CN1544469A - Anticoagulation raticide immunogen synthesis method - Google Patents
Anticoagulation raticide immunogen synthesis method Download PDFInfo
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- CN1544469A CN1544469A CNA2003101063866A CN200310106386A CN1544469A CN 1544469 A CN1544469 A CN 1544469A CN A2003101063866 A CNA2003101063866 A CN A2003101063866A CN 200310106386 A CN200310106386 A CN 200310106386A CN 1544469 A CN1544469 A CN 1544469A
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- succinate
- coumarin
- rodenticide
- sodium hydroxide
- synthetic method
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- 230000002163 immunogen Effects 0.000 title claims abstract description 22
- 230000010100 anticoagulation Effects 0.000 title claims description 24
- 238000001308 synthesis method Methods 0.000 title 1
- 238000004108 freeze drying Methods 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 10
- 238000010168 coupling process Methods 0.000 claims abstract description 7
- 230000008878 coupling Effects 0.000 claims abstract description 5
- 238000005859 coupling reaction Methods 0.000 claims abstract description 5
- 150000008064 anhydrides Chemical class 0.000 claims abstract description 4
- 238000005886 esterification reaction Methods 0.000 claims abstract description 4
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 claims abstract description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 45
- 239000000126 substance Substances 0.000 claims description 31
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 29
- 229940098773 bovine serum albumin Drugs 0.000 claims description 26
- 239000003128 rodenticide Substances 0.000 claims description 23
- VXIXUWQIVKSKSA-UHFFFAOYSA-N benzotetronic acid Natural products C1=CC=CC2=C1OC(=O)C=C2O VXIXUWQIVKSKSA-UHFFFAOYSA-N 0.000 claims description 18
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 14
- 238000010189 synthetic method Methods 0.000 claims description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 12
- MJKVTPMWOKAVMS-UHFFFAOYSA-N 3-hydroxy-1-benzopyran-2-one Chemical class C1=CC=C2OC(=O)C(O)=CC2=C1 MJKVTPMWOKAVMS-UHFFFAOYSA-N 0.000 claims description 11
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- IMFACGCPASFAPR-UHFFFAOYSA-N tributylamine Chemical compound CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 claims description 10
- PQLFROTZSIMBKR-UHFFFAOYSA-N ethenyl carbonochloridate Chemical compound ClC(=O)OC=C PQLFROTZSIMBKR-UHFFFAOYSA-N 0.000 claims description 9
- 239000012154 double-distilled water Substances 0.000 claims description 8
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 7
- 239000013256 coordination polymer Substances 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- WLVHQRFJNIDAQD-UHFFFAOYSA-N C(CCC(=O)O)(=O)O.C12(C(=O)CC(CC1)C2(C)C)C Chemical compound C(CCC(=O)O)(=O)O.C12(C(=O)CC(CC1)C2(C)C)C WLVHQRFJNIDAQD-UHFFFAOYSA-N 0.000 claims description 4
- 241000522215 Dipteryx odorata Species 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- RINCXYDBBGOEEQ-UHFFFAOYSA-N succinic anhydride Chemical compound O=C1CCC(=O)O1 RINCXYDBBGOEEQ-UHFFFAOYSA-N 0.000 claims description 4
- 102000009027 Albumins Human genes 0.000 claims description 3
- 108010088751 Albumins Proteins 0.000 claims description 3
- 229920002307 Dextran Polymers 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 230000032050 esterification Effects 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 235000013555 soy sauce Nutrition 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 239000007795 chemical reaction product Substances 0.000 claims description 2
- OWBBAPRUYLEWRR-UHFFFAOYSA-N 4-hydroxycoumarin Chemical compound C1=CC=C2OC(O)=CC(=O)C2=C1 OWBBAPRUYLEWRR-UHFFFAOYSA-N 0.000 claims 2
