A carboxin hapten, carboxin monoclonal antibody and its hybridoma cell strain and application

Abstract

This invention discloses a hapten, an artificial antigen, an anti-hypoxanthin monoclonal antibody, a hybridoma cell line, and their applications. The hapten is 4-(2-methyl-5,6-dihydro-1,4-oxothiacyclohexene-3-carboxamido)benzoic acid. Hypoxanthin cell line 6E6A7 was deposited at the Guangdong Provincial Microbial Culture Collection Center on July 23, 2026, with accession number GDMCC No:68508. The monoclonal antibody secreted by this hybridoma cell line can specifically bind to hypoxanthin, exhibiting an IC50 concentration of 68.6%. 50 The concentration was 22.61 ng / mL, and the cross-reactivity with both oxycarboxim and carboxim sulfoxide was less than 0.12%. This antibody can be used to prepare an enzyme-linked immunosorbent assay (ELISA) kit for carboxim and to detect carboxim residues in wheat samples.

Description

Technical Field

[0001] This invention belongs to the field of pesticide residue immunoassay technology, specifically relating to a carbendazim hapten, an anti-carbendazim monoclonal antibody, its hybridoma cell line, and its application. Background Technology

[0002] Carboxin is a systemic carboxamide fungicide, belonging to the succinate dehydrogenase inhibitor class. It primarily works by acting on the mitochondrial respiratory chain complex II of pathogenic fungi, inhibiting their energy metabolism. Carboxin is mainly used to control wheat smut, rust, sorghum smut, corn smut, and some cotton seedling diseases.

[0003] The use of carbendazim in agricultural production may result in its residues in grains and other agricultural products. Current methods for detecting carbendazim residues typically employ instrumental analysis methods such as gas chromatography, high-performance liquid chromatography, or liquid chromatography-tandem mass spectrometry. While these methods offer advantages such as high accuracy and strong quantitative capabilities, they generally require complex sample pretreatment processes and specialized equipment. They also demand a high level of expertise from the testing environment and operators, and are relatively costly and time-consuming, making them insufficient for meeting the needs of rapid screening of large numbers of samples and on-site testing at the grassroots level.

[0004] Coriolis is a small molecule compound that possesses only reactivity but not complete immunogenicity, and cannot directly induce the production of corresponding specific antibodies in animals. To prepare anti-coriolis antibodies, it is usually necessary to introduce an active group capable of coupling with a carrier protein while retaining the main characteristic structure of coriolis, obtaining a coriolis hapten. This hapten is then coupled with a carrier protein to form a coriolis artificial antigen.

[0005] The structure of the hapten, its coupling site, and the way it connects to the carrier protein can all affect the exposure of the characteristic structure of oxychloride in artificial antigens, thus affecting the sensitivity and specificity of the resulting antibody in recognizing oxychloride. Oxychloride is structurally similar to compounds such as oxychloride sulfoxide and oxychloride sulfoxide. Therefore, obtaining monoclonal antibodies that can both sensitively recognize oxychloride and effectively distinguish these structural analogs is crucial for establishing a oxychloride-specific immunoassay method.

[0006] Therefore, it is necessary to provide a structurally defined hapten of oxychloride and an artificial antigen prepared therefrom, and to screen hybridoma cell lines that can stably secrete anti-oxychloride monoclonal antibodies based on the artificial antigen, and to further establish an enzyme-linked immunosorbent assay kit and sample detection method for oxychloride. Summary of the Invention

[0007] The purpose of this invention is to provide a hybridoma cell line capable of stably secreting anti-carboxin monoclonal antibodies and the monoclonal antibodies secreted therein, as well as carboxin hapten and artificial antigen for preparing the monoclonal antibodies, the application of the monoclonal antibodies in carboxin immunoassay, an enzyme-linked immunosorbent assay kit containing the monoclonal antibodies, and a method for detecting carboxin residues in wheat samples, thereby achieving rapid, sensitive and specific detection of carboxin residues.

[0008] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a hybridoma cell line 6E6A7 that secretes an anti-carboxin monoclonal antibody. The taxonomic name of the hybridoma cell line 6E6A7 is mouse monoclonal antibody hybridoma cell line. It was deposited on July 23, 2026 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC NO: 68508. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0009] Furthermore, the hybridoma cell line 6E6A7 was obtained by fusing mouse spleen cells immunized with oxytocin immunogen with myeloma cells, followed by selective culture, positive screening, and subcloning.

[0010] Secondly, the present invention provides an anti-carboxin monoclonal antibody, which is secreted by the hybridoma cell line 6E6A7 and can specifically bind to carboxin.

[0011] Furthermore, the heavy chain subtype of the anti-carboxin monoclonal antibody is IgG2a, and the light chain type is κ chain.

[0012] Furthermore, under the indirect competitive enzyme-linked immunosorbent assay (ELISA) conditions established in this invention, the half-maximum inhibitory concentration (IC50) of the anti-carboxin monoclonal antibody against carboxin is [value missing]. 50 It was 22.61 ng / mL.

[0013] Furthermore, the cross-reactivity of the anti-carboxin monoclonal antibody to both carboxin oxide and carboxin sulfoxide is less than 0.12%.

[0014] Furthermore, the cross-reactivity of the anti-carbendazim monoclonal antibody against chlorantraniliprole, phorate, methomyl, chlorothalonil, tricyclazole, triazophos, cyazofamid, and flufenoxuron is less than 0.12%.

[0015] Thirdly, the present invention provides a chlorothalonil hapten, wherein the chlorothalonil hapten is 4-(2-methyl-5,6-dihydro-1,4-oxothiacyclohexene-3-carboxamido)benzoic acid, having the structure shown in formula (I):

[0016] Formula (I).

[0017] Fourthly, the present invention also provides a method for preparing the thiazolinone hapten, comprising reacting 2-methyl-5,6-dihydro-1,4-oxothiacyclohexene-3-carboxyl chloride with 4-aminobenzoic acid in the presence of pyridine and in N,N-dimethylformamide as solvent with an amidation reaction, followed by separation and purification to obtain the thiazolinone hapten.

[0018] Furthermore, the amidation reaction was carried out at room temperature for 16 h.

[0019] Furthermore, the reaction product is concentrated under reduced pressure and then separated and purified using preparative high-performance liquid chromatography.

[0020] Fifthly, the present invention also provides a carboxin artificial antigen, which is obtained by covalently coupling the carboxyl group of the carboxin hapten to the amino group of a carrier protein.

[0021] Furthermore, the carrier protein is bovine serum albumin or ovalbumin.

[0022] Furthermore, the conjugate of the carboxin hapten and bovine serum albumin serves as an immunogen, and the conjugate of the carboxin hapten and ovalbumin serves as a coating antigen.

[0023] Furthermore, the thiamethoxam hapten is activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide before being coupled to the carrier protein.

[0024] In a sixth aspect, the present invention provides a carboxin enzyme-linked immunosorbent assay kit, the kit comprising an ELISA plate coated with carboxin-coated antigen, the anti-carboxin monoclonal antibody, horseradish peroxidase-labeled anti-mouse immunoglobulin antibody, carboxin standard, TMB chromogenic solution, stop solution, washing solution and sample diluent.

[0025] Furthermore, the thiamethoxam coating antigen is a conjugate of the thiamethoxam hapten and ovalbumin.

[0026] Seventhly, the present invention also provides a method for detecting carboxin residues in wheat samples, comprising the following steps: (1) Extract and purify the wheat sample to obtain wheat sample extract; (2) The wheat sample extract was subjected to indirect competitive enzyme-linked immunosorbent assay (ELISA) using the chlorothalonil ELISA kit described in the sixth aspect, and the absorbance value of the colorimetric reaction was measured. (3) Prepare a series of malondioxin matrix-matched standard solutions of different concentrations using blank wheat matrix extract, establish a matrix-matched standard curve, substitute the absorbance value measured in step (2) into the standard curve, and calculate the malondioxin content in the wheat sample.

[0027] Further, step (1) includes: crushing the wheat sample and passing it through a 40-mesh standard sieve, weighing 5 g of the crushed wheat sample, adding 10 mL of water, vortexing and mixing and letting stand for 30 min; adding 15 mL of acetonitrile, shaking for 1 min, and centrifuging at 4200 r / min for 5 min; taking 2 mL of the supernatant and adding it to a centrifuge tube containing 300 mg of anhydrous magnesium sulfate, 100 mg of C18 adsorbent and 50 mg of primary and secondary amine adsorbent (PSA), vortexing and mixing for 1 min, and centrifuging at 4200 r / min for 5 min; collecting the supernatant, blowing it dry with nitrogen, reconstituted with 1 mL of 0.01 mol / L PBS buffer and filtering to obtain the wheat sample extract.

