CN109438424A - Ribavirin haptens and artificial antigen and the preparation method and application thereof - Google Patents
Ribavirin haptens and artificial antigen and the preparation method and application thereof Download PDFInfo
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- CN109438424A CN109438424A CN201811367056.5A CN201811367056A CN109438424A CN 109438424 A CN109438424 A CN 109438424A CN 201811367056 A CN201811367056 A CN 201811367056A CN 109438424 A CN109438424 A CN 109438424A
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- Prior art keywords
- ribavirin
- haptens
- formula
- preparation
- reaction
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- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 title claims abstract description 111
- 229960000329 ribavirin Drugs 0.000 title claims abstract description 97
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 title claims abstract description 93
- 239000000427 antigen Substances 0.000 title claims abstract description 35
- 102000036639 antigens Human genes 0.000 title claims abstract description 35
- 108091007433 antigens Proteins 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 238000001514 detection method Methods 0.000 claims abstract description 17
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 14
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 14
- 238000006243 chemical reaction Methods 0.000 claims description 32
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 150000001875 compounds Chemical class 0.000 claims description 18
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 16
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 16
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 12
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 12
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 12
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 10
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 6
- 238000010168 coupling process Methods 0.000 claims description 6
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims description 6
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 6
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 6
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 6
- DYHSDKLCOJIUFX-UHFFFAOYSA-N tert-butoxycarbonyl anhydride Chemical compound CC(C)(C)OC(=O)OC(=O)OC(C)(C)C DYHSDKLCOJIUFX-UHFFFAOYSA-N 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 230000008878 coupling Effects 0.000 claims description 5
- 238000005859 coupling reaction Methods 0.000 claims description 5
- 239000012043 crude product Substances 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 239000012265 solid product Substances 0.000 claims description 5
- 230000005526 G1 to G0 transition Effects 0.000 claims description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- HEWZVZIVELJPQZ-UHFFFAOYSA-N 2,2-dimethoxypropane Chemical compound COC(C)(C)OC HEWZVZIVELJPQZ-UHFFFAOYSA-N 0.000 claims description 3
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims description 3
- BBYDXOIZLAWGSL-UHFFFAOYSA-N 4-fluorobenzoic acid Chemical compound OC(=O)C1=CC=C(F)C=C1 BBYDXOIZLAWGSL-UHFFFAOYSA-N 0.000 claims description 3
- DXRFZHILMCWCNG-UHFFFAOYSA-N N,N-dimethyl-1,8-naphthyridin-2-amine Chemical compound C1=CC=NC2=NC(N(C)C)=CC=C21 DXRFZHILMCWCNG-UHFFFAOYSA-N 0.000 claims description 3
- FJDQFPXHSGXQBY-UHFFFAOYSA-L caesium carbonate Chemical compound [Cs+].[Cs+].[O-]C([O-])=O FJDQFPXHSGXQBY-UHFFFAOYSA-L 0.000 claims description 3
- 229910000024 caesium carbonate Inorganic materials 0.000 claims description 3
- 239000012230 colorless oil Substances 0.000 claims description 3
- 238000002425 crystallisation Methods 0.000 claims description 3
- 230000008025 crystallization Effects 0.000 claims description 3
- 150000002148 esters Chemical class 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 239000002516 radical scavenger Substances 0.000 claims description 3
- 229940014800 succinic anhydride Drugs 0.000 claims description 3
- -1 thyroprotein Proteins 0.000 claims description 3
- 238000005292 vacuum distillation Methods 0.000 claims description 3
- LBLYYCQCTBFVLH-UHFFFAOYSA-N 2-Methylbenzenesulfonic acid Chemical compound CC1=CC=CC=C1S(O)(=O)=O LBLYYCQCTBFVLH-UHFFFAOYSA-N 0.000 claims description 2
- 108010058846 Ovalbumin Proteins 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 229940092253 ovalbumin Drugs 0.000 claims description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 2
- 235000011152 sodium sulphate Nutrition 0.000 claims description 2
- 108091006905 Human Serum Albumin Proteins 0.000 claims 1
- 102000008100 Human Serum Albumin Human genes 0.000 claims 1
