JP2016180665A - METHOD FOR DETECTING CARDIAC FAILURE WITH β-ANP - Google Patents
METHOD FOR DETECTING CARDIAC FAILURE WITH β-ANP Download PDFInfo
- Publication number
- JP2016180665A JP2016180665A JP2015060777A JP2015060777A JP2016180665A JP 2016180665 A JP2016180665 A JP 2016180665A JP 2015060777 A JP2015060777 A JP 2015060777A JP 2015060777 A JP2015060777 A JP 2015060777A JP 2016180665 A JP2016180665 A JP 2016180665A
- Authority
- JP
- Japan
- Prior art keywords
- anp
- total
- heart failure
- antibody
- solution
- 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.)
- Granted
Links
- 102000002723 Atrial Natriuretic Factor Human genes 0.000 title claims abstract description 179
- 101800001288 Atrial natriuretic factor Proteins 0.000 title claims abstract description 179
- 206010019280 Heart failures Diseases 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 39
- 239000003153 chemical reaction reagent Substances 0.000 claims description 16
- 238000001514 detection method Methods 0.000 claims description 7
- 101800001890 Atrial natriuretic peptide Proteins 0.000 abstract description 61
- 238000011282 treatment Methods 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 12
- NSQLIUXCMFBZME-MPVJKSABSA-N carperitide Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 NSQLIUXCMFBZME-MPVJKSABSA-N 0.000 description 59
- 239000000243 solution Substances 0.000 description 32
- 210000002381 plasma Anatomy 0.000 description 24
- 238000005259 measurement Methods 0.000 description 16
- 230000027455 binding Effects 0.000 description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 10
- 238000003127 radioimmunoassay Methods 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 206010007556 Cardiac failure acute Diseases 0.000 description 9
- 239000000872 buffer Substances 0.000 description 9
- 210000004408 hybridoma Anatomy 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- 239000000427 antigen Substances 0.000 description 8
- 108091007433 antigens Proteins 0.000 description 8
- 102000036639 antigens Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 238000012216 screening Methods 0.000 description 8
- 102400001263 NT-proBNP Human genes 0.000 description 7
- 238000003018 immunoassay Methods 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 238000000691 measurement method Methods 0.000 description 7
- 108010008064 pro-brain natriuretic peptide (1-76) Proteins 0.000 description 7
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- 235000018102 proteins Nutrition 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 238000004007 reversed phase HPLC Methods 0.000 description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 6
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 6
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 6
- 101800000407 Brain natriuretic peptide 32 Proteins 0.000 description 6
- 102400000667 Brain natriuretic peptide 32 Human genes 0.000 description 6
- 101800002247 Brain natriuretic peptide 45 Proteins 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- 125000003275 alpha amino acid group Chemical group 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 239000002202 Polyethylene glycol Substances 0.000 description 5
- 230000000903 blocking effect Effects 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 239000012228 culture supernatant Substances 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- 229920001223 polyethylene glycol Polymers 0.000 description 5
- 206010003445 Ascites Diseases 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 239000000539 dimer Substances 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 108010039627 Aprotinin Proteins 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 3
- 239000013504 Triton X-100 Substances 0.000 description 3
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 3
- 229960004405 aprotinin Drugs 0.000 description 3
- 230000036772 blood pressure Effects 0.000 description 3
- 230000000747 cardiac effect Effects 0.000 description 3
- 229940109239 creatinine Drugs 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 3
- 210000001165 lymph node Anatomy 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 3
- 230000007170 pathology Effects 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000002861 ventricular Effects 0.000 description 3
- ZOOGRGPOEVQQDX-UUOKFMHZSA-N 3',5'-cyclic GMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 ZOOGRGPOEVQQDX-UUOKFMHZSA-N 0.000 description 2
- PQSUYGKTWSAVDQ-ZVIOFETBSA-N Aldosterone Chemical compound C([C@@]1([C@@H](C(=O)CO)CC[C@H]1[C@@H]1CC2)C=O)[C@H](O)[C@@H]1[C@]1(C)C2=CC(=O)CC1 PQSUYGKTWSAVDQ-ZVIOFETBSA-N 0.000 description 2
- PQSUYGKTWSAVDQ-UHFFFAOYSA-N Aldosterone Natural products C1CC2C3CCC(C(=O)CO)C3(C=O)CC(O)C2C2(C)C1=CC(=O)CC2 PQSUYGKTWSAVDQ-UHFFFAOYSA-N 0.000 description 2
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 206010020772 Hypertension Diseases 0.000 description 2
- 108010023244 Lactoperoxidase Proteins 0.000 description 2
- 102000045576 Lactoperoxidases Human genes 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 102100028255 Renin Human genes 0.000 description 2
- 108090000783 Renin Proteins 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 241000473945 Theria <moth genus> Species 0.000 description 2
- 229960002478 aldosterone Drugs 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000001746 atrial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- ZOOGRGPOEVQQDX-UHFFFAOYSA-N cyclic GMP Natural products O1C2COP(O)(=O)OC2C(O)C1N1C=NC2=C1NC(N)=NC2=O ZOOGRGPOEVQQDX-UHFFFAOYSA-N 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 239000013024 dilution buffer Substances 0.000 description 2
- 230000001882 diuretic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000024924 glomerular filtration Effects 0.000 description 2
- 210000002837 heart atrium Anatomy 0.000 description 2
- 230000000004 hemodynamic effect Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000003907 kidney function Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 229940057428 lactoperoxidase Drugs 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- 238000004904 shortening Methods 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- ZMXRHHJWXLKMTL-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate Chemical compound COCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCC(=O)ON1C(=O)CCC1=O ZMXRHHJWXLKMTL-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000011735 C3H mouse Methods 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 238000001061 Dunnett's test Methods 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- GHAZCVNUKKZTLG-UHFFFAOYSA-N N-ethyl-succinimide Natural products CCN1C(=O)CCC1=O GHAZCVNUKKZTLG-UHFFFAOYSA-N 0.000 description 1
- HDFGOPSGAURCEO-UHFFFAOYSA-N N-ethylmaleimide Chemical compound CCN1C(=O)C=CC1=O HDFGOPSGAURCEO-UHFFFAOYSA-N 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 229910021536 Zeolite Inorganic materials 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000011545 carbonate/bicarbonate buffer Substances 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- UKWLRLAKGMZXJC-QIECWBMSSA-L disodium;[4-chloro-3-[(3r,5s)-1-chloro-3'-methoxyspiro[adamantane-4,4'-dioxetane]-3'-yl]phenyl] phosphate Chemical compound [Na+].[Na+].O1OC2([C@@H]3CC4C[C@H]2CC(Cl)(C4)C3)C1(OC)C1=CC(OP([O-])([O-])=O)=CC=C1Cl UKWLRLAKGMZXJC-QIECWBMSSA-L 0.000 description 1
- 125000002228 disulfide group Chemical group 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000011984 electrochemiluminescence immunoassay Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 230000001452 natriuretic effect Effects 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004648 relaxation of smooth muscle Effects 0.000 description 1
- 230000036454 renin-angiotensin system Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 238000009666 routine test Methods 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000012134 supernatant fraction Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000002820 sympathetic nervous system Anatomy 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 230000000304 vasodilatating effect Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 239000010457 zeolite Substances 0.000 description 1
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
本発明は心不全の検出方法及びそのキットに関する。 The present invention relates to a method for detecting heart failure and a kit thereof.
心房性ナトリウム利尿ペプチド（ＡＮＰ：ａｔｒｉａｌ ｎａｔｒｉｕｒｅｔｉｃ ｐｅｐｔｉｄｅ）は松尾・寒川らにより１９８４年に報告されたペプチドホルモンで（非特許文献１参照）、心臓で産生され強力な利尿、ナトリウム利尿活性と平滑筋弛緩活性を持つ。ＡＮＰは、ヒトばかりでなく、げっ歯類以外の哺乳類では同一のアミノ酸配列を有し、例えばヒト、ウシ、ブタのＡＮＰは同一である。ＡＮＰには、分子量約３０００のα型、α型の逆平行二量体であるβ型、α型の前駆物質であるγ型の３種類の分子型が確認されている（図１参照）。 Atrial natriuretic peptide (ANP) is a peptide hormone reported in 1984 by Matsuo and Samukawa et al. (See Non-Patent Document 1), a strong diuresis produced in the heart, natriuretic activity and smooth muscle relaxation. Has activity. ANP has the same amino acid sequence not only in humans but also in mammals other than rodents. For example, ANP in humans, cows and pigs are identical. ANP has been confirmed to have three molecular types: α type having a molecular weight of about 3000, β type which is an antiparallel dimer of α type, and γ type which is a precursor of α type (see FIG. 1).
