CN102002103A - Pesticide trichlorfon-resistant specific antibody - Google Patents
Pesticide trichlorfon-resistant specific antibody Download PDFInfo
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Abstract
The invention discloses a method for preparing artificial antigen and antibody of pesticide trichlorfon, and relates to a method for preparing artificial hapten, antigen and antibody which keep the structure of the pesticide trichlorfon to the greatest degree. The active ester of the pesticide trichlorfon hapten prepared by a chemical synthesis method and a carrier protein keyhole hemocyanin are connected to synthesize the artificial antigen, and the antibody is prepared by performing animal immunization and blood exaction on the artificial antigen, separating the antiserum and purifying. By designing and synthesizing hapten and artificial antigen with the trichlorfon structure, the method highlights a specific antigenic determinant of the pesticide molecule and overcomes the difficulty in chemical synthesis, and a high-affinity antibody is induced by an immune animal. Compared with other like methods, the method has the characteristics of specificity, sensitivity, accuracy, rapidness, convenience, low cost and the like, and the designed and synthesized hapten lays a foundation for preparing a high-specificity antibody.
Description
Technical field
The present invention relates to select a kind ofly have-COOH, again maximum possible comprise the Trichlorphon original structure compound as the Trichlorphon haptens, and haptens made antigen and then produces antibody; And this type of haptens, antigenic synthetic and preparation method for antibody.The invention belongs to biological technical field.
Background technology
Along with modern agricultural development, agricultural chemicals is being brought into play more and more important effect on the one hand in agriculture production, on the other hand, the unreasonable use of agricultural chemicals has also caused the serious pesticide residue problem that exceeds standard, and has a strong impact on the healthy of people and export of farm produce trade.Trichlorphon is a kind of water miscible sterilant, and pure product are white crystalline powder, to having showed outstanding selection toxicity between higher animal and insect.And Trichlorphon still is the precursor substance of SD-1750, and in alkaline medium, easily the dehydrochlorination molecular transposition becomes the bigger SD-1750 of virulence.There is comparatively strict standard in China for the residual quantity of Trichlorphon in agricultural-food.Regulation according to GB2763-2005: paddy≤0.1mg/mL, wheat≤0.1mg/mL, fruit≤0.1mg/mL, vegetables≤0.1mg/mL.
Yet Trichlorphon agricultural chemicals and meta-bolites thereof because its molecular weight is 257.5, less than 1000dolton (dalton), generally adopt physical chemistry methods such as gas-chromatography (GC), liquid chromatography (HPLC) or mass spectrum to its residual detection traditionally at present.Though these traditional physico-chemical analysis method sensitivity are higher, and it is comparatively accurate, but because their general complex operation complexity, cost is higher, analysis speed is slow, thus be difficult to satisfy the needs of actual analysis, therefore press for development a kind of simple, fast, the sensitive analytical technology.
And a kind of just quick, sensitive, simple to operate, detection technique that expense is low of immuno analytical method.Its ultimate principle is thought: the immune response of antigen-antibody relates to intermolecular three-dimensional arrangement, electric charge, hydrogen bond and Van der Waals force effect etc. are all multifactor, 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 it and set 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 Trichlorphon.
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 molecules analyte specific antibody, utilize the amplification of the marker of the specificity immunology reaction of antigen-antibody and easy detected identification, 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 Trichlorphon haptens as shown below
, then haptens being connected synthetic artificial antigen with carrier proteins, immune animal obtains antibody.
(1) Trichlorphon raw material [(2,2,2-three chloro-1-hydroxyethyls) chlorooxon] reacts in anhydrous pyridine with Succinic anhydried; generate compound 2,2,2-three chloro-1-(dimethoxy phosphoryl)-ethyl monoesters succsinic acid; feed ratio is 1: 1～10, room temperature reaction 12～36 hours.After reaction finishes, remove the solution pickling of pyridine with concentrated hydrochloric acid and water 1: 2～20, use ethyl acetate extraction, anhydrous sodium sulphate dewaters, and steaming removes ethyl acetate and gets target product.Adopt recrystallization method to carry out purifying, obtain purer ground target product.
