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Development of time-resolved fluoroimmunoassay based on fluorescence resonance energy transfer and its application to forensic medicine and sience

基于荧光共振能量转移的时间分辨荧光免疫分析技术的发展及其在法医学和科学中的应用

基本信息

批准号：
15590588
负责人：
KIMURA Hiroko
金额：
$ 2.24万
依托单位：
Juntendo Unversity
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

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

1.建立了一种基于荧光共振能量转移（FRET）从铕（Eu）到菁染料（Cy5）的快速、简便、均匀的时间分辨荧光免疫测定（TR-FIA）方法，用于甲基苯丙胺的定量。在这个实验中，Eu螯合物被标记为甲基苯丙胺和牛血清白蛋白（MA-BSA）的偶联物，作为能量供体，Cy5被标记为抗ma作为能量受体。免疫复合物中两个标签之间的接近允许能量从兴奋的Eu^<3+>供体转移。因此，通过时间分辨法测量Cy5的敏化发射，可以在不分离步骤的均质溶液中在30分钟内测量MA-BSA和anti-MA的免疫复合物。通过竞争性免疫分析法，MA可在0.1 ~ 1000 ng/mL范围内测定。5种不同浓度下，组内变异为5.4% ~ 14.8%。当检测尿液或血清样品时，观察到Eu荧光猝灭，但受体与受体的比值不断依赖于样品的稀释。对20份尿样进行了分析，结果与气相色谱法的结果具有良好的相关性（r=0.94）。使用Eu-Cy5能量转移的均相分析省去了任何洗涤程序，适用于常见滥用药物的筛选。建立了一种新的IL-22免疫检测方法，采用入侵法和FRET系统。本实验IL-22的检出限为0.2 ~ 0.9 pg/ ml。研究了脂质过氧化物4-羟基壬烯醛（HNE）的异相型TR-FIA。通过本实验，我们发现腹腔注射低剂量细菌脂多糖（0.5mg/kg）可使血清HNE水平升高28倍，并在20分钟达到峰值。观察LPS注射后大鼠肠黏膜HNE的变化。小肠HNE在注射LPS后20分钟出现小高峰，2 ~ 4小时持续升高。免疫组织化学显示，注射LPS后，浆细胞中抗iga和抗hne抗体的标记增强，标记物在粘膜下组织中扩散。少