CN113788814A

CN113788814A - Hapten for detecting content of isoprothiolane, preparation method and application thereof

Info

Publication number: CN113788814A
Application number: CN202111203671.4A
Authority: CN
Inventors: 王春琼; 陈丹; 张燕; 秦世春; 李苓; 蔡洁云; 张轲; 张晓伟; 彭丽娟; 孙浩巍; 杨艺敏; 张庆刚
Original assignee: Yunnan tobacco quality supervision and inspection station
Current assignee: Yunnan tobacco quality supervision and inspection station
Priority date: 2021-10-15
Filing date: 2021-10-15
Publication date: 2021-12-14
Anticipated expiration: 2041-10-15
Also published as: CN113788814B

Abstract

The invention discloses a hapten for detecting the content of isoprothiolane, a preparation method and application thereof, in particular relates to a hapten for enzyme-linked immunosorbent assay of isoprothiolane, and also discloses a preparation method of the hapten and application thereof in the aspects of antibodies and kits. The enzyme linked immunosorbent assay kit rapid detection product established based on the isoprothiolane hapten is convenient to use, low in detection cost, efficient, accurate and rapid in detection method, capable of detecting a large number of samples simultaneously, and suitable for field monitoring of agricultural products, particularly isoprothiolane in tobacco, and screening of a large number of samples.

Description

Hapten for detecting content of isoprothiolane, preparation method and application thereof

Technical Field

The invention relates to the technical field of pesticide residue detection, in particular to a hapten for detecting the content of isoprothiolane, a preparation method and application thereof.

Background

Isoprothiolane (Isoprothiolane), also known as Isoprothiolane and Fushiyi, has a chemical name of 1, 3-dithiolane-2-isopropylidene malonate, is a high-efficiency systemic fungicide, has a narrow fungicide spectrum, is a specific drug for preventing and treating rice blast, particularly has an excellent effect on panicle neck blast, can be systemically transmitted through roots, stems and leaves of plants, and can reach the heart and leaves. It is also effective on rice seedling plague and sclerotinia scleotiorum, and can be used for treating rice planthopper and leafhopper in large area, and has regulating effect on plant growth. However, with the application of the isoprothiolane in large quantities, the environment can be polluted, the residual quantity of the isoprothiolane in agricultural products is increased, and the isoprothiolane has potential threat to human health. The residual quantity of isoprothiolane in various agricultural products needs to be effectively and accurately detected.

The problem of isoprothiolane residue in fruits, vegetables and tobacco products is receiving more and more attention. China sets the maximum residue limit standard of isoprothiolane for different crops, wherein the maximum residue limit of watermelons is 0.1mg/kg, the maximum residue limit of rice is 1mg/kg, and the maximum residue limit of tobacco is 2 mg/kg.

At present, only detection reports of residual quantity of the bactericides in grains, fruits and vegetables appear in the existing documents, and no report about a measuring method of the residual quantity in tobacco leaves is seen. The existing detection methods for isoprothiolane residue at home and abroad mainly comprise gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), Gas Chromatography (GC) and the like. The instrument analysis method has the advantages of high detection sensitivity, strong specificity and the like, but the pretreatment of a detection sample is complicated and time-consuming, the sample needs to be extracted and purified, and meanwhile, the instrument detection method needs expensive large-scale instruments and equipment, is equipped with professional detection technicians for operation and management, cannot carry out large-scale rapid detection on site, is poor in timeliness and is difficult to popularize.

The immunological detection and analysis technology has the advantages of high sensitivity, high specificity, rapidness, simple and convenient operation and the like, is widely applied to the field of drug residue detection, and has many advantages compared with an instrument detection method. However, the prior art lacks a detection reagent with specific reaction to the isoprothiolane, which hinders the application of an immunoassay method in the isoprothiolane detection.

When an immunological detection method is established and the detection method is applied to detecting the isoprothiolane, the key technology is that an antibody with strong specificity and high sensitivity can be obtained, and the goal is to realize, on the premise that a proper isoprothiolane hapten needs to be synthesized and prepared. However, at present, no relevant report aiming at the isoprothiolane hapten exists in China.

Disclosure of Invention

The invention provides a hapten for detecting the content of isoprothiolane, a preparation method and application thereof, which are used for solving the technical problems that an antibody with specific reaction on isoprothiolane components and a hapten needed by a synthetic antibody are lacked in the prior art.

