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

Abstract

The invention relates to a hybridoma cell strain secreting TBHQ monoclonal antibody, which is named as monoclonal cell strain and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 12 months and 16 days in 2021, wherein the preservation address is the North Chen Silu No. 1,3 in the Chaoyang area of Beijing city, and the preservation number is CGMCC No.45016. The hybridoma cell strain can efficiently and stably secrete TBHQ monoclonal antibody, has better sensitivity and specificity when being applied to the immunoassay detection of TBHQ, and has IC ₅₀ The value is 1.37ng/mL, the crossing rate of TBHQ analogues is less than 10%, and the method can be used for establishing an immune detection method of the TBHQ content in oil products and other foods containing TBHQ, and has practical application value.

Description

Hybridoma cell strain secreting TBHQ monoclonal antibody and application thereof

Technical Field

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

Background

TBHQ is a novel synthetic antioxidant with good antioxidant effect, is especially suitable for the antioxidant of vegetable oil, and can improve the antioxidant stability of edible oil products by 3-5 times. However, it has been found that long-term consumption of foods containing an excessive amount of synthetic antioxidants adversely affects the human body, and thus, almost all countries have strict restrictions on the addition of synthetic antioxidants to processed foods, such as: the addition amount of the synthetic antioxidant is not more than 0.1g/kg or 0.2g/kg specified in GB 2760-2014 in China. Therefore, there is a need to establish a rapid, efficient method for detecting TBHQ in food products.

TBHQ content analysis methods include a gas chromatography-mass spectrometry (GC-MS), a High Performance Liquid Chromatography (HPLC), a liquid chromatography-mass spectrometry (LC-MS) and other instrument methods, such as Zhang Fengmei and the like (TBHQ [ J ] in soybean oil is measured by liquid phase microextraction-high performance liquid chromatography based on eutectic solvents; the university of Henan university of industry (Nature science edition), 2017,38 (5): 32-36.DOI: 10.3969/j.issn.1673-2383.2017.05.007.) the method comprises the steps of directly extracting tert-butylhydroquinone (TBHQ) in grease by acetonitrile, and then analyzing and measuring by gas chromatography, wherein the result shows that the minimum detection limit of TBHQ is 5mg/kg, the relative standard deviation is 2.5% -3.2%, the linear range is 1-200 mu g/mL, and the linear correlation coefficient is more than 0.999; zhang Kangdi et al, by measuring TBHQ in soybean oil by liquid phase microextraction-high performance liquid chromatography based on a eutectic solvent, found that the mass ratio of choline chloride/ethylene glycol was 1:2, the eutectic solvent has higher extraction rate to TBHQ in soybean oil, and the optimal extraction conditions are as follows: the dosage of the eutectic solvent is 400 mu L, the dosage of the oil is 0.15g, the extraction temperature is 50 ℃, the extraction time is 30min, the dosage of the n-hexane is 2mL, and the detection limit is 0.02 mu g/mL; xiong Weilin (solid phase extraction-high performance liquid chromatography for measuring TBHQ content [ J ]. Chinese grease in vegetable oil, 2017,42 (10): 143-145.DOI: 10.3969/j.issn.1003-7969.2017.10.031.) to establish neutral alumina solid phase extraction-high performance liquid chromatography for TBHQ in vegetable oil, and performing standard recovery rate and precision test with TBHQ standard solution 10, 25 μg/mL and 50 μg/mL respectively, wherein the standard recovery rate is 100.0% -103.0%, and RSD is 2.0% -6.0%; patent CN202011529362.1 discloses a method for detecting TBHQ by surface enhanced Raman spectrum, patent CN201811039067.0 discloses a method for detecting TBHQ in biodiesel, patent CN200910064791.3 discloses a method for rapidly detecting TBHQ in edible oil, but the above detection methods have the defects of time consumption, complicated steps, incapability of rapid on-site detection, high cost and the like, and the detection limit is only milligram or microgram level, so that the establishment of a rapid and simple TBHQ detection method has important significance. The enzyme-linked immunosorbent assay (ELISA) is a very efficient, sensitive and rapid detection method, is suitable for on-site rapid detection of a large number of samples, and provides a novel detection path for TBHQ detection.

Disclosure of Invention

In order to solve the technical problems, the invention provides a hybridoma cell strain secreting TBHQ monoclonal antibody, and detection of TBHQ is performed by using the strain and monoclonal antibody generated by the strain.

The first object of the present invention is to provide a hybridoma cell strain secreting TBHQ monoclonal antibody, named monoclonal cell strain, which is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 12 months and 16 days in 2021, wherein the preservation address is No. 3 of North Chen West Lu No. 1 in the Korean region of Beijing city, and the preservation number is CGMCC No.45016.

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

s1: preparing Cheng Fushi adjuvant from TBHQ immunogen, and injecting Freund's adjuvant into immunized animal; complete Freund's adjuvant is adopted for primary immunization, incomplete Freund's adjuvant is adopted for boosting immunization, and TBHQ immunogen is adopted for sprint immunization;

the TBHQ immunogen is prepared from a TBHQ hapten, and the structure of the TBHQ hapten is shown as a formula I;

s2: the immune animals subjected to the immunization process are subjected to blood sampling, the serum immune titer and the immune inhibition capacity of the immune animals are detected, and immune animals with high TBHQ antibody content in serum are screened out;

s3: fusing and culturing spleen cells and myeloma cells of the immune animals screened in the step S2, detecting positive cell holes, determining the inhibition effect of the positive cell holes, and subcloning the positive cell holes with the best inhibition effect to obtain hybridoma cell strains secreting TBHQ monoclonal antibodies;

further, the TBHQ immunogen is obtained by coupling bovine serum albumin after activation of TBHQ hapten. In particular, the method comprises the steps of,

dissolving TBHQ hapten in DMF, adding EDC and NHS, and stirring for 5-8h at 20-30 ℃ to obtain a reaction solution A; dissolving BSA in borate buffer to obtain solution B; adding the reaction solution A into the solution B, reacting for 5-10h to obtain a conjugate TBHQ-COOH-BSA mixed solution, and separating complete antigen and unconjugated small molecule hapten by dialysis to obtain an immunogen TBHQ-COOH-BSA.

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

dissolving 3-tert-butyl-4-hydroxybenzoic acid, glycine tert-butyl ester, carbodiimide and N-hydroxysulfosuccinimide in DMF, and mixing the solution with CH ₂ Cl ₂ Dilution, washing with brine, na ₂ SO ₄ And (5) drying. The organic layer was removed, and the resulting residue was purified by silica gel column (CH ₂ Cl ₂ Methanol=20:1) chromatography, the resulting compound was dissolved in CH ₂ Cl ₂ And trifluoroacetic acid to obtain TBHQ hapten TBHQ-COOH.

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

Further, the interval between the first immunization and the first boost is 28-30 days, the interval between the boosts is 20-22 days, and the interval between the last boost and the sprint immunization is 18-21 days.

Further, in step S1, freund' S adjuvant is injected subcutaneously into the immunized animal via the back.

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

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

Further, paraffin oil is injected into the abdominal cavity of the immunized animal, hybridoma cell strain with the preservation number of CGMCC No.45016 is injected into the abdominal cavity, ascites is collected after injection, and the ascites is purified, so that the TBHQ monoclonal antibody is obtained.

The third object of the invention is to provide the application of the hybridoma cell strain or the TBHQ monoclonal antibody in detecting TBHQ, and the application of the hybridoma cell strain or the TBHQ monoclonal antibody in the analysis and detection of TBHQ residues in the safety detection of foods (especially oil products).

A TBHQ detection kit comprising the hybridoma cell line and/or a TBHQ monoclonal antibody.

Further, the TBHQ detection kit also comprises a TBHQ coating antigen, wherein the TBHQ coating antigen is obtained by coupling chicken ovalbumin after TBHQ hapten activation.

By means of the scheme, the invention has at least the following advantages:

the TBHQ monoclonal antibody hybridoma cell strain provided by the invention can efficiently and stably secrete TBHQ monoclonal antibody, has better specificity (the crossing rate of TBHQ analogue is less than 10%) and detection sensitivity (IC) ₅₀ A value of 1.37 ng/mL) provides an immunological method for detecting TBHQ levels in oil preparations and other TBHQ-containing foods. The TBHQ monoclonal antibody hybridoma cell strain and the monoclonal antibody secreted by the same canThe kit for detecting TBHQ is prepared, and has practical application value.

The foregoing description is only an overview of the present invention, and is presented in terms of preferred embodiments of the present invention and the following detailed description of the invention in conjunction with the accompanying drawings.

Preservation of biological materials

The monoclonal cell strain is preserved in China general microbiological culture Collection center (CGMCC) No.45016 in the year 2021, 12 and 16, and has a preservation address of North Chen Xiyu No. 1 and 3 in the Korean region of Beijing city.

Drawings

In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings.

FIG. 1 shows the standard curve of TBHQ inhibition by the TBHQ monoclonal antibodies of the present invention.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

The following examples relate to the following media:

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, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B12.005, sodium bicarbonate 2000.

The reagents involved in the following examples were as follows:

carbonate Buffer (CBS): weighing Na ₂ CO ₃ 1.59g，NaHCO ₃ 2.93g, respectively dissolving in a small amount of double distilled water, mixing, adding double distilled water to about 800mL, mixing, adjusting pH to 9.6, adding double distilled water to 1000mL, and storing at 4deg.C for use;

phosphate Buffer (PBS): 8.00g NaCl,0.2g KCl,0.2g KH ₂ PO ₄ ，2.9g Na ₂ HPO ₄ ·12H ₂ O is dissolved in 800mL of pure water, pH is regulated to 7.2-7.4 by NaOH or HCl, and volume is regulated to 1000mL;

PBST: PBS containing 0.05% Tween 20;

antibody dilution: a wash buffer containing 0.1% gelatin;

TMB color development liquid: and (3) solution A: na (Na) ₂ HPO ₄ ^. 12H ₂ 18.43g of O, 9.33g of citric acid and pure water to 1000mL; and (2) liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. The volume ratio of the solution B is 1:5, mixing to obtain TMB color development liquid, and mixing immediately.

The detection method involved in the following examples is as follows:

the detection method of the TBHQ inhibition rate comprises the following steps: the most appropriate antigen and antibody concentrations in the ic-ELISA were selected by a checkerboard assay. The antigen was diluted to 0.03,0.1,0.3 and 1 μg/mL with Carbonate Buffer (CBS) and the antibody was diluted to 0.03,0.1,0.3 and 1 μg/mL with antibody dilution. After selecting the optimal working point, diluting the TBHQ standard substance to concentrations of 0,0.04,0.12,0.37,1.11,3.33, 10 and 30ng/mL, and performing the steps of IC-ELISA, finally mapping with OriginPro 8.5 (the result is shown in FIG. 1), obtaining a TBHQ standard inhibition curve, and calculating IC ₅₀ 。

EXAMPLE 1 Synthesis of TBHQ hapten

160mg of compound 1 3-tert-butyl-4-hydroxybenzoic acid, 200mg of compound 2-glycine tert-butyl ester, 65mg of carbodiimide and 36mg of N-hydroxysulfosuccinimide were dissolved in 8mL of N, N-dimethylformamide. The mixture was treated with 10mL of CH ₂ Cl ₂ Dilute, wash with 5mL brine, na ₂ SO ₄ And (5) drying. The organic layer was then removed from the solvent, and the residue was purified by silica gel column (CH ₂ Cl ₂ Methanol=20:1) to give compound 3. Compound 3 was dissolved in 3mL CH ₂ Cl ₂ And 1mL of trifluoroacetic acid, 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

5.50mg of hapten is weighed and dissolved in 800 mu L of DMF, 9.08mg EDC,5.12mg NHS is added, and the mixture is stirred at room temperature for 6 hours to obtain a reaction solution A; 10mg of BSA was weighed and dissolved in 0.1M borate buffer to give solution B; then, dropwise adding the reaction solution A into the solution B, reacting for 8 hours at room temperature to obtain a conjugate TBHQ-COOH-BSA mixed solution, and separating the complete antigen and the unconjugated small molecule hapten by dialysis to obtain the immunogen TBHQ-COOH-BSA.

