WO1999009058A1 - Isolated osteocalcin fragments - Google Patents

Isolated osteocalcin fragments Download PDF

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WO1999009058A1
WO1999009058A1 PCT/FI1998/000550 FI9800550W WO9909058A1 WO 1999009058 A1 WO1999009058 A1 WO 1999009058A1 FI 9800550 W FI9800550 W FI 9800550W WO 9909058 A1 WO9909058 A1 WO 9909058A1
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Prior art keywords
fragment
amino acid
hoc
osteocalcin
carboxylated
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PCT/FI1998/000550
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English (en)
French (fr)
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Jukka Hellman
Sanna-Maria KÄKÖNEN
Matti Karp
Timo Lövgren
H. Kalervo VÄÄNÄNEN
Kim Pettersson
Original Assignee
Jukka Hellman
Kaekoenen Sanna Maria
Matti Karp
Loevgren Timo
Vaeaenaenen H Kalervo
Kim Pettersson
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Application filed by Jukka Hellman, Kaekoenen Sanna Maria, Matti Karp, Loevgren Timo, Vaeaenaenen H Kalervo, Kim Pettersson filed Critical Jukka Hellman
Priority to JP2000509736A priority Critical patent/JP2001514875A/ja
Priority to EP98929457A priority patent/EP1003778A1/en
Publication of WO1999009058A1 publication Critical patent/WO1999009058A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • This invention relates to an isolated osteocalcin fragment derived from human urine, a monoclonal antibody or recombinant antibody fragment capable to bind said fragment, a cell line producing said monoclonal antibody, and an immunoassay for quantitative determination of said fragment. Furthermore, the invention concerns a method for the measurement of the rate of bone turnover (formation and/or resorption) and/or for the investigation of metabolic bone disorders.
  • hOC Human osteocalcin
  • BGP bone Gla protein
  • Peptide bonds between arginine residues 19 and 20 and between residues 43 and 44 are susceptible to tryptic hydrolysis leading to peptides 1-19, 20- 43, 45-49, 1-43, and 20-49 which may be the main products of hOC breakdown in the circulation (Farrugia and Melick, Calcif Tissue Int 1986; 39:234-8, Hellman et al. J Bone Miner Res 1996; 11 : 1 165-75 and Garnero et al. J Bone Miner Res 1994; 9:255-4).
  • Multiple immunoreactive forms of hOC have been discovered in circulation (Garaero et al. J Bone Miner Res 1994; 9:255-4) and also in urine (Taylor et al.
  • the fragments of hOC can be produced either during osteoclastic degradation of bone matrix or as the result of the catabolic breakdown of the circulating protein after synthesis by osteoblasts. There is evidence that the production of some of the fragments found in urine occurs before renal clearance and is not a result of it (Taylor et al. J Clin Endocrin Metab. 1990; 70:467-72). Because of the rapid clearance from the circulation by glomerular filtration, the shOC (serum human OC) could reflect the acute changes in bone metabolism, while some of the uhOC (urine human OC) fragments might serve as an index of long term changes (Price et al. J Biol Chem 1980; 256: 12760-6).
  • the first reported measurement of urine osteocalcin (Taylor et al. J Clin Endocrin Metab. 1990; 70:467 - 72) is based on competitive RIA utilizing polyclonal guinea pig antihuman OC antibodies for recognizing the immunoreactive OC fragments (Taylor et al. Metabolism 1988; 37:872-7.).
  • the assay is said to be specific for the midmolecule epitope of hOC molecule according to information obtained from crossreactivity tests with tryptic fragments and synthetic peptide. The probable epitope for polyclonal antibody recognition is determined quite widely by the authors.
  • the binding site of antisera is located in the midmolecule of the protein and probably involves amino acid 19 and at least a portion of the N-terminal sequence of the 20-43 tryptic digest fragment prior to amino acid 37.
  • the assay is unable to distinquish decarboxylated hOC from carboxylated hOC in other words it is not dependent on the ⁇ -carboxylation degree of glutamic acids 17, 21 and 24 in hOC. Furthermore the detailed characterization of the fragments detected by the assay is missing. In addition, this assay is not suitable for routine measurement of urine because the desalting of urine samples before measurement is inevitable for the proper function of RIA.
