CN107337690A - Organophosphor haptens and wide range polyspecific antigen, antibody and its preparation method and application - Google Patents
Organophosphor haptens and wide range polyspecific antigen, antibody and its preparation method and application Download PDFInfo
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- CN107337690A CN107337690A CN201710424121.2A CN201710424121A CN107337690A CN 107337690 A CN107337690 A CN 107337690A CN 201710424121 A CN201710424121 A CN 201710424121A CN 107337690 A CN107337690 A CN 107337690A
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- organophosphor
- haptens
- wide range
- reaction solution
- polyspecific
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- 239000000427 antigen Substances 0.000 title claims abstract description 50
- 102000036639 antigens Human genes 0.000 title claims abstract description 50
- 108091007433 antigens Proteins 0.000 title claims abstract description 50
- 238000002360 preparation method Methods 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 15
- 239000003987 organophosphate pesticide Substances 0.000 claims abstract description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 58
- 239000000243 solution Substances 0.000 claims description 52
- 238000006243 chemical reaction Methods 0.000 claims description 44
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 34
- 238000003756 stirring Methods 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 230000004044 response Effects 0.000 claims description 20
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- 239000005457 ice water Substances 0.000 claims description 18
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims description 16
- 239000012074 organic phase Substances 0.000 claims description 12
- 102000014914 Carrier Proteins Human genes 0.000 claims description 10
- 108010078791 Carrier Proteins Proteins 0.000 claims description 10
- 229940090248 4-hydroxybenzoic acid Drugs 0.000 claims description 8
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 8
- 230000035484 reaction time Effects 0.000 claims description 8
- WQYSXVGEZYESBR-UHFFFAOYSA-N thiophosphoryl chloride Chemical compound ClP(Cl)(Cl)=S WQYSXVGEZYESBR-UHFFFAOYSA-N 0.000 claims description 8
- 239000008055 phosphate buffer solution Substances 0.000 claims description 7
- 238000005057 refrigeration Methods 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- 230000033228 biological regulation Effects 0.000 claims description 4
- LCPVQAHEFVXVKT-UHFFFAOYSA-N 2-(2,4-difluorophenoxy)pyridin-3-amine Chemical compound NC1=CC=CN=C1OC1=CC=C(F)C=C1F LCPVQAHEFVXVKT-UHFFFAOYSA-N 0.000 claims description 3
- 238000005352 clarification Methods 0.000 claims description 3
- 230000008878 coupling Effects 0.000 claims description 3
- 238000010168 coupling process Methods 0.000 claims description 3
- 238000005859 coupling reaction Methods 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- CHQMHPLRPQMAMX-UHFFFAOYSA-L sodium persulfate Substances [Na+].[Na+].[O-]S(=O)(=O)OOS([O-])(=O)=O CHQMHPLRPQMAMX-UHFFFAOYSA-L 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 2
- 230000032050 esterification Effects 0.000 claims description 2
- 238000005886 esterification reaction Methods 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 12
- 238000003018 immunoassay Methods 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 230000007812 deficiency Effects 0.000 abstract description 2
- 235000011121 sodium hydroxide Nutrition 0.000 description 18
- 238000002965 ELISA Methods 0.000 description 6
- 235000019441 ethanol Nutrition 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 239000003905 agrochemical Substances 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 102000003914 Cholinesterases Human genes 0.000 description 3
- 108090000322 Cholinesterases Proteins 0.000 description 3
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 3
- 229960004373 acetylcholine Drugs 0.000 description 3
- 229940048961 cholinesterase Drugs 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- GRVDJDISBSALJP-UHFFFAOYSA-N methyloxidanyl Chemical group [O]C GRVDJDISBSALJP-UHFFFAOYSA-N 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- KCPJLBUDMSTBRT-UHFFFAOYSA-N [P].ClSCl Chemical class [P].ClSCl KCPJLBUDMSTBRT-UHFFFAOYSA-N 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- XFBJRFNXPUCPKU-UHFFFAOYSA-N chloro-dimethoxy-sulfanylidene-$l^{5}-phosphane Chemical class COP(Cl)(=S)OC XFBJRFNXPUCPKU-UHFFFAOYSA-N 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- -1 ester compound Chemical class 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 125000000954 2-hydroxyethyl group Chemical group [H]C([*])([H])C([H])([H])O[H] 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 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
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000005374 Poisoning Diseases 0.000 description 1
