US20110159607A1 - Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof - Google Patents
Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof Download PDFInfo
- Publication number
- US20110159607A1 US20110159607A1 US12/737,050 US73705009A US2011159607A1 US 20110159607 A1 US20110159607 A1 US 20110159607A1 US 73705009 A US73705009 A US 73705009A US 2011159607 A1 US2011159607 A1 US 2011159607A1
- Authority
- US
- United States
- Prior art keywords
- peptide
- osteoarthritis
- concentration
- determining
- same
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 201000008482 osteoarthritis Diseases 0.000 title claims abstract description 80
- 201000010099 disease Diseases 0.000 title claims abstract description 47
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 47
- 230000032683 aging Effects 0.000 title claims abstract description 45
- 239000000090 biomarker Substances 0.000 title abstract description 33
- 239000012472 biological sample Substances 0.000 claims abstract description 22
- 238000011282 treatment Methods 0.000 claims abstract description 22
- 238000004393 prognosis Methods 0.000 claims abstract description 8
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 71
- 102000004169 proteins and genes Human genes 0.000 claims description 43
- 108090000623 proteins and genes Proteins 0.000 claims description 43
- 239000012634 fragment Substances 0.000 claims description 33
- 238000000034 method Methods 0.000 claims description 32
- 239000000523 sample Substances 0.000 claims description 16
- 210000002700 urine Anatomy 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 8
- 238000003018 immunoassay Methods 0.000 claims description 6
- 108091033319 polynucleotide Proteins 0.000 claims description 5
- 102000040430 polynucleotide Human genes 0.000 claims description 5
- 239000002157 polynucleotide Substances 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 210000002381 plasma Anatomy 0.000 claims description 3
- 210000001258 synovial membrane Anatomy 0.000 claims description 3
- 150000001408 amides Chemical class 0.000 claims description 2
- 210000004369 blood Anatomy 0.000 claims description 2
- 239000008280 blood Substances 0.000 claims description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 claims description 2
- 210000002751 lymph Anatomy 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000002207 metabolite Substances 0.000 claims description 2
- 235000013336 milk Nutrition 0.000 claims description 2
- 239000008267 milk Substances 0.000 claims description 2
- 210000004080 milk Anatomy 0.000 claims description 2
- 210000003296 saliva Anatomy 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 210000002966 serum Anatomy 0.000 claims description 2
- 210000004243 sweat Anatomy 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 8
- 238000012544 monitoring process Methods 0.000 abstract description 6
- 238000003745 diagnosis Methods 0.000 abstract description 5
- 235000018102 proteins Nutrition 0.000 description 40
- 239000000499 gel Substances 0.000 description 28
- 150000001413 amino acids Chemical group 0.000 description 16
- 238000002349 difference gel electrophoresis Methods 0.000 description 13
- 239000002243 precursor Substances 0.000 description 12
- 101710091916 Leukocyte elastase inhibitor Proteins 0.000 description 11
- 102100030635 Leukocyte elastase inhibitor Human genes 0.000 description 11
- 102100022712 Alpha-1-antitrypsin Human genes 0.000 description 7
- 102100035792 Kininogen-1 Human genes 0.000 description 7
- 101710111227 Kininogen-1 Proteins 0.000 description 7
- 238000003556 assay Methods 0.000 description 7
- 238000004949 mass spectrometry Methods 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 6
- 239000003001 serine protease inhibitor Substances 0.000 description 6
- 102000008847 Serpin Human genes 0.000 description 5
- 108050000761 Serpin Proteins 0.000 description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 108010033276 Peptide Fragments Proteins 0.000 description 4
- 102000007079 Peptide Fragments Human genes 0.000 description 4
- 102100035187 Polymeric immunoglobulin receptor Human genes 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 210000000845 cartilage Anatomy 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 206010061818 Disease progression Diseases 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 241000219061 Rheum Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000001188 articular cartilage Anatomy 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 230000005750 disease progression Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 2
- 102100031814 EGF-containing fibulin-like extracellular matrix protein 1 Human genes 0.000 description 2
- 101710176517 EGF-containing fibulin-like extracellular matrix protein 1 Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 206010056740 Genital discharge Diseases 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101001010513 Homo sapiens Leukocyte elastase inhibitor Proteins 0.000 description 2
- 101150095279 PIGR gene Proteins 0.000 description 2
- 108010046644 Polymeric Immunoglobulin Receptors Proteins 0.000 description 2
- 102000012479 Serine Proteases Human genes 0.000 description 2
- 108010022999 Serine Proteases Proteins 0.000 description 2
- 102100036383 Serpin B3 Human genes 0.000 description 2
- 101710156166 Serpin B3 Proteins 0.000 description 2
- 238000000692 Student's t-test Methods 0.000 description 2
- 102100021144 Zinc-alpha-2-glycoprotein Human genes 0.000 description 2
- 108010000711 Zn-Alpha-2-Glycoprotein Proteins 0.000 description 2
- 108010050122 alpha 1-Antitrypsin Proteins 0.000 description 2
- 229940024142 alpha 1-antitrypsin Drugs 0.000 description 2
- 206010003246 arthritis Diseases 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 102000055165 human SERPINB1 Human genes 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000001155 isoelectric focusing Methods 0.000 description 2
- 239000003591 leukocyte elastase inhibitor Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 239000013074 reference sample Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 210000005065 subchondral bone plate Anatomy 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 1
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- 102000004881 Angiotensinogen Human genes 0.000 description 1
- 108090001067 Angiotensinogen Proteins 0.000 description 1
- 101710199744 Anionic trypsin-2 Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000005927 Cysteine Proteases Human genes 0.000 description 1
- 108010005843 Cysteine Proteases Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 101000609762 Gallus gallus Ovalbumin Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000001214 HSP47 Heat-Shock Proteins Human genes 0.000 description 1
- 108010055039 HSP47 Heat-Shock Proteins Proteins 0.000 description 1
- 101000797623 Homo sapiens Protein AMBP Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 208000012659 Joint disease Diseases 0.000 description 1
- 102000010631 Kininogens Human genes 0.000 description 1
- 108010077861 Kininogens Proteins 0.000 description 1
- 108010028275 Leukocyte Elastase Proteins 0.000 description 1
- 102000016799 Leukocyte elastase Human genes 0.000 description 1
- 102000009112 Mannose-Binding Lectin Human genes 0.000 description 1
- 108010087870 Mannose-Binding Lectin Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010006519 Molecular Chaperones Proteins 0.000 description 1
- 102000005431 Molecular Chaperones Human genes 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102100032859 Protein AMBP Human genes 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 101710151381 Serine protease 2 Proteins 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102100034392 Trypsin-2 Human genes 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000007818 agglutination assay Methods 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 229940019748 antifibrinolytic proteinase inhibitors Drugs 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000002869 basic local alignment search tool Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 238000004422 calculation algorithm Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 239000006167 equilibration buffer Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000011540 hip replacement Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 208000037821 progressive disease Diseases 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000029865 regulation of blood pressure Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
- 229960003147 reserpine Drugs 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- 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
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- 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
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/105—Osteoarthritis, e.g. cartilage alteration, hypertrophy of bone
Definitions
- the present invention relates to a biomarker for osteoarthritis and/or other ageing-related diseases.
- the invention relates to a method of diagnosing osteoarthritis, and/or other ageing-related diseases, by determining the level of a biomarker in a biological sample.
- the invention also relates to the use of a biomarker found in biological samples to monitor the efficacy of a treatment for osteoarthritis, and/or other ageing-related diseases, and to determine the prognosis for an individual diagnosed with osteoarthritis, and/or another ageing-related disease.
- Osteoarthritis is a progressive disorder characterized by destruction of articular cartilage and subchondral bone, and by synovial changes.
- diagnosis of osteoarthritis is based on clinical and radiographic changes which occur late during disease progression. More specifically, diagnosis is based on cartilage integrity, which as articular cartilage is invisible on radiographs must be assessed indirectly from the spacing between subchondral bone ends in a joint. This method does not allow detection of early structural damage, and is cumbersome to use in daily practice.
- Biochemical markers of bone, synovium or cartilage turnover have been proposed as potential tools for the diagnosis, prognosis and treatment monitoring of osteoarthritis (Garnero, P., et al., Ann Rheum Dis, 2001. 60 (6): p. 619-26; Bruyere, O., et al., J Rheumatol, 2003. 30 (5): p. 1043-50; and Wu, J., et al., Arthritis Rheum, 2007. 56 (11): p. 3675-84). More specifically, Wu, J., et al. (Arthritis Rheum, 2007. 56 (11): p. 3675-84) describe potential molecular mediators and biomarkers of osteoarthritis in cartilage tissue.
