CN113736743A

CN113736743A - Hybridoma cell strain secreting monoclonal antibody against bisamide compounds and application thereof

Info

Publication number: CN113736743A
Application number: CN202111082512.3A
Authority: CN
Inventors: 胥传来; 郭鑫; 匡华; 刘丽强; 宋珊珊; 胡拥明; 吴爱红; 徐丽广; 郝昌龙; 孙茂忠; 马伟
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-09-15
Filing date: 2021-09-15
Publication date: 2021-12-03
Anticipated expiration: 2041-09-15
Also published as: CN113736743B

Abstract

The invention provides a hybridoma cell strain secreting monoclonal antibodies against bisamide compounds and application thereof, belonging to the technical field of immunochemistry. The preservation number of the hybridoma cell strain is as follows: CGMCC No. 22322. The invention mixes and emulsifies complete antigen and Freund's adjuvant, carries out subcutaneous immunization on mice, and leads the mice to have high titer and low IC₅₀The spleen cells of the mouse are fused with myeloma cells of the mouse by a PEG method, and the hybrid cells after the fusion of the two cells are screened out by adopting a selective culture medium; screening the cells by an indirect competitive enzyme-linked immunosorbent assay for three timesSubcloning to finally obtain a monoclonal antibody hybridoma cell strain. The monoclonal antibody secreted by the cell strain has good detection sensitivity on the chlorantraniliprole, the cyantraniliprole, the chlorantraniliprole and the tetrachloro-cyantraniliprole, and can be used for detecting the residues of the four pesticides in food.

Description

Hybridoma cell strain secreting monoclonal antibody against bisamide compounds and application thereof

Technical Field

The invention belongs to the technical field of immunochemistry, and particularly relates to a hybridoma cell strain secreting monoclonal antibodies against bisamide compounds and application thereof.

Background

Cyclobromantraniliprole (cycloaniliprole), cyantraniliprole (cyantraniliprole), chlorantraniliprole (chlorantraniliprole) and tetrachlorantraniliprole (tetrachlorantraniliprole) belong to the bisamide insecticides. The bisamide pesticide belongs to a ryanodine receptor activator, has excellent control effect on lepidoptera pests and the like on crops such as rice and the like, is safe to non-target organisms, and has no cross resistance with the prior pesticides with other action modes. Currently, 4 varieties are commercialized, including flubendiamide marketed in 2006, chlorantraniliprole marketed in 2007, cyantraniliprole marketed in 2013, and chlorantraniliprole marketed in 2014.

The chlorantraniliprole is a lead compound of the DuPont company which takes the flubendiamide as a lead compound, and is discovered through deep lead optimization research, has certain systemic conductivity and wider insecticidal spectrum, and can be popularized and applied in a large area because the chlorantraniliprole has obtained all certificates for registration, sale and application of pesticides. The chemical structure of chlorantraniliprole has a brand new insecticidal principle which any other insecticides do not have, and the chlorantraniliprole can efficiently activate receptors of the animalia spinosa. Excessive release of calcium ions in calcium stores in cells leads to paralysis and death of insects, and has high activity to larvae of lepidoptera pests, wide insecticidal spectrum and good persistence. According to the current test result, the activity of the compound preparation on target pests is 10-100 times higher than that of other products, and the compound preparation can cause the disorder of the mating process of some lepidoptera insects, and researches prove that the compound preparation can reduce the egg laying rate of various noctuidae pests, and the characteristics are actually the comprehensive embodiment of the functions of permeability, conductivity, chemical stability, high insecticidal activity, immediate feeding stopping of the pests and the like due to the biological characteristics of good persistence and rain wash resistance. It is therefore decided that they have a longer and more stable and crop-protecting action than the other insecticides most currently in use. At present, the pesticide is registered on preventing and controlling main pests of rice, can quickly protect the growth of the rice, has special effect particularly on other pests with resistance to rice pesticides, such as cnaphalocrocis medinalis guenee, chilo suppressalis, tryporyza incertulas and sesamiella inferens guenee, and also has good preventing and controlling effect on rice gall midge, rice weevil and rice weevil. The pesticide is of micro-toxicity level, is very safe for pesticide application personnel, and is also very safe for beneficial insects, fishes and shrimps in the rice field. The lasting period can reach more than 15 days, no residual influence is caused on agricultural products, and the pesticide has good mixing performance with other pesticides.