ＡＮＰは、心臓、特に心房から分泌されるホルモンで、利尿作用、Ｎａ利尿作用、血管拡張作用、レニン・アンジオテンシン・アルドステロン系や交感神経系の抑制作用など多様な生物活性を有し、体液量、血圧の調節に重要な役割を担っている（非特許文献２，３参照）。
ANP is a hormone secreted from the heart, particularly the atrium, and has various biological activities such as diuretic action, Na diuretic action, vasodilatory action, renin-angiotensin-aldosterone system and sympathetic nervous system suppressive action, It plays an important role in regulating blood pressure (see Non-Patent
ＡＮＰは心血行動態的負荷、特に心房内圧の増加が主要な産生、分泌刺激になると考えられ、体液量あるいは心房内圧の増加する各種心疾患、腎疾患において血中濃度の増加が認められることから、これら病態の把握、重症度の指標として極めて有用であり、ルーチンの検査項目として日常臨床に応用されている（非特許文献４〜６参照）。
ANP is considered to be a major production and secretion stimulus due to an increase in cardiac hemodynamic load, especially atrial pressure, and an increase in blood concentration is observed in various heart diseases and renal diseases in which body fluid volume or atrial pressure increases. It is extremely useful as an index of grasping and severity of these pathological conditions, and is applied to daily clinical practice as a routine test item (see Non-Patent
α−ＡＮＰは血中に存在するＡＮＰ類の中心となる分子種であり、２８アミノ酸残基からなり、Ｎ末端から７番目のＣｙｓと２３番目のＣｙｓが分子内でジスルフィド結合し、その間の配列が環状構造をなしている。一方、β−ＡＮＰは、２分子のα−ＡＮＰが分子間でジスルフィド結合した、α−ＡＮＰの逆平行二量体である（特許文献１参照）。γ−ＡＮＰは１２６アミノ酸残基からなり、そのＣ末端にα−ＡＮＰ配列を有する。健常者においては、γ−ＡＮＰは心房中に保存されており、分泌時にα−ＡＮＰとＮ末端ペプチドに切断され、血中ではこの２つの分子で存在する（非特許文献７参照）。 α-ANP is a molecular species that is the center of ANPs present in the blood, consists of 28 amino acid residues, and the 7th Cys and 23rd Cys from the N-terminus are disulfide-bonded in the molecule, and the sequence between them. Has a ring structure. On the other hand, β-ANP is an antiparallel dimer of α-ANP in which two molecules of α-ANP are disulfide bonded between molecules (see Patent Document 1). γ-ANP consists of 126 amino acid residues and has an α-ANP sequence at its C-terminus. In healthy individuals, γ-ANP is conserved in the atria, cleaved into α-ANP and an N-terminal peptide during secretion, and exists in these two molecules in blood (see Non-Patent Document 7).
ＡＮＰの測定は、酵素やラジオアイソトープで標識した抗体と、担体に固定化した固定化抗体とでサンドイッチされる免疫学的測定法が利用されており、特に臨床検査薬として広く医療現場で使用されており（非特許文献９参照）、α型、β型、γ型の３種類の総和が測定されるものである。 An ANP measurement uses an immunoassay method in which an antibody labeled with an enzyme or radioisotope and an immobilized antibody immobilized on a carrier are sandwiched. Particularly, it is widely used as a clinical test drug in the medical field. (See Non-Patent Document 9), and the total of three types of α type, β type, and γ type is measured.
α−ＡＮＰの有用性は多く報告されているが、β−ＡＮＰとγ−ＡＮＰについても重症心不全症例で増加していることが報告されている（非特許文献８参照）。特にβ−ＡＮＰの動向は顕著であり、健常者では確認されない様式であるが、心不全の重篤度に合わせて出現することが確認され、心不全病態との関係が確認されている（非特許文献１０，１１参照）。心不全治療前後におけるＡＮＰの存在様式を検討すると、心不全時は，α−、γ−ＡＮＰの他に、健常人では認めなかったβ−ＡＮＰと思われる中分子型ＡＮＰが出現し、心不全治療による血行動態・臨床症状が改善した後には、ＡＮＰ濃度の低下とともにこのβ−ＡＮＰの割合が減少あるいは消失している。 Although the usefulness of α-ANP has been reported a lot, β-ANP and γ-ANP have also been reported to increase in cases of severe heart failure (see Non-Patent Document 8). In particular, the trend of β-ANP is remarkable, and it is a mode that is not confirmed in healthy individuals, but it has been confirmed that it appears in accordance with the severity of heart failure, and its relationship with heart failure pathology has been confirmed (Non-Patent Document). 10, 11). When the existence pattern of ANP before and after heart failure treatment was examined, in addition to α- and γ-ANP, medium molecular type ANP that seems to be β-ANP, which was not observed in healthy individuals, appeared during heart failure. After the kinetic / clinical symptoms improve, the ratio of β-ANP decreases or disappears with a decrease in ANP concentration.
治療により心不全が改善する過程における血中ＡＮＰ濃度と存在様式の推移では、血中ＡＮＰ濃度は治療により漸減し、存在様式は重症心不全時はβ−ＡＮＰがピークを形成していたが、治療によりβ−ＡＮＰは漸減し、血行動態および臨床症状が改善した時点では存在様式はβ−ＡＮＰの割合は著しく減少あるいは消失し、心不全病態とβ−ＡＮＰの関係が示されている（非特許文献１１参照）。しかしながらこれらの報告では具体的にβ−ＡＮＰ値のカットオフ値、参考基準値あるいは総ＡＮＰに対する存在比率などを用いて心不全の病態や治療効果を見ていない。加えてα型、β型、γ型の各分子型の共通領域（即ちα−ＡＮＰ）を用いる抗体と逆相高速液体クロマト（ＲＰ−ＨＰＬＣ）法を用いた測定法により確認しているために、定量性に乏しく操作による変動も大きい。 In the process of improvement of heart failure by treatment, the blood ANP concentration gradually changed with the treatment, and the presence pattern showed that β-ANP peaked during severe heart failure. β-ANP gradually decreases, and at the time when hemodynamics and clinical symptoms are improved, the proportion of β-ANP is significantly reduced or disappeared, indicating the relationship between heart failure pathology and β-ANP (Non-patent Document 11). reference). However, these reports do not specifically examine the pathological condition or therapeutic effect of heart failure using the cut-off value of β-ANP value, the reference standard value, or the abundance ratio with respect to total ANP. In addition, because it is confirmed by a measurement method using an antibody using a common region (namely, α-ANP) of each molecular type of α-type, β-type, and γ-type and reverse-phase high-performance liquid chromatography (RP-HPLC) method Quantities are poor and fluctuations due to operation are large.
このように、従来は、β−ＡＮＰの分子型に対し特異的な抗体や測定方法は見出されていなかったために、β−ＡＮＰの総ＡＮＰに対する存在比率から心不全の状態を判断する例はなかった。 Thus, conventionally, since no antibody or measurement method specific to the molecular type of β-ANP has been found, there is no example of determining the state of heart failure from the abundance ratio of β-ANP to the total ANP. It was.
β−ＡＮＰは、心不全の重篤度に合わせて出現することが確認され、心不全病態との関係が確認されている。しかし市販されているＡＮＰを測定するサンドイッチ免疫測定法においては、α型、α型の２量体であるβ型、α型の前駆物質とされるγ型の３種類の分子型も測定され、β−ＡＮＰのみを特異的に測定する方法は一般化されていない。 β-ANP has been confirmed to appear in accordance with the severity of heart failure, and the relationship with the heart failure pathology has been confirmed. However, in the sandwich immunoassay for measuring ANP that is commercially available, three types of molecules, α-type, β-type that is a dimer of α-type, and γ-type that is a precursor of α-type, are also measured. A method for specifically measuring only β-ANP has not been generalized.
β−ＡＮＰに特異的な測定系については、α−ＡＮＰのホモダイマーであることから、同一のモノクローナル抗体を用いてβ−ＡＮＰを測定する測定系は報告されている（特許文献２参照）。しかし、β−ＡＮＰに特異的な抗体を用いているわけではなく、α−ＡＮＰ濃度が高くなると競合阻害を受けることになり測定系によっては感度が低くなることがあり、依然、高感度にβ−ＡＮＰのみを測定するためには、課題が残っている（特許文献２参照）。 Since the measurement system specific for β-ANP is a homodimer of α-ANP, a measurement system for measuring β-ANP using the same monoclonal antibody has been reported (see Patent Document 2). However, an antibody specific for β-ANP is not used, and when the α-ANP concentration becomes high, competitive inhibition occurs and the sensitivity may be lowered depending on the measurement system. -A problem remains to measure only ANP (see Patent Document 2).
総ＡＮＰおよびβ−ＡＮＰが心不全の重症例で高値を示し、治療により改善すると低下することが報告されているが、これらの報告では具体的にβ−ＡＮＰ値のカットオフ値、参考基準値あるいは総ＡＮＰに対する存在比率などを用いて心不全の病態や治療効果を見ていない。加えてα型、β型、γ型の各分子型の共通領域（即ちα−ＡＮＰ）を用いる抗体と逆相高速液体クロマト（ＲＰ−ＨＰＬＣ）法を用いた測定法により確認しているために定量性に乏しく操作による変動も大きいために診断には利用されていない。 Total ANP and β-ANP have been reported to be high in severe cases of heart failure and decreased with improvement in treatment. In these reports, the cut-off value of β-ANP value, reference standard value or We do not look at the pathophysiology or therapeutic effect of heart failure using the ratio of total ANP. In addition, because it is confirmed by a measurement method using an antibody using a common region (namely, α-ANP) of each molecular type of α-type, β-type, and γ-type and reverse-phase high-performance liquid chromatography (RP-HPLC) method It is not used for diagnosis because of its poor quantitativeness and large variation due to operation.
このように、従来は、β−ＡＮＰの分子型に非常に特異的な抗体や測定方法は見出されていなかったために、β−ＡＮＰと総ＡＮＰとの存在比から心不全の状態を判断する例はなかった。 Thus, since no antibody or measurement method very specific to the molecular type of β-ANP has been found in the past, an example of determining the state of heart failure from the abundance ratio of β-ANP and total ANP There was no.