[0028] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention obtained a hybridoma cell line 6E6A7 that can stably secrete monoclonal antibodies against oxychloride, providing a stable source of monoclonal antibodies for oxychloride immunoassay.

[0029] (2) The monoclonal antibody secreted by hybridoma cell line 6E6A7 can specifically bind to oxychloride. Under indirect competitive enzyme-linked immunosorbent assay (ELISA) conditions, the IC50 of this antibody against oxychloride is [value missing]. 50 The concentration was 22.61 ng / mL, and the cross-reactivity with oxychloride, oxychloride sulfoxide, and other tested pesticides was less than 0.12%, indicating good recognition sensitivity and specificity.

[0030] (3) The hapten provided by the present invention retains the oxothiocyclic amide characteristic structure of oxysulfuric acid and sets a carboxyl coupling site at the para position of the benzene ring, which can form a stable artificial antigen with the carrier protein, providing an immunogen and coating antigen for the preparation of anti-oxysulfuric acid monoclonal antibody.

[0031] (4) The indirect competitive enzyme-linked immunosorbent assay (ELISA) method established using the aforementioned anti-carboxin monoclonal antibody can be used to detect carboxin in wheat matrix. At spiking levels of 50, 200, and 500 ng / mL, the average recoveries obtained by ELISA were 78.30%–88.90%, with relative standard deviations of 9.11%–12.05%, demonstrating good accuracy and repeatability. Attached Figure Description

[0032] Figure 1 This is a synthetic route diagram for the hapten of chlorothalonil.

[0033] Figure 2 This is a high-performance liquid chromatography (HPLC) chromatogram of the purity of the thiazolinone hapten.

[0034] Figure 3 The images show the ultraviolet absorption spectra of the thiazolinone hapten, carrier protein, and artificial thiazolinone antigen. In the images, A represents the ultraviolet absorption spectra of the thiazolinone hapten, bovine serum albumin, and the thiazolinone hapten-bovine serum albumin conjugate, while B represents the ultraviolet absorption spectra of the thiazolinone hapten, ovalbumin, and the thiazolinone hapten-ovalbumin conjugate.

[0035] Figure 4 The standard curve for indirect competitive enzyme-linked immunosorbent assay (ELISA) of carboxin monoclonal antibody against carboxin is shown.

[0036] Figure 5 This is a matrix-matched standard curve for the indirect competitive enzyme-linked immunosorbent assay (ELISA) of carboxin in wheat matrix. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments. It should be noted that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Reagents, materials, and instruments not specifically described are all obtainable through conventional commercial channels; experimental methods not specifically described are all conventional methods in the art.

[0038] Example 1: Preparation of the carboxin hapten At room temperature, 2-methyl-5,6-dihydro-1,4-oxothiacyclohexene-3-carboxyl chloride (B1-4, 3.60 g, 20.1 mmol) was dissolved in DMF (18.0 mL), and the resulting solution was slowly added dropwise to a DMF (18.0 mL) solution of 4-aminobenzoic acid (3.32 g, 24.2 mmol) and pyridine (3.18 g, 40.2 mmol). After the addition was complete, the reaction mixture was stirred at room temperature for 16 h.

[0039] After the reaction was completed, the reaction solution was concentrated under reduced pressure. The residue was purified by preparative high-performance liquid chromatography to obtain 0.61 g of white solid 4-(2-methyl-5,6-dihydro-1,4-oxothiacyclohexene-3-carboxamido)benzoic acid, with a yield of 10.9%. Two batches were prepared in parallel under the same feed amount and reaction conditions, yielding a total of 1.20 g of the target product. This is the thiamethoxam hapten shown in formula (I), denoted as H1, and its synthetic route is as follows. Figure 1 As shown.

[0040]

[0041] Formula (I) The hapten for thiocarboxamide retains the characteristic structure of thiocarboxamide and sets a carboxyl coupling site at the para position of the benzene ring, providing a reaction site for subsequent coupling with carrier protein to prepare thiocarboxamide artificial antigen.

