CN114292335B - Hybridoma cell strain secreting TBHQ monoclonal antibody and application thereof - Google Patents

Abstract

The present invention relates to a hybridoma cell strain secreting TBHQ monoclonal antibody, named as the monoclonal cell strain, which was preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on December 16, 2021, and the preservation address is Chaoyang District, Beijing No. 3, No. 1 Yard, Beichen West Road, the preservation number is CGMCC No. 45016. The hybridoma cell line of the present invention can efficiently and stably secrete TBHQ monoclonal antibody, has good sensitivity and specificity when applied to the immunoassay detection of TBHQ, and the _IC50 value is 1.37ng/mL, and the crossover rate to TBHQ analogues Less than 10%, it can be used to establish an immunological detection method for TBHQ content in oil products and other foods containing TBHQ, and has practical application value.

Description

A hybridoma cell line secreting TBHQ monoclonal antibody and its application

technical field

The invention relates to the technical field of immunoassay, in particular to a hybridoma cell line secreting TBHQ monoclonal antibody and application thereof.

Background technique

TBHQ is a newly synthesized antioxidant with good anti-oxidation effect, especially suitable for anti-oxidation of vegetable oil, which can increase the anti-oxidation stability of edible oil products by 3-5 times. However, studies have found that long-term consumption of foods containing excessive synthetic antioxidants will have adverse effects on the human body, so almost all countries have strict restrictions on the addition of synthetic antioxidants in processed foods, such as: my country's GB 2760-2014 stipulates synthetic antioxidants The amount added should not exceed 0.1g/kg or 0.2g/kg. Therefore, it is necessary to establish a rapid and efficient detection method for TBHQ in food.

TBHQ content analysis methods include instrumental methods such as gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), such as Zhang Fengmei et al. - Determination of TBHQ in Soybean Oil by High Performance Liquid Chromatography[J]. Journal of Henan University of Technology (Natural Science Edition), 2017, 38(5): 32-36. DOI: 10.3969/j.issn.1673-2383.2017.05.007 .) Utilize acetonitrile to directly extract tertiary butyl hydroquinone (TBHQ) in the oil, then utilize gas chromatography to analyze and measure, the result shows that the minimum detection limit of TBHQ is 5mg/kg, and the relative standard deviation is 2.5%～3.2 %, the linear range is 1-200μg/mL, and the linear correlation coefficients are all greater than 0.999; Zhang Kangdi et al. determined TBHQ in soybean oil by liquid phase microextraction-high performance liquid chromatography based on deep eutectic solvent, and found that choline chloride/ The deep eutectic solvent composed of ethylene glycol has a high extraction rate for TBHQ in soybean oil when the molar ratio of substances is 1:2. The extraction temperature is 50°C, the extraction time is 30min, the amount of n-hexane is 2mL, and the detection limit is 0.02μg/mL; Xiong Weilin et al. (10): 143-145.DOI: 10.3969/j.issn.1003-7969.2017.10.031.) established a neutral alumina solid-phase extraction-high performance liquid chromatography analysis method for TBHQ in vegetable oil, using TBHQ standard solution 10 , 25μg/mL and 50μg/mL for the standard recovery and precision tests, the results show that the standard recovery is between 100.0% and 103.0%, and the RSD is 2.0% to 6.0%; patent CN202011529362.1 discloses a surface The method for detecting TBHQ by enhanced Raman spectroscopy, patent CN201811039067.0 discloses a method for the determination of TBHQ in biodiesel, and patent CN200910064791.3 discloses a rapid detection method for TBHQ in edible oils and fats, but the above detection methods are time-consuming , cumbersome steps, inability to perform on-site rapid detection, high cost, and the detection limit is only at the milligram or microgram level. Therefore, it is of great significance to establish a fast and simple TBHQ detection method. Enzyme-linked immunoassay (ELISA) is an extremely efficient, sensitive and rapid detection method, suitable for on-site rapid detection of a large number of samples, and provides a new detection method for TBHQ detection.

