Hybridoma cell strain secreting mepiquat chloride monoclonal antibody and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain secreting a mepiquat chloride monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of immunodetection. The hybridoma cell strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on April 17, 2025, the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.46517. The monoclonal antibody secreted by the hybridoma cell strain has good sensitivity and specificity to mepiquat chloride, the IC50 value of the mepiquat chloride is 4.981 ng / mL, structural analogues and functional analogues of the mepiquat chloride, such as chlormequat chloride, paclobutrazol and uniconazole, have no cross reaction, the cross reaction rate is smaller than 1%, and the monoclonal antibody secreted by the hybridoma cell strain can be used for preparing the mepiquat chloride monoclonal antibody. Therefore, low-concentration mepiquat chloride can be accurately detected.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay technology, in particular to a hybridoma cell strain secreting mepiquat monoclone antibody and application thereof. BACKGROUND

[0002] Mepiquat, CAS No. 115302-91-7, molecular weight 114.21, is a quaternary ammonium salt plant growth regulator, also known as mepiquat chloride, mainly used for controlling crop overgrowth, such as cotton, wheat, corn, promoting anti-lodging, and increasing yield. However, although mepiquat chloride is lowly toxic, it will leave residues through absorption-metabolism-accumulation in crops, which will directly threaten human health, agricultural product trade and ecological environment if not detected and controlled. In order to control this risk, countries and regions have formulated clear residual limit standards: the National Food Safety Standard Maximum Residue Limits of Pesticides in Food (GB 2763) of China stipulates that the residual limit of mepiquat chloride in wheat is 0.5 mg / kg, and in cotton seeds is 0.2 mg / kg; the Health and Safety Executive (HSE) of the United Kingdom requires that the limit of artificially cultivated edible fungi (except Pleurotus ostreatus) is 0.09 mg / kg, and the limit of Pleurotus ostreatus is relaxed to 30 mg / kg due to metabolic characteristics, which provides clear basis for the safety access of agricultural products.

[0003] At present, the methods for determining the content of mepiquat 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), which require professional equipment and operators, and the detection cost is high, which is difficult to meet the needs of instant and batch detection. The enzyme-linked immunoassay (ELISA) technology provides a new solution for the detection of mepiquat. This technology can realize the trace detection of mepiquat with high sensitivity and fast detection speed, and the operation is relatively simple, which greatly improves the flexibility and timeliness of detection, and is expected to solve the problems of traditional detection methods and promote the development of mepiquat detection technology, and provide more efficient technical support for drug residue detection. The premise of using enzyme-linked immunoassay to detect mepiquat is to obtain a monoclonal antibody that can accurately detect mepiquat, therefore, it is crucial to find a monoclonal antibody that can accurately detect mepiquat, and the existing technology does not have a monoclonal antibody for mepiquat. SUMMARY

[0004] In order to solve the above technical problems, the present application provides a hybridoma cell strain secreting mepiquat monoclone antibody and application thereof. The monoclonal antibody secreted by the hybridoma cell strain of the present application has good sensitivity and specificity to mepiquat, and the IC 50The value is 4.981 ng / mL, and there is no cross reaction with structural analogues and functional analogues of piperphos such as chlormequat, paclobutrazol and uniconazole, so that low concentration of piperphos can be accurately detected.

[0005] The present application is realized by the following technical solutions:

[0006] The first object of the present application is to provide a hybridoma cell strain secreting piperphos monoclonal antibody, which is preserved in China General Microbiological Culture Collection Center on April 17, 2025, and the address is No. 1, Beichen West Road, Haidian District, Beijing, and the preservation number is CGMCC No. 46517.

[0007] Further, the hybridoma cell strain is obtained by immunizing animals with a complete antigen prepared from a piperphos hapten.

[0008] Further, the complete antigen is obtained by coupling the hapten with a carrier protein.

[0009] The second object of the present application is to provide a piperphos monoclonal antibody, which is secreted by the hybridoma cell strain.

[0010] Further, the piperphos monoclonal antibody is obtained by immunizing animals with a piperphos complete antigen.

[0011] Further, the piperphos complete antigen is obtained by coupling the piperphos hapten with a carrier protein.

[0012] Further, the structure of the piperphos hapten is as follows:

[0013]

[0014] Further, the carrier protein includes keyhole limpet hemocyanin and / or chicken egg white albumin.

[0015] The third object of the present application is to provide a composition, which includes the hybridoma cell strain and / or the piperphos monoclonal antibody.

[0016] The fourth object of the present application is to provide a kit, which includes one or more of the hybridoma cell strain, the piperphos monoclonal antibody and the composition.

[0017] The fifth object of the present application is to provide a test strip, which includes one or more of the hybridoma cell strain, the piperphos monoclonal antibody and the composition.

[0018] The sixth object of the present application is to provide the application of the hybridoma cell strain, the monoclonal antibody of pipermethyl, the composition, the kit or the test strip in detecting pipermethyl.

[0019] The above technical solutions of the present application have the following advantages compared with the prior art:

[0020] The present application provides a hybridoma cell strain secreting a monoclonal antibody of pipermethyl and its application. The present application first isolates a hybridoma cell strain capable of accurately detecting low-concentration pipermethyl. Specifically, the monoclonal antibody secreted by the hybridoma cell strain of the present application has good sensitivity and specificity to pipermethyl, wherein the IC 50 value of the monoclonal antibody to pipermethyl is 4.981 ng / mL, and the monoclonal antibody has no cross-reaction to structural analogues and functional analogues of pipermethyl, such as chlormequat, paclobutrazol and uniconazole, so that the monoclonal antibody can accurately detect low-concentration pipermethyl.

[0021] Preservation of biological material:

[0022] A hybridoma cell strain LBZC secreting a monoclonal antibody of pipermethyl has been preserved in the China General Microbiological Culture Collection Center (CGMCC), located at No. 1, Yihuangyuan, Beichen West Road, Chaoyang District, Beijing, China, and classified as a monoclonal cell strain. The preservation date is April 17, 2025, and the preservation number is CGMCC No. 46517. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which:

[0024] Figure 1 is the inhibition standard curve of the monoclonal antibody of pipermethyl to pipermethyl. DETAILED DESCRIPTION

[0025] The present application will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present application and implement it. However, the embodiments are not limiting to the present application.

[0026] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0027] (1) The culture medium involved in the following examples is as follows:

[0028] 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.

[0029] (2) The reagents involved in the following examples are as follows:

[0030] 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.

[0031] Phosphate-buffered saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O, dissolved in 800mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000mL with ultrapure water.

[0032] Washing buffer (PBST): Add 0.5 mL of Tween-20 to 1000 mL of 0.01 mol / L pH 7.4 PBS solution;

[0033] PBST: PBS containing 0.05% Tween-20;

[0034] Antibody diluent: a washing solution containing 0.1% gelatin;

[0035] TMB colorimetric solution: Solution A: 18.43g Na2HPO4·12H2O, 9.33g citric acid, diluted to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solutions A and B in a volume ratio of 5:1 to obtain the TMB colorimetric solution. Mix fresh before use.

[0036] (3) The detection methods involved in the following embodiments are as follows:

[0037] Mepiquat inhibition rate assay: 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, mepiquat 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, the mepiquat standard inhibition curve was obtained using Origin 2024, and the IC50 was calculated. 50 .

[0038] Example 1: Preparation of Hapten

[0039] 5 g of 3-bromopyridine was dissolved in methanol, and 4.5 g of methylpentyl-4-enoate and 10 mL of triethylamine were added. The mixture was stirred at 110 °C for 12 h to remove the organic solvent. The solution was then extracted with ethyl acetate and dried to obtain an oily substance. The product was subsequently dissolved in methanol, and 8 g of iodomethane was added. The mixture was stirred at 110 °C for 12 h to remove the organic solvent. The solution was then extracted with ethyl acetate and dried to obtain a yellow oily substance. The product was then dissolved in methanol and added to sodium borohydride solution at 0 °C. The mixture was stirred for 6 h, then diluted with water and extracted with ethyl acetate to obtain a yellow solid. The yellow solid was dissolved in methanol and stirred with palladium on carbon at 60 °C for 6 h to remove the organic solvent. The solution was then extracted with ethyl acetate and dried to obtain a yellow solid. The yellow solid was then dissolved in solvent, and 2 M sodium hydroxide solution was added. The mixture was stirred at 60 °C for 6 h to remove the organic solvent. The solution was then extracted with ethyl acetate and dried to obtain the mepiternium hapten JPW-COOH. The reaction equation is as follows:

[0040]

[0041] Example 2: Preparation of Immunogen

[0042] Weigh 8.3 mg of mepiquat hapten JPW-COOH and dissolve it in 300 μL of N,N-dimethylformamide.

[0043] In DMF, 6.4 mg of N-hydroxysuccinimide was added under stirring at room temperature and reacted for 15 min. Then, 10.3 mg of 1-ethylcarbodiimide hydrochloride was added and reacted at room temperature for 6 h. The resulting mixture was called solution A. Then, 6 mg of keyhole hemocyanin (KLH) was weighed and dissolved in 3 mL of carbonate buffer, which was called solution B. Solution A was slowly added dropwise to solution B and reacted at room temperature for 24 h with stirring. The mixture was then dialyzed against 0.01 mol / L phosphate buffered PBS for 3 days to obtain the conjugate JPW-COOH-KLH, which was stored at -20℃ for later use.

[0044] Example 3: Preparation of the coating agent

[0045] Weigh 5.5 mg of the prepared mepimuntane hapten JPW-COOH and dissolve it in 300 μL of N,N-dimethylformamide (DMF). Add 4.3 mg of N-hydroxysuccinimide and react for 15 min with stirring at room temperature. Then add 6.9 mg of 1-ethylcarbodiimide hydrochloride and react for 6 h. The resulting mixture is called solution A. Then weigh 6 mg of ovalbumin (OVA) 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 with stirring for 12 h. Dialyze the mixture with 0.01 mol / L phosphate-buffered saline (PBS) for 3 days to obtain the conjugate JPW-COOH-OVA, which is then stored at -20 °C for later use.

[0046] Example 4: Preparation of hybridoma cell lines

[0047] 1. Immunization of mice: Healthy BALB / c mice aged 6–8 weeks were selected for immunization. Mepiquat immunogen was emulsified with an equal volume of Freund's adjuvant and administered subcutaneously via the back of the 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.

[0048] 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:

[0049] 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. Under aseptic conditions, 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 it (1200 rpm, 8 minutes). Wash the spleen cells three times with RPMI-1640 medium. After the last centrifugation, dilute the spleen cells to a certain volume, count them, and set them aside for later use.

[0050] 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.

[0051] 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.

[0052] (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, mepiquat chloride was used as a standard, and its inhibitory effect on positive cells was determined using icELISA. Cell wells that showed good inhibition of the mepiquat chloride standard were selected, and subcloning was performed using the limiting dilution method. The same method was used for detection, repeated three times, to obtain the cell line.

[0053] Example 5: Preparation and Identification of Monoclonal Antibodies

[0054] 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 0.01M PBS solution (pH 7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

[0055] The IC50 of the monoclonal antibody against mepiquat chloride was determined using the icELISA method. 50 The concentration was 4.981 ng / mL, and its IC50 for functional analogues was verified. 50 and cross-reactivity rate, the cross-reactivity rate with related fungicide analogues is less than 1%, where the cross-reactivity rate = (IC50 of mepiquat chloride) / (IC50 of mepiquat chloride) 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 mepiquat chloride, as shown in Table 1.

[0056] Table 1. Sensitivity and specificity of monoclonal antibodies against mepiquat chloride.

[0057] IC 50 (ng / mL) Cross-reactivity (%) Mepiquat 4.981 100 Chlormequat >50 <1 Paclobutrazol >50 <1 Uniconazole >50 <1

[0058] (2) Antibody application

[0059] The monoclonal antibody prepared from hybridoma cell lines via in vivo ascites fluid was used in a spiking and recovery assay for mepiquat chloride. The specific steps are as follows:

[0060] a. Coating: The coating proto-mepiride-OVA was serially diluted with 0.05M pH 9.6 carbonate buffer starting from 1 μg / mL, 100 μL / well, and reacted at 37℃ for 2 h;

[0061] b. Washing: Pour off the solution in the plate and wash with washing solution 3 times, 3 minutes each time;

[0062] c. Sealing: After patting dry, add 200 μL / well sealing solution and react at 37℃ for 2 h. Wash and dry for later use.

[0063] 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 1:3000 diluted HRP-goat anti-mouse IgG at 100 μL / well and incubate at 37°C for 30 min.

[0064] 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.

[0065] 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.

[0066] The standard curve of mepiquat monoclonal antibody inhibition against mepiquat is as follows: Figure 1 As shown, the IC50 of the monoclonal antibody against mepiquat chloride was determined using icELISA. 50 The concentration was 4.981 ng / mL, indicating good sensitivity to mepiquat chloride and suitable for immunoassay detection of mepiquat chloride.

[0067] 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 secreting mepiquat monoclonal antibodies, characterized in that, The hybridoma cell line 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 CGMCCNo.46517.

2. A mepiquat chloride monoclonal antibody, characterized in that, Produced by the hybridoma cell line of claim 1.

3. The mepiquat chloride monoclonal antibody according to claim 1, characterized in that, The mepiston monoclonal antibody was obtained by immunizing animals with the mepiston complete antigen.

4. The mepiquat chloride monoclonal antibody according to claim 3, characterized in that, The mepillon complete antigen is obtained by conjugating the mepillon hapten with a carrier protein.

5. The mepiquaton monoclonal antibody according to claim 4, characterized in that, The structural formula of the mepiron hapten is shown below:

6. The mepiquaton monoclonal antibody according to claim 4, characterized in that, The carrier proteins include keyhole hemocyanin and / or oocyte albumin.

7. A composition, characterized in that, The composition comprises the hybridoma cell line of claim 1 and / or the mepiternium monoclonal antibody of any one of claims 2-6.

8. A reagent kit, characterized in that, The kit comprises one or more of the hybridoma cell line of claim 1, the mepiternium monoclonal antibody of any one of claims 2-6, and the composition of claim 7.

9. A test strip, characterized in that, The test strip comprises one or more of the hybridoma cell line of claim 1, the mepiternium monoclonal antibody of any one of claims 2-6, and the composition of claim 7.

10. The use of the hybridoma cell line of claim 1, the mepiquat monoclonal antibody of any one of claims 2-6, the composition of claim 7, the kit of claim 8, or the test strip of claim 9 in the detection of mepiquat; the use does not relate to the diagnosis and treatment of disease.

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