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IL1β and IL1β are central players of the immune response, displaying roles in inflammation both at local and systemic levels. Despite they seem to display very similar functions, these proteins are encoded by two independent genes sharing only ~30% identity. IL1β is synthesized as a 31 kDa precursor that is cleaved by Caspase-1 (ICE) into the active 17 kDa form, and eventually released into the extracellular space. Its production has been reported in many cell types including brain and, importantly, monocytic and periferal blood mononuclear cells. After binding to its receptor, IL-1RI, IL1β triggers a cascade of kinase signaling pathways that lead to the activation of transcription factors like NFκB and AP-1, eventually activating the expression of genes such as MIP-2 and C-reactive protein.
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|
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.
Breast cancer tumors can adapt to immune cell infiltration by responding to the increased concentration of interferon gamma (IFN-ɣ) and other cytokines secreted by subsets of T lymphocytes with the upregulation of the immune checkpoint proteins such as Programmed cell death ligand 1 (PD-L1). These checkpoint proteins allow the tumors to evade immune targeting and reduce the immune response, thus promoting tumor progression.
In this application note, you will learn:
Various cytokines are secreted during an active immune response that can have modulatory effects on target cell populations, including interferon gamma (IFN-ɣ), tumor necrosis factor alpha (TNFa) and several interleukins.
In this application note, you will learn how we investigated:
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.