Cyenopyrafen hapten, antigen and antibody as well as preparation method and application thereof

Abstract

The invention discloses a cyenopyrafen hapten, a cyenopyrafen antigen, a cyenopyrafen antibody, a preparation method and an application thereof. The preparation method comprises the following steps: reacting a hydrolysate B with an ester group-containing compound to obtain a carboxyl-containing hapten compound with a structural formula shown as A1; carrying out first coupling reaction on the hapten compound A1 and N-hydroxysuccinimide to obtain a precursor compound; the precursor compound and a carrier protein are subjected to a second coupling reaction to obtain an antigen with a structural formula shown as A2, and # imgabs1 # can be further prepared into a cyenopyrafen antibody with good specificity and high sensitivity through the preparation method, so that rapid, accurate and low-cost detection of cyenopyrafen is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of cypermethrin detection reagents, and in particular to cypermethrin hapten, antigen and antibody, and preparation methods and applications thereof. Background Art

[0002] Cyenopyrafen, commonly known as cyenopyrafen, is a new pyrazole acaricide developed by Nissan Chemical in 2009. The pure product is white crystals with a melting point of 93-94°C and is stable for 14 days at 54°C. Its main formulations include suspension concentrate, emulsifiable concentrate, and powder. Its chemical name is (E)-2-(4-tert-butylphenyl)-2-cyano-1(1,3,4-trimethylpyrazol-5-yl)enyl 2,2-dimethylpropionate, and its structural formula is as follows:

[0003]

[0004] A contact acaricide, cypermethrin is activated through metabolism into its hydroxyl form. This hydroxyl form inhibits mitochondria by disrupting Complex II (succinate dehydrogenase) in the respiratory electron transport chain. Following hydrolysis of the tert-butyl ester, it exhibits a strong inhibitory effect on mitochondrial protein Complex II, hindering electron transport and disrupting oxidative phosphorylation. It is effective in controlling various mite pests on fruits, citrus fruits, tea leaves, and vegetables.

[0005] On the other hand, some existing studies believe that cypermethrin is a type of pesticide that may be carcinogenic to humans. Research on cypermethrin at home and abroad mainly focuses on the analysis of its original drug and formulations and its field efficacy, and there is little research on its residual degradation in crops.

[0006] At present, the main methods for detecting the residues of cypermethrin in agricultural products are gas chromatography, liquid chromatography and gas chromatography-mass spectrometry. Although instrumental detection methods are highly sensitive, accurate and reliable, they have high requirements for sample pretreatment process, long detection time, and the required instruments are large and expensive, making them unsuitable for large-scale sample determination. Summary of the Invention

[0007] The purpose of the present invention is to solve the above-mentioned problems existing in the prior art and provide a cypermethrin hapten, antigen and antibody and their preparation methods and applications, which can achieve quantitative detection of the target substance cypermethrin through specific binding between the antibody and the antigen, with high sensitivity and selectivity, and at the same time be simple, rapid and low-cost.

[0008] The technical solution adopted by the present invention is as follows: a pyraclostrobin hapten, characterized in that it is a compound having a structure as shown in formula (A1):

[0009]

[0010] Wherein, R is selected from -(CH2) m -or-X-(CH2) a -, X is selected from any one of an aromatic ring and an aromatic heterocycle;

[0011] n is selected from integers of 0-6, m is selected from integers of 0-6, and a is selected from integers of 0-6.

[0012] The present invention also provides a method for preparing the above-mentioned cypermethrin hapten, comprising mixing a compound represented by formula (B) and a compound represented by formula (C) in an organic solvent to react to obtain a cypermethrin hapten represented by formula (A1);

[0013] Y-(CH2) n -R-COO-(CH2)b-CH3 formula (C);

[0014] Wherein, R is selected from -(CH2) m -or-X-(CH2) a -, X is selected from any one of an aromatic ring and an aromatic heterocycle; Y is selected from any one of Cl, Br and I, n is selected from an integer of 0-6, m is selected from an integer of 0-6, a is selected from an integer of 0-6, and b is selected from an integer of 0-6.

[0015] Preferably, the compound represented by formula (C) is one or more of methyl chloroformate, ethyl chloroformate, ethyl chloroacetate, butyl chloroacetate, methyl chloropropionate, methyl chlorobutyrate, ethyl chlorobutyrate, propyl chlorobutyrate, methyl bromoacetate, ethyl bromoacetate, butyl bromoacetate, methyl bromobutyrate, ethyl bromobutyrate, butyl bromobutyrate, ethyl iodoacetate, methyl iodobutyrate, ethyl iodobutyrate, methyl p-chlorobenzoate, ethyl p-chlorophenylacetate, ethyl o-chlorophenylacetate, ethyl m-chlorophenylacetate, methyl p-bromophenylacetate, ethyl p-bromophenylpropionate, methyl p-bromomethylbenzoate, ethyl p-bromomethylbenzoate, methyl m-bromobenzoate, methyl m-bromobenzoate, ethyl m-bromobenzoate, methyl p-iodobenzoate, ethyl p-iodophenylacetate, ethyl o-iodobenzoate; and the organic solvent is one or more of pyridine, N,N-dimethylformamide, dimethyl sulfoxide, and tetrahydrofuran.

[0016] Preferably, the molar ratio of the compound represented by formula (C) to the compound represented by formula (B) is (0.1-1):0.1; the temperature of the mixing reaction is 0-100° C., and the reaction time is 6-48 hours.

[0017] The present invention also provides a cypermethrin antigen, which is a compound having a structure as shown in formula (A2):

[0018]

[0019] Wherein, R is selected from -(CH2) m -or-X-(CH2) a -, X is selected from any one of an aromatic ring and an aromatic heterocycle;

[0020] n is selected from an integer of 0-6, m is selected from an integer of 0-6, and a is selected from an integer of 0-6;

[0021] Protein is a carrier protein, and the carrier protein is one or more of bovine serum albumin, ovalbumin, and hemocyanin.

[0022] The present invention also provides a method for preparing the above-mentioned cypermethrin antigen, comprising subjecting the compound of the above-mentioned formula (A1) to a first coupling reaction with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to obtain a compound of formula (E); and subjecting the compound of formula (E) to a second coupling reaction with a carrier protein to obtain a cypermethrin antigen of the structure shown in formula (A2).

[0023] Preferably, the molar ratio of the compound represented by formula (A1), N-hydroxysuccinimide and the dicyclohexylcarbodiimide is 1:(1~5):(1~5); the molar ratio of the compound represented by formula (A1), N-hydroxysuccinimide and the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:(1~5):(1~5); the molar ratio of the compound represented by formula E to the carrier protein is (5~30):1.

[0024] Preferably, the temperature of the first coupling reaction is 0-50°C, the reaction time is 4-24 hours; the temperature of the second coupling reaction is 0-50°C, the reaction time is 8-36 hours, and the reaction pH is 5-9; the second coupling reaction is carried out in a solution of a carrier protein, and the solution of the carrier protein is obtained by adding the carrier protein to a buffer solution; the buffer solution is selected from one or more of a carbonate buffer, a phosphate buffer, a borate buffer, and a 4-hydroxyethylpiperazineethanesulfonic acid buffer; the pH value of the buffer is 4-10; after the second coupling reaction, a mixed system is obtained, and the mixed system is dialyzed, and the dialysate is a phosphate buffer solution with a pH value of 4-10 and a concentration of 0.01-0.2 mol / L.

[0025] The present invention also provides a cypermethrin antibody, which is prepared using the cypermethrin hapten or cypermethrin antigen as an immunogen.

[0026] The present invention also provides the use of the above-mentioned cypermethrin hapten or cypermethrin antigen or cypermethrin antibody in the preparation of cypermethrin detection products, wherein the cypermethrin detection products include enzyme-linked immunosorbent assay kits, luminescent immunoassay kits, and immunoaffinity chromatography column detection reagents.

[0027] The beneficial effects of the present invention are:

[0028] (1) The preparation method of the pyraclostrobin hapten and antigen in the present invention is simple and clear, with low synthesis cost and good synthesis effect.

[0029] (2) The cypermethrin antibodies obtained by immunization with the cypermethrin antigen of the present invention have good specificity and a low minimum detection limit.

[0030] (3) The cypermethrin antigen and antibody of the present invention have high sensitivity and strong selectivity in the rapid immunoassay of cypermethrin and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the synthesis routes of cypermethrin-ovalbumin (cypermethrin-OVA) and cypermethrin-bovine serum albumin (cypermethrin-OBSA) antigens in Example 1. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0033] Example 1

[0034] Preparation of cypermethrin-ovalbumin (cypermethrin-OVA) antigen

[0035] Through the attached Figure 1 The preparation of the antigen is carried out by the synthetic route shown, which specifically includes:

[0036] (1) Synthesis of the compound represented by formula I

[0037] 0.3 g of hydrolyzate B was added to a 50 mL three-necked flask, 40 mL of acetonitrile was added to dissolve it, 0.23 g of ethyl 4-bromobutyrate was added dropwise, 0.30 g of anhydrous potassium carbonate was added, and the mixture was stirred and heated to 100 degrees. The mixture was reacted for 24 hours, and then rotary evaporated to dryness. The mixture was extracted with ethyl acetate (3×50 mL) and dried over anhydrous magnesium sulfate. Rotary desolventization and column chromatography were performed to obtain the compound shown in formula I. 1 H-NMR, 13 C-NMR and mass spectrometry characterization data are as follows:

[0038] 1H NMR (400MHz, DMSO) δ7.69(d,J=8.5Hz,2H),7.49(d,J=8.5Hz,2H),4.02(q,J=7.1Hz,2H),3.72(s,3H), 3.70(m,2H),2.33(m,2H),2.15(s,3H),1.98(s,3H),1.86(m,2H),1.30(s,9H),1.15(q,J=7.1Hz,3H).

[0039] 13 C NMR (101MHz, DMSO) δ172.62,157.09,151.55,146.22,132.55,128.45,128.11,125.90,118 .47,116.39,98.76,70.57,60.36,37.14,34.88,31.40,30.19,24.84,14.48,11.82,8.51.

[0040] ESI-MS: m / z calcd for C 25 H 34 N3O3(M+H + )424.26,found 424.30.

[0041] Its structural formula is as follows:

[0042]

[0043] (2) Synthesis of the compound represented by formula II

[0044] In a 50 mL three-necked flask, add 0.5 g of the compound of formula I and 0.3 g of lithium hydroxide, dissolve them in 20 mL of methanol and 5 mL of water, and stir at room temperature for 8 hours. After the reaction is complete, remove the methanol by rotary evaporation, then add 20 mL of water, adjust the pH value to 3 with concentrated hydrochloric acid, filter and dry to obtain the compound of formula II. 1 H-NMR, 13 C-NMR and mass spectrometry characterization data are as follows:

[0045] 1 H NMR (400MHz, DMSO) δ12.19(s,1H),7.69(d,J=8.6Hz,2H),7.50(d,J=8.6Hz,2H),3.73(s,3H ),3.70(m,2H),2.28(t,J=7.3Hz,3H),2.15(s,3H),1.98(s,3H),1.84(m,2H),1.30(s,9H).

[0046] 13 C NMR(101MHz,DMSO)δ174.18,157.18,151.55,146.21,132.61,128.46,128.08,1 25.93,118.50,116.38,98.64,70.71,37.13,34.90,31.42,30.19,11.84,8.52.

[0047] ESI-MS: m / z calcd for C 23 H 30 N3O3(M+H + )396.23,found 396.40.

[0048] Its structural formula is as follows:

[0049]

[0050] (3) Coupling reaction

[0051] 0.03 mmol of the pyraclostrobin hapten of formula II obtained in step (2), 0.045 mmol of NHS, and 0.033 mmol of DCC were weighed and dissolved in 1 mL of anhydrous DMF. After stirring at room temperature (25°C) for 6 hours, the reaction solution was centrifuged at 8000 rpm for 5 minutes, and the supernatant was collected to obtain the compound of formula III:

[0052]

[0053] (4) The compound of formula III obtained in step (3) was slowly added dropwise to a carrier protein OVA solution, wherein the carrier protein solution was prepared by dissolving 20 mg of OVA in 5 mL of phosphate buffer (PBS) with a pH value of 7.4 and mixing the mixture. The molar ratio of the compound of formula III to the carrier protein was 20:1, and the mixture was stirred at 4°C overnight.

[0054] (5) Dialysis: The reaction solution obtained in step (4) was dialyzed for three days with a PBS solution having a pH value of 7.4 and a concentration of 0.01 mol / L. The dialyzed reaction product solution (pyraclostrobin-OVA) was diluted to a 1 mg / mL solution and stored at -40°C until use.

[0055] In the above process, the PBS solution was prepared as follows: NaCl, KH2PO4 and Na2HPO4·12H2O were dissolved in water at a mass ratio of 8.0:0.2:2.96, and the volume was adjusted to 1 L with water.

[0056] In the above process, dialysis can remove unreacted pyraclostrobin hapten and other small molecules to obtain the conjugate of pyraclostrobin and OVA shown in Formula III-1, i.e., the pyraclostrobin antigen shown in Formula A2:

[0057]

[0058]

[0059] Example 2

[0060] Preparation of cypermethrin-bovine serum albumin (cypermethrin-BSA) antigen

[0061] 1) The compound of Formula II obtained in step 2) of Example 1 was slowly added dropwise to a carrier protein solution (the carrier protein solution was prepared by dissolving 157.5 mg of BSA in 10 mL of phosphate buffered saline (PBS) at a pH of 7.4) at a molar ratio of 15:1. The mixture was stirred at 4°C overnight.

[0062] 2) Dialysis: The reaction solution obtained in step 1) was dialyzed against PBS (pH 7.4, 0.01 mol / L) for three days. The dialyzed reaction product (fenpyraclostrobin-BSA) was diluted to a 1 mg / mL solution and frozen at -40°C until use. Dialysis removes unreacted fenpyraclostrobin hapten and other small molecules, yielding the fenpyraclostrobin-BSA conjugate of Formula III-2, i.e., the fenpyraclostrobin antigen of Formula A2.

[0063]

[0064] The PBS solution was prepared as follows: NaCl, KH2PO4, and Na2HPO4·12H2O were dissolved in water at a mass ratio of 8.0:0.2:2.96, and the volume was adjusted to 1 L with water.

[0065] Example 3

[0066] Preparation of antibodies using cypermethrin-bovine serum albumin (cypermethrin-BSA) antigen

[0067] 1) 6-8 week old Balb / c mice were used as experimental animals.

[0068] (2) Basic Immunization: The diluted 1 mg / mL cypermethrin-BSA antigen solution obtained in Example 2 was sterile filtered and added with an equal volume of Freund's complete adjuvant. The mixture was stirred thoroughly to emulsify the solution until it did not diffuse when dropped into water. The emulsified complete antigen was injected into Balb / c mice at multiple points, including the peritoneal cavity and the back, with each mouse receiving a dose of 0.1 mg of antigen.

[0069] (3) Booster immunization: Two weeks after the primary immunization, take 1 mL of the diluted cypermethrin-BSA antigen solution and add 1 mL of Freund's incomplete adjuvant. Stir thoroughly to emulsify until it does not spread when dropped into water. Inject the emulsified antigen into Balb / c mice at multiple points in the abdominal cavity and back subcutaneously. The injection dose for each mouse is 0.1 mg of antigen.

[0070] After the fifth immunization, blood was collected from the mouse eye sockets. The blood was allowed to stand at room temperature for 1 hour and then in a refrigerator at 4°C for 2 hours. The blood was then centrifuged at 8000 rpm for 5 minutes to separate the antiserum, thereby obtaining the cypermethrin antibody, which was used for the following tests.

[0071] Example 4

[0072] Antibody effect detection

[0073] The antibodies obtained in Example 3 were tested using the following buffers:

[0074] Coating buffer: 0.05 M carbonate buffer, pH 9.6.

[0075] Phosphate buffer (PBS) (pH 7.4): Weigh 4.0 g NaCl, 0.1 g KH2PO4, and 1.48 g Na2HPO4·12H2O, and dilute to 500 mL with distilled water to obtain a 0.01 M phosphate buffer solution with a pH of 7.4.

[0076] Sample diluent PBSTG: 0.5 mL Tween 20, 0.5 g gelatin and 500 mL 0.1 M PBS buffer with a pH of 7.4 were mixed.

[0077] Stop buffer: 2.0 M sulfuric acid aqueous solution.

[0078] Washing solution: composed of NaCl, KH2PO4, Na2HPO4·12H2O, Tween-20 and water; the concentration of NaCl in the washing solution is 8.0 g / L, the concentration of KH2PO4 in the washing solution is 0.2 g / L, the concentration of Na2HPO4·12H2O in the washing solution is 2.96 g / L, and the volume percentage of Tween-20 in the washing solution is 1:1000.

[0079] The testing process includes:

[0080] (1) Preparation of cypermethrin-OVA coated antigen solution

[0081] After the diluted 1 mg / mL cypermethrin-OVA antigen prepared in Example 1 was completely thawed, it was diluted with coating buffer at a gradient of 1:500, 1:1000, 1:2000, and 1:4000 to obtain coating antigen solutions of cypermethrin-OVA with different concentrations.

[0082] (2) Preparation of cypermethrin standard solution

[0083] Weigh 10 mg of cypermethrin standard sample and dissolve it in 10 mL of anhydrous methanol to obtain a 1.0 mg / mL cypermethrin standard solution; use sample diluent to prepare a 1000 ng / mL cypermethrin standard solution from the obtained 1.0 mg / mL cypermethrin standard solution.

[0084] (3) Preparation of cypermethrin-BSA antibody dilution solution

[0085] The cypermethrin antibody prepared in Example 3 was diluted with the sample diluent at a gradient of 1:4000, 1:8000, 1:16000, and 1:32000 to obtain a cypermethrin antibody dilution solution.

[0086] (4) Antigen and antibody checkerboard experiment

[0087] Coating: Add 100 μL of cypermethrin-OVA coating antigen solution to each well of a 96-well ELISA plate, incubate at 37°C for 3 hours, and wash four times with washing solution.

[0088] Blocking: Weigh 5 g of skim milk powder and dissolve it in 100 mL of sample diluent to obtain 5% blocking solution. Add 200 μL of blocking solution to each well of a 96-well ELISA plate. Block in a 37°C wet chamber for 1 h, discard the blocking solution, and wash three times.

[0089] Competition: Add 50 μl of sample diluent to each well of the zero well and 50 μl of cypermethrin standard solution to each well of the inhibition well. Add cypermethrin antibody dilution to the ELISA plate (50 μl / well), incubate in a humidified chamber at 37°C for 30 minutes, and wash the plate four times.

[0090] Add enzyme-labeled secondary antibody: dilute 0.1 mg / mL goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson, catalog number 79556) 1000-fold in 0.01 M PBSTG, pH 7.4. Add 100 μL to each well and incubate in a humidified chamber at 37°C for 30 min. Wash the plate four times.

[0091] Color development: Add single-component TMB color development solution to the ELISA plate, 100 μl per well, and develop in the dark for 15 minutes.

[0092] Stop: Add 50 μL of stop buffer to each well and measure the OD value of each well at 450 nm using a microplate reader.

[0093] The results are shown in Table 1:

[0094] Table 1. Serum titer detection of cypermethrin-resistant mice (single-component TMB colorimetric solution for 15 min at room temperature, 1000 ng standard sample for inhibition)

[0095]

[0096] Note: I represents the inhibition well in the ELISA plate, and C represents the control well in the ELISA plate.

[0097] The results showed that the obtained cypermethrin-BSA can be used as an immunogen to prepare antibodies for detecting cypermethrin.

[0098] The description and drawings of the present invention are considered to be illustrative rather than restrictive. On the basis of the present invention, those skilled in the art can make some substitutions and modifications to some of the technical features according to the disclosed technical content without creative work, and all of them are within the scope of protection of the present invention.

Claims

1. A pyraclostrobin hapten, characterized in that: It is a compound having a structure as shown in formula (A1): Wherein, R is selected from -(CH2) m -or-X-(CH2) a -, X is selected from any one of an aromatic ring and an aromatic heterocycle; n is selected from an integer of 0-6, m is selected from an integer of 0-6, and a is selected from an integer of 0-6.

2. The method for preparing a pyraclostrobin hapten according to claim 1, wherein: The compound represented by formula (B) and the compound represented by formula (C) are mixed and reacted in an organic solvent to obtain the pyraclostrobin hapten represented by formula (A1); Y-(CH2) n -R-COO-(CH2) b -CH3 formula (C); Wherein, R is selected from -(CH2) m -or-X-(CH2) a -, X is selected from any one of an aromatic ring and an aromatic heterocycle; Y is selected from any one of Cl, Br and I, n is selected from an integer of 0-6, m is selected from an integer of 0-6, a is selected from an integer of 0-6, and b is selected from an integer of 0-6.

3. The preparation method according to claim 2, characterized in that The compound represented by formula (C) is one or more of methyl chloroformate, ethyl chloroformate, ethyl chloroacetate, butyl chloroacetate, methyl chloropropionate, methyl chlorobutyrate, ethyl chlorobutyrate, propyl chlorobutyrate, methyl bromoacetate, ethyl bromoacetate, butyl bromoacetate, methyl bromobutyrate, ethyl bromobutyrate, butyl bromobutyrate, ethyl iodoacetate, methyl iodobutyrate, ethyl iodobutyrate, methyl p-chlorobenzoate, ethyl p-chlorophenylacetate, ethyl o-chlorophenylacetate, ethyl m-chlorophenylacetate, methyl p-bromophenylacetate, ethyl p-bromophenylpropionate, methyl p-bromomethylbenzoate, ethyl p-bromomethylbenzoate, methyl m-bromobenzoate, methyl m-bromobenzoate, ethyl m-bromobenzoate, methyl p-iodobenzoate, ethyl p-iodophenylacetate, ethyl p-iodophenylacetate, and methyl o-iodobenzoate; and the organic solvent is one or more of pyridine, N,N-dimethylformamide, dimethyl sulfoxide, and tetrahydrofuran.

4. The preparation method according to claim 2, characterized in that The molar ratio of the compound represented by formula (C) to the compound represented by formula (B) is (0.1-1):0.1; the temperature of the mixed reaction is 0-100° C., and the reaction time is 6-48 hours.

5. A cypermethrin antigen, characterized in that: It is a compound having a structure as shown in formula (A2): Wherein, R is selected from -(CH2) m -or-X-(CH2) a -, X is selected from any one of an aromatic ring and an aromatic heterocycle; n is selected from an integer of 0-6, m is selected from an integer of 0-6, and a is selected from an integer of 0-6; Protein is a carrier protein, and the carrier protein is one or more of bovine serum albumin, ovalbumin, and hemocyanin.

6. The method for preparing a cypermethrin antigen according to claim 5, wherein: A first coupling reaction is performed on the compound of formula (A1) as claimed in claim 1 and N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to obtain a compound represented by formula (E); a second coupling reaction is performed on the compound represented by formula (E) and a carrier protein to obtain a cypermethrin antigen represented by formula (A2).

7. The preparation method according to claim 6, characterized in that The molar ratio of the compound represented by formula (A1), N-hydroxysuccinimide and the dicyclohexylcarbodiimide is 1:(1~5):(1~5); the molar ratio of the compound represented by formula (A1), N-hydroxysuccinimide and the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:(1~5):(1~5); the molar ratio of the compound represented by formula E to the carrier protein is (5~30):

1.

8. The preparation method according to claim 6, characterized in that The temperature of the first coupling reaction is 0-50°C, the reaction time is 4-24 hours; the temperature of the second coupling reaction is 0-50°C, the reaction time is 8-36 hours, and the reaction pH value is 5-9; the second coupling reaction is carried out in a carrier protein solution, and the carrier protein solution is obtained by adding the carrier protein to a buffer solution; the buffer solution is selected from one or more of carbonate buffer, phosphate buffer, borate buffer, and 4-hydroxyethylpiperazineethanesulfonic acid buffer; the pH value of the buffer is 4-10; after the second coupling reaction, a mixed system is obtained, and the mixed system is dialyzed, and the dialysate is a phosphate buffer solution with a pH value of 4-10 and a concentration of 0.01-0.2 mol / L.

9. A cypermethrin antibody, characterized in that: The immunogen is prepared by using the cypermethrin hapten according to claim 1 or the cypermethrin antigen according to claim 5 as an immunogen.

10. Use of the pyraclostrobin hapten according to claim 1, the pyraclostrobin antigen according to claim 5, or the pyraclostrobin antibody according to claim 9 in preparing a pyraclostrobin detection product, characterized in that: The cypermethrin detection products include enzyme-linked immunosorbent assay kits, luminescent immunoassay kits, and immunoaffinity chromatography column detection reagents.

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