CN114181108B - Dendritic multiple hapten, artificial antigen and preparation method thereof - Google Patents
Dendritic multiple hapten, artificial antigen and preparation method thereof Download PDFInfo
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- 239000000427 antigen Substances 0.000 title claims abstract description 58
- 102000036639 antigens Human genes 0.000 title claims abstract description 57
- 108091007433 antigens Proteins 0.000 title claims abstract description 57
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 17
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 17
- 150000007524 organic acids Chemical class 0.000 claims abstract description 7
- 238000009833 condensation Methods 0.000 claims abstract description 6
- 230000005494 condensation Effects 0.000 claims abstract description 6
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 5
- 239000004952 Polyamide Substances 0.000 claims abstract description 4
- 230000018044 dehydration Effects 0.000 claims abstract description 4
- 238000006297 dehydration reaction Methods 0.000 claims abstract description 4
- 229920002647 polyamide Polymers 0.000 claims abstract description 4
- 238000010976 amide bond formation reaction Methods 0.000 claims abstract description 3
- 150000001412 amines Chemical class 0.000 claims abstract description 3
- 238000006482 condensation reaction Methods 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 32
- VHRGRCVQAFMJIZ-UHFFFAOYSA-N cadaverine Chemical compound NCCCCCN VHRGRCVQAFMJIZ-UHFFFAOYSA-N 0.000 claims description 24
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 15
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical group OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 claims description 11
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 6
- BAPJBEWLBFYGME-UHFFFAOYSA-N Methyl acrylate Chemical compound COC(=O)C=C BAPJBEWLBFYGME-UHFFFAOYSA-N 0.000 claims description 6
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- 125000003277 amino group Chemical group 0.000 claims description 6
- 229940098773 bovine serum albumin Drugs 0.000 claims description 6
- 239000002608 ionic liquid Substances 0.000 claims description 6
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- 229920000962 poly(amidoamine) Polymers 0.000 claims description 6
- 229940014800 succinic anhydride Drugs 0.000 claims description 6
- 150000002148 esters Chemical class 0.000 claims description 5
- 229940092253 ovalbumin Drugs 0.000 claims description 5
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 4
- 230000009471 action Effects 0.000 claims description 4
- 150000001408 amides Chemical class 0.000 claims description 4
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 3
- 239000000412 dendrimer Substances 0.000 claims description 3
- 229920000736 dendritic polymer Polymers 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 2
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- 238000010168 coupling process Methods 0.000 description 12
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- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 description 12
- 238000001243 protein synthesis Methods 0.000 description 11
- 238000001514 detection method Methods 0.000 description 10
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical group CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- MKYBYDHXWVHEJW-UHFFFAOYSA-N N-[1-oxo-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propan-2-yl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(C(C)NC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 MKYBYDHXWVHEJW-UHFFFAOYSA-N 0.000 description 7
- 235000013305 food Nutrition 0.000 description 7
- 150000003384 small molecules Chemical class 0.000 description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
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- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
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- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
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- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
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- 238000012360 testing method Methods 0.000 description 2
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 1
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- BHHGXPLMPWCGHP-UHFFFAOYSA-N Phenethylamine Chemical compound NCCC1=CC=CC=C1 BHHGXPLMPWCGHP-UHFFFAOYSA-N 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
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- 239000010839 body fluid Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
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- 229960001340 histamine Drugs 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
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- 238000005406 washing Methods 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C249/00—Preparation of compounds containing nitrogen atoms doubly-bound to a carbon skeleton
- C07C249/02—Preparation of compounds containing nitrogen atoms doubly-bound to a carbon skeleton of compounds containing imino groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/14—Preparation of carboxylic acid amides by formation of carboxamide groups together with reactions not involving the carboxamide groups
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
- C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
- C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
- C07C237/12—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by carboxyl groups
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C251/00—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton
- C07C251/02—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups
- C07C251/04—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups having carbon atoms of imino groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C251/06—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups having carbon atoms of imino groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of a saturated carbon skeleton
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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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- 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
- C07K14/77—Ovalbumin
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- Genetics & Genomics (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a dendritic multiple hapten, an artificial antigen and a preparation method thereof, and relates to the technical field of biology. The dendritic multiple hapten is prepared by carrying out aldehyde-amine condensation or amide bond formation on an amino-containing micromolecular compound and organic acid/anhydride, a hapten product is prepared, the hapten product and polyamide amine are subjected to dehydration condensation, and a carrier protein and the dendritic multiple hapten are subjected to dehydration condensation reaction, so that the dendritic artificial antigen is obtained. The preparation method provided by the invention has the advantages of easy acquisition, environmental friendliness, low preparation cost of artificial antigen, simplicity and convenience in operation and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a dendritic multiple hapten, an artificial antigen and a preparation method thereof.
