WO2001001136A1 - Procede et kit de mesure des reticulations pyridinium totales - Google Patents

Procede et kit de mesure des reticulations pyridinium totales Download PDF

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Publication number
WO2001001136A1
WO2001001136A1 PCT/US2000/017849 US0017849W WO0101136A1 WO 2001001136 A1 WO2001001136 A1 WO 2001001136A1 US 0017849 W US0017849 W US 0017849W WO 0101136 A1 WO0101136 A1 WO 0101136A1
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Prior art keywords
sample
crosslinks
free
peptide
pyridinium
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PCT/US2000/017849
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English (en)
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Stephen L. Weitz
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Quidel Corporation
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Priority to AU57764/00A priority Critical patent/AU5776400A/en
Publication of WO2001001136A1 publication Critical patent/WO2001001136A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Definitions

  • the present invention relates to methods, reagents, and kits for measuring levels of total pyridinoline and/or deoxypyridinoline crosslinks in a fluid sample.
  • 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.
  • 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.
  • decades of study have failed to demonstrate much utility for this metabolite, in part because of substantial metabolism of hydroxyproline in the liver.
  • 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 ( ⁇ l (I)) and 933 ( ⁇ 2 (I)) (Bonde et al . , 1994; Hanson and Eyre, 1996; Knott and Bailey, 1998).
  • pyridinium crosslinks have been reported to occur between (i) an N-telopeptide ⁇ l(I) site ( 9N position) in a first collagen fibril, (ii) an N- telopeptide ⁇ 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).
  • Pyd and Dpd 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) .
  • 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)
  • Pyd may be preferred for assessing breakdown of cartilage, e.g., in rheumatoid arthritis .
  • total levels of Pyd and Dpd have been measured as indicators of collagen degradation using the acid hydrolysis method of Gunja-Smith and Boucek
  • This method utilizes an acid hydrolysis step, typically in the presence of 6 N HC1 at 108°C for 24 hours, to separate the crosslinking moieties from attached collagen polypeptide chains, followed by measurement of the hydrolysed Pyd or Dpd.
  • telopeptide fragments of collagen see PCT Publications No. WO 89/04491 and WO 91/08478 (Eyre), WO 95/08115 (Osteometer) , and WO 94/14844 (Baylink) .
  • One difficulty with the foregoing telopeptide assays is that the measured peptides can be excreted as a spectrum of peptide variants, potentially reducing the precision of the assay.
  • the levels of peptides can vary significantly over time in the same patient and among different patients, hindering assay reliability.
  • these methods do not distinguish Pyd from Dpd-containing fragments .
  • the peptide-free forms of Pyd and Dpd can be measured in non-hydrolyzed samples as taught in PCT Publication No. WO 91/10141 (Robins), for example .
  • the measurement of total Pyd and Dpd crosslinks after sample acid hydrolysis has continued to be used as a urinary indicator of collagen degradation.
  • this method has been limited due to the caustic conditions employed, and the relatively long incubation times necessary for sample conversion.
  • current HC1 methods frequently are limited in their sensitivity so that dilute samples, such as serum or plasma, cannot be reliably assayed.
  • the present invention includes, in one aspect, a method for forming peptide-free pyridinium crosslinks.
  • a sample having one or more collagen peptides containing pyridinium crosslinks selected from the group consisting of pyridinoline, deoxypyridinoline, or both is mixed with at least one acid reagent having a pKa less than or equal to 2 and (ii) at least one reducing agent, such that the acid reagent and reducing agent may be separate compounds or may be combined in a single compound.
  • the reducing agent contains a containing a free thiol group.
  • the method is effective to produce a pyridinium crosslink mixture in which at least 80% of total pyridinium crosslinks in the hydrolysed sample are present as the peptide-free forms.
  • at least 90%, and more preferably, at least 95% of the pyridinium crosslink mixture after proteolysis are present as the peptide-free forms (Pyd and/or Dpd) .
  • the heating step is performed simultaneously on a plurality of samples using a multiwell microtiter plate format.
  • the acid reagent is any acidic compound that has a pKa less than or equal to 2, and that is capable of cleaving peptide bonds while leaving the structure of the pyridinium crosslinks, Pyd and Dpd, intact.
  • the acid reagent comprises HC1.
  • the acid reagent and reducing agent together comprise a mercaptoalkylsulfonic acid, which may be present, for example, at a concentration of from about 0.1 M to about 2 M.
  • a preferred mercaptoalkylsulfonic acid is mercaptoethanesulfonic acid (Mesna) .
  • the present invention may be used with a variety of samples.
