CN103159648A - Bensulfuron-methyl universal hapten, artificial antigen, preparation method and application thereof - Google Patents
Bensulfuron-methyl universal hapten, artificial antigen, preparation method and application thereof Download PDFInfo
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Abstract
Belonging to the technical fields of immunodetection and biochemical engineering, the invention relates to a bensulfuron-methyl universal hapten, an artificial antigen, a preparation method and application thereof. The artificial antigen prepared from the bensulfuron-methyl universal hapten provided in the invention can be immunized to obtain an antibody, which can achieve spectral identification of bensulfuron-methyl pesticide molecular with the artificial hapten structure, so that the indirect Elisa method adopting the artificial antigen and the antibody prepared therefrom can realize a wide detectable spectrum range. In the invention, the preparation of the artificial antigen employs an improved mixed anhydride method, which has high coupling efficiency and is simple.
Description
Technical field
The invention belongs to immunodetection and technical field of biochemical industry, relate to a kind of benbbensulfuronmethyl universal hapten, artificial antigen and preparation method thereof and application.
Background technology
The grand class weedicide of sulphur is a maximum class weedicide in the world at present, since E.I.Du Pont Company develop first nineteen eighty-two cornfield herbicidal chlorine sulphur grand after, by the derivatization method such as modifying on sulfonylurea two ends and urea bridge, existing nearly 40 kind commercializations at present.The grand class weedicide of sulphur is by suppressing acetolactate synthestase (ALS)/acetic acid hydroxy acid synthetic enzyme (AHAS), thereby destroys protein synthesis, disturb DNA synthetic and cell fission and growth, is that the weeds normal growth is damaged and dead
[1]It because of efficient, consumption is few, toxicity low and the short gourd, fruit and vegetables such as the field crop such as paddy rice, wheat, corn and peanut, potato that are widely used in of half-life
[2]But the grand compounds of sulphur because of not volatile, be difficult for photodissociation, organic adsorption index low, with the characteristics of soil pH rising Increased Plasma Half-life, cause also to force us will strengthen its residual study on monitoring to the harm of local farm crop and environment and the raising of the frequent report of loss event and public's awareness of environment protection and health.European Union has been 0.05ppm to making minimum limit standard in tap water and fruits and vegetables.
The grand class herbicide residue of traditional sulphur detection method has the methods such as gas-chromatography, liquid chromatography and capillary electrophoresis.Vapor-phase chromatography needs derivatize because the ureide derivative volatility is low with thermally labile; Though liquid chromatography coordinates the methods such as mass spectrum and solid-phase microextraction to carry out sensitive and accurate detection to sulfonylurea herbicide in sample, have lot of documents research report both at home and abroad, toxic reagent application and analysis cost are high; Menne H J etc. with capillary electrophoresis method to the discovery of analyzing and researching of the grand class weedicide of sulphur in pedotheque, because sample size is little, extracting solution needs high density concentrated, thereby limited application (the GARCIA F of capillary electrophoresis in toxicological analysis, HENION J.Fast capillary electrophoresis-ion spray mass spectrometric determination of sulfonylureas[J] .J Chromatogr A, 1992,606 (2): 237-247; MENNE H J, JANOWITZ K, BERGER B M.Comparison of capillary electrophoresis and liquid chromatography for determination of sulfonylurea herbicides in soil[J] .J AOAC Int, 1999,82 (6): 1534-1540).immunological detection, its principle is on the basis that successfully prepares the benbbensulfuronmethyl artificial antigen, utilize indirect enzyme-linked immunosorbent detection method (ELISA) to come the content of benbbensulfuronmethyl in test sample, the method has highly sensitive, operation is fast succinct, need not the advantages such as precision instrument and professional be on the scene, development fast in the residual analyzing and testing of agriculture, document and patent research report (KELLEY M M that the residual elisa assay of the grand class agriculture of sulphur is also arranged recently, ZAHNOW E W, PETERSEN W C, TOY S T.Chlorsulfuron determination in soil extracts by enzyme immunoassay[J] .J Agric Food Chem, 1985, 33 (5): 962-965, ZHAO J, YI G X, HE S P, et al.Development of monoclonal antibody-based enzyme linked immunosorbent assay for the herbicide chlorimuron-ethyl[J] .J Agric Food Chem, 2006,54 (14): 4948-4953, SCHLAEPPI D, RAMSTEINER D.Immunological detection of triasulfuron:European, WO/1988/009798A[P] .1997-01-15).
