Intended use
This immunoassay kit allows for the specific measurement of rat interleukin 8 (IL-8) concentrations in cell culture supernates, serum and plasma.
Introduction
Interleukin 8 (IL-8), a member of the neutrophil-specific CXC subfamily of chemokines, is a potent neutrophil chemotactic and activating factor. It is a primary inflammatory cytokine produced by many cells (including monocytes/macrophages, T cells, neutrophils, fibroblasts, endothelial cells, keratinocytes, hepatocytes, astrocytes and chondrocytes) in response to proinflammatory stimuli such as IL-1, TNF, LPS and viruses. Its function is, in part, to attract neutrophils to the site of inflammation and to activate them.
The IL-8 cDNA sequence predicts a protein of 99 amino acids. Removal of a 22-residue signal peptide generates a mature protein of 77 amino acids (~ 8 kDa). Further proteolysis of the N-terminal end leads to a variant form with 72 amino acids; full activation of IL-8 may require cleavage to the 72 amino acid form. IL-8 can form non-covalent dimers in solution, especially at high concentrations, but dimerization is not necessary for biological activity.
IL-8 binds to two seven-transmembrane, G protein-coupled receptors, CXCR1 and CXCR2, as well as to the non-signalling Duffy antigen on red-blood cells. The Duffy antigen may play a role in regulating IL-8 activity on functional receptors.
Test principle
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-8 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-8 present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for IL-8 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, an enhanced luminol/peroxide substrate solution is added to the wells and light is produced in proportion to the amount of IL-8 bound in the initial step. A microplate luminometer is used to measure the intensity of the light emitted.
