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 b 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-1b 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.
Sensitivity: <30pg/ml
Range: 30-1600pg/ml
Kit Components:
1. MCP-3 Microtiter Plate, 1x96 wells
2. MCP-3 Conjugate, 1x15ml
3. MCP-3 Standard, 2x1 vials
4. Calibrator Diluent I, 1x22ml
5. Calibrator Diluent II, 1x 22ml
6. Sample Diluent, 1x12ml
7. Wash Buffer (20X), 1x60ml
8. Substrate A, 1x11ml
9. Substrate B, 1x11ml
10. Stop Solution, 1x14ml, 2N Suphuric Acid (H2SO4).
Storage and Stabilty:
Store components at -20 degrees C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.�
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