Abréu-Vélez Ana María, Yepes Maria Mercedes, Patiño Pablo Javier, Bollag Wendy B, Montoya Fernando
Institute for Molecular Medicine and Genetics, Medical College of Georgia, CB 2803, 1120 15th Street, GA 30912-2630, Augusta, USA,
Arch Dermatol Res. 2004 Mar;295(10):434-41. doi: 10.1007/s00403-003-0441-4. Epub 2004 Jan 17.
We recently described a new variant of endemic pemphigus foliaceus (EPF) in El Bagre, Colombia, that resembles Senear-Usher syndrome and identified autoantibodies to desmoglein 1 (Dsg1), as well as to multiple known and unknown antigens including plectins, in the serum of these patients. Here, we developed a cost-effective ELISA assay capable of detecting the heterogeneous antibody population observed in these EPF patients, and useful for serum epidemiological studies. A protein extract obtained from trypsin-digested fresh bovine skin and further purified on a concanavalin A matrix was used as antigen. This extract contains an important conformational epitope (a 45 kDa tryptic fragment of the Dsg1 ectodomain), which is recognized by antibodies in serum from patients with all varieties of pemphigus foliaceus (PF), and from half of those with pemphigus vulgaris with active clinical disease. The cut-off and threshold values were normalized using human serum obtained from both endemic and non-endemic areas for PF. The efficiency of this ELISA was tested using 600 serum samples from controls and patients diagnosed with EPF, non-endemic PF and other bullous diseases. The overall sensitivity and specificity of the assay were determined to be 95% and 72%, respectively, with reproducibilities of 98% (intraassay) and 95% (interassay). Comparing the ELISA with other tests to detect EPF autoantibodies, this ELISA was the most sensitive, followed by direct immunofluorescence (DIF), indirect immunofluorescence using anti-IgG4 monoclonal antibodies and immunoprecipitation (IP), respectively. The most specific assay was IP, followed by DIF. Immunoblotting to Dsg1 exhibited both poor sensitivity and poor specificity, although plectins were well visualized. We conclude that this ELISA is an excellent tool for field serological studies, allowing testing of multiple serum samples simultaneously and for detecting, with appropriate restriction and sensitivity, the heterogeneous antibody population seen in patients with this variant of EPF. Finally, autoantibody serum levels obtained with this ELISA correlated well with the clinical activity and extent of disease in patients with El Bagre EPF.
我们最近描述了哥伦比亚埃尔巴格雷地区一种类似Senear-Usher综合征的地方性落叶型天疱疮（EPF）新变体，并在这些患者的血清中鉴定出了针对桥粒芯糖蛋白1（Dsg1）以及包括网蛋白在内的多种已知和未知抗原的自身抗体。在此，我们开发了一种经济高效的酶联免疫吸附测定（ELISA）方法，该方法能够检测在这些EPF患者中观察到的异质性抗体群体，并且对血清流行病学研究有用。从经胰蛋白酶消化的新鲜牛皮中获得并在伴刀豆球蛋白A基质上进一步纯化的蛋白质提取物用作抗原。该提取物包含一个重要的构象表位（Dsg1胞外域的一个45 kDa胰蛋白酶片段），所有类型落叶型天疱疮（PF）患者以及一半有活动性临床疾病的寻常型天疱疮患者血清中的抗体均可识别该表位。使用从PF的地方性和非地方性地区获得的人血清对临界值和阈值进行标准化。使用来自对照以及被诊断为EPF、非地方性PF和其他大疱性疾病患者的600份血清样本对该ELISA的效率进行了测试。该测定的总体敏感性和特异性分别确定为95%和72%，重复性分别为98%（批内）和95%（批间）。将该ELISA与其他检测EPF自身抗体的试验进行比较，该ELISA最为敏感，其次分别是直接免疫荧光（DIF）、使用抗IgG4单克隆抗体的间接免疫荧光和免疫沉淀（IP）。最具特异性的试验是IP，其次是DIF。对Dsg1进行免疫印迹时，敏感性和特异性均较差，尽管网蛋白能清晰显示。我们得出结论，这种ELISA是现场血清学研究的一种优秀工具，能够同时检测多个血清样本，并以适当的局限性和敏感性检测在这种EPF变体患者中所见的异质性抗体群体。最后，用这种ELISA获得 的自身抗体血清水平与埃尔巴格雷EPF患者的临床活动度和疾病范围密切相关。
