Monoclonal hybridoma cell strain for broad-spectrum recognition of organophosphorus pesticide and antibody thereof

Abstract

The invention discloses a monoclonal hybridoma cell strain capable of broad-spectrum recognition of 14 organophosphorus pesticides and an antibody of the monoclonal hybridoma cell strain, and belongs to the field of food safety. The monoclonal antibody is secreted by a hybridoma cell strain with the preservation number of CCTCC NO: C2024359, the amino acid sequence of a heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO.2, and the amino acid sequence of a light chain variable region of the monoclonal antibody is as shown in SEQ ID NO.4; according to the application, a hybridoma cell strain is obtained by designing and synthesizing a hapten with a common structure, carrying out animal immunization and screening by utilizing a hybridoma cloning technology; the monoclonal antibody secreted by the monoclonal antibody can be used for broad-spectrum recognition of 14 organophosphorus pesticides, is good in stability and wide in recognition spectrum, and can be used for on-site rapid screening and detection of various organophosphorus pesticide residues in the environment and food.

Description

Technical Field

[0001] The present invention relates to the field of food safety, and particularly to a hybridoma cell line secreting monoclonal antibodies that broadly recognize 14 kinds of organophosphorus pesticides and a preparation method thereof. Technical Background

[0002] Organophosphorus pesticides (OPs) are a class of organic compounds containing phosphorus elements, including phosphate esters, thiophosphate esters, dithiophosphate esters, phosphoric acid amines, etc. Most organophosphorus pesticides belong to phosphoric (phosphine) acid esters and thiophosphate esters. Due to their large variety, good insecticidal effect, and low price, organophosphorus pesticides are still important agents used in agricultural production. Some highly toxic and extremely toxic organophosphorus pesticides have been prohibited or restricted, but there are still situations of illegal and excessive use in production, which have an impact on food safety and the environment. At the same time, the toxic action mechanisms of organophosphorus pesticides are the same, and their cumulative toxicity in food residues poses a threat to people's physical health. The simultaneous and rapid screening and detection of multiple residual organophosphorus pesticides in agricultural products / food face technical challenges.

[0003] Currently, the conventional methods for detecting OPs residues mainly include enzyme inhibition method, chromatography, spectroscopy, immunoassay, etc. Huo et al. (Detection of organophosphorus pesticides by a fluorescent sensing assay coupled with enzyme inhibition, Analytical Chemistry, 2024) developed a fluorescent sensing detection method for quantitative detection of glyphosate by combining the enzyme inhibition method, with a detection range of 0.2 - 1.8 mg / L and a detection limit as low as 0.015 mg / L. Wang et al. (Determination of 33 organophosphorus pesticide residues in green tea by QuEChERS-gas chromatography-tandem mass spectrometry (Chinese Tea Leaves, 2021)) established a method for detecting 33 organophosphorus pesticides in tea by using gas chromatography-tandem mass spectrometry, and the quantitative limit for each pesticide was 0.01 mg / kg. The qualitative and quantitative detection methods based on large-scale precision instruments have good accuracy, but they are time-consuming, have high detection costs, and require professional technical requirements for operators, which greatly limits their rapid identification and response to food safety. The immunological detection method based on antibodies has the characteristics of simple operation, rapidity, on-site operation, high sensitivity, and accurate qualitative determination, which is a favorable supplement to the legal inspection methods, especially has obvious advantages in the rapid screening and detection of prohibited / restricted pesticides and has been widely applied.

[0004] Enzyme-linked immunosorbent assay (ELISA) is a rapid detection method based on antigen / antibody recognition reactions. It is highly specific, sensitive, simple, fast, economical and efficient. This technique includes antibodies that specifically recognize corresponding targets, coating antigens, as well as substrates and chromogenic reagents. Among them, specific antibodies are the core recognition elements. At present, there have been some reports on monoclonal antibodies that specifically recognize organophosphorus compounds, such as monoclonal antibodies against diethoxy thiophosphate organophosphorus pesticides (DPPs); or monoclonal antibodies that recognize the common structural features of some organophosphorus pesticides, such as monoclonal antibodies that recognize the phosphate group (parathion, methyl parathion, fenitrothion, etc.). However, the recognition range of these antibodies is still relatively narrow and cannot meet the needs of rapid detection of multiple OPs, especially the rapid screening of prohibited / restricted organophosphorus pesticides and the rapid identification of their possible residual cumulative toxicity. Therefore, it is crucial to prepare monoclonal antibodies with better broad-spectrum specificity for organophosphorus. Summary of the Invention