- MUUGCDGGIVCSDT-UHFFFAOYSA-N [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O Chemical compound [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O MUUGCDGGIVCSDT-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 230000001713 cholinergic effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 125000001891 dimethoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 125000005909 ethyl alcohol group Chemical group 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000000568 immunological adjuvant Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/16—Esters of thiophosphoric acids or thiophosphorous acids
- C07F9/165—Esters of thiophosphoric acids
- C07F9/18—Esters of thiophosphoric acids with hydroxyaryl compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/16—Esters of thiophosphoric acids or thiophosphorous acids
- C07F9/165—Esters of thiophosphoric acids
- C07F9/17—Esters of thiophosphoric acids with hydroxyalkyl compounds without further substituents on alkyl
-
- 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
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2496/00—Reference solutions for assays of biological material
- G01N2496/45—Reference solutions for assays of biological material containing protease inhibitors, e.g. sulfonylfluorides, chloromethylketones or organophosphates
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of organophosphor haptens and organophosphor wide range polyspecific artificial antigen, antibody and preparation method and application.The present invention overcomes the technical deficiency of existing organophosphorus pesticide immunoassay method, provide a kind of immunoassay thinking of organophosphor wide range polyspecific, for organophosphorus pesticide, the method degree of accuracy and sensitivity are fine, detection available for organophosphor in agricultural product and food samples, general wide range polyspecific detection is realized, it is very convenient quick in actual use.The organophosphor wide range polyspecific antigen has formula（Ⅲ）Shown structure：（Ⅲ）.
Description
Technical field
It is more more particularly, to a kind of organophosphor haptens, organophosphor wide range the present invention relates to technical field of food safety
Specific artificial antigen, antibody and preparation method and application.
Background technology
Organophosphorus pesticide category organophosphorus ester compound, there is wide spectrum, efficient, the small and mode of action of amount is more, uses
Conveniently, it is that current dosage is maximum the advantages that half-life short, the most wide agricultural chemicals of application surface.Yet with production, transport and
Abused in agricultural, cause serious environmental problem.Organophosphorus pesticide all has stronger toxic side effect to people, animal, can pass through
The approach such as skin, breathing and stomach absorption enter in people, animal body, and inhibitory action is produced to the activity of internal cholinesterase, because
It can be combined rapidly with cholinesterase, form phosphorylated cholinesterase, lose the ability of catalyzing hydrolysis acetylcholine, as a result be made big
The acetylcholine of amount is accumulated in vivo, causes acetylcholine to be in state of being overexcited for the cholinergic nerve of transmitting medium and goes out
Existing poisoning symptom.
The detection method of existing organophosphorus pesticide mainly includes instrument detection method and immunodetection, wherein immunodetection
Due to having the advantages that high sensitivity, high specificity, pre-treatment is simple, round of visits is short, is detected suitable for batch samples,
As the focus of Recent study.
Organophosphor can substantially be divided into two classes, methoxyl organophosphorus in structure（CH3O）2P（X）Y and ethyoxyl organophosphor
（C2H5O）2P（X）Y, X represent O or S, Y represent a series of chemical constitutions.In actual use, can not be in the very first time
Recognize that agricultural chemicals that needs detect is any or the residual of which group agricultural.
Because being limited by specific thinking set is limited to, the existing immuno analytical method in this area is led to for a long time
It is often either the specific detection particularly for ethyoxyl organophosphor or to have particularly for methoxyl organophosphorus for methoxyl group
The immunization detection of the one of which agricultural chemicals of machine phosphorus insecticide, that is to say, that immunoassay method is only applicable to a kind of or a certain small
The detection of class agricultural chemicals, it is difficult to the detection needs for meet high flux, remaining more, it should be made in the design of antigen with more general
Property, avoid the useless duplication of labour.Therefore a kind of quick determination method with more characteristic organophosphors is developed with important meaning
Justice.
The content of the invention
The technical problem to be solved in the present invention is to overcome the technology of existing organophosphorus pesticide immunoassay method
Deficiency, there is provided how special a kind of organophosphor haptens I, the organophosphor haptens I can be applied to prepare organophosphor wide range
Property antigen.
