CN112813034B - Hybridoma cell strain capable of secreting oxadixyl monoclonal antibody and application of hybridoma cell strain - Google Patents

Abstract

A hybridoma cell strain secreting a oxadixyl monoclonal antibody and application thereof belong to the field of food safety immunodetection. The hybridoma cell strain AAB2 secreting the oxadixyl monoclonal antibody has been preserved in China general microbiological culture Collection center (CGMCC), is named as a monoclonal cell strain in a classified manner, and has a preservation date of 2020 and 4-23 days, and a preservation number of CGMCC No.19677. Firstly, synthesizing oxadixyl hapten, preparing oxadixyl complete antigen, immunizing BALB/c mice, and taking high titer and low IC ₅₀ And fusing the mouse spleen cells with myeloma cells by a PEG method, and obtaining the hybridoma cell strain AAB2 by screening and three times of subcloning by an indirect competitive enzyme-linked immunosorbent assay. The monoclonal antibody secreted by the cell strain has better specificity and detection sensitivity on the oxadixyl.

Description

A hybridoma cell line secreting oxaxanthine monoclonal antibody and its application

technical field

The invention relates to a hybridoma cell strain secreting oxaxyl monoclonal antibody and its application, and belongs to the field of food safety immune detection.

Background technique

Oxalaxyl is a systemic fungicide with protective and curative effects, long duration, and special effects on downy mildew pathogens. The control object is used to control downy mildew pathogens such as tobacco, cucumber, grape, and vegetable downy mildew, blight, etc., and can also treat a variety of secondary diseases such as brown spot, black rot, etc. Specific diseases such as tobacco black shank, tomato late blight, cucumber downy mildew, eggplant cotton blight, pepper blight, potato late blight, cabbage downy mildew, grape downy mildew.

There have been a few reports at home and abroad about the detection methods of oxalaxyl residues in animal tissues, mainly using high performance liquid chromatography-fluorescence detector and liquid chromatography-tandem mass spectrometry. The extraction methods are also different, such as liquid-liquid extraction, liquid-solid extraction, etc. Instrumental detection methods can carry out quantitative analysis and have lower detection limits, but usually require expensive instruments and complicated operations, and long pretreatment and detection time, which seriously restrict the popularization of these detection methods.

Immunoassay methods have the characteristics of low cost, high throughput, high sensitivity, and relatively low requirements for technicians, so they are suitable for rapid screening of a large number of samples.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a hybridoma cell line that secretes oxalaxyl monoclonal antibody and its application. Oxalaxyl enzyme-linked immunosorbent assay method, or the establishment of a rapid detection method for colloidal gold immunochromatographic test strips.

The technical solution of the present invention is a hybridoma cell line AAB2 that secretes oxalaxyl monoclonal antibody, which has been deposited in the General Microbiology Center of the China Microorganism Culture Collection and Management Committee, CGMCC, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Institute of Microbiology, Chinese Academy of Sciences, classified as monoclonal cell line, deposited on April 23, 2020, and deposited as CGMCC No.19677.

The basic steps for the preparation of the AAB2 cell line provided by the present invention are:

(1) Synthesis of hapten: A carboxyl group is derived on the basis of the structure of oxaxyl through chemical synthesis, so as to facilitate the connection of carrier proteins.

(2) Preparation and identification of immunogens: Oaxaxyl derivatives were used as raw materials, linked to the amino group of the protein carrier by the activated ester method. After the reaction, the complete antigen and the unconjugated small molecule hapten were separated by dialysis. Antigens were identified by UV absorption scanning method;

(3) Immunization of mice: BALB/c mice aged 6-8 weeks were selected for immunization. After the immunogen and Freund's adjuvant were completely emulsified, the mice were immunized by subcutaneous multi-point injection. The first immunization was with Freund's complete adjuvant, and the booster immunization was with Freund's incomplete adjuvant. The immunization dose for the sprint immunization was the previous immunization dose. Half of the immunogens were mixed with normal saline and injected directly into the abdominal cavity; the interval between each immunization was three weeks. After the third immunization, blood was collected at intervals of one week to detect serum titer and inhibition;

(4) Cell fusion and establishment of cell lines: mouse splenocytes and mouse myeloma cells were fused by polyethylene glycol (PEG 4000) method, cultured in HAT medium, positive cell wells were detected by indirect ELISA, and further The inhibitory effect of positive cell wells was determined by indirect competitive ELISA method, and the positive cell wells with the best inhibition were subcloned three times by limiting dilution method, and finally the hybridoma cell line AAB2 was obtained by screening;

(5) Identification of the properties of hybridoma cell lines: The mouse monoclonal antibody Ig class/subclass identification was determined with an enzyme-labeled secondary antibody kit; IC ₅₀ value, cross-reaction rate and affinity were determined by ELISA.

