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K. Yoneda, M. Nakano, K. Mori, S. Kinoshita, K. Tashiro; Successful Quantitation of TGF-beta3 in Disease-Related Human Aqueous Humor. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5543.
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© ARVO (1962-2015); The Authors (2016-present)
Transforming growth factor (TGF)-ß controls versatile cellular processes. Although the proportion of all three TGF-ß isoforms, TGF-ß1, -ß2, and -ß3, was affecting to the pathogenic situation, TGF-ß3 has never been assessed in human aqueous humor (AH). To investigate the pathogenesis of ocular diseases, we established a quantitative enzyme-linked immunosorbent assay (ELISA) of TGF-ß3 and quantitated its concentration in human AH together with the concentration of TGF-ß1 and -ß2.
AH samples were collected from 149 eyes of patients: pseudoexfoliation syndrome (PE) (n = 17), primary open angle glaucoma (POAG) (n = 46), chronic angle closure glaucoma (CACG) (n = 18), proliferative diabetic retinopathy (PDR) (n=23) and cataract (n = 45) as a control, with the IRB approval and informed consent. The concentration of TGF-ß3 in AH were quantitated by a double-antibody sandwich ELISA by screening the best combination of antibodies with a modification in TGF-ß3 activation procedure.
We established a quatitative TGF-ß3 ELISA (CV: 2%, detection limit: 0.75 pg/ml, cross reactivity against the other TGF-ß isoforms: less than 0.1%, recovery rate in AH: 99.7 ± 3.7%) as well as TGF-ß1 and -ß2 ELISA. During establishing TGF-ß3 ELISA, we revealed that the prolonged activation time was necessary to accurately quantitate TGF-ß3 in human AH. Using these ELISAs, we determined absolute concentration of all three TGF-ß isoforms in each AH sample and discovered significantly high concentration of TGF-ß3 and TGF-ß2 in PE eyes (46.6 ± 19.9 pg/ml, control: 4.3 ± 5.7 pg/ml) and in CACG eyes (3134 ± 700.5 pg/ml, control: 2232 ± 627.7 pg/ml), respectively.
We have succeeded to establish a quantitative ELISA for TGF-ß3 in human AH. Moreover, combining the system with quantitative TGF-ß1 and -ß2 ELISA, we quantitated the concentration of all three TGF-ß isoforms in AH obtained from one eye. Consequently, a complete set of the quantitative TGF-ß ELISAs enabled us to investigate and discuss further about the relationship between the proportion of three isoforms and those concerned diseases.
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