阅读说明：本技术 一种新的异丙威半抗原、完全抗原及其制备与应用 (New isoprocarb hapten and complete antigen as well as preparation and application thereof ) 是由 胡江 冯艳武 卫大群 郭志雄 葛怀娜 于兴华 于 2021-12-06 设计创作，主要内容包括：本发明涉及抗原合成技术领域,公开了一种新的异丙威半抗原、完全抗原及其制备与应用。本发明以3-异丙基-4-羟基苯甲酸为起始原料,一步法得到3-异丙基-4-甲基氨基甲酰氧基苯甲酸,将其作为异丙威半抗原与载体蛋白偶联获得完全抗原,本发明制备方法简单,直接在苯环上引入活性的羧基,完整的保留了异丙威氨基甲酸酯和异丙基的分子结构,具有较高的特异性和灵敏度。(The invention relates to the technical field of antigen synthesis, and discloses a novel isoprocarb hapten, a novel isoprocarb complete antigen, and preparation and application thereof. The invention takes 3-isopropyl-4-hydroxybenzoic acid as the starting material, 3-isopropyl-4-methyl carbamoyloxybenzoic acid is obtained by one-step method, and is coupled with carrier protein to obtain complete antigen as isoprocarb hapten.)

1. A novel isoprocarb hapten, having the structural formula shown in formula I:

。

2. a method of preparing isoprocarb hapten as claimed in claim 1, comprising the steps of:

s11, mixing 3-isopropyl-4-hydroxybenzoic acid with a solvent to obtain a mixed solution;

s12, adding triethylamine and trimethyl bromosilane into the mixed solution obtained in the step S11 for reaction;

s13, directly reacting the reaction product obtained in the step S12 with methyl isocyanate without separation, and hydrolyzing and acidifying to obtain 3-isopropyl-4-methyl carbamoyloxybenzoic acid, namely the isoprocarb hapten.

3. The method for preparing isoprocarb hapten according to claim 2, wherein the feeding molar ratio of the 3-isopropyl-4-hydroxybenzoic acid, the trimethylbromosilane, the methyl isocyanate and the triethylamine is 1: 1: 1: 2.

4. a novel isoprocarb complete antigen, which is obtained by coupling the isoprocarb hapten as claimed in claim 1 with a carrier protein, and the structural formula of which is shown in formula II:

wherein, in the step (A),is a carrier protein.

5. A method for preparing isoprocarb complete antigen according to claim 4, which comprises the following steps:

reacting and dialyzing the isoprocarb hapten solution and the carrier protein solution at room temperature to obtain the isoprocarb complete antigen.

6. The method for preparing isoprocarb complete antigen according to claim 5, wherein the mass ratio of the isoprocarb hapten to the carrier protein is 1:1-1: 20; the reaction time of the reaction is 4-24 h.

7. The method for preparing isoprocarb complete antigen according to claim 6, wherein the isoprocarb hapten solution is obtained by the following method one or method two:

the method comprises the following steps: dissolving isoprocarb hapten in DMF, adding DCC and NHS to react for 12-24h to obtain isoprocarb hapten solution;

the second method comprises the following steps: dissolving isoprocarb hapten in DMF, adding n-butylamine and isobutyl chloroformate, and reacting for 0.5-2h to obtain the isoprocarb hapten solution.

8. An isoprocarb antibody obtained by immunizing an animal with one of the isoprocarb hapten as set forth in claim 1, the isoprocarb hapten prepared by the preparation method as set forth in claim 2 or claim 3, the isoprocarb complete antigen as set forth in claim 4, the isoprocarb complete antigen prepared by the preparation method as set forth in claim 5 or claim 6 or claim 7.

9. An enzyme-linked immunoassay kit for detecting isoprocarb, wherein the kit comprises one of the isoprocarb complete antigen of claim 4, the isoprocarb complete antigen prepared by the preparation method of claim 5, or claim 6 or claim 7, or the isoprocarb antibody of claim 8.

10. Use of the isoprocarb hapten of claim 1, the isoprocarb complete antigen of claim 4 or the isoprocarb antibody of claim 8 in the detection of isoprocarb or in the preparation of a product for detecting isoprocarb.

