A scintillation proximity assay for human interleukin-5 (hIL-5) high-affinity binding in insect cells coexpressing hIL-5 receptor alpha and beta subunits.
The high-affinity receptor for human interleukin-5 (hIL-5) is composed of alpha and beta subunits. A baculovirus expression system was established in Sf9 cells capable of expressing hIL-5 receptor alpha and beta subunits simultaneously. By using wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads to capture 125I-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 x 10(5) sites/cell. This is the first study in which the high-affinity Kd value 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.
Department of Allergy, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey, 07033, USA.
J
J
Zhang
P
P
Wu
R
R
Kuvelkar
J L
JL
Schwartz
R W
RW
Egan
M M
MM
Billah
P
P
Wang
D000818:Animals; D016367:Baculoviridae; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D015848:Interleukin-5 / Q000378:metabolism*; D037102:Lectins / Q000378:metabolism; D011485:Protein Binding; D018123:Receptors, Interleukin / Q000235:genetics* / Q000378:metabolism; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012588:Scintillation Counting; D014162:Transfection
D015848:Interleukin-5; D037102:Lectins; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins