Omethoate hapten as well as preparation method and application thereof

Abstract

The invention relates to an omethoate hapten and a preparation method and application thereof, and the structure of the omethoate hapten is shown as a formula I. The omethoate hapten and the omethoate antigen provided by the invention are simple in synthesis method, high in purity and high in yield, and have great value for preparation of an omethoate antibody and detection of omethoate drug residues. The detection limit of the omethoate enzyme linked immunosorbent assay kit developed by the invention in cowpeas, lettuces and eggplants is 0.5 mu g / kg, the recovery rate of each addition concentration is 87.73-113.34%, the intra-batch variation coefficient is less than 10%, and the inter-batch variation coefficient is less than 15%.

Description

Technical Field

[0001] This invention belongs to the field of pesticide residue detection in food, and relates to an omethoate hapten and antigen, as well as their preparation method and application. Background Technology

[0002] Omethoate, also known as dimethoate, has the chemical name O,O-dimethyl-S-(N-methylcarbamoylmethyl)thiophosphate. It is a highly toxic organophosphate insecticide with strong contact and systemic action, widely used to control various pests on fruits, vegetables, and other agricultural products. However, omethoate can be absorbed into the plant through the stems and leaves and transported to various parts of the plant, easily resulting in residues in vegetables, fruits, and other agricultural products. To ensure the safety of agricultural products, human and animal health, and the ecological environment, on December 25, 2023, the Ministry of Agriculture and Rural Affairs of the People's Republic of China issued Announcement No. 736, banning four highly toxic pesticides: omethoate, carbofuran, methomyl, and aldicarb.

[0003] Despite this, the illegal use and abuse of omethoate persists in fruit and vegetable cultivation, meaning the problem of omethoate residues in agricultural products remains unresolved. Consuming food with excessive omethoate residues can easily lead to acute or chronic poisoning, causing symptoms such as dizziness, headache, nausea, vomiting, abdominal pain, and diarrhea. In severe cases, it can even cause difficulty breathing, rapid heartbeat, convulsions, coma, and death. Furthermore, omethoate is metabolized slowly in the body, easily leading to cumulative poisoning, and can also be secreted into breast milk, posing a potential threat to breastfeeding women and infants.

[0004] Therefore, accurate and timely detection of omethoate residues in fruits, vegetables, and other foods is particularly important. Currently, omethoate detection still relies primarily on laboratory instruments, which are cumbersome, require expensive equipment, and are time-consuming. To better meet the needs of fruit and vegetable testing in my country and improve food safety assurance, it is necessary to develop convenient, rapid, and accurate detection methods. Immunochemical analysis has significant advantages in the qualitative and quantitative analysis of antigens and antibodies. It is simple to operate, low in cost, highly sensitive, and suitable for large-scale sample analysis, effectively compensating for the shortcomings of laboratory testing. Currently available rapid immunoassay products for omethoate in fruits and vegetables have detection limits of 1-2 mg / kg, far exceeding the maximum residue limit of 0.02 mg / kg stipulated in GB2763-2021. Therefore, there is an urgent need for an innovative omethoate hapten structure and a highly sensitive, specific antibody for the development of rapid immunoassay methods / products for omethoate that meet national limits. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an omethoate hapten, its preparation method, and its application.

[0006] The first objective of this invention is to provide an omethoate hapten, the structure of which is shown in Formula I:

[0007]

[0008] A second objective of this invention is to provide a method for preparing the omethoate hapten of Formula I, comprising the following steps:

[0009] 1389.4 μL of mercaptoacetic acid was placed in a 50 mL round-bottom flask, and 25 mL of dichloromethane was added. After stirring until dissolved, the mixture was cooled to 0–4 °C in an ice bath. 2782 μL of triethylamine was added, followed by 2156.5 μL of O,O-dimethylphosphoryl chloride. The mixture was stirred at 0–4 °C for 3 hours. The salt was removed by filtration, and the filtrate was concentrated under reduced pressure. The solid was dissolved in 20 mL of dichloromethane. Column chromatography was performed using silica gel, with a mixture of petroleum ether and ethyl acetate (1:1 v / v) as the eluent. The main product was collected and concentrated under reduced pressure to give approximately 2800 mg of O,O-dimethyl-S-(carboxymethyl)thiophosphate oil.

[0010] 1000 mg of O,O-dimethyl-S-(carboxymethyl)thiophosphate and 25 mL of dichloromethane were placed in a 50 mL round-bottom flask and stirred until homogeneous. 690.5 mg of N-hydroxysuccinimide (NHS) and 1150.2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were added, and the mixture was stirred at room temperature (20-25°C) for 3 hours. 600 mg of 4-aminobutyric acid solid powder was added, and the mixture was stirred at room temperature (20-25°C) for 12 hours. Column chromatography was performed using silica gel, with a mixture of petroleum ether and ethyl acetate (volume ratio 1:2) as the eluent. The main product was collected and concentrated under reduced pressure to obtain approximately 990 mg of carboxyl-modified omethoate hapten (Formula I) in oil.

[0011] The synthetic route for preparing the above-mentioned omethoate hapten is as follows:

[0012]

[0013] A third objective of this invention is to provide an omethoate antigen, obtained by conjugating the compound of formula I with a carrier protein.

[0014] Further, the carrier protein is selected from at least one of bovine thyroglobulin, bovine serum albumin, rabbit serum albumin, human serum albumin, and ovalbumin. Preferably, it is bovine serum albumin (BSA) or ovalbumin (OVA).

[0015] The molar ratio of the omethoate hapten (Formula I) to the carrier protein is 8.36:1.

[0016] In one specific embodiment of the present invention, the omethoate antigen is prepared by a method comprising the following steps:

[0017] (1) Dissolve the omethoate hapten described in Formula I in dimethylformamide (DMF), then add a carboxyl activator to activate it, and stir magnetically at 20-25℃ for 2-3 hours to obtain solution A;

[0018] The activator is a combination of EDC and NHS. Further, the ratio of omethoate hapten, DMF, EDC, and NHS in Formula I is 15.5 mg: 1.5 mL: 23.4 mg: 9.3 mg.

[0019] (2) Place the carrier protein in 0.1-0.2M sodium bicarbonate buffer and stir until fully dissolved to obtain solution B; the ratio of the carrier protein to sodium bicarbonate buffer is 33.6-50 mg: 3.5 mL;

[0020] Preferably, when the carrier protein is bovine serum albumin (BSA), the ratio of bovine serum albumin (BSA) to sodium bicarbonate buffer is 50 mg: 3.5 mL; when the carrier protein is ovalbumin (OVA), the ratio of ovalbumin (OVA) to carbonate buffer is 33.6 mg: 3.5 mL.

[0021] (3) Under conditions of 0-4℃ and stirring at 500-1000 rpm, solution A is added dropwise to solution B and stirred at 500-1000 rpm for 24 h. The mixture is then dialyzed with 10 mM phosphate buffer at 4-10℃ for 3-5 days to obtain the omethoate antigen.

[0022] Preferably, the pH of the 10mM phosphate buffer is 7.0-7.5.

[0023] The fourth objective of this invention is to provide an omethoate antibody obtained by immunizing animals with the above-mentioned omethoate antigen.

[0024] Furthermore, the antibody is selected from monoclonal antibodies, polyclonal antibodies, or antiserum.

[0025] A fifth object of the present invention is to provide any of the following uses of the omethoate hapten represented by Formula I above:

[0026] (i) Use in the preparation of omethoate antigen or omethoate antibody;

[0027] (ii) Use in the preparation of equipment / reagents for the detection of omethoate;

[0028] (iii) Other related uses in the detection of omethoate.

[0029] The equipment and / or reagents for detecting omethoate include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) kits and immunochromatographic assay cards.

[0030] The sixth objective of this invention is to provide a method for detecting omethoate using the aforementioned omethoate antibody.

[0031] Furthermore, the detection methods include enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, time-resolved immunochromatography, chemiluminescent immunoassay, and suspension array method.

[0032] The omethoate hapten and the omethoate antigen provided by this invention have a simple synthesis method, high purity, and high yield, and are of great value for the preparation of omethoate antibodies and the detection of omethoate drug residues. Attached Figure Description

[0033] Figure 1 This is the mass spectrum of the omethoate hapten shown in Formula I obtained in Example 1.

[0034] Figure 2 The image shown is a MALDI-TOF-MS image of BSA in Example 2.

