Use of scintillation proximity assay to measure radioligand binding to immobilized receptors without separation of bound from free ligand.
Scintillation proximity assay (SPA) is a bead-based homogeneous assay technology that removes the need for a filtration step to separate bound from free ligand in a receptor binding assay. SPA allows the rapid and sensitive assay of a wide range of molecular interactions in a homogeneous system and is routinely used for radioligand binding assays, particularly in drug screening applications where high throughput is required. Existing filter binding assays may be readily converted to SPA assays or assays may be directly developed in SPA format. This chapter describes the development of SPA radioligand binding assays detailing the choice of isotope, selection of SPA bead type, optimization of SPA bead and receptor ratio, optimization of assay buffer, selection of assay format, and assay validation including saturation binding, competition binding, and association/dissociation binding studies using SPA.
GE Healthcare Biosciences, Cardiff, Wales, UK.
Jenny
J
Berry
Molly
M
Price-Jones
Barbara
B
Killian
D000818:Animals; D019921:Biotinylation; D002021:Buffers; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism*; D007554:Isotopes; D007700:Kinetics; D008024:Ligands; D008863:Microspheres; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods*; D015203:Reproducibility of Results; D012588:Scintillation Counting
D002021:Buffers; D055767:Immobilized Proteins; D007554:Isotopes; D008024:Ligands