Method of assaying type III collagen degradation

Abstract

A method of assaying collagen fragments in body fluids, including bringing a sample of body fluid in contact with at least one immunological binding partner for the collagen fragments, said binding partner being immunoreactive with synthetic peptides, the sequences of which are essentially derived from collagen and containing potential sites for cross-linking. The immunological binding partners are incorporated, either as whole antibodies or as immunologically active fragments thereof, in an assay for quantitative determination of collagen fragments in the sample. In addition to being contacted with the immunological binding partner(s), the sample may be brought into direct contact with the corresponding synthetic peptide. The invention further comprises a test kit and specific means for carrying out the method. The structure of specific peptides is also described.

What is claimed is:  
     
         1 . A method of determining collagen fragments in a body fluid, wherein a sample of said body fluid is brought into contact with at least one immunological binding partner for the collagen fragments, said binding partner being an antibody immunoreactive with synthetic peptides the sequences of which are essentially derived from collagen and containing potential sites for cross-linking, said immunological binding partners being incorporated, either as whole antibodies or as immunologically active fragments thereof, in an assay for quantitative determination of collagen fragments in the sample.  
     
     
         2 . A method according to  claim 1 , wherein the sample of body fluid in addition to the contacting with the immunological binding partner(s) is brought into direct contact with the corresponding synthetic peptide containing potential sites for cross-linking.  
     
     
         3 . A method according to  claim 1 , wherein the immunological binding partner has been raised by immunization with the corresponding synthetic peptide containing potential sites for cross-linking.  
     
     
         4 . A method according to  claim 2 , wherein the immunological binding partner has been raised by immunization with the corresponding synthetic peptide containing potential sites for cross-linking.  
     
     
         5 . A method according to  claim 1 , wherein the potential sites for cross-linking comprise lysine or hydroxylysine residues incorporated in the peptide sequence.  
     
     
         6 . A method according to  claim 1 , wherein the sequence of the synthetic peptide is derived from the sequence of human collagen type I and consisting essentially of one of the following structures 
 Asp-Glu-Lys-Ser-Thr-Gly-Gly    Glu-Lys-Ala-His-Asp-Gly-Gly-Arg    Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly    Gly-Met-Lys-Gly-His-Arg    Gly-Ile-Lys-Gly-His-Arg    Gly-Phe-Lys-Gly-Ile-Arg    or Gly-Leu-Pro-Gly-Leu-Lys-Gly-His-Asn.    
     
     
         7 . A method according to  claim 2 , wherein the sequence of the synthetic peptide is derived from the sequence of human collagen type I and consisting essentially of one of the following structures 
 Asp-Glu-Lys-Ser-Thr-Gly-Gly    Glu-Lys-Ala-His-Asp-Gly-Gly-Arg    Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly    Gly-Met-Lys-Gly-His-Arg    Gly-Ile-Lys-Gly-His-Arg    Gly-Phe-Lys-Gly-Ile-Arg    or Gly-Leu-Pro-Gly-Leu-Lys-Gly-His-Asn.    
     
     
         8 . A method according to  claim 1 , wherein the sequence of the synthetic peptide is derived from the sequence of human collagen type II and consisting essentially of one of the following structures 
 Glu-Lys-Gly-Pro-Asp    Gly-Val-Lys    Pro-Gly-Val-Lys-Gly    Pro-Gly-Pro-Lys-Gly-Glu    Gly-Gln-Lys-Gly-Glu-Pro    or Gly-Asp-Ile-Lys-Asp-Ile-Val.    
     
     
         9 . A method according to  claim 2 , wherein the sequence of the synthetic peptide is derived from the sequence of human collagen type II and consisting essentially of one of the following structures 
 Glu-Lys-Gly-Pro-Asp    Gly-Val-Lys    Pro-Gly-Val-Lys-Gly    Pro-Gly-Pro-Lys-Gly-Glu    Gly-Gln-Lys-Gly-Glu-Pro    or Gly-Asp-Ile-Lys-Asp-Ile-Val.    
     
     
         10 . A method according to  claim 1 , wherein the sequence of the synthetic peptide is derived from the sequence of human collagen type III and consisting essentially of one of the following structures: 
 Asp-Val-Lys-Ser-Gly-Val    Glu-Lys-Ala-Gly-Gly-Phe-Ala    Gly-Phe-Pro-Gly-Met-Lys-Gly-His-Arg    or Gly-Ala-Ala-Gly-Ile-Lys-Gly-His-Arg.    
     
