CN106674138B - A kind of novel haptens of Tebuconazole and its synthetic method and application - Google Patents

A kind of novel haptens of Tebuconazole and its synthetic method and application Download PDF

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CN106674138B
CN106674138B CN201611019736.9A CN201611019736A CN106674138B CN 106674138 B CN106674138 B CN 106674138B CN 201611019736 A CN201611019736 A CN 201611019736A CN 106674138 B CN106674138 B CN 106674138B
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陈秀金
李兆周
王耀
高红丽
李智丽
曹力
李道敏
胥传来
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Henan University of Science and Technology
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    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
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    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

The present invention relates to a kind of novel haptens of Tebuconazole and its synthetic method and application, the structural formula of the novel haptens of Tebuconazole is：.For the novel haptens of Tebuconazole using Tebuconazole as raw material, synthetic route is as follows：First Tebuconazole is dissolved in anhydrous methylene chloride, after dissolution, triethylamine is added, is stirred under ice bath, add diethyl succinate list acyl chlorides, be stirred overnight at room temperature, is washed, is dried, filtered, be concentrated to dryness to obtain intermediate product；Then plus the aqueous solution of a hydronium(ion) lithia intermediate product is dissolved in anhydrous methanol and tetrahydrofuran mixed solution, is stirred overnight at room temperature, under ice bath, pH value is adjusted, using reverse phase column purification, concentrated freeze-dried, recycling obtains colorless oil solid, the as novel haptens of Tebuconazole.The novel synthesis of semiantigen of the method for the present invention Tebuconazole is simple, prepares Tebuconazole antibody using the haptens, the high sensitivity of gained antibody can be used for establishing the enzyme-linked immunosorbent assay of Tebuconazole residue detection.

Description

A kind of novel haptens of Tebuconazole and its synthetic method and application
Technical field
The present invention relates to a kind of haptens, the novel haptens of specifically a kind of Tebuconazole and its synthetic method and answer With.
Background technique
Tebuconazole, the entitled Tebuconazole of English, is a kind of ethoxy triazole derivative, belongs to less toxic, efficient, wide spectrum Triazole type systemic fungicide is sterol demethylation inhibitors, ergosterol in the cell membrane by destroying and preventing germ Biosynthesis keeps pathogen lethal because that cannot form cell membrane, can effectively prevent the rust and powdery mildew of cereal crop；Apple The diseases such as the leaf spot of fruit and banana, ring spot and scab.However, long-term be used for field diseases prevention and treatment for Tebuconazole in large quantities When, since its lasting period is long, cause serious " 3R "（Resistance Resistance, second rice crop season Resurgence and residual Residue）Problem；In addition, research also found that Tebuconazole has certain lethal and teratogenic effect to Zebrafish Embryo, it is right Mouse has toxicity accumulation and hereditary effect, Environmental Protection Agency（Enviroment Protection Agency, EPA） Tebuconazole is classified as " mankind are potentially carcinogenic substance ", therefore, needs to establish the side for detecting Tebuconazole fungicide quickly, with sensitivity Method.
Currently, the detection remaining method of Tebuconazole mainly has high performance liquid chromatography, gas chromatography and gas chromatography combined Use mass spectrography.Although instrument analytical method is sensitive, accurate and favorable reproducibility, the pre-treatment step of sample is cumbersome, instrument and equipment Valuableness needs professional to operate, and testing cost is high, needs to consume a large amount of toxic organic solvents, is not particularly suitable for a large amount of samples The quick screening of product.In recent years, researcher also uses fluorescent spectrometry and molecular imprinting electrochemical sensor in agricultural product penta Azoles alcohol residual is detected, and has the advantages that rapid sensitive, but the testing cost of both methods is higher, stability is poor.And enzyme Linked immune analysis method has the advantages that high-throughput, testing cost is low and fast and convenient, can just make up the deficiency of the above method. Up to the present, domestic not reported about the enzyme-linked immunosorbent assay of Tebuconazole residue detection still.And it is enzyme-linked to establish Tebuconazole The key of immunoassay method is the design and synthesis of its haptens.
