CAJ | 학술논문

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3 E12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。
목적：건립장도병독71형（ EV71）특이성검측적면역형광방법용우병독분리배양후적EV71감정，위EV71생물학특정연구화역묘주적사선전정기출。방법：이EV71 VP1위면역원제비단극륭항체，용경측서감정적장도병독독주건립EV71특이성면역형광방법；장불동지구수집적수족구병환인적림상표본진행병독분리，이용EV71특이성단항진행분리배양후적병독감정。결과：제비획득2주단항，경감정기중1주3 E12위EV71특이성단항；이차단항건립적면역형광방법대EV71감염세포정양성반응，이여가살기병독A16화애가병독6등비EV71장도병독불반응。종104빈수족구병림상표본중공분리도35주병독，산생장도병독전형적세포병변；용3E12특이성단항면역형광감정분리병독，기중29주위EV71，여RT-PCR험증결과완전일치。결론：성공제비획득EV71특이성단극륭항체，이차단항건립적EV71특이성면역형광방법민감성고、특이성강，가용우분리배양EV71적감정。
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 .