Hybridoma cell strain secreting isoxadifen-ethyl monoclonal antibody and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain secreting a isoxadifen-ethyl monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of immunodetection. The hybridoma cell strain capable of secreting the isoxadifen-ethyl acid monoclonal antibody is screened for the first time, the monoclonal antibody separated from the hybridoma cell strain has good sensitivity and specificity to isoxadifen-ethyl acid, and the IC50 value of the monoclonal antibody is 0.2579 ng / mL; and the isoxadifen-ethyl derivative has no cross reaction on isoxadifen-ethyl structural analogues such as DDT, pyroxasulfone, oxaflutole, topramezone, pyribenzoxim and the like. Therefore, the monoclonal antibody disclosed by the invention can be used for establishing an immunological detection method of isoxadifen-ethyl so as to detect residues of isoxadifen-ethyl in food.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a hybridoma cell line that secretes a monoclonal antibody against bis(oxazolyl) acid and its application. Background Technology

[0002] Bismuthoxazol is a herbicide safener specifically designed to protect crops from herbicide damage. Primarily used in corn and rice paddies, it protects crops and enhances weed control. Studies have shown that it simultaneously upregulates the activity of key enzyme systems in crops, including cytochrome P450 monooxygenase, glutathione S-transferase, and hydrolases, thereby accelerating the oxidation, conjugation, and hydrolysis of herbicide molecules, converting them into non-toxic or low-toxic products for storage. It can enter the human body through skin contact, inhalation, or ingestion. Environmental residues and toxicity: Bismuthoxazol readily remains in soil and water bodies, exhibiting high toxicity to aquatic organisms (such as fish) and posing a threat to non-target organisms such as bees and birds. Its high mobility may also pose a risk of groundwater pollution. Due to its long persistence, it can accumulate in soil. Furthermore, it may cause long-term adverse effects in aquatic environments. Although these substances are classified as priority toxic pollutants by the U.S. Environmental Protection Agency, their pure forms or combinations with other substances are used in various herbicides in many parts of the world. To mitigate the negative health effects of bis(oxazolyl)amine, many countries have established maximum residue limits (MRLs) for its use in agricultural products. The European Union's Regulation 396 / 2005 sets the maximum residue limit for bis(oxazolyl)amine at 0.05 mg / kg. Detecting the presence of bis(oxazolyl)amine residues in agricultural products is crucial for protecting human health.

[0003] Currently, the detection methods for bis(oxazolonic acid) are mainly instrumental, with commonly used methods including high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), high-performance liquid chromatography-mass spectrometry (HPLC-MS), and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). Although these chromatographic methods have high sensitivity and specificity, they also have some drawbacks, such as the need for thorough sample purification, high solvent consumption, expensive equipment, and skilled technicians. Therefore, a rapid and simple method for detecting bis(oxazolonic acid) residues is needed.

[0004] Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method. It requires simple sample pretreatment, involves few purification steps, has a large analytical capacity, low detection cost, and is easy to operate, making it suitable for rapid on-site detection of large numbers of samples. Therefore, it is widely used in drug residue analysis. However, the prerequisite for using ELISA to detect bis(benzoxazolonic acid) is obtaining a monoclonal antibody with high specificity and sensitivity to bis(benzoxazolonic acid). Currently, such a monoclonal antibody is lacking. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a highly specific and highly sensitive bisbenzoxazole monoclonal antibody in the prior art.

[0006] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes a monoclonal antibody against bis(benzoxazolyl) acid and its applications. This invention is the first to screen a hybridoma cell line capable of secreting a monoclonal antibody against bis(benzoxazolyl) acid. The monoclonal antibody isolated from this hybridoma cell line exhibits good sensitivity and specificity to bis(benzoxazolyl) acid, with an IC50 value of [missing information]. 50 The value was 0.2579 ng / mL, and it showed no cross-reactivity with dibenzoxazole acid structural analogs such as DDT, sulfonamide, benzyloxazine, benzoxazine, and pyrimimethoxazole. Therefore, the monoclonal antibody of this invention can be used to establish an immunological detection method for dibenzoxazole acid to detect its residues in food.

[0007] The first objective of this invention is to provide a hybridoma cell line, characterized in that the hybridoma cell line is named a monoclonal cell line and has the accession number CGMCC No. 46735.

[0008] Furthermore, the hybridoma cell line is obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structural formula of the hapten is shown in Formula I:

[0009] .

[0010] Furthermore, the complete antigen is obtained from the hapten coupled with a carrier protein.

[0011] Furthermore, the carrier protein includes keyhole hemocyanin.

