CN103073634B - Specific antibody against herbicide anilofos - Google Patents
Specific antibody against herbicide anilofos Download PDFInfo
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- CN103073634B CN103073634B CN201110326286.9A CN201110326286A CN103073634B CN 103073634 B CN103073634 B CN 103073634B CN 201110326286 A CN201110326286 A CN 201110326286A CN 103073634 B CN103073634 B CN 103073634B
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Abstract
The invention provides preparation of a semi-antigen and an artificial antigen and an antibody against herbicide anilofos, which relates to the semi-antigen, the artificial antigen and the antibody which have a basic structure of the herbicide anilofos. According to the invention, the antibody with good specificity to the herbicide anilofos is prepared by using an easy and convenient method. The antibody is prepared through the following steps: synthesizing the semi-antigen from N-chloracetyl-N-isopropyl-4-chloroaniline and mercaptoacetic acid; respectively connecting the semi-antigen with bovine serum albumin (BSA) and horseradish peroxidase (HRP) to synthesize the artificial antigen and an enzyme-labeled antigen; and subjecting the artificial antigen to animal immunization, blood drawing, separation of antiserum and purification so as to prepare the antibody. The antibody is stable and has good specificity and sensitivity; the synthetic method for the antibody is simple; and the antibody can be used for rapid immunodetection of residual herbicide anilofos in the environment, agricultural products and foodstuffs and has a good application prospect.
Description
Technical field
The present invention relates to select compound a kind of have-COOH, that maximum possible comprises anilofos original structure again as anilofos haptens, and haptens is made to antigen and then produced antibody; And the synthetic and preparation method for antibody of this type of haptens, antigen.The invention belongs to biological technical field.
Background technology
Weedicide is a kind of agricultural chemicals type growing up gradually over nearly 20 years, and along with the development of chemical industry, the kind of weedicide also increases gradually.Weedicide in China's development and operation has also reached tens of kinds.Some weedicide has teratogenesis, and the security of weedicide has caused people's concern, should note pollution and the person poultry toxicity problems in role of weedicide to environment.Use the weedicide can be residual in grain etc., make people and animals' generation chronic hazard effect by food chain.In Japan positive list system " agriculture chemical residues limitation food volume/medicine volume in food ", the detection of anilofos in rice (brown rice) is limited to 0.05mg/kg; In " agricultural chemical residues detection method enlarged edition 1 in food ", the makings of anilofos mensuration lower bound is 0.025mg/kg; In " People's Republic of China's inspection and quarantining for import/export industry standard " (2008-09-04 issues 2009-03-16 and implements), using gas chromatography-mass spectrography to measure the wherein mensuration lower bound of anilofos to 110 kinds of pesticide residue in food in rice, brown rice, barley, wheat and maize 5 is 0.01mg/kg.
But, anilofos is because its molecular weight is 367.84, be less than 1000dolton (dalton), generally adopt traditionally the physical chemistry methods such as gas-chromatography (GC), liquid chromatography (HPLC) or GC-MS coupling to its residual detection.Although these traditional physico-chemical analysis method sensitivity are higher, and comparatively accurate, but due to their general complex operation complexity, cost is higher, analysis speed is slow, so be difficult to meet the needs of actual analysis, therefore in the urgent need to developing a kind of simple, quick, sensitive analytical technology.
And immuno analytical method a kind of quick, sensitive, simple to operate, detection technique that expense is low just.Its ultimate principle is thought: the immune response of antigen-antibody relates to intermolecular three-dimensional arrangement, the factors such as electric charge, hydrogen bond and Van der Waals force effect, there is high specificity and susceptibility, follow the law of mass action, not only can in body, carry out, also can externally carry out, these features can be utilized sets up immune analysis method, can reach traditional physico-chemical analysis technology be beyond one's reach selectivity and sensitivity.So immunoassay provides a new analyzing and testing approach for the residual research of anilofos.
Summary of the invention
The present invention designs and has synthesized small molecules target analytes haptens, and with carrier protein coupling, prepare effective artificial antigen, immune animal is prepared small molecule analysis thing specific antibody, utilize the specificity immunology reaction of antigen-antibody, thereby ultramicron small molecules target compound in the detection sample of qualitative, quantitative, can be used for sample and measures.The key of this technical study is the preparation of haptenic molecular designing, synthetic and holoantigen and antibody.
The present invention is that to reach the designed technical scheme of above object be synthetic molecules structural formula anilofos haptens as shown below, then haptens is connected to synthetic artificial antigen with carrier proteins, and immune animal, obtains antibody.
(1) N-chloracetyl-N-sec.-propyl-4-chloroaniline (being called for short CCPA) is synthetic
Raw material N-sec.-propyl p-Chlorobenzoic acid amide is in dry toluene to react at 1: 1: 1 with triethylamine, chloroacetyl chloride according to feed ratio, maintains the temperature at 110 DEG C of backflow 5-6h.After completion of the reaction, filter, wash with benzene triethylamine hydrochloride to the filtrate water generating and be washed till neutrality, anhydrous sodium sulfate drying.Product is crossed silicagel column purifying (eluent ethyl acetate: normal hexane=1: 10, every 10ml normal hexane adds 200 μ l glacial acetic acids), TLC follows the tracks of silicagel column purifying, collects target components and revolves the majority of organic solvent evaporating wherein, room temperature is cooling, adularescent crystallization.Target compound is separated out in normal hexane washing.
(2) haptenic synthetic
Thiovanic acid reacts according to 1: 1 feed ratio of mol ratio with CCAP in sodium hydroxide solution, and backflow 2h, uses concentrated hydrochloric acid acidifying, filters, and washing, obtains target product.
(3) haptens ultrasonic 2h in DMSO dissolves.Again according to mol ratio haptens: N-hydroxy-succinamide (NHS): N, N-dicyclohexylcarbodiimide (DCC) is 1: 5: 2 stirring reaction 24h, the centrifugal precipitation of going out, obtains Acibenzolar liquid.
(4) artificial antigen is synthetic: above-mentioned Acibenzolar liquid is dropwise joined in bovine serum albumin (BSA) solution lentamente, and 4 DEG C of stirring reactions spend the night.Reaction product dialysis three days in 4 DEG C, the phosphate buffered saline buffer (PBS) of pH 7.4, obtains immunogen.
(5) preparation of coating antigen
According to mol ratio haptens: N-hydroxy-succinamide (NHS): N, N-dicyclohexylcarbodiimide (DCC) is 1: 5: 2 stirring reaction 24h, the centrifugal precipitation of going out, obtains Acibenzolar liquid.
Acibenzolar liquid is dropwise joined in ovalbumin (OVA) solution lentamente, and 4 DEG C of stirring reactions spend the night.Reaction product dialysis three days in 4 DEG C, the phosphate buffered saline buffer (PBS) of pH 7.4, makes coating antigen.
(6) preparation method of anilofos enzyme-labelled antigen
Take 1.02784mg haptens in a brown vial, add 150 μ l dimethyl sulfoxide (DMSO) (DMSO), ultrasonic 2h, haptens dissolves, take respectively 1.96mgN-N-Hydroxysuccinimide (NHS), 1.4045mgN, N-dicyclohexylcarbodiimide (DCC) adds in above-mentioned haptenic DMSO solution, stirring reaction 24h again, the centrifugal precipitation of going out, obtains Acibenzolar liquid; Then the horseradish peroxidase (HRP) that accurately takes 5mg is dissolved in the NaHCO of 2ml
3in solution, above-mentioned activated ester solution is slowly joined in ice bath in HRP solution, under 4 DEG C of conditions, stir and spend the night.With pH7.4 phosphate buffered saline buffer dialysis 3 days, add Thiomersalate, 4 DEG C of Refrigerator stores afterwards;
(7) preparation of antibody and purifying:
Immune animal is selected 2 of male New Zealand large ear rabbits, and at 3 months monthly ages, 1.5 kilograms of left and right of body weight, are numbered respectively No. 1, No. 2.Immunity takes subcutaneous and intramuscular injection jointly to carry out.
Immune programme for children: initial immunity: for improving the immunity of antigen, adopt the immunogen by Freund's complete adjuvant emulsification, every rabbit of initial immunity is used 1mg artificial antigen, uses the NaCl solution dilution of 1mL 0.9% to 1g L
-1, with immunity after the emulsification of Freund's complete adjuvant equal-volume.Booster immunization: carry out booster immunization in after initial immunity the 14th day and 28 days, dosage is 0.5mg, and the NaCl that is dissolved in 1mL 0.9% is diluted to 1gL
-1, then mix and carry out emulsification with the Freund's incomplete adjuvant of equivalent.Once every 14 days booster immunizations, be total to immunity 6 times later.Since the 2nd booster immunization, after each immune 8-10 days, animal pilot production blood is carried out to serum titer mensuration and avidity mensuration.After last immunity, adopt femoral artery blood-collecting method to adopt whole blood to animal, after centrifugal treating, collect whole serum, take immunoaffinity chromatography method to carry out antibody purification, after the sodium azide of gained antibody interpolation 0.1% (W/V), store for future use in 4 DEG C.
Advantage of the present invention and positively effect
1. the present invention has farthest retained the chemical structure of anilofos, for domestic and international pioneering new compound, go immune animal maximum possible to retain the molecular structure of original anilofos with immunizing antigen prepared by this haptens, this has the antibody of high degree of specificity that guarantee is provided for obtaining to anilofos.
2. on the haptenic basis of synthetic anilofos, synthetic its Acibenzolar can improve the connection rate of haptens and high molecular weight protein greatly.
3. the present invention, has the features such as special, sensitive, accurate, quick, cheap, and designed, synthetic haptens is that the good antibody of preparation specificity is laid a good foundation.
4. through verification experimental verification, above-mentioned haptens, its synthetic method is simpler, and main raw material used is cheap, easily obtain, and all can buy in general chemical reagents corporation.
5. the present invention is through the above-mentioned haptens of verification experimental verification, its simple synthetic method, and combined coefficient is high, and reactions steps is few, has improved the controllability of reaction; In addition, extraction, the purification process of synthetic product are easy, are easy to popularize.
Embodiment
Following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Embodiment 1:
Synthesizing of 1.N-chloracetyl-N-sec.-propyl-4-chloroaniline (being called for short CCPA)
It is the synthetic intermediate product of haptens that the present invention selects CCAP, and its molecular structural formula is:
Concrete preparation method is: take 5.859g (0.035mol) N-sec.-propyl p-Chlorobenzoic acid amide, 28ml dry toluene, 3.57g (0.035mol) triethylamine and be placed in round-bottomed flask, under stirring at room temperature, utilize constant pressure funnel dropwise to drip wherein 3.92g (0.035mol) chloroacetyl chloride (controlling the speed dripping makes temperature remain on 65 DEG C below).After dropwising, maintain the temperature at 110 DEG C of backflow 5-6h.Cooling, filter, the triethylamine hydrochloride generating with benzene washing, filtrate water is washed till neutrality, and anhydrous sodium sulfate drying filters, and obtains a dark solution, carries out Mass Spectrometric Identification.Product is crossed silicagel column purifying (eluent ethyl acetate: normal hexane=1: 10, every 10ml normal hexane adds 200 μ l glacial acetic acids), TLC follows the tracks of silicagel column purifying, collects target components and revolves the majority of organic solvent evaporating wherein, room temperature is cooling, adularescent crystallization.Normal hexane washs the white crystal of separating out.It is 245 that product carries out its molecular weight of Mass Spectrometric Identification.
2. haptenic synthetic
Concrete preparation method is: take 2.25g dissolution of sodium hydroxide in 18ml water, add wherein 132.65mg (0.00144mol) Thiovanic acid, stir after 15min, drop into wherein the CCPA of 354.24mg (0.00144mol), backflow 2h, cooling, be acidified to pH=1. with concentrated hydrochloric acid and filter, washing, obtains white solid.It is 301 that product carries out its molecular weight of Mass Spectrometric Identification.
3. haptens Acibenzolar is synthetic
Concrete preparation method is: takes 4.568mg (0.01515mmol) haptens in a brown vial, adds 350 μ l DMSO, and ultrasonic 2h, haptens dissolves.Take respectively again 8.711mg (0.07575mmol) N-hydroxy-succinamide (NHS), 6.242mg (0.0303mmol) N, N-dicyclohexylcarbodiimide (DCC) adds in above-mentioned haptenic DMSO solution, stirring reaction 24h, the centrifugal precipitation of going out, obtains Acibenzolar liquid.
4. artificial antigen is synthetic:
Adopt haptens active ester method to be connected to bovine serum albumin (BSA) upper, synthetic artificial antigen, its molecular structural formula is:
Concrete preparation method is: the above-mentioned Acibenzolar liquid preparing is dropwise joined in bovine serum albumin (BSA) solution (20mg BSA is dissolved in the PBS damping fluid of 4ml pH=7.4) lentamente, and 4 DEG C of reactions are spent the night.Reaction product dialysis three days in 4 DEG C, the phosphate buffered saline buffer (PBS) of pH 7.4, the then volume of accurate measuring protein conjugate solution, measures concentration, packing ,-20 DEG C of preservations.
The qualification of artificial antigen:
In the ratio of synthetic anilofos immunizing antigen reaction used carrier albumen and coupled product, carry out ultraviolet (200nm～400nm) sweep measuring.There is maximum absorption band at 274nm place in conjugate haptens-BSA, and the maximum absorption band of BSA is respectively 280, and both exist obvious variation, shows the synthetic success of artificial antigen.
5. the preparation of coating antigen
Concrete building-up process is: takes 6.80mg (0.0226mmol) haptens in a brown vial, adds 400 μ l DMSO, and ultrasonic 2h, haptens dissolves.Take respectively again 12.98mg (0.1128mmol) N-hydroxy-succinamide (NHS), 9.3mg (0.045mmol) N, N-dicyclohexylcarbodiimide (DCC) adds in above-mentioned haptenic DMSO solution, stirring reaction 24h, the centrifugal precipitation of going out, obtains Acibenzolar liquid.
Above-mentioned Acibenzolar liquid is dropwise joined in ovalbumin (OVA) solution (20mg OVA is dissolved in the PBS damping fluid of 4ml pH=7.4) lentamente, and 4 DEG C of reactions are spent the night.Reaction product dialysis three days in 4 DEG C, the phosphate buffered saline buffer (PBS) of pH 7.4, the then volume of accurate measuring protein conjugate solution, measures concentration, packing ,-20 DEG C of preservations.
6. the preparation of antibody and purifying:
Immune animal is selected 2 of New Zealand's large ear rabbits, and at 3 months monthly ages, 1.5 kilograms of left and right of body weight, raise in standard test Animal House, and Continuous Observation 7 days is determined after physical appearance normally and carried out immunity.Immunity takes subcutaneous and intramuscular injection jointly to carry out.
Immune programme for children: initial immunity: for improving the immunity of antigen, adopt the immunogen by Freund's complete adjuvant emulsification, every rabbit of initial immunity is used 1mg artificial antigen, uses the NaCl solution dilution of 1mL 0.9% to 1g L
-1, with immunity after the emulsification of Freund's complete adjuvant equal-volume.Booster immunization: carry out booster immunization in after initial immunity the 14th day and 28 days, dosage is 0.5mg, and the NaCl that is dissolved in 1mL 0.9% is diluted to 1gL
-1, then mix and carry out emulsification with the Freund's incomplete adjuvant of equivalent.Once every 14 days booster immunizations, be total to immunity 6 times later.Since the 2nd booster immunization, after each immune 8-10 days, animal pilot production blood is carried out to serum titer mensuration and avidity mensuration.After last immunity, adopt femoral artery blood-collecting method to adopt whole blood to animal, after centrifugal treating, collect whole serum, take immunoaffinity chromatography method to carry out antibody purification, after the sodium azide of gained antibody interpolation 0.1% (W/V), store for future use in 4 DEG C.
