Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins (1 - 3). They are secreted as zymogens (Pro-MMPs) that are activated by a variety of proteinases or by reaction with organic mercurials. They are inhibited by specific tissue inhibitors of metalloproteinases (TIMPs) and by 2-macroglobulin (3 - 6). The regulation of MMP activity is important in tissue remodeling, inflammation, tumor growth and metastasis (3, 7 - 9).
Human MMP-9 (also known as gelatinase B) is secreted as a 92 kDa zymogen (2, 3). Cleavage of Pro-MMP-9 at or near residue 87 results in the active enzyme with a mass of approximately 82 kDa (1). MMP-9 has three fibronectin type II domains, a hemopexin-like domain and a proline-rich type V collagen-homologous domain (1 - 3). Pro-MMP-9 can be activated by MMP-3 (5) or by certain bacterial proteinases (10). MMP-9 is inhibited by ?2-macroglobulin or by TIMP-1 (3 - 6), which binds to Pro-MMP-9 as well as to active MMP-9 (3). In vitro treatment of Pro-MMP-9 with 4-aminophenylmercuric acid (APMA) produces not only the 82 kDa active enzyme but also a C-terminal truncated form of approximately 65 kDa with the activity comparable to that of the 82 kDa form (11).
Pro-MMP-9 is secreted by monocytes, macrophages, neutrophils, keratinocytes, fibroblasts, osteoclasts, chondrocytes, skeletal muscle satellite cells, endothelial cells, and various tumor cells(1, 7- 21). Pro-MMP-9 expression is upregulated by TGF-?1, IL-1?, TGF-?, PDGF-AB, TNF-?, and IL-1? (7, 15, 17). Substrates for MMP-9 include denatured collagen type I (gelatin), native collagens type IV, V, VII, X and XI, fibrinogen, vitronectin, IL-1?, and entactin, a molecule that bridges laminin and type IV collagen (3, 4, 6, 13, 21 - 23).
For the quantitative determination of human active (82 kDa) and Pro- (92 kDa) Matrix Metalloproteinase 9 (MMP-9) concentrations in cell culture supernates, serum, and plasma.
Principle of the assay: This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for MMP-9 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any MMP-9 present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for MMP-9 is added to the wells and binds to the combination of capture antibody-MMP-9 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 colored product is formed in proportion to the amount of MMP-9 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 MMP-9 standard dilutions and MMP-9 sample concentration determined.
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