Hybridoma cell strain capable of secreting anti-napropamide monoclonal antibody and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain capable of secreting a napropamide-resistant monoclonal antibody and application of the hybridoma cell strain, and belongs to the field of food safety immunodetection. The hybridoma cell strain Tau4 secreting the napropamide cloned antibody is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation date is October 31, 2024, and the preservation number is CGMCC No.46242. The hybridoma cell strain Tau4 secreting the napropamide cloned antibody has the advantages that the hybridoma cell strain Tau4 secreting the napropamide The napropamide-resistant monoclonal antibody secreted by the cell strain is used for analyzing and detecting napropamide residues in food safety detection. The napropamide-resistant monoclonal antibody cell strain obtained by the invention can be used for immunoassay detection, and has better detection sensitivity and specificity (IC50 value is 1.13 ng / mL) on napropamide. The result of the invention can be used for preparing an immunodetection kit and a colloidal gold test strip of napropamide, and provides a powerful detection method and means for detecting the residual quantity of napropamide in foods such as fruits, vegetables and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety immunoassay, in particular to a hybridoma cell line secreting anti-napropamide monoclonal antibody and its application. Background Art

[0002] Napropamide is a broad-spectrum herbicide with good control effects on various weeds. Napropamide is a glyphosate herbicide that can inhibit the growth of weeds by disrupting the amino acid synthesis pathway in plants. This chemical substance has a high selectivity for broad-leaved herbaceous plants and some trailing plants, and can effectively control spring weeds. Its main mechanism of action is to inhibit the amino acid synthesis of weeds, interfere with their growth and development process, so as to achieve the effect of weed control. Under normal circumstances, the effective period of napropamide herbicide is usually 2-3 years. This means that napropamide can maintain its herbicidal activity within this time period under good storage conditions and when unopened. Napropamide has a long residual time in the soil, which may cause long-term effects on the soil ecosystem and affect soil biodiversity. It may enter water bodies through rainwater runoff and irrigation water, causing toxicity to aquatic organisms and affecting the balance of the aquatic ecosystem. At the same time, it may volatilize into the air during spraying, affecting air quality and may pose hazards to human health and animals and plants. After inhaling napropamide, symptoms such as coughing, expectoration, and chest tightness may occur. In severe cases, it may lead to dyspnea and pulmonary edema. Long-term exposure to napropamide may cause damage or death of nerve cells, resulting in symptoms such as decreased cognitive function and impaired motor coordination, and may affect the activity of germ cells, leading to a decrease in sperm count and motility. Long-term exposure may cause infertility or other reproductive problems.

[0003] Currently, the detection methods for napropamide residues are mainly instrumental analysis methods, including gas chromatography, gas chromatography-mass spectrometry tandem method, etc. These methods have reliable results and high sensitivity, and there are relevant technical standards for reference. However, due to the need for expensive instruments, specialized operators, and complex sample pretreatment, high cost, and long time, they cannot better meet the requirements of rapid and simple on-site detection. Therefore, it is of great significance to establish a rapid and simple detection method for napropamide. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method, suitable for on-site rapid detection of a large number of samples, providing a new detection approach for napropamide. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a hybridoma cell line secreting anti-napropamide monoclonal antibody and its application. The antibody secreted by this cell line has high detection sensitivity for napropamide and can be used to establish an immunological detection method for napropamide.

[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 secreting napropamide monoclonal antibody. The hybridoma cell line was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on October 31, 2024, with the deposit number of CGMCC No. 46242, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named as monoclonal cell line.

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

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

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

[0010]

[0011] In one embodiment of the present invention, the carrier protein is selected from one or more of bovine serum albumin, ovalbumin and keyhole limpet hemocyanin.

[0012] The second object of the present invention is to provide a napropamide monoclonal antibody, which is secreted by the above-mentioned hybridoma cell line.

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

[0014] The fourth object of the present invention is to provide a kit for detecting napropamide, which is characterized in that the kit includes one or more of the above-mentioned hybridoma cell line, the above-mentioned napropamide monoclonal antibody and the above-mentioned composition.

