A specific immunoassay for monitoring human bone resorption: Quantitation of type I collagen cross-linked N-telopeptides in urine

TLDR

The assay aims to study normal bone metabolism, diagnose and monitor bone disease, and evaluate antiresorption therapies. The authors isolated pyridinoline‑cross‑linked peptides from urine, produced monoclonal antibodies against the N‑telopeptide domain, and developed a sensitive inhibition ELISA to quantify these bone‑derived peptides directly from untreated urine. The high‑affinity antibody selectively binds the N‑telopeptide cross‑link peptides, and the ELISA correlates strongly with hydroxyproline and total pyridinoline in Paget’s disease, demonstrating a rapid, specific index of bone resorption superior to total pyridinoline analysis.

Abstract

Abstract Peptides of low molecular weight that contain pyridinoline cross-links were isolated from adolescent human urine. A fraction was selected that was enriched in the N-telopeptide-to-helix intermolecular cross-linking domain of bone type I collagen. Mouse monoclonal antibodies were generated against these urinary peptides conjugated to a carrier protein as immunogen. A high-affinity antibody was identified that specifically bound to the trivalent peptides derived from the N-telopeptide-to-helix pyridinoline cross-linking site in type I collagen of human bone. This was confirmed by the direct isolation from human bone collagen of similar fragments recognized selectively by the antibody. A sensitive inhibition ELISA was established on microtiter plates that could quantify the bone-derived peptides in human urine. The assay, which can be run directly on untreated urine, was thoroughly tested against samples from normal subjects and from patients with metabolic bone disease. For example, strong correlations with urinary hydroxyproline and total pyridinoline cross-links were found in patients with Paget's disease of bone. The method shows considerable promise as a rapid and specific index of human bone resorption rates, with greatly improved specificity and convenience over total pyridinoline analysis. Potential applications include the study of normal metabolism, the diagnosis and monitoring of bone disease, and evaluating the effectiveness of antiresorption therapies.