Kimura Hiroko, Matsumoto Kazuko, Mukaida Masahiro
Department of Forensic Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Tokyo, Japan.
J Anal Toxicol. 2005 Nov-Dec;29(8):799-804. doi: 10.1093/jat/29.8.799.
A rapid and simple homogeneous time-resolved fluoroimmunoassay based on fluorescence resonance energy transfer 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 permits energy transfer from the excited Eu(3+) donor. Therefore, by measuring the sensitized emission of Cy5 with the time-resolved assay, immunocomplex of MA-BSA and anti-MA can be measured in the homogeneous solution without separation steps within 30 min. By a competitive immunoassay, MA could be assayed in the range 0.1-1,000 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-donor 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 time-saving without any washing procedures and is suitable for screening drugs that are commonly abused.
已开发出一种基于铕（Eu）到花菁染料（Cy5）的荧光共振能量转移的快速、简单的均相时间分辨荧光免疫分析法，用于定量测定甲基苯丙胺。在该分析方法中，铕螯合物标记到甲基苯丙胺与牛血清白蛋白的偶联物（MA-BSA）上作为能量供体，而Cy5标记到抗甲基苯丙胺抗体上作为能量受体。免疫复合物中两个标记物的紧密接近使得能够从激发态的Eu(3+)供体进行能量转移。因此，通过时间分辨分析法测量Cy5的敏化发射，可以在均相溶液中测量MA-BSA与抗甲基苯丙胺抗体的免疫复合物，无需分离步骤，30分钟内即可完成。通过竞争性免疫分析，甲基苯丙胺的检测范围为0.1 - 1000 ng/mL。在5种不同浓度下，批内变异为5.4 - 14.8%。检测尿液或血清样本时，观察到铕荧光的猝灭，但受体与供体的比例始终取决于样本的稀释度。检测了20份尿液样本，数据显示与气相色谱法获得的数据具有良好的相关性（r = 0.94）。使用Eu-Cy5能量转移的均相分析方法无需任何洗涤步骤，节省时间，适用于筛查常见滥用药物。
