Hybridoma cell strain secreting monoclonal antibody of triketone herbicide and metabolite of triketone herbicide and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain capable of secreting monoclonal antibodies of triketone herbicides and metabolites of the triketone herbicides and application of the hybridoma cell strain, and belongs to the technical field of food safety immunodetection. The hybridoma cell strain HXJA secreting the monoclonal antibody of the triketone herbicide and the metabolite of the triketone herbicide is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.46231. The hybridoma cell strain obtained by the invention belongs to monoclonal cell strains. The monoclonal antibody cell strain of the triketone herbicide and the metabolite of the triketone herbicide can be used for immunoassay detection, has better detection sensitivity (IC50 value is 1.13 ng / mL) and broad-spectrum specificity on mesotrione, and can be used for detecting the residual quantity of the triketone herbicide and the metabolite of the triketone herbicide in brown rice and soybeans; the invention provides a raw material for immunological detection of triketone herbicide and metabolite residues thereof in food, and has practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety immunoassay, and particularly to a hybridoma cell line secreting monoclonal antibodies against triketone herbicides and their metabolites and its application. Background Art

[0002] Mesotrione (2-(4-methylsulfonyl-2-nitro-benzoyl) cyclohexane-1,3-dione), also known as methylsulfonyltrizone or Mestone, with the chemical formula C 14 H 13 NO7S, molecular weight of 339.32, CAS number 104206-82-8, is a new triketone herbicide developed by Zeneca Agrochemicals. It can be used for pre-emergence or post-emergence control of annual broadleaf weeds and some gramineous weeds in corn fields, including most important field broadleaf weeds such as Xanthium sibiricum, Abutilon theophrasti, Chenopodium album, Amaranthus retroflexus, Polygonum spp., Solanum nigrum, Ambrosia trifida, etc. and some gramineous weeds such as young Echinochloa crusgalli, Digitaria sanguinalis, Setaria viridis, Brachiaria eruciformis, etc. Studies have found that mesotrione is relatively stable in water with a long half-life, and its metabolites are prone to accumulate and remain in soil and water bodies, posing a potential poisoning risk to aquatic organisms and humans. Therefore, most countries such as the European Union, China, and the United States have successively prohibited or clearly stipulated limit values.

[0003] At present, there are few studies on analytical methods for determining the content of mesotrione. The main instrumental methods include gas chromatography-tandem mass spectrometry (GC-MS / MS), high performance liquid chromatography (HPLC-UV), liquid chromatography-tandem mass spectrometry (LC-MS / MS), etc. These detection methods have disadvantages such as time-consuming, high cost, cumbersome operation steps, and inability to perform on-site rapid detection. Therefore, it is of great significance to establish a rapid and simple detection method for mesotrione and its metabolites. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method, suitable for on-site rapid detection of a large number of samples, providing a new detection approach for the detection of mesotrione and its metabolites. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the deficiencies of the above-mentioned instruments for detecting triketone herbicides and their metabolites in the prior art.

[0005] To solve the above technical problem, the present invention provides a hybridoma cell line secreting monoclonal antibodies against triketone herbicides and their metabolites and its application. The antibody secreted by this cell line has high detection sensitivity for triketone herbicides and their metabolites, and can be used to establish an immunological detection method for triketone herbicides and their metabolites.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a hybridoma cell line secreting monoclonal antibodies against triketone herbicides and their metabolites. The hybridoma cell line was deposited with the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on October 31, 2024, with the deposit number CGMCC No. 46231 and the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified as a monoclonal cell line.

[0008] In one embodiment of the present invention, the triketone herbicide is selected from mesotrione and / or sulcotrione;

[0009] and / or, the metabolite is at least one of 4-methylsulfonyl-2-nitrobenzoic acid, 2-amino-4-(methylsulfonyl)benzoic acid, sulcotrione, and 2-chloro-4-methylsulfonylbenzoic acid.

[0010] The second object of the present invention is to provide a monoclonal antibody against triketone herbicides and their metabolites, which is secreted by the above-mentioned hybridoma cell line.

