CN110616195B

CN110616195B - Metformin monoclonal antibody hybridoma cell strain and application thereof

Info

Publication number: CN110616195B
Application number: CN201910961111.1A
Authority: CN
Inventors: 胥传来; 许晓昕; 匡华; 徐丽广; 孙茂忠; 刘丽强; 吴晓玲; 宋珊珊; 胡拥明
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-10-11
Filing date: 2019-10-11
Publication date: 2021-05-04
Anticipated expiration: 2039-10-11
Also published as: CN110616195A

Abstract

A metformin monoclonal antibody hybridoma cell strain and application thereof belong to the field of food safety immunodetection. The metformin monoclonal antibody hybridoma cell strain Tcf 1A6 prepared by the invention has been preserved in the China general microbiological culture Collection center (CGMCC), the preservation date is 3 and 7 days in 2019, and the preservation number is CGMCC No. 17399. The invention mixes and emulsifies the complete antigen of the metformin and an equivalent Freund's adjuvant, and screens hybrid cells after the fusion of two kinds of cells by injecting immune BALB/c mice subcutaneously at multiple points on the neck and back; and screening cells by an indirect competitive enzyme-linked immunosorbent assay and subcloning for three times to finally obtain a monoclonal antibody hybridoma cell strain Tcf 1A 6. The monoclonal antibody secreted by the cell strain Tcf 1A6 provided by the invention has better specificity and detection sensitivity (IC) on MET₅₀The value is 1 ng/mL), can realize the detection of the MET residual quantity in the liver and the feed of chicken and chicken, provides raw materials for the immunodetection of the MET residual quantity in food, and has practical application value.

Description

Metformin monoclonal antibody hybridoma cell strain and application thereof

Technical Field

The invention relates to a metformin monoclonal antibody hybridoma cell strain and application thereof, and belongs to the field of food safety immunodetection.

Background

Metformin (MET) is a hypoglycemic agent and has an inhibitory effect on type ii diabetes, particularly obese type ii diabetes, for which simple diet control and physical exercise therapy are ineffective. The metformin and insulin are used together, so that the dosage of insulin can be reduced, and hypoglycemia can be prevented. It can be used together with sulfonylurea hypoglycemic agent, and has synergistic effect. Metformin can promote the utilization of glucose by peripheral tissue cells (muscles and the like), and inhibit the action of hepatic gluconeogenesis, thereby reducing hepatic glucose output, and inhibiting the glucose uptake by intestinal wall cells.

In recent years, particular attention has been paid to various chronic diseases caused by the low-level intake of metformin residues for a long period of time, the induction of drug resistance by pathogenic bacteria, and the like. Metformin causes diabetes and dizziness, and acute fever. Has gastrointestinal tract reaction. Nausea, abdominal pain, diarrhea and other digestive tract symptoms are rarely seen, and the dosage of the composition is reduced when the composition is taken at meal or after meal. The product can be used for treating diseases without stopping administration, and can disappear automatically. In the case of excess, lactic acidosis may occur due to accumulation. The product can reduce the absorption of vitamin B12 by human body after long-term use. The residual amount in the food should not exceed 5 mug/mL according to the pharmacopoeia residual solvent assay requirement. Therefore, the establishment of a method for quickly and effectively detecting the content of the metformin has important significance and market value. The detection is carried out by adopting a high performance liquid chromatography method, the detection method is relatively complicated and complex, the detection limit is too high, in order to maintain the benefits of vast consumers, a high-efficiency and quick detection method aiming at MET is needed to be established, the enzyme-linked immunosorbent assay (ELISA) pretreatment is simple, the cost is low, the quick detection of a large number of samples can be realized, and the requirement on the purity of the samples during detection is not high. Therefore, it is necessary to establish an efficient immunological detection method, and an important prerequisite for establishing the method is to screen a monoclonal monomer with high specificity for metformin.

Disclosure of Invention

The invention aims to provide a metformin monoclonal antibody hybridoma cell strain, and an antibody prepared from the cell strain has good specificity and detection sensitivity on metformin, and can be used for establishing an immunological detection method of metformin.

According to the technical scheme, a metformin monoclonal antibody hybridoma cell strain Tcf 1A6 is preserved in China general microbiological culture Collection center, and the address is as follows: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, is classified and named as monoclonal cell strain with the preservation date of 2019, 3 months and 7 days and the preservation number of CGMCC No. 17399.

The metformin monoclonal antibody is secreted and generated by a metformin monoclonal antibody hybridoma cell strain Tcf 1A6 with the preservation number of CGMCC No. 17399.

The application of the metformin monoclonal antibody is used for analyzing and detecting metformin residues in food safety detection.

