CN102875671A - Clothianidin antigen, antibody and application thereof - Google Patents
Clothianidin antigen, antibody and application thereof Download PDFInfo
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Abstract
The invention relates to a clothianidin antigen, an antibody and an application thereof, and belongs to the technical field of immunochemistry analysis. The clothianidin antigen and the antibody of the invention are specially used for clothianidin specific polyclonal antibody preparation, enzyme-linked immunoassay (ELISA), and high-sensitivity and rapid detection of clothianidin residues in environment and agricultural products. A hapten is synthesized by substituting a chlorine atom on a thiazole ring of clothianidin, has a chemical name of 3-(5-((3-methyl-2-nitroguanidine)-yl methyl)thiazole-2-mercapto)propionic acid, and is coupled with bovine serum albumin and ovalbumin to prepare an antigen and an envelope antigen. Newzealand white rabbit is immunized by the immunizing antigen to obtain the specific polyclonal antibody of clothianidin. The established ELISA linear scope is 1.1 microgram/L-2 mg/L, and the detection limit is 1.1 microgram/L. The hapten synthetic technology of the invention is simple and feasible, high in antibody specificity, and suitable for detection and on-site monitoring of mass samples in environment and agricultural products.
Description
One, technical field
The present invention relates to clothianidin antigen, antibody and application thereof, belong to fields of immunochemistry analysis.Be exclusively used in the preparation of clothianidin polyclonal antibody, and in environment and agricultural-food the residual ELISA adsorption analysis method (ELISA) of clothianidin.
Two, background technology
Clothianidin (clothianidin), chemical name 1-(2-chloro-1,3-thiazoles-5-ylmethyl)-3-methyl-2-nitroguanidine lives chemical weapons field/Bayer by Japan and develops jointly, and obtains registration, chemical structure See Figure, molecular formula: C December calendar year 2001 in Japan
6H
8CIN
5O
2S, relative molecular mass: 249.68, fusing point: 176.8 ℃.Clothianidin is the novel nicotinamide insecticide that contains thiazole ring, and Main Function is in the nAChR of insect nerve system postsynaptic membrane.Clothianidin is widely used in preventing and treating Hemiptera, Coleoptera, Diptera and some lepidoptera pest of the crops such as hazard rice, vegetables, orchard, tealeaves with the insecticidal activity of its novel mechanism of action, broad-spectrum high efficacy, using method flexibly.People eat the generation that the agricultural-food that polluted by clothianidin can cause the disorderly of endocrine system even cause cancer for a long time.In addition, clothianidin is to honeybee and the high poison of silkworm.Thereby, need a kind of sensitivity, fast, method for detecting residue optionally.
The maximum maximum permission quantity of the clothianidin of U.S.'s issue Vegetable and fruit is 0.05mg/kg.The clothianidin method for detecting residue adopts instrument analytical method at present, is used in conjunction analytical method etc. such as liquid phase chromatographic analysis method, gas chromatography, liquid phase-mass spectrum.These instrument analytical methods are sensitive and accurate, but detecting instrument is expensive, sample pre-treatments is more loaded down with trivial details, detect the needs of wasting time and energy, be difficult to satisfy the fast and convenient detection of a large amount of samples.
Immunologic detection method has advantages of fast, cheap, easy, sensitive, special, in a large amount of sample rapid screening and field monitoring, demonstrating unique advantage.ELISA is a kind of immune labeled determination techniques that enzymic catalytic reaction and immune response are combined, and the highly sensitive of enzymic catalytic reaction and the high specific of antigen antibody reaction are arranged, and very large advantage is arranged aspect sensitivity.ELISA has obtained ripe application as a kind of detection method of rapid sensitive in many small molecules immunoassays.By chemosynthesis clothianidin haptens and artificial antigen, preparation is set up the clothianidin immunological detection method for the specific antibody of clothianidin.Finishing of this inventive method will solve the clothianidin haptens synthetic, and the gordian techniquies such as antibody preparation are set up the ELISA Fast Detection Technique of clothianidin.This invention not only is food safety detection, and provides effective technique means and detection method for the entry and exit detection of agricultural products in China etc., the water area monitoring of environmental monitoring department.Sustainable development and food-safety problem to agricultural products in China have important practical significance and important society, economic worth.There is not yet at present the immunology research report of relevant clothianidin both at home and abroad.
Three, summary of the invention
Technical problem
The object of the present invention is to provide a kind of ELISA adsorption analysis method of clothianidin, to clothianidin in environment and the agricultural-food residual carry out accurately, sensitive, fast, the simple detection.