(2), compound 2; 2; 2-three chloro-1-(dimethoxy phosphoryl)-ethyl monoesters succsinic acid and N-hydroxy-succinamide (being NHS) N-hydroxy-succinamide (being NHS) react in anhydrous methylene chloride; stirring reaction is after 0.5～2 hour under the ice bath; under nitrogen protection, dropwise drip the anhydrous tetrahydro furan that is dissolved with DCC; feed ratio is 1: 2～4: 2～5, continues stirring reaction 1～4h under ice bath, rises to stirred overnight at room temperature then naturally.After the termination reaction, remove by filter white precipitate (being DCU).Obtain further perfect Trichlorphon artificial semiantigen Acibenzolar { [2,2,2-three chloro-1-(dimethoxy phosphoryl) B carbonyl] succsinic acid (2,5-dioxo cyclopentyl) ester } through this reaction.
Carry out column chromatography and carry out purifying by preparing suitable polarity developping agent.Wherein the developping agent proportioning is: ethyl acetate: normal hexane=2～6: 1, add about 1% glacial acetic acid again.
The present invention also provides above-mentioned Trichlorphon haptenic purposes, is the raw material as the antigen system of animal immune.
(3), artificial antigen is synthetic: get haptens [2,2,2-three chloro-1-(dimethoxy phosphoryl) B carbonyl] succsinic acid (2,5-dioxo cyclopentyl) ester and be dissolved among an amount of DMF; Hemocyanin is dissolved in the dipotassium hydrogen phosphate solution, in the stirring, under ice bath, is added dropwise to the haptens reaction solution in the hemocyanin solution, dropwising 3～5 ℃ of following stirring reactions in back spends the night, the dialysis tubing of then reaction solution being packed into is fully dialysed with 0.01mol/L PBS solution, obtains DBC-KLH;
(4), the preparation of coating antigen
Be connected with oralbumin (OVA) with the Trichlorphon Acibenzolar and promptly make envelope antigen, be used for bag and reacted.
(5), the preparation of antibody and purifying:
Immune animal is selected 2 of Japan 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 antigenic immunity, adopt by Freund's complete adjuvant emulsive immunogen, dosage is 1mg, is dissolved in the NaCl of 1mL 0.9%, mixes with the Freund's complete adjuvant of 1mL and carries out emulsification.Booster immunization: carried out booster immunization in the 14th day and 28 days behind the initial immunity, dosage is 0.5mg, is dissolved in the NaCl of 1mL 0.9%, mixes with the Freund's incomplete adjuvant of 1mL and carries out emulsification.Later on once, be total to immunity 6 times every 28 days booster immunizations.Since the 2nd booster immunization, each immunity after 10 days animal pilot production blood is carried out serum titer mensuration and avidity is measured.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).
The step of serum titer detection method:
1) the Trichlorphon coating antigen for preparing is dissolved in pH 9.6Na
2CO
3-NaHCO
3Damping fluid in, with Trichlorphon coating antigen dilution be 1.0 μ g/100 μ L as coating buffer, the every hole of 96 hole microwell plates adds 100 μ L, room temperature is placed and is spent the night or 37 ℃ of constant temperature incubations 2～3 hours, with PBST is phosphate buffered saline buffer 0.05% (V/V), Tween20 washing lotion washing three times;
2) every hole adds 200 μ L, 1% bovine serum albumins (BSA)/PBS confining liquid, seals 1 hour, with washing lotion washing four times;
3) the Trichlorphon antiserum(antisera) of various extension rates is joined in separately the micropore, the parallel application of sample in three holes, concussion mixing 5～10min, room temperature reaction 1～2 hour;
4) wash microwell plate 3～4 times with washing lotion, liquid in the hole is got rid of, buckle with thieving paper and do, add 100 microlitre goat-anti rabbit ELIAS secondary antibody to every micropore, 37 ℃ were reacted 30 minutes;
5) wash microwell plate 3～4 times with washing lotion, liquid in the hole is got rid of, buckle with thieving paper and do, add the mixed solution of substrate A and substrate B, the proportioning of the mixed solution of this substrate A and substrate B is 14.6: 0.45, and reaction is 0.5 hour under the room temperature;
6) add stop buffer with termination reaction in each micropore, read absorbance with microplate reader, the serum dilution that according to light absorption value is at 1 o'clock is drawn from 24 days to the 120th day Trichlorphon antibody titer curve of immunity for tiring.
