CN108410824B

CN108410824B - Azepinone monoclonal antibody hybridoma cell strain and application thereof

Info

Publication number: CN108410824B
Application number: CN201810209891.XA
Authority: CN
Inventors: 匡华; 林璐; 胥传来; 徐丽广; 马伟; 刘丽强; 吴晓玲; 宋珊珊; 胡拥明
Original assignee: Wuxi Determine Bio Tech Co ltd; Jiangnan University
Current assignee: Wuxi Determine Bio Tech Co ltd; Jiangnan University
Priority date: 2018-03-14
Filing date: 2018-03-14
Publication date: 2020-03-06
Anticipated expiration: 2038-03-14
Also published as: CN108410824A

Abstract

The invention discloses an azapirone monoclonal antibody hybridoma cell strain and application thereof, and belongs to the field of food safety immunodetection. The invention prepares the azapirone complete antigen, and completely mixes and emulsifies the azapirone complete antigen and an equivalent Freund's adjuvant, and immunizes a BALB/c mouse through back subcutaneous injection to obtain a hybridoma cell strain CGMCC No.14694 for generating the azapirone antibody. The monoclonal antibody secreted by the cell strain has better specificity and detection sensitivity (IC) on the azaperone₅₀The value was 0.5 ng/mL). The achievement of the invention can be used for establishing an immunodetection method for the azaperone residue in animal tissues, and has practical application value.

Description

Azepinone monoclonal antibody hybridoma cell strain and application thereof

Technical Field

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

Background

Azapirone (also known as Azaperone, abbreviated as Azap) is a butyrophenone neuroleptic. After the medicine is injected into animals through muscles, the stress can be eliminated, the activity is reduced, the animals are indifferent to the environment and are in a quiet state for a long time, the phenomena that the animals attack and fight each other due to 'gathering stress' and in the mixed group are avoided, the death rate caused by stress and trauma is reduced, and therefore the medicine is often used for the animals such as pigs and the like in long-distance transportation. The maximum residual limit value (MRL) of the azapirone in the muscle, the skin, the fat, the liver and the kidney of the pig specified by the maximum residual limit standard in animal food in China is 60mg/kg, 60mg/kg, 100mg/kg and 100mg/kg respectively. Therefore, the establishment of a rapid and effective method for detecting the content of the azapirone has important significance and market value.

The enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method, however, the obtaining of a monoclonal antibody with high affinity and high specificity is a prerequisite for immunological detection, and the synthesis of artificial antigen is an important step. The reports on the detection method of the residual azapirone are less, so that the establishment of a rapid and simple azapirone detection method has important significance. The enzyme-linked immunosorbent assay (ELISA) is an extremely high-efficiency, sensitive and rapid detection method, has low requirement on the purity of a sample during detection, is simple and convenient to operate, and is suitable for the field rapid detection of a large number of samples. Establishing an efficient immunological detection method, and screening a monoclonal antibody with high specificity is an important prerequisite.

Disclosure of Invention

The invention aims to provide a azapirone monoclonal antibody hybridoma cell strain which is preserved in China general microbiological culture Collection center (CGMCC) on 9 and 5 days in 2017, wherein the preservation number is CGMCC No.14694, and the preservation address is No. 3 of Beijing Shangyang district Beichen Xilu No. 1. The antibody prepared by the cell strain has better specificity and detection sensitivity to the azaperone, and can be used for establishing an immunological detection method of the azaperone.

The second purpose of the invention is to provide an azapirone monoclonal antibody which is secreted and produced by the azapirone monoclonal antibody hybridoma cell strain with the preservation number of CGMCC No. 14694.

The application of the azapirone monoclonal antibody is used for analyzing and detecting the residual azapirone in animal tissues.

