Abstract
Abstract: :
Purpose: Autoantigens hold a critical key in our understanding of the etiology and diagnosis of autoimmune uveitis. Identification of autoantigens with antibody from the sera of uveitis patients presents a formidable challenge as the majority of antibodies in patient sera may not be uveitis–relevant. In this study, we have developed and optimized the system of subtractive phage biopanning for more efficient identication of autoantigens. Methods: IgG was purified from patient and control sera using protein G columns. A human eye cDNA phage–display library was pre–adsorbed with control IgG, followed by selection with patient IgG. Four rounds of subtractive phage biopanning were performed with phage enrichment monitored by PCR. Excessive amounts of replication–deficient mutant phage were used during biopanning to reduce non–specific phage binding. The binding affinity of phage clones, as defined by the preferential binding of isolated phages vs. wild–type phage to patient IgG, and their patient specificity, as defined by the binding of phage clones to patient IgG vs. control IgG, were analyzed by phage–based binding assay and dot blot. Results: The excessive amount of replication–deficient mutant phage substantially reduced non–specific phage enrichment, resulting in a mean 226–fold improvement in binding affinity for enriched phage clones. Several phage clones specifically enriched with patient IgG were identified by PCR and isolated. Their binding affinity and patient specificity were verified by the binding assay and dot blot. Several candidate autoantigens were identified by sequencing the cDNA inserts of these phage clones. Their immunoreactivities are currently under further investigation. Conclusions: The novel optimized system of subtractive phage display is an efficient and sensitive approach for impartial identification of previously unknown autoantigens in uveitis patients. Moreover, the replication–deficient mutant phage can substantially improve the ability of the phage biopanning to identify uveitis–relevant autoantigens.
Keywords: autoimmune disease • uveitis-clinical/animal model • gene screening