G145R变异HBsAg免疫检测方法的建立及其应用特征的实验研究

Establishment of detecting G145R mutant HBsAg by enzyme-linked immunosorbent assay and experimental study of its clinical ap- plication

目的：建立G145R变异HBsAg检测ELISA，并探讨其应用特征。方法：采用SPA亲和层析纯化抗体；以抗G145R变异HBsAgMcAb包被，羊抗HBs-HRP示踪，建立双抗体夹心ELISA；将该法初步应用于临床标本的检测，并将阳性标本以PCR、DNA直接测序、雅培Abbott试剂等方法做对比分析，探讨其临床检测特征。结果：该法能特异检测G145R变异HBsAg，而与野生型HBsAg无交叉反应，灵敏度约为2．0μg/L；平均批内及批间CV分别为（8．87±3.04）％和（8．3±2．95）％。该法从206例特定HBV感染相关标本中检出阳性18份，阳性率为8、7％；18份阳性标本中HBVDNA阳性6例，直接测序检出1126A及M133T各一例；雅培Abbott试剂检测该6份标本HBsAg含量在（4．62～211）IU／ml间。结论：建立了一种快速、简便的G145R变异HBsAg检测ELISA，该法也能检测部分具有类似抗原性漂移的其它逃逸变异HBsAg。

Objective To develop enzyme-linked immunosorbent assay for G145R mutant HBsAg detection, and preliminarily investigate it＇s characteristic in clinical serological detection. Metheds SPA affinity chromatograph was used to purify antibody. The sandwich ELISA was developed based on the anti-G145R HBsAg McAb and peroxidase-labeled goat anti-HBs antibody which were used as coating and tracing antibody respectively. The experiments of superseding, competitive inhibition and neutralization were chosen to confirm specificity of this assay. To evaluate the clinical application characteristic of this assay, the positive sera which were confirmed in this assay were detected for HBV DNA, ＂a＂ determinant mutation and HBsAg by PCR, DNA direct sequencing and Abbott chemiluminescence, respectively. Resuits This assay can detect the G145R mutant r-HBsAg with high specificity, the sensitivity is 2. 0μg/L. The intra-assay and inter-assay coefficient of variation were （ 8. 87 ± 3.04 ） % and （ 8.3 ±2.95 ） %. 206 HBV-associated specimens were tested with this assay, the rate of positive was 8.7%. There were 6 out of 18 specimens that were picked out by this ELISA, but none of the 6 specimens was confirmed to be G145R mutant. The 6 specimens were positive in HBsAg detection by Abbott chemiluminescence. Conclusion A quick and convenient ELISA for G145R mutant HBsAg was established successfully. And it can also detect some other kinds of escape mutants that caused the similar protein structure as the G145R mutant.