Monocyte Chemotactic Protein (MCP-3) is a novel chemokine that has been recently purified from human osteosarcoma cell line.1 It was shown that MCP-3 is produced by both tumors cells1 and leukocytes.2,3 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.6,7 Murine MCP-3 Marc/Fic chemokines that have been previously cloned are thought to be homologues of human MCP-3.3,4 MCP-3 cDNA cloning2 and structural analysis revealed that this 76 amino acid (a.a.) polypeptide with a molar mass of 8.5kD belongs to a family of small inflammatory proteins, characterized by four conserved cysteine residues and is localized on human chromosome 17.1,4,5 MCP-3 is designated a C-C or intercrine ß cytokine.13 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.6,7 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.7
It has been suggested that MCP-3 binds multiple C-C receptors such as MCP-1 on monocytes and basophils,8,9 MIP-1a on neutrophils, basophils, and eosinophils9 and RANTES on basophils and eosinophils(9,10). Evidence suggests that MCP-3 does indeed use mutiple receptors (MCP-1, RANTES, and MIP-1a) and binds with MIP-1ß receptor as well as other unique binding sites.6,7 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.6,11 Also, MCP-3 promotes exocytosis of eosinophil granule proteins and stimulates histamine release from human basophils.4,12 In vivo, MCP-3 induces the selective infiltration of monocytes on intradermal injection in rabbits.1 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.10 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.
This MCP-3 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 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 color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 2nm.
In order to measure the concentration of MCP-3 in the samples, this kit includes two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant testing.) According to the 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.
Storage and Stabilty:Store components at -20°C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
