Hybridoma cell strain secreting monoclonal antibody of fenoxaprop-ethyl compounds
By preparing hybridoma cell lines that secrete monoclonal antibodies against quizalofop-p-ethyl compounds, the problems of complex and costly detection methods in existing technologies have been solved, achieving high sensitivity and specificity for the detection of quizalofop-p-ethyl compounds, which is suitable for rapid detection of agricultural products.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2024-12-04
- Publication Date
- 2026-06-09
Smart Images
Figure CN120118850B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of immunochemical technology, and in particular to hybridoma cell lines that secrete monoclonal antibodies against quizalofop-p-ethyl compounds. Background Technology
[0002] The main compounds in the quizalofop-p-ethyl class include quizalofop-p-ethyl, quizalofop-p-ethyl, and quizalofop-p-ethyl. Quizalofop-p-ethyl belongs to the heterocyclic phenoxypropionic acid class of herbicides. It primarily inhibits fatty acid synthesis by acting as a suppressor of acetyl-CoA carboxylase, thus hindering the synthesis of oleic acid, linoleic acid, linolenic acid, the waxy layer, and the cuticle. This leads to rapid destruction of the plant's membrane structure, increased permeability, and ultimately plant death. The herbicide is absorbed through the stems and leaves and translocated to the meristems and root growth points. Its action is rapid; growth ceases 2-3 days after application, and within 5-6 days, the central leaves turn purple, the meristems turn brown, and the leaves gradually wither. It is a selective foliar herbicide, and quizalofop-p-ethyl is its degradation product. Quizalofop-p-ethyl is mainly used to control wild oats, barnyard grass, foxtail grass, oats, barnyard grass, free-growing corn, and crabgrass, among others. Studies have shown that quizalofop-p-ethyl compounds are highly toxic to aquatic organisms, and direct or indirect water pollution can also affect fish. They induce high acute and developmental toxicity in zebrafish embryos. Furthermore, hepatotoxicity of quizalofop-p-ethyl compounds has been observed in long-term studies in mice. Quizalofop-p-ethyl compounds are also considered to have reproductive toxicity and severely affect sperm motility patterns in pigs. To mitigate the negative impacts of quizalofop-p-ethyl compounds on human health, many countries have established maximum residue limits (MRLs) for their use in agricultural products. In China, the MRLs for quizalofop-p-ethyl range from 0.02 mg / kg in cottonseed to 0.5 mg / kg in rapeseed. Detecting quizalofop-p-ethyl compound residues in agricultural products is crucial for protecting human health.
[0003] Currently, the detection methods for quizalofop-p-ethyl compounds 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 still have some drawbacks, such as the need for thorough sample purification, high solvent consumption, expensive equipment, and skilled technicians. Therefore, a rapid and simple method for detecting quizalofop-p-ethyl compound residues is needed.
[0004] Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method. It requires simple sample pretreatment, involves few purification steps, has a large analytical capacity, low detection cost, and is easy to operate, making it suitable for rapid on-site detection of large numbers of samples. Therefore, it is widely used in drug residue analysis. However, the prerequisite for using ELISA to detect quizalofop-p-ethyl compounds is obtaining monoclonal antibodies with high specificity and sensitivity to these compounds. Therefore, finding a monoclonal antibody with high specificity and sensitivity to quizalofop-p-ethyl compounds is crucial. However, the specificity and sensitivity of existing monoclonal antibodies for quizalofop-p-ethyl compounds remain relatively low. Summary of the Invention
[0005] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a monoclonal antibody with high specificity and high sensitivity to quizalofop-p-ethyl compounds in the prior art.
[0006] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes monoclonal antibodies against quizalofop-p-ethyl compounds. The monoclonal antibodies secreted by this hybridoma cell line exhibit good sensitivity and specificity against quizalofop-p-ethyl compounds, wherein the IC50 of quizalofop-p-ethyl... 50 The value was 0.53 ng / mL, and the IC50 of oxadiazon was... 50 The value was 0.46 ng / mL, and the IC50 of oxazolidinyl was... 50 The value is 0.84 ng / mL, and there is no cross-reactivity with structural analogs of quizalofop-P-ethyl, such as quizalofop-P-ethyl, clodinafop-P-ethyl, pyrifluquinazon, and quizalofop-P-ethyl, thus enabling accurate detection of low concentrations of quizalofop-P-ethyl compounds.