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

[0016] The fifth object of the present invention is to provide a test strip for detecting napropamide, which includes one or more of the above-mentioned hybridoma cell line, the above-mentioned napropamide monoclonal antibody and the above-mentioned composition.

[0017] The sixth object of the present invention is to provide a chip for detecting napropamide, which includes one or more of the above-mentioned hybridoma cell line, the above-mentioned napropamide monoclonal antibody and the above-mentioned composition.

[0018] The seventh object of the present invention is to provide the use of the hybridoma cell line, the napropamide monoclonal antibody, the composition, the kit or the test strip in detecting napropamide.

[0019] The application of the monoclonal antibody against napropamide is used for the analysis and detection of napropamide residue in food safety monitoring.

[0020] In one embodiment of the present invention, the preparation method of the napropamide hapten is as follows:

[0021] Potassium carbonate and methyl 2-chloropropionate are added to an organic solution of 6-bromonaphthalene to obtain a suspension; after stirring the obtained suspension, solid-liquid separation is carried out to take the solid phase, and methyl 2-[(6-bromonaphthalen-1-yl)oxy]propionate is obtained;

[0022] Water and LiOH are added to the alcoholic solution of the obtained methyl 2-[(6-bromonaphthalen-1-yl)oxy]propionate, stirred at room temperature, the reaction mixture is concentrated under reduced pressure, and diluted with water; after adjusting the p, filtration is carried out, and drying is carried out to obtain 2-[(6-bromonaphthalen-1-yl)oxy]propionic acid;

[0023] At 0 °C, oxalyl chloride and dimethylformamide are added to an organic solution of 2-[(6-bromonaphthalen-1-yl)oxy]propionic acid, stirred at room temperature, diethylamine is added at 0 °C, the obtained suspension is stirred at room temperature, solid-liquid separation is carried out to take the solid phase, and 2-[(6-bromonaphthalen-1-yl)oxy] is obtained;

[0024] Pd(dppf)Cl2DCM, potassium carbonate, methyl (4E)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pent-4-enoate and water are added to the solution of 2-[(6-bromonaphthalen-1-yl)oxy], the obtained suspension is stirred overnight, solid-liquid separation is carried out to take the solid phase, and a solution of methyl (4E)-5-{5-[1-(diethylcarbamoyl)ethoxy]naphthalen-2-yl}pent-4-enoate salt is obtained

[0025] Pd / C is added to the alcoholic solution of methyl (4E)-5-{5-[1-(diethylcarbamoyl)ethoxy]naphthalen-2-yl}pent-4-enoate, the obtained suspension is stirred at room temperature under hydrogen, and the filtrate is concentrated under reduced pressure to obtain methyl 5-{5-[1-(diethylcarbamoyl)ethoxy]naphthalen-2-yl}pentanoate.

[0026] LiOH and water are added to the alcoholic solution of methyl 5-{5-[1-(diethylcarbamoyl)ethoxy]naphthalen-2-yl}pentanoate, the obtained solution is stirred at room temperature, concentrated under reduced pressure, diluted, and extracted to obtain the napropamide hapten.

[0027] In one embodiment of the present invention, the preparation of the complete antigen of napropamide: The hapten of napropamide was coupled with carrier proteins BSA and OVA respectively by the carbodiimide method to prepare the immunogen napropamide-BSA and the coating antigen napropamide-OVA, and the complete antigen was separated from the uncoupled hapten by dialysis.

[0028] The above technical solution of the present invention has the following advantages compared with the prior art:

[0029] The monoclonal antibody secreted by the hybridoma cell line Tau4 provided by the present invention has good specificity and detection sensitivity for napropamide (IC 50 values are both 1.13 ng / mL), and can realize the detection of napropamide residues in foods such as fruits and vegetables, providing raw materials for the immunological detection of napropamide residues in foods, and having practical application value.

