Chlorantraniliprole hapten, complete antigen and application of chlorantraniliprole hapten and complete antigen

Abstract

The invention relates to a chlorantraniliprole hapten, a complete antigen and application of the chlorantraniliprole hapten and the complete antigen. When the chlorantraniliprole artificial antigen and the monoclonal antibody are used for ELISA detection, the specificity is high, the IC50 value is 0.058 [mu] g / L, the detection limit of chlorantraniliprole in a standard solution reaches 0.6 [mu] g / L, and the detection limit of a sample to be detected reaches 2 g / kg; the cross reaction rates with cyantraniliprole, tetrachlorantraniliprole and cyclaniliprole are respectively 20%, 12% and 12%, and the material specificity is greatly improved. By utilizing the chlorantraniliprole hapten, the complete antigen and the antibody, the purpose of rapidly and accurately detecting chlorantraniliprole in a sample can be achieved.

Description

Technical Field

[0001] This application relates to the field of food safety testing technology, and in particular to a chlorantraniliprole hapten, a complete antigen, an antibody, and their synthesis and application. Background Technology

[0002] Chlorantraniliprole (CAP), developed by DuPont in 2008, is an o-aminophthalamide insecticide used to control lepidopteran insects. Within just eight years, it became a best-selling insecticide, with sales reaching $1.3 billion. As a novel broad-spectrum insecticide, it effectively activates ryanodine receptors in lepidopteran pests, opening calcium ion channels and causing excessive calcium loss from cells, thereby disrupting the physiological balance of the pests and leading to their death. Chlorantraniliprole has shown good efficacy in controlling pests and diseases in fruits and vegetables due to its good persistence, low toxicity, and high efficiency.

[0003] Given the widespread use of chlorantraniliprole in edible agricultural products, the National Food Safety Standard for Maximum Residue Limits of Pesticides in Food (GB 2763—2021) stipulates that the provisional maximum residue limits for chlorantraniliprole in fruits and vegetables are 0.01–30 mg / kg. Fruits and vegetables are a major component of the diet of Chinese residents, and chlorantraniliprole residues in them can accumulate in organisms through the food chain, posing a threat to human health. Studies have shown that chlorantraniliprole has been proven to promote obesity and heart problems by affecting key obesity genes and disrupting protein secondary structure. Therefore, detecting chlorantraniliprole residues in food is crucial.

[0004] Currently, methods for detecting chlorantraniliprole include gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), and immunoassay. GC-MS and LC-MS / MS methods are sensitive and accurate, but require expensive equipment and time-consuming sample pretreatment.

[0005] Immunoassay has become an important technique for pesticide residue detection due to its simplicity, low cost, and high specificity. Its core lies in the preparation of specific antibodies, which relies on the design of highly efficient haptens. Currently, there are few reports on the preparation of monoclonal antibodies against chlorantraniliprole, and the antibodies do exhibit poor specificity, with cross-contamination rates exceeding 60%. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a chlorantraniliprole hapten, a complete antigen, and their applications.

[0007] A novel chlorantraniliprole hapten has been developed. By optimizing the hapten design to prepare complete antigens and specific antibodies, the specificity and sensitivity of detection can be improved, which is currently in demand. Based on this, a high-performance immunoassay device can be developed to improve practicality, detection efficiency, and accuracy.

[0008] The first objective of this invention is to provide a chlorantraniliprole hapten.

[0009] A second object of the present invention is to provide a method for preparing a compound with the structure shown in formula (I).

[0010] A third objective of this invention is to provide the use of the chlorantraniliprole hapten in the preparation of chlorantraniliprole complete antigen.

[0011] The fourth objective of this invention is to provide a complete chlorantraniliprole antigen.

[0012] The fifth objective of this invention is to provide another complete chlorantraniliprole antigen.

[0013] A sixth object of the present invention is to provide a composition of an immunogen and a coating antigen.

[0014] The seventh object of the present invention is to provide a method for immunoassay of chlorantraniliprole.

[0015] The eighth object of the present invention is to provide a product for the immunoassay of chlorantraniliprole.

[0016] A ninth object of the present invention is to provide the use of the chlorantraniliprole hapten, the chlorantraniliprole complete antigen, the other chlorantraniliprole complete antigen, and / or the composition in the preparation of products for immunodetection of chlorantraniliprole or in the establishment of methods for immunodetection of chlorantraniliprole.

[0017] To achieve the above objectives, the present invention is implemented through the following technical solution: This claim seeks to protect a chlorantraniliprole hapten, the structure of which is shown in formula (I): .

[0018] The hapten prepared in this invention retains the three core structures of chlorantraniliprole: the 3-bromopyrazole ring, the 3-chloropyridine ring, and the 2-amino-5-chloro-N,3-dimethylbenzamide ring. Furthermore, the designed coupling arm is a straight chain with an active group, ensuring that the small molecule structure of the prepared artificial antigen retains its original characteristics. This is more conducive to presenting the structural features of chlorantraniliprole drugs and improves the immunogenicity of the antigen. Furthermore, it can be coupled beforehand using a carrier protein modified with γ-aminobutyric acid or directly to the protein.

[0019] Its functionality is achieved through the following features: (1) Ortho-position configuration optimization: This invention provides a longer spacer arm by pre-coupling the carrier protein modified with γ-aminobutyric acid with the hapten, avoiding the shielding of the chlorantraniliprole core structure. Furthermore, the arm of this invention is located in the middle of the chlorantraniliprole structure, significantly exposing the core structure of chlorantraniliprole (2-amino-5-chloro-N,3-dimethylbenzamide, 3-bromopyrazole ring, and its 3-chloropyridine ring) while ensuring the stability of the chlorantraniliprole hapten structure and ensuring that the electron cloud distribution of the chlorantraniliprole hapten structure is similar to that of the chlorantraniliprole pesticide structure. This design introduces an amide bond at the arm to prevent the long spacer arm from causing folding of the hapten molecule configuration, allowing the hapten molecule to be close to the carrier protein surface and thus shielded, thereby improving the immune recognition efficiency of the antigen epitope.

[0020] (2) Spacer arm length and carboxyl modification: This invention provides higher reactivity and more efficient coupling with carrier proteins by incorporating short chain arms. By using different methods of protein coupling, a complete antigen with a long chain arm can be obtained for immunization, better exposing the group, or a complete antigen with a short chain arm can be obtained for detection, appropriately creating structural shielding and obtaining better detection sensitivity.

[0021] This invention exposes the core structure of chlorantraniliprole and enhances the efficiency of the immune response by optimizing its spatial configuration and chemical properties. This design not only simplifies the synthesis pathway of the hapten but also significantly improves the specificity and sensitivity of the antibody while maintaining chemical stability, laying the foundation for improved performance in immunoassays.