EXAMPLE 3 Synthesis of TBHQ coating antigen

1.92mg of TBHQ hapten, 4.54mg of 1-ethylcarbodiimide hydrochloride and 2.35mg of N-hydroxysuccinimide are weighed, 600 mu L of anhydrous N, N-dimethylformamide is used for dissolution, and A1 solution is obtained and stirred at room temperature for reaction for 6 hours. Weighing 10mg of chicken ovalbumin OVA, dissolving in 2mL of 0.1M borate buffer solution to obtain a B1 solution, dropwise adding the A1 solution into the B1 solution at room temperature, and reacting for 8 hours at room temperature to obtain a conjugate TBHQ-COOH-OVA mixed solution, and separating a coating antigen and unconjugated small molecule hapten through dialysis. The coating antigen is used for detecting mouse serum titer and inhibition in the preparation process of the monoclonal antibody, is not directly used for mice, and is necessary for preparing the monoclonal antibody.

EXAMPLE 4 preparation of hybridoma cell lines secreting TBHQ monoclonal antibodies

1. Animal immunization: healthy BALB/c mice of 6-8 weeks of age were selected for immunization. BALB/c mice were immunized by subcutaneous injection on the back after mixing TBHQ complete antigen with equivalent Freund's adjuvant for emulsification. The first immunization was with complete Freund's adjuvant, followed by incomplete Freund's adjuvant. The interval between the first immunization and the second boost was 28 days, and the interval between the multiple boosts was 21 days. Blood was collected 7 days after the third immunization (mice were bled 5ul+995ul antibody diluent = antisera), mice serum titers and inhibition were determined using ic-ELISA, mice with high titers were selected for sprint immunization 21 days after the fifth immunization, i.e. intraperitoneal injection, requiring halving of the dosage of the wash-out and no adjuvant.

2. Cell fusion: three days after sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, as follows:

a. taking eyeball and blood, killing a mouse by a cervical dislocation method, immediately putting the mouse into 75% alcohol for disinfection, soaking for about 5min, taking out spleen of the mouse by aseptic operation, moderately grinding the spleen by a rubber head of a syringe, obtaining spleen cell suspension by a 200-mesh cell screen, collecting, centrifuging (1200 rpm,8 min), washing the spleen cells for three times by using an RPMI-1640 culture medium, and diluting the spleen cells to a certain volume and counting for later use after the last centrifugation;

b. collecting SP2/0 cells: SP2/0 tumor cells were cultured in 10% FBS (fetal bovine serum) RPMI-1640 medium at 5% CO 7-10 days prior to fusion ₂ In an incubator. The number of SP2/0 tumor cells before fusion reaches 1 to 4 multiplied by 10 ⁷ Ensuring SP2/0 tumor cells to be in logarithmic growth phase before fusion. During fusion, collecting tumor cells, suspending in RPMI-1640 basic culture solution, and performing cell count;

c. the fusion process was 7min. 1min, 1mL of PEG 1500 was added dropwise to the cells from slow to fast; and (2) standing for 2 min. Dripping 1mL of RPMI-1640 culture medium in the period of 1min for 3min and 4 min; dripping 2mL of RPMI-1640 culture medium in the period of 1min at the 5 th and 6 th min; at 7min, 1mL of RPMI-1640 medium was added dropwise every 10 s. Then, the mixture was incubated at 37℃for 5min. Centrifuging (800 rpm,8 min), discarding supernatant, re-suspending in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50 XHAT, adding 200 μl/well to 96-well cell plate, and standing at 37deg.C and 5% CO ₂ IncubatorIs cultured.

