A hybridoma cell line that secretes monoclonal antibodies against dihydrazide compounds

Abstract

This invention relates to a hybridoma cell line that secretes monoclonal antibodies against diacylhydrazine compounds. The hybridoma cell line of this invention was deposited on April 18, 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. 45916. The monoclonal antibodies secreted by the hybridoma cell line of this invention exhibit good sensitivity and specificity to diacylhydrazine compounds, wherein the IC50 of tebufenozide is [not specified in the original text]. 50 The value was 0.176 ng / mL, and the IC50 of methoxyfenozide was... 50 The value was 0.44 ng / mL, and the IC50 of hydrazine was... 50 The value was 2.48 ng / mL, and the IC50 of cyclophosphamide was... 50 The value was 1.36 ng / mL, and the IC50 of chlorantraniliprole was... 50 With a value of 1.68 ng / mL, it can efficiently and rapidly detect dihydrazide pesticides.

Description

Technical Field

[0001] This invention relates to the field of immunochemistry, and in particular to a hybridoma cell line that secretes monoclonal antibodies against diacylhydrazine compounds. Background Technology

[0002] Dimethyl chlorpyrifos pesticides are nonsteroidal insect growth regulators commonly used in agriculture to control lepidopteran larvae. Dimethyl chlorpyrifos pesticides include products such as chlorfenapyr, tebufenozide, chlorfenapyr, methoxyfenozide, and cyclofenozide. Although dimethyl chlorpyrifos pesticides are considered environmentally friendly insecticides due to their high specificity, their residues in agricultural products may pose potential health risks to humans. Studies have shown that tebufenozide exhibits indiscriminate cytotoxicity in both insect and human cells and can induce G1 / S cell cycle arrest via the mitochondrial pathway, ultimately leading to apoptosis. To mitigate the negative health effects of tebufenozide on humans, many countries have established maximum residue limits (MRLs) for its use in agricultural products. In China, the maximum residue limits (MRLs) for tebufenozide range from 0.05 mg / kg in lilies to 20 mg / kg in leafy vegetables; the MRLs for methoxyfenozide range from 0.02 mg / kg in corn to 20 mg / kg in dried chili peppers; the MRL for phosmet is 20 mg / kg in grains; and the MRL for cyclofenozide is 2 mg / kg in grains. Detecting the presence of diazide pesticide residues in agricultural products is crucial for protecting human health.

[0003] Currently, the detection methods for dihydrazide pesticides 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 skilled technicians. Therefore, a rapid and simple method for detecting dihydrazide pesticide 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 dihydrazide pesticides is obtaining a monoclonal antibody with high specificity and sensitivity to these pesticides. Therefore, the preparation of a monoclonal antibody with high specificity and sensitivity to dihydrazide pesticides is crucial. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the lack of monoclonal antibodies with high specificity and high sensitivity to dihydrazide pesticides in the prior art.

[0006] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes monoclonal antibodies against diacylhydrazine compounds. This hybridoma cell line was deposited on April 18, 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. 45916. The monoclonal antibodies secreted by this hybridoma cell line exhibit good sensitivity and specificity against diacylhydrazine compounds, such as chlorfenapyr, tebufenozide, chlorfenapyr, methoxyfenozide, and cyclofenozide, enabling efficient and rapid detection of diacylhydrazine compounds.

[0007] The first objective of this invention is to provide a hybridoma cell line that was deposited on April 18, 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. 45916.

[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 above-mentioned 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 dihydrazide compounds.

[0013] Furthermore, the dihydrazide compounds include chlorfenapyr, tebufenozide, chlorfenapyr, methoxyfenozide, and cyclofenozide.

[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 an application of a monoclonal antibody in the detection of dihydrazide compounds.

[0016] A fifth objective of this invention is to provide a detection product for dihydrazide 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, including chicken ovalbumin.

[0019] The beneficial effects of this invention are:

[0020] The monoclonal antibodies secreted by the hybridoma cell line described in this invention exhibit high sensitivity and specificity to diacylhydrazine compounds, wherein the IC50 of tebufenozide is... 50 The value was 0.176 ng / mL, and the IC50 of methoxyfenozide was... 50 The value was 0.44 ng / mL, and the IC50 of hydrazine was... 50 The value was 2.48 ng / mL, and the IC50 of cyclophosphamide was... 50 The value was 1.36 ng / mL, and the IC50 of chlorantraniliprole was... 50 The concentration was 1.68 ng / mL, thus allowing the detection of trace amounts of dihydrazide compounds in the sample. This invention provides an immunological method for detecting the content of dihydrazide pesticides in samples, and has practical application value.

[0021] Preservation of biological materials

[0022] The monoclonal cell line Plau was deposited on April 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45916, 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 standard inhibition curve for monoclonal antibodies against dihydrazide 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): Dissolve 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, and 2.9g Na2HPO4·12H2O in 800mL of pure water, adjust the pH to 7.2-7.4 with NaOH or HCl, and bring the volume to 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 dihydrazide 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 diluent. After selecting the optimal operating point, the dihydrazide compound standard was diluted to eight concentrations (0, 0.0098, 0.039, 0.156, 0.625, 2.5, 10, and 40 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 dihydrazide compounds were obtained, and IC50 was calculated. 50 .

[0036] Example 1: Synthesis of diazid haptens

[0037] Since dihydrazide compounds are small molecules and lack immunogenicity, they cannot stimulate an immune response in mice to produce antibodies. Therefore, they need to be coupled to proteins using protein conjugation technology to acquire immunogenicity. Commonly used active groups in protein conjugation technology include amino, carboxyl, hydroxyl, and thiol groups. However, since dihydrazide compounds do not contain these active groups in their molecular structure, dihydrazide compounds are designed for derivatization.

[0038] Benzoic acid, 3,5-diMethyl-,1-(1,1-diMethylethyl)hydrazide (CAS: 162752-59-2, 200 mg) was dissolved in DMF (15 mL), and dimethyl terephthalate (100 mg) was slowly added. The resulting mixture was stirred at 75 °C for 7 h, then concentrated to give a pale yellow viscous substance. The substance was treated with water and extracted with ethyl acetate, then dried over NaSO4, concentrated on a rotary evaporator, and purified on a silica gel column to give a pale yellow solid. The pale yellow solid was dissolved in anhydrous methanol (20 mL) at 25 °C, and NaOH (2 M, 20 mL) was slowly added. The mixture was stirred for 10 min and then heated to 45 °C overnight. The mixture was filtered through a diatomaceous earth filter, and the solid was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (100 mL) and washed successively with water (3 × 100 mL) and brine (100 mL). The organic layer was separated, dried with sodium sulfate, filtered, and concentrated under vacuum to obtain a hapten of a dihydrazide compound as shown in Formula I (60 mg, 8%).

[0039]

[0040] Example 2: Synthesis of complete antigens from dihydrazide compounds

[0041] Weigh 6 mg of a dihydrazide 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 dihydrazide hapten solution. Stir at room temperature for 6-8 h to activate the hapten. Take 6 mg of keyhole hemocyanin (KLH), add it to 3 mL of 0.01 M carbonate buffer (CBS), and dissolve it completely. Slowly add the activated hapten to the diluted KLH solution and stir overnight at room temperature. Then dialyze with 0.01 M PBS to remove unreacted small molecules, obtaining a relatively pure complete antigen, which is then identified by UV absorption scanning.

[0042] Example 3: Synthesis of coating agents of dihydrazide compounds

[0043] 3.2 mg of a dihydrazide 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 same 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.

[0044] Example 4: Preparation of hybridoma cell lines secreting monoclonal antibodies against diacylhydrazine compounds

[0045] (1) Acquisition of immunity in animals

[0046] The complete antigen of a dihydrazide compound 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.

[0047] (2) Cell fusion

[0048] 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:

[0049] 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.

[0050] 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.

[0051] 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.

[0052] (3) Cell screening and cell line establishment

[0053] 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.

[0054] The screening process consists of two steps: First, positive cell wells are selected using ic-ELISA. Second, dihydrazide compounds are used as standards, and the inhibitory effect on positive cells is determined using ic-ELISA.

[0055] Cell wells that showed good inhibition of dihydrazide compounds were selected, and subcloning was performed using the limiting dilution method. The cells were then tested using the same method after seven days.

[0056] At least three subcloning operations were performed using the method described above to finally obtain a monoclonal antibody cell line containing dihydrazide compounds.

[0057] Example 5: Preparation and Identification of Monoclonal Antibodies Based on Dihydrazide Compounds

[0058] 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 diazid hybridoma cells starting on day 7, and the ascites fluid was purified for antibody using the caprylic acid-saturated ammonium sulfate method.

