Hybridoma cell strain secreting napropamide monoclonal antibody and application of hybridoma cell strain

Abstract

The invention provides a hybridoma cell strain secreting a napropamide monoclonal antibody and application of the hybridoma cell strain, and relates to the technical field of immunodetection. The monoclonal antibody secreted by the hybridoma cell strain disclosed by the invention has good sensitivity to the napropamide, and the IC50 of the monoclonal antibody disclosed by the invention to the napropamide is 1.13 ng / mL, so that the monoclonal antibody disclosed by the invention can be used for detecting trace napropamide. In addition, the monoclonal antibody disclosed by the invention has no cross reaction on structural analogues of the napropamide, such as alachlor, acetochlor, propachlor, propanil, diquat and diuron, so that the monoclonal antibody disclosed by the invention can be used for accurately detecting the low-concentration napropamide.

Description

Technical Field

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

[0002] Napropamide is a broad-spectrum herbicide that has a good control effect on many weeds. Napropamide is a glyphosate-based herbicide that can inhibit the growth of weeds by disrupting the amino acid synthesis pathway in plants. The chemical is highly selective for broad-leaved herbs and some creeping plants, which can effectively control spring weeds. Its main mechanism of action is to inhibit the amino acid synthesis of weeds and interfere with their growth and development processes, thereby achieving the effect of weed control. Under normal circumstances, the effective period of naproxamide herbicides is usually 2-3 years. This means that if the storage conditions are good and it is unopened, naproxamide can maintain its herbicidal activity during this period. Napropamide remains in the soil for a long time, which may have a long-term impact on the soil ecosystem and affect soil biodiversity. Or it can enter the water body through rainwater runoff and irrigation water, causing poisoning to aquatic organisms and affecting the balance of aquatic ecosystems. At the same time, it may volatilize into the air during spraying, affecting air quality and causing harm to human health and animals and plants. After inhaling propamide, symptoms such as coughing, sputum, and chest tightness may occur. In severe cases, it may cause difficulty breathing and pulmonary edema. Long-term exposure to propamide may cause damage or death of nerve cells, causing symptoms such as decreased cognitive function and motor coordination disorders. It may affect the activity of germ cells, leading to a decrease in sperm count and motility. Long-term exposure may cause infertility or other fertility problems.

[0003] At present, the detection method for the residue of propamide is mainly instrumental analysis method, 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 pre-treatment, high cost, and long time, it cannot better meet the requirements of fast and easy on-site detection. Therefore, it is of great significance to establish a fast and easy detection method for propamide. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method, which is suitable for rapid on-site detection of a large number of samples and provides a new detection method for propamide. The premise of using enzyme-linked immunosorbent assay to detect propamide is to obtain a monoclonal antibody with high sensitivity to propamide, and the prior art lacks a monoclonal antibody with high sensitivity and high specificity to propamide. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art of lacking a monoclonal antibody with high sensitivity and high specificity to propamide.

[0005] In order to solve the above technical problems, the present invention provides a hybridoma cell line secreting a monoclonal antibody against naproxen and its application. The monoclonal antibody secreted by the hybridoma cell line of the present invention has good sensitivity to naproxen, and the IC value of the monoclonal antibody of the present invention to naproxen is 50 The monoclonal antibody of the present invention has no cross reaction to the structural analogs of naproxen, such as alachlor, acetochlor, chlorfenapyr, propanil, diquat and diuron, so the monoclonal antibody of the present invention can accurately detect low concentrations of naproxen.

[0006] The first object of the present invention is to provide a hybridoma cell strain, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on October 31, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.46229.

[0007] Furthermore, the hybridoma cell line is obtained by immunizing an animal with a complete antigen prepared by a hapten, wherein the structural formula of the hapten is as shown in Formula I:

[0008]

[0009] Furthermore, the complete antigen is obtained by coupling the hapten to a carrier protein.

[0010] Furthermore, the carrier protein includes bovine serum albumin.

[0011] The second object of the present invention is to provide an application of the hybridoma cell line mentioned above in detecting propamide.

[0012] The third object of the present invention is to provide a monoclonal antibody secreted by the hybridoma cell line mentioned above.

[0013] The fourth object of the present invention is to provide a use of the above monoclonal antibody in detecting propamide.

[0014] A fifth object of the present invention is to provide a detection product for propamide, which comprises the above-mentioned monoclonal antibody.

[0015] Furthermore, the detection product also includes a coating source.

