Monoclonal antibodies, enzyme-linked immunosorbent assay, and kit for detecting eight type II pyrethroids

Abstract

This invention belongs to the fields of drug residue analysis and immunology, and relates to a broad-spectrum monoclonal antibody capable of recognizing eight type II pyrethroid drugs, and an enzyme-linked immunosorbent assay (ELISA) method and kit for detecting type II pyrethroid drugs. The monoclonal antibody of this invention is secreted by the hybridoma cell line PYR / 1D2 with accession number CCTCC NO: C2021228. Compared with existing technologies, the monoclonal antibody prepared by this invention can simultaneously recognize eight type II pyrethroid drugs, including cypermethrin, lambda-cyhalothrin, deltamethrin, cyhalothrin, cyhalothrin, cypermethrin, deltamethrin, and fenvalerate, and has the advantages of simple sample pretreatment, high sensitivity, accuracy, and good precision.

Description

Technical Field

[0001] This invention belongs to the fields of drug residue analysis and immunology technology, specifically relating to a monoclonal antibody that identifies eight type II pyrethroid drugs, namely cypermethrin, lambda-cyhalothrin, deltamethrin, lambda-cyhalothrin, lambda-cyhalothrin, cypermethrin, deltamethrin, and fenvalerate, as well as an enzyme-linked immunosorbent assay (ELISA) method and kit for detecting the eight type II pyrethroid drugs. Background Technology

[0002] Type II pyrethroid insecticides are a class of drugs composed of benzene rings and heterocycles, often containing aromatic rings, amide bonds, and heterocycles in their structure. Representative members include cypermethrin, deltamethrin, cyhalothrin, lambda-cyhalothrin, cypermethrin, deltamethrin, and fenvalerate. These insecticides are widely used for pest control in crops, animals, the environment, and household hygiene, as well as for malaria control. However, excessive use leads to increased Type II pyrethroid residues in fruits, vegetables, animals, and the environment, posing serious harm and risks to the ecological environment and human health. Studies have shown that long-term low-dose exposure to pyrethroid pesticides can damage the male reproductive system (Koureas M. Systematic review of biomonitoring studies to determine the association between exposure to organophosphorus and pyrethroid insecticides and human health outcomes. Toxicol Lett, 2012, 210(2):155-168), cause hearing impairment in adolescents (Xu HD, et al. Association between pyrethroid pesticide exposure and hearing loss in adolescents. Environ Res, 2020, 187:109640), and cognitive developmental disorders in children (De Joode BVW, et al. Pesticide exposure and neurodevelopment in children aged 6-9 years from Talamanca, Costa Rica. Cortex, 2016, 85:137-150), and can also cause damage to lymph nodes and spleen, as well as carcinogenic effects (Zisis Rah, Bayu Rustika. Toxicity and health hazards of pyrethroid pesticides. Science Insights, 2022, 41(6):733-739, etc.

[0003] The main detection methods for type II pyrethroid insecticides include instrumental methods and enzyme-linked immunosorbent assays (ELISA). Instrumental methods include gas chromatography (GC), high-performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS). Instrumental methods offer high sensitivity, accuracy, and precision, but they require expensive equipment and complex sample pretreatment processes such as extraction, purification, and derivatization. They also require skilled personnel and are unsuitable for large-scale sample screening and on-site testing. In contrast, enzyme-linked immunosorbent assays (ELISA) offer advantages such as simple operation, no need for complex sample preparation, high sensitivity, high throughput, and low cost, making them suitable for rapid screening of large batches of samples.

[0004] Currently, reports on the preparation of type II pyrethroid antibodies show that antibodies capable of simultaneously recognizing multiple type II pyrethroid drugs are relatively few, limiting their application in drug residue detection. Patent application CN111269139 A discloses a polyclonal antibody that can recognize lambda-cyhalothrin, with a limit of detection of 10 μg / L and cross-reactivity of less than 10% with deltamethrin, cypermethrin, flufenoxuron, and fenvalerate. Patent application CN 104327186 A discloses a polyclonal antibody that can recognize bifenthrin, with a sensitivity of 50 μg / L and cross-reactivity of less than 0.7% with cypermethrin, fenvalerate, and other pyrethroids. Patent application CN 113024415 A discloses a monoclonal antibody that can recognize cypermethrin, with a sensitivity of 0.1 μg / L. Patent application CN 110776567 A discloses a monoclonal antibody that can only recognize cypermethrin, and its IC50 value for cypermethrin is [not specified]. 50 With a concentration of 4.5 μg / L, it can be used for the determination of spiked recovery rates in samples from vegetables and fruits. While the antibodies disclosed in these four patent applications have high sensitivity, they only exhibit high recognition ability for one type of pyrethroid drug. Furthermore, the first three patents do not include methods for determining spiked recovery rates in samples, limiting their application in the detection of multiple pyrethroid drug residues in samples. Patent application CN102393465 A discloses a monoclonal antibody that can recognize deltamethrin, cypermethrin, fenvalerate, and bifenthrin, with an IC50 of 4.5 μg / L for the pyrethroid metabolite m-phenoxybenzoic acid (PBA). 50 The concentration was 570 μg / L, and the IC50 concentration for deltamethrin, cypermethrin, fenvalerate, and bifenthrin was [not specified]. 50 The concentrations were 790, 740, 630, 800, and 997 μg / L, respectively. The antibody had low sensitivity and was not used for the detection of residual compounds of this type in actual samples. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody and enzyme-linked immunosorbent assay (ELISA) kit that can simultaneously identify eight common type II pyrethroid insecticides, including cypermethrin, lambda-cyhalothrin, deltamethrin, cyhalothrin, cyhalothrin, cyhalothrin, deltamethrin, and fenvalerate. Furthermore, this kit is used to establish an ELISA detection method for residues of these eight type II pyrethroid insecticides in samples (milk, celery, leeks, and sewage, etc.), providing strong technical support for the monitoring of pyrethroid insecticide residues in food and the environment.