Technical Field

The invention relates to the technical field of antigen synthesis, in particular to a novel isoprocarb hapten, a novel isoprocarb complete antigen, and preparation and application thereof.

Background

Isoprocarb (MIPC) also called as chlorpheniramine maleate powder and cicada slough powder, and the molecular formula is C₁₁H₁₅NO₂The chemical name is 2-isopropylphenyl-N-methyl carbamate, which is one of carbamate insecticides and has a structural formula shown in the formula III:. Isoprocarb can paralyze and kill insects by inhibiting the biological activity of acetylcholinesterase in the insects, and is widely applied to fruits, vegetables, food, tobacco and the like. However, such pesticides are also highly toxic and remain in agricultural products or the environment, which may cause a persistent health hazard to the human body. In order to guarantee human health and monitor the pesticide exposure level, the establishment of a simple, reliable and high-sensitivity detection method for detecting isoprocarb is of great significance.

The immunoassay is an analysis technique based on a specific and reversible binding reaction between an antigen and an antibody. The immune reaction relates to the comprehensive action of highly complementary stereo structures, static electricity, hydrogen bonds, van der waals force and the like between antigen and antibody molecules, has selectivity and sensitivity which are difficult to achieve by any single physical and chemical analysis technology, has the advantages of consistent sensitivity with conventional instrument analysis, suitability for field screening, simplicity, rapidness, low cost, less required amount of samples and the like, and is considered as a competitive and challenging rapid detection technology in the 21 st century. The world Food and Agriculture Organization (FAO) has recommended this technology to many countries. The American Chemical Society (ACS) lists immunoassay and chromatography as the main technologies for residue analysis of pesticides, veterinary drugs and fishery drugs.

The invention patent with publication number CN106588699A discloses isoprocarb hapten, artificial antigen, antibody and preparation method and application thereof, wherein the structural formula of the isoprocarb hapten is shown as formula IV:(ii) a The structural formula of the isoprocarb artificial antigen is shown as a formula V:the linear range of the antibody prepared based on the hapten and the artificial antigen is 1.88-82.8 ng/mL, the lowest detection limit is 0.54ng/mL, the half-inhibition concentration is 11.7ng/mL, the specificity, the sensitivity and the accuracy of the antibody are high, and the antibody has a wide application prospect in the aspect of detecting the residue of isoprocarb in agricultural products.

The hapten prepared by directly introducing active carboxyl on a benzene ring and completely reserving the molecular structures of isoprocarb carbamate and isopropyl is not reported at present.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a novel isoprocarb hapten, a novel isoprocarb complete antigen, and preparation and application thereof.

The first purpose of the invention is to provide a novel isoprocarb hapten.

The second purpose of the invention is to provide a preparation method of the isoprocarb hapten.

The third purpose of the invention is to provide a new isoprocarb complete antigen.

The fourth purpose of the invention is to provide a preparation method of the isoprocarb complete antigen.

The fifth purpose of the invention is to provide an isoprocarb antibody.

The sixth purpose of the invention is to provide the application of the isoprocarb hapten, the isoprocarb complete antigen or the isoprocarb antibody in detecting isoprocarb or preparing products for detecting isoprocarb.

The seventh purpose of the invention is to provide an enzyme-linked immunoassay kit containing the isoprocarb complete antigen or the isoprocarb antibody.

In order to achieve the purpose, the invention is realized by the following technical scheme:

a novel isoprocarb hapten having the formula shown in formula I:

。

the preparation method of the isoprocarb hapten comprises the following steps:

s11, mixing 3-isopropyl-4-hydroxybenzoic acid with a solvent to obtain a mixed solution;

s12, adding triethylamine and trimethyl bromosilane into the mixed solution obtained in the step S11, taking triethylamine as an acid-binding agent, and reacting with trimethyl bromosilane;

s13, directly reacting the reaction product obtained in the step S12 with methyl isocyanate without separation, and hydrolyzing and acidifying to obtain 3-isopropyl-4-methyl carbamoyloxybenzoic acid, namely the isoprocarb hapten.

