肠道病毒68型新型中和试验方法的建立及其初步应用

发布时间：2018-05-16 04:37

本文选题：肠道病毒型(EV-D) + 中和试验　；参考：《病毒学报》2017年05期

【摘要】：针对肠道病毒68型(EV-D68)建立一种新型快速、高效的中和试验方法,并初步评价其在EV-D68流行病学研究中的应用价值。以灭活EV-D68病毒免疫Balb/c小鼠,利用杂交瘤技术制备其单克隆抗体;筛选反应性好、特异性强的抗EV-D68单克隆抗体作为检测抗体,基于酶联免疫斑点检测技术(ELISPOT)建立EV-D68高效中和试验方法;进一步比较该方法与传统中和试验方法 Nt-CPE的一致性,并使用该方法对临床血清样本进行检测。共获得10株分泌抗EV-D68单克隆抗体的杂交瘤细胞株,选取一株性能最优的15C5进行生产纯化,鉴定其为IgG3亚型;以15C5为检测抗体,建立了快速检测EV-D68中和抗体的Nt-ELISPOT方法;与Nt-CPE方法比较,Nt-ELISPOT方法将检测时间由5~7d缩短至1d以内,并且两种方法检测结果一致性良好;应用所建立的Nt-ELISPOT方法对厦门地区146份人群血清样本进行检测,显示血清样本EV-D68中和抗体阳性率为98.6%(144/146),提示厦门地区可能存在过EV-D68的流行。本研究基于ELISPOT建立的新型EV-D68中和试验方法快速可靠,适合通量检测,可以应用于EV-D68中和抗体水平的血清学调查,为EV-D68感染的防控工作提供帮助。
[Abstract]:A new rapid and efficient neutralization test for enterovirus 68 (EV-D68) was established and its application value in epidemiological study of EV-D68 was preliminarily evaluated. Balb/c mice were immunized with inactivated EV-D68 virus, and their monoclonal antibodies were prepared by hybridoma technique. The high efficiency neutralization test method of EV-D68 was established based on Elisa dot assay (Elisa), and the consistency of this method with the traditional neutralization test method (Nt-CPE) was compared, and the clinical serum samples were detected by this method. Ten hybridoma cell lines secreting monoclonal antibodies against EV-D68 were obtained, and a 15C5 with the best performance was selected for production and purification, which was identified as IgG3 subtype, and a Nt-ELISPOT method for rapid detection of neutralizing antibodies of EV-D68 was established with 15C5 as antibody. Compared with Nt-CPE method, Nt-Elispot method shortened the detection time from 5 days to less than 1 day, and the results of the two methods were in good agreement, and the established Nt-ELISPOT method was used to detect 146 human serum samples in Xiamen area. The results showed that the positive rate of EV-D68 neutralizing antibody in serum samples was 98.6% 14.4% 146G, which suggested that there might be an epidemic of EV-D68 in Xiamen area. The new EV-D68 neutralization test method based on ELISPOT is fast and reliable and suitable for flux detection. It can be applied to the serological investigation of EV-D68 neutralizing antibody level and provide help for the prevention and control of EV-D68 infection.
【作者单位】：国家传染病诊断试剂与疫苗工程技术研究中心厦门大学生命科学学院;分子疫苗学和分子诊断学国家重点实验室厦门大学公共卫生学院;厦门市疾病预防与控制中心;
【基金】：国家自然科学基金(项目号:31670933),题目:手足口病肠道病毒跨血清型广谱中和抗体的表位结构与作用机制研究;国家自然科学基金(项目号:81601762),题目:人补体调节蛋白CD59在水痘-带状疱疹病毒免疫逃逸中的作用研究福建省自然科学基金(项目号:2015J05073),题目:柯萨奇病毒A16型(CA16)广谱中和表位的结构基础~~
【分类号】：R446.6;R512.5

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