CN110669736A

CN110669736A - Analgin monoclonal antibody hybridoma cell strain CBD and application thereof

Info

Publication number: CN110669736A
Application number: CN201911146280.6A
Authority: CN
Inventors: 胥传来; 李月; 匡华; 徐丽广; 马伟; 刘丽强; 吴晓玲; 宋珊珊; 胡拥明
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-11-21
Filing date: 2019-11-21
Publication date: 2020-01-10

Abstract

An analgin monoclonal antibody hybridoma cell strain CBD and application thereof, belonging to the field of food safety immunoassay. The invention obtains an analgin monoclonal antibody hybridoma cell strain CBD through screening, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 18511. And the method is used for detecting the residue of analgin and metabolites thereof in food. The monoclonal antibody secreted by the cell strain has better affinity and detection sensitivity to four main metabolites of analgin, (MAA: IC)₅₀=8.1 ng/mL，AA：IC₅₀=8.8 ng/mL，FA：IC₅₀=0.9 ng/mL，AAA：IC₅₀=0.6 ng/mL), can be used for meat products,The detection of the residual quantity of the analgin metabolite in the dairy products and the like provides raw materials for the immunodetection of the residual quantity of the analgin metabolite in the food, and has practical application value.

Description

Analgin monoclonal antibody hybridoma cell strain CBD and application thereof

Technical Field

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

Background

Analgin (dipyrone, abbreviated as Dip) is one of antipyretic analgesics commonly used in clinic, belongs to pyrazolone antipyretic analgesics, and has been used for treatment for centuries since clinical administration. Analgin can be rapidly hydrolyzed into 4-Methylaminoantipyrine (MAA) as an active product in vivo, and then enters systemic circulation; MAA is metabolized in vivo to 4-formamidoantipyrine (4-formamidoantipyrine, FA) and 4-aminoantipyrine (4-aminoantipyrine, AA); AA can be continuously metabolized into 4-acetaminoantipyrine (AAA), and the specific mechanism is shown in figure 1.

Analgin has strong antipyretic and analgesic effects, is widely used in many countries at present, but can cause severe reactions such as granulocytopenia, aplastic anemia, severe allergy, shock collapse and the like. Due to its toxic side effects and yet uncertain pharmacodynamic action mechanism, some developed countries in the world have made a stop or restriction on the use of analgin, such as the drug is banned in the us, sweden for use in food-borne animals, and the japanese positive list also puts it in a uniform standard, whereas the european union has a restriction of 50 mug/kg (calculated as MAA content) in cow's milk.

The currently reported methods for assaying analgin or its analogs include spectrophotometry, flow injection chemiluminescence photometry, Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), etc., however, these instrumental methods all require complicated pre-treatment, are time-consuming and labor-consuming, and are not suitable for rapid detection of large amounts of samples. In order to maintain the benefits of the consumers, an efficient and rapid detection method for analgin is needed to be established, the enzyme-linked immunosorbent assay (ELISA) pretreatment is simple, the cost is low, the rapid 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 high specificity monoclonal monomer aiming at analgin.

Disclosure of Invention

The invention aims to provide an analgin monoclonal antibody hybridoma cell strain CBD and application thereof, and an ELISA method is established on the basis.

The technical scheme of the invention is that an analgin monoclonal antibody hybridoma cell strain CBD is preserved in China general microbiological culture collection center (CGMCC) of China Committee for culture Collection of microorganisms, and is classified and named as a monoclonal cell strain by the microbiological research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Kyoto-Yang, wherein the preservation date is 2019, 10 months and 14 days, and the preservation number is CGMCC No. 18511.

The analgin monoclonal antibody is secreted and generated by the analgin monoclonal antibody hybridoma cell strain CBD with the preservation number of CGMCC No. 18511.

The application of the analgin monoclonal antibody is used for detecting the analgin and metabolite residues thereof in food.

In one embodiment of the invention, the application is for the analytical detection of residues of metabolites of analgin in milk.

In one embodiment of the invention, the application is to prepare a reagent or a detection kit for detecting analgin metabolite by an ELISA competition method.

The preparation method of the analgin monoclonal antibody hybridoma cell strain CBD comprises the steps of synthesizing an analgin complete antigen, mixing and emulsifying the complete antigen and an equivalent amount of Freund's adjuvant, immunizing a mouse, and taking a high affinity (OD)₄₅₀>1.5), high sensitivity (IC)₅₀<200 ng/mL) of the immune mouse spleen cells and myeloma cells are fused, serum is detected by ic-ELISA, and subclones are screened to obtain hybridoma cells.

