Bead-based Scintillation Proximity Assays

Comprising a mix-and-read technique akin to homogenous fluorescence-based detections is the scintillation proximity assay (SPA). Only when radiolabeled molecules of interest are bonded to the surface of the beads, resulting in the emission of detectable light, does this stimulation event take place. The distance between unbound radiolabeled molecules and the bead prevents them from stimulating the emission of light. Creative Biolabs provides kinase drug discovery services using bead-based scintillation proximity assays for kinase profiling.

The Key Technologies of Bead-based Scintillation Proximity Assays

• The scintillation proximity materials

The basis for all SPAs is the physical phenomenon known as scintillation. Particles are released as a result of the ground state return. Fluorophores, which absorb these photons and glow at a wavelength that is "red-shifted" and more "tuned" to the peak sensitivity of the detector, are commonly doped into scintillation materials. Traditionally, liquids made of aromatic hydrocarbons were utilized as scintillation materials in bioassays. It was necessary to wash these bioassays before adding the scintillation liquid and counting. Crystals of yttrium oxide (YOx), polyvinyl toluene (PVT), yttrium silicate (YSi), and polystyrene (PS) are employed as the scintillant in SPA technology. To detect the decay particles immediately in the bioassay without the need for wash procedures, these materials are functionalized with affinity tags.

• The scintillation detectors

The original HTS SPA readers relied on a PMT's ability to detect photon emissions. For these PMT counters to count enough photons for statistical significance, it took about 1-2 minutes per well. By putting PMTs above and below the MTP wells and only counting counts that happened simultaneously in both PMTs, a coincidence correction factor may be obtained. This function helped to eliminate static electricity and cosmic radiation background signals. To account for the variations in sensitivity caused by many factors, including physical alignment and sensitivity, a correction factor was applied to each PMT.

• The radioactive tracer materials

The creation of an SPA requires radiolabeled tracers. The most often utilized isotopes are ¹⁴C, ³⁵S, ¹²⁵I, ³³P, and ³H. For kinase assays or any other assay requiring phosphotransfer from a nucleoside triphosphate, ³³P is a great radionuclide. ³³P is comparatively harmless since thin plastic can obstruct its decomposition. It can decompose on its own after being stored for around a year because it breaks down so quickly. ³³P decay exhibits a large non-proximity effect in SPAs due to its lengthy travel distance.

Fig.1. Principle of SPA.¹

The "red-shifted" assays are comparatively free of artifacts, and SPAs are quite sensitive. There are many different compounds that can be labeled, and they can be mechanized and downsized. Owing to these advantages, SPAs have established a solid reputation in the kinase HTS screening. SPAs have the advantage of being highly customizable to different drug targets in HTS; nevertheless, their inability to handle radioactive materials without specific infrastructure is a constraint. Please contact us for more information about our bead-based scintillation proximity assays to meet the special requirements of our clients.

Reference

1. Glickman, J. Fraser, Andres Schmid, and Sandrine Ferrand. "Scintillation proximity assays in high-throughput screening." Assay and Drug Development Technologies 6.3 (2008): 433-455.