The invention provides a hapten for detecting the content of isoprothiolane, which has a chemical formula as follows: 2- (2- (1, 3-diisopropyl ether-1, 3-dioxopropan-2-ylidene) -1, 3-dithiolane) acetic acid; the chemical structural formula is as follows:

the hapten with the structural formula can be processed according to a common method in the prior art to prepare an antibody, and the antibody can generate a specific reaction on isoprothiolane contained in various pretreated agricultural product samples. According to the characteristics, after a solution is prepared for a isoprothiolane standard substance, after isoprothiolane samples with different concentrations are obtained through gradient dilution, an enzyme-labeling instrument is adopted to measure the OD value of each sample at 450nm to obtain a standard curve, the sample to be detected is pretreated, the OD value at the same wavelength is measured, and the corresponding concentration is obtained through the standard curve, so that the quantitative, effective and rapid detection of the isoprothiolane content in the sample is realized. The hapten product provided by the invention is shown in figure 2.

For example, for the pretreatment of the sample, taking tobacco leaves as an example, the method specifically comprises the following steps: homogenizing the tobacco leaf sample with a homogenizer; weighing 1.0g +/-0.05 g of homogenized tobacco leaf sample into a 50mL polystyrene centrifuge tube, adding 5mL of methanol solution, oscillating for 5min by using an oscillator, uniformly mixing, and centrifuging for 10min at 3000g of room temperature (20-25 ℃); transferring 10 mu L of supernatant into a 2mL polystyrene centrifuge tube, adding 1990 mu L complex solution, whirling by a vortex instrument for 30s, and mixing uniformly; 50 μ L were taken for analysis (dilution factor: 1000).

The invention also provides a preparation method of the hapten, which comprises the following steps:

under a strong alkali reaction environment, carrying out condensation reaction on diisopropyl malonate serving as a raw material, carbon disulfide and 3, 4-dichlorobutyric acid to obtain the hapten for detecting the isoprothiolane content.

The hapten with higher purity can be obtained by adopting the reaction. The reagents and reaction conditions used in the preparation method can be selected according to the reagents and conditions required by the existing condensation reaction.

Preferably, the method comprises the following steps:

1) after uniformly stirring diisopropyl malonate and carbon disulfide at room temperature, reinforcing alkali solution, stirring for 1h at room temperature, adding 3, 4-dichlorobutyric acid, and supplementing strong alkali solution, tetrabutylammonium bromide and acetonitrile to obtain raw material solution;

2) heating and refluxing the raw material liquid for 4 hours, and stopping the reaction to obtain a reaction liquid;

3) and carrying out rotary evaporation, purification and separation on the obtained reaction solution to obtain the hapten for detecting the content of isoprothiolane.

The reaction equation is as shown in formula (2):

from the above, this reaction can occur normally, and a hapten having the above structure is produced.

Preferably, the rotary steaming operation in the step 3) comprises the following steps: and removing acetonitrile and carbon disulfide in the reaction liquid by rotary evaporation, adding water and an acid solution, and adjusting the pH value of the reaction liquid to 6.

Preferably, the step of separating in step 3) comprises the steps of: extracting the reaction solution for three times by using ethyl acetate as a solvent, merging the obtained organic phases, washing the organic phases with water, drying and evaporating anhydrous sodium sulfate to obtain yellow oily substances, loading the yellow oily substances on a silica gel column, eluting, separating and purifying by using a petroleum ether-ethyl acetate mixed solution with a volume ratio of 3:1 to obtain the hapten for detecting the isoprothiolane content.

Preferably, the strong alkaline solution is 2mol/L KOH solution; the acid solution is 6mol/L HCl solution; the volume of solvent used for each extraction was 100 mL.

To achieve an efficient application to haptens, the following applications can be made:

the invention also provides an antigen for detecting the content of isoprothiolane, which is a conjugate obtained by coupling the hapten and carrier protein.

The coupling method of the hapten and the carrier protein used in the invention can be a method for preparing the antigen by the hapten in the prior art, for example, the coupling method by taking bovine serum albumin as the carrier can be as follows:

taking 12.9mg of the hapten, adding 1mL of tetrahydrofuran for dissolving and clarifying, adding 8.5mg of N-hydroxysuccinimide (NHS) and 14.8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), fully dissolving and uniformly mixing, and reacting at room temperature for 2 hours to obtain a hapten activation solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.1mol/L CB9.18mL for dissolving to obtain a solution B; dropwise adding the solution A into the solution B, reacting at room temperature for 4h, stopping the reaction, dialyzing and purifying with 0.02mol/L PBS buffer solution for 3 days, changing the solution 3 times per day, centrifuging to obtain the artificial antigen of isoprothiolane coupled with bovine serum albumin, subpackaging, and storing at-20 ℃.

The coupling method using ovalbumin as a carrier comprises the following steps:

taking 7.7mg of the isoprothiolane hapten, adding 1mL of dimethyl sulfoxide (DMSO) for dissolving and clarifying, adding 5.1mg of NHS and 9.7mg of EDC, fully dissolving and uniformly mixing, and reacting at room temperature for 2 hours to obtain hapten activating solution A; dissolving Ovalbumin (OVA)50mg in 0.1mol/L CB9.17mL to obtain solution B; dropwise adding the solution A into the solution B, reacting at room temperature for 4h, stopping the reaction, dialyzing and purifying with 0.02mol/L PBS buffer solution for 3 days, changing the solution 3 times per day, centrifuging to obtain the artificial antigen of isoprothiolane coupled with ovalbumin, subpackaging, and storing at-20 ℃.

Preferably, the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin. The detection function of the hapten can be effectively exerted by adopting the carrier proteins.

The invention also provides an antibody for detecting the content of isoprothiolane, which is obtained by immunizing animals by the antigen; the antibody can generate specific immune reaction with isoprothiolane. The antibody to be used for the production of the above antigen may be an animal immunization method commonly used in the art, and includes, for example, the following steps:

(1) animal immunization: the above antigen conjugated with bovine serum albumin was injected into Balb/c mice at an immunizing dose of 150. mu.g/mouse to produce polyclonal antibody serum.

(2) Cell fusion and cloning: after the measurement result of the mouse serum is higher, spleen cells are taken and fused with SP2/0 myeloma cells according to the ratio of 8:1, the cell supernatant is measured by adopting indirect competition ELISA, and positive holes are screened. Cloning the positive hole by using a limiting dilution method until obtaining a hybridoma cell strain secreting the monoclonal antibody.

(3) CellsFreezing and recovering: preparing 1 × 10 by using freezing medium for monoclonal hybridoma cell strain of isoprothiolane⁹Cell suspension per mL, preserved for long period 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.

(4) Production and purification of monoclonal antibodies: injecting sterilized paraffin oil 0.5 mL/mouse abdominal cavity into Balb/c mouse, injecting isoprothiolane 6X 10 monoclonal hybridoma cell strain into abdominal cavity 7 days later⁵Ascites were collected 7 days later. Purifying ascites by octanoic acid-saturated ammonium sulfate method, and storing at-20 deg.C.

(5) Determination of monoclonal antibody titer: the titer of the antibody is measured to be 1 (64000-120000) by an indirect competition ELISA method.

Indirect competitive ELISA method: the elisa plate was coated with the above antigen coupled to ovalbumin.

Preferably, the antibody is a isoprothiolane monoclonal antibody.

The invention also provides a rice blast enzyme-linked immunoassay kit, and the antibody is the antibody. The kit is prepared according to the existing method, and the specific method can be obtained by searching the existing immune kit production method by a person skilled in the art.

Preferably, the detection sample of the isoprothiolane enzyme-linked immunosorbent assay kit is a pretreated tobacco sample. The detection antibody has high sensitivity and accuracy in detection of isoprothiolane contained in tobacco.

Preferably, the immunoassay kit comprises: an ELISA plate and working solution; the ELISA plate is the antigen; the working solution is the antibody liquid marked by horseradish peroxidase.

Preferably, the immunoassay kit comprises: a rice blast standard substance solution with gradient concentration, a substrate developing A, B solution, a stop solution, a washing solution and a redissolution;

the rice blast standard solution with gradient concentration has gradient concentration of 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L and 8.1 μ g/L;

substrate color development A, B liquid: the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;

the stop solution is 2mol/L sulfuric acid;

the washing solution is pH7.2, and contains 0.8% -1.2% of Tween-20, 0.01-0.03% of thimerosal preservative and 0.1-0.3 mol/L of carbonate buffer solution;

the pH of the compound solution is 7.6, and the compound solution contains 8-12% of casein and 0.1-0.3 mol/L of phosphate buffer solution.