  • the sample is a blood sample, such as a plasma or serum sample.
  • the sample is a urine sample.
  • the selected peptide-free pyridinium crosslinks can be measured by any method available in the art.
  • the crosslinks are measured by immunoassay using suitably specific antibodies.
  • the antibodies may be monoclonal or polyclonal, or can be provided as Fc fragments or the like.
  • the crosslinks are measured chromatographically, by capillary electrophoresis, or by chemical derivatization followed by spectrophotometric or fluorescence detection.
  • the sample is preferably a body fluid sample, and more preferably is serum or urine.
  • the invention also includes a method for measuring a level of pyridinoline and/or deoxypyridinoline crosslinks in a sample.
  • a sample having one or more collagen peptides containing pyridinium crosslinks selected from the group consisting of pyridinoline, deoxypyridinoline, or both is mixed with (i) at least one acid reagent having a pKa less than or equal to 2 and (ii) at least one reducing agent, such that the acid reagent and reducing agent may be separate compounds or may be combined in a single compound.
  • the level (s) of the selected peptide-free pyridinium crosslinks in the mixture are measured.
  • the invention includes a method of screening for or monitoring bone and/or cartilage collagen degradation activity in a mammalian subject.
  • a sample having one or more collagen peptides containing pyridinium crosslinks selected from the group consisting of pyridinoline, deoxypyridinoline, or both is mixed with (i) at least one acid reagent having a pKa less than or equal to 2 and (ii) at least one reducing agent, such that the acid reagent and reducing agent may be separate compounds or may be combined in a single compound.
  • the level of selected peptide-free pyridinium crosslinks in the mixture is measured.
  • a measured level that is above that characteristic of normal subjects indicates the presence of an elevated rate of bone or cartilage collagen degradation in the subject.
  • the invention includes a kit for measuring collagen pyridinium crosslinks in a sample.
  • the kit preferably includes (i) one or more acid reagents having a pKa less than or equal to 2, (ii) at least one reducing agent, such that the acid reagent and reducing agent may be separate compounds or may be combined in a single compound.
  • the kit may also include a binding partner that is specific for peptide-free pyridinoline and/or peptide-free deoxypyridinoline.
  • the kit may additionally include one or more of the following: a predetermined amount of peptide-free pyridinoline, peptide-free deoxypyridinoline, or both; a urine or blood sample containing a known amount of Pyd and/or Dpd crosslinks; a stock solution of an alkaline base; and a buffer; all of which may be provided in suitable containers.
  • a predetermined amount of peptide-free pyridinoline, peptide-free deoxypyridinoline, or both a urine or blood sample containing a known amount of Pyd and/or Dpd crosslinks
  • a stock solution of an alkaline base and a buffer; all of which may be provided in suitable containers.
  • the kit is particularly useful for practicing methods described above.
  • the present invention is based in part on the applicant' s discovery of sample hydrolysis conditions that can significantly increase the recovery of peptide-free Pyd and Dpd crosslinks relative to previous methods, particularly for samples derived from blood, such as serum and plasma.
  • the invention can thus provide significantly improved sensitivity, accuracy, and rapidity in methods where the total levels of Pyd and Dpd crosslinks are of interest .
  • pyridinoline refers to the compound shown at I below, where the pyridinium ring nitrogen derives from the ⁇ -amino group of a hydroxylysyl residue .
  • Dpd refers to the compound shown at II below, where the pyridinium ring nitrogen derives from the ⁇ - amino group of a lysyl residue.
  • Free crosslinks and “peptide-free crosslinks” refer to compound I, compound II, or both, i.e., pyridinoline and/or deoxypyridinoline crosslink species, free from covalently attached amino acids, peptides, and glycosyl groups .
  • "Glycosylated pyridinoline” or “glyco-Pyd” refers to glycosylated forms of compound I, which may be peptide- free or peptide-bound, wherein glycosyl groups are covalently bound to the aliphatic hydroxyl group.
  • Two exemplary glyco-Pyd compounds are Gal -Pyd and Glc-Gal-Pyd.
  • N-Pyd refers to native (non-hydrolysed) , non- glycosylated, peptide-free Pyd.
  • N-Dpd refers to native (non- hydrolysed), peptide-free Dpd.
  • Pyd-peptides refers to peptide forms of compound I, which may or may not contain glycosylated forms of Pyd, in which one or more of the three Pyd amino acid moieties are linked by peptide linkages to additional amino acid residues from collagen.
  • Dpd-peptides refers to peptide-bound forms of compound II, in which one or more of the three Dpd amino acid moieties are linked via peptide linkages to additional amino acid residues.
