Identification of Biomarkers of Autoimmunity in T1D by Novel Tools

描述（由申请人提供）：该提案旨在通过创新方法鉴定1型糖尿病（T1 D）的新型生物标志物。筛选从人胰岛构建的噬菌体cDNA表达文库的方法，所述人胰岛具有来自具有临床前和临床T1 D的受试者的血清，随后在微阵列上验证分离的克隆，这是用于鉴定大量推定的自身抗原的有用策略。该研究方案包括以下步骤：（a）利用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库。一个新的噬菌体展示载体（pPAO）在一个参与实验室开发的将用于此目的。该载体被设计为通过选择符合读码框的编码序列来优化抗原肽的拯救。(b)cDNA噬菌体文库的选择基于从具有临床前和临床T1 D的各个阶段的受试者获得的血清中存在的抗体。(c)通过（i）通过蛋白质微阵列和经典免疫测定，使用来自具有β细胞自身免疫体征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源的原始cDNA克隆，对大量分离的抗原片段进行表征。(d)通过用来自疾病不同阶段的充分表征的受试者的血清进行挑战，验证分离抗原的一般抗原特性。该步骤将鉴定提供最佳免疫识别谱的肽。(e)构建具有诊断和预后能力的选定抗原集的蛋白质微阵列。 T1 D是一种慢性疾病，影响越来越多的年轻人。本研究的目的是构建一个蛋白质微阵列，用于检测T1 D相关的自身抗体。这种微阵列有望改善T1 D的诊断，根据自身免疫反应特异性的差异鉴定疾病的亚型，定义可能具有预后价值的自身抗体特征，并促进监测疾病进展或对治疗的反应。

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.