- 241001465754 Metazoa Species 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 15
- 239000011248 coating agent Substances 0.000 description 12
- 238000000576 coating method Methods 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 4
- 244000144977 poultry Species 0.000 description 4
- 235000013594 poultry meat Nutrition 0.000 description 4
- 238000012797 qualification Methods 0.000 description 4
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 244000144972 livestock Species 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003443 antiviral agent Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 2
- 238000011587 new zealand white rabbit Methods 0.000 description 2
- 230000005311 nuclear magnetism Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- ZEWJFUNFEABPGL-UHFFFAOYSA-N 1,2,4-triazole-3-carboxamide Chemical class NC(=O)C=1N=CNN=1 ZEWJFUNFEABPGL-UHFFFAOYSA-N 0.000 description 1
- 241001269238 Data Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- KJHOZAZQWVKILO-UHFFFAOYSA-N N-(diaminomethylidene)-4-morpholinecarboximidamide Chemical compound NC(N)=NC(=N)N1CCOCC1 KJHOZAZQWVKILO-UHFFFAOYSA-N 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- DKNWSYNQZKUICI-UHFFFAOYSA-N amantadine Chemical compound C1C(C2)CC3CC2CC1(N)C3 DKNWSYNQZKUICI-UHFFFAOYSA-N 0.000 description 1
- 229960003805 amantadine Drugs 0.000 description 1
- 238000003975 animal breeding Methods 0.000 description 1
- 230000002155 anti-virotic effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 208000037798 influenza B Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229960005389 moroxydine Drugs 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 235000013613 poultry product Nutrition 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000002212 purine nucleoside Substances 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
- 229940100050 virazole Drugs 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Gastroenterology & Hepatology (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to Ribavirin haptens and artificial antigens and the preparation method and application thereof.Shown in the structure such as formula (I) or formula (II) of the Ribavirin haptens:
Description
Technical field
The invention belongs to technical field of biochemical industry, specifically, be related to Ribavirin haptens and artificial antigen and its
Preparation method and application.
Background technique
Ribavirin (Ribavirin, RBV), also known as virazole, ribavirin are a kind of purine nucleosides of synthesis
Like object.It is researched and developed within 1972, is thrown in Mexico's Initial Public Offering, China's official approval in 1980 by ICN company, the U.S. within 1974
It produces.Ribavirin is a kind of broad-spectrum antiviral drug, has inhibiting effect, A type, influenza B to a variety of RNA virus and DNA virus
Virus is most sensitive, and mechanism of action is that it is metabolized as Ribavirin group (1,2,4- triazole -3- carboxylic acid amides) in vivo, which can
To inhibit varial polymerases, the synthesis of viral interference RNA, thus the duplication of blocking virus.In addition, Ribavirin can also induce
T cell Phenotypic Change, to enhance the humoral immune reaction of T cell mediation.
Ribavirin is playing extensive antivirus action simultaneously, and toxic side effect is also of increasing concern, for a long time,
In order to prevent livestock and poultry influenza, Ribavirin is also widely used for during livestock and poultry cultivation.In order to guarantee mankind's drug safety and
Animal epidemic prevention safety, the Ministry of Agriculture in 2005 issues No. 560 bulletin, forbids Ribavirin, amantadine, moroxydine etc. antiviral
Drug uses in animal-breeding.However Ribavirin is still generally existing the livestock and poultry cultivation process Misuse the phenomenon that, poultry
Poultry product quality safety is by certain threat.
Currently, the detection means of Ribavirin is mainly liquid chromatogram (LC), LC-MS (LC-MS/MS) method, but by
It is expensive in large-scale instrument, complicated for operation, portability is poor, it is difficult to realize the detection of production line, seriously constrain for benefit
Effective supervision that Ba Weilin is used.Based on the immuno analytical method of Ag-Ab specific reaction, have detection speed fast, at
This low advantage, therefore immune hapten molecule is designed, it is aobvious to develop a kind of simple, quick, for detecting Ribavirin antibody
It obtains particularly important.
Summary of the invention
The object of the present invention is to provide Ribavirin haptens and artificial antigens and the preparation method and application thereof.