- 210000002700 urine Anatomy 0.000 abstract description 3
- 239000003146 anticoagulant agent Substances 0.000 abstract 2
- 229940127219 anticoagulant drug Drugs 0.000 abstract 2
- 229920001503 Glucan Polymers 0.000 abstract 1
- 238000007385 chemical modification Methods 0.000 abstract 1
- 150000002148 esters Chemical class 0.000 abstract 1
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 17
- 238000004458 analytical method Methods 0.000 description 10
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 6
- HSHNITRMYYLLCV-UHFFFAOYSA-N 4-methylumbelliferone Chemical compound C1=C(O)C=CC2=C1OC(=O)C=C2C HSHNITRMYYLLCV-UHFFFAOYSA-N 0.000 description 5
- 238000001819 mass spectrum Methods 0.000 description 5
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 4
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- CJIJXIFQYOPWTF-UHFFFAOYSA-N 7-hydroxycoumarin Natural products O1C(=O)C=CC2=CC(O)=CC=C21 CJIJXIFQYOPWTF-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- PCMORTLOPMLEFB-ONEGZZNKSA-N sinapic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC(OC)=C1O PCMORTLOPMLEFB-ONEGZZNKSA-N 0.000 description 2
- PCMORTLOPMLEFB-UHFFFAOYSA-N sinapinic acid Natural products COC1=CC(C=CC(O)=O)=CC(OC)=C1O PCMORTLOPMLEFB-UHFFFAOYSA-N 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 2
- ORHBXUUXSCNDEV-UHFFFAOYSA-N umbelliferone Chemical compound C1=CC(=O)OC2=CC(O)=CC=C21 ORHBXUUXSCNDEV-UHFFFAOYSA-N 0.000 description 2
- HFTAFOQKODTIJY-UHFFFAOYSA-N umbelliferone Natural products Cc1cc2C=CC(=O)Oc2cc1OCC=CC(C)(C)O HFTAFOQKODTIJY-UHFFFAOYSA-N 0.000 description 2
- 206010000383 Accidental poisoning Diseases 0.000 description 1
- IZJBEFCHFXZWRN-UHFFFAOYSA-N CCCCN(CCCC)CCCC.CCCCN(CCCC)CCCC Chemical compound CCCCN(CCCC)CCCC.CCCCN(CCCC)CCCC IZJBEFCHFXZWRN-UHFFFAOYSA-N 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- UYJNMEUJYZFHFO-UHFFFAOYSA-N ClC(=O)OCC.ClC(=O)OC=C Chemical compound ClC(=O)OCC.ClC(=O)OC=C UYJNMEUJYZFHFO-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 238000003891 environmental analysis Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 231100000567 intoxicating Toxicity 0.000 description 1
- 230000002673 intoxicating effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 235000021251 pulses Nutrition 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- -1 tonka bean camphor amber acid ester Chemical class 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a process for synthesizing anticoagulant raticide immunogen for rapidly and accurately detecting anticoagulant raticide component in urine comprising the steps of, using 4-hydroxyl coumarine as haptene, obtaining coumarin-succinic ester by using esterification reaction for chemical modification, proceeding coupling through isobutyl chlorocarbonate in mixed anhydride method, purifying through glucan G50 gel column, and freeze-drying.
Description
One, technical field
The invention belongs to biomedicine field, relate to a kind of immune analysis method, the immunogenic synthetic method of specifically a kind of anticoagulation class rodenticide.
Two, background technology
In recent years, the use rodenticide is poisoned or the incident of accidental poisoning happens occasionally, and has had a strong impact on stabilizing of society and stablizing of people's lives.Detect poisoner's urine rapidly and accurately, determining the poisonous substance title, in time to hospital rescue the provider to the qualitative foundation that provides of event property is provided, be the direction of current relevant departments research.The intoxicating phenomenon of anticoagulation class rodenticide happens occasionally at present, therefore checks anticoagulation class rodenticide (providing the result within the 10min) to have important and practical meanings accurately and rapidly.
