摘要
目的建立抗BPl80NCl6AIgE抗体酶联免疫吸附试验（ELISA）检测方法，评价其在大疱性类天疱疮（BP）诊断中的意义。方法采用pGEX一2T原核表达GST—BPl80NCl6A融合蛋白，并用谷胱甘肽亲和层析法进行纯化。采用棋盘滴定法确定抗原包被浓度，明确二抗及待测标本最佳稀释度，确定最佳实验条件。对2011年2月至2012年10月就诊于北京协和医院的56例BP患者、24名健康对照以及18例天疱疮、1例Stevens—Johnson综合征（SJS）患者血清进行检测，根据受试者工作特征（ROC）曲线确定最优阳性界值并评价其敏感度和特异度。结果确定抗原包被浓度为500μg/ml、酶标二抗最佳稀释倍数为1：1000，血清标本采用原液及1：10为最佳实验条件。根据ROC曲线判断最优阳性界值为0．549，实验的敏感度为71．4％、特异度为100％。天疱疮和SJS患者血清中此抗体均为阴性。结论抗BPl80NCl6AIgE抗体检测方法具有较高的特异度以及相对较高的敏感度，在BP的诊断及发病机制的研究中具有重要意义。
Objective To establish a method of detecting circulating immunoglobulin E （IgE） autoantibodies for BPISONC16A and evaluate its significance in bullous pemphigoid （BP）. Methods GST-NCI6A fusion proteins were expressed in Escherichia coli using the pGEX-2T expression system and purified by glutathione affinity chromatography. For optimal working conditions of enzyme-linked immunoabsorbent assay （ELISA）, checkerboard titrations were performed with serial dilutions of antigen. Also optimized dilution of secondary antibody was confirmed. Sera samples from 56 patients with BP, 24 healthy controls, 18 with pemphigus and 1 with Stevens-Johnson syndrome at our hospital during February 2011 to October 2012 were exanfined by the modified ELISA approach. The optimal cut-off point for a positive result was selected with receiver operating characteristic analysis. Results The optimized ELISA was performed with plates coated with 500 μg/ml GST-NC16A. And the optimal dilutions of sera samples and secondary antibody were 1：10 and 1：1000 respectively. According to the established cut-off value （0. 549） , 40 of 56 BP patients and none of controls had detectable levels of BP180NCI6A IgE. Conclusion The established EHSA provides a highly specific tool for the detection of IgE anti-BP180NC16A in BP patients.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2013年第28期2244-2247,共4页
National Medical Journal of China
基金
国家自然科学基金（81071301）
教育部新世纪优秀人才计划（NCET-10-0964）
