CN113956355B - 抗人脑利钠肽(bnp)兔单克隆抗体及其应用 - Google Patents
抗人脑利钠肽(bnp)兔单克隆抗体及其应用 Download PDFInfo
- Publication number
- CN113956355B CN113956355B CN202110822760.0A CN202110822760A CN113956355B CN 113956355 B CN113956355 B CN 113956355B CN 202110822760 A CN202110822760 A CN 202110822760A CN 113956355 B CN113956355 B CN 113956355B
- Authority
- CN
- China
- Prior art keywords
- bnp
- antibody
- amino acid
- rabbit monoclonal
- seq
- 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.)
- Active
Links
- 241000283973 Oryctolagus cuniculus Species 0.000 title claims abstract description 101
- 101800000407 Brain natriuretic peptide 32 Proteins 0.000 title claims abstract description 85
- 101800002247 Brain natriuretic peptide 45 Proteins 0.000 title claims abstract description 85
- 102400000667 Brain natriuretic peptide 32 Human genes 0.000 title claims abstract description 84
- HPNRHPKXQZSDFX-OAQDCNSJSA-N nesiritide Chemical compound C([C@H]1C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)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(N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)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)CNC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CO)C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 HPNRHPKXQZSDFX-OAQDCNSJSA-N 0.000 title claims abstract description 81
- 150000001413 amino acids Chemical group 0.000 claims abstract description 24
- 238000003118 sandwich ELISA Methods 0.000 claims abstract description 11
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 101500026735 Homo sapiens Brain natriuretic peptide 32 Proteins 0.000 claims abstract description 8
- 238000003018 immunoassay Methods 0.000 claims description 6
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 10
- 229920001184 polypeptide Polymers 0.000 abstract description 8
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 8
- 210000003719 b-lymphocyte Anatomy 0.000 abstract description 5
- 238000003745 diagnosis Methods 0.000 abstract description 5
- 201000010099 disease Diseases 0.000 abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 5
- 238000012216 screening Methods 0.000 abstract description 4
- 238000005215 recombination Methods 0.000 abstract description 3
- 238000010369 molecular cloning Methods 0.000 abstract description 2
- 230000006798 recombination Effects 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 40
- 125000003275 alpha amino acid group Chemical group 0.000 description 18
- 238000000034 method Methods 0.000 description 17
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 description 15
- 235000001014 amino acid Nutrition 0.000 description 13
- 239000000427 antigen Substances 0.000 description 13
- 102000036639 antigens Human genes 0.000 description 13
- 108091007433 antigens Proteins 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- 238000002965 ELISA Methods 0.000 description 10
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 239000011248 coating agent Substances 0.000 description 8
- 238000000576 coating method Methods 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- 238000011161 development Methods 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 206010019280 Heart failures Diseases 0.000 description 6
- 230000003053 immunization Effects 0.000 description 6
- 238000002649 immunization Methods 0.000 description 6
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 6
- 239000012089 stop solution Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 241001529936 Murinae Species 0.000 description 5
- 108010089804 glycyl-threonine Proteins 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- JBGSZRYCXBPWGX-BQBZGAKWSA-N Ala-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CCCN=C(N)N JBGSZRYCXBPWGX-BQBZGAKWSA-N 0.000 description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 4
- QESXLSQLQHHTIX-RHYQMDGZSA-N Leu-Val-Thr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QESXLSQLQHHTIX-RHYQMDGZSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- OTQSTOXRUBVWAP-NRPADANISA-N Gln-Ser-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O OTQSTOXRUBVWAP-NRPADANISA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 102400001263 NT-proBNP Human genes 0.000 description 3
- 102100036836 Natriuretic peptides B Human genes 0.000 description 3
- 101710187802 Natriuretic peptides B Proteins 0.000 description 3
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 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 3
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 108010008064 pro-brain natriuretic peptide (1-76) Proteins 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000004393 prognosis Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- PRLPSDIHSRITSF-UNQGMJICSA-N Arg-Phe-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PRLPSDIHSRITSF-UNQGMJICSA-N 0.000 description 2
- KRXIWXCXOARFNT-ZLUOBGJFSA-N Asp-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O KRXIWXCXOARFNT-ZLUOBGJFSA-N 0.000 description 2