In order to achieve the object of the present invention, in a first aspect, the present invention provide Ribavirin haptens, structure such as formula (I) or
Shown in formula (II):
Second aspect, the present invention provide the preparation method of the haptens, when the Ribavirin haptens is formula (I) institute
When showing compound, preparation method includes the following steps:
1) 48.0g Ribavirin, 6.17g p-methyl benzenesulfonic acid and 39.2mL 2,2- dimethoxy propane are dissolved in 500mL third
In ketone, mixed liquor is in 60 DEG C of reaction 3h；The sodium bicarbonate that 1mL 5% is added terminates reaction；After scavenger precipitation, collects and concentration is filtered
Liquid obtains formula (III) compound after column chromatographic purifying；
2) 35.0g parafluorobenzoic acid, 15.0g dimethylamino naphthyridine are dissolved in the 500mL tert-butyl alcohol and are cooled to 0 DEG C；To mixed
It closes and adds 107.50g di-tert-butyl dicarbonate in liquid, 16h is then stirred at room temperature, obtains colorless oil as product, this is colourless
Oil product and 15.0g formula (III) compound and 60.0g cesium carbonate are dissolved in 300mL dimethyl sulfoxide, 110 DEG C of continuous heatings
16h；Dimethyl sulfoxide is removed by vacuum distillation；200mL water is added into residue, makes to be extracted with dichloromethane, and use sulphur
It is concentrated after sour sodium is dry, residue is purified by column chromatography, and obtained 6.0g white solid product is molten at room temperature
Solution stirs 16h in the mixed liquor 100mL of hydrochloric acid and tetrahydrofuran, with sodium bicarbonate tune pH to 7-9, obtains after filtering mixture
To crude product；Crude product further with methanol crystallization to get.
Wherein, step 2) progress column chromatographs mobile phase used and is mixed by methanol and methylene chloride by 50:1 volume ratio,
Stationary phase used is the silica gel of 200-300 mesh.
The mixed liquor of the hydrochloric acid and tetrahydrofuran is by the hydrochloric acid of tetrahydrofuran and concentration 12mol/L by 9:1 volume ratio
It mixes.
When the Ribavirin haptens is compound shown in formula (II), the preparation method is as follows:
1.0g Ribavirin is put into bis- neck bottle of 100mL, acetone 20mL is added, 10min is stirred at room temperature, puts into 50mg pairs
Toluenesulfonic acid, 60 DEG C of reaction 4h, after reaction system is spin-dried for, residue is chromatographed with column and is purified, and obtains white solid production
Object is added in microwave tube, and 3mL pyridine is added, and 10min is stirred at room temperature, and puts into succinic anhydride 426mg, 120 DEG C of reaction 20min, will
The product that reaction system is spin-dried for obtaining after rear pillar chromatographic purifying is transferred in 100mL flask, and 15mL pyridine is added, is stirred at room temperature
10min, put into 5mL trifluoroacetic acid, 50 DEG C of reaction 12h, by reaction system be spin-dried for get.
Wherein, it carries out column and chromatographs mobile phase used to be to be mixed by methanol and methylene chloride by 1:20 volume ratio, it is used
Stationary phase is the silica gel of 200-300 mesh.
The third aspect, the present invention provide Ribavirin artificial antigen, are by the Ribavirin haptens and carrier protein
It is obtained after coupling.The Ribavirin artificial antigen can be used as immunogene and can also be used as coating antigen.
Wherein, the carrier protein is selected from bovine serum albumin(BSA), ovalbumin, keyhole limpet hemocyanin, thyroprotein, people
Seralbumin；It is preferred that bovine serum albumin(BSA), keyhole limpet hemocyanin.
Fourth aspect, the present invention provide the preparation method of the artificial antigen, using active ester method by carrier protein couplet
In the carboxyl carbon of the haptens described in claim 1.
Preferably, the coupling Mole Ratio of compound shown in formula (I) and carrier protein is 9.6:1.Compound shown in formula (II)
Coupling Mole Ratio with carrier protein is 7.0:1.
5th aspect, the present invention provide the specific antibody prepared by the Ribavirin artificial antigen, including polyclonal
Antibody and monoclonal antibody, preferably polyclonal antibody.The polyclonal antibody can pass through Ribavirin artificial antigen immunization experiment
Animal (such as new zealand white rabbit) collects serum purifying and obtains.