  • Pyridinium-peptides and “peptide-bound forms” refer to Pyd-containing peptides and/or Dpd-containing peptides .
  • Pyd crosslinks refers collectively to peptide-free Pyd, glycosylated peptide-free Pyd, and Pyd-containing peptides.
  • Dpd crosslinks refers collectively to peptide-free Dpd and Dpd-containing peptides.
  • Pyridinium crosslinks refers to Pyd crosslinks and Dpd crosslinks.
  • Total Pyd or “T-Pyd” refers to the total quantity (or concentration) of Pyd crosslinks present in a sample.
  • total Dpd or “T-Dpd” refers to the total quantity of Dpd crosslinks that are present.
  • mamal has its standard meaning, and includes humans, dogs, cats, horses, cows, sheep, pigs, rabbits, guinea pigs, rats, and mice, for example.
  • Peptide and polypeptide are used interchangeably to denote chemical entities containing two or more amino acids (of which Pyd and Dpd are examples) linked by one or more peptide bonds .
  • Body fluid refers to any body fluid that contains Pyd and/or Dpd crosslinks.
  • exemplary body fluids include blood, serum, plasma, urine, saliva, synovial fluid, cerebrospinal fluid, and sweat, which may be subjected to one or more purification steps prior to hydrolysis or analysis .
  • Bone resorption abnormality or “bone resorption condition” refers to a condition characterized by an elevated level of bone degradation (resorption) in a mammalian subject.
  • Bone resorption conditions include osteoporosis, osteoarthritis, rheumatoid arthritis, primary hyperparathyroidism, hyperthyroidism, Paget ' s disease, bone cancers (e.g., metastases in bone), osteomalacia, rickets, renal osteodystrophy, and drug- induced osteopenia, for example.
  • pyridinium crosslinks from collagen occur in body fluids as heterogeneous mixtures of pyridinium-containing peptides as well as peptide-free forms, wherein the peptide forms may contain a variety of attached amino acid residues and polypeptide chains of varying lengths and compositions.
  • the present invention provides an improved method for converting all or substantially all of the many peptide forms to the peptide-free (and non-glycosylated) crosslinks conveniently and in high yield.
  • the invention includes a method for forming peptide-free pyridinium crosslinks.
  • a sample having one or more collagen peptides containing pyridinium crosslinks selected from the group consisting of pyridinoline, deoxypyridinoline, or both is mixed with at least one acid reagent having a pKa less than or equal to 2 and (ii) at least one reducing agent (which preferably contains a free thiol) , such that the acid reagent and reducing agent may be separate compounds or may be combined in a single compound.
  • the mixture is then heated for a time sufficient to convert substantially all of the pyridinium crosslinks to their peptide-free forms .
  • the method is effective to produce a pyridinium crosslink mixture in which at least 80% of total pyridinium crosslinks in the hydrolysed sample are present as the peptide-free forms. Preferably at least 90%, and more preferably, at least 95% of the pyridinium crosslink mixture after proteolysis are present as the peptide-free forms (Pyd and/or Dpd) .
  • the hydrolysis mixtures can be placed in heat resistant vials with air tight caps.
  • the heating step is performed simultaneously on a plurality of samples using a multiwell microtiter plate format, such as a heat resistant 96-well microtiter plate of the type used for the polymerase chain reaction (PCR) .
  • the acid reagent is any acidic compound that has a pKa less than or equal to 2, and that is capable of cleaving peptide bonds while leaving the structure of the pyridinium crosslinks, Pyd and Dpd, intact.
  • Exemplary acid reagents for use in the invention include HC1, organic sulfonic acids, sulfuric acid, organic phosphonic acids, and phosphoric acid, for example, which are typically provided as aqueous concentrates .
  • Organic sulfonic acids are preferably of the form RS0 2 H, where R is a linear, branched, or cyclic Ci to C 5 alkyl group which may include at least one polar group, such as a hydroxyl or amino group, to impart greater water solubility; or R is a C ⁇ to C ⁇ aryl or arylalkyl group such as phenyl, p- methylphenyl, m-methylphenyl, benzyl, or phenethyl.
  • organic phosphonic acids are preferably of the form RP0 3 H 2 , where R is an alkyl, aryl, or arylalkyl group as just described.
  • Preferred acid reagents include HC1 and Ci to C 5 alkylsulfonic acids, such as methane sulfonic acid and ethane sulfonic acid, for example.
