摘要
目的：建立一种新型的快速、高通量的埃可病毒25型（ECHO25）中和抗体检测方法,并初步评价其在ECHO25中和抗体筛选和血清流行病学调查中的应用价值。方法：应用免疫荧光方法筛选ECHO25高亲和性抗体并将其作为检测单抗,结合酶联免疫斑点检测技术（ELISPOT）建立ECHO25中和抗体检测方法;使用不同效价的血清评价该方法的准确性;采用所建立的中和方法对ECHO25单克隆抗体、临床血清样品进行检测。结果：建立了快速检测ECHO25中和抗体的Nt-ELISPOT方法,以ECHO25单克隆抗体5B9作为检测抗体;相比经典的中和实验方法 Nt-CPE,该方法可显著缩短检测时间（从5～7 d缩短至1 d以内）,检测结果具有较好的一致性;采用所建立的Nt-ELISPOT方法首次筛选获得3株对ECHO25具有较好中和能力的单克隆抗体;临床血清样品检测结果显示厦门地区可能存在ECHO25的流行。结论：该方法可以应用于中和抗体筛选和血清学的临床辅助诊断,为ECHO25的防治研究提供支持。
Objective： To establish a rapid and high-throughput neutralization test for echovirus 25（ECHO25）, and evaluate its application in neutralizing antibody screening and seroepidemiological surveys. Methods： Immuno- fluorescence assay was applied to screen a high affinity antibody, which was used as the detection antibody for ECHO25, and a rapid neutralization test was established based on enzyme-linked immunospot assay （Nt- ELISPOT）. The accuracy of this method was evaluated by detecting serum samples with different titer. Monoclonal antibodies against ECHO25 and clinical serum samples were detected via the established neutralization test. Re- suits： A rapid method to detect neutralizing antibody against ECHO25 was established and an anti-ECHO25 anti- body, 5B9, was used as the detection antibody. The detection period could be shortened significantly compared with the classical neutralization test（Nt-CPE）（from five to seven days to less than one day）, and the Nt- ELISPOT had good consistency with the Nt-CPE. Meanwhile, three neutralizing antibodies for ECHO25 were screened firstly by this method. The detection results of clinical serum samples showed that infection of ECHO25 might be popular in Xiamen. Conclusion： This method can be used in neutralizing antibody screening and seroepi- demiological surveys, and it may provide support for the control of ECHO25.
出处
《生物技术通讯》
CAS
2016年第1期52-57,共6页
Letters in Biotechnology
基金
国家自然科学基金(81371817
81401669)
关键词
埃可病毒25型
中和实验
酶联免疫斑点法
中和抗体
echovirus 25
neutralization teat
enzyme-linked immunospot assay
neutralizing antibody
