Hybridoma cell strain capable of secreting Xiawei monoclonal antibody and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain capable of secreting a monoclonal antibody of Xiawei and application of the hybridoma cell strain, and belongs to the technical field of immunochemistry. The hybridoma cell strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on April 17, 2025, the preservation number is CGMCC No.46507, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the hybridoma cell strain is classified and named as a monoclonal cell strain. The monoclonal antibody secreted by the hybridoma cell strain disclosed by the invention has better detection sensitivity (IC50 value is 0.47 ng / mL) to acarb, and can be used for residual detection of acarb.

Description

Technical Field

[0001] The present invention relates to the technical field of immune detection, in particular to a hybridoma cell line secreting a Mioxiewei monoclonal antibody and an application thereof. Background Art

[0002] Aminocarb (AMI), commonly known as Aminocarb (AMI), is a carbamate insecticide with a unique insecticidal mechanism, including stomach poisoning, contact killing, feeding repellent, repellent, and inhibition or interference with pest growth and development. It is highly effective against pests such as red spider mites, white spiders, whiteflies, aphids, and scale insects on citrus, lemons, and apples. For vegetable pests such as cabbage worms, aphids, and diamondback moths, the most effective treatment is when applied to young larvae. Although Aminocarb is relatively low in toxicity, its half-life in soil is 7-30 days, and its extensive use can have environmental impacts. Therefore, accurate quantitative assessment of Aminocarb in the environment is essential.

[0003] Currently, the detection of chlorpyrifos is primarily instrumental, with gas chromatography, liquid chromatography, and gas chromatography-mass spectrometry being the most common methods. While these chromatography-based methods offer high sensitivity and specificity, they have drawbacks, such as the need for thorough sample cleanup, high solvent consumption, expensive equipment, and the need for skilled technicians. Therefore, a rapid and simple method for detecting chlorpyrifos residues is needed.

[0004] Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method. During the detection, the sample pre-treatment is simple, the purification steps are few, the analysis capacity is large, the detection cost is low and the operation is simple. It is suitable for on-site rapid detection of a large number of samples and has therefore been widely used in drug residue analysis. The premise of using the enzyme-linked immunosorbent assay to detect chloramphenicol is to obtain a monoclonal antibody with high specificity and high sensitivity to chloramphenicol. Therefore, it is very important to find a method for preparing a monoclonal antibody with high specificity and high sensitivity to chloramphenicol. The inventors attempted to prepare chloramphenicol monoclonal antibodies using hybridoma cells. However, in the process of preparing hybridoma cell lines that can secrete chloramphenicol monoclonal antibodies, how to prepare chloramphenicol haptens and complete antigens and how to make mice produce a strong immune effect still need further research; how to make the prepared hybridoma cell lines able to successfully secrete chloramphenicol monoclonal antibodies still need further research; how to make the secreted chloramphenicol monoclonal antibodies have strong specificity and high sensitivity also need further research. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a hybridoma cell line secreting a monoclonal antibody against Mioxetine and its application. The hybridoma cell line of the present invention secretes a monoclonal antibody against Mioxetine with good detection sensitivity (IC 50The value was 0.47 ng / mL), which can be used to establish an immunological detection method for cypermethrin and detect the residues of cypermethrin in soil.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a hybridoma cell line that secretes a monoclonal antibody against cypermethrin. 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. 46507 and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified as a monoclonal cell line.

[0008] In one embodiment of the present invention, the hybridoma cell line is obtained by immunizing mice with the complete antigen of Mirephrenia.

[0009] In one embodiment of the present invention, the complete Mioxiewei antigen is obtained by coupling Mioxiewei hapten with a carrier protein.

[0010] In one embodiment of the present invention, the carrier protein comprises keyhole limpet hemocyanin or chicken ovalbumin.

[0011] In one embodiment of the present invention, the structural formula of the Miehaiwei hapten is:

[0012]

[0013] In one embodiment of the present invention, the structural formula of the complete antigen of Miehaiwei is:

[0014]

[0015] The second purpose of the present invention is to provide the use of the hybridoma cell line in detecting Miehaiwei.