6th aspect, the present invention provide the following any of the Ribavirin haptens or the Ribavirin artificial antigen
Using:
1. preparing the application in anti-Ribavirin specific antibody；
2. detecting the application in anti-Ribavirin specific antibody.
7th aspect, the present invention provide the Ribavirin detection reagent prepared by the specific antibody or kit.
Eighth aspect, the present invention provide following any application of the specific antibody:
(1) application in detection Ribavirin；
(2) application in the immuno-chromatographic test paper strip for preparing Ribavirin；
(3) application in the colloidal gold colloidal gold detection test paper strip for preparing Ribavirin.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
Present invention firstly discloses two kinds of new Ribavirin haptens, artificial antigens and preparation method thereof, with the benefit
Animal is immunized in Ba Weilin artificial antigen, and potency height, the specific antibody of high sensitivity can be obtained.Ribavirin provided by the invention
Haptens and its antibody of preparation, the Ribavirin detection method to establish quick, easy, inexpensive, sensitive, special provide newly
Means.
Ribavirin antibody (how anti-) is prepared using the conjugate of haptens provided by the invention and carrier protein, was prepared
Journey is simple, economical, and the detection sensitivity of antibody is high up to 0.31ng/mL, practical value.The present invention has in detection of veterinary drugs in food
There is good application prospect.
Detailed description of the invention
Fig. 1 is the flow chart of the preparation of Ribavirin haptens shown in formula (I) in the embodiment of the present invention 1.
Fig. 2 is the flow chart of the preparation of Ribavirin haptens shown in formula (II) in the embodiment of the present invention 1.
Fig. 3 is the mass spectrogram of Ribavirin haptens shown in formula (I) in the embodiment of the present invention 1.
Fig. 4 is the mass spectrogram of Ribavirin haptens shown in formula (II) in the embodiment of the present invention 1.
Fig. 5 is the nuclear magnetic spectrogram of Ribavirin haptens shown in formula (I) in the embodiment of the present invention 1.
Fig. 6 is the nuclear magnetic spectrogram of Ribavirin haptens shown in formula (II) in the embodiment of the present invention 1.
Fig. 7 is the artificial antigen structure chart of the preparation of Ribavirin haptens shown in formula (I) in the embodiment of the present invention 2.
Fig. 8 is the artificial antigen structure chart of the preparation of Ribavirin haptens shown in formula (II) in the embodiment of the present invention 2.
Fig. 9 be the embodiment of the present invention 2 in RBV 1.-BSA MALDI-TOF-MS figure.
Figure 10 be the embodiment of the present invention 2 in RBV 2.-BSA MALDI-TOF-MS figure.
Figure 11 is the canonical plotting for detecting Ribavirin in the embodiment of the present invention 4 using polyclonal antibody.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.Used in embodiment
PBS buffer solution is the PBS buffer solution of pH7.4,0.01M.Carbonate buffer solution used in embodiment be pH9.6,
The sodium carbonate buffer of 0.05mol/L.
NHS is the abbreviation of n-hydroxysuccinimide.EDC is 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide salt
The abbreviation of hydrochlorate.DMF is the abbreviation of N,N-dimethylformamide.NHS, EDC, bovine serum albumin(BSA) (Albuminfrom
Bovine serum, BSA), keyhole limpet hemocyanin (Keyhole Limpet Hemocyanin, KLH) and Freund help completely
Agent, incomplete Freund's adjuvant are purchased from Sigma company.Column chromatographs the silica gel that stationary phase used is 200-300 mesh.
The preparation and characterization of 1 Ribavirin haptens of embodiment
One, the preparation of Ribavirin haptens
1, the preparation of Ribavirin haptens shown in formula (I)
48.0g Ribavirin, 6.17g p-methyl benzenesulfonic acid and 39.2mL 2,2- dimethoxy propane are dissolved in 500mL acetone
In, mixed liquor is in 60 DEG C of reaction 3h.The sodium bicarbonate that 1mL 5% is added terminates reaction.After scavenger precipitation, collects and concentration is filtered
Liquid.Intermediate product shown in formula (III) is obtained after column chromatographic purifying.
