EV71抗原检测方法的建立及初步临床应用

Establishment and preliminary clinical application of EV71 antigen detection method

目的建立EV71抗原双抗体夹心检测方法,并初步探讨其在临床血清样品检测中的应用。方法用抗EV71病毒单克隆抗体包被酶联反应板,加入待检标本反应后,用HRP标记的抗EV71病毒单克隆抗体进行检测,建立EV71抗原双抗体夹心检测方法;采用抗人IgM单克隆抗体包被96孔酶联板,辣根酶标记的EV71抗原,建立ELISA捕获方法检测抗EV71-IgM。2种方法同时检测230份体检人员血清,确定方法的cut-off(CO)值,并检测140份手足口病患者血清,采用Graraph Pad Prism 4软件作图并绘制ROC曲线。结果EV71抗原检测方法的CO值为0.168,抗EV71-IgM ELISA检测方法的CO值为0.173。在140例手足口病患者血清中,EV71抗原检测方法的阳性检出率为45.00%,抗EV71-IgM ELISA检测方法的阳性检出率为42.86%,其中共同阳性患者45例,单独EV71抗原阳性患者18例,单独抗EV71-IgM抗体阳性患者15例,如果2种方法联合检测,其阳性检出率可提高到55.71%。结论ELISA检测EV71抗原诊断HFMD具有很好的准确性,结合EV71-IgM抗体捕获检测技术,可进一步提高灵敏度,具有很好的临床应用前景。

Objective To establish a double-antibody sandwich assay for EV71 antigen and to discuss its application in the detection of clinical serum samples.Methods The anti-EV71 virus monoclonal antibody was coated with the enzyme-linked reaction plate,and after the test sample was added,the HRP-labeled anti-EV71 virus monoclonal antibody was used for detection,and the EV71 antigen double antibody sandwich detection method was established;the anti-human IgM monoclonal antibody was used to coat the 96-well enzyme-linked plate,horseradish-labeled EV71 antigen,and an ELISA capture method was established to detect anti-EV71-IgM.Two methods were used to simultaneously detect the serum of 230 patients,determine the CO value of the method,and detect the serum of 140 patients with HFMD.The data were plotted using the Graraph Pad Prism 4 software and the ROC curve was drawn.Results The CO value of EV71 antigen detection method was0.168,and the CO value of anti-EV71-IgM ELISA was 0.173.In the serum of 140 HFMD patients,the positive detection rate of EV71 antigen detection method was 45.00%.The positive detection rate of anti-EV71-IgM ELISA was 42.86%.45 HFMD patients were all positive for EV71 antigen and anti-EV71 IgM antibody,18 HFMD patients were positive for EV71 antigen alone,and 15 HFMD patients were positive for anti-EV71 IgM antibody.If the two methods are combined,the positive detection rate can reach to 55.71%.Conclusion The accuracy of ELISA detection for HFMD with EV71 antigen is good.Combined with EV71-IgM antibody capture detection technology,it can further improve the sensitivity and has a good clinical application prospect.