[0005] The object of the present invention is to provide a monoclonal antibody that broadly recognizes organophosphorus pesticides, its hybridoma cell line and ELISA detection application. The organophosphorus monoclonal antibody secreted by this hybridoma cell line has good broad-spectrum properties (simultaneously recognizing 14 organophosphorus pesticides) and detection sensitivity (IC 50 value 1.25 - 1221.09 ng / mL), and can be used for the established immunological detection method to detect residues of multiple prohibited / restricted organophosphorus pesticides in food.

[0006] Specifically, the above object of the invention is achieved through the following solutions:

[0007] First, the present application provides a monoclonal antibody that broadly recognizes organophosphorus pesticides, which is secreted by a hybridoma cell line with the preservation number of CCTCC NO: C2024359.

[0008] The nucleotide sequences of the heavy chain variable region and the light chain variable region of this antibody are shown in SEQ IN NO.1 and SEQ INNO.3 in sequence, and the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ IN NO.2 and SEQ INNO.4 in sequence.

[0009] Second, the present application provides a hybridoma cell line with the preservation number of CCTCC NO: C2024359;

[0010] This cell line was preserved in the China Center for Type Culture Collection on January 15, 2025. The address is Wuhan University, Wuchang District, Wuhan City, Hubei Province, with the postal code 430072. The applicant named this cell line as Hybridoma cell line 5 - 10D - 6D.

[0011] Third, the present application provides the above monoclonal antibody as a detection antibody to establish an indirect competitive ELISA (enzyme-linked immunosorbent assay) to detect multiple organophosphorus pesticides; the above organophosphorus pesticides include but are not limited to at least one of parathion, coumaphos, triazophos, phoxim, benthion, ethion, methyl parathion, phorate, quinalphos, demeton, chlorfenapyr, phosphamidon, phosphamidon, ethoprophos, and cadusafos. The organophosphorus monoclonal antibody has good broad spectrum and sensitivity to these 14 organophosphorus pesticides (IC 50 is 1.25-1221.09ng / mL).

[0012] Fourth, the present application provides an ELSIA kit for detecting multiple organophosphorus pesticides, the kit comprising the above-mentioned monoclonal antibody.

[0013] The hybridoma cell line with the accession number CCTCC NO: C2024359 was obtained by screening using the following method:

[0014] 1) Synthesis of haptens. Organophosphorus pesticides are family compounds with certain common structural characteristics. Compounds with common structural characteristics were synthesized through organic synthesis technology. After mass spectrometry and nuclear magnetic resonance identification, a hapten compound BPB with a carboxyl group and common structural characteristics of organophosphorus pesticides was obtained.

[0015] 1) Animal immunization. The synthesized hapten was coupled with KLH to prepare the immunogen. After the immunogen and adjuvant were completely emulsified, BALB / c mice were immunized multiple times by subcutaneous injection on the back. The mice that had undergone the above immunization were cut off from their tails to collect blood. The antibody immune induction in the mouse serum was monitored by indirect ELISA. Mice with high serum titer and good recognition effect were selected for intraperitoneal injection shock immunization in preparation for subsequent cell fusion.

[0016] 2) Cell fusion and hybridoma cell screening. Immunized BALB / c mouse spleen cells and myeloma cells SP2 / 0 were fused by the polyethylene glycol (PEG2000) method, and the fused hybridoma cells were cultured in HAT medium. The recognition effect of the supernatant of positive cell wells on organophosphorus pesticides was monitored by indirect ELISA, and positive clones were screened. Then, subclones were screened by limiting dilution method, and finally monoclonal hybridoma cell lines were obtained.