Preferably, the feeding molar ratio of the 3-isopropyl-4-hydroxybenzoic acid to the trimethyl bromosilane to the methyl isocyanate to the triethylamine is 1: 1: 1: 2.

preferably, the solvent is one or more of tetrahydrofuran, dioxane, dichloromethane, chloroform, Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile, ethyl acetate, preferably tetrahydrofuran.

The invention also provides a novel isoprocarb complete antigen, which is obtained by coupling the isoprocarb hapten and carrier protein, and the structural formula of the antigen is shown as the formula II:

wherein, in the step (A),is a carrier protein.

Preferably, the mass ratio of the isoprocarb hapten to the carrier protein is 1:1-1: 20.

Preferably, the carrier protein is one of Bovine Serum Albumin (BSA), egg serum albumin (OVA) or Keyhole Limpet Hemocyanin (KLH).

The preparation method of the isoprocarb complete antigen comprises the following steps:

reacting and dialyzing the isoprocarb hapten solution and the carrier protein solution at room temperature to obtain the isoprocarb complete antigen.

Preferably, the reaction time of the reaction is 4 to 24 hours.

Preferably, the dialysis is dialysis with PBS buffer at 4 ℃ for three days.

Preferably, the carrier protein solution is a solution of carrier protein dissolved in a carbonate buffer solution; the carbonate buffer solution was 0.1M, pH = 9.6.

Preferably, the isoprocarb hapten solution is obtained by the following method I or method II:

the method comprises the following steps: dissolving isoprocarb hapten in Dimethylformamide (DMF), adding Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) to react for 12-24h to obtain isoprocarb hapten solution;

the second method comprises the following steps: dissolving isoprocarb hapten in DMF, adding n-butylamine and isobutyl chloroformate, and reacting for 0.5-2h to obtain the isoprocarb hapten solution.

More preferably, the molar ratio of isoprocarb hapten, DCC and NHS is 1:1.0-2.0: 1.0-2.0.

More preferably, the molar ratio of isoprocarb hapten, n-butylamine and isobutyl chloroformate is 1: 1.2:1.2.

The invention also provides an isoprocarb antibody, which is obtained by immunizing an animal with one of the isoprocarb hapten, the isoprocarb complete antigen or the isoprocarb complete antigen prepared by the preparation method.

Preferably, the isoprocarb antibody is isoprocarb polyclonal antibody, isoprocarb monoclonal antibody or isoprocarb genetically engineered antibody, more preferably isoprocarb monoclonal antibody.

The application of the isoprocarb hapten, the isoprocarb complete antigen or the isoprocarb antibody in the detection of isoprocarb or the preparation of products for detecting isoprocarb also belongs to the protection scope of the invention.

The invention also provides an enzyme-linked immunoassay kit containing the isoprocarb complete antigen or the isoprocarb antibody, and the kit contains the isoprocarb complete antigen or the isoprocarb antibody.

Preferably, the kit comprises: the kit comprises an enzyme label plate coated with isoprocarb complete antigen, an enzyme-labeled secondary antibody, isoprocarb standard working solution, substrate color development solution, stop solution, washing solution and complex solution.

Wherein, the coated isoprocarb complete antigen is a conjugate of isoprocarb hapten and carrier protein.

The labeled enzyme of the enzyme-labeled secondary antibody is horseradish peroxidase (HRP).

The working solution of the isoprocarb standard substance is 6 bottles of concentrated solution of the isoprocarb standard substance (the solvent of the concentrated solution is methanol), and the concentrations are respectively 0 mug/L, 10 mug/L, 30 mug/L, 90 mug/L, 270 mug/L and 810 mug/L.

The substrate color development liquid comprises a liquid A and a liquid B, wherein the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine.

The stop solution is a 2mol/L sulfuric acid solution.

The washing solution is pH 7.2, contains 0.01% Tween, 3g/L sodium azide preservative, 0.01mol/L phosphate buffer solution, wherein the percentage is weight volume percentage.

The complex solution is phosphate buffer solution with the pH value of 7.0 and 0.02mol/L, and the percentage is weight volume percentage.

