CN114317448B

CN114317448B - Hybridoma cell strain secreting anti-etoxazole monoclonal antibody and application thereof

Info

Publication number: CN114317448B
Application number: CN202111624469.9A
Authority: CN
Inventors: 胥传来; 刘杰; 匡华; 徐丽广; 孙茂忠; 刘丽强; 郝昌龙; 宋珊珊; 胡拥明; 吴爱红; 郭玲玲; 胥欣欣; 倪萍; 毕雪威; 郭鹏飞
Original assignee: Wuxi Determine Bio Tech Co ltd
Current assignee: Wuxi Determine Bio Tech Co ltd
Priority date: 2021-12-28
Filing date: 2021-12-28
Publication date: 2023-11-28
Anticipated expiration: 2041-12-28
Also published as: CN114317448A

Abstract

The invention discloses a hybridoma cell strain secreting anti-etoxazole monoclonal antibodies and application thereof, wherein the hybridoma cell strain AZX is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45015. The hybridoma cell strain AZX can secrete etoxazole with good affinity and high sensitivity, has 50% inhibition concentration IC50 of etoxazole of 4.04ng/mL, can be used for preparing an immunodetection kit and a colloidal gold test strip of etoxazole, and provides a powerful detection method and means for detecting etoxazole in food.

Description

Hybridoma cell strain secreting anti-etoxazole monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of food safety immunodetection, and particularly relates to a hybridoma cell strain secreting anti-etoxazole monoclonal antibodies and application thereof.

Background

Etoxazole (etoxazole) is a novel special acaricide for oxazoles newly developed by Sumitomo chemistry. Has excellent control effect on spider mites of crops such as citrus, cotton, apples, flowers, vegetables and the like, tetranychus urticae, panonychus, tetranychus urticae, tetranychus cinnabarinus and the like. The method has the effects of inhibiting embryo formation of mite eggs and the process of molting from young mites to adult mites, and is effective on eggs and young mites and ineffective on adult mites. The mite is killed by inhibiting embryo formation of mite eggs and molting process from young mites to adult mites, has the effects of contact killing and stomach toxicity, has no systemic property, has stronger permeability and is resistant to rain wash. It has been reported that etoxazole has a negative effect on human peripheral blood lymphocytes, and can cause genetic toxicity and cytotoxicity, thus potentially threatening human health.

There are few reports about the detection method of Guan Yiman azole drug residue in animal tissue, mainly using high performance liquid chromatography-fluorescence detector detection method and liquid chromatography-tandem mass spectrometry. The extraction methods are also different, and include liquid-liquid extraction, liquid-solid extraction, etc. The instrument detection method can perform quantitative analysis and has lower detection limit, but expensive instruments and complex operation are usually required, and the pretreatment and detection time are long, so that the popularization of the detection methods is severely restricted. The immunoassay method has the characteristics of low cost, high flux, high sensitivity, low relative requirements on technicians and the like, so that the method is suitable for rapid screening of a large number of samples. The invention aims to provide a preparation method of a monoclonal antibody hybridoma cell strain with higher affinity and detection sensitivity to etoxazole. Lays a foundation for research and development and popularization of indirect competition ELISA kits and colloidal gold test strips.

Disclosure of Invention

The invention aims to: in order to overcome the defects in the prior art, the invention provides an anti-etoxazole monoclonal antibody hybridoma cell strain and application thereof, and a monoclonal antibody prepared by the cell strain has better affinity and sensitivity to etoxazole, and can be used for establishing an etoxazole enzyme-linked immunosorbent assay method or a colloidal gold immunochromatographic test strip rapid detection method.

The technical scheme is as follows: in order to achieve the above purpose, the invention adopts the following technical scheme:

the first object of the present invention is to provide a hybridoma cell strain secreting and secreting anti-etoxazole monoclonal antibody, which is preserved in the China general microbiological culture Collection center, called monoclonal cell strain AZX for short, with a preservation number of CGMCC No.45015 and a preservation address of Beicheng Kong region, beicheng Xiyu No. 1, 3, national academy of sciences of China.

The second object of the invention is to provide an anti-etoxazole monoclonal antibody which is secreted by a monoclonal cell strain AZX with the preservation number of CGMCC No.45015.