Cyantraniliprole is a second generation ryanodine receptor inhibitor pesticide successfully developed by DuPont company after chlorantraniliprole, is prepared by changing various polar groups on a benzene ring, has higher efficiency, is more widely applicable to crops, and can effectively control lepidoptera, hemiptera and coleopteran pests.

Shenyang chemical research institute Co., Ltd uses chlorantraniliprole as a lead compound, and discovers a compound chlorantraniliprole with high insecticidal activity by carrying out structural modification on a benzene ring substituent and a pyrazole substituent in the structure of the chlorantraniliprole. It can be used for preventing and controlling chilo suppressalis, cnaphalocrocis medinalis and the like on rice and lepidoptera pests such as diamond back moth, cabbage caterpillar and the like on vegetables.

The cyclic bromoantraniliprole is an insecticide with a bisamide structure developed by Japan stone original industry company, has broad-spectrum insecticidal activity, can be used for numerous crops such as fruit trees, vegetables, potatoes, tea trees, soybeans, cotton and the like, and can be used for preventing and treating lepidoptera, coleopteran, thysanoptera, diptera and homoptera pests. According to the action mechanism classification of the international Insecticide Resistance Action Committee (IRAC), the main action site of the cyclic bromoantraniliprole is a ryanodine receptor allosteric, and unlike other bisamide insecticides, the agent is not completely a ryanodine receptor agent.

At present, methods for detecting the cyclic bromoantraniliprole, the cyantraniliprole, the chlorantraniliprole and the tetrachloro-insect amide mainly adopt instrument detection, and commonly adopt gas chromatography, liquid chromatography and gas chromatography-mass spectrometry. Despite the high sensitivity and specificity of these chromatography-based methods, there are some disadvantages, such as the need for thorough sample purification, high solvent consumption, expensive equipment and skilled technicians. Therefore, a rapid and simple method for analyzing the residue of the cyclic bromoantraniliprole, the cyantraniliprole, the chlorantraniliprole and the tetrachloro-cyantraniliprole is needed.

The enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method, has the advantages of simple pretreatment of a sample during detection, few purification steps, large analysis capacity, low detection cost and simple and convenient operation, and is suitable for field rapid detection of a large number of samples, so the ELISA is widely applied to pesticide residue analysis. The precondition of using the enzyme-linked immunosorbent assay for detection is that a monoclonal antibody with high specificity and high sensitivity to a target detection object is obtained, so that the method for preparing the monoclonal antibody with high specificity and high sensitivity to the cyclic bromoxynil amide, the cyantraniliprole, the chlorantraniliprole and the tetrachloro cyantraniliprole is very key. In the process of preparing hybridoma cell strains capable of secreting monoclonal antibodies against cyromaniliprole, cyantraniliprole, chlorantraniliprole and tetrachloro-cyantraniliprole, how to prepare hapten and complete antigen and how to make mice generate strong immunity, how to make the prepared hybridoma cell strains capable of successfully secreting monoclonal antibodies against cyromaniliprole, cyantraniliprole, chlorantraniliprole and tetrachloro-traniliprole, how to make the secreted monoclonal antibodies have strong specificity and high sensitivity, and further research is needed.

Disclosure of Invention

In order to solve the technical problems, the invention provides a hybridoma cell strain secreting monoclonal antibodies against bisamide compounds and application thereof.

A hybridoma cell strain secreting monoclonal antibodies against bisamide compounds is preserved in China general microbiological culture Collection center at 2021, 05 and 13 months, wherein the preservation address is No. 3 of the Xilu No. 1 of Beijing Korean district, and the preservation number is CGMCC No. 22322.

