Mouse MCP-1 ELISA Kit

Principle of the Assay

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for MCP-1 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any MCP-1 present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for MCP-1 is added to the wells and binds to the combination of capture antibody-MCP-1 in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A coloured product is formed in proportion to the amount of MCP-1 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven MCP-1 standard dilutions and MCP-1 sample concentration determined.

For Use with serum, plasma and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

The mouse JE gene was originally described as a platelet-derived growth factor-inducible gene in mouse fibroblasts. The protein encoded by mouse JE was found to belong to the large CC chemokine family of inflammatory and immunoregulatory cytokines. Among CC chemokine family members, JE is functionally and structurally most closely related to the MCP/eotaxin subfamily of proteins. Within this MCP/eotaxin subfamily, five human (MCP-1, 2, 3, 4 and eotaxin) and four mouse (JE/MCP-1, MARC/MCP-3, MCP-5, and eotaxin) proteins have been identified. At the amino acid (aa) sequence level, mature human MCP-1 shows 55%, 59%, and 66% identity with the analogous regions of mouse JE, MARC, and MCP-5, respectively. Although JE has been presumed to be the mouse homolog of human MCP-1, the more recently isolated mouse MCP-5 is actually more homologous and may be considered to be a second human MCP-1 homolog.

Mouse MCP-1 cDNA encodes a 148 aa residue precursor protein with a predicted 23 aa residue signal peptide that is cleaved to generate a putative mature protein of 125 aa residues. Compared to mature human MCP-1, mouse MCP-1 has a 49 aa residue carboxy-terminal extension that is rich in serine and threonine residues. Recombinant MCP-1 expressed in CHO cells as well as natural MCP-1 purified from mouse astrocytes and a mouse thymic epithelial cell line, were shown to be approximately 30 kDa glycoproteins with multiple O-linked oligosaccharide chains added to the 49 aa residue C-terminal domain. Nonetheless, the natural form of MCP-1 produced by virus-stimulated mouse L929 fibroblasts occurs as a non-glycosylated 7-8 kDa protein that lacks the C-terminal domain. The carboxy-terminal domain has been found not to be required for mouse MCP-1 activity. Besides fibroblasts, astrocytes and epithelial cells, mouse MCP-1 has been found to be expressed in macrophages, mast cells, endothelial cells, osteoblasts and ameloblasts. The expression of mouse MCP-1 is induced after stimulation with inflammatory stimuli including viruses, LPS, and cytokines such as TNF-α, IL-1, IFN-γ, and PDGF.

Mouse MCP-1 is a potent chemoattractant for monocytes/macrophages and lymphocytes. It has also been shown to be involved in the regulation of Th1/Th2 lymphocyte differentiation, enhancing Th2 development by increasing IL-4 production and inhibiting IL-12 production. The activities of mouse MCP-1 have been shown to be mediated by the mouse CC chemokine receptor CCR2, a G protein-coupled, seven transmembrane domain receptors. Mouse CCR2 cDNA encodes a 373 aa residue protein that shows the highest (80%) overall identity at the aa sequence level with the human MCP-1 receptor, CCR2B. The gene for mouse CCR2 has been mapped to mouse chromosome 9, in close proximity with mouse CCR1 and CCR3. High levels of mouse CCR2 expression have been detected in monocytes/macrophages.

GENE ID 20296
SWISS PROT P10148
SYNONYMS JE; HC11; MCAF; MCP1; MCP-1; Scya2; Sigje; SMC-CF; AI323594

Materials Supplied

Kit Components 96 Wells Quantity/Size
Aluminium pouches with a Microwell Plate coated with monoclonal antibody to mouse MCP-1(8﹡12) 1 plate
Mouse MCP-1 Standard lyophilized, 1000 pg/ml upon reconstitution 2 vials
Concentrated Biotin-Conjugate anti-mouse MCP-1 monoclonal antibody 2 vials
Streptavidin-HRP solution 2 vials
Standard /sample Diluent 1 bottle
Biotin-Conjugate antibody Diluent 1 bottle
Streptavidin-HRP Diluent 1 bottle
Wash Buffer Concentrate 20x (PBS with 1% Tween-20) 1 bottle
Substrate Solution 1 vial
Stop Solution 1 vial
Adhesive Films 4 pieces
Product data sheet 1 copy

Storage

Storage Store at 2 - 8°C

Performance Characteristics

REPEATABILITY The coefficient of variation of both intra-assay and inter-assay were less than 10%.
SENSITIVITY The minimum detectable dose was 7pg/mL.
ASSAY RANGE 15.6-1000 pg/mL
SPECIFICITY This assay recognizes both natural and recombinant mouse MCP-1. The factors listed below were prepared at 50ng/ml in Standard /sample Diluent and assayed for cross-reactivity and no significant cross-reactivity or interference was observed.
Factors assayed for cross-reactivity
Recombinant human IL-1α, IL-1β, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, GM-CSF, M-CSF, SCF, TNF-α, IFN-r, VEGF
Recombinant mouse IL-2 CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL7/MCP-3, CCL7/MCP-3, CCL11/Eotaxin, CXCL1/GROα, CXCL1/GROα, CXCL2/GROβ, CXCL8/IL-8