AlphaLISA® no-wash immunoassay kit for detection of human TNFα in serum, buffered solution or cell culture medium.
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Tumor necrosis factor alpha (TNFa) is a multifunctional proinflammatory cytokine synthesized mainly by nucleated blood cells as a 233 aa type II transmembrane protein which is cleaved by ADAM17 between aa 76-77 to form a soluble homotrimeric complex. TNFa plays a role in lipid metabolism, coagulation, and endothelial function and has been associated with cancer, infection and inflammation (including inflammatory bowel disease), ischemia/reperfusion injury and heart failure, and insulin resistance.
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 aim of this work was to compare the performance of the AlphaLISA® amplified luminescent proximity homogeneous assay technology platform and an alternative time-resolved fluorescenceresonance energy transfer (TR-FRET) technology assay platform for the detection of five biomarkers.
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.
The aim of this work was to compare the performance of AlphaLISA® amplified luminescent proximity homogeneous assay technology platform and an alternative time-resolved fluorescence resonance energy transfer (TR-FRET) technology assay platform for the detection of cytokines in high-thoughput screening. Many cytokines are important biomarkers for inflammation. As they are often studied using cell-based assays, components in the sample matrix could affect the performance of homogeneous assays.
Enzyme-linked Immunosorbent Assay is the most widely Kits adopted method for detection and quantification of cytokines and other biomarkers. This traditional technology offers good,selectivity, sensitivity and assay versatility; however, it has certain disadvantages such as limited dynamic range and low throughput due to the numerous wash steps.