Technical scheme
The residual ELISA detection method of clothianidin of the present invention comprises:
The preparation of 1 artificial semiantigen
8mmol (0.45g) KOH is dissolved in the 20mL ethanol, adds 4mmol (0.42g) β-mercaptopropionic acid and stir, add until completely dissolved 4mmol (1.02g) clothianidin, 80 ℃ of stirring and refluxing 2h.After reaction finishes, filtering reacting liquid, filtrate decompression concentrates to get faint yellow solid.Add 50mL water, regulate pH=2.0 with the hydrochloric acid of 1mol/L.Use ethyl acetate extraction, organic phase is through washing and anhydrous sodium sulfate drying, and concentrating under reduced pressure gets the white powder material, through recrystallizing methanol, gets white crystal.Clothianidin artificial semiantigen chemistry 3-(5-((3-methyl-2-nitroguanidine)-ylmethyl) thiazole-2-sulfydryl) propionic acid by name, molecular structure is as follows:
The preparation of 2 artificial antigens
Adopt carbodlimide method that haptens and bovine serum albumin (BSA) coupling are prepared immunizing antigen, adopt mixed anhydride method that haptens and ovalbumin (OVA) coupling are prepared envelope antigen.Its molecular structure is as follows:
The preparation of 3 antibody
With the immunizing antigen immunity new zealand white rabbit for preparing, immunizing dose is 1.0-1.5mg/kg (by the BSA amount), and immunity is five times altogether.First immunisation is by isopyknic immunizing antigen and Freund's complete adjuvant emulsification, and equal-volume immunizing antigen and Freund's incomplete adjuvant emulsification are used later in the back subcutaneous injection.From for the third time immunity, ear edge vein exploitating blood after each immunity, mensuration is tired.Until immune serum tire stable after, the heart blood sampling.Adopt sad-ammonium sulfate salting-out process purified blood serum, lyophilize obtains the antibody lyophilized powder.
4 immune analysis methods are set up and are used
Clothianidin antibody to preparation is tired, the analysis of specificity and sensitivity, sets up the ELISA of clothianidin.The enzyme immunoassay method of setting up is applied to the residues detection of clothianidin in environmental sample and the agricultural-food.
Beneficial effect
Advantage of the present invention and positively effect show:
1 Nover practical: clothianidin antigen is synthetic to have important practical value and practical significance with antibody production techniques.The synthetic clothianidin artificial antigen of first design produces specific polyclonal antibody through immune animal, for the residual efficient real-time analysis of clothianidin provides a kind of new method.Traditional agricultural chemicals instrumental analysis pretreatment process is loaded down with trivial details, cost is high, and length consuming time with an organic solvent easily causes environmental pollution in a large number, and operator quality is had relatively high expectations, be difficult to satisfy the agricultural product security analysis to demands quick, easy, accurate, a large amount of detections.Fast (only needing 2-3 hour) simple to operate, analysis cost are low, large, safe and reliable, the easy penetration and promotion of analysis capacity and clothianidin immune analysis method provided by the invention has, be specially adapted to batch samples and detect and field monitoring, can complement one another with the traditional instrument analytical procedure.
2 accuracy are high: clothianidin antibody is purified, and to make tiring behind the lyophilized powder be 4 * 10
6Antibodies specific identification clothianidin except having certain cross reaction with MTI-446, does not have obvious cross reaction with other nicotinic insecticides.Thereby as can be known, prepared antibodies specific is strong, rapidly and accurately the residual quantity of analyzing and testing clothianidin in sample.Use this immunization method clothianidin in actual sample the rate of recovery be 80.36-116.35%, the variation coefficient is lower than 10%, meets the retention analysis standard.
3 is highly sensitive: concentration (IC in the inhibition of the ELISA of foundation
50) be 0.046mg/L, detectability (IC
10, LOD) being 1.1 μ g/L, linearity range is 1.1 μ g/L-2mg/L.
