CN112458062B - Ethyl maltol monoclonal antibody hybridoma cell strain and application thereof - Google Patents
Ethyl maltol monoclonal antibody hybridoma cell strain and application thereof Download PDFInfo
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Abstract
An ethyl maltol monoclonal antibody hybridoma cell strain and application thereof, belonging to the field of food safety immunoassay. The invention discloses an ethyl maltol monoclonal antibody hybridoma cell strain ABC01 which is deposited 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, wherein the preservation date is 2019, 11 and 28 days, and the preservation number is CGMCC No. 19178. The invention mixes and emulsifies the complete antigen of the ethyl maltol and an equivalent amount of Freund's adjuvant, and immunizes BALB/c mice through subcutaneous multi-point injection on the neck and the back. And screening cells by an indirect competitive enzyme-linked immunosorbent assay and subcloning for three times to finally obtain the monoclonal antibody hybridoma cell strain. The monoclonal antibody secreted by the cell strain has good specificity and detection sensitivity on ethyl maltol, can realize detection on the residual quantity of the ethyl maltol in foods such as grease, milk and the like, provides a raw material for immunodetection of the residual quantity of the ethyl maltol in the foods, and has practical application value.
Description
Technical Field
The invention relates to an ethyl maltol monoclonal antibody hybridoma cell strain ABC01 and application thereof, and belongs to the field of food safety immunodetection.
Background
Ethyl Maltol (EM) is an essence and flavor that can be artificially synthesized, and is widely used in the fields of sweetening, flavoring, keeping fragrance and covering up peculiar smell in the fields of food, beverage, cigarette, daily cosmetics and the like due to the characteristics of low amount, high efficiency, safety and no toxicity. Although ethyl maltol is an ideal edible spice additive allowed to be used by the national regulation, the national regulation also has a clear regulation on the application range, wherein the plant oil, pasteurized milk, sterilized milk and fermented milk can not be added with essence and spice, and the ethyl maltol is also listed as an edible vegetable oil detection item in 'national food safety supervision and sampling inspection implementation rules' in 2020. Considering that ingestion of large amounts also leads to headache, vomiting and may affect liver and kidney function, the food and agricultural organization and the world health organization food additive agency of the united nations stipulates: the daily intake of ethyl maltol in human body is not more than 2 mg/kg-1The guide dosage in food processing is 100-200 mug/kg-1. In recent years, some illegal merchants illegally add ethyl maltol to food for flavoring and toning under the drive of interests to achieve the purpose of being secondary and goodFor the purpose of falsely and truly, for example, adding ethyl maltol into common oil and fat to act as high-grade oil, adding ethyl maltol into milk to cover up the fishy smell of the milk, and the like, the 'essence milk' and 'essential oil' not only disturb the normal development of the industry, but also seriously damage the health rights and interests of consumers. Therefore, the establishment of a method for quickly and effectively detecting the content of ethyl maltol has important significance and market value.
At present, the ethyl maltol in the food is mainly detected by adopting an instrumental analysis method, which comprises a high performance liquid chromatography, a gas-liquid chromatography, a liquid-mass combined method, a gas-mass combined method, an ultraviolet spectroscopy method, a Raman spectroscopy method and the like. The methods have higher sensitivity and stability, but the instruments and equipment are expensive, the requirement on the professional performance of operators is high, the detection process is more complicated and complicated, and the detection requirement of field analysis of a large number of samples cannot be met. In recent years, an immunoassay technology based on antigen-antibody specific binding is rapidly developed in the field of food safety detection and analysis, has strong anti-matrix interference capability, can simplify the sample pretreatment process to a certain extent, has the characteristics of high throughput, low cost and short time consumption, and does not need professionals. Establishing an efficient and rapid enzyme-linked immunosorbent assay (ELISA) method aiming at EM is necessary for maintaining the development order of the food industry and guaranteeing the rights and interests of people for life health, and an important premise for establishing the method is to screen a high-sensitivity specific monoclonal antibody aiming at ethyl maltol.
Disclosure of Invention
The invention aims to overcome the defects and provide an ethyl maltol monoclonal antibody hybridoma cell strain and application thereof.
The technical scheme of the invention is that an ethyl maltol monoclonal antibody hybridoma cell strain ABC01 is deposited in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms, and the institute of microbiology, China academy of sciences, No. 3, Xilu No.1, Beijing, Tokyo, Kyoho is classified and named as a monoclonal cell strain, the deposition date is 2019, 11 months and 28 days, and the deposition number is CGMCC No. 19178.