Summary of the invention
Object of the present invention is to deficiency to solve above-mentioned technical problem, a kind of novel haptens of Tebuconazole and its synthesis are provided Methods and applications, the synthetic method is simple, prepares Tebuconazole antibody, gained antibody using the synthesized novel haptens of Tebuconazole High sensitivity, half-inhibitory concentration（IC50）For 1.46 ± 0.32 ng/mL；High specificity, the cross reacting rate with its analog 10% is below,
A kind of novel haptens of Tebuconazole, structural formula are：。
The novel haptens of Tebuconazole is characterized by the hydrogen of LC-MS technology and nuclear magnetic resonance spectrum and carbon spectrum, As a result it is:
The novel haptens of Tebuconazole is chemically reacted by 2 steps and is obtained, synthetic route is such as using Tebuconazole as raw material Under：
Wherein DCM represents methylene chloride；Et3N represents triethylamine；LiOH-H2O represents Lithium hydroxide monohydrate；MeOH represents first Alcohol；THF represents tetrahydrofuran.
The synthetic method of the novel haptens of Tebuconazole, includes the following steps：
（1）The synthesis of intermediate product
Tebuconazole (184 mg, 0.6 mmol) is first dissolved in anhydrous methylene chloride (CH2Cl2, 15 mL) in, dissolution Afterwards, triethylamine is added（C6H15N, 730 mg, 7.2 mmol）, stir after five minutes under ice bath, add diethyl succinate Single acyl chlorides（C6H9ClO3, 988 mg, 6 mmol）, it is stirred overnight, is then successively washed with water, saturated brine at room temperature, with nothing The filtering of aqueous sodium persulfate dry methylene chloride, reduction vaporization are concentrated to dryness, obtain about 400 mg of intermediate product, be directly used in next step water Solution.The structure of the product is confirmed by LC-MS technology.
The saturated brine is saturation NaCl aqueous solution.It is described successively to be washed with water, saturated brine, that is, use 15 mL water Washing 3 times, then washed 3 times using saturated brine.
（2）The synthesis of the novel haptens of Tebuconazole
By 400 mg of crude product（About 0.6 mmol）It is dissolved in anhydrous methanol-tetrahydrofuran (9 mL-9 mL) mixed liquor, Aqueous solution (the LiOHH of a hydronium(ion) lithia is added2O, 252 mg, 6 mmol, 3 mL H2O), it is stirred overnight at room temperature, Organic solvent is removed, under ice bath, the pH of solution is transferred to by 2.0-3.0 using 1 mol/L hydrochloric acid, using reverse phase column purification, flowing It is 40% acetonitrile solution that phase, which is volumetric concentration, concentrated freeze-dried, and recycling obtains colorless oil solid (70mg), yield 29%.
Application of the novel haptens of Tebuconazole in preparation immunogene, includes the following steps：
（1）Weigh Tebuconazole haptens（4.1 mg, 0.01 mmoL）It is added to N-N- dimethylformamide（DMF, 1 mL） In, after stirring and dissolving, the 1- ethyl-carboddiimide hydrochloride of addition（EDC, 4.5 mg）And n-hydroxysuccinimide（NHS, 4.4 mg）, react at room temperature 6 hours, obtain A liquid；
（2）Take hemocyanin（KLH, 20.92 mg/mL, 0.3 mL）In the borate buffer solution being added to（0.2 mol/ L, 3.5 mL, pH 8.8）, stir and evenly mix, obtain B liquid；Under ice bath, A liquid is added dropwise in B liquid, is stirred to react at 4 DEG C 8-10 hours；
（3）Using phosphate buffer solution（0.01mol/L, pH 7.2）It dialyses 3 days to reaction solution, replacement one in every 6 hours Secondary dialyzate is replaced 8 times；The packing of resulting Tebuconazole immunogene, freeze with it is spare.
The novel haptens of Tebuconazole can be applied to prepare anti-Tebuconazole polyclonal antibody.
The novel haptens of Tebuconazole can be applied to the immunoassay of Tebuconazole residue detection in agricultural product.
The novel haptens of Tebuconazole can be applied to prepare anti-Tebuconazole polyclonal antibody.
