CN1086229C - High-efficient affinity film chromatography medium and its synthesis method - Google Patents
High-efficient affinity film chromatography medium and its synthesis method Download PDFInfo
- Publication number
- CN1086229C CN1086229C CN 97111908 CN97111908A CN1086229C CN 1086229 C CN1086229 C CN 1086229C CN 97111908 CN97111908 CN 97111908 CN 97111908 A CN97111908 A CN 97111908A CN 1086229 C CN1086229 C CN 1086229C
- Authority
- CN
- China
- Prior art keywords
- medium
- aglucon
- reaction
- affinity
- preparation
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The present invention relates to an efficient affinity film chromatogram medium. A wood cellulose compound film is used as a substrate, and a metal ion, triazine dye or protein ligand is loaded in a solid mode to obtain the affinity film chromatogram medium. An affinity film chromatogram column which is prepared by the medium is matched with a liquid phase chromatograph to directly test the content and the activity of big molecules, such as protein, enzymes, etc., and testing time is only 2 to 7 minutes. A usual ELISA method for testing the activity of the enzymes generally needs several hours. Because the chromatogram process of an affinity film analytical column for an efficient liquid phase chromatogram can be finished for 1 to 3 minutes, the efficient affinity film chromatogram medium can be used for on-line analysis and can be used for preparing fewer samples.
Description
Technical field
The present invention is a kind of high-efficient affinity film chromatography medium and synthetic method thereof that liquid-phase chromatographic analysis affinity film chromatography post is used that be used for.The affinity column that this medium is filled can with the supporting use of high performance liquid chromatograph, the biologically active material is measured fast; Also can from form complicated sample, extract and prepare one or more active components.
Background technology
In recent years, growing along with life science, biotechnology, pharmacy and medical skill, also more and more higher to the requirement of the macromolecular analysis of active bio, separation and purifying.For analysis, separation and the preparation of biomacromolecule (as protein, nucleic acid etc.), used method has the Perfusion Chromatography that liquid phase chromatography, classical affinity chromatography, high-effective affinity chromatography method, the vast Perseptive Biosystem of the high performance membrane chromatography and U.S. company released in 1992 usually.
Nowadays liquid phase chromatography still is to separate and analyze the dominant technology of various albumen and peptide matters.Liquid chromatography selectivity height, sensitivity, be easy to quantitatively, for the preparation of low amounts of product, purity, productive rate and activity are all higher.But classical HPLC also has its limitation: pressure drop is excessive, is difficult to the mass preparation of sample; One time analytical cycle is long, is difficult to be applied in the on-line analysis; This has also limited the method improvement of liquid chromatography and has optimized research simultaneously; For the selective adsorption of certain material in the lean solution, liquid chromatography can not realize.
Affinity chromatography is that biomacromolecule is separated the most effective means with purifying, and it has, and specificity is good, purifying multiple advantages of higher.Classical affinity chromatography matrix has agarose, glucosan, cellulose, silica gel, polyamide, fritted glass and various high molecular polymer.Wherein, agarose is to use the earliest, most widely used affinity media, advantage such as it has that bio-compatibility is good, structure homogeneous and non-specific adsorption are low, but because agarose is a kind of gel-like substance, very easily produce irreversible transformation, even by crosslinked increase intensity, the pressure that it can bear is also very limited, and this has limited its application greatly.The pressure that matrix such as fritted glass can bear is bigger, but the reactive group that the fritted glass surface can be utilized to connect aglucon very little.And affinity chromatography once to analyze the required time longer, this has just limited it to the application in the on-line analysis of time requirement strictness, and its improvement is had several directions: the one, and grain diameter is reduced, thereby reduce the evolving path, reduce analysis time, make it become high-effective affinity chromatography.Matrix commonly used in the high-effective affinity chromatography is the silica gel of better mechanical property, but the silicon hydroxyl on silica gel surface has part ion character, make that silica gel surface non-specific adsorption is stronger, and the tolerant pH value scope of silica gel institute is narrower; And increased on-stream pressure greatly when reducing granularity, it is unpractiaca preparing on a large scale in this way.Another direction of improvement affinity chromatography technology is a membrane chromatography.Membrane chromatography is that modification is carried out to reach optionally absorption in the surface of microporous barrier or ultra filtration membrane, and the evolving path of sample molecule is short in the membrane chromatography, can realize analyzing fast; But general membrane chromatography adsorption capacity is low.Membrane chromatography has relatively large cross-sectional area and less height, so pressure drop is lower; But, be difficult to realize the absorption fully of sample, thereby reduced productive rate because the height of bed is less; And the ratio of film system bed outer volume is higher, has increased the peak diffusion.These shortcomings make that the resolution of membrane chromatography is lower.
The POROS series chromatography matrix that is used for Perfusion Chromatography is close to perfection, and its skeleton is the very tygon divinylbenzene of homogeneous of aperture, its surface coverage one deck polyhydroxy hydrophilic polymer.With this matrix is that the chromatographic process of carrier can be finished within 5 minutes at 30 seconds mostly.This medium is used for ion-exchange chromatography now more.The shortcoming of this class matrix is very expensive.
Summary of the invention
The object of the present invention is to provide a kind of efficient analysis and affinity film chromatography medium that separates and synthetic method thereof of being suitable for.Can directly measure general only the need with the liquid chromatograph coupling by the prepared affinity film chromatography post of this medium with 2～7 minutes to macromolecular content such as albumen, enzyme and activity; And the ELISA method that is commonly used to measure enzymatic activity generally need be used several hrs.Because this shortest can in 1～3 minute, the finishing of chromatographic process that is used for the film affinity analyzing post of high performance liquid chromatography, thereby can be applied to on-line analysis.It also can be applicable to the preparation of sample in a small amount, and the cost of this analytical column is 1/50th of a POROS post.
Provided by the inventionly be suitable for the film medium that high performance liquid chromatography affinity membrane analytical column is used, it is characterized in that with the Wood Fiber Composite film as matrix, immobilized again aglucon and being suitable for of obtaining perform an analysis and the affinity film chromatography medium that separates.Supported aglucon can be according to analyzing and needs employing metallic ion, triasine dyes or the protein Preparation of sample separation become the metal-chelating high-efficient affinity film chromatography medium, triasine dyes or albumen are the high-effective affinity chromatography medium of aglucon.
The synthetic method of film medium of the present invention comprises the steps:
1. glytidyl methacrylate or acrylic acid epoxy propyl ester carry out self-polymeric reaction and make polymkeric substance;
2. polymkeric substance and lignose carry out graft reaction and make cellulose composite membrane;
3. composite membrane carries out ring-opening reaction;
4. film medium is made in the immobilized reaction of aglucon.
Specifically at first adopting glytidyl methacrylate or acrylic acid epoxy propyl ester to carry out self-polymeric reaction in film medium synthetic makes polymkeric substance, carries out graft reaction with xylogen again, so just made cellulose composite membrane.Autohemagglutination and graft reaction carry out in aqueous medium, and temperature of reaction is 50～90 ℃, should add initiating agent in the polyreaction, for example azoisobutyronitrile, n-BuLi or ammonium persulfate and sodium thiosulfate, and the initiating agent addition is 1～5% of a polymerization single polymerization monomer weight.For affinity chromatographic medium of metal chelating, add chelating aglucons such as imines ethane diacid and carry out ring-opening reaction; Adding metal ion solution then reacts; For triasine dyes class aglucon, need composite membrane acidolysis open loop is become adjacent hydroxyl, and then with the dyestuff bonding; Contain hydroxyl or amino aglucon for its itself, after needing the composite membrane acidolysis become adjacent hydroxyl, become aldehyde radical with sodium periodate oxidation again, and then with the aglucon bonding.Other condition in the film medium synthetic method of the present invention all can be carried out with reference to ordinary skill.As stated above Zhi Bei lignose composite membrane medium have that swelling is little, permeability good, withstand voltage height, not yielding and be easy to make the characteristics of film.
In sum, the present invention has following advantage:
1. analysis speed is fast, and chromatographic process only needs 1～7 minute, can be applicable to on-line analysis.
2. be easy to quantitatively, utilize liquid chromatography instrument signal recorder can easily carry out the quantitative of sample.
3. post forces down, and the normal running center pillar is pressed generally below 100psi.
4. cost is low, compares with the Perfusion chromatographic column that is used for similar sample analysis abroad, and cost is lower than 1/50th of imported product price.
5. column volume is little, and the post inner volume has only 0.5mL.
6. non-specific adsorption is little, and the non-specific adsorption of high-efficient affinity film chromatography post detects not come out with high performance liquid chromatograph.
Adopt analysis provided by the present invention with affinity media synthetic method and protein quantification assay method, can carry out quantitative measurement to biological active components such as albumen, enzymes quickly and easily.Can be applicable to on-line analysis, also can be used for specimen preparation in a small amount.This method has very application prospects in industries such as Biochemical Research, clinical medicine.
Description of drawings
The spectrogram that Fig. 1, copper ion chelating highly effective affinity film chromatography post are analyzed IgG.Wherein detecting wavelength is 280nm, and flow velocity is the 1.0ml/ branch, and sample size is 20ul.
The spectrogram that Fig. 2, triasine dyes Cibacron Blue F3GA high-efficient affinity film chromatography post are analyzed IgG.Wherein detecting wavelength is 280nm, and flow velocity is the 1.0ml/ branch, and sample size is 20ul.
The spectrogram that Fig. 3, albumin A high-efficient affinity film chromatography post are analyzed IgG.Wherein detecting wavelength is 280nm, and flow velocity is the 1.0ml/ branch, and sample size is 20ul.
The spectrogram of Fig. 4, albumin A high-efficient affinity film chromatography post non-specific adsorption BSA, first spectrum peak is the BSA that goes up sample, the non-specific adsorption of chromatographic column detects to come out substantially.
Embodiment
Be further described below by the application of example medium synthetic method of the present invention and chromatographic column.
Example 1: the synthetic and application of the efficient affinity film chromatography medium of imines ethane diacid (IDA) metal-chelating.
The building-up process of A, the efficient affinity film chromatography medium of metal-chelating is as follows:
1) the lignose composite membrane is synthetic
Carry out self-polymeric reaction with methyl-prop diluted acid epoxy propyl ester, be reflected in the aqueous medium and carry out, add polymerization single polymerization monomer and weigh 2% azoisobutyronitrile and make initiating agent, in the reaction 4 hours down of 80～85 ℃ of temperature.Under 75～80 ℃, add lignose and carried out graft reaction 3 hours behind the self-polymeric reaction, obtain the lignose composite membrane with the deionized water wash product.
2) cellulose composite membrane ring-opening reaction: the ring-opening reaction thing is imines ethane diacid (IDA), and the pH value of ring-opening reaction is controlled at 10～12, and temperature of reaction is 55～60 ℃.Reacted 5 hours.After stopping reaction, to neutral, use alcohol immersion with a large amount of deionized water rinsings then, the residue of flush away fiber surface is used deionized water rinsing again.
3) copper ion is immobilized: add the copper-bath (pH4.0) of 2% (W/V), reacted 2 hours, be washed till no copper ion with a large amount of deionized waters.The content that attracts spectroscopic methodology to record copper ion in the medium with atom is 29.4 μ mol/g.