The kit prepared from the components has high detection efficiency and good detection accuracy, and is suitable for field large-scale detection.

The invention can produce the beneficial effects that:

1) the hapten for detecting the content of the isoprothiolane can reserve the characteristic structure of the isoprothiolane to the greatest extent, and after the hapten is used for preparing an artificial antibody, the antibody has the characteristics of high sensitivity and strong specificity on the detection of the isoprothiolane.

2) The hapten for detecting the content of the isoprothiolane provided by the invention is an antibody prepared by taking the hapten as a raw material, and the hapten is taken as the raw material, so that the hapten shows stronger immunogenicity for detecting the residue of the isoprothiolane; the hapten is used as a raw material to prepare an artificial antigen system suitable for animal immunization for immunizing animals, the titer, the specificity and the affinity of the obtained antibody are good, the detection sensitivity aiming at the isoprothiolane residual sample can reach 0.1 mu g/L, and the detection requirement of the existing national standard on the isoprothiolane residual quantity in agricultural products is met.

3) The antibody and the kit for detecting the content of isoprothiolane provided by the invention have the advantages that the detection operation is simple, large-scale instrument analysis equipment does not need to be purchased, the detection of a sample can be completed only through the kit, and the antibody and the kit are suitable for field large-scale detection.

4) The enzyme linked immunosorbent assay kit is prepared from the obtained antibody, is convenient to use, low in detection cost, efficient, accurate and rapid in detection method, can be used for simultaneously detecting a large number of samples, and is suitable for field monitoring of isoprothiolane in tobacco and screening of a large number of samples.

Drawings

FIG. 1 is a standard curve drawn by using the antibody kit provided by the invention to detect a isoprothiolane standard sample gradient dilution solution in the application of the antibody kit in tobacco sample detection;

FIG. 2 is a photograph of a hapten product synthetically prepared by the method provided in example 1 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings. The hapten preparation antigen, the antigen preparation antibody and the antibody preparation kit of the invention are not described in detail and are performed according to the methods disclosed in the prior art, and the details are not repeated here.

Examples

Materials and instruments used in the following examples were all commercially available as indicated otherwise.

Example 1

The preparation of the hapten comprises the following steps:

taking 1.88g of diisopropyl malonate, adding 20mL of carbon disulfide, stirring at room temperature, fully and uniformly mixing, adding 13mL of 2mol/L KOH solution, stirring at room temperature for 1h, adding 1.56g of 3, 4-dichlorobutyric acid, supplementing 5mL of 2mol/L KOH solution, 3.8g of tetrabutylammonium bromide and 80mL of acetonitrile, heating and refluxing for 4h, stopping the reaction, carrying out rotary evaporation, removing the acetonitrile and the carbon disulfide, adding 80mL of water, adding 6mol/L HCl to adjust the pH value to 6, adding 100mL multiplied by 3 of ethyl acetate, extracting for three times, combining organic phases, washing with water, drying and evaporating anhydrous sodium sulfate to dryness to obtain yellow oily substances, loading on a silica gel column, eluting, separating and purifying by using a petroleum ether-ethyl acetate mixed solution with the volume ratio of 3:1 to obtain 0.76g of acetic acid-isoprothiolane, namely the isoprothiolane hapten. The hapten product obtained in this example is a white powder material as shown in fig. 2.

Example 2

The preparation of the antigen for detecting the content of isoprothiolane comprises the following steps:

dissolving 12.9mg of hapten prepared in example 1 in 1mL of tetrahydrofuran for clarification, adding 8.5mg of N-hydroxysuccinimide (NHS) and 14.8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), fully dissolving and uniformly mixing, and reacting at room temperature for 2 hours to obtain a hapten activation solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.1mol/L CB9.18mL for dissolving to obtain a solution B; dropwise adding the solution A into the solution B, reacting at room temperature for 4h, stopping the reaction, dialyzing and purifying with 0.02mol/L PBS buffer solution for 3 days, changing the solution 3 times per day, centrifuging to obtain the artificial antigen of isoprothiolane coupled with bovine serum albumin, subpackaging, and storing at-20 ℃.