[0016] Furthermore, the coating agent is prepared by coupling a hapten to a carrier protein, wherein the carrier protein comprises chicken ovalbumin.

[0017] Beneficial effects of the present invention:

[0018] The present invention provides a hybridoma cell line secreting a monoclonal antibody against naproxen and its application. The monoclonal antibody secreted by the hybridoma cell line of the present invention has good sensitivity to naproxen, and the IC value of the monoclonal antibody of the present invention to naproxen is 50 The monoclonal antibody of the present invention has no cross reaction to the structural analogs of naproxen, such as alachlor, acetochlor, chlorfenapyr, propanil, diquat and diuron, so the monoclonal antibody of the present invention can accurately detect low concentrations of naproxen.

[0019] Biomaterial Deposit

[0020] Monoclonal cell line DAZ, the monoclonal cell line was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 31, 2024, with the deposit number CGMCC No.46229, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0022] Figure 1 is the standard inhibition curve of the naproxenamide monoclonal antibody of the present invention;

[0023] Figure 2 The invention discloses a process for preparing the propamide hapten of the invention. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0025] 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 respectively and mix them, add double distilled water to about 800 mL and mix well, adjust the pH to 9.6, add double distilled water to 1000 mL, and store at 4°C for later use.

[0026] Phosphate buffered saline (PBS): 8.0 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;

[0027] Washing solution (PBST): add 0.5 mL of Tween-20 to 1000 mL of 0.01 mol / L pH 7.4 PBS solution;

[0028] PBST: PBS containing 0.05% Tween-20;

[0029] Antibody diluent: Wash buffer containing 0.1% gelatin;

[0030] TMB colorimetric solution: Solution A: 18.43g Na2HPO4·12H2O, 9.33g citric acid, dilute to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mixing solution A and solution B in a volume ratio of 1:5 is TMB. The colorimetric solution should be mixed before use.

[0031] Example 1: Preparation of hapten

[0032] Potassium carbonate (1.24 g, 8.97 mmol) and methyl 2-chloropropionate (989 mg, 8.07 mmol) were added to a solution of 6-bromonaphthalene (1000 mg, 4.48 mmol) in ACN (15 mL). The final suspension was stirred at 70°C for 16 hours. The solvent was removed under reduced pressure and purified by silica gel column chromatography (PE:EA=10:1) to obtain 1.13 g of product 2.

[0033] To a solution of product 2, methyl 2-[(6-bromonaphthalene-1-yl)oxy]propanoate (1130 mg, 3.65 mmol) in MeOH (10 mL), water (5 mL) and LiOH (460 mg, 10.9 mmol) were added. The final solution was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure and the residue was diluted with water (20 mL). The solution was adjusted to pH = 4 with HCl (1 M) and then filtered. The filter cake was dried under reduced pressure to obtain product 3: 2-[(6-bromonaphthalene-1-yl)oxy]propanoic acid.

[0034] At 0°C, oxalyl chloride (1.08, 8.5 mmol) and DMF (0.1 mL) were added to a DCE (15 mL) solution of 2-[(6-bromonaphthalene-1-yl)oxy]propionic acid (1 g, 3.4 mmol). The mixture was stirred at room temperature for 1 hour, and diethylamine (1.24 g, 16.9 mmol) was added at 0°C. The final suspension was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (PE:EA=2:1) ​​to obtain intermediate product 4: 2-[(6-bromonaphthalene-1-yl)oxy].

[0035] 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)pent-4-enoate (0.89 g, 3.7 mmol) and water (3 mL) were added to the 2-[(6-bromonaphthalene-1-yl)oxy] solution. 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 light yellow product 6: methyl (4E)-5-{5-[1-(diethylcarbamoyl)ethoxy]naphthalene-2-yl}pent-4-enoate.

[0036] Pd / C (0.2 g, 10%, wet) was added to a solution of methyl (4E)-5-{5-[1-(diethylcarbamoyl)ethoxy]naphthalen-2-yl}pentane-4-enoate (1.15 g, 3 mmol) in MeOH (20 mL). The final suspension was stirred at room temperature for 3 hours under hydrogen. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (PE:EA=2:1) ​​to give intermediate 7: methyl 5-{5-[1-(diethylcarbamoyl)ethoxy]naphthalen-2-yl}pentane methyl ester.

