A Screening Method for Estrogens Using an Array-Type DNA Glass Slide
スポンサーリンク
概要
- 論文の詳細を見る
A new screening assay was described for the determination of endocrine disrupting chemicals (EDCs), such as synthetic estrogens, with an array-type DNA glass slide having characteristics of 1) a high sample throughput, 2) a compact size allowing a small sample volume, and 3) a sensitive determination based on the estrogen-dependent binding of the human estrogen receptor α (hERα) with its estrogen responsive element (ERE; Vit. A<SUB>2</SUB> gene promoter). We devised a glass slide on which a thin agarose gel was mounted. Avidin was then covalently immobilized on each well of the glass slide after the gel was activated by a NaIO<SUB>4</SUB> solution. Also, the biotinylated ERE as a DNA probe was immobilized on the gel layer through avidin-biotin binding. After the estrogen-dependent binding of a yellow fluorescent protein-fused hERα (YFP-hERα) to ERE on the gel layer, the fluorescence intensity of YFP-hERα quantitatively extracted into the gel was directly determined with a fluorescence microplate reader. Pre-incubation of YFP-hERα with estrogen at 37°C for 30min enhanced the estrogen-dependent hERα-ERE binding. The determined hormonal activities of estrogens on the interaction of YFP-hERα with ERE were as follows in their decreasing order: diethylstilbestrol (DES) > 17β-estradiol (E<SUB>2</SUB>) ≈ ethynylestradiol (EE<SUB>2</SUB>) > 4-hydroxy tamoxifen (OHT) > clomiphene (Clo). The present method provides a sensitive estrogen-dependent dose-response curve down to ∼10<SUP>-13</SUP> M in the case of DES. This method will become a competitive alternative to the conventional <I>in vitro</I> assays, such as a DNA-binding assay using radioisotopes.
- 社団法人日本分析化学会の論文
社団法人日本分析化学会 | 論文
- 微生物学的試験法による畜産物中に残留する抗菌性物質の高感度測定法(安心と安全に役立つ分析化学)
- 液体クロマトグラフィー/質量分析法による缶飲料中のビスフェノールAの定量 (環境ホルモンと分析化学)
- 高速液体クロマトグラフィーによるL-シトルリン測定を指標としたマクロファージの一酸化窒素産生の評価 ( バイオサイエンスと分析化学)
- 小型定電流電解電量/光度滴定装置の開発と食用油の過酸化物価定量への応用
- 固定化ヒスタミンオキシダーゼを用いる吸光検出フローインジェクション分析による魚醤中のヒスタミンの定量(安心と安全に役立つ分析化学)