Advantage of the present invention and positively effect are:
1. the present invention has farthest kept the chemical structure of Trichlorphon, be the new compound of initiating both at home and abroad, immunizing antigen with this haptens preparation goes the immune animal maximum possible to keep the molecular structure of original Trichlorphon, and this has the antibody of high degree of specificity that assurance is provided for obtaining to Chlorpyrifos 94.
2. on the haptenic basis of synthetic Trichlorphon, synthetic its Acibenzolar can improve the connection rate of haptens and high molecular weight protein greatly.
3. the present invention has characteristics such as special, sensitive, accurate, quick, cheapness, 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 simple, and used main raw material such as Trichlorphon, and cheap, the easy acquisition of pyridine all can be buied in general chemical reagents corporation.
5. the present invention is through the above-mentioned haptens of verification experimental verification, its simple synthetic method, and the combined coefficient height, 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
Below in conjunction with accompanying drawing, the embodiment of the invention is described further; Following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Embodiment 1:
1. haptenic synthetic
The present invention selects 2,2, and 2-three chloro-1-(dimethoxy phosphoryl)-ethyl monoesters succsinic acid is a haptens, and its molecular weight is 234, and molecular structural formula is:
Concrete preparation method is: Trichlorphon raw material [(2,2,2-three chloro-1-hydroxyethyls) chlorooxon] 257mg (1mol) joins 20mL and contains in the anhydrous pyridine solution of 100mg (about 1mmol) Succinic anhydried, stirring at room reaction 10～22h under the air-isolation condition, question response stops the back and carry out decompressing and extracting under 35 ℃ of (± 5 ℃) agitation condition, removes pyridine; Use the solution pickling of concentrated hydrochloric acid and the ratio 1: 6～10 of water then, carry out acidifying; Then extract, product is dissolved in the organic phase, merge organic phase with ethyl acetate; Anhydrous sodium sulphate dewaters, and back solution is transferred in the dry round-bottomed flask of having weighed with dewatering; The reduced pressure backspin steams removes ethyl acetate.Through the synthetic Trichlorphon artificial semiantigen [2,2,2-three chloro-1-(dimethoxy phosphoryl)-ethyl monoesters succsinic acid] of this step reaction.Adopt recrystallization method to carry out purifying, promptly add the higher tetrahydrofuran (THF) (THF) of an amount of polarity to revolving to steam to the product of doing earlier, add-on is dissolved fully with product and is as the criterion, add the lower sherwood oil of polarity then, and put it into refrigerator, product is separated out under cold condition, reach the recrystallization purifying purpose, identify that by mass spectrum obtain purer target product, its mass spectrum in the accompanying drawings.