Example 3

taking 7.7mg of isoprothiolane hapten prepared in example 1, adding 1mL of dimethyl sulfoxide (DMSO) for dissolving and clarifying, adding 5.1mg of NHS and 9.7mg of EDC, fully dissolving and uniformly mixing, and reacting at room temperature for 2h to obtain hapten activating solution A; dissolving Ovalbumin (OVA)50mg in 0.1mol/L CB9.17mL to obtain solution B; dropwise adding the solution A into the solution B, reacting at room temperature for 4h, stopping the reaction, dialyzing and purifying with 0.02mol/L PBS buffer solution for 3 days, changing the solution 3 times per day, centrifuging to obtain the artificial antigen of isoprothiolane coupled with ovalbumin, subpackaging, and storing at-20 ℃.

Example 4

Preparing a isoprothiolane antibody, comprising the steps of:

(1) animal immunization: the artificial antigen of isoprothiolane coupled with bovine serum albumin prepared in example 2 was injected into Balb/c mice at an immunization dose of 150. mu.g/mouse to generate polyclonal antibody serum.

(3) Freezing and recovering cells: preparing 1 × 10 by using freezing medium for monoclonal hybridoma cell strain of isoprothiolane⁹Cell suspension per mL, preserved for long period 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.

(4) Generation of monoclonal antibodiesProduction and purification: injecting sterilized paraffin oil 0.5 mL/mouse abdominal cavity into Balb/c mouse, injecting isoprothiolane 6X 10 monoclonal hybridoma cell strain into abdominal cavity 7 days later⁵Ascites were collected 7 days later. Purifying ascites by octanoic acid-saturated ammonium sulfate method, and storing at-20 deg.C.

Indirect competitive ELISA method: coating an enzyme label plate with the artificial antigen of isoprothiolane coupled with ovalbumin prepared in example 3, adding a isoprothiolane standard substance solution and a monoclonal antibody working solution, reacting at 4 ℃ for 30min, pouring out liquid in pores, washing with a PBST (basic-particle system test) washing solution for 3-5 times, and patting dry with absorbent paper; adding a goat anti-mouse anti-antibody marked by horseradish peroxidase, reacting for 30min at 25 ℃, taking out and repeating the plate washing step; adding a substrate color developing solution, reacting for 15min at 25 ℃, and adding a stop solution to stop the reaction; the microplate reader was set to measure the absorbance value per well at a wavelength of 450 nm.

Example 5

Preparing a isoprothiolane enzyme linked immunosorbent assay kit, wherein the obtained kit comprises:

(1) an ELISA plate coated with an artificial antigen of isoprothiolane coupled with ovalbumin;

(2) a working solution of a isoprothiolane monoclonal antibody marked by horseradish peroxidase;

(3) 6 bottles of isoprothiolane standard substance solution with the concentrations of 0 mug/L, 0.1 mug/L, 0.3 mug/L, 0.9 mug/L, 2.7 mug/L and 8.1 mug/L respectively;

(4) the substrate color development liquid consists of 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 pH7.2, and contains 0.8% -1.2% of Tween-20, 0.01-0.03% of thimerosal preservative and 0.1-0.3 mol/L of carbonate buffer solution;

(7) the complex solution is pH7.6, and contains 8% -12% casein and 0.1-0.3 mol/L phosphate buffer solution.

The main reagents of the kit are provided in the form of working solution, and the detection method is convenient and easy to implement and has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like.

The application of the obtained antibody kit in the detection of tobacco leaf samples:

an application of a isoprothiolane enzyme linked immunosorbent assay kit for detecting isoprothiolane in tobacco comprises the following steps:

1. sample pretreatment

Homogenizing the tobacco leaf sample with a homogenizer; weighing 1.0g +/-0.05 g of homogenized tobacco leaf sample into a 50mL polystyrene centrifuge tube, adding 5mL of methanol solution, oscillating for 5min by using an oscillator, uniformly mixing, and centrifuging for 10min at 3000g of room temperature (20-250 ℃); transferring 10 mu L of supernatant into a 2mL polystyrene centrifuge tube, adding 1990 mu L complex solution, whirling by a vortex instrument for 30s, and mixing uniformly; 50 μ L were taken for analysis (dilution factor: 1000).

