This human Monocyte Chemotactic Protein-3 (MCP-3) ELISA kit applies a technique called a quantitative sandwich immunoassay. This human MCP-3 ELISA kit is a 2.5 hour solid phase immunoassay readily applicable to measure MCP-3 in serum, plasma, cell culture supernatant, and other biological fluids in the range of 0 to 1600 pg/mL. Monocyte Chemotactic Protein(MCP-3) is a novel chemokine that has been recently purified from human osteosarcoma cell line. It was shown that MCP-3 is produced by both tumours cellsand leukocytes.MCP-3 can bind and activate a vast diversity of inflammatory cell types through interaction with multiple leukocyte receptors as well as it’s own receptor. This sandwich human MCP-3 ELISA kit can recognises both natural and recombinant human MCP-3. This kit exhibits no significant cross-reactivity with human IL-8, IL-1ß, SAA, RANTES, MCAF, EGF, TNF-α, TGF-ß, M-CSF, GM-CSF, FGF, and EPO.
The microtiter plate provided in this human MCP-3 ELISA kit has been pre-coated with a monoclonal antibody specific for MCP-3. Standards or samples are then added to the appropriate microtiter plate wells and incubated. MCP-3 if present, will bind and become immobilised by the antibody pre-coated on the wells. The microtiter plate wells of this human MCP-3 ELISA kit are thoroughly washed to remove unbound MCP-3 and other components of sample. In order to quantitate the amount of MCP-3 present in the sample, a standardised preparation of horseradish peroxidase (HRP)-conjugated polyclonal antibody specific for MCP-3 is added to each well to "sandwich" the MCP-3 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 and 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 MCP-3 and enzyme-conjugated antibody will exhibit a change in colour. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm.
In order to measure the concentration of MCP-3 in the samples, this human MCP-3 ELISA kit includes two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant testing). According to this human MCP-3 ELISA kit system, the provided standard is diluted (2-fold) with 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.) versus MCP-3 concentration (pg/mL). The concentration of MCP-3 in the samples is then determined by comparing the O.D. of the samples to the standard curve. The minimum detectable dose of MCP-3 was determined using human MCP-3 ELISA kit by adding two standard deviations to the mean optical density value of 20 zero standard replicates and calculating the corresponding concentration from the standard curve. The minimum detectable dose using a standard curve generated with Calibrator Diluent I is 30 pg/mL and using Calibrator Diluent II is 20 pg/mL.
This human MCP-3 ELISA Kit is to be used for the in vitro quantitative determination of human monocyte chemotactic protein-3 (MCP-3) concentrations in serum, plasma, cell culture supernatant, and other biological fluids. This human MCP-3 ELISA kit showed no cross-reactivity with other cytokines tested. MCP-3 may play a role in certain diseases, therefore this human MCP-3 ELISA kit is expected to be effectively used for further investigations into the relationship between MCP-3 and various pathological conditions. This human MCP-3 ELISA kit assay is calibrated against a highly purified E. Coli-expressed 76-amino acid polypeptide form of recombinant human MCP-3. This human MCP-3 ELISA kit is intended for LABORATORY RESEARCH USE ONLY and is not for use in diagnostic or therapeutic procedures. Fourteen apparently healthy, normal individuals were evaluated in this assay. The MCP-3 concentration of serum/plasma samples were less than 30 pg/mL. The MCP-3 concentration of urine samples were less than 20 pg/mL.
MCP-3 is designated a C-C or intercrine ß cytokine.MCP-3 can indeed use a wide variety of binding sites and activate many inflammatory cells. In vitro, both MCP-3 and MCP-1 can activate T-cells, monocytes, and basophils, but only the first can activate eosinophils. These cytokines show 71% a.a. homology. MCP-3 was also revealed to have 30% a.a homology with MIP-1 and RANTES which can also activate eosinophils plus all cell types mentioned above.MCP-3 seems to be the only C-C chemokine that regularly induces neutrophil migration. It is also a potent chemoattractant for human dendritic cells.
It has been suggested that MCP-3 binds multiple C-C receptors such as MCP-1 on monocytes and basophils, MIP-1a on neutrophils, basophils, and eosinophilsand RANTES on basophils and eosinophils. Evidence suggests that MCP-3 does indeed use multiple receptors (MCP-1, RANTES, and MIP-1a) and binds with MIP-1ß receptor as well as other unique binding sites. MCP-3 was discovered to be the strongest C-C chemokine in inducing the migration of C-C CKR1 transfected cells. It was shown that MCP-3 binds with C-C CKR1 receptor with greater affinity than MIP-1a or Rantes, which mainly activate it. Also, MCP-3 promotes exocytosis of eosinophil granule proteins and stimulates histamine release from human basophils. In vivo, MCP-3 induces the selective infiltration of monocytes on intradermal injection in rabbits.Since MCP-3 acts on a variety of inflammatory cells and utilizes multiple receptors for its function, characterization and isolation of the shared as well as unique receptors for MCP-3 will provide further insights into the pathophysiological roles of MCP-3. C-C chemokines are mediators of a number of pathological conditions such as chronic inflammation, tumor, allergy, as well as atherosclerosis. Since the binding and signaling of MCP-3 is most promiscuous, the development of antibody or antagonist which can block MCP-3 through binding to the receptor or ligand may prove to be useful in the treatment of diseases mediated by a number of C-C chemokines.
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