The preparation process of the ELISA plate is preferably as follows: diluting the coating source to 2 μ g/ml with a coating buffer (carbonate buffer with pH = 9.6), adding 100 μ l to each well, incubating at 4 ℃ in the dark for 16 h, decanting off the liquid in the wells, washing with a washing solution for 1 time, standing for 30s, patting to dryness, adding 200 μ l of a blocking solution to each well, incubating at 37 ℃ in the dark for 2h, decanting off the liquid in the wells, drying, and storing in an aluminum film vacuum seal.

The invention also provides application of the kit in detection of isoprocarb or structural analogues thereof.

Specifically, the application is to detect isoprocarb by using the kit, and the method comprises the following steps:

s21, sample adding: taking out the ELISA plate coated with the isoprocarb complete antigen, adding isoprocarb standard solutions with different concentrations into standard wells (the isoprocarb standard solution is obtained by diluting an isoprocarb standard working solution, specifically, 0.02mol of phosphate buffer solution (pH = 7.2) is diluted into the isoprocarb standard solution in a ratio of 9:1, and the concentrations after dilution are respectively 0 mug/L, 1 mug/L, 3 mug/L, 9 mug/L, 27 mug/L and 81 mug/L), adding samples into sample wells, then adding isoprocarb antibodies and ELISA secondary antibodies into each well, and lightly shaking and uniformly mixing:

s22, incubation: covering a cover plate film, and reacting for 45min in a dark environment at 25 ℃;

s23, washing: carefully uncovering the microplate membrane, spin-drying liquid in the pores, fully washing for 4-5 times by using 250 mul/pore of washing liquid at an interval of 10 s each time, and patting the elisa plate by using absorbent paper (bubbles which are not removed after patting the elisa plate are slightly punctured by using an unused gun head):

s24, color development: adding the substrate color developing solution A, adding the substrate color developing solution B, slightly oscillating and uniformly mixing, covering a plate with a cover plate film, and then developing for 15 min in a dark environment at 25 ℃:

s25, terminating: adding stop solution into each hole, and lightly shaking and uniformly mixing;

s26, reading value: setting the detection wavelength of 450 nm and the reference wavelength of 620 nm by a microplate reader, and determining the absorbance value of each hole.

The invention has the following beneficial effects:

the invention synthesizes the isoprocarb hapten by a one-step method, the isoprocarb hapten reserves the characteristic structures of carbamate and isopropyl of isoprocarb, active carboxyl is directly introduced on a benzene ring for being coupled with carrier protein to obtain a complete antigen, the method is simple and easy to operate, and the obtained complete antigen has higher specificity and sensitivity.

Drawings

Figure 1 is a scheme showing the synthesis scheme of isoprocarb hapten and isoprocarb complete antigen.

Detailed Description

EXAMPLE 1 Synthesis of isoprocarb hapten

The synthetic route of isoprocarb hapten is shown in figure 1, and the structural formula is shown in formula I:

。

the preparation method comprises the following steps:

200 g of 3-isopropyl-4-hydroxybenzoic acid was weighed, dissolved in 10 ml of tetrahydrofuran solvent, 286. mu.l of triethylamine was added, stirring was carried out for 10 min, 143. mu.l of trimethylbromosilane was added dropwise, stirring was continued for 30 min, then 107. mu.l of methyl isocyanate was added dropwise, and stirring was carried out at room temperature overnight. Then adding 1.0 ml of water for quenching reaction, concentrating the reaction solution, adding 25 ml of water and 2 ml of 6M NaOH into the residue, extracting for 1 time by adding 15 ml of methyl tert-butyl ether, acidifying the water phase by using 6M HCl until the pH is = 3-4, filtering, washing a filter cake by using 10 ml of water, and drying in vacuum to obtain solid 3-isopropyl-4-methylcarbamoyloxybenzoic acid (isoprocarb hapten), wherein the purity of the synthesized isoprocarb hapten is 99.8%.

EXAMPLE 2 Synthesis of isoprocarb hapten

The difference from example 1 is that the solvent used is dioxane, and the rest of the setup is the same as example 1. The purity of the synthesized isoprocarb hapten was 99.3%.

