Amitraz hapten, antigen, antibody, detection device and their preparation and application

Abstract

The present invention discloses amitraz hapten, antigen, antibody, detection device and preparation and application thereof, and relates to amitraz hapten, antigen, antibody, colloidal gold detection device and preparation and application thereof for detecting amitraz. The present invention provides a method for preparing artificial antigen by synthesizing amitraz hapten and coupling antigen with carrier protein, as well as antibodies specific to amitraz produced by the body of experimental animals after immunization, and the IC 50 The value was 5.22 ng / mL, and the linear detection range for amitraz was 1.82-14.96 ng / mL, which meets the limit for amitraz in the national standard GB 2763-2021, "National Food Safety Standard Maximum Limits of Pesticide Residues in Food." Therefore, the present invention can be applied to the rapid detection of amitraz residues in agricultural products.

Description

Technical Field

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

[0002] Amitraz is a broad-spectrum formamidine insecticide and acaricide. It is toxic to humans and livestock and can suppress the human central nervous system. There is no specific treatment for poisoning. Its main hydrolysis metabolite, 2,4-dimethylaniline, is highly toxic and poses multiple potential risks to humans, including carcinogenicity and mutagenicity. Currently, commonly used methods for the detection and analysis of amitraz include gas chromatography, gas chromatography-mass spectrometry (GC-MS), liquid chromatography (LC-MS), and liquid chromatography-mass spectrometry (LC-MS). The current national standard method for the determination of amitraz in fruits and vegetables uses the GB / T 5009.143-2003 method, which utilizes a packed column for gas chromatography separation and requires pretreatment with hydrolysis in a reflux chamber. The hydrolysis products must then be derivatized before determination. This method is significantly time-consuming and presents significant challenges and challenges in detection. Furthermore, these methods require repeated acid adjustments and extractions during sample pretreatment, resulting in complex processes that affect the accuracy of the results. High performance liquid chromatography has certain defects. Amitraz is easily hydrolyzed during the determination process, which makes the determination results inaccurate.

[0003] In recent years, research on immunoassay technology for pesticide and veterinary drug residue analysis has increased, but there is no immunoassay method for amitraz. Based on the acid and alkali resistance of amitraz, the present invention uses a novel synthesis method to design and develop a new amitraz hapten, prepare a highly sensitive antibody, and establish a corresponding rapid amitraz detection method based on this antibody, enabling rapid immunological detection of amitraz residues in fruits, vegetables, and animal tissues. Summary of the Invention

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

[0005] According to one aspect of the present invention, a bimethamidine hapten is provided, the structure of which is shown in formula (I):

[0006] , formula (Ⅰ).

[0007] According to another aspect of the present invention, there is provided a method for preparing amitraz hapten, comprising the following steps:

[0008] S1, 2-(3-methyl-4-nitrophenyl)acetic acid and 2-(trimethylsilyl)ethanol are dissolved in N,N-dimethylformamide, and esterification reaction is carried out under the action of condensing agent EDC.HCl and catalyst imidazole to obtain intermediate 1 with an arm. The structural formula of intermediate 1 is shown in reaction formula (II):

[0009] , formula (II);

[0010] S2, the intermediate 1 is dissolved in ethanol, and hydrazine hydrate, palladium carbon, and ferric chloride hexahydrate are added, and the nitro group reduction reaction occurs under heating at 65°C to obtain the intermediate 2. The structural formula of the intermediate 2 is shown in reaction formula (III):

[0011] , formula (III);

[0012] S3, dissolving the intermediate 2, cyanamide and triethyl orthoformate in toluene, and reacting at 80°C for 2h to obtain the intermediate 3. The structural formula of the intermediate 3 is shown in reaction formula (IV):

[0013] , formula (IV);

[0014] S4, dissolving the intermediate 3 in tetrahydrofuran, adding triethylamine, methylamine hydrochloride and distilled water, and reacting at room temperature for 3 hours to obtain intermediate 4, the structural formula of intermediate 4 is shown in reaction formula (V):

[0015] , formula (V);

[0016] S5, dissolving the intermediate 4 and 2,4-dimethylphenyl isocyanide in toluene, adding cuprous oxide as a catalyst, and refluxing for 3 hours to obtain the intermediate 5, the structure of which is shown in reaction formula (VI):

[0017] , formula (VI);

[0018] S6, dissolving the intermediate 5 in anhydrous tetrahydrofuran, adding tetrabutylammonium fluoride under stirring, and reacting at room temperature to obtain amphetamine hapten with the structural formula shown in formula (I). The reaction of this step is shown in reaction formula (VII):

[0019] , formula (VII).

[0020] According to another aspect of the present invention, a conjugate of the amitraz hapten according to claim 1 and a carrier protein is provided, wherein the carrier protein is bovine serum albumin, human serum albumin, chicken ovalbumin or hemocyanin.

[0021] According to a fourth aspect of the present invention, there is provided a non-disease diagnosis application of amitraz hapten or amitraz antigen in immunological detection of amitraz.

[0022] According to a fifth aspect of the present invention, there is provided a biformamidine antibody prepared by immunizing an animal with a biformamidine antigen, wherein the biformamidine antibody is a biformamidine monoclonal antibody.

[0023] According to a sixth aspect of the present invention, there is provided a use of amitraz antibodies in immunological detection of amitraz for non-disease diagnosis.

[0024] According to the seventh aspect of the present invention, a biformazidine colloidal gold chromatography detection device is provided, comprising 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 a biformazidine antigen, and the reaction cup contains a biformazidine antibody labeled with colloidal gold.

[0025] According to an eighth aspect of the present invention, a method for detecting amitraz in a sample is provided. The method uses an amitraz colloidal gold chromatography detection device to detect amitraz in the sample, and the sample is an agricultural product.

[0026] The present invention has the following beneficial effects: The method for preparing amitraz hapten provided by the present invention uses readily available chemical reagents, has a simple operation process, concise and effective synthesis steps, a high reaction yield, and a low detection cost. The present invention provides an artificial antigen prepared by coupling amitraz hapten with a carrier protein, and an antibody specifically against amitraz produced by the body of an experimental animal after immunization, which has a high titer. An ELISA standard curve for amitraz antibodies is established, and its IC value is 0. 50 The value is 5.22 ng / mL, and the linear detection range is 1.82-14.96 ng / mL, which complies with the limit provisions of the latest national standard GB 2763-2021 "National Food Safety Standard Maximum Pesticide Residue Limits in Food" for amitraz. The present invention utilizes the principle of chromatographic immunocolloidal gold to qualitatively detect the content of amitraz in the sample by colorimetry between the test line and the quality control line in the test strip. It does not require the use of large instruments such as liquid chromatography or mass spectrometry to achieve the purpose of rapid detection, and has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, and long shelf life. Therefore, the present invention can be applied to the rapid detection of amitraz residues in agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure is a mass spectrum of amitraz hapten according to one embodiment of the present invention.

[0028] Figure 2 The figure is a standard curve of an ELISA based on amitraz antibody according to one embodiment of the present invention. DETAILED DESCRIPTION

[0029] The present invention will be further described in detail through specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention by those skilled in the art are all within the scope of the appended claims. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents available on the conventional market.

[0030] Example 1 Synthesis and Identification of Amitraz Hapten

[0031] The preparation method of amitraz hapten comprises the following steps:

[0032] 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 EDC-HCl (condensing agent) and 0.3 g of imidazole (catalyst), and stir the mixture at room temperature overnight. After the reaction, add 150 mL of distilled water, extract twice with ethyl acetate, combine the mixture, dry over anhydrous sodium sulfate, evaporate to dryness, and purify by column chromatography to obtain 6.1 g of intermediate 1 with an arm.

[0033] S2, 5.0 g of intermediate 1 was dissolved in 150 mL of ethanol, and 2.3 g of hydrazine hydrate, 0.5 g of palladium on carbon, and 0.5 g of ferric chloride hexahydrate were added. The mixture was heated at 65°C for 45 min, and the precipitate was filtered. The ethanol phase was evaporated to dryness, diluted with water, and extracted twice with ethyl acetate. The ethyl acetate phases were combined, dried over anhydrous sodium sulfate, evaporated to dryness, and purified by column chromatography to obtain 3.8 g of intermediate 2;

[0034] S3, weigh 2.6 g of intermediate 2, 0.45 g of cyanamide and 1.6 g of triethyl orthoformate and dissolve them in 20 ml of toluene. React at 80°C for 2 h. Evaporate the solvent and directly purify by column to obtain 2.9 g of intermediate 3.

[0035] S4, 2.5 g of intermediate 3 was dissolved in 30 mL of tetrahydrofuran, and 2 mL of triethylamine, 0.6 g of methylamine hydrochloride and 5 mL of distilled water were added. The mixture was reacted at room temperature for 3 h. After the solvent was evaporated, 30 mL of water was added, and the mixture was extracted twice with ethyl acetate. The mixture was combined and dried over anhydrous sodium sulfate to evaporate the solvent to obtain 2.2 g of intermediate 4, which was directly used for the next step without purification;

[0036] S5, 2.0 g of intermediate 4 and 0.85 g of 2,4-dimethylphenyl isocyanide were dissolved in 20 mL of toluene, 0.1 g of cuprous oxide was added as a catalyst, and the mixture was refluxed for 3 h, the solvent was evaporated, and column purification was performed to obtain 2.4 g of intermediate 5;

[0037] S6, 1.2 g of intermediate 5 was dissolved in 30 mL of anhydrous tetrahydrofuran, 2.5 g of tetrabutylammonium fluoride was added with stirring, the reaction was carried out at room temperature for 7 h, evaporated to dryness, diluted with water, extracted twice with ethyl acetate, and the ethyl acetate was combined. The ethyl acetate phase was dried over anhydrous sodium sulfate, evaporated to dryness, and purified by column chromatography to obtain 0.76 g of amitraz hapten, the structural formula of which is shown in Formula I, with a yield of 82%.

[0038] The amitraz hapten prepared in this example was identified by mass spectrometry. The mass spectrum obtained is shown in the accompanying drawings. Figure 1 From the mass spectrum, we can see that the molecular ion peak of the amitraz hapten is EI-MS (positive) m / z: 360.2 [M+Na] + 、697.2[2M+Na] + All of them are target molecular ion peaks, which are consistent with the molecular weight of the compound of 337.1.

[0039] The amitraz hapten prepared by the present invention introduces a linker arm structure and an active group for coupling with a macromolecule on the basis of retaining the basic structure of amitraz, which is conducive to its coupling with the macromolecule and can fully expose its own molecular structure and the basic structure of amitraz with a smaller molecular weight after coupling, thereby avoiding being masked by the macromolecule and affecting the recognition of the animal body.

[0040] Example 2 Synthesis of Amitraz Immune Antigen and Amitraz Coated Antigen

[0041] The method for preparing amitraz immune antigen comprises the following steps:

[0042] 34 mg of amitraz hapten was weighed and dissolved in 2.5 mL of DMF. 14 mg of NHS and 30 mg of EDC-HCl were added and allowed to react at room temperature for 6 h to prepare an activation solution. 50 mg of bovine lactoferrin (LF) was dissolved in 5 mL of 0.1 M boric acid buffer (pH 9.0). 2 mL of DMF and 0.7 mL of the activation solution were added and allowed to react at room temperature for 4 h. The solution was then dialyzed against PBS (0.01 mol / L phosphate buffer, pH 7.4), with the solution changed every 4 h for 7-8 times. After dialysis, the solution was centrifuged at 4000 rpm for 5 min. The supernatant was collected to prepare the amitraz immunizing antigen, i.e., the amitraz hapten-LF conjugate, and stored at -20°C. Similarly, the amitraz coating antigen was prepared by replacing LF with BSA to prepare the amitraz coating antigen, i.e., the amitraz hapten-BSA conjugate, and stored at -20°C.

[0043] Example 3 Preparation and purification of amitraz monoclonal antibody

[0044] 3.1 Animal immunization

[0045] Healthy 6-8 week old BALB / c mice were selected for immunization. The amitraz immunization antigen obtained in Example 2 was mixed and emulsified with an equal amount of Freund's adjuvant, and then the BALB / c mice were immunized by multiple subcutaneous injections on the back of the neck (except for sprint immunization). Complete Freund's adjuvant was used for the first immunization at a dose of 180 μg / mouse; after a lapse of 4 weeks, a booster immunization was performed at a dose of 90 μg / mouse, mixed and emulsified with incomplete Freund's adjuvant, and the intervals between subsequent booster immunizations were 3 weeks; for sprint immunization, the dose was halved to 45 μg / mouse, and the complete antigen was diluted with physiological saline and injected intraperitoneally into the mice. After the third immunization, the mice were tail-cut and blood was collected for testing, and the titer and IC of the mouse serum were detected by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). 50 , select high titer, IC 50 Low mice were fused;

[0046] 3.2 Cell fusion and cloning

[0047] Splenocytes of immunized BALB / c mice were obtained and fused with SP2 / 0 myeloma cells at a ratio of 10:1 to obtain a biformazidine monoclonal hybridoma cell line that stably secreted biformazidine monoclonal antibodies.

[0048] 3.3 Cell cryopreservation and thawing

[0049] The amitraz monoclonal hybridoma cells were cryopreserved to prepare 5×10 6 Cell suspensions of 100 cells / mL can be stored in liquid nitrogen for a long time. When thawing, remove the cryovials and immediately place them in a 37°C water bath for rapid thawing. After centrifugation to remove the cryopreservation solution, transfer the cells to culture bottles for culturing.

[0050] 3.4 Preparation, purification and performance determination of monoclonal antibodies

[0051] Incremental culture method: Place the amitraz monoclonal hybridoma cells in cell culture medium and culture at 37°C. Purify the resulting culture medium using the caprylic acid-saturated ammonium sulfate method to obtain the amitraz monoclonal antibody and store it at -20°C. The performance of the antibody is determined by indirect ELISA. An ELISA standard curve for the amitraz antibody is established, see the attached figure in the specification. Figure 2 , its IC 50 The linear range for amitraz was 1.82-14.96 ng / mL.

[0052] Example 4 Preparation of amitraz colloidal gold chromatography detection device

[0053] 4.1 Preparation of colloidal gold solution

[0054] Dilute a 1% chloroauric acid solution to 0.01% (mass fraction) with double-distilled deionized water. Place 100 mL of the 0.01% chloroauric acid solution in a conical flask and heat to boiling using a constant-temperature electromagnetic stirrer. Add 2.0 mL of a 1% trisodium citrate solution while continuing to stir at a constant temperature until the solution turns a translucent red. Cool to room temperature, then return to the original volume with deionized water to obtain a colloidal gold solution. Store at 4°C. The prepared colloidal gold solution should be pure, translucent, and free of precipitates and floating matter.

[0055] 4.2 Preparation of amitraz monoclonal antibody-colloidal gold label

[0056] Under magnetic stirring, adjust the pH of the colloidal gold solution to 7.2 with 0.2 mol / L potassium carbonate. Add the amitraz monoclonal antibody prepared in Example 3 to the colloidal gold solution at a standard concentration of 20-60 μg of amitraz monoclonal antibody per milliliter of colloidal gold solution. Continue stirring and mixing for 30 minutes. After standing for 10 minutes, add 10% bovine serum albumin (BSA) solution to a volume percentage of 1% in the colloidal gold solution. Allow to stand for 10 minutes. Centrifuge at 12,000 rpm at 4°C for 40 minutes, discard the supernatant, and resuspend the precipitate in a reconstitution buffer with a volume of 1 / 10 the initial colloidal gold solution to prepare the amitraz monoclonal antibody-colloidal gold label. Store at 4°C until use.

[0057] Reconstitution buffer: 0.02 mol / L phosphate buffer containing 0.3%-0.5% bovine serum albumin (Volume Percentage), 0.1%-0.3% Tween-20 (Material Percentage), 3%-6% trehalose (Material Percentage), pH = 7.2;

[0058] 4.3 Preparation of microwell reaction cups

[0059] Add 100 μL of amitraz monoclonal antibody-colloidal gold label to a microporous reaction cup, place in a freeze dryer, pre-freeze at a cold trap temperature of -50°C for 3 hours, and then vacuum dry for 6 hours. Then, take out the microporous reaction cup containing freeze-dried amitraz monoclonal antibody-colloidal gold label, and seal it for storage. The freeze-dried amount of amitraz monoclonal antibody-colloidal gold label is 0.20-0.50 μg / ml.

[0060] 4.4 Preparation of sample absorption pad

[0061] Soak the sample absorption pad in 0.02 mol / L phosphate buffer containing bovine serum albumin for 2 hours, then dry it at 50°C for 2 hours before use. The pH of the 0.02 mol / L phosphate buffer is 7.2, and the volume percentage of bovine serum albumin is 1.0%.

[0062] 4.5 Preparation of reaction membrane

[0063] Coating process: The amitraz-coated antigen (mitraz hapten-BSA) prepared in Example 2 was diluted to a concentration of 10 mg / mL in phosphate buffer and coated onto the detection zone (zone T) of the nitrocellulose membrane using a gold-labeled gold-spraying device at a coating concentration of 0.5 mg / mL. A commercial goat anti-mouse antibody (product) was diluted to a concentration of 10 mg / mL in phosphate buffer (0.01 mol / L, pH 7.4) and coated onto the quality control zone (zone C) of the nitrocellulose membrane using a gold-labeled gold-spraying device at a coating concentration of 1.0 mg / mL. The coated reaction membrane was dried at 50°C for 6 hours.

[0064] 4.6 Preparation of amitraz colloidal gold chromatography detection device

[0065] 4.6.1 Assembly of test strips

[0066] A sample absorption pad, a reaction membrane, and a water-absorbing pad are sequentially attached to a base plate. The base plate is a PVC base plate, the sample absorption pad is filter paper, the water-absorbing pad is filter paper, and the reaction membrane is a 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-absorbing pad, the beginning of the sample absorption pad is aligned with the beginning of the base plate, and the end of the water-absorbing pad is aligned with the end of the base plate.

[0067] 4.6.2 Assembly of the Amitraz Colloidal Gold Chromatography Test Kit

[0068] Assemble the test strip obtained in step 4.6.1 above and the microporous reaction cup obtained in step 4.3 above into a test strip box, store it at 2-8°C, and the shelf life is 12 months.

[0069] Example 5 A method for detecting amitraz in a sample

[0070] 5.1 Sample pretreatment

[0071] 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 extract (sample extract: 7 mL of 0.1 M pH 8.5 PBS and 1 mL of ethanol), mix well, centrifuge at 3000 rpm for 1 min at room temperature (20-25°C), and collect the supernatant as the test solution;

[0072] 5.2 Determination steps

[0073] Pipette 200 μl of the test solution into the microporous reaction cup of the amitraz colloidal gold chromatography detection device prepared in Example 4 and pipette up and down 5-10 times to mix thoroughly. Incubate at room temperature for 3 minutes. Insert the test strip into the reaction cup and incubate at room temperature for 3 minutes. Remove the test strip, gently scrape off the sample pad at the bottom of the strip, and read the result.

[0074] 5.3 Result determination

[0075] The results are determined by comparing the color depth of the control line (C line) and the test line (T line).

[0076] Positive: When the control area (C) shows a band and the test area (T) does not show color, it is judged as positive, that is, the sample contains amitraz, which is indicated by "+";

[0077] Negative: When both the control area (C) and the test area (T) show bands, it is judged as negative, that is, there is no amitraz in the sample, indicated by "-";

[0078] Invalid: When no band is displayed in the quality control area (C), the test strip is considered invalid.

[0079] Example 6 Sensitivity and False Negative Rate of Amitraz Colloidal Gold Chromatography Detection Device

[0080] Apples, pears, cucumbers, and tomatoes tested to be free of amitraz were selected as blank samples. Based on the maximum residue limit (MRL) of 0.5 mg / kg for apples, pears, cucumbers, and tomatoes stipulated in GB 2763-2021, the method detection limit (LDL) for apples, pears, cucumbers, and tomatoes was set at 0.5 mg / kg, the concentration of concern. The sensitivity and false negative rate were assessed using spiked samples at 1 and 2 times the concentration of concern. Fifty samples were spiked at each of the two concentration levels and tested according to the method described in Example 5. The results are shown in Table 1 below.

[0081] Table 1 Sensitivity and false negative rate of amitraz colloidal gold chromatography detection device

[0082]

[0083] As can be seen from Table 1, the detection sensitivity of the detection method in this embodiment for the amitraz residue in the sample is ≥95%, and the false negative rate is ≤5%.

[0084] Example 7 False Positives and Specificity of the Amitraz Colloidal Gold Chromatography Detection Device

[0085] Blank samples of apples, pears, cucumbers, and tomatoes were spiked with blank matrix to prepare 50 samples at two concentration levels (0.5 times the detection limit, blank matrix). The samples were tested using the detection method described in Example 5. The test results are shown in Table 2 below.

[0086] Table 2 False positive test results of amitraz colloidal gold chromatography detection device

[0087]

[0088] As shown in Table 2, the specificity of the detection method in this embodiment is ≥95%, and the false positive rate is ≤5%. The above results indicate that the colloidal gold chromatography detection device for detecting amitraz of the present invention has good specificity and can accurately detect amitraz in vegetable and fruit samples. Therefore, it can be used to quickly detect amitraz residues in vegetable and fruit samples.

[0089] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. Amitraz hapten, characterized in that Its structure is shown in formula (I): , formula (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 esterified in the presence of a condensing agent, EDC-HCl, and a catalyst, imidazole, to obtain an intermediate 1 having an arm. The structure of the intermediate 1 is shown in reaction formula (II): , formula (II); S2. The intermediate 1 is dissolved in ethanol, and hydrazine hydrate, palladium on carbon, and ferric chloride hexahydrate are added. The nitro group is reduced under heating at 65°C to obtain the 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 were dissolved in toluene and reacted at 80°C for 2h 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, and triethylamine, methylamine hydrochloride and distilled water are added. After reacting at room temperature for 3 hours, the intermediate 4 is obtained. The structural formula of the intermediate 4 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 the reaction is refluxed for 3 hours to obtain the intermediate 5. The structure of the intermediate 5 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 hapten having the structural formula (I) as shown in formula (VII). 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.

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