[0017] 3) Ascites preparation and antibody purification. 8-10 week old BALB / c mice were taken and each mouse was intraperitoneally injected with 1 mL of incomplete adjuvant. Seven days later, each mouse was intraperitoneally injected with 1×10 6The obtained hybridoma cells were injected, and ascites was collected 7 days later. The ascites was purified by Protein G column to obtain monoclonal antibodies, which were stored at -20°C. The obtained monoclonal antibodies were identified for antibody subtypes, and an ELISA method was established to determine the recognition specificity, broad-spectrum property, and sensitivity of the antibodies to organophosphorus pesticides.

[0018] In this application, a hybridoma cell line was obtained and preserved. The monoclonal antibodies secreted by this cell line can broadly recognize 14 organophosphorus pesticides. Using this antibody as the detection antibody, an indirect competitive ELISA method was established to achieve rapid detection of multiple organophosphorus pesticide residues in the environment and food. Brief Description of the Drawings

[0019] Figure 1 It is for the synthesis and verification of BPB hapten. A is the synthesis step, B is the 1H NMR spectrum, and C is the mass spectrum.

[0020] Figure 2 It is the UV characterization diagrams of the synthesized complete antigens BPB-KLH and BPB-OVA.

[0021] Figure 3 It is the electrophoresis diagram of the purified monoclonal antibody and its identification.

[0022] Figure 4 It is the recognition characteristics of the monoclonal antibody to organophosphorus pesticides.

[0023] Figure 5 、 Figure 6 It is the recognition inhibition standard curves of the monoclonal antibody to hapten and 14 organophosphorus pesticides;

[0024] Among them, A-H respectively represent hapten, parathion, coumaphos, triazophos, phoxim, EPN, disulfoton, methyl parathion. I-O respectively represent phorate, quinalphos, demeton, dioxathion, phosalone, ethoprophos, cadusafos.

[0025] Figure 7 It is the schematic diagram of the amino acid sequence structure of the heavy chain variable region of the antibody.

[0026] Figure 8 It is the schematic diagram of the amino acid sequence structure of the light chain variable region of the antibody. Detailed Embodiments

[0027] Unless otherwise specified, the raw materials and reagents involved in the following examples were obtained through market channels.

[0028] 1640 Incomplete Medium: 100 mL fetal bovine serum was melted at room temperature and inactivated at 56 °C for 0.5 h. Weigh 0.5 g of sodium bicarbonate, 0.15 g of L-glutamine, 1.2 g of Hepes, and 0.05 g of sodium pyruvate, and add them to the melted 5 mL penicillin G and streptomycin solution until dissolved. After filtering through a membrane, add it to 500 mL of RPMI 1640 and mix well.

[0029] Synthesis of Hapten BPB in Example 1

[0030] The synthesis route adopted in this example is as shown in the appendix Figure 1 and the structure of the organophosphorus hapten (BPB) is synthesized, mainly targeting the common chemical structure developed and designed for organophosphorus pesticides of the diethyl thiophosphate type. The specific steps are as follows:

[0031] Diethoxyphosphinous chloride (3): Under the protection of N2, 17 mL of P(OEt)3 (0.1 mol) was added dropwise to 4.7 mL of PCl3 (0.05 mol), and the mixture was slowly stirred at room temperature for 1 h, and then the mixture was heated to 140 °C and stirred for 1 h. The reaction product was concentrated under reduced pressure to obtain product 3.

[0032] Diethoxythiophosphoryl chloride (4): Dissolve 6.39 g (40 mmol) of product 3 in 6 mL of toluene, add 0.1 mL (0.17 g, 1.0 mmol) of TiCL4 to it, heat the mixture to 80 °C, stir under the protection of N2, and add 1.34 g of sulfur powder to it, which was added within 30 min. After adding the sulfur powder, the mixture became a yellow solution, and it was continuously stirred at 80 °C for 6 h. The reaction product was cooled to 0 °C, and the solvent was removed by distillation under reduced pressure to obtain 7.01 g of product 4.

