報告書番号

U01064

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４) --夾雑物質の影響を排除したモニタリング法の開発--

タイトル（英文）

Application of biological function to sensing 4- An immunoassay for monitoring to avoid effects of coexisting substances in environmental sample on measurement -

概要 （図表や脚注は「報告書全文」に掲載しております）

環境試料において夾雑物質の影響を排除し、測定しうる迅速・簡易なモニタリング法を開発した。本法では試料中の物質を経時的に蛍光強度の時間変化として検出できる。蛍光性抗エストラジオール抗体を用いた女性ホルモン（エストラジオール）の検出をモデルにモニタリング手法を評価した結果、強度の時間変化から、エストラジオールを４pM（1ppt）から３nM（800ppt）の範囲で定量できた。なお、分析時間を従来法の20分間から試料あたり約２分間まで短縮できた。また、 ２種類の抗体（対照抗体と測定抗体）と内部標準物質を用い、夾雑物質の影響の排除する二つの機能をモニタリング法に付与した。その結果、夾雑物質の影響を排除し、環境試料においてエストラジオールを正確に測定できた。

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. Numerous assays applying biological functions have been described as highly sensitive and rapid method to detect estrogenic chemicals. However, several subjects have not been solved in the measurement of the environmental samples with the bio-sensing methods. The difficulties in the assays are summarized as how to estimate the effect of the substances in the samples on the activities of biological component for the detection and reflect the estimated effect to the measured values. The purpose of this study is to establish an assay for monitoring to avoid effects of coexisting substances in environmental sample on measurement. The method for monitoring of the analyte is established on the basis of a time-dependent change of the fluorescence intensity with antibody-antigen reactions. The binding mixture of fluorescence-labeled antibody and antigen is drawn past the cell containing the coated antigen. A small portion of the unbound antibody to the analyte is captured in the cell, and the captured antibody is represented as a linear time-dependent change of fluorescence intensity (Slope). While, the liganded antibody in the sample is passed through without the capturing. The slope directly relates to the proportion of the unbound antibody to the analyte in the sample. Thus, it is possible to monitor rapidly and easily the analyte at the real time from the scale of the slope in the assay. The monitoring method was modeled with an anti-estradiol antibody in the detection of estradiol. The slope was reflected to the concentration of estradiol, therefore, the quantification was possible in the range from 4pM to 3nM with the slopes. The required time for each solution is only 2 minutes. Two different abilities to avoid effect of coexisting substances in the environmental samples was added to the monitoring method by using dual antibodies with internal standards as control antibody (antigen) and detective antibody (antigen). The control antibody and control antigen estimates the effect of the coexisting substances in the sample to antibody-antigen reaction to correct the reaction between the detective antibody and the analyte. The analyte is finally quantified as the fitted value with a series of the assay responses in the presence of the detective antigen at the varied concentrations. This assay is applied to environmental samples with the exogenous estradiol. The anti-biotin antibody and biotin are correctly served as control for reaction between anti-estradiol antibody and the spiked estradiol in the samples. The assay to avoid effects of coexisting substances on measurement achieved the direct quantification of estradiol in the environmental samples.

報告者

キーワード