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Use of an antineoepitope antibody for identification of type-II collagen degradation in equine articular cartilage

R. Clark BillinghurstEquine Orthopaedic Research Laboratory, the Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.

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 DVM, PhD
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Elizabeth M. BuxtonEquine Orthopaedic Research Laboratory, the Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.

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Michael G. EdwardsEquine Orthopaedic Research Laboratory, the Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.
Present address is College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI 53706.

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Megan S. McGrawEquine Orthopaedic Research Laboratory, the Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.

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C. Wayne McIlwraithEquine Orthopaedic Research Laboratory, the Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.

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 BVSc, PhD

Abstract

Objective—To develop an antibody that specifically recognizes collagenase-cleaved type-II collagen in equine articular cartilage.

Sample Population—Cartilage specimens from horses euthanatized for problems unrelated to the musculoskeletal system.

Procedure—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 and Clinical Relevance—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. (Am J Vet Res 2001;62:1031–1039)

Abstract

Objective—To develop an antibody that specifically recognizes collagenase-cleaved type-II collagen in equine articular cartilage.

Sample Population—Cartilage specimens from horses euthanatized for problems unrelated to the musculoskeletal system.

Procedure—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 and Clinical Relevance—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. (Am J Vet Res 2001;62:1031–1039)