EXAMPLE 3 Synthesis of isoprocarb hapten

The difference from example 1 is that the solvent used is dichloromethane, and the rest of the setup is the same as example 1. The purity of the synthesized isoprocarb hapten was 99.5%.

Example 4 Synthesis of isoprocarb hapten

The difference from example 1 is that chloroform was used as the solvent, and the rest of the setup was the same as example 1. The purity of the synthesized isoprocarb hapten was 99.1%.

Example 5 Synthesis of isoprocarb hapten

The difference from example 1 is that DMF is used as solvent and the rest of the setup is the same as example 1. The purity of the synthesized isoprocarb hapten was 98.6%.

Example 6 Synthesis of isoprocarb hapten

The difference from example 1 is that DMSO is used as the solvent, and the rest of the setup is the same as example 1. The purity of the synthesized isoprocarb hapten was 99.3%.

Example 7 Synthesis of isoprocarb hapten

The difference from example 1 is that the solvents used are acetonitrile and ethyl acetate in a volume ratio of 1:1, and the rest of the setup is the same as example 1. The purity of the synthesized isoprocarb hapten was 99.2%.

Example 8 Synthesis of isoprocarb complete antigen (isoprocarb BSA complete antigen)

The synthetic route of isoprocarb complete antigen is shown in figure 1, and the structural formula is shown in formula II:

wherein, in the step (A),is a carrier protein.

The preparation method comprises the following steps:

10 mg of isoprocarb hapten obtained in example 1 was weighed and dissolved in 200. mu.l of DMF, and 5.9 mg of NHS and 17 mg of DCC were added thereto, and stirred at room temperature overnight for 12 to 24 hours to obtain an active ester solution, i.e., an isoprocarb hapten solution. Weighing 50 mg of BSA, dissolving in 4 ml of carbonate buffer solution (pH = 9.6), centrifuging the active ester solution, taking the supernatant, dropping the supernatant into the BSA solution, wherein the mass ratio of the isoprocarb hapten to the carrier protein is 1:5, and standing at room temperature overnight for 4-24h to obtain the isoprocarb BSA complete antigen solution. Dialyzing the isoprocarb BSA complete antigen solution with a PBS buffer solution at 4 ℃ for three days, taking out the dialysate after the dialysis is finished, and freeze-drying to obtain the isoprocarb BSA complete antigen, and storing at-40 ℃.

Example 9 Synthesis of isoprocarb complete antigen (isoprocarb OVA complete antigen)

The difference from example 8 is that substitution of the specifically prepared BSA solution with OVA solution resulted in the complete antigen isoprocarb OVA.

EXAMPLE 10 Synthesis of isoprocarb complete antigen (isoprocarb KLH complete antigen)

The difference from example 8 is that substitution of the specifically prepared BSA solution with KLH solution gave the isoprocarb KLH complete antigen.

EXAMPLE 11 Synthesis of isoprocarb complete antigen (isoprocarb BSA complete antigen)

The synthetic route of isoprocarb complete antigen is shown in figure 1, and the structural formula is shown in formula II:

wherein, in the step (A),is a carrier protein.

The preparation method comprises the following steps:

5 mg of isoprocarb hapten obtained in example 1 was weighed and dissolved in 150. mu.l of DMF, 8.2 mg of DCC and 3 mg of NHS were added, and the mixture was stirred at room temperature overnight for 12 to 24 hours to obtain an active ester solution, i.e., an isoprocarb hapten solution. Weighing 50 mg of BSA, dissolving in 4 ml of carbonate buffer solution (pH = 9.6), centrifuging the active ester solution, taking the supernatant, dropping the supernatant into the BSA solution, wherein the mass ratio of the isoprocarb hapten to the carrier protein is 1:10, and standing at room temperature overnight for 4-24h to obtain the isoprocarb BSA complete antigen solution. Dialyzing the isoprocarb BSA complete antigen solution with a PBS buffer solution at 4 ℃ for three days, taking out the dialysate after the dialysis is finished, and freeze-drying to obtain the isoprocarb BSA complete antigen, and storing at-40 ℃.