[0022] Further claims are made for a method for preparing a compound with the structure shown in formula (I), the method comprising the following steps: S1. Aromatic amide compound a and haloester compound b are subjected to a substitution reaction under alkaline conditions to obtain the first intermediate product compound; S2. The first intermediate compound c is reacted with heterocyclic aromatic carboxylic acid compound d in an organic solvent to give the second intermediate compound e; S3. Hydrolyze the second intermediate compound e in an alkaline solution, adjust the pH to precipitate a solid, and obtain chlorantraniliprole hapten.

[0023] Preferably, S1. Aromatic amide compound a (2-amino-5-chloro-N,3-dimethylbenzamide, CAS: 890707-28-5) and haloester compound b (ethyl bromoacetate, CAS: 105-36-2) are subjected to a substitution reaction under alkaline conditions to obtain the first intermediate compound c; S2. The first intermediate compound c is reacted with a heterocyclic aromatic carboxylic acid compound d (3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid, CAS: 500011-86-9) in an organic solvent to give the second intermediate compound e; S3. The second intermediate compound e is hydrolyzed in an alkaline solution, and the pH is adjusted to precipitate a solid, yielding chlorantraniliprole hapten.

[0024] More preferably, the substitution reaction described in step S1 is carried out in a polar aprotic solvent.

[0025] More preferably, the polar aprotic solvent is N,N-dimethylformamide (DMF).

[0026] More preferably, in step S1, the ratio of compound a to compound b is (700-900) mg : (1500-3000) mg.

[0027] More preferably, in step S1, the ratio of compound a to compound b is 800 mg : 2698 mg.

[0028] More preferably, the alkaline conditions described in step S1 are pH = 8.5 to 10.5.

[0029] More preferably, the alkaline condition described in step S1 is pH = 9.

[0030] Optionally, the alkaline conditions described in step S1 can be controlled by adjusting the amount of potassium carbonate added.

[0031] More preferably, in step S2, the ratio of compound c to compound d is (200-500) mg : (230-638) mg.

[0032] More preferably, in step S2, the ratio of compound c to compound d is 300 mg : 480 mg.

[0033] More preferably, the amide reaction in step S2 is carried out in a nonpolar to moderately polar solvent environment.

[0034] More preferably, the nonpolar to moderately polar solvent environment is provided by a mixed solvent of toluene and thionyl chloride.

[0035] More preferably, the alkaline solution in step S3 is an aqueous solution of lithium hydroxide.

[0036] More preferably, the alkaline solution in step S3 is a 4-8 mol / L lithium hydroxide aqueous solution (most preferably 6 mol / L).

[0037] More preferably, in step S3, adjusting the pH to precipitate solids involves adjusting the pH to 4-5.

[0038] As an alternative implementation, the method for preparing the above-mentioned chlorantraniliprole hapten includes the following steps: S1. Compound a was thoroughly mixed with N,N-dimethylformamide, potassium carbonate, and compound b, and reacted at 80°C for 2 to 8 hours. After the reaction was complete, purified water was added, and the aqueous phase was extracted twice with n-hexane. The n-hexane layer was discarded, and the aqueous phase was extracted three more times with ethyl acetate. The organic phase was desolventized under reduced pressure to obtain compound c.

[0039] S2. Compound d was dissolved in a mixed solution of toluene and thionyl chloride, and compound c was added and stirred at 60°C for 4–8 h. After the reaction was complete, purified water was added, the aqueous phase was extracted three times with ethyl acetate, and the organic phase was washed with saturated NaHCO3 solution. The organic phase was desolventized under reduced pressure to obtain compound e.

[0040] S3. Dissolve compound e in methanol, then add 6 M lithium hydroxide aqueous solution, react at room temperature for 10-12 h, add purified water, and extract twice with ethyl acetate. Discard the ethyl acetate layer, adjust the pH of the aqueous phase to 4-5 with 4 M hydrochloric acid, and the solid precipitates. Filter and dry to obtain chlorantraniliprole hapten.

[0041] More preferably, in step S1, the ratio of compound a, N,N-dimethylformamide, potassium carbonate and compound b is (700-900) mg : (3.5-5.5) mL : (480-1280) mg : (1.3-5.6) mL.

[0042] Most preferably, in step S1, the ratio of compound a, N,N-dimethylformamide, potassium carbonate, and compound b is 800 mg : 4 mL : 836 mg : 2698 mg.

[0043] More preferably, the ratio of toluene to thionyl chloride in step S2 is (0.5-2):(0.5-4).

[0044] Most preferably, the ratio of toluene to thionyl chloride in step S2 is 1:1. And the application of the chlorantraniliprole hapten in the preparation of chlorantraniliprole complete antigen.

[0045] This invention also claims protection for a chlorantraniliprole complete antigen, which is formed by direct coupling of the chlorantraniliprole hapten with a carrier protein, and its structure is shown in formula (II): .

[0046] As an alternative implementation, the method for preparing the chlorantraniliprole complete antigen includes the following steps: S1. Dissolve the above chlorantraniliprole hapten in dimethylformamide (DMF), add EDC and N-hydroxysuccinimide (NHS) to react and obtain the hapten activated ester; S2. Add the hapten activated ester to the carrier protein solution; S3. Dialyze the solution obtained in step S2 to obtain the complete chlorantraniliprole antigen.

[0047] And another chlorantraniliprole complete antigen, which is formed by coupling the aforementioned chlorantraniliprole hapten with a carrier protein pre-modified with γ-aminobutyric acid, and its structure is shown in formula (III): .

[0048] As an alternative implementation, the method for preparing the chlorantraniliprole complete antigen 2 includes the following steps: S1. Gamma-aminobutyric acid is dissolved in dimethylformamide (DMF), and EDC and N-hydroxysuccinimide (NHS) are added to react and obtain activated γ-aminobutyric acid ester; S2. Add the activated γ-aminobutyric acid ester to the carrier protein solution; S3. Dialyze the solution obtained in step S2 to obtain the γ-aminobutyric acid modified carrier protein; S4. Dissolve the above chlorantraniliprole hapten in dimethylformamide (DMF), add EDC and N-hydroxysuccinimide (NHS) to react and obtain the hapten activated ester; S5. Add the hapten activated ester to the γ-aminobutyric acid modified carrier protein solution; S6. Dialyze the solution obtained in step S5 to obtain the complete chlorantraniliprole antigen.

[0049] Preferably, the carrier protein is selected from one of the following: bovine serum albumin, lactoferrin, ovalbumin, human serum albumin, and hemocyanin.

[0050] The present invention also claims a composition of an immunogen and a coating antigen, comprising chlorantraniliprole complete antigen as shown in formula (II) as a coating antigen and chlorantraniliprole complete antigen as shown in formula (III) as an immunogen.

