ELISA差减分析--一种简便实用的免疫逃逸变异HBsAg检测新方法

ELISA differential analysis:a novel convenient and practical method for detecting immune escape mutant HBsAg

目的:立足现有实验技术与试剂,建立一项血清免疫逃逸变异HBsAg检测的新方法。方法:以市售ELISA试剂筛选其检测信号在灰区范围的HBV感染者;采用2种不同免疫逃逸变异HBsAg检测能力的ELISA试剂对选留血清进行检测,以"ELISA差减分析"方式确认免疫逃逸变异HBsAg阳性者;以中和实验及雅培化学发光试剂验证检测结果的特异性;并对部分免疫逃逸变异HBsAg阳性者以PCR及直接测序做HBV DNA分析。结果:自10231例受检者中筛选HBsAgELISA检测信号灰区标本244例;复核检测显示G6ELISA、市售ELISA两者对野生HBsAg检测敏感性分别为0.0625ng/ml,与0.125ng/ml;检测阳性率分别为16.80%(G6ELISA)及5.73%(市售ELISA)。ELISA差减分析确定免疫逃逸变异HBsAg阳性者27例,阳性率为11.07%。对部分免疫逃逸变异HBsAg阳性血清做进一步考核,抗HBs中和强度为(84.07%±6.32%),中和率100%(13/13);雅培化学发光试剂复核阳性率为94.4%(17/18,阳性者中包括两份单项G6-ELISA检测最低阳性信号标本);HBV DNA阳性率36.9%(7/19),略低于同期HBsAg低信号阳性者(6/14,42.9%,P<0.05);直接测序发现其中"a"区变异3例,分别为P142L、T126A及M133T/K160T;S蛋白亲水区非"a"决定簇变异1例,变异类型为L109Q。结论:ELISA差减分析法能有效地用于部分免疫逃逸变异HBsAg的临床诊断,其诊断能力视所依托试剂免疫逃逸变异检测能力不同而有显著差别。

Objective：To design a novel method to detect the immune escape mutant HBsAg in serum on the basis of available experimental technique and reagents.Methods：Using the market ELISA to screen the patients＇ sera that were infected by HBV and the ELISA signals were in grey area.Applying two ELISA kits capable of screening the immune escape mutant HBsAg,the selected sera were detected.The positive number of the immune escape mutant HBsAg was comfirmed by the ELISA differential analysis.The specificity of the assay was checked through both neutralising experiment and Abbott chemiluminescence kits.Then a part of the positive sera of the immune escape mutant HBsAg were detected by PCR and direct sequencing to analyze viral DNA.Results：There were 244 samples in gray area when screened for 10,231 samples using market ELISA.The results showed that sensibility of the two kinds of ELISA （G6-ELISA,market ELISA） was 0.125 ng/ml and 0.25 ng/ml,respectively.The detected positive rate was 16.80%（G6 ELISA） and 5.73%（ELISA）,respectively.The positive number of the immune escape mutant HBsAg,which were detected by the ELISA differential analysis was 27.The positive rate was 11.07%.Partial positive serum samples of the immune escape mutant HBsAg were detected further.The neutralising intensity of anti-HBs was 84.07%±6.32%,and neutralising rate reached 100%（13/13）.The positive rate of Abbott chemiluminescence kits was 94.4%（17/18）,including two samples with the lowest signals in G6-ELISA.The positive rate of HBV DNA was 36.9%（7/19）,which was slightly lower than the synchronization lower signal positive of HBsAg.The direct sequence showed that 3 mutants were found in ＇a＇ determinant （P142L,T126A and M133T/K160T） and 1 mutant in the hydrophilic area of S protein but not in ＇a＇determinant （L109Q）.Conclusion：ELISA differential analysis can be applied to diagnose partial immune escape mutant HBsAg effectually.The diagnosis capability of this method depends on the detecting capability of the regents to the immune escape mutant HBsAg.