本発明の目的は、β−ＡＮＰと総ＡＮＰとの比から、心不全の進行または心不全治療の効果を判定することができる心不全の検出方法を提供することである。 An object of the present invention is to provide a heart failure detection method capable of determining the progress of heart failure or the effect of heart failure treatment from the ratio of β-ANP and total ANP.
本発明者らは、α−ＡＮＰの逆平行ダイマーが特異的につくるジスルフィド結合を含むアミノ酸配列や構造をエピトープとして認識する抗体を獲得し、それを利用するβ−ＡＮＰの高感度測定法を完成している（特願２０１４−２１４１０３号）。そして本発明者らは、上記課題に関し鋭意検討した結果、本発明に到達した。 The present inventors have obtained an antibody that recognizes an amino acid sequence or structure containing a disulfide bond specifically produced by an antiparallel dimer of α-ANP as an epitope, and completed a highly sensitive measurement method of β-ANP using this antibody. (Japanese Patent Application No. 2014-214103). And as a result of intensive studies on the above problems, the present inventors have reached the present invention.
即ち本発明は以下のとおりである。
（１）試料中のβ−ＡＮＰと総ＡＮＰとの比を求めることを特徴とする、心不全の検出方法。
（２）請求項１に記載の方法において、β−ＡＮＰと総ＡＮＰとの比が、総ＡＮＰに対するβ−ＡＮＰの比（β−ＡＮＰ／総ＡＮＰ）である方法。
（３）β−ＡＮＰを特異的に測定する試薬および総ＡＮＰを測定する試薬を含むことを特徴とする、心不全の検出キット。
以下、本発明を更に詳細に説明する。
That is, the present invention is as follows.
(1) A method for detecting heart failure, wherein a ratio of β-ANP and total ANP in a sample is determined.
(2) The method according to
(3) A detection kit for heart failure, comprising a reagent that specifically measures β-ANP and a reagent that measures total ANP.
Hereinafter, the present invention will be described in more detail.
本発明において、総ＡＮＰとは、α−ＡＮＰ、β−ＡＮＰ及びγ−ＡＮＰを合計したものを表す。総ＡＮＰの測定方法としては特に限定はなく、例えば市販の免疫測定試薬等を用いて行うことができる。例えばα−ＡＮＰ、β−ＡＮＰ、γ−ＡＮＰの共通部位を認識する抗体を組み合わせた測定系を用いることができ、市販キットのように例えばα−ＡＮＰの環状部位とＣ末端部分を認識するものでも良い。 In the present invention, the total ANP represents the sum of α-ANP, β-ANP and γ-ANP. The method for measuring total ANP is not particularly limited, and for example, a commercially available immunoassay reagent can be used. For example, a measurement system combining an antibody that recognizes the common site of α-ANP, β-ANP, and γ-ANP can be used, such as a commercially available kit that recognizes the cyclic site and C-terminal part of α-ANP, for example. But it ’s okay.
一方、β−ＡＮＰの測定方法も特に限定されるものではなく、例えばβ−ＡＮＰを特異的に測定する方法でもよく、また総ＡＮＰの測定値からα−ＡＮＰとγ−ＡＮＰの測定値を差し引いてもよい。β−ＡＮＰを特異的に測定する方法としては特に限定されるものではないが、β−ＡＮＰを特異的に認識することができ、α−ＡＮＰやγ−ＡＮＰを実質的に認識しない方法があげられる。好ましくはβ−ＡＮＰのジスルフィド結合を含むアミノ酸配列を認識する抗体を用いた測定系、さらに好ましくはβ−ＡＮＰのジスルフィド結合とその周辺のアミノ酸配列を認識する抗体を用いた測定系が選択される。 On the other hand, the β-ANP measurement method is not particularly limited. For example, β-ANP may be specifically measured, and the α-ANP and γ-ANP measurement values are subtracted from the total ANP measurement value. May be. The method for specifically measuring β-ANP is not particularly limited, but a method that can specifically recognize β-ANP and does not substantially recognize α-ANP or γ-ANP is mentioned. It is done. Preferably, a measurement system using an antibody recognizing an amino acid sequence containing a β-ANP disulfide bond, more preferably a measurement system using an antibody recognizing a β-ANP disulfide bond and its surrounding amino acid sequence is selected. .
β−ＡＮＰや総ＡＮＰを測定する方法として、免疫学的測定方法を用いる場合は、標識を用いることができる。標識としては、１２５Ｉ、３Ｈなどの放射性物質、西洋わさびペルオキシダーゼ、β−Ｄ−ガラクトシダーゼ、アルカリホスファターゼ（ＡＬＰ）などの酵素、フルオレッセインなどの蛍光物質、金コロイド、セレンコロイド、ルシフェリンなどの発光又は発色物質などが用いられ、標識された抗原あるいは抗体が試薬として用いられている。また、直接これらの物質を検出に用いる物質に標識せず、ビオチン−アビジン等を利用して間接的に標識してもよい。抗原に結合した標識物質を検出することは、例えば、公知の酵素免疫測定法（ＥＩＡ、ＥＬＩＳＡ）、放射免疫測定法（ＲＩＡ）、蛍光免疫測定法（ＦＩＡ）、発光免疫測定法（ＬＩＡ）又は発光酵素免疫測定法（ＣＬＥＩＡ）等により行うことができる。 As a method for measuring β-ANP and total ANP, a label can be used when an immunological measurement method is used. Examples of labels include radioactive substances such as 125 I and 3 H, enzymes such as horseradish peroxidase, β-D-galactosidase and alkaline phosphatase (ALP), fluorescent substances such as fluorescein, colloidal gold, selenium colloid and luciferin. Luminescent or coloring substances are used, and labeled antigens or antibodies are used as reagents. Alternatively, these substances may be directly labeled using biotin-avidin or the like without being labeled with a substance used for detection. Detecting a labeled substance bound to an antigen can be performed by, for example, a known enzyme immunoassay (EIA, ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), luminescence immunoassay (LIA) or It can be performed by a luminescent enzyme immunoassay (CLEIA) or the like.
またβ−ＡＮＰや総ＡＮＰを測定する方法として、免疫学的測定方法を用いる場合は、不溶性担体を用いることができる。不溶性担体に関しては、よく知られているガラス、ポリスチレン、ポリプロピレン、デキストランなどの物質からなるビーズ、チューブ、プレート、磁性微粒子など用いることができ、反応後にＢ／Ｆ分離可能な担体が好ましく、その材質などは問わない。また、不溶性担体と抗体（あるいはレセプター、結合蛋白質）との結合は、物理的結合あるいは化学的に中間体を介した結合等、Ｂ／Ｆ分離時に結合能が失われない方法が好ましい。 Moreover, when using an immunological measuring method as a method for measuring β-ANP and total ANP, an insoluble carrier can be used. As for the insoluble carrier, beads, tubes, plates, magnetic fine particles, etc. made of well-known substances such as glass, polystyrene, polypropylene, dextran, etc. can be used. It doesn't matter. The insoluble carrier and the antibody (or receptor or binding protein) are preferably bound by a method that does not lose the binding ability during B / F separation, such as physical binding or chemical binding via an intermediate.
本発明における試料としては被験体由来の血液等が挙げられ、ＥＤＴＡ・アプロチニン血漿を用いるのが最も好ましい。 Examples of the sample in the present invention include blood derived from a subject, and it is most preferable to use EDTA / aprotinin plasma.
本発明では、β−ＡＮＰと総ＡＮＰとの比を求める。これは例えば、総ＡＮＰに対するβ−ＡＮＰの比（β−ＡＮＰ／総ＡＮＰ）であってもよく、またβ−ＡＮＰに対する総ＡＮＰの比（総ＡＮＰ／β−ＡＮＰ）であってもよい。 In the present invention, the ratio between β-ANP and total ANP is determined. This may be, for example, the ratio of β-ANP to total ANP (β-ANP / total ANP), or the ratio of total ANP to β-ANP (total ANP / β-ANP).
例えば本願実施例で用いた測定系の場合は、β−ＡＮＰ／総ＡＮＰの値が１０％以上の場合は心不全と判定される。 For example, in the case of the measurement system used in the examples of the present application, when the value of β-ANP / total ANP is 10% or more, it is determined as heart failure.
このようにして本発明の方法により心不全を検出することができ、それにより心不全又は心不全治療の効果を判定することができる。 In this way, heart failure can be detected by the method of the present invention, whereby the heart failure or the effect of heart failure treatment can be determined.
またβ−ＡＮＰを特異的に測定する試薬および総ＡＮＰを測定する試薬を含むキットで、本発明の心不全の検出を行うこともできる。β−ＡＮＰを特異的に測定する試薬および総ＡＮＰを測定する試薬としては、前述のものを使用することができる。 In addition, the detection of heart failure of the present invention can be performed with a kit containing a reagent that specifically measures β-ANP and a reagent that measures total ANP. As the reagent for specifically measuring β-ANP and the reagent for measuring total ANP, those described above can be used.
本発明により、β−ＡＮＰと総ＡＮＰとの比から心不全の進行または心不全治療の効果を判定することが可能となった。この方法は心不全の診断に有用である。 According to the present invention, it is possible to determine the progress of heart failure or the effect of treatment for heart failure from the ratio of β-ANP and total ANP. This method is useful for the diagnosis of heart failure.