The preparation of the metformin monoclonal antibody hybridoma cell strain Tcf 1A6 provided by the invention comprises the following basic steps: reacting metformin and p-aldehyde benzoic acid to obtain a product with carboxyl, namely hapten 4cPME, and coupling the hapten 4cPME and carrier protein by using a carbodiimide method to obtain the metformin artificial antigen; the method comprises the following steps:

(1) synthesis of hapten 4cPME, the synthetic route is as follows:

；

adding 0.65 g of compound p-aldehyde benzoic acid (4-CBA) into a 25 mL round-bottom flask, and adding 10mL of pure water and 3mL of 1M HCl solution into the round-bottom flask; under the stirring of a magnetic stirrer, slowly dropwise adding DMF into the system until the 4-CBA is completely dissolved;

② adding 1g of metformin hydrochloride (MET. HCl) to dissolve, placing the mixture in a water bath kettle at 60 ℃ for magnetic stirring, connecting a condensing device for refluxing overnight to generate white turbidity;

thirdly, centrifuging the reaction solution at 5000rpm for 10min, discarding the supernatant, placing the white solid on filter paper, filtering and washing the white solid for 3 times by pure water, and finally placing the white solid in a 60 ℃ oven for drying to obtain a product hapten 4 cPME;

(2) preparation of complete antigen 4 cpMET-BSA: 1.8mg of MET derivative 4cPME (the molar ratio of 4cPME to Bovine Serum Albumin (BSA) is 30: 1) is weighed and dissolved in 300 mu L N, N-Dimethylformamide (DMF), stirred for reaction for 10min, after full dissolution, 1.3mg of N-hydroxysuccinimide (NHS) and 2.1mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) are added in sequence, and activation reaction is carried out at room temperature for 4-6h to obtain solution A. 6mg of BSA was dissolved in 2mL of 0.01M carbonate buffer (CB, pH = 9.0) (referred to as solution B), and solution A was slowly added dropwise to solution B, followed by coupling reaction at room temperature overnight; dialyzing with 0.01M PBS solution, removing unreacted small molecule hapten to obtain complete antigen 4 cpME-BSA, and identifying by ultraviolet absorption scanning method;

(3) immunization of mice: after mixing and emulsifying the 4 cPME-BSA complete antigen and an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous multipoint injection on the back of the neck (except for the puncture immunization). Complete Freund adjuvant is used for the first immunization, and the dosage is 100 mug/mouse; incomplete Freund's adjuvant is used for multiple times of boosting immunization, and the dosage is reduced by half to be 50 mu g/mouse; the thorny immunity does not use an adjuvant, and the thorny immunity is directly diluted by normal saline and injected into the abdominal cavity, and the dosage is reduced by half to obtain 25 mu g/mouse. The interval between the first immunization and the second boosting immunization is one month, the interval between the multiple boosting immunizations is 21 days, and the interval between the sprint immunization and the last boosting immunization is 18-21 days. The immune effect of the mouse is observed by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), namely the titer and inhibition of the mouse serum are detected;

(4) cell fusion and cell line establishment: fusing mouse spleen cells and mouse myeloma cells by a polyethylene glycol (PEG 4000) method, screening hybridoma cells by using a selective medium (HAT medium), and performing cell culture by using an HT medium. And detecting the positive cell holes by using an ic-ELISA method after one week of fusion, further determining the inhibition effect of the positive cell holes by using the ic-ELISA method, performing subcloning on the positive cell holes with better inhibition by using a limiting dilution method, and detecting, selecting and subcloning again after one week. Carrying out subcloning for three times according to the method to obtain a monoclonal hybridoma cell strain Tcf 1A6 of the high-secretion specific antibody of MET;

(5) 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 cell strain Tcf 1A6 provided by the invention has better specificity and detection sensitivity (IC) on MET₅₀The value is 1 ng/mL), can realize the detection of the MET residual quantity in the liver and the feed of chicken and chicken, provides raw materials for the immunodetection of the MET residual quantity in food, and has practical application value.

Biological material sample preservation: a metformin monoclonal antibody hybridoma cell strain Tcf 1A6, which is preserved in China general microbiological culture Collection center, and has the address: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, is classified and named as monoclonal cell strain with the preservation date of 2019, 3 months and 7 days and the preservation number of CGMCC No. 17399.

Drawings

FIG. 1 is a standard curve for inhibition of the Tcf 1A6 monoclonal antibody.

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 a metformin complete antigen, cell fusion and HAT selective culture medium culture are carried out, and cell supernatant is screened by ic-ELISA, so that a hybridoma cell strain Tcf 1A6 with high secretion specificity antibody for metformin is finally obtained.

