A hybridoma cell line secreting antifungal monoclonal antibodies and its application

Abstract

This invention relates to a hybridoma cell line that secretes a monoclonal antibody against azoxystrobin and its application, belonging to the field of immunochemistry. The hybridoma cell line of this invention was deposited on November 12, 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. 46742, and classified as a monoclonal cell line. The monoclonal antibody secreted by the invented hybridoma cell line exhibits good detection sensitivity (IC50) against azoxystrobin. 50 With a value of 0.049 ng / mL, it can be used for the detection of antimicrobial residues.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a hybridoma cell line that secretes a bactericidal monoclonal antibody and its application. Background Technology

[0002] Dimethoprim, also known as trimethoprim, is an antimicrobial drug widely used in veterinary clinical practice, especially in poultry farming where it is commonly used as an anticoccidial and anti-Leukocytozoon gargarizans drug. Although dimethoprim plays an important role in controlling bacterial and parasitic infections, it may leave residues in chicken meat.

[0003] In actual farming practices, some farmers may overuse antibiotics to increase economic benefits. Antimicrobial agents are typically added at a concentration of 0.01% through drinking water or 0.02% mixed with feed. Without strict control of dosage and withdrawal periods, this method can easily lead to drug residues in chicken meat. Furthermore, antimicrobial agents should not be used alone; they are often used in combination with sulfonamides or other antibiotics at a 1:5 ratio. While this enhances the antibacterial effect, it may also increase the risk of residues. Antimicrobial residues in chicken meat not only affect food safety but may also lead to antibiotic resistance. The overuse of antimicrobial agents may cause bacteria to develop resistance, resulting in decreased effectiveness of antibiotic treatment. This not only affects animal health but also poses a potential threat to public health. In actual farming practices, farmers or farming enterprises may have irrational medication habits. For example, over-reliance on antibiotics for disease prevention rather than treatment, failure to follow drug instructions, and lack of relevant veterinary guidance all increase the risk of drug residues. In addition, some farmers may deliberately shorten the withdrawal period due to economic incentives, leading to excessive drug residues in chicken meat.

[0004] Dimethoate has a certain degree of fat solubility, which allows it to accumulate in adipose tissue. If chickens ingest dimethoate and their metabolism or excretion functions are impaired, this will increase the bioaccumulation of dimethoate in their bodies. This bioaccumulation not only results in drug residues in the product but may also affect the overall health of poultry, leading to further drug residues.

[0005] To reduce the negative impacts of dichlorvos residues on human health and the environment, many countries and regions have imposed strict regulations on its residue levels. Nevertheless, current methods for detecting dichlorvos residues still primarily rely on traditional instrumental analytical techniques, such as high-performance liquid chromatography (HPLC), fluorescence spectroscopy, and LC-MS / MS. While these instrumental methods offer high sensitivity and specificity, they also have some drawbacks, such as requiring complex sample pretreatment processes, expensive equipment, cumbersome operation, and highly specialized technical personnel.

[0006] To address these issues, there is an urgent need to develop rapid, simple, low-cost methods suitable for on-site detection. Enzyme-linked immunosorbent assay (ELISA), as a commonly used immunoassay method, has been widely applied in food safety, environmental monitoring, and other fields due to its ease of operation, rapid detection speed, and low cost. The key to detecting antimicrobial residues using ELISA lies in obtaining highly specific and sensitive monoclonal antibodies.

[0007] Monoclonal antibodies can specifically bind to the antiviral molecule, enabling its efficient and sensitive detection. The preparation of monoclonal antibodies against antiviral typically requires hybridoma technology. Although there have been some advancements in existing techniques, successfully preparing monoclonal antibodies with high specificity and sensitivity remains a challenge in this field. Key issues include how to effectively design and prepare the complete antigen of antiviral, how to induce a strong immune response in mice through immunogenicity, and how to ensure that the final antibody exhibits good specificity and high sensitivity during detection.

