Anti-trifloxystrobin monoclonal antibody and application thereof

Abstract

The invention discloses an anti-trifloxystrobin monoclonal antibody and an application thereof. A light chain variable region of the anti-trifloxystrobin monoclonal antibody has an amino acid sequence as shown in SEQ ID No.1, and a heavy chain variable region of the anti-trifloxystrobin monoclonal antibody has an amino acid sequence as shown in SEQ ID No.2. The monoclonal antibody has good affinity to trifloxystrobin, the half inhibitory concentration (IC50) of the monoclonal antibody is 4.55 ng / mL, the detection limit (IC10) of the monoclonal antibody is 1.34 ng / mL, the linear range (IC20-IC80) of the monoclonal antibody is 2.11-9.83 ng / mL, the cross reaction rate of the monoclonal antibody to structural analogues such as enestroburin, picoxystrobin and metoxystrobin is smaller than 1%, and the monoclonal antibody is high in specificity and has the advantages of being good in thermal stability, high in organic solvent and acid-base tolerance and high in sensitivity. The method is suitable for detecting trifloxystrobin residues in different environments.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular immunology and immune detection, and more specifically, relates to an anti-trifloxystrobin monoclonal antibody and application thereof. Background Art

[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 circulation, and exerting an antibacterial effect. Trifloxystrobin can be used in combination with a variety of fungicides and has good activity against almost all fungal diseases. It has the characteristics of a broad fungicidal spectrum, strong systemic activity, no cross-resistance, and good compatibility. Therefore, it is widely used in agriculture to control diseases of cereals, fruits, vegetables, etc. However, unscientific application methods, such as excessive application, illegal use of banned high-residue and highly toxic pesticides, and inadequate safety precautions during application, can cause 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 trifloxystrobin. Trifloxystrobin exhibits high levels of developmental toxicity, biotoxicity induction, and genotoxicity to fish, aquatic organisms, and algae. In humans, it can cause potential harm, including eye and respiratory irritation, weakness, dizziness, skin redness, and chest pain. Beyond physiological toxicity, the potential environmental impact of trifloxystrobin is also a concern. Trifloxystrobin is stable in water and has a long half-life, and its application can contaminate soil and rivers. Currently, China's GB 2763-2016 "National Food Safety Standard: Maximum Residue Limits of Pesticides in Food" stipulates a maximum permissible residue limit (MRL) of 40 mg / kg for various foods. For most fruits, vegetables, and grains, the MRLs in Europe and Russia range from 0.05 to 1.5 mg / kg. Therefore, for the sake of public health, strengthened monitoring and testing of trifloxystrobin in food and the environment is crucial.

[0003] Currently, the main methods for detecting trifloxystrobin include instrumental methods such as HPLC-MS / MS, GC-MS-MS / MS, and UPLC-MS / MS. While these instrumental methods offer high accuracy and sensitivity, they are cumbersome and time-consuming. Furthermore, the high testing and maintenance costs of large-scale instruments limit their application in large-scale, on-site screening. Immunoassays, based on antigen-antibody specific recognition, offer the advantages of rapidity, sensitivity, and high throughput, and have become a common method for screening large numbers of samples. Antibodies are the most important biological components in immunoassays. Chinese patent CN117623980A discloses a monoclonal antibody against trifloxystrobin with excellent potency, specificity, and affinity, achieving a detection sensitivity of up to 0.02 μg / L for trifloxystrobin. However, its thermal stability, organic solvent tolerance, and acid-base tolerance are limited, making it unsuitable for detecting trifloxystrobin under diverse environmental conditions. Therefore, providing an antibody with strong specificity, thermal stability, strong organic solvent and acid-base tolerance, and high sensitivity is crucial to meet future trifloxystrobin detection needs. Summary of the Invention

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

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

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

[0007] The fourth object of the present invention is to provide a recombinant cell containing the recombinant vector.