[0051] As a specific embodiment, the composition of an immunogen and a coating antigen contains chlorantraniliprole complete antigen with the structure shown in formula (II) as a coating antigen and chlorantraniliprole complete antigen with the structure shown in formula (III) as an immunogen, wherein the carrier protein of the coating antigen is bovine serum albumin and the carrier protein of the immunogen is lactoferrin.

[0052] A method for immunoassay of chlorantraniliprole using the aforementioned composition is also claimed.

[0053] It also claims protection for a product for the immunoassay of chlorantraniliprole, which is prepared using or contains the composition.

[0054] The product is one or more of the following, including but not limited to enzyme immunoassay, immunochromatography, immunosensing, and immunogold.

[0055] As an alternative implementation, the preparation method of the chlorantraniliprole colloidal gold immunochromatographic detection device includes the following steps: S1. Preparation of reaction membrane: Spray a solution containing the above-mentioned chlorantraniliprole complete antigen onto the detection zone (T line) of the nitrocellulose membrane (NC membrane), and spray sheep-derived immunoglobulin (sheep anti-mouse IgG) onto the control zone (C line). S2. Preparation of colloidal gold-labeled microwells: Colloidal gold was prepared by sodium citrate reduction method. The above-mentioned chlorantraniliprole monoclonal antibody was conjugated with colloidal gold, bovine serum albumin was added for blocking, the precipitate was collected by centrifugation, and the precipitate was reconstituted and dispensed into microwells. S3. Preparation of sample pads: Soak blank sample pads in buffer solution and then dry them for later use; S4. Assemble the test card: Stack the sample pad obtained in step S3, the reaction membrane obtained in step S1, and the absorbent pad on the PVC board in sequence, cut them into test strips, and insert them into the test card case.

[0056] Specifically, a chlorantraniliprole complete antigen solution of 0.05–0.4 mg / mL is sprayed in the detection area (T line), and a sheep-derived immunoglobulin (sheep anti-mouse IgG) solution of 0.05–0.2 mg / mL is sprayed in the control area (C line).

[0057] Preferably, a 0.1 mg / mL chlorantraniliprole complete antigen solution is sprayed in the detection area (T line), and a 0.1 mg / mL sheep-derived immunoglobulin (sheep anti-mouse IgG) solution is sprayed in the control area (C line).

[0058] The method of using the chlorantraniliprole colloidal gold immunochromatographic assay device of the above-mentioned optional implementation scheme includes the following steps: S1. Preparation of test solution: Add the sample to be tested to a phosphate-containing buffer solution, mix well, and let stand. Take the supernatant and mix it with the phosphate buffer solution to prepare the test solution. S2. Detection: Add the test solution to the colloidal gold-labeled microwells of the chlorantraniliprole colloidal gold immunochromatographic assay device, mix to reconstitute the colloidal gold-labeled antibody, let stand for 3–7 minutes, then transfer the solution from the colloidal gold-labeled microwells to the sample wells of the chlorantraniliprole colloidal gold immunochromatographic assay device test card; start timing after sample addition, observe the results within 5–8 minutes and determine the presence or absence of chlorantraniliprole residue based on the color development results; results after 8 minutes are invalid; the result interpretation criteria are as follows: (1) Negative (-): The T line shows stronger color than the C line or shows no significant difference in color between the two lines; (2) Positive (+): The T line is significantly weaker than the C line or the T line is not visible; (3) Invalid: No C line appears, the operation process is incorrect or the test strip has expired.

[0059] Sensitivity tests on chlorantraniliprole standard solutions and test samples showed that the chlorantraniliprole colloidal gold immunochromatographic detection device of the above-mentioned optional implementation scheme had a detection limit of 0.6 μg / L for chlorantraniliprole in the standard solution and a detection limit of up to 2 µg / kg for chlorantraniliprole in the test sample. The chlorantraniliprole colloidal gold immunochromatographic detection device of the present invention has a low detection limit for chlorantraniliprole in samples, meeting the requirements for trace detection and demonstrating excellent detection performance.

[0060] Through stability testing, the chlorantraniliprole colloidal gold immunochromatographic detection device of the above-mentioned optional implementation scheme has good stability under storage conditions. It can still accurately detect chlorantraniliprole residues after storage and can be stably stored at room temperature for more than one year, which can meet the market requirements in storage and transportation.

[0061] Finally, the use of the chlorantraniliprole hapten, the chlorantraniliprole complete antigen, the chlorantraniliprole complete antigen, and / or the composition in the preparation of products for immunodetection of chlorantraniliprole or in the establishment of methods for immunodetection of chlorantraniliprole is also within the scope of protection of this invention.

[0062] Compared with the prior art, the present invention has the following beneficial effects: (1) High specificity. This invention prepares complete antigens and antibodies based on novel chlorantraniliprole haptens. The antibodies have high affinity and their IC50 is high. 50The value was 0.058 μg / L. The colloidal gold immunochromatographic test strip prepared in this invention showed sensitivities of chlorantraniliprole, bromocyanamide, tetrachlorantraniliprole, and cyclobrombutamide in standard solutions of 0.6 µg / kg, 3.0 µg / kg, 5.0 µg / kg, and 5.0 µg / kg, respectively, with cross-reactivity rates of 100%, 20%, 12%, and 12%, respectively. The chlorantraniliprole immunoassay device constructed thus reduces nonspecific reactions and improves detection accuracy.

[0063] (2) High sensitivity. The chlorantraniliprole immunoassay device prepared based on the novel chlorantraniliprole hapten of this invention has a detection limit of 0.6 μg / L for chlorantraniliprole in standard solution and a detection limit of 2 µg / kg for test sample, which greatly improves the sensitivity and significantly enhances the detection capability of low concentration chlorantraniliprole residues.

[0064] (3) Strong anti-interference ability. Through the optimized design of the chlorantraniliprole hapten structure and the enhancement of antibody specificity, the chlorantraniliprole immunoassay device maintains stable performance under the test sample environment. The invention significantly reduces the influence of matrix effect, and the detection limit of the test sample reaches 2µg / kg, ensuring the reliability of practical application.

[0065] (4) Good stability. The chlorantraniliprole immunoassay device prepared based on the novel chlorantraniliprole hapten of this invention can be stably stored at room temperature for more than one year, and can still accurately detect chlorantraniliprole residues after storage.

[0066] (5) Fast and portable. The chlorantraniliprole immunoassay device prepared based on the novel chlorantraniliprole hapten of this invention has a detection time of only 5 to 8 minutes, requires no laboratory instrument support, and is easy to operate.

