Technologies for Radioactive Assays

3.27.3.1.1 Filtration assay

Filtration assays, which are usually performed in the 96-well format, mainly because no higher density filtration unit is available, are used for HTS for ligand binding to cell membranes and proteins. The binding reactions are performed in polypropylene or polystyrene plates and the reaction mix is then transferred onto the filter plates via a vacuum system (Millipore, Billerica, MA, USA; TomTec, Hampton, CT, USA; PerkinElmer, Welleslay, MA, USA; Brandel, Gaithersburg, MD, USA). After washing and drying, filter plates have to be sealed on the back side, scintillation liquid has to be added, and the plates have to be sealed on the top before they are measured in a liquid scintillation counter (MicroBeta; TopCount, both PerkinElmer). Recently, new developments have shown that the 384-well format also can be applied either using Millipore's MultiScreenHTS 384-well filter plates, as described in a case study in association with Sanofi-Aventis. This case study describes an inhibition filtration assay for identification of inhibitors for FabD enzyme (malonyl-coenzyme A: acyl carrier protein (APC) transacylase). More than 270000 compounds were screened. Substantial time saving, high reproducibility with Z' values reaching 0.75, and a low background make this type of filtration assay attractive for screening campaigns. As an alternative, 384-well filtration assays can be performed using a specific type of the Brandel Harvester (Brandel, Gaithersburg, MD, USA), which allows the reaction to be run in a 384-well plate and harvests the reaction mix into four 96-well filter plates. The advantage of this system is the usage of any commercially available 96-well filter plate. Systems from Brandel and Millipore can be fully integrated in automation units. Radioactive filtration assays are often the method of choice for certain assays, not only because of simplicity but also because of the quality of data generated, particularly in binding assays, where other labels could interfere with binding. Additionally, the influence of colored compounds or quenching effects is reduced to a minimum, since the test compound is washed out before the addition of scintillation liquid.

3.27.3.1.2 Scintillation proximity assay (SPA) technology

SPA technology uses coated resin beads that contain a scintillant. Upon binding of an isotopically labeled assay component to the beads, there is an increased likelihood that particles from radioactive decay cause light emission from the scintillant compared to unbound components (Figure 4). The light output can be quantified by a photo multiplier tube (PMT)-based scintillation counter or by a charge coupled device (CCD)-based image reader. For the two reader principles Amersham developed two different types of beads: the SPA scintillation beads (PMT) and the SPA imaging beads (CCD). Choice of bead type depends on different parameters: (1) instrument, (2) throughput, (3) format, and (4) emission. There are some advantages to using the SPA imaging beads such as higher throughput and adaptation to a 1536-well plate format. The emission lies in the red region (615 nm), which reduces color quenching caused by yellow/ orange/red compounds, usually represented in a higher portion than blue compounds in screening compound collections. The characteristics of the radioisotope are important in SPA assays and the shorter the path length of the decay particle the more suitable the radioisotope. Tritium and iodine-125 are most suitable, but SPA has also been successfully applied using carbon-14, sulfur-35, and phosphorus-33. The homogeneous SPA technology is open to many applications (receptor-ligand binding, radioimmunoassay, signal transduction/molecular interactions) and screening of many different target classes (receptors, kinases, transferases, proteases, nucleases, lipid modifying enzymes, DNA/ RNA modifying enzymes). A large variety of beads coated with different coupling molecules like WGA (wheat germ agglutinine), glutathione, copper chelate, nickel chelate, and streptavidin and innumerable application notes are available as well as many publications on the subject.14-17

Recently, PerkinElmer (Welleslay, MA, USA) developed FlashBlue GPCR beads, which are based on the same principle as that described for the SPA beads from Amersham. Bound radioisotope induces emission of blue light that can be detected by liquid scintillation counters like TopCount and MicroBeta as well as by luminescence counters in 96-well and 384-well formats. FlashBlue GPCR beads are specifically designed for high-throughput homogeneous

SPA bead (scintillant)

Bound

Bound radioligand

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