Methyl isofenphos hapten, artificial antigen and antibody, and preparation method and application thereof

Abstract

The application discloses methyl isofenphos hapten, artificial antigen and antibody and a preparation method and application thereof, and belongs to the technical field of agricultural product quality safety detection. The methyl isofenphos hapten has a structure as shown in formula I, 6-carboxyhexyloxy connecting arms are introduced at the alkoxy side (P-O-C site) of the phosphorus atom of a methyl isofenphos mother nucleus, and three characteristic functional groups of phosphoric acyl, thiophosphoric amide and isopropoxy carbonyl are completely reserved. The hapten is coupled with bovine serum albumin (BSA) or bovine thyroid globulin (BTG) by an active ester method to prepare artificial antigen, and a monoclonal antibody is obtained after Balb / c mice are immunized. The antibody has an IC 50 of 6.09 ng / mL, a linear range of 1.16-30.49 ng / mL, and a cross reactivity of less than 1% to 11 kinds of structural similar pesticides. The application avoids the damage to a sensitive thiophosphoric amide bond, solves the problem of epitope distortion caused by the modification of the -NH site in the prior art, and is suitable for high-specificity residual screening. Formula I

Description

Technical Field

[0001] This invention belongs to the field of agricultural product quality and safety testing technology. Specifically, it relates to a methyl isofenphos hapten, an artificial antigen and an antibody, as well as their preparation methods and applications. Background Technology

[0002] Isofenphos-methyl is a broad-spectrum soil organophosphorus pesticide widely used in agriculture. Its chemical name is O-methyl-O-[(2-isopropoxyformyl)phenyl]-N-isopropylthiophosphoramide. It has a strong toxic effect on underground pests and can control grubs, mole crickets, cutworms, wireworms, and can also be used to control armyworms, aphids, tobacco budworms, peach fruit moths, and spider mites. According to Announcement No. 536 of the Ministry of Agriculture and Rural Affairs of my country, methyl isofenphos is a highly toxic pesticide and its use on vegetables, fruit trees, tea, and medicinal herbs is prohibited. It has strong contact, stomach poison, and systemic effects. Long-term or large-scale exposure can harm human health and inhibit the activity of acetylcholinesterase, leading to the accumulation of acetylcholine in the body and producing highly toxic effects on the central nervous system, respiratory system, and skin. Meanwhile, methyl isofenphos has a long degradation half-life, and most of the pesticide residues remain in the soil after application and are difficult to degrade. These residues may migrate and spread through the food chain, disrupting the agricultural ecological balance and threatening the quality and safety of agricultural products and the development of green agriculture. Therefore, the monitoring and control of methyl isofenphos residues is of great significance for ensuring agricultural ecological security and promoting the construction of green agriculture.

[0003] To reduce the impact of methyl isofenphos pesticides on human health, the agricultural ecological environment, and the quality of agricultural products, it is urgent to establish efficient detection methods and a sound regulatory system. Currently, the main methods for detecting methyl isofenphos residues both domestically and internationally include gas chromatography, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), surface-enhanced Raman spectroscopy (SMR), near-infrared spectroscopy (NIRS), and high-performance thin-layer chromatography (HPLC). While these methods possess high sensitivity, accuracy, stability, and repeatability, enabling rapid quantitative detection of methyl isofenphos residues, they also suffer from drawbacks such as reliance on complex, sophisticated, and expensive instruments; high detection costs; cumbersome operating procedures; long detection cycles; high technical requirements for operators; strict sample pretreatment requirements; insufficient environmental friendliness; and unsuitability for on-site or batch testing. Immunoassay based on antigen-antibody specific binding reactions, which has been developed in recent years, has advantages such as simple operation, low cost, high throughput, high detection sensitivity, and strong portability. It is suitable for rapid on-site screening and can also meet the needs of large-scale laboratory testing. It effectively makes up for the shortcomings of traditional instrument detection methods and provides a new technical path for the detection of methyl isofenphos residues. It plays an important supporting role in strengthening the supervision of agricultural inputs, ensuring agricultural ecological security, and promoting the development of green agriculture.

[0004] Immunoassay, based on the highly specific recognition reaction between antibodies and antigens, achieves highly selective detection of pesticide residues and has become a major analytical method in fields such as agricultural ecological environment monitoring and agricultural product quality and safety analysis, showing broad application prospects in pesticide residue detection. When establishing and promoting the application of immunoassay methods for methyl isofenphos residues, the core technology lies in obtaining antibodies with high specificity and sensitivity, and the preparation of suitable methyl isofenphos haptens is a prerequisite for achieving this goal. Studies have shown that the phosphoryl group and thiophosphoramide structure of methyl isofenphos form its core framework, with the isopropoxycarbonyl group being its characteristic structure, playing a crucial role in the specificity of the immunoassay reaction. Existing invention patents CN115925740A, CN110760004A, and CN114292818A all employ a technique of derivatizing the -NH group of methyl isofenphos into a spacer arm with -COOH, and then coupling it with a carrier protein. This modification method destroys the thiophosphoramide structure of methyl isofenphos (i.e., the P=SN bond is broken), failing to retain its specific substituent groups, easily leading to a significant decrease in hapten specificity, thus affecting the accuracy and reliability of immunoassay. In addition, this type of method requires the direct use of highly toxic methyl isofenphos technical as a starting material, which poses problems such as high operational risks and significant environmental risks.