以下、実施例により本発明をさらに詳細に説明するが、本発明は本実施例により限定されるものではない。また以下の試験では、健常者以外は被験者に本研究の説明文書に基づく説明を行い、文書で同意が得られた被験者の検体を用いて行われた。 EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by a present Example. In the following tests, non-healthy subjects were given explanations to the subjects based on the explanatory documents of this study, and the subjects were given consent from the samples.
［対象被験者］
本実験に使用した検体としては、健常者１０例、心不全患者（急性心不全患者）６０例の血漿を用いた。急性心不全患者は、（１）入院時、（２）入院後１〜２日後、（３）入院後５〜８日後、（４）退院時の治療経過にあわせて採血した。健常者１０例は、男性５例、女性５例で、年齢は４０．３±１３．５歳（ｍｅａｎ±ＳＤ）を用いた。急性心不全患者６０例は、男性４３例、女性１７例で、年齢は７０．７±１２．５歳（ｍｅａｎ±ＳＤ）を用いた。急性心不全患者６０例の入院時および退院時におけるニューヨーク心臓協会（ＮＹＨＡ）による分類、体重、平均血圧、心臓超音波検査（左室内径短縮率）を表１に示す。血漿ＢＮＰ濃度は市販のＣＬＥＩＡキット（ルミパルスＧ１２００、富士レビオ社）を用いてＥＤＴＡ・アプロチニン血漿を測定した。血清ＮＴ−ｐｒｏＢＮＰ濃度は市販の電気化学発光免疫測定法キット（エクルーシス試薬ＮＴ−ｐｒｏＢＮＰ ＩＩ、ロシュ社）を用いて測定した。血漿レニン活性は、ラジオイムノアッセイ二抗体法を用いてＥＤＴＡ血漿を測定した。血漿アルドステロン濃度は、ラジオイムノアッセイ固相法を用いてＥＤＴＡ血漿を測定した。サイクリックＧＭＰは、サクシニル化したＥＤＴＡ血漿をラジオイムノアッセイ法を用いて測定した。血清尿素窒素濃度、血清クレアチニン濃度は、汎用生化学自動分析装置（Ｌａｂｏｓｐｅｃｔ ００８、日立製作所）を用いて測定した。
[Target subjects]
As samples used in this experiment, plasmas of 10 healthy subjects and 60 heart failure patients (acute heart failure patients) were used. Patients with acute heart failure were (1) hospitalized, (2) 1-2 days after hospitalization, (3) 5-8 days after hospitalization, and (4) blood collected according to the course of treatment at discharge. Ten healthy subjects were 5 males and 5 females, and the age was 40.3 ± 13.5 years (mean ± SD). Sixty patients with acute heart failure were 43 males and 17 females, and the age was 70.7 ± 12.5 years (mean ± SD). Table 1 shows the classification, body weight, mean blood pressure, and cardiac ultrasonography (left ventricular diameter shortening rate) according to the New York Heart Association (NYHA) at the time of hospitalization and discharge of 60 patients with acute heart failure. The plasma BNP concentration was determined by measuring EDTA / aprotinin plasma using a commercially available CLEIA kit (Lumipulse G1200, Fujirebio). Serum NT-proBNP concentration was measured using a commercially available electrochemiluminescence immunoassay kit (Eccursis Reagent NT-proBNP II, Roche). Plasma renin activity was measured in EDTA plasma using the radioimmunoassay two-antibody method. Plasma aldosterone concentrations were measured in EDTA plasma using a radioimmunoassay solid phase method. Cyclic GMP was measured using radioimmunoassay on succinylated EDTA plasma. Serum urea nitrogen concentration and serum creatinine concentration were measured using a general-purpose biochemical automatic analyzer (Labospect 008, Hitachi, Ltd.).
［ヒト血漿抽出物の調製］
ヒト正常血漿は市販品（ＥＤＴＡ−２Ｎａ、コージンバイオ社 １２２７１４４０）を使用した。血漿抽出物の調製には固相抽出カートリッジ（Ｓｅｐ−Ｐａｋ Ｃ１８ Ｐｌｕｓ、Ｗａｔｅｒｓ社 ＷＡＴ０２０５１５）を用いた。具体的には、固相抽出カートリッジを５ｍＬの６０％ アセトニトリル／０．１％トリフルオロ酢酸（ＴＦＡ）溶液にて洗浄し、５ｍＬの０．１％ＴＦＡ溶液にて平衡化した後、３００μＬの血漿を添加した。５ｍＬの１０％ アセトニトリル／０．１％ ＴＦＡ溶液にて２回洗浄した後、６ｍＬの４０％ アセトニトリル／０．１％ ＴＦＡ溶液にて溶出した。溶出液を濃縮した後、凍結乾燥し、反応液に溶解して測定に用いた。
[Preparation of human plasma extract]
As human normal plasma, a commercially available product (EDTA-2Na, Kojin Bio Inc. 12271440) was used. A solid phase extraction cartridge (Sep-Pak C18 Plus, Waters WAT020515) was used for the preparation of the plasma extract. Specifically, the solid-phase extraction cartridge was washed with 5 mL of 60% acetonitrile / 0.1% trifluoroacetic acid (TFA) solution, equilibrated with 5 mL of 0.1% TFA solution, and then 300 μL of plasma. Was added. After washing twice with 5 mL of 10% acetonitrile / 0.1% TFA solution, elution was performed with 6 mL of 40% acetonitrile / 0.1% TFA solution. The eluate was concentrated, freeze-dried, dissolved in the reaction solution and used for measurement.
（１）β−ＡＮＰの測定試薬の調製
［実験用試薬等］
・固相化用緩衝液：５０ｍＭ炭酸−重炭酸緩衝液（ｐＨ９．５）
・ＰＥＧ化試薬溶液：５μＭ ｍｅｔｈｙｌ−ＰＥＧ１２−ＮＨＳ ｅｓｔｅｒ（Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ社 ２２６８５）、ＰＢＳ（ｐＨ７．４）
・ブロッキング溶液：２５ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．４）、１５０ｍＭ ＮａＣｌ、２％ ＢｌｏｃｋＡｃｅ（ＤＳファーマバイオメディカル社 ＵＫ−Ｂ８０）、５％ ウマ血清、２０％ スクロース
・洗浄液：２５ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．４）、１５０ｍＭ ＮａＣｌ、０．０５％ Ｔｒｉｔｏｎ Ｘ−１００、０．０５％ ＮａＮ３
・反応液：２５ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．４）、１５０ｍＭ ＮａＣｌ、０．５ｍＭ ＥＤＴＡ−２Ｎａ、５％ ＢＳＡ、０．０５％ Ｔｒｉｔｏｎ Ｘ−１００、５００ＫＩＵ／ｍＬ アプロチニン（和光純薬社）、０．０５％ ＮａＮ３
・検出抗体希釈用緩衝液：２５ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．４）、１５０ｍＭ ＮａＣｌ、０．４％ ＢｌｏｃｋＡｃｅ、０．０５％ ＮａＮ３
・化学発光基質：ＣＤＰ−Ｓｔａｒ ｗｉｔｈ Ｅｍｅｒａｌｄ ＩＩ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社 Ｔ２２１６）
［ＡＬＰ標識＃３２−３の調製］
β−ＡＮＰのジスルフィド結合を含むアミノ酸配列を認識するマウスモノクローナル抗体＃３２−３（図２参照）のＡＬＰ標識にはＡｌｋａｌｉｎｅ Ｐｈｏｓｐｈａｔａｓｅ Ｌａｂｅｌｉｎｇ Ｋｉｔ−ＮＨ２（同仁化学社 Ｌ１２）を用いた。標識および標識抗体の精製は製造業者のマニュアルに従い行った。なおマウスモノクローナル抗体＃３２−３の製法は、後述の参考例に記載した。
(1) Preparation of β-ANP measurement reagent [Experimental reagent, etc.]
-Buffer for solid phase: 50 mM carbonate-bicarbonate buffer (pH 9.5)
PEGylation reagent solution: 5 μM methyl-PEG 12 -NHS ester (Thermo Scientific 22865), PBS (pH 7.4)
Blocking solution: 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2% BlockAce (DS Pharma Biomedical UK-B80), 5% horse serum, 20% sucrose. Washing solution: 25 mM Tris-HCl (pH 7.4) ), 150 mM NaCl, 0.05% Triton X-100, 0.05% NaN 3
Reaction solution: 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5 mM EDTA-2Na, 5% BSA, 0.05% Triton X-100, 500 KIU / mL aprotinin (Wako Pure Chemical Industries), 0. 05% NaN 3
Detection antibody dilution buffer: 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.4% BlockAce, 0.05% NaN 3
Chemiluminescent substrate: CDP-Star with Emerald II (Applied Biosystems T2216)
[Preparation of ALP Label # 32-3]
Alkaline Phosphatase Labeling Kit-NH 2 (Dojin Chemical Co., Ltd. L12) was used for ALP labeling of mouse monoclonal antibody # 32-3 (see FIG. 2) that recognizes an amino acid sequence containing a β-ANP disulfide bond. Purification of the label and labeled antibody was performed according to the manufacturer's manual. The production method of mouse monoclonal antibody # 32-3 was described in Reference Examples described later.