  • the reducing agent can be any reducing reagent that can withstand the elevated temperature used for sample hydrolysis (e.g., up to 150°C) and which leaves intact the structure of the pyridinium crosslinks, Pyd and Dpd.
  • the reducing agent is provided as a small molecule having a molecular weight less than 1000 daltons.
  • the reducing agent contains a thiol group or is a mild borohydride, such as sodium borohydride.
  • the reducing agent is an alkyl thiol of the form RSH, wherein R is a Ci to C 5 alkyl group which may include one or more polar groups, such as hydroxyl and amino.
  • Preferred reducing agents include 2- mercaptoethanol (BME) , dithiothreitol (DTT) , dithioerythreitol, 2-mercaptoethylamine, cysteine, thio- glycolic acid, thioacetic acid, and mercaptoalkylsulfonic acids such as Mesna.
  • the acid reagent and reducing agent can be combined in a single molecule, such as a Ci to C 5 mercaptoalkylsulfonic acid, of which mercaptoethanesulfonic acid (Mesna) is a preferred example .
  • a Ci to C 5 mercaptoalkylsulfonic acid of which mercaptoethanesulfonic acid (Mesna) is a preferred example .
  • the appropriate concentrations of the acid reagent and reducing agent used in the hydrolysis mixture are dependent on the temperature and duration of the hydrolysis step. Generally, lower concentrations can be used with higher hydrolysis temperatures or a longer duration of hydrolysis.
  • the acid reagent is present in the hydrolysis mixture at a concentration of from about 1 M to about 6 M, and preferably from about 1 M to about 3 M.
  • the reducing agent is typically present at a concentration of from about 0.1 M to about 6 M, preferably from about 0.1 M to about 3 M.
  • the acid reagent and reducing agent together include from about 1 M to about 6 M HC1 and from about 0.1 to about 2 M of a mercaptoalkylsulfonic acid, which is preferably mercaptoethanesulfonic acid (Mesna) .
  • a mercaptoalkylsulfonic acid which is preferably mercaptoethanesulfonic acid (Mesna) .
  • the present invention can be used with any sample that is suspected of having pyridinium-containing peptides.
  • the sample is preferably a tissue sample or a body fluid sample, such as urine, blood (e.g., serum or plasma), or saliva, for example.
  • body fluids such as synovial fluid, cerebrospinal fluid, and sweat are also contemplated.
  • the method may also be used with tissue and cell samples, such as from bone, cartilage, and muscle, and also tissue culture supernatants .
  • the sample may be subjected to one or more pre-purification steps to remove extraneous materials, if desired.
  • crosslink-containing samples and reaction mixtures are preferably protected from light until measurement is complete, e.g., using amber-colored containers and low light conditions, to minimize photo-degradation of the pyridinium moieties.
  • the sample is preferably reacted with the acid reagent and reducing agent under conditions effective to cleave the crosslinks from substantially all of any attached collagen amino acids, peptides, and glycosyl groups.
  • the heating temperature for hydrolysis is usually at or above about 80°C, typically from about 80°C to about 150°C, preferably from about 95°C to about 150°C, and more preferably from about 95°C to about 120°C.
  • the hydrolysis can be conducted overnight, e.g., for a period of 16 to 20 hours. More generally, the heating step can be performed for from 15 minutes to 7 days, and usually for from 1 hour to 24 hours.
  • the hydrolysis mixture Prior to heating, the hydrolysis mixture can be allowed to stand at ambient temperature (e.g., 4°C to 30°C) to allow non-pyridinium polypeptides and other sample constituents to precipitate from the mixture.
  • ambient temperature e.g. 4°C to 30°C
  • precipitation is nearly instantaneous or occurs within a few minutes.
  • precipitation may occur more slowly or not at all, particularly for dilute samples and samples that are less complex in their compositions than serum.
  • an incubation time of from 1 minute to 60 minutes, preferably from 2 minutes to 15 minutes can be used for precipitation.
  • This precipitation step can be useful to remove substances that do no contain the pyridinium crosslinks of interest, and also to enrich the mixture for pyridinium crosslink species to simplify later analysis.
  • the supernatant can be heated as above to effect sample hydrolysis .
  • the acid reagent and reducing agent are provided as stock solutions that provide desired final concentrations upon admixture with the sample.
  • the stock solution (s) of acid reagent and reducing agent should be at least twice as concentrated as the desired final concentrations. More generally, the sample can be mixed with the acid reagent and reducing agent in any proportion that is suitable for the purposes of the user.
  • Hydrolysis is complete when the desired amount of cleavage has occurred.