  • the concentration of serum osteocalcin is increased.
  • the level of increase is partly dependent on the differences in the osteocalcin assays employed or the population studied, but is generally about 30 -50 % above the premenopausal values (Ravn et al. Bone 1996; 19:291-8, Bonde et al. J Clin Endocrinol Metab 1995; 80:864-8, Garnero et al. J Bone Miner Res 1996; 11:337-49, Akesson et al. J Bone Miner Res 1995; 1823-9).
  • HRT hormon replacement therapy
  • This invention relates to an isolated osteocalcin fragment derived from human urine, said fragment being characterized in that at least one of the glutamic acids in the position 17, 21 and 24 of the amino acid sequence
  • the invention relates to a monoclonal antibody or recombinant antibody fragment having the capability of binding the human gamma- carboxylated osteocalcin fragment as defined above.
  • the invention concerns a cell line producing said monoclonal antibody.
  • the invention relates to an immunoassay for quantitative determination of a gamma-carboxylated osteocalcin fragment defined above, said immunoassay being characterized in that a sample containing said fragment is exposed to a monoclonal antibody or recombinant antibody fragment which binds said gamma-carboxylated osteocalcin fragment.
  • the invention relates to a method for the measurement of the rate of bone turnover (formation and/or resorption) and/or for the investigation of metabolic bone disorders in an individual, said method being based on the quantitative determination of an osteocalcin fragment as defined above.
  • Figure 1 A shows the nucleic acid and the a ino acid sequences of the synthetic human osteocalcin insert.
  • Figure 1 B shows plasmid vector pGEX-3X (Pharmacia). The arrow indicates the Smal-ligation site of the hOC insert. The pfXa (protease factor Xa) cleavage site is located after the Ile-Glu-Gly-Arg -sequence.
  • Figure 2 A shows SDS-PAGE and Figure 2 B shows Western blotting analysis of various osteocalcin forms.
  • Lane 1. Low molecular weight (kDa) markers, lane 2.
  • Affinity-purified GST (glutathione S-transferase), lane 3. hOC purified from human bone, lane 4.
  • bOC bovine osteocalcin
  • Affinity-purified GST-rhOC (GST- recombinant human osteocalcin fusion protein), lane 6.
  • Chromatographically purified rhOC recombinant human osteocalcin i.e. the cleavage product from incubation of GST-rhOC with pfXa.
  • Figure 3 is a schematic representation of the approximate epitopes recognized by the Mabs used in two-site hOC assays.
  • the molecule has been divided into four epitope areas each of which is being recognized by different Mabs. Circled numbers indicate the number of the hOC specific immunoassay.
  • Amino- and carboxyterminal amino acids have been marked as 1 and 49, respectively.
  • the protease-sensitive sites have been indicated as R-R (arginine-arginine), the three Gla-residues are shown as well as the disulphide bridge (C-C).
  • Figure 4 shows the determination of immunoreactive uhOC fragments in normal pubertal urine.
  • Urine was subjected to immunoaffmity chromatography and solid phase extraction before HPLC fractionation. The fractions were measured for immunoreactive material with hOC specific assay #7. The squares refer to osteocalcin and the triangles to acetonitrile.
  • Figures 5 A to 5 E show characteristics of the immunoreactive hOC fragments isolated from normal pubertal urine.
  • Fig. 5 A Mass analysis of the most prominent fragment 44 isolated from urine spanning the amino acid residues 7-30 (Gly- Asp).
  • Fig. 5 B Mass analysis of the urine hOC fragment 46 spanning the amino acid residues 6-30 (Leu- Asp).
  • Fig. 5 C Mass analysis of the urine hOC fragment 43.
  • Fig. 5 D Mass analysis of the urine hOC fragment 47.
  • Fig. 5 E Characteristics of the hOC fragments isolated in urine and characteristics of intact hOC.
