Research paper
The assessment of cartilage degradation in vivo: development of an immunoassay for the measurement in body fluids of type II collagen cleaved by collagenases

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Abstract

A monoclonal antibody has been developed which recognizes a neoepitope in type II collagen which is generated by the intrahelical cleavage of collagenases. Antibody reactivity is directed at the carboxyl-terminus of the TCA or 3/4 piece of the degraded α1(II) chain. Reactivity is dependent upon hydroxylation of proline. Evidence is provided suggesting that epitope binding involves the recognition of a conformational neoepitope. Using an ELISA, we show that this neoepitope can be detected in urines and sera of nonarthritic persons and patients with rheumatoid arthritis (RA). An increased content is observed in the sera and urines of patients. The assay may be of value in studying cartilage type II degradation both in vitro and in vivo such as in those with arthritis.

Introduction

Arthritis involves the progressive loss of normal joint structure and function resulting from the erosion and loss of articular cartilages in diseases such as rheumatoid arthritis (RA) and osteoarthritis. The consequences of this destructive process can be viewed radiologically by examining joint space narrowing. It is of importance to be able to accurately detect the loss of articular cartilage as a process not only as an outcome. This may be possible by a methodology that permits measurement in body fluids of degradation products resulting from the cleavage of cartilage-specific macromolecules which are components of the extracellular matrix and are progressively destroyed in arthritis (Poole, 2001, Poole, 2003). One of these molecules is type II collagen. This is the major component of hyaline cartilage matrix that forms an extensive fibrillar network that endows cartilage with its tensile strength (Poole, 2001).

We have shown in rheumatoid arthritis (Dodge and Poole, 1989) and osteoarthritis (Hollander et al., 1994, Billinghurst et al., 1997, Dahlberg et al., 2000, Wu et al., 2002) that degradation of type II collagen is increased, involving excessive cleavage of this molecule by collagenases. As a consequence, cleavage products produced by collagenases are excessively released from cartilage and can be measured in culture medium (Billinghurst et al., 1997, Dahlberg et al., 2000). Previously, we prepared an antibody that recognizes the cleavage of type II collagen by collagenases in cartilage and which was used to demonstrate increased collagenase activity both in cartilage (Billinghurst et al., 1997) and in culture media (Dahlberg et al., 2000, Billinghurst et al., 1997). Although articular cartilages do not contain type I collagen, this antibody cross-reacted with this molecule when cleaved by collagenases (Billinghurst et al., 1997).

To enable us to selectively detect cleavage of only type II collagen, we have prepared a monoclonal antibody which provides this specificity. In this paper, we describe the specificity of the antibody C2C (previously known as COL2-3/4CLong mono) and its use in an ELISA to detect collagenase-generated cleavage products of type II collagen in body fluids in health and arthritis.

Section snippets

Patients, body fluids and general assays

We have studied body fluid samples from patients with rheumatoid arthritis (RA) as well as from a nonarthritic population of hospital employees and friends of patients who volunteered blood samples. Informed consent was obtained after the nature and possible consequences of the studies had been fully explained. Patients were classified in accordance with the guidelines of the American College of Rheumatology (Arnett et al., 1987). The ages, sexes and clinical characteristics of patients and

Monoclonal antibody C2C (COL2-3/4 CLong mono) and its specificity

An antibody with an IgG1 isotype was identified by Western blotting. It reacted strongly with the α chain of type II collagen but only following cleavage by collagenases MMP-1, or -13 (Fig. 2B, lane 5, 6 and 7). Reactivity was with the TCA or 3/4 piece. There was no reactivity with the α chain or the TCB or 1/4 piece observed by Coomassie staining (Fig. 2A). Secondary degradation products of the TCA piece of type II collagen can be seen in the Western blot in (Fig. 2B) lanes 6 and 7. The

Discussion

The immunoassay described here offers the opportunity to examine in vivo the degradation of articular cartilage by measuring degradation products resulting from the cleavage of cartilage type II collagen by collagenase. A prior report of such an assay that is specific for the cleavage of the triple helix of type II collagen by collagenase has already been described (Downs et al., 2001). This is a sandwich assay that employs a monoclonal antibody to the collagenase-generated cleavage neoepitope

Acknowledgements

This study was funded by Shriners Hospitals for Children, National Institute of Ageing, National Institutes of Health, Canadian Institutes of Health and Canadian Arthritis Network (to ARP). RCB received a fellowship from the Fonds de la Recherche en Santé du Québec.

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Present address: St. Lawrence College, Kingston, Ontario, Canada.

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