[0008] Therefore, developing a novel monoclonal antibody detection method, especially one capable of accurately and rapidly detecting dichlorvos residues, is of great practical significance. This would not only improve the efficiency and accuracy of dichlorvos residue detection but also provide more convenient and low-cost detection methods for fields such as veterinary drug monitoring, food safety, and drug research. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes a monoclonal antibody against azoxystrobin and its applications. The monoclonal antibody against azoxystrobin secreted by the hybridoma cell line of this invention exhibits good detection sensitivity (IC50) against azoxystrobin. 50 With a value of 0.049 ng / mL, it can be used to establish an immunological detection method for dichlorvos to detect dichlorvos residues in chicken meat.

[0010] The technical solution of the present invention is as follows:

[0011] The first objective of this invention is to provide a hybridoma cell line that secretes a monoclonal antibody against cytotoxic bacteria. This hybridoma cell line was deposited on November 12, 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. 46742.

[0012] The second objective of this invention is to provide a bactericidal monoclonal antibody secreted by the hybridoma cell line.

[0013] The preparation method of the above-mentioned antibacterial monoclonal antibody is as follows: BALB / c mice are injected intraperitoneally with paraffin oil, followed by intraperitoneal injection of hybridoma cell line. Ascites fluid is collected after injection, purified, and the obtained monoclonal antibody is stored at low temperature.

[0014] In one embodiment of the present invention, the antimicrobial monoclonal antibody is obtained by immunizing animals with antimicrobial complete antigen.

[0015] In one embodiment of the present invention, the antibacterial complete antigen is obtained by conjugating the antibacterial hapten with a carrier protein.

[0016] In one embodiment of the present invention, the structural formula of the antibacterial hapten is shown below:

[0017] .

[0018] In one embodiment of the present invention, the carrier protein includes keyhole hemocyanin and / or bovine serum albumin.

[0019] A third object of the present invention is to provide a composition comprising the hybridoma cell line described herein and / or the antibacterial monoclonal antibody described herein.

[0020] A fourth objective of this invention is to provide a kit comprising one or more of the hybridoma cell line, the antibacterial monoclonal antibody, and the composition described herein.

[0021] A fifth objective of this invention is to provide a test strip comprising one or more of the aforementioned hybridoma cell line, the aforementioned antibacterial monoclonal antibody, and the aforementioned composition.

[0022] The sixth objective of this invention is to provide the application of the hybridoma cell line, the antimicrobial monoclonal antibody, the composition, the kit, or the test strip in the detection of antimicrobial; the application does not involve the diagnosis or treatment of diseases.

[0023] The seventh objective of this invention is to provide a method for preparing the hybridoma cell line that secretes the antibacterial monoclonal antibody, comprising the following steps:

[0024] Step 1: Use the succinic anhydride method to derive antibacterial agent into an antibacterial hapten that is more likely to elicit a sensitive immune response;

[0025] Step 2: Prepare the obtained hapten into a complete hapten, and emulsify the obtained complete hapten with Freund's adjuvant or incomplete Freund's adjuvant to prepare an immunogen;

[0026] Step 3: The obtained immunogen was injected subcutaneously into BALB / c mice via the back for multiple immunizations. The first immunization used complete Freund's adjuvant, and the booster immunization used incomplete Freund's adjuvant.

[0027] Step 4: Collect blood from mice that have undergone the above immunization process, and detect the serum immune titer and immunosuppressive ability of mice by indirect ELISA to screen out mice with high sensitivity of antibacterial antibodies in their serum.

[0028] Step 5: The selected mice were immunized by intraperitoneal injection using the complete antigen of nitroglycerin without Freund's adjuvant.

[0029] Step 6: Fuse spleen cells and myeloma cells from BALB / c mice after sprint immunization. The fused cells are cultured in HAT medium and screened. Positive cell pores are detected by indirect ELISA. The inhibitory effect of positive cell pores is further determined by indirect competitive ELISA. The positive cell pores with the best inhibition are subcloned by limiting dilution method. Finally, hybridoma cell lines that can secrete highly sensitive antimicrobial monoclonal antibodies are screened out.

