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Alessandro Iannaccone, Allison Umfress, Indira Neeli, Lauryn Murphy, Pratheebha Krishnamurthy, Nataliya Lenchik, Ivan C. Gerling, Marko Z. Radic; Evidence for Auto-Antibodies (auto-Abs) Recognizing Human Macular Tissue Antigens in Serum Samples of Patients with Age-Related Macular Degeneration (AMD). Invest. Ophthalmol. Vis. Sci. 2012;53(14):2272.
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To report on auto-Abs recognizing human macular neuroretinal (nRet), retinal pigment epithelium (RPE), Bruch’s membrane (BM) and choroidal (Ch) antigens detected in AMD patients by Western blot (WB).
Sera from 131 AMD [2/3 early (AREDS cat. 3) AMD, 1/3 advanced (AREDS cat. 4) AMD] and 245 unaffected subjects, all ≥70 years old, were screened for presence of auto-Abs by WB. The nRet/RPE/BM/Ch homogenates were obtained from full-thickness, 10mm diameter, human donor eye macular punches. Fifteen µg of nRet/RPE/BM/Ch protein lysate were separated on SDS-PAGE, transferred to nitrocellulose membrane, incubated with AMD or control sera, developed with anti-human IgG-HRP and chemiluminescence, and exposed for 5, 15 and 30 sec to obtain estimates of the number of the immunoreactive (IR) bands and their relative intensity. A 6-step (grades 0-5) classification method was developed and applied. Presence and intensity of IR bands were assessed every 2kDa, and the information was entered in a database for analysis. Odds ratios (OR), 95% confidence intervals, Χ2 statistics, and p-values were calculated utilizing the Benjamini-Hochberg (B-H) method (α=0.05) to correct for multiple comparisons and limit the false discovery rate. The specificity (Sp) of the detected bands was also calculated.
WBs for auto-Abs against nRet/RPE/BM/Ch tissue antigens in AMD and control samples revealed the presence of multiple IR bands. Compared to control samples, AMD sera exhibited autoreactivity: a) more frequently, for the dataset as a whole (Χ2 =35.67; p=0.02x10-8) and, specifically, for 11 distinct 2-kDa IR intervals (3 between 26- and 32-kDa, 7 between 36- and 50-kDa and one at 98-100 kDa); and b) more intensely in 13 instances, 12 of which coincided with the more frequent IR intervals. The 30-32 kDa IR interval was highly significant for all levels of analysis. The highest OR estimate was 4.84, the lowest 2.04, and all clearly excluded the null 1.0 value (p-value cut-off significance level by B-H method = 0.0055). Most of the significant IR bands exhibited Sp >0.84, and 11 of them has Sp ≥0.90.
Consistent with the growing body of evidence supporting a role for inflammation and the immune system in AMD pathogenesis, reactivity in AMD was observed more often and more intensely than in control sera at several distinct IR intervals by WB criteria. The binding was highly statistically significant and highly specific. As in primary and cancer-associated autoimmune retinopathies, autoimmunity in AMD is not specific for a single antigen, and different IR patterns are observed in different patients. Auto-Abs may play a contributory or causal role in AMD pathogenesis.
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