[0011] The third object of the present invention is to provide a hapten of triketone herbicides and their metabolites for preparing the above-mentioned hybridoma cell line. The structural formula of the hapten of triketone herbicides and their metabolites is:

[0012]

[0013] The fourth object of the present invention is to provide a composition for detecting triketone herbicides and their metabolites, which contains the above-mentioned hybridoma cell line and / or the monoclonal antibody against triketone herbicides and their metabolites.

[0014] The fifth object of the present invention is to provide a kit for detecting triketone herbicides and their metabolites, which contains at least one of the above-mentioned hybridoma cell line, the monoclonal antibody against triketone herbicides and their metabolites, and the above-mentioned composition.

[0015] In one embodiment of the present invention, the kit is selected from an enzyme-linked immunosorbent assay kit, a fluorescence immunoassay kit, or a chemiluminescent immunoassay kit.

[0016] The sixth object of the present invention is to provide a test strip for detecting triketone herbicides and their metabolites, which contains at least one of the above-mentioned hybridoma cell line, the monoclonal antibody against triketone herbicides and their metabolites, and the above-mentioned composition.

[0017] The seventh object of the present invention is to provide a chip for detecting triketone herbicides and their metabolites, which contains at least one of the above-mentioned hybridoma cell line, the monoclonal antibody against triketone herbicides and their metabolites, and the above-mentioned composition.

[0018] The eighth object of the present invention is to provide the application of the hybridoma cell line, the monoclonal antibody against triketone herbicides and their metabolites, the composition, the kit, the test strip or the chip in the detection of triketone herbicides and their metabolites. In particular, it is applied to the analysis and detection of the residues of triketone herbicides and their metabolites in food safety detection.

[0019] The present invention also provides a preparation method of a hybridoma cell line secreting monoclonal antibodies against triketone herbicides and their metabolites, including the following steps,

[0020] S1. Prepare the hapten of triketone herbicides and their metabolites and the complete antigen of triketone herbicides and their metabolites, and prepare the complete antigen of triketone herbicides and their metabolites into Freund's complete adjuvant containing antigen and Freund's incomplete adjuvant containing antigen;

[0021] S2. Perform the first immunization, booster immunization and boost immunization on the immunized animal. The first immunization uses the Freund's complete adjuvant containing antigen described in S1, the booster immunization uses the Freund's incomplete adjuvant containing antigen described in S1, and the boost immunization uses the complete antigen of triketone herbicides and their metabolites described in S1;

[0022] S3. Take the spleen cells and myeloma cells of the immunized animal after the boost immunization in S2 for cell fusion to obtain the hybridoma cell line of the monoclonal antibody against triketone herbicides and their metabolites.

[0023] In an embodiment of the present invention, in S1, the structural formula of the hapten of triketone herbicides and their metabolites is as follows:

[0024]

[0025] In an embodiment of the present invention, in S1, the preparation method of the hapten of triketone herbicides and their metabolites specifically includes the following steps: Weigh 128 mg of mesotrione and dissolve it in 1.5 mL of concentrated hydrochloric acid, add 85 mg of stannous chloride, stir and react at 60 °C for 6 hours. After the reaction is completed, add 10 mL of ice water and adjust the pH to 8 with 5 mol / L sodium hydroxide solution, extract with 20 mL of dichloromethane three times, combine the organic phases, dry with anhydrous magnesium sulfate, concentrate under reduced pressure, and separate and purify by column chromatography to obtain the hapten of triketone herbicides and their metabolites.

[0026] In an embodiment of the present invention, in S1, the preparation method of the complete antigen of triketone herbicides and their metabolites includes the following steps: Mix and dissolve the hapten of triketone herbicides and their metabolites, sodium nitrite and hydrochloric acid in a solvent, react to obtain a mixed solution, and then add the mixed solution to a carrier protein solution for reaction to obtain the complete antigen of triketone herbicides and their metabolites.

[0027] In one embodiment of the present invention, the carrier protein is chicken ovalbumin (OVA) and / or keyhole limpet hemocyanin (KLH).

