CN105037525A - Tris(2,3-dibromopropyl) isocyanurate (TBC) artificial immunogen TBC-BSA (bovine serum albumin) and preparation and usage thereof - Google Patents
Tris(2,3-dibromopropyl) isocyanurate (TBC) artificial immunogen TBC-BSA (bovine serum albumin) and preparation and usage thereof Download PDFInfo
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- CN105037525A CN105037525A CN201510483198.8A CN201510483198A CN105037525A CN 105037525 A CN105037525 A CN 105037525A CN 201510483198 A CN201510483198 A CN 201510483198A CN 105037525 A CN105037525 A CN 105037525A
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- tbc
- bsa
- dibromopropyl
- acid ester
- isocyanuric acid
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- 230000002163 immunogen Effects 0.000 title claims abstract description 58
- 238000002360 preparation method Methods 0.000 title claims abstract description 29
- 108091003079 Bovine Serum Albumin Proteins 0.000 title claims abstract description 7
- 229940098773 bovine serum albumin Drugs 0.000 title claims abstract description 7
- NZUPFZNVGSWLQC-UHFFFAOYSA-N 1,3,5-tris(2,3-dibromopropyl)-1,3,5-triazinane-2,4,6-trione Chemical compound BrCC(Br)CN1C(=O)N(CC(Br)CBr)C(=O)N(CC(Br)CBr)C1=O NZUPFZNVGSWLQC-UHFFFAOYSA-N 0.000 title abstract description 6
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 10
- 230000008878 coupling Effects 0.000 claims abstract description 9
- 238000010168 coupling process Methods 0.000 claims abstract description 9
- 238000005859 coupling reaction Methods 0.000 claims abstract description 9
- -1 (2,3-dibromopropyl) isocyanuric acid ester Chemical class 0.000 claims description 69
- 238000006243 chemical reaction Methods 0.000 claims description 45
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 20
- 238000003756 stirring Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 238000005119 centrifugation Methods 0.000 claims description 12
- ZFSLODLOARCGLH-UHFFFAOYSA-N isocyanuric acid Natural products OC1=NC(O)=NC(O)=N1 ZFSLODLOARCGLH-UHFFFAOYSA-N 0.000 claims description 12
- RNFJDJUURJAICM-UHFFFAOYSA-N 2,2,4,4,6,6-hexaphenoxy-1,3,5-triaza-2$l^{5},4$l^{5},6$l^{5}-triphosphacyclohexa-1,3,5-triene Chemical class N=1P(OC=2C=CC=CC=2)(OC=2C=CC=CC=2)=NP(OC=2C=CC=CC=2)(OC=2C=CC=CC=2)=NP=1(OC=1C=CC=CC=1)OC1=CC=CC=C1 RNFJDJUURJAICM-UHFFFAOYSA-N 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 10
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 10
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 150000001875 compounds Chemical class 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 6
- 238000001556 precipitation Methods 0.000 claims description 6
- 229960003280 cupric chloride Drugs 0.000 claims description 5
- 239000002994 raw material Substances 0.000 claims description 5
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims description 4
- 239000012888 bovine serum Substances 0.000 claims description 4
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 4
- 229910052794 bromium Inorganic materials 0.000 claims description 4
- 238000001953 recrystallisation Methods 0.000 claims description 4
- 239000000010 aprotic solvent Substances 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 229920006395 saturated elastomer Polymers 0.000 claims description 3
- FIDRAVVQGKNYQK-UHFFFAOYSA-N 1,2,3,4-tetrahydrotriazine Chemical compound C1NNNC=C1 FIDRAVVQGKNYQK-UHFFFAOYSA-N 0.000 claims description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 2
- 239000012295 chemical reaction liquid Substances 0.000 claims description 2
- SFHYNDMGZXWXBU-LIMNOBDPSA-N 6-amino-2-[[(e)-(3-formylphenyl)methylideneamino]carbamoylamino]-1,3-dioxobenzo[de]isoquinoline-5,8-disulfonic acid Chemical compound O=C1C(C2=3)=CC(S(O)(=O)=O)=CC=3C(N)=C(S(O)(=O)=O)C=C2C(=O)N1NC(=O)N\N=C\C1=CC=CC(C=O)=C1 SFHYNDMGZXWXBU-LIMNOBDPSA-N 0.000 claims 1
- 239000003960 organic solvent Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 241001465754 Metazoa Species 0.000 abstract description 5
- 230000036039 immunity Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 29
- 239000000427 antigen Substances 0.000 description 14
- 102000036639 antigens Human genes 0.000 description 14
- 108091007433 antigens Proteins 0.000 description 14
- 230000015572 biosynthetic process Effects 0.000 description 11
- 238000003786 synthesis reaction Methods 0.000 description 11
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 238000003018 immunoassay Methods 0.000 description 9
- 239000000047 product Substances 0.000 description 8
- 238000012797 qualification Methods 0.000 description 8
- 102000014914 Carrier Proteins Human genes 0.000 description 6
- 108010078791 Carrier Proteins Proteins 0.000 description 6
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 6
- 239000003063 flame retardant Substances 0.000 description 6
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
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- 238000011534 incubation Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 125000001246 bromo group Chemical group Br* 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- MPTQRFCYZCXJFQ-UHFFFAOYSA-L copper(II) chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Cu+2] MPTQRFCYZCXJFQ-UHFFFAOYSA-L 0.000 description 4
- 230000003203 everyday effect Effects 0.000 description 4
- 238000002329 infrared spectrum Methods 0.000 description 4
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- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 4
- 230000001681 protective effect Effects 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000006287 biotinylation Effects 0.000 description 3
- 238000007413 biotinylation Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 230000000875 corresponding effect Effects 0.000 description 3
