抗BP180N C16A IgG亚型检测方法的建立以及在大疱性类天疱疮中的意义

Development of a method to detect anti-BP180NC16A IgG subclasses and the significance of anti- BP180NC16A IgG in bullous pemphigoid

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BP180NC16AIgG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500μg／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℃20min。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BP180NC16AELISA检测法特异性强、重复性好，是检测BP和妊娠疱疹患者抗BP180NC16A抗体亚型的半定量方法。

Objective 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）-BP180NC16A 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-BP180NC16A 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 GST- BP180NC16A 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 IgG1 and IgG3. Conclusion The developed ELISA is a highly specific and reproducible semiquantitative method for the detection of anti-BP180NC16A IgG subclasses in patients with BP and PG.