Uniconazole monoclonal antibody and application thereof

Abstract

The invention discloses a uniconazole monoclonal antibody and application thereof, and relates to the technical field of biochemistry. The amino acid sequences of complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody are respectively as shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7; the amino acid sequences of complementary determining regions CDR1, CDR2 and CDR3 of the light chain are respectively shown as SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10; the invention also provides a gene for coding the amino acid sequence, and a uniconazole hapten and a uniconazole antigen which are used for preparing the monoclonal antibody. The invention also provides application of the antibody in detection of uniconazole. The uniconazole monoclonal antibody disclosed by the invention has the beneficial effects that the uniconazole monoclonal antibody with high sensitivity and high specificity is disclosed, and the uniconazole monoclonal antibody has relatively excellent detection sensitivity and specificity on uniconazole.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and in particular relates to a uniconazole monoclonal antibody and an application thereof. Background Art

[0002] Uniconazole belongs to the triazole class of compounds and is a plant growth regulator. It has the functions of sterilization and weed control, lodging resistance, and improving drought tolerance and crop yield. It is commonly used in crops such as rice, wheat, soybeans, peanuts, and rapeseed. It is reported that uniconazole has certain side effects on the endocrine system of humans and animals, has the risk of teratogenicity, and may even cause mutation and poisoning of organs in the body. Therefore, the safety of the use of uniconazole has received widespread attention. Many countries have successively formulated the residue limit standards for uniconazole. For example, Japan stipulates that the residue limit requirement for uniconazole in some fruits and vegetables is 0.05-0.5 mg / kg; my country's food safety standard "GB2763-2021" stipulates that the maximum residue of uniconazole in brown rice is 0.1 mg / kg, and the maximum residue of uniconazole in wheat, soybeans, peanut kernels, and rapeseed is 0.05 mg / kg, and the daily allowable intake is 0.02 mg / kg.

[0003] At present, the methods for detecting chloramphenicol at home and abroad mainly include instrumental analysis methods such as gas chromatography, liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. These methods are cumbersome and complicated, time-consuming and labor-intensive, have high requirements for detection personnel, and are not suitable for on-site detection. The immunoassay is a detection method based on the specific binding mechanism between antigens and antibodies. This method has the advantages of rapid operation, high sensitivity, and strong specificity. It has been widely used in the detection of harmful substances such as pesticide residues and mycotoxins. Summary of the invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art, to provide a clofosconazole monoclonal antibody and its application, and specifically to provide a clofosconazole monoclonal antibody, an encoding gene, a clofosconazole hapten, a clofosconazole antigen and its application. The clofosconazole monoclonal antibody of the present invention has high sensitivity and specificity to clofosconazole, and can be used for immunological analysis of clofosconazole pesticide residues by indirect competition.

[0005] The technical solution of the present invention is as follows: The first aspect of the present invention provides a uniconazole monoclonal antibody, wherein the uniconazole monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region 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 variable region are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively.

[0006] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.1.

[0007] Preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID No.2.

[0008] The second aspect of the present invention provides a gene encoding the uniconazole monoclonal antibody, wherein: The nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 are shown in SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13, respectively; The nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 are shown in SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, respectively.

[0009] Preferably, the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; The third aspect of the present invention provides a hapten for preparing the hapten monoclonal antibody, wherein the hapten has the following structural formula: .

[0010] The fourth aspect of the present invention provides a method for preparing a hapten of uniconazole, comprising the following steps: S1, reacting 1,2,4-1H-triazole with 5-bromo-3,3-dimethyl-4-oxopentanoic acid to obtain compound 1 after purification; S2, reacting the compound 1 with acetic anhydride, potassium carbonate and 4-chlorobenzaldehyde, and purifying to obtain the uniconazole hapten; Wherein, the compound 1 has the following structural formula: .

[0011] Preferably, the addition ratio of the 1,2,4-1H-triazole and 5-bromo-3,3-dimethyl-4-oxopentanoic acid is 1.5 g to 2.5 g: 4 mL to 6 mL; The addition ratio of the compound 1, acetic anhydride, potassium carbonate and 4-chlorobenzaldehyde is 0.5 g~1.5 g: 4 mL~6 mL: 1.5 g~2.5 g: 0.6 g~1.0 g.

