Stenroos K, Hurskainen P, Blomberg K, Lindqvist C
Abo Akademi University, Department of Biochemistry and Pharmacy, Turku, Finland.
Immunol Lett. 1997 Jun;58(1):15-8. doi: 10.1016/s0165-2478(97)02719-3.
A time-resolved fluorometric, solid phase, receptor ligand interaction assay is described. The assay consists of wells coated with anti-human IL-2 receptor alpha (hIL-2R alpha) monoclonal antibodies (mAb), europium labelled hIL2 (Eu-IL-2) and human recombinant IL-2 receptor alpha subunits expressed in the baculovirus expression vector system (BEVS). In the assay hIL-2R alpha-Eu-IL-2 complexes bind to the solid phase mAb. Receptor bound Eu is dissociated into an enhancer solution where it forms highly fluorescent complexes. The fluorescence is measured in a time-resolved fluorometer. The Kd value calculated from the saturation curve is in good agreement with previously reported values for the low affinity type of IL-2R, making the described assay a simple and nonradioactive alternative for measurement of soluble hIL-2R alpha in biological systems. Furthermore this assay format provides convenient separation of bound ligand from unbound and is therefore suitable for high throughput screenings.
描述了一种时间分辨荧光、固相受体-配体相互作用测定法。该测定法由包被有抗人白细胞介素-2受体α（hIL-2Rα）单克隆抗体（mAb）的孔、铕标记的hIL2（Eu-IL-2）以及在杆状病毒表达载体系统（BEVS）中表达的人重组白细胞介素-2受体α亚基组成。在该测定法中，hIL-2Rα-Eu-IL-2复合物与固相mAb结合。结合受体的铕解离到增强剂溶液中，在其中形成高荧光复合物。在时间分辨荧光计中测量荧光。根据饱和曲线计算的Kd值与先前报道的低亲和力型IL-2R的值高度一致，使得所描述的测定法成为生物系统中可溶性hIL-2Rα测量的一种简单且无放射性的替代方法。此外，这种测定形式便于将结合的配体与未结合的配体分离，因此适用于高通量筛选。
