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 detection test strip developed by the invention on omethoate in cowpea, leaf lettuce and eggplant is 0.5 mu g / kg, and the false positive rate and the false negative rate are both 0.

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, thus easily leaving residues in vegetables, fruits, and other agricultural products.

[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 dissolved in 25 mL of dichloromethane and stirred until dissolved. The mixture was then cooled to 0–4 °C in an ice bath, and 2782 μL of triethylamine was added. 2156.5 μL of O,O-dimethylphosphoryl chloride was added dropwise, and 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 and dissolved in dichloromethane. Column chromatography was performed using silica gel, with a 1:1 mixture of petroleum ether and ethyl acetate as the eluent. The major product was collected and concentrated under reduced pressure to give 2800 mg of O,O-dimethyl-S-(carboxymethyl)thiophosphate oil.

[0010] 1000 mg of O,O-dimethyl-S-(carboxymethyl)thiophosphate was added to 25 ml of dichloromethane 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 (20-25°C) for 3 hours, 756 mg of 4-aminomethylbenzoic acid solid powder was added and stirred at room temperature for 12 hours. Column chromatography was performed using silica gel, eluting with a mixture of petroleum ether and ethyl acetate (v / v 1:1.5). The main product was collected and concentrated under reduced pressure to obtain 1160 mg of benzoic acid-modified omethoate hapten (Formula I) in oil.

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

[0012]

[0013] The third objective of this invention is to provide an omethoate antigen, which is 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 9.87:1.

[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 17.3 mg: 1.5 mL: 26.9 mg: 10.8 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. 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 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. Elution was performed using petroleum ether:ethyl acetate = 1:1. The main product was collected and concentrated under reduced pressure to obtain 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, 756 mg of 4-aminomethylbenzoic acid solid powder was added and stirred at room temperature for 12 hours. 7000 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 = 1:1.5. The main product was collected and concentrated under reduced pressure to obtain 1160 mg of benzoic acid-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 17.3 mg of omethoate hapten in 1.5 mL of DMF, stir at 200 rpm for 10 min, add 26.9 mg of EDC to dissolve, then add 10.8 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 17.3 mg of omethoate hapten in 1.5 mL of DMF, stir at 200 rpm for 10 min, add 26.9 mg of EDC to dissolve, then add 10.8 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 4.53 mg / mL and 4.85 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 = (68059.841 - 64771.297) / 333.29 = 9.87.

[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, the cell line with the highest antibody titer and the best inhibition was 6H8, with an IC50 value of [missing value]. 50 It was 0.047 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 10.39 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 methyl parathion, parathion, dimethoate, phosphamidon, phosphamidon, and malathion. The results are shown in Table 2. Cross-reactivity (%) = IC50 50(氧乐果) / IC 50(类似物) ×100%.

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

[0065]

[0066] As shown in Table 2, the cross-reactivity of the antibody obtained from the omethoate hapten provided by this invention with methyl parathion, parathion, dimethoate, phosphamidon, phosphamidon, and malathion is all <10%. This indicates that the antibody has high specificity for omethoate, while its recognition ability for other organophosphorus compounds is relatively low. This means that the antibody is not easily interfered with by these compounds when detecting omethoate, thereby improving the accuracy and reliability of the detection.

[0067] Example 4: Establishment of a rapid detection method for omethoate colloidal gold

[0068] 1. Preparation of colloidal gold solution

[0069] Colloidal gold solution was prepared by the trisodium citrate reduction method. 100 mL of pure water was added to a round-bottom flask and heated to boiling. Then, 2.0 mL of chloroauric acid (2%) was added. After the solution boiled again, 1.0 mL of trisodium citrate solution (1%) was added. The mixture was stirred and heated for 10 min, then allowed to cool naturally to room temperature. The solution was then restored to its original volume with distilled water and stored at 4°C for later use.

[0070] 2. Preparation of colloidal gold probes

[0071] Adjust the pH of 1 mL colloidal gold solution by adding 20 μL of 0.1 M K₂CO₃. Add 80 μL of omethoate monoclonal antibody solution diluted 5000 times with pure water to the colloidal gold solution, invert to mix, and let stand for 10 min for labeling. Add 100 μL of BSA solution (10%), mix well, and let stand for 10 min for blocking. Centrifuge at 10000 rpm for 10 min at 4 °C, discard the supernatant, resuspend the precipitate in a resuspending solution to 200 μL, sonicate to mix the reconstitution solution, and store at 4 °C for later use. The reconstitution solution is 0.01 M Tris-HCl (pH 8.5), 10% sucrose, 1% BSA, and 0.5% PVP-40.

[0072] 3. Preparation of the coating stock solution

[0073] Omethoate was coated with 20,000, 40,000, 80,000, and 160,000 dilutions, and then sprayed onto nitrocellulose membranes at a concentration of 0.8 μL / cm. After drying at 37°C for 2 hours, the membranes were cut into test strips, and the color intensity and inhibition effect were observed. When omethoate was coated with an 80,000-fold dilution, the color development was clear, and the sensitivity was also good.

