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融合蛋白，以保存这些蛋白的三维结构。ELISA法证实融合蛋白能与以人促冻蛋白1为靶点的人叶性天疱疮血清和以人促冻蛋白3为靶点的人寻常型天疱疮血清发生反应。用ELISA法测定这些蛋白与6例寻常型天疱疮、1例副肿瘤性天疱疮、1例素食性天疱疮、44例叶状天疱疮患者血清的效价。54只狗的正常狗血清和患有其他皮肤病如demodemo病和脓皮病的狗血清作为对照。这些疾病的诊断是基于以下发现，包括临床特征，组织病理学发现，如角膜下大脓疱伴大量中性粒细胞/嗜酸性粒细胞和棘溶性角化细胞，通过直接和间接免疫荧光试验发现细胞间荧光沉积，通过活角化细胞染色发现血清中存在自身抗体。6例寻常型天疱疮血清中有4例用重组粘粒蛋白3进行ELISA检测，其效价明显高于对照血清。然而，叶状天疱疮血清与重组犬粘粒蛋白1的反应并不一致。因此，利用重组犬粘粒蛋白3的酶联免疫吸附试验（ELISA）对寻常型天疱疮进行血清学诊断似乎是成功的，但对于叶状天疱疮，ELISA目前还不是诊断犬叶状天疱疮的实用工具。

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.