propiconazole hapten, artificial antigen, polyclonal antibody, and preparation method and application thereof

Abstract

This invention relates to a prothioconazole hapten, artificial antigen, polyclonal antibody, and their preparation methods and applications. The structural formula of the prothioconazole hapten is shown in formula (1). When the prothioconazole hapten, artificial antigen, and polyclonal antibody of this invention are used for indirect competitive enzyme-linked immunosorbent assay (ELISA) to detect prothioconazole residues, the sample pretreatment is simple, the detection results are accurate, the detection time is short and the cost is low. At the same time, the operation is simple and samples can be processed in batches, which is conducive to the development of field work.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a prothioconazole hapten, an artificial antigen, a polyclonal antibody, and their preparation methods and applications. Background Technology

[0002] Prothioconazole is a novel triazole thione fungicide. Its mechanism of action is to inhibit the demethylation of lanosterol precursors, lanosterol, or 2,4-methylenedihydrolanosterol at the 14-position in fungi, thus acting as a demethylation inhibitor. Prothioconazole is mainly used to control numerous diseases in cereal, wheat, and legume crops. It not only possesses excellent systemic activity and superior protective, curative, and eradicative activity against crops, but also has a long-lasting effect. Compared to other triazole fungicides, prothioconazole exhibits a broader spectrum of fungicidal activity.

[0003] However, pesticide residues are a significant factor affecting the quality and safety of agricultural products. With increasing consumer demand and international trade volume, the issue of pesticide residues in agricultural products has received growing attention. Prothioconazole is highly susceptible to degradation in the environment, transforming into dethioconazole (Desthio), which is teratogenic and poses potential harm to the environment and human health. To strictly control the quality of agricultural products, GB 2763-2021, the National Food Safety Standard for Maximum Residue Limits of Pesticides in Food, stipulates that the maximum residue limit for prothioconazole (dethioconazole) is 0.02 mg / kg-1 mg / kg in grains, oilseeds, and fats; 0.02 mg / kg-0.2 mg / kg in vegetables; 1.5 mg / kg in fruits; and 0.3 mg / kg in sugar beets.

[0004] Traditional methods for detecting prothioconazole residues mainly include instrumental analytical methods such as gas chromatography, liquid chromatography, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), ultra-high performance liquid chromatography (UHPLC), and UHPLC-MS. While these instrumental methods can achieve accurate qualitative and quantitative analysis of prothioconazole residues, they suffer from drawbacks such as expensive instruments, complex sample pretreatment, time-consuming detection, high costs, and the inability to process samples in batches. Furthermore, they require specialized operators, which is inconvenient for field operations. Summary of the Invention

[0005] Therefore, it is necessary to address the above-mentioned problems by providing a prothioconazole hapten, artificial antigen, polyclonal antibody, their preparation methods, and applications. When the prothioconazole hapten, artificial antigen, and polyclonal antibody are used in the indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of prothioconazole residues, the sample pretreatment is simple, the detection results are accurate, the detection time is short, and the cost is low. At the same time, the operation is simple, and samples can be processed in batches, which is beneficial to the field work.

[0006] A prothioconazole hapten, the structural formula of which is shown in formula (1):

[0007]

[0008] A method for preparing the prothioconazole hapten as described above includes the following steps:

[0009] Prothioconazole was subjected to a substitution reaction with a haloester with the structural formula shown in formula (2) under the action of a catalyst to obtain the first intermediate with the structural formula shown in formula (3).

[0010] The first intermediate was hydrolyzed with lithium hydroxide to obtain the prothioconazole hapten with the structural formula shown in formula (1).

[0011]

[0012] Where R represents a halogen.

[0013] A prothioconazole artificial antigen is a conjugate of the prothioconazole hapten as described above and a carrier protein.

[0014] In one embodiment, the carrier protein is selected from bovine serum albumin, hemocyanin, or chicken ovalbumin.

[0015] In one embodiment, the conjugate of the prothioconazole hapten with bovine serum albumin or hemocyanin serves as an immunogen.

[0016] In one embodiment, the conjugate of the prothioconazole hapten and chicken ovalbumin is used as the coating agent.

[0017] A method for preparing the prothioconazole artificial antigen as described above includes the following steps:

[0018] The prothioconazole hapten as described in claim 1 is reacted with N-hydroxysuccinimide to generate a second intermediate with the structural formula shown in formula (4);

[0019] The second intermediate was coupled to the carrier protein to obtain the prothioconazole artificial antigen.

[0020]

[0021] A prothioconazole polyclonal antibody was obtained by immunizing host animals with an emulsified prothioconazole artificial antigen as described above.

