The AlphaLISA Human IL8 Detection Kit is designed for the quantitative determination of human IL-8 in serum, buffered solution or cell culture medium using a homogeneous (no wash steps, no separation steps) assay.
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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||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.
Immunoassays are a mainstay for the quantification of a variety of bio-molecular analytesin drug discovery, drug development, and life sciences research laboratories. While ELISAs have traditionally been the most popular form of immunoassay, they are limited by the need to perform multiple wash steps.
Immunoassays are used for detection and quantification of low analyte concentrations. Enzyme Linked-Immuno-Sorbent Assay (ELISA) technology is the most widely adopted assays method for performing sandwich or competition based immunoassays.
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.
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.