[0012] A second objective of this invention is to provide an application of the above-described hybridoma cell line in the detection of bis(oxazol) acid.

[0013] A third objective of this invention is to provide a monoclonal antibody secreted by the aforementioned hybridoma cell line.

[0014] A fourth objective of this invention is to provide an application of the above-described monoclonal antibody in the detection of bis(oxazolium) acid.

[0015] A fifth object of the present invention is to provide a detection product for bis(oxazolidine) acid, the detection product comprising the above-mentioned monoclonal antibody.

[0016] Furthermore, the test product also includes a coating agent.

[0017] Furthermore, the coating is prepared from a hapten-conjugated carrier protein, wherein the carrier protein includes chicken ovalbumin.

[0018] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0019] The monoclonal antibody isolated from the hybridoma cell line described in this invention exhibits good sensitivity and specificity to bis(oxazolyl) acid, with an IC50 value of [missing information]. 50 The value was 0.2579 ng / mL, and it showed no cross-reactivity with dibenzoxazole acid structural analogs such as DDT, sulfonamide, benzyloxazine, benzoxazine, and pyrimimethoxazole. Therefore, the monoclonal antibody of this invention can be used to establish an immunological detection method for dibenzoxazole acid to detect its residues in food.

[0020] Preservation of biological materials

[0021] The monoclonal cell line NTZ was deposited on November 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46735, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0022] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0023] Figure 1 This is the standard curve of the inhibition of bisphenyloxazolidine monoclonal antibody against bisphenyloxazolidine in this invention. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0025] The culture media involved in the following examples are as follows:

[0026] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cysteine ​​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, Para-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.

[0027] The reagents involved in the following examples are as follows:

[0028] Carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800 mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000 mL. Store at 4°C for later use.

[0029] Phosphate-buffered saline (PBS): Dissolve 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, and 2.9g Na2HPO4·12H2O in 800 mL of pure water, adjust the pH to 7.2-7.4 with NaOH or HCl, and bring the volume to 1000 mL.

[0030] PBST: PBS containing 0.05% Tween 20;

[0031] Antibody dilution buffer: PBS containing 0.1% gelatin;

[0032] TMB colorimetric solution: Solution A: 18.43 g Na2HPO4·12H2O, 9.33 g citric acid, diluted to 1000 mL with pure water; Solution B: 60 mg TMB dissolved in 100 mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution, and mix again before use.

[0033] The detection methods involved in the following embodiments are as follows:

[0034] Method for detecting the inhibition rate of bis(oxazolidine)-C: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The antigen was diluted to 0.01, 0.03, 0.1, and 0.3 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03, 0.1, 0.3, and 1 μg / mL with antibody diluent. After selecting the optimal operating point, the bis(oxazolidine)-C standard was diluted to eight concentrations (0, 0.021, 0.062, 0.185, 0.56, 1.67, 5, and 15 ng / mL). Following the ic-ELISA procedure, the results were plotted using OriginPro 8.5 (see results below). Figure 1 (As shown), the standard inhibition curve of bis(oxazolidine) was obtained, and the IC50 was calculated. 50 .

[0035] Example 1: Synthesis of complete bis(oxazolyl) acid antigen

[0036] Weigh 6 mg of bis(oxazolyl) hapten and 4.4 mg of N-hydroxysuccinimide (NHS), dissolve them in 200 μL of N,N-dimethylformamide (DMF), and stir at room temperature for 10 min. Then weigh 7.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and add it to the bis(oxazolyl) hapten solution, stirring at room temperature for 6-8 h to activate it. Take 6 mg of keyhole hemocyanin (KLH), add it to 3 mL of 0.01 M carbonate buffer (CBS), dissolve it thoroughly, and slowly add the activated hapten to the diluted KLH solution, stirring overnight at room temperature. Then dialyze with 0.01 M PBS to remove unreacted small molecules, obtaining a relatively pure complete antigen, which is then identified by UV absorption scanning.

[0037] Example 2: Synthesis of the bis(oxazolyl) acid coating agent

[0038] 3.2 mg of bis(oxazolyl) hapten and 2.4 mg of N-hydroxysuccinimide (NHS) were dissolved in 200 μL of anhydrous N,N-dimethylformamide (DMF) and reacted with stirring at room temperature for 10 min. 4.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in the above solution and reacted with stirring at room temperature for 6–8 h to obtain the hapten activation solution. 6 mg of chicken ovalbumin (OVA) was dissolved in carbonate buffer (CBS). The hapten activation solution was slowly added to the protein dilution solution and stirred overnight at room temperature. Then, the reaction solution was dialyzed against 0.01 M PBS to remove unreacted small molecules, yielding the coating antigen.

[0039] Example 3: Preparation of hybridoma cell lines secreting biphenyloxazolium monoclonal antibodies

[0040] 1. Acquisition of immunity in animals

[0041] The complete antigen of bis(oxazolidinedionate) was emulsified with an equal amount of Freund's adjuvant and then administered to BALB / c mice via subcutaneous injection at multiple sites on the back of the neck (except for sprint immunization). The first immunization used complete Freund's adjuvant at a dose of 100 μg / mouse; subsequent booster immunizations used incomplete Freund's adjuvant at a dose halved to 50 μg / mouse; sprint immunizations did not use adjuvants, but were directly diluted with physiological saline and injected intraperitoneally at a dose halved to 25 μg / mouse. The interval between the first and second booster immunizations was one month, the interval between multiple booster immunizations was 21 days, and the interval between the sprint immunization and the last booster immunization was 18-21 days. The immunization effect in mice was observed by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), i.e., the titer and inhibition of mouse serum were detected.

[0042] 2. Cell fusion

[0043] Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 4000) method, with the following specific steps:

[0044] a. After euthanizing the mouse by tail dislocation and cervical dislocation, immediately disinfect the mouse in 75% alcohol for about 5 minutes. Under aseptic conditions, remove the spleen and grind it moderately with the rubber tip of a syringe and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect the suspension, centrifuge (1200 rpm, 8 min), wash the spleen cells three times with RPMI-1640 medium, and after the last centrifugation, dilute the spleen cells to a certain volume, count them, and set them aside for later use.

[0045] b. Collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells are expanded in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The number of SP2 / 0 tumor cells should reach 1-4 × 10⁶ cells before fusion. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, tumor cells are collected and suspended in RPMI-1640 basal culture medium for cell counting during fusion.

[0046] c. Fusion process (7 min): At min 1, add 1 mL of PEG 4000 to the cells dropwise, gradually increasing the speed; at min 2, allow to stand; at min 3 and min 4, add 1 mL of RPMI-1640 medium dropwise over 1 min; at min 5 and min 6, add 2 mL of RPMI-1640 medium dropwise over 1 min; at min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s. Except for min 2, continuously agitate the solution. Then incubate at 37°C for 5 min; centrifuge (800 rpm, 8 min), discard the supernatant, and resuspend in RPMI-1640 selection medium containing 20% ​​fetal bovine serum and 2% 50×HAT. Add 200 μL / well to a 96-well cell plate and incubate at 37°C in a 5% CO2 incubator.

[0047] 3. Cell selection and cell line establishment

[0048] On day 3 after cell fusion, the fused cells were partially replaced with RPMI-1640 selection medium. On day 5, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On day 7, the cell supernatant was collected for screening.

[0049] The screening process consists of two steps: First, positive cell wells are selected using ic-ELISA. Second, bis(oxazolamide) is used as a standard, and the inhibitory effect on positive cells is determined using ic-ELISA.

[0050] Cell wells that showed good inhibition of the bis(oxazol) acid standard were selected, and subcloning was performed using the limiting dilution method. The cells were then tested using the same method after seven days.

[0051] At least three subcloning operations were performed using the method described above to finally obtain the bis(oxazol) monoclonal antibody cell line.

[0052] Example 5: Preparation and Identification of Bisbenoxazolic Acid Monoclonal Antibody

[0053] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6 Ascites fluid was collected from the bis(oxazol) hybridoma cells starting from day 7, and the ascites fluid was purified for antibody using the caprylic acid-saturated ammonium sulfate method.

[0054] Under slightly acidic conditions, octanoic acid can precipitate other proteins in the ascites fluid besides IgG immunoglobulin. After centrifugation, the precipitate is discarded. Then, an equal volume of saturated ammonium sulfate solution is used to precipitate IgG-type monoclonal antibodies. After centrifugation, the supernatant is discarded. The antibody is dissolved in 0.01M PBS solution (pH 7.4), dialyzed to desalt, and finally purified monoclonal antibodies are obtained and stored at -20 °C.

[0055] Using an indirect competitive ELISA, the monoclonal antibody against bisphenyloxazine showed good detection sensitivity (IC50) for bisphenyloxazine. 50 With a value of 0.2579 ng / mL, it can be used for the immunoassay of bis(oxazolidinedionate).

