Date of Award

3-30-2007

Document Type

Thesis

Degree Name

Medical Doctor (MD)

First Advisor

M. Bruce Shields MD

Abstract

Autoimmune uveitis is a group of ocular inflammatory disorders with unknown causes. Identification of disease-relevant autoantigens using antibodies from the sera of uveitis patients is important for our understanding of disease mechanisms, but presents a formidable challenge due to antibody heterogeneity. Herein, we report the development of a novel system of subtractive phage display to overcome these challenges. We characterize TRB2 expression in uveitis-susceptible tissue and further optimize the system for autoantigen identification by competitive immunoblotting. To verify TRB2 expression in the eye, recombinant TRB2 was expressed as a glutathione-S-transferase fusion protein and purified. Anti-TRB2 antibody was affinity purified using immobilized TRB2. Western blot analysis with purified TRB2 indicated that TRB2 was recognized by uveitic patient IgG, but not by control human IgG. TRB2 expression was detected in the ciliary body by immunohistochemistry using affinity purified TRB2 specific antibody. To optimize the phage display system, we report the novel use of mutant phage during biopanning and the development of competitive immunoblotting. In competitive immunoblotting, all phage clones binding to common antibodies were enriched and used to block disease-irrelevant antibodies, facilitating the detection and isolation of patient-specific phage clones. Of 88 phage clones isolated by competitive immunoblotting, sixteen have been verified by dot blotting. These clones are under further investigation for their roles in uveitis. In conclusion, we demonstrate that subtractive phage display can identify autoantigens in an unbiased way using uveitis patient serum. Furthermore, we have successfully verified TRB2 expression in uveitis-susceptible ocular tissue, suggesting that phage display can identify disease-relevant autoantigens using patient polyclonal antibodies. We also demonstrate that use of mutant phage and competitive immunoblotting can improve the accuracy of isolating patient-specific autoantigens. Identification of autoantigens by this phage display system will advance our understanding of disease mechanisms and facilitate disease diagnosis.

Comments

This thesis is restricted to Yale network users only. This thesis is permanently embargoed from public release.

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