Amitraz hapten, amitraz antigen, amitraz antibody, amitraz detection device and preparation and application thereof

Abstract

The invention discloses amitraz hapten, antigen, antibody and detection device as well as preparation and application thereof, and relates to amitraz hapten, antigen, antibody and colloidal gold detection device as well as preparation and application thereof in amitraz detection. The invention provides a method for preparing an artificial antigen from amitraz hapten synthesis and antigen coupling carrier protein, and a specific amitraz antibody generated by the body of an immune experimental animal after the immune experimental animal, the IC50 value of the antibody is 5.22 ng / mL, the linear detection range of amitraz is 1.82-14.96 ng / mL, and the detection result shows that the antibody can be used for detecting amitraz. And the amitraz composition accords with the limit regulation of amitraz in the national standard GB 2763-2021 National Food Safety Standard Maximum Pesticide Residue Limit in Food. Therefore, the method can be applied to rapid detection of the residual content of amitraz in agricultural products.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection. More specifically, the present invention relates to an amitraz hapten, antigen, antibody, detection device and their preparation and application. Background Art

[0002] Amitraz is a broad-spectrum formamidine insecticide and acaricide, which is toxic to humans and livestock to a certain extent. It can inhibit the human central nervous system, and there is no specific drug for poisoning. Its main hydrolysis metabolite, 2,4-dimethylaniline, is highly toxic and has various potential hazards to the human body such as carcinogenesis and mutagenesis. At present, the commonly used detection and analysis methods for amitraz include gas chromatography, gas chromatography-mass spectrometry, liquid chromatography, and liquid chromatography-mass spectrometry. The current determination of amitraz in fruits and vegetables uses the national standard method of "GB / T 5009.143-2003". This method is a gas chromatography method that uses a packed column for chromatographic separation. The pretreatment requires a reflux device for hydrolysis, and the hydrolysis product has to be derivatized before determination. The method is seriously lagging behind, bringing many inconveniences and troubles to the detection work. Moreover, these methods need to repeatedly adjust the acid and repeatedly extract in the sample pretreatment, and the process is complex, affecting the detection accuracy of the results. The high-performance liquid chromatography method also has certain defects. Amitraz is extremely easy to hydrolyze during the determination process, making the determination result inaccurate.

[0003] In recent years, the research on immunoassay technology in the analysis of pesticide and veterinary drug residues has increased, but there is no immunoassay method for amitraz. According to the characteristics of amitraz being intolerant to acid and alkali, etc., the present invention designs and develops a new amitraz hapten by using a new synthesis method, prepares an antibody with high sensitivity, and thus establishes a corresponding rapid detection method for amitraz, realizing the rapid detection of residual amitraz in fruits, vegetables and animal tissues by immunological methods. Summary of the Invention

[0004] The purpose of the present invention is to provide an amitraz hapten, antigen, antibody, detection device and their preparation and application.

[0005] According to one aspect of the present invention, there is provided an amitraz hapten, whose structure is shown in formula (Ⅰ): , formula (Ⅰ).

[0006] According to another aspect of the present invention, there is provided a method for preparing an amitraz hapten, comprising the following steps: S1, dissolving 2-(3-methyl-4-nitrophenyl)acetic acid and 2-(trimethylsilyl)ethanol in N,N-dimethylformamide, and carrying out an esterification reaction under the action of a condensing agent EDC.HCl and a catalyst imidazole to obtain an intermediate 1 with an arm, and the structural formula of the intermediate 1 is shown in reaction formula (Ⅱ): , formula (II); S2, dissolve Intermediate 1 in ethanol, add hydrazine hydrate, palladium carbon, and ferric chloride hexahydrate, and carry out a nitro reduction reaction under heating at 65°C to obtain Intermediate 2. The structural formula of Intermediate 2 is as shown in Reaction Formula (III): , formula (III); S3, dissolve Intermediate 2, cyanamide, and triethyl orthoformate in toluene, and react at 80°C for 2 h to obtain Intermediate 3. The structural formula of Intermediate 3 is as shown in Reaction Formula (IV): , formula (IV); S4, dissolve Intermediate 3 in tetrahydrofuran, add triethylamine, methylamine hydrochloride, and distilled water, and react at room temperature for 3 h to obtain Intermediate 4. The structural formula of Intermediate 4 is as shown in Reaction Formula (V): , formula (V); S5, dissolve Intermediate 4 and 2,4-dimethylphenyl isocyanide in toluene, add cuprous oxide as a catalyst, and reflux for 3 h to obtain Intermediate 5. The structural formula of Intermediate 5 is as shown in Reaction Formula (VI): , formula (VI); S6, dissolve Intermediate 5 in anhydrous tetrahydrofuran, add tetrabutylammonium fluoride under stirring, and react at room temperature to obtain the amitraz hapten with the structural formula as shown in formula (I). The reaction of this step is shown in Reaction Formula (VII): , formula (VII).