［＃１３１−７固相化プレートの作製］
本ＣＬＥＩＡ法では、ヒトα−ＡＮＰの１３〜１７残基目をエピトープとするウサギポリクローナル抗体＃１３１−７（Ａｎａｌ． Ｂｉｏｃｈｅｍ．， ４６１；１０−１６，２０１４）（図２参照）を固相化抗体として用いた。ウサギポリクローナル抗体＃１３１−７の製法は、後述の参考例に記載した。固相化プレートの作製では、プレートへの非特異的吸着および固相化抗体のＦｃ領域への血漿由来成分の結合を低減するため、従来のＣＬＥＩＡ法と比較して、固相化抗体のＦｃ領域を標的としたポリエチレングリコール修飾（ＰＥＧ化）を追加した方法（Ａｎａｌ． Ｂｉｏｃｈｅｍ．， ４６１；１０−１６，２０１４）を採用した。具体的には、固相化用緩衝液に溶解した＃１３１−７（３μｇ／ｍＬ、１５０μＬ）を９６穴プレート（Ｆｌｕｏｒｏ−Ｎｕｎｃ Ｍａｘｉ−Ｓｏｒｐ、Ｎｕｎｃ社 ４３７７９６）に添加し、４℃で２４時間インキュベートした。抗体溶液を除去し、ＰＥＧ化試薬溶液（１００μＬ）を添加し、室温で３０分間インキュベートした。ＰＥＧ化試薬溶液を除去し、ブロッキング溶液（２００μＬ）を添加し、室温で２時間インキュベートした。ブロッキング溶液を除去した＃１３１−７固相化プレートをデシケーターにより乾燥させ、酸素吸収剤（アズワン社 １−６６５５−０２）およびゼオライト乾燥剤（アズワン社 １−６６５５−０３）と同封して密閉し、使用時まで−２０℃にて保存した。
[Production of # 131-7 solid-phase plate]
In the present CLEIA method, a rabbit polyclonal antibody # 131-7 (Anal. Biochem., 461; 10-16, 2014) (see FIG. 2) having epitopes 13 to 17 as human α-ANP as an epitope is immobilized. Used as an antibody. The production method of rabbit polyclonal antibody # 131-7 was described in Reference Examples described later. In the preparation of a solid-phased plate, in order to reduce non-specific adsorption to the plate and the binding of plasma-derived components to the Fc region of the solid-phased antibody, the Fc of the solid-phased antibody was compared with the conventional CLEIA method. A method (Anal. Biochem., 461; 10-16, 2014) in which polyethylene glycol modification (PEGylation) targeting the region was added was employed. Specifically, # 131-7 (3 μg / mL, 150 μL) dissolved in an immobilization buffer was added to a 96-well plate (Fluoro-Nunc Maxi-Sorp, Nunc 437796) for 24 hours at 4 ° C. Incubated. The antibody solution was removed, PEGylation reagent solution (100 μL) was added and incubated for 30 minutes at room temperature. The PEGylation reagent solution was removed, blocking solution (200 μL) was added and incubated for 2 hours at room temperature. The # 131-7 solid phase-immobilized plate from which the blocking solution has been removed is dried with a desiccator and sealed with an oxygen absorbent (ASONE 1-6655-02) and a zeolite desiccant (ASONE 1-6655-03). Stored at -20 ° C until use.
［ＣＬＥＩＡ法を用いた測定手順］
マイクロプレートウォッシャー（バイオテック社 ＡＭＷ−８Ｒ）を用いて＃１３１−７固相化プレートを洗浄液（３５０μＬ）で３回洗浄した。反応液（５０μＬ）を各ウェルに添加した後、標準β−ＡＮＰ溶液（反応液に溶解した定量済の合成β−ＡＮＰ溶液、ペプチド研究所）またはヒト血漿抽出物溶液（５０μＬ）を添加し、マイクロプレートシェーカー（日伸理化社 Ｎ−７０４）を用いて振盪撹拌しながら４℃で２４時間インキュベートした。上記と同様に３回洗浄した後、検出抗体希釈用緩衝液で０．２ｎｇ／ｍＬに希釈したＡＬＰ標識＃３２−３溶液（１００μＬ）を添加し、マイクロプレートシェーカーを用いて振盪撹拌しながら室温で１時間インキュベートした。上記と同様に４回洗浄した後、化学発光基質（１００μＬ）を添加し、室温で２０分間インキュベートした。マイクロプレートルミノメーター（ＳｐｅｃｔｒａＭａｘ Ｌ、Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅｓ社）により１秒間に生ずる発光量を測定した。各試料は異なる２ウェルで個別に測定し、その平均値より定量値を算出した。
[Measurement Procedure Using CLEIA Method]
Using a microplate washer (Biotech AMW-8R), the # 131-7 solid-phased plate was washed three times with a washing solution (350 μL). After adding reaction solution (50 μL) to each well, standard β-ANP solution (quantitative synthetic β-ANP solution dissolved in reaction solution, Peptide Institute) or human plasma extract solution (50 μL) is added, The mixture was incubated at 4 ° C. for 24 hours with shaking and stirring using a microplate shaker (Nissen Rika Co., Ltd. N-704). After washing three times in the same manner as described above, ALP-labeled # 32-3 solution (100 μL) diluted to 0.2 ng / mL with a detection antibody dilution buffer was added, and the mixture was stirred at room temperature while stirring using a microplate shaker And incubated for 1 hour. After washing 4 times as above, chemiluminescent substrate (100 μL) was added and incubated at room temperature for 20 minutes. The amount of luminescence generated per second was measured with a microplate luminometer (SpectraMax L, Molecular Devices). Each sample was individually measured in two different wells, and a quantitative value was calculated from the average value.
（２）総ＡＮＰ測定
総ＡＮＰの測定は、市販のＣＬＥＩＡキット（ＭＩ０２ シオノギ ＡＮＰ、シオノギ製薬社）を用いて測定した。
(2) Total ANP measurement Total ANP measurement was performed using a commercially available CLEIA kit (MI02 Shionogi ANP, Shionogi Pharmaceutical Co., Ltd.).
（３）β−ＡＮＰ／総ＡＮＰの比の算出
健常者１０例、急性心不全患者６０例のβ−ＡＮＰと総ＡＮＰを測定した。その測定結果からβ−ＡＮＰ／総ＡＮＰの比を算出した。
(3) Calculation of ratio of β-ANP / total ANP β-ANP and total ANP of 10 healthy subjects and 60 acute heart failure patients were measured. The ratio of β-ANP / total ANP was calculated from the measurement results.
（４）考察
健常者、急性心不全患者におけるβ−ＡＮＰ／総ＡＮＰを表１（６０例；入院時と退院時の比較）と図４（４７例；１回目〜４回目の経時的変動）に示す。
(4) Discussion β-ANP / total ANP in healthy subjects and patients with acute heart failure are shown in Table 1 (60 cases; comparison between hospitalization and discharge) and FIG. 4 (47 cases; first to fourth changes over time). Show.
急性心不全患者の総ＡＮＰとβ−ＡＮＰは治療経過とともに低下していたが、退院時（４回目）ではどちらの値も健常者よりも高い値を示していた。一方、β−ＡＮＰ／総ＡＮＰは健常者と同等の値を示していた。退院時には、心臓超音波検査における左室内径短縮率の上昇、ＮＹＨＡ分類の低下、体重の低下、平均血圧の低下で示されるように、心不全重症度が軽減したと判断されたことから、本方法で測定したβ−ＡＮＰ／総ＡＮＰが心不全の改善に相関していることが示された。また、β−ＡＮＰおよびβ−ＡＮＰ／総ＡＮＰは、既存の心不全マーカーであるＢＮＰやＮＴ−ｐｒｏＢＮＰとは異なる経時的変動を示した。腎機能の指標（血清尿素窒素、血清クレアチニン、推算糸球体濾過量）が入院時と退院時では同等であったことから（表１）、上記のＡＮＰ分子型の血漿濃度の変動は、腎機能の変化によるものではないと示された。本実施例の結果と文献等の研究に基づくと、総ＡＮＰは心不全の増悪に伴って増加してくるが、β−ＡＮＰの挙動が他のＡＮＰやＢＮＰ、ＮＴ−ｐｒｏＢＮＰとは異なる挙動を示すことから、β−ＡＮＰ／総ＡＮＰを観察することにより、心不全の進行または心不全治療の効果を判定することができる。また、ＢＮＰやＮＴ−ｐｒｏＢＮＰと併用することにより、心不全の進行の評価精度、心不全治療の効果判定の精度を上げることができると考えられる。 The total ANP and β-ANP of patients with acute heart failure decreased with the course of treatment, but both values were higher than those of healthy subjects at the time of discharge (fourth time). On the other hand, β-ANP / total ANP showed a value equivalent to that of healthy subjects. At the time of discharge, it was determined that the severity of heart failure was reduced as indicated by increased left ventricular diameter shortening rate, decreased NYHA classification, decreased body weight, and decreased mean blood pressure in cardiac ultrasonography. It was shown that β-ANP / total ANP measured in (1) correlated with improvement of heart failure. In addition, β-ANP and β-ANP / total ANP showed temporal changes different from those of existing heart failure markers BNP and NT-proBNP. Since the indicators of renal function (serum urea nitrogen, serum creatinine, estimated glomerular filtration rate) were the same at the time of hospitalization and at the time of discharge (Table 1), fluctuations in the plasma concentration of the above ANP molecular type It was shown that it was not due to changes in Based on the results of this example and studies in the literature, the total ANP increases with worsening heart failure, but the behavior of β-ANP is different from that of other ANP, BNP, and NT-proBNP. Therefore, by observing β-ANP / total ANP, the progression of heart failure or the effect of heart failure treatment can be determined. Moreover, it is thought that the combined use with BNP or NT-proBNP can improve the evaluation accuracy of the progression of heart failure and the accuracy of the effect determination of the heart failure treatment.