Example 12 Synthesis of isoprocarb complete antigen (isoprocarb BSA complete antigen)

The synthetic route of isoprocarb complete antigen is shown in figure 1, and the structural formula is shown in formula II:

wherein, in the step (A),is a carrier protein.

The preparation method comprises the following steps:

10 mg of isoprocarb hapten obtained in example 1 was weighed and dissolved in 200. mu.l of DMF, 18.1 mg of DCC and 5.8 mg of NHS were added, and the mixture was stirred at room temperature overnight for 12 to 24 hours to obtain an active ester solution, i.e., an isoprocarb hapten solution. Weighing 20 mg of BSA, dissolving in 2 ml of carbonate buffer solution (pH = 9.6), centrifuging the active ester solution, taking the supernatant, dripping the supernatant into the BSA solution, wherein the mass ratio of the isoprocarb hapten to the carrier protein is 1:2, and standing at room temperature overnight for 4-24h to obtain the isoprocarb BSA complete antigen solution. Dialyzing the isoprocarb BSA complete antigen solution with a PBS buffer solution at 4 ℃ for three days, taking out the dialysate after the dialysis is finished, and freeze-drying to obtain the isoprocarb BSA complete antigen, and storing at-40 ℃.

Example 13 Synthesis of isoprocarb complete antigen (isoprocarb BSA complete antigen)

The synthetic route of isoprocarb complete antigen is shown in figure 1, and the structural formula is shown in formula II:

wherein, in the step (A),is a carrier protein.

The preparation method comprises the following steps:

10 mg of isoprocarb hapten obtained in example 1 is weighed and dissolved in 250 ul of DMF, the mixture is cooled in ice bath, 10 ul of n-butylamine is added, 5.5 ul of isobutyl chloroformate is added, and the reaction is continued for 1 hour in ice bath to obtain the isoprocarb hapten solution. Weighing 50 mg of BSA, dissolving in 4 ml of carbonate buffer solution (pH = 9.6), slowly dropping isoprocarb hapten solution, wherein the mass ratio of isoprocarb hapten to carrier protein is 1:5, and reacting at room temperature in a dark place for 4h to obtain the isoprocarb BSA complete antigen solution. The isoprocarb BSA complete antigen solution is dialyzed by PBS buffer solution for three days at 4 ℃ to obtain isoprocarb BSA complete antigen, and the isoprocarb BSA complete antigen is stored at-40 ℃.

Example 14 Synthesis of isoprocarb complete antigen (isoprocarb BSA complete antigen)

The difference from example 13 was that the reaction time in ice bath was 0.5 h, the reaction time in room temperature and light shielding was 4h, and the rest of the setup was the same as example 13.

Example 15 Synthesis of isoprocarb complete antigen (isoprocarb BSA complete antigen)

The difference from example 13 was that the reaction time in ice bath was 1 hour, the reaction time in room temperature and light shielding was 16 hours, and the rest of the setup was the same as example 13.

Example 16 Synthesis of isoprocarb complete antigen (isoprocarb BSA complete antigen)

The difference from example 13 was that the reaction time in ice bath was 2 hours, the reaction time in room temperature and light shielding was 24 hours, and the rest of the setup was the same as example 13.

Example 17 Synthesis of isoprocarb complete antigen (isoprocarb OVA complete antigen)

The difference from example 13 is that substitution of the specifically prepared BSA solution with OVA solution resulted in the complete antigen isoprocarb OVA.

EXAMPLE 18 Synthesis of isoprocarb complete antigen (isoprocarb KLH complete antigen)

The difference from example 13 is that substitution of the specifically prepared BSA solution with KLH solution gave the isoprocarb KLH complete antigen.

Example 19 Synthesis of isoprocarb complete antigen (isoprocarb BSA complete antigen)

The difference from example 13 is that 100 mg of BSA was weighed and dissolved in 8 ml of carbonate buffer (pH = 9.6) at a 1:10 ratio by mass of isoprocarb hapten to carrier protein, and the rest of the setup was the same as example 13.

Example 20 Synthesis of isoprocarb complete antigen (isoprocarb BSA complete antigen)

The difference from example 13 is that 20 mg of BSA was weighed and dissolved in 2 ml of carbonate buffer (pH = 9.6) with a mass ratio of isoprocarb hapten to carrier protein of 1:2, and the rest of the setup was the same as example 13.

