| Project/Area Number |
15590588
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Legal medicine
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| Research Institution | Juntendo Unversity |
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Principal Investigator |
KIMURA Hiroko Juntendo University, Department of Forensic Medicine, Lecturer, 医学部, 講師 (00053299)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUMOTO Kazuko Waseda University, Department of Chemistry, Professor, 理工学部, 教授 (60111457)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | fluorescence resonance energy transfer (FRET) / time-resolved fluoroimmunoassay / time-resolved fluoromicroscopy / methamphetamine / 4-hydroxy nonenal / IL-22 / ニトロチロシン / リポポリサッカライド / FRET / 時間分解蛍光測定 / ハイドロキシノネナール / LPS / 酸化ストレス / ユウロピウム錯体 / ホモジニアスイムノアッセイ / 4-ヒドロキシノネナール / 過酸化脂質 / 敗血症 |
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| Research Abstract |
1.A rapid and simple homogeneous time-resolved fluoroimmunoassay (TR-FIA) based on fluorescence resonance energy transfer (FRET) from europium (Eu) to cyanine dye (Cy5) has been developed for the quantitation of methamphetamine. In this assay, Eu chelate was labeled to a conjugate of methamphetamine and bovine serum albumin (MA-BSA), as an energy donor, and Cy5 was labeled to anti-MA as an energy acceptor. The close proximity between the two labels in the immunocomplex permit energy transfer from the excited Eu^<3+> donor. Therefore, by measuring the sensitized emission of Cy5 with time-resolved assay, immunocomplex of MA-BSA and anti-MA can be measured in the homogeneous solution without separation steps within 30 minuets. By a competitive immunoassay, MA could be assayed in the range 0.1-1000 ng/mL. The intra-assay variations were 5.4%-14.8% at 5 different concentrations. When urine or serum samples were examined, the quenching of Eu fluorescence was observed but the acceptor-to-dono
… More
r ratio constantly depended upon the dilution of samples. Twenty urine samples were assayed and the data showed a good correlation to those obtained by gas chromatography (r=0.94). The homogeneous assay using Eu-Cy5 energy transfer is tune-saving without any washing procedures and is suitable for screening drugs that are commonyl abused.
2.New imunnoassay for IL-22 using invader assay and FRET system was developed. In this assay, the detection limit of IL-22 was 0.2-0.9 pg/mL.
3.Hetorogeneous type of TR-FIA for a lipid peroxide 4-hydroxynonenal (HNE) was developed. By this assay, we found that a low dose of bacterial lipo-polysaccharide (LPS), injected intra-peritoneally (0.5mg/kg), increased serum HNE level by 28-folds, with a peak at 20 min. HNE in the rat intestinal mucosa after LPS injection was also measured. HNE in the intestine was increased with a small peak at 20 min followed by a sustained elevation at 2〜4 hours after injection of LPS. Immunohistochemistry demonstrated enhanced labeling with anti-IgA and anti-HNE antibodies in the plasma cells followed by diffusion of the labeled materials in the submucosal tissue after LPS injection. Less
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