Conventional immune analysis method mainly utilizes mark radio isotope, enzyme, fluorescent substance etc. on antibody, behind determined antigen and traget antibody specific reaction, washing plate separates, the radio isotope by measuring mark or the signal of fluorescent substance, or measure the signal that marker enzyme acts on electroactive substance that substrate produces, luminophore etc. and measure.In recent decades, immuno analytical method combines characteristics such as the sensitivity, convenience of technology such as specific recognition reaction between antigen, antibody molecule and electrochemistry, spectroscopy, acoustics, with its highly selective and high sensitivity to analyte, become clinical, biological chemistry, one of every field important analysis means such as environmental analysis.In all these methods, no matter which kind of material mark is, all need to obtain high specificity, highly sensitive monoclonal antibody, and the acquisition of monoclonal antibody needs the immunogen of high specificity.Micromolecular compound itself does not possess immunogenicity, and is therefore immunogenic synthetic most important.Immunogenic synthetic normally with micromolecular compound and bovine serum albumin BSA connection, the position of connection directly influences the specificity of monoclonal antibody.
Three, summary of the invention
The purpose of this invention is to provide a kind of immunogenic synthetic method of anticoagulation class rodenticide that can detect anticoagulation class rodenticide composition in poisoner's urine rapidly and accurately, it is in time rescued to hospital foundation is provided, give simultaneously the qualitative provider of poisoning character to.
The objective of the invention is to be achieved through the following technical solutions: the immunogenic synthetic method of a kind of anticoagulation class rodenticide is characterized in that it may further comprise the steps:
A) the Hydroxycoumarin compounds utilizes esterification to carry out chemically modified and obtains tonka bean camphor-succinate as haptens;
Hydroxycoumarin in the pyridine medium with succinyl oxide esterification formula is:
B) with steps A) tonka bean camphor-succinate of obtaining carries out coupling by the isobutyl chlorocarbonate method of mixed anhydride method;
Hydroxycoumarin and bovine serum albumin (BSA) coupling process is:
C) with step B) reaction product that obtains is by dextran G50 gel column purifying, and the immunogenic binding substances of anticoagulation class rodenticide is prepared in freeze-drying.
Anticoagulation class rodenticide metabolite in vivo is mainly the Hydroxycoumarin compounds, as umbelliferone, 4 hydroxy coumarin.Consider representativeness, selecting the haptens of anticoagulation class rodenticide is 4 hydroxy coumarin, in steps A) described in the Hydroxycoumarin compounds be 4 hydroxy coumarin.
With steps A) in 4 hydroxy coumarin 1～2g in reaction flask, add succinyl oxide, in 100～120 ℃ of pyridine media, refluxed 3～5 hours, dry pyridine solution with rotatory evaporator again, chloroform extraction, chloroform layer is washed 2～4 times with vapor enrichment, and obtaining product is color of soy sauce oily body note legumin-succinate.
Step B) raw materials used bovine serum albumin Bovine Serum Albumin (BSA), Tributylamine Tri-n-butylamine, Vinyl chloroformate Ethyl Chloroformate, dioxane and the sodium hydroxide solution of comprising in.Wherein, described bovine serum albumin (BSA) is the BR level, albumin content 95%, molecular weight 67366.4; Tributylamine is a molecular weight 185.34, and CP is pure, and content is no less than 99%; Vinyl chloroformate is a molecular weight 108.53, and CP is pure, and content is no less than 96.5%; Dioxane is the AR level, vapor enrichment; Sodium hydroxide solution is 1M.The concrete operations step is:
(1), at first bovine serum albumin is dissolved in the double distilled water, transfer to pH 8.5 afterreactions with sodium hydroxide solution;
(2), then coumarin-4-succinate is dissolved in the double distilled dioxane solvent, adds Vinyl chloroformate and Tributylamine, ice bath reaction again;
(3) solution that at last (2) is obtained is slowly poured in the solution of (1) and careful the stirring, adds sodium hydroxide solution and transfers to pH 8.5, adds double distilled water, and overall agent is reacted in 5 ℃ of refrigerators.