The key point of enzyme-linked immunoassay method is that preparation has immunogenic artificial antigen, and the preparation of high-titer antigen is the important prerequisite that obtains specific antibody and guarantee the immunodetection susceptibility, and wherein haptenic preparation is again the key of antigen preparation.Benbbensulfuronmethyl does not have immunogenicity (molecular weight is less than 1000) because molecular weight is little, can not produce in animal body antibody, must prepare artificial antigen with the macromolecular carrier coupling, the generation of ability inducing specific antibody.For this reason, must manage first with benbbensulfuronmethyl molecule and carrier protein covalent coupling benbbensulfuronmethyl artificial antigen.Existing synthetic benbbensulfuronmethyl artificial antigen has kept whole molecular structures (phenyl ring part, heterocyclic moiety and urea bridge portion) of benbbensulfuronmethyl, as the record in the patent of invention of 200910234686.X, its immunity preparation and antibody only can identify a kind of benbbensulfuronmethyl molecule.
On the method for preparing coupling benbbensulfuronmethyl artificial antigen, usually the active ester method that adopts in prior art carries out coupling with the grand compounds of sulphur and carrier proteins, and Zhang Lijie (Zhang Lijie, Zhou Yingxia, Wang Lei, Deng. the synthetic and evaluation [J] of the general antigen of organophosphorus pesticide. agricultural chemicals, 2010,49 (3): 164-166,169) if etc. the people study and find that there is decomposed in long Acibenzolar of reaction times, its productive rate can descend on the contrary, and the polarity of the solvent that adopts in simultaneous reactions also can exert an influence to the productive rate of Acibenzolar.The patent No. is that the patent of invention of 200910234686.X has proposed a kind of bensulfuron-methyl artificial immunogen BE-BSA and preparation and purposes, what this piece patent documentation was selected on artificial antigen is synthetic is active ester method, the method is consuming time, poor efficiency, more than the linked reaction of an antigen and carrier proteins just needs 5d.
Summary of the invention
The present invention provides preparation method and the application of a kind of benbbensulfuronmethyl artificial antigen according to the demand in above-mentioned field.The present invention adopts mixed anhydride method to utilize free carboxy and isobutyl chlorocarbonate on hapten molecule first to form the mixed acid anhydride intermediate under the anhydrous condition that tri-n-butylamine exists, then carry out coupling with carrier proteins again, verify through testing data, this antigen has higher immunogenicity, the antiserum(antisera) for preparing and benbbensulfuronmethyl class material have good affinity, and productive rate height of the present invention is effective, simple and efficient.
A kind of benbbensulfuronmethyl haptens has following molecular structure:
A kind of artificial antigen of benbbensulfuronmethyl is characterized in that: use the molecule of following structure,
Described artificial antigen, described carrier proteins refer to bovine serum albumin BSA or Protalbinic acid OVA, and the antigen for preparing is respectively immunogen and coating antigen.
The preparation method of the artificial antigen that a kind of benbbensulfuronmethyl is general comprises being prepared as follows step:
(1) synthetic universal hapten;
(2) described universal hapten by with carrier protein couplet;
The synthetic route of described synthetic universal hapten is:
The preparation process of described universal hapten is: amidate action occurs in 2-methyl-formiate benzyl sulphonamide and succinyl oxide under DBU/HCl catalysis in acetonitrile, purifying gets universal hapten Hapten, described amidate action carries out in 22 ℃ of water-baths, reaction times is 2h, described catalyzer DBU dropwise adds in 10min, described purifying refers to reaction soln is poured in distilled water, the ethyl acetate washing, remove organic phase, HCl regulates the rear ethyl acetate extraction of using of water pH to 2, merges the anhydrous MgSO of organic phase
4Drying, solvent evaporated, re-crystallizing in ethyl acetate gets Hapten.
