Patent ID: 6509450
Filing Date: 2003-01-21
Classification: C07K,G01N,Y10S

Abstract:
An immunoassay kit for the quantification of degradation products of carboxy-terminal telopeptides of type I collagen in a human serum sample, comprising an antibody that is characterized by binding to at least one peptide derived from the carboxy- terminal telopeptide domain of type I collagen and having a 3-hydroxypyridinium cross-link, wherein the peptide is isolatable from a urine sample of a patient with active Paget's disease by a process which comprises the following steps:dialyzing the urine sample in reduced porosity dialysis tubing (>3,500), and freeze-drying the non-diffusat; chromatographing the dried material on a column of polyacrylamide gel with a weight exclusion limit of 2,000 in 10% acetic acid at room temperature, identifying a region of effluent the contains 3-hydroxypyridinium cross-linked peptides by measuring the fluorescence of collected fractions at 297 nm excitation/395 nm emission, and freez-drying the pooled fluorescent fractions; chromatographing the dried material on a column of polyacrylamide gel with a weight exclusion limit of 4,000 eluted at 10% acetic acid, identifying two overlapping fraction pools by fluorescence of the eluant as measured above, and freeze-drying the earlier fraction pool; chromatographing the dried material by ion-exchange HPLC on a DEAE-5-PW column eluted with a gradient of NaCl (0-0.2 M) in 0.02M Tris-HCl, pH 7.5 containing 10% (v/v) acetonitrile, identifying carboxy-terminal type I collagen telopeptide peaks that elute between 0.08M and 0.15M NaCl by fluorescence as measured above, and freeze-drying each peak; chromatographing each dried peak on a C-18 reverse phase HPLC column eluted with a gradient (0-10%) of a acetonitrile:n-propanol (3.1 v/v) in 0.01M trifluoroacetic acid to isolate the individual peptides; selecting isolated peptides that contain a 3-hydroxypyridinum cross-link derived from the carboxy-terminal telopeptide domain of type I collagen; and confirming that the antibody binds to at least one of the selected isolated peptides.