CAJ | 학술논문

目的建立抗BP180NC16A IgE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-2T原核表达GST-BP180NC16A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500 μg/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BP180NC16A IgE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.
목적 건립항BP180NC16A IgE항체매련면역흡부시험(ELISA)검측방법,평개기재대포성류천포창(BP)진단중적의의.방법 채용pGEX-2T원핵표체GST-BP180NC16A융합단백,병용곡광감태친화층석법진행순화.채용기반적정법학정항원포피농도,명학이항급대측표본최가희석도,학정최가실험조건.대2011년2월지2012년10월취진우북경협화의원적56례BP환자、24명건강대조이급18례천포창、1례Stevens-Johnson종합정(SJS)환자혈청진행검측,근거수시자공작특정(ROC)곡선학정최우양성계치병평개기민감도화특이도.결과 학정항원포피농도위500 μg/ml、매표이항최가희석배수위1∶1000,혈청표본채용원액급1∶10위최가실험조건.근거ROC곡선판단최우양성계치위0.549,실험적민감도위71.4％、특이도위100％.천포창화SJS환자혈청중차항체균위음성.결론 항BP180NC16A IgE항체검측방법구유교고적특이도이급상대교고적민감도,재BP적진단급발병궤제적연구중구유중요의의.
Objective To establish a method of detecting circulating immunoglobulin E (IgE) autoantibodies for BP180NC16A and evaluate its significance in bullous pemphigoid (BP).Methods GST-NC16A 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 examined 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 BP180NC16A IgE.Conclusion The established ELISA provides a highly specific tool for the detection of IgE anti-BP180NC16A in BP patients.