Human CXCL8/IL-8 Immunoassay for the quantitative determination of human interleukin 8 (IL-8) concentrations in cell culture supernates, serum, and plasma.
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 toproinflammatory 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. (For reviews, see references 1-6.) The human 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 (~8kD). 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. The Interleukin 8, Human (IL-8) BioAssay™ 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. Autoantibodies to IL-8, as well as immune complexes of IL-8 and these antibodies, have been reported in the serum of some normal subjects (7). It remains to be determined if these autoantibodies may have the potential to interfere with the measurement of serum IL-8 by ELISA.
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, 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.
Kit Components:IL-8 Microplate-96 well polystyrene microplate (12 strips of 8 wells) coated with a mouse monoclonal antibody against IL-8
IL-8 Conjugate-11.5ml/vial of a polyclonal antibody against IL-8 conjugated to horseradish peroxidase, with red dye and preservatives
IL-8 Standard-10ng/vial of recombinant human IL-8 in a buffered protein base with preservatives, lyophilized. Assay Diluent -11ml/vial of a buffered protein base with preservatives
Calibrator Diluent Concentrate (5X)-21ml/vial of aconcentrated buffered protein base with preservatives. For cell culture supernate samples
Calibrator Diluent-21ml/vial of animal serum with preservatives. For serum/plasma samples. Wash Buffer Concentrate-21ml/vial of a 25-fold concentrated solution of buffered surfactant with preservatives
Color Reagent A-12.5ml/vial of stabilized hydrogen peroxide
Color Reagent B-12.5ml/vial of stabilized chromogen (TMB, tetramethylbenzidine). Stop Solution-6ml/vial of 2 N sulfuric acid
Plate Covers-Adhesive strips. 4 strips
Contains sufficient materials to run an ELISA on one 96 well plate.
Storage and Stability:Store components at 4°C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Materials Required but not supplied:Microplate reader capable of measuring absorbance at 450nm with the correction wavelength set at 540nm or 570nm
Pipettes and pipette tips
Deionized or distilled water
Squirt bottle, manifold dispenser, or automated microplate washer