[0022] The application of the above-mentioned prothioconazole hapten, the above-mentioned prothioconazole artificial antigen, and the above-mentioned prothioconazole polyclonal antibody in the indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of prothioconazole.

[0023] In one embodiment, the prothioconazole artificial antigen serves as the coating antigen, and the prothioconazole polyclonal antibody serves as the detection antibody.

[0024] The prothioconazole hapten provided by this invention has a moderately long linker arm. On the one hand, this allows the hapten to be fully exposed on the surface of the carrier protein when it is coupled with the carrier protein. On the other hand, the linker arm will not fold due to its own or external influences, allowing the hapten to fully unfold and enabling the artificial antigen to be recognized by the immune body to the greatest extent. At the same time, since the active group of the prothioconazole hapten is attached to a sulfur atom, it is far from the characteristic structure of the hapten molecule, which is conducive to the exposure of the characteristic structure of the hapten molecule, resulting in higher specificity and sensitivity of the antibodies produced when immunizing the host animal.

[0025] Therefore, when using the prothioconazole hapten, artificial antigen, and polyclonal antibody of this invention for the indirect competitive enzyme-linked immunosorbent assay (ELISA) to detect prothioconazole, the polyclonal antibody provides accurate detection results due to its high specificity and sensitivity. Furthermore, the procedure only requires the prothioconazole artificial antigen and prothioconazole antibody to react with the sample, and the concentration of prothioconazole in the sample is calculated based on the absorbance value. This method is simple to operate, has a short detection time, and is inexpensive, making it beneficial for field work.

[0026] In addition, when detecting prothioconazole using indirect competitive enzyme-linked immunosorbent assay (ELISA), sample pretreatment only requires centrifugation and dilution, which is simple and allows for the simultaneous reaction of multiple samples, thus enabling batch processing of samples. Attached Figure Description

[0027] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the first intermediate in this invention;

[0028] Figure 2 This is the 1H NMR spectrum of the prothioconazole hapten in this invention. Detailed Implementation

[0029] To facilitate understanding of the present invention, a more complete description will be given below with reference to relevant embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0031] The present invention provides a prothioconazole hapten, the structural formula of which is shown in formula (1):

[0032]

[0033] The prothioconazole hapten provided by this invention has a moderately long linker arm. On the one hand, this allows the hapten to be fully exposed on the surface of the carrier protein when it is coupled with the carrier protein. On the other hand, the linker arm will not fold due to its own or external influences, allowing the hapten to fully unfold and enabling the artificial antigen to be recognized by the immune body to the greatest extent. At the same time, since the active group of the prothioconazole hapten is attached to a sulfur atom, it is far from the characteristic structure of the hapten molecule, which is conducive to the exposure of the characteristic structure of the hapten molecule, resulting in higher specificity and sensitivity of the antibodies produced when immunizing the host animal.

[0034] The present invention also provides a method for preparing the prothioconazole hapten as described above, comprising the following steps:

[0035] S11, thiophanate-methyl and a haloester with the structural formula shown in formula (2) are subjected to a substitution reaction under the action of a catalyst to obtain the first intermediate with the structural formula shown in formula (3).

[0036] S12, the first intermediate is hydrolyzed with lithium hydroxide to obtain the prothioconazole hapten with the structural formula shown in formula (1).

[0037]

[0038] Wherein, R is a halogen element, selected from chlorine, bromine, iodine, etc., and further, the haloester is preferably methyl 3-bromopropionate.

[0039] Taking methyl 3-bromopropionate as an example, the reaction formula for step S11 is as follows:

[0040]

[0041] In one embodiment, the catalyst is selected from at least one of potassium iodide or cesium carbonate.

[0042] To better generate the first intermediate, the molar ratio of prothioconazole to the haloester with the structural formula shown in formula (2) is 1:1 to 1:4, preferably 1:2. In the step of carrying out the substitution reaction of prothioconazole and the haloester with the structural formula shown in formula (2) under the action of a catalyst, the reaction temperature is 50℃ to 70℃, preferably 60℃.

[0043] After the reaction is completed, the first intermediate with the structure shown in formula (3) is separated by silica gel column chromatography. The preferred mobile phase for column chromatography is petroleum ether and ethyl acetate.

[0044] The reaction formula for step S12 is:

[0045]

[0046] In order to better generate prothioconazole hapten, in one embodiment, the mass ratio of the first intermediate to lithium hydroxide is 1:1-1:5, preferably 1:3, and in the step of hydrolyzing the first intermediate with lithium hydroxide, the reaction temperature is -10℃ to 10℃, preferably 0℃.

[0047] Since the prothioconazole hapten is only immunoreactive and not immunogenic, and cannot stimulate the body to produce corresponding antibodies on its own, it is necessary to couple, bind, or cross-link the prothioconazole hapten with a carrier protein to produce an artificial prothioconazole antigen that is both immunoreactive and immunogenic.

