CN108148130A - A kind of application of the preparation method and NOTA of heavy metal Hg artificial antigen in heavy metal Hg artificial antigen reagent is prepared - Google Patents
A kind of application of the preparation method and NOTA of heavy metal Hg artificial antigen in heavy metal Hg artificial antigen reagent is prepared Download PDFInfo
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- CN108148130A CN108148130A CN201810132707.6A CN201810132707A CN108148130A CN 108148130 A CN108148130 A CN 108148130A CN 201810132707 A CN201810132707 A CN 201810132707A CN 108148130 A CN108148130 A CN 108148130A
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- 229910001385 heavy metal Inorganic materials 0.000 title claims abstract description 48
- 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
- 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
- 239000012279 sodium borohydride Substances 0.000 claims abstract description 14
- 229910000033 sodium borohydride Inorganic materials 0.000 claims abstract description 14
- 210000000991 chicken egg Anatomy 0.000 claims abstract description 8
- 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 156
- 238000006243 chemical reaction Methods 0.000 claims description 71
- 239000003643 water by type Substances 0.000 claims description 24
- 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
- 238000002156 mixing Methods 0.000 claims description 13
- VRJVVIKEWDDYOG-UHFFFAOYSA-N mercury;nitric acid Chemical compound [Hg].O[N+]([O-])=O VRJVVIKEWDDYOG-UHFFFAOYSA-N 0.000 claims description 12
- 150000002823 nitrates Chemical class 0.000 claims description 12
- 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 12
- 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
- 238000012545 processing Methods 0.000 abstract description 5
- BQPIGGFYSBELGY-UHFFFAOYSA-N mercury(2+) Chemical compound [Hg+2] BQPIGGFYSBELGY-UHFFFAOYSA-N 0.000 abstract description 2
- 241000283690 Bos taurus Species 0.000 abstract 1
- 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
- 229910052751 metal Inorganic materials 0.000 description 13
- 239000002184 metal Substances 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- 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
- 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
- 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
- 210000004681 ovum Anatomy 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
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 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
- LTMRRSWNXVJMBA-UHFFFAOYSA-L 2,2-diethylpropanedioate Chemical compound CCC(CC)(C([O-])=O)C([O-])=O LTMRRSWNXVJMBA-UHFFFAOYSA-L 0.000 description 5
- 235000014103 egg white Nutrition 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 229910052753 mercury Inorganic materials 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000013522 chelant Substances 0.000 description 4
- 150000001875 compounds Chemical class 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
- 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
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- IYXGSMUGOJNHAZ-UHFFFAOYSA-N Ethyl malonate Chemical class CCOC(=O)CC(=O)OCC IYXGSMUGOJNHAZ-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
- 239000008280 blood Substances 0.000 description 2
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 2
- XIEPJMXMMWZAAV-UHFFFAOYSA-N cadmium nitrate Chemical class [Cd+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O XIEPJMXMMWZAAV-UHFFFAOYSA-N 0.000 description 2
- AJVCUHHHRPBRHU-UHFFFAOYSA-N cadmium nitric acid Chemical compound [Cd].[N+](=O)(O)[O-] AJVCUHHHRPBRHU-UHFFFAOYSA-N 0.000 description 2
- 230000001808 coupling effect Effects 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000002354 inductively-coupled plasma atomic emission spectroscopy Methods 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 239000000203 mixture Substances 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
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-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
- 210000004369 blood Anatomy 0.000 description 1
- 229910052793 cadmium Inorganic materials 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
- 235000013399 edible fruits 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
- -1 mercury ions Chemical class 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
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of preparation method of heavy metal Hg 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, mercury ion chelating agent complexes and carrier proteins Bovine pure protein B SA or chicken egg white OVA are coupled, prepare artificial antigen, this method increases sodium borohydride processing step on original traditional infrastructure, improves coupling efficiency and antiserum titre.The invention also discloses applications of the NOTA in heavy metal Hg artificial antigen reagent is prepared.
Description
Technical field
The invention belongs to heavy metal ion immunochemical technique fields, and in particular to a kind of system of heavy metal Hg artificial antigen
The application of Preparation Method and NOTA in heavy metal Hg artificial antigen reagent is prepared.
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 very big harm.Therefore, strengthen 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, different heavy metals is due to its attribute difference, and the mode of processing is also not exactly the same, example
Such as, heavy metal Hg is a kind of hypertoxicity element, and micro mercury can generate very big toxicity, therefore, the processing for mercury element
Whether thoroughly and be particularly important completely.
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.
