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Interleukin 8 (IL8 or CXCL8), a member of the ELR+ CXC chemokine family, is a 8.4 kDa polypeptide that forms homodimers in vivo. IL8 is secreted by several types of cells: fibroblasts, monocytes, macrophages and endothelial cells, among many others, in response to inflammatory stimuli. It is a chemoattractant and activator for neutrophils, directing them from periferal blood to the site of inflammation. It is also a potent angiogenic factor promoting endothelial and epithelial migration and proliferation in several cancers, and is associated with metastasis. It signals through two specific G protein-coupled receptors, CXCR1 and CXCR2, sharing ~77% identity.
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.
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.
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.