The third object of the invention is to provide an anti-etoxazole hapten, which is an etoxazole derivative with carboxyl, and the structure of the anti-etoxazole hapten is shown as the following formula (I):

the fourth object of the invention is to provide a synthesis method of the anti-etoxazole hapten, which comprises the following steps:

s1, dissolving a compound 1, namely 1-tertiary butyl-3-methoxybenzene, in methylene dichloride, cooling to-5-0 ℃, adding chloroacetyl chloride and anhydrous aluminum chloride, reacting, and extracting and separating to obtain a yellow oily compound 2;

s2, dissolving the compound 2 in ethanol water solution at room temperature, adding sodium formate, stirring for reaction, and removing the solvent to obtain a pale white solid compound 3;

s3, dissolving the compound 3 in a methanol solution of sodium acetate, adding hydroxylamine hydrochloride, heating to 50-60 ℃ and keeping for 2-4 hours, cooling to room temperature, concentrating, extracting and concentrating to obtain a pale yellow solid compound 4;

s4, dissolving the compound 4 in ethanol and tetrahydrofuran, adding a catalyst, hydrogenating, and filtering the catalyst to obtain a light brown solid compound 5;

s5, dissolving the compound 5 and triethylamine in tetrahydrofuran, cooling and stirring, adding 4-bromo-2, 6-difluorobenzoyl chloride, stirring for 5-8 hours at room temperature, filtering and concentrating to obtain a brown solid compound 6;

s6, dissolving the compound 6 and thionyl chloride in benzene, stirring and refluxing in an oil bath for 2-4 hours, and concentrating and filtering to obtain a colorless solid compound 7;

s7, dissolving the compound 7, palladium acetate and 2-dicyclohexylphosphine-2 ',6' -dimethoxy-1, 1' -biphenyl in degassed tetrahydrofuran, stirring at room temperature under nitrogen, adding a tetrahydrofuran solution of ethyl 4-bromobutyrate, stirring at 50-60 ℃ for 5-8 hours, extracting and collecting an organic phase, and obtaining a white solid compound 8 after chromatography;

s8, dissolving the compound 8 in a THF aqueous solution, adding NaOH, stirring at room temperature for 12-18h, concentrating, acidifying to pH of 2-3, extracting an organic layer, and purifying to obtain an off-white solid target compound, namely the etoxazole hapten.

The carboxyl of the hapten prepared by the invention is positioned on the benzene ring at the side of the etoxazole, and the carboxyl of the connecting arm with proper length is derived on the basis of the etoxazole structure on the basis of not changing the etoxazole structure. From the perspective of hapten design principle, the hapten coupling protein can better realize the whole structure exposure of etoxazole due to the superiority of carboxyl position, so that monoclonal antibodies with higher sensitivity and specificity can be generated.

In one embodiment of the present invention, in the step S1, the mass ratio of the compound 1 to chloroacetyl chloride is 20: 15.21.

In one embodiment of the present invention, in the step S2, a volume ratio of ethanol to water in the aqueous ethanol solution is 7:3, adding sodium formate, and stirring for 12-18 hours at 80-100 ℃.

In one embodiment of the present invention, in the step S4, the catalyst comprises palladium on carbon, and the hydrogenation is performed at room temperature and atmospheric pressure for 18 to 24 hours.

In one embodiment of the present invention, in the step S5, the mass ratio of the compound 5, triethylamine and 4-bromo-2, 6-difluorobenzoyl chloride is 4.5:2.88:6.3.

in one embodiment of the present invention, in the step S6, the mass ratio of the compound 6 to thionyl chloride is 3.5: 4.87.

In one embodiment of the present invention, in the step S7, the mass ratio of the palladium acetate compound 7 to the 2-dicyclohexylphosphine-2 ',6' -dimethoxy-1, 1' -biphenyl is 1.5:269:550, the concentration of the tetrahydrofuran solution of the ethyl 4-bromobutyrate is 0.6mol/L.

