A New Monoclonal Antibody ELISA for Detection and Characterization of C-telopeptide Fragments of Type I Collagen in Urine

Abstract

Clinical studies have shown that measurements of urine concentration of degradation products of C-terminal telopeptides of type I collagen provide a selective marker to assess bone resorption. Assays for C-telopeptide fragments have been described using antibodies generated against an eight amino acid synthetic peptide EKAHDGGR. In this study, we describe development of a rat monoclonal antibody against a synthetic linear peptide, DFSFLPQPPQEKAHDGGR, which we isolated and characterized from Paget's urine by chromatographic methods. An ELISA procedure was developed to evaluate the efficacy of this monoclonal to measure bone resorption and characterize urinary degradation products of C-terminal type I collagen. The measuring range of ELISA was 10-1,000 ng/ml with a detection limit of 10 ng/ml. Averaged intraassay and interassay CVs were <7% (n = 8) and <11% (n = 3), respectively. Mean spike and dilution recoveries range between 90 and 116%. In assessing the clinical performance, we observed approximately 48% higher values for postmenopausal patients (n = 20) compared with premenopausal women (n= 19), Z score = 2.7 (P = 0.0091). The discriminatory power to assess subtle changes in bone turnover between pre- and postmenopausal women compares well with the commercially available markers of bone resorption. Comparison of C-telopeptide concentration in a population including normal and osteoporotic patients (n = 74) shows a strong correlation with DPYR r = 0.95 (P < 0.001). In addition, in postmenopausal osteoporotic patients, a 44% (P < 0.05) decrease in C-telopeptide concentration was observed after 3 months of treatment with Alendronate, as compared with basal values. The antibody developed in this study displays a slightly different epitope than that reported previously using eight amino acid residues EKAHDGGR. In addition, isolation and characterization of circulating forms of C-telopeptide from healthy children by immunoaffinity purification revealed the presence of two novel urinary fragments corresponding to amino acid sequence EKAHDGGR and EKAHDG, in addition to the previously known two fragments composed of two alpha-1 chains linked together by a pyridinium crosslinks. The origin of the fragment EKAHDG could not be explained by the known mechanisms involved in collagen degradation, indicating that it could be a product of renal handling of C-telopeptide fragments generated from bone. The preliminary clinical data suggest that the new assay may be useful for further investigation of the clinical importance of measurement of these type I collagen degradation products.