CN107759547A - A kind of carbofuran half-antigen, comlete antigen and preparation method and application - Google Patents
A kind of carbofuran half-antigen, comlete antigen and preparation method and application Download PDFInfo
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- CN107759547A CN107759547A CN201711049977.2A CN201711049977A CN107759547A CN 107759547 A CN107759547 A CN 107759547A CN 201711049977 A CN201711049977 A CN 201711049977A CN 107759547 A CN107759547 A CN 107759547A
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- Prior art keywords
- antigen
- liquid
- carbofuran
- furadan
- comlete
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- DUEPRVBVGDRKAG-UHFFFAOYSA-N carbofuran Chemical compound CNC(=O)OC1=CC=CC2=C1OC(C)(C)C2 DUEPRVBVGDRKAG-UHFFFAOYSA-N 0.000 title claims abstract description 77
- 239000000427 antigen Substances 0.000 title claims abstract description 70
- 102000036639 antigens Human genes 0.000 title claims abstract description 34
- 108091007433 antigens Proteins 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 8
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 8
- WJGPNUBJBMCRQH-UHFFFAOYSA-N 2,2-dimethyl-2,3-dihydro-1-benzofuran-7-ol Chemical compound C1=CC(O)=C2OC(C)(C)CC2=C1 WJGPNUBJBMCRQH-UHFFFAOYSA-N 0.000 claims abstract description 7
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 claims abstract description 7
- QCTBMLYLENLHLA-UHFFFAOYSA-N aminomethylbenzoic acid Chemical compound NCC1=CC=C(C(O)=O)C=C1 QCTBMLYLENLHLA-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000000126 substance Substances 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 41
- 239000000203 mixture Substances 0.000 claims description 22
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 21
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 15
- 239000007787 solid Substances 0.000 claims description 13
- UJMGZPCKYHBCKU-UHFFFAOYSA-N 2,2-dimethyl-2,3-dihydrobenzofuran Chemical class C1=CC=C2OC(C)(C)CC2=C1 UJMGZPCKYHBCKU-UHFFFAOYSA-N 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 150000002148 esters Chemical class 0.000 claims description 10
- 239000012074 organic phase Substances 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 8
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 8
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 7
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 6
- 235000013305 food Nutrition 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 claims description 5
- 229910000041 hydrogen chloride Inorganic materials 0.000 claims description 5
- 238000010791 quenching Methods 0.000 claims description 5
- 230000000171 quenching effect Effects 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 4
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims 1
- 125000002252 acyl group Chemical group 0.000 claims 1
- 239000000385 dialysis solution Substances 0.000 claims 1
- 239000013067 intermediate product Substances 0.000 abstract description 4
- 230000005847 immunogenicity Effects 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 238000003018 immunoassay Methods 0.000 abstract 1
- 238000002965 ELISA Methods 0.000 description 6
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 6
- 230000031700 light absorption Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 238000001953 recrystallisation Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- BWCJVGMZEQDOMY-UHFFFAOYSA-N 2-methyl-2,3-dihydro-1-benzofuran Chemical class C1=CC=C2OC(C)CC2=C1 BWCJVGMZEQDOMY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000005711 Benzoic acid Substances 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 239000003905 agrochemical Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 235000010233 benzoic acid Nutrition 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 150000002118 epoxides Chemical class 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- -1 nitrite ions Chemical class 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 235000005254 Allium ampeloprasum Nutrition 0.000 description 1
- 240000006108 Allium ampeloprasum Species 0.000 description 1
- 241001124076 Aphididae Species 0.000 description 1
- 241000952610 Aphis glycines Species 0.000 description 1
- 241001498622 Cixius wagneri Species 0.000 description 1
- 241000008892 Cnaphalocrocis patnalis Species 0.000 description 1
- 241001364569 Cofana spectra Species 0.000 description 1
- 206010051625 Conjunctival hyperaemia Diseases 0.000 description 1
- 206010012442 Dermatitis contact Diseases 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 241000255967 Helicoverpa zea Species 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 241000966204 Lissorhoptrus oryzophilus Species 0.000 description 1
- 241000244206 Nematoda Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000346285 Ostrinia furnacalis Species 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 206010039424 Salivary hypersecretion Diseases 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 241001161749 Stenchaetothrips biformis Species 0.000 description 1
- 208000003443 Unconsciousness Diseases 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000000507 anthelmentic effect Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 208000010247 contact dermatitis Diseases 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- WJJMNDUMQPNECX-UHFFFAOYSA-N dipicolinic acid Chemical group OC(=O)C1=CC=CC(C(O)=O)=N1 WJJMNDUMQPNECX-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000012055 fruits and vegetables Nutrition 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 231100000567 intoxicating Toxicity 0.000 description 1
- 230000002673 intoxicating effect Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 201000005404 rubella Diseases 0.000 description 1
- 208000026451 salivation Diseases 0.000 description 1
- 210000004894 snout Anatomy 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
- C07D307/86—Benzo [b] furans; Hydrogenated benzo [b] furans with an oxygen atom directly attached in position 7
-
- 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
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of carbofuran half-antigen, comlete antigen and preparation method and application, the carbofuran half-antigen reacts generation intermediate product using benzofuranol as raw material, with isobutyl chlorocarbonate, then by intermediate product and 4（Amino methyl）Benzoic acid reacts in wet chemical obtains carbofuran half-antigen.Carbofuran half-antigen and carrier protein couplet are obtained into Furadan comlete antigen.Immune zoopery shows that artificial antigen prepared by the present invention has good immunogenicity.The carbofuran half-antigen and antigen of the present invention detects available for Furadan immunoassay, and application prospect is very wide.
Description
Technical field
The invention belongs to technical field of biochemical industry, and in particular to a kind of carbofuran half-antigen, comlete antigen and preparation side
Method and application.
Background technology
Furadan (Carbofuran), 2,3- dihydro -2,2- dimethyl -7- benzofuranyl-N- methyl carbamates,
It is a kind of efficient, agricultural chemicals of wide spectrum, has preventing and treating to endangering grain, veterinary antibiotics and the insect of industrial crops, acarid, nematode
Effect.Preventing and treating the snout moth's larva of rice, rice thripses, rice leaf roller, planthopper, rice leafhopper, Lissorhoptrus oryzophilus Kuschel, corn borer, corn rootwom,
Once the extensive use such as cotten aphid, bollworm, soybean aphid, eating-core bean worm.Furadan is mainly that interior inhale is killed to the intoxicating mode of insect
Worm acts on.Medicament imposes on the foundation part of soil crop, with moisture conveying to leaves and stemses after Root Absorption, enters after insect's food-taking
Polypide, reach the purpose for controlling worm.
Although the anthelminthic effect of Furadan is notable, Furadan has potential pathogenic to animal, human body, can cause
There is drop in blood pressure, unconsciousness in giddy, headache, vomiting, hidrosis, salivation, myosis, eye-blurred, severe patient, and skin goes out
Existing contact dermatitis such as rubella, local erubescence swell and itch, conjunctival congestion, shed tears, be uncomfortable in chest, expiratory dyspnea etc..By Chinese agricultural chemicals poison
Property grade scale, Furadan belong to high-toxic pesticide, it is impossible to on fruit and vegetables.The countries and regions such as the U.S., Europe are all tight
Lattice limit residual quantity of the Furadan in food.
At present, the pesticide residue detection method of Furadan includes liquid chromatography, Liquid Chromatography/Mass Spectrometry, Enzyme-linked Immunosorbent Assay point
Analysis method etc..Wherein, enzyme linked immunosorbent assay is the residual detection method of agriculture conventional in recent years, and the most important condition for establishing this method is to set
Meter synthesizes effective haptens and comlete antigen.
The content of the invention
It is an object of the present invention to provide a kind of carbofuran half-antigen and Furadan comlete antigen, can effectively pierce
Swash immunized animal and produce high sensitivity, the antibody of high specificity.
It is another object of the present invention to provide the preparation side of above-mentioned carbofuran half-antigen and Furadan comlete antigen
Method, this method are simple and easy.
The carbofuran half-antigen and Furadan comlete antigen that the present invention is prepared can be used for immune point of follow-up Furadan
Analysis method is established, the detection applied to Furadan in food.
To achieve the above object, the present invention uses following technical scheme：
A kind of carbofuran half-antigen, its molecular structure are：
A kind of Furadan comlete antigen, its molecular structure are：
The preparation method of the carbofuran half-antigen, comprises the following steps：
1) 1 part of benzofuranol is dissolved in 1~4 part of dichloromethane, first adds 1~1.3 part of isobutyl chlorocarbonate, then add
Enter 1~2 part of hydrogen chloride for absorbing generation of pyridine, reaction temperature is -5~5 DEG C, reacts to obtain mixture A；
2) add 1~4 part of water quenching to go out said mixture A, after liquid-liquid layering, take organic phase drying to be concentrated to give one light yellow
Liquid, as 2,2- dimethyl -2,3- Dihydrobenzofuranes -7- isobutyl group carbonic esters；
3) 1 part of 2,2- dimethyl -2,3- Dihydrobenzofuranes -7- isobutyl group carbonic ester is dissolved in 1~4 part of tetrahydrofuran
In, 1~2 part of 4- (amino methyl) benzoic acid and 1~4 part of wet chemical are added, reaction temperature is -5~5 DEG C, is reacted
Mixture B；
4) after said mixture B being carried out into liquid-liquid layering, take organic phase to dry and be concentrated to give a light yellow solid, it is light yellow
Solid obtains a white solid with 1~2 part of ethyl alcohol recrystallization, as described carbofuran half-antigen, the entitled 4- (((((2,2- bis- of chemistry
Methyl -2,3- Dihydrobenzofuranes -7- bases) epoxide) carbonyl) amino) methyl) benzoic acid.
The Furadan comlete antigen is prepared by carbofuran half-antigen, and its preparation method comprises the following steps：
1) carbofuran half-antigen is dissolved in dimethylformamide (DMF), adds dicyclohexylcarbodiimide (DCC) and N-
HOSu NHS (NHS), being stirred at room temperature 18-24 hours obtains A liquid；
Wherein, carbofuran half-antigen, DMF, DCC and NHS amount ratio are 20 μm of ol: 1mL: 60 μm of ol: 60 μm of ol；
2) carrier protein is dissolved in carbonate buffer solution, obtains B liquid；Wherein, carrier protein and carbonate buffer solution
Amount ratio is 0.4 μm ol: 3mL；
3) above-mentioned A drops are added in B liquid and stir 10-12 hours in 4 DEG C, obtain C liquid；
4) C liquid is placed in bag filter and dialysed in phosphate buffer, change liquid once within every 3 hours, dialysed 6 times altogether, collected
Solution in bag filter, as immunogen solution, i.e., described Furadan comlete antigen, -20 DEG C of preservations.
The carrier protein is bovine serum albumin (BSA).
The present invention uses above technical scheme, using benzofuranol as raw material, reacts generation intermediate product with isobutyl chlorocarbonate,
Intermediate product and 4- (amino methyl) benzoic acid are reacted in wet chemical again and obtain carbofuran half-antigen, by Furadan
Haptens obtains Furadan comlete antigen with carrier protein couplet.The carbofuran half-antigen and Furadan comlete antigen of the present invention
Preparation method it is simple and easy, carbofuran half-antigen, the Furadan comlete antigen being prepared, can effectively stimulate by immune dynamic
Thing produces high sensitivity, the antibody of high specificity, can be applied to the detection of Furadan in food.
Embodiment
The present invention is further described below in conjunction with specific embodiment.The reagent of embodiment unless otherwise indicated, is all
Conventional use for laboratory reagent purchased in market.
Embodiment 1
A kind of preparation method of carbofuran half-antigen, comprises the following steps：
1) 1 part of benzofuranol is dissolved in 2.5 parts of dichloromethane, first adds 1.2 parts of isobutyl chlorocarbonate, add pyrrole
1.5 parts of hydrogen chloride for absorbing generation of pyridine, reaction temperature is 0 DEG C, reacts to obtain mixture A；
2) add 2.5 parts of water quenchings to go out said mixture A, after liquid-liquid layering, take organic phase to dry and be concentrated to give a light yellow liquid
Body, as 2,2- dimethyl -2,3- Dihydrobenzofuranes -7- isobutyl group carbonic esters；
3) by 1 part 2,2- dimethyl -2,3- Dihydrobenzofuranes -7- isobutyl group carbonic esters are dissolved in 2.5 parts of tetrahydrofurans,
1.5 parts of 4- (amino methyl) benzoic acid and 1~4 part of wet chemical are added, reaction temperature is 0 DEG C, reacts to obtain mixture B；
4) after said mixture B being carried out into liquid-liquid layering, take organic phase to dry and be concentrated to give a light yellow solid, it is light yellow
Solid obtains a white solid with 1.5 parts of ethyl alcohol recrystallizations, as described carbofuran half-antigen, the entitled 4- (((((2,2- bis- of chemistry
Methyl -2,3- Dihydrobenzofuranes -7- bases) epoxide) carbonyl) amino) methyl) benzoic acid, molecular structure is：
Embodiment 2
A kind of preparation method of carbofuran half-antigen, comprises the following steps：
1) 1 part of benzofuranol is dissolved in 1 part of dichloromethane, first adds 1 part of isobutyl chlorocarbonate, add pyridine 1
Part absorbs the hydrogen chloride of generation, and reaction temperature is -5 DEG C, reacts to obtain mixture A；
2) add 1 part of water quenching to go out said mixture A, after liquid-liquid layering, take organic phase to dry and be concentrated to give a light yellow liquid
Body, as 2,2- dimethyl -2,3- Dihydrobenzofuranes -7- isobutyl group carbonic esters；
3) by 1 part 2,2- dimethyl -2,3- Dihydrobenzofuranes -7- isobutyl group carbonic esters are dissolved in 1 part of tetrahydrofuran, are added
Enter 1 part of 4- (amino methyl) benzoic acid and 1 part of wet chemical, reaction temperature is -5 DEG C, reacts to obtain mixture B；
4) after said mixture B being carried out into liquid-liquid layering, take organic phase to dry and be concentrated to give a light yellow solid, it is light yellow
Solid obtains a white solid with 1 part of ethyl alcohol recrystallization, as described carbofuran half-antigen.
Embodiment 3
A kind of preparation method of carbofuran half-antigen, comprises the following steps：
1) 1 part of benzofuranol is dissolved in 4 parts of dichloromethane, first adds 1.3 parts of isobutyl chlorocarbonate, add pyridine
2 parts of hydrogen chloride for absorbing generation, reaction temperature are 5 DEG C, react to obtain mixture A；
2) add 4 parts of water quenchings to go out said mixture A, after liquid-liquid layering, take organic phase to dry and be concentrated to give a light yellow liquid
Body, as 2,2- dimethyl -2,3- Dihydrobenzofuranes -7- isobutyl group carbonic esters；
3) by 1 part 2,2- dimethyl -2,3- Dihydrobenzofuranes -7- isobutyl group carbonic esters are dissolved in 4 parts of tetrahydrofurans, are added
Enter 2 parts of 4- (amino methyl) benzoic acid and 1~4 part of wet chemical, reaction temperature is 5 DEG C, reacts to obtain mixture B；
4) after said mixture B being carried out into liquid-liquid layering, take organic phase to dry and be concentrated to give a light yellow solid, it is light yellow
Solid obtains a white solid with 2 parts of ethyl alcohol recrystallizations, as described carbofuran half-antigen.
Embodiment 4
Furadan comlete antigen is prepared with the carbofuran half-antigen of embodiment 1, comprised the following steps：
1) 20 μm of ol carbofuran half-antigens are dissolved in 1mL DMF, add 60 μm of ol DCC and 60 μm of ol NHS, room temperature are stirred
Mix 24 hours and obtain A liquid；
2) 0.4 μm of ol BSA is dissolved in 3mL carbonate buffer solutions, obtains B liquid；
3) above-mentioned A drops are added in B liquid and stirred 12 hours in 4 DEG C, obtain C liquid；
4) C liquid is placed in bag filter and dialysed in phosphate buffer, change liquid once within every 3 hours, dialysed 6 times altogether, collected
Solution in bag filter, as immunogen solution, i.e., described Furadan comlete antigen, -20 DEG C of preservations.
Embodiment 5
Furadan comlete antigen is prepared with the carbofuran half-antigen of embodiment 2, comprised the following steps：
1) 20 μm of ol carbofuran half-antigens are dissolved in 1mL DMF, add 60 μm of ol DCC and 60 μm of ol NHS, room temperature are stirred
Mix 24 hours and obtain A liquid；
2) 0.4 μm of ol BSA is dissolved in 3mL carbonate buffer solutions, obtains B liquid；
3) above-mentioned A drops are added in B liquid and stirred 10 hours in 4 DEG C, obtain C liquid；
4) C liquid is placed in bag filter and dialysed in phosphate buffer, change liquid once within every 3 hours, dialysed 6 times altogether, collected
Solution in bag filter, as immunogen solution, i.e., described Furadan comlete antigen, -20 DEG C of preservations.
Embodiment 6
The identification of Furadan comlete antigen
The Furadan comlete antigen prepared with embodiment 4 is immunized Bal b/c mouse, 3 exempt from after collection antiserum detected,
Judge whether prepared comlete antigen has immunogenicity.
1) ELISA Plate is added after Furadan comlete antigen prepared by embodiment 4 being diluted into 2,1,0.5,0.25 μ g/mL, often
The μ L of hole 100, each concentration are coated with 6 holes, take out after 37 DEG C of incubation 3h, cleaned with PBST；
2) mice serum of above-mentioned collection is taken, dilutes 1000,2000,4000,8000,16000,32000 times, Mei Gekang
Original content hole adds a hole, per the μ L of hole 100.Take out after ELISA Plate then is placed in into 37 DEG C of incubation 0.5h, cleaned with PBST；
3) 0.5 μ g/mL goat anti-mouse antibody is added into enzyme mark hole, per the μ L of hole 100, ELISA Plate is then placed in 37 DEG C
Take out after incubating 0.5h, cleaned with PBST；
4) 100 μ L OPD nitrite ions are added into enzyme mark hole, color development at room temperature 15min, read 492nm light absorption values；
5) experimental result is shown, light absorption value increases with the extension rate of antigen, serum, and the rule being gradually reduced is presented, small
Mouse serum titer is between 16000-32000 times.
Embodiment 6
The detection of Furadan, comprises the following steps in food：
1) food (leek, potato, sweet potato etc.) 2g is weighed, is homogenized, after shaking extraction 30min with ethyl acetate 5mL, from
The heart takes 2mL supernatants, is dried up with nitrogen, adds 2mL PBS dissolved residues, as prepare liquid；
2) ELISA Plate is added after Furadan comlete antigen prepared by embodiment 4 being diluted into 0.5 μ g/mL, per the μ L of hole 100,
Take out after 37 DEG C of incubation 3h, cleaned with PBST；
3) 50 μ L prepare liquids are separately added into enzyme mark hole, then, mice serum prepared by Example 5, dilution 4000
Times, 50 μ L are added per hole.Take out after ELISA Plate then is placed in into 37 DEG C of incubation 0.5h, cleaned with PBST；
4) 0.5 μ g/mL goat anti-mouse antibody is added into enzyme mark hole, per the μ L of hole 100, ELISA Plate is then placed in 37 DEG C
Take out after incubating 0.5h, cleaned with PBST；
5) 100 μ L OPD nitrite ions are added into enzyme mark hole, color development at room temperature 15min, read 492nm light absorption values；
6) more different samples determine light absorption values, and Furadan residual quantity is higher in sample, and light absorption value is lower；Residual quantity is got over
Low, light absorption value is higher.
Claims (6)
- A kind of 1. carbofuran half-antigen, it is characterised in that：Its molecular structure is：
- A kind of 2. preparation method of carbofuran half-antigen as claimed in claim 1, it is characterised in that：It comprises the following steps：1) 1 part of benzofuranol is dissolved in 1~4 part of dichloromethane, first adds 1~1.3 part of isobutyl chlorocarbonate, add pyrrole 1~2 part of hydrogen chloride for absorbing generation of pyridine, reaction temperature is -5~5 DEG C, reacts to obtain mixture A；2) adding 1~4 part of water quenching to go out said mixture A, after liquid-liquid layering, take organic phase to dry concentration, obtained liquid is 2, 2- dimethyl -2,3- Dihydrobenzofuranes -7- isobutyl group carbonic esters；3) by 1 part 2,2- dimethyl -2,3- Dihydrobenzofuranes -7- isobutyl group carbonic esters are dissolved in 1~4 part of tetrahydrofuran, are added Enter 1~2 part of 4- (amino methyl) benzoic acid and 1~4 part of wet chemical, reaction temperature is -5~5 DEG C, reacts to obtain mixture B；4) after said mixture B being carried out into liquid-liquid layering, organic phase is taken to dry concentration, 1~2 part of second of the solid being concentrated to give Alcohol recrystallizes, and obtained product is the carbofuran half-antigen.
- A kind of 3. Furadan comlete antigen, it is characterised in that：Its molecular structure is：
- A kind of 4. application of Furadan comlete antigen as claimed in claim 3, it is characterised in that：The Furadan comlete antigen Detection applied to Furadan in food.
- A kind of 5. preparation method of Furadan comlete antigen as claimed in claim 3, it is characterised in that：The Furadan is complete Antigen is prepared by the carbofuran half-antigen described in claim 1, is comprised the following steps：1) carbofuran half-antigen is dissolved in dimethylformamide, adds dicyclohexylcarbodiimide and N- hydroxysuccinimidyls acyl is sub- Amine, being stirred at room temperature 18-24 hours obtains A liquid；Wherein, the amount ratio of carbofuran half-antigen, dimethylformamide, dicyclohexylcarbodiimide and n-hydroxysuccinimide For 20 μm of ol: 1mL: 60 μm of ol: 60 μm of ol；2) carrier protein is dissolved in carbonate buffer solution, obtains B liquid；Wherein, the dosage of carrier protein and carbonate buffer solution Than for 0.4 μm ol: 3mL；3) above-mentioned A drops are added in B liquid and stir 10-12 hours in 4 DEG C, obtain C liquid；4) C liquid is placed in bag filter and dialysed in phosphate buffer, change liquid once within every 3 hours, dialysed 6 times altogether, collect dialysis Solution in bag, as Furadan comlete antigen.
- A kind of 6. preparation method of Furadan comlete antigen according to claim 5, it is characterised in that：The carrier protein For bovine serum albumin.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112028786A (en) * | 2020-08-12 | 2020-12-04 | 华南农业大学 | Tyramine hapten, antigen and antibody, and preparation method and application thereof |
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|---|---|---|---|---|
| JPH08231591A (en) * | 1995-02-28 | 1996-09-10 | Otsuka Chem Co Ltd | Hapten, antigen, antibody specific to carbamate agent and detection of carbamate agent |
| CN1384103A (en) * | 2001-04-30 | 2002-12-11 | 浙江大学 | Carbofuran half-antigen, antigen and antibody and their prepn process |
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2017
- 2017-10-31 CN CN201711049977.2A patent/CN107759547A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08231591A (en) * | 1995-02-28 | 1996-09-10 | Otsuka Chem Co Ltd | Hapten, antigen, antibody specific to carbamate agent and detection of carbamate agent |
| CN1384103A (en) * | 2001-04-30 | 2002-12-11 | 浙江大学 | Carbofuran half-antigen, antigen and antibody and their prepn process |
Non-Patent Citations (2)
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| ANTONIO ABAD 等: "Development of Monoclonal Antibody-Based Immunoassays to the N-Methylcarbamate Pesticide Carbofuran", 《J. AGRIC. FOOD CHEM.》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112028786A (en) * | 2020-08-12 | 2020-12-04 | 华南农业大学 | Tyramine hapten, antigen and antibody, and preparation method and application thereof |
| CN112028786B (en) * | 2020-08-12 | 2022-02-11 | 华南农业大学 | Tyramine hapten, antigen and antibody, and preparation method and application thereof |
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