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 telopeptides of type I collagen (ICTP), cross-linked with the helical domain of another type I collagen molecule, after isolation from human femoral bone. The cross-linked peptide was liberated by digesting insoluble, denatured bone collagen either with bacterial collagenase or with trypsin, and purified by two successive reversed-phase separations on HPLC, with monitoring of pyridinoline-specific fluorescence. The purity of the peptide was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its origin in the type I collagen fibers was determined by amino-terminal amino acid sequencing. Polyclonal antibodies and a separation reagent containing second antibody and polyethylene glycol are used in the RIA. An immunologically identical, somewhat larger 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.
Risteli
J
J
Department of Medical Biochemistry, University of Oulu, Finland.
Elomaa
I
I
Niemi
S
S
Novamo
A
A
Risteli
L
L
D000328:Adult; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000097:blood* / Q000737:chemistry; D001842:Bone and Bones / Q000378:metabolism*; D003094:Collagen / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008875:Middle Aged; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000097:blood* / Q000737:chemistry; D011863:Radioimmunoassay; D012016:Reference Values
D000596:Amino Acids; D010446:Peptide Fragments; C015484:pyridinoline; D003094:Collagen; D017364:Collagenases