A preparation method of a hybridoma cell strain secreting monoclonal antibodies against bisamide compounds comprises the following steps:

s1: preparing a bisamide compound hapten, preparing a bisamide compound complete antigen by using the obtained bisamide compound hapten, preparing the obtained bisamide compound complete antigen and a complete Freund adjuvant to obtain an immunogen 1, and emulsifying the obtained bisamide compound complete antigen and the complete Freund adjuvant to obtain an immunogen 2;

s2: subcutaneously immunizing a mouse by the immunogen 1 obtained in the S1;

s3: the immunized mice in S2 are boosted by using immunogen 2 in S1, and are pricked and immunized by using the complete antigen of the bisamide compound;

s4: three days before cell fusion, performing thrust immunization through intraperitoneal injection, wherein the thrust immunization is performed by adopting complete antigen without Freund adjuvant;

s5: taking splenocytes and myeloma cells of the mice after the sprint immunization in S4, carrying out a cell fusion experiment, culturing the fused cells by an HAT culture medium, detecting positive cell pores by indirect ELISA, further determining the inhibition effect of the positive cell pores by an indirect competition ELISA method, subcloning the positive cell pores with the best inhibition by a limiting dilution method, and finally screening out the hybridoma cell strains capable of secreting monoclonal antibodies against the cyclic bromoantraniliprole, the chlorantraniliprole and the tetrachloro diamide to obtain the hybridoma cell strains.

In one embodiment of the present invention, the structural formula of the bisamide hapten in S1 is as follows:

the invention also provides a preparation method of the bisamide hapten, which comprises the following steps:

step 1: mixing 2-amino-3-bromo-5-chlorobenzoic acid and thionyl chloride in an organic solvent, and carrying out reflux reaction to obtain a reaction solution;

step 2: mixing triethylamine and tert-butyl 4-aminobutyrate in an organic solvent to obtain a mixture;

and step 3: and (3) adding the reaction liquid obtained in the step (1) into the mixture obtained in the step (2), uniformly mixing and stirring, reacting, and separating and purifying to obtain the bisamide compound hapten.

In one embodiment of the present invention, the complete antigenic structural formula of the bisamide compound in S1 is:

the invention also provides a preparation method of the bisamide complete antigen, which comprises the following steps:

stirring and reacting bisamide compound hapten and N-hydroxysuccinimide (NHS) in an organic solvent, adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution, and stirring and reacting to obtain a solution A; diluting bovine serum albumin with Carbonate Buffer Solution (CBS) to obtain solution B; adding the solution A into the solution B for reaction to obtain a reaction solution; and (4) dialyzing the reaction solution by using Phosphate Buffer Solution (PBS) to obtain the complete antigen of the bisamide compound.

In one embodiment of the present invention, the first immunization and the booster immunization are separated by one month, the booster immunization is separated by 21 days, and the booster immunization and the sprint immunization are separated by 18 to 21 days in S2 and S4.

In one embodiment of the invention, the primary immunization dose is 100. mu.g/mouse, the booster immunization dose is 50. mu.g/mouse, and the sprint immunization dose is 25. mu.g/mouse in S2 and S4.

In one embodiment of the invention, the immunization process described in S2 and S4 comprises 1 first immunization, 4 booster immunizations, and 1 sprint immunization.

In one embodiment of the present invention, the cell fusion in step 5 is performed 3 days after the completion of the sprint immunization.

In one embodiment of the present invention, the cell fusion in step 5 is performed by a polyethylene glycol (PEG4000) method.

In one embodiment of the present invention, the medium in the step 5 is RPMI-1640 medium.

In one embodiment of the present invention, the number of subclonings in step 5 is 3.

The bisamide compound monoclonal antibody is secreted by the hybridoma cell strain.

In one embodiment of the invention, the bisamide compound is cyclic bromoantraniliprole, cyantraniliprole, chlorantraniliprole or tetrachloro-antraniliprole.

A preparation method of a bisamide compound monoclonal antibody comprises the steps of taking a mouse, injecting paraffin oil into the abdominal cavity, then injecting the hybridoma cell strain into the abdominal cavity, collecting ascites after injection, purifying the ascites to obtain the bisamide compound monoclonal antibody, and storing the bisamide compound monoclonal antibody at a low temperature.

