囊型棘球蚴病Antigen 5 iELISA检测方法的建立和应用

Establishment and application of iELISA detection method for Antigen 5 of echinococcosis

目的 基于Antigen 5(Ag5)建立一种具有较高灵敏度和特异度的检测囊型棘球蚴病(CE)的间接酶联免疫吸附试验(iELISA)法。方法 构建pGEX-6p-2-Ag5表达载体,经表达及亲和纯化后的重组蛋白作为包被抗原,经过标准品血清的检测,确定其临界值。对184例CE患者按照感染时期,包囊数量、状态,是否接受药物及手术治疗分组,利用建立的iELISA法进行检测,并与商品化试剂盒方法比较。结果 当临界吸光值(A)比值为0.46时,该iELISA法具有最佳的灵敏度(91.6%)和特异度(96.3%)。处于过渡期(CE3a、CE3b)的CE患者血清中含有较高水平的特异性抗体。iELISA方法对于CE4和CE5期患者的阳性率分别为83.3%和63.0%,明显高于商品化试剂盒检测的阳性率(P<0.05);iELISA方法对于健康人血清的检测,阳性率为2.4%。另外,活性包囊、多包囊以及经过药物治疗的CE患者具有更高的阳性率。结论 该研究建立的iELISA法具有较高的灵敏度及特异度,为CE患者的临床诊断提供了一种有价值的检测手段。

Objective To establish an indirect ELISA(iELISA)method with high sensitivity and specificity for detecting cystic echinococcosis(CE)based on Antigen 5(Ag5).Methods The pGEX-6p-2-Ag5 expression vector was constructed,and the recombinant proteins of Ag5 after expression and affinity purification served as the coating antigen.The critical value was determined by the standard serum detection.A total of 184 cases of CE were grouped according to the infection time,cyst number,status,whether receiving medication or operation treatment.The detection was performed by using the established iELISA and the results were compared with a commercial method.Results iELISA had the optimal sensitivity(91.6%)and specificity(96.3%)when the critical value absorbance(A)ratio was 0.46.The serum during the transitional stage(CE3a、CE3b)in the patients with CE contained the higher level of specific antibody.For the patients with stage CE4 and CE5,the positive rates of iELISA were 83.3%and 63.0%respectively,which were significantly higher than those of the commercial method.The positive rate of serum from healthy people detected by iELISA was 2.4%.In addition,the patients with active cyst,multiple cyst and medication treatment had higher positive rate.Conclusion iELISA established by this study has higher sensitivity and specificity and provide a valuable detection means for the clinical diagnosis of the patients with CE.