Peptide protein binding assay using ImageFlashPlates or Imaging Beads.

Abstract

Imaging devices used for the measurement of radioligand-receptor binding assays are typically based on charge-coupled device (CCD) cameras, which are more sensitive for red-shifted scintillation. In the past, red-shifted scintillants had only been integrated into microspheres, referred to as scintillation proximity assay (SPA) Imaging Beads. More recently, ImageFlashPlates have been developed that emit light at 615 nm when exposed to beta-radiation. In this article, we report the establishment of peptide-protein binding assays using either streptavidin-coated ImageFlashPlates or Imaging Beads in a low volume 384-well format. In these assays, we employed a biotinylated peptide X and a [33P]-phosphorylated protein Y as the binding partner. The FlashPlates required a washing step, the bead-filled microtiter plates (MTPs) needed a centrifugation step for optimal performance in the scintillation measurements. Both the peptide X-loaded FlashPlates and the beads displayed saturable binding of [33P]-phosphorylated protein Y with a similar scintillation efficiency. A KD value of about 30 nmol/l was measured using the bead-based assay. Due to the washing step in the FlashPlate experiment, approximately two-thirds of the [33P]-phosphorylated protein Y were withdrawn from equilibrium binding. This resulted in correspondingly lower scintillation signals for the FlashPlate experiment. For this reason, the FlashPlate produced a Z' value of 0.64 that was lower than the Z' value of 0.87 for the beads. Using a reference inhibitor in a competition assay produced similar IC50 values for the bead-based assay as for the FlashPlate. Depending on the local automation environment either the centrifugation step for the beads or the washing step for the FlashPlates may be considered more or less of a challenge. Low volume 384-well high-throughput screening (HTS) applicable assay formats are achievable using either the ImageFlashPlates or the Imaging Beads.