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Interleukin 6 (IL6) is a ~22 kDa pleiotropic cytokine that acts not only on the immune system, but also affects many physiological events in various organs. IL6 exerts pro- or anti-inflammatory effects, depending on the target cell analyzed and the in vivo environmental circumstances. IL6 is a differentiation and proliferation factor for B and T cells, and acts as a migration factor on monocytic cells. It is the major activator of acute-phase protein expression in the liver, a hematopoietic factor, and acts as a survival factor on neuronal cells. IL6 signals through binding to the gp130/ IL-6R receptor complex, leading to the activation of JAK/STAT, MAPK and PI3K cascades.
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|
The introduction of enzyme-linked immunosorbent assays (ELISAs) in the early 1970’s offered researchers a non-radiometric immunoassay platform without compromising sensitivity. Over the last 50 years scientists have made huge strides in disease research and drug discovery and a demand for greater assay throughput and sensitivity has evolved. In response, more robust immunoassays have been developed to address some of the limitations of the standard, colorimetric ELISA.
Find out about the most common limitations of traditional ELISAs and how different ELISA alternative technologies address these limitations.
Pyrogens are substances that cause fever in humans and animals. They can be endogenous or exogenous. One of the characteristics of endogenous pyrogens is their ability to induce cytokine production (IL-1ß, IL-6, IL-8, and TNFa) by the body's immune system. The detection of these pyrogens in pharmaceutical products is an essential regulatory requirement to ensure product quality and patient safety.
In recent years, the Monocyte Activation Test (MAT) has become the most attractive and relevant method for humans. This method enables the detection and quantification of substances with a pyrogenic effect, i.e. those causing a febrile response, and does not require the use of animals.
This application note demonstrates the usefulness of the AlphaLISA® IL-6 kit for the quantification of pyrogens and the ease of use of a homogeneous, wash-free assay with low sample volumes.
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.