報告書番号

U99040

タイトル（和文）

生体機能を利用したセンシング (その２) --抗原抗体反応を用いた外因性内分泌かく乱物質の測定法の開発--

タイトル（英文）

Application of biological function to sensing (Part2)- Immunoassay using fluorescence flow spectrophotometer for environmental estrogens -

概要 （図表や脚注は「報告書全文」に掲載しております）

生体の抗原抗体反応を利用し、環境ホルモン向けの測定法について検討した。フロー式蛍光光度計を用いて抗体と女性ホルモンとの平衡結合状態を測定することにより、女性ホルモン濃度を決定した。本法は抗体の抗原結合能力の全てを検出に利用することができ、定量濃度範囲はエストリオールとエストラジオールでそれぞれ、1ppt～100ppt、0.5ppt～100pptであった。本法により、女性ホルモンを簡便かつ高感度に計測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 endocrine systems in human and the other organisms. The chemicals such as endocrine disrupters called as environmental hormones became to be a problem in the world. We developed the assay system applying estrogen receptor to detect estrogenic activities of chemicals. The assay was more suitable for the detection of activities of the chemicals than for the quantification. To monitor the chemicals in environment, a method capable of detecting chemicals with high sensitivity will be important in near future. We are now developing a method to quantify the estrogenic chemicals including female hormones using antigen-antibody reactions.Here we report that optimized detection of female hormones was accomplished with a flow-based, immunoassay that used microspheres as the solid phase. The estriol was immobilized onto the 95 micrometer PMMA beads in a diameter by absorption coating. The coated beads were packed into the glass flow cell having the 50 micrometer mesh stopper. The equilibrated mixture of fluoresce-labeled anti-estriol monoclonal antibody and sample was drawn through bead pack containing immobilized estriol. In the case that the sample did not contain estriol, free antibody present in the mixture was bound by the immobilized estriol because there was no binding partner in the mixture. The bound antibody was quantified directly by their label intensity under the excitation light. The quantification was also possible by indirect label with subsequent flow of fluorescence-conjugated anti-species secondary antibody, which could bind to the bound anti-estriol antibody to the immobilized estriol. In the case that the sample contained estriol, a part of antibody present in the mixtures was bound to estriol under a steady state. The amount of the bound antibody to the immobilized estriol was decreased when the equilibrated mixture was drawn through bead pack. Fluorescent signals were proportional to the concentration of the free antibody and were also proportional to the concentration of estriol in a mixture. The optimum achievable dynamic range of this immunoassay was 1 ppt ‐ 100 ppt. Because the proportion of free anti-estriol antibody in the mixture was controlled by the Kd governing its binding to estriol, when the concentration of the antibody was below the Kd, the smallest detectable estriol concentration approached the theoretical limit of detectability achievable with this antibody. Those results provided the evidences that the developed method exploiting the function of an antibody in the binding could sense female hormones within few minutes with high detectability.

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