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，分为寻常型天疱疮(PV)和叶型天疱疮(PF)。天疱疮患者存在循环抗DSG1和/或抗Dsg3自身抗体。我们以前已经开发了使用杆状病毒表达的重组DSG1和Dsg3作为天疱疮诊断工具的ELISA。本研究的目的是通过大量的血清样本来评估这些ELISA在临床上的实际应用。用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份其他非天疱疮患者血清和179份正常对照血清。截断值由受试者工作特性(ROC)图确定。然而，BP和胶原病的部分疾控血清超过了临界值。灰色地带的引入有助于减少假阳性血清的数量。此外，在所研究的11名患者中，各自的DSG1和Dsg3酶联免疫吸附试验分数显示出与疾病活动性平行波动的时间进程。

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.