[0067] In summary, this invention significantly improves the immunoassay efficiency of antibodies and the performance of immunoassay devices by optimizing the spatial configuration and chemical properties of a novel chlorantraniliprole hapten structure. The resulting chlorantraniliprole immunoassay device integrates high sensitivity, strong specificity, anti-interference capability, and rapid portability, overcoming the shortcomings of existing technologies and providing an efficient and reliable solution for rapid screening of chlorantraniliprole in samples. It demonstrates significant technological advancement and broad application prospects. Attached Figure Description

[0068] Figure 1 The mass spectrum of the chlorantraniliprole hapten of the present invention.

[0069] Figure 2 The synthetic route of chlorantraniliprole complete antigen 1 of the present invention.

[0070] Figure 3The synthetic route of chlorantraniliprole complete antigen 2 of the present invention.

[0071] Figure 4 The following are UV scan images of chlorantraniliprole hapten, chlorantraniliprole complete antigen-BSA, and BSA in Example 2 of the present invention.

[0072] Figure 5 The chlorantraniliprole hapten, chlorantraniliprole complete antigen-LF, and LF ultraviolet scan images in Example 2 of the present invention.

[0073] Figure 6 A standard ELISA curve of chlorantraniliprole monoclonal antibody according to one embodiment of the present invention.

[0074] Figure 7 A schematic diagram of the result judgment criteria for the chlorantraniliprole colloidal gold immunochromatographic detection device of the present invention. Detailed Implementation

[0075] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0076] Example 1: Preparation and Identification of Chlorantraniliprole Hapten I. Experimental Methods 1. Preparation of chlorantraniliprole hapten The synthesis reaction formula is as follows: , , .

[0077] The specific synthesis steps are as follows: S1. Take 800 mg of compound a (4.03 mmol, CAS: 890707-28-5) and place it in a 50 mL round-bottom flask. Then add 4 mL of DMF (N,N-dimethylformamide), 836 mg of potassium carbonate (6.03 mmol), and 2698 mg of compound b (16.13 mmol, CAS: 105-36-2) in sequence. After stirring and mixing thoroughly, react at 80 °C for 2 h to 8 h. After the reaction is complete, add 50 mL of purified water. Extract the aqueous phase twice with n-hexane and discard the n-hexane layer. Extract the aqueous phase three times with ethyl acetate. Remove the solvent from the organic phase under reduced pressure to obtain 738 mg of crude compound c. S2. 480 mg of compound d (1.59 mmol, 3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid, CAS: 500011-86-9) was dissolved in 10 mL of a 1:1 mixture of toluene and thionyl chloride. 10 µL of LDMF was added, and the mixture was reacted at 60 °C for 1–2 h. After the reaction was complete, the mixture was evaporated to dryness under reduced pressure. The residue was dissolved in 10 mL of dichloromethane and slowly added dropwise under ice bath to a mixture containing 304 mg of triethylamine (4.77 mmol, CAS: 121-44-8), 300 mg of crude compound c, and 10 mL of dichloromethane. After the addition was complete, the mixture was brought to room temperature and stirred at room temperature for 4–8 h. After the reaction was complete, purified water was added, and the aqueous phase was extracted three times with ethyl acetate. The organic phase was washed with saturated NaHCO3 solution, and the organic phase was desolventized under reduced pressure. The residue was purified by column chromatography to give 160 mg of compound e. S3. Dissolve 160 mg of compound e in 3 mL of methanol, then add 3 mL of 6 mol / L lithium hydroxide aqueous solution, react at room temperature for 10–12 h, add 70 mL of purified water, extract twice with 30 mL of dichloromethane, adjust the pH of the aqueous phase to 4–5 with 4 M hydrochloric acid, precipitate the solid, filter and dry to obtain 106 mg of chlorantraniliprole hapten.

[0078] 2. Identification of chlorantraniliprole hapten Mass spectrometry and nuclear magnetic resonance were used to detect the chlorantraniliprole hapten.

[0079] II. Experimental Results Mass spectrum detection results as follows Figure 1 As shown. According to Figure 1 The molecular ion peak of the chlorantraniliprole hapten in ESI negative ion mode is [MH]. -The m / z value is 538 (the theoretical molecular weight of the chlorantraniliprole hapten is 538.98 Da), consistent with the theoretical value. The 1H NMR results are shown below: 1H NMR (500 MHz, CDCl3) δ 12.25 (s, 1H), 8.53 (dd, J = 3.6, 1.8 Hz, 1H), 7.86 (q, J = 5.2 Hz, 1H), 7.73 (d, J = 2.3 Hz, 1H), 7.68 (dd, J = 7.8, 1.8 Hz, 1H), 7.43 (dd, J = 7.9, 3.5 Hz, 1H), 7.40 (s, 1H), 7.37 (d, J = 2.0 Hz, 1H), 5.02 (s, 1H), 2.91 (d, J = 1H). 5.2 Hz, 3H), 2.35 s, 3H. This demonstrates the synthesis of the chlorantraniliprole hapten represented by formula (I). .

[0080] Example 2 Preparation of chlorantraniliprole complete antigen I. Experimental Methods 1. The synthetic routes for chlorantraniliprole complete antigen 1 and chlorantraniliprole complete antigen 2, as shown in structural formula (II), are as follows: Figure 2 , .

[0081] (1) The specific preparation method of chlorantraniliprole complete antigen 1 is as follows: S1. Take 5 mg of the chlorantraniliprole hapten prepared in Example 1, dissolve it in 0.1 mL of dimethylformamide (DMF), stir thoroughly, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain the hapten activated ester; S2. Weigh 30 mg of bovine serum albumin (BSA) and dissolve it completely in 5 mL of 0.1 mol / L CB solution to form a bovine serum albumin carrier solution. Under stirring, slowly add the hapten activated ester obtained in step S1 dropwise to the bovine serum albumin carrier solution and stir at room temperature for 16–24 h. S3. Dialyze the solution obtained in step S2 with 0.01 mol / L PBS solution at room temperature for 3 days, changing the dialysis solution 3 times a day to remove unreacted small molecules, to obtain chlorantraniliprole complete antigen 1 (i.e. chlorantraniliprole hapten-BSA conjugate), aliquot and store at 4°C for later use.

[0082] (2) The specific preparation method of chlorantraniliprole complete antigen 2 is as follows: S1. Take 5 mg of the chlorantraniliprole hapten prepared in Example 1, dissolve it in 0.3 mL of dimethylformamide (DMF), stir thoroughly, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain the hapten activated ester; S2. Weigh 50 mg of ovalbumin (OVA) and dissolve it completely in 5 mL of 0.1 mol / L CB solution to form an ovalbumin carrier solution. Under stirring, slowly add the hapten activated ester obtained in step S1 dropwise to the ovalbumin carrier solution and stir at room temperature for 16–24 h. S3. Dialyze the solution obtained in step S2 with 0.01 mol / L PBS solution at room temperature for 3 days, changing the dialysis solution 3 times a day to remove unreacted small molecules, to obtain chlorantraniliprole complete antigen 2 (i.e. chlorantraniliprole hapten-OVA conjugate), aliquot and store at 4°C for later use.