  • Figure 6 shows the difference between the hOC concentrations in serum and urine between pubertal and adult samples as measured by the hOC IFMAs. hOC concentration in urine and serum samples was clearly higher in pubertal girls than in premenopausal women as measured with the hOC immunoassays.
  • Figures 7 A and 7 B demonstrate hOC levels in pre-, postmenopausal and postmenopausal with HRT groups of women measured with the IFMAs.
  • the concentrations have been obtained in serum (Fig. 7 A) and urine (Fig. 7 B) samples of adult females. In the urine samples the differences between menopausal groups were more obvious than in the serum samples even as measured with the same combination of Mabs.
  • Figures 8 A to 8 D show correlations between different assays measured in urine and serum samples from adult female panel.
  • Fig. 8 A Correlation between uhOC as measured by the assays #4 and #7.
  • Fig. 8 B Correlation between uhOC as measured by the assays #7 and #9.
  • Fig. 8 C Correlation between serum and urine samples as measured by the assay #7.
  • Fig. 8 D Correlation between serum and urine samples as measured by the assay #4. DETAILED DESCRIPTION OF THE INVENTION
  • the isolated osteocalcin fragment derived from human urine is a fragment spanning i) from the amino acid in position 7 to the amino acid in position 30, or ii) from the amino acid in position 6 to the amino acid in position 30 of the amino acid sequence
  • the preferred monoclonal antibody or recombinant antibody fragment has a specificity to epitopes that have been identified on the gamma-carboxylated fragment of osteocalcin, wherein said fragment spans either i) from the amino acid in position 7 to the amino acid in position 30, or ii) from the amino acid in position 6 to the amino acid in position 30 of the amino acid sequence described above, and that all three glutamic acids in the positions 17, 21 and 24 of said sequence are gamma-carboxylated.
  • the preferred immunoassay employs a monoclonal antibody or recombinant antibody fragment having the said specificity.
  • the preferred immunoassay is a non-competitive immunoassay employing at least two different monoclonal antibodies or recombinant antibody fragments.
  • the non-competitive immunoassay is preferably carried out in either a one-step or a two-step incubation procedure.
  • Particularly preferable immunoassays are those where the two monoclonal antibodies employed are
  • All the disclosed assays are highly sensitive and are based on widely characterized reagents. Difference in hOC concentration between the premenopausal and the pubertal group was clearly higher in urine samples than in serum samples. All the hOC assays discriminated the menopausal groups effectively using either serum or urine specimens. Due to their ability to detect different hOC forms, these assays should be of interest in monitoring various disease states, particularly of bone metabolism diseases. The assays are thus especially useful in methods for the measurement of the rate of bone turnover (formation and/or resorption) and/or for the investigation of metabolic bone disorders.
  • L-broth culture medium contained 10 g/1 Bacto® Tryptone (Difco laboratories, Michigan, USA), 5 g/1 Bacto® Yeast extract (Difco) and 5 g/1 NaCl, pH 7.4. lsopropyl-1 -thio- ⁇ -D-galactoside, IPTG (Sigma Chemical CO, USA) was used for induction.
  • PBS buffer consisted of 150 mM NaCl, 16 mM Na HP04, 4 mM NaH 2 PO/i, pH 7.3.
  • PMSF and reduced glutathione were obtained from Sigma and protease factor Xa, pfXa from New England Biolabs.
  • Glutathione Sepharose® 4B column (bed volume 8 ml) was obtained from Pharmacia.
  • the size separation of the proteins was done with SDS-PAGE 25 % gradient Phastgel and using Low molecular weight markers for standardization (Pharmacia).
  • Bovine osteocalcin (bOC) was obtained from Biodesign International, Kennebunkport, ME.
  • a commercial anti-bOC Mab BD (Biodesign) was used as a primary antibody and a horseradish peroxidase linked anti-mouse immunoglobulin raised in sheep was used as a second antibody (Amersham, Buckinghamshire, England). ECL Western blotting reagents (Amersham) were used for visualization according to manufacturer's suggestions.
  • PhastTransfer Semi-dry Transfer Kit (Pharmacia) according to PhastSystem manual.