Background
In recent years, food safety problems caused by chemical pests in foods have been particularly serious. Strengthening food safety detection has become one of the important means for controlling the entry of contaminated food into the market and ensuring consumer safety. Currently, detection means for chemical harmful substances in foods mainly comprise instrument detection methods based on chromatographic techniques and chromatographic and mass spectrometry combined techniques. Although the instrument method detection is accurate and reliable, expensive equipment, professional operators, complex sample pretreatment and high detection cost are required, and the requirement of on-site detection of a large number of samples cannot be met. The immune detection technology based on antigen-antibody specific reaction has the advantages of short analysis time, simple operation, high sensitivity, good specificity, low detection cost and the like, and is quite in line with the characteristics of dispersion and large quantity and wide range of agricultural product and food production and management in China.
However, a number of test results showed that: if the molecular weight of the target analyte is less than 500Da, it is not directly handled and submitted by antigen presenting cells and recognized directly by specific B and T cells, it is difficult or even impossible to obtain high quality antibodies or to conduct a series of immunological studies. Most small molecule substances do not have groups such as-COOH, -NH-attached to macromolecular carriers such as proteins or the like 2 Functional groups such as-OH and-SH, so hapten which protrudes out of specific parts of the molecular three-dimensional structure must be synthesized and connected with a macromolecular carrier to form an effective artificial conjugate, so that animals can be immunized to generate specific antibodies aiming at the target small molecular compound. The conjugate of such hapten and macromolecular carrier is called artificial antigen. The preparation of artificial antigens is not arbitrary and may greatly affect the properties of the corresponding antibodies, including binding sites, binding patterns, carrier species, and any structural differences between hapten and analyte of interest. It is therefore the key to determine the quality of antibodies raised specifically and to establish immunochemical analysis methods.
Although small molecule compounds are not immunogenic, they are reactive, i.e. have the ability to react immunologically with the corresponding antibodies, and can be quantified in vitro, following the law of mass action. If the label is introduced to amplify and display the reaction, the method has the specificity and sensitivity of immune reaction, has the dual characteristics that the label is easy to identify and detect, can analyze ultra-trace small molecular compounds in complex samples such as environment, food, human body fluid and the like, and has better selectivity and sensitivity. The operation is simple and quick, and the cost is low.
The patent application with the publication number of CN106831534B discloses a phenolphthalein hapten, an artificial antigen and a preparation method thereof, and the detection of whether the phenolphthalein component is contained in a Chinese patent medicine or a health care product is unknown, and the current or traditional detection method needs to bring an extracted sample back to a laboratory for large-scale instrument analysis, so that the time is consumed and the cost is high. Therefore, the hapten is introduced with a connecting arm structure and active groups for coupling macromolecules on the basis of retaining the basic structure of phenolphthalein, so that the coupling of the hapten and the macromolecules is facilitated, the molecular structure and the basic structure of phenolphthalein with smaller molecular weight can be fully exposed after the coupling, and the influence of the masking of the phenolphthalein by the macromolecules on the recognition of animal organisms is avoided. The hapten and the protein are coupled by an active esterification method to prepare an artificial antigen, the coupling ratio can reach 18.2, and the hapten and the protein can be used for immunodetection of phenolphthalein.
Therefore, the technical bottleneck of ' difficult preparation of high affinity antibody against ' low molecular compound ' is broken through, a rapid immunoassay method is established and screening application is carried out, and the method has important theoretical and practical significance.
Disclosure of Invention
The invention aims to provide a dendritic multiple hapten, an artificial antigen and a preparation method thereof, which can enhance the antigen immunity of a small molecular compound.
The invention provides a preparation method of a dendritic multiple hapten, which comprises the steps of carrying out aldehyde-amine condensation or amide bond formation by using a small molecular compound containing amino and organic acid/anhydride to prepare a hapten product, and carrying out dehydration condensation on the hapten product and polyamide to obtain the dendritic multiple hapten, wherein the molecular weight of the small molecular compound is less than 500Da.
Preferably, the organic acid/anhydride is glyoxylic acid, maleic anhydride or succinic anhydride.
