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

1.建立了基于铕(Eu)-菁染料(Cy5)荧光共振能量转移(FRET)的均相时间分辨荧光免疫分析法(TRFIA)快速测定甲基苯丙胺的方法。在本实验中，将Eu络合物标记到甲基苯丙胺和牛血清白蛋白(MA-BSA)的结合物上作为能量供体，将Cy5标记到抗MA上作为能量受体。免疫复合体中两个标记物之间的紧密联系允许激发的Eu3+&gt；供体的能量转移。因此，通过用时间分辨方法测量Cy5的增敏发射，可以在30分钟内无需分离步骤就能在均一溶液中测定MA-BSA和抗MA免疫复合物。通过竞争免疫分析，MA的检测范围为0.1-1000 ng/mL。5种不同浓度的批内变异系数在5.4%~14.8%之间。当检测尿样或血清样品时，观察到Eu的荧光猝灭，但受体与DONO的…更大的R比始终取决于样品的稀释程度。对20份尿样进行了测定，结果与气相色谱测定结果具有良好的相关性(r=0.94)。采用Eu-Cy5能量转移的均相检测方法不需要任何洗涤步骤，省时，适用于常见滥用药物的筛选。2.建立了入侵者检测和FRET系统检测IL-22的新方法。在本方法中，IL-22的检出限为0.2-0.9pg/mL.建立了过氧化脂质4-羟基壬烯醛(HNE)的异构型RT-FIA。通过本实验，我们发现，小剂量的细菌脂多糖(LPS)腹腔注射(0.5 mg/kg)可使血清HNE水平增加28倍，并在20min达到高峰。同时测定注射内毒素后大鼠肠黏膜组织中HNE的含量。肠内HNE升高，20min达高峰，2~4h持续升高。免疫组织化学显示，注射脂多糖后，浆细胞中抗IgA和抗HNE抗体的标记增强，标记物质在黏膜下组织中弥散分布。较少

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