- XEDQMTWEYFBOIK-ACZMJKKPSA-N Asp-Ala-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O XEDQMTWEYFBOIK-ACZMJKKPSA-N 0.000 description 2
- -1 CTNT Proteins 0.000 description 2
- IZUNQDRIAOLWCN-YUMQZZPRSA-N Cys-Leu-Gly Chemical compound CC(C)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CS)N IZUNQDRIAOLWCN-YUMQZZPRSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- FJAYYNIXQNERSO-ACZMJKKPSA-N Gln-Cys-Asp Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)O)C(=O)O)N FJAYYNIXQNERSO-ACZMJKKPSA-N 0.000 description 2
- MADFVRSKEIEZHZ-DCAQKATOSA-N Gln-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)N)N MADFVRSKEIEZHZ-DCAQKATOSA-N 0.000 description 2
- JXFLPKSDLDEOQK-JHEQGTHGSA-N Gln-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCC(N)=O JXFLPKSDLDEOQK-JHEQGTHGSA-N 0.000 description 2
- IQACOVZVOMVILH-FXQIFTODSA-N Glu-Glu-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O IQACOVZVOMVILH-FXQIFTODSA-N 0.000 description 2
- JVZLZVJTIXVIHK-SXNHZJKMSA-N Glu-Trp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CCC(=O)O)N JVZLZVJTIXVIHK-SXNHZJKMSA-N 0.000 description 2
- HQRHFUYMGCHHJS-LURJTMIESA-N Gly-Gly-Arg Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N HQRHFUYMGCHHJS-LURJTMIESA-N 0.000 description 2
- GGAPHLIUUTVYMX-QWRGUYRKSA-N Gly-Phe-Ser Chemical compound OC[C@@H](C([O-])=O)NC(=O)[C@@H](NC(=O)C[NH3+])CC1=CC=CC=C1 GGAPHLIUUTVYMX-QWRGUYRKSA-N 0.000 description 2
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 2
- CUVBTVWFVIIDOC-YEPSODPASA-N Gly-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)CN CUVBTVWFVIIDOC-YEPSODPASA-N 0.000 description 2
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 2
- 108010065920 Insulin Lispro Proteins 0.000 description 2
- UGTHTQWIQKEDEH-BQBZGAKWSA-N L-alanyl-L-prolylglycine zwitterion Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UGTHTQWIQKEDEH-BQBZGAKWSA-N 0.000 description 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- VGPCJSXPPOQPBK-YUMQZZPRSA-N Leu-Gly-Ser Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O VGPCJSXPPOQPBK-YUMQZZPRSA-N 0.000 description 2
- FKQPWMZLIIATBA-AJNGGQMLSA-N Leu-Lys-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FKQPWMZLIIATBA-AJNGGQMLSA-N 0.000 description 2
- QWWPYKKLXWOITQ-VOAKCMCISA-N Leu-Thr-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(C)C QWWPYKKLXWOITQ-VOAKCMCISA-N 0.000 description 2
- UWKNTTJNVSYXPC-CIUDSAMLSA-N Lys-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN UWKNTTJNVSYXPC-CIUDSAMLSA-N 0.000 description 2
- GQFDWEDHOQRNLC-QWRGUYRKSA-N Lys-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN GQFDWEDHOQRNLC-QWRGUYRKSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- UNBFGVQVQGXXCK-KKUMJFAQSA-N Phe-Ser-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O UNBFGVQVQGXXCK-KKUMJFAQSA-N 0.000 description 2
- JMVQDLDPDBXAAX-YUMQZZPRSA-N Pro-Gly-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 JMVQDLDPDBXAAX-YUMQZZPRSA-N 0.000 description 2
- AFXCXDQNRXTSBD-FJXKBIBVSA-N Pro-Gly-Thr Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O AFXCXDQNRXTSBD-FJXKBIBVSA-N 0.000 description 2
- GOMUXSCOIWIJFP-GUBZILKMSA-N Pro-Ser-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GOMUXSCOIWIJFP-GUBZILKMSA-N 0.000 description 2
- 101100537532 Rattus norvegicus Tnni3 gene Proteins 0.000 description 2
- FMDHKPRACUXATF-ACZMJKKPSA-N Ser-Gln-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O FMDHKPRACUXATF-ACZMJKKPSA-N 0.000 description 2
- XXXAXOWMBOKTRN-XPUUQOCRSA-N Ser-Gly-Val Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O XXXAXOWMBOKTRN-XPUUQOCRSA-N 0.000 description 2
- FLONGDPORFIVQW-XGEHTFHBSA-N Ser-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO FLONGDPORFIVQW-XGEHTFHBSA-N 0.000 description 2
- HHJFMHQYEAAOBM-ZLUOBGJFSA-N Ser-Ser-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O HHJFMHQYEAAOBM-ZLUOBGJFSA-N 0.000 description 2
- CAJFZCICSVBOJK-SHGPDSBTSA-N Thr-Ala-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAJFZCICSVBOJK-SHGPDSBTSA-N 0.000 description 2
- UZJDBCHMIQXLOQ-HEIBUPTGSA-N Thr-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N)O UZJDBCHMIQXLOQ-HEIBUPTGSA-N 0.000 description 2
- LGNBRHZANHMZHK-NUMRIWBASA-N Thr-Glu-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O LGNBRHZANHMZHK-NUMRIWBASA-N 0.000 description 2
- RRRRCRYTLZVCEN-HJGDQZAQSA-N Thr-Leu-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O RRRRCRYTLZVCEN-HJGDQZAQSA-N 0.000 description 2
- VRUFCJZQDACGLH-UVOCVTCTSA-N Thr-Leu-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VRUFCJZQDACGLH-UVOCVTCTSA-N 0.000 description 2
- RVMNUBQWPVOUKH-HEIBUPTGSA-N Thr-Ser-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O RVMNUBQWPVOUKH-HEIBUPTGSA-N 0.000 description 2
- YOPQYBJJNSIQGZ-JNPHEJMOSA-N Thr-Tyr-Tyr Chemical compound C([C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 YOPQYBJJNSIQGZ-JNPHEJMOSA-N 0.000 description 2