[0048] Therefore, the present invention also provides a prothioconazole artificial antigen, which is a conjugate of the prothioconazole hapten as described above and a carrier protein.

[0049] The carrier protein includes macromolecular proteins or non-antigenic polylysine. In one embodiment, the carrier protein is selected from bovine serum albumin, hemocyanin, or chicken ovalbumin.

[0050] In one embodiment, the conjugate of the prothioconazole hapten with bovine serum albumin or hemocyanin is used as an immunogen to immunize the host animal and induce it to produce specific antibodies.

[0051] In one embodiment, the conjugate of the prothioconazole hapten and chicken ovalbumin is used as a coating agent to attach to an ELISA plate in an enzyme-linked immunosorbent assay (ELISA).

[0052] The present invention also provides a method for preparing the prothioconazole artificial antigen as described above, comprising the following steps:

[0053] S21, the prothioconazole hapten as described in claim 1 is reacted with N-hydroxysuccinimide to generate a second intermediate with the structural formula shown in formula (4);

[0054] S22, the second intermediate is coupled with the carrier protein to obtain the prothioconazole artificial antigen;

[0055]

[0056] The carrier protein is selected from bovine serum albumin, hemocyanin, or chicken ovalbumin. Taking bovine serum albumin (BSA) as an example, the reaction formula between prothioconazole hapten and bovine serum albumin is as follows:

[0057]

[0058] The present invention also provides a prothioconazole polyclonal antibody, which is obtained by emulsifying a host animal with the prothioconazole artificial antigen as described above.

[0059] When artificial antigens enter the host animal, they stimulate the reticuloendothelial cell system, causing a large proliferation of lymphocytes in the lymph nodes and spleen, which in turn produces specific antibodies. Since the antigen molecule contains multiple antigenic epitopes with different antigenic specificities, the specific antibodies produced contain immunoglobulins targeting multiple different antigenic epitopes, resulting in polyclonal antibodies.

[0060] To enhance the body's immune response to artificial antigens, Freund's complete adjuvant can be mixed and emulsified with the artificial antigen and then injected into the animal during the immunization of the host animal.

[0061] Understandably, monoclonal antibodies can also be obtained by manufacturing and culturing hybridoma cells and collecting individual cells.

[0062] The present invention also provides the application of the above-mentioned prothioconazole hapten, the above-mentioned prothioconazole artificial antigen, and the above-mentioned prothioconazole polyclonal antibody in the indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of prothioconazole.

[0063] In one embodiment, the prothioconazole artificial antigen serves as the coating antigen, and the prothioconazole antibody serves as the detection antibody.

[0064] The prothioconazole hapten, artificial antigen, and polyclonal antibody of this invention, when used in the indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of prothioconazole, yield accurate results due to the high specificity and sensitivity of the polyclonal antibody. Furthermore, only the prothioconazole artificial antigen and antibody need to react with the sample; the concentration of prothioconazole in the sample is calculated based on the absorbance value. This method is simple to operate, has a short detection time, and is inexpensive, making it beneficial for field work.

[0065] In addition, when detecting prothioconazole using indirect competitive enzyme-linked immunosorbent assay (ELISA), sample pretreatment only requires centrifugation and dilution, which is simple and allows for the simultaneous reaction of multiple samples, thus enabling batch processing of samples.

[0066] The following specific examples will further illustrate the prothioconazole hapten, artificial antigen, polyclonal antibody, their preparation methods, and applications.

[0067] Synthesis and identification of prothioconazole hapten:

[0068] 0.86 g of 2 mmol / L prothioconazole technical, 0.0336 g of 0.2 mmol / L potassium iodide, 0.9771 g of 3 mmol / L cesium carbonate, and 0.42 g of 4 mmol / L methyl 3-bromopropionate were dissolved in 10 mL of N,N-dimethylformamide. The mixture was stirred at 60 °C for 20 h. After the reaction was complete, 40 mL of saturated brine was added, and the mixture was extracted three times with 50 mL of ethyl acetate. The organic phase was then concentrated under reduced pressure. The concentrated organic phase was then purified by rapid column chromatography on a silica gel column using petroleum ether / ethyl acetate as the eluent to obtain the first intermediate.

[0069] 430 mg of the 1 mmol / L first intermediate was added to a mixture of 6 mL methanol and 2 mL water. Then, 72 mg of a 3 mmol / L lithium hydroxide aqueous solution was slowly added at 0 °C. The mixture was allowed to react overnight at 25 °C. After the reaction was complete, the product was purified by rapid column chromatography to obtain the prothioconazole hapten.