The mixed anhydride method preparation is adopted in the coupling of described universal hapten and carrier proteins, and step is as follows:
(a) preparation A liquid: Hapten is dissolved in tetrahydrofuran (THF), adds while stirring tri-n-butylamine, and is cooling, add isobutyl chlorocarbonate, stir, be A liquid, described Hapten is dissolved in tetrahydrofuran (THF) to carry out under 4 ℃, describedly coolingly refers to be cooled to 0 ℃, and described stirring refers to stir 60min under ice bath;
(b) preparation B liquid: BSA or OVA are dissolved in borate buffer solution, are B liquid, and described borate buffer solution concentration is 0.2mol/L, pH=9.0;
(c) the A drop is added in B liquid, stirring is spent the night, and reaction solution is dialysed in the PBS damping fluid, changes liquid, dialyzate is centrifugal, get the supernatant liquor lyophilize, preserve described PBS pH of buffer=7.4, described dialysis is 4 ℃ of dialysis 3d, the described liquid that changes refers to change 3 times/d of liquid, described centrifugal be the centrifugal 5min of 10000r/min, the temperature of described preservation is-20 ℃.
Described artificial antigen as immunogen for the preparation of the antibody that detects benbbensulfuronmethyl or the application in antiserum(antisera).
Described artificial antigen adopt indirect Elisa method detect benbbensulfuronmethyl in as the application of coating antigen.
The coated concentration of described coating antigen is 5.0mgL
-1, antiserum(antisera) dilution 3.2 * 10 in the Elisa method indirectly
4Doubly.
The antiserum(antisera) that described artificial antigen prepares as the immunogen immune animal, antibody or hybridoma.
The preparation of high-titer antigen is the important prerequisite that obtains specific antibody and guarantee the immunodetection susceptibility, and wherein haptenic preparation is again the key of antigen preparation.Benbbensulfuronmethyl does not have immunogenicity because molecular weight is little, can not produce in animal body antibody.So being preparation, the key point of enzyme-linked immunoassay method has immunogenic artificial antigen.The present invention has the common trait of the herbicide molecular structure of the grand class of sulphur take 2-methyl-formiate benzyl sulphonamide and succinyl oxide as the haptens of raw material preparation, compare with disclosed benbbensulfuronmethyl haptens structure in prior art, phenyl ring and urea bridge portion that haptens of the present invention is just got the benbbensulfuronmethyl molecule consist of universal hapten, can detect simultaneously grand class agricultural chemicals of multiple sulphur such as benbbensulfuronmethyl, AC322140, the phonetic sulphurs of chlorine, as embodiment 8.The antiserum(antisera) that this artificial antigen prepares as the immunogen immune animal can be to having the material specific binding of this molecular characterization, i.e. the grand class herbicide molecular of specific recognition sulphur.
For can with the coupling of carrier proteins molecule, need to introduce connecting arm and carboxyl, the synthetic route that antigen of the present invention adopts is based on has considered that connecting arm and carboxyl introduce the selection of position to the impact of the three-D space structure of synthetic product, avoid having affected due to the introducing of connecting arm the space conformation of the molecular structure characteristic of synthetic product, to guarantee determining that as antigen the structure in site is unaffected as far as possible.
Artificial antigen of the present invention is adopted the mixed anhydride method preparation, utilize free carboxy and isobutyl chlorocarbonate on hapten molecule first to form the mixed acid anhydride intermediate under the anhydrous condition that tri-n-butylamine exists, then carry out coupling with carrier proteins again, obtain good result.Select at the carrier proteins of hapten conjugation artificial immunization antigen, rabbit anteserum albumen (RSA), human serum protein (HSA), keyhole hemocyanin (KLH), thyroglobulin (TG) etc. are arranged.That experiment is adopted is BSA, because its physicochemical property are stable, and contains than high-lysine content and free amino group group.The present invention successfully synthesizes artificial antigen, provides the foundation for the immunoassay of benbbensulfuronmethyl detects.