The fifth object of the present invention is to provide an anti-etoxazole complete antigen, wherein the structural formula of the anti-etoxazole complete antigen is shown as the following formula (II):

the sixth object of the present invention is to provide a method for synthesizing the anti-etoxazole complete antigen, which comprises the following steps: dissolving the etoxazole hapten, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in N, N-dimethylformamide, stirring at room temperature, adding the activated liquid after 6-8h of activation into a carrier protein solution dropwise, stirring at room temperature for reaction overnight, and dialyzing to obtain the etoxazole complete antigen;

the anti-etoxazole hapten is an etoxazole derivative with carboxyl, and the structure is shown as the following formula (I):

the carrier protein solution is prepared by dissolving carrier proteins in carbonate buffer solution, wherein the carrier proteins comprise any one of KLH, BSA, OVA.

In one embodiment of the invention, the mass ratio of the etoxazole hapten, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 4.5:5.0:3.7.

in one embodiment of the invention, the carrier protein solution is prepared from 15mg of carrier protein dissolved in 3mL of 0.05M carbonate buffer at pH 9.6.

In one embodiment of the present invention, the preparation of the YY cell line provided by the present invention comprises the following basic steps:

(1) Synthesis of anti-etoxazole hapten: the synthesis of the target compound, as shown in formula (I), is structurally equivalent to the derivatization of carboxyl groups based on etoxazole, so as to facilitate the ligation of carrier proteins.

(2) Preparation and identification of immunogens: the etoxazole hapten is used as a raw material, is connected with amino groups of a protein carrier through an activated ester method, and after the reaction is finished, the complete antigen and unconjugated small molecule hapten are separated through dialysis, and the complete antigen is identified through an ultraviolet absorption scanning method;

(3) Immunization of mice: BALB/c mice of 6-8 weeks of age were selected for immunization. After the immunogen and the Freund's adjuvant are completely emulsified, the mice are immunized by subcutaneous multipoint injection, the Freund's complete adjuvant is adopted for primary immunization, the Freund's incomplete adjuvant is adopted for boosting immunization, the immunization dose is half of the previous immunization dose during sprint immunization, and the mice are directly injected into the abdominal cavity after being uniformly mixed with normal saline; each immunization interval was three weeks. After the third immunization, blood sampling is carried out at intervals of one week to detect serum titer and inhibition;

(4) Cell fusion and cell strain establishment: fusing the spleen cells of the mice and myeloma cells of the mice by a polyethylene glycol (PEG 2000) method, culturing by a HAT culture medium, detecting positive cell holes by using an indirect ELISA (enzyme-linked immunosorbent assay), further measuring the inhibition effect of the positive cell holes by using an indirect competition ELISA method, subcloning the positive cell holes with the best inhibition for three times by using a limiting dilution method, and finally screening to obtain hybridoma cell strains YY;

(5) Identification of hybridoma cell line properties: adopting enzyme-labeled secondary antibody suit determination for mouse monoclonal antibody Ig class/subclass identification; determination of IC50 values, cross-reactivity and affinity was by ELISA.

The seventh object of the invention is to provide the application of the anti-etoxazole monoclonal antibody, which is an immune detection method for establishing the anti-etoxazole content and is applied to the detection of the anti-etoxazole in food.

In one embodiment of the invention, the anti-etoxazole monoclonal antibody is used for preparing a detection main body for analyzing and detecting the residual amount of the anti-etoxazole in food safety detection.

In one embodiment of the present invention, the detection body includes a reagent, a detection plate, and a kit.

Preservation of biological material samples: the hybridoma cell strain secreting the anti-etoxazole monoclonal antibody is preserved in China General Microbiological Culture Center (CGMCC) of China Committee for culture Collection of microorganisms, and is named as monoclonal cell strain AZX in China institute of sciences of China, no. 3 of North-West-Lu No. 1, the Korean area of Beijing, and the preservation date is 2021, 12 months and 16 days, and the preservation number is CGMCC No.45015.

The beneficial effects are that: compared with the prior art, the hybridoma cell strain secreting the anti-etoxazole monoclonal antibody and the application thereof have the following advantages:

(1) The anti-etoxazole monoclonal antibody obtained by the invention has better detection sensitivity and affinity for etoxazole, and the 50% inhibition concentration IC50 for etoxazole is 4.04ng/mL;

(2) The anti-etoxazole monoclonal antibody can be used for preparing an etoxazole immunodetection kit and a colloidal gold test strip, and provides a powerful detection method and means for detecting etoxazole in food;

(3) The new method for synthesizing the etoxazole hapten and the immunogen is provided, the synthesis steps are simplified and effective, and the thought and the method for synthesizing the immunogen are provided for the research of people in future.