[0028] In one embodiment of the present invention, the solvent is N,N-dimethylformamide (DMF).

[0029] In one embodiment of the present invention, in S1, the antigen-containing Freund's complete adjuvant is an emulsion formed by mixing Freund's complete adjuvant and a complete antigen of triketone herbicides and their metabolites in equal volumes.

[0030] In one embodiment of the present invention, in S1, the antigen-containing Freund's incomplete adjuvant is an emulsion formed by mixing Freund's incomplete adjuvant and a complete antigen of triketone herbicides and their metabolites in equal volumes.

[0031] In one embodiment of the present invention, in S1, the entire immunization process includes 1 primary immunization, 3 - 5 booster immunizations, and 1 boost immunization.

[0032] In one embodiment of the present invention, in S2, during the entire immunization process, the interval between the primary immunization and the booster immunization is 28 - 31 days, the interval between booster immunizations is 20 - 22 days, and the interval between the booster immunization and the boost immunization is 18 - 21 days.

[0033] In one embodiment of the present invention, in S2, the primary immunization and the booster immunization are administered into the immunized animal through subcutaneous injection in the back; the boost immunization is administered into the immunized animal through intraperitoneal injection.

[0034] In one embodiment of the present invention, in S2, during the booster immunization process, blood is collected from the immunized animal, and the serum titer and inhibition rate are detected by indirect competitive enzyme-linked immunosorbent assay (icELISA) to screen out immunized animals with high titer and good inhibition.

[0035] In one embodiment of the present invention, the blood collection is performed on the 6 - 8th day after the end of the booster immunization process.

[0036] In one embodiment of the present invention, in S3, cell fusion is to culture the fused cells in HAT medium, detect positive cell wells by indirect ELISA, and further determine the inhibition effect of positive cell wells by indirect competitive ELISA method. The positive cell wells with the best inhibition are subcloned by the limiting dilution method to obtain hybridoma cell lines.

[0037] In one embodiment of the present invention, the HAT medium is RPMI - 1640 medium.

[0038] In one embodiment of the present invention, the number of subcloning is 2 - 4 times.

[0039] In one embodiment of the present invention, in S3, the cell fusion is carried out by the polyethylene glycol (PEG 1500) method.

[0040] In one embodiment of the present invention, in S3, the cell fusion is carried out 2 - 4 days after the booster immunization ends.

[0041] The present invention also provides a method for preparing a monoclonal antibody against a triketone herbicide and its metabolite, comprising the following steps: injecting sterile paraffin oil into the abdominal cavity of an immunized animal, then injecting the hybridoma cell line with the preservation number CGMCC No. 46231 into the abdominal cavity. After injection, ascites is collected, and the ascites is purified to obtain the monoclonal antibody against the triketone herbicide and its metabolite, which is stored at low temperature.

[0042] The above technical solution of the present invention has the following advantages compared with the prior art:

[0043] The present invention provides a hybridoma cell line secreting a monoclonal antibody against a triketone herbicide and its metabolite and its application. The monoclonal antibody secreted by the hybridoma cell line provided by the present invention has good broad-spectrum specificity for the triketone herbicide and its metabolite (the cross-reactivity rates with mesotrione, mesotrione metabolites MNBA and AMBA, SUL and its metabolite CMBA are 100%, 46.2%, 44.4%, 51.7% and 33.7% respectively, and the cross-reactivity rates with other herbicide pesticides such as nitrofen, acifluorfen, oxyfluorfen, fomesafen, dicamba and lactofen are less than 1%) and detection sensitivity (IC 50 values are 1.13 ng / mL respectively), and can realize the detection of the residues of triketone herbicides and their metabolites in brown rice and soybeans, providing raw materials for the immunological detection of triketone herbicides and their metabolites residues in food, and having practical application value.