[0007] The first objective of this invention is to provide a hybridoma cell line that was deposited on October 31, 2024, 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. 46225.
[0008] Furthermore, the hybridoma cell line is obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structural formula of the hapten is shown in Formula I:
[0009]
[0010] Furthermore, the complete antigen is obtained from the hapten coupled with a carrier protein.
[0011] Furthermore, the carrier protein includes keyhole hemocyanin.
[0012] A second objective of this invention is to provide an application of the above-mentioned hybridoma cell line in the detection of quizalofop-p-ethyl compounds.
[0013] Furthermore, the quizalofop-p-ethyl compounds include quizalofop-p-ethyl, quizalofop-p-ethyl, and quizalofop-p-ethyl.
[0014] A third objective of this invention is to provide a monoclonal antibody secreted by the aforementioned hybridoma cell line.
[0015] A fourth objective of this invention is to provide the application of the above-mentioned monoclonal antibody in the detection of quizalofop-p-ethyl compounds.
[0016] A fifth objective of this invention is to provide a detection product for quizalofop-p-ethyl compounds, the detection product comprising the aforementioned monoclonal antibody.
[0017] Furthermore, the test product also includes a coating agent.
[0018] Furthermore, the coating is prepared from a hapten-conjugated carrier protein, wherein the carrier protein includes chicken ovalbumin.
[0019] The beneficial effects of this invention are:
[0020] The monoclonal antibody secreted by the hybridoma cell line described in this invention exhibits high sensitivity and specificity against penoxsulam-like compounds. Specifically, the IC50 of penoxsulam... 50 The value was 0.53 ng / mL, and the IC50 of oxadiazon was... 50 The value was 0.46 ng / mL, and the IC50 of oxazolidinyl was... 50 The concentration was 0.84 ng / mL, therefore, trace amounts of quizalofop-p-ethyl compounds in the sample could be detected. Furthermore, the monoclonal antibody of this invention showed no cross-reactivity with structural analogs of quizalofop-p-ethyl compounds, such as quizalofop-p-ethyl, clodinafop-propargyl, pyrifluquinazon, and quizalofop-p-ethyl, thus exhibiting good specificity. This invention provides an immunological method for detecting the content of quizalofop-p-ethyl compounds in samples, and has practical application value.
[0021] Preservation of biological materials
[0022] The monoclonal cell line OLZ was deposited on October 31, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46225, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description
[0023] 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, wherein...
[0024] Figure 1 This is the inhibition standard curve of monoclonal antibodies against quizalofop-p-ethyl compounds. Detailed Implementation
[0025] 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.
[0026] The culture media involved in the following examples are as follows:
[0027] 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.
[0028] The reagents involved in the following examples are as follows:
[0029] Carbonate buffer (CBS): Weigh 1.59g of Na2CO3 and 2.93g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000mL. Store at 4℃ for later use.
[0030] Phosphate-buffered saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O, dissolved in 800mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000mL.
[0031] PBST: PBS containing 0.05% Tween 20;
[0032] Antibody dilution buffer: PBS containing 0.1% gelatin;
[0033] TMB colorimetric solution: Solution A: 18.43g Na2HPO4·12H2O, 9.33g citric acid, diluted to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution. Mix again before use.
[0034] The detection methods involved in the following embodiments are as follows:
[0035] Method for detecting the inhibition rate of quizalofop-p-ethyl compounds: 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 dilution buffer. After selecting the optimal operating point, the quizalofop-p-ethyl compound standard was diluted to eight concentrations (0, 0.041, 0.123, 0.37, 1.11, 3.33, 10, and 30 ng / mL). Following the ic-ELISA procedure, the results were plotted using OriginPro 8.5 (see results below). Figure 1 (As shown), standard inhibition curves of quizalofop-p-ethyl compounds were obtained, and IC50 was calculated. 50 .
