CN109425734A - A kind of complex immunity affinity column of bisphenol-A and the like and its preparation and application - Google Patents
A kind of complex immunity affinity column of bisphenol-A and the like and its preparation and application Download PDFInfo
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- CN109425734A CN109425734A CN201710770548.8A CN201710770548A CN109425734A CN 109425734 A CN109425734 A CN 109425734A CN 201710770548 A CN201710770548 A CN 201710770548A CN 109425734 A CN109425734 A CN 109425734A
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- bisphenol
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- IISBACLAFKSPIT-UHFFFAOYSA-N bisphenol A Chemical compound C=1C=C(O)C=CC=1C(C)(C)C1=CC=C(O)C=C1 IISBACLAFKSPIT-UHFFFAOYSA-N 0.000 title claims abstract description 156
- 229940106691 bisphenol a Drugs 0.000 title claims abstract description 73
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 230000036039 immunity Effects 0.000 title description 6
- 239000000427 antigen Substances 0.000 claims abstract description 30
- 102000036639 antigens Human genes 0.000 claims abstract description 30
- 108091007433 antigens Proteins 0.000 claims abstract description 30
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 12
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 12
- 235000013305 food Nutrition 0.000 claims abstract description 12
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 57
- 239000000243 solution Substances 0.000 claims description 42
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 30
- 238000006243 chemical reaction Methods 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 20
- 239000000463 material Substances 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 15
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 239000000741 silica gel Substances 0.000 claims description 12
- 229910002027 silica gel Inorganic materials 0.000 claims description 12
- 229960001866 silicon dioxide Drugs 0.000 claims description 12
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 10
- PXKLMJQFEQBVLD-UHFFFAOYSA-N bisphenol F Chemical compound C1=CC(O)=CC=C1CC1=CC=C(O)C=C1 PXKLMJQFEQBVLD-UHFFFAOYSA-N 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 10
- 241001465754 Metazoa Species 0.000 claims description 8
- 108010058846 Ovalbumin Proteins 0.000 claims description 8
- 229940092253 ovalbumin Drugs 0.000 claims description 8
- 210000002966 serum Anatomy 0.000 claims description 8
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 7
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 238000011068 loading method Methods 0.000 claims description 6
- BXGTVNLGPMZLAZ-UHFFFAOYSA-N n'-ethylmethanediimine;hydrochloride Chemical compound Cl.CCN=C=N BXGTVNLGPMZLAZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000012074 organic phase Substances 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 5
- 238000000502 dialysis Methods 0.000 claims description 5
- VPWNQTHUCYMVMZ-UHFFFAOYSA-N 4,4'-sulfonyldiphenol Chemical compound C1=CC(O)=CC=C1S(=O)(=O)C1=CC=C(O)C=C1 VPWNQTHUCYMVMZ-UHFFFAOYSA-N 0.000 claims description 4
- 229930185605 Bisphenol Natural products 0.000 claims description 4
- 238000013019 agitation Methods 0.000 claims description 4
- 235000013405 beer Nutrition 0.000 claims description 4
- ZFVMWEVVKGLCIJ-UHFFFAOYSA-N bisphenol AF Chemical compound C1=CC(O)=CC=C1C(C(F)(F)F)(C(F)(F)F)C1=CC=C(O)C=C1 ZFVMWEVVKGLCIJ-UHFFFAOYSA-N 0.000 claims description 4
- 235000013336 milk Nutrition 0.000 claims description 4
- 239000008267 milk Substances 0.000 claims description 4
- 210000004080 milk Anatomy 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- GRHQDJDRGZFIPO-UHFFFAOYSA-N 4-bromobutanoic acid Chemical compound OC(=O)CCCBr GRHQDJDRGZFIPO-UHFFFAOYSA-N 0.000 claims description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 240000007124 Brassica oleracea Species 0.000 claims description 3
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 claims description 3
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- 235000007164 Oryza sativa Nutrition 0.000 claims description 3
- 229920002684 Sepharose Polymers 0.000 claims description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- 238000001261 affinity purification Methods 0.000 claims description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims description 3
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- 235000009566 rice Nutrition 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N sodium azide Substances [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 2
- 244000141359 Malus pumila Species 0.000 claims description 2
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- 235000015277 pork Nutrition 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims 2
- LCPVQAHEFVXVKT-UHFFFAOYSA-N 2-(2,4-difluorophenoxy)pyridin-3-amine Chemical compound NC1=CC=CN=C1OC1=CC=C(F)C=C1F LCPVQAHEFVXVKT-UHFFFAOYSA-N 0.000 claims 1
- 108010088751 Albumins Proteins 0.000 claims 1
- 102000009027 Albumins Human genes 0.000 claims 1
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- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 claims 1
- CHQMHPLRPQMAMX-UHFFFAOYSA-L sodium persulfate Substances [Na+].[Na+].[O-]S(=O)(=O)OOS([O-])(=O)=O CHQMHPLRPQMAMX-UHFFFAOYSA-L 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 7
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- 210000004027 cell Anatomy 0.000 description 7
- 230000003053 immunization Effects 0.000 description 7
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses the immune affinity columns and its preparation method and application that a kind of bisphenol-A and the like purifies simultaneously, are by immune affinity column made of bisphenol-A shown in any one in Formulas I (1 ~ 5) and the like haptens and the resulting antigen of carrier protein couplet, immune gained Antibody preparation the present invention is based on provided bisphenol-A and the like artificial antigen.The immune affinity column that bisphenol-A provided by the present invention and the like purifies simultaneously only need to be extracted once, so that it may so that a variety of components to be measured is obtained high purification, can be completely eliminated the matrix effect of sample substantially, greatly improve purification efficiency.And combined with LC-MS/MS detection technique, it is used for the exposure assessment of food incretion interferent.
