Hybridoma cell strain secreting pyrimorph monoclonal antibody and application thereof

Abstract

The invention relates to a hybridoma cell strain capable of secreting pyrimorph monoclonal antibody and application of the hybridoma cell strain, and belongs to the field of food safety immunodetection. The hybridoma cell strain DBL capable of secreting the pyrimorph monoclonal antibody is preserved in the China General Microbiological Culture Collection Center on April 17, 2025, the preservation number is CGMCC No.46510, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the hybridoma cell strain DBL is classified and named as a monoclonal cell strain. The pyrimorph monoclonal antibody secreted by the strain is used for analyzing and detecting pyrimorph residues in food safety detection. The pyrimorph monoclonal antibody cell strain obtained by the invention can be used for immunoassay detection, and has better detection sensitivity (IC50 value is 104.49 ng / mL) to pyrimorph.

Description

Technical Field

[0001] The present invention relates to the field of food safety immunoassay, in particular to a hybridoma cell line secreting pyrimorph monoclonal antibodies and applications thereof. Background Art

[0002] Pyrimorph is a new agricultural fungicide with excellent efficacy against plant diseases caused by oomycetes. It can be used to control a variety of plant diseases, including pepper blight, tomato late blight, and cucumber downy mildew. It also has potential to treat grape downy mildew, potato blight, and rice and cotton damping-off. Its efficacy is superior to its similar agent, dimethomorph. Compared with similar domestic and international products, it boasts high efficacy, a broad spectrum of activity, and a low cost, making it an ideal pesticide for controlling oomycete diseases. Its mechanism of action suggests that this compound inhibits energy synthesis in harmful pathogens by inhibiting complex III of the cellular respiratory chain and also affects the distribution of cell wall synthesis substances. This makes it a multi-target pesticide with a low risk of resistance. Pyrimorph is widely used in crop cultivation, but consuming excessive residues in fruits and vegetables can be harmful to humans, necessitating a rapid detection method for its effectiveness.

[0003] For the detection of pyrifos pesticide residues, high performance liquid chromatography, gas chromatography, gas or liquid chromatography-mass spectrometry are often used. However, these methods have shortcomings such as complex sample pre-treatment and long detection time, and are not suitable for rapid detection of a large number of samples. In order to safeguard the interests of consumers, it is necessary to establish a high-efficiency and rapid detection method for pyrifos. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method. During detection, the pre-treatment of the sample is simple, the purification steps are few, the analysis capacity is large, the detection cost is low and it is easy to operate. It is suitable for on-site rapid detection of a large number of samples and has therefore been widely used in pesticide residue analysis. The prerequisite for using ELISA to detect pyrifos is to obtain a monoclonal antibody with high specificity and high sensitivity to pyrifos. Therefore, it is very critical to find a method for preparing a monoclonal antibody with high specificity and high sensitivity to pyrifos. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a hybridoma cell line secreting a monoclonal antibody against pyrifos and its application. The monoclonal antibody against pyrifos secreted by the hybridoma cell line has good specificity and detection sensitivity (IC 50 The value was 104.49 ng / mL), which can be used to establish an immunological detection method for butylpyroximate and detect butylpyroximate residues in food.

[0005] The present invention is achieved through the following technical solutions:

[0006] The first object of the present invention is to provide a hybridoma cell line that secretes a monoclonal antibody against pyrimethamine. The hybridoma cell line was deposited in the General Microbiology Center of the China Culture Collection Administration on April 17, 2025, with a deposit number of CGMCC No. 46510 and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified as a monoclonal cell line.

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

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

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

[0010]

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

[0012] The second object of the present invention is to provide a monoclonal antibody against pyrifos, which is secreted and produced by the hybridoma cell line.

[0013] The third object of the present invention is to provide a composition for detecting pyrifos, which comprises the hybridoma cell line and / or the pyrifos monoclonal antibody.

[0014] The fourth object of the present invention is to provide a kit for detecting pyrifos, which comprises one or more of the hybridoma cell line, the pyrifos monoclonal antibody and the composition.

[0015] In one embodiment of the present invention, the kit is selected from an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

[0016] The fifth object of the present invention is to provide a test strip for detecting pyrifos, wherein the test strip comprises one or more of the hybridoma cell line, the pyrifos monoclonal antibody and the composition.

[0017] The sixth object of the present invention is to provide the use of the hybridoma cell line, the pyrifos monoclonal antibody, the composition, the kit or the test strip in detecting pyrifos.

[0018] The present invention also provides a method for preparing the hybridoma cell line secreting the pyrimorph monoclonal antibody, comprising the following steps:

[0019] (1) using the pyrimorph hapten to prepare the pyrimorph complete antigen, and preparing the obtained pyrimorph complete antigen into Freund's adjuvant containing the antigen and incomplete Freund's adjuvant containing the antigen;

[0020] (2) The obtained Freund's adjuvant was injected subcutaneously into the back of BALB / c mice for multiple immunizations, with complete Freund's adjuvant used for the first immunization and incomplete Freund's adjuvant used for the booster immunization;

[0021] (3) collecting blood from the mice that have undergone the above immunization process, detecting the immune titer and immunosuppressive ability of the mouse serum by indirect ELISA, and screening out the immunized mice with high levels of pyrimethamine antibodies in the serum;

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

[0023] (5) The spleen cells of BALB / c mice after sprint immunization were fused with myeloma cells, the fused cells were cultured in culture medium, the positive cell wells were detected by indirect ELISA, and the inhibitory effect of the positive cell wells was further determined by indirect competitive ELISA. The positive cell wells with the best inhibition were subcloned by limiting dilution method, and finally the hybridoma cell line that could secrete the pyrimorph monoclonal antibody was screened;

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

[0025]

[0026] The structural formula of the pyrimorph 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 first 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 is collected on the 7th day after the third 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 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 subcloning is performed three times.

[0036] The present invention provides a method for preparing the above-mentioned pyrimorph monoclonal antibody, which specifically comprises: taking BALB / c mice, injecting paraffin oil into the abdominal cavity, and then injecting the hybridoma cell line DBL with a deposit number of CGMCC No. 46510 into the abdominal cavity, collecting ascites after the injection, purifying the ascites, and storing the obtained monoclonal antibody at low temperature.

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

[0038] The present invention provides a hybridoma cell line secreting a monoclonal antibody against pyrifos and its application. The pyrifos monoclonal antibody cell line obtained by the present invention can be used for immunoassay detection and has a good detection sensitivity (IC 50 The value was 104.49 ng / mL).

[0039] Deposit of biological material samples: A hybridoma cell line DBL that secretes the monoclonal antibody against pyrimethamine was deposited at the General Microbiology Center of the China Culture Collection Administration on April 17, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 46510 and the classification name as a monoclonal cell line. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0041] Figure 1 This is a standard inhibition curve of the pyrimorph monoclonal antibody prepared in the present invention against pyrimorph. DETAILED DESCRIPTION

[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 the present invention and implement it. However, the embodiments 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 all commercially available unless otherwise specified.

[0044] The culture medium involved in the following examples is as follows:

[0045] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine 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, p-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 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them in a small amount of double-distilled water, mix them, add double-distilled water to about 800 mL, adjust the pH to 9.6, add double-distilled water to 1000 mL, and store at 4°C until used.

[0048] Phosphate buffered saline (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, dissolved in 800 mL pure water, adjusted to pH 7.2–7.4 with NaOH or HCl, and made up to 1000 mL.

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

[0050] Antibody diluent: PBS supplemented with 0.1% gelatin.

[0051] TMB colorimetric solution: Solution A: Na2HPO4 . 18.43g of 12H2O, 9.33g of citric acid, and dilute to 1000mL with pure water. Solution B: Dissolve 60mg of TMB in 100mL of ethylene glycol. Mix Solution A and Solution B in a ratio of 5:1 to create the TMB colorimetric solution. Mix immediately before use.

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

[0053] Method for detecting the inhibition rate of pyrifoline: The most appropriate antigen and antibody concentrations for ic-ELISA were selected by a checkerboard test. 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 diluent. After selecting the optimal working point, the pyrifoline standard was diluted to a gradient concentration of 0, 1.23, 3.70, 11.11, 33.33, 100, 300, and 900 ng / mL. The ic-ELISA procedure was followed, and the graph was generated using OriginPro8.5 (the results are shown in the figure). Figure 1 As shown), obtain the standard inhibition curve of pyrimorph and calculate IC 50 .

[0054] Example 1: Preparation of pyrimorph hapten

[0055] Because small molecules are not immunogenic and cannot stimulate mice to produce an immune response and subsequently antibodies, they must be coupled to proteins through protein conjugation technology to make them immunogenic. Commonly used active groups in protein conjugation technology include amino, carboxyl, hydroxyl, and sulfhydryl groups. Because the hydroxyl group of pyrimorph is very important for antibody recognition, it cannot be directly used for conjugation with proteins. In this invention, rhein was selected as the hapten.

[0056] The structure of the pyrimorph hapten used in the present invention is as follows:

[0057]

[0058] Example 2: Synthesis of complete pyrimorph antigen

[0059] 3.6 mg of butylpyromorph hapten (DBL-COOH) and 4.2 mg of N-hydroxysuccinimide (NHS) were weighed and dissolved in 300 μL of N,N-dimethylformamide (DMF) and stirred at room temperature for 10 min. 6.9 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was then weighed and fully dissolved in 100 μL of DMF. The mixture was then added to the DBL-COOH solution and stirred at room temperature for 4-6 h (referred to as Solution A). 6 mg of KLH was diluted to 3 mg / mL in 0.05 M carbonate buffer (CBS) (referred to as Solution B). Solution A was then slowly added dropwise to Solution B and allowed to react at room temperature overnight. The solution was then dialyzed against 0.01 M PBS to remove unreacted small molecule hapten, yielding the complete antigen, DBL-COOH-KLH, which was identified by UV absorption scanning.

[0060] Example 3: Synthesis of pyrimorph coating

[0061] 5.7 mg of daptomycin hapten (DBL-COOH) and 4.9 mg of N-hydroxysuccinimide (NHS) were dissolved in 300 μL of anhydrous N,N-dimethylformamide (DMF) and stirred at room temperature for 10 minutes to obtain a daptomycin 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 stirred at room temperature for 4-6 hours 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 react and obtain a reaction solution. The reaction solution was dialyzed with PBS solution to remove unreacted small molecule hapten to obtain the coating source (DBL-COOH-OVA).

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

[0063] 1. Immunization of Animals: After emulsification, the complete pyrimorph antigen was mixed with an equal amount of Freund's adjuvant and immunized BALB / c mice by multiple subcutaneous injections at the back of the neck (except for booster immunizations). Complete Freund's adjuvant was used for the first immunization at a dose of 100 μg per mouse. Incomplete Freund's adjuvant was used for multiple booster immunizations, and the dose was halved to 50 μg per mouse. For booster immunizations, no adjuvant was used and the mice were directly diluted with normal saline and injected intraperitoneally, and the dose was further halved to 25 μg per mouse. The interval between the first and second booster immunizations was one month, between multiple booster immunizations was 21 days, and between the booster immunization and the final booster immunization was 18-21 days. The immune response of mice was assessed by indirect competitive enzyme-linked immunosorbent assay (IC-ELISA), i.e., the titer and inhibition of mouse serum were measured.

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

[0065] a. Blood was collected by tail amputation. Mice were sacrificed by cervical dislocation and immediately disinfected in 75% alcohol for about 5 minutes. The spleen of the mouse was removed aseptically. The spleen was moderately ground with the rubber tip of a syringe and passed through a 200-mesh cell sieve to obtain a splenocyte suspension. The suspension was collected and centrifuged (1200 rpm, 8 minutes). The splenocytes were washed three times with RPMI-1640 medium. After the final centrifugation, the splenocytes were diluted to a certain volume, counted, and set aside.

[0066] b. Collect 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. Before fusion, the number of SP2 / 0 tumor cells should reach 1-4×10 7 , ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. When fusion occurs, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counts are performed;

[0067] c. Fusion process 7 min: In the first minute, 1 mL of PEG 1500 was added dropwise to the cells from slow to fast addition; in the second minute, the cells were allowed to stand; in the third and fourth minutes, 1 mL of RPMI-1640 medium was added dropwise over 1 min; in the fifth and sixth minutes, 2 mL of RPMI-1640 medium was added dropwise over 1 min; in the seventh minute, 1 mL of RPMI-1640 medium was added dropwise every 10 s; the cells were then incubated at 37°C for 5 min; centrifuged (800 rpm, 8 min), the supernatant was discarded, and the cells were resuspended in RPMI-1640 screening medium containing 20% ​​fetal bovine serum and 2% 50× HAT, and 200 μL / well were added to a 96-well cell plate and cultured in a 37°C, 5% CO2 incubator.

[0068] 3. Cell screening and cell line establishment: On the third day after cell fusion, the fused cells were half-replaced with RPMI-1640 screening medium. On the fifth day, the medium was fully replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On the seventh day, the cell supernatant was collected for screening.

[0069] The screening was divided into two steps: the first step was to screen the positive cell wells using the ic-ELISA method, and the second step was to use pyrimorph as a standard to measure the inhibitory effect of the positive cells using the ic-ELISA method;

[0070] The cell wells that showed good inhibition of the pyrimorph standard were selected and subcloned using the limiting dilution method, and then tested using the same method seven days later;

[0071] Subcloning was performed three times according to the above method, and finally the pyrimorph monoclonal antibody cell line DBL was obtained.

[0072] Example 5: Preparation and identification of monoclonal antibodies against pyrimorph

[0073] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil per mouse; 7 days later, 1×10 6 The ascites of the pyrimorph hybridoma cells were collected from the seventh day and the antibodies were purified by the octanoic acid-saturated ammonium sulfate method.

[0074] Under slightly acidic conditions, octanoic acid can precipitate other miscellaneous proteins in the ascites except IgG immunoglobulin, and then centrifuge and discard the precipitate; then use an equal amount of saturated ammonium sulfate solution to precipitate IgG type monoclonal antibodies, centrifuge, discard the supernatant, dissolve it with 0.01M PBS solution (pH 7.4), dialyze and desalt, and finally obtain the purified monoclonal antibody and store it at -20°C.

[0075] The IC of the monoclonal antibody against pyrimethamine was determined using an indirect competitive ELISA. 50 The value was 104.49 ng / mL, and its IC 50 The cross-reactivity rate is less than 1% for related analogs (azoxystrobin, cyazolin, mandiprop-1, pyraclostrobin and fluthiazolin). The cross-reactivity value calculation formula is: (IC 50 / Other analog ICs 50 )×100%, with the crossover of sensitivity to pyrimorph being 100%. It can be seen that the antibody has a good sensitivity to pyrimorph and can be used for pyrimorph immunoassay detection.

[0076] Example 6: Application of pyrimethamine monoclonal antibody

[0077] The monoclonal antibody produced by hybridoma cell lines in vivo through ascites was used in the ELISA spike-recovery test of pyrimorph. The specific steps are as follows:

[0078] (1) Coat a 96-well microtiter plate with 100 μL of the coating agent diluted in carbonate buffer (CBS) at a concentration of 0.3 μg / mL per well. Incubate at 37°C for 2 h. Wash the plate three times with PBST solution, 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, at 37°C for 2 h, wash the plate three times with PBST, 200 μL per well each time, for 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 of pyrifos standard solutions using phosphate buffered saline (PBS). Add the standard solutions and the sample extracts to the blocked ELISA plate, 50 μL per well. Repeat for three wells for each sample. Then, add 50 μL of anti-pyrifos monoclonal antibody diluted 1:32,000 to each well. Incubate at 37°C for 0.5 h, then wash the plate and pat dry.

[0081] (4) Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 in PBS containing 0.1% gelatin to each well, react at 37°C for 0.5 h, and then wash the plate and pat dry.

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

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

[0084] Cucumber was selected as the test sample.

[0085] Three 20g negative cucumber samples were spiked with a standard solution of pyrimorph to create positive samples at concentrations of 50ng / mL, 100ng / mL, and 200ng / mL, respectively. Each sample was then extracted with 20mL of 20% methanol. After vortexing for 2 minutes, the samples were centrifuged at 8000 rpm for 10 minutes. The supernatant was used for subsequent ELISA testing.

[0086] Indirect competitive ELISA was used to conduct a spike-in recovery experiment. The corresponding recovery rates for the three spiked concentrations of positive samples were 94.2%, 106.7% and 95.8%, respectively.

[0087] The standard curve of inhibition of pyrimorph monoclonal antibody against pyrimorph is shown in Figure 2. Figure 1 As shown, the IC of the monoclonal antibody against pyrimethamine was determined by IC-ELISA. 50 The value was 104.49 ng / mL, indicating that the antibody had good sensitivity to butylpyroxithromycin and could be used for immunoassay detection of butylpyroxithromycin.

[0088] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A hybridoma cell line secreting a monoclonal antibody against pyrifos, characterized in that: The hybridoma cell line was deposited in the General Microbiology Center of the China Culture Collection Administration on April 17, 2025, with the deposit number CGMCC No. 46510, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named as a monoclonal cell line.

2. The hybridoma cell line according to claim 1, wherein The hybridoma cell line is obtained by immunizing mice with a complete pyrimorph antigen.

3. The hybridoma cell line according to claim 1, characterized in that The pyrimorph complete antigen is obtained by coupling the pyrimorph hapten with a carrier protein.

4. The hybridoma cell line according to claim 1, wherein The structural formula of the pyrimorph hapten is:

5. A monoclonal antibody against pyrimorph, characterized in that: The protein is secreted and produced by the hybridoma cell line according to any one of claims 1 to 4.

6. A composition for detecting pyrimorph, characterized in that The composition comprises the hybridoma cell line according to any one of claims 1 to 4 and / or the pyrimethamine monoclonal antibody according to claim 5.

7. A kit for detecting pyrimorph, characterized in that: The kit comprises one or more of the hybridoma cell line according to any one of claims 1 to 4, the pyrimorph monoclonal antibody according to claim 5, and the composition according to claim 6.

8. The kit according to claim 7, characterized in that The kit is selected from an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

9. A test strip for detecting pyrimorph, characterized in that: The test strip comprises one or more of the hybridoma cell line according to any one of claims 1 to 4, the pyrimorph monoclonal antibody according to claim 5, and the composition according to claim 6.

10. Use of the hybridoma cell line according to any one of claims 1 to 4, the monoclonal antibody against butylpyroximate according to claim 5, the composition according to claim 6, the kit according to claim 7 or 8, or the test strip according to claim 9 in detecting butylpyroximate.

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