35.0g parafluorobenzoic acid, 15.0g dimethylamino naphthyridine are dissolved in the 500mL tert-butyl alcohol and are cooled to 0 DEG C.It is mixing
107.50g di-tert-butyl dicarbonate is added in liquid, and 16h is then stirred at room temperature.It is purified, is obtained a kind of colourless with column chromatography
Intermediate product shown in oil product and 15.0g formula (III) and 60.0g cesium carbonate are dissolved in 300mL dimethyl sulfoxide by oil product
In, 110 DEG C of continuous heating 16h.Dimethyl sulfoxide is removed by vacuum distillation.200mL water is added in residue, uses dichloro
Methane extraction, and with being concentrated after sodium sulphate drying, residue is the column of ethanol/methylene (volume ratio 50:1) by mobile phase
Chromatography purified, will obtain 6.0g white solid product at room temperature with hydrochloric acid/tetrahydrofuran (12mol/L concentrated hydrochloric acid
10ml and tetrahydrofuran 90ml) mixed liquor stir 16h.With sodium bicarbonate tune pH=9, crude product is obtained after filtering mixture.
Crude product further with methanol crystallization 3 times, obtain the pure products of 2.6g white solid, as Ribavirin shown in formula (I)
Haptens, yield 55%.Specific synthetic route is shown in Fig. 1.
2, the preparation of Ribavirin haptens shown in formula (II)
It weighs 1.0g Ribavirin to be put into bis- neck bottle of 100ml, acetone 20mL is added, 10min is stirred at room temperature, put into 50mg
P-methyl benzenesulfonic acid, 60 DEG C of reaction 4h, after reaction system is spin-dried for, residue is ethanol/methylene (volume ratio by mobile phase
Column chromatography 1:20) is purified, and is obtained white solid product and is added in microwave tube, 3mL pyridine is added, is stirred at room temperature
10min, puts into succinic anhydride 426mg, and reaction system is spin-dried for the product obtained after rear pillar chromatographic purifying by 120 DEG C of reaction 20min
It is added in 100mL flask, 15mL pyridine is added, 10min is stirred at room temperature, put into 5mL trifluoroacetic acid, 50 DEG C of reaction 12h will react
System is spin-dried for obtaining target product being Ribavirin haptens shown in formula (II).Specific synthetic route is shown in Fig. 2.
Two, the characterization of Ribavirin haptens
1, Mass Spectrometric Identification
Ribavirin haptens (molecular formula C shown in the formula (I) of step 115H16N4O7) Mass Spectrometric Identification result: MS m/
z[M+H]+Theoretical value: 364.3；Measured value: 365.0, it matches with the molecular weight of target product, mass spectrum is shown in Fig. 3.
Ribavirin haptens (molecular formula C shown in the formula (II) of step 112H16N4O8) Mass Spectrometric Identification result: MS
m/z[M+H]+Theoretical value: 344.3；Measured value: 343.1, it matches with the molecular weight of target product, mass spectrum is shown in Fig. 4.
2, nuclear magnetic resonance is identified
The nuclear-magnetism qualification result of Ribavirin haptens shown in the formula (I) of step 1:1H NMR(400MHz,CDCl3),
1.35(s,3H),1.52(s,9H),1.53(s,3H).4.16-4.27(m,2H),4.60-4.63(m,1H),5.08-5.10(m,
1H), 5.30 (d, J=2H, 1H), 6.33 (s, 1H), 6.91 (d, J=7.2Hz, 2H), 7.70 (s, br, 1H), 7.79 (d, J=
8.8Hz, 2H), 8.01 (s, br, 1H), 8.79 (s, 1H) nuclear magnetic datas show that the compound of above method synthesis is that target produces
Object, magnetic resonance qualification result are shown in Fig. 5.
The nuclear-magnetism qualification result of Ribavirin haptens shown in the formula (II) of step 1:1H NMR(400MHz,CD3OD), δ
8.68(s,1H),5.93(s,4H),4.55-4.54(m,1H),4.46-4.39(m,2H),4.26-4.25(m,2H),2.59
(brs, 4H) nuclear magnetic data shows that the compound of above method synthesis is target product, and magnetic resonance qualification result is shown in Fig. 6.
