Balb / c mouse source hybridoma cell strain secreting anti-thiram pesticide broad-spectrum monoclonal antibody and gold nanoflower immunochromatography test strip for detecting thiram pesticide

Abstract

The invention discloses a Balb / c mouse source hybridoma cell strain secreting an anti-thiram pesticide broad-spectrum monoclonal antibody and a gold nanoflower immunochromatography test strip for detecting thiram pesticides. The preservation number of the Balb / c mouse source hybridoma cell strain in the China General Microbiological Culture Collection Center is CGMCC (China General Microbiological Culture Collection Center) No.46545. The gold nanoflower immunochromatographic test strip for detecting the thiram pesticides is prepared by preparing a gold nanoflower immunoprobe from the thiram pesticide-resistant broad-spectrum monoclonal antibody secreted by the Balb / c mouse murine hybridoma cell strain and finally preparing the gold nanoflower immunochromatographic test strip for detecting the thiram pesticides. The gold nanoflower immunochromatography test strip is short in determination time, easy to operate and low in cost, has excellent specificity and sensitivity, and can simply, conveniently and quickly detect thiram pesticides.

Description

Technical Field

[0001] This invention belongs to the field of rapid detection of pesticide residues, and relates to a Balb / c mouse hybridoma cell line that secretes broad-spectrum monoclonal antibodies against thiram-type pesticides and a gold nanoflower immunochromatographic test strip for detecting thiram-type pesticides. Background Technology

[0002] Thiram-based pesticides (such as thiram, thiram, and ferrous thiram) are a class of dithiocarbamate fungicides and were among the first organosulfur fungicides to be marketed. They are protective contact fungicides that primarily form a film on the plant surface to prevent pathogen infection. They are non-systemic, do not penetrate plant tissue or seeds, and inhibit spore germination or the initial growth of pathogenic hyphae on the surface. They also inhibit fungal enzyme systems, disrupt enzymes containing -SH groups within pathogens (such as pyruvate dehydrogenase), and interfere with energy metabolism. Due to their low cost and good fungicidal effect, they are widely used on seeds, vegetables, and fruits, primarily for controlling damping-off, anthracnose, and scab in citrus and other fruit tree seedlings, and gray mold in dried chili peppers. These pesticides are available in powder, wettable powder, and aqueous suspension formulations and can be mixed with other fungicides, hence their large-scale use worldwide.

[0003] Thiram-based pesticides readily decompose into metabolites such as carbon disulfide (CS2) and ethylenediamine, and their residual period is significantly affected by environmental factors (light, temperature, pH). Thiram-based pesticides are metabolized in the body into toxic metabolites dimethyl dithiocarbamate and carbon disulfide. Extensive evidence indicates that thiram-based pesticides can affect various organs in animals, including but not limited to: tibial cartilage dysplasia, reduced fertility, neurodegeneration, immune damage, embryonic maldevelopment, and even death due to cardiac arrest. Thiram-based pesticides exhibit moderate toxicity to poultry; levels of 100-500 ppm in the diets of hens, quails, and partridges at one-third of their concentration inhibit egg production and lead to soft eggshells, stunted growth, and leg abnormalities. The LC50 (48h) of thiram-based pesticides in fish is 0.1-0.2 mg / L. Establishing a rapid and effective detection system for monitoring thiram-based pesticide residues in the environment and food and agricultural products has significant application value and practical significance for source control of food quality.

[0004] Currently, conventional methods for quantitative and qualitative monitoring of thiram-type pesticide residues and their metabolites mainly employ high-performance liquid chromatography (HPLC), gas chromatography (GC), HPLC-MS, and GC-MS. While these methods offer good reproducibility and accuracy, their cumbersome sample pretreatment steps, susceptibility to matrix interference, limitations on analyte sensitivity, and high equipment requirements and time-consuming sample processing hinder rapid detection of large batches of samples, making widespread application difficult. Immunological detection systems based on the specific immune properties of antigen-antibody reactions and signal probe amplification strategies using nanomaterial labeling offer advantages such as ease of operation, short detection time, speed, and high sensitivity, and are widely used in food safety and in vitro diagnostics. Therefore, high-quality antibodies and signal amplification probes are crucial. AuNF nanoflowers, with their large surface area, multi-branching, and colloidal stability, can significantly improve the sensitivity of immunochromatographic bands.

[0005] Therefore, this patent uses thiram as an antigen to synthesize a thiram hapten with a carboxyl group, and introduces a linker arm on the carboxyl group to couple it with a carrier protein (BSA / BSA) to prepare a complete antigen. By combining animal immunization and cell fusion technologies, a high-affinity and broad-spectrum specific monoclonal antibody against thiram-like pesticides is developed. Based on the nanomaterial-coupled antibody reaction mechanism and immunochromatographic principle, a nanoflower immunoassay system for thiram-like pesticide residues is constructed to achieve rapid, sensitive, and accurate identification and monitoring of thiram-like pesticide residues in the field of food and agricultural product quality and safety. Summary of the Invention

[0006] In view of the above background, the present invention aims to provide a Balb / c mouse hybridoma cell line that secretes broad-spectrum monoclonal antibodies against thiram-type pesticides and a gold nanoflower immunochromatographic test strip for detecting thiram-type pesticides.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A Balb / c mouse hybridoma cell line secreting a broad-spectrum monoclonal antibody against thiram-type pesticides was deposited on June 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46545.

[0008] A broad-spectrum monoclonal antibody against thiram-type pesticides, wherein the broad-spectrum monoclonal antibody against thiram-type pesticides is secreted by the Balb / c mouse hybridoma cell line described above.

[0009] The above-mentioned Balb / c mouse hybridoma cell line or broad-spectrum monoclonal antibody against thiram-type pesticides is used in the preparation of products for detecting thiram-type pesticides.

[0010] A gold nanoflower immunochromatographic test strip for detecting thiram-type pesticides, comprising the following components: a plastic shell, a sample pad, an immunoprobe conjugation pad, an absorbent pad, a nitrocellulose membrane, and a PVC base plate; the immunoprobe conjugation pad is coated with a gold nanoflower immunoprobe labeled with a broad-spectrum monoclonal antibody against thiram-type pesticides; the broad-spectrum monoclonal antibody against thiram-type pesticides is secreted by Balb / c mouse hybridoma cell line; the Balb / c mouse hybridoma cell line was deposited on June 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46545.

[0011] The preparation method of the above-mentioned gold nanoflower immunochromatographic test strip for detecting thiram-type pesticides includes the following steps: (1) Preparation of colloidal gold: Colloidal gold was prepared by citric acid reduction method; (2) Preparation of gold nanoflowers: Using the colloidal gold prepared in step (1) as gold seeds, gold nanoflowers were prepared by seed growth method. (3) Preparation of gold nanoflower immunoprobe labeled with broad-spectrum monoclonal antibody against thiram-type pesticides: 7.5 μL of 0.1M potassium carbonate and 15 μg of broad-spectrum monoclonal antibody against thiram-type pesticides were added to each 1 mL of gold nanoflowers prepared in step (2). After the system stabilized, BSA was added to block the unbound sites. Finally, PEG20000 was added, and after centrifugation and resuspending, the gold nanoflower immunoprobe labeled with broad-spectrum monoclonal antibody against thiram-type pesticides was obtained. (4) Pretreatment of immunoprobe binding pads, sample pads and absorbent pads: Cut untreated immunoprobe binding pads into strips 1 cm wide, place them in a large petri dish, soak them in the pre-prepared blocking solution, and incubate them in a 37°C constant temperature incubator for 2 hours. After removing them from the incubator, filter out the blocking solution and continue to dry them in the incubator at 37°C. Then seal them at room temperature. For subsequent use, cut them to a length of 1 cm and a width of 4 mm. The composition of the blocking solution is: 5 wt% BSA + 1 vol% Tween-20. The sample pads do not need to be treated and should be cut to a length of 1 cm and a width of 4 mm for subsequent use. The absorbent pads do not need to be treated and should be cut to a length of 1.5 cm and a width of 4 mm for subsequent use. (5) Streaking on nitrocellulose membrane: Use goat anti-mouse secondary antibody to streak C lines on nitrocellulose membrane, and use complete antigen TMTD-BSA synthesized by active lipid method to streak T lines on nitrocellulose membrane. The distance between C and T lines on the same nitrocellulose membrane is 0.6 cm. (6) Preparation of immune probe binding pad: The gold nanoflower immune probe labeled with the broad-spectrum monoclonal antibody against thiram-type pesticides prepared in step (3) was dropped onto the pretreated immune probe binding pad and dried at 37°C for later use. (7) Assembly of immunochromatographic test strips: The nitrocellulose membrane after scribing in step (5), the immunoprobe binding pad prepared in step (6), the sample pad pretreated in step (4) and the absorbent pad are assembled on a PVC base plate; the immunoprobe binding pad and the sample pad are overlapped and pasted, with a 2mm gap between the same end, and the absorbent pad and the nitrocellulose membrane overlap by 2mm at both ends. The plastic outer shell is covered, dried and sealed, and stored at 4℃.

[0012] The preparation method of the broad-spectrum monoclonal antibody against thiram-type pesticides in step (3) is as follows: Balb / c mouse hybridoma cell line in the logarithmic growth phase is injected into the peritoneal cavity of paraffin-sensitized Balb / c mice. When the mouse abdomen is distended, the ascites is extracted and purified to obtain the broad-spectrum monoclonal antibody against thiram-type pesticides.

[0013] The detection principle of the gold nanoflower immunochromatographic test strip for detecting thiram-type pesticides described in this invention is as follows: When the test strip is first used for detection, the prepared gold nanoflower immunoprobe is first dropped onto the immunoprobe binding pad, and after drying, it is then followed according to the schematic diagram (…). Figure 3 The sample is assembled using the following method, and then dropped onto the sample pad. The liquid flows towards the absorbent paper via chromatography. When it reaches the T-line, any gold nanoflower immunoprobes that have not yet bound to the antigen in the sample bind completely to the antigen at the T-line and remain there, resulting in color development at the T-line. The liquid continues to flow forward. When it reaches the C-line, both those bound to the antigen and those not bound to the sample bind to the C-line until saturation. The bound gold nanoflower immunoprobes remain at the C-line, resulting in color development at the C-line. The complete colorimetric reaction takes approximately 10 minutes.

[0014] The beneficial effects of this invention are: This invention is the first to propose a gold nanoflower immunochromatographic test strip for detecting thiram-like pesticides, utilizing gold nanoflowers combined with a broad-spectrum monoclonal antibody against thiram-like pesticides. The gold nanoflower immunochromatographic test strip provided by this invention achieves a limit of detection of 156 ng / mL for thiram-like pesticides, requires no special instruments, and takes only 10 minutes to detect, meeting the highest sensitivity requirements in national standards. Attached Figure Description

[0015] Figure 1 Subtypes and affinity of broad-spectrum monoclonal antibodies against thiram-type pesticides.

[0016] Figure 2Cross-reactivity of broad-spectrum monoclonal antibodies against thiram-based pesticides. TMTD; Asomate; Ferbam; Mancozeb; Zineb; Metiram; Propineb; Carbendazim; Chlorothalonil.

[0017] Figure 3 Schematic diagram of the working mode of the gold nanoflower immunoassay strip.

[0018] Figure 4 Selection of the optimal streaking concentration (mg / mL) for the C line of the gold nanoflower immunochromatographic test strip.

[0019] Figure 5 Selection of the optimal streak concentration (μg / mL) for the T line of the gold nanoflower immunochromatographic test strip.

[0020] Figure 6 Determination of the optimal probe amount (μL) for gold nanoflower immunochromatographic test strips.

[0021] Figure 7 Specificity determination of gold nanoflower immunochromatographic test strips.

[0022] Figure 8 , Figure 9 , Figure 10 Sensitivity determination of gold nanoflower immunochromatographic test strips. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments. The illustrative embodiments and descriptions herein are used to explain the present invention, but are not intended to limit the present invention.

[0024] Example 1: Preparation of Balb / c mouse hybridoma cell line. The specific preparation process of the Balb / c mouse hybridoma cell line is as follows: I. Preparation of Hapten Synthesis of Compound 2: 4-(methylamine)butyrate (1.53 g, 10 mmol) was added to a solution of sodium hydroxide (1.2 g, 30 mmol) in water (50 mL), followed by the dropwise addition of carbon disulfide (0.91 g, 12 mmol) at room temperature. After stirring at room temperature for 1 hour, the mixture was heated to 40°C and stirred at this temperature for 2 hours. After the reaction was complete, the mixture was cooled to room temperature, and the solvent was evaporated under vacuum to obtain crude Compound 2. This crude compound was used directly in the next step without further purification.

[0025] Synthesis of compound 3: (1) Compound 2 (2.37 g, 10 mmol) was dissolved in a mixed solvent of methanol (45 mL) and dichloromethane (15 mL), and sodium dimethyl dithiocarbamate hydrate (4 g, 22.3 mmol) was added. The mixture was stirred overnight at room temperature. (2) 1 M HCl (100 mL) was added to the reaction solution, and the mixture was stirred thoroughly. The solution was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, dried with anhydrous sodium sulfate, and the solvent was evaporated under vacuum to obtain the crude product. The crude product was then purified by silica gel column chromatography to obtain a white solid compound 3 (2.54 g, yield 77.8%). 1 H NMR (400MHz, DMSO-d6): δ12.16(s,1H),5.24-5.21(m,2H),4.00(t,J=8.0Hz,1H),3.72(t ,J=8.0Hz,1H),3.46(s,3H),3.36-3.27(m,6H),2.28-2.22(m,2H),1.88-1.80(m,2H)ppm. Synthesis of compound 4: Compound 3 (940 mg, 2.9 mmol) was dissolved in dichloromethane (100 mL), and then N-hydroxysuccinimide (334 mg, 2.9 mmol) and dicyclohexylcarbodiimide (597 mg, 2.9 mmol) were added. After stirring at room temperature overnight, the insoluble matter was removed by filtration, and the mixture was purified by rotary evaporation and pulping to obtain compound 4 (1.1 g, 93%).

[0026] II. Complete Antigen Synthesis The complete antigen was synthesized using the active lipid method. The specific steps were as follows: First, 4.18 mg of the thiram-type pesticide thiram (TMTD), 5.52 mg of N-hydroxysuccinimide, and 8.48 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were dissolved in 0.4 mL of dimethylformamide and reacted in a constant temperature shaker at 20 °C and 110 r / min for 12 h. Then, the system that had been fully reacted in the previous step was added dropwise to 3.6 mL of 5 mg / mL bovine serum albumin (BSA) solution (in 0.01 M, pH 8.5 PBS). Next, the fully reacted system from the previous step was added dropwise to 3.6 mL of 5 mg / mL BSA solution (solvent: 0.01 M, pH 8.5 PBS). The mixture was reacted in a constant temperature shaker at 25 °C and 110 r / min for 6 h. The reaction solution was then transferred to a dialysis bag and dialyzed in 0.01 M PBS (pH 7.4) at 4 °C for 3 days to obtain the complete antigen TMTD-BSA.

[0027] The preparation method of the complete antigen TMTD-OVA is basically the same as that of TMTD-BSA, except that BSA in the preparation step is replaced with chicken ovalbumin (OVA).

[0028] III. Animal Immunization TMTD-OVA was selected as the immunogen, diluted to an appropriate concentration with 0.01M PBS (pH 7.4), and then emulsified with an equal volume of Freund's complete adjuvant. Six-week-old Balb / c mice that had been acclimatized to their environment for one week were immunized with the fully emulsified emulsion. The same batch of mice was then immunized every two weeks with the same immunogen and an emulsion containing Freund's incomplete adjuvant. Starting with the second immunization, TMTD-BSA was used as the coating antigen for each immunization. The antibody titer and specific recognition ability against thiram-based pesticides in the mouse tail vein serum were measured using ELISA. When the serum titer reached 1:8000 and showed good specificity, the spleen of the mouse was harvested for cell fusion after the initial immunization.

[0029] ELISA specific steps: Add 100 μL of the coated antigen diluted to the optimal concentration to an ELISA plate and incubate at 37°C for 2 h or 4°C for 12 h. After washing, block with blocking buffer (200 μL / well) and incubate at 37°C for 2 h or 4°C for 12 h. Add diluted antibody or antiserum to the plate (it can be added directly when determining titer; when determining specificity, take an appropriate amount of hapten and antibody or antiserum dilution and incubate at 37°C for 1 h), and incubate at 37°C for 50 min. After washing, add 100 μL of diluted goat anti-mouse IgG-HRP and incubate at 37°C for 50 min. After washing, add 100 μL / well of chromogenic buffer and incubate at 37°C for 15 min. Finally, add 50 μL of 2M H2SO4 stop solution and measure the optical density (OD) of each well at 450 nm. 450nm ).

[0030] IV. Cell Fusion Methods Mouse spleen cells were fused with mouse myeloma cells (SP2 / 0) using the PEG fusion method. After multiple subcloning screenings, a hybridoma cell line that stably secretes high-quality antibodies was obtained.

[0031] Required materials: alcohol swabs (5-6), Dissection board (1 piece) 5mL syringes (2) 50mL centrifuge tubes (4) 15mL centrifuge tubes (2) Three cutters and three tweezers (1 set) Cell culture dishes (3) Filters (2) Filter cartridge nozzles (1 box) 1mL pipette tips (≥10) Waste liquid tank.

[0032] On the day of cell fusion, prepare in advance one bottle of DMEM (>250mL), one tube of DMEM (approximately 40mL), one bottle of 20% FBS-DMEM with added HAT (>200mL, referred to as HAT medium), one mL of PEG1450 (all the above reagents have been preheated to 37°C before use), and 40°C sterile water (>300mL, placed in a beaker and preheated in a water bath). The specific steps for cell fusion are as follows: (1) Take out mice that have been immunized by shock from the mouse room, take blood from their eyeballs, place them in a preheated 37°C water bath, and then euthanize them by dislocation (after fusion, centrifuge them to obtain serum and store it at -20°C), soak them in 75% alcohol, and introduce them into the intercellular space; (2) Turn on the clean bench, light the alcohol lamp, tear open the outer packaging of the three sets of tweezers, absorb as much alcohol as possible from the mouse's fur, and fix the mouse on the dissection board. (3) After being burned by the flame of the alcohol lamp, the first set of scissors cuts open the fur, the second set of scissors opens the peritoneum, and the third set of scissors removes the spleen and removes the surface fat tissue. (4) Take a culture dish and use a 5mL syringe to draw a small amount of DMEM into the lid and the bottom of the dish respectively. Clean the spleen in the lid and then place the spleen in the bottom of the dish. (5) Open the 50mL centrifuge tube, place the filter screen at the centrifuge tube opening, and moisten the filter screen with a little DMEM. (6) Take a syringe, first puncture the spleen with the needle, then press the spleen fully with the handle to allow the spleen cells to enter the culture medium, use a 1mL pipette to draw DMEM through the filter and add it to the centrifuge tube, then repeat the process of taking DMEM to rinse the bottom of the dish several times, adding it through the filter to the centrifuge tube, and replenishing the culture medium to 40mL. (7) Centrifuge the spleen cell suspension at 1100 rpm and room temperature for 7 min (the centrifugation conditions in subsequent steps are the same as this step). At this time, take out SP2 / 0 from the incubator, add a little DMEM to a 50 mL centrifuge tube, blow off SP2 / 0 and add it to the centrifuge tube, and add DMEM to make up to 40 mL for later use. (8) After centrifugation, remove the spleen cells, discard the supernatant, resuspend the precipitate with DMEM and add fluid to 40 mL, and centrifuge the spleen cell suspension together with SP2 / 0 suspension. (9) After centrifugation, compare the amount of precipitate in the two tubes, discard the supernatant, take an appropriate amount of DMEM to resuspend the cells and count them. After calculating the number of cells, take an appropriate amount of SP2 / 0 suspension at a ratio of 1 / 3 to 1 / 10 and add it to the spleen cell suspension. Add DMEM to 40 mL, mix the cell suspension and centrifuge. (10) After centrifugation, place the beaker, PEG1450 and preheated single tube of DMEM into the clean bench, discard the mixed cell supernatant, tap the bottom of the centrifuge tube to disperse the cells, loosen the caps of the two tubes, place the cell precipitate in the centrifuge tube below the liquid surface in the beaker, add PEG1450 dropwise into the centrifuge tube, gently shake the bottom of the tube while adding, and add it within 1 minute, starting slowly and then quickly. (11) After adding PEG1450, let it stand in a water bath for 1 minute, then add DMEM in stages to the centrifuge tube, starting slowly and then quickly (add 1 mL in the first minute, 3 mL in the second minute, 5 mL in the third minute, and 10 mL in the fourth minute). After adding, add DMEM to 40 mL and let it stand at 37°C for 10 minutes. (12) After centrifugation, prepare several cell culture plates, take out the HAT medium for fusion and pour a small amount into the cell culture dish. After centrifugation, discard the supernatant, take an appropriate amount of HAT medium from the dish to resuspend the fused cell pellet, add the resuspended liquid to the whole bottle of HAT medium, let it stand and mix well, plate it, and place it in a carbon dioxide incubator at 37°C and 5% CO2 concentration for culture.

[0033] V. Antibody Characterization After a week of culture, positive cell wells with high titers and good specificity were screened using ELISA. After multiple rounds of subclonal screening, a stable Balb / c mouse hybridoma cell line secreting a high-affinity broad-spectrum monoclonal antibody against thiram-type pesticides was obtained, namely the monoclonal antibody cell line: 5G2.

[0034] The obtained Balb / c mouse hybridoma cell line 5G2 was deposited on June 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46545.

[0035] Example 2: Preparation of broad-spectrum monoclonal antibodies against thiram-type pesticides 1. Preparation of ascites (1) Take a 1mL sterile syringe and inject 500μL of paraffin into the peritoneal cavity of Balb / c mice around 12 weeks old to sensitize them. When injecting, insert the syringe vertically into the back of the mouse's peritoneal cavity. Disinfect the injection site with alcohol before and after injection to avoid bleeding. (2) One week after sensitization, take Balb / c mouse hybridoma cell line 5G2 that has just grown to the logarithmic phase and has filled about 80% of the culture dish, discard the supernatant, use a pipette to take 1 mL of fresh 1640 medium to wash the cells from the bottom of the culture dish, mix them evenly, use a 1 mL sterile syringe to draw the cell suspension from one well of a 6-well plate, and then vertically inject it into the peritoneal cavity of the sensitized mouse. Note that the needle must be inserted into the peritoneal cavity accurately and without obstruction, and should not be injected into the peritoneal tissue to avoid tumor formation and waste of the mouse. (3) About a week after injecting cells into the peritoneal cavity of mice, under normal circumstances, the abdominal distension of mice can be clearly observed, and the mice are listless and have messy fur. At this time, it is necessary to start preparing to collect ascites. (4) Prepare sterile injection needles, 1.5mL EP tubes, alcohol swabs, etc. Hold the mouse with one hand and wipe the blood collection site with alcohol swabs. With the other hand, carefully insert the sterile injection needle into the side of the abdominal cavity. Place the sterile injection needle against the EP tube to collect the ascites. You can see the ascites flowing into the tube along the sterile injection needle. The color is reddish, which may be due to excessive ascites production and hemolysis. However, it does not affect the properties of the antibodies in the ascites. When collecting ascites, the mouse should be fixed to prevent the mouse from struggling and causing ascites loss. Each mouse can only collect about 1.5mL of ascites at a time. After the tail blood is collected, put the mouse back into the cage. Ascites can continue to be produced. Ascites can be collected again the next day until the mouse dies. (5) After the mouse ascites fluid was balanced, it was centrifuged at 12000 rpm for 20 min in a 4℃ centrifuge. After taking it out, it can be observed that the liquid in the centrifuge tube is divided into three layers. The top layer is a layer of fat. Carefully take out the middle layer of ascites fluid, put it into a new 1.5mL EP tube, label it, and store it in a -20℃ refrigerator.

[0036] 2. Ascites purification Ascites fluid was diluted 1:10 with equilibration buffer (Na2HPO4 3.5814 g / L, NaCl 4.383 g / L; pH=7.0). The diluted ascites fluid was filtered through a 0.45 μm filter and then passed through a Protein G affinity chromatography column. The procedure was performed according to the instructions for commercial Protein G affinity chromatography. The purified antibody was dialyzed in a dialysis bag with PBS (NaCl 8.0 g / L, KCl 0.2 g / L, Na2HPO4 1.44 g / L, KH2PO4 0.24 g / L; pH=7.4) for 3 days, followed by dialyzed with ultrapure water for 1 day. Finally, the antibody was concentrated using PEG 20000 to obtain a broad-spectrum monoclonal antibody against thiram-type pesticides. The broad-spectrum monoclonal antibody against thiram-type pesticides was stored at -80°C for later use.

[0037] This study used a cell subtype assay kit to determine the subtypes of the obtained broad-spectrum monoclonal antibodies against thiram-type pesticides. The results are as follows: Figure 1 As shown, the heavy chain subtype of the broad-spectrum monoclonal antibody against thiram-like pesticides secreted by the Balb / c mouse hybridoma cell line 5G2 is IgG1, and the light chain subtype is Lamda.

[0038] The affinity of the obtained broad-spectrum monoclonal antibody against thiram-type pesticides was identified using ELISA. The detection antigen TMTD-BSA was diluted to 10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, and 0.625 μg / mL using coating buffer and coated onto enzyme-linked immunosorbent assay (ELISA) strips. The monoclonal antibody was then diluted 12 times from 1:1000 using 5% PBSM. 100 μL of each antibody dilution was added to an ELISA strip coated with the same concentration of antigen. After incubation for 1 hour, the strips were washed with PBST, and a 1:8000 dilution of secondary antibody was added. After incubation for another hour, color development was observed, and the assay was terminated. The absorbance at OD450 was measured using an ELISA reader, and the affinity constant K of the 5G2 antibody was calculated using Origin 8 software. aff =1.22×10 10 M -1 It belongs to the category of high-affinity antibodies.

[0039] Cross-reaction experiments were performed using icELISA. The purchased analogue standards were dissolved and diluted to 50 μg / mL with PBS. 200 μL of each diluted standard solution was added to a 1.5 mL EP tube, followed by serial dilution with PBS, for a total of 12 dilutions. After each dilution, 100 μL of liquid was aspirated from the last tube, leaving 100 μL of liquid in each EP tube. The antibody was then diluted with 5% PBSM to the next higher dilution factor corresponding to the absorbance of 1 point measured in the affinity experiment. After thorough dilution, 100 μL of antibody diluent was added to each EP tube and mixed thoroughly by pipetting. 5% PBSM was used as a negative control, and 100 μL of antibody diluent mixed with 100 μL PBS served as a positive control. All EP tubes were then incubated at 37°C for 1 hour. After incubation, all reaction solutions from the EP tubes were transferred to pre-coated enzyme-labeled strips using a pipette. The remaining steps of the ELISA method were then followed to complete the experiment, and the data were analyzed. Figure 2 As shown, the broad-spectrum monoclonal antibody against thiram-like pesticides secreted by the Balb / c mouse hybridoma cell line 5G2 showed cross-reactivity with thiram, arsine thiram, and ferric thiram, indicating that the antibody can simultaneously recognize the residues of the three thiram-like pesticides.

[0040] Example 3: Preparation of gold nanoflower immunoprobes 1. Preparation of colloidal gold Dissolve 1g of chloroauric acid in 100mL of deionized water to obtain a 1% chloroauric acid aqueous solution; add 1mL of the chloroauric acid aqueous solution to a clean flask containing 100mL of deionized water, mix well and heat. When it is about to boil, quickly add 1.5mL of 1wt% trisodium citrate solution and continue heating until the solution turns purple-red. Heat for another 3-5 minutes to allow the solution to stabilize fully and then let it cool to room temperature. Then dilute to 100mL with deionized water to obtain a colloidal gold solution (DLS (average particle size 26.8nm (PDI=0.386)), Zeta potential (-12.1±1.1mV)).

[0041] 2. Preparation of gold nanoflowers Add 750 μL of 1% chloroauric acid aqueous solution to 100 mL of deionized water and stir slowly. Then, add 500 μL of the colloidal gold solution prepared in the previous step and 300 μL of 1 wt% trisodium citrate solution and mix well. Adjust the pH with 1M sodium hydroxide solution to control the pH to 7.0–10.0 before and after the reaction. After waiting for 30 seconds, add 1 mL of 0.03M hydroquinone solution and increase the stirring speed until the solution color changes from clear pale yellow to clear dark blue. After it is completely stable, the gold nanoflower solution (DLS (average particle size 68.8 nm (PDI = 0.465)), Zeta potential (10.8 ± 0.6 mV)) is obtained and stored at 4 °C for later use.

[0042] 3. Preparation of gold nanoflower immunoprobe labeled with broad-spectrum monoclonal antibody against thiram-like pesticides: Take 10 mL of gold nanoflower solution, add 75 μL of 0.1 M potassium carbonate and 150 μL of 1 mg / mL Balb / c mouse hybridoma cell line 5G2 containing broad-spectrum monoclonal antibody against thiram-like pesticides in sequence under ice bath conditions, stir continuously for 45 min, add 2.5 mL of 5 wt% BSA aqueous solution, stir continuously for 30 min, and finally add 1.388 mL of... A 1 wt% PEG20000 aqueous solution was continuously stirred for 30 min to obtain a gold nanoflower immunoprobe solution. The solution was centrifuged at low speed for 15 min at room temperature, and the precipitate was discarded to remove a small portion of unstable probes. The solution was then centrifuged at 12000 r / min for 40 min at 4℃, and the supernatant was discarded. Each 1 mL of the precipitate was resuspended in 20 μL of resuspension buffer (0.01 M Tris-HCl + 1 wt% BSA + 10 wt% trehalose, pH = 8.5) to obtain a gold nanoflower immunoprobe labeled with a broad-spectrum monoclonal antibody against thiram-type pesticides. The solution was stored at 4℃ for later use.

[0043] Example 4: Preparation of gold nanoflower immunochromatographic test strips 1. Pretreatment of immunoprobe binding pads, sample pads, and absorbent pads Untreated immunoprobe binding pads (purchased from Shanghai Jieyi Biotechnology, model: GL0194) were cut into strips 1cm wide and placed in a large petri dish. They were then soaked in pre-prepared blocking solution and incubated at 37℃ for 2 hours. After removal from the incubator, the blocking solution was filtered out, and the pads were dried at 37℃ (checking every hour until completely dry). After drying, they were sealed at room temperature. For subsequent use, the pads were cut to 1cm in length and 4mm in width. The blocking solution consisted of 5wt% BSA + 1vol% Tween-20. Sample pads (purchased from Shanghai Jieyi Biotechnology, model: GL-b01) required no treatment and were cut to 1cm in length and 4mm in width. Absorbent pads (purchased from Shanghai Jieyi Biotechnology, model: H5076) required no treatment and were cut to 1.5cm in length and 4mm in width.

[0044] 2. Selection of the optimal concentration for line C of the gold nanoflower immunochromatographic test strip 1 mg / mL of goat anti-mouse secondary antibody (purchased from Beijing Bio-Sen Biotechnology Co., Ltd., product number: bs-0293G) was diluted with 0.01M PBS at pH 7.4 according to the final concentration of C line, resulting in five concentrations of 0.5, 0.7, 0.9, 1, and 1.5 mg / mL. 10 μL of each concentration was taken and streaked onto a nitrocellulose membrane. When streaking with a gold sputtering instrument, the streaking speed was 0.2 μL / cm, and each nitrocellulose membrane was streaked 3 times. After the nitrocellulose membrane was dried in a 37°C incubator for 10 minutes, it was cut into strips 4 mm wide. Next, 3 μL of gold nanoflower immunoprobe labeled with a broad-spectrum monoclonal antibody against thiram-type pesticides was added to each of the five probe binding pads. After drying, the immunochromatographic test strips were assembled (assembly method described in the invention description). 100 μL of 0.01M PBS (pH = 7.4) was slowly added to the sample pad, and the mixture was allowed to react at room temperature for 10 minutes. The color intensity of the C line on the test strip was observed. Finally, based on observation, 0.9 mg / mL was selected as the optimal concentration for the C line. Figure 4 ).

[0045] 3. Selection of the optimal concentration of the T line on gold nanoflower immunochromatographic test strips The complete antigen TMTD-BSA was diluted with 0.01M PBS (pH=7.4) according to the final concentration of the T line, resulting in five concentrations of 20, 30, 40, 50, and 90 μg / mL. 10 μL of each concentration was taken and streaked on a nitrocellulose membrane. When streaking with a gold sputtering instrument, the streaking speed was 0.2 μL / cm, and each nitrocellulose membrane was streaked 3 times. After the nitrocellulose membrane was dried in a 37°C incubator for 10 minutes, it was cut into strips 4 mm wide. Next, 3 μL of gold nanoflower immunoprobe labeled with a broad-spectrum monoclonal antibody against thiram-type pesticides was added to each of the five probe binding pads. After drying, the immunochromatographic test strips were assembled (assembly method described in the invention description). 100 μL of 0.01M PBS (pH = 7.4) was slowly added to the sample pad, and the mixture was allowed to react at room temperature for 10 minutes. The color intensity of the T line on the test strip was observed. Finally, based on observation, 50 μg / mL was selected as the optimal T line concentration. Figure 5 ).

[0046] 4. Determination of the optimal probe amount for gold nanoflower immunochromatographic test strips Based on the optimized C-line streaking concentration (0.9 mg / mL) and T-line streaking concentration (50 μg / mL) mentioned above, streaks were simultaneously applied to nitrocellulose membranes. After streaking, the nitrocellulose membranes were dried in a 37°C incubator for 10 min, then cut into strips 4 mm wide. Next, 1, 2, 3, and 4 μL of gold nanoflower immunoprobe labeled with a broad-spectrum monoclonal antibody against thiram-like pesticides were added to five groups of probe binding pads, respectively. After drying, the immunochromatographic test strips were assembled (assembly method described in the invention description). 100 μL of 0.01 M PBS (pH = 7.4) was slowly added to the sample pads, and the mixture was allowed to react at room temperature for 10 min. The color intensity of the C and T lines on the test strips was observed. Finally, based on observation, 2 μL was selected as the optimal amount of gold nanoflower immunoprobe for application. Figure 6 ).

[0047] Example 5: Performance Verification of Gold Nanoflower Immunochromatographic Test Strips 1. Specificity determination of gold nanoflower immunochromatographic test strips Based on the optimized C-line concentration (0.9 mg / mL) and T-line concentration (50 μg / mL) mentioned above, streaks were simultaneously applied to nitrocellulose membranes. The streaked nitrocellulose membranes were then dried in a 37°C incubator for 10 min before being cut into strips. Next, 2 μL of gold nanoflower immunoprobe labeled with a broad-spectrum monoclonal antibody against thiram-like pesticides was added to each probe binding pad. After drying, the immunochromatographic test strips were assembled (assembly method described in the invention description). Thiram-like pesticides, mancozeb, propineb, zineb, carbendazim, and chlorothalonil were diluted to a final concentration of 10 μg / mL using 0.01 M PBS (pH = 7.4), with 0.01 M PBS (pH = 7.4) used as a negative control. 100 μL of each was added to the sample pad of the test strip, and the strips were incubated at room temperature for 10 min. The disappearance of the T-line was observed to determine the specificity of the test strips. The results showed that the test strip only exhibited competitive reactions to thiram, arsenic, and ferric thiram, with the T line disappearing, while no significant cross-reactivity was observed with other analogues, indicating that the test strip had good specificity. Figures 7 to 10 ).

[0048] 2. Sensitivity determination of gold nanoflower immunochromatographic test strips Based on the optimized C-line streaking concentration (0.9 mg / mL) and T-line streaking concentration (50 μg / mL) mentioned above, streaks were simultaneously applied to nitrocellulose membranes. The streaked nitrocellulose membranes were then dried in a 37°C incubator for 10 min before being cut into strips. Next, 2 μL of gold nanoflower immunoprobe labeled with a broad-spectrum monoclonal antibody against thiram-like pesticides was added to each probe binding pad. After drying, the immunochromatographic test strips were assembled (assembly method described in the invention description). Thiram, thiram, and ferrous thiram were diluted with 0.01 M PBS (pH = 7.4) to final concentrations of 20 μg / mL, 10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, 0.3125 μg / mL, and 0.15625 μg / mL, respectively. 0.01 M PBS (pH = 7.4) was used as a negative control. Subsequently, 100 μL of each line was added to the sample pad of the test strip and allowed to react at room temperature for 10 min. The disappearance of the T line was observed, and the color intensity of the T and C lines was detected using an immunochromatographic quantitative analyzer. The T / C value was calculated to determine the detection range of the test strip. The results showed that when the TMTD concentration in the sample solution was 10 ng / mL, the T line disappeared significantly, indicating that the broad-spectrum monoclonal antibody against thiram-like pesticides in the gold nanoparticle immunoprobe was completely bound to thiram, arsenic, and ferric sulfate in the sample solution. With halving of the concentration, the T line gradually darkened. When the concentration of thiram, arsenic, and ferric sulfate in the sample solution was 0.156 μg / mL, the color intensity of the T line was still significantly lower than that of the C line, and the corresponding T / C value was also significantly lower than that of the negative control. Therefore, the detection limit of this immunochromatographic test strip for thiram, arsenic, and ferric sulfate was 0.156 μg / mL. Figures 8 to 10 ).

[0049] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A Balb / c mouse hybridoma cell line that secretes a broad-spectrum monoclonal antibody against thiram-type pesticides, characterized in that: The Balb / c mouse hybridoma cell line was deposited on June 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46545.

2. A broad-spectrum monoclonal antibody against thiram-type pesticides, characterized in that: The broad-spectrum monoclonal antibody against thiram-type pesticides is secreted by the Balb / c mouse hybridoma cell line described in claim 1.

3. The use of the Balb / c mouse hybridoma cell line of claim 1 or the broad-spectrum monoclonal antibody against thiram-like pesticides of claim 2 in the preparation of products for detecting thiram-like pesticides.

4. A gold nanoparticle immunochromatographic test strip for detecting thiram-type pesticides, characterized in that: The gold nanoflower immunochromatographic test strip includes a gold nanoflower immunoprobe, which is formed by electrostatic adsorption of gold nanoflower and a broad-spectrum monoclonal antibody against thiram-like pesticides; the broad-spectrum monoclonal antibody against thiram-like pesticides is secreted by the Balb / c mouse hybridoma cell line described in claim 1.

5. The gold nanoflower immunochromatographic test strip according to claim 4, characterized in that: The preparation method of the broad-spectrum monoclonal antibody against thiram-type pesticides includes the following steps: injecting Balb / c mouse hybridoma cell line into Balb / c mice that have been pre-sensitized with paraffin, collecting ascites fluid, and purifying it to obtain the broad-spectrum monoclonal antibody against thiram-type pesticides.

6. The method for preparing the gold nanoflower immunochromatographic test strip according to any one of claims 4 to 5, characterized in that: Includes the following steps: (1) Preparation of colloidal gold: Colloidal gold was prepared by citric acid reduction method; (2) Preparation of gold nanoflowers: Using the colloidal gold prepared in step (1) as gold seeds, gold nanoflowers were prepared by seed growth method; (3) Preparation of gold nanoflower immunoprobe: 75 μL of 0.1 M potassium carbonate and 150 μL of 1 mg / mL broad-spectrum monoclonal antibody against thiram-like pesticides were added to every 10 mL of gold nanoflowers prepared in step (2). After the system stabilized, BSA was added to block the unbound sites, and finally PEG20000 was added. After centrifugation and resuspending, gold nanoflower immunoprobes labeled with broad-spectrum monoclonal antibody against thiram-like pesticides were obtained. (4) Pretreatment of immunoprobe binding pads, sample pads and absorbent pads: Cut the untreated immunoprobe binding pads into strips 1 cm wide, place them in a large petri dish, soak them in the pre-prepared blocking solution, and incubate them in a 37°C constant temperature incubator for 2 hours. After removing them from the incubator, filter out the blocking solution and continue to dry them in the incubator at 37°C. Then seal them at room temperature. For subsequent use, cut them to a length of 1 cm and a width of 4 mm. The sample pads do not need to be treated. For subsequent use, cut them to a length of 1 cm and a width of 4 mm. The absorbent pads do not need to be treated. For subsequent use, cut them to a length of 1.5 cm and a width of 4 mm. (5) Streaking on nitrocellulose membrane: Use goat anti-mouse secondary antibody to streak C line on nitrocellulose membrane, and use complete antigen TMTD-BSA synthesized by active lipid method to streak T line on nitrocellulose membrane. The distance between C line and T line on the same nitrocellulose membrane is 0.6 cm. (6) Preparation of immune probe binding pad: The gold nanoflower immune probe labeled with the broad-spectrum monoclonal antibody against thiram-type pesticides prepared in step (3) was dropped onto the pretreated immune probe binding pad and dried at 37°C for later use. (7) Assembly of immunochromatographic test strips: The nitrocellulose membrane after scribing in step (5), the immunoprobe binding pad prepared in step (6), the sample pad pretreated in step (4) and the absorbent pad are assembled on the base plate; the immunoprobe binding pad and the sample pad are overlapped and pasted, with a 2mm gap between the same end, and the absorbent pad and the nitrocellulose membrane overlap by 2mm at both ends. The plastic outer shell is covered, dried and sealed, and stored at 4℃.

7. The preparation method according to claim 6, characterized in that: The composition of the sealing solution is: 5wt% BSA + 1vol% Tween-20.

8. The preparation method according to claim 6, characterized in that: The concentration indicated by line C is 0.9 mg / mL.

9. The preparation method according to claim 6, characterized in that: The concentration indicated by the T line is 50 μg / mL.

10. The preparation method according to claim 6, characterized in that: The amount of gold nanoflower immunoprobe labeled with a broad-spectrum monoclonal antibody against thiram-type pesticides added was 2 μL.

Images