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These AlphaLISA Toolbox Acceptor beads enable antibody binding studies or the detection of mouse IgG2b antibodies from various sources in various matrices. The IgG2 subtypes are the second most prevalent isotype and is responsible for thymus (TH1) mediated immune response to carbohydrate antigens.
These beads can be used in conjunction with Alpha Donor beads for use in AlphaLISA no-wash assays for isotyping or antibody binding studies. In a typical AlphaLISA assay, 1 mg of Acceptor beads is sufficient to run 1,000-2,000 wells using a 50 µL reaction volume.
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.
|Antibody Conjugates||Anti-mouse IgG2b|
|Bead Type or Core Bead Type||AlphaLISA Acceptor|
|Experimental Type||In vitro|
|Product Brand Name||AlphaLISA|
|Shipping Condition||Blue Ice|
|Unit Size||250 µg|
The introduction of enzyme-linked immunosorbent assays (ELISAs) in the early 1970’s offered researchers a non-radiometric immunoassay platform without compromising sensitivity. Over the last 50 years scientists have made huge strides in disease research and drug discovery and a demand for greater assay throughput and sensitivity has evolved. In response, more robust immunoassays have been developed to address some of the limitations of the standard, colorimetric ELISA.
Find out about the most common limitations of traditional ELISAs and how different ELISA alternative technologies address these limitations.
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.
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.
Many laboratories developing and producing antibodies still rely on traditional enzyme-linked immunosorbent assay (ELISA) to perform clonal selection and characterization. Whilst well established, ELISAs can lack sensitivity and reproducibility, and are difficult to automate.
In this white paper, you will learn how Alpha Technologies can provide a faster and simpler workflow for antibody detection and characterization. It will take you through key steps such as antibody clonal selection, measuring antibody affinity and characterizing antibodies, providing examples and demonstrating the advantages Alpha technology can provide as an alternative to ELISA.
Download the white paper to discover how to advance your antibody detection and characterization.