Oxaxyl monoclonal antibody, which is secreted and produced by the hybridoma cell line AAB2 of the CGMCC No. 19677.

The preparation method of oxalaxyl hapten, its synthetic route is as follows:

Proceed as follows:

(1) Add pyridine and 2-chloroethyl carbon chloride to an aqueous solution of 2,6-dimethylphenylhydrazine hydrochloride at low temperature, and stir the reaction mixture at room temperature; the mixture is extracted with ethyl acetate, The organic layer was washed with water and brine, dried over Na ₂ SO ₄ and concentrated to give compound 2 as an off-white solid;

(2) Benzyl 4-(2-chloro-2-oxoethoxy)butyrate and triethanolamine TEA were added to the THF solution of compound 2, and stirred; the mixture was washed with saturated NH ₄ Cl and ethyl acetate EA Extraction; the mixture was concentrated and purified by TLC to give compound 3 as a yellow oil;

(3) To a suspension of NaH in toluene, compound 3 was added in portions, and the mixture was stirred at room temperature; the mixture was purified by TLC to give compound 4 as a colorless oil;

(4) adding palladium-carbon catalyst Pd-C to the MeOH solution of compound 4; stirring the mixture at room temperature, then filtering the mixture; concentrating the filtrate and purifying by preparative TLC to obtain the target compound in the form of a yellow gel, namely It is the hapten of oxalaxyl.

Specific steps are as follows:

(1) 87.2 mmol of pyridine and 29.1 mmol of 2-chloroethyl chlorocarbochloride were added to 29.1 mmol of an aqueous solution of 2,6-dimethylphenylhydrazine hydrochloride at 0-5 °C, and the reaction mixture was mixed with Stir at room temperature for 2 h; the mixture was extracted with ethyl acetate, the organic layer was washed with water and brine, dried over Na ₂ SO ₄ and concentrated to give compound 2 as an off-white solid;

(2) 9.1 mmol of benzyl 4-(2-chloro-2-oxoethoxy)butyrate and 27.3 mmol of triethanolamine TEA were added to 9.1 mmol of compound 2 in 40 mL of THF; the reaction mixture was stirred at 65°C 2h; the mixture was washed with saturated _NH4Cl and extracted with ethyl acetate EA; the mixture was concentrated and purified by TLC to give compound 3 as a yellow oil;

(3) To a suspension of 2.1 mmol NaH in 450 mL of toluene, 2.1 mmol of compound 3 was added in portions; the mixture was stirred at room temperature for 15 min; the mixture was purified by TLC to give compound 4 as a colorless oil;

(4) 120 mg of palladium-carbon catalyst Pd-C was added to 1.8 mmol of compound 4 in 40 mL of MeOH; the mixture was stirred at room temperature for 1 h, then the mixture was filtered, the filtrate was concentrated and purified by TLC to obtain the yellow gel-like target compound, that is, the hapten of oxalaxyl.

Compound a in the above-mentioned synthetic route, namely the preparation route of 4-(2-chloro-2-oxoethoxy) benzyl butyrate is as follows:

The process is as follows:

(1) Dess-Martin periodinane was added in portions to the DCM solution of compound 5,2-(4-hydroxybutoxy)acetate tert-butyl, to the mixture was added a solution of Na ₂ S ₂ O ₃ and NaHCO 3 , and then Extraction with DCM, concentration of the organic layer, and purification by chromatography gave compound 6;

(2) adding the NaClO ₂ aqueous solution to the monohydrate NaH ₂ PO ₄ aqueous solution; adding the aqueous solution to the precooled solution in 2-methylbut-2-ene and compound 6 in tert-butanol; placing the mixture at a low temperature Under stirring, water was added to the mixture and extracted with EA; the mixture was dried over Na ₂ SO ₄ and concentrated to give compound 7;

(3) To the DMF solution of compound 7, bromomethylbenzene and K ₂ CO ₃ were added; the mixture was stirred at room temperature for 1 h and then water was added, and extracted with ethyl acetate; the organic layer was washed with water and brine, and dried over Na ₂ SO ₄ , concentrated and purified by chromatography to give compound 8;

(4) TFA was added to the DCM solution of compound 8, and the mixture was stirred at room temperature for 1 h; the mixture was concentrated and water was added; the mixture was extracted with DCM, and the organic layer was dried over Na ₂ SO ₄ and concentrated to obtain compound 9;

(5) (COCl) ₂ was added dropwise to the DCM solution of compound 9 at low temperature, and the mixture was stirred at room temperature for 1 h; the mixture was concentrated to obtain benzyl 4-(2-chloro-2-oxoethoxy)butyric acid ester.

Compound a, namely the preparation method of benzyl 4-(2-chloro-2-oxoethoxy) butyrate, its synthetic route is as shown above: from compound 5, 2-(4-hydroxybutoxy) acetic acid tertiary Dess-Martin periodinane (22.9 g, 53.9 mmol) was added portionwise to a solution of butyl ester ﹙﹚ in DCM (350 mL), a solution of Na ₂ S ₂ O ₃ and NaHCO ₃ was added to the mixture, extracted with DCM, and the organic layer and purified by chromatography to give compound 6 (8 g, 80.8%).

Aqueous NaClO ₂ (80%, 29.9 g, 237.6 mmol) was added to a solution of NaH ₂ PO ₄ monohydrate (12.4 g, 79.2 mmol) in water (360 mL). The aqueous solution was added to a precooled (0°C) solution of 2-methylbut-2-ene (41.6 g, 594.1 mmol) and compound 6 (8.0 g, 39.6 mmol) in t-BuOH (240 mL). The mixture was stirred at 0 °C for 0.5 h, water was added to the mixture, and extracted with EA. The mixture was dried _{over Na2SO4} and concentrated to give compound 7 (7.0 g, crude).

To a solution of compound 7 (7.0 g, 32.1 mmol) in DMF (70 mL) was added bromomethylbenzene (8.2 g, 48.2 mmol) and K2CO3 ₍ 13.3 _g , 96.3 mmol). The mixture was stirred at room temperature for 1 h, then water was added and extracted with ethyl acetate; the organic layer was washed with water and brine, dried _{over Na2SO4} , concentrated and purified by chromatography to give compound 8 (6.0 g, 60.6%).

To a solution of compound 8 (6.0 g, 19.5 mmol) in DCM (30.0 mL) was added TFA (18 mL). The mixture was stirred at room temperature for 1 h. The mixture was concentrated and water was added. The mixture was extracted with DCM. The organic layer was dried _{over Na2SO4} and concentrated to give compound 9 (4.5 g, 91.8%).

To a solution of compound 9 (4.5 g, 17.9 mmol) in DCM (45.0 mL) at 0 °C was added (COCl) ₂ (3.4 g, 26.8 mmol) dropwise. The mixture was stirred at room temperature for 1 h. The mixture was concentrated to give compound a, benzyl 4-(2-chloro-2-oxoethoxy)butyrate (4.0 g, 83.3%).

The preparation method of oxalaxyl complete antigen, including oxalaxyl immunogen and oxalaxyl coated original;

The preparation method of the immunogen is as follows: taking the oxalaxyl hapten, then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS, Use N,N-dimethylformamide DMF to dissolve, stir and activate at room temperature to obtain an activation solution; take another bovine serum albumin BSA and dissolve it in carbonate buffer solution CB; add the activation solution dropwise to the BSA solution, and stir at room temperature to react After overnight, use PBS buffer to dialyze for storage, and finally obtain the oxaxyl immunogen;

The preparation method of the original coating is as follows: taking the oxalaxyl hapten, then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS , use N,N-dimethylformamide DMF to dissolve, stir and activate at room temperature to obtain an activation solution; take another chicken ovalbumin OVA and dissolve it in carbonate buffer solution CB; add the activation solution dropwise to the OVA solution, stir at room temperature After overnight reaction, PBS buffer was dialyzed for storage in aliquots to obtain oxaxyl-coated original.