[0006] The above objective is achieved through the following technical solution:

[0007] 1. This invention provides haptens for detecting type II pyrethroid insecticides cypermethrin, lambda-cyhalothrin, deltamethrin, cyhalothrin, cyhalothrin, cypermethrin, deltamethrin, and fenvalerate, comprising hapten A and hapten B, with the following structural formula:

[0008]

[0009] 2. This invention provides a method for preparing the above two haptens, which includes the following steps:

[0010] (1) Synthesizing hapten A using ethyl chrysanthemate as a raw material

[0011] Ethyl chrysanthemate was weighed and dissolved in distilled water. Potassium permanganate was added and the mixture was reacted for 12 hours. Then, 5 mol / L sulfuric acid solution was added. The mixture was extracted with ethyl acetate, washed with saturated brine, and dried overnight with anhydrous sodium sulfate to obtain 3,3-dimethyl-1,3-cyclopropanedicarboxylic acid. 3,3-Dimethyl-1,3-cyclopropanedicarboxylic acid was reacted with sulfoxide for 10–12 hours to obtain the product 3,3-dimethyl-1,3-cyclopropanedicarboxylic acid. (S)-(3-phenoxyphenyl)hydroxyacetonitrile was added to the reaction mixture, and the reaction was monitored by TLC. After the reaction was complete, saturated ammonium chloride solution and dichloromethane were added to the system, and the mixture was allowed to stand for separation. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain hapten A.

[0012] (2) Synthesis of hapten B using 3-methoxybenzaldehyde as a raw material

[0013] 3-Methoxybenzaldehyde was weighed and dissolved in anhydrous methanol. Sodium borohydride was added, and the reaction was allowed to proceed for 2–3 hours, monitored by TLC until complete. After removing methanol by evaporation, the product was extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate overnight to obtain an oily product. The oily product was dissolved in anhydrous pyridine, succinate was added, and the reaction was allowed to proceed for 12 hours. The product was then purified by silica gel column chromatography to obtain a colorless oily product (hapten B).

[0014] 3. This invention also provides a monoclonal antibody capable of simultaneously recognizing eight type II pyrethroid insecticides: cypermethrin, lambda-cyhalothrin, deltamethrin, cyhalothrin, cyhalothrin, cyhalothrin, deltamethrin, and fenvalerate. This is achieved through the following technical solution:

[0015] (1) Immunogen A-DCC-KLH was obtained by conjugating the hapten A with keyhole hemocyanin (KLH) using the carbodiimide method (DCC).

[0016] (2) Immunogen A-DCC-KLH was administered to mice via subcutaneous injection on the back at different doses. After cell fusion and screening, hybridoma cell line PYR / 1D2 was obtained and deposited in the China Center for Type Culture Collection, accession number CCTCCNO:C2021228.

[0017] (3) Monoclonal antibodies were prepared using the hybridoma cell line PYR / 1D2 with accession number CCTCC NO:C2021228.

[0018] 4. The present invention utilizes the monoclonal antibody to prepare an enzyme-linked immunosorbent assay kit for detecting type II pyrethroid drugs cypermethrin, lambda-cyhalothrin, cypermethrin, lambda-cyhalothrin, lambda-cyhalothrin, cypermethrin, deltamethrin, and fenvalerate.

[0019] The kit consists of:

[0020] (1) The solid support is coated with the original B-DCC-BSA.

[0021] (2) Cypermethrin standard solution.

[0022] (3) Working solution of monoclonal antibody secreted by hybridoma cell line PYR / 1D2 with accession number CCTCC NO:C2021228.

