MMP-9 ELISA Assay Kit

$390.00

The Eagle Biosciences Human Matrix Metalloproteinase 9 (MMP-9) ELISA Assay Kit (enzyme-linked immunoassay kit) is intended for the quantitative determination of human Matrix Metalloproteinase 9 (MMP-9) concentrations in cell culture supernates, serum, and plasma. The Eagle Biosciences Human Matrix Metalloproteinase 9 (MMP-9) ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

SKU: MM931-K01 Categories: , ,

MMP-9 ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: 31 pg/ml
Dynamic Range: 62.5 – 2000 pg/ml
Incubation Time: 3.5 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl

Product manufactured in the USA

Additional Information

Assay Principle

The Eagle Biosciences Human Matrix Metalloproteinase 9 (MMP-9) ELISA Assay Kit 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.

  1. Prepare all reagents and working standards as directed in the previous sections.
  2. Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
  3. Add 100 µl of Standard, control, or sample, per well. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37°C.
  4. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350 µl) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
  5. Add 100 µl of the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37°C.
  6. Repeat the aspiration/wash.
  7. Add 100 µl of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at 37°C . Avoid placing the plate in direct light.
  8. Repeat the aspiration/wash.
  9. Add 100 µl of Substrate Solution to each well. Incubate for 10-20 minutes at 37°C. Avoid placing the plate in direct light.
  10. Add 100 µl of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  11. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.(optionally 630nm as the reference wave length;610-650nm is acceptable) 

Assay Background

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 α2-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-β1, IL-1β, TGF-α, PDGF-AB, TNF-α, and IL-1α (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-1β, and entactin, a molecule that bridges laminin and type IV collagen (3, 4, 6, 13, 21 – 23).

Manual

Product Manual


Publications

References

1.    Boring, L.et al.( 1996). J Biol Chem.271:7551.
2.    Capsoni, F. et al.( 1989) J Immunol Methods.120:125.
3.    Hsu, S.M. et al. (1981). Am J Clin Pathol.75:734.
4.    Hsu, S.M. et al. (1981)J Histochem Cytochem. 29:577.
5.    Matsushima, K. et al. (1989)Cytokine.1:2.
6.    Peri, G. et al. (1994)J Immunol Methods. 174:249.
7.    Prussin, C. and Metcalfe, D.D. (1995) J Immunol Methods.188:117.
8.    Rollins, B.J. et al.( 1989) Mol Cell Biol. 9:4687.

Citations

SERIN, SO;OKUTURLAR, Y;Acta Medica Mediterranea, 31:1271.