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Colony-stimulating factors (CSFs) are proteins necessary for the survival, proliferation, and differentiation of hematopoietic progenitor cells. The human Granulocyte/Macrophage colony-stimulating factor (GM-CSF) is a ~23 kDa glycosylated protein (144-aa), encoded by the CSF2 gene. It has been originally purified from culture media conditioned by lung tissue from endotoxin injected mice, having the capacity to stimulate the formation of both granulocyte and macrophage colonies.
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|
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.
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.