Drawings

FIG. 1 is a synthetic route for etoxazole hapten in an embodiment of the invention.

FIG. 2 is a standard inhibition curve of etoxazole monoclonal antibodies in examples of the invention.

Detailed Description

The invention will be further described with reference to the drawings and examples. The invention will be better understood from the following examples. However, it will be readily understood by those skilled in the art that the specific material ratios, process conditions and results thereof described in the examples are illustrative of the present invention and should not be construed as limiting the invention described in detail in the claims.

According to the invention, the whole etoxazole antigen is used for immunizing a mouse, the cell fusion is carried out, the HAT selective medium is used for culturing, and the cell supernatant is screened by indirect ELISA and indirect competition ELISA, so that the monoclonal antibody hybridoma cell strain with better affinity and sensitivity to etoxazole is finally obtained.

Example 1: preparation of anti-etoxazole monoclonal antibody hybridoma cell strain YY

1. Synthesis of hapten:

the synthesis route of etoxazole hapten shown in the attached figure 1 comprises the following steps:

s1, 20g of compound 1 is dissolved in 200mL of dichloromethane solution, the solution is placed in a three-necked flask and cooled to-5 ℃, and 15.21g of chloroacetyl chloride is slowly added into the three-necked flask while stirring, so that the compound 1 is dissolved in dichloromethane. The reaction flask was charged with anhydrous aluminum chloride in three portions while maintaining the low temperature. After 1 hour, the reaction was substantially completed, and the reaction mixture was poured into 300mL of hydrochloric acid-ice water, and the mixture was stirred for half an hour until the reaction became pale yellow green. Dichloromethane was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and the organic solvent was distilled off to give compound 2 (25 g,87.45% yield) as a yellow oil.

S2, mixing at room temperature in 7: to a stirred solution of compound 2 (18 g,70.6 mol) in ethanol water was added sodium formate (24.03 g,353.28 mmol) and stirred at 100℃for 12 hours. The solvent was removed in vacuo and the resulting crude product was diluted with water and the precipitate filtered through a fritted funnel, washed with water and the solid dried under high vacuum to give compound 3 (13.5 g,80.8% yield) as an off-white solid.

S3 7.65g hydroxylamine hydrochloride was added to a methanol solution of compound 3 (13 g,55.01 mmol) and sodium acetate (9.03 g,110.03 mmol). The solution was heated to 50 ℃ for 2 hours. Cooled to 25 ℃ and concentrated. To the residue was added water and toluene, the layers were separated and the aqueous phase was extracted with toluene. The organic extracts were combined and the solution was concentrated with water to give compound 4 (11.5 g,83.18% yield) as a pale yellow solid.

S4, mixing Compound 4 (10 g,39.79 mmol) in ethanol and tetrahydrofuran (ethanol 200mL, tetrahydrofuran 200 mL) with 4g of palladium on carbon as catalyst, hydrogenating the resulting mixture at 23℃and atmospheric pressure for 18 hours, filtering off the catalyst, washing with ethanol, evaporating the filtrate entirely and drying under high vacuum to give Compound 5 (8.5 g,90.01% yield) as a light brown solid.

S5A mixture of 4.5 g of Compound 5, 2.88 g of triethylamine and 100ml of tetrahydrofuran was cooled and stirred, 6.3 g of 4-bromo-2, 6-difluorobenzoyl chloride was slowly added, and the mixture was stirred at room temperature for 5 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give compound 6 (6.5 g,75.13% yield) as a brown solid.

