CN105527434B - One kind is used for the kit of diacetyl spermine (DAS) detection - Google Patents
One kind is used for the kit of diacetyl spermine (DAS) detection Download PDFInfo
- Publication number
- CN105527434B CN105527434B CN201511020936.1A CN201511020936A CN105527434B CN 105527434 B CN105527434 B CN 105527434B CN 201511020936 A CN201511020936 A CN 201511020936A CN 105527434 B CN105527434 B CN 105527434B
- Authority
- CN
- China
- Prior art keywords
- das
- spermine
- diacetyl
- room temperature
- added
- 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
- 238000001514 detection method Methods 0.000 title claims abstract description 56
- NQNXERHVLXYXRO-UHFFFAOYSA-N Diacetylspermine Chemical compound Cl.Cl.CC(=O)NCCCNCCCCNCCCNC(C)=O NQNXERHVLXYXRO-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 claims abstract description 25
- 239000003153 chemical reaction reagent Substances 0.000 claims description 56
- 239000007788 liquid Substances 0.000 claims description 50
- 238000006243 chemical reaction Methods 0.000 claims description 32
- 239000000047 product Substances 0.000 claims description 30
- 230000008878 coupling Effects 0.000 claims description 24
- 238000010168 coupling process Methods 0.000 claims description 24
- 238000005859 coupling reaction Methods 0.000 claims description 24
- 102000014914 Carrier Proteins Human genes 0.000 claims description 22
- 108010078791 Carrier Proteins Proteins 0.000 claims description 22
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 claims description 22
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 19
- 150000001718 carbodiimides Chemical class 0.000 claims description 18
- 239000013078 crystal Substances 0.000 claims description 18
- 239000013598 vector Substances 0.000 claims description 17
- GUNURVWAJRRUAV-UHFFFAOYSA-N N(1)-acetylspermine Chemical compound CC(=O)NCCCNCCCCNCCCN GUNURVWAJRRUAV-UHFFFAOYSA-N 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- -1 N-hydroxy-succinamide ester Chemical class 0.000 claims description 12
- PVGATNRYUYNBHO-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-(2,5-dioxopyrrol-1-yl)butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O PVGATNRYUYNBHO-UHFFFAOYSA-N 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 11
- 229940063675 spermine Drugs 0.000 claims description 11
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000002105 nanoparticle Substances 0.000 claims description 10
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 9
- 238000001994 activation Methods 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 241001465754 Metazoa Species 0.000 claims description 7
- 239000007822 coupling agent Substances 0.000 claims description 6
- 238000003908 quality control method Methods 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 6
- 239000008118 PEG 6000 Substances 0.000 claims description 5
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- 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 claims description 5
- 239000007983 Tris buffer Substances 0.000 claims description 5
- 230000033228 biological regulation Effects 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 229920000136 polysorbate Polymers 0.000 claims description 4
- VGCXGMAHQTYDJK-UHFFFAOYSA-N Chloroacetyl chloride Chemical compound ClCC(Cl)=O VGCXGMAHQTYDJK-UHFFFAOYSA-N 0.000 claims description 3
- 238000012986 modification Methods 0.000 claims description 3
- 230000004048 modification Effects 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 239000000376 reactant Substances 0.000 claims description 3
- DCQLBQHPZCXPLE-UHFFFAOYSA-N 2-acetyl-2-sulfanylbutanedioic acid Chemical compound CC(=O)C(S)(C(O)=O)CC(O)=O DCQLBQHPZCXPLE-UHFFFAOYSA-N 0.000 claims description 2
- 102100033620 Calponin-1 Human genes 0.000 claims description 2
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 claims description 2
- 101000652736 Homo sapiens Transgelin Proteins 0.000 claims description 2
- 230000006957 competitive inhibition Effects 0.000 claims description 2
- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 claims description 2
- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 claims description 2
- 229960002317 succinimide Drugs 0.000 claims description 2
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 claims description 2
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 claims 2
- 125000002252 acyl group Chemical group 0.000 claims 1
- 239000012514 monoclonal antibody product Substances 0.000 claims 1
- 238000002965 ELISA Methods 0.000 abstract description 18
- 239000000427 antigen Substances 0.000 abstract description 15
- 102000036639 antigens Human genes 0.000 abstract description 15
- 108091007433 antigens Proteins 0.000 abstract description 15
- 238000012360 testing method Methods 0.000 abstract description 12
- 238000002360 preparation method Methods 0.000 abstract description 8
- NPDTUDWGJMBVEP-UHFFFAOYSA-N N(1),N(12)-diacetylspermine Chemical compound CC(=O)NCCCNCCCCNCCCNC(C)=O NPDTUDWGJMBVEP-UHFFFAOYSA-N 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 42
- 239000000243 solution Substances 0.000 description 29
- 241000699666 Mus <mouse, genus> Species 0.000 description 16
- 230000000694 effects Effects 0.000 description 15
- 235000015097 nutrients Nutrition 0.000 description 14
- 210000002700 urine Anatomy 0.000 description 14
- 210000004408 hybridoma Anatomy 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 239000000203 mixture Substances 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 10
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 229920000768 polyamine Polymers 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 206010009944 Colon cancer Diseases 0.000 description 8
- 239000012980 RPMI-1640 medium Substances 0.000 description 8
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 8
- 239000007853 buffer solution Substances 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 7
- 206010035226 Plasma cell myeloma Diseases 0.000 description 7
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 7
- 201000000050 myeloid neoplasm Diseases 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 230000007910 cell fusion Effects 0.000 description 5
- 239000007795 chemical reaction product Substances 0.000 description 5
- 208000029742 colonic neoplasm Diseases 0.000 description 5
- 239000000385 dialysis solution Substances 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 206010003445 Ascites Diseases 0.000 description 4
- 241001494479 Pecora Species 0.000 description 4
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 4
- 102000006601 Thymidine Kinase Human genes 0.000 description 4
- 108020004440 Thymidine kinase Proteins 0.000 description 4
- 210000000683 abdominal cavity Anatomy 0.000 description 4
- 235000011054 acetic acid Nutrition 0.000 description 4
- 210000003719 b-lymphocyte Anatomy 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 239000010931 gold Substances 0.000 description 4
- 229910052737 gold Inorganic materials 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 210000002381 plasma Anatomy 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 4
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 3
- 102000018251 Hypoxanthine Phosphoribosyltransferase Human genes 0.000 description 3
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 3
- 239000007987 MES buffer Substances 0.000 description 3
- 210000001015 abdomen Anatomy 0.000 description 3
- 150000004075 acetic anhydrides Chemical class 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 201000001275 rectum cancer Diseases 0.000 description 3
- 210000004988 splenocyte Anatomy 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- QSJXEFYPDANLFS-UHFFFAOYSA-N Diacetyl Chemical group CC(=O)C(C)=O QSJXEFYPDANLFS-UHFFFAOYSA-N 0.000 description 2
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 2
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 2
- 102000052812 Ornithine decarboxylases Human genes 0.000 description 2
- 108700005126 Ornithine decarboxylases Proteins 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 208000015634 Rectal Neoplasms Diseases 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 229920002892 amber Polymers 0.000 description 2
- 229960003896 aminopterin Drugs 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 238000010241 blood sampling Methods 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- VHRGRCVQAFMJIZ-UHFFFAOYSA-N cadaverine Chemical compound NCCCCCN VHRGRCVQAFMJIZ-UHFFFAOYSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 201000002313 intestinal cancer Diseases 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- ZSBDPRIWBYHIAF-UHFFFAOYSA-N n-acetylacetamide Chemical compound CC(=O)NC(C)=O ZSBDPRIWBYHIAF-UHFFFAOYSA-N 0.000 description 2
- 229960003104 ornithine Drugs 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- KIDHWZJUCRJVML-UHFFFAOYSA-N putrescine Chemical compound NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 description 2
- 206010038038 rectal cancer Diseases 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- 229960005137 succinic acid Drugs 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010052358 Colorectal cancer metastatic Diseases 0.000 description 1
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- 201000003741 Gastrointestinal carcinoma Diseases 0.000 description 1
- 208000005016 Intestinal Neoplasms Diseases 0.000 description 1
- 238000012449 Kunming mouse Methods 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 241000393460 Peromyscus nasutus Species 0.000 description 1
- 239000005700 Putrescine Substances 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 150000001243 acetic acids Chemical class 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- PPQRONHOSHZGFQ-LMVFSUKVSA-N aldehydo-D-ribose 5-phosphate Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PPQRONHOSHZGFQ-LMVFSUKVSA-N 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 125000004063 butyryl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 210000000038 chest Anatomy 0.000 description 1
- 230000009668 clonal growth Effects 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 150000002169 ethanolamines Chemical class 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000037189 immune system physiology Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 210000004279 orbit Anatomy 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 235000015320 potassium carbonate Nutrition 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000000192 social effect Effects 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57419—Specifically defined cancers of colon
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Hospice & Palliative Care (AREA)
- Biotechnology (AREA)
- Oncology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to detection diacetyl spermine (N1, N12Diacetylspermine, abbreviation DAS) content method, specifically a kind of kit for being used for diacetyl spermine (DAS) content detection, the method for production and preparation scheme of antigen, the preparation method of antibody and kit that kit uses.Kit of the present invention optimizes DAS detection method of content, compared with conventional ELISA method, have easy to operate, detection speed is fast, precision is high, reproducible, testing result is not influenceed by operation, can high-volume test sample the advantage such as use.