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

1) synthesis of hapten Azap: adding 4-phenylbutyric acid methyl ester, 4-chlorobutyryl chloride and aluminum chloride into carbon disulfide, heating to room temperature, and stirring overnight; adding ice water into the reaction solution, extracting with dichloromethane, drying with anhydrous sodium sulfate, and concentrating to obtain a yellow oily intermediate product 2;

adding the intermediate product 2, potassium iodide and potassium carbonate into an acetonitrile solution, adding 1- (2-pyridyl) piperazine, and heating the reaction solution to 70 ℃ for overnight; adding ice water into the reaction solution, extracting with dichloromethane, drying with anhydrous sodium sulfate, and concentrating; purifying by a silica gel column to obtain a yellow oily intermediate product 3;

adding tetrahydrofuran and water into the intermediate product 3 and lithium hydroxide; heating the reaction solution to room temperature, and stirring overnight; acidifying with hydrochloric acid until pH is 6, washing with saturated saline solution, drying with anhydrous sodium sulfate, and concentrating to obtain white solid, to obtain hapten Azap;

2) preparation of complete antigen:

preparation of immunogen Azap-KLH: weighing hapten Azap, 1-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide, and dissolving with anhydrous N, N-dimethylformamide to obtain solution A; dissolving keyhole limpet hemocyanin KLH with boric acid buffer solution to obtain solution B, dropwise adding the solution A into the solution B at room temperature, reacting at room temperature overnight to obtain conjugate Azap-KLH mixed solution, and dialyzing to separate complete antigen and unconjugated small molecular hapten to obtain immunogen Azap-KLH.

The fourth purpose of the invention is to provide a method for preparing the azapirone monoclonal antibody hybridoma cell strain, which mainly comprises the following steps:

(1) immunization of mice: mixing and emulsifying the immunogen Azap-KLH and an equivalent amount of Freund's adjuvant, and injecting the mixture to immunize BALB/c mice through back subcutaneous injection; complete Freund adjuvant is used for the first immunization, incomplete Freund adjuvant is used for the multiple times of boosting immunization, the interval between the first immunization and the second boosting immunization is 28 days, the interval between the multiple times of boosting immunization is 21 days, and the final time of sprint immunization is carried out by Azap-KLH complete antigen (without adjuvant); detecting serum titer and inhibition by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA);

(2) cell fusion and cell line establishment: fusing mouse splenocytes and mouse myeloma cells by a polyethylene glycol (PEG 4000) method, culturing by HAT culture medium, detecting positive cell holes by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), further determining the inhibition effect of the positive cell holes by the ic-ELISA, carrying out three times of subcloning on the positive cell holes with the best inhibition effect by a limiting dilution method, and finally screening to obtain an Azap monoclonal antibody hybridoma cell strain;

(3) and (3) identification of the properties of hybridoma cell strains: sensitivity and specificity were determined by ic-ELISA.

The invention has the beneficial effects that: the monoclonal antibody secreted by the azapirone monoclonal antibody hybridoma cell strain has better specificity and detection sensitivity (IC) on the azapirone₅₀A value of 0.5ng/mL) provides an immunological method for the residual detection of azapirone in animal tissues. The azapirone monoclonal antibody hybridoma cell strain and the monoclonal antibody secreted by the azapirone monoclonal antibody hybridoma cell strain can be prepared into a kit for detecting azapirone, and have practical application values.

Biological material preservation

A monoclonal cell strain is preserved in China general microbiological culture Collection center (CGMCC) at 9.5.2017, with the preservation number of CGMCC No.14694 and the preservation address of No. 3 of Xilu 1 Beichen of the Korean district in Beijing.

Description of the drawings:

FIG. 1 is a standard curve of the inhibition of Azap by the monoclonal antibody 1A 8.

Detailed Description

The following examples are provided as further illustration of the invention and are not to be construed as limitations or limitations of the invention. The invention is further illustrated by the following examples.

According to the invention, a mouse is immunized by the Azap complete antigen, cell fusion and HAT selective culture medium culture are carried out, and cell supernatant is screened by ic-ELISA, so that the monoclonal antibody hybridoma cell strain with good specificity and sensitivity to Azap is finally obtained.