[0037] LiOH (267 mg, 6.4 mmol) and H2O (4 mL) were added to a solution of methyl 5-{5-[1-(diethylcarbamoyl)ethoxy]naphthalene-2-yl}pentanoate (818 mg, 2.1 mmol) in MeOH (10 mL). The final solution was stirred at room temperature for 3 hours. 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), PE (40 mL) was added and stirred for 10 minutes. The filtered cake was dried at room temperature to obtain the hapten (284 mg) of the formula I:

[0038]

[0039] Example 2: Preparation of complete antigen

[0040] Preparation of immunogens: Preparation of immunogens: Weigh 24.36 mg of the prepared enantiopyramide hapten, dissolve it in 400 μL DMF and stir slowly, then add 15.63 mg N-hydroxysuccinimide and 13.42 mg 1-ethylcarbodiimide hydrochloride, react at room temperature for 4-6 hours to obtain a mixture; then weigh 10 mg bovine serum albumin BSA and dissolve it in 3 mL carbonate buffer, slowly add the obtained mixture to the bovine serum albumin solution, react at room temperature for 12 hours under stirring, dialyze with 0.01 mol / L phosphate buffer PBS for 3 days to obtain the conjugate, and freeze it at -20°C for later use.

[0041] Example 3: Preparation of coating

[0042] 17.31 mg of the prepared propamide hapten was weighed, dissolved in 400 μL DMF and slowly stirred, 9.56 mg N-hydroxysuccinimide and 7.25 mg 1-ethylcarbodiimide hydrochloride were added in sequence, and the mixture was reacted at room temperature for 4-6 hours to obtain a mixture; then 10 mg of chicken ovalbumin OVA was weighed and dissolved in 2 mL of carbonate buffer, and the obtained mixture was slowly dripped into the chicken ovalbumin solution, reacted at room temperature for 12 hours under stirring, and dialyzed with 0.01 mol / L phosphate buffer PBS for 3 days to obtain a conjugate, which was frozen at -20°C for later use.

[0043] Example 4: Obtaining hybridoma cell lines

[0044] 1. Immunization of mice: Select healthy 6-8 week old BALB / c mice for immunization. Take the complete antigen of enaziramide and mix it with an equal amount of Freund's adjuvant for emulsification, and then immunize the BALB / c mice by subcutaneous injection on the back. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used thereafter. There was an interval of 28 days between the first immunization and the second booster immunization, and an interval of 21 days between multiple booster immunizations. Blood was collected 7 days after the third immunization (5 μL of blood collected from the tail of the mouse + 995 μL of antibody diluent = antiserum), and the mouse serum titer and inhibition were determined by ic-ELISA. Mice with high titer and good inhibition were selected, and a sprint immunization was performed 21 days after the fifth immunization, and the sprint dose was required to be halved and not contain any adjuvant.

[0045] 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:

[0046] (1) Eyeballs were removed to obtain blood. Mice were killed by cervical dislocation and immediately placed in 75% alcohol for disinfection. The spleens were soaked for about 5 minutes. The spleens were removed by aseptic operation. The spleens were moderately ground with the rubber tip of a syringe and passed through a 200-mesh cell sieve to obtain a spleen cell suspension. The suspensions were collected and centrifuged (1200 rpm, 8 minutes). The spleen cells were washed three times with RPMI-1640 medium. After the last centrifugation, the spleen cells were diluted to a certain volume, counted, and set aside.

[0047] (2) Collecting mouse myeloma SP2 / 0 cells: 7-10 days before fusion, the 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 , to 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;

[0048] (3) The fusion process lasted 7 minutes. In the first minute, 1 mL of PEG 1500 was added to the cells from slow to fast speed; in the second minute, the cells were allowed to stand. In the third and fourth minutes, 1 mL of RPMI-1640 medium was added within 1 minute; in the fifth and sixth minutes, 2 mL of RPMI-1640 medium was added within 1 minute; in the seventh minute, 1 mL of RPMI-1640 medium was added every 10 seconds. Then, the cells were incubated at 37°C for 5 minutes. Centrifuged (800 rpm, 8 minutes), the supernatant was discarded, and the cells were resuspended in RPMI-1640 screening culture medium containing 20% ​​fetal bovine serum and 2% 50×HAT, and added to a 96-well cell plate at 200 μL / well, and cultured in a 37°C, 5% CO2 incubator.

[0049] 3. Cell screening and cell line establishment: On the third day of cell fusion, the fused cells were half-changed with RPMI-1640 screening culture medium, and on the fifth day, the RPMI-1640 transition culture medium containing 20% ​​fetal bovine serum and 1% 100×HT was fully replaced. On the seventh day, the cell supernatant was taken for screening. The screening was divided into two steps: the first step was to screen the positive cell wells with ic-ELISA, and the second step was to select propamide as the standard and use ic-ELISA to measure the inhibitory effect of positive cells. The cell wells that had good inhibition on the propamide standard were selected, subcloned by limiting dilution method, and tested by the same method. Repeat three times to obtain hybridoma cell lines.

[0050] Example 5: Preparation and identification of monoclonal antibodies

[0051] 8-10 week old BALB / c mice were taken and each mouse was intraperitoneally injected with 1 mL of sterile paraffin oil. Seven days later, each mouse was intraperitoneally injected with 1×10 6 Hybridoma cells, ascites was collected from the seventh day, and the ascites was purified by the caprylic acid-ammonium sulfate method. Under slightly acidic conditions, caprylic acid can precipitate other miscellaneous proteins except IgG immunoglobulin in the ascites, 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 with 0.01M PBS solution (pH7.4), dialyze and desalt, and finally obtain the purified monoclonal antibody and store it at -20℃.

[0052] The most suitable antigen and antibody concentrations in the indirect competitive ELISA method 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 0.01, 0.03, 0.1 and 0.3 μg / mL with antibody diluent. After selecting the best working point, the naproxenamide standard was diluted to 8 concentrations (0, 0.11, 0.33, 1, 3, 9, 27 and 81 ng / mL), and the ic-ELISA operation steps were followed. Finally, Origin 9.0 was used to draw the graph (the results are shown in Figure 1 As shown), the standard inhibition curve of naproxenamide was obtained to determine the IC value of the monoclonal antibody to naproxenamide. 50 The IC value of the drug against propamide was 1.13 ng / mL. 50 The specific cross-reaction rates are shown in Table 1.

[0053] Table 1 IC values ​​of monoclonal antibodies against propamide and its structural analogs 50 Cross-reaction rate

[0054]

[0055] Antibody application: The monoclonal antibody prepared by hybridoma cell line through ascites in vivo was applied to the addition recovery test of propamide. The specific steps are as follows:

[0056] (1) Coating: The original Naprofil-OVA was diluted serially starting from 1 μg / mL with 0.05 M pH 9.6 carbonate buffer, 100 μL / well, and reacted at 37°C for 2 h.

[0057] (2) Washing: Pour off the solution in the plate and wash with washing solution three times, each time for 3 minutes.

[0058] (3) Blocking: After patting dry, add 200 μL / well blocking solution and react at 37°C for 2 h. Wash and dry for later use.

[0059] (4) Sample addition: dilute the antiserum (blood is collected from mice by tail amputation, and then diluted with antibody diluent to obtain the antiserum) from 1:1000 onwards and add to the coated wells of each dilution at 100 μL / well and react at 37°C for 30 min. After thorough washing, add 1:3000 diluted HRP-sheep anti-mouse IgG at 100 μL / well and react at 37°C for 30 min.

[0060] (5) Color development: Take out the ELISA plate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37°C in the dark for 15 min.

[0061] (6) Termination and measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using an ELISA reader. 450 value.

[0062] Determination of IC of monoclonal antibodies against naproxenamide by IC-ELISA 50 It is 1.13 ng / mL, indicating that it has good sensitivity to propamide and can be used for propamide immunoassay detection.

[0063] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled 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 methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A hybridoma cell line, characterized in that The hybridoma cell line was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on October 31, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.46229.

2. The hybridoma cell line according to claim 1, characterized in that The hybridoma cell line is obtained by immunizing an animal with a complete antigen prepared by a hapten, wherein the structural formula of the hapten is shown in Formula I:

3. The hybridoma cell line according to claim 2, characterized in that The complete antigen is obtained by coupling the hapten to a carrier protein.

4. The hybridoma cell line according to claim 3, characterized in that The carrier protein includes bovine serum albumin.

5. Use of the hybridoma cell line according to any one of claims 1 to 4 in detecting propamide.

6. A monoclonal antibody secreted by the hybridoma cell line according to any one of claims 1 to 4.

7. Use of the monoclonal antibody according to claim 6 in detecting naproxenamide.

8. A detection product for propamide, characterized in that: The detection product comprises the monoclonal antibody according to claim 6.

9. The detection product according to claim 8, characterized in that: The detection product also includes a coating source.

10. The detection product according to claim 9, characterized in that: The coating is prepared by coupling hapten with carrier protein, wherein the carrier protein includes chicken ovalbumin.

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