Immunology detection technology is with detection speed is fast, analysis capacity is big, low-cost, the simple portable, user of instrument
Member's technology is of less demanding, easily universal and promote, 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, since heavy metal ion is with 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 preparing 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 again with carrier protein couplet prepare artificial antigen 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 with metal ion by carboxylic acid group and nitrogen atom bonding it is more more stable than complex compound
Metal-EDTA chelates, and bifunctional chelating agent should have there are two act on, 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 the opposite sex 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 Hg artificial antigen
Method.The affinity of antibody height obtained after animal, high specificity, antiserum titre height is immunized in the heavy metal Hg 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 metal mercury ions 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
Antigen and carrier protein couplet add in diethyl malonate, and the heavy metal Hg artificial antigen of generation is made more to stablize, antiserum effect
Valency is high.
To achieve these goals, the present invention uses following technical scheme：
A kind of preparation method of heavy metal Hg 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), 2mL 0.01M, pH7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution are dissolved in, NOTA chelas are made
Mixture solution, this solution are A liquid；
It weighs 121.73mg mercuric nitrates to be dissolved in 5mL ultra-pure waters, is made a concentration of 7.5 × 10-2The nitric acid mercury 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, it is rear to add in 150-200 μ L sodium borohydride solutions
(20mg is dissolved in 200 μ l ultra-pure waters), is protected from light 1h at room temperature；
Reaction solution is first dialysed 3-5 times with the bag filter of 8KD, then centrifuges 3- with the ultra-filtration centrifuge tube 7000-9000rpm of 30KD
5 times, with the 0.01M of 5-10mL, the HEPES solution of pH7.4 redissolves, rear to dispense, and heavy metal Hg people is made in -20 DEG C of Cord bloods
Work antigen.
The identification of artificial antigen：
UV scanning and SDS-PAGE is taken to identify its coupling effect, weight is measured using ICP-MS methods and Bradford methods
Metal ion and protein concentration calculate artificial antigen coupling combine than.
The preparation method of heavy metal Hg 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
Middle addition diethyl malonate after glutaraldehyde method coupling, the heavy metal Hg artificial antigen of preparation are more stablized, and antiserum titre is high.
Description of the drawings
Fig. 1 Hg-NOTA-BSA and Hg-NOTA-OVA UV scanning collection of illustrative plates.
Fig. 2 Hg-NOTA-BSA and Hg-NOTA-OVA electrophoresis patterns.
The structure chart of Fig. 3 NOTA, DTPA and EDTA.
Specific embodiment
In order to which the purpose of the present invention, technical solution, technique effect is 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, are dissolved in 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4), are made
For into 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, it is rear to add in 150-200 μ L sodium borohydride solutions
(20mg is dissolved in 200 μ l ultra-pure waters), is protected from light 1h at room temperature；
Reaction solution is first dialysed 3 times with the bag filter of 8KD, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, is used
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 21 by measuring content of beary metal and conjugate protein concentration calculations incorporated ratio:1, coupling efficiency is high.
With reference to than higher, illustrating that the quantity that heavy metal ion is coupled on a protein molecular is more, coupling efficiency is also higher, with reference to than for
21：1, then it represents that 21 heavy metal ion are combined on a protein molecular.
Comparative example 1
7mg NOTA are weighed, are dissolved in 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4), are made
For into 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, reaction solution is first dialysed 3 times with the bag filter of 8 KD,
It is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD again, with the 0.01M of 5ml, the HEPES solution of pH7.4 redissolves, 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 8:1.
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 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 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, it is rear to add in 150-200 μ L sodium borohydride solutions
(20mg is dissolved in 200 μ l ultra-pure waters), is protected from light 1h at room temperature；
Reaction solution is first dialysed 3 times with the bag filter of 8KD, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, is used
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 the display of UV scanning collection of illustrative plates, is measured and conjugate egg by content of beary metal
White concentration mensuration calculations incorporated ratio is 7:1.
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 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 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, it is rear to add in 150-200 μ L sodium borohydride solutions
(20mg is dissolved in 200 μ l ultra-pure waters), is protected from light 1h at room temperature；
Reaction solution is first dialysed 3 times with the bag filter of 8KD, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, is used
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 the display of UV scanning collection of illustrative plates, is measured and conjugate egg by content of beary metal
White concentration mensuration calculations incorporated ratio is 7:1.
Comparative example 4
7mg NOTA are weighed, are dissolved in 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4), are made
For into 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 is first dialysed 3 times with the bag filter of 8KD, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, is used
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 15:1.
Embodiment 2
7mg NOTA are weighed, are dissolved in 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4), are made
For into 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, it is rear to add in 150-200 μ L sodium borohydride solutions
(20mg is dissolved in 200 μ l ultra-pure waters), is protected from light 1h at room temperature；
Reaction solution is first dialysed 3 times with the bag filter of 8KD, 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.
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 36:1, coupling effect
Rate is high.With reference to than higher, illustrating that the quantity that heavy metal ion is coupled on a protein molecular is more, Conjugate ratio is also higher, such as ties
Composition and division in a proportion is 36：1, then it represents that 36 heavy metal ion are combined on a protein molecular.
Comparative example 5
7mg NOTA are weighed, are dissolved in 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4), are made
For into 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 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 is first dialysed 3 times with the bag filter of 8KD, 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.
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 7:1.
Comparative example 6
7mg DTPA are weighed, are dissolved in 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4), are made
For into DTPA 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 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, it is rear to add in 150-200 μ L sodium borohydride solutions
(20mg is dissolved in 200 μ l ultra-pure waters), is protected from light 1h at room temperature；
Reaction solution is first dialysed 3 times with the bag filter of 8KD, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, is used
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 6:1.
Comparative example 7
7mg EDTA are weighed, are dissolved in 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4), are made
For into EDTA 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 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, it is rear to add in 150-200 μ L sodium borohydride solutions
(20mg is dissolved in 200 μ l ultra-pure waters), is protected from light 1h at room temperature；
Reaction solution is first dialysed 3 times with the bag filter of 8KD, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, is used
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 6:1.
Comparative example 8
7mg NOTA are weighed, are dissolved in 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4), are made
For into 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 is first dialysed 3 times with the bag filter of 8KD, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, is used
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 10:1.
Comparative example 9
7mg NOTA are weighed, are dissolved in 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4), are made
For into 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 to add in 150-200 μ L sodium borohydride solutions
(20mg is dissolved in 200 μ l ultra-pure waters), is protected from light 1h at room temperature；
Reaction solution is first dialysed 3 times with the bag filter of 8KD, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, is used
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 15:1.
Comparative example 10
7mg NOTA are weighed, are dissolved in 2ml 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) solution (0.01M, pH7.4), are made
For into 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 to add in 150-200 μ L sodium borohydride solutions
(20mg is dissolved in 200 μ l ultra-pure waters), is protected from light 1h at room temperature；
Reaction solution is first dialysed 3 times with the bag filter of 8KD, then is centrifuged 5 times with the ultra-filtration centrifuge tube 8000rpm of 30KD, is used
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.
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 strengthened exempting from interval of 21 days
Epidemic disease, is total to booster immunization 3 times, and booster immunization is emulsified using Freund's incomplete adjuvant, and metering is immunized as 150 μ g/ mouse, finally carries out
End is exempted from, and end exempts to carry out in a manner that antigen is directly injected intraperitoneally, and metering is immunized as 300 μ g/ mouse, 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, with reference to 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
Processing is gone, but the combination ratio and antiserum titre of comparative example 2,3,9, less than embodiment 1, this illustrates OVA, BSA and chelating agent
NOTA and the chelate of mercury ion generation are relatively 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 employs sodium borohydride
It is handled, with reference to than, higher than comparative example 1,2 and 3, but being below embodiment 1, this illustrates sodium borohydride with antiserum titre
Coupling efficiency, which can be improved, can simultaneously improve antiserum titre, 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 mercury metal, and the combination ratio of OVA is obvious
Combination ratio higher than BSA, therefore, mercury metal are more suitable for handling using the albumen of the clear class of ovum gallinaceum, and the combination ratio of OVA can be with
Up to 36:1, and antiserum titre is up to 268000, method using the present invention can play mercury metal processing effect well
Fruit.
In addition, from the point of view of comparative example 9 and comparative example 10, cadmium metal is using the chelating agent identical with the present invention and identical
Under treatment conditions, with reference to than that will be far below mercury with potency, so as to illustrate, different heavy metals is needed using different reactions
Condition is handled, for heavy metal Hg, in the case where chelating agent is NOTA and reducing agent is sodium borohydride, using chicken
Egg white albuminoid can obtain optimization process effect.
Claims (2)
1. a kind of preparation method of heavy metal Hg 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）, 2 mL, 0.01 M, pH7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution are dissolved in, it is molten that NOTA chelating agents are made
Liquid, this solution are A liquid；
It weighs 121.73mg mercuric nitrates to be dissolved in 5 mL ultra-pure waters, is made a concentration of 7.5 × 10-2The nitric acid mercury 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 to add in 150-200 μ L sodium borohydride solutions（20
Mg is dissolved in 200 μ l ultra-pure waters）, 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
Secondary, with 0.01 M of 5-10 mL, the HEPES solution of pH7.4 redissolves, rear to dispense, and heavy metal Hg 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 Hg people
Application in work antigenic agents.
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