[0056] Cross-reactivity experiments were conducted with dibenzoxazolone structural analogs (DDT, sulfonamide, benzyloxazol, benzoxazolone, and pyrimimethoxam). The results showed that the absorbance values ​​of the enzyme-labeled plate for dibenzoxazolone in each well did not differ significantly with the dilution concentration of the standard. Specific results are shown in Table 1. This indicates that the monoclonal antibody has a very low cross-reactivity rate with other structural analogs but exhibits good sensitivity to dibenzoxazolone.

[0057] Cross-reactivity rate (%) = (dibenzoxazole acid IC50) 50 ) / (Similar IC 50 )×100%

[0058] Table 1 Cross-reactivity of bis(oxazolyl) acid monoclonal antibodies

[0059]

[0060] Example 6: Application of Bisbenoxazol Acid Monoclonal Antibody

[0061] The monoclonal antibody prepared from hybridoma cell lines via in vivo ascites fluid was used in an ELISA addition and recovery assay for bis(oxazolidine) acid. The specific steps are as follows:

[0062] (1) Coat a 96-well microplate with 0.1 μg / mL of the coating stock diluted with carbonate buffer (CBS), 100 μL per well, dry at 37℃ for 2 h, wash the plate three times with PBST washing buffer, 200 μL per well each time, for 3 min each time, and pat dry.

[0063] (2) Block with CBS containing 0.2% gelatin, 200 μL per well, dry at 37℃ for 2 h, wash the plate three times with PBST washing solution, 200 μL per well each time, 3 min each time, and pat dry;

[0064] (3) Prepare 0, 0.021, 0.062, 0.185, 0.56, 1.67, 5 and 15 ng / mL bis(oxazol) standard solutions using phosphate-buffered saline (PBS). Add the standard solutions and the extracts of the samples to be tested to the sealed microplates, 50 μL per well, and repeat each sample in 3 wells. Then add 50 μL of bis(oxazol) monoclonal antibody diluted to 0.1 μg / mL to each well. After reacting at 37℃ for 30 min, wash the plate and pat dry.

[0065] (4) Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 with PBS containing 0.1% gelatin to each well, react at 37°C for 30 min, then wash the plate and pat dry.

[0066] (5) Add 100 μL of TMB colorimetric solution to each well, develop the color at 37℃ for 15 min, then add 50 μL of 2 M H2SO4 stop solution to each well, and measure the absorbance at 450 nm.

[0067] (6) Addition of recovery and sample pretreatment:

[0068] Corn was selected as the test sample.

[0069] After pulverizing the sample, it was passed through a 20-mesh standard sieve. Three 20 g portions of the sample were weighed, and 0.05 ppb, 0.2 ppb, and 1 ppb of bis(oxazolyl) acid standard were added to each portion respectively (based on the antibody linear range and IC50). 50 (Set the concentration), add 10 mL of water, vortex mix, let stand for 30 min, and filter. Add 50 mL of acetone to the sample, shake on an electric shaker for 30 min, filter through rapid qualitative filter paper into a beaker, extract the residue again with 30 mL of acetone using the above method, wash the residue twice with 30 mL of acetone, combine the washings in a beaker, concentrate to near dryness in a 50℃ water bath, and reconstitute with 5 mL of 10% acetone PBS solution (i.e., dilute five times to reduce the influence of the sample matrix).

[0070] Spiking and recovery experiments were performed using indirect competitive ELISA, and the recoveries of bis(oxazolidine) ranged from 85.37% to 102.2%.

[0071] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A hybridoma cell line, characterized in that, The hybridoma cell line was named a monoclonal cell line and its accession number was CGMCC No. 46735.

2. The hybridoma cell line according to claim 1, characterized in that, The hybridoma cell line was obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structural formula of the hapten is shown in Formula I: 。 3. The hybridoma cell line according to claim 2, characterized in that, The complete antigen is obtained by conjugating the hapten to a carrier protein.

4. The hybridoma cell line according to claim 3, characterized in that, The carrier protein includes keyhole hemocyanin.

5. The use of the hybridoma cell line according to any one of claims 1-4 in the detection of bis(oxazol) acid.

6. A monoclonal antibody secreted by a hybridoma cell line according to any one of claims 1-4.

7. The use of the monoclonal antibody according to claim 6 in the detection of bis(oxazol) acid.

8. A detection product for bis(benzoxazolidine) acid, characterized in that, The detection product includes the monoclonal antibody as described in claim 6.

9. The testing product according to claim 8, characterized in that, The tested products also include coating agents.

10. The testing product according to claim 9, characterized in that, The coating is prepared from a hapten-coupled carrier protein, wherein the carrier protein includes chicken ovalbumin.

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