[0042] The purity and structure of the obtained thiamethoxam hapten were determined by high performance liquid chromatography (HPLC), mass spectrometry (LC-MS), and hydrogen nuclear magnetic resonance spectroscopy (1H-NMR).

[0043] The high-performance liquid chromatography (HPLC) detection conditions were as follows: a SunFire C18 column (5 μm, 4.6 mm × 150 mm), a mobile phase of 50% acetonitrile aqueous solution containing 0.03% trifluoroacetic acid (TFA) (acetonitrile / water = 50:50, v / v), a flow rate of 1 mL / min, and a detection wavelength of 214 nm. The retention time of the obtained oxychloride hapten was 7.006 min, and the purity was 98.009%. Figure 2 As shown.

[0044] Liquid chromatography-mass spectrometry (LC-MS) was performed in electrospray ionization positive ion mode (ESI⁺), with a retention time (Rt) of 1.462 min; the measured quasi-molecular ion peak was 280.2 m / z, assigned to [M+H]. + .

[0045] 1 H NMR (400 MHz, DMSO-d6) δ 12.62 (br s, 1H), 10.16 (s, 1H), 7.90~7.88 (m, 2H), 7.75~7.73 (m, 2H), 4.31 (t, J=4.4 Hz, 2H), 3.07 (t, J=4.4 Hz, 2H), 2.01 (s, 3H).

[0046] The results of high performance liquid chromatography, liquid chromatography-mass spectrometry and nuclear magnetic resonance hydrogen spectroscopy show that the purity, molecular weight and structure of the obtained product are consistent with the thiazolinone hapten shown in formula (I).

[0047] Example 2 Synthesis of Artificial Antigen of Coriolis Acid Conjugates of thiamethoxam hapten with bovine serum albumin (BSA) and ovalbumin (OVA) were prepared using the carbodiimide method. After activation with EDC·HCl and NHS, the carboxyl group of the thiamethoxam hapten reacted with the amino group of the carrier protein to form an amide bond. The molar ratio of hapten to carrier protein was 50:1, and the molar ratio of hapten, NHS, and EDC·HCl was 1:2:2.

[0048] To prepare the immunoantigen, 4.20 mg of carboxin hapten was dissolved in 0.10 mL of DMF. Then, 3.46 mg of NHS dissolved in 0.10 mL of DMF and 5.77 mg of EDC·HCl dissolved in 0.30 mL of DMF were added separately. The mixture was stirred overnight at 4 °C and 400–600 rpm. After centrifuging the reaction solution at 6000 rpm for 5 min, the supernatant was slowly added dropwise to a PBS solution containing 20.00 mg of BSA. The mixture was stirred at room temperature for 8 h to obtain the carboxin hapten-BSA conjugate.

[0049] To prepare the coating antigen, 6.28 mg of thiamethoxam hapten was dissolved in 0.10 mL of DMF, and then activated with 5.17 mg NHS and 8.62 mg EDC·HCl, respectively. The reaction conditions were the same as above. After centrifugation, the supernatant was slowly added dropwise to 20.00 mg OVA in PBS solution, and the mixture was stirred at room temperature for 8 h to obtain the thiamethoxam hapten-OVA conjugate.

[0050] The reaction products were dialyzed in 0.01 mol / L PBS, with the dialysate changed every 3–4 hours, for a total of 4 dialyses. After dialysis, the products were aliquoted and stored at −20℃.

[0051] Ultraviolet full-wavelength scans were performed on the hapten of carboxin, bovine serum albumin, ovalbumin, carboxin hapten-bovine serum albumin conjugate, and carboxin hapten-ovalbumin conjugate.

[0052] The results of the ultraviolet full-wavelength scan are as follows Figure 3 As shown, the characteristic absorption peak of the thiamethoxam hapten (H1) is located at 267.5 nm, while the characteristic absorption peaks of BSA and OVA are located at 279.0 nm and 280.0 nm, respectively; the characteristic absorption peaks of the H1–BSA and H1–OVA conjugates are located at 273.5 nm and 273.0 nm, respectively. Compared with the corresponding carrier proteins, the characteristic absorption peaks of both conjugates are shifted, and the absorption peak shape and absorption intensity change significantly; the absorption peaks of the conjugates are located between the characteristic absorption peaks of the hapten and the carrier protein, indicating that the thiamethoxam hapten has been successfully conjugated with BSA and OVA.

