Development of disease activity monitoring assay system by ELISA using recombinant pemphigus antigens

天疱疮是一种自身免疫性水疱性疾病，有两种主要亚型，寻常型天疱疮（PV）和落叶型天疱疮（PF）。天疱疮患者存在循环抗Dsg 1和/或抗Dsg 3 IgG自身抗体。我们以前已经开发了ELISA使用重组Dsg 1和Dsg 3杆状病毒表达作为天疱疮的诊断工具。本研究的目的是评价这些ELISA在临床上大量血清样本的实际应用。我们使用了81份PV血清，48份PF血清，114份大疱性类天疱疮（BP）血清，124份胶原病血清，8份其他非天疱疮大疱性疾病血清和179份正常对照血清。通过受试者工作特征（ROC）图确定截止值。48份PF血清中47份（97.9%）Dsg 1 ELISA阳性，81份PV血清中79份（97.5%）Dsg 3 ELISA阳性，179份正常人血清中Dsg 1和Dsg 3 ELISA阳性分别为2份（1.1%）和4份（2.2%）。然而，BP和胶原病的一些疾病对照血清超过了临界值。灰色区域的引入有助于减少这些假阳性血清的数量。此外，在研究的11例患者中，Dsg 1和Dsg 3 ELISA评分显示出沿着时间进程与疾病活动平行波动。我们提出了一个基于ELISA反应性的天疱疮诊断标准：如果血清对Dsg 3呈阳性，则表明诊断为PV，而不管对Dsg 1的反应性如何;如果血清Dsg 3阴性而Dsg 1阳性，则表明PF的诊断。我们的结论是，这些Dsg 1和Dsg 3 ELISA提供了一种简单，敏感性和高度特异性的检测方法用于诊断PV和PF患者，这些ELISA可能是监测疾病活动的有价值的工具。

Pemphigus is an autoimmune blistering disease and has two major subtypes, pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Patients with pemphigus have circulating anti-Dsg1 and/or anti-Dsg3 IgG autoantibodies. We have previously developed ELISAs using recombinant Dsg1 and Dsg3 expressed by baculovirus as a diagnostic tool for pemphigus. The purpose of this study was to evaluate the practical application of these ELISAs for clinical use with a large number of serum samples. We used 81 PV sera, 48 PF sera, 114 bullous pemphigoid (BP) sera, 124 collagen disease sera, 8 sera of other non-pemphigus bullous diseases and 179 normal control sera. A cutoff value was determined by Receiver-Operating-Characteristic (ROC) plots. 47 of 48 PF sera (97.9%) were positive in the Dsg1 ELISA and 79 of 81 PV sera (97.5%) were positive in the Dsg3 ELISA, while only 2(1.1%) and 4(2.2%) of 179 normal sera were positive in Dsg1 and Dsg3 ELISAs, respectively. However, some disease control sera of BP arid collagen diseases exceeded the cutoff value. Introduction of a gray zone helped to decrease the number of those false positive sera. Furthermore, in 11 patients studied, the respective Dsg1 and Dsg3 ELISA scores showed parallel fluctuation with the disease activity along the time course. We proposed a diagnostic criteria for pemphigus based on ELISA reactivity ; if a serum is positive against Dsg3 it indicates diagnosis of PV, regardless of reactivity against Dsg1 ; if a serum is negative for Dsg3 and positive for Dsg1, it indicates diagnosis of PF.We concluded that these Dsg1 and Dsg3 ELISAs provide a simple, sensitive and highly specific assay for the diagnosis of patients with PV and PF and that these ELISAs may be a valuable tool to monitor the disease activity.