A Screening Method for Estrogens Using an Array-Type DNA Glass Slide
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- KIM Sung Bae
- Department of Chemistry, School of Science, The University of Tokyo
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- OZAWA Takeaki
- Department of Chemistry, School of Science, The University of Tokyo
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- UMEZAWA Yoshio
- Department of Chemistry, School of Science, The University of Tokyo
書誌事項
- タイトル別名
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- Screening Method for Estrogens Using an Array Type DNA Glass Slide
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抄録
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. A2 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 NaIO4 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 (E2) ≈ ethynylestradiol (EE2) > 4-hydroxy tamoxifen (OHT) > clomiphene (Clo). The present method provides a sensitive estrogen-dependent dose-response curve down to ∼10-13 M in the case of DES. This method will become a competitive alternative to the conventional in vitro assays, such as a DNA-binding assay using radioisotopes.
収録刊行物
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- Analytical Sciences
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Analytical Sciences 19 (4), 499-504, 2003
社団法人 日本分析化学会
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詳細情報 詳細情報について
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- CRID
- 1390282679233624320
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- NII論文ID
- 130004440753
- 80015917930
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- NII書誌ID
- AA10500785
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- COI
- 1:CAS:528:DC%2BD3sXjt1WgtLY%3D
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- ISSN
- 13482246
- 09106340
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- NDL書誌ID
- 6507381
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- PubMed
- 12725382
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- 本文言語コード
- en
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- データソース種別
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- JaLC
- NDL
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- PubMed
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