The preparation and characterization of 2 Ribavirin artificial antigen of embodiment
In the immunogene and the preparation method of coating antigen, difference is the usage type of carrier protein, described immune
Body protein published originally mainly uses KLH, and the coating antigen carrier protein mainly uses BSA, and coupling method used is active ester method.By
The artificial antigen structure of the preparation of Ribavirin haptens shown in formula (I) and formula (II) is shown in Fig. 7, Fig. 8.
One, the synthesis and identification of Ribavirin coating antigen
1, the preparation of Ribavirin coating antigen
(1) compound shown in (I) that 20mg embodiment 1 is prepared is dissolved in 2mL DMF, be added 10mg NHS and
10mg DCC, is stirred overnight at room temperature, obtains solution I.
(2) 7mg BSA is added in 7mL PBS buffer solution, is sufficiently dissolved, as solution II.
(3) solution I is slowly added dropwise into solution II, slow 4 DEG C of stirrings are packed into bag filter afterwards for 24 hours, 4 in physiological saline
DEG C dialysis 72h (water is changed 6 times in centre), obtain Ribavirin coating original solution, -20 DEG C preservation.Compound synthesis shown in formula (I)
Ribavirin coating antigen abbreviation RBV 1.-BSA.
(4) using the Ribavirin coating antigen of compound synthesis shown in the same method preparation formula (II) of above-mentioned three step, letter
Claim RBV 2.-BSA.
2, the identification of Ribavirin coating antigen
With Matrix-assisted laser desorption ionization (Matrix-Assisted Laser Desorption/
Ionization Time of Flight Mass Spectrometry, MALDI-TOF-MS) method measures RBV 1.-BSA respectively
Solution and RBV 2. in-BSA solution BSA and haptens combination ratio.As a result see Fig. 9, Figure 10.
In conjunction with than={ M (conjugate)-M (protein) }/M (haptens)
The molecular weight of BSA is 65700.3, and the molecular weight of formula (I) haptens is 364.3, is analyzed by mass spectrum peak-peak even
The molecular weight for joining object is 69212.9, is computed and show that the combination ratio of BSA and haptens are 9.6, i.e. a RBV 1. BSA in-BSA
9.6 haptens are averagely coupled on molecule.
The molecular weight of formula (II) haptens is 344.3, and the molecular weight by mass spectrum peak-peak analysis conjugate is
68119.3, it is computed and show that the combination ratio of BSA and haptens are 7.0, is i.e. 2. RBV is averagely coupled in-BSA on a BSA molecule
7.0 haptens.
Two, the synthesis of Ribavirin immunogene
1, the preparation of Ribavirin immunogene
BSA is replaced with KLH, the 1 of other same step 2.
Compound synthesis Ribavirin immunogene shown in formula (I) and formula (II) distinguishes abbreviation RBV 1.-KLH, RBV 2.-KLH.
The sero-fast preparation of 3 Ribavirin of embodiment
2. 2 groups of 3-4 monthly ages, weight 1.5- is immunized in-KLH solution respectively by RBV prepared by embodiment 2 1.-KLH, RBV
The Female New Zealand White Rabbit of 2.0kg, every group 2.By each immunogene normal saline dilution to 1mg/mL, helped with equivalent Freund
Agent emulsification.Head exempts from using Freund's complete adjuvant, the intradermal multi-point injection of the nape of the neck, and immunizing dose is 1mg/.Reinforced after 4 weeks
It is immune, every 4 weeks plus exempt from 1 time, altogether plus exempts from 3 times, adjuvant is changed to incomplete Freund's adjuvant, and immunizing dose is constant, is changed to the nape of the neck
Subcutaneous multi-point injection.After the 4th is 1 week immune, largely taken a blood sample with the method for Culling heart blood.After taking blood, by 37 DEG C of standing 2h of blood,
Then it stands overnight for 4 DEG C, then 3000rpm is centrifuged 20min, collects supernatant, as antiserum, and -20 DEG C of packing save.
The sero-fast measurement of 4 Ribavirin of embodiment
One, antiserum titre is detected using indirect ELISA method, specific steps are as follows:
1) it is coated with: the antigen in embodiment 2 is carried out again with 0.05M, pH9.6 carbonate buffer solution since 10 μ g/mL
Than dilution, 100 holes μ L/, 37 DEG C of reaction 2h.
