Key words: SPA Imaging Beads • LEADSeeker • cAMP • receptor binding assay
SPA Imaging Assays are homogeneous immunoassays based on the coupling of one assay component to an SPA Imaging Bead in order to generate a signal. Polystyrene (PS) SPA Imaging Beads coated with donkey anti-rabbit antibody enable the immobilization of rabbit secondary antibodies during the assay. The Donkey Anti-Rabbit PS SPA Imaging Beads bind to the Fc region of the secondary antibody, leaving the antigen binding Fab region unhindered. Application data is demonstrated here for the utilization of Donkey Anti-Rabbit PS SPA Imaging Beads in a cAMP assay.
SPA Immunoassay principle
Binding radiolabeled ligands to antibodies immobilized on the surface of an SPA Imaging Bead brings the radiolabel into close proximity with scintillant incorporated within the bead. The emitted radiation (beta particles for tritium or Auger electrons for iodine-125) then stimulates the scintillant to emit light (Fig 1). Unbound radiolabeled ligand is not in close enough proximity to the scintillant for energy transfer to occur and no signal is generated. Light emitted by stimulated SPA Imaging Beads can be detected by platforms such as the LEADseeker™ Multimodality Imaging System.
Adrenergic receptors are linked to adenylate cyclase via either inhibitory or stimulatory G proteins. These receptors, when activated by agonist or antagonist occupancy of ligand binding sites, affect the intracellular generation of cAMP. In response to receptor binding, adenylate cyclase converts AMP to cAMP, which exerts its effect by activating a protein kinase capable of phosphorylating specific substrates.
Here, we describe a direct screening 384-well assay that enables the direct extraction and assay of cAMP from c