Identification of Biomarkers of Autoimmunity in T1D by Novel Tools

描述（由申请人提供）：本提案旨在通过创新方法鉴定1型糖尿病（T1D）的新型生物标志物。用临床前和临床T1D患者的血清筛选人类胰岛构建的噬菌体cDNA表达文库，然后在微阵列上验证分离的克隆，是鉴定大量假定的自身抗原的有用策略。研究计划包括以下步骤：(a)利用从器官供体获得的人胰岛生成的cdna构建抗原噬菌体展示文库。其中一个参与实验室开发的一种新的噬菌体展示载体（pPAO）将用于这一目的。该载体通过选择帧内编码序列来优化抗原肽的修复。(b)基于临床前和临床不同阶段T1D患者血清中存在的抗体选择cDNA噬菌体文库。(c)对大量分离的抗原片段进行鉴定，方法是：(i)通过蛋白质微阵列和经典免疫分析，利用来自具有β细胞自身免疫迹象的受试者的血清抗体，确认筛选的抗原片段的反应性；（ii）自动测序和数据库筛选，以鉴定所有阳性片段来自的原始cDNA克隆。(d)用疾病不同阶段特征明确的受试者的血清进行挑战，验证分离抗原的一般抗原特性。这一步将确定肽提供免疫识别的最佳配置文件。(e)构建具有诊断和预后能力的选定抗原的蛋白质微阵列。

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.