Wherein the organic acid/anhydride means an organic acid or anhydride.
Preferably, the small molecule compound is cadaverine or sulfanilamide.
The preparation method of the dendritic multiple artificial antigen is convenient and simple to operate, and is suitable for small molecular compounds containing amino.
Specifically, the synthesis method of the polyamidoamine comprises the following steps:
firstly, adding concentrated sulfuric acid into N, N-dimethylformamide under ice bath, reacting to obtain amide ionic liquid, and then reacting with ethylenediamine and methyl acrylate to obtain the polyamidoamine.
Specifically, the hapten product is synthesized as follows:
hapten products are prepared from small molecule compounds containing amino groups and having a molecular weight of less than 500Da and glyoxylic acid, maleic anhydride or succinic anhydride, as shown in figure 2.
Preferably, the catalyst is triethylamine.
The invention also provides the dendritic multiple hapten prepared by the preparation method of the dendritic multiple hapten, and the molecular structural formula of the dendritic multiple hapten is shown in figure 3.
The dendritic multiple hapten is an artificially constructed dendritic multiple hapten, the structural characteristics of functional groups of small molecules are reserved to the greatest extent, small molecular compounds are exposed on the surface of carrier proteins, the dendritic multiple hapten is a high-density hapten, and the dendritic multiple artificial antigen prepared from the hapten can exert the best structure-activity advantage and increase the antigen immunogenicity of the small molecular compounds.
The invention also provides a dendritic artificial antigen, which is a conjugate obtained by connecting dendritic multiple hapten and carrier protein through amide bonds, and the molecular formula of the conjugate is shown in figure 4.
Preferably, the carrier protein is bovine serum albumin or ovalbumin.
The invention also provides a preparation method of the dendritic artificial antigen, which is to obtain the dendritic artificial antigen by the reaction of the dendritic multiple hapten and the amino group on the carrier protein.
Preferably, the carrier protein is bovine serum albumin or ovalbumin.
Specifically, the preparation method comprises the following steps:
the dendritic multiple half-antigen is reacted with N-hydroxysuccinimide under the action of dicyclohexylcarbodiimide to generate an activated ester derivative, and the activated ester derivative reacts with amino groups on carrier protein to form a conjugate which is obtained by amide bond connection, namely the dendritic artificial antigen.
Preferably, the carrier protein is bovine serum albumin or ovalbumin.
The dendritic multiple artificial antigen prepared by the invention is obtained by coupling bovine serum albumin and ovalbumin serving as carrier proteins with dendritic multiple hapten, and has good immune activity.
The dendritic multiple artificial antigen prepared by the invention not only improves the density of target small molecules, but also exposes the target small molecules on the surface of carrier protein, thereby being beneficial to increasing the immunogenicity of the antigen and preparing high-quality antibodies.
The dendritic multiple artificial antigen prepared by the invention can be used for animal immunity experiments, corresponding antibody preparation and the like, and provides a new means for researching the immunity direction of small molecules containing amino.
The invention has the beneficial effects that: the preparation method provided by the invention has the advantages of easy acquisition, environmental friendliness, low preparation cost of artificial antigen, simplicity and convenience in operation and the like.
Drawings
FIG. 1 is a polyamide amine (NPAMAM) 4 ) Coupled with hapten product (H) to form dendritic multiple hapten (H-NPAMAM) 4 ) A method process diagram; wherein A is NPAMAM 4 Coupling with hapten product H1 to form H1-NPAMAM 4 Is a process of (1); b is NPAMAM 4 Coupling with hapten product H2 to form H2-NPAMAM 4 Is a process of (1); c is NPAMAM 4 Coupling with hapten product H3 to form H3-NPAMAM 4 Is a process of (2).
FIG. 2 is a process diagram of a method for reacting a small molecule compound with glyoxylate to give a hapten product (H1), reacting a small molecule compound with maleic anhydride to give a hapten product (H2), and reacting a small molecule compound with succinic anhydride to give a hapten product (H3).
FIG. 3 is a molecular formula diagram of a dendrimer hapten; wherein A is NPAMAM 4 Coupling with hapten product H1H1-NPAMAM is formed by combining 4 The method comprises the steps of carrying out a first treatment on the surface of the B is NPAMAM 4 Coupling with hapten product H2 to form H2-NPAMAM 4 The method comprises the steps of carrying out a first treatment on the surface of the C is NPAMAM 4 Coupling with hapten product H3 to form H3-NPAMAM 4 。
FIG. 4 is a molecular formula diagram of a dendritic artificial antigen; wherein A is an artificial antigen H1-NPAMAM 4 -Protein formula; b is artificial antigen H2-NPAMAM 4 -Protein formula; c is artificial antigen H3-NPAMAM 4 -Protein formula.
