Principle of the Assay
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-1β has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-1β 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-1β is added to the wells and binds to the combination of capture antibody- IL-1β 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-1β 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-1β standard dilutions and IL-1β sample concentration determined.
For Use with serum, plasma and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.
The Interleukin 1 (IL-1) family of proteins consists of the classic members IL-1α, IL-1β, and IL-1ra, plus IL-18, IL-33, and IL-1F5-10. IL-1α and IL-1β bind to the same cell surface receptors and share biological functions. IL-1 is not produced by unstimulated cells of healthy mice with the exception of skin keratinocytes, some epithelial cells, and certain cells of the central nervous system. In response to inflammatory agents, infections, or microbial endotoxins, however, a dramatic increase in the production of IL-1 by macrophages and various other cell types is observed. IL-1β plays a central role in immune and inflammatory responses, bone remodeling, fever, carbohydrate metabolism, and GH/IGF-I physiology. Inappropriate or prolonged production of IL-1 has been implicated in a variety of pathological conditions including sepsis, rheumatoid arthritis, inflammatory bowel disease, acute and chronic myelogenous leukemia, insulin-dependent diabetes mellitus, atherosclerosis, neuronal injury, and aging-related diseases.
IL-1α and IL-1β are structurally related polypeptides that show approximately 25% homology at the amino acid level. Both are synthesized as 31 kDa precursors that are subsequently cleaved into mature proteins of approximately 17.5 kDa. Cleavage of the IL-1β precursor by Caspase-1/ICE is a key step in the inflammatory response. Neither IL-1α nor IL-1β contains a typical hydrophobic signal peptide, but evidence suggests that these factors can be secreted by non-classical pathways. A portion of unprocessed IL-1α can be presented on the cell membrane and may retain biological activity. The precursor form of IL-1β, unlike the IL-1α precursor, shows little or no biological activity in comparison to the processed form. Both unprocessed and mature forms of IL-1β are exported from the cell.
IL-1α and IL-1β exert their effects through immunoglobulin superfamily receptors that additionally bind IL-1ra. The 80 kDa transmembrane type I receptor (IL-1 RI) is expressed on T cells, fibroblasts, keratinocytes, endothelial cells, synovial lining cells, chondrocytes, and hepatocytes. The 68 kDa transmembrane type II receptor (IL-1 RII) is expressed on B cells, neutrophils, and bone marrow cells. The two IL-1 receptor types show approximately 28% homology in their extracellular domains but differ significantly in that the type II receptor has a cytoplasmic domain of only 29 amino acids (aa), whereas the type I receptor has a 217 aa cytoplasmic domain. IL-1 RII does not appear to signal in response to IL-1 and may function as a decoy receptor that attenuates IL-1 function. The IL-1 receptor accessory protein (IL-1 RAcP) associates with IL-1 RI and is required for IL-1 RI signal transduction. IL-1ra is a secreted molecule that functions as a competitive inhibitor of IL-1. Soluble forms of both IL-1 RI and IL-1 RII have been detected in human plasma, synovial fluids, and the conditioned media of several human cell lines. In addition, IL-1 binding proteins that resemble soluble IL-1 RII are encoded by vaccinia and cowpox viruses.
|Kit Components||96 Wells Quantity/Size|
|Aluminium pouches with a Microwell Plate coated with antibody to rat IL-1β (8-12)||1 plate|
|Rat IL-1β Standard lyophilized, 4000pg/ml upon reconstitution||2 vials|
|concentrated Biotin-Conjugate anti-rat IL-1β 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|
|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 15pg/mL.|
|ASSAY RANGE||62.50 - 4000 pg/mL|
|SPECIFICITY||This assay recognizes both natural and recombinant rat IL-1β. 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 rat||IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-, IFN-γ|
|Recombinant human||IL-1, IL-2, IL-6|
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