Examples of multiple platforms for homogeneous radioactive binding assays include SPA® beads (Amersham Biosciences), LEADseeker® beads (Amersham Biosciences), FlashBlue® beads (PerkinElmer), and FlashPlate® (PerkinElmer). All these formats are scintillation proximity assays (SPAs). In this type of assay, an isotope-labeled ligand is brought into close proximity to a scintillant embedded in beads or plate plastics by binding to a receptor immobilized onto the beads or plates.17
SPA is a simple assay format and is well suited to automation because it requires no separation step. The scintillant in SPA beads is incorporated into fluomicro-spheres. SPA beads are made of either polyvinyl toluene (PVT) or yttrium silicate (Ysi). Beads are available with several surface coatings to immobilize membranes including PVT-WGA (wheat germ agglutinin), Ysi-WGA, PVT-PEI (polyethylen-imine)-WGA, and Ysi-poly-D-lysine. Beads coated with WGA bind cell membranes through surface carbohydrates.
Beads coated with poly-D-lysine, which rely on the negative charge of the membrane, are also used for certain receptors. The choice of bead type depends on the nature of the radioligand. If the ligand is a glycoprotein, WGA-coated SPA beads should be avoided because they interact with the ligand and the background noise in the assay will increase. Similarly, poly-D-lysine-coated beads should not be used with negatively charged ligands.
The capacity of WGA beads for crude membranes is approximately 10 to 30 mg membrane protein/mg bead; 1 mg of poly-D-lysine-coated beads can bind 10 |mg membrane protein. Ysi beads are denser than PVT and PS (polystyrene) beads and settle very quickly in aqueous buffers; care must be taken when pipetting to ensure the even distribution of solids. Assays using Ysi beads must be shaken in order to facilitate equilibrium. SPA beads are typically added to the assay pre-coupled with membranes. Alternatively, beads can be added together with receptor and ligand or they can also be added after the reaction has reached equilibrium.
SPA beads emit light in the blue region of the emission spectrum between 400 and 450 nm and the light is measured using a standard photomultiplier tube-based reader such as a PerkinElmer TopCount® or MicroBeta®. An SPA-based receptor binding assay should be allowed to reach equilibrium before counting is performed and this results in a longer incubation period. Because the incubation time is fairly long, care should be taken during assay development to ensure the stability of the assay components.
Unfortunately, colored compounds can cause a reduction in counting efficiency during screening and this produces quenching of the signal. This color quenching is in addition to chemical quenching (interference in the process of energy transfer between solvent and fluorescent molecules) and physical quenching (orientation of bound labeled ligands is far away from bead). Quench correction protocols that use quenching agents have been developed, but these protocols do not fully correct for this problem.
An improvement over the first generation of SPA bead is the LEADseeker® bead of Amersham Biosciences.18 LEADseeker beads are optimized to read in CCD-based imagers such as Amersham's LEADseeker or the Viewlux® of PerkinElmer.
Yellow and red compounds do not quench the light emitted in the red region. Blue compounds can absorb the red light output, but the number of blue compounds in the compound libraries is relatively rare — and hence less of an issue for screening. The LEADseeker beads consist of two types, one composed of polystyrene (PS) and the other of yttrium oxide (Yox). For receptor binding assays, these beads typically have wheat germ agglutinin coatings. Assay development using LEADseeker beads is similar to the traditional SPA format. Importantly, the final readout for these beads is in a CCD-based imaging platform, so reading takes less time than required for a PMT-based system. Plates containing LEADseeker beads must be dark-adapted before reading to eliminate the phosphorescence innate to the plastic material of the plates. Also, the LEADseeker system is much more amenable to miniaturization and assays can be easily converted into 1536-well plates.
FlashPlates (PerkinElmer) are similar to SPA beads but instead of incorporating the scintillant directly into beads, it is incorporated into the plastic of the plate well. Available plates contain 96 or 384 wells with different coatings, similar to SPA beads. PerkinElmer also provides ScreenReady Target® FlashPlates for certain receptors, where the receptor is already immobilized onto the FlashPlate. ScreenReady Targets are ready-to-use, fully validated microtiter plate assays for homogeneous GPCR receptor binding assays using 125I or 3H radioligands. Also like SPA imaging beads, imaging flash plates (Imageplate®) are available from PerkinElmer. Note that these homogeneous formats are not suitable for high throughput screening if both receptor density and ligand affinity are low.
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