  • Synthetic human osteocalcin oligomers (Fig. 1A), each containing 88 nucleic acids (8 of them being complementary to each other) were hybridized at RT after phosphorylation of the ends.
  • the single-stranded ends were filled by 1 U of Klenow polymerase and 100 ⁇ M of each deoxynucleotide (30 minutes incubation at 37 °C) to create a 160 base pair human osteocalcin insert, which contains a stop codon and a PstI restriction enzyme site at the 3' end of the gene (Fig. 1A).
  • the blunt-ended insert was ligated in Smal-digested, dephosphorylated prokaryotic expression vector, pGEX-3X (Fig. IB).
  • the recombinant plasmid was transformed into E.
  • the sonicate was clarified by centrifugation (10,000g, 20 min, 4 °C) and filtered through a 0.45 ⁇ m filter before applying it to an equilibrated Glutathione Sepharose® 4B column according to manufacturer's suggestions.
  • the eluate containing the GST-rhOC fusion protein was incubated with pfXa in 20 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl 2 , pH 8.0, using a protease to substrate ratio of 0.5 % (w/w) for 30 min at RT to release the rhOC portion (Nagai and Th ⁇ gersen Nature 1984; 309:810-2). Then rhOC was separated from the mixture reverse phase chromatography using a C8 RP-300 column. The optimization of the rhOC purification has been described in detail by Kak ⁇ nen et al. (1996).
  • the expression vector contained the entire sequence of the hOC gene fused in frame to the 3' end of the Schistosoma japonicum glutathione S-transferase gene (Smith and Johnson Gene 1988;. 67:31-40) as verified by nucleic acid sequencing.
  • the resulting fusion protein contains three additional amino acids between the protease factor Xa cleavage site and the first amino terminal tyrosine of the hOC gene (Fig. IB
  • the molecular mass of the reverse phase HPLC purified rhOC was 6068.1 as determined by mass spectrometry.
  • rhOC migrates similarly both to hOC purified from human femurs and bOC on SDS-PAGE (Fig. 2A, lanes 3, 4 and 6, respectively).
  • Mab BD binds to hOC and bOC (Fig. 2B, lanes 3 and 4, respectively) and also to the both recombinant forms, GST-rhOC and rhOC (Fig. 2B, lanes 5 and 6, respectively).
  • a 64 kDa band is also observed from the fusion protein sample (Fig. 2B, lane 5) which probably indicates a dimeric form of GST-rhOC.
  • Mab BD does not bind to GST or to any of the molecular weight markers (Fig. 2B, lanes 1 and 2, respectively). 2. Production of the osteocalcin monoclonal antibodies
  • Bovine osteocalcin was obtained from Biodesign International, Kennebunkport, ME.
  • Freund's complete adjuvant Fca
  • Freund's incomplete adjuvant Fa
  • the Optimem-1 with glutamax-1, Dulbecco's Modified Eagle Medium (DMEM, 10 x liquid), L-glutamine, sodiumpyruvate (tissue culture tested), sodiumbicarbonate (tissue culture grade) and penicillin-streptomycin (P/S) solution were purchased from Gibco BRL, Life Technologies, Grand Island, NY, Hepes from Boehringer Mannheim, Germany and the fetal bovine serum from Hyclone, Logan, UT and were used as components in culture mediums. Reagents and equipment used for screening the hOC specific hybroma cell lines were obtained from Wallac Oy, Turku, Finland except the Europium-labeled hOC was prepared according to Hellman et al. (J Bone Miner Res 1996; 1 1 : 1165-75).
  • Pristane (2,6, 10,14-tetramethyl pentadecan) used for the production of Mabs as ascitic fluid was obtained from Aldrich-Chemie, Steinhiem, Germany).
  • Tecnomouse hollow-fiber bioreactor used for the large scale production of Mabs was obtained from Integra Biosciences AG, Wallisellen, Switzerland and Protein A agarose Affigel ® for the purification of the Mabs was from Bio-Rad, Richmond, CA.
