CN108409853A - The application of a kind of preparation method and NOTA of heavy metal cadmium artificial antigen in preparing heavy metal cadmium artificial antigen reagent - Google Patents
The application of a kind of preparation method and NOTA of heavy metal cadmium artificial antigen in preparing heavy metal cadmium artificial antigen reagent Download PDFInfo
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- CN108409853A CN108409853A CN201810132944.2A CN201810132944A CN108409853A CN 108409853 A CN108409853 A CN 108409853A CN 201810132944 A CN201810132944 A CN 201810132944A CN 108409853 A CN108409853 A CN 108409853A
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- 229910001385 heavy metal Inorganic materials 0.000 title claims abstract description 47
- JHALWMSZGCVVEM-UHFFFAOYSA-N 2-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]acetic acid Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CC1 JHALWMSZGCVVEM-UHFFFAOYSA-N 0.000 title claims abstract description 30
- 239000000427 antigen Substances 0.000 title claims abstract description 30
- 102000036639 antigens Human genes 0.000 title claims abstract description 30
- 108091007433 antigens Proteins 0.000 title claims abstract description 30
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 title claims abstract description 24
- 229910052793 cadmium Inorganic materials 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 239000003153 chemical reaction reagent Substances 0.000 title abstract description 3
- 239000002738 chelating agent Substances 0.000 claims abstract description 29
- LTMRRSWNXVJMBA-UHFFFAOYSA-L 2,2-diethylpropanedioate Chemical compound CCC(CC)(C([O-])=O)C([O-])=O LTMRRSWNXVJMBA-UHFFFAOYSA-L 0.000 claims abstract description 8
- 210000000991 chicken egg Anatomy 0.000 claims abstract description 7
- 239000002253 acid Substances 0.000 claims abstract description 6
- 150000007513 acids Chemical class 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims description 163
- 238000006243 chemical reaction Methods 0.000 claims description 70
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 17
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 16
- 210000004700 fetal blood Anatomy 0.000 claims description 14
- 238000000108 ultra-filtration Methods 0.000 claims description 14
- 239000003643 water by type Substances 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 13
- XIEPJMXMMWZAAV-UHFFFAOYSA-N cadmium nitrate Chemical class [Cd+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O XIEPJMXMMWZAAV-UHFFFAOYSA-N 0.000 claims description 12
- AJVCUHHHRPBRHU-UHFFFAOYSA-N cadmium nitric acid Chemical compound [Cd].[N+](=O)(O)[O-] AJVCUHHHRPBRHU-UHFFFAOYSA-N 0.000 claims description 12
- 235000019441 ethanol Nutrition 0.000 claims description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 8
- 229940098773 bovine serum albumin Drugs 0.000 claims description 8
- 230000000890 antigenic effect Effects 0.000 claims description 6
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical class NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims 10
- 239000007995 HEPES buffer Substances 0.000 claims 3
- 238000000034 method Methods 0.000 abstract description 11
- 230000008878 coupling Effects 0.000 abstract description 9
- 238000010168 coupling process Methods 0.000 abstract description 9
- 238000005859 coupling reaction Methods 0.000 abstract description 9
- 102000014914 Carrier Proteins Human genes 0.000 abstract description 5
- 108010078791 Carrier Proteins Proteins 0.000 abstract description 5
- WLZRMCYVCSSEQC-UHFFFAOYSA-N cadmium(2+) Chemical compound [Cd+2] WLZRMCYVCSSEQC-UHFFFAOYSA-N 0.000 abstract description 3
- 238000012545 processing Methods 0.000 abstract description 3
- 241000283690 Bos taurus Species 0.000 abstract description 2
- 108010042653 IgA receptor Proteins 0.000 abstract 1
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 abstract 1
- 150000001448 anilines Chemical class 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 19
- 150000002500 ions Chemical class 0.000 description 15
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 13
- 238000013019 agitation Methods 0.000 description 12
- 238000004364 calculation method Methods 0.000 description 12
- 238000001514 detection method Methods 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- 229910052751 metal Inorganic materials 0.000 description 11
- 239000002184 metal Substances 0.000 description 11
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 9
- IYXGSMUGOJNHAZ-UHFFFAOYSA-N Ethyl malonate Chemical class CCOC(=O)CC(=O)OCC IYXGSMUGOJNHAZ-UHFFFAOYSA-N 0.000 description 9
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 7
- 230000001588 bifunctional effect Effects 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 239000013522 chelant Substances 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 235000014103 egg white Nutrition 0.000 description 4
- 239000012279 sodium borohydride Substances 0.000 description 4
- 229910000033 sodium borohydride Inorganic materials 0.000 description 4
- WDLRUFUQRNWCPK-UHFFFAOYSA-N Tetraxetan Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC1 WDLRUFUQRNWCPK-UHFFFAOYSA-N 0.000 description 3
- 238000003968 anodic stripping voltammetry Methods 0.000 description 3