The novel haptens of Tebuconazole can be applied to the immunoassay of Tebuconazole residue detection in agricultural product.
Beneficial effect is：
The novel synthesis of semiantigen of the method for the present invention Tebuconazole is simple, and synthesized Tebuconazole fungicide novel half is anti- Original prepares Tebuconazole antibody, gained antibody high sensitivity, half-inhibitory concentration using the haptens（IC50）It is 1.46 ± 0.32 ng/mL；High specificity is below 10% with the cross reacting rate of its analog, can be used for establishing the enzyme-linked of Tebuconazole residue detection Immunoassay provides raw material to establish the immunoassay method of Tebuconazole residue detection in agricultural product.
Detailed description of the invention
Fig. 1 is the liquid chromatogram of intermediate product；
Fig. 2 is the mass spectrogram of intermediate product；
The liquid chromatogram of Fig. 3 Tebuconazole haptens；
The mass spectrogram of Fig. 4 Tebuconazole haptens；
The nucleus magnetic hydrogen spectrum figure of Fig. 5 Tebuconazole haptens；
The nuclear-magnetism carbon spectrogram of Fig. 6 Tebuconazole haptens；
The UV scanning figure of Fig. 7 various concentration Tebuconazole haptens；
The ultraviolet phenogram of Fig. 8 Tebuconazole immunogene；
The ultraviolet phenogram of Fig. 9 Tebuconazole coating antigen；
The standard suppression curve figure of Figure 10 measurement Tebuconazole.
Specific embodiment
A kind of synthetic method of the novel haptens of Tebuconazole is chemically reacted using Tebuconazole as raw material by 2 steps, obtains penta The novel haptens of azoles alcohol.Its synthetic route is as follows：
Explanation：DCM represents methylene chloride；Et3N represents triethylamine；LiOH-H2O represents Lithium hydroxide monohydrate；MeOH is represented Methanol；THF represents tetrahydrofuran.
Its synthesis step is：
（1）The synthesis of haptens
1., the synthesis of crude product
Tebuconazole (184 mg, 0.6 mmol) is first dissolved in anhydrous methylene chloride (CH2Cl2, 15 mL) in, dissolution Afterwards, triethylamine is added（C6H15N, 730 mg, 7.2 mmol）, stir after five minutes under ice bath, add diethyl succinate Single acyl chlorides（C6H9ClO3, 988 mg, 6 mmol）, it is stirred overnight, is then successively washed with water, saturated brine at room temperature, with nothing The filtering of aqueous sodium persulfate dry methylene chloride, reduction vaporization are concentrated to dryness, and obtain intermediate product（About 400 mg）, it is directly used in next step Hydrolysis.The structure of the product is confirmed by LC-MS technology.Shown in the result is shown in Figure 1 and Fig. 2, LCMS (ESI) m/z = 564.8/566.8 ([M+H]+)。
The saturated brine is saturation NaCl aqueous solution.It is described successively to be washed with water, saturated brine, that is, use 15 mL water Washing 3 times, then washed 3 times using saturated brine.
2. the synthesis of the novel haptens of Tebuconazole
Crude product (400 mg, about 0.6 mmol) is dissolved in anhydrous methanol-tetrahydrofuran (9 mL-9 mL) mixed liquor, Aqueous solution (the LiOHH of a hydronium(ion) lithia is added2O, 252 mg, 6 mmol, 3 mL H2O), it is stirred overnight at room temperature, Organic solvent is removed, under ice bath, the pH of solution is transferred to by 2.0-3.0 using 1 mol/L hydrochloric acid, using reverse phase column purification, flowing It is mutually the acetonitrile solution that volumetric concentration is 40%, concentrated freeze-dried, recycling obtains colorless oil solid (70mg), yield 29%.
The structure of the product is characterized by the hydrogen of LC-MS technology and nuclear magnetic resonance spectrum and carbon spectrum.As a result see Fig. 3- Shown in Fig. 6:
（2）The preparation of immunogene
Weigh Tebuconazole haptens（4.1 mg, 0.01 mmoL）It is added to N-N- dimethylformamide（DMF, 1 mL）In, After stirring and dissolving, the 1- ethyl-carboddiimide hydrochloride of addition（EDC, 4.5 mg）And n-hydroxysuccinimide（NHS, 4.4 mg）, react at room temperature 6 hours, obtain A liquid.
