The AlphaLISA® Human Interleukin-1β (IL1β) Detection Kit is designed for detection and quantitation of human IL-1β in serum, buffered solution or cell culture medium in a homogeneous (no-wash steps, no separation steps) assay.
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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||5,000 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.