A hybridoma cell strain secreting monoclone antibody of monochloro-phenoxy acetic acid and its application

Abstract

The application provides a hybridoma cell strain secreting monoclone antibody of diquat and application thereof, and relates to the technical field of immune detection. The monoclone antibody secreted by the hybridoma cell strain has good sensitivity to diquat, the IC 50 1.13 ng / mL, so that the monoclone antibody can detect trace diquat. In addition, the monoclone antibody has no cross reaction to structural analogs of diquat, such as alachlor, acetochlor, butachlor, propanil, diquat and diquat, so that the monoclone antibody can accurately detect low concentration diquat.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay, in particular to a hybridoma cell strain secreting monoclone antibody of napropamide and application thereof. BACKGROUND

[0002] Napropamide is a broad-spectrum herbicide with good control effect 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 has high selectivity for broad-leaved herbaceous plants and some climbing plants, and can effectively control spring weeds. Its main mechanism of action is to inhibit the synthesis of amino acids in weeds, interfering with their growth and development process, thereby achieving the effect of weeding. Under normal circumstances, the persistence period of napropamide herbicide is usually 2-3 years. This means that under good storage conditions and without opening, napropamide can maintain its weeding activity within this period. Napropamide has a long residual time in the soil, which can have a long-term impact on the soil ecosystem and affect soil biodiversity. Or through rainwater runoff and irrigation water into water bodies, causing harm 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 humans, animals and plants. After inhaling napropamide, symptoms such as coughing, expectoration, and chest tightness may occur. In severe cases, it may cause respiratory difficulty and pulmonary edema. Long-term exposure to napropamide can cause damage or death to nerve cells, causing cognitive function decline, motor coordination disorders, and other symptoms, and may affect reproductive cell activity, leading to a decrease in sperm count and motility, and long-term exposure may cause infertility or other reproductive problems.

[0003] Currently, the main method for detecting napropamide residues is instrumental analysis, including gas chromatography, gas chromatography-mass spectrometry, etc. These methods are reliable and highly sensitive, and there are relevant technical standards for reference. However, due to the need for expensive equipment, specialized personnel, 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 method for detecting napropamide. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method suitable for on-site rapid detection of large quantities of samples, providing a new detection approach for napropamide. The premise of using enzyme-linked immunosorbent assay to detect napropamide is to obtain a monoclonal antibody with high sensitivity to napropamide, and there is a lack of a monoclonal antibody with high sensitivity and high specificity to napropamide in the prior art. SUMMARY

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

[0005] To solve the above technical problems, the application provides a hybridoma cell strain secreting monoclone antibody of diquat and application thereof. The monoclone antibody secreted by the hybridoma cell strain of the application has good sensitivity to diquat. The IC50 of the monoclone antibody of the application to diquat is 1.13 ng / mL, so the monoclone antibody of the application can detect trace diquat. In addition, the monoclone antibody of the application has no cross reaction to structural analogues of diquat, such as alachlor, acetochlor, butachlor, propanil, diquat and diquat, so the monoclone antibody of the application can accurately detect low concentration of diquat. 50 To solve the above technical problems, the application provides a hybridoma cell strain secreting monoclone antibody of diquat and application thereof. The monoclone antibody secreted by the hybridoma cell strain of the application has good sensitivity to diquat. The IC50 of the monoclone antibody of the application to diquat is 1.13 ng / mL, so the monoclone antibody of the application can detect trace diquat. In addition, the monoclone antibody of the application has no cross reaction to structural analogues of diquat, such as alachlor, acetochlor, butachlor, propanil, diquat and diquat, so the monoclone antibody of the application can accurately detect low concentration of diquat.

[0006] The first object of the application is to provide a hybridoma cell strain, which is preserved in China General Microbiological Culture Collection Center on October 31, 2024, the address of the center is No. 1, Beichen West Road, Haidian District, Beijing, and the preservation number is CGMCC No. 46229.

[0007] Further, the hybridoma cell strain is obtained by immunizing animals with complete antigens prepared from haptens, wherein the structure of the haptens is shown in Formula I.

[0008]

[0009] Further, the complete antigens are obtained by coupling carrier proteins with the haptens.

[0010] Further, the carrier proteins include bovine serum albumin.

[0011] The second object of the application is to provide application of the above hybridoma cell strain in detection of diquat.

[0012] The third object of the application is to provide monoclone antibody secreted by the above hybridoma cell strain.

[0013] The fourth object of the application is to provide application of the above monoclone antibody in detection of diquat.

[0014] The fifth object of the application is to provide a detection product of diquat, which comprises the above monoclone antibody.

[0015] Further, the detection product further comprises a coating agent.

[0016] Further, the coating agent is prepared by coupling carrier proteins with haptens, wherein the carrier proteins include chicken egg white protein.

[0017] The application has the following beneficial effects:

[0018] The application provides a hybridoma cell strain secreting monoclone antibody of monochloroacetic acid and application thereof. The monoclone antibody secreted by the hybridoma cell strain of the application has good sensitivity to monochloroacetic acid, and the IC 50 1.13 ng / mL, so that the monoclone antibody of the application can detect trace monochloroacetic acid. In addition, the monoclone antibody of the application has no cross reaction to structural analogues of monochloroacetic acid, such as alachlor, acetochlor, butachlor, propanil, monochloroacetic acid and monochloroacetic acid, so that the monoclone antibody of the application can accurately detect low concentration of monochloroacetic acid.

[0019] Biological material preservation

[0020] The monoclone cell strain DAZ is preserved in China General Microbiological Culture Collection Center on October 31, 2024, with a preservation number of CGMCC No.46229 and a preservation address of No.3, Beichen West Road, Haidian District, Beijing. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to make the content of the application more easily understood, the application will be further described in detail below according to specific embodiments of the application and in combination with the drawings, in which

[0022] Figure 1 is a standard inhibition curve of the monoclone antibody of monochloroacetic acid of the application;

[0023] Figure 2 is a preparation process of the monochloroacetic acid hapten of the application. DETAILED DESCRIPTION

[0024] The application will be further described below in combination with the drawings and specific embodiments, so that those skilled in the art can better understand the application and implement it, but the embodiments are not used as limitation of the application.

[0025] Carbonate buffer solution (CBS): 1.59 g of Na2CO3 and 2.93 g of NaHCO3 are weighed, respectively dissolved in a small amount of double distilled water, mixed, double distilled water is added to about 800 mL, mixed, the pH value is adjusted to 9.6, double distilled water is added to constant volume to 1000 mL, and stored at 4℃ for standby.

[0026] Phosphate buffer solution (PBS): 8.0 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4 and 2.9 g of Na2HPO4·12H2O are dissolved in 800 mL of pure water, the pH value is adjusted to 7.2-7.4 by NaOH or HCl, and the constant volume is added to 1000 mL;

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

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

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

[0030] TMB developing solution: A solution: Na2HPO4·12H2O 18.43 g, citric acid 9.33 g, and pure water to 1000 mL; B solution: 60 mg of TMB was dissolved in 100 mL of ethylene glycol. A and B solutions were mixed in a volume ratio of 1:5 to obtain TMB, and the developing solution was mixed immediately 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 the product 2 was 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-bromonaphthalen-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 HC1 (1M), and then filtered. The filter cake was dried under reduced pressure to obtain product 3: 2-[(6-bromonaphthalen-1-yl)oxy]propanoic acid.

[0034] Oxalyl chloride (1.08, 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) at 0 °C. Stirring at room temperature for 1 hour, 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 intermediate product 4: 2-[(6-bromonaphthalen-1-yl)oxy] was purified by silica gel column chromatography (PE:EA=2:1) to obtain 1.13 g of product 2.

[0035] To a solution of 2-[(6-bromonaphthalen-1-yl)oxy] (1.15 g, 3 mmol) in DMF (20 mL) was added Pd(dppf)Cl2DCM (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). The final suspension was stirred at 90 °C overnight. The solvent was removed under reduced pressure and the residue was purified by column chromatography on silica gel (PE:EA = 2:1) to give the pale yellow product 6: methyl (4E)-5-{5-[1-(diethylcarbamoyl)ethoxy]naphthalen-2-yl}pent-4-enoate.

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

[0037] To a solution of methyl 5-{5-[1-(diethylcarbamoyl)ethoxy]naphthalen-2-yl}pentanoate (818 mg, 2.1 mmol) in MeOH (10 mL) was added LiOH (267 mg, 6.4 mmol) and H2O (4 mL). 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 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 x 50 mL). The organic layer was washed with brine (50 mL) and dried over Na2SO4. The solvent was removed under reduced pressure to give a white solid. The crude product was dissolved in EA (10 mL) and PE (40 mL) was added and stirred for 10 minutes. The filter cake was dried at room temperature to give the nicosulfuron hapten (284 mg) as shown in Formula I:

[0038]

[0039] Example 2: Preparation of the complete antigen

[0040] Preparation of immunogen: Preparation of immunogen: 24.36 mg of the prepared diquat hapten was weighed and dissolved in 400 μL of DMF with slow stirring, then 15.63 mg of N-hydroxysuccinimide and 13.42 mg of 1-ethylcarbodiimide hydrochloride were added, and the mixture was reacted at room temperature for 4-6 h. Then 10 mg of bovine serum albumin BSA was weighed and dissolved in 3 mL of carbonate buffer, and the obtained mixture was slowly added to the bovine serum albumin solution, and the mixture was reacted at room temperature for 12 h with stirring, and then dialyzed against 0.01 mol / L phosphate buffer PBS for 3 days to obtain the conjugate, which was stored at -20℃ for standby.

[0041] Example 3: Preparation of coating agent

[0042] 17.31 mg of the prepared diquat hapten was weighed and dissolved in 400 μL of DMF with slow stirring, then 9.56 mg of N-hydroxysuccinimide and 7.25 mg of 1-ethylcarbodiimide hydrochloride were added in turn, and the mixture was reacted at room temperature for 4-6 h. Then 10 mg of chicken ovalbumin OVA was weighed and dissolved in 2 mL of carbonate buffer, and the obtained mixture was slowly added dropwise to the chicken ovalbumin solution, and the mixture was reacted at room temperature for 12 h with stirring, and then dialyzed against 0.01 mol / L phosphate buffer PBS for 3 d to obtain the conjugate, which was stored at -20℃ for standby.

[0043] Example 4: Obtaining of hybridoma cell strain

[0044] 1. Immunization of mice: Healthy 6-8 week old BALB / c mice were selected for immunization. The diquat complete antigen was mixed and emulsified with an equal amount of Freund's adjuvant, and then the BALB / c mice were immunized by subcutaneous injection on the back. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used thereafter. The interval between the first immunization and the second booster immunization was 28 days, and the interval between multiple booster immunizations was 21 days. Blood was collected 7 days after the third immunization (mouse tail blood 5 μL + 995 μL antibody diluent = antiserum), and the mouse serum titer and inhibition were determined using ic-ELISA. Mice with high titer and good inhibition were selected for a boost immunization 21 days after the fifth immunization, and the boost immunization was performed by intraperitoneal injection, with the requirement that the dose be halved and without any adjuvant.

[0045] 2. Cell fusion: Three days after the boost immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, and the specific steps were as follows:

[0046] (1) Enucleation of the eye to collect blood, after the mice were killed by cervical dislocation, immediately put into 75% alcohol disinfection, soak for about 5 min, aseptic operation to remove the spleen of mice, with a syringe rubber head moderate grinding and through 200 mesh cell screen to get spleen cell suspension, collection, and centrifugation (1200 rpm, 8 min), washing the spleen cells three times with RPMI-1640 medium, after the last centrifugation, the spleen cells were diluted to a certain volume, count, standby;

[0047] (2) Collection of mouse myeloma SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were cultured with 10% FBS (fetal bovine serum) RPMI-1640 medium in a 5% CO2 incubator. Before fusion, the number of SP2 / 0 tumor cells is required to reach 1-4×10 7 , to ensure that the SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. At the time of fusion, the tumor cells were collected and suspended in RPMI-1640 base culture solution for cell counting;

[0048] (3) Fusion process 7 min. In the first minute, 1 mL of PEG 1500 was added to the cells from slow to fast; in the second minute, stand still. In the third and fourth minutes, 1 mL of RPMI-1640 medium was added within 1 min; in the fifth and sixth minutes, 2 mL of RPMI-1640 medium was added within 1 min; in the seventh minute, 1 mL of RPMI-1640 medium was added every 10 seconds. Then 37°C water bath for 5 min. Centrifugation (800 rpm, 8 min), discard the supernatant, resuspended in RPMI-1640 screening medium containing 20% fetal bovine serum, 2% 50×HAT, 200 μL / well into a 96-well cell plate, and incubated in a 37°C, 5% CO2 incubator.

[0049] 3. Cell screening and cell strain establishment: On the third day of cell fusion, the fusion cells were subjected to RPMI-1640 screening medium semi-replacement, and on the fifth day, the cells were subjected to full replacement with RPMI-1640 transition medium containing 20% fetal bovine serum and 1% 100×HT, and on the seventh day, the cell supernatant was collected for screening. Screening is divided into two steps: first, positive cell wells are screened out by ic-ELISA, and second, naptalam is used as a standard and ic-ELISA is used to determine the inhibition effect of positive cells. Cell wells with good inhibition to naptalam standard are selected, and subcloning is performed by limiting dilution, and detection is performed by the same method. Repeat three times to obtain hybridoma cell strains.

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

[0051] 8-10 weeks old BALB / c mice were taken, and each mouse was injected with 1 mL of sterile paraffin oil intraperitoneally; 7 days later, each mouse was injected with 1 x 10 6 Hybridoma cells, from the seventh day, the ascites were collected, and the ascites were purified by caprylic acid-ammonium sulfate method. In the weak acid condition, n-octanoic acid can precipitate other impurities in the ascites except IgG immunoglobulin, and then centrifuged, and the precipitate was discarded; then the same amount of saturated ammonium sulfate solution was used to precipitate IgG type monoclonal antibody, centrifuged, and the supernatant was discarded. After being dissolved in 0.01M PBS solution (pH 7.4), dialysis and desalting, the purified monoclonal antibody was finally obtained and stored at -20℃.

[0052] The most suitable antigen and antibody concentration in the indirect competitive ELISA method was selected by the 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 standard sample of dimethomorph 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 for plotting (the results are shown in Figure 1 The IC 50 of the monoclonal antibody to dimethomorph was 1.13 ng / mL, and its IC 50 and cross-reactivity to dimethomorph and the like were verified, as shown in Table 1.

[0053] Table 1 IC 50 and cross-reactivity of monoclonal antibody to dimethomorph and its structural analogs

[0054]

[0055] Antibody application: the monoclonal antibody prepared by hybridoma cell line in vivo ascites was applied to the addition recovery test of dimethomorph, and the specific steps were as follows:

[0056] (1) Coating: the coating original dimethomorph-OVA was diluted by 100 μL / well from 1 μg / mL with 0.05M pH 9.6 carbonate buffer, and reacted at 37℃ for 2h.

[0057] (2) Washing: the solution in the plate was poured off, and washed with washing solution for 3 times, 3 min each time.

[0058] (3) Blocking: after being patted dry, 200 μL / well of blocking solution was added, and reacted at 37℃ for 2h. After washing, it was dried and ready for use.

[0059] (4) Add sample: the anti-serum (mouse tail blood after the antibody diluent dilution corresponding multiple after the anti-serum) from 1:1000 start multiple dilution, and add to each dilution coated hole, 100 μL / hole, 37 ℃ reaction 30 min; after washing, add 1:3000 dilution of HRP-goat anti-mouse IgG, 100 μL / hole, 37 ℃ reaction 30 min.

[0060] (5) Color development: the enzyme-labeled plate is taken out, washed thoroughly, and 100 μL of TMB color developing liquid is added to each hole, 37 ℃ reaction for 15 min in the dark.

[0061] (6) Termination and determination: 50 μL of termination liquid is added to each hole to terminate the reaction, and then the OD value of each hole is determined by an enzyme-labeled instrument. 450

[0062] The ic-ELISA determination of monoclonal antibody on the IC of alachlor 50 is 1.13 ng / mL, which shows that it has good sensitivity to alachlor and can be used for the immune analysis and determination of alachlor.

[0063] Obviously, the above examples are only examples for clearly illustrating, and are not limited to the embodiments. Other different forms of changes or variations can be made on the basis of the above description for those skilled in the art. Here, all the embodiments are not required to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.​

Claims

1. A hybridoma cell line, characterized in that, The hybridoma cell strain is preserved in China General Microbiological Culture Collection Center on October 31, 2024, and the address is No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 46229.

2. Application of the hybridoma cell strain of claim 1 in the preparation of a monoclonal antibody.

3. A monoclonal antibody secreted by the hybridoma cell strain of claim 1.

4. Application of the monoclonal antibody of claim 3 in the detection of diquat.

5. A detection product of chlorpropham, characterized by, The detection product comprises the monoclonal antibody of claim 3.

6. The test product according to claim 5, characterized in that The detection product further comprises a coating agent.

7. The test product of claim 6, wherein, The coating agent is prepared from a hapten coupled carrier protein, wherein the carrier protein comprises chicken egg white protein.

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