Hybridoma cell strain secreting monoclone antibody of bensulfuron methyl and its application

Abstract

The present application relates to a hybridoma cell strain secreting a dimethomorph monoclonal antibody and its application, and belongs to the field of food safety immune detection. The hybridoma cell strain DBL secreting a dimethomorph monoclonal antibody has been preserved in the China General Microbiological Culture Collection Center on April 17, 2025, with a preservation number of CGMCC No. 46510, a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing, and a classification designation of a monoclonal cell strain. The dimethomorph monoclonal antibody obtained by using the strain to secrete can be used for the analysis and detection of dimethomorph residues in food safety detection. The dimethomorph monoclonal antibody cell strain obtained by the present application can be used for immune analysis and detection, and has good detection sensitivity (IC 50 value of 104.49 ng / mL) for dimethomorph.

Description

Technical Field

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

[0002] Pyrimorph is a novel agricultural fungicide with excellent efficacy against plant diseases caused by oomycetes. It can be used to control various plant diseases such as pepper blight, tomato late blight, and cucumber downy mildew, and can also control grape downy mildew, potato blight, and rice and cotton damping-off. Its efficacy is superior to that of similar drugs like dimethomorph. Compared with similar products at home and abroad, it features high efficiency, a broad fungicidal spectrum, and low cost, making it an ideal pesticide for controlling oomycete diseases. The mechanism of action indicates that this compound can inhibit the energy synthesis of harmful pathogens by inhibiting respiratory chain complex III, and can also affect the distribution of cell wall synthetic substances, making it a multi-target pesticide with low resistance risk. Pyrimorph is now widely used in crop cultivation, but consuming fruits and vegetables with excessive residues can be harmful to human health; therefore, a rapid detection method is needed to detect it.

[0003] For the detection of butylpyraclostrobin pesticide residues, high-performance liquid chromatography (HPLC), gas chromatography (GC), and GC-MS / MS are commonly used. However, these methods have drawbacks such as complex sample pretreatment and long detection times, making them unsuitable for rapid detection of large numbers of samples. To protect the interests of consumers, it is necessary to develop a highly efficient and rapid detection method for butylpyraclostrobin. 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 has been widely used in pesticide residue analysis. However, the prerequisite for using ELISA to detect butylpyraclostrobin is obtaining a monoclonal antibody with high specificity and sensitivity to butylpyraclostrobin. Therefore, finding a method to prepare a monoclonal antibody with high specificity and sensitivity to butylpyraclostrobin is crucial. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes a monoclonal antibody against butylpyrroline and its applications. The monoclonal antibody against butylpyrroline secreted by this hybridoma cell line exhibits good specificity and detection sensitivity (IC50) for butylpyrroline. 50 The value is 104.49 ng / mL, which can be used to establish an immunological detection method for butylpyrrole to detect butylpyrrole residues in food.

[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, which was deposited on April 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46510, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as a monoclonal cell line.

[0007] In one embodiment of the present invention, the hybridoma cell line is obtained by immunizing mice with buprofen complete antigen.

[0008] In one embodiment of the present invention, the butylpyrroline complete antigen is obtained by conjugating the butylpyrroline hapten with a carrier protein.

[0009] In one embodiment of the present invention, the structural formula of the butylpyrroline hapten is:

[0010]

[0011] In one embodiment of the present invention, the carrier protein includes keyhole hemocyanin and / or chicken ovalbumin.

[0012] A second objective of this invention is to provide a butylpyrroline monoclonal antibody secreted by the hybridoma cell line described above.

[0013] A third objective of this invention is to provide a composition for detecting butylpyrroline, the composition comprising the hybridoma cell line and / or the butylpyrroline monoclonal antibody.

[0014] A fourth objective of this invention is to provide a kit for detecting butylpyrroline, the kit comprising one or more of the hybridoma cell line, the butylpyrroline monoclonal antibody, and the composition described herein.

[0015] In one embodiment of the present invention, the kit is selected from enzyme-linked immunosorbent assay (ELISA) kits, fluorescence immunoassay kits, or chemiluminescent immunoassay kits.

[0016] A fifth objective of this invention is to provide a test strip for detecting butylpyrroline, the test strip comprising one or more of the hybridoma cell line, the butylpyrroline monoclonal antibody, and the composition described herein.

[0017] A sixth objective of this invention is to provide the application of the hybridoma cell line, the butylpyrroline monoclonal antibody, the composition, the kit, or the test strip in the detection of butylpyrroline.

[0018] This invention also provides a method for preparing the hybridoma cell line that secretes the above-mentioned butylpyridomorph monoclonal antibody, comprising the following steps:

[0019] (1) Use butylpyrroline hapten to prepare butylpyrroline complete antigen, and prepare the obtained butylpyrroline complete antigen into antigen-containing Freund's adjuvant and antigen-containing incomplete Freund's adjuvant.

