Monoclonal cell stably secreting anti-benzhydrazide monoclonal antibody and its application

Abstract

This invention discloses a monoclonal cell line 1B1 that stably secretes anti-butyrylhydrazine monoclonal antibody and its applications. The monoclonal cell line 1B1 has the accession number CGMCC No. 46765 at the China General Microbiological Culture Collection Center. This monoclonal cell line 1B1 can stably secrete anti-butyrylhydrazine monoclonal antibody, the heavy chain isoform of which is IgG1, the light chain isoform of which is Kappa, and the affinity constant Kaff = 8.85 × 10⁻⁶. 9 L / mol; Based on this monoclonal antibody, this invention establishes an ELISA detection method for butyrylhydrazine. The limit of detection in the buffer system is 1.26 μg / mL, and the limit of detection in actual samples (such as broad beans, peanuts, etc.) is 1.35~2.52 μg / mL. The ELISA detection method provided by this invention has a short measurement time, is simple to operate, and is low in cost. It has excellent specificity and sensitivity, and can quickly and accurately detect butyrylhydrazine residues in actual samples. It is suitable for rapid batch screening and quantitative detection of butyrylhydrazine residues and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of rapid detection of pesticide residues and relates to a monoclonal cell that stably secretes anti-butyrylhydrazine monoclonal antibody and its application. Background Technology

[0002] Daminozide (CAS Registry No. 1596-84-5) is a commonly used plant growth regulator, widely applied in fruits, vegetables, and other crops to inhibit growth and improve quality. However, excessive use of daminozide can lead to its residues in the environment and agricultural products, potentially posing health risks, such as carcinogenicity, once it enters the human body through the food chain. Therefore, establishing a rapid and sensitive detection method for daminozide is crucial for ensuring food safety.

[0003] Currently, the detection of butyrylhydrazine mainly relies on instrumental methods such as gas chromatography and high-performance liquid chromatography. While these methods are accurate, they are time-consuming, costly, and require specialized operators. Immunologically based ELISA detection methods offer advantages such as speed, high throughput, and low cost; however, ELISA methods specifically for butyrylhydrazine have not yet been reported. This invention fills this technological gap by screening for high-affinity monoclonal antibodies. Summary of the Invention

[0004] In view of the above background, the present invention aims to provide a monoclonal cell that stably secretes anti-butyrylhydrazine monoclonal antibody and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A monoclonal cell line 1B1 that stably secretes anti-butyrylhydrazine monoclonal antibody was deposited on December 4, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46765.

[0007] An anti-butyrylhydrazine monoclonal antibody, said anti-butyrylhydrazine monoclonal antibody being secreted by the aforementioned monoclonal cell 1B1.

[0008] Furthermore, the preparation method of the above-mentioned anti-butyrylhydrazine monoclonal antibody includes the following steps: injecting monoclonal cells 1B1 into Balb / c mice that have been pre-sensitized with paraffin, collecting ascites fluid, and purifying it by centrifugation, filtration, Protein G affinity chromatography, dialysis, and PEG20000 concentration to obtain the anti-butyrylhydrazine monoclonal antibody.

[0009] The application of the aforementioned monoclonal cell 1B1 or anti-butyrylhydrazine monoclonal antibody in the detection of butyrylhydrazine.

[0010] The above-mentioned monoclonal cell 1B1 or anti-butyrylhydrazine monoclonal antibody is used in the preparation of products for detecting butyrylhydrazine.

[0011] An ELISA detection method for detecting butyrylhydrazine, wherein the method uses the above-mentioned anti-butyrylhydrazine monoclonal antibody as the detection antibody and detects butyrylhydrazine residues in the sample by enzyme-linked immunosorbent assay.

[0012] Furthermore, the above ELISA detection method includes the following steps:

[0013] (1) Coating: Dilute the coating antigen with 0.05M carbonate buffer, coat 100μL per well of the microplate, incubate at 37℃ for 2h or 4℃ overnight, discard the liquid in the well and wash with PBST washing solution.

[0014] (2) Blocking: Add 200 μL of 0.01 M PBS buffer containing 5 wt% BSA and 1 vol% Tween-20 to each well to block the microplate. Incubate at 37°C for 2 h or at 4°C overnight. Wash with PBST washing solution after blocking.