[0023] (4) Horseradish catalase-labeled sheep anti-mouse IgG antibody (HRP-IgG) working solution.

[0024] (5) 10× concentrated phosphate buffer.

[0025] (6) 10× concentrated washing solution.

[0026] (7) Two bottles of substrate solution, namely substrate solution A and substrate solution B.

[0027] (8) Termination solution.

[0028] 5. This invention utilizes the aforementioned kit to establish an enzyme-linked immunosorbent assay (ELISA) method for detecting residues of type II pyrethroid insecticides, including cypermethrin, lambda-cyhalothrin, deltamethrin, cyhalothrin, cyhalothrin, cyhalothrin, deltamethrin, and fenvalerate, in samples. The method comprises the following steps:

[0029] (1) The hapten B was coupled with bovine serum albumin (BSA) using the carbodiimide method (DCC) to obtain coated hapten B-DCC-BSA;

[0030] (2) The solid support is coated with the original B-DCC-BSA obtained in step (1);

[0031] (3) First, the sample was processed, and then the sample was detected using a monoclonal antibody prepared from the hybridoma cell line PYR / 1D2 with accession number CCTCC NO:C2021228.

[0032] This invention uses the aforementioned monoclonal antibody and coating agent as core reagents, combined with other conventional reagents, to produce an enzyme-linked immunosorbent assay (ELISA) kit capable of detecting eight type II pyrethroid insecticides: cypermethrin, lambda-cyhalothrin, deltamethrin, cyhalothrin, cyhalothrin, cyhalothrin, cypermethrin, deltamethrin, and fenvalerate. Combined with the aforementioned ELISA method, it enables the detection of residues of these eight type II pyrethroid insecticides in samples (such as milk, celery, leeks, and sewage).

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] 1. The hapten A designed in this invention is significantly different from existing technologies (see Table 1). Specifically, patent application CN111269139 A modifies the "-CN" of lambda-cyhalothrin by introducing a linker arm "-NH2"; patent application CN104327186 A uses a linker arm "-COOH" to replace the cyclopropane of bifenthrin to synthesize the hapten; patent application CN 113024415 A introduces a linker arm onto the cyclopropane of lambda-cyhalothrin; patent application CN 110776567 A introduces "-NH2" into the m-phenoxybenzyl group of cypermethrin to expose the antigenic determinant; and patent application CN 102393465... Patent application A directly uses m-phenoxybenzoic acid as a hapten, while this invention introduces a linker arm "-COOH" onto the cyclopropane of cypermethrin, replacing the halogen element of cypermethrin, thus fully exposing the common structure of pyrethroid drugs, thereby improving the broad spectrum of monoclonal antibodies as well as their sensitivity and specificity to pyrethroid drugs.

[0035] 2. The synthetic routes for hapten A and hapten B in this invention differ from existing technologies. This invention uses ethyl chrysanthemate and 3-methoxybenzaldehyde as reagents to synthesize haptens de novo, and these are all commonly used raw materials and reagents with low toxicity and minimal environmental pollution. The reaction equipment and conditions are easy to implement, and the experimental instruments used, such as flasks, constant temperature water baths, and chromatography columns, are inexpensive and simple to operate.

[0036] 3. The monoclonal antibody prepared in this invention can simultaneously recognize eight type II pyrethroid insecticides, including cypermethrin, lambda-cyhalothrin, deltamethrin, cyhalothrin, deltamethrin, cypermethrin, deltamethrin, and fenvalerate. It exhibits high sensitivity and a high IC50 value for these eight type II pyrethroids. 50 The values ​​were 130.07 μg / L, 199.60 μg / L, 215.49 μg / L, 241.67 μg / L, 226.86 μg / L, 220.32 μg / L, 591.23 μg / L, and 763.13 μg / L, respectively.

[0037] 4. The enzyme-linked immunosorbent assay (ELISA) method and kit established in this invention are sensitive, accurate, and precise. The sample pretreatment is simple, and it is suitable for the detection of multiple residues of eight type II pyrethroid pesticides in samples (milk, celery, leeks, and sewage).

[0038] Table 1. Positive effects of the present invention compared with the prior art

[0039] Attached Figure Description

[0040] Figure 1 This is a standard curve showing the inhibition of cypermethrin standard by the monoclonal antibody of the present invention, where the X-axis represents the logarithm of the concentration of the cypermethrin standard solution, and the Y-axis represents the OD value of the cypermethrin standard solution. 450nm Value divided by "0" hole OD 450nm Value (B / B0).

[0041] Preservation Information

[0042] Deposit date: August 13, 2021;

[0043] Name of depositary institution: China Center for Type Culture Collection;

[0044] Accession number: CCTCC NO:C2021228;

[0045] Address of the depositary institution: Wuhan University, Wuhan, China;

[0046] Classification and nomenclature: Hybridoma cell line PYR / 1D2. Detailed Implementation

[0047] The present invention will now be described in more detail through specific embodiments to facilitate understanding of the technical solution of the present invention, but this is not intended to limit the scope of protection of the present invention.

[0048] Example 1: Preparation of type II pyrethroid hapten

[0049] (1) Preparation of hapten A

[0050] ① Synthesis of 3,3-dimethyl-1,3-cyclopropanedicarboxylic acid chloride

[0051]

[0052] Ethyl chrysanthemate (19.6 g, 0.1 mol) was dissolved in 200 mL of distilled water, and potassium permanganate (31.6 g, 0.2 mol) was added in portions. The reaction was allowed to proceed for 12 h, followed by the addition of 50 mL of 5 mol / L sulfuric acid solution and a further 6 h reaction. Then, 200 mL of ethyl acetate was added to the system, the organic phase was separated, and the aqueous phase was extracted again. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate overnight, and purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 4:1, v / v) to give 15 g of the intermediate product 3,3-dimethyl-1,3-cyclopropanedicarboxylic acid. 7.9 g (50 mmol) of 3,3-dimethyl-1,3-cyclopropanedicarboxylic acid was weighed, dissolved in 100 mL of dichloromethane, and reacted with sulfoxide (24.5 g, 0.4 mol) to give the product 3,3-dimethyl-1,3-cyclopropanedicarboxylic acid chloride.

[0053] ② Synthesis of (RS)-cyano-3-phenoxybenzyl-2,2,3,3-tetramethylcyclopropanecarboxylic acid

[0054]

[0055] Weigh 3,3-dimethyl-1,3-cyclopropanedicarboxylic acid chloride (3.86 g, 20 mmol) obtained in step ①, dissolve it in 50 mL of dichloromethane, and slowly add 10 mL of (S)-(3-phenoxyphenyl)hydroxyacetonitrile. Monitor the reaction by TLC until complete, add 50 mL of saturated ammonium chloride solution to the system, and allow it to stand for separation. Wash with saturated brine, dry with anhydrous sodium sulfate to recover the thallium, and purify by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 4:1, v / v) to obtain 1.5 g of hapten A.

[0056] (2) Synthesis of hapten B

[0057]

[0058] Weigh 19.8 g (0.1 mol) of 3-methoxybenzaldehyde and dissolve it in 100 mL of anhydrous methanol. Add sodium borohydride (7.6 g, 0.2 mol) in portions and react for 2 h. Monitor the reaction by TLC until complete. After removing methanol by evaporation and extracting with ethyl acetate, wash with saturated brine and dry overnight with anhydrous sodium sulfate to obtain 35 g of oily product.

[0059] The oily product (2.0 g, 10 mmol) was dissolved in 50 mL of anhydrous pyridine, and succinic acid (1.3 g, 13 mmol) was added. The reaction was carried out for 12 h, the pyridine was removed by evaporation, and the product was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 4:1, v / v) to give 2.1 g of colorless oily product (hapten B).

[0060] Example 2: Preparation of immunogen and coating agent

[0061] (1) Preparation of immunogen (A-DCC-KLH)

[0062] Weigh 15 mg of hapten A and dissolve it in 1 mL of N,N-dimethylformamide (DMF). Add 10 mg of N-hydroxysuccinimide (NHS) and 20 mg of N,N-dicyclohexylcarbodiimide (DCC). Stir at room temperature and react overnight in the dark. After the reaction is complete, filter to obtain the filtrate, which is solution A. Weigh 10 mg of KLH and slowly dissolve it in 8 mL of PBS, which is solution B. Under ice bath conditions, slowly add solution A dropwise to solution B and stir to react overnight. Dialyze the reaction solution with PBS at 4 °C for 5 days, changing the dialysate 3 times a day. Centrifuge at 8000 r / min for 10 min and retain the supernatant to obtain the immunogen (A-DCC-KLH). The synthesis was confirmed by UV scanning, and the coupling ratio was calculated to be 6.5. Store at -20 °C for later use.

[0063] (2) Preparation of the coating agent (B-DCC-BSA)

[0064] Weigh out four 50mg portions of hapten B and dissolve them in 0.7mL of DMF. Add 28.9mg of N-hydroxysuccinimide (NHS) and 51.9mg of N,N-dicyclohexylcarbodiimide (DCC) to each portion. Stir at room temperature and react overnight in the dark. After the reaction is complete, filter to obtain the filtrate, which is solution C. 55.7, 74.3, 111.5, and 222.9 mg of BSA were weighed and dissolved in 8 mL of PBS, respectively (this is solution D). Solution C was slowly added dropwise to solution D, and the mixture was stirred and reacted overnight. The mixture was dialyzed in PBS at 4 °C for 5 days, with the dialysate changed three times a day. After dialyzing, the mixture was centrifuged at 8000 r / min for 10 min, and the supernatant was retained to obtain coated antigens (B-DCC-BSA) with feed ratios (Hapten B:BSA) of 200:1, 150:1, 100:1, and 50:1, respectively. The synthesis was confirmed to be successful by UV scanning, and the coupling ratios were 13.4, 17.4, 15.3, and 16.0, respectively. The mixture was stored at -20 °C for later use.

[0065] Example 3: Screening of hybridoma cell lines

[0066] (1) Mouse immunization

[0067] Six- to eight-week-old SPF-grade female Balb / c mice were immunized with the immunogen A-DCC-KLH prepared by the inventors. Immunization procedure: The initial immunization dose was 100 μg / mouse, and the immunogen A-DCC-KLH was emulsified with Freund's complete adjuvant and injected subcutaneously at multiple sites on the neck and back of the mouse. Three weeks later, booster immunizations were performed using the same dose, with a two-week interval between booster immunizations. The immunogen was emulsified with Freund's incomplete adjuvant. Blood was collected from the tail 6-8 days after each booster immunization. Serum titers and specificity were detected by indirect competitive ELISA. Mice with high titers and strong specificity were selected for cell fusion.

[0068] (2) Cell fusion and screening

[0069] Mouse immune spleen cells and myeloma cells (SP 2 / 0) were fused using 50% polyethylene glycol (PEG 1450). Hybridoma cells were cultured in HAT selective medium, and cell wells with the highest titer and best inhibitory effect were selected for subcloning. Cells were then cultured in HT selective medium, and cell colonies with high supernatant titer, good morphology, and monocolony growth were selected for further subcloning until the positive rate reached 100%. After four subcloning processes, the hybridoma cell line PYR / 1D2, which secretes anti-type II pyrethroid monoclonal antibodies, was finally obtained and deposited at the China Center for Type Culture Collection (CCTCC) (Wuhan University, Wuhan, China) on August 13, 2021, with accession number CCTCC NO: C2021228.

[0070] Example 4: Preparation and Detection of Monoclonal Antibodies

[0071] Each mouse was pretreated with 0.5 mL of liquid paraffin via intraperitoneal injection; 7 days later, each mouse was injected intraperitoneally with 0.5 mL of a suspension of the logarithmic growth phase hybridoma cell line PYR / 1D2 (1×10⁻⁶ cells). 6 (cell / mL); After 7 days, the ascites production in mice was observed daily. When the mouse abdomen was significantly distended, the abdominal skin was disinfected with an alcohol swab before collecting the ascites. The collected ascites was centrifuged at 8000 r / min for 10 min, the surface oil layer was removed, the supernatant was collected, and the monoclonal antibody was obtained. Glycerol was added, mixed well, and aliquoted, and stored at -20℃. The monoclonal antibody obtained in this invention was identified as the IgG1 subtype according to the instructions of the mouse monoclonal antibody rapid ELISA subtype detection kit. The result was that the light chain was Kappa chain.

[0072] Example 5: Establishment of an Indirect Competitive ELISA Method

[0073] (1) Preliminary determination of coating agent and antibody dilution factor

[0074] The coating antigens with different feed ratios were diluted to different folds, and then titrated with antibodies of different dilutions using a checkerboard titration method to screen for the optimal pairing (see Table 2). Based on the screened pairings, an indirect competitive ELISA was performed, using OD values ​​from well "0". 450nm Value close to 2.0, IC 50 The lowest value corresponds to the optimal coating concentration. The following combinations were initially selected as the preferred combinations for further optimization: ① coating agent feed ratio 200:1, coating agent dilution factor 1:2000, antibody dilution factor 1:2400; ② coating agent feed ratio 150:1, coating agent dilution factor 1:2000, antibody dilution factor 1:4800; ③ coating agent feed ratio 100:1, coating agent dilution factor 1:1550, antibody dilution factor 1:3600.

[0075] Table 2. Preliminary screening using coating antigen and antibody dilution factor

[0076]

[0077]

[0078] (2) Optimization of optimal coating agent concentration and antibody dilution factor

[0079] Using the three initially determined optimal original dilutions to coat the ELISA plate, an arithmetic gradient of antibody dilutions was designed, and the IC50 was calculated. 50 Table 3 shows that when the original coating ratio is 100:1, the original coating dilution is 1:1550, and the antibody dilution is 1:3600, and when the original coating ratio is 150:1, the original coating dilution is 1:2000, and the antibody dilution is 1:5000, the IC50 values ​​are...50 The values ​​were both relatively low, at 161.8 μg / L and 168.8 μg / L, respectively. Adhering to the principle of conserving antibody, a coating agent feed ratio of 150:1 and a coating agent dilution factor of 1:2000 were selected as the optimal coating agent concentration and optimal antibody dilution factor, i.e., a coating concentration of 1.94 μg / mL and an antibody dilution factor of 1:5000.

[0080] Table 3. Optimal Coating Agent and Antibody Dilute Fold Ratio Optimization

[0081]

[0082] (3) Establishment of standard curve and sensitivity testing

[0083] Cypermethrin standard solution was diluted with PBS (20% DMSO) to eight concentrations: 0 μg / L, 31.25 μg / L, 62.5 μg / L, 125 μg / L, 250 μg / L, 500 μg / L, 1000 μg / L, and 2000 μg / L. The concentrations were determined using an indirect competitive ELISA method. A standard curve was plotted with the logarithm of the cypermethrin concentration on the x-axis and B / B0 on ​​the y-axis, and the IC50 was calculated. 50 The ELISACalc software was used for fitting. The regression equation for the standard curve is y = (AD) / [1 + (1000x / C)]. ^B ]+D, A=0.9738, B=8.7437, C=2.0995, D=0.0969, R 2 =0.9986, IC 50 The value was 129.05±2.69μg / L (n=5), which proves that the antibody has high sensitivity to cypermethrin and can be used for immunoassay detection.

[0084] (4) Monoclonal antibody specificity detection

[0085] The cross-reactivity of the monoclonal antibody secreted by the hybridoma cell line PYR / 1D2 with cypermethrin was 100%. The cross-reactivity rates with lambda-cyhalothrin, cypermethrin, deltamethrin, cypermethrin, cypermethrin, cypermethrin, deltamethrin, and fenvalerate were 65.2%, 60.4%, 57.3%, 53.8%, 59.0%, 22.0%, and 17.0%, respectively. The cross-reactivity with S-fenvalerate, permethrin, carbamates, and organophosphates was less than 0.01%.

[0086] Table 4. Cross-reactivity of monoclonal antibodies secreted by hybridoma cell line PYR / 1D2 against type II pyrethroids

[0087] Drug Name <![CDATA[IC 50 (μg / L)]]> Cross-reactivity rate (%) Linear range (μg / L) Cypermethrin 130.07 100.0 31.25～2000 High-efficiency cypermethrin 199.60 65.2 62.5～4000 Cypermethrin 215.49 60.4 62.5～4000 Cypermethrin 220.32 59.0 62.5～4000 High-efficiency cyhalothrin 226.86 57.3 62.5～4000 High-efficiency cypermethrin 241.67 53.8 62.5～4000 deltamethrin 591.23 22.0 62.5～4000 Cypermethrin 763.13 17.0 62.5～4000 S-cypermethrin >1000 <0.01 - Permethrin >1000 <0.01 - Carbamates >1000 <0.01 - Organophosphorus >1000 <0.01 -

[0088] Example 6: Assembly of the enzyme-linked immunosorbent assay kit

[0089] 6.1 Components of the reagent kit:

[0090] (1) A solid-phase carrier coated with the original B-DCC-BSA (96-well polystyrene microplate).

[0091] (2) Eight bottles of cypermethrin standard solution with concentrations of 0 μg / L, 31.25 μg / L, 62.5 μg / L, 125 μg / L, 250 μg / L, 500 μg / L, 1000 μg / L and 2000 μg / L, respectively.

[0092] (3) Working solution of monoclonal antibody secreted by hybridoma cell line PYR / 1D2 with accession number CCTCC NO:C2021228.

[0093] (4) Horseradish catalase-labeled sheep anti-mouse IgG antibody (HRP-IgG) working solution.

[0094] (5) 10× concentrated phosphate buffer.

[0095] (6) 10× concentrated washing solution.

[0096] (7) Two bottles of substrate solution, namely substrate solution A and substrate solution B.

[0097] (8) Termination solution.

[0098] 6.2 Preparation of reagents

[0099] (1) 10× concentrated phosphate buffer: NaCl 80.0g, KH2PO4 2.0g, Na2HPO4·12H2O 29.0g, KCl 2.0g, add triple-distilled water to 1000mL.

[0100] (2) 10× concentrated washing solution: NaCl 80.0g, KH2PO4 2.0g, Na2HPO4·12H2O 29.0g, KCl 2.0g, Tween-20 5mL, add triple-distilled water to 1000mL.

[0101] (3) Substrate solution A: 160 mg of 3,3',5',5-tetramethylbenzidine TMB and 10 mL of dimethylacetamide.

[0102] (4) Substrate solution B: 13.70 g citric acid, 10.14 g trisodium citrate, 282 mg urea peroxide, and triple-distilled water to 1000 mL.

[0103] (5) Termination solution: 2 mol / L sulfuric acid solution.

[0104] (6) Coating solution: Accurately weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, add about 800 mL of triple-distilled water, adjust the pH value to 9.6, and add water to make up to 1 L.

[0105] (7) Washing solution: Dilute 10× concentrated washing solution with triple-distilled water 10 times before use.

[0106] (8) Blocking solution: Accurately weigh 8g of ovalbumin, add 800mL of PBS buffer, and stir until the protein is completely dissolved.

[0107] (9) HRP-IgG working solution: Dilute HRP-IgG with antibody diluent to 5000 times according to the required amount each time, mix well, and store at 2-8℃.

[0108] (10) Antibody working solution: Take an appropriate amount of monoclonal antibody secreted by hybridoma cell line PYR / 1D2 with accession number CCTCC NO:C2021228, dilute it with antibody diluent at 1:5000, and store at 2-8℃.

[0109] 6.3 Preparation of ELISA plates

[0110] Dilute B-DCC-BSA to 1.94 μg / mL with coating buffer, add 100 μL to each well of a 96-well microplate, and incubate overnight at 4°C. Discard the coating buffer, add 250 μL of washing buffer to each well and wash 3 times, pat dry, then add 250 μL of blocking buffer to each well and incubate at 37°C for 2 hours. Discard the liquid in the wells, wash 3 times with washing buffer, pat dry, and vacuum seal with aluminum foil for storage.

[0111] Example 7: Application of the enzyme-linked immunosorbent assay kit

[0112] 7.1 Preparation of reagents

[0113] (1) Phosphate buffer: Dilute the 10× concentrated phosphate buffer provided in the kit with triple-distilled water 10 times before use.

[0114] (2) Washing solution: Dilute the 10× concentrated washing solution provided in the kit with triple-distilled water 10 times before use.

[0115] (3) Sample dilution solution: Mix PBS and DMSO at a volume ratio of 4:1 according to the required amount each time.

[0116] (4) Substrate mixture: According to the required amount each time, mix the prepared substrate solution A and substrate solution B at a volume ratio of 1:100 and use immediately.

[0117] 7.2 Sample Pretreatment

[0118] Milk: Measure 4 mL of milk, add 1 mL of dimethyl sulfoxide, vortex for 5 min, then centrifuge at 8000 rpm for 10 min at 4℃. Take the intermediate layer for analysis. (Dilution factor: 1.2)

[0119] Celery and leeks: Weigh 10g of homogenized celery, leeks, or apple sample, add 10mL of acetonitrile, vortex thoroughly for 5min, then add 1g of sodium chloride and 4g of magnesium sulfate. Vortex for 5min, centrifuge at 6000r / min for 5min, collect 1mL of the supernatant, and dry under nitrogen. Redissolve the residue in 1mL of sample diluent before analysis. (Dilution factor: 1.0)

[0120] Wastewater: Measure 4 mL of wastewater, add 1 mL of dimethyl sulfoxide, vortex thoroughly, and then analyze. (Dilution factor 1.2)

[0121] 7.3 Application steps of enzyme-linked immunosorbent assay (ELISA) kit

[0122] (1) Sample addition: Add 50 μL of cypermethrin series concentration standard solution or sample solution to the microplate wells, then add 50 μL of antibody working solution, place in a humidified chamber, and incubate at 37℃ for 60 min;

[0123] (2) Washing: Pour out the liquid in the hole, add 250μL of washing solution to each hole and wash three times, then pat dry;

[0124] (3) Add 100 μL of HRP-IgG antibody working solution (working concentration 1:5000) to each well, place in a humidified chamber and incubate at 37°C for 60 min;

[0125] (4) Washing: Pour out the liquid in the hole, add 250μL of washing solution to each hole and wash three times, then pat dry;

[0126] (5) Color development: Add 100 μL of substrate mixture to each well, place in a humidified chamber, and incubate at 37°C for 15 min.

[0127] (6) Termination: Add 50 μL of termination solution to each well;

[0128] (7) Measurement: The optical density (OD value) of each well was measured at 450 nm using an ELISA reader.

[0129] 7.4 Result Determination

[0130] Standard curve: based on the measured OD of the standard. 450nm Value divided by "0" hole OD 450nm A standard curve was plotted with the B / B0 value (B / B0) on the ordinate and the logarithm of the cypermethrin concentration on the abscissa. A linear regression was then performed to derive the regression equation, as follows: Figure 1 As shown.

[0131] Calculation of cypermethrin concentration in the sample: Calculate the inhibition rate of the sample (OD of the obtained sample). 450nm Value divided by "0" hole OD 450nm The concentrations of cypermethrin in wastewater, milk, celery, leeks, and apple samples were calculated by substituting the values ​​into the regression equation of the standard curve and multiplying by the dilution factor. The dilution factor for cypermethrin in milk and wastewater samples was 1.2, and the dilution factor for celery, leeks, and apple samples was 1.0.

[0132] Example 8: Sensitivity, precision, accuracy, and repeatability tests of the kit

[0133] (1) Sensitivity test of the kit

[0134] The OD of 20 0 μg / L standard solutions was detected. 450nm Substitute the values ​​into the standard curve, calculate the concentrations of 20 blank assays, and determine the mean and standard deviation. Calculate the Z-value using the formula Z = C + 3SD. The limit of detection (LOD) for this ELISA method is 23.90 μg / L. The OD values ​​of 20 blank tissue samples were then measured. 450nm The value was calculated based on the regression equation of the standard curve to determine the corresponding cypermethrin concentration, and then the average cypermethrin concentration was calculated. and standard deviation (SD), according to the formula Calculate the LOD (Limit of Quantification) of the sample; Table 5 details the LOD and LOQ of the eight type II pyrethroids in the samples.

[0135] Table 5. LODs and LOQs of type II pyrethroid insecticides in different matrix samples

[0136]

[0137]

[0138] (2) Precision test of the kit

[0139] In establishing the standard curve, five replicates were performed within the plate and five replicates were performed between the plates. The coefficients of variation were calculated for both within and between the plates. The results showed that the coefficient of variation within the plates was less than 9.47%, and the coefficient of variation between the plates was less than 8.02%. The intra-plate and inter-plate variability rates of the standard curve were within 15%, indicating that the established ic-ELISA method has good precision.

[0140] (3) Accuracy and repeatability tests of the kit

[0141] Eight type II pyrethroid pesticide standards were added to samples at four concentrations: 1×LOQ, 2×LOQ, 4×LOQ, and the maximum residue limit (MRL). The samples were analyzed with five replicates within each plate and three replicates between plates. The recoveries of these pesticides in wastewater ranged from 80.1% to 106.7%, with intra- and inter-batch coefficients of variation (COP) less than 14.8%. In milk, the recoveries ranged from 82.8% to 106.2%, with COP less than 14.0%. In celery, the recoveries ranged from 82.6% to 102.3%, with COP less than 13.4%. In leeks, the recoveries ranged from 81.9% to 112.4%, with COP less than 10.8%.

[0142] Table 6. Recovery rates and coefficients of variation of pyrethroid pesticides in samples

[0143]

[0144]

[0145] Note: MRL stands for Maximum Residue Limit.

[0146] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of the present invention should be included within the scope of the present invention.

Claims

1. The application of a hapten in the detection of type II pyrethroid insecticides, including cypermethrin, lambda-cyhalothrin, deltamethrin, cyhalothrin, cyhalothrin, cypermethrin, deltamethrin, and fenvalerate, characterized in that... The hapten includes hapten A and hapten B. The molecular structural formula of hapten A is: ； The molecular structural formula of hapten B is: ； (1) Hapten A was conjugated with keyhole hemocyanin to obtain an immunogen; mice were then immunized with the immunogen, and hybridoma cell lines were obtained through cell fusion and screening; then monoclonal antibodies were prepared using the hybridoma cell lines. (2) Hapten B is conjugated with bovine serum albumin to obtain a coating antigen; then the coating antigen is used to coat a solid-phase carrier; then the sample is detected by enzyme-linked immunosorbent assay (ELISA) using the monoclonal antibody.

2. An enzyme-linked immunosorbent assay (ELISA) kit for detecting type II pyrethroid insecticides, including cypermethrin, lambda-cyhalothrin, deltamethrin, cyhalothrin, cyhalothrin, cyhalothrin, deltamethrin, and fenvalerate, characterized in that, The kit consists of: (1) A solid-phase carrier coated with a coating antigen; wherein the coating antigen is obtained by coupling the hapten B of claim 1 with bovine serum albumin; (2) Cypermethrin standard solution; (3) Monoclonal antibody working solution; the monoclonal antibody is secreted by the hybridoma cell line PYR / 1D2; the preparation method of the hybridoma cell line includes: ① conjugating the hapten A in claim 1 with keyhole hemocyanin to obtain an immunogen; ② immunizing mice with the immunogen obtained in step ①, and obtaining a hybridoma cell line through cell fusion and screening; the preservation number of the hybridoma cell line is CCTCC NO: C2021228; (4) Working solution of horseradish catalase-labeled goat anti-mouse IgG antibody; (5) 10× concentrated phosphate buffer; (6) 10× concentrated detergent; (7) Substrate A and substrate B; (8) Termination solution.

3. The application of the kit according to claim 2 in detecting residues of type II pyrethroid insecticides such as cypermethrin, lambda-cyhalothrin, deltamethrin, cyhalothrin, cyhalothrin, cyhalothrin, cypermethrin, deltamethrin, and fenvalerate in samples.

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