[0012] The fifth aspect of the present invention provides a uniconazole antigen for preparing the uniconazole monoclonal antibody, wherein the uniconazole antigen has the following structural formula: ; Wherein, the BSA is bovine serum albumin, and the OVA is ovalbumin.

[0013] The sixth aspect of the present invention provides the use of the uniconazole monoclonal antibody or the gene or the uniconazole hapten or the uniconazole antigen in detecting uniconazole.

[0014] Preferably, the uniconazole monoclonal antibody is used to detect the uniconazole content in the sample to be tested by an indirect competitive ELISA method.

[0015] The present invention has at least one of the following beneficial effects: The invention firstly prepares a nifeconazole hapten by chemical synthesis; then obtains a nifeconazole antigen by coupling a carrier protein to the nifeconazole hapten; and finally prepares a nifeconazole monoclonal antibody by immunizing an animal with the nifeconazole antigen.

[0016] The experimental results show that the chloranil monoclonal antibody provided by the present invention has high sensitivity and specificity to chloranil, and the chloranil monoclonal antibody can be used for immunological analysis of chloranil pesticide residues by indirect competition, thereby achieving highly sensitive, accurate and rapid detection of chloranil residues in food, which is of great significance for ensuring food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG1 is a synthetic route diagram of the chemically synthesized immune antigen and detection antigen in Example 1; FIG2 is an SDS-PAGE electrophoretogram of the uniconazole monoclonal antibody of Example 2; FIG. 3 is a standard curve diagram of the indirect competitive ELISA based on the uniconazole monoclonal antibody in Example 3. DETAILED DESCRIPTION

[0018] 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 in conjunction with 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 used to limit the present invention.

[0019] Example 1: Preparation of artificial antigen of oxadiazole Add 5 g of potassium carbonate to 20 mL of acetone and stir at room temperature. Add 2 g of 1,2,4-1H-triazole to the solution and stir for 30 minutes. Add 5 mL of cold 5-bromo-3,3-dimethyl-4-oxopentanoic acid to the mixture and stir at room temperature for 2.5 h, then add 10 mL of water to terminate the reaction, and then add 70 mL of water to dissolve potassium carbonate. After removing acetone by rotary evaporation, acidify with hydrochloric acid, extract with ethyl acetate (60 mL × 3), wash the organic layer with saturated saline solution (15 mL × 3), and add anhydrous sodium sulfate to dry. Filter and concentrate the obtained product under reduced pressure, and purify it by silica gel column chromatography with hexane-ethyl acetate (7:3) to obtain compound 1.

[0020] Add 5 mL of acetic anhydride to 1 g of compound 1, add 2 g of potassium carbonate and 0.8 g of ‌4-chlorobenzaldehyde and stir at room temperature for 3.5 h. Add 20 mL of water to terminate the reaction, acidify with hydrochloric acid, and then add 10 mL of ethyl acetate to dissolve the product. The organic layer is extracted with ethyl acetate (2 mL×3), washed with water (20 mL×3), and then dried over anhydrous sodium sulfate. After vacuum concentration, purify by silica gel column chromatography with hexane-ethyl acetate (4:1), dissolve the purified substance in 10 mL of ethyl acetate, and irradiate with 365 nm ultraviolet light for 5 hours. The reaction solution is vacuum concentrated to obtain compound 2, i.e., the hapten of oxadiazole. 100 μL 10 mg / mL oxadiazole hapten (DMF solution) was added with 1 mg EDC and 2 mg NHS, and activated at room temperature for 8 hours to obtain the activated hapten. The carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) were prepared with PBS to 3.68 mg / mL and 2.5 mg / mL, respectively. 1.5 mL of the carrier protein solution was added to the above activated hapten, stirred at 4 ° C for 12 hours, and then dialyzed with PBS for 3 days to obtain the immune antigen oxadiazole-BSA and the detection antigen oxadiazole-OVA.

[0021] Example 2: Preparation of monoclonal antibody against cloxacillin 1. Animal immunization Three female Balb / c mice aged 6-8 weeks were selected, and the same volume of uniconazole-BSA was mixed with Freund's adjuvant as the immunogen, vortexed until the immunogen was completely emulsified, and the mice were immunized by multiple subcutaneous injections in the abdomen. Freund's complete adjuvant was used for the first immunization, and the immunogen dose was 120μg / mouse; Freund's incomplete adjuvant was used for subsequent immunization, and the immunogen dose was gradually reduced, with immunization once every 3 weeks, for a total of 3 immunizations. One week after the second immunization, the tail blood of the mice was collected, and the serum antibody titer and inhibition rate in the tail blood were determined by indirect ELISA. The immunogen was directly used to boost the highest titer mice three days before fusion, and the immunization dose was 40μg / mouse.

