A hybridoma cell strain secreting a monoclone antibody of thiophanate-methyl and its application

Abstract

The present application relates to a kind of secreting thiophanate-methyl monoclonal antibody hybridoma cell strain and its application, belong to immune detection technical field.The present application has screened and prepared a kind of hybridoma cell strain capable of secreting thiophanate-methyl monoclonal antibody, the monoclonal antibody secreted by the hybridoma cell strain of the present application has good sensitivity and specificity to thiophanate-methyl, IC 50 Value is 119.44 ng / mL, and the cross reactivity of the analog (thiophanate-methyl, thiabendazole, macaroni, thirham) of thiophanate-methyl is less than 10%, so low concentration thiophanate-methyl can be accurately detected.

Description

Technical Field

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

[0002] Thiophanate-methyl, chemically known as 1,2-bis-(methoxycarbonyl-2-thiourea)benzene, is converted into carbendazim after being absorbed by plants. It primarily interferes with fungal hyphae formation, affects fungal cell division, causes cell wall poisoning, and results in malformed germ tubes from spore germination, thus killing the pathogen. Its effective period is 5-7 days. It is mainly used for foliar spraying, but can also be used for soil treatment. It can be mixed with hymexazol to control root and soil diseases with significant effects.

[0003] Currently, the detection methods for thiophanate-methyl are mainly instrumental, with gas chromatography, liquid chromatography, and gas chromatography-mass spectrometry being commonly used. Although these chromatographic methods offer high sensitivity and specificity, they also have 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 thiophanate-methyl 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 thiophanate-methyl is obtaining a monoclonal antibody with high specificity and sensitivity to thiophanate-methyl. Therefore, finding a method to prepare a monoclonal antibody with high specificity and sensitivity to thiophanate-methyl is crucial. However, further research is needed on how to prepare thiophanate-methyl hapten and complete antigen, how to induce a strong immune response in mice, how to ensure the successful secretion of thiophanate-methyl monoclonal antibodies by the prepared hybridoma cell lines, and how to ensure that the secreted thiophanate-methyl monoclonal antibodies have high specificity and sensitivity. Summary of the Invention

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

[0006] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes monoclonal antibodies against thiophanate-methyl and its applications. This invention has screened and prepared a hybridoma cell line capable of secreting monoclonal antibodies against thiophanate-methyl. The monoclonal antibody secreted by this hybridoma cell line exhibits good sensitivity and specificity to thiophanate-methyl, with an IC50 value of [missing value]. 50 The value was 119.44 ng / mL, and the cross-reactivity with thiophanate-methyl analogues (thiophanate, carbendazim, thiamethoxam, and thiamethoxam) was less than 10%, thus enabling accurate detection of low concentrations of thiophanate-methyl.

[0007] The first objective of this invention is to provide a hybridoma cell line that secretes a monoclonal antibody against thiophanate-methyl, the hybridoma cell line being proposed to be taxonomically named a monoclonal cell line and having the accession number CGMCC NO.46814.

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

[0009] .

[0010] Furthermore, the complete antigen is obtained by conjugating the first hapten to a carrier protein.

[0011] Furthermore, the carrier protein includes bovine serum albumin.

[0012] A second objective of this invention is to provide an application of the above-mentioned hybridoma cell line in the detection of thiophanate-methyl.

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

[0014] The fourth objective of this invention is to provide an application of the above-mentioned monoclonal antibody in the detection of thiophanate-methyl.

[0015] A fifth objective of this invention is to provide a detection product for thiophanate-methyl, the detection product comprising the aforementioned monoclonal antibody.

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

[0017] Furthermore, the coating is prepared by conjugating a second hapten with a carrier protein; the carrier protein includes ovalbumin, and the structural formula of the second hapten is shown in Formula II:

[0018] .

[0019] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0020] The monoclonal antibody secreted by the hybridoma cell line of the present invention exhibits good sensitivity and specificity against thiophanate-methyl, IC50. 50 The value was 119.44 ng / mL, and the cross-reactivity with thiophanate-methyl analogues (thiophanate-methyl, carbendazim, thiamethoxam, and thiamethoxam) was all less than 10%, thus enabling accurate detection of low concentrations of thiophanate-methyl.

[0021] Preservation of biological materials

[0022] The cell line APZL is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. It is classified as a monoclonal cell line, deposited on April 24, 2026, with accession number CGMCC No. 46814. Attached Figure Description

[0023] 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.

[0024] Figure 1 This is the inhibition standard curve of the methylthiophanate monoclonal antibody of the present invention. Detailed Implementation

[0025] 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.

[0026] The culture media involved in the following examples are as follows:

[0027] 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.

[0028] The reagents involved in the following examples are as follows:

[0029] 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.

[0030] Phosphate buffered saline (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, dissolved in 800 mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000 mL.

[0031] PBST: PBS containing 0.05% Tween 20;

[0032] Antibody dilution buffer: PBS containing 0.1% gelatin;

[0033] 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.

[0034] The detection methods involved in the following embodiments are as follows:

[0035] Method for detecting the inhibition rate of thiophanate-methyl: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. Complete antigen 2 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, thiophanate-methyl standards were diluted to eight concentrations (0, 5.55, 16.67, 50, 150, 450, 1350, and 4050 ng / mL). Following the ic-ELISA procedure, the results were plotted using OriginPro 8.5 (see results below). Figure 1 (As shown), obtain the standard inhibition curve of thiophanate-methyl and calculate IC50. 50 .