2. the haptens Acibenzolar is synthetic
Concrete preparation method is: in the 100mL round-bottomed flask; Trichlorphon haptens [2; 2; 2-three chloro-1-(dimethoxy phosphoryl)-ethyl monoesters succsinic acid] 257mg (about 1mol) joins 10mL and is dissolved with in the anhydrous methylene chloride of 127mg (about 1.1mol) N-hydroxy-succinamide (being NHS); under the ice bath behind the stirring reaction 30min; under nitrogen protection, dropwise drip 2.5mL and be dissolved with the anhydrous tetrahydro furan of 227mg (about 1.1mol) DCC; continuation is stirring reaction 1～4h under ice bath, rises to stirred overnight at room temperature then naturally.After the termination reaction; remove by filter white precipitate (being DCU); obtain further perfect Trichlorphon artificial semiantigen Acibenzolar { [2 through this step reaction; 2; 2-three chloro-1-(dimethoxy phosphoryl) B carbonyl] succsinic acid (2,5-dioxo cyclopentyl) ester } carry out column chromatography and carry out purifying by preparing suitable polarity developping agent.Wherein the developping agent proportioning is: ethyl acetate: normal hexane=6: 1 adds about 1% glacial acetic acid again.Consumption is followed successively by ethyl acetate 180mL, normal hexane 30mL, glacial acetic acid 2mL.The dress post adopts 3g, 200～300 order silica gel, and revolve steaming to doing, to carry out the dry method application of sample after in the Acibenzolar after product, adding the same specification silica gel of 0.3g.The target product molecular weight is 454, through mass spectrogram analysis, has this material monomer to add the sodium peak and diploid adds the sodium peak, proves synthetic this material, and mass spectrum in the accompanying drawings.
3, artificial antigen is synthetic
Adopt haptens Acibenzolar { [2,2,2-three chloro-1-(dimethoxy phosphoryl) B carbonyl] succsinic acid (2,5-dioxo cyclopentyl) ester } to be connected on the hemocyanin (KLH) by active ester method, synthetic artificial antigen, its molecular structural formula is:
Concrete preparation method is: get 2mg{[2,2,2-three chloro-1-(dimethoxy phosphoryl) B carbonyl] succsinic acid (2,5-dioxo cyclopentyl) } ester is dissolved among the 200ulDMF, is A liquid.Take by weighing 10mg KLH and be dissolved in the 4mL 0.13mol/L sodium hydrogen carbonate solution (pH 8.1), be B liquid.Stir down, A liquid is dropwise joined B liquid, 4 ℃ of following stirring reactions spend the night.The dialysis tubing of then reaction solution being packed into is under 4 ℃, the 0.01mol/L PBS solution of pH 7.4 fully dialyses, and accurately measures the volume of protein conjugate solution then, measures concentration and binding ratio, packing ,-20 ℃ of preservations.
The evaluation of artificial antigen:
In the ratio of synthetic Trichlorphon immunizing antigen reaction used carrier albumen and coupled product, carry out the ultraviolet (sweep measuring of 200nm～400nm).Conjugate DBC-KLH maximum absorption band occurs at the 251nm place, and the maximum absorption band of KLH is respectively 279, and there is obvious variation in both, shows the synthetic success of artificial antigen DBC-KLH.
4, envelope antigen is synthetic
Be connected with oralbumin with haptens Acibenzolar { [2,2,2-three chloro-1-(dimethoxy phosphoryl) B carbonyl] succsinic acid (2,5-dioxo cyclopentyl) } and promptly make envelope antigen, be used for coated elisa plate.Its specific practice is synthetic with artificial antigen.
5, the preparation of antibody and purifying:
Immune animal is selected 2 of Japan large ear rabbits, and at 3 months monthly ages, about 1.5 kilograms of body weight are raised in the standard test Animal House, observe continuously 4 days, determines to carry out immunity after physical appearance normally.Immunity is taked subcutaneous and intramuscular injection is carried out jointly.
Initial immunity: taking by weighing dosage is 1mg, is dissolved in the NaCl of 1mL 0.9%, mixes with the Freund's complete adjuvant of 1mL and carries out emulsification, and emulsion is used for immunity.