Another technical problem to be solved by the present invention is that a kind of organophosphor haptens II is provided, the organophosphor haptens II
It can be applied to prepare organophosphor wide range polyspecific antigen.
The main technical problem to be solved in the present invention is to provide a kind of organophosphor wide range polyspecific antigen.
The invention solves an also technical problem be to provide the preparation method of the organophosphor wide range polyspecific antigen.
A present invention also technical problems to be solved are to provide the application of the organophosphor wide range polyspecific antigen.
The purpose of the present invention is achieved by the following technical programs：
A kind of organophosphor haptens I for being applied to prepare organophosphor wide range polyspecific antigen is provided, it has formula（Ⅰ）Shown
Structure：
（Ⅰ）.
Present invention simultaneously provides the preparation method of the organophosphor haptens I, comprise the following steps：
S01. in ice-water bath, absolute methanol is slowly added dropwise into phosphorus thiochloride, is reacted, reaction solution is then poured into frozen water
Middle shaking twice, is removed a layer organic phase and is dried with anhydrous sodium sulfate, and product refrigeration is standby；
S02. under ice-water bath, the product of step S01 preparations is slowly added dropwise into powdered sodium hydroxide and absolute methanol mixed liquor
Reacted；After question response terminates, the shaking of cold hydrochloric acid is added into reaction solution once, is washed with water, with anhydrous sodium sulfate drying,
Product refrigeration is standby；
S03. under ice-water bath, P-hydroxybenzoic acid and sodium hydroxide are dissolved in methanol, stirring until completely dissolved, is slowly dripped
The product for adding step S02 to prepare is reacted；After question response terminates, reaction solution is concentrated, diluted, regulation pH value is highly basic
Property, then extracted with dichloromethane, water intaking mutually adjusts pH to be then extracted with ethyl acetate, wash, take organic phase to carry out to highly acid
It is concentrated and dried, then crosses post, that is, organophosphor haptens I is made.
Preferably, the mass ratio of phosphorus thiochloride and absolute methanol described in step S01 is according to 1:50 determine.
Preferably, the mass ratio of sodium hydroxide and absolute methanol described in step S02 is according to 1:50 determine.
Preferably, the mass ratio of the mixed liquor of sodium hydroxide and absolute methanol described in step S02 and the S01 products prepared is pressed
According to 1:250 determine.
Preferably, the concentration of cold hydrochloric acid described in step S02 is 1 mol/L.
Preferably, in step S03, the quality of product prepared by P-hydroxybenzoic acid, sodium hydroxide, methanol and step S02
Than according to 1:4:50:1.2 determine.
Preferably, step S03 is to adjust pH value with the sodium hydroxide of mass concentration 10% as 9.
Preferably, step S03 is to adjust pH value with 1 mol/L hydrochloric acid solutions as 3 or so.
A kind of organophosphor haptens II for being applied to prepare organophosphor wide range polyspecific antigen is provided, it has formula（Ⅱ）
Shown structure：
（Ⅱ）.
Present invention simultaneously provides the preparation method of the organophosphor haptens II, comprise the following steps：
S11. in ice-water bath, absolute ethyl alcohol is slowly added dropwise into phosphorus thiochloride, is reacted, reaction solution is then poured into frozen water
Middle shaking twice, is removed a layer organic phase and is dried with anhydrous sodium sulfate, and product refrigeration is standby.
S12. under ice-water bath, the product of step S04 preparations is slowly added dropwise into sodium hydroxide and absolute ethyl alcohol mixed liquor
Reacted.After question response terminates, cold dilute hydrochloric acid shaking is added into reaction solution once, is then washed with water once, finally with nothing
Aqueous sodium persulfate is dried, and product refrigeration is standby.
S13. under ice-water bath, into P-hydroxybenzoic acid and sodium hydroxide mixed liquor, absolute ethyl alcohol is added, it is slowly molten
Clearly, the product that step S04 preparations are then slowly added dropwise is reacted；After question response terminates, reaction solution is concentrated, diluted,
Regulation pH value is strong basicity, is then extracted with dichloromethane, and water intaking mutually adjusts pH to be then extracted with ethyl acetate, water to highly acid
Wash, take organic phase to be concentrated and dried, then cross post, that is, organophosphor haptens II is made.
