肠道病毒71型手足口病ELISA诊断试剂盒研制与临床应用

目的 研制早期快速检测抗肠道病毒71型(EV71)抗体的ELISA血清学诊断性试剂盒,评价其临床应用价值.方法 将表达纯化的EV71重组蛋白VP1作为包被抗原,建立EV71型手足口病间接ELISA的血清学检测方法 .通过与逆转录(RT)PCR方法 、EV71病毒分离试验和微量血清中和试验比较,评价抗.EV71 IgM和抗-EV71 IgG血清学诊断方法 在EV71型手足口病的诊断价值.结果 与RT-PCR比较,抗-EV71 IgM敏感度、特异度、阳性预测值和阴性预测值分别为83%、85%、81%和87%;抗.EV71 IgG敏感度、特异度、阳性预测值和阴性预测值分别为72%、74%、68%和77%.与EV71病毒分离方法 比较,抗-EV71 IgM敏感度、特异度分别为85%和97%;抗-EV71 IgG敏感度、特异度分别为75%和77%.通过直线相关分析发现,抗-EV71 IgG抗体滴度和中和抗体滴度呈显著正相关(r=0.72,P＜0.05).EV71型手足口病患儿恢复期血清抗IgG滴度较急性期升高(P＜0.01),但抗IgM滴度差异无统计学意义(P＞0.05).结论 利用VP1重组蛋白作为包被抗原,成功开发ELISA检测人血清抗-EV71 IgM和抗-EV71 IgG抗体的诊断试剂盒.

Development of a serological ELISA kit for detection of EV71 infection associated with hand-foot and mouth disease and its clinical application

Objective To develop an ELISA(Enzyme-Linked Immunosorbent Assay)diagnostic kit for early rapid detection of sarum anti-EV71 antibody and evaluate its clinical application value.Methods Recombinant protein VP1 of EV71 were prepared and purified as an immobilized antigen for establishment of an indirect ELISA for detection of serum anti-EV71 IgM and anti-EV71 IgG.Compared with RT-PCR.isolation of EV71 and micro-neutralizing assay.the clinical application value of anti-EV71 IgM and anti-EV71 ISG in the diagnosis of EV71 disease was evaluated.Results In comparison with RT-PCR.the sensitivity,specificity,positive predictive value and negative predictive value of anti-EV71 IgM antibody were 83%,85%,81%and 87%,respectively.The sensitivity,specificity,positive predictive value and negative predictive value of anti-EV71 IgG antibody were 72%,74%,68%and 77%.respectively.Compared with viral isolation assay.the sensitivity and specificity of anti-EV71 IgM antibody were 85%and 97%,respectively.The sensitivity and specificity of anti-EV71 IsG antibody were 75%and 77%,respectively.In addition.the titers of anti-EV71 IgG antibody were significantly correlated with the titers of neutralizing antibody to EV71 by linear regression analysis(r=0.72,P＜0.05).Finally,the serum titers of anti-IgG from patients with EV71 associated hand food and mouth disease at convalescent stage exhibited significantly higher than that of the same patients at acute stage(P＜0.01),but the titers of anti-IgM had no significant difference(P＞0.05).Conclusions With VP1 recombinant protein used as an immobilized antigen,an indirect ELISA diagnostic kit was successfully develooed for detection of serum anti-human EV71 IgM and anti-human EV71 IgG antibodies.