[0033] Methyl 4-hydroxybenzoate (5): Dissolve 2 mmol of 4-hydroxybenzoic acid in 3.0 mL of dried DMF, and then add 2.4 mmol of NaHCO3 to it and stir to dissolve at room temperature. Then add 3 mmol of CH3I to the mixture, heat it to 80 °C, and monitor the reaction by thin-layer chromatography. After that, add the reaction mixture to 10 mL of water and extract it with ethyl acetate. The organic layer was washed with 5% NaHCO3 and 5% NaCl, dried over anhydrous Na2SO4, and finally distilled under reduced pressure to remove the organic solvent. The product was purified by silica gel column chromatography, and the eluent was ethyl acetate - acetone (5:1) to obtain the intermediate 5.

[0034] O,O-Diethyl-O-(4-carboxylate phenyl)-thiophosphate (6): 0.24 g (1.57 mmol) of product 4 was added to 3 mL of butanone. 0.50 g (2.65 mmol) of diethoxythiophosphoryl chloride and 5 g of finely ground K2CO3 were added thereto. The mixture was stirred at 80 °C for 24 h. After the product was concentrated under reduced pressure, it was purified by silica gel column chromatography. The eluent was n-hexane-ethyl acetate (5:1), and 0.362 g of product 6 was obtained.

[0035] O,O-Diethyl-O-(4-carboxylate phenyl)-thiophosphate (hapten BPB): 0.304 g (1.0 mmol) of product 6 was dissolved in 60 mL of ethanol. 25 mL of 1 M KOH solution was added thereto with stirring at room temperature. After stirring for 30 min, 30 mL of 1 M HCl was added to the mixture. The reaction mixture was extracted with ethyl acetate. The organic layer was washed with 1 M HCl (10 mL × 2) and dried over anhydrous magnesium sulfate. The solvent was recovered by concentration under reduced pressure. The product was purified by silica gel column chromatography. The eluent was n-hexane-ethyl acetate-acetic acid (50:25:2), and the thin layer chromatography R f = 0.57. The final purified product was 70 mg, which was the target organophosphorus hapten.

[0036] Example 2 Preparation of Immunogen and Coating Antigen

[0037] In this example, the immunogen and coating antigen were respectively obtained by coupling the hapten with the carrier proteins KLH and OVA by the active ester method. The specific preparation method is as follows:

[0038] 2.1) Weigh 29.0 mg (0.1 mmol) of the hapten prepared in Example 1 and add it to 2 mL of DMF. After mixing, add 13.8 mg (0.12 mmol) of NHS and 30.9 mg (0.15 mmol) of DCC respectively, and stir at room temperature overnight. The next day, the reaction mixture was centrifuged at 12000 g for 10 min, and the supernatant was collected.

[0039] 2.2) Weigh 135 mg (3×10 -3 mmol) of OVA or 160 mg (0.46×10 -3 mmol) of KLH and dissolve them in 6 mL of PBS with magnetic stirring. Then, slowly add the supernatant collected in 2.1 thereto, and continue to stir at room temperature for 6 h. The reaction mixture was centrifuged at 12000 g for 10 min, and the supernatant was taken.

[0040] 2.3) Transfer the supernatant into a dialysis bag (molecular weight cut-off 8000 - 14000 Da), and dialyze it with stirring in 2 L of PBS (0.01 M, pH 7.4) at 4°C for 3 days, changing the PBS (0.01 M, pH 7.4) dialysis fluid 3 - 4 times a day to obtain the immunogen (BPB-KLH) and the coating antigen (BPB-OVA), which are stored at -20°C for later use.

[0041] The hapten BPB, the carrier proteins KLH and OVA, and their conjugates BPB-KLH and BPB-OVA were scanned by ultraviolet spectrophotometry at wavelengths of 200 - 400 nm, and their ultraviolet absorption spectra are as shown in the appendix Figure 2 as follows.

[0042] Example 3 Animal Immunization and Monitoring of Polyclonal Antiserum

[0043] After 1 week of adaptive feeding of female Balb / c mice at 5 - 6 weeks old, the mice were immunized according to the immunization protocol in Table 1, with an interval of 2 weeks between each immunization.