[0083] 2. The synthetic routes for chlorantraniliprole complete antigen 3, chlorantraniliprole complete antigen 4, and chlorantraniliprole complete antigen 5, as shown in formula (III), are as follows: Figure 3 .

[0084] (1) The specific preparation method of chlorantraniliprole complete antigen 3 is as follows: S1. Take 50 mg of γ-aminobutyric acid, dissolve it in 0.4 mL of dimethylformamide (DMF), stir thoroughly, add 50 mg of EDC and 50 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain activated γ-aminobutyric acid ester; S2. Weigh 200 mg of lactoferrin (LF) and dissolve it completely in 4 mL of 0.1 mol / L CB solution to form a lactoferrin carrier solution. Under stirring, slowly add the γ-aminobutyric acid activated ester obtained in step S1 dropwise to the lactoferrin carrier solution and stir at room temperature for 16–24 h. S3. Dialyze the solution obtained in step S2 with 0.01 mol / L CB solution at room temperature for 3 days, changing the dialysate 3 times a day to remove unreacted small molecules, to obtain γ-aminobutyric acid modified lactoferrin, make up to 20 mL, dispense and store at 4℃ for later use. S4. Take 5 mg of the chlorantraniliprole hapten prepared in Example 1, dissolve it in 0.1 mL of dimethylformamide (DMF), stir thoroughly, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain the hapten activated ester; S5. Take 3 mL of γ-aminobutyric acid modified lactoferrin, and slowly add the hapten activated ester obtained in step S4 dropwise to the γ-aminobutyric acid modified lactoferrin solution while stirring, and stir at room temperature for 16-24 h. S6. Dialyze the solution obtained in step S5 with 0.01 mol / L PBS solution at room temperature for 3 days, changing the dialysis solution 3 times a day to remove unreacted small molecules, and obtain chlorantraniliprole complete antigen 3 (i.e. chlorantraniliprole hapten-γ-aminobutyric acid modified LF conjugate), aliquot and store at 4°C for later use.

[0085] (2) The specific preparation method of chlorantraniliprole complete antigen 4 is as follows: S1. Take 50 mg of γ-aminobutyric acid, dissolve it in 0.4 mL of dimethylformamide (DMF), stir thoroughly, add 50 mg of EDC and 50 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain activated γ-aminobutyric acid ester; S2. Weigh 240 mg of human serum albumin (HSA) and dissolve it completely in 3 mL of 0.1 mol / L CB solution to form a human serum albumin carrier solution. Under stirring, slowly add the γ-aminobutyric acid activated ester obtained in step S1 dropwise to the human serum albumin carrier solution and stir at room temperature for 16–24 h. S3. Dialyze the solution obtained in step S2 with 0.01 mol / L CB solution at room temperature for 3 days, changing the dialysate 3 times a day to remove unreacted small molecules, to obtain γ-aminobutyric acid modified human serum albumin, make up to 9 mL, aliquot and store at 4℃ for later use. S4. Take 5 mg of the chlorantraniliprole hapten prepared in Example 1, dissolve it in 0.1 mL of dimethylformamide (DMF), stir thoroughly, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain the hapten activated ester; S5. Take 3 mL of γ-aminobutyric acid modified human serum albumin, and slowly add the hapten activated ester obtained in step S4 dropwise to the γ-aminobutyric acid modified human serum albumin carrier solution while stirring, and stir at room temperature for 16-24 h; S6. Dialyze the solution obtained in step S5 with 0.01 mol / L PBS solution at room temperature for 3 days, changing the dialysis solution 3 times a day to remove unreacted small molecules, and obtain chlorantraniliprole complete antigen 4 (i.e. chlorantraniliprole hapten-γ-aminobutyric acid modified HSA conjugate), aliquot and store at 4℃ for later use.

[0086] (3) The specific preparation method of chlorantraniliprole complete antigen 5 is as follows: S1. Take 50 mg of γ-aminobutyric acid, dissolve it in 0.4 mL of dimethylformamide (DMF), stir thoroughly, add 50 mg of EDC and 50 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain activated γ-aminobutyric acid ester; S2. Weigh 240 mg of hemocyanin (KLH) and dissolve it completely in 3 mL of 0.1 mol / L CB solution to form a human serum albumin carrier solution. Under stirring, slowly add the γ-aminobutyric acid activated ester obtained in step S1 dropwise to the hemocyanin (KLH) carrier solution and stir at room temperature for 16–24 h. S3. Dialyze the solution obtained in step S2 with 0.01 mol / L CB solution at room temperature for 3 days, changing the dialysate 3 times a day to remove unreacted small molecules, and obtain γ-aminobutyric acid modified hemocyanin. Make up to 9 mL, dispense into aliquots and store at 4℃ for later use. S4. Take 5 mg of the chlorantraniliprole hapten prepared in Example 1, dissolve it in 0.1 mL of dimethylformamide (DMF), stir thoroughly, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain the hapten activated ester; S5. Take 3 mL of γ-aminobutyric acid modified hemocyanin, and slowly add the hapten activated ester obtained in step S4 dropwise to the γ-aminobutyric acid modified hemocyanin carrier solution while stirring, and stir at room temperature for 16-24 h. S6. Dialyze the solution obtained in step S5 with 0.01 mol / L PBS solution at room temperature for 3 days, changing the dialysis solution 3 times a day to remove unreacted small molecules, and obtain chlorantraniliprole complete antigen 5 (i.e. chlorantraniliprole hapten-γ-aminobutyric acid modified hemocyanin conjugate), aliquot and store at 4°C for later use.

[0087] II. Experimental Results Taking complete antigen 1 and complete antigen 3 in Example 2 as examples, ultraviolet scanning (190-400 nm) was performed on two carrier proteins, chlorantraniliprole hapten, complete antigen 1 and complete antigen 3.

[0088] The results are as follows Figure 4 and Figure 5 As shown; from Figure 4 and Figure 5 It can be seen that the UV characteristic absorption peak of the complete antigen is shifted to varying degrees relative to the chlorantraniliprole hapten and the carrier protein. Furthermore, it was found that the chlorantraniliprole complete antigen possesses the characteristic absorption peaks of both the chlorantraniliprole hapten and the carrier protein, indicating that the chlorantraniliprole hapten and the carrier protein were successfully coupled, and the benzo[a]chlorantraniliprole complete antigen was successfully prepared.

