CN107955070B - Improved synthesis method of heavy metal copper artificial antigen and application of NOTA in preparation of heavy metal copper artificial antigen reagent - Google Patents
Improved synthesis method of heavy metal copper artificial antigen and application of NOTA in preparation of heavy metal copper artificial antigen reagent Download PDFInfo
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- 229910001385 heavy metal Inorganic materials 0.000 title claims abstract description 49
- 239000000427 antigen Substances 0.000 title claims abstract description 27
- 102000036639 antigens Human genes 0.000 title claims abstract description 27
- 108091007433 antigens Proteins 0.000 title claims abstract description 27
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 title claims abstract description 14
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- 238000002360 preparation method Methods 0.000 title claims abstract description 12
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- 238000001308 synthesis method Methods 0.000 title description 2
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- 238000006243 chemical reaction Methods 0.000 claims description 60
- XTVVROIMIGLXTD-UHFFFAOYSA-N copper(II) nitrate Chemical compound [Cu+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O XTVVROIMIGLXTD-UHFFFAOYSA-N 0.000 claims description 20
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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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Abstract
The invention provides a synthetic method for preparing a novel bifunctional chelating agent NOTA-based heavy metal copper artificial antigen, which takes 2-S- (4-aminobenzene) -1,4,7 triazacyclononane-1, 4, 7-triacetic acid as a chelating agent, and couples a copper ion chelating agent compound with carrier proteins bovine serum albumin BSA and chicken ovalbumin OVA to prepare the artificial antigen. The invention also provides application of NOTA in preparation of the artificial antigen of heavy metal copper.
Description
Technical Field
The invention relates to a synthesis technology of an artificial antigen aiming at heavy metal copper by adopting a novel bifunctional chelating agent NOTA as a connecting carrier, belongs to the technical field of artificial antigen preparation in immunochemistry technology, and can be used for the subsequent preparation of high-specificity anti-heavy metal copper antibodies and the research of immunoassay technology, and the detection of trace and constant heavy metal copper in environmental samples.
Background
Heavy metal pollution mainly refers to pollutants such as lead, cadmium, mercury, nickel, chromium, arsenic, zinc, copper and the like. 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. According to the first national pollution source census result, the generation amount of five heavy metals such as lead, mercury, cadmium, chromium, arsenic and the like in the national wastewater in 2007 is 2.54 ten thousand tons, and the emission amount is 897.3 tons. The emission of the five heavy metal pollutants in the atmosphere is about 9500 tons. The generation amount of the waste containing the five heavy metals listed in the national hazardous waste record is 1690 ten thousand tons, the pollution situation of the heavy metals in the ecological environment of China is very severe, pollution events occur frequently, statistics shows that 11 serious heavy metal pollution events, such as the pollution accident of Jing chromium in southwestern cloud, the cadmium pollution accident of Longjiang river in Kyowa, the standard exceeding event of blood lead of children and the like, occur nationally in 2011 to 8 months, the influence range of the heavy metal pollution event on the surrounding ecological environment is wide, the harm to the life health of people is large, and the social high attention is attracted. 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 a new situation are particularly urgent.
The traditional heavy metal detection method mostly 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 Diethylene Triamine Pentacetic 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 in open-loop and straight-chain structures and are combined with heavy metal ions, and the complex of the heavy metal ions and the chelating agents as an antigen recognition site is simpler in spatial structure and weak in characteristic functional group characteristics, so that the prepared antigenic determinant has weak antigenic characteristics, and the preparation efficiency of the antibody with high specificity and high sensitivity is directly influenced.
Therefore, it is required to provide a new chelating agent for preparing heavy metal antigen, so that the produced antibody has strong specificity, and is beneficial to preparing heavy metal resistant monoclonal antibody with higher affinity and stronger specificity.
Disclosure of Invention
The novel bifunctional chelating agent NOTA is selected, has the characteristics of a three-nitrogen closed-loop structure, can better combine heavy metal ions structurally, can better show a composite three-dimensional structure of heavy metal copper ions in a spatial structure, is used as an antigenic determinant, and is favorable for preparing heavy metal resistant monoclonal antibodies with higher affinity and stronger specificity.
The invention provides a synthetic method for preparing a novel bifunctional chelating agent NOTA-based heavy metal copper artificial antigen, which adds a step of reducing by using sodium borohydride on the original traditional basis and improves the coupling efficiency.
The technical scheme of the invention is as follows: a synthetic method of a heavy metal copper artificial antigen takes 2-S- (4-aminophenyl) -1,4,7 triazacyclononane-1, 4, 7-triacetic acid as a chelating agent, and a copper ion chelating agent compound is coupled with carrier proteins of Bovine Serum Albumin (BSA) and chicken Ovalbumin (OVA) to prepare the artificial antigen.
The preparation steps are as follows:
(1) synthesis of heavy metal artificial antigen:
weighing 5-8 mg of NOTA, dissolving in 2ml of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01 mol/L, pH7.4) to prepare a NOTA chelating agent solution, wherein the reaction solution is solution A;
70.33mg of copper nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X10-2M, copper nitrate solution, wherein the reaction solution is solution B;
absorbing 170 and 200 mu l of B liquid, dropwise adding the B liquid into the A liquid, and reacting for 3-5h at room temperature in a dark place, wherein the reaction liquid is C liquid;
adding 700 mu l of 600-700 mM glutaraldehyde solution drop by drop into the solution C, and reacting overnight at room temperature in a dark place, wherein the reaction solution is solution D;
weighing 25-30 mg bovine serum albumin BSA or chicken ovalbumin OVA, dissolving in 3ml HEPES, and magnetically stirring at room temperature to mix uniformly, wherein the reaction solution is solution E;
dropwise adding the solution D into the solution E, reacting for 24h at room temperature in a dark place, then dropwise adding 150 and 200 mu l of sodium borohydride solution (20 mg dissolved in 200 mu l of ultrapure water), and reacting for 1h at room temperature in a dark place;
the reaction solution is dialyzed for 3-5 times by using a dialysis bag with 8KD, then centrifuged for 3-5 times by using an ultrafiltration centrifugal tube with 30KD at 7000-8000 rpm, re-dissolved by using 5-10 ml of HEPES solution with 0.01M and pH7.4, and then subpackaged for low-temperature storage at-20 ℃.
(2) Identification of artificial antigen:
the coupling effect is identified by SDS-PAGE, and the coupling and binding ratio of the artificial antigen is determined by ultraviolet scanning and ICP-MS.
Description of the drawings:
FIG. 1: UV scanning spectra of Cu-NOTA-BSA and Cu-NOTA-OVA.
FIG. 2: Cu-NOTA-BSA and Cu-NOTA-OVA electropherograms.
FIG. 3: NOTA, DTPA and EDTA structural diagrams.
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 6mg of NOTA, dissolving the NOTA in 2ml of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01 mol/L, pH7.4) to prepare a NOTA chelating agent solution, wherein the reaction solution is solution A;
70.33mg of copper nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X10-2M, copper nitrate solution, wherein the reaction solution is solution B;
adding 185 mul of the solution B into the solution A dropwise, and reacting for 3 hours at room temperature in a dark place to obtain a reaction solution C;
adding 650 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) A, 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 at room temperature in a dark place for 24 hours, then dropwise adding 200 mu l of sodium borohydride solution (20 mg dissolved in 200 mu l of ultrapure water), and reacting at room temperature in a dark place for 1 hour;
the reaction solution is dialyzed for 3 times by using an 8KD dialysis bag, then centrifuged for 4 times by using a 30KD ultrafiltration centrifuge tube at 7500rpm, redissolved by using 8ml of 0.01M HEPES solution with pH of 7.4, and then subpackaged and stored 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 calculated by the conjugate protein concentration measurement and ICP-MS measurement is 38:1, and the coupling efficiency is high (one chelate is coupled with 38 complexes of heavy metal ions and the chelator).
Example 2
Weighing 7 mg of NOTA, dissolving in 2ml of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01 mol/L, pH7.4) to prepare a NOTA chelating agent solution, wherein the reaction solution is solution A;
70.33mg of copper nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X10-2M, copper nitrate solution, wherein the reaction solution is solution B;
dripping 200 mul of the solution B into the solution A, and reacting for 5 hours at room temperature in a dark place, wherein the reaction solution is solution C;
dropwise adding 700 ul of 20mM glutaraldehyde solution into the solution C, and reacting overnight at room temperature in a dark place to obtain a reaction solution D;
weighing 20mg of 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 at room temperature in a dark place for 24h, then dropwise adding 200 ul of sodium borohydride solution (20 mg dissolved in 200 mul of ultrapure water), and reacting at room temperature in a dark place for 1 h;
the reaction solution is dialyzed for 5 times by a dialysis bag with 8KD, then centrifuged for 5 times by an ultrafiltration centrifugal tube with 30KD at 8000 rpm, redissolved by 6 ml of HEPES solution with 0.01M and pH of 7.4, 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 calculated by the conjugate protein concentration measurement and ICP-MS measurement is 35:1, and the coupling efficiency is high. (higher binding ratio indicates that the amount of heavy metal ions coupled to one protein molecule is larger, and the coupling rate is higher, and if the binding ratio is 35:1, 35 heavy metal ions are coupled to one protein molecule.)
Example 3
Weighing 7 mg of NOTA, dissolving in 2ml of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01 mol/L, pH7.4) to prepare a NOTA chelating agent solution, wherein the reaction solution is solution A;
70.33mg of copper nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X10-2M, copper nitrate solution, wherein the reaction solution is solution B;
dripping 200 mul of the solution B into the solution A, and reacting for 5 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 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, and reacting at room temperature in a dark place for 24 hours to obtain a solution F;
the reaction solution F is dialyzed for 5 times by using a dialysis bag with 8KD, then is centrifuged for 5 times by using an ultrafiltration centrifugal tube with 30KD at 8000 rpm, is redissolved by using 6 ml of HEPES solution with 0.01M and pH7.4, and is subpackaged and stored at the low temperature of-20 ℃.
The success of coupling is shown by SDS-PAGE experiments and ultraviolet scanning, the binding ratio is calculated to be 9:1 by the protein concentration determination of the conjugate and the ICP-MS determination, and the coupling efficiency is general. The sodium borohydride can improve the coupling efficiency.
Example 4
Weighing 6mg DTPA, dissolving in 2ml 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01 mol/L, pH7.4), and preparing into DTPA chelating agent solution A;
70.33mg of copper nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X10-2M, copper nitrate solution, wherein the reaction solution is solution B;
adding 185 mul of the solution B into the solution A dropwise, and reacting for 3 hours at room temperature in a dark place to obtain a reaction solution C;
adding 650 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 at room temperature in a dark place for 24 hours, then dropwise adding 200 mu l of sodium borohydride solution (20 mg dissolved in 200 mu l of ultrapure water), and reacting at room temperature in a dark place for 1 hour;
the reaction solution is dialyzed for 3 times by using an 8KD dialysis bag, then centrifuged for 4 times by using a 30KD ultrafiltration centrifuge tube at 7500rpm, redissolved by using 8ml of 0.01M HEPES solution with pH of 7.4, and then subpackaged and stored at the 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 12:1 by conjugate protein concentration determination and ICP-MS determination.
Example 5
Weighing 6mg DTPA, dissolving in 2ml 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01 mol/L, pH7.4), and preparing into DTPA chelating agent solution A;
70.33mg of copper nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X10-2M, copper nitrate solution, wherein the reaction solution is solution B;
adding 185 mul of the solution B into the solution A dropwise, and reacting for 3 hours at room temperature in a dark place to obtain a reaction solution C;
adding 650 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 at room temperature in a dark place for 24 hours, then dropwise adding 200 mu l of sodium borohydride solution (20 mg dissolved in 200 mu l of ultrapure water), and reacting at room temperature in a dark place for 1 hour;
the reaction solution is dialyzed for 3 times by using an 8KD dialysis bag, then centrifuged for 4 times by using a 30KD ultrafiltration centrifuge tube at 7500rpm, redissolved by using 8ml of 0.01M HEPES solution with pH of 7.4, and then subpackaged and stored at the 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 10:1 by conjugate protein concentration determination and ICP-MS determination.
Example 6
Weighing 6mg of EDTA, dissolving in 2ml of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01 mol/L, pH7.4), and preparing into EDTA chelating agent solution, wherein the reaction solution is solution A;
70.33mg of copper nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X10-2M, copper nitrate solution, wherein the reaction solution is solution B;
adding 185 mul of the solution B into the solution A dropwise, and reacting for 3 hours at room temperature in a dark place to obtain a reaction solution C;
adding 650 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 at room temperature in a dark place for 24 hours, then dropwise adding 200 mu l of sodium borohydride solution (20 mg dissolved in 200 mu l of ultrapure water), and reacting at room temperature in a dark place for 1 hour;
the reaction solution is dialyzed for 3 times by using an 8KD dialysis bag, then centrifuged for 4 times by using a 30KD ultrafiltration centrifuge tube at 7500rpm, redissolved by using 8ml of 0.01M HEPES solution with pH of 7.4, and then subpackaged and stored at the 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.
Example 7
Weighing 6mg of EDTA, dissolving in 2ml of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) solution (0.01 mol/L, pH7.4), and preparing into EDTA chelating agent solution, wherein the reaction solution is solution A;
70.33mg of copper nitrate are weighed and dissolvedIn 5ml of ultrapure water, 7.5X10-2M, copper nitrate solution, wherein the reaction solution is solution B;
adding 185 mul of the solution B into the solution A dropwise, and reacting for 3 hours at room temperature in a dark place to obtain a reaction solution C;
adding 650 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 at room temperature in a dark place for 24 hours, then dropwise adding 200 mu l of sodium borohydride solution (20 mg dissolved in 200 mu l of ultrapure water), and reacting at room temperature in a dark place for 1 hour;
the reaction solution is dialyzed for 3 times by using an 8KD dialysis bag, then centrifuged for 4 times by using a 30KD ultrafiltration centrifuge tube at 7500rpm, redissolved by using 8ml of 0.01M HEPES solution with pH of 7.4, and then subpackaged and stored at the 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 10:1 by conjugate protein concentration determination and ICP-MS determination.
EXAMPLE 8
BALB/C mice were immunized with the antigens prepared in examples 1, 3, 4 and 6, respectively, with a complete adjuvant emulsified antigen for the primary immunization, measured as 200. mu.g/mouse, followed by a booster immunization 3 times at intervals of 21 days, with an incomplete adjuvant emulsified booster immunization, measured as 75. mu.g/mouse, and finally with a final immunization, performed by direct intraperitoneal injection of the antigen, measured as 100. mu.g/mouse, and then blood was collected for the measurement of the titer of the multiple antisera, with the following results:
| immunity antigen | Binding ratio | Detection of antigens | Binding ratio | Antiserum potency |
| Cu-NOTA-BSA example 1 | 38：1 | Cu-NOTA-OVA example 2 | 35：1 | 512000 |
| Cu-NOTA-BSA example 3 | 9：1 | Cu-NOTA-OVA example 2 | 9：1 | 128000 |
| Cu-DTPA-BSA example 4 | 12：1 | Cu-DTPA-OVA example 5 | 10：1 | 128000 |
| Cu-EDTA-BSA example 6 | 11：1 | Cu-EDTA-OVA example 7 | 10：1 | 128000 |
The data show that the titer of the example 1 is the highest, and the antiserum titer of the example 3 is remarkably reduced due to the lack of the reaction of the sodium borohydride solution, which indicates that the sodium borohydride solution not only can improve the coupling efficiency, but also can remarkably improve the titer of the antiserum. Similarly, the antiserum titers in examples 4 and 6 show that the affinities of different chelates for the same antibody are significantly reduced, and although sodium borohydride solution is also used for treatment, the chelators adopted in examples 4 and 6 belong to an open-loop direct chain type binding mode, so that the formation of characteristic epitopes is influenced, heavy metal epitopes cannot be better represented, the antigen specificity and the binding degree are not enough, the preparation of specific antibodies is not as good as those in example 1, and the superiority of the NOTA chelators is reflected.
Claims (2)
1. An improved method for synthesizing heavy metal copper artificial antigen is characterized in that 2-S- (4-aminophenyl) -1,4,7 triazacyclononane-1, 4, 7-triacetic acid is taken as a chelating agent, copper ions are coupled with carrier protein bovine serum albumin BSA, and the coupling method is as follows:
weighing 6mg of 2-S- (4-aminobenzene) -1,4,7 triazacyclononane-1, 4, 7-triacetic acid, dissolving in 2ml of 0.01 mol/L4-hydroxyethyl piperazine ethanesulfonic acid solution with pH of 7.4, and preparing a chelating agent solution, wherein the reaction solution is solution A;
70.33mg of copper nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X 10-2M, copper nitrate solution, wherein the reaction solution is solution B;
adding 185 mul of the solution B into the solution A dropwise, and reacting for 3 hours at room temperature in a dark place to obtain a reaction solution C;
adding 650 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, dissolving in 3ml of 4-hydroxyethyl piperazine ethanesulfonic acid solution, and magnetically stirring and uniformly mixing 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 dropwise adding 200 mu l of sodium borohydride solution, and reacting at room temperature in a dark place for 1 hour; the preparation method of the sodium borohydride solution comprises the steps of dissolving 20mg of sodium borohydride in 200 mul of ultrapure water;
dialyzing the reaction solution for 3 times by using an 8KD dialysis bag, centrifuging for 4 times by using a 30KD ultrafiltration centrifuge tube 7500rpm, redissolving by using 8ml of 0.01 mol/L4-hydroxyethyl piperazine ethanesulfonic acid solution with pH of 7.4, and subpackaging at-20 ℃ for low-temperature storage.
The application of 2-S- (4-aminophenyl) -1,4,7 triazacyclononane-1, 4, 7-triacetic acid in preparing heavy metal copper artificial antigen reagent is characterized in that 2-S- (4-aminophenyl) -1,4,7 triazacyclononane-1, 4, 7-triacetic acid is taken as a chelating agent, copper ions are coupled with carrier protein bovine serum albumin BSA, and the coupling method is as follows:
weighing 6mg of 2-S- (4-aminobenzene) -1,4,7 triazacyclononane-1, 4, 7-triacetic acid, dissolving in 2ml of 0.01 mol/L4-hydroxyethyl piperazine ethanesulfonic acid solution with pH of 7.4, and preparing a chelating agent solution, wherein the reaction solution is solution A;
70.33mg of copper nitrate was weighed and dissolved in 5ml of ultrapure water to prepare 7.5X 10-2M, copper nitrate solution, wherein the reaction solution is solution B;
adding 185 mul of the solution B into the solution A dropwise, and reacting for 3 hours at room temperature in a dark place to obtain a reaction solution C;
adding 650 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, dissolving in 3ml of 4-hydroxyethyl piperazine ethanesulfonic acid solution, and magnetically stirring and uniformly mixing at room temperature to obtain a reaction solution E;
dropwise adding the solution D into the solution E, reacting for 24 hours at room temperature in a dark place, then dropwise adding 200 mu l of sodium borohydride solution, and reacting for 1 hour at room temperature in a dark place, wherein the sodium borohydride solution is prepared by dissolving 20mg of sodium borohydride in 200 mu l of ultrapure water;
dialyzing the reaction solution for 3 times by using an 8KD dialysis bag, centrifuging for 4 times by using a 30KD ultrafiltration centrifuge tube 7500rpm, redissolving by using 8ml of 0.01 mol/L4-hydroxyethyl piperazine ethanesulfonic acid solution with pH of 7.4, and subpackaging at-20 ℃ for low-temperature storage.
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| [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] targeting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues;Prasanphanich AF 等;《Proceedings of the National Academy of Sciences of the United States of America》;20070712;第104卷(第30期);第12462-12467页 * |
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