Yan L¹, Joachim SC², Erb C3, Pfeiffer N², Grus FH²
1: Department of Ophthalmology, Baoshan Central hospital, 201900, Shanghai, China
2: Experimental Ophthalmology, Department of Ophthalmology, Johannes Gutenberg University, Mainz, Germany
3: Department of Ophthalmology, Schlosspark Klinik, Berlin, Germany
Purpose: Previous studies on antibody profiles generated by Western blotting showed that different antibody repertoires exist between glaucoma patients and control subjects. But it is disadvantageous that Western blotting is time consuming, susceptible to variation, and limited in sensitivity.
The aim of this study was to find a faster and more precise method of antibody profiling. Therefore, we developed a bead-based immunoassay to detect antibody repertoires against retinal antigens in serum samples and to explore the immunological component of primary open-angle glaucoma (POAG).
Methods: Serum samples of 44 POAG patients and 48 control subjects were analyzed. Magnetic Protein G beads (Dynal, Oslo, Norway) were used to covalently bind serum antibodies. These bead-antibody complexes were incubated with homogenized retinal antigens. After several washing steps the bound antigens were eluted with glycine through a pH-shift. The antigen elutions were measured on ProteinChips by SELDI-TOF mass spectrometry (Biorad, Hercules, Ca, USA) on two different chip surfaces: a weak cation exchange surface (CM10) and a reversed phase chemistry hydrophobic chip (H50). The patterns were analyzed by multivariate statistical techniques.
Results: Complex antibody profiles were detected with significantly different immunoreactive spectra between POAG patients and control subjects (P<0.01). Not only up-, but also several down-regulated regions in the antibody spectra were present in POAG patients. These results are consistent with other studies on antibody repertoires of glaucoma.
Conclusion: The significant differences antibody patterns of POAG patients imply an autoimmune mechanism at least in some glaucoma patients. This study increases the information on the immunological component of glaucoma. The bead-based assay was shown to be an effective and promising tool for antibody-antigen-profiling, though it still needs to be developed further.
Key words: Glaucoma, Mass spectrometry, Autoimmunity, Antibody-antigen, SELDI-TOF
Supported by a grant from the German research community (Gr1463/4-1)
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