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Interferons (IFNs) activity has been discovered due to their antiviral effects. In humans, there are three families of IFNs: IFN type I (IFN-α, ß, ω, ε, and κ), IFN type II (one single representative, IFN-γ), and IFN type III (IFN-λ1-3). Antigens and mitogens stimulate in Natural Killer (NK) and activated helper T lymphocytes (Th1) the production of IFN-γ. Human IFN-γ is a 140 amino acids polypeptide that shows multiple effects; it induces the production of cytokines, upregulates the expression of class I and II MHC antigens, and leukocyte adhesion molecules. It also activates macrophages, enhances the secretion of immunoglobulins by B cells, and potentiates Th1 cell expansion. Response to IFN-γ is mediated by the hetetodimeric IFN-γ Receptor, triggering a signalling cascade involving JAK1, JAK2, and STAT1. Importantly, IFNs have been proved to be effective in the treatment of several viral infections and cancers.
AlphaLISA technology allows the detection of molecules of interest in a no-wash, highly sensitive, quantitative assay. In an AlphaLISA assay, a biotinylated anti-analyte antibody binds to the Streptavidin-coated Donor beads while another anti-analyte antibody is conjugated to AlphaLISA Acceptor beads. In the presence of the analyte, the beads come into close proximity. The excitation of the Donor beads causes the release of singlet oxygen molecules that triggers a cascade of energy transfer in the Acceptor beads, resulting in a sharp peak of light emission at 615 nm.
|Assay Target Class||Cytokine|
|Experimental Type||In vitro|
|Product Brand Name||AlphaLISA|
|Shipping Condition||Blue Ice|
|Unit Size||5,000 assay points|
Immunoassays are a mainstay for the quantification of a variety of bio-molecular analytesin drug discovery, drug development, and life sciences research laboratories. While ELISAs have traditionally been the most popular form of immunoassay, they are limited by the need to perform multiple wash steps.
For the detection of three biomarkers in complex sample matrices, the AlphaLISA and Electrochemiluminescent (ECL) assay technologies were shown to have similar: Assay windows (linear dynamic range), Lower and upper detection limit, Intra-and inter-assay precision (lower % CV) The advantages of using AlphaLISA over ECL are: Shorter total assay duration No wash steps No shaking Lower sample volume requirement for equivalent performance Less expensive instrument and plates required
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.