[0022] 2. Preparation and screening of hybridoma cells The selected immune mice were killed by pulling the neck, and the spleen was taken out and ground on the clean bench. Mouse myeloma cells SP2 / 0 and spleen cells were mixed at a ratio of 1:10, and preheated 50% PEG was added for cell fusion, and then added dropwise to a 96-well cell culture plate with feeder cells. After fusion, the cell state was observed under a microscope, and semi-quantitative medium was replaced on the 5th day after fusion, and the culture was continued until the 7th day, after which the full medium was replaced.

[0023] About 7 to 10 days after cell fusion, the cell supernatant in the cell culture plate was aspirated, and the secretion of antibodies in the cell supernatant was determined by indirect ELISA, and the positive cell plate wells were screened, and subcloning was performed by limiting dilution. The cells were coated with uniconazole-OVA, the blank control wells were PBS, the negative control was the culture medium, and the positive control was the eye blood serum of the immunized mouse. When the positive rate of the selected monoclonal cells in the 96-well cell culture plate reached 100%, it was determined to be a positive monoclonal cell, and it was frozen and expanded in time.

[0024] 3. Preparation and purification of monoclonal antibody ascites Balb / c mice of about 8 weeks old were injected with liquid paraffin one week in advance for pre-stimulation to promote the secretion and accumulation of nutrients in the mouse peritoneal cavity. The obtained hybridoma cell line was expanded to the required number, centrifuged, carefully washed, resuspended with sterile 75% saline, and injected into the abdomen of the mouse by intraperitoneal injection. After the abdomen of the mouse was significantly swollen in about one week, ascites was collected, centrifuged at 10000r / min for 10 minutes, and the supernatant was collected as monoclonal antibody ascites.

[0025] The collected ascites was purified using a Protein G affinity chromatography column. Before loading, the ascites was filtered through a microporous membrane and used as the loading solution. The Protein G column was equilibrated with a binding buffer (0.15M NaCl, 20mM Na2HPO4, pH7.4). The filtered ascites was loaded onto the chromatography column. After equilibration, the elution buffer (0.1M citric acid, pH2.5~3.0) was used for elution, and the antibody-rich eluate was collected. The eluate was acidic, and the pH should be immediately adjusted to neutral using a neutralization buffer (1M Tris-HCl, pH9.0) to prevent the inactivation of the antibody. SDS-PAGE electrophoresis was then used for identification to obtain the clopidogrel monoclonal antibody. Please refer to Figure 2 ,Depend on Figure 2 It can be seen that the band size of the oxadiazole monoclonal antibody is about 160 kDa (non-reduced), and the band sizes of the heavy chain and light chain are 50 kDa and 22 kDa (reduced), respectively. The band size is close to the molecular weight, which is in line with expectations.

[0026] 4. Determination of the heavy and light chain variable regions of the monoclonal antibody against clonostatin The hybridoma cell line that can stably secrete the monoclonal antibody of clofosinate was cultured to 1×10 6 Cells / mL, discard the supernatant of cell waste liquid, add 1mL Trizol reagent, shake gently, and then lyse in an ice bath for 10min, add 200 μL chloroform, shake upside down for 30s, and place on ice for 10min; centrifuge at 4℃ and 12000g for 15min, aspirate the supernatant into a new centrifuge tube, add an equal volume of pre-cooled isopropanol, gently turn upside down, and place on ice for 10min; centrifuge at 4℃ and 12000g for 10min, discard the supernatant, add 1mL 75% ethanol, turn upside down to suspend the precipitate in ethanol, let it stand for 5min, centrifuge at 4℃ and 12000g for 5min, discard the supernatant, dry it in a clean bench for ventilation for 10min, add 100μL enzyme-free sterile water to resuspend the precipitate, and obtain total RNA. Using 5μg total RNA as a template, reverse transcription was performed according to the instructions of the HifairIII1st Strand cDNA Synthesis Kit. The upstream and downstream primers of the heavy and light chain variable regions of the monoclonal antibody were designed respectively: ggggatatccaccatgracttcgggytgagctkggtttt (upstream primer of heavy chain), ctttacccggagaccgggagatggtcT (downstream primer of heavy chain), gacattswgatgacmcagtctcca (upstream primer of light chain), gccgcggcctgcaaagactcactttattga (downstream primer of light chain). The cDNA obtained by reverse transcription was amplified, and the reaction parameters were: 94℃ 5min, 94℃ 10s, 60℃ 20s, 72℃ 30s, a total of 25 cycles, and then 72℃ 5min. The obtained amplification products were identified by 1% agarose gel, and the obtained products were sequenced.

