Antibodies to imidacloprid and uses thereof

Abstract

The present application relates to the field of biotechnology, in particular to an antibody against diquat and application thereof. The present application overcomes technical barriers, and a new hapten molecule is used to prepare an antibody with high affinity to diquat, and cross interference with non-selective herbicides such as paraquat, glufosinate-ammonium and glyphosate with similar structures to diquat can be avoided, which has great significance for the immune detection of diquat.

Description

[0001] Invention Name

[0002] Antibodies against cypermethrin and their applications Technical Field

[0003] This invention relates to the field of biotechnology, specifically to antibodies against cypermethrin and their applications. Background Technology

[0004] With the development of agriculture and animal husbandry, people have expanded the scope and amount of various pesticides and veterinary drugs used due to the need to prevent and control pests and diseases, thereby increasing agricultural and livestock output. However, this has also brought about some problems of pesticide residues polluting agricultural products and feed, posing a threat to human health and becoming a common problem worldwide.

[0005] Diquat (DQ), chemically known as 1,1'-ethylidene-2,2'-bipyridine dibromide, is a colorless to pale yellow crystal with a vapor pressure <0.01 MPa. Its solubility in water (20℃) is 700 g / L, it is slightly soluble in ethanol and carbonyl solvents, and insoluble in non-polar organic solvents. It is stable in acidic and neutral solutions but unstable under alkaline conditions. First developed and applied globally in the UK in 1957, it has been widely used worldwide due to its low cost, rapid onset of action, and low pollution. In recent years, with the ban on paraquat aqueous solution in the domestic market, the sales volume of diquat in the domestic herbicide market has surged. Diquat is generally used as a systemic contact herbicide, rapidly absorbed by green plant tissues and quickly loses its activity upon contact with soil. It is used for weed control in fields, orchards, non-cultivated land, and before harvest, and can also be used to prune the stems and leaves of potatoes and sweet potatoes.

[0006] Pesticide residues refer to the total amount of pesticide residues, toxic metabolites, gaseous degradation products, and impurities that remain in organisms, harvested crops, soil, water bodies, and the atmosphere after pesticide application and are not decomposed within a certain period. Of the pesticides applied to crops, some adhere to the crops, while some are dispersed into the soil, air, and water environment. Some of the pesticide residues in the environment are then absorbed by plants. Residual pesticides can directly reach humans and livestock through plant fruits, water, or the atmosphere, or ultimately be transferred to humans and livestock through the environment and the food chain, causing harm to humans, the environment, and the market economy.

[0007] With increasing dosage of diquat, reports of diquat poisoning have also increased significantly. Diquat poisoning is more common in rural areas of developing countries, and there is currently a lack of statistical data on its incidence in China. Although diquat is less toxic than paraquat, it still has a high mortality rate. Studies have found that diquat can damage the kidneys, lungs, and heart, especially causing acute severe renal failure in the early stages, and severe poisoning can lead to multiple organ failure. Diquat is a toxic herbicide; the acute oral LD50 in rats is [not specified].50 The acute oral LD50 in mice is 231 mg / kg. 50 The concentration is 125 mg / kg. Through cyclic redox reactions, it disrupts the balance between oxidation and antioxidation in the body, tilting the balance towards oxidation. This induces inflammatory infiltration of neutrophils, leading to the secretion of large amounts of lysosomal enzymes and the production of large amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS), triggering oxidative stress. Excessive ROS and RNS products damage cellular DNA, biomembrane lipids, proteins, and other macromolecules, initiating a series of damages, primarily lipid peroxidation. This alters the fluidity and permeability of cell membrane structures, ultimately resulting in changes in cell structure and function, cell damage, and death. The gastrointestinal tract and kidneys are most severely affected, followed by the liver, lungs, and heart. Therefore, establishing a rapid detection method for diquat is of significant clinical importance.

[0008] Countries worldwide, especially developed countries, place great importance on pesticide residue issues and have established increasingly stringent limits for pesticide residues in various agricultural products. Many countries use pesticide residue limits as technical barriers to restrict the import of agricultural products and protect agricultural production. The application market for diquat monoclonal antibodies includes, but is not limited to: ① detecting diquat residues in agricultural products and water quality to ensure food safety, reduce pesticide poisoning incidents, and benefit agricultural development. ② detecting diquat residues in soil and the environment to guide the rational use of diquat and maintain the ecological environment. ③ detecting diquat poisoning and guiding treatment.

[0009] Currently, methods for detecting diquat include chemical analysis, ultraviolet spectrophotometry, high-performance liquid chromatography (HPLC), capillary electrophoresis-mass spectrometry (CES-MS), and liquid chromatography-mass spectrometry (LC-MS). Chemical analysis detects diquat content through chemical reactions, but its sensitivity is low and it cannot perform trace analysis. Ultraviolet spectrophotometry determines concentration by measuring the absorption spectrum of diquat, but it also cannot perform trace analysis. Mass spectrometry can detect diquat with a sensitivity below 20 ng / mL, but the instruments are expensive, time-consuming, and the operation is cumbersome, requiring professional personnel, making it difficult to promote. Immunological detection technology has advantages such as being economical, rapid, low in technical requirements, simple to operate, and capable of on-site detection. The key to this method is the preparation of monoclonal antibodies that specifically recognize diquat. However, since small molecule monoclonal antibodies (molecular weight less than 10,000) are not immunogenic, they need to be conjugated with carrier proteins before they can be prepared by immunization, which makes antibody preparation difficult. At the same time, non-selective herbicides such as paraquat, glufosinate, and glyphosate have structures similar to diquat, which can easily lead to cross-interference between antibodies. Therefore, there are currently no antibodies that specifically recognize diquat. Summary of the Invention

[0010] To address the aforementioned problems, this invention proposes a novel anti-diquat antibody that can bind to diquat molecules with high specificity and without cross-interference.

[0011] Specifically as follows:

[0012] Antibodies that specifically bind to compound I

[0013]

[0014] Formula I

[0015] The characteristic feature is that the antibody also binds to dichlorvos.

[0016] Preferably, the antibody is a monoclonal antibody.

[0017] Preferably, the antibody according to claim 1 or 2 is characterized in that the antibody has the following complementarity-determining region:

[0018] a) Heavy chain variable region CDR1 as listed in SEQ ID NO:1;

[0019] b) Heavy chain variable region CDR2 as listed in SEQ ID NO:2;

[0020] c) Heavy chain variable region CDR3 as listed in SEQ ID NO:3;

[0021] d) Light chain variable region CDR1 as listed in SEQ ID NO:4;

[0022] e) the light chain variable region CDR2 as listed in SEQ ID NO:5; and

[0023] f) Light chain variable region CDR3 as listed in SEQ ID NO:6.

[0024] Preferably, the antibody has a heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:8.

[0025] Preferably, the antibody does not bind to paraquat, glufosinate or glyphosate.

[0026] Another aspect of the present invention provides a method for preparing antibodies, the method comprising the following steps:

[0027] (1) Prepare a complete antigen using the compound of Formula I;

[0028] (2) Immunize experimental animals with the complete antigen to induce B cells to produce antibodies that bind to the complete antigen;

[0029] (3) Alternatively, by hybridoma technology or by B-cell PCR technology, a monoclonal antibody that binds to the complete antigen produced by B-cells is obtained;

[0030] (4) Select the antibodies from step (3) that bind to paraquat and select those that do not bind to paraquat, glufosinate, or glyphosate.

[0031] The compound of Formula I of this invention is a hapten and cannot be directly used to immunize animals. It needs to be processed into a complete antigen before it can be used to immunize experimental animals.

[0032] On the other hand, the present invention provides a kit that contains the antibody.

[0033] On the other hand, the present invention provides a conjugate comprising the antibody covalently linked to a chemical label or a biological label.

[0034] On the other hand, the present invention provides a conjugate formed by coupling the antibody or the conjugate with a solid or semi-solid medium.

[0035] On the other hand, the present invention provides the use of the antibody, the conjugate, or the conjugate described herein in the preparation of a product for detecting the expression of dichlorvos.

[0036] On the other hand, the present invention provides the use of the compound of Formula I in the preparation of anti-diphtheria antibody.

[0037] Terminology Explanation:

[0038] The "antibody" described in this invention includes various forms of antibody structures, including but not limited to complete antibodies and antibody fragments. Antibodies according to the invention are preferably goat, sheep, mouse, rabbit, or rat antibodies, chimeric antibodies, or further genetically engineered antibodies, as long as they retain the characteristic properties according to the invention. The properties according to the invention can be either monoclonal or polyclonal antibodies.

[0039] An "antibody fragment" comprises a portion of a complete antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single-chain antibody molecules; scFv, sc(Fv)2; biantibodies; and multispecific antibodies formed from antibody fragments.

[0040] As used herein, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to formulations of antibody molecules consisting of a single amino acid. This refers to antibodies obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical except for the possibility of small amounts of possible mutations (e.g., naturally occurring mutations). Therefore, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete antibodies. In some embodiments, such monoclonal antibodies generally comprise antibodies containing a polypeptide sequence that binds to a target, wherein said target-binding polypeptide sequence is obtained by a method comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection method may be to select a unique clone from a plurality of clones, such as a library of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that the selected target-binding sequence can be further modified, for example, to increase affinity for the target, to humanize the target-binding sequence, to improve its production in cell cultures, to reduce its immunogenicity in vivo, to produce multispecific antibodies, etc., and antibodies containing modified target-binding sequences are also monoclonal antibodies of the present invention. Unlike polyclonal antibody formulations, which typically consist of different antibodies targeting different determinants (epitopes), monoclonal antibody formulations contain each monoclonal antibody targeting a single determinant on the antigen. In addition to their specificity, monoclonal antibody formulations are advantageous because they are generally not contaminated by other immunoglobulins.

[0041] Monoclonal antibodies in this article specifically include “chimeric” antibodies, wherein a portion of the heavy and / or light chains is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of one or more chains is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, provided they exhibit the desired biological activity.

[0042] In the context of antibodies, the term "analyte-specific binding" refers to the immunospecific interaction between an antibody and its target epitope on the analyte; that is, the binding of the antibody to the epitope on the analyte. The concept of analyte-specific binding of an antibody through its epitope on the analyte is fully apparent to those skilled in the art.

[0043] A "hapten" is a small molecule (e.g., an insecticide, fungicide, drug, hormone, toxin, synthetic peptide, etc.) that does not directly induce an immune response, such as antibody formation. Techniques for generating antibodies against haptens by conjugating them to immunogenic carriers (e.g., antigenic macromolecules) have been established. For the purposes of this disclosure, Formula I is a hapten that does not elicit an immune response until and unless conjugated to an immunogenic carrier such as a protein. Once an antibody is formed, it can bind to the hapten. The resulting antibody can be used in many fields, particularly in the development of immunodiagnostic kits or biosensors. In the context of the disclosure of compounds according to Formula I, a hapten that is part of the compound is understood to be covalently coupled to the remaining part of the compound, wherein the hapten portion is capable of eliciting an immune response only when attached to an immunogenic carrier such as...

[0044] The term "laboratory animal" refers to a non-human animal. In one embodiment, the laboratory animal is selected from rats, mice, hamsters, rabbits, camels, llamas, non-human primates, sheep, dogs, cattle, chickens, amphibians, sharks, and reptiles. In one embodiment, the laboratory animal is a mouse.

[0045] Beneficial effects: A novel hapten is used to prepare a highly specific monoclonal antibody against diquat. The antibody described in this invention specifically binds to the hapten and diquat, and can avoid cross-interference from non-selective herbicides such as paraquat, glufosinate, and glyphosate, which have similar structures to diquat. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0048] As used herein, the terms “comprising,” “including,” “having,” “may,” and their variations are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures.

[0049] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0050] Example 1: Immunogen Preparation

[0051] (1) Preparation of hapten

[0052] Since diquat is a small molecule and lacks immunogenicity, it cannot stimulate an immune response in mice to produce antibodies. Therefore, it is necessary to conjugate diquat to a protein using protein coupling technology to acquire immunogenicity. Commonly used active groups in protein coupling technology include amino, carboxyl, hydroxyl, and thiol groups. However, since the diquat molecule does not contain such active groups, derivatization is required. We designed and synthesized compound 5 as a hapten of diquat, and the specific synthetic route is as follows.

[0053]

[0054] Synthesis of Compound 2

[0055]

[0056] Dry diisopropylamine (1 mL, 7.1 mmol) was dissolved in ultra-dry tetrahydrofuran (50 mL), purged three times with nitrogen, and then kept under a nitrogen atmosphere. The mixture was cooled to -78°C using a dry ice-acetone bath. Butyllithium (1.6 M n-hexane solution, 3.8 mL, 6.1 mmol) was slowly added dropwise to the reaction mixture while maintaining an internal temperature below -70°C. After the addition was complete, the mixture was stirred at -70°C for 30 minutes. Compound 1 (681 mg, 4 mmol) was dissolved in ultra-dry tetrahydrofuran (15 mL) and slowly added dropwise to the reaction mixture while maintaining an internal temperature below -70°C. After the addition was complete, the mixture was stirred at -70°C for 60 minutes. 1,3-Dibromopropane (4 g, 20 mmol) was dissolved in ultra-dry tetrahydrofuran (15 mL, 6.1 mmol). The solution was slowly added dropwise to the above reaction mixture (mL), keeping the internal temperature below -70°C. After the addition was complete, the cold bath was not removed, and the mixture was allowed to warm naturally to room temperature and stirred overnight. A saturated sodium bicarbonate aqueous solution was carefully added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed once with saturated brine, and dried over anhydrous sodium sulfate. The organic phases were concentrated under reduced pressure and vacuum at 45°C. The residue was purified by column chromatography to give approximately 926 mg of white solid. MS Found: [M+1]+=292.19; 1H NMR (300MHz, CDCl3): 8.67 (d, 1H), 8.51 (d, 1 H), 8.36 (d, 1 H), 8.31 (d, 1 H), 7.81 (d, 1 H), 7.64 (dd, 1H), 7.31-7.29 (m, 1 H), 3.41 (t, 2 H), 2.70 (t, 2 H), 1.91 (m, 2 H), 1.70 (m, 2 H)

[0057] Synthesis of Compound 3

[0058]

[0059] Diethyl malonate (0.72 g, 4.5 mmol) was dissolved in ultradry tetrahydrofuran (20 mL), purged three times with nitrogen, and then kept under a nitrogen atmosphere. The mixture was cooled to 0°C using an ice-water bath. Sodium hydride (60%, 0.18 g, 4.5 mmol) was added to the reaction mixture in one go, and the mixture was stirred at 0–20°C for 30 minutes. Compound 2 (926 mg, 3 mmol) was dissolved in ultradry tetrahydrofuran (10 mL) and slowly added dropwise to the reaction mixture. After the addition was complete, the reaction mixture was heated to 60°C and stirred at 60°C for 3 hours. The reaction mixture was cooled to room temperature and poured into saturated brine. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The mixture was washed once with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure and vacuum at 45°C. The residue was purified by column chromatography to give about 1 g of white solid. MS Found: [M+1]+=371.45

[0060] Synthesis of Compound 4

[0061]

[0062] Compound 3 (1 g, 2.7 mmol) was dissolved in ethanol (20 mL), and 6N hydrochloric acid aqueous solution (20 mL) was added. The reaction mixture was heated to 80 °C and stirred at 80 °C for 6 hours. The reaction solution was cooled to room temperature and then concentrated under reduced pressure at 45 °C. The residue was slurried with acetone to give approximately 700 mg of white solid. MS Found: [M+1]+=257.31

[0063] Synthesis of Compound 5

[0064]

[0065] Compound 4 (700 mg, 2.1 mmol) obtained in the previous step was dispersed in dibromoethane (10 mL); the mixture was purged with nitrogen three times and maintained under a nitrogen atmosphere; the reaction mixture was heated to 125 °C and stirred at 125 °C for 6 hours; the reaction solution was cooled to room temperature and filtered, and the filter cake was washed with a small amount of dichloromethane; the filter cake was slurried with acetone and filtered, and dried under reduced pressure at 45 °C; the residue was purified by Pre-HPLC to give 326 mg of white solid. MS Found: [M+1]+=284.36

[0066] (2) Preparation of complete antigen

[0067] Preparation of complete antigen DQ-BSA: Weigh 1.5 mg of diquat hapten (compound 5) and dissolve it in 300 μl of 20 mM phosphate buffer (PBS, pH 7.2). Dissolve 0.87 mg of N-hydroxysuccinimide (NHS) in 87 μL of 20 mM PBS (pH 7.2). Weigh 1.92 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and dissolve it in 192 μL of 20 mM PBS (pH 7.2). Mix the three solutions and stir at room temperature for 30 min to obtain solution A. Dissolve 2.25 mg of BSA in 450 μL of 20 mM PBS (resulting in solution B). Slowly add solution A dropwise to solution B and react at room temperature for 4 hours. Then, use 20 mM PBS... Unreacted small molecule haptens were removed by dialysis with PBS solution to obtain the complete antigen DQ-BSA, which was then identified by ultraviolet full-wavelength scanning.

[0068] Example 2 Antibody Preparation

[0069] (1) Mouse immunization and antibody detection

[0070] Freund's complete adjuvant was emulsified with an equal volume of DQ-BSA protein at a concentration of 2 mg / mL. Six 6-8 week old SPF-grade female BALB / c mice were selected and immunized via subcutaneous injection in the back, with an immunization dose of 0.1 mg / mouse. Two weeks after the initial immunization, the antigen protein was emulsified with Freund's incomplete adjuvant and injected again via paw or subcutaneous injection in the back, with each mouse receiving 50 μg of antigen protein. Two weeks later, blood was collected via tail vein, the supernatant was collected by centrifugation, and serum titer was detected by ELISA. Immunization was repeated every two weeks, and serum titer was detected by ELISA. A screening serum titer of 10 was performed. 6 The spleens of the mice were used to isolate B lymphocytes for cell fusion.

[0071] (2) Hybridoma cell fusion

[0072] Spleens from mice that met the tail blood titer criteria were collected, ground, and lysed to prepare B cells. B cells were then mixed with SP2 / 0 myeloma cells at a 1:1 ratio and electrofused to obtain hybridoma cells. The hybridoma cells were then added to 1.2% methylcellulose semi-solid medium, followed by the addition of HAT, anti-mycoplasma drugs, and feeder cells. The mixture was thoroughly mixed and plated. After culturing for 3-4 days, DMEM complete medium was added, and the cells were cultured continuously for 13 days. The cell supernatant was then used for ELISA antibody screening.

[0073] (3) Antibody screening

[0074] The objective of this invention is to screen for antibodies that have a high affinity for diquat and no cross-contamination with other herbicides. Therefore, we designed the following screening scheme:

[0075] ① Dilute the whole antigen DQ-BSA to 1 μg / mL with coating buffer (0.05 M pH 9.5 carbonate and bicarbonate buffer), add 100 μL / well to the microplate, and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20 (wash buffer).

[0076] ② Dilute the culture supernatant of the hybridoma cells to be tested with diluent (1% BSA, 0.1% PBST) at a ratio of 1:1, and add 50 μL / well to the microplate; at the same time, dilute different concentrations of diquat or paraquat with diluent, with a diquat concentration of 1 μg / mL and a paraquat concentration of 100 μg / mL. Use diluent without paraquat and diquat as a control, add 50 μL / well to the microplate, and incubate at 37°C for 30 minutes.

[0077] ③ After washing the plate three times with washing buffer, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing the plate three more times, add the colorimetric solution and incubate for about 5 minutes (depending on the strength of the reaction). Then add 2 M sulfuric acid to stop the reaction and read the OD450 absorbance value.

[0078] The results are as follows: Cell line C showed a significantly weaker colorimetric signal value, indicating that it did not recognize the full antigen DQ-BSA and did not meet the screening criteria. Cell lines A, B, and D (control wells) showed high colorimetric intensity values. After adding diquat to cell line A, its signal value decreased significantly (by 89%), indicating that diquat competitively binds to the antibody in the cell supernatant with the full antigen DQ-BSA. The antibody in the supernatant of cell line A showed strong affinity for diquat. Furthermore, the addition of high concentrations of paraquat, glufosinate, and glyphosate did not significantly decrease the signal value (signal values ​​decreased by 11%, 3%, and increased by 6%, respectively), indicating that paraquat and glufosinate... Glyphosate did not competitively bind to the antibody in the cell supernatant with the whole antigen DQ-BSA, indicating good antibody specificity and meeting the screening criteria. Cell line B, after the addition of diquat, did not show a significant decrease in signal value (signal value decreased by 37%), indicating that diquat did not competitively bind to the antibody in the cell supernatant with the whole antigen DQ-BSA. This antibody has low affinity for diquat and therefore does not meet the screening criteria. Cell line D, after the addition of paraquat, showed a significant decrease in signal value (signal value decreased by 73%), indicating that paraquat competitively binds to the antibody in the cell supernatant with the whole antigen DQ-BSA. This antibody has poor specificity and does not meet the screening criteria.

[0079] In summary, cell line A is selected.

[0080] Table 1 Cell line screening

[0081]

[0082] (4) Identification of monoclonal antibody subtypes and cloning of gene sequences

[0083] The SBA Clonotyping System-HRP kit from Southern Biothech was used to identify the heavy and light chain isotypes of monoclonal antibodies, following the manufacturer's instructions. The specific procedure was as follows:

[0084] ① Dilute the capture antibody to 1 μg / mL with coating buffer (0.05 M pH 9.5 carbonate and bicarbonate buffer), add 100 μL / well to the microplate, and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20 (wash buffer).

[0085] ② Dilute the culture supernatant of the hybridoma cells (cell line A) to be tested with diluent (1% BSA, 0.1% PBST) at a ratio of 1:1, add 100 μL / well to the ELISA plate, and incubate at 37°C for 30 minutes.

[0086] ③ Dilute the corresponding enzyme-labeled antibodies (Ig A-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lambda-HRP) 1:3000 with diluent. After washing the plate three times with washing buffer, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing the plate three more times, add chromogenic buffer and wait approximately 5 minutes (depending on the reaction strength). Then, add 2M sulfuric acid to stop the reaction and read the OD450 absorbance. Identification showed that the heavy chain subtype of antibody A was IgG1, and the light chain was Kappa (the wells corresponding to IgG1 and Kappa showed color development).

[0087] Based on the antibody subtype results, the antibody gene sequence was cloned using a RACE-based method. Hybridoma cells in good growth condition were collected, and total RNA was obtained from the hybridoma cells using a total RNA extraction kit. The mRNA was reverse transcribed into cDNA according to the Takara SMARTer RACE instruction manual, and the full-length sequence of the target antibody was amplified.

[0088] (5) Production and purification of monoclonal antibodies

[0089] Two groups of 6-8 week old BALB / c mice were selected, and 500 μL of paraffin oil was injected intraperitoneally to suppress the immune response. One week after injection, 0.5 mL of hybridoma cells (cell line A) was injected intraperitoneally into one group of mice, with a cell count of approximately 1 × 10⁶. Ascites fluid collection began two weeks later. The collected ascites fluid was purified by ammonium sulfate precipitation and affinity purification of protein A to obtain the target antibody A.

[0090] Example 3 Antibody Performance Experiment

[0091] ① Dilute the whole antigen DQ-BSA to 1 μg / mL with coating buffer (0.05 M pH 9.5 carbonate and bicarbonate buffer), add 100 μL / well to the microplate, and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20 (wash buffer).

[0092] ② Dilute the purified antibody A to 0.1 ug / ml with diluent and add 50 μL / well to the microplate. At the same time, dilute different concentrations of diquat, paraquat, glufosinate, and glyphosate (non-selective herbicide) with diluent to final concentrations of 500 ng / ml, 167 ng / ml, 56 ng / ml, 19 ng / ml, 6 ng / ml, 2 ng / ml, and 1 ng / ml, respectively. Do not add non-selective herbicide as a control. Add 50 μL / well to the microplate and incubate at 37℃ for 30 minutes.

[0093] ③ After washing the plate three times with washing buffer, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing the plate three more times, add the colorimetric solution and incubate for about 5 minutes (depending on the strength of the reaction). Then add 2 M sulfuric acid to stop the reaction and read the OD450 absorbance value.

[0094] Table 2 Antibody Performance Validation

[0095]

[0096] The results are shown in Table 2. Antibody A was identified as having a high affinity for the whole antigen DQ-BSA (color development value of 1.7 when the herbicide concentration was 0). After adding diquat to the reaction system, the color development value decreased with increasing diquat concentration, indicating that diquat competitively binds to the site of antibody A against the whole antigen DQ-BSA, leading to a decrease in color development. However, the color development value did not change after adding other non-selective herbicides such as paraquat, glufosinate, and glyphosate to the reaction system, indicating that these herbicides do not affect the binding of antibody A to the whole antigen DQ-BSA, and antibody A does not recognize paraquat, glufosinate, or glyphosate. Therefore, antibody A can specifically recognize the non-selective herbicide diquat and is not affected by other non-selective herbicides such as paraquat.

[0097] Example 4: Antibody Stability Verification

[0098] ① Take 1 mL of antibody A and store it at 4℃ for 14 days and at 37℃ for 14 days respectively.

[0099] ② Dilute the whole antigen DQ-BSA to 1 μg / mL with coating buffer (0.05 M pH 9.5 carbonate and bicarbonate buffer), add 100 μL / well to the microplate, and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20 (wash buffer).

[0100] ③ Dilute antibody A stored at 4℃ for 14 days and antibody A accelerated at 37℃ for 14 days with diluent to 1000 ng / ml, 333 ng / ml, 111 ng / ml, 37 ng / ml, 12 ng / ml, 4 ng / ml and 1 ng / ml respectively. Add 100 μL / well to the microplate and add 100 μl of diluent as a blank control. Incubate at 37℃ for 30 minutes.

[0101] ③ After washing the plate three times with washing buffer, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing the plate three more times, add the colorimetric solution and incubate for about 5 minutes (depending on the strength of the reaction). Then add 2 M sulfuric acid to stop the reaction and read the OD450 absorbance value.

[0102] Table 3. Antibody stability verification

[0103]

[0104] The results are shown in Table 3. After 14 days of accelerated treatment at 37℃, the ELISA titer of antibody A was consistent with that of antibody A stored at 4℃ for 14 days, indicating that antibody A is relatively stable.

[0105] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. An antibody against dichlorvos, characterized in that, The antibody has the following complementarity-determining regions: a) Heavy chain variable region CDR1 as listed in SEQ ID NO:1; b) Heavy chain variable region CDR2 as listed in SEQ ID NO:2; c) Heavy chain variable region CDR3 as listed in SEQ ID NO:3; d) Light chain variable region CDR1 as listed in SEQ ID NO:4; e) Light chain variable region CDR2 as listed in SEQ ID NO:5; f) Light chain variable region CDR3 as listed in SEQ ID NO:

6.

2. The antibody as described in claim 1, characterized in that, The antibody specifically binds to compounds of formula I; Formula I.

3. The antibody as described in claim 1, characterized in that, The antibody does not bind to paraquat, glufosinate or glyphosate.

4. A test kit for detecting dichlorvos, characterized in that, The kit contains the antibody as described in any one of claims 1-3.

5. A conjugate comprising the antibody of any one of claims 1-3 covalently linked to a chemical or biological marker.

6. The use of the antibody according to any one of claims 1-3, the kit according to claim 4, or the conjugate according to claim 5 in the preparation of a product for detecting diquat.

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