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N. Bohm, S. C. Joachim, W. Haass, N. Pfeiffer, F. H. Grus; Protein Micro-Arrays as an Effective Method for Antibody Profiling in Glaucoma. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1570.
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Changes in antibody profiles against retinal and optic nerve antigens in sera of glaucoma patients were detected in multiple studies, referring to an autoimmune involvement in the pathogenesis of the disease. Interestingly, not only elevated antibody reactivities, but also down-regulated regions have been shown in glaucoma patients compared to healthy controls. However, many studies concerning typical autoimmune diseases rather demonstrated up-regulated immunoreactivities. In this study, we used highly sensitive protein micro-arrays in a high-throughput compatible approach for serum antibody profiling.
For the analysis of antibody reactivities, sera of patients with primary open-angle glaucoma (POAG; n=70) and healthy controls (n=70) were used. The protein arrays were prepared by spotting highly purified antigens in quadruplicate spots onto special nitrocellulose-coated slides. Approx. 100 different antigens were used for each customized protein micro-array. The arrays were incubated with patients’ sera (1:250) for 12h. For visualization of the antibody-antigen-reactions the arrays were treated with a secondary fluorescence labeled antibody (Cy5), followed by scanning the slides with a high-resolution confocal-scanner. The fluorescence signals emitted from secondary antibodies were digitized and the spot intensities were compared using multivariate statistical techniques.
Consistent with previous studies analyzing antibody patterns of glaucoma patients, we could detect multiple differences in antibody reactivities between POAG patients and healthy subjects. Interestingly, POAG patients showed many significantly decreased immunoreactivities against e.g. VEGF, Jo-1, HSP 70, spectrin, and β2-adrenergic receptor (P<0.006). Based on these antibody patterns we were able to differentiate between POAG and healthy subjects with a specificity and sensitivity of approx. 90% (ROC-curve r=0.91).
As a result of the protein micro-array approach we could confirm both up- and down-regulations of antibody reactivities in sera of glaucoma patients, but detected a much higher rate of down-regulations than in previous studies. These findings suggest a considerable damage in protective natural autoimmunity of glaucoma patients. Further studies need to verify changes of the natural antibody patterns during the disease process, e.g. in longitudinal studies.
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