[0044] Table 1 Mouse Immunization Protocol

[0045]

[0046] Preparation of mouse serum: Use sterile scissors to quickly cut off the tip of the tail of the mice after 1 week of adaptive feeding, collect the plasma, let the collected mouse plasma stand at room temperature for 2 h, then centrifuge at 4°C and 10000 g for 10 min to take the supernatant as the negative serum; collect the serum from the immunized mice using the same method as above as the polyclonal antiserum, which is stored at -20°C for later use.

[0047] Monitoring of polyclonal antiserum: Start monitoring the titer of mouse serum one week after the third immunization. The indirect non-competitive ELISA method was used to evaluate the titer of the polyclonal antiserum (the titer is defined as the dilution multiple of the polyclonal antiserum when the OD 450 value is around 1.0), and an indirect competitive ELISA method was established to determine the recognition effect of the mouse polyclonal antiserum on a mixed standard of various organophosphorus pesticides. The specific operation steps are as follows: ① Coating: Dilute the coating antigen concentration to 0.1 μg / mL with CBS buffer, add 100 μL per well to the 96-well enzyme-labeled wells, and incubate overnight at 4°C; ② Washing: Add 300 μL / well of PBST washing solution, repeat the washing 3 times, and pat the enzyme-labeled wells dry on absorbent paper after washing; ③ Blocking: Add 5% skim milk powder (0.5 g skim milk powder added to 10 mL of PBS) to the enzyme-labeled wells, 250 μL / well, incubate at 37°C for 2 h; ④ Washing: The same as step ②; ⑤ Sampling: Dilute the polyclonal antiserum in a certain proportion, and use PBS as the blank group to eliminate background interference, 100 μL / well, incubate at 37°C for 1 h; ⑥ Washing: The same as step ②; ⑦ Adding enzyme-labeled secondary antibody: Dilute the enzyme-labeled secondary antibody 5×10 3times, 100 μL / well, incubate at 37 °C for 1 h; ⑧ Washing: Wash 6 times with PBST, 300 μL / well, and pat dry the enzyme-labeled wells on absorbent paper after washing; ⑨ Color development: Add 100 μL of TMB color development solution to each well, react at 37 °C in the dark for 15 min; ⑩ Terminate the reaction: Add 2 mol / L H2SO4 to the enzyme-labeled wells, 50 μL / well, and read the OD 450 value.

[0048] The specific steps of the indirect competitive ELISA method are similar to those of the indirect non-competitive ELISA method, only different in the sample addition step. In the indirect competitive ELISA, at the ⑤th step of sample addition, the spiked competition group is 50 μL of polyclonal antiserum and 50 μL of the compound to be detected (organophosphorus pesticides or haptens), where the polyclonal antiserum is diluted with PBS, and the compound to be detected is diluted with 5% methanol-PBS buffer; the negative control group is 50 μL of negative serum and 50 μL of 5% methanol-PBS buffer; the blank control group is 50 μL of PBS buffer and 50 μL of 5% methanol-PBS buffer.

[0049] Example 4 Cell Fusion and Subcloning

[0050] 4.1) Cell fusion: Mix splenocytes and SP2 / 0 cells at a ratio of 1:6, make up the volume to 30 mL with incomplete 1640 medium, centrifuge at 3000 g for 10 min, discard the supernatant, gently disperse the cells and place them in a 37 °C water bath. Slowly add 1 mL of 50% PEG preheated to 37 °C along the centrifuge tube wall, shake clockwise while adding, and finish adding within 1 min, then let it stand for 1 min. Add 15 - 30 mL of incomplete 1640 medium to terminate the PEG reaction, finish adding within 5 min, and make up the volume to 40 mL. After standing at 37 °C in the incubator for 10 min, centrifuge at 800 rpm for 5 min, discard the supernatant. Add 3 mL of HAT medium and pipette the cells to make them suspended, make up the volume to 60 mL, and mix well. Aliquot 100 μL / well into a 96-well culture plate containing feeder cells, and culture in a 37 °C incubator.

