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Alessandro Iannaccone, Indira Neeli, Pratheebha Krishnamurthy, Nataliya Lenchik, Ivan C. Gerling, Marko Z. Radic; Autoreactivity Against Human Macular Tissue Antigens in Sera from Age-Related Macular Degeneration (AMD) Patients. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4016.
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To report on Western blot (WB) analyses performed with serum samples collected from AMD and unaffected controls on macular tissue homogenates inclusive of neuroretina (nRet), retinal pigment epithelium (RPE), Bruch’s membrane (BM) and choroid (Ch) to screen for the presence of auto-antibodies as potential disease biomarkers in AMD.
Serum samples from White subjects with AMD (most of whom with AREDS category 3 early AMD) and unaffected controls, all ≥70 years old, is being screened for the presence of auto-Abs by standard WB techniques. The nRet/RPE/BM/Ch homogenates were obtained through dissection of human donor eyes, collecting full-thickness macular punches, 10mm in diameter. Fifteen µg of nRet/RPE/BM/Ch protein lysate were loaded on SDS-PAGE gel lanes, separated, transferred to nitrocellulose membrane, incubated with either AMD or control sera, developed using a chemiluminescent substrate, and exposed for 5, 15 and 45 sec to obtain estimates of the number of the reactive bands and their relative intensity. A 5-step (grades 0-4) classification method was developed and applied. Presence and intensity of the bands was assessed every 2kDa, and the information entered in a database for analysis. Odds ratios (OR), 95% confidence interval (CI), Χ2 statistic, and p-values were calculated (only <0.05 reported here).
WBs for auto-Abs against nRet/RPE/BM/Ch tissue antigens in AMD and control samples revealed the presence of multiple bands, some of which seen also in controls. In AMD samples, more frequent bands were seen in the 24-26kDa (OR: 3.25), 32-34kDa (OR: 6.50; 95%CI 1.05-40.13, Χ2=4.55, p=0.03), 38-40kDa (OR: 4.73), 40-42kDa (OR: 4.00), 48-50kDa (OR: 3.00), and 58-60kDa ranges (OR: 5.00; 95%CI 1.03-24.28; Χ2=4.21, p=0.04); more intense bands were seen in the 32-34kDa (OR: 4.19) and 58-60kDa ranges (OR: 3.25) when using the cut-off grade of ≥3, and in the 46-48kDa range for bands grade ≥2 (OR: 5.25).
Auto-Abs against human macular nRet/RPE/BM/Ch tissue antigens are present in AMD sera more often and more intensely than in control samples. Bands in six specific regions appear more common and/or intense in AMD sera and, therefore, of high interest for AMD biomarkers research. Replication studies and mass spectrometry analyses are in progress to verify these findings and characterize the identity of the antigens involved in autoreactivity in AMD.
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