[0027] After sequencing, the encoding V HNucleotide sequence: caggtccaactacagcagcctggggctgaactggtgaggcctggggcttcagtgaagttgtcctgcaaggcttctggctacaccttcaccgtctactatatctactgggtgaaacagaggcctggacaaggccttgagtggattggggggattcatcctagaaacggtggttcttattacaatgcgaagttcaggaacagggccacactgactgtagacaaatcctccaacacagcctacatgcaattcagcagcctgacatctgaggactctgcggtctattactgtacaagagggatttactacgatggtaaattcggagagcgtgctatggactactggggtcaaggaacctctgtcaccgtctcctca (SEQ ID No. 3); Encoding V L Nucleotide sequence: gacattgtgatgacccagactccaaacactttgtctgttaccattggacagccagcttccatttcttgcaagtcaagtcagagcctcttatatagtgatggaaaaacctatttgcattggttattacagagtccaggccagtctccaaagctcctaatctatctggtgtctaaactggaatctggagtccctgacagattcagtggcagtggatcagggacagattttacactgaaaatcagcagagtggaggctgaggatttgggggtttattactgcgtgcaagctgcacatctcccccatacgttcgggtcggggaccaagctggaaataaaa (SEQ ID No. 4).

[0028] The nucleotide sequence encoding the amino acid shown in SEQ ID No.5 is: ggctacaccttcaccgtctactata (SEQ ID No.11); the nucleotide sequence encoding the amino acid shown in SEQ ID No.6 is: attcatcctagaaacggtggttct (SEQ ID No.12); the nucleotide sequence encoding the amino acid shown in SEQ ID No.7 is: acaagagggatttactacgatggtaaattcggagagcgtgctatggactac (SEQ ID No.13); The nucleotide sequence encoding the amino acid shown in SEQ ID No.8 is: cagagcctcttatatagtgatggaaaaacctat (SEQ ID No.14); the nucleotide sequence encoding the amino acid shown in SEQ ID No.9 is: ctggtgtct (SEQ ID No.15); the nucleotide sequence encoding the amino acid shown in SEQ ID No.10 is: gtgcaagctgcacatctcccccatacg (SEQ ID No.16).

[0029] The amino acid sequence obtained by translating the nucleotide sequence using Snapgene, wherein the uniconazole monoclonal antibody contains a V H The heavy chain variable region and the name V L The light chain variable region of V H Amino acid sequence: QVQLQQPGAELVRPGASVKLSCKASGYTFTVYYIYWVKQRPGQGLEWIGGIHPRNGGSYYNAKFRNRATLTVDKSSNTAYMQFSSLTSEDSAVYYCTRGIYYDGKFGERAMDYWGQGTSVTVSS (SEQID No. 1); V L Amino acid sequence: DIVMTQTPNTLSVTIGQPASISCKSSQSLLYSDGKTYLHWLLQSPGQSPKLLIYLVSKLESGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCVQAAHLPHTFGSGTKLEIK (SEQ ID No. 2); V H and V L They are all composed of complementary determining regions and framework regions, and the complementary determining regions are all composed of CDR1, CDR2 and CDR3; V HThe amino acid sequence of CDR1: GYTFTVYY (SEQ ID No. 5); V H The amino acid sequence of CDR2: IHPRNGGS (SEQ ID No. 6); V H The amino acid sequence of CDR3: TRGIYYDGKFGERAMDY (SEQ ID No. 7); V L The amino acid sequence of CDR1: QSLLYSDGKTY (SEQ ID No. 8); V L The amino acid sequence of CDR2: LVS (SEQ ID No. 9); V L The amino acid sequence of CDR3: VQAAHLPHT (SEQ ID No.10).

