Interleukin 8, Human (IL-8) BioAssay(TM) ELISA Kit

Human CXCL8/IL-8 Immunoassay for the quantitative determination of human interleukin 8 (IL-8) concentrations in cell culture supernates, serum, and plasma.\n\nInterleukin 8 (IL-8), a member of the neutrophil-specific CXC subfamily of chemokines, is a\npotent neutrophil chemotactic and activating factor. It is a primary inflammatory cytokine\nproduced by many cells (including monocytes/macrophages, T cells, neutrophils, fibroblasts,\nendothelial cells, keratinocytes, hepatocytes, astrocytes and chondrocytes) in response to\nproinflammatory stimuli such as IL-1, TNF, LPS and viruses. Its function is, in part, to attract\nneutrophils to the site of inflammation and to activate them. (For reviews, see references\n1-6.)\n\nThe human IL-8 cDNA sequence predicts a protein of 99 amino acids. Removal of a\n22 residue signal peptide generates a mature protein of 77 amino acids (~8kD). Further\nproteolysis of the N-terminal end leads to a variant form with 72 amino acids; full activation of\nIL-8 may require cleavage to the 72 amino acid form. IL-8 can form non-covalent dimers in\nsolution, especially at high concentrations, but dimerization is not necessary for biological\nactivity.\n\nIL-8 binds to two seven-transmembrane, G protein-coupled receptors, CXCR1 and CXCR2,\nas well as to the non-signalling Duffy antigen on red blood cells. The Duffy antigen may play a\nrole in regulating IL-8 activity on functional receptors. The Interleukin 8, Human (IL-8) BioAssay(TM) ELISA Kit is a 3.5 hour solid phase ELISA designed to measure human IL-8 in cell culture supernates, serum and plasma. It is based on antibodies raised against the 72 amino acid variant of human IL-8 derived from E. coli. It is calibrated with the same recombinant factor. This immunoassay accurately quantitates recombinant human IL-8. Measurement of natural human IL-8 or the 77 amino acid variant of human IL-8 gave results parallel to the standard curves obtained using the E. coli-expressed kit standards. These results indicate that the Immunoassay kit can be used to determine relative mass values for natural human IL-8.\n\nAutoantibodies to IL-8, as well as immune complexes of IL-8 and these antibodies, have been\nreported in the serum of some normal subjects (7). It remains to be determined if these\nautoantibodies may have the potential to interfere with the measurement of serum IL-8 by\nELISA.\n\nPrinciple:\nThis 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, a substrate solution is added to the wells and color develops in proportion to the amount of IL-8 bound in the initial step. The color development is stopped and the intensity of the color is measured.\n\nKit Components:\nIL-8 Microplate-96 well polystyrene microplate (12 strips of 8 wells) coated with a mouse monoclonal antibody against IL-8.\nIL-8 Conjugate-11.5ml/vial of a polyclonal antibody against IL-8 conjugated to horseradish peroxidase, with red dye and preservatives.\nIL-8 Standard-10ng/vial of recombinant human IL-8 in a buffered protein base with preservatives, lyophilized. \nAssay Diluent -11ml/vial of a buffered protein base with preservatives.\nCalibrator Diluent Concentrate (5X)-21ml/vial of a\nconcentrated buffered protein base with preservatives. For cell culture supernate samples.\nCalibrator Diluent-21ml/vial of animal serum with preservatives. For serum/plasma samples. \nWash Buffer Concentrate-21ml/vial of a 25-fold concentrated solution of buffered surfactant with preservatives.\nColor Reagent A-12.5ml/vial of stabilized hydrogen peroxide.\nColor Reagent B-12.5ml/vial of stabilized chromogen (TMB, tetramethylbenzidine). \nStop Solution-6ml/vial of 2 N sulfuric acid.\nPlate Covers-Adhesive strips. 4 strips\n\nContains sufficient materials to run an ELISA on one 96 well plate.\n\nStorage and Stability:\nStore components at 4 degrees C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

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