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An AlphaLISA assay using these Acceptor beads allows the detection of human IgG4 in buffer, cell culture media, serum and plasma in a highly sensitive, quantitative, reproducible and user-friendly mode. These beads (along with a suitable Donor bead) permit antibody binding studies or detection of human IgG4 in different sample matrices, including monkey serum.
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.
Immunoglobulin G (IgG), a major effector molecule of the humoral immune response, accounts for about 75% of the total immunoglobulins in plasma of healthy individuals whereas IgM, IgA, IgD and IgE, each of which has characteristic properties and functions, constitute the remaining 25%. 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 to treat 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 animals, and commercial manufacturing.
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 IgG4|
|Bead Type or Core Bead Type||AlphaLISA Acceptor|
|Experimental Type||In vitro|
|Product Brand Name||AlphaLISA|
|Shipping Condition||Blue Ice|
|Unit Size||5 mg|
Product brochure for the Alpha Technology, a versatile, no wash, homogeneous assay technology that's suitable for a broad range of applications.
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.
Biomarker levels were measured directly in CulturPlates-96 and-384 in a simple, fast, all-in-one-well AlphaLISA assay format. The elimination of transfer and wash steps simplifies cellular assays, reduces variability and significantly reduces hands-on time and costs associated with consumables. Integral plasma membrane (EGFR) and secreted solubleproteins (TNFa, IL1ß, IL6, IL8) were successfully determined, on adherent or suspension cells, using the standard AlphaLISA Immunoassay buffer.
The AlphaLISA® assay is a homogeneous immunoassay alternative to classical ELISA. AlphaLISA assays were originally utilized to detect analytesin cell cultures upernatants or serum/plasma samples.
Enzyme-linked Immunosorbent Assay is the most widely Kits adopted method for detection and quantification of cytokines and other biomarkers. This traditional technology offers good,selectivity, sensitivity and assay versatility; however, it has certain disadvantages such as limited dynamic range and low throughput due to the numerous wash steps.
Alpha technology is homogeneous and non-radiometric with distinct features that makes it enabling in comparison to other proximity assays. Alpha technologies represent powerful means of detecting and characterizing a wide range of proteins including antibodies.