Hybridoma cell strain secreting azamiphenanthridine monoclonal antibody and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain capable of secreting an azamiphenanthridine monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of safe immunodetection. The hybridoma cell strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on November 12, 2025, the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.46743. The azaphenanthridine monoclonal antibody disclosed by the invention is secreted by the hybridoma cell strain, and the monoclonal antibody secreted by the hybridoma cell strain has better detection sensitivity (IC50 value is 0.529 ng / mL) to azaphenanthridine, and can be used for residue detection of azaphenanthridine.

Description

Technical Field

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

[0002] Isometamidium (ISM) is a long-acting anti-trypsin drug composed of a group of complex isomers. It works by inhibiting trypanosome DNA and RNA polymerases, thus hindering nucleic acid synthesis. It is widely used as a preventative and therapeutic chemical drug for the prevention and treatment of parasitic diseases in animals such as cattle and sheep, and can also be used for the prevention and treatment of sleeping sickness in animals. However, while isometamidium is effective in preventing and treating parasitic diseases in cattle and sheep, it also has side effects such as carcinogenicity, teratogenicity, and mutagenicity. The misuse of this type of drug not only affects the animals themselves, but its residues in animal-derived foods also pose a potential threat to human health. To mitigate the negative impacts of isometamidium on human health, many countries have established maximum residue limits (MRLs) for it in animal-derived products. In China, the MRL for isometamidium is set at 0.1 mg / kg. Therefore, detecting the residue levels of isometamidium in animal-derived agricultural products is crucial for protecting human health.

[0003] Currently, the detection methods for phenanthrene dichloride are mainly instrumental, with commonly used methods including high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), high-performance liquid chromatography-mass spectrometry (HPLC-MS), and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). Although these chromatographic methods have high sensitivity and specificity, they also have some drawbacks, such as the need for thorough sample purification, high solvent consumption, expensive equipment, and high skill requirements for operators. Therefore, there is an urgent need to establish a rapid and simple method for the detection of phenanthrene dichloride residues.

[0004] Enzyme-linked immunosorbent assay (ELISA) is a highly efficient, sensitive, and rapid detection method. It requires simple sample pretreatment, involves few purification steps, has high analytical throughput, low detection cost, and is easy to operate, making it suitable for rapid on-site detection of large numbers of samples. Therefore, it has been widely used in the field of drug residue analysis. However, a prerequisite for using ELISA to detect amphetrimidine is obtaining a monoclonal antibody with high specificity and sensitivity to amphetrimidine. Therefore, establishing a method for preparing a highly specific and sensitive monoclonal antibody against amphetrimidine is crucial. The inventors attempted to prepare amphetrimidine monoclonal antibodies using hybridoma cell technology. However, several key issues remain to be addressed in the preparation of hybridoma cell lines capable of secreting amphetrimidine monoclonal antibodies: the preparation methods for amphetrimidine haptens and complete antigens; how to induce a strong immune response in mice; how to ensure that the prepared hybridoma cell lines can stably secrete amphetrimidine monoclonal antibodies; and how to improve the specificity and sensitivity of the secreted monoclonal antibodies. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes a monoclonal antibody against phenanthridine and its applications. The monoclonal antibody against phenanthridine secreted by this hybridoma cell line exhibits good detection sensitivity (IC50) for phenanthridine. 50 The values ​​were 0.529 ng / mL, which can be used to establish an immunological detection method for phenanthridine to detect phenanthridine residues in food.

[0006] This invention is achieved through the following technical solution:

[0007] The first objective of this invention is to provide a hybridoma cell line that secretes a monoclonal antibody of phenanthridine, which 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. 46743.

[0008] A second objective of this invention is to provide a phenanthridine monoclonal antibody secreted by the hybridoma cell line.

[0009] In one embodiment of the present invention, the azirmetidine monoclonal antibody is obtained by immunizing animals with the azirmetidine complete antigen.

