CN108410824B - A hybridoma cell line with azaperidone monoclonal antibody and its application - Google Patents

Abstract

The invention discloses an azapirone monoclonal antibody hybridoma cell strain and an application thereof, and belongs to the field of food safety immune detection. The present invention prepares azaperidone complete antigen, mixes it with an equal amount of Freund's adjuvant and emulsifies it completely, and immunizes BALB/c mice by subcutaneous injection on the back to obtain a hybridoma cell line CGMCC No. .14694. The monoclonal antibody secreted by this cell line has good specificity and detection sensitivity for azaperidone (IC ₅₀ value is 0.5ng/mL). The results of the invention can be used to establish an immunological detection method for azaperidone residues in animal tissues, and have practical application value.

Description

A hybridoma cell line with azaperidone monoclonal antibody and its application

technical field

The invention relates to an azapirone monoclonal antibody hybridoma cell strain and its application, and belongs to the technical field of food safety immunological detection.

Background technique

Azaperone (also known as Azaperone, or Azap for short) is a butyrylphenone neuroleptic. After intramuscular injection of this drug, animals can eliminate tension, reduce activity, indifference to the environment and stay in a quiet state for a long time, which helps to avoid the phenomenon of animals attacking and fighting each other due to "living stress" and when mixing groups, reducing coping. Therefore, the drug is often given to animals such as pigs during long-distance transportation. my country's maximum residue limit standard for animal food stipulates that the maximum residue limit value (MRL) of azaperidone in muscle, skin and fat, liver and kidney of pigs is 60mg/kg, 60mg/kg, 100mg/kg, 100mg/kg respectively . Therefore, it is of great significance and market value to establish a rapid and effective method for detecting the content of azaperidone.

Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method. However, obtaining high-affinity and high-specificity monoclonal antibodies is a prerequisite for immunological detection, and the synthesis of artificial antigens is an important step. There are few reports on the residual detection methods of azaperidone, so it is of great significance to establish a fast and simple detection method for azaperidone. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method. It does not require high sample purity and is easy to operate. It is suitable for on-site rapid detection of a large number of samples. The establishment of efficient immunological detection methods and the screening of highly specific monoclonal antibodies are important prerequisites.

SUMMARY OF THE INVENTION

The purpose of the present invention is to firstly provide an azaperidone monoclonal antibody hybridoma cell line, which has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee on September 5, 2017, and the preservation number is CGMCC No. 14694. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The antibody prepared from the cell line has good specificity and detection sensitivity to azaperidone, and can be used to establish an immunological detection method for azaperidone.

The second object of the present invention is to provide an azaperidone monoclonal antibody, which is secreted and produced by the azaperidone monoclonal antibody hybridoma cell line whose deposit number is CGMCC No. 14694.

The application of the azaperidone monoclonal antibody is used for the analysis and detection of azaperidone residues in animal tissues.

The third object of the present invention is to provide a method for preparing the azaperidone immunogen, which mainly comprises the following steps:

1) Synthesis of the hapten Azap: methyl 4-phenylbutyrate, 4-chlorobutyryl chloride, and aluminum chloride were added to carbon disulfide, and the temperature was raised to room temperature and stirred overnight; ice water was added to the reaction solution, and dichloromethane was extracted. , dried over anhydrous sodium sulfate, and concentrated to obtain a yellow oily intermediate product 2;

The intermediate product 2, potassium iodide and potassium carbonate, were added to the acetonitrile solution, 1-(2-pyridyl)piperazine was added, and the reaction solution was heated to 70°C overnight; ice water was added to the reaction solution, extracted with dichloromethane, and anhydrous dried over sodium sulfate, concentrated; purified by silica gel column to obtain yellow oily intermediate product 3;

The intermediate product 3, lithium hydroxide, was added with tetrahydrofuran and water; the reaction solution was warmed to room temperature and stirred overnight; acidified with hydrochloric acid to pH 6, washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a white solid, which was prepared by hapten Azap;

2) Preparation of complete antigen:

Preparation of immunogen Azap-KLH: Weigh hapten Azap, 1-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide, dissolve with anhydrous N,N-dimethylformamide to obtain A solution; take keyhole limpet hemocyanin KLH, dissolve it with boric acid buffer solution to obtain solution B, at room temperature, add solution A to solution B dropwise, and react at room temperature overnight to obtain the conjugate Azap-KLH mixed solution, pass through The complete antigen and unconjugated small molecule hapten were separated by dialysis to obtain the immunogen Azap-KLH.

