的工具，包括RT-PCR和酶免疫测定，由于得出结果所需的大量时间、设备和专门知识，造成了一些限制。可用于护理点的免疫层析测定具有较差的灵敏度和特异性，特别是对于基因组I诺如病毒，因此需要用更灵敏的测试方法确认结果。因此，显然需要新的试剂来帮助实现快速和可靠的结果。在这项研究中，我们已经确定了两个新的单链抗体（scFv）命名为NJT-R3-A2和NJT-R3-A3，有效地检测GI. 1和GI. 7病毒样颗粒（VLP），通过选择针对GI. 1主要衣壳蛋白的P-结构域的噬菌体展示文库。GI. 1和GI. 7的每种scFv的检测限分别为0.1和0.2 ng，以及6.25和25 ng。他们在多种诊断形式中以强特异性检测VLP，包括ELISA和基于膜的斑点印迹，以及在诺如病毒阴性粪便悬浮液的背景下。scFv还有效地检测诺如病毒阳性临床粪便样品中的天然病毒体。纯化的scFv分别以27 nM和49 nM的平衡常数（KD）值结合GI. 1和GI. 7 VLP。总体而言，本文鉴定和表征的基于噬菌体的scFv试剂显示出用于以具有强特异性和灵敏度的多种诊断测定形式检测GI. 1和GI. 7诺如病毒的实用性，表明有希望整合到现有的即时检测中以改善未来的诊断。
Norovirus infections commonly lead to outbreaks of acute gastroenteritis and spread quickly, resulting in many health and economic challenges prior to diagnosis. Rapid and reliable diagnostic tests are therefore essential to identify infections and to guide the appropriate clinical responses at the point-of-care. Existing tools, including RT-PCR and enzyme immunoassays, pose several limitations based on the significant time, equipment and expertise required to elicit results. lmmunochromatographic assays available for use at the point-of-care have poor sensitivity and specificity, especially for genogroup I noroviruses, thus requiring confirmation of results with more sensitive testing methods. Therefore, there is a clear need for novel reagents to help achieve quick and reliable results. In this study, we have identified two novel single-chain antibodies (scFvs) named NJT-R3-A2 and NJT-R3-A3 that effectively detect GI.1 and GI.7 virus-like particles (VLPs) through selection of a phage display library against the P-domain of the GI.1 major capsid protein. The limits of detection by each scFv for GI.1 and GI.7 are 0.1 and 0.2 ng, and 6.25 and 25 ng, respectively. They detect VLPs with strong specificity in multiple diagnostic formats, including ELISAs and membrane-based dot blots, and in the context of norovirus-negative stool suspensions. The scFvs also detect native virions effectively in norovirus-positive clinical stool samples. Purified scFvs bind to GI.1 and GI.7 VLPs with equilibrium constant (KD) values of 27 nM and 49 nM, respectively. Overall, the phage-based scFv reagents identified and characterized here show utility for detecting GI.1 and GI.7 noroviruses in multiple diagnostic assay formats with strong specificity and sensitivity, indicating promise for integration into existing point-of-care tests to improve future diagnostics.