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Wei Li, Hui Wang, Nora Caberoy, Gabriela S. Alvarado, Rui Chen, Janet L. Davis; Global Mapping Of Autoantigens For Uveitis Patient By ORF Phage Display. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2923.
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© ARVO (1962-2015); The Authors (2016-present)
Autoantigens hold a key to understanding pathogenesis of autuoimmunity, and are valuable biomarkers for disease diagnosis. We developed open reading frame (ORF) phage display to identify autoantigens with minimal reading frame problem. However, it is time-consuming and labor-intensive to identify autoantigens by screening individual phage clones in large scales. The purpose of this study is to globally identify patient-specific autoantigens for autoimmune uveitis by a new technology of open reading frame phage display and next generation DNA sequencing (OPD-NGS).
IgG was purified from the sera of a patient with acute anterior uveitis and a healthy control. Purified IgG was immobilized on ELISA plates for multi-round selection with an ORF phage display cDNA library of adult eye. Total bound phages were quantified at each round by plaque assay. After 3 rounds of selection, cDNA inserts of enriched phages were amplified by PCR and analyzed by NGS. The sequencing data were blasted against NCBI GenBank database to identify all enriched IgG-binding proteins. Patient-specific autoantigens were globally identified by comparing entire OPD-NGS datasets for patient and control IgG. Identified autoantigens were isolated, verified for their binding activity to patient and control IgG, and quantified by plaque assay.
Multi-rounds of selection resulted in ~429-fold and ~404-fold increase in total bound phages for patient and control IgG, respectively. NGS sequenced all enriched phage clones in each sample. Data analysis identified two large sets of IgG-binding proteins for patient and control. Comparative analysis between these two datasets globally identified a long list of patient-specific IgG-binding proteins, which were specifically enriched with patient IgG, but not with control IgG. All non-specific binding proteins to Fc domain were enriched with both patient and control IgG, and were eliminated by the comparative analysis. The enrichment frequencies of identified proteins in NGS analysis were the equivalents of their IgG-binding activities. Several identified IgG-binding proteins were verified by phage binding assay.
These data demonstrated that OPD-NGS is a powerful technology for global identification of all patient-specific autoantigens. Identified autoantigens will facilitate delineation of underlying immunological mechanisms of autoimmune uveitis.
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