In above operation steps, bovine serum albumin is that 0.512g, double distilled water are that 100ml, sodium hydroxide solution are that 1M, reaction times are 30min in (1); (2) coumarin-4-succinate is that 0.3g, double distilled dioxane solvent are that 25ml, Vinyl chloroformate are that 97 μ l, Tributylamine are that 178 μ l, ice bath reaction times are 30min in; (3) sodium hydroxide solution is 1M in, and overall agent is 200ml, and the reaction times is 8 hours in the refrigerator.
The above coumarin-4 that makes by the present invention-succinate haptens-BSA binding substances is carried out immunogen to be identified:
1, uv analysis method
Coumarin-4 behind the purifying-succinate haptens-BSA binding substances is measured with ultraviolet spectrometer, measure coumarin-4-succinate and BSA respectively with ultraviolet spectrometer simultaneously, whether with the checking binding substances is coumarin-4-succinate haptens-BSA binding substances, the coumarin-4 really-succinate haptens-BSA binding substances of checking result proof.
2, infrared analysis
Coumarin-4-succinate behind the freeze-drying purifying-BSA binding substances, pressing potassium bromide troche carries out infrared analysis, analyzes BSA and tonka bean camphor amber acid ester simultaneously, in order to judge whether binding substances, the binding substances of the succinate of tonka bean camphor really of judged result proof for the tonka bean camphor succinate.
3, flight mass spectrum method
Adopt substance assistant laser desorpted flight time mass spectrum (MALDI-TOF-MS) to analyze haptens and be attached to number on the BSA molecule, haptens is attached to number on the BSA molecule usually to be influenced antibody and produces.The author that past has thinks that the haptens of receiving on the BSA molecule is many as far as possible.Experimental result shows that too much haptens can not obtain expected result.Too much or the very few ability that produces antibody of bringing out that all may reduce of haptens.
Advantage of the present invention is: 1, the position of antigen and BSA connection directly influences the specificity of monoclonal antibody, the present invention is chosen in the unessential hydroxy position of identification and connects, the anticoagulation class rodenticide immunogen high specificity that synthesizes, can be further immune animal, for the monoclonal antibody of preparing high specificity is laid a good foundation; 2, synthetic method is simple, easy to operate among the present invention, does not need special plant and instrument.
Four, embodiment
The immunogenic synthetic method of a kind of anticoagulation class rodenticide of the present invention, anticoagulation class rodenticide metabolite in vivo is mainly the Hydroxycoumarin compounds, as umbelliferone, 4 hydroxy coumarin.Consider representativeness, selecting the haptens of anticoagulation class rodenticide is 4 hydroxy coumarin.It may further comprise the steps:
A) coumarin-4-succinate (Coumarin-4-hemisuccinate) is synthetic
Disubstituted-4-hydroxy tonka bean camphor 1.6g adds the 3.0g succinyl oxide in three mouthfuls of reaction flasks, refluxed 4 hours in 110 ℃ of pyridine media, dries pyridine solution with rotatory evaporator, chloroform extraction, and chloroform layer is washed 3 times with vapor enrichment, and product is a color of soy sauce oily body.
Coumarin-4-succinate synthetic route is as follows:
4 hydroxy coumarin Succinic anhydried coumarin-7-succinate
(4-Hydroxycoumarin) Coumarin-4-hemisuccinate
(4-O-carboxymethylcoumarin)
B) coupling of coumarin-4-succinate-BAS binding substances
Raw material is:
Bovine serum albumin Bovine Serum Albumin (BSA), BR level, content 95% (albumin), molecular weight 67366.4.Tributylamine Tri-n-butylamine, molecular weight 185.34, CP is pure, and content is no less than 99%.Vinyl chloroformate Ethyl Chloroformate, molecular weight 108.53, CP is pure, and content is no less than 96.5.Dioxane AR level, vapor enrichment.The 1M sodium hydroxide solution.
