What is AlphaScreen?

AlphaScreen® (ALPHA for Amplified Luminescent Proximity Homogeneous Assay) is bead-based chemistry used to study interactions between molecules in a microplate. Originally developed as a methodology for diagnostic assays called LOCI® (Luminescent Oxygen Channeling Immunoassay) in 19941, today the AlphaScreen Technology (Alpha Technology) includes AlphaScreen, AlphaLISA®, and AlphaPlexTM. It is mainly used in the life sciences for screening purposes. 

The fundamental principle of the AlphaScreen technology relies on binding two different molecules of interest to specific beads. In case of interaction between the two molecules and the resulting proximity of the two beads, an energy transfer from one bead to the other takes place. This results in the production of a chemiluminescent signal. The AlphaScreen technology is mainly used in high-throughput screening assays to assess biomolecular interactions, the formation/depletion of a substrate or product, post-translational modifications and to quantify analytes.

Besides interaction assays (including ligand/receptor, protein/protein, protein/DNA), the AlphaScreen technology can also be applied to GPCR functional assays (second messenger detection), enzymatic assays, and immunoassays. 

AlphaScreen technology

AlphaScreen assays are based on two types of hydrogel-coated beads, called donor and acceptor beads. The two bead types contain different chemicals that are key to the generation of the luminous signal. The donor bead contains a photosensitizer, which upon excitation by light at 680 nm, converts oxygen (O2) into an exciting form, singlet oxygen (1O2). Singlet oxygen molecules have a reduced lifetime (4 microseconds half-life) and can diffuse approximately 200 nm in solution before falling back to the ground state. 

In the absence of acceptor beads, the singlet oxygen molecules fall back to the ground state without producing any light signal. In case an acceptor bead is within 200 nm, energy is transferred from the singlet oxygens to the bead, resulting in light production. AlphaScreen acceptor beads contain three chemical dyes, thioxene, anthracene, and rubrene. Singlet oxygen molecules initially react with thioxene to generate light. This is transferred to anthracene and to rubrene and results in broad light emission from 520 nm to 620 nm.2 The half-life of the signal decay reaction is 0.3 seconds.

In binding assays, one binding partner (e.g. a receptor) is linked to the donor, while the other (e.g. a ligand) to the acceptor beads. When receptor and ligand interact, chemical energy is transferred from one bead type to the other and a luminous signal is produced (fig. 1). AlphaScreen can also be employed for competition or cleavage assays. In these cases, a reduction in signal intensity is measured.  

 

Fig. 1: basic principle of AlphaScreen. Left: the donor and acceptor beads are not in proximity. Singlet oxygen molecules decay with no signal generation. Right: biological interactions bring beads into proximity. Singlet oxygen molecules reach the acceptor bead and a light signal in the 520-620 nm range is generated.

AlphaLISA 

AlphaLISA is a further development of the AlphaScreen technology that relies on the same donor beads but uses a different type of acceptor beads. In AlphaLISA beads, anthracene and rubrene are substituted by europium chelates. Excited europium emits light at 615 nm with a much narrower wavelength bandwidth than AlphaScreen (fig. 2). Hence, AlphaLISA emission is less susceptible to compound interference and can be employed for the detection of analytes in biological samples like cell culture supernatants, cell lysates, serum, and plasma.

 

Fig. 2: Comparison of the emission spectra of AlphaScreen and AlphaLISA. Because of its narrow peak, AlphaLISA is mainly used for the detection of analytes in cell culture supernatants, cell lysates, serum and plasma.

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