CN112028989B - Chlorogenic acid artificial antigen and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a chlorogenic acid artificial antigen which is characterized by comprising the following structures:
the preparation method comprises the following steps:
Description
Technical Field
The invention belongs to the field of biological medical treatment, and particularly relates to a preparation method and application of an artificial antigen of chlorogenic acid.
Background
Chlorogenic acid (CGA) has a molecular weight of 354.31, a CAS number of 327-97-9, and a structural formula:
chlorogenic acid is dephenolic acid generated from Caffeic acid (Caffeic acid) and Quinic acid (Quinic 1-hydroxyhexahydro gallic acid), is a phenylpropanoid compound generated by plants in aerobic respiration process, widely exists in plants of eucommia, caprifoliaceae, lonicera and Compositae, is an important bioactive substance, and has the effects of resisting bacteria, resisting virus, increasing leukocyte, protecting liver, benefiting gallbladder, resisting tumor, lowering blood pressure, reducing blood fat, removing free radicals, exciting central nervous system and the like. The research on the biological activity of chlorogenic acid by modern science is deeply carried out in a plurality of fields such as food, health care, medicine, daily chemical industry and the like. Therefore, the search for a suitable method for measuring the content of chlorogenic acid becomes an important link for the research of the traditional Chinese medicinal materials.
At present, the detection methods of chlorogenic acid at home and abroad mainly comprise: ultraviolet spectrophotometry, high performance liquid chromatography, gas chromatography, etc. These conventional instrumental analysis methods have the advantages of accurate, stable and reliable detection results, but require expensive instruments, complex operations and complex sample pretreatment, and are not suitable for rapid detection of a large number of samples. The immunoassay method has the advantages of simplicity, convenience, rapidness, sensitivity, good specificity and the like, and has important significance for detecting chlorogenic acid. In order to establish a rapid immunoassay method for chlorogenic acid, a chlorogenic acid artificial antigen and a preparation method thereof are needed to be provided.
CN105237633 discloses a chlorogenic acid complete antigen and a preparation method and application thereof, but the antiserum titer of the prepared antigen after immunizing animals needs to be improved, and the sensitivity for immunodetection is unknown.
Therefore, the application develops a novel chlorogenic acid artificial antigen and a preparation method thereof, the preparation method is simple and convenient in process, and immune animals can generate high-titer antibodies, so that the chlorogenic acid artificial antigen can be used for quickly detecting chlorogenic acid, and the detection sensitivity of the chlorogenic acid is improved.
Disclosure of Invention
The technical problem to be solved by the invention is to provide the chlorogenic acid artificial antigen which has high sensitivity and simple preparation method and can be used for quickly detecting the chlorogenic acid, and the preparation method and the application thereof.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a chlorogenic acid artificial antigen has the following structure:
a method for artificial antigen synthesis of chlorogenic acid, comprising the steps of:
(1) Weighing 30mg of bovine serum albumin or ovalbumin and 6mg of 6-aminocaproic acid, adding 8mL of 2- (N-morpholine) ethanesulfonic acid (MES) solution for dissolving, adding 25mg of EDAC, and continuously stirring at room temperature for reacting for 12h;
(2) After the reaction is finished, putting the reaction solution into a dialysis bag, and dialyzing for 72 hours at 4 ℃ by PBS to obtain an aminated carrier protein solution;
(3) Weighing chlorogenic acid 10mg, adding DMF to 0.5mL for dissolving, adding NHS 15mg and EDAC 16mg, stirring overnight at 16 deg.C, and stirring for 12 hr to obtain chlorogenic acid activated intermediate;
(4) On the next day, dropwise adding the chlorogenic acid activation intermediate product into the aminated carrier protein solution while stirring on a magnetic stirrer, and stirring overnight for reaction, namely continuously stirring for 12 hours;
(5) The next day the overnight stirred reaction was placed in a treated dialysis bag and dialyzed in 0.01M PBS (pH = 7.4) at 4 ℃ in a refrigerator for 72h, changing the dialysate every 8 h;
(6) And after the dialysis is finished, centrifuging the liquid in the dialysis bag, and removing precipitates to obtain the chlorogenic acid artificial antigen.
A method for preparing a chlorogenic acid antibody by using a chlorogenic acid artificial antigen, comprising the following steps:
(1) Synthesizing artificial antigen of chlorogenic acid, wherein the immune antigen is synthesized by adopting chlorogenic acid and bovine serum albumin, and the detection antigen is synthesized by adopting chlorogenic acid and ovalbumin, namely the artificial antigen of chlorogenic acid is prepared by the method;
(2) Selecting a healthy female mouse or a female Leiheng chicken as an experimental object, injecting the immune antigen, collecting eggs produced by the poultry or carrying out cell fusion by using mouse spleen cells to obtain hybridoma cells, and further obtaining a monoclonal antibody (Zhulixin, establishing an immunoaffinity column and detecting Sudan red I [ D ]. Nanchang university, 2011.);
(3) IgG in mouse ascites and IgY in egg are purified by using octanoic acid-ammonium sulfate method (Shushu bead, shuxu Yang, dunhun Duzhou, etc.. Preparation of monoclonal antibody immunoaffinity column of ochratoxin A [ J ]. Food science, 2009 (09): 172-175.) or protein M affinity Chromatography (Jiang X, thirumai D, zhang X. Affinity purification of egg yolk immunoglobulin (IgY) using a human mycoplasma protein. Journal of Chromatography B,2016, s1012-1013; the IgG and IgY are antibodies to chlorogenic acid.
The method for preparing the immune egg comprises the following steps: the first immunization injection is carried out on 20-week-old poultry, the immunization dose is 350 mug/mouse, and after 21 days of the first immunization, 4 times of boosting immunization are carried out, the interval time is 21 days, and the immunization dose is 300 mug/mouse.
It is further described that the antigen is administered to the mice by a first immunization injection into 6-8 week-old female mice at an immunization dose of 60. Mu.g/female, followed by 3 booster immunizations 21 days after the first immunization, at an interval of 21 days at an immunization dose of 50. Mu.g/female, followed by a fourth booster immunizations at an immunization dose of 30. Mu.g/female.
The invention has the beneficial effects that the antibody can be effectively enhanced, specifically, the anti-chlorogenic acid IgY starts to generate the antibody about 20 days after the priming, and the antibody titer reaches the highest about 90 days after the priming, namely 1:256000, after cessation of the booster immunization, the antibody titer was maintained for more than one month.
Compared with the prior art, the chlorogenic acid antigen developed by the invention has higher immunogenicity, the generated antibody has high specificity and sensitivity, the detection effect is good, and the method for preparing the antigen has simple steps and is simple and convenient.
Drawings
FIG. 1 is a reaction equation for synthesizing artificial antigen of chlorogenic acid according to the present invention;
FIG. 2 is a UV spectrum of chlorogenic acid artificial antigen;
FIG. 3 is SDS-PAGE of IgG antibody against chlorogenic acid purified by octanoic acid-ammonium sulfate method;
FIG. 4 is a SDS-PAGE electrophoresis of purified anti-chlorogenic IgG antibodies;
FIG. 5 is a competition plot of chlorogenic acid.
FIG. 6 is a structural formula of chlorogenic acid artificial antigen.
Detailed Description
Preferred embodiments of the present invention are described in detail below.
Example 1: the invention relates to a method for preparing chlorogenic acid artificial antigen, which comprises the following steps:
(1) Weighing 30mg of bovine serum albumin or ovalbumin and 6mg of 6-aminocaproic acid, adding 8mL of 0.01M MES solution for dissolving, adding 25mg of EDAC, and stirring at 25 ℃ for reaction for 12h;
(2) After the reaction was completed, the reaction solution was placed in a dialysis bag (Mw < 8000-14000) and dialyzed at 4 ℃ against 0.01M PBS (pH = 7.4) for 72h to obtain an aminated carrier protein solution;
(3) Weighing 10mg of chlorogenic acid, adding DMF0.5mL for dissolving, adding NHS 15mg and EDAC 16mg, stirring overnight at 16 ℃, continuously stirring for 12h, centrifuging the reaction solution at 6000r/min for 10min, removing precipitate, and preparing a chlorogenic acid activation intermediate product;
(4) On the next day, dropwise adding the chlorogenic acid activation intermediate product into the aminated carrier protein solution while stirring on a magnetic stirrer, stirring overnight for reaction, and continuously stirring for 12h;
(5) The next day the overnight stirred reaction was placed in a treated dialysis bag and dialyzed in 0.01M PBS (pH = 7.4) in a refrigerator at 4 ℃ for 72h, changing the dialysate every 8 h;
(6) And after the dialysis is finished, centrifuging the liquid in the dialysis bag, and removing precipitates to obtain the chlorogenic acid artificial antigen.
The specific reaction equation is shown in FIG. 1.
The embodiment of the invention respectively carries out ultraviolet scanning on chlorogenic acid, BSA and chlorogenic acid artificial antigen at 200-800 nm. FIG. 2 is the ultraviolet scan before and after the preparation of chlorogenic acid artificial antigen. As shown in figure 2, the ultraviolet absorption peak of the chlorogenic acid artificial antigen has a certain shift compared with BSA and chlorogenic acid, which indicates that the chlorogenic acid and the carrier protein are successfully coupled, and the chlorogenic acid artificial antigen is successfully prepared by the embodiment of the invention.
Further, a method of preparing chlorogenic acid antibodies, comprising the steps of:
(1) Preparing chlorogenic acid artificial antigen, wherein the immune antigen is synthesized by adopting chlorogenic acid and bovine serum albumin, and the detection antigen is synthesized by adopting chlorogenic acid and ovalbumin;
(2) Selecting a healthy female mouse or a female Leiheng chicken as an experimental object, injecting the immune antigen, collecting eggs produced by the poultry or carrying out cell fusion by using mouse spleen cells to obtain hybridoma cells, and further obtaining a monoclonal antibody (Zhulixin, establishing an immunoaffinity column and detecting Sudan red I [ D ]. Nanchang university, 2011.);
(3) Purifying by caprylic acid-ammonium sulfate method or protein M affinity chromatography to obtain IgG in mouse ascites and IgY in egg; the IgG and IgY are antibodies to chlorogenic acid.
The method for preparing the immune egg comprises the following steps: the method comprises the steps of performing primary immunization injection on 20-week-old poultry, wherein the immunization dose is 350 mu g/poultry, performing secondary immunization 21 days after the primary immunization, the immunization dose is 300 mu g, performing tertiary immunization 42 days after the secondary immunization, the immunization dose is 300 mu g, performing quartic immunization 63 days after the tertiary immunization, the immunization dose is 300 mu g, performing quintic immunization 84 days after the quartic immunization, and the immunization dose is 300 mu g.
Further, the method for immunizing the mice by the antigen comprises the following steps of carrying out primary immunization injection on 6-8 week-old female balb/c mice, wherein the immunization dose is 60 mu g/mouse, carrying out secondary immunization 21 days after the primary immunization, the immunization dose is 50 mu g, carrying out three times of immunization 42 days after the secondary immunization, the immunization dose is 50 mu g, carrying out four times of immunization 63 days after the three times of immunization, the immunization dose is 50 mu g, carrying out five times of immunization 84 days after the four times of immunization, and the immunization dose is 30 mu g.
As shown in fig. 2 to 5, for further explanation, chlorogenic acid antibody titers were measured:
(1) Coating an enzyme label plate: preparing 2 mu g/mL chlorogenic acid-ovalbumin solution by using CBS solution, adding a 96-well enzyme label plate into 100 mu L/well, incubating overnight at 37 ℃, removing liquid in the well, and washing the plate for 4 times by using PBST;
(2) And (3) sealing: blocking the wells with 2% BSA, incubating at 37 ℃ for 2h, discarding the liquid in the wells, and washing the plates with PBST 4 times;
(3) Addition of purified IgY or IgG: at first every hole add 100 mu L's PBS bottoming, take out the IgY solution that separation and purification obtained, resume to room temperature after, dilute with diluent PBS1, take 100 mu L to add first hole, suck 100 mu L after the blowing-sucking mixing and add the second hole, in proper order multiple dilution to 1. Extracting antibody 100 μ L from blank egg or mouse serum as Negative Control (NC), PBS 100 μ L as Blank Control (BC), incubating at 37 deg.C for 40min, discarding liquid in the well, and washing plate;
(4) Adding an enzyme-labeled secondary antibody: diluting goat anti-chicken IgY or rabbit anti-mouse IgG labeled by horseradish peroxidase with PBS1:5000, adding 100 mu L per hole, incubating at 37 ℃ for 40min, discarding liquid in the hole, and washing the plate;
(5) Color development: adding freshly prepared TMB-H 2 O 2 Placing 100 mu L of substrate liquid in each hole at room temperature in a dark place for 8-10 minutes, discarding liquid in the hole, and washing the plate;
(6) And (4) terminating: finally, 50. Mu.L of 2M H was added 2 SO 4 Stopping reaction, measuring the OD value of the reaction product by using an enzyme-linked immunosorbent assay at the wavelength of 450nm, and repeating the step three times for each hole to obtain an average value;
(7) And (4) judging a result: determining OD of sample well 450 Value (P) and negative control well OD 450 When the P/N ratio of the value (N) is more than or equal to 2.1, the antibody is judged to be positive, and the maximum dilution multiple of the corresponding antibody is the titer of the antibody.
The results show that the anti-chlorogenic acid-IgY starts to produce the antibody about 20 days after the priming, and the antibody titer reaches the highest value about 90 days after the priming, namely 1:256000, after cessation of booster immunization, the antibody titer was maintained for more than one month. The anti-chlorogenic acid-IgG titer is as high as 1.
The invention also detects the purity of the antibody of the antigen immunized animal prepared in the example 1, and the method comprises the following steps: preparing 5% concentrated gel and 12% separation gel, diluting the prepared purified antibody to 1mg/Ml, adjusting the voltage of the concentrated gel to 90V and the voltage of the separation gel to 120V, performing polyacrylamide gel electrophoresis (SDS-PAGE), dyeing with Coomassie brilliant blue, and scanning an electrophoresis pattern after decoloring. FIG. 3 is an SDS-PAGE picture of purified chlorogenic acid IgG, which shows clear staining band and high aggregation degree, indicating that the purity of the produced chlorogenic acid antibody is high.
While the foregoing is directed to the preferred embodiment of the present invention, it is noted that various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention.
Claims (1)
1. A method for preparing chlorogenic acid artificial antigen is characterized by comprising the following steps:
(1) Weighing carrier protein and 6-aminocaproic acid, adding 2- (N-morpholine) ethanesulfonic acid (MES) solution for dissolving, adding carbodiimide hydrochloride (EDAC), and continuously stirring for reacting for 12h at room temperature;
(2) After the reaction is finished, putting the reaction solution into a dialysis bag, and dialyzing for 72 hours at 4 ℃ by PBS to obtain an aminated carrier protein solution;
(3) Weighing chlorogenic acid, adding DMF to dissolve, adding NHS and EDAC, stirring at 16 deg.C overnight to obtain chlorogenic acid activated intermediate;
(4) Dropwise adding the chlorogenic acid activated intermediate product into the aminated carrier protein solution while stirring on a magnetic stirrer, and stirring overnight;
(5) The next day, placing the overnight stirred reaction solution in a treated dialysis bag, dialyzing in 0.01M PBS at 4 ℃ for 72h, and changing the dialysate every 8 h;
(6) After dialysis, the liquid in the dialysis bag is centrifuged, and precipitates are removed to obtain the chlorogenic acid artificial antigen, which has the following structure:
in the step (1), 30mg of bovine serum albumin or ovalbumin is used as the carrier protein; 6mg of 6-aminocaproic acid; 2- (N-morpholine) ethanesulfonic acid (MES) solution (8mL), EDAC 25mg;
in the step (3), the chlorogenic acid is 10mg; DMF0.5 mL; NHS 15mg; EDAC 16mg;
the pH of PBS was 7.4.
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| CN101486762A (en) * | 2008-01-18 | 2009-07-22 | 北京中医药大学 | Antibody, method and reagent kit for detecting and determining chlorogenic acid |
| CN105237633A (en) * | 2015-10-15 | 2016-01-13 | 深圳市药品检验所 | Chlorogenic acid complete antigen, preparation method and application thereof |
| EP2987802A1 (en) * | 2014-08-22 | 2016-02-24 | Stern Enzym GmbH & Co. KG | An enzyme with chlorogenic acid esterase activity and feruloyl esterase activity |
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| CN101486762A (en) * | 2008-01-18 | 2009-07-22 | 北京中医药大学 | Antibody, method and reagent kit for detecting and determining chlorogenic acid |
| EP2987802A1 (en) * | 2014-08-22 | 2016-02-24 | Stern Enzym GmbH & Co. KG | An enzyme with chlorogenic acid esterase activity and feruloyl esterase activity |
| CN105237633A (en) * | 2015-10-15 | 2016-01-13 | 深圳市药品检验所 | Chlorogenic acid complete antigen, preparation method and application thereof |
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