Preferably, the mass ratio of phosphorus thiochloride and absolute ethyl alcohol described in step S11 is according to 1:50 determine.
Preferably, the mass ratio of sodium hydroxide and absolute ethyl alcohol described in step S12 is according to 1:50 determine.
The mass ratio of product prepared by the mixed liquor of sodium hydroxide and absolute ethyl alcohol described in step S12 and S11 is according to 1:250
It is determined that.
Preferably, in step S13, the quality of product prepared by P-hydroxybenzoic acid, sodium hydroxide, ethanol and step S11
Than according to 1:4:50:1.2 determine.
Preferably, step S13 is to adjust pH value with the sodium hydroxide of mass concentration 10% as 9.
Preferably, step S13 is to adjust pH value with 1 mol/L hydrochloric acid solutions as 3 or so.
A kind of organophosphor wide range polyspecific antigen is provided, it has formula（Ⅲ）Shown structure：
（Ⅲ）.
Preferably, the carrier protein is bovine serum albumin(BSA), human serum albumins, hemocyanin or ovalbumin.
The present invention provides the preparation method of the organophosphor wide range polyspecific antigen, is to use active ester method by organophosphor
Haptens I and carrier protein are first coupled, and the product after coupling then is continued through into active esterification process and organophosphor half is anti-
Original II is coupled, and prepares organophosphor wide range polyspecific antigen.
Specifically, the preparation method of the organophosphor wide range polyspecific antigen, comprises the following steps：
S1. organophosphor haptens I is dissolved in DMF, formed mixed solution, then add EDC and NHS stir at room temperature into
Row reaction, after question response terminates, reaction solution is added in the phosphate buffer solution of carrier protein, is continued at room temperature anti-
Should, after reaction terminates, reaction solution is dialysed at room temperature, that is, organophosphor comlete antigen I is made；
S2. organophosphor haptens II is dissolved in DMF, forms mixed solution, then added EDC and NHS, stir at room temperature
Reacted；
S3. organophosphor antigens I made from step S1 is dissolved in DMF solution, then adds reaction solution prepared by step S2, in
Reacted, after question response terminates, reaction solution is dialysed at room temperature, that is, the organophosphor wide range polyspecific is made and resists
It is former.
Preferably, the mass ratio of organophosphor haptens I described in step S1 and carrier protein is 12:1.
Preferably, the reaction time of organophosphor haptens I described in step S1 and EDC and NHS is 8~24h.
Preferably, the reaction time of organophosphor haptens I described in step S1 and carrier protein is 6~24h.
Preferably, the reaction time described in step S2 is 8~12h.
Preferably, the mass ratio of organophosphor haptens II described in step S3 and organophosphor haptens I is 13.5:1.
Preferably, the reaction time described in step S3 is 6~8h.
In above-mentioned preparation method, DMF, EDC and NHS mass ratio are according to 10:2:1 determines.
In above-mentioned preparation method, the composition of the phosphate buffer solution of carrier protein dissolves for 50mg BSA 6mL PBS
After add 3.75mlDMF, dosage is 50 mg.
Present invention simultaneously provides how special the organophosphor wide range being prepared using the organophosphor wide range polyspecific antigen is
Heterogenetic antibody.
The present invention provides the organophosphor wide range polyspecific antigen and the antibody answering in organophosphorus pesticide is detected
With.
Beneficial effects of the present invention：
The present invention breaks the special attitude towards sex that this area is limited to for a long time, there is provided a kind of organophosphor wide range polyspecific is exempted from
Epidemic disease analytical mathematics, for organophosphorus pesticide, realize general wide range polyspecific detection.
To realize the object of the invention, invention also provides two kinds of haptens, and the preparation of the haptens is provided
Method, based on described two haptens, the organophosphor wide range polyspecific antigen is prepared very simple and easyly, for this
Invention detection organophosphor lays technical foundation.
The invention provides a kind of new organophosphor wide range polyspecific antigen, based on the antigen, can be achieved to a variety of
Detect, have broad application prospects while organophosphorus pesticide.
Preparation method mild condition of the present invention, it is easy to spread.
Brief description of the drawings
The syntheti c route figure of Fig. 1 organophosphors haptens I.
The mass spectrogram of Fig. 2 organophosphors haptens I.
The syntheti c route figure of Fig. 3 organophosphors haptens II.
The mass spectrogram of Fig. 4 organophosphors haptens II.
Fig. 5 ethyl parathion standard curves.
Embodiment
The inventive method is further illustrated with reference to specific embodiment.Following being given for example only property of embodiment explanations, no
It is understood that as limitation of the present invention.Unless stated otherwise, biomaterial, the reagent raw material used in following embodiments is conventional
The biomaterial and reagent raw material that purchased in market or commercial sources obtain, unless stated otherwise, the method that is used in following embodiments and
Equipment is method and apparatus commonly used in the art.
The preparation of the haptens I of embodiment 1
The syntheti c route of organophosphor haptens I is as shown in Figure 1.
S01.60mL phosphorus thiochlorides stir 10min under ice-water bath, and 100mL absolute methanols are then slowly added dropwise, and carry out anti-
Should, after question response terminates, reaction solution is poured into frozen water and shaken twice, removed a layer organic phase and be dried with anhydrous sodium sulfate,
Obtain O- methyl thio-phosphoryl chlorine；
S02. 2.7g sodium hydroxides are taken, 13.6mL methanol stirs 15min under ice-water bath, is then slowly added dropwise obtained by 10g S01
O- methyl thio-phosphoryl chlorine, after question response terminates, cold dilute hydrochloric acid shaking is added into reaction solution once, washing once, then with nothing
Aqueous sodium persulfate is dried.
S03. 2g P-hydroxybenzoic acid is taken, 1.16g sodium hydroxides, 5min is stirred under ice-water bath, then adds 30mL first
Alcohol, slowly dissolved clarification.Then 2.8g dimethoxy thiophosphoryl chlorides are slowly added dropwise, react 1h.After question response terminates, concentration reaction
Liquid, then plus water is diluted, and is adjusted pH to 9 or so with 10% sodium hydroxide, is then extracted with dichloromethane, hydrochloric acid is mutually used in water intaking
Solution adjusts pH to 3 or so, is then extracted with ethyl acetate, and washes, takes organic phase to be concentrated and dried, and then crosses post, that is, is made
Organophosphor haptens I.The mass spectrogram of organophosphor haptens I is as shown in Figure 2.Prove that it has formula（Ⅰ）Shown structure is：
（Ⅰ）.
The preparation of the haptens II of embodiment 2
The syntheti c route of organophosphor haptens II is as shown in Figure 3.
S01.60mL phosphorus thiochlorides stir 10min under ice-water bath, and 100mL absolute ethyl alcohols are then slowly added dropwise, and carry out anti-
Should, after question response terminates, reaction solution is poured into frozen water and shaken twice, removed a layer organic phase and be dried with anhydrous sodium sulfate.
Dimethoxy thiophosphoryl chloride
S02. 2.7g sodium hydroxides are taken, 13.6mL ethanol stirs 15min under ice-water bath, 10g diethoxies are then slowly added dropwise
Thiophosphoryl chloride, after question response terminates, cold dilute hydrochloric acid shaking is added into reaction solution once, washing once, then uses anhydrous slufuric acid
Sodium is dried.
S03. 2g P-hydroxybenzoic acid is taken, 1.16g sodium hydroxides, 5min is stirred under ice-water bath, then adds 30mL first
Alcohol（Ethanol）, slowly dissolved clarification.Then 2.8g dimethoxys are slowly added dropwise（Ethyoxyl）Thiophosphoryl chloride, react 1h.Question response knot
Shu Hou, concentration of reaction solution, then plus water is diluted, and is adjusted PH to 9 or so with 10% sodium hydroxide, is then extracted with dichloromethane,
Water intaking mutually adjusts pH to 3 or so with hydrochloric acid solution, is then extracted with ethyl acetate, and washes, takes organic phase to be concentrated and dried, then
Post is crossed, that is, organophosphor haptens II is made.The mass spectrogram of organophosphor haptens II is as shown in Figure 4.Prove that it has formula（Ⅱ）Institute
The structure shown is：
（Ⅱ）.
The preparation of the organophosphor wide range polyspecific antigen of embodiment 3
S1. weigh the 26mg of organophosphor haptens I made from embodiment 1 to be dissolved in 1mL DMF, stirring adds 36mg EDC and 25mg
NHS, at room temperature stirring reaction 8h.After question response terminates, 0.25mL reaction solutions are taken to be added to 50mg pH7.4 BSA phosphate
In cushioning liquid, stirring reaction 6h at room temperature.Reaction solution is fitted into bag filter, dialysed with pH7.4 phosphate buffer solution
3d, changes dialyzate 2~3 times daily, and organophosphor comlete antigen I is made.
S2. weigh the 26mg of organophosphor haptens II made from embodiment 2 to be dissolved in 1mL DMF, stirring adds 36mg EDC
With 25mg NHS, at room temperature stirring reaction 8h.
S3. organophosphor comlete antigen I is dissolved in 5mL DMF solutions；Reaction solution obtained by 0.25mL steps S2 is taken to be added to
In the DMF solution of organophosphor comlete antigen I, at room temperature after stirring reaction 6h, reaction solution is loaded into bag filter, with pH7.4's
Phosphate buffer solution dialysis 3d, changes liquid 2~3 times, that is, organophosphor wide range polyspecific antigen is made daily.
The preparation of the organophosphor wide range polyspecific antigen of embodiment 4
S1. weigh the 26mg of organophosphor haptens I made from embodiment 1 to be dissolved in 1mL DMF, stirring adds 36mg EDC and 25mg
NHS, at room temperature stirring reaction 12h.After question response terminates, 0.25mL reaction solutions are taken to be added to 50mg pH7.4 BSA phosphoric acid
In salt buffer solution, stirring reaction 8h at room temperature.Reaction solution is fitted into bag filter, it is saturating with pH7.4 phosphate buffer solution
3d is analysed, changes dialyzate daily 2~3 times, organophosphor comlete antigen I is made.
S2. weigh the 26mg of organophosphor haptens II made from embodiment 2 to be dissolved in 1mL DMF, stirring adds 36mg EDC
With 25mg NHS, at room temperature stirring reaction 12h.
S3. organophosphor comlete antigen I is dissolved in 5mL DMF solutions；Reaction solution obtained by 0.25mL steps S2 is taken to be added to
In the DMF solution of organophosphor comlete antigen I, at room temperature after stirring reaction 6h, reaction solution is loaded into bag filter, with pH7.4's
Phosphate buffer solution dialysis 3d, changes liquid 2~3 times, that is, organophosphor wide range polyspecific antigen is made daily.
Organophosphor wide range polyspecific antigen made from embodiment 3 and embodiment 4 is demonstrate,proved using ultraviolet full wavelength scanner
Bright organophosphor wide range polyspecific antigen of the present invention has formula（Ⅲ）Shown structure：
（Ⅲ）.
The preparation and identification of the antibody of embodiment 5
Preparation method for antibody described in the present embodiment is conventional with reference to this area.
Mixed organophosphor wide range multispecific immune is former with isodose immunologic adjuvant（1st time immune Freund is complete
Adjuvant, the later military incomplete Freund's adjuvant of booster immunization）, after being emulsified completely with agitator, back multiple spot immune health great Bai
Rabbit, serum titer and inhibiting rate are determined using indirect ELISA later booster immunization every two weeks once, after titer plateaus, then
Booster immunization is once.10 days Culling heart bloods after last time is immune, centrifugation retain serum.Using octanoic acid-ammonium sulfate salting-out process pair
Serum is purified to obtain the antibody of high specific.
The foundation of the enzyme-linked immunoassay method of embodiment 6
Determine the working concentration of antigen and antibody by square formation titration, the working concentration of coating antigen is 0.5 μ g/mL, organophosphor
The extension rate of antibody is 1/40000.
Experimental solutions are done with organic phosphorus solution of various concentrations, are at war with gradient dilution, using 9 groups of parallel laboratory tests（n
=3）.
Indirect Competitive ELISA method：It is with the coated elisa plate of above-mentioned working concentration, experimental solutions are same with antibody-solutions
When be added in ELISA Plate aperture, while blank well and negative control hole are set, 37 DEG C of incubation 40min, liquid in hole is poured out, uses
Cleaning solution is washed 5 times, and ELISA Plate is upside down on blotting paper and patted：Add the ELIAS secondary antibody solution diluted, 37 DEG C of incubations
20min, washed 5 times, patted dry with cleaning solution：Substrate chromophoric solution is added, 37 DEG C of incubation 10min, adds terminate liquid color development stopping,
Light absorption value OD is determined at wavelength 450nm with ELIASA, using light absorption value OD as ordinate, with the log10 of OPS experimental solutions concentration
It is worth for abscissa, draws semilog canonical plotting, see the curve (ethyl parathions of organophosphor ELISA competition tests shown in accompanying drawing 5
Exemplified by).
As a result show that standard curve has complete reverse-s shape shape, and have upper mounting plate and lower platform, standard curve it is parallel
Determine number 3 times, experimental repeatability is good, and relative standard deviation is but within 15%.
Standard curve is drawn by same method, calculates minimum detection limit (LOD value), the half suppression of other organophosphorus pesticides
Amount (IC processed50Value）, the range of linearity is shown in Table 1.It can illustrate the inventive method degree of accuracy and sensitivity from the testing result of table 1
Very well, the detection available for organophosphor in agricultural product and food samples is very convenient quick in actual use.
1 antigen of the present invention of table, the minimum detection limit of antibody test organophosphorus pesticide, half amount of suppression and range of linearity value
Claims (9)
1. a kind of organophosphor haptens I for being applied to prepare organophosphor wide range polyspecific antigen, it is characterised in that it has formula
（Ⅰ）Shown structure：
（Ⅰ）.
2. the preparation method of organophosphor haptens I described in claim 1, it is characterised in that comprise the following steps：
S01. in ice-water bath, absolute methanol is slowly added dropwise into phosphorus thiochloride, is reacted, reaction solution is then poured into frozen water
Middle shaking twice, is removed a layer organic phase and is dried with anhydrous sodium sulfate, and product refrigeration is standby；
S02. under ice-water bath, in the mixed liquor prepared to powdered sodium hydroxide and absolute methanol, step S01 preparations are slowly added dropwise
Product reacted；After question response terminates, cold 1 mol/L hydrochloric acid shaking is added into reaction solution once, is washed with water, with nothing
Aqueous sodium persulfate is dried, and product refrigeration is standby；
S03. under ice-water bath, P-hydroxybenzoic acid and sodium hydroxide mixed solution are added in absolute methanol, stirring has been treated
After fully dissolved, the product that step S02 preparations are slowly added dropwise is reacted；After question response terminates, reaction solution is concentrated, it is dilute
To release, regulation pH value is strong basicity, is then extracted with dichloromethane, and water intaking mutually adjusts pH to be then extracted with ethyl acetate to highly acid,
Washing, takes organic phase to be concentrated and dried, and then crosses post, that is, organophosphor haptens I is made.
3. a kind of organophosphor haptens II for being applied to prepare organophosphor wide range polyspecific antigen, it is characterised in that it has
Formula（Ⅱ）Shown structure：
（Ⅱ）.
4. the preparation method of organophosphor haptens II described in claim 3, it is characterised in that comprise the following steps：
S11. in ice-water bath, absolute ethyl alcohol is slowly added dropwise into phosphorus thiochloride, is reacted, reaction solution is then poured into frozen water
Middle shaking twice, is removed a layer organic phase and is dried with anhydrous sodium sulfate, and product refrigeration is standby；S12. under ice-water bath, Xiang Fen
In shape sodium hydroxide and absolute ethyl alcohol mixed liquor, the product that step S04 preparations are slowly added dropwise is reacted, after question response terminates,
Cold 1 mol/L hydrochloric acid shaking is added into reaction solution once, is then washed with water once, finally with anhydrous sodium sulfate drying, product
Refrigerate standby；
S13. under ice-water bath, P-hydroxybenzoic acid and sodium hydroxide mixed solution are added in absolute ethyl alcohol, slowly dissolved clarification,
Then the product that step S04 preparations are slowly added dropwise is reacted；After question response terminates, reaction solution is concentrated, diluted, regulation
PH value is strong basicity, is then extracted with dichloromethane, and water intaking mutually adjusts pH to be then extracted with ethyl acetate to highly acid, wash, take
Organic phase is concentrated and dried, and then crosses post, that is, organophosphor haptens II is made.