S6A mixture of 3.5g of Compound 6, 4.87g of thionyl chloride and 50ml of benzene was stirred at reflux on an oil bath for 2 hours. The reaction solution was brought to room temperature and concentrated under reduced pressure; to the concentrate was added 100ml of ethyl acetate; the mixture was washed with saturated aqueous sodium hydrogencarbonate solution and saturated brine solution in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. To the concentrate was added 50ml of methanol and 10ml of 20% aqueous sodium hydroxide solution in this order, followed by stirring at 70℃for 30 minutes. The reaction mixture was concentrated under reduced pressure, 100ml of benzene was added to the concentrated solution, and the mixture was washed with saturated brine and dried over anhydrous sodium sulfate. The dried solution was concentrated under reduced pressure, and the concentrated solution was purified by silica gel column chromatography (mobile phase: hexane: ethyl acetate=7:3). The purified product was dissolved in 50ml of hexane by heating, and the solution was left overnight at room temperature. The precipitated crystals were collected by filtration to give compound 7 (2.2 g,65.44% yield) as a colorless solid.

S7 to a 100mL dry round bottom flask was added 1.5g of Compound 7, 269mg of palladium acetate and 550mg of 2-dicyclohexylphosphine-2 ',6' -dimethoxy-1, 1' -biphenol (S-Phos). Then 30m degassed tetrahydrofuran was added and stirred at room temperature under nitrogen for 5 minutes. Then 30mL of a 0.6mol/L solution of ethyl 4-bromobutyrate (4-bromoxyrate) in tetrahydrofuran was added, and the mixture was stirred at 50℃for 5 hours. The organic phase was collected, dried over sodium sulfate, and the filtrate was concentrated under reduced pressure, and the residue was chromatographed on silica gel (0-50% ethyl acetate in hexane) to give compound 8 (930 mg,55.35% yield) as a white solid.

S8, naOH was added to a mixed solution of Compound 8 (900 mg,1.90 mmol) in Tetrahydrofuran (THF) and water at room temperature and the reaction mixture was stirred at room temperature for 12h. The reaction mixture was concentrated and the residue was acidified to pH 2 with 1.0M aqueous HCl and the combined organic layers were extracted with ethyl acetate (EtOAc) with Na ₂ SO ₄ Drying, filtering and concentrating the filtrate to provide a crude product; purification using preparative HPLC gave the target compound anti-etoxazole hapten (550 mg,64.9% yield) as an off-white solid.

2. Complete antigen synthesis: taking 4.5mg of the hapten, adding 5.0mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 3.7mg of N-hydroxysuccinimide (NHS), dissolving by using N, N-Dimethylformamide (DMF), stirring at room temperature, and activating for 6 hours; another 15mg of Keyhole Limpet Hemocyanin (KLH) was dissolved in 3mL, 0.05M, pH9.6 Carbonate Buffer (CBS) solution; and (3) dropwise adding the activating solution into a BSA solution, stirring at room temperature for reaction overnight, taking out the immunogen, dialyzing with PBS for 3 days, and subpackaging at-20 ℃ for storage to obtain the etoxazole complete antigen.

3. Animal immunization: healthy BALB/c mice of 6-8 weeks of age were selected for immunization. After the etoxazole complete antigen (1 mg/mL) was emulsified uniformly with an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous multi-point injection, 100. Mu.L each. The first immunization adopts Freund's complete adjuvant, the boost immunization adopts Freund's incomplete adjuvant, the immunization dose is half of the previous immunization dose during the sprint immunization, and the mixture is uniformly mixed with normal saline and then directly injected into the abdominal cavity; each immunization interval was three weeks. After the third immunization, blood sampling is carried out at intervals of one week to detect serum titer and inhibition; mice with the best inhibition were selected and were challenged 18 days after five days to prepare for fusion.

4. Cell fusion: after three days of sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 2000) method, as follows:

(1) Taking a mouse spleen in a sterile mode, grinding and passing through a 200-mesh cell screen to obtain spleen cell suspension, and performing cell counting;

(2) Collecting SP2/0 cells, suspending in RPMI-1640 basic culture solution, and performing cell count;

(3) Mixing spleen cells and SP2/0 cells according to a counting ratio of 2-10:1, centrifuging, fusing with PEG for 1min, adding RPMI-1640 basic culture solution from slow to fast, centrifuging, suspending in RPMI-1640 screening culture solution containing 20% fetal calf serum and 2% 50 XHAT, adding into 96-well cell culture plate, and placing at 37deg.C and 5% CO ₂ Is cultured in an incubator of (a).