- the method used required articular cartilage to be obtained, which requires an invasive procedure and provides only a limited amount of tissue.
- the method is therefore costly, time consuming and unsuitable for routine diagnostic testing, or for monitoring disease progression, or for determining the therapeutic effect of a treatment.
- the present invention provides a method for (i) diagnosing osteoarthritis and/or another ageing-related disease, (ii) determining the prognosis for a patient with osteoarthritis and/or another ageing-related disease, and (iii) monitoring the efficacy of treatment for osteoarthritis and/or another ageing-related disease, using readily availables which are simple to obtain and allow for rapid and cost effective use.
- Reference herein to ageing-related diseases includes osteoporosis, and other degenerative diseases.
- the present invention provides a method of determining the osteoarthritis status of a subject, and/or the status of another ageing-related disease in a subject, comprising the steps of:
- the present invention provides a method of diagnosing osteoarthritis and/or another ageing-related disease in a subject, comprising the steps of:
- the present invention provides a method of determining the prognosis for a subject with osteoarthritis and/or another ageing-related disease, comprising the steps of:
- the present invention provides a method of determining the efficacy of a treatment for osteoarthritis and/or another ageing-related disease in a subject, comprising the steps of:
- the concentration of a peptide with the same sequence as the sequence of Seq ID no: 1 is determined.
- the concentration of a peptide substantially the same as the sequence of Seq ID no: 1 may be determined.
- the concentration of a peptide fragment having a sequence the same, or substantially the same, as part of the sequence of Seq ID no: 1 may be determined.
- the concentration of a fragment having a sequence the same, or substantially the same, as part of the sequence of Seq ID no: 1 is determined, the fragment represents an epitope within the sequence of Seq ID no: 1.
- the peptide fragment is at least 10, preferably at least 20, more preferably at least 30 amino acids long.
- a free fragment which is intended to refer to a polypeptide, a peptide or otherwise released from mammalian serpine B1 molecule by an oxidative or enzymatic processing.
- a free fragment is different from a native protein by its structure and configuration and may undergo modification such as phosphorylation, glycosylation or any other post-traductional modification resulting of a pathological mechanism.
- the free fragment as well as the peptide or peptide fragment contributes to the identification of the pathologic status of osteoarthritis patient.
- An epitope is a binding site of an antibody on an antigen.
- a linear epitope will be at least about 7 amino acids in length, and may be at least 8, at least 9, at least 10, at least 11, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, or at least 30 amino acid residues in length.
- antibodies may also recognise conformational determinants formed by non-contiguous residues on an antigen, and an epitope can therefore require a larger fragment of the antigen to be present for binding, e.g. a domain.
- sequence substantially the same as all or part of the sequence of Seq ID no: 1
- sequence identity with all or part of the sequence of Seq ID No:1.
- sequence is the same or substantially the same as at least about 10, 15, 20, 25, 30, 35, 40, 45 or more consecutive amino acids on the sequence of Seq ID No:1
- Homology or sequence identity of two or more amino acid sequences can be measured by using a homology scoring algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool).
- NCBI BLAST National Center for Biotechnology Information Basic Local Alignment Search Tool
- the UWGCC Package provides the BESTFIT program which can be used to calculate sequence identity between two or more sequences (e.g. used on its default setting) (Devereux et al (1984) Nucleic Acids Research 12 p 387-395).
- the sequence of Seq ID no:1 represents a fragment of the serpin B1 protein.
- the fragment may be a degradation product of serpin B1.
- the peptide comprising a sequence the same or substantially the same as the sequence of Seq ID no: 1 or a part thereof is preferably differentially present in the sample from a subject with osteoarthritis or another ageing-related disease compared to a normal subject.
- a peptide comprising the same or substantially the same sequence as Seq ID no: 1 or a fragment thereof, which is measured in step (i) and/or step (iii) in any method of the invention, is also referred to herein as the biomarker or the biomarker peptide.
- osteoarthritis status refers to any distinguishable manifestation of osteoarthritis or the ageing-related disease, including diseased and non-diseased.
- osteoarthritis status includes, without limitation, the presence or absence of osteoarthritis in a subject, the risk of a subject developing osteoarthritis, the stage of the disease, the progression of the disease, and the effectiveness or response of a subject to treatment for osteoarthritis.
- Any method of the invention may be used in conjunction with the assessment of clinical symptoms and/or imaging results and/or the concentration of one or more other biomarkers.
- the biological sample may comprise urine, whole blood, blood serum, blood plasma, synovium, sweat, cerebrospinal fluid, mucous, saliva, lymph, bronchial aspirates, milk and the like.
- the biological sample is urine.
- Biological sample such as urine, have the advantage of being abundant and easily accessible.
- a further advantage of using a biological sample, compared to a tissue such as cartilage, is that the progression of osteoarthritis or another ageing-related disease, and/or the therapeutic effect of a treatment, may be monitored by taking and testing samples as often as necessary without the need for invasive procedures.
- the concentration of the biomarker peptide in a sample may be determined by any suitable assay.
- a suitable assay may include one or more of the following methods, an enzyme assay, an immunoassay, mass spectrometry, HPLC, electrophoresis or an antibody microarray, or any combination thereof. If an immunoassay is used it may be an enzyme linked immunoassay (ELISA), a sandwich assay, a competitive assay, a radioimmunoassay, a Western Blot, an immunoassay using a biosensor, an immunoprecipitation assay, an agglutination assay, a turbidity assay or a nephlelometric assay. If mass spectrometry is used it may be Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry.
- MALDI-TOF Matrix Assisted Laser Desorption/Ionization Time-of-Flight
- the concentration of the biomarker peptide is determined using an immunoassay which uses one or more antibodies directed to the specific biomarker peptide to determine the concentration of the biomarker peptide in the sample.
- one or more antibodies are used to determine the concentration of a biomarker peptide in a sample
- the one or more antibodies may be synthetic, monoclonal, polyclonal, oligoclonal, bispecific, chimeric and/or humanised.
- One or more of the antibodies used may comprise a tag or a label.
- the tag or label may be selected from the group comprising a radioactive, a fluorescent, a chemiluminescent, a dye, an enzyme, or a histidine tag or label, or any other suitable label or tag known in the art.
- the reference value, to which the determined concentration of the biomarker peptide is compared is the concentration of the same peptide in one or more “normal” subjects that do not have any detectable osteoarthritis and/or other ageing-related disease, or any clinical symptoms of osteoarthritis and/or other ageing-related disease, and have so called “normal values” of the biomarker peptide.
- the reference value may be a previous value for the biomarker peptide obtained from a specific subject. This kind of reference value may be used if the method is to be used to monitor progression of osteoarthritis and/or another ageing-related disease, or to monitor the response of a subject to a particular treatment.
- an increase or a decrease in the concentration of the biomarker may be indicative of the osteoarthritis status, and/or the status of another ageing-related disease, in the subject.
- an increase or a decrease in the concentration of the biomarker may be indicative, or diagnostic, of osteoarthritis in the subject.
- a decrease in the concentration of the biomarker, preferably of a peptide with the sequence of Seq ID no:1, in a sample is diagnostic of osteoarthritis.
- an at least about 5 fold or more increase in the concentration of a peptide with the sequence of Seq ID no:1, or sequence substantially the same as Seq ID no:1, or a fragment thereof, in a sample compared to a reference sample from a normal subject is diagnostic of osteoarthritis.
- an at least about 5.8 fold, decrease in the concentration of a peptide with the sequence of Seq ID no: 1, or sequence substantially the same as Seq ID no: 1, or a fragment thereof, in a sample compared to a reference sample from a normal subject is diagnostic of osteoarthritis.
- the method of the invention may also be used to monitor osteoarthritis progression, and/or the progression of another ageing-related disease, in a subject. Furthermore, the method of the invention may be used to monitor the efficacy of a treatment for osteoarthritis, and/or another ageing-related disease, following administration of the treatment to a subject. Efficacy of a treatment may be monitored by analysing samples taken from a subject at various time points following initiation of the treatment. By monitoring changes in the concentration of the biomarker peptide over time and comparing these concentrations to normal and/or reference values, efficacy of the treatment may be determined.
- reference concentrations may include the initial concentration of the biomarker peptide in the subject, or the concentration of the biomarker peptide in the subject when they were last tested, or both.
