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。目前还没有专门的检测方法来诊断这种疾病。 在没有其他原因的情况下，在受累器官的组织活检中证实非干酪化性肉芽肿， 目前诊断结节病的技术水平。我们建议测试的假设，整体免疫力 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 在这种疾病中存在各种抗体/自身抗体。结节病和肺结核 临床和病理上的相似性尽管分离出结核分枝杆菌的各种成分， (MTB)从结节病组织中，结节病患者对结核菌素皮肤试验（PPD）呈阴性反应。相比之下 对结节病潜伏性结核感染者（LTBI）的PPD反应为迟发型超敏反应 反应利用高通量的方法，我们开发了一个复杂的cDNA文库，来自组织， 结节病患者。我们构建了一个微阵列平台，从这个cDNA文库含有大量的 结节病克隆，并用来自结节病患者、对照和 其他呼吸道疾病。我们确定了一组具有高灵敏度和特异性的生物标志物/分类器 可以区分结节病患者的血清，健康对照其他呼吸道疾病。 因此，我们的技术使我们能够测试结节病是由免疫系统疾病引发的假设。 一组特异性抗原，其不同于LTBI抗原。此外，这些特异性抗原肽是 能够在结节病外周单核细胞（PBMC）中体外诱导肉芽肿。为了验证这一 假设，我们将选择性地用不同结节病血清对我们的T7噬菌体cDNA展示文库进行生物筛选， LTBI受试者选择多样化的克隆以增加抗原库，以构建全面的 抗原微阵列平台。其次，我们建议使用来自多元化人群的大样本量， 结节病患者和健康对照以及LTBI受试者。我们想扩大测试 从病例和对照中独立获得的血清的生物活性，以鉴定一组诊断性 生物标志物/分类器，其可以区分结节病和健康对照以及潜伏性TB。 此外，我们将在独立的验证集中验证分类器，并确定 发现的生物标志物/分类器可以确定临床结果，特别是在进行性疾病中， 当然了在目的3中，我们将合成抗原肽以开发ELISA来确定 识别抗原。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外周单核细胞。总体目标是确定具体的 结节病中引发肉芽肿形成的抗原以及对抗原的免疫应答如何导致 结节病和潜伏性TB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.