[0059] 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℃.

[0060] Using an indirect competitive ELISA, monoclonal antibodies against diazinon compounds were found to have good detection sensitivity (IC50) for tebufenozide, methoxyfenozide, cyprofenozide, cyclofenozide, and chlorfenapyr. 50 The values ​​were 0.176 ng / mL, 0.44 ng / mL, 2.48 ng / mL, 1.36 ng / mL, and 1.68 ng / mL, respectively, and can be used for the immunoassay detection of dihydrazide compounds.

[0061] Cross-reactivity experiments were conducted on dihydrazides and their structural analogs (benzoylhydrazide, p-aminohippuric acid, 4-biphenylcarboxylic acid hydrazide). The formula for calculating the cross-reactivity rate is: Cross-reactivity rate (%) = (tebufenozide IC50) / (benzylhydrazide) 50 ) / (Similar IC 50 The results were calculated as follows: the absorbance values ​​in each well of the ELISA plate corresponding to benzoyl hydrazine, p-aminohippuric acid, and 4-biphenylcarboxylic acid hydrazine showed no significant difference with the dilution concentration of the standard. Specific results are shown in Table 1. This indicates that the monoclonal antibody has a very low cross-reactivity rate to other structural analogs and good specificity to tebufenozide, methoxyfenozide, cyprofenozide, cyclofenozide, and chlorfenapyr.

[0062] Table 1. Cross-reactivity results of diacidhydrazide compounds and their structural analogs

[0063]

[0064]

[0065] Example 6: Application of monoclonal antibodies against dihydrazide compounds

[0066] Monoclonal antibodies prepared from hybridoma cell lines via in vivo ascites fluid were used in an ELISA addition and recovery assay for dihydrazide compounds. The specific steps are as follows:

[0067] 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.

[0068] 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.

[0069] Prepare standard solutions of dihydrazide compounds at concentrations of 0, 0.037, 0.111, 0.333, 1, 3, 9, and 27 ng / mL using phosphate-buffered saline (PBS). Add the standard solutions and the extracts of the samples to be tested to pre-blocked ELISA plates, 50 μL per well, with each sample repeated in 3 wells. Then add 50 μL of dihydrazide compound monoclonal antibody diluted to 0.1 μg / mL to each well. Incubate at 37°C for 30 min, then wash and blot dry.

[0070] 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.

[0071] Add 100 μL of TMB colorimetric solution to each well, develop the color at 37℃ for 15 min, then add 50 μL of 2M H2SO4 stop solution to each well, and measure the absorbance at 450 nm.

[0072] Recovery and Sample Pretreatment: Rice was selected as the test sample. The sample was pulverized and passed through a 20-mesh standard sieve. Three 20g portions were weighed, and 5ppb, 10ppb, and 20ppb of diazid compound standards were added to each sample respectively (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).

[0073] The recovery rates of spiking cyclophosphamide were 96%-104%, methoxyfenozide 98.3%-113%, chlorfenapyr 93.8%-102.8%, cyclophosphamide 97%-105%, and chlorfenapyr 97.3%-112%, respectively, using indirect competitive ELISA.

[0074] 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 April 18, 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. 45916.

2. The hybridoma cell line according to claim 1, characterized in that, The hybridoma cell line was obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structural formula of the hapten is shown in Formula I: 。 3. The hybridoma cell line according to claim 2, characterized in that, The complete antigen is obtained from the hapten coupled with the carrier protein.

4. The hybridoma cell line according to claim 3, characterized in that, The carrier protein includes keyhole hemocyanin.

5. A monoclonal antibody secreted by a hybridoma cell line according to any one of claims 1-4.

6. The use of the monoclonal antibody according to claim 5 in the detection of dihydrazide compounds, characterized in that, The dihydrazide compounds are chlorfenapyr, tebufenozide, chlorfenapyr, methoxyfenozide, and cyclofenozide.

7. A detection product for dihydrazide compounds, characterized in that, The detection product includes the monoclonal antibody as described in claim 5, and the dihydrazide compound is chlorfenapyr, tebufenozide, chlorfenapyr, methoxyfenozide, and cyclofenozide.

8. The testing product according to claim 7, characterized in that, The tested products also include coating agents.

9. The testing product according to claim 8, characterized in that, The coating is prepared by a hapten-coupled carrier protein represented by Formula I; the carrier protein includes chicken ovalbumin. 。

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