Further, the preparation method of the oxalaxyl immunogen, the steps are as follows: take 4.5mg oxalaxyl hapten, add 5.0mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt EDC and 3.7 mg N-hydroxysuccinimide NHS; then the above materials were dissolved in N,N-dimethylformamide DMF, stirred at room temperature, and activated for 6 h to obtain an activation solution; another 15 mg of bovine serum albumin BSA was dissolved In 3mL, 0.05M, pH9.6 carbonate buffer solution CB; add the prepared activation solution dropwise to the BSA solution, stir overnight at room temperature, dialyze with PBS buffer for 3 days, and store at -20 ℃ , to obtain the oxaraxim immunogen.

Further, the preparation method of the oxalaxyl coating original, the steps are as follows: get 3mg oxalaxyl hapten, then add 5.0mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Salt EDC and 3.7mg N-hydroxysuccinimide NHS were dissolved in N,N-dimethylformamide DMF, stirred at room temperature, and activated for 6h to obtain an activation solution; another 10mg of chicken ovalbumin OVA was dissolved in 3mL, 0.05M , in the carbonate buffer solution CB solution with pH 9.6; the activation solution was added dropwise to the OVA solution, after stirring at room temperature overnight, dialyzed with PBS buffer for 3 days, and stored at -20 °C in separate packs to obtain Oxalaxyl packs been original.

The application of the oxalaxyl monoclonal antibody is used for the detection of oxalaxyl residues in food.

Further, an immunoassay kit for oxaxyl and a colloidal gold test strip were prepared by using the oxaxyl monoclonal antibody for detection.

Beneficial effects of the present invention: the monoclonal antibody secreted by the hybridoma cell line AAB2 provided by the present invention has good specificity and detection sensitivity to oxalaxyl (oxalaxyl IC ₅₀ =3.68 μg/L, metalaxyl, The IC ₅₀ of dimethomorph and procymidyl are all >500μg/L), which provides the necessary hybridoma cell line secreting monoclonal antibody for the detection of oxalaxyl residues in food, which can be used to detect The kit of oxalaxyl and the colloidal gold test strip have practical application value.

Preservation of biological material samples: A hybridoma cell line AAB2 that secretes oxalaxyl monoclonal antibody has been deposited in CGMCC, General Microbiology Center of China Microorganism Culture Collection Administration, address No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Academy of Sciences, classified as monoclonal cell line, deposited on April 23, 2020, and deposited as CGMCC No.19677.

Description of drawings

Figure 1. Standard inhibition curve of oxaxyl monoclonal antibody.

Figure 2. Liquid phase mass spectrogram of the oxalaxyl hapten.

Detailed ways

The following embodiments of the present invention are only used as a further description of the content of the present invention, and cannot be used as a limitation or scope of the present invention. The present invention will be further described below through examples.

In the present invention, mice are immunized with the complete antigen of oxalaxyl, cultured in HAT selective medium through cell fusion, and the cell supernatant is screened through indirect ELISA and indirect competitive ELISA, so as to finally obtain oxalaxyl with good affinity and sensitivity. Monoclonal antibody hybridoma cell line AAB2.

Example 1 Synthesis of oxaxyl hapten

1. Synthesis of compound a:

Compound a, the preparation method of 4-(2-chloro-2-oxoethoxy) butyric acid benzyl ester, its synthetic route is shown in the above formula, from compound 5, 2-(4-hydroxybutoxy) acetic acid tertiary Dess-Martin periodinane (22.9 g, 53.9 mmol) was added in portions to a solution of butyl ester ( 350 mL) in DCM, a solution of Na ₂ S ₂ O ₃ and NaHCO ₃ was added to the mixture, extracted with DCM, and the organic layer was concentrated , and purified by chromatography to give compound 6 (8 g, 80.8%). Aqueous NaClO ₂ (80%, 29.9 g, 237.6 mmol) was added to NaH ₂ PO ₄ monohydrate (12.4 g, 79.2 mmol) in water (360 mL). ) solution. The aqueous solution was added to a precooled (0°C) solution of 2-methylbut-2-ene (41.6 g, 594.1 mmol) and compound 6 (8.0 g, 39.6 mmol) in t-BuOH (240 mL). The mixture was stirred at 0 °C for 0.5 h, water was added to the mixture, and extracted with EA. The mixture was dried over Na ₂ SO ₄ and concentrated to give compound 7 (7.0 g, crude). To compound 7 (7.0 g, 32.1 g) mmol) in DMF (70 mL) was added bromomethylbenzene (8.2 g, 48.2 mmol) and K2CO3 ₍ 13.3 _g , 96.3 mmol). The mixture was stirred at room temperature for 1 h then water was added and extracted with ethyl acetate. The organic layer was washed with water and brine, dried _{over Na2SO4} , concentrated and purified by chromatography to give compound 8 (6.0 g, 60.6%). To a solution of compound 8 (6.0 g, 19.5 mmol) in DCM (30.0 mL) TFA (18 mL) was added. The mixture was stirred at room temperature. 1 h. The mixture was concentrated and water was added. The mixture was extracted with DCM. The organic layer was dried _{over Na2SO4} and concentrated to give compound 9 (4.5 g, 91.8%). To a solution of compound 9 (4.5 g, 17.9 mmol) in DCM (45.0 mL) was added dropwise (COCl) ₂ (3.4 g, 26.8 mmol) at 0° C. The mixture was stirred at room temperature for 1 h. The mixture was concentrated to give compound a, Benzyl 4-(2-chloro-2-oxoethoxy)butyrate (4.0 g, 83.3%).