  • the duration of hydrolysis can be selected according to prior experiments to determine the rate of cleavage for the selected sample type and proteolytic conditions, by incubating the sample with acid and reducing agent and withdrawing aliquots periodically to measure the levels of peptide-free pyridinium crosslinks as a function of time.
  • hydrolysis is allowed to occur for between 15 minutes and 24 hours, depending on the concentration of acid and the temperature of the reaction mixture, although longer and shorter time periods are also contemplated.
  • the hydrolysis mixture is allowed to cool.
  • the hydrolysis mixture is neutralized with a alkaline base stock solution, such as aqueous sodium hydroxide or potassium hydroxide.
  • the base stock solution is highly concentration, e.g., is a 1 to 10 N solution of base, to help minimize sample dilution.
  • a buffer can be added after neutralization to ensure that the final pH of the mixture after hydrolysis is not too extreme, i.e., not to acidic or basic for subsequent manipulation.
  • the pH of the buffer may vary, but is usually from 5 to 9, more preferably from 6 to 8.
  • the buffer stock is sufficiently concentrated to produce an adequate buffering capacity to overcome any excess acid or base in the mixture.
  • the buffer stock can contain buffer at a concentration of from 50 to 1000 mM, preferably from 100 to 500 mM.
  • Exemplary buffers include Tris, MES, TES, and phosphate.
  • the level (s) of the selected peptide-free pyridinium crosslinks can be measured by any suitable technique, such as immunoassay, chromatography, or capillary electrophoresis .
  • Pyd and Dpd may be measured by immunoassay techniques employing antibodies specific for peptide-free Pyd, Dpd, or both.
  • the antibodies may be monoclonal or polyclonal, as described further below. With regard to specificity, the antibodies should be sufficiently specific for the selected crosslinks (Pyd and/or Dpd) to avoid spurious results due to binding other components in the sample.
  • Binding affinity can be determined by known methods, e.g., by Scatchard analysis using an ELISA assay (Campbell, 1991; Segel, 1976) . Accordingly, in one embodiment, the antibody has a binding affinity constant for the selected free crosslink species of greater than about 1 x 10 7 /molar, and preferably greater than about 1 x 10 8 /molar.
  • the screening process for antibodies may be based on additional binding criteria, such as low affinities for amino acids, polypeptides, and/or other possible sample components.
  • additional binding criteria such as low affinities for amino acids, polypeptides, and/or other possible sample components.
  • a high affinity for the selected free crosslinks (Pyd and/or Dpd) in combination with a relatively low affinity for Pyd or Dpd peptide forms is thus one additional criterion that can be used in the screening process.
  • the antibodies have a ratio of reactivity toward the selected peptide-free pyridinium crosslink (s) and urinary pyridinium peptides larger than 1,000 daltons in molecular weight, of greater than about 3:1, more preferably greater than about 5:1.
  • the antibodies can be tested for cross-reactivity with free amino acids.
  • an amino acid mixture comprising all 20 standard amino acids at selected concentrations can be used, such as the amino acid mixture employed in Example 10 of WO 94/03814.
  • the antibodies for use in the invention may be specific for Pyd, Dpd, or both, including antibodies which are specific for one and have moderate crossreactivity (e.g., 40%) with the other.
  • the antibody is highly specific for Pyd, the antibody preferably has a ratio of reactivity toward peptide-free pyridinoline and petpide-free deoxypyridinoline of greater than about 5:1, preferably greater than about 20:1, and more preferably greater than about 100:1.
  • the antibody preferably has a ratio of reactivity toward peptide-free deoxypyridinoline and peptide-free pyridinoline of greater than about 5:1, preferably greater than about 25:1, and more preferably greater than about 100:1.
  • the antibody preferably has a ratio of reactivity toward peptide-free pyridinoline and peptide- free deoxypyridinoline of between about 2:1 and 1:2. Antibodies having such properties, and methods for preparing them, are disclosed in PCT Pub. Nos. WO 96/27134 (Kung et al .
  • Reaction of sample with specific antibodies can be carried out by any of a variety of immunoassay configurations known in the art, including homogeneous and heterogeneous assay formats. Any appropriate technique for detection can be used, such as radioimmunoassay, coupled enzymes, UV-VIS absorbance, fluorescence, chemiluminescence, or an EMIT configuration.
  • One preferred reporter is alkaline phosphatase, which can react with a p-nitrophenylphosphate substrate to produce a colored product having a strong absorption peak at 405 nm.
  • the reporter is horse radish peroxidase.
  • a biotin-labeled second antibody in combination with a reporter-labeled streptavidin may be employed, such as a biotin-labeled second antibody in combination with a reporter-labeled streptavidin .
