Development of serological diagnosing methods for canine pemphigus by ELISA using fusion protein of canine desmosomal proteins

对犬桥粒蛋白的胞外部分桥粒蛋白1、桥粒蛋白3和桥粒蛋白1进行了克隆和测序，并通过杆状病毒表达制备了融合蛋白，以保存这些蛋白的三维结构。经酶联免疫吸附试验证实，融合蛋白可与靶向于桥粒蛋白1的叶状天疱疮血清和靶向于桥粒蛋白3的寻常型天疱疮血清反应。用ELISA法测定了6例寻常型天疱疮、1例副肿瘤性天疱疮、1例植物天疱疮、44例叶型天疱疮患者的血清。以54只正常犬血清和蠕形螨病、脓皮病等皮肤病犬血清为对照。诊断依据包括：特征性的临床表现、组织病理学表现，如角膜下含大量中性粒细胞/嗜酸性粒细胞和棘细胞松解性角质形成细胞的大脓疱，直接和间接免疫荧光试验检测细胞间荧光沉积，活体角质形成细胞染色检测血清中是否存在自身抗体。重组桥粒蛋白3在6份寻常型天疱疮患者血清中有4份的效价显著高于对照血清。然而，重组犬桥粒蛋白1与叶状天疱疮血清的反应不一致。因此，利用重组桥粒蛋白3的重组蛋白建立的ELISA诊断寻常型天疱疮的血清学方法似乎是成功的，但对于叶型天疱疮，目前还不是一种实用的诊断工具。

The extraxcellular portion of canine desmosomal proteins, desmoglein 1, desmoglein3 and desmocollin 1 were cloned and sequenced, and fusion proteins were made by baculovirus expression in order to conserve the three dimensional structure of these proteins. Fusion proteins were confirmed to react with human pemphigus foliaceus sera which target human desmoglein 1, with human pemphigus vulgaris sera which target human desmoglein 3 by ELISA. These proteins were then measured the titer by ELISA with the sera from dogs with 6 pemphigus vulgaris, 1 paraneoplastic pemphigus, 1 pemphigus vegetans, 44 pemphigus foliaceus. Normal dog sera from 54 dogs and sera from dogs with the other dermatological disorders such as demodicosis and pyoderma were used as controls. Diagnosis of these disorders was based the following findings including characteristic clinical features, histopathological findings such as subcorneal large pustules with abundant neutrophils/eosinophils and acantholytic keratinocytes, intercellular fluorescent deposits by direct and indirect immunofluorescence test, the existence of autoantibodies in sera by living keratinocyte staining. Four out of six sera with pemphigus vulgaris showed a significant high titer in ELISA using recombinant desmoglein 3 compared with control sera. However, sera from pemphigus foliaceus were not consistently reacted with recombinant canine desmoglein 1.As a result, the serological diagnosing method for pemphigus vulgaris by ELISA using recombinant proteins of canine desmoglein 3 seems to be successful, but for pemphigus foliaceus, the ELISA is not yet a practical tool for the diagnosis of canine pemphigus foliaceus at the present time.