2. Detection with a kit

Adding 50 mu L of a rice blast standard substance solution or a sample solution into micropores of an ELISA plate coated with a rice blast artificial antigen coupled with ovalbumin, then adding 50 mu L/hole of a rice blast monoclonal antibody working solution labeled with horseradish peroxidase, lightly oscillating and uniformly mixing, covering the plate with a cover plate film, reacting for 30min in a dark place at 25 ℃, pouring out liquid in the hole, adding 250 mu L of washing solution into each hole, pouring out the liquid in the hole after 30s, repeating the operation of washing the plate for 5 times, and patting the plate dry with absorbent paper; adding 50 mu L of substrate color development liquid A, namely carbamide peroxide, and 50 mu L of substrate color development liquid B, namely Tetramethylbenzidine (TMB) into each hole, slightly oscillating and mixing uniformly, covering a cover plate by using a cover plate film, then placing the cover plate in a 25 ℃ dark place for color development for 15min, adding 50 mu L of stop solution 2mol/L sulfuric acid into each hole, slightly oscillating and mixing uniformly, setting the wavelength at 450nm by using an enzyme-linked immunosorbent assay, and measuring the absorbance value (OD value) of each hole.

3. Analysis of detection results

Dividing the average absorbance (B) obtained for each concentration of the standard solution by the absorbance value (B) of the first standard solution (0 standard)₀) And then multiplied by 100 percent to obtain the percent absorbance value. A standard curve chart is drawn by taking the logarithmic value of the concentration (mu g/L) of the isoprothiolane standard substance as an X axis and the percent absorbance value as a Y axis, and is shown in figure 1. Calculating the percent absorbance value of the sample solution by the same method, and reading the corresponding sample from the standard curveAnd multiplying the concentration by the corresponding dilution times to obtain the actual concentration of the isoprothiolane in the sample.

The kit can detect the minimum amount of the substance to be detected:

sensitivity: the lowest amount of the substance to be detected can be detected by applying the method, and the sensitivity of the quantitative kit can be generally understood as the concentration value of the standard substance with the smallest concentration or the corresponding lowest concentration of the standard substance when a standard curve is established, except for the '0' standard substance in the standard substance of the kit. Therefore, the sensitivity of the method is 0.1 mug/L.

And (3) determining the detection limit of the kit:

detection limit: generally refers to the minimum detection quantity of the actual sample measured by the kit product, and the theory is defined as: and (3) measuring 20 negative (blank) samples according to a reasonable pretreatment method, calculating the average value X and the Standard Deviation (SD), and obtaining the result according to the formula X +3SD, namely the detection (lower) limit of the sample. Therefore, the detection limit of the kit on the tobacco leaf sample is 100 mug/kg according to the method.

And (3) determining the accuracy and precision of the kit:

accuracy and precision: the accuracy of the ELISA assay is expressed as recovery and the precision as coefficient of variation. Taking a blank tobacco leaf sample, and carrying out an addition recovery test on the blank tobacco leaf sample by using isoprothiolane with the concentrations of 100, 200 and 400 mu g/kg, wherein the result shows that the recovery rate of the method on the tobacco leaf sample is 100% +/-20%, the intra-batch variation coefficient is less than 10%, and the inter-batch variation coefficient is less than 15%.

The test result of the kit is compared with the instrument method:

20 parts of the collected tobacco leaves to be roasted and the tobacco leaves primary roasted are respectively taken, and the samples are numbered from No. 1 to No. 20, the kit provided by the invention is used for carrying out comparative detection with an instrument detection method, and the instrument detection method refers to: GB/T20769-2008 fruit and vegetable residues determination liquid chromatogram-tandem mass spectrometry (the detection limit of isoprothiolane is 0.46mg/kg) of 450 pesticides and related chemicals.

Taking the maximum residue limit (2mg/kg) of isoprothiolane in the standard YQ 50-2014 "maximum residue limit of tobacco leaf pesticides" of China tobacco general company enterprise as a positive judgment line, and taking the maximum residue limit (2mg/kg) of isoprothiolane as undetected, wherein the undetected maximum residue limit is represented by "-"; samples above 2mg/kg are expressed by practical results, and the results are shown in tables 1-2 below.

