CN113968865A - Hapten and artificial antigen of integripetal tailferone and preparation method and application thereof - Google Patents
Hapten and artificial antigen of integripetal tailferone and preparation method and application thereof Download PDFInfo
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- CN113968865A CN113968865A CN202010714570.2A CN202010714570A CN113968865A CN 113968865 A CN113968865 A CN 113968865A CN 202010714570 A CN202010714570 A CN 202010714570A CN 113968865 A CN113968865 A CN 113968865A
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- senecine
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
- C07D491/18—Bridged systems
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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Abstract
The invention discloses a hapten and an artificial antigen of cepharanthine, and a preparation method and application thereof. The structural formula of the hapten of the senecine is shown as a formula I, and the senecine can be prepared by the following method: the compound is obtained by reacting the senecine with succinic anhydride under the catalysis of 4-dimethylamino pyridine. Coupling carrier protein to carboxyl carbon of hapten by active ester method to obtain the artificial antigen of the esmolephrine. The invention also provides a monoclonal antibody of the trinexandrine, which is obtained by immunizing experimental animals with the artificial antigen of the trinexandrine through cell fusion, screening and in vitro induction. The monoclonal antibody of the senecine can be used for preparing a senecine detection reagent or a kit. The invention has good application prospect in food safety detection.
Description
Technical Field
The invention relates to a hapten and an artificial antigen of cepharanthine, and a preparation method and application thereof, and belongs to the field of food safety detection.
Background
The cepharanthine is one of pyrrolizidine alkaloids. In 2017, 10 and 27, the list of carcinogens published by the international cancer research institution of the world health organization is preliminarily collated for reference, and the senecine is in the list of 3 types of carcinogens. Pyrrolizidine alkaloids are secondary metabolites of various flowering plants, are the most important phytotoxic components known at present after aristolochic acid, and are natural toxins affecting livestock, wild animals and human beings. The pyrrolizidine alkaloids are mainly distributed in plants of Compositae, Boraginaceae, Leguminosae, etc., and the main target organ is liver, and can cause hemorrhagic necrosis of liver cell, liver megaloblastic disease, venous occlusion, etc., and also has lung toxicity, carcinogenesis, mutagenic effect, neurotoxicity, etc. Because the pyrrolizidine alkaloids are widely distributed, the pyrrolizidine alkaloids can cause great harm to human or livestock life through indirect food pollution such as pollution of honey, grains, milk, pasture and the like or directly through traditional herbal medicines, tonics, tea and the like containing the pyrrolizidine alkaloids.
Corresponding regulations are established in many countries or organizations aiming at the extensive existence and harm of pyrrolizidine alkaloids in human foods and natural medicines. Some Chinese medicine varieties in new pharmacopoeia of China really contain pyrrolizidine alkaloids. Except for individual varieties (such as groundsel), the control of toxic components is strengthened in the quality standard, and related researches are less for varieties with dual purposes of medicine and food. The existing method for detecting pyrrolizidine alkaloids in animal and plant foods is mainly an instrument method, and comprises high performance liquid chromatography, a gas chromatography-mass spectrometry combined method, a liquid chromatography-tandem mass spectrometry method and the like, although the chromatography technology can carry out qualitative, quantitative, high-efficiency and high-sensitivity detection, the method can not realize field detection and is difficult to popularize. Compared with the chromatographic technology, the antigen-antibody-based specific binding has the advantages of sensitivity, rapidness, specificity, simplicity and the like, and is widely applied to food safety detection in recent years.
At present, immunological detection of pyrrolizidine alkaloids is mostly instrument detection, and the development of domestic related pyrrolizidine alkaloid detection kits is relatively laggard. Therefore, the design of an immune hapten molecule and the development of a simple and quick antibody for detecting various types of the senecine are particularly important; and moreover, theoretical and technical bases are provided for rapid detection of pyrrolizidine toxic alkaloids in import and export foods in China, and food safety and human health are guaranteed.
