Identification of Biomarkers of Autoimmunity in T1D by Novel Tools

描述(由申请人提供)：这项提案旨在通过创新的方法识别1型糖尿病(T1D)的新生物标志物。从临床前和临床T1D患者的血清中筛选人胰岛构建的噬菌体表达文库，然后在微阵列上验证分离克隆的方法是鉴定大量假定自身抗原的有用策略。研究建议包括以下步骤：(A)从器官捐赠者获得的人胰岛产生的cDNA构建抗原噬菌体展示文库。其中一个参与实验室开发的新型噬菌体展示载体(PPAO)将用于这一目的。该载体被设计成通过选择帧编码序列来优化抗原肽的拯救。(B)根据从临床前和临床T1D不同阶段的受试者血清中存在的抗体来选择cDNA噬菌体文库。(C)对大量分离的抗原片段进行鉴定，方法是：(1)使用蛋白质微阵列和经典免疫分析中有β细胞自身免疫迹象的受试者的血清抗体，确认筛选出的抗原片段的反应性；(2)自动测序和数据库筛选，以确定所有阳性片段的原始cDNA克隆。(D)在疾病的不同阶段用具有良好特征的受试者的血清挑战，以确认分离抗原的一般抗原性。这一步将确定提供最佳免疫识别特征的多肽。(E)用选定的一组具有诊断和预测能力的抗原构建蛋白质微阵列。 T1D是一种慢性病，影响着越来越多的年轻人。本研究方案旨在构建检测与T1D相关的自身抗体的蛋白质微阵列。这种微阵列有望提高对T1D的诊断，根据自身免疫反应的特异性差异识别疾病的亚型，确定可能具有预后价值的自身抗体特征，并促进对疾病进展或治疗反应的监测。

DESCRIPTION (provided by applicant): This proposal aims at the identification of novel biomarkers in type 1 diabetes (T1D) through innovative approaches. The approach of screening a phage cDNA expression library constructed from human pancreatic islets with sera from subjects with preclinical and clinical T1D followed by validation of the isolated clones on microarrays is a useful strategy for the identification of a high number of putative autoantigens. The research proposal comprises the following steps: (a) The construction of antigen phage display libraries from cDNAs generated from human pancreatic islets obtained from organ donors. A new phage display vector (pPAO) developed in one of the participating laboratories will be used for this purpose. This vector has been designed to optimize the rescue of antigenic peptides by selecting in frame coding sequences. (b) The selection of the cDNA phage library based on antibodies present in sera obtained from subjects with various stages of preclinical and clinical T1D. (c) The characterization of a large number of the isolated antigen fragments by (i) confirmation of the reactivity of the screened antigen fragments using serum antibodies from subjects with signs of beta-cell autoimmunity by protein microarray and classical immunoassays; (ii) the automated sequencing and data bank screening for the identification of the original cDNA clones from which all the positive fragments derive. (d) The validation of the general antigenic properties of the isolated antigens by challenging with sera from well characterized subjects at different stages of the disease. This step will identify the peptides providing the best profile of immune recognition. (e) The construction of a protein microarray with the selected set of antigens with diagnostic and prognostic capacity. T1D is a chronic disease affecting an increasing number of young people. This research proposal aims at the construction of a protein microarray for the detection of autoantibodies relevant for T1D. Such a microarray is expected to improve the diagnosis of T1D, to identify subtypes of the disease based on differences in the specificity of the autoimmune response, to define autoantibody signatures that may have prognostic value, and to facilitate the monitoring of disease progression or response to therapy.