The polyclonal antibody for preparing is measured by indirect competitive ELISA (CI-ELISA) haptenic inhibiting rate.Carry out according to best antigen-antibody working concentration and optimal conditions, with haptens PBST (0.01mol/LPBS, pH=7.4, containing 0.05%Tween-20) compound concentration is respectively 0,0.001,0.01,0.1,1,10,50, the standardized solution of 100mg/L, drawing standard suppresses curve, calculates concentration (IC in inhibition
50) and lowest detectable limit (IC
10).
The artificial antigen Hapten-BSA of the present invention's preparation stimulating animal well produces antibody, and simultaneously due to the difference of animal individual, tiring that each mouse produces is slightly different.
Carry out indirect competitive ELISA according to antigen-antibody best effort concentration, from suppress curve know Hapten as the competition thing can be with coating antigen be combined serum competitively antibody, suppressed the combination of antibody and coating antigen.From take haptens as research object, the regression equation of foundation can learn that antibody has stronger avidity to haptens.
Synthetic method of the present invention is optimized on the basis of foregoing invention and is more succinct, the borate buffer solution of the multiplex pH=8.0 of carrier proteins B liquid in the existing mixed anhydride method of commonly using, and mixed anhydride method of the present invention adopts the borate buffer solution of pH=9.0, solvability to carrier proteins of the present invention is better, is conducive to the higher in conjunction with productive rate of haptens and carrier proteins.
Description of drawings
Fig. 1 is the grand compounds molecular structure of sulphur
Wherein: R=CH
3, alkyl; R
1=CH
3, OCH
3, Cl, NHCH
3R
2=CH
2, OCH
3, Cl, CH
2OF
2, OCH
2CH
3, CH
2SePH; X=N, CH; Y=Cl, CH
3, CF
3, SCH
3, OCH
3, CH
2Cl, SO
2CH
3, COOCH
3, COOC
2H
5, OCH
2CH
2Cl, OCH
2CH
2OCH
3, SO
2N (CH
3)
2, SO
2NHOCH
3
Fig. 2 is the universal hapten synthetic route chart
Fig. 3 is Hapten TLC detected result figure
A:2-methyl-formiate benzyl sulphonamide B wherein: universal hapten
Fig. 4 is universal hapten 1
1H nuclear magnetic spectrum figure
Fig. 5 is Hapten, carrier proteins and conjugate uv absorption spectra thereof
Fig. 6 is the polyclonal antibody figure that tires
Wherein: X-coordinate is the antiserum(antisera) extension rate; Ordinate zou is absorbance OD
Fig. 7 is the indirect competitive ELISA graphic representation
Embodiment
The invention provides following embodiment is in order further to understand better the present invention; be not limited to described preferred forms; content of the present invention and protection domain are not construed as limiting; other any or akin products identical with the present invention that anyone draws under enlightenment of the present invention are within all dropping on protection scope of the present invention.
Preparation and the evaluation of embodiment 1 universal hapten
200mg 2-methyl-formiate benzyl sulphonamide and 87mg succinyl oxide are dissolved in the 5mL acetonitrile, be placed in 22 ℃ of water-baths, enter in 10min, 265.5mg DBU dropwise being added, after stirring 2h, reaction soln is poured in 50mL distilled water, the washing of 50mL ethyl acetate, remove organic phase, and the rear use of HCl adjusting water pH to 2 ethyl acetate extraction (2 * 50mL), merge the anhydrous MgSO of organic phase
4Drying, solvent evaporated, re-crystallizing in ethyl acetate gets Hapten, and first through thin-layer chromatography (TLC) Preliminary detection, rear nuclear-magnetism is identified.