[0044] Biological material preservation

[0045] A hybridoma cell line HXJA secreting a monoclonal antibody against a triketone herbicide and its metabolite has been deposited in the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing 100101. It is classified and named as a monoclonal cell line. The deposit date is October 31, 2024, and the deposit number is CGMCC No. 46231. Brief description of the drawings

[0046] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in conjunction with the drawings, wherein,

[0047] Figure 1This is the inhibition standard curve of the monoclonal antibody against triketone herbicides and their metabolites of the present invention for mesotrione. Specific Embodiments

[0048] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0049] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0050] (1) The culture media involved in the following embodiments are as follows:

[0051] RPMI-1640 culture medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23.19, L-valine 20, p-aminobenzoic acid 1, calcium nitrate 100, anhydrous magnesium sulfate 48.84, anhydrous sodium dihydrogen phosphate 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, D-calcium pantothenate 0.25, folic acid 1, i-inositol 35, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B12 0.005, sodium bicarbonate 2000.

[0052] (2) The reagents involved in the following embodiments are as follows:

[0053] Carbonate buffer solution (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them separately in a small amount of double-distilled water, then mix them, add double-distilled water to about 800 mL and mix well, adjust the pH value to 9.6, add double-distilled water to a volume of 1000 mL, and store at 4°C for later use;

[0054] Phosphate buffer solution (PBS): 8.00 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, 2.9 g of Na2HPO4·12H2O, dissolve them in 800 mL of pure water, adjust the pH to 7.2 - 7.4 with NaOH or HCl, and make up to 1000 mL with ultrapure water;

[0055] Washing solution (PBST): Add 0.5 mL of Tween-20 to 1000 mL of 0.01 mol / L PBS solution with pH 7.4;

[0056] PBST: PBS containing 0.05% Tween-20;

[0057] Antibody diluent: Washing solution containing 0.1% gelatin;

[0058] TMB chromogenic solution: Solution A: 18.43 g of Na2HPO4·12H2O and 9.33 g of citric acid, make up the volume to 1000 mL with pure water; Solution B: Dissolve 60 mg of TMB in 100 mL of ethylene glycol. Mix Solution A and Solution B in a volume ratio of 5:1 to obtain the TMB chromogenic solution, and mix it immediately before use.

[0059] (3) The detection methods involved in the following examples are as follows:

[0060] Detection method for the inhibition rate of mesotrione: Select the most suitable antigen and antibody concentrations in ic-ELISA through checkerboard experiments. Dilute the antigen to 1, 0.3, 0.1, and 0.03 μg / mL with carbonate buffer solution (CBS), and dilute the antibody to 1, 0.3, 0.1, and 0.03 μg / mL with antibody diluent. After selecting the optimal working point, dilute the mesotrione standard product to concentrations such as 15, 6, 3, 1.5, 0.75, 0.3, 0.15, and 0 ng / mL, follow the ic-ELISA operation steps, and finally plot the graph with OriginPro 8.5 to obtain the mesotrione standard inhibition curve and calculate IC 50 。

[0061] Example

[0062] This example provides a hybridoma cell line secreting monoclonal antibodies against triketone herbicides and their metabolites and a preparation method thereof, which specifically includes the following steps:

[0063] (1) Preparation of complete antigen

[0064] a. Synthesis of hapten, the reaction equation is as follows:

[0065]

[0066] A brief description of the derivation steps is as follows:

[0067] Preparation of triketone herbicide and its metabolite hapten: Weigh 128 mg of mesotrione and dissolve it in 1.5 mL of concentrated hydrochloric acid. Add 85 mg of stannous chloride and stir at 60 °C for 6 hours. After the reaction is completed, add 10 mL of ice water and adjust the pH to 8 with 5 mol / L sodium hydroxide solution. Extract with 20 mL of dichloromethane three times and combine the organic phases. Dry with anhydrous magnesium sulfate, concentrate under reduced pressure, and separate and purify by column chromatography to obtain the triketone herbicide and its metabolite hapten MES-NH2.