Detailed Description
Example 1
Synthetic route for hapten (1):
the synthesis scheme of hapten is shown in FIG. 2.
3.0g cadaverine is weighed and dissolved in 100mL of dimethyl sulfoxide, then 20mL of glyoxylic acid is added dropwise, after the dropwise addition is finished, stirring and heating are carried out, the temperature is slowly increased to 65 ℃, and stirring and reaction are continued for 12h. After the reaction is finished, cooling to room temperature, and carrying out suction filtration to obtain a hapten H1 product. The hapten H1 product was dried and weighed to 3.01g with an average yield of 74.8%.
Dendritic multiple hapten (H1-NPAMAM) 4 ) The synthetic route is as follows:
the synthesis route of dendritic multiple hapten is shown in figure 1, and the molecular formula is shown in figure 3.
a. Synthesizing amide ionic liquid: adding 10mL of N, N-dimethylformamide into a round-bottom flask, adding 16.8mL of concentrated sulfuric acid with the concentration of 12M under ice bath, stirring and incubating at 80 ℃ for 24 hours, washing with toluene, rotary evaporating, and vacuum drying to obtain light yellow transparent viscous ionic liquid;
b. synthesis of polyamidoamine (NPAMAM): adding 10mL of ethylenediamine, 14.5mL of methyl acrylate and the ionic liquid into a round-bottom flask, stirring at room temperature for reaction for 24 hours to obtain polyamidoamine, and extracting and spin-drying after the reaction is completed;
c. dendritic multiple hapten (H1-NPAMAM) 4 ) Is synthesized by the following steps: 3.8g of hapten H1 product and 1.0g of polyamidoamine are taken and dissolved in 100mL of dimethyl sulfoxide, 1g of EDCI and 1g of HOBT are added as condensing agents, and the mixture is stirred at room temperature for 2H, and after the reaction is completed, the mixture is extracted and dried by spinning.
Artificial antigen H1-NPAMAM 4 -Protein synthesis route:
the artificial antigen molecular formula is shown in fig. 4.
Carboxyl-containing dendritic multiple hapten (H1-NPAMAM) 4 ) Under the action of Dicyclohexylcarbodiimide (DCC), it reacts with N-hydroxysuccinimide to form activated ester derivatives, which react with the amino groups on the carrier protein to form amide-linked conjugates.
Weighing synthetic dendritic multiple hapten (H1-NPAMAM) 4 ) 1mM in 1mL of N, N-Dimethylformamide (DMF), 0.42mM of N-hydroxysuccinimide (NHS in 1mL of DMF) and 72mg of N, N-dicyclohexylcarbodiimide (DCC in 1mL of DMF) were added, and the mixture was stirred at room temperature for 1h, and after overnight at 4℃a precipitate was formed.
The next day 12000 Xg, 30min, centrifugation at 4℃and after centrifugation the supernatant was added to 15mL of a PBS solution of carrier protein (BSA or OVA) at a concentration of 4mg/mL and reacted at 4℃for 5 hours. After the reaction, the reaction solution is filled into a dialysis bag, unconjugated small molecules are removed by PBS dialysis for 2 days, and dendritic multiple hapten (H1-NPAMAM) is detected by ultraviolet spectrophotometry 4 ) The coupling ratio to BSA was 81:1 and to OVA was 53:1.
Preparation of cadaverine polyclonal antibodies:
artificial antigen group immune antigen H1-NPAMAM synthesized by above 4 The BSA solution is fully emulsified with equivalent Freund's complete adjuvant and then used for immunizing mice, and cadaverine polyclonal antibodies are prepared; the control group was cadaverine and equivalent Freund's complete adjuvant were fully emulsified for immunization of mice. The method comprises the following steps: about 200g of Balb/c mice with the age of 8 weeks are taken, and H1-NPAMAM is injected subcutaneously into each back in a multipoint manner 4 Mixing BSA solution with adjuvant antigen 0.1mL, and multi-point subcutaneous injection of cadaverine and adjuvant mixture 0.1mL per back of control group. Mice were immunized 2 times after 2 weeks, 3 times after 3 weeks, and 4 times after 3 weeks.