The ethyl maltol monoclonal antibody is secreted and produced by the monoclonal cell strain ABC01 with the preservation number of CGMCC No. 19178.
The application of the ethyl maltol monoclonal antibody is used for analyzing and detecting ethyl maltol residue in food safety detection.
The preparation of the ethyl maltol monoclonal antibody hybridoma cell strain ABC01 provided by the invention comprises the following basic steps:
(1) derivation of hapten:
synthesis of Ethyl maltol hapten EM-1: reacting ethyl maltol (CAS: 4940-11-8) with ethyl bromobutyrate, and hydrolyzing with NaOH to obtain ethyl maltol hapten EM-1;
the reaction process is as follows:
the method comprises the following specific steps: 200mg of ethyl maltol was dissolved in 5mL of acetone, 600mg of ethyl 4-bromobutyrate, 800mg of potassium carbonate and 14.2mg of potassium iodide were added, and after refluxing the solution in a boiling water bath for 24 hours, the organic solvent was removed by a vacuum rotary evaporator to obtain a pale yellow precipitate. Dissolving the precipitate with 0.5M 20% methanol-NaOH aqueous solution, stirring in 80 deg.C water bath for 1 hr, hydrolyzing ethyl 4-bromobutyrate attached to ethyl maltol to 4-bromobutyrate, returning the solution to room temperature, adjusting to neutral with 0.5M HCl, extracting with 15mL ethyl acetate for three times, collecting organic phase, and adding anhydrous Na2SO4Drying, and finally removing the organic solvent by using a vacuum rotary evaporator to obtain the ethyl maltol hapten EM-1.
A method for synthesizing ethyl maltol hapten EM-2 comprises reacting kojic acid (CAS: 501-30-4) with succinic anhydride to obtain ethyl maltol hapten EM-2;
the reaction process is as follows:
the method comprises the following steps: 200mg of kojic acid is dissolved in 5mL of pyridine, 200mg of succinic anhydride and 200mg of dimethylaminopyridine are added, the solution is stirred and reacted for 4 hours at the temperature of 80 ℃, and the organic solvent is dried by nitrogen. The precipitated product was dissolved in 5mL of 0.01M HCl solution, and then extracted by adding 5mL of ethyl acetate, and the organic phase was collected. And repeating the extraction operation for three times, mixing the organic phases, and blow-drying the organic phases by using nitrogen to obtain the ethyl maltol hapten EM-2.
(2) The complete antigens EM-1-EDC-KLH and EM-2-IC-BSA were prepared as follows:
synthesis of complete antigen EM-1-EDC-KLH: 1.51mg of ethyl maltol hapten EM-1 is weighed and dissolved in 300 mu L N of N-Dimethylformamide (DMF), then 3.1mg of N-hydroxysuccinimide (NHS) is added, stirring reaction is carried out for 15min, then 5.1mg of 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride (EDC) is added, and stirring reaction is carried out for 4-6h while keeping room temperature to obtain solution A. 10mg of KLH 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, and the reaction was stirred at room temperature overnight; then dialyzing with 0.01M phosphate buffer solution (PBS, pH = 7.4) to remove small molecules not participating in the reaction, obtaining complete antigen EM-1-EDC-KLH, and identifying by ultraviolet absorption scanning method;
synthesis of coatingen EM-2-IC-BSA: 2.17 mg of ethyl maltol hapten EM-2 is weighed and dissolved in 300 mu L of anhydrous N, N-Dimethylformamide (DMF), then 8.1 mu L of tri-N-butylamine is added, after stirring in ice bath for 15min, 5.6 mu L of Isobutyl Chloroformate (IC) is added, and stirring reaction is continuously carried out in ice bath for 1h to obtain solution A. Dissolving 10mg of BSA in 2mL of 0.01M carbonate buffer solution (CB, pH =9.0) (referred to as solution B), slowly adding the solution A into the solution B dropwise, and stirring in an ice bath for reaction for 4-6 h; then dialyzing with 0.01M phosphate buffer solution (PBS, pH = 7.4) solution, removing small molecules which do not participate in the reaction to obtain coating source EM-2-IC-BSA, and identifying by an ultraviolet absorption scanning method;