Disclosure of Invention
The invention aims to provide an aleuridrimine hapten, an artificial antigen, and preparation methods and applications thereof.
The structural formula of the cepharanthine hapten provided by the invention is shown as the formula I:
the cepharanthine hapten can be prepared according to the following preparation method:
the compound is obtained by reacting the senecine with succinic anhydride under the catalysis of 4-dimethylamino pyridine.
Specifically, the molar ratio of the senecine to the 4-dimethylaminopyridine is 1: 1-2;
the molar ratio of the senecine to the succinic anhydride is 1: 1.2 to 1.5;
the reaction temperature is room temperature (about 25 ℃), and the reaction time is 4-5 h.
The reaction is carried out in dichloromethane;
after the reaction, the reaction mixture can be collected through a silica gel column and then dried under nitrogen at 35 ℃.
The invention also provides an artificial antigen of the cepharanthine, which is obtained by coupling the cepharanthine hapten with carrier protein;
the carrier protein is selected from any one of bovine serum albumin, ovalbumin and keyhole limpet hemocyanin, and is preferably bovine serum albumin or ovalbumin.
The molar coupling ratio of the concurrinine hapten to the carrier protein is about 5: 1;
the artificial antigen of the cepharanthine can be used as an immunogen or a coating antigen.
The method can adopt an active ester method to couple the carrier protein to the carboxyl carbon of the hapten to obtain the artificial antigen of the esmolephrine, and comprises the following specific steps: dissolving the cepharanthine hapten, N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in N, N-Dimethylformamide (DMF), and stirring at room temperature overnight to obtain solution A; dissolving the carrier protein in a carbonate buffer solution to obtain a solution B; dropwise adding the solution A into the solution B, and stirring at room temperature for reaction overnight to obtain a mixed solution of the conjugate; and dialyzing the mixed solution in PBS for three days to obtain the artificial antigen of the senecine.
The said cepharanthine hapten or the said artificial cepharanthine antigen has the following applications:
1) preparing an anti-senecine antibody;
2) detecting the residue of senecine in food or medicine.
The invention also provides a monoclonal antibody of the trinexandrine, which is obtained by immunizing experimental animals (female BALB/c mice) with the artificial antigen of the trinexandrine through cell fusion, screening and in vitro induction.
The monoclonal antibody of the senecine can be used for preparing a senecine detection reagent or a kit.
The monoclonal antibody of the cepharanthine has the following applications:
1) immunodetection of the senecine;
2) preparing the senecine immunochromatographic test strip;
3) and (3) preparing the colloidal gold test strip of the senecine.
The invention provides a trinexandrine hapten, an artificial antigen and a preparation method thereof, and the trinexandrine artificial antigen is adopted to immunize animals, so that broad-spectrum antibodies which have high titer and high sensitivity and can simultaneously Recognize Trinexandrine (RTS), latifoline (PLA), Senecine (SEN), Integerrimine (INT), Rosmarinine (ROS), Senecine (SEV), Neolatifoline (NEO), gynura seguinine (Gynine, GYN) and Monocrotaline (MCT) can be obtained. The invention provides a new means for establishing a method for rapidly, simply, conveniently, cheaply and sensitively simultaneously detecting residues of various pyrrolizidine alkaloids.
The hapten is simple in preparation method, the detection sensitivity of the monoclonal antibody prepared from the conjugate of the hapten and the carrier protein to RTS, PLA, SEN, INT, ROS, SEV, NEO, CYN and MCT can respectively reach 0.86, 0.75, 0.43, 0.59, 19.32, 56.32, 129.34, 184.61 and 1003.95ng/mL, and the practical value is high. The invention has good application prospect in food safety detection.
Drawings
FIG. 1 is a reaction equation for preparing an inventive haptens of senecine of formula I.
FIG. 2 is a mass spectrum of the inventive cepharanthine hapten of formula I.
FIG. 3 is a MALDI-TOF-MS diagram of the conjugate of the hapten and bovine serum albumin of the present invention, wherein FIG. 3(A) is a MALDI-TOF-MS diagram of bovine serum albumin, and FIG. 3(B) is a MALDI-TOF-MS diagram of the conjugate.
FIG. 4 is a graph showing the standard curves for detecting 9 types of bakelite according to the present invention using a monoclonal antibody, wherein FIG. 4(A) to FIG. 4(I) show the standard curves for detecting RTS, SEN, INT, PLA, SEV, ROS, CYN, NEO, and MCT in this order.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the quantitative tests in the following examples, three replicates were set up and the results averaged. The PBS buffers used in the examples were all PBS buffer with pH7.4 and 0.01M, and the carbonate buffers were all sodium carbonate buffer with pH 9.6 and 0.05 mol/L.
In the following examples, NHS is an abbreviation for N-hydroxysuccinimide, EDC is an abbreviation for 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and DMF is an abbreviation for N, N-dimethylformamide. NHS, EDC, Bovine Serum Albumin (BSA), Keyhole Limpet Hemocyanin (KLH), Ovalbumin (OVA), and freund's complete adjuvant, freund's incomplete adjuvant were purchased from Sigma.
Example 1 preparation and characterization of the haptens of integerrimine
Preparation of monochorine and senecine hapten
The preparation of the cepharanthine hapten shown in formula I is shown in figure 1.
(1) Completely dissolving 35.1mg of trinexandrine and 2.2mg of 4-Dimethylaminopyridine (DMAP) in 10mL of dichloromethane, wherein the molar ratio of the trinexandrine to the 4-dimethylaminopyridine is 1: 2;
(2) adding 10.7mg of succinic anhydride to the reaction solution, stirring at 600r/min for 5 hours at room temperature, and collecting the compound through a silica gel column, wherein the molar ratio of the senecine to the succinic anhydride is 1: 1.2.
(3) the resulting compound was blown dry at 35 ℃ under nitrogen to yield 37.9mg (84%) of the cepharanthine hapten.
Characterization of the hapten of senecine
Mass spectrum identification: the mass spectrum identification result of the cepharanthine hapten shown in the formula I: MS M/z [ M + H ]]+Theoretical value: 451.2 of the total weight of the mixture; measured value: 452.45, which corresponds to the molecular weight of the target product, and the mass spectrum is shown in FIG. 2.
Example 2 preparation and characterization of Artificial antigen of integerrimine
The preparation method of immunogen and coating antigen is characterized by that the different is the application type of carrier protein, the immunogen carrier protein mainly adopts BSA, and the coating antigen carrier protein mainly adopts OVA, and its coupling method is active ester method.
Synthesis and identification of artificial antigen of integerrimine
1. Preparation of artificial antigen of cepharanthine
(1) And (3) accurately weighing 9mg of hapten, 8mg of EDC and 5mg of NHS according to the feeding ratio of 1:100 of the hapten to BSA, respectively dissolving in 2mL of DMF, and placing on a magnetic stirrer to stir at room temperature overnight to obtain solution A.
(2) Then, 22mg of BSA and OVA were dissolved in 5mL of 0.05M carbonate buffer solution, respectively, to obtain solution B
(3) Slowly dripping the solution A into the solution B, and stirring at room temperature for overnight reaction to obtain a mixed solution of the conjugate; dialyzing the mixed solution in 0.01M PBS for three days to obtain the artificial antigen of the cepharanthine, and storing at-20 ℃. RTS-HS-BSA and RTS-HS-OVA for short.
2. Identification of artificial antigen of cepharanthine
The binding ratio of BSA to hapten in the RTS-HS-BSA solution was determined by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS). The results are shown in FIG. 3.
Binding ratio { M (conjugate) -M (protein) }/M (hapten)
The molecular weight of BSA is 64771.30, the molecular weight of the hapten of formula (I) is 452.45, the molecular weight of the conjugate by mass spectrometry highest peak assay is 66991.13, and the binding ratio of BSA to hapten is calculated to be 4.9: 1, RTS-HS-BSA on average 4.9 haptens were coupled to one BSA molecule.
