Alpha Screen

The amplified luminescent proximity homogeneous assay screen (Alpha Screen™) developed by BioSignal, a Packard Bioscience Company, is a nonradioactive proximity assay using two proprietary 200 nm diameter latex donor and acceptor beads [64]. The beads are coated with hydrogel to prevent nonspecific interactions and self-aggregation and to provide a functionalized surface for cova-lent attachment and to retain dyes. The donor beads contain a photosensitizer that absorbs light at 680 nm (laser excitation) and then converts ambient molecular oxygen to the excited singlet state that has a short lifetime of 4 |sec, allowing it to diffuse up to 200 nm in aqueous solution. When the acceptor bead is brought into close proximity (within 200 nm) by a molecular binding event, the singlet oxygen reacts with the thioxene derivative of the acceptor beads, generating chemiluminescence at 370 nm, which is immediately transferred to fluorescent acceptors in the same beads. The fluorophores then emit light at 600 nm with high yield (Fig. 18). The half-life of the decay reaction is 0.3 sec, allowing to operate in time-resolved mode. The singlet oxygen does not react with the unbound acceptor beads; they do not emit light.

1. Instrumentation

The AlphaQuest-AD microplate analyzer from Packard is a single fiber-optic detector that can be used for AlphaScreen assays in 96-well plates as well as 384- and 1536-well high density plates (Fig. 19). The AlphaQuest-HTS is a high speed detection system with four simultaneous detectors. Using efficient fiberoptic bundles, AlphaQuest-HTS provides high speed measurement of 96-, 384-, and 1536-well microplates with a throughput of up to 100,000 samples per day.

2. Applications

AlphaScreen™ is a nonradioactive homogeneous assay technology. AlphaScreen has been applied to several different classes of assays including protein tyrosine kinases, serine-threonine kinases, proteases, helicases, receptor binding assays, protein-protein interactions, protein-DNA interactions, immunoassays for de-

Figure 18 Principle of amplified luminescent proximity homogeneous assay (Alpha) screen. (A) On laser excitation, the chemical signal generated by the donor bead cannot be detected if the acceptor bead is not in close proximity. (B) When acceptor and donor beads are brought into close proximity by specific biological interaction, amplified signal is generated in AlphaScreen. (Courtesy of BioSignal, a Packard Bioscience company.)

Figure 18 Principle of amplified luminescent proximity homogeneous assay (Alpha) screen. (A) On laser excitation, the chemical signal generated by the donor bead cannot be detected if the acceptor bead is not in close proximity. (B) When acceptor and donor beads are brought into close proximity by specific biological interaction, amplified signal is generated in AlphaScreen. (Courtesy of BioSignal, a Packard Bioscience company.)

Emission 3ifurcated

Emission 3ifurcated

Figure 19 Line drawing of AlphaQuest AD, an AlphaScreen microplate reader. (Courtesy of BioSignal, a Packard Bioscience company.)

termining small molecules and hormone levels, and cAMP functional assays for GPCRs (Fig. 20).

3. Comments

As each donor bead contains a high concentration of photosensitizers, each donor bead emits up to 60,000 singlet oxygen molecules per second, which results in a very high amplification. Excitation at a longer wavelength (680 nm) and emission at a lower wavelength (500-600 nm) minimize nonspecific excitation and reduce background. The beads are the optimal size, too small to settle in aqueous buffers and large enough to be centrifuged. The effective distance between the donor and acceptor (R0 value) is large, ~ 200 nm, which overcomes the assay limitations of other FRET based systems with weak interactions. Because of the long lifetime (0.3 s) of the AlphaScreen fluorescence signal, measurements are

Gpcr Functional Assay

Figure 20 AlphaScreen cyclic AMP assay. (A) Schematic view of AlphaScreen cAMP assay (a whole cell GPCR functional assay) is based on the competition of endogenous cAMP with biotin-cAMP for cAMP antibody coated acceptor beads. (Courtesy of BioSignal A Packard BioScience Company.) (B) Standard curve of cAMP in AlphaScreen assay. (C) Activation of adenyl cyclase by forskolin in CHO-k1 cells expressing a GPCR. EC50 obtained for forskolin in this assay was 4.4 ||M.

Figure 20 AlphaScreen cyclic AMP assay. (A) Schematic view of AlphaScreen cAMP assay (a whole cell GPCR functional assay) is based on the competition of endogenous cAMP with biotin-cAMP for cAMP antibody coated acceptor beads. (Courtesy of BioSignal A Packard BioScience Company.) (B) Standard curve of cAMP in AlphaScreen assay. (C) Activation of adenyl cyclase by forskolin in CHO-k1 cells expressing a GPCR. EC50 obtained for forskolin in this assay was 4.4 ||M.

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