Hybridoma cell strain secreting metconazole and ipconazole monoclonal antibody and application thereof

Abstract

The invention relates to a hybridoma cell strain secreting metconazole and ipconazole monoclonal antibodies and application of the hybridoma cell strain, and belongs to the technical field of immunodetection. The monoclonal antibody secreted by the hybridoma cell strain has good sensitivity to metconazole and ipconazole, the IC50 of the metconazole is 4.296 ng / mL, the IC50 of the ipconazole is 4.394 ng / mL, and structural analogues of the metconazole and the ipconazole, such as tebuconazole, flusilazole, difenoconazole, fenbuconazole, myclobutanil, diniconazole, propiconazole and epoxiconazole, are free of cross reaction, so that the monoclonal antibody secreted by the hybridoma cell strain has the advantages of high sensitivity, high sensitivity and the like. Therefore, low-concentration metconazole and ipconazole can be accurately detected at the same time.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a hybridoma cell line that secretes monoclonal antibodies against tebuconazole and tebuconazole and its applications. Background Technology

[0002] Ipconazole and tebuconazole are triazole fungicides that control plant fungal diseases by inhibiting the synthesis of ergosterol in fungal cell membranes, thereby disrupting the structure and function of fungi. Ipconazole, with its unique epoxy group in its molecular structure, exhibits stronger fungicidal activity, longer-lasting effect, and excellent systemic translocation. Tebuconazole, with its azole ring and unique side chain, possesses bidirectional systemic translocation, allowing it to be absorbed by crop roots and translocated upwards, as well as absorbed through leaves and diffused throughout the plant. It also exhibits strong inhibitory activity against fungi and a long-lasting effect.

[0003] Currently, the main methods for determining the content of tebuconazole and tebuconazole include ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS), gas chromatography-tandem mass spectrometry (GC-MS / MS), and high performance liquid chromatography (HPLC-UV). These methods require specialized equipment and operators, and their high cost makes them unsuitable for real-time, batch detection. Enzyme-linked immunosorbent assay (ELISA) technology offers a new solution for the detection of tebuconazole and tebuconazole. This technology, with its high sensitivity and fast detection speed, can achieve trace detection of tebuconazole and tebuconazole, and its operation is relatively simple, greatly improving the flexibility and timeliness of detection. It is expected to solve the problems of traditional detection methods, promote the development of tebuconazole and tebuconazole detection technology, and provide more efficient technical support for drug residue detection. However, the prerequisite for using ELISA to simultaneously detect tebuconazole and tebuconazole is to obtain monoclonal antibodies with high sensitivity and specificity to tebuconazole and tebuconazole. Currently, existing technologies lack monoclonal antibodies with high sensitivity and specificity to tebuconazole and tebuconazole. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a monoclonal antibody that is highly sensitive and specific to both tebuconazole and styraxazole in the prior art.

[0005] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes monoclonal antibodies against tebuconazole and tebuconazole, and its applications. The monoclonal antibodies secreted by the hybridoma cell line of this invention exhibit good sensitivity to tebuconazole and tebuconazole, with an IC50 of [missing value] for tebuconazole. 50 The IC50 concentration of 4.296 ng / mL was 4.296 ng / mL, and the IC50 concentration of tebuconazole was... 50The concentration was 4.394 ng / mL, and it showed no cross-reactivity with structural analogs of tebuconazole and styraconazole, such as tebuconazole, flusilazole, difenoconazole, cyproconazole, tebuconazole, propiconazole, and flutriafol. Therefore, it can simultaneously and accurately detect low concentrations of tebuconazole and styraconazole.

[0006] The first objective of this invention is to provide a monoclonal cell line that was deposited on April 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46523.

[0007] 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:

[0008] .

[0009] Furthermore, the complete antigen is obtained from the hapten coupled with a carrier protein.

[0010] Furthermore, the carrier protein includes keyhole hemocyanin.

[0011] A second objective of this invention is to provide an application of the above-mentioned hybridoma cell line in the detection of tebuconazole and tebuconazole.

[0012] A third objective of this invention is to provide a monoclonal antibody secreted by the aforementioned hybridoma cell line.

[0013] A fourth objective of this invention is to provide an application of the above-mentioned monoclonal antibody in the detection of tebuconazole and tebuconazole.

[0014] A fifth objective of this invention is to provide a detection product for tebuconazole and tebuconazole, the detection product comprising the aforementioned monoclonal antibody.

[0015] Furthermore, the test product includes a coating agent.

[0016] Furthermore, the coating is prepared from a hapten-conjugated carrier protein, wherein the carrier protein includes chicken oocyte albumin.

[0017] The beneficial effects of this invention are:

[0018] This invention marks the first isolation of a hybridoma cell line capable of accurately detecting low concentrations of tebuconazole and tebuconazole. Specifically, the monoclonal antibody secreted by this hybridoma cell line exhibits good sensitivity to both tebuconazole and tebuconazole, with an IC50 value of [missing information - likely related to tebuconazole]. 50 The IC50 concentration of 4.296 ng / mL was 4.296 ng / mL, and the IC50 concentration of tebuconazole was... 50The concentration was 4.394 ng / mL, and it showed no cross-reactivity with structural analogues of tebuconazole and styraxazole, such as tebuconazole, flusilazole, difenoconazole, cyproconazole, tebuconazole, propiconazole, and flutriafol. Therefore, it can simultaneously detect low concentrations of tebuconazole and styraxazole.

[0019] Preservation of biological materials

[0020] The monoclonal cell line XDFJ was deposited on April 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46523, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0021] 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.

[0022] Figure 1 This is the synthetic route for the hapten of the present invention;

[0023] Figure 2 This is the standard curve of inhibition of tebuconazole and styraxone by the monoclonal antibodies of the present invention against tebuconazole and styraxone. 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] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0026] (1) 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] (2) 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 buffer (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 with ultrapure water.

[0031] Washing buffer (PBST): Add 0.5 mL of Tween-20 to 1000 mL of 0.01 mol / L pH 7.4 PBS solution;

[0032] PBST: PBS containing 0.05% Tween-20;

[0033] Antibody diluent: a washing solution containing 0.1% gelatin;

[0034] 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 volume ratio of 5:1 to obtain the TMB colorimetric solution. Mix fresh before use.

[0035] (3) The detection methods involved in the following embodiments are as follows:

[0036] Methods for detecting the inhibition rates of tebuconazole and tebuconazole: The optimal antigen and antibody concentrations for icELISA were selected using a checkerboard assay. The antigen was diluted to 1, 0.3, 0.1, and 0.03 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 1, 0.3, 0.1, and 0.03 μg / mL with antibody diluent. After selecting the optimal operating point, tebuconazole and tebuconazole standards were diluted to concentrations of 300, 100, 33.33, 11.11, 3.70, 1.23, and 0.41 ng / mL, following the icELISA procedure. Finally, Origin 2024 was used to plot the inhibition curves of tebuconazole and tebuconazole standards, and the IC50 was calculated. 50 .

[0037] Example 1: Preparation of Hapten

[0038] Weigh 200 mg of tebuconazole and dissolve it in 300 μL of N,N-dimethylformamide until the system is clear. Then, add 615 mg of bromobutyric acid and 432 mg of potassium carbonate sequentially, and stir the mixture at 60°C for 12 hours. After the reaction is complete, cool to 25°C, filter, wash, and dry to obtain the hapten. The reaction route is as follows: Figure 1 As shown.

[0039] Example 2: Preparation of Immunogen

[0040] Weigh 12.1 mg of hapten and dissolve it in 300 μL of N,N-dimethylformamide. Add 7.9 mg of N-hydroxysuccinimide and react for 15 min with stirring at room temperature. Then add 13.4 mg of 1-ethylcarbodiimide hydrochloride and react at room temperature for 6 h. The resulting mixture is called solution A. Then weigh 5 mg of keyhole hemocyanin (KLH) and dissolve it in 2 mL of carbonate buffer, which is called solution B. Slowly add solution A to solution B and react at room temperature with stirring for 24 h. Dialyze the mixture with phosphate buffer (PBS) for 3 days to obtain the conjugate tebuconazole-KLH, which is stored at -20℃ for later use.

[0041] Example 3: Preparation of the coating agent

[0042] Weigh 6.9 mg of hapten and dissolve it in 300 μL of tebuconazole hapten. Add 7.7 mg of N-hydroxysuccinimide and react for 15 min with stirring at room temperature. Then add 9.2 mg of 1-ethylcarbodiimide hydrochloride and react for 4 h. The resulting mixture is called solution A. Then weigh 5 mg of ovalbumin (OVA) and dissolve it in 2 mL of carbonate buffer. This is called solution B. Slowly add solution A to solution B and react with stirring at room temperature for 24 h. Dialyze the mixture with 0.01 mol / L PBS for 3 days to obtain the conjugate tebuconazole-OVA. Store at -20℃ for later use.

[0043] Example 4: Preparation of hybridoma cell lines

[0044] 1. Immunization of mice: Healthy 6-8 week old BALB / c mice were selected for immunization. Imidacloprid and tebuconazole immunogens were mixed with an equal volume of Freund's adjuvant and emulsified, then injected subcutaneously into the back of each BALB / c mouse. The first immunization used complete Freund's adjuvant, and subsequent immunizations used incomplete Freund's adjuvant. The interval between the first and second booster immunizations was 28 days, and the interval between multiple booster immunizations was 21 days. Seven days after the third immunization, blood was collected (5 μL of blood from tail amputation + 995 μL of antibody diluent = antiserum). Serum titers and inhibition were measured using icELISA. Mice with high titers and good inhibition were selected for a sprint immunization 21 days after the fifth immunization, administered intraperitoneally. The sprint dose was halved and contained no adjuvant.

[0045] 2. Cell fusion: Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 1500) method. The specific steps are as follows:

[0046] a. After euthanizing the mice by enucleation and cervical dislocation-assisted carbon dioxide euthanasia, immediately disinfect the mice in 75% alcohol for about 5 minutes. Aseptically remove the spleens of the mice, grind them moderately with the rubber tip of a syringe and pass them through a 200-mesh cell sieve to obtain a spleen cell suspension. After collection, centrifuge (1200 rpm, 8 min), then 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 them aside for later use.

[0047] b. Collection of murine myeloma SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The required number of SP2 / 0 tumor cells before fusion was (1-4) × 10⁻⁶. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. During fusion, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counting is performed.

[0048] c. Fusion process (7 min). At min 1, add 1 mL of PEG 1500 dropwise to the cells, 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 seconds. Then incubate at 37°C for 5-8 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.

[0049] (6) Cell screening and cell line establishment: On day 3 of cell fusion, the fused cells were partially replaced with RPMI-1640 screening 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. The screening consisted of two steps: First, positive cell wells were selected using icELISA. Second, tebuconazole and tebuconazole were used as standards, and the inhibitory effect on positive cells was determined by icELISA. Cell wells that showed good inhibition against both tebuconazole and tebuconazole standards were selected, and subcloning was performed using the limiting dilution method. The same method was used for detection, repeated three times, to obtain cell lines.

[0050] Example 5: Preparation and Identification of Monoclonal Antibodies

[0051] BALB / c mice aged 6-8 weeks were injected intraperitoneally with 0.5 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6 Hybridoma cells were used, and ascites fluid was collected starting on day 7. The ascites fluid was purified using the caprylic acid-ammonium sulfate method. Under slightly acidic conditions, caprylic acid precipitates other proteins in the ascites fluid besides IgG immunoglobulins. The precipitate was then discarded by centrifugation. IgG-type monoclonal antibodies were then precipitated with an equal volume of saturated ammonium sulfate solution, centrifuged, and the supernatant was discarded. The precipitate was dissolved in 0.01 M PBS solution (pH 7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

[0052] The IC50 of the monoclonal antibody against tebuconazole was determined using the icELISA method. 50 The IC50 concentration of 4.296 ng / mL was 4.296 ng / mL, and the IC50 concentration of tebuconazole was... 50 The concentration was 4.394 ng / mL, and its IC50 for functional analogues was verified. 50The cross-reactivity rate is less than 1% for related fungicide analogues, where the cross-reactivity rate = (IC50 of tebuconazole and tebuconazole) / (IC50 of tebuconazole and tebuconazole). 50 ICs of similar types 50 The cross-reactivity ratio (CRR) is calculated as 100%. Based on the CRR values, it can be seen that the antibody has high sensitivity to tebuconazole and tebuconazole, as shown in Table 1.

[0053] Table 1. Sensitivity and specificity of monoclonal antibodies against tebuconazole and fenbendazole.

[0054]

[0055] (2) Antibody application

[0056] Monoclonal antibodies prepared from hybridoma cell lines via in vivo ascites fluid were used in a spiking and recovery assay for tebuconazole and tebuconazole. The specific steps are as follows:

[0057] a. Coating: The coated protozocacillin-OVA was serially diluted with 0.05 M pH 9.6 carbonate buffer starting from 1 µg / mL, 100 μL / well, and reacted at 37℃ for 2 h;

[0058] b. Washing: Pour off the solution in the plate and wash with washing solution 3 times, 3 minutes each time;

[0059] c. Sealing: After patting dry, add 200 μL / well sealing solution and react at 37℃ for 2 h. Wash and dry for later use.

[0060] d. Sample addition: Serially dilute the antiserum (antiserum obtained by diluting mouse tail blood with antibody diluent) starting from 1:1000 and add it to each well of the coated sample at 100 μL / well. Incubate at 37°C for 30 min. After thorough washing, add HRP-goat anti-mouse IgG diluted 1:3000 at 100 μL / well and incubate at 37°C for 30 min.

[0061] e. Color development: Remove the microplate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37°C in the dark for 15 min.

[0062] f. Termination and Measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 value.

[0063] For recovery rates, barley and oat samples purchased from a local supermarket in Wuxi were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). LC-MS / MS analysis showed no detectable residues of tebuconazole or fenbendazole in either sample. Therefore, in this embodiment, the recovery rate was determined by adding tebuconazole and fenbendazole. Specifically, as shown in Table 2, the average recovery rate of LC-MS / MS was 85.9-116.8%, with a relative standard deviation (RSD) of 1.54-7.59%; the recovery rate of the indirect competitive ELISA method was 94.5-108.1%, with an RSD of 2.03-6.74%. This indicates that the method based on tebuconazole and fenbendazole monoclonal antibodies for detection is relatively accurate and can be used for immunoassay detection of tebuconazole and fenbendazole residues in barley and oats.

[0064] Table 2. Recovery rates of monoclonal antibodies against septica and tebuconazole in barley and oats.

[0065]

[0066] 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 cells were named monoclonal cell lines and their accession number was CGMCC No. 46523.

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 tebuconazole and tebuconazole.

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 tebuconazole and tebuconazole.

8. A detection product for tebuconazole and tebuconazole, 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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