Amplified Luminescent Proximity Homogeneous Assay (Alpha) Technology is a bead-based detection mode which measures the interaction of two molecules bioconjugated to donor and acceptor beads.
When excited at 680 nm, the donor bead produces singlet oxygen. If the acceptor bead is within a 200 nm distance, the singlet oxygen transfers energy to the donor resulting in production of light between 520 and 620 nm. The amount of produced light is directly proportionate to the amount of bound donor-acceptor beads.
AlphaLISA differs from AlphaScreen mainly in the acceptor beads which emit in a sharp peak centered at 615 nm. AlphaLISA is better suited for serum/plasma samples as the narrow emission spectrum yields less noise and interference.
Multiplexing Alpha Assays
Measuring two analytes in one sample well is performed by using two different kinds of acceptor beads each one bound to an analyte specific antibody. Optical resolution is achieved by the use of different fluorophores that emit in different wavelength ranges. The following combinations are possible:
- AlphaLISA® and AlphaPlex™545
- AlphaLISA® and AlphaPlex™645
- AlphaPlex™545 and AlphaPlex™645
Alpha Technology detection with BMG LABTECH microplate readers
AlphaScreen/AlphaLISA detection can be performed on the PHERAstar® FSX, CLARIOstar®, FLUOstar® and POLARstar® Omega. Since a high intensity excitation source is beneficial to the assay, the CLARIOstar and PHERAstar FSX have dedicated, solid-state lasers which produce high-intensity light at 680 nm – perfect for Alpha Technology. In addition, the PHERAstar FSX offers AlphaPlex specific optic modules that allow the measurement of two Alpha signals simultaneously.
In the Omega series readers, acceptor beads are excited by a high energy xenon flash lamp.