The fourth object of the present invention is to provide a method for preparing the azaperidone monoclonal antibody hybridoma cell line, which mainly comprises the following steps:

(1) Immunization of mice: After mixing and emulsification of the immunogen Azap-KLH with an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous injection on the back; complete Freund's adjuvant was used for the first immunization, and multiple booster immunizations were used to immunize BALB/c mice. Incomplete Freund's adjuvant, 28 days between the first immunization and the second boost, 21 days between multiple boosts, the last sprint with Azap-KLH complete antigen (without adjuvant); by indirect Serum titer and inhibition were detected by competitive enzyme-linked immunosorbent assay (ic-ELISA);

(2) Cell fusion and establishment of cell lines: mouse spleen cells and mouse myeloma cells were fused by polyethylene glycol (PEG 4000) method, cultured in HAT medium, and indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was used. ) to detect the positive cell wells, and further use ic-ELISA to determine the inhibitory effect of the positive cell wells. The positive cell wells with the best inhibitory effect were subcloned three times by the limiting dilution method, and finally the Azap monoclonal antibody hybridoma cell line was obtained by screening. ;

(3) Characterization of hybridoma cell lines: Sensitivity and specificity were determined by ic-ELISA.

Beneficial effects of the present invention: the monoclonal antibody secreted by the azaperidone monoclonal antibody hybridoma cell line provided by the present invention has good specificity and detection sensitivity for azaperidone (IC ₅₀ value is 0.5ng/mL), An immunological method is provided for the detection of azaperidone residues in animal tissues. The azaperidone monoclonal antibody hybridoma cell line and the monoclonal antibody secreted by the azaperidone monoclonal antibody provided by the invention can be made into a kit for detecting azaperidone, and have practical application value.

biological material preservation

A monoclonal cell line has been deposited in the General Microbiology Center of the China Microbial Culture Collection and Management Committee on September 5, 2017, the preservation number is CGMCC No. 14694, and the preservation address is 3, No. 1, Beichen West Road, Chaoyang District, Beijing. No.

Description of drawings:

Figure 1 is a standard curve for the inhibition of Azap by 1A8 monoclonal antibody.

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, monoclonal antibody hybridoma cells with better specificity and sensitivity to Azap are finally obtained by immunizing mice with Azap complete antigen, culturing in HAT selective medium through cell fusion, and screening cell supernatant through ic-ELISA. strains.

Example 1: Preparation of hybridoma cell line 1A8

(1) Preparation of complete antigen:

The hapten synthesis route is as follows:

a, 1.00g of methyl 4-phenylbutyrate (compound 1), 1.19g of 4-chlorobutyryl chloride, and 1.50g of aluminum chloride were added to 10mL of carbon disulfide, and the temperature was raised to room temperature and stirred overnight; ice was added to the reaction solution Water, dichloromethane extraction, drying over anhydrous sodium sulfate, and concentration to obtain yellow oily intermediate product 2;

1.20 g of intermediate product 2, 98.5 mg of potassium iodide, and 1.17 g of potassium carbonate were added to 12 mL of acetonitrile solution, 700 mg of 1-(2-pyridyl)piperazine was added, and the reaction solution was heated to 70 °C overnight; ice water was added to the reaction solution. , extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated; purified by silica gel column to obtain yellow oily intermediate product 3;

360 mg of intermediate product 3, 90.2 mg of lithium hydroxide, 3 mL of tetrahydrofuran and 1 mL of water were added; the reaction solution was warmed to room temperature and stirred overnight; acidified to pH 6 with hydrochloric acid, washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain White solid, prepared hapten Azap;

b. Weigh 1.87 mg of Azap, 2.18 mg of 1-ethylcarbodiimide hydrochloride, and 1.32 mg of N-hydroxysuccinimide, and dissolve them with 400 μL of anhydrous N,N-dimethylformamide to obtain A1 solution, Stir at room temperature for 6-8h. Get keyhole limpet hemocyanin KLH 10mg (Azap and KLH molar ratio is 1500:1), dissolve with 4mL boric acid buffer solution, obtain B1 solution, at room temperature, add A1 solution dropwise to B1 solution, react at room temperature overnight, That is, the Azap-KLH (1500:1) mixture of the conjugate is obtained, and the complete antigen and the unconjugated small molecule hapten are separated by dialysis;

According to the above steps, complete antigens with Azap and KLH molar ratios of 3000:1 and 4500:1 were prepared respectively to obtain the conjugates Azap-KLH (3000:1) and Azap-KLH (4500:1).