- 229910000042 hydrogen bromide Inorganic materials 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 125000000467 secondary amino group Chemical group [H]N([*:1])[*:2] 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 125000006416 CBr Chemical group BrC* 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
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- 231100000704 bioconcentration Toxicity 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
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- 230000002860 competitive effect Effects 0.000 description 2
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- 238000011587 new zealand white rabbit Methods 0.000 description 2
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- 239000002957 persistent organic pollutant Substances 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 238000003825 pressing Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 150000003384 small molecules Chemical group 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical compound C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 description 1
- FXXCUGLLZLHZFF-UHFFFAOYSA-N 1-(2,3-dibromopropyl)-1,3,5-triazinane-2,4,6-trione Chemical class BrCC(Br)CN1C(=O)NC(=O)NC1=O FXXCUGLLZLHZFF-UHFFFAOYSA-N 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 229920002430 Fibre-reinforced plastic Polymers 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 239000012901 Milli-Q water Substances 0.000 description 1
- 241000283977 Oryctolagus Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 229920000122 acrylonitrile butadiene styrene Polymers 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
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- 150000001336 alkenes Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
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- 210000004369 blood Anatomy 0.000 description 1
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- 239000000356 contaminant Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
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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
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
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- Chemical & Material Sciences (AREA)
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- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
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- Gastroenterology & Hepatology (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a tris(2,3-dibromopropyl) isocyanurate (TBC) artificial immunogen TBC-BSA (bovine serum albumin) and preparation and usage thereof. A structural formula of the TBC-BSA is shown in the description. The preparation method comprises the steps of coupling TBC hapten with a protein molecule BSA. The immunogen preparation method is simple, good in stability, low in cost and easy for industrialized production. TBC-BSA is prepared to a specific antibody by immune animals, and lays a foundation for establishing a immunity detection method.
Description
Technical field
The present invention relates to a kind of TBC artificial immunogen TBC-BSA and preparation thereof and purposes, specifically a kind of for emerging persistence organic pollutant---TBC artificial immunogen TBC-BSA of three (2,3-dibromopropyl) isocyanuric acid ester immunodetection and its production and use.
Background technology
Brominated flame-retardant (BrominatedFlameRetardant, BFRs), makes its slack-off a kind of auxiliary agent that burns after referring to make polymkeric substance to be not easy to catch fire or catch fire, and is one of output and the maximum organic fire-retardant of consumption in the world at present.Along with multiple brominated flame-retardant is prohibited production and application at majority state and area gradually, new brominated flame-retardant product is gone out to fill a hole in the market by Development and Production gradually, three (2,3-dibromopropyl) isocyanuric acid ester (Tris (2,3-dibromopropyl) isocyanurate, TBC) be exactly a representative of new brominated flame-retardant.TBC is the one in brominated flame-retardant, containing six bromine atoms in molecular structure, also belongs to the agent of triazine fire-retardant.As the new variety of fire retardant, TBC has excellent flame retardant properties, thermostability, the not easily advantage such as photodissociation, low smokiness, is widely used in the goods such as fibre reinforced plastics, agricultural polyurethane film, polyolefine, polyvinyl chloride (PVC), polyphenyl alkene, ABS resin unsaturated polyester, synthetic rubber and synthon.In above-mentioned materials or product, the content of TBC fire retardant is generally at 5-10%w/w.China produces from middle 1980s, use TBC product, and manufacturer concentrates on southeastern coast and the Yangtze valley.
TBC fire retardant also has the deficiency of brominated flame-retardant, and there is certain environmental problem: as discharged toxic gas hydrogen bromide during cracking, and the moisture that hydrogen bromide easily absorbs in air forms Hydrogen bromide, has very strong corrodibility, there is secondary harm.Bioconcentration and air free-radical oxidn transformation period result display TBC have the persistence similar with persistence organic pollutant, long range propagation and can the physicochemical property such as bioconcentration, and are enriched in cerebral tissue by hemato encephalic barrier.Although the production and application of present stage TBC is also not applied to any restriction, it has been included into Canadian environmental threat ecological material (LowerEcologicalConcern) and has screened in register, as the identification of a kind of high priority chemical.