In one embodiment of the invention, the preparation method of the anti-bisamide monoclonal antibody comprises the steps of taking BALB/c mice 8-10 weeks old, injecting 1mL of paraffin oil into the abdominal cavity of each mouse, and injecting 1 x 10 paraffin oil into the abdominal cavity of each mouse 7 days later⁶The hybridoma cell strain is obtained by collecting ascites from day 7Purifying by octanoic acid-ammonium sulfate method, and storing at-20 deg.C.

The application of the anti-bisamide compound monoclonal antibody in identifying the cyclic bromoantraniliprole, the cyantraniliprole, the chlorantraniliprole and the tetrachloro-insect amide.

A composition comprising the anti-bisamide monoclonal antibody.

The composition is applied to detection of the cyclic bromoxydiamide, cyantraniliprole, chlorantraniliprole and tetrachloro-cyantraniliprole.

A kit, which comprises the anti-bisamide monoclonal antibody.

The kit is applied to detection of the cyclic bromoxynil, the cyantraniliprole, the chlorantraniliprole and the tetrachloro-insect amide.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the monoclonal antibodies of anti-cyromaniliprole, cyantraniliprole, chlorantraniliprole and tetrachloro-cyantraniliprole secreted by the hybridoma cell strain have good detection sensitivity (IC) on cyromaniliprole, cyantraniliprole, chlorantraniliprole and tetrachloro-traniliprole₅₀Values of 0.137ng/mL, 0.11ng/mL and 0.165ng/mL) respectively, can be used for establishing an immunological detection method for the cyclic bromine worm amide, the cyanogen bromide amide, the chlorantraniliprole and the tetrachloro worm amide, and detecting the residues of the cyclic bromine worm amide, the cyanogen bromide amide, the chlorantraniliprole and the tetrachloro worm amide in the food.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the present disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1 is a standard curve for inhibition of monoclonal antibodies against chlorantraniliprole, cyantraniliprole, chlorantraniliprole and tetrachloro-cyantraniliprole according to the invention against chlorantraniliprole, cyantraniliprole, chlorantraniliprole and tetrachloro-cyantraniliprole.

FIG. 2 is a nuclear magnetic hydrogen spectrum of Hapten1 of the bisamide compound in example 1 of the invention.

FIG. 3 is a mass spectrum of Hapten1 of bisamide compound in example 1 of the invention.

FIG. 4 is a nuclear magnetic hydrogen spectrum of Hapten2 of the bisamide compound in example 1 of the invention.

FIG. 5 is a mass spectrum of the Hapten Hapten2 of the bisamide compound in example 1 of the present invention.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The media involved in the following examples are as follows:

RPMI-1640 medium (mg/L): l-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine 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, p-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, calcium D-pantothenate 0.25, Folic acid 1, i-inositol 35, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B120.005, and sodium bicarbonate 2000.

The reagents involved in the following examples are as follows:

carbonate Buffer (CBS): weighing Na₂CO₃ 1.59 g，NaHCO₃2.93 g, respectively dissolving in a small amount of double distilled water, mixing, adding the double distilled water to about 800mL, uniformly mixing, adjusting the pH value to 9.6, adding the double distilled water to a constant volume of 1000mL, and storing at 4 ℃ for later use.

Phosphate Buffered Saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH₂PO₄，2.9g Na₂HPO₄·12H₂Dissolving O in 800mL of pure water, adjusting the pH value to 7.2-7.4 by using NaOH or HCl, and fixing the volume to 1000 mL;

PBST: PBS containing 0.05% tween 20;

antibody dilution: PBS containing 0.1% gelatin;

TMB color development liquid: solution A: na (Na)₂HPO_4.12H₂18.43g of O, 9.33g of citric acid and pure water to reach the constant volume of 1000 mL; and B, liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. The liquid B is prepared according to the following steps: 1 to obtain the TMB color developing solution which is mixed at present.

