A pyraclostrobin hapten, an artificial antigen and its application

Abstract

This invention discloses a pyraclostrobin hapten, an artificial antigen, and their applications. The hapten prepared by this invention avoids the common backbone of structural analogs, fully exposes the unique characteristic structure of pyraclostrobin, and prepares highly specific and sensitive pyraclostrobin antibodies. Simultaneously, it simplifies the synthesis steps of the pyraclostrobin hapten, provides two novel pyraclostrobin haptens, and establishes an indirect competitive ELISA detection method for pyraclostrobin. The limit of detection (LOD) is 0.23 ng / mL, the quantitative detection range is 0.57–12.57 ng / mL, and there is no cross-reactivity with pyraclostrobin analogs. This method can be widely used for large-scale, rapid, and accurate screening of pyraclostrobin.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, specifically to a pyraclostrobin hapten, an artificial antigen, and their applications. Background Technology

[0002] Pyraclostrobin, also known as cymoxanil / pyraclostrobin, is a white to light beige odorless crystalline powder, sparingly soluble in water but soluble in organic solvents such as acetonitrile, methanol, ethyl acetate, and acetone. Pyraclostrobin is a novel methoxyacrylate fungicide that inhibits mitochondrial respiration, ultimately leading to cell death. It possesses protective, curative, and transdermal effects, and is primarily used to control various fungal diseases in crops. It shows good control efficacy against grape powdery mildew, loquat angular leaf spot, citrus scab, and wheat powdery mildew.

[0003] Currently, the detection of pyraclostrobin mainly focuses on samples such as bananas, citrus fruits, grapes, and cucumbers. Analytical methods primarily include gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC). Existing technologies CN111961002A and CN112114147A disclose haptens, antigens, and antibodies for pyraclostrobin detection, but due to the common skeleton of methoxyacrylate fungicides (such as...),... Figure 1 As shown in the diagram (the red highlighted portion represents the common backbone), the hapten design of the aforementioned patent incorporates this common backbone, causing antibodies to non-specifically recognize this type of structural analog. In addition, the aforementioned patent also suffers from having numerous synthesis steps.

[0004] Therefore, when establishing an immunological detection method and applying it to rapidly detect the pyraclostrobin content in fruits and vegetables, the key technology lies in obtaining antibodies with high specificity and sensitivity. To achieve this goal, the prerequisite is to synthesize and prepare a suitable pyraclostrobin hapten. Summary of the Invention

[0005] To address the lack of a rapid, sensitive, and convenient immunoassay method for pyraclostrobin in existing technologies, this invention provides a pyraclostrobin hapten, an artificial antigen, and their applications.

[0006] The first objective of this invention is to provide two pyraclostrobin haptens.

[0007] A second objective of this invention is to provide a method for preparing the pyraclostrobin hapten.

[0008] The third objective of this invention is to provide two artificial antigens for pyraclostrobin.

[0009] A fourth object of the present invention is to provide the use of the pyraclostrobin artificial antigen in the preparation of antibodies against pyraclostrobin.

[0010] The fifth objective of this invention is to provide a pyraclostrobin artificial antigen combination.

[0011] A sixth object of the present invention is to provide the application of the pyraclostrobin artificial antigen combination in the preparation of a kit for detecting pyraclostrobin.

[0012] The seventh object of the present invention is to provide a kit for detecting pyraclostrobin.

[0013] To achieve the above objectives, the present invention is implemented through the following solution:

[0014] The key to immunoassay methods lies in designing suitable haptens to prepare antibodies with high sensitivity and specificity. When designing haptens, it is better to avoid retaining the common backbone of structural analogs and instead expose the unique characteristic structure of the target analyte, which is a better strategy for improving antibody specificity. This invention employs a one-step strategy to introduce a linear spacer arm, which fully exposes the characteristic structure of pyraclostrobin while avoiding the common backbone of structural analogs, thus preparing a highly sensitive and specific pyraclostrobin hapten. It also provides a simpler and more practical approach to the synthesis of pyraclostrobin haptens.

[0015] A pyraclostrobin hapten, namely pyraclostrobin hapten PYE, has the structural formula shown in formula (I).

[0016]

[0017] The pyrazol-methyl hapten PYE was named systematically as 4-((2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)phenyl)amino)butanoic acid, i.e. 4-((2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)phenyl)amino)butanoic acid.

[0018] Another pyraclostrobin hapten, namely pyraclostrobin hapten PYG, has the structural formula shown in formula (II).

[0019]

[0020]

[0021] The pyrazol-methyl hapten PYE was named systematically as (2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)phenyl)aminoacetic acid, i.e. (2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)phenyl)glycine.

[0022] The use of any of the pyraclostrobin haptens in the preparation of pyraclostrobin artificial antigens should also be within the scope of protection of this invention.

[0023] The preparation method of the compound with the structural formula shown in formula (I), namely the pyraclostrobin hapten PYE, includes the following steps:

[0024] 2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)aniline, ethyl 4-bromobutyrate, potassium carbonate and sodium iodide are reacted with acetonitrile to a complete extent. The resulting reactants are then fully hydrolyzed under alkaline conditions, and the pH is adjusted to acidic to obtain the final product.

[0025]

[0026] Preferably, the molar ratio of 2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)aniline, ethyl 4-bromobutyrate, potassium carbonate and sodium iodide is 1::(1~2):(0.5~1):(0.1~1).

[0027] More preferably, the molar ratio of 2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)aniline, ethyl 4-bromobutyrate, potassium carbonate and sodium iodide is 1:1.5:0.5:0.1.

[0028] Preferably, 2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)aniline, ethyl 4-bromobutyrate, potassium carbonate, and sodium iodide are fully reacted with acetonitrile; the reactants are separated and purified, and then fully hydrolyzed under alkaline conditions. After separation and purification by column chromatography, the pH is adjusted to 1-2, and the product is dried to obtain the final product.

[0029] Preferably, the alkaline environment includes lithium hydroxide and methanol.

[0030] More preferably, the alkaline environment consists of lithium hydroxide, methanol, and water.