[0016] The third object of the present invention is to provide a monoclonal antibody against Miewei, which is secreted by the hybridoma cell line.

[0017] The present invention also provides a method for preparing the above-mentioned monoclonal antibody of Miehaiwei, which specifically comprises: taking BALB / c mice, injecting paraffin oil into the peritoneal cavity, and then injecting the hybridoma cell line with the deposit number of CGMCC No.46507 into the peritoneal cavity, collecting ascites after the injection, purifying the ascites, and storing the obtained monoclonal antibody at low temperature.

[0018] The fourth object of the present invention is to provide a composition comprising the hybridoma cell line and / or the Mioxim monoclonal antibody.

[0019] The fifth object of the present invention is to provide a colloidal gold test strip, which comprises the hybridoma cell line, the Mioxin monoclonal antibody or the composition.

[0020] In one embodiment of the present invention, the colloidal gold test strip comprises a sample pad, a colloidal gold conjugate pad, a nitrocellulose membrane and a water absorbent pad, the nitrocellulose membrane is sequentially provided with a detection line and a quality control line, and the colloidal gold conjugate pad is coated with the Mioxetine monoclonal antibody.

[0021] The sixth object of the present invention is to provide a kit comprising the hybridoma cell line, the Mioxin monoclonal antibody or the composition.

[0022] 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.

[0023] The seventh object of the present invention is to provide a chip, which comprises the hybridoma cell line, the Mioxin monoclonal antibody or the composition.

[0024] The eighth object of the present invention is to provide the use of the monoclonal antibody to fenvalerate, the composition, the colloidal gold test strip, the kit or the chip in detecting fenvalerate.

[0025] The present invention also provides a method for preparing the hybridoma cell line secreting the Miehuiwei monoclonal antibody, comprising the following steps:

[0026] Step 1: preparing the hapten and complete antigen of Bihayi, and emulsifying the obtained complete antigen of Bihayi with Freund's adjuvant or incomplete Freund's adjuvant to prepare an immunogen;

[0027] Step 2: The obtained immunogen 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;

[0028] Step 3: Blood was collected from the mice that had undergone the above immunization process, and the immune titer and immunosuppressive capacity of the mouse serum were detected by indirect ELISA to screen out mice with high sensitivity of the Mieheiwei antibody in the serum;

[0029] Step 4: The screened mice were immunized by intraperitoneal injection using complete antigen of Metformin without Freund's adjuvant;

[0030] Step 5: The spleen cells of the BALB / c mice after sprint immunization were fused with myeloma cells. The fused cells were screened and cultured in HAT 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 highly sensitive monoclonal antibody against Metformin was screened out.

[0031] In one embodiment of the present invention, in step 1, the molecular formula of the Miehaiwei hapten is as follows:

[0032]

[0033] 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.

[0034] 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.

[0035] 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;

[0036] In one embodiment of the present invention, in step 3, the blood is collected on the 7th day after the third immunization process.

[0037] In one embodiment of the present invention, in step 5, the cell fusion is performed 3 days after the end of the sprint immunization.

[0038] In one embodiment of the present invention, in step 5, the cell fusion is performed by polyethylene glycol (PEG4000) method.

[0039] In one embodiment of the present invention, in step 5, the culture medium is RPMI-1640 culture medium.

[0040] In one embodiment of the present invention, in step 5, the number of subcloning is 4 times.

[0041] The above technical solution of the present invention has the following advantages over the prior art:

[0042] (1) The monoclonal antibody against Miehaiwei obtained by the present invention has a good detection sensitivity (IC 50 value was 0.47 ng / mL);

[0043] (2) The Miehaiwei monoclonal antibody cell line obtained in the present invention can be used for immunoassay detection.

[0044] Deposit of biological material samples: A hybridoma cell line YMF that secretes the monoclonal antibody against Metformin was deposited on April 17, 2025, at the General Microbiology Center of the China Culture Collection Administration. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit number is CGMCC No. 46507, and the classification name is a monoclonal cell line. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] 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:

[0046] Figure 1 This is the standard curve of inhibition of the Mioxiewei monoclonal antibody of the present invention on Mioxiewei. DETAILED DESCRIPTION

[0047] 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.