[0020] (2) The Freund's adjuvant was injected into BALB / c mice via subcutaneous injection on the back for multiple immunizations. The first immunization used complete Freund's adjuvant, and the booster immunization used incomplete Freund's adjuvant.

[0021] (3) Blood was collected from mice that had undergone the above immunization process. The serum immune titer and immunosuppressive ability of the mice were detected by indirect ELISA. Mice with high levels of butylpyrroline antibody in their serum were screened to obtain immunization.

[0022] (4) The selected mice were given a final booster immunization with incomplete Freund's adjuvant, and then sprint immunization was performed by intraperitoneal injection. The sprint immunization was performed using complete antigen of butylpyrroline without Freund's adjuvant.

[0023] (5) Fusing spleen cells and myeloma cells from BALB / c mice after sprint immunization, culturing the fused cells in a culture medium, detecting positive cell pores using indirect ELISA, and further determining the inhibitory effect of positive cell pores using indirect competitive ELISA, subcloning the positive cell pores with the best inhibition using limiting dilution method, and finally screening out hybridoma cell lines that can secrete butylpyrroline monoclonal antibody.

[0024] In one embodiment of the present invention, in step (1), the structural formula of the butylpyrroline hapten is as follows:

[0025]

[0026] The structural formula of the butylpyrroline complete antigen is as follows:

[0027]

[0028] In one embodiment of the present invention, in steps (2) and (4), the interval between the first immunization and the booster immunization is one month; the interval between the booster immunizations is 21 days; and the interval between the booster immunization and the sprint immunization is 18 to 21 days.

[0029] In one embodiment of the present invention, in steps (2) and (4), the initial immunization dose is 100 μg / animal; the booster immunization dose is 50 μg / animal; and the sprint immunization dose is 25 μg / animal.

[0030] In one embodiment of the present invention, in steps (2) and (4), the immunization process includes one initial immunization, four booster immunizations and one sprint immunization.

[0031] In one embodiment of the present invention, in step (3), the blood collection is performed on the 7th day after the end of the 3rd immunization process.

[0032] In one embodiment of the present invention, in step (5), the cell fusion is performed 3 days after the end of the sprint immunization.

[0033] In one embodiment of the present invention, in step (5), the cell fusion is performed by the polyethylene glycol (PEG4000) method.

[0034] In one embodiment of the present invention, in step (5), the culture medium is RPMI-1640 culture medium.

[0035] In one embodiment of the present invention, in step (5), the number of subcloning operations is 3.

[0036] The present invention provides a method for preparing the above-mentioned butylpyrroline monoclonal antibody, specifically as follows: BALB / c mice are injected intraperitoneally with paraffin oil, and then injected intraperitoneally with hybridoma cell line DBL with preservation number CGMCC No.46510. Ascites fluid is collected after injection, the ascites fluid is purified, and the obtained monoclonal antibody is stored at low temperature.

[0037] The technical solution of the present invention has the following advantages compared with the prior art:

[0038] This invention provides a hybridoma cell line secreting a monoclonal antibody against butylpyridoxine and its applications. The butylpyridoxine monoclonal antibody cell line obtained by this invention can be used for immunoassay detection, and it has good detection sensitivity (IC50) for butylpyridoxine. 50 The value was 104.49 ng / mL.

[0039] Biological material sample deposit: A hybridoma cell line DBL secreting buprofen monoclonal antibody 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, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 46510 and classified as a monoclonal cell line. Attached Figure Description

[0040] 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, wherein:

[0041] Figure 1 This is a standard inhibition curve of the butylpyrroline monoclonal antibody prepared in this invention against butylpyrroline. Detailed Implementation

[0042] 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.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0044] The culture media involved in the following examples are as follows:

[0045] 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.

[0046] The reagents involved in the following examples are as follows:

[0047] Carbonate buffer (CBS): Weigh 1.59g of Na2CO3 and 2.93g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000mL. Store at 4℃ for later use.

[0048] 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.

[0049] PBST: PBS containing 0.05% Tween 20;

[0050] Antibody dilution solution: PBS with 0.1% gelatin added.

[0051] TMB colorimetric solution: Solution A: Na2HPO4 . 12H₂O 18.43g, citric acid 9.33g, diluted to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution, mix fresh before use.

[0052] The detection methods involved in the following embodiments are as follows:

[0053] Method for detecting the inhibition rate of butylpyramidoxime: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The antigen was diluted to 0.03, 0.1, 0.3, and 1 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03, 0.1, 0.3, and 1 μg / mL with antibody dilution buffer. After selecting the optimal operating point, the butylpyramidoxime standard was diluted to a gradient concentration of 0, 1.23, 3.70, 11.11, 33.33, 100, 300, and 900 ng / mL, following the ic-ELISA procedure. Finally, graphs were plotted using OriginPro 8.5 (results are shown in the figure). Figure 1 (As shown), the standard inhibition curve of butylpyridoxine was obtained, and the IC50 was calculated. 50 .

[0054] Example 1: Preparation of butylpyrroline hapten

[0055] Since small molecules lack immunogenicity and cannot stimulate an immune response in mice to produce antibodies, protein conjugation technology is required to couple them to proteins to acquire immunogenicity. Commonly used reactive groups in protein conjugation techniques include amino, carboxyl, hydroxyl, and thiol groups. Because the hydroxyl group of butylpyrroline is crucial for antibody recognition, its hydroxyl group cannot be directly used for protein conjugation. In this invention, rhein was selected as a hapten.

[0056] The structure of the butylpyrroline hapten used in this invention is as follows:

[0057]

[0058] Example 2: Synthesis of Butyroxymorphine Complete Antigen

[0059] Weigh 3.6 mg of butylpyrroline hapten (DBL-COOH) and 4.2 mg of N-hydroxysuccinimide (NHS), dissolve them in 300 μL of N,N-dimethylformamide (DMF), and stir at room temperature for 10 min. Then weigh 6.9 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), dissolve it thoroughly in 100 μL of DMF, and add it to the DBL-COOH solution. Stir at room temperature for 4-6 h (referred to as solution A). Take 6 mg of KLH, dilute it to 3 mg / mL with 0.05 M carbonate buffer (CBS) (referred to as solution B), and slowly add solution A dropwise to solution B. React at room temperature overnight. Then dialyze with 0.01 M PBS to remove unreacted small molecule hapten to obtain the complete antigen DBL-COOH-KLH, which is identified by ultraviolet absorption scanning.

[0060] Example 3: Synthesis of the butylpyrroline coated origin

[0061] 5.7 mg of butylpyrroline hapten (DBL-COOH) and 4.9 mg of N-hydroxysuccinimide (NHS) were dissolved in 300 μL of anhydrous N,N-dimethylformamide (DMF) and reacted with stirring at room temperature for 10 min to obtain a butylpyrroline hapten (DBL-COOH) solution. 11.46 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in 100 μL of anhydrous DMF and added to the DBL-COOH solution. The mixture was reacted with stirring at room temperature for 4-6 h to obtain solution A. 6 mg of chicken ovalbumin (OVA) was diluted with 1 mL of 0.05 M carbonate buffer (CBS) to obtain solution B. Solution A was slowly added dropwise to solution B to obtain the reaction solution. The reaction solution was dialyzed with PBS to remove unreacted small molecule hapten to obtain the coating antigen (DBL-COOH-OVA).

[0062] Example 4: Preparation of hybridoma cell lines secreting butylpyrroline monoclonal antibodies

[0063] 1. Obtaining animal immunization: The complete antigen of buprofen was emulsified with an equal volume of Freund's adjuvant and administered to BALB / c mice via subcutaneous injection at multiple sites on the back of the neck (except for sprint immunization). The initial immunization used complete Freund's adjuvant at a dose of 100 μg / mouse. For multiple booster immunizations, incomplete Freund's adjuvant was used at half the dose (50 μg / mouse). For sprint immunization, no adjuvant was used; the adjuvant was diluted directly with physiological saline and injected intraperitoneally at a dose halved (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 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), i.e., the titer and inhibition of mouse serum were detected.

[0064] 2. Cell fusion: Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 4000) method. The specific steps are as follows:

[0065] a. After euthanizing the mouse by tail dislocation and cervical dislocation, immediately disinfect the mouse in 75% alcohol for about 5 minutes. Under aseptic conditions, remove the spleen 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.

[0066] b. Collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells are cultured 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 / year 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.

[0067] c. Fusion process (7 min): Min 1, add 1 mL of PEG 1500 to the cells dropwise from slow to fast; Min 2, let stand; Min 3 and Min 4, add 1 mL of RPMI-1640 medium dropwise over 1 min; Min 5 and Min 6, add 2 mL of RPMI-1640 medium dropwise over 1 min; Min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s; then incubate at 37°C for 5 min; centrifuge (800 rpm, 8 min), discard the supernatant, 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;

[0068] 3. Cell screening and cell line establishment: On the 3rd day of cell fusion, the fused cells were screened with RPMI-1640 medium with half medium replacement. On the 5th day, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On the 7th day, the cell supernatant was collected for screening.

[0069] The screening process consists of two steps: First, positive cell wells are selected using ic-ELISA; second, butylpyridoxine is used as a standard, and the inhibitory effect on positive cells is determined using ic-ELISA.

[0070] Cell wells that showed good inhibition of butylpyraclone standard were selected, and subcloning was performed using the limiting dilution method. The same method was used to detect the cells after seven days.

[0071] Three subclonings were performed using the method described above, and the DBL monoclonal antibody cell line was finally obtained.

[0072] Example 5: Preparation and Identification of Butyrmorpholine Monoclonal Antibody

[0073] 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 was collected from butylpyrroline hybridoma cells starting on day 7, and the ascites was purified for antibody purification using the caprylic acid-saturated ammonium sulfate method.

[0074] 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℃.

[0075] The IC50 of the butylpyridinium monoclonal antibody was determined using an indirect competitive ELISA. 50 The value was 104.49 ng / mL, and its IC50 for analogues was verified. 50 The cross-reactivity rates with related analogues (azoxystrobin, cyazofamid, dimethomorph, pyraclostrobin, and fluoxastrobin) were all less than 1%. The cross-reactivity value was calculated using the formula: (IC50 of pyraclostrobin) 50 ICs of other similar types 50 The cross-value is calculated as 100% (1) × 100%, with the cross-value for sensitivity to pyrmorpholine as 100%. It can be seen that this antibody has excellent sensitivity to butylpyrmorpholine and can be used for the immunoassay detection of butylpyrmorpholine.

[0076] Example 6: Application of butylpyrroline monoclonal antibody

[0077] The monoclonal antibody prepared from hybridoma cell lines via in vivo ascites fluid was used in an ELISA addition and recovery assay for butylpyridoxine. The specific steps are as follows:

[0078] (1) Coat a 96-well microplate with 0.3 μg / mL of the coating stock diluted with carbonate buffer (CBS), 100 μL per well, and coat at 37℃ for 2 h. Then wash the plate three times with PBST washing buffer, 200 μL per well each time, for 3 min each time, and pat dry.

[0079] (2) Block with CBS containing 0.2% gelatin, 200 μL per well, block at 37°C 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;

[0080] (3) Prepare 0 ng / mL, 1.23 ng / mL, 3.70 ng / mL, 11.11 ng / mL, 33.33 ng / mL, 100 ng / mL, 300 ng / mL and 900 ng / mL butylpyrroline 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 anti-butylpyrroline monoclonal antibody diluted 1:32000 to each well. After reacting at 37℃ for 0.5 h, wash the plate and pat dry.

[0081] (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 0.5 h, then wash and pat dry.

[0082] (5) Add 100 μL of TMB colorimetric solution to each well, develop the color at 37℃ for 15 min, then add 50 μL of 2M H2SO4 stop solution to each well, and measure the absorbance at 450 nm.

[0083] (6) Add recovery and sample pretreatment:

[0084] Cucumbers were selected as the test sample.

[0085] Take three 20g negative cucumber samples and add butylpyrroline standard solution to them, artificially adding it to the positive samples at concentrations of 50ng / mL, 100ng / mL, and 200ng / mL, respectively. Then, add 20mL of 20% methanol solution for extraction, vortex for 2 minutes, and centrifuge at 8000 rpm for 10 minutes. Collect the supernatant for subsequent ELISA detection.

[0086] Indirect competitive ELISA was used for additive recovery experiments. The recovery rates for the three concentrations of positive samples were 94.2%, 106.7%, and 95.8%, respectively.

[0087] The standard curve of inhibition of butylpyridamole monoclonal antibody against butylpyridamole is as follows: Figure 1 As shown, the IC50 of butylpyrroline monoclonal antibody was determined by ic-ELISA. 50 The value was 104.49 ng / mL, indicating that the antibody has good sensitivity to butylpyrroline and can be used for the immunoassay detection of butylpyrroline.

[0088] 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 a monoclonal antibody against butylpyridoxine, characterized in that, The hybridoma cell line was deposited on April 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46510. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and it is classified as a monoclonal cell line.

2. A butylpyrroline monoclonal antibody, characterized in that, Produced by the hybridoma cell line described in claim 1.

3. A composition for detecting butylpyridoxine, characterized in that, The composition comprises the hybridoma cell line of claim 1 and / or the butylpyrroline monoclonal antibody of claim 2.

4. A kit for detecting butylpyrroline, characterized in that, The kit comprises one or more of the hybridoma cell line of claim 1, the butylpyrroline monoclonal antibody of claim 2, and the composition of claim 3.

5. The reagent kit according to claim 4, characterized in that, The kit is selected from enzyme-linked immunosorbent assay (ELISA) kits, fluorescence immunoassay kits, or chemiluminescence immunoassay kits.

6. A test strip for detecting butylpyrroline, characterized in that, The test strip comprises one or more of the hybridoma cell line of claim 1, the butylpyrroline monoclonal antibody of claim 2, and the composition of claim 3.

7. The use of the hybridoma cell line of claim 1, the butylpyrroline monoclonal antibody of claim 2, the composition of claim 3, the kit of claim 4 or 5, or the test strip of claim 6 in the detection of butylpyrroline.

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