Claims for Patent: 6,132,976

Summary for Patent: 6,132,976

Title:	Immunoassays for the measurement of collagen denaturation and cleavage in cartilage
Abstract:	The present invention relates to a method for detecting cartilage degradation in a biological sample by identifying the presence of unwound type II collagen in the biological sample, said method comprising: contacting the biological sample with a monoclonal antibody which does not bind to native helical collagen but which does bind to an epitope on unwound type II collagen chains or fragments thereof, wherein said epitope has the following sequence (SEQ ID NO: 4): detecting the presence of the bound monoclonal antibody. The present invention also relates to a method for detecting collagenase induced cartilage degradation in a biological sample by identifying the presence of an epitope on type II collagen which only becomes detectable following cleavage of said collagen by collagenase, said method comprising: contacting the biological sample with a monoclonal antibody which binds to said epitope on type II collagen chains or fragments thereof containing said epitope; and detecting the presence of said monoclonal antibody bound to the type II collagen and fragments.
Inventor(s):	Poole; Anthony Robin (Baie d\'Urfe, CA), Hollander; Anthony Peter (Greystones, GB), Billinghurst; R. Clark (Fort Collins, CO)
Assignee:	Shriners Hospitals for Children (Tampa, FL)
Application Number:	09/010,999
Patent Claims:	1. A method for detecting cartilage degradation in a biological sample by identifying the presence of unwound type II collagen in the biological sample, said method comprising: contacting the biological sample with a monoclonal antibody which does not bind to native helical collagen but which does bind to an epitope on unwound type II collagen chains or fragments thereof, wherein said epitope has the following sequence (SEQ ID NO: 4): detecting the presence of the bound monoclonal antibody. 2. The method according to claim 1 wherein said monoclonal antibody is produced by the cell line ATCC HB 11202. 3. The method according to claim 1, wherein said biological sample is a biological fluid. 4. A method for the determination of cartilage degradation in a biological sample by quantifying the amount of unwound type II collagen in the biological sample, said method comprising: (a) contacting the biological sample with a first enzyme which cleaves unwound type II collagen chains in the biological sample into collagen fragments without cleaving an antibody-reactive epitope on said unwound type II collagen chains; (b) extracting the collagen fragments produced by said enzyme from said biological sample to produce an enzyme extract and remaining biological sample; (c) treating said remaining biological sample with a second enzyme to solubilize and unwind remaining native collagen contained therein without degrading the epitope to produce a solubilized biological sample; (d) separately contacting said extract and said solubilized biological sample with a monoclonal antibody which binds an epitope on unwound type II collagen chains or fragments thereof containing said epitope, wherein said monoclonal antibody does not bind to native helical collagen; (e) determining the amount of unwound type II collagen and fragments thereof in said extract and said solubilized biological sample by separately determining the amount of bound monoclonal antibody; and (f) quantifying the amount of type II collagen that is unwound in said sample by comparing the amount of unwound type II collagen in said extract to the amount of type II collagen in said solubilized biological sample. 5. The method of claim 4, further comprising the step of determining the amount of wound collagen extracted by the first enzyme, said method comprising, (a) removing an aliquot of said enzyme extract; (b) treating said aliquot with said second enzyme; (c) contacting said aliquot with a monoclonal antibody which binds an epitope on unwound type II collagen chains or fragments thereof containing said epitope, wherein said monoclonal antibody does not bind to native helical collagen; and (d) determining the amount of unwound type II collagen and fragments thereof in said aliquot by determing the amount of bound monoclonal antibody. 6. The method according to claim 4, wherein said first enzyme is chymotrypsin. 7. The method according to claim 4, wherein said second enzyme is proteinase K. 8. A method according to claim 4, wherein said monoclonal antibody is produced by the cell line ATCC HB 11202. 9. A method for measuring total type II collagen content in a biological sample, said method comprising: (a) treating the biological sample to solubilize and unwind collagen contained therein, without degrading an epitope that binds to a monoclonal antibody to produce a solubilized biological sample containing unwound collagen; (b) measuring the amount of unwound collagen present in said solubilized biological sample by contacting said solubilized biological sample with the monoclonal antibody which binds an epitope on unwound type II collagen chains or fragments thereof containing said epitope, wherein said monoclonal antibody does not bind to native helical collagen; and (c) determining the amount of said unwound collagen and fragments thereof bound to said monoclonal antibody. 