- FYBFTPLPAXZBOY-KKHAAJSZSA-N Thr-Val-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O FYBFTPLPAXZBOY-KKHAAJSZSA-N 0.000 description 2
- HYVLNORXQGKONN-NUTKFTJISA-N Trp-Ala-Lys Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O)=CNC2=C1 HYVLNORXQGKONN-NUTKFTJISA-N 0.000 description 2
- LGEYOIQBBIPHQN-UWJYBYFXSA-N Tyr-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 LGEYOIQBBIPHQN-UWJYBYFXSA-N 0.000 description 2
- UPODKYBYUBTWSV-BZSNNMDCSA-N Tyr-Phe-Cys Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CS)C(O)=O)C1=CC=C(O)C=C1 UPODKYBYUBTWSV-BZSNNMDCSA-N 0.000 description 2
- JIODCDXKCJRMEH-NHCYSSNCSA-N Val-Arg-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N JIODCDXKCJRMEH-NHCYSSNCSA-N 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 108010062266 glycyl-glycyl-argininal Proteins 0.000 description 2
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 108010064235 lysylglycine Proteins 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000011587 new zealand white rabbit Methods 0.000 description 2
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 239000012898 sample dilution Substances 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- 108010041801 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase Proteins 0.000 description 1
- VOZKAJLKRJDJLL-UHFFFAOYSA-N 2,4-diaminotoluene Chemical compound CC1=CC=C(N)C=C1N VOZKAJLKRJDJLL-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 101800003158 5 kDa peptide Proteins 0.000 description 1
- YIGLXQRFQVWFEY-NRPADANISA-N Ala-Gln-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O YIGLXQRFQVWFEY-NRPADANISA-N 0.000 description 1
- RTZCUEHYUQZIDE-WHFBIAKZSA-N Ala-Ser-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RTZCUEHYUQZIDE-WHFBIAKZSA-N 0.000 description 1
- QDGMZAOSMNGBLP-MRFFXTKBSA-N Ala-Trp-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)N QDGMZAOSMNGBLP-MRFFXTKBSA-N 0.000 description 1
- VHAQSYHSDKERBS-XPUUQOCRSA-N Ala-Val-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O VHAQSYHSDKERBS-XPUUQOCRSA-N 0.000 description 1
- 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 1
- 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 1
- GIVATXIGCXFQQA-FXQIFTODSA-N Arg-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N GIVATXIGCXFQQA-FXQIFTODSA-N 0.000 description 1
- XYOVHPDDWCEUDY-CIUDSAMLSA-N Asn-Ala-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O XYOVHPDDWCEUDY-CIUDSAMLSA-N 0.000 description 1
- DAYDURRBMDCCFL-AAEUAGOBSA-N Asn-Trp-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC(=O)N)N DAYDURRBMDCCFL-AAEUAGOBSA-N 0.000 description 1
- RDLYUKRPEJERMM-XIRDDKMYSA-N Asn-Trp-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(C)C)C(O)=O RDLYUKRPEJERMM-XIRDDKMYSA-N 0.000 description 1
- QJHOOKBAHRJPPX-QWRGUYRKSA-N Asp-Phe-Gly Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=CC=C1 QJHOOKBAHRJPPX-QWRGUYRKSA-N 0.000 description 1
- 101800001288 Atrial natriuretic factor Proteins 0.000 description 1
- 101800001890 Atrial natriuretic peptide Proteins 0.000 description 1
- BVFQOPGFOQVZTE-ACZMJKKPSA-N Cys-Gln-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O BVFQOPGFOQVZTE-ACZMJKKPSA-N 0.000 description 1
- HHABWQIFXZPZCK-ACZMJKKPSA-N Cys-Gln-Ser Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CS)N HHABWQIFXZPZCK-ACZMJKKPSA-N 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- LPIKVBWNNVFHCQ-GUBZILKMSA-N Gln-Ser-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O LPIKVBWNNVFHCQ-GUBZILKMSA-N 0.000 description 1
- GMAGZGCAYLQBKF-NHCYSSNCSA-N Glu-Met-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(O)=O GMAGZGCAYLQBKF-NHCYSSNCSA-N 0.000 description 1
- LSYFGBRDBIQYAQ-FHWLQOOXSA-N Glu-Tyr-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LSYFGBRDBIQYAQ-FHWLQOOXSA-N 0.000 description 1
- UQJNXZSSGQIPIQ-FBCQKBJTSA-N Gly-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)CN UQJNXZSSGQIPIQ-FBCQKBJTSA-N 0.000 description 1
- WCORRBXVISTKQL-WHFBIAKZSA-N Gly-Ser-Ser Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WCORRBXVISTKQL-WHFBIAKZSA-N 0.000 description 1
- DBUNZBWUWCIELX-JHEQGTHGSA-N Gly-Thr-Glu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O DBUNZBWUWCIELX-JHEQGTHGSA-N 0.000 description 1
- PYFIQROSWQERAS-LBPRGKRZSA-N Gly-Trp-Gly Chemical compound C1=CC=C2C(C[C@H](NC(=O)CN)C(=O)NCC(O)=O)=CNC2=C1 PYFIQROSWQERAS-LBPRGKRZSA-N 0.000 description 1
- DCQMJRSOGCYKTR-GHCJXIJMSA-N Ile-Asp-Ser Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O DCQMJRSOGCYKTR-GHCJXIJMSA-N 0.000 description 1
- JHNJNTMTZHEDLJ-NAKRPEOUSA-N Ile-Ser-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O JHNJNTMTZHEDLJ-NAKRPEOUSA-N 0.000 description 1
- JZNVOBUNTWNZPW-GHCJXIJMSA-N Ile-Ser-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)O)C(=O)O)N JZNVOBUNTWNZPW-GHCJXIJMSA-N 0.000 description 1
- ZLFNNVATRMCAKN-ZKWXMUAHSA-N Ile-Ser-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)NCC(=O)O)N ZLFNNVATRMCAKN-ZKWXMUAHSA-N 0.000 description 1
- VGSPNSSCMOHRRR-BJDJZHNGSA-N Ile-Ser-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N VGSPNSSCMOHRRR-BJDJZHNGSA-N 0.000 description 1