[0070] The above intermediate product was identified by 1H NMR spectroscopy, and the result was: 1H NMR (600 MHz) z,CDCl3)δ7.87(s,1H),7.53(d,J=7.2Hz,1H),7.35(d,J=7.8Hz,1H),7.23-7.18(m,2H),4.77(d,J=14.4Hz,1H),4.64(s,1H),3.90(d,J=14.4Hz,1 H),3.70(s,3H),3.60(d,J=13.8Hz,1H),3.50-3.43(m,2H),3.08(d,J=14 .4Hz,1H),2.83-2.81(m,2H),0.97-0.77(m,2H),0.60(t,J=9.0Hz,2H), such as Figure 1 As shown, this proves that the first intermediate was successfully coupled.

[0071] The above-mentioned prothioconazole hapten was identified by 1H NMR spectroscopy. The results were as follows: 1H NMR (600MHz, CDCl3) δ 7.98 (s, 1H), 7.54 (dd, J = 1.2, 7.2Hz, 1H), 7.34 (d, J = 7.8Hz, 1H), 7.22-7.18 (m, 2H), 4.76 (d, J = 14.4Hz, 1H), 4.55 (s, 1H), 4.51 (d, J = 14.4Hz, 1H), 4.26 (t, J = 6.0Hz, 2H), 3.58 (d, J = 14.4Hz, 1H), 3.14 (d, J = 14.4Hz, 1H), 2.98-2.96 (m, 2H), 0.96-0.69 (m, 4H). Figure 2 As shown, this proves that the prothioconazole hapten conjugation was successful.

[0072] Synthesis and identification of prothioconazole artificial antigen:

[0073] Immunogen preparation: Weigh 86 mg of 0.2 mmol / L prothioconazole hapten and dissolve it in 1 mL of N,N-dimethylformamide. Then add 0.6 mmol / L of N-hydroxysuccinimide and stir at 25 °C for 15 min. After the reaction, add 0.3 mmol / L of dicyclohexylcarbodiimide and react overnight at 25 °C. Then centrifuge at 12000 r / min for 5 min. Take 0.5 mL of the supernatant and slowly add it dropwise to 10 mL of 10 mg / mL bovine serum albumin carbonate buffer solution. Seal and magnetically stir at 25 °C for 4 h. After the reaction is complete, place the solution in a dialysis bag and dialyze with stirring at 4 °C. Dialyze three times with ultrapure water, then dialyze with phosphate buffer for three days, changing the dialysate every 4 h, for a total of 10 dialysis cycles. After dialysis, remove the dialysate to obtain the prothioconazole immunogen, aliquot it, and store it at -20 °C.

[0074] Preparation of the coating antigen: Weigh 0.2 mmol / L of prothioconazole hapten and 10 mL of 10 mg / mL chicken ovalbumin and conjugate them. The synthesis method is the same as the immunogen method described above to obtain the prothioconazole coated antigen.

[0075] Preparation of prothioconazole polyclonal antibody:

[0076] First immunization: Take 5-month-old New Zealand white rabbits, dilute the above immunogen with physiological saline, mix it with an equal volume of Freund's complete adjuvant, emulsify it thoroughly, and inject it subcutaneously into the New Zealand white rabbits. The injection dose is 1 mg / kg per rabbit.

[0077] Second to fourth immunization: Three weeks after the first immunization, the immunogen was removed and fully emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at a dose of 1.5 mg / kg per animal. After that, immunization was performed every two weeks for a total of three immunizations.

[0078] Booster immunization: One week after the fourth immunization, blood was collected from the marginal ear vein of New Zealand white rabbits to measure the titer and inhibition. New Zealand white rabbits with effective titers and good inhibitory effects were given booster immunization: New Zealand white rabbits were given booster immunization 7 days before the heart blood collection, and the same dose of immunogen as the fourth immunization was injected into the thigh intramuscularly. Blood was collected from the heart 7 days later to prepare serum.

[0079] Purification of polyclonal antibodies: Polyclonal antibodies were purified using the caprylic acid-ammonium sulfate method. The serum was diluted with acetate buffer at a volume ratio of 1:4. The pH was then adjusted to 4.5. Caprylic acid was added dropwise at 25°C with stirring. The initial serum-caprylic acid volume ratio was 1 mL:75 μL. Stirring continued for 30 min, followed by standing at 4°C for 2 h. The mixture was then centrifuged at 10000 rpm for 30 min at the same temperature, and the precipitate was discarded. The supernatant was filtered through filter paper, and 1 / 10 the volume of the supernatant was added to adjust the pH to 7.4. Pre-cool at 4℃ for 20 min, then slowly add 0.277 g / mL ammonium sulfate until the ammonium sulfate saturation is 45%. After standing for 2 h, centrifuge at 12000 r / min for 30 min, discard the supernatant, dissolve the precipitate with phosphate buffer, and then dialyze with phosphate buffer containing ethylenediaminetetraacetic acid at 4℃ for 3 days, changing the medium 4 times a day. After dialysis, remove the dialysate to obtain prothioconazole polyclonal antibody, aliquot and store at -20℃.