[0007] According to another aspect of the present invention, there is provided an amitraz antigen which is a conjugate of the amitraz hapten as claimed in claim 1 and a carrier protein, and the carrier protein is bovine serum albumin, human serum albumin, chicken ovalbumin or hemocyanin.

[0008] According to the fourth aspect of the present invention, there is provided the use of the amitraz hapten or amitraz antigen in the immunological detection of amitraz for non-disease diagnosis.

[0009] According to the fifth aspect of the present invention, there is provided an amitraz antibody which is prepared by immunizing an animal with the amitraz antigen, and the amitraz antibody is an amitraz monoclonal antibody.

[0010] According to the sixth aspect of the present invention, there is provided the use of the amitraz antibody in the immunological detection of amitraz for non-disease diagnosis.

[0011] According to the seventh aspect of the present invention, there is provided a amitraz colloidal gold chromatography detection device, comprising a test strip and a reaction cup. The test strip includes a reaction membrane, on which a detection area and a quality control area are provided. The detection area is coated with amitraz antigen, and the reaction cup contains colloidal gold-labeled amitraz antibody.

[0012] According to the eighth aspect of the present invention, there is provided a method for detecting amitraz in a sample. This method uses an amitraz colloidal gold chromatography detection device to detect amitraz in the sample, and the sample is an agricultural product.

[0013] Advantages of the present invention: The preparation method of the amitraz hapten provided by the present invention uses chemical reagents that are easily obtained, has a simple operation process, concise and effective synthesis steps, a relatively high reaction yield, and a low detection cost. The present invention provides an artificial antigen prepared by conjugating the amitraz hapten with a carrier protein, and the specific antibody against amitraz produced in the body after immunizing experimental animals has a high titer. For establishing an ELISA standard curve for the amitraz antibody, its IC 50 value is 5.22 ng / mL, and the linear detection range is 1.82 - 14.96 ng / mL, which meets the limit regulations of amitraz in the newly promulgated national standard GB 2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Foods". The present invention utilizes the principle of chromatographic immune colloidal gold, and qualitatively detects the content of amitraz in the sample by color comparison between the detection line and the quality control line in the test strip, without the need to use large-scale instruments such as liquid chromatography or mass spectrometry, achieving the purpose of rapid detection, and having the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, long shelf life, etc. Therefore, the present invention can be applied to the rapid detection of amitraz residue content in agricultural products. Description of the Drawings

[0014] Figure 1 It is the mass spectrum of the amitraz hapten of an embodiment of the present invention.

[0015] Figure 2 It is the ELISA standard curve based on the amitraz antibody of an embodiment of the present invention. Detailed Description of the Invention

[0016] The present invention is further described in detail through specific implementation cases. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art all fall within the scope defined by the appended claims of this application. Without special instructions, all raw materials and reagents of the present invention are raw materials and reagents that can be purchased in the conventional market.

