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. It is 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 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 (~ 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.

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. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for IL-8 is added to the wells and binds to the combination of capture antibody-IL-8 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-8 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-8 standard dilutions and IL-8 sample concentration determined.

Figure 1:Schematic diagram of the assay

1. Aluminium pouches with a Microwell Plate coated with monoclonal antibody to human IL-8 (812)

2. 2 vials human IL-8 Standard lyophilized, 2000 pg/ml upon reconstitution

3. 2 vials concentrated Biotin-Conjugate anti-human IL-8 antibody

4. 2 vials Streptavidin-HRP solution,

5. 1 bottle Standard /sample Diluent

6. 1 bottle Biotin-Conjugate antibody Diluent

7. 1 bottle Streptavidin-HRP Diluent

8. 1 bottle Wash Buffer Concentrate 20x (PBS with 1% Tween-20)

9. 1 vial Substrate Solution

10. 1 vial Stop Solution

11. 4 pieces Adhesive Films

12. package insert

NOTE: [96 Tests]

Unopened Kit：Store at 2 -8° C. Do not use past kit expiration date. opened/ReconstitutedReagents：Please refer to the datasheets for detail information.

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