[0080] Potency and inhibition detection: Serum supernatant from New Zealand white rabbits was used for screening using indirect non-competitive enzyme-linked immunosorbent assay (ELISA) and indirect competitive ELISA.

[0081] Indirect non-competitive enzyme-linked immunosorbent assay (ELISA): Coating: Dilute the prothioconazole coating agent 1000 times with coating buffer and add 50 μL to each well of the ELISA plate. Incubate at 37°C for 2 h.

[0082] Sealing: Add 100 μL of 1% gelatin to each well and incubate at 37°C for 1.5 h.

[0083] Add prothioconazole polyclonal antibody solution: Add 50 μL of serum supernatant diluted with phosphate buffer at different ratios to each well, incubate at 37°C for 1 h, and set up positive and negative controls in parallel.

[0084] Add enzyme-labeled secondary antibody: Add 50 μL of enzyme-labeled antibody diluted 1:10000 with phosphate buffer to each well and incubate at 37°C for 1 h.

[0085] Washing: After each step of the above reaction, wash 5 times with phosphate buffer and pat dry with absorbent paper.

[0086] Color development: Add 50 μL of freshly prepared substrate color development solution to each well and incubate at 37°C for 15 min.

[0087] Termination: Add 25 μL of 2 mol / L sulfuric acid solution to each well.

[0088] Absorbance measurement: The absorbance of each well at a wavelength of 450 nm was measured using an ELISA reader.

[0089] The potency is the maximum dilution of a positive reaction.

[0090] The titer of the prothioconazole polyclonal antibody was measured to be 1:512000.

[0091] Indirect competitive enzyme-linked immunosorbent assay (ELISA): Coating: Dilute the prothioconazole coating agent with coating buffer and add 50 μL to each well of the ELISA plate. Incubate at 37°C for 2 h.

[0092] Sealing: Add 100 μL of 1% gelatin to each well and incubate at 37°C for 1.5 h.

[0093] Add analytes and primary antibodies: Add 25 μL of prothioconazole standard solution or the sample solution to be tested to each well; then add 25 μL of prothioconazole polyclonal antibody solution diluted with phosphate buffer to each well. Incubate at 37°C for 1 h, and set up positive and negative controls in parallel.

[0094] Add enzyme-labeled secondary antibody: Add 50 μL of enzyme-labeled antibody diluted 1:10000 with phosphate buffer to each well and incubate at 37°C for 1 h.

[0095] Washing: After each step of the above reaction, wash 5 times with phosphate buffer and pat dry with absorbent paper.

[0096] Color development: Add 50 μL of freshly prepared substrate color development solution to each well and incubate at 37°C for 15 min.

[0097] Termination: Add 25 μL of 2 mol / L sulfuric acid solution to each well.

[0098] Absorbance measurement: The absorbance of each well at a wavelength of 450 nm was measured using an ELISA reader.

[0099] Determination of polyclonal antibody specificity: Seven prothioconazole analogue pesticides—dethioconazole, hexaconazole, tebuconazole, triadimefon, uniconazole, paclobutrazol, and tebuconazole standards—were tested using an indirect competitive enzyme-linked immunosorbent assay (ELISA). The specificity of the polyclonal antibodies was assessed by calculating the cross-reactivity rates of prothioconazole with each analogue. The calculation formula is as follows:

[0100]

[0101] In equation (1), IC 50 The half-inhibition concentration (WIC) represents the analyte concentration at which the binding rate is 50%.

[0102] The results showed that the cross-reactivity rates of the analogues were as follows: prothioconazole 100%, dethioconazole 69%, tebuconazole 0.12%, hexaconazole <0.1%, triazole <0.1%, uniconazole <0.1%, paclobutrazol <0.1%, and tebuconazole <0.1%. The antibody of this invention showed no cross-reactivity with hexaconazole, tebuconazole, triazole, uniconazole, paclobutrazol, and tebuconazole analogues, but specifically bound to prothioconazole and its metabolite dethioconazole.