[0010] Specifically, BALB / c mice were injected intraperitoneally with paraffin oil, followed by an intraperitoneal injection of hybridoma cell lines. Ascites fluid was collected after the injection, purified, and the obtained monoclonal antibody was stored at low temperature.

[0011] In one embodiment of the present invention, the azirmetidine complete antigen is obtained by conjugating the azirmetidine hapten with a carrier protein.

[0012] In one embodiment of the present invention, the structural formula of the phenanthridine hapten is shown below:

[0013] .

[0014] In one embodiment of the present invention, the carrier protein includes bovine serum albumin and / or chicken oocyte albumin.

[0015] A third objective of this invention is to provide a composition comprising the hybridoma cell line and / or the azirmonidine monoclonal antibody.

[0016] A fourth objective of this invention is to provide a kit comprising one or more of the hybridoma cell line, the azirmonidine monoclonal antibody, and the composition described herein.

[0017] A fifth objective of this invention is to provide a test strip comprising one or more of the aforementioned hybridoma cell line, the aforementioned ammonia-phenanthridine monoclonal antibody, and the aforementioned composition.

[0018] The sixth objective of this invention is to provide the application of the hybridoma cell line, the azirmonidine monoclonal antibody, the composition, the kit, or the test strip in the detection of azirmonidine; the application does not involve the diagnosis or treatment of diseases.

[0019] The seventh objective of this invention is to provide a method for preparing a hybridoma cell line that secretes a phenanthridine monoclonal antibody, comprising the following steps:

[0020] Step 1: Prepare the complete azirmetidine antigen by emulsifying the obtained complete azirmetidine antigen with Freund's adjuvant or incomplete Freund's adjuvant to prepare an immunogen;

[0021] Step 2: 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.

[0022] Step 3: Prepare the azirmonidine-coated antigen, collect blood from mice that have undergone the above immunization process, and detect the serum immunotiter and immunosuppressive ability of mice by indirect ELISA to screen out mice with high sensitivity of azirmonidine antibody in serum.

[0023] Step 4: The selected mice were immunized by intraperitoneal injection using the complete antigen of azirmonidine without Freund's adjuvant.

[0024] Step 5: 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 amphetrimidine monoclonal antibodies are screened out.

[0025] The molecular formula of the phenanthridine complete antigen in step 1 is as follows:

[0026] .

[0027] In one embodiment of the present invention, the interval between the first immunization and the booster immunization in steps 2 and 4 is one month, the interval between booster immunizations is 21 days, and the interval between booster immunization and the sprint immunization is 18 to 21 days.

[0028] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0029] This invention provides a hybridoma cell line that secretes a monoclonal antibody against aziramine and its applications. The monoclonal antibody against aziramine obtained by this invention has good detection sensitivity (IC50) for aziramine. 50 The value was 0.529 ng / mL); the phenanthridine monoclonal antibody cell line obtained in this invention can be used for immunoassay detection.

[0030] Preservation of biological materials:

[0031] A hybridoma cell line, YSFCM, that secretes a monoclonal antibody against phenanthridine 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. 46743. Attached Figure Description

[0032] 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.

[0033] Figure 1 This is a standard curve showing the inhibition of phenanthridine by the monoclonal antibody against phenanthridine in this invention. Detailed Implementation

[0034] 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.

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

[0036] (1) The culture media involved in the following examples are as follows:

[0037] 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.

[0038] (2) The reagents involved in the following examples are as follows:

[0039] 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.

[0040] 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.

[0041] PBST: PBS containing 0.05% Tween 20;

[0042] Antibody dilution buffer: PBS containing 0.1% gelatin;

[0043] 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.