  • Bovine osteocalcin was coupled to keyhole limpet hemocyanin (KLH) as described (Young et al. Prostaglandines 1982; 23:603-13).
  • KLH keyhole limpet hemocyanin
  • Two three-month-old Balb/c male mice were i.p. immunized with 50 ⁇ g of bOC-KLH antigen mixed with Fca. The mice were boostered with the same amount of antigen in Fia. The final booster dose, 10 ⁇ g bOC-KLH in PBS, was given intravenously.
  • splenocytes were fused to mouse myeloma cells SP 2/0 as described in more detail earlier (Lilja et al. Clin Chem 1991 ; 37: 1618-25).
  • the hybridomas were grown in Optimem-1 with glutamax-1 containing 20 % of fetal bovine serum.
  • the hOC specific Mabs were screened with immunofluorometric assay (IFMA) using rabbit antimouse Ig microtitration wells and hOC labeled with Europium as described earlier (Matikainen 1995).
  • IFMA immunofluorometric assay
  • the positive hybridomas were cloned at least twice by the limiting dilution method.
  • Optimem-1 with glutamax-1 supplemented with 20 % of fetal bovine serum was used as culture medium.
  • Cell lines 2H9F 1 1 F8 (2H9) and 6F9G4E10 (6F9) obtained from the GST-rhOC immunization and 3G8E1F11 (3G8), 1C4B 1D7E7 (1C4) and 3H8H2D2A12F12 (3H8) obtained from bOC immunizations were selected for further characterization.
  • the Mabs were produced as ascites fluid in Balb/c mice primed with pristane and in Tecnomouse hollow-fiber bioreactor.
  • DMEM (1 x solution) supplemented with L-glutamine, Hepes, sodiumpyruvate, sodiumbicarbonate and P/S was used as a culture medium in intracapillary circulation.
  • Produced Mabs were purified by Protein A agarose chromatography using Affigel * purification kit according to manufacturer's suggestions.
  • the streptavidin-coated microtiter plates were obtained from Wallac Oy and biotinylated rat antimouse Ig subclass specific Mabs from Serotec, Oxford, England.
  • the synthetic osteocalcin peptide 7-19 containing Glu at residue 17 was purchased from Bachem, Switzerland and bovine osteocalcin (bOC) was obtained from Biodesign International, Kennebunkport, ME.
  • Osteocalcin from human femurs was purified by modifying a previously described method (Gundberg et al. Meth Enzymol 1984; 107:516-544) as explained in detail by Hellman et al. (1996).
  • Mabs were characterized for their binding to Eu-labeled intact hOC, bOC and tryptic or synthetic peptides as described (Hellman et al. 1996).
  • the antibodies 1 C4 and 3H8 obtained by immunization with bOC conjugated to KLH recognized the tryptic 20-43 fragment.
  • labeling of intact bOC and hOC abolished their immunoreactivity with 3G8, suggesting either that intact Tyr (1) is needed for efficient binding of that the Eu-chelate causes steric hindrance.
  • Hybridoma Immunoglobulin Eu-labeled OC forms recognized one Immunogen class H-chain " Eu-hOC Eu hOC l-19 Eu-hOC 7-19 Ku-hOC 15-31 Eu-hOC 2 Q- 3 Eu-bOC
  • the Mabs 3G8, 2H9 and 6F9 were biotinylated with BITC and the Mabs 2H9, 6F9, 1C4 and 3H8 were labeled with europium(III) chelate in reaction conditions previously described (Hellman et al, 1996).
  • the two-site immunoassay utilized time-resolved fluorometry using lanthanide chelate labels, like europium, as a detection system (Soini and Lovgren CRC Crit Rev Anal Chem. 1987; 18: 105-53).
  • the assays # 4 (bio-2H9/Eu-6F9), #7 (bio-6F9/Eu-lC4) and #9 (bio- 6F9/Eu-3H8) were able to detect the full length hOC and also the large NH 2 - terminal fragment.
  • Assay #4 measured both ⁇ -carboxylated and fully decarboxylated form of hOC.