Four, embodiment
Synthesizing of 1 artificial semiantigen
Chlorine atom on the clothianidin molecule thiazole ring is replaced, and synthetic haptens with four atom connecting arms had both kept the clothianidin characteristic group to greatest extent, had formed again the C-terminal group, can follow preferably albumen coupling.Behind the product purification by mass spectrum (ESI) and proton nmr spectra (
1H-NMR) identify.Clothianidin artificial semiantigen chemistry 3-(5-((3-methyl-2-nitroguanidine)-ylmethyl) thiazole-2-sulfydryl) propionic acid by name, molecular structure is as follows:
1.1 artificial semiantigen is synthetic
8mmol (0.45g) KOH is dissolved in the 20mL ethanol, adds 4mmol (0.42g) β-mercaptopropionic acid and stir, add until completely dissolved 4mmol (1.02g) clothianidin, 80 ℃ of stirring and refluxing 2h.After reaction finishes, filtering reacting liquid, filtrate decompression concentrates to get faint yellow solid.Add 50mL water, regulate pH=2.0 with the hydrochloric acid of 1mol/L.Use ethyl acetate extraction, organic phase is through washing and anhydrous sodium sulfate drying, and concentrating under reduced pressure gets the white powder material, through recrystallizing methanol, gets white crystal.
1.2 the evaluation of clothianidin artificial semiantigen
With the haptens behind the purifying through ESI-MS,
1H-NMR measures, to identify its molecular structure.Mass spectrum and nucleus magnetic resonance spectrum information are as follows: ESI-MS, m/z, 320[M+H]
+And 342[M+Na]
+ 1H-NMR(400MHz，d
6-DMSO)，δ：2.68-2.71(t，2H，CH
2COO)，2.79-2.80(s，3H，CH
3)，3.13-3.34(t，2H，SCH
2)，4.50-4.51(d，2H，NHCH
2)，7.60(s，1H，thiazol-4-H)，7.92(s，1H，NH)，9.13(s，1H，NH)，12.44(s，1H，COOH)。
Artificial semiantigen melting range behind the purifying: 158.5 ℃-160.2 ℃.
From above information comprehensive analysis as can be known, the product that is synthesized is the target artificial semiantigen.
Synthesizing of 2 artificial antigens
The molecular structural formula of clothianidin artificial antigen is as follows:
2.1 immunizing antigen is synthetic
The synthetic employing carbodlimide method of immunizing antigen.63.8mg (0.2mmol) clothianidin haptens is dissolved in the N of 1mL, in the dinethylformamide (DMF), then the N-hydroxy-succinamide (NHS) that in solution, adds 69.0mg (0.6mmol), stirring reaction 15min under the room temperature, add the dicyclohexylcarbodiimide (DCC) of 62.7mg (0.3mmol), stirring reaction spends the night under the room temperature again.Centrifugal, get supernatant liquor 0.5mL, supernatant liquor is slowly joined in the carbonate buffer solution (CBS, 0.1mol/L, pH9.6) of 10mg/mL bovine serum albumin (BSA), react 4h under the magnetic agitation.Solution after reaction the finished good dialysis tubing of pre-treatment of packing into, use first distill water dialysis 3 times (interval 2-3h) under 4 ℃, then use phosphate buffer soln (PBS, 0.01mol/L, pH7.4) dialysis 72h, change liquid 3-5 every day, namely gets immunizing antigen, packing is stored in-20 ℃ the refrigerator.
2.2 envelope antigen is synthetic
The synthetic employing mixed anhydride method of envelope antigen.With 79.8mg (0.25mmol) haptens, be dissolved in the N of 1mL, in the dinethylformamide (DMF), then in solution, add while stirring 60 μ L tri-n-butylamines and 30 μ L isobutyl chlorocarbonates, react 1h under the room temperature, slowly join again among the CBS (0.1mol/L, pH9.6) of 10mg/mL ovalbumin (OVA) of 15mL, react 2h under the magnetic agitation, after question response was finished, the dialysis tubing of packing into was used first distill water dialysis 3 times (interval 2-3h) under 4 ℃, then use PBS (0.01mol/L, pH7.4) dialysis 72h, change liquid 3-5 every day, namely gets envelope antigen, packing is stored in-20 ℃ the refrigerator for subsequent use.
2.3 the evaluation of artificial antigen
Haptens, carrier proteins and conjugate are carried out ultraviolet (200nm～400nm) scanning.See that from uv atlas the conjugate ultra-violet absorption spectrum than carrier proteins and haptenic absorption spectrum obvious variation has occured, have carrier proteins and haptenic characteristic ultraviolet absorption, the success of haptens and carrier protein couplet is described.Obtain the combination of immunizing antigen and envelope antigen than being respectively 15: 1 and 8: 1 according to their molar absorptivity estimations under the 280nm wavelength.