     
         11 . A method according to  claim 2 , wherein the sequence of the synthetic peptide is derived from the sequence of human collagen type III and consisting essentially of one of the following structures: 
 Asp-Val-Lys-Ser-Gly-Val    Glu-Lys-Ala-Gly-Gly-Phe-Ala    Gly-Phe-Pro-Gly-Met-Lys-Gly-His-Arg    or Gly-Ala-Ala-Gly-Ile-Lys-Gly-His-Arg.    
     
     
         12 . A method according to  claim 1 , wherein the body fluid is human urine, blood, serum or synovial fluid.  
     
     
         13 . A cell line that produces a monoclonal antibody binding to any of the synthetic peptides used in the method according to  claim 1 .  
     
     
         14 . A cell line that produces a monoclonal antibody binding to any of the synthetic peptides used in the method according to  claim 2 .  
     
     
         15 . A monoclonal antibody produced by the cell line according to  claim 13 .  
     
     
         16 . An immunological binding partner raised by immunization with synthetic peptides selected from the group consisting of sequences derived from the sequence of human collagen type I and consisting essentially of one of the following structures 
 Asp-Glu-Lys-Ser-Thr-Gly-Gly    Glu-Lys-Ala-His-Asp-Gly-Gly-Arg    Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly    Gly-Met-Lys-Gly-His-Arg    Gly-Ile-Lys-Gly-His-Arg    Gly-Phe-Lys-Gly-Ile-Arg    and Gly-Leu-Pro-Gly-Leu-Lys-Gly-His-Asn,    sequences derived from the sequence of human collagen type II and consisting essentially of one of the following structures    Glu-Lys-Gly-Pro-Asp    Gly-Val-Lys    Pro-Gly-Val-Lys-Gly    Pro-Gly-Pro-Lys-Gly-Glu    Gly-Gln-Lys-Gly-Glu-Pro    and Gly-Asp-Ile-Lys-Asp-Ile-Val, and    sequences derived from the sequence of human collagen type III and consisting essentially of one of the following structures:    Asp-Val-Lys-Ser-Gly-Val    Glu-Lys-Ala-Gly-Gly-Phe-Ala    Gly-Phe-Pro-Gly-Met-Lys-Gly-His-Arg    and Gly-Ala-Ala-Gly-Ile-Lys-Gly-His-Arg,    and said polyclonal antibody being used for the preparation of an immunological binding partner.    
     
     
         17 . A test kit for quantitating the amount of collagen fragments in a body fluid, said kit comprising at least one immunological binding partner immunoreactive with a synthetic peptide having a sequence essentially derived from collagen and containing one or more potential sites for cross-linking.  
     
     
         18 . A method of diagnosing the presence of disorders associated with the metabolism of collagen in a patient, said method comprising the steps of: 
 (a) contacting a sample of body fluid collected from said patient with at least one immunological binding partner for collagen fragments, said binding partner being an antibody or a fragment thereof which is immunoreactive with synthetic peptides the sequences of which are essentially derived from collagen and containing potential sites for cross-linking,    (b) detecting the quantity of said antibody bound to collagen fragments in the sample, and    (c) comparing said quantity to a previously established standard based on binding of said antibody in samples originating from control subjects not suffering from any disorder associated with the metabolism of collagen.    
     
     
         19 . A method according to  claim 18 , wherein the antibody has been raised by immunization with the corresponding synthetic peptide containing potential sites for cross-linking.  
     
     
         20 . The method according to  claim 18 , which includes combining the determination of collagen fragments with other markers of the metabolism of bone tissue, connective tissue and other tissues in order to diagnose a given disorder.  
     
     
         21 . A method for determining whether a susceptible subject is suffering from collagen degradation comprising the steps of: 
 (a) contacting a sample of body fluid collected from said subject with an antibody that is specifically immunochemically reactive with an antigenic determinant of said collagen, said antigenic determinant consisting essentially of (i) a potentially crosslinkable site of the molecule of said collagen or (ii) an antigenic determinant essentially immediately adjacent to a potentially crosslinkable site on said collagen molecule;    (b) detecting the quantity of said antibody bound to collagen fragments in said sample; and    (c) comparing said quantity to a previously established standard based on binding of said antibody in samples from control subjects not suffering from collagen degradation.    
     
     
         22 . A method for determining the presence of collagen degradation products in a body fluid comprising the steps of: 
 (a) contacting a sample of said body fluid with an antibody that is specifically immunochemically reactive with an antigenic determinant of said collagen, said antigenic determinant consisting essentially of (i) a potentially crosslinkable site of the molecule of said collagen or (ii) an antigenic determinant essentially immediately adjacent to a potentially crosslinkable site on said collagen molecule; and    (b) detecting the quantity of said antibody bound to collagen fragments in said sample.