MMP-13 ELISA Assay Kit

$775.00

The MMP-13 ELISA Assay Kit provides a highly sensitive and specific quantitative determination of activated human MMP-13 in serum, synovial fluid, and cell culture supernatants. The calibration curve covers the range from 32 pg/ml to 2000 pg/ml. The sensitivity of the assay is 7 pg/ml.TheMMP-13 ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

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MMP-13 ELISA Assay Kit

The MMP-13 ELISA Assay Kit is For Research Use Only

Size: 1 x 96 wells
Sensitivity: 7 pg/mL
Dynamic Range: 32 pg/ml to 2000 pg/ml
Incubation Time: 4.25 hours
Sample Type: Serum, Synovial fluid, cell culture supernatants
Sample Size: 10 µL
Controls Included


Assay Background

Matrix Metalloproteinases (MMPs) comprise a family of secreted and membrane-bound endopeptidases which hydrolyse extracellular matrix proteins. Based on their preferred substrates and on structural features MMPs can be divided into collagenases, gelatinases, stromelysins and membrane-type matrix metalloproteinases. Their activity is tightly regulated by inhibitors.

Collagenase 3 (MMP-13) is a secreted 452 residue protein which is released from cells as an inactive zymogen and activated extracellularly by removal of its propeptide. Cleavage of the 84 residue propeptide can be catalysed by other MMPs such as MMP-2 and MMP-14, or by other proteases like plasmin.

Under normal conditions, MMP-13 is expressed during embryogenesis (fetal bone development) and is present only at very low levels in adult tissue. However, this enzyme is reported to be involved in the development and metastasis of breast and lung carcinomas, chondrosarcomas and osteosarcomas, head and neck carcinomas and some forms of skin cancer. Additionally, this enzyme plays an important role in degenerative bone diseases including osteoarthritis and rheumatoid arthritis.

Our MMP-13 test kit enables the user to measure the concentration of this putative marker enzyme in body fluids such as serum and synovial fluid with high sensitivity. The ELISA MMP-13 is specific for the activated enzyme.


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Additional Information

Assay Principle


This Eagle Biosciences MMP-13 ELISA Assay Kit is a quantitative assay is based on a two site sandwich format. A highly specific monoclonal antibody for the activated form of MMP-13 is immobilised on the plate. MMP-13 will be bound to the wells, other components of the sample are removed by aspiration and washing of the plate. The analyte is detected in two steps using a secondary biotin-labeled antibody and a highly polymerised streptavidin-peroxidase conjugate. Any excess is removed by aspiration and washing after each detection step. The amount of peroxidase bound to each well is determined by the addition of TMB Substrate. The reaction is stopped by adding the Stop Solution, and the resultant colour is read in a microtiter plate reader at 450 nm. The concentration of activated MMP-13 in a sample is determined by interpolation from the standard curve.

One kit contains reagents for 96 determinations, thus allowing the measurement of one standard curve and 40 samples in duplicate.

Assay Procedure


  1. Prepare reagents and standards as described in the sections above. For assessing serum samples, prepare the standard using the Serum Standard Diluent. Remind that it is necessary to equilibrate the reagents to room temperature before use.
  2. Prepare the unknown samples as described above by appropriate dilution with Assay Buffer.
  3. Prepare the Microtiter plate by inserting the required amount of wells into the frame. Note that you need 16 wells for the standard curve.
  4. Pipette 100 µl of the reconstituted standards in duplicate in the wells using a clean pipette tip for each standard. Assay Buffer serves as zero blank.
  5. Pipette 100 µl of the prepared unknown samples in duplicate into the wells.
  6. Seal the plate with the provided foil and incubate on a shaker at room temperature for exactly 120 minutes.
  7. Aspirate and wash the wells 4 times with Wash Buffer. Take care that all wells are completely filled and emptied at each wash.
  8. Blot the plate on paper towels to remove residual fluid from the plate.
  9. Add 100 µl of diluted Biotinylated Antibody into each well.
  10. Seal the plate and incubate on a shaker at room temperature for exactly 90 minutes.
  11. Aspirate and wash the wells 4 times with Wash Buffer. Take care that all wells are completely filled and emptied at each wash.
  12. Blot the plate on paper towels to remove residual fluid from the plate.
  13. Add 100 µl of diluted Conjugate Solution into each well.
  14. Seal the plate with the provided foil and incubate on a shaker at room temperature for exactly 30 minutes.
  15. Aspirate and wash the wells 6 times with Wash Buffer. Take care that all wells are completely filled and emptied at each wash.
  16. Blot the plate on paper towels to remove residual fluid from the plate.
  17. Add 100 µl of Substrate Solution to each well.
  18. Seal the plate with foil provided and incubate in the dark at room temperature for exactly 15 minutes.
  19. Stop the reaction by adding 100 µl of Stop Solution to each well.
  20. Read the plate at 450 nm (540, 570 or 620 nm reference filter) within 30 minutes. Reading of the plate without reference may yield higher absorbances and thus may be less accurate.

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References

  • Nagase, H. and Woessner, F. Jr. (1999) J. Biol. Chem. 274, 21491-21494.
  • Brew, K. et al. (2000) Biochim Biophys. Acta 1477, 267.283.
  • Knauper, V. et al. (1996) J. Biol. Chem. 271, 17124-17131.
  • Pendas, AM. et al (2000) Clin. Chim. Acta 291, 137-155.
  • Bluteau, G. et al. (2001) Biochim. Biophys. Acta 1526, 147-158
  • Westhoff, CS. et al. (1999) Arthritis Rheum. 42, 1517-1527.

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