[0089] Example 3: Preparation of chlorantraniliprole monoclonal antibody I. Experimental Methods Using the chlorantraniliprole complete antigen 3 (i.e., chlorantraniliprole hapten-γ-aminobutyric acid modified LF conjugate) prepared in Example 2 as the immunization antigen, chlorantraniliprole monoclonal antibodies were prepared.

[0090] The specific method is as follows: S1. After emulsifying chlorantraniliprole complete antigen with an equal volume of Freund's adjuvant, BALB / C mice were subcutaneously immunized. Each mouse received 100 μg of immunization per immunization, with an interval of 2 weeks between immunizations, for a total of 3 immunizations. S2. After the third immunization, take tail vein blood from mice to test serum antibody titer. If the antibody titer is lower than 1:8000, administer a booster immunization of 100 μg every 7 days until the titer stabilizes above 1:32000. S3. Subcutaneous booster immunization was performed using 0.01 M PBS solution containing 100 μg of chlorantraniliprole complete antigen. Five days later, mouse spleen cells were collected and fused with SP20 mouse myeloma cells. S4. The fused cells were screened in HAT medium, and after 5 days they were cultured in complete medium (fetal bovine serum, basal medium, streptomycin-penicillin). S5. Using the chlorantraniliprole complete antigen 1 (chlorantraniliprole hapten-BSA conjugate) prepared in Example 2 as the coating antigen, the titer and inhibition of the cell supernatant were tested by enzyme-linked immunosorbent assay (ELISA). Positive cells with OD450>1.0 and an inhibition rate of more than 85% when the chlorantraniliprole drug concentration was 2 μg / mL were screened. The cells were cultured three times using the limiting dilution method to confirm that the positive cells were hybridoma cells secreting chlorantraniliprole monoclonal antibodies. S6. After expanding and culturing hybridoma cells, use 1×10 6 Injecting one cell / mouse into the peritoneal cavity of mice to induce ascites, the ascites was purified by octanoic acid-ammonium sulfate precipitation to obtain chlorantraniliprole monoclonal antibody.

[0091] Example 4: Performance of chlorantraniliprole monoclonal antibody I. Experimental Methods Using the chlorantraniliprole complete antigen 1 (chlorantraniliprole hapten-BSA conjugate) prepared in Example 2 as the coating antigen, and taking the chlorantraniliprole monoclonal antibody prepared in Example 3 as an example, the performance of the chlorantraniliprole monoclonal antibody was evaluated by enzyme-linked immunosorbent assay (ELISA). The specific method is as follows: S1. Using carbonate buffer (pH = 9.6) as the coating dilution buffer, dilute chlorantraniliprole complete antigen 1 to 0.2 μg / mL, add 100 μL / well to polystyrene microplates, coat overnight at 4°C, spin dry, add 280 μL / well of 1% BSA solution, block in phosphate buffer containing 1% BSA at 37°C for 1 h, spin dry, and vacuum package for storage. S2. Dilute the chlorantraniliprole monoclonal antibody to 0.1 μg / mL with phosphate buffer containing 0.05% sodium azide (pH = 7.4) and store at 4°C for later use; S3. Add 30 μL / well of the chlorantraniliprole monoclonal antibody solution from step S2 to the blank mixing wells, then add 90 μL / well of the chlorantraniliprole standard solution (0-0.32 μg / L), mix well, and react at 37°C for 5 min. Then transfer 100 μL of the liquid from the mixing wells to the microplate coated with the complete chlorantraniliprole antigen from step S1, react at 37°C for 0.5 h, and then dry. S4. Add 280 μL of washing buffer to each well, wash 3 times, pat dry, then add 100 μL of enzyme-labeled secondary antibody to each well, and react at 37℃ for 0.5 h. S5. After washing three times, pat dry. Add 100 μL of TMB chromogenic solution to each well and react at 37°C for 15 min. Stop the reaction by adding 50 μL of 2 M sulfuric acid solution to each well. Set the microplate reader to 450 nm and measure the OD value of each well. II. Experimental Results The results are shown in Table 1.

[0092] Table 1. Concentration and OD value (450 nm) of chlorantraniliprole standard solution

[0093] Using the data in Table 1, a standard curve was plotted by fitting a four-parameter logistic curve using ELISA Calc software. Figure 6The linear equation of the standard curve is: y = (A - D) / [1 + (x / C)^B] + D, r² = 0.99901, A = 1.07216, B = 1.17809, C = 0.05309, D = 0.05581. Where X is the concentration of the chlorantraniliprole standard solution, and y is the OD value. The IC50 of the chlorantraniliprole monoclonal antibody was calculated. 50 The concentration was 0.058 μg / L, showing a linear relationship between 0.02 and 0.32 μg / L, demonstrating that the chlorantraniliprole monoclonal antibody prepared in this invention possesses high affinity and specificity, significantly improving the detection performance of chlorantraniliprole. This performance stems from the optimized design of the novel hapten structure, providing a foundation for the high sensitivity of the chlorantraniliprole immunoassay device.

[0094] Example 5: Preparation of a chlorantraniliprole colloidal gold immunochromatographic detection device S1. Preparation of reaction membranes coated with chlorantraniliprole complete antigen and mouse IgG. Using nitrocellulose membrane (NC membrane) as the substrate, 0.1 mg / mL of chlorantraniliprole complete antigen 1 solution prepared in Example 2 was sprayed onto the detection zone (T line), and 0.1 mg / mL of sheep-derived immunoglobulin (sheep anti-mouse IgG) solution was sprayed onto the control zone (C line). Both solutions were prepared with coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 MPBS buffer at pH = 7.6), and the spraying volume was 1.0 μL / cm. The detection zone and control zone were spaced 2.5 mm apart. The membranes were dried in an oven at 45°C for 12 hours and stored at 20–25°C and ≤ 35% humidity.

[0095] S2. Preparation of microwells containing colloidal gold-labeled chlorantraniliprole monoclonal antibody S2.1 Preparation of Colloidal Gold Take 1g of chloroauric acid, dissolve it in pure water by sonication, and then bring the volume to 100 mL. Take 4 mL of the above solution into 100 mL of pure water, heat to boiling, add 1.2 mL of 0.06% sodium citrate solution for reduction, and continue heating for 10 minutes. After heating, cool to 25℃ and then add pure water to the original volume to obtain a colloidal gold solution. Store it away from light. All glassware used should be soaked overnight in a mixture of potassium permanganate and sulfuric acid, and then washed and dried before use. S2.2 Labeling of chlorantraniliprole monoclonal antibody Colloidal gold solution was dispensed in 1 mL vials, and the pH was adjusted dropwise to different values ​​within the range of 7.0–9.0 using 0.1 mol / L K₂CO₃ solution to optimize the binding of the antibody to the colloidal gold. 5 μg of the chlorantraniliprole monoclonal antibody prepared in Example 3 was added to the colloidal gold solution at each pH, ​​and the reaction was carried out at 25°C for 5 minutes. The color change of the solution was observed, and the lowest pH value that maintained a red color was selected as the optimal pH. The optimal pH was approximately pH 6.8 when 5 μL of 0.1 mol / L K₂CO₃ solution was added. 10 μL of 10% bovine serum albumin (BSA) solution was added for blocking, and the mixture was centrifuged at 4°C and 12,000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was collected. S2.3 Preparation of colloidal gold labeled micropores The precipitate (i.e. labeled antibody) was reconstituted with colloidal gold diluent containing 0.02 M Tris, 2% bovine serum albumin, 0.05% thimerosal, and 5% sucrose. The solution was aliquoted into microwells at 10 μL / well and dried at 37°C for 16 hours before storage.