[0017] Example 1 Synthesis and Identification of Amitraz Hapten Preparation method of amitraz hapten, comprising the following steps: S1, Weigh 4.0 g of 2-(3-methyl-4-nitrophenyl)acetic acid and 2.5 g of 2-(trimethylsilyl)ethanol, dissolve them in 30 mL of DMF, add 5.0 g of condensing agent EDC.HCl and 0.3 g of catalyst imidazole, stir and react overnight at room temperature. After the reaction is completed, add 150 ml of distilled water, extract twice with ethyl acetate and combine. After drying over anhydrous sodium sulfate, evaporate to dryness, and purify by column chromatography to obtain 6.1 g of intermediate 1 with an arm; S2, Take 5.0 g of intermediate 1 and dissolve it in 150 mL of ethanol, add 2.3 g of hydrazine hydrate, 0.5 g of palladium carbon, and 0.5 g of ferric chloride hexahydrate, react at 65 °C for 45 min, filter the precipitate, evaporate the ethanol phase to dryness, dilute with water, extract twice with ethyl acetate, then combine the ethyl acetate. After drying the ethyl acetate phase over anhydrous sodium sulfate, evaporate to dryness, and purify by column chromatography to obtain 3.8 g of intermediate 2; S3, Weigh 2.6 g of intermediate 2, 0.45 g of cyanamide and 1.6 g of triethyl orthoformate, dissolve them in 20 ml of toluene, react at 80 °C for 2 h, evaporate the solvent and directly purify by column chromatography to obtain 2.9 g of intermediate 3; S4, Dissolve 2.5 g of intermediate 3 in 30 mL of tetrahydrofuran, add 2 mL of triethylamine, 0.6 g of methylamine hydrochloride and 5 mL of distilled water, react at room temperature for 3 h, distill out the solvent, add 30 mL of water, extract twice with ethyl acetate, then combine and dry over anhydrous sodium sulfate and evaporate the solvent to obtain 2.2 g of intermediate 4, which can be directly used for the next reaction without purification; S5, Dissolve 2.0 g of intermediate 4 and 0.85 g of 2,4-dimethylphenyl isocyanide in 20 mL of toluene, add 0.1 g of cuprous oxide as a catalyst, reflux and react for 3 h, directly evaporate the solvent and purify by column chromatography to obtain 2.4 g of intermediate 5; S6, Dissolve 1.2 g of intermediate 5 in 30 mL of anhydrous tetrahydrofuran, add 2.5 g of tetrabutylammonium fluoride under stirring, react at room temperature for 7 h, evaporate to dryness, dilute with water, extract twice with ethyl acetate, then combine the ethyl acetate. After drying the ethyl acetate phase over anhydrous sodium sulfate, evaporate to dryness, and purify by column chromatography to obtain 0.76 g of amitraz hapten, the structural formula of which is shown in Formula I, and the yield is 82%.

[0018] The amitraz hapten prepared in this example was identified by mass spectrometry, and the obtained mass spectrum is shown in the accompanying drawings of the specification Figure 1 . It can be seen from the mass spectrum that the molecular ion peaks of the amitraz hapten are EI-MS(positive) m / z: 360.2 [M+Na] + 、697.2 [2M+Na] + Both are the target molecular ion peaks, which are consistent with the molecular weight 337.1 of this compound.

[0019] The amitraz hapten prepared by the present invention introduces a linker structure and an active group for coupling with macromolecules on the basis of retaining the basic structure of amitraz. This not only facilitates its coupling with macromolecules, but also fully exposes the basic structure of amitraz itself with a small molecular structure and molecular weight after coupling, avoiding its being masked by macromolecules and affecting the recognition of the animal body.

[0020] Example 2 Synthesis of Amitraz Immunoantigen and Amitraz Coating Antigen The preparation method of amitraz immunoantigen includes the following steps: Weigh 34 mg of amitraz hapten, dissolve it in 2.5 mL of DMF, add 14 mg of NHS and 30 mg of EDC·HCl, and react at room temperature for 6 h to prepare an activation solution; take 50 mg of bovine lactoferrin (LF) and dissolve it in 5 ml of 0.1 M boric acid buffer solution with pH 9.0, add 2 mL of DMF and 0.7 mL of the above activation solution, react at room temperature for 4 h, then dialyze with PBS (phosphate buffer solution with 0.01 mol / L and pH = 7.4), change the solution once every 4 h, change the solution 7 - 8 times, centrifuge at 4000 r / min for 5 min after dialysis, take the supernatant, and prepare the amitraz immunoantigen, that is, the amitraz hapten - LF conjugate, and store it at -20°C. Similarly, prepare the amitraz coating antigen, replace LF with BSA, prepare the amitraz coating antigen, that is, the amitraz hapten - BSA conjugate, and store it at -20°C.

