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

This IL-8 ELISA is a 2.5 hour solid phase immunoassay readily applicable to measure IL-8 levels in serum, plasma, cell culture supernatant and other biological fluids in the range of 50-1600pg/ml. It showed no crossreactivity with human IL-1b, EGF, MCAF, RANTES, and SAA. This IL-8 ELISA is expected to be effectively used for further investigations into the relationship between IL-8 and various diseases. Principle: This IL-8 enzyme-linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal specific for IL-8. Standards or samples are then added to the appropriate microtiter plate wells and incubated. IL-8, if present, will bind and become immobilized by the antibody pre-coated on the wells. The microtiter plate wells are thoroughly washed to remove unbound IL-8 and other components of sample. In order to quantitate the amount of IL-8 present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated polyclonal antibody specific for IL-8 is added to each well to "sandwich" the IL-8 immobilized during the first incubation. The microtiter plate then undergoes a second incubation. The wells are thoroughly washed to remove all unbound HRP-conjugated antibodies. A TMB (3,3'5,5' tetramethyl- benzidine) substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain IL-8 and enzyme-conjugated antibody will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulfuric acid solution. The color change is measured spectrophotometrically at a wavelength of 450nm ± 2nm. In order to measure the concentration of IL-8 in the samples, this kit contains two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant/ urine testing). According to the testing system, the provided standard is diluted (2-fold) with the appropriate Calibrator Diluent and assayed at the same time as the samples. This allows the operator to produce a standard curve of Optical Density (O.D.) vs. IL-8 concentration (pg/ml). The concentration of IL-8 in the samples is then determined by comparing the O.D. of the samples to the standard curve. Introduction: Interleukin-8 (IL-8) is known as neutrophil attractant/activating protein (NAP-1), monocyte-derived neutrophil-activating peptide (MONAP), monocyte-derived neutrophil chemotactic factor (MDNCF), T lymphocyte chemotactic factor (TCF) and leukocyte adhesion inhibitor (LAI) It is a member of the chemokine superfamily which selectively chemoattract and activate specific leukocyte subpopulation. All of these cytokines have four conserved cysteines, and two subfamilies can be distinguished according to the position of the first two cysteines, which either are separated by on amino acid (C-X-C proteins) or are adjacent (CC-protein). The members of the two subfamilies differ in their target cell selectivity and the chromosomal location of their genes (chromosome 4 for the C-X-C proteins and chromosome 17 for the C-C proteins). IL-8 belongs to the C-X-C subfamily along with platelet factor 4 (PF4), platelet basic protein (PBP), connective-tissue-activating peptide III (CTAPIII), b-thromboglobulin, neutrophil-activating peptide-2 (NAP-2), ENA-78, three closely related MGSA/CRO gene products (GRO-a, GRO-b, GRO-g), and g-interferon-inducible protein (g-IP-10). The members of the C-C chemokines are mainly chemotactic for monocytes, whereas the C-X-C chemokines, except for IP10 and PF4, chemoattract and activate neutrophils. In addition to the effect on neutrophils, IL-8 has been reported to be a less potent chemoattractant for T lymphocytes. IL-8 is produced by many cells in response to inflammatory stimuli such as IL-1b or TNF-a and to various types of mitogen, lectins, crystals, viruses, phorbol esters (PMA). Many cell types that produce IL-8 in response to these stimuli is probably far from complete but includes, monocytes/macrophages, T lymphocytes, neutrophils, fibroblasts, keratinocytes, hepacytes, chondrocytes, endothelial cells, glioblatoma cells, and mesothelial cells. , The IL-8 predominant form secreted by stimulated monocytes has 72 residues (MW 8385), whereas the predominant form secreted by IL-1 stimulated endothelial cells has 77 residues (MW 8922). These variants have similar biological activities, although the 72-residue form of IL-8 appears to be 2-10 fold more potent than the 77-residue form, depending on the type of assay used. Various non-infectious diseases of man are known to be associated with neutrophilia and/or neutrophil infiltration into organs. Example of some of these human diseases include rheumatoid arthritis, gouty arthritis, psoriasis, glomerulonephritis, adult respiratory distress syndrome, immune vasculitis, inflammatory bowel disease, ischemia-reperfusion syndrome (including myocardial infarction and multiple organ failure), chorioretinitis, cystic fibrosis, septic shock, acute meningococcal infections, alcoholic hepatitis and mediterranean fever (8). In the joint fluids from rheumatoid arthritis, gouty arthritis, psoriatic scale, plasma from adult respiratory syndrome caused by sepsis, and serum from nephrotic syndrome, the presence of IL-8 protein has been directly proved. The peripheral blood mononuclear cells (PBMC) obtained from patients undergoing an asthmatic attack have been shown to spontaneously produce IL-8-like molecules in vitro. The production of IL-8 in many other activities which contributes to these human diseases, whereas systemic inflammatory reactions such as fever, acute phase protein induction, and other cytokine induction are not known to be triggered by IL-8. Development of an accurate immunoassay for the quantitative determination of human IL-8 levels in cell culture supernatant, serum, plasma and other biological fluids is expected to be effectively used for the further investigation on the relationship of IL-8 with various inflammatory diseases , Sensitivity:, >10pg/ml using Calibrator Diluent I, >4pg/ml using Calibrator Diluent II, Detection Range: 50-1600pg/ml, Kit Components: I8430-01A: Microtiter Plate, 1x96 wells, I8430-01B: Anti-human IL-8 (HRP), 1x15ml , *I8430-01C: IL-8 Standard, Recombinant, 2x1vial, I8430-01D: Calibrator Diluent I, 1x22ml, I8430-01E: Calibrator Diluent II, 1x22ml, I8430-01F: Wash Buffer (20X), 1x60ml, I8430-01G: Substrate A, 1x11ml, I8430-01H: Substrate B, 1x11ml, I8430-01J: Stop Solution, 1x14ml, Storage and Stability: Store all kit components except *I8430-01C at 4°C. Stable for 6 months. Store *I8430-01C powder at 4°C, liquid at -20°C (30 days). For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.