2. Synthesis of oxalaxyl hapten: its synthetic route is as follows:

To an aqueous solution of compound 1,2,6-dimethylphenylhydrazine hydrochloride (5.0 g, 29.1 mmol) at 0-5 °C was added pyridine (6.9 g, 87.2 mmol) and 2-chlorocarbon chloride ethyl ester (4.1 g, 29.1 mmol) and the reaction mixture was stirred at room temperature for 2 h. The mixture was extracted with ethyl acetate, the organic layer was washed with water and brine, dried _{over Na2SO4} and concentrated to give compound 2 (2.2 g, 31.4%) as an off-white solid. To a solution of compound 2 (2.2 g, 9.1 mmol) in THF (40 mL) was added compound a (2.5 g, 9.1 mmol) and TEA (2.8 g, 27.3 mmol). The reaction mixture was stirred at 65 °C for 2 h. The mixture was washed with saturated _NH4Cl and extracted with ethyl acetate. The mixture was concentrated and purified by prep-TLC to give compound 3 (1.0 g, 23.3%) as a yellow oil. To a suspension of NaH (84 mg, 2.1 mmol) in toluene (450 mL) was added compound 3 (1.0 g, 2.1 mmol) portionwise. The mixture was stirred at room temperature for 15 min. The mixture was purified by preparative TLC to give compound 4 (800 mg, 86.8%) as a colorless oil. To a solution of compound 4 (800 mg, 1.8 mmol) in MeOH (40 mL) was added Pd-C (120 mg). The mixture was stirred at room temperature for 1 h, then the mixture was filtered, and the filtrate was concentrated and purified by preparative TLC to give the target compound (550 mg, 86.5%) as a yellow gel, the hapten of oxaxyl.

The liquid phase mass spectrogram of oxalaxyl hapten is shown in Figure 2; The retention time of the chromatography was 2.37 minutes, and the corresponding mass spectrogram detected fragments with molecular weights of 350.9 and 33.9 Da in negative ion mode, which were consistent with the structure of the oxalaxyl hapten, thus proving the successful derivatization of the oxalaxyl hapten.

Example 2 Synthesis of the complete antigen of oxaxyl:

The preparation method of oxalaxyl immunogen: take 4.5 mg of the above oxalaxyl hapten, then add 5.0 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 3.7mg NHS (N-hydroxysuccinimide) was dissolved in DMF (N,N-dimethylformamide), stirred at room temperature, and activated for 6h; another 15mg BSA (bovine serum albumin) was dissolved in 3mL, 0.05M, CB (carbonate buffer solution) solution with pH 9.6; the above activation solution was added dropwise to the BSA solution, after stirring at room temperature overnight, the immunogen was taken out for PBS dialysis for 3 days, and stored in aliquots at -20°C.

The preparation method of oxalaxyl-coated original: take 3mg of the above oxalaxyl hapten, then add 5.0mg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 3.7mg of NHS (N-hydroxysuccinimide) was dissolved in DMF (N,N-dimethylformamide), stirred at room temperature, and activated for 6h; another 10mg of OVA (chicken ovalbumin) was dissolved in 3mL, 0.05M, CB (carbonate buffer solution) solution with pH 9.6; the above activation solution was added dropwise to the OVA solution, after stirring at room temperature overnight, the immunogen was taken out for PBS dialysis for 3 days, and stored in aliquots at -20°C.

