Mini Sample Rat TIMP-1 (Tissue Inhibitors of Metalloproteinase 1) ELISA Kit (E-MSEL-R0001)

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to TIMP-1 . Standards or samples are added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for TIMP-1 and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain TIMP-1 , biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 50 nm. The OD value is proportional to the concentration of TIMP-1 . You can calculate the concentration of TIMP-1 in the samples by comparing the OD of the samples to the standard curve.

Matrix metalloproteinases (MMPs), also called matrixins, constitute a family of zinc and calciumdependent endopeptidases that function in the breakdown of extracellular matrix and in theprocessing of a variety of biological molecules. They play an important role in many normalphysiological processes such as embryonic development, morphogenesis, reproduction andtissue remodeling (1). They also participate in many pathological processes such as arthritis,cancer and cardiovascular disease (2). While the amounts of newly synthesized MMPs areregulated mainly at the levels of transcription, the proteolytic activities of existing MMPs arecontrolled through both the activation of proenzymes or zymogens and the inhibition ofactive enzymes by endogenous inhibitors such as α2-macroglobulins and tissue inhibitors ofmetalloproteinases (TIMPs).The mammalian TIMP family includes four members that share structural similarity (3). All TIMPproteins have 12 conserved cysteine residues that form six intrachain disulfide bonds, resultingin an extremely stable protein with six loops. The TIMP protein has two structurally andfunctionally distinct domains: the N-terminal domain consisting of loops 1-3 that areresponsible for tight, but non-covalent binding to the active MMPs in a 1:1 stoichiometry; andthe C-terminal domain consisting of loops 4-6 that enhances the enzyme-inhibitor interactions.In the case of TIMP-1, the C-terminal domain has also been shown to bind the hemopexin-likedomain of pro-MMP-9. TIMP-1 stimulates erythropoiesis, inhibits angiogenesis and is an antiapoptotic agent for B cells. These TIMP-1 functions may be independent of MMP inhibition(4-6).Rat TIMP-1 is a 28-35 kDa secreted glycoprotein (6, 7). The protein is synthesized as a 217 aminoacid (aa) precursor that contains a 23 aa signal peptide and a 194 aa mature form (8). RatTIMP-1 shares 85%, 70%, 68%, and 66% aa sequence identity with its mouse, human, porcine,and canine counterparts, respectively (9-13). Among the three known rat TIMPs, TIMP-1 shares40% and 35% aa identity with TIMP-2 and TIMP-3, respectively (14, 15). TIMP-1 is widelyexpressed in many cells such as fibroblasts, osteoblasts, endothelial cells, granulosa cells,dendritic cells, vascular smooth muscle cells, adipocytes, and monocytes (6, 16-19).The Quantikine® Rat TIMP-1 Immunoassay is a 4.5 hour solid-phase ELISA designed to measurerat TIMP-1 levels in cell culture supernates, serum, and plasma. It contains NS0-expressedrecombinant rat TIMP-1 and antibodies raised against the recombinant protein. Natural ratTIMP-1 showed dose-response curves that were parallel to the standard curves obtained usingthe Quantikine® kit standards. These results indicate that this kit can be used to determine .relative levels of natural rat TIMP-1.