[0048] 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.

[0049] The culture medium involved in the following examples is as follows:

[0050] 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.

[0051] The reagents involved in the following examples are as follows:

[0052] 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.

[0053] 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.

[0054] PBST: PBS containing 0.05% Tween 20;

[0055] Antibody diluent: PBS containing 0.1% gelatin;

[0056] 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. Re-mix before use.

[0057] The detection methods involved in the following embodiments are as follows:

[0058] Method for detecting the inhibition rate of cypermethrin: The most suitable antigen and antibody concentrations in ic-ELISA were selected by chessboard test. The antigen was diluted to 0.01, 0.03, 0.1 and 0.3 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03 μg / mL, 0.1 μg / mL, 0.3 μg / mL and 1 μg / mL with antibody diluent. After selecting the optimal working point, the cypermethrin standard was diluted to 8 concentrations (0, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL and 10 ng / mL), and the ic-ELISA operation steps were followed. Finally, the graph was made using OriginPro 8.5 (the results are shown in Figure 2). Figure 1 As shown), obtain the standard inhibition curve of cypermethrin and calculate IC 50 .

[0059] Example 1: Synthesis of the hapten Miehaiwei

[0060] Since the small molecule of Mirexed is not immunogenic and cannot stimulate mice to produce an immune response and then produce antibodies, Mirexed needs to be coupled to the protein through protein coupling technology to make it immunogenic; the active groups commonly used in protein coupling technology include amino, carboxyl, hydroxyl, thiol, etc. Since the molecular structure of Mirexed does not contain these active groups, Mirexed is derivatized.

[0061] Fe (2.87 g), NH4Cl (2.75 g), and 4-(benzyloxy)-2-methyl-1-nitrobenzene (2.5 g) were added to 20 mL of ethanol and 3 mL of water, stirred at 90°C for 16 h, and the precipitated solid was removed by filtration using celite. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by reverse phase column chromatography using a gradient elution of acetonitrile in water from 5% to 95%. The desired fractions were combined and concentrated to obtain product A. tert-Butyl 5-bromovalerate (2.22 g), potassium carbonate (2.6 g), and product A (2.01 g) were added to a 30 mL dimethylformamide solution, stirred at 60°C for 16 h, and the mixture was extracted with ethyl acetate to obtain product B as a white solid. Product B (2.98 g) was dissolved in acetonitrile solution, and formaldehyde (726 mg) was added, followed by stirring at 25°C for 30 min. Sodium cyanoborohydride (1.01 g) was then added to the mixture, and stirring was continued at 25°C for 4 hours. The mixture was extracted with ethyl acetate to obtain product C as a white solid. Product C (1.0 g) was dissolved in 30 mL of methanol and Pd / C (200 mg) was added. The mixture was stirred at 25°C under a H2 atmosphere for 16 hours to obtain product D. Product D (746 mg) was dissolved in 20 mL of tetrahydrofuran and potassium tert-butoxide (284 mg) was added. The reaction mixture was stirred at 0°C under a nitrogen atmosphere for 30 minutes. Methylcarbamic acid chloride (475 mg) was added to the mixture, and stirring was continued at 25°C under a nitrogen atmosphere for 16 hours. The reaction mixture was extracted with ethyl acetate to obtain product E. Product E (400 mg) was dissolved in 2 mL of dioxane and 6 mL of hydrochloric acid / dioxane was added. The mixture was stirred at room temperature for 3 hours to obtain product F as a brown oil, the hapten of mirbak.

[0062] Example 2: Synthesis of complete antigen of Miehaiwei

[0063] 8 mg of the hapten methylphenidate (MMP) and 6.21 mg of N-hydroxysuccinimide (NHS) were dissolved in 200 μL of N,N-dimethylformamide (DMF) and stirred at room temperature for 10 minutes. 10.36 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was then added to the MMP hapten solution and stirred at room temperature for 6-8 hours for activation. 6 mg of keyhole limpet hemocyanin (KLH) was added to 3 mL of 0.01 M carbonate buffer (CBS) and thoroughly dissolved. The activated hapten was then slowly added to the KLH solution and stirred at room temperature overnight. The purified complete antigen was then dialyzed against 0.01 M PBS to remove unreacted small molecules. The purified antigen was then identified by UV absorption scanning.

[0064] Example 3: Synthesis of the original coating of Mioxit

[0065] Dissolve 3.54 mg of the hapten bisulphite and 2.76 mg of N-hydroxysuccinimide (NHS) in 200 μL of anhydrous N,N-dimethylformamide (DMF) and stir at room temperature for 10 minutes. Dissolve 4.6 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in this solution and stir at room temperature for 6-8 hours to obtain a hapten activation solution. Dissolve 6 mg of chicken ovalbumin (OVA) in carbonate buffer (CBS). Slowly add the hapten activation solution to the protein dilution solution and stir at room temperature overnight. Then, dialyze the reaction solution against 0.01 M PBS to remove unreacted small molecules to obtain the coating source.

[0066] Example 4: Preparation of hybridoma cell lines secreting monoclonal antibodies to Miewei

[0067] 1. Acquisition of animal immunity

[0068] After emulsifying the complete antigen of Mihaiwei and an equal amount of Freund's adjuvant, BALB / c mice were immunized by subcutaneous injection at multiple points on the back of the neck (except for the boost immunization). 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 boost immunizations, and the dose was halved to 50 μg per mouse. No adjuvant was used for the boost immunization, and the mouse was directly diluted with normal saline and injected intraperitoneally, with the dose further halved to 25 μg per mouse. The interval between the first and second boost immunizations was one month, the interval between multiple boost immunizations was 21 days, and the interval between the boost immunization and the final boost immunization was 18-21 days. The immune effect of the mice was observed by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), i.e., the titer and inhibition of the mouse serum were detected.

[0069] 2. Cell fusion

[0070] 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:

[0071] 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.

[0072] b. Collect SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were expanded 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;

[0073] c. Fusion process (7 min): In the first minute, add 1 mL of PEG 4000 dropwise to the cells in a gradual, increasing order. In the second minute, allow the cells to stand. In the third and fourth minutes, add 1 mL of RPMI-1640 medium dropwise over 1 minute. In the fifth and sixth minutes, add 2 mL of RPMI-1640 medium dropwise over 1 minute. In the seventh minute, add 1 mL of RPMI-1640 medium dropwise every 10 seconds. Except for the second minute, shake the solution continuously. Then, incubate at 37°C for 5 minutes. Centrifuge (800 rpm, 8 minutes), discard the supernatant, and resuspend the cells in RPMI-1640 selection medium supplemented with 20% fetal bovine serum and 2% 50× HAT. Plate 200 μL / well of the cells into a 96-well plate and incubate in a 37°C, 5% CO2 incubator.

[0074] 3. Cell screening and cell line establishment

[0075] On the third day after cell fusion, the fused cells were subjected to a half-change of RPMI-1640 screening culture medium. On the fifth day, the culture medium was fully replaced with RPMI-1640 transition culture medium containing 20% ​​fetal bovine serum and 1% 100×HT. On the seventh day, the cell supernatant was collected for screening.

[0076] The screening is divided into two steps: the first step is to screen out the positive cell wells using the ic-ELISA method, and the second step is to use cypermethrin as the standard and use the ic-ELISA method to determine the inhibitory effect on the positive cells.

[0077] The cell wells with better inhibition to the standard of Metformin were selected and subcloned using the limiting dilution method. The cells were then tested using the same method seven days later.

[0078] Subcloning was performed at least three times according to the above method to finally obtain the Miohewei monoclonal antibody cell line.

[0079] Example 5: Preparation and identification of monoclonal antibodies to Miehaiwei

[0080] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil. Seven days later, 1×10 6 The ascites of the hybridoma cells were collected from the seventh day and the antibodies were purified by the caprylic acid-saturated ammonium sulfate method.

[0081] 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.

[0082] The IC of the monoclonal antibody against Miheiwei was measured using an indirect competitive ELISA. 50 The value was 0.47 ng / mL, indicating that it had good sensitivity to chlorpyrifos and could be used for chlorpyrifos immunoassay detection.

[0083] Verify its IC values ​​for analogues (aldicarb, methomyl, cypermethrin, pirimicarb and carbofuran) 50 and cross-reaction rate, the cross-reaction rate of related analogs is less than 1%, wherein the cross-reaction rate = (IC 50 / analog IC 50 )×100%. According to the cross-reaction rate, it can be seen that the antibody has a high sensitivity and specificity to Miehaiwei, as shown in Table 1:

[0084] Table 1 IC values ​​of monoclonal antibodies against mirbane and its analogs 50 and cross-reaction rate

[0085] <![CDATA[IC 50 (ng / mL)]]> Cross-reaction rate (%) Eliminate harm 0.47 100 Aldicarb 987.12 ＜1 Methomyl 672.91 ＜1 Metformin 356.35 ＜1 Pirimicarb 469.24 ＜1 Carbofuran 200.39 ＜1

[0086] Example 6: Application of Miehaiwei Monoclonal Antibody

[0087] The monoclonal antibodies prepared from the hybridoma cell line in vivo using ascites were applied to the ELISA spike-recovery test of Mioxetine. The specific steps are as follows:

[0088] (1) Coat a 96-well microtiter plate with 100 μL of the coating agent diluted in carbonate buffer (CBS) at a concentration of 0.1 μg / mL per well. Bake 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.

[0089] (2) Block with CBS containing 0.2% gelatin, 200 μL per well, bake at 37°C for 2 h, wash the plate three times with PBST, 200 μL per well each time, 3 min each time, and pat dry;

[0090] (3) Prepare 0, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL and 10 ng / mL of standard solutions of chlorpyrifos, respectively, with phosphate buffered saline (PBS). Add the standard solutions and the sample extracts to the blocked ELISA plate, 50 μL per well, and repeat 3 wells for each sample. Then add 50 μL of chlorpyrifos monoclonal antibody diluted to 0.1 μg / mL to each well, react at 37°C for 30 min, wash the plate and pat dry.

[0091] (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 30 min, and then wash the plate and pat dry.

[0092] (5) Add 100 μL of TMB colorimetric solution to each well, 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.

[0093] (6) Add recycling:

[0094] Soil was selected as the test sample.

[0095] Add 0.1ppb, 0.5ppb, and 2.5ppb of the standard solution of cypermethrin to the samples (according to the linear range of the antibody and IC 50 The concentration of the spiked product was set and the mixture was shaken on an electric shaker. The recovery rates of spiked product were 113%, 116%, and 87% respectively using indirect competitive ELISA.

[0096] 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 Miewei, 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. 46507, 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. Use of the hybridoma cell line according to claim 1 in detecting Miehaiwei.

3. A monoclonal antibody against Miehaiwei, characterized in that: The hybridoma cell line according to claim 1 is secreted.

4. A composition, characterized in that The composition comprises the hybridoma cell line according to claim 1 and / or the Mioxan monoclonal antibody according to claim 3.

5. A colloidal gold test strip, characterized in that: The colloidal gold test strip comprises the hybridoma cell line according to claim 1, the Mihewei monoclonal antibody according to claim 3, or the composition according to claim 4.

6. The colloidal gold test strip according to claim 5, characterized in that The colloidal gold test strip comprises a sample pad, a colloidal gold binding pad, a nitrocellulose membrane and a water absorbent pad; a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane; and the colloidal gold binding pad is coated with the Mieheiwei monoclonal antibody according to claim 3.

7. A kit, characterized in that The kit comprises the hybridoma cell line according to claim 1, the Mioxin monoclonal antibody according to claim 3 or the composition according to claim 4.

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 chip, characterized in that: The chip comprises the hybridoma cell line according to claim 1, the Mioxin monoclonal antibody according to claim 3, or the composition according to claim 4.

10. Use of the monoclonal antibody against fenvalerate according to claim 3, the composition according to claim 4, the colloidal gold test strips according to claims 5-6, the kits according to claims 7-8 or the chip according to claim 9 in detecting fenvalerate.

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