[0103] Establishing the standard curve:

[0104] Standard curve establishment: Under optimal analytical conditions, the analyte was detected using an indirect competitive enzyme-linked immunosorbent assay (ELISA) procedure. The working concentrations of the prothioconazole standard solution were 0.05 ng / mL, 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 250 ng / mL, 500 ng / mL, 1000 ng / mL, 2500 ng / mL, 5000 ng / mL, and 10000 ng / mL. The experimental results were processed and analyzed using Origin data processing software. A standard inhibition curve for the detection of prothioconazole was established by fitting a logistic regression curve with prothioconazole concentration as the x-axis and binding rate (B / B0) as the y-axis. Plotting the binding rate (B / B0) on the ordinate and the logarithm of the concentration of prothioconazole standard solution on the abscissa, a linear regression analysis of the inhibition curve was performed to obtain the linear regression equation for the standard curve, along with the linear equation and coefficient of determination. Ultimately, the linear range of prothioconazole was found to be 5 ng / mL–500 ng / mL, with a half-maximal inhibitory concentration (IC50) of [missing value]. 50 =102.28 ng / mL, limit of detection (LOD) 10 The concentration was 10.74 ng / mL.

[0105] Wherein, B0 is the OD value of the control well without the addition of prothioconazole, and B is the OD value of the well with the addition of prothioconazole.

[0106] The detection limit is the theoretically detectable minimum value calculated based on the standard curve, and is generally the concentration of the inhibitory substance corresponding to a binding rate of 10%.

[0107] Recycling experiment:

[0108] This embodiment uses pond water and wheat grains as subjects to investigate the effect of the matrix on the detection of prothioconazole residues. Pond water samples were taken from a small pond in the Smart Plaza of Anhui Agricultural University, and wheat grains were purchased from the Hefei Agricultural Products Market in Anhui Province for suspension and recovery studies. The samples had been verified to be free of prothioconazole using ultra-high performance liquid chromatography-tandem mass spectrometry. Prothioconazole was added to both pond water and wheat grains for suspension and recovery experiments to evaluate the accuracy of the indirect competitive enzyme-linked immunosorbent assay (ELISA).

[0109] Sample preparation: The obtained pond water sample was filtered through a 0.45 μm water system filter membrane to obtain the matrix extract.

[0110] The purchased wheat grain sample was added to a homogenizer to homogenize it. 1 g of the homogenized sample was weighed into a 50 mL centrifuge tube, and 5 mL of phosphate buffer containing 5% methanol was added as the extraction solvent. The mixture was then vortexed for 1 min. The mixture was filtered through filter paper, and the filtrate was centrifuged at 4000 rpm for 10 min to remove the precipitate and obtain the matrix extract.

[0111] Matrix effect evaluation: The matrix extract was diluted to reduce the impact of the matrix effect on the detection method. Pond water samples that had been verified to be free of prothioconazole residues were processed according to the sample preparation method. Different volumes of phosphate buffer and methanol were added to dilute the pond water samples to 2, 5, 10, and 15 times. Prothioconazole standard solutions of different concentrations were then prepared using these solutions containing the sample matrix extract as solvents, and the inhibition rate was determined by indirect competitive enzyme-linked immunosorbent assay (ELISA). The data were compared with those obtained from prothioconazole standard solutions prepared from solutions without the extract, and the closest dilution factor (i.e., pond water diluted 5 times) was taken as the matrix dilution factor for that sample.

[0112] Wheat grain samples that have been verified to be free of prothioconazole residues were processed according to the sample preparation method. Different volumes of phosphate buffer and methanol were added to dilute the wheat grain matrix solution to 20, 30, 40, and 50 times. Prothioconazole standard solutions of different concentrations were then prepared using these solutions containing the sample matrix extract as solvents, and the inhibition rate was determined by indirect competitive enzyme-linked immunosorbent assay (ELISA). The data were compared with those obtained from prothioconazole standard solutions prepared from solutions without extracts, and the closest dilution factor, i.e., a 30-fold dilution of the wheat grains, was taken as the matrix dilution factor for that sample.

[0113] Determination of recovery rate and instrument validation: Recovery experiments were conducted using manual spiking to assess the accuracy and precision of the analytical method, with the recovery rate of prothioconazole as the criterion. Prothioconazole standards at concentrations ranging from 25 ng / mL to 100 ng / mL were added to pond water samples to obtain extracts. These extracts were then diluted several times, and the recovery rate was determined using indirect competitive enzyme-linked immunosorbent assay (ELISA).

[0114] Add 25 ng / mL to 100 ng / mL of prothioconazole standard to wheat grain samples, process the extracts as described above, dilute them by a certain factor, and determine the recovery rate using indirect competitive enzyme-linked immunosorbent assay (ELISA).

[0115] Analysis of test results: The recovery rate of prothioconazole in pond water was 101.8%-104.7%, and the recovery rate of prothioconazole in wheat grains was 81.9%-94.2%, both of which met the recovery rate standards.