[0030] Biological material preservation: A hybridoma cell line Tau4 secreting monoclonal antibody against napropamide has been preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, abbreviated as CGMCC. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. It is classified and named as a monoclonal cell line. The preservation date is October 31, 2024, and the preservation number is CGMCC No. 46242. Description of the drawings

[0031] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in combination with the drawings, wherein,

[0032] Figure 1 is the inhibition standard curve of the Tau4 monoclonal antibody of the present invention against napropamide. Detailed implementation manners

[0033] The following further illustrates the present invention in combination with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and can implement it, but the examples given do not limit the present invention.

[0034] The present invention immunized mice with the complete antigen of napropamide, through cell fusion, culture in HAT selective medium, and screened the cell supernatant by ic-ELISA, and finally obtained the hybridoma cell line Tau4 of monoclonal antibody with high sensitivity to napropamide.

[0035] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0036] (1) The media involved in the following examples are as follows:

[0037] 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.

[0038] (2) The reagents involved in the following examples are as follows:

[0039] Carbonate Buffer Solution (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them separately in a small amount of double - distilled water, then mix them, add double - distilled water to about 800 mL and mix well. Adjust the pH value to 9.6, and make up the volume to 1000 mL with double - distilled water. Store at 4°C for later use.

[0040] Phosphate Buffer Solution (PBS): Dissolve 8.0 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, and 2.9 g of Na2HPO4·12H2O in 800 mL of pure water. Adjust the pH to 7.2 - 7.4 with NaOH or HCl, and make up the volume to 1000 mL;

[0041] Washing Solution (PBST): Add 0.5 mL of Tween - 20 to 1000 mL of 0.01 mol / L PBS solution with pH 7.4;

[0042] PBST: PBS containing 0.05% Tween - 20;

[0043] Antibody Diluent: Washing buffer containing 0.1% gelatin;

[0044] TMB Chromogenic Solution: Solution A: Dissolve 18.43 g of Na2HPO4·12H2O and 9.33 g of citric acid in pure water and make up the volume to 1000 mL; Solution B: Dissolve 60 mg of TMB in 100 mL of ethylene glycol. Mix Solution A and Solution B in a volume ratio of 1:5 to obtain TMB;

[0045] Chromogenic Solution, mix it immediately before use.

[0046] (3) The detection methods involved in the following examples are as follows:

[0047] Inhibition rate detection method: Select the most suitable antigen and antibody concentrations in ic-ELISA through checkerboard titration. Dilute the antigen to 1 μg / mL, 0.3 μg / mL, 0.1 μg / mL, and 0.03 μg / mL with carbonate buffer solution (CBS), and dilute the antibody to 1 μg / mL, 0.3 μg / mL, 0.1 μg / mL, and 0.03 μg / mL with antibody diluent. After selecting the optimal working point, dilute the standards to concentrations such as 15 ng / mL, 6 ng / mL, 3 ng / mL, 1.5 ng / mL, 0.75 ng / mL, 0.3 ng / mL, 0.15 ng / mL, and 0 ng / mL. According to the operating steps of ic-ELISA, finally plot the graph with OriginPro8.5 to obtain the standard inhibition curve and calculate the IC 50 .

[0048] Preparation of hybridoma cell line Tau4 in the example

[0049] 1. Preparation of napropamide hapten, and the reaction equation is as follows:

[0050]

[0051] The derivation steps are as follows:

[0052] Add potassium carbonate (1.24 g, 8.97 mmol) and methyl 2-chloropropionate (989 mg, 8.07 mmol) to a solution of 6-bromonaphthalene (1000 mg, 4.48 mmol) in ACN (15 mL). Stir the final suspension at 70 °C for 16 h. Remove the solvent under reduced pressure and purify by silica gel column chromatography (PE:EA = 10:1) to obtain 1.13 g (81.5%) of a pale yellow gum, namely methyl 2-[(6-bromonaphthalen-1-yl)oxy]propionate.

[0053] Add water (5 mL) and LiOH (460 mg, 10.9 mmol) to a solution of methyl 2-[(6-bromonaphthalen-1-yl)oxy]propionate (1130 mg, 3.65 mmol) in MeOH (10 mL). Stir the final solution at room temperature for 3 h. Concentrate the reaction mixture under reduced pressure, and dilute the residue with water (20 mL). Adjust the pH of the solution to 4 with HCl (1 M), and then filter. Dry the filter cake under reduced pressure to obtain the desired product (1 g, 92.7%), namely 2-[(6-bromonaphthalen-1-yl)oxy]propionic acid.

[0054] At 0 °C, oxalyl chloride (1.08 g, 8.5 mmol) and DMF (0.1 mL) were added to a solution of 2-[(6-bromonaphthalen-1-yl)oxy]propanoic acid (1 g, 3.4 mmol) in DCE (15 mL). The mixture was stirred at room temperature for 1 h, and diethylamine (1.24 g, 16.9 mmol) was added at 0 °C. The final suspension was stirred at room temperature for 1 h. The solvent was removed under reduced pressure and purified by silica gel column chromatography (PE:EA = 2:1) to obtain a colorless gum (1.1 g, 92.7%), namely 2-[(6-bromonaphthalen-1-yl)oxy].

[0055] To the solution of 2-[(6-bromonaphthalen-1-yl)oxy], Pd(dppf)Cl2 DCM (0.25 g, 0.31 mmol), potassium carbonate (1.29 g, 9.3 mmol), methyl (4E)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)penta-4-enoate (0.89 g, 3.7 mmol) and water (3 mL) were added. The final suspension was stirred at 90 °C overnight. The solvent was removed under reduced pressure and purified by silica gel column chromatography (PE:EA = 2:1) to obtain a pale yellow gum (1.1 g, 93.5%), namely methyl (4E)-5-{5-[1-(diethylcarbamoyl)ethoxy]naphthalen-2-yl}penta-4-enoate solution.

[0056] To a solution of methyl (4E)-5-{5-[1-(diethylcarbamoyl)ethoxy]naphthalen-2-yl}penta-4-enoate (1.15 g, 3 mmol) in MeOH (20 mL), Pd / C (0.2 g, 10%, wet) was added. The final suspension was stirred at room temperature under hydrogen for 3 h. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (PE:EA = 2:1) to obtain a colorless gum (818 mg, 70%), namely methyl 5-{5-[1-(diethylcarbamoyl)ethoxy]naphthalen-2-yl}pentanoate.

[0057] To a solution of methyl 5-{5-[1-(diethylcarbamoyl)ethoxy]naphthalen-2-yl}pentanoate (818 mg, 2.1 mmol) in MeOH (10 mL), LiOH (267 mg, 6.4 mmol) and H2O (4 mL) were added. The final solution was stirred at room temperature for 3 h. The reaction mixture was concentrated under reduced pressure, the residue was diluted with 50 mL of water, and extracted with 30 mL of EA. The pH of the water was adjusted to 3 with 1M HCl, and then extracted with EA (2 × 50 mL). The organic layer was washed with brine (50 mL) and dried over Na2SO4. Under reduced pressure, the solvent was removed to obtain a white solid. The crude product was dissolved in EA (10 mL), and PE (40 mL) was added and stirred for 10 min. The filtered cake was dried at room temperature to obtain the desired product (284 mg, 36%) as a white solid, namely the hapten of napropamide.

[0058] 2. Preparation of the coating antigen:

[0059] Weigh 17.31 mg of the prepared napropamide hapten, dissolve it in 400 μL of DMF and stir slowly. Then, add 9.56 mg of N-hydroxysuccinimide and 7.25 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in sequence, and react at room temperature for 4 - 6 h to obtain a mixture. Then, weigh 10 mg of chicken ovalbumin OVA and dissolve it in 2 mL of carbonate buffer. Slowly drip the obtained mixture into the chicken ovalbumin solution, and react at room temperature for 12 h with stirring. Dialyze it with 0.01 mol / L phosphate buffer PBS for 3 d to obtain the conjugate, and store it at -20 °C for later use.

[0060] 3. Immunization of mice: Select healthy BALB / c mice at 6 - 8 weeks old for immunization. Mix the complete antigen of napropamide with an equal amount of Freund's adjuvant and emulsify it, then immunize BALB / c mice by subcutaneous injection in the back. Use complete Freund's adjuvant for the first immunization and incomplete Freund's adjuvant for subsequent immunizations. The interval between the first immunization and the second booster immunization is 28 days, and the interval between multiple booster immunizations is 21 days. Collect blood 7 days after the third immunization (5 μL of mouse tail blood + 995 μL of antibody diluent = antiserum), and use ic-ELISA to measure the titer and inhibition of mouse serum. Select mice with high titer and good inhibition, and perform a boost immunization 21 days after the fifth immunization by intraperitoneal injection, requiring the boost immunization dose to be halved and without any adjuvant.

[0061] 4. Cell fusion: Three days after the boost immunization, perform cell fusion according to the conventional PEG (polyethylene glycol, molecular weight 4000) method. The specific steps are as follows:

[0062] (1) Draw blood from the eyeballs, immediately put the mice to death by cervical dislocation after drawing blood, then disinfect them in 75% alcohol and soak for about 5 min. Under sterile conditions, take out the spleens of the mice, moderately grind them with the rubber head of a syringe and obtain a spleen cell suspension through a 200-mesh cell sieve. Collect the suspension and centrifuge it (1200 rpm, 8 min). Wash the spleen cells three times with RPMI-1640 medium. After the last centrifugation, dilute the spleen cells to a certain volume, count them, and keep them for later use;

[0063] (2) Collect murine myeloma SP2 / 0 cells: Seven to ten days before fusion, culture SP2 / 0 tumor cells in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. Before fusion, the number of SP2 / 0 tumor cells is required to reach 1 - 4×10 7 , ensuring that the SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. At the time of fusion, collect the tumor cells, suspend them in RPMI-1640 basal culture medium, and perform cell counting;

[0064] (3) The fusion process takes 7 minutes. In the first minute, 1 mL of PEG 1500 is added dropwise to the cells from slow to fast; in the second minute, leave it standing. In the third and fourth minutes, 1 mL of RPMI-1640 medium is added dropwise within 1 minute; in the fifth and sixth minutes, 2 mL of RPMI-1640 medium is added dropwise within 1 minute; in the seventh minute, 1 mL of RPMI-1640 medium is added dropwise every 10 seconds. Then incubate at 37 °C for 5 minutes. Centrifuge (800 rpm, 8 minutes), discard the supernatant, resuspend in RPMI-1640 screening culture medium containing 20% fetal bovine serum and 2% of 50×HAT, and add it to a 96-well cell plate at 200 μL / well, and culture it in an incubator at 37 °C and 5% CO2.

[0065] 5. Cell screening and cell line establishment: On the 3rd day after cell fusion, half of the culture medium of the fused cells is replaced with RPMI-1640 screening culture medium, and on the 5th day, a complete medium change is carried out with RPMI-1640 transition culture medium containing 20% fetal bovine serum and 1% of 100×HT. On the 7th day, the cell supernatant is taken for screening. The screening is carried out in two steps: First, positive cell wells are screened out by ic-ELISA, and second, napropamide is used as a standard product, and the inhibitory effect of positive cells is measured by ic-ELISA. Select cell wells with good inhibition against the napropamide standard product, and subclone by the limiting dilution method, and detect by the same method. Repeat three times to obtain cell line Tau4.

[0066] 6. Preparation and identification of monoclonal antibodies: Take 8-10-week-old BALB / c mice, and inject 1 mL of sterile paraffin oil intraperitoneally into each mouse; 7 days later, inject 1×10 6 hybridoma cells into each mouse intraperitoneally. Ascites is collected starting from the seventh day, and the ascites is purified by the caprylic acid-ammonium sulfate method. Under acidic conditions, caprylic acid can precipitate other miscellaneous proteins in the ascites except IgG immunoglobulins, and then centrifuge and discard the precipitate; then precipitate the monoclonal antibody of IgG type with an equal saturation ammonium sulfate solution, centrifuge and discard the supernatant, dissolve it with 0.01 M PBS solution (pH 7.4), and dialyze to desalt. Finally, the purified monoclonal antibody is stored at -20 °C.

[0067] Using the indirect competitive ELISA method, the IC 50 of the monoclonal antibody against napropamide is 1.13 ng / mL, and its IC 50 against napropamide and the cross-reactivity rate are verified. The cross-reactivity rates of other herbicide pesticides such as alachlor, acetochlor, propachlor, propanil, diquat, and diuron are less than 1%. It can be seen that the antibody has high sensitivity and specificity against napropamide, as shown in Table 1 specifically:

[0068] Table 1 IC of monoclonal antibody Tau4 against acetochlor, acetalochlor, propachlor, propanil, diquat, and diuron 50 and cross-reactivity rate

[0069] <![CDATA[IC 50 (ng / mL)]]> Cross-reactivity rate Napropamide 1.13 100％ Alachlor >100 <1％ Acetochlor >100 <1％ Propachlor >100 <1％ Propanil >100 <1％ Diquat >100 <1％ Diuron >100 <1％

[0070] 7. Antibody application: The monoclonal antibody prepared from the hybridoma cell line Tau4 through in vivo ascites was applied to the recovery test of propachlor. The specific steps are as follows:

[0071] (1) Coating: The coating antigen propachlor-OVA was serially diluted from 1 μg / mL with 0.05 M pH 9.6 carbonate buffer, 100 μL / well, and reacted at 37 °C for 2 h.

[0072] (2) Washing: Pour out the solution in the plate and wash it 3 times with the washing solution, 3 min each time.

[0073] (3) Blocking: After patting dry, add 200 μL / well of the blocking solution and react at 37 °C for 2 h. After washing, dry it for standby.

[0074] (4) Sample addition: The antiserum (after collecting blood from the mouse's tail and diluting it with the antibody diluent to the corresponding multiple, it is the antiserum) was serially diluted starting from 1:1000 and added to the coated wells at each dilution, 100 μL / well, and reacted at 37 °C for 30 min; after thorough washing, add HRP-goat anti-mouse IgG diluted 1:3000, 100 μL / well, and react at 37 °C for 30 min.

[0075] (5) Color development: Take out the enzyme-labeled plate, after thorough washing, add 100 μL of TMB color development solution to each well and react at 37 °C in the dark for 15 min.

[0076] (6) Termination and determination: Add 50 μL of the termination solution to each well to terminate the reaction, and then measure the OD450 value of each well with an enzyme-labeled instrument.

[0077] The recovery rate measured by ic-ELISA is greater than 95%, which can be used for the immunoassay detection of propachlor.

[0078] The inhibition standard curve of Tau4 monoclonal antibody against propachlor is as Figure 1 shown.

[0079] Obviously, the above embodiments are only examples clearly described and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A hybridoma cell line secreting napropamide monoclonal antibody, characterized in that, The hybridoma cell line was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on October 31, 2024, with the deposit number CGMCC No. 46242, and the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The taxonomic name is monoclonal cell line.

2. The hybridoma cell line according to claim 1, wherein The hybridoma cell line was obtained by immunizing mice with the napropamide complete antigen.

3. The hybridoma cell line according to claim 2, characterized in that, The napropamide complete antigen was obtained by conjugating the napropamide hapten with a carrier protein.

4. The hybridoma cell line according to claim 3, characterized in that, The structural formula of the napropamide hapten is:

5. Monoclonal antibody against napropamide, characterized in that, It is secreted and produced by the hybridoma cell line described in any one of claims 1-4.

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

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

8. The kit according to claim 7, wherein The kit is selected from an enzyme-linked immunosorbent assay kit, a fluorescence immunoassay kit, or a chemiluminescence immunoassay kit.

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

10. Use of the hybridoma cell line described in any one of claims 1-4, the napropamide monoclonal antibody described in claim 5, the composition described in claim 6, the kit described in claim 7 or 8, or the test strip described in claim 9 for detecting napropamide.

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