Example 3 preparation of monoclonal antibody to Execine
8 BALB/c female mice with 6-8 weeks old are immunized with the RTS-HS-BSA artificial antigen prepared in example 2. Each immunogen was diluted to 1mg/mL with 0.01M PBS and emulsified with an equal amount of Freund's adjuvant to form a water-in-oil structure. Except for Freund complete adjuvant for primary immunization, Freund incomplete adjuvant is uniformly adopted for other booster immunizations, the immunization dose is 100 mu g/mouse, and the neck and the back are injected in multiple points in the skin. Booster immunizations were performed 4 weeks later and were given 1 time every 3 weeks for 2 total immunizations, followed by multiple subcutaneous injections at the back and neck. Centrifuging orbital blood of each mouse 7-10 days after the two immunizations, taking supernatant for determination, selecting mice with good antiserum titer and inhibition, taking splenocytes to fuse with myeloma cells, performing three times of single cell mass subcloning, performing strain determination and expanded culture, obtaining antibodies by adopting an in vivo induction mode, purifying by using a Protein A immunoaffinity column, and finally storing at-20 ℃ for later use.
Example 4 determination of the antiserum to integerrimine
Method for detecting antiserum titer by adopting indirect ELISA (enzyme-linked immunosorbent assay)
The specific operation steps are as follows:
(1) coating: the artificial antigen RTS-HS-OVA in example 2 was diluted in 0.05M, pH 9.6.6 carbonate buffer from 15. mu.g/mL in two fold, 100. mu.L/well, incubated in a 37 ℃ incubator for 2h, and spun-dried.
(2) Washing: washing with 280 μ L/hole for 2 times, and spin-drying.
(3) And (3) sealing: sealing solution of 150 μ L/hole, incubating in a constant temperature incubator at 37 deg.C for 1h, and drying.
(4) Sample adding: adding 50 mu L of PBS into each hole, then diluting antiserum by multiple times from 1:1000, adding 50 mu L/hole into the coated holes of each dilution, placing in a constant-temperature incubator at 37 ℃ for incubation for 30min, washing for 3 times, and then spin-drying; adding HRP-labeled goat anti-mouse antibody diluted at a ratio of 1:5000, 100 μ L/well, incubating in a constant temperature incubator at 37 deg.C for 30min, washing for 5 times, and drying.
(5) Color development: 100. mu.L of TMB developing solution was added to each well, and the reaction was carried out at 37 ℃ for 15min in the absence of light.
(6) Stop and assay 50. mu.L stop solution (2M H) was added to each well2SO4) Then the O of each well is determined by a microplate readerD450The value is obtained.
(7) And (4) interpretation of results: by OD450The highest dilution factor of the serum corresponding to the value which is more than or equal to 2.1 times of that of the negative control hole (namely P/N is more than or equal to 2.1) is the ELISA titer of the serum.
Second, minimum detection limit, half inhibition and detection of specificity
The specific operation steps are as follows:
(1) the indirect ELISA method is used for determining that RTS-HS-OVA is used as a coating antigen, RTS-HS-KLH immune mice are used for cell fusion to produce antibodies, and OD is used450The corresponding antigen and antibody concentrations are the optimal working concentrations at values around 1.5.
(2) Coating: the coating antigen was diluted 20000 times with coating buffer at 100. mu.L/well and incubated in a 37 ℃ incubator for 2 h.
(3) Washing and sealing: the procedure was the same as for the indirect ELISA method described above.
