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Tatsuya Yoshitake, Tomoaki Murakami, Kiyoshi Suzuma, Shin Yoshitake, Akihito Uji, Masahiro Fujimoto, Yuko Miwa, Nagahisa Yoshimura; Identification of Anti-Hexokinase-1 Antibody in the Sera from Patients with Diabetic Macular Edema. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3251.
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© ARVO (1962-2015); The Authors (2016-present)
Blood-retinal barrier (BRB) guarantees the sequestration of retinal antigens from immune system in healthy eyes. In contrast, diabetic macular edema (DME) disrupts inner and outer BRB, although it remains ill-defined how autoimmune responses to retinal antigens are induced and concomitantly have influences on the damages on retinal parenchyma. Here, we screened autoantibodies and identified retinal antigens for serum IgG from DME patients.
We performed Western blotting using porcine retinal lysates for screening of anti-retinal antibodies in DME serum. Combination of immunopercipitation and mass spectrometry was performed to identify the targets of the autoantibodies. We confirmed the immunoreactivity of autoantibodies using Western blot using human recombinant proteins and immunostaining of retinal sections from C57BL/6 mice with serum IgG and antigen-specific antibodies. We measured titers of the autoantibodies by enzyme-linked immunosorbent assay (ELISA).
Screening by Western blotting showed approximately 100-kDa band which was detected in sera from 34 (82.9%) of 41 DME patients. Combination of immunoprecipitation and mass spectrometry identified hexokinase-1 (predicted molecular weight=105-kDa) as one of autoantigens. Immunoblotting confirmed that IgG from DME sera containing higher titer of anti-hexokinase-1 antibody was reactive to human recombinant hexokinase-1, Immunostaining with the serum IgG was to some extent colocalized to hexokinase-1 in the outer plexiform layer or the inner retinal layers. The quantitative analyses using ELISA demonstrated that the titer of anti-hexokinase-1 antibody in DME sera (0.267±0.204 [A.U.]) was significantly higher than that in diabetic patients without diabetic retinopathy (DR) (0.139±0.094 [A.U.]; P=0.003) or DR patients without DME (0.180±0.127 [A.U.]; P=0.020).
We identified hexokinase-1 as one of autoantigens for antibodies from DME sera, suggesting its potential feasibility as a novel serum biomarker for diagnosis of DME.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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