Preparation and Evaluation of Recombinant Severe Fever with Thrombocytopenia Syndrome Virus Nucleocapsid Protein for Detection of Total Antibodies in Human and Animal Sera by Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay
Preparation and Evaluation of Recombinant Severe Fever with Thrombocytopenia Syndrome Virus Nucleocapsid Protein for Detection of Total Antibodies in Human and Animal Sera by Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay
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DOI：
10.1128/jcm.01319-11
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发表时间：
2012-02-01
影响因子：
9.4
通讯作者：
Wang, Hua
中科院分区：
文献类型：
--
作者：
Jiao, Yongjun;Zeng, Xiaoyan;Wang, Hua
最近在中国出现的经证实由发热伴血小板减少综合征病毒（SFTSV）引起的人感染引起全球关注。在流行病学调查中，迫切需要安全的诊断免疫试剂来测定人类和动物的血清阳性率。本文报道了SFTSV病毒核衣壳蛋白的克隆与表达。建立了以n蛋白为基础的双抗原夹心酶联免疫吸附试验（ELISA）检测人、动物血清中总抗体的方法。我们认为，由于双抗原夹心ELISA检测总抗体的灵敏度高于传统间接ELISA，可用于检测不同动物的sftsv特异性抗体。采用血清中和试验验证该ELISA系统的性能。所有中和试验呈阳性的人和动物血清在夹心ELISA中也呈阳性，并且血清中和滴度与ELISA读数之间存在高度相关性。交叉反应性评价表明，该体系对SFTSV具有高度特异性；所有汉坦病毒和登革热病毒确诊患者样本均为阴性。在该ELISA中，中国安徽和湖北两省确诊的sftsv人血清和动物血清与N蛋白反应，表明不同地理位置的病毒分离株之间没有主要的抗原差异，并且该检测在全国范围内适用。ELISA结果显示，3.6%的人血清样本和47.7%的动物现场血清样本中SFTSV抗体阳性，表明SFTSV在中国广泛流行。这种检测方法操作简单，不存在生物危害风险，不需要复杂的设备，可用于疾病监测项目，特别是用于筛选来自不同动物物种的大量样本。
The recent emergence of the human infection confirmed to be caused by severe fever with thrombocytopenia syndrome virus (SFTSV) in China is of global concern. Safe diagnostic immunoreagents for determination of human and animal seroprevalence in epidemiological investigations are urgently needed. This paper describes the cloning and expression of the nucleocapsid (N) protein of SFTSV. An N-protein-based double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) system was set up to detect the total antibodies in human and animal sera. We reasoned that as the double-antigen sandwich ELISA detected total antibodies with a higher sensitivity than traditional indirect ELISA, it could be used to detect SFTSV-specific antibodies from different animal species. The serum neutralization test was used to validate the performance of this ELISA system. All human and animal sera that tested positive in the neutralization test were also positive in the sandwich ELISA, and there was a high correlation between serum neutralizing titers and ELISA readings. Cross-reactivity was evaluated, and the system was found to be highly specific to SFTSV; all hantavirus- and dengue virus-confirmed patient samples were negative. SFTSV-confirmed human and animal sera from both Anhui and Hubei Provinces in China reacted with N protein in this ELISA, suggesting no major antigenic variation between geographically disparate virus isolates and the suitability of this assay in nationwide application. ELISA results showed that 3.6% of the human serum samples and 47.7% of the animal field serum samples were positive for SFTSV antibodies, indicating that SFTSV has circulated widely in China. This assay, which is simple to operate, poses no biohazard risk, does not require sophisticated equipment, and can be used in disease surveillance programs, particularly in the screening of large numbers of samples from various animal species.