- 238000001479 atomic absorption spectroscopy Methods 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 230000001808 coupling effect Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000002354 inductively-coupled plasma atomic emission spectroscopy Methods 0.000 description 2
- 229910052753 mercury Inorganic materials 0.000 description 2
- VRJVVIKEWDDYOG-UHFFFAOYSA-N mercury;nitric acid Chemical compound [Hg].O[N+]([O-])=O VRJVVIKEWDDYOG-UHFFFAOYSA-N 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 150000002823 nitrates Chemical class 0.000 description 2
- 229910052757 nitrogen Chemical group 0.000 description 2
- 231100000572 poisoning Toxicity 0.000 description 2
- 230000000607 poisoning effect Effects 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- BDDLHHRCDSJVKV-UHFFFAOYSA-N 7028-40-2 Chemical compound CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O BDDLHHRCDSJVKV-UHFFFAOYSA-N 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 241000040710 Chela Species 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- VIZORQUEIQEFRT-UHFFFAOYSA-N Diethyl adipate Chemical compound CCOC(=O)CCCCC(=O)OCC VIZORQUEIQEFRT-UHFFFAOYSA-N 0.000 description 1
- RPNUMPOLZDHAAY-UHFFFAOYSA-N Diethylenetriamine Chemical compound NCCNCCN RPNUMPOLZDHAAY-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 229910052785 arsenic Inorganic materials 0.000 description 1
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 125000002843 carboxylic acid group Chemical group 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229910052804 chromium Inorganic materials 0.000 description 1
- 239000011651 chromium Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 229960001484 edetic acid Drugs 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- 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
-
- 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
Abstract
The invention discloses a kind of preparation methods of heavy metal cadmium artificial antigen, with 2 S (4 aminobenzenes）Isosorbide-5-Nitrae, 7 7-triazacyclononane Isosorbide-5-Nitraes, 7 triacetic acids（p‑NH2Bn NOTA, abbreviation NOTA）For chelating agent, cadmium ion chelating agent complexes and the pure protein B SA of carrier proteins Bovine or chicken egg white OVA are coupled, artificial antigen is prepared, this method increases diethyl malonate processing step on original traditional infrastructure, improves coupling efficiency and antiserum titre.The invention also discloses applications of the NOTA in preparing heavy metal cadmium artificial antigen reagent.
Description
Technical field
The invention belongs to heavy metal ion immunochemical technique fields, and in particular to a kind of system of heavy metal cadmium artificial antigen
The application of Preparation Method and NOTA in preparing heavy metal cadmium artificial antigen reagent.
Background technology
Heavy metal pollution refers mainly to pollution of the pollutants such as lead, cadmium, mercury, nickel, chromium, arsenic, zinc, copper to environment.Heavy metal point
Cloth is extensive, is difficult to degrade, and can enter human body by big gas and water, food chain, have an effect, make with vivo protein and various enzymes
They lose activity, and are enriched in certain organs, if it exceeds the limit that human body is resistant to, can cause human body acute or slow
Property poisoning, have carcinogenic, teratogenesis and mutagenesis, to human body have prodigious harm.Therefore, reinforce heavy metal environment,
Residue detection becomes the important means for ensureing heavy metal safety, and the research of new detecting technique under the new situation in agricultural product and food
Seem particularly urgent with exploitation, still, for different heavy metals due to its attribute difference, the mode of processing is also not exactly the same.
Traditional heavy metal detection method mainly uses atomic absorption spectroscopy (Atomic Absorption
Spectroscopy, AAS), inductive coupling plasma emission spectrum (InductiveLy CoupLedpLasma Atomic
Emission Spectroscopy, ICP-AES), anodic stripping voltammetry (Anodic Stripping VoLtammetry,
ASV), chromatography and various combination detection methods.Although these methods can carry out the heavy metal ion in various environmental samples
Effectively analysis, but large-scale instrument is needed mostly, analysis method is of high cost, and sample is needed by resolution, and analysis time is long, is unsuitable for
The field quick detection of heavy metal, it is difficult to adapt to the spot check of environment and market product, manufacturing enterprise checks oneself and product disengaging
The requirement that mouth speeds passage through customs, will not targetedly be handled according to the property of heavy metal.
Immunology detection technology is fast with detection speed, analysis capacity is big, low-cost, the simple portable, user of instrument
Member's technology is of less demanding, is easy universal and promotes, the advantages that high sensitivity and selectivity are strong, is especially suitable for scene screening and a large amount of
The quick analysis of sample, it has also become the 21 century most competitive detection and analysis technology with challenge.It is opened based on the technology
A series of detection products of hair, such as ELISA detection kit, colloidal gold strip, immunosensor are widely used to now
The quick detection of field sample and a large amount of samples.
The key of heavy metal ion immune detection is the preparation of preventing from heavy metal specific antibody, and prepared by specific antibody
The crucial synthesis for being high-quality heavy metal immunogene again.On the one hand, due to heavy metal ion carry charge, can in animal body
Strong irreversible reaction occurs for biomolecule, and animal poisoning is caused to be reacted；On the other hand, the molecular weight of heavy metal is low, does not have
Have immunogenicity, it and carrier protein couplet need to could be formed complete immunogene, but due to heavy metal ion directly with
Albumen connects, and can make protein denaturation, therefore, bifunctional chelating agent need to be utilized to chelate heavy metal ion, prepare metal-chelant
Compound, by the compound again with prepare comlete antigen after albumen coupling, and then immune animal prepares specific antibody.It takes
That a kind of bifunctional chelating agent of open loop type is chelated, after prepare artificial antigen with carrier protein couplet again and carry out animal immune
With the preparation of antibody, different immuno analytical method and method are established on this basis.
The key for preparing heavy metal immunogene is the selection of bifunctional chelating agent, currently used bifunctional chelating agent master
If ethylenediamine tetra-acetic acid (ethyLenediamie tetraacetic acid, EDTA) or diethylene triamine pentacetic acid (DTPA)
The derivative and other structures with chelating function of (diethyLene triamine penLaacetic acid, DTPA)
Analog, belongs to the chelating agent of chain type open loop structure, and chelate is that have ring by what central ion and multidentate ligand were combined into
The complex of shape structure.For example, EDTA formed by carboxylic acid group and nitrogen atom bonding with metal ion it is more more stable than complex compound
Metal-EDTA chelates, and bifunctional chelating agent should have there are two effect, in addition to can specificity chelating heavy metal ion it
Outside, moreover it is possible to carrier protein couplet, form immunogene, for follow-up immunization animal, prepare antibody.These conventional chelating agents by
Then the compound of the structure of open loop type and straight chain type, heavy metal ion and chelating agent is as antigen recognition site in space structure
Upper fairly simple, characteristic group's feature is not strong, causes prepared antigenic determinant antigenic characteristic not strong, directly affects Gao Te
The preparation efficiency of anisotropic and highly sensitive antibody.
Invention content
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of preparation sides of heavy metal cadmium artificial antigen
Method.The affinity of antibody height obtained after animal, high specificity, antiserum titre height is immunized in the heavy metal cadmium artificial antigen of preparation.
New-type bifunctional chelating agent 2-S- (4- aminobenzenes)-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acids (p-NH2-
Bn-NOTA, abbreviation NOTA) there are three nitrogen closed loop configuration features, heavy metal ion can be preferably combined, and on space structure
Preferably the complex three-dimensional structures of heavy metal cadmium ion can be shown as antigenic determinant, antigen property becomes apparent from, and has
Conducive to preparing affinity higher, the stronger heavy metal monoclonal of specificity and polyclonal antibody, and glutaraldehyde method is by amino half
Diethyl malonate is added in antigen and carrier protein couplet, and the heavy metal cadmium artificial antigen of generation is made more to stablize, antiserum effect
Valence is high.
To achieve the goals above, the present invention uses following technical scheme：
A kind of preparation method of heavy metal cadmium artificial antigen, includes the following steps：
Weigh 5-8mg 2-S- (4- aminobenzenes)-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acids (p-NH2-Bn-
NOTA, abbreviation NOTA), it is dissolved in 2mL 0.01M, pH7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution, NOTA chelas are made
Mixture solution, this solution are A liquid；
It weighs 88.66mg cadmium nitrates to be dissolved in 5mL ultra-pure waters, is made a concentration of 7.5 × 10-2The nitric acid cadmium solution of M,
This solution is B liquid；
The B liquid of 150-200 μ L is added in A liquid, is protected from light 3-5h at room temperature, this reaction solution is C liquid；
The glutaraldehyde solution of 500-800 μ L, 20mM is added dropwise into C liquid, room temperature is protected from light overnight, this reaction solution is
D liquid；
It weighs 20-30mg bovine serum albumin(BSA)s BSA or chicken egg white OVA and is dissolved in 3 mL HEPES, at room temperature magnetic force
It stirs and evenly mixs, this reaction solution is E liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, rear addition 150-200 μ L diethyl malonates are molten
Liquid (84.6mg is dissolved in 2 00 μ L ethyl alcohol), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3-5 times, then centrifuges 3- with the ultra-filtration centrifuge tube 7000-9000rpm of 30KD
It 5 times, is redissolved with the HEPES solution of the 0.01M of 5-10mL, pH7.4, rear to dispense, heavy metal cadmium people is made in -20 DEG C of Cord bloods
Work antigen.
The identification of artificial antigen：
It takes UV scanning and SDS-PAGE to identify its coupling effect, weight is measured using ICP-MS methods and Bradford methods
Metal ion and albumen concentration calculate artificial antigen coupling combine than.
The preparation method of heavy metal cadmium artificial antigen of the present invention selects bifunctional chelating agent NOTA, can preferably combine a huge sum of money
Belong to ion, as antigenic determinant, the heavy metal monoclonal and polyclonal antibody affinity of preparation are high, high specificity, and
The heavy metal cadmium artificial antigen of middle addition diethyl malonate after glutaraldehyde method coupling, preparation is more stablized, and antiserum titre is high.
Description of the drawings
Fig. 1 Cd-NOTA-BSA and Cd-NOTA-OVA ultraviolet scanning atlases.
Fig. 2 Cd-NOTA-BSA and Cd-NOTA-OVA electrophoresis patterns.
The structure chart of Fig. 3 NOTA, DTPA and EDTA.
Specific implementation mode
In order to make the purpose of the present invention, technical solution, technique effect be more clearly understood, by following embodiment to this hair
It is bright to be further elaborated.Description below for specific embodiment is only used for explaining the present invention, does not limit this hair
It is bright.
Embodiment 1
7mg NOTA are weighed, 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4) is dissolved in, are made
For at NOTA chelating agent solutions, this reaction solution is A liquid；
It weighs 88.66mg cadmium nitrates to be dissolved in 5ml ultra-pure waters, is prepared into 7.5 × 10-2The nitric acid cadmium solution of M, this is anti-
It is B liquid to answer liquid；
The B liquid for drawing 180 μ l is added dropwise in A liquid, is protected from light 3h at room temperature, this reaction solution is C liquid；
The glutaraldehyde solution of 700 μ l, 20mM is added dropwise into C liquid, room temperature is protected from light overnight, this reaction solution is D liquid；
It weighs 20mg bovine serum albumin(BSA)s (BSA) and is dissolved in 4ml HEPES, at room temperature magnetic agitation mixing, this reaction solution
For E liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, rear addition 150-200 μ L diethyl malonates are molten
Liquid (84.6mg is dissolved in 2 00 μ L ethyl alcohol), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3 times, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, uses
The HEPES solution of the 0.01M of 5ml, pH7.4 redissolve, rear to dispense, -20 DEG C of Cord bloods.
Show conjugate electrophoretic band than single albumen one with lag trailing phenomenon, point of conjugate through SDS-PAGE
Son amount is bigger than single molecular weight of albumen, illustrates to be coupled successfully, and in addition UV scanning shows that maximum absorption wavelength changes, into one
Step illustrates to be coupled successfully.It is 27 by measuring content of beary metal and conjugate albumen concentration calculations incorporated ratio:1, coupling efficiency is high.
In conjunction with than higher, illustrating that the quantity for being coupled heavy metal ion on a protein molecular is more, coupling efficiency is also higher, in conjunction with than for
27：1, then it represents that 27 heavy metal ion are combined on a protein molecular.
Comparative example 1
7mg NOTA are weighed, 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4) is dissolved in, are made
For at NOTA chelating agent solutions, this reaction solution is A liquid；
It weighs 88.66mg cadmium nitrates to be dissolved in 5ml ultra-pure waters, is prepared into 7.5 × 10-2The nitric acid cadmium solution of M, this is anti-
It is B liquid to answer liquid；
The B liquid for drawing 180 μ l is added dropwise in A liquid, is protected from light 3h at room temperature, this reaction solution is C liquid；
The glutaraldehyde solution of 700 μ l, 20mM is added dropwise into C liquid, room temperature is protected from light overnight, this reaction solution is D liquid；
It weighs 20mg bovine serum albumin(BSA)s (BSA) and is dissolved in 4ml HEPES, at room temperature magnetic agitation mixing, this reaction solution
For E liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, reaction solution is first dialysed 3 times with the bag filter of 8 KD,
It uses the ultra-filtration centrifuge tube 8000rpm of 30KD to centrifuge again 5 times, is redissolved with the HEPES solution of the 0.01M of 5ml, pH7.4, rear point
Dress, -20 DEG C of Cord bloods.
Show conjugate electrophoretic band than single albumen one with lag trailing phenomenon, point of conjugate through SDS-PAGE
Son amount is bigger than single molecular weight of albumen, illustrates to be coupled successfully, and in addition UV scanning shows that maximum absorption wavelength changes, into one
Step illustrates to be coupled successfully.It is measured by content of beary metal and conjugate determination of protein concentration calculations incorporated ratio is 9:1.
Comparative example 2
Weigh 7mg p-NH2- Bn-DTPA (hereinafter referred to as DTPA) is dissolved in 2ml 4- hydroxyethyl piperazineethanesulfonic acids
(HEPES) solution (0.01M, pH7.4) is prepared into DTPA chelating agent solutions, this reaction solution is A liquid；
It weighs 88.66mg cadmium nitrates to be dissolved in 5ml ultra-pure waters, is prepared into 7.5 × 10-2The nitric acid cadmium solution of M, this is anti-
It is B liquid to answer liquid；
The B liquid for drawing 190 μ l is added dropwise in A liquid, is protected from light 3h at room temperature, this reaction solution is C liquid；
The glutaraldehyde solution of 680 μ l, 20mM is added dropwise into C liquid, room temperature is protected from light overnight, this reaction solution is D liquid；
It weighs 20mg bovine serum albumin(BSA)s BSA and is dissolved in 3ml HEPES, at room temperature magnetic agitation mixing, this reaction solution is E
Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, rear addition 150-200 μ L diethyl malonates are molten
Liquid (84.6mg is dissolved in 2 00 μ L ethyl alcohol), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3 times, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, uses
The HEPES solution of the 0.01M of 5ml, pH7.4 redissolve, rear to dispense, -20 DEG C of Cord bloods.
It is coupled successfully through SDS-PAGE experiments and ultraviolet scanning atlas display, passes through conjugate determination of protein concentration and ICP-
MS measure and calculations are combined than being 10:1.
Comparative example 3
Weigh 7mg p-NH2- Bn-EDTA (hereinafter referred to as EDTA) is dissolved in 2ml 4- hydroxyethyl piperazineethanesulfonic acids
(HEPES) solution (0.01M, pH7.4) is prepared into EDTA chelating agent solutions, this reaction solution is A liquid；
It weighs 88.66mg cadmium nitrates to be dissolved in 5ml ultra-pure waters, is prepared into 7.5 × 10-2The nitric acid cadmium solution of M, this is anti-
It is B liquid to answer liquid；
The B liquid for drawing 180 μ l is added dropwise in A liquid, is protected from light 3h at room temperature, this reaction solution is C liquid；
The glutaraldehyde solution of 680 μ l, 20mM is added dropwise into C liquid, room temperature is protected from light overnight, this reaction solution is D liquid；
It weighs 20mg bovine serum albumin(BSA)s BSA and is dissolved in 3ml HEPES, at room temperature magnetic agitation mixing, this reaction solution is E
Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, rear addition 150-200 μ L diethyl malonates are molten
Liquid (84.6mg is dissolved in 2 00 μ L ethyl alcohol), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3 times, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, uses
The HEPES solution of the 0.01M of 5ml, pH7.4 redissolve, rear to dispense, -20 DEG C of Cord bloods.
It is coupled successfully through SDS-PAGE experiments and ultraviolet scanning atlas display, is measured and conjugate egg by content of beary metal
White concentration mensuration calculations incorporated ratio is 9:1.
Comparative example 4
7mg NOTA are weighed, 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4) is dissolved in, are made
For at NOTA chelating agent solutions, this reaction solution is A liquid；
It weighs 88.66mg cadmium nitrates to be dissolved in 5ml ultra-pure waters, is prepared into 7.5 × 10-2The nitric acid cadmium solution of M, this is anti-
It is B liquid to answer liquid；
The B liquid for drawing 180 μ l is added dropwise in A liquid, is protected from light 3h at room temperature, this reaction solution is C liquid；
The glutaraldehyde solution of 700 μ l, 20mM is added dropwise into C liquid, room temperature is protected from light overnight, this reaction solution is D liquid；
It weighs 20mg bovine serum albumin(BSA)s (BSA) and is dissolved in 4ml HEPES, at room temperature magnetic agitation mixing, this reaction solution
For E liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, it is rear that 150-200 μ L sodium borohydride solutions are added
(20mg is dissolved in 2 00 μ L pure water), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3 times, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, uses
The HEPES solution of the 0.01M of 5ml, pH7.4 redissolve, rear to dispense, -20 DEG C of Cord bloods.
Show conjugate electrophoretic band than single albumen one with lag trailing phenomenon, point of conjugate through SDS-PAGE
Son amount is bigger than single molecular weight of albumen, illustrates to be coupled successfully；In addition UV scanning shows that maximum absorption wavelength changes, into one
Step illustrates to be coupled successfully.It is measured by content of beary metal and conjugate determination of protein concentration calculations incorporated ratio is 23:1.
Embodiment 2
7mg NOTA are weighed, 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4) is dissolved in, are made
For at DOTA chelator solution, this reaction solution is A liquid；
It weighs 88.66mg cadmium nitrates to be dissolved in 5ml ultra-pure waters, is prepared into 7.5 × 10-2The nitric acid cadmium solution of M, this is anti-
It is B liquid to answer liquid；
The B liquid for drawing 180 μ l is added dropwise in A liquid, is protected from light 5h at room temperature, this reaction solution is C liquid；
The glutaraldehyde solution of 700 μ l, 20mM is added dropwise into C liquid, room temperature is protected from light overnight, this reaction solution is D liquid；
It weighs 20mg chicken egg whites OVA and is dissolved in 4ml HEPES, at room temperature magnetic agitation mixing, this reaction solution is E
Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, rear addition 150-200 μ L diethyl malonates are molten
Liquid (84.6mg is dissolved in 2 00 μ L ethyl alcohol), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3 times, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, uses
The HEPES solution of the 0.01M of 5ml, pH7.4 redissolve, rear to dispense, -20 DEG C of Cord bloods.
Show conjugate electrophoretic band than single albumen one with lag trailing phenomenon, point of conjugate through SDS-PAGE
Son amount is bigger than single molecular weight of albumen, illustrates to be coupled successfully, and in addition UV scanning shows that maximum absorption wavelength changes, into one
Step illustrates to be coupled successfully.It is measured by content of beary metal and conjugate determination of protein concentration calculations incorporated ratio is 22:1, coupling effect
Rate is high.In conjunction with than higher, illustrating that the quantity for being coupled heavy metal ion on a protein molecular is more, Conjugate ratio is also higher, such as ties
Composition and division in a proportion is 22：1, then it represents that 22 heavy metal ion are combined on a protein molecular.
Comparative example 5
7mg NOTA are weighed, 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4) is dissolved in, are made
For at NOTA chelating agent solutions, this reaction solution is A liquid；
It weighs 88.66mg cadmium nitrates to be dissolved in 5ml ultra-pure waters, is prepared into 7.5 × 10-2The nitric acid cadmium solution of M, this is anti-
It is B liquid to answer liquid；
The B liquid for drawing 180 μ l is added dropwise in A liquid, is protected from light 5h at room temperature, this reaction solution is C liquid；
The glutaraldehyde solution of 700 μ l, 20mM is added dropwise into C liquid, room temperature is protected from light overnight, this reaction solution is D liquid；
It weighs 20mg chicken egg whites OVA and is dissolved in 4ml HEPES, at room temperature magnetic agitation mixing, this reaction solution is E
Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours；
Reaction solution first uses the bag filter of 8KD to dialyse 3 times, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, uses
The HEPES solution of the 0.01M of 5ml, pH7.4 redissolve, rear to dispense, -20 DEG C of Cord bloods.
Show conjugate electrophoretic band than single albumen one with lag trailing phenomenon, point of conjugate through SDS-PAGE
Son amount is bigger than single molecular weight of albumen, illustrates to be coupled successfully, and in addition UV scanning shows that maximum absorption wavelength changes, into one
Step illustrates to be coupled successfully.It is measured by content of beary metal and conjugate determination of protein concentration calculations incorporated ratio is 6:1.
Comparative example 6
7mg DTPA are weighed, 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4) is dissolved in, are made
For at DTPA chelating agent solutions, this reaction solution is A liquid；
It weighs 88.66mg cadmium nitrates to be dissolved in 5ml ultra-pure waters, is prepared into 7.5 × 10-2The nitric acid cadmium solution of M, this is anti-
It is B liquid to answer liquid；
The B liquid for drawing 190 μ l is added dropwise in A liquid, is protected from light 3h at room temperature, this reaction solution is C liquid；
The glutaraldehyde solution of 680 μ l, 20mM is added dropwise into C liquid, room temperature is protected from light overnight, this reaction solution is D liquid；
It weighs 20mg ovalbumins OVA and is dissolved in 3ml HEPES, at room temperature magnetic agitation mixing, this reaction solution is E liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, rear addition 150-200 μ L diethyl malonates are molten
Liquid (84.6mg is dissolved in 2 00 μ L ethyl alcohol), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3 times, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, uses
The HEPES solution of the 0.01M of 5ml, pH7.4 redissolve, rear to dispense, -20 DEG C of Cord bloods.
It is coupled successfully through SDS-PAGE experiments and UV scanning display, is measured by content of beary metal and conjugate albumen is dense
Measure and calculation is spent to combine than being 7:1.
Comparative example 7
7mg EDTA are weighed, 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4) is dissolved in, are made
For at EDTA chelating agent solutions, this reaction solution is A liquid；
It weighs 88.66mg cadmium nitrates to be dissolved in 5ml ultra-pure waters, is prepared into 7.5 × 10-2The nitric acid cadmium solution of M, this is anti-
It is B liquid to answer liquid；
The B liquid for drawing 180 μ l is added dropwise in A liquid, is protected from light 3h at room temperature, this reaction solution is C liquid；
The glutaraldehyde solution of 690 μ l, 20mM is added dropwise into C liquid, room temperature is protected from light overnight, this reaction solution is D liquid；
It weighs 20mg ovalbumins OVA and is dissolved in 3ml HEPES, at room temperature magnetic agitation mixing, this reaction solution is E liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, rear addition 150-200 μ L diethyl malonates are molten
Liquid (84.6mg is dissolved in 2 00 μ L ethyl alcohol), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3 times, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, uses 5ml
0.01M, the HEPES solution of pH7.4 redissolves, rear to dispense, -20 DEG C of Cord bloods.
It is coupled successfully through SDS-PAGE experiments and UV scanning display, is measured by content of beary metal and conjugate albumen is dense
Measure and calculation is spent to combine than being 5:1.
Comparative example 8
7mg NOTA are weighed, 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4) is dissolved in, are made
For at DOTA chelator solution, this reaction solution is A liquid；
It weighs 88.66mg cadmium nitrates to be dissolved in 5ml ultra-pure waters, is prepared into 7.5 × 10-2The nitric acid cadmium solution of M, this is anti-
It is B liquid to answer liquid；
The B liquid for drawing 180 μ l is added dropwise in A liquid, is protected from light 5h at room temperature, this reaction solution is C liquid；
The glutaraldehyde solution of 700 μ l, 20mM is added dropwise into C liquid, room temperature is protected from light overnight, this reaction solution is D liquid；
It weighs 20mg chicken egg whites OVA and is dissolved in 4ml HEPES, at room temperature magnetic agitation mixing, this reaction solution is E
Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, it is rear that 150-200 μ L sodium borohydride solutions are added
(20mg is dissolved in 2 00 μ L pure water), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3 times, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, uses
The HEPES solution of the 0.01M of 5ml, pH7.4 redissolve, rear to dispense, -20 DEG C of Cord bloods.
Show conjugate electrophoretic band than single albumen one with lag trailing phenomenon, point of conjugate through SDS-PAGE
Son amount is bigger than single molecular weight of albumen, illustrates to be coupled successfully, and in addition UV scanning shows that maximum absorption wavelength changes, into one
Step illustrates to be coupled successfully.It is measured by content of beary metal and conjugate determination of protein concentration calculations incorporated ratio is 19:1.
Comparative example 9
7mg NOTA are weighed, 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4) is dissolved in, are made
For at NOTA chelating agent solutions, this reaction solution is A liquid；
It weighs 121.73mg mercuric nitrates to be dissolved in 5ml ultra-pure waters, is prepared into 7.5 × 10-2The nitric acid mercury solution of M, this is anti-
It is B liquid to answer liquid；
The B liquid for drawing 180 μ l is added dropwise in A liquid, is protected from light 3h at room temperature, this reaction solution is C liquid；
The glutaraldehyde solution of 700 μ l, 20mM is added dropwise into C liquid, room temperature is protected from light overnight, this reaction solution is D liquid；
It weighs 20mg bovine serum albumin(BSA)s (BSA) and is dissolved in 4ml HEPES, at room temperature magnetic agitation mixing, this reaction solution
For E liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, rear addition 150-200 μ L diethyl malonates are molten
Liquid (84.6mg is dissolved in 2 00 μ L ethyl alcohol), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3 times, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, uses
The HEPES solution of the 0.01M of 5ml, pH7.4 redissolve, rear to dispense, -20 DEG C of Cord bloods.
It is coupled successfully through SDS-PAGE experiments and UV scanning display, is measured by content of beary metal and conjugate albumen is dense
Measure and calculation is spent to combine than being 21:1.
Comparative example 10
7mg NOTA are weighed, 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4) is dissolved in, are made
For at DOTA chelator solution, this reaction solution is A liquid；
It weighs 121.73mg mercuric nitrates to be dissolved in 5ml ultra-pure waters, is prepared into 7.5 × 10-2The nitric acid mercury solution of M, this is anti-
It is B liquid to answer liquid；
The B liquid for drawing 180 μ l is added dropwise in A liquid, is protected from light 5h at room temperature, this reaction solution is C liquid；
The glutaraldehyde solution of 700 μ l, 20mM is added dropwise into C liquid, room temperature is protected from light overnight, this reaction solution is D liquid；
It weighs 20mg chicken egg whites OVA and is dissolved in 4ml HEPES, at room temperature magnetic agitation mixing, this reaction solution is E
Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, rear addition 150-200 μ L diethyl malonates are molten
Liquid (84.6mg is dissolved in 2 00 μ L ethyl alcohol), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3 times, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, uses
The HEPES solution of the 0.01M of 5ml, pH7.4 redissolve, rear to dispense, -20 DEG C of Cord bloods.
It is coupled successfully through SDS-PAGE experiments and UV scanning display, is measured by content of beary metal and conjugate albumen is dense
Measure and calculation is spent to combine than being 19:1.
Embodiment 3
Antigen prepared by embodiment 1, comparative example 1,2,3,4 and 9 is respectively immunized BALB/C mouse, exempts from for the first time
Epidemic disease emulsifies antigen using Freund's complete adjuvant, measures as 250 μ g/ mouse (using albumen as measurement unit), after reinforced exempting from interval of 21 days
Epidemic disease, total booster immunization 3 times, booster immunization is emulsified using Freund's incomplete adjuvant, and it is 150 μ g/ mouse that metering, which is immunized, is finally carried out
End is exempted from, and end exempts to carry out in such a way that antigen is directly injected intraperitoneally, and it is 300 μ g/ mouse that metering, which is immunized, is taken a blood sample afterwards into how anti-blood
Clear titration, detection antigen is respectively embodiment 2, comparative example 5,6,7,8 and 10, as a result as follows：
Data above shows that the combination ratio of embodiment 1 and antiserum titre are higher, due to lacking in comparative example 1 and third
The reaction step of diethyl adipate, in conjunction with than being significantly reduced with antiserum titre, this illustrates that diethyl malonate not only can be with
Coupling efficiency is improved, antiserum titre can also be significantly improved.Although comparative example 2,3 and 9 also use diethyl malonate into
Go processing, but the combination ratio and antiserum titre of comparative example 2,3,9 are less than embodiment 1, this illustrates OVA, BSA and chelating agent
The chelate that NOTA and cadmium ion generate relatively is stablized, and preferably heavy-metal antigen determinant can be shown, and antigen is exempted from
More preferably, the antibody specificity of preparation is strong for epidemic disease characteristic, and chelating agent NOTA is better than DTPA and EDTA.Comparative example 4 uses sodium borohydride
It is handled, in conjunction with than being higher than comparative example 1,2 and 3 with antiserum titre, but is below embodiment 1, this illustrates sodium borohydride
Coupling efficiency can be improved and antiserum titre can be improved, but effect is inferior to diethyl malonate.
From the point of view of the comparison of two groups of embodiments of BSA and OVA, for cadmium metal, and the combination ratio of BSA is obvious
Combination ratio higher than OVA, therefore, cadmium metal is more suitable for handling using the albumen of cow's serum class.
From the point of view of comparative example 9 and comparative example 10, cadmium metal is with mercury metal in identical chelating agent and identical treatment conditions
Under, it is clear that cadmium is combined than that will be far above mercury with potency.
For cadmium metal, in the case where chelating agent is NOTA and reducing agent is diethyl malonate, using ox blood
Clear albuminoid BSA can obtain optimization process effect.
Claims (2)
1. a kind of preparation method of heavy metal cadmium artificial antigen, which is characterized in that include the following steps：
Weigh 5-8 mg 2-S- (4- aminobenzenes）Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acids（p-NH2- Bn-NOTA, letter
Claim NOTA）, it is dissolved in 2 mL, 0.01 M, pH7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution, it is molten that NOTA chelating agents are made
Liquid, this solution are A liquid；
It weighs 88.66 mg cadmium nitrates to be dissolved in 5 mL ultra-pure waters, is made a concentration of 7.5 × 10-2The nitric acid cadmium solution of M, this is molten
Liquid is B liquid；
The B liquid of 150-200 μ L is added in A liquid, is protected from light 3-5h at room temperature, this reaction solution is C liquid；
500-800 μ L, the glutaraldehyde solution of 20 mM are added dropwise into C liquid, room temperature is protected from light overnight, this reaction solution is D
Liquid；
It weighs 20-30 mg bovine serum albumin(BSA)s BSA or chicken egg white OVA and is dissolved in 3 mL HEPES, magnetic force stirs at room temperature
Mixing is mixed, this reaction solution is E liquid；
D liquid is added dropwise in E liquid, is protected from light 24 h at room temperature, it is rear that 150-200 μ L diethyl malonate solution is added
（84.6 mg are dissolved in 2 00 μ L ethyl alcohol）, it is protected from light 1h at room temperature；
Reaction solution is first dialysed 3-5 times with the bag filter of 8 KD, then centrifuges 3-5 with the ultra-filtration centrifuge tube 7000-9000 rpm of 30 KD
It is secondary, it is redissolved with the HEPES solution of 0.01 M of 5-10 mL, pH7.4, rear to dispense, heavy metal cadmium is made in -20 DEG C of Cord bloods
Artificial antigen.
2.2-S- (4- aminobenzenes）Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acids（Abbreviation NOTA）Preparing heavy metal cadmium people
Application in work antigenic agents.
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