[0031] Specifically, the preparation method of pyraclostrobin hapten PYE includes the following steps:

[0032] 2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)aniline (CAS No.: 222838-33-7) (0.67 mmol) and ethyl 4-bromobutyrate (1.005 mmol) were dissolved in acetonitrile, and potassium carbonate (0.33 mmol) and sodium iodide (0.067 mmol) were added. The mixture was reacted overnight (12 h) at 50 °C under nitrogen protection. The reactants were separated and purified, and then hydrolyzed with 5 mL of 1 mol / L lithium hydroxide solution (LiOH-H2O) and 10 mL of 50% v / v methanol aqueous solution at room temperature (27 °C) for 3-5 h. The mixture was then separated and purified by column chromatography (stationary phase: silica gel, mobile phase: petroleum ether: ethyl acetate volume ratio = 10:1), and the pH was adjusted to 1-2. After drying, the hapten PYE was obtained.

[0033] The preparation method of the compound with the structural formula shown in formula (II), namely the pyraclostrobin hapten PYG, includes the following steps:

[0034] 2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)aniline, ethyl bromoacetate, potassium carbonate and sodium iodide are reacted with acetonitrile to a complete extent. The resulting reactants are then fully hydrolyzed under alkaline conditions, and the pH is adjusted to acidic to obtain the final product.

[0035]

[0036] Preferably, the molar ratio of 2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)aniline, ethyl bromoacetate, potassium carbonate and sodium iodide is 1:(1-2):(0.5-1):(0.1-1).

[0037] More preferably, the molar ratio of 2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)aniline, ethyl bromoacetate, potassium carbonate and sodium iodide is 1::1.5:0.5:0.1.

[0038] Preferably, 2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)aniline, ethyl bromoacetate, potassium carbonate, and sodium iodide are reacted with acetonitrile to a sufficient degree; the reactants are separated and purified, and then fully hydrolyzed under alkaline conditions. After separation and purification by column chromatography, the pH is adjusted to 1-2, and the product is dried to obtain the final product.

[0039] Preferably, the alkaline environment includes lithium hydroxide and methanol.

[0040] More preferably, the alkaline environment consists of lithium hydroxide, methanol, and water.

[0041] Specifically, the preparation method of the pyraclostrobin hapten PYG includes the following steps:

[0042] 2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)aniline (CAS No.: 222838-33-7) (0.67 mmol): ethyl bromoacetate (1.005 mmol) was dissolved in acetonitrile, and potassium carbonate (0.33 mmol) and sodium iodide (0.067 mmol) were added. The mixture was reacted overnight (12 h) at 60 °C under nitrogen protection. The reactants were separated and purified, and then hydrolyzed with 5 mL of 1 mol / L lithium hydroxide solution (LiOH-H2O) and 10 mL of 50% v / v methanol aqueous solution at room temperature (27 °C) for 3-5 h. The mixture was then separated and purified by column chromatography (stationary phase: silica gel, mobile phase: petroleum ether: ethyl acetate volume ratio = 10:1), and the pH was adjusted to 1-2. After drying, the hapten PYG was obtained.

[0043] A pyraclostrobin artificial antigen, obtained by conjugating the pyraclostrobin hapten PYE with a carrier protein, has the structural formula shown in formula (III).

[0044]

[0045] Wherein, P is a carrier protein, and the carrier protein is bovine serum albumin, lactoferrin, or chicken ovalbumin.

[0046] Preferably, the carrier protein is bovine serum albumin.

[0047] The method for preparing pyraclostrobin artificial antigen with the structural formula shown in formula (Ⅲ) involves coupling the pyraclostrobin hapten PYE with a carrier protein via an active ester method.

[0048]

[0049] Wherein, P is a carrier protein, and the carrier protein is bovine serum albumin, lactoferrin, or chicken ovalbumin.

[0050] Preferably, the active ester method includes the following steps:

[0051] S1. Pyraclostrobin hapten PYE, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N,N-dimethylformamide were reacted thoroughly to obtain solution A; the carrier protein was dissolved in phosphate buffer to obtain solution B.

[0052] S2. After solution A and solution B have reacted completely, they are then dialyzed to obtain the final product.

[0053] More preferably, in step S1, the molar ratio of pyraclostrobin hapten PYE, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(0.5-1.5):(1-3).

[0054] More preferably, in step S1, the molar ratio of pyraclostrobin hapten PYE, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:0.8:1.9.

[0055] Preferably, in step S1, the mass-to-volume ratio of the carrier protein to the phosphate buffer is (8 mg to 12 mg): (1 mL to 2 mL).

[0056] More preferably, in step S1, the mass-to-volume ratio of the carrier protein to the phosphate buffer is 10 mg: 1 mL.

[0057] Preferably, in step S1, the mass ratio of the pyraclostrobin hapten PYE to the carrier protein is (1-2):(1-4).

[0058] More preferably, in step S1, the mass ratio of the pyraclostrobin hapten PYE to the carrier protein is 2:3.

[0059] Specifically, the preparation method of pyraclostrobin artificial antigen with the structural formula shown in formula (Ⅲ) includes the following steps:

[0060] Pyraclostrobin hapten PYE (1 mol) was dissolved in 50–200 μL of N,N-dimethylformamide (DMF) with N-hydroxysuccinimide (NHS) (0.8 mol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (1.9 mol). The solution was stirred in the dark at room temperature (27°C) for 2–4 h to obtain the activated pyraclostrobin hapten PYE solution, denoted as solution A. Carrier protein (10 mg) was added to 1 mL of PBS buffer (0.01 mol / L, pH = 7.4) to obtain the diluted carrier protein solution, denoted as solution B. Solution A was slowly added dropwise to solution B, and the reaction was carried out at 4°C for 12 h. The solution was dialyzed against PBS buffer for 3 days, 3 times a day. The final product was obtained after dialysis.

[0061]

[0062] Wherein, P is a carrier protein, and the carrier protein is bovine serum albumin, lactoferrin, or chicken ovalbumin.

[0063] Another pyraclostrobin artificial antigen, obtained by conjugating the pyraclostrobin hapten PYG with a carrier protein, has the structural formula shown in (Ⅳ).

[0064]

[0065] Wherein, P is a carrier protein, and the carrier protein is bovine serum albumin, lactoferrin, or chicken ovalbumin.

[0066] Preferably, the carrier protein is lactoferrin or chicken ovalbumin.

[0067] More preferably, the carrier protein is chicken ovalbumin.

[0068] The method for preparing pyraclostrobin artificial antigen with the structural formula shown in (Ⅳ) involves coupling the pyraclostrobin hapten PYG with a carrier protein via an active ester method.

[0069]

[0070] Wherein, P is a carrier protein, and the carrier protein is bovine serum albumin, lactoferrin, or chicken ovalbumin.

[0071] Preferably, the active ester method includes the following steps:

[0072] S1. Pyraclostrobin hapten PYG, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N,N-dimethylformamide were reacted thoroughly to obtain solution A; the carrier protein was dissolved in phosphate buffer to obtain solution B.

[0073] S2. After solution A and solution B have reacted completely, they are then dialyzed to obtain the final product.

[0074] More preferably, in step S1, the molar ratio of pyraclostrobin hapten PYG, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(0.5-1.5):(1-3).

[0075] More preferably, in step S1, the molar ratio of pyraclostrobin hapten PYG, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:0.8:1.9.

[0076] Preferably, in step S1, the mass-to-volume ratio of the carrier protein to the phosphate buffer is (8 mg to 12 mg): (1 mL to 2 mL).

[0077] More preferably, in step S1, the mass-to-volume ratio of the carrier protein to the phosphate buffer is 10 mg: 1 mL.

[0078] Preferably, in step S1, the mass ratio of the pyraclostrobin hapten PYG to the carrier protein is (1-2):(1-4).

[0079] More preferably, in step S1, the mass ratio of the pyraclostrobin hapten PYE to the carrier protein is 2:3.

[0080] Specifically, the preparation method of pyraclostrobin artificial antigen with the structural formula shown in formula (Ⅲ) includes the following steps:

[0081] Pyraclostrobin hapten PYG (1 mol) was dissolved in 50–200 μL of N,N-dimethylformamide (DMF) with N-hydroxysuccinimide (NHS) (0.8 mol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (1.9 mol). The mixture was stirred in the dark at room temperature (27°C) for 2–4 h to obtain the activated pyraclostrobin hapten PYE solution, denoted as solution A. Carrier protein (10 mg) was added to 1 mL of PBS buffer (0.01 mol / L, pH = 7.4) to obtain the diluted carrier protein solution, denoted as solution B. Solution A was slowly added dropwise to solution B, and the reaction was carried out at 4°C for 12 h. The solution was dialyzed against PBS buffer for 3 days, 3 times a day. The final product was obtained after dialysis.

[0082]

[0083] Wherein, P is a carrier protein, and the carrier protein is bovine serum albumin, lactoferrin, or chicken ovalbumin.

[0084] The use of any of the pyraclostrobin artificial antigens in the preparation of pyraclostrobin antibodies should also be within the scope of protection of this invention.

[0085] A pyraclostrobin antibody is prepared by immunizing animals with any of the pyraclostrobin artificial antigens.

[0086] Preferably, the pyraclostrobin artificial antigen is obtained by conjugating the pyraclostrobin hapten PYE with a carrier protein.

[0087] More preferably, the carrier protein is bovine serum albumin, namely pyraclostrobin artificial antigen PYE-BSA.

[0088] Preferably, the pyraclostrobin antibody is a monoclonal antibody. Hybridoma cells are obtained by immunizing an animal with any of the pyraclostrobin artificial antigens, the obtained hybridoma cells are cultured and collected for animal immunization to obtain ascites, and after identification and purification, the pyraclostrobin monoclonal antibody is obtained.

[0089] More preferably, if the pyraclostrobin antibody is a monoclonal antibody, then hybridoma cells are obtained by immunizing animals with the pyraclostrobin artificial antigen PYE-BSA, the obtained hybridoma cells are cultured and collected for animal immunization to obtain ascites, and after identification and purification, the pyraclostrobin monoclonal antibody is obtained.

[0090] The application of any of the pyraclostrobin antibodies in the detection of pyraclostrobin should also be within the scope of protection of this invention, wherein the detection is for non-disease treatment diagnosis purposes.

[0091] The use of any of the pyraclostrobin antibodies in the preparation of kits for detecting pyraclostrobin should also be within the scope of protection of this invention.

[0092] A pyraclostrobin artificial antigen combination comprises an immunogen and a coating antigen, wherein the immunogen is a pyraclostrobin artificial antigen with the structural formula shown in formula (III); and the coating antigen is a pyraclostrobin artificial antigen with the structural formula shown in formula (III) or the structural formula shown in formula (IV).

[0093]

[0094]

[0095] Wherein, P is a carrier protein, and the carrier protein is bovine serum albumin, lactoferrin, or chicken ovalbumin.

[0096] Preferably, the carrier protein of the immunogen is bovine serum albumin, that is, the immunogen is pyraclostrobin artificial antigen PYE-BSA.

[0097] Preferably, the carrier protein of the coating antigen is lactoferrin or chicken egg albumin, that is, the coating antigen is pyraclostrobin artificial antigen PYG-BSA, PYG-LF, PYE-OVA or PYG-OVA.

[0098] More preferably, the coating is a pyraclostrobin artificial antigen with the structural formula shown in (Ⅳ);

[0099]

[0100] Wherein, P is a carrier protein, which is lactoferrin or chicken egg albumin, i.e., the coating antigen is pyraclostrobin artificial antigen PYG-LF or PYG-OVA.

[0101] More preferably, the carrier protein is chicken ovalbumin, i.e., the coating antigen is pyraclostrobin artificial antigen PYG-OVA.

[0102] Most preferably, the carrier protein of the immunogen is bovine serum albumin, i.e., the immunogen is pyraclostrobin artificial antigen PYE-BSA; the carrier protein of the coating antigen is chicken ovalbumin with the structural formula shown in (Ⅳ) as pyraclostrobin artificial antigen, i.e., the coating antigen is pyraclostrobin artificial antigen PYG-OVA.

[0103]

[0104] Wherein, P is the carrier protein.

[0105] The application of any of the pyraclostrobin artificial antigen combinations in the detection of pyraclostrobin should also be within the scope of protection of this invention, wherein the detection is for non-disease treatment diagnosis purposes.

[0106] An immunoassay method for detecting pyraclostrobin, using any of the pyraclostrobin artificial antigen combinations for detection, said detection being for the purpose of non-disease treatment diagnosis.

[0107] The use of any of the described pyraclostrobin artificial antigen combinations in the preparation of kits for detecting pyraclostrobin should also be within the scope of protection of this invention.

[0108] A kit for detecting pyraclostrobin includes an immunogen and a coating antigen, wherein the immunogen is a pyraclostrobin artificial antigen with the structural formula shown in formula (III); and the coating antigen is a pyraclostrobin artificial antigen with the structural formula shown in formula (III) or the structural formula shown in formula (IV).