EXAMPLE 1 preparation of hybridoma cell line Tcf 1A6

(1) Synthesis of complete antigen: 1.8mg of MET derivative 4cPME (the molar ratio of 4cPME to Bovine Serum Albumin (BSA) is 30: 1) is weighed and dissolved in 300 mu L N, N-Dimethylformamide (DMF), stirred for reaction for 10min, after full dissolution, 1.3mg of N-hydroxysuccinimide (NHS) and 2.1mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) are added in sequence, and activation reaction is carried out at room temperature for 4-6h to obtain solution A. 6mg of BSA was dissolved in 2mL of 0.01M carbonate buffer (CB, pH = 9.0) (referred to as solution B), and solution A was slowly added dropwise to solution B, followed by coupling reaction at room temperature overnight; dialyzing with 0.01M PBS solution, removing unreacted small molecule hapten to obtain complete antigen 4 cpME-BSA, and identifying by ultraviolet absorption scanning method;

(2) animal immunization: after mixing and emulsifying the 4cPME complete antigen and an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous multipoint injection on the back of the neck (except for the puncture immunization). Complete Freund adjuvant is used for the first immunization, and the dosage is 100 mug/mouse; incomplete Freund's adjuvant is used for multiple times of boosting immunization, and the dosage is reduced by half to be 50 mu g/mouse; the thorny immunity does not use an adjuvant, and the thorny immunity is directly diluted by normal saline and injected into the abdominal cavity, and the dosage is reduced by half to obtain 25 mu g/mouse. The interval between the first immunization and the second boosting immunization is one month, the interval between the multiple boosting immunizations is 21 days, and the interval between the sprint immunization and the last boosting immunization is 18-21 days. The immune effect of the mouse is observed by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), namely the titer and inhibition of the mouse serum are detected;

(3) 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. the method comprises the following steps of (1) taking eyeballs of a mouse, taking blood, killing the mouse by a cervical vertebra dislocation method, immediately placing the mouse into 75% alcohol for disinfection, soaking for about 5min, taking out the spleen of the mouse by aseptic operation, properly grinding the spleen by using a syringe rubber head, passing through a 200-mesh cell screen to obtain a spleen cell suspension, collecting, centrifuging (1200 rpm, 8 min), 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. collection of SP_2/0Cell: 7-10 days before fusion, SP is added_2/0Tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) at 5% CO₂Culturing in an incubator. Pre-fusion requirements SP_2/0Number of neoplastic cells up to (1-4) x 10⁷Ensuring SP before fusion_2/0The tumor cells are in logarithmic growth phase. 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, dripping 1mL of PEG4000 into cells from slow to fast; standing for 2 min. Dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4 min; dropping 2mL of RPMI-1640 culture medium within 1min at 5min and 6 min; at 7min, 1mL of RPMI-1640 medium was added dropwise every 10 s. Then, the mixture is incubated at 37 ℃ for 5 min. Centrifuging (800 rpm, 10 min), discarding the supernatant, gently tapping the cells, adding RPMI-1640 selective medium (HAT medium) containing 20% fetal bovine serum and 2%50 XHAT to the supernatant, adding the mixture to a 96-well cell plate at 200. mu.L/well, and standing at 37 ℃ with 5% CO₂Culturing in an incubator;

(4) cell screening and cell strain establishment: half-changing the fused cells with HAT medium on day 3 after cell fusion; on day 5, the whole culture medium was changed with RPMI-1640 medium (HT medium) containing 20% fetal bovine serum and 1% 100 × HT; cell supernatants were taken on day 7 for screening. The screening is divided into two steps: firstly, screening positive cell holes by using an ic-ELISA method, secondly, selecting metformin as a standard substance, and measuring the inhibition effect of the positive cells by using the ic-ELISA method. And selecting cell pores with better inhibition on the metformin standard substance, performing subcloning by adopting a limiting dilution method, and detecting by using the same method after seven days. Carrying out subcloning for three times according to the method to finally obtain a metformin monoclonal antibody cell strain Tcf 1A 6;

(5) preparation and identification of monoclonal antibody: collecting 8-10 week old BALB/c mouseInjecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 10⁶Metformin hybridoma cells, ascites fluid was collected from the seventh day, and antibody purification was performed on the ascites fluid by the octanoic acid-saturated ammonium sulfate method. Under the condition of partial acid, the caprylic acid can precipitate other hybrid proteins except IgG immunoglobulin in the ascites, then the centrifugation is carried out, and the precipitate is discarded; precipitating IgG type monoclonal antibody with ammonium sulfate solution of equal saturation degree, centrifuging, discarding supernatant, dissolving with 0.01M PBS solution (pH7.4), dialyzing, desalting, and storing at-20 deg.C;