[0030] In step 1, the molecular formula of the antibacterial agent is as follows:

[0031] .

[0032] In step 2, the molecular formula of the antibacterial hapten is as follows:

[0033] .

[0034] In one embodiment of the present invention, in steps 3 and 5, there is a one-month interval between the first immunization and the booster immunization, a 21-day interval between booster immunizations, and an 18-21-day interval between booster immunizations and the final immunization.

[0035] In one embodiment of the present invention, in steps 3 and 5, the initial immunization dose is 100 μg / animal, the booster immunization dose is 50 μg / animal, and the final immunization dose is 25 μg / animal.

[0036] In one embodiment of the present invention, steps 3 and 5, the immunization process, includes one initial immunization, four booster immunizations and one sprint immunization.

[0037] In one embodiment of the present invention, in step 4, blood is collected on the 7th day after the end of the 3rd immunization process.

[0038] In one embodiment of the present invention, in step 6, cell fusion is performed 3 days after the end of the sprint immunization.

[0039] In one embodiment of the present invention, in step 6, cell fusion is performed by the polyethylene glycol (PEG4000) method.

[0040] In one embodiment of the present invention, in step 6, the culture medium is RPMI-1640 medium.

[0041] In one embodiment of the present invention, in step 6, the number of subcloning operations is 4.

[0042] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0043] This invention provides a hybridoma cell line that secretes a monoclonal antibody against azoxystrobin and its application. The monoclonal antibody against azoxystrobin obtained by this invention has good detection sensitivity (IC50) for azoxystrobin. 50 (Value: 0.049 ng / mL); the antibacterial monoclonal antibody cell line obtained in this invention can be used for immunoassay detection.

[0044] Preservation of biological materials:

[0045] A hybridoma cell line, YMSF, secreting a monoclonal antibody against cypermethrin, has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. It has been classified as a monoclonal cell line, deposited on November 12, 2025, with accession number CGMCC No. 46742. Attached Figure Description

[0046] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0047] Figure 1 This is the standard curve of the inhibition of antimicrobial monoclonal antibody against antimicrobial in this invention. Detailed Implementation

[0048] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0050] The culture media involved in the following examples are as follows:

[0051] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cysteine ​​dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5. L-Tyrosine 23.19, L-Valine 20, Para-aminobenzoic acid 1, Calcium nitrate 100, Anhydrous magnesium sulfate 48.84, Anhydrous sodium dihydrogen phosphate 676.13, Potassium chloride 400, Sodium chloride 6000, Glucose 2000, Reduced glutathione 1, Phenol red 5, L-Glutamine 300, Biotin 0.2, D-Calcium pantothenate 0.25, Folic acid 1, I-Inositol 35, Nicotinamide 1, Choline chloride 3, Pyridoxine hydrochloride 1, Riboflavin 0.2, Thiamine hydrochloride 1, Vitamin B12 0.005, Sodium bicarbonate 2000.

[0052] The reagents involved in the following examples are as follows:

[0053] Carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800 mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000 mL. Store at 4°C for later use.

[0054] Phosphate-buffered saline (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, dissolved in 800 mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000 mL.

[0055] PBST: PBS containing 0.05% Tween 20;

[0056] Antibody dilution buffer: PBS containing 0.1% gelatin;

[0057] TMB colorimetric solution: Solution A: Na2HPO4 . 12H₂O 18.43 g, citric acid 9.33 g, diluted to 1000 mL with pure water; Solution B: 60 mg TMB dissolved in 100 mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution, and mix again before use.

