Hybridoma cell strain secreting monoclonal antibody of jinyaoxiong and its application

Abstract

The present application relates to a kind of hybridoma cell strains secreting monoclone antibody of tin oxoline and its application, belong to the field of immune detection technology.The hybridoma cell strains secreting monoclone antibody of tin oxoline of the present application is preserved in China Microorganism Strain Preservation Management Committee General Microorganism Center on April 24, 2026, and the preservation address is No.3, 1st Courtyard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.46835.The monoclone antibody secreted by the hybridoma cell strains of the present application has good sensitivity and specificity to tin oxoline, wherein the IC 50 Value of tin oxoline is 5.461 ng / mL, and there is no cross reaction to structural analog and functional analog of tin oxoline, such as maleic hydrazine, benzenediazine, thidiazuron, paclobutrazol and uniconazole, and the cross reaction rate is less than 1%, so low concentration tin oxoline 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 tinorelin monoclonal antibody and its application. Background Technology

[0002] Jinaolin, CAS number 130561-48-7, chemical name 1-(4-chlorophenyl)-1,4-dihydro-5-(2-methoxyethoxy)-4-ketozoline-3-carboxylic acid, belongs to the benzopyridazine class of compounds. It is mainly used as a plant growth regulator, particularly as a chemical emasculator for wheat. As a chemical emasculator applied in the field, Jinaolin can leave residues through crop absorption, metabolism, or environmental diffusion. Without monitoring and control, it will directly threaten human health and ecological safety.

[0003] Currently, methods for determining the content of tinorelin mainly 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 tinorelin detection. This technology, with its high sensitivity and fast detection speed, can achieve trace detection of tinorelin, 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 tinorelin detection technology, and provide more efficient technical support for drug residue detection. The prerequisite for using ELISA to detect tinorelin is obtaining a monoclonal antibody that can accurately detect tinorelin. Therefore, finding a monoclonal antibody that can accurately detect tinorelin is crucial, but current technology does not yet have a monoclonal antibody specifically targeting tinorelin. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes a monoclonal antibody against zim-olin and its applications. The monoclonal antibody secreted by the hybridoma cell line of this invention exhibits good sensitivity and specificity to zim-olin, with an IC50 value of [missing information - likely related to IC50 or similar]. 50 The value was 5.461 ng / mL, and there was no cross-reactivity with structural and functional analogues of jinolin, such as cyhalothrin, benzimidazine, thiabendazole, paclobutrazol, and uniconazole. Therefore, it can accurately detect low concentrations of jinolin.

[0005] This invention is achieved through the following technical solution:

[0006] The first objective of this invention is to provide a hybridoma cell line that secretes a monoclonal antibody, said hybridoma cell line was deposited on April 24, 2026, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46835.

[0007] In one embodiment of the present invention, the hybridoma cell line is obtained by immunizing animals with a complete antigen prepared from the phytoalkaloid hapten;

[0008] In one embodiment of the invention, the complete antigen is obtained from the hapten-coupled carrier protein.

[0009] In one embodiment of the present invention, the carrier protein includes keyhole hemocyanin.

[0010] A second objective of this invention is to provide the application of the hybridoma cell line in the preparation of tinorelin monoclonal antibodies.

[0011] A third objective of this invention is to provide a zimoline monoclonal antibody secreted by the hybridoma cell line.

[0012] A fourth objective of this invention is to provide the application of the hybridoma cell line or the tinoreline monoclonal antibody in the detection of tinoreline.

[0013] The fifth objective of this invention is to provide a detection product, characterized in that the detection product contains the aforementioned jin-aoline monoclonal antibody.

[0014] In one embodiment of the present invention, the detection product further includes a coating agent.

[0015] In one embodiment of the present invention, the coating antigen is obtained by conjugating the zimoline hapten with a carrier protein.

[0016] In one embodiment of the present invention, the structural formula of the ginsenoline hapten is shown below:

[0017] .

[0018] In one embodiment of the present invention, the carrier protein includes chicken oocyte albumin.

[0019] The sixth object of the present invention is to provide the application of the aforementioned detection product in the detection of linolenic acid.

[0020] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0021] This invention provides a hybridoma cell line that secretes a monoclonal antibody against zim-olin and its application. This invention is the first to isolate a hybridoma cell line capable of accurately detecting low concentrations of zim-olin. Specifically, the monoclonal antibody secreted by this hybridoma cell line exhibits good sensitivity and specificity to zim-olin, with an IC50 value of [missing information - likely related to IC50]. 50 The value was 5.461 ng / mL, and there was no cross-reactivity with structural analogs of jinolin, such as cyhalothrin, benzimidazine, thiabendazole, paclobutrazol, and uniconazole (plant growth regulators / cyhalothrin analogs), thus enabling accurate detection of low concentrations of jinolin.

[0022] Preservation of biological materials

[0023] A hybridoma cell line ISKZ1A3 that secretes a monoclonal antibody against ginsenosides has been 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 has been classified as a monoclonal cell line, deposited on April 24, 2026, with accession number CGMCC No. 46835. Attached Figure Description

[0024] 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.

[0025] Figure 1 This is the standard curve of inhibition of jinolin by the monoclonal antibody of jinolin of the present invention. Detailed Implementation

[0026] 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.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0028] (1) The culture media involved in the following examples are as follows:

[0029] 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.

[0030] (2) The reagents involved in the following examples are as follows:

[0031] 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.

[0032] Phosphate buffer (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 with ultrapure water.

[0033] Washing buffer (PBST): Add 0.5 mL of Tween-20 to 1000 mL of 0.01 mol / L pH 7.4 PBS solution;

[0034] PBST: PBS containing 0.05% Tween-20;

[0035] Antibody diluent: a washing solution containing 0.1% gelatin;

[0036] 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.

[0037] (3) The detection methods involved in the following embodiments are as follows:

[0038] The method for detecting the inhibition rate of jin-aoline was as follows: The optimal antigen and antibody concentrations for icELISA were selected using a checkerboard assay. The antigen was diluted to 1 μg / mL, 0.3 μg / mL, 0.1 μg / mL, and 0.03 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 1 μg / mL, 0.3 μg / mL, 0.1 μg / mL, and 0.03 μg / mL with antibody dilution buffer. After selecting the optimal operating point, the jin-aoline standard was diluted to concentrations of 100 ng / mL, 33.33 ng / mL, 11.11 ng / mL, 3.70 ng / mL, 1.23 ng / mL, 0.41 ng / mL, and 0.13 ng / mL, following the icELISA procedure. Finally, Origin 2024 was used to plot the jin-aoline standard inhibition curve, and the IC50 was calculated. 50 .

[0039] Example 1: Preparation of Immunogen

[0040] Weigh 6.4 mg of jinaolin (CAS: 130561-48-7) and dissolve it in 300 μL of DMF. Add 4.5 mg of N-hydroxysuccinimide under stirring at room temperature and react for 15 min. Then add 8.7 mg of 1-ethylcarbodiimide hydrochloride and react at room temperature for 6 h. The resulting mixture is called solution A. Then weigh 6 mg of keyhole hemocyanin (KLH) and dissolve it in 3 mL of carbonate buffer, which is called solution B. Slowly add solution A to solution B and react at room temperature for 24 h with stirring. Dialyze the mixture with phosphate-buffered saline (PBS) for 3 days to obtain the conjugate jinaolin-KLH, which is stored at -20℃ for later use.

[0041] Example 2: Preparation of the coating agent

[0042] Weigh 5.2 mg of jin-oline and dissolve it in 300 μL of DMF. Add 3.7 mg of N-hydroxysuccinimide and react for 15 min with stirring at room temperature. Then add 5.8 mg of 1-ethylcarbodiimide hydrochloride and react for 4 h. The resulting mixture is called solution A. Then weigh 6 mg of ovalbumin (OVA) and dissolve it in 33 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 0.01 mol / L phosphate-buffered saline (PBS) for 3 days to obtain the conjugate jin-oline-OVA, which is stored at -20℃ for later use.

[0043] Example 3: Preparation of hybridoma cell lines

[0044] 1. Immunization of mice: Healthy 6-8 week old BALB / c mice were selected for immunization. The immunogen, ginsenoside R, was emulsified with an equal volume of Freund's adjuvant and administered subcutaneously to 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 immunization 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. Collect the suspension and centrifuge (1200 rpm, 8 min). 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] 3. Cell Screening and Cell Line Establishment: On day 3 of 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. Screening consisted of two steps: First, positive cell wells were selected using icELISA. Second, using azithromycin as a standard, the inhibitory effect on positive cells was determined by icELISA. Cell wells showing good inhibition against the azithromycin standard were selected, and subcloning was performed using the limiting dilution method. The same method was repeated three times to obtain the cell line.

[0050] Example 4: 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 standard curve of inhibition of zimolin by monoclonal antibody is as follows: Figure 1 As shown, the IC50 of the monoclonal antibody against jinolin was determined using icELISA. 50 The concentration was 5.461 ng / mL, indicating good sensitivity to linopyrin, making it suitable for immunoassay detection of linopyrin. The IC50 for structural and functional analogues was also verified. 50and cross-reactivity rate, the cross-reactivity rate with related fungicide analogues is less than 1%, where the cross-reactivity rate = (IC50 of aziridine) / (IC50 of aziridine) 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 this antibody has high sensitivity and specificity to linolenic acid, as shown in Table 1.

[0053] Table 1. Sensitivity and specificity of monoclonal antibodies to zizoline.

[0054]

[0055] (2) Antibody application

[0056] Monoclonal antibodies prepared from hybridoma cell lines via in vivo ascites fluid were used in a recovery assay for ginsenosides in wheat. The specific steps are as follows, and the results are shown in Table 2:

[0057] a. Coating: The coated protozoanol-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: The antiserum (antiserum obtained by diluting mouse tail blood with antibody diluent) was serially diluted from 1:1000 to each well, 100 μL / well, and incubated at 37°C for 30 min; after thorough washing, HRP-goat anti-mouse IgG diluted 1:3000 was added, 100 μL / well, and incubated 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] g. Add recovered and tested samples.

[0064] The pretreatment for indirect competitive ELISA involves pulverizing and sieving the sample, sealing it, and storing it at 4°C in a light-protected environment. Weigh 2.0 g (accurate to 0.01 g) of pulverized blank wheat matrix powder and place it in a 10 mL polypropylene centrifuge tube. Add the corresponding concentration of Jinaolin standard working solution, vortex for 1 min, and let stand at room temperature for 30 min to allow the target analyte to fully wet and adsorb onto the matrix. Then, precisely add 6 mL of methanol-water solution (v:v = 1:1) to the centrifuge tube as the extraction buffer, and tighten the cap. Extract at 25°C and 3000 rpm for 5 min. Centrifuge at 5000 rpm for 5 min at room temperature, and after separation, collect the upper organic extract supernatant. Take 100 μL of the supernatant and dilute it 3-fold with 400 μL of PBS buffer (0.01 mol / L, pH 7.4). Vortex to mix well. This is the sample solution for indirect competitive ELISA and can be used directly for indirect competitive ELISA detection.

[0065] LC-MS / MS was used as a control: 2.0 g (accurate to 0.01 g) of pulverized blank wheat matrix powder was accurately weighed and placed in a 10 mL polypropylene centrifuge tube. The corresponding concentration of Jinaolin standard working solution was added, vortexed for 1 min, and allowed to stand at room temperature for 30 min. 6 mL of methanol-water (v:v=1:1) extraction solution was accurately added, the tube was tightly capped, and extraction was performed at 25℃ and 3000 r / min for 5 min with shaking. The tube was then centrifuged at 5000 r / min at room temperature for 5 min. After the two phases separated, all the upper supernatant was collected. All the supernatant was transferred to a nitrogen blow-off tube and dried to near dryness in a 40℃ water bath under nitrogen. The solution was then vortexed for 3 min with 1 mL of initial mobile phase (methanol-ultrapure water = 20:80, containing 0.1% formic acid). The solution was filtered through a 0.22 μm organic phase microporous membrane into a LC vial for LC-MS / MS analysis.

[0066] The LC-MS / MS instrument detection conditions were as follows: Liquid chromatography conditions: Column: C18 reversed-phase column (4.6 mm × 150 mm, 5 μm); Column temperature: 30℃; Mobile phase A: ultrapure water (0.1% formic acid); Mobile phase B: methanol (0.1% formic acid); Flow rate: 0.8 mL / min; Injection volume: 10 μL; Gradient elution program: 0–3 min, phase B 20% to 85%; 3–6 min, phase B 85% held; 6–7 min, phase B 85% reduced to 20%; 7–10 min, phase B 20% equilibrate the column. Mass spectrometry conditions: Ionization mode: ESI electrospray positive ion mode; Detection mode: MRM multiple reaction monitoring; Drying gas temperature 350℃, drying gas flow rate 10 L / min, nebulizer gas pressure 45 psi, sheath gas temperature 380℃.

[0067] Table 2 Recovery rates of monoclonal antibodies against zeanoline in wheat

[0068]

[0069] Table 2 shows that the recoveries of the indirect competitive ELISA method in the spiked recovery experiment were 96.3%, 105.8%, and 104.2%, respectively. The recoveries of the LC-MS / MS method in the spiked recovery experiment were 90.6%, 91.3%, and 94.9%, respectively. At the three spiked levels in the wheat matrix, the spiked recoveries of both the LC-MS / MS method and the indirect competitive ELISA method were within reasonable ranges, and the results of the two methods showed good agreement. This demonstrates that the indirect competitive ELISA method established in this study has reliable accuracy and precision and can be used for the rapid detection of phytocyanin residues in wheat samples.

[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 a monoclonal antibody against zimoline, characterized in that, The hybridoma cell line was deposited on April 24, 2026, 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. 46835.

2. The use of the hybridoma cell line of claim 1 in the preparation of tinopyrin monoclonal antibody.

3. A monoclonal antibody containing aziridine, characterized in that, Produced by the hybridoma cell line of claim 1.

4. The use of the hybridoma cell line of claim 1 or the monoclonal antibody of tinopyrin of claim 3 in the detection of tinopyrin.

5. A testing product, characterized in that, The test product contains the jinolin monoclonal antibody as described in claim 3.

6. The testing product according to claim 5, characterized in that, The tested products also include coating agents.

7. The testing product according to claim 5, characterized in that, The coating is obtained by coupling the zinoline hapten with a carrier protein.

8. The testing product according to claim 7, characterized in that, The structural formula of the zimoline hapten is shown below: 。 9. The testing product according to claim 7, characterized in that, The carrier protein includes chicken oocyte albumin.

10. The use of the detection product according to any one of claims 5-9 in the detection of linolenic acid.

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