Human IL-8 ELISA Kit

Principle of the Assay

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.

For Use with serum, plasma and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

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.

GENE ID 3576
SWISS PROT P10145
SYNONYMS CXCL8; NAF; GCP1; LECT; LUCT; NAP1; GCP-1; LYNAP; MDNCF; MONAP; NAP-1

Materials Supplied

Kit Components 96 Wells Quantity/Size
Aluminium pouches with a Microwell Plate coated with monoclonal antibody to human IL-8 (8﹡12) 1 plate
Human IL-8 Standard lyophilized, 2000 pg/ml upon reconstitution 2 vials
Concentrated Biotin-Conjugate anti-human IL-8 monoclonal antibody 2 vials
Streptavidin-HRP solution 2 vials
Standard /sample Diluent 1 bottle
Biotin-Conjugate antibody Diluent 1 bottle
Streptavidin-HRP Diluent 1 bottle
Wash Buffer Concentrate 20x (PBS with 1% Tween-20) 1 bottle
Substrate Solution 1 vial
Stop Solution 1 vial
Adhesive Films 4 pieces
Product data sheet 1 copy

Storage

Storage Store at 2 - 8°C

Performance Characteristics

REPEATABILITY The coefficient of variation of both intra-assay and inter-assay were less than 10%.
SENSITIVITY The minimum detectable dose was 7pg/mL.
ASSAY RANGE 15.6 - 1000 pg/mL
SPECIFICITY This assay recognizes both natural and recombinant human IL-8. The factors listed below were prepared at 50ng/ml in Standard /sample Diluent and assayed for cross-reactivity and no significant cross-reactivity or interference was observed.
Factors assayed for cross-reactivity
Recombinant human ENA-78, BLC/BCA-1, GCP-2, GROα, GROβ, GROγ, IP-10, I-TAC, MIG, NAP-2, PF4, SDF-1α, SDF-1β
Recombinant mouse BLC/BCA-1, GCP-2, CRG-2, KC, MIG, SDF-1β