Booster immunization: carried out booster immunization in the 14th day and 28 days behind the initial immunity, take by weighing 0.5mg, be dissolved in the NaCl of 1mL 0.9%, mix with the Freund's incomplete adjuvant of 1mL and carry out emulsification, emulsion carries out immunity.Since the 4th immunity once, be total to immunity 6 times every 28 days booster immunizations.Since the 2nd booster immunization, each immunity is carried out serum titer to animal pilot production blood after 10 days and is measured.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 proteinA-SepharoseCL-4B immune affinity chromatographic column to carry out antibody purification, store for future use in 4 ℃ behind the sodium azide of gained antibody interpolation 0.1% (W/V).
6, the step of serum titer detection method:
Bag quilt: the Trichlorphon coating antigen for preparing is dissolved in pH 9.6Na
2CO
3-NaHCO
3Damping fluid in, with Trichlorphon coating antigen dilution be 1.0 μ g/100 μ L as coating buffer, the every hole of 96 hole microwell plates adds 100 μ L, room temperature is placed and is spent the night or 37 ℃ of constant temperature incubations 2～3 hours, with PBST is phosphate buffered saline buffer 0.05% (V/V), Tween20 washing lotion washing three times;
Sealing: every hole adds 200 μ L, 1% bovine serum albumins (BSA)/PBS confining liquid, seals 1 hour, with washing lotion washing four times;
Application of sample: the Trichlorphon antiserum(antisera) of various extension rates is joined in separately the micropore, the parallel application of sample in three holes, concussion mixing 5～10min, room temperature reaction 1～2 hour;
Add ELIAS secondary antibody: wash microwell plate 3～4 times with washing lotion, liquid in the hole is got rid of, buckle with thieving paper and do, add 100 microlitre goat-anti rabbit ELIAS secondary antibody to every micropore, 37 ℃ were reacted 30 minutes;
Add substrate: wash microwell plate 3～4 times with washing lotion, liquid in the hole is got rid of, buckle with thieving paper and do, chromogenic substrate uses tetramethyl benzidine (TMB).Every hole adds 150 microlitre tetramethyl benzidine-hydrogen peroxide solutions (the 5mg tetramethyl benzidine is dissolved in the 1ml substrate buffer solution), and reaction is 0.5 hour under the room temperature;
Stop: add stop buffer with termination reaction in each micropore, read absorbance with microplate reader, the serum dilution that according to light absorption value is at 1 o'clock is for tiring.
Claims (4)
1. the artificial antigen of agricultural chemicals Trichlorphon and antibody is characterized in that using molecular formula to be
2; 2; 2-three chloro-1-(dimethoxy phosphoryl)-ethyl monoesters succsinic acid is that haptens is connected synthetic artificial antigen with hemocyanin; be connected synthetic enzyme-labelled antigen with horseradish peroxidase; wherein artificial antigen passes through immune new zealand white rabbit again; get blood, isolate the antiserum(antisera) purifying and make antibody.
2. the preparation method of the described agricultural chemicals Trichlorphon of claim 1 artificial antigen is characterized in that it being to make with following step:
(1) haptens 2,2,2-three chloro-1-(dimethoxy phosphoryl)-ethyl monoesters succsinic acid synthetic
Trichlorphon raw material [(2,2,2-three chloro-1-hydroxyethyls) chlorooxon] 300mg joins 20mL and contains in the anhydrous pyridine solution of 70mg Succinic anhydried, stirring at room reaction 10～22h under the air-isolation condition, question response stops the back and carry out decompressing and extracting under 30 ℃ of agitation condition, removes pyridine; Use the concentrated hydrochloric acid and the solution acid washing back of the ratio 1: 6～10 of water to extract then with ethyl acetate, merging the organic phase anhydrous sodium sulphate dewaters, the steaming of reduced pressure backspin is removed ethyl acetate and can be synthesized Trichlorphon haptens [2,2,2-three chloro-1-(dimethoxy phosphoryl)-ethyl monoesters succsinic acid];
(2) the haptens Acibenzolar is synthetic
