隐匿性HBV感染相关S基因突变对HBsAg检测的影响及其机制

目的 探讨隐匿性HBV感染(OBI)相关S基因突变对多种抗-HBs及HBsAg临床检测试剂的反应性.方法 9种代表性S基因突变株(M1-M9,其中M1-M5为本课题组首次报道)分离自1例OBI患者和3例OBI献血人员血清.分别采用野生型及突变型S基因重组质粒转染中国仓鼠卵巢细胞,分析9种突变株HBsAg表达及其突变蛋白对抗体和检测试剂反应的影响.结果 采用HBsAg罗氏诊断试剂Elecsys定量检测9种突变株胞内HBsAg水平,均较野生株的水平明显下降;相同样品用抗-His抗体检测显示仅M1、M6和M7的HBsAg-His融合蛋白表达水平与野生株比较明显降低.6种抗-HBs与各突变HBsAg的反应性结果(S/CO值)显示,与野生株相比,多数突变株与6种抗-HBs的反应性明显降低,M1、M4、M7和M9产生的HBsAg与抗体4及M9产生的HBsAg与抗体6反应性最差(S/CO＜1).6种商品化HBsAg临床试剂检测结果显示,多数突变株与6种HBsAg试剂反应性较野生株明显降低(P＜0.05),ELISA试剂D、E和F漏检率分别为11.1％、22.2％和55.6％.结论 本实验涉及的突变HBsAg与抗-HBs的结合力降低是引起OBI表现的主要原因之一.当前临床常用HBsAg检测试剂对突变HBsAg的检测能力存在明显不足,亟待提高.

Analysis of the influence of nine occult HBV infection-related S-gene mutations on HBsAg detection

Objective To determine the reactivity of occult HBV infection (OBI)-related S-gene mutations to various anti-HBs antibodies (Abs) and HBsAg detection reagents.Methods Nine representative S-gene mutations M1-M9 (including 5 novel mutations M1-M5) from 1 OBI patient and 3 OBI blood donors were investigated.S-gene recombinant plasmids harboring 9 mutants or wild-type sequences constructed before were transfected into Chinese hamster ovary (CHO) cells,respectively.HBsAg expression levels and reactivity with various anti-HBs Abs and regular HBsAg detection reagents of all the mutants were analyzed.Results Compared to wild-type strain,intracellular HBsAg levels of the 9 mutants were obviously reduced upon Roche quantitative Elecsys assay.In contrast,analysis of the same samples using anti-His-tag Ab showed that the levels of HBsAg-Histag fusion protein were significantly reduced only in mutants M1,M6,and M7.The results of reactivity of mutant HBsAg against 6 anti-HBs Abs (S/CO values) showed that poor reactivity was observed for most mutants.Specifically,M1-,M4-,M7-,and M9-produced HBsAg with Ab4,and M9-produced HBsAg with Ab6 had the worst reactivity (S/CO＜1) compared to the wild-type.The results of reactivity of mutant HBsAg with 6 commercial HBsAg detection reagents showed that the reactivity for most mutants was significantly lower than that of the wild-type (P＜0.05).The miss rates of ELISA reagents D,E,and F were 11.1％,22.2％,and 55.6％,respectively.Conclusions Lower-affinity of studied mutant HBsAg with anti-HBs is one of the major causes of OBI presentation.The regular HBsAg detection reagents used currently in clinic show a significant deficiency in detection of the mutant HBsAg and thus need to be improved.