[0058] The detection methods involved in the following embodiments are as follows:

[0059] Method for detecting the inhibition rate of antimicrobial agents: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The antigen was diluted to 0.01, 0.03, 0.1, and 0.3 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03, 0.1, 0.3, and 1 μg / mL with antibody diluent. After selecting the optimal operating point, the antimicrobial standard was diluted to eight concentrations (0.005, 0.016, 0.049, 0.148, 0.444, 1.333, and 4 ng / mL). Following the ic-ELISA procedure, the results were plotted using OriginPro 8.5 (see results below). Figure 1 As shown), the double-antibacterial standard inhibition curve was obtained, and the IC50 was calculated. 50 .

[0060] Example 1: Preparation of the antibacterial hapten

[0061] Weigh 10 mg of chlorfenapyr small molecules and 50 mg of butylboronic acid, add 1 mL of pyridine, and dissolve by vigorous stirring at room temperature in the dark. Then add 4.2 mg of succinic anhydride and 5 mg of DAMP, stir vigorously at 60 °C for 6 h, and then add 100 μL of ultrapure water to terminate the reaction. Dry the product with nitrogen, and then extract it five times with chloroform. Take the lower layer (chloroform layer), dry it with nitrogen, and obtain the chlorfenapyr hapten.

[0062] The molecular formula of the small molecule of the antibacterial agent is as follows:

[0063] .

[0064] The molecular formula of the antibacterial hapten is as follows:

[0065] .

[0066] Example 2: Synthesis of complete antigen of antibacterial agent

[0067] Weigh 3.66 mg of nitrocellulose hapten and 2.3 mg of N-hydroxysuccinimide (NHS), dissolve them in 200 μL of N,N-dimethylformamide (DMF), and stir at room temperature for 10 min. Then weigh 4.27 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and add it to the nitrocellulose hapten solution. Stir at room temperature for 6-8 h to activate the hapten. Take 6 mg of keyhole hemocyanin (KLH), add it to 3 mL of 0.01 M carbonate buffer (CBS), and dissolve it completely. Slowly add the activated hapten to the diluted KLH solution and stir overnight at room temperature. Then dialyze with 0.01 M PBS to remove unreacted small molecules to obtain a relatively pure complete antigen, which is then identified by UV absorption scanning.

[0068] Example 3: Synthesis of the antibacterial coating agent

[0069] 2.51 mg of nitrofurazone hapten and 2.1 mg of N-hydroxysuccinimide (NHS) were dissolved in 200 μL of anhydrous N,N-dimethylformamide (DMF) and reacted with stirring at room temperature for 10 min. 3.44 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in the above solution and reacted with stirring at room temperature for 6–8 h to obtain the hapten activation solution. 6 mg of bovine serum albumin (BSA) was dissolved in carbonate buffer (CBS). The hapten activation solution was slowly added to the protein dilution solution and stirred overnight at room temperature. Then, the reaction solution was dialyzed against 0.01 M PBS to remove unreacted small molecules, yielding the coating antigen.

[0070] Example 4: Preparation of hybridoma cell lines secreting antibacterial monoclonal antibodies

[0071] 1. Acquisition of immunity in animals

[0072] After emulsifying a mixture of complete antifungal antigen and an equal volume of Freund's adjuvant, BALB / c mice were immunized by subcutaneous injection at multiple sites on the back of the neck (except for sprint immunization). The first immunization used complete Freund's adjuvant at a dose of 100 μg / mouse; subsequent booster immunizations used incomplete Freund's adjuvant at half the dose of 50 μg / mouse; sprint immunizations did not use adjuvant, but were directly diluted with physiological saline and injected intraperitoneally at a dose of half the dose of 25 μg / mouse. The interval between the first and second booster immunizations was one month, the interval between multiple booster immunizations was 21 days, and the interval between the sprint immunization and the last booster immunization was 18-21 days. The immunization effect in mice was observed by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), i.e., the titer and inhibition of mouse serum were detected.

