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 coloured 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.
For Use with serum, plasma and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. 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. The regulation of MMP activity is important in tissue remodeling, inflammation, tumor growth and metastasis.
Human MMP-9 (also known as gelatinase B) is secreted as a 92 kDa zymogen. Cleavage of Pro-MMP-9 at or near residue 87 results in the active enzyme with a mass of approximately 82 kDa. MMP-9 has three fibronectin type II domains, a hemopexin-like domain and a proline-rich type V collagen-homologous domain. Pro-MMP-9 can be activated by MMP-3 or by certain bacterial proteinases. MMP-9 is inhibited by α2-macroglobulin or by TIMP-1, which binds to Pro-MMP-9 as well as to active MMP-9. 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.
Pro-MMP-9 is secreted by monocytes, macrophages, neutrophils, keratinocytes, fibroblasts, osteoclasts, chondrocytes, skeletal muscle satellite cells, endothelial cells, and various tumor cells. Pro-MMP-9 expression is upregulated by TGF-β1, IL-1β, TGF-α, PDGF-AB, TNF-α, and IL-1α. Substrates for MMP-9 include denatured collagen type I (gelatin), native collagens type IV, V, VII, X and XI, fibrinogen, vitronectin, IL-1β, and entactin, a molecule that bridges laminin and type IV collagen.
|SYNONYMS||GELB; CLG4B; MMP-9; MANDP2|
|Kit Components||96 Wells Quantity/Size|
|Aluminium pouches with a Microwell Plate coated with monoclonal antibody to human MMP-9 (8﹡12)||1 plate|
|Human MMP-9 Standard lyophilized, 4000 pg/ml upon reconstitution||2 vials|
|Concentrated Biotin-Conjugate anti-human MMP-9 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||Store at 2 - 8°C|
|REPEATABILITY||The coefficient of variation of both intra-assay and inter-assay were less than 10%.|
|SENSITIVITY||The minimum detectable dose was 30pg/mL.|
|ASSAY RANGE||62.50 - 4000 pg/mL|
|SPECIFICITY||This assay recognizes both natural and recombinant human MMP-9. The factors listed below were prepared at 200ng/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||MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-10, MMP-12|
|Recombinant mouse||MMP-2, MMP-3, MMP-9, TIMP-1|
Data Analysis Assistance
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