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. ? ?