The preparation of 3 antibody
3.1 immune new zealand white rabbit prepares antiserum(antisera)
Back subcutaneous injection immunity new zealand white rabbit, initial immunity equivalent Freund's complete adjuvant (FCA) emulsification immunogen, immunizing dose is 1.0mg/kg (by the BSA amount), use Freund's incomplete adjuvant (FIA) emulsification booster immunization after 3 weeks, immunizing dose is 1.5mg/kg, 2 weeks of later every interval add exempts from once, adds altogether and exempts from 4 times.Concrete immunization protocol sees the following form.
In front 1 week of fundamental immunity, rabbit ear edge vein gathers negative blood, and preparation serum is as negative control.From for the third time immunity, ear edge vein exploitating blood after each immunity is with sero-fast the tiring of indirect non-competing Enzyme-Linked Immunosorbent Assay (extension rate of serum is and tires).
Wait tire qualified after, heart blood sampling.Blood is loaded in the centrifuge tube, leaves standstill 30min in 37 ℃ of water-baths, after blood coagulation, with pin blood cell is peeled off centrifugal tube wall, places 4 ℃ of refrigerator overnight, and centrifugal 10min (4000r/min) isolates serum.
3.2 the purifying of antibody
Adopt the caprylic acid-ammonium antibody purification.Get anti-blood and disappear, by 1: 4 (V/V) dilution, regulate pH to 4.5 with the 0.06mol/L acetate buffer; Drip while stirring sad (75 μ L/mL serum amount) under the room temperature, continue to stir 30min, leave standstill 2h (4 ℃), (4 ℃ 10000r/min), discard precipitation to centrifugal 30min.With 1: 10 dilution supernatant liquor, adjust pH to 7.4 with PBS (0.1mol/L, pH7.4), precooling 15min (4 ℃) slowly adds (NH
4)
2SO
40.277g/mL, continue stirring reaction 30min, leave standstill 2-3h (4 ℃); Centrifugal 30min (4 ℃, 12000r/min), abandon supernatant liquor, throw out uses the PBS that contains EDTA (74.4mg/L) in 4 ℃ of dialysis 3 days with a small amount of PBS (0.01mol/L, pH7.4) dissolving, and change liquid 3-5 every day.Finally by packing and lyophilize, get lyophilized powder ,-20 ℃ of preservations are stand-by.
3.3 antibody titer
Tiring behind the purified antibody lyophilized powder of making is 4 * 10
6
The foundation of 4 clothianidin ELISA
4.1 Method And Principle
Adopt the indirect competition immune analysis method.The mixture that pesticide molecule and macromolecular carrier (such as protein) coupling are made is adsorbed on the solid phase carrier (96 hole enzyme plate) as envelope antigen, is prepared into solid phase antigen, then adds agricultural chemicals to be measured and corresponding antibodies.Solid phase antigen, agricultural chemicals to be measured, with the antibody association reaction that is at war with, pesticide concentration to be measured is many, and the antibody that is bonded on the solid phase antigen is just few, otherwise it is many to be combined in the antibody of solid phase antigen, add after the reaction ELIAS secondary antibody (can only be combined in solid phase antigen on antibody combine), develop the color with substrate at last and measured, when one timing of antibody amount, the pesticide volume to be measured of adding is more, the antibody of being combined with solid phase antigen is just fewer, colour developing just weakens, and combination rate reduces, otherwise, then colour developing strengthens, combination rate raises, thereby can according to the combination rate of typical curve and the sample to be checked of known quantity agricultural chemicals, extrapolate the concentration of agricultural chemicals to be measured.
4.2 antigen-antibody working concentration
Definite square formation volumetry of using of ELISA antigen-antibody working concentration, selection OD value are 1.0 o'clock antigen-antibody weaker concn.Through experiment, envelope antigen concentration 0.25 μ g/mL, antibody concentration 0.3 μ g/mL is as the suitableeest working concentration
4.3 indirect competition immune response program
4.3.1 coated
With CBS damping fluid (0.05mol/L, pH9.6) envelope antigen is diluted to optimal concentration, 100 μ L/ holes add 96 hole enzyme plate (Maxisorp
TMThe transparent polyethylene plate), 4 ℃ of coated spending the night;
4.3.2 sealing
Take out coated good enzyme plate, discard coating buffer, after phosphate buffer soln (PBST) washing with 0.5% tween 20, add 5.0% the skimmed milk confining liquid 200 μ L/ holes of diluting with PBS buffered soln, incubation 30min in 37 ℃ of incubators.
4.3.3 application of sample
Learn from else's experience enzyme plate after sealing and the washing adds clothianidin reference liquid or the testing sample extracting solution 50 μ L/ holes of series concentration, adds antibody diluent 50 μ L/ holes again, blank and negative control is set simultaneously, 37 ℃ of incubation 1h.
