基于热分离法制备的人表皮侧抗原的Western印迹实验对大疱性类天疱疮的诊断价值评估

Evaluation of the value of Western blot analysis based on human epidermal extracts prepared by thermal separation in the diagnosis of bullous pemphigoid

目的通过热分离方法制备人表皮侧蛋白,评价表皮侧蛋白Western印迹在大疱性类天疱疮(BP)诊断中的价值。方法热分离健康人皮肤制备人表皮侧抗原,以此为底物,对2015年1月至2017年8月在中国医学科学院皮肤病医院住院的22例BP患者及25例非BP对照血清行Western印迹检测,同时对所有受试者血清行BP180 NC16A ELISA检测。应用统计软件SPSS22.0进行统计学分析。计数资料采用χ2检验及Fisher精确检验法分析。结果表皮侧蛋白Western印迹、BP180 NC16A ELISA诊断BP的敏感性分别为86.36%(95% CI为64.03%~96.41%)、95.45%(95% CI为75.11%~99.76%)(P=0.294),特异性分别为100%(95% CI为83.42%~100%)、92%(95% CI为75.11%~99.76%)(P=0.149)。BP患者表皮侧Western印迹条带分析显示,4例见相对分子质量(RMM)为230 000的蛋白条带,18例见180 000的蛋白条带,1例见120 000和1例见97 000蛋白条带,且为180 000蛋白条带的BP患者BP180 NC16A ELISA结果均大于50 U/ml。结论对BP患者行表皮侧蛋白Western印迹检测,主要阳性条带RMM为180 000;表皮侧蛋白Western印迹诊断BP的敏感性低于BP180NC16A ELISA,当患者自身抗体滴度较低时表皮侧蛋白Western印迹结果往往为阴性。

Objective To prepare human epidermal extracts by thermal separation, and to evaluate the value of epidermal extract-based Western blot analysis in the diagnosis of bullous pemphigoid (BP) .Methods Human epidermal extracts were prepared by thermal separation from circumcised foreskins of healthy males. Serum samples were obtained from 22 inpatients with BP and 25 inpatients without BP in Hospital for Skin Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College between January 2015 and August 2017. These serum samples were subjected to Western blot analysis with epidermal extracts as substrates, as well as to BP180-NC16A enzyme-linked immunosorbent assay (ELISA) . Statistical analysis was carried out using chi-square test and Fisher′s exact test with the SPSS22.0 software.Results The sensitivities of epidermal extract-based Western blot analysis and BP180-NC16A ELISA in the diagnosis of BP were 86.36% (95%CI: 64.03%-96.41%) and 95.45% (95%CI: 75.11%-99.76%) respectively (χ2= 1.10, P= 0.294) , and the specificities were 100% (95% CI: 83.42%-100%) and 92% (95% CI: 75.11%-99.76%) respectively (χ^2=20.8, P=0.149) . Epidermal extract-based Western blot analysis in the 22 patients with BP showed a protein band with relative molecular mass (RMM) of 230 000 in 4 patients, a protein band with RMM of 180 000 in 18, a protein band with RMM of 120 000 in 1, and a protein band with RMM of 97 000 in 1. The BP180-NC16A ELISA showed that the antibody titers were more than 50 U/ml in the BP patients with protein bands of RMM of 180 000.Conclusions The epidermal extract-based Western blot analysis mainly showed the protein band with RMM of 180 000 in the patients with BP. The sensitivity of the epidermal extract-based Western blot analysis was lower than that of the BP180-NC16A ELISA, and the epidermal extract-based Western blot analysis tends to be negative when the titer of the autoantibody is low.