- kits for use in determining the osteoarthritis status, or the status of another ageing-related disease, in a subject comprising at least one agent for determining the concentration of a peptide comprising the same or substantially the same amino acid sequence as the sequence of Seq ID No: 1, or a part thereof, in a sample provided by the subject.
- the kit may be used to diagnose osteoarthritis and/or another ageing-related disease in a subject.
- the kit may alternatively be used to monitor disease progression or the efficacy of a treatment administered to a subject with osteoarthritis and/or another ageing-related disease.
- the agent may be an enzyme, an antibody, a protein probe, a metabolite or any other suitable composition.
- the agent for determining the concentration of one or more biomarker proteins is preferably labelled.
- the kit may also comprise means for detecting the label.
- the kit may comprise one or more capture agents for capturing the peptide comprising the same or substantially the same amino acid sequence as the sequence of Seq ID No:1, or a part thereof, in a sample provided by the subject.
- the capture agent may be one or more antibodies.
- the capture agent may be an antibody according to an aspect of the invention.
- the kit may comprise two antibodies for use in a sandwich assay to determine the concentration of a peptide comprising the same or substantially the same amino acid sequence as the sequence of Seq ID No:1, or a part thereof.
- the kit comprises two antibodies, each directed to a different epitope on the peptide comprising the same or substantially the same amino acid sequence as the sequence of Seq ID No:1, or a part thereof.
- One antibody is preferably the capture antibody, and the other may comprise a label to allow its detection.
- the capture agent may be attached to a solid support.
- the solid support may be a chip, a microtitre plate, a bead or a resin.
- the kit may comprise instructions for suitable operational parameters in the form of a label or separate insert.
- the instructions may inform a user about how to collect the sample, and/or how to wash the capture agent.
- the kit may comprise samples of the biomarker peptide to be detected.
- the samples of the biomarker peptide may be used as a standard for calibration and comparison.
- the kit may also comprise instructions to compare the concentration of the biomarker peptide detected in a sample with a calibration sample or chart.
- the kit may also include instructions indicating what concentration of the biomarker peptide is diagnostic of osteoarthritis and/or another ageing-related disease.
- the invention provides the use of the determination of the concentration of the biomarker peptide in a biological sample of as a means of assessing the osteoarthritis status in a subject or as a means of assessing the status of another ageing-related disease in a subject.
- the present invention provides the use of a biological sample, such as urine, as a source of at least one biomarker for the prognosis of osteoarthritis progression and/or another ageing-related disease, for diagnosing osteoarthritis and/or another ageing-related disease and for monitoring the effect of a treatment for osteoarthritis and/or another ageing-related disease.
- a biological sample such as urine
- the invention provides a peptide, which comprises the same or substantially the same amino acid sequence as the amino acid sequence of Seq ID NO: 1, or a part thereof, or its amide, or a salt thereof.
- the peptide of the invention has a sequence the same as that of Seq ID No: 1.
- the invention provides a polynucleotide encoding a peptide according to the invention.
- the polynucleotide is a DNA molecule.
- the invention provides a recombinant vector, which comprises a polynucleotide of the invention.
- the invention provides transformant, which is transformed with the recombinant vector of the invention.
- the invention provides the use of a peptide according to the invention in the manufacture of an antibody.
- the invention provides an antibody specific for a peptide according to the invention.
- the invention provides an antibody specific for a peptide having the sequence of Seq ID No: 1.
- An antibody according to the invention may be synthetic, monoclonal, polyclonal, oligoclonal, bispecific, chimeric or humanised.
- the antibody may be complete or a fragment thereof, such as, Fv, Fab and F(ab) 2 fragments.
- Methods of generating antibodies are well known in the art, and may include immunisation of suitable animals, such as, a rabbit, mouse, sheep or goat, with the peptide of interest (or an immunogenic fragment thereof) or recombinant techniques.
- FIG. 1 A shows an enlargement of a portion of a 2D-DIGE map from osteoarthritis (OA) subject (right) and non-osteoarthritis (NO) subject (left).
- FIG. 1 B shows a representation of spot 351 volume variation between OA subjects (right) and non-osteoarthritis subjects (left).
- FIG. 2 shows a graphic view of serpin B1 abundance modification based on the spot volume decrease shown in FIG. 1B .
- FIG. 3 A shows the result of a mass spectrometry analysis of tryptic fragments of the peptide of serpin B1 recovered from spot 351 detailed in FIG. 1A , FIG. 1B and FIG. 2 .
- FIG. 3 B shows the sequence of human serpin B1 containing the fragments shown in FIG. 3A . (SEQ ID NO 3)
- FIG. 4 shows the protein sequence of SEQ ID No. 1.
- FIG. 5 shows the protein sequence of SEQ ID No. 2.
- 2D-DIGE two dimensional difference gel electrophoresis—Marouga et al, (2005) Anal Bioanal Chem 382 (3): 669-78) methodology is a powerful tool for investigating protein expression profiles in multiple sets of samples.
- 2D-DIGE was used to study the protein expression profiles in urine samples from subjects with serious osteoarthritis and from healthy young subjects. Proteins in the samples to be compared were labelled with either Cy3 or Cy5 CyDye DIGE Fluors.
- the Cy2 CyDye DIGE Fluor was used to label a pooled sample comprising equal amounts of each of the samples within the study, and this used as an internal standard. The use of this internal standard ensured that all proteins present in the samples were represented, assisting both inter- and intra-gel matching.
- Urine samples were collected from 10 women undergoing hip replacement surgery due to severe osteoarthritis. Control samples were obtained from 5 healthy women (25.6 ⁇ 2.6 years) without articular degeneration. The urine samples were concentrated 100 ⁇ by ultracentrifugation on Amicon Ultra-15 (Millipore, USA). Proteins were purified by precipitation using PlusOne 2D Clean-up kit (GE Healthcare, Sweden). Albumin depletion from urine samples was performed using affinity columns according to the Montage Albumin Deplete Kit (Millipore, USA) manual utilisation.
- the purified proteins were labelled on lysine residues with Cy3 or Cy5 CyDye DIGE Fluors.
- the samples were minimal labelled which means that the ratio of dye to protein used was such that each protein molecule was labelled with only one dye molecule.
- Three gels were made as shown in Table 1. Proteins from different samples were labelled with Cy3 or Cy5 and loaded on the same gel. On the first and second gels, proteins from NO (normal) samples were labelled with Cy3 CyDye DIGE Fluor whereas proteins from osteoarthritis (OA) samples were labelled with Cy5 CyDye DIGE Fluor.
- proteins from NO samples were labelled with Cy5 CyDye DIGE Fluor and proteins from OA samples were labelled with Cy3 CyDye DIGE Fluor on the third gel.
- An internal standard (MIX) comprising equal amounts of NO and OA samples was labelled with Cy2 CyDye DIGE Fluor and loaded on each gel.
- OA means samples from subject with osteoarthritis
- NO means control samples from normal subjects.
- Protein samples (37.5 ⁇ g) labelled with Cy3, Cy5 or Cy2 DIGE Fluor were separated by 2D electrophoresis using an IEF (ioselectric focusing) buffer (8 M urea, 2% CHAPS, 0.5% immobilized pH gradient [IPG] buffer, 1% DTT, and trace of bromophenol blue) which was loaded into an immobiline DryStrip (pH 3-10 NL, 24 cm) (GE Healthcare, Sweden).
- IEF ioselectric focusing
- equilibration buffer I 375 mM Tris-Cl [pH 8.8], 6 M urea, 20% glycerol, 2% SDS, and 130 mM DTT
- II 375 mM Tris-Cl [pH 8.8], 6 M urea, 20% glycerol, 2% SDS, and 135 mM IAA.
- the second dimension was run according to the Ettan DALTsix Electrophoresis Unit operating manual (GE Healthcare, Sweden).
- a 12.5% SDS-polyacrylamide slab gel 24 cm was used for the second-dimension gel electrophoresis.
- the IPG strips were placed on the surface of the second-dimension gel.
- the gels were then placed in SDS electrophoresis buffer (25 mM Tris base, 192 mM glycine, 0.1% SDS) and run overnight at 1.5 W per gel.
- DeCyder 2D v6.5 software (GE Healthcare, Sweden) was used for simultaneous comparison of abundance changes across sample groups.
- the DeCyder differential in-gel analysis (DIA) module generated ratios for each protein “spot” by comparing Cy3 and Cy5 signals to the Cy2 control signal.
- the DeCyder biological variation analysis module matched all protein spot maps from the gels and normalized the DIA-generated Cy3:Cy2 and Cy5:Cy2 ratios relative to the Cy2 signals for each resolved feature separately. This enabled the calculation of average abundance changes across all three samples within each test group, and the application of univariate statistical analyses (Student's t-test, ANOVA).