[0035] Figure 3 The image shows the MALDI-TOF-MS image of the omethoate-BSA complex in Example 2. Detailed Implementation

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] Example 1: Preparation of Omethoate Hapten

[0039] 1389.4 μL of mercaptoacetic acid was added to a 50 mL flask, followed by 25 mL of dichloromethane. The mixture was stirred until dissolved and then cooled to 0-4 °C in an ice bath. 2782 μL of triethylamine was added, and 2156.5 μL of O,O-dimethylphosphoryl chloride was added dropwise. The mixture was stirred at 0-4 °C for 3 hours. The salt was removed by filtration, and the filtrate was concentrated under reduced pressure. The filtrate was dissolved in 20 mL of dichloromethane, and 6000 mg of 100-200 mesh silica gel was added and mixed. The mixture was then packed into a 200-300 mesh silica gel column for chromatography. The elution was performed using petroleum ether:ethyl acetate = 1:1. The main product was collected and concentrated under reduced pressure to obtain approximately 2800 mg of O,O-dimethyl-S-(carboxymethyl)thiophosphate oil.

[0040] 1000 mg of O,O-dimethyl-S-(carboxymethyl)thiophosphate and 25 mL of dichloromethane were added to a 50 mL flask and stirred until homogeneous. Then, 690.5 mg of N-hydroxysuccinimide (NHS) and 1150.2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were added. After stirring at room temperature for 3 hours, 600 mg of 4-aminobutyric acid solid powder was added and stirred at room temperature for 12 hours. 5000 mg of 100-200 mesh silica gel was added and mixed. The sample was then packed into a 200-300 mesh silica gel column for chromatography. Elution was performed using petroleum ether:ethyl acetate = 1:2. The main product was collected and concentrated under reduced pressure to obtain approximately 990 mg of carboxyl-modified omethoate hapten.

[0041]

[0042] Figure 1 The mass spectrum of the omethoate hapten shown in Formula I obtained in Example 1 shows that its molecular weight is correct.

[0043] Example 2: Preparation and structural identification of omethoate artificial antigen

[0044] 1. Synthesis of immunogens

[0045] (1) Dissolve 15.5 mg of omethoate hapten in 1.5 mL of DMF, stir at 200 rpm for 10 min, add 23.4 mg of EDC to dissolve, then add 9.3 mg of NHS, and activate at room temperature for 2-3 h.

[0046] (2) Weigh 50 mg BSA and dissolve it in 3.5 mL of 0.1 M sodium bicarbonate solution. Stir at 200 rpm for 10 min to fully dissolve it. Cool it in an ice bath at 0-4 °C. Add the reaction solution from step 1 dropwise (1 mL / min) while stirring at 1000 rpm. Stir at 500 rpm for 24 h.

[0047] (3) The reaction product was placed in a dialysis bag rinsed with distilled water, and dialyzed for 3 days with 1L 0.01M PBS (1×, pH 7.2) at 4℃ and stirred (100rpm). The solution was changed 3 times a day. The dialysis product was centrifuged at 5000rpm for 6min, aliquoted, and stored at -20℃ for later use.

[0048] 2. Synthesis of coating antigens

[0049] (1) Dissolve 15.5 mg of omethoate hapten in 1.5 mL of DMF, stir at 200 rpm for 10 min, add 23.4 mg of EDC to dissolve, then add 9.3 mg of NHS and activate at room temperature for 2-3 h.

[0050] (2) Weigh 33.6 mg of OVA and dissolve it in 3.5 mL of 0.1 M sodium bicarbonate solution. Stir at 200 rpm for 10 min to ensure complete dissolution. Cool the solution in an ice bath to 0-4 °C. Add the reaction solution from step 1 dropwise (1 mL / min) while stirring at 1000 rpm. Stir at 500 rpm for 24 h.

[0051] (3) The reaction product was placed in a dialysis bag rinsed with distilled water, and dialyzed for 3 days with 1L 0.01M PBS (1×, pH 7.2) at 4℃ and stirred (100rpm). The solution was changed 3 times a day. The dialysis product was centrifuged at 5000rpm for 6min, aliquoted, and stored at -20℃ for later use.

[0052] 3. Identification of antigens

[0053] (1) The concentrations of the synthesized immunogen and coating agent were determined by ultraviolet absorption method. The results for the immunogen and coating agent were 2.59 mg / mL and 2.32 mg / mL, respectively.

[0054] (2) The immunogen was identified using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). The identification results were obtained by... Figure 2 (Results of BSA carrier protein MALDI-TOF detection) and Figure 3 (Results of immunogenic omethoate-BSA MALDI-TOF detection) are shown; the calculated molar ratio of omethoate hapten (Formula I) to bovine serum albumin (BSA) is: R = (67155.818 - 64771.297) / 285.25 = 8.36.