Coupling process is:
(1) BSA 0.512g is dissolved in the 100ml double distilled water, transfers pH 8.5 with the 1M sodium hydroxide solution, reaction 30min.
(2) coumarin-4-succinate 0.3g is dissolved in the double distilled dioxane solvent of 25ml, adds 97 μ l Vinyl chloroformates, and 178 μ l Tributylamines are at ice bath reaction 30min.
(3) slowly pour into the solution of (2) in the solution of (1) and careful the stirring, add the 1M sodium hydroxide solution and transfer pH 8.5, add double distilled water, making overall agent was 200ml, 5 ℃ of refrigerators reactions 8 hours.
Coumarin-4-succinate-BSA coupling (mixed anhydride method) is as follows:
Tonka bean camphor succinate tonka bean camphor succinate-BSA binding substances
C) the freeze-drying purifying that anhydrates
Reacted solution is placed on freeze-drying in the Freeze Drying Equipment.Freeze dried binding substances is crossed dextran G50 post with the dissolving of 2ml phosphate buffer soln, receives the liquid at second peak, and receiving liquid freeze-drying in the freeze-drying machine is finished product anticoagulation mouse medicine binding substances.
Coumarin-4-succinate-immunogenic evaluation of BSA binding substances that above employing the present invention is made:
1, flight mass spectrum method
Coumarin-4 behind tonka bean camphor succinate-BSA binding substances purifying-succinate haptens-BSA binding substances is a medium with the sinapinic acid, carries out flight mass spectrum and measures.Because the molecular weight of binding substances is different with the molecular weight of BSA, the flight velocity in flight mass spectrum is different, according to the nucleo plasmic relation (m/z) of binding substances and BSA, can calculate the mol ratio that combines of carrier proteins and medicine in the immunogen.
Adopt JMS-LDI1700 time-of-fight mass spectrometry (TOF), N
2Laser apparatus, wavelength 337nm, pulse 3ns, detector voltage-4.75kv, matrix: 10
3The multiple sinapinic acid contains 10% trifluoroacetic acid aqueous solution.The molecular weight of coumarin-4-succinate is 262, and the molecular weight of the actual BSA of recording is 67366.4, and BSA-coumarin-7-succinate molecular weight is 70839.5.
2, ultra-violet analysis
Coumarin-4-succinate haptens-BSA binding substances (not freeze-drying) behind the dialysis purifying is measured with ultraviolet spectrometer, measure the UV spectrum of coumarin-4-succinate and BSA simultaneously respectively, if on BSA,, just do not have the absorption peak of coumarin-4-succinate not in conjunction with coumarin-4-succinate.Verify with this whether binding substances is coumarin-4-succinate-BSA binding substances, the coumarin-4 really-succinate haptens-BSA binding substances of checking result proof.
3, infrared analysis
Coumarin-4-succinate behind the purifying-BSA binding substances freeze-drying, pressing potassium bromide troche carries out infrared analysis, analyzes BSA and coumarin-4-succinate simultaneously, in order to judge whether binding substances, the binding substances of the succinate of tonka bean camphor really of judged result proof for coumarin-4-succinate.
Claims (7)
1, the immunogenic synthetic method of a kind of anticoagulation class rodenticide is characterized in that it may further comprise the steps:
A) use the Hydroxycoumarin compounds as haptens, utilize esterification to carry out chemically modified and obtain tonka bean camphor-succinate;
B) with steps A) tonka bean camphor-succinate of obtaining carries out coupling by the isobutyl chlorocarbonate method of mixed anhydride method;
C) with step B) reaction product that obtains is by dextran G50 gel column purifying, and the immunogenic binding substances of anticoagulation class rodenticide is prepared in freeze-drying.
2, the immunogenic synthetic method of anticoagulation class rodenticide according to claim 1, it is characterized in that: the compounds of Hydroxycoumarin steps A) is a 4 hydroxy coumarin.
