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Y. Kim, X. Jiang, N. Caberoy, D. Maiguel, J. Davis, W. Li; Identification of Autoantigens With Serum Immunoreactivity Specific to Uveitis Patients by Phage Display. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4754. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Autoantigens not only hold a key for our understanding of the pathogenesis of autuoimmunity, but also can be used as biomarkers for disease diagnosis. Phage display technology is a sensitive and efficient approach to identify autoantigens by screening cDNA library. However, conventional phage display cDNA library often resulted in identification of unnatural short peptides due to reading frame problems for the cDNA library fusion proteins. The purpose of this study is to develop a phage display cDNA library with open reading frame (ORF) and demonstrate its application for autoantigen identification in uveitis patients.
cDNA library was constructed from mouse eyes by random priming method and fused to T7 phage capsid as fusion proteins with a biotinylation peptide epitope at the C-terminus of the fusion proteins. ORF phage display cDNA library was enriched by streptavidin selection. To identify patient-specific autoantigens by subtractive phage display, the ORF library was pre-adsorbed with control human IgG and selected by purified IgG from patients with acute anterior uveitis (AAU). After four rounds of selection, individual phage clones with higher binding activity than control phage were isolated and identified by DNA sequencing. Their binding specificity to control IgG vs. patient IgG was further verified by phage binding assay and ELISA.
The ORF library expressed mouse eye proteins with 86% - 95% of them in the correct reading frame vs. ~3% for conventional phage display libraries. After four rounds of the subtractive selection, phage binding activity to patient IgG typically increased 100 - 1000 folds. Several phage clones with ORF were identified with 30 -100 fold higher binding activity to patient IgG vs. control IgG by phage binding assay. Their patient-specific binding activities were further verified by ELISA.
This new phage display cDNA library has substantially improved the efficiency to identify patient-specific candidate autoantigens in the correct reading frame by subtractive phage display.
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