[0036] Example 1: Synthesis of hapten from quizalofop-p-ethyl compounds
[0037] Since the small molecule of quizalofop-P-ethyl is not immunogenic and cannot stimulate an immune response in mice to produce antibodies, it is necessary to conjugate quizalofop-P-ethyl to a protein using protein conjugation technology to acquire immunogenicity. Commonly used reactive groups in protein conjugation technology include amino, carboxyl, hydroxyl, and thiol groups. However, since the molecular structure of quizalofop-P-ethyl does not contain these reactive groups, the analogue Fenoxaprop acid (CAS: 95617-09-7) was found as a hapten for immunization. The structural formula of the hapten is shown in Formula I.
[0038]
[0039] Example 2: Synthesis of complete antigens from quizalofop-p-ethyl compounds
[0040] Weigh 6 mg of quizalofop-P-ethyl hapten and 4.4 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 7.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and add it to the quizalofop-P-ethyl hapten solution, stirring at room temperature for 6-8 h to activate it. Take 6 mg of keyhole hemocyanin (KLH), add it to 3 mL of 0.01 M carbonate buffer (CBS), dissolve it completely, and slowly add the activated hapten to the diluted KLH solution, stirring overnight at room temperature. Then dialyze with 0.01 M PBS to remove unreacted small molecules, obtaining a relatively pure complete antigen, which is then identified by UV absorption scanning.
[0041] Example 3: Synthesis of coating agents for quizalofop-p-ethyl compounds
[0042] 3.2 mg of quizalofop-P-ethyl hapten and 2.4 mg of N-hydroxysuccinimide (NHS) were dissolved in 200 μL of anhydrous N,N-dimethylformamide (DMF) and reacted with the solution at room temperature for 10 min. 4.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in the above solution and reacted with the solution at room temperature for 6-8 h to obtain the hapten activation solution. 6 mg of chicken ovalbumin (OVA) was dissolved in carbonate buffer (CBS). The hapten activation solution was slowly added to the protein dilution solution and stirred overnight at room temperature. The reaction solution was then dialyzed against 0.01 M PBS to remove unreacted small molecules, yielding the coating antigen.
[0043] Example 4: Preparation of hybridoma cell lines secreting monoclonal antibodies against quizalofop-p-ethyl compounds
[0044] 1. Acquisition of immunity in animals
[0045] The complete antigen of quizalofop-P-ethyl compounds 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 neck and back (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.
[0046] 2. Cell fusion
[0047] 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:
[0048] 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 minutes), 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.
[0049] 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 / year 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.
[0050] 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.
[0051] 3. Cell selection and cell line establishment
[0052] 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.
[0053] The screening process consists of two steps: First, positive cell wells are selected using ic-ELISA. Second, quizalofop-p-ethyl is used as a standard, and the inhibitory effect on positive cells is determined using ic-ELISA.
[0054] Cell wells that showed good inhibition of the quizalofop-p-ethyl standard were selected, and subcloning was performed using the limiting dilution method. The results were then tested using the same method after seven days.
[0055] At least three subcloning operations were performed using the method described above to finally obtain monoclonal antibody cell lines containing quizalofop-p-ethyl compounds.
[0056] Example 5: Preparation and identification of monoclonal antibodies against quizalofop-p-ethyl compounds
[0057] 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 hybridoma cells containing quizalofop-p-ethyl compounds starting on day 7, and the ascites fluid was purified for antibody purification using the caprylic acid-saturated ammonium sulfate method.
[0058] 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 antibodies are dissolved in 0.01M PBS solution (pH 7.4), dialyzed to desalt, and finally purified monoclonal antibodies are obtained and stored at -20℃.
[0059] Using an indirect competitive ELISA, monoclonal antibodies against quizalofop-p-ethyl compounds were found to have good detection sensitivity for these compounds (IC50 of quizalofop-p-ethyl). 50 The value was 0.53 ng / mL, and the IC50 of oxadiazon was... 50 The value was 0.46 ng / mL, and the IC50 of oxazolidinyl was... 50 With a value of 0.84 ng / mL, it can be used for the immunoassay detection of quizalofop-p-ethyl compounds.