The detection methods referred to in the following examples are as follows:

the method for detecting the inhibition rates of the cyclic bromoantraniliprole, the cyantraniliprole, the chlorantraniliprole and the tetrachloro-insect amide comprises the following steps: the most suitable antigen and antibody concentrations in the ic-ELISA were selected by a checkerboard assay. The antigen was diluted to 0.01,0.03,0.1 and 0.3. mu.g/mL with Carbonate Buffer (CBS) and the antibody was diluted to 0.01,0.03,0.1 and 0.3. mu.g/mL with antibody diluent. After selecting the optimal working point, diluting the standard substances of the cyromaniliprole, the cyantraniliprole, the chlorantraniliprole and the tetrachloro cyantraniliprole to 8 concentrations (0, 0.006, 0.02, 0.06, 0.2, 0.6, 1.8, 5.4ng/mL), obtaining standard inhibition curves of the cyromaniliprole, the cyantraniliprole, the chlorantraniliprole and the tetrachloro traniliprole according to the operation steps of the IC-ELISA, and finally drawing by using originPro8.5 (the result is shown in figure 1), calculating the IC standard inhibition curves of the cyromaniliprole, the cyantraniliprole, the chlorantraniliprole and the tetrachloro traniliprole, and calculating the IC standard inhibition curves of the cyromaniliprole, the cyantraniliprole and the tetrachloro traniliprole₅₀。

Example 1: synthesis of Hapten (Hapten1)

Because the small molecules have no immunogenicity and can not stimulate mice to generate immune response so as to generate antibodies, the small molecules need to be coupled to proteins by a protein connection technology so as to obtain the immunogenicity; active groups commonly used in the protein coupling technology comprise amino, carboxyl, hydroxyl, sulfydryl and the like, and in order to obtain a monoclonal antibody capable of specifically recognizing the cyclic bromoxynil, the cyantraniliprole, the chlorantraniliprole and the tetrachloro-cyantraniliprole, a good hapten needs to be designed and derived, and specific derivation steps are shown as follows.

The method comprises the following steps: synthesis of tert-butyl 4- (2-amino-3-bromo-5-chlorobenzoylamino) butyrate

Adding 1.0g (4.0mmol) of 2-amino-3-bromo-5-chlorobenzoic acid, 10mL of toluene and 0.80mL (12.0mmol) of thionyl chloride into a reaction flask, refluxing for 3 hours, removing the solvent, and adding 5mL of tetrahydrofuran for later use; the solution was dropped into a reaction flask containing 10mL of tetrahydrofuran, 1.7mL (12.0mmol) of triethylamine, and 2.3g (12.0mmol) of tert-butyl 4-aminobutyrate, and the mixture was stirred at room temperature for 0.5 hour, and TLC showed that the reaction was complete. After the solvent was removed, 50mL of water and 200mL of ethyl acetate were added to conduct separation and extraction. The organic phase was washed with 100mL of saturated brine and with anhydrous Na₂SO₄Drying and removing under reduced pressure. The residue was purified by column chromatography to give 1.4g of a white solid (tert-butyl 4- (2-amino-3-bromo-5-chlorobenzoylamino) butyrate).

Step two: synthesis of Hapten Hapten1

1.0g (3.3mmol) of 3-bromo-1- (3-chloropyridin-2-yl) -1H-pyrazole-5-carboxylic acid, 10mL of toluene and 0.68mL (10mmol) of thionyl chloride were added to the reaction flask, refluxed for 3H, the solvent was removed, and 5mL of tetrahydrofuran was added; the above-mentioned stock solution was dropped into a reaction flask containing 10mL of tetrahydrofuran, 1.39mL (10mmol) of triethylamine and 480mg (1.1mmol) of tert-butyl 4- (2-amino-5-chlorobenzoylamino) butyrate, and stirred at room temperature for 0.5 hour, and TLC showed that the reaction was completed. After removing the solvent, adding 50mL of water and 200mL of ethyl acetate for separation and extraction; the organic phase was washed with 100mL of saturated brine and with anhydrous Na₂SO₄Drying and removing under reduced pressure. The residue was purified by column chromatography to give the crude product, which was dissolved in a mixed solution of 5ml tfa and DCM (V/V ═ 1:1) and stirred at room temperature for 0.5 h. The solvent was removed under reduced pressure and the residue was purified by column chromatography to give 190mg of product. The characterization result is as follows:¹H NMR(400MHz,Methanol-d4)δ8.44(dd,J＝4.7,1.6Hz,1H),8.03(dd,J＝8.1,1.6Hz,1H),7.75(d,J＝2.3Hz,1H),7.52(dd,J＝8.1,4.7Hz,1H),7.48(d,J＝2.3Hz,1H),7.32(s,1H),3.28-3.24(m,2H),2.29(t,J＝7.3Hz,2H),1.84-1.71(m,2H).ESI-MS:C₂₀H₁₅Br₂Cl₂N₅O₄+Na⁺required 639.8766, found 639.76 (see FIGS. 2-3 for nuclear magnetic spectra)