(3) immunization of mice: after mixing and emulsifying complete EM-1-EDC-KLH (2 mg/mL) antigen with an equal volume of Freund's adjuvant, BALB/c mice were immunized by subcutaneous multipoint injection at the back of the neck (except for the puncture immunization). Complete Freund adjuvant is used for the first immunization, and the dosage is 80 mug/mouse; multiple boosts with incomplete Freund's adjuvant and dose reduction to 50 μ g/mouse; the thorny immunization does not use an adjuvant, and the thorny immunization is directly carried out intraperitoneal injection after the complete antigen is diluted by normal saline, and the dosage is 25 mu g per mouse. The interval of each immunization is 18-21 days, and the seventh day after the sprint immunization is used for tail-breaking blood collection of the mice, and the titer and the inhibition of the serum of the mice are detected by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), so that the immunization effect is evaluated, and one mouse with the best effect is selected for cell fusion;
(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 screening out positive cell holes capable of generating antibodies 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, carrying out subcloning on the positive cell holes with better inhibition by using a limiting dilution method, and detecting, selecting and subcloning again after one week. After the three times of subcloning according to the method, a monoclonal hybridoma cell strain ABC01 capable of stably secreting anti-EM high-specificity antibody is obtained;
(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 ABC01 has better specificity and detection sensitivity (IC) on EM50The value is 0.78 ng/mL), the detection of the ethyl maltol content in foods such as oil, milk and the like can be realized, raw materials are provided for the immunodetection of the ethyl maltol in the foods, and the method has practical application value.
Biological material sample preservation: an ethyl maltol monoclonal antibody hybridoma cell strain ABC01 which is deposited in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms, China academy of sciences microorganism research institute No. 3, Xilu No.1, Beijing, Tokyo, Yangyang, is classified and named as a monoclonal cell strain, the deposition date is 2019, 11 months and 28 days, and the deposition number is CGMCC No. 19178.
Drawings
FIG. 1A standard curve for the inhibition of the ABC01 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 hybridoma cell strain with high secretion specificity antibody for ethyl maltol is finally obtained by immunizing a mouse with the ethyl maltol complete antigen, performing cell fusion, culturing in HAT selective medium, and screening cell supernatant through ic-ELISA.
EXAMPLE 1 preparation of hybridoma cell line ABC01
(1) Synthesis of hapten EM-1: 200mg of ethyl maltol was dissolved in 5mL of acetone, 600mg of ethyl 4-bromobutyrate, 800mg of potassium carbonate and 14.2mg of potassium iodide were added, and after refluxing the solution in a boiling water bath for 24 hours, the organic solvent was removed by a vacuum rotary evaporator to obtain a pale yellow precipitate.
Dissolving the precipitate with 0.5M 20% methanol-NaOH aqueous solution, stirring in 80 deg.C water bath for 1 hr, hydrolyzing ethyl 4-bromobutyrate attached to ethyl maltol to 4-bromobutyrate, returning the solution to room temperature, adjusting to neutral with 0.5M HCl, extracting with 15mL ethyl acetate for three times, collecting organic phase, and adding anhydrous Na2SO4Drying, and finally removing the organic solvent by using a vacuum rotary evaporator to obtain the ethyl maltol hapten EM-1.