[0109]

[0110] Wherein, P is a carrier protein, and the carrier protein is bovine serum albumin, lactoferrin, or chicken ovalbumin.

[0111] Preferably, the carrier protein of the immunogen is bovine serum albumin, that is, the immunogen is pyraclostrobin artificial antigen PYE-BSA.

[0112] Preferably, the carrier protein of the coating antigen is lactoferrin or chicken egg albumin, that is, the coating antigen is pyraclostrobin artificial antigen PYG-BSA, PYG-LF, PYE-OVA or PYG-OVA.

[0113] More preferably, the coating is a pyraclostrobin artificial antigen with the structural formula shown in (Ⅳ);

[0114]

[0115] Wherein, P is a carrier protein, which is lactoferrin or chicken egg albumin, i.e., the coating antigen is pyraclostrobin artificial antigen PYG-LF or PYG-OVA.

[0116] More preferably, the carrier protein is chicken ovalbumin, i.e., the coating antigen is pyraclostrobin artificial antigen PYG-OVA.

[0117] Most preferably, the carrier protein of the immunogen is bovine serum albumin, i.e., the immunogen is pyraclostrobin artificial antigen PYE-BSA; the carrier protein of the coating antigen is chicken ovalbumin with the structural formula shown in (Ⅳ) as pyraclostrobin artificial antigen, i.e., the coating antigen is pyraclostrobin artificial antigen PYG-OVA.

[0118]

[0119] Wherein, P is a carrier protein, and the carrier protein is chicken ovalbumin.

[0120] Preferably, the kit contains an antibody obtained by immunizing an animal with the immunogen.

[0121] More preferably, if the antibody is a monoclonal antibody, it is obtained by immunizing mice with the pyraclostrobin hapten PYE-BSA conjugated to bovine serum albumin, screening the cells after cell fusion, injecting the antibody into the mice intraperitoneally, and collecting the ascites fluid.

[0122] Preferably, the kit further comprises an enzyme-labeled plate, pyraclostrobin standard, and substrate chromogenic solution.

[0123] More preferably, the enzyme-labeled plate is coated with the coating antigen.

[0124] More preferably, the enzyme-labeled plate is coated with a coating antigen, which is pyraclostrobin artificial antigen PYG-BSA, PYG-LF, PYE-OVA or PYG-OVA.

[0125] More preferably, the enzyme-labeled plate is coated with a coating antigen, which is pyraclostrobin artificial antigen PYG-LF or PYG-OVA.

[0126] Most preferably, the enzyme-labeled plate is coated with a coating antigen, which is pyraclostrobin artificial antigen PYG-OVA.

[0127] More preferably, the substrate colorimetric solution comprises urea peroxide and tetramethylbenzidine.

[0128] More preferably, the kit further comprises a stop solution, a washing solution, a blocking solution, a secondary antibody, and an antibody diluent.

[0129] More preferably, the termination liquid fraction is 1 mol / L to 3 mol / L H2SO4.

[0130] Most preferably, the termination liquid fraction is 2 mol / L H2SO4.

[0131] More preferably, the washing solution is a phosphate buffer containing 0.5% to 1.0% Tween-20 by volume, 0.01‰ to 0.03‰ sodium azide preservative by mass, and 0.1 mol / L to 0.3 mol / L, with a pH of 7.2 to 7.6.

[0132] Most preferably, the washing solution is a phosphate buffer containing 0.5% Tween-20 by volume, 0.01% sodium azide preservative by mass, and 0.1 mol / L, with a pH of 7.4.

[0133] More preferably, the blocking solution is a phosphate buffer containing 1 wt% to 6 wt% casein and 0.1 mol / L to 0.3 mol / L, with a pH of 7.1 to 7.5.

[0134] Most preferably, the blocking solution is a phosphate buffer containing 6 wt% casein and 0.1 mol / L, with a pH of 7.4.

[0135] More preferably, the secondary antibody is a horseradish peroxidase-labeled goat anti-mouse secondary antibody.

[0136] More preferably, the antibody diluent is a phosphate buffer containing 0.5%–1.0% Tween-20 by volume, 0.01‰–0.03‰ sodium azide preservative by mass, and 0.1 mol / L–0.3 mol / L, with a pH of 7.2–7.6.

[0137] Most preferably, the antibody diluent is 0.5% Tween-20, 0.01% sodium azide preservative, 0.1 mol / L phosphate buffer, and has a pH of 7.4.

[0138] Compared with the prior art, the present invention has the following beneficial effects:

[0139] This invention simplifies the synthesis steps of pyraclostrobin haptens, provides two novel pyraclostrobin haptens, prepares pyraclostrobin-specific antibodies with high titer, strong specificity and high affinity, and establishes an indirect competitive ELISA detection method for pyraclostrobin. The limit of detection (LOD) is 0.23 ng / mL, the quantitative detection range is 0.57–12.57 ng / mL, there is no cross-reactivity with pyraclostrobin analogues, and the detection limit is lower, the sensitivity is higher and the specificity is stronger. Attached Figure Description

[0140] Figure 1 It refers to pyraclostrobin and its structural analogues (the red part is the common skeleton).

[0141] Figure 2 This describes the synthetic route for the pyraclostrobin hapten PYE.

[0142] Figure 3 This describes the synthetic route for the pyraclostrobin hapten PYG.

[0143] Figure 4This is a UV scan image of the identification results of pyraclostrobin artificial antigen PYE-BSA.

[0144] Figure 5 This is a UV scan image of the identification results of the artificial antigen PYE-LF of pyraclostrobin.

[0145] Figure 6 This is a UV scan image of the identification results of the artificial antigen PYE-OVA of pyraclostrobin.

[0146] Figure 7 This is a UV scan image of the identification results of the artificial antigen PYG-BSA of pyraclostrobin.

[0147] Figure 8 This is a UV scan image of the identification results of the artificial antigen PYG-LF of pyraclostrobin.

[0148] Figure 9 This is a UV scanning identification result of the artificial antigen PYG-OVA of pyraclostrobin.

[0149] Figure 10 The standard curve for indirect competitive ELISA using monoclonal antibodies against pyraclostrobin. Detailed Implementation

[0150] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0151] Example 1: Synthesis and Identification of Pyraclostrobin Hapten

[0152] I. Synthesis and Identification of Pyraclostrobin Hapten (PYE)

[0153] 1. Synthesis of pyraclostrobin hapten PYE

[0154] The synthetic route of pyraclostrobin hapten PYE is as follows: Figure 2 As shown, the specific steps are as follows:

[0155] 2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)aniline (CAS No.: 222838-33-7) (0.67 mmol) and ethyl 4-bromobutyrate (1.005 mmol) were dissolved in acetonitrile, and potassium carbonate (0.33 mmol) and sodium iodide (0.067 mmol) were added. The mixture was reacted overnight (12 h) at 50 °C under nitrogen protection. The reactants were separated and purified, and then hydrolyzed with 5 mL of 1 mol / L lithium hydroxide solution (LiOH-H2O) and 10 mL of 50% v / v methanol aqueous solution at room temperature (27 °C) for 3-5 h. The mixture was then separated and purified by column chromatography (stationary phase: silica gel, mobile phase: petroleum ether: ethyl acetate volume ratio = 10:1), and the pH was adjusted to 1-2. After drying, the hapten PYE was obtained.

[0156] 2. Identification of pyraclostrobin hapten PYE

[0157] The 1H NMR spectrum of the pyraclostrobin hapten PYE is as follows: 1 H NMR(600MHz, DMSO-d6)8.32(d,J=3.1Hz,1H),7.67-7.44(m,2H),7.41-7.32(m,2H),7.21-6 .82(m,4H),6.78(m,1H),5.27-5.15(m,2H),3.42(s,2H),2.41(s,2H),,1.91-1.52(m,2H).

[0158] The mass spectrometry results for the pyraclostrobin hapten PYE were as follows: MS: C 20 H 20 N3O3Cl: 385.12, ESI-[M+H]+: 386.12.

[0159] The mass spectrometry and NMR results show that the relative molecular mass of the hapten PYE is 385.12 Da. Structural identification using electrospray ionization mass spectrometry (ESI-MS) revealed the corresponding molecular ion peak in its positive mass spectrum, indicating that the pyraclostrobin hapten PYE was successfully prepared. Its structural formula is shown in formula (Ⅰ).

[0160]

[0161] The pyrazol-methyl hapten PYE was named systematically as 4-((2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)phenyl)amino)butanoic acid, i.e. 4-((2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)phenyl)amino)butanoic acid.

[0162] II. Synthesis and Identification of Pyraclostrobin Hapten PYG

[0163] 1. Synthesis of pyraclostrobin hapten PYG

[0164] The synthetic route of pyraclostrobin hapten PYG is as follows: Figure 3 As shown, the specific steps are as follows:

[0165] 2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)aniline (CAS No.: 222838-33-7) (0.67 mmol): ethyl bromoacetate (1.005 mmol) was dissolved in acetonitrile, and potassium carbonate (0.33 mmol) and sodium iodide (0.067 mmol) were added. The mixture was reacted overnight (12 h) at 60 °C under nitrogen protection. The reactants were separated and purified, and then hydrolyzed with 5 mL of 1 mol / L lithium hydroxide solution (LiOH-H2O) and 10 mL of 50% v / v methanol aqueous solution at room temperature (27 °C) for 3-5 h. The mixture was then separated and purified by column chromatography (stationary phase: silica gel, mobile phase: petroleum ether: ethyl acetate volume ratio = 10:1), and the pH was adjusted to 1-2. After drying, the hapten PYG was obtained.

[0166] 2. Identification of pyraclostrobin hapten PYG

[0167] The 1H NMR spectrum of the pyraclostrobin hapten PYG is as follows: 1 H NMR(600MHz,DMSO-d6)8.40(d,J=2.7Hz,1H),7.57-7.45(m,2H),7.39-7.2 5(m,2H),7.15-6.72(m,4H),6.68(m,1H),5.24-5.12(s,2H),4.02(s,2H).

[0168] The mass spectrometry results for the pyraclostrobin hapten PYG were as follows: MS: C 18 H 16 N3O3Cl: 357.09, ESI-[M+H]+: 358.09.

[0169] The mass spectrometry and NMR results show that the relative molecular mass of the hapten PYG is 357.09 Da. Structural identification using electrospray ionization mass spectrometry (ESI-MS) revealed the corresponding molecular ion peak in its positive mass spectrum, indicating that the pyraclostrobin hapten PYG was successfully prepared. Its structural formula is shown in formula (II).

[0170]

[0171] The pyrazol-methyl hapten PYG was named systematically as: 4-((2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)phenyl)aminoacetic acid; that is, (2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)phenyl)glycine.

[0172] Example 2: Synthesis and Identification of Pyraclostrobin Artificial Antigen

[0173] 1. Synthesis of pyraclostrobin artificial antigen

[0174] The pyraclostrobin hapten PYE and pyraclostrobin hapten PYG prepared in Example 1 were conjugated with bovine serum albumin (BSA), lactoferrin (LF), or chicken ovalbumin (OVA) via the active ester method. The specific steps are as follows:

[0175] Weigh 1 mol of the pyraclostrobin hapten PYE prepared in Example 1 and dissolve it in 200 μL of N,N-dimethylformamide (DMF) with 0.8 mol of N-hydroxysuccinimide (NHS) and 1.9 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). Stir at room temperature (27°C) in the dark for 4 h to obtain the activated pyraclostrobin hapten PYE solution, denoted as solution A. Add 10 mg of BSA to 1 mL of PBS buffer (0.01 mol / L, pH = 7.4) to obtain the carrier protein dilution solution, denoted as solution B. Slowly add solution A dropwise to solution B and react at 4°C for 12 h. Dialyze with PBS buffer for 3 days, 3 times a day. After dialysis, the pyraclostrobin artificial antigen PYE-BSA is obtained. Then aliquot into centrifuge tubes and store at -20°C for use. The PBS buffer formula is as follows: Na2HPO4·12H2O 2.90g, NaCl 8.50g, KCl 0.20g, KH2PO4 0.20g, and distilled water is added to bring the volume to 1000mL.

[0176] The only difference in the above method is that the pyraclostrobin hapten PYG is used instead of PYE to prepare the pyraclostrobin artificial antigen PYG-BSA; LF is used instead of BSA to prepare the pyraclostrobin artificial antigens PYE-LF and PYG-LF; and OVA is used instead of BSA to prepare the pyraclostrobin artificial antigens PYE-OVA and PYG-OVA.

[0177] 2. Identification of pyraclostrobin artificial antigen

[0178] BSA, OVA, LF, pyraclostrobin hapten PYE, PYG, pyraclostrobin artificial antigen PYE-BSA, PYG-BSA, PYE-LF, PYG-LF, PYE-OVA and PYG-OVA were scanned and identified using the ultraviolet full-wavelength method (200-350 nm).

[0179] like Figures 4-6 As shown, by comparing the highest absorbance values ​​of each substance before and after conjugation, it was found that the absorption curves of PYE-BSA, PYE-LF, and PYE-OVA were significantly different from those of the carrier proteins BSA, LF, and OVA. PYE has a characteristic peak at 330 nm, while after conjugation with the carrier protein, the absorption peaks of PYE-BSA, PYE-LF, and PYE-OVA at 335 nm and 280 nm showed significant shifts relative to the curves of the carrier protein and the hapten PYE. Since the unreacted drug and other small molecule components were completely removed by dialysis during the post-conjugation dialysis process, the drug characteristic peaks appearing in the conjugated products are contributed by the protein-bound drug molecules, indicating that the reaction products are complexes of the carrier protein and PYE, and that the conjugation of PYE-BSA, PYE-LF, and PYE-OVA was successful.

[0180] like Figures 7-9 As shown, by comparing the highest absorbance values ​​of each substance before and after coupling, it was found that the absorption curves of PYG-BSA, PYG-LF, and PYG-OVA were significantly different from those of the carrier proteins BSA, LF, and OVA. The hapten PYG showed strong absorption above 300 nm, while after coupling with the carrier protein, the absorption peaks of PYG-BSA, PYG-LF, and PYG-OVA were significantly higher than those of OVA at 320 nm and 280 nm, and the curves relative to the hapten PYG showed significant shifts. Since unreacted drugs and other small molecules were completely removed during the dialysis process of the coupling reaction, the characteristic drug peaks appearing in the coupling products were contributed by protein-bound drug molecules, indicating that the reaction products are complexes of the carrier protein and the hapten PYG, and that the coupling of PYG-BSA, PYG-LF, and PYG-OVA was successful.

[0181] The above results demonstrate that the present invention successfully prepared pyraclostrobin artificial antigens PYE-BSA, PYE-LF, and PYE-OVA, the structural formulas of which are shown in formula (III).

[0182]

[0183] Where P represents the carrier protein BSA, LF, or OVA.

[0184] This invention also successfully prepared pyraclostrobin artificial antigens PYG-BSA, PYG-LF, and PYG-OVA, whose structural formulas are shown in formula (Ⅳ).

[0185]

[0186] Where P represents the carrier protein BSA, LF, or OVA.

[0187] Example 3: Preparation of monoclonal antibodies for detecting pyraclostrobin

[0188] Female Balb / c mice were immunized with the pyraclostrobin artificial antigens PYE-BSA, PYE-LF, PYG-BSA, and PYG-LF prepared in Example 2. The pyraclostrobin artificial antigens PYE-BSA (or PYE-LF, PYG-BSA, or PYG-LF) were emulsified with an equal volume of adjuvant (full Freund's adjuvant for the initial immunization, and incomplete Freund's adjuvant for booster immunizations). Mice were then immunized via subcutaneous injection at multiple sites in the abdomen. One week after each booster immunization, blood was collected from the tail to determine the antiserum titer. Once the titer stabilized, mice with the best immunization response were selected for a booster immunization. Three days later, spleen cells from the mice were fused with myeloma cells. The selected positive hybridoma cells were then injected intraperitoneally into the mice, and ascites fluid was collected to obtain monoclonal antibodies against the four different immunogens.

[0189] Example 4: Optimization of the combination of pyraclostrobin immunogen and coating agent

[0190] I. Experimental Methods

[0191] Using the pyraclostrobin artificial antigens PYE-BSA, PYG-BSA, PYE-LF, PYG-LF, PYE-OVA, and PYG-OVA prepared in Example 2 as coating antigens, the titers and inhibition rates of the antiserum obtained by immunizing Balb / c mice were detected by ELISA using monoclonal antibodies against four different immunogens prepared in Example 3. The specific operating steps are as follows:

[0192] 1. Dilute each coating agent to a concentration of 250 ng / mL with coating buffer (0.05 M carbonate buffer, pH 9.6), and coat a 96-well microplate with 100 μL / well. Incubate overnight (12 h) at 37°C in a water bath. Discard the coating buffer and wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)).

[0193] 2. Add 120 μL of blocking solution (PBST containing 6 wt% skim milk powder) to each well, block at 37°C for 3 hours, discard the blocking solution, plate, and dry in a drying oven at 37°C for later use.

[0194] 3. The monoclonal antibody prepared in Example 3 was diluted with PBST at ratios of 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000 and 1:256000, respectively. Blank control wells (replaced with PBST) were also set up. The 1 mg / mL pyraclostrobin standard was diluted 1000 times with PBST to a concentration of 1 μg / mL.

[0195] 4. Potency column setup: First, add 50 μL of PBST to each well. Then, add 50 μL of pyraclostrobin monoclonal antibody at different serial dilutions to each well. Do not add antibody to the last well; replace it with 50 μL of PBST.

[0196] 5. Inhibition column setup: First, add 50 μL of pyraclostrobin standard to each well. Then, add 50 μL of pyraclostrobin monoclonal antibody diluted at different ratios to each well. Do not add antibody to the last well; instead, add 50 μL of PBST.

[0197] 6. Incubate at 37℃ for 40 minutes, wash 5 times, and then plate.

[0198] 7. Add goat anti-mouse secondary antibody IgG-HRP (5000-fold dilution), incubate at 37℃ for 30 minutes, wash 5 times, and plate.

[0199] 8. Add the color developing solution and incubate at 37°C for 10 minutes.

[0200] 9. Terminate the reaction by adding 10% v / v H2SO4 and read the OD value at 450 nm; calculate the titer and inhibition rate. The titer is the OD value. 450 The antibody dilution factor corresponding to approximately 1.0 is: inhibition rate = (OD value of titer - OD value of inhibition) / OD value of inhibition × 100%.

[0201] II. Experimental Results

[0202] Table 1. ELISA detection results of different combinations of immunogens and coating antigens.

[0203]

[0204]

[0205] As can be seen from Table 1, the antisera produced by the artificial antigens PYE-BSA, PYE-LF, PYG-BSA, and PYG-LF of pyraclostrobin all have certain titers, and the obtained antisera all have different degrees of inhibitory effect on the target analyte pyraclostrobin.

[0206] Combinations of immunogens and coating agents numbered 1, 2, 3, and 4 all exhibited antiserum titers higher than 1:8000 and inhibition rates higher than 38%, demonstrating good antibody sensitivity and specific recognition of the target analyte, pyraclostrobin. Among these, the combination of immunogen and coating agent number 2 (PYE-BSA as the immunogen and PYG-OVA as the coating agent) showed the highest antiserum titer and inhibition rate, making it the optimal combination.

[0207] Example 5: Establishment of an indirect competitive ELISA method for detecting pyraclostrobin.

[0208] I. An indirect competitive ELISA method for detecting pyraclostrobin, comprising the following steps:

[0209] 1. Using the pyraclostrobin artificial antigens PYE-LF, PYG-LF, PYE-OVA or PYG-OVA prepared in Example 2 as coating agents, dilute with coating buffer to 62.5 ng / mL, coat 96-well microplates, add 100 μL to each well, incubate at 37°C overnight (12 h), discard the coating buffer, and wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v));

[0210] 2. Add 120 μL of blocking solution (PBST containing 6 wt% skim milk powder) to each well, block at 37°C for 3 h, discard the blocking solution, plate, dry at 37°C for 30 min, and then pack in a resealable bag for later use.

[0211] 3. The monoclonal antibody prepared in Example 3 using pyraclostrobin artificial antigen PYE-BSA as an immunogen was diluted with PBST at a ratio of 1:4000 to obtain the antibody dilution solution; and the pyraclostrobin standard was diluted to 1000 ng / mL, 166.67 ng / mL, 27.27 ng / mL, 4.63 ng / mL, 0.77 ng / mL, 0.13 ng / mL, 0.02 ng / mL, 0.003 ng / mL, 0.0006 ng / mL and 0.0001 ng / mL to obtain the pyraclostrobin standard dilution solution.

[0212] 4. Add 50 μL of dilution buffer of pyraclostrobin standard at different concentrations to each row (three sets in parallel), then add 50 μL of antibody dilution buffer per well, incubate at 37℃ for 40 min, wash five times, and pat dry.

[0213] 5. Add 100 μL / well of goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST), incubate at 37℃ for 30 min, wash five times, and pat dry;

[0214] 6. Add 100 μL of colorimetric reagent per well and develop for 10 min;

[0215] 7. Add 50 μL of 10% H2SO4 solution to terminate the reaction and read the OD value at 450 nm.

[0216] 8. ELISA standard curve plotting: Plot B / B0 as the ordinate (B represents the absorbance OD of different concentrations of pyraclostrobin standards). 450 B0 is the absorbance value (OD) of the blank control well. 450 The logarithm of the standard concentration is used as the abscissa. The Logistic function is used for curve fitting to obtain the formula of the standard curve and the standard curve is prepared.

[0217] II. Test Results

[0218] The standard curve prepared using the combination of the most effective immunogen and coating agent in Example 4 (i.e., PYE-BSA as the immunogen and PYG-OVA as the coating agent) is shown below. Figure 10 As shown, the limit of detection (LOD) of the antibody used to detect pyraclostrobin is 0.23 ng / mL, and the quantitative detection range is 0.57 ng / mL to 12.57 ng / mL. This indicates that the antibody prepared by this invention for detecting pyraclostrobin can meet the detection requirements, and has high recognition ability, strong specificity, and high detection sensitivity for pyraclostrobin.

[0219] Example 6: Specificity evaluation of antibodies used to detect pyraclostrobin.

[0220] 1. Experimental Methods

[0221] Azoxystrobin, pyraclostrobin, oxadiazon, azoxystrobin, acetamiprid, bensulfuron-methyl, pyraclostrobin, and eugenol are all analogs of pyraclostrobin. The specificity of the antibody prepared in this invention for detecting pyraclostrobin was determined by cross-reactivity experiments.

[0222] The method of Example 5 was followed, except that the pyraclostrobin standard was replaced with standards for azoxystrobin, pyraclostrobin, oxadiazon, azoxystrobin, fenpyroxime, pyraclostrobin, pyraclostrobin, and eugenol, and the tests were performed at the same dilution factor to obtain the IC50 values ​​for each structurally similar compound. 50Value. Calculate the cross-reactivity (CR) of pyraclostrobin using the following formula: CR(%) = IC50 50 (Pyraclostrobin) / IC 50 (Structural analogue) × 100%, the smaller the cross-reactivity rate, the stronger the specificity.

[0223] 2. Experimental Results

[0224] Table 2. Cross-reactivity results of pyraclostrobin monoclonal antibodies with pyraclostrobin and its analogues.

[0225]

[0226] Note: NR indicates no reaction, meaning the antibody does not recognize the analogue.

[0227] Table 2 shows that the monoclonal antibody used to detect pyraclostrobin has a cross-reactivity of 100% with pyraclostrobin, IC50... 50 The concentration was 2.69 ng / mL, and there was no cross-contamination with pyraclostrobin analogues such as azoxystrobin, pyraclostrobin, oxadiazon, azoxystrobin, acetamiprid, benzopyrethroid, azoxystrobin, and eugenol. This indicates that the antibody used to detect pyraclostrobin has high recognition ability and strong specificity for pyraclostrobin, and can effectively eliminate the interference of pyraclostrobin analogues such as azoxystrobin, pyraclostrobin, oxadiazon, azoxystrobin, acetamiprid, benzopyrethroid, azoxystrobin, and eugenol on the detection of pyraclostrobin. It can be used specifically for the detection of pyraclostrobin.

[0228] Example 7: An ELISA kit for detecting pyraclostrobin

[0229] 1. Composition

[0230] (1) ELISA plates coated with the coating antigen:

[0231] The enzyme-labeled plate was prepared by the following method:

[0232] Using the pyraclostrobin artificial antigens PYE-LF, PYG-LF, PYE-OVA, or PYG-OVA prepared in Example 2 as coating agents, the antigens were diluted to 62.5 ng / mL with coating buffer and coated with 100 μL of each agent in a 96-well microplate. The plates were incubated overnight (12 h) at 37°C. The coating buffer was discarded, and the plates were washed twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)) for 30 s each time. The plates were then blotted dry, and 200 μL of blocking buffer was added to each well. The plates were incubated at 25°C in the dark for 2 h. The liquid in the wells was discarded, and the plates were blotted dry. After drying, the plates were vacuum-sealed with aluminum foil. The coating buffer was 0.05 mol / L carbonate buffer (pH 9.6), and the blocking buffer was 0.1 mol / L phosphate buffer (pH 7.1–7.5) containing 6 wt% casein.

[0233] (2) Standard products:

[0234] Pyraclostrobin standard solution: 8 concentration gradients, namely 1000 μg / L, 200 μg / L, 40 μg / L, 8 μg / L, 1.6 μg / L, 0.32 μg / L, 0.064 μg / L and 0.0128 μg / L;

[0235] (3) Pyraclostrobin antibody:

[0236] Example 3: Monoclonal antibody prepared using pyraclostrobin artificial antigen PYE-BSA as an immunogen;

[0237] (4) Secondary antibody:

[0238] Horseradish peroxidase-labeled goat anti-mouse secondary antibody;

[0239] (5) Substrate colorimetric solution: composed of solution A and solution B, where solution A is urea peroxide and solution B is tetramethylbenzidine;

[0240] (6) Termination solution

[0241] 2 mol / L H2SO4;

[0242] (7) Washing liquid

[0243] The pH value is 7.4, and it contains 0.5% Tween-20, 0.01‰ sodium azide preservative, and 0.1 mol / L phosphate buffer, where the percentages are weight-volume percentages.

[0244] (8) Antibody diluent

[0245] The solution used to dilute pyraclostrobin antibodies and secondary antibodies is a 0.1 mol / L phosphate buffer solution containing 0.5% Tween-20 and 0.01% sodium azide preservative, at pH 7.4.

[0246] 2. Instructions for use

[0247] (1) Sample testing

[0248] Number the wells corresponding to the samples and standards in this kit sequentially. Perform two parallel wells for each sample and standard, and record the positions of the standard and sample wells. Dilute the pyraclostrobin antibody with the required amount of diluent at a volume ratio of 1:64000 (i.e., add 1 part pyraclostrobin antibody to 64000 parts antibody diluent; prepare fresh before use) to obtain the pyraclostrobin antibody working solution. Dilute the secondary antibody with the required amount of diluent at a volume ratio of 1:5000 (i.e., add 1 part secondary antibody to 5000 parts antibody diluent; prepare fresh before use) to obtain the secondary antibody working solution.

[0249] Add 50 μL of standard or sample to the corresponding well, then add 50 μL of pyraclostrobin antibody working solution to the corresponding well. Gently shake to mix, cover with a cover film, and incubate at 25°C in the dark for 40 min. Shake off the liquid in the wells, and add 250 μL of washing buffer per well. Wash thoroughly 4–5 times, with 10-second intervals between each wash. Discard the washing buffer from the wells and pat dry with absorbent paper (any remaining air bubbles can be punctured with an unused pipette tip).

[0250] Add 100 μL of secondary antibody working solution per well to the corresponding microwell, gently shake to mix, cover with a cover plate and incubate at 25°C in the dark for 30 min. Shake off the liquid in the wells, add 250 μL of washing solution per well. Wash thoroughly 4–5 times, with 10-second intervals between each wash. Discard the washing solution and working solution in the wells, and pat dry with absorbent paper (any remaining air bubbles can be punctured with an unused pipette tip).

[0251] Add 50 μL of substrate development solution A per well, then add 50 μL of substrate development solution B per well, gently shake to mix, cover with a cover plate and incubate at 25°C in the dark for 10 min.

[0252] Add 50 μL of stop solution per well, gently shake to mix, set the microplate reader to 450 nm, and measure the OD value of each well.

[0253] (2) Plotting the standard curve

[0254] Plot B / B0 as the ordinate (B represents the absorbance OD of different concentrations of pyraclostrobin standards). 450 B0 is the absorbance value (OD) of the blank control well. 450 The logarithm of the standard concentration is used as the abscissa. The Logistic function is used for curve fitting to obtain the formula of the standard curve and the standard curve is prepared.

[0255] (3) Calculation of sample concentration

[0256] The absorbance OD value of the sample was measured. 450 Substitute the values ​​into the above calculation formula to calculate the percentage absorbance of the sample; substitute the percentage absorbance of the sample into the formula of the above standard curve to obtain the concentration of the sample, and then multiply it by the corresponding dilution factor to obtain the actual concentration of pyraclostrobin in the test sample.

[0257] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A pyraclostrobin hapten, characterized in that, The structural formula is shown as formula (I), Formula (I).

2. A pyraclostrobin hapten, characterized in that, The structural formula is shown as formula (II), Formula (II).

3. Process for the preparation of a compound of formula (I) characterized in that, The method comprises the following steps: 2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl) aniline, ethyl 4-bromobutyrate, potassium carbonate and sodium iodide are fully reacted with acetonitrile, and the obtained reaction product is fully hydrolyzed in an alkaline environment, and then the pH is adjusted to be acidic, thereby obtaining the product; Formula (I).

4. A process for the preparation of a compound of formula (II) ###0002### (II) characterized in that, The method comprises the following steps: 2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl) aniline, ethyl 4-bromobutyrate, potassium carbonate and sodium iodide are fully reacted with acetonitrile, and the obtained reaction product is fully hydrolyzed in an alkaline environment, and then the pH is adjusted to be acidic, thereby obtaining the product; Formula (II).

5. A synthetic antigen of pyraclostrobin, characterized in that, The pyraclostrobin hapten conjugated carrier protein is obtained from the pyraclostrobin hapten conjugated carrier protein according to claim 1, and the structural formula is shown as formula (III), Formula (III), wherein P is a carrier protein, and the carrier protein is bovine serum albumin, lactoferrin or chicken egg white protein.

6. A synthetic antigen of pyraclostrobin, characterized in that, The pyraclostrobin hapten conjugated carrier protein is obtained from the pyraclostrobin hapten conjugated carrier protein according to claim 2, and the structural formula is shown as formula (IV), Formula (IV), wherein P is a carrier protein, and the carrier protein is bovine serum albumin, lactoferrin or chicken egg white protein.

7. The pyraclostrobin artificial antigen according to claim 5 or claim 6 is used for preparing an antibody of pyraclostrobin.

8. A combination of artificial antigens of pyraclostrobin, characterized in that, The immunogen is the pyraclostrobin artificial antigen according to claim 5, and the coating antigen is the pyraclostrobin artificial antigen according to claim 5 or claim 6.

9. The pyraclostrobin artificial antigen combination according to claim 8 is used for preparing a kit for detecting pyraclostrobin.

10. A kit for detecting pyraclostrobin, characterized by The kit comprises the pyraclostrobin artificial antigen combination according to claim 8.

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