- Protein spots were cut out of the polyacrylamide gel and washed twice for 5 minutes with an ammonium hydrogenocarbonate (50 mM)-acetonitrile mix (1:1). Gel spots were incubated in dithiothreitol (10 mM), NH 4 HCO 3 (50 mM), for 40 min in a 56° C. water bath. Proteins in the gel spots were alkylated for 1 h in the dark with iodoacetamide (55 mM) in NH 4 HCO 3 (50 mM). The gel spots were then washed twice with an ammonium hydrogenocarbonate (50 mM)-acetonitrile mix (1:1), dehydrated with acetonitrile, and then dried for 15 minutes at room temperature.
- an ammonium hydrogenocarbonate 50 mM
- the gel spots were then rehydrated for 10 minutes on ice with modified trypsin (10 ng/ ⁇ l) in NH 4 HCO 3 (25 mM) and then incubated overnight at 37° C. Hydrolysis of peptides was stopped in TFA (1%)-ACN (5%) solution. The gel spots were then sonicated twice for 1 minute in order to release protein fragments out of the isolated gel spots. Protein fragments in solution were freeze-dried. The identity of proteins was determined by tandem mass spectrometry (MS-MS spectrometry). The Mowse score (Pappin et al (1993) Curr Biol June 1; 3 (6):327-32) gave the fidelity of identification.
- Proteins isolated from the urine samples and labelled with Cy3 or Cy5 were separated by two-dimensional electrophoresis. The first separation was performed with an isoelectric focusing range of pH 3-10 NL and a load of 37.5 ⁇ g of protein. 372 spots of proteins were matched between all gels. Spots with a modification of intensity between OA and NO with a ratio superior to 1.5 (t-test: p ⁇ 0.05) were selected for protein identification using mass spectrometry.
- Table 2 shows the results of analysis of these spots, and details the size of the spot, the Mowse score (which is ⁇ 10 log (P) where P is the probability that the observed match is a random event), the abundance ratio, the name of the protein identified in the spot and the accession number for the identified protein in the Swiss Prot database.
- FIG. 1A there is shown an enlargement of the area around spot 351 on the 2D-DIGE map of proteins extracted from urine from osteoarthritis subjects (right) and a NO subjects (left).
- spot 351 the same spot (spot 351) is represented by volume variation of the spot between samples from OA subjects (right) and NO subjects (left).
- FIG. 2 A graphic view of the abundance modification based on the spot 351 volume decrease is also shown in FIG. 2 .
- Tryptic fragments from spot 351 were identified by mass spectrometry analysis as shown in FIG. 3A . These fragments were identified as being fragments from the protein serpin B1, as shown in FIG. 3B .
- FIG. 3B shows the protein sequence derived by the translation of the mRNA of human serpin B1 and in bold the specific sequence identified by mass spectrometry. Each tryptic fragment studied was given a score which is ⁇ 10 log(P) where P is the probability that the observed match is a random event. Individual ion scores >26 indicate identity or extensive homology.
- serpins are a superfamily of serine protease inhibitors (SERine Protease Inhibitors). In human, serpin family is divided, into nine clades (A-I). These proteins are involved in a diverse set of processes including blood coagulation, inflammation and complement activation, cell migration, pro-hormone activation and matrix remodelling. They undergo a unique and dramatic conformational change when they inhibit target proteases. Serpines inhibit protease with substrate-like irreversible inhibitory mechanism. Not all serpins functions as proteinase inhibitors. Some of them act as chaperone proteins (heat shock protein 47) or are involved in blood pressure regulation (angiotensinogen).
- chaperone proteins heat shock protein 47
- angiotensinogen angiotensinogen
- Clade B represents a particular set of serpines, ov-serpines, with amino acid similarities among chicken ovalbumin. Most serpins target serine protease, however, some serpines from clade B can inhibit serine or cysteine protease or both.
- the serpin B1 or LEI leukocyte elastase inhibitor
- LEI leukocyte elastase inhibitor
- the elastase is also involved in the pathogenesis of inflammatory diseases such as rheumatoid arthritis and has been proposed as biomarker of chronic joint diseases (Kleesiek et al., 1986; Momohara et al, 1997).
- administration of recombinant serpin B1 has a protective role against proteases released by neutrophils (Rees et al. 1999, Hirche et al., 2004; Rubio et al., 2004; and Yasumaki al., 2006).
- serpin B3 is associated with squamous cell carcinoma and serves as a diagnostic marker for some cancers (Pemberton et al., 1997).
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Rheumatology (AREA)
- Rehabilitation Therapy (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the identification of a biomarker whose abundance in biological samples is changed in subjects with osteoarthritis and/or other ageing-related diseases. The biomarker has applications in the diagnosis of osteoarthritis and/or other ageing-related diseases, in determining the prognosis for an individual diagnosed with osteoarthritis and/or other ageing-related diseases, and in monitoring the efficacy of treatment for osteoarthritis and/or other ageing-related diseases.
Description
- The present invention relates to a biomarker for osteoarthritis and/or other ageing-related diseases. In particular, the invention relates to a method of diagnosing osteoarthritis, and/or other ageing-related diseases, by determining the level of a biomarker in a biological sample. The invention also relates to the use of a biomarker found in biological samples to monitor the efficacy of a treatment for osteoarthritis, and/or other ageing-related diseases, and to determine the prognosis for an individual diagnosed with osteoarthritis, and/or another ageing-related disease.
- Osteoarthritis is a progressive disorder characterized by destruction of articular cartilage and subchondral bone, and by synovial changes. Currently the diagnosis of osteoarthritis is based on clinical and radiographic changes which occur late during disease progression. More specifically, diagnosis is based on cartilage integrity, which as articular cartilage is invisible on radiographs must be assessed indirectly from the spacing between subchondral bone ends in a joint. This method does not allow detection of early structural damage, and is cumbersome to use in daily practice.
- Biochemical markers of bone, synovium or cartilage turnover have been proposed as potential tools for the diagnosis, prognosis and treatment monitoring of osteoarthritis (Garnero, P., et al., Ann Rheum Dis, 2001. 60 (6): p. 619-26; Bruyere, O., et al., J Rheumatol, 2003. 30 (5): p. 1043-50; and Wu, J., et al., Arthritis Rheum, 2007. 56 (11): p. 3675-84). More specifically, Wu, J., et al. (Arthritis Rheum, 2007. 56 (11): p. 3675-84) describe potential molecular mediators and biomarkers of osteoarthritis in cartilage tissue. The method used required articular cartilage to be obtained, which requires an invasive procedure and provides only a limited amount of tissue. The method is therefore costly, time consuming and unsuitable for routine diagnostic testing, or for monitoring disease progression, or for determining the therapeutic effect of a treatment.
- There therefore remains a need for a simple, rapid and effective method for the diagnosis of osteoarthritis and/or other ageing-related diseases, and/or to monitor the efficacy of treatments for osteoarthritis and/or other ageing-related diseases, and/or to determine the prognosis for a patient diagnosed with osteoarthritis and/or other ageing-related diseases.
- The present invention provides a method for (i) diagnosing osteoarthritis and/or another ageing-related disease, (ii) determining the prognosis for a patient with osteoarthritis and/or another ageing-related disease, and (iii) monitoring the efficacy of treatment for osteoarthritis and/or another ageing-related disease, using readily availables which are simple to obtain and allow for rapid and cost effective use.
- Reference herein to ageing-related diseases includes osteoporosis, and other degenerative diseases.
- According to one aspect, the present invention provides a method of determining the osteoarthritis status of a subject, and/or the status of another ageing-related disease in a subject, comprising the steps of:
-
- (i) determining the concentration in a biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof;
- (ii) comparing the peptide concentration determined in step (i) with one or more reference values.
- According to another aspect, the present invention provides a method of diagnosing osteoarthritis and/or another ageing-related disease in a subject, comprising the steps of:
-
- (i) determining the concentration in a biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof;
- (ii) comparing the peptide concentration determined in step (i) with one or more reference values.
- According to yet another aspect, the present invention provides a method of determining the prognosis for a subject with osteoarthritis and/or another ageing-related disease, comprising the steps of:
-
- (i) determining the concentration in a biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof;
- (ii) comparing the peptide levels determined in step (i) with one or more reference values.