Example 1: preparation of hybridoma cell line 1A8

(1) Preparation of complete antigen:

the hapten synthetic 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 mixture was warmed to room temperature and stirred overnight; adding ice water into the reaction solution, extracting with dichloromethane, drying with anhydrous sodium sulfate, and concentrating to obtain a yellow oily intermediate product 2;

adding 1.20g of intermediate product 2, 98.5mg of potassium iodide and 1.17g of potassium carbonate into 12mL of acetonitrile solution, adding 700mg of 1- (2-pyridyl) piperazine, and heating the reaction solution to 70 ℃ overnight; adding ice water into the reaction solution, extracting with dichloromethane, drying with anhydrous sodium sulfate, and concentrating; purifying by a silica gel column to obtain a yellow oily intermediate product 3;

adding 360mg of intermediate product 3 and 90.2mg of lithium hydroxide into 3mL of tetrahydrofuran and 1mL of water; heating the reaction solution to room temperature, and stirring overnight; acidifying with hydrochloric acid until pH is 6, washing with saturated saline solution, drying with anhydrous sodium sulfate, and concentrating to obtain white solid, to obtain hapten Azap;

b. 1.87mg of Azap, 2.18mg of 1-ethylcarbodiimide hydrochloride and 1.32mg of N-hydroxysuccinimide were weighed and dissolved in 400. mu.L of anhydrous N, N-dimethylformamide to obtain A1 solution, which was stirred at room temperature for trans-6-8 hours. Dissolving keyhole limpet hemocyanin KLH 10mg (molar ratio of Azap to KLH is 1500:1) with 4mL of boric acid buffer solution to obtain B1 liquid, dropwise adding A1 liquid into B1 liquid at room temperature, reacting overnight at room temperature to obtain conjugate Azap-KLH (1500:1) mixed liquid, and separating complete antigen and unconjugated small molecular hapten through dialysis;

complete antigens were prepared in a molar ratio of Azap to KLH of 3000:1 and 4500:1, respectively, according to the above procedure to give conjugates Azap-KLH (3000:1) and Azap-KLH (4500: 1).

(2) Preparation of coating antigen Azap-OVA:

1.3mg of Azap, 2.0mg of 1-ethylcarbodiimide hydrochloride and 1.2mg of N-hydroxysuccinimide were weighed, and dissolved in 300. mu.L of anhydrous N, N-dimethylformamide to obtain solution A2, which was stirred at room temperature for reverse 6-8 hours. Weighing 5mg of chicken egg white albumin OVA (the molar ratio of Azap to OVA is 30:1), dissolving the 5mg of chicken egg white albumin OVA in 2mL of boric acid buffer solution to obtain B2 solution, dropwise adding the A2 solution into the B2 solution at room temperature, reacting overnight at room temperature to obtain conjugate Azap-OVA mixed solution, and separating the envelope antigen and the unconjugated small molecule hapten through dialysis. The coating antigen is used for detecting the serum titer and inhibition of the mouse in the monoclonal antibody preparation process, is not directly used for the mouse, and is necessary for preparing the monoclonal antibody.

(3) Animal immunization: healthy BALB/c mice 6-8 weeks old were selected for immunization. Three different molar ratios of Azap complete antigen were mixed with equal amounts of Freund's adjuvant and emulsified, and then BALB/c mice were immunized by back subcutaneous injection, respectively. 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 boosting immunization is 28 days, and the interval between the boosting immunization is 21 days. Blood was collected 7 days after the third immunization (mice were bled with tail cut 5ul +995ul of antibody diluent as antiserum), serum titer and inhibition of mice were measured using ic-ELISA, mice with high titer inhibition were selected, and 21 days after the fourth immunization, immunization by spiking, i.e. intraperitoneal injection, required that the dose of the boost was halved and no adjuvant was included.

Selection of spurted immune mice: the serum titer and inhibition rate of the mice were determined by ic-ELISA and were selected for the next experiment when the antiserum was diluted at 27K and the coating concentration was 0.1. mu.g/mL, and the titers of the mice immunized with the immunogens Azap-KLH 1500:1, Azap-KLH 3000:1 and Azap-KLH 4500:1, respectively, were 1.719,1.822 and 1.279, respectively, and the inhibition rates of the mice added with 200ppbAzap standards were 82%, 79% and 75%, respectively.

(4) 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. taking eyeballs and blood, immediately putting the mice into 75% alcohol for disinfection after the mice are killed by a cervical vertebra dislocation method, soaking for about 5min, taking out the spleen of the mice by 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% CO₂An incubator. Before fusion, the number of SP2/0 tumor cells is required to reach 1-4 multiplied by 10⁷Ensuring that SP2/0 tumor cells are in logarithmic growth phase before fusion. During fusion, tumor cells are collected and suspended in RPMI-1640 basic culture solution for cell counting;

c. the fusion process is 7 min. 1min, 1mL of PEG 1500 is added to the cells from slow to fast; standing for 2 min. Dropping 1mLRPMI-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 medium was added dropwise every 10 s. Then, the mixture is incubated at 37 ℃ for 5min. 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.

