CN114181108A - Dendritic multiple hapten, artificial antigen and preparation method - Google Patents

Dendritic multiple hapten, artificial antigen and preparation method Download PDF

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CN114181108A
CN114181108A CN202111358974.3A CN202111358974A CN114181108A CN 114181108 A CN114181108 A CN 114181108A CN 202111358974 A CN202111358974 A CN 202111358974A CN 114181108 A CN114181108 A CN 114181108A
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hapten
dendritic
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carrier protein
npamam
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CN114181108B (en
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傅玲琳
王彦波
王翀
周瑾茹
陈善君
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Zhejiang Gongshang University
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    • 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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Abstract

The invention discloses a dendritic multiple hapten, an artificial antigen and a preparation method thereof, relating to the technical field of biology. The dendritic multiple hapten is obtained by carrying out aldehyde-amine condensation or amide bond formation on a micromolecule compound containing amino and organic acid/acid anhydride to prepare a hapten product, carrying out dehydration condensation on the hapten product and polyamidoamine, and carrying out dehydration condensation reaction on carrier protein and dendritic multiple hapten to obtain the dendritic artificial antigen. The preparation method provided by the invention has the advantages of easy acquisition, environmental friendliness, low artificial antigen preparation cost, simple and convenient operation and the like.

Description

Dendritic multiple hapten, artificial antigen and preparation method
Technical Field
The invention relates to the field of biotechnology, 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 become particularly serious. Strengthening food safety detection becomes one of important means for controlling contaminated food to enter the market and guaranteeing the safety of consumers. At present, the detection means for chemical harmful substances in food mainly takes an instrument detection method based on a chromatographic technique and a chromatographic and mass spectrometry combined technique as a main method. Although the instrumental detection is accurate and reliable, expensive equipment, professional operators, complex sample pretreatment and high detection cost are required, and the requirements of field detection of large-batch samples cannot be met. The immunoassay 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 very in line with the characteristics of scattered operation and large quantity and wide range of agricultural product and food production in China.
However, a number of experimental results have shown that: if the target analyte has a molecular weight of less than 500Da, it cannot be directly handled and presented by antigen presenting cells and recognized directly by specific B and T cells, and it is difficult or even impossible to obtain high quality antibodies or perform a series of immunological studies. Most small molecule substances do not have groups, e.g., -COOH, -NH, for attachment to macromolecular carriers, e.g., proteins2Functional groups such as-OH and-SH, therefore, hapten which protrudes out of the specific site of the three-dimensional structure of the molecule must be synthesized and connected with a macromolecular carrier to form an effective artificial conjugate, so that the animal can be immunized to generate specific antibody aiming at the target small molecular compoundAnd (3) a body. The conjugate of this hapten with a macromolecular carrier is called an artificial antigen. The artificial antigen is not prepared arbitrarily, and any structural differences, including binding sites, binding modes, carrier types, and haptens and target analytes, may greatly affect the properties of the corresponding antibodies. Therefore, it is critical to determine the quality of antibodies specific to it and to establish immunochemical assays.
Although small molecule compounds are not immunogenic, they are reactogenic, i.e., have the ability to immunologically react with the corresponding antibody, and can be quantitated in vitro, following the law of mass action. If the label is introduced to show the reaction in an amplifying way, the kit has the specificity and the sensitivity of immunoreaction, has the double characteristics that the label is easy to identify and detect, can analyze ultra-low molecular weight compounds in complex samples such as environment, food, human body fluid and the like, and has better selectivity and sensitivity. Simple and quick operation and low cost.
The patent application with the publication number of CN106831534B discloses a phenolphthalein hapten, an artificial antigen and a preparation method thereof, wherein the phenolphthalein component is not known in Chinese patent medicine or health care products, and the conventional detection method is adopted at present, so that the extracted sample needs to be taken back to a laboratory for analysis by a large instrument, which consumes long time and has high cost. Therefore, the hapten of the invention introduces a connecting arm structure and an active group for coupling macromolecules on the basis of keeping the basic structure of phenolphthalein, thereby being beneficial to the coupling of the hapten and the macromolecules, fully exposing the molecular structure and the basic structure of phenolphthalein with smaller molecular weight after the coupling, and avoiding the influence on the recognition of animal organisms caused by the masking of the hapten by the macromolecules. The hapten and protein are coupled by an active esterification method to prepare the artificial antigen, the coupling ratio can reach 18.2, and the artificial antigen can be used for immunoassay of phenolphthalein.
Therefore, the technical bottleneck that the preparation of the high-affinity antibody of the anti-low molecular compound is difficult is broken through, the rapid immunoassay method is established and the 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 small molecular compounds.
The invention provides a preparation method of dendritic multiple hapten, which comprises the steps of firstly carrying out aldehyde-amine condensation or amide bond formation on a small molecular compound containing amino and organic acid/acid anhydride to prepare a hapten product, and carrying out dehydration condensation on the hapten product and polyamidoamine to obtain the dendritic multiple hapten, wherein the molecular weight of the small molecular compound is less than 500 Da.
Preferably, the organic acid/anhydride is glyoxylic acid, maleic anhydride or succinic anhydride.
Wherein, the organic acid/acid anhydride means an organic acid or acid anhydride.
Preferably, the small molecule compound is cadaverine or sulfonamide.
The dendritic multiple artificial antigen is convenient and fast in preparation method, simple and convenient to operate and suitable for amino-containing small molecular compounds.
Specifically, the synthesis method of the polyamide amine comprises the following steps:
adding concentrated sulfuric acid into N, N-dimethylformamide in an ice bath, reacting to obtain amide ionic liquid, and reacting with ethylenediamine and methyl acrylate to obtain the polyamidoamine.
Specifically, the synthesis method of the hapten product is as follows:
hapten products are prepared from amino-containing small-molecule compounds with molecular weight less than 500Da and glyoxylic acid, maleic anhydride or succinic anhydride, and the process is shown in figure 2.
Preferably, the catalyst is triethylamine.
The invention also provides a 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 artificially constructed dendritic multiple hapten, the structural characteristics of functional groups of small molecules are retained to the maximum extent by the hapten, the small molecule compound is exposed on the surface of the carrier protein, the dendritic multiple hapten is high-density hapten, and the dendritic multiple artificial antigen prepared by the hapten can exert the optimal structure-effect advantage and increase the antigen immunogenicity of the small molecule compound.
The invention also provides a dendritic artificial antigen which is a conjugate obtained by connecting dendritic multiple hapten and carrier protein by amide bond, and the molecular formula 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 reacting dendritic multiple hapten with amino on carrier protein.
Preferably, the carrier protein is bovine serum albumin or ovalbumin.
Specifically, the preparation method comprises the following steps:
the dendritic multiple hapten and N-hydroxysuccinimide are subjected to an activated ester reaction under the action of dicyclohexylcarbodiimide to generate an activated ester derivative, and the activated ester derivative reacts with amino on carrier protein to form a coupling compound 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 haptens, and has good immunocompetence.
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, thus being beneficial to increasing the immunogenicity of the antigen and preparing high-quality antibody.
The dendritic multiple artificial antigen prepared by the invention can be used for animal immunity experiments, preparation of corresponding antibodies and the like, and provides a new means for research on the immune direction of amino-containing micromolecules.
The invention has the beneficial effects that: the preparation method provided by the invention has the advantages of easy acquisition, environmental friendliness, low artificial antigen preparation cost, simple and convenient operation and the like.
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FIG. 1 shows polyamidoamine (NPAMAM)4) Coupled with hapten product (H) to form dendritic multiple hapten (H-NPAMAM)4) A method process diagram; wherein A is NPAMAM4Coupled with hapten product H1 to form H1-NPAMAM4The process of (2); b is NPAMAM4Coupled with hapten product H2 to form H2-NPAMAM4The process of (2); c is NPAMAM4Coupled with hapten product H3 to form H3-NPAMAM4The process of (1).
FIG. 2 is a process diagram of a method for obtaining a hapten product (H1) by reacting a small molecular compound with glyoxylic acid, obtaining a hapten product (H2) by reacting a small molecular compound with maleic anhydride, and obtaining a hapten product (H3) by reacting a small molecular compound with succinic anhydride.
FIG. 3 is a graph of the molecular formula of a dendrimer multiplex hapten; wherein A is NPAMAM4Coupled with hapten product H1 to form H1-NPAMAM4(ii) a B is NPAMAM4Coupled with hapten product H2 to form H2-NPAMAM4(ii) a C is NPAMAM4Coupled with hapten product H3 to form H3-NPAMAM4。
FIG. 4 is a graph of the molecular formula of dendritic artificial antigens; wherein A is artificial antigen H1-NPAMAM4-Protein formula; b is artificial antigen H2-NPAMAM4-Protein formula; c is artificial antigen H3-NPAMAM4-Protein formula.
Detailed Description
Example 1
Synthetic route for hapten (1):
the reaction scheme for hapten synthesis is shown in FIG. 2.
Weighing 3.0g of cadaverine, dissolving in 100mL of dimethyl sulfoxide, dropwise adding 20mL of glyoxylic acid, stirring and heating after dropwise adding, slowly heating to 65 ℃, and continuously stirring and reacting for 12 hours. After the reaction is finished, cooling to room temperature, and then carrying out suction filtration to obtain a hapten H1 product. The hapten H1 product weighed 3.01g after drying, and the average yield was 74.8%.
Dendritic multiple hapten (H1-NPAMAM)4) The synthetic route is as follows:
the dendritic multiple hapten synthetic route is shown in figure 1, and the molecular formula is shown in figure 3.
a. Synthesis of amide ionic liquid: adding 10mL of N, N-dimethylformamide into a round-bottom flask, adding 16.8mL of 12M concentrated sulfuric acid under ice bath, stirring and incubating at 80 ℃ for 24h, washing with toluene, performing rotary evaporation, and performing vacuum drying to obtain a 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 and reacting at room temperature for 24 hours to obtain polyamidoamine, and extracting and spin-drying after the reaction is finished;
c. dendritic multiple hapten (H1-NPAMAM)4) The synthesis of (2): 3.8g of hapten H1 and 1.0g of polyamidoamine are dissolved in 100mL of dimethyl sulfoxide, 1g of EDCI and 1g of HOBT are added as condensing agents, the mixture is stirred and reacted for 2 hours at room temperature, and after the reaction is finished, extraction and spin drying are carried out.
Artificial antigen H1-NPAMAM4-Protein synthetic route:
the molecular formula of the artificial antigen is shown in figure 4.
Dendritic multiple hapten containing carboxyl (H1-NPAMAM)4) Reacting with N-hydroxysuccinimide under the action of Dicyclohexylcarbodiimide (DCC) to generate an activated ester derivative, and reacting with the amino group on the carrier protein to form a coupling compound connected by an amide bond.
Weighing the synthesized 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 1 hour to produce a precipitate at 4 ℃ overnight.
The next day 12000 Xg, 30min, centrifugation at 4 ℃ and addition of the supernatant to 15mL of a 4mg/mL PBS solution of carrier protein (BSA or OVA) at 4 ℃ for 5 hours. After the reaction is finished, the reaction solution is filled into a dialysis bag, the dialysis bag is dialyzed by PBS for 2 days to remove the unconjugated micromolecules, and the dendritic multiple hapten (H1-NPAMAM) is detected by an ultraviolet spectrophotometry4) Coupling with BSA at a coupling ratio of 81:1, coupling with OVAThe ratio was 53: 1.
Preparation of cadaverine polyclonal antibody:
artificial antigen group Using the above synthetic immune antigen H1-NPAMAM4The BSA solution is fully emulsified with equivalent Freund's complete adjuvant and then used for immunizing mice to prepare cadaverine polyclonal antibody; the control group was used to immunize mice after fully emulsifying cadaverine with an equivalent amount of complete freund adjuvant. The method comprises the following steps: 200g of Balb/c mice aged about 8 weeks are injected with H1-NPAMAM subcutaneously at multiple points on each back4-BSA solution mixed with adjuvant antigen 0.1mL, control group injected subcutaneously with cadaverine and adjuvant mixture 0.1mL at multiple points per back. Mice were immunized 2 weeks later, 3 weeks later, and 3 weeks later, 4 weeks later.
On day 10 after the 4 th immunization, 0.2mL of blood was taken from the canthus, left to stand at room temperature for 2 hours, centrifuged, and the serum was diluted by several fold and the antiserum titer was checked by an indirect enzyme-linked immunosorbent assay (ELISA). The serum titer of the artificial antigen group is measured to be more than or equal to 50000:1, and the serum of the control group does not produce cadaverine antibody. A large amount of antiserum is prepared by collecting blood from mouse decapitation, and the antiserum is cadaverine polyclonal antibody and is frozen and stored after being subpackaged.
The results show that immunogen H1-NPAMAM4BSA midpoint titer (OD)492nm1) is 13200:1, and the end-point titer (P/N ≥ 2) is 70000: 1.
Determination of specificity of the artificial antibody:
the test is carried out by taking spermine, spermidine, histamine, beta-phenylethylamine and other related compounds as small molecule competitors and adopting the established ELISA method. Regression calculations were performed based on the inhibition curves to calculate the concentration of competitor IC50 that produced 50% inhibition (or binding) and the relative ratio of each competitor to hapten (H1-NPAMAM)4) The results of the cross-reactivity measurements are shown in Table 1.
TABLE 1 antibody specificity and Cross-reactivity
Figure BDA0003358306250000061
The results show selected relevant combinations for this exampleNone of the substances showed cross-reactivity to the antibody, and all four competitors had IC50 greater than 105ng/mL indicates that the prepared cadaverine polyclonal antibody has higher specific recognition capability.
Example 2
Synthetic route for hapten (2):
3.0g of cadaverine and 3.6g of maleic anhydride were weighed out and dissolved in 100mL of dichloromethane, 10mL of triethylamine was added as an acid-binding agent, and the reaction was stirred at room temperature for 5 hours. After the reaction is finished, the hapten H2 product is obtained by suction filtration.
Dendritic multiple hapten (H2-NPAMAM)4) The synthetic route is as follows:
dendritic multiple hapten (H2-NPAMAM)4) The synthetic procedure of (1) was the same as that of the dendritic multiple hapten (H1-NPAMAM)4) The synthesis step (2).
Artificial antigen H2-NPAMAM4-Protein synthetic route:
artificial antigen H2-NPAMAM4Synthesis of Protein the same procedure as for the artificial antigen H1-NPAMAM in example 14-a Protein synthesis step.
Example 3
Synthetic route for hapten (3):
in the synthesis of hapten (3), the reactants and the synthesis steps were the same as those in the synthesis of hapten (2) in example 2 except that the reactants were cadaverine 3.0g and succinic anhydride 3.6g, and the reaction was terminated to obtain hapten H3.
Dendritic multiple hapten (H3-NPAMA M4) The synthetic route is as follows:
dendritic multiple hapten (H3-NPAMAM)4) The synthetic procedure of (1) was the same as that of the antigen dendritic multiple hapten (H1-NPAMA M)4) The synthesis step (2).
Artificial antigen H3-NPAMAM4-Protein synthetic route:
artificial antigen H3-NPAMAM4Synthesis of Protein the same procedure as for the artificial antigen H1-NPAMA M in example 14-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, stirring and heating are carried out after the addition, the temperature is slowly raised to 65 ℃, and stirring and reaction are continuously carried out for 12 hours. After the reaction is finished, cooling to room temperature, and then carrying out suction filtration to obtain a hapten H4 product.
Dendritic multiple hapten H4-NPAMA M4The synthetic route is as follows:
dendritic multiple hapten (H4-NPAMA M4) The procedure of synthesis of (1) was the same as in example 1.
Artificial antigen H4-NPAMAM4-Protein synthetic route:
artificial antigen H4-NPAMAM4Synthesis of Protein the same procedure as for the artificial antigen H1-NPAMA M in example 14-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 and reacted for 5 hours at room temperature. After the reaction is finished, the hapten H5 product is obtained by suction filtration
Dendritic multiple hapten synthetic route:
dendritic multiple hapten (H5-NPAMA M4) The procedure of synthesis of (1) was the same as in example 1.
Antigen H5-NPAMAM4-Protein synthetic route:
artificial antigen H5-NPAMAM4Synthesis of Protein the same procedure as for the artificial antigen H1-NPAMA M in example 14-a Protein synthesis step.
Example 6
Synthesis of hapten (6):
the synthesis of hapten (2) in example 2 was performed except that the reactants were 3.0g of sulfonamide and 3.6g of succinic anhydride, and the H6 product was obtained after the reaction.
Dendritic multiple hapten synthetic route:
dendritic multiple hapten (H6-NPAMA M4) The procedure of synthesis of (1) was the same as in example 1.
Antigen H6-NPAMAM4-Protein synthetic route：
Artificial antigen H6-NPAMAM4Synthesis of Protein the same procedure as for the artificial antigen H1-NPAMA M in example 14-a Protein synthesis step.

Claims (10)

1. A preparation method of dendritic multiple hapten is characterized in that small molecular compounds containing amino and organic acid/acid anhydride are subjected to aldehyde-amine condensation or amide bond formation to prepare hapten products, the hapten products and polyamidoamine are subjected to dehydration condensation to obtain the dendritic multiple hapten, and the molecular weight of the small molecular compounds is less than 500 Da.
2. The method of claim 1, wherein the organic acid/anhydride is glyoxylic acid, maleic anhydride or succinic anhydride.
3. The method of claim 1, wherein the small molecule compound is cadaverine or sulfonamide.
4. The method of claim 1, wherein the polyamidoamine is obtained by synthesizing the amide ionic liquid from N, N-dimethylformamide and then reacting with ethylenediamine and methyl acrylate.
5. A dendritic multiple hapten prepared by the process for preparing a dendritic multiple hapten as claimed in any one of claims 1 to 4.
6. A dendritic artificial antigen which is a conjugate obtained by linking the dendritic multiple hapten according to claim 5 with a carrier protein through an amide bond.
7. The dendritic artificial antigen of claim 6, wherein the carrier protein is bovine serum albumin or ovalbumin.
8. The method for producing a dendritic artificial antigen according to claim 6, wherein the dendritic multiple hapten is condensed with an amino group on the carrier protein to obtain the dendritic artificial antigen.
9. The method of claim 8, wherein the carrier protein is bovine serum albumin or ovalbumin.
10. The method for preparing dendritic artificial antigen as claimed in claim 8, wherein the dendritic multiple hapten is firstly reacted with N-hydroxysuccinimide under the action of dicyclohexylcarbodiimide to generate an activated ester derivative, and the activated ester derivative is reacted with the amino group on the carrier protein to form a conjugate which is connected by an amide bond, namely the dendritic artificial antigen.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030224447A1 (en) * 2002-01-31 2003-12-04 Randox Laboratories Limited Haptens, immunogens, antibodies and conjugates to ketamine and its metabolites
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)
CN111675670A (en) * 2020-05-25 2020-09-18 华南农业大学 Citric acid-based dendritic hapten CAA, dendritic antigen and heavy chain antibody for directly detecting AMOZ and preparation method thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030224447A1 (en) * 2002-01-31 2003-12-04 Randox Laboratories Limited Haptens, immunogens, antibodies and conjugates to ketamine and its metabolites
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)
CN111675670A (en) * 2020-05-25 2020-09-18 华南农业大学 Citric acid-based dendritic hapten CAA, dendritic antigen and heavy chain antibody for directly detecting AMOZ and preparation method thereof

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