- According to a further aspect, the present invention provides a method of determining the efficacy of a treatment for osteoarthritis and/or another ageing-related disease in a subject, comprising the steps of:
-
- (i) determining the concentration in a biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof;
- (ii) administering a treatment for osteoarthritis and/or another ageing related disease to the subject;
- (iii) determining after treatment, the concentration in another biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof;
- (iv) comparing the peptide concentrations determined in step (i) and (iii) with one another, and optionally with one or more reference values.
- Preferably, in any method of the invention, the concentration of a peptide with the same sequence as the sequence of Seq ID no: 1 is determined. Alternatively, the concentration of a peptide substantially the same as the sequence of Seq ID no: 1 may be determined.
- Alternatively, the concentration of a peptide fragment having a sequence the same, or substantially the same, as part of the sequence of Seq ID no: 1 may be determined. Preferably if the concentration of a fragment having a sequence the same, or substantially the same, as part of the sequence of Seq ID no: 1 is determined, the fragment represents an epitope within the sequence of Seq ID no: 1. Preferably the peptide fragment is at least 10, preferably at least 20, more preferably at least 30 amino acids long.
- Alternatively to a peptide or a peptide fragment one may also consider a free fragment which is intended to refer to a polypeptide, a peptide or otherwise released from mammalian serpine B1 molecule by an oxidative or enzymatic processing. A free fragment is different from a native protein by its structure and configuration and may undergo modification such as phosphorylation, glycosylation or any other post-traductional modification resulting of a pathological mechanism. The free fragment as well as the peptide or peptide fragment contributes to the identification of the pathologic status of osteoarthritis patient.
- An epitope is a binding site of an antibody on an antigen. In a peptide antigen, generally a linear epitope will be at least about 7 amino acids in length, and may be at least 8, at least 9, at least 10, at least 11, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, or at least 30 amino acid residues in length. However, antibodies may also recognise conformational determinants formed by non-contiguous residues on an antigen, and an epitope can therefore require a larger fragment of the antigen to be present for binding, e.g. a domain.
- Reference herein to “a sequence substantially the same as” all or part of the sequence of Seq ID no: 1, refers to a peptide with a sequence which has at least 80%, preferably at least 90%, more preferably at least 95% or 98%, sequence identity with all or part of the sequence of Seq ID No:1. Preferably the sequence is the same or substantially the same as at least about 10, 15, 20, 25, 30, 35, 40, 45 or more consecutive amino acids on the sequence of Seq ID No:1
- Homology or sequence identity of two or more amino acid sequences can be measured by using a homology scoring algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool). Alternatively, the UWGCC Package provides the BESTFIT program which can be used to calculate sequence identity between two or more sequences (e.g. used on its default setting) (Devereux et al (1984) Nucleic Acids Research 12 p 387-395).
- The sequence of Seq ID no:1 represents a fragment of the serpin B1 protein. The fragment may be a degradation product of serpin B1.
- In any method of the invention the peptide comprising a sequence the same or substantially the same as the sequence of Seq ID no: 1 or a part thereof is preferably differentially present in the sample from a subject with osteoarthritis or another ageing-related disease compared to a normal subject.
- A peptide comprising the same or substantially the same sequence as Seq ID no: 1 or a fragment thereof, which is measured in step (i) and/or step (iii) in any method of the invention, is also referred to herein as the biomarker or the biomarker peptide.
- Reference to the “osteoarthritis status” or to the “status of an ageing-related disease” refers to any distinguishable manifestation of osteoarthritis or the ageing-related disease, including diseased and non-diseased. For example, osteoarthritis status includes, without limitation, the presence or absence of osteoarthritis in a subject, the risk of a subject developing osteoarthritis, the stage of the disease, the progression of the disease, and the effectiveness or response of a subject to treatment for osteoarthritis.
- Any method of the invention may be used in conjunction with the assessment of clinical symptoms and/or imaging results and/or the concentration of one or more other biomarkers.
- Preferably all methods of the invention are carried out in vitro.
- The biological sample may comprise urine, whole blood, blood serum, blood plasma, synovium, sweat, cerebrospinal fluid, mucous, saliva, lymph, bronchial aspirates, milk and the like. Preferably the biological sample is urine.
- Biological sample, such as urine, have the advantage of being abundant and easily accessible.
- A further advantage of using a biological sample, compared to a tissue such as cartilage, is that the progression of osteoarthritis or another ageing-related disease, and/or the therapeutic effect of a treatment, may be monitored by taking and testing samples as often as necessary without the need for invasive procedures.
- The concentration of the biomarker peptide in a sample may be determined by any suitable assay. A suitable assay may include one or more of the following methods, an enzyme assay, an immunoassay, mass spectrometry, HPLC, electrophoresis or an antibody microarray, or any combination thereof. If an immunoassay is used it may be an enzyme linked immunoassay (ELISA), a sandwich assay, a competitive assay, a radioimmunoassay, a Western Blot, an immunoassay using a biosensor, an immunoprecipitation assay, an agglutination assay, a turbidity assay or a nephlelometric assay. If mass spectrometry is used it may be Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry.
- Preferably the concentration of the biomarker peptide is determined using an immunoassay which uses one or more antibodies directed to the specific biomarker peptide to determine the concentration of the biomarker peptide in the sample.
- If one or more antibodies are used to determine the concentration of a biomarker peptide in a sample the one or more antibodies may be synthetic, monoclonal, polyclonal, oligoclonal, bispecific, chimeric and/or humanised.
- One or more of the antibodies used may comprise a tag or a label. The tag or label may be selected from the group comprising a radioactive, a fluorescent, a chemiluminescent, a dye, an enzyme, or a histidine tag or label, or any other suitable label or tag known in the art.
- Preferably the reference value, to which the determined concentration of the biomarker peptide is compared, is the concentration of the same peptide in one or more “normal” subjects that do not have any detectable osteoarthritis and/or other ageing-related disease, or any clinical symptoms of osteoarthritis and/or other ageing-related disease, and have so called “normal values” of the biomarker peptide.
- Alternatively, the reference value may be a previous value for the biomarker peptide obtained from a specific subject. This kind of reference value may be used if the method is to be used to monitor progression of osteoarthritis and/or another ageing-related disease, or to monitor the response of a subject to a particular treatment.
- When the determined concentration of the biomarker is compared with a reference value, an increase or a decrease in the concentration of the biomarker may be indicative of the osteoarthritis status, and/or the status of another ageing-related disease, in the subject.
- More specifically an increase or a decrease in the concentration of the biomarker may be indicative, or diagnostic, of osteoarthritis in the subject. Preferably, a decrease in the concentration of the biomarker, preferably of a peptide with the sequence of Seq ID no:1, in a sample is diagnostic of osteoarthritis.
- Preferably an at least about 5 fold or more increase in the concentration of a peptide with the sequence of Seq ID no:1, or sequence substantially the same as Seq ID no:1, or a fragment thereof, in a sample compared to a reference sample from a normal subject is diagnostic of osteoarthritis. Preferably, an at least about 5.8 fold, decrease in the concentration of a peptide with the sequence of Seq ID no: 1, or sequence substantially the same as Seq ID no: 1, or a fragment thereof, in a sample compared to a reference sample from a normal subject is diagnostic of osteoarthritis.
- The method of the invention may also be used to monitor osteoarthritis progression, and/or the progression of another ageing-related disease, in a subject. Furthermore, the method of the invention may be used to monitor the efficacy of a treatment for osteoarthritis, and/or another ageing-related disease, following administration of the treatment to a subject. Efficacy of a treatment may be monitored by analysing samples taken from a subject at various time points following initiation of the treatment. By monitoring changes in the concentration of the biomarker peptide over time and comparing these concentrations to normal and/or reference values, efficacy of the treatment may be determined.
- In this case reference concentrations may include the initial concentration of the biomarker peptide in the subject, or the concentration of the biomarker peptide in the subject when they were last tested, or both.
- According to another aspect of the invention there is provided a kit for use in determining the osteoarthritis status, or the status of another ageing-related disease, in a subject comprising at least one agent for determining the concentration of a peptide comprising the same or substantially the same amino acid sequence as the sequence of Seq ID No: 1, or a part thereof, in a sample provided by the subject.
- The kit may be used to diagnose osteoarthritis and/or another ageing-related disease in a subject. The kit may alternatively be used to monitor disease progression or the efficacy of a treatment administered to a subject with osteoarthritis and/or another ageing-related disease.
- The agent may be an enzyme, an antibody, a protein probe, a metabolite or any other suitable composition.
- The agent for determining the concentration of one or more biomarker proteins is preferably labelled. The kit may also comprise means for detecting the label.
