Profiling of alopecia areata autoantigens based on protein microarray technology
Profiling of alopecia areata autoantigens based on protein microarray technology
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DOI：
10.1074/mcp.t500004-mcp200
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发表时间：
2005-09-01
影响因子：
7
通讯作者：
Cahill, DJ
中科院分区：
文献类型：
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作者：
Lueking, A;Huber, O;Cahill, DJ
蛋白质生物芯片作为研究工具和诊断在未来复杂样品的并行处理方面具有巨大的潜力。为了生成蛋白质生物芯片，已经开发了用于 cDNA 表达文库生产、高通量蛋白质表达、大规模蛋白质分析和蛋白质微阵列生成的高度自动化技术。利用这项技术，我们在这里提出了一种策略来识别与斑秃发病机制有关的潜在自身抗原，斑秃是一种导致头皮毛发迅速脱落的慢性疾病。对于参与这一过程的推定自身抗原知之甚少。通过将蛋白质微阵列技术与大型 cDNA 表达文库相结合，我们针对由 37,200 个冗余重组人类蛋白质组成的人类蛋白质阵列，分析了斑秃患者血清的自身抗体库。将患者血清孵育获得的数据集与临床健康人的对照血清以及抗人 IgG 抗体的背景孵育进行比较。根据这些结果，生成了较小的蛋白质子集，并在高度敏感的蛋白质微阵列上进行定性和定量验证，以鉴定新的斑秃相关自身抗原。通过蛋白质芯片技术鉴定出8种自身抗原，并通过Western blot分析成功证实。这些自身抗原排列在蛋白质微阵列上，以生成与疾病相关的蛋白质芯片。为了确认所获得结果的特异性，在疾病相关蛋白芯片上另外检查了牛皮癣或手足湿疹以及皮肤过敏患者的血清。通过使用斑秃作为自身免疫性疾病的模型，我们的研究表明蛋白质微阵列技术具有识别和评估自身抗原以及诊断（例如区分斑秃与其他皮肤病）的潜力。
Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases.