CN106404963A - A method of preparing a mycotoxin immuno affinity column adopting resin as a carrier - Google Patents
A method of preparing a mycotoxin immuno affinity column adopting resin as a carrier Download PDFInfo
- Publication number
- CN106404963A CN106404963A CN201610585477.XA CN201610585477A CN106404963A CN 106404963 A CN106404963 A CN 106404963A CN 201610585477 A CN201610585477 A CN 201610585477A CN 106404963 A CN106404963 A CN 106404963A
- Authority
- CN
- China
- Prior art keywords
- zea
- don
- affinity column
- antibody
- mycotoxin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/56—Packing methods or coating methods
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/56—Packing methods or coating methods
- G01N2030/562—Packing methods or coating methods packing
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
A method of preparing a mycotoxin (DON, ZEA, T-2 and HT-2) four-in-one immuno affinity column adopting poly(glycidyl methacrylate)-divinylbenzene as a matrix is provided and belongs to preparation techniques. The method includes antibody preparing and purifying, matrix preparing, ligand coupling, and column filling. The column is novel in design, advanced in process, unique in method and wide in use. Problems such as matrix interferences, low specificity and a high cost of a traditional enriching process of fusarium toxins are overcome, and simultaneous purification of multiple targets is achieved. The column is high in specificity, high in mechanical strength and wide in pH suitable range, thus greatly increasing the using efficiency of the immuno affinity column, and the column is easy to operate, low in production cost and high in practicality and popularization performance.
Description
Technical field
The present invention relates to a kind of mycotoxin with poly (glycidylmethacrylate--co-ethylene dimethacrylate)-divinylbenzene as substrate
The preparation method of (DON, ZEA, T-2, HT-2) four-in-one immune affinity column, belongs to technology of preparing.
Background technology
Fusarium toxin is the secondary metabolite being produced by mycotoxin, has very big danger to the health of people and domestic animal
Evil, is widely present in various cereal crops and converted productss, is more common in the mouldy grain of storage in wet environment.Often from
Be detected in contaminated grain and feedstuff has 4 kinds：T-2 toxin, HT-2 toxin, deoxynivalenol DON and jade
Zearlenone ZEA.Mensure analysis method for fusarium toxin in cereal product is mainly chromatography, mostly need to be in conjunction with front
Process step, such as Solid-Phase Extraction (SPE).However, current SPE is the surface reservation based on physics and chemistry function, divide except obtaining target
Analysis beyond the region of objective existence is easily mixed with other matrix components, and the shortcoming of conventional solid phase extraction techniques is low to target analytes selectivity.Immunity
Affinity column is then a kind of high-efficient purification technology with high selectivity, using the specific recognition of antigen-antibody be implemented in combination with list
Individual compound or the selective extraction of a class compound.Immune affinity column is easy and simple to handle, reusable.In China although exempting from
Epidemic disease affinity solid phase abstraction technique is widely used in many laboratorys, but Solid-Phase Extraction material still relies primarily on import.Mesh
Immune affinity column present on front market, mostly based on Sepharose4B curdlan substrate, supplier is single for its filler
One, use cost is high.Select other substrate as filler, become the research direction of immune affinity column from now on.Polymethyl
Sour polyglycidyl-divinylbenzene (glycidyl methacrylate-divinylbenzene, abbreviation GMA-DVB) material is not subject to
The restriction of pH, mechanical strength is big.A kind of efficient reaping hook bacterium with poly (glycidylmethacrylate--co-ethylene dimethacrylate)-divinylbenzene as substrate
Plain multiple target (DON, ZEA, T-2, HT-2) immune affinity column, overcomes polysaccharide substrate immune affinity column bad mechanical strength, pH
The narrow defect with high cost of range of application, enables to purify while several mycotoxin again, greatlys save time and cost.
Content of the invention
The present invention provide a kind of mycotoxin with poly (glycidylmethacrylate--co-ethylene dimethacrylate)-divinylbenzene as substrate (DON,
ZEA, T-2, HT-2) four-in-one immune affinity column preparation method, above-mentioned difficulties can be overcome.
The present invention employs the following technical solutions to realize：One kind is with poly (glycidylmethacrylate--co-ethylene dimethacrylate)-divinylbenzene
The preparation method of mycotoxin (DON, ZEA, T-2, HT-2) the four-in-one immune affinity column for substrate, it includes the preparation of antibody
With purification, host material preparation, ligand coupling, dress four steps of post it is characterised in that：Take glycidyl third in proportion
Ester and divinylbenzene monomer, add 0.1～0.3g potassium peroxydisulfate KBS as the initiator of reaction, are allowed to be completely dissolved in two kinds
As oil phase in monomer.Add 4～5 times of monomer mass to go in the there-necked flask equipped with gyrator, thermometer and condensing tube
Ionized water, adds the gelatin of total Water 0.5% and 5% sodium chloride, starts and stir and be slowly heated, being warmed up to 50 DEG C will mix
Good oil phase is added in three mouthfuls of burnings, and logical nitrogen, to remove the oxygen in solution, is then warmed up to 60～80 DEG C of holding 2h, then rises
Temperature continues reaction to 85 DEG C, until resin sizing, is finally warmed up to 90 DEG C and keeps 2h.By the resin filter of synthesis, use hot water
Wash away gelatin, cool down to obtain poly (glycidylmethacrylate--co-ethylene dimethacrylate)-divinylbenzene, 37 DEG C of vacuum drying 24h.Take polymethylacrylic acid ring
Oxygen propyl ester-divinylbenzene swelling 12h in dimethyl sulfoxide, adds the there-necked flask equipped with gyrator, thermometer and condensing tube
In, and add excessive 1,6- hexamethylene diamine and boron trifluoride diethyl etherate as catalyst, 80 DEG C, 300r/min reacts 8h.Add second two
Alcohol continues reaction 3h, closes unreacted epoxy radicals.After reaction terminates, filter, washed three times with ethanol and deionized water are each, obtain
Amination poly (glycidylmethacrylate--co-ethylene dimethacrylate)-divinylbenzene, 37 DEG C of vacuum drying 24h.Take amination poly (glycidylmethacrylate--co-ethylene dimethacrylate)-
Divinylbenzene microspheres, with the PBS dispersion of pH 7.0, ultrasonic 30min, add excessive 25% glutaraldehyde, in 29 DEG C shake
Swing oscillating reactionss in water-bath (200r/min) 3h, stop activation.Then use the disodium hydrogen phosphate-phosphorus of the 0.2mol/L of pH 5.0
Acid dihydride sodium buffer (PBS) cleans 3 times and dissolves in this buffer, obtains resin suspension.Take 1～5mg DON antibody,
0.1～1mg T-2, HT-2 antibody, 0.2～1mg ZEA antibody, above-mentioned antibody are added 50%PBS- glycerite, then take
State the resin material 1mL (about 0.3g) activating, both are mixed, oscillating reactionss 12h in 25 DEG C of shaking bath pot.Using wet
Method fills post, and every pillar loads 1mL gel, after pillar installs, with the 0.01%NaN of the aseptic filtration of 5 times of bed volumes3-PBS
Cross post, and use 0.01%NaN3- PBS preserves.
Advantages of the present invention and good effect：
Modern design, technique is advanced, and method is unique, and purposes is wide.Not only solve fusarium toxin in traditional enrichment process
Present in matrix interference, specificity be low, high cost the problems such as, and realize multiple target and purify simultaneously, high specificity, machinery is strong
Degree is high, and pH is applied widely, drastically increases the service efficiency of immune affinity column.This poly (glycidylmethacrylate--co-ethylene dimethacrylate)-two
The preparation method of Ethenylbenzene fusarium toxin multi-objective immune affinity column makes the multiple target of fusarium toxin purify becomes efficient, fast
Fast, convenient, and high practicability, generalization.
Brief description
Legend be the mycotoxin with poly (glycidylmethacrylate--co-ethylene dimethacrylate)-divinylbenzene as substrate for the present invention (DON, ZEA,
T-2, HT-2) four-in-one immune affinity column preparation method schematic flow diagram.
Specific embodiment
Specific embodiment is provided by legend.What legend was given is a kind of with poly (glycidylmethacrylate--co-ethylene dimethacrylate)-divinylbenzene
The process chart of mycotoxin (DON, ZEA, T-2, HT-2) the four-in-one immune affinity column preparation for substrate, it includes antibody
Preparation and purification 1, host material preparation 2, ligand coupling 3, dress post 4.
Claims (3)
1. a kind of mycotoxin (DON, ZEA, T-2, HT-2) four with poly (glycidylmethacrylate--co-ethylene dimethacrylate)-divinylbenzene as substrate
The preparation method of unification immune affinity column, it includes the preparation and purification of antibody, host material preparation, ligand coupling, dress post four
Individual step it is characterised in that：Amino poly (glycidylmethacrylate--co-ethylene dimethacrylate)-divinylbenzene after 1mL activation and 1～5mg DON resist
Body, 0.1～1mg T-2, HT-2 antibody, 0.2～1mg ZEA antibody coupling.
2. mycotoxin with poly (glycidylmethacrylate--co-ethylene dimethacrylate) as substrate according to claim 1 (DON, ZEA, T-2,
HT-2) four-in-one immune affinity column preparation technology it is characterised in that：Its substrate is aggregated, amination, the poly- methyl-prop of activation
Olefin(e) acid polyglycidyl-divinylbenzene.
3. mycotoxin with poly (glycidylmethacrylate--co-ethylene dimethacrylate)-divinylbenzene as substrate according to claim 1 (DON,
ZEA, T-2, HT-2) four-in-one immune affinity column preparation technology it is characterised in that：The situation of antibody coupling include DON, ZEA,
Tri- kinds of antibody couplings of T-2；Tri- kinds of antibody couplings of DON, ZEA, HT-2；4 kinds of antibody coupling (some feelings of DON, ZEA, T-2, HT-2
Under condition, a kind of antibody of T-2 or HT-2 can be simultaneously used for purifying two kinds of mycotoxins of T-2 and HT-2).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610585477.XA CN106404963B (en) | 2016-07-25 | 2016-07-25 | Preparation method of mycotoxin immunoaffinity column using resin as carrier |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610585477.XA CN106404963B (en) | 2016-07-25 | 2016-07-25 | Preparation method of mycotoxin immunoaffinity column using resin as carrier |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106404963A true CN106404963A (en) | 2017-02-15 |
| CN106404963B CN106404963B (en) | 2023-06-16 |
Family
ID=58004608
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610585477.XA Active CN106404963B (en) | 2016-07-25 | 2016-07-25 | Preparation method of mycotoxin immunoaffinity column using resin as carrier |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106404963B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101869778A (en) * | 2009-04-24 | 2010-10-27 | 西安瑞仁生物技术有限公司 | Composite GMA (Glycidyl Methacrylate)/ZrO2 copolymer microsphere as well as preparation and application thereof |
| CN104785225A (en) * | 2015-04-15 | 2015-07-22 | 浙江大学 | Method for preparing antiphase weak anion exchange mixed mode chromatographic stationary phase by using organic polymer as substrate |
| CN104931695A (en) * | 2015-07-02 | 2015-09-23 | 北京农学院 | Immunoaffinity column for simultaneous analysis of four mycotoxins (DON, ZEA, T-2, HT-2) and preparation method thereof |
-
2016
- 2016-07-25 CN CN201610585477.XA patent/CN106404963B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101869778A (en) * | 2009-04-24 | 2010-10-27 | 西安瑞仁生物技术有限公司 | Composite GMA (Glycidyl Methacrylate)/ZrO2 copolymer microsphere as well as preparation and application thereof |
| CN104785225A (en) * | 2015-04-15 | 2015-07-22 | 浙江大学 | Method for preparing antiphase weak anion exchange mixed mode chromatographic stationary phase by using organic polymer as substrate |
| CN104931695A (en) * | 2015-07-02 | 2015-09-23 | 北京农学院 | Immunoaffinity column for simultaneous analysis of four mycotoxins (DON, ZEA, T-2, HT-2) and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| GÜNTHER STECHER 等: "Evaluation of extraction methods for the simultaneous analysis of simple and macrocyclic trichothecenes" * |
| PEIWU LI 等: "ADVANCED HYPHENATED CHROMATOGRAPHIC-MASS SPECTROMETRY IN MYCOTOXIN DETERMINATION: CURRENT STATUS AND PROSPECTS" * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106404963B (en) | 2023-06-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105424931B (en) | Helicobacter Pylori urease antibody IgM, IgG associating device for fast detecting and preparation method thereof | |
| CN101650366B (en) | Quick test paper for detecting enterovirus and method for preparing same | |
| CN106370868B (en) | Spr sensor of detection Microcystin based on aptamer signal amplification strategy and its preparation method and application | |
| CN107001432A (en) | The immunoglobulin binding polypeptide of mutation | |
| CN104356308B (en) | A kind of preparation method of bovine serum albumin molecular surface imprinting polymer microsphere | |
| CN103007846B (en) | Method for preparing protein loaded magnetic microsphere | |
| CN106226513A (en) | A kind of method of immunomagnetic beads detection by quantitative related antigen and application thereof | |
| CN105651999A (en) | Molybdenum disulfide-based sensor and preparation method and application thereof | |
| CN105131329B (en) | A kind of preparation method and application of the polyvinyl alcohol crosslinked affinity membrane of macropore chitosan of chelated metal ions | |
| CN108371942A (en) | A kind of composite magnetic nano-particle Fe3O4@Au/MPA/NTA-Ni2+And its it prepares and applies | |
| WO2017206713A1 (en) | Method for coupling magnetic particles with antibody molecules | |
| CN103301820B (en) | Core-shell type Rhodamine B molecular imprinting solid-phase extraction magnetic material, and preparation method and application thereof | |
| CN105498721B (en) | A kind of aflatoxin molecular engram material and preparation method thereof | |
| CN108982842A (en) | A kind of zearalenone pretreatment reagent kit using immunomagnetic bead technique | |
| CN102626609A (en) | Organic-inorganic hybrid protein molecular engram capillary tube monolithic column | |
| JP2011232098A (en) | Measuring method for immunoglobulin amount in solution | |
| CN104277189B (en) | A kind of preparation method of organic inorganic hybridization integral material | |
| CN106404963A (en) | A method of preparing a mycotoxin immuno affinity column adopting resin as a carrier | |
| CN113893833A (en) | Preparation method and application of molecularly imprinted composite two-dimensional material for high-throughput identification of aflatoxin | |
| CN102879580A (en) | Method for detecting trace protein | |
| CN104931695A (en) | Immunoaffinity column for simultaneous analysis of four mycotoxins (DON, ZEA, T-2, HT-2) and preparation method thereof | |
| CN108160056A (en) | A kind of nylon membrane preparation method for adsorbing heavy metal | |
| CN101787143B (en) | Method for preparing layer-by-layer self-assembled protein-imprinted polymer of chitosan | |
| CN106947038B (en) | Molecular imprinting stirring rod and preparation method thereof | |
| CN106000363B (en) | A kind of preparation method of phenyl boric acid hydrophilic silica gels material |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |