CN103073634A - Specific antibody against herbicide anilofos - Google Patents
Specific antibody against herbicide anilofos Download PDFInfo
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- CN103073634A CN103073634A CN2011103262869A CN201110326286A CN103073634A CN 103073634 A CN103073634 A CN 103073634A CN 2011103262869 A CN2011103262869 A CN 2011103262869A CN 201110326286 A CN201110326286 A CN 201110326286A CN 103073634 A CN103073634 A CN 103073634A
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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 a kind ofly have-COOH, again maximum possible comprise the anilofos original structure compound as the anilofos haptens, and haptens made antigen and then produces 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 that grows 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 notice that weedicide is to pollution and person poultry toxicity's problems in role of environment.Use the weedicide can be residual in grain etc., make people and animals' generation chronic hazard effect by food chain.The detection of anilofos in rice (brown rice) is limited to 0.05mg/kg in the Japan positive list system " in the food agriculture chemical residues limit the quantity of food volume/medicine volume "; The makings of anilofos mensuration lower bound is 0.025mg/kg in " agricultural chemical residues detection method enlarged edition 1 in the food "; The mensuration lower bound of anilofos is 0.01mg/kg to use gas chromatography-mass spectrography to measure wherein to 110 kinds of pesticide residue in the food in rice, brown rice, barley, the wheat and maize 5 in " People's Republic of China's inspection and quarantining for import/export industry standard " (2008-09-04 issue 2009-03-16 implements).
Yet, anilofos is because its molecular weight is 367.84, 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 because their general complex operations are complicated, cost is higher, analysis speed is slow, thus be difficult to satisfy 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, the detection technique that sensitive, simple to operate, 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, have high specificity and susceptibility, follow the law of mass action, but not only carry out in the body, also can externally carry out, these characteristics 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 the carrier protein coupling, prepare effective artificial antigen, the immune animal preparation is to 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 purpose be synthetic molecules structural formula anilofos haptens as shown below, then haptens is connected 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 and triethylamine, chloroacetyl chloride are in dry toluene to react at 1: 1: 1 according to feed ratio, maintain the temperature at 110 ℃ of backflow 5-6h.React complete after, filter, triethylamine hydrochloride to the filtrate water that generates with the benzene washing is 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 the silicagel column purifying, collects target components and revolves the majority of organic solvent that evaporates wherein, the room temperature cooling, the adularescent crystallization.Target compound is separated out in the normal hexane washing.
(2) haptenic synthetic
Thiovanic acid and CCAP react in sodium hydroxide solution according to 1: 1 feed ratio of mol ratio, and backflow 2h uses the concentrated hydrochloric acid acidifying, filter, and washing obtains target product.
(3) haptens ultrasonic 2h dissolving in DMSO.Again according to the mol ratio haptens: N-hydroxy-succinamide (NHS): N, N-dicyclohexylcarbodiimide (DCC) are 1: 5: 2 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 in bovine serum albumin (BSA) solution lentamente, and 4 ℃ of stirring reactions spend the night.Reaction product dialysis three days in 4 ℃, the phosphate buffered saline buffer (PBS) of pH 7.4 obtains immunogen.
(5) preparation of coating antigen
According to the mol ratio haptens: N-hydroxy-succinamide (NHS): N, N-dicyclohexylcarbodiimide (DCC) are 1: 5: 2 stirring reaction 24h, and the centrifugal precipitation of going out obtains Acibenzolar liquid.
Acibenzolar liquid is dropwise joined in ovalbumin (OVA) solution lentamente, and 4 ℃ of stirring reactions spend the night.Reaction product dialysis three days in 4 ℃, the phosphate buffered saline buffer (PBS) of pH 7.4 makes coating antigen.
(6) preparation method of anilofos enzyme-labelled antigen
Take by weighing the 1.02784mg haptens in a brown vial, add 150 μ l dimethyl sulfoxide (DMSO) (DMSO), ultrasonic 2h, the haptens dissolving, take by weighing respectively 1.96mg N-hydroxy-succinamide (NHS), 1.4045mg N, N-dicyclohexylcarbodiimide (DCC) adds in the 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 by weighing 5mg is dissolved in the NaHCO of 2ml
3In the solution, above-mentioned activated ester solution is slowly joined in ice bath in the HRP solution, stir under 4 ℃ of conditions and spend the night.Rear with pH7.4 phosphate buffered saline buffer dialysis 3 days, add Thiomersalate, 4 ℃ of Refrigerator stores;
(7) preparation of antibody and purifying:
Immune animal is selected 2 of male New Zealand large ear rabbits, and at 3 months monthly ages, about 1.5 kilograms of body weight are numbered respectively No. 1, No. 2.Immunity is taked subcutaneous and intramuscular injection is carried out jointly.
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 the 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: carried out booster immunization in the 14th day and 28 days behind the initial immunity, dosage is 0.5mg, and the NaCl that is dissolved in 1mL 0.9% is diluted to 1gL
-1, mix with the Freund's incomplete adjuvant of equivalent again and carry out emulsification.Later on every 14 days booster immunizations once, be total to immunity 6 times.Since the 2nd booster immunization, after each immune 8-10 days animal pilot production blood is carried out serum titer mensuration and avidity mensuration.Adopt the femoral artery blood-collecting method that animal is adopted whole blood after the last immunity, after centrifugal treating, collect whole serum, take the immunoaffinity chromatography method to carry out antibody purification, store for future use in 4 ℃ behind the sodium azide of gained antibody interpolation 0.1% (W/V).
Advantage of the present invention and positively effect
1. the present invention has farthest kept the chemical structure of anilofos, be pioneering new compound both at home and abroad, immunizing antigen with this haptens preparation goes the immune animal maximum possible to keep the molecular structure of original anilofos, and this has the antibody of high degree of specificity that assurance 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 characteristics such as special, sensitive, accurate, quick, cheap, and designed, synthetic haptens is laid a good foundation for the good antibody of preparation specificity.
4. through verification experimental verification, above-mentioned haptens, its synthetic method is simpler, and cheap, the easy acquisition of used main raw material, 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:
1.N-chloracetyl-N-sec.-propyl-4-chloroaniline (being called for short CCPA) is synthetic
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 by weighing 5.859g (0.035mol) N-sec.-propyl p-Chlorobenzoic acid amide, 28ml dry toluene, 3.57g (0.035mol) triethylamine and place round-bottomed flask, under stirring at room, utilize constant pressure funnel to wherein dropwise dripping 3.92g (0.035mol) chloroacetyl chloride (speed that control drips remains on below 65 ℃ temperature).After dropwising, maintain the temperature at 110 ℃ of backflow 5-6h.Cooling is filtered, and with the triethylamine hydrochloride that the benzene washing generates, 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 the silicagel column purifying, collects target components and revolves the majority of organic solvent that evaporates wherein, the room temperature cooling, the adularescent crystallization.The white crystal that the normal hexane washing is separated out.It is 245 that product carries out its molecular weight of Mass Spectrometric Identification.
2. haptenic synthetic
Concrete preparation method is: take by weighing the 2.25g dissolution of sodium hydroxide in 18ml water, to wherein adding 132.65mg (0.00144mol) Thiovanic acid, after stirring 15min, to the CCPA that wherein drops into 354.24mg (0.00144mol), backflow 2h, cooling is acidified to pH=1. with concentrated hydrochloric acid and filters, washing obtains white solid.It is 301 that product carries out its molecular weight of Mass Spectrometric Identification.
3. haptens activates ester synthesis
Concrete preparation method is: take by weighing 4.568mg (0.01515mmol) haptens in a brown vial, add 350 μ l DMSO, ultrasonic 2h, haptens dissolving.Take by weighing respectively again 8.711mg (0.07575mmol) N-hydroxy-succinamide (NHS), 6.242mg (0.0303mmol) N, N-dicyclohexylcarbodiimide (DCC) adds in the above-mentioned haptenic DMSO solution, stirring reaction 24h, the centrifugal precipitation of going out obtains Acibenzolar liquid.
4. artificial antigen is synthetic:
Adopt the haptens active ester method to be connected on the bovine serum albumin (BSA), synthetic artificial antigen, its molecular structural formula is:
Concrete preparation method is: the above-mentioned Acibenzolar liquid for 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 ℃ of reactions are spent the night.Reaction product dialysis three days in 4 ℃, the phosphate buffered saline buffer (PBS) of pH 7.4, then the volume of accurate measuring protein conjugate solution is measured concentration, packing ,-20 ℃ of preservations.
The evaluation of artificial antigen:
In the ratio of synthetic anilofos immunizing antigen reaction used carrier albumen and coupled product, carry out the ultraviolet (sweep measuring of 200mn～400nm).Conjugate haptens-BSA maximum absorption band occurs at the 274nm place, and the maximum absorption band of BSA is respectively 280, and there is obvious variation in both, shows the synthetic success of artificial antigen.
5. the preparation of coating antigen
Concrete building-up process is: take by weighing 6.80mg (0.0226mmol) haptens in a brown vial, add 400 μ l DMSO, ultrasonic 2h, haptens dissolving.Take by weighing respectively again 12.98mg (0.1128mmol) N-hydroxy-succinamide (NHS), 9.3mg (0.045mmol) N, N-dicyclohexylcarbodiimide (DCC) adds in the 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 ℃ of reactions are spent the night.Reaction product dialysis three days in 4 ℃, the phosphate buffered saline buffer (PBS) of pH 7.4, then the volume of accurate measuring protein conjugate solution is measured concentration, packing ,-20 ℃ 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, about 1.5 kilograms of body weight are raised in the standard test Animal House, and Continuous Observation 7 days is determined to carry out immunity after physical appearance normally.Immunity is taked subcutaneous and intramuscular injection is carried out jointly.
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 the 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: carried out booster immunization in the 14th day and 28 days behind the initial immunity, dosage is 0.5mg, and the NaCl that is dissolved in 1mL 0.9% is diluted to 1gL
-1, mix with the Freund's incomplete adjuvant of equivalent again and carry out emulsification.Later on every 14 days booster immunizations once, be total to immunity 6 times.Since the 2nd booster immunization, after each immune 8-10 days animal pilot production blood is carried out serum titer mensuration and avidity mensuration.Adopt the femoral artery blood-collecting method that animal is adopted whole blood after the last immunity, after centrifugal treating, collect whole serum, take the immunoaffinity chromatography method to carry out antibody purification, store for future use in 4 ℃ behind the sodium azide of gained antibody interpolation 0.1% (W/V).
Claims (4)
1. the artificial antigen of herbicide anilofos and antibody is characterized in that using molecular formula to be
Haptens be connected synthetic artificial antigen with bovine serum albumin, is connected with horseradish peroxidase and synthesizes enzyme-labelled antigen, wherein artificial antigen passes through immune new zealand rabbit again, gets blood, isolates the antiserum(antisera) purifying and makes antibody;
2. the preparation method of herbicide anilofos artificial antigen claimed in claim 1 it is characterized in that making with following step:
(1) N-chloracetyl-N-sec.-propyl-4-chloroaniline (CCPA) is synthetic
Take by weighing 5.859g N-sec.-propyl p-Chlorobenzoic acid amide, the 28ml dry toluene, 3.57g triethylamine places round-bottomed flask, under stirring at room, utilize constant pressure funnel to wherein dropwise dripping the 3.92g chloroacetyl chloride, the speed that control drips remains on below 65 ℃ temperature, after dropwising, maintain the temperature at 110 ℃ of backflow 5-6h, cooling, filter, with the triethylamine hydrochloride that the benzene washing generates, filtrate water is washed till neutrality, anhydrous sodium sulfate drying, filter, obtain a dark solution, dark solution is crossed the silicagel column purifying, collect target components and revolve the majority of organic solvent that evaporates wherein, the room temperature cooling, the white crystals of separating out is N-chloracetyl-N-sec.-propyl-4-chloroaniline;
(2) haptenic synthetic
Take by weighing the 2.25g dissolution of sodium hydroxide in 18ml water, to wherein adding the 132.65mg Thiovanic acid, stir 15min after, to the CCPA that wherein drops into 354.24mg, backflow 2h, cooling, be acidified to pH=1. with concentrated hydrochloric acid and filter, washing obtains haptenic white solid;
(3) take by weighing the 4.568mg haptens in a brown vial, add 350 μ l dimethyl sulfoxide (DMSO) (DMSO), ultrasonic 2h, take by weighing respectively again 8.711mg N-hydroxy-succinamide (NHS), 6.242mg N after the haptens dissolving, N-dicyclohexylcarbodiimide (DCC) adds in the above-mentioned haptenic DMSO solution, stirring reaction 24h, the centrifugal precipitation of going out obtains Acibenzolar liquid;
