| What's New |
| Promotions |
| Events/Invites |
Scintillation Proximity Assays (SPA) in GPCR Research
Despite numerous advances in nonradioactive technologies, radioligand binding assays remain popular for analysis of ligand-receptor interaction in High Throughput Screening (HTS) and Structure Activity Relationship (SAR) studies in GPCR research.
A well-established radioligand binding assay is Scintillation Proximity Assay (SPA), which due to its homogenous nature (no separation of bound and unbound radiotracers) is easily miniaturized and automated for HTS applications. SPA’s homogeneity allows binding reactions to be assayed without the cumbersome washing or filtration procedures normally used to separate bound from free fractions.
Assays are performed using radioactive labels that emit short range (10 ?m) electrons in water. When bound close to a solid scintillator surface by the binding reaction, they produce photons detectable with a scintillation counter. Electrons emitted from labeled molecules not bound close to the surface dissipate their energy in the medium and are not detected. The bound fraction is detected without separation of the solution from the solid support.
Radioisotopes 3H and 125I, commonly used in ligand binding assays and radioimmunoassay, emit electrons with the low energies required for SPA.
An additional feature of SPA is that progress of binding reactions can be monitored in time, and that the bound fraction can be measured while in equilibrium with the free fraction.
DEVELOPING SCINTILLATION BINDING ASSAYS
Building robust assays is contingent upon meeting the following four criteria:
- Ligand depletion must be less than 10%
- S/N must be greater than 4
- Z’ factor must be higher than 0.5
- Signal stability must be greater than 4
Meeting these requirements is dependent upon careful management of the following parameters.
1) Reagents: Receptors and Radioligands
Quality reagents are crucial in the successful development of SPA. PerkinElmer offers access to the most extensive portfolio of pharmacologically characterized GPCR membrane preparations. Membrane Target Systems® are quality-assured frozen membranes from cells that express recombinant or endogenous receptors.
A pioneer in the development of receptor ligand binding tools, PerkinElmer offers over 400 NEN® 3H and 125I ligands. Each of PerkinElmer’s ultra-pure radioligands is fully characterized for pharmacological action and validated in receptor binding assay.
2) Assay Format
The radioligand used and the final throughput required will deeply impact the selection of the assay format.
Different SPA bead types are commercially available: PVT (polyvinyltoluene) and YtSi with different surface coupling molecules. Typically five bead types are compared in order to maximize the receptor capture on the beads with minimal interaction of the radioligand.
In order to optimize the assay (especially for HTS research), amounts of membranes and beads are kept at a minimum level, with bound radioligand kept below 10% to avoid depletion.
Several assay plate formats are commercially available. Final throughput will govern the selection of 96 or 384 well plates.
The order of addition of the different reagents in the assay may affect the assay performance as well as the ease of automation.
3) Assay Conditions
Typically, receptor binding assays are performed at room temperature. Assay buffers are kept as simple as possible and the buffer optimization is an iterative process. Depending on the individual receptor-ligand system, different reagents may be required to achieve optimal assay performance.
Experimentation will determine when a stable signal is obtained and how long it can be maintained. The stable signal is a combination of the reaction between receptor and ligand reaching steady state and bead settling down.
Most compounds are stored in organic solvent, DMSO being the most common. It is therefore necessary to investigate the potential interference of the solvent in the receptor-ligand reaction.
4) Assay Miniaturization and Robotic Assembly
When setting up an HTS assay, decreasing volume (and therefore the material used in the reaction) and automation, while maintaining the robustness of the assay as much as possible are major concerns.
5) Pharmacology
A saturation binding experiment will estimate affinity of the radioligand for the receptor (Kd) and the maximal number of receptor binding sites (Bmax). Also, a heterologous competition binding experiment to determine the affinity of reference ligands of the receptor (Ki) is performed. Fixed amounts of receptor, radioligand and beads are incubated in presence of increasing amounts of cold ligands in the optimum assay conditions.
CONCLUSION
Beside traditional filtration and FlashPlate® assays, PerkinElmer has developed SPA binding conditions for a large number of receptors. Custom development of a SPA assay can be performed utilizing PerkinElmer’s OnPoint Reagent Service team.
Click to download a recent application note, Scintillation Proximity Assay (SPA):Custom Assay Development and Membrane Validation