[0073] 2. Cell fusion

[0074] Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 4000) method, with the following specific steps:

[0075] a. After euthanizing the mouse by tail dislocation and cervical dislocation, immediately disinfect the mouse in 75% alcohol for about 5 minutes. Under aseptic conditions, remove the spleen and grind it moderately with the rubber tip of a syringe and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect the suspension, centrifuge (1200 rpm, 8 min), wash the spleen cells three times with RPMI-1640 medium, and after the last centrifugation, dilute the spleen cells to a certain volume, count them, and set them aside for later use.

[0076] b. Collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells are expanded in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The number of SP2 / 0 tumor cells should reach 1-4 × 10⁻⁶ cells before fusion. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, tumor cells are collected and suspended in RPMI-1640 basal culture medium for cell counting during fusion.

[0077] c. Fusion process (7 min): At min 1, add 1 mL of PEG 4000 to the cells dropwise, gradually increasing the speed; at min 2, allow to stand; at min 3 and min 4, add 1 mL of RPMI-1640 medium dropwise over 1 min; at min 5 and min 6, add 2 mL of RPMI-1640 medium dropwise over 1 min; at min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s. Except for min 2, continuously agitate the solution. Then incubate at 37°C for 5 min; centrifuge (800 rpm, 8 min), discard the supernatant, resuspend in RPMI-1640 selection medium containing 20% ​​fetal bovine serum and 2% 50×HAT, add 200 μL / well to a 96-well cell plate, and incubate at 37°C in a 5% CO2 incubator.

[0078] 3. Cell selection and cell line establishment

[0079] On day 3 after cell fusion, the fused cells were partially replaced with RPMI-1640 selection medium. On day 5, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On day 7, the cell supernatant was collected for screening.

[0080] The screening process consists of two steps: First, positive cell wells are selected using the ic-ELISA method. Second, the inhibitory effect of the antibacterial agent on the positive cells is determined using the ic-ELISA method, with antibacterial agent as the standard.

[0081] Cell wells that showed good inhibition of the antibacterial standard were selected, and subcloning was performed using the limiting dilution method. The same method was used for detection seven days later.

[0082] At least three subclonings were performed using the method described above to finally obtain the antibacterial monoclonal antibody cell line.

[0083] Example 5: Preparation and Identification of Antimicrobial Monoclonal Antibody

[0084] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6Starting from day 7, ascites fluid was collected from the antibacterial hybridoma cells, and the ascites fluid was purified for antibody purification using the caprylic acid-saturated ammonium sulfate method.

[0085] Under slightly acidic conditions, octanoic acid can precipitate other proteins in the ascites fluid besides IgG immunoglobulin. After centrifugation, the precipitate is discarded. Then, an equal volume of saturated ammonium sulfate solution is used to precipitate IgG-type monoclonal antibodies. After centrifugation, the supernatant is discarded. The antibody is dissolved in 0.01M PBS solution (pH 7.4), dialyzed to desalt, and finally purified monoclonal antibodies are obtained and stored at -20 °C.

[0086] Using an indirect competitive ELISA, the monoclonal antibody against dichlorvos showed good detection sensitivity and specificity (IC50) for dichlorvos. 50 With a value of 0.049 ng / mL, it can be used for immunoassay of antimicrobial agents.

[0087] Cross-reactivity experiments were conducted on the structural analogues of dichlorvos (dichlorvos, trimethoprim, and omeprine). The results showed that the absorbance values ​​in each well of the ELISA plate corresponding to trimethoprim and omeprine did not differ significantly with the dilution concentration of the standard. The specific results are shown in Table 1.

[0088] This result indicates that the monoclonal antibody has a low cross-reactivity rate with other structural analogs and good specificity for antimicrobial agents.

