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JP Morgan, PJ Tighe, HS Dua; Discovery of Novel Autoantigens in Endogenous Uveitis using a Human Retinal cDNA Expression Library . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1547.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Several retinal proteins have been proposed as autoantigens in autoimmune uveitis because of their ability to induce Experimental Autoimmune Uveitis in animals. These proteins have subsequently been studied for a role in human disease. This approach risks placing undue emphasis on antigens that may be primarily relevant to non-humans. We sought to address this problem by constructing a human retinal cDNA expression library and screening it with sera from uveitis patients and controls. Methods: Purified human retinal mRNA was used to construct a cDNA library using a random primer strategy. This was inserted into T7 phage vectors and packaged. A "micro-biopanning" technique was used to screen the library with sera from 37 uveitis patients and 42 controls, producing 79 independent phage pools. PCR`s using a modified T7 primer enabled identification of recombinant phage, which were DNA sequenced. Results: Comparison of DNA sequences with established databases has identified 11 affinity selected retinal protein fragments, derived from both uveitis and control groups. Several of these fragments represent segments of various coupling proteins. The proteins have been expressed and purified. The specificities of these fragments to patient/control sera are currently being assessed by ELISA. Conclusion: The above approach has the potential to identify retinal autoantigens relevant to human uveitis, independent of data based on animal experiments. We have successfully constructed a human, retinal cDNA expression library and micro-biopanned it with uveitis and control sera. This strategy has already yielded a number of interesting retinal protein fragments.
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