- WLRJHVNFGAOYPS-HJPIBITLSA-N Ile-Ser-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N WLRJHVNFGAOYPS-HJPIBITLSA-N 0.000 description 1
- KGCLIYGPQXUNLO-IUCAKERBSA-N Leu-Gly-Glu Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O KGCLIYGPQXUNLO-IUCAKERBSA-N 0.000 description 1
- HRTRLSRYZZKPCO-BJDJZHNGSA-N Leu-Ile-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O HRTRLSRYZZKPCO-BJDJZHNGSA-N 0.000 description 1
- NRFGTHFONZYFNY-MGHWNKPDSA-N Leu-Ile-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NRFGTHFONZYFNY-MGHWNKPDSA-N 0.000 description 1
- SIGZKCWZEBFNAK-QAETUUGQSA-N Leu-Ser-Ser-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 SIGZKCWZEBFNAK-QAETUUGQSA-N 0.000 description 1
- LCNASHSOFMRYFO-WDCWCFNPSA-N Leu-Thr-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(N)=O LCNASHSOFMRYFO-WDCWCFNPSA-N 0.000 description 1
- HGLKOTPFWOMPOB-MEYUZBJRSA-N Leu-Thr-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HGLKOTPFWOMPOB-MEYUZBJRSA-N 0.000 description 1
- QWTGQXGNNMIUCW-BPUTZDHNSA-N Met-Asn-Trp Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O QWTGQXGNNMIUCW-BPUTZDHNSA-N 0.000 description 1
- MNGBICITWAPGAS-BPUTZDHNSA-N Met-Ser-Trp Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O MNGBICITWAPGAS-BPUTZDHNSA-N 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 102000015731 Peptide Hormones Human genes 0.000 description 1
- 108010038988 Peptide Hormones Proteins 0.000 description 1
- NAXPHWZXEXNDIW-JTQLQIEISA-N Phe-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 NAXPHWZXEXNDIW-JTQLQIEISA-N 0.000 description 1
- SMFGCTXUBWEPKM-KBPBESRZSA-N Phe-Leu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 SMFGCTXUBWEPKM-KBPBESRZSA-N 0.000 description 1
- MRYUJHGPZQNOAD-IHRRRGAJSA-N Pro-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@@H]1CCCN1 MRYUJHGPZQNOAD-IHRRRGAJSA-N 0.000 description 1
- PCWLNNZTBJTZRN-AVGNSLFASA-N Pro-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 PCWLNNZTBJTZRN-AVGNSLFASA-N 0.000 description 1
- PZZJMBYSYAKYPK-UWJYBYFXSA-N Ser-Ala-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O PZZJMBYSYAKYPK-UWJYBYFXSA-N 0.000 description 1
- SFTZWNJFZYOLBD-ZDLURKLDSA-N Ser-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO SFTZWNJFZYOLBD-ZDLURKLDSA-N 0.000 description 1
- SRSPTFBENMJHMR-WHFBIAKZSA-N Ser-Ser-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SRSPTFBENMJHMR-WHFBIAKZSA-N 0.000 description 1
- VAIWUNAAPZZGRI-IHPCNDPISA-N Ser-Trp-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)[C@H](CO)N VAIWUNAAPZZGRI-IHPCNDPISA-N 0.000 description 1
- PIQRHJQWEPWFJG-UWJYBYFXSA-N Ser-Tyr-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O PIQRHJQWEPWFJG-UWJYBYFXSA-N 0.000 description 1
- GSCVDSBEYVGMJQ-SRVKXCTJSA-N Ser-Tyr-Asp Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CO)N)O GSCVDSBEYVGMJQ-SRVKXCTJSA-N 0.000 description 1
- JGUWRQWULDWNCM-FXQIFTODSA-N Ser-Val-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O JGUWRQWULDWNCM-FXQIFTODSA-N 0.000 description 1
- XXNLGZRRSKPSGF-HTUGSXCWSA-N Thr-Gln-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N)O XXNLGZRRSKPSGF-HTUGSXCWSA-N 0.000 description 1
- OQCXTUQTKQFDCX-HTUGSXCWSA-N Thr-Glu-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N)O OQCXTUQTKQFDCX-HTUGSXCWSA-N 0.000 description 1
- PAXANSWUSVPFNK-IUKAMOBKSA-N Thr-Ile-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N PAXANSWUSVPFNK-IUKAMOBKSA-N 0.000 description 1
- FQPDRTDDEZXCEC-SVSWQMSJSA-N Thr-Ile-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O FQPDRTDDEZXCEC-SVSWQMSJSA-N 0.000 description 1
- IHAPJUHCZXBPHR-WZLNRYEVSA-N Thr-Ile-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N IHAPJUHCZXBPHR-WZLNRYEVSA-N 0.000 description 1
- KERCOYANYUPLHJ-XGEHTFHBSA-N Thr-Pro-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O KERCOYANYUPLHJ-XGEHTFHBSA-N 0.000 description 1
- DANHCMVVXDXOHN-SRVKXCTJSA-N Tyr-Asp-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 DANHCMVVXDXOHN-SRVKXCTJSA-N 0.000 description 1
- HGEHWFGAKHSIDY-SRVKXCTJSA-N Tyr-Asp-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N)O HGEHWFGAKHSIDY-SRVKXCTJSA-N 0.000 description 1
- KIJLSRYAUGGZIN-CFMVVWHZSA-N Tyr-Ile-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O KIJLSRYAUGGZIN-CFMVVWHZSA-N 0.000 description 1
- GITNQBVCEQBDQC-KKUMJFAQSA-N Tyr-Lys-Asn Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O GITNQBVCEQBDQC-KKUMJFAQSA-N 0.000 description 1
- OJCISMMNNUNNJA-BZSNNMDCSA-N Tyr-Tyr-Asp Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(O)=O)C(O)=O)C1=CC=C(O)C=C1 OJCISMMNNUNNJA-BZSNNMDCSA-N 0.000 description 1
- 108010001957 Ularitide Proteins 0.000 description 1
- 102400001279 Urodilatin Human genes 0.000 description 1
- UGFMVXRXULGLNO-XPUUQOCRSA-N Val-Ser-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O UGFMVXRXULGLNO-XPUUQOCRSA-N 0.000 description 1
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 description 1