[0036] Example 1: Synthesis of thiophanate-methyl hapten

[0037] Since thiophanate-methyl small molecules are not immunogenic and cannot stimulate mice to produce an immune response and thus antibodies, thiophanate-methyl needs to be conjugated to proteins using protein conjugation technology to acquire immunogenicity. Commonly used active groups in protein conjugation technology include amino, carboxyl, hydroxyl, and thiol groups. However, since the thiophanate-methyl molecule does not contain these active groups, derivatization is required to obtain a suitable hapten.

[0038] 100 mg of methyl [(2-aminophenyl)carbonyl]carbamate was dissolved in 10 mL of pyridine, and 60 mg of succinic anhydride was added. The mixture was reacted at 70 °C for 12 hours to obtain hapten 1 (as shown in Formula I).

[0039]

[0040] 100 mg of methyl carbamate [(2-aminophenyl)carbonyl]carbamate was dissolved in 10 mL of pyridine, and 100 mg of phenyl 3-carboxyisothiocyanate was added. The mixture was reacted at room temperature for 12 hours to obtain hapten 2 (as shown in Formula II).

[0041] .

[0042] Example 2: Synthesis of complete thiophanate-methyl antigen

[0043] Weigh 5 mg of thiophanate-methyl hapten No. 1 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 thiophanate-methyl hapten solution, stirring at room temperature for 6-8 h to activate it. Take 6 mg of bovine serum albumin (BSA), add it to 3 mL of 0.01 M carbonate buffer (CBS), dissolve it completely, and slowly add the activated hapten to the diluted BSA 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 ultraviolet absorption scanning.

[0044] Example 3: Synthesis of thiophanate-methyl coating agent

[0045] 3 mg of thiophanate-methyl hapten 2 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 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.

[0046] Example 4: Preparation of hybridoma cell lines secreting monoclonal antibodies against thiophanate-methyl

[0047] 1. Acquisition of immunity in animals

[0048] Methylthiophanate complete antigen No. 1 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 neck and back (except for sprint immunization). The initial immunization used complete Freund's adjuvant at a dose of 100 μg / mouse; subsequent booster immunizations used incomplete Freund's adjuvant at half the dose (50 μg / mouse); sprint immunizations did not use adjuvant, but were directly diluted with physiological saline and injected intraperitoneally at a dose halved again (25 μg / mouse). The interval between the initial 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 final booster immunization was 18-21 days. The immunization effect in mice was observed using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), which detected the serum titer and inhibition.

[0049] 2. Cell fusion

[0050] 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:

[0051] a. After euthanizing the mouse by tail dislocation and cervical dislocation, immediately disinfect the mouse in 75% alcohol for about 5 minutes. Aseptically remove the spleen of the mouse, 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.

[0052] 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.

[0053] 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.

[0054] 3. Cell selection and cell line establishment

[0055] 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.

[0056] The screening process consists of two steps: First, positive cell wells are selected using the ic-ELISA method. Second, thiophanate-methyl is used as a standard, and the inhibitory effect on positive cells is determined using the ic-ELISA method.

[0057] Cell wells that showed good inhibition against thiophanate-methyl standards were selected, and subcloning was performed using the limiting dilution method. The same method was used for detection seven days later.

[0058] At least three subcloning operations were performed using the method described above to finally obtain the methylthiophanate monoclonal antibody cell line.

[0059] Example 5: Preparation of thiophanate-methyl monoclonal antibody

[0060] 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 methylthiophanate hybridoma cells starting on day 7, and the ascites fluid was purified for antibody using the caprylic acid-saturated ammonium sulfate method.

[0061] 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.

[0062] Example 6: Application of methyl thiophanate monoclonal antibody

[0063] The monoclonal antibody prepared from hybridoma cell lines via in vivo ascites fluid was applied to the ELISA assay for thiophanate-methyl. The specific steps are as follows:

[0064] (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.

[0065] (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;

[0066] (3) Prepare 0, 5.55, 16.67, 50, 150, 450, 1350 and 4050 ng / mL thiophanate-methyl standard solutions with phosphate buffer (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 thiophanate-methyl monoclonal antibody diluted to 0.1 μg / mL to each well. After reacting at 37℃ for 30 min, wash the plate and pat dry.

[0067] (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.

[0068] (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.

[0069] The IC50 of thiophanate-methyl monoclonal antibody was determined using an indirect competitive ELISA. 50 The value was 119.44 ng / mL, indicating good sensitivity to thiophanate-methyl and its suitability for immunoassay detection. Cross-reactivity tests were performed on thiophanate-methyl analogues (thiophanate-methyl, carbendazim, thiamethoxam, and thiamethoxam), and the cross-reactivity rates were all less than 10%, demonstrating excellent specificity for thiophanate-methyl.

[0070] 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 that secretes monoclonal antibodies against methylthiophanate, characterized in that, The proposed taxonomic name for the hybridoma cell line is monoclonal cell line, and its accession number is CGMCC NO.46814.

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 the first hapten as shown in Formula I: 。 3. The hybridoma cell line according to claim 1, characterized in that, The complete antigen is obtained by conjugating the first hapten to a carrier protein.

4. The hybridoma cell line according to claim 1, characterized in that, The carrier protein includes bovine serum albumin.

5. The use of the hybridoma cell line according to any one of claims 1-4 in the detection of thiophanate-methyl.

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 thiophanate-methyl.

8. A detection product for thiophanate-methyl, 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 second hapten-coupled carrier protein with the structural formula shown in Formula II; the carrier protein includes ovalbumin: 。

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