(2) Preparation of coated original Azap-OVA:

Weigh 1.3 mg of Azap, 2.0 mg of 1-ethylcarbodiimide hydrochloride, and 1.2 mg of N-hydroxysuccinimide, dissolve with 300 μL of anhydrous N,N-dimethylformamide to obtain solution A2, and stir at room temperature Reverse 6-8h. Weigh 5mg chicken egg albumin OVA (the molar ratio of Azap to OVA is 30:1), dissolve it in 2mL boric acid buffer solution to obtain B2 solution, at room temperature, add A2 solution dropwise to B2 solution, and react at room temperature Overnight, the conjugate Azap-OVA mixture was obtained, and the coated and unconjugated small molecule haptens were separated by dialysis. The coating was originally used for the detection of mouse serum titer and inhibition during the preparation of monoclonal antibodies. It is not directly used in mice and is necessary for the preparation of monoclonal antibodies.

(3) Animal immunization: healthy BALB/c mice aged 6-8 weeks were selected for immunization. Three different molar ratios of Azap complete antigen were mixed and emulsified with the same amount of Freund's adjuvant, and then BALB/c mice were immunized by subcutaneous injection on the back. Complete Freund's adjuvant was used for the first immunization, followed by incomplete Freund's adjuvant. The interval between the first immunization and the second booster immunization was 28 days, and the interval between multiple booster immunizations was 21 days. Blood was collected 7 days after the third immunization (5ul+995ul antibody diluent = antiserum) from tail-broken blood, and ic-ELISA was used to determine the serum titer and inhibition of mice. Select mice with high titers and good inhibition. 21 days after the four immunizations, the sprint immunization was administered intraperitoneally, requiring a halved dose without any adjuvant.

Selection of sprint immunized mice: The serum titer and inhibition rate of mice were measured by ic-ELISA, and it was found that when the dilution factor of the antiserum was 27K and the concentration of the original coating was 0.1 μg/mL, the immunogen Azap-KLH 1500:1 was used. , Azap-KLH 3000:1 and Azap-KLH 4500:1 immunized mice with titers of 1.719, 1.822 and 1.279 respectively, and the inhibition rates were 82%, 79% and 75% respectively after adding 200ppb Azap standard. The titer and inhibition rate of the mice immunized with the immunogen Azap-KLH 1500:1 were the highest, so this mouse was selected for the next experiment.

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

a. Take the blood from the eyeballs. After the mice were killed by cervical dislocation, they were immediately put into 75% alcohol for disinfection, soaked for about 5 minutes, and the spleen of the mice was taken out aseptically. The spleen cell suspension was obtained by netting, collected, centrifuged (1200 rpm, 8 min), washed three times with RPMI-1640 medium, and after the last centrifugation, the spleen cells were diluted to a certain volume, counted, and used for later use;

b. Collection of SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were treated with RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO ₂ incubator. Before fusion, the number of SP2/0 tumor cells was required to reach 1-4×10 ⁷ to ensure that the SP2/0 tumor cells were in the logarithmic growth phase before fusion. During fusion, tumor cells were collected, suspended in RPMI-1640 basal medium, and counted;

c. The fusion process is 7 minutes. For the 1st minute, 1 mL of PEG 1500 was added dropwise to the cells from slow to fast; for the 2nd minute, let stand. At the 3rd and 4th minutes, 1 mL of RPMI-1640 medium was added dropwise within 1 minute; at the 5th and 6th minutes, 2 mL of RPMI-1640 medium was added dropwise within 1 minute; at the 7th minute, 1 mL of RPMI-1640 medium was added dropwise every 10s. Then incubate at 37°C for 5min. Centrifuge (800rpm, 8min), discard the supernatant, resuspend in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50×HAT, add 200 μL/well to a 96-well cell plate, and place at 37 °C, 5% CO ₂ incubator.