At present, the research about TBC mainly concentrates in the toxicity of the environmental areas such as water body, settling, air and organism, but the detection method aspect research of sample is less.At present to three (2,3-dibromopropyl) detection means of isocyanuric acid ester is mainly the instrument such as gas-chromatography and high performance liquid chromatography detection method, although these methods accurately and reliably, there is very high requirement to the pretreatment process of sample and the professional of operator.Just because of the process of these methods is complicated, consuming time, instrument price is expensive and be not suitable for promoting the use of, be also unfavorable in environmental pollution accident field quick detection.For overcoming these shortcomings, seek the main direction of studying that a kind of quick, easy, sensitive and economical and practical analytical procedure just becomes environmental monitoring field.Therefore, conduct a research and set up effectively, the detection method of brominated flame-retardant and standard reliably, and to be ensured by the regulation of being correlated with, become a very important and urgent job.
The immunoassay that the sixties in 20th century grows up is based on antigen and the specificity of antibody, the analytical technology of reversibility association reaction.Immunoassay has the unrivaled selectivity of conventional physical and chemical analysis technology and high sensitivity, is applicable to very much the analysis of trace components in complex dielectrics.Therefore the high specificity, the advantage such as highly sensitive, method is quick and easy, analysis throughput is large, testing cost is low that have of immunoassay, makes these class methods can meet simply, detects the requirement of persistence organic pollutant fast, delicately.K.B.Mullis and R.K.Saiki etc. of Cetus company of the U.S. in 1985 has invented a kind of specific DNA Amplification Technologies, becomes PolymeraseChainReaction, is called for short PCR, i.e. polymerase chain reaction.Immuno analytical method is combined with round pcr by Sano in 1992 etc., develops fluorescence immunoassay round pcr.Fluorescence immunoassay round pcr has had the high specificity of immunoassay and round pcr highly sensitive concurrently, and (detectability is up to 10
-11~ 10
-12g/L) feature, can be used for contaminant trace species in testing environment.
At present, there is no the relevant report detecting TBC with fluorescence immunoassay round pcr.And to prepare qualified TBC artificial immunogen be the prerequisite that biological immune obtains the anti-TBC antibody of high specific, set up the key of TBC immunoassay analysis especially, this will have important using value and theoretical significance.
Summary of the invention
For defect of the prior art, the object of this invention is to provide a kind of step simple, speed is fast, productive rate high three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen (TBC-BSA) preparation method, obtain specific antibody for immunity and utilize artificial antigen, antibody sets up immunologic detection method and lays the foundation.
The object of the invention is to be achieved through the following technical solutions:
The invention provides a kind of artificial immunogen TBC-BSA of three (2,3-dibromopropyl) isocyanuric acid ester, structural formula is as shown in formula I:
formula I; BSA is bovine serum albumin.
Present invention also offers the preparation method of a kind of artificial immunogen TBC-BSA of three (2,3-dibromopropyl) isocyanuric acid ester, comprise the following steps:
Adopt glutaraldehyde method by TBC haptens
artificial immunogen TBC-BSA is prepared with protein molecule BSA coupling.
Preferably, the haptenic preparation method of described TBC comprises the steps:
A1,2,4,6-tri-(allyloxy)-1,3,5-triazines, water, cupric chloride be dissolved in forming reactions liquid a after toluene, heating reflux reaction, to raw material point disappears, obtains formula II compound;
A2, by gained formula II compound
be dissolved in forming reactions liquid b in methylene dichloride, in reaction solution b, add bromine, keeping back flow reaction, to reacting completely, obtaining TBC haptens.TBC haptenization formal name used at school is called two (2,3-dibromopropyl) isocyanuric acid ester, and molecular formula is C
9h
10br
4n
3o
3, molecular weight 527.78.
Preferably, in described A1, the mol ratio of 2,4,6-tri-(allyloxy)-1,3,5-triazines, water, cupric chloride is 1:1:45 ~ 50, and the temperature of reaction solution a is 95 ~ 100 DEG C; In described A2, the mol ratio of formula II compound and bromine is 1:2 ~ 2.5.Ideally, 2,4, the mol ratio of 6-tri-(allyloxy)-1,3,5-triazines, water, Copper dichloride dihydrate is 1:1:40, but carry out to the right smoothly to react, Copper dichloride dihydrate need excessive but can not exceed desirable consumption 15% return stirring heating when, 2,4,6-tri-(allyloxy)-1,3,5-triazines is in boiling state at 95 DEG C, so 100 DEG C are more in boiling state, and reaction is carried out to the right.