4.3.4 add ELIAS secondary antibody
Discard liquid in the hole, use the PBST solution washing.Add 3000 times of diluents of goat-anti rabbit, the 100 μ L/ holes of horseradish peroxidase-labeled, 37 ℃ of incubation 1h discard liquid in the hole, use the PBST solution washing.
4.3.5 color reaction
Add tetramethyl benzidine (TMB)-H
2O
2Substrate solution 100 μ L/ holes, incubation 15min in 37 ℃ of incubators is with 50 μ L/ hole 2mol/L H
2SO
4Termination reaction.At iMark
TMMeasure the light absorption value (A) under the 490nm wavelength on the microplate reader.
4.4 typical curve and sensitivity
Logarithm mapping according to combination rate and clothianidin concentration namely obtains typical curve, calculates concentration (IC in the inhibition
50) and lowest detectable limit (IC
10, LOD).Combination rate (B/Bo, %) is calculated with following formula:
B/Bo(％)＝[(Ax-Amin)/(Amax-Amin)]×100
In the formula: the light absorption value when Ax is not dosing, the light absorption value of the negative contrast of Amax, Amin are the light absorption value of blank.
The linear equation of typical curve is: B/Bo (%)=-24.549LogC+17.168
Clothianidin concentration is in 1.1 μ g/L-2mg/L scopes, and is linear, and relation conefficient is R
2=0.9975, IC
50Be 0.046mg/L, LOD is 1.1 μ g/L.
4.5 the specificity of antibody
The ability that the specificity of antibody refers to its homospecificity antigen combination and comparison with this antigen-analogues ability.Cross reaction commonly used is as the major criterion of estimating.Cross reaction is less, and the specificity of antibody is better.
The antibody of preparation does not have obvious cross reaction (CR%＜0.77%) except with MTI-446 (CR%=11.8%) certain cross reaction being arranged with other nicotinic insecticides.Thereby as can be known, prepared antibodies specific is strong, can be used for the analysis of clothianidin.
5 samples add to be analyzed
5.1 extracting method
5.1.1 water sample
Water sample (or after filtration) interpolation clothianidin is made three different concns levels, and (0.01,0.05, sample 0.5mg/kg) repeats 3 times, and contrast is set.Mixing, after placement is spent the night, but direct-detection
5.1.2 soil or agricultural-food sample (wild cabbage, rice, corn, tomato)
Take by weighing the sample 10g after the pulverizing, in the triangular flask of packing into.The mode that clothianidin adds is identical with water sample.Mixing, placement is spent the night, and adds 10mL water and 50mL acetonitrile mechanical shaking extraction 1 hour in sample.Vibrate complete after, extracting solution Büchner funnel suction filtration fills filtrate pouring in the tool plug graduated cylinder of 5g sodium-chlor, thermal agitation makes acetonitrile and water stratification, leaves standstill 10min, gets 25mL to Florence flask.With concentrated near the doing of rotatory evaporator, with the PBS constant volume that contains 20% methyl alcohol.
5.2 the elisa assay of sample
The sample analysis step is with reference to 4.3.By analysis as can be known, the clothianidin rate of recovery of this ELISA is 85.1-112.2%, and average coefficient of variation is 1.1-8.4%.
The clothianidin residues detection carries out with reference to 5.1 and 5.2 methods in the actual sample.
The residual immune analysis method of clothianidin that the present invention sets up meets clothianidin retention analysis standard.The method can be used for the residue detection of clothianidin in environment and the agricultural-food, and pre-treating process is simple than instrument analytical method, is fit to mass detection and field monitoring.
Claims (3)
1. the clothianidin polyclonal antibody is obtained by immune new zealand white rabbit.
2. the preparation method of the described clothianidin antibody of claim 1 comprises:
1) artificial semiantigen is characterized in that its molecular structural formula is:
2) artificial semiantigen preparation
8mmol (0.45g) KOH is dissolved in the 20mL ethanol, adds 4mmol (0.42g) β-mercaptopropionic acid and stir, add until completely dissolved 4mmol (1.02g) clothianidin, 80 ℃ of stirring and refluxing 2h.After reaction finishes, filtering reacting liquid, filtrate decompression concentrates to get faint yellow solid.Add 50mL water, regulate pH=2.0 with the hydrochloric acid of 1mol/L.Use ethyl acetate extraction, organic phase is through washing and anhydrous sodium sulfate drying, and concentrating under reduced pressure gets the white powder material, through recrystallizing methanol, gets white crystal.Chemical name: 3-(5-((3-methyl-2-nitroguanidine)-ylmethyl) thiazole-2-sulfydryl) propionic acid, relative molecular mass: 319, melting range: 158.5 ℃-160.2 ℃.