B, utilize the film medium of A, preparation membrane chromatography post, the membrane chromatography rod structure that is adopted for and use the axia flow pattern that high performance liquid chromatograph is complementary always, the film bag forms for filling layer by layer.With copper ion chelating highly effective affinity film chromatography post immunoglobulin G in the solution (IgG) is analyzed: two solution that carry out gradient elution are respectively: solution A: 50mM phosphate buffer (PBS) contains 1M NaCl (pH8.2); Solution B: solution A adds the 100mM imidazoles.
Gather spectrogram such as the Fig. 1 of IgG in the copper ion chelating highly effective affinity film chromatography analytical column analytical solution with the liquid chromatography workstation, wherein first peak is in the sample copper ion not to be shown active component, and second peak is the active immune globulin G component that elutes.Can be easy to immunoglobulin G active component in the sample is carried out quantitatively by chromatographic work station.
Metal-chelating high-efficient affinity film chromatography post also can be used for many albumen that metallic ion had specific effect such as analyst's seralbumin, clotting factor, collagen, interferon, urokinase.
Example 2: triasine dyes (Cibacron Blue F3GA) is the synthetic and application of the high-efficient affinity film chromatography medium of aglucon.
A, triasine dyes (Cibacron Blue F3GA) are that the building-up process of high-efficient affinity film chromatography medium of aglucon is as follows:
1) utilize example 1 identical method to synthesize the lignose composite membrane.
2) cellulose composite membrane ring-opening reaction: cellulose composite membrane is carried out the acidolysis open loop.0.5M hydrochloric acid is added in the composite membrane matrix, make that the epoxide group acidolysis in the composite membrane becomes two adjacent hydroxyls, 72 hours reaction time.The reaction back is extremely neutral with a large amount of distilled water flushings.
3) the triasine dyes aglucon is immobilized: at first triasine dyes is dissolved in the carbonic acid buffer (the pH value is 11.0), concentration is 1%.Reaction time is 2 hours.After having reacted, affinity media is cleaned with distilled water.The content of immobilized affinity ligand be 3～5mg/g medium.
B, utilize the film medium of A, preparation membrane chromatography post, the membrane chromatography rod structure that is adopted for and use the axia flow pattern that high performance liquid chromatograph is complementary always, the film bag forms for filling layer by layer.With triasine dyes high-efficient affinity film chromatography post immunoglobulin G in the solution (IgG) is analyzed: two solution that carry out gradient elution are respectively: solution A: 10mM phosphate buffer (PBS); Solution B: solution A adds 1MNaCl (pH7.0).
Gather spectrogram such as the Fig. 2 of IgG in the triasine dyes high-efficient affinity film chromatography post analytical solution with the liquid chromatography workstation, wherein first peak is in the sample triasine dyes not to be shown active component, and second peak is active immune globulin G component.Can be easy to the active group of immunoglobulin G in the sample is carried out quantitatively by chromatographic work station.
Triasine dyes high-efficient affinity film chromatography post also can be used for many albumen that triasine dyes had specific effect such as analyst's seralbumin, interferon.
Example 3: with albumin A (Protein A) is the synthetic and application of the high-efficient affinity film chromatography medium of aglucon.
A: albumin A is that the building-up process of high-efficient affinity film chromatography medium of aglucon is as follows:
1) utilize example 1 identical method to synthesize the lignose composite membrane.
2) cellulose composite membrane ring-opening reaction: with triasine dyes high-efficient affinity film chromatography post.
3) albumin A (Protein A) aglucon is immobilized: at first the adjacent hydroxyl on the composite membrane of acidolysis open loop is oxidized to aldehyde radical with sodium periodate solution, the concentration of sodium periodate solution is 2% (w/w), and oxidization time is 10 minutes.Rinse film well with a large amount of distilled water the reaction back.Albumin A is dissolved in the 0.2M borate buffer (the pH value is 8.2), and concentration is 3mg/ml.Albumin A solution is carried out covalent bonding by pillar, add reductive agent itrile group sodium borohydride simultaneously, the reaction time is 12 hours.After having reacted, affinity media is cleaned with distilled water.The content of immobilized affinity ligand be 8～10mg/g medium.Utilize this bonding method can also immobilized other albumen, amino acid etc. contains the aglucon of amino or hydroxyl.
B, utilize the film medium of A, preparation membrane chromatography post, the membrane chromatography rod structure that is adopted for and use the axia flow pattern that high performance liquid chromatograph is complementary always, the film bag forms for filling layer by layer.With albumin A high-efficient affinity film chromatography post immunoglobulin G in the solution (IgG) is analyzed: two solution that carry out gradient elution are respectively: solution A: 50mM phosphate buffer (PBS) contains 0.15M NaCl, and the pH value is 7.0; Solution B: 0.2M glycocoll-hydrochloride buffer, pH value are 2.3.
Gather spectrogram such as the Fig. 3 of IgG active component in the albumin A high-efficient affinity film chromatography post analytical solution with the liquid chromatography workstation, wherein first peak is in the sample albumin A not to be shown active component, second peak is active immune globulin G component, and the 3rd peak is the solvent peak.Can be easy to carry out quantitatively by chromatographic work station in the sample albumin A being shown active igg fraction.
Albumin A high-efficient affinity film chromatography post also can be used for analyzing and various albumin A (Protein A) is shown active immunoglobulin G and various monoclonal, polyclonal antibody.
Example 4: the non-specific adsorption of high-efficient affinity film chromatography post.
A certain amount of bovine serum albumin(BSA) (BSA) is gone up sample to albumin A efficient affinity membrane analytical column, to investigate the non-specific adsorption of this affinity media, the spectrogram of gained such as Fig. 4, first peak is the BSA spectrum peak of sample among the figure, as seen from the figure, the efficient affinity membrane analytical column detects not come out to the absorption of BSA, and as seen its non-specific adsorption is very low.
Claims (6)
1. high-efficient affinity film chromatography medium is characterized in that with the lignose composite membrane as matrix, immobilized again aglucon and the affinity film chromatography medium that obtains; The supported aglucon of institute adopts metallic ion, triasine dyes or protein Preparation to become metal chelated affinity membrane chromatographic medium, and triasine dyes or albumen are the media for affinity chromatography of aglucon.
2. preparation method who prepares the described medium of claim 1, comprise the organism self-polymeric reaction, the polymer graft reaction, the immobilized reaction of composite membrane ring-opening reaction and aglucon,--grafting--open loop--aglucon is immobilized to occur in sequence reaction, and self-polymeric reaction is that glytidyl methacrylate or acrylic acid epoxy propyl ester carry out self-polymeric reaction and make polymkeric substance to it is characterized in that organism is by autohemagglutination; Graft reaction is that polymkeric substance and lignose carry out graft reaction and make cellulose composite membrane; Autohemagglutination and graft reaction carry out in aqueous medium, and temperature of reaction is 50～90 ℃, should add initiating agent in the polyreaction, and the initiating agent addition is 1～5% of a polymerization single polymerization monomer weight.
3. according to the described preparation method of claim 2, it is characterized in that initiating agent is azoisobutyronitrile, n-BuLi or ammonium persulfate and sodium thiosulfate.
4. according to the described preparation method of claim 2, it is characterized in that in the preparation of affinity chromatographic medium of metal chelating, add imines ethane diacid chelating aglucon and carry out ring-opening reaction; Adding metal ion solution then reacts.
5. according to the described preparation method of claim 2, it is characterized in that needing composite membrane acidolysis open loop is become adjacent hydroxyl in the medium preparation of triasine dyes class aglucon, and then with the dyestuff bonding.