  • a known volume, typically 10-50 ⁇ L, of sample is added to an analyte-coated solid support, e.g., the wells in a microtitre plate, and sample addition is followed by addition of a known volume, typically 50-200 ⁇ L, of analyte-specific antibody of a known dilution.
  • the mixture on the solid support surface is then incubated, preferably under conditions effective to achieve equilibrium between the antibody binding to sample crosslinks and surface-bound analyte (e.g., overnight at 2-8°C or at room temperature for several hours) .
  • the solid support is washed several times to remove antibody not specifically bound to the support, and is then incubated with an enzyme-labeled anti-IgG antibody effective to bind specifically to support-bound antibody.
  • an enzyme-labeled anti-IgG antibody effective to bind specifically to support-bound antibody.
  • the enzyme-labeled antibody can be goat anti-rabbit IgG conjugated with alkaline phosphatase.
  • the enzyme-labeled antibody can be a goat anti-mouse IgG derivatized with alkaline phosphatase .
  • the support is again washed to remove non-specifically bound material, and the level of enzyme bound to the support is determined by addition of enzyme substrate, with spectrophotometric determination of converted substrate.
  • standards containing a range of analyte concentrations are added in duplicate to some of the wells, to generate a standard curve. Up to 40 samples are then added in duplicate to remaining wells, and the wells are then assayed as above.
  • the immunoassay utilizes a competitive, heterogeneous immunoassay format in which the reporter label for detection of the immunocomplex is directly attached to either a competitor analyte or to the analyte-specific antibody.
  • the analyte-specific antibodies are immobilized on a solid support, and enzyme- labeled analyte is added to compete with peptide-free crosslinks in the sample, for binding to the immobilized antibodies.
  • the enzyme label can be alkaline phosphatase or horse-radish peroxidase, for example.
  • analyte is immobilized on a solid support to compete with peptide-free crosslinks in the sample for binding to non-immobilized enzyme-labeled antibody.
  • the crosslinks can be measured using an automated immunoassay cassette, as described in PCT Pub. No. WO 98/37416 (Jones) .
  • the crosslinks can be measured by fluorescence detection based on their intrinsic fluorescence properties. Pyd and Dpd strongly fluoresce with peak emission at 390-400 nm when subjected to an excitation source at about 297 nm. Chromatographic (Black et al., 1988; Eyre et al . , 1984b, James et al . , 1993) and capillary electrophoresis techniques (James et al . , 1991) for measuring Pyd and Dpd have been described. The crosslinks may also be measured based on UV-absorbance properties as described by Colwell et al. (1992).
  • the level of measured crosslinks may be normalized using a measured level of creatinine or any equivalent thereof, by conventional methods .
  • Blood samples may be converted to serum or plasma by known methods, and may be subjected to further pre-processing if desired.
  • serum can be passed through a spin-filter having a defined molecular weight cutoff to remove proteins of a selected size from the sample prior to assay.
  • the hydrolysis method of the invention can be adapted to a method of screening for or monitoring bone and/or cartilage collagen degradation activity in a mammalian subject.
  • a non-hydrolysed body fluid sample is contacted with an acid and reducing agent as described above under conditions effective to cleave peptide-bound pyridinium crosslink species from one or more attached collagen amino acids and peptides.
  • the level of peptide-free pyridinoline and/or deoxypyridinoline is measured, wherein a measured level that is above that characteristic of normal subjects indicates the presence of an elevated rate of bone or cartilage collagen degradation in the subject.
  • control group may be tailored according to the characteristics of the population to be tested. For example, the control group may be limited to a particular age group, e.g. 25-55 year old males, or 25-44 year old premenopausal females, to obtain baseline levels. Other parameters of interest may include the subjects' weight, race, or gender for example.
  • the determined average or range of free crosslinks in the normal subjects is then used as a benchmark for detecting above-normal levels indicative of an abnormally elevated rate of collagen degradation.
  • the invention can also be applied as a prognostic indicator for likelihood of bone fracture, such that the likelihood of fracture increases in proportion to the degree of elevation of the measured crosslink level.
  • the invention also includes a kit for measuring collagen pyridinium crosslinks in a sample.
  • the kit preferably includes a strong acid which can effectively cleave the amino acid groups of pyridinoline and deoxypyridinoline crosslinks from attached collagen amino acids and peptides, and also a mild reducing agent such as discussed above.
  • Preferred acids include HC1 and sulfonic acids, such as methane sulfonic acid or ethane sulfonic acid.
