Principle of the Assay
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-33 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-33 present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for IL-33 is added to the wells and binds to the combination of capture antibody- IL-33 in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A coloured product is formed in proportion to the amount of IL-33 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven IL-33 standard dilutions and IL-33 sample concentration determined.
For Use with serum, plasma and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.
Interleukin-33 (IL-33), also known as NF-HEV and DVS 27, is a 30 kDa pro-inflammatory protein that plays an important role in Th2-biased immune responses and cardiac pathology. Human IL-33 is synthesized as a 270 amino acid (aa) molecule with an N-terminal nuclear localization signal, a helix-turn-helix motif, and a C-terminal region with structural homology to IL-1 family cytokines. Full length IL-33 interacts with nuclear chromatin, binds NFκB, and inhibits pro-inflammatory NFκB transactivation. Cleavage of full-length IL-33 leads to the extracellular release of an 18-20 kDa C-terminal fragment known as mature IL-33. Cathepsin G, Elastase, and Proteinase 3 can each cleave full length IL-33, giving rise to N-terminal heterogeneity of the mature form. IL-33 can be inactivated by further cleavage at several sites by Proteinase 3 and Caspase-1. Additional isoforms of human IL-33 with internal deletions are generated by alternative splicing. Mature human IL-33 shares 57% and 59% aa sequence identity with mouse and rat IL-33, respectively.
IL-33 binds the transmembrane receptor ST2/IL-1 R4 which subsequently associates with IL-1 RAcP to enable IL-33 dependent signaling. IL-1 RAcP is a shared signaling subunit that also associates with the receptors IL-1 RI, IL-1 RII, IL-1 R6, and SCF R/c-kit. A soluble isoform of ST2 retains the ability to bind IL-33 and blocks ST2-dependent responses. Soluble IL-1 RAcP enhances the decoy function of soluble ST2. IL-33 binding to transmembrane ST2 induces the association of ST2 with existing IL-1 RAcP/SCF R complexes. Activation of either ST2 or SCF R by their respective ligands can induce signal transduction through the other receptor subunit. IL-33 signaling through ST2 additionally triggers VE-Cadherin phosphorylation and internalization on vascular endothelial cells which leads to increased vascular permeability, vessel sprouting, and tubule formation.
IL-33 exerts multiple effects on immune system function. It acts on Th2 cells, basophils, and mast cells to induce their migration to sites of inflammation and production of Th2 cytokines. IL-33 also promotes the expansion of regulatory T cells and alternately activated macrophages while attenuating Th17 cell expansion and activation. IL-33 contributes to infection clearance by enhancing neutrophil sensitization to TLR and Dectin-1 signaling, phagocytic activity, and migration to sites of infection. It is upregulated in a wide variety of cells under inflammatory conditions. Full length IL-33 is also found at elevated levels in bronchiolar lavage fluid during pulmonary fibrosis.
The full-length protein is classified as an alarmin due to its release from physically damaged or necrotic cells and its ability to trigger inflammatory and anti-viral CD8+ T cell responses. Like mature IL-33, the full-length protein activates ST2 and promotes mast cell activation and neutrophil infiltration. IL-33 induces both protective and pathologic actions in the heart. It counteracts cardiac myocyte hypertrophy and responsiveness to angiotensin II and phenylephrine. It is induced in cardiac fibroblasts by mechanical stress and circulates at elevated levels during chronic heart failure (as does the full-length form). The soluble ST2 receptor is elevated in the serum of heart failure as well as asthma patients. IL-33 inhibits the development of atherosclerotic plaques and induces the production of anti-oxidized LDL antibodies. It can also enhance eosinophilic perimyocarditis and impair heart function. In other settings, IL-33 limits neutrophil infiltration and circulating inflammatory chemokine levels following hepatic ischemia/reperfusion injury but exacerbates CD4+ T cell infiltration and tissue damage following cisplatin-induced acute kidney injury.
|SYNONYMS||DVS27; IL1F11; NF-HEV; NFEHEV; C9orf26|
|Kit Components||96 Wells Quantity/Size|
|Aluminium pouches with a Microwell Plate coated with monoclonal antibody to human IL-33 (8﹡12)||1 plate|
|Human IL-33 Standard lyophilized, 1500 pg/ml upon reconstitution||2 vials|
|Concentrated Biotin-Conjugate anti-human IL-33 monoclonal antibody||2 vials|
|Streptavidin-HRP solution||2 vials|
|Standard /sample Diluent||1 bottle|
|Biotin-Conjugate antibody Diluent||1 bottle|
|Streptavidin-HRP Diluent||1 bottle|
|Wash Buffer Concentrate 20x (PBS with 1% Tween-20)||1 bottle|
|Substrate Solution||1 vial|
|Stop Solution||1 vial|
|Adhesive Films||4 pieces|
|Product data sheet||1 copy|
|Storage||Store at 2 - 8°C|
|REPEATABILITY||The coefficient of variation of both intra-assay and inter-assay were less than 10%.|
|SENSITIVITY||The minimum detectable dose was 11pg/mL.|
|ASSAY RANGE||23.43 - 1500 pg/mL|
|SPECIFICITY||This assay recognizes both natural and recombinant human IL-33. The factors listed below were prepared at 50ng/ml in Standard /sample Diluent and assayed for cross-reactivity and no significant cross-reactivity or interference was observed.|
Factors assayed for cross-reactivity
|Recombinant human||IL-18, IL-1b F7, IL-1α, IL-1β, IL-18 Rα, IL-18 Rβ, IL-36 a, IL-36 β, IL-37, IL-38, ST2/IL-1 R4|
|Recombinant mouse||IL-36a, IL-1β, IL-36 β, IL-18|
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