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F. H. Grus, J. Storf, D. Wuenschig, S. C. Joachim, N. Pfeiffer; Analysis of Antibody Patterns in Glaucoma Patients by Means of Protein Micro-Arrays. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5616.
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In previous studies changes in the antibody profiles against ocular antigens have been shown in the sera of glaucoma patients and these findings suggest a role for autoimmune involvement in the pathogenesis of glaucoma in some patients. All studies could consistently demonstrate up- and downregulations in immunoreactivities in glaucoma patients compared to controls. However, all these studies have in common that they used crude protein extracts from retina or optic nerve. It was the aim of this study to analyze the immunoreactivities in the sera of glaucoma patients against the most important purified antigens identified in previous studies by customized protein micro-arrays.
Sera of patients with primary open angle glaucoma (n=50) and healthy controls (n=50) were used. The protein arrays were prepared by spotting the antigens onto special nitrocellulose-coated slides. Up to 80 different antigens were used in each customized protein micro-array. The arrays were incubated overnight with the sera of patients (1:25) and the antibody-antigen-reactions were visualized by chloronaphthol staining. After digitizing, the spot intensities were compared and analyzed by multivariate statistical techniques.
Using protein micro-arrays, we were able to detect immunoreactivities in the sera of patients and healthy controls against the purified antigens such GFAP, GST, HSP 27, HSP70, HSP60, alpha-fodrin, and α-Crystallin. The statistical analysis revealed a significant difference (P<0.01) between the antibody reactivities of glaucoma patients and healthy subjects. Furthermore, we could confirm both up- and downregulations in the sera of glaucoma patients compared to healthy subjects as we could demonstrate in our previous studies using antigen mixtures from retina and optic nerve. Based on these antibody reactivities, we were able to detect glaucoma patients with a sensitivity and specificity of approx. 90%.
In this first pilot study, we were able to measure significant changes in the immunoreactivities in the sera of glaucoma patients against purified antigens using protein micro-arrays. The use of purified antigens spotted onto protein micro-arrays might be an important step towards a robust antibody profiling in glaucoma patients suitable to be used in clinical routine.
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