Development and application of a monoclonal-antibody-based blocking ELISA for detection of Japanese encephalitis virus NS1 antibodies in swine
Development and application of a monoclonal-antibody-based blocking ELISA for detection of Japanese encephalitis virus NS1 antibodies in swine
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DOI：
10.1007/s00705-019-04218-9
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发表时间：
2019-03
影响因子：
2.7
通讯作者：
Dengyuan Zhou;Chao Pei;Kelu Yang;Jing Ye;Shengfeng Wan;Qiu-Yu Li;Luping Zhang;Huanchun Chen;S. Cao;Yunfeng Song
中科院分区：
文献类型：
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作者：
Dengyuan Zhou;Chao Pei;Kelu Yang;Jing Ye;Shengfeng Wan;Qiu-Yu Li;Luping Zhang;Huanchun Chen;S. Cao;Yunfeng Song
日本脑炎病毒（JEV）是一种由库蚊传播的人畜共患病原体，是人类病毒性脑炎的主要病因。猪是乙脑病毒的主要扩增宿主，猪感染乙脑病毒可导致母猪繁殖失败；在公猪中，它可以引起睾丸炎和不育。猪乙脑病毒的流行是对人类健康的持续威胁。在猪群中监测乙脑病毒感染的实用诊断方法对于控制猪和人之间的疾病至关重要。本研究通过间接ELISA法鉴定出一种高亲和力的抗乙脑病毒NS1单克隆抗体（mAb），并利用其开发阻断型ELISA （bELISA）。棋盘滴定法测定最佳NS1蛋白包被浓度（2 μg/mL）和mAb工作浓度（1 μg/mL）。110份jev抗体阴性血清样品，确定34.03%的抑制率为阴性的临界值。通过bELISA， 80%的新接种猪在免疫后7天检测到血清转化，而通过基于包膜蛋白的商业化iELISA， 20%的新接种猪检测到血清转化。当我们使用这两种方法测试157个油田样品时，发现bELISA和商用iELISA的一致性为98.7%。对2014 - 2016年采集的猪血清进行流行病学调查发现，未接种疫苗的商品猪平均乙脑病毒血清阳性率为8.1%，接种疫苗的公猪和母猪平均乙脑病毒血清阳性率为67.6%。
Japanese encephalitis virus (JEV) is a zoonotic pathogen transmitted byCulexmosquitoes and is the leading cause of viral encephalitis in humans. JEV infection of swine, which are the main amplifying hosts for JEV, can cause reproductive failure in sows; in boars it can cause testitis and infertility. The prevalence of JEV in swine is a continuous threat to human health. A practical diagnostic method for monitoring JEV infection in swine herds is essential for control of the disease in both swine and humans. Here, we have identified a high-affinity anti-JEV NS1 monoclonal antibody (mAb) by indirect ELISA and utilized it for the development of a blocking ELISA (bELISA). The optimal NS1 protein coating concentration (2 μg/mL) and mAb working concentration (1 μg/mL) were determined by checkerboard titration. One hundred ten JEV-antibody-negative serum samples were used to establish 34.03% inhibition as the cutoff value for a negative result. By the bELISA, seroconversion in 80% of newly JEV-vaccinated pigs was detected by 7 days post-immunization, while by the commercial envelope-protein-based iELISA, seroconversion was detected in 20% of the newly vaccinated pigs. We found 98.7% agreement between the bELISA and the commercial iELISA when we tested 157 field samples using both methods. From an epidemiological survey of swine serum collected between 2014 and 2016, we found that the average JEV seropositive rate in unvaccinated commodity pigs was 8.1%, and in vaccinated boars and sows, it was 67.6%.