[0005] Therefore, there is an urgent need to develop a hapten design strategy that eliminates the need for methyl isofenphos technical and precisely preserves its pharmacophore without disrupting the thiophosphoramide bond. This strategy would address the shortcomings of existing technologies, such as high cross-reactivity and poor specificity due to epitope distortion. This is of great significance for optimizing immunoassay results, ensuring the quality and safety of agricultural products, and promoting the development of green and ecological agriculture. Summary of the Invention

[0006] The purpose of this invention is to provide a methyl isofenphos hapten, an artificial antigen, an antibody, and their preparation methods and applications. The methyl isofenphos hapten introduces a carboxyl-containing carbon chain linker arm into the alkoxy side (POC site) of the phosphorus atom of the methyl isofenphos nucleus, thus fully preserving the three major characteristic functional groups: phosphoryl group, thiophosphoramide, and isopropoxycarbonyl group. This avoids chemical modification of the sensitive thiophosphoramide bond, thereby achieving immune presentation of the complete pharmacophore of methyl isofenphos.

[0007] To achieve the above-mentioned objective, the present invention provides a methyl isofenphos hapten, wherein the methyl isofenphos hapten is 6-(((2-(isopropoxycarbonyl)phenoxy)(isopropylamino)thiophosphoryl)oxy)hexanoic acid, which has the structure shown in Formula I.

[0008]

[0009] Formula I

[0010] The methyl isofenphos hapten provided by this invention introduces a 6-carboxyhexyloxy linker arm into the alkoxy side of the phosphorus atom of methyl isofenphos, thereby enabling it to be coupled with a carrier protein to obtain an artificial antigen for immunization. This hapten completely retains all the characteristic groups of methyl isofenphos, with only minimal changes to the original structural features of methyl isofenphos. At the same time, after coupling with the carrier protein, the unique phosphoryl group, thiophosphoramide structure, and isopropoxycarbonyl structure of methyl isofenphos are highlighted, laying the foundation for subsequent stimulation of animal immune responses to produce antibodies with stronger specificity and higher sensitivity.

[0011] The present invention also provides a method for preparing the above-mentioned methyl isofenphos hapten, which is to obtain intermediate 1 by phosphorylation reaction of isopropyl salicylate and thiophosphoryl chloride under triethylamine-mediated reaction, to obtain intermediate 2 by amination substitution reaction of intermediate 1 and isopropylamine, to obtain intermediate 2 by nucleophilic substitution reaction of intermediate 2 and 6-tert-butoxy-6-oxohexanol under sodium hydride, to obtain intermediate 3, and finally to obtain the target hapten by formic acid-mediated deprotection reaction of intermediate 3.

[0012] Furthermore, the preparation method includes the following steps:

[0013] (1) Under nitrogen protection and -10℃ conditions, isopropyl salicylate and thiophosphoric chloride were dissolved in dichloromethane, triethylamine was added dropwise, and the temperature was raised to 20℃ and stirred for 12 h to obtain isopropyl-2-thiophosphoric oxybenzoate, i.e. methyl isofenphos intermediate 1.

[0014] (2) Under nitrogen protection and at 0°C, triethylamine and isopropylamine were added to a dichloromethane solution of methyl isofenphos intermediate 1, stirred at 0°C for 2 h, concentrated under reduced pressure, and purified by silica gel column chromatography with a gradient of 0-10% ethyl acetate / n-hexane to obtain isopropyl-2-[chloro-(isopropylamino)thiophosphoryl]oxybenzoate, i.e. methyl isofenphos intermediate 2;

[0015] (3) Under nitrogen protection and at 0°C, sodium hydride was added to the tetrahydrofuran solution of 6-tert-butoxy-6-oxohexanol. After stirring for 15 min, the tetrahydrofuran solution of methyl isofenphos intermediate 2 was added dropwise. After stirring for 1 h, the solution was quenched with saturated ammonium chloride, extracted with ethyl acetate, washed with saturated brine, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and then purified by silica gel column chromatography with a gradient of 5% to 7% ethyl acetate / n-hexane to obtain isopropyl-2-[(6-tert-butoxy-6-oxohexoxy)-(isopropylamino)thiophosphoryl]oxybenzoate, i.e., methyl isofenphos intermediate 3;

[0016] (4) Methyl isofenphos intermediate 3 was dissolved in formic acid, stirred at 20°C for 1 h, quenched with water at 0°C, extracted with ethyl acetate, washed with saturated brine, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and first obtained crude product by 25%–35% ethyl acetate / n-hexane gradient silica gel column chromatography, and then purified by preparative high performance liquid chromatography to obtain the methyl isofenphos hapten.

[0017] In step (1), the ratio of isopropyl salicylate:thiophosphoryl chloride:triethylamine is 1:1.3:2; in step (2), the ratio of methyl isofenphos intermediate 1:triethylamine:isopropylamine is 1:1:1.1; and in step (3), the ratio of methyl isofenphos intermediate 2:6-tert-butoxy-6-oxohexanol:sodium hydride is 1:1:0.9.

[0018] Based on the structural characteristics of methyl isofenphos, this invention rationally designs a synthetic route to construct the methyl isofenphos skeleton by reverse engineering from isopropyl salicylate and simultaneously introduces a linker arm. It eliminates the need for the use of highly toxic methyl isofenphos technical, ensures safe and mild reaction operation, maintains high stability of intermediates in each step, achieves 100% purity of the final product, and has controllable yield, making it suitable for large-scale production.

[0019] This invention also provides a methyl isofenphos artificial antigen, which is a conjugate obtained by coupling a carrier protein with the above-mentioned methyl isofenphos hapten. The methyl isofenphos artificial antigen can be used as an immunogen or as a coating antigen.

[0020] Furthermore, the carrier protein is bovine serum albumin or bovine thyroglobulin.

[0021] More specifically, such as: immunogens formed by methyl isofenphos hapten-bovine thyroglobulin (BTG); coating antigens formed by methyl isofenphos hapten-bovine serum albumin (BSA).

[0022] The present invention also provides a method for preparing the above-mentioned methyl isofenphos artificial antigen, comprising:

[0023] S1: Dissolve the methyl isofenphos hapten in dimethyl sulfoxide, add EDC and NHS, stir at room temperature in the dark for 3 h to obtain activated hapten solution A; wherein the molar ratio of methyl isofenphos hapten: EDC: NHS is 1:2:2.

[0024] S2: Dissolve the carrier protein in pH 9.6 carbonate buffer at a concentration of 5-10 mg / mL to obtain solution B;

[0025] S3: Add solution A dropwise to solution B under stirring in an ice bath, adjust the pH to 9.6 with NaOH, react overnight in the dark, and then dialyze with 0.01mol / L PBS for 3 days, changing the solution 3 times a day to obtain the artificial antigen.

[0026] The present invention also provides a methyl isofenphos antibody, which is obtained by animal immunization with the above-mentioned methyl isofenphos artificial antigen, and can produce a specific immune response with methyl isofenphos.

[0027] Furthermore, the methyl isofenphos antibody is a monoclonal antibody. Additionally, the methyl isofenphos antibody can be prepared using methods conventional in the art.

[0028] In one specific implementation, the methyl isofenphos antibody is a murine monoclonal antibody specifically targeting the methyl isofenphos artificial antigen of the aforementioned methyl isofenphos hapten.

[0029] IC50 of methyl isofenphos antibody against methyl isofenphos obtained using the methyl isofenphos artificial antigen of the present invention 50 The concentration was 6.09 ng / mL, the linear detection range was 1.16-30.49 ng / mL, and the cross-reactivity with methamidophos, profenofos, methyl parathion, triazophos, pyrazophos, fenthion, chlorpyrifos, acephate, DDT, hexachlorocyclohexane, and trichlorfon was less than 1%.

[0030] The present invention also provides the application of water-based methyl isofenphos antibody in the preparation and detection of methyl isofenphos residues, including the methyl isofenphos hapten, methyl isofenphos complete antigen and methyl isofenphos antibody.

[0031] This invention induces antibody production in immunized animals using an artificial antigen of methyl isofenphos, which is then used in the immunoassay of methyl isofenphos. The immunoassay of methyl isofenphos includes, but is not limited to, a methyl isofenphos ELISA kit.

[0032] The beneficial effects of this invention are:

[0033] (1) The epitope is intact, and the antibody specificity is significantly improved. The methyl isofenphos hapten provided by the present invention not only retains the characteristic structure of methyl isofenphos to the greatest extent, thus significantly enhancing the immunogenicity of the methyl isofenphos hapten, but also has a carboxyl group that can be coupled with the carrier protein. Immunizing animals with the methyl isofenphos artificial antigen obtained by coupling the methyl isofenphos hapten with the carrier protein is more conducive to stimulating the animal's immune response to produce antibodies with stronger specificity and higher sensitivity, thus providing a foundation for the subsequent establishment of various immunoassay methods for methyl isofenphos.

[0034] (2) The synthetic route is safe and does not require highly toxic active ingredients. This invention discloses a method for preparing methyl isofenphos hapten. The raw materials used are readily available, the reaction operation is relatively simple, the reaction conditions are easy to control, the purity and yield of the prepared methyl isofenphos hapten are high, and the destruction of the thiophosphoramide bond is avoided, thus solving the problem of epitope distortion caused by the modification of the -NH site in the prior art.

[0035] (3) Excellent detection performance and strong practicality. The methyl isofenphos antibody obtained using the methyl isofenphos artificial antigen of this invention has good titer, specificity, and affinity. The constructed indirect competitive enzyme-linked immunosorbent assay method has a high sensitivity (IC50) for methyl isofenphos. 50 The antibody has a detection range of 1.16-30.49 ng / mL and a detection limit of 6.09 ng / mL, exhibiting a wide linear range and high sensitivity, meeting the needs of rapid screening. Furthermore, its cross-reactivity with 11 structurally similar pesticides (methamidophos, profenofos, methyl parathion, triazophos, pyraclostrobin, fenthion, chlorpyrifos, acephate, DDT, hexachlorocyclohexane, and trichlorfon) is less than 1%, significantly superior to existing antibodies with high cross-reactivity due to -NH modification. This antibody is particularly suitable for high-specificity regulatory scenarios with extremely low tolerance for false positives, such as those involving exported agricultural products. Attached Figure Description

[0036] Figure 1 This is the synthetic route for the methyl isofenphos hapten of this invention.

[0037] Figure 2 The mass spectrometry spectrum of the methyl isofenphos hapten prepared in Example 1 is shown.

[0038] Figure 3 The image shows the UV identification of the methyl isofenphos artificial antigen prepared in Examples 2 and 3.

[0039] Figure 4 This is a competitive standard curve of ic-ELISA for the methyl isofenphos antibody obtained in Example 4. Detailed Implementation

[0040] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0041] Example 1

[0042] A method for preparing methyl isofenphos hapten, the preparation method is as follows: Figure 1 As shown, the steps are as follows:

[0043] Under nitrogen protection and at -10°C, 44.95 mmol of isopropyl salicylate and 58.44 mmol of thiophosphoryl chloride were dissolved in 150 mL of dichloromethane, followed by the dropwise addition of 89.90 mmol of triethylamine. After the addition was complete, the temperature was raised to 20°C and the reaction was stirred at a constant temperature for 12 h. Thin-layer chromatography (developing solvent: hexane: ethyl acetate = 5:1, Rf = 0.45) showed that the raw material isopropyl salicylate was completely consumed, yielding crude methyl isofenphos intermediate 1, isopropyl-2-thiophosphoryloxybenzoate.

[0044] Under nitrogen protection and at 0°C, 44.71 mmol of triethylamine and 49.18 mmol of isopropylamine were added to 150 mL of a dichloromethane solution containing 44.71 mmol of isopropyl-2-thiophosphoryloxybenzoate (methyl isofenphos intermediate 1), and the mixture was stirred at 0°C for 2 h. The reaction progress was monitored by thin-layer chromatography (developing solvent: hexane: ethyl acetate = 5:1, Rf = 0.40). After the reaction solution was concentrated under reduced pressure, it was purified by silica gel column chromatography (eluent: 0-10% ethyl acetate / hexane gradient, flow rate 100 mL / min) to obtain methyl isofenphos intermediate 2, isopropyl-2-[chloro-(isopropylamino)thiophosphoryl]oxybenzoate (6.25 mmol, yield 13.99%).

[0045] At 0 °C, 8.49 mmol of 60% sodium hydride was added to 20 mL of a tetrahydrofuran solution containing 5.66 mmol of 6-tert-butoxy-6-oxohexanol, and the mixture was stirred for 15 min under nitrogen protection. Subsequently, 10 mL of a tetrahydrofuran solution containing 5.66 mmol of isopropyl-2-[chloro-(isopropylamino)thiophosphoryl]oxybenzoate (methyl isofenphos intermediate 2) was added dropwise, and the mixture was stirred at 0 °C for 1 h. After monitoring the consumption of the reactants by LC-MS, the reaction was quenched with saturated ammonium chloride solution, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then purified by silica gel column chromatography (eluent: 5%–7% ethyl acetate / n-hexane gradient, flow rate 100 mL / min) to obtain a yellow oily compound, methyl isofenphos intermediate 3, and isopropyl-2-[(6-tert-butoxy-6-oxohexyl)-(isopropylamino)thiophosphoryl]oxybenzoate (2.33... (mmol, yield 41.10%, purity 98.61%).

[0046] 2.36 mmol of isopropyl-2-[(6-tert-butoxy-6-oxohexyloxy)-(isopropylamino)thiophosphoryl]oxybenzoate (methyl isofenphos intermediate 3) was dissolved in 15 mL of formic acid and stirred at 20 °C for 1 h. After monitoring the formation of the target product by LC-MS, the reaction was quenched with water at 0 °C, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to silica gel column chromatography (eluting with a gradient of 25%–35% ethyl acetate / n-hexane, flow rate 100 mL / min) to obtain crude methyl isofenphos hapten. 300 mg of crude methyl isofenphos hapten was subjected to preparative HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm, mobile phase: 10 mM ammonium bicarbonate aqueous solution-acetonitrile, eluted with a gradient of 15%–45% acetonitrile for 8 hours). The product was purified by (min) to obtain the light yellow oily target product methyl isofenphos hapten 6-(((2-(isopropoxycarbonyl)phenoxy)(isopropylamino)thiophosphoryl)oxy)hexanoic acid (247.98 μmol, yield 10.51%, purity 100%).

[0047] Mass spectrometry results as follows Figure 2 The results show that the ion peak with a mass-to-charge ratio (m / z) of 430.1 in the positive ion mode ESI ionization full scan corresponds to the quasi-molecular ion peak [M+H]+, which is consistent with the theoretical molecular weight of the target hapten of 431.15, proving that the methyl isofenphos hapten was successfully synthesized.

[0048] Example 2

[0049] A method for preparing a methyl isofenphos artificial antigen (BSA conjugate) includes the following steps:

[0050] Take 78.5 mg of the methyl isofenphos hapten prepared in Example 1, dissolve it in 1 mL of DMSO, add 46.1 mg of NHS and stir at room temperature in the dark for 15 min, then add 62.1 mg of EDC dropwise and stir for 3 h in the dark to obtain hapten solution A; take 83.8 mg of BSA and dissolve it in 5 mL of 0.05 mol / L carbonate buffer to obtain solution B; add solution A dropwise to solution B, react at 4℃ for 4 h, and purify by dialysis with 0.01 mol / L PBS for 3 days, changing the dialysis solution 3 times a day to obtain methyl isofenphos artificial antigen conjugated with bovine serum albumin, aliquot it into 1 mg / mL and store at -20℃.

[0051] Example 3

[0052] A method for preparing a methyl isofenphos artificial antigen (BTG conjugate) includes the following steps:

[0053] Take 78.5 mg of the methyl isofenphos hapten prepared in Example 1, dissolve it in 1 mL of DMSO, add 46.1 mg of NHS and stir at room temperature in the dark for 15 min, then add 62.1 mg of EDC dropwise and stir in the dark for 3 h to obtain hapten solution A; take 16.5 mg of BTG and dissolve it in 1 mL of 0.05 mol / L carbonate buffer to obtain solution B; add solution A dropwise to solution B, react at 4℃ for 4 h, and purify by dialysis with 0.01 mol / L PBS for 3 days, changing the dialysis solution 3 times a day to obtain the methyl isofenphos artificial antigen conjugated with bovine thyroglobulin, aliquot it into 1 mg / mL and store at -20℃.

[0054] like Figure 3 As shown, all synthesized artificial antigens exhibit characteristic absorption peaks of haptens and carrier proteins, or their absorption peaks shift relative to the original carrier protein, indicating that all artificial antigen synthesis was successful.

[0055] Example 4

[0056] A methyl isofenphos antibody, the preparation method of which is as follows:

[0057] 1. Animal immunization

[0058] Twelve healthy 6-8 week old female Balb / c mice (divided into groups A and B, with 6 mice in each group) were used for the initial immunization. Each mouse received a subcutaneous injection at multiple sites on the back of the neck using Freund's complete adjuvant emulsified with 200 μg of methyl isofenphos artificial antigen conjugated with bovine thyroglobulin. Subsequent booster immunizations were administered every two weeks via subcutaneous injection at multiple sites on the back of the neck, using Freund's incomplete adjuvant emulsification. The final immunization used physiological saline instead of Freund's incomplete adjuvant via intraperitoneal injection, with the same injection dose as the previous immunizations. Specific immunization procedures are shown in Table 1.

[0059] Seven days after the third, fourth, and booster immunizations, orbital blood was collected from mice, and serum titers were determined by ELISA. The specific steps are as follows:

[0060] (1) Dilute the methyl isofenphos artificial antigen conjugated with bovine serum albumin at a ratio of 1:1000 with 0.05 mol / L pH 9.6 carbonate buffer, coat each well with 100 μL, incubate at 37°C for 3 h or at 4°C for 12 h, discard the coating solution, wash 3 times with PBST, and pat dry.

[0061] (2) Add 250 μL of blocking solution to each well, react at 37℃ for 1 h, then discard the blocking solution, wash 3 times with PBST, and pat dry.

[0062] (3) Add 100 μL of antiserum diluted with PBS to each well, react at 37°C for 30 min, then discard the reaction solution, wash 3 times with PBST, with an interval of 30 s each time, and pat dry.

[0063] (4) Add 100 μL / well of horseradish peroxidase-labeled goat anti-mouse anti-antibody (1:5000) diluted in PBS with 2% skim milk powder, react at 37℃ for 30 min, wash 3 times with PBST, with an interval of 30 s each time, and pat dry.

[0064] (5) Add 100 μL of the substrate TMB colorimetric solution to each well, react at 37℃ in the dark for 15 min, and then add 50 μL of 2mol / L H2SO4 solution to each well to terminate the reaction.

[0065] (6) The OD value at a wavelength of 450 nm was measured using an ELISA reader, and the OD value of the sample wells was used as the reference value. 450 A dilution factor close to 1 is used as the titer of positive serum.

[0066] Table 1. Immunization schedule for mice

[0067]

[0068] 2. Cell fusion

[0069] (1) Preparation of feeder cells: One day before cell fusion, select 6-8 week old blank Balb / c mice of the same strain used for immunization, euthanize them by cervical dislocation, immerse them in 75% alcohol for 5 min, and then place them in a laminar flow hood, abdomen up, in a petri dish or fixed on a dissecting board. Soak the autoclaved forceps and scissors in alcohol and then sterilize them by heating them with an alcohol lamp. Using the sterilized forceps, lift the mouse abdomen, cut open the abdominal skin without damaging the peritoneum to avoid leakage and contamination of peritoneal fluid, and completely expose the peritoneum to the operating table. Wipe the peritoneum with an alcohol swab for disinfection. Draw 5 mL of DMEM basal culture medium preheated to 37℃ into the mouse peritoneal cavity with a syringe, gently withdraw the syringe, and shake the mouse's legs and tail a few times. Aspirate the peritoneal fluid with the original syringe and inject it into a centrifuge tube. Repeat this operation 3-4 times. Centrifuge at 1000 r / min for 10 min and discard the supernatant. Resuspend the cells in 50 mL of preheated HAT complete culture medium at 37°C, mix thoroughly, and then seed the feeder cells into 96-well cell culture plates at 100 μL / well. Cover the plates and incubate at 37°C in a 5% CO2 incubator.

[0070] (2) Spleen cell preparation: Three days after booster immunization, immunized Balb / c mice were euthanized by euthanasia after blood was collected from the orbital cavity and the cells were immersed in 75% alcohol for 5 minutes. The mice were then transferred to a clean bench, fixed abdominally in a paraffin tray, and the forceps and scissors were sterilized using an alcohol lamp. The abdomen was lifted with the forceps, and the abdominal skin and peritoneum were cut open to fully expose the internal organs and facilitate spleen removal. After locating the spleen, the surrounding tissue was pinched with forceps to avoid damaging the spleen, and the cells were quickly transferred to a clean culture dish. After removing the adhering fat and connective tissue around the spleen, avoiding damage, the spleen was cleaned and transferred to DMEM culture medium. Using a syringe needle, make 10-20 holes on both sides of the spleen, inject DMEM into the spleen and blow out spleen cells. Repeat this process until the spleen turns white to ensure that the spleen cells are completely blown out. After mixing, transfer to a 50mL centrifuge tube, add DMEM culture medium to 45mL, centrifuge at 1500 r / min for 5 min, discard the supernatant, and scrape the cells in a clean bench to fully disperse them. Then, resuspend the spleen cells in DMEM culture medium and count them under a microscope.

[0071] (3) Preparation of myeloma cells: Take 3 bottles of myeloma cells in good growth condition (number of live cells >95%), blow them off completely, transfer them to 50 mL centrifuge tubes, add DMEM culture medium to 45 mL, centrifuge at 1500 r / min for 5 min, discard the supernatant, scrape the cells in a clean bench to make them fully dispersed, and then resuspend the spleen cells in DMEM culture medium and count them under a microscope.

[0072] (4) Cell mixing: After cell counting, add the resuspended spleen cells to the tumor cells according to the ratio of myeloma cells to spleen cells of 1:8, mix by pipetting, centrifuge at 1500 r / min for 5 min, discard the supernatant, leaving 1-2 drops of DMEM in the cells. Scrape the bottom of the centrifuge tube on a clean bench to mix the cells.

[0073] (5) Cell fusion: Centrifuge the mixed cells, discard the supernatant, and tap the precipitated cell pellet into a paste. Place the centrifuge tube containing the mixed cell pellet in a 40°C water bath for 1 minute to preheat. Using a pipette, slowly add 1 ml of preheated PEG solution (40°C) dropwise over 1 minute, touching the bottom. During the addition, gently stir with the tip of the pipette or by slowly rotating the centrifuge tube. After adding the PEG, pipette two or three times, being careful not to generate air bubbles. Keep the centrifuge tube in the 40°C water bath for 1 minute, and slowly add 45 ml of preheated 37°C DMEM medium along the tube wall to dilute it, thus stopping the PEG reaction. Centrifuge at 800 rpm for 5 minutes, discard the supernatant, and remove any residual PEG. Resuspend the cells in 45 mL of HAT complete culture medium and seed them in 96-well cell culture plates containing feeder cells, 100 μL per well, and place in an incubator.

[0074] 3. Cell line screening

[0075] When the cells grow to 1 / 3 to 1 / 2 of the bottom of the well, antibody detection can be performed. ELISA is used to screen culture wells with hybridoma cell growth. The screening process involves two steps: first, indirect ELISA is used to screen for positive cells; second, methyl isofenphos is used as a standard, and indirect competitive ELISA is used to determine the inhibitory effect on the positive cells. Wells showing good inhibition of the methyl isofenphos standard are selected, and subcloning is performed using the limiting dilution method, followed by detection using the same method. This process is repeated three times to obtain a cell line that stably secretes methyl isofenphos monoclonal antibodies.

[0076] 4. Preparation of ascites

[0077] Liquid paraffin was injected into 6-8 week old Balb / c mice, 500 μL per mouse. Ten days later, hybridoma cells in the logarithmic growth phase were collected in DMEM basal medium and counted using a hemocytometer and microscope, with a cell concentration of approximately 1.0 × 10⁻⁶. 6 ~1.5×10 6 Within the range of cells / mL. 0.5 mL of hybridoma cells were injected into the peritoneal cavity of each mouse. Observe the mice for abdominal distension after one week. Collect ascites fluid from the peritoneal cavity using a sterile syringe, repeating this process every one to two days until the mouse dies naturally. Centrifuge at 12000 r / min for 5 min at 4℃, collect the supernatant, and remove the floating fat and protein membranes from the upper layer of the ascites fluid.

[0078] 5. Antibody purification

[0079] Monoclonal antibodies were purified using the caprylic acid-ammonium sulfate method: The centrifuged mouse ascites fluid was taken out of the -20℃ freezer, thawed, and the volume was measured and placed in a beaker. Four times the volume of the ascites fluid was added with 60 mmol / L acetate buffer, and the mixture was slowly stirred on a magnetic stirrer at 4℃. The pH was adjusted to 4.5 with 0.1 mol / L NaOH. 33 μL / mL of caprylic acid was slowly added according to the volume of mouse ascites fluid. After stirring for 30 min, the mixture was placed in a freezer at 4℃ and allowed to stand for 1 h. Centrifuge at 10000 r / min for 30 min at 4℃. Take the supernatant and place it on a magnetic stirrer at 4℃, maintaining slow stirring. Add 1 / 10 of the supernatant volume of 0.1 mol / L PBS, adjust the pH to 7.4 with 1 mol / L NaOH, and pre-cool in a refrigerator at 4℃ for 15 min. Then, slowly add 0.277 g / mL of ammonium sulfate according to the total volume. After the addition is complete, continue stirring for 30 min and let it stand at 4℃ for 3 h. Centrifuge at 12000 r / min for 20 min at 4℃, discard the supernatant and collect the white precipitate at the bottom. Gently pipette with 1 / 10 of the original sample volume of pre-cooled 0.01 mol / L PBS buffer until the precipitate is completely dissolved. Put the reconstituted antibody solution into a dialysis bag and dialyze in a large amount of PBS at 4℃ for 3 days (changing the buffer 3-4 times during this period).

[0080] Example 5

[0081] Establishment of a standard curve for antibody detection of methyl isofenphos and determination of antibody cross-reactivity

[0082] The working concentrations of the coating agent and the methyl isofenphos antibody were determined using a matrix titration method. The working concentration of the coating agent was 500 ng / mL, and the working concentration of the methyl isofenphos antibody was 100 ng / mL. Other organochlorine pesticides, including methamidophos, profenofos, methyl parathion, triazophos, pyraclostrobin, fenthion, chlorpyrifos, acephate, DDT, hexachlorocyclohexane, and trichlorfon, were tested in triplicate (n=3).

[0083] The antibody indirect competitive ELISA detection procedure is the same as the ELISA method in Example 4 for determining mouse serum titers. It is important to note that methyl isofenphos is diluted into a series of gradient standard solutions, diluted to 600, 400, 300, 200, 150, 100, 75, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, and 0.390625 ng / mL, respectively. Add 50 μL to each well, followed by 50 μL of methyl isofenphos antibody. For antibody cross-reactivity, 11 structurally similar pesticides were selected as controls: methamidophos, profenofos, methyl parathion, triazophos, pyrazophos, fenthion, chlorpyrifos, acephate, DDT, hexachlorocyclohexane, and trichlorfon. The IC50 of each compound was determined using the same method described above. 50Value. The cross-reactivity rate (CR%) is calculated using the formula: CR% = (methyl isofenphos IC50) / (methyl isofenphos IC50) 50 / Chemical to be tested IC 50 ) × 100%.

[0084] The IC50 of the suppression curve was calculated using the four-parameter fitting module of Prism 9. 20 IC 50 IC 80 Values, see standard curve Figure 4 The standard curve IC obtained under the optimal conditions. 50 The effective value was 6.09 ng / mL, and the linear detection range was 1.16–30.49 ng / mL. The cross-reactivity of the methyl isofenphos monoclonal antibody against methyl isofenphos and other organochlorine pesticides was 100% for methyl isofenphos, and <1% for methamidophos, profenofos, methyl parathion, triazophos, pyraclostrobin, fenthion, chlorpyrifos, acephate, DDT, hexachlorocyclohexane, and trichlorfon. This indicates that the antibody has extremely high specificity and a very low risk of false positives, making it suitable for high-specificity residue screening scenarios.

Claims

1. A methyl isofenphos hapten, characterized in that, The methyl isofenphos hapten is 6-(((2-(isopropoxycarbonyl)phenoxy)(isopropylamino)thiophosphoryl)oxy)hexanoic acid, which has the structure shown in Formula I. Furthermore, the methyl isofenphos hapten retains the phosphoryl group, thiophosphoramide, and isopropoxycarbonyl group structures completely by introducing a 6-carboxyhexyloxy linker arm to the alkoxy side of the phosphorus atom in the methyl isofenphos parent nucleus. Formula I.

2. The method for preparing the methyl isofenphos hapten according to claim 1, characterized in that, Includes the following steps: (1) Under nitrogen protection and -10℃ conditions, isopropyl salicylate and thiophosphoric chloride were dissolved in dichloromethane, triethylamine was added dropwise, and the temperature was raised to 20℃ and stirred for 12 h to obtain isopropyl-2-thiophosphoric oxybenzoate, i.e. methyl isofenphos intermediate 1. (2) Under nitrogen protection and at 0°C, triethylamine and isopropylamine were added to a dichloromethane solution of methyl isofenphos intermediate 1, stirred at 0°C for 2 h, concentrated under reduced pressure, and purified by silica gel column chromatography with a gradient of 0-10% ethyl acetate / n-hexane to obtain isopropyl-2-[chloro-(isopropylamino)thiophosphoryl]oxybenzoate, i.e. methyl isofenphos intermediate 2; (3) Under nitrogen protection and at 0°C, sodium hydride was added to the tetrahydrofuran solution of 6-tert-butoxy-6-oxohexanol. After stirring for 15 min, the tetrahydrofuran solution of methyl isofenphos intermediate 2 was added dropwise. After stirring for 1 h, the solution was quenched with saturated ammonium chloride, extracted with ethyl acetate, washed with saturated brine, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and then purified by silica gel column chromatography with a gradient of 5% to 7% ethyl acetate / n-hexane to obtain isopropyl-2-[(6-tert-butoxy-6-oxohexoxy)-(isopropylamino)thiophosphoryl]oxybenzoate, i.e., methyl isofenphos intermediate 3; (4) Methyl isofenphos intermediate 3 was dissolved in formic acid, stirred at 20°C for 1 h, quenched with water at 0°C, extracted with ethyl acetate, washed with saturated brine, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and first obtained crude product by 25%–35% ethyl acetate / n-hexane gradient silica gel column chromatography, and then purified by preparative high performance liquid chromatography to obtain the methyl isofenphos hapten. In step (1), the ratio of isopropyl salicylate:thiophosphoryl chloride:triethylamine is 1:1.3:2; in step (2), the ratio of methyl isofenphos intermediate 1:triethylamine:isopropylamine is 1:1:1.1; and in step (3), the ratio of methyl isofenphos intermediate 2:6-tert-butoxy-6-oxohexanol:sodium hydride is 1:1:0.

9.

3. A methyl isofenphos artificial antigen prepared from the methyl isofenphos hapten of claim 1, characterized in that, The methyl isofenphos artificial antigen is prepared by coupling the methyl isofenphos hapten with a carrier protein via an active ester method, wherein the carrier protein is bovine serum albumin or bovine thyroglobulin.

4. The method for preparing methyl isofenphos artificial antigen according to claim 3, characterized in that, include: S1: Dissolve the methyl isofenphos hapten of claim 1 in dimethyl sulfoxide (DMSO), add carbodiimide (EDC) and N-hydroxysuccinimide (NHS), stir at room temperature in the dark for 3 h to obtain activated hapten solution A; wherein the molar ratio of methyl isofenphos hapten:EDC:NHS is 1:2:

2. S2: Dissolve the carrier protein in pH 9.6 carbonate buffer at a concentration of 5-10 mg / mL to obtain solution B; S3: Add solution A dropwise to solution B under stirring in an ice bath, adjust the pH to 9.6 with NaOH, react overnight in the dark, and then dialyze with 0.01 mol / L PBS for 3 days, changing the solution 3 times a day to obtain the artificial antigen.

5. A methyl isofenphos antibody prepared from the methyl isofenphos artificial antigen of claim 3, characterized in that, The methyl isofenphos antibody was prepared by immunizing animals with the methyl isofenphos artificial antigen and is a mouse-derived monoclonal antibody. Its IC50 against methyl isofenphos is... 50 The concentration was 6.09 ng / mL, the linear detection range was 1.16-30.49 ng / mL, and the cross-reactivity with methamidophos, profenofos, methyl parathion, triazophos, pyrazophos, fenthion, chlorpyrifos, acephate, DDT, hexachlorocyclohexane, and trichlorfon was less than 1%.

6. The method for preparing methyl isofenphos antibody according to claim 5, characterized in that, include: (1) Balb / c mice were immunized with the methyl isofenphos artificial antigen, and spleen cells were fused with myeloma cells after booster immunization; (2) Positive hybridoma cell wells were initially screened using the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), and then positive wells with high inhibitory activity were screened using methyl isofenphos standard ic-ELISA. (3) Using the limiting dilution method, hybridoma cell lines that stably secrete antibodies were obtained through three subclonings; (4) After preparing ascites, the octanoic acid-ammonium sulfate method is used to purify it to obtain methyl isofenphos monoclonal antibody.

7. The application of the methyl isofenphos antibody according to claim 5 in the preparation of an enzyme-linked immunosorbent assay (ELISA) for detecting methyl isofenphos residues.

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