On day 10 after the 4 th immunization, 0.2mL of blood is taken from the canthus, the mixture is kept stand at room temperature for 2 hours and centrifuged, serum is taken for dilution by multiple ratio, and the antiserum titer is checked by an indirect enzyme-linked immunosorbent assay (ELISA) method. The serum titer of the artificial antigen group is measured to be more than or equal to 50000:1, and cadaverine antibodies are not generated in the serum of the control group. A large amount of antisera, namely cadaverine polyclonal antibody, is prepared by taking blood from broken ends of mice, and is frozen after split charging.
The results showed that the immunogen H1-NPAMAM 4 Mid-titer in BSA (OD 492nm =1) 13200:1, endpoint potency (P/n+.2) 70000:1.
Determination of artificial antibody specificity:
related compounds such as spermine, spermidine, histamine and beta-phenethylamine are respectively used as small molecule competitors, and the established ELISA method is adopted for testing. Regression calculations were performed based on the inhibition curves to calculate the concentration of competitor IC50 that resulted in 50% inhibition (or binding) and the relative amounts of each competitor to hapten (H1-NPAMAM) 4 ) The cross-reactivity of (2) is shown in Table 1.
TABLE 1 antibody specificity and Cross-reactivity
The results indicate that none of the related compounds selected in this example show cross-reactivity to antibodies, and that the IC50 of each of the four competitors is greater than 10 5 ng/mL, the prepared cadaverine polyclonal antibody has higher specific recognition capability.
Example 2
Synthetic route for hapten (2):
3.0g cadaverine and 3.6g maleic anhydride were weighed out and dissolved in 100mL methylene chloride, 10mL triethylamine was added as an acid-binding agent, and the reaction was stirred at room temperature for 5 hours. And after the reaction is finished, suction filtration is carried out to obtain a hapten H2 product.
Dendritic multiple hapten (H2-NPAMAM) 4 ) The synthetic route is as follows:
dendritic multiple hapten (H2-NPAMAM) 4 ) The synthesis procedure is the same as that of the dendritic multiple hapten (H1-NPAMAM) in example 1 4 ) Is a synthesis step of (a).
Artificial antigen H2-NPAMAM 4 -Protein synthesis route:
artificial antigen H2-NPAMAM 4 The procedure for the synthesis of Protein was the same as that for the artificial antigen H1-NPAMAM in example 1 4 -a Protein synthesis step.
Example 3
Synthetic route for hapten (3):
in the synthesis of hapten (3), except that reactants are 3.0g cadaverine and 3.6g succinic anhydride, the other reactants and the synthesis steps are the same as those of hapten (2) in example 2, and hapten H3 product is obtained after the reaction.
Dendritic multiple hapten (H3-NPAMA M) 4 ) The synthetic route is as follows:
dendritic multiple hapten (H3-NPAMAM) 4 ) The synthesis procedure is the same as that of the antigen dendritic multiple hapten (H1-NPAMA M) in example 1 4 ) Is a synthesis step of (a).
Artificial antigen H3-NPAMAM 4 -Protein synthesis route:
artificial antigen H3-NPAMAM 4 The procedure for the synthesis of Protein was the same as that for the artificial antigen H1-NPAMA M in example 1 4 -a Protein synthesis step.
Example 4
Synthetic route for hapten (4):
3.0g of sulfanilamide is weighed and dissolved in 100mL of dimethyl sulfoxide, then 20mL of glyoxylic acid is added dropwise, after the dropwise addition is finished, stirring and heating are carried out, the temperature is slowly increased to 65 ℃, and stirring and reaction are continued for 12h. And after the reaction is finished, cooling to room temperature, and carrying out suction filtration to obtain a hapten H4 product.
Dendritic multiple hapten H4-NPAMA M 4 The synthetic route is as follows:
dendritic multiple hapten (H4-NPAMA M) 4 ) The synthesis procedure of (2) is the same as that of example 1.
Artificial antigen H4-NPAMAM 4 -Protein synthesis route:
artificial antigen H4-NPAMAM 4 The procedure for the synthesis of Protein was the same as that for the artificial antigen H1-NPAMA M in example 1 4 -a Protein synthesis step.
Example 5
Synthetic route for hapten (5):
3.0g of sulfanilamide and 3.6g of maleic anhydride are weighed and dissolved in 100mL of dichloromethane, 10mL of triethylamine is added as an acid binding agent, and the mixture is stirred at room temperature for reaction for 5 hours. Suction filtering after the reaction is finished to obtain hapten H5 products
Dendritic multiple hapten synthetic routes:
dendritic multiple hapten (H5-NPAMA M) 4 ) The synthesis procedure of (2) is the same as that of example 1.