Description
Technical field
The present invention relates to detection diacetyl spermine (N1, N12- Diacetylspermine, abbreviation DAS) content method, tool
Saying for body is a kind of kit for being used for diacetyl spermine (DAS) detection.
Background technology
Colorectal cancer includes colon cancer and the carcinoma of the rectum, and colorectal cancer comes the 3rd in the global incidence of disease in malignant tumour
Position, the incidence of disease in China comes the 5th, and the intestinal cancer incidence of disease of China male is 16/100000ths, and women is 100,000/
14, it is one of most common tumour.Westerner is mainly based on colon, and 70% to 80% patient suffers from for colon cancer, directly
Patients with bowel cancer accounts for 20% to 30%.But on the contrary, rectal cancer patient accounts for 60% to 70%, colon cancer accounts for 30% to 40% in China.Closely
Year researchs and analyses discovery, and the rectum cancer morbidity in China is held essentially constant, and colon cancer disease rates are but rising.Although change
Treatment is effective to metastatic colorectal cancer patients, but prognosis is poor.Therefore, prepared by research is used to detect colorectal cancer patients tumor markers
The kit of content has important social effect and scientific value.
Polyamines includes putrescine, cadaverine, spermidine and spermine, is a kind of low molecular weight organic for having bioactivity nitrogenous
The general name of compound.It is considered as the material generated after 1-3 hydrogen atom is substituted by alkyl or aryl in amino molecule.Can be by original
Core and eukaryotic produce.The tissue of fast-growth generally has active polyamines synthesis system, contains substantial amounts of polyamines.When internal
During in the presence of such tissue, in urine the synthesis of polyamines can increase.Previous research shows that compared with Healthy People cancer patient urinates
The content increase of polyamines in liquid.However, follow-up research shows that polyamine content that is total and dissociating is big due to generating in urine
The false positive and negative findings of amount, thus cannot function as reliable tumor markers.
DAS molecular formula are C14H30N4O2, molecular weight 286.41, be a kind of diacetyl derivative of polyamines, in vivo by
Ornithine is acted on through ornithine decarboxylase and generated.Diacetyl spermine is the metabolite of tumour cell, is excreted in urine.Cell cancer
After change, the secretion increase of acetyl group polyamines, cause diacetyl spermine concentration in tumor patient urine significantly raised.DAS early stage inspection
Survey is completed by high performance liquid chromatography (HPLC), was established the enzyme-linked of DAS content detections suitable for urine later and is exempted from
Epidemic disease adsorption system.The detection for having DAS expression quantity in studies have shown that urine can be used as colon cancer, breast cancer and liver cancer effective
Novel tumor markers.So far, the ELISA detection method for DAS content detections in urine has obtained extensive clinical research.
But ELISA detection method equally exists complex operation, it is impossible to the problems such as batch detection.What the present invention developed utilizes full-automatic biochemical
The turbidimetry detection kit of DAS contents in analyzer detection clinical samples, be it is a kind of it is more more convenient than ELISA detection method,
Efficiently detection technique, once great amount of samples can be detected, and its high sensitivity, it is simple to operate.
The content of the invention：
Present invention aims at provide a kind of diacetyl spermine (DAS) immunoturbidimetry vitro detection kit.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of diacetyl spermine (DAS) immunoturbidimetry vitro detection kit, including R1 reagents, R2 reagents, standard items
And quality-control product, the R1 reagents are by diacetyl spermine antibody coupling on nano particle, with liquid form after addition protection liquid
Or the reagent that the solid form after freezing preserves；Described R2 reagents are that acetyl spermine (DAS) molecule is passed through into coupling reagent
Be coupled on carrier protein, with liquid form or it is lyophilized after the reagent that preserves of solid forms.
The diacetyl spermine antibody is to be coupled at institute on carrier protein by coupling reagent with acetyl spermine (DAS) molecule
The product obtained is as immunogene, then immune animal obtains polyclonal antibody or obtains monoclonal antibody through screening.
Protection liquid is by weight percentage in the R1：0.1-0.5% Tris, 0.5-2% PVP40,0.5-1%
BSA, 0.1-0.5% PEG20000,2-5% sucrose, 0.01-0.8% TWEEN20,0.02-0.1% Sodium azide,
0.85-1.5% NaCl, 1-4% PEG 6000, surplus are water.
Described sample is including but not limited to serum, blood plasma, urine etc..The nano particle includes but is not limited to nanometer
Gold, microballoon etc..The carrier protein includes but is not limited to seralbumin (BSA), poly-D-lysine, ovalbumin etc..
The coupling reagent is two sub- amide-type of carbon or maleimide；Wherein, two sub- amide-type of carbon is EDC, 1- second
Base -3- (3- dimethyl aminopropyls)-carbodiimides or N-hydroxy-succinamide ester class (NHS esters)；Maleimide is 4-
Maleimidobutyric acid-N- succinimide esters (GMBS) or 4- (N- maleimidomethyls) hexamethylene -1- carboxylic acid ambers
Amber imide ester (SMCC).
Diacetyl spermine (DAS) the molecular conjugate vectors albumen is：With carbodiimide/n-hydroxysuccinimide
(EDC/NHS) coupling reagent is used as, the carrier protein after activation process is added to the pH=6.5- of the 0.01M-0.1M to excess
In 8.0 PBS, water miscible carbodiimide (EDC) and n-hydroxysuccinimide (NHS) are added, is mixed to as clear as crystal,
Then stand 15-30 minutes at room temperature；Excessive acetyl spermine is added after standing, is mixed to as clear as crystal, regulation system pH value
To 10-12,4-6 hours are reacted at room temperature；Through products therefrom of dialysing after reaction, diacetyl spermine (DAS) molecule coupling labeled load is produced
Body protein.
Specially：Diacetyl spermine (DAS) the molecular conjugate vectors albumen is sub- with carbodiimide/N- hydroxysuccinimidyls acyl
Amine (EDC/NHS) is used as coupling reagent, and the 6-15mg carrier proteins after activation process are added to 0.01M-0.1M pH=
In 6.5-8.0PBS 3-8mL, water miscible carbodiimide (EDC) 7-10mg and n-hydroxysuccinimide (NHS) 3- are added
5mg, mix to as clear as crystal, then stand 15-30 minutes at room temperature；Acetyl spermine is added after standing, is mixed to as clear as crystal,
Regulation system pH value is adjusted to 10-12, reacts 4-6 hours at room temperature；Through products therefrom of dialysing after reaction, diacetyl spermine is produced
(DAS) molecular conjugate vectors albumen.
Diacetyl spermine (DAS) the molecular conjugate vectors albumen is：With carbodiimide/n-hydroxysuccinimide
(EDC/NHS) it is used as coupling reagent, the carrier protein after activation process adds the pH=6.5-8.0 of the 0.01M-0.1M to excess
PBS in, add water miscible carbodiimide (EDC) and n-hydroxysuccinimide (NHS), mix to as clear as crystal, and
Stand 15-30 minutes at room temperature afterwards；Spermine is added after standing, is mixed to as clear as crystal, regulation system pH value is adjusted to 10-12, room
Temperature lower reaction 4-6 hours；Through dialysing products therefrom in 2-8 DEG C after reaction, the acylating agent added into reaction system, then again
1-3 hours are reacted at 2-8 DEG C, reactant is producing diacetyl spermine (DAS) molecular conjugate vectors albumen through dialysing.