Description
Technical field
The invention belongs to endocrine disruptors purifications, detection field in food, are related to a kind of while purifying bisphenol-A and its class
Like the complex immunity affinity column and its preparation method and application of object.
Background technique
In recent years, the case of congenital malformation, hypoplasia and dementia that fetus and baby occur is commonplace.Much grind
Studying carefully the main reason for showing to cause these phenomenons is interference of the endocrine system of the mankind by chemical substances some in environment,
Cause the unbalance of internal natural hormone level.These chemical substances that can interfere with hormone in vivo balance are typically all the mankind in life
The waste for producing and generating and discharge in living, because of referred to herein as environment incretion interferent (Environmental endocrine
Disrupting chemicals, EDCs).Bisphenol-A (Bisphenol A, BPA) is typical environment incretion interferent, because
For its chemical property stabilization, high temperature resistant, it is heat-resisting, there is good ductility, and simple production process is low in cost, wide
It is general to be used for packaging material for food, food storage containers and plastic plasticizer etc..Because of its increasingly specific estrogenic activity, toxicity
The pollution condition learned effect and be seriously, and limited and use in various fields by many countries.Based on such situation, Hen Duoshang
The substitute of BPA is found one after another by family, wherein the BPA class such as bisphenol AF, bisphenol S, Bisphenol F, bisphenol b (BPAF, BPS, BPF and BPB)
It is most like object application.There is their presence in plastic products, thermo-sensitive material, rubber product, textile.Due to BPS, BPF,
BPB and BPAF chemical property and structure are similar to BPA, they can simulate estrogen, in conjunction with estrogen receptor, induce a system
Column estrogen effect.In addition, they show the poisonous effect of varying strength in terms of reproduction, development, heredity and nerve.
Therefore, Accurate Determining is carried out for carrying out exposure assessment to the residual level of these incretion interferents in food
It is very important.Bisphenol-A and the like there are many analysis test method, mainly have at present gas chromatography it is gentle-matter connection
Usage, liquid chromatography and liquid-mass chromatography method etc..Sample-pretreating method mainly includes that liquid-liquid extraction, Solid Phase Extraction, matrix are solid
Phase dispersion extraction and accelerated solvent extraction etc..These methods are there are poor selectivity, organic solvent usage amount is big, complicated for operation and not
With the disadvantages of poor reproducibility, therefore, it is necessary to establish a kind of good selectivity, high sensitivity and easy to operate, quick sample between matrix
Product pre-treating method.
Summary of the invention
The object of the present invention is to provide the complex immunity affinity columns and preparation method thereof for purifying bisphenol-A and the like simultaneously
And purposes.
The complex immunity affinity column of bisphenol-A provided by the present invention and the like, half including bisphenol-A and the like
Antigen.The haptens can be for such as any one of following formula I (1) into (5):
Formulas I (1)
Formulas I (2)
Formulas I (3)
Formulas I (4)
Formulas I (5)
The Formulas I (1) provided by the present invention for preparing specifically may include following steps to the method for (5):
Will in any one raw material in bisphenol-A, bisphenol AF, bisphenol S, Bisphenol F, bisphenol b be added dimethylformamide (DMF),
NaH, the raw material, DMF, NaH proportion be 1000mg:7ml:1400mg, required reaction temperature be ice bath react, there is no gas
Bubble removes ice bath when emerging, then 40 min of temperature reaction obtain reaction solution；The addition 200mg 4- bromobutyrate in reaction solution, 40
It is reacted under the conditions of DEG C oil bath, 25ml ethyl acetate extracts 2 times, and combined ethyl acetate phase is evaporated, and 12mL ethyl acetate is added will be residual
Object is stayed to dissolve, the silica gel for weighing 2 times of weight is added thereto, and revolving is dry, prepares silicagel column loading.