- The kit may comprise one or more capture agents for capturing the peptide comprising the same or substantially the same amino acid sequence as the sequence of Seq ID No:1, or a part thereof, in a sample provided by the subject. The capture agent may be one or more antibodies. The capture agent may be an antibody according to an aspect of the invention.
- The kit may comprise two antibodies for use in a sandwich assay to determine the concentration of a peptide comprising the same or substantially the same amino acid sequence as the sequence of Seq ID No:1, or a part thereof. Preferably the kit comprises two antibodies, each directed to a different epitope on the peptide comprising the same or substantially the same amino acid sequence as the sequence of Seq ID No:1, or a part thereof. One antibody is preferably the capture antibody, and the other may comprise a label to allow its detection.
- The capture agent may be attached to a solid support. The solid support may be a chip, a microtitre plate, a bead or a resin.
- The kit may comprise instructions for suitable operational parameters in the form of a label or separate insert. The instructions may inform a user about how to collect the sample, and/or how to wash the capture agent.
- The kit may comprise samples of the biomarker peptide to be detected. The samples of the biomarker peptide may be used as a standard for calibration and comparison. The kit may also comprise instructions to compare the concentration of the biomarker peptide detected in a sample with a calibration sample or chart. The kit may also include instructions indicating what concentration of the biomarker peptide is diagnostic of osteoarthritis and/or another ageing-related disease.
- According to a yet further aspect, the invention provides the use of the determination of the concentration of the biomarker peptide in a biological sample of as a means of assessing the osteoarthritis status in a subject or as a means of assessing the status of another ageing-related disease in a subject.
- According to a yet further aspect, the present invention provides the use of a biological sample, such as urine, as a source of at least one biomarker for the prognosis of osteoarthritis progression and/or another ageing-related disease, for diagnosing osteoarthritis and/or another ageing-related disease and for monitoring the effect of a treatment for osteoarthritis and/or another ageing-related disease.
- According to another aspect the invention provides a peptide, which comprises the same or substantially the same amino acid sequence as the amino acid sequence of Seq ID NO: 1, or a part thereof, or its amide, or a salt thereof.
- Preferably the peptide of the invention has a sequence the same as that of Seq ID No: 1.
- According to a further aspect, the invention provides a polynucleotide encoding a peptide according to the invention. Preferably, the polynucleotide is a DNA molecule.
- According to a yet further aspect the invention provides a recombinant vector, which comprises a polynucleotide of the invention.
- According to another aspect the invention provides transformant, which is transformed with the recombinant vector of the invention.
- In a further aspect, the invention provides the use of a peptide according to the invention in the manufacture of an antibody.
- It a yet further aspect, the invention provides an antibody specific for a peptide according to the invention. In particular, the invention provides an antibody specific for a peptide having the sequence of Seq ID No: 1.
- An antibody according to the invention may be synthetic, monoclonal, polyclonal, oligoclonal, bispecific, chimeric or humanised. The antibody may be complete or a fragment thereof, such as, Fv, Fab and F(ab)2 fragments. Methods of generating antibodies are well known in the art, and may include immunisation of suitable animals, such as, a rabbit, mouse, sheep or goat, with the peptide of interest (or an immunogenic fragment thereof) or recombinant techniques.
- The skilled man will appreciate that preferred features of any one embodiment and/or aspect of the invention may be applied to all other embodiments and/or aspects of the invention.
- Embodiments of the invention will now be described merely by way of example with reference to the accompanying figures in which:
- FIG. 1A—shows an enlargement of a portion of a 2D-DIGE map from osteoarthritis (OA) subject (right) and non-osteoarthritis (NO) subject (left).
- FIG. 1B—shows a representation of
spot 351 volume variation between OA subjects (right) and non-osteoarthritis subjects (left). - FIG. 2—shows a graphic view of serpin B1 abundance modification based on the spot volume decrease shown in
FIG. 1B . - FIG. 3A—shows the result of a mass spectrometry analysis of tryptic fragments of the peptide of serpin B1 recovered from
spot 351 detailed inFIG. 1A ,FIG. 1B andFIG. 2 . - FIG. 3B—shows the sequence of human serpin B1 containing the fragments shown in
FIG. 3A . (SEQ ID NO 3) - FIG. 4—shows the protein sequence of SEQ ID No. 1.
- FIG. 5—shows the protein sequence of SEQ ID No. 2.
- 2D-DIGE (two dimensional difference gel electrophoresis—Marouga et al, (2005) Anal Bioanal Chem 382 (3): 669-78) methodology is a powerful tool for investigating protein expression profiles in multiple sets of samples.
- In this example, 2D-DIGE was used to study the protein expression profiles in urine samples from subjects with serious osteoarthritis and from healthy young subjects. Proteins in the samples to be compared were labelled with either Cy3 or Cy5 CyDye DIGE Fluors. The Cy2 CyDye DIGE Fluor was used to label a pooled sample comprising equal amounts of each of the samples within the study, and this used as an internal standard. The use of this internal standard ensured that all proteins present in the samples were represented, assisting both inter- and intra-gel matching.
- Urine samples were collected from 10 women undergoing hip replacement surgery due to severe osteoarthritis. Control samples were obtained from 5 healthy women (25.6±2.6 years) without articular degeneration. The urine samples were concentrated 100× by ultracentrifugation on Amicon Ultra-15 (Millipore, USA). Proteins were purified by precipitation using PlusOne 2D Clean-up kit (GE Healthcare, Sweden). Albumin depletion from urine samples was performed using affinity columns according to the Montage Albumin Deplete Kit (Millipore, USA) manual utilisation.
- Labelling of Proteins with Cy3 and Cy5 Dyes
- In all experiments, the purified proteins were labelled on lysine residues with Cy3 or Cy5 CyDye DIGE Fluors. The samples were minimal labelled which means that the ratio of dye to protein used was such that each protein molecule was labelled with only one dye molecule. Three gels were made as shown in Table 1. Proteins from different samples were labelled with Cy3 or Cy5 and loaded on the same gel. On the first and second gels, proteins from NO (normal) samples were labelled with Cy3 CyDye DIGE Fluor whereas proteins from osteoarthritis (OA) samples were labelled with Cy5 CyDye DIGE Fluor. Conversely, proteins from NO samples were labelled with Cy5 CyDye DIGE Fluor and proteins from OA samples were labelled with Cy3 CyDye DIGE Fluor on the third gel. An internal standard (MIX) comprising equal amounts of NO and OA samples was labelled with Cy2 CyDye DIGE Fluor and loaded on each gel.
-
TABLE 1 Gel 1Gel 2 Gel 3Cy3 NO NO OA Cy5 OA OA NO Cy2 MIX MIX MIX - In Table 1 OA means samples from subject with osteoarthritis, and NO means control samples from normal subjects.
- Protein samples (37.5 μg) labelled with Cy3, Cy5 or Cy2 DIGE Fluor were separated by 2D electrophoresis using an IEF (ioselectric focusing) buffer (8 M urea, 2% CHAPS, 0.5% immobilized pH gradient [IPG] buffer, 1% DTT, and trace of bromophenol blue) which was loaded into an immobiline DryStrip (pH 3-10 NL, 24 cm) (GE Healthcare, Sweden). The first dimension isoelectric focusing was performed for 70,000 Vhr using a Protean IEF Cell (Biorad) at 20° C. Next, the gels were equilibrated for 12 minutes in equilibration buffer I (375 mM Tris-Cl [pH 8.8], 6 M urea, 20% glycerol, 2% SDS, and 130 mM DTT) and II (375 mM Tris-Cl [pH 8.8], 6 M urea, 20% glycerol, 2% SDS, and 135 mM IAA). The second dimension was run according to the Ettan DALTsix Electrophoresis Unit operating manual (GE Healthcare, Sweden). A 12.5% SDS-polyacrylamide slab gel (24 cm) was used for the second-dimension gel electrophoresis. The IPG strips were placed on the surface of the second-dimension gel. The gels were then placed in SDS electrophoresis buffer (25 mM Tris base, 192 mM glycine, 0.1% SDS) and run overnight at 1.5 W per gel.
- Gels were scanned while still between two low-fluorescence glass plates using a Typhoon 9400 fluorescent scanner and saved in .gel format using ImageQuant software (GE Healthcare, Sweden). The excitation wavelengths for Cy3 and Cy5 are 550 nm and 645 nm, and the emission wavelengths are 570 nm and 670 nm for Cy3 and Cy5, respectively. The excitation/emission wavelength of Cy2 is around 489/505 nm. Image analysis was performed on DeCyder™ software (GE Healthcare, Sweden). Interesting spots with differential fluorescent intensity between Cy3 and Cy5 were picked out the gel in order to allow protein identification after post-staining with Coomassie Blue.