Specially：Diacetyl spermine (DAS) the molecular conjugate vectors albumen is：With carbodiimide/N- hydroxysuccinimidyl acyls
When imines (EDC/NHS) is used as coupling reagent, the 6-15mg carrier proteins after activation process are added to 0.01M-0.1M pH
In=6.5-8.0PBS 3-8mL, water miscible carbodiimide (EDC) 7-10mg and n-hydroxysuccinimide (NHS) are added
3-5mg, mix to as clear as crystal, then stand 15-30 minutes at room temperature；Spermine is added after standing, is mixed to as clear as crystal, tune
Section system pH value is adjusted to 10-12, reacts 4-6 hours at room temperature；Through dialysing products therefrom in 2-8 DEG C after reaction, to 3-10mL
40 μ L-80 μ L acylating agent is added in reaction system, 1-3 hours are then reacted at 2-8 DEG C, reactant produces through dialysing
Diacetyl spermine (DAS) molecular conjugate vectors albumen.
Diacetyl spermine (DAS) the molecular conjugate vectors albumen is with N- [γ-maleimide butyryl oxygen] succinyl
Imines ester (GMBS) is used as coupling reagent, by acylating agent (acetic anhydride or anhydrous acetic acid or chloroacetic chloride) and spermine at 2-8 DEG C it is anti-
60-90min is answered, is mixed after reaction with coupling agent (GMBS) in 0.01M-0.1M pH=6.5-8.0 PBS, at room temperature instead
3-4 hours are answered, obtain acetyl spermine-GMBS coupled products；By carrier protein and acetyl mercapto butanedioic acid 0.01M-0.1M's
Mixed in pH=6.5-8.0 PBS, react 3-5 hours at room temperature；0.1M azanols are added after reaction, react 30-35 points at room temperature
Clock removes deacetylate；Dialysed in 0.01M-0.1M pH=6.5-8.0 PBS, obtain the BSA of sulfhydrylation modification；By acetyl spermine-
The BSA that GMBS coupled products are modified with above-mentioned sulfhydrylation is mixed, and reacts 4-6 hours at room temperature；0.01M-0.1M pH=6.5-
Dialysed in 8.0 PBS to obtain diacetyl spermine (DAS) molecular conjugate vectors albumen.
Specially：Diacetyl spermine (DAS) the molecular conjugate vectors albumen is：With N- [γ-maleimide butyryl
Oxygen] succinimide ester (GMBS) is used as coupling reagent, 10-15 μ L acylating agent reacted at 2-8 DEG C with 15-25mg spermine
60-90min, after reaction with 2-4mg coupling agents (GMBS) 0.01M-0.1M pH=6.5-8.0PBS 3-5mL cushioning liquid
Middle mixing, 3-4 hours are reacted at room temperature, obtain acetyl spermine-GMBS coupled products；By the second of 8-15mg carrier proteins and 3-7mg
Acyl mercapto succinic acid mixes in 0.01M-0.1M pH=6.5-8.0PBS 3-5mL cushioning liquid, and it is small to react 3-5 at room temperature
When；0.1M azanols are added after reaction, 30-35 minutes is reacted at room temperature and removes deacetylate；0.01M-0.1M pH=6.5-
Dialysed in 8.0PBS buffer solutions, obtain the BSA of sulfhydrylation modification；Acetyl spermine-GMBS coupled products and above-mentioned sulfhydrylation are modified
BSA mixing, at room temperature react 4-6 hours；Being dialysed in 0.01M-0.1M pH=6.5-8.0PBS buffer solutions, it is smart to obtain diacetyl
Amine (DAS) molecular conjugate vectors albumen.
The carrier protein activation process is that carrier protein 0.01M-0.1M pH=6.5-8.0PBS buffer solutions is molten
Solution, adds MES after dissolving, is placed in 2-8 DEG C of environment and reacts half an hour.
.The acylating agent includes acetic anhydride, anhydrous acetic acid or chloroacetic chloride.
Pass through the diacetyl spermine in immunoturbidimetry A competitive inhibition method detection sample using the kit.
Specifically, diacetyl spermine (N1, N12- Diacetylspermine, abbreviation DAS) molecular formula is C14H30N4O2, point
Son amount 286.41, is a kind of diacetyl derivative of polyamines, is acted on generating through ornithine decarboxylase by ornithine in vivo.
DAS sheets are haptens, do not possess immunogenicity, only with reactionogenicity, can not made in itself using DAS as small-molecule substance
DAS antibody is prepared for immunogene, DAS need to be prepared into complete antigen by coupling agent and carrier protein couplet, by complete
Antigen-immunized animal is only possible to obtain corresponding DAS antibody.
Its detection method principle is：The DAS antigens of carrier protein couplet can be with the anti-DAS monoclonal antibodies on nano particle
Carry out specific binding and form immune complex, turbidity occur；The competitive knots of the DAS of DAS and carrier protein couplet in urine
Close DAS monoclonal antibodies.Solution turbidity is determined by Biochemical Analyzer.The content of turbidity and DAS in urine is inversely proportional.Pass through survey
Determine standard items, establish the standard curve of a degree of reaction corresponding concentration, you can DAS concentration values in measure urine sample.
The desired use of DAS kits is that Quantitative in vitro detects diacetyl spermine (N1, N12- in human urine
Diacetylspermine, DAS) content.
Advantage for present invention：
The content of vitro detection kit of the present invention include for DAS monoclonal antibody specific and
Reagent needed for nanoparticle label measure, is combined with novel nano particle immunologic detection method, for detecting human-body biological sample
The vitro detection of the DAS contents of product, open the new method of DAS content detections, compared with ELISA method, have it is easy to operate,
Detection speed is fast, precision is high, reproducible, testing result the advantage such as is not influenceed by operation.
Brief description of the drawings
Fig. 1 is standard curve provided in an embodiment of the present invention.
Fig. 2 detects sample DAS content balances to be provided in an embodiment of the present invention using immunoturbidimetry of the present invention and ELISA
Figure.
Fig. 3 is provided in an embodiment of the present invention related to ELISA detections sample DAS contents using immunoturbidimetry of the present invention
Property.
Embodiment
With reference to specific embodiment, the invention will be further described, but the invention is not restricted to specific examples below.
Embodiment 1
The preparation of the DAS antigens of BSA couplings：
1.BSA activation process：Precise bovine serum albumin(BSA) (BSA) 66mg, buffered with 0.01M pH=7.2PBS
Liquid 5ml dissolves；10mgMES is weighed, is added in above-mentioned lysate, is placed in 2-8 DEG C of environment and reacts half an hour；
2. measuring activated b SA product 10mg, it is placed in a clean 10ml test tube, adds 0.01M pH=7.2PBS to body
Product 4ml, adds EDC/NHS (7mg/3mg), mixes to as clear as crystal, stands 25 minutes at room temperature；
3. accurately measuring acetyl spermine 5mg, above-mentioned reaction system is added, is mixed to as clear as crystal, pH value is adjusted to 10, room temperature
Lower reaction 5 hours；
4. reaction product is dialysed 48 hours in 0.01M pH=7.2PBS dialyzates 1000ml, liquid is changed 4 times in centre；
5. final concentration is adjusted to 2mg/ml with 0.01M pH=7.2PBS；
6. being sub-packed in clean EP pipes (0.5ml/ pipes), saved backup in -20 DEG C of refrigerators.
Embodiment 2
1.BSA activation process：Precise bovine serum albumin(BSA) (BSA) 66mg, with 0.05MpH=8.0PBS buffer solutions
5ml dissolves；10mg MES are weighed, are added in above-mentioned lysate, is placed in 2-8 DEG C of environment and reacts half an hour；
2. measuring activated b SA product 10mg, it is placed in clean 10ml test tubes, adds 0.05M pH=8.0PBS to volume
4ml, EDC/NHS (10mg/5mg) is added, mixed to as clear as crystal, stand 15 minutes at room temperature；
3. measuring spermine 10mg, add to above-mentioned reaction system, mix to as clear as crystal, pH value modulation 11.2, at room temperature
Reaction 4 hours；
4. reaction product is dialysed 48 hours in 0.05M pH=8.0PBS dialyzates 1000ml, liquid is changed 4 times in centre；
5. in 2-8 DEG C of environment, 60 μ l acetic anhydrides are added into reaction system, are reacted 1 hour under 2-8 DEG C of environment；
6. reaction product is dialysed 48 hours in 0.05M pH=8.0PBS dialyzates 1000ml, liquid is changed 4 times in centre；
7. adjusting final concentration to 2mg/ml with 0.05M pH=8.0PBS, it is sub-packed in clean EP pipes (0.5ml/ pipes), -20
Saved backup in DEG C refrigerator.