6. according to the described preparation method of claim 2, it is characterized in that containing albumen and do in the medium preparation of aglucon, after needing the composite membrane acidolysis become adjacent hydroxyl, become aldehyde radical with sodium periodate oxidation again, and then with the aglucon bonding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 97111908 CN1086229C (en) | 1997-06-25 | 1997-06-25 | High-efficient affinity film chromatography medium and its synthesis method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 97111908 CN1086229C (en) | 1997-06-25 | 1997-06-25 | High-efficient affinity film chromatography medium and its synthesis method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1203363A CN1203363A (en) | 1998-12-30 |
| CN1086229C true CN1086229C (en) | 2002-06-12 |
Family
ID=5171942
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 97111908 Expired - Fee Related CN1086229C (en) | 1997-06-25 | 1997-06-25 | High-efficient affinity film chromatography medium and its synthesis method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1086229C (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103364356B (en) * | 2013-07-24 | 2016-04-13 | 西北大学 | A kind of method measuring adsorption of metal ions steadiness parameter on fixing metal affinity column |
| CN103586008A (en) * | 2013-10-24 | 2014-02-19 | 中国科学院过程工程研究所 | Affinity chromatography medium and preparation method and application thereof |
| WO2017012886A1 (en) * | 2015-07-17 | 2017-01-26 | Ares Trading S.A. | Methods for modulating production profiles of recombinant proteins |
| EP3395422B1 (en) * | 2017-04-24 | 2022-01-26 | Biotage AB | Removal of metal ions from essential oils |
-
1997
- 1997-06-25 CN CN 97111908 patent/CN1086229C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1203363A (en) | 1998-12-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Křenková et al. | Immobilized microfluidic enzymatic reactors | |
| JP4303885B2 (en) | Free solution ligand interaction molecule separation method | |
| Sportsman et al. | Chromatographic properties of silica-immobilized antibodies | |
| EP2404162B1 (en) | Detection system and method for high sensitivity fluorescent assays | |
| US6358692B1 (en) | High speed, automated, continuous flow, multi-dimensional molecular selection and analysis | |
| Gunasena et al. | Organic monoliths for hydrophilic interaction electrochromatography/chromatography and immunoaffinity chromatography | |
| CN1563989A (en) | Method of detecting small molecule compound and its special biochip | |
| US20110124118A1 (en) | Microfluidic structure for detecting biomolecule and microfluidic device comprising the same | |
| US20070048795A1 (en) | Immunoaffinity separation and analysis compositions and methods | |
| CA2829549A1 (en) | Method for recognition and quantification of multiple analytes in a single analysis | |
| CN1086229C (en) | High-efficient affinity film chromatography medium and its synthesis method | |
| WO2005049653A1 (en) | Affinity separation composition and method | |
| US20080176254A1 (en) | Affinity separation compositions and methods | |
| CN101046479A (en) | Process of preparing human serum base matter containing no target protein | |
| JPH063449B2 (en) | Stable immobilized hapten reagent for use in heterogeneous immunoassay, method for producing the same and use thereof | |
| Bayer et al. | Avidin column as a highly efficient and stable alternative for immobilization of ligands for affinity chromatography | |
| EP1239285A1 (en) | Improved homogeneous immunoassay method | |
| US20150105280A1 (en) | Selector based recognition and quantification system and method for multiple analytes in a single analysis | |
| CA2503601A1 (en) | A protein chips detecting system which can simultaneously detect multi target | |
| JPH08201383A (en) | Method for determining sugar chain structure | |
| EP1406089A1 (en) | A method for selectively staining water soluble protein using reactive dye | |
| CN116046506B (en) | Separation and purification method of natural high-purity glycosylated hemoglobin HbA1c and hemoglobin HbA0 | |
| Trinh et al. | Impact of double cryogelation process on a macroporous dye‐affinity hydrogel | |
| US5306615A (en) | Immunoassays and nucleic acid assays with liquid exchange supports | |
| US20140066605A1 (en) | Purification of biological conjugates by size exclusion chromatography |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20020612 Termination date: 20120625 |