[0089] Cross-reactivity rate (%) = (IC50 of antibacterial agent) 50 ) / (Similar IC 50 )×100%

[0090] Table 1 Cross-reactivity of antimicrobial monoclonal antibodies

[0091]

[0092] Example 6: Application of antibacterial monoclonal antibody

[0093] The monoclonal antibody prepared from hybridoma cell lines via in vivo ascites fluid was applied to the ELISA addition and recovery assay of antibacterial agent. The specific steps are as follows:

[0094] (1) Coat a 96-well microplate with 0.1 μg / mL of the coating stock diluted with carbonate buffer (CBS), 100 μL per well, dry at 37℃ for 2 h, wash the plate three times with PBST washing buffer, 200 μL per well each time, for 3 min each time, and pat dry.

[0095] (2) Block with CBS containing 0.2% gelatin, 200 μL per well, dry at 37℃ for 2 h, wash the plate three times with PBST washing solution, 200 μL per well each time, 3 min each time, and pat dry;

[0096] (3) Prepare standard solutions of 0.005 ng / mL, 0.016 ng / mL, 0.049 ng / mL, 0.148 ng / mL, 0.444 ng / mL, 1.333 ng / mL and 4 ng / mL of chlorhexidine using phosphate buffer (PBS). Add the standard solutions and the extracts of the samples to be tested to the sealed microplates, 50 μL per well, and repeat each sample in 3 wells. Then add 50 μL of chlorhexidine monoclonal antibody diluted to 0.1 μg / mL to each well. Incubate at 37°C for 30 min, then wash and dry the plate.

[0097] (4) Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 with PBS containing 0.1% gelatin to each well, react at 37℃ for 30 min, then wash the plate and pat dry.

[0098] (5) Add 100 μL of TMB colorimetric solution to each well, develop the color at 37℃ for 15 min, then add 50 μL of 2 M H2SO4 stop solution to each well, and measure the absorbance at 450 nm.

[0099] (6) Addition of recovery and sample pretreatment:

[0100] Chicken was selected as the test sample.

[0101] After the chicken meat was minced, it was placed in 10 mL of pure water, 40 mg of trypsin was added, and the mixture was enzymatically hydrolyzed at 40 °C for 3 h. Then, 20 mL of ethyl acetate was added and stirred for 2 min. 10 mL of the supernatant was taken and dried under nitrogen. The resulting dry product was resuspended in suspension buffer and used for subsequent ELISA experiments and test strips.

[0102] The recovery rate of antimicrobial agent was 98.7%–106.2% when spiking was performed using an indirect competitive ELISA test.

[0103] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A hybridoma cell line that secretes a monoclonal antibody against a fungicide, characterized in that, The hybridoma cell line was deposited on November 12, 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. 46742.

2. A bactericide monoclonal antibody, characterized in that, Produced by the hybridoma cell line of claim 1.

3. The antibacterial monoclonal antibody according to claim 1, characterized in that, The antimicrobial monoclonal antibody was obtained by immunizing animals with the antimicrobial complete antigen.

4. The antibacterial monoclonal antibody according to claim 3, characterized in that, The complete antifungal antigen is obtained by conjugating the antifungal hapten with a carrier protein.

5. The antibacterial monoclonal antibody according to claim 4, characterized in that, The structural formula of the antibacterial net hapten is shown below: 。 6. The antibacterial monoclonal antibody according to claim 4, characterized in that, The carrier proteins include keyhole hemocyanin and / or bovine serum albumin.

7. A composition, characterized in that, The composition comprises the hybridoma cell line of claim 1 and / or the antimicrobial monoclonal antibody of any one of claims 2-6.

8. A reagent kit, characterized in that, The kit comprises one or more of the hybridoma cell line of claim 1, the antimicrobial monoclonal antibody of any one of claims 2-6, and the composition of claim 7.

9. A test strip, characterized in that, The test strip comprises one or more of the hybridoma cell line of claim 1, the antimicrobial monoclonal antibody of any one of claims 2-6, and the composition of claim 7.

10. The use of the hybridoma cell line of claim 1, the antimicrobial monoclonal antibody of any one of claims 2-6, the composition of claim 7, the kit of claim 8, or the test strip of claim 9 in the detection of antimicrobial; the use does not relate to the diagnosis and treatment of disease.

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