5.1 coating: diluting the coated MET-OVA with 0.05M carbonate buffer solution (pH9.6) at 3 times from 1 μ g/mL, 100 μ L/well, and reacting at 37 deg.C for 2 h;

5.2 washing: the plate solution was decanted and washed 3 times for 3min each with washing solution;

5.3 sealing: after patting to dryness, 200. mu.L/well blocking solution was added and reacted at 37 ℃ for 2 hours. Drying for later use after washing;

5.4 sample adding: diluting antiserum from 1:1000 times, adding into coated wells of each dilution, reacting at 37 deg.C for 30min at 100 μ L/well; after fully washing, adding HRP-goat anti-mouse IgG diluted by 1:3000, 100 mu L/hole, and reacting for 30min at 37 ℃;

5.5 color development: taking out the ELISA plate, fully washing, adding 100 mu L of TMB color development solution into each hole, and reacting for 15min at 37 ℃ in a dark place;

5.6 termination and determination: the reaction was stopped by adding 50. mu.L of a stop buffer to each well, and the OD 450 value of each well was measured by a microplate reader.

Determination of IC of the monoclonal antibody metformin by IC-ELISA₅₀Comprises the following steps: 1ng/mL, which indicates that the reagent has good sensitivity to metformin and can be used for immunoassay detection of metformin.

Solution preparation:

carbonate Buffer (CBS): weighing Na₂CO₃ 1.59 g，NaHCO₃2.93 g of the mixture is respectively dissolved in a small amount of double distilled water and then mixed, the double distilled water is added to about 800mL and mixed evenly, the pH value is adjusted to 9.6, the double distilled water is added to the volume of 1000mL, and the mixture is stored for later use at 4 ℃;

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

PBST: PBS containing 0.05% tween 20;

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. And the volume ratio of the solution B to the solution B is 5: 1 to obtain the TMB color developing solution which is mixed at present.

Claims

1. A metformin monoclonal antibody hybridoma cell strain Tcf 1A6, which is preserved in China general microbiological culture Collection center, and has the address: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, is classified and named as monoclonal cell strain with the preservation date of 2019, 3 months and 7 days and the preservation number of CGMCC No. 17399.

2. A metformin monoclonal antibody characterized by: the metformin monoclonal antibody hybridoma cell strain Tcf 1A6 with the preservation number of CGMCC No.17399 as claimed in claim 1 is secreted and produced.

3. The use of the metformin monoclonal antibody according to claim 2, characterized in that: the method is used for analyzing and detecting the residual metformin in food safety detection.

4. The preparation method of the metformin complete antigen 4 cPME-BSA is characterized by comprising the following steps: weighing 1.8mg MET derivative 4cPME, wherein the molar ratio of 4cPME to bovine serum albumin BSA is 30: dissolving the mixture in 300 mu LN, N-dimethylformamide DMF, stirring and reacting for 10min, after full dissolution, adding 1.3mg of N-hydroxysuccinimide NHS and 2.1mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC in sequence, and activating and reacting for 4-6h at room temperature to obtain solution A; 6mg of BSA was dissolved in 2mL of 0.01M carbonate buffer CB (pH = 9.0) to obtain solution B; slowly adding the solution A into the solution B drop by drop, and performing room-temperature coupling reaction overnight; dialyzing with 0.01M PBS solution, removing unreacted small molecule hapten to obtain complete antigen 4 cpME-BSA, and identifying by ultraviolet absorption scanning method;

the structural formula of the 4cPME is as follows:

(ii) a Named as p-metformin benzoic acid.

5. The method for preparing metformin complete antigen 4cpMET-BSA according to claim 4, wherein the 4cpMET is synthesized by the following route:

；

adding 0.65 g of p-aldehyde benzoic acid 4-CBA compound into a 25 mL round-bottom flask, and adding 10mL of pure water and 3mL of 1M HCl solution into the round-bottom flask; under the stirring of a magnetic stirrer, slowly dropwise adding DMF into the system until the 4-CBA is completely dissolved;

adding 1g of metformin hydrochloride MET & HCl for dissolution, placing the mixture in a water bath kettle at the temperature of 60 ℃ for magnetic stirring, connecting a condensing device for refluxing overnight, and generating white turbidity;

thirdly, the reaction solution is centrifuged for 10min at 5000rpm, the supernatant is discarded, the white solid is placed on filter paper, filtered and washed for 3 times by pure water, and finally placed in a 60 ℃ oven for drying, and the product hapten 4cPME is obtained.