The use of AlphaScreen technology in HTS: current status

Abstract

AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) is versatile assay technology developed to measuring analytes using a homogenous protocol. This technology is an example of a bead-based proximity assay and was developed from a diagnostic assay technology known as LOCI (Luminescent Oxygen Channeling Assay). Here, singlet oxygen molecules, generated by high energy irradiation of Donor beads, travel over a constrained distance (approx. 200 nm) to Acceptor beads. This results in excitation of a cascading series of chemical reactions, ultimately causing generation of a chemiluminescent signal.In the past decade, a wide variety of applications has been reported, ranging from detection of analytes involved in cell signaling, including protein:protein, protein:peptide, protein:small molecule or peptide:peptide interactions. Numerous homogeneous HTS-optimized assays have been reported using the approach, including generation of second messengers (such as accumulation of cyclic AMP, cyclic GMP, inositol [1, 4, 5] trisphosphate or phosphorylated ERK) from liganded GPCRs or tyrosine kinase receptors, post-translational modification of proteins (such as proteolytic cleavage, phosphorylation, ubiquination and sumoylation) as well as protein-protein and protein-nucleic acid interactions.Recently, the basic AlphaScreen technology was extended in that the chemistry of the Acceptor bead was modified such that emitted light is more intense and spectrally defined, thereby markedly reducing interference from biological fluid matrices (such as trace hemolysis in serum and plasma). In this format, referred to as AlphaLISA, it provides an alternative technology to classical ELISA assays and is suitable for high throughput automated fluid dispensing and detection systems.Collectively, AlphaScreen and AlphaLISA technologies provide a facile assay platform with which one can quantitate complex cellular processes using simple no-wash microtiter plate based assays. They provide the means by which large compound libraries can be screened in a high throughput fashion at a diverse range of therapeutically important targets, often not readily undertaken using other homogeneous assay technologies. This review assesses the current status of the technology in drug discovery, in general, and high throughput screening (HTS), in particular.

Fig. (1). AlphaScreen/AlphaLISA Assay Principle

AlphaScreen/AlphaLISA assays are bead based proximity assays, based upon an oxygen channeling technology. When the Donor (blue bead), which contains phthalocyanine, is laser excited (at 680 nm) ambient oxygen is converted to singlet oxygen. This is a highly amplified reaction since approx. 60,000 singlet oxygen molecules can be generated and travel at least 200 nm in aqueous solution before decay. Consequently, if the Donor and Acceptor (gold beads) beads are within that proximity, energy transfer occurs. Singlet oxygen molecules react with chemicals in the Acceptor beads to produce a luminescent response. If the Acceptor bead contains Europium, as in the AlphaLISA assay, an intense luminescence is emitted at a wavelength of 615 nm.

Fig. (2). Comparative protocols of ELISA vs. AlphaLISA assays

In these schematic protocols, panel on the left illustrates the multiple wash and separation steps required in a typical ELISA experiment. By contrast, the use of the AlphaLISA allows experiments to be conducted using simple mix and read protocols.

Fig. (3). Surefire assay principle

The AlphaScreen platform can be used to detect analytes in a crude cell lysate using protocols developed into the Surefire format. Here, the levels of a phosphorylated protein (such as phosphoERK_1/2 can be quantified using the approach, by use of specific antibodies to the phosphoprotein, without wash and separation steps as in a typical ELISA assay.