IL-8 Human ELISA Assay
The IL-8 Human ELISA Assay is For Research Use Only
Size: 1×96 wells
Sensitivity: 4 pg/mL
Dynamic Range: 31.25 – 1000 pg/ml
Incubation Time: 3.5 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl
Alternative Names: Interleukin 8, IL8
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 (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 (~ 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-signaling Duffy antigen on red-blood cells. The Duffy antigen may play a role in regulating IL-8 activity on functional receptors.
Related Products to IL-8 Human ELISA Assay
Human IL-9 ELISA Kit
The Eagle Biosciences Human Interleukin 8 (IL-8) ELISA 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 colored 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.
- Prepare all reagents and working standards as directed in the previous sections.
- Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
- Add 100 µL of Standard, control, or sample, per well. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37C.
- Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350 µL) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
- Add 100 µLof the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37C.
- Repeat the aspiration/wash.
- Add 100 µL of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at 37C. Avoid placing the plate in direct light.
- Repeat the aspiration/wash.
- Add 100 µL of Substrate Solution to each well. Incubate for 10-20 minutes at 37C. Avoid placing the plate in direct light.
- Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm. (optionally 630nm as the reference wave length;610-650nm is acceptable)
Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.
1. Oppenheim, J.J. (1991) Annu. Rev. Immunol. 9:617.
2. Miller, M.D. and M.S. Krangel (1992) Crit. Rev. Immunol. 12:17.
3. Matsushima, K. et al. (1992) Chem. Immunol. 51:236.
4. Taub, D.D. and J.J. Oppenheim (1993) Cytokine 5:175.
5. Vaddi, K. et al. (1997) The Chemokine Facts Book, Academic Press, p. 23.
6. Hack, C.E. et al. (1997) Adv. Immunol. 66:101.
7. Baggio liniM. Chemok ines in patho logy and medicine. J Intern Med, 2001.250 (2)91.