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去唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGPR)是肝细胞膜表面特有的一种内吞性受体,H1是参与内吞功能的主要亚基。自身免疫性肝炎(autoimmune-hepatitis,AIH)患者存在抗-ASGPR的自身抗体。本研究以去唾液酸糖蛋白受体H1亚基在大肠杆菌中诱导表达,用纯化的重组去唾液酸糖蛋白受体H1亚单位rH1作为检测抗原,建立间接法rH1-IgG-ELISA,检测抗去唾液酸糖蛋白受体IgG抗体,观察其阳性和阴性符合率,并对rH1-IgG-ELISA检测的精确度、灵敏度和特异性进行评价。结果显示制备的rH1重组蛋白纯度在90%以上;以该重组抗原建立的rH1-IgG-ELISA的最佳检测条件为:重组抗原rH1的包被浓度为6μg/ml,血清稀释度1:100,酶标记的羊抗人IgG 1:3000稀释;用rH1-IgGELISA对混合ASGPR阳性和阴性血清的重复检测表明:IgG阳性血清的检测值的变异系数(CV值)为9.9%,IgG阴性血清的CV值为9.7%;灵敏度检测表明血清稀释度在1:50~1:200均可检出阳性;特异性试验的抑制率为63.5%;rH1-IgG-ELISA与总符合率为87.36%,其中阳性和阴性符合率分别为82%(41/50)和93.33%(42/45)。故建立的rH1-IgGELISA具有较好的灵敏性和特异性,与提供的ASGPR阳性血清有较高的符合率,表明该法具有较好的AIH诊断价值,可进一步推广应用。
Asialoglycoprotein receptor (ASGPR) is an endocytic receptor specific on the surface of hepatocytes. H1 is the major subunit involved in endocytosis. Anti-ASGPR autoantibodies exist in patients with autoimmune-hepatitis (AIH). In this study, the expression of asialoglycoprotein H1 subunit was induced in E.coli. The purified rH1 recombinant asialoglycoprotein receptor H1 subunit rH1 was used as detection antigen to establish indirect ELISA rH1-IgG-ELISA. IgG antibodies against sialoglycoprotein receptor were assayed for positive and negative coincidence rates and the accuracy, sensitivity and specificity of the rH1-IgG-ELISA assay were evaluated. The results showed that the purity of rH1 recombinant protein was over 90%. The optimum conditions of rH1-IgG-ELISA assay were as follows: the concentration of recombinant rH1 was 6μg / ml, the dilution of serum was 1:100, Repeated detection of mixed ASGPR-positive and -negative sera with rHl-IgG ELISA showed a coefficient of variation (CV) of 9.9% for the detection of IgG-positive sera, a CV of IgG-negative sera The value of which was 9.7%. The sensitivity test showed that the serum dilution was positive at 1: 50-1: 200; the inhibition rate of specificity test was 63.5%; and the total coincidence rate of rH1-IgG-ELISA was 87.36% And negative coincidence rates were 82% (41/50) and 93.33% (42/45), respectively. Therefore, the establishment of rH1-IgGELISA has good sensitivity and specificity, and provide a high coincidence rate of ASGPR-positive sera, indicating that the method has a good diagnostic value of AIH, which can be further promoted and applied.