title: Use of an antineoepitope antibody for identification of type-II collagen degradation in equine articular cartilage.
abstract: OBJECTIVE
  To develop an antibody that specifically recognizes collagenase-cleaved type-II collagen in equine articular cartilage.
  
  
  METHODS
  Cartilage specimens from horses euthanatized for problems unrelated to the musculoskeletal system.
  
  
  METHODS
  A peptide was synthesized representing the carboxy- (C-) terminus (neoepitope) of the equine type-II collagen fragment created by mammalian collagenases. This peptide was used to produce a polyclonal antibody, characterized by western analysis for reactivity to native and collagenase-cleaved equine collagens. The antibody was evaluated as an antineoepitope antibody by ELISA, using peptides +/- an amino acid at the C-terminus of the immunizing peptide. Collagen cleavage was assayed from equine articular cartilage cultured with interleukin-1 (IL-1), +/- a synthetic MMP inhibitor, BAY 12-9566. Cartilage specimens from osteoarthritic and nonarthritic joints were compared for antibody staining.
  
  
  RESULTS
  An antibody, 234CEQ, recognized only collagenase-generated 3/4-length fragments of equine type-II collagen. This was a true antineoepitope antibody, as altering the C-terminus of the immunizing peptide significantly decreased competition for binding in an inhibition ELISA. The IL-1-induced release of type-II collagen fragments from articular cartilage was prevented with the MMP inhibitor. Cartilage from an osteoarthritic joint of a horse had increased staining with the 234CEQ antibody, compared with normal articular cartilage.
  
  
  CONCLUSIONS
  We generated an antineoepitope antibody recognizing collagenase-cleaved type-II collagen of horses. This antibody detects increases in type-II collagen cleavage in diseased equine articular cartilage. The 234CEQ antibody has the potential to aid in the early diagnosis of arthritis and to monitor treatment responses.
authors.affiliation: Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins 80523, USA.
authors.forename: R C
authors.initials: RC
authors.lastname: Billinghurst
authors.forename: E M
authors.initials: EM
authors.lastname: Buxton
authors.forename: M G
authors.initials: MG
authors.lastname: Edwards
authors.forename: M S
authors.initials: MS
authors.lastname: McGraw
authors.forename: C W
authors.initials: CW
authors.lastname: McIlwraith
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000970:Antineoplastic Agents / Q000276:immunology; D001713:Biphenyl Compounds; D015153:Blotting, Western / Q000662:veterinary; D002358:Cartilage, Articular / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D024043:Collagen Type II / Q000276:immunology* / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D000939:Epitopes / Q000276:immunology*; D005260:Female; D006734:Horse Diseases / Q000276:immunology / Q000378:metabolism; D006736:Horses / Q000276:immunology* / Q000378:metabolism; D007150:Immunohistochemistry / Q000662:veterinary; D019278:Matrix Metalloproteinase 3 / Q000737:chemistry; D009930:Organic Chemicals; D010003:Osteoarthritis / Q000276:immunology / Q000378:metabolism / Q000662:veterinary; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D010654:Phenylbutyrates; D011509:Proteoglycans / Q000276:immunology / Q000378:metabolism; D011817:Rabbits
chemical_list: D000906:Antibodies; D000970:Antineoplastic Agents; D001713:Biphenyl Compounds; D024043:Collagen Type II; D000939:Epitopes; D009930:Organic Chemicals; D010446:Peptide Fragments; D010654:Phenylbutyrates; D011509:Proteoglycans; C113281:Bay 12-9566; D017364:Collagenases; D019278:Matrix Metalloproteinase 3