（５）参考例：マウスモノクローナル抗体＃３２−３及びウサギポリクローナル抗体＃１３１−７の製法
（５−１）モノクローナル抗体産生ハイブリドーマの調製とモノクローナル抗体の産生
［抗原の調製］
固相法で合成、精製した還元型ヒトα−ＡＮＰ（シグマジェノシス社製、２．１ｍｇ）を、ジスルフィド結合を形成しない状態で、マレイミド活性化キーホールリンペットヘモシアニン（３ｍｇ、Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ社 ７７６０５）と結合した。これを透析し、生理的食塩水で１ｍｇ／ｍＬの溶液とした。
(5) Reference Example: Production Method of Mouse Monoclonal Antibody # 32-3 and Rabbit Polyclonal Antibody # 131-7 (5-1) Preparation of Monoclonal Antibody-Producing Hybridoma and Production of Monoclonal Antibody [Preparation of Antigen]
Reduced human α-ANP (manufactured by Sigma Genosys, 2.1 mg) synthesized and purified by a solid-phase method is maleimide-activated keyhole limpet hemocyanin (3 mg, Thermo Scientific 77605) without forming a disulfide bond. ). This was dialyzed and made into a 1 mg / mL solution with physiological saline.
［モノクローナル抗体産生ハイブリドーマの調製］
抗原溶液１００μＬとアジュバント１００μＬ（Ｆｒｅｕｎｄ ｃｏｍｐｌｅｔｅ ａｄｊｕｖａｎｔ，三菱化学ヤトロン社 ＲＭ６０６−１）を十分に混合して安定したエマルジョンにして、４週齢のＣ３Ｈ系マウス（４匹）の足の裏に各５０μＬずつ免疫した。これを合計５回、３日おきに実施した。最終免疫の３日後にマウス両足からリンパ節を収集し、リンパ節内の細胞を回収した。リンパ節由来細胞と増殖させたミエローマ細胞（Ｐ３Ｕ１）を２：１〜１０：１の割合で混和し、遠心して回収後、５０％ポリエチレングリコールを加えて細胞を融合させた。
[Preparation of monoclonal antibody-producing hybridoma]
100 μL of antigen solution and 100 μL of adjuvant (Freund complete adjuvant, Mitsubishi Chemical Yatron RM606-1) were mixed thoroughly to form a stable emulsion, 50 μL each on the sole of 4 week old C3H mice (4 mice) I was immunized. This was performed a total of 5 times every 3 days. Three days after the final immunization, lymph nodes were collected from both mouse feet, and cells in the lymph nodes were collected. Lymph node-derived cells and proliferated myeloma cells (P3U1) were mixed at a ratio of 2: 1 to 10: 1, collected by centrifugation, and then 50% polyethylene glycol was added to fuse the cells.
無血清培地で洗浄後、レスキュー用ｓｕｐｐｌｅｍｅｎｔ含有の１５％ウシ胎児血清含有ＨＡＴ培地に懸濁し、９６穴プレート３枚に播種した。１〜２週間後に、ハイブリドーマのコロニー形成を確認し、各ウェルから培養上清を一部回収した。 After washing with serum-free medium, the suspension was suspended in 15% fetal bovine serum-containing HAT medium containing rescue supplements and seeded in three 96-well plates. After 1 to 2 weeks, formation of hybridoma colonies was confirmed, and a portion of the culture supernatant was collected from each well.
［ハイブリドーマの１次スクリーニング］
１次スクリーニングは標準的なＥＬＩＳＡ法（Ｍｅｔｈｏｄｓ ｉｎ Ｉｍｍｕｎｏｄｉａｇｎｏｓｉｓ ２ｎｄ Ｅｄｉｔｉｏｎ， Ｒｏｓｅ ａｎｄ Ｂｉｇａｚｚｉ， ｅｄｓ．， Ｊｏｈｎ Ｗｉｌｅｙ ａｎｄ Ｓｏｎｓ， １９８０； Ｃａｍｐｂｅｌｌ， ｅｔ ａｌ．， Ｍｅｔｈｏｄｓ ａｎｄ Ｉｍｍｕｎｏｌｏｇｙ， Ｗ． Ａ． Ｂｅｎｊａｍｉｎ， Ｉｎｃ．， １９６４； Ｏｅｌｌｅｒｉｃｈ， Ｍ．， Ｊ． Ｃｌｉｎ． Ｃｈｅｍ． Ｃｌｉｎ． Ｂｉｏｃｈｅｍ． ２２：８９５−９０４， １９８４； 羊土社 タンパク質実験ノート（下）岡田雅人編集 改訂第４版 ２０１１年１１月１日発行）で実施した。抗原を１μｇ／ｍＬに希釈後、９６穴プレート（ＮＵＮＣ ４６８６６７）に分注し、４℃で一晩、静置した。抗原溶液を除去後、Ｂｌｏｃｋｉｎｇ Ｂｕｆｆｅｒを１００μＬ／ｗｅｌｌで分注し、ブロッキングを行った。培養上清５０μＬを各ウェルに添加して反応させた。洗浄後、西洋わさびペルオキシダーゼで標識した抗マウスＩｇＧヤギ抗体と反応後、発色剤を添加し、４５０ｎｍの吸光度を測定した。ヘモシアニンに対する吸光度が０．２以下で、抗原に対する特異的な吸光度を０．２以上有するクローンを陽性として選択した。
[Primary screening of hybridoma]
Primary screening was performed using standard ELISA methods (Methods in Immunodiagnosis 2nd Edition, Rose and Bigazzi, eds., John Wiley and Sons, 1980; Campbell, et al., Et al., Meth. Oellerich, M., J. Clin. Chem. Clin. Biochem.22: 895-904, 1984; Yochisha Protein Experiment Notes (below) edited by Masato Okada, revised 4th edition, issued on November 1, 2011) did. The antigen was diluted to 1 μg / mL, dispensed into a 96-well plate (NUNC 466667), and allowed to stand at 4 ° C. overnight. After removing the antigen solution, Blocking Buffer was dispensed at 100 μL / well for blocking. 50 μL of the culture supernatant was added to each well for reaction. After washing, after reacting with anti-mouse IgG goat antibody labeled with horseradish peroxidase, a color former was added and the absorbance at 450 nm was measured. A clone having an absorbance for hemocyanin of 0.2 or less and a specific absorbance for antigen of 0.2 or more was selected as positive.
［ハイブリドーマの２次スクリーニング］
ヒトα−ＡＮＰ（ペプチド研究所製）を還元後、カルボキシアミドメチル（ＣＡＭ）化し、ＲＰ−ＨＰＬＣで精製した。精製したＣＡＭ−α−ＡＮＰをラクトペルオキシダーゼ法によりヨード１２５（１２５Ｉ）で標識し、ＲＰ−ＨＰＬＣで精製することにより、１分子の１２５Ｉで標識されたペプチドを調製した。以下の反応には、ＲＩＡ用標準バッファー（ＲＩＡバッファー、５０ ｍＭリン酸緩衝液、８０ｍＭ ＮａＣｌ、２５ｍＭ ＥＤＴＡ、０．０５％ ＮａＮ３、０．５％ Ｎ−エチルマレイミド処理済ＢＳＡ（ＳＩＧＭＡ−Ａｌｄｒｉｃｈ社 Ａ７８８８）、０．５％ Ｔｒｉｔｏｎ Ｘ−１００、ｐＨ７．４）（Ｋａｔａｆｕｃｈｉ Ｔ， ｅｔ ａｌ．， Ｊ． Ｂｉｏｌ． Ｃｈｅｍ．， ２７８：１２０４６−１２０５４， ２００３）を使用した。
[Secondary screening of hybridomas]
Human α-ANP (manufactured by Peptide Institute) was reduced, converted to carboxyamidomethyl (CAM), and purified by RP-HPLC. The purified CAM-α-ANP was labeled with iodo125 ( 125 I) by the lactoperoxidase method and purified by RP-HPLC to prepare one molecule of 125 I-labeled peptide. For the following reaction, RIA standard buffer (RIA buffer, 50 mM phosphate buffer, 80 mM NaCl, 25 mM EDTA, 0.05% NaN 3 , 0.5% N-ethylmaleimide-treated BSA (SIGMA-Aldrich) A7888), 0.5% Triton X-100, pH 7.4) (Katafuchi T, et al., J. Biol. Chem., 278: 12046-12504, 2003).
