纯化与未纯化重组PVA表位EC1-2在检测抗PVA抗体中的比较

Comparing of Detecting Anti PVA Antibody in Substrates of Purified and Unpurified Recombinant EC1-2 of PVA

目的 比较纯化与未纯化重组寻常型天疱疮抗原 ( pemphigusvulgarisantigen ,PVA)的细胞外区 (extracel lulardomain ,EC) 1 2表位 (EC1 2 )在检测抗PVA抗体中的差异 ,以提高检测抗PVA抗体的敏感性。方法 在构建EC1 2重组质粒的基础上进行融合蛋白的表达 ,并依据亲和层析的原理进行该融合蛋白的纯化。采用免疫印迹方法分别将纯化前后的EC1 2融合蛋白应用于寻常型天疱疮 (PV)患者的血清学检测。结果 2 2例被测PV患者血清中 ,用纯化前EC1 2融合蛋白进行检测的阳性率为 5 4.5 % ,用纯化后的融合蛋白进行检测的阳性率为 77.3 %。结论 以纯化后的EC1 2融合蛋白为抗原底物 ,其抗PVA抗体的检出率显著高于纯化前 ,为临床特异性诊断PV提供了新方法。

Objective To increase the sensitivity and specificity of detecting the anti pemphigus vulgaris antigen (PVA) antibody, the discriminations were compared between purified recombinant EC1-2 epitopes of PVA and unpurified recombinant EC1-2 epitopes.Methods After constructing the PVA EC1-2 recombinant plasmid, EC1-2 fusion proteins were expressed and purified by glutathione affinity chromatography.The sera from the patients with pemphigus vulgaris (PV) were tested respectively in the substrates of purified EC1-2 fusion proteins or unpurified EC1-2 fusion proteins with the method of immunoblotting (IBT).Results 22 PV patients, the positive rates were 77.3% in the substrates of purified EC1-2 fusion proteins but 54.5% in the substrates of unpurified.Conclusion The purified EC1-2 fusion proteins are more sensitive in detecting the anti-PVA antibody. This study will provide a new method in the specific serum diagnosis of PV.