[0053] Example 3 Animal Immunization Four 6-8 week old female BALB / c mice were selected for immunization.

[0054] For the initial immunization, the oxycortisone immunogen prepared in Example 2 was thoroughly emulsified with Freund's complete adjuvant at a volume ratio of 1:1, and injected subcutaneously at multiple points on the back of the neck of mice, with an immunization dose of 0.1 mg / mouse. A second immunization was performed 21 days after the initial immunization, using an emulsion of oxycortisone immunogen and Freund's incomplete adjuvant, with an immunization dose of 0.1 mg / mouse.

[0055] Thereafter, booster immunizations were administered every 14 days, with a third and fourth immunization administered sequentially. The immunization dose was 0.1 mg / animal, using Freund's incomplete adjuvant.

[0056] Four days prior to cell fusion, animals underwent a sprint immunization at a dose of 0.2 mg per animal via intraperitoneal injection without adjuvant. The immunization schedule is shown in Table 1.

[0057] Table 1. Immunization strategy for mice

[0058] Example 4: Determination of serum titer and inhibition rate in immunized mice The oxycorticosteroid-coated antigen (OVA-hapten) prepared in Example 2 was diluted to 0.5 μg / mL with carbonate buffer and added to a 96-well microplate, 100 μL per well, and incubated overnight at 4°C.

[0059] Discard the coating solution and wash the ELISA plate twice with PBST. Add 300 μL of 2% skim milk powder solution to each well and block at 37°C for 1 h. After blocking, wash four times with PBST.

[0060] Set up control wells and inhibition wells in the ELISA plate. Add 50 μL of 0.5 μg / mL carboxin standard to the inhibition wells, and add an equal volume of sample dilution to the control wells.

[0061] The serum from immunized mice was serially diluted using sample dilution buffer. 50 μL of serum dilution buffer was added to each well, and the mixture was incubated at 37°C for 30 min. After washing three times with PBST, 100 μL of enzyme-labeled secondary antibody diluted 1:5000 was added to each well, and the mixture was incubated at 37°C for 1 h.

[0062] Wash three times with PBST, add 100 μL of TMB chromogenic solution to each well, and develop the color for 15 min in the dark. Then, add 50 μL of 0.2 mol / L H2SO4 to each well to terminate the reaction, and measure the absorbance at 450 nm.

[0063] and with OD 450The highest serum dilution factor corresponding to a value of 1.0 was taken as the serum titer. The inhibition rate was calculated using the following formula: Inhibition rate (%) = (OD value of control group - OD value of sample group) / OD value of control group × 100%. Mice with good immune responses were screened based on serum antibody titer and inhibitory activity. Spleen cells were isolated from mice that met the criteria and used for subsequent cell fusion experiments.

[0064] Example 5: Preparation and screening of hybridoma cell line 6E6A7 Four days before cell fusion, mice with high serum titers and good competitive inhibitory effects against oxytocin were selected for sprint immunization. Sprint immunization was performed via intraperitoneal injection, with each mouse receiving 0.2 mg of oxytocin immunogen.

[0065] Four days after sprint immunization, the spleens of mice were removed under aseptic conditions, and spleen cell suspensions were prepared. SP2 / 0-Ag14 cells were used for myeloma cells and collected after reaching the logarithmic growth phase. Spleen cells and myeloma cells were mixed at a cell ratio of 10:1, and PEG1450 was used as a fusion agent for 2 minutes to achieve cell fusion.

[0066] After fusion, the cells were seeded into 96-well cell culture plates and cultured in a 37°C, 5% CO2 incubator, using HAT medium for selection. When the hybridoma cell clones grew to about 1 / 10 of the bottom area of ​​the culture wells, the HAT medium was replaced with HT medium, and cultured for another 3 days.

[0067] The antibody titer of cell culture supernatant was detected by ic-ELISA, and its recognition ability and sensitivity to carboxin were evaluated by ic-ELISA. The selected positive wells were subcloned using the limiting dilution method. After multiple rounds of screening, a positive hybridoma cell line capable of stably secreting anti-carboxin monoclonal antibody was finally obtained and named 6E6A7.

[0068] The taxonomic name of hybridoma cell line 6E6A7 is mouse monoclonal antibody hybridoma cell line. It was deposited at Guangdong Provincial Microbial Culture Collection Center (GDMCC) on July 23, 2026, with accession number GDMCC NO: 68508. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0069] Example 6: Preparation and Subtype Identification of Anti-Carbendazim Monoclonal Antibody Hybridoma cell line 6E6A7 with good growth status was selected for expanded culture, and ascites antibody was prepared using an in vivo ascites induction method. Six- to eight-week-old female BALB / c mice were pretreated with sterile paraffin oil via intraperitoneal injection approximately one week prior to the treatment. After the hybridoma cell line 6E6A7 was in good culture condition, the original culture medium was discarded, and the cells were resuspended in preheated DMEM medium by pipetting and washing. The cell concentration was adjusted after centrifugation and washing. Subsequently, the cell suspension was injected intraperitoneally into the paraffin oil-pretreated mice, with approximately 10 cells per mouse. 6 1 cell. After injection, the mice were observed. When the abdomen of the mice was significantly distended after 10 days, the ascites fluid was collected, placed at room temperature for 0.5 h, and then left to stand overnight at 4℃. After centrifugation at 10000 r / min and 4℃ for 10 min, the clear supernatant ascites fluid was collected for subsequent purification.

[0070] Ascites fluid purification was performed using the ammonium sulfate salting-out method. A certain volume of clear ascites fluid was taken, diluted with an equal volume of PBS buffer, and placed in an ice bath. Two volumes of saturated ammonium sulfate were slowly added dropwise under low-speed magnetic stirring. After standing at 4°C for 4 hours, the precipitate was collected by centrifugation. The precipitate was thoroughly dissolved in PBS, and saturated ammonium sulfate was added again for a second salting-out process. After standing at 4°C, the antibody precipitate was collected by centrifugation. Finally, the antibody protein was dissolved in PBS and placed in a dialysis bag for thorough dialyzing with PBS and PB (phosphate buffer, sodium chloride-free) to remove small molecule impurities such as ammonium sulfate. The dialyzed anti-carboxin monoclonal antibody was aliquoted and mixed with glycerol to prepare an antibody stock solution, which was stored at -20°C.

[0071] The obtained anti-carboxin monoclonal antibody was identified using an antibody subtype identification kit. The results showed that the heavy chain subtype of the anti-carboxin monoclonal antibody was IgG2a, and the light chain type was κ chain.

[0072] Example 7 Sensitivity determination of oxytocin monoclonal antibody The recognition ability of the anti-carboxin monoclonal antibody prepared in Example 6 against carboxin was evaluated using an indirect competitive enzyme-linked immunosorbent assay (ELISA).

[0073] The carboxin-coated antigen prepared in Example 2 was coated onto an ELISA plate. Different concentrations of carboxin standard and anti-carboxin monoclonal antibody were added, allowing free carboxin to competitively bind to the monoclonal antibody against the carboxin-coated antigen immobilized on the ELISA plate. After incubation with enzyme-labeled secondary antibody, washing, color development, and termination of the reaction, the absorbance of each well was measured at a wavelength of 450 nm.

[0074] The relative binding rate was calculated as B / B0 = A / A0, where A is the absorbance value measured with the corresponding concentration of carboxin standard added, and A0 is the absorbance value measured without the addition of carboxin standard. A standard curve for the indirect competitive enzyme-linked immunosorbent assay (ELISA) of carboxin was established by fitting a four-parameter Logistic equation with the logarithm of the carboxin standard concentration on the x-axis and B / B0 on ​​the y-axis. The results are shown below. Figure 4 As shown.

[0075] The results showed that the half-maximal inhibitory concentration (IC50) of the anti-carboxin monoclonal antibody against carboxin was [value missing]. 50 The concentration was 22.61 ng / mL, and the IC50 concentration was... 20 The concentration was 2.21 ng / mL, and the IC50 concentration was 2.21 ng / mL. 20 ~IC 80 The corresponding concentration range of carboxin is 2.21–230 ng / mL.

[0076] Example 8 Specificity determination of oxytetracycline monoclonal antibody The cross-reactivity of anti-carbendazim monoclonal antibody with carbendazim, structural analogues and other common pesticides was detected by indirect competitive enzyme-linked immunosorbent assay (ELISA).

[0077] The cross-reactivity rate is calculated using the following formula: Cross-reactivity rate (%) = IC 50 (Corrosion-resistant agent) / IC 50 (Compound to be tested) × 100%.

