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-ELIS-POT方法将检测时间由5~7d缩短至ld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。We wished to establish a novel, rapid and efficient neutralization test; for enterovirus D68 (EV-D68) and evaluate its application in; seroepidemiological studies. Balb/c mice were immunized with inactivated; EV-D68. Monoclonal antibodies against EV-D68 were prepared by the; hybridoma method. Anti-EV-D68 monoclonal antibodies with high affinity; and specificity were selected as detection antibodies for the; establishment of a rapid neutralization test for EV-D68 using the; enzyme-linked immunospot (ELISPOT) assay. Also, the consistency between; the established method and conventional one, neutralization test based; on the inhibition of cytopathic effects (Nt-CPE),was investigated.; Finally, the established method was evaluated with clinical serum; samples. Ten hybridoma cell lines secreting monoclonal antibodies; against EV-D68 were obtained, and the best of them, 15C5, was selected; for the production and characterization of monoclonal antibodies.; Results showed that 15C5 was an immunoglobulin(Ig) G3 subclass. A rapid; method for the detection of neutralizing antibodies against EV-D68,; Nt-ELISPOT, was established using 15C5 as the detection antibody.; Nt-ELISPOT could shorten the detection time substantially from 5~7 days; in conventional Nt-CPE to <1 day, and there was good consistency between; the two methods. The established Nt-ELISPOT was applied to 146 clinical; serum samples from Xiamen, China, and the positive detection rate was; 98. 6% (144/146),suggesting that EV-D68 infection might be prevalent in; Xiamen. The established novel neutralization test for EV-D68 based on; ELISPOT was rapid and reliable. This method could be used to detect; EV-D68 neutralizing antibodies in seroepidemiological studies, thereby; providing support for the prevention and control of EV-D68 infection.国家自然科学基金; 国家自然科学基金; 福建省自然科学基