Preferably, described cupric chloride is Copper dichloride dihydrate.
Preferably, after described steps A 2 also comprises and reacting completely, in reaction solution b, saturated Na is added
2s
2o
3solution disappears to red, and extractive reaction liquid is separated, the removal of impurity, dry, the haptenic step of recrystallization TBC.
Preferably, TBC haptens and protein molecule BSA coupling are prepared artificial immunogen TBC-BSA and are specifically comprised the following steps by described employing glutaraldehyde method:
B, TBC haptens is dissolved in aprotic solvent, forms TBC haptens solution; Bovine serum albumin BSA is dissolved in PBS, forms bovine serum albumen solution; By forming reactions liquid c in described TBC haptens solution instillation bovine serum albumen solution;
C, in reaction solution c, add glutaraldehyde, stirring reaction;
After D, reaction terminate, centrifugation supernatant liquor, loads supernatant liquor in dialysis tubing and dialyses, and namely must obtain three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen TBC-BSA after centrifugation precipitation.
The present invention adopts glutaraldehyde method to synthesize three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen, and its reaction equation is as follows:
In above-mentioned reaction equation, intermediate product
the aldehyde radical of one end is combined with the amino of bovine serum albumin BSA one end, and coupling is dewatered, and generates the artificial holoantigen of TBC
Preferably, the mol ratio of described TBC haptens, glutaraldehyde, BSA is 1:30 ~ 50:0.005 ~ 0.05.
Preferably, in step C, described churning time is 18 ~ 24 hours.
For abundant reaction, rule of thumb, when the mol ratio that haptens and glutaraldehyde, BSA react is 1:50:0.005, stirring reaction 18 hours is just passable, reacts just need 24 hours when the mol ratio that haptens and glutaraldehyde, BSA react is 1:30:0.05.
Preferably, in step B, described aprotic solvent is DMF or methyl-sulphoxide; Described is carry out under 0 ~ 4 DEG C of low-temperature magnetic stirs by TBC haptens solution instillation BSA solution.All needs keeps the long-time reaction of protein-active all to need to carry out at low temperatures, experience show 0 ~ 4 DEG C most suitable.
Present invention also offers the purposes of a kind of artificial immunogen TBC-BSA of three (2,3-dibromopropyl) isocyanuric acid ester, it is characterized in that, for the detection of trace brominated flame-retardant three (2,3-dibromopropyl) isocyanuric acid ester.
The sign of artificial immunogen:
The artificial immunogen of preparation is suitably diluted, making its absorbancy between 0.1 ~ 2, take PBS as blank, measures TBC haptens and TBC-BSA light absorption value with ultraviolet spectrophotometer respectively at 200 ~ 500nm, draw uv absorption spectra, and according to following formulae discovery coupling ratio:
In formula: OD
conjugate---conjugate absorbancy; OD
protein---protein light absorption value; OD
hapten---haptens light absorption value; C
hapten---haptens concentration; C
protein---protein concn; M
hapten---hapten molecule amount; M
protein---protein molecular weight.
Of the present invention three (2,3-dibromopropyl) application of isocyanuric acid ester artificial immunogen TBC-BSA, by immune animal preparation anti-three (2,3-dibromopropyl) isocyanuric acid ester specific antibody, can with three (2,3-dibromopropyl) isocyanuric acid ester generation specific immune response immunoglobulin IgG reaction, for the detection of trace brominated flame-retardant three (2,3-dibromopropyl) isocyanuric acid ester in the products such as the environmental samples such as water body, soil, air and electronic product, textile printing and dyeing product, building decoration.
Due to three (2, 3-dibromopropyl) isocyanuric acid ester is small-molecule substance, only there is reactionogenicity and there is no immunogenicity, and this molecule there is no the amino that directly can be combined with protein molecule, the functional groups such as secondary amino group, therefore the present invention is by selection 2, 4, 6-tri-(allyloxy)-1, 3, 5-triazine is raw material, through the TBC haptens of two-step reaction preparation with secondary amino group active group, then with glutaraldehyde method, this is prepared artificial immunogen containing amino haptens and protein molecule coupling, the immunogen of preparation is immune to new zealand white rabbit, obtain anti-three (2 of high specific, 3-dibromopropyl) isocyanuric acid ester polyclonal antibody, this antibody will be used for setting up for three (2, 3-dibromopropyl) immune analysis method of isocyanuric acid ester.
Compared with prior art, the present invention has following beneficial effect:
(1) antigen is practical: the preparation of three (2,3-dibromopropyl) isocyanuric acid ester antigen and antibody preparation have important practical value and realistic meaning.Utilize the method in this patent, successfully prepare three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen TBC-BSA first.This artificial immunogen remains three (2,3-dibromopropyl) structure of isocyanuric acid ester, there is the antigenic determinant for three (2,3-dibromopropyl) isocyanuric acid ester, for preparation specificity is good, the high antibody and set up Immuno-PCR and provide guarantee of tiring.