Antigen H5-NPAMAM 4 -Protein synthesis route:
artificial antigen H5-NPAMAM 4 The procedure for the synthesis of Protein was the same as that for the artificial antigen H1-NPAMA M in example 1 4 -a Protein synthesis step.
Example 6
Synthesis of hapten (6):
the synthesis of hapten (2) in example 2 was repeated except that the reactants were 3.0g of sulfonamide and 3.6g of succinic anhydride, and the reaction was completed to obtain H6 product.
Dendritic multiple hapten synthetic routes:
dendritic multiple hapten (H6-NPAMA M) 4 ) The synthesis procedure of (2) is the same as that of example 1.
Antigen H6-NPAMAM 4 -Protein synthesis route:
artificial antigen H6-NPAMAM 4 The procedure for the synthesis of Protein was the same as that for the artificial antigen H1-NPAMA M in example 1 4 -a Protein synthesis step.
Claims (8)
1. A preparation method of a dendritic multiple hapten is characterized in that cadaverine containing amino and organic acid/anhydride are subjected to aldehyde-amine condensation or amide bond formation to prepare a hapten product, and the hapten product and polyamide amine are subjected to dehydration condensation to obtain the dendritic multiple hapten;
the organic acid/anhydride is glyoxylic acid, maleic anhydride or succinic anhydride.
2. The method for preparing the dendritic multiple hapten according to claim 1, wherein the polyamidoamine is obtained by synthesizing an amide ionic liquid from N, N-dimethylformamide and then reacting the amide ionic liquid with ethylenediamine and methyl acrylate.
3. A dendrimer hapten prepared by the method for preparing a dendrimer hapten according to claim 1 or 2.
4. A dendritic artificial antigen, wherein the dendritic artificial antigen is a conjugate obtained by amide bond connection of the dendritic multiple hapten and a carrier protein according to claim 3.
5. The dendritic artificial antigen of claim 4, wherein the carrier protein is bovine serum albumin or egg albumin.
6. The method for preparing the dendritic artificial antigen according to claim 4, wherein the dendritic multiple hapten and the amino group on the carrier protein are subjected to condensation reaction to obtain the dendritic artificial antigen.
7. The method of claim 6, wherein the carrier protein is bovine serum albumin or ovalbumin.
8. The method for preparing the dendritic artificial antigen according to claim 6, wherein the dendritic multiple hapten is reacted with N-hydroxysuccinimide to generate an activated ester derivative under the action of dicyclohexylcarbodiimide, and the activated ester derivative reacts with amino groups on carrier protein to form a conjugate obtained by amide bond connection, namely the dendritic artificial antigen.
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| CN102917699A (en) * | 2009-10-13 | 2013-02-06 | 密执安大学评议会 | Dendrimer compositions and methods of synthesis |
| CN103951630A (en) * | 2014-04-17 | 2014-07-30 | 华南农业大学 | Arborescence half antigen capable of directly detecting furaltadone metabolite AMOZ, arborescence antigen and application of arborescence half antigen |
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| CN105527434A (en) * | 2015-12-31 | 2016-04-27 | 辽宁迈迪生物科技有限公司 | A kit used for detecting N1,N<12>-diacetylspermine (DAS) |
| CN111675670A (en) * | 2020-05-25 | 2020-09-18 | 华南农业大学 | A kind of citrate-based dendritic hapten CAA, dendritic antigen, heavy chain antibody and preparation method for directly detecting AMOZ |
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| CN102917699A (en) * | 2009-10-13 | 2013-02-06 | 密执安大学评议会 | Dendrimer compositions and methods of synthesis |
| CN103951630A (en) * | 2014-04-17 | 2014-07-30 | 华南农业大学 | Arborescence half antigen capable of directly detecting furaltadone metabolite AMOZ, arborescence antigen and application of arborescence half antigen |
| CN104744294A (en) * | 2014-07-15 | 2015-07-01 | 广州城市职业学院 | Preparation method and application of di(alpha-ethyl hexyl) phthalate (DEHP) half antigen, artificial antigen and antibody |
| CN105527434A (en) * | 2015-12-31 | 2016-04-27 | 辽宁迈迪生物科技有限公司 | A kit used for detecting N1,N<12>-diacetylspermine (DAS) |
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