Silica gel sample that reaction solution is mixed with silica gel is loaded using dry sample sample-adding method, with petroleum ether: acetic acid second
The elution of ester system, collects leacheate, collects product revolving.
Product is dissolved with 7 mL methanol, and 2 M NaOH solutions are added by the amount of 2 times of substances, heat at 40 DEG C, hydrolysis is complete
Afterwards, pH to 2 is adjusted with the HCl of 6M.Reaction solution is extracted twice with 50mL ethyl acetate, collects organic phase, anhydrous slufuric acid is added
Sodium (anhydrous sodium sulfate is added by the amount of 10% organic phase volume), 1 h of magnetic agitation after addition.Filtering, revolving are dry.It obtains described
Haptens.
The antigen that resulting bisphenol-A and the like is constructed on the basis of the haptens also belongs to protection of the invention
Range.
The bisphenol-A and the like antigen, for will in described bisphenol-A and the like haptens Formulas I (1-5) it is any
A kind of and resulting antigen of carrier protein couplet.In one embodiment of the invention, the carrier protein is specially cow's serum
Albumin (BSA) or ovalbumin (OVA).
The preparation method of the bisphenol-A and the like antigen also belongs to protection scope of the present invention.
The preparation method of the bisphenol-A and the like antigen, specifically may include following steps: by the bisphenol-A and its
Analog haptens (any one in Formulas I (1-5)) and carrier protein are coupled by amido bond, obtain the bisphenol-A and its class
Like object antigen.
In the present invention, described bisphenol-A and the like antigen is specifically to obtain according to the method preparation included the following steps
:
(1) bisphenol-A and the like haptens (any one in Formulas I (1-5)) is dissolved in dimethylformamide (DMF)
In, 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and n-hydroxysuccinimide is then added
(NHS), 20-25 DEG C of magnetic agitation reacts 2-3h, obtains solution I；
Wherein, described bisphenol-A and the like haptens (any one in Formulas I (1-5)), the dimethylformamide (DMF),
1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC), the n-hydroxysuccinimide (NHS)
Proportion is 9.55mg:1.5mL:21.5mg:13mg.
(2) carrier protein is placed in 0.1M sodium bicarbonate buffer liquid, 200rpm stirs 10min, sufficiently dissolves, obtains
To solution II；The proportion of the carrier protein and the 0.1M sodium bicarbonate buffer liquid is 33.6-50mg:3.5mL；
Wherein, if the carrier protein is bovine serum albumin(BSA) (BSA), the bovine serum albumin(BSA) (BSA) and the 0.1M
The proportion of sodium bicarbonate buffer liquid is 50mg:3.5mL；If the carrier protein is ovalbumin (OVA), the ovalbumin
It (OVA) is 33.6mg:3.5mL with the proportion of the 0.1M carbonic acid buffer；
(3) solution I and the solution II are mixed, specially under the conditions of 0-4 DEG C, under 1000rpm stirring, by solution I
It is added dropwise in the solution II, 500rpm is stirred to react for 24 hours, obtains solution III；
(4) phosphate buffer (0.01M PBS, pH7.2) is used, in 4 DEG C to the solution III stirring dialysis 3 days, obtained described
Bisphenol-A and the like antigen.
Protection scope of the present invention is also belonged to using antibody prepared by the bisphenol-A and the like antigen.The antibody
It can be polyclonal antibody, monoclonal antibody or antiserum.
The bisphenol-A and the like haptens (any one in Formulas I (1-5)) or the bisphenol-A and the like are anti-
Application of the original in purification bisphenol-A and the like also belongs to protection scope of the present invention.
