Monoclonal antibody capable of specifically recognizing paraquat and application thereof

Abstract

This invention relates to a monoclonal antibody capable of specifically recognizing paraquat and its applications, belonging to the field of immunoassay technology. The monoclonal antibody is produced by hybridoma cells with accession number GDMCC No: 68912. The monoclonal antibody of this invention exhibits high affinity and specificity for paraquat, high tolerance to non-physiological environments, and can be used to establish immunological detection methods for paraquat. It is suitable for rapid and sensitive detection of paraquat residues in agricultural products and environmental samples, providing a core reagent for on-site detection and batch screening of paraquat residues, and is of great significance for ensuring the quality and safety of agricultural products and the safety of the ecological environment. This invention establishes immunological analysis methods (icELISA and LFIA methods) for paraquat residues in different samples such as animal-derived foods, biological samples, and fruits and vegetables, providing an efficient and reliable technical means for monitoring paraquat residues.

Description

Technical Field

[0001] This invention belongs to the field of antibody and pesticide residue immunoassay technology, specifically relating to a monoclonal antibody that can specifically identify paraquat and its application. Background Technology

[0002] Paraquat (PQ) is a highly effective, broad-spectrum organic heterocyclic quaternary ammonium herbicide that was once widely used in agricultural production due to its excellent weed-controlling effect. However, paraquat is extremely toxic to humans, and there is no specific antidote, making it one of the herbicides with the highest mortality rate from acute poisoning in humans.

[0003] Methods for detecting phosphoric acid (PQ) residues in food and environmental samples are mainly divided into two categories: instrumental analysis methods and immunological analysis methods. Instrumental analysis methods (such as liquid chromatography-mass spectrometry) offer high accuracy and precision, but suffer from drawbacks such as expensive equipment, complex sample pretreatment, long detection cycles, and reliance on specialized operators, making them unsuitable for large-scale screening at the grassroots level and in the field. In contrast, immunological methods, relying on the specific recognition mechanism of antigens and antibodies, offer advantages such as ease of operation, low detection costs, and suitability for large-scale field screening, and have become an important development direction for rapid detection of PQ residues. Within immunological methods, antibodies are the core determinant of sensitivity and practicality; insufficient recognition performance and stability of the antibody itself cannot be fundamentally compensated for by simply optimizing the reaction system and detection conditions.

[0004] Currently, some progress has been made in the immunological analysis of paraquat. For example, Ouyang Zicheng et al. established ELISA and colloidal gold immunochromatographic assay (LFIA) methods, Sun Xiulan et al. constructed a direct competitive colloidal gold immunochromatographic assay system, and Fu et al. developed an LFIA test strip that can detect PQ residues in drinking water and real human samples within 10 minutes. However, the above studies did not take into account the interference of organic solvents in actual sample pretreatment systems on the immunological reaction, which limits the applicability of immunological methods in the detection of actual samples.

[0005] In the detection of paraquat residues, it is often necessary to introduce acidified organic solvents such as methanol during sample pretreatment. For example, the standard SN / T 0293—2014, "Determination of Paraquat and Diquat Residues in Exported Plant-Derived Foods by Liquid Chromatography-Mass Spectrometry / Mass Spectrometry," specifies that plant-derived food samples should be pre-extracted using methanol-0.1 mol / L HCl (1:9, volume ratio). However, organic solvents and acidic systems can weaken the binding ability between antigens and antibodies, reducing detection sensitivity and even leading to false negatives or false positives. Furthermore, the complex composition of different foods or biological samples may cause varying degrees of matrix effects, further affecting the detection results. Therefore, whether antibodies can maintain stable binding activity in acidified systems containing organic solvents and in non-physiological environments such as high salt, wide pH, and high urea is crucial for the practical application of immunological methods in real-world sample detection.

[0006] In summary, developing a monoclonal antibody against paraquat with high affinity and good tolerance under non-physiological environments (organic solvents, urea, salt ions, pH, etc.) is of great significance for solving the problem of insufficient binding capacity of existing antibodies in practical sample pretreatment systems. Furthermore, providing a matching immunological detection method with this antibody as the core recognition element is of great importance for establishing a convenient, accurate, and applicable immunological method for detecting paraquat residues in food and environmental samples. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the present invention provides a monoclonal antibody that can specifically identify paraquat and its application in the detection of paraquat.

[0008] The technical solution of this invention mainly includes the following: A mouse hybridoma cell, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No. 68912), with a deposit date of August 28, 2026, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, is classified and named as follows: Murine hybridoma cell line .

[0009] The method for obtaining the mouse hybridoma cells includes the following steps: (1) Synthesis of complete antigen: The hapten 1-(4-carboxybutyl)-1′-methyl-[4,4'-bipyridine]-1,1'-dionium was conjugated with bovine serum albumin using the active ester method to obtain the coating antigen PH-Q-BSA; the hapten 1-(4-carboxybutyl)-1′-methyl-[4,4'-bipyridine]-1,1'-dionium was conjugated with bovine thyroglobulin using the active ester method to obtain the immunogen PH-Q-BTG. (2) Mouse immunization: Mice were immunized with the immunogen PH-Q-BTG four times. The first immunization was performed by subcutaneous injection of the immunogen and Freund's complete adjuvant after emulsification in the neck and back. The subsequent three immunizations were performed by subcutaneous injection of the immunogen and Freund's incomplete adjuvant after emulsification in the neck and back. Mouse serum was collected after the final immunization. PH-Q-BSA was used as the coating agent. Paraquat standard solution and serum were diluted with acidified methanol working solution, and mouse serum was detected by icELISA. The acidified methanol working solution was prepared by diluting acidified methanol stock solution 30 times with phosphate buffer. The acidified methanol stock solution was a methanol and 0.1 mol / L HCl solution with a volume ratio of 10:90. (3) Cell fusion Mice with high tolerance and affinity to acidified methanol were selected and subjected to a shock immunization via intraperitoneal injection of the immunogen PH-Q-BTG. Hybridoma cells were prepared by combining mouse spleen lymphocytes with Sp2 / 0 myeloma cells using hybridoma technology and then subcloned. Using PH-Q-BSA as the coating agent, paraquat standard solution and hybridoma cell supernatant were diluted with acidified methanol working solution. The mouse hybridoma cell supernatant was detected by icELISA, and the cell line least affected by acidified methanol was selected to obtain the hybridoma cell line.

[0010] Preferably, in a specific embodiment of the present invention, hapten 1-(4-carboxybutyl)-1′-methyl-[4,4'-bipyridine]-1,1'-dionium, 1-ethyl-(3-dimethylaminopropyl), carbodiimide N-hydroxysuccinamide, and bovine serum albumin are taken in a mass ratio of 6.47:13.13:8.230:26.613. The hapten 1-(4-carboxybutyl)-1′-methyl-[4,4'-bipyridine]-1,1'-dionium is coupled with bovine serum albumin using the active ester method to obtain the coated antigen PH-Q-BSA. Preferably, in a specific embodiment of the present invention, the hapten 1-(4-carboxybutyl)-1′-methyl-[4,4'-bipyridine]-1,1'-dionium, 1-ethyl-(3-dimethylaminopropyl)carbodiimide N-hydroxysuccinamide, and bovine thyroglobulin are taken in a mass ratio of 3.649:7.717:4.633:14.941 and the hapten 1-(4-carboxybutyl)-1′-methyl-[4,4'-bipyridine]-1,1'-dionium is coupled with bovine thyroglobulin using the active ester method to obtain the immunogen PH-Q-BTG; A monoclonal antibody capable of specifically recognizing paraquat, said monoclonal antibody being secreted by mouse hybridoma cells with accession number GDMCC No: 68912.

[0011] The monoclonal antibody was prepared by the following method: The hybridoma cell line was injected into the peritoneal cavity of mice, and antibodies were prepared by in vivo induction. The obtained antibodies were purified to remove impurities and obtain monoclonal antibodies.

[0012] The application of the monoclonal antibody includes at least one of the following (1) to (2): (1) Application in the detection of paraquat; (2) Application in the preparation of paraquat detection reagent.

[0013] An icELISA method for detecting paraquat residues includes the following steps: S1. Dilute the coating agent (preferably PH-Q-BSA) with coating buffer, incubate in an ELISA plate; add blocking buffer to block, and wash. The coating solution is CB buffer (i.e., carbonate buffer, Na2CO3 6.36g, NaHCO3 11.6g, pure water to 4L, adjusted pH=9.6) or phosphate buffer; the incubation conditions are 4℃ for 16h or 25℃ to 37℃ for 2h; the blocking solution is at least one of No. 1 blocking solution and 2-5% (v / v) skim milk, and each 2L of No. 1 blocking solution contains 100mL fetal bovine serum, 100g sucrose, 5g casein, 11.6g Na2HPO4·12H2O, 1.186g NaH2PO4·2H2O, and 1mL Proclin 300 preservative, and the blocking time is 60-120min; S2. Add the sample solution to be tested and the stock solution of the monoclonal antibody to each well, and incubate them together to carry out a competitive reaction; The conditions for the competitive reaction are incubation at 4~37℃ for 30~60 min; S3. Wash; add enzyme-labeled secondary antibody dilution solution that specifically binds to the monoclonal antibody, and incubate; the dilution of the enzyme-labeled secondary antibody dilution solution is 1000~7000 times, and the secondary antibody reaction time is 15~60 min; S4. Add the colorimetric solution to the microplate to initiate the colorimetric reaction, and then add the stop solution to terminate the reaction; S5. Measure the OD of each well. 450nm The value was used to calculate the paraquat residue in the sample based on the standard curve.

[0014] Preferably, the coating solution in S1 is a carbonate buffer solution; the incubation conditions are 25°C for 2 hours; the blocking solution is No. 1 blocking solution; and the blocking time is 60 minutes. The conditions for the competitive reaction described in S2 are incubation at 4°C for 60 min; The enzyme-labeled secondary antibody dilution solution described in S3 is diluted 5000 times, and the secondary antibody reaction time is 30 minutes.

[0015] An LFIA method for detecting paraquat residues includes the following steps: (1) Preparation of colloidal gold probe: Take colloidal gold solution and adjust pH; then add stock solution of the monoclonal antibody and vortex mix. After reaction, block treatment is performed (e.g., add 10% BSA aqueous solution to block for 30 min); centrifuge, discard supernatant, resuspend precipitate to obtain colloidal gold probe solution; (2) Preparation of detection membrane: The coating agent (preferably PH-Q-BSA) and goat anti-mouse secondary antibody are diluted and applied to nitrocellulose membrane using a membrane scribing instrument to serve as the detection line (T line) and the quality control line (C line) respectively. After drying, they are ready for use. (3) Assemble the test strips: Assemble the sample pad, the nitrocellulose membrane obtained in step (2) and the absorbent filter paper on the PVC substrate according to the conventional test strip assembly method, and then use the cutting equipment to cut them into test strips of uniform width. (4) Detection: Mix the sample solution to be tested with the colloidal gold probe solution obtained in step (1) to obtain a mixture; vertically immerse the sample end of the prepared test strip into the mixture to drive the liquid chromatography reaction; after the reaction is completed, take out the test strip and observe the color development state of the detection line and the control line with the naked eye. By visually comparing the chromatography results with the negative control group, the test result of the sample can be determined.

[0016] Preferably, in step (1), 25-30 μL of 0.1 M K2CO3 is added to 1 mL of colloidal gold solution in a gradient to adjust the pH; the concentration of the monoclonal antibody stock solution is 1 mg / mL, and the volume ratio of the colloidal gold solution to the monoclonal antibody stock solution is 1 mL: 8-12 μL.

[0017] Preferably, the volume ratio of the sample solution to be tested to the colloidal gold probe solution obtained in step (1) is 200:6.

[0018] Preferably, the sample to be tested includes at least one of skim milk, pig urine, cucumber, and apple.

[0019] The beneficial effects of this invention are: This invention yields mAbs with high specificity, high affinity, and good binding activity even in acidified methanol solutions of a certain concentration. These antibodies exhibit good tolerance under non-physiological conditions, tolerating 40% methanol, 10% acetonitrile, 4M urea, 580mM salt ions, and pH 7-11.

[0020] By optimizing icELISA reaction conditions and evaluating detection performance under non-physiological environments, icELISA methods for detecting PQ in skim milk, pig urine, cucumber, and apple were established. Samples extracted with acidified methanol (10% methanol, 90% 0.1 mol / L HCl) only required dilution before direct detection. The LODs in the four matrices were 0.13 µg / L, 0.35 µg / L, 1.68 µg / kg, and 0.86 µg / kg, respectively. The recoveries ranged from 87.5% to 127.2%, and the inter-batch coefficient of variation was less than 14%.

[0021] A colloidal gold probe, mAb 6C10, was prepared using colloidal gold nanoparticles as the signal element and mAb 6C10 as the recognition element. Through optimization of the colloidal gold probe, a lateral flow immunochromatographic method (LFIA) was established for analysis of skim milk, pig urine, cucumber, and apple. Samples extracted with acidified methanol (10% methanol, 90% 0.1 mol / L HCl) only required dilution before direct detection. The LODs of this method in the four samples were 1.63 μg / L, 9.76 μg / L, 6.55 μg / kg, and 3.27 μg / kg, respectively. This method allows for qualitative analysis of results within 10 minutes, demonstrating its potential for rapid on-site detection. Attached Figure Description

[0022] Figure 1 Effects of acidified methanol on serum A at different dilution ratios max / IC 50 The impact.

[0023] Figure 2 Results of the effect of acidified methanol on hybridoma cell supernatant.

[0024] Figure 3 Results of mAbs' tolerance to methanol solution. (a) mAb 5G5, (b) mAb 6C1, (c) mAb 6C10. In the figure, the horizontal axis represents concentration, and the vertical axis represents A. max Value, IC 50 Value, A max / IC 50 Value, the red line represents A. max Value, the black line represents IC. 50 Value, the blue line represents A. max / IC 50 value.

[0025] Figure 4 : mAbs tolerance results in acetonitrile solution. (a) mAb 5G5, (b) mAb 6C1, (c) mAb 6C10. In the figure, the horizontal axis represents concentration, and the vertical axis represents A. max Value, IC 50 Value, Amax / IC 50 Value, the red line represents A. max Value, the black line represents IC. 50 Value, the blue line represents A. max / IC 50 value.

[0026] Figure 5 : mAb urea tolerance results. (a) mAb 5G5, (b) mAb 6C1, (c) mAb 6C10. In the figure, the horizontal axis represents concentration, and the vertical axis represents A. max Value, IC 50 Value, A max / IC 50 Value, the red line represents A. max Value, the black line represents IC. 50 Value, the blue line represents A. max / IC 50 value.

[0027] Figure 6 : Sample matrix inhibition curves. (a) pig urine, (b) skim milk, (c) apple, (d) cucumber.

[0028] Figure 7 pH optimization for colloidal gold probe labeling.

[0029] Figure 8 Optimization of antibody labeling amount. (a) is a photograph of the test strips with different antibody labeling amounts; (b) is a graph showing the quantitative results of T-line optical density and inhibition rate under different antibody labeling amounts. The curve T / T0 shown in (b) represents the inhibition rate (T / T0×100%) of different antibody labeling amounts when the PQ concentration is 1 μg / L; where T: T-line signal of the spiked strip, the T-line optical density measured by the reader after chromatography when the sample contains PQ; T0: T-line signal of the blank strip, the T-line optical density measured under the same conditions when PQ is not present (0 µg / L, pure buffer). The same applies below.

[0030] Figure 9 Optimization of colloidal gold probe dosage. (a) is a photograph of the test strips with different colloidal gold probe dosages; (b) is a graph showing the quantitative results of T-line optical density and inhibition rate with different colloidal gold probe dosages. The curve T / T0 shown in (b) represents the inhibition rate (T / T0×100%) of different colloidal gold probe dosages when the PQ concentration is 1 μg / L.

[0031] Figure 10Optimization of the original coating dilution factor. (a) is a photograph of the test strips at different original coating dilution factors; (b) is a graph showing the quantitative results of the T-line optical density and inhibition rate at different original coating dilution factors. The curve T / T0 shown in (b) represents the inhibition rate (T / T0×100%) at different original coating dilution factors when the PQ concentration is 1 μg / L.

[0032] Figure 11 : PQ (paraquat) test card standard curve.

[0033] Figure 12 : Specificity test results of PQ (paraquat) test card. Detailed Implementation

[0034] To facilitate a clearer understanding of the technical content of this invention by those skilled in the art, the invention will be further described below in conjunction with specific embodiments.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0036] Example 1: Preparation of Monoclonal Antibodies for Paraquat Tolerance to Methanol 1.1 Test Methods The hapten 1-(4-carboxybutyl)-1′-methyl-[4,4'-bipyridine]-1,1'-diium (PH-Q) used in this experiment was kindly provided by Professor Shen Yudong's laboratory at the College of Food Science, South China Agricultural University. The English name of PH-Q is 1-(4-carboxybutyl)-1'-methyl-[4,4'-bipyridine]-1,1'-diium. Relevant literature describing this hapten includes "Ultrasensitive and rapid colorimetric detection of paraquat via a high-specific VHH nanobody," https: / / doi.org / 10.1016 / j.bios.2022.114089 and patent document CN 117209610 A. The hapten PAR-BBA was provided by Beijing Weideweikang Biotechnology Co., Ltd.

[0037] 1.1.1 Complete synthesis and identification of PQ antigen 1.1.1.1 Complete antigen synthesis The carrier protein coupling is performed using the active ester method, and the specific procedure is as follows: (1) Accurately weigh the paraquat hapten raw material according to the amount of feed in Table 1-1 below, dissolve it in 2 mL of DMSO (dimethyl sulfoxide) solution, sonicate until completely dissolved, weigh NHS and EDC, add them to the above DMSO solution and place them on a magnetic stirrer to activate the reaction for 3 h (500 rpm) in the dark. (2) Weigh the protein according to the feeding amount and dissolve it in 5 mL of CB buffer (Na2CO3 6.36 g, NaHCO3 11.6 g, and dilute to 4 L with pure water, then adjust). In a solution with pH=9.6, stir at 500 rpm until completely dissolved; slowly drop the solution prepared in step (1) into the protein solution (maintain magnetic stirring at 4℃ and 500 rpm throughout the process), and couple at room temperature in the dark for 24 hours after the addition is completed; (3) Check for leaks and clean the dialysis bag with pure water. Transfer the coupled solution into the dialysis bag and dialyze with 0.01M PBS buffer (KCl 0.8g, NaCl 34g, Na2HPO4·12H2O 11.6g, NaH2PO4·2H2O 2.36g, pure water to a final volume of 4L, pH adjusted to 7.4) for 72 hours. Change the dialysate every 8 hours. After dialysis, collect the solution from the dialysis bag and store it separately at -20℃.

[0038] Table 1-1 Proportions of PH-Q-BSA-coated raw materials

[0039] Table 1-2 Ratio of Raw Materials for PAR-BBA-BSA Coating

[0040] Table 1-3 PH-Q-BTG Immunogen Synthesis Material Ratio

[0041] 1.1.1.2 Determination of protein concentration of complete antigen The concentration of complete antigen protein was determined using a Nano Drop UV spectrophotometer. After background correction with PBS buffer as a blank, the absorbance (A) of the sample was measured. 280 Each sample was measured in triplicate, and the average value was used as the final concentration.

[0042] 1.1.1.3 Determination of complete antigen-conjugation ratio The synthesized complete antigen was analyzed using MALDI-TOF-MS. Since BTG is a mixture of multiple proteins and does not have a fixed molecular weight, this experiment only identified and analyzed the complete antigen conjugated with BSA protein. Given that the complete antigen is formed by the covalent bonding of the hapten and carrier protein, the relative molecular mass of its conjugation product will be significantly higher than that of the free hapten. Therefore, detecting the increase in molecular weight before and after the reaction is a standard strategy for verifying the efficiency of the conjugation reaction and the successful construction of the complete antigen. The conjugation ratio was calculated using Equation 1-1.

[0043] Coupling ratio = (BSA conjugate molecular weight) BSA molecular weight) / Hapten molecular weight (Formula 1-1) 1.1.2 Animal Immunization and Serum Identification 1.1.2.1 Immunogen emulsification The prepared immunogen was diluted with PBS buffer to a concentration of 1 mg / mL. Before emulsification, two 5 mL screw-type syringes were used to draw 1 mL of immunogen solution and an equal volume of Freund's adjuvant. The side containing the adjuvant was pushed out to expel gas from the tube. The two syringes were connected using a metal two-way valve. The first emulsification was performed by transferring the aqueous phase (immunogen) into the oil phase (adjuvant), forming a water-in-oil emulsion.

[0044] 1.1.2.2 Mouse Immunization Mice were immunized by injection according to the immunization protocol designed in Table 1-4. The first four immunizations were administered via subcutaneous injection at multiple sites on the back of the neck, with 4-6 injection sites per mouse. For a pre-cell fusion shock immunization, an intraperitoneal injection was administered 5 days prior to immunization.

[0045] Table 1-4 Mouse Immunization Protocols

[0046] 1.1.2.3 Serum monitoring (1) On the 7th day after each immunization, 150 μL-200 μL of blood was collected from the orbital venous plexus of mice. The collected whole blood was allowed to stand at 37℃ for 30 min, centrifuged at 4000 rpm for 10 min, and the supernatant was taken for testing.

[0047] Determination of optimal working concentration: Serum and coated antigen were cross-tested at multiple concentration gradients, and the optimal working concentrations of antigen and serum were screened using a checkerboard method. The inhibition rate of the second immunization serum of mice was measured by icELISA (Formula 1-2, where B0 is the OD value without standard and B is the OD value with standard). The antigen-antibody concentration at which B / B0 was lowest was selected as the optimal working concentration.

[0048] Inhibition rate % = (1 - B / B0) × 100% (Formula 1-2) Serum affinity determination: Based on the optimized original coating concentration and serum dilution factor, the absorbance values ​​of a series of standard concentrations at 450 nm wavelength were measured using icELISA. The standard concentrations were plotted on the x-axis, and the OD values ​​on the y-axis. A four-parameter logistic regression curve was fitted using Origin Pro software to establish a standard curve for quantitative detection. The regression equation was established, and its mathematical model is shown in Equations 1-3, where A1 and A2 represent the absorbance values ​​at the minimum and maximum standard concentrations, respectively, and X0 represents the half-maximum inhibitory concentration (IC50). 50 ), p represents the slope of the curve: Y=A2+(A1 A2) / [1+(x / x0) p ] (Formula 1-3) (2) icELISA detection: Coating: Dilute the coating agent to the determined concentration using CB buffer, add 100 μL / well to a 96-well microplate, and incubate at 37°C for 2 h.

[0049] Washing: After incubation, discard the liquid in the wells, wash once with 1×PBST washing buffer (immunohistochemical washing buffer), and then discard the washing buffer in the wells.

[0050] Blocking: Add 150 μL of blocking solution to each well (100 mL calf serum, 100 g sucrose, 5 g casein, 11.6 g Na2HPO4·12H2O, 1.186 g NaH2PO4·2H2O, 1 mL Proclin 300 preservative, and dilute to 2 L with pure water), block at 37℃ for 1 h, then wash once and discard the liquid in the well.

[0051] Competitive reaction: Add 50 μL of standard solution diluted in PBS buffer (or PBS buffer containing acidified methanol) to each well. Add 50 μL of serum diluted in PBS buffer (or PBS buffer containing acidified methanol) to each well, and incubate at 37°C for 30 min.

[0052] Washing: Shake off the liquid in the microplate, wash twice with 1×PBST washing solution, and shake off the liquid again.

[0053] Enzyme-labeled secondary antibody: Goat anti-mouse enzyme-labeled secondary antibody was diluted 5000 times with enzyme dilution buffer (Na2HPO4·12H2O 8.7g, NaH2PO4·2H2O 1.305g, glycerol 100mL, fetal bovine serum 400mL, Proclin 300 preservative 2mL, and purified water to a final volume of 2L). 100 μL / well was added to a 96-well microplate and reacted at 37℃ for 30 min.

[0054] Washing: Shake off the liquid in the microplate, wash twice with 1×PBST washing solution, and shake off the liquid again.

[0055] Color development: Prepare fresh color development solution, mix equal volumes of solution A and solution B according to the required amount, add 100 μL / well to the microplate, and perform color development reaction at 37℃ for 10 min.

[0056] Termination: Add 50 μL of stop solution to each well to terminate the reaction.

[0057] Reading: Set the detection wavelength of the microplate reader to 450 nm. Place the microplate into the microplate reader, read the absorbance (OD) value, and record the data.

[0058] 1.1.3 Construction of Pressure Screening Strategy for Acidified Methanol The pressure screening solution system referenced the pretreatment extract in the recommended detection method SN / T 0293 of the latest Chinese national food safety standard, "National Food Safety Standard - Maximum Residue Limits for Pesticides in Food" (GB2763-2026). This standard specifies the pretreatment extract as methanol-0.1 mol / L HCl, with a methanol to 0.1 mol / L HCl volume ratio of 1:9 (i.e., 900 mL of 0.1 mol / L HCl is added to 100 mL of methanol solution and mixed thoroughly). In this strategy, the effect of acidified methanol on antibody binding performance is quantified as an "impact value" to evaluate antibody tolerance. The impact value is defined as A under acidified methanol conditions. max / IC 50 The absolute value of the relative change (or B / B0) relative to the PBS buffer control is used to calculate the serum stage effect value according to Formula 1-4. The hybridoma cell supernatant stage effect value is calculated according to Formula 1-5.

[0059] (Formula 1-4) (Formula 1-5) Among them, A max This indicates the OD value of serum without spiking. 450nm IC50 value, i.e., the maximum binding capacity between antigen and antibody; 50 B represents the half-maximal inhibitory concentration (IC50); B represents the OD500 of hybridoma cell supernatant spiked with the supernatant. 450nm The value, B0, represents the OD value without a scalar. 450nm The smaller the influence value, the more stable the antibody's binding ability and the higher its tolerance under acidified methanol conditions; the larger the influence value, the more significant the interference of acidified methanol on the antibody's performance.

[0060] 1.1.3.1 Determination of Acidified Methanol Concentration The acidified methanol solution was serially diluted 15, 20, 25, 30, and 35-fold using PBS buffer. Serum and standards were diluted with PBS buffer containing acidified methanol, and the serum from the last immunized mice was tested by icELISA. The PBS-buffered diluted serum and standards served as standard controls. A specific antibody against mouse serum was selected. max / IC 50 The maximum acidified methanol concentration (i.e., the lowest dilution factor) with an absolute impact value of less than 80% was used as the concentration for subsequent pressure screening.

[0061] 1.1.3.2 Identification of Acidified Methanol Tolerance in Serum and Cell Supernatant The serum from mice obtained from the last immunization was analyzed by icELISA using a determined concentration of acidified methanol. A... max / IC 50 The absolute value of the relative change (B / B0) relative to the PBS buffer control was used as the basis for assessing acidified methanol tolerance. The serum with the highest affinity and A was selected. max / IC 50 Cell fusion was performed on mice with minimal effects from acidified methanol. After cell fusion, a predetermined pressure screening concentration of acidified methanol was used as a sample buffer to analyze the cell supernatant. The effect of acidified methanol on the B / B0 ratio of the cell supernatant was used as a screening index, and hybridoma cells with an absolute B / B0 effect on the hybridoma cell supernatant of less than 70% were selected for subcloning.

[0062] 1.1.4 Preparation of mAbs 1.1.4.1 Shock Immunization in Mice Based on the previous serum titer assessment, candidates with high tolerance to acidified methanol (i.e., A) were selected. max / IC 50 Less affected) and has the highest affinity (i.e., IC) 50 Mice with the lowest values ​​were given a shock immunization 5 days before cell fusion. The immunogen was prepared into a 1 mg / mL solution using PBS buffer and administered intraperitoneally at a dose of 0.5 mL per mouse (containing 200-500 μg of immunogen). No adjuvant was used at this stage.

[0063] 1.1.4.2 Myeloma cell resuscitation SP2 / 0 myeloma cells were removed, revived, cultured in expanded culture, centrifuged, and the cell pellet was resuspended in 10 mL of DMEM medium for later use.

[0064] 1.1.4.3 Preparation of feeder cells On the night before or the day of cell fusion, healthy blank mice aged 8 weeks or older were euthanized by cervical dislocation, disinfected, and their peritoneum was aseptically exposed. The peritoneum was then injected intraperitoneally with a mixture of 20% (v / v) fetal bovine serum and 1×HAT (HAT Media Supplement (50×) Hybri-Max). TM Peritoneal macrophages were collected by gently massaging and then aspirating after being placed in DMEM complete medium containing 1% (v / v) penicillin-streptomycin-amphoteric B (100X) from Sigma-Aldrich and Sigma-Aldrich, and then used for subsequent 96-well plate formation.

[0065] 1.1.4.4 Preparation of mouse spleen lymphocytes Mice that underwent shock immunization on day 4 prior to fusion were subjected to enucleation to collect blood for serum separation (as a positive control for subsequent testing). The mice were then euthanized by cervical dislocation, and the spleens were aseptically harvested after disinfection. The spleens were ground, passed through a cell sieve, and replenished with DMEM culture medium to a volume of 30 mL. The cells were centrifuged at 1000 rpm for 5 min to collect the spleen cell pellet. The cells were gently lifted and approximately 1 mL of erythrocyte lysis buffer was added. Lysis was carried out for 5 min, during which clumped cells were aspirated with a pipette until no large clumps of spleen cells remained. 10 mL of DMEM was then added for further processing.

[0066] 1.1.4.5 Cell Fusion (1) Take myeloma cell suspension and spleen cell suspension, mix them at a ratio of spleen cell number: myeloma cell number = 5:1, add DMEM medium preheated to 37℃ to a total volume of 30 mL, mix thoroughly by pipetting; centrifuge at 1000 rpm for 5 min, discard the supernatant, tap the bottom of the tube gently to loosen and evenly disperse the cells at the bottom; (2) Preheat the 800 μL PEG (polyethylene glycol) and DMEM culture medium in advance. Add 800 μL PEG dropwise within 30 seconds while rotating the centrifuge tube. Stir with a pipette without aspirating liquid within 30 seconds while rotating the centrifuge tube. The entire fusion process is carried out at 37°C. Therefore, a small beaker containing water can be preheated in a water bath. (3) Add preheated DMEM medium to terminate the fusion reaction. Add 1 mL every 15 seconds for the first 45 seconds, for a total of 3 mL. Add 4 mL quickly for the next 15 seconds. Then continuously add DMEM medium until 30 mL is reached. At this point, the centrifuge tube can be added without rotating it. (4) Centrifuge at 800 rpm for 7 min, discard the supernatant, gently tap the bottom of the tube, and add 20% (v / v) fetal bovine serum and 1×HAT (HAT Media Supplement (50×) Hybri-Max) TMCells were resuspended in DMEM complete medium containing 1% (v / v) penicillin-streptomycin-amphoteric B (100X) from Sigma-Aldrich and dispensed into 96-well plates containing 100 μL of feeder cells using a pipette, with 200 μL added to each well. (5) Place the cell culture plate containing fused cells in a constant temperature incubator at 37°C and 5% CO2 for culture.

[0067] 1.1.4.6 Hybridoma Screening On days 5-7 of cell growth, a randomly selected plate was used for preliminary experiments to determine the optimal working concentration of the coating agent. The remaining wells were then analyzed using icELISA, with priority given to positive wells showing deep staining and a clear number of clones. Based on this, an acidified methanol pressure screening strategy (see Section 1.1.3) was introduced for specific rescreening: on day 7, the plate was coated with the determined concentration of the coating agent, and the sample loading system was adjusted to 50 µL supernatant + 50 µL PBS buffer containing acidified methanol. By setting a gradient of standard concentrations, the difference in inhibition rate between the cell supernatant and the acidified methanol pressure environment was compared, and cell lines less affected by acidified methanol were ultimately selected for subcloning.

[0068] 1.1.4.7 Cell Cloning and Cryopreservation (1) 24 hours before the subcloning experiment, the method described in 1.1.4.3 was followed, using a mixture containing 16% (v / v) fetal bovine serum and 1×HT (HT Media Supplement (50×) Hybri-Max). TM Feeder cells were prepared using DMEM complete medium (Sigma-Aldrich). (2) Subcloning was performed on positive wells using a 3-fold serial dilution method. During microscopic observation, if the cell density was too high and the boundaries were blurred, the single well was divided into 8 quadrants for localization. The supernatant from the cell-free growth area was aspirated and cryopreserved as a backup to prevent clone loss; (3) If the culture medium in the well is consumed too much due to the detection, 100 μL of DMEM complete culture medium containing 16% (v / v) fetal bovine serum and 1×HT should be slowly added along the well wall. Use the reverse pipetting method to aspirate the target cell cluster into the pre-placed feeder cells in well A1. After confirming the successful transfer of cells under a microscope, perform a 2-3 fold serial dilution of the cells in well H1. (4) The feeder cells were resuspended and plated using a serial dilution method: First, DMEM complete medium containing 16% (v / v) fetal bovine serum and 1×HT was added to the first well of a 96-well plate to a final volume of 300µL; then, the cell suspension was repeatedly aspirated and pipetted using an 8-line pipette to mix it, and 100µL was dispensed into the second and third wells respectively. This process was repeated to complete the entire plate operation, ensuring that the total volume of liquid in all cell wells was uniformly replenished to 300µL.

[0069] (5) Place the processed cell plates in an incubator at 37°C and 5% CO2 for 7-10 days, and then carry out the next round of cell screening and cloning. (6) After completing the clone screening, immediately freeze the backup cells.

[0070] 1.1.4.8 Cell line establishment and scale-up culture (1) After three consecutive subclonings of hybridoma single-cell clusters, the cell line tends to be stable and can be further established. (2) Expand the culture of single-cell clusters after establishment.

[0071] 1.1.4.9 Production of mAbs mAbs were prepared intraperitoneally in mice using an in vivo induction method.

[0072] Healthy female Balb / c mice over 10 weeks old were selected, and after intraperitoneal injection of liquid paraffin for 7 days, each mouse was inoculated with approximately 1×10 6 Hybridoma cells were collected, and ascites was collected when the abdomen became significantly distended. After centrifugation to remove the fat layer, the clear middle liquid was collected, aliquoted, and cryopreserved.

[0073] 1.1.4.10 Purification of mAbs mAbs (mouse ascites) obtained through in vivo induction contain a large amount of impurities and require further purification.

[0074] The antibody was diluted with PBS buffer at a volume ratio of 1:3.5, filtered, loaded onto a pre-equilibrated Protein A affinity column, washed, and then eluted with citric acid (pH=3). Fractions with concentrations higher than 0.1 mg / mL were collected. The collected antibody solution was then dialyzed. The antibody solution was transferred to a dialysis bag and placed in 0.01M PBS buffer (pH=7.4) with stirring for 72 hours, changing the dialysis buffer every 8 hours. After dialysis, the solution in the dialysis bag was collected, and the antibody concentration was measured using a Nano Drop UV spectrophotometer. The aliquots were stored at -20°C.

[0075] 1.1.5 PQ mAbs Performance Characterization 1.1.5.1 Antibody subtype identification The prepared mAb subtypes were identified using the mouse monoclonal antibody subtype identification kit from Beijing Bio-Long Immunotherapy Co., Ltd.

[0076] 1.1.5.2 Antibody Affinity Assay The prepared mAbs were cross-tested with PH-Q-BSA and PAR-BBA-BSA using a checkerboard method. The optimal coating agent for each antibody was determined using icELISA, and then the OD values ​​at different coating agents and antibody dilutions were compared. 450nm Value and IC 50 The optimal dilution ratio of the coating antigen to the antibody was determined. After determining the optimal concentration of the antigen-antibody pair, a standard curve for the prepared mAbs was established, and the IC50 value corresponding to the mAb secreted by each hybridoma cell line was calculated. 50 The value is used as a standard for evaluating antibody affinity.

[0077] 1.1.5.3 Antibody Specificity Assay Following the icELISA procedure in section 1.1.2, standard curves for mAbs were established using Diquat (DQ), a structural analog of PQ, and PQ technical grade as standards. The IC50 values ​​of mAbs for different standards were calculated. 50 Value. Using PQ as the reference drug, the cross-reactivity (CR) of different mAbs was calculated according to the formula.

[0078] CR%=IC 50 (PQ) / IC 50 (DQ) × 100% (Formula 1-6) 1.1.5.4 Antibody-Acidified Methanol Tolerance Assay Acidified methanol (methanol-0.1 mol / L HCl, volume ratio 10:90) was diluted 20, 30, 40, 50, and 100 times with PBS buffer, respectively. The diluted acidified methanol solution was used as a sample diluent to dilute antibodies and standards secreted by three hybridoma cell lines. PBS sample diluent was used as a control group. max IC 50 A max / IC 50 As a detection indicator, the antibody's tolerance to acidified methanol at different dilution ratios was determined.

[0079] 1.2 Experimental Results and Analysis 1.2.1 Identification of PQ complete antigen synthesis The concentrations of the synthesized coating precursors PH-Q-BSA, PAR-BBA-BSA, and immunogen PH-Q-BTG were determined using a Nano Drop UV spectrophotometer. The results are shown in Tables 1-5. The concentrations of the synthesized coating precursors PH-Q-BSA and PAR-BBA-BSA were 9.96 mg / mL and 1.22 mg / mL, respectively, and the concentration of the immunogen PH-Q-BTG was 4.12 mg / mL. MALDI-TOF-MS was used to determine the conjugation ratios of BSA, PH-Q-BSA, and PAR-BBA-BSA. The results showed that the conjugation ratios of the synthesized coating precursors PH-Q-BSA and PAR-BBA-BSA were 21:1 and 5.2:1, respectively, confirming successful conjugation. Since BTG is a mixture of multiple proteins and does not have a fixed molecular weight, this experiment used the shift in the peak concentration of antibody and BSA obtained by Nano Drop UV spectrophotometer as a criterion for judging whether the synthesis was successful. The results showed that the peak of PH-Q-BTG shifted compared to PH-Q, proving that the coupling was successful.

[0080] Table 1-5 Concentration of Artificially Synthesized Complete Antigen Proteins

[0081] Table 1-6 Results of the All-Antigen-Conjugation Ratio Determination

[0082] 1.2.2 Results of mouse serum monitoring Seven healthy Balb / c mice aged 6-8 weeks were immunized with the immunogen PH-Q-BTG. The mice were numbered 2-#1 to 2-#7. Due to individual differences among the mice, different immunization responses were observed. Serum titers and inhibition rates were monitored on day 7 after each booster immunization using icELISA, and serum IC50 was calculated. 50 The results showed that the serum affinity of mice 2-#2 decreased after the fourth immunization, manifested as worse inhibition, with the B / B0 ratio increasing from 32.14% to 34.61%. The serum affinity of the other six mice all showed some degree of improvement after the fourth immunization. The optimal IC50 value was monitored in seven mice after the fourth immunization. 50 The concentration ranged from 1.52 to 29.71 μg / L, with the highest affinity observed in the serum of mice 2-#4, and an IC50 concentration of [missing value]. 50 The concentration was 1.52 μg / L, and the inhibition was the best. After four immunizations, the B / B0 ratio was 15.80%.

[0083] Table 1-7 Results of mouse serum monitoring

[0084] Note: The average serum titer was measured at a dilution of 80,000 times to the original coating; B / B0 was the result obtained by adding 20 μg / L paraquat standard to the serum of mice after the third and fourth immunizations.

[0085] 1.2.3 Construction of the pressure screening strategy for acidified methanol 1.2.3.1 Determination of Acidified Methanol Concentration After serially diluting acidified methanol (10% methanol, 90% 0.1 mol / L HCl, pH 1.16) with PBS buffer, the serum from the last immune test of seven mice was analyzed. max / IC 50 The effect of acidified methanol concentration on serum was evaluated, and the acidified methanol concentration used for subsequent screening was determined as the effect on A. max / IC 50 The maximum acidified methanol concentration that has an absolute impact of less than 80% is considered. Results are as follows: Figure 1 As shown, the serum of each mouse was affected differently by different concentrations of acidified methanol. For example, in an acidified methanol solution containing 0.67% methanol and 6% 0.1 mol / L HCl (i.e., diluted 15 times), mice A of types 2-#1, 2-#2, 2-#5, 2-#6, and 2-#7... max / IC 50 The impact was significant, ranging from 88.17% to 94.58%, with acidified methanol at this concentration affecting mice A in models 2-#1 and 2-#7. max / IC 50 The levels of acidified methanol decreased by 93.55% and 94.58% respectively, almost completely disrupting the binding of antigen and antibody. However, in mice such as 2-#3 and 2-#4, the effects of this concentration of acidified methanol were still below 80%, at 69.24% and 72.94% respectively. Therefore, the effects on the antigen-antibody binding were selected from 7 mice. max / IC 50 The acidified methanol concentration (0.33% methanol, 3% 0.1 mol / L HCl) with an impact of less than 80% was used as the subsequent screening concentration.

[0086] Figure 1 (b) represents the A values ​​of 7 mice in acidified methanol solutions of different concentrations. max / IC 50 The fluctuations were observed, with PBS buffer serving as a negative control. The figure shows that mice 2-#4 exhibited A levels in PBS buffer. max / IC 50 The value was significantly higher than that of the other six mice, indicating that this mouse was the most sensitive. Furthermore, although different concentrations of acidified methanol had some impact, the sensitivity fluctuations were smaller and more stable compared to mice 2-4. Therefore, the mouse with the highest serum affinity and the best sensitivity in different concentrations of acidified methanol solutions was ultimately selected. max / IC 50 Mice #2-#4 with relatively small fluctuations were fused.

[0087] 1.2.3.3 Acidified methanol pressure screening of hybridoma cells The effects of acidified methanol (0.33% methanol, 3% 0.1 mol / L HCl) on the inhibition rate of hybridoma cell supernatant were determined starting from the second subcloning. Cell clusters with an inhibition rate less than 70% affected by acidified methanol were screened for cloning. A total of over 400 wells of cell supernatant were analyzed across five subcloning trials. The results are shown in Figure 2. Using the average effect as a standard, it can be concluded that the effect of acidified methanol on cell supernatant decreased progressively after each subcloning trial. For example, the effect value of cell supernatant after the fifth subcloning trial was significantly lower than that after the second subcloning trial (closer to 0%). The specific effects of cell pore detection are shown in Tables 1-8. As the number of cell subclonings under pressure screening increased, the number of cell pores with less than 70% influence of acidified methanol on the B / B0 ratio of hybridoma cell supernatant increased from 23 to 57, and the number of cell pores with less than 30% influence on the B / B0 ratio of hybridoma cell supernatant increased from 2 to 21. The lowest influence rate of cell pores was 0.13% during the fifth subcloning, indicating that the acidified methanol pressure screening strategy can play a certain screening role on cell supernatant.

[0088] Table 1-8 Statistical analysis of the effects of acidified methanol on hybridoma cell supernatant

[0089] Three hybridoma cell lines, 5G5, 6C1, and 6C10, were finally obtained through screening. The 6C10 cell line has been deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No. 68912), with a deposit date of August 28, 2026. The deposit address is Building 59, 5th Floor, No. 100 Xianlie Middle Road, Guangzhou. The cell line has been classified and named as follows: Murine hybridoma cell line .

[0090] 1.2.4 Performance Characterization of PQ mAbs This study obtained mAbs secreted by three hybridoma cell lines through in vivo induction and evaluated the subtypes, affinity, specificity, and tolerance to acidified methanol of the obtained antibodies.

[0091] 1.2.4.1 Antibody subtype identification The light chains of the mAbs secreted by the three hybridoma cell lines were all of the Kappa (κ) type, and the heavy chain subtype was IgG2a.

[0092] 1.2.4.2 Antibody Affinity Assay The checkerboard method was used to screen for coating agents. Results showed that the synthesized coating agent PAR-BBA-BSA had low titers, and the antigen-antibody reaction was weak or nonexistent at different dilutions. Therefore, PH-Q-BSA was selected as the coating agent for subsequent antibody detection. Based on this, icELISA standard curves for mAbs secreted by three hybridoma cell lines were established, and the IC50 of each mAb was calculated. 50 The values ​​are shown in Table 1-9. Based on serum screening, the antibodies obtained through screening showed an approximately 20-fold increase in affinity, IC50... 50 The concentration decreased from 1.52 μg / L to 0.050-0.072 μg / L.

[0093] Table 1-9 Results of Antibody Affinity Assay

[0094] Note: OD 零标 This refers to the OD value without the addition of standard substances.

[0095] 1.2.4.3 Antibody Specificity Assay The cross-reactivity of the three mAbs and the PQ structural analog DQ was determined using icELISA. The cross-reactivity rate (CR) of the monoclonal antibodies was calculated according to formula 1-6. The results are shown in Table 1-10. As can be seen from the results, the CR of the three prepared mAbs with DQ is less than 0.001%, and there is no cross-reactivity with DQ, indicating that the antibodies have good specificity.

[0096] Table 1-10 Results of Antibody Specificity Detection

[0097] Note: IC in the table 50 The units are all μg / L.

[0098] 1.2.4.4 Antibody-Acidified Methanol Tolerance Assay The tolerance of three mAbs to different concentrations of acidified methanol was tested. Acidified methanol solutions (10% methanol, 90% 0.1 mol / L HCl, pH 1.16) were serially diluted (20, 30, 40, 50, 100 times) with PBS buffer. The PBS buffer group served as a negative control. Results showed that different dilutions of acidified methanol had little effect on the antigen-antibody binding activity of mAbs 5G5 and 6C10. Specifically, as the dilution factor of acidified methanol decreased, the antigen-antibody binding activity of mAbs 5G5 and 6C10 decreased. max The value change was not significant, A max The value remained stable at around 2.0; among them, acidified methanol diluted 20 times had a more significant effect on mAb 6C1, manifested as A maxThe value decreased significantly, from 1.9 to 0.5. Furthermore, the IC50 values ​​of different concentrations of acidified methanol for the three mAbs were also significantly reduced. 50 This had a certain impact, as evidenced by the IC value decreasing with decreasing dilution factor. 50 The value increases, with the acidified methanol solution, which is only diluted 20 times, showing the greatest impact. 50 The average value increased from 0.06 μg / L to 0.98 μg / L, while the IC50 value decreased when acidified methanol was diluted 30-100 times. 50 The influence of the value gradually decreases, IC 50 The average concentration increased from 0.06 to 0.2 μg / L. Compared to the serum phase, the antibody showed improved tolerance to acidified methanol and increased its affinity for it (IC50). 50 The serum concentration increased from 1.52 μg / L to 0.2 μg / L.

[0099] In summary, this embodiment synthesized two coating antigens, PH-Q-BSA and PAR-BBA-BSA, and one immunogen, PH-Q-BTG. The conjugation ratios of the two coating antigens were 21:1 and 5.2:1, respectively. After screening, the PH-Q-BSA coating antigen showed better efficacy than the PAR-BBA-BSA coating antigen. The immunogen PH-Q-BTG showed good immunization efficacy, with significantly increased serum affinity in mice after four immunizations. The optimal IC50 concentration in mice (2-#4) was [not specified]. 50 =1.52 μg / L. Acidified methanol stress screening (0.33% methanol, 3% 0.1mol / L HCl) was performed in both serum monitoring and hybridoma cell supernatant detection stages. After five cloning cycles, the influence of acidified methanol on the hybridoma cell supernatant gradually decreased, approaching 0%, preliminarily demonstrating the effectiveness of the acidified methanol stress screening strategy. Through fixed-line expansion culture, three hybridoma cell lines (5G5, 6C10, and 6C1) were finally obtained. The antibodies obtained from these three hybridoma cell lines were characterized. All three antibodies showed good binding only to the homologous coating antigen. All three antibodies exhibited good titers and sensitivity, with IC50 values ​​of 1.52 μg / L. 50 All values ​​were within the range of 0.05 ± 0.02 μg / L. None of the three antibodies showed cross-reactivity with their structural analogue DQ, and all exhibited good antigen-antibody binding activity in acidified methanol solutions of a certain concentration. After comprehensive consideration, the three antibodies were simultaneously optimized, and the optimal antibody was selected based on the optimization results for establishing the icELISA method.

[0100] Example 2: Establishment of an icELISA method for the detection of paraquat residues 2.1 Test Methods 2.1.1 Optimization of icELISA reaction conditions (1) Optimization of wrapping conditions Based on the optimal working concentration of antigen and antibody established in Example 1, solid-phase coating conditions were screened. Three coating modes were investigated: incubation at room temperature for 2 h, incubation at 37°C for 2 h, and overnight (16 h) at 4°C. The signal-to-weight ratio (Ag / V) under different conditions was calculated. max / IC 50 The optimal coating scheme is selected when the ratio reaches its peak value.

[0101] (2) Coating fluid optimization Based on the optimized coating conditions described above, CB buffer and PBS buffer were used as the original coating diluents for coating, with the remaining steps unchanged. The Aij of mAbs in the two coating solutions was calculated. max / IC 50 Ratio, choose A max / IC 50 The coating solution at which the maximum value is achieved is taken as the optimal coating solution.

[0102] (3) Optimization of sealing fluid Under the established optimal coating conditions and solutions, the mAbs were blocked using 2% (w / v) skim milk aqueous solution, 5% (w / v) skim milk aqueous solution, and Blocking Solution #1 (100 mL calf serum, 100 g sucrose, 5 g casein, 11.6 g Na2HPO4·12H2O, 1.186 g NaH2PO4·2H2O, 1 mL Proclin 300 preservative, and diluted to 2 L with pure water). The A of mAbs under the three different blocking solutions was calculated. max / IC 50 Ratio, choose A max / IC 50 The sealing liquid at which the maximum value is reached is taken as the optimal sealing liquid.

[0103] (4) Optimization of closing time Based on the aforementioned optimization results, the closure time was set to 60 minutes, 90 minutes, and 120 minutes, respectively, and the A of mAbs was measured at each time period. max / IC 50 The ratio is used, and the closure time corresponding to the highest ratio is taken as the optimal condition.

[0104] (5) Optimization of competitive reaction conditions After optimizing the above steps, three competing reaction methods were compared: reaction at room temperature for 30 minutes, reaction at 37°C for 30 minutes, and reaction at 4°C for 60 minutes. The A of mAbs under each condition was calculated. max / IC 50 The ratio is used to select the condition with the largest ratio as the final competing response.

[0105] (6) Optimization of secondary antibody dilution After determining the optimal enzyme dilution, goat anti-mouse secondary antibody was diluted 1000-fold, 3000-fold, 5000-fold, and 7000-fold using this dilution. The Ab of mAbs at different dilutions was calculated. max / IC 50 Ratio, choose A max / IC 50 The dilution of the secondary antibody at which the maximum value is achieved is taken as the optimal dilution.

[0106] (7) Optimization of secondary antibody reaction time Based on the aforementioned optimizations, the secondary antibody was added and reacted for 15 minutes, 30 minutes, and 60 minutes, respectively. The activity of mAbs at each time point was compared. max / IC 50 The ratio is used to determine the optimal secondary antibody reaction time corresponding to the highest ratio.

[0107] 2.1.2 Antibody tolerance test to non-physiological environment The tolerance of the detection antibodies mAb 5G5, mAb 6C1, and mAb 6C10 to pH, salt ion concentration, organic solvents, and urea was determined.

[0108] 2.1.2.1 pH tolerance test Under optimized experimental conditions, antibody working solutions and a series of standard solutions were prepared using PBS buffer at pH values ​​of 5, 6, 7, 8, 9, 10, and 11. The maximum reaction value (A) was determined under each pH condition. max ), and calculate the corresponding A max / IC 50 The ratio was used to evaluate the effect of buffer pH on antibody binding characteristics and detection performance.

[0109] 2.1.2.2 Salt ion concentration tolerance test Under optimal conditions, salt ions were diluted with PB buffer to concentrations of 0, 72.5, 145, 290, 580, 1160, 2320 mM and 5 M, respectively. These diluted salt ion solutions were then used to dilute antibodies and standards at various concentrations. A at different salt ion concentrations was then detected. max Values ​​and calculations of A at different salt ion concentrations. max / IC 50 The ratio is used to determine the effect of different salt ion concentrations on antibody performance and detection results.

[0110] 2.1.2.3 Organic Solvent Tolerance Test Under the established optimal conditions, the reaction buffer was replaced with PBS buffer containing 60% (v / v), 40%, 20%, 10%, 5%, 2.5%, and 1.25% methanol, and PBS buffer containing 40%, 20%, 10%, 5%, 2.5%, 1.25%, and 0.63% acetonitrile, respectively. Antibodies and standards were diluted with these buffers. The detection of A in organic solvents... max Value and calculate A in different organic solvent contents. max / IC 50 The ratio was used to assess the impact of different organic solvent contents on antibody performance and detection results.

[0111] 2.1.2.4 Urea Tolerance Test Under optimal conditions, antibodies and standards were diluted with PBS buffer containing 8 M, 4 M, 2 M, 1 M, 0.4 M, 0.2 M, and 0.1 M urea, respectively. A was then detected in solutions with different urea concentrations. max Value and calculate A in solutions with different urea concentrations. max / IC 50 The ratio was used to analyze the impact of changes in urea concentration on antibody performance and detection results.

[0112] 2.1.3 Establishment of the icELISA method 2.1.3.1 Establishment of the standard curve Based on the optimized conditions, the target PQ was used as a standard to establish a standard curve for the icELISA method. The PQ standard was diluted to 0.0002 μg / L, 0.002 μg / L, 0.02 μg / L, 0.2 μg / L, 2 μg / L, 20 μg / L, and 200 μg / L as working solutions for competitive reactions. Standard curves were constructed and IC50 was calculated using Origin Pro software. 50 Value, in IC 20 - IC 80 The value serves as the quantitative detection range for the established method.

[0113] 2.1.3.2 Determination of Pretreatment Conditions (1) Pretreatment methods for skim milk and pig urine samples Take 1 mL of skim milk and pig urine samples, and extract them with 4 mL of methanol-0.1 mol / L HCl, respectively. Then take 1 mL, 0.75 mL, and 0.4 mL of the sample extracts, respectively, and add 7 mL, 8.25 mL, and 7.6 mL of PBS buffer, respectively, for 8, 12, and 20-fold dilution. Vortex mix for 2 min and use for icELISA detection.

[0114] (2) Pretreatment methods for cucumber and apple samples Fresh cucumber and apple samples were selected and processed using organic solvent extraction. 1 g of homogenized sample was accurately weighed and added to 4 mL of a methanol-0.1 mol / L HCl mixture. The mixture was homogenized thoroughly for 1 min to separate the target analyte from impurities. After centrifugation at 4000 rpm for 5 min, the supernatant was collected. 1 mL, 0.75 mL, and 0.4 mL of the extract were accurately pipetted and diluted with 7 mL, 8.25 mL, and 7.6 mL of PBS buffer, respectively, for 8-fold, 12-fold, and 20-fold serial dilutions. The mixture was vortexed for 2 min before analysis.

[0115] Sample solutions obtained after pretreatment of blank samples (skim milk, pig urine, cucumber, and apple) were used as diluents to dilute PQ standard and antibody to working concentrations, respectively. Inhibition curves for each sample matrix were established. By fitting the curves to the PBS standard curve, the curve with the highest overlap was selected as the successfully fitted curve. The sample pretreatment conditions corresponding to this curve are the pretreatment conditions that can eliminate matrix effects. See section 2.1.3.2 for details of the sample pretreatment method.

[0116] 2.1.3.3 Sensitivity and Quantitative Detection Range Based on the sample matrix inhibition curves established in section 2.1.3.2, the IC50 values ​​corresponding to each sample matrix were determined. 10 The actual limit of detection (LOD) of this method is calculated by multiplying the value by its corresponding dilution factor and then using IC50. 20 -IC 80 Multiply by the corresponding dilution factor to obtain the quantitative detection range.

[0117] 2.1.3.4 Accuracy and Precision Within the linear detection range of the samples, standard substance concentrations at low, medium, and high gradients were selected for spiking experiments. The samples were then processed according to the method in 2.1.3.2, a PBS standard curve was set up for control, and icELISA was performed under the determined optimized conditions. The recovery rate was obtained using Equation 2-1 and used as an indicator to evaluate the accuracy of the method.

[0118] The precision of the method was validated using spiking and recovery experiments. Spiking and recovery experiments were repeated for each sample at different concentrations, with five replicates for each concentration gradient. The intra-assay CV and inter-assay CV were calculated using Equation 2-2, and the calculated CVs were used as indicators of the method's accuracy.

[0119] Recovery rate (%) = (Actual detected concentration / Added concentration) × 100% (Formula 2-1) CV(%) = (Standard deviation of measured concentration / Average of measured concentration) × 100% (Formula 2-2) 2.2 Experimental Results and Analysis 2.2.1 Optimization of icELISA reaction conditions (1) Optimization of wrapping conditions When mAb 5G5 and 6C1 are coated at 37°C for 2 hours, A max / IC 50 Maximum, mAb 6C10 coated under ambient temperature for 2 hours, A max / IC 50 The maximum value was selected, so this condition was chosen for subsequent experiments.

[0120] (2) Coating fluid optimization When all three mAbs were coated with CB buffer, A max / IC 50 The maximum value was found, therefore CB buffer was chosen for coating.

[0121] (3) Optimization of sealing fluid When sealing with #1 sealing solution, A max / IC 50 The maximum value was found in 2% skim milk powder and 5% skim milk powder, which significantly reduced the OD value and interfered with the icELISA reaction. Therefore, blocking solution #1 was selected for subsequent blocking.

[0122] (4) Optimization of closing time Based on the A of 3 types of mAbs max / IC 50 In peak cases, mAb 5G5 and 6C10 were blocked for 60 minutes, while mAb 6C1 was blocked for 120 minutes.

[0123] (5) Optimization of competitive reaction conditions Based on the A of 3 types of mAbs max / IC 50 At peak values, all three mAbs showed the best response at 4℃ for 60 min, exhibiting the characteristic of A. max / IC 50 The value is the largest.

[0124] (6) Optimization of secondary antibody dilution All three mAbs showed A at a secondary antibody dilution of 5000. max / IC 50 The value was the highest, so the secondary antibody was diluted 5000 times for further optimization.

[0125] (7) Optimization of secondary antibody reaction time The reaction time of the secondary antibody was then optimized by incubating it for 15 min, 30 min, and 60 min. The results showed that 15 min would decrease the OD value, while 60 min would increase the OD value to 3.0. Therefore, the optimal result was that all three antibodies should be incubated for 30 min.

[0126] Table 2-1 Summary of mAb 5G5 Condition Optimization Results

[0127] Table 2-2 Summary of Optimization Results for mAb 6C1 Conditions

[0128] Table 2-3 Summary of Optimization Results for mAb 6C10 Conditions

[0129] 2.2.2 Antibody tolerance test to non-physiological environment In actual sample testing, different food or biological samples can cause varying degrees of matrix effects, thus affecting the accuracy and sensitivity of the detection. To comprehensively evaluate the non-physiological environmental performance of antibodies, this study systematically tested the pH tolerance, salt ion tolerance, and organic solvent tolerance of antibodies. Furthermore, considering that urine is a commonly used biomonitoring sample after paraquat exposure, its complex composition and high urea concentration may affect antibody binding; therefore, the urea tolerance of antibodies was also measured to ensure that antibodies maintain stable binding capacity and sensitivity in urine sample testing.

[0130] 2.2.2.1 pH tolerance test The three antibodies exhibited similar pH tolerance patterns, maintaining good detection performance in neutral to alkaline environments (pH > 7), and demonstrating excellent antibody affinity, particularly at pH 10. max / IC 50 Its IC is much larger than other test groups. 50 As low as 0.005 μg / L; while in acidic environments with pH < 7, although A max The impact of fluctuations was relatively low, but the antibody detection performance deteriorated significantly, with its IC50 value decreasing. 50 >4 μg / L. Overall results show that mAb 5G5, 6C1, and 6C10 all have pH tolerance of 7-11.

[0131] 2.2.2.2 Salt ion concentration tolerance test As salt concentration increases, A max / IC 50 All three antibodies showed a decreasing trend, indicating a decrease in antibody sensitivity. The A values ​​of the three antibodies at a salt concentration of 72.5 mM were...max / IC 50 It reaches its peak value, demonstrating optimal detection performance; while under 5 M high-salt conditions, A max / IC 50 Significant decline, IC 50 The highest concentration of salt was observed, indicating the lowest antibody affinity. This suggests that all three antibodies are intolerant to high-salt environments, with their activity being inhibited to some extent under high-salt conditions. Overall, the salt ion tolerance concentration for mAbs 5G5, 6C1, and 6C10 was 580 mM.

[0132] 2.2.2.3 Methanol Tolerance Test The results are as follows Figure 3 As shown, with increasing methanol concentration, the A of the three antibodies... max / IC 50 The curve shows an upward trend, A max It remained stable within the range of 1.5-2.7, with 6C10 A max Higher than 5G5 and 6C1 antibodies; in addition, the IC50 of the three antibodies is higher. 50 The effect of methanol concentration was not significant, fluctuating within the range of 0.01-0.06 μg / L. Overall, all three antibodies showed good tolerance to methanol solution, tolerating 40% methanol concentration. However, at 60% methanol concentration, antigen-antibody binding was completely disrupted, resulting in low or no color development; therefore, this was not shown in the figure. The overall results show that mAbs 5G5, 6C1, and 6C10 all tolerated a methanol concentration of 40%.

[0133] 2.2.2.4 Acetonitrile tolerance test The results are as follows Figure 4 As shown, with increasing acetonitrile concentration, all three antibodies could tolerate 10% acetonitrile and maintain stable A concentrations. max / IC 50 The values ​​indicate that antibody sensitivity and binding ability are largely unaffected under low concentration organic solvent conditions. mAb5G5 exhibits stronger tolerance to acetonitrile, maintaining good binding performance even at 20% acetonitrile concentrations; mAb 6C10 and 6C1 show improved binding performance even at acetonitrile concentrations exceeding 10%. max / IC 50 The sensitivity was significantly affected, with a noticeable decrease. Overall results showed that the acetonitrile tolerance concentrations for mAb5G5, 6C1, and 6C10 were 20%, 10%, and 10%, respectively.

[0134] 2.2.2.5 Urea Tolerance Test The results are as follows Figure 5 As shown, with the increase of urea concentration, the A of the three antibodies... max The values ​​all showed a downward trend, with the 6C1 antibody exhibiting poor tolerance to urea; at a urea concentration of 2M, A...max / IC 50 The sensitivity of mAb 5G5 initially decreased significantly at a urea concentration of 4M; however, the sensitivity of A... max The decrease is more significant below 1.0, A max / IC 50 It is 16.08; mAb 6C10 at a urea concentration of 4M A max It can still maintain a level above 1.5, and A max / IC 50 The value was 20.26, which was higher than that of mAb 5G5. Overall, the urea tolerance concentrations for mAb 5G5, 6C1, and 6C10 were 4M, 2M, and 4M, respectively.

[0135] 2.2.3 Establishment of the icELISA method 2.2.3.1 Establishment of the Standard Curve Based on the optimal conditions summarized in Tables 2-1, 2-2, and 2-3, standard curves for 5G5, 6C10, and 6C1 are established. The ICs for mAb5G5, 6C10, and 6C1 are also described. 50 The concentrations were 0.046, 0.041, and 0.063 μg / L, respectively. 2 All values ​​were greater than 0.99, indicating good correlation with the curve fit. Based on the optimization and antibody performance testing results, mAb 6C10, which has high sensitivity and good tolerance to organic solvents, was ultimately selected for actual sample testing.

[0136] 2.2.3.2 Determination of Pretreatment Conditions Figure 6 Inhibition curves of mAb 6C10 were established for sample matrices in pig urine, skim milk, apple, and cucumber. The results showed that the matrix effect was low when the sample matrices were diluted 20-fold with PBS buffer after extraction, and the results showed good fit with the PBS standard curve, indicating that the pretreatment method basically eliminated the matrix effect caused by the sample.

[0137] 2.2.3.3 Sensitivity and Quantitative Detection Range IC50 of the sample matrix inhibition curve established above 10 The actual limit of detection (LOD) of this method is calculated by multiplying the value by its corresponding dilution factor and then using IC50. 20 -IC 80Multiply by the corresponding dilution factor to obtain the quantitative detection range. The LOD of mAb6C10 in pig urine is 0.35 μg / L, and the quantitative detection range is 0.93-25.19 μg / L; the LOD in skim milk is 0.13 μg / L, and the quantitative detection range is 0.38-12.61 μg / L; the LOD in apple is 0.86 μg / kg, and the quantitative detection range is 1.84-25.07 μg / kg; and the LOD in cucumber is 1.68 μg / kg, and the quantitative detection range is 3.47-41.14 μg / kg.

[0138] 2.2.3.4 Accuracy and Precision Three concentrations (high, medium, and low) were selected within the linear detection range of the matrix inhibition curve for each sample for matrix addition and recovery experiments. Based on the detection range of the matrix inhibition curve, the addition concentrations were set as follows: 2, 5, and 10 μg / L for pig urine; 2, 5, and 10 μg / L for skim milk; 5, 10, and 20 μg / kg for apples; and 10, 20, and 30 μg / kg for cucumbers. As shown in Table 2-4, the spiked recovery rate of pig urine samples was 89.5%-116.9%, with an intra-batch CV of 2.3-17.3% and an inter-batch CV of 7.73-10.1%; the spiked recovery rate of skim milk samples was 98.5-120.5%, with an intra-batch CV of 2.5-17.8% and an inter-batch CV of 3.21-10.28%; the spiked recovery rate of apple samples was 87.5-127.2%, with an intra-batch CV of 6.4-15.5% and an inter-batch CV of 3.43-10.32%; and the spiked recovery rate of cucumber samples was 90.1-124%, with an intra-batch CV of 3.1-19.8% and an inter-batch CV of 11.11-13.54%.

[0139] Table 2-4 Statistics on PQ Spiked Recovery

[0140] Note: If the sample is liquid, the corresponding unit is μg / L; if the sample is solid, the corresponding unit is μg / kg.

[0141] In summary, this embodiment optimized the conditions and evaluated the performance of three antibodies, ultimately selecting mAb 6C10 to establish an icELISA method for detecting PQ residues. Optimal conditions included: CB buffer coating at room temperature for 2 hours, blocking at 37°C for 60 minutes, competitive reaction at 4°C for 60 minutes, and secondary antibody dilution at 5000-fold with a reaction time of 30 minutes. The antibody was resistant to pH 7-11, 580 mM salt concentration, 40% methanol, 10% acetonitrile, and 4 M urea. The method's IC50... 50The concentration was 0.041 μg / L, and the quantitative detection range was 0.011–0.144 μg / L. It was stable in skim milk, pig urine, cucumber, and apple matrices, with LODs of 0.13 μg / L, 0.35 μg / L, 1.68 μg / kg, and 0.86 μg / kg, respectively. The recoveries ranged from 87.5% to 127.2%, with a CV of less than 14%, indicating good recovery and method stability. The icELISA method established in this study meets the requirements for rapid detection of PQ in fruits, vegetables, skim milk, and pig urine in terms of sensitivity, accuracy, and stability.

[0142] Example 3: Establishment of an LFIA method for the detection of paraquat residues Colloidal gold probes were prepared based on the prepared mAb 6C10. By optimizing the pH of the colloidal gold probe, the amount of antibody labeling, and the amount of colloidal gold probe used, a PQ colloidal gold immunochromatographic method was established, and the accuracy and precision of the detection method were evaluated.

[0143] 3.1 Test Methods 3.1.1 Preparation of colloidal gold probes Rinse multiple 1.5 mL centrifuge tubes with pure water, add 1 mL of colloidal gold solution to each tube, then add an appropriate amount of 0.1 M potassium carbonate dropwise, quickly vortex to mix and adjust to the required pH; then add antibody diluted with 0.5% (w / v) BSA, vortex to mix, and incubate at room temperature for 30 min. Then add 30 μL of 10% (w / v) BSA aqueous solution to each tube, vortex to mix and continue blocking for 30 min; after the treatment is completed, centrifuge at 1000 rpm for 10 min at 4℃, discard the supernatant, and resuspend the precipitate in 200 μL of reconstitution solution (provided by Beijing Weideweikang Biotechnology Co., Ltd.); finally, vortex the labeled colloidal gold probe thoroughly and store at 4℃ for later use.

[0144] 3.1.2 Optimization of Colloidal Gold Probes 3.1.2.1 pH Optimization of Colloidal Gold Probe For specific details, refer to the colloidal gold preparation steps in 3.1.1. Add 0, 5, 10, 15, 20, 25, 30, and 35 μL of 0.1 M K2CO3 solution respectively, adjust the pH of the solution system, and keep other steps unchanged. Then observe the color change of the colloidal gold solution, read the colloidal gold test card using a reading instrument, and select the K2CO3 labeling amount with the largest negative T line color development value as the optimal condition.

[0145] 3.1.2.2 Optimization of antibody labeling amount Based on the optimization results in 3.1.2.1, 8 μL, 10 μL, and 12 μL of antibody were added to the tubes, respectively, and PQ standard solutions with concentrations of 0, 1, and 5 ng / mL were used for detection. The optimal antibody labeling amount was determined based on the final color development and inhibition results.

[0146] 3.1.2.3 Optimization of Colloidal Gold Probe Dosage The dosage of colloidal gold probe was optimized using a gradient approach, with series volumes ranging from 3 to 8 μL (1 μL intervals). PQ standard solutions at concentrations of 0, 1, and 5 ng / mL were added to each system for measurement. The optimal dosage of colloidal gold was determined by comprehensively evaluating the final colorimetric intensity and the inhibitory effect of the target analyte on the chromatography process in different groups.

[0147] 3.1.3 Construction of Colloidal Gold Immunochromatographic Method 3.1.3.1 Determination of antigen coating concentration The original PH-Q-BSA coating was serially diluted (50×, 100×, 200×, 400×, 800×) and the goat anti-mouse secondary antibody (Jackson Immuno Research Laboratories, USA) was diluted 10×. These were then applied to nitrocellulose membranes as T and C lines using a streak scribing machine. The nitrocellulose membranes were placed in a 37°C incubator for at least 12 hours to dry completely. After dehydration, the membranes were integrated with absorbent paper, sample pads, and other components. The membranes were then processed into uniformly wide test strips using a strip cutter. The performance of the test strips was evaluated using PQ standard solutions containing 0, 1, and 5 ng / mL. The optimal working concentration for antigen coating was determined based on the intensity of the colorimetric signal after chromatography and the inhibitory effect of the target analyte on the immune response.

[0148] 3.1.3.2 Assembly and Testing of Colloidal Gold Test Card Referring to the experimental parameters and optimal reaction system established in Chapter 3.1.2, the pretreated sample pad, nitrocellulose membrane, and absorbent filter paper were sequentially bonded to a PVC substrate, and then cut into strips with a width of 3.8 mm using a cutting device. The specific detection procedure is as follows: (1) Measure the colloidal gold probe after condition optimization and place it in the reaction system (sample to be tested) containing 200 µL phosphate buffer. Gently pipette several times to ensure uniform dispersion of the system. Incubate at room temperature for 3 min to obtain the mixture. (2) Immerse the sample end of the prepared test strip vertically below the surface of the mixed solution and drive the liquid chromatography reaction for 5 min; (3) After the reaction is complete, take out the test strip and observe the color development of the test line (T line) and the control line (C line) with the naked eye. By visually comparing the results with the chromatography results of the negative control group, the qualitative result of the sample can be determined.

[0149] 3.1.4 Establishment of the LFIA Method 3.1.4.1 Establishment of the standard curve Based on the optimization results, a standard curve for the LFIA method was established. The specific procedures were as follows: PQ standards were serially diluted using PBS buffer to prepare a series of standard solutions with concentration gradients of 0, 0.032, 0.064, 0.32, 1.6, 8, 40, and 200 μg / L, and a blank control group was simultaneously established. After the chromatographic reaction, the color development results were observed visually: Under the premise that the control line (C line) showed normal color development, the concentration of the analyte corresponding to a significant decrease in color of the detection line (T line) compared to the blank group was defined as the visual limit of detection (vLOD). Furthermore, the optical density ratio of the T line to the C line was read using a matching strip reader to achieve quantitative signal analysis. The standard curve was plotted using the Logistic equation in Origin Pro software, with the standard concentration as the x-axis and the T / C line optical density ratio as the y-axis. The IC was calculated according to formula 3-1. 10 The value is used as the LOD of the established method, in IC 20 - IC 80 The value is used as the linear detection range of the established method. In the equation, A1 represents the maximum colorimetric value without the addition of standards, P represents the slope of the curve distortion point, and X0 represents the IC50 value of the analyte. 50 The value, A2, represents the minimum color development value when the amount of standard added is infinite.

[0150] Y=A2+(A1 A2) / [1+(x / x0) p ] (Formula 3-1) Visual detection limit (vLOD): The concentration value at which the color intensity of the T-line changes significantly when observed visually, based on the processing method and conditions determined for each sample.

[0151] 3.1.4.2 Determination of Pretreatment Conditions (1) Pretreatment methods for skim milk and pig urine samples Weigh 1 mL of skim milk and pig urine samples, and add 4 mL of methanol-0.1 mol / L HCl to homogenize and extract for 1 min. Then take 1 mL, 0.5 mL, and 0.25 mL of the sample extracts respectively, add 3 mL, 3.5 mL, and 3.75 mL of PBS buffer respectively, and dilute 4, 8, and 16 times. Vortex mix for 2 min and use for LFIA detection.

[0152] (2) Pretreatment methods for cucumber and apple samples Weigh 1 g of cucumber and apple samples, add 4 mL of methanol-0.1 mol / L HCl, and homogenize for 1 min. Centrifuge at 4000 rpm for 5 min, and transfer the supernatant to a 10 mL centrifuge tube. Then, take 1 mL, 0.5 mL, and 0.25 mL of the sample extracts respectively, add 4 mL, 4.5 mL, and 4.75 mL of PBS buffer respectively, and dilute 5, 10, and 20 times. Vortex to mix for 2 min, and use for LFIA detection.

[0153] Based on optimized experimental parameters, PQ standards were serially diluted to the required working solution concentration. Sample solutions obtained after pretreatment of blank samples (skim milk, pig urine, cucumber, and apple) were used as reaction solutions for detection, thereby establishing interference inhibition curves for each matrix. These curves were then fitted and analyzed against the standard curve in a PBS buffer system. The sample pretreatment method with the highest goodness of fit (i.e., best overlap) was identified as the optimal treatment condition for effectively eliminating matrix interference.

[0154] 3.1.4.3 Determination of the detection limit Based on the sample matrix inhibition curves established in 3.1.4.2, the IC50 values ​​corresponding to each sample matrix were determined. 10 The actual limit of detection (LOD) of this method is calculated by multiplying the value by its corresponding dilution factor. At the same time, the visual limit of detection (vLOD) is determined by visual inspection: under the premise that the quality control line (C line) is normally colored, the concentration of the analyte corresponding to the point where the color of the detection line (T line) fades significantly compared to the blank control group (the color is significantly weakened to the interpretation threshold) is defined as the visual limit of detection of the matrix sample.

[0155] 3.1.4.4 Specificity determination The concentration of PQ was diluted to 10 ng / mL using PBS buffer, and the concentration of its structural analog DQ was diluted to 1000 μg / L. A PBS negative control was also set up. The T-line color development was observed visually, and the T / C value results were interpreted using instruments.

[0156] 3.1.4.5 Accuracy and Precision Within the linear detection range of the sample, standard substance concentrations at low, medium, and high gradients were selected for spiking experiments. The sample pretreatment procedure described in Section 3.1.4.2 was followed, and the established LFIA method was used to quantitatively analyze the residues in the actual samples. The spiked recovery rate was calculated based on Formula 3-2, and this recovery rate was used as the core indicator for evaluating the accuracy of the method.

[0157] Recovery rate (%) = (Actual detected concentration / Added concentration) × 100% (Formula 3-2) Repeatability validation experiments were conducted using test strips from three different production batches. Spike recovery tests were performed on each sample matrix, with five replicate measurements for each concentration gradient. Intra-batch and inter-batch coefficients of variation (CV) were calculated using formula 3-3 to assess the precision of the method.

[0158] CV(%) = (Standard deviation of measured concentration / Average of measured concentration) × 100% (Formula 3-3) 3.2 Experimental Results and Analysis 3.2.1 Optimization of colloidal gold probes 3.2.1.1 pH Optimization of Colloidal Gold Probe See results Figure 7 The color of the colloidal gold solution changed from deep purple to wine red with increasing K₂CO₃ addition, and the color intensity of the T-line (detection line) gradually increased. Quantitative analysis of the signal values ​​revealed that the T-line reached its peak color when the K₂CO₃ addition was 25 μL. Therefore, 25 μL was determined to be the optimal pH adjustment condition for this system.

[0159] 3.2.1.2 Optimization of antibody labeling amount See results Figure 8 At 6 μL, the colloidal gold probe showed significant aggregation, preventing chromatography from being completed, and therefore no colorimetric development was achieved. The results indicate that when the antibody labeling amount was set to 10 μL, the test strip exhibited the most ideal colorimetric sensitivity and competitive inhibition effect. Because this amount yielded the best immunochromatographic performance, it was used as the fixed antibody labeling amount in subsequent condition optimization.

[0160] 3.2.1.3 Optimization of colloidal gold probe dosage See results Figure 9 When the amount of labeled material added was 6 μL, the T-line signal intensity and suppression effect of the chromatographic bands reached the optimal balance. Therefore, this volume was selected as the fixation amount of the colloidal gold probe in subsequent experiments.

[0161] 3.2.2 Establishment of a colloidal gold immunochromatographic method 3.2.2.1 Optimization of antigen coating concentration Experimental results show that ( Figure 10 When the coating antigen is diluted 400 times, the T-line is clearly visible and the inhibition effect is optimal. Therefore, this concentration was selected as the working concentration of the coating antigen in subsequent optimizations.

[0162] 3.2.3 Establishment of the LFIA Method 3.2.3.1 Establishment of the standard curve After optimization, the detection performance of the PQ test card is shown in the figure. Figure 11 And Table 3-1, where R 2 The value of 0.99 indicates a good curve fit. The results show that the vLOD of LFIA for PQ in the buffer system is 0.32 μg / L, the LOD is 0.049 μg / L, and the linear detection range is within the range of IC50. 20 -IC 80 The concentration ranges from 0.10 to 1.19 μg / L, and the detection is sensitive.

[0163] Table 3-1 Standard Curve Parameters of PQ Test Card

[0164] 3.2.3.2 Determination of Pretreatment Conditions The results showed that when pig urine and skim milk samples were diluted 16 times with PBS buffer after extraction, and apple and cucumber samples were diluted 20 times with PBS buffer after extraction, the matrix inhibition curves and PBS standard curves of the samples had a higher degree of overlap than those of other dilution factors. This indicated that the pretreatment method at this point essentially eliminated the matrix effect of the samples.

[0165] 3.2.3.3 Determination of the detection limit IC using the above matrix curve 10 The LOD of PQ in the actual sample was obtained by multiplying it by the corresponding sample matrix dilution factor, and the vLOD of the sample was obtained by visually interpreting the concentration value at which a significant change in the T-line occurred. The results showed that the LODs in skim milk, pig urine, apple, and cucumber were 1.63 μg / L, 9.76 μg / L, 6.55 μg / kg, and 3.27 μg / kg, respectively, and the vLODs were 25 μg / L, 25 μg / L, 32 μg / kg, and 32 μg / kg, respectively.

[0166] 3.2.3.4 Specificity determination Under determined optimal conditions, the specificity of colloidal gold detection cards for the detection of DQ, a PQ structural analogue, was investigated. For example... Figure 12As shown, the T / C value of the negative control group was approximately 1.7, while the T / C value of PQ at a concentration of 10 ng / mL was approximately 0.05, significantly lower than that of the negative control group. For the PQ structural analog DQ, the T / C value of the detection card at a concentration of 1000 ng / mL was approximately 1.06, with little difference from the negative control group, indicating that the established LFIA has good specificity and no cross-reactivity with its structural analog DQ.

[0167] 3.2.3.5 Accuracy and Precision Within the linear detection range of the matrix inhibition curve for each sample, three concentration points (high, medium, and low) were selected for sample matrix spike recovery experiments. Based on the quantitative detection range of the matrix inhibition curves established for different samples, the following concentrations were set for spiked samples: 40, 50, and 100 μg / L for pig urine; 10, 20, and 30 μg / L for skim milk; 20, 40, and 60 μg / kg for apples; and 20, 40, and 60 μg / kg for cucumbers.

[0168] The results showed that the spiked recovery rate for pig urine samples was 70.16%-114.8%, with an inter-batch CV of 7.33%-11.33%; the spiked recovery rate for skim milk samples was 88%-121%, with an inter-batch CV of 6.40%-11.61%; the spiked recovery rate for apple samples was 79.5%-103.8%, with an inter-batch CV of 8.33%-10.90%; and the spiked recovery rate for cucumber samples was 78.4%-109.6%, with an inter-batch CV of 8.61%-11.27%.

[0169] In summary, this embodiment establishes an LFIA method based on colloidal gold materials for the detection of PQ in skim milk, pig urine, and fruits and vegetables.

[0170] (1) Based on the previous icELISA optimization results, a mAb 6C10 colloidal gold probe was prepared using colloidal gold nanoparticles as signal elements and mAb 6C10 as recognition elements. Homologously coated original PH-Q-BSA was used as the solid-phase trap for the detection line (T line). On this basis, the coating concentration of the NC membrane and the acid-base environment of the reaction system were optimized, and the optimal reaction conditions were established as follows: original coating dilution of 400×, K2CO3 volume for pH adjustment of 25µL, antibody volume of 10µL, and colloidal gold probe volume of 6µL.

[0171] (2) An LFIA method for detecting PQ residues was established under optimized conditions, with a vLOD value of 0.32 μg / L. It did not recognize its structural analogue DQ, demonstrating good specificity. The method showed good detection performance in skim milk, pig urine, and fruit and vegetable samples, with vLOD values ​​of 25 µg / L, 25 µg / L, 32 µg / kg, and 32 µg / kg, respectively. The recoveries ranged from 70.16% to 121%, the inter-batch CV was less than 12%, and the detection limit met the national standard requirements.

[0172] The results show that the LFIA method based on mAb 6C10 that we established can qualitatively judge the results within 10 minutes and can be applied to the rapid detection of PQ in animal-derived foods, plant-derived foods and biological samples.

[0173] The above description is only a partial embodiment of the technical solution of the present invention. These embodiments are merely illustrative examples intended to help those skilled in the art understand the core concept of the present invention, and are not exhaustive enumerations of all embodiments of the present invention. The scope of protection of the present invention should be determined by the claims. Any adaptive modifications, equivalent substitutions of technical features, further optimizations and improvements made based on the basic principles and concepts disclosed in the present invention are all within the scope of protection of the present invention.

Claims

1. A mouse hybridoma cell, characterized in that, The mouse hybridoma cells are preserved at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 68912.

2. A monoclonal antibody capable of specifically recognizing paraquat, characterized in that, The monoclonal antibody is secreted by the mouse hybridoma cells described in claim 1.

3. The method of producing the monoclonal antibody according to claim 2, characterized by, Includes the following steps: The hybridoma cells described in claim 1 are injected into the peritoneal cavity of mice, and antibodies are prepared by in vivo induction. The obtained antibodies are then purified to remove impurities and obtain monoclonal antibodies.

4. The use of a monoclonal antibody according to claim 2, characterized in that, Includes at least one of the following (1) to (2): (1) Application in the detection of paraquat; (2) Application in the preparation of paraquat detection reagent.

5. An icELISA method for detection of paraquat residues, characterized by, Includes the following steps: S1. Dilute the coating agent with coating buffer and incubate in an ELISA plate; add blocking buffer to block, and wash. The coating solution is a carbonate buffer or a phosphate buffer; the coating precursor is PH-Q-BSA, which is obtained by the following method: the hapten 1-(4-carboxybutyl)-1′-methyl-[4,4'-bipyridine]-1,1'-dionium is coupled with bovine serum albumin using the active ester method to obtain the coating precursor PH-Q-BSA; the incubation conditions are 4℃ for 16h or 25℃ to 37℃ for 2h; the blocking solution is at least one of No. 1 blocking solution and 2-5% (v / v) skim milk, and each 2L of No. 1 blocking solution contains 100 mL of calf serum, 100g of sucrose, 5g of casein, 11.6g of Na2HPO4·12H2O, 1.186g of NaH2PO4·2H2O, and 1 mL of Proclin 300 preservative, and the blocking time is 60-120min; S2. Add the sample solution to be tested and the stock solution of the monoclonal antibody as described in claim 2 to each well, and incubate them together to carry out a competitive reaction; The conditions for the competitive reaction are incubation at 4~37℃ for 30~60 min; S3. Wash; add enzyme-labeled secondary antibody dilution solution that specifically binds to the monoclonal antibody, and incubate; the dilution of the enzyme-labeled secondary antibody dilution solution is 1000~7000 times, and the secondary antibody reaction time is 15~60 min; S4. Add the colorimetric solution to the microplate to initiate the colorimetric reaction, and then add the stop solution to terminate the reaction; S5. Measure OD of each well 450nm Values, and calculate the amount of paraquat in the sample from the standard curve.

6. The icELISA method according to claim 5, characterized in that, The coating solution in S1 is carbonate buffer; the incubation conditions are 25°C for 2 hours; the blocking solution is No. 1 blocking solution; the blocking time is 60 minutes. The conditions for the competitive reaction described in S2 are incubation at 4°C for 60 min; The enzyme-labeled secondary antibody dilution solution described in S3 is diluted 5000 times, and the secondary antibody reaction time is 30 minutes.

7. A LFIA method for detection of paraquat residues, characterized by, Includes the following steps: (1) Preparation of colloidal gold probe: Take colloidal gold solution and adjust pH; then add stock solution of monoclonal antibody as described in claim 2 and vortex mix well. After reaction, perform blocking treatment; centrifuge, discard supernatant, resuspend precipitate to obtain colloidal gold probe solution; (2) Preparation of detection membrane: The coating agent and the goat anti-mouse secondary antibody were diluted separately and applied to a nitrocellulose membrane using a membrane scribing instrument as the detection line and the quality control line, respectively. After drying, they were ready for use. The coating agent was PH-Q-BSA, which was obtained by the following method: The hapten 1-(4-carboxybutyl)-1′-methyl-[4,4'-bipyridine]-1,1'-dionium was coupled with bovine serum albumin by active ester method to obtain the coating agent PH-Q-BSA. (3) Assemble the test strips: Assemble the sample pad, the nitrocellulose membrane obtained in step (2) and the absorbent filter paper on the PVC substrate, and then use a cutting device to cut them into test strips of uniform width. (4) Detection: Mix the sample solution to be tested with the colloidal gold probe solution obtained in step (1) to obtain a mixture; vertically immerse the sample end of the prepared test strip into the mixture to drive the liquid chromatography reaction; after the reaction is completed, take out the test strip and observe the color development state of the detection line and the control line with the naked eye. By visually comparing the chromatography results with the negative control group, the test result of the sample can be determined.

8. The LFIA method according to claim 7, characterized in that, In step (1), 25-30 μL of 0.1M K2CO3 is added to 1 mL of colloidal gold solution to adjust the pH; the concentration of the monoclonal antibody stock solution is 1 mg / mL, and the volume ratio of the colloidal gold solution to the monoclonal antibody stock solution is 1 mL: 8-12 μL.

9. The icELISA method according to claim 5 or the LFIA method according to claim 7, characterized in that, The samples to be tested include at least one of skim milk, pig urine, cucumber, and apple.

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