Identification of autoantigens in autoimmune uveitis by subtractive phage biopanning

Abstract

Abstract: : Purpose: Although several autoantigens have been identified and demonstrated to play a pathogenic role in experimental autoimmune uveitis (EAU), identification of autoantigens in uveitis patients by conventional approaches, such as Western blot, has been proved to be technically challenging and time–consuming. In this study, we overcome the difficulty by establishing a system for the rapid identification of autoantigens in uveitis patients with subtractive phage display technique. Methods: Human eye mRNA was purified and used to construct cDNA library in bacteriophage T7 vector using both random primer and oligoT primer strategies. Packaged phage library was characterized by the isolation of rhodopsin–expressing phages using anti–rhodopsin mAb. To identify autoantigens, the library was screened by a "subtractive biopanning" technique, using purified patient IgG vs. control IgG. Clonal phages enriched by patient IgG was isolated and analyzed for their binding specificities. Results: We have identified 7 phage clones specifically enriched by the pooled IgG from 14 uveitis patients and additional 4 clones by the IgG from uveitis patients. Most of the clones encode for peptide fragments expressed in human eye. Two of the clones have been demonstrated for their specificities with 65 – 200 fold increase in their binding activity to patient IgG vs. control IgG. Other clones are currently under investigation for their specificities. Conclusions: A phage display human eye cDNA library was successfully generated and characterized. We established a system of subtractive phage biopanning for the rapid identification of uveitis–associated autoantigens or antigenic epitopes. Identified autoantigens were verified for their specific binding activities to patient IgG.

Keywords: autoimmune disease • uveitis–clinical/animal model • inflammation