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

Objective-To develop an antibody that specifically recognizes collagenase-c leaved 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, chara cterized by western analysis for reactivity to native and collagenase-cleav ed equine collagens. The antibody was evaluated as an antineoepitope antibo dy by ELISA, using peptides +/- an amino acid at the C-terminus of the immu nizing peptide. Collagen cleavage was assayed from equine articular cartila ge cultured with interleukin-1 (IL-1), +/- a synthetic MMP inhibitor, BAY 1 2-9566. Cartilage specimens from osteoarthritic and nonarthritic joints wer e compared for antibody staining. Results-An antibody, 234CEQ, recognized only collagenase-generated 3/4-leng th fragments of equine type-II collagen. This was a true antineoepitope ant ibody, as altering the C-terminus of the immunizing peptide significantly d ecreased competition for binding in an inhibition ELISA. The IL-1-induced r elease of type-it 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 artic ular cartilage. Conclusions and Clinical Relevance-We generated an antineoepitope antibody recognizing collagenase-cleaved type-II collagen of horses. This antibody d etects increases in type-II collagen cleavage in diseased equine articular cartilage. The 234CEQ antibody has the potential to aid in the early diagno sis of arthritis and to monitor treatment responses.