Patent ID: 6204367
Filing Date: 2001-03-20
Classification: C07K,G01N,Y10S

Abstract:
), and freeze-drying the non-diffusate;chromatographing the dried material on a Bio-Gel P2 column in 10% acetic acid at room temperature, identifying a region of effluent that contains 3-hydroxypyridinium cross-linked peptides by measuring the fluorescence of collected fractions at 297 nm excitation/395 nm emission, and freeze-drying the pooled fluorescent fractions;chromatographing the dried material on a Bio-Gel P-4 column 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 TSK DEAE-5-PW column eluted with a gradient of NaCl (0-0.2M) 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-hydroxypyridinium cross-link derived from the carboxy-terminal telopeptide domain of type I collagen; andconfirming that the antibody binds to at least one of the selected isolated peptides.