[0030] Example 3: Indirect competitive ELISA for determination of uniconazole The detection antigen OVA was diluted to 1 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, the plate was washed 3 times with PBST, 5% skim milk was added to the wells of the ELISA plate at 300 μL / well, and blocked at 37°C for 2 h. After blocking, the liquid in the wells was discarded, the plate was washed 3 times with PBST, the OVA pesticide standard was graded diluted from 100 ng / mL with 5% methanol-PBS solution at 50 μL / well, and 100 ng / mL of OVA monoclonal antibody was added at 50 μL / well. At the same time, set up zero standard wells (replace the uniconazole standard with 5% methanol-PBS, and other conditions are the same) and blank control wells (replace the added antibody solution with PBS, and other conditions are the same), and incubate at 37°C for 30 min; after the incubation, wash the plate 3 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 3 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 to terminate the reaction, 50 μL / well; finally, quickly place the ELISA plate in an ELISA reader and measure its OD value at a wavelength of 450 / 630 nm.

[0031] See also Figure 3 The above ELISA experimental results show that its half inhibitory concentration (IC 50) is 4.1 ng / mL, and the linear range is 0.78-25 ng / mL. The oxadiazole monoclonal antibody has high sensitivity and a wide linear range for oxadiazole detection.

[0032] Example 4: Specific response based on monoclonal antibodies Based on the monoclonal antibody prepared in Example 2 of the present invention, the present invention selected structural analogs and functional analogs of uniconazole (see Table 1) and diluted them in a gradient manner with 5% methanol-PBS, and conducted indirect competitive ELISA detection with the monoclonal antibody to establish a standard curve to obtain IC 50 , and calculate the cross-reaction rate CR (%) according to the formula = IC 50 (Analyte) / IC 50 (analog)*100%.

[0033] Table 1 Cross-reaction rate The results of the indirect competitive ELISA cross-reaction experiment on the analogs of uniconazole are shown in Table 1. From the results shown in Table 1, it can be seen that the present invention utilizes Figure 1 The cross-reaction rates of the monoclonal antibodies prepared from the hapten and the structural and functional analogs of uniconazole were less than 0.8%, indicating that the uniconazole monoclonal antibodies obtained can recognize uniconazole with high specificity.

[0034] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A monoclonal antibody to clonal antibody, characterized in that: The uniconazole monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region 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 variable region are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively.

2. The uniconazole 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, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

3. The gene encoding the uniconazole monoclonal antibody according to any one of claims 1 to 2, characterized in that: The nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 are shown in SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13, respectively; The nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 are shown in SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, respectively.

4. The gene according to claim 3, characterized in that The nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

5. A hapten of uniconazole, characterized in that: Used for preparing the uniconazole monoclonal antibody according to any one of claims 1 to 2, wherein the uniconazole hapten has the following structural formula: 。 6. The method for preparing the uniconazole hapten according to claim 5, characterized in that: The steps include: S1, reacting 1,2,4-1H-triazole with 5-bromo-3,3-dimethyl-4-oxopentanoic acid to obtain compound 1 after purification; S2, reacting the compound 1 with acetic anhydride, potassium carbonate and 4-chlorobenzaldehyde, and purifying to obtain the uniconazole hapten; Wherein, the compound 1 has the following structural formula: 。 7. The preparation method according to claim 6, characterized in that: The addition ratio of the 1,2,4-1H-triazole and 5-bromo-3,3-dimethyl-4-oxopentanoic acid is 1.5 g to 2.5 g: 4 mL to 6 mL; The addition ratio of the compound 1, acetic anhydride, potassium carbonate and 4-chlorobenzaldehyde is 0.5 g~1.5 g: 4 mL~6 mL: 1.5 g~2.5 g: 0.6 g~1.0 g.

8. A uniconazole antigen, characterized in that Used for preparing the uniconazole monoclonal antibody according to any one of claims 1 to 2, wherein the uniconazole antigen has the following structural formula: ； Wherein, the BSA is bovine serum albumin, and the OVA is ovalbumin.

9. Use of the uniconazole monoclonal antibody according to any one of claims 1 to 2, or the gene according to any one of claims 4 to 5, or the uniconazole hapten according to claim 5, or the uniconazole antigen according to claim 8 in detecting uniconazole.

10. The use according to claim 9, characterized in that: The indirect competitive ELISA method is used to detect the content of uniconazole in the sample to be tested.

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