報告書番号

U98064

タイトル（和文）

生体機能を利用したセンシング (その１) --受容体を用いた外因性内分泌かく乱物質の蛍光測定法の開発--

タイトル（英文）

Application of biological function to sensing 1Receptor assay using fluorescence flow spectrophotometer for environmental hormones

概要 （図表や脚注は「報告書全文」に掲載しております）

生体の女性ホルモン受容体機能を利用し、内分泌かく乱物質である環境ホルモン向けの検出法について検討した。フロー式蛍光光度計を用いて受容体と天然ホルモンとの結合阻害から受容体と結合しうる化学物質を測定した。その結果、標準物質として選んだ数種の天然ホルモンでピコモルオーダーの検出が可能であった。また、４種の化学物質（タモキシフェン、ノニルフエノール、ビスフェノールA、ジエチルベスステロール）でピコモルからミリモルでの定量が可能であった。本法により、受容体結合性を持つ物質を簡便かつ高感度に検出できると考えられた。

概要 （英文）

It is growing consensus that several chemicals have estrogenic activity by interacting with development and functions of endocrine systems in human and the other organisms. The chemicals such as endocrine disrupters called as environmental hormones became to be world wide problem for our future. It is expecting that those chemicals may be contaminated into the environment at quite low concentration. Therefore, a universal method which can detect large number of the chemicals having estrogenic activity with high sensitivity is desired at present. In order to get a better insight of environmental estrogens, we are now developing a method for detecting and screening of estrogenic chemicals using the function of estrogen receptor with fluorescence.We reported here the new method can detect several compounds including female hormones at pico molar order. The function of human estrogen receptor in the binding to the ligands was applied to this assay system. The assay was established as following. The estrogen receptor was immobilized onto the 95 micrometer PMMA beads in a diameter by absorption coating. The coated beads was packed into the glass flow cell having the 50 micrometer mesh stopper. When the estrodiol conjugated with BSA-CY-5 fluorescence dye(E2-BSA-CY5) was flew through the flow cell, E2-BSA-CY5 was captured onto the beads surface due to the binding to the receptor. The free E2-BSA-CY5 between the beads particles were washed out by switching the flow to the binding buffer. The binding profile could observe on time as a fluorescence intensity from the beads pack by using the light and detector system including adequate excitation and emission filters. The bound E2-BSA-CY5 to the receptor related functionally to the fluorescence intensity after the washing. The concentration of the receptor as the beads packing and the continuous capturing of the E2-BSA-CY5 by the flow system was useful to improve the detectable concentration of the analyte. In the case that the chemicals capable of binding to the receptor was mixed with E2-BSA-CY5, the fluorescence intensity decreased by binding of the chemicals to the receptor instead of that of E2-BSA-CY5 when compared with the case without the chemicals. The decrease of the fluorescence intensity could be expressed functionally as the concentration of the compound. As results, it was possible to detect both estrodiol and estriol at pM order. Tamoxifen and DES were also detected at the same order. In contrast, nonylphenol and bisphenol A was detectable at the range from nM to M. Those results are rising the possibility that this fluorescence detection applying the receptor function could measure quickly the chemicals having the affinity to the receptor with high detectable level.

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