MCP-3 ELISA KIT
INTENDED USE
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 MCP-3 ELISA Kit is intended for LABORATORY RESEARCH USE ONLY and is not for use in diagnostic or therapeutic procedures.
INTRODUCTION
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.Murine MCP-3 Marc/Fic chemokines that have been previously cloned are thought to be homologues of human MCP-3.MCP-3 cDNA cloning and structural analysis revealed that this 76 amino acid (a.a.) polypeptide with a molar mass of 8.5 kDa belongs to a family of small inflammatory proteins, characterized by four conserved cysteine residues and is localized on human chromosome 17. 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.
PRINCIPLE OF THE ASSAY
MCP-3 ELISA Kit
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 MCP-3 ELISA Kit is intended for LABORATORY RESEARCH USE ONLY and is not for use in diagnostic or therapeutic procedures. This 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. MCP-3 ELISA Kit showed no cross-reactivity with other cytokines tested. MCP-3 may play a role in certain diseases, therefore this MCP-3 ELISA Kit is expected to be effectively used for further investigations into the relationship between MCP-3 and various pathological conditions. This MCP-3 ELISA Kit applies a technique called a quantitative sandwich immunoassay.
The microtiter plate provided in this 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 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 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 the MCP-3 ELISA Kit testing 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.
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