5. Cell screening and cell strain establishment: the cells were subjected to half-replacement of the RPMI-1640 selection medium on day 3 of cell fusion, full-replacement with a 100 XHT-containing RPMI-1640 transition medium containing 20% fetal bovine serum and 1% on day 5, and cell supernatants were collected on day 7 for selection.

Screening is carried out in two steps: the first step is to screen out positive cell holes by indirect ELISA, and the second step is to use etoxazole as a standard substance, and to measure the inhibition effect of the positive cells by indirect competition ELISA. Selecting cell holes with better inhibition on etoxazole, subcloning by adopting a limiting dilution method, and detecting by adopting the same method. Cell lines were obtained by repeating the procedure three times.

The obtained cell strain is preserved in a biological material sample, and is preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (CGMCC), the national academy of sciences of China, including national academy of sciences of China, no. 3, north Chen West Lu 1, korea, beijing, and the preservation date 2021, 12 months, 16 days, and the preservation number of CGMCC No.45015.

6. Preparation and identification of monoclonal antibodies: taking 8-10 week-old BALB/c mice, and injecting paraffin oil into the abdominal cavity of each mouse by 1mL; intraperitoneal injection of 1X 10 per mouse after 7 days ⁶ Hybridoma cells, collecting ascites from day 7, purifying the ascites by octanoic acid-saturated ammonium sulfate method, and storing the obtained monoclonal antibody at-20deg.C.

Immunoglobulin subtype identification was performed on the monoclonal antibodies obtained by ascites purification using a mouse monoclonal antibody subtype identification kit, the subtype of which was IgG2b type, as shown in table 1.

TABLE 1 subtype identification of etoxazole monoclonal antibodies

Antibody subclasses	OD value
		IgA	0.113
IgG1	0.209
		IgG2a	0.203
IgG2b	2.001
		IgG3	0.302
IgM	0.187

Sensitivity to chlorfenapyr was measured by ic-ELISA as shown in FIG. 2, according to the standard equation y=0.144+ 1.531/(1+ [ x/4.04)] ^1.166 ) IC for determining monoclonal antibody of trichlorfon by IC-ELISA ₅₀ The antibody has high sensitivity, and can be used for immunoassay detection of etoxazole, wherein the detection limit is 4.04ng/mL.

Further, it was verified that it was against IC such as pyridaben ₅₀ And the cross-reactivity, specifically shown in table 2, the monoclonal antibodies secreted by the cell lines were found to be associated with other acaricides such as: the pyridaben, the cyromazine and the spirodiclofen have no cross reaction and have good specific recognition on the etoxazole.

TABLE 2 IC of etoxazole monoclonal antibodies to etoxazole, pyridaben, cyromazine, spirodiclofen ₅₀ Cross-reactivity ratio

	IC ₅₀ (μg/L)	Cross reaction rate
			Etoxazole	4.04	100％
Pyridaben medicine	＞500	＜5％
			Acarus killing medicine	＞500	＜5％
Spirodiclofen	＞500	＜5％

Example 2 antibody application:

the monoclonal antibody prepared from the hybridoma cell strain YY prepared in the example 1 through in vivo ascites is applied to an etoxazole ELISA (enzyme-Linked immunosorbent assay) additive recovery test, and the specific steps are as follows:

(1) Coating 0.1 mu g/mL etoxazole diluted by Carbonate Buffer (CBS) as a coating raw material, coating a 96-well ELISA plate, coating 100 mu L of each well at 37 ℃ for 2 hours, washing the plate three times by using PBST washing liquid, 200 mu L of each well for 3min, and drying;

(2) Blocking with CBS containing 0.2% gelatin, blocking at 37deg.C for 2 hr, washing the plate with PBST wash solution three times, 200 μl each time, 3min each time, and drying;

(3) 0,0.02,0.05,0.1,0.2,0.5,1,2. Mu.g/L etoxazole standard solutions were prepared with Phosphate Buffered Saline (PBS), respectively. Respectively adding the standard solution and the sample extracting solution to be detected into the sealed ELISA plate, wherein each hole is 50 mu L, each sample is repeated for 3 holes, and then 50 mu L of 1 is added into each hole: after 16000 diluted anti-etoxazole monoclonal antibody reacts for half an hour at 37 ℃, washing the plate and beating;

