Stenroos K, Hurskainen P, Eriksson S, Hemmilä I, Blomberg K, Lindqvist C
Department of Biology, Abo Akademi University, Turku, Finland.
Cytokine. 1998 Jul;10(7):495-9. doi: 10.1006/cyto.1997.0321.
A homogeneous receptor-ligand assay based on fluorescence resonance energy transfer is described. In the assay, recombinant human interleukin 2 (IL-2) and a monoclonal antibody against the human IL-2 receptor alpha chain were labelled with a highly fluorescent europium chelate and Cy5, respectively. As a result of a successful receptor-ligand complex formation, these labels are brought into close proximity, which will thereby allow an energy transfer to occur from the donor (europium) to the acceptor (Cy5), upon excitation of the donor. Utilization of specific non-neutralizing antibodies made it possible to use crude hIL-2R alpha membranes prepared from recombinant baculovirus-infected Sf9 insect cells. The specific energy transfer was measured at the emission wavelength of Cy5 using a time-resolved fluorometer. The data presented, demonstrate that this assay design can be utilized for saturation as well as competitive binding experiments in addition to regular receptor titrations. As a rapid simple homogeneous assay it is particularily suitable for high throughput screening analyses.
描述了一种基于荧光共振能量转移的均相受体-配体测定法。在该测定法中，重组人白细胞介素2（IL-2）和抗人IL-2受体α链的单克隆抗体分别用高荧光铕螯合物和Cy5进行标记。由于成功形成了受体-配体复合物，这些标记物彼此靠近，从而在供体（铕）被激发时，能使能量从供体转移至受体（Cy5）。使用特异性非中和抗体使得能够使用从重组杆状病毒感染的Sf9昆虫细胞制备的粗制hIL-2Rα膜。使用时间分辨荧光计在Cy5的发射波长处测量特异性能量转移。所呈现的数据表明，除了常规的受体滴定外，该测定设计还可用于饱和以及竞争性结合实验。作为一种快速简单的均相测定法，它特别适用于高通量筛选分析。
