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G.V. Jirawuthiworavong, S.A. Morales, K. Shimazaki, J.R. Heckenlively, G.M. Seigel, L.K. Gordon; A Flow Cytometric Assay for the Detection of Pro–Apoptotic Antiretinal Antibodies in Human Serum . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2684.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Autoantibodies against retinal antigens are implicated in immune–mediated vision loss. These antibodies are described in cancer–associated retinopathy (CAR), melanoma–associated retinopathy, and a subset of patients with retinitis pigmentosa. In CAR, antibodies to recoverin enter retinal cells inducing apoptosis in vitro and in vivo. Currently, no standardized assay exists for the detection of pathogenic antiretinal antibodies. We describe a new cytotoxicity assay using a retinal precursor cell line, R28, to detect the presence of pro–apoptotic antiretinal antibodies in serum of patients with presumed autoimmune retinopathy. Methods: R28 cells, an immortalized rat retinal precursor cell line, was used as a target for antiretinal antibodies. Cells were plated at a density of 2–3x105 cells/well and exposed for 72 h to purified human IgG of patient's sera at a concentration of 25 to 400ug/ml. Normal human IgG Fab fragments and normal human whole IgG were used as controls. Annexin V, a cell surface marker for early apoptosis, was detected using an Annexin V–FITC Apoptosis Detection Kit . Analysis was performed with flow cytometry and analyzed with CellQuest. Results: As compared to the untreated controls, the mean fluorescence intensity (MFI) of Annexin V–FITC was unchanged in the cells exposed to normal whole human IgG or IgG Fab fragments. However, the MFI of cells treated with human IgG from all five patients with autoimmune retinopathy was significantly increased, indicating early apoptosis. One serum was tested at multiple IgG concentrations, documenting a dose–dependent response. Conclusions: Purified IgG from five patients with presumed autoimmune retinopathy induces R28 cell death via apoptosis as evidenced by an Annexin V assay. The analysis of this pro–apoptotic activity is rapid and may represent a new alternative screening method for patients with suspected autoimmune retinopathy. Additional studies are required to further characterize and validate these findings.
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