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AlphaLISA Acceptor beads conjugated to anti-HA (hemagglutinin) antibody. These beads can be used to capture HA-tagged proteins and peptides, and can be used in conjunction with Alpha Donor beads to create AlphaLISA no-wash assays for:
In a typical AlphaLISA assay, 1 mg of AlphaLISA Acceptor beads is sufficient to run 1,000-2,000 wells using a 50 µL final reaction volume. Bead concentration can be adjusted for optimal performance.
AlphaScreen® and AlphaLISA® are bead-based assay technologies used to study biomolecular interactions in a microplate format. The acronym "Alpha" stands for amplified luminescent proximity homogeneous assay. As the name implies, some of the key features of these technologies are that they are non-radioactive, homogeneous proximity assays. Binding of molecules captured on the beads leads to an energy transfer from one bead to the other, ultimately producing a luminescent/fluorescent signal. To understand how a signal is produced, one must begin with an understanding of the beads. AlphaScreen and AlphaLISA assays require two bead types: Donor beads and Acceptor beads. Each bead type contains a different proprietary mixture of chemicals, which are key elements of the AlphaScreen technology. Donor beads contain a photosensitizer, phthalocyanine, which converts ambient oxygen to an excited and reactive form of O2, singlet oxygen, upon illumination at 680 nm. Please note that singlet oxygen is not a radical; it is molecular oxygen with a single excited electron. Like other excited molecules, singlet oxygen has a limited lifetime prior to falling back to ground state. Within its 4 µsec half-life, singlet oxygen can diffuse approximately 200 nm in solution. If an Acceptor bead is within that proximity, energy is transferred from the singlet oxygen to thioxene derivatives within the Acceptor bead, subsequently culminating in light production at 520-620 nm (AlphaScreen) or at 615 nm (AlphaLISA). In the absence of an Acceptor bead, singlet oxygen falls to ground state and no signal is produced. This proximity-dependent chemical energy transfer is the basis for AlphaScreen's homogeneous nature.
|Experimental Type||In vitro|
|Product Brand Name||AlphaLISA|
|Shipping Condition||Blue Ice|
|Unit Size||5 mg|
Anti-inflammatory monoclonal antibody drugs that specifically target TNFα, such as Humira®, have been highly successful in the market. As patents expire on these top-selling drugs, effort has been placed on developing biosimilars. Biosimilars differ from small molecule generic drugs in that their chemical structure does not have to be exactly the same as the patented drug. Therefore, the FDA has stringent requirements for proving that the biosimilars have the same efficacy and safety profile as the patented drug. Companies that develop biosimilars are tasked with proving that the biosimilar shows equivalent pharmacokinetics as the patented drug.
Proving “biosimilarity” involves comparing parameters such as overall exposure, absorption, half-life, and clearance time using patient samples. Sensitive, robust, and fast assays are needed to measure these parameters. Traditional methods for detecting and quantifying these drugs in patient samples include time-consuming, wash-based ELISA and MSD methods. In contrast, AlphaLISA allows for fast, no-wash, high-throughput detection and quantification of the drug of interest in a variety of sample matrices. Here, we demonstrate the application of AlphaLISA for detecting biosimilars targeting TNFα.
The interactions and bindingof proteins are implicated in a large number of biological processes. The needfor an efficient, highly sensitive assay to study large protein interactions is increasingly important. Alpha Technology is a highly flexible, homogeneous, no-wash assay ideal for the measurement of protein interactions and complexes as large as 200 nm in size
Alpha has been used to study a wide variety of interactions, including protein:protein, protein:peptide, protein:DNA, protein:RNA, protein:carbohydrate, protein:small molecule, receptor:ligand, and nuclear receptor:ligand interactions. Both cell-based and biochemical interactions have been monitored, and applications such as phage display, ELISA, and EMSA (electrophoretic mobility shift assay) have been adapted to Alpha.
This guide presents the simple conversion of an ELISA or other immunoassay to an AlphaLISA® immunoassay.
AlphaScreen® and AlphaLISA® are bead-based assay technologies used to study biomolecular interactions in a microplate format. The acronym “Alpha” stands for Amplified Luminescent Proximity Homogeneous Assay. The assay does not require any washing steps. Binding of proteins or other binding partners captured on the beads leads to an energy transfer from one bead to the other, ultimately producing a luminescent signal.
Binding events between biomolecules are important components of biological processes and a number of these biomolecular interactions have been targeted for the development of novel therapeutic drugs. p53 is a transcription factor and tumor suppressor protein that is activated in response to cellular stress, and MDM2 was identified as a negative regulator that binds to p53 and tags it for ubiquitination and subsequent degradation.
The p53-MDM2 protein-protein interaction has been an excellent target for therapeutic drugs and therefore makes a good model system for developing an AlphaLISA assay to screen for inhibitors of the interaction. In this technical note, we show how to develop an assay to screen for inhibitors and how to measure a dissociation constant for moderate binding protein-protein interaction using AlphaLISA®.