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

Preparation of specific monoclonal antibody against enterovirus 71 and its application in the virus identification

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 EV 71 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 EV 71-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 A 16 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 EV 71 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 EV 71 isolated from cell culture .