Example 2: synthesis of complete antigens

Weighing 2.8mg Hapten (Hapten1) and 1.6mg N-hydroxysuccinimide (NHS), dissolving in 300 μ LN, N-Dimethylformamide (DMF), and stirring at room temperature for reaction for 10 min; then, 2.6mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was weighed, dissolved sufficiently in 100. mu.L of DMF, added to the hapten solution, and reacted for 6 to 8 hours at room temperature with stirring (referred to as solution A). Taking 10mg BSA, diluting to 5mg/mL (called B liquid) by using 0.01M Carbonate Buffer Solution (CBS), slowly adding the A liquid into the B liquid drop by drop, and reacting at room temperature overnight; then dialyzing with 0.01M PBS solution, removing unreacted small molecule hapten to obtain complete antigen, and identifying by ultraviolet absorption scanning method.

Example 3: synthesis of coatingen

To improve sensitivity, a new Hapten for coating (Hapten2) was designed with the following structure:

the synthesis of Hapten (Hapten2) was similar to Hapten (Hapten 1). The coating source synthesis comprises the following specific steps: dissolving 3.6mg Hapten (Hapten2) and 2.3mg N-hydroxysuccinimide (NHS) in 300 μ L anhydrous N, N-Dimethylformamide (DMF), and stirring at room temperature for 10min to obtain Hapten solution; dissolving 3.8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in 100 mu L of anhydrous DMF, adding the solution into a hapten solution, and stirring at room temperature to react for 6-8h to obtain a solution A; diluting 10mg of egg albumin (OVA) with 1mL of Carbonate Buffer Solution (CBS) with the concentration of 0.01mol/L to obtain solution B; slowly adding the solution A into the solution B dropwise for reaction to obtain a reaction solution; dialyzing the reaction solution with PBS solution to remove unreacted small moleculesHapten to obtain coating antigen. The characterization data for the Hapten (Hapten2) are: 1H NMR (400MHz, DMSO-d6) δ 12.52(s,1H),12.13(s,1H),9.02(s,1H),8.54(dd, J ═ 4.7,1.6Hz,1H), 8.30-8.15 (m,2H),7.93(d, J ═ 2.4Hz,1H),7.67(dd, J ═ 8.1,4.7Hz,1H),7.55(dd, J ═ 8.9,2.4Hz,1H),7.19(s,1H),3.33(q, J ═ 6.0Hz,2H),2.32(t, J ═ 7.3Hz,2H),1.79(p, J ═ 7.2Hz,2H), ESI-MS: C₂₀H₁₆BrCl₂N₅O₄+H⁺required 539.9841, found 539.89. (see FIGS. 4-5 for nuclear magnetic characterization).

Example 4: preparation of hybridoma cell strain secreting monoclonal antibodies against chlorantraniliprole, cyantraniliprole, chlorantraniliprole and tetrachloro-cyantraniliprole

1. Obtaining immunity of animals

Mixing and emulsifying a complete antigen and an equivalent amount of Freund's adjuvant, and performing neck-back subcutaneous multipoint injection immunization (except for sprint immunization) on a BALB/c mouse; complete Freund adjuvant is used for the first immunization, and the dosage is 100 ug/mouse; incomplete Freund's adjuvant is used for multiple times of boosting immunization, and the dosage is reduced by half to be 50 ug/mouse; the thorny immunity does not use an adjuvant, the thorny immunity is directly diluted by normal saline and then injected into the abdominal cavity, and the dosage is reduced by half to obtain 25 ug/mouse; one month is separated between the first immunization and the second boosting immunization, 21 days are separated between the multiple boosting immunizations, and 18-21 days are separated between the sprint immunization and the last boosting immunization; the titer and inhibition of the mouse serum are detected by observing the immune effect of the mouse by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).