[0015] (3) Competitive reaction: Add 50 μL of the sample to be tested to each well, then immediately add 50 μL of anti-butyrylhydrazine monoclonal antibody working solution, mix well and incubate at 37°C for 50 min, then wash with PBST washing solution;

[0016] (4) Add enzyme-labeled secondary antibody: Add 100 μL of 1:8000 diluted goat anti-mouse IgG-HRP to each well, incubate at 37°C for 50 min, and wash with PBST washing solution;

[0017] (5) Color development: Add 100 μL of TMB color development solution to each well and incubate at 37°C in the dark for 15 min;

[0018] (6) Termination and detection: Add 50 μL of 2M H2SO4 stop solution to each well and measure the OD value at a wavelength of 450 nm;

[0019] The working concentration of the coating antigen is 5 μg / mL;

[0020] The working concentration of the anti-butyrylhydrazine monoclonal antibody is 0.5 μg / mL.

[0021] The above-mentioned ELISA detection method is applied to the detection of butyrylhydrazine residues in actual samples.

[0022] The beneficial effects of this invention are as follows:

[0023] This invention provides for the first time a stable monoclonal cell line 1B1 that secretes anti-butyrylhydrazine monoclonal antibody and establishes an ELISA detection method. The ELISA method has a short detection time, is simple to operate, and has low cost. The detection limit for butyrylhydrazine in the buffer system is 1.26 μg / mL, and the detection limit in actual samples (such as broad beans, peanuts, etc.) is 1.35~2.52 μg / mL. It also has high specificity and is suitable for screening large batches of samples. Attached Figure Description

[0024] Figure 1 Synthetic route of butyrylhydrazine hapten.

[0025] Figure 2 HPLC purity analysis chromatogram of butyrylhydrazine hapten.

[0026] Figure 3 Mass spectrum of butyrylhydrazine hapten.

[0027] Figure 4 Butyrylhydrazine hapten 1 H-NMR spectrum.

[0028] Figure 5 Affinity curves of anti-butyrylhydrazine monoclonal antibodies.

[0029] Figure 6 : Subtype of anti-butyrylhydrazine monoclonal antibody.

[0030] Figure 7 : Standard curve of ELISA method.

[0031] Figure 8 : Specific assay results of the ELISA method.

[0032] Figure 9 , Figure 10 , Figure 11 , Figure 12 ELISA actual sample standard curve. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but they do not constitute a limitation of the present invention.

[0034] Example 1: Preparation of mouse hybridoma cells 1B1

[0035] The specific preparation process of mouse hybridoma cells 1B1 is as follows:

[0036] I. Preparation of Butyrylhydrazine Hapten

[0037] 7-Aminoheptanoic acid (substrate 1), benzyl alcohol (2 eq.), and p-toluenesulfonic acid (1.1 eq.) were dissolved in toluene and refluxed at 110 °C for 24 hours under a nitrogen atmosphere. After cooling to room temperature, petroleum ether was added to precipitate the solid. The precipitate was filtered and collected to obtain intermediate 2, which did not require further purification. Intermediate 2 was dissolved in dichloromethane with butyrylhydrazine (1.1 eq.), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.1 eq.), 1-hydroxybenzotriazole (1.1 eq.), and N,N-diisopropylethylamine (2.5 eq.) and reacted at room temperature under nitrogen protection for 12 hours. The reaction solution was acidified with 0.5 M hydrochloric acid, extracted with ethyl acetate, and the organic phase was concentrated to obtain intermediate 3, which did not require further purification. Intermediate 3 was dissolved in methanol, and 10% (by weight) palladium on carbon was added as a catalyst. The reaction was hydrogenated at room temperature under a hydrogen atmosphere for 12 hours. The reaction solution was separated by silica gel column chromatography and eluted with dichloromethane / methanol (volume ratio 5:1) to obtain the target product, butyrylhydrazine hapten (product 4).

[0038] Figure 1 This is the synthetic route for butyrylhydrazine hapten. Figure 2 This is the HPLC purity analysis chromatogram of butyrylhydrazine hapten. Figure 3 This is the mass spectrum of the butyrylhydrazine hapten. Figure 4 butyrylhydrazine hapten 1 H-NMR spectrum.