TABLE 1 comparison of the results of the detection (mg/kg) of the reagent kit and the instrument method for the tobacco leaf samples to be roasted after harvest

Sample numbering

1

2

3

4

5

6

7

8

9

10

Reagent kit

－

7.9

－

2.6

－

LC-MS/MS

－

8.4

－

3.3

－

Sample numbering

11

12

13

14

15

16

17

18

19

20

Reagent kit

11.5

－

LC-MS/MS

12.7

－

TABLE 2 comparison of assay results (mg/kg) of kit and instrument method for flue-cured tobacco leaf samples

Sample numbering

1

2

3

4

5

6

7

8

9

10

Reagent kit

－

3.4

－

LC-MS/MS

－

4.1

－

Sample numbering

11

12

13

14

15

16

17

18

19

20

Reagent kit

－

10.3

－

2.2

－

LC-MS/MS

－

9.5

－

2.8

－

As can be seen from the above tables 1-2, the detection result of the kit is basically consistent with the detection result of the instrument, and the error is within an acceptable range. The kit shelf life conditions were:

the kit provided by the invention can be stored for at least 12 months at 2-8 ℃ by determination.

According to the detection results, the antibody prepared from the hapten and the kit have a relatively accurate detection effect in detecting the residual quantity of the oryzalin in the tobacco leaves, and the kit provided by the invention can be used for effectively detecting various pretreated agricultural products. .

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.

Claims

1. A hapten for detecting the content of isoprothiolane is characterized by having a chemical formula as follows: 2- (2- (1, 3-diisopropyl ether-1, 3-dioxopropan-2-ylidene) -1, 3-dithiolane) acetic acid; the chemical structural formula is as follows:

2. a method of preparing a hapten as described in claim 1 comprising the steps of:

3. The method of claim 2, comprising the steps of:

4. The preparation method according to claim 2, wherein the rotary evaporation operation in the step 3) comprises the steps of: removing acetonitrile and carbon disulfide in the reaction solution by rotary evaporation, adding water and an acid solution, and adjusting the pH value of the reaction solution to 6;

5. An antigen for detecting isoprothiolane content, which is a conjugate prepared by the method according to any one of claims 2 to 4 or obtained by coupling the hapten according to claim 1 to a carrier protein.

6. The antigen for detecting isoprothiolane content according to claim 5, wherein the carrier protein is bovine serum albumin, ovalbumin, human serum albumin, or hemocyanin.

7. An antibody for detecting isoprothiolane content, which is an antibody obtained by immunizing an animal with the antigen according to any one of claims 5 or 6; the antibody can generate specific immune reaction with isoprothiolane.

8. An enzyme-linked immunosorbent assay kit for detecting isoprothiolane, characterized in that the antibody used is the antibody for detecting the isoprothiolane content according to claim 7.

9. The enzyme-linked immunosorbent assay kit for rice blast according to claim 8, wherein the detection sample of the enzyme-linked immunosorbent assay kit for rice blast is a pretreated tobacco sample.

10. The enzyme-linked immunosorbent assay kit for rice blast according to claim 8, wherein the enzyme-linked immunosorbent assay kit for rice blast comprises: an ELISA plate and working solution; the ELISA plate is the antigen; the working solution is the antibody liquid marked by horseradish peroxidase.

11. The enzyme-linked immunosorbent assay kit for rice blast according to claim 8, wherein the enzyme-linked immunosorbent assay kit for rice blast comprises: a rice blast standard substance solution with gradient concentration, a substrate developing A, B solution, a stop solution, a washing solution and a redissolution;

the gradient concentration of the isoprothiolane standard substance solution with gradient concentration is 0 mug/L, 0.1 mug/L, 0.3 mug/L, 0.9 mug/L, 2.7 mug/L and 8.1 mug/L;

the substrate develops color A, B liquid: the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;

the stop solution is 2mol/L sulfuric acid;

the washing solution is pH7.2, and contains 0.8-1.2% of Tween-20, 0.01-0.03% of thimerosal preservative and 0.1-0.3 mol/L of carbonate buffer solution;

the complex solution is pH7.6, contains 8-12% casein and 0.1-0.3 mol/L phosphate buffer solution.