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G.N. Alberti, M. Garate, Z. Cao, D. Zoukhri, M. Goldstein, H.K. Wu, N. Panjwani; Normal Human Tears Contain IgA Antibodies Against Acanthamoeba Mannose–Binding Protein . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4969.
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Purpose: Acanthamoeba keratitis is a sight–threatening corneal infection of low–incidence. Exposure to the pathogen is common, however most people exposed to Acanthamoeba never develop keratitis. Previous studies have shown that Acanthamoebae express a mannose–binding protein (MBP), which mediates the adhesion of the pathogen to the corneal epithelial surface. The goal of this study was to determine if there are specific IgA antibodies against the Acanthamoeba MBP in human tears that might confer this protection. Methods: Unstimulated tears were collected from normal human subjects with surgical spears and microcapillary tubes (N=7). Tear protein level quantification was made using the Bradford method. Analysis of anti–MBP in tears was carried out using ELISA. Briefly, microtiter wells were coated with recombinant MBP (1µg/ml, overnight at room temperature). Nonspecific binding sites were blocked with 5% BSA in PBS (1 hour, 37ºC) and the wells were sequentially incubated with tears (1:40–1:400 dilution in PBS–0.5% BSA; 37ºC, 1 hour) and peroxidase–conjugated antihuman IgA (1:2000, 1 h, 37ºC). Finally the reactions were developed using a TMB substrate kit (Vector Labs) and the optical density was measured at 405 nm. Control wells were processed the same way except that instead of tear samples, dilution buffer was used. Results: All seven tear samples of normal individuals were found to contain significant levels of anti–MBP IgA. Significantly higher optical density over background values was detected in all tear samples when analyzed at 1:40 and 1:80 dilutions. Conclusions: IgA antibodies against Acanthamoeba MBP are present in human tears. These data suggest that healthy individuals normally have protective antibodies against Acanthamoeba keratitis, and that lack of these antibodies may predispose an individual to infection. It is hoped that this study will help find ways to determine a patient’s risk for infection, and develop effective immunization strategies for high–risk patients.
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