  • the #9 and #7 assays distinctively preferred the carboxylated form of hOC. Determination of the affinity constants of labeled Mabs were done according to Hellman et al. 1996 using Scatchard analysis (Scatchard, Ann NY Acad Sci 1949; 51 :660-72). Characteristics of the assays are summarized in Table 2.
  • the hOC specific antibodies could be utilized in competitive assays as a capture antibody.
  • hOC fragments in urine compete with the Eu-labeled hOC for binding to the limited number of capture Mabs.
  • Mabs 2H9, 1 C4 and 3H8 also Eu-labeled bOC could be used due to crossreactivities explained in Table 1.
  • hOC and CPYhOC Carboxy-peptidase Y digested hOC
  • DTPA diethylenetriaminepentaaceticacid
  • TSA-buffer 50 mM Tris-HCl, 150 mM NaCl, 15 mM NaN 3 , pH 7.75
  • Materials and equipment used in the OC IFMAs have been listed in the section 3.
  • the plates were shaken for 2 h at RT followed by washing six times with Delfia ® Wash Solution. To detect the Eu fluorescence, 200 ⁇ l of Delfia ® Enhancement solution per well was added. Prior to the measurement with the Delfia ® Research Fluorometer the plates were shaken 30 min at RT.
  • the lower limit of the detection of the different assays were determined based on two standard deviations of the background signal produced by the standard diluent and was under 0.1 ⁇ g/L for each assay.
  • the developed IFMAs showed a linear response of over four orders of magnitude and were highly reproducible.
  • the calibration curve for assay #4 was prepared using purified hOC purified hOC in 7.5 % (w/v) DTPA-treated BSA in TSA buffer as a standard covering the range from 0.05 to 16 ng/ml.
  • Carboxy-peptidase Y digested hOC (CPY hOC) in the same diluent covering the range from 0.05 to 16 ng/ml was used for standardization for the assays #7 and #9.
  • Carboxy-peptidase Y digested hOC (CPY hOC) used for standardization of the urine IFMAs was produced as explained in section 3. Immuno affinity chromatography coupled with purified 6F9Mab (section 2) using Affi-Gel Hz Ixnmunoaffinity kit from Bio-Rad, a C-18 solid phase extraction cartridge (Millipore) and a C-4 reverse phase HPLC column (Vydac, Hesperia, CA, U.S.A.) were used for the isolation of uhOC fragments.
  • Matrix assisted laser deso ⁇ tion MALDI-TOF mass spectrometer (LASERMAT R , Thermo Bioanalysis Ltd., U.K.) was employed for mass determinations and a protein sequencer (Applied Biosystems model All A) equipped with an on-line Applied Biosystems model 120A PTH amino acid analyzer was used for the NH 2 -terminal amino acid sequence analyses.
  • Urine pool was collected in the morning from one healthy male volunteer aged 13 years and stored at +4 °C. Within three hours the pool was aliquoted and frozen at - 70 °C. Later it was stored at -20 °C. Urine pool was thawed, centrifuged and filtrated before subjected to any isolation steps.
  • Immunoreactive uhOC in both urine pool and different steps of isolation process was measured with a two-site immunoassay recognizing, not only the intact hOC, but also the N-terminal mid-fragment of hOC (amino acid residues 1 - 43) (Fig. 3).
  • the assay used monoclonal antibodies (Mabs) 6F9 and 1 C4 as a capture and tracer Mab, respectively (combination #7).
  • the calibration curve was prepared using carboxy-peptidase Y digested hOC (CPY hOC) as a standard.
  • the amount of immunoreactive uhOC in puberty urine pool was 100 ng/ml.
  • the assays #9 (Mab combination 6F9/3H8) and #4 (Mab combination 2H9/6F9) were utilized to determine the immunoreactive fragments as described in section 4.
  • the sample containing the immunoreactive fragments was applied onto a Vydac C-4 (2.1 mm x 150 mm) reverse phase HPLC column, eluted with an acetonitrile gradient (Fig. 4) and the peak fractions detected at 276 nm were collected manually.