Embodiment 3
1. the synthesis of acetyl spermine：Using 20mg spermine and 10 μ L anhydrous acetic acids as synthesis material, 1mL 0.1M pH are added
In=7.2PBS, reacted 60 minutes in 2-8 DEG C of environment, do not stop to stir and evenly mix during reaction；
2. by above-mentioned resulting acetyl spermine and coupling agent 4mg GMBS in 2mL 0.1M pH=7.2PBS cushioning liquid
Middle mixing, react 3 hours at room temperature；
3. 10mg BSA are mixed in 2mL 0.1MPBS cushioning liquid with 4mg acetyl mercaptos butanedioic acid, reflect at room temperature
3 hours；10 μ L 0.1M azanols are added, reacts 30 minutes remove deacetylate at room temperature；In 0.1M pH=7.2PBS buffer solutions thoroughly
Analysis 4 hours, remove reaction impurities；
4. acetyl spermine-GMBS coupled products are mixed with the BSA that above-mentioned sulfhydrylation is modified, react 4 hours at room temperature；
Dialysed 24 hours in 1000mL 0.1M pH=7.2PBS buffer solutions, -20 DEG C save backup.
Embodiment 4
1. the synthesis of acetyl spermine：Using 20mg spermine and 10 μ L acetic anhydrides or acetic acid as synthesis material, in 2-8 DEG C of environment
The 0.01M pH=7.2PBS for being added to 1mL react 60 minutes, do not stop to stir and evenly mix during reaction；
2. taking activated b SA product 10mg, it is placed in clean 10ml test tubes, adds in 4mL 0.01M pH=7.2PBS,
EDC/NHS (8mg/4mg) is added, is mixed to as clear as crystal, stands 45 minutes at room temperature；
3. the acetyl spermine for taking the above method to synthesize, adding reaction system, mix to as clear as crystal, pH value is adjusted to 11, room
The lower reaction of temperature more than 4 hours；
4. reaction product is dialysed 48 hours in 0.01M pH=7.2PBS dialyzates 1000ml, liquid is changed 4 times in centre；
5. final concentration is adjusted to 2mg/ml with 0.01M pH=7.2PBS；
6. being sub-packed in clean EP pipes (0.5ml/ pipes), saved backup in -20 DEG C of refrigerators.
Embodiment 5
1. weigh spermine 20mg, it is mixed in 1mL 0.01M pH=6.5PBS cushioning liquid with 4mg coupling agents GMBS
Close, react 1.5 hours at room temperature；
2. activated b SA 10mg and 4mg acetyl mercaptos butanedioic acid are mixed in 2mL 0.01M pH=6.5PBS cushioning liquid
Close, react 3 hours at room temperature；10 μ L 0.1M azanols are added, reacts 30 minutes remove deacetylate at room temperature；0.01MM PBS delay
Dialysed 4 hours in fliud flushing 1000mL, remove reaction impurities；
3. spermine-GMBS coupled products are mixed with the BSA that above-mentioned sulfhydrylation is modified, react 4 hours at room temperature；
4.0.01M pH=6.5PBS buffer solutions 1, dialyse 24 hours in 000ml, liquid is changed 2 times in centre；
5. in 2-8 DEG C of environment, 60 μ l acetic anhydrides are added into reaction system, 2-8 DEG C is reacted 1 hour；
6. reaction product is dialysed 48 hours in 0.01M pH=6.5PBS dialyzates 1000ml, liquid is changed 4 times in centre；
7. adjusting final concentration to 2mg/ml with 0.01M pH=6.5PBS, it is sub-packed in clean EP pipes (0.5ml/ pipes), -20
Saved backup in DEG C refrigerator.
Embodiment 6
Polyclonal antibody or the monoclonal antibody through screening gained.
Preparation method for antibody process is as follows：
1. above-mentioned prepared antigen is used to be obtained as the animal such as immunogene, immune sheep, rabbit, mouse after being repeatedly immunized
Plasma/serum containing anti-DAS antibody or the effect B cell that anti-DAS antibody can be expressed；
2. after obtaining plasma/serum, polyclonal antibody needed for kit developing is obtained by Protein G column chromatography；
3. after obtaining the effect B cell that can express anti-DAS antibody, B cell is merged with myeloma cell, screened,
Obtain has the hybridoma cell strain for expressing anti-DAS antibody and infinite multiplication function, after being enlarged culture, enrichment of cell simultaneously
Supernatant, purified by protein A chromatography and obtain monoclonal antibody.
Embodiment 7
The preparation of anti-DAS antibody
First, immunogene is that (concrete operations refer to implementation to product of the acetyl spermine molecule coupling labeled obtained by carrier protein BSA
Example 1- embodiments 5)
Concentration：2mg/ml
2nd, animal selection is with being immunized
(1), animal selects
Selection and the homologous Balb/c Healthy female mouse of myeloma cell used, mouse age was at 6~10 weeks.To avoid mouse
React dead in bad or immunologic process, while 5 mouse, numbering 1-5 is immunized.
(2), immunization protocol
1st, initial immunity
300ug antigens are dissolved in PBS, add isometric Freund's complete adjuvant (CFA), are emulsified into Water-In-Oil shape, are used
Abdominal cavity and dorsal sc multi-point injection, 0.5ml/ only, after three weeks carry out second and are immunized；
2nd, it is immune for the second time
300ug antigens are dissolved in PBS, adds isometric freund 's incomplete adjuvant (IFA), is emulsified into Water-In-Oil shape, adopts
With abdominal cavity and dorsal sc multi-point injection, it is immune that 0.5ml/ only, after three weeks carries out third time；
3rd, third time is immune
300 μ g antigens are dissolved in PBS, adds isometric freund 's incomplete adjuvant (IFA), is emulsified into Water-In-Oil shape, adopts
With abdominal cavity and dorsal sc multi-point injection, only, blood sampling detection antiserum is active after one week by 0.5ml/；
4th, antiserum Activity determination (ELISA method detection antiserum activity)
4.1 experimental procedure
4.1.1 antigen coat
Pure antigen concentration 2mg/ml, 100 μ l are taken to add in each hole of the enzyme-linked detection plate of polystyrene after being diluted with coating buffer, 4
DEG C overnight after, wash liquid 3 times.
4.1.2 closing
Add 200 μ l confining liquids per hole, 4 DEG C overnight or 37 DEG C after two hours, washs 3 times, pats dry.It is standby to put 4 DEG C of refrigerators preservations
With.
4.1.3 ELISA is detected
1) when detecting serum titer, doubling dilution is done to serum, 100 μ l/ holes, using Normal Mouse Serum as negative control,
37 DEG C of incubation 30min, wash 3 times, pat dry.
2) add sheep anti mouse-HRP, the μ l/ holes of potency 105,100,37 DEG C of incubation 30min, wash 3 times, pat dry.
3) develop the color：Add each 50 μ l/ holes of A, B liquid, 37 DEG C of colour developing 15min.
4) terminate：Add the μ l/ holes of terminate liquid 50.
5) reading：With 450nm, 630nm dual wavelengths determine each hole OD values, with the ratio (P/N) with negative control hole OD values
It is limited more than 2.1, as the critical point for being judged as the positive or determination potency.
After testing, 5 mouse activity intensities are：4#>5#>1#>3#>2#, booster immunization 4# mouse after two weeks.
5th, booster immunization
50ug antigens are mixed with 0.5mlPBS, in tail vein injection 4# Mice Bodies, cell fusion is carried out after three days.
3rd, cell fusion
(1), prepared by feeder cells
1st, reagent consumptive material：
1.1 RPMI-1640 complete culture solutions；
The endless full nutrient solutions of 1.2 RPMI-1640；
1.3 HAT nutrient solutions；
1.4 centrifuge；
1.5 other materials：Cell diluent, blood cell counting plate, cover glass, sterile centrifugation tube, 96 porocyte culture plates,
Eye scissors, tweezers, big tweezers, plate, Dispensable 1 ml syringe, disposable 10ml syringes.
