摘要目的 建立抗BP180NC16A IgG亚型的检测方法,并探讨其在大疱性类天疱疮(BP)中的意义.方法 原核表达GST-NC16A融合蛋白,并采用亲和层析法纯化.优化ELISA关键环节,建立抗BP180NC16A IgG各亚型的ELISA检测方法,并对10例未经治疗的BP、5例妊娠疱疹、1例成人线状IgA大疱性皮病、2例天疱疮患者血清分别进行检测.结果 通过方阵测定法确定GST-NC16A融合蛋白的包被浓度为500 μg/L,包被条件为4℃12h,血清稀释倍数为1∶100,酶标二抗为1∶2000,孵育条件为37℃1h,底物反应条件37℃20 min.10例大疱性类天疱疮患者10例IgG1阳性,9例IgG2阳性,5例IgG3阳性,9例IgG4阳性.2例寻常型天疱疮、1例成人线状IgA大疱性皮病均阴性.5例妊娠疱疹所有亚型均阳性,以IgG1和IgG3亚型为主.结论 抗BPI80NCI6A ELISA检测法特异性强、重复性好,是检测BP和妊娠疱疹患者抗BP180NC 16A抗体亚型的半定量方法.

abstractsObjective To develop an assay to quantitatively detect anti-BP180NC16A IgG subclasses and to assess the significance of anti-BP180NC16A lgG in bullous pemphigoid (BP).Methods The Glutathione S-transferase (GST)-BPI80NC16A fusion protein was expressed in E.coli system and purified by affinity chromatography.An improved enzyme-linked immunoabsorbent assay (ELISA) was developed and used to detect anti-BP180NCI6A IgG subclasses in serum samples from 10 patients with BP,5 patients with pemphigoid gestationis (PG),1 patient with linear IgA bullous dermatosis (LIBD) and 2 patients with pemphigus.Results The optimal condition for the ELISA was determined by cross assay as follows:the concentration of GSTBP180NC16A fusion protein for coating,500 μg/L; the condition for coating,4 ℃ for 12 hours; the dilution ratio of sera and secondary antibody,1∶ 100 and 1 ∶ 2000 respectively; the condition for incubation,37 ℃ for 1 hour;,the condition for the enzyme-substrate reaction,37 ℃ for 20 minutes.Of the 10 patients with BP,all were positive for anti-BP180NC16A IgG1,9 for IgG2,5 for IgG3,and 9 for IgG4.Anti-BP180NC16A IgG was undetected in any of the serum samples from 2 patients with pemphigus vulgaris or 1 patient with adult LIBD.All the 5 sera from patients with PG; were positive for all the anti-BP180NC16A IgG subclasses,which were predominated by IgGl and IgG3.Conclusion The developed ELISA is a highly specific and reproducible semiquantitative method for the detection of anti-BP180NCI6A IgG subclasses in patients with BP and PG.