[0078] The test results are shown in Table 2: Table 2 Cross-reactivity rates of monoclonal antibodies against oxychloride

[0079] The results showed that the anti-carboxin monoclonal antibody had an IC50 of carboxin. 50 The concentration was 22.61 ng / mL, while the IC50 for oxychloride, oxychloride sulfoxide, and other tested pesticides was [missing information]. 50 All values ​​were greater than 20,000 ng / mL. Based on the cross-reactivity formula, the cross-reactivity rates of the tested compounds were all less than 0.12%, indicating that the antibody has good recognition specificity for oxychloride.

[0080] Example 9: Preparation of the chlorothalonil enzyme-linked immunosorbent assay kit This embodiment provides a chlorothalonil enzyme-linked immunosorbent assay kit, which includes: (1) ELISA plates coated with oxytetracycline-coated antigen; (2) Anti-carbendazim monoclonal antibody; (3) Enzyme-labeled secondary antibody; (4) Standard product of carbendazim; (5) TMB colorimetric solution; (6) Termination solution; (7) Washing solution; (8) Sample diluent.

[0081] The oxycortisone coating antigen was the oxycortisone hapten-oval albumin conjugate prepared in Example 2. The oxycortisone coating antigen was diluted to 0.5 μg / mL with carbonate buffer and added to an ELISA plate, 100 μL per well, and incubated overnight at 4°C.

[0082] The working dilution of the anti-carboxin monoclonal antibody is 1:1600; the working dilution of the enzyme-labeled secondary antibody is 1:5000; and the concentration gradient of the carboxin standard is 0.97–500 ng / mL.

[0083] During the assay, the standard solution of carboxin or the extract of the test sample and the anti-carboxin monoclonal antibody were added to the ELISA plate and reacted competitively at 37℃ for 30 min. After washing, 100 μL of enzyme-labeled secondary antibody was added to each well and incubated at 37℃ for 1 h. After washing again, 100 μL of TMB chromogenic solution was added to each well and the plate was incubated in the dark for 15 min. The reaction was then terminated by adding 50 μL of 0.2 mol / L H2SO4, and the absorbance was measured at 450 nm. Different concentrations of carboxin matrix-matched standard solutions were prepared using a blank matrix, and a matrix standard curve was plotted. The results are shown below. Figure 5 As shown. The IC50 of the obtained wheat matrix standard curve. 50 The concentration was 74.20 ng / mL, and the IC50 concentration was... 20 and IC 80 The corresponding concentrations of carboxin were 9.56 ng / mL and 575.92 ng / mL, respectively. The carboxin content in the test samples was calculated based on the established wheat matrix standard curve.

[0084] Example 10 Spiked Recovery Test of Carboxin in Wheat Samples The blank wheat sample was pulverized and passed through a 40-mesh standard sieve. 5 g of the pulverized wheat sample was weighed, and 10 mL of water was added. The mixture was vortexed and allowed to stand for 30 min. 15 mL of acetonitrile and one ceramic homogenant were added, and the mixture was vigorously shaken for 1 min. The mixture was then centrifuged at 4200 r / min for 5 min, and the supernatant was collected. 2 mL of the supernatant was quantitatively pipetted into a centrifuge tube containing 300 mg of anhydrous magnesium sulfate, 100 mg of C18 adsorbent, and 50 mg of primary and secondary amine adsorbent (PSA). The mixture was vortexed for 1 min and centrifuged at 4200 r / min for 5 min. The supernatant was collected, dried under nitrogen, reconstituted with 1 mL of 0.01 mol / L PBS buffer, and filtered to obtain the blank wheat matrix extract.

[0085] A standard of carboxin was added to the purified blank wheat matrix extract to achieve concentrations of 50, 200, and 500 ng / mL, with five parallel samples for each concentration. The carboxin content in each spiked sample was determined using the carboxin enzyme-linked immunosorbent assay kit prepared in Example 9 and liquid chromatography-tandem mass spectrometry, respectively.

[0086] Liquid chromatography-tandem mass spectrometry was used to prepare a matrix-matched standard solution of carboxin using a blank wheat matrix. A matrix standard curve was established with carboxin concentration as the x-axis and peak area as the y-axis for quantitative analysis of carboxin content.

[0087] Spike recovery rate is calculated using the following formula: Spiked recovery rate (%) = (Measured content of spiked sample - Measured content of blank sample) / Theoretical amount × 100%.