[0116] To verify the accuracy of the test results, pond water and wheat grain samples were added with prothioconazole standard and then verified by ultra-high performance liquid chromatography-tandem mass spectrometry.

[0117] Weigh 5.0 g of wheat grain sample into a 50 mL centrifuge tube, add 20 mL of acetonitrile, shake for 20 min, centrifuge at 5000 r / min, transfer 1 mL of supernatant to a 5 mL centrifuge tube, blow dry with nitrogen, dilute to volume with 2 mL of chromatographic methanol, add 100 mg of anhydrous magnesium sulfate and 50 mg of C18 packing material, shake, centrifuge at 5000 r / min, collect the supernatant in a sample vial, and detect by high performance liquid chromatography-tandem mass spectrometry. The recovery rate of wheat grain matrix was 87.3%-103.0%.

[0118] Pond water samples were filtered through a 0.22 μm filter membrane. 11 mg of a magnetite@carbon@zirconium 1,4-carboxybenzene metal-organic framework was uniformly dispersed in 40 mL of water using an ultrasonic mixer at room temperature until adsorption equilibrium was reached. The adsorbent was then collected in the solution using an external magnet, washed twice with 5 mL of ultrapure water, and 1 mL of acetonitrile was added. The mixture was rotated and collected, then purged with nitrogen and resuspended in 200 μL of mobile phase. After filtration through a membrane, the solution was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). The recovery rate of the pond water matrix was found to be 95.93%–96.72%.

[0119] The detection conditions for high performance liquid chromatography-tandem mass spectrometry are as follows.

[0120] Chromatographic conditions: ACQUITY column (50 mm × 2.1 mm, 1.7 μm), column temperature 40 ℃; mobile phase A was 0.1% formic acid in water, mobile phase B was acetonitrile, flow rate was 0.2 mL / min, total run time was 5 min, and sample injection volume was 5 μL.

[0121] Mass spectrometry conditions: Performed in multiple reaction monitoring (MRM) mode using a point spray ionization source in positive ion mode, with a capillary voltage of 3.0 kV, and ion source and desolvation temperatures of 150 °C and 500 °C, respectively; nitrogen was used as the nebulizer gas at a flow rate of 3.0 L / min. The conical gas flow rate was 150.0 L / h, the desolvation gas flow rate was 1000.0 L / h, and argon (99.99%) was used as the collision gas at a pressure of 2 × 10⁻⁶. -3 The mass spectrometry parameters were measured in mbar, with a cone voltage of 16.0 V and collision voltages of 30.0 V and 12.0 V, respectively. The relative molecular masses, ion pairs, transport charges, and collision energies analyzed by mass spectrometry are shown in Table 1.

[0122] Table 1

[0123]

[0124] Wheat grain and pond water samples were pretreated according to the sample preparation method in the recovery experiment, and then detected by indirect competitive enzyme-linked immunosorbent assay.

[0125] Wheat grain and pond water samples were pretreated according to the instrument injection method used in the recovery experiment, and then analyzed by high-performance liquid chromatography-tandem mass spectrometry. The specific pretreatment method was as follows: 2.0 g of sample was weighed into a 50 mL centrifuge tube, prothioconazole standard was added and allowed to stand for 30 min, then 5 mL of ultrapure water and 10 mL of acetonitrile were added, vortexed for 2 min, ultrasonically extracted for 15 min, 4.0 g of analytical grade sodium chloride was added, vortexed for 1 min, centrifuged at 4000 rpm for 5 min, and 1 mL of the supernatant was filtered through a 0.22 μm organic phase microporous membrane and added to a sample vial for analysis.

[0126] The two test results were compared, and the final results are shown in Table 2.

[0127] Table 2

[0128]

[0129] As shown in Table 2, the detection results of indirect competitive enzyme-linked immunosorbent assay (ELISA) and instrumental analysis both met the recovery rate standard.

[0130] Furthermore, in the indirect competitive enzyme-linked immunosorbent assay (ELISA), the test solution can be obtained using simple sample pretreatment and extraction methods. Through experimental optimization, pond water samples only need to be diluted through a membrane before testing. Wheat grains only require one shaking centrifugation followed by dilution before testing. Each ELISA plate has 96 wells, allowing for simultaneous testing of multiple samples. Completing one plate takes approximately 2.5 hours.

[0131] In ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), wheat grain matrix undergoes complex pretreatment steps including shaking, centrifugation, nitrogen blowing, and purification followed by shaking and centrifugation to obtain the analyte. The pretreated sample is then added to the injection vial, and a single injection takes 5 minutes. Environmental water samples undergo multiple pretreatment steps, including magnetic solid-phase extraction and nitrogen blowing, before being added to the injection vial. A single injection (one sample) takes 18.5 minutes. Therefore, although the instrument's sample pretreatment method is relatively simple, it still requires at least one hour for sample pretreatment.

[0132] The comparison shows that the sample pretreatment in this invention is simple, requires little time, can be performed in large batches, and does not require professional personnel.

[0133] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0134] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A prothioconazole hapten, characterized in that, Its structural formula is shown in equation (1):

2. A method for preparing the prothioconazole hapten as described in claim 1, characterized in that, Includes the following steps: Prothioconazole is subjected to a substitution reaction with a haloester with the structural formula shown in formula (2) under the action of a catalyst to obtain a first intermediate with the structural formula shown in formula (3), wherein the catalyst is selected from at least one of potassium iodide or cesium carbonate. The first intermediate was hydrolyzed with lithium hydroxide to obtain the prothioconazole hapten with the structural formula shown in formula (1). Where R represents a halogen.

3. A prothioconazole artificial antigen, characterized in that, The conjugate of prothioconazole hapten and carrier protein as described in claim 1, wherein the carrier protein is selected from bovine serum albumin, hemocyanin or chicken ovalbumin.

4. The prothioconazole artificial antigen according to claim 3, characterized in that, The conjugate of the prothioconazole hapten with bovine serum albumin or hemocyanin serves as an immunogen.

5. The prothioconazole artificial antigen according to claim 3, characterized in that, The conjugate of prothioconazole hapten and chicken ovalbumin serves as the coating agent.

6. A method for preparing prothioconazole artificial antigen as described in any one of claims 3-5, characterized in that, Includes the following steps: The prothioconazole hapten as described in claim 1 is reacted with N-hydroxysuccinimide to generate a second intermediate with the structural formula shown in formula (4); The second intermediate was coupled to the carrier protein to obtain the prothioconazole artificial antigen.

7. A prothioconazole polyclonal antibody, characterized in that, Obtained by immunizing host animals with the prothioconazole artificial antigen emulsified as described in any one of claims 3-5; The preparation method of the prothioconazole polyclonal antibody is as follows: First immunization: Take 5-month-old New Zealand white rabbits, dilute the above immunogen with physiological saline, mix it with an equal volume of Freund's complete adjuvant, emulsify it thoroughly, and inject it subcutaneously into the New Zealand white rabbits. The injection dose is 1 mg / kg per rabbit. Second to fourth immunization: Three weeks after the first immunization, the immunogen and an equal volume of Freund's complete adjuvant were fully emulsified and injected subcutaneously at a dose of 1.5 mg / kg per animal. After that, immunization was carried out every 2 weeks for a total of 3 immunizations. Boosting immunization: One week after the fourth immunization, blood was collected from the marginal ear vein of New Zealand white rabbits to measure the titer and inhibition. New Zealand white rabbits with effective titers and good inhibitory effects were given booster immunization: New Zealand white rabbits were given booster immunization 7 days before heart blood collection. The same dose of immunogen as the fourth immunization was injected into the thigh intramuscularly. Heart blood was collected 7 days later to prepare serum. Purification of polyclonal antibodies: Polyclonal antibodies were purified using the caprylic acid-ammonium sulfate method. The above serum was taken and diluted with acetate buffer at a volume ratio of 1:

4. The pH was then adjusted to 4.

5. Caprylic acid was added dropwise while stirring at 25°C. Before dilution, the volume ratio of serum to caprylic acid was 1 mL:75 μL. Stirring was continued for 30 min, and the mixture was allowed to stand at 4°C for 2 h. Then, it was centrifuged at 10000 r / min for 30 min at the same temperature, and the precipitate was discarded. The supernatant was filtered through filter paper, and 0.1% of the volume of the supernatant was added. Adjust the pH to 7.4 using 1 mol / L phosphate buffer; pre-cool at 4°C for 20 min, then slowly add 0.277 g / mL ammonium sulfate until the ammonium sulfate saturation is 45%. After standing for 2 h, centrifuge at 12000 r / min for 30 min, discard the supernatant, dissolve the precipitate with phosphate buffer, and then dialyze with phosphate buffer containing ethylenediaminetetraacetic acid at 4°C for 3 days, changing the medium 4 times a day. After dialysis, remove the dialysate to obtain prothioconazole polyclonal antibody, aliquot and store at -20°C.

8. The application of a prothioconazole hapten as described in claim 1, a prothioconazole artificial antigen as described in any one of claims 3-5, and a prothioconazole polyclonal antibody as described in claim 7 in the agricultural detection of prothioconazole using an indirect competitive enzyme-linked immunosorbent assay (ELISA).

9. The application according to claim 8, characterized in that, The prothioconazole artificial antigen is used as the coating antigen, and the prothioconazole polyclonal antibody is used as the detection antibody.

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