- VTIAEOKFUJJBTC-YDHLFZDLSA-N Val-Tyr-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N VTIAEOKFUJJBTC-YDHLFZDLSA-N 0.000 description 1
- RLVTVHSDKHBFQP-ULQDDVLXSA-N Val-Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)CC1=CC=C(O)C=C1 RLVTVHSDKHBFQP-ULQDDVLXSA-N 0.000 description 1
- AOILQMZPNLUXCM-AVGNSLFASA-N Val-Val-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN AOILQMZPNLUXCM-AVGNSLFASA-N 0.000 description 1
- 108010081404 acein-2 Proteins 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010041407 alanylaspartic acid Proteins 0.000 description 1
- 229960002478 aldosterone Drugs 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 230000000916 dilatatory effect Effects 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000005002 finish coating Substances 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 108010010147 glycylglutamine Proteins 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 108010053037 kyotorphin Proteins 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000027939 micturition Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- LWGJTAZLEJHCPA-UHFFFAOYSA-N n-(2-chloroethyl)-n-nitrosomorpholine-4-carboxamide Chemical compound ClCCN(N=O)C(=O)N1CCOCC1 LWGJTAZLEJHCPA-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- IUCCYQIEZNQWRS-DWWHXVEHSA-N ularitide 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](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](C)NC(=O)[C@@H](N)[C@@H](C)O)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 IUCCYQIEZNQWRS-DWWHXVEHSA-N 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 108010009962 valyltyrosine Proteins 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- 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
- G01N2333/575—Hormones
- G01N2333/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Endocrinology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及生物技术领域，公开了兔抗人脑利钠肽(BNP)单克隆抗体OTIR5G8和OTIR5D11,所述的抗体是利用分选特异B细胞并培养筛选及分子克隆重组产生。所述兔单克隆抗体OTIR5G8和OTIR5D11的免疫原分别为人脑利钠肽(BNP)特定表位合成多肽，其OTIR5G8抗体轻链(VL)的氨基酸序列如SEQIDNO.1所示；OTIR5G8抗体的重链(VH)的氨基酸序列如SEQIDNo.2所示。其OTIR5D11轻链(VL)的氨基酸序列如SEQIDNO.11所示；OTIR5D11的重链(VH)的氨基酸序列如SEQIDNo.12所示。本发明的抗人脑利钠肽(BNP)兔单克隆抗体可应用于检测BNP的各种免疫检测试剂盒的制备，包括但不限于双抗体夹心ELISA或化学发光法试剂盒的制备，为与BNP相关的疾病诊断提供辅助手段，也为下一步工程抗体的制备提供基础。
Description
技术领域
本发明属于生物技术领域，尤其涉及抗人脑利钠肽(BNP)兔单克隆抗体及其在免疫检测方面应用。
背景技术
人BNP由proBNP裂解而来，proBNP裂解后C端为BNP，N端为NT-proBNP。BNP氨基酸序列即为proBNP的77-108氨基酸序列，氨基酸序列则通常记为1-32，是一种3.5KDa的肽段。BNP的理论等电点为10.95。
心力衰竭是一种复杂的临床症状群，是各种心脏病的严重阶段。发病率高，5年存活率与恶性肿瘤相仿。BNP和NT-proB-NP均是心衰诊断及预后的生物标志物。BNP具有利钠利尿，抗醛固酮作用，舒张血管，降低血压作用。BNP属于一类具有类似环状结构的多肽激素家族，该家族还包括ANP、CNP和尿扩张素。该家族的典型结构为一个由一对半胱氨酸之间二硫键所形成的17个氨基酸的环状结构。人BNP的二硫键位置为C10和C26。
BNP是检测心功能衰竭和冠脉病变的重要指标，对临床病情的评估具有重要价值。BNP诊断心力衰竭敏感而且特异，可作为呼吸困难鉴别诊断的一个指标。随着心衰程度的加重，BNP水平逐渐升高。BNP水平在评估心力衰竭的预后和危险分层中具有重要的标志物作用。BNP水平越高，其预后越差。在健康成年人体内，血浆BNP的参考上限为35pg/ml。
中国专利关于BNP兔单克隆抗体的条目基本没有，申请公布号CN 101878225 A涉及特异性性识别序列FGRKMDR的鼠单克隆抗体，由登录号CNCM I-3746杂交瘤产生，不是兔单克隆抗体，同国际专利申请号US20100209939A1。国际专利申请号US2006/0183154 A1(HUMAN RING SPECIFIC ANTIBODIES)公布了hBNP鼠单克隆抗体及多克隆抗体、嵌合抗体，识别BNP的环状结构，由杂交瘤3-631-436产生，未提到兔单克隆抗体及抗体CDR序列。国际专利申请号US 6,677,124B2(MONOCLONAL ANYIBODY RECOGNIZING C-TERMINUS HBNP)公布了C-terminus of hBNP鼠单克隆抗体，由杂交瘤BC203,FERM BP-3515产生，未提到兔单克隆抗体及抗体CDR序列。国际专利申请号US 9,255,930 B2(BNP-SP ANTIBODIES)公布了BNP-SP(17-26)peptide的鼠单克隆抗体及鼠重组抗体细胞系，未提到兔单克隆抗体及抗体CDR序列。
发明内容
本发明的目的是提供特异性好、亲和力高的抗BNP兔单克隆抗体OTIR5G8和OTIR5D11及其在双抗体夹心ELISA或化学发光法制备的免疫检测试剂盒中的应用，为与BNP相关的疾病提供辅助诊断，也为下一步工程抗体的制备提供基础。
所述兔单克隆抗体为兔单克隆抗体OTIR5G8或兔单克隆抗体OTIR5D11。
所述兔单克隆抗体OTIR5G8以26-32aa合成多肽为免疫原，兔单克隆抗体OTIR5D11以11-26aa合成多肽为免疫原。通过免疫注射新西兰大白兔，取免疫兔子外周血单核细胞(PMBCs)，分选特异性B细胞培养、筛选并利用分子克隆重组技术获得。
所述兔单克隆抗体OTIR5G8，其轻链可变区(VL)含110aa，其氨基酸序列如SEQ IDNO.1所示；所述抗体的重链(VH)含108aa，其氨基酸序列如SEQ ID No.2所示。
所述兔单克隆抗体OTIR5G8，其中VL区域包括3个抗原决定簇：CDR1、CDR2和CDR3，抗原决定簇的区域相应地分别为27aa-34aa，52aa-54aa和91aa-100aa。其氨基酸序列分别如SEQ ID No.3-5所示。
所述兔单克隆抗体OTIR5G8，其中VH区域包括3个抗原决定簇：CDR1、CDR2和CDR3，抗原决定簇的区域相应地分别为25aa-32aa，50aa-56aa和93aa-97aa。其氨基酸残基序列分别如SEQ ID No.6-8所示。
所述兔单克隆抗体OTIR5D11，其轻链可变区(VL)含109aa，其氨基酸序列如SEQ IDNO.11所示；所述抗体的重链(VH)含107aa，其氨基酸序列如SEQ ID No.12所示。
所述兔单克隆抗体OTIR5D11，其中VL区域包括3个抗原决定簇：CDR1、CDR2和CDR3，抗原决定簇的区域相应地分别为27aa-34aa，52aa-54aa和91aa-99aa。其氨基酸序列分别如SEQ ID No.13-15所示。
所述兔单克隆抗体OTIR5D11，其中VH区域包括3个抗原决定簇：CDR1、CDR2和CDR3，抗原决定簇的区域相应地分别为25aa-32aa，50aa-56aa和93aa-97aa。其氨基酸残基序列分别如SEQIDNo.16-18所示。
所述的兔单克隆抗体包括兔单克隆抗体OTIR5G8或兔单克隆抗体OTIR5D11可以与BNP高特异性结合，可通过本领域技术人员所公知的的方法，制备成检测BNP的各种免疫检测试剂盒。尤其是，应用于双抗体夹心ELISA或化学发光法制备的免疫检测试剂盒中。双抗体夹心ELISA检测BNP标准抗原，检测灵敏度能达到31.25pg/ml；板式化学发光法检测BNP标准抗原，检测灵敏度能达到10pg/ml。
附图说明
图1为兔单克隆抗体OTIR5G8和OTIR5D11重链和轻链全长扩增产物的电泳图，M为DNA分子量Marker。
图2为双抗体夹心ELISA法检测BNP血浆样本，横坐标为BNP血浆样本中BNP浓度，纵坐标为检测的OD值。
图3为双抗体夹心ELISA法检测BNP标准抗原，横坐标为BNP标准抗原浓度，纵坐标为检测的OD值。标准曲线的R2＝0.9962，线性检测范围0-250pg/mL，推出样品浓度计算公式：y＝0.0082x–0.0076。
图4为板式化学发光检测BNP标准抗原，横坐标为BNP标准抗原浓度，纵坐标为检测的化学发光读数。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件、实验室手册中所述的条件或按照制造厂商所建议的条件。
实施例1:抗BNP兔单克隆抗体的制备
一、BNP免疫原的制备
根据BNP1-32aa序列设计2个免疫原多肽，2个多肽序列分别为26-32aa：CKVLRRH及10-26aa：CFGRKMDRISSSSGLGC，多肽纯度在90％以上，达到了制备单抗的纯度要求。多肽由第三方中肽生化有限公司合成。
二、动物免疫