- DeCyder 2D v6.5 software (GE Healthcare, Sweden) was used for simultaneous comparison of abundance changes across sample groups. The DeCyder differential in-gel analysis (DIA) module generated ratios for each protein “spot” by comparing Cy3 and Cy5 signals to the Cy2 control signal. The DeCyder biological variation analysis module matched all protein spot maps from the gels and normalized the DIA-generated Cy3:Cy2 and Cy5:Cy2 ratios relative to the Cy2 signals for each resolved feature separately. This enabled the calculation of average abundance changes across all three samples within each test group, and the application of univariate statistical analyses (Student's t-test, ANOVA).
- Protein spots were cut out of the polyacrylamide gel and washed twice for 5 minutes with an ammonium hydrogenocarbonate (50 mM)-acetonitrile mix (1:1). Gel spots were incubated in dithiothreitol (10 mM), NH4HCO3 (50 mM), for 40 min in a 56° C. water bath. Proteins in the gel spots were alkylated for 1 h in the dark with iodoacetamide (55 mM) in NH4HCO3 (50 mM). The gel spots were then washed twice with an ammonium hydrogenocarbonate (50 mM)-acetonitrile mix (1:1), dehydrated with acetonitrile, and then dried for 15 minutes at room temperature. The gel spots were then rehydrated for 10 minutes on ice with modified trypsin (10 ng/μl) in NH4HCO3 (25 mM) and then incubated overnight at 37° C. Hydrolysis of peptides was stopped in TFA (1%)-ACN (5%) solution. The gel spots were then sonicated twice for 1 minute in order to release protein fragments out of the isolated gel spots. Protein fragments in solution were freeze-dried. The identity of proteins was determined by tandem mass spectrometry (MS-MS spectrometry). The Mowse score (Pappin et al (1993) Curr Biol June 1; 3 (6):327-32) gave the fidelity of identification.
- Proteins isolated from the urine samples and labelled with Cy3 or Cy5 were separated by two-dimensional electrophoresis. The first separation was performed with an isoelectric focusing range of pH 3-10 NL and a load of 37.5 μg of protein. 372 spots of proteins were matched between all gels. Spots with a modification of intensity between OA and NO with a ratio superior to 1.5 (t-test: p<0.05) were selected for protein identification using mass spectrometry. Table 2 shows the results of analysis of these spots, and details the size of the spot, the Mowse score (which is −10 log (P) where P is the probability that the observed match is a random event), the abundance ratio, the name of the protein identified in the spot and the accession number for the identified protein in the Swiss Prot database.
-
TABLE 2 Abun- dance Sequence ratio Spot coverage Mowse (OA/ Swiss-Prot no (%) score NO) Protein description Accession 40 9 390 1.83 Poly-Ig receptor P01833 (PIGR) 43 5 159 1.6 Poly-Ig receptor P01833 (PIGR) 75 10 340 −1.68 Transferrin P02787 219 11 334 −1.7 Kininogen-1 precursor P01042 6 55 Alpha 1 anti-trypsin P01009 (A1AT) 222 15 349 −1.64 Kininogen-1 precursor P01042 7 99 A1AT P01009 223 13 405 −1.89 Kininogen-1 precursor P01042 16 311 A1AT P01009 226 18 334 −1.73 A1AT P01009 6 219 Kininogen-1 precursor P01042 262 13 368 −1.91 Kininogen-1 precursor P01042 6 61 A1AT P01009 267 10 304 −1.98 Kininogen-1 precursor P01042 269 5 143 −1.83 Kininogen-1 precursor P01042 343 189 4.18 Beta-actine 348 21 192 2 Zn-α-2-glycoprotein P25311 precursor 349 8 195 −2.44 Serpin B3 P29508 351 6 115 −5.84 Serpin B1 P30740 352 5 130 2.2 Fibulin-3 Q12805 73 Apoptosis-inducing Q9BRQ8 factor 2 (two proteins identified in the same spot) 356 8 110 2.01 Zn-α-2-glycoprotein P25311 2 45 precursor Q12805 FIBULIN3 386 10 197 1.54 GP36b Q12907 398 5 187 2.28 AMBP protein P02760 precursor 485 8 289 −2.3 Mannan-binding lectin O00187 serine protease 2 E.C precursor 3.4.21.104 - As can be seen from Table 2, various proteins with significant changes in concentration in samples from osteoarthritis compared to samples from normal subjects were identified. Some of the proteins identified are implicated in the inflammatory process, for example, the kininogen precursor or alpha-1-antitrypsin. This observation coincides with the pathology of osteoarthritis.
- A significant decrease in the concentration of a specific serpin B1 fragment was observed in osteoarthritis subjects compared to normal subject, as shown in Table 2 and
FIGS. 1A and 1B . InFIG. 1A there is shown an enlargement of the area aroundspot 351 on the 2D-DIGE map of proteins extracted from urine from osteoarthritis subjects (right) and a NO subjects (left). InFIG. 1B the same spot (spot 351) is represented by volume variation of the spot between samples from OA subjects (right) and NO subjects (left). A graphic view of the abundance modification based on thespot 351 volume decrease is also shown inFIG. 2 . - Tryptic fragments from
spot 351 were identified by mass spectrometry analysis as shown inFIG. 3A . These fragments were identified as being fragments from the protein serpin B1, as shown inFIG. 3B .FIG. 3B shows the protein sequence derived by the translation of the mRNA of human serpin B1 and in bold the specific sequence identified by mass spectrometry. Each tryptic fragment studied was given a score which is −10 log(P) where P is the probability that the observed match is a random event. Individual ion scores >26 indicate identity or extensive homology. - The serpins are a superfamily of serine protease inhibitors (SERine Protease Inhibitors). In human, serpin family is divided, into nine clades (A-I). These proteins are involved in a diverse set of processes including blood coagulation, inflammation and complement activation, cell migration, pro-hormone activation and matrix remodelling. They undergo a unique and dramatic conformational change when they inhibit target proteases. Serpines inhibit protease with substrate-like irreversible inhibitory mechanism. Not all serpins functions as proteinase inhibitors. Some of them act as chaperone proteins (heat shock protein 47) or are involved in blood pressure regulation (angiotensinogen).
- Clade B represents a particular set of serpines, ov-serpines, with amino acid similarities among chicken ovalbumin. Most serpins target serine protease, however, some serpines from clade B can inhibit serine or cysteine protease or both.
- The serpin B1 or LEI (leukocyte elastase inhibitor) is involved in regulating the degradation of the extracellular matrix through its action on neutrophil elastase. The elastase is also involved in the pathogenesis of inflammatory diseases such as rheumatoid arthritis and has been proposed as biomarker of chronic joint diseases (Kleesiek et al., 1986; Momohara et al, 1997). In the lung, administration of recombinant serpin B1 has a protective role against proteases released by neutrophils (Rees et al. 1999, Hirche et al., 2004; Rubio et al., 2004; and Yasumaki al., 2006).
- An increased amount of serpin in plasma may be associated with a particular pathology. For example, serpin B3 is associated with squamous cell carcinoma and serves as a diagnostic marker for some cancers (Pemberton et al., 1997).
Claims (17)
1. A method of determining the osteoarthritis status of a subject, and/or the status of another ageing-related disease in a subject, comprising the steps of:
(i) determining the concentration in a biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof;
(ii) comparing the peptide concentration determined in step (i) with
one or more reference values.
2. A method of diagnosing osteoarthritis and/or another ageing-related disease in a subject, comprising the steps of:
(i) determining the concentration in a biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof;
(ii) comparing the peptide concentration determined in step (i) with
one or more reference values.
3. A method of determining the prognosis for a subject with osteoarthritis and/or another ageing-related disease, comprising the steps of:
(i) determining the concentration in a biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof;
(ii) comparing the peptide concentration determined in step (i) with
one or more reference values.
4. A method of determining the efficacy of a treatment for osteoarthritis and/or another ageing-related disease in a subject, comprising the steps of:
(i) determining the concentration in a biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof;
(ii) administering a treatment for osteoarthritis and/or another ageing related disease to the subject;
(iii) determining after treatment the concentration in another biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof;
(iv) comparing the peptide concentrations determined in step (i) and (iii) with one another, and optionally with one or more reference values.