１次スクリーニング陽性クローンの培養上清を，１／１０希釈より３倍希釈系列液を各１００μＬ作製し、これに約２０，０００ｃｐｍの１２５Ｉ標識ＣＡＭ−α−ＡＮＰ（１２５Ｉ−ＣＡＭ−α−ＡＮＰ）を含む溶液１００μＬ、ＲＩＡバッファー１００μＬを添加、撹拌し、４℃で４０時間静置した。抗体に結合した１２５Ｉ−ＣＡＭ−α−ＡＮＰの放射活性量をポリエチレングリコール分離法で分離し測定した（Ｋａｔａｆｕｃｈｉ Ｔ， ｅｔ ａｌ． 同上文献）。具体的には、リン酸緩衝生理食塩水（ＰＢＳ、５０ｍＭリン酸緩衝液、８０ｍＭ ＮａＣｌ、０．０５％ ＮａＮ３、ｐＨ７．４）を用いて作製した１００μＬの１％ ウシγ−グロブリン（Ｓｉｇｍａ−Ａｌｄｒｉｃｈ社 Ｇ５００９）溶液および５００μの２３％ポリエチレングリコール（＃６０００、ナカライテスク社 ２８２５４−８５）溶液を加え、混合し、氷上で１０分間静置した後、遠心分離（４℃、３０００ｒｐｍ、１５分間）し、上清を除去した。得られた沈殿の１分間の放射活性をγカウンター（ＡＲＣ−１０００Ｍ、Ａｌｏｋａ社）にて測定した。希釈された培養上清でも１２５Ｉ−ＣＡＭ−α−ＡＮＰに対する結合能力のあるクローン５種選択した。 The culture supernatant of the primary screening positive clone was prepared by 100 μL each of a 3-fold diluted serial solution from 1/10 dilution, and about 20,000 cpm of 125 I-labeled CAM-α-ANP ( 125 I-CAM-α- 100 μL of a solution containing ANP) and 100 μL of RIA buffer were added, stirred, and left at 4 ° C. for 40 hours. The amount of radioactivity of 125 I-CAM-α-ANP bound to the antibody was measured by separation using a polyethylene glycol separation method (Katafuchi T, et al., Ibid.). Specifically, 100 μL of 1% bovine γ-globulin (Sigma-Aldrich) prepared using phosphate buffered saline (PBS, 50 mM phosphate buffer, 80 mM NaCl, 0.05% NaN3, pH 7.4). G5009) solution and 500μ 23% polyethylene glycol (# 6000, Nacalai Tesque 28254-85) solution were added, mixed, allowed to stand on ice for 10 minutes, and then centrifuged (4 ° C., 3000 rpm, 15 minutes). The supernatant was removed. The radioactivity of the resulting precipitate for 1 minute was measured with a γ counter (ARC-1000M, Aloka). Five clones capable of binding to 125 I-CAM-α-ANP were selected from the diluted culture supernatant.
［限界希釈法によるモノクローン化］
上記５種のクローンを増殖し、対数増殖期の状態でハイブリドーマを分散させ、培地で希釈後、９６穴プレートに播種した。１〜２週間後にハイブリドーマのシングルコロニーの形成が確認された段階で、各ウェルから培養上清をサンプリングし、上述の「ハイブリドーマの１次スクリーニング」に記載した方法に従い、活性を評価した。抗原に対する特異的な吸光度の強いクローンを各３種、合計１５種を選択した。
[Monocloning by limiting dilution method]
The above five clones were grown, hybridomas were dispersed in a logarithmic growth phase, diluted with a medium, and then seeded in a 96-well plate. After the formation of a single colony of hybridoma was confirmed after 1 to 2 weeks, the culture supernatant was sampled from each well, and the activity was evaluated according to the method described in the above-mentioned “primary screening of hybridoma”. Three clones each having a strong specific absorbance against the antigen were selected, for a total of 15 clones.
［ハイブリドーマの３次スクリーニング］
ＣＡＭ−α−ＡＮＰに加えて、α−ＡＮＰ、β−ＡＮＰ（ペプチド研究所製）をそれぞれラクトペルオキシダーゼ法により１２５Ｉで標識後、ＲＰ−ＨＰＬＣで精製し、１分子の１２５Ｉで標識されたペプチドを調製した（１２５Ｉ−α−ＡＮＰ、１２５Ｉ−β−ＡＮＰ）。
[Third screening of hybridomas]
In addition to CAM-α-ANP, α-ANP and β-ANP (manufactured by Peptide Institute) were each labeled with 125 I by the lactoperoxidase method, then purified by RP-HPLC and labeled with one molecule of 125 I. Peptides were prepared ( 125 I-α-ANP, 125 I-β-ANP).
上記５種のクローンより調製した各３種のクローンについて、連続した希釈液を作製し、１２５Ｉ−ＣＡＭ−α−ＡＮＰに対する結合能力を評価し、２次スクリーニングより得られた５種のクローンより得られた各３種のクローンの中で、最も結合能力の高いクローンを選択した。 For each of the three clones prepared from the above five clones, serial dilutions were prepared, the binding ability to 125 I-CAM-α-ANP was evaluated, and the five clones obtained from the secondary screening were used. Among the three clones obtained, the clone with the highest binding ability was selected.
これら５種について、連続した希釈液を作製し、１２５Ｉ−α−ＡＮＰ、１２５Ｉ−β−ＡＮＰを用いて２次スクリーニングに記載した方法に従い、各ペプチドに対する結合能力を評価した。 About these 5 types, the serial dilution liquid was produced and the binding ability with respect to each peptide was evaluated according to the method described in the secondary screening using 125 I- (alpha) -ANP and 125 I- (beta) -ANP.
次に、通常のＲＩＡ法に従い、１２５Ｉ−β−ＡＮＰ、１２５Ｉ−ＣＡＭ−α−ＡＮＰ、１２５Ｉ−α−ＡＮＰをトレーサーとして、それぞれについてβ−ＡＮＰ、α−ＡＮＰ、ＣＡＭ−α−ＡＮＰの標準曲線あるいは交差活性曲線を作成し、各クローンの特異性、感度を評価した。その結果、β−ＡＮＰに対して強い結合活性を有し、α−ＡＮＰ、ＣＡＭ−α−ＡＮＰとの交差性が少なく、かつＲＩＡ法において高感度にβ−ＡＮＰを測定できるクローン（＃３２−３）を選択した。 Next, according to a normal RIA method, 125 I-β-ANP, 125 I-CAM-α-ANP, 125 I-α-ANP are used as tracers, and β-ANP, α-ANP, and CAM-α-ANP are respectively used as tracers. A standard curve or cross-activity curve was prepared, and the specificity and sensitivity of each clone were evaluated. As a result, a clone having a strong binding activity to β-ANP, a low cross-reactivity with α-ANP and CAM-α-ANP, and capable of measuring β-ANP with high sensitivity by the RIA method (# 32- 3) was selected.
［モノクローナル抗体＃３２−３の産生］
腹水採取用にはヌードマウスを使用し、アジュバントとしてプリスタンを腹腔に注射して１週間後のマウスを用いた。選定したクローン（＃３２−３）を増殖、培養し、ＰＢＳにて懸濁し、上記のマウスに１匹当たり約１×１０６細胞を腹腔に注射した。２週間後ごろになると腹水がたまるので、経過をよく観察して腹水を複数回にわたり回収した。回収する容器には、予め保存用抗凝固剤（ＡＣＤ液）を添加して凝固を抑制した。遠心により血球成分や不要物を除去し、上清を凍結保存した。
[Production of monoclonal antibody # 32-3]
Nude mice were used for collecting ascites, and mice one week after injection of pristane as an adjuvant were used. The selected clone (# 32-3) was grown, cultured, suspended in PBS, and about 1 × 10 6 cells per mouse were injected into the peritoneal cavity. Ascites accumulated around 2 weeks, and the ascites was collected several times with careful observation. A preservative anticoagulant (ACD solution) was added to the container to be collected in advance to suppress coagulation. Blood cell components and unnecessary substances were removed by centrifugation, and the supernatant was stored frozen.
（５−２）抗体の精製
［精製モノクローナル抗体＃３２−３の調製］
腹水の上清画分からのモノクローナル抗体の精製にはＡｆｆｉ Ｇｅｌ Ｐｒｏｔｅｉｎ Ａ（ＢｉｏＲａｄ社 １５３−６１５３）を用いたプロテインＡ結合アフィニティーカラムを使用した。アフィニティーカラムへのサンプルの結合、洗浄、溶出は製造業者のマニュアルに従った。溶出液を５００μＬごとに、予め１Ｍトリス塩酸緩衝液（ｐＨ９．０）１６０μＬを加えた１．５ｍＬ容チューブに回収した。精製モノクローナル抗体を含む画分を、ＰＢＳ中で４℃、オーバーナイトにて透析した。精製した溶液中のタンパク質量をＢＣＡ Ｐｒｏｔｅｉｎ Ａｓｓａｙ Ｋｉｔ（Ｐｉｅｒｃｅ社 ２３２２７）を用いて定量した。
(5-2) Purification of antibody [Preparation of purified monoclonal antibody # 32-3]
A protein A-binding affinity column using Affi Gel Protein A (BioRad 153-1615) was used for purification of the monoclonal antibody from the ascites supernatant fraction. Binding of the sample to the affinity column, washing and elution were in accordance with the manufacturer's manual. The eluate was collected in a 1.5 mL tube to which 160 μL of 1M Tris-HCl buffer (pH 9.0) was added in advance every 500 μL. The fraction containing the purified monoclonal antibody was dialyzed overnight at 4 ° C. in PBS. The amount of protein in the purified solution was quantified using BCA Protein Assay Kit (Pierce 23227).