5. a kind of organophosphor wide range polyspecific antigen, it is characterised in that it has formula（Ⅲ）Shown structure：
（Ⅲ）.
6. the preparation method of organophosphor wide range polyspecific antigen described in claim 5, it is characterised in that be to use active ester method
The organophosphor haptens I of claim 1 or 2 and carrier protein are first coupled, then continue to lead to by the product after coupling
Cross active esterification process to be coupled with the organophosphor haptens II of claim 3 or 4, how special prepare organophosphor wide range
Property antigen.
7. the preparation method of organophosphor wide range polyspecific antigen according to claim 6, it is characterised in that including following step
Suddenly：
S1. the organophosphor haptens I of claim 1 or 2 is dissolved in DMF, formed mixed solution, then add EDC and
NHS is stirred at room temperature to be reacted, and after question response terminates, reaction solution is added to the phosphate buffer solution of carrier protein
In, continue to react at room temperature, after reaction terminates, reaction solution is dialysed at room temperature, that is, organophosphor comlete antigen I is made；
S2. the organophosphor haptens II of claim 3 or 4 is dissolved in DMF, formed mixed solution, then add EDC and
NHS, at room temperature stirring are reacted；
S3. organophosphor antigens I made from step S1 is dissolved in DMF solution, then adds reaction solution prepared by step S2, in
Reacted, after question response terminates, reaction solution is dialysed at room temperature, that is, the organophosphor wide range polyspecific is made and resists
It is former.
8. the preparation method of organophosphor wide range polyspecific antigen according to claim 7, it is characterised in that described in step S1
The coupling ratio of organophosphor haptens I and carrier protein is 20:1；
The reaction time of organophosphor haptens I described in step S1 and EDC and NHS is 8~24h；
The reaction time of organophosphor haptens I described in step S1 and carrier protein is 6~24h；
Reaction time described in step S2 is 8~12h；
The mass ratio of organophosphor haptens II described in step S3 and organophosphor haptens I is 13.5:1；
Reaction time described in step S3 is 6~8h；
Described DMF, EDC and NHS mass ratio are according to 10:2:1 determines.
9. any one of the claim 5~8 organophosphor wide range polyspecific antigen, using described in any one of claim 5~8
Application of the antibody that organophosphor wide range polyspecific antigen is prepared in organophosphorus pesticide is detected.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100644205B1 (en) * | 2005-06-30 | 2006-11-10 | 이용태 | Haptens and antibodies for the class-specific determination of organophosphorus pesticides and the immunoassay method using the same |
| CN101475587A (en) * | 2009-01-09 | 2009-07-08 | 华南农业大学 | Diethoxy thiophosphate organophosphorus pesticide hapten and preparation thereof |
-
2017
- 2017-06-07 CN CN201710424121.2A patent/CN107337690B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100644205B1 (en) * | 2005-06-30 | 2006-11-10 | 이용태 | Haptens and antibodies for the class-specific determination of organophosphorus pesticides and the immunoassay method using the same |
| CN101475587A (en) * | 2009-01-09 | 2009-07-08 | 华南农业大学 | Diethoxy thiophosphate organophosphorus pesticide hapten and preparation thereof |
Non-Patent Citations (4)
| Title |
|---|
| PANPAN JIANG等: ""Preparation and Identifcation of Generic Antigens and Broad-Spectrum Monoclonal Antibodies for Detection of Organophosphorus Pesticides"", 《JOURNAL OF AOAC INTERNATIONAL》 * |
| RIKA KODAKA等: ""Comparative Metabolism of Organophosphorus Pesticides in Water-Sediment Systems"", 《J.PESTIC.SCI》 * |
| 尹志刚主编: "《有机磷化合物》", 31 March 2011, 北京：化学工业出版社 * |
| 徐振林 等: "有机磷农药半抗原设计及多特异性抗体识别机制研究", 《中国科学》 * |
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