RADIOIMMUNOASSAY FOR THE PYRIDINOLINE CROSS-LINKED CARBOXY-TERMINAL TELOPEPTIDE OF TYPE-I COLLAGEN - A NEW SERUM MARKER OF BONE-COLLAGEN DEGRADATION

We developed a radioimmunoassay (RIA) for the carboxy-terminal telopep tides of type I collagen (ICTP), cross-linked with the helical domain of another type I collagen molecule, after isolation from human femora l bone. The cross-linked peptide was liberated by digesting insoluble, denatured bone collagen either with bacterial collagenase or with try psin, and purified by two successive reversed-phase separations on HPL C, with monitoring of pyridinoline-specific fluorescence. The purity o f the peptide was verified by sodium dodecyl sulfate-polyacrylamide ge l electrophoresis, and its origin in the type I collagen fibers was de termined by amino-terminal amino acid sequencing. Polyclonal antibodie s and a separation reagent containing second antibody and polyethylene glycol are used in the RIA. An immunologically identical, somewhat la rger antigen is present in human serum; its concentration increases in multiple myeloma and in rheumatoid arthritis. The ICTP antigen seems to be cleared from the circulation by the kidneys, because glomerular filtration rates that are two-thirds of normal or less are associated with increased circulating ICTP concentrations. The CVs of the method are between 3% and 8% for a wide range of concentrations. The analysis of 40 serum samples can be completed in 4 h.