10. The method according to claim 4, wherein said biological sample is osteoarthritic cartilage. 11. The method according to claim 4, wherein said biological sample is intervertebral discs. 12. A kit for the measurement of cartilage degradation products in a biological sample, said kit comprising: (a) a monoclonal antibody which does not bind to native helical type II collagen but which does bind to an epitope on unwound type II collagen chains or fragments thereof containing said epitope; (b) a solid support for binding proteins; (c) a labelled antibody to measure the binding of monoclonal antibody to said unwound collagen; and (d) an enzyme which solubilizes and unwinds native helical type II collagen without degrading the antibody reactive epitope on unwound type II collagen. 13. The kit according to claim 12, wherein said enzyme is proteinase K. 14. The kit according to claim 12, further comprising: a second enzyme which selectively cleaves unwound type II collagen chains in said biological sample without cleaving the antibody-reactive epitope on said unwound type II collagen chains. 15. The kit according to claim 14, wherein said second enzyme is chymotrypsin. 16. The kit according to claim 12, wherein said monoclonal antibody is produced by the hybridoma cell line ATCC HB 11202. 17. A monoclonal antibody which binds to an epitope on unwound type II collagen chains or fragments thereof containing said epitope, wherein said epitope has the following sequence (SEQ ID NO:4): 18. The monoclonal antibody produced by the hybridoma cell line ATCC HB 11202. 19. A cell line producing a monoclonal antibody which does not bind to native helical collagen type II but which does bind to an epitope on unwound collagen type II chains or fragments thereof containing said epitope, said epitope having the following sequence (SEQ ID NO:4): 20. 20. A cell line according to claim 19, which has all the identifying characteristics of ATCC HB 11202. 21. The method according to claim 1, wherein said monoclonal antibody detects approximately the same amount of collagen as that detected by measuring hydroxyproline content. 22. The method according to claim 4, wherein said monoclonal antibody detects approximately the same amount of collagen as that detected by measuring hydroxyproline content. 23. The kit according to claim 12, wherein said monoclonal antibody detects approximately the same amount of collagen as that detected by measuring hydroxyproline content. 24. The method according to claim 4, wherein the biological sample is a biological fluid. 25. The method according to claim 4, wherein said biological sample is a biological tissue. 26. A method for detecting collagenase induced cartilage degradation in a biological sample by identifying the presence of an epitope on type II collagen which only becomes detectable following cleavage of said collagen by collagenase, said method comprising: contacting the biological sample with a monoclonal antibody which binds to said epitope on type II collagen chains or fragments thereof containing said epitope; and detecting the presence of said monoclonal antibody bound to the type II collagen and fragments. 27. The method of claim 26, wherein said epitope has the following sequence (SEQ ID NO: 7): 28. The method of claim 26, wherein said biological sample is a biological fluid. 29. The method of claim 28, wherein said biological fluid is selected from the group consisting of synovial fluid, serum and urine. 30. The method of claim 26, wherein said monoclonal antibody is produced by the cell line ATCC HB-12429. 31. A kit for detecting collagenase induced cartilage degradation in a biological sample, said kit comprising: (a) a monoclonal antibody which binds to an epitope on type II collagen, or fragments thereof, which only becomes detectable following cleavage of said collagen by collagenase; (b) a solid support for binding proteins; and (c) a labelled antibody to measure the binding of monoclonal antibody to said unwound collagen. 32. A kit according to claim 31, wherein said monoclonal antibody is produced by the hybridoma cell line ATCC HB-12429. 33. A monoclonal antibody which binds to an epitope on type II collagen, or fragments thereof, which epitope only becomes detectable following cleavage of said collagen by collagenase. 34. The monoclonal antibody of claim 33, produced by the hybridoma cell line ATCC HB-12429. 35. A cell line producing a monoclonal antibody which binds to an epitope on type II collagen, or fragments thereof, which epitope only becomes detectable following cleavage of said collagen by collagenase. 36. The method according to claim 26, wherein said biological sample is articular cartilage. 37. The method according to claim 36, wherein said articular cartilage is in vivo.

Details for Patent 6,132,976

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2012-12-04