Description

Heparan sulfate is a polysaccharide which is ubiquitously present in mammals as cell membrane component.
Heparan sulfate functions as anchor for lipoprotein lipase on the surface of capillary endothelial cells and also participates in mutual recognition, prevention of cell infiltration, intercellular adhesion and so on.
Heparanase of mammal and heparitinase of bacteria is known as heparan sulfate degrading enzyme.
The activity of mammalian heparanase is found in liver, spleen, skin, placenta, and platelet of mammals, and also found in tumor tissues such as melanoma, lymphoma, sarcoma, fibrosarcoma and colon cancer. It has been reported that this enzyme activity in high-invasive cancer cells was considerably higher than that in low-invasive cells^{1) 2)}, and the correlation of heparan sulfate degrading enzyme activity with malignancy of cancer cells is attracting interest.^{1) - 4)}
Takara's Heparan Degrading Enzyme Assay Kit is designed to measure the activity of heparan degrading enzyme in cultured cells, tissues or serum and to allow screening of inhibitors for heparan sulfate degrading enzyme, without using RI, utilizing the property that heparin-like molecules and bFGF (basic fibroblast growth factor) combine each other. One of the features of this kit is that it utilizes domain-oriented capture (DOC) method which employs uniquely prepared CBD-FGF. CBD-FGF is a fusion protein of cell-binding domain of human fibronectin and human fibroblast growth factor (U.S. Patent 532701 is owned by Takara). This CBD-FGF is bound on a microtiterplate supplied in this kit, with captured by anti-fibronectin antibody having epitope in CBD region.
In addition, biotinylated heparan sulfate is used as a substrate of the enzyme in this kit. Since only undegraded substrate can combine to CBD-FGF, the detection of the remaining undegraded substrate by avidin-peroxidase realizes highsensitive non-RI measurement.
The duration of a series of assay is around 100 minutes, including 45 min of enzyme degradation reaction. Tiny enzyme assay can be detected by extending the time of enzyme degradation reaction. As the correlation of heparan sulfate degrading enzyme activity with malignancy of cancer cells is attracting interest, this kit can also be utilized for screening of its inhibitor.



Figure 1 Principle of measurement

Storage

4°C

Principle

• Reaction of biotinylated heparan sulfate and sample
• Transfer of the reactant into well of CBD-FGF immobilized 96-well plate
• Reaction of remaining undegraded biotinylated heparan sulfate bound to CBD-FGF with avidin POD conjugate
• Color development by POD substrate

Features

• No radioactive isotopes are required.
• 96-well plate allows simultaneous assay of many samples.
• Any tissue, cell or serum regardless of mammalian species can be used as sample.
• Applicable for research of interaction between bFGF and heparin-like substances or other ligands.

Kit components

1. CBD-FGF immobilized microtiterplate (96 wells: 8 well × 12 strips)	1 plate
2. Biotinylated heparan sulfate in reaction buffer (labeling substrate)	1 vial (for 5.5 ml)
3. Reaction buffer for dilution	1 vial (11 ml)
4. Extraction buffer	1 vial (11 ml)
5. Standard	1 vial (for 250 µl)
6. Avidin POD conjugate	1 vial (for 11 ml)
7. POD substrate	1 vial (12 ml)

Note

This kit does not contain stop solution (1N sulfric acid). It should be prepared before starting protocol.

Detection sensitivity

more than 0.1 U/ml

Assay range

Prepare calibration curve at each measurement. (Typical example of calibration curve is shown in Figure 2.)


Figure 2 Typical example of calibration curve