3) artificial antigen preparation
Artificial antigen is characterised in that, claim adopts carbodlimide method that haptens and bovine serum albumin (BSA) coupling are prepared immunizing antigen, and employing mixed anhydride method and ovalbumin (OVA) coupling prepare envelope antigen.Its molecular structural formula is:
4) immunity
With claim 23) described clothianidin immunizing antigen immunity new zealand white rabbit prepares polyclonal antibody.
3. the polyclonal antibody enzyme-linked immunosorbent assay of the described clothianidin of claim 1 (ELISA) is set up, and the application in the clothianidin residual quantity in testing environment and agricultural-food.
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| CN107473961A (en) * | 2017-06-19 | 2017-12-15 | 宁波市农业科学研究院 | TrinexAN_SNacethyl artificial antigen, specific antibody preparation method and its usage |
| CN108840848A (en) * | 2018-05-30 | 2018-11-20 | 中国烟草总公司郑州烟草研究院 | A kind of preparation method and application of cumarin haptens and antigen |
| CN108948188A (en) * | 2018-08-16 | 2018-12-07 | 江南大学 | One plant of hybridoma cell strain J6 for secreting clothianidin monoclonal antibody and its application |
| CN112062852A (en) * | 2020-07-28 | 2020-12-11 | 浙江大学 | Variable region sequence of anti-clothianidin and dinotefuran broad-spectrum antibody and preparation of recombinant complete antibody thereof |
| CN112194725A (en) * | 2020-04-26 | 2021-01-08 | 浙江大学 | Variable region sequence of specific anti-clothianidin antibody and preparation and application of recombinant complete antibody thereof |
| CN112595850A (en) * | 2020-11-18 | 2021-04-02 | 北京勤邦生物技术有限公司 | Application of clothianidin artificial antigen in enzyme linked immunosorbent assay kit |
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| CN105296520B (en) * | 2015-12-04 | 2019-01-15 | 中国科学院福建物质结构研究所 | A kind of preparation method and its enzyme-linked immunologic detecting kit of human tumor antigen 3H11Ag |
| CN105296520A (en) * | 2015-12-04 | 2016-02-03 | 中国科学院福建物质结构研究所 | Preparation method and enzyme-linked immunosorbent assay kit of human tumor antigen 3H11Ag |
| CN107473961A (en) * | 2017-06-19 | 2017-12-15 | 宁波市农业科学研究院 | TrinexAN_SNacethyl artificial antigen, specific antibody preparation method and its usage |
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| CN108948188B (en) * | 2018-08-16 | 2021-05-04 | 江南大学 | Hybridoma cell strain J6 secreting clothianidin monoclonal antibody and application thereof |
| CN108948188A (en) * | 2018-08-16 | 2018-12-07 | 江南大学 | One plant of hybridoma cell strain J6 for secreting clothianidin monoclonal antibody and its application |
| CN112194725A (en) * | 2020-04-26 | 2021-01-08 | 浙江大学 | Variable region sequence of specific anti-clothianidin antibody and preparation and application of recombinant complete antibody thereof |
| WO2021218053A1 (en) * | 2020-04-26 | 2021-11-04 | 浙江大学 | Variable region sequence of specific anti-clothianidin antibody, and preparation and application of anti-clothianidin recombinant intact antibody |
| CN112194725B (en) * | 2020-04-26 | 2022-04-19 | 浙江大学 | Variable region sequence of specific anti-clothianidin antibody and preparation and application of recombinant complete antibody thereof |
| US11884743B2 (en) | 2020-04-26 | 2024-01-30 | Zhejiang University | Preparation and application of an intact recombinant antibody specific to clothianidin based on the identified variable region sequence |
| CN112062852B (en) * | 2020-07-28 | 2021-10-12 | 浙江大学 | Variable region sequence of anti-clothianidin and dinotefuran broad-spectrum antibody and preparation of recombinant complete antibody thereof |
| CN112062852A (en) * | 2020-07-28 | 2020-12-11 | 浙江大学 | Variable region sequence of anti-clothianidin and dinotefuran broad-spectrum antibody and preparation of recombinant complete antibody thereof |
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Application publication date: 20130116 |