肠道病毒71型特异性单克隆抗体的制备及其在病毒鉴定中的应用-东南大学学报医学版

肠道病毒71型特异性单克隆抗体的制备及其在病毒鉴定中的应用

单位：东南大学医学院, 江苏南京 210009

分类号：R512.5

出版年·卷·期（页码）：2013·32·第六期（698-703）

摘要：

目的：建立肠道病毒71型（EV71）特异性检测的免疫荧光方法用于病毒分离培养后的EV71鉴定，为EV71生物学特征研究和疫苗株的筛选奠定基础。方法：以EV71 VP1为免疫原制备单克隆抗体，用经测序鉴定的肠道病毒毒株建立EV71特异性免疫荧光方法；将不同地区收集的手足口病患儿的临床标本进行病毒分离，利用EV71特异性单抗进行分离培养后的病毒鉴定。结果：制备获得2株单抗，经鉴定其中1株3E12为EV71特异性单抗；以此单抗建立的免疫荧光方法对EV71感染细胞呈阳性反应，而与柯萨基病毒A16和埃可病毒6等非EV71肠道病毒不反应。从104份手足口病临床标本中共分离到35株病毒，产生肠道病毒典型的细胞病变；用3E12特异性单抗免疫荧光鉴定分离病毒，其中29株为EV71，与RT-PCR验证结果完全一致。结论：成功制备获得EV71特异性单克隆抗体，以此单抗建立的EV71特异性免疫荧光方法敏感性高、特异性强，可用于分离培养EV71的鉴定。

Objective: To establish a specific immunofluorescence assay for identification of enterovirus 71 (EV71) after isolation from cell culture and lay the foundation for EV71 pathogen biology research and vaccine strains screening. Methods: Monoclonal antibodies (McAb) were prepared against EV71 VP1. An EV71-specific McAb and identified enteroviruses were used for establishment of an EV71-specific immunoflurescence assay. The assay was applied for EV71 identification after virus isolation from clinical samples collected from patients with hand, foot and mouth disease (HFMD) at different areas of China. Results: Two McAbs, designated 3E12 and 5C1, against EV71 VP1 were obtained. The McAb 3E12 was EV71-specific. The immunofluorescence assay established based on 3E12 was identified to detect EV71 only. Other enterovirus strains such as coxsackievirus A16 and ECHO virus 6 were not detected by the assay. Thirty-five virus strains were isolated with typical cytopathogenic effect of enterovirus in cell culture from 104 HFMD specimens. Twenty-nine isolated virus strains were identified as EV71 by the EV71-specific immunofluorescence assay, which was the same as the result tested by EV71 RT-PCR. Conclusion: The EV71-specific McAb is successfully prepared and used for establishment of the immunofluorescence assay with high sensitivity and specificity for identification of EV71 isolated from cell culture.

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