上述合成的BNP多肽以完全弗氏佐剂乳化，采用皮下注射方法免疫2kg左右的新西兰大白兔，免疫剂量为500μg/只，间隔两周后进行第二次免疫，以不完全弗氏佐剂乳化，免疫剂量为250μg/只。免疫两次后取尾血以ELISA法梯度稀释测定血清效价；依据ELISA效价128000时的OD450大于1.0为标准，根据结果判定是否收集PBMCs或是继续免疫，选取抗体效价最高的兔子进行PBMCs收集。
三、PBMCs分离、特异性B细胞分选、克隆重组将兔仰卧固定在手术台上，将心脏部位被毛减掉，用酒精消毒皮肤，选择心搏最明显处用50ml注射器穿刺，针头刺入心脏后即有血液涌入注射器，取得所需血量后迅速将针头拔出，将注射器中的全血转入无菌50ml管中，与等量的PBS混匀后逐滴缓慢的加入到淋巴细胞分离液上方，室温400×g离心30分钟，离心后，液面由上至下分为四层：黄色血浆层、白色薄膜层即单个核细胞层、分离液层及红细胞层，小心吸取单个核细胞层并用PBS洗涤去除血小板和淋巴细胞分离液后即可获得兔PBMCs。从兔PBMCs中继续分选抗原特异性的B细胞进行培养，培养后的B细胞上清用抗原包被的ELISA板筛选阳性克隆。阳性克隆的细胞收集裂解后提取RNA并反转录成cDNA,天然配对的兔单克隆抗体轻重链全长序列从对应阳性克隆的cDNA中被扩增出来，通过克隆重组方法构建兔单克隆抗体表达载体，并经测序确定序列。扩增的全长PCR产物结果如图1。
四、单克隆抗体的制备和纯化为了获得多株识别人BNP蛋白的兔单克隆抗体，本发明将兔单克隆抗体重链、轻链基因装载在表达载体上，将质粒转染KEK293细胞，转染120-144小时获得培养上清中含有重组的识别人BNP蛋白的兔单克隆抗体。收取细胞悬液，离心取上清，亲和层析法进行抗体纯化。以BCA法测定纯化后的单抗浓度，再分装、冻干。
实施例2:抗BNP兔单克隆抗体OTIR5G8或OTIR5D11的鉴定
一、兔单克隆抗体的特异性鉴定
采用间接ELSA法检测。以BNP、NT-proBNP、CTNI、CTNT、BSA等蛋白包被酶标板，浓度为2μg/ml，4℃过夜，以含1％BSA的PBST封闭酶标板，加104倍稀释的纯化抗体，37℃反应50min，PBST洗板3次，加HRP-羊抗兔Ig二抗，37℃反应50min，PBST洗板5次，加TMB显色10min，加终止液，酶标仪测A450。
结果显示，NT-proBNP、CTNI、CTNT、BSA与兔单克隆抗体OTIR5G8或OTIR5D11反应均为阴性，OD450均小于0.1；BNP与OTIR5G8或OTIR5D11抗体反应呈阳性，OD450均值2.85，说明本发明的抗BNP兔单克隆抗体OTIR5G8或OTIR5D11对BNP具有高度特异性。
二、兔单克隆抗体的效价
采用倍比稀释法稀释兔单克隆抗体OTIR5G8或OTIR5D11，用间接ELISA测抗体效价，结果显示本发明涉及的兔单克隆抗体OTIR5G8或OTIR5D11效价分别为6.6×106或1.3×107。
三、抗体配对
为了挑选包被抗体和检测抗体的最佳组合，采用棋盘组合，将26-32aa表位兔单克隆抗体与10-26aa表位兔单克隆抗体相互配对。基本步骤：多株单克隆抗体分别包被酶标板，4℃过夜。第二天取出酶标板，PBST洗涤一次，1％BSA溶液37℃封闭2小时，PBST洗涤3次；每孔加入BNP全长多肽100μl，浓度分别为5000、2500、1250、625、312.5、156.25、78.125和0pg/ml，37℃孵育1小时；孵育完成后取出酶标板，PBST洗涤3次，分别加入HRP标记的上述多株抗体作为检测抗体，37℃孵育1小时。PBST洗涤5次，加入TMB底物，37℃显色10min。取出后加终止液，在酶标仪上测定OD450读数。根据样品的OD值与阴性对照的背景值，选出最为理想的兔单克隆抗体对，配对筛选结果见表1。本发明涉及的抗体OTIR5G8作为包被抗体，抗体OTIR5D11作为检测抗体最佳。
表1抗体配对实验筛选结果
四、对BNP血浆样本的检测
7个BNP血浆样本，BNP浓度分别为39.7pg/ml、108pg/ml、287pg/ml、425.6pg/ml、1125.7pg/ml、2046.4pg/ml、3287.3pg/ml，以OTIR5G8抗体为包被抗体、OTIR5D11抗体为检测抗体，用上述检测方法检测7个BNP血浆样本，检测结果见表2。本发明所述的兔单克隆抗体可检测不同浓度的BNP血浆样本且检测结果与血浆BNP浓度呈正比。依据表2制作BNP血浆样本检测图，见图2。
表2.双抗体夹心ELISA法检测BNP血浆样本
| 包被抗体 | 5G8 |
| pg/ml | OD450nm |
| 39.7 | 0.273 |
| 108 | 0.951 |
| 287 | 1.366 |
| 425.6 | 1.74 |
| 1125.7 | 2.803 |
| 2046.3 | 3.481 |
| 3287.3 | 3.597 |
| 0 | 0.026 |
| 检测抗体 | 5D11 |
实施例3:兔单克隆抗体OTIR5G8或OTIR5D11可变区基因与氨基酸序列分析
分别以OTIR5G8或OTIR5D11抗体的重组质粒为DNA模板，根据模板上轻链及重链5'端载体序列设计轻链可变区及重链可变区测序引物，采用测序仪ABI3730进行测序。经测序获得兔单克隆抗体OTIR5G8和OTIR5D11轻链及重链可变区核苷酸序列。
利用互联网，在http://www.imgt.org上使用IMGT/V-QUEST分析软件，分别将轻链与重链的核苷酸序列进行测序结果数据分析，得到兔单克隆抗体OTIR5G8的轻链氨基酸序列如SEQIDNO.1所示，重链氨基酸序列如SEQIDNO.2所示。VL全长为110个氨基酸，其FR的4个结构域氨基酸数分别为26、17、36和10，CDR的3个结构域氨基酸数分别为8、3和10，CDR1、CDR2和CDR3的区域相应地分别为27aa-34aa，52aa-54aa和91aa-100aa，其氨基酸序列分别:QSVYKNNW、RAS、LGSYDCSSAD。
通过分析，所述兔单克隆抗体OTIR5G8VH全长为108个氨基酸，其FR的4个结构域氨基酸数分别为24、17、36和11，CDR的3个结构域氨基酸数分别为8、7和5，CDR1、CDR2和CDR3分别为25aa-32aa，50aa-56aa和93aa-97aa，其氨基酸序列分别为GFSLISYD、LGSGSSA、ARGTG。
通过分析，所述兔单克隆抗体OTIR5D11的轻链氨基酸序列如SEQIDNO.11所示，重链氨基酸序列如SEQIDNO.12所示。VL全长为109个氨基酸，其FR的4个结构域氨基酸数分别为26、17、36和10，CDR的3个结构域氨基酸数分别为8、3和9，CDR1、CDR2和CDR3的区域相应地分别为27aa-34aa，52aa-54aa和91aa-99aa，其氨基酸序列分别:QSVYDNNA、KAS、LGEYYDDAE。
通过分析，所述兔单克隆抗体OTIR5D11VH全长为107个氨基酸，其FR的4个结构域氨基酸数分别为24、17、36和10，CDR的3个结构域氨基酸数分别为8、7和5，CDR1、CDR2和CDR3分别为25aa-32aa，50aa-56aa和93aa-97aa，其氨基酸序列分别为GFSLSSYA、IDSSGTI、ARGMN。
实施例4:抗BNP兔单克隆抗体OTIR5G8或OTIR5D11用于制备双抗体夹心ELISA检测试剂盒
采用本领域技术人员所公知的基于ELISA双抗体夹心法原理的检测技术，制作BNP检测试剂盒，用于临床上与BNP相关疾病辅助诊断。
一、试剂盒组成
1.包被OTIR5G8抗体的板条：用PBS缓冲液稀释抗体至5μg/ml，包被到微孔板上，每孔100μl，4℃孵育过夜，以PBST洗涤3次，甩干；加含有1％BSA、5％蔗糖、0.05％Proclin300的PBS进行封闭，37℃反应2h，弃去孔内封闭液，甩干；将包被板置于37℃烘箱2h，即完成包被，以铝箔袋密封，存放于4℃保存备用。
2.试剂配制
1.酶结合物配制采用简易过碘酸钠法标记抗BNP兔单克隆抗体OTIR5D11，滴定工作浓度，配制成1:20的浓缩液，稀释液为含1％BSA、5％甘油、0.05％Proclin300的PBS，过滤除菌。
2.洗涤缓冲液为常规的pH7.4的PBST，含0.05％Proclin300，配制成20倍浓缩液。
3.酶的底物液(显色液)单组分TMB(从市场采购)。
4.样本稀释液含1％BSA、0.05％Proclin300的PBS，过滤除菌。
5.终止液用10mlHCl(36-38％)加入110ml蒸馏水中，混合过程需缓慢进行，配置成1N的HCl。
6.标准品合成全长BNP多肽，以含1％BSA、5％蔗糖、10％甘油、0.05％Proclin300的PBS为稀释液，将样品稀释为20μg/ml，过滤除菌，无菌分装。
二、检测操作要点
1、为保证检测结果的准确性，建议标准品及样本均设双孔测定。每次检测均需做标准曲线。
2、如标本中待测物质含量过高，请先用样本稀释液进行稀释，以使样本符合试剂盒的检测范围，最后计算时再乘以相应的稀释倍数。
3、加样：加样时，请使用一次性的洁净吸头，避免交叉污染。加样时应尽量轻缓，避免起泡，将样本加于酶标板孔底部，切勿沿孔壁加样。
4、温育：为防止样本蒸发或污染，温育过程中酶标板必须覆上板贴，实验过程中酶标板应避免处于干燥的状态。温育过程中应随时观察温箱温度是否恒定于37℃，及时调整。温育过程中，温箱不易开启太多次，以免影响温度平衡。
5、洗涤：洗涤过程非常重要，不充分的洗涤易造成假阳性。
6、显色：为保证实验结果的准确性，底物反应时间到后应尽快加入终止液。可在加入底物溶液后每隔一段时间观察一下显色情况以控制反应时间(比如每隔10分钟)。当肉眼可见标准品前3-4孔有明显梯度蓝色，后3-4孔显色不明显时，即可加入终止液终止反应，此时蓝色立刻变为黄色。终止液的加入顺序应尽量与底物溶液的加入顺序相同。
7、底物溶液应为浅蓝色或无色，如果颜色严重变深则必须弃用。底物溶液易受污染，请避光妥善保存。
三、标准BNP抗原不同稀释度标准曲线的确定
上述制备的检测BNP蛋白双抗体夹心ELISA检测试剂盒从4度冰箱取出，平衡到室温。将标准品用PBS从20μg/ml稀释到250pg/ml，制备成0、3.9、7.8、15.625、31.25、62.5、125、250pg/ml共8个系列浓度标准品，按照上述的检测方法在每孔中加入标准品100μl，进行温育，然后弃去液体，加入HRP标记的检测抗体工作液进行温育，弃去孔内液体，甩干后每孔加入底物溶液，避光显色后每孔加入终止溶液，在反应终止后5分钟内用酶标仪在450nm波长依序测量各孔的光密度OD值做标准曲线，结果见表3。依据表3制作BNP检测标准曲线，见附图3。标准曲线的R2＝0.9962，线性检测范围0-250pg/mL，推出样品浓度计算公式：y＝0.0082x–0.0076。检测灵敏度能达到31.25pg/ml。
表3.双抗体夹心ELISA法检测BNP标准品
| 包被抗体 | 5G8 |
| pg/ml | OD450nm |
| 250 | 2.004 |
| 125 | 1.112 |
| 62.5 | 0.529 |
| 31.25 | 0.224 |
| 15.625 | 0.086 |
| 7.8125 | 0.033 |
| 3.90625 | 0.024 |
| 0 | 0.010 |
| 检测抗体 | 5D11 |
实施例5：抗BNP兔单克隆抗体OTIR5G8或OTIR5D11用于板式化学发光免疫检测
采用本领域技术人员所公知的基于双抗体夹心法原理的板式化学发光免疫检测技术，制作BNP检测试剂盒，用于临床上与BNP相关疾病辅助诊断。
一、检测原理与方法
