Patent EA-003901-B1 (EA)
Title: Method of detecting of mammalian heparanase activity and a kit for realization of method
Inventor: FREEMAN CRAIG GEOFFREY
IPC: C12Q1/34
Filed: 1997-07-18
Abstract: 1. A method for the detection of mammalian heparanase activity in a sample, which comprises the steps of: a) contacting the sample to be tested with a heparanase substrate for a time and under conditions sufficient for heparanase in the sample to degrade the heparanase substrate; b) separating degradation products from undegraded or partially degraded heparanase substrate by binding the undegraded or partially degraded heparanase substrate with a heparan sulphate-binding protein which is a histidine-rich glycoprotein; and c) detecting separated degradation products to indicate heparanase activity in the sample. 2. A method according to claim 1, wherein said sample is mammalian sera, selected from human and non-human mammalian sera. 3. A method according to claim 1, wherein said sample is cell and tissue homogenates or extracts. 4. A method according to any of claims 1-3, wherein said heparanase substrate is a labelled substrate. 5. A method according to claim 4, wherein said heparanase substrate is a radiolabelled substrate. 6. A method according to any of claims 1-5, wherein said heparanase substrate is a heparan sulphate. 7. A method according to claim 6, wherein said heparan sulphate is porcine mucosal heparan sulphate or bovine kidney heparan sulphate. 8. A method according to claim 6, wherein said heparan sulphate is <3>H porcine mucosal heparan sulphate or <3>H bovine kidney heparan sulphate. 9. A method according to any of claims 1-8, wherein said heparan sulphate-binding protein is glycoprotein with high content of histidine. 10. A method according to claim 9, wherein said glycoprotein with high content of histidine is chicken glycoprotein with high content of histidine. 11. A method according to any of claims 1-10 wherein said heparan sulphate-binding protein is immobilized. 12. A method according to claim 11 wherein said heparan sulphate-binding protein is immobilized by binding to agarose beads. 13. A method according to any of claims 1-12, wherein said sample is contacted with said heparanase substrate at a temperature in tie range of 35 degree C. to about 40 degree C for a period of from 2 to 48 hours at a pH in the range from 4.2 to 7.5, preferably approximately at 5.1. 14. A kit for the detection of mammalian heparanase activity in a sample according to any of claims 1-13, which comprises in compartmentalized state: a) heparanase substrate; b) a heparan sulphate-binding protein; and c) optionally, the instructions for realizing of method according to invention by any of claims 1-13. 15. The kit according to claim 14, wherein said heparanase substrate is a labeled substrate. 16. The kit according to claim 15, wherein said heparanase substrate is a radiolabelled substrate. 17. The kit according to any of claims 14-16, wherein said heparanase substrate is a heparan sulphate. 18. The kit according to claim 17, wherein said heparan sulphate is porcine mucosal heparan sulphate or bovine kidney heparan sulphate. 19. The method according to claim 18, wherein said heparan sulphate is <3>H porcine mucosal heparan sulphate or <3>H bovine kidney heparan sulphate. 20. A method according to any of claims 14-19, wherein said heparan sulphate-binding protein is glycoprotein with high content of histidine. 21. A method according to claim 20, wherein said glycoprotein with high content of histidine is chicken glycoprotein with high content of histidine. 22. The method according to any of claims 14-21, wherein said chicken histidine-rich glycoprotein is immobilized. 23. The kit according to claim 22, wherein said chicken histidine-rich glycoprotein is immobilized by binding to agarose beads.