  • Preferred reducing agents include 2-mercaptoethanol (BME) , dithiothreitol (DTT) , 2-mercaptoethylamine, and cysteine.
  • the strong acid and reducing agent are combined in a single molecule, such as a mercaptoalkylsulfonic acid, of which mercaptoethanesulfonic acid is a preferred example.
  • An alkaline base stock solution such as aqueous sodium hydroxide or potassium hydroxide, can also be included for neutralizing the acid hydrolysis mixture after hydrolysis has been completed.
  • the kit can also include a buffer for neutralizing excess acid or base, such as described above, and/or a hydrolysis control containing a known amount of Pyd or Ddp crosslinks, such as urine or blood (e.g., plasma or serum) .
  • the kit may further include a binding partner, such as an antibody or any equivalent thereof, that is specific for the pyridinoline and/or deoxypyridinoline.
  • the kit may additionally include a predetermined amount of peptide-free pyridinium crosslinks selected from the group consisting of peptide-free pyridinoline, deoxypyridinoline, or both, for generating a standard curve.
  • the kit may include an antibody of the type described above, and any other suitable reagents for carrying out the assay.
  • the immunoassay format may be heterogeneous or homogeneous, and can have a competitive or non-competitive format such as discussed above.
  • the kit may further include instructions for conducting the steps of an assay method for the selected crosslinks.
  • the present invention is useful for converting heterogeneous pyridinium crosslink mixtures to a more homogeneous form (i.e., peptide-free) in which pyridinium crosslinks are easier to quantify.
  • a substantial proportion preferably greater than about 80%, more preferably greater than 90% or 95%) of the crosslinks is provided in peptide-free form
  • the method provides a useful measure of the total level of the selected pyridinium crosslinks in the sample.
  • the invention also provides a method of screening for or monitoring bone and/or cartilage collagen degradation activity in a mammalian subject.
  • the method may be used in a screening embodiment or to detect (diagnose) non- invasively the presence of a bone or cartilage disorder characterized by above-normal collagen degradation.
  • Exemplary disorders for which the invention may be used include osteoporosis, osteoarthritis, rheumatoid arthritis, and conditions related to the progress of benign and malignant tumors of bone, and metastatic cancers that have migrated to bone cells from elsewhere in the body, e.g., from prostate or breast initial tumors.
  • Other conditions of interest include osteomalacial diseases, rickets, abnormal growth in children, renal osteodystrophy, and drug-induced osteopenia.
  • the method may also be used to monitor the progress of an ongoing bone collagen disorder over time, or to monitor a subject's response to therapeutic treatment.
  • a number of anti-resorptive therapies are now under development or are already available for which the invention will be useful, such as alendronate and pamidronate-based therapeutic regimens.
  • the method may be used in the context of metastatic cancer conditions, to determine whether a primary cancer has spread to the subject's bone tissue, and whether a subject is responding to treatment. It will be appreciated that the method may also be used with other diagnostic methods, such as radiographic techniques, ultrasound, and assays directed to other indicators of bone resorption and/or formation status, to provide a fuller picture of the subject's status.
  • the invention provides a simple method for converting peptide-bound pyridinium crosslinks in a sample to peptide-free forms with high crosslink yield.
  • the resultant peptide-free crosslinks can then be measured easily and conveniently by available methods.
  • the invention thus provides an improvement for measuring total pyridinium crosslinks in a sample.
  • TDpd Assay Buffer 500 mM Na phosphate buffer (pH 7.2, 25 mL)
  • TDpd Controls 0.5 mL of human urine or bovine serum in lyophilized form, containing known amounts of Pyd and/or Dpd crosslinks
  • Serum samples are collected using standard venipuncture techniques and should be processed to avoid hemolysis. Serum samples may be kept refrigerated (2-8°C) for storage of less than 4 days, or frozen at ⁇ -20°C for longer periods. Samples should not be subjected to more than 3 freeze/thaw cycles. Prolonged exposure to light, especially direct sunlight, should be avoided. Usually, samples are not affected by normal, artificial laboratory lighting .
  • the lyophilized serum control material is reconstituted by adding 0.5 mL of deionized water to the vial of lyophilized material and allowing it to stand for 30 minutes, followed by vortexing before use. Unused reconstituted hydrolysis control solution can be stored at 2-8°C for up to 2 weeks, or at ⁇ -20°C for longer periods .
  • Urine Hydrolysis Samples Urine samples are collected using preservative-free first morning void (FMV) or second morning void (SMV) collections. It is recommended that collections be made prior to 10:00 a.m. to obviate any potential influence of diurnal variation. Urine samples are preferably refrigerated at 2-8°C for storage of less than 7 days, or frozen below -20°C for longer periods. Samples should not be subjected to more than 5 freeze/thaw cycles .