[0055] Example 3: Preparation of monoclonal antibodies by immunizing animals with omethoate artificial antigen

[0056] The specific steps for preparing omethoate monoclonal antibody using conventional methods are as follows:

[0057] I. Animal Immunization

[0058] The immunogen prepared in Example 2 was dissolved in physiological saline at a dose of 100 μg / mouse and mixed with an equal volume of Freund's complete adjuvant. The mixture was then subcutaneously injected into the neck and back of 6-8 week old Balb / c female mice. On days 7, 14, and 28 after the initial immunization, an additional immunization was administered by mixing an equal volume of the immunogen with Freund's incomplete adjuvant. Three days before fusion, an additional immunization was administered by 100 μg / mouse of the immune complex without Freund's adjuvant.

[0059] II. Cell Fusion and Cloning

[0060] Following standard procedures, spleen cells from immunized mice were mixed with myeloma cells (SP2 / 0) in the logarithmic growth phase. Preheated fusion agent (PEG4000) was then slowly added over 45 seconds for fusion. The cells were then suspended in HAT medium and homogenized. An appropriate amount of feeder cells was added, and the mixture was cultured in 96-well plates at 37°C in a 5% CO2 incubator. After 5 days, the medium was partially replaced with HT medium, and after 9 days, the medium was completely replaced.

[0061] After cell fusion, when the cells reached 1 / 4 of the culture well area, a stepwise screening method was used to screen hybridoma cells. Initial selection used indirect ELISA, screening 834 positive wells. The selected positive wells were then further screened using indirect competitive ELISA. Wells with strong positive results and high inhibition rates were selected for subclonal screening. After multiple subclonal screenings, the positive rate exceeded 100%, ultimately yielding multiple hybridoma cell lines that secreted omethoate monoclonal antibodies. Among them, 9C2 showed the highest antibody titer and the best inhibition. 50 It was 0.056 ng / mL.

[0062] Omethoate monoclonal antibody was prepared using an in vivo ascites induction method in mice. 8–10-week-old Balb / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin per mouse, and 7–10 days later, hybridoma cells were intraperitoneally injected with 1–2 × 10⁻⁶ cells. 5 Ascites fluid was collected from each mouse 7–10 days later. The fluid was centrifuged at 5000 rpm for 10 min at 4°C to remove the upper oil layer and the lower precipitate. The omethoate monoclonal antibody was then purified using the caprylic acid-saturated ammonium sulfate method and stored at -20°C. The concentration of the purified omethoate monoclonal antibody was determined to be 4.22 mg / mL.

[0063] The cross-reactivity of the monoclonal antibody prepared in this invention to omethoate structural / functional analogues was tested using an indirect competitive ELISA method, including: omethoate, methyl parathion, parathion, phosphamidon, phorate, and malathion. The results are shown in Table 2. Cross-reactivity rate (%) = IC50 50(氧乐果) / IC 50(类似物) ×100%.

[0064] Table 1. Results of cross-reactivity test of omethoate monoclonal antibody

[0065] name Cross-reactivity rate (%) Omethoate 100 Lego 37.5 Long-lasting phosphorus 21.59 Phosphamide 7.14 Malathion 6.19 Methyl parathion <1 parathion <1

[0066] As shown in Table 2, the cross-reactivity rates of the antibody obtained from the omethoate hapten provided in this invention against dimethoate, phosmet, phosphamidon, and malathion were 37.5%, 21.59%, 7.14%, and 6.19%, respectively, while the cross-reactivity rates against methyl parathion and parathion were relatively low. This indicates that the antibody has high specificity for omethoate and also has some recognition ability against other organophosphorus compounds with similar structures, although the degree of recognition varies. This finding is of great significance for further research and development of detection methods and technologies for organophosphorus compounds, especially in the fields of pesticide residue detection and environmental monitoring, where it may have potential applications.

[0067] Example 4: Establishment of an enzyme-linked immunosorbent assay (ELISA) method using omethoate.

[0068] The optimal dosage of the coating antigen and omethoate monoclonal antibody, as well as the most suitable coating solution, were determined using a checkerboard method. The specific procedure is as follows:

[0069] 1) Antigen coating: The omethoate antigen was diluted to a series of concentrations (1:8000, 1:16000, 1:32000, 1:64000 and 1:128000) using coating buffer, and 100 μL of each was added to a 96-well plate. The plates were incubated overnight at 4°C. The coating buffer was 0.05 M carbonate buffer (pH 9.6).

[0070] 2) Washing: Pour out the liquid in the wells, wash once with washing solution (PBST), 280 μL / well, and pat dry on absorbent paper.

