Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-01-07
2004-04-06
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007910, C435S007940, C435S810000, C435S975000, C436S512000, C436S151000, C530S387100, C530S387900, C530S389100, C530S391100
Reexamination Certificate
active
06716593
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods, reagents, and kits for measuring levels of total pyridinoline and/or deoxypyridinoline in fluid samples, after proteolytic digestion to remove attached amino acid residues. The invention also relates to diagnostic methods for assessing collagen degradation rates in mammals, particularly humans, and to the diagnosis and monitoring of medical conditions associated with abnormal collagen metabolism.
References
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BACKGROUND OF THE INVENTION
Disease conditions associated with elevated rates of collagen breakdown, especially in bone and cartilage, are responsible for considerable medical expenditures on the elderly and result in significant pain, impaired mobility, and other reductions in quality of life. Among the more common disease conditions are osteoporosis, osteoarthritis, rheumatoid arthritis, Paget's disease, metabolic bone disease, and conditions related to the progress of benign and malignant tumors in bone tissue. Other conditions associated with elevated collagen breakdown rates include osteomalacial diseases, rickets, abnormal growth in children, renal osteodystrophy, and drug-induced osteopenia. Abnormalities in bone metabolism are also often side effects of thyroid treatment and thyroid conditions per se, such as primary hypothyroidism, thyrotoxicosis and Cushing's disease.
The prevalence of collagen-related diseases of bone and cartilage has motivated a search for biochemical methods for detecting and monitoring such diseases. Various methods for diagnosing abnormalities of bone and cartilage degradation have been proposed. For example, hydroxyproline is a major constituent of the helical regions of collagens and was an early target for study as a potential marker for collagen synthesis and degradation. However, decades of study have failed to demonstrate much utility for this metabolite, in part because of substantial metabolism of hydroxyproline in the liver.
More recently, a number of assays have been proposed based on measuring certain 3-hydroxypyridinium crosslinking species known as pyridinoline (Pyd) and deoxypyridinoline (Dpd), that are excreted in urine in peptide-free and peptide bound forms. These crosslinking species are formed in collagen by condensation of the side chains derived from either three hydroxylysyl residues (for pyridinoline), or two hydroxylysyl residues and one lysyl residues (for deoxypyridinoline) (Robins, 1982, Eyre, 1984a; Eyre, 1987).
Crosslinking sites have been identified in collagen types I, II and III, and for collagen type I include lysyl/hydroxylysyl residues at positions 9N (in the N-telopeptide), 16C (in C-telopeptide), and helical residues 87, 930(&agr;1(I)) and 933(&agr;2(I)) (Bonde et al., 1994; Hanson and Eyre, 1996; Knott and Bailey, 1998). For example, in type I collagen, pyridinium crosslinks have been reported to occur between (i) an N-telopeptide &agr;1(I) site (9N position) in a first collagen fibril, (ii) an N-telopeptide &agr;2(I) site (9N position) from a second collagen fibril, and (iii) an internal helical site from a third collagen fibril (930H or 933H); and also between two C-telopeptide sites (16C from different chains) and another helical site (87H) (Hanson et al., 1992).
The relative abundances of Pyd and Dpd are tissue-dependent. Pyd has been found in cortical bone, trabecular bone, invertebral disc, articular cartilage, aorta, and ligament tissues. Dpd is present in trace quantities in articular cartilage and absent from invertebral disc, but is present in the other tissues just mentioned, albeit at lower frequencies than Pyd. Pyd and Dpd are absent from the collagens in normal skin, and from immature and newly synthesized collagens (Robins et al., 1990). Thus, although Pyd and Dpd are both useful in assays for measuring bone collagen degradation, Dpd appears to be more specific with respect to bone collagen (e.g., Knott et al., 1998), while Pyd may be preferred for assessing breakdown of cartilage, e.g., in rheumatoid arthritis.
For nearly 20 years, total levels of Pyd and Dpd have been measured as indicators of collagen degradation using the acid hydrolysis method of Gunja-Smith and Boucek (1981). This method utilizes an acid hydrolysis step to separate the crosslinking moieties from attached collagen polypeptide chains, followed by measurement of the hydrolysed Pyd or Dpd. Although effective to convert myriad peptide fragments to discrete peptide-free forms, the acid hydrolysis step is inconvenient, particularly for automated testing, due to the lengthy sample preparation and caustic conditions employed (e.g., 3 to 12 N HCl for up to 20 hours).
Others have investigated assays based on measuring telopeptides of collagen. For example, PCT Publications No. WO 89/04491 and WO 91/08478 (Eyre) disclose a method of detecting collagen degradation by quantitating certain 3-hydroxypyridinium-containing peptides derived from the N- and C-terminal telopeptides of type I collagen. PCT Pub. No. WO 95/08115 (Osteometer) discloses an assay for collagen fragments in a biological fluid based on the measurement of collagen-derived peptides which contain potential pyridinium crosslinking sites. PCT Pub. No. WO 94/14844 (Baylink) discloses an immunoassay method for assessing bone collagen degradation. The method employs antibodies raised against an 18 amino acid peptide consisting of residues 5 through 22 of the &agr;1 C-telopeptide region, with the requirement that the antibodies bind to a contiguous sequence of at least six amino acids.
One difficulty with the foregoing telopeptide assays is that the measured peptides can be excreted as a spectrum of peptide variants, rather than as a single, discrete species, potentially reducing the precision of the assay. Second, it has been found that the levels of peptides can vary significantly over time in the same patient and among different patients, hindering assay reliability. Third, these methods do not distinguish Pyd from Dpd-containing fragments.
In PCT Publication WO 91/10141, it was disclosed that Pyd and Dpd are surprisingly present in peptide-free form as a substantial portion of excreted pyridinium crosslinks in biological fluids such as urine and serum, and that the levels of these native (i.e., non-hydrolysed) peptide-free forms are useful as indicators of collagen degradation rates in humans for a number of collagen disorders. Despite the absence of collagen peptides, the peptide-free crosslinks are specific for collagen degradation because the pyridinium crosslinks do not occur outside of collagen. Thus, in contrast to collagen peptide based assays, this method provides discrete metab
Brady Jeffrey D.
Robins Simon P.
Cook Lisa V.
Dehlinger Peter J.
Le Long V.
Mohr Judy M.
Perkins Coie LLP
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