H?NMR(CD
3OD)：δ7.91(d，1H，J＝7.5Hz?Ar)，7.61-7.42(m，3H，Ar)，4.78(s，2H，CH
2SO
2)，3.85(s，3H，OCH
3)，2.48(m，2H，COCH
2CH
2COOH)，2.43(m，2H，COCH
2CH
2COOH)。See Fig. 4.Wherein a little assorted peak has appearred respectively in 5.0-5.5 and 11-13 place, and this may be incomplete owing to crossing the pillar purifying, but the peak of target substance occurs, and prove that product is exactly universal hapten, and the coupling that can carry out artificial antigen prepares.
16.45mg Hapten is dissolved in the 1mL tetrahydrofuran (THF), adds while stirring 13 μ L tri-n-butylamines, after being cooled to 0 ℃, adds 7 μ L isobutyl chlorocarbonates, stirs 60min under ice bath, is A liquid.Take 33.5mg BSA (or 21.5mg OVA) and be dissolved in borate buffer solution (0.2mol/L, Ph=9.0), be B liquid.4 ℃ slowly add to the A drop in B liquid, and magnetic agitation is spent the night.Next day, with reaction solution 4 ℃ of dialysis 3d in PBS damping fluid (pH=7.4), change 3 times/d of liquid, the centrifugal 5min of dialyzate 10000r/min gets the supernatant liquor lyophilize ,-20 ℃ of preservations.
Ultraviolet scanner scans sample after dialysing at the 190-400nm wavelength, identifies the coupling situation.According to charateristic avsorption band wavelength and the light absorption value of each material, calculate coupling ratio according to following formula
[13]
Wherein: D, B, C are respectively universal hapten, BSA (or OVA), reaction product; CD, CB are the molconcentration of universal hapten and BSA (or OVA); A is absorbance; K is molar extinction coefficient (K=A * molecular weight/concentration); Dm, bm are respectively the maximum absorption wavelength of universal hapten and BSA (or OVA).
The Balb/c mouse is observed and to get tail blood a week after without difference and preserve and make negative control.With immunogenic normal saline solution and Freund's complete adjuvant balanced mix, the subcutaneous multi-point injection immune mouse of the complete postabdomen of emulsification, immunizing dose is 100 μ g/0.2ml.Using Freund's incomplete adjuvant after two weeks instead is emulsifying agent, with same dose and method booster immunization mouse.Immunologic process often biweekly, the immunity 4 times.After the 3rd immunity, 10d gets mouse tail blood, and indirect non-competing ELISA surveys and tires.
Polyclonal antibody is measured by indirect competitive ELISA (CI-ELISA) the inhibiting rate of universal hapten.Carry out according to best antigen-antibody working concentration and optimal conditions, with universal hapten PBST (0.01mol/LPBS, pH=7.4, containing 0.05%Tween-20) compound concentration is respectively 0,0.001,0.01,0.1,1,10,50, the standardized solution of 100mg/L, drawing standard suppresses curve, calculates concentration (IC in inhibition
50) and lowest detectable limit (IC
10).
Embodiment 4 universal haptens are identified
Raw material 2-methyl-formiate benzyl sulphonamide is obtained white solid after synthetic route reaction, with 2-methyl-formiate benzyl sulphonamide with obtain white solid solution and carry out TLC and detect, developping agent is methyl alcohol: methylene dichloride: acetic acid=1: 9: 0.1 (v/v/v), result such as Fig. 3, the phosphor dot that obtains white solid obviously is different from the raw material point, calculates white solid R
f=0.48, tentatively obtaining white solid is target product Hapten, but also need synthesize further checking.
Universal hapten Hapten deuterated methanol (CD
3OD) dissolution with solvents, through the nucleus magnetic hydrogen spectrum qualification result as shown in Figure 4.
As seen from the figure,
1H NMR (CD
3OD): δ 7.91 (d, 1H, J=7.5Hz Ar), 7.61-7.42 (m, 3H, Ar), 4.78 (s, 2H, CH
2SO
2), 3.85 (s, 3H, OCH
3), 2.48 (m, 2H, COCH
2CH
2COOH), 2.43 (m, 2H, COCH
2CH
2COOH), wherein a little assorted peak has appearred respectively in 5.0-5.5 and 11-13 place, and this may be incomplete owing to crossing the pillar purifying, but the peak of target substance occurs, and prove that product is exactly universal hapten, and the coupling that can carry out artificial antigen prepares.
Be 1mgmL-1 with Hapten-BSA, Hapten-OVA, Hapten and carrier proteins with the PBS dilution, take PBS as blank, carry out UV scanning at 190-400nm, result such as Fig. 5
As can be seen from Figure, the charateristic avsorption band of Hapten, BSA and Hapten-BSA is respectively 269nm, 278nm and 275nm, and the charateristic avsorption band of OVA and Hapten 1-OVA is respectively 276nm and 271nm.The ultra-violet absorption spectrum of conjugate is different from Hapten and BSA and OVA, can infer that thus some group of conjugate is modified by universal hapten, still keeps the character of carrier proteins simultaneously, and successful coupling of universal hapten is described.The coupling ratio that calculates Hapten and BSA and OVA according to formula was respectively 8: 1 and 13: 1, so can illustrate tentatively that Hapten and BSA and OVA coupling synthesize successfully.
Embodiment 6 antibody titers are measured
After 5 immunity, indirect non-competing ELISA detects the antiserum titre of every mouse, take negative serum as blank, and positive sero-fast the tiring of the antiserum(antisera) extension rate greater than blank more than 2 times.Measurement result is as shown in Figure 6: mouse all can produce the positive antiserum(antisera) of more efficient valency, wherein 2 and No. 5 mouse to tire be 6.4 * 10
4, and 1,3 and No. 4 mouse tire reach 1: 1.28 * 10
5Result show synthetic artificial antigen Hapten-BSA well stimulating animal produce antibody, but due to the difference of animal individual, tiring that each mouse produces is slightly different.
Embodiment 7 affinity of antibodies are measured
The tire antiserum(antisera) of No. 4 higher mouse of selection with indirect non-competing ELISA method defined antigen antibody best effort concentration is: coating antigen concentration is 5.0mgL
-1, serum diluting multiple is 32000 times.Carry out the indirect competitive ELISA method with determined antigen-antibody best effort concentration, draw and suppress curve as shown in Figure 7:
As shown in Figure 7, the concentration of inhibiting rate and Hapten is obvious positive correlation, shows that Hapten is as competing thing at 0.001-100mgL
-1Antibody that can be in coating antigen is combined serum has competitively suppressed the combination of antibody and coating antigen.Take universal hapten as research object, setting up regression equation is y=0.172x+0.5513 (R
2=0.9923), IC
50=0.5032mgL
-1, I
10=0.0024mgL
-1, this shows that antibody has stronger avidity to universal hapten.
The detection test of embodiment 8. artificial antigens of the present invention.
With the determined antigen-antibody best effort of embodiment 7 concentration, detect antibody that antigen of the present invention prepares to the specificity of the pesticide molecules such as benbbensulfuronmethyl, ethoxysulfuron, AC322140 and chlorimuronethyl by indirect Elisa method, result is as shown in table 1, the analogue of antiserum(antisera) to the structure of antigen with the present invention preparation, have obvious cross reaction as benbbensulfuronmethyl, ethoxysulfuron, AC322140 and chlorimuronethyl etc., and azimsulfuron and the imidazoles sulphur crown with textural difference do not had relatively poor reaction.
Table 1
Claims (10)
1. benbbensulfuronmethyl haptens has following molecular structure:
2. the artificial antigen of a benbbensulfuronmethyl is characterized in that: use the molecule of following structure,
3. artificial antigen according to claim 2, described carrier proteins refers to bovine serum albumin BSA or Protalbinic acid OVA, the antigen for preparing is respectively immunogen and coating antigen.
4. the preparation method of the general artificial antigen of a benbbensulfuronmethyl comprises being prepared as follows step:
(1) synthetic universal hapten;
(2) described universal hapten by with carrier protein couplet;
The synthetic route of described synthetic universal hapten is:
5. preparation method according to claim 4, the preparation process of described universal hapten is: amidate action occurs in 2-methyl-formiate benzyl sulphonamide and succinyl oxide under DBU/HCl catalysis in acetonitrile, purifying gets universal hapten Hapten, described amidate action carries out in 22 ℃ of water-baths, reaction times is 2h, described catalyzer DBU dropwise adds in 10min, described purifying refers to reaction soln is poured in distilled water, the ethyl acetate washing, remove organic phase, HCl regulates the rear ethyl acetate extraction of using of water pH to 2, merges the anhydrous MgSO of organic phase
4Drying, solvent evaporated, re-crystallizing in ethyl acetate gets Hapten.
6. preparation method according to claim 4, the mixed anhydride method preparation is adopted in the coupling of described universal hapten and carrier proteins, and step is as follows:
(a) preparation A liquid: Hapten is dissolved in tetrahydrofuran (THF), adds while stirring tri-n-butylamine, and is cooling, add isobutyl chlorocarbonate, stir, be A liquid, described Hapten is dissolved in tetrahydrofuran (THF) to carry out under 4 ℃, describedly coolingly refers to be cooled to 0 ℃, and described stirring refers to stir 60min under ice bath;
(b) preparation B liquid: BSA or OVA are dissolved in borate buffer solution, are B liquid, and described borate buffer solution concentration is 0.2mol/L, pH=9.0;
(c) the A drop is added in B liquid, stirring is spent the night, and reaction solution is dialysed in the PBS damping fluid, changes liquid, dialyzate is centrifugal, get the supernatant liquor lyophilize, preserve described PBS pH of buffer=7.4, described dialysis is 4 ℃ of dialysis 3d, the described liquid that changes refers to change 3 times/d of liquid, described centrifugal be the centrifugal 5min of 10000r/min, the temperature of described preservation is-20 ℃.
Artificial antigen claimed in claim 2 as immunogen for the preparation of the antibody that detects benbbensulfuronmethyl or the application in antiserum(antisera).
Artificial antigen claimed in claim 2 adopt indirect Elisa method detect benbbensulfuronmethyl in as the application of coating antigen.
9. application according to claim 8, the coated concentration of described coating antigen is 5.0mgL
-1, antiserum(antisera) dilution 3.2 * 10 in the Elisa method indirectly
4Doubly.
10. the antiserum(antisera), antibody or the hybridoma that prepare as the immunogen immune animal of artificial antigen claimed in claim 2.
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| CN103483274A (en) * | 2013-09-25 | 2014-01-01 | 江苏快达农化股份有限公司 | Method for preparing bensulfuron methyl |
| CN105572348A (en) * | 2015-12-23 | 2016-05-11 | 中国烟草总公司郑州烟草研究院 | Enzyme linked immunosorbent assay kit for detecting triadimenol and application of enzyme linked immunosorbent assay kit |
| CN106083662A (en) * | 2016-05-21 | 2016-11-09 | 魏东 | Sulfabenz formaldehyde derivatives preparation method |
| CN111763172A (en) * | 2020-07-09 | 2020-10-13 | 中国农业科学院农业质量标准与检测技术研究所 | Halosulfuron-methyl hapten and complete antigen as well as preparation method and application thereof |
| CN112250604A (en) * | 2020-09-25 | 2021-01-22 | 华南农业大学 | Hapten and artificial antigen for broad-spectrum detection of sulfonylurea drugs, antibody and application thereof |
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| CN105572348A (en) * | 2015-12-23 | 2016-05-11 | 中国烟草总公司郑州烟草研究院 | Enzyme linked immunosorbent assay kit for detecting triadimenol and application of enzyme linked immunosorbent assay kit |
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| CN111763172A (en) * | 2020-07-09 | 2020-10-13 | 中国农业科学院农业质量标准与检测技术研究所 | Halosulfuron-methyl hapten and complete antigen as well as preparation method and application thereof |
| CN111763172B (en) * | 2020-07-09 | 2022-04-08 | 中国农业科学院农业质量标准与检测技术研究所 | Halosulfuron-methyl hapten and complete antigen as well as preparation method and application thereof |
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