PerkinElmer Inc, Wallac Oy, P.O. Box 10, 20101 Turku, Finland.
Anal Bioanal Chem. 2011 Feb;399(4):1677-82. doi: 10.1007/s00216-010-4485-y. Epub 2010 Dec 15.
The limitation of current dissociative fluorescence enhancement techniques is that the lanthanide chelate structures used as molecular probes are not stable enough in one-step assays with high concentrations of complexones or metal ions in the reaction mixture since these substances interfere with lanthanide chelate conjugated to the detector molecule. Lanthanide chelates of diethylenetriaminepentaacetic acid (DTPA) are extremely stable, and we used EuDTPA derivatives conjugated to antibodies as tracers in one-step immunoassays containing high concentrations of complexones or metal ions. Enhancement solutions based on different β-diketones were developed and tested for their fluorescence-enhancing capability in immunoassays with EuDTPA-labelled antibodies. Characteristics tested were fluorescence intensity, analytical sensitivity, kinetics of complex formation and signal stability. Formation of fluorescent complexes is fast (5 min) in the presented enhancement solution with EuDTPA probes withstanding strong complexones (ethylenediaminetetra acetate (EDTA) up to 100 mM) or metal ions (up to 200 μM) in the reaction mixture, the signal is intensive, stable for 4 h and the analytical sensitivity with Eu is 40 fmol/L, Tb 130 fmol/L, Sm 2.1 pmol/L and Dy 8.5 pmol/L. With the improved fluorescence enhancement technique, EDTA and citrate plasma samples as well as samples containing relatively high concentrations of metal ions can be analysed using a one-step immunoassay format also at elevated temperatures. It facilitates four-plexing, is based on one chelate structure for detector molecule labelling and is suitable for immunoassays due to the wide dynamic range and the analytical sensitivity.
当前的荧光解离增强技术的局限性在于，在反应混合物中存在高浓度的络合剂或金属离子的一步测定中，用作分子探针的镧系螯合物结构不够稳定，因为这些物质会干扰与检测分子偶联的镧系螯合物。乙二胺四乙酸（DTPA）的镧系螯合物非常稳定，我们将 EuDTPA 衍生物与抗体偶联作为示踪剂用于含有高浓度络合剂或金属离子的一步免疫测定中。开发了基于不同β-二酮的增强溶液，并在含有 EuDTPA 标记抗体的免疫测定中测试了它们的荧光增强能力。测试的特性包括荧光强度、分析灵敏度、络合物形成动力学和信号稳定性。在所提出的增强溶液中，EuDTPA 探针形成荧光络合物的速度很快（5 分钟），能够耐受反应混合物中强络合剂（乙二胺四乙酸（EDTA）高达 100 mM）或金属离子（高达 200 μM），信号强度高，稳定 4 小时，Eu 的分析灵敏度为 40 fmol/L，Tb 为 130 fmol/L，Sm 为 2.1 pmol/L，Dy 为 8.5 pmol/L。使用改进的荧光增强技术，即使在高温下，也可以使用一步免疫测定格式分析 EDTA 和柠檬酸盐血浆样品以及含有相对高浓度金属离子的样品。它促进了四聚化，基于一种螯合物结构用于检测分子标记，并且由于宽动态范围和分析灵敏度，适用于免疫测定。