  • the sample contained multiple immunoreactive fragments of OC, which eluted between 70 min (35% AcN), and 82 min (48% AcN) (Fig.4).
  • Fractions containing 1.4 - 19.5 ⁇ g/ml of immunoreactive uhOC were subjected to further analysis. Characterization of osteocalcin fragments
  • HPLC fractions were analyzed by the two-site immunoassays #7, #9 and #4 as before and fractions containing immunoreactive uhOC fragments were subjected to MALD1-TOF mass spectrometry and N-terminal amino acid sequencing.
  • the molecular masses of the prominent ions in mass spectrometry were 2778, 2814, 2930 and 3005.
  • the sequence obtained from fraction 44 matches with hOC starting from residue Gly(7). Taking into account the experimental mass 2814, the fragment spans residues 7-30, with ⁇ -carboxylated residues at positions 17, 21 and 24 giving a calculated mass of 2812 (Fig.
  • ⁇ - carboxylation of the Glu residues is further supported by the fact that ⁇ -carboxylated Glu residues are known not to give signal using the sequencing technique in question (Caims et al. (1991) Anal Biochem. 199, 93-97).
  • Fraction 46 was subjected to trypsin digestion to demonstrate that the fragment can be cleaved as expected (after Arg residue).
  • the determined N-terminal sequence matches with hOC starting from residue Leu(6).
  • the determined mass of the N- terminal part of the fragment cleaved with trypsin was 1566 and in accordance with the expected mass mass (1565 with ⁇ -carboxylated Glu 17).
  • the fragment spans residues 6-30 of hOC with three ⁇ -carboxylated residues as above (calculated mass 2925).
  • the ion species with masses 2778 (Fig. 5 C) and 3005 (Fig. 5 D) represent close structural variants of the same hOC region, based on immunoreactivity, cliromatographic behaviour and molecular mass. Such structural variability can be caused by partial lack of ⁇ -carboxylation or additional 1 -2 residues, and/or combinations of both.
  • the characteristics of the fragments have been described in figure 5 E.
  • immunoassay #1 also assay #9 recognize effectively these forms of urine osteocalcin.
  • Epitope of #4 differs slightly from epitope recognized by the combination #7 and #9 because the combination could not recognize the 7-30 fragment but could detect the fragment which mass was 3005.
  • Urine may contain shorter hOC fragments which remain to be characterized.
  • FSH concentration of serum samples was measured by Delfia ® hFSH assay (Wallac, Turku, Finland).
  • the creatinine concentration in urine samples was measured using AU OLYMPUS 510 equipment according to manufacturer's protocols. The protocols of the shOC and uhOC assays have been described in section 5.
  • Serum samples were measured with intact hOC assay specific for full-length hOC (#2), with total hOC assay recognizing not only the intact hOC, but also the N- terminal midfragment of hOC (#4) and with two assays dependent on the degree of ⁇ -carboxylation of the glutamic acids in hOC (#7 and #9).
  • the urine samples were measured with two ⁇ -carboxylation dependent assay (#7 and #9) and also with assay #4.
  • the urine osteocalcin values used for analysis have been corrected for creatinine.
  • Table 4 Percentual differences and statistical significancies between menopausal groups as measured by hOC IFMAs in serum and urine samples.
  • the assays definitely detected different forms of circulating hOC, their performance in measuring the serum panel was almost identical.
  • the IFMAs were even more effective in discriminating postmenopausal group from premenopausal group and also postmenopausal group under HRT from postmenopausal control group when measured in urine samples.

