報告書番号

U01011

タイトル（和文）

生体機能を利用した微量物質のセンシング (その３) --複数の抗体を利用した検出濃度範囲と検出種の拡大--

タイトル（英文）

Application of biological function to sensing 3 Extension of dynamic range and detectable antigen with dual immunoassay

概要 （図表や脚注は「報告書全文」に掲載しております）

多種・多様の環境ホルモンの簡易・高感度検出法を目指し、複数の抗体を利用して検出濃度範囲や検出物質を拡大する多重同時検出法を開発した。検出濃度範囲の拡大では、同じ抗原に対して結合の強さが異なる抗体を組み合わせた。強く結合する抗体が薄い範囲で働き、弱く結合する抗体が濃い範囲で働くので、従来は困難であった一度に広い濃度範囲での検出が可能となった。検出物質の拡大では、異なる特定の抗原に結合する抗体を組み合わせた。抗体は各々の抗原の検出に働くので、一度に複数の抗原が検出できた。なお、検出物質は女性ホルモンを対象とした場合、2種類の抗体を同時に使用することで6種類（エストリオール、エストリオール-3-硫酸、エストリオール-3-グルクロン酸、エストラジオール、エストラジオール-3-硫酸、エストラジオール-3-グルクロン酸）を同時に、かつ高感度に検出することができた。

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 worldwide problem. We developed the assay system applying either an estrogen receptor or an antibody to detect estrogenic chemicals. The considerable numbers of chemicals to be detected created another demand to develop multi-detection system for monitoring of many chemicals in the environment. The purpose of this study is to establish multi detection assay using dual antibodies with broad dynamic range. The dual assay was modeled with female hormones such as estriol and estradiol and the corresponding antibodies.Antibody exhibits binding affinity towards the corresponding antigen. It was possible to extend dynamic range and detectable antigen in a single assay operation with combinatory use of the two antibodies. To extend the range, the two antibodies exhibiting different affinities towards the same antigen were used together in an assay. The antibody having high affinity quantified the antigen at low concentration range. The other antibody having low affinity quantified the antigen at high concentration range. The binding of each antibody was simultaneously and independently occurred to quantify the antigen at entire concentration range. Thus, it was possible to extend dynamic range. To extend detectable antigen, dual assay was performed with two antibodies towards the different antigens. Since each antibody independently bound to the corresponding antigen, simultaneous detection of the two antigens was possible. The dual system was established with two different anti-estriol antibodies exhibiting the different affinities. In the assay with a single antibody, the antibody having high affinity quantified estriol in the range from 1 pM to 1000 pM. The other antibody having low affinity quantified in the range from 1000 pM to 1000 nM. In contrast, in the dual assay with both antibodies, the dynamic range was extended from 1 pM to 1000 nM. It is well known that the narrow dynamic range was one of drawbacks in immunoassay system. The established dual assay provided a novel procedure to achieve broad dynamic range. The dual assay was performed with anti-estriol and anti-estradiol antibody. The combinatory use of two antibodies made it possible to quantify both antigens not only as total concentrations but also as estradiol and estriol concentrations. The dynamic range of each female hormone was from 1 pM to 1000 pM. It was possible to extend the number of detectable antigen up to six. The dual assay provided multi-detection ability for the two antigens in a single assay operation.

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