[0039] II. Complete Antigen Synthesis

[0040] The complete antigen was synthesized using the carbodiimide method. The specific steps were as follows: First, 8 mg of butyrylhydrazine hapten, 30 mg of N-hydroxysuccinimide, and 30 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were dissolved in 2 mL of DMSO and reacted at 20 °C and 110 r / min for 12 h. Then, the above reaction system was added dropwise to 7 mL of 5.71 mg / mL BSA solution (0.01 M, pH 8.5 PBS buffer) and reacted at 25 °C and 110 r / min for 6 h. After the reaction was completed, the reaction system was transferred to a dialysis bag and dialyzed at 4 °C and 0.01 M PBS buffer (pH 7.4) for 3 days to finally obtain the complete antigen, denoted as butyrylhydrazine-BSA (Alar-BSA).

[0041] The preparation method of the complete antigen Alar-OVA is basically the same as that of Alar-BSA, except that bovine serum albumin (BSA) is replaced with chicken ovalbumin (OVA) in the preparation steps.

[0042] III. Animal Immunization

[0043] Alar-BSA was selected as the immunogen, diluted to an appropriate concentration with 0.01M PBS buffer (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 rearing environment for one week were given a primary immunization. Following the primary immunization, booster immunizations were administered every two weeks using the same immunogen and an emulsion of Freund's incomplete adjuvant. From the second immunization onwards, after each immunization, Alar-OVA was used as the coating antigen, and the antibody titer in the mouse tail vein serum and the serum's specific recognition ability of butyrylhydrazine were measured using ELISA. When the serum antibody titer reached 1:8000 and showed good specificity, the mice were given a pulse immunization, and their spleens were harvested after the pulse immunization for subsequent cell fusion experiments.

[0044] ELISA specific steps:

[0045] Add 100 μL of the coating antigen Alar-OVA diluted to the optimal concentration to each well of the ELISA plate and incubate at 37°C for 2 h or at 4°C for 12 h. After incubation, wash the ELISA plate and add 200 μL of blocking buffer (0.01 M PBS buffer (pH 7.4) containing 5 wt% BSA and 1 vol% Tween-20) to each well for blocking. Incubate at 37°C for 2 h or at 4°C for 12 h. After blocking, add diluted antibody or antiserum to each well for titer determination. If performing a specificity assay, add an appropriate amount of butyrylhydrazine standard or its structural analogue to each well first, then immediately add the antibody or antiserum dilution, mix well, and incubate at 37°C for 50 min. After incubation, wash the ELISA plate and add 100 μL of goat anti-mouse IgG-HRP diluted 1:8000 to each well. Incubate at 37°C for 50 min. After washing the ELISA plate again, add 100 μL of... Incubate with TMB chromogenic solution 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 a wavelength of 450 nm. 450nm ).

[0046] IV. Cell Fusion Methods

[0047] 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 can stably secrete high-quality antibodies was obtained.

[0048] (a) Required materials:

[0049] Alcohol swabs (5-6)

[0050] Dissection board (1 piece)

[0051] 5mL syringes (2)

[0052] 50mL centrifuge tubes (4)

[0053] 15mL centrifuge tubes (2)

[0054] Three scissors and three tweezers (1 set)

[0055] Cell culture dishes (3)

[0056] Filters (2)

[0057] Filter cartridge nozzles (1 box)

[0058] 1mL pipette tips (≥10)

[0059] Waste liquid tank.

[0060] (II) Preliminary Preparations

[0061] On the day of cell fusion, prepare the following reagents in advance and preheat them to 37°C: 1 bottle of DMEM medium (>250mL), 1 tube of DMEM medium (about 40mL), 1 bottle of 20% FBS-DMEM medium with added HAT (>200mL, referred to as HAT medium), 1mL of PEG1450; at the same time, prepare 40°C sterile water (>300mL), put it in a beaker and preheat it in a water bath.

[0062] (III) Specific Steps

[0063] (1) Remove mice that have undergone shock immunization from the mouse room, collect blood from their eyeballs and place them in a water bath preheated to 37°C. Then, euthanize the mice by dislocation of their necks (after the fusion experiment, centrifuge the collected blood from their eyeballs and store the obtained serum at -20°C for later use); after euthanizing the mice, disinfect them by immersing them in 75% alcohol and then transfer them into the intercellular space.

[0064] (2) Turn on the clean bench and light the alcohol lamp, tear open the outer packaging of the three scissors and three forceps, use sterile gauze to absorb as much alcohol as possible from the surface of the mouse fur, and fix the mouse on the dissection board.