Because analgin and its metabolite belong to small molecule and have no immunogenicity, can not stimulate mice to produce immune response, and then produce antibody, it needs to couple analgin or its metabolite to protein through protein connection technology, so that it can obtain immunogenicity; the active groups commonly used in the protein coupling technology are amino, carboxyl, hydroxyl, sulfydryl and the like, and in view of preparing an antibody capable of detecting four metabolites of analgin simultaneously, a hapten needs to be designed, so that the structure of the hapten has the greatest similarity with the four metabolites, and the hapten contains the active groups. These reactive groups are not contained in the molecular formula and thus derivatization is required.

In one embodiment of the invention, hapten is synthesized as H1-HS from 4-aminoantipyrine (H1) and succinic anhydride. The synthetic route is as follows:

the method comprises the following steps: hapten is synthesized by taking 4-aminoantipyrine (H1) and succinic anhydride as starting materials and is marked as H1-HS. Weighing H150 mg, 4-dimethylaminopyridine 175 mg and succinic anhydride 300 mg, dissolving in 20 mL of anhydrous pyridine, and refluxing at 60 ℃ for 12H. The reaction solution was concentrated by evaporation, the residue was dissolved in 10 mL of water and extracted three times with 5 mL of ethyl acetate, and the organic phase was collected. And drying the organic phase by nitrogen to obtain a white solid, namely H1-HS, and identifying the structure by nuclear magnetism and mass spectrometry.

In one embodiment of the present invention, the synthetic route of the coated antigen H1-BSA is as follows:

the preparation method of the coating source comprises the following specific steps: weighing H12.03 mg, dissolving in 500 μ L of ultrapure water, adding 60 μ L of 2.5% glutaraldehyde solution, and stirring in ice bath for 15min to obtain reaction solution A. 5mg of BSA was weighed and dissolved in 1mL of 0.01M phosphate buffer solution (PBS, pH = 7.4) to obtain solution B. And dropwise adding the solution A into the solution B while stirring, and stirring at room temperature for reaction for 2 hours to obtain a coupling mixed solution. Taking a dialysis bag (protein retention: 3kDa) of 10cm, boiling for 5min, cooling to room temperature, adding the coupling mixed solution into the dialysis bag, dialyzing for 3 days by using PBS (pH = 7.4) and 0.01M, replacing the dialyzate every 6H, removing redundant small molecular compounds to obtain an artificial antigen H1-BSA, and identifying by adopting an ultraviolet absorption scanning method.

The synthesis route of the immunogen is as follows:

the immunogen preparation steps are as follows: weighing 1.1 mg of hapten H1-HS and 1.3 mg of N-hydroxysuccinimide (NHS), dissolving in 300 mu L N N-Dimethylformamide (DMF), and stirring at room temperature for 10 min; weighing 2.2 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), fully dissolving with 50 μ L of 2- (n-morpholine) ethyl sulfonic acid buffer solution (MES) with pH = 4.6 and 0.01M, adding into the H1-HS solution, and stirring at room temperature for 6-8H to obtain solution A; dissolving 5mg of KLH in 0.01M PBS to obtain solution B; and slowly adding the solution A into the solution B dropwise, and stirring at room temperature for reaction overnight to obtain a conjugate mixed solution. Dialyzing the mixed solution in 0.01M PBS solution for three days to finally obtain the artificial antigen H1-HS-KLH, and identifying by adopting an ultraviolet absorption scanning method.

Immunization of mice: mixing and emulsifying analgin complete antigen H1-HS-KLH and equivalent Freund's adjuvant, and performing neck-back subcutaneous multipoint injection immunization (except for sprint immunization) on BALB/c mice, wherein the dose of the complete Freund's adjuvant for the first immunization 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. Detecting the affinity and sensitivity of the mouse serum by ic-ELISA;

cell fusion and cell line establishment: fusing mouse spleen cells and mouse myeloma cells by a polyethylene glycol (PEG 1500) 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 CBD of the analgin high-secretion specific antibody;

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

The invention also provides a detection reagent or a detection kit containing the monoclonal antibody.

The invention has the beneficial effects that: the monoclonal antibody secreted by the monoclonal antibody hybridoma cell strain CBD provided by the invention has good affinity and detection sensitivity (MAA: IC) for four main metabolites of analgin₅₀= 8.1 ng/mL，AA：IC₅₀= 8.8 ng/mL，FA：IC₅₀= 0.9 ng/mL，AAA：IC₅₀=0.6 ng/mL), can realize the detection of residual quantity of analgin metabolite in meat products, dairy products and the like, provides raw materials for immunoassay of residual analgin metabolite in food, and has practical application value.