[0071] 3) Blocking: Add 150 μL of blocking solution per well, incubate at 37°C for 2 hours, and then remove and pat dry.

[0072] 4) Sample addition: Add 50 μL / well of a series of concentrations of omethoate standard (0 μg / L, 0.05 μg / L, 0.15 μg / L, 0.45 μg / L, 1.35 μg / L, 4.05 μg / L), then add 50 μL / well of appropriately diluted omethoate monoclonal antibody and react at 25℃ for 30 min. The dilution gradient of the tested omethoate monoclonal antibody includes: 1:10000, 1:20000, 1:40000, 1:80000 and 1:160000.

[0073] 5) Washing: Pour out the liquid in the wells, wash 3 times with washing solution (PBST), 280 μL / well, and pat dry on absorbent paper.

[0074] 6) Add horseradish peroxidase-labeled secondary antibody: Add 100 μL / well of HRP-goat anti-mouse IgG (1:5000 dilution) and react at 25℃ for 30 min.

[0075] 7) Washing: Pour out the liquid in the well, wash 3 times with washing solution, 280μL / well, and pat dry on absorbent paper.

[0076] 8) Color development: Add freshly prepared TMB substrate solution, 100 μL / well, and react at 37°C in the dark for 10 min.

[0077] 9) Termination: Add 2 mol / L H2SO4 as the termination solution, 50 μL / well.

[0078] 10) Measurement: Read the OD of each well using a microplate reader. 450 nm Value (dual wavelength: 630nm is the reference filter wavelength).

[0079] result The optimal dilution of omethoate coating was 1:32000, and the optimal dilution of omethoate monoclonal antibody was 1:40000, as determined by the checkerboard method.

[0080] Example 5: Application of omethoate enzyme-linked immunosorbent assay (ELISA)

[0081] 1. Sample pretreatment

[0082] Weigh 2.00±0.05g of chopped cowpeas, romaine lettuce, and eggplant samples into a 50mL graduated centrifuge tube; add 3mL of sample diluent, invert and mix (50 times / min), wash the sample thoroughly for 2min, and let stand for 1min to obtain the test solution. The sample diluent consists of 9g sodium chloride, 4.50g disodium hydrogen phosphate dodecahydrate, and 0.40g sodium dihydrogen phosphate dihydrate, dissolved in water and diluted to a final volume of 1000mL.

[0083] 2. Testing Steps

[0084] 1) Add 50 μL of omethoate standard working solution of various concentrations (0 μg / L, 0.05 μg / L, 0.15 μg / L, 0.45 μg / L, 1.35 μg / L, 4.05 μg / L) or the sample solution to each well of the kit coated using the method in Example 4, and then add 50 μL of omethoate monoclonal antibody. Shake to mix and react at 25°C for 30 min. Record the position of each standard and sample, and perform duplicate wells.

[0085] 2) Discard the liquid in the wells, wash 3 times with washing solution (PBST), 280 μL / well, and pat dry on absorbent paper.

[0086] 3) Add 100 μL of HRP-goat anti-mouse IgG (1:5000 dilution) to each well and react at 25°C for 30 min.

[0087] 4) Repeat step 2.

[0088] 5) Immediately add freshly prepared TMB substrate solution, 100 μL / well, and react at 37°C in the dark for 10 min.

[0089] 6) Add stop solution, 50 μL / well.

[0090] 7) Read the OD of each well using a microplate reader within 5 minutes after termination. 450 nm Value (dual wavelength: 630nm is the reference filter wavelength).

[0091] 8) Result calculation or judgment

[0092] Calculate the percentage absorbance value: Divide the average absorbance value of each standard (or the sample to be tested) by the absorbance value of the zero standard (the standard with a concentration of 0 μg / L), and multiply by 100% to obtain the percentage of absorbance corresponding to each standard, i.e., the percentage absorbance value.

[0093] Constructing a standard curve: Plot a standard curve with the percentage absorbance of each standard on the ordinate and the corresponding omethoate concentration on the abscissa.

[0094] To calculate the omethoate content in a sample: Substitute the percentage absorbance value of the sample to be tested into the standard curve equation to obtain the concentration of the sample to be tested. Then multiply it by the dilution factor of the corresponding sample to obtain the actual omethoate content in the original sample to be tested.

[0095] Example 6: Evaluation and application of the omethoate enzyme-linked immunosorbent assay (ELISA) method

[0096] 1. Determination of the lowest detection limit

[0097] Blank samples of cowpeas, romaine lettuce, and eggplant were tested. The measured values ​​were obtained according to the standard curve, and the average value was calculated. The value was then added to three times the standard deviation to obtain the limit of detection (LOD).