(4) mu.L of PBS 1 with 0.1% gelatin was added to each well: after a reaction of 3000 dilution of HRP-marked goat anti-mouse IgG secondary antibody at 37 ℃ for half an hour, washing the plate and beating;

(5) 100 mu L TMB color developing solution is added into each hole, and after color development is carried out for 15min at 37 ℃,50 mu L2M H is added into each hole ₂ SO ₄ Stop solution, measuring light absorption value at 450 nm;

(6) And (3) adding and recycling and sample pretreatment: fresh cucumber samples of 5g are taken, and three different doses of etoxazole standard substances of 5ng, 10ng and 20ng are added. The mixture was placed in a 50mL centrifuge tube, 1mL of 50% potassium hydroxide solution was slowly dropped, the mixture was sufficiently shaken on a vortex mixer, 20mL of ethyl acetate was slowly dropped, the mixture was shaken on the vortex mixer for 10 minutes, and the mixture was then placed in a centrifuge and centrifuged at 3000r/min for 5 minutes. 4mL of supernatant was removed from the tube and dried with nitrogen, 1mL of PBS containing 10% methanol was added for reconstitution, and 50. Mu.L was taken for detection. The recovery rate of the indirect competition ELISA is 90.2%, 103.1% and 99.3% respectively, so that the antibody has good recovery rate and accuracy in actual sample detection, and can be applied to etoxazole residue detection in food samples.

Configuration of relevant solutions in this example:

carbonate Buffer (CBS): weighing Na ₂ CO ₃ 1.59g，NaHCO ₃ 2.93g, respectively dissolving in a small amount of double distilled water, mixing, adding double distilled water to about 800mL, mixing, adjusting pH to 9.6, adding double distilled water to 1000mL, and storing at 4deg.C for use;

phosphate Buffer (PBS): 8.00g NaCl,0.2g KCl,0.24g KH ₂ PO ₄ ，3.62g Na ₂ HPO ₄ ·12 H ₂ O is dissolved in 800mL of pure water, pH is regulated to 7.2-7.4 by NaOH or HCl, and volume is regulated to 1000mL;

PBST: PBS containing 0.05% tween 20;

TMB color development liquid: and (3) solution A: na (Na) ₂ HPO ₄ ·12H ₂ 18.43g of O, 9.33g of citric acid and pure water to 1000mL; and (2) liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. The volume ratio of the solution B is 1:5, mixing to obtain TMB color development liquid, and mixing immediately.

Anti-etoxazole single gram generated by YY secretion of hybridoma cell strainThe long antibody has better affinity and higher sensitivity to etoxazole, and particularly has 50% inhibition concentration IC to etoxazole ₅₀ The method reaches 4.04ng/mL, can be used for preparing an immunodetection kit of etoxazole, a colloidal gold test strip, a detection plate, a reagent and other detection bodies, is used for analyzing and detecting the residual trichlorfon in food safety detection, and particularly provides a powerful detection method and means for detecting etoxazole in food.

The foregoing is only a preferred embodiment of the invention, it being noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.

Claims

1. Hybridoma cell strain secreting anti-etoxazole monoclonal antibody, which is preserved in China general microbiological culture Collection center, called monoclonal cell strain AZX for short, with preservation number of CGMCC No.45015 and preservation address of North Chen West Lu No. 1, north Chao in the Chao of Beijing city, and China academy of sciences of microorganisms.

2. An anti-etoxazole monoclonal antibody which is secreted by the monoclonal cell strain AZX with the preservation number of CGMCC No.45015 according to claim 1.

3. The application of the anti-etoxazole monoclonal antibody as claimed in claim 2, wherein an immunodetection method for the anti-etoxazole content is established and is applied to the detection of the anti-etoxazole in food.

4. The use of the anti-etoxazole monoclonal antibody according to claim 3, wherein the anti-etoxazole monoclonal antibody is used for preparing a detection body for analysis and detection of the residual amount of the anti-etoxazole in food safety detection.

5. The use of anti-etoxazole monoclonal antibody according to claim 4, wherein said test body comprises a reagent, a test plate, a kit.