[0074] 4. Preparation of Omethoate Colloidal Gold Test Strips

[0075] The suspension obtained in step 2 was sprayed into micropores to form gold-labeled micropores. The omethoate-coated stock solution prepared in step 3 was sprayed onto the T-line of the nitrocellulose membrane (NC membrane), and goat anti-mouse antibody was sprayed onto the C-line. The sample pad, NC membrane, and absorbent paper were assembled and cut into strips using standard methods.

[0076] Example 5: Application of the rapid detection method for omethoate colloidal gold

[0077] 1. Sample pretreatment

[0078] 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 (amplitude 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.

[0079] 2. Testing Steps

[0080] Add 120 μL of the test solution from step 1 of this embodiment to the gold label microwell. Gently blow with a dropper for 30 seconds to completely dissolve the red substance at the bottom of the gold label microwell. Let it stand horizontally and wait for the reaction to proceed for 2 minutes. Then, aspirate the red solution from the gold label microwell and gently blow for 10 seconds. Insert the test strip into the gold label microwell to fully immerse the sample pad in the liquid. Read the results after 5-8 minutes. Results obtained at other times are invalid.

[0081] Result judgment criteria:

[0082] Negative: The T line is darker than or the same as the C line, indicating that the concentration of omethoate pesticide residue in the sample is below the detection limit or there is no omethoate pesticide residue.

[0083] Positive: The T line is lighter than the C line, indicating that the concentration of omethoate pesticide residue in the sample is higher than the detection limit; the lighter the T line is than the C line, the higher the concentration of omethoate pesticide residue in the sample.

[0084] Invalid: If line C does not develop color, the test strip is considered invalid regardless of whether line T develops color.

[0085] Example 6: Evaluation and application of the rapid detection method for omethoate colloidal gold

[0086] 1. Determination of the lowest detection limit

[0087] Samples containing omethoate were tested using test strips from three different batches. Three concentrations were set for each batch, with 60 samples of each concentration tested using the test strips.

[0088] Table 2. Detection Limit of Omethoate Colloidal Gold Test Strip

[0089]

[0090]

[0091] As shown in Table 2, samples of cowpea, romaine lettuce, and eggplant with omethoate standard solution added at 0.25 μg / kg were all negative; samples of cowpea, romaine lettuce, and eggplant with omethoate standard solution added at 0.5 μg / kg and 1.0 μg / kg were all positive. The results indicate that the detection limit of this test strip for omethoate in cowpea, romaine lettuce, and eggplant is 0.5 μg / kg.

[0092] 2. False positive rate and false negative rate test

[0093] Three batches of test strips were used to test negative samples (60 samples of each type). Samples with positive results were tested again and retested using instrumental methods. The number of false positives was counted, and the false positive rate was calculated. The false positive rate was calculated using the following formula: False positive rate (%) = Number of positive samples detected by test strips / Total number of negative samples × 100%.

[0094] Three batches of test strips were used to test positive samples with a spiking concentration of LOD (60 samples of each type). Suspected negative samples were tested again and retested using instrumental methods. The number of false negatives was counted, and the false negative rate was calculated. The false negative rate was calculated using the following formula: False negative rate (%) = (Number of negative samples detected by test strips / Total number of positive samples) × 100%.

[0095] The results are shown in Table 3. In the verification of false positive and false negative rates, the omethoate test strips showed a result of 0%.

[0096] Table 3. Results of False Negative and False Positive Rates of Test Strips

[0097]

[0098]

[0099] 4. Sample determination

[0100] Blind samples (20 of each type) were tested and measured using test strips after pretreatment.

[0101] The blind sample consists of negative and positive samples at different concentration levels. The negative sample is the sample that has been confirmed as negative by the reference method, and the positive sample is the negative sample added to the specified concentration. The content of the drug to be tested in the sample is confirmed by the reference method, which is LC-MS / MS.

[0102] Table 4. Results of omethoate sample determination (μg / kg)

[0103]

[0104] In the blind sample testing capability validation of cowpeas, romaine lettuce, and eggplant, all blind samples with omethoate content of 0.5 μg / kg, 1.0 μg / kg, and 3.0 μg / kg were positive (detection rate 100%). The test strips correctly identified all omethoate-negative samples. Therefore, the rapid omethoate test strip has good accuracy and can be used for the detection of omethoate in vegetables.

Claims

1. An omethoate hapten, characterized in that, The structure is shown in Equation 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 was added to 25 mL of dichloromethane and stirred until homogeneous. Then, 690.5 mg of NHS and 1150.2 mg of EDC were added. The mixture was stirred at 20-25 °C for 3 h. Then, 756 mg of 4-aminomethylbenzoic acid solid powder was added and stirred at 20-25 °C for 12 h. Column chromatography was performed using silica gel, eluting with a mixture of petroleum ether and ethyl acetate at a volume ratio of 1:1.

5. The main product was collected and concentrated under reduced pressure to obtain 1160 mg of benzoic acid-modified omethoate hapten oil as shown in 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 9.87:

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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