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Human Interleukin 17 (IL17 or IL17A) is a homodimer formed of two ~15 kDa subunits produced by a subset of T helper cells named Th17. It is a proinflammatory cytokine that enhances T cell priming and stimulates macrophages, fibroblasts, endothelial and epithelial cells to produce multiple mediators of inflammation like IL1, IL6, TNF-a, NOS-2, metalloproteases, and chemokines. IL17 has been implicated in the proinflammatory patterns associated with joint inflammation and rheumatoid arthritis (RA) in mouse and human models. It is also critical for neutrophil activation and migration, and induces IL8, a key chemokine for neutrophils. IL17 signals through IL-17R, which in mice has at least two members, IL-17RA, and IL-17RC. Recent studies suggest that the IL17 pathway may be a novel therapeutic target for the treatment of chronic inflammatory diseases like asthma and RA.
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||500 assay points|
Immunoassays are a mainstay for the quantification of a variety of biomolecular analytes in drug discovery, drug development, and life sciences research. AlphaLISA® proves advantageous to ELISAs, offering a novel, homogenous immunoassay technology that eliminates wash steps. Using the JANUS® Automated Workstation in combination with AlphaLISA provides a solution to easily preparing assays that can be tailored to the needs of the laboratory. Moreover, the JANUS workstation can be easily set-up to process different assay types in a multi-user, multi-assay environment, thus providing a flexible automation solution.
In this application note, we demonstrate the performance of the JANUS Workstation in automating four AlphaLISA cytokine assays: IL1b, TNFa, IL17, IFNg.
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.