摘要目的 建立并优化大疱性类天疱疮(BP)抗BP180NC16A IgG抗体亚型的酶联免疫吸附试验(ELISA)检测方法,评估其敏感性和特异性.方法 通过在大肠杆菌中表达并纯化GSTBP180NC16A重组蛋白,建立抗BP180NC16A IgG亚型的ELISA检测方法,检测2009至2012年北京协和医院136例BP患者血清中抗体水平,以20名健康体检者血清作为对照;阳性界值为健康对照血清测得的均值加3倍标准差,高于该界值的判断为阳性.并利用Western印迹法检测其中4种抗体均阳性的患者血清,和ELISA方法进行比较.结果 ELISA法检测的特异性IgG1、IgG2、IgG3和IgG4的阳性界值分别为0.113、0.196、0.154和0.120,20名健康对照4种IgG亚型检测值均低于阳性界值水平,使本检测方法特异性较好.136例患者抗BP180NC16A IgG1、IgG2、IgG3及IgG4抗体亚型阳性率分别为67.6％ (92/136)、45.6％ (62/136)、50.7％(69/136)和54.4％(74/136);其中4种IgG亚型均阳性者29例,占21.3％;至少一种IgG亚型为阳性者112例,占82.4％,即4种亚型综合敏感度为82.4％.而Western印迹检测20例经ELISA证实4种IgG亚型抗体均阳性的患者血清显示IgG1和Igc4阳性人数分别为15、14例.结论 本研究建立的ELISA方法的特异性较好,敏感性尚需进一步提高.

abstractsObjective To evaluate the distribution of four IgG subclasses targeting NC16A domain of BP180 in bullous pemphigoid (BP) patients by developing and optimizing a detection method of antiBP180NC16A IgG subclasses so as to assess its sensitivity and specificity.Methods Enzyme-linked immunosorbent assay (ELISA) was developed with recombinant GST-BP180NC16A proteins generated by a bacterial expression system.And 136 BP sera and 20 healthy control sera from our hospital between 2009 and 2012 were tested by ELISA,and the cutoff value of four IgG subclasses was set at an A reading corresponding to the mean value plus 3 times of standard deviation of 20 healthy controls sera.Western blot was also used to detect the IgG subclasses in patients with four positive IgG subclasses by ELISA.Results The cutoff value of specific IgG1,IgG2,IgG3 and IgG4 were 0.113,0.196,0.154 and 0.120.The values of four IgG subclasses from 20 healthy controls were lower than the corresponding cutoff value,making the detection system good specificity.The positive rates of anti-BP180NC16A IgG1,IgG2,IgG3 and IgG4 antibody were 67.6％ (92/136),45.6％ (62/136),50.7％ (69/136) and 54.4％ (74/136) respectively in 136 BP sera.All four IgG subclasses were positive in 29 BP sera,accounting for 21.3％.The number of BP sera positive for at least one IgG subclass were 112,accounting for 82.4％,indicating that the combined sensitivity of four IgG subclasses was 82.4％.Western blot revealed that the number of positivity was 15 and 14 for IgG1 and IgG4 respectively in 20 BP patients with four IgG subclasses positive with ELISA.Conclusion The specificity of ELISA is excellent while its sensitivity needs further improvements.