用不同基因型戊型肝炎病毒重组蛋白建立酶联免疫吸附测定一步法  

Establishment of enzyme-linked immunosorbent assay one-step assay based on recombinant proteins derived from different genotypes of hepatitis E virus

作　　者：周镇先[1] 丁福[2] 董晨[2] 龚希平[1] 耿全林[1] 孟继鸿[2] 

机构地区：[1]东南大学医学院附属南京市第二医院检验科,南京210003 [2]东南大学临床科学研究中心

出　　处：《中华传染病杂志》2009年第3期152-155,共4页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金资助项目（30271231、30671858）;国家863高技术基金资助项目（2006AA02A235）;江苏省自然科学基金资助项目（KB2006098）;江苏省高技术基金资助项目（BG2006607）;江苏省社会发展基金资助项目（BS2004506）

摘　　要：目的以7种不同基因型和亚型的HEV重组蛋白作为包被抗原，建立HEV抗体检测EuSA一步法。方法通过方阵滴定法确定包被抗原浓度，确定临界值，并进行敏感度、特异度和热稳定性试验。结果7种重组抗原混合物（Mix166）最佳包被浓度为1．5mg／L。抗-HEV IgG检测试剂的批内、批间变异系数分别为8．67％和10．85％，抗一HEV IgM检测试剂的批内、批间变异系数分别为4．56％和5．99％。一步法检测50份HEV RNA阳性血清的HEV IgG和HEV IgM抗体，阳性率均为94％。一步法检测674份健康者血清，52份抗-HEVIgG阳性．3份抗HEV IgM阳性。一步法检测HEV墨西哥株攻击黑猩猩后收集的系列血清发现，病毒攻击后1～6周抗-HEVIgM阳性，2～76周抗-HEV IgG阳性，而进口试剂盒缺乏对抗-HEV墨西哥株IgG、IgM的反应性。结论Mix166作为包被抗原建立的HEV抗体ELISA一步法具有较好的敏感性和特异性，可用于HEV感染的诊断。Objective To establish an anti-hepatitis E virus （HEV） enzyme-linked immunosorbent assay （ELISA） one step assay based on seven glutathione S transferase （GST）-fusion recombinant proteins derived from different HEV genotypes and subtypes. Methods Concentration of the coating antigen was optimized by block titration. The cut off values were determined for anti HEV IgG and IgM, respectively. Assay sensitivity, specificity and reproducibility were investigated using samples with confirmed anti HEV positive. Results An optimal concentration of mixture of recombinant proteins （Mix166） was 1.5 mg/L for antigen coating. Coefficient of variations （CV） of anti-HEV within-run and between-run were 8.67% and 10.85%, respectively. CV of anti HEV IgM within-run and between-run were 4.56% and 5.99%, respectively. Positive rates of anti-HEV IgG and IgM were both 94% for 50 HEV-polymerase chain reaction （PCR） positive sera tested with the one step assay. Using one-step assay to detect 674 serum samples from healthy people, 52 samples were found anti-HEV IgG positive and 3 samples were anti-HEV IgM positive. A series of serum specimens collected at different time points until 76 weeks from a chimpanzee challenged with HEV Mexican strain were anti-HEV IgM positive during week 1--6 and anti-HEV IgG positive during week 2--76 determined by the one step ELISA. However, import ELISA kits were lack of both the IgM and IgG reactivity to all of the serial chimpanzee sera. Conclusions The sensitivity and specificity of anti- HEV ELISA one-step assay based on the Mix166 antigen are high and could be used for the diagnosis of HEV infection.

关 键 词：肝炎病毒 戊型 重组 遗传 酶联免疫吸附测定 基因型 

分 类 号：R686[医药卫生—骨科学]