Hybridoma cell strain secreting monoclonal antibody of chlorinated nitrogen aminophenazone and its application

Abstract

The present application relates to a hybridoma cell strain secreting a chlorinated nitrogen aminophenazone monoclonal antibody and its application, and belongs to the technical field of immune detection. The hybridoma cell strain secreting a chlorinated nitrogen aminophenazone monoclonal antibody was preserved in the China General Microbiological Culture Collection Center on April 24, 2026, and the address of the preservation is No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 46821. The monoclonal antibody secreted by the hybridoma cell strain of the present application has good detection sensitivity (IC 50 Value is 0.27 ng / mL) to chlorinated nitrogen aminophenazone, and can be used for the residual detection of chlorinated nitrogen aminophenazone.

Description

Technical Field

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

[0002] Amphenanthridine chloride is an antiparasitic drug widely used in veterinary clinical practice, especially in cattle farming where it is often used as an antitrypanosomal drug and is a core veterinary drug for the prevention and treatment of bovine trypanosomiasis. Although amphenanthridine chloride plays an important role in controlling trypanosomiasis infection in livestock and ensuring healthy livestock farming, the potential residue problem in animal-derived foods such as beef and milk remains a key focus of food safety management.

[0003] In actual livestock farming, some farmers may engage in irrational drug use in an attempt to reduce disease losses and improve farming efficiency. Amphenanthridine chloride is often administered via deep intramuscular injection. Without strict control over dosage, frequency, and withdrawal period, this method of administration can easily lead to drug accumulation in livestock, resulting in excessive drug residues in animal-derived products. Furthermore, in clinical farming, to enhance antiparasitic effects, it is occasionally used in combination with other antitrypanosomal drugs. While this can strengthen disease prevention and control, it also further increases the potential risk of drug residues in animals. Amphenanthridine chloride residues in livestock products not only directly affect food safety and quality but may also induce drug resistance in parasites. The abuse of this drug can lead to trypanosomal resistance, significantly reducing the effectiveness of treatments. This not only hinders the effectiveness of livestock disease control and affects the healthy development of the farming industry but also poses a potential threat to human public health and safety through the food chain. In actual farming practices, irregular drug use is prevalent among farmers and farming enterprises. For example, blindly using drugs for disease prevention rather than targeted treatment, failing to strictly follow the instructions for veterinary drug administration, and lacking professional veterinary technical guidance can all significantly increase the risk of drug residues.

[0004] Amphenanthridine chloride possesses a certain degree of lipid solubility and tissue accumulation, allowing it to accumulate in large quantities in animal tissues and organs such as the liver, kidneys, and fat. If livestock and poultry have impaired liver metabolism or kidney excretion functions after ingesting amphenanthridine chloride, the efficiency of drug metabolism and excretion will be significantly reduced, further exacerbating the bioaccumulation of the drug in the body. This continuous bioaccumulation not only results in drug residues in edible livestock and poultry tissues and raw milk but also damages the health of the livestock and poultry's own organs, triggering drug metabolism disorders and creating a more severe vicious cycle of residues.

[0005] To strictly control the negative impacts of chlorpyrifos residues on human health and the food industry, many countries and regions around the world have established strict standards for the residue limits of this drug in animal-derived foods. my country has also clearly stipulated the maximum residue limit for it in bovine tissues. Even so, current detection methods for chlorpyrifos residues still mainly rely on traditional large-scale instrumental analytical techniques, primarily including high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). While these instrumental detection methods possess high detection sensitivity and result specificity, they also have many limitations. For example, they require cumbersome and complex sample pretreatment procedures, have high costs for purchasing and maintaining detection equipment, and involve complex and tedious experimental procedures that must be operated by specialized technicians, making them difficult to meet the practical needs of rapid on-site screening.

[0006] To overcome the application challenges of traditional detection methods, there is an urgent need to develop rapid, simple, low-cost residue detection technologies suitable for large-scale on-site screening. Enzyme-linked immunosorbent assay (ELISA), as a mature immunoassay technique, has been widely adopted in the field of food safety and veterinary drug residue detection due to its advantages such as simple operation, fast detection speed, low detection cost, and suitability for large-scale sample screening. The core prerequisite for using ELISA to detect chlorinated amphenanthridine residues is the preparation of highly specific and sensitive monoclonal antibodies.

[0007] Therefore, developing a novel monoclonal antibody-based method for detecting chloramine-methyl phenanthrene residues, especially establishing an immunoassay technique for accurate, rapid, and low-cost detection of this drug residue, has significant practical application value. This will not only comprehensively improve the efficiency and accuracy of chloramine-methyl phenanthrene residue detection but also provide more convenient, efficient, and low-cost detection technology support for related fields such as the regulation of veterinary drug use in livestock farming, the control of animal food safety, and the research and development of antiparasitic veterinary drugs, thus contributing to the improvement of the food safety and quality assurance system. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes a monoclonal antibody against chloramine and its applications. The monoclonal antibody against chloramine secreted by this hybridoma cell line exhibits good detection sensitivity (IC50) for chloramine. 50 With a value of 0.27 ng / mL, it can be used to establish an immunological detection method for chloramine phenanthridine to detect chloramine phenanthridine residues in beef.