[0021] Example 3 Preparation and Purification of Amitraz Monoclonal Antibody 3.1 Animal Immunization Select healthy BALB / c mice at 6 - 8 weeks old for immunization. After mixing and emulsifying the amitraz immunizing antigen obtained in Example 2 with an equal amount of Freund's adjuvant, perform multiple subcutaneous injections on the neck and back of BALB / c mice for immunization (except for the booster immunization). Use complete Freund's adjuvant for the first immunization, with a dose of 180 μg / mouse; perform a booster immunization after 4 weeks, with a dose of 90 μg / mouse, mix and emulsify with incomplete Freund's adjuvant, and the time interval for subsequent booster immunizations is 3 weeks; the dose is halved again during the booster immunization, to 45 μg / mouse, dilute the complete antigen with physiological saline, and perform intraperitoneal injection on the mice. Tail bleeding can be performed for detection after the third immunization of the mice, and the titer of the mouse serum and IC 50 , select mice with high titer and low IC 50 for cell fusion; 3.2 Cell Fusion and Cloning Immune BALB / c mouse spleen cells were taken and fused with SP2 / 0 myeloma cells at a ratio of 10:1. A amitraz monoclonal hybridoma cell line that stably secretes amitraz monoclonal antibody was screened out; 3.3 Cell cryopreservation and resuscitation The amitraz monoclonal hybridoma cells were made into a cell suspension of 5×10 6 cells / mL with cryopreservation solution and stored in liquid nitrogen for a long time. When resuscitating, take out the cryopreservation tube, immediately put it into a 37°C water bath for rapid thawing. After centrifuging to remove the cryopreservation solution, transfer it into a culture flask for culture; 3.4 Preparation, purification and performance determination of monoclonal antibody Incremental culture method: The amitraz monoclonal hybridoma cells were placed in cell culture medium and cultured at 37°C. The obtained culture solution was purified by the octanoic acid-ammonium sulfate saturation method to obtain amitraz monoclonal antibody, which was stored at -20°C. The performance of the antibody was determined by the indirect ELISA method. An ELISA standard curve for the amitraz antibody was established, as shown in the attached drawings of the specification Figure 2 and its IC 50 value was 5.22 ng / mL, and the linear range for amitraz was 1.82 - 14.96 ng / mL.

[0022] Example 4 Preparation of amitraz colloidal gold chromatography detection device 4.1 Preparation of colloidal gold solution The 1% chloroauric acid solution by mass fraction was diluted to 0.01% (mass fraction) with double-distilled deionized water. Take 100 mL of 0.01% chloroauric acid solution and place it in a conical flask. Heat it to boiling with a constant-temperature electromagnetic stirrer. Under continuous high temperature and continuous stirring, add 2.0 mL of 1% trisodium citrate solution. Continue to stir evenly and heat until the solution turns into a bright red color and then stop. After cooling to room temperature, restore the volume to the original volume with deionized water to obtain the colloidal gold solution, which was stored at 4°C. The prepared colloidal gold solution was pure, transparent, without precipitation and floating substances; 4.2 Preparation of amitraz monoclonal antibody-colloidal gold label Under magnetic stirring, the pH value of the colloidal gold solution was adjusted to 7.2 with 0.2 mol / L potassium carbonate. According to the standard of adding 20 - 60 μg of amitraz monoclonal antibody per milliliter of colloidal gold solution, the amitraz monoclonal antibody prepared in Example 3 was added to the colloidal gold solution. Continue to stir and mix evenly for 30 min, then let it stand for 10 min. Then add 10% bovine serum albumin (BSA) solution to make its volume percentage content in the colloidal gold solution 1%, and let it stand for 10 min. Centrifuge at 12000 rpm at 4°C for 40 min, discard the supernatant, and resuspend the precipitate with a reconstitution buffer with a volume of 1 / 10 of the initial volume of the colloidal gold solution to prepare the amitraz monoclonal antibody-colloidal gold label, which was stored at 4°C for standby; Reconstitution buffer: 0.02 mol / L phosphate buffer with 0.3% - 0.5% (v / v) bovine serum albumin, 0.1% - 0.3% (w / w) Tween - 20, 3% - 6% (w / w) trehalose, and pH = 7.2; 4.3 Preparation of microtiter reaction cups Add 100 μL of amitraz monoclonal antibody - colloidal gold conjugate to the microtiter reaction cups, place them in a freeze - dryer, pre - freeze for 3 h at a cold trap temperature of - 50 °C, and then vacuum - dry for 6 h. Then take them out to obtain microtiter reaction cups with freeze - dried amitraz monoclonal antibody - colloidal gold conjugate, and store them sealed. The freeze - dried amount of amitraz monoclonal antibody - colloidal gold conjugate is 0.20 - 0.50 μg / ml; 4.4 Preparation of sample absorption pads Soak the sample absorption pads in 0.02 mol / L phosphate buffer containing bovine serum albumin for 2 h, and dry them at 50 °C for 2 h for standby. The pH of the 0.02 mol / L phosphate buffer is 7.2, and the volume percentage of bovine serum albumin is 1.0%; 4.5 Preparation of reaction membranes Coating process: Dilute the amitraz coating antigen (amitraz hapten - BSA) prepared in Example 2 with phosphate buffer to a concentration of 10 mg / mL, and coat it on the test area (T area) of the nitrocellulose membrane using a gold - labeled membrane - spraying instrument, with a coating concentration of 0.5 mg / ml; Dilute the goat anti - mouse antibody (commercial product) with 0.01 mol / L phosphate buffer with pH = 7.4 to a concentration of 10 mg / mL, and coat it on the quality control area (C area) of the nitrocellulose membrane using a gold - labeled membrane - spraying instrument, with a coating concentration of 1.0 mg / mL. Place the coated reaction membrane at 50 °C and dry it for 6 h.

[0023] 4.6 Preparation of amitraz colloidal gold chromatography detection device 4.6.1 Assembly of test strips Paste the sample absorption pad, reaction membrane, and water absorption pad on the bottom plate in sequence. Among them, the bottom plate is a PVC bottom plate, the sample absorption pad is filter paper for absorption, the water absorption pad is filter paper for water absorption, and the reaction membrane is nitrocellulose membrane. The end of the sample absorption pad is connected to the beginning of the reaction membrane, the end of the reaction membrane is connected to the beginning of the water absorption pad, the beginning of the sample absorption pad is aligned with the beginning of the bottom plate, and the end of the water absorption pad is aligned with the end of the bottom plate.

[0024] 4.6.2 Assembly of amitraz colloidal gold chromatography test strip cassette Assemble the test strip obtained in step 4.6.1 and the microtiter reaction cup obtained in step 4.3 into a test strip cassette, and store it in an environment of 2 - 8 °C, with a validity period of 12 months.

[0025] Example 5 A method for detecting amitraz in samples 5.1 Sample pretreatment Weigh 2.0 g ± 0.01 g of homogenized vegetable or fruit sample into a 50 mL polystyrene centrifuge tube, add 8 mL of sample extraction solution (sample extraction solution: 7 mL of 0.1 M pH 8.5 PBS and 1 mL of ethanol are mixed evenly), mix well, centrifuge at 3000 r / min for 1 min at room temperature (20 - 25 °C), and take the supernatant as the test solution; 5.2 Measurement steps Pipette 200 μl of the above-mentioned test solution into the micro-well reaction cup of the amitraz colloidal gold chromatography detection device prepared in Example 4, and aspirate up and down 5 - 10 times to mix evenly. Incubate at room temperature for 3 min, insert the test strip into the reaction cup, incubate at room temperature for 3 min, take out the test strip, gently scrape the sample pad at the lower end of the test strip, and perform result interpretation; 5.3 Result determination The result is determined by comparing the color intensity of the control line (C line) and the test line (T line).

[0026] Positive: When a band appears in the quality control area (C) and no color appears in the test area (T), it is judged as positive, that is, the sample contains amitraz, and it is represented by "+"; Negative: When bands appear in both the quality control area (C) and the test area (T), it is judged as negative, that is, the sample does not contain amitraz, and it is represented by "-"; Invalid: When no band appears in the quality control area (C), it is judged that the test strip is invalid.

[0027] Example 6 Sensitivity and false negative rate of the amitraz colloidal gold chromatography detection device Select apples, pears, cucumbers, and tomatoes that have been tested and do not contain amitraz as blank samples. According to the "GB 2763 - 2021", the maximum residue limit (MRL) of apples, pears, cucumbers, and tomatoes is 0.5 mg / kg. Set the method detection limit of apples, pears, cucumbers, and tomatoes to 0.5 mg / kg, that is, the attention concentration. The addition levels are 1 times the attention concentration and 2 times the attention concentration, respectively, to investigate the sensitivity and false negative rate. For the samples at two addition concentration levels, 50 specimens for each concentration level are detected according to the detection method in Example 5, and the detection results are shown in Table 1 below.

[0028] Table 1 Detection results of sensitivity and false negative rate of the amitraz colloidal gold chromatography detection device

[0029] As can be seen from Table 1, the detection sensitivity of the detection method in this example for the amitraz residue in the sample is ≥ 95%, and the false negative rate is ≤ 5%.

[0030] Example 7 False Positive and Specificity of Amitraz Colloidal Gold Chromatographic Detection Device Blank samples of apples, pears, cucumbers, and tomatoes were selected and prepared into 50 samples each at 2 concentration levels (0.5 times the detection limit, blank matrix) by spiking the blank matrix. The samples were detected using the detection method in Example 5, and the detection results are shown in Table 2 below.

[0031] Table 2 False Positive Detection Results of Amitraz Colloidal Gold Chromatographic Detection Device

[0032] As can be seen from Table 2, the specificity of the detection method in this example is ≥95%, and the false positive rate is ≤5%. The above results indicate that the colloidal gold chromatographic detection device for detecting amitraz of the present invention has good specificity and can accurately detect amitraz in vegetable and fruit samples. Therefore, the residue of amitraz in vegetable and fruit samples can be rapidly detected.

[0033] The above are only some embodiments of the present invention. For those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention.

Claims

1. Amitraz hapten, characterized in that Its structure is shown in formula (I): ，（Ⅰ）。 2. The method for preparing the amitraz hapten according to claim 1, characterized in that: The following steps are involved: S1. 2-(3-methyl-4-nitrophenyl)acetic acid and 2-(trimethylsilyl)ethanol are dissolved in N,N-dimethylformamide, and an esterification reaction is carried out under the action of a condensing agent EDC.HCl and a catalyst imidazole to obtain an intermediate 1 with an arm. The structural formula of the intermediate 1 is shown in reaction formula (II): , formula (II); S2. The intermediate 1 is dissolved in ethanol, and hydrazine hydrate, palladium carbon, and ferric chloride hexahydrate are added, and a nitro reduction reaction is carried out at 65° C. to obtain an intermediate 2. The structural formula of the intermediate 2 is shown in reaction formula (III): , formula (III); S3. The intermediate 2, cyanamide and triethyl orthoformate are dissolved in toluene and reacted at 80° C. for 2 h to obtain the intermediate 3. The structural formula of the intermediate 3 is shown in reaction formula (IV): , formula (IV); S4. The intermediate 3 is dissolved in tetrahydrofuran, triethylamine, methylamine hydrochloride and distilled water are added, and the reaction is carried out at room temperature for 3 hours to obtain the intermediate 4, the structure of which is shown in reaction formula (V): , formula (V); S5. The intermediate 4 and 2,4-dimethylphenyl isocyanide are dissolved in toluene, cuprous oxide is added as a catalyst, and refluxed for 3 hours to obtain the intermediate 5, the structure of which is shown in reaction formula (VI): , formula (VI); S6. The intermediate 5 is dissolved in anhydrous tetrahydrofuran, tetrabutylammonium fluoride is added under stirring, and the reaction is carried out at room temperature to obtain a biformamidine hapten having a structural formula as shown in formula (I). The reaction of this step is shown in reaction formula (VII): , formula (VII).

3. Amitraz antigen, characterized in that The amitraz antigen is a conjugate of the amitraz hapten according to claim 1 and a carrier protein, and the carrier protein is bovine serum albumin, human serum albumin, chicken ovalbumin or hemocyanin.

4. Use of the amitraz hapten according to claim 1 or the amitraz antigen according to claim 3 in immunological detection of amitraz for non-disease diagnosis.

5. A biformazidine antibody, characterized in that The amitraz antibody is prepared by immunizing an animal with the amitraz antigen according to claim 3, and the amitraz antibody is a amitraz monoclonal antibody.

6. Use of the amitraz antibody according to claim 5 in immunological detection of amitraz for non-disease diagnosis.

7. A biformamidine colloidal gold chromatography detection device, characterized in that: The invention comprises a test strip and a reaction cup, wherein the test strip comprises a reaction membrane, the reaction membrane is provided with a detection area and a quality control area, the detection area is coated with the amitraz antigen as claimed in claim 3, and the reaction cup contains the amitraz antibody as claimed in claim 5 marked with colloidal gold.

8. A method for detecting amitraz in a sample, characterized in that: The method adopts the amitraz colloidal gold chromatography detection device described in claim 7 to detect amitraz in a sample, wherein the sample is an agricultural product.

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