The preparation for the immune affinity column that bisphenol-A and the like purifies simultaneously includes the following steps:
(1) column material is mixed up and down, and the wet column material of Sepharose 4B for taking 4ml NHS to activate is added in reaction tube, to natural subsidence
Volume is 3ml bed volume afterwards, and gravity column acts on trickle, washed off in column bed with the pre-cooling 1mM HCl of 10 times of bed volumes
Organic solvent, add the coupling buffer of 2 times of column volumes into column material, siphon away liquid with syringe back suction rapidly；
(2) column tube bottom end is sealed, antibody is formed to antibody-solutions in 0.2M NaHCO3buffer and is added in column material, covered
Good upper cover mixes column material and solution up and down；
(3) 3 times of column beds are washed with 0.1M Tris-HCl pH8.0 respectively until trickle to dripless outflow after reaction
Volume, then change 0.1 mol/L acetic acid/sodium acetate (containing 0.5 mol/L NaCl), pH4.0 wash 3 times of bed volumes, then with 20 mM
PBS washes 5 times of bed volumes of column；
(4) plus containing 0.05% NaN3PBS buffer solution in 4 DEG C of preservation column material；As needed prepare different capabilities bisphenol-A and its
The affinity purification column that analog purifies simultaneously.
Bisphenol-A provided by the present invention and the like haptens and the bisphenol-A and the like antigen, synthesis
Method is simple, with high purity, yield is high, preparation and bisphenol-A for bisphenol-A and the like antibody and the like drug
Residue detection, immunologic purging have substantial worth.According to immune affinity column made of the haptens, antigen and Antibody preparation,
It only need to once extract, so that it may so that a variety of components to be measured is obtained high purification, the matrix effect of sample can be completely eliminated substantially, greatly
It is big to improve purification efficiency.And combined with LC-MS/MS detection technique, it is used for the exposure assessment of food incretion interferent.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation of embodiment 1, bisphenol-A and the like haptens
Will in any one raw material in bisphenol-A, bisphenol AF, bisphenol S, Bisphenol F, bisphenol b be added dimethylformamide (DMF),
NaH, the raw material, DMF, NaH proportion be 1000mg:7ml:1400mg, required reaction temperature be ice bath react, there is no gas
Bubble removes ice bath when emerging, then 40 min of temperature reaction obtain reaction solution；The addition 200mg 4- bromobutyrate in reaction solution, 40
It is reacted under the conditions of DEG C oil bath, 25ml ethyl acetate extracts 2 times, and combined ethyl acetate phase is evaporated, and 12mL ethyl acetate is added will be residual
Object is stayed to dissolve, the silica gel for weighing 2 times of weight is added thereto, and revolving is dry, prepares silicagel column loading.
Silica gel sample that reaction solution is mixed with silica gel is loaded using dry sample sample-adding method, with petroleum ether: acetic acid second
The elution of ester system, collects leacheate, collects product revolving.
Product is dissolved with 7 mL methanol, and 2 M NaOH solutions are added by the amount of 2 times of substances, heat at 40 DEG C, hydrolysis is complete
Afterwards, pH to 2 is adjusted with the HCl of 6M.Reaction solution is extracted twice with 50mL ethyl acetate, collects organic phase, anhydrous slufuric acid is added
Sodium (anhydrous sodium sulfate is added by the amount of 10% organic phase volume), 1 h of magnetic agitation after addition.Filtering, revolving are dry.It obtains described
Haptens Formulas I (1-5).
Formulas I (1)
Formulas I (2)
Formulas I (3)
Formulas I (4)
Formulas I (5)
The preparation of embodiment 2, bisphenol-A and the like artificial antigen and Structural Identification
1, the synthesis of immunogene
(1) 9.55mg bisphenol-A and the like haptens (any one in Formulas I (1-5)) 1.5mL DMF is dissolved,
200rpm stirs 10min, adds NHS 13mg after EDC 21.5mg dissolution is added, and (500rpm) activation 2-3h is stirred at room temperature.
(2) it weighs BSA 50mg to be dissolved in 3.5mL 0.1M sodium bicarbonate solution, 200rpm stirs 10min, makes it sufficiently
Dissolution, ice bath cool down 0-4 DEG C, and under 1000rpm stirring, step 1 reaction solution is added dropwise (1mL/min), and 500rpm stirring is anti-
Answer for 24 hours
(3) reaction product is packed into the clean bag filter of distilled water flushing (10cm), 1L0.01M PBS(1 ×, pH7.2) 4 DEG C of stirrings
(100rpm) dialysis 3d changes liquid 3 times (each primary in the morning, afternoon and evening) daily, total to change liquid 9 times, will dialysis product 5000rpm centrifugation
The packing of 6min, 1.5mL/ pipe, antigen is numbered, -20 DEG C save backup.
2, the synthesis of coating antigen
(1) 9.55mg bisphenol-A and the like haptens (any one in Formulas I (1-5)) 1.5mL DMF is dissolved,
200rpm stirs 10min, adds NHS 13mg after EDC 21.5mg dissolution is added, and (500rpm) activation 2-3h is stirred at room temperature.
(2) it weighs OVA 33.6mg to be dissolved in 3.5mL 0.1M sodium bicarbonate solution, 200rpm stirs 10min, fills it
Divide dissolution, ice bath cools down 0-4 DEG C, under 1000rpm stirring, step 1 reaction solution is added dropwise (1mL/min), 500rpm stirring
Reaction is for 24 hours.
(3) reaction product is packed into the clean bag filter of distilled water flushing (10cm), 1L0.01M PBS(1 ×, pH7.2) 4 DEG C
Stir (100rpm) to dialyse 3d, change liquid daily 3 times (in the morning, afternoon and evening each primary), it is total to change liquid 9 times, will dialysis product 5000rpm from
The packing of heart 6min, 1.5mL/ pipe, antigen is numbered, -20 DEG C save backup.
Embodiment 3, bisphenol-A and the like artificial antigen are immunized animal and prepare monoclonal antibody
One, animal immune
The immunogene (bisphenol-A and the like (any one in Formulas I (1-5))-BSA) prepared with embodiment 2 is by 100 μ g/
Only, it is mixed in equal volume with physiological saline solution immunogene with Freund's complete adjuvant, immune 6 ~ 8 week old are subcutaneously injected in the nape of the neck
Balb/c female mice is mixed after initial immunity, each supplementary immunization on the the 7th, 14,28 day in equal volume with immunogene and incomplete Freund's adjuvant
Once, first 3 days are merged with 100 μ of immune complex g/, Freund's adjuvant is not added, and supplementary immunization is primary again.
Two, cell fusion and clone
It carries out according to a conventional method, takes the splenocyte of immune mouse and the murine myeloma cell (SP2/0) for being in logarithmic growth phase
Mixing, the fusion agent (PEG4000) that preheating is then slowly added in 45s are merged, and are suspended uniformly with HAT culture medium, then
Suitable feeder cells are added, are incubated at 96 well culture plates, in 37 DEG C, 5%CO2It is cultivated in incubator, HT culture medium is used after 5 days
Partly change liquid, 9 days whens carry out changing liquid entirely.
After cell fusion, when cell grows to the 1/4 of culture hole area, hybridoma is screened using substep screening method.
Primary election uses indirect ELISA method, (uses its best peridium concentration of square matrix method conventional titration and positive serum in advance with envelope antigen
Dilution) coated elisa plate, measured hole culture supernatant is added, is incubated for, sheep anti-mouse igg-HRP and IgM-HRP is added after cleaning,
OPD carries out chromogenic reaction.The positive Kong Zaiyong indirect competitive ELISA method screening filtered out, first by cell conditioned medium and 100 μ g/
Bisphenol-A of mL and the like mixes in equal volume, and 37 DEG C of water-baths act on 30min, is then added in the ELISA Plate being coated with.Simultaneously
Bisphenol-A and the like is replaced to compare with PBS, remaining step is same as above.If the OD after bisphenol-A and the like blocking450nm
Value drops to the 50% of control wells hereinafter, being then judged to the positive, is all positive hole through 2 ~ 3 detections, uses limiting dilution assay immediately
Carry out subcloning.
Three, the preparation and purifying of monoclonal antibody
The hybridoma that 2 ~ 3 subclones are built after strain is expanded into culture, supernatant is collected with indirect ELISA and measures potency, freeze
It deposits；And take 8 ~ 10 week old Balb/c mouse peritoneal injecting fluid paraffin 0.5mL/ only, hybridoma 1 is injected intraperitoneally after 7 ~ 10 days
Mouse ascites are extracted after ~ 2 × 105/, 7 ~ 10 days.Cell conditioned medium or ascites are collected, its potency is measured using indirect ELISA method
(being indicated when measurement potency with the cell conditioned medium of P/N > 2.1 or ascites maximum dilution multiple), the results showed that the potency of cell conditioned medium
For 1:10000, the potency of ascites is 1:50000.Then, it is purified with octanoic acid-saturated ammonium sulfate method, is put after purification
Enter -20 DEG C of environment to save.
Embodiment 4, bisphenol-A and the like artificial antigen are immunized animal and prepare antiserum
One, animal immune
Use the bisphenol-A of the acquisition of step embodiment 2 and the like artificial antigen " bisphenol-A and the like-BSA " as immunogene
Immune new zealand white rabbit.Each immunizing dose is 100 ~ 200 μ g, and immunization ways are double shoulder and the subcutaneous multiple spot note of rear thigh
Penetrate, each region with about 1/4 immunogene.By immunogene normal saline dilution when head exempts from, then not exclusively helped with Freund
Agent carries out 1:1(volume ratio) it is mixed and made into emulsifier, took same dose immunogene that isometric Freund is added not exclusively to help at interval of 2 weeks
Booster immunization is primary after agent mixing and emulsifying, is added altogether after exempting from 3 times using this mode, and interval takes same dose immunogene to add in 3~4 weeks again
Not formula Freund's incomplete adjuvant carries out final immunization, and arteria auricularis takes blood examination to survey antibody titer.Arteria carotis is used after final immunization 7-10 days
Bloodletting, every rabbit can obtain blood 100-120mL or so, and the blood taken is placed 3 ~ 4 hours in 4 DEG C of refrigerators, and centrifugation separates bleeding
Clearly.
Two, antiserum titre measures
It is specific as follows using the antibody titer of one gained serum of indirect elisa method determination step:
1) it is coated with: " bisphenol-A and the like-OVA " solution that 100 μ L concentration are 2 μ g/mL being added in 96 hole elisa Plates and (uses
Coating buffer is diluted), while the control of not envelope antigen is set, 4 DEG C of coatings overnight, are washed 3 times with PBS buffer solution.
Be coated with buffer: (solvent is water to the sodium carbonate-bicarbonate buffer of pH9.6,0.05M, and solute and its concentration are such as
Under: Na2CO31.59g/L and NaHCO32.93g/L).
2) it closes: the confining liquid in 150 holes μ L/ is added, in 37 DEG C of incubation 2h, abandon confining liquid, wash 3 times, pat dry.It is placed in 4
DEG C refrigerator saves backup.
Confining liquid: contain 0.5%(volumn concentration) calf serum, 3%(3g/100mL) casein phosphate it is slow
Fliud flushing, pH7.4.
3) add sample to be tested: drawing the 100 μ l of test serum of different dilutions, be added in corresponding ELISA Plate, 37 DEG C incubate
30min is educated, board-washing 4 times, is patted dry.
The control of non-immunized rabbit anteserum is set simultaneously；The control (negative control hole) of sample to be tested is replaced with PBS.
4) add ELIAS secondary antibody: the goat anti-rabbit igg antibody for taking horseradish peroxidase to mark dilutes for 1:5000 times by volume
Afterwards, 100 hole μ l/, 37 DEG C are incubated for 20 to 30min, wash 4 times, pat dry.
5) it develops the color: 20 × TMB is diluted to 1 × TMB, be added by 100 holes μ l/, 37 DEG C of colour developing 15-30min.
6) it terminates: terminate liquid (2MH is added2SO4) 50 holes μ l/.
7) it reads: each hole OD value is measured with 450nm Single wavelength, (to replace pair of sample to be tested with PBS with negative control hole
According to) ratio (P/N) of OD value is greater than 2.1 and is limited, as the critical point for being judged as serum titer.
ELISA result judgement method: it is indicated with the serum maximum dilution multiple of P/N > 2.1.
The result shows that the antibody titer in serum is 1:16000.
The preparation for the immune affinity column that embodiment 5, bisphenol-A and the like purify simultaneously
1, column material is mixed up and down, and the wet column material of Sepharose 4B for taking 4ml NHS to activate is added in reaction tube, to natural subsidence
Volume is 3ml bed volume afterwards, and gravity column acts on trickle, washed off in column bed with the pre-cooling 1mM HCl of 10 times of bed volumes
Organic solvent, add the coupling buffer of 2 times of column volumes into column material, siphon away liquid with syringe back suction rapidly；
2, column tube bottom end is sealed, antibody is formed to antibody-solutions in 0.2M NaHCO3buffer and is added in column material, is covered
Upper cover mixes column material and solution up and down；
3,3 times of column bed bodies are washed with 0.1M Tris-HCl pH8.0 respectively until trickle to dripless outflow after reaction
Product, then change 0.1 mol/L acetic acid/sodium acetate (containing 0.5 mol/L NaCl), pH4.0 wash 3 times of bed volumes, then with 20 mM
PBS washes 5 times of bed volumes of column；
4, plus containing 0.05% NaN3PBS buffer solution in 4 DEG C of preservation column material；As needed prepare different capabilities bisphenol-A and its
The affinity purification column that analog purifies simultaneously.