2nd, experimental animal
Balb/c or Kunming healthy mice (male and female) 1,6~10 week old, 18~22 grams of body weight.
3rd, experimental method
Balb/c or kunming mice are drawn neck dislocation to put to death by 3.1, are soaked in 75% alcohol 5 minutes, are put into ultra-clean work immediately
Make in platform, belly is put in plate upward；
3.2 play mouse part skin with ophthalmic tweezers sub-folder, cut an osculum with scissors, pay attention to being sure not to break peritonaeum, in order to avoid abdomen
Chamber liquid outflows, and then does blunt separation to upper and lower both sides with scissors, fully exposes peritonaeum, and wiping peritonaeum with cotton ball soaked in alcohol sterilizes；
3.3 draw endless full nutrient solution with syringe, inject mouse peritoneal, and syringe stop is motionless, rock mouse or anti-
Multiple suction draws back intraperitoneal liquid several times, with original annotation emitter, injects centrifuge tube；
3.41000rpm centrifuging 10min, supernatant is abandoned；
3.5 are resuspended cell with 10ml complete culture solutions, count, and adjustment cell concentration is 200,000 every milliliter, 100 μ l/ holes
96 porocyte culture plates are added drop-wise to, 37 DEG C is put, is cultivated in 5%CO2 incubators standby；
The growth conditions of 3.6 observation feeder cells, general well-grown feeder cells and macrophage are in fusiformis or more
It is angular, cell is bright, refractivity is strong.
(2), cell fusion
1st, reagent consumptive material
1.1 ophthalmology tweezers, scissors number handle, fixed plate
1.2 37 DEG C of warm water
1.3 alcolhol burners, centrifuge tube, centrifuge, plate
1.4 fusion agent：50%PEG, W=4000 (37 DEG C of preheatings)
The endless full nutrient solutions of 1.5 RPMI-1640 (37 DEG C of preheatings)
1.6 RPMI-1640 complete culture solutions (37 DEG C of preheatings)
1.7 HAT cultures solution additive (37 DEG C of preheatings)
2nd, splenocyte suspension is prepared
Booster immunization 4# mouse are taken, dislocates and puts to death after eye socket blood sampling, are invaded in 75% alcohol after bubble sterilizes under aseptic condition
Spleen is taken out, splenocyte suspension is made, is centrifuged, 1000rpm, 10min, endless full nutrient solution wash twice, and count, stand-by；
3rd, myeloma cell's suspension is prepared
Take the good (viable count of 3 bottles of growth conditions>95%) myeloma cell, using sterile dropper to thin in blake bottle
Born of the same parents carry out pressure-vaccum, and then cell suspension is transferred in 50ml centrifuge tubes, centrifuge, 1000rpm, 10min, use incomplete culture
Liquid washes twice, and counts, stand-by；
4th, cell fusion
4.1 are mixed myeloma cell and splenocyte by the quantitative proportion of 1: 10 or 1: 5, use 50ml centrifuge tubes
Centrifugation, cannots be used up full nutrient solution and washes 1 time, 1000rpm, 10min；
4.2 abandon supernatant, with dropper exhaust residual liquid, in order to avoid influence PEG concentration；
4.3 gently attack centrifuge tube bottoms, make cell precipitation loosen slightly；
4.4 merge at room temperature
1. adding the 1ml 50%PEG (molecular weight 4000) of preheating in 1 minute, side edged rocks；
2. effect 90 seconds；
3. plus the endless full nutrient solutions of RPMI-1640 of preheating, termination PEG are acted on, and are separately added into 1ml, 2ml at intervals of two minutes,
The endless full nutrient solutions of RPMI-1640 of 3ml, 4ml, 5ml and 10ml preheating；
4.5 centrifugations, 1000rpm, 10min；
4.6 abandon supernatant, are first gently suspended cell with RPMI1640 complete culture solutions of the 6ml or so containing 20% calf serum,
Make sure to keep in mind firmly to blow and beat, in case the cell for making to be merged scatters；
The quantity of 96 well culture plates used in 4.7 bases, add complete culture solution (RPMI-1640 complete culture solutions, per 50ml
Addition 1mlHAT cultures solution additive in complete culture solution)；
Cell suspension after fusion is added 96 orifice plates containing feeder cells, 100 μ l/ holes, 37 DEG C, 5%CO2 trainings by 4.8
Support case culture；
4th, filtering hybridoma
(1), HAT selects hybridoma
HAT Selective agar mediums contain hypoxanthine, aminopterin-induced syndrome and thymidine, and wherein aminopterin-induced syndrome can blocking dna conjunction
Into main path.After main path blocks, by according to emergent approach, i.e. in HGPRT, (enzyme hypoxanthine guanine ribose phosphate shifts
Enzyme) and TK (thymidine kinase) effects under, using thymidine and hypoxanthine synthetic DNA, lack one of which, DNA is synthesized not
It can occur.Myeloma cell line for hybridization forms auxotrophic cell caused by selection by through toxic medicament induction,
Into the cell without TK or HGPRT, thus single or fusion myeloma cell in HAT nutrient solutions by death.Although B cell has
HGPRT and TK, but under the conditions of being generally incubated in vitro, long-term surviving is especially difficult under individual cells environment and propagation passes
Generation, thus only hybridoma could in HAT nutrient solutions growth and breeding.
(2), cell culture, change liquid
Daystart after cell fusion, cell is examined, recorded the growth conditions of cell, nutrient solution has
Pollution-free, feeder cells situations.3~5 days HAT nutrient solutions of culture change liquid once, change within 10 days the culture of HT nutrient solutions to 20 days, change
1640 complete culture solutions.
(3), screen the hybridoma of secretory antibody (with one of culture plate accompanying drawing)
When hybridoma colonies grow into a certain size after fusion, nutrient solution starts to turn yellow, and can start screening antibodies
Activity.Typically conventional has：Immunofluorescence, radio-immunity (RIA), histochemical method and ELISA (ELISA) method etc..
It is most frequently ELISA method with method.When collecting supernatant, carried out within 3-4 days after liquid should be changed in last time, so as to antibody accumulation.Supernatant
Can be without dilution or 1：5-1：Antibody activity is surveyed again after 10 dilutions, and the anti-of weakly positive can be screened out when being screened with dilution antibody
Body, can also remove the false positive caused by high background, and detection method is as follows：
1st, antiserum Activity determination (ELISA method detection antiserum activity)
1.1 antigen coat
Pure antigen concentration 2mg/ml, 100 μ l are taken to add in each hole of the enzyme-linked detection plate of polystyrene after being diluted with coating buffer, 4
DEG C overnight after, wash liquid 3 times.
1.2 closing
Add 200 μ l confining liquids per hole, 4 DEG C overnight or 37 DEG C after two hours, washs 3 times, pats dry.It is standby to put 4 DEG C of refrigerators preservations
With.
1.3 ELISA are detected
1) cell conditioned medium of absorption culture hole to be detected, 100 μ l/ holes, using not celliferous culture medium as negative control, with
Positive serum 37 DEG C of incubation 30min, is washed 3 times, patted dry as positive control.
2) add sheep anti mouse-HRP, the μ l/ holes of potency 105,100,37 DEG C of incubation 30min, wash 3 times, pat dry.
3) develop the color：Add each 50 μ l/ holes of A, B liquid, 37 DEG C of colour developing 15min.
4) terminate：Add the μ l/ holes of terminate liquid 50.
5) reading：With 450nm, 630nm dual wavelengths determine each hole OD values, with the ratio (P/N) with negative control hole OD values
It is limited more than 2.1 (referring to table 1).