Biological material sample preservation: an analgin monoclonal antibody hybridoma cell strain CBD is deposited in China general microbiological culture collection center (CGMCC), China academy of sciences (China institute of microbiology, institute of sciences, No. 3, Xilu 1, North Cheng, south China, Beijing, and the Yangyang area, and is classified and named as a monoclonal cell strain, the preservation date is 2019, 10 and 14 days, and the preservation number is CGMCC No. 18511.

Drawings

FIG. 1 shows the mechanism of analgin hydrolysis in vivo.

FIG. 2-a is a standard detection curve for CBD monoclonal antibodies against AA and MAA.

FIG. 2-b is a standard detection curve for CBD monoclonal antibodies against FA and AAA.

Detailed Description

Solution preparation:

carbonate Buffer (CBS): weighing Na₂CO₃1.59 g，NaHCO₃2.93 g, respectively dissolving in a small amount of double distilled water, mixing, adding the double distilled water to about 800 mL, uniformly mixing, adjusting the pH value to 9.6, adding the double distilled water to a constant volume of 1000 mL, and storing at 4 ℃ for later use.

Phosphate Buffered Saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH₂PO₄，2.9g Na₂HPO₄·12 H₂Dissolving O in 800 mL 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 ^.12H₂18.43 g of O, 9.33 g of citric acid and pure water to reach the constant volume of 1000 mL; and B, liquid B: 60mg of TMB was dissolved in 100 mL 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.

Example 1 Synthesis of Analgin complete antigen

(1) Derivation of hapten: 4-aminoantipyrine and succinic anhydride are used for derivatization to obtain hapten H1-HS containing active groups (-COOH). Weighing H150 mg, 4-dimethylaminopyridine 175 mg and succinic anhydride 300 mg, dissolving in 20 mL of anhydrous pyridine, and refluxing at 60 ℃ for 12H. The reaction solution was concentrated by evaporation, the residue was dissolved in 10 mL of water and extracted three times with 5 mL of ethyl acetate, and the organic phase was collected. Drying the organic phase by nitrogen to obtain a white solid, namely H1-HS, and identifying the structure by nuclear magnetism and mass spectrometry;

(2) preparation of complete antigen:

coating source: weighing H12.03 mg, dissolving in 500 μ L of ultrapure water, adding 60 μ L of 2.5% glutaraldehyde solution, and stirring in ice bath for 15min to obtain reaction solution A. 5mg of BSA was weighed and dissolved in 1mL of 0.01M phosphate buffer solution (PBS, pH = 7.4) to obtain solution B. And dropwise adding the solution A into the solution B while stirring, and stirring at room temperature for reaction for 2 hours to obtain a coupling mixed solution. Taking a dialysis bag (protein retention: 3kDa) of 10cm, boiling for 5min, cooling to room temperature, adding the coupling mixed solution into the dialysis bag, dialyzing for 3 days by using PBS (pH = 7.4) and 0.01M, replacing dialyzate every 6H, removing redundant small molecular compounds to obtain an artificial antigen H1-BSA, and identifying by adopting an ultraviolet absorption scanning method;

immunogen: weighing 1.1 mg of hapten H1-HS and 1.3 mg of N-hydroxysuccinimide (NHS) obtained in the step (1), dissolving the hapten in 300 mu L N N-Dimethylformamide (DMF), and stirring at room temperature for reaction for 10 min; weighing 2.2 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), fully dissolving with 50 μ L of 2- (n-morpholine) ethyl sulfonic acid buffer solution (MES) with pH = 4.6 and 0.01M, adding into the H1-HS solution, and stirring at room temperature for 6-8H to obtain solution A; dissolving 5mg of KLH in 0.01M PBS to obtain solution B; and slowly adding the solution A into the solution B dropwise, and stirring at room temperature for reaction overnight to obtain a conjugate mixed solution. Dialyzing the mixed solution in 0.01M PBS solution for three days to finally obtain the artificial antigen H1-HS-KLH, and identifying by adopting an ultraviolet absorption scanning method.

Example 2 preparation of monoclonal antibody hybridoma cell line CBD

(1) Immunization of mice: mixing and emulsifying analgin complete antigen H1-HS-KLH and an equivalent amount of Freund adjuvant, and performing neck-back subcutaneous multipoint injection immunization (except puncture immunization) on BALB/c mice; 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. Detecting the affinity and sensitivity of the mouse serum by ic-ELISA;

(2) cell fusion: after three days of the spurting immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight 1500) 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 Xg, 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. 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% CO₂Culturing in an incubator. The number of SP2/0 tumor cells required to reach (1-4) × 10 before fusion⁷Ensuring 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 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 x g, 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 ℃ under 5% CO₂Culturing in an incubator.

