A novel T7 phage display technology to detect sarcoidosis specific antigens

结节病是一种病因不明的炎症性疾病，在全世界范围内发生，其特征是 不同器官的肉芽肿形成。目前还没有开发出诊断这种疾病的特定测试。 在无其他原因的受累器官的组织活检中确认为非干酪性肉芽肿 目前诊断结节病的最新技术。我们建议检验一种假设，即总体免疫 在结节病的发病机制中起着重要作用，因为免疫功能异常和 在这种疾病中会出现各种抗体/自身抗体。结节病和结核病 临床和病理相似之处。尽管分离出了结核分枝杆菌的各种成分 (MTB)结节病患者对结核菌素皮肤试验(PPD)的反应为阴性。相比之下， 结节病潜伏感染结核患者(LTBI)对PPD迟发性超敏反应的反应 反应。利用高通量方法，我们成功地构建了一个来源于日本血吸虫组织的复杂的cdna文库。 结节病患者。我们利用该文库构建了包含大量基因的微阵列平台 结节病克隆并用结节病患者血清免疫筛选该平台，对照和 其他呼吸道疾病。我们确定了一组具有高度敏感性和特异性的生物标志物/分类器 这可以区分结节病患者的血清和健康对照的其他呼吸道疾病。 因此，我们的技术允许我们测试这样的假设，即结节病是一种由 一组不同于LTBI抗原的特异性抗原。此外，这些特定的抗原肽是 在体外能够在结节病外周单个核细胞(PBMC)中诱导肉芽肿。为了测试这一点 假设，我们将有选择地用不同结节病和 LTBI受试者选择多样化克隆增加抗原库构建综合 抗原微阵列平台。其次，我们建议使用来自不同人群的大样本。 结节病患者和健康对照以及LTBI受试者。我们想扩大测试范围 独立于病例和对照获得的血清的生物反应性，以识别一组诊断 生物标志物/分类器，可以区分结节病和健康对照以及潜伏的结核病。 此外，我们将验证独立验证集中的分类器，并确定是否 已发现的生物标志物/分类可以决定临床结果，特别是在进展性疾病中 当然了。在目标3中，我们将合成抗原肽，以建立一种测定病毒滴度的酶联免疫吸附试验 识别出的抗原。最后，我们测试这些克隆是否能够在体外产生肉芽肿 潜伏的健康对照和结节病患者外周血单核细胞。总体目标是定义特定的 在结节病中启动肉芽肿形成的抗原以及对抗原的免疫反应如何导致 结节病和潜伏性结核表型。

Sarcoidosis is an inflammatory disease of unknown etiology that occurs worldwide and is characterized by granuloma formation in different organs. No specific test has been developed to diagnose this disease. Confirmation of non-caseating granuloma in tissue biopsy of involved organs in the absence of other causes is the current state of the art for diagnosing sarcoidosis. We propose to test the hypothesis that overall immunity plays a prominent role in the pathogenesis of sarcoidosis, since abnormalities of the immune function and the presence of various antibodies/autoantibodies occurs in this disorder. Sarcoidosis and tuberculosis have clinical and pathological similarities. Despite isolation of various components of Mycobacterium tuberculosis (MTB) from sarcoidosis tissues, sarcoidosis subjects react to tuberculin skin test (PPD) negatively. In contrast to sarcoidosis latently infected individuals with TB (LTBI) respond to PPD with delayed type hypersensitivity reaction. Using a high throughput method, we developed a complex cDNA library derived from tissues of sarcoidosis patients. We constructed a microarray platform from this cDNA library containing large numbers of sarcoidosis clones and immunoscreened this platform with sera from patients with sarcoidosis, controls and other respiratory diseases. We identified a panel of biomarkers/classifiers with high sensitivity and specificity that can discriminate between sera of patients with sarcoidosis, healthy controls other respiratory diseases. Thus, our technology allows us to test the hypothesis that sarcoidosis is an immune disorder triggered by a group of specific antigens, which differ from LTBI antigens. Furthermore, these specific antigen peptides are capable of inducing granuloma in vitro in sarcoidosis peripheral mononuclear cells (PBMCs). To test this hypothesis, we will selectively biopan our T7 phage cDNA display library with sera of diverse sarcoidosis and LTBI subjects to select diversified clones to increase the antigen repertoire to construct a comprehensive antigen microarray platform. Second, we propose to use a large sample size from a diversified population of sarcoidosis patients and healthy controls as well as subjects with LTBI. We would like to expand test the bioreactivity of sera obtained independently from cases and controls to identify a panel of diagnostic biomarkers/classifiers, which can discriminate between sarcoidosis and healthy controls and latent TB. Furthermore, we will validate the classifiers in independent validation sets and determine whether the discovered biomarkers/classifiers can determine the clinical outcome, especially in the progressive disease course. In Aim 3, we will synthesize the antigen peptides to develop an ELISA to determine the titers of identified antigens. Finally, we test whether these clones are capable of generating granuloma in vitro using latent, healthy controls and sarcoidosis peripheral mononuclear cells. The overall goal is to define the specific antigens initiating granuloma formation in sarcoidosis and how the immunological response to antigen leads to sarcoidosis and latent TB phenotypes.