[0088] The test results are shown in Table 3.

[0089] Table 3. Spiked recovery results of carboxin in wheat matrix extract

[0090] The results showed that, at spiking levels of 50, 200, and 500 ng / mL, the average recoveries obtained using the phosmet ELISA kit were 78.30%–88.90%, with relative standard deviations (RSDs) of 9.11%–12.05%; while the average recoveries obtained using liquid chromatography-tandem mass spectrometry (LC-MS / MS) were 83.57%–95.71%, with RSDs of 5.43%–8.21%. The results from both methods showed consistent trends, indicating that the established indirect competitive ELISA method has good accuracy and repeatability under wheat matrix conditions and can be used for the detection of phosmet in wheat extracts obtained from the above pretreatment.

Claims

1. A hybridoma cell line 6E6A7 that secretes a monoclonal antibody against carboxin, characterized in that, The hybridoma cell line 6E6A7 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 23, 2026, with accession number GDMCC NO:68508.

2. A monoclonal antibody against carboxin, characterized in that, The anti-carboxin monoclonal antibody is secreted by the hybridoma cell line 6E6A7 described in claim 1 and is capable of specifically binding to carboxin.

3. The anti-carboxin monoclonal antibody according to claim 2, characterized in that, The heavy chain subtype of the anti-carboxin monoclonal antibody is IgG2a, and the light chain type is κ chain.

4. The anti-carboxin monoclonal antibody according to claim 2, characterized in that, When detected using an indirect competitive enzyme-linked immunosorbent assay (ELISA), the half-maximal inhibitory concentration (IC50) of the anti-carboxin monoclonal antibody against carboxin is [value missing]. 50 The concentration was 22.61 ng / mL, and the cross-reactivity with both oxycarboxin and carboxin sulfoxide was less than 0.12%.

5. A hapten for carboxin, characterized in that, The hapten of the carboxin is 4-(2-methyl-5,6-dihydro-1,4-oxothiacyclohexene-3-carboxamido)benzoic acid.

6. A chlorothalonil artificial antigen, characterized in that, The thiazolinone artificial antigen is a conjugate formed by the covalent coupling of the thiazolinone hapten of claim 5 with the amino group of the carrier protein.

7. The artificial antigen of carboxin according to claim 6, characterized in that, The carrier protein is bovine serum albumin or ovalbumin.

8. A carbendazim enzyme-linked immunosorbent assay kit, characterized in that, The ELISA plate is coated with a carboxin-coated antigen, the anti-carboxin monoclonal antibody as described in claim 2, a horseradish peroxidase-labeled anti-mouse immunoglobulin antibody, carboxin standard, TMB chromogenic solution, stop solution, washing solution, and sample dilution solution; the carboxin-coated antigen is the carboxin artificial antigen as described in claim 6, and the carrier protein is ovalbumin.

9. A method for detecting carboxin residues in wheat samples, characterized in that, Includes the following steps: (1) Extract and purify the wheat sample to obtain wheat sample extract; (2) The wheat sample extract was subjected to indirect competitive enzyme-linked immunosorbent assay (ELISA) using the chlorothalonil ELISA kit described in claim 8, and the absorbance value of the colorimetric reaction was measured. (3) Prepare a series of malondioxin matrix-matched standard solutions of different concentrations using blank wheat matrix extract, establish a matrix-matched standard curve, substitute the absorbance value measured in step (2) into the standard curve, and calculate the malondioxin content in the wheat sample.

10. The method according to claim 9, characterized in that, Step (1) includes: crushing the wheat sample and passing it through a 40-mesh standard sieve; weighing 5 g of the crushed wheat sample; adding 10 mL of water; vortexing and mixing; and letting it stand for 30 min; adding 15 mL of acetonitrile; shaking for 1 min; centrifuging at 4200 r / min for 5 min; and collecting the supernatant; adding 2 mL of the supernatant to a centrifuge tube containing 300 mg of anhydrous magnesium sulfate, 100 mg of C18 adsorbent, and 50 mg of primary and secondary amine adsorbent; vortexing and mixing for 1 min; and centrifuging at 4200 r / min for 5 min; collecting the supernatant; blowing it dry with nitrogen; reconstituted with 1 mL of 0.01 mol / L PBS buffer; and filtering to obtain the wheat sample extract.

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