Table 1
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | 0.010 | 0.020 | 0.015 | 0.020 | 0.040 | 0.020 | 0.060 | 0.020 | 0.973 | 1.132 | 0.020 | 0.006 |
| B | 0.008 | 0.020 | 0.010 | 0.008 | 0.007 | 0.008 | 0.041 | 0.008 | 0.079 | 0.080 | 0.020 | 0.008 |
| C | 0.050 | 0.035 | 0.030 | 0.044 | 0.063 | 0.020 | 0.004 | 0.023 | 0.050 | 0.014 | 1.257 | 0.891 |
| D | 0.024 | 0.010 | 0.016 | 1.453 | 0.010 | 0.023 | 0.030 | 0.010 | 0.010 | 0.055 | 0.018 | 0.019 |
| E | 0.017 | 0.019 | 0.022 | 0.010 | 0.060 | 0.010 | 0.050 | 0.010 | 0.041 | 0.007 | 0.025 | 0.023 |
| F | 0.017 | 0.011 | 0.015 | 0.006 | 0.007 | 0.011 | 0.050 | 0.031 | 0.020 | 0.062 | 0.010 | 0.010 |
| G | 0.050 | 0.050 | 0.008 | 0.035 | 0.050 | 0.886 | 0.008 | 0.984 | 0.062 | 0.042 | 0.009 | 0.006 |
| H | 0.046 | 0.008 | 0.005 | 0.008 | 1.266 | 0.008 | 0.030 | 0.052 | 0.006 | 1.102 | 0.005 | 0.008 |
2nd, antiserum Activity determination (colloidal gold strip detection)
2.1 test strips make：
Mark gold pad：525nm collaurums, add K2CO3 and sheep anti-mouse igg by 6/25 volume ratio, shaking table shakes 10min, adds
10% casein, 10min is closed, centrifuged, redissolved, paving gold, drained；
Coating：ASP-PLL, 12mg/mL, 5 times are diluted with 1*PBS, 30uL/ plates is coated with, draws a line；
Assembling：It is followed successively by from top to bottom in PVC board：Blotting paper, NC films, gold pad (2 layers of 6mm), sample-adding pad；
Slitting：3mm/ bars are cut；
2.2 detection：
50-100ul cell supernatants are loaded, static 20min, contrast chromogenic line colour developing is strong and weak, judges in cell culture medium
Antibody activity is strong and weak, and colour developing is stronger, and antibody activity is higher.
By above-mentioned, the testing results of two methods warp it was found that, enzyme-linked detection numerical value is high, colloidal gold strip detection
Line colour developing is deep, and testing result is consistent, therefore later antibody screening uses colloidal gold strip method.
5th, the cloning (limiting dilution assay) of hybridoma
Fused cell is subcloned after antibody activity is screened, and picks out monoclonal cell strain, and step is as follows：
1st, 100 μ l feeder cells suspension (containing about 2.0 × 104 abdominal cavities are sequentially added per hole in 96 porocyte culture plates
Cell)；
2nd, hybridoma suspension to be cloned is prepared, 10 cell/ml are diluted to the HT culture mediums containing 10% serum,
It is 0.1ml cell suspensions per hole inoculum concentration, i.e., contains 1 cell per hole；
3rd, 37 DEG C, 5%CO2 cultivate 7~10 days, occur it is macroscopic clone or can be observed under inverted microscope
It is covered with the area of bottom hole 1/3 i.e. detectable antibody to hybridoma；The hole of only single clonal growth is marked, takes supernatant to make antibody
Detection,
4th, antibody test positive hole is taken to separate cell, and transferred species expands culture in 24 orifice plates, and freeze；
5th, appeal 1-4 steps 2 time are repeated, it containing cell hole is that positive monoclonal cell expands to choose all after cloning
Big culture, conservation freeze-stored cell.
The finished product cell line numbering finally filtered out：5D4A7A10
6th, the production of monoclonal antibody
Cell after conservation is further continued for expand culture, carries out a large amount of preparations of monoclonal antibody.
1st, adult Balb/c mouse (3 monthly age) intraperitoneal inoculation atoleine, every 0.3~0.5ml of mouse, is used after 7 days；
2nd, the hybridoma of exponential phase is collected, with brine twice, 1500r/min centrifugations 10min；
3rd, sample, with Trypan Blue, living cell counting, again with normal saline into 1 × 106 cell/ml's
Suspension
4th, the hybridoma of exponential phase is in every mouse peritoneal injection 1ml；
5th, after being spaced 5 days, the production of mouse ascites is observed daily, as belly substantially expands, when touching, skin
There is tension, put to death mouse, gather ascites；
6th, 4000r/min centrifuges ascites 15min, removes cell component and other sediments, collects supernatant, dispenses, -70
DEG C freeze standby；
7th, the ascites for being 5D4A7A10 numbering, 10ml, is purified using protein A chromatography altogether, and carries out desalting processing,
It is anti-DAS antibody after purification.
Embodiment 8
Kit, including R1 reagents, R2 reagents, standard items and quality-control product, the R1 reagents are by diacetyl spermine antibody
The reagent that solid form after being coupled on nano particle liquid form after rear addition protection liquid or freezing preserves.Described R2
Consolidating after acetyl spermine (DAS) molecule is coupled at rear liquid form on carrier protein or freezed by coupling reagent during reagent
The reagent that volume morphing preserves, is prepared by above-described embodiment 1-5.The diacetyl spermine antibody is by acetyl spermine (DAS)
The product that molecule is coupled on carrier protein gained by coupling reagent be used as immunogene, be immunized animal gained polyclonal antibody or
Monoclonal antibody (being prepared by above-described embodiment 7) through screening gained.
Described sample is including but not limited to serum, blood plasma, urine.The nano particle include but is not limited to nanogold,
Microballoon.
It is described protection liquid be by weight percentage：0.1-0.5% Tris, 0.5-2% PVP 40,0.5-1%
BSA, 0.1-0.5% PEG20000,2-5% sucrose, 0.01-0.8% TWEEN 20,0.02-0.1% Sodium azide,
0.85-1.5% NaCl, 1-4% PEG 6000, surplus are water.
The preparation of the R1 reagents：
1. preparing 50mM pH6.0 MES buffer solutions, for activating nano particle, make its final concentration of 1%w/v.
2. every milliliter of nano particle suspension adds 20mg EDC, 20min is incubated at room temperature.
3.12000rpm centrifuges 30min, with isometric coating buffer solution (100mM Hepes pH7.0 antibody containing DAS
100 μ g/mL) suspend, it is incubated at room temperature 2-5 hours.
4. adding 2.5 μ L monoethanolamines in every milliliter of reaction solution, persistently stir and be incubated at room temperature 10min.
5.12000rpm centrifuges 30min, is suspended with protection liquid.
R2 reagents are the DAS antigens of 800ng/mL BSA couplings；
Standard items are the DAS standard items through water gradient dilution, gradient dilution concentration is 0nM, 250nM, 500nM, 750nM,
5 concentration of 1000nM.
R1 and R2 volume ratio is=150：40
Detection
Vitro detection is carried out using the vitro detection kit of above-described embodiment, and contrasted with ELISA detection method：
(1) 40 biological samples (i.e. human urine) are gathered immediately in 30 minutes, centrifuging and taking supernatant conduct under the conditions of 4 DEG C
Measuring samples, be placed in ice chest 1 hour using or -20 DEG C save backup；
(2) reagent R1, R2 is taken out to place at room temperature to equalized temperature；
(3) R1, R2 reagent and DAS standard items are placed on the corresponding position of Biochemical Analyzer, formulate the standard curve (He of table 2
Referring to Fig. 1)；
Table 2
| Standard curve | 1 | 2 | 3 | 4 | 5 |
| Concentration (nM) | 0 | 250 | 500 | 750 | 1000 |
| Degree of reaction | 0.4625 | 0.3545 | 0.2139 | 0.1280 | 0.0844 |
(4) DAS quality-control products are detected by automatic clinical chemistry analyzer, set and add the μ L of 150 μ L, R2 reagent of R1 reagents 40,
The μ L of sample 5 are added, as first point detect, and be that second point detects, and using 2 differences as detected value, passes through mark at the 22nd point at the 45th point
Directrix curve returns, and determines testing result validity (referring to table 3)；
Table 3
(5) DAS samples are detected with automatic clinical chemistry analyzer, sets and add the μ L of 150 μ L, R2 reagent of R1 reagents 40, add
The μ L of sample 5, the 22nd point is detected as first point, and the 45th point is that second point detects, and using 2 differences as detected value, it is bent to pass through standard
Line returns and draws final result.
(6) DAS pattern detections result is detected referring to table 4, Fig. 2 using mode of the present invention and ELISA method, passes through ELISA
Method measured value is abscissa, and this law measured value is ordinate, and it is as shown in Figure 3 to do straight line using related software.
The two methods of table 4 detect DAS content results
By table 4 and Fig. 2 it is visible by two methods it can be seen from ratio to detect DAS content trend consistent, can be with by Fig. 3
Find out, linear equation y=0.5714x-68.630, r2=0.9272 correlation coefficient r=0.9629 (shown in Fig. 3), as a result table
Bright, the DAS values of two methods measure are closely related.And the inventive method detection DAS biochemical reagents box and ELISA method phase
Than detection time is only 10-15min, is saved the substantial amounts of time, easy to operate, quick.