(2) Preparation of complete antigen EM-1-EDC-KLH: 1.51mg of ethyl maltol hapten EM-1 is weighed and dissolved in 300 mu L N of N-Dimethylformamide (DMF), then 3.1mg of N-hydroxysuccinimide (NHS) is added, stirring reaction is carried out for 15min, then 5.1mg of 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride (EDC) is added, and stirring reaction is carried out for 4-6h while keeping room temperature to obtain solution A. 10mg of KLH 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, and the reaction was stirred at room temperature overnight; then dialyzing with 0.01M phosphate buffer solution (PBS, pH = 7.4) to remove small molecules not participating in the reaction, obtaining complete antigen EM-1-EDC-KLH, and identifying by ultraviolet absorption scanning method;
(3) immunization of mice: after mixing and emulsifying complete EM-1-EDC-KLH (2 mg/mL) antigen with an equal volume of Freund's adjuvant, BALB/c mice were immunized by subcutaneous multipoint injection at the back of the neck (except for the puncture immunization). Complete Freund adjuvant is used for the first immunization, and the dosage is 80 mug/mouse; multiple boosts with incomplete Freund's adjuvant and dose reduction to 50 μ g/mouse; the thorny immunization does not use an adjuvant, and the thorny immunization is directly carried out intraperitoneal injection after the complete antigen is diluted by normal saline, and the dosage is 25 mu g per mouse. The interval of each immunization is 18-21 days, and the seventh day after the sprint immunization is used for tail-breaking blood collection of the mice, and the titer and the inhibition of the serum of the mice are detected by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), so that the immunization effect is evaluated, and one mouse with the best effect is selected for cell fusion;
(4) cell fusion: after three days of the spurting immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight is 4000) method, and the specific steps are as follows:
a. the method comprises the following steps of taking eyeballs of a mouse, taking blood, killing the mouse by a cervical vertebra dislocation method, immediately putting the mouse into 75% alcohol by volume concentration for soaking and sterilizing for about 5min, taking out the spleen of the mouse by aseptic operation, appropriately 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. collecting SP2/0 tumor 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. Before fusion, the number of SP2/0 tumor cells is required to reach (1-4) x 107Ensuring that SP2/0 tumor cells are in logarithmic growth phase before fusion. During fusion, tumor cells are collected and suspended in RPMI-1640 basic culture solution for cell counting;
c. the fusion process is 7 min: at 1min, 1mL of PEG 4000 was run from slow to fastDropping into the cells; standing for 2 min. Dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4min respectively; dripping 2mL of RPMI-1640 culture medium within 1min at 5min and 6min respectively; 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;
(5) 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: in the first step, positive cell holes are screened by an ic-ELISA method, in the second step, ethyl maltol is selected as a standard substance, and the inhibition effect of positive cells is measured by the ic-ELISA method. And selecting a cell hole with better inhibition on the ethyl maltol standard, performing subcloning by using a limiting dilution method, and detecting by using the same method after seven days. And subcloning for three times according to the method to finally obtain the cell strain ABC01 capable of stably secreting the anti-ethyl maltol monoclonal antibody.
(6) Preparation and identification of monoclonal antibody: taking BALB/c mice 8-10 weeks old, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 106 Ethyl maltol 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 ℃.
6.1 coating: diluting the coating source EM-2-IC-BSA by 3 times from 1 microgram/mL by using 0.05M carbonate buffer solution with pH9.6, reacting at the temperature of 37 ℃ for 2 hours by using 100 mu L/hole;
6.2 washing: the plate solution was decanted and washed 3 times for 3min each with washing solution;
6.3 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;
6.4 sample adding: diluting the antiserum by 3 times from 1:1000, adding the diluted antiserum into coated wells of each dilution, reacting at 37 ℃ for 30min at 100 mu L/well; after fully washing, adding HRP-goat anti-mouse antibody IgG diluted by 1:3000, 100 mu L/hole, and reacting for 30min at 37 ℃;
6.5 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;
6.6 termination and determination: the reaction was stopped by adding 50. mu.L of 2M sulfuric acid stop solution to each well, and the OD of each well was measured by a microplate reader450The value is obtained.
Determination of IC of monoclonal antibody Ethyl Maltol by IC-ELISA50Comprises the following steps: 0.78ng/mL, which shows that the reagent has good sensitivity to ethyl maltol and can be used for immunoassay detection of ethyl maltol.
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 (3)
1. An ethyl maltol monoclonal antibody hybridoma cell strain ABC01 which is deposited in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms, China academy of sciences microorganism research institute No. 3, Xilu No.1, Beijing, Tokyo, Yangyang, is classified and named as a monoclonal cell strain, the deposition date is 2019, 11 months and 28 days, and the deposition number is CGMCC No. 19178.
2. The monoclonal antibody against ethyl maltol is secreted by the monoclonal cell strain ABC01 with the preservation number of CGMCC No.19178 according to claim 1.
3. The use of the monoclonal antibody against ethyl maltol as claimed in claim 2, wherein: the method is used for analyzing and detecting the ethyl maltol residue in food safety detection.
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| CN110441512A (en) * | 2019-07-23 | 2019-11-12 | 重庆市食品药品检验检测研究院 | A kind of colloidal gold immunochromatographimethod detection device of ethylmaltol haptens and ethylmaltol |
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| CN112458062A (en) | 2021-03-09 |
| WO2022116782A1 (en) | 2022-06-09 |
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