采用基于双抗体夹心法原理的板式化学发光免疫检测技术。在不透光的白色微孔板内包被抗BNP的兔单克隆抗体OTIR5G8，将封闭好的包被板放置到科斯迈全自动发化学发光仪上，设置仪器程序，将标准品或待测标本与辣根过氧化物酶(HRP)标记的兔单克隆抗体OTIR5D11同时进行温育，则三者形成抗体-抗原-抗体复合物。洗涤除去未结合的物质，加入北京科跃中楷化学发光底物，反应10min后科斯迈全自动发化学发光仪显示化学发光值。依据信噪比大于2.0判读结果为阳性。发光值的大小反映了结合的酶标抗体的量，它与标本中的BNP的浓度成正比。根据所测定的标准品的发光值绘制标准曲线,待测标本中的BNP浓度值即可从标准曲线上获得。
二、检测BNP的板式化学发光检测试剂盒组成
1.包被OTIR5G8抗体的板条：同实施例4。
2.试剂配制
1.酶结合物配制同实施例4。
2.洗涤缓冲液同实施例4。。
3.化学发光显色液A液和B液，购于北京科跃中楷生物技术有限公司。
4.样本稀释液含1％BSA、0.05％Proclin300的PBST，过滤除菌。
5.标准品合成全长BNP多肽，以含1％BSA、5％蔗糖、10％甘油、0.05％Proclin300的PBS为稀释液，将样品稀释为5μg/ml，过滤除菌，无菌分装。
三、对BNP标准抗原的检测
把BNP标准抗原进行梯度稀释，分别为0pg/ml、10pg/ml、30pg/ml、100pg/ml、300pg/ml、1000pg/ml，以OTIR5G8抗体为包被抗体、OTIR5D11抗体为检测抗体，用上述检测方法检测6个梯度稀释的抗原，检测结果见表4。根据信噪比大于2.0判定，本发明所述的兔单克隆抗体用于板式化学发光免疫检测试剂，最低灵敏度为10pg/ml，线性范围为10-1000pg/ml。依据表4制作BNP检测标准曲线，见图4
表4.板式化学发光法检测BNP标准抗原
序列表
<110> 无锡傲锐东源生物科技有限公司
<120>抗人脑利钠肽（BNP）兔单克隆抗体及其应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 110
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 1
Ala Gln Val Leu Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Lys Asn
20 25 30
Asn Trp Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Leu Lys Arg
35 40 45
Leu Thr Tyr Arg Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Val
65 70 75 80
Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Ser Tyr Asp Cys
85 90 95
Ser Ser Ala Asp Phe Gly Gly Gly Thr Glu Met Val Val Lys
100 105 110
<210> 2
<211> 108
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 2
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ile Ser Tyr Asp
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45
Phe Leu Gly Ser Gly Ser Ser Ala Tyr Tyr Ala Ser Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Arg Thr Ser Thr Thr Val Asp Leu Lys Ile Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Thr
85 90 95
Gly Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105
<210> 3
<211> 8
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 3
Gln Ser Val Tyr Lys Asn Asn Trp
1 5
<210> 4
<211> 3
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 4
Arg Ala Ser
1
<210> 5
<211> 10
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 5
Leu Gly Ser Tyr Asp Cys Ser Ser Ala Asp
1 5 10
<210> 6
<211> 8
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 6
Gly Phe Ser Leu Ile Ser Tyr Asp
1 5
<210> 7
<211> 7
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 7
Leu Gly Ser Gly Ser Ser Ala
1 5
<210> 8
<211> 5
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 8
Ala Arg Gly Thr Gly
1 5
<110> 无锡傲锐东源生物科技有限公司
<120>抗人脑利钠肽（BNP）兔单克隆抗体及其应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 11
<211> 109
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 11
Ala Ala Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Ser Cys Gln Ser Ser Gln Ser Val Tyr Asp Asn
20 25 30
Asn Ala Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg
35 40 45
Leu Ile Tyr Lys Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val
65 70 75 80
Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Glu Tyr Tyr Asp
85 90 95
Asp Ala Glu Phe Gly Gly Gly Thr Glu Val Val Val Lys
100 105
<210> 12
<211> 107
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 12
Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala
20 25 30
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45
Tyr Ile Asp Ser Ser Gly Thr Ile Tyr Tyr Ala Ser Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Met
85 90 95
Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105
<210> 13
<211> 8
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 13
Gln Ser Val Tyr Asp Asn Asn Ala
1 5
<210> 14
<211> 3
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 14
Lys Ala Ser
1
<210> 15
<211> 9
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 15
Leu Gly Glu Tyr Tyr Asp Asp Ala Glu
1 5
<210> 16
<211> 8
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 16
Gly Phe Ser Leu Ser Ser Tyr Ala
1 5
<210> 17
<211> 7
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 17
Ile Asp Ser Ser Gly Thr Ile
1 5
<210> 18
<211> 5
<212> PRT
<213> Oryctolaguscuniculus (rabbit)
<400> 18
Ala Arg Gly Met Asn
1 5
Claims (6)
1.抗人脑利钠肽（BNP）的兔单克隆抗体，其特征在于：其中轻链可变区包括3个抗原决定簇：CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID No.3-5所示；其中重链可变区包括3个抗原决定簇：CDR1、CDR2和CDR3，其氨基酸序列分别如 SEQ ID No.6-8所示。
2.根据权利要求1所述抗人脑利钠肽（BNP）兔单克隆抗体，其特征在于:其轻链可变区氨基酸序列如SEQ ID NO.1所示；其重链可变区氨基酸序列如SEQ IDNo.2所示。
3.抗人脑利钠肽（BNP）的兔单克隆抗体，其特征在于：其中轻链可变区包括3个抗原决定簇：CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID No.13-15所示；其中重链可变区包括3个抗原决定簇：CDR1、CDR2和CDR3，其氨基酸序列分别如SEQ ID No.16-18所示。
4.根据权利要求3所述抗人脑利钠肽（BNP）兔单克隆抗体，其特征在于:其轻链可变区氨基酸序列如SEQ ID NO.11所示；其重链可变区氨基酸序列如SEQ IDNo.12所示。
5.权利要求1-4任一项所述的抗人脑利钠肽（BNP）兔单克隆抗体的应用，其特征在于:用于人脑利钠肽（BNP）免疫检测试剂盒的制备。
6.根据权利要求5所述的抗人脑利钠肽（BNP）兔单克隆抗体的应用，其特征在于：权利要求5所述试剂盒为双抗体夹心ELISA试剂盒或化学发光免疫检测试剂盒。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110822760.0A CN113956355B (zh) | 2021-07-26 | 2021-07-26 | 抗人脑利钠肽(bnp)兔单克隆抗体及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110822760.0A CN113956355B (zh) | 2021-07-26 | 2021-07-26 | 抗人脑利钠肽(bnp)兔单克隆抗体及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113956355A CN113956355A (zh) | 2022-01-21 |