Embodiment 9
Kit includes R1 reagents, R2 reagents, standard items and quality-control product.
The preparation of the R1 reagents, the 0.2M K for taking 1mL nanogold to add 7 μ L2CO3；15 μ g DAS antibody are added after mixing,
Mix and stand 10min；Again plus 10 μ L confining liquids are closed, and mix stand 10min again；Centrifuge 10min, rotating speed 8000r/
Min, abandons supernatant, and precipitation 1mL R1 protection liquid redissolves.
R1 protection liquid be by weight percentage：0.3% Tris, 2% PVP 40,1% BSA, 0.1%
PEG20000,5% sucrose, 0.1%TWEEN 20,0.04% Sodium azide, 1% NaCl, 4% PEG 6000, surplus are
Water.
R2 reagents are the DAS antigens of 800ng/mLBSA couplings；
Standard items are the DAS standard items through water gradient dilution, gradient dilution concentration is 0nM, 250nM, 500nM, 750nM,
5 concentration of 1000nM.
R1 and R2 volume ratio is=150：40
Embodiment 10
Kit includes R1 reagents, R2 reagents, redissolves liquid, standard items and quality-control product.
It is prepared by the R1 reagents：It is to be dispensed after being prepared according to R1 in embodiment 8, is put into freeze drier and is frozen
It is dry, R1 reagents are redissolved with liquid is redissolved again during use.
It is described redissolution liquid be：R1 protection liquid be by weight percentage：0.3% Tris, 2% PVP40,1%
BSA, 0.1% PEG20000,5% sucrose, 0.1%TWEEN 20,0.04% Sodium azide, 1% NaCl, 4% PEG
6000, surplus is water.
The other compositions are same as Example 8.
The inventive method and comparative example detection DAS pattern detection results are as shown in table 5 below.
Table 5
Shown by the above results, R1 reagents are more or less the same with lyophilized form detection DAS containing numerical quantity in liquid form,
Meet product requirement.
Claims (6)
1. a kind of diacetyl spermine (DAS) immunoturbidimetry vitro detection kit, including R1 reagents, R2 reagents, standard items and
Quality-control product, it is characterised in that：The R1 reagents be by diacetyl spermine antibody coupling on nano particle, and add protection liquid
Afterwards, with liquid form or it is lyophilized after the reagent that preserves of solid form；Described R2 reagents are by acetyl spermine (DAS) molecule
Be coupled at by coupling reagent on carrier protein, with liquid form or it is lyophilized after the reagent that preserves of solid forms；
The diacetyl spermine antibody be with acetyl spermine (DAS) molecule as obtained by being coupled on carrier protein coupling reagent
As immunogene, immune animal obtains polyclonal antibody or through screening to obtain monoclonal antibody product；
Protection liquid is by weight percentage in the R1：0.1-0.5% Tris, 0.5-2% PVP 40,0.5-1%
BSA, 0.1-0.5% PEG20000,2-5% sucrose, 0.01-0.8% TWEEN 20,0.02-0.1% Sodium azide,
0.85-1.5% NaCl, 1-4% PEG 6000, surplus are water.
2. diacetyl spermine (DAS) immunoturbidimetry vitro detection kit as described in claim 1, it is characterised in that：Institute
It is two sub- amide-type of carbon or maleimide to state coupling reagent；Wherein, two sub- amide-type of carbon is EDC, 1- ethyls -3- (3- bis-
Methyl aminopropyl)-carbodiimide or N-hydroxy-succinamide ester class (NHS esters)；Maleimide is 4- maleimides
Base butyric acid-N- succinimide esters (GMBS) or 4- (N- maleimidomethyls) hexamethylene -1- carboxylic acid succinimide esters
(SMCC)。
3. diacetyl spermine (DAS) immunoturbidimetry vitro detection kit as described in claim 1 or 2, it is characterised in that：
Diacetyl spermine (DAS) the molecular conjugate vectors albumen be using carbodiimide/n-hydroxysuccinimide (EDC/NHS) as
Coupling reagent, the carrier protein after activation process is added into excessive 0.01M-0.1M pH=6.5-8.0 PBS, then
Water miscible carbodiimide (EDC) and n-hydroxysuccinimide (NHS) are added, is mixed to as clear as crystal, it is then quiet at room temperature
Put 15-30 minutes；Excessive acetyl spermine is added after standing, is mixed to as clear as crystal, regulation system pH value is adjusted to 10-12, room
Temperature lower reaction 4-6 hours；Through products therefrom of dialysing after reaction, diacetyl spermine (DAS) molecular conjugate vectors albumen is produced.
4. diacetyl spermine (DAS) immunoturbidimetry vitro detection kit as described in claim 1 or 2, it is characterised in that：
Diacetyl spermine (DAS) the molecular conjugate vectors albumen be using carbodiimide/n-hydroxysuccinimide (EDC/NHS) as
Coupling reagent, the carrier protein after activation process is added into excessive 0.01M-0.1M pH=6.5-8.0 PBS, then
Water miscible carbodiimide (EDC) and n-hydroxysuccinimide (NHS) are added, is mixed to as clear as crystal, it is then quiet at room temperature
Put 15-30 minutes；Spermine is added after standing, is mixed to as clear as crystal, regulation system pH value is adjusted to 10-12, reacts 4-6 at room temperature
Hour；Through dialysing products therefrom in 2-8 DEG C after reaction, acylating agent is added into reaction system, is then reacted at 2-8 DEG C
1-3 hours, reactant produce diacetyl spermine (DAS) molecular conjugate vectors albumen through dialysing.
5. diacetyl spermine (DAS) immunoturbidimetry vitro detection kit as described in claim 1 or 2, it is characterised in that：
Diacetyl spermine (DAS) the molecular conjugate vectors albumen is with N- [γ-maleimide butyryl oxygen] succinimide ester
(GMBS) coupling reagent is used as, acylating agent (acetic anhydride or anhydrous acetic acid or chloroacetic chloride) and spermine are reacted into 60- at 2-8 DEG C
90min, mixed after reaction with coupling agent (GMBS) in 0.01M-0.1M pH=6.5-8.0 PBS, react 3-4 at room temperature
Hour, obtain acetyl spermine-GMBS coupled products；By carrier protein and acetyl mercapto butanedioic acid 0.01M-0.1M pH=6.5-
Mixed in 8.0 PBS, react 3-5 hours at room temperature；After reaction add 0.1M azanols, at room temperature react 30-35 minutes remove second
Acyl group；Dialysed in 0.01M-0.1M pH=6.5-8.0 PBS, obtain the BSA of sulfhydrylation modification；Acetyl spermine-GMBS is coupled
The BSA that product is modified with above-mentioned sulfhydrylation is mixed, and reacts 4-6 hours at room temperature；0.01M-0.1M pH=6.5-8.0 PBS
In dialyse to obtain diacetyl spermine (DAS) molecular conjugate vectors albumen.