[0068] b. Weigh 8 mg of the prepared MES-NH2 and dissolve it in 200 μL of DMF. Add 76.8 μL of 1 mol / L hydrochloric acid solution under stirring at 0 °C and react for 30 minutes. Then add 20 μL of 10% sodium nitrite solution and react for 1 hour. The resulting mixture is called solution A. Then weigh 10 mg of keyhole limpet hemocyanin KLH and dissolve it in 2 mL of carbonate buffer solution, which is called solution B. Slowly drip solution A into solution B and react at room temperature with stirring for 12 h. Dialyze with 0.01 mol / L phosphate buffer PBS for 3 days to obtain the conjugate mesotrione-KLH, and store it at -20 °C for later use.

[0069] (2) Preparation of coating antigen:

[0070] Weigh 4 mg of the prepared MES-NH2 and dissolve it in 200 μL of DMF. Add 38.4 μL of 1 mol / L hydrochloric acid solution under stirring at 0 °C and react for 30 minutes. Then add 10 μL of 10% sodium nitrite solution and react for 1 hour. The resulting mixture is called solution A. Then weigh 10 mg of ovalbumin OVA and dissolve it in 2 mL of carbonate buffer solution, which is called solution B. Slowly drip solution A into solution B and react at room temperature with stirring for 12 h. Dialyze with 0.01 mol / L phosphate buffer PBS for 3 days to obtain the conjugate mesotrione-OVA, and store it at -20 °C for later use.

[0071] (3) Immunization of mice: Select healthy BALB / c mice aged 6 - 8 weeks for immunization. Mix the triketone herbicide and its metabolite complete antigen with an equal amount of Freund's adjuvant and emulsify it. Then immunize BALB / c mice by subcutaneous injection in the back. Use complete Freund's adjuvant for the first immunization and incomplete Freund's adjuvant for subsequent immunizations. The interval between the first immunization and the second booster immunization is 28 days, and the interval between multiple booster immunizations is 21 days. Collect blood 7 days after the third immunization (5 μL of mouse tail blood + 995 μL of antibody diluent = antiserum), and use ic-ELISA to measure the titer and inhibition of mouse serum. Select mice with high titer and good inhibition, and conduct a boost immunization 21 days after the fifth immunization by intraperitoneal injection. The boost immunization dose is required to be halved and without any adjuvant.

[0072] (4) Cell fusion: Three days after the boost immunization, perform cell fusion according to the conventional PEG (polyethylene glycol, molecular weight 1500) method. The specific steps are as follows:

[0073] a. Eyeball enucleation and blood collection: After sacrificing the mice by cervical dislocation, immediately place them in 75% alcohol for disinfection and soak for about 5 minutes. Under sterile conditions, remove the spleens of the mice, moderately grind them with the rubber head of a syringe, and obtain a spleen cell suspension through a 200-mesh cell sieve. Collect the suspension and centrifuge it (1200 rpm, 8 minutes). Wash the spleen cells three times with RPMI-1640 medium. After the last centrifugation, dilute the spleen cells to a certain volume, count them, and set aside for use;

[0074] b. Collection of murine myeloma SP2 / 0 cells: Seven to ten days before fusion, culture the SP2 / 0 tumor cells in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. Before fusion, it is required that the number of SP2 / 0 tumor cells reaches (1 - 4)×10 7 , ensuring that the SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. At the time of fusion, collect the tumor cells, suspend them in RPMI-1640 basal culture medium, and perform cell counting;

[0075] c. The fusion process takes 7 minutes. In the first minute, slowly add 1 mL of PEG 1500 dropwise to the cells at an increasing rate; in the second minute, let it stand still. In the third and fourth minutes, add 1 mL of RPMI-1640 medium dropwise within 1 minute; in the fifth and sixth minutes, add 2 mL of RPMI-1640 medium dropwise within 1 minute; in the seventh minute, add 1 mL of RPMI-1640 medium every 10 seconds. Then incubate at 37°C for 5 minutes. Centrifuge (800 rpm, 8 minutes), discard the supernatant, resuspend the cells in RPMI-1640 screening culture medium containing 20% fetal bovine serum and 2% of 50×HAT, and add 200 μL / well to a 96-well cell plate, and culture it in a 37°C, 5% CO2 incubator.