(4) Preparing standard solutions respectively: preparing a stock solution of 2mg/mL by using a PBS solution with 0.01mol/L and pH7.4, and diluting the stock solution with a PBS solution with 0.01mol/L and pH7.4 in a multiple ratio to obtain a gradient of 0, 0.04, 0.11, 0.33, 1, 3, 9, 27ng/mL RTS, PLA, SEN and INT standard solutions with a gradient of 0, 0.14, 0.41, 1.23, 3.70, 11.11, 33.33 and 100ng/mL ROS and SEV standard solutions with a gradient of 0, 1.4, 4.1, 12.3, 37, 111.11, 333.33 and 1000ng/mL NEO and CYN standard solutions with a gradient of 0, 13.71, 41.15, 123.46, 370.37, 1111.11, 3333.33 and 10000ng/mL MCT standard solutions with a gradient of 0, 1.4, 12.3, 37, 111.11, 333.33 and 1000ng/mL
(5) Sample adding: each well was added with 50. mu.L of each concentration standard diluted at a double rate, and then 50. mu.L of the antibody at the optimum dilution rate per well was added thereto, and the reaction was carried out at 37 ℃ for 30 min. After thorough washing, 1: HRP-goat anti-mouse IgG at 5000 dilution, 100. mu.L/well, reacted at 37 ℃ for 30 min.
(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) Stopping and measuring by adding 50. mu.L of a stopping solution to each well to stop the reaction, and then measuring the OD of each well by using a microplate reader450The value is obtained.
8) Data ofAnd (3) treatment: the OD corresponding to each concentration is determined by taking the concentration of each standard as the abscissa450nmValues are plotted as ordinate against a four parameter log fit using Origin software, as shown in FIG. 4, by calculating IC50The value (median inhibitory concentration) determines whether the antibody recognizes pyrrolizidine alkaloids.
The results show that the detection sensitivity of the obtained monoclonal antibody to RTS, PLA, SEN, INT, ROS, SEV, NEO, CYN and MCT can reach 0.86, 0.75, 0.43, 0.59, 19.32, 56.32, 129.34, 184.61 and 1003.95ng/mL respectively.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. A cepharanthine hapten of formula I;
2. the method of claim 1, comprising the steps of:
the compound is obtained by reacting the senecine with succinic anhydride under the catalysis of 4-dimethylamino pyridine.
3. The method of claim 2, wherein: the molar ratio of the senecine to the 4-dimethylaminopyridine is 1: 1-2;
the molar ratio of the senecine to the succinic anhydride is 1: 1.2 to 1.5;
the reaction temperature is room temperature, and the reaction time is 4-5 h;
the reaction was carried out in dichloromethane.
4. An artificial antigen of the esmolephrine, which is obtained by coupling the semiantigen of the esmolephrine of claim 1 with a carrier protein;
the carrier protein is selected from any one of bovine serum albumin, ovalbumin and keyhole limpet hemocyanin.
5. The method for preparing the artificial antigen of the esmolephrine as claimed in claim 4, comprising the steps of:
coupling the carrier protein to the carboxyl carbon of the hapten of claim 1 by an active ester method.
6. The use of the penoxerine hapten of claim 1 or the artificial antigen of penoxerine of claim 4 in 1) or 2) below:
1) preparing an anti-senecine antibody;
2) detecting the residue of senecine in food or medicine.
7. A monoclonal antibody to eslerotine prepared from the artificial antigen of eslerotine of claim 4.
8. An assay reagent or kit for the detection of cepharanthine prepared from a monoclonal antibody to cepharanthine according to claim 7.
9. The use of a monoclonal antibody to the eskaline of claim 7 in any one of the following 1) -3):
1) immunodetection of the senecine;
2) preparing an immunochromatographic test strip of the senecine;
3) and (3) preparing the colloidal gold test strip of the senecine.
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Non-Patent Citations (2)
| Title |
|---|
| ERHARD ROEDER，等: "Analysis of Pyrrolizidine Alkaloids: A Competitive Enzyme-Linked lmmunoassay (ELISA) for the Quantitative Determination of Some Toxic Pyrrolizidine Alkaloids", 《NATURAL TOXINS》 * |
| 张小龙，等: "天然药物中生物碱类化合物人工抗原的合成及其鉴定方法", 《世界科学技术-中医药现代化》 * |
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