| CN113956355B true CN113956355B (zh) | 2023-06-23 |
Family
ID=79460425
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110822760.0A Active CN113956355B (zh) | 2021-07-26 | 2021-07-26 | 抗人脑利钠肽(bnp)兔单克隆抗体及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113956355B (zh) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117430702B (zh) * | 2022-07-21 | 2024-06-21 | 东莞市朋志生物科技有限公司 | 抗b型尿钠肽抗体或其功能性片段、检测b型尿钠肽的试剂和试剂盒 |
| CN118406146B (zh) * | 2022-07-22 | 2025-06-24 | 东莞市朋志生物科技有限公司 | 抗b型尿钠肽抗体或其功能性片段、检测b型尿钠肽的试剂和试剂盒 |
| CN118702815A (zh) * | 2023-10-31 | 2024-09-27 | 菲鹏生物股份有限公司 | 一种抗NT-proBNP的抗体及其应用 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101878225A (zh) * | 2007-08-03 | 2010-11-03 | 比奥-拉德巴斯德公司 | 新型bnp(1-32)表位以及针对所述表位的抗体 |
| CN110616192A (zh) * | 2019-08-09 | 2019-12-27 | 无锡傲锐东源生物科技有限公司 | 一种抗人神经丝轻链（nefl）的单克隆抗体及其应用 |
| CN110684740A (zh) * | 2019-08-09 | 2020-01-14 | 无锡傲锐东源生物科技有限公司 | 一种抗人泛素羧基末端水解酶-1(uch-l1)的单克隆抗体及其应用 |
| CN112608385A (zh) * | 2020-12-18 | 2021-04-06 | 杭州贤至生物科技有限公司 | 一种犬脑钠肽（bnp）单克隆抗体的制备 |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU1981597A (en) * | 1996-03-04 | 1997-09-22 | Scios Inc. | Assay and reagents for quantifying hbnp |
| US20050033031A1 (en) * | 2003-08-07 | 2005-02-10 | Couto Fernando Jose Rebelo Do | Methods for humanizing rabbit monoclonal antibodies |
| FI20075251A0 (fi) * | 2007-04-13 | 2007-04-13 | Hytest Oy | Immunomääritys epästabiilien antigeenien määrittämiseksi |
| WO2011051350A1 (en) * | 2009-10-27 | 2011-05-05 | Ucb Pharma S.A. | Function modifying nav 1.7 antibodies |
| EP3265113A4 (en) * | 2015-03-06 | 2019-02-20 | The Board of Regents of The University of Texas System | ANTI-LRB ANTIBODIES AND THEIR USE FOR THE DETECTION AND TREATMENT OF CANCER |
| US10906971B2 (en) * | 2015-06-26 | 2021-02-02 | Sanofi Biotechnology SAS | Monoclonal anti-IL-1RAcP antibodies |
| CN110352351B (zh) * | 2016-10-18 | 2022-08-16 | 积水医疗株式会社 | 使用抗人bnp片段(4-32)抗体的免疫测定方法 |
| CN108593941A (zh) * | 2018-04-26 | 2018-09-28 | 北京丹大生物技术有限公司 | 一种NT-proBNP免疫法测定试剂制备和检测方法 |
| CN109678958B (zh) * | 2019-01-31 | 2022-03-18 | 重庆探生科技有限公司 | 一种人NT-proBNP特异性重组羊单克隆抗体及其制备方法和应用 |
| CN112175080B (zh) * | 2020-10-22 | 2021-05-25 | 优睿赛思(武汉)生物科技有限公司 | 抗人白介素-6高亲和力兔单克隆抗体及应用 |
-
2021
- 2021-07-26 CN CN202110822760.0A patent/CN113956355B/zh active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101878225A (zh) * | 2007-08-03 | 2010-11-03 | 比奥-拉德巴斯德公司 | 新型bnp(1-32)表位以及针对所述表位的抗体 |
| CN110616192A (zh) * | 2019-08-09 | 2019-12-27 | 无锡傲锐东源生物科技有限公司 | 一种抗人神经丝轻链（nefl）的单克隆抗体及其应用 |
| CN110684740A (zh) * | 2019-08-09 | 2020-01-14 | 无锡傲锐东源生物科技有限公司 | 一种抗人泛素羧基末端水解酶-1(uch-l1)的单克隆抗体及其应用 |
| CN112608385A (zh) * | 2020-12-18 | 2021-04-06 | 杭州贤至生物科技有限公司 | 一种犬脑钠肽（bnp）单克隆抗体的制备 |
Non-Patent Citations (6)
| Title |
|---|
| AAB94699.1: IgM heavy chain VDJ region, partial [Oryctolagus cuniculus];GenBank;《GenBank》;标题、PROTEIN、CDS、ORIGIN部分 * |
| AAF14433.1: immunoglobulin kappa light chain, partial [Oryctolagus cuniculus];GenBank;《GenBank》;标题、PROTEIN、CDS、ORIGIN部分 * |
| BAQ55252.1: immunoglobulin light chain [Oryctolagus cuniculus];GenBank;《GenBank》;标题、PROTEIN、CDS、ORIGIN部分 * |
| Monoclonal antibody against brain natriuretic peptide and characterization of brain natriuretic peptide-transgenic mice;M Nakagawa等;《Journal of hypertension》;第19卷(第03期);第475-483页 * |
| 兔单克隆抗体的发展及应用前景;李婷婷、崔慧斐;《食品与药品》;第14卷(第09期);第373-376页 * |
| 抗人N端脑钠肽前体抗原表位分析及单克隆抗体的研制;侯亚璐等;《生物技术通讯》;第30卷(第06期);第762-767页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113956355A (zh) | 2022-01-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113956355B (zh) | 抗人脑利钠肽(bnp)兔单克隆抗体及其应用 | |
| CN107383192B (zh) | 半乳糖凝集素-3检测试剂盒 | |
| JPH09511915A (ja) | ヒト心室ミオシン軽鎖に対するモノクローナル抗体 | |
| JP5458011B2 (ja) | 新規ｂｎｐ（１−３２）エピトープおよび前記エピトープに対する抗体 | |
| WO2013035799A1 (ja) | ペリオスチンの特定領域に結合する抗体及びこれを用いたペリオスチンの測定方法 | |
| EP1664769B1 (en) | Assay for detecting atrial and brain natriuretic peptide prohormones | |
| WO2023061388A1 (zh) | 半乳糖凝集素-3的免疫测定 | |
| CN114181908B (zh) | 抗人s100b蛋白鼠单克隆抗体及其应用 | |
| CN117624351B (zh) | 一种抗人磷酸化tau181兔单克隆抗体及其应用 | |
| CN119192368B (zh) | 用于检测叶酸及其结合蛋白复合物的单克隆抗体或其抗原结合片段、其制备方法及应用 | |
| CN113817062B (zh) | 抗人羟基类固醇17-β脱氢酶13（HSD17B13）兔单克隆抗体及其应用 | |
| CN109142738A (zh) | Ecm1作为血清学检测肝纤维化的标志物及其应用 | |
| CN107478848B (zh) | 定量检测人NT-proBNP的试剂盒及其制备方法 | |
| CN117624354B (zh) | 一种抗人乙酰化tau281兔单克隆抗体及其应用 | |
| CN118772273A (zh) | 一种睾酮夹心法兔单克隆抗体mAb1及其应用 | |
| CN113150143A (zh) | 一种用于检测血清中g17含量的配对抗体及其应用 | |
| CN107746430B (zh) | 一种gp73 c端抗原的制备及其应用 | |
| WO2022221877A2 (en) | Lateral flow analysis and breast cancer | |
| CN113912719A (zh) | 检测小鼠白介素6的单克隆抗体及其制备方法和应用 | |
| CN117624350B (zh) | 一种抗人磷酸化tau217兔单克隆抗体及其应用 | |
| CN117024586B (zh) | 一种抗体组合物及应用于检测血液中ykl-40浓度的方法 | |
| CN114456265B (zh) | 一种抗hfabp单克隆抗体及其应用 | |
| JP6508996B2 (ja) | β−ＡＮＰによる心不全の検出方法 | |
| CN114609394B (zh) | 抗缪勒氏管激素检测试剂盒、单克隆抗体、杂交瘤细胞株 | |
| CN113999302B (zh) | 用于体外诊断的新型冠状病毒核衣壳蛋白抗体 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |