CN113493432A - Synthetic method and application of vomitoxin hapten and artificial antigen - Google Patents
Synthetic method and application of vomitoxin hapten and artificial antigen Download PDFInfo
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Abstract
The invention discloses a vomitoxin hapten which has the following molecular structure:
the invention also discloses the hapten, the artificial antigen, a method for preparing the antibody by using the artificial antigen, and application of the hapten and the antibody. The antibody prepared by the invention has good specificity and high sensitivity, and is similar to a structureThe cross reaction is low. Therefore, the antigen and the prepared antibody of the invention can be used for establishing an immunological detection method, thereby being used for quickly detecting vomitoxin in food.
Description
Technical Field
The invention belongs to the field of food safety detection, and particularly relates to a synthetic method and application of vomitoxin hapten and artificial antigen.
Background
Vomitoxin (vomitoxin), also known as Deoxynivalenol (DON), is mainly produced by fusarium such as fusarium graminearum, fusarium oxysporum, fusarium moniliforme, fusarium sporotrichioides, fusarium pinorum, fusarium nivale and the like. In addition, strains of the genus cephalosporium, the genus painterl, the genus trichoderma, etc. can produce the toxin. The trichothecene toxins are over 150 kinds, are strong immunosuppressants, and cause the typical symptom that the feed intake is reduced, so the toxins are also called as feed antifeedant toxins. Vomitoxin (DON) is one of the most important toxins, mainly from Fusarium species, especially Fusarium graminearum and Fusarium yellow. It is also known as Vomitoxin (VT) because it can cause vomiting in pigs. Because of their highly cytotoxic and immunosuppressive properties, they pose a threat to the health of humans and animals, especially with a significant impact on immune function. Depending on the dose of DON and the exposure time, immunosuppression or immunostimulation may be induced. When people ingest food contaminated by DON, acute poisoning symptoms such as anorexia, emesis, diarrhea, fever, unstable standing, and slow response can be caused, and in severe cases, hematopoietic system is damaged to cause death. Because the grain proportion in the traditional Chinese diet habit is greatly higher than that in the west, the harm of vomitoxin is more prominent. In 1998, vomitoxin was listed as a class 3 carcinogen in an evaluation report published by the international cancer research institute. The European Union requires vomitoxin to be less than 1.0 mg/kg; chinese feed requirements are less than 1 ppm.
Most of the existing detection methods are instrument detection, such as thin layer chromatography, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry and the like. Although the instrument detection method has higher accuracy, the sample pretreatment is complicated, the detection time is long, and the instrument is expensive, so that the wide application of the method is limited. Enzyme-Linked Immunosorbent Assay (ELISA) is an ultramicro Assay method established by combining an immunological technique with a modern test means, has the characteristics of low cost, high speed, high sensitivity and simple instrument and equipment, and can be operated on site. Is suitable for rapid analysis of large-batch samples. The basis of the immunological detection method is the preparation of an antibody with high specificity and high affinity, and the vomitoxin serving as a small molecular compound does not have immunogenicity, so that the structural modification and the synthesis of a whole antigen of a hapten are one of the key and difficult points for establishing an immunological rapid detection technology.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a synthetic method and application of vomitoxin hapten and artificial antigen.
In order to achieve the purpose, the invention provides the following technical scheme:
a vomitoxin hapten, wherein the molecular structure of the hapten is as follows:
further, the preparation method of the vomitoxin hapten comprises the following steps:
(1) taking a 5mL single-mouth bottle, and weighing 10mg of vomitoxin standard substance and ZnCl22mg of gamma-aminobutyric acid and 2mL of dichloromethane are added, and the mixture is stirred at room temperature for reaction overnight;
(2) and after TLC detection reaction is completed, adding 5mL of water, extracting for 3 times by using 5mL of dichloromethane, drying the organic phase by using anhydrous sodium sulfate, and then, spinning the organic phase to obtain the vomitoxin hapten.
An artificial antigen for vomitoxin, which is prepared by coupling the hapten and carrier protein of claim 1.
Further, the preparation method of the vomitoxin artificial antigen comprises the following steps:
(1) dissolving 4mg of the hapten, 3.5 mg of NHS and 5mg of EDC of the claim 1 in 200 mu L of DMF solution, and reacting for 2 h;
(2) and (2) dissolving 20 mg of carrier protein into 2mL of buffer solution with the pH value of 9.6, dropwise adding the reaction solution obtained in the step (1) into the carrier protein solution, and reacting overnight to obtain the vomitoxin artificial antigen.
Furthermore, in the preparation method of the vomitoxin artificial antigen, the carrier protein is BSA, OVA or KLH.
The vomitoxin antibody which is prepared by immunizing a mouse by adopting the artificial antigen of the vomitoxin and can generate specific immunoreaction with the vomitoxin.
The invention relates to an application of vomitoxin antibody in detecting the residual quantity of vomitoxin in food.
Has the advantages that: the invention provides a synthetic method and application of vomitoxin hapten and artificial antigen, and the detection limit of a monoclonal antibody obtained after immunizing a mouse by using the artificial antigen prepared from the hapten synthesized by the method is 1 ng/mL. The antibody prepared by the invention has good specificity, high sensitivity and low cross reaction with structural analogues. Therefore, the antigen and the prepared antibody of the invention can be used for establishing an immunological detection method, thereby being used for quickly detecting vomitoxin in food.
Drawings
FIG. 1 is a scheme showing the synthesis of emetic toxin haptens.
Detailed Description
The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
EXAMPLE 1 Synthesis and identification of haptens
The preparation method of the vomitoxin hapten comprises the following steps:
(1) get 5mL single-necked bottleWeighing vomitoxin standard substance 10mg and ZnCl22mg of gamma-aminobutyric acid and 2mL of dichloromethane are added, and the mixture is stirred at room temperature for reaction overnight;
(2) and after TLC detection reaction is completed, adding 5mL of water, extracting for 3 times by using 5mL of dichloromethane, drying the organic phase by using anhydrous sodium sulfate, and then, spinning the organic phase to obtain the vomitoxin hapten.
The synthetic route is as follows:
and (3) nuclear magnetic resonance identification results: 1H NMR (300 MHz, CDCl3) δ 6.62 (dq, J =5.9, 1.4Hz, 1H), 5.31 (dt, J =11.2, 4.4Hz, 1H), 4.81 (s, 1H), 4.78 (s, 2H), 4.74 (d, J =5.9Hz, 1H), 3.99 (d, J =4.5Hz, 1H), 3.86 (d, J =11.5Hz, 1H), 3.77 (br, 1H), 3.76 (d, J =11.5Hz, 1H), 3.22 (d, J =4.1Hz, 1H), 3.14 (d, J =4.1Hz, 1H), 2.80 (dd, J =15.7, 4.3Hz, 1H), 2.5(m, 2H), 2.25 (m, 3H), 1.91 (m, 3H), 1H, 71 (br, 71H), 1H) 1.17 (s, 3H); 13C NMR (75 MHz, CDCl3) Δ 201.3, 182.5, 140.0, 136.9, 88.8, 86.0, 78.8, 71.0, 67.0, 62.1, 50.4, 50.2, 49.9, 47.5, 42.3, 35.9, 25.9, 18.0, 16.5 HRMS (ESI) Calcd for C19H30NO8+ (M + H) + 400.1966, found: 400.1983.
Example 2 Synthesis of Artificial antigen
1. Synthesis of immunogens
(1) Dissolving 4mg of the hapten, 3.5 mg of NHS and 5mg of EDC of the claim 1 in 200 mu L of DMF solution, and reacting for 2 h;
(2) and (2) dissolving 20 mg of BSA in 2mL of buffer solution with pH9.6, dropwise adding the reaction solution obtained in the step (1) into the carrier protein solution, and reacting overnight to obtain the vomitoxin artificial antigen.
2. Synthesis of coatingen
(1) Dissolving 4mg of the hapten, 3.5 mg of NHS and 5mg of EDC of the claim 1 in 200 mu L of DMF solution, and reacting for 2 h;
(2) and (2) dissolving 20 mg of OVA in 2mL of buffer solution with pH value of 9.6, dropwise adding the reaction solution obtained in the step (1) into the carrier protein solution, and reacting overnight to obtain the vomitoxin artificial antigen.
EXAMPLE 3 monoclonal antibody preparation
A vomitoxin monoclonal antibody is prepared by immunizing a BALB/c mouse with 6-8 weeks old with the immunogen prepared in the embodiment 2, and is used for detecting the residual quantity of vomitoxin in food.
(1) Mouse immunization: Balb/C female mice were immunized with the complete antigen as the immunogen. Diluting the complete antigen to 1mg/mL by using sterile physiological saline, taking a proper amount of diluent, mixing the diluent with the quick immunologic adjuvant with the same volume, uniformly oscillating, and performing intramuscular single-point injection according to the dose of 10 mu g/mouse. Three weeks later, a second immunization was performed in the same dose and manner, and the mice were tail-bled seven days after immunization and titer was checked.
(2) Cell fusion: after separating serum from the collected mouse tail blood, the titer and inhibition of the serum are determined by indirect ELISA and indirect competitive ELISA. Selecting mice with high titer and good inhibition for cell fusion. Mice were immunized by shock 18 days after the last immunization, three days later spleens were removed by aseptic manipulation, and a spleen single cell suspension was prepared and mixed with mouse myeloma cells SP2/0 cells grown in logarithmic phase at a ratio of 10: 1. The supernatant liquid was removed by centrifugation, and after cells were scattered, cell fusion was carried out by the polyethylene glycol method.
(3) Subcloning: and 3 days and 5 days after fusion, respectively carrying out liquid change of 50% and 90% HAT culture solution, and carrying out screening on cell supernatant on 7-9 days. Positive cell supernatants were selected, assayed for inhibition by ELISA, and the best inhibited cell wells selected for subcloning. Subcloning was performed by limiting dilution method, with 0.5-2 cells per well, and subcloning was repeated three times. The HT culture solution is used for the first subcloning, the 1640 culture solution is used for the second time, the serum content in all the culture solutions is 15%, and finally the required monoclonal antibody cell strain is obtained.
(4) Freezing and recovering cells for purification: and (3) performing expanded culture on the obtained cell strain, freezing and preserving the seed, continuously culturing the rest cells, preparing the monoclonal antibody by an in vivo ascites method, and purifying by an ammonium caprylate method or a protein G method.
Example 4 construction of vomitoxin monoclonal antibody enzyme-linked immunosorbent assay method
Firstly, detecting the serum titer by adopting an indirect ELISA method, and specifically comprising the following operation steps:
1. coating: the coating source prepared in example 2 was diluted in 0.05M pH9.6 carbonate buffer starting from 5. mu.g/mL in two fold, 100. mu.L/well, reacted at 37 ℃ for 2 h.
2. Washing: the plate was decanted, spun-dried and washed 3 times with washing solution for 3min each time.
3. And (3) sealing: after patting dry, adding 200 mu L/hole sealing liquid, reacting for 2h at 37 ℃, washing and drying for later use.
4. Sample adding: diluting the monoclonal antibody obtained in example 3 from 1:1000 in a multiple ratio, adding the diluted monoclonal antibody into coated wells of each dilution, reacting at 37 ℃ for 1h at 100 mu L/well; after washing well, HRP-goat anti-mouse IgG diluted 1:3000 was added at 100. mu.L/well and reacted at 37 ℃ for 1 h.
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 100. mu.L of a stop buffer to each well, and the OD450 value of each well was measured by a microplate reader.
Second, detection of minimum detection Limit and specificity
The specific operation steps are as follows:
1. the working concentrations of the coating antigen and the antibody are determined by the indirect ELISA matrix titration method, and the corresponding antigen and antibody concentrations are the optimal working concentrations when the OD450 value is about 1.5.
2. Coating: the coating source was diluted to the optimal working concentration with coating buffer, 100. mu.L/well, and reacted at 37 ℃ for 2 h.
3. Washing and sealing: the procedure was the same as for the indirect ELISA method described above.
4. Preparing a vomitoxin standard solution: the vomitoxin standard substance is prepared into 1mg/mL mother solution by using 0.01mol/L PBS solution with pH7.4, and then diluted into a series of concentrations by using 0.01mol/L PBS solution with pH7.4.
5. Sample adding: each well was added with 50. mu.L of the diluted standard, followed by 50. mu.L/well of the antibody at the optimum dilution, and reacted at 37 ℃ for 1 hour. After washing well, HRP-goat anti-mouse IgG diluted 1:3000 was added at 100. mu.L/well and reacted at 37 ℃ for 1 h.
6. And (3) color development reaction: 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.
7. Termination and measurement: the reaction was stopped by adding 100. mu.L of a stop buffer to each well, and the OD450 value of each well was measured by a microplate reader. 8. Data processing: the logarithm of each concentration of emetic toxin was plotted on the abscissa and the corresponding OD value was plotted on the ordinate to prepare a standard curve, and the median inhibitory concentration (IC 50, i.e., the concentration of the standard at which the OD450 value decreased from the value corresponding to the zero standard solution to 50%) was calculated to determine whether the antiserum had specificity for emetic toxin.
Determination of antibody cross-reactivity:
IC of vomitoxin monoclonal antibody measured by indirect competitive ELISA50And the cross-reaction rate with its analogues (3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, nivalenol and fusarenone X).
Cross reaction rate (%) = IC50(vomitoxin)/IC50(analogue), experiments were repeated 3 times and the results averaged.
The result shows that the detection limit of the vomitoxin monoclonal antibody is 1ng/mL, and the cross reaction rate of each analogue is less than 0.1%.
Comparative example 1
Firstly, preparing vomitoxin hapten by using a succinic anhydride method: the coupling of the carboxyl groups to the carrier protein is achieved by first linking (blocking) the hydroxyl groups on the 7, 15 carbons with butyl boronic acid, then linking succinic anhydride on the 3 carbon to introduce the carboxyl groups, and finally removing the butyl boronic acid.
(1) Preparation of DON-BBA: dissolving 10mgDON in 5mL of anhydrous pyridine, weighing 25mg of butyl boric acid, adding the butyl boric acid into the reaction solution, and stirring the mixture at room temperature overnight to obtain an intermediate product;
(2) preparation of BBA-DON-HS: adding 12mg of succinic anhydride into a reaction bottle under the stirring condition, sealing the reaction bottle, and reacting in a 100C oil bath for 3 hours to obtain an intermediate product;
(3) preparation of 3-HS-DON: pyridine is blown to dryness by nitrogen at room temperature, 1mL of double distilled water is added, then the residue 3-HS-DON is dissolved in 1mL of ethyl acetate, after 15min of ultrasonic treatment, 1500 g of the solution is centrifuged for 3min, and the supernatant is taken. Dissolving the precipitate with ethyl acetate, repeating the above operation, combining the supernatant, and rotary evaporating to obtain the final product, i.e. the hemisuccinate of DON.
Preparation of artificial antigen
The artificial immunogen and the coatingen were prepared using the same method as in example 2.
Preparation of monoclonal antibody
The DON monoclonal antibody was prepared using the same method as example 3.
Detection of four, minimum detection Limit and specificity
The enzyme-linked immunoassay method was constructed in the same manner as in example 4, and the limit of detection of the vomitoxin monoclonal antibody measured by the method was 10ng/mL, and the cross-reactivity of each analog was less than 0.1%.