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

[0009] The above-mentioned object of the present invention is achieved through the following technical solutions:

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

[0011] Specifically, the monoclonal antibody includes four framework regions (FR1, FR2, FR3, FR4) and three complementary determining regions (CDR1, CDR2, CDR3) of the light chain variable region, and the four framework regions and three complementary determining regions of the heavy chain variable region are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The monoclonal antibody has a good affinity for trifloxystrobin, and its half inhibitory concentration (IC50) is 2.50 ) was 4.55 ng / mL, and the detection limit (IC 10 ) was 1.34 ng / mL, and the linear range (IC 20 ~IC 80 ) is 2.11-9.83 ng / mL, and the cross-reaction rate to structural analogues such as oxathiostrobin, picoxystrobin, and benoxystrobin is less than 1%. It has strong specificity, good thermal stability, strong tolerance to organic solvents and acid and alkali, and high sensitivity. It is suitable for the detection of oxathiostrobin residues in different environments.

[0012] The present invention also provides a gene encoding the anti-trifloxystrobin monoclonal antibody. 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 the present invention, the monoclonal antibody is obtained by immunizing an animal with an artificial antigen obtained by coupling trifloxystrobin hapten with a carrier protein as an immunogen, and then screening to obtain the above-mentioned anti-trifloxystrobin monoclonal antibody with strong specificity, good thermal stability, strong tolerance to organic solvents and acid and alkali, and high sensitivity.

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

[0015]

[0016] The trifloxystrobin hapten fully exposes the characteristic structure of trifloxystrobin, and the carbon chain length of the linker arm introduced into the hapten is appropriate. After being connected to the carrier protein, it has no effect on the antigen structure, which is conducive to the mouse immune system to effectively recognize the characteristic structure of the hapten and effectively improve the immunogenicity of the trifloxystrobin hapten. Therefore, highly specific antibodies can be prepared.

[0017] Furthermore, the trifloxystrobin hapten is obtained by condensing the carboxyl site of the ester group on trifloxystrobin with 6-aminocaproic acid. Specifically, the trifloxystrobin hapten preparation method includes the following steps:

[0018] S1: dissolving trifloxystrobin and sodium hydroxide in a mixed solution of methanol and water to cause a hydrolysis reaction, adding an ice-water mixture to terminate the reaction, and adjusting the pH with hydrochloric acid.

[0019] S2: The reaction solution obtained in S1 was extracted, and the organic phase was collected, washed, dried, and filtered. The filtrate was subjected to reduced pressure distillation to remove the solvent, and the residue was purified by silica gel column chromatography to obtain a white solid, which was the hydrolyzed product TF-H1.

[0020] S3: Dissolve TF-H1, EDC, and NHS in tetrahydrofuran and react overnight (referred to as liquid A). Dissolve 6-aminohexanoic acid in water (referred to as liquid B). Slowly add liquid B dropwise to liquid A to initiate a condensation reaction. Remove excess tetrahydrofuran by rotary evaporation. Add an ice-water mixture to terminate the reaction. Adjust the pH with hydrochloric acid.

[0021] S4: The reaction solution obtained in S3 is extracted and the organic phase is collected. After washing, drying, and filtering, the filtrate is subjected to reduced pressure distillation to remove the solvent. 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 feeding ratio of trifloxystrobin to sodium hydroxide is 1:4.

[0023] Preferably, the hydrolysis reaction conditions in step S1 are 60° C. for 2 h.

[0024] Preferably, in step S3, the feed ratio of trifloxystrobin 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 trifloxystrobin artificial antigen is shown in formula (II):

[0027]

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

[0029] S1: Dissolve the trifloxystrobin hapten TF-H3 in an organic solvent, then add NHS and EDC, and stir at 4°C for 6-12 hours, which is called solution A.

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

[0031] S3: The reaction solution obtained in S2 was dialyzed at 4°C for 3 days, with the dialysate changed twice a day to obtain the artificial antigen.

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

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

[0034] More preferably, the feed ratio of trifloxystrobin 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 trifloxystrobin hapten in step S2 is 1:80.

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

[0037] As an embodiment, the method for preparing the monoclonal antibody of the present invention specifically comprises the following steps:

[0038] (1) Preparation of trifloxystrobin hapten: Synthesis of trifloxystrobin artificial antigen from trifloxystrobin hapten. The trifloxystrobin artificial antigen was mixed with equal amounts of complete Freund's adjuvant, emulsified, and then injected subcutaneously or intraperitoneally into BALB / c mice for multiple immunizations. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for the booster immunization. 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 the mice that had undergone the above immunization process, and the immune titer and immunosuppressive ability of the mouse serum were detected by IC-ELISA to screen out the immunized mice with the best recognition of trifloxystrobin in the serum.

[0040] (3) The screened mice were given a final booster immunization with incomplete Freund's adjuvant. Three days before cell fusion, 200 μL of oxathiocarb-methyl artificial antigen without Freund's adjuvant was directly injected into the mouse peritoneal cavity for shock immunization.

[0041] (4) The spleen cells of BALB / c mice after shock immunization were fused with myeloma cells, the fused cells were cultured in culture medium, the positive cell wells were detected by ic-ELISA, and the inhibitory effect of the positive cell wells was further determined by ic-ELISA. The positive cell wells with the best inhibition were subcloned by limiting dilution method, and finally a hybridoma cell line that can secrete trifloxystrobin monoclonal antibodies was screened out. The hybridoma cells were inoculated into female BALB / c mice for culture to obtain a high concentration of ascites that can produce the above-mentioned monoclonal antibodies, and the ascites was purified to obtain anti-trifloxystrobin monoclonal antibodies.

[0042] Since the present invention provides the amino acid sequence of the anti-trifloxystrobin monoclonal antibody and the gene sequence encoding the monoclonal antibody, as a preferred embodiment, those skilled in the art can, based on this, obtain the monoclonal antibody described herein using known recombinant DNA techniques. Therefore, any recombinant vector or recombinant cell that can be used to prepare the monoclonal antibody described herein is also within the scope of protection of the present invention.

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

[0044] The present invention also provides a recombinant cell, which contains the recombinant vector.

[0045] The present invention also provides use of the anti-trifloxystrobin monoclonal antibody, the gene encoding the anti-trifloxystrobin monoclonal antibody, the recombinant vector or the recombinant cell in preparing a product for detecting trifloxystrobin.

[0046] The present invention also provides a kit for detecting trifloxystrobin, which contains the anti-trifloxystrobin monoclonal antibody.

[0047] The present invention also provides use of any of the above-mentioned anti-trifloxystrobin monoclonal antibodies in detecting trifloxystrobin.

[0048] The present invention also provides a method for detecting trifloxystrobin, comprising using a complete antigen obtained by coupling trifloxystrobin hapten with a carrier protein as a coating source, and using the above-mentioned anti-trifloxystrobin monoclonal antibody as a detection antibody for detection; the structural formula of the trifloxystrobin 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] The present invention provides an anti-trifloxystrobin monoclonal antibody, wherein the light chain variable region of the anti-trifloxystrobin 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 has a good affinity for trifloxystrobin, and its half inhibitory concentration (IC50) is 2. 50 ) was 4.55 ng / mL, and the detection limit (IC 10 ) was 1.34 ng / mL, and the linear range (IC 20 ~IC 80 ) is 2.11-9.83 ng / mL, and the cross-reaction rate to structural analogues such as oxathiostrobin, picoxystrobin, and benoxystrobin is less than 1%. It has strong specificity, good thermal stability, strong tolerance to organic solvents and acid and alkali, and high sensitivity. It is suitable for the detection of oxathiostrobin residues in different environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Flow chart for the preparation of trifloxystrobin hapten, artificial antigen and monoclonal antibody.

[0056] Figure 2 This is the mass spectrometry identification diagram of trifloxystrobin hapten TF-H1.

[0057] Figure 3 This is the mass spectrometry identification diagram of trifloxystrobin hapten TF-H3.

[0058] Figure 4 This is the UV scanning identification curve of trifloxystrobin artificial antigen, carrier protein and trifloxystrobin hapten.

[0059] Figure 5 Schematic diagram of the amino acid sequence and structural division of the light chain variable region of the anti-trifloxystrobin monoclonal antibody TF-A1-H7.

[0060] Figure 6 Schematic diagram of the amino acid sequence and structural division of the heavy chain variable region of the anti-trifloxystrobin monoclonal antibody TF-A1-H7.

[0061] Figure 7 This is the standard curve for indirect competition ELISA using the anti-trifloxystrobin monoclonal antibody TF-A1-H7.

[0062] Figure 8 This is a graph showing the organic solvent tolerance of the anti-trifloxystrobin monoclonal antibody TF-A1-H7.

[0063] Figure 9 This is a graph showing the thermal stability results of the anti-trifloxystrobin monoclonal antibody TF-A1-H7.

[0064] Figure 10 This is a graph showing the acid-base tolerance results of the anti-trifloxystrobin monoclonal antibody TF-A1-H7. DETAILED DESCRIPTION

[0065] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples 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 the art.

