The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. A method for determining the absolute rate of bone resorption, which comprises quantitating the concentration of peptide fragments in a body fluid, the peptide fragments being derived from bone collagen resorption and having a 3-hydroxypyridinium cross-link.
2. The method of claim 1, wherein the 3-hydroxypyridinium cross-link is lysyl pyridinoline.
3. The method of claim 1, wherein the 3-hydroxypyridinium cross-link is hydroxylysyl pyridinoline.
4. The method of claim 1, wherein the body fluid is urine.
5. The method of claim 1, wherein the quantitating step comprises contacting the body fluid with an immunological binding partner specific to a peptide fragment having a 3-hydroxypyridinium cross-link derived from bone collagen resorption.
6. The method of claim 5, further comprising, prior to the contacting step, purifying the body fluid.
7. The method of claim 6, wherein the purifying step comprises a procedure selected from the group consisting of cartridge adsorption and elution, molecular sieve chromatography, dialysis, ion exchange, alumina chromatography, and hydroxyapatite chromatography.
8. The method of claim 1, wherein the quantitating step comprises those selected from the group consisting of ultraviolet absorbance spectroscopy, natural fluorescence spectroscopy and electrochemical titration.
9. The method of claim 1, wherein the quantitating step comprises electrochemical titration.
10. The method of claim 9, further comprising prior to the electro-chemical titration step, purifying the body fluid.
11. The method of claim 10, wherein the purifying step comprises a procedure selected from the group consisting of dialysis, ion exchange chromatography, alumina chromatography, molecular sieve chromatography, and hydroxyapatite chromatography.
12. The method of claim 10, wherein the purifying step comprises adsorbing the body fluid on an ion-exchange adsorption filter, eluting the ion-exchange adsorption filter and collecting the eluate containing peptide fragments having a 3-hydroxypyridinium cross-link.
13. The method of claim 10, further comprising, after purifying the body fluid, resolving the peptide fragments having a 3-hydroxypyridinium cross-link.
14. The method of claim 1, wherein the quantitating step comprises fluorometric measurement.
15. The method of claim 14, further comprising, prior to the fluorometric measurement step, purifying the body fluid.
16. The method of claim 15, further comprising, after purifying the body fluid, hydrolyzing the peptide fragments thereby forming a hydrolyzate.
17. The method of claim 16, comprising, after hydrolyzing the peptide fragments, resolving the hydrolyzate.
18. The method of claim 17, wherein the resolving step comprises chromatographic resolution.
19. The method of claim 18, wherein the chromatographic resolution step comprises high performance liquid chromatography.
20. The method of claim 15, wherein the purifying step comprises:
(a) dialyzing an aliquot of urine against an aqueous buffered solution, thereby producing a partially purified peptide fragment containing non-diffusate,
(b) lyophilizing the non-diffusate,
(c) dissolving the lyophilized non-diffusate in an ion pairing solution,
(d) adsorbing the non-diffusate onto an affinity chromatography column,
(e) washing the adsorbed non-diffusate with a volume of ion pairing solution, and
(f) eluting the affinity column containing the peptide fragments with an eluting solution.
21. The method of claim 20, further comprising the step, prior to adsorbing the non-diffusate, contacting the non-diffusate with a molecular sieving column.
22. A peptide fragment derived from bone collagen and obtained from a body fluid, substantially free of other human peptides.
23. The peptide fragment of claim 22, containing a 3-hydroxypyridinium cross-link.
24. The peptide fragment of claim 22, containing a lysyl pyridinoline cross-link.
25. The peptide fragment of claim 22, containing a hydroxylysyl pyridinoline cross-link.
26. The peptide fragment of claim 22, comprising an amino acid sequence that derives from the aminoterminal telopeptide domain of bone type I collagen linked through a 3-hydroxypyridinium cross-link, the peptide fragment having an amino acid composition (Asx)2-(Glx)2-(Gly)5-Val-Tyr-Ser-Thr and having one residue of hydroxylysyl pyridinoline or lysyl pyridinoline.
where
Asx is the amino acid Asp or Asn, and
Glx is the amino acid Glu or Gln.
27. The peptide fragment of claim 22, comprising the amino acid sequence:
6
is hydroxylysyl pyridinoline or lysyl pyridinoline, and
Gln is glutamine or wholly cyclized pyrrolidone carboxylic acid.
28. The peptide fragment of claim 22, comprising an amino acid sequence that derives from the carboxyterminal telopeptide domain of bone type I collagen.
29. The peptide fragment of claim 28, wherein the amino acid sequence contains a lysyl pyridinoline cross-link.
30. The peptide fragment of claim 28, wherein the amino acid sequence contains a hydroxylysyl pyridinoline cross-link.
31. The peptide fragment of claim 28, comprising the amino acid sequence:
7
is hydroxylysyl pyridinoline or lysyl pyridinoline.
32. The peptide fragment of claim 22, wherein the body fluid is selected from the group consisting of urine, synovial fluid, and serum.
33. The peptide fragment of claim 32, wherein the body fluid is urine.
34. A fused cell hybrid which produces monoclonal antibodies specific for the peptide fragment of claim 22.
35. A monoclonal antibody produced by the fused cell hybrid of claim 34.
36. The monoclonal antibody of claim 35, coupled to a detectable marker.
37. The monoclonal antibody of claim 36, wherein the detectable marker is selected from the group consisting of enzymes, chromophores, fluorophores, coenzymes, enzyme inhibitors, chemiluminescent materials, paramagnetic metals, spin labels, and radionuclides.
38. A test kit for quantitating the amount of peptide fragments in a body fluid, the peptide fragment being derived from bone collagen resorption and having a 3-hydroxypyridinium cross-link, comprising the monoclonal antibody of claim 35.
39. The test kit of claim 38, wherein the monoclonal antibody is coupled to a detectable marker.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
What is claimed:
1. A nucleic acid sequence encoding HC gp-39L.
2. A protein encoded by the a nucleic acid sequence comprising the sequence of claim 1.
3. A nucleic acid sequence comprising the sequence of SEQ ID NO: 1.
4. A method of diagnosing a tissue remodeling disorder related to expression of a mutated HC gp-39L protein in a host comprising:
(a) synthesizing PCR primers complementary to a nucleic acid encoding a normal HC gp-39L protein;
(b) obtaining nucleic acids from cells of a host;
(c) amplifying the obtained nucleic acids with the synthesized PCR primers to obtain an amplified product; and
(d) comparing the amplified product to a normal genotype encoding the normal HC gp-39L protein to detect any mutations in the amplified product.
5. The method of claim 4 wherein the HC gp-39L comprises the polypeptide sequence of SEQ ID NO: 1.
6. A method of detecting altered expression of a HC gp-39L protein in a host comprising:
(a) preparing a first antibody specific to a HC gp-39L protein;
(b) preparing a reporter antibody against the first antibody, said reported antibody linked to an attached detectable reagent;
(c) obtaining a sample from a host;
(d) incubating said sample on a solid support which binds any proteins in said sample;
(e) incubating said first antibody with any bound protein on said solid support so that said first antibody attaches to any HC ap-39L proteins bound to said solid support;
(f) contacting said reporter antibody linked to a detectable reagent with said attached first antibody on said solid support;
(g) detecting said detectable reagent; and
(h) determining an amount of HC gp-39L protein in said sample.
7. The method of claim 6 wherein the HC gp-39L comprises the polypeptide sequence of SEQ ID NO: I