A monoclonal antibody against trifloxystrobin and application thereof

Abstract

The application discloses an anti-cyprodinil monoclonal antibody and application thereof. The light chain variable region of the anti-cyprodinil monoclonal antibody has an amino acid sequence shown in SEQ ID No. 1, and the heavy chain variable region has an amino acid sequence shown in SEQ ID No. 2. The monoclonal antibody has good affinity to cyprodinil, the half-inhibitory concentration (IC 50 ) is 4.55 ng / mL, the detection limit (IC 10 ) is 1.34 ng / mL, the linear range (IC 20 ~ IC 80 ) is 2.11~9.83 ng / mL, the cross-reaction rate of structural analogs such as fenamidone, bifenox and pheonox is less than 1%, the monoclonal antibody has the characteristics of good thermal stability, strong resistance to organic solvents and acid and alkali, high sensitivity, and is suitable for detecting cyprodinil residues in different environments.

Description

Technical Field

[0001] This invention belongs to the field of molecular immunology and immunodetection technology, and more specifically, relates to an anti-oxime ester monoclonal antibody and its application. Background Technology

[0002] Trifloxystrobin (TF) is a fluoromethoxyacrylate fungicide that inhibits mitochondrial respiration by blocking electron transfer between cytochrome b and c1, thereby disrupting ATP production and energy cycling, and exerting its antibacterial effect. Trifloxystrobin can be used in combination with various fungicides and exhibits good activity against almost all fungal diseases. It possesses broad-spectrum fungicidal activity, strong systemic properties, no cross-resistance, and good compatibility, making it widely used in agriculture for disease control in grains, fruits, and vegetables. However, improper application methods, such as excessive application, illegal use of prohibited high-residue and highly toxic pesticides, and inadequate safety precautions during application, can lead to excessive fungicide residues in the environment and agricultural products, posing potential hazards to humans, animals, and the environment. Numerous researchers have investigated the physiological toxicity of azoxystrobin. Azoxystrobin exhibits high toxicity to fish, aquatic organisms, and algae, including developmental toxicity, induced biochemical toxicity, and genotoxicity. It can cause potential harm to humans, such as eye and respiratory irritation, weakness, dizziness, skin redness, and chest pain. Besides physiological toxicity, the potential environmental pollution caused by azoxystrobin is also a significant concern. Azoxystrobin is stable in water but has a long half-life, and its spraying process can pollute soil and rivers. Currently, China's GB 2763-2016 "National Food Safety Standard for Maximum Residue Limits of Pesticides in Food" stipulates a maximum allowable residue limit (MRL) of 40 mg / kg for various food products. For most fruits, vegetables, and grains, the MRL values ​​in Europe and Russia range from 0.05 to 1.5 mg / kg. Therefore, for public health, strengthening the monitoring and detection of azoxystrobin in food and the environment is particularly necessary.

[0003] Currently, the main methods for detecting oximeprost include instrumental methods such as HPLC-MS / MS, GC-MS-MS / MS, and UPLC-MS / MS. While instrumental methods offer high accuracy and sensitivity, they are cumbersome and time-consuming to operate, and the high cost of large-scale instruments for testing and maintenance limits their application in large-scale on-site screening. Immunoassay methods based on antigen-antibody specific recognition, however, offer advantages such as speed, sensitivity, and high throughput, and have become commonly used in large-volume sample screening. Antibodies are the most important biological element in immunoassay methods. Chinese patent CN117623980A discloses a monoclonal antibody against oximeprost, which exhibits good titer, specificity, and affinity, with a detection sensitivity of 0.02 μg / L for oximeprost. However, its thermal stability, organic solvent tolerance, and acid-base tolerance are poor, making it unsuitable for detecting oximeprost under various environmental conditions. Therefore, providing an antibody with high specificity, good thermal stability, strong tolerance to organic solvents and acid-base conditions, and high sensitivity is crucial to meeting future needs for oximeprost detection. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an anti-oxime ester monoclonal antibody.

[0005] A second object of the present invention is to provide a gene encoding the anti-oxime ester monoclonal antibody.

[0006] A third object of the present invention is to provide a recombinant vector containing the said gene.

[0007] A fourth object of the present invention is to provide recombinant cells containing the recombinant vector.

[0008] A fifth object of the present invention is to provide the use of the anti-oxime ester monoclonal antibody, the gene, the recombinant vector, or the recombinant cell.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] An anti-oxime ester monoclonal antibody, wherein the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 2.

[0011] Specifically, the monoclonal antibody comprises four framework regions (FR1, FR2, FR3, FR4) and three complementarity-determining regions (CDR1, CDR2, CDR3) in the light chain variable region, and the four framework regions and three complementarity-determining regions in the heavy chain variable region are arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The monoclonal antibody exhibits good affinity for oxime ester, with a half-maximal inhibitory concentration (IC50) of [missing value].50 The concentration was 4.55 ng / mL, and the limit of detection (IC50) was 4.55 ng 10 The concentration was 1.34 ng / mL, and the linear range was (IC50) 20 ~IC 80 The concentration ranges from 2.11 to 9.83 ng / mL. The cross-reactivity rate with structural analogs such as oxadiazon, azoxystrobin, and phenoxystrobin is less than 1%. It has high specificity, good thermal stability, strong tolerance to organic solvents and acids and alkalis, and high sensitivity. It is suitable for detecting oxadiazon residues under different environments.

[0012] The present invention also provides a gene encoding the anti-oxime ester monoclonal antibody, wherein the nucleotide sequence encoding the light chain variable region is shown in SEQ ID No. 3, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No. 4.

[0013] In this invention, the monoclonal antibody is obtained by immunizing animals with an artificial antigen obtained by conjugating the oxime ester hapten with a carrier protein, and then screening to obtain the above-mentioned anti-oxime ester monoclonal antibody with high specificity, good thermal stability, strong tolerance to organic solvents and acid and alkali, and high sensitivity.

[0014] Furthermore, the structural formula of the oxime ester hapten is shown in formula (I):

[0015]

[0016] The oxime ester hapten fully exposes the characteristic structure of oxime ester, and the carbon chain length of the linker arm introduced by the hapten is appropriate. After the linker protein is linked, it does not affect the antigen structure, which is conducive to the mouse immune system effectively recognizing the characteristic structure of the hapten and effectively improving the immunogenicity of the oxime ester hapten. Therefore, a highly specific antibody can be prepared.

[0017] Further, the oxime hapten is obtained by condensing it with 6-aminohexanoic acid, starting from the carboxyl site after hydrolysis of the ester group on oxime ester. Specifically, the preparation method of the oxime hapten includes the following steps:

[0018] S1: Hydrolyze oxime ester and sodium hydroxide in a mixture of methanol and water. Terminate the reaction by adding an ice-water mixture and adjust the pH with hydrochloric acid.

[0019] S2: Extract the reaction solution obtained from S1, collect the organic phase, wash, dry, filter, and remove the solvent by vacuum distillation of the filtrate. The residue is purified by silica gel column chromatography to obtain a white solid, which is the hydrolysis product TF-H1.

[0020] S3: Dissolve TF-H1, EDC, and NHS in tetrahydrofuran and react overnight. This solution is labeled A. Dissolve 6-aminohexanoic acid in water and label it B. Slowly add solution B to solution A to induce a condensation reaction. Remove excess tetrahydrofuran by rotary evaporation. Terminate the reaction by adding an ice-water mixture and adjust the pH with hydrochloric acid.

[0021] S4: Extract the organic phase from the reaction solution obtained in S3, wash, dry, filter, and remove the solvent by vacuum distillation of the filtrate. The residue is purified by silica gel column chromatography to obtain a white solid, which is the hapten TF-H3.

[0022] Preferably, in step S1, the ratio of oxime ester to sodium hydroxide is 1:4.

[0023] Preferably, the hydrolysis reaction conditions in step S1 are 60℃ for 2 hours.

[0024] Preferably, in step S3, the ratio of oxime ester to EDC, NHS and 6-aminocaproic acid is 0.5:1:1:1.

[0025] Preferably, the condensation reaction conditions in step S3 are room temperature and 12 hours.

[0026] Furthermore, the structural formula of the oxime ester artificial antigen is shown in formula (II):

[0027]

[0028] Further, the carrier protein is bovine serum albumin (BSA) or chicken ovalbumin (OVA). The artificial antigen is prepared by conjugating the hapten TF-H3 with the carrier protein using an active esterification method, specifically including the following steps:

[0029] S1: Dissolve the oxime ester hapten TF-H3 in an organic solvent, then add NHS and EDC, and stir at 4°C for 6-12 hours to obtain solution A;

[0030] S2: Weigh the carrier protein and dissolve it in a carbonate buffer solution, which is called solution B; add solution A dropwise to solution B and react at 4°C.

[0031] S3: Dialyze the reaction solution obtained in S2 at 4°C for 3 days, changing the dialysis solution twice a day to obtain the artificial antigen.

[0032] Preferably, the organic solvent in step S1 is DMF.

[0033] Preferably, the ratio of oxime ester hapten TF-H3, NHS and EDC in step S1 is 1:1 to 2:1 to 2.

[0034] More preferably, the ratio of the oxime ester hapten TF-H3, NHS and EDC in step S1 is 1:1.5:1.5.

[0035] Preferably, the molar ratio of the carrier protein to the oxime ester hapten in step S2 is 1:80.

[0036] Preferably, the reaction in step S2 is carried out for 8 hours.

[0037] As one possible implementation method, the preparation method of the monoclonal antibody of the present invention specifically includes the following steps:

[0038] (1) Preparation of oxime ester hapten: Oxime ester artificial antigen was synthesized from oxime ester hapten. The oxime ester artificial antigen was mixed with an equal amount of complete Freund's adjuvant, emulsified, and the emulsified immunogen was injected subcutaneously or intraperitoneally into BALB / c mice for multiple immunizations. The first immunization used complete Freund's adjuvant, and the booster immunization used incomplete Freund's adjuvant. The interval between the first and second immunizations was two weeks, and the interval between each subsequent immunization was three weeks.

[0039] (2) Blood was collected from the tail vein of mice that had undergone the above immunization process. The serum immune titer and immunosuppressive ability of the mice were detected by ic-ELISA. The immunized mice with the best recognition of oxime ester in the serum were screened out.

[0040] (3) The selected mice were given a final booster immunization with incomplete Freund's adjuvant. Three days before cell fusion, the mice were directly injected intraperitoneally with 200 μL of oxime ester artificial antigen without Freund's adjuvant for shock immunization.

[0041] (4) Fusing spleen cells and myeloma cells from BALB / c mice after shock immunization, culturing the fused cells in culture medium, detecting positive cell pores using ic-ELISA, and further measuring the inhibitory effect of positive cell pores using ic-ELISA, subcloning the positive cell pores with the best inhibition using limiting dilution method, and finally screening out hybridoma cell lines that can secrete oxime ester monoclonal antibodies, inoculating the hybridoma cells into female BALB / c mice for culture, obtaining high concentrations of ascites fluid that can produce the above monoclonal antibodies, and purifying the ascites fluid to obtain anti-oxime ester monoclonal antibodies.

[0042] Since the present invention has provided the amino acid sequence of the anti-oxime ester monoclonal antibody and the gene sequence encoding the monoclonal antibody, as a preferred embodiment, those skilled in the art can obtain the monoclonal antibody described in this application using known recombinant DNA techniques. Therefore, any recombinant vectors or recombinant cells that can be used to prepare the monoclonal antibody described in this invention should also be within the scope of protection of this invention.

[0043] Therefore, the present invention also provides a recombinant vector containing the gene encoding the anti-oxime ester monoclonal antibody.

[0044] The present invention also provides a recombinant cell containing the above-mentioned recombinant vector.

[0045] The present invention also provides the use of the anti-oxime ester monoclonal antibody, the gene encoding the anti-oxime ester monoclonal antibody, the recombinant vector or the recombinant cell in the preparation of products for detecting oxime ester.

[0046] The present invention also provides a kit for detecting oxime ester, the kit containing the above-mentioned anti-oxime ester monoclonal antibody.

[0047] The present invention also includes the application of any of the above-described anti-oxime ester monoclonal antibodies in the detection of oxime esters.

[0048] This invention also provides a method for detecting oxime ester, which involves using a complete antigen obtained by conjugating an oxime ester hapten with a carrier protein as a coating antigen, and using the aforementioned anti-oxime ester monoclonal antibody as a detection antibody for detection; the structural formula of the oxime ester hapten is shown in formula (I):

[0049]

[0050] Furthermore, the structural formula of the complete antigen is shown in formula (II):

[0051]

[0052] Preferably, the carrier protein is chicken ovalbumin.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] This invention provides an anti-oximetromethorphan monoclonal antibody, wherein the light chain variable region of the anti-oximetromethorphan monoclonal antibody has the amino acid sequence shown in SEQ ID No. 1, and the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 2. The monoclonal antibody exhibits good affinity for oximetromethorphan, and its half-maximal inhibitory concentration (IC50) is [not specified]. 50 The concentration was 4.55 ng / mL, and the limit of detection (IC50) was 4.55 ng 10 The concentration was 1.34 ng / mL, and the linear range was (IC50) 20 ~IC 80 The concentration ranges from 2.11 to 9.83 ng / mL. The cross-reactivity rate with structural analogs such as oxadiazon, azoxystrobin, and phenoxystrobin is less than 1%. It has high specificity, good thermal stability, strong tolerance to organic solvents and acids and alkalis, and high sensitivity. It is suitable for detecting oxadiazon residues under different environments. Attached Figure Description

[0055] Figure 1 This is a flowchart illustrating the preparation of oxime ester hapten, artificial antigen, and monoclonal antibody.

[0056] Figure 2 This is a mass spectrometry chromatogram for identifying the oxime ester hapten TF-H1.

[0057] Figure 3 This is a mass spectrometry diagram for identifying the oxime ester hapten TF-H3.

[0058] Figure 4 Ultraviolet scanning identification curves for oximeprobamate artificial antigen, carrier protein, and oximeprobamate hapten.

[0059] Figure 5 This is a schematic diagram of the amino acid sequence and structural division of the variable region of the light chain of the anti-oxime ester monoclonal antibody TF-A1-H7.

[0060] Figure 6 This is a schematic diagram showing the amino acid sequence and structural division of the variable region of the heavy chain of the anti-oxime ester monoclonal antibody TF-A1-H7.

[0061] Figure 7 The standard curve for the indirect competitive ELISA of the anti-oxime ester monoclonal antibody TF-A1-H7 is shown.

[0062] Figure 8 Figure showing the organic solvent tolerance results of the anti-oxime ester monoclonal antibody TF-A1-H7.

[0063] Figure 9 The graph shows the thermostability results of the anti-oxime ester monoclonal antibody TF-A1-H7.

[0064] Figure 10 The figure shows the acid-base tolerance results of the anti-oxime ester monoclonal antibody TF-A1-H7. Detailed Implementation

[0065] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0066] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0067] The following examples include the synthesis of oximeprobamate hapten and oximeprobamate artificial antigen, and the preparation of anti-oximeprobamate monoclonal antibodies, the process of which is as follows: Figure 1 As shown.

[0068] Example 1: Synthesis and Identification of Oxime Propionate Hapten

[0069] 1. Synthesis of oxime ester hapten

[0070] (1) Dissolve 1 mmol (408.37 mg) of oxime ester in 5 mL of methanol, add 4 mmol (160 mg) of NaOH dissolved in 2 mL of H2O, react at 60 °C for 2 h, and monitor by TLC. Remove methanol by rotary evaporation, add an appropriate amount of ice-water mixture, adjust the pH to about 3-4 with 1 M hydrochloric acid, and extract with ethyl acetate 3-4 times. Combine the organic phases, wash 2-3 times with saturated brine, dry with anhydrous sodium sulfate, remove the solvent by rotary evaporation, and obtain the hapten TF-H1. After purification by silica gel column chromatography, the compound shown in formula (I) can be obtained.

[0071]

[0072] (2) Dissolve 1 mmol (394.11 mg) of TF-H1 in 3 mL of THF, add 2 mmol of EDC and 2 mmol of NHS, stir overnight at room temperature, and monitor by TLC. After the reaction is complete, add an appropriate amount of ice-water mixture, extract with ethyl acetate 3-4 times, collect and combine the organic layers, wash with saturated brine 2-3 times, dry with anhydrous sodium sulfate, and remove the solvent by vacuum distillation. Redissolve in 2 mL of THF, add dropwise 10 mL of a mixture of 2 mmol (262.34 mg) of 6-aminocaproic acid and 1.2 mmol (100.81 mg) of NaHCO3, monitor by TLC, after the reaction is complete, add an appropriate amount of ice-water mixture, adjust the pH of the mixture to about 3-4 with 6M HCl, and extract with ethyl acetate 3-4 times. Combine the organic phases, wash with saturated brine 3-4 times, dry with anhydrous sodium sulfate, remove the solvent by rotary evaporation, which is the hapten TF-H3. After purification by silica gel column chromatography, the compound shown in formula (II) can be obtained.

[0073]

[0074] 2. Identification

[0075] Mass spectrometry was used to identify the oxime ester hapten.

[0076] The ESI-MS identification results of the haptens shown in formulas (I) and (II) are as follows: Figure 2 and Figure 3 As shown, the molecular weights of the compounds represented by formulas (I) and (II) are 394.11 and 507.20, respectively, and the ESI-MS identification results are consistent with the molecular weights of the compounds.

[0077] Example 2: Synthesis and Identification of Oxime Propylene Artificial Antigen

[0078] 1. Coupling of oxime ester artificial antigen and carrier protein

[0079] (1) Weigh 15 mg of hapten, 8 mg of NHS and 13 mg of EDC and dissolve them in 1 mL of DMF. Stir overnight at 4°C.

[0080] (2) Weigh 10 mg of bovine serum albumin and dissolve it in 3 mL of CB buffer;

[0081] (3) Add the solution obtained in step (1) dropwise to the solution obtained in step (2) and stir overnight at 4°C;

[0082] (4) Dialyze with PBS buffer for two days, three times a day, and obtain oxime ester artificial antigen after dialysis.

[0083] The formula for CB buffer is: 2.65g NaHCO3, 1.65g Na2CO3, and distilled water to a final volume of 1000mL; the formula for PBS buffer is: 8.5g NaCl, 2.9g NaHPO4·12H2O, 0.2g KH2PO4, 0.2g KCl, and distilled water to a final volume of 1000mL.

[0084] Similarly, the process of obtaining the artificial antigen of oxime ester by replacing bovine serum albumin with chicken egg albumin as the carrier protein is the same as the preparation process described above.

[0085] 2. Identification

[0086] The oxime hapten, oxime hapten, and carrier protein were subjected to a full-wavelength ultraviolet scan, and the results are as follows: Figure 4 As shown. The oxadiazon hapten, oxadiazon artificial antigen, BSA, and OVA were identified by ultraviolet (200–800 nm) scanning. Comparing the shifts of each substance before and after coupling, the curves of the oxadiazon hapten, oxadiazon artificial antigen, and carrier protein were significantly different. The oxadiazon artificial antigen showed an absorption peak at approximately 280 nm, indicating that the oxadiazon hapten was successfully coupled with the carrier protein to obtain the artificial antigen.

[0087] Example 3: Preparation of Oxime Propionate Monoclonal Antibody

[0088] (1) Three 4-week-old female BALB / c mice were immunized. Equal volumes of oximeprotein artificial antigen and complete Freund's adjuvant were mixed, emulsified, and the emulsified immunogen was injected subcutaneously or intraperitoneally into the BALB / c mice for multiple immunizations. The first immunization used complete Freund's adjuvant, and the booster immunization used incomplete Freund's adjuvant. The interval between the first and second immunizations was two weeks, and the interval between each subsequent immunization was three weeks. One week after the third immunization, blood was collected from the tail vein of the mice. The serum immunotiter and immunosuppressive capacity of the mice were detected by ic-ELISA. The immunized mice with the best serum recognition of oximeprotein were selected, and the spleen cells of the mice with the best immunization effect were used for cell fusion.

[0089] (2) One day before cell fusion, a blank Balb / c mouse older than 8 weeks was sacrificed, its abdomen was exposed, and the abdominal skin was separated using sterile scissors and forceps. 5 mL of complete culture medium was pipetted into the peritoneal cavity of the mouse, and after several pipetting motions, it was transferred to a 100 mL culture flask. Complete culture medium was added to the culture flask to bring the volume to 80 mL, and the cells in the culture flask were gently shaken. 100 μL of the culture medium was added to 8 wells of a 96-well plate, and the plates were cultured in an incubator (5% CO2, 37°C) to obtain feeder cells. Three days before cell fusion, 200 μL of immunogen (1 mg / mL) was directly injected into the peritoneal cavity of the mouse for shock immunization.

[0090] (3) Spleen cells from mice with the best immunogenicity were mixed with myeloma cells from mice in the logarithmic growth phase (SP2 / 0). Preheated fusion agent (PEG 4000) was slowly added over 45 seconds for fusion. The cells were then resuspended in HAT medium and a suitable amount of feeder cells were added. The mixture was cultured in 96-well plates at 37°C in a 5% CO2 incubator. After 5 days, the medium was partially replaced with HT medium, and after 9 days, the medium was completely replaced. When the cells in the plates reached 1 / 3 of the well area, positive wells were screened using ic-ELISA.

[0091] (4) During screening, the artificial antigen E1-H1-OVA prepared in Example 2 was used as the coating agent. The coating agent concentration was 1000 ng / mL, and 100 μL was coated per well. The concentration of oxime ester was 100 ng / mL. Positive wells were further screened using ic-ELISA. The limiting dilution method was used to reduce the number of cells to approximately one per well. On the third day of culture, the cell growth in the wells was observed. When the cell cluster size reached 1 / 4 of the well area, 50 μL of cell supernatant was taken for detection. After 2-3 rounds of subcloning, cell lines with 100% positive titers and comparable inhibitory effects were obtained. These were the positive monoclonal cell lines. The cell lines were numbered and named. Finally, the positive monoclonal cell lines were expanded for monoclonal antibody preparation. The obtained monoclonal antibodies were subjected to tolerance tests. The monoclonal antibody TF-A1-H7, which has high specificity, good thermal stability, strong tolerance to organic solvents and acid and alkali, and high sensitivity, was screened.

[0092] (5) The screening steps are as follows:

[0093] S1. Coating: Dilute the artificial antigen of formula (III) prepared in Example 2 to 1000 ng / mL with CB buffer (pH 9.6), add 100 μL to each well of the ELISA plate, and incubate overnight at 37°C.

[0094] S2. Washing: Wash the plate twice with a plate washer, adding 300μL of washing solution to each well, and spin dry any residual liquid in the well;

[0095] S3. Sealing: Add 120 μL of sealing solution to each well, seal at 37°C for 3 hours, spin dry the liquid in the well, invert and dry in a 37°C oven for 1 hour, and store at 4°C.

[0096] S4. Sample addition and incubation: Add 50 μL of PBS buffer to the titer well and 50 μL of 100 ng / mL oxime ester to the inhibition well. Add 50 μL of cell supernatant to both the titer and inhibition wells. Incubate at 37°C for 40 min. Wash the plate 5 times with a plate washer and shake off the liquid in the wells.

[0097] S5. Add secondary antibody: Use PBS as the diluent for HRP-goat anti-mouse secondary antibody, dilute 5000 times, add 100 μL of HRP-goat anti-mouse secondary antibody to each well, incubate at 37℃ for 30 min, wash the plate 5 times with a plate washer, and spin dry the liquid in the wells.

[0098] S6. Color development: Mix 10 mL of H2O2 substrate buffer with 550 μL of TMB color development solution, add 100 μL to each well, incubate at 37 °C for 10 min, and add 50 μL of 10% H2SO4 stop solution to each well.

[0099] S7. Measurement: Measure the OD of each well using a microplate reader. 450nm The absorbance value;

[0100] S8. Calculate the inhibition rate: Inhibition rate = (Absorbance of the potent aperture - Absorbance of the inhibition aperture) / Absorbance of the potent aperture × 100%.

[0101] Example 4: Sequencing and determination of the amino acid sequence of the gene encoding the anti-oxime ester monoclonal antibody TF-A1-H7.

[0102] 1. Total RNA extraction

[0103] Total RNA was extracted using the Trizol reagent method from Guangzhou Jiebes Biotechnology Co., Ltd.

[0104] The specific steps are as follows:

[0105] Take approximately 1×10 6Centrifuge cells at 3000 rpm for 5 min, collect cells into centrifuge tubes, and carefully discard all culture medium supernatant. Gently tap the bottom of the centrifuge tube to loosen the cell pellet, immediately add 2 mL of lysis buffer (TRNsol), disperse the pellet, and collect it into a 2 mL centrifuge tube. Add 0.2 mL of chloroform to each 1 mL of the lysis buffer. Cap the centrifuge tube, gently shake up and down for 15 s, incubate on ice for 5 min, and centrifuge at 12000 rpm for 10 min at room temperature. Transfer the upper aqueous phase to a new centrifuge tube, slowly add 0.7 volumes of anhydrous ethanol, and mix well; transfer the resulting solution and pellet together into a GBC adsorption column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid; add 500 μL of Wash Buffer I to the GBC adsorption column, centrifuge for 1 min, and discard the waste liquid; add 600 μL of Wash Buffer II to the GBC adsorption column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid. Centrifuge at 12000 rpm for 1 min, discard the waste liquid, and allow the column to air dry under a clean bench with the lid open to remove any residual washing solution. Transfer the GBC adsorption column to a new centrifuge tube, add 30–100 μL of LNase-free ddH2O, incubate at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min to obtain the RNA solution.

[0106] 2. cDNA Synthesis

[0107] Using RNA as a template, cDNA first-strand synthesis was performed according to the instructions of the Takara First-Strand Reverse Transcription Kit. Specifically, 3 μg of RNA was used as a template, and 1 μL of Oligo(dT) 18 primer was added to a 1.5 mL nuclease-free centrifuge tube. RNase-free ddH2O was added to a total volume of 12 μL, and the mixture was incubated at 65°C for 5 min, followed by cooling on ice for 2 min. After the reaction, 4 μL of 5×Reaction Buffer, 1 μL of RNase inhibitor, 2 μL of 10 mM dNTP Mix, and 1 μL of M-MuLV reverse transcriptase (200 U / μL) were added to a total volume of 20 μL. The mixture was incubated at 42°C for 60 min, followed by incubation at 70°C for 5 min to inactivate the reverse transcriptase. The reaction mixture was then stored at -80°C for later use.

[0108] 3. Amplification of antibody variable region genes

[0109] (1) PCR cloning of V H V L Gene

[0110] Using cDNA synthesized by reverse transcription as a template, genes in the variable regions of the antibody heavy and light chains were cloned using universal primers. The universal primers are shown in Table 1.

[0111] Table 1. Universal primers for amplifying the variable regions of the heavy and light chains of single-chain antibodies.

[0112]

[0113]

[0114] Using the first strand of cDNA as a template, the variable region of the light chain was amplified using the LB18 / LF2 primer set. The reaction system and specific parameters are shown in Table 2. After PCR, the bands were identified by gel electrophoresis at 120V for 25min. The bands in the range of 300bp to 500bp were recovered by gel excision using the kit.

[0115] Table 2. PCR amplification system and reaction conditions for the light chain variable region

[0116]

[0117] ② Cloning of heavy chain variable region genes

[0118] Using the first strand of cDNA as a template, the variable region of the heavy chain was amplified using the HB15 / HF2 primer set. The reaction system and specific parameters are shown in Table 3. After PCR, the bands were identified by gel electrophoresis at 120V for 25min. The bands in the range of 300bp to 500bp were recovered by gel excision using a kit.

[0119] Table 3. PCR amplification system and reaction conditions for the heavy chain variable region

[0120]

[0121] (2) DNA gel recovery kit was used to recover the amplified VH and VL genes.

[0122] The OMEGA gel extraction kit was used to recover PCR gel products. The specific steps are as follows: Cut off the target band with a clean blade and weigh it. Add an equal volume of Binding Buffer (XP2). Incubate at 55–60°C for 10 minutes until the gel is completely melted, shaking occasionally every 2–3 minutes. Transfer the melted gel to a HiBind DNA column and centrifuge at 10,000 rpm for 1 minute. Discard the liquid in the collection tube, add 300 μL of Binding Buffer (XP2), and centrifuge at 10,000 rpm for 1 minute. Discard the liquid in the collection tube, add 700 μL of SPW Wash Buffer (add ethanol to SPW Wash Buffer before first use), and centrifuge at 10,000 rpm for 1 minute. Repeat the addition of SPW Wash Buffer... Wash with buffer; discard the liquid in the collection tube, centrifuge at 10000 rpm for 2 min; transfer the adsorption membrane to a 1.5 mL centrifuge tube, add 15 μL of sterile water, centrifuge at 10000 rpm for 2 min, collect the liquid in the tube, measure the DNA concentration using a nanodrop micro-UV spectrophotometer, and store at -20℃. The recovered V... H V L Genes were ligated into an 18-T Vector Cloning vector (TakaRa).

[0123] (3) Transformation

[0124] Competent DH5α cells were placed on ice and allowed to thaw for 5 minutes. 5 μL of the target vector was added, gently mixed, and incubated on ice for 25 minutes. The cells were then heat-shocked in a 42°C water bath for 45 seconds, immediately returned to ice, and incubated for 2 minutes. 400 μL of preheated LB broth (37°C) was added, and the cells were incubated at 37°C and 250 rpm for 1 hour. 100 μL of the culture was then evenly spread onto LB-A plates and incubated upside down at 37°C overnight. The following day, multiple single colonies were randomly selected from the plates for colony PCR and DNA sequencing identification and analysis.

[0125] 4. Antibody heavy chain and light chain gene sequence analysis

[0126] After adjusting the sequencing results using DNAman software, the complete forward sequence was obtained and input into IMGT (https: / / www.imgt.org / IMGT_vquest / analysis) in FASTA format for murine antibody variable region gene sequence analysis. The amino acid sequences of the heavy chain variable region (HF2-HB15) and the light chain variable region (LF2-LB18) were obtained through analysis.

[0127] 5. Experimental Results

[0128] The amino acid sequences of the light chain variable region and heavy chain variable region of the monoclonal antibody TF-A1-H7 prepared according to Example 3 are as follows: Figure 5 and Figure 6 As shown.

[0129] The amino acid sequence of the variable region of the light chain of the monoclonal antibody TF-A1-H7 is shown in SEQ ID No. 1:

[0130] DIVMTQSPKFMSTSVRDRVSITCKASRNVLSPVTWYQQKPRQFPKTLIYLVSNRHTGVPDPFRGSGSGSDFTLTISSVQSEDPADYFCLHLWNLPYTFGAGTKLEIK

[0131] The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody TF-A1-H7 is shown in SEQ ID No. 2:

[0132] EVQVEESGGGLVQPGASMKLSCAASGSTVSGAWMDWVRQYPEKRLEWVAEVSINPATFYAESVKGRFSTSRDDSKRSVYLHMNNLRDEDTGIYYCFSLHYGNDYWGQGTTLTVSS

[0133] The nucleotide sequence encoding the light chain variable region of the monoclonal antibody TF-A1-H7 is shown in SEQ ID No. 3:

[0134] GACATTGTAATGACACAGTCTCCAAAATTCATGTCCACATCGGTGCGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCGGAATGTTCTTTCTCCTGTAACCTGGTATCAACAGAAACCACGGCAGTTTCCTAAAACACTGATTTACTTGGTTTCCAACCG GCATACTGGAGTCCCTGATCCCTTCAGGGGCATGGATCTGGGTCAGATTTCACTCTCACCATTAGCAGTGTGCAATCTGAAGACCCGGCAGATTATTTCTGTCTGCACCTTTGGAATCTTCCGTACACATTCGGAGCGGGGACCAAGCTGGAAATAAAC

[0135] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody TF-A1-H7 is shown in SEQ ID No. 4:

[0136] GAAGTGCAAGTTGAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGCATCCATGAAACTCTCTTGTGCTGCCTCTGGATCCACTGTTAGTGGCGCCTGGATGGACTGGGTCCCGCCAGTATCCAGAGAAGCGGCTTGAGTGGGTTGCTGAAGTTAGCATTAATCCTGCAACATT CTATGCTGAGTCTGTGAAAGGGAGGTTCAGCACCTCAAGAGATGATTCCAAAAGGAGTGTCTACCTGCACATGAATAACTTAAGAGATGAAGACACTGGCATTTATTACTGCTTCAGTCTTCACTACGGCAATGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCGA

[0137] Example 5: Establishment of a standard curve for indirect competitive ELISA based on monoclonal antibodies

[0138] 1. Wrapping and sealing

[0139] Dilute the TF-H3-OVA coating agent to 1000 ng / mL with CB buffer, add 100 μL of the coating agent to each well, and coat overnight at 37°C. Wash the plate twice with a plate washer, spin dry, add 120 μL of blocking buffer to each well, block at 37°C for 3 h, spin dry the blocking buffer, dry at 37°C for 1 h, and store in a sealed bag at 4°C for later use.

[0140] 2. Establishment of the standard curve

[0141] Add 50 μL of PBS buffer to the titer wells and 50 μL of 100 ng / mL oxime ester to the inhibition wells. Add 50 μL of cell supernatant to both titer and inhibition wells. Incubate at 37°C for 40 min, wash the plate 5 times, and remove excess liquid from the wells. Dilute HRP-goat anti-mouse secondary antibody 5000 times with PBS, add 100 μL of HRP-goat anti-mouse secondary antibody to each well, incubate at 37°C for 30 min, wash the plate 5 times, and remove excess liquid from the wells. Mix 10 mL of H2O2 substrate buffer with 550 μL of TMB chromogenic solution, add 100 μL to each well, incubate at 37°C for 10 min, and add 50 μL of 10% H2SO4 stop solution to each well. Measure the OD of each well using a microplate reader. 450nm The absorbance value; calculation: B / B0 = OD of the well with added oxime procymidone drug. 450nm / OD of valence pore 450nm A standard curve was constructed with the logarithm of the concentration of azoxystrobin standard as the x-axis and B / B0 as the y-axis.

[0142] 3. Experimental Results

[0143] The standard curve of ic-ELISA based on monoclonal antibodies is shown below. Figure 7 As shown, the standard curve exhibits an S-shape, indicating good linear correlation. The half-maximal inhibitory concentration (IC50) for azoxystrobin is [value missing]. 50 The concentration was 4.55 ng / mL, and the limit of detection (IC50) was 4.55 ng / mL. 10 The concentration was 1.34 ng / mL, and the linear range was (IC50) 20 ~IC 80 The concentrations ranged from 2.11 to 9.83 ng / mL, indicating high detection sensitivity and a wide linear range.

[0144] Example 6: Determination of Organic Solvent Tolerance of Oxime Propylene Monoclonal Antibody TF-A1-H7

[0145] (1) Experimental methods

[0146] The oxime protonate monoclonal antibody TF-A1-H7 was diluted to the same working concentration with solutions containing 0%, 10%, 20%, 30%, 40%, 50%, methanol, acetonitrile, and ethanol, respectively, to determine the antibody-antigen binding ability. The antibody-antigen binding ability diluted with 0% organic solvent was taken as 100%, and the tolerance of oxime protonate monoclonal antibody TF-A1-H7 to methanol, acetonitrile, and ethanol was evaluated. The specific method is as follows:

[0147] Add 50 μL of diluted oxime ester monoclonal antibody TF-A1-H7 and 50 μL of PBS to the pre-packaged ELISA plate, incubate at 37°C for 40 min, wash the plate five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 μL of HRP-labeled goat anti-mouse secondary antibody diluted 1:5000, incubate at 37°C for 30 min, wash the plate five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 μL of LMB substrate solution, and develop the color at 37°C in the dark for 10 min; add 50 μL of stop solution (10% H2SO4, v / v) to stop the reaction; read the absorbance at 450 nm using an ELISA reader.

[0148] (2) Experimental Results

[0149] The activity curves of TF-A1-H7 oxime ester monoclonal antibody with different ratios of organic solvent / PBS as diluents are shown in the figure below. Figure 8As shown in the figure, the oximetrozine monoclonal antibody TF-A1-H7 exhibits good tolerance to methanol. At methanol concentrations below 30%, TF-A1-H7 shows good activity, exceeding 89%. Furthermore, methanol concentrations below 10% enhance antibody activity. At concentrations of acetonitrile and ethanol below 10%, antibody activity is consistently above 50%. These results indicate that the oximetrozine monoclonal antibody TF-A1-H7 possesses a certain degree of tolerance to organic solvents, and at appropriate concentrations, methanol can enhance antibody activity.

[0150] Example 7: Determination of the thermal stability of TF-A1-H7 oxime ester monoclonal antibody

[0151] (1) Experimental methods

[0152] The oxime proton monoclonal antibody TF-A1-H7 was treated at 25℃, 35℃, 45℃, 55℃, 65℃, and 75℃ for 5 min, respectively, to determine its binding ability. The binding ability at 25℃ was taken as 100%, and the antibody binding ability of oxime proton monoclonal antibody TF-A1-H7 under different heat treatment temperatures was evaluated. The specific method is as follows:

[0153] 50 μL of diluted oxime ester monoclonal antibody TF-A1-H7 was incubated at 25℃, 35℃, 45℃, 55℃, 65℃, and 75℃ for 5 min each time. 50 μL of PBS was then added to each well, and the plates were incubated at 37℃ for 40 min. The plates were washed five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), the liquid in the wells was dried, and 100 μL of HRP-labeled goat anti-mouse secondary antibody diluted 1:5000 was added. The plates were incubated at 37℃ for 30 min, and then washed five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)). Wash the plate five times with PBS (0.06% Tween-20 (v / v)), pat dry the liquid in the wells, add 100 μL of LMB substrate solution, and incubate at 37°C in the dark for 10 min; add 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; read the absorbance at 450 nm using a microplate reader.

[0154] (2) Experimental Results

[0155] The activity curves of TF-A1-H7 oxime ester monoclonal antibody after heat treatment at different temperatures are shown in the figure below. Figure 9 As shown in the figure, the oxime ester monoclonal antibody TF-A1-H7 exhibits good thermostability. The antibody binding capacity is greater than 91% after heat treatment below 65℃.

[0156] Example 8: Determination of acid-base tolerance of oxime ester monoclonal antibody TF-A1-H7

[0157] (1) Experimental methods

[0158] PBS was prepared into buffer systems with different pH values ​​(1.4, 3.4, 5.4, 7.4, 9.4, and 11.4). These buffers were used to dilute the oxime protonate monoclonal antibody TF-A1-H7 to the same working concentration for antibody binding assay. The antibody binding capacity at pH 7.4 was taken as 100% to evaluate the antibody binding capacity of oxime protonate monoclonal antibody TF-A1-H7 under different acid-base conditions. The specific method is as follows:

[0159] The oxime ester monoclonal antibody TF-A1-H7 was diluted with PBS at pH 1.4, 3.4, 5.4, 7.4, 9.4, and 11.4, respectively. 50 μL of each diluted solution was added to 50 μL of PBS in pre-packaged microplates. The plates were incubated at 37°C for 40 min. The plates were washed five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), and the liquid in the wells was patted dry. 100 μL of HRP-labeled goat anti-mouse secondary antibody diluted 1:5000 was added, and the plates were incubated at 37°C for 30 min. The plates were then washed five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)). Wash the plate five times with PBS (0.06% Tween-20 (v / v)), pat dry the liquid in the wells, add 100 μL of LMB substrate solution, and incubate at 37°C in the dark for 10 min; add 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; read the absorbance at 450 nm using a microplate reader.

[0160] (2) Experimental Results

[0161] The activity curves of the oxime ester monoclonal antibody TF-A1-H7 under different pH conditions are shown in the figure below. Figure 10 As shown in the figure, the oxime ester monoclonal antibody TF-A1-H7 exhibits good acid and alkali tolerance within the pH range of 3.4 to 9.4.

[0162] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody against oxime ester, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

2.

2. The gene encoding the anti-oxime ester monoclonal antibody of claim 1, characterized in that, The nucleotide sequence encoding the light chain variable region is shown in SEQ ID No. 3, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No.

4.

3. A recombinant vector, characterized in that, It contains the gene described in claim 2.

4. A recombinant cell, characterized in that, It contains the recombinant vector as described in claim 3.

5. The use of the anti-oxime ester monoclonal antibody of claim 1, the gene of claim 2, the recombinant vector of claim 3, or the recombinant cell of claim 4 in the preparation of products for detecting oxime ester.

6. A kit for detecting oxime esters, characterized in that, It contains the anti-oxime ester monoclonal antibody as described in claim 1.

7. The use of the anti-oxime ester monoclonal antibody according to claim 1 in the detection of oxime ester for non-disease diagnostic purposes.

8. A method for detecting oxime esters for non-disease diagnostic purposes, characterized in that, The complete antigen obtained by conjugating the oxime hapten with a carrier protein is used as the coating antigen, and the anti-oxime hapten monoclonal antibody according to claim 1 is used as the detection antibody for detection; the structural formula of the oxime hapten is shown in formula (I): Equation (I).

9. The method according to claim 8, characterized in that, The structural formula of the complete antigen is shown in formula (II): Equation (II).

10. The method according to claim 8, characterized in that, The carrier protein is chicken ovalbumin.

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