肠道病毒EV71 IgM抗体参考品的建立

Establishment of the reference panel for EV71 IgM antibodies testing

目的建立肠道病毒EV71 IgM抗体检测用血清国家盘。 方法 通过收集手足口病感染早期患者的咽拭子和血清,对咽拭子病毒进行分离、鉴定、基因分型,通过血清中和试验及酶联免疫吸附试验（捕获法）的验证,确定EV71 IgM抗体阳性样品,组建EV71 IgM抗体国家参考品盘,并经3家实验室对国家参考品进行协同标定和确认。结果 12名患儿的咽拭子中均分离到EV71病毒,在RD细胞上引起细胞病变。 通过套式PCR扩增、序列测定后进化树分析确认均为流行的C4基因亚型 。每名患儿的双份血清均可中和EV71病毒,抗体效价为1∶512 － 1∶2048。 通过捕获法进行EV71 IgM抗体检测结果均为阳性 。 以此12份样品为原料制备EV71 IgM抗体阳性参考品,同时以12份EV71 IgM抗体阴性血清制备阴性参考品,建立了由12份阳性参考品、12 份阴性参考品、1 份最低检出限参考品和1份精密性参考品组成的肠道病毒EV71 IgM抗体参考血清盘。 通过3家实验室协同标定,各实验室间差异均无统计学意义。 结论 建立了EV71 IgM抗体检测参考血清盘,为相关检测试剂的质量控制和评价提供了标准。

Objective To develop the new national reference panel for testing intestinal virus EV71 IgM antibody. Methods The serum samples and throat swabs from HFMD patients were collected,and the swab samples were used for further virus isolating,identifying and genotyping. The EV71 IgM antibody positive samples were verified by neutralization test and ELISA（ capture method） to establish EV71 IgM antobody positive samples and construct the national reference panel. Results The EV71 virus from swabs were all isolated in 12 patients. These virus resulted in the pathological changes of RD cells. The isolated virus were confirmed as epidemic G4 subtype virus by the nested PCR,sequencing and phylogenetic tree analysis. The double samples from each patient could neutralize the EV71 virus. The antibody titers ranged from 1∶ 512 to 1∶ 2 048,and the tested results of IgM antibodies against EV71 virus were all positive. The EV71 IgM antibody positive reference was developed using 12 positive serum samples as raw materials,and at the same time,the negative reference for EV71 IgM antibody testing was also developed. New national reference panel for testing intestinal virus EV71 IgM antibody was tested by ELISA in the different laboratories. Conclusions The testing panel was suitable for the evaluation of IgM antibody against EV71 virus. It could be used as the reference of EV71 IgM diagnostic reagents. The sensitivity and accuracy of the panel was in line with the pharmacopeia requirements of the national reference materials of the P. R. China.