Take hemocyanin（KLH, 20.92 mg/mL, 0.3 mL）In the borate buffer solution being added to（0.2 mol/L, 3.5 mL, pH 8.8）, stir and evenly mix, obtain B liquid.Under ice bath, A liquid is added dropwise in B liquid, is stirred to react 8- at 4 DEG C 10 hours.
Using phosphate buffer solution（0.01mol/L, pH 7.2）It dialyses 3 days to reaction solution, replacement in every 6 hours is primary Dialyzate is replaced 8 times.The packing of resulting Tebuconazole immunogene, freeze with it is spare.
（3）The preparation of coating antigen
Weigh Tebuconazole haptens（4.1 mg, 0.01 mmol）It is dissolved in N-N- dimethylformamide（DMF, 1 mL）, then plus Enter tri-n-butylamine（8μL）And isobutyl chlorocarbonate（5 μL）, react at room temperature 30 minutes, obtain A liquid.
Weigh bovine serum albumin(BSA)（20 mg）Or chicken egg white（15 mg）It is added to borate buffer solution（0.2 mol/L, 5.0 mL, pH 8.8）, stir and evenly mix, obtain B liquid.Then A liquid is added dropwise in B liquid under stirring, it is anti-at 4 DEG C It answers 6 hours.
Using phosphate buffer solution（0.01mol/L, pH 7.2）It dialyses 3 days to reaction solution, replacement in every 6 hours is primary Dialyzate is replaced 8 times.The packing of resulting Tebuconazole coating antigen, freeze with it is spare.
（2）The identification of artificial antigen
The determination of Tebuconazole haptens maximum absorption wavelength：The Tebuconazole haptens methanol solution of 1 mg/mL is first prepared, is passed through Dilution is crossed, a series of Tebuconazole hapten concentration is prepared（0,25,50,75 μ g/mL）, scanning wavelength 200-500 nm's is ultraviolet Line.Ultraviolet determination result is shown in Fig. 7, and as can be seen from the results, a length of 247 nm of the maximum absorption wave of Tebuconazole haptens, each concentration is done 3 Duplicate Samples.
The calculating of molar absorption coefficient (ε)：The calculation formula ε=light absorption value under maximum absorption wavelength/of molar absorption coefficient rubs That concentration.This experimental calculation obtains Tebuconazole antigen ε=4029.3 L/moL.
The measurement of conjugate protein concentration：Because resulting antigenic solution is the clear solution of brown color, therefore protein concentration Calculation formula:
C=M/V
M is the quality of carrier protein, g；V is the volume after antigen dialysis, mL；C is the protein concentration of antigen.Therefore, exist After antigen dialysis, the total volume of antigen directly after measurement dialysis.By can be calculated, the protein concentration of antigen is 3.72mg mL-1。
The measurement of coupling ratio：The aqueous solution of 0.2 mg/mL carrier protein is prepared, antigen is diluted to 0.2 using aqueous solution Mg/mL measures the light absorption value at 278 nm respectively, the light absorption value measured be A1, A2, then coupling ratio r be：
R=((A2-A1)/ε)/(200 × 10-3The molecular mass of/carrier protein)
Wherein ε is molar absorption coefficient（L/moL）, this experimental calculation obtains：r≈15.It follows that haptens and carrier egg White ratio is 15：1.
The synthesis and identification of 1 Tebuconazole haptens of embodiment
Step 1: the synthesis of intermediate product
Tebuconazole (184 mg, 0.6 mmol) is dissolved in anhydrous methylene chloride (CH first2Cl2, 15 mL) in, then Triethylamine is added（C6H15N, 730 mg, 7.2 mmol）, ice bath stirring after five minutes, be added diethyl succinate list acyl chlorides （C6H9ClO3, 988 mg, 6 mmol）, it is stirred overnight, is successively washed with water, saturated brine at room temperature（Respectively 15 mL × 3）, after anhydrous sodium sulfate dry methylene chloride, filtering, reduction vaporization is concentrated to dryness, and obtains intermediate product（About 400mg）, directly It connects for next one-step hydrolysis.The product structure is characterized by LC-MS technology.
