CN108264552B - Preparation method of heavy metal zinc artificial antigen and application of DOTA in preparation of heavy metal zinc artificial antigen reagent - Google Patents
Preparation method of heavy metal zinc artificial antigen and application of DOTA in preparation of heavy metal zinc artificial antigen reagent Download PDFInfo
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- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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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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Abstract
The invention discloses a preparation method of a heavy metal zinc artificial antigen, which is prepared from 2-S- (4-aminobenzene) -1,4,7, 10-tetraazacyclononane-1, 4,7, 10-tetraacetic acid (p-NH)2-Bn-DOTA, DOTA for short) as a chelating agent, and coupling the zinc ion chelating agent compound with carrier protein bovine serum albumin BSA or chicken ovalbumin OVA to prepare the artificial antigen. The invention also discloses application of the DOTA in preparation of the heavy metal zinc artificial antigen reagent.
Description
Technical Field
The invention belongs to the technical field of heavy metal ion immunochemistry, and particularly relates to a preparation method of a heavy metal zinc artificial antigen and application of DOTA in preparation of a heavy metal zinc artificial antigen reagent.
Background
Heavy metal pollution mainly refers to the pollution of pollutants such as lead, cadmium, mercury, nickel, chromium, arsenic, zinc, copper and the like to the environment. Heavy metals are widely distributed and difficult to degrade, can enter a human body through atmosphere, water and food chains, and react with proteins and various enzymes in the human body to lose activity, and are enriched in certain organs, if the heavy metals exceed the tolerance limit of the human body, acute or chronic poisoning of the human body can be caused, and the heavy metals have carcinogenic, teratogenic and mutagenic effects and great harm to the human body. Different heavy metals have different properties, so the treatment modes are not completely the same, for example, the chemical properties of metal zinc are relatively active and are easy to react with elements in the surrounding environment to cause secondary pollution. Therefore, the enhancement of the detection of the heavy metal residues in the environment, agricultural products and food becomes an important means for guaranteeing the safety of the heavy metal, and the research and development of a new detection technology under the new situation are particularly urgent.
The traditional heavy metal detection method mainly adopts Atomic Absorption Spectroscopy (AAS), InductiveLy coupled plasma Emission Spectroscopy (ICP-AES), Anodic Stripping VoLtammetry (ASV), chromatography and various combined detection methods. Although the methods can effectively analyze the heavy metal ions in various environmental samples, most of the methods need large-scale instruments, the analysis method has high cost, the samples need to be digested, the analysis time is long, the method is not suitable for the field rapid detection of the heavy metals, and the method is difficult to adapt to the requirements of the field spot check of the environment and market products, the self-check of production enterprises and the rapid clearance of product import and export.
The immunological detection technology has the advantages of high detection speed, large analysis capacity, low cost, simple and portable instrument, low technical requirement of users, easy popularization and promotion, high sensitivity, strong selectivity and the like, is particularly suitable for field screening and rapid analysis of a large number of samples, and becomes the most competitive and challenging detection and analysis technology in the 21 st century. A series of detection products developed on the basis of the technology, such as ELISA detection kits, colloidal gold test strips, immunosensors and the like, are widely applied to rapid detection of on-site samples and a large number of samples.
The key of the heavy metal ion immunodetection lies in the preparation of anti-heavy metal specific antibodies, and the key of the specific antibodies lies in the synthesis of high-quality heavy metal immunogens. On one hand, the heavy metal ions have charges and can generate strong irreversible reaction with biological molecules in the animal body to cause animal poisoning reaction; on the other hand, heavy metal has low molecular weight and no immunogenicity, and can form complete immunogen by coupling with carrier protein, but because heavy metal ions are directly connected with protein, the protein is denatured, so a bifunctional chelating agent is required to chelate the heavy metal ions to prepare a metal-chelating agent complex, and the complex is coupled with the protein to prepare a complete antigen, so that an immune animal can prepare a specific antibody. An open-loop bifunctional chelating agent is adopted for chelation, and then the chelating agent is coupled with carrier protein to prepare artificial antigen for animal immunization and antibody preparation, and different immunoassay technologies and methods are established on the basis.
The key point for preparing heavy metal immunogen lies in the selection of bifunctional chelating agent, the commonly used bifunctional chelating agent at present mainly is Ethylene Diamine Tetraacetic Acid (EDTA) or diethylenetriamine pentaacetic acid (DTPA) derivative and other structural analogues with chelating function, and belongs to a chelating agent with chain type open-loop structure, and the chelate is a complex with ring structure formed by combining central ion and polydentate ligand. For example, EDTA and metal ions are bonded through carboxylic acid groups and nitrogen atoms to form a metal-EDTA chelate which is more stable than a complex, and the bifunctional chelating agent has two functions, can specifically chelate heavy metal ions, and can be coupled with carrier protein to form immunogen for subsequent immunization of animals and preparation of antibodies. The conventional chelating agents are of open-loop and straight-chain structures, and the complex of the heavy metal ions and the chelating agent is simpler in spatial structure as an antigen recognition site, and the characteristic functional group is not strong, so that the prepared antigenic determinant has weak antigenic property, and the preparation efficiency of the antibody with high specificity and high sensitivity is directly influenced.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method of a heavy metal zinc artificial antigen. The antibody obtained by immunizing animals with the prepared heavy metal zinc artificial antigen has high affinity, strong specificity and high antiserum titer.
Novel bifunctional chelating agent 2-S- (4-aminobenzene) -1,4,7,10 tetraazacyclononane-1, 4,7, 10-tetraacetic acid (p-NH)2-Bn-DOTA, DOTA for short) has the characteristics of a four-nitrogen closed-loop structure, can better combine heavy metal ions, can better show a composite three-dimensional structure of heavy metal zinc ions as an antigenic determinant in a spatial structure, has more obvious antigenic characteristics, and is beneficial to preparing heavy metal monoclonal and polyclonal antibodies with higher affinity and stronger specificity, and an amino hapten is coupled with carrier protein by a glutaraldehyde method, and diethyl malonate is added, so that the generated heavy metal zinc artificial antigen is more stable, and the antiserum titer is high.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a heavy metal zinc artificial antigen comprises the following steps:
weighing 5-8mg of 2-S- (4-aminobenzene) -1,4,7, 10-tetraazacyclononane-1, 4,7, 10-tetraacetic acid (p-NH)2-Bn-DOTA, hereinafter referred to as DOTA), dissolved in 2mL of 0.01M HEPES solution of hydroxyethyl piperazine ethanesulfonic acid (pH7.44) to prepare a DOTA chelating agent solution, which is solution A;
111.56mg of zinc nitrate was weighed and dissolved in 5mL of ultrapure water to obtain a solution having a concentration of 7.5X 10-2M is zinc nitrate solution, and the solution is B solution;
adding 150-200 mu L of liquid B into the liquid A, and reacting for 3-5h at room temperature in a dark place, wherein the reaction liquid is liquid C;
adding 800 mu L of 20mM glutaraldehyde solution into the solution C drop by drop, and reacting overnight at room temperature in a dark place, wherein the reaction solution is solution D;
weighing 20-30mg of bovine serum albumin BSA or chicken ovalbumin OVA, dissolving in 3mL of HEPES, and uniformly mixing by magnetic stirring at room temperature to obtain a reaction solution E;
dropwise adding the solution D into the solution E, reacting for 24h at room temperature in a dark place, then adding 150-200 mu L diethyl malonate solution (84.6mg dissolved in 200 mu L ethanol), and reacting for 1h at room temperature in a dark place;
dialyzing the reaction solution by using a dialysis bag with 8KD for 3-5 times, centrifuging by using an ultrafiltration centrifugal tube 7000 with 30KD at 9000rpm for 3-5 times, redissolving by using 5-10mL of HEPES solution with 0.01M and pH7.4, and subpackaging at low temperature of-20 ℃ to prepare the heavy metal zinc artificial antigen.
Identification of artificial antigen:
ultraviolet scanning and SDS-PAGE are adopted to identify the coupling effect, and ICP-MS method and Bradford method are adopted to determine the concentration of heavy metal ions and protein to calculate the coupling and binding ratio of the artificial antigen.
The preparation method of the heavy metal zinc artificial antigen selects the bifunctional chelating agent DOTA, can better combine heavy metal ions as an antigenic determinant, and the prepared heavy metal monoclonal and polyclonal antibodies have high affinity and strong specificity.
Drawings
FIG. 1 UV scanning spectra of Zn-DOTA-BSA and Zn-DOTA-OVA.