[0096] S3. Prepare the sample pad The cut 30×30 cm blank sample pads were immersed in 0.05 M PBS buffer containing 0.05% Tween 20, 1% sucrose, 0.5% PVP40, and 0.05% sodium azide for 5 min. After immersion, the pads were removed and dried at 37°C for 16 h. They were then stored at 20–25°C and ≤ 35% humidity.

[0097] S4. Assemble the colloidal gold chromatography detection card The reaction membrane prepared in step S1 is stacked in the middle of the PVC board backing; the sample pad prepared in step S3 is stacked on the side of the reaction membrane near the detection area, and the absorbent pad is stacked on the side near the control area; the two ends of the reaction membrane are connected to the sample pad and the absorbent pad respectively, and the connection parts of the sample pad, reaction membrane and absorbent pad overlap by 1-2 mm, and are attached to the PVC board; the base plate is cut into 3 mm wide test strips using a strip cutter and inserted into the detection card with a sample application hole, with the sample application hole aligned with the sample pad area of ​​the test strip.

[0098] Example 6: Preparation of a chlorantraniliprole colloidal gold immunochromatographic detection device S1. Preparation of reaction membrane Using nitrocellulose membranes (NC membranes) as the substrate, a 0.05 mg / mL solution of chlorantraniliprole complete antigen was sprayed onto the detection zone (T line), and a 0.1 mg / mL solution of sheep-derived immunoglobulin (sheep anti-mouse IgG) was sprayed onto the control zone (C line). Both solutions were prepared using coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 M PBS buffer at pH 7.6), with a spraying volume of 0.8 μL / cm. The detection and control zones were spaced 2.5 mm apart. The membranes were dried in an oven at 45°C for 12 hours and stored under constant temperature and humidity conditions.

[0099] The other steps (S2 to S4) are the same as S2 to S4 in Example 10.

[0100] Example 7: Preparation of a chlorantraniliprole colloidal gold immunochromatographic detection device S1. Preparation of reaction membrane Using nitrocellulose membranes (NC membranes) as the substrate, a 0.2 mg / mL solution of chlorantraniliprole complete antigen was sprayed onto the detection zone (T line), and a 0.08 mg / mL solution of sheep-derived immunoglobulin (sheep anti-mouse IgG) was sprayed onto the control zone (C line). Both solutions were prepared using coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 M PBS buffer at pH 7.6), with a spraying volume of 1.0 μL / cm. The detection and control zones were spaced 2.5 mm apart. The membranes were dried in an oven at 45°C for 12 hours and stored under constant temperature and humidity conditions.

[0101] The other steps (S2 to S4) are the same as S2 to S4 in Example 10.

[0102] Example 8: Preparation of a chlorantraniliprole colloidal gold immunochromatographic detection device S1. Preparation of reaction membrane Using nitrocellulose membranes (NC membranes) as the substrate, a 0.3 mg / mL solution of chlorantraniliprole complete antigen was sprayed onto the detection zone (T line), and a 0.12 mg / mL solution of sheep-derived immunoglobulin (sheep anti-mouse IgG) was sprayed onto the control zone (C line). Both solutions were prepared using coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 M PBS buffer at pH 7.6), with a spraying volume of 1.2 μL / cm. The detection and control zones were spaced 2.5 mm apart. The membranes were dried in an oven at 45°C for 12 hours and stored under constant temperature and humidity conditions.

[0103] The other steps (S2 to S4) are the same as S2 to S4 in Example 10.

[0104] Example 9: Preparation of a chlorantraniliprole colloidal gold immunochromatographic detection device S1. Preparation of reaction membrane Using nitrocellulose membranes (NC membranes) as the substrate, a 0.25 mg / mL solution of chlorantraniliprole complete antigen was sprayed onto the detection zone (T line), and a 0.15 mg / mL solution of sheep-derived immunoglobulin (sheep anti-mouse IgG) was sprayed onto the control zone (C line). Both solutions were prepared using coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 M PBS buffer at pH 7.6), with a spraying volume of 0.9 μL / cm. The detection and control zones were spaced 2.5 mm apart. The membranes were dried in an oven at 45°C for 12 hours and stored under constant temperature and humidity conditions.

[0105] The other steps (S2 to S4) are the same as S2 to S4 in Example 10.

[0106] Example 10: Preparation of a chlorantraniliprole colloidal gold immunochromatographic detection device S1. Preparation of reaction membrane Using nitrocellulose membranes (NC membranes) as the substrate, a 0.2 mg / mL solution of chlorantraniliprole complete antigen was sprayed onto the detection zone (T line), and a 0.2 mg / mL solution of sheep-derived immunoglobulin (sheep anti-mouse IgG) was sprayed onto the control zone (C line). Both solutions were prepared using coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 M PBS buffer at pH 7.6), with a spraying volume of 0.9 μL / cm. The detection and control zones were spaced 2.5 mm apart. The membranes were dried in an oven at 45°C for 12 hours and stored under constant temperature and humidity conditions.

[0107] The other steps (S2 to S4) are the same as S2 to S4 in Example 10.

[0108] Example 11: Detection method, sensitivity, and specificity of chlorantraniliprole colloidal gold immunochromatographic assay device I. Experimental Methods 1. Detection of chlorantraniliprole standard solution samples Prepare a series of chlorantraniliprole standard solutions with different concentrations (0, 0.3, 0.6, 1.2, and 2.4 μg / L) using 0.01 M PBS buffer. Then, add 100 μL of each concentration of chlorantraniliprole standard solution to the colloidal gold-labeled microwells of the chlorantraniliprole colloidal gold immunochromatographic detection device prepared in Example 6, and repeatedly pipet to reconstitute the solution. After standing for 5 min, transfer the solution from the colloidal gold-labeled microwells to the sample wells of the detection card of the chlorantraniliprole colloidal gold immunochromatographic detection device of this invention. Start timing after sample addition, and observe the results after 5–8 min (the signal intensity is uniform during this time period; the specific reading time can be adjusted according to the ambient temperature, for example, reading the result at 6 minutes). Readings after 8 min are invalid. The detection experiment is set up with 3 replicates.