(5) Cell screening and cell strain 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: in the first step, positive cell holes are screened by ic-ELISA, in the second step, Azap is selected as a standard substance, and the inhibition effect of positive cells is measured by ic-ELISA. And selecting cell wells with good inhibition to the Azap standard, performing subcloning by using a limiting dilution method, and detecting by using the same method. This was repeated three times to obtain cell line 1A 8.

(6) Preparation and identification of monoclonal antibody: 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 the ascites fluid was purified by the octanoic acid-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, and the supernatant was discarded, dissolved in a 0.01M PBS solution (pH7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.

Example 2: IC of Azap monoclonal antibody₅₀Measurement of (2)

Carbonate Buffer (CBS): weighing Na₂CO₃1.59g，NaHCO₃2.93g, 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;

wash solution (PBST): adding 0.5mL of Tween-20 into 1000mL of 0.01mol/L PBS solution with pH of 7.4;

PBST: PBS containing 0.05% tween 20;

antibody dilution: wash buffer containing 0.1% gelatin;

TMB color development liquid: solution A: na (Na)₂HPO₄.12H₂18.43g 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. Mixing the solution B at a ratio of 1:5 to obtain TMB.

The color developing liquid is mixed at present.

(1) Coating: the coating antigen Azap-OVA was diluted in 0.05M carbonate buffer pH 9.6 at 1. mu.g/mL in duplicate, 100. mu.L/well, and reacted at 37 ℃ for 2 h.

(2) Washing: the plate solution was decanted and washed 3 times for 3min each with washing solution.

(3) And (3) sealing: after patting to dryness, 200. mu.L/well blocking solution was added and reacted at 37 ℃ for 2 hours. And drying after washing for later use.

(4) Sample adding: diluting antiserum (which is obtained by diluting the antiserum by a corresponding multiple with an antibody diluent after tail-cutting blood collection of a mouse) from 1:1000 by multiple, adding the diluted antiserum into coated holes of each dilution, reacting at the temperature of 100 mu L/hole for 30 min; after washing sufficiently, HRP-goat anti-mouse IgG diluted at a ratio of 1:3000 was added thereto at 100. mu.L/well, and the reaction was carried out at 37 ℃ for 30 min.

(5) Color development: the ELISA plate was removed, washed thoroughly, and 100. mu.L of TMB developing solution was added to each well, and the reaction was carried out at 37 ℃ in the dark for 15min.

(6) Termination and measurement: the reaction was stopped by adding 50. mu.L of a stop buffer to each well, and the OD450 value of each well was measured by a microplate reader.

Determination of IC of monoclonal antibody Azap by IC-ELISA₅₀The concentration is 0.5ng/mL, which indicates that the kit has good sensitivity to Azap and can be used for Azap immunoassay detection.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A monoclonal cell strain is preserved in China general microbiological culture Collection center (CGMCC) at 9.5.2017, with the preservation number of CGMCC No.14694 and the preservation address of No. 3 of Xilu 1 Beichen of the Korean district in Beijing.

2. A method for detecting azapirone, which comprises using the monoclonal cell line of claim 1 to produce a secreted azapirone monoclonal antibody.

3. An azapirone monoclonal antibody secreted by the hybridoma cells of claim 1.

4. The use of the azapirone monoclonal antibody of claim 3 for the analytical detection of residual azapirone in food safety testing.

5. A kit comprising the monoclonal cell strain of claim 1.

6. The kit of claim 5, wherein the kit is used for the analytical detection of azapirone residues in food safety tests.

7. Use of the kit of claim 5 or 6 for detecting azapirone content.

8. A kit comprising the azapirone monoclonal antibody of claim 3.

9. The kit of claim 8, wherein the kit is used for the analytical detection of azapirone residues in food safety tests.

10. Use of the kit of claim 8 or 9 for detecting azapirone content.