Example 3 Preparation of Oxaxyl Monoclonal Antibody Hybridoma Cell Line AAB2

(1) Animal immunization: healthy BALB/c mice aged 6-8 weeks were selected for immunization. BALB/c mice were immunized by subcutaneous multi-point injection, each 100 μL, after taking the complete oxalaxyl antigen (1 mg/mL) and emulsified with equal amount of Freund’s adjuvant. Freund's complete adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for booster immunization. The immunization dose during sprint immunization was half of the previous immunization dose. It was mixed with normal saline and injected directly into the abdominal cavity; the interval between each immunization was three weeks. . After the third immunization, blood was collected at an interval of one week to detect serum titer and inhibition; the mice with the best inhibition were selected and sprinted 18 days after the fifth immunization to prepare for fusion.

(2) Cell fusion: After three days of sprint immunization, cell fusion was carried out according to the conventional PEG (polyethylene glycol, molecular weight 4000) method. The specific steps are as follows:

a. Aseptically take the mouse spleen, grind and pass through a 200-mesh cell screen to obtain a spleen cell suspension, and count the cells;

b. Collect SP2/0 cells, suspend them in RPMI-1640 basal medium, and count the cells;

c. Mix spleen cells and SP2/0 cells according to the counting ratio of 2 to 10:1, fuse with PEG after centrifugation, for 1 min, then add RPMI-1640 basal medium from slow to fast, and suspend after centrifugation. 20% fetal bovine serum, 2% 50×HAT RPMI-1640 screening medium, added to a 96-well cell culture plate, and cultured in a 37°C, 5% CO ₂ incubator.

(3) Cell selection and cell line establishment: on the 3rd day of cell fusion, RPMI-1640 was used to screen the fused cells and the medium was half-changed, and on the 5th day, RPMI containing 20% fetal bovine serum and 1% 100×HT was used The -1640 transition medium was completely replaced, and the cell supernatant was taken for screening on the 7th day. The screening is divided into two steps: in the first step, indirect ELISA is used to screen out positive cell wells, and in the second step, oxaxan is used as the standard substance, and the inhibitory effect of positive cells is determined by indirect competitive ELISA. Cell wells with better inhibition of oxaxan were selected, subcloned by limiting dilution method, and detected by the same method. Repeat three times to obtain cell line AAB2.

(4) Preparation and identification of monoclonal antibodies: 8-10 weeks old BALB/c mice were taken, and each mouse was injected with 1 mL of paraffin oil; 7 days later, 1×10 ⁶ hybridoma cells were injected into each mouse The ascites was collected on the 7th day, and the ascites was purified by the octanoic acid-saturated ammonium sulfate method, and the obtained monoclonal antibody was stored at -20°C.

The monoclonal antibody obtained by purification of ascites was subjected to immunoglobulin subtype identification using a mouse monoclonal antibody subtype identification kit, and its subtype was IgG2b, as shown in Table 1.

Table 1. Isolation of Oxaxyl Monoclonal Antibodies

Antibody Subtype Subclass OD value IgA 0.103 IgG1 0.129 IgG2a 0.103 IgG2b 2.341 IgG3 0.212 IgM 0.164

Using the indirect competitive ELISA method, the IC ₅₀ of the monoclonal antibody to oxaxyl was determined to be 3.68 μg/L, and the IC ₅₀ and cross-reaction rate of the monoclonal antibody to metalaxyl were verified, as shown in Table 2.

