Abstract
The high-affinity receptor for human interleukin-5 (hIL-5) is composed of α and β subunits. A baculovirus expression system was established in Sf9 cells capable of expressing hIL-5 receptor α and β subunits simultaneously. By using wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads to capture125I-labeled hIL-5-bound Sf9 cells, a SPA was developed and used to measure hIL-5 high-affinity binding. The hIL-5 receptors expressed in the Sf9 cells represented a single class of high-affinity binding sites with a dissociation constant (Kd) of 0.24 nM and a density of 2.95 × 105sites/cell. This is the first study in which the high-affinityKdvalue similar to that for hIL-5 binding to human eosinophils was achieved using a recombinant expression system. The SPA compared favorably with the filter binding assay with regard to various binding parameters. We also found that several lectins, when coated on SPA beads, were even more effective than WGA-coated SPA beads for capturing the insect cells. We conclude that the baculovirus expression system was highly efficient in producing the high-affinity hIL-5 receptors and that the SPA was a simple and sensitive assay that could be readily adapted into a high-throughput screening format. The SPA described here could be a prototype for binding assays for other multimeric receptors.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.