3, the immunogenic synthetic method of anticoagulation class rodenticide according to claim 2, it is characterized in that: with steps A) in 4 hydroxy coumarin 1～2g place reaction flask, add succinyl oxide, in 100～120 ℃ of pyridine media, refluxed 3～5 hours, dry pyridine solution with rotatory evaporator again, chloroform extraction, chloroform layer is washed 2～4 times with vapor enrichment, and obtaining product is color of soy sauce oily body note legumin-succinate.
4, the immunogenic synthetic method of anticoagulation class rodenticide according to claim 2 is characterized in that: raw materials used bovine serum albumin, Tributylamine, Vinyl chloroformate, dioxane and the sodium hydroxide solution of comprising step B).
5, the immunogenic synthetic method of anticoagulation class rodenticide according to claim 4, it is characterized in that: described bovine serum albumin is the BR level, albumin content 95%, molecular weight 67366.4; Tributylamine is a molecular weight 185.34, and CP is pure, and content is no less than 99%; Vinyl chloroformate is a molecular weight 108.53, and CP is pure, and content is no less than 96.5%; Dioxane is the AR level, vapor enrichment; Sodium hydroxide solution is 1M.
6, the immunogenic synthetic method of anticoagulation class rodenticide according to claim 5 is characterized in that:
(1) at first bovine serum albumin is dissolved in the double distilled water, transfers to pH 8.5 afterreactions with sodium hydroxide solution;
(2) then coumarin-4-succinate is dissolved in the double distilled dioxane solvent, adds Vinyl chloroformate and Tributylamine, ice bath reaction again;
(3) solution that at last (2) is obtained is slowly poured in the solution of (1) and careful the stirring, adds sodium hydroxide solution and transfers to pH 8.5, adds double distilled water, and overall agent is reacted in 5 ℃ of refrigerators.
7, the immunogenic synthetic method of anticoagulation class rodenticide according to claim 6 is characterized in that: bovine serum albumin is that 0.512g, double distilled water are that 100ml, sodium hydroxide solution are that 1M, reaction times are 30min in (1); (2) coumarin-4-succinate is that 0.3g, double distilled dioxane solvent are that 25ml, Vinyl chloroformate are that 97 μ l, Tributylamine are that 178 μ l, ice bath reaction times are 30min in; (3) sodium hydroxide solution is 1M in, and overall agent is 200ml, and the reaction times is 8 hours in the refrigerator.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102875512A (en) * | 2012-10-29 | 2013-01-16 | 公安部物证鉴定中心 | Preparation method of anticoagulant raticide brodifacoum hapten and holoantigen |
| CN102898410A (en) * | 2012-10-29 | 2013-01-30 | 公安部物证鉴定中心 | Method for preparing anti-coagulation raticide bromadiolone half antigen and complete antigen |
| CN109824645A (en) * | 2019-02-28 | 2019-05-31 | 中国农业大学 | Warfarin haptens and artificial antigen and the preparation method and application thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897025B (en) * | 2019-02-28 | 2021-01-01 | 中国农业大学 | Anticoagulant raticide hapten and artificial antigen as well as preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102875512A (en) * | 2012-10-29 | 2013-01-16 | 公安部物证鉴定中心 | Preparation method of anticoagulant raticide brodifacoum hapten and holoantigen |
| CN102898410A (en) * | 2012-10-29 | 2013-01-30 | 公安部物证鉴定中心 | Method for preparing anti-coagulation raticide bromadiolone half antigen and complete antigen |
| CN102875512B (en) * | 2012-10-29 | 2014-12-17 | 公安部物证鉴定中心 | Preparation method of anticoagulant raticide brodifacoum hapten and holoantigen |
| CN109824645A (en) * | 2019-02-28 | 2019-05-31 | 中国农业大学 | Warfarin haptens and artificial antigen and the preparation method and application thereof |
| CN109824645B (en) * | 2019-02-28 | 2020-10-27 | 中国农业大学 | Warfarin hapten and artificial antigen as well as preparation method and application thereof |
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