3. Cell screening and cell strain establishment: the cells were subjected to half-replacement of the RPMI-1640 selection medium on day 3 of cell fusion, full replacement with a 100 XHT RPMI-1640 transition medium containing 20% fetal bovine serum and 1% on day 5, and cell supernatants were collected on day 7 for selection. Screening is carried out in two steps: the first step is to screen out positive cell holes by using ic-ELISA, and the second step is to select TBHQ as a standard substance, and to measure the inhibition effect of positive cells by using ic-ELISA. Cell holes with better inhibition on TBHQ standard are selected, subcloning is carried out by adopting a limiting dilution method, and detection is carried out by adopting the same method. The cell line of TBHQ monoclonal antibody is obtained by repeating the steps three times.

EXAMPLE 5 preparation and identification of TBHQ monoclonal antibodies

Taking 8-10 week old BALB/c mice, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; after 7 days, 1X 106 hybridoma cells were intraperitoneally injected into each mouse, and ascites was collected from the seventh day and purified by the octanoic acid-ammonium sulfate method. Under the condition of meta-acid, the n-octanoic acid can precipitate other hetero proteins except IgG immunoglobulin in ascites, and then the mixture is centrifuged and the precipitate is discarded; precipitating the monoclonal antibody of the IgG type by using an ammonium sulfate solution with equivalent saturation, centrifuging, discarding the supernatant, dissolving by using a 0.01M PBS solution (pH 7.4), dialyzing for desalting, and finally obtaining the purified monoclonal antibody, and storing at-20 ℃.

Determination of IC of TBHQ monoclonal antibody using indirect competition ELISA ₅₀ The value is 1.37ng/mL, the crossing rate of the TBHQ analogue is less than 10%, and the detection method has good sensitivity and specificity to TBHQ and can be used for TBHQ immunoassay detection. Wherein the crossing rate= (IC of TBHQ ₅₀ IC of analog ₅₀ ) X 100%) IC of TBHQ analog ₅₀ The values are given in the following table.

TABLE 1 IC of TBHQ analog ₅₀ Value of

EXAMPLE 6 use of TBHQ monoclonal antibodies

The monoclonal antibody prepared in example 5 was applied to an ELISA additive recovery assay for TBHQ, and the specific procedure was as follows:

(1) Coating: the coating stock TBHQ-OVA was diluted in 0.05M carbonate buffer pH9.6 at a 1. Mu.g/mL ratio of 100. Mu.L/well and reacted at 37℃for 2 hours.

(2) Washing: the plate solution was poured off and washed 3 times with wash solution for 3min each.

(3) Closing: after drying, 200. Mu.L/well of blocking solution was added thereto and reacted at 37℃for 2 hours. And (5) drying for standby after washing.

(4) Sample adding: the antiserum (after tail breaking and blood sampling of the mice, the antiserum is diluted by corresponding times by antibody diluent) is diluted by a ratio of 1:1000, and is added into the coating holes of each dilution, 100 mu L/hole and reacted for 30min at 37 ℃; after extensive washing, HRP-goat anti-mouse IgG diluted 1:3000 was added, 100. Mu.L/well, and reacted at 37℃for 30 minutes.

(5) Color development: and taking out the ELISA plate, fully washing, adding 100 mu L of TMB color developing solution into each hole, and carrying out light-shielding reaction for 15min at 37 ℃.

(6) Termination and measurement: 50. Mu.L of stop solution was added to each well to terminate the reaction, and the OD450 value of each well was measured by using a microplate reader.

The standard curve of TBHQ inhibition by TBHQ monoclonal antibody is shown in FIG. 1, and it can be seen that IC of TBHQ monoclonal antibody is determined by IC-ELISA ₅₀ The value is 1.37ng/mL, which shows that the antibody has better sensitivity to TBHQ and can be used for the immunoassay detection of TBHQ.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope 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.

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