[0009] The technical solution of the present invention is as follows:

[0010] The first objective of this invention is to provide a hybridoma cell line that secretes a monoclonal antibody against nitric acid phenanthridine, which was deposited on April 24, 2026, 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. 46821.

[0011] A second objective of this invention is to provide the application of the hybridoma cell line in the preparation of chlorinated phenanthridine monoclonal antibodies.

[0012] A third objective of this invention is to provide a monoclonal antibody containing chlorinated phenanthridine, which is secreted by the hybridoma cell line.

[0013] The fourth objective of this invention is to provide the application of the hybridoma cell line or the monoclonal antibody against chloramine in the detection of chloramine; the application does not involve the diagnosis or treatment of diseases.

[0014] The fifth objective of this invention is to provide a detection product containing the aforementioned chloramine-aminophenanthridine monoclonal antibody.

[0015] In one embodiment of the present invention, the detection product further includes a coating agent.

[0016] In one embodiment of the present invention, the coating antigen is obtained by activating the nitric acid phenanthridine hapten and then coupling it with a carrier protein.

[0017] In one embodiment of the present invention, the structural formula of the chlorinated phenanthridine hapten is shown below:

[0018] .

[0019] In one embodiment of the present invention, the carrier protein is selected from at least one of chicken ovalbumin, keyhole hemocyanin, bovine serum albumin, human serum albumin, lactoferrin, horseradish peroxidase, thyroglobulin, immunoglobulin, and hormones.

[0020] The sixth objective of this invention is to provide the application of the aforementioned detection product in the detection of nitric acid phenanthridine; the application does not involve the diagnosis and treatment of diseases.

[0021] This invention also provides a method for preparing the hybridoma cell line that secretes the above-mentioned monoclonal antibody against nitric acid phenanthridine, comprising the following steps:

[0022] Step 1: Prepare complete immunogenic antigen and coating antigen of amphenanthridine chloride using amphenanthridine chloride hapten; emulsify the obtained complete immunogenic antigen of amphenanthridine chloride with Freund's adjuvant or incomplete Freund's adjuvant to prepare an immunogen;

[0023] 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.

[0024] Step 3: Blood was collected from mice that had undergone the above immunization process. The mice were coated with nitric acid phenanthridine and their serum immunogenicity and immunosuppressive ability were detected by indirect ELISA. Mice with high sensitivity of nitric acid phenanthridine antibody in serum were screened.

[0025] Step 4: The selected mice were immunized by intraperitoneal injection using the complete antigen of chlorophenanthridine chloride without Freund's adjuvant.

[0026] Step 5: Fuse spleen cells and myeloma cells from BALB / c mice after sprint immunization. The fused cells are then screened and cultured in HAT medium. Positive cell pores are detected using indirect ELISA, and the inhibitory effect of positive cell pores is further determined using indirect competitive ELISA. The positive cell pores with the best inhibition are subcloned using limiting dilution, and finally, hybridoma cell lines that can secrete highly sensitive monoclonal antibodies against chlorophenanthridine chloride are screened out.

[0027] In one embodiment of the present invention, in step 1, the molecular formula of the chlorophenanthridine hapten is as follows:

[0028] .

[0029] The molecular formula of the nitrogen chloride-aminophenanthridine immunogen is as follows:

[0030] .

[0031] In one embodiment of the present invention, the method for preparing the chlorophenanthridine complete antigen includes the following steps: dissolving the chlorophenanthridine hapten and 50% glutaraldehyde in anhydrous N,N-dimethylformamide (DMF), stirring and reacting to obtain an activated chlorophenanthridine hapten solution, i.e., solution A; diluting keyhole hemocyanin with carbonate buffer solution (CBS) to obtain solution B; slowly adding solution A to solution B to react and obtain a reaction solution; dialyzing the reaction solution with phosphate buffered saline (PBS) to obtain the complete antigen.

[0032] In one embodiment of the present invention, the molecular formula of the nitrogen chloride-aminophenanthrene-coated antigen is as follows:

[0033] .

[0034] In one embodiment of the present invention, the preparation method of the chlorophenanthridine hapten is as follows: the chlorophenanthridine hapten is dissolved in anhydrous N,N-dimethylformamide (DMF), and then NaNO2 is dissolved and added dropwise. The reaction is carried out in an ice bath in the dark with stirring to obtain an activated chlorophenanthridine hapten solution, i.e., solution A; bovine serum albumin is diluted with carbonate buffer (CBS) to obtain solution B; solution A is slowly added to solution B to carry out the reaction to obtain a reaction solution; the reaction solution is dialyzed with phosphate buffer (PBS) to obtain the immunogen.