[0065] (3) After sterilizing the three shears and three forceps by burning them with an alcohol lamp flame, use the first set of shears and forceps to cut open the mouse fur, use the second set of shears and forceps to open the peritoneum, use the third set of shears and forceps to remove the spleen, and remove the adipose tissue attached to the surface of the spleen.

[0066] (4) Take one cell culture dish, use a 5mL syringe to draw a small amount of DMEM culture medium and add it to the lid and bottom of the dish respectively. After cleaning the spleen in the lid, transfer it to the bottom of the dish.

[0067] (5) Take a 50mL centrifuge tube, place the filter screen at the mouth of the centrifuge tube, and moisten the filter screen with a little DMEM culture medium;

[0068] (6) Take a syringe, first puncture the spleen with the needle, then press the spleen fully with the bottom of the syringe handle to release the spleen cells into the culture medium; use a 1 mL pipette to draw up DMEM culture medium, and add it to the centrifuge tube through the filter screen. Repeat the process of rinsing the bottom of the dish with DMEM culture medium several times, and add the rinsing solution to the centrifuge tube through the filter screen. Finally, use DMEM culture medium to fill the liquid in the centrifuge tube to 40 mL.

[0069] (7) Centrifuge the spleen cell suspension at 1100 rpm at room temperature for 7 min (the conditions for all subsequent centrifugation steps are the same); at the same time, take out the SP2 / 0 cells from the incubator, add a small amount of DMEM medium to another 50 mL centrifuge tube in advance, use a pipette to blow the SP2 / 0 cells to detach them, transfer them into the centrifuge tube, and add DMEM medium to make up to 40 mL for later use;

[0070] (8) After centrifugation, remove the spleen cell centrifuge tube, discard the supernatant, resuspend the cell pellet in DMEM medium and add to 40 mL, then centrifuge the spleen cell suspension with the prepared SP2 / 0 cell suspension.

[0071] (9) After centrifugation, compare the cell pellet volume in the two tubes and discard the supernatant; take an appropriate amount of DMEM medium to resuspend the cells and count them. According to the counting results, take an appropriate amount of SP2 / 0 cell suspension at a ratio of 1 / 3 to 1 / 10 and add it to the spleen cell suspension. Add DMEM medium to 40 mL, mix the cell suspension thoroughly and then centrifuge.

[0072] (10) After centrifugation, place the preheated beaker, PEG1450 and single tube of DMEM culture medium into the clean bench, discard the supernatant of mixed cells, tap the bottom of the centrifuge tube to disperse the cell pellet, loosen the caps of the two centrifuge tubes, immerse the cell pellet at the bottom of the centrifuge tube into the 40°C sterile water 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.

[0073] (11) After adding PEG1450, place the centrifuge tube in a water bath and let it stand for 1 minute; then add the single tube of DMEM medium in stages, slowly at first 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, use DMEM medium to make up the liquid in the centrifuge tube to 40 mL and place it at 37℃ for 10 minutes.

[0074] (12) Centrifuge after standing; at the same time, prepare several cell culture plates, take out HAT medium and pour a small amount into the cell culture dish; after centrifugation, discard the supernatant, take an appropriate amount of HAT medium from the culture dish to resuspend the fused cell pellet, add the resuspended liquid to the whole bottle of HAT medium, let it stand and mix well in time, plate it, and place it in a carbon dioxide incubator at 37℃ and 5% CO2 concentration for culture.

[0075] V. Antibody Characterization

[0076] After one week of cell culture, positive cell wells with high antibody titers and good specificity were screened using ELISA. After multiple rounds of subclonal screening, a Balb / c mouse hybridoma cell line 1B1 was obtained, which can stably secrete high-affinity anti-butyrylhydrazine monoclonal antibody, i.e., monoclonal cell 1B1 that stably secretes anti-butyrylhydrazine monoclonal antibody.

[0077] The monoclonal cell 1B1 that stably secretes anti-butyrylhydrazine monoclonal antibody was deposited on December 4, 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. 46765.

[0078] Example 2: Preparation of anti-butyrylhydrazide monoclonal antibody

[0079] 1. Preparation of ascites

[0080] (1) Take a 1mL sterile syringe and inject 500μL of paraffin into the peritoneal cavity of Balb / c mice at about 12 weeks of age to sensitize them. When injecting, insert the syringe vertically into the posterior side of the mouse's peritoneal cavity. Disinfect the injection site with alcohol swabs before and after the injection. Avoid bleeding during the operation and ensure a sterile environment.

