CN108982841A - A kind of sulfamido Sample pretreatment kit based on immunomagnetic beads - Google Patents
A kind of sulfamido Sample pretreatment kit based on immunomagnetic beads Download PDFInfo
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- CN108982841A CN108982841A CN201811115421.3A CN201811115421A CN108982841A CN 108982841 A CN108982841 A CN 108982841A CN 201811115421 A CN201811115421 A CN 201811115421A CN 108982841 A CN108982841 A CN 108982841A
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- sulfamido
- immunomagnetic beads
- sample
- storehouse
- cleaning
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- 239000011324 bead Substances 0.000 title claims abstract description 99
- 238000004140 cleaning Methods 0.000 claims abstract description 30
- 239000007788 liquid Substances 0.000 claims abstract description 14
- 238000010828 elution Methods 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- 239000007853 buffer solution Substances 0.000 claims description 19
- 239000000872 buffer Substances 0.000 claims description 18
- 239000006228 supernatant Substances 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 16
- 230000004913 activation Effects 0.000 claims description 12
- 238000000746 purification Methods 0.000 claims description 12
- 238000005859 coupling reaction Methods 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 239000007977 PBT buffer Substances 0.000 claims description 8
- 239000012190 activator Substances 0.000 claims description 8
- 230000008878 coupling Effects 0.000 claims description 8
- 238000010168 coupling process Methods 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 239000003480 eluent Substances 0.000 claims description 6
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 claims description 5
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- -1 cistosulfa Chemical compound 0.000 claims description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- 239000004005 microsphere Substances 0.000 claims description 3
- 238000007885 magnetic separation Methods 0.000 claims description 2
- 238000012986 modification Methods 0.000 claims description 2
- 230000004048 modification Effects 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 238000002604 ultrasonography Methods 0.000 claims description 2
- 229940124530 sulfonamide Drugs 0.000 claims 4
- 239000003153 chemical reaction reagent Substances 0.000 claims 2
- NHZLNPMOSADWGC-UHFFFAOYSA-N 4-amino-N-(2-quinoxalinyl)benzenesulfonamide Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=CN=C(C=CC=C2)C2=N1 NHZLNPMOSADWGC-UHFFFAOYSA-N 0.000 claims 1
- GWBPFRGXNGPPMF-UHFFFAOYSA-N N-[4-[(4-nitrophenyl)sulfamoyl]phenyl]acetamide Chemical compound C1=CC(NC(=O)C)=CC=C1S(=O)(=O)NC1=CC=C([N+]([O-])=O)C=C1 GWBPFRGXNGPPMF-UHFFFAOYSA-N 0.000 claims 1
- ZCQWOFVYLHDMMC-UHFFFAOYSA-N Oxazole Chemical compound C1=COC=N1 ZCQWOFVYLHDMMC-UHFFFAOYSA-N 0.000 claims 1
- NHUHCSRWZMLRLA-UHFFFAOYSA-N Sulfisoxazole Chemical compound CC1=NOC(NS(=O)(=O)C=2C=CC(N)=CC=2)=C1C NHUHCSRWZMLRLA-UHFFFAOYSA-N 0.000 claims 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims 1
- 238000012412 chemical coupling Methods 0.000 claims 1
- 125000003700 epoxy group Chemical group 0.000 claims 1
- 108091008104 nucleic acid aptamers Proteins 0.000 claims 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims 1
- FRLJPXLANPCKLP-UHFFFAOYSA-N sodium;[4-[(5-methoxypyrimidin-2-yl)sulfamoyl]phenyl]azanide Chemical compound [Na+].N1=CC(OC)=CN=C1NS(=O)(=O)C1=CC=C([NH-])C=C1 FRLJPXLANPCKLP-UHFFFAOYSA-N 0.000 claims 1
- 229960004730 sulfabenzamide Drugs 0.000 claims 1
- PBCZLFBEBARBBI-UHFFFAOYSA-N sulfabenzamide Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC(=O)C1=CC=CC=C1 PBCZLFBEBARBBI-UHFFFAOYSA-N 0.000 claims 1
- 229960002673 sulfacetamide Drugs 0.000 claims 1
- SKIVFJLNDNKQPD-UHFFFAOYSA-N sulfacetamide Chemical compound CC(=O)NS(=O)(=O)C1=CC=C(N)C=C1 SKIVFJLNDNKQPD-UHFFFAOYSA-N 0.000 claims 1
- 229960004306 sulfadiazine Drugs 0.000 claims 1
- SEEPANYCNGTZFQ-UHFFFAOYSA-N sulfadiazine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=NC=CC=N1 SEEPANYCNGTZFQ-UHFFFAOYSA-N 0.000 claims 1
- 229960002135 sulfadimidine Drugs 0.000 claims 1
- 229960000654 sulfafurazole Drugs 0.000 claims 1
- ASWVTGNCAZCNNR-UHFFFAOYSA-N sulfamethazine Chemical compound CC1=CC(C)=NC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 ASWVTGNCAZCNNR-UHFFFAOYSA-N 0.000 claims 1
- 229960005404 sulfamethoxazole Drugs 0.000 claims 1
- GPTONYMQFTZPKC-UHFFFAOYSA-N sulfamethoxydiazine Chemical compound N1=CC(OC)=CN=C1NS(=O)(=O)C1=CC=C(N)C=C1 GPTONYMQFTZPKC-UHFFFAOYSA-N 0.000 claims 1
- VLYWMPOKSSWJAL-UHFFFAOYSA-N sulfamethoxypyridazine Chemical compound N1=NC(OC)=CC=C1NS(=O)(=O)C1=CC=C(N)C=C1 VLYWMPOKSSWJAL-UHFFFAOYSA-N 0.000 claims 1
- 229960004936 sulfamethoxypyridazine Drugs 0.000 claims 1
- 229960002229 sulfametoxydiazine Drugs 0.000 claims 1
- 229950004215 sulfanitran Drugs 0.000 claims 1
- QWCJHSGMANYXCW-UHFFFAOYSA-N sulfaphenazole Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=CC=NN1C1=CC=CC=C1 QWCJHSGMANYXCW-UHFFFAOYSA-N 0.000 claims 1
- 229960003097 sulfaquinoxaline Drugs 0.000 claims 1
- 229960001544 sulfathiazole Drugs 0.000 claims 1
- JNMRHUJNCSQMMB-UHFFFAOYSA-N sulfathiazole Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=NC=CS1 JNMRHUJNCSQMMB-UHFFFAOYSA-N 0.000 claims 1
- JLKIGFTWXXRPMT-UHFFFAOYSA-N sulphamethoxazole Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 JLKIGFTWXXRPMT-UHFFFAOYSA-N 0.000 claims 1
- 241001465754 Metazoa Species 0.000 abstract description 7
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- 235000013622 meat product Nutrition 0.000 abstract description 2
- 238000002203 pretreatment Methods 0.000 abstract description 2
- 238000012549 training Methods 0.000 abstract description 2
- 238000011017 operating method Methods 0.000 abstract 1
- 238000001514 detection method Methods 0.000 description 10
- 239000000427 antigen Substances 0.000 description 8
- 102000036639 antigens Human genes 0.000 description 8
- 108091007433 antigens Proteins 0.000 description 8
- 229940079593 drug Drugs 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 239000007790 solid phase Substances 0.000 description 6
- 235000013372 meat Nutrition 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- 230000009849 deactivation Effects 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 230000005389 magnetism Effects 0.000 description 3
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- 239000011534 wash buffer Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 208000031295 Animal disease Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
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- 238000009835 boiling Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
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- 125000000524 functional group Chemical group 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 206010011509 Crystalluria Diseases 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 231100000703 Maximum Residue Limit Toxicity 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
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- 238000004458 analytical method Methods 0.000 description 1
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- 230000027455 binding Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
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- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
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- 238000011010 flushing procedure Methods 0.000 description 1
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
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- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Nanotechnology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides the preparation and application of a kind of sulfamido Sample pretreatment kit based on immunomagnetic beads, which is mainly made of several parts such as sample storehouse, agent bin, cleaning storehouse, elution storehouses.The operating method is efficiently quick, easy to operate, does not need the professional operator of large-scale instrument and Special Training, sample does not need special pre-treatment, can be widely applied in the products such as animal tissue, aquatic products, meat products the enrichment of sulfamido with separate, to the sulfamido sample for being lower than detectable limit, it is sufficiently spread in a liquid by the enrichment and immunomagnetic beads of immunomagnetic beads so that combined surface area expands, it reacts more thorough, so as to change detectable limit, the phenomenon that improving the sensitivity detected, avoiding missing inspection.
Description
Technical field
The invention belongs to biological detection analysis fields, and in particular to a kind of sulfamido Sample pretreatment based on immunomagnetic beads
Kit.
Background technique
Sulfa drugs is a kind of artificial synthesized, the broad spectrum antibiotic with P-aminobenzene-sulfonamide structure, to most
Several gram-positive bacterias is all inhibited.Since its property is stable, quality is more, price is low, using simple, in liberal supply
Etc. advantages, control drug as feed addictive or Animal diseases in veterinary clinic and animal husbandry and be used widely,
For preventing and treating the chemotherapeutic agent of bacterial infection disease.In veterinary clinic, can be used for preventing and treating Animal diseases,
Treatment urinary tract infection, stranguria syndrome, meningitis etc. have significant curative effect, furthermore former to the various globidiosis of poultry, Cattell leucocyte
Parasitosis etc. also has better effects.In husbandry sector, feed addictive can be used as, promote the growth of livestock and poultry, improve the product of product
Matter.At the same time, some illegal businessman take in pursue high interests, the phenomenon that abuse sulfa drugs occur, so that this
A little drugs generate excessive accumulation in animal foodstuff.Food of the people after edible pollution, side effects of pharmaceutical drugs occur therewith,
People's urinary system function may be will affect, cause crystalluria, the reaction such as blood urine, it may appear that allergic reaction, kidney damage, leucocyte
Disease etc. is reduced, there is certain carcinogenicity.It endangers caused by the residual of sulfa drugs to ecological environmental pollution and human health
One of the problem of potential threat that evil is constituted has been concerned, has become mankind's urgent need to resolve.In order to guarantee human food safety and
The stabilization of ecological environment, China provide that maximum residue limit of the sulfa drugs in edible food is 100 μ g/kg.
Currently, immunologic detection method is so that antigen or antibody is integrated to certain surface of solid phase carriers, and keep its immune work
Property.In measurement, by inspection sample (measuring antigen or antibody therein) and enzyme-labelled antigen or antibody by different steps with consolidate
The antigen or antibody of phase carrier surface react.Make the antigen antibody complex formed on solid phase carrier and its with the method for washing
He separates substance, finally combines the enzyme amount on solid phase carrier directly related with the amount of tested substance in sample, therefore can be according to face
The depth of colour response does qualitative or quantitative analysis.Since the catalysis frequency of enzyme is very high, thus can greatly iodine effect, thus
Measuring method is set to reach very high sensitivity.But there are problems to have for presently used solid phase carrier: differences between batches are not easily-controllable
System, packaging operation is complicated, and the degree of automation is not high, is not suitable for the operation of high-volume sample.
Immunomagnetic beads are by the magnetic microsphere and the nanometer materials that are combined into of immunoligand as carrier.Magnetic microsphere
Carrier is usually the magnetic bead for having the chemical functional groups such as amino, carboxyl, hydroxyl or sulfydryl, the functional group and different immunoligands
Such as activated protein, antibody, antigen, Avidin, biotin, which combine, forms immunomagnetic beads.Magnetic Microspheres-Carrier has superparamagnetism
Feature when immunomagnetic beads can be made to be placed in magnetic field, shows its magnetism, and when removing from magnetic field, magnetism is eliminated, and immunomagnetic beads divide again
It dissipates.As solid phase carrier, antibody and corresponding antigens thereon occurs specific binding and forms antigen-immunomagnetic beads of coated antibody
Antibody-bead complexes, this compound, rapidly to magnetic field movement, separate compound with other substances under magnetic fields,
Achieve the purpose that separate specific antigen, this separation method fast, specific height, high sensitivity, repeatability with detection speed
The advantages such as good.The magnetism separate method favorable repeatability of immunomagnetic beads, it is easy to operate, it is not required to expensive instrument and equipment, does not influence quilt
The biological character and function of biomaterial, and the performance special due to magnetic bead are separated, immune detection can be realized and be automated,
For the detection of extensive sample, kinetics is fast compared with traditional immunization detection method, and has bigger solid phase binding
Surface.
Summary of the invention
The purpose of the invention is to make up the shortcomings of the prior art, a kind of sulfamido for immunomagnetic beads is provided
Sample pretreatment kit is enriched in purification for sulfamido in sample, to solve the operation of sulfamido specimen sample purification separation
The technical problems such as complicated, low separation efficiency.Immunomagnetic beads are after appropriate activator (EDC-NHS combination) processing, so that magnetic
Bead surface has affinity ligand, is coupled, is obtained under the appropriate reaction conditions by affinity chromatography and sulfamido antibody
Sulfamido antibody specificity immunomagnetic beads are enriched with, are inhaled using solution of the sulfamido antibody specificity immunomagnetic beads to sulfamido
It is attached, to be enriched with and purify sulfamido in sample, so as to subsequent measurement.
For achieving the above object, The technical solution adopted by the invention is as follows:
A kind of sulfamido Sample pretreatment kit based on immunomagnetic beads, the kit by sample storehouse, agent bin, cleaning storehouse,
Elute several part compositions such as storehouse.Sample storehouse is used to place the pretreatment solution of sample, and agent bin is exempted from for placing immunomagnetic beads
Sulfamido antibody is coupled on epidemic disease magnetic bead, cleaning storehouse is mainly used for elution of bound object for placing cleaning solution cleaning, elution storehouse.
Sulfamido Sample pretreatment kit based on above-mentioned immunomagnetic beads, mainly include the following:
(1) Sample pretreatment solution is put in sample storehouse first, anti-sulfamido antibody immune magnetic beads are put into agent bin, and rotation is mixed
It is even, then immunomagnetic beads are added in sample storehouse, rotation is resuspended, rotation capture 30min at 37 DEG C, to realize farthest
The sulfamido in sample is captured, Magneto separate discards supernatant liquid；The immunomagnetic beads for capturing sample are used into cleaning in cleaning storehouse
Liquid cleaning, jog are resuspended, and Magneto separate abandons supernatant；This process is 2-3 times repeatable.After the completion of immunomagnetic ca pture sulfamido, then pass through
Magneto separate, to realize the enrichment purification of sulfamido sample；Supernatant after removing Magneto separate, precipitating are to capture sulfamido
Immunomagnetic beads；
(2) elution of immunomagnetic beads
It is eluted eluent is added in elution storehouse in immunomagnetic beads obtained in step (1), room temperature shakes several seconds, magnetic
Separation, supernatant is the sulfamido sample after enrichment purification；
In above-mentioned steps, it is preferable that the optimum reaction condition of immunomagnetic beads enrichment purification sulfamido is, at 37 DEG C, pH7.4,
Rotation capture 30min in the PBS buffer solution containing methanol 10% of 0.02mo1/L；
In above-mentioned steps, it is preferable that the capture buffer of use is preferably pH7.4, and the PBS containing methanol 10% of 0.02mo1/L is slow
Fliud flushing；
In above-mentioned steps, it is preferable that the cleaning solution used is pH7.4 PBS buffer solution；
In above-mentioned steps, it is preferable that eluent uses anhydrous methanol, and concentration of the immunomagnetic beads in eluent uses 5-10mg/
mL；Room temperature, which is shaken 1-5 minutes, to be advisable；
Preferably, in step (1) anti-sulfamido antibody specificity immunomagnetic beads specific the preparation method comprises the following steps:
S1 cleaning: taking 0.4mg magnetic bead in 2mL centrifuge tube respectively, using 200 μ L PBT(0.02mo1/L, pH7.4, contains 0.05%
Tween-20 it) cleans 2 times, is resuspended after washing with 250 μ L PBS buffer solution (0.02mo1/L, pH7.4) ultrasonic disperses；
S2 activation: EDC and NHS that 200 μ L are prepared with PBS buffer solution (0.02mo1/L, pH7.4), 37 DEG C of activation are separately added into
2h keeps suspended state；
S3 coupling: Magneto separate sucks supernatant, and ibid three times, ultrasound is resuspended the washing of PBT buffer.Again respectively by the anti-of 200 μ g
Sulfamido antibody is added in the magnetic bead activated, and 37 DEG C of coupling reaction 5h, 15 r/min rotations keep suspended state；
S4 closing: Magneto separate takes out supernatant, and the ethanolamine solutions and BSA confining liquid of 100 μ L are added, and 37 DEG C of closing 2h are kept
Suspended state；
S5 save: wash magnetic bead 3 times with PBT, Magneto separate, abandoning supernatant, with 250 μ LPBS buffers (0.02mo1/L, pH7.4,
Containing 0.02%NaN3And 0.5%BSA) be resuspended, it is saved in 4 DEG C；
Preferably, activator described in above-mentioned steps is EDC and NHS, and concentration is 1-5mg/mL, molten with activation buffer
Solution；
Preferably, in the above-mentioned method for preparing immunomagnetic beads, the preferred pH7.4 of coupling buffer, the PBS buffer solution of 0.02mo1/L；
Cleaning solution is preferably containing the pH7.4 of 0.05%Tween-20, the PBT buffer of 0.02mo1/L；Activator is prepared with PBS buffer solution；
The 5%BSA solution that confining liquid is preferably prepared with PBS buffer solution.
The beneficial effect of the present invention compared with the existing technology is:
1. the present invention using anti-sulfamido antibody immune magnetic beads be enriched with sulfamido, can the short time rapidly, be selectively separating in
By the sulfamido enrichment and purifying in sample, avoids largely using organic solvent, effectively reduces the interference of background material,
Improve the accuracy and accuracy of detection；
2. applying immunomagnetic bead technique, without carrying out purification step after extracting, this is easy to operate, facilitates feasible；
3. the operation does not need the professional of large-scale instrument and Special Training, sample does not need special pre-treatment, can answer extensively
For the sulfamido in feed, meat products, animal tissue purifying, be enriched with and separate.
Detailed description of the invention
A kind of structural schematic diagram of the sulfamido Sample pretreatment kit based on immunomagnetic beads of attached drawing 1
Description of symbols 1- sample storehouse, 2- agent bin, 3- cleaning storehouse, 4- cleaning storehouse, 5- cleaning storehouse, 6- cleaning storehouse, 7- elution
Storehouse.
Specific embodiment
The present invention is described in further details below by embodiment, these embodiments are only used to illustrate the present invention, and
It does not limit the scope of the invention, the specific embodiment of the invention is as follows.
Embodiment 1
One, the preparation of anti-sulfamido antibody immune magnetic beads:
1. magnetic bead is taken to be put into centrifuge tube, activation buffer is added and washs magnetic bead, repeatedly for three times, every minor tick 3 minutes, except deactivation
Each 200 μ L of activator EDC and NHS is added after changing buffer, mixes, is incubated for 2 hours in 37 DEG C of rotations, carries out magnetic bead activation；
2. being washed magnetic bead three times with coupling buffer, every minor tick 3 minutes, sulfamido antibody is added, mixes, is incubated for 3 in 37 DEG C
Hour, it is prepared into sulfamido antibody immune magnetic beads；
3. washing immunomagnetic beads repeatedly for three times with washing buffer, every minor tick 3 minutes.It is incubated for 2 hours with confining liquid in 37 DEG C,
It is placed in save in buffer and save for use for 4 DEG C；
Two, the application that anti-sulfamido immunomagnetic beads are enriched with sulfamido in animal tissue's sample:
1, animal tissue's sample is smashed to pieces with meat pulper, or minces (pulpous state) with knife；
2, the tissue sample that 5.0 g are smashed to pieces is weighed, is fitted into the cryovial of 5 mL, tightening pipe lid (not allow water to enter pipe
It is interior)；
3, the cryovial equipped with sample is boiled in boiling water take out after ten minutes (meat well-done subject to) be cooled to room temperature it is spare.It is made
The even liquid of sample, is put into sample storehouse；
4, immunomagnetic beads are added in the sample of suitable concentration, are incubated for 30 minutes after mixing well in 37 DEG C, after effect, set
In capturing magnetic bead on magnetic frame, supernatant is removed, in cleaning storehouse with buffer solution for cleaning 3 times, is dissociated in elution storehouse with anhydrous methanol
Obtained meoh eluate nitrogen is blown concentration by the sulfamido on immunomagnetic beads out, and gained sample is sulfamido (sample after purification
It freezes, remains to carry out identification detection to sulfamido using ELISA).
Embodiment 2
One, the preparation of anti-sulfamido antibody immune magnetic beads:
1. magnetic bead is taken to be put into centrifuge tube, activation buffer is added and washs magnetic bead, repeatedly for three times, every minor tick 5 minutes, except deactivation
Each 180 μ L of activator EDC and NHS is added after changing buffer, mixes, is incubated for 2 hours in 37 DEG C of rotations, carries out magnetic bead activation；
2. being washed magnetic bead three times with coupling buffer, every minor tick 3 minutes, sulfamido antibody is added, mixes, is incubated for 3 in 37 DEG C
Hour, it is prepared into sulfamido antibody immune magnetic beads；
3. washing immunomagnetic beads repeatedly for three times with washing buffer, every minor tick 3 minutes.It is incubated for 2 hours with confining liquid in 37 DEG C,
It is placed in save in buffer and save for use for 4 DEG C；
Two, the application that anti-sulfamido immunomagnetic beads are enriched with sulfamido in Feed Sample:
1, the Feed Sample for weighing 2.0 g crushing, is fitted into the centrifuge tube of 5 mL, certain sample diluting liquid is added, be put in shake
It swings device to shake 10 minutes, centrifuging and taking supernatant；
2, immunomagnetic beads are added in the sample of suitable concentration, are incubated for 30 minutes after mixing well in 37 DEG C, after effect, set
In capturing magnetic bead on magnetic frame, supernatant is removed, in cleaning storehouse with buffer solution for cleaning 2 times, is dissociated in elution storehouse with anhydrous methanol
Obtained meoh eluate nitrogen is blown concentration by the sulfamido on immunomagnetic beads out, and gained sample is sulfamido (sample after purification
It freezes, remains to carry out identification detection to sulfamido using ELISA).
Embodiment 3
One, the preparation of anti-sulfamido antibody immune magnetic beads:
1. magnetic bead is taken to be put into centrifuge tube, activation buffer is added and washs magnetic bead, repeatedly for three times, every minor tick 4 minutes, except deactivation
Each 250 μ L of activator EDC and NHS is added after changing buffer, mixes, is incubated for 4 hours in 37 DEG C of rotations, carries out magnetic bead activation；
2. being washed magnetic bead three times with coupling buffer, every minor tick 4 minutes, sulfamido antibody is added, mixes, is incubated for 4 in 37 DEG C
Hour, it is prepared into sulfamido antibody immune magnetic beads；
3. washing immunomagnetic beads repeatedly for three times with washing buffer, every minor tick 4 minutes.It is incubated for 4 hours with confining liquid in 37 DEG C,
It is placed in save in buffer and save for use for 4 DEG C；
Two, the application that anti-sulfamido immunomagnetic beads are enriched with sulfamido in sample:
1, animal tissue's sample is smashed to pieces with meat pulper, or minces (pulpous state) with knife；
2, the tissue sample that 5.0 g are smashed to pieces is weighed, is fitted into the cryovial of 5 mL, tightening pipe lid (not allow water to enter pipe
It is interior)；
3, the cryovial equipped with sample is boiled in boiling water take out after ten minutes (meat well-done subject to) be cooled to room temperature it is spare.It is made
The even liquid of sample, is put into sample storehouse；
4, immunomagnetic beads are added in the sample of suitable concentration, are incubated for 2 hours after mixing well in 37 DEG C, after effect, set
In capturing magnetic bead on magnetic frame, supernatant is removed, in cleaning storehouse with buffer solution for cleaning 3 times, is dissociated in elution storehouse with anhydrous methanol
Out on immunomagnetic beads, obtained meoh eluate nitrogen is blown into concentration, gained sample be after purification sulfamido (sample freezes,
It remains to carry out identification detection to sulfamido using ELISA).
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned
The all possible combination of each technical characteristic in embodiment is described, as long as however, thinking the group of these technical characteristics
It closes and contradiction is not present, be all considered as the range of this specification record.
Only several embodiments of the present invention are expressed for above embodiment, and the description thereof is more specific and detailed, but not
Therefore it can understand the limitation to the scope of the patents, it is noted that for those of ordinary skill in the art, do not taking off
Under the premise of from present inventive concept, various modifications and improvements can be made, these are all belonged to the scope of protection of the present invention.
Claims (8)
1. a kind of sulfamido Sample pretreatment kit based on immunomagnetic beads, which comprises the following steps: the reagent
Box is made of several parts such as sample storehouse, agent bin, cleaning storehouse, elution storehouses；Sample storehouse is used to place the pretreatment solution of sample,
Agent bin has been coupled sulfamido antibody, cleaning storehouse is washed for placing cleaning solution cleaning for placing immunomagnetic beads on immunomagnetic beads
De- storehouse is mainly used for elution of reactive conjugate；Step is main include the following:
(1) sample is collected in the capture of immunomagnetic beads
Sample pretreatment solution is put in sample storehouse first, anti-sulfamido antibody immune magnetic beads are put into agent bin, and rotation mixes,
Then immunomagnetic beads are added in sample storehouse, rotation is resuspended, and rotation capture 30min, is farthest caught at 37 DEG C with realizing
The sulfamido in sample is obtained, Magneto separate discards supernatant liquid；The immunomagnetic beads for capturing sample are used into cleaning solution in cleaning storehouse
Cleaning, jog are resuspended, and Magneto separate abandons supernatant；This process is 2-3 times repeatable；
After the completion of immunomagnetic ca pture sulfamido, then through Magneto separate, to realize the enrichment purification of sulfamido sample；Remove magnetic point
Supernatant from after, precipitating are the immunomagnetic beads for capturing sulfamido；
(2) elution of immunomagnetic beads
It is eluted eluent is added in elution storehouse in immunomagnetic beads obtained in step (1), room temperature shakes several seconds, magnetic
Separation, supernatant is the sulfamido sample after enrichment purification, can be used for subsequent measurement.
2. a kind of sulfamido Sample pretreatment kit based on immunomagnetic beads according to claim 1, it is characterised in that
The optimum reaction condition of the immunomagnetic beads enrichment purification sulfamido is, at 37 DEG C, pH7.4's, 0.02mo1/L contains methanol
Rotation capture 30min in 10% PBS buffer solution.
3. a kind of sulfamido Sample pretreatment kit based on immunomagnetic beads according to claim 1, it is characterised in that
The capture buffer of the use is preferably pH7.4, the PBS buffer solution containing methanol 10% of 0.02mo1/L.
4. a kind of sulfamido Sample pretreatment kit based on immunomagnetic beads according to claim 1, it is characterised in that
The eluent uses anhydrous methanol, and concentration of the immunomagnetic beads in eluent uses 5-10mg/mL；Room temperature shakes 1-5 points
Clock is advisable.
5. a kind of sulfamido Sample pretreatment kit based on immunomagnetic beads according to claim 1, it is characterised in that
Preferably, in step (1) anti-sulfamido antibody specificity immunomagnetic beads specific the preparation method comprises the following steps:
S1 cleaning: taking 0.4mg magnetic bead in 2mL centrifuge tube respectively, using 200 μ L PBT(0.02mo1/L, pH7.4, contains 0.05%
Tween-20 it) cleans 2 times, is resuspended after washing with 250 μ L PBS buffer solution (0.02mo1/L, pH7.4) ultrasonic disperses；
S2 activation: EDC and NHS that 200 μ L are prepared with PBS buffer solution (0.02mo1/L, pH7.4), 37 DEG C of activation are separately added into
2h keeps suspended state；
S3 coupling: Magneto separate sucks supernatant, and ibid three times, ultrasound is resuspended the washing of PBT buffer；
The anti-sulfamido antibody of 200 μ g is added in the magnetic bead activated respectively again, 37 DEG C of coupling reaction 5h, 15 r/min rotations
Turn to keep suspended state；
S4 closing: Magneto separate takes out supernatant, and the ethanolamine solutions and BSA confining liquid of 100 μ L are added, and 37 DEG C of closing 2h are kept
Suspended state；
S5 save: wash magnetic bead 3 times with PBT, Magneto separate, abandoning supernatant, with 250 μ LPBS buffers (0.02mo1/L, pH7.4,
Containing 0.02%NaN3 and 0.5%BSA) it is resuspended, it is saved in 4 DEG C；
Preferably, activator described in above-mentioned steps is EDC and NHS, and concentration is 1-5mg/mL, molten with activation buffer
Solution；
Preferably, in the above-mentioned method for preparing immunomagnetic beads, the preferred pH7.4 of coupling buffer, the PBS buffer solution of 0.02mo1/L；
Cleaning solution is preferably containing the pH7.4 of 0.05%Tween-20, the PBT buffer of 0.02mo1/L；Activator is prepared with PBS buffer solution；
The 5%BSA solution that confining liquid is preferably prepared with PBS buffer solution.
6. a kind of sulfamido Sample pretreatment kit based on immunomagnetic beads according to claim 1, it is characterised in that
The immunomagnetic beads pass through chemical coupling method by active group in magnetic microsphere surface modification, such as amino (- NH2), carboxyl (-
COOH) or epoxy group etc., for the chemical reagent used for EDC and NHS, concentration is 1-5mg/mL.
7. a kind of sulfamido Sample pretreatment kit based on immunomagnetic beads according to claim 1, it is characterised in that
The sulfamido antibody that has been coupled of the immunomagnetic beads coupling is sulfamido monoclonal antibody, sulfamido polyclonal antibody, nucleic acid
Aptamers etc..
8. a kind of sulfamido Sample pretreatment kit based on immunomagnetic beads according to claim 1, it is characterised in that
The sulfamido mainly includes sulfacetamide, sulphadiazine, sulphathiazole, sulfapryidine, methylsulfidine, sulfanilamide (SN) two
First oxazole, sulfadimidine, sulfamethoxypyridazine, cistosulfa, sulfamethoxazole, sulfametoxydiazine sodium, sulfanilamide (SN)
Sulfafurazole, sulfabenzamide, sulfaphenazolum, sulfaquinoxaline, sulfanitran, bacteresulf, sulfametoxydiazine etc..
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Application publication date: 20181211 |