[0051] 4.2) Screening and subcloning of positive wells: After one week, measure the cell well supernatant by ELISA, and the cell wells with inhibitory effects on organophosphorus pesticides are positive cell wells. Take 0.5 - 2 μL of cell suspension (about 100 cells) according to the cell number, dilute and disperse it in a new 96-well plate with medium for subcloning screening, and detect the antibody situation in the cell well supernatant to further screen positive cell wells. The subsequent subcloning steps are the same as above until monoclonal cells are obtained. The applicant named the multiple organophosphorus monoclonal hybridoma cell lines obtained as 6-3G-5H, 5-10D-6D, 2-4C-11D, B1.

[0052] Example 5 Ascites Preparation, Identification and Purification

[0053] 5.1) Ascites preparation: Female Balb / c mice that have given birth are sensitized by multiple-point injection of Freund's incomplete adjuvant in the abdomen, 1 mL per mouse. Approximately 5 - 7 days later, each mouse is injected intraperitoneally with approximately 10^6 cells. Approximately 7 - 14 days later, the abdomen of the mouse becomes enlarged, and ascites is collected by inserting a 10 mL sterile syringe needle into the abdomen. The collected ascites is centrifuged at 2000 rpm for 10 min at 4°C, and the clear ascites in the middle is aspirated and stored at -20°C for later use.

[0054] 5.2) Subtype identification: In this experiment, a commercial subtype kit (Mouse mAb isotype ELISA Kit) was used for identification, and the detection process was carried out according to the product manual.

[0055] 5.3) Antibody purification: The steps of purifying antibodies with a Protein G column are conventional methods in the art, as disclosed in the literature "Affinity chromatography: A versatile technique for antibody purification" (Sushrut Arora. Methods. 2017. 3. 84 - 89). The specific purification steps in this example are as follows: Ascites was purified using a Protein G column. After the ascites diluted with the binding buffer was passed through the column 3 times repeatedly, the purification column was rinsed 3 times with the binding buffer and the elution buffer in sequence, and the eluate containing the antibody was collected, which was the purified antibody obtained. Then, the column was washed 3 times with 5 column volumes of the binding buffer and the purification column was blocked with 20% ethanol.

[0056] SDS-PAGE electrophoresis verification: A 10% protein gel was loaded into the electrophoresis tank, and an appropriate amount of SDS protein buffer was added. The antibody was treated with 4× protein loading buffer, and 6 μL of Marker (160 - 20 kDa) was added as a reference. The electrophoresis conditions were (constant voltage 140 V for 90 min). After completion, the protein gel was taken out, and the protein precast gel was stained in the SDS-PAGE instant blue staining solution for 20 min. Clear blue bands as shown in Figure 4 could be seen on the lanes of the protein precast gel. Figure 4 In the SDS-PAGE results of the purified antibody shown, lane 1: ascites antibody; lane 2: Marker; lane 3: B1 antibody; lane 4: 2 - 4C - 11D antibody; lane 5: 5 - 10D - 6D antibody; lane 6: 6 - 3G - 5H antibody.

[0057] Example 6 Antibody performance identification

[0058] 6.1) Establish an indirect competitive ELISA method

[0059] The specific operation is similar to the indirect competitive ELISA method in Example 3, only different in coating and sample addition. Coating: Dilute the coating antigen BPB-OVA (prepared in Example 2) with CBS buffer to 1 μg / mL, add 100 μL / well to a 96-well ELISA plate, and incubate at 37 °C for 2 h. Sample addition: Dilute the monoclonal antibody against organophosphorus (1 mg / mL) 1200-fold with PBS buffer and add it to the ELISA plate, 50 μL / well. At the same time, add 50 μL of different concentrations of organophosphorus pesticides or haptens, set three parallels, incubate at 37 °C for 1 h, and the subsequent process is the same as the steps in Example 3. The optimal working concentration of the antigen for the established indirect competitive ELISA is 0.1 μg / mL, and the optimal working concentration of the antibody is 0.188 μg / mL.