FIG. 2 Zn-DOTA-BSA and Zn-DOTA-OVA electropherograms.
FIG. 3 structural diagrams of DOTA, DTPA and EDTA.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more apparent, the present invention is further described in detail by the following examples. The following description of specific embodiments is intended to be illustrative of the invention and is not intended to be limiting.
Example 1
Weighing 7mg DOTA, dissolving in 2ml 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01M, pH7.4), and making into DOTA chelating agent solution, which is solution A;
111.56mg of zinc nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X 10-2M, zinc nitrate solution, wherein the reaction solution is solution B;
sucking 180 mu l of the solution B, dropwise adding the solution B into the solution A, and reacting for 3 hours at room temperature in a dark place to obtain a reaction solution C;
adding 700 mu l of 20mM glutaraldehyde solution into the solution C dropwise, and reacting overnight at room temperature in a dark place to obtain a reaction solution D;
weighing 20mg of Bovine Serum Albumin (BSA) and dissolving in 4ml of HEPES, and uniformly mixing by magnetic stirring at room temperature to obtain a reaction solution E;
dropwise adding the solution D into the solution E, reacting for 24h at room temperature in a dark place, then adding 150-200 mu L diethyl malonate solution (84.6mg dissolved in 200 mu L ethanol), and reacting for 1h at room temperature in a dark place;
the reaction solution is dialyzed for 3 times by a dialysis bag with 8KD, then centrifuged for 5 times by an ultrafiltration centrifugal tube with 30KD at 8000rpm, redissolved by 5ml of HEPES solution with 0.01M and pH7.4, and then subpackaged for preservation at low temperature of-20 ℃.
SDS-PAGE shows that the electrophoretic band of the conjugate has a hysteresis tailing phenomenon compared with a single protein band, the molecular weight of the conjugate is larger than that of a single protein, which indicates the successful coupling, and in addition, ultraviolet scanning shows that the maximum absorption wavelength is changed, which further indicates the successful coupling. The binding ratio is calculated to be 23:1 by measuring the content of heavy metal and the concentration of conjugate protein, and the coupling efficiency is high. The higher the binding ratio, the greater the amount of heavy metal ions coupled to a protein molecule, the higher the coupling efficiency, the binding ratio is 23:1, it means that 23 heavy metal ions are combined on one protein molecule.
Comparative example 1
Weighing 7mg DOTA, dissolving in 2ml 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01M, pH7.4), and making into DOTA chelating agent solution, which is solution A;
111.56mg of zinc nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X 10-2M, zinc nitrate solution, wherein the reaction solution is solution B;
sucking 180 mu l of the solution B, dropwise adding the solution B into the solution A, and reacting for 3 hours at room temperature in a dark place to obtain a reaction solution C;
adding 700 mu l of 20mM glutaraldehyde solution into the solution C dropwise, and reacting overnight at room temperature in a dark place to obtain a reaction solution D;
weighing 20mg of Bovine Serum Albumin (BSA) and dissolving in 4ml of HEPES, and uniformly mixing by magnetic stirring at room temperature to obtain a reaction solution E;
and dropwise adding the solution D into the solution E, reacting at room temperature in a dark place for 24h, dialyzing the reaction solution for 3 times by using a dialysis bag with the volume ratio of 8KD, centrifuging for 5 times by using an ultrafiltration centrifugal tube with the volume ratio of 30KD at 8000rpm, redissolving by using 5ml of HEPES solution with the volume ratio of 0.01M and the pH value of 7.4, and subpackaging at the low temperature of-20 ℃.
SDS-PAGE shows that the electrophoretic band of the conjugate has a hysteresis tailing phenomenon compared with a single protein band, the molecular weight of the conjugate is larger than that of a single protein, which indicates the successful coupling, and in addition, ultraviolet scanning shows that the maximum absorption wavelength is changed, which further indicates the successful coupling. The binding ratio was calculated to be 12:1 by heavy metal content determination and conjugate protein concentration determination.
Comparative example 2
Weighing 7mg of p-NH2-Bn-DTPA (hereinafter referred to as DTPA) dissolved in 2ml of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) solution (0.01M, pH7.4) to prepare a DTPA chelating agent solution, wherein the reaction solution is solution A;
111.56mg of zinc nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X 10-2M, zinc nitrate solution, wherein the reaction solution is solution B;
sucking 190 mu l of the solution B, dropwise adding the solution B into the solution A, and reacting for 3 hours at room temperature in a dark place to obtain a reaction solution C;
adding 680 mu l of 20mM glutaraldehyde solution into the solution C dropwise, and reacting overnight at room temperature in a dark place to obtain a reaction solution D;
weighing 20mg bovine serum albumin BSA and dissolving in 3ml HEPES, and uniformly mixing by magnetic stirring at room temperature to obtain a reaction solution E;
dropwise adding the solution D into the solution E, reacting for 24h at room temperature in a dark place, then adding 150-200 mu L diethyl malonate solution (84.6mg dissolved in 200 mu L ethanol), and reacting for 1h at room temperature in a dark place;
the reaction solution is dialyzed for 3 times by a dialysis bag with 8KD, then centrifuged for 5 times by an ultrafiltration centrifugal tube with 30KD at 8000rpm, redissolved by 5ml of HEPES solution with 0.01M and pH7.4, and then subpackaged for preservation at low temperature of-20 ℃.
The success of the coupling was shown by SDS-PAGE experiments and UV scanning, and the binding ratio was calculated to be 11:1 by conjugate protein concentration determination and ICP-MS determination.
Comparative example 3
Weighing 7mg of p-NH2-Bn-EDTA (hereinafter, EDTA) dissolved in 2ml of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) solution (0.01M, pH7.4) to prepare an EDTA chelating agent solution, the reaction solution being solution A;
111.56mg of zinc nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X 10-2M, zinc nitrate solution, wherein the reaction solution is solution B;
sucking 180 mu l of the solution B, dropwise adding the solution B into the solution A, and reacting for 3 hours at room temperature in a dark place to obtain a reaction solution C;
adding 680 mu l of 20mM glutaraldehyde solution into the solution C dropwise, and reacting overnight at room temperature in a dark place to obtain a reaction solution D;
weighing 20mg bovine serum albumin BSA and dissolving in 3ml HEPES, and uniformly mixing by magnetic stirring at room temperature to obtain a reaction solution E;
dropwise adding the solution D into the solution E, reacting for 24h at room temperature in a dark place, then adding 150-200 mu L diethyl malonate solution (84.6mg dissolved in 200 mu L ethanol), and reacting for 1h at room temperature in a dark place;
the reaction solution is dialyzed for 3 times by a dialysis bag with 8KD, then centrifuged for 5 times by an ultrafiltration centrifugal tube with 30KD at 8000rpm, redissolved by 5ml of HEPES solution with 0.01M and pH7.4, and then subpackaged for preservation at low temperature of-20 ℃.
SDS-PAGE experiments and ultraviolet scanning maps show that the coupling is successful, and the binding ratio is calculated to be 10:1 through heavy metal content determination and conjugate protein concentration determination.
Comparative example 4
Weighing 7mg DOTA, dissolving in 2ml 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01M, pH7.4), and making into DOTA chelating agent solution, which is solution A;
111.56mg of zinc nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X 10-2M, zinc nitrate solution, wherein the reaction solution is solution B;
sucking 180 mu l of the solution B, dropwise adding the solution B into the solution A, and reacting for 3 hours at room temperature in a dark place to obtain a reaction solution C;
adding 700 mu l of 20mM glutaraldehyde solution into the solution C dropwise, and reacting overnight at room temperature in a dark place to obtain a reaction solution D;
weighing 20mg of Bovine Serum Albumin (BSA) and dissolving in 4ml of HEPES, and uniformly mixing by magnetic stirring at room temperature to obtain a reaction solution E;
dropwise adding the solution D into the solution E, reacting for 24h at room temperature in a dark place, then adding 150 and 200 mu L sodium borohydride solution (20mg dissolved in 200 mu L pure water), and reacting for 1h at room temperature in a dark place;
the reaction solution is dialyzed for 3 times by a dialysis bag with 8KD, then centrifuged for 5 times by an ultrafiltration centrifugal tube with 30KD at 8000rpm, redissolved by 5ml of HEPES solution with 0.01M and pH7.4, and then subpackaged for preservation at low temperature of-20 ℃.
SDS-PAGE shows that the electrophoretic band of the conjugate has a lagging tailing phenomenon compared with a single protein band, and the molecular weight of the conjugate is larger than that of a single protein, thereby indicating that the conjugation is successful; in addition, the UV scanning showed a change in the maximum absorption wavelength, further indicating the success of the coupling. The binding ratio was calculated to be 20:1 by heavy metal content determination and conjugate protein concentration determination.
Example 2
Weighing 7mg DOTA, dissolving in 2ml 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01M, pH7.4), and making into DOTA chelating agent solution, which is solution A;
111.56mg of zinc nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X 10-2M, zinc nitrate solution, wherein the reaction solution is solution B;
sucking 180 mu l of the solution B, dropwise adding the solution B into the solution A, and reacting for 5 hours at room temperature in a dark place to obtain a reaction solution C;
adding 700 mu l of 20mM glutaraldehyde solution into the solution C dropwise, and reacting overnight at room temperature in a dark place to obtain a reaction solution D;
weighing 20mg of chicken ovalbumin OVA, dissolving in 4ml of HEPES, and uniformly mixing by magnetic stirring at room temperature to obtain a reaction solution E;
dropwise adding the solution D into the solution E, reacting for 24h at room temperature in a dark place, then adding 150-200 mu L diethyl malonate solution (84.6mg dissolved in 200 mu L ethanol), and reacting for 1h at room temperature in a dark place;
the reaction solution is dialyzed for 3 times by a dialysis bag with 8KD, then centrifuged for 5 times by an ultrafiltration centrifugal tube with 30KD at 8000rpm, redissolved by 5ml of HEPES solution with 0.01M and pH7.4, and then subpackaged for preservation at low temperature of-20 ℃.
SDS-PAGE shows that the electrophoretic band of the conjugate has a hysteresis tailing phenomenon compared with a single protein band, the molecular weight of the conjugate is larger than that of a single protein, which indicates the successful coupling, and in addition, ultraviolet scanning shows that the maximum absorption wavelength is changed, which further indicates the successful coupling. The binding ratio is calculated to be 30:1 by measuring the content of heavy metal and the concentration of conjugate protein, and the coupling efficiency is high. The higher binding ratio indicates that the amount of heavy metal ions coupled to one protein molecule is larger, and the coupling rate is higher, such as the binding ratio of 30:1, it means that 30 heavy metal ions are combined on one protein molecule.
Comparative example 5
Weighing 7mg DOTA, dissolving in 2ml 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01M, pH7.4), and making into DOTA chelating agent solution, which is solution A;
111.56mg of zinc nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X 10-2M, zinc nitrate solution, wherein the reaction solution is solution B;
sucking 180 mu l of the solution B, dropwise adding the solution B into the solution A, and reacting for 5 hours at room temperature in a dark place to obtain a reaction solution C;
adding 700 mu l of 20mM glutaraldehyde solution into the solution C dropwise, and reacting overnight at room temperature in a dark place to obtain a reaction solution D;
weighing 20mg of chicken ovalbumin OVA, dissolving in 4ml of HEPES, and uniformly mixing by magnetic stirring at room temperature to obtain a reaction solution E;
dropwise adding the solution D into the solution E, and reacting at room temperature in a dark place for 24 hours;
the reaction solution is dialyzed for 3 times by a dialysis bag with 8KD, then centrifuged for 5 times by an ultrafiltration centrifugal tube with 30KD at 8000rpm, redissolved by 5ml of HEPES solution with 0.01M and pH7.4, and then subpackaged for preservation at low temperature of-20 ℃.
SDS-PAGE shows that the electrophoretic band of the conjugate has a hysteresis tailing phenomenon compared with a single protein band, the molecular weight of the conjugate is larger than that of a single protein, which indicates the successful coupling, and in addition, ultraviolet scanning shows that the maximum absorption wavelength is changed, which further indicates the successful coupling. The binding ratio was calculated to be 8:1 by heavy metal content determination and conjugate protein concentration determination.
Comparative example 6
Weighing 7mg DTPA, dissolving in 2ml 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01M, pH7.4), and preparing into DTPA chelating agent solution A;
111.56mg of zinc nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X 10-2M, zinc nitrate solution, wherein the reaction solution is solution B;
sucking 190 mu l of the solution B, dropwise adding the solution B into the solution A, and reacting for 3 hours at room temperature in a dark place to obtain a reaction solution C;
adding 680 mu l of 20mM glutaraldehyde solution into the solution C dropwise, and reacting overnight at room temperature in a dark place to obtain a reaction solution D;
weighing 20mg ovalbumin OVA, dissolving in 3ml HEPES, and uniformly mixing by magnetic stirring at room temperature to obtain a reaction solution E;
dropwise adding the solution D into the solution E, reacting for 24h at room temperature in a dark place, then adding 150-200 mu L diethyl malonate solution (84.6mg dissolved in 200 mu L ethanol), and reacting for 1h at room temperature in a dark place;
the reaction solution is dialyzed for 3 times by a dialysis bag with 8KD, then centrifuged for 5 times by an ultrafiltration centrifugal tube with 30KD at 8000rpm, redissolved by 5ml of HEPES solution with 0.01M and pH7.4, and then subpackaged for preservation at low temperature of-20 ℃.
SDS-PAGE experiments and ultraviolet scanning show that the coupling is successful, and the binding ratio is calculated to be 7:1 by heavy metal content determination and conjugate protein concentration determination.
Comparative example 7
Weighing 7mg of EDTA, dissolving in 2ml of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01M, pH7.4), and preparing into EDTA chelating agent solution A;
111.56mg of zinc nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X 10-2M, zinc nitrate solution, wherein the reaction solution is solution B;
sucking 180 mu l of the solution B, dropwise adding the solution B into the solution A, and reacting for 3 hours at room temperature in a dark place to obtain a reaction solution C;
690 mu l of 20mM glutaraldehyde solution is added into the solution C drop by drop, and the reaction is carried out overnight at room temperature in a dark place, and the reaction solution is solution D;
weighing 20mg ovalbumin OVA, dissolving in 3ml HEPES, and uniformly mixing by magnetic stirring at room temperature to obtain a reaction solution E;
dropwise adding the solution D into the solution E, reacting for 24h at room temperature in a dark place, then adding 150-200 mu L diethyl malonate solution (84.6mg dissolved in 200 mu L ethanol), and reacting for 1h at room temperature in a dark place;
the reaction solution is dialyzed for 3 times by a dialysis bag with 8KD, then centrifuged for 5 times by an ultrafiltration centrifugal tube with 30KD at 8000rpm, redissolved by 5ml of HEPES solution with 0.01M and pH7.4, and then subpackaged for preservation at low temperature of-20 ℃.
SDS-PAGE experiments and ultraviolet scanning show that the coupling is successful, and the binding ratio is calculated to be 6:1 through heavy metal content determination and conjugate protein concentration determination.
Comparative example 8
Weighing 7mg DOTA, dissolving in 2ml 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01M, pH7.4), and making into DOTA chelating agent solution, which is solution A;
111.56mg of zinc nitrate is weighed and dissolved in 5ml of ultrapure waterIs prepared to 7.5 multiplied by 10-2M, zinc nitrate solution, wherein the reaction solution is solution B;
sucking 180 mu l of the solution B, dropwise adding the solution B into the solution A, and reacting for 5 hours at room temperature in a dark place to obtain a reaction solution C;
adding 700 mu l of 20mM glutaraldehyde solution into the solution C dropwise, and reacting overnight at room temperature in a dark place to obtain a reaction solution D;
weighing 20mg of chicken ovalbumin OVA, dissolving in 4ml of HEPES, and uniformly mixing by magnetic stirring at room temperature to obtain a reaction solution E;
dropwise adding the solution D into the solution E, reacting for 24h at room temperature in a dark place, then adding 150 and 200 mu L sodium borohydride solution (20mg dissolved in 200 mu L pure water), and reacting for 1h at room temperature in a dark place;
the reaction solution is dialyzed for 3 times by a dialysis bag with 8KD, then centrifuged for 5 times by an ultrafiltration centrifugal tube with 30KD at 8000rpm, redissolved by 5ml of HEPES solution with 0.01M and pH7.4, and then subpackaged for preservation at low temperature of-20 ℃.
SDS-PAGE shows that the electrophoretic band of the conjugate has a hysteresis tailing phenomenon compared with a single protein band, the molecular weight of the conjugate is larger than that of a single protein, which indicates the successful coupling, and in addition, ultraviolet scanning shows that the maximum absorption wavelength is changed, which further indicates the successful coupling. The binding ratio was calculated to be 18:1 by heavy metal content determination and conjugate protein concentration determination.
Comparative example 9
Weighing 7mg of NOTA, dissolving in 2ml of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01M, pH7.4) to prepare a NOTA chelating agent solution, wherein the reaction solution is solution A;
111.56mg of zinc nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X 10-2M, zinc nitrate solution, wherein the reaction solution is solution B;
sucking 180 mu l of the solution B, dropwise adding the solution B into the solution A, and reacting for 3 hours at room temperature in a dark place to obtain a reaction solution C;
adding 700 mu l of 20mM glutaraldehyde solution into the solution C dropwise, and reacting overnight at room temperature in a dark place to obtain a reaction solution D;
weighing 20mg of Bovine Serum Albumin (BSA) and dissolving in 4ml of HEPES, and uniformly mixing by magnetic stirring at room temperature to obtain a reaction solution E;
dropwise adding the solution D into the solution E, reacting for 24h at room temperature in a dark place, then adding 150-200 mu L diethyl malonate solution (84.6mg dissolved in 200 mu L ethanol), and reacting for 1h at room temperature in a dark place;
the reaction solution is dialyzed for 3 times by a dialysis bag with 8KD, then centrifuged for 5 times by an ultrafiltration centrifugal tube with 30KD at 8000rpm, redissolved by 5ml of HEPES solution with 0.01M and pH7.4, and then subpackaged for preservation at low temperature of-20 ℃.
SDS-PAGE shows that the electrophoretic band of the conjugate has a hysteresis tailing phenomenon compared with a single protein band, the molecular weight of the conjugate is larger than that of a single protein, which indicates the successful coupling, and in addition, ultraviolet scanning shows that the maximum absorption wavelength is changed, which further indicates the successful coupling. The binding ratio is calculated to be 18:1 by measuring the content of heavy metal and the concentration of conjugate protein, and the coupling efficiency is high. The higher the binding ratio, the greater the amount of heavy metal ions coupled to a protein molecule, the higher the coupling efficiency, the binding ratio is 18:1, 18 heavy metal ions are combined on one protein molecule.
Comparative example 10
Weighing 7mg DOTA, dissolving in 2ml 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01M, pH7.4), and making into DOTA chelating agent solution, which is solution A;
88.66mg of cadmium nitrate is weighed and dissolved in 5ml of ultrapure water to prepare 7.5 multiplied by 10-2Cadmium nitrate solution of M, wherein the reaction solution is solution B;
sucking 180 mu l of the solution B, dropwise adding the solution B into the solution A, and reacting for 5 hours at room temperature in a dark place to obtain a reaction solution C;
adding 700 mu l of 20mM glutaraldehyde solution into the solution C dropwise, and reacting overnight at room temperature in a dark place to obtain a reaction solution D;
weighing 20mg of chicken ovalbumin OVA, dissolving in 4ml of HEPES, and uniformly mixing by magnetic stirring at room temperature to obtain a reaction solution E;
dropwise adding the solution D into the solution E, reacting for 24h at room temperature in a dark place, then adding 150-200 mu L diethyl malonate solution (84.6mg dissolved in 200 mu L ethanol), and reacting for 1h at room temperature in a dark place;
the reaction solution is dialyzed for 3 times by a dialysis bag with 8KD, then centrifuged for 5 times by an ultrafiltration centrifugal tube with 30KD at 8000rpm, redissolved by 5ml of HEPES solution with 0.01M and pH7.4, and then subpackaged for preservation at low temperature of-20 ℃.
SDS-PAGE shows that the electrophoretic band of the conjugate has a hysteresis tailing phenomenon compared with a single protein band, the molecular weight of the conjugate is larger than that of a single protein, which indicates the successful coupling, and in addition, ultraviolet scanning shows that the maximum absorption wavelength is changed, which further indicates the successful coupling. The binding ratio is calculated to be 11:1 by measuring the content of heavy metal and the concentration of conjugate protein, and the coupling efficiency is high. The higher the binding ratio, the more the number of heavy metal ions coupled on one protein molecule is, the higher the coupling ratio is, for example, the binding ratio is 11:1, it means that 11 heavy metal ions are combined on one protein molecule.
Example 3
BALB/C mice were immunized with the antigens prepared in example 1, comparative examples 1, 2, 3, 4 and 9, respectively, and the primary immunization was performed with a complete adjuvant emulsified antigen measured at 250. mu.g/mouse (protein as a unit), followed by a booster immunization every 21 days for 3 times, and the booster immunization was performed with an incomplete adjuvant emulsified at 150. mu.g/mouse, and finally, a final immunization was performed, which was performed by direct intraperitoneal injection of the antigen measured at 300. mu.g/mouse, followed by blood collection for titer determination of multiple antisera, and the test antigens were example 2, comparative examples 5, 6, 7, 8 and 10, respectively, and the results were as follows:
the data show that the binding ratio and the antiserum titer of example 1 are both high, and the binding ratio and the antiserum titer are significantly reduced due to the absence of the reaction step with diethyl malonate in comparative example 1, which indicates that diethyl malonate can not only improve the coupling efficiency, but also significantly improve the antiserum titer. Although comparative examples 2, 3 and 9 were also treated with diethyl malonate, the binding ratio of comparative examples 2, 3 and 9 was lower than that of example 1, which indicates that the chelate formed by OVA, BSA, chelating agents DOTA and zinc ions is more stable and can better display heavy metal antigenic determinants, the antigen immunity characteristic is better, the prepared antibody specificity is stronger, and the chelating agent DOTA is better than NOTA, DTPA and EDTA. Comparative example 4 was treated with sodium borohydride, which has a higher binding ratio and higher antiserum titer than comparative examples 1, 2 and 3, but a lower binding ratio than example 1, indicating that sodium borohydride can increase coupling efficiency and can increase antiserum titer, but the effect is inferior to diethyl malonate. This indicates that the combination of heavy metal zinc and chelating agent DOTA and diethyl malonate is stable and the antiserum titer is high. Example 2 compared to comparative example 10, illustrates the different metal properties for different metals, even though the binding ratio of zinc metal to cadmium and the same chelator are different under the same treatment conditions, and the antiserum titer is different. Obviously, the combination ratio of zinc and DOTA and the antiserum titer are far higher than those of cadmium, which indicates that different heavy metal elements need to be selected in a targeted manner to obtain the best treatment effect.
From a comparison of the two groups of examples of BSA and OVA, the binding ratio to OVA is clearly higher for metallic zinc than for BSA, so that the best effect is obtained with the treatment of metallic zinc with ovalbumin OVA under conditions in which the chelating agent is DOTA and the reducing agent is diethyl malonate.
Claims (1)
1. The preparation method of the heavy metal zinc artificial antigen is characterized by comprising the following steps:
weighing 7mg of 2-S- (4-aminobenzene) -1,4,7, 10-tetraazacyclononane-1, 4,7, 10-tetraacetic acid (DOTA), and dissolving in 2mL of 0.01M HEPES solution (pH7.44-hydroxyethylpiperazine ethanesulfonic acid) to prepare a DOTA chelating agent solution which is solution A;
111.56mg of zinc nitrate was weighed and dissolved in 5mL of ultrapure water to obtain a solution having a concentration of 7.5X 10-2M is zinc nitrate solution, and the solution is B solution;
adding 180 mu L of the solution B into the solution A, and reacting for 3 hours at room temperature in a dark place, wherein the reaction solution is solution C;
adding 700 mu L of 20mM glutaraldehyde solution into the solution C dropwise, and reacting overnight at room temperature in a dark place to obtain a reaction solution D;
weighing 20mg of bovine serum albumin BSA or chicken ovalbumin OVA, dissolving in 4mL of HEPES, and uniformly mixing by magnetic stirring at room temperature to obtain a reaction solution E;
dropwise adding the solution D into the solution E, reacting at room temperature in a dark place for 24 hours, then adding 150 mu L of diethyl malonate solution, and reacting at room temperature in a dark place for 1 hour; the diethyl malonate solution is prepared by dissolving 84.6mg of diethyl malonate in 200 mu L of ethanol;
dialyzing the reaction solution by using a dialysis bag with 8KD for 3 times, centrifuging the reaction solution by using an ultrafiltration centrifugal tube with 30KD at 8000rpm for 5 times, redissolving the reaction solution by using 5mL of HEPES solution with 0.01M and pH7.4, subpackaging the re-dissolved reaction solution, and storing the re-dissolved reaction solution at the low temperature of-20 ℃ to prepare the heavy metal zinc artificial antigen.
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