(2) Antigen Stability is good: three (2,3-dibromopropyl) isocyanuric acid ester artificial antigen of this method synthesis has good stability, can preserve 9 months unchangeability, can preserve 3 years under-20 DEG C of environment under 0-4 DEG C of environment.
(3) antigen technology of preparing is simple and feasible: the whole preparation process of antigen is without the need to special plant and instrument, with low cost, easy commercial scale production.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is three (2,3-dibromopropyl) isocyanuric acid ester hapten infrared spectra;
Fig. 2 is the haptenic nmr spectrum of three (2,3-dibromopropyl) isocyanuric acid esters;
Fig. 3 is three (2,3-dibromopropyl) isocyanuric acid ester hapten, carrier proteins and artificial immunogen uv absorption spectra;
Fig. 4 is the change of the anti-TBC antibody titer that immunize New Zealand White Rabbit produces;
Fig. 5 is the amplification curve that direct competition method detects three (2,3-dibromopropyl) isocyanuric acid ester Immuno-PCR;
Fig. 6 is the standard working curve that direct competitive Immuno-PCR detects three (2,3-dibromopropyl) isocyanuric acid ester;
Wherein: solid line to be log concentration be-2 ~ 1 typical curve; Dotted line to be log concentration be-4 ~ 2 typical curve.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
the embodiment 1 three haptenic synthesis of (2,3-dibromopropyl) isocyanuric acid ester and qualification
The haptenic synthesis of (1) three (2,3-dibromopropyl) isocyanuric acid ester
Raw material 2,4,6-tri-(allyloxy)-1, the mol ratio of 3,5-triazine, water, Copper dichloride dihydrate is 1:1:45, joins in round-bottomed flask after being dissolved in toluene, 95 ~ 100 DEG C of reflux, till thin plate chromatography (developping agent, normal hexane: ethyl acetate=5:1 (v:v)) detects that raw material point disappears, after naturally cooling to room temperature, reaction solution is transferred to underpressure distillation in Rotary Evaporators and removes desolventizing; Use mixed solution (v:v=2:1) recrystallization of methylene dichloride and sherwood oil subsequently, after suction filtration, obtain shallow green powder shape solid; Solid dilute hydrochloric acid repeated washing green solid is until color becomes white, be washed with distilled water to weakly acidic pH again, getting 0.5g after pressing and stoving is dissolved in 20mL methylene dichloride, 120 μ L bromines are dropwise added under heated condition, keep back flow reaction, thin plate chromatography (developping agent, normal hexane: ethyl acetate=5:1 (v:v)) monitoring reaction is until react completely; In reaction system, saturated Na is added after being cooled to room temperature
2s
2o
3solution is until reaction system liquid redness disappears, with 100mL water and 150mL (50mL × 3 time) methylene dichloride re-extract reaction solution, be separated and retain organic phase, and repeatedly wash to remove impurity with saturated nacl aqueous solution, again by anhydrous sodium sulfate drying organic phase to remove unnecessary moisture, crude product ethyl acetate/normal hexane (v:v=1:1) recrystallization obtains acicular crystals and TBC haptens, productive rate 47.1%; Fusing point: 128-131 DEG C.
The haptenic qualification of (2) three (2,3-dibromopropyl) isocyanuric acid ester
Obtained haptens is carried out infrared spectra and nucleus magnetic resonance sign.
Three (2,3-dibromopropyl) the isocyanuric acid ester hapten getting above-mentioned synthesis is a little, and make Infrared spectrum scanning with after pressing potassium bromide troche, as shown in Figure 1, result is as follows: IR (KBr) [ν (cm
-1)]: 1671.98 (C=O stretches), 1430.92 (O-H in-plane bendings); 3320.82 (swollen amine N-H stretches), 1295.93,1315.21 (C-N fragrance is flexible); 2989.12 (C-H stretches), 1648.84 ~ 1432.85 (C=C phenyl ring skeletal vibrations), 910 ~ 665 (C-H out-of-plane bendings), 621.08 (C-Br stretches).There are the group charateristic avsorption bands such as obvious-COOH ,-NH, phenyl ring, C-Br in the infrared spectra of TBC hapten molecule, demonstrate in reaction and successfully secondary amino group is connected in TBC molecular structure.
Get three (2,3-dibromopropyl) isocyanuric acid ester hapten a little, use CDCl
3dissolve, concentration range is roughly 10-20mg/0.5mL.As shown in the nucleus magnetic resonance figure of Fig. 2, detected result is
1hNMR (CDCl
3) δ (ppm): 9.22 (1HN-H), 4.42 (1HR-CH-Br), 3.77,3.88 (2H-CH
2-Br), 1.44,1.28 (2H-CH
2-).
From infrared, nuclear-magnetism characterization result analysis above, synthesized product is target compound.
the synthesis of embodiment 2 three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen
The synthesis of (1) three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen
0.1mmolTBC haptens is dissolved in 1mLN, and in dinethylformamide, 120mgBSA is dissolved in 10mLPBS; Under 0 DEG C of low-temperature magnetic stirs, dropwise by TBC haptens solution instillation BSA solution, drip and terminate to add 120 μ L25% glutaraldehyde in backward reaction system, make the mol ratio of TBC haptens, glutaraldehyde, BSA be 1:30:0.005, continue low temperature and stir 18 hours; After reaction terminates, low-temperature centrifugation separation of supernatant, loads supernatant liquor in dialysis tubing, dialyses 3 days with PBS, changes water every day 3 times, obtains three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen TBC-BSA after centrifugation precipitation.After ultraviolet-visible spectrophotometer qualification, packing in a small amount ,-20 DEG C of freezen protective.
the synthesis of embodiment 3 three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen
The synthesis of (1) three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen
0.1mmolTBC haptens is dissolved in 1mLN, and in dinethylformamide, 240mgBSA is dissolved in 10mLPBS; Under 2 DEG C of low-temperature magnetic stir, dropwise by TBC haptens solution instillation BSA solution, drip and terminate to add 160 μ L25% glutaraldehyde in backward reaction system, make the mol ratio of TBC haptens, glutaraldehyde, BSA be 1:40:0.01, continue low temperature and stir 20 hours; After reaction terminates, low-temperature centrifugation separation of supernatant, loads supernatant liquor in dialysis tubing, dialyses 3 days with PBS, changes water every day 3 times, obtains three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen TBC-BSA after centrifugation precipitation.After ultraviolet-visible spectrophotometer qualification, packing in a small amount ,-20 DEG C of freezen protective.
the synthesis of embodiment 4 three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen
The synthesis of (1) three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen
0.1mmolTBC haptens is dissolved in 1mLN, and in dinethylformamide, 1200mgBSA is dissolved in 10mLPBS; Under 3 DEG C of low-temperature magnetic stir, dropwise by TBC haptens solution instillation BSA solution, drip and terminate to add 200 μ L25% glutaraldehyde in backward reaction system, make the mol ratio of TBC haptens, glutaraldehyde, BSA be 1:50:0.05, continue low temperature and stir 24 hours; After reaction terminates, low-temperature centrifugation separation of supernatant, loads supernatant liquor in dialysis tubing, dialyses 3 days with PBS, changes water every day 3 times, obtains three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen TBC-BSA after centrifugation precipitation.After ultraviolet-visible spectrophotometer qualification, packing in a small amount ,-20 DEG C of freezen protective.
the qualification of embodiment 5 three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen
The method can carrying out artificial immunogen qualification has chromatography, infrared spectroscopy, ultraviolet spectroscopy, SDS-PAGE gel electrophoresis etc.Wherein the most frequently used method is ultraviolet visible spectrometry.In general, as long as the ultraviolet-visible spectrum of artificial immunogen is different with haptenic UV spectrum from year albumen, get final product the success of side light haptens and protein molecule, artificial immunogen synthesizes successfully.Because according to Law of Lambert-Beer, when wavelength one timing, the uv-absorbing of mixture has additive properties.Adopt the long ultraviolet spectrophotometer of all-wave to scan haptens, carrier proteins and artificial immunogen, determine that whether coupling is successful according to the position of charateristic avsorption band.The structure that results of elemental analyses further demonstrates product is consistent with designed route.
From the ultraviolet spectrogram of Fig. 3, TBC haptens has charateristic avsorption band at 281nm place, carrier proteins BSA 227, there is charateristic avsorption band at 278nm place, and artificial immunogen TBC-BSA has charateristic avsorption band at 256 places, and conjugate has the Absorption Characteristics of TBC haptens and carrier proteins.Immunogen contains the charateristic avsorption band of haptens and carrier proteins, and haptens charateristic avsorption band there occurs blue shift, and this may be subject to carrier proteins caused by the impact of about 270nm absorption peak.The appearance of absorption peak blue-shifted phenomenon, illustrate that TBC hapten conjugation is to carrier proteins surface, artificial immunogen is successfully prepared.Calculate the combination of TBC and BSA in TBC-BSA than being 36:1.
the mensuration of embodiment 6 three (2,3-dibromopropyl) isocyanuric acid ester immunogen protein concentration
Immunogen protein concentration is measured according to Coomassie brilliant blue G250 method.Coomassie brilliant blue G250 is red when free, and is cyanic colours after protein bound, and the former obtained the maximum absorption is at 465nm, and the latter is at 595nm place.Take BSA as reference material configuration different concns standardized solution, after reacting with a certain amount of Coomassie brilliant blue G250, measure solution absorbance value at 595nm place respectively.Standardized solution is good in 0-150 μ g/mL scope internal linear relation.In the immunogen solution of dilution certain multiple, add coomassie brilliant blue staining, measure absorbance at phase co-wavelength place, compare with typical curve afterwards, the immunogen protein concentration obtaining preparation is 5.34mg/mL.These immunogens both may be used for immune animal, and the immune response produced by animal obtains the mono-clonal that can be used for doing immunoassay or polyclonal antibody, can use again as Immune competition object.
the experimental verification of embodiment 7 animal immune
Select 2 adult healthy male New Zealand rabbits, the artificial immunogen synthesized is after fully mixing with Freund's complete adjuvant, neck and back point-like injecting immune are carried out to White Rabbit, after 7 booster immunizations, adopt indirect enzyme-linked immunosorbent assay antiserum titre, its antiserum titre reaches 100000 all, and Fig. 4 is shown in change of specifically tiring.Test display cross reaction is all not obvious, illustrates that its specificity is better.
embodiment 8 direct competitive Immuno-PCR detects three (2,3-dibromopropyl) isocyanuric acid ester
In order to strengthen the adsorptivity of PCR pipe, the glutaraldehyde with 0.8% carries out pre-treatment to PCR pipe, and every hole adds 20 μ L glutaraldehyde solutions, and incubation 5 hours at 37 DEG C, then uses milli-Q water 3 times, dry for subsequent use; Be buffered liquid (0.05MpH9.60 carbonate buffer solution) with bag and artificial coating antigen (TBC-OVA) is diluted to proper concn, join and carry out bag quilt in the PCR pipe of glutaraldehyde process, every hole 20 μ L, wraps at 4 DEG C and is spent the night; Outwell coating antigen, every hole adds 200 μ L washingss (0.01MpH7.40PBST) and washs, vibrate after 3 minutes and dry, add 200 μ L confining liquids (phosphate buffer soln containing 3% oralbumin) after washing three times, incubation 1 hour at 37 DEG C; Wash plate three times, anti-for biotinylation TBC polyclonal antibody is diluted to proper concn, every hole adds 10 μ L biotinylated antibodies and 10 μ LTBC small molecules, adds 20 μ LPBS in blank control wells, incubation 30 minutes at 37 DEG C; Wash plate three times and dry, with 0.01MpH7.40PBS, avidin being diluted to suitable multiple, every hole adds 20 μ L, incubation 30 minutes at 37 DEG C; Wash plate three times and dry, with 0.01MpH7.40PBS, biotinylation DNA being diluted to suitable multiple, every hole adds 20 μ L, incubation 30 minutes at 37 DEG C; Use elutriant repeated washing five times after outwelling solution, then use ultrapure water repeated washing five times; Finally add amplimer and fluorescent substance, adopt the amplification system of 20 μ L, every hole adds 10 μ LPCR test kit mixture, and upstream and downstream primer respectively adds 0.25 μ L, ultrapure water 9.5 μ L; When adding pcr amplification reagent, in Fluorescence PCR instrument, carry out (the pcr amplification program: 94 DEG C of denaturation 4min that increases; 94 DEG C of 20s, 55 DEG C of 20s, 72 DEG C of 20s, carry out 30 circulations with this; 72 DEG C extend 3min).The Ct value measured and the amount of determined antigen are inversely proportional to.Antigen and corresponding Ct value according to adding concentration known make typical curve, thus can obtain the concentration of three (2,3-dibromopropyl) isocyanuric acid ester in corresponding testing sample.The typical curve of its amplification curve measured and foundation as shown in Figure 5 and Figure 6.
Biotinylation DNA used by experiment, (Details as Follows for double-stranded DNA: 5 '-Bio-GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA AGCTT-3 ' and 5 '-Bio-AAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCG AATTC-3 ') and corresponding upstream primer, (5 '-GTAAAACGACGGCCAGT-3 ') and downstream primer, (5 '-CAGGAAACAGCTATGAC-3 ') by raw work biotechnology, the preparation synthesis of (Shanghai) limited-liability company.
The preparation method of described artificial coating antigen TBC-OVA is as follows: 0.1mmolTBC haptens is dissolved in 1mLN, and in dinethylformamide, 120mgOVA is dissolved in 10mLPBS; Under 0 DEG C of low-temperature magnetic stirs, dropwise by TBC haptens solution instillation OVA solution, drip and terminate to add 120 μ L25% glutaraldehyde in backward reaction system, make the mol ratio of TBC haptens, glutaraldehyde, OVA be 1:30:0.005, continue low temperature and stir 18 hours; After reaction terminates, low-temperature centrifugation separation of supernatant, loads supernatant liquor in dialysis tubing, dialyses 3 days with PBS, changes water every day 3 times, obtains the artificial coating antigen TBC-OVA of three (2,3-dibromopropyl) isocyanuric acid ester after centrifugation precipitation.After ultraviolet-visible spectrophotometer qualification, packing in a small amount ,-20 DEG C of freezen protective.
When TBC concentration is at 0.1pg/L-100pg/L, linear dependence is better, and linear equation is Ct=1.071lgC+14.3 (R
2=0.974, n=5), detect and be limited to 0.97pg/L.Compared with traditional instrument analytical procedure, sensitivity improves 1000-5000 doubly.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (10)
1. the artificial immunogen TBC-BSA of one kind three (2,3-dibromopropyl) isocyanuric acid ester, is characterized in that, structural formula is such as formula shown in (I):
2. the preparation method of the artificial immunogen TBC-BSA of three (2,3-dibromopropyl) as claimed in claim 1 isocyanuric acid ester, is characterized in that, said method comprising the steps of:
Adopt glutaraldehyde method by TBC haptens
artificial immunogen TBC-BSA is prepared with protein molecule BSA coupling.
3. the preparation method of the artificial immunogen TBC-BSA of three (2,3-dibromopropyl) isocyanuric acid ester as claimed in claim 2, it is characterized in that, the haptenic preparation method of described TBC comprises the steps:
A1,2,4,6-tri-(allyloxy)-1,3,5-triazines, water, cupric chloride be dissolved in forming reactions liquid a after toluene, heating reflux reaction, to raw material point disappears, obtains formula (II) compound
A2, gained formula (II) compound is dissolved in forming reactions liquid b in methylene dichloride, in reaction solution b, adds bromine, keeping back flow reaction, to reacting completely, obtaining TBC haptens.
4. as claimed in claim 3 three (2,3-dibromopropyl) preparation method of artificial immunogen TBC-BSA of isocyanuric acid ester, it is characterized in that, in described steps A 1,2,4,6-tri-(allyloxy)-1, the mol ratio of 3,5-triazine, water, cupric chloride is 1:1:45 ~ 50, and the temperature of reaction solution a is 95 ~ 100 DEG C; In described steps A 2, the mol ratio of formula (II) compound and bromine is 1:2 ~ 2.5.
5. the preparation method of the artificial immunogen TBC-BSA of three (2,3-dibromopropyl) isocyanuric acid ester as claimed in claim 3, is characterized in that, after described steps A 2 also comprises and reacting completely, in reaction solution b, adds saturated Na
2s
2o
3solution disappears to red, and extractive reaction liquid is separated, the removal of impurity, dry, the haptenic step of recrystallization TBC.
6. as claimed in claim 2 three (2,3-dibromopropyl) preparation method of artificial immunogen TBC-BSA of isocyanuric acid ester, it is characterized in that, TBC haptens and protein molecule BSA coupling are prepared artificial immunogen TBC-BSA and are specifically comprised the following steps by described employing glutaraldehyde method:
B, TBC haptens is dissolved in aprotic organic solvent, forms TBC haptens solution; Bovine serum albumin BSA is dissolved in PBS, forms bovine serum albumen solution; By forming reactions liquid c in described TBC haptens solution instillation bovine serum albumen solution;
C, in reaction solution c, add glutaraldehyde, stirring reaction;
After D, reaction terminate, centrifugation, loads supernatant liquor in dialysis tubing and dialyses, and namely must obtain three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen TBC-BSA after centrifugation precipitation.
7. as claimed in claim 6 three (2,3-dibromopropyl) preparation method of artificial immunogen TBC-BSA of isocyanuric acid ester, it is characterized in that, the mol ratio of described TBC haptens, glutaraldehyde, bovine serum albumin BSA is 1:30 ~ 50:0.005 ~ 0.05.
8. the preparation method of the artificial immunogen TBC-BSA of three (2,3-dibromopropyl) isocyanuric acid ester as claimed in claim 6, it is characterized in that, in step C, described churning time is 18 ~ 24 hours.
9. as claimed in claim 6 three (2,3-dibromopropyl) preparation method of artificial immunogen TBC-BSA of isocyanuric acid ester, it is characterized in that, in step B, described aprotic solvent is N, dinethylformamide or methyl-sulphoxide, described is carry out under 0 ~ 4 DEG C of low-temperature magnetic stirs by TBC haptens solution instillation BSA solution.
10. the purposes of the artificial immunogen TBC-BSA of three (2,3-dibromopropyl) as claimed in claim 1 isocyanuric acid ester, is characterized in that, for the detection of trace brominated flame-retardant three (2,3-dibromopropyl) isocyanuric acid ester.
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| CN107501409A (en) * | 2017-09-06 | 2017-12-22 | 杨蕾 | A kind of preparation method of uric acid artificial antigen |
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