[0035] In one embodiment of the present invention, the preparation method of the chlorinated amphenanthridine monoclonal antibody is as follows: BALB / c mice are injected intraperitoneally with paraffin oil, and then injected intraperitoneally with a hybridoma cell line with accession number CGMCC No.46821. Ascites fluid is collected after injection, the ascites fluid is purified, and the obtained monoclonal antibody is stored at low temperature.

[0036] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0037] (1) The monoclonal antibody against chloramine-aminophenanthridine obtained in this invention has good detection sensitivity (IC50) for chloramine-aminophenanthridine. 50 The value was 0.27 ng / mL.

[0038] (2) In this invention, the monoclonal antibody obtained by immunization with the glutaraldehyde-conjugated amphenanthridine chloride immunogen exhibits superior performance when detected by ELISA using the diazo-conjugated coating antigen. The reason is that the glutaraldehyde conjugation site is located on the amidine side chain of the amphenanthridine chloride molecule. This cross-linking method mainly modifies the amino group of the side chain. Although it masks some side chain epitopes to a certain extent, it retains the immunogenicity of the core characteristic structures such as the phenanthridine core and aromatic ring. The induced antibody mainly targets the dominant epitopes in the core region of the molecule. On the other hand, the diazo conjugation site is located on the benzene ring of the side chain of the molecule, which can completely expose the core dominant epitopes recognized by the antibody, significantly improving the specificity and signal-to-noise ratio of the detection, and ultimately achieving a better detection effect.

[0039] (3) The monoclonal antibody cell line of chlorinated phenanthridine obtained in this invention can be used for immunoassay detection.

[0040] Preservation of biological materials

[0041] A hybridoma cell line secreting a monoclonal antibody against chlorinated aminophenidin 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 April 24, 2025, with accession number CGMCC No. 46821. Attached Figure Description

[0042] 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.

[0043] Figure 1 This is the standard curve of inhibition of nitric acid phenanthridine monoclonal antibody against nitric acid phenanthridine in this invention. Detailed Implementation

[0044] 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.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0046] The culture media involved in the following examples are as follows:

[0047] 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.

[0048] The reagents involved in the following examples are as follows:

[0049] 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.

[0050] 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.

[0051] PBST: PBS containing 0.05% Tween 20;

[0052] Antibody dilution buffer: PBS containing 0.1% gelatin;

[0053] 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.

[0054] The detection methods involved in the following embodiments are as follows:

[0055] Method for detecting the inhibition rate of nitric acid phenanthridine: 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, the nitric acid phenanthridine standard was diluted to eight concentrations (0.004 ng / mL, 0.012 ng / mL, 0.037 ng / mL, 0.111 ng / mL, 0.333 ng / mL, 1 ng / mL, and 3 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 nitric acid phenanthridine chloride was obtained, and the IC50 was calculated. 50 .

[0056] Example 1: Synthesis of the complete antigen of nitric acid phenanthridine chloride

[0057] 2.98 mg of nitrochlorophenanthridine (CAS: 34301-55-8) was weighed and dissolved in 200 μL of N,N-dimethylformamide (DMF) and stirred at room temperature. Then, 2 μL of 50% glutaraldehyde solution was added to the nitrochlorophenanthridine hapten solution, and the mixture was stirred at room temperature for 4 h to activate it. 6 mg of keyhole hemocyanin (KLH) was added to 3 mL of 0.01 M carbonate buffer (CBS) and dissolved thoroughly. The activated hapten was slowly added to the diluted KLH solution, and the mixture was stirred overnight at room temperature. The solution was then dialyzed against 0.01 M PBS to remove unreacted small molecules, yielding a relatively pure complete antigen, which was then identified by UV absorption scanning.

[0058] Example 2: Synthesis of the completely coated precursor of nitrogen chloride and phenanthrene chloride

[0059] 2.23 mg of nitrochlorophenanthridine (CAS: 34301-55-8) was dissolved in 200 μL of anhydrous N,N-dimethylformamide (DMF). 4.8 mg of NaNO2 was dissolved and added to the solution. The mixture was stirred in an ice bath in the dark for 4 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.

[0060] Example 3: Preparation of hybridoma cell lines secreting monoclonal antibodies against nitric acid phenanthridine chloride

[0061] 1. Acquisition of immunity in animals

[0062] The complete antigen of nitrochlorophenanthridine 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; subsequent booster immunizations used incomplete Freund's adjuvant at half the dose of 50 μg / mouse; sprint immunizations did not use adjuvants, 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 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.

[0063] 2. Cell fusion

[0064] 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:

[0065] 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.