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PCT/FI1998/000550 1997-08-15 1998-06-24 Isolated osteocalcin fragments WO1999009058A1 (en)

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JP2000509736A JP2001514875A (ja) 1997-08-15 1998-06-24 単離オステオカルシンフラグメント
EP98929457A EP1003778A1 (en) 1997-08-15 1998-06-24 Isolated osteocalcin fragments

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FI973371A FI973371A0 (fi) 1997-08-15 1997-08-15 Nya isolerade peptider
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000058732A1 (en) * 1999-03-29 2000-10-05 Kaekoenen Sanna Maria Method for prediction of bone fractures by osteocalcin measurements
WO2001013123A2 (de) * 1999-08-14 2001-02-22 november Aktiengesellschaft Gesellschaft für Molekulare Medizin Verfahren zur indirekten bestimmung des blutgerinnungsstatus
WO2001064748A1 (en) * 2000-03-02 2001-09-07 Protease Ab Antibodies binding a gamma carboxyglutamic acid displaying epitope
WO2002077639A2 (en) * 2001-03-23 2002-10-03 Osteometer Biotech A/S Osteocalcin fragments assay to monitor bone resorption
US20220175888A1 (en) * 2018-04-30 2022-06-09 National Institute Of Immunology Carboxylated osteocalcin for treatment of amyloidosis or diseases associated with abnormal protein folding

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0557663A1 (en) * 1992-02-27 1993-09-01 Delmas, Pierre, Dr. Assessment of bone fragility and prediction of osteoporotic fracture risk using a quantitative determination of circulating under-carboxylated osteocalcin
EP0834740A1 (en) * 1996-04-10 1998-04-08 Eisai Co., Ltd. Anti-glu17-osteocalcin antibody

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0557663A1 (en) * 1992-02-27 1993-09-01 Delmas, Pierre, Dr. Assessment of bone fragility and prediction of osteoporotic fracture risk using a quantitative determination of circulating under-carboxylated osteocalcin
EP0834740A1 (en) * 1996-04-10 1998-04-08 Eisai Co., Ltd. Anti-glu17-osteocalcin antibody

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* Cited by examiner, † Cited by third party
Title
JOURNAL OF BONE AND MINERAL RESEARCH, Volume 11, No. 8, 1996, JUKKA HELLMAN et al., "Epitope Mapping of Nine Monoclonal Antibodies Against Osteocalcin: Combinations into Two-Site Assays Affect Both Assay Specificity and Sample Stability", pages 1165-1175. *
PEPTIDE RESEARCH, Volume 7, No. 4, 1994, M. NAKAO et al., "Synthesis of Human Osteocalcins: gamma-Carboxyglutamic Acid at Position 17 is Essential for a Calcium-Dependent Conformational Transition", pages 171-174. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000058732A1 (en) * 1999-03-29 2000-10-05 Kaekoenen Sanna Maria Method for prediction of bone fractures by osteocalcin measurements
US6967081B1 (en) 1999-03-29 2005-11-22 Kaekoenen Sanna-Maria Method for prediction of bone fractures by osteocalcin measurements
WO2001013123A2 (de) * 1999-08-14 2001-02-22 november Aktiengesellschaft Gesellschaft für Molekulare Medizin Verfahren zur indirekten bestimmung des blutgerinnungsstatus
WO2001013123A3 (de) * 1999-08-14 2001-08-30 November Ag Molekulare Medizin Verfahren zur indirekten bestimmung des blutgerinnungsstatus
US7303884B1 (en) 1999-08-14 2007-12-04 november Aktiengesellschaft Gesellschaft für Molekulare Medizin Method for indirectly determining the blood-clotting status
WO2001064748A1 (en) * 2000-03-02 2001-09-07 Protease Ab Antibodies binding a gamma carboxyglutamic acid displaying epitope
US7439025B2 (en) 2000-03-02 2008-10-21 Protease Ab Antibodies binding a gamma carboxyglutamic acid displaying epitope
WO2002077639A2 (en) * 2001-03-23 2002-10-03 Osteometer Biotech A/S Osteocalcin fragments assay to monitor bone resorption
WO2002077639A3 (en) * 2001-03-23 2003-09-04 Osteometer Biotech As Osteocalcin fragments assay to monitor bone resorption
US20220175888A1 (en) * 2018-04-30 2022-06-09 National Institute Of Immunology Carboxylated osteocalcin for treatment of amyloidosis or diseases associated with abnormal protein folding

Also Published As

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FI973371A0 (fi) 1997-08-15
EP1003778A1 (en) 2000-05-31
JP2001514875A (ja) 2001-09-18

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