[0098] Table 2. Statistical table of blank sample determination results (μg / kg)

[0099]

[0100] The results are shown in Table 3. To prevent false positives, the detection limit for omethoate in cowpeas, romaine lettuce and eggplant in this kit is 0.5 μg / kg.

[0101] 3. Determination of method accuracy and precision

[0102] Accuracy refers to the degree of agreement between the measured value and the true value. In ELISA assays, accuracy is often expressed as recovery rate, while precision is often expressed as coefficient of variation. Blank samples of cowpea, romaine lettuce, and eggplant were added with 0.5 μg / kg and 1.0 μg / kg omethoate standards, respectively, with five replicates for each. The assays were performed using three batches of the kit, and the recovery rate and intra- and inter-batch coefficients of variation were calculated.

[0103] Table 3. Accuracy and Precision Test Results

[0104]

[0105]

[0106] The results are shown in Table 3. The recoveries of the spiked concentrations of cowpea, romaine lettuce, and eggplant samples ranged from 87.73% to 113.34%, with intra-batch coefficients of variation less than 10% and inter-batch coefficients of variation less than 15%.

[0107] 4. Actual sample determination using omethoate enzyme-linked immunosorbent assay (ELISA).

[0108] Ten vegetable samples were analyzed using ELISA and UPLC-MS / MS respectively, as shown in Table 5. The results of ELISA and UPLC-MS / MS showed good overall consistency.

[0109] Table 5. Actual sample determination using the omethoate enzyme-linked immunosorbent assay (ELISA) method.

[0110]

Claims

1. An omethoate hapten, characterized in that, The structure is shown in Equation I: I。 2. The method for preparing the omethoate hapten of Formula I as described in claim 1, characterized in that, The synthesis route is as follows: 。 3. The preparation method according to claim 2, characterized in that, Includes the following steps: 1389.4 μL of mercaptoacetic acid was placed in a 50 mL round-bottom flask, and 25 mL of dichloromethane was added. After stirring until dissolved, the mixture was cooled to 0-4 °C in an ice bath. 2782 μL of triethylamine was added, and 2156.5 μL of O,O-dimethylphosphoryl chloride was added dropwise. The mixture was stirred at 0-4 °C for 3 h. The mixture was filtered to remove salts, and the filtrate was concentrated under reduced pressure. 20 mL of dichloromethane was added to dissolve the solid. Column chromatography was performed using silica gel, with a 1:1 volume ratio of petroleum ether and ethyl acetate as the eluent. The main product was collected and concentrated under reduced pressure to obtain approximately 2800 mg of O,O-dimethyl-S-(carboxymethyl)thiophosphate oil. 1000 mg of O,O-dimethyl-S-(carboxymethyl)thiophosphate and 25 mL of dichloromethane were placed in a 50 mL round-bottom flask and stirred until homogeneous. 690.5 mg of NHS and 1150.2 mg of EDC were added, and the mixture was stirred at 20-25 °C for 3 h. 600 mg of 4-aminobutyric acid solid powder was added, and the mixture was stirred at 20-25 °C for 12 h. Column chromatography was performed using silica gel with a 1:2 volume ratio of petroleum ether and ethyl acetate as the eluent. The main product was collected and concentrated under reduced pressure to obtain approximately 990 mg of the omethoate hapten oil of Formula I.

4. An omethoate antigen, characterized in that, It is obtained by coupling the compound of formula I as described in claim 1 with a carrier protein.

5. The omethoate antigen according to claim 4, characterized in that... The carrier protein is selected from at least one of bovine thyroglobulin, bovine serum albumin, rabbit serum albumin, human serum albumin, and ovalbumin; The molar ratio of the omethoate hapten of Formula I to the carrier protein is 8.36:

1.

6. An omethoate antibody, characterized in that, Obtained by immunizing animals with the omethoate antigen as described in claim 4 or 5.

7. The omethoate antibody according to claim 6, characterized in that, The antibody is selected from monoclonal antibodies, polyclonal antibodies, or antiserum.

8. Any of the following applications of the omethoate hapten of Formula I as described in claim 1: (i) Use in the preparation of omethoate antigen or omethoate antibody; (ii) Use in the preparation of equipment or reagents for the detection of omethoate; (iii) Other related uses in the detection of omethoate.

9. A method for detecting omethoate, using the omethoate antibody as described in claim 6 or 7.

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