Table 2 IC ₅₀ and cross-reaction rate of oxalaxyl monoclonal antibody to oxalaxyl, metalaxyl, dimethomorph and procymidone

IC₅₀(μg/L) cross-reaction rate hoarfrost 3.68 100% metalaxyl ＞500 <5% Dimethomorph ＞500 <5% Procymid ＞500 <5%

Application Example 1

The monoclonal antibody prepared by the hybridoma cell line AAB2 through in vivo ascites was applied to the oxalaxyl ELISA addition recovery test, and the specific steps are as follows:

(1) Coat 96-well microtiter plate with 0.1 μg/mL oxaxyl diluted with carbonate buffer (CBS), 100 μL per well, coat at 37°C for 2 hours, and wash with PBST lotion Plate three times, 200 μL per well each time, 3 min each time, and pat dry;

(2) Block with CBS containing 0.2% gelatin, 200 μL per well, block at 37°C for 2 h, wash the plate three times with PBST washing solution, 200 μL per well for 3 min each time, and pat dry;

(3) 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1, and 2 μg/L oxaxyl standard solutions were prepared with phosphate buffered saline (PBS). Add the standard solution and the extraction solution of the sample to be tested to the sealed microtiter plate, 50 μL per well, repeat 3 wells for each sample, and then add 50 μL 1:16000 dilution of antioxaxyl monoclonal to each well. Antibody, after 30min reaction at 37℃, wash plate and pat dry;

(4) Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted with PBS containing 0.1% gelatin at 1:3000 to each well, react at 37°C for 30 minutes, wash the plate and pat dry;

(5) Add 100 μL of TMB chromogenic solution to each well, and after 15 minutes of color development at 37°C, add 50 μL of 2M H ₂ SO ₄ stop solution to each well, and measure the absorbance at 450 nm;

(6) Addition recovery and sample pretreatment: 5 g of fresh cucumber samples were taken, and three different doses of oxaxyl standard were added, 5 ng, 10 ng and 20 ng respectively. Put it in a 50mL centrifuge tube, slowly drop 1mL of 50% potassium hydroxide solution, shake it fully on a vortex mixer, slowly drop 20mL of ethyl acetate, shake it on a vortex mixer for 10min, and then put it into a centrifuge Centrifuge at 3000r/min for 5min. Pipette 4 mL of supernatant into another centrifuge tube, dry with nitrogen, add 1 mL of PBS containing 10% methanol to reconstitute, and take 50 μL for detection. Indirect competition ELISA was used to add recovery test, and the recovery rates were 90.2%, 103.1% and 99.3%, respectively.

Configuration of the solution:

Carbonate buffer solution (CBS): Weigh Na ₂ CO ₃ 1.59g, NaHCO ₃ 2.93g, respectively dissolve in a small amount of double-distilled water, mix, add double-distilled water to about 800mL and mix well, adjust pH to 9.6, add Make up to 1000mL with double distilled water and store at 4°C for later use;

Phosphate Buffered Saline (PBS): 8.00g NaCl, 0.2g KCl, 0.24g KH ₂ PO ₄ , 3.62g Na ₂ HPO ₄ ·12H ₂ O, dissolved in 800 mL of pure water, and adjusted to pH 7.2～7 with NaOH or HCl 7.4, dilute to 1000mL;

PBST: PBS with 0.05% Tween.20;

TMB color developing solution: Solution A: Na ₂ HPO ₄ ·12H ₂ O 18.43 g, citric acid 9.33 g, and purified water to 1000 mL; Solution B: 60 mg of TMB was dissolved in 100 mL of ethylene glycol. A and B liquids are mixed according to the volume ratio of 1:5, which is the TMB color developing liquid.

To sum up, the above are only preferred embodiments of the present invention, and are not intended to limit the scope of implementation of the present invention. That is, all equivalent changes and modifications made according to the content of the application scope of the present invention shall fall within the technical scope of the present invention.

Claims (4)

1. A hybridoma cell strain AAB2 secreting a oxadixyl monoclonal antibody is deposited in China general microbiological culture Collection center (CGMCC), china academy of sciences microorganism institute 3, west Lu No.1 Hospital, kogyo, beijing, and the south Korean district, and is classified and named as a monoclonal cell strain, wherein the preservation date is 2020, 4 and 23 days, and the preservation number is CGMCC No.19677.

2. The oxadixyl monoclonal antibody is characterized in that: the hybridoma cell strain AAB2 with the preservation number of CGMCC No.19677 as claimed in claim 1 secretes and produces the hybridoma cell strain AAB2.

3. Use of a monoclonal antibody against oxadixyl as claimed in claim 2, characterized in that: the method is used for detecting the residual amount of the oxadixyl in the food.

4. Use of a monoclonal antibody against oxadixyl according to claim 3, characterized in that: an immunodetection kit for preparing the oxadixyl by adopting the oxadixyl monoclonal antibody and a colloidal gold test strip are used for detection.

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