In conclusion, the detection limit of the monoclonal antibody prepared by the artificial antigen is obviously lower than that of the antibody prepared by the comparative example 1, so that the synthesis method of the hapten disclosed by the invention has a better technical effect than that of the hapten prepared by the comparative example 1, the sensitivity of the antibody prepared by the invention on DON detection is greatly improved, and the detection application of the antibody on an actual sample is more facilitated.
Claims (7)
1. A vomitoxin hapten, wherein the molecular structure of the hapten is as follows:
2. the method of producing the vomitoxin hapten of claim 1, comprising the steps of:
(1) taking a 5mL single-mouth bottle, and weighing 10mg of vomitoxin standard substance and ZnCl22mg of gamma-aminobutyric acid and 2mL of dichloromethane are added, and the mixture is stirred at room temperature for reaction overnight;
(2) and after TLC detection reaction is completed, adding 5mL of water, extracting for 3 times by using 5mL of dichloromethane, drying the organic phase by using anhydrous sodium sulfate, and then, spinning the organic phase to obtain the vomitoxin hapten.
3. An artificial vomitoxin antigen prepared by coupling the hapten of claim 1 with a carrier protein.
4. The method for producing the vomitoxin artificial antigen according to claim 1, comprising the steps of:
(1) dissolving 4mg of the hapten, 3.5 mg of NHS and 5mg of EDC of the claim 1 in 200 mu L of DMF solution, and reacting for 2 h;
(2) and (2) dissolving 20 mg of carrier protein into 2mL of buffer solution with the pH value of 9.6, dropwise adding the reaction solution obtained in the step (1) into the carrier protein solution, and reacting overnight to obtain the vomitoxin artificial antigen.
5. The method for producing an artificial antigen for vomitoxin according to claim 3, wherein the carrier protein is BSA, OVA or KLH.
6. An vomitoxin antibody specifically immunoreactive with vomitoxin, prepared by immunizing a mouse with the artificial vomitoxin antigen of claim 3.
7. Use of the vomitoxin antibody according to claim 6 for detecting the residual amount of vomitoxin in food.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102080067A (en) * | 2009-11-26 | 2011-06-01 | 北京维德维康生物技术有限公司 | Method for detecting deoxynivalenol and special reagent kit thereof |
| CN102161623A (en) * | 2010-02-23 | 2011-08-24 | 蓬莱红卫化工有限公司 | Synthesis method of alkylhydroxyethylimide |
| CN105277423A (en) * | 2015-09-07 | 2016-01-27 | 北京勤邦生物技术有限公司 | Immunomagnetic bead used for vomitoxin enrichment purifying and preparation method and application thereof |
| CN109776563A (en) * | 2019-01-18 | 2019-05-21 | 国家食品安全风险评估中心 | Vomitoxin haptens and preparation method thereof, artificial antigen, kit and vomitoxin detection method |
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- 2021-06-24 CN CN202110704775.7A patent/CN113493432A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102080067A (en) * | 2009-11-26 | 2011-06-01 | 北京维德维康生物技术有限公司 | Method for detecting deoxynivalenol and special reagent kit thereof |
| CN102161623A (en) * | 2010-02-23 | 2011-08-24 | 蓬莱红卫化工有限公司 | Synthesis method of alkylhydroxyethylimide |
| CN105277423A (en) * | 2015-09-07 | 2016-01-27 | 北京勤邦生物技术有限公司 | Immunomagnetic bead used for vomitoxin enrichment purifying and preparation method and application thereof |
| CN109776563A (en) * | 2019-01-18 | 2019-05-21 | 国家食品安全风险评估中心 | Vomitoxin haptens and preparation method thereof, artificial antigen, kit and vomitoxin detection method |
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| Title |
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| 张勋: "真菌毒素类高灵敏高通量快速检测方法研究", 《中国博士学位论文全文数据库 工程科技I辑》, vol. 2020, no. 2014, pages 024 - 111 * |
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