(5) Cell screening and establishment of cell lines: on the 3rd day of cell fusion, RPMI-1640 was used to screen the fused cells, and the medium was half-changed. The RPMI-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, ic-ELISA is used to screen out positive cell wells, and in the second step, Azap is used as the standard substance, and the inhibitory effect of positive cells is determined by ic-ELISA. Cell wells with better inhibition of Azap standard were selected, subcloned by limiting dilution method, and detected by the same method. This was repeated three times to obtain cell line 1A8.

(6) Preparation and identification of monoclonal antibodies: Take 8-10 week old BALB/c mice, inject 1 mL of sterile paraffin oil into each mouse intraperitoneally; 7 days later, inject 1×10 ⁶ hybridoma cells into each mouse intraperitoneally , the ascites was collected from the seventh day, and the ascites was purified by the caprylic acid-ammonium sulfate method. Under partial acid conditions, n-octanoic acid can precipitate other impurity proteins except IgG immunoglobulin in ascites, then centrifuge and discard the precipitate; then use ammonium sulfate solution of equal saturation to precipitate IgG-type monoclonal antibody, centrifuge, discard The supernatant was dissolved in 0.01M PBS solution (pH 7.4), and then dialyzed for desalting, and finally the purified monoclonal antibody was obtained and stored at -20°C.

Example 2: Determination of the _IC50 of Azap monoclonal antibody

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 1000 mL with double-distilled water and store at 4°C for later use.

Phosphate buffered saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH ₂ PO ₄ , 2.9g Na ₂ HPO ₄ ·12H ₂ O, dissolved in 800 mL of pure water, adjusted to pH 7.2～ 7.4, dilute to 1000mL;

Washing solution (PBST): 0.5 mL of Tween-20 was added to 1000 mL of 0.01 mol/L pH 7.4 PBS solution;

PBST: PBS containing 0.05% Tween 20;

Antibody Diluent: Wash buffer containing 0.1% gelatin;

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

The color developing solution is used and mixed now.

(1) Coating: The coated original Azap-OVA was diluted with 0.05M pH 9.6 carbonate buffer from 1 μg/mL, 100 μL/well, and reacted at 37°C for 2 h.

(2) Washing: Pour off the solution in the plate, and wash with washing solution 3 times, 3min each time.

(3) Blocking: After patting dry, add 200 μL/well of blocking solution, and react at 37° C. for 2 h. Dry after washing.

(4) Sampling: The antiserum (antiserum after the mouse is tail-broken for blood collection, diluted with the antibody diluent to the corresponding multiples) is diluted from 1:1000, and added to the coated wells of each dilution. 100 μL/well, react at 37 °C for 30 min; after thorough washing, add 1:3000 diluted HRP-goat anti-mouse IgG, 100 μL/well, and react at 37 °C for 30 min.

(5) Color development: Take out the ELISA plate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37°C for 15 minutes in the dark.

(6) Termination and determination: 50 μL of termination solution was added to each well to terminate the reaction, and then the OD450 value of each well was measured with a microplate reader.

The IC ₅₀ of the monoclonal antibody Azap determined by ic-ELISA was 0.5 ng/mL, indicating that it has a good sensitivity to Azap and can be used for Azap immunoassay detection.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (10)

1. A monoclonal cell line has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee on September 5, 2017, with the preservation number of CGMCC No. 14694, and the preservation address is No. 1, Beichen West Road, Chaoyang District, Beijing. Courtyard No. 3. 2 . A method for detecting azaperidone, characterized in that the method utilizes the azaperidone monoclonal antibody secreted and produced by the monoclonal cell line of claim 1 . 3 . An azapirone monoclonal antibody, characterized in that it is secreted and produced by the hybridoma cell of claim 1 . 4. the application of the azaperidone monoclonal antibody according to claim 3, is characterized in that, is used for the analysis and detection of azaperidone residue in food safety detection. 5 . A kit comprising the monoclonal cell line of claim 1 . 6 . 6 . The kit according to claim 5 , wherein the kit is used for the analysis and detection of azaperidone residues in food safety detection. 7 . 7. The application of the kit according to claim 5 or 6 in detecting the content of azaperidone. 8. A kit, characterized in that it contains the azaperidone monoclonal antibody of claim 3. 9 . The kit according to claim 8 , wherein the kit is used for the analysis and detection of azaperidone residues in food safety detection. 10 . 10. The application of the kit according to claim 8 or 9 in detecting the content of azaperidone.

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