Test examples

(a) Preparation of isoprocarb monoclonal antibody

Animal immunization: the immunogens prepared by the methods of examples 8, 11, 12 and 13 were injected into Balb/c (10-week) mice at an immunization dose of 100. mu.g/mouse, respectively, to generate antiserum.

Cell fusion and cloning: after the serum determination result of the mouse is higher, spleen cells are taken and fused with FO myeloma cells according to the proportion of 10:1 (quantity ratio), indirect competition ELISA is adopted to determine cell supernatant, and positive holes are screened. Cloning the positive hole by using a limiting dilution method until obtaining a hybridoma cell strain secreting the isoprocarb monoclonal antibody.

Freezing and recovering cells: preparation of monoclonal hybridoma cell line into 1 × 10 frozen stock solution⁶Cell suspension per ml, preserved for long periods in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.

Production and purification of monoclonal antibodies: adopting a method of inducing monoclonal antibodies in animals, and collecting ascites after 7 days. Ascites was purified by proteinG purification column and stored at-20 ℃.

(b) Preparation of enzyme-labeled Secondary antibody

The goat is taken as an immune animal, and the isoprocarb monoclonal antibody is taken as immunogen to immunize the goat without the pathogen, so as to obtain the isoprocarb anti-antibody. Coupling the isoprocarb anti-antibody with horseradish peroxidase (HRP) to obtain an enzyme-labeled secondary antibody.

(c) Preparation of ELISA plates

Diluting the coating source to 2 μ g/ml with a coating buffer (carbonate buffer with pH = 9.6), adding 100 μ l to each well, incubating at 4 ℃ in the dark for 16 h, decanting off the liquid in the wells, washing with a washing solution for 1 time, standing for 30s, patting to dryness, adding 200 μ l of a blocking solution to each well, incubating at 37 ℃ in the dark for 2h, decanting off the liquid in the wells, drying, and storing in an aluminum film vacuum seal.

(d) Construction of enzyme-linked immunoassay kit for detecting isoprocarb

An enzyme-linked immunoassay kit for detecting isoprocarb is constructed, and comprises the following components:

(1) an ELISA plate coated with isoprocarb complete antigen;

(2) the concentration of the isoprocarb standard substance working solution is 0 mug/L, 10 mug/L, 30 mug/L, 90 mug/L, 270 mug/L and 810 mug/L respectively (concentrated solution solvent methanol), when in use, the isoprocarb standard substance working solution is diluted by 0.02mol phosphate buffer solution (pH = 7.2) at a ratio of 9:1, and the concentration after dilution is 0 mug/L, 1 mug/L, 3 mug/L, 9 mug/L, 27 mug/L and 81 mug/L respectively;

(3) isoprocarb anti-antibody labeled with horseradish peroxidase;

(4) the substrate color development liquid comprises a liquid A and a liquid B, wherein the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;

(5) the stop solution is 2mol/L sulfuric acid;

(6) the washing solution is pH 7.2, contains 0.01% Tween, 3g/L sodium azide antiseptic, 0.01mol/L phosphate buffer solution, wherein the percentage is weight volume percentage;

(7) the compound solution is phosphate buffer solution with pH value of 7.0 and 0.02mol/L, and the percentage is weight volume percentage.

(e) Detection of isoprocarb enzyme-linked immunoassay kit

(1) Detection with a kit

Adding 50 mul of standard solution/sample into the corresponding micropore, adding 50 mul/hole of isoprocarb antibody, finally adding 50 mul/hole of enzyme-labeled secondary antibody, mixing by gentle oscillation, covering with a cover plate, and reacting for 45min in a dark environment at 25 ℃. Carefully uncovering the microplate membrane, spin-drying liquid in the pores, fully washing with 250 mu l/pore of washing liquid for 4-5 times at intervals of 10 s each time, and patting the elisa plate with absorbent paper (bubbles which are not removed after patting are slightly punctured by an unused gun head). Adding 50 mul/hole of substrate color development liquid A, adding 50 mul/hole of substrate color development liquid B, mixing by gentle oscillation, covering with cover plate, and developing at 25 deg.C in dark environment for 15 min. Add stop solution 50. mu.l/well, mix by gentle shaking. Setting the detection wavelength of a microplate reader to be 450 nm and the reference wavelength to be 620 nm, and determining the absorbance value of each hole.