Contents of the invention

In order to solve the above technical problems, the present invention provides a hybridoma cell strain secreting TBHQ monoclonal antibody, and the detection of TBHQ is carried out by using the strain and the monoclonal antibody produced by it.

The first purpose of the present invention is to provide a hybridoma cell line secreting TBHQ monoclonal antibody, named as monoclonal cell line, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Management Committee on December 16, 2021. The address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 45016.

Further, the preparation method of the above-mentioned hybridoma cell strain comprises the following steps:

S1: Prepare TBHQ immunogen into Freund's adjuvant, and then inject Freund's adjuvant into immunized animals; use complete Freund's adjuvant for the first immunization, use incomplete Freund's adjuvant for booster immunization, and use TBHQ immunogen for sprint immunization ;

Wherein, the TBHQ immunogen is prepared from the TBHQ hapten, and the structure of the TBHQ hapten is shown in formula I;

S2: Collect blood from the immunized animals that have undergone the above immunization process, detect the serum immune titer and immunosuppressive ability of the immunized animals, and screen out the immunized animals with high TBHQ antibody content in the serum;

S3: The splenocytes and myeloma cells of the immunized animal screened in step S2 are fused and cultured, the positive cell wells are detected and the inhibitory effect of the positive cell wells is determined, and the positive cell wells with the best inhibitory effect are subcloned to obtain A hybridoma cell line secreting TBHQ monoclonal antibody;

Further, the TBHQ immunogen is obtained by coupling the TBHQ hapten with bovine serum albumin after activation. specifically,

Dissolve TBHQ hapten in DMF, add EDC and NHS, stir at 20-30°C for 5-8h to obtain reaction solution A; dissolve BSA in borate buffer solution to obtain solution B; add reaction solution A to Put into solution B and react for 5-10 hours to obtain the conjugate TBHQ-COOH-BSA mixture, and separate the complete antigen and unconjugated small molecule hapten by dialysis to obtain the immunogen TBHQ-COOH-BSA.

Further, the preparation method of TBHQ hapten comprises the following steps:

Dissolve 3-tert-butyl-4 _- hydroxybenzoic acid, tert-butyl glycine, carbodiimide and N-hydroxysulfosuccinimide in DMF, dilute the mixture with _CH2Cl2 , wash with brine, Na _2SO4 dry _. The organic layer was removed, and the obtained residue was purified by silica gel column chromatography (CH ₂ Cl ₂ :methanol=20:1). The obtained compound was dissolved in CH ₂ Cl ₂ and trifluoroacetic acid to obtain TBHQ hapten TBHQ-COOH.

Further, using N,N-dimethylformamide (DMF), 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) Activated TBHQ hapten.

Further, the interval between the first immunization and the first booster immunization is 28-30 days, the interval between the booster immunizations is 20-22 days, and the interval between the last booster immunization and the rush immunization is 18-21 days.

Further, in step S1, Freund's adjuvant is subcutaneously injected into the immunized animal through the back.

Further, in step S3, the fused cells are cultured on HAT medium.

The second object of the present invention is to provide a TBHQ monoclonal antibody secreted by the hybridoma cell line with deposit number CGMCC No.45016.

Further, paraffin oil was injected intraperitoneally into the immunized animal, and then the hybridoma cell line with the preservation number CGMCC No. 45016 was injected intraperitoneally, ascites was collected after injection, and the ascites was purified to obtain the above-mentioned TBHQ monoclonal antibody.

The third object of the present invention is to provide the application of the hybridoma cell line or TBHQ monoclonal antibody in the detection of TBHQ, which is applied to the analysis and detection of TBHQ residues in the safety detection of food (especially oil products).

A TBHQ detection kit comprising the above-mentioned hybridoma cell line and/or TBHQ monoclonal antibody.

Further, the TBHQ detection kit also includes a TBHQ coating agent, which is obtained by coupling the TBHQ hapten to chicken ovalbumin after activating the TBHQ hapten.