(3) 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: the first step is to screen out positive cell holes by an ic-ELISA method, the second step is to select analgin as a standard substance, and the inhibition effect of the positive cells is measured by the ic-ELISA method. And selecting a cell well with good inhibition on an analgin metabolite MAA standard substance, performing subcloning by using a limiting dilution method, and detecting by using the same method after seven days. And carrying out subcloning for three times according to the method to finally obtain the analgin monoclonal antibody cell strain CBD.

(4) 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 10⁶Analgin hybridoma, ascites was collected from the seventh day, and antibody purification was performed on the ascites 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; then, the IgG type monoclonal antibody was precipitated with an ammonium sulfate solution of the same saturation, centrifuged, the supernatant was discarded, and the supernatant was dissolved in a 0.01M PBS solution (pH 7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.

Example 3 Analgin monoclonal antibody sensitivity assay

(1) Coating: the original coatingen H1-BSA was diluted 3-fold with 0.05M carbonate buffer pH9.6 from 1. mu.g/mL, 100. mu.L/well, reacted at 37 ℃ for 2H.

(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 the monoclonal antibody by a certain multiple, adding the diluted monoclonal antibody into a plate coated with H1-BSA, incubating at 100 mu L/well for 30min at 37 ℃; after washing sufficiently, a certain dilution of horse radish peroxidase-labeled goat anti-mouse IgG was added at 100. mu.L/well, and incubated 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 15 min.

(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.

The standard inhibition curves of CBD against the four analgin metabolites were obtained using OriginPro 8.5 for mapping.

The cross-over of the monoclonal antibodies prepared to the analgin metabolite is shown in table 1.

TABLE 1 Cross-over of monoclonal antibodies to Analgin metabolites

Detection of MAA, y = 0.17. + -. 0.007 + (1.68. + -. 0.016-0.17. + -. 0.007)/[1 + (x/8.08. + -. 0.116)^1.25±0.029]，R²= 0.999，IC₅₀The concentration is 8.1 ng/mL, and the detection limit (the addition amount corresponding to 20-80% of inhibition rate) is 2.66-24.51 ng/mL.

Detection of AA, y = 0.17. + -. 0.017 + (1.77. + -. 0.017-0.17. + -. 0.017)/[1 + (x/8.67. + -. 0.334)^1.14±0.049]，R²= 0.999，IC₅₀The concentration is 8.8 ng/mL, and the detection limit is 2.58-29.13 ng/mL.

The standard detection curve for the monoclonal antibodies against AA and MAA is shown in FIG. 2-a.

Detection of FA, y = 0.266. + -. 0.021 + (1.77. + -. 0.007-0.266. + -. 0.021)/[1 + (x/0.98. + -. 0.0279)^1.19±0.041]，R²= 0.989，IC₅₀0.9 ng/mL and the detection limit is 0.30-3.12.

Detection of AAA, y = 0.21. + -. 0.039 + (1.91. + -. 0.021-0.21. + -. 0.039)/[1 + (x/0.82. + -. 0.0360)^1.35±0.097]，R²= 0.999，IC₅₀The concentration is 0.6 ng/mL, and the detection limit is 0.29-2.3 ng/mL.

The standard detection curve for monoclonal antibodies against FA and AAA is shown in FIG. 2-b.

The monoclonal antibody secreted by the monoclonal cell strain CBD has good sensitivity to the analgin metabolite and can be used for immunoassay detection of the analgin metabolite.

Example 4 milk was listed and the residual amount of the metabolite of analgin was determined

The negative milk samples were added with analgin four metabolites and with a mixture of the four metabolites, mixed well and diluted 10-fold with PBS, followed by simultaneous labeling and actual detection according to the procedure of example 3, with the results shown in table 2. The recovery rate is 80.4-108.8%, the interference of the matrix is less, and the result is more ideal.

TABLE 2 milk addition test

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. An analgin monoclonal antibody hybridoma cell strain CBD is deposited in China general microbiological culture collection center (CGMCC), China academy of sciences (China institute of microbiology, institute of sciences, No. 3, Xilu 1, North Cheng, south China, Beijing, and the Yangyang area, and is classified and named as a monoclonal cell strain, the preservation date is 2019, 10 and 14 days, and the preservation number is CGMCC No. 18511.

2. An analgin monoclonal antibody, characterized in that: the antibody is secreted and produced by the analgin monoclonal antibody hybridoma cell strain CBD with the preservation number of CGMCC No.18511 as claimed in claim 1.

3. The use of the monoclonal antibody to analgin of claim 2, wherein: the method is used for detecting the residues of analgin and metabolites thereof in food.

4. The use of the monoclonal antibody to analgin according to claim 3, wherein: the analgin monoclonal antibody is applied to a reagent for detecting analgin and metabolites thereof by an ELISA competition method.

5. A detection reagent or a detection kit comprising the analgin monoclonal antibody of claim 2.