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Heparan degrading enzyme detection

Heparan sulfate is a complex and ubiquitous polysaccharide that is a component of mammalian cell membranes. It has been identified in mammalian liver, spleen, skin, placenta, and platelets as well as in cancerous tissues such as melanomas, lymphomas, sarcomas, fibrosarcomas, and colon cancer. Heparan sulfate has additionally been found to play an important defensive role in tumor cell invasion. The activity of heparan degrading enzyme (heparanase), an enzyme that cleaves heparan sulfate, is reported to be considerably higher in invasive cancer than normal cells. Thus, the correlation of heparan sulfate degrading enzyme activity with cancer malignancy is of much research interest.

This Heparan Degrading Enzyme Assay Kit provides a 96-well non-radioactive assay format for the measurement of heparan sulfate degradation in cultured cells, tissues, or serum, as well as for the screening of heparan sulfate degradation enzyme inhibitors. This kit is based on the ability of heparin-like molecules to bind bFGF (basic fibroblast growth factor).

When heparan sulfate is degraded, it loses its ability to bind bFGF. Thus, the enzymatic activity of a sample can be quantitated by comparing the amount of bFGF-bound undegraded heparan sulfate with control heparanase-free total-bound heparan sulfate. A unique feature of the Heparan Degrading Enzyme ELISA is the use of CBD-FGF, a fusion protein composed of the human fibronectin cell-binding domain and human fibroblast growth factor. CBD-FGF is immobilized on the surface of microtiter ELISA plates by an anti-fibronectin antibody that binds the CBD epitope. Termed DOC (Domain Oriented Capture), this solid phase attachment method enables the formation of a natural 3D bFGF structure for enhanced accessibility to and binding of heparan sulfate. Detection occurs via a colorimetric assay method that uses biotinylated heparan sulfate as the substrate.

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Cat. #	Product	Size	Price	License	Quantity	Details
MK412	Heparan Degrading Enzyme Assay Kit	96 Assays	USD $685.00
Takara's Heparan Degrading Enzyme Assay Kit provides a 96-well non-radioactive assay for the measurement of heparan degrading enzyme activity in cultured cells, tissues, or serum as well as screening of heparan sulfate degrading enzyme inhibitors. This kit is based upon the property of heparin-like molecules to bind bFGF (basic fibroblast growth factor). When heparan sulfate is degraded by heparan sulfate degrading enzyme, it loses its ability to bind to bFGF. Thus, the enzymic activity of a sample can be quantitated by comparing the amount of bFGF-bound undegraded heparan sulfate of the sample to total-bound heparan sulfate of a heparanase-free control. A unique feature of this kit is the use of CBD-FGF, a fusion protein composed of human fibronectin cell-binding domain and human fibroblast growth factor. CBD-FGF is immobilized on the surface of Takara's microtiter plate by an anti-fibronectin antibody containing an epitope in the CBD region. Termed DOC (Domain Oriented Capture), this solid phase attachment method allows a natural 3-dimensional bFGF structure for enhanced accessibility and binding to heparan sulfate. Detection is by a colorimetric method using biotinylated heparan sulfate as the substrate. Notice to purchaser Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Documents Components Resources

Overview

Safe: No radioactive isotopes required
96-well format allows simultaneous assay of many samples
Any tissue, cell, or serum, regardless of mammalian species, can be used
Enables interaction studies of bFGF to heparin-like substances and other ligands
Unique solid phase attachment method creates a natural 3D bFGF structure for enhanced accessibility and binding to heparan sulfate

More Information

Applications

Quantitative measurement of heparan sulfate degradation in mammals
Screening of heparan sulfate degradation enzyme inhibitors
Heparan degrading enzyme ELISA

Components

Immobilized CBD-FGF 96-well microtiter plate
Biotinylated heparan sulfate in reaction buffer (5.5 ml)
Reaction buffer (for dilution; 11 ml)
Extraction buffer (11 ml)
Standard (lyophilized; 250 ml)
Avidin-peroxidase conjugate (lyophilized; 11 ml)
Peroxidase substrate (12 ml)

Storage

2–8°C

Performance characteristics

Range of assay: 0.1–4.2 U/ml
Assay sensitivity: 0.1 U/ml
Assay duration: 2–3 hours
Specificity: All animals
Test specimen type: Tissue, cultured cells, serum

Product citations

Foxall, C., Holme, K. R., Liang, W. & Wei, Z. An enzyme-linked immunosorbent assay using biotinylated heparan sulfate to evaluate the interactions of heparin-like molecules and basic fibroblast growth factor. Anal. Biochem. 231, 366–73 (1995).

Freeman, C. & Parish, C. R. Human platelet heparanase: purification, characterization and catalytic activity. Biochem. J. 1341–50 (1998).

Hashi, H., Hatai, M., Kimizuka, F., Kato, I. & Yaoi, Y. Angiogenic activity of a fusion protein of the cell-binding domain of fibronectin and basic fibroblast growth factor. Cell Struct. Funct. 19, 37–47 (1994).

He, X. et al. Hypoxia increases heparanase-dependent tumor cell invasion, which can be inhibited by antiheparanase antibodies. Cancer Res. 64, 3928–33 (2004).

Katayama, M. et al. Isolation and characterization of two monoclonal antibodies that recognize remote epitopes on the cell-binding domain of human fibronectin. Exp. Cell Res. 185, 229–36 (1989).

Nakajima, M. et al. Suramin. A potent inhibitor of melanoma heparanase and invasion. J. Biol. Chem. 266, 9661–6 (1991).

Nakajima, M., Irimura, T., Di Ferrante, D., Di Ferrante, N. & Nicolson, G. L. Heparan sulfate degradation: relation to tumor invasive and metastatic properties of mouse B16 melanoma sublines. Science 220, 611–3 (1983).

Nakajima, M., Irimura, T., Di Ferrante, N. & Nicolson, G. L. Metastatic melanoma cell heparanase. Characterization of heparan sulfate degradation fragments produced by B16 melanoma endoglucuronidase. J. Biol. Chem. 259, 2283–90 (1984).

Nicolson, G. L. et al. Cancer cell heparanase activity associated with invasion and metastasis. Adv. Enzyme Regul. 38, 19–32 (1998).

Ricoveri, W. & Cappelletti, R. Heparan sulfate endoglycosidase and metastatic potential in murine fibrosarcoma and melanoma. Cancer Res. 46, 3855–61 (1986).

Addtional product information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.

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