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

[0067] The following examples include the synthesis of trifloxystrobin hapten, trifloxystrobin artificial antigen and the preparation of anti-trifloxystrobin monoclonal antibody. Figure 1 shown.

[0068] Example 1 Synthesis and Identification of Trifloxystrobin Hapten

[0069] 1. Synthesis of trifloxystrobin hapten

[0070] (1) 1 mmol (408.37 mg) of trifloxystrobin was dissolved in 5 mL of methanol, and 4 mmol (160 mg) of NaOH was added and dissolved in 2 mL of H2O. The mixture was reacted at 60°C for 2 h and monitored by TLC. The methanol was removed by rotary evaporation, and an appropriate amount of ice-water mixture was added. The pH was adjusted to about 3-4 with 1 M hydrochloric acid, and the mixture was extracted 3-4 times with ethyl acetate. The organic phases were combined, washed 2-3 times with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the hapten TF-H1. After purification on a silica gel column, the compound represented by formula (I) was obtained.

[0071]

[0072] (2) 1 mmol (394.11 mg) of TF-H1 was dissolved in 3 mL of THF, and 2 mmol of EDC and 2 mmol of NHS were added. The mixture was stirred at room temperature overnight and monitored by TLC. After the reaction was completed, an appropriate amount of ice-water mixture was added, and the mixture was extracted with ethyl acetate 3 to 4 times. The organic layers were collected and combined, washed with saturated brine 2 to 3 times, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The mixture was redissolved in 2 mL of THF, and 10 mL of a mixture of 2 mmol (262.34 mg) of 6-aminohexanoic acid and 1.2 mmol (100.81 mg) of NaHCO3 was added dropwise. The mixture was monitored by TLC. After the reaction was completed, an appropriate amount of ice-water mixture was added, and the pH of the mixture was adjusted to about 3 to 4 with 6 M HCl. The mixture was extracted with ethyl acetate 3 to 4 times. The organic phases were combined, washed with saturated brine 3 to 4 times, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the hapten TF-H3. After purification on a silica gel column, the compound represented by formula (II) was obtained.

[0073]

[0074] 2. Identification

[0075] The trifloxystrobin hapten was identified by mass spectrometry.

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

[0077] Example 2 Synthesis and Identification of Trifloxystrobin Artificial Antigen

[0078] 1. Conjugation of trifloxystrobin artificial antigen and carrier protein

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

[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 at 4°C overnight;

[0082] (4) The cell was dialyzed with PBS buffer for two days, three times a day, and the artificial antigen of trifloxystrobin was obtained after the dialysis.

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

[0084] Similarly, the process of obtaining the artificial antigen of trifloxystrobin by using chicken ovalbumin instead of bovine serum albumin as the carrier protein is the same as the above preparation process.

[0085] 2. Identification

[0086] Take trifloxystrobin hapten, trifloxystrobin artificial antigen and carrier protein to perform UV full wavelength scanning, the results are as follows Figure 4 As shown in Figure 2, the trifloxystrobin hapten, trifloxystrobin artificial antigen, BSA, and OVA were each subjected to UV scanning (200-800 nm) for identification. Comparison of the offsets of each substance before and after conjugation revealed that the curves for the trifloxystrobin hapten, the trifloxystrobin artificial antigen, and the carrier protein were significantly different. The trifloxystrobin artificial antigen exhibited a protein absorption peak at approximately 280 nm, indicating that the trifloxystrobin hapten and carrier protein were successfully conjugated to yield the artificial antigen.

[0087] Example 3 Preparation of trifloxystrobin monoclonal antibody

[0088] (1) Three 4-week-old female BALB / c mice were immunized. The artificial antigen of trifloxystrobin was mixed with complete Freund's adjuvant in equal amounts. After emulsification, the emulsified immunogen was injected into the BALB / c mice subcutaneously or intraperitoneally for multiple immunizations. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for the booster immunization. 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, the mice were blood sampled from the tail vein. The immune titer and immunosuppressive ability of the mouse serum were tested by ic-ELISA. The immunized mice with the best recognition of trifloxystrobin in the serum were screened, and the spleen cells of the mice with the best immunization effect were taken for cell fusion.

[0089] (2) One day before cell fusion, a blank Balb / c mouse over 8 weeks old was sacrificed, the abdomen of the mouse was exposed, the abdominal skin was separated with sterile scissors and forceps, 5 mL of complete culture medium was pipetted into the mouse abdominal cavity, and after blowing back and forth several times, it was aspirated and placed in a 100 mL culture flask. Complete culture medium was added to the culture flask to make up to 80 mL, and the cells in the culture flask were gently shaken. 100 μL per well was added to 8 96-well plates, and finally placed in an incubator (5% CO2, 37°C) to culture to obtain feeder layer cells. Three days before cell fusion, 200 μL of immunogen (1 mg / mL) was directly injected into the mouse abdominal cavity for shock immunization.

[0090] (3) Take the spleen cells of the mouse with the best immune effect and mix them with mouse myeloma cells (SP2 / 0) in the logarithmic growth phase. Then, slowly add preheated fusion agent (PEG 4000) within 45 seconds for fusion. Suspend evenly with HAT medium. Then add an appropriate amount of feeder cells and culture in a 96-well culture plate. Incubate in a 37°C, 5% CO2 incubator. After 5 days, half of the medium is replaced with HT medium. After 9 days, the medium is fully replaced. When the cells in the plate grow to 1 / 3 of the culture well area, use ic-ELISA to screen the cell-positive wells.

[0091] (4) During screening, the artificial antigen E1-H1-OVA prepared in Example 2 was used as the coating agent at a coating agent concentration of 1000 ng / mL, 100 μL of which was coated per well, and the concentration of trifloxystrobin was 100 ng / mL. Positive wells were further screened using ic-ELISA, using the limiting dilution method to approximately one cell 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 to 3 rounds of subcloning, a cell line with 100% positive results and comparable titer and inhibitory effect was obtained, which was a positive monoclonal cell line. The cell lines were numbered and named. Finally, the positive monoclonal cell line was expanded and cultured for the preparation of monoclonal antibodies. The obtained monoclonal antibodies were subjected to tolerance experiments, and the monoclonal antibody TF-A1-H7 was screened to have strong specificity, good thermal stability, strong tolerance to organic solvents and acid-base, and high sensitivity.

[0092] (5) The screening steps are:

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

[0094] S2. Wash: Wash the plate twice using a plate washer, adding 300 μL of wash buffer to each well and swishing to remove any residual liquid.

[0095] S3. Blocking: Add 120 μL of blocking solution to each well and block at 37°C for 3 h. Shake dry the wells and dry them upside down in a 37°C oven for 1 h. Store at 4°C.

[0096] S4. Sample loading and incubation: Add 50 μL of PBS buffer to the titer wells and 50 μL of 100 ng / mL trifloxystrobin to the inhibition wells. Add 50 μL of cell supernatant to both the titer and inhibition wells. Incubate at 37°C for 40 min. Wash the plate five times with a microplate washer and spin dry.

[0097] S5. Add secondary antibody: Dilute HRP-goat anti-mouse secondary antibody 5000-fold in PBS. Add 100 μL of HRP-goat anti-mouse secondary antibody to each well. Incubate at 37°C for 30 min. Wash the plate five times using a microplate washer and spin dry.

[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. Determination: Measure the OD of each well using a microplate reader 450nm Absorbance value;

[0100] S8. Calculate the inhibition rate: Inhibition rate = (absorbance value of the titer well - absorbance value of the inhibition well) / absorbance value of the titer well × 100%.

[0101] Example 4 Sequencing of the Gene Encoding Anti-Trifloxystrobin Monoclonal Antibody TF-A1-H7 and Determination of the Amino Acid Sequence

[0102] 1. Total RNA Extraction

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

[0104] The specific steps are as follows:

[0105] Take about 1×10 6Cells were centrifuged at 3000 rpm for 5 minutes. The cells were collected into a centrifuge tube and all the supernatant culture medium was carefully discarded. Gently tap the bottom of the centrifuge tube to loosen the cell pellet. Immediately add 2 mL of lysis buffer (TRNsol), blow off the pellet, and collect it in a 2 mL centrifuge tube. Add 0.2 mL of chloroform for every 1 mL of the above lysis buffer. Cover the centrifuge tube and shake it up and down slowly for 15 seconds. Incubate on ice for 5 minutes and centrifuge at 12000 rpm for 10 minutes at room temperature. Transfer the upper aqueous phase to a new centrifuge tube and slowly add 0.7 volumes of anhydrous ethanol to mix. Transfer the resulting solution and the pellet to a GBC adsorption column and centrifuge at 12000 rpm for 30 seconds. Discard the waste solution. Add 500 μL of Wash Buffer I to the GBC adsorption column and centrifuge for 1 minute. Discard the waste solution. Add 600 μL of Wash Buffer II to the GBC adsorption column and centrifuge at 12000 rpm for 30 seconds. Discard the waste solution. Centrifuge at 12,000 rpm for 1 minute, discard the waste liquid, and open the lid under a clean bench to dry any remaining rinse solution in the adsorption column. Transfer the GBC adsorption column to a new centrifuge tube, add 30-100 μL of RNase-free ddH2O, incubate at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute to obtain the RNA solution.

[0106] 2. Synthesis of cDNA

[0107] Using RNA as a template, the first-strand cDNA was synthesized according to the instructions of Takara's first-strand reverse transcription kit. The specific method is to use 3μg RNA as a template, 1μL Oligo (dT) 18 primer, add RNase-free ddH2O to a total volume of 12μL in a 1.5mL nuclease-free centrifuge tube, incubate at 65℃ for 5 minutes, and cool in an ice bath for 2 minutes. After the reaction, add 4μL 5× Reaction Buffer, 1μL RNase inhibitor, 2μL 10mM dNTP Mix, and 1μL M-MuLV reverse transcriptase (200U / μL) to the system, and incubate at 42℃ for 60 minutes, incubate at 70℃ for 5 minutes to inactivate the reverse transcriptase, and store at -80℃ for use.

[0108] 3. Antibody variable region gene amplification

[0109] (1) PCR cloning V H 、V L Gene

[0110] The cDNA synthesized by reverse transcription was used as a template and universal primers were used to clone the genes of the variable regions of the antibody heavy chain and light chain. The universal primers are shown in Table 1.

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

[0112]

[0113]

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

[0115] Table 2 Light chain variable region PCR amplification system and reaction conditions

[0116]

[0117] ② Heavy chain variable region gene cloning

[0118] Using the first-strand cDNA as a template, the HB15 / HF2 primer set was used to amplify the heavy chain variable region. The reaction system and specific parameters are shown in Table 3. After PCR, gel electrophoresis was performed at 120V for 25 minutes to identify bands. The gel was cut using a kit to recover bands within the range of 300 bp to 500 bp.

[0119] Table 3 Heavy chain variable region PCR amplification system and reaction conditions

[0120]

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

[0122] The PCR gel products were recovered using the OMEGA gel extraction kit. The specific steps are as follows: cut the target band with a clean blade and weigh it, add an equal volume of Binding Buffer (XP2), and place it in a 55-60°C water bath for 10 minutes until the gel is completely melted, shaking every 2-3 minutes; transfer all the melted gel to the HiBind DNA column and centrifuge at 10,000 rpm for 1 minute; pour out the liquid in the collection tube, add 300 μL Binding Buffer (XP2), and centrifuge at 10,000 rpm for 1 minute; pour out the liquid in the collection tube, add 700 μL SPW Wash Buffer (add ethanol when using SPW Wash Buffer for the first time), and centrifuge at 10,000 rpm for 1 minute; repeat the addition of SPW Wash Buffer. Buffer wash; discard the liquid in the collection tube, centrifuge at 10000rpm for 2min; transfer the adsorption membrane to a 1.5mL centrifuge tube, add 15μL of sterile water, centrifuge at 10000rpm for 2min, collect the liquid in the tube, measure the DNA concentration with a micro-UV spectrophotometer (Nanodrop), and store at -20℃. H 、V L The gene was ligated into 18-T Vector Cloning vector (TakaRa).

[0123] (3) Conversion

[0124] Place competent DH5α cells on ice. After 5 minutes, allow the cells to thaw. Add 5 μL of the target vector, mix gently, and let it rest on ice for 25 minutes. Heat shock the cells in a 42°C water bath for 45 seconds. Immediately return the cells to ice and let them rest for 2 minutes. Add 400 μL of LB liquid medium preheated at 37°C and incubate at 37°C, 250 rpm, for 1 hour. Spread 100 μL of the culture evenly on an LB-A plate and incubate inverted at 37°C overnight. The next day, randomly select several single colonies from the plate for colony PCR and DNA sequencing.

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

[0126] The sequencing results were adjusted using DNAman software to obtain complete forward sequences, which were then imported into IMGT (https: / / www.imgt.org / IMGT_vquest / analysis) in FASTA format for mouse antibody variable region gene sequence analysis. The translated amino acid sequences of the heavy chain variable region (HF2-HB15) and 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 the heavy chain variable region of the monoclonal antibody TF-A1-H7 prepared in Example 3 are shown in FIG. Figure 5 and Figure 6 shown.

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

[0130] DIVMTQSPKFMSTSVRDRVSITCKASRNVLSPVTWYQQKPRQFPKTLIYLVSNRHTGVPDPFRGSGSGSDFTLTISSVQSEDPADYFCLHLWNLPYTFGAGTKLEIK

[0131] The amino acid sequence of the heavy chain variable region 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 indirect competitive ELISA standard curve based on monoclonal antibodies

[0138] 1. Coating and sealing

[0139] Dilute the TF-H3-OVA coating agent to 1000 ng / mL in 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 solution to each well, and block at 37°C for 3 hours. Spin dry the blocking solution, dry at 37°C for 1 hour, and store in a sealed bag at 4°C until ready to use.

[0140] 2. Establishment of standard curve

[0141] 50 μL PBS buffer was added to the titer wells, 50 μL 100 ng / mL trifloxystrobin was added to the inhibition wells, 50 μL cell supernatant was added to both the titer wells and the inhibition wells, the cells were incubated at 37°C for 40 min, the plates were washed 5 times with a plate washer, and the liquid in the wells was dried; PBS was used as the diluent for the HRP-goat anti-mouse secondary antibody, diluted 5000-fold, 100 μL HRP-goat anti-mouse secondary antibody was added to each well, the cells were incubated at 37°C for 30 min, the plates were washed 5 times with a plate washer, and the liquid in the wells was dried; 10 mL H2O2 substrate buffer was mixed with 550 μL TMB color development solution, 100 μL was added to each well, the cells were incubated at 37°C for 10 min, and 50 μL 10% H2SO4 stop solution was added to each well; the OD value of each well was measured using a microplate reader. 450nm Calculation: B / B0 = OD of the well containing trifloxystrobin 450nm OD of the titer well 450nm A standard curve was established with the logarithm of the concentration of the trifloxystrobin standard as the horizontal axis and B / B0 as the vertical axis.

[0142] 3. Experimental results

[0143] The standard curve of ic-ELISA established based on monoclonal antibodies is shown in the figure below. Figure 7 As shown in the figure, it can be seen that the standard curve is S-shaped and has a good linear correlation. The half-inhibitory concentration (IC 50 ) was 4.55 ng / mL, and the detection limit (IC 10 ) was 1.34 ng / mL, and the linear range (IC 20 ~IC 80 ) were 2.11~9.83 ng / mL, with high detection sensitivity and wide linear range.

[0144] Example 6 Determination of organic solvent tolerance of trifloxystrobin monoclonal antibody TF-A1-H7

[0145] (1) Experimental methods

[0146] The trifloxystrobin 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 capacity. The antibody-antigen binding capacity of the solution containing 0% organic solvent was taken as 100% to evaluate the tolerance of the trifloxystrobin monoclonal antibody TF-A1-H7 to methanol, acetonitrile, and ethanol. The specific method is as follows:

[0147] Add 50 μL of diluted trifloxystrobin monoclonal antibody TF-A1-H7 and 50 μL of PBS to the coated ELISA plate, incubate at 37°C for 40 min, wash the plate five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), pat dry the liquid in the wells, add 100 μL of 1:5000 diluted HRP-labeled goat anti-mouse secondary antibody, incubate at 37°C for 30 min, wash the plate five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), pat dry the liquid in the wells, add 100 μL of TMB substrate solution, and develop 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 value at 450 nm using a microplate reader.

[0148] (2) Experimental results

[0149] The activity curves of trifloxystrobin monoclonal antibody TF-A1-H7 when different ratios of organic solvent / PBS were used as diluents are shown in Figure 2. Figure 8As shown in the figure, the trifloxystrobin monoclonal antibody TF-A1-H7 exhibits good tolerance to methanol. At methanol concentrations below 30%, the trifloxystrobin monoclonal antibody TF-A1-H7 exhibits excellent activity, exceeding 89%. Furthermore, activity is significantly enhanced when the methanol content is below 10%. Furthermore, the antibody activity is above 50% when the concentrations of acetonitrile and ethanol are below 10%. These results demonstrate that the trifloxystrobin monoclonal antibody TF-A1-H7 exhibits a certain degree of tolerance to organic solvents, and that methanol, at appropriate concentrations, enhances the antibody's activity.

[0150] Example 7 Thermal Stability Determination of Trifloxystrobin Monoclonal Antibody TF-A1-H7

[0151] (1) Experimental methods

[0152] The binding capacity of the trifloxystrobin monoclonal antibody TF-A1-H7 was measured by heating it at 25°C, 35°C, 45°C, 55°C, 65°C, and 75°C for 5 minutes. The binding capacity of the antibody at 25°C was taken as 100%, and the binding capacity of the trifloxystrobin monoclonal antibody TF-A1-H7 after heat treatment at different temperatures was evaluated. The specific method is as follows:

[0153] 50 μL of diluted trifloxystrobin monoclonal antibody TF-A1-H7 was placed at 25°C, 35°C, 45°C, 55°C, 65°C and 75°C for 5 min, and 50 μL of PBS was added to the coated ELISA plate, incubated at 37°C for 40 min, washed five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), patted dry, and 100 μL of 1:5000 diluted HRP-labeled goat anti-mouse secondary antibody was added, incubated at 37°C for 30 min, and washed with PBST (0.01 M The plate was washed five times with PBS, 0.06% Tween-20 (v / v), the liquid in the wells was patted dry, 100 μL of TMB substrate solution was added, and the color was developed at 37°C in the dark for 10 min; 50 μL of stop solution (10% H2SO4, v / v) was added to terminate the reaction; and the absorbance at 450 nm was read using a microplate reader.

[0154] (2) Experimental results

[0155] The activity curves of trifloxystrobin monoclonal antibody TF-A1-H7 when the antibody was heat treated at different temperatures are shown in the figure below. Figure 9 As shown in the figure, it can be seen that the trifloxystrobin monoclonal antibody TF-A1-H7 has good thermal stability. When heat-treated below 65°C, the antibody binding capacity is greater than 91%.

[0156] Example 8 Determination of Acid-Base Tolerance of Trifloxystrobin 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). This was used as the diluent to dilute the anti-trifloxystrobin monoclonal antibody TF-A1-H7 to the same working concentration to determine the antibody binding capacity. The antibody binding capacity at pH 7.4 was taken as 100%, and the antibody binding capacity of the anti-trifloxystrobin monoclonal antibody TF-A1-H7 under different acidic and alkaline conditions was evaluated. The specific method was as follows:

[0159] The trifloxystrobin 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 the diluted solution was added to the coated ELISA plate, incubated at 37°C for 40 min, washed five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), the liquid in the well was patted dry, 100 μL of HRP-labeled goat anti-mouse secondary antibody diluted 1:5000 was added, incubated at 37°C for 30 min, and washed with PBST (0.01 M The plate was washed five times with PBS, 0.06% Tween-20 (v / v), the liquid in the wells was patted dry, 100 μL of TMB substrate solution was added, and the color was developed at 37°C in the dark for 10 min; 50 μL of stop solution (10% H2SO4, v / v) was added to terminate the reaction; and the absorbance at 450 nm was read using a microplate reader.

[0160] (2) Experimental results

[0161] The activity curves of trifloxystrobin monoclonal antibody TF-A1-H7 under different pH environments are shown in Figure 2. Figure 10 As shown in the figure, it can be seen that within the pH range of 3.4 to 9.4, the trifloxystrobin monoclonal antibody TF-A1-H7 has good acid and base tolerance.

[0162] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An anti-trifloxystrobin monoclonal antibody, 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-trifloxystrobin monoclonal antibody according to 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 Containing the gene according to claim 2.

4. A recombinant cell, characterized in that Contains the recombinant vector according to claim 3.

5. Use of the anti-trifloxystrobin monoclonal antibody according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3, or the recombinant cell according to claim 4 in the preparation of a product for detecting trifloxystrobin.

6. A kit for detecting trifloxystrobin, characterized in that: Contains the anti-trifloxystrobin monoclonal antibody according to claim 1.

7. Use of the anti-trifloxystrobin monoclonal antibody according to claim 1 in detecting trifloxystrobin.

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

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

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

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