2. Cell fusion

After three days of the spurting immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight is 4000) method, and the specific steps are as follows:

a. cutting the tail, taking blood, immediately putting the mouse into 75% alcohol for disinfection after the mouse is killed by a cervical vertebra dislocation method, soaking for about 5min, taking out the spleen of the mouse through aseptic operation, properly grinding the spleen by using a rubber head of an injector, passing through a 200-mesh cell screen to obtain a spleen cell suspension, collecting, centrifuging (1200rpm, 8min), washing the spleen cells for three times by using an RPMI-1640 culture medium, diluting the spleen cells to a certain volume after the last centrifugation, and counting for later use;

b. collecting SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) at 5% CO₂In an incubator, the number of SP2/0 tumor cells is required to reach 1-4 multiplied by 10 before fusion⁷Ensuring SP2/0 tumor cells to be in logarithmic growth phase before fusion, collecting the tumor cells during fusion, suspending the tumor cells in RPMI-1640 basic culture solution, and counting the cells;

c. the fusion process is 7 min: 1min, dripping 1mL of PEG into the cells from slow to fast; standing for 2 min; dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4 min; dropping 2mL of RPMI-1640 culture medium within 1min at 5min and 6 min; at 7min, 1mL of RPMI-1640 culture medium is added dropwise every 10 s; then carrying out warm bath at 37 ℃ for 5 min; centrifuging (800rpm, 8min), discarding supernatant, resuspending in RPMI-1640 screening medium containing 20% fetal calf serum, 2% 50 × HAT, adding to 96-well cell plate at 200 μ L/well, standing at 37 deg.C and 5% CO₂Culturing in an incubator.

3. Cell screening and cell line establishment

On day 3 of cell fusion, the fused cells were subjected to RPMI-1640 screening medium half-replacement, on day 5, to total-replacement with RPMI-1640 medium containing 20% fetal bovine serum and 1% 100 XHT, and on day 7, cell supernatants were collected for screening.

The screening is divided into two steps: firstly, screening out positive cell holes by using an ic-ELISA method, and secondly, selecting and using the cyromonamide, the cyantraniliprole, the chlorantraniliprole and the tetrachloro-insect amide as standard substances, and determining the inhibition effect of the positive cells by using the ic-ELISA method.

Selecting cell pores which have better inhibition on the standard substances of the chlorantraniliprole, the cyantraniliprole, the chlorantraniliprole and the tetrachloro cyantraniliprole, performing subcloning by adopting a limiting dilution method, and detecting by using the same method after seven days.

And carrying out subcloning for three times according to the method to finally obtain the cell strain capable of secreting monoclonal antibodies for specifically recognizing the chlorantraniliprole, the cyantraniliprole, the chlorantraniliprole and the tetrachloro-cyantraniliprole.

Example 5: preparation and characterization of monoclonal antibodies

Taking BALB/c mice 8-10 weeks old, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 10⁶Hybridoma cells, ascites fluid was collected from the seventh day, and antibody purification was performed on the ascites fluid by the octanoic acid-saturated ammonium sulfate method.

Under the condition of partial acid, the caprylic acid can precipitate other hybrid proteins except IgG immunoglobulin in the ascites, then the centrifugation is carried out, and the precipitate is discarded; then, the IgG type monoclonal antibody was precipitated with an ammonium sulfate solution of the same saturation, centrifuged, the supernatant was discarded, and the supernatant was dissolved in a 0.01M PBS solution (pH 7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.