2) it washs: solution in plate is inclined, dry, and washed 3 times with cleaning solution, each 3min.
3) it closes: after patting dry, 200 hole μ L/ confining liquids, 37 DEG C of reaction 2h is added.It is dried for standby after washing.
4) it is loaded: antiserum being subjected to doubling dilution since 1:1000, and is added in the coating hole of various dilutions,
100 holes μ L/, 37 DEG C of reaction 1h；Sufficiently after washing, the diluted HRP- goat anti-rabbit igg of 1:3000 is added, 100 holes μ L/, 37 DEG C anti-
Answer 1h.
5) it develops the color: ELISA Plate is taken out, sufficiently after washing, the TMB developing solution of 100 μ L is added in every hole, and 37 DEG C are protected from light
15min。
6) terminate and measure: 100 μ L terminate liquids are added to terminate reaction in every hole, and the OD in each hole is then measured with microplate reader450
Value.
7) result interpretation: with OD450Serum highest corresponding to 2.1 times of value >=negative control hole (i.e. P/N >=2.1) is dilute
Release the ELISA potency that multiple is serum.
Two, minimum detection limit, half inhibition and the detection of specificity
Specific steps are as follows:
1) above-mentioned indirect ELISA method is used to determine that 1.-BSA is as coating antigen using RBV, with RBV 1.-KLH immune rabbit
The serum of acquisition is as antibody, with OD450Being worth the antigen and antibody concentration corresponding at 1.5 or so is most suitable working concentration.
2) it is coated with: coating antigen being used and is coated with 20000 times of buffer dilution, 100 holes μ L/, 37 DEG C of reaction 2h.
3) wash and close: method is operated with above-mentioned indirect elisa method.
4) match ribavirin standard solution: the PBS solution of ribavirin standard product 0.01mol/L, pH7.4 are configured to
The mother liquor of 5mg/mL, then, before sample-adding, then with 0.01mol/L, the PBS solution doubling dilution of pH7.4 is at needing concentration (RBV
Concentration be respectively 0.005ng/mL, 0.05ng/mL, 0.25ng/mL, 0.5ng/mL, 5ng/mL, 50ng/mL).
5) be loaded: each concentration standards of Ribavirin of 50 μ L doubling dilutions are added in every hole, then add 50 holes μ L/ most
The antiserum of suitable extension rate, 37 DEG C of reaction 1h.Sufficiently after washing, the diluted HRP- goat anti-rabbit igg of 1:3000,100 μ L/ are added
Hole, 37 DEG C of reaction 1h.
6) chromogenic reaction: ELISA Plate is taken out, and sufficiently after washing, the TMB developing solution of 100 μ L is added in every hole, and 37 DEG C are protected from light
React 15min.
7) terminate and measure: 100 μ L terminate liquids are added to terminate reaction in every hole, and the OD in each hole is then measured with microplate reader450
Value.
8) data processing: using the logarithm of each concentration of Ribavirin as abscissa, with the corresponding OD value of each concentration of Ribavirin
Standard curve is drawn according to four parameter logistic fits and passes through meter as shown in figure 11 using 8.5 software of Origin for ordinate
Calculate IC50Value (half-inhibitory concentration) determines whether antiserum has specificity to Ribavirin.