(4) artificial antigen is synthetic
Above-mentioned Acibenzolar liquid dropwise joined lentamente by 20mg bovine serum albumin (BSA) be dissolved in the solution that configures in the PBS damping fluid of 4ml pH=7.4,4 ℃ of reactions are spent the night, reaction product dialysis three days in 4 ℃, the phosphate buffered saline buffer (PBS) of pH 7.4, then the volume of accurate measuring protein conjugate solution, measure concentration, packing ,-20 ℃ of preservations;
3. the preparation method of anilofos enzyme-labelled antigen claimed in claim 1 it is characterized in that making with following step:
Take by weighing the 1.02784mg haptens in a brown vial, add 150 μ l dimethyl sulfoxide (DMSO) (DMSO), ultrasonic 2h, the haptens dissolving, take by weighing respectively 1.96mg N-hydroxy-succinamide (NHS), 1.4045mg N, N-dicyclohexylcarbodiimide (DCC) adds in the 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 by weighing 5mg is dissolved in the NaHCO of 2ml
3In the solution, above-mentioned activated ester solution is slowly joined in ice bath in the HRP solution, stir under 4 ℃ of conditions and spend the night.Rear with pH7.4 phosphate buffered saline buffer dialysis 3 days, add Thiomersalate, 4 ℃ of Refrigerator stores;
4. the preparation method of herbicide anilofos antibody claimed in claim 1 it is characterized in that making with following step:
Immune animal is selected male new zealand white rabbit, immunization method adopts subcutaneous and intramuscular injection, carry out booster immunization after just exempting from four times, booster immunization respectively at initial immunity after 2 weeks, 4 weeks, immunity is four times after 6 weeks and 8 weeks, immunity was got blood by the auricular vein of rabbit in rear 9 days, carried out bioactivity, and specific practice is:
Initial immunity: get 1mg and be dissolved in the solution that 0.9% NaCl solution and Freund's complete adjuvant equal-volume prepare according to the synthetic artificial antigen of the described method of claim 2, carry out animal immune;
Booster immunization: be dissolved in the solution that 0.9% NaCl solution and Freund's incomplete adjuvant equal-volume are prepared with the above-mentioned artificial antigen of 0.5mg, carry out animal immune;
Antibody purification: the periodic monitor animal's antibody is tired, when antibody reaches suitable tiring to certain envelope antigen, gather blood, and centrifugal acquisition antiserum(antisera), use G-Sepharose albumen affinity column antagonistic Serum to carry out purifying, obtain the specific antibody of anti-anilofos.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105675858A (en) * | 2016-03-15 | 2016-06-15 | 中国烟草总公司郑州烟草研究院 | Enzyme-linked immunosorbent assay kit for detecting quinclorac and application of enzyme-linked immunosorbent assay kit |
| CN109265364A (en) * | 2018-09-21 | 2019-01-25 | 中国烟草总公司郑州烟草研究院 | A kind of preparation and application of pendimethalin haptens and antigen |
| CN110054576A (en) * | 2019-04-15 | 2019-07-26 | 华南农业大学 | A kind of isopropyl methoxalamine chirality haptens, artificial antigen and antibody and its preparation method and application |
| 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 |
| CN113698430A (en) * | 2021-10-29 | 2021-11-26 | 潍坊新绿化工有限公司 | Preparation method of herbicide anilofos |
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| 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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| 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 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105675858A (en) * | 2016-03-15 | 2016-06-15 | 中国烟草总公司郑州烟草研究院 | Enzyme-linked immunosorbent assay kit for detecting quinclorac and application of enzyme-linked immunosorbent assay kit |
| CN109265364A (en) * | 2018-09-21 | 2019-01-25 | 中国烟草总公司郑州烟草研究院 | A kind of preparation and application of pendimethalin haptens and antigen |
| 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 |
| CN110054576A (en) * | 2019-04-15 | 2019-07-26 | 华南农业大学 | A kind of isopropyl methoxalamine chirality haptens, artificial antigen and antibody and its preparation method and application |
| 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 |
| CN113698430A (en) * | 2021-10-29 | 2021-11-26 | 潍坊新绿化工有限公司 | Preparation method of herbicide anilofos |
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| CN103073634B (en) | 2014-07-02 |
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