The application for the immune affinity column that embodiment 6, bisphenol-A and the like purify simultaneously
One, sample pre-treatments
12 kinds of diet sample substrates are studied in the present invention, are divided into three classes according to its separate sources.Respectively plant-derived food
Product, animal derived food and beer.
For plant-derived sample (flour, rice, apple, potato and cabbage), sample after taking 1 g homogeneous in
In 10 mL centrifuge tubes, 30 s are vibrated after 5 mL acetonitriles are added, and after 20 min of ultrasonic extraction, 10000 rpm take after being centrifuged 10 min
Supernatant liquor repeats to extract primary, combined extract in another 15 mL centrifuge tube, with 5 mL acetonitriles.Nitrogen is slow at 40 DEG C
Slow drying purifies after being redissolved with 5 mL methanol and PBS mixed liquor (15:85, v/v) to IAC loading.The pre-treatment of peanut oil
It is identical with animal derived food pre-treating method.
For animal derived sample (grass carp, egg, milk, pork and baby milk powder), sample after taking 1 g homogeneous in
In 10 mL centrifuge tubes, it is added after 5 mL acetonitriles and 3 mLPBS and vibrates 30 s, after 20 min of ultrasonic extraction, 10000 rpm centrifugation
It taking supernatant liquor in another 10 mL centrifuge tube after 10 min, is put into after freezing 3 h at -20 DEG C, it is seen that liquid level is divided into three layers,
Top layer is acetonitrile layer, and centre is the fat being precipitated, and lower layer is PBS layers.Take acetonitrile layer in another 10 mL centrifuge tube, 40
Nitrogen slowly dries up at DEG C, after being redissolved with 5 mL methanol and PBS mixed liquor (15:85, v/v), purifies to IAC loading.
For beer sample, first by 30 min of its ultrasonic degassing, taking 10 mL beer samples and PBS, 1:1 dilutes by volume
Afterwards, pH to 7.5 is adjusted with 1 M sodium hydroxide solution, mixed liquor waits for that IAC loading purifies.
Two, the detection of actual sample, IAC-UPLC-MS/MS measure the dense of BPA and its metabolin in 15 parts of actual samples
Degree.
| Sample type | Sample number into spectrum | BPA and its metabolite concentration (μ g kg-1) |
| Rice | 1 | 0.04 |
| 2 | n.d.a | |
| 3 | 0.26 | |
| Grass carp | 4 | 0.59 |
| 5 | 1.77 | |
| 6 | 1.58 | |
| Milk | 7 | 0.09 |
| 8 | 0.17 | |
| 9 | 0.26 | |
| Egg | 10 | 0.30 |
| 11 | 0.25 | |
| 12 | 0.40 | |
| Cabbage | 13 | n.d. |
| 14 | 0.02 | |
| 15 | 0.06 |
N.d is lower than detection limit.
Claims (9)
1. the immune affinity column that a kind of bisphenol-A and the like purifies simultaneously, which is characterized in that including structure in Formulas I (1-5)
Any one haptens:
Formulas I (1)
Formulas I (2)
Formulas I (3)
Formulas I (4)
Formulas I (5).
2. the immune affinity column that a kind of bisphenol-A according to claim 1 and the like purifies simultaneously, it is characterised in that:
The preparation method of the haptens includes step are as follows:
Will in any one raw material in bisphenol-A, bisphenol AF, bisphenol S, Bisphenol F, bisphenol b be added dimethylformamide (DMF),
NaH, the raw material, DMF, NaH proportion be 1000mg:7ml:1400mg, required reaction temperature be ice bath react, there is no gas
Bubble removes ice bath when emerging, then 40 min of temperature reaction obtain reaction solution；The addition 200mg 4- bromobutyrate in reaction solution, 40
It is reacted under the conditions of DEG C oil bath, 25ml ethyl acetate extracts 2 times, and combined ethyl acetate phase is evaporated, and 12mL ethyl acetate is added will be residual
Object is stayed to dissolve, the silica gel for weighing 2 times of weight is added thereto, and revolving is dry, prepares silicagel column loading；
Silica gel sample that reaction solution is mixed with silica gel is loaded using dry sample sample-adding method, with petroleum ether: ethyl acetate body
System's elution, collects leacheate, collects product revolving；
Product is dissolved with 7 mL methanol, and 2 M NaOH solutions are added by the amount of 2 times of substances, heat at 40 DEG C, after hydrolysis completely, use
The HCl of 6M adjusts pH to 2；Reaction solution is extracted twice with 50mL ethyl acetate, collects organic phase, anhydrous sodium sulfate (nothing is added
Aqueous sodium persulfate is added by the amount of 10% organic phase volume), 1 h of magnetic agitation after addition；Filtering, revolving is dry, obtains Formulas I (1-5) institute
State compound.
3. the immune affinity column that a kind of bisphenol-A according to claim 1 and the like purifies simultaneously, it is characterised in that:
It further include bisphenol-A and the like antigen, for by compound described in claim 1 and the resulting antigen of carrier protein couplet.
4. the immune affinity column that a kind of bisphenol-A according to claim 3 and the like purifies simultaneously, it is characterised in that:
The carrier protein can be bovine serum albumin(BSA), ovalbumin, human serum albumins, hemocyanin, mouse haemocyanin, first shape
Gland albumen or rabbit serum proteins.
5. the immune affinity column that a kind of bisphenol-A according to claim 3 or 4 and the like purifies simultaneously, feature exist
In: the preparation method of described bisphenol-A and the like antigen includes the following steps: compound described in claim 1 and carries
Body protein is coupled by amido bond, obtains the bisphenol-A and the like antigen.
6. the immune affinity column that a kind of bisphenol-A according to claim 5 and the like purifies simultaneously, it is characterised in that:
The preparation method of described bisphenol-A and the like antigen includes the following steps:
(1) bisphenol-A and the like haptens (Formulas I) is dissolved in dimethylformamide (DMF), 1- is then added
(3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and n-hydroxysuccinimide (NHS), 20-25 DEG C of magnetic force
It is stirred to react 2-3h, obtains solution I；
Wherein, described bisphenol-A and the like haptens (Formulas I), the dimethylformamide (DMF), the 1- (3- diformazan ammonia
Base propyl) -3- ethyl-carbodiimide hydrochloride (EDC), the n-hydroxysuccinimide (NHS) proportion be 9.55mg:
1.5mL:21.5mg:13mg；
(2) carrier protein is placed in 0.1M sodium bicarbonate buffer liquid, 200rpm stirs 10min, sufficiently dissolves, obtains molten
Liquid II；The proportion of the carrier protein and the 0.1M sodium bicarbonate buffer liquid is 33.6-50mg:3.5mL；
(3) solution I and the solution II are mixed, specially under the conditions of 0-4 DEG C, under 1000rpm stirring, by solution I
It is added dropwise in the solution II, 500rpm is stirred to react for 24 hours, obtains solution III；
(4) phosphate buffer (0.01M PBS, pH7.2) is used, in 4 DEG C to the solution III stirring dialysis 3 days, obtained described
Bisphenol-A and the like antigen.
7. the immune affinity column that a kind of bisphenol-A according to claim 1 and the like purifies simultaneously, it is characterised in that:
The compound or the bisphenol-A and the like antigen can prepare bisphenol-A and the like antibody.
8. the immune affinity column that a kind of bisphenol-A and the like purifies simultaneously, is obtained with the following method form:
(1) column material is mixed up and down, and the wet column material of Sepharose 4B for taking 4ml NHS to activate is added in reaction tube, to natural subsidence
Volume is 3ml bed volume afterwards, and gravity column acts on trickle, washed off in column bed with the pre-cooling 1mM HCl of 10 times of bed volumes
Organic solvent, add the coupling buffer of 2 times of column volumes into column material, siphon away liquid with syringe back suction rapidly；
(2) column tube bottom end is sealed, antibody is formed to antibody-solutions in 0.2M NaHCO3buffer and is added in column material, covered
Good upper cover mixes column material and solution up and down；
(3) 3 times of column beds are washed with 0.1M Tris-HCl pH8.0 respectively until trickle to dripless outflow after reaction
Volume, then change 0.1 mol/L acetic acid/sodium acetate (containing 0.5 mol/L NaCl), pH4.0 wash 3 times of bed volumes, then with 20 mM
PBS washes 5 times of bed volumes of column；
(4) plus containing 0.05% NaN3PBS buffer solution in 4 DEG C of preservation column material；As needed prepare different capabilities bisphenol-A and its
The affinity purification column that analog purifies simultaneously.
9. the immune affinity column that a kind of bisphenol-A according to claim 1 and the like purifies simultaneously, which is characterized in that
It can be used for the plant-derived food such as flour, rice, apple, potato and cabbage, grass carp, egg, milk, pork and infant
It is purified while bisphenol-A and the like in the food such as the animal derived foods such as milk powder and beer.
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