A specific immunoassay for monitoring human bone resorption: quantitation of type I collagen cross-linked N-telopeptides in urine.
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.
Hanson
D A
DA
Department of Orthopaedics, University of Washington, Seattle.
Weis
M A
MA
Bollen
A M
AM
Maslan
S L
SL
Singer
F R
FR
Eyre
D R
DR
D000293:Adolescent; D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000652:urine*; D000911:Antibodies, Monoclonal; D001851:Bone Diseases, Metabolic / Q000652:urine*; D001862:Bone Resorption / Q000652:urine*; D002648:Child; D002675:Child, Preschool; D003094:Collagen / Q000737:chemistry / Q000652:urine*; D003404:Creatinine / Q000652:urine; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006909:Hydroxyproline / Q000652:urine; D007223:Infant; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010001:Osteitis Deformans / Q000652:urine; D010455:Peptides / Q000737:chemistry / Q000276:immunology / Q000652:urine*
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D010455:Peptides; C015484:pyridinoline; D003094:Collagen; D003404:Creatinine; D006909:Hydroxyproline