5. The method of claim 1 wherein the biological sample is selected from the group comprising urine, whole blood, blood serum, blood plasma, synovium, sweat, cerebrospinal fluid, mucous, saliva, lymph, bronchial aspirates and milk.
6. The method of claim 1 wherein the concentration of the peptide is determined by using an immunoassay.
7. The method of claim 1 wherein the reference value is the concentration of the peptide in biological samples of one or more normal subjects.
8. A kit for use in determining the osteoarthritis status, or the status of another ageing-related disease, in a subject comprising at least one agent for determining the concentration of a peptide comprising the same or substantially the same amino acid sequence as the sequence of Seq ID No:1, or a part thereof, in a biological sample.
9. The kit according to claim 8 wherein the agent is selected from the group comprising an enzyme, an antibody, a protein probe, a metabolite or any other suitable composition.
10. The kit according to claim 8 wherein the agent comprises two antibodies directed to different epitopes on the peptide.
11. The kit according to claim 10 for use in determining the concentration of the peptide by using a sandwich immunoassay.
12. An isolated peptide which comprises the same or substantially the same amino acid sequence as the amino acid sequence of Seq ID NO: 1, or a part thereof, or its amide, or a salt thereof.
13. The polynucleotide encoding a peptide according to claim 12 .
14. The recombinant vector comprising a polynucleotide according to claim 13 .
15. The transformant transformed with a recombinant vector according to claim 14 .
16. The of a peptide according to claim 12 in the manufacture of an antibody.
17. The antibody specific for a peptide according to claim 12 .
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP08157521A EP2131199A1 (en) | 2008-06-03 | 2008-06-03 | Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof |
EP08157521.9 | 2008-06-03 | ||
EP09153804.1 | 2009-02-26 | ||
EP09153804 | 2009-02-26 | ||
PCT/EP2009/052396 WO2009146957A1 (en) | 2008-06-03 | 2009-02-27 | Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof. |
Publications (1)
Publication Number | Publication Date |
---|---|
US20110159607A1 true US20110159607A1 (en) | 2011-06-30 |
Family
ID=40613112
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/737,050 Abandoned US20110159607A1 (en) | 2008-06-03 | 2009-02-27 | Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof |
US12/737,051 Expired - Fee Related US8771968B2 (en) | 2008-06-03 | 2009-02-27 | Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof |
US14/316,921 Expired - Fee Related US9052313B2 (en) | 2008-06-03 | 2014-06-27 | Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof |
Family Applications After (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/737,051 Expired - Fee Related US8771968B2 (en) | 2008-06-03 | 2009-02-27 | Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof |
US14/316,921 Expired - Fee Related US9052313B2 (en) | 2008-06-03 | 2014-06-27 | Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof |
Country Status (5)
Country | Link |
---|---|
US (3) | US20110159607A1 (en) |
EP (3) | EP2286240B1 (en) |
DK (1) | DK2286240T3 (en) |
ES (1) | ES2565838T3 (en) |
WO (2) | WO2009146957A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DK2286240T3 (en) * | 2008-06-03 | 2016-04-11 | Université de Liège | Biomarker of osteoarthritis and use thereof |
CN105755146A (en) * | 2016-04-29 | 2016-07-13 | 北京致成生物医学科技有限公司 | Application of SERPINB3 gene in preparing osteoarthritis diagnosis preparation |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030236392A1 (en) * | 2001-11-05 | 2003-12-25 | Helix Research Institute | Novel full length cDNA |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7011952B2 (en) | 2000-02-22 | 2006-03-14 | University Of Iowa Research Foundation | Diagnostics and therapeutics for macular degeneration-related disorders |
JP2005511002A (en) * | 2001-05-11 | 2005-04-28 | ワイス | Transgenic animal model of bone mass modulation |
WO2003063689A2 (en) * | 2002-01-29 | 2003-08-07 | Quark Biotech, Inc. | Islr gene and its association with osteoarthritis and other bone and cartilage disorders, expression products derived therefrom, and uses thereof |
WO2006004066A1 (en) * | 2004-07-02 | 2006-01-12 | Locomogene, Inc. | S1-5-containing protein preparation |
WO2006006477A1 (en) * | 2004-07-09 | 2006-01-19 | Shionogi & Co., Ltd. | Polypeptide participating in bone disease or joint disease and dna thereof |
US20060094054A1 (en) | 2004-11-04 | 2006-05-04 | National Jewish Medical And Research Center | Fibulin-3 and uses thereof |
AU2006261641B2 (en) * | 2005-06-17 | 2011-09-29 | The Brigham And Women's Hospital, Inc. | Protein profile for osteoarthritis |
EP2121747A2 (en) * | 2006-07-27 | 2009-11-25 | Oxford Genome Sciences (UK) Limited | New protein isoforms and uses thereof |
DK2286240T3 (en) * | 2008-06-03 | 2016-04-11 | Université de Liège | Biomarker of osteoarthritis and use thereof |
-
2009
- 2009-02-27 DK DK09757339.8T patent/DK2286240T3/en active
- 2009-02-27 US US12/737,050 patent/US20110159607A1/en not_active Abandoned
- 2009-02-27 US US12/737,051 patent/US8771968B2/en not_active Expired - Fee Related
- 2009-02-27 EP EP09757339.8A patent/EP2286240B1/en not_active Not-in-force
- 2009-02-27 EP EP15180470.5A patent/EP2975411A1/en not_active Ceased
- 2009-02-27 WO PCT/EP2009/052396 patent/WO2009146957A1/en active Application Filing
- 2009-02-27 EP EP20090757340 patent/EP2297582A1/en not_active Withdrawn
- 2009-02-27 ES ES09757339.8T patent/ES2565838T3/en active Active
- 2009-02-27 WO PCT/EP2009/052392 patent/WO2009146956A1/en active Application Filing
-
2014
- 2014-06-27 US US14/316,921 patent/US9052313B2/en not_active Expired - Fee Related
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030236392A1 (en) * | 2001-11-05 | 2003-12-25 | Helix Research Institute | Novel full length cDNA |
Also Published As
Publication number | Publication date |
---|---|
WO2009146957A1 (en) | 2009-12-10 |
ES2565838T3 (en) | 2016-04-07 |
EP2286240B1 (en) | 2016-01-06 |
EP2975411A1 (en) | 2016-01-20 |
EP2297582A1 (en) | 2011-03-23 |
US9052313B2 (en) | 2015-06-09 |
EP2286240A1 (en) | 2011-02-23 |
US20150037823A1 (en) | 2015-02-05 |
US20110159514A1 (en) | 2011-06-30 |
US8771968B2 (en) | 2014-07-08 |
DK2286240T3 (en) | 2016-04-11 |
WO2009146956A1 (en) | 2009-12-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20230117596A1 (en) | Clinical diagnosis of non-alcoholic fatty liver disease using a panel of human blood protein biomarkers | |
WO2013172105A1 (en) | Marker for detecting pancreatic cancer | |
Xuan et al. | Proteomic study reveals plasma protein changes in congenital heart diseases | |
AU2011217734B2 (en) | Protein biomarkers for obstructive airways diseases | |
Snipsøyr et al. | Towards identification of novel putative biomarkers for infective endocarditis by serum proteomic analysis | |
Gibson et al. | Comparative analysis of synovial fluid and plasma proteomes in juvenile arthritis–proteomic patterns of joint inflammation in early stage disease | |
US9052313B2 (en) | Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof | |
US20090061457A1 (en) | Apolipoprotein E3 protein as a biomarker of Parkinson's disease | |
AU2013285362B2 (en) | Tropomyosin isoforms related to Alzheimers disease and Mild Cognitive Impairment | |
EP2131199A1 (en) | Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof | |
TWI397687B (en) | Use of biomarkers associated with nephropathy and kits used therein | |
US20140030734A1 (en) | Biomarkers for Diseases of the Central Nervous System | |
EP1938100A2 (en) | Identification of novel protein targets on the surface of stressed cells | |
KR101257307B1 (en) | Marker proteins for the diagnosis, therapy and monitoring of Atopic dermatitis from human plasma | |
CA2817851A1 (en) | Albumin-bound protein/peptide complex as a biomarker for disease | |
Filip et al. | Proteomics of Body Fluids | |
JP2022082068A (en) | Method for detecting infant atopic dermatitis | |
ES2370671B1 (en) | METHOD OF FORECAST AND DIAGNOSIS OF THE AORTIC STENOSIS THAT INCLUDES ALPHA-1-ANTICHEMIOTRIPSINE AS A MARKER. | |
JP2011158261A (en) | Method for detecting progressive degree of osteoarthritis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |