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Nataliya Lenchik, Francesco Giorgianni, Sarka Beranova-Giorgianni, Ivan Gerling, Marko Radic, Alessandro Iannaccone; Identification of Human Macular Tissue Antigens Recognized by Serum Auto-Antibodies (auto-Abs) in Patients with Age-Related Macular Degeneration (AMD). Invest. Ophthalmol. Vis. Sci. 2013;54(15):4103.
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To report on the first results of a 2-D gel electrophoresis (DE) and mass spectrometry (MS)-based approach to identifying human macular tissue antigens recognized by serum auto-Abs found in AMD patients.
2-DE, MS and database screening were used to identify proteins obtained through immunoprecipitation. Macular tissue lysates were obtained from full-thickness, 10mm diameter, human donor eye macular punches. 150 µg lysate were immunoprecipitated with AMD or control sera obtained from a collection of 131 AMD samples and 245 unaffected subjects, all previously tested for auto-Abs by Western blots (WBs). By choosing appropriate sample amount, using pre-cast IPG dry strips (pH3-10) and casting 12% equal gel, stained by Sypro Ruby, 2-DE images were obtained and a steady 2-DE technique was established. Protein spots were analyzed by Progenesis Same Spots software (v 2.0). The program generated a list of proteins ranked by spot intensity. Spots corresponding to the bands previously identified as differentially reactive by WBs in the same AMD sera were cut out and analyzed by MS.
Among the 22 differentially reactive spots in 2-DE gels that were identified, 11 of which unique to AMD samples, preliminary analyses have thus far identified reactivity against the following targets: a 38-kDa secreted protein that plays a role in immune system regulation and apoptosis inhibition; the N-terminal peptide of another secreted protein, normally cleaved into various isoforms with distinct biological activities, that is implicated in neural neuroprotection, cell replication, and lysosomal activity; and a 39-kDa protein known to be normally involved in retinal cell development, morphogenesis and differentiation. Neither target had thus far been implicated in the growing body of evidence for an inflammatory/immune-mediated pathogenesis of AMD. Additional studies are in progress on these and other samples.
2-DE and MS are effective tools to identify human macular tissue antigens recognized by serum auto-Abs observed in AMD patients. The utilization of this approach, combined with in vivo validation studies, offers the opportunity to test hypotheses about the role played by the autoreactivities observed in AMD patients, to understand better AMD pathogenesis, and to develop, characterize and test treatments on new models of the disease.
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