[0109] 2. Result Determination The criteria for determining the results are as follows: Figure 7 As shown, specifically: (1) Negative (-): The T line shows stronger color than the C line or shows no significant difference in color between the two lines; (2) Positive (+): The T line is significantly weaker than the C line or the T line is not visible; (3) Invalid: No C line appears, the operation process is incorrect or the test strip has expired.

[0110] II. Experimental Results The detection results are shown in Table 2. Table 2 shows that the chlorantraniliprole colloidal gold immunochromatographic detection device prepared in this invention has a sensitivity of 0.6 μg / L for chlorantraniliprole.

[0111] Table 2. Determination results of standard solutions of different concentrations

[0112] The colloidal gold immunochromatographic test strip prepared in this invention exhibits sensitivities of 0.6 µg / kg, 3.0 µg / kg, 5.0 µg / kg, and 5.0 µg / kg to chlorantraniliprole, bromocyanamide, tetrachlorantraniliprole, and cyclochlorantraniliprole in standard solutions, respectively, with cross-reactivity rates of 100%, 20%, 12%, and 12%, respectively. This product shows strong specificity with cross-reactivity rates of less than 25% to other similar compounds.

[0113] Example 12 Detection of spiked fruit and vegetable samples using a chlorantraniliprole colloidal gold immunochromatographic assay device I. Experimental Methods Lettuce, cowpeas, cucumbers, romaine lettuce, Chinese cabbage, chives, rapeseed, spinach, celery, green peppers, and apples were selected as blank samples. The blank samples of these 11 fruits and vegetables were subjected to gradient spiking with chlorantraniliprole standard solution at 0, 1, 2, 4, and 8 μg / kg. The experiment was conducted in triplicate.

[0114] According to GB 2763-2021 Maximum Residue Limits for Pesticides in Food and GB2763.1-2022-CKJ Maximum Residue Limits for Pesticides, take 2g of fruit and vegetable samples. Leafy vegetables are cut into pieces about 1 cm square. For tubers, take cross-sectional samples or their epidermis. Place them in a 50 mL centrifuge tube, add 4 mL of 0.01 M PB buffer, and shake vigorously for 2 min. After the centrifuge tube stands for 2 min, the supernatant is the test solution. Add 100 μL of the test solution to the colloidal gold-labeled microwell of the chlorantraniliprole colloidal gold immunochromatographic detection device of the present invention. Repeatedly blow and mix to reconstitute the solution evenly. After standing for 5 min, transfer the solution in the colloidal gold-labeled microwell to the sample well of the detection card of the chlorantraniliprole colloidal gold immunochromatographic detection device of the present invention. Start timing after adding the sample. After 5-8 min, observe the results according to the judgment criteria (same as Example 11).

[0115] II. Experimental Results The test results are shown in Table 3.

[0116] Table 3. Detection Limits for Spiked Samples

[0117] Table 3 shows that a chlorantraniliprole concentration below 2 μg / kg in the test sample is negative, while a concentration above or equal to 2 μg / kg is positive. A chlorantraniliprole concentration above or equal to 5 μg / kg in all test samples is positive. Therefore, the detection limit of the chlorantraniliprole colloidal gold immunochromatographic detection device prepared in this invention is 2 μg / kg.

[0118] Example 13 Stability of the chlorantraniliprole colloidal gold immunochromatographic assay device I. Experimental Methods The chlorantraniliprole colloidal gold immunochromatographic assay device prepared in Example 6 was continuously placed at 45°C for 60 days to simulate one year of long-term storage. Negative results and color changes in the chlorantraniliprole standard solution were detected on days 0, 5, 10, 20, 30, 40, 50, and 60, with three replicates. The specific detection method was the same as in Example 11. This was to assess its stability and determine its shelf life.

[0119] II. Experimental Results The test results are shown in Table 4.

[0120] Table 4. Stability test results of the chlorantraniliprole colloidal gold immunochromatographic assay device

[0121] Table 4 shows that after the chlorantraniliprole colloidal gold immunochromatographic detection device of the present invention was sealed and stored at 45°C for 60 days, the T / C colorimetric depth of the detection card did not change significantly. The results demonstrate that the chlorantraniliprole colloidal gold immunochromatographic detection device prepared in Example 6 of the present invention has good stability, can still accurately detect chlorantraniliprole residues after storage, and can be stably stored at room temperature for more than one year, meeting market requirements for storage and transportation.

Claims

1. A chlorantraniliprole hapten, characterized in that, Its structure is shown in equation (I): 。 2. A method for preparing a compound with the structure shown in formula (I), characterized in that, The preparation method includes the following steps: S1. Aromatic amide compound a and haloester compound b are subjected to a substitution reaction under alkaline conditions to obtain the first intermediate product compound; S2. The first intermediate compound c is reacted with heterocyclic aromatic carboxylic acid compound d in an organic solvent to give the second intermediate compound e; S3. Hydrolyze the second intermediate compound e in an alkaline solution, adjust the pH to precipitate a solid, and obtain chlorantraniliprole hapten.

3. The use of the chlorantraniliprole hapten according to claim 1 in the preparation of chlorantraniliprole complete antigen.

4. A chlorantraniliprole complete antigen, characterized in that, It is formed by direct coupling of the chlorantraniliprole hapten of claim 1 with a carrier protein, and its structure is shown in formula (II): 。 5. A chlorantraniliprole complete antigen, characterized in that, It is formed by direct coupling of the chlorantraniliprole hapten of claim 1 with a carrier protein, and its structure is shown in formula (III): 。 6. The chlorantraniliprole complete antigen according to claim 5, characterized in that, The carrier protein is selected from one of the following: bovine serum albumin, lactoferrin, ovalbumin, human serum albumin, and hemocyanin.

7. A composition of an immunogen and a coating antigen, characterized in that, It contains the chlorantraniliprole complete antigen as described in claim 5 as a coating antigen, and the chlorantraniliprole complete antigen as described in claim 6 as an immunogen.

8. A method for immunoassay of chlorantraniliprole, characterized in that, The composition of claim 7 is used.

9. A product for the immunoassay of chlorantraniliprole, characterized in that, The composition of claim 7 is prepared or contains the composition of claim 7.

10. The use of the chlorantraniliprole hapten of claim 1, the chlorantraniliprole complete antigen of claim 4, the chlorantraniliprole complete antigen of claim 5, and / or the composition of claim 7 in the preparation of products for the immunodetection of chlorantraniliprole or in the establishment of methods for the immunodetection of chlorantraniliprole.

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