[0076] (5) Cell screening and cell line establishment: On the 3rd day after cell fusion, perform a semi-medium change of the fusion cells with RPMI-1640 screening culture medium. On the 5th day, perform a full medium change with RPMI-1640 transition culture medium containing 20% fetal bovine serum and 1% of 100×HT. On the 7th day, take the cell supernatant for screening. The screening is carried out in two steps: First, use ic-ELISA to screen out positive cell wells. Second, use mesotrione as a standard product and use ic-ELISA to measure the inhibitory effect on the positive cells. Select cell wells that have good inhibition on the mesotrione standard product, and perform subcloning by the limiting dilution method, and detect them by the same method, repeating three times to obtain a cell line.

[0077] Test Example

[0078] (1) Preparation and identification of monoclonal antibodies

[0079] 8 - 10 - week - old BALB / c mice were intraperitoneally injected with 1 mL of sterile paraffin oil per mouse; 7 days later, each mouse was intraperitoneally injected with 1×10 6 hybridoma cells. Ascites were collected starting from the 7th day, and the ascites were purified by the caprylic acid - ammonium sulfate method. Under acidic conditions, caprylic acid can precipitate other miscellaneous proteins in the ascites except for IgG immunoglobulins, and then centrifuged to discard the precipitate; then, an ammonium sulfate solution with an equal saturation was used to precipitate the monoclonal antibody of the IgG type, centrifuged to discard the supernatant, dissolved in 0.01M PBS solution (pH 7.4), and dialyzed to desalt. Finally, the purified monoclonal antibody was stored at - 20°C.

[0080] Using the indirect competitive ELISA method, the IC 50 of the monoclonal antibody against mesotrione was determined to be 1.13 ng / mL, and its IC 50 against metabolites and analogs and the cross - reaction rate were verified. The cross - reaction rates of the monoclonal antibody against the mesotrione metabolites MNBA and AMBA, SUL and its metabolite CMBA were 46.2%, 44.4%, 51.7% and 33.7% respectively. The cross - reaction rates with other herbicide pesticides such as nitrofen, acifluorfen, oxyfluorfen, fomesafen, dicamba and lactofen were less than 1%. Among them, the cross - reaction rate = (IC 50 of mesotrione / IC 50 of metabolite or analog) × 100%. According to the cross - reaction rate values, it can be seen that the antibody has high sensitivity and specificity to mesotrione and its metabolites, as shown in Table 1 specifically.

[0081] Table 1 shows the IC 50 and cross - reaction rates of the monoclonal antibody against mesotrione and its metabolites 4 - mesosulfonyl - 2 - nitrobenzoic acid MNBA and 2 - amino - 4 - (methylsulfonyl) benzoic acid AMBA, sulcotrione SUL and its metabolite 2 - chloro - 4 - mesosulfonylbenzoic acid CMBA, and other herbicide pesticides such as nitrofen, acifluorfen, oxyfluorfen, fomesafen, dicamba and lactofen:

[0082] Table 1

[0083]

[0084] (2) Antibody application

[0085] The monoclonal antibody prepared from the hybridoma cell line through in - vivo ascites was applied to the spiking and recovery test of mesotrione. The specific steps are as follows:

[0086] a. Coating: The coating antigen mesotrione - OVA was serially diluted starting from 1 μg / mL with 0.05M pH 9.6 carbonate buffer, 100 μL per well, and reacted at 37°C for 2 h.

[0087] b. Washing: Pour out the solution in the plate and wash it 3 times with the washing solution for 3 minutes each time.

[0088] c. Blocking: After patting dry, add 200 μL / well of the blocking solution and react at 37 °C for 2 h. After washing, dry it for standby.

[0089] d. Sample addition: Dilute the antiserum (after collecting blood from the mouse's tail tip and diluting it by the corresponding multiple with the antibody diluent, it becomes the antiserum) serially from 1:1000 and add it to the coated wells at 100 μL / well, and react at 37 °C for 30 min; after thorough washing, add HRP-goat anti-mouse IgG diluted 1:3000 at 100 μL / well and react at 37 °C for 30 min.