By means of the above solution, the present invention has at least the following advantages:

The TBHQ monoclonal antibody hybridoma cell line provided by the present invention can efficiently and stably secrete TBHQ monoclonal antibody, and has good specificity (less than 10% to TBHQ analogue crossover rate) and detection sensitivity (IC50 value of _TBHQ ) to TBHQ 1.37ng/mL), providing an immunological method for the detection of TBHQ content in oil products and other foods containing TBHQ. The TBHQ monoclonal antibody hybridoma cell line and the monoclonal antibody secreted by the invention can be made into a kit for detecting TBHQ, which has practical application value.

The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the preferred embodiments of the present invention are described below with detailed drawings.

biological material deposit

Monoclonal cell strain, the monoclonal cell strain has been preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on December 16, 2021, the preservation number is CGMCC No.45016, and the preservation address is Beichen West Road, Chaoyang District, Beijing Courtyard No. 1, No. 3.

Description of drawings

In order to make the content of the present invention more clearly understood, the present invention will be further described in detail below according to the specific embodiments of the present invention and in conjunction with the accompanying drawings.

Figure 1 is the standard curve of inhibition of TBHQ by the TBHQ monoclonal antibody of the present invention.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the examples given are not intended to limit the present invention.

The medium involved in the following examples is as follows:

RPMI-1640 medium (mg/L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine dihydrochloride 65.15, L-glutamic acid 20 , Glycine 10, L-Histidine 15, L-Hydroxyproline 20, L-Isoleucine 50, L-Leucine 50, L-Lysine Hydrochloride 40, L-Methionine 15. L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23.19, L-valine 20, p-aminobenzoic acid 1, calcium nitrate 100, anhydrous magnesium sulfate 48.84, anhydrous sodium dihydrogen phosphate 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L- Glutamine 300, biotin 0.2, D-calcium pantothenate 0.25, folic acid 1, i-inositol 35, niacinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1 , vitamin B12 0.005, sodium bicarbonate 2000.

The reagents involved in the following examples are as follows:

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

Phosphate buffer 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 800mL pure water, adjust the pH to 7.2~ with NaOH or HCl 7.4, set the volume to 1000mL;

PBST: PBS containing 0.05% Tween 20;

Antibody diluent: wash buffer containing 0.1% gelatin;

TMB chromogenic solution: A solution: Na ₂ HPO ₄ ^. 12H ₂ O 18.43g, citric acid 9.33g, pure water to 1000mL; B solution: 60mg TMB dissolved in 100mL ethylene glycol. A and B liquids are mixed at a volume ratio of 1:5 to form TMB chromogenic liquid, which is ready-to-use and mixed.

The detection methods involved in the following examples are as follows:

TBHQ inhibition rate detection method: choose the most appropriate antigen and antibody concentration in ic-ELISA by checkerboard test. Antigen was diluted to 0.03, 0.1, 0.3 and 1 μg/mL with carbonate buffered saline (CBS), and antibody was diluted to 0.03, 0.1, 0.3 and 1 μg/mL with antibody diluent. After selecting the best working point, dilute the TBHQ standard to 0, 0.04, 0.12, 0.37, 1.11, 3.33, 10 and 30ng/mL concentrations, follow the ic-ELISA operation steps, and finally use OriginPro 8.5 to make a graph (the result is shown in Figure 1 shown), obtain the standard inhibition curve of TBHQ, and calculate IC ₅₀ .

Synthesis of embodiment 1 TBHQ hapten

Dissolve 160 mg of compound 1 3-tert-butyl-4-hydroxybenzoic acid, 200 mg of compound 2 tert-butyl glycine, 65 mg of carbodiimide and 36 mg of N-hydroxysulfosuccinimide in 8 mL of N,N-dimethyl Formamide is dissolving. The _mixture was diluted with 10 mL _CH2Cl2 , washed with 5 mL brine and _dried over _Na2SO4 . Then the organic layer in the solvent was removed, and the resulting residue was purified by silica gel column chromatography (CH ₂ Cl ₂ :methanol=20:1) to obtain compound 3. Compound 3 was dissolved in 3 mL CH ₂ Cl ₂ and 1 mL trifluoroacetic acid, and the mixture was stirred at room temperature overnight. Finally, the solution was concentrated to give compound 4TBHQ-COOH. The hapten synthesis route is as follows:

Example 2 Synthesis of TBHQ immunogen

Weigh 5.50 mg of hapten and dissolve it in 800 μL DMF, then add 9.08 mg EDC and 5.12 mg NHS, and stir the mixture at room temperature for 6 hours to obtain reaction solution A; weigh 10 mg BSA and dissolve it in 0.1 M borate buffer , to obtain solution B; then, add reaction solution A to solution B drop by drop, and react at room temperature for 8 hours to obtain the conjugate TBHQ-COOH-BSA mixture, which is separated from the complete antigen and unconjugated small The molecular hapten yields the immunogen TBHQ-COOH-BSA.

Example 3 Synthesis of TBHQ Coating Progen

Weigh 1.92mg of TBHQ hapten, 4.54mg of 1-ethylcarbodiimide hydrochloride, and 2.35mg of N-hydroxysuccinimide, dissolve in 600μL of anhydrous N,N-dimethylformamide to obtain A1 solution , Stir the reaction at room temperature for 6h. Weigh 10 mg of chicken ovalbumin OVA, dissolve it in 2 mL of 0.1M borate buffer solution to obtain B1 solution, add A1 solution dropwise to B1 solution at room temperature, and react at room temperature for 8 hours to obtain the conjugate The TBHQ-COOH-OVA mixture was separated by dialysis from the original coating and the unconjugated small molecule hapten. The original coating is used for the detection of mouse serum titer and inhibition during the preparation of monoclonal antibodies, and is not directly used in mice, and is necessary for the preparation of monoclonal antibodies.

The preparation of the hybridoma cell line of embodiment 4 secreting TBHQ monoclonal antibody

1. Animal immunization: healthy BALB/c mice aged 6-8 weeks were selected for immunization. After mixing and emulsifying the complete TBHQ antigen with an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous injection in 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) after the third immunization, and ic-ELISA was used to measure the titer and inhibition of the mouse serum. 21 days after the five immunizations, sprint immunization, intraperitoneal injection, requires half of the sprint dose without any adjuvant.

2. Cell fusion: Three days after the sprint immunization, perform cell fusion according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, the specific steps are as follows:

a. Pick eyeballs to take blood, kill mice by cervical dislocation, put them into 75% alcohol immediately for disinfection, soak for about 5 minutes, take out the spleen of the mice aseptically, use the rubber tip of the syringe to grind moderately, and pass through a 200-mesh cell sieve The splenocyte suspension was obtained, collected, and centrifuged (1200rpm, 8min), and the splenocytes were washed three times with RPMI-1640 medium. After the last centrifugation, the splenocytes were diluted to a certain volume, counted, and set aside;

b. Collection of SP2/0 cells: 7-10 days before fusion, the SP2/0 tumor cells were placed in a 5% CO ₂ incubator with RPMI-1640 medium containing 10% FBS (fetal bovine serum). The number of SP2/0 tumor cells is required to reach 1-4×10 ⁷ before fusion, and it is ensured that the SP2/0 tumor cells are in the logarithmic growth phase before fusion. When fused, tumor cells were collected, suspended in RPMI-1640 basal culture medium, and cell counted;

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

3. Cell screening and cell line establishment: On the 3rd day of cell fusion, perform half-change of RPMI-1640 screening culture medium on the fused cells, and use RPMI containing 20% fetal bovine serum and 1% 100×HT on the 5th day -1640 transition culture medium was completely changed, and the cell supernatant was collected on the 7th day for screening. The screening is divided into two steps: the first step is to use ic-ELISA to screen positive cell wells, and the second step is to select TBHQ as a standard, and use ic-ELISA to measure the inhibitory effect on positive cells. Select cell wells that have good inhibition to TBHQ standard, subclone by limiting dilution method, and detect by the same method. Repeat three times to obtain TBHQ monoclonal antibody cell lines.

Example 5 Preparation and Identification of TBHQ Monoclonal Antibody

Take BALB/c mice aged 8-10 weeks, and inject 1 mL of sterile paraffin oil into each mouse; 7 days later, inject 1×106 hybridoma cells into each mouse, collect ascites from the seventh day, pass the ascites through Caprylic acid - ammonium sulfate purification. Under acidic conditions, n-octanoic acid can precipitate other miscellaneous proteins in ascites except IgG immunoglobulin, then centrifuge, and discard the precipitate; then use an equal amount of saturated ammonium sulfate solution to precipitate IgG-type monoclonal antibodies, centrifuge, and discard The supernatant was dissolved with 0.01M PBS solution (pH 7.4), dialyzed and desalted, and finally the purified monoclonal antibody was stored at -20°C.

Using indirect competition ELISA, the measured _IC50 value of TBHQ monoclonal antibody is 1.37ng/mL, and the crossover rate to TBHQ analogues is less than 10%, indicating that it has good sensitivity and specificity for TBHQ, and can be used for TBHQ immunoassay detection. Wherein, crossover rate=(IC ₅₀ of TBHQ/IC ₅₀ of analogue)×100%), and the IC ₅₀ value of TBHQ analogue is shown in the table below.

Table 1 _IC50 values of TBHQ analogues

Example 6 Application of TBHQ Monoclonal Antibody

The monoclonal antibody prepared in Example 5 was applied to the ELISA addition recovery test of TBHQ, and the specific steps were as follows:

(1) Coating: The coated original TBHQ-OVA was diluted with 0.05M pH9.6 carbonate buffer starting from 1 μg/mL, 100 μL/well, and reacted at 37°C for 2 hours.

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

(3) Blocking: After patting dry, add 200 μL/well blocking solution and react at 37°C for 2 hours. Wash and tumble dry for later use.

(4) Adding samples: Dilute the antiserum (after blood collection by docking the tail of the mouse, dilute the corresponding multiple with antibody diluent) from 1:1000, and add it to the coated wells of each dilution, 100 μL/well, react at 37°C for 30 min; after washing thoroughly, add 1:3000 diluted HRP-goat anti-mouse IgG, 100 μL/well, react at 37°C for 30 min.

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

(6) Termination and measurement: 50 μL of stop 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 standard curve of inhibition of TBHQ monoclonal antibody against TBHQ is shown in Figure 1. It can be seen that the _IC50 value of TBHQ monoclonal antibody measured by ic-ELISA is 1.37ng/mL, indicating that the antibody has good sensitivity to TBHQ and can be used for TBHQ immunoassay detection.

Apparently, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation. For those of ordinary skill in the art, on the basis of the above description, other changes or changes in various forms can also be made. It is not necessary and impossible to exhaustively list all the implementation manners here. However, the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.

Claims (8)

1. A hybridoma cell line secreting a TBHQ monoclonal antibody, characterized in that: the hybridoma cell strain is named as a monoclonal cell strain and is preserved in China general microbiological culture Collection center (China Committee for culture Collection) for 12 months and 16 days in 2021, wherein the preservation address is number 3 of North Chen Xili No. 1 in the Korean region of Beijing city, and the preservation number is CGMCC No.45016.

2. Use of the hybridoma cell line of claim 1 for detecting TBHQ.

3. A TBHQ monoclonal antibody, characterized by: the TBHQ monoclonal antibody is secreted by the hybridoma cell strain of claim 1.

4. Use of the monoclonal antibody of claim 3 for detecting TBHQ.

5. A TBHQ assay kit, characterized in that: the TBHQ detection kit comprises the TBHQ monoclonal antibody of claim 3.

6. The TBHQ assay kit of claim 5, wherein: the TBHQ detection kit also comprises a TBHQ coating antigen.

7. The TBHQ assay kit of claim 6, wherein: the TBHQ coating antigen is obtained by coupling a TBHQ hapten and chicken ovalbumin; the TBHQ hapten has a structure shown in a formula I:

。

8. use of a TBHQ assay kit as claimed in any one of claims 5 to 7 for the detection of TBHQ.

Images