[0066] 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 × 10⁶ 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.

[0067] 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.

[0068] 3. Cell selection and cell line establishment

[0069] 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.

[0070] The screening process consists of two steps: First, positive cell wells are selected using ic-ELISA. Second, nitric acid phenanthridine chloride is used as a standard, and the inhibitory effect on positive cells is determined using ic-ELISA.

[0071] Cell wells that showed good inhibition of the nitric acid 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.

[0072] At least three subcloning operations were performed using the method described above to finally obtain the chloramine-aminophenidine monoclonal antibody cell line.

[0073] Example 4: Preparation and Identification of Monoclonal Antibody for Nitrochloride and Amphenanthridine

[0074] 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 nitric chloride-aminophenanthrene hybridoma cells starting from day 7, and the ascites fluid was purified for antibody using the octanoic acid-saturated ammonium sulfate method.

[0075] 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.

[0076] Using an indirect competitive ELISA, the monoclonal antibody against chlorophenanthridine chloride showed good sensitivity and specificity (IC50) for detecting chlorophenanthridine chloride. 50 With a value of 0.27 ng / mL, it can be used for the immunoassay of nitric acid phenanthridine.

[0077] Cross-reactivity experiments were conducted on the structural analogues of nitric acid phenanthridine (ethidium bromide and triazine). The results showed that the absorbance values ​​in each well of the ELISA plate corresponding to etidium bromide and triazine did not differ significantly with the dilution concentration of the standard. The specific results are shown in Table 1.

[0078] Table 1 Cross-reactivity of nitric acid phenanthridine monoclonal antibodies

[0079]

[0080] These results indicate that the monoclonal antibody has a low cross-reactivity with other structural analogs and good specificity for nitric acid phenanthridine.

[0081] Cross-reactivity rate (%) = (nitrogen chloride, phenanthrene chloride, IC50) 50 ) / (Similar IC 50 )×100%

[0082] Example 5: Application of monoclonal antibody against nitric acid phenanthridine

[0083] The monoclonal antibody prepared from hybridoma cell lines via in vivo ascites fluid was used in an ELISA addition and recovery assay for nitric acid phenanthridine chloride. The specific steps are as follows:

[0084] (1) Coat a 96-well microplate with 100 μL of the coating agent (prepared in Example 2) diluted with carbonate buffer (CBS) at a concentration of 0.1 μg / mL. After drying at 37°C for 2 h, wash the plate three times with PBST washing buffer, 200 μL per well each time, for 3 min each time, and then pat dry.

[0085] (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;

[0086] (3) Prepare 0.004 ng / mL, 0.012 ng / mL, 0.037 ng / mL, 0.111 ng / mL, 0.333 ng / mL, 1 ng / mL and 3 ng / mL nitrifamine chloride standard solutions with PBS buffer. 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 nitrifamine chloride monoclonal antibody diluted to 0.1 μg / mL to each well. After reacting at 37℃ for 30 min, wash the plate and pat dry.

[0087] (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.

[0088] (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.

[0089] (6) Addition of recovery and sample pretreatment:

[0090] Beef was chosen as the sample for testing.

[0091] After the beef 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.

[0092] Spiking recovery experiments were conducted using indirect competitive ELISA, and the recoveries of chlorpyrifos were 95.7%–103.6%.

[0093] 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 nitric acid phenanthridine, characterized in that, The hybridoma cell line was deposited on April 24, 2026, 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. 46821.

2. The use of the hybridoma cell line of claim 1 in the preparation of chlorinated phenanthridine monoclonal antibody.

3. A monoclonal antibody against chlorinated aminophenanthridine, characterized in that, Produced by the hybridoma cell line of claim 1.

4. The use of the hybridoma cell line of claim 1 or the monoclonal antibody of chloramine-aminophenanthridine of claim 3 in the detection of chloramine-aminophenanthridine; the use does not involve the diagnosis and treatment of diseases.

5. A testing product, characterized in that, The test product contains the chlorinated phenanthridine monoclonal antibody as described in claim 3.

6. The testing product according to claim 5, characterized in that, The tested products also include coating agents.

7. The testing product according to claim 5, characterized in that, The coating is obtained by activating the chlorinated phenanthridine hapten and then coupling it with a carrier protein.

8. The testing product according to claim 7, characterized in that, The structural formula of the nitrogen chloride phenanthridine hapten is shown below: 。 9. The testing product according to claim 7, characterized in that, The carrier protein is selected from at least one of chicken ovalbumin, keyhole hemocyanin, bovine serum albumin, human serum albumin, lactoferrin, horseradish peroxidase, thyroglobulin, immunoglobulin, and hormones.

10. The use of the detection product according to any one of claims 5-9 in the detection of chloramine-3-aminophenidine; the use does not involve the diagnosis and treatment of disease.

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