Step 2: the synthesis of Tebuconazole haptens
Intermediate product (400 mg, 0.6 mmol) is dissolved in anhydrous methanol-tetrahydrofuran (9 mL：9 mL) mixed liquor In, the aqueous solution (LiOHH of a hydronium(ion) lithia is added2O, 252 mg, 6 mmol, 3 mL water), it was stirred at room temperature Night removes organic solvent.Under ice bath, the pH of solution is transferred to by 2.0-3.0 using 1 mol/L hydrochloric acid, using reverse phase column purification, stream It is 40% acetonitrile solution that dynamic phase, which is volumetric concentration, concentrated freeze-dried, and recycling obtains colorless oil solid (about 70mg), yield 29%. The product structure is analyzed using the hydrogen of LC-MS technology and nuclear magnetic resonance spectrum and carbon spectrum.The structure of Tebuconazole haptens is such as Under,
The preparation and identification of 2 Tebuconazole antigen of embodiment
1, Tebuconazole haptens is used into active ester method（EDC method）And hemocyanin（KLH）Coupling is used as immunogene, tool Precursor reactant route is as follows：
Tebuconazole haptens is used into mixed anhydride method and chicken egg white（OVA）Or bovine serum albumin(BSA)（BSA）Coupling, As coating antigen, specific reaction route is as follows：
Explanation：Wherein Protein is protein.
2, the purifying of artificial antigen
Desalting and purifying is carried out to artificial antigen using dialysis.
3, the identification of artificial antigen
The measurement of conjugate protein concentration：Antigen prepared by this method is the clear solution of brown color, protein concentration Calculation formula is：
C=M/V
M is the quality of carrier protein, g；V is the volume after antigen dialysis, mL；C is the protein concentration of antigen.Therefore, exist After antigen dialysis, the total volume of antigen directly after measurement dialysis.By can be calculated, the protein concentration of antigen is 3.72 mg/ mL。
Coupling ratio measurement：The aqueous solution of 0.2 mg/mL carrier protein is prepared, antigen is diluted to 0.2 mg/ using distilled water ML, measures the survey light absorption value at 278 nm respectively, and the light absorption value measured is A1, A2.The calculation formula of coupling ratio r is：
；Wherein ε is molar absorption coefficient（L/moL）, this experiment It is calculated：Therefore r ≈ 15 estimates the coupling ratio 15 of Tebuconazole immunogene according to above-mentioned formula：1.
The potency of polyclonal antibody is 1:16000, half-inhibitory concentration（IC50）For 1.46 ± 0.32 ng/mL, with its class It is lower than 10% like the cross reacting rate of object；It generates in accordance with the following steps：
The preparation of step primary antibody Tebuconazole polyclonal antibody
1, animal immune：Selecting weight for the new zealand white rabbit of 2.0 kg or so is experimental animal, and every 2 are one group, Totally 3 groups.Immunogene is the conjugate that Tebuconazole haptens and hemocyanin are prepared by active ester method.Just exempt from complete using Fu Shi Adjuvant emulsion is immunized by the way of the multi-point injection of back after immunogene emulsification, and just exempting from dosage is 1 mg/kg weight（With Protein content meter）, carry out after four weeks reinforcing exempting from.Reinforcement exempts to be emulsified using Freund's incomplete adjuvant, 0.65 mg/kg of immunizing dose Weight was reinforced exempting from primary every 3 weeks.
2, sero-fast screening：4th time immune 7-10 days latter, by auricle outer rim venous blood collection, after blood sampling, at room temperature 30 minutes are stood, centrifugation（5000 rpm, 10 min）, supernatant is sucked out, obtains antiserum.Using indirect competitive enzyme-linked immunosorbent Method measures sero-fast potency and inhibition, select the serum that potency is high and has inhibited, to selecting by way of Culling heart blood Rabbit blood sampling is collected in 50 mL sterilizing plastic centrifuge tube, prepares antiserum according to the method described above.