[0090] e. Color development: Take out the enzyme-linked immunosorbent assay (ELISA) plate, after thorough washing, add 100 μL of the TMB color development solution to each well and react at 37 °C in the dark for 15 min.

[0091] f. Termination and measurement: Add 50 μL of the termination solution to each well to terminate the reaction, and then measure the OD value of each well with an ELISA reader. 450 Value.

[0092] The inhibition standard curve of the monoclonal antibody against triketone herbicides and their metabolites to mesotrione is as Figure 1 shown. The IC of the monoclonal antibody to mesotrione determined by ic-ELISA 50 is 1.13 ng / mL, indicating good sensitivity to mesotrione. For the recovery rate, brown rice and soybean samples purchased from a local supermarket in Wuxi were detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS). After LC-MS / MS detection, no mesotrione residues were detected in the three samples. Therefore, in this implementation case, the recovery rate was determined by adding mesotrione. Specifically, as shown in Table 2, the average recovery rate of LC-MS / MS is 84 - 109.44%, and the relative standard deviation (RSD) is 2.59 - 8.79%; the recovery rate determined by indirect competitive ELISA is 86.53 - 96.89%, and the RSD is 3.93 - 8.97%. It shows that the method for detecting mesotrione based on the monoclonal antibody against triketone herbicides and their metabolites is relatively accurate and can be used for the immunoassay detection of mesotrione pesticide residues in agricultural products.

[0093] Table 2 shows the spiked recovery rates of the monoclonal antibody to mesotrione in brown rice and soybeans:

[0094] Table 2

[0095]

[0096]

[0097] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A hybridoma cell line secreting monoclonal antibodies against triketone herbicides and their metabolites, characterized in that: The hybridoma cell line was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on October 31, 2024, with the deposit number CGMCC No.46231. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named as a monoclonal cell line.

2. The hybridoma cell line according to claim 1, characterized in that The triketone herbicide is selected from mesotrione and / or sulcotrione; And / or, the metabolite is at least one of 4-methylsulfonyl-2-nitrobenzoic acid, 2-amino-4-(methylsulfonyl)benzoic acid, sulcotrione and 2-chloro-4-methylsulfonylbenzoic acid.

3. A triketone herbicide and a monoclonal antibody to its metabolites, characterized in that: The hybridoma cell line is secreted and produced by the hybridoma cell line according to claim 1 or 2.

4. A triketone herbicide and its metabolite hapten, used for preparing the hybridoma cell line according to claim 1 or 2, characterized in that: The structural formula of the triketone herbicide and its metabolite hapten is:

5. A composition for detecting triketone herbicides and their metabolites, characterized in that: The composition contains the hybridoma cell line according to claim 1 or 2 and / or the monoclonal antibody against the triketone herbicide and its metabolites according to claim 3.

6. A kit for detecting triketone herbicides and their metabolites, characterized in that: The kit contains at least one of the hybridoma cell line according to claim 1 or 2, the monoclonal antibody against triketone herbicides and their metabolites according to claim 3, and the composition according to claim 5.

7. The kit according to claim 6, characterized in that The kit is selected from an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

8. A test strip for detecting triketone herbicides and their metabolites, characterized in that: The test strip contains at least one of the hybridoma cell line according to claim 1 or 2, the monoclonal antibody against the triketone herbicide and its metabolites according to claim 3, and the composition according to claim 5.

9. A chip for detecting triketone herbicides and their metabolites, characterized in that: The chip contains at least one of the hybridoma cell line according to claim 1 or 2, the monoclonal antibody against triketone herbicides and their metabolites according to claim 3, and the composition according to claim 5.

10. Use of the hybridoma cell line according to claim 1 or 2, the monoclonal antibody against triketone herbicides and their metabolites according to claim 3, the composition according to claim 5, the kit according to claim 6 or 7, the test strip according to claim 8 or the chip according to claim 9 in detecting triketone herbicides and their metabolites.

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