(2) Analysis of detection results

The percent absorbance of the standard or sample is equal to the absorbance of the standard or sample divided by the absorbance of the first standard (0 standard) and multiplied by 100% to obtain the percent absorbance of the standard or sample. And drawing a standard curve graph by taking the percent absorbance of the standard substance as an ordinate and taking the logarithm of the concentration (mu g/L) of the isoprocarb standard substance as an abscissa. And substituting the percent absorbance of the sample into the standard curve, reading the corresponding concentration of the sample from the standard curve, and multiplying the corresponding dilution times to obtain the actual concentration of the isoprocarb in the sample.

(3) Specificity detection

Preparing different concentration standard curves of alachlor, diethofencarb and isobutrol, namely 0 mu g/L, 1 mu g/L, 3 mu g/L, 9 mu g/L, 27 mu g/L and 81 mu g/L respectively, detecting by using the isoprocarb enzyme-linked immunoassay kit, obtaining IC50 values of the alachlor, the diethofencarb and the isobutrol according to the absorbance value, and then calculating the cross reaction rate according to the following formula:

the results are shown in the following table 1, and the smaller the cross reaction rate with other drugs is, the better the detection specificity of the isoprocarb enzyme-linked immunoassay kit for isoprocarb is.

TABLE 1 Cross-reactivity of alachlor, diethofencarb, isobutrol

As can be seen from table 1, the results of the cross-reactivity of alachlor, diethofencarb, and isobutramine measured by the isoprocarb elisa kits prepared in examples 8, 11, 12, and 13 are all less than 1%, and the results indicate that the isoprocarb antibody has good specificity, is not easy to have the phenomena of false positive, false detection, and the like, and can be applied to the screening detection of isoprocarb residues in actual samples.

(4) Detection of precision

Precision determination of standard solutions

10 kits were extracted from each of 3 different kit lots prepared in example 8, and the absorbance values of 10 microwell assay standard solutions (containing 10. mu.g/L isoprocarb) were immediately extracted from each microplate, and the coefficient of variation was calculated as: coefficient of Variation (CV) × (ratio of standard deviation of each parallel sample to average of each parallel sample) × 100%. The experiment was repeated three times and the results are shown in table 2 below:

table 2 concentration and coefficient of variation of standard solution tested by isoprocarb elisa kit prepared in example 8:

as can be seen from Table 2, the coefficient of variation of the isoprocarb ELISA kit standard solution (containing 10. mu.g/L isoprocarb) prepared in example 8 is between 3.2% and 5.7%, and the coefficient of variation of the isoprocarb ELISA kit standard solution (containing 10. mu.g/L isoprocarb) prepared in examples 11, 12 and 13 is similar to that of example 8, which all meet the national standard for kit precision.

(5) Sensitivity detection

The lowest detection limit detection is performed on the isoprocarb enzyme-linked immunoassay kit prepared in examples 8, 11, 12 and 13, and the results are shown in the following table 3:

TABLE 3 detection results of lowest detection limit of isoprocarb ELISA kit prepared in examples 8, 11, 12 and 13

As can be seen from Table 3, the lowest detection limit of the isoprocarb ELISA kits prepared in examples 8, 11, 12 and 13 is lower than the lowest detection limit (0.54 ng/mL) of the antibodies prepared from the hapten and the artificial antigen disclosed in the invention patent with publication number CN106588699A, which indicates that the isoprocarb ELISA kits prepared in examples 8, 11, 12 and 13 have higher sensitivity.

In conclusion, the 3-isopropyl-4-hydroxybenzoic acid is used as a raw material, the 3-isopropyl-4-methylcarbamoyloxybenzoic acid is obtained by a one-step method and is used as a hapten to be coupled with carrier protein to obtain a complete antigen.

Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the invention, and such equivalent modifications or substitutions are included in the scope defined by the claims of the present application.