In the 100mL round-bottomed flask, Trichlorphon haptens [2,2,2-three chloro-1-(dimethoxy phosphoryl)-ethyl monoesters succsinic acid] 300mg joins 10mL and is dissolved with in the anhydrous methylene chloride of 167mgN-N-Hydroxysuccinimide, under the ice bath behind the stirring reaction 30-60min, under nitrogen protection, dropwise drip the anhydrous tetrahydro furan that 3.5mL is dissolved with 207mgDCC, continuation is stirring reaction 1～4h under ice bath, naturally rise to stirred overnight at room temperature then, after the termination reaction, remove by filter white precipitate, obtain Trichlorphon haptens Acibenzolar { [2,2,2-three chloro-1-(dimethoxy phosphoryl) B carbonyl] succsinic acid (2,5-dioxo cyclopentyl) ester };
(3) artificial antigen is synthetic
Get haptens Acibenzolar [2; 2; 2-three chloro-1-(dimethoxy phosphoryl) B carbonyl] succsinic acid (2; 5-dioxo cyclopentyl) the 5mg ester is dissolved among an amount of 250ul DMF and is configured to A liquid, the 15mg hemocyanin is dissolved in be configured to B liquid in the 3mL dipotassium hydrogen phosphate solution, is added dropwise to A liquid in the B liquid under ice bath; dropwising 3～5 ℃ of following stirring reactions in back spends the night; the dialysis tubing of then reaction solution being packed into is fully dialysed with 0.01mol/L PBS solution, obtains artificial antigen.
3. the preparation method of the described agricultural chemicals Trichlorphon of claim 1 enzyme-labelled antigen is characterized in that it being to make with following step:
The weighting profit requires that institute's synthetic haptens Acibenzolar 1.67mg is dissolved in 2ml dimethyl formamide wiring solution-forming A in 2,0.51mg horseradish peroxidase is dissolved in the 0.2mol/L phosphoric acid buffer wiring solution-forming B of 2ml pH 7.4, under ice bath, add solution A in the solution B slowly then, reaction is spent the night under 4 ℃ after stirring, at last reaction solution is inserted in the dialysis tubing, 0.2mol/L phosphoric acid buffer dialysis with pH7.4 under 4 ℃ can obtain enzyme-labelled antigen in three days, was used for color reaction.
4. the preparation method of the described agricultural chemicals Trichlorphon of claim 1 antibody is characterized in that it being to make with following step:
Immune animal is selected female new zealand white rabbit for use, 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 and 6 week back immunity three times, after this carried out the 5th immunity at interval in one month, immunity was got blood by the auricular vein of rabbit in back 9 days, the detection of tiring, 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 prepared, carry out animal immune according to the described method synthetic of claim 2 artificial antigen;
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-pesticide meta-tolyl-N-methylcarbamate.
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| CN2009100703415A CN102002103A (en) | 2009-09-03 | 2009-09-03 | Pesticide trichlorfon-resistant specific antibody |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109608479A (en) * | 2018-11-07 | 2019-04-12 | 中国农业大学 | The preparation method and application of a kind of Tetramine haptens and monoclonal antibody |
| CN113896743A (en) * | 2021-11-04 | 2022-01-07 | 深圳市易瑞生物技术股份有限公司 | Dipterex hapten, preparation method thereof, antigen, antibody and application thereof |
-
2009
- 2009-09-03 CN CN2009100703415A patent/CN102002103A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109608479A (en) * | 2018-11-07 | 2019-04-12 | 中国农业大学 | The preparation method and application of a kind of Tetramine haptens and monoclonal antibody |
| CN113896743A (en) * | 2021-11-04 | 2022-01-07 | 深圳市易瑞生物技术股份有限公司 | Dipterex hapten, preparation method thereof, antigen, antibody and application thereof |
| CN113896743B (en) * | 2021-11-04 | 2024-03-15 | 深圳市易瑞生物技术股份有限公司 | Dipterex hapten, preparation method thereof, antigen, antibody and application thereof |
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Application publication date: 20110406 |