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Immunoglobulin G (IgG) is a major effector molecule of the humoral immune response and accounts for about 75% of the total immunoglobulins in plasma of healthy individuals. The remainder 25% comprises IgM, IgA, IgD and IgE, each of which has characteristic properties and functions. The basic IgG molecule has a four-chain structure, comprising two identical heavy (H) chains and two identical light (L) chains, linked together by inter-chain disulfide bonds. Four IgG subclasses have been identified: IgG1, IgG2, IgG3 and IgG4.
Biotherapeutic antibody drugs, usually IgG1 or IgG4 molecules, are becoming increasingly important for treating debilitating diseases such as cancer and autoimmune disorders. Drug levels need to be accurately measured at various stages of drug development, including early antibody discovery, preclinical research in vivo, and commercial manufacturing.
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-human IgG1|
|Bead Type or Core Bead Type||AlphaLISA Acceptor|
|Experimental Type||In vitro|
|Product Brand Name||AlphaLISA|
|Shipping Condition||Blue Ice|
|Unit Size||25 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.
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.