Using indirect competitive ELISA, the IC of the monoclonal antibody to the chlorantraniliprole, cyantraniliprole, chlorantraniliprole and chlorantraniliprole is measured₅₀0.137ng/mL, 0.11ng/mL and 0.165ng/mL, which shows that the sensitivity is good, and simultaneously other amide compounds are detected, IC₅₀All are more than 5ppb, have good specificity, and can be used for immunoassay detection as shown in Table 1.

TABLE 1 Cross-reactivity of monoclonal antibodies

Example 6: application of monoclonal antibody

The monoclonal antibody prepared from the hybridoma cell strain through in vivo ascites is applied to an ELISA test, and the specific steps are as follows:

coating a 96-well enzyme label plate with coating antigen with the concentration of 0.1 mu g/mL diluted by Carbonate Buffer Solution (CBS), coating 100 mu L of the enzyme label plate in each well at 37 ℃ for 2h, washing the plate with PBST washing liquor three times, wherein 200 mu L of the washing liquor is used in each well for 3min, and drying by beating;

sealing with CBS containing 0.2% gelatin, sealing at 37 deg.C for 2 hr with 200 μ L per well, washing the plate with PBST lotion for three times, each time with 200 μ L per well, each time for 3min, and drying;

respectively preparing series gradient standard solutions by using Phosphate Buffer Solution (PBS), respectively adding the standard solutions into the closed enzyme label plate, wherein 50 mu L of each well is used for making three wells in parallel, then adding 50 mu L of monoclonal antibody diluted to 0.03 mu g/mL into each well, reacting for 0.5h at 37 ℃, washing the plate and drying;

adding 100 μ L of HRP-labeled goat anti-mouse IgG secondary antibody diluted with PBS containing 0.1% gelatin at a ratio of 1:3000 into each well, reacting at 37 ℃ for 0.5h, washing and drying;

adding 100 μ L of TMB developing solution into each well, developing at 37 deg.C for 15min, and adding 50 μ L of H2M into each well₂SO₄Stop solution, absorbance at 450 nm.

Using the absorbance measured at 450nm as ordinate and standard substance concentration as abscissa, and using originPro8.5 as graph (the result is shown in FIG. 1), obtain standard inhibition curves of Cyromonamide, Cyantraniliprole, Chlorantraniliprole and Tetrachlorantraniliprole, and their corresponding IC₅₀The values are 0.137ng/mL, 0.11ng/mL and 0.165ng/mL respectively, which indicates good sensitivity.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. A hybridoma cell strain secreting monoclonal antibodies against bisamide compounds is characterized in that the hybridoma cell strain is preserved in China general microbiological culture Collection center at 2021, 05 and 13 months, wherein the preservation address is No. 3 of Xilu No. 1 of Beijing Korean district, and the preservation number is CGMCC No. 22322.

2. A preparation method of a hybridoma cell strain secreting monoclonal antibodies against bisamide compounds is characterized by comprising the following steps,

s2: subcutaneously immunizing a mouse by the immunogen 1 obtained in the S1;

s4: and (3) taking spleen cells and myeloma cells of the immunized mouse punched in S3, and carrying out cell fusion to obtain the hybridoma cell strain.

3. The method according to claim 2, wherein the hapten of the bisamide compound in S1 has a structural formula as follows:

4. an anti-bisamide compound monoclonal antibody, which is secreted by the hybridoma cell line of claim 1.

5. The monoclonal antibody against bisamide compounds according to claim 4, wherein the bisamide compound is cyromanilide, cyantraniliprole, chlorantraniliprole or tetrachlorantraniliprole.

6. The use of the monoclonal antibody against bisamide compounds according to claim 4 for recognizing cyclic bromoantraniliprole, cyantraniliprole, chlorantraniliprole or chlorantraniliprole.

7. A composition comprising the monoclonal antibody against a bisamide-based compound according to claim 5.

8. Use of the composition of claim 7 for detecting cyromaniliprole, cyantraniliprole, chlorantraniliprole, and tetrachlorantraniliprole.

9. A kit comprising the monoclonal antibody against a bisamide-based compound according to claim 5.

10. The use of the kit of claim 9 for detecting cimetifugamide, cyantraniliprole, chlorantraniliprole, and tetrachlorantraniliprole.