Description
Features:
No-wash steps, no separation steps
Ease-of-use: few addition steps, fast assay development
Broad range of affinities: detect strong or weak interactions, from pM to mM affinity
Distance: measure very large protein or antibody complexes – spanning up to 200 nm or more
High avidity: multiple binding sites on each bead enables use of nanomolar concentrations of antibodies or proteins, as well as use of low affinity binders
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.
