The Nc1 Fragment Of Type X Collagen Measured In Serum As A Potential Biomarker Of Osteoarthritis

Purpose: The chondrocytes taking a terminal differentiation route of hypertrophy, invasion of blood vessels from the subchondral bone, apoptosis, and calcification of cartilage has been observed in experimental models of OA and human osteoarthritis (OA). Hypertrophy-like changes in chondrocytes are recently believed to play a role in the initiation and progression of cartilage degeneration. Type X collagen, a well-known marker of hypertrophy chondrocytes, plays a role in facilitating and regulating endochondral ossification of cartilage. We aimed to develop an immunoassay that detects the released NC1 fragment of type X collagen in the serum of healthy donors and OA patients. Methods: A sandwich chemiluminescence immunoassay, designated COL10NC, detecting the cleaved NC1 domain of type X collagen was developed. A biotinylated antibody against a newly discovered cathepsin K-generated neoepitope 479GIATKG (one amino acid apart from the well-known cleavage site 480IATKG generated by collagenase) was employed as a capture. A horseradish peroxidase (HRP) labeled antibody to target the C-terminus of the NC1 domain was used as a detector. The COL10NC assay was validated to assure the reliability of the measurement. To characterize the biological relevance of the assay, 69 serum samples from normally growing infants and children from birth to 17years were used. The assay was further measured in healthy adult serum samples (n=20) and commercially available samples from OA patients (n=8). Results: The COL10NC displays good technical performance. The intra-plate variation is 3.1% and the inter-plate variation is 15.1%. The low limit of quantification (LLOQ) is 3.42 pM and the upper limit of quantification is 55.6 pM. The minimum required dilution is 1+1. The assay has an acceptable matrix-matrix spike recovery and dilution-of-linearity recovery of range 80-120%. The COL10NC levels are stable at up to four freeze-thaw cycles in serum samples. The COL10NC levels were negatively associated with age, which corresponds with the growth of long bones (endochondral ossification process) is highest in young infants and drops substantially after adolescence (Figure 1). Nineteen of twenty healthy donors had levels of COL10NC under the detection limit. The mean (95%CI) of COL10NC in the eight OA serum samples was 4.50 (3.65-6.34) pM. Conclusions: There is an inverse correlation between COL10NC levels and age in the normal growing infants and children. The COL10NC level is elevated in the serum samples from OA patients compared to the normal adult. Overall, the COL10NC has the potential to serve as a biomarker of osteoarthritis.