[0060] Cross-reactivity experiments were conducted on structural analogs of quizalofop-P-ethyl (quizalofop-P-ethyl, clodinafop-P-ethyl, pyrifluquinazon, and quizalofop-P-ethyl). The results showed that the absorbance values of the microplate for the structural analogs did not differ significantly with the dilution concentration of the standard. The specific results are shown in Table 1. This indicates that the cross-reactivity rate of the monoclonal antibody to other structural analogs is very low.
[0061] Cross-reactivity rate (%) = (Synoxazone / Clostridium perfringens IC50) 50 ) / (Similar IC 50 )×100%
[0062] Example 6: Application of monoclonal antibody against quizalofop-p-ethyl
[0063] The monoclonal antibody prepared from hybridoma cell lines via in vivo ascites fluid was applied to the ELISA addition and recovery assay for quizalofop-p-ethyl. The specific steps are as follows:
[0064] Coat 96-well microplates with 0.1 μg / mL of the coating stock diluted with carbonate buffer (CBS), 100 μL per well. After drying at 37°C for 2 h, wash the plates three times with PBST washing buffer, 200 μL per well each time, for 3 min each time, and pat dry.
[0065] Block with CBS containing 0.2% gelatin, 200 μL per well, dry at 37°C for 2 h, wash three times with PBST washing solution, 200 μL per well each time, 3 min each time, and pat dry.
[0066] Prepare standard solutions of 0, 0.037, 0.111, 0.333, 1, 3, 9 and 27 ng / mL of quizalofop-P-ethyl using phosphate-buffered saline (PBS). Add the standard solutions and the extracts of the samples to be tested to the pre-blocked microplates, 50 μL per well, with each sample repeated in 3 wells. Then add 50 μL of monoclonal antibody of quizalofop-P-ethyl diluted to 0.1 μg / mL to each well. Incubate at 37°C for 30 min, then wash and blot dry.
[0067] 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°C for 30 min, then wash and blot dry.
[0068] Add 100 μL of TMB colorimetric solution to each well, develop at 37℃ for 15 min, then add 50 μL of 2M H2SO4 stop solution to each well, and measure the absorbance at 450 nm.
[0069] Add recycling and sample pretreatment:
[0070] Rice was selected as the test sample.
[0071] After being pulverized, the sample was passed through a 20-mesh standard sieve. Three 20g portions of the sample were weighed, and 0.1 ppb, 0.5 ppb, and 2 ppb of quizalofop-p-ethyl standard were added to each portion (based on the antibody linear range and IC50). 50 (Set the concentration), add 10 mL of water, vortex mix, let stand for 30 min, and filter. Add 50 mL of acetone to the sample, shake on an electric shaker for 30 min, filter through rapid qualitative filter paper into a beaker, extract the residue again with 30 mL of acetone using the above method, wash the residue twice with 30 mL of acetone, combine the washings in a beaker, concentrate to near dryness in a 50℃ water bath, and reconstitute with 5 mL of 10% acetone-PBS solution (i.e., diluted five times to reduce the influence of the sample matrix).
[0072] Spiking recovery tests were conducted using indirect competitive ELISA, and the recoveries were 93.2%-108% for quizalofop-p-ethyl, 91.3%-118% for quizalofop-p-ethyl, and 96.9%-119.8% for quizalofop-p-ethyl.
[0073] Table 1. Specificity detection results of monoclonal antibodies against quizalofop-p-ethyl compounds.
[0074]
[0075] 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, characterized in that, The hybridoma cell line was deposited on October 31, 2024, 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. 46225.
2. The application of the hybridoma cell line according to claim 1 in the detection of quizalofop-p-ethyl compounds, characterized in that, The compounds in the quizalofop-p-ethyl class are quizalofop-p-ethyl, quizalofop-p-ethyl, and / or quizalofop-p-ethyl.
3. A monoclonal antibody secreted by the hybridoma cell line of claim 1.
4. The application of the monoclonal antibody according to claim 3 in the detection of quizalofop-p-ethyl compounds, characterized in that, The compounds in the quizalofop-p-ethyl class are quizalofop-p-ethyl, quizalofop-p-ethyl, and / or quizalofop-p-ethyl.
5. A detection product for quizalofop-p-ethyl compounds, characterized in that, The detection product includes the 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 6, characterized in that, The coating is prepared from a hapten-coupled carrier protein, wherein the carrier protein includes chicken ovalbumin.