［精製ポリクローナル抗体＃１３１−７の調製］
ポリクローナル抗体＃１３１−７は、Ａ． Ｓａｓａｋｉ， ｅｔ ａｌ． Ｈｙｐｅｒｔｅｎｓｉｏｎ １０；３０８−３１２， １９８７に記載の方法で調製した。得られたウサギ抗血清にキャリアタンパク質として使用したサイログロブリン（Ｓｉｇｍａ−Ａｌｄｒｉｃｈ社 Ｔ１００１、１７ｍｇ／ｍＬ抗血清）およびアジュバント（Ｍ． Ｂｕｔｙｒｉｃｕｍ、ＤＩＦＣＯ社 ５２６−０２６５１、１０ｍｇ／ｍＬ抗血清）を加え、ローテーターにより撹拌しながら４℃、一晩インキュベートした後、遠心分離（４℃、１３，０００×ｇ、１５分間）し、上清を回収した。上清は直ちに上述のプロテインＡ結合アフィニティーカラムによる精製に供した。アフィニティーカラムへのサンプルの結合、洗浄、溶出は製造業者のマニュアルに従った。溶出液を５００μＬごとに、予め１Ｍトリス塩酸緩衝液（ｐＨ９．０）１６０μＬを加えた１．５ｍＬ容チューブに回収した。精製ポリクローナル抗体を含む画分を、ＰＢＳ中で４℃、一晩、透析した。精製した溶液中のタンパク質量をＢＣＡ Ｐｒｏｔｅｉｎ Ａｓｓａｙ Ｋｉｔを用いて定量した。このようにして精製ポリクローナル抗体＃１３１−７を得た。これはヒトα−ＡＮＰの１３〜１７残基目をエピトープとするウサギポリクロ―ナル抗体である（Ｎａｇａｉ Ｃ， Ｍｉｎａｍｉｎｏ Ｎ．， Ａｎａｌ． Ｂｉｏｃｈｅｍ．， ４６１：１０−１６， ２０１４）。
[Preparation of purified polyclonal antibody # 131-7]
Polyclonal antibody # 131-7 was prepared from A. Sasaki, et al.
（５−３）モノクローナル抗体の特性
ヒトα−ＡＮＰおよびβ−ＡＮＰに対する精製モノクローナル抗体＃３２−３の親和定数はＲＩＡ法を用いて算出した。具体的には、ＲＩＡバッファーを用いて精製モノクローナル抗体の５倍希釈系列液（２００μＬ）を作製し、試験管内にて１２５Ｉで標識したα−ＡＮＰおよびβ−ＡＮＰ（２０，０００ｃｐｍ、１００μＬ）と混合し、４℃で４０時間インキュベートした。抗体に結合した１２５Ｉ−α−ＡＮＰまたは１２５Ｉ−β−ＡＮＰの放射活性量を上述のポリエチレングリコール分離法で分離し、測定した。１２５Ｉ−α−ＡＮＰまたは１２５Ｉ−β−ＡＮＰの結合量が５０％となる抗体濃度をＫｄ値として算出したところ、モノクローナル抗体＃３２−３はヒトα−ＡＮＰに対して１．３４×１０−８Ｍ、ヒトβ−ＡＮＰに対して１．６９×１０−１１ＭのＫｄ値を示し、この抗体がβ−ＡＮＰを選択的に認識することが実証された。
(5-3) Characteristics of monoclonal antibody The affinity constant of purified monoclonal antibody # 32-3 for human α-ANP and β-ANP was calculated using the RIA method. Specifically, a 5-fold diluted series solution (200 μL) of a purified monoclonal antibody was prepared using an RIA buffer, and α-ANP and β-ANP (20,000 cpm, 100 μL) labeled with 125 I in a test tube were used. Mix and incubate at 4 ° C. for 40 hours. The amount of radioactivity of 125 I-α-ANP or 125 I-β-ANP bound to the antibody was separated by the above-mentioned polyethylene glycol separation method and measured. The antibody concentration at which the binding amount of 125 I-α-ANP or 125 I-β-ANP was 50% was calculated as the K d value. Monoclonal antibody # 32-3 was found to be 1.34 × against human α-ANP. A K d value of 1.69 × 10 −11 M was shown against 10 −8 M, human β-ANP, demonstrating that this antibody selectively recognizes β-ANP.
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2015060777A JP6508996B2 (en) | 2015-03-24 | 2015-03-24 | Method of detecting heart failure by β-ANP |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2015060777A JP6508996B2 (en) | 2015-03-24 | 2015-03-24 | Method of detecting heart failure by β-ANP |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2016180665A true JP2016180665A (en) | 2016-10-13 |
| JP6508996B2 JP6508996B2 (en) | 2019-05-08 |
Family
ID=57130971
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2015060777A Active JP6508996B2 (en) | 2015-03-24 | 2015-03-24 | Method of detecting heart failure by β-ANP |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6508996B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2020535448A (en) * | 2017-09-25 | 2020-12-03 | ユニバーシティー オブ フロリダ リサーチ ファンデーション， インク． | Immunity assay for detection of RAN protein |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6461500A (en) * | 1987-09-01 | 1989-03-08 | Shionogi & Co | Alpha-hanp recognizing monoclonal antibody and immunologically measuring reagent for alpha-hanp |
| JPH01221399A (en) * | 1988-02-29 | 1989-09-04 | Shionogi & Co Ltd | Monoclonal antibody capable of recognizing alpha-anp immunological measurement method of alpha-anp |
| JPH0242359A (en) * | 1988-06-30 | 1990-02-13 | Shionogi & Co Ltd | Immunoassay |
| JPH02276591A (en) * | 1989-04-19 | 1990-11-13 | Shionogi & Co Ltd | Monoclonal antibody recognizing c end side of anp |
| JPH034169A (en) * | 1989-05-31 | 1991-01-10 | Green Cross Corp:The | Mehtod for immunoassay of hanp antibody and hanp |
| US20120065075A1 (en) * | 2010-07-02 | 2012-03-15 | Prognomix Inc. | Method of determining a predisposition to atrial fibrillation (af) in a subject |
-
2015
- 2015-03-24 JP JP2015060777A patent/JP6508996B2/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6461500A (en) * | 1987-09-01 | 1989-03-08 | Shionogi & Co | Alpha-hanp recognizing monoclonal antibody and immunologically measuring reagent for alpha-hanp |
| JPH01221399A (en) * | 1988-02-29 | 1989-09-04 | Shionogi & Co Ltd | Monoclonal antibody capable of recognizing alpha-anp immunological measurement method of alpha-anp |
| JPH0242359A (en) * | 1988-06-30 | 1990-02-13 | Shionogi & Co Ltd | Immunoassay |
| JPH02276591A (en) * | 1989-04-19 | 1990-11-13 | Shionogi & Co Ltd | Monoclonal antibody recognizing c end side of anp |
| JPH034169A (en) * | 1989-05-31 | 1991-01-10 | Green Cross Corp:The | Mehtod for immunoassay of hanp antibody and hanp |
| US20120065075A1 (en) * | 2010-07-02 | 2012-03-15 | Prognomix Inc. | Method of determining a predisposition to atrial fibrillation (af) in a subject |
Non-Patent Citations (2)
| Title |
|---|
| NAGAI, C. ET AL.: "Direct chemiluminescent enzyme immunoassay for atrial natriuretic peptide in mammalian plasma using", ANALYTICAL BIOCHEMISTRY, vol. 461, JPN6018031681, 4 June 2014 (2014-06-04), pages 10 - 16, XP029012487, ISSN: 0004009323, DOI: 10.1016/j.ab.2014.05.022 * |
| 黒澤利郎ほか: "心疾患における心房性ナトリウム利尿ペプチド（ANP）濃度とMolecular formの検討", 北里医学, vol. 18, JPN6018037986, 1988, pages 503 - 513, ISSN: 0003888240 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2020535448A (en) * | 2017-09-25 | 2020-12-03 | ユニバーシティー オブ フロリダ リサーチ ファンデーション， インク． | Immunity assay for detection of RAN protein |
Also Published As
| Publication number | Publication date |
|---|---|
| JP6508996B2 (en) | 2019-05-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11340239B2 (en) | BNP (1-32) epitope and antibodies directed against said epitope | |
| RU2673455C2 (en) | Adrenomedullin to guide therapy of blood pressure decline | |
| CN104067130B (en) | Adrenomedullin assays and methods for determining mature adrenomedullin | |
| US20050244904A1 (en) | Diagnostics based on signal peptide detection | |
| JP2006519754A (en) | Identification of thyroid-stimulating hormone receptor autoantibodies using affinity purified antibodies | |
| ES2661516T3 (en) | Biomarkers | |
| AU2017294549C1 (en) | Adrenomedullin for assessing congestion in a subject with acute heart failure | |
| US9994631B2 (en) | Signal biomarkers | |
| WO2016140063A1 (en) | Method for measuring oxytocin with high sensitivity | |
| RU2607588C2 (en) | Method of producing agent, binding with pre-vasopressin or its fragments | |
| JP6508996B2 (en) | Method of detecting heart failure by β-ANP | |
| JP2016079146A (en) | SPECIFIC METHOD FOR MEASUREMENT OF β-ANP | |
| JPH01221399A (en) | Monoclonal antibody capable of recognizing alpha-anp immunological measurement method of alpha-anp | |
| JP4423426B2 (en) | Method of using increased concentration of adrenomedullin precursor C-terminal peptide as an indicator of cardiovascular disease or inflammatory disease | |
| US20090081714A1 (en) | Assays |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20180219 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20180912 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20181002 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20181128 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20190402 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20190402 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6508996 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |