A broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides and its application

Abstract

The present invention discloses a broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides and its application, relating to the field of biochemical technology. The antibody includes a heavy chain and a light chain. The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain are shown as SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively; the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain are shown as SEQ ID NO.8, SEQ ID NO.15, and SEQ ID NO.9, respectively. The present invention also provides applications of the antibody. The beneficial effects of the present invention are the disclosure of a monoclonal antibody against phenoxycarboxylic acid pesticides with high affinity and sensitivity, and the excellent detection sensitivity for phenoxycarboxylic acid pesticides under the condition of cooperating with the heterologous detection antigen MCPA-OVA.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and in particular to a broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides and applications thereof. Background Art

[0002] Phenoxycarboxylic acid (PCAs) pesticides, as first-generation selective herbicides, are widely used to eliminate broadleaf weeds in agriculture. Different PCAs have different substituents and positions on the benzene ring of the PCAs molecule, as well as the number of carbon atoms in the carboxylic acid, resulting in different types of PCAs. Currently, the two most widely used PCAs in my country are 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA). These two types are the primary products produced by domestic companies, accounting for over 80% of total PCAs production. To date, PCAs remain an important herbicide.

[0003] Due to the widespread use of PCAs, their residues can enter the human body through the food chain, posing a serious food safety hazard. Therefore, rapid screening and detection of PCAs is crucial. Currently, immunoassays based on specific antigen-antibody recognition and binding play a crucial role in the accurate and rapid detection of small molecule pesticides. Therefore, developing high-affinity, sensitive, broad-spectrum monoclonal antibodies against PCAs is crucial for the sensitive and accurate detection of PCAs.

[0004] At the same time, studies have shown that, in order to achieve better detection sensitivity, selecting suitable heterologous detection antigens—that is, antigens in which the hapten used in the detection antigen is different from the hapten used in the immunizing antigen (including heterogeneous haptens, heterogeneous linker sites, and heterogeneous linker arms)—can make the affinity between the detection antigen and the antibody slightly lower than the affinity between the natural hapten and the antibody, thereby improving the sensitivity of the immunoassay. Therefore, under the condition that the antibody remains unchanged, selecting suitable heterologous detection antigens is a feasible method to improve the performance of immunoassays for the target being tested. Summary of the Invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide a broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides and its application, so as to obtain a broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides with high affinity and sensitivity, especially having strong recognition ability for 2,4-D, 2,4-DB, MCPA and 2,4,5-T.

[0006] The technical solutions of the present invention are as follows:

[0007] The first aspect of the present invention provides a broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides, comprising a heavy chain and a light chain.

[0008] The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain are shown in SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, respectively;

[0009] The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID NO. 8, SEQ ID NO. 15 and SEQ ID NO. 9, respectively.

[0010] Optionally, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1.

[0011] Optionally, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0012] The second aspect of the present invention provides a gene encoding the broad-spectrum monoclonal antibody according to claim 1.

[0013] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 5 is shown in SEQ ID NO. 10;

[0014] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 6 is shown in SEQ ID NO. 11;

[0015] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 7 is shown in SEQ ID NO. 12;

[0016] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 8 is shown in SEQ ID NO. 13;

[0017] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 15 is shown in SEQ ID NO. 16;

[0018] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 9 is shown in SEQ ID NO. 14.

[0019] Optionally, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1 is shown in SEQ ID NO. 3.

[0020] Optionally, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2 is shown in SEQ ID NO. 4.

[0021] A third aspect of the present invention provides the use of the broad-spectrum monoclonal antibody or the gene in detecting phenoxycarboxylic acid pesticide residues.

[0022] Optionally, the broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides is used in an indirect competitive method with heterologous detection antigens to achieve rapid qualitative detection of PCAs pesticides in the sample to be tested.

[0023] Optionally, in the application of the above-mentioned broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides, the indirect competition method is enzyme-linked immunosorbent assay (ELSIA).

[0024] The fourth aspect of the present invention provides a detection reagent, a kit or a test strip for detecting phenoxycarboxylic acid pesticide residues, comprising the above-mentioned broad-spectrum monoclonal antibody.

[0025] A fifth aspect of the present invention provides a method for detecting phenoxycarboxylic acid pesticide residues, using the above-mentioned broad-spectrum monoclonal antibody.

[0026] Optionally, the broad-spectrum monoclonal antibody is used to detect phenoxycarboxylic acid pesticide residues in the sample to be tested by an indirect competitive method using a heterologous detection antigen;

[0027] Wherein, the heterologous detection antigen is MCPA-OVA.

[0028] The present invention has at least one of the following beneficial effects:

[0029] The broad-spectrum monoclonal antibodies against phenoxycarboxylic acid pesticides provided by the present invention have experimental results showing that the broad-spectrum monoclonal antibodies against phenoxycarboxylic acid pesticides have high affinity and sensitivity to 2,4-D, 2,4-DB, MCPA, and 2,4,5-T. The broad-spectrum monoclonal antibodies against phenoxycarboxylic acid pesticides can be used for immunological analysis of PCAs pesticide residues through an indirect competitive approach, achieving highly sensitive, accurate, and rapid detection of PCAs pesticide residues in food, which is of great significance for ensuring food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The UV characterization diagram of chemically synthesized immune antigen and heterologous detection antigen;

[0031] Figure 2 This is the SDS-PAGE electrophoresis diagram of the broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides;

[0032] Figure 3 This is a subtype identification diagram of a broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides;

[0033] Figure 4The standard curve of the indirect competitive ELISA based on a broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides. DETAILED DESCRIPTION

[0034] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0035] The present invention provides a broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides, comprising a heavy chain variable region amino acid sequence: EVQLQQAGPELVKPGASVKISCKASGYSFTGYNMHWVKQSHVKSLEWIGRINLSNGATDYNQSFRDKATLTVDKSSSTAYMDLHSLTSEDSAVYYCVDQGLFSAYWGQGTLVTVSA (SEQ ID NO. 1) and a light chain variable region amino acid sequence: DIVMTQTPSSLSASLGERVSLTCRASQDIGNRLHWLQQEPDGTIKRLIYATSNLDSGVPRRFSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPYTFGGGTKLEIK (SEQ ID NO. 2).

[0036] The above-mentioned broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides, wherein the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1 is: gaggtccagctgcaacaggctggacctgaactggtgaagcctggggcttcagtgaagatatcctgcaaggcttctggttactcattcactggctacaacatgcactgggtgaaacaaagccatgtgaagagccttgagtggattggacgtattaatctttctaatggtgctactgactacaaccagagtttcagggacaaggccaccttgactgtagataagtcctccagcacagcctacatggacctccacagcctgacatctgaagactctgcagtctattactgtgtagaccaaggactttttagcgcttactggggccaagggactctggtcacagtctctgca (SEQ ID NO. 3).

[0037] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2 is: gacattgtgatgacccagactccatcctccttatctgcctctctgggagaaagagtcagtctcacttgtcgggcaagtcaggacattggtaataggttacactggcttcagcaggaaccagatggaactattaaacgcctgatctacgccacatccaatttagattctggtgtccccagaaggttcagtggcagtaggtctgggtcagattattctctcaccatcagcagccttgagtctgaagattttgtagactattactgtctacaatatgctagttctccgtacacgttcggaggggggaccaagctggaaataaaa (SEQ ID NO. 4).

[0038] The above-mentioned broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain of the capture antibody are respectively:

[0039] CDR1: GYSFTGYN (SEQ ID NO.5);

[0040] CDR2: INLSNGAT (SEQ ID NO.6);

[0041] CDR3: VDQGLFSAY (SEQ ID NO.7);

[0042] The above-mentioned broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain of the capture antibody are respectively:

[0043] CDR1: QDIGNR (SEQ ID NO.8)

[0044] CDR2: ATS (SEQ ID NO. 15)

[0045] CDR3:LQYASSPYT (SEQ ID NO.9)

[0046] The above-mentioned broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides, wherein the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 5 is: ggttactcattcactggctacaac (SEQ ID NO. 10);

[0047] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 6 is: attaatctttctaatggtgctact (SEQ ID NO. 11);

[0048] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 7 is: gtagaccaaggactttttagcgcttac (SEQ ID NO. 12);

[0049] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 8 is: caggacattggtaatagg (SEQ ID NO. 13);

[0050] The nucleotide sequence encoding the amino acid sequence GTS is: gccacatcc (SEQ ID NO .16);

[0051] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 9 is: ctacaatatgctagttctccgtacacg (SEQ ID NO. 14);

[0052] The present invention also provides the application of the broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides, wherein the broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides is used in an indirect competitive method with heterologous detection antigens to achieve rapid qualitative detection of PCAs pesticides in a sample to be tested.

[0053] In the application of the above-mentioned broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides, the indirect competition method is enzyme-linked immunosorbent assay (ELSIA).

[0054] The present invention is further described in detail below with reference to specific examples, but the present invention is not limited to the following specific examples.

[0055] If no specific technology or scheme is specified in the examples, the technology or scheme described in the literature in this field is followed. The reagents or instruments used in the examples without indicating the manufacturer are all conventional products that can be purchased through regular channels.

[0056] Example 1 Synthesis of Immunizing Antigens and Heterologous Detection Antigens

[0057] 5 mM 5-aminovaleric acid powder and 5 mM 4-chlorophenoxyacetic acid powder were weighed and dissolved in 5 mL of tetrahydrofuran (v:v = 4:1). 200 μL of 2 M NaOH solution was then slowly added dropwise. After thorough stirring, the mixture was allowed to react at room temperature for 12 h. After completion of the reaction, the mixture was extracted twice with 5 mL of ethyl acetate. The extract was then washed with 5 mL of 0.1 M HCl, saturated NaHCO₃ solution, ultrapure water, and saturated NaCl solution, followed by drying over anhydrous NaSO₄. The resulting solution was rotary evaporated to remove the organic solvent to obtain the target hapten: 4-chlorophenoxyacetamide butyric acid.

[0058] The hapten was conjugated to the carrier protein using the activated ester method. 50 μM 4-chlorophenoxyacetamidobutyric acid, 60 μM N-hydroxysuccinimide, and 60 μM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were accurately weighed and dissolved in 600 μL of dimethylformamide. The mixture was reacted at room temperature for 8 h. After the reaction, the mixture was centrifuged at 5000 rpm for 10 min, the precipitate was removed, and the supernatant was the target activated ester, designated Solution 1. 20 mg of bovine serum albumin (BSA) was weighed and dissolved in 2 mL of carbonate buffer (pH 9.6), designated Solution 2. With stirring, 150 μL of Solution 1 was slowly added dropwise to Solution 2. The reaction was allowed to react at room temperature for 6 h. After the reaction, the reaction solution was placed in a dialysis bag and dialyzed against phosphate buffered saline (PBS) (pH 7.4) at 4°C for three days to obtain the immunogen 4-chlorophenoxyacetamidobutyric acid-BSA.

[0059] The synthesis method of the heterologous coating antigen is consistent with the above method, that is, 4-chlorophenoxyacetamide butyric acid is replaced by MCPA and BSA is replaced by ovalbumin (OVA), and the heterologous detection antigen MCPA-OVA can be obtained.

[0060] Example 2 Preparation of broad-spectrum monoclonal antibodies against phenoxycarboxylic acid pesticides

[0061] 1) Animal immunization:

[0062] After mixing equal volumes of the immunogen and adjuvant, the mixture was vortexed until the immunogen was completely emulsified, and immunization was performed by multiple subcutaneous injections in the abdomen. Each mouse was immunized four times, with the initial immunization using Freund's complete adjuvant and subsequent immunizations using Freund's incomplete adjuvant, with a 21-day interval between immunizations. The four immunization doses were 100 μg / mouse, 80 μg / mouse, 60 μg / mouse, and 40 μg / mouse, respectively, and immunization was performed by multiple subcutaneous injections. The booster dose was 40 μg / mouse, and immunization was performed by intraperitoneal injection.

[0063] 2) Preparation and screening of hybridoma cells

[0064] Selected immunized mice were sacrificed by cervical dislocation. The spleens were removed and ground in a clean bench. Mouse myeloma SP2 / 0 cells were mixed with spleen cells at a ratio of 1:10. Cell fusion was achieved by adding preheated 50% PEG and the cells were then added dropwise to a 96-well cell culture plate containing feeder cells. After fusion, the cells were observed microscopically. Semi-quantitative medium exchange was performed on the fifth day after fusion, and culture was continued until the seventh day, when full medium exchange was performed.

[0065] About 7-10 days after cell fusion, aspirate the cell supernatant from the cell culture plate and assay the antibody secretion in the supernatant using an indirect ELISA. Positive cells are screened for subcloning using limiting dilution. The test antigen is coated, with blank controls containing PBS, negative controls containing culture medium, and positive controls containing post-immunization mouse eye serum. When the positive rate of selected monoclonal cells reaches 100% in the 96-well cell culture plate, they are considered positive clones and can be cryopreserved and expanded as appropriate.

[0066] 3) Preparation, purification and identification of monoclonal antibodies

[0067] Eight-week-old Balb / c mice were pre-stimulated with liquid paraffin (0.5 mL / mouse) one week in advance to promote nutrient secretion and accumulation in the mouse peritoneal cavity. The screened monoclonal hybridoma cell line was expanded to the desired number, centrifuged at 1000 rpm for 7 minutes, carefully washed, resuspended in sterile 75% saline, and injected intraperitoneally into the mouse abdomen. Approximately one week after the mouse abdomen became noticeably distended, ascites was collected and centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected as purified ascites.

[0068] The collected ascites was purified using a Protein G affinity chromatography column. Prior to loading, the ascites was filtered through a 0.22 μm microporous membrane and an equal volume of equilibration buffer (0.15 M NaCl, 20 mM Na₂HPO₄, pH 7.4) was added as the loading fluid. The Protein G column was equilibrated with two column volumes of ddH₂O and then two column volumes of equilibration buffer. The loading fluid was applied to the Protein G column and adsorbed at 4°C for 10 minutes. The flow-through was collected and then reapplied to the Protein G column for 10 minutes at 4°C. This was repeated three times. After complete adsorption, the antibody was washed with equilibration buffer to remove nonspecific proteins and impurities. After washing, the antibody-rich eluate was eluted with elution buffer (0.1 M citric acid, pH 2.5-3.0). The eluate was acidic and should be immediately adjusted to pH 7.4 with neutralization buffer (1 M Tris-HCl, pH 8.5) to prevent antibody inactivation. Finally, the molecular weight and purity of the monoclonal antibody were determined by SDS-PAGE (refer to Figure 2 ), use the mouse monoclonal antibody Ig class / subclass identification enzyme-linked secondary antibody ready-to-use kit to determine the subtype of the monoclonal antibody (see Figure 3 ).

[0069] 4) Sequence determination of the heavy and light chain variable regions of monoclonal antibodies

[0070] The hybridoma cell line that can stably secrete MCPA monoclonal antibody was cultured to 1×10 6 Cells were plated at 1 mL / mL. The supernatant was discarded, 1 mL of Trizol reagent was added, and the tube was gently vortexed followed by 200 μL of chloroform. The tube was shaken end over end for 30 seconds and placed on ice for 10 minutes. The tube was centrifuged at 12,000 g for 15 minutes at 4°C. The supernatant was transferred to a new enzyme-free centrifuge tube, an equal volume of isopropanol was added, and the tube was placed on ice for 10 minutes. The tube was centrifuged at 12,000 g for 10 minutes at 4°C, the supernatant discarded, 1 mL of 75% ethanol was added, and the tube was centrifuged at 7,500 g for 5 minutes at 4°C. The tube was dried in a clean hood with low-speed ventilation for 5 minutes. 30–50 μL of enzyme-free sterile water was added and mixed to dissolve RNA. Reverse transcription was performed using 1 μg of total RNA as a template according to the instructions of the HifairIII 1st Strand cDNA Synthesis Kit. Upstream and downstream primers for the heavy and light chain variable regions of the monoclonal antibody were designed, respectively. The heavy chain upstream primer sequence was GGGGATATCCACCATGGRATGSAGCTGKGTMATSCTCTT; the heavy chain downstream primer sequence was CCGTTTACCAGGAGAGTGGGAGAG; the light chain upstream primer sequence was GGGGATATCCACCATGGAGACAGACACACTCCTGCTAT; and the light chain downstream primer sequence was GCCGCGGCCTGCAAAGACTCACTTTATTGA. Reverse-transcribed cDNA was amplified using PCR primers synthesized by Shanghai Sangon Biotechnology Co., Ltd. The heavy and light chain PCR reaction conditions were: 94°C for 5 minutes, 94°C for 10 seconds, 55°C for 20 seconds, and 72°C for 30 seconds, for a total of 25 cycles, followed by 72°C for 5 minutes. The amplified products were identified on 1% agarose gel and sequenced.

[0071] After sequencing, the monoclonal antibody has the heavy chain variable region amino acid sequence shown in SEQ ID NO. 1 and the light chain variable region amino acid sequence shown in SEQ ID NO. 2.

[0072] Example 3 Establishment of an indirect competitive ELISA standard curve based on a broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides

[0073] The heterologous detection antigen MCPA-OVA was diluted to 0.5 μg / mL with 1× PBS solution and added to a 96-well ELISA plate at 100 μL / well and coated at 37°C for 2 h. After coating, the liquid in the wells was discarded and the plate was washed three times with PBST. 5% skim milk was added to the ELISA plate wells at 300 μL / well and blocked at 37°C for 2 h. After blocking, the liquid in the wells was discarded and the plate was washed three times with PBST. PCAs pesticide standards were serially diluted from 10 μg / mL with 5% methanol-PBS solution at 50 μL / well, and 125 ng / mL of a broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides was added at 50 μL / well. At the same time, set up zero standard wells (replace the PCAs standard with 5% methanol-PBS, other conditions are the same) and blank control wells (replace the added antibody solution with PBS, other conditions are the same), and incubate at 37°C for 30 min; after the incubation, wash the plate three times with PBST, add HRP enzyme-labeled goat anti-mouse IgG antibody (1:5000), 100 μL / well, and incubate at 37°C for 30 min; after the incubation, discard the liquid in the wells, wash the plate three times with PBST, add TMB color development solution, 100 μL / well, react at 37°C in the dark for 7 min, and then add 2 M sulfuric acid, 50 μL / well to terminate the reaction; finally, quickly place the microplate in a microplate reader and measure its OD value at 450 / 630 nm wavelength.

[0074] See also Figure 4 The above experimental results show that the broad-spectrum monoclonal antibody against phenoxycarboxylic acid has high sensitivity to 2,4-D, 2,4-DB, MCPA and 2,4,5-T, among which the half-maximal inhibitory concentration (IC 50 ) was 34.17 ng / mL, IC 10 The IC value for 2,4-DB is 6.98 ng / mL. 50 is 200.5 ng / mL, IC 10 is 12.44 ng / mL; the IC of MCPA 50 was 45.29 ng / mL, IC 10 The IC value for 2,4,5-T was 2.74 ng / mL. 50 was 286.3 ng / mL, IC 10 The IC value for 2,4-DP is 25.79 ng / mL. 50 is 3630 ng / mL; the IC 50 It is 1193 ng / mL.

[0075] The present invention prepares broad-spectrum monoclonal antibodies against PCAs, which exhibit strong binding to some PCAs pesticides. This allows for the development of immunoassays based on these monoclonal antibodies against PCAs pesticides, which are widely applicable to the detection of various PCAs pesticide residues and provide opportunities for the development of detection kits for multiple PCAs pesticide residues.

[0076] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A broad-spectrum monoclonal antibody against phenoxycarboxylic acid pesticides, comprising a heavy chain and a light chain, characterized in that: The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain are shown in SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, respectively; The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID NO. 8, SEQ ID NO. 15 and SEQ ID NO. 9, respectively.

2. The broad-spectrum monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

1.

3. The broad-spectrum monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

4. The gene encoding the broad-spectrum monoclonal antibody according to any one of claims 1 to 3, characterized in that: The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 5 is shown in SEQ ID NO. 10; The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 6 is shown in SEQ ID NO. 11; The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 7 is shown in SEQ ID NO. 12; The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 8 is shown in SEQ ID NO. 13; The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 15 is shown in SEQ ID NO. 16; The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 9 is shown in SEQ ID NO.

14.

5. The gene according to claim 4, characterized in that The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1 is shown in SEQ ID NO.

3.

6. The gene according to claim 4, characterized in that The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2 is shown in SEQ ID NO.

4.

7. Use of the broad-spectrum monoclonal antibody according to any one of claims 1 to 3 or the gene according to any one of claims 4 to 6 in detecting phenoxycarboxylic acid pesticide residues, characterized in that: The phenoxycarboxylic acid pesticide is at least one of 2,4-D, 2,4-DB, MCPA and 2,4,5-T.

8. A detection reagent, kit or test strip for detecting phenoxycarboxylic acid pesticide residues, characterized in that: The invention comprises the broad-spectrum monoclonal antibody according to any one of claims 1 to 3.

9. A method for detecting phenoxycarboxylic acid pesticide residues, characterized in that: The broad-spectrum monoclonal antibody according to any one of claims 1 to 3 is used for detection, wherein the phenoxycarboxylic acid pesticide is at least one of 2,4-D, 2,4-DB, MCPA and 2,4,5-T.

10. The detection method according to claim 9, characterized in that: The broad-spectrum monoclonal antibody is used to detect phenoxycarboxylic acid pesticide residues in the sample to be tested by an indirect competitive method using a heterologous detection antigen; Wherein, the heterologous detection antigen is MCPA-OVA.

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