The results show that for rabbit antiserum titre up to 50000, detection is limited to 0.02ng/mL, IC after four exempt from50Value is
0.31ng/mL, linear detection range 0.06-1.67ng/mL.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. Ribavirin haptens, which is characterized in that shown in its structure such as formula (I) or formula (II):
2. the preparation method of haptens described in claim 1, which is characterized in that when the Ribavirin haptens is formula (I) institute
When showing compound, preparation method includes the following steps:
1) 48.0g Ribavirin, 6.17g p-methyl benzenesulfonic acid and 39.2mL 2,2- dimethoxy propane are dissolved in 500mL acetone
In, mixed liquor is in 60 DEG C of reaction 3h；The sodium bicarbonate that 1mL 5% is added terminates reaction；After scavenger precipitation, collects and concentration is filtered
Liquid obtains formula (III) compound after column chromatographic purifying；
2) 35.0g parafluorobenzoic acid, 15.0g dimethylamino naphthyridine are dissolved in the 500mL tert-butyl alcohol and are cooled to 0 DEG C；To mixed liquor
Middle addition 107.50g di-tert-butyl dicarbonate, is then stirred at room temperature 16h, obtains colorless oil as product, by the colorless oil
Product and 15.0g formula (III) compound and 60.0g cesium carbonate are dissolved in 300mL dimethyl sulfoxide, 110 DEG C of continuous heating 16h；
Dimethyl sulfoxide is removed by vacuum distillation；200mL water is added into residue, makes to be extracted with dichloromethane, and use sodium sulphate
It is concentrated after drying, residue is purified by column chromatography, and obtained 6.0g white solid product is dissolved at room temperature
In the mixed liquor 100mL of hydrochloric acid and tetrahydrofuran, 16h is stirred, with sodium bicarbonate tune pH to 7-9, is obtained slightly after filtering mixture
Product；Crude product further with methanol crystallization to get；
Wherein, step 2) progress column chromatographs mobile phase used and is mixed by methanol and methylene chloride by 50:1 volume ratio, used
Stationary phase is the silica gel of 200-300 mesh；The mixed liquor of the hydrochloric acid and tetrahydrofuran is by tetrahydrofuran and concentration 12mol/L
Hydrochloric acid is mixed by 9:1 volume ratio.
3. the preparation method of haptens described in claim 1, which is characterized in that when the Ribavirin haptens is formula (II) institute
When showing compound, the preparation method is as follows:
1.0g Ribavirin is put into bis- neck bottle of 100mL, acetone 20mL is added, 10min is stirred at room temperature, puts into 50mg to toluene
Sulfonic acid, 60 DEG C of reaction 4h, after reaction system is spin-dried for, residue is chromatographed with column and is purified, and is obtained white solid product and is added
Enter in microwave tube, 3mL pyridine is added, 10min is stirred at room temperature, put into succinic anhydride 426mg, 120 DEG C of reaction 20min will react
The product that system is spin-dried for obtaining after rear pillar chromatographic purifying is transferred in 100mL flask, and 15mL pyridine is added, 10min is stirred at room temperature,
Put into 5mL trifluoroacetic acid, 50 DEG C of reaction 12h, by reaction system be spin-dried for get；
Wherein, it carries out column and chromatographs mobile phase used to be to be mixed by methanol and methylene chloride by 1:20 volume ratio, fixation used
It is mutually the silica gel of 200-300 mesh.
4. Ribavirin artificial antigen, which is characterized in that Ribavirin haptens and carrier protein couplet as described in claim 1
After obtain；
Wherein, the carrier protein is selected from bovine serum albumin(BSA), ovalbumin, keyhole limpet hemocyanin, thyroprotein, human serum
Albumin；It is preferred that bovine serum albumin(BSA), keyhole limpet hemocyanin.
5. the preparation method of artificial antigen described in claim 4, which is characterized in that using active ester method by carrier protein couplet in
In the carboxyl carbon of haptens described in claim 1.
6. method described in claim 5, which is characterized in that the coupling Mole Ratio of compound shown in formula (I) and carrier protein is
9.6:1；The coupling Mole Ratio of compound shown in formula (II) and carrier protein is 7.0:1.
7. following any application of artificial antigen described in haptens or claim 4 described in claim 1:
1. preparing the application in anti-Ribavirin specific antibody；
2. detecting the application in anti-Ribavirin specific antibody.
8. the specific antibody of the preparation of the artificial antigen as described in claim 4, including polyclonal antibody and monoclonal antibody, preferably
Polyclonal antibody.
9. the Ribavirin detection reagent or kit of the preparation of the specific antibody as described in claim 8.
10. following any application of specific antibody described in claim 8:
(1) application in detection Ribavirin；
(2) application in the immuno-chromatographic test paper strip for preparing Ribavirin；
(3) application in the colloidal gold colloidal gold detection test paper strip for preparing Ribavirin.
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| CN112462071A (en) * | 2020-11-13 | 2021-03-09 | 北京元恩生物技术有限公司 | Special enzyme-linked immunoassay kit for ribavirin |
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