[0044] (3) The detection methods involved in the following embodiments are as follows:

[0045] Method for detecting aziramine inhibition rate: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The antigen was diluted to 0.01 μg / mL, 0.03 μg / mL, 0.1 μg / mL, and 0.3 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03 μg / mL, 0.1 μg / mL, 0.3 μg / mL, and 1 μg / mL with antibody dilution buffer. After selecting the optimal operating point, aziramine standards were diluted to eight concentrations (0, 0.027 ng / mL, 0.082 ng / mL, 0.247 ng / mL, 0.741 ng / mL, 2.22 ng / mL, 6.67 ng / mL, and 20 ng / mL). Following the ic-ELISA procedure, the results were plotted using OriginPro 8.5 (see results below). Figure 1 (As shown), the standard inhibition curve of nitrophenanthridine was obtained, and the IC50 was calculated. 50 .

[0046] Example 1: Synthesis of the complete antigen of azirmonidine

[0047] Weigh 1.50 mg of phenanthridine (CAS: 20438-03-3) hapten and dissolve it in a reaction flask containing 400 μL of DMF. After the phenanthridine hapten dissolves, add 16 μL of 1 M HCl solution dropwise to the reaction flask to adjust the pH to between 2 and 3. React at 0℃ for 20 min, then add 18 μL of freshly prepared NaNO2 solution dropwise to the reaction flask. React at 0℃ for 3 h to obtain the activated solution. The molar ratio of the reactants is phenanthridine hapten:HCl:NaNO2 = 1:3:15. Weigh 6 mg of BSA into another reaction flask and dissolve it in 2 mL of CBS solution. At 37°C, the activation solution was slowly added dropwise to the BSA CBS solution for coupling reaction. During the addition of the activation solution, 2 M NaOH solution was added dropwise to the reaction flask to adjust the pH of the solution to between 8 and 9 until the activation solution was completely added. At this point, the color of the solution in the reaction flask should change from colorless to orange or orange-yellow. After coupling for 12 h, the reaction solution was obtained, in which the molar ratio of BSA to phenanthridine was BSA:phenanthridine = 60:1. At 37°C, the reaction solution was dialyzed in potassium-free PBS solution to remove small molecules that could not be coupled to BSA. After 72 h of dialyzing, the immunogen free of uncoupled small molecules was obtained.

[0048] Example 2: Synthesis of the N-aminophenanthrene-coated antigen

[0049] Weigh 3.71 mg of phenanthridine hapten and dissolve it in a reaction flask containing 400 μL of DMF. After the phenanthridine hapten dissolves, add 40 μL of 1 M HCl solution dropwise to the reaction flask to adjust the pH to between 2 and 3. React at 0°C for 20 min. Then, add 46 μL of freshly prepared NaNO2 solution dropwise to the reaction flask and react at 0°C for 3 h to obtain the activated solution. The molar ratio of the reactants is phenanthridine hapten:HCl:NaNO2 = 1:3:15. Weigh 10 mg of OVA into another reaction flask and dissolve it in 2 mL of CBS solution. At 37°C, the activation solution was slowly added dropwise to the OVA CBS solution for coupling reaction. During the addition of the activation solution, 2 M NaOH solution was added dropwise to the reaction flask to adjust the pH of the solution to between 8 and 9 until the activation solution was completely added. At this point, the color of the solution in the reaction flask should change from colorless to orange or orange-yellow. After coupling for 12 h, the reaction solution was obtained, in which the molar ratio of OVA to phenanthridine was OVA:phenanthridine = 60:1. At 37°C, the reaction solution was dialyzed in potassium-free PBS solution to remove small molecules that could not be coupled to OVA. After dialysis for 72 h, the coated antigen free of uncoupled small molecules was obtained.

[0050] Example 3: Preparation of hybridoma cell lines secreting phenanthridine monoclonal antibodies

[0051] 1. Acquisition of immunity in animals

[0052] The complete antigen of azirfenidone was emulsified with an equal amount of Freund's adjuvant and then administered to BALB / c mice via 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. For multiple booster immunizations, incomplete Freund's adjuvant was used at a dose halved to 50 μg / mouse. For sprint immunization, no adjuvant was used; the adjuvant was diluted directly with physiological saline and injected intraperitoneally at a dose halved to 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 sprint immunizations 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.