3, the purifying of antibody
It is purified using ammonium sulfate precipitation method.
Step 2: the measurement of Tebuconazole antibody characteristic
1, the measurement of antibody titer
Using Indirect cELISA（ic-ELISA）The potency for measuring Tebuconazole antibody, show that the potency of antibody is 1:16000.Using Tebuconazole haptens-bovine serum albumin(BSA) conjugate coated elisa plate；After closing, doubling dilution is added One antiserum reacts 30 minutes at 37 DEG C；After washing pats dry, ELIAS secondary antibody is added, is reacted 30 minutes at 37 DEG C；Washing pats dry Afterwards, it develops the color 15 minutes at 37 DEG C, terminates.Light absorption value under 450 nm is measured using microplate reader.
2, the measurement of sensitivity
Using Indirect cELISA（ic-ELISA）Antibody is measured to the inhibitory effect of Tebuconazole pesticide.Selection packet It is 0.3 μ g/mL by original content, the protein concentration of antibody is 0.04 μ g/mL, and measurement result carries out four ginsengs with 8.5 software of origin Number regression fit, the result is shown in Figure 10.According to the regression equation of fitting, the half-inhibitory concentration of Tebuconazole antibody, i.e. IC are calculated50For 1.46±0.32 ng/mL；The range of linearity of standard curve is between 0-40 ng/mL.
3, specific measurement
Using Indirect cELISA measurement Tebuconazole antibody to its analog（Triadimenol, triazolone, olefin conversion, Nitrile bacterium azoles and bitertanol）Inhibiting effect.According to the half of the four parametric regression equation calculation Tebuconazole analogs of Figure 10 Inhibition concentration recycles following formula to calculate the cross reacting rate of Tebuconazole analog.
Cross reacting rate（CR, %）= IC50（Tebuconazole）/ IC50（Tebuconazole analog）.The intersection of Tebuconazole antibody is anti- That answers the results are shown in Table 1.
Table 1：

Claims (7)

1. a kind of Tebuconazole haptens, it is characterised in that：Its structural formula is：
2. the synthetic method of Tebuconazole haptens as described in claim 1, it is characterised in that：The Tebuconazole haptens is with penta azoles Alcohol is raw material, chemically reacts and obtains by 2 steps, and synthetic route is as follows：
Wherein DCM represents methylene chloride；Et3N represents triethylamine；LiOH-H2O represents Lithium hydroxide monohydrate；MeOH represents methanol； THF represents tetrahydrofuran.
3. the synthetic method of Tebuconazole haptens as claimed in claim 2, it is characterised in that：The conjunction of every 70mg Tebuconazole haptens At including the following steps：
(1) synthesis of intermediate product
First 0.6mmol Tebuconazole is dissolved in 15mL anhydrous methylene chloride, after dissolution, adds 7.2mmol triethylamine, under ice bath Stirring after five minutes, adds 6mmol diethyl succinate list acyl chlorides, is stirred overnight at room temperature, then successively with water, saturation chlorine Change sodium water solution washing, it is dry with anhydrous sodium sulfate, it is filtered with methylene chloride, reduction vaporization is concentrated to dryness, and obtains intermediate product, directly It connects for next one-step hydrolysis；
(2) synthesis of Tebuconazole haptens
It is 1 that crude product, which is dissolved in 18mL volume ratio,：In 1 anhydrous methanol and tetrahydrofuran mixed solution, then plus 3-4mL The aqueous solution of a hydronium(ion) lithia of 0.002mmol/L is stirred overnight at room temperature, and obtains mixed solution C；Under ice bath, use The pH value of mixed solution C is transferred to 2.0-3.0 by 1mol/L hydrochloric acid, and using reverse phase column purification, mobile phase is that volumetric concentration is 40% Acetonitrile solution, concentrated freeze-dried, recycling obtains colorless oil solid, as Tebuconazole haptens.
4. application of the Tebuconazole haptens as described in claim 1 in preparation immunogene.