6. diacetyl spermine (DAS) immunoturbidimetry vitro detection kit as described in claim 1, it is characterised in that：Profit
Pass through the diacetyl spermine in immunoturbidimetry A competitive inhibition method detection sample with the kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511020936.1A CN105527434B (en) | 2015-12-31 | 2015-12-31 | One kind is used for the kit of diacetyl spermine (DAS) detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511020936.1A CN105527434B (en) | 2015-12-31 | 2015-12-31 | One kind is used for the kit of diacetyl spermine (DAS) detection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105527434A CN105527434A (en) | 2016-04-27 |
| CN105527434B true CN105527434B (en) | 2017-12-19 |
Family
ID=55769786
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201511020936.1A Active CN105527434B (en) | 2015-12-31 | 2015-12-31 | One kind is used for the kit of diacetyl spermine (DAS) detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105527434B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106771125A (en) * | 2016-11-25 | 2017-05-31 | 南方医科大学 | A kind of time resolution immunity detection reagent of real-time monitoring anthracycline chemotherapy medicine blood concentration |
| CN108614107A (en) * | 2016-12-12 | 2018-10-02 | 上海复星长征医学科学有限公司 | A kind of methotrexate (MTX) latex immunoturbidimetry assay kit and preparation method thereof |
| CN107300613A (en) * | 2017-06-27 | 2017-10-27 | 深圳市优圣康生物科技有限公司 | A kind of biomarker, the method for sampling, modeling method and application thereof |
| CN108362892B (en) * | 2018-01-18 | 2020-04-14 | 河南省科学院生物研究所有限责任公司 | Procalcitonin colloidal gold immunoturbidimetry detection reagent |
| CN108226483B (en) * | 2018-01-19 | 2020-06-26 | 辽宁迈迪生物科技股份有限公司 | DAS detection kit and preparation method and application thereof |
| CN109870579B (en) * | 2019-02-03 | 2022-02-11 | 辽宁迈迪生物科技股份有限公司 | Monoclonal antibody protection solution, preparation method and application thereof, reagent using monoclonal antibody protection solution and immunohistochemical kit |
| CN114181108B (en) * | 2021-11-17 | 2024-01-16 | 浙江工商大学 | Dendritic multiple hapten, artificial antigen and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102628868A (en) * | 2011-12-30 | 2012-08-08 | 北京九强生物技术股份有限公司 | Latex enhanced immunoturbidimetry kit for detection of asymmetric dimethylarginine content |
| CN102680712A (en) * | 2012-06-18 | 2012-09-19 | 王钊 | Competitive latex-particle-enhanced immunoturbidimetric assay kit for BNP (B-type natriuretic peptide) and preparation method thereof |
| CN104316685A (en) * | 2014-10-10 | 2015-01-28 | 辽宁迈迪生物科技有限公司 | Diacetyl spermine detection kit and preparation method and application thereof |
| CN104991077A (en) * | 2015-07-29 | 2015-10-21 | 安徽省煦棠医疗科技有限公司 | Troponin I competition turbidimetry detecting kit |
| CN105044351A (en) * | 2015-07-01 | 2015-11-11 | 南京普朗医疗设备有限公司 | Antistreptolysin O immunological turbidimetry reagent and preparation method thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1610127B1 (en) * | 2003-03-11 | 2012-04-25 | CRC Inc. | N1,n12-diacetylspermine as tumor marker |
| JP4490197B2 (en) * | 2004-07-26 | 2010-06-23 | アルフレッサファーマ株式会社 | Diacetylspermine measurement method and reagent kit |
| US8354238B2 (en) * | 2007-12-07 | 2013-01-15 | Alfresa Pharma Corporation | Method of immunoassaying specimen using aggregation reaction of microparticles and assay kit |
| CN101819202B (en) * | 2010-05-06 | 2012-12-26 | 上海太阳生物技术有限公司 | D-Dimer measuring kit (latex immunonephelometry method) |
| CN104198724B (en) * | 2014-08-14 | 2016-05-11 | 上海睿康生物科技有限公司 | Fibrin or fibrinogen degradation product (FDP) detection kit |
| CN104198725B (en) * | 2014-08-14 | 2016-04-06 | 上海睿康生物科技有限公司 | Cyclic citrullinated peptid detection kit |
-
2015
- 2015-12-31 CN CN201511020936.1A patent/CN105527434B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102628868A (en) * | 2011-12-30 | 2012-08-08 | 北京九强生物技术股份有限公司 | Latex enhanced immunoturbidimetry kit for detection of asymmetric dimethylarginine content |
| CN102680712A (en) * | 2012-06-18 | 2012-09-19 | 王钊 | Competitive latex-particle-enhanced immunoturbidimetric assay kit for BNP (B-type natriuretic peptide) and preparation method thereof |
| CN104316685A (en) * | 2014-10-10 | 2015-01-28 | 辽宁迈迪生物科技有限公司 | Diacetyl spermine detection kit and preparation method and application thereof |
| CN105044351A (en) * | 2015-07-01 | 2015-11-11 | 南京普朗医疗设备有限公司 | Antistreptolysin O immunological turbidimetry reagent and preparation method thereof |
| CN104991077A (en) * | 2015-07-29 | 2015-10-21 | 安徽省煦棠医疗科技有限公司 | Troponin I competition turbidimetry detecting kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105527434A (en) | 2016-04-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105527434B (en) | One kind is used for the kit of diacetyl spermine (DAS) detection | |
| CN103073642B (en) | CRP monoclonal antibody nanometer latex microsphere composition and preparation technology | |
| CN100445746C (en) | Method for detecting 19-nortestosterone and special enzyme-linked immune reagent kit thereof | |
| CN101983971A (en) | Preparation method of anti-fluoroquinolone rabbit monoclonal antibody and application thereof | |
| CN104762267B (en) | Hybridoma AFB1 2A4 and its caused aflatoxin B1 monoclonal antibody | |
| CN107422112B (en) | It is a kind of to detect the immune reagent kit of ethopabate, preparation method and application | |
| CN107192821A (en) | A kind of modified latex immunoturbidimetry for improving helicobacter pylori pathogenicity bacterial strain Antibody positive rate determines kit | |
| CN109387627A (en) | A kind of Reagent Protocol of screening for cancer and early diagnosis based on placenta sample chondroitin sulfate A (CSA) | |
| CN100488983C (en) | Technology for preparing colorless malachite green complete antigen and monoclonal antibody | |
| CN107677807A (en) | A kind of kitasamycin magnetic immunochemiluminescence detection kit | |
| CN102336831A (en) | Anti-sildenafil specific antibody | |
| CN103389373B (en) | A kind of test paper and application thereof detecting sulfa drugs | |
| CN106443025B (en) | A kind of immune-gold labeled card of universal detection FQNS and preparation method thereof | |
| CN105628923B (en) | A kind of sxemiquantitative immunity colloidal gold test paper strip and its application | |
| CN104388392A (en) | Enrofloxacin monoclonal antibody as well as preparation method and application thereof | |
| CN111500546A (en) | Cell strain secreting four subtype antibodies against aflatoxin, antibody secreted by cell strain and immunochromatography detection card | |
| CN114752568B (en) | Furosemide monoclonal antibody, hybridoma cell strain and application | |
| CN106381289A (en) | Monoclonal antibody of resisting 25-hydroxyvitamin D3 as well as preparation cell lines and method of monoclonal antibody | |
| CN110713986A (en) | Vitamin B1Monoclonal antibody hybridoma cell strain CBDD and application thereof | |
| CN104789535B (en) | Anti- methadone metabolin EDDP hybridoma cell strains and its preparation method and application | |
| CN107058240A (en) | One strain of hybridoma strain AB1 and its 2,4,5 trichlorophenoxyacetic acid monoclonal antibodies of generation | |
| CN103698519A (en) | Chemiluminescence detection kit for furaltadone metabolite and applications of the kit | |
| CN113151188A (en) | Hybridoma cell strain capable of secreting diphenhydramine monoclonal antibody and application thereof | |
| CN101275947A (en) | Sulfalene oxazole monoclonal antibody reagent kit and detecting method | |
| CN106771216B (en) | Improve the method and its application of immunoreagent detection specificity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: 117004 Liaoning Province, Benxi City Economic Development Zone Cladrastis road 166 Patentee after: Liaoning Maidi biological Polytron Technologies Inc Address before: 117004 Liaoning city of Benxi province Cladrastis Economic Development Zone Benxi Road, building 166 Patentee before: Liaoning Maidi Biologic Technology Co., Ltd. |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A kit for detection of diacetyl spermine (DAS) Effective date of registration: 20190505 Granted publication date: 20171219 Pledgee: Chaoyang Bank Co., Ltd. Benxi Branch Pledgor: Liaoning Maidi biological Polytron Technologies Inc Registration number: 2019210000013 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20200513 Granted publication date: 20171219 Pledgee: Chaoyang Bank Co., Ltd. Benxi Branch Pledgor: LIAONING MEDI BIOLOGICAL TECHNOLOGY Co.,Ltd. Registration number: 2019210000013 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A kit for the detection of diacetyl spermine (DAS) Effective date of registration: 20200514 Granted publication date: 20171219 Pledgee: Chaoyang Bank Co., Ltd. Benxi Branch Pledgor: LIAONING MEDI BIOLOGICAL TECHNOLOGY Co.,Ltd. Registration number: Y2020210000020 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20210601 Granted publication date: 20171219 Pledgee: Chaoyang Bank Co.,Ltd. Benxi Branch Pledgor: LIAONING MEDI BIOLOGICAL TECHNOLOGY Co.,Ltd. Registration number: Y2020210000020 |