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1. (WO1991008478) METHODS OF DETECTING COLLAGEN DEGRADATION $i(IN VIVO)
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The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of determining bone resorption, comprising the step of contacting a body fluid with at least one specific binding partner to a first peptide consisting essentially of the structure

Asp-Glu-K-Ser-Thr-Gly-Gly
Gl n-Tyr-Asp-Gly-K-Gly-Val -Gly
K

where K
K

I
K
is hydroxylysyl pyridinoline or lysyl pyridinoline, and Gin is glutamine or pyrrolidine carboxylic acid, and to a second peptide having the above formula except that the pyridinium ring of the cross-linking amino acid has been cleaved.

2. A method according to Claim 1, wherein said body fluid is urine, blood, serum, or synovial fluid.

3. A method according to Claim 1, comprising contacting said body fluid with a first specific binding partner to said first peptide and with a second specific binding partner to said second peptide.

4. A kit for determining bone resorption, comprising a specific binding partner to a first peptide consisting essentially of the structure

Asp-Glu-K-Ser-Thr-Gly-Gly
Gln-Tyr-Asp-Gly-K-Gly-Val-Gly
K

where K
I

K

I
K is hydroxylysyl pyridinoline or lysyl pyridinoline, and Gin is glutamine or pyrrolidine carboxylic acid, and to a second peptide having the above formula except that the pyridinium ring of the cross-linking amino acid has been cleaved.

5. A method of determining cartilage degradation in vivo, comprising quantitating in a body fluid the concentrations of a first peptide comprising a C-terminal type II collagen telopeptide containing a hydroxylysyl pyridinoline cross-link which has an intact pyridinium ring, and a second peptide comprising a C-terminal type II collagen telopeptide containing a hydroxylysyl pyridinoline cross-link which has a cleaved pyridinium ring.

6. A method of determining cartilage degradation according to Claim 5, wherein the body fluid is urine, blood, serum, or synovial fluid.

7. A method of determining cartilage degradation according to Claim 5, wherein the first and second peptides have the structure:
Glu-Hyl-Gly-Pro-Asp
Glu-Hyl-Gly-Pro-Asp
Gly-Val-Hyl
Hyl
wherein | is hydroxylysyl pyridinoline, said first and second
Hyl
I
Hyl
peptides containing an intact or a cleaved pyridinium ring, respectively.

8. A method of determining cartilage degradation according to Claim 5, wherein the first and second peptides have the structure:
Glu-Hyl-Gly-Pro-Asp-Pro-Leu
Glu-Hyl-Gly-Pro-Asp
Gly-Val-Hyl

Gl u-Hy 1 -Gly-Pro-Asp-Pro
Glu-Hyl-Gly-Pro-Asp
Gly-Val-Hyl , or

Hyl
wherein | is hydroxylysyl pyridinoline, said first and second
Hyl
Hyl
peptides containing an intact or a cleaved pyridinium ring, respectively.

9. A method of determining collagenous connective tissue degradation in vivo, comprising quantitating in a body fluid the concentrations of a first peptide comprising a type III collagen telopeptide containing a 3-hydroxypyridinium cross-link, and a second peptide comprising a type III collagen telopeptide containing a 3-hydroxypyridinium cross-link which has a cleaved pyridinium ring.

10. The method according to Claim 9, wherein the 3-hydroxypyridinium cross-link is hydroxylysyl pyridinoline.

11. The method according to Claim 9, wherein the body fluid is urine, blood, serum, or synovial fluid.

12. The method according to Claim 9, wherein the type III collagen telopeptide is:
Asp-Val -Hyl -Ser-Gly-Val
Asp-Val-Hyl-Ser-Gly-Val
Hyl , or


Hyl
wherein | is hydroxylysyl pyridinoline, said first and second
Hyl
I
Hyl
peptides containing an intact or a cleaved pyridinium ring, respectively.

13. A kit for carrying out the method of any of Claims 5-12, comprising at least one specific binding partner to said first and second peptides.

14. A cell line that produces a monoclonal antibody that binds to first and second peptides consisting essentially of the structure
Asp-Glu-K-Ser-Thr-Gly-Gly
Gln-Tyr-Asp-Gly-K-Gly-Val -Gly
K

wherein
K

I K

I
K
is hydroxylysyl pyridinoline or lysyl pyridinoline, and Gin is glutamine or pyrrolidine carboxylic acid, and wherein the pyridinium ring of the cross-linking amino acid in the first and second peptides is closed and open, respectively.

15. The cell line of Claim 14, having the identifying characteristics of ATCC HB 10611 (1H11).

16. A monoclonal antibody produced by the cell line of Claim 14.

17. A monoclonal antibody produced by the cell line of Claim 15/

18. A method of determining cartilage degradation in vivo, comprising quantitating the concentration of a peptide in a body fluid, said peptide comprising a C-terminal type II collagen telopeptide containing a hydroxylysyl pyridinoline cross-link.

19. A method of determining cartilage degradation according to Claim 18, wherein the body fluid is urine, blood, serum, or synovial fluid.

20. A method of determining cartilage degradation according to Claim 18, wherein the detecting step comprises contacting the body fluid with a specific binding partner to said C-terminal type II collagen telopeptide.

21. A method of determining cartilage degradation according to Claim 18, wherein the C-terminal type II collagen telopeptide is:
Glu-Hyl-Gly-Pro-Asp
Glu-Hyl-Gly-Pro-Asp
Gly-Val -Hyl
Hyl
wherein | is hydroxylysyl pyridinoline.
Hyl
Hyl

22. A method of determining cartilage degradation according to Claim 18, wherein the C-terminal type II collagen telopeptide is:
Gl u-Hyl -Gly-Pro-Asp-Pro-Leu
Glu-Hyl-Gly-Pro-Asp
Gly-Val-Hyl

Glu-Hyl -Gly-Pro-Asp-Pro
Glu-Hyl-Gly-Pro-Asp
Gly-Val-Hyl , or

Glu-Hyl -Gly-Pro-Asp
Glu-Hyl-Gly-Pro-Asp
Val-Hyl
Hyl
wherein | is hydroxylysyl pyridinoline.
Hyl
I
Hyl

23. A method of determining collagenous connective tissue degradation in vivo, comprising quantitating the concentration of a peptide in a body fluid, said peptide comprising a type III collagen telopeptide containing a 3-hydroxypyridinium cross-link.

24. The method according to Claim 23, wherein the 3-hydroxypyridinium cross-link is hydroxylysyl pyridinoline.

25. The method according to Claim 23, wherein the body fluid is urine, blood, serum, or synovial fluid.

26. The method according to Claim 23, wherein the detecting step comprises contacting the body fluid with a specific binding partner to said type III collagen cross-linked telopeptide.

27. The method according to Claim 23, wherein the type III collagen telopeptide is:
Asp-Val -Hyl -Ser-G ly-Val
Asp-Val -Hyl -Ser-Gly-Val
Hyl , or



Hyl
wherein | is hydroxylysyl pyridinoline.
Hyl
I
Hyl

28. A peptide comprising an amino acid sequence of a C-terminal type II collagen telopeptide containing a 3-hydroxypyridinium cross-link, the peptide having an amino acid composition of 2 Asx, 2 Glx, 9 Gly, 7 Pro, 5 Leu, 3 Arg, 1 His, 1 Val, and 1 hydroxylysyl pyridinoline residue, wherein
each Asx is independently selected from the group consisting of Asp and Asn, and each Glx is independently selected from the group consisting of Glu and Gin,
or the peptide has an amino acid composition given above less from 1 to 21 amino acids.

29. A peptide according to Claim 28, having the amino acid sequence

Glu-Hyl-Gly-Pro-Asp
I
Glu-Hyl-Gly-Pro-Asp
I
Gly-Val-Hyl

Hyl
wherein | is hydroxylysyl pyridinoline.
Hyl
I
Hyl

30. A peptide according to Claim 28, having the amino acid sequence





Hyl
wherein | is hydroxylysyl pyridinoline.
Hyl
Hyl

31. A peptide comprising an amino acid sequence of a type III collagen telopeptide and having a 3-hydroxypyridinium cross-link, the peptide having an amino acid composition of
2 Glx, 4 Tyr, 4 Ser, 2 Asx, 4 Val, 5 Gly, 2 Ala, 1 He, 1 His, 1 Arg, and one hydroxylysyl pyridinoline residue,
or
13 Gly, 2 He, 2 Glx, 4 Ala, 3 Phe, 1 Pro, 1 Met, 1 His, 1 Arg, and one hydroxylysyl pyridinoline residue, wherein
each Asx is independently selected from the group consisting of Asp and Asn, and
each Glx is independently selected from the group consisting of Glu and Gin,
or the peptide has an amino acid composition given above less from 1 to 18 amino acids.

32. A peptide according to Claim 31, having the amino acid sequence

Asp-Val -Hyl -Ser-Gly-Val
I
Asp-Val -H l -Ser-Gly-Val
I
Hyl , or

Glu-Hyl-Ala-Gly-Gly-Phe
I
Glu-Hyl-Al a-Gly-Gly-Phe
I
Hyl

Hyl
I
wherein Hyl is hydroxylysyl pyridinoline.
Hyl

33. A test kit for carrying out the method of Claim 18, comprising a specific binding partner to a C-terminal type II collagen telopeptide containing a hydroxylysyl pyridinoline cross-link.

34. A test kit for carrying out the method of Claim 23, comprising a specific binding partner to a type III collagen telopeptide containing a 3-hydroxypyridinium cross-link.

35. A test kit comprising one or more immunological binding partners that specifically bind to a C-terminal type II collagen telopeptide containing a hydroxylysyl pyridinoline cross-link, or a type III collagen telopeptide containing a hydroxylysyl pyridinoline cross-link.

36. In a method of analyzing body fluids for the presence of an analyte indicative of a physiological condition, comprising the steps of contacting the body fluid with a specific binding partner for said analyte, and detecting any binding that occurs between the analyte and the specific binding partner, the improvement comprising contacting the body fluid with at least one specific binding partner to a cross-linked telopeptide having a sequence identical to that of a cross-linked telopeptide produced in vivo upon degradation of type II, or type III collagen.