5. application of the Tebuconazole haptens as claimed in claim 4 in preparation immunogene, it is characterised in that：Every penta azoles of 4.1mg Alcohol haptens prepares immunogene, includes the following steps：
(1) it weighs Tebuconazole haptens 4.1mg to be added in 1mL N-N '-dimethylformamide, after stirring and dissolving, be added 4.5mg 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and 4.4mg n-hydroxysuccinimide, room temperature are anti- It answers 6 hours, obtains A liquid；
(2) taking 0.3mL concentration is 20.92mg/mL hemocyanin solution, and being added to 3.5mL concentration is 0.2mol/L, pH value 8.8 Borate buffer solution in, stir and evenly mix, obtain B liquid；Under ice bath, A liquid is added dropwise in B liquid, is stirred at 4 DEG C anti- It answers 8-10 hours, obtains reaction solution；
(3) use concentration for 0.01mol/L, the phosphate buffer solution that pH value is 7.2 dialyses to reaction solution 3 days, and every 6 hours more Change a dialyzate, replace 8 times, obtain Tebuconazole immunogene, dispense, freeze with it is spare.
6. Tebuconazole haptens as described in claim 1 is preparing the application in anti-Tebuconazole polyclonal antibody.
7. Tebuconazole haptens as described in claim 1 is applied in the immunoassay of Tebuconazole residue detection in agricultural product.
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CN110054576B (en) * 2019-04-15 2021-03-12 华南农业大学 Metolachlor chiral hapten, artificial antigen and antibody as well as preparation method and application thereof
CN110845429A (en) * 2019-10-31 2020-02-28 北京勤邦生物技术有限公司 Tebuconazole hapten, artificial antigen and antibody as well as preparation method and application thereof

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1164229A (en) * 1994-11-21 1997-11-05 拜尔公司 Microbicidal triazolyl derivatives
CN1217717A (en) * 1996-05-02 1999-05-26 拜尔公司 Acyl mercapto-triazolyl derivative and use thereof as microbiocides
CN1226238A (en) * 1996-05-22 1999-08-18 拜尔公司 Sulphonyl-mercapto-triazolyl derivative and their use as microbiocides
WO2010146113A1 (en) * 2009-06-18 2010-12-23 Basf Se Antifungal 1, 2, 4-triazolyl derivatives having a 5- sulfur substituent
WO2011113820A1 (en) * 2010-03-16 2011-09-22 Basf Se A process using grignard reagents
WO2012025506A1 (en) * 2010-08-26 2012-03-01 Bayer Cropscience Ag 5-iodo-triazole derivatives
CN102595903A (en) * 2009-06-18 2012-07-18 巴斯夫欧洲公司 Fungicidal mixtures
CN105061584A (en) * 2015-07-16 2015-11-18 河南科技大学 synthesis method and application of tebuconazole artificial antigen

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1164229A (en) * 1994-11-21 1997-11-05 拜尔公司 Microbicidal triazolyl derivatives
CN1217717A (en) * 1996-05-02 1999-05-26 拜尔公司 Acyl mercapto-triazolyl derivative and use thereof as microbiocides
CN1226238A (en) * 1996-05-22 1999-08-18 拜尔公司 Sulphonyl-mercapto-triazolyl derivative and their use as microbiocides
WO2010146113A1 (en) * 2009-06-18 2010-12-23 Basf Se Antifungal 1, 2, 4-triazolyl derivatives having a 5- sulfur substituent
CN102595903A (en) * 2009-06-18 2012-07-18 巴斯夫欧洲公司 Fungicidal mixtures
WO2011113820A1 (en) * 2010-03-16 2011-09-22 Basf Se A process using grignard reagents
WO2012025506A1 (en) * 2010-08-26 2012-03-01 Bayer Cropscience Ag 5-iodo-triazole derivatives
CN105061584A (en) * 2015-07-16 2015-11-18 河南科技大学 synthesis method and application of tebuconazole artificial antigen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Identification and characterization of tebuconazole transformation products in soil by combining suspect screening and molecular typology;Veronika Storck等;《Environmental Pollution》;20151107;第208卷;第537-545页 *

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