  • FMV first morning void
  • SMV second morning void
  • the lyophilized urine control material is reconstituted by adding 0.5 mL of deionized water to the vial of lyophilized material and allowing it to stand for 30 minutes, followed by vortexing before use. Unused reconstituted hydrolysis control solution can be stored at 2-8°C for up to 2 weeks, or at ⁇ -20°C for longer periods .
  • PCR thermocycler If a Metra Hydrolysis Block is used, the lid is placed over the block and sealed with screws, and the sealed block is placed on an insulator (e.g., flat plastic test tube holder, oven hotpad, etc.) in an oven to avoid direct contact of the hydrolysis block with the oven shelf. Heat at 99°C for 18-20 hours.
  • an insulator e.g., flat plastic test tube holder, oven hotpad, etc.
  • the following procedure can be used in conjunction with a Pyrilinks-D assay kit and reagents available from Metra Biosystems, Inc. (Part No. 8007) .
  • the Pyrilinks assay is a competitive enzyme immunoassay in a microtiter strip-well format utilizing a monoclonal antibody coated on the strip to capture Dpd. Dpd in the sampe competes with conjugated Dpd-alkaline phosphatase for the antibody and the reaction is detected with a pNPP substrate.
  • Wash Buffer 1:10 with deionized water Store at room temperature (20-28°C) . Use IX Wash Buffer within 24 hours of preparation, or else store at 2-8°C for greater longevity.
  • Enzyme Conjugate Prepare Enzyme Conjugate within 2 hours of use. Reconstitute each required vial of Enzyme
  • the Substrate Buffer should be brought to room temperature (20-28°C) before beginning the assay (two hours to overnight recommended) .
  • Dpd standard solutions are prepared that contain Dpd purified from bovine bone in 10 mM phosphoric acid containing sodium azide (0.05% w/v) . The Dpd concentrations are 0, 1, 3, 10, 30, and 100 nM. Dilute the low and high control solutions by mixing 50 ⁇ L of each control with 100 ⁇ L of deionized water. Add 50 ⁇ L of each control solution (diluted if necessary) to containers or a dilution plate. Then add 50 ⁇ L of Acid Reagent followed by 25 ⁇ L of TDPD Assay Buffer and 25 ⁇ L of Base Reagent. Close lid and vortex. Use directly for the urine and serum assays.
  • Controls Reconstitute Urine or Serum Hydrolysis Control with 0.5 mL deionized water and let stand for 30 minutes. Store reconstituted control at 4°C for 2 weeks or -20°C indefinitely.
  • D) / (1+ (x/C) ⁇ B) +D) can be used to analyze Serum or Urine Total Dpd assay results.

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Abstract

La présente invention concerne un procédé de formation de réticulations pyridinium exemptes de peptide. Dans ce procédé, un échantillon contenant un ou plusieurs peptides collagène à réticulations pyridinium est mélangé avec: a) au moins un réactif acide présentant un pKa inférieur ou égal à 2 et b) au moins un agent réducteur, contenant de préférence un thiol libre, de sorte que le réactif acide et l'agent réducteur puissent être des composés séparés ou combinés en un seul composé, ce mélange étant chauffé pendant un temps suffisamment long pour transformer sensiblement la totalité des réticulations pyridinium en leur forme exempte de peptide. Ce procédé permet d'obtenir des rendements de réticulation accrus et une sensibilité améliorée par rapport aux procédés connus, en particulier pour les échantillons sanguins. En outre, cette invention comprend un procédé de mesure des réticulations pyridinoline et/ou desoxypyridinoline dans un échantillon, des procédés de détermination du taux de dégradation de collagène chez un sujet, ainsi que des kits et des réactifs correspondants.
PCT/US2000/017849 1999-06-30 2000-06-28 Procede et kit de mesure des reticulations pyridinium totales WO2001001136A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10851336B2 (en) 2014-06-18 2020-12-01 Luminex Corporation Apparatus and methods for magnetic mixing

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994003814A2 (fr) * 1992-07-31 1994-02-17 Metra Biosystems, Inc. Procede et materiel de reticulation en presence d'ions pyridium

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994003814A2 (fr) * 1992-07-31 1994-02-17 Metra Biosystems, Inc. Procede et materiel de reticulation en presence d'ions pyridium

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10851336B2 (en) 2014-06-18 2020-12-01 Luminex Corporation Apparatus and methods for magnetic mixing

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