Claims (1)
1. a preparation method for the artificial antigen of herbicide anilofos, is characterized in that, uses molecular formula to be
Haptens be connected synthetic artificial antigen with bovine serum albumin, concrete preparation is made up of following step:
(1) N-chloracetyl-N-sec.-propyl-4-chloroaniline is synthetic
Take 5.859g N-sec.-propyl p-Chlorobenzoic acid amide, 28ml dry toluene, 3.57g triethylamine is placed in round-bottomed flask, under stirring at room temperature, utilize constant pressure funnel dropwise to drip wherein 3.92g chloroacetyl chloride, controlling the speed dripping remains on below 65 DEG C temperature, after dropwising, maintain the temperature at 110 DEG C of backflow 5-6h, cooling, filter, the triethylamine hydrochloride generating with benzene washing, filtrate water is washed till neutrality, anhydrous sodium sulfate drying, filter, obtain a dark solution, dark solution is crossed to silicagel column purifying, collect target components and revolve the majority of organic solvent evaporating wherein, room temperature is cooling, the white crystals of separating out is N-chloracetyl-N-sec.-propyl-4-chloroaniline,
(2) haptenic synthetic
Take 2.25g dissolution of sodium hydroxide in 18ml water, add wherein 132.65mg Thiovanic acid, stir after 15min, drop into wherein the CCPA of 354.24mg, backflow 2h, cooling, be acidified to pH=1. with concentrated hydrochloric acid and filter, washing, obtains white solid;
(3) take 4.568mg haptens in a brown vial, add 350 μ l DMSO, ultrasonic 2h, haptens dissolves.Take respectively 8.711mgN-N-Hydroxysuccinimide, 6.242mgN, N-dicyclohexylcarbodiimide adds in above-mentioned haptenic DMSO solution again, stirring reaction 24h, and the centrifugal precipitation of going out, obtains Acibenzolar liquid;
(4) artificial antigen is synthetic:
Above-mentioned Acibenzolar liquid is dropwise joined lentamente by 20mg bovine serum albumin and is dissolved in the solution configuring in the PBS damping fluid of 4mlpH=7.4,4 DEG C of reactions are spent the night, reaction product is dialysed three days in 4 DEG C, the phosphate buffered saline buffer of pH7.4, then the volume of accurate measuring protein conjugate solution, measure concentration, packing ,-20 DEG C of preservations.
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| CN105675858B (en) * | 2016-03-15 | 2017-07-21 | 中国烟草总公司郑州烟草研究院 | Detect enzyme linked immunological kit and its application of dichloro quinolinic acid |
| CN109265364B (en) * | 2018-09-21 | 2021-02-02 | 中国烟草总公司郑州烟草研究院 | Preparation and application of pendimethalin hapten and antigen |
| CN110054576B (en) * | 2019-04-15 | 2021-03-12 | 华南农业大学 | Metolachlor chiral hapten, artificial antigen and antibody as well as preparation method and application thereof |
| CN110156651A (en) * | 2019-04-17 | 2019-08-23 | 深圳市易瑞生物技术股份有限公司 | A kind of propargite haptens and its synthetic method and application |
| CN113698430B (en) * | 2021-10-29 | 2022-01-25 | 潍坊新绿化工有限公司 | Preparation method of herbicide anilofos |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005030736A1 (en) * | 2003-09-29 | 2005-04-07 | Isagro Ricerca S.R.L. | Derivatives of 1,3-diones having a herbicidal activity |
| WO2005074685A1 (en) * | 2004-02-06 | 2005-08-18 | Bayer Cropscience Gmbh | Plant protection compositions and use thereof |
| CN101396016A (en) * | 2008-11-06 | 2009-04-01 | 蔡国庆 | Effervescent granule containing pyrazosulfuron and anilofos and preparation method thereof |
| CN101971859A (en) * | 2010-10-22 | 2011-02-16 | 吉林金秋农药有限公司 | Weedicide composition, preparation method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005030736A1 (en) * | 2003-09-29 | 2005-04-07 | Isagro Ricerca S.R.L. | Derivatives of 1,3-diones having a herbicidal activity |
| WO2005074685A1 (en) * | 2004-02-06 | 2005-08-18 | Bayer Cropscience Gmbh | Plant protection compositions and use thereof |
| CN101396016A (en) * | 2008-11-06 | 2009-04-01 | 蔡国庆 | Effervescent granule containing pyrazosulfuron and anilofos and preparation method thereof |
| CN101971859A (en) * | 2010-10-22 | 2011-02-16 | 吉林金秋农药有限公司 | Weedicide composition, preparation method and application thereof |
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