[0053] 2. Cell fusion

[0054] 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:

[0055] 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.

[0056] 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.

[0057] 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, and 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.

[0058] 3. Cell selection and cell line establishment

[0059] 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.

[0060] The screening process consists of two steps: First, positive cell wells are selected using ic-ELISA. Second, azirmonidine is used as a standard, and the inhibitory effect on positive cells is determined using ic-ELISA.

[0061] Cell wells that showed good inhibition of the phenanthridine standard were selected, and subcloning was performed using the limiting dilution method. The cells were then tested using the same method after seven days.

[0062] At least three subclonings were performed using the method described above to finally obtain the azirmonidine monoclonal antibody cell line.

[0063] Example 4: Preparation and Identification of Azaminephenanthridine Monoclonal Antibody

[0064] 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 6 Ascites fluid was collected from the phenanthrene hybridoma cells starting on day 7, and the ascites fluid was purified for antibody using the octanoic acid-saturated ammonium sulfate method.

[0065] 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.

[0066] Using an indirect competitive ELISA, the monoclonal antibody against azaminephenanthridine showed good detection sensitivity (IC50) for azaminephenanthridine. 50 The values ​​were 0.529 ng / mL, which can be used for the immunoassay of phenanthridine.

[0067] Cross-reactivity experiments were conducted on phenanthridine analogues (bromophenanthridine, quinpyrimidine, and trypanosamine). The results showed that the absorbance values ​​of phenanthridine in each well of the ELISA plate did not differ significantly with the dilution concentration of the standard. The specific results are shown in Table 1.

[0068] Table 1 Cross-reactivity of phenanthridine monoclonal antibodies

[0069]

[0070] This result indicates that the monoclonal antibody has a low cross-reactivity rate with other structural analogs and good sensitivity to azirmonidine.

[0071] Cross-reactivity rate (%) = (N-aminophenanthrene IC50) 50 ) / (Similar IC 50 )×100%

[0072] Example 5: Application of Azaminephenanthridine Monoclonal Antibody

[0073] The monoclonal antibody prepared from hybridoma cell lines via in vivo ascites fluid was used in an ELISA addition and recovery assay for azirmonidine. The specific steps are as follows:

[0074] (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.

[0075] (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;

[0076] (3) Prepare 0, 0.0274 ng / mL, 0.0823 ng / mL, 0.247 ng / mL, 0.741 ng / mL, 2.22 ng / mL, 6.67 ng / mL and 20 ng / mL aziridine standard solutions with phosphate buffer (PBS). Add the standard solutions and the extract of the sample to be tested to the sealed microplate, 50 μL per well, and repeat each sample in 3 wells. Then add 50 μL of aziridine monoclonal antibody diluted to 0.1 μg / mL to each well. After reacting at 37℃ for 30 min, wash the plate and pat dry.

[0077] (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.

[0078] (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.

[0079] (6) Addition of recovery and sample pretreatment:

[0080] Milk was chosen as the test sample.

[0081] Add 0.1 ppb, 1 ppb, and 5 ppb of azirmonidine standard to 2 mL of milk sample, respectively (based on antibody linearity range and IC50). 50 (Set the concentration to be added), add 10 mL of PBS buffer, vortex to mix, let stand for 30 min, and then filter.

[0082] Spiking and recovery experiments were conducted using indirect competitive ELISA, and the recoveries of azirmonidine ranged from 94.34% to 118.83%.

[0083] 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 phenanthridine, 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. 46743.

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

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

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

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

7. A composition, characterized in that, The composition comprises the hybridoma cell line of claim 1 and / or the azirmonidine 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 azirmonidine 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 azirmonidine 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 monoclonal antibody of azirmonidine 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 azirmonidine; the use does not relate to the diagnosis and treatment of disease.

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