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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||500 assay points|
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.
While fundamental knowledge about tumor immunology has exploded recently, a new therapeutic approach to cancer is taking off: immunotherapy. Instead of directly attacking tumor cells, the idea is to help the immune system recognize and destroy them.
The use of CAR-T cells (Chimeric Antigen Receptor-T Cells), a new avenue of immunotherapy, consists in genetically modifying the patient's immune cells to arm them against a tumor. Concretely, T lymphocytes are taken from the patient's blood and modified in vitro. This leads to their expression of specific surface receptors, which recognize a tumor antigen. Once modified, these CAR-T cells are multiplied and re-injected into the patient's body in large quantities. There they go on to destroy cancer cells after binding to the tumor antigen, releasing a mixture of cytokines and pro-inflammatory chemokines.
This application note focuses on detecting cytokine and chemokine secretion using two orthogonal no-wash immunoassays, AlphaLISA® and HTRF®, in an in vitro co-culture model with CAR-T cells and CD19 positive Raji cells targeting tumors.
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.