[0081] (2) One week after sensitization, Balb / c mouse hybridoma cell line 1B1, which is in the logarithmic growth phase and has a coverage of about 80% of the culture dish, was selected and the culture supernatant was discarded. 1 mL of fresh 1640 medium was pipetted to wash the cells from the bottom of the culture dish and the mixture was blown evenly to form a cell suspension. Then, 1 mL of the cell suspension was drawn from one well of a 6-well plate using a sterile syringe and injected vertically into the peritoneal cavity of the sensitized mouse.

[0082] (3) About one week after cell injection, observe the condition of the mice. Under normal circumstances, the mice will be obviously distended in the abdomen, and the mice will be listless and have messy fur. At this time, it is necessary to start preparing to collect ascites.

[0083] (4) Prepare sterile injection needles, 1.5mL EP tubes, alcohol swabs, etc. in advance. During the operation, hold the mouse with one hand and wipe the peritoneal fluid collection site of the mouse with an alcohol swab with the other hand; then carefully insert the sterile injection needle into one side of the peritoneal cavity, and place the needle end against the EP tube to collect the ascites fluid. During the collection process, the ascites fluid may appear light red, which may be due to mild hemolysis caused by excessive ascites production. This does not affect the activity and properties of the antibodies in the ascites fluid. The mouse should be fixed when collecting ascites fluid to prevent it from struggling and causing the ascites fluid to be lost. Each mouse can collect about 1.5mL of ascites fluid at a time. After collection, put the mouse back in the cage. Ascites fluid can be collected again every other day until the mouse dies naturally.

[0084] (5) After the collected mouse ascites fluid was balanced, it was placed in a centrifuge at 4℃ and centrifuged at 12000 rpm for 20 min. After centrifugation, the centrifuge tube was removed, and it could be observed that the liquid in the centrifuge tube was divided into three layers: the uppermost layer was the fat layer, the middle layer was the target ascites fluid layer, and the lower layer was the sediment layer. The middle layer of ascites fluid was carefully aspirated with a pipette and transferred to a new 1.5 mL EP tube. The tube was labeled and stored in a -20℃ freezer for later use.

[0085] 2. Ascites purification

[0086] The preserved ascites fluid was diluted 1:10 with equilibration buffer (Na₂HPO₄ 3.5814 g / L, NaCl 4.383 g / L; pH=7.0). After removing impurities, the diluted ascites fluid was filtered through a 0.45 μm filter and then loaded onto a Protein G affinity chromatography column. Purification was performed according to the instructions for commercial Protein G affinity chromatography columns. The purified antibody was placed in a dialysis bag and dialyzed sequentially with PBS buffer (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 and then with ultrapure water for 1 day. After dialysis, the antibody was concentrated using PEG20000 to obtain the anti-butyrylhydrazine monoclonal antibody, which was stored at -80°C for later use.

[0087] like Figure 5 and Figure 6 As shown, the anti-butyrylhydrazine monoclonal antibody secreted by Balb / c mouse hybridoma cell line 1B1 has a heavy chain isoform of IgG1 and a light chain isoform of Kappa, with an affinity constant of Kaff = 8.85 × 10⁻⁶. 9 L / mol, which is a high-affinity antibody.

[0088] Example 3: Establishment of ELISA detection method

[0089] 1. Reagent pre-preparation

[0090] Coating buffer: 0.05M carbonate buffer (pH 9.6)

[0091] Washing buffer (PBST): 0.01M PBS buffer (pH 7.4) containing 0.05 vol% Tween-20.

[0092] Blocking solution: 0.01M PBS buffer (pH 7.4) containing 5wt% BSA and 1vol% Tween-20.

[0093] Coating original working solution: Dilute Alar-BSA to 5 μg / mL with coating buffer.

[0094] Antibody working solution: Dilute the purified anti-butyrylhydrazide monoclonal antibody to 0.5 μg / mL with 0.01M PBS (pH 7.4).

[0095] Butyrylhydrazine standard gradient: diluted to different concentrations with 0.01M PBS (pH 7.4).

[0096] Enzyme-labeled secondary antibody: Goat anti-mouse IgG-HRP, diluted 1:8000 with 0.01M PBS (pH 7.4).

[0097] Developing solution: TMB developing solution; Stop solution: 2M H2SO4 solution

[0098] 2. Operating Procedures

[0099] (1) Antigen coating

[0100] Take the diluted 5 μg / mL Alar-BSA coating agent, add 100 μL to each well of the microplate, and incubate at 37°C for 2 hours or at 4°C overnight.

[0101] (2) Board washing

[0102] Discard any remaining liquid in the wells, add 250-300 μL of PBST washing buffer to each well, let stand for 3 minutes, then discard the liquid completely. Invert the microplate onto clean absorbent paper and pat dry repeatedly. Repeat the washing process 3-5 times.

[0103] (3) Sealing treatment

[0104] Add 200 μL of blocking solution to each well to completely cover the bottom of the well, and incubate at 37°C for 2 hours or at 4°C overnight. After sealing, strictly follow the washing procedure in (2) and pat dry for later use.

[0105] (4) Competitive response

[0106] A stepwise sample addition and equal volume mixing method was adopted: first, 50 μL of butyrylhydrazine standard solution of different concentrations was added to each well, and then 50 μL of antibody working solution was added immediately to ensure that the total volume of each well was 100 μL; the ELISA plate was gently shaken to mix the liquid and incubated at 37℃ for 50 min to complete the competitive binding of free antigen and coated antigen.

[0107] (5) Second washing

[0108] Follow the washing procedure in (2) again to thoroughly wash away any unbound free antibodies and standards, and pat the ELISA plate dry.

[0109] (6) Add enzyme-labeled secondary antibody

[0110] Add 100 μL of diluted goat anti-mouse IgG-HRP secondary antibody to each well and incubate at 37°C for 50 min to ensure that the secondary antibody binds fully and specifically to the solid-phase antibody.

[0111] (7) Three washes

[0112] Repeat the washing procedure in (2) to wash away unbound free secondary antibody and avoid nonspecific color development.

[0113] (8) Color development in the dark

[0114] Add 100 μL of TMB colorimetric solution to each well and incubate at 37°C in the dark for 15 minutes. Stop incubation immediately after a clear blue color appears in the blank well.

[0115] (9) Reaction terminated

[0116] Add 50 μL of 2M H2SO4 stop solution to each well quickly, gently shake to mix, and the liquid in the well will change from blue to yellow, thus terminating the colorimetric reaction.

[0117] 3. Measurement and Result Calculation

[0118] After termination, place the ELISA plate into the microplate reader and measure the optical density (OD) of each well at a wavelength of 450 nm. 450nm Data processing was performed using the indirect competitive ELISA indicator B / B0: where B0 is the OD of the butyrylhydrazine blank control well. 450nm Value, B is the OD value of each concentration standard well and the sample well. 450nm The ratio B / B0 was calculated as the relative absorbance. A standard curve was plotted with the concentration of butyrylhydrazine standard on the x-axis and the corresponding B / B0 value on the y-axis. After fitting the regression equation, the sample could be quantitatively analyzed. Statistical calculations showed that the detection limit of this method was 1.26 μg / mL. Figure 7 ).

[0119] Example 4: Performance Validation of the ELISA Method

[0120] 1. Specificity assay: Butyrylhydrazine structural analogs were selected for cross-reactivity experiments. The specific ELISA procedure was as described in Example 1, where the concentration of the coating antigen Alar-BSA was 5 μg / mL and the working concentration of the anti-butyrylhydrazine monoclonal antibody was 0.5 μg / mL. For specificity assays, appropriate amounts of butyrylhydrazine standard (Alar) or its structural analogs (oxalic acid dihydrazine, carbamate dihydrazine, adipic acid dihydrazine, succinic acid, isoniazid) were first incubated with the antibody working solution at 37°C for 1 hour before being added to the ELISA plate. The detection results showed ( Figure 8 The established method only exhibits a specific reaction with butyrylhydrazine and shows no significant cross-reaction with other analogs, indicating that the method has good specificity.

[0121] 2. Sensitivity determination: See Example 3.

[0122] 3. Actual Sample Testing: Broad beans, peanuts, apples, and grapes were selected as actual test samples. After homogenization, 5g of each sample was accurately weighed and placed in a clean centrifuge tube. 20mL of 30vol% ethanol solution was added to the tube, and the mixture was vortexed to ensure thorough dispersion. The mixture was centrifuged at 1100r / min for 10min. After centrifugation, the clear supernatant was collected and filtered through a 0.22μm filter membrane to remove impurities. The collected filtrate was the sample extract to be tested. The above sample extract was used for ELISA testing, following the specific operating steps described in Example 3 above. During the testing process, the sample extract to be tested and serially diluted butyrylhydrazine standard were simultaneously added to the corresponding wells of the ELISA plate, with blank control wells also included. The plate was then placed in an ELISA reader, and the optical density (OD) of each well was measured at a wavelength of 450nm. 450nm Data processing was performed using the indirect competitive ELISA indicator B / B0: where B0 is the OD of the butyrylhydrazine blank control well. 450nm Value, B is the OD value of each concentration standard well and the sample well. 450nm The ratio B / B0 was calculated as the relative absorbance. A standard curve was plotted with the concentration of butyrylhydrazine standard on the x-axis and the corresponding B / B0 value on the y-axis. After fitting the regression equation, the sample could be quantitatively analyzed. Statistical calculations showed that the limits of detection for butyrylhydrazine in broad bean, peanut, apple, and grape matrices were 2.52 μg / mL, 2.27 μg / mL, 1.35 μg / mL, and 1.78 μg / mL, respectively. Figures 9 to 12 ).

[0123] 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 monoclonal cell line 1B1 that stably secretes anti-butyrylhydrazine monoclonal antibody, characterized in that: The monoclonal cell 1B1 was deposited on December 4, 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. 46765.

2. A monoclonal antibody against butyrylhydrazine, characterized in that: The anti-butyrylhydrazine monoclonal antibody is secreted by monoclonal cell 1B1 as described in claim 1.

3. The anti-butyrylhydrazine monoclonal antibody according to claim 2, characterized in that: The method for preparing the anti-butyrylhydrazine monoclonal antibody includes the following steps: injecting monoclonal cells 1B1 into Balb / c mice that have been presensitized with paraffin, collecting ascites fluid, and purifying it by centrifugation, filtration, Protein G affinity chromatography, dialysis, and PEG20000 concentration to obtain the anti-butyrylhydrazine monoclonal antibody.

4. The use of the monoclonal cell 1B1 of claim 1 or the anti-butyrylhydrazine monoclonal antibody of claim 2 in the detection of butyrylhydrazine.

5. The use of the monoclonal cell 1B1 of claim 1 or the anti-butyrylhydrazine monoclonal antibody of claim 2 in the preparation of products for detecting butyrylhydrazine.

6. An ELISA detection method for detecting butyrylhydrazine, characterized in that: The method uses the anti-butyrylhydrazine monoclonal antibody as described in claim 2 as the detection antibody, and detects butyrylhydrazine residues in the sample by enzyme-linked immunosorbent assay.

7. The ELISA detection method according to claim 6, characterized in that: Includes the following steps: (1) Coating: Dilute the coating antigen with 0.05M carbonate buffer, coat 100μL per well of the microplate, incubate at 37℃ for 2h or 4℃ overnight, discard the liquid in the well and wash with PBST washing solution. (2) Blocking: Add 200 μL of 0.01 M PBS buffer containing 5 wt% BSA and 1 vol% Tween-20 to each well to block the microplate. Incubate at 37°C for 2 h or at 4°C overnight. Wash with PBST washing solution after blocking. (3) Competitive reaction: Add 50 μL of the sample to be tested to each well, then immediately add 50 μL of anti-butyrylhydrazine monoclonal antibody working solution, mix well and incubate at 37°C for 50 min, then wash with PBST washing solution; (4) Add enzyme-labeled secondary antibody: Add 100 μL of 1:8000 diluted goat anti-mouse IgG-HRP to each well, incubate at 37°C for 50 min, and wash with PBST washing solution; (5) Color development: Add 100 μL of TMB color development solution to each well and incubate at 37°C in the dark for 15 min; (6) Termination and detection: Add 50 μL of 2M H2SO4 stop solution to each well and measure the OD value at a wavelength of 450 nm.

8. The ELISA detection method according to claim 7, characterized in that: The working concentration of the coating antigen is 5 μg / mL.

9. The ELISA detection method according to claim 7, characterized in that: The working concentration of the anti-butyrylhydrazine monoclonal antibody is 0.5 μg / mL.

10. The application of the ELISA detection method according to claim 6 in the detection of butyrylhydrazine residues in actual samples.

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