[0060] 6.2) Identification of antibody recognition specificity and sensitivity performance

[0061] Measure the IC 50 value of the antibody against the hapten in Example 1, and define its cross-reactivity as 100%. Dilute 41 organophosphorus pesticides with 5% methanol-PBS to gradient concentrations. Use the established indirect competitive ELISA method to measure the detection sensitivity and recognition effect of the monoclonal antibody against 41 organophosphorus pesticides (ethoprophos, cadusafos, isofenphos-methyl, phosphamidon, isocarbophos, terbufos, profenofos, quinalphos, parathion, triazophos, coumaphos, disulfoton, bromophos-ethyl, demephion, phoxim, chlorpyrifos, phorate, diazinon, phosalone, malathion, dichlorvos, monocrotophos, methamidophos, paraoxon, bromophos, fenthion, trichlorfon, demeton, omethoate, phosfolan, sulfotep, methyl parathion, chlorpyrifos-methyl, fenitrothion, EPN, kitazin P, acephate, pirimiphos-methyl, dimethoate, chlorpyrifos-ethyl), all of the above pesticides are standard products, purchased from Nanjing Taipuri Company), as Figure 5 shown, where the abscissa represents different organophosphorus pesticides and the ordinate represents the inhibition rate of different antibodies against the same pesticide. The monoclonal antibody secreted by the cell line numbered 5-10D-6D has the best recognition effect on 41 organophosphorus pesticides.

[0062] The 5-10D-6D antibody can recognize 14 organophosphorus pesticides, including: parathion, coumaphos, triazophos, phoxim, EPN, disulfoton, methyl parathion, phorate, quinalphos, demeton, demephion, phosalone, ethoprophos, cadusafos. The standard curves are as Figure 5 , Figure 6 shown, the IC 50 is 1.25 - 1221.09 ng / mL, and it has the best recognition effect on parathion (IC 50 = 1.25 ng / mL). The sensitivity (IC 50 ) and specificity (cross-reactivity) of the monoclonal antibody 5-10D-6D against 14 organophosphorus pesticides are shown in Table 2.

[0063] Table 2 Detection results of monoclonal antibody 5-10D-6D for organophosphorus pesticides

[0064]

[0065] The applicant deposited the obtained 5-10D-6D monoclonal cell line at the China Center for Type Culture Collection (CCTCC) on January 15, 2025. The address is Wuhan University, Wuchang District, Wuhan City, Hubei Province, with a postal code of 430072, and the deposit number is CCTCC NO: C2024359.

[0066] The antibody variable region gene sequence of the 5-10D-6D monoclonal cell line was sequenced, and the heavy and light chain variable regions (CDR regions) were analyzed by abysis( http: / / www.abysis.org / abysis / )). The heavy chain variable region of this antibody includes CDR-H1, CDR-H2, and CDR-H3, and their nucleotide and amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively. The amino acid sequence structure of the heavy chain variable region is as Figure 7 shown; the light chain variable region includes CDR-L1, CDR-L2, and CDR-L3, and the nucleotide and amino acid sequences are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively. The sequence structure of the light chain variable region is as Figure 8 shown.

Claims

1. A monoclonal antibody for broad-spectrum recognition of organophosphorus pesticides, characterized in that, The monoclonal antibody is secreted by a hybridoma cell line with the preservation number of CCTCC NO: C2024359.

2. The monoclonal antibody according to claim 1, characterized in that, The nucleotide sequences of the heavy chain variable region and the light chain variable region of the antibody are shown in SEQ IN NO.1 and SEQ IN NO.3 respectively.

3. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are shown in SEQ IN NO.2 and SEQ IN NO.4 respectively.

4. The application of the monoclonal antibody according to claim 1 in detecting organophosphorus pesticides.

5. The application according to claim 4, wherein Using the monoclonal antibody as a detection antibody, detect organophosphorus pesticides by ELISA method.

6. The application according to claim 4, characterized in that The organophosphorus pesticides include at least one of parathion, coumaphos, triazophos, phoxim, EPN, disulfoton, methyl parathion, phorate, quinalphos, demeton, dioxathion, phosalone, abamectin, cadusafos.

7. A hybridoma cell line with the preservation number of CCTCC NO: C2024359, which was preserved in the China Center for Type Culture Collection on January 15, 2025.

8. An ELSIA kit for detecting organophosphorus pesticides, which includes a monoclonal antibody secreted by a hybridoma cell line with the preservation number of CCTCC NO: C2024359.

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