CN103230784A - Composite continuous bed cryogel and preparation thereof, and application in separating IgG and albumin - Google Patents
Composite continuous bed cryogel and preparation thereof, and application in separating IgG and albumin Download PDFInfo
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- CN103230784A CN103230784A CN2013101517260A CN201310151726A CN103230784A CN 103230784 A CN103230784 A CN 103230784A CN 2013101517260 A CN2013101517260 A CN 2013101517260A CN 201310151726 A CN201310151726 A CN 201310151726A CN 103230784 A CN103230784 A CN 103230784A
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Abstract
The invention discloses a composite continuous bed cryogel and a preparation method thereof, and application in quickly separating human serum IgG and albumin. The pore size of the composite continuous bed cryogel is 0.1-300 mu m, and the porosity is 80-95%; and the composite continuous bed cryogel has a hydrophobic benzyl-anion exchange tertiary amino functional group disclosed as Formula (I). The composite continuous bed cryogel polymer chain simultaneously contains amino ion-exchange group and benzyl group with certain hydrophobic functions, so the cryogel can form multi-point adsorption with different competitivenesses with the IgG macromolecule and albumin; and thus, after the composite continuous bed cryogel is sequentially decomposed after being eluted by saliferous eluates with different concentrations. The composite continuous bed cryogel has the advantages of high separation purity and favorable separating properties. The chromatography method has the advantages of fewer steps and quick separation process, is simple to operate, and can implement quick separation, thereby having wide application prospects in the aspect of serum protein separation; and the eluates are buffer solution can be easily prepared. In Formula (I), n is a positive integer.
Description
(1) technical field
The present invention relates to a kind of compound brilliant glue continuous bed medium and preparation method thereof, and separating immune globulin IgG and the sero-abluminous application fast from human serum of this compound brilliant glue continuous bed medium.
(2) background technology
Human serum antibody and albumin have important application at numerous areas such as biotechnology, clinical medicine and diagnosis, and serum is to obtain IgG and albuminous main source at present.Existing separation method as saltout, methods such as precipitation with alcohol, metal ion affinity chromatography, dye affinity chromatography, Protein A affinity chromatography, amino acid aglucon affinity chromatography are studying aspect IgG and the albumin separating, the part technology has obtained application.But, these methods usually run into such as affinity ligand leakage easily, aglucon after target protein is combined, enter eluent and be difficult for to remove, purification procedures is many, process time is long, IgG and albumin lose problems such as biologically active easily in separation process.In recent years, mixing mode expanded bed medium also begins for IgG and albuminous adsorbing separation, but because the influence that bed disperses and the research of efficient separating medium relatively lags behind, when being directly used in from human serum IgG with albuminous a large amount of separation, still needing and to carry out the work of the system of going deep into.Therefore, research separates IgG and albuminous new method fast from human serum, significant.
The crystal glue chromatography bio-separation method that occurs is separating medium with the brilliant glue with super large hole recently, fast separating bio macromolecular substances from zymotic fluid.Document (Bereli N., et al., Materials Science and Engineering2010,30:323 – 329) is once with triasine dyes Cibacron Blue F3GA and Cu
2+Affine crystal glue chromatography method is separated IgG and albumin, but these affinity medias selectively little to these two kinds of target proteins, what obtain is the mixture of these two kinds of albumen, and being difficult to separately becomes independently product with the two, and the adsorption capacity of column is lower.Document (Alkan et al.; Biochemical Engineering Journal2009; 45:201 – 208) once used the affine crystal gel medium chromatography of Protein A IgG; but because ten fens costlinesses of Protein A; and existing aglucon to leak and the problem of pollution target protein, scale separates and is difficult to realize.
(3) summary of the invention
The object of the invention provides a kind of compound brilliant glue continuous bed medium and preparation and application thereof, and described compound brilliant glue continuous bed medium is suitable for immune globulin antibody (IgG) and albumin in the quick separation of human serum.
The technical solution used in the present invention is:
The invention provides a kind of compound brilliant glue continuous bed medium, described compound brilliant glue continuous bed pore size of media 0.1～300 μ m, porosity 80～95%, described compound brilliant glue continuous bed medium has the functional group of hydrophobic benzyl shown in the formula (I)-anion exchange uncle amino:
N is positive integer in the formula (I);
Described compound brilliant glue continuous bed medium is that hydrophobic benzyl-anion exchange uncle amino functional group is immobilized in the skeleton of compound brilliant glue continuous bed dielectric matrix;
Described compound brilliant glue continuous bed dielectric matrix prepares as follows: saturated cellulose grain is added among the mixed aqueous solution A of hydroxyethyl methacrylate and polyethyleneglycol diacrylate, under the effect of catalyst, through crystallization pore and polymerisation, make described compound brilliant glue continuous bed dielectric matrix; Described saturated cellulose grain be cellulose powder is adsorbed in the mixed aqueous solution B of hydroxyethyl methacrylate and polyethyleneglycol diacrylate saturated, preferred 300～1500 μ m of described cellulose powder particle diameter＜2mm(); Described catalyst is ammonium persulfate with the tetramethyl diethylamine with the mixing of mass ratio 1:1～3, described catalyst quality consumption be among the mixed aqueous solution A hydroxyethyl methacrylate and polyethyleneglycol diacrylate gross mass 0.5～1%; Hydroxyethyl methacrylate and polyethyleneglycol diacrylate mass ratio are 3:1 among the described mixed aqueous solution A, and hydroxyethyl methacrylate and polyethyleneglycol diacrylate total mass concentration are 15% among the mixed aqueous solution A; Described saturated cellulose grain quality consumption is counted 3.6～6.4g/ml with the volumetric usage of mixed aqueous solution A; Described mixed aqueous solution B forms identical with mixed aqueous solution A.
Further, the temperature of described crystallization pore and polymerisation is-15 ℃, and the reaction time is 20h.
Further again, compound brilliant glue continuous bed medium of the present invention makes as follows: can be raw material with the monomer of compound brilliant glue continuous bed dielectric matrix generation graft reaction, with Cu
3+Deionized water solution (preferred K
5[Cu (HIO
6)
2] aqueous solution) be catalyst, immobilized in compound brilliant glue continuous bed dielectric matrix by carrying out graft reaction with described compound brilliant glue continuous bed dielectric matrix, namely obtain described compound brilliant glue continuous bed medium; The monomer of described graft reaction is N, N, N-trimethyl-ethylene base benzene first ammonium chloride; The monomer of described graft reaction adds with the form of 0.5mol/L monomer solution, and the volumetric usage of described monomer solution is 5 times of compound brilliant glue continuous bed dielectric matrix volume, described Cu
3+The concentration of deionized water solution is 0.037M, Cu
3+The deionized water solution volumetric usage is 3 times of compound brilliant glue continuous bed dielectric matrix volume; The temperature of described graft reaction is 40～55 ℃, and the reaction time is 0.5～4h.
Cellulose grain in the described compound crystal gel medium matrix is embedded in the poly hydroxy ethyl acrylate, the sub-micron that is suitable for the IgG Molecular Adsorption is in a large number arranged to several microns fine pore in the particle; And the poly hydroxy ethyl acrylate skeleton mainly forms the super large hole that size reaches several microns to hundreds of microns, other impurity and the foreign protein be convenient in the serum pass through fast smoothly, the cellulose grain consumption accounts for the mass percent about 28～40% of matrix scaffold polymer, cellulose powder particle size＜2mm scope, average grain diameter 800～900 μ m.
The present invention also provides the application of a kind of described compound brilliant glue continuous bed medium in quick separation of human serum IgG and albumin, described being applied as: with the buffer solution (Na of preferred pH7.2～7.8,20～50mM of human serum with pH7.2～7.8
2HPO
4/ NaH
2PO
4Buffer solution) the human serum dilution is made in dilution, be that chromatographic column separates with compound brilliant glue continuous bed medium then, (namely use the NaCl final concentration 50～100mM of the buffer solution preparation of pH7.2～7.8, the eluent of 150～200mM, 1M, the Na of preferred pH7.2～7.8,20～50mM with the NaCl eluent of 50～100mM, 150～200mM, 1M respectively
2HPO
4/ NaH
2PO
4The outflow liquid (it doesn't matter for peak shape, as long as there is the peak to occur just beginning to collect) of eluting peak appears in wash-out buffer solution) when collecting the NaCl eluent wash-out of 50～100mM, namely get human serum albumins through dialysis and freeze drying; Occur the outflow liquid of eluting peak when collecting the NaCl eluent wash-out of 150～200mM, namely get IgG through dialysis and freeze drying.
Further, described human serum is made the human serum dilution for 6～15 times with the phosphate buffer dilution of pH7.2～7.8,20～50mM.
Further, the application optimum condition of the compound brilliant glue continuous bed medium of the present invention in quick separation of human serum IgG and albumin is as follows: with the phosphate (Na of human serum with 20mM, pH7.8
2HPO
4/ NaH
2PO
4) buffer solution dilutes 15 times, is that chromatographic column separates with compound brilliant glue continuous bed medium, with sample on the speed of 1cm/min, uses the phosphate (Na of 20mM, pH7.8 then
2HPO
4/ NaH
2PO
4) buffer solution washes to balance, the eluent that is respectively 100mM, 150mM, 1M with the NaCl final concentration of the phosphate buffer of 20mM, pH7.8 preparation wash-out successively again, elution flow rate is 1cm/min, the outflow liquid that occurs eluting peak when collecting NaCl final concentration 100mM eluent wash-out, namely get human serum albumins through dialysis and freeze drying (preferably the MWCO50000 dialysis membrane is with the deionized water 24h that dialyses, then at-60 ℃ of following freeze drying 24h); The outflow liquid that occurs eluting peak when collecting NaCl final concentration 150mM eluent wash-out namely gets immune globulin antibody IgG through dialysis and freeze drying (preferably the MWCO100000 dialysis membrane is with the deionized water 24h that dialyses, then at-65 ℃ of following freeze drying 24h); Flow velocity by compound brilliant glue continuous bed medium post in the chromatography process is 1cm/min.
Mixed aqueous solution A of the present invention and mixed aqueous solution B are mixed aqueous solution, name for ease of the amount difference of distinguishing the used mixed aqueous solution of different step, and letter itself does not have implication.
Human serum antibody provided by the invention and albuminous fast separating process and compound crystal gel medium thereof have following characteristics:
(1) matrix of compound brilliant glue continuous bed medium provided by the invention is cellulose grain and poly hydroxy ethyl acrylate, the extensive use in clinical and medical diagnosis of these two kinds of materials, its biocompatibility and biological safety are good, very stable under the serum isolating environment, can reuse, make the human serum IgG and the albumin product that adopt its adsorbing separation to obtain have good security.
(2) compound brilliant glue continuous bed medium provided by the invention is different with existing ion-exchange or affine crystal gel medium, cellulose grain is embedded in the poly hydroxy ethyl acrylate with the porous particle form in the compound brilliant glue continuous bed medium of the present invention, form the multilevel pore network that possesses diffusion mass transfer absorption aperture and convective mass transfer super big hole simultaneously, be suitable for the big molecule of serum IgG and albuminous adsorption chromatography.
(3) contain the benzyl of amino-type ion-exchange group and certain hydrophobic function in the compound brilliant glue continuous bed dielectric polymers chain provided by the invention simultaneously, help the big molecule of crystal gel medium and IgG and albumin to form different competitive multiple spot absorption, make it contain the salt eluent stepwise elution through variable concentrations, resolved successively, the separation purity height, separating property is good.
(4) chromatography method step provided by the invention is few, simple to operate, and used eluent and buffer solution all are easy to configuration, and separation process is rapid, can realize quick separation, is having broad application prospects aspect the separation of haemocyanin.
(4) specific embodiment
Fig. 1 is the sem photograph of inside of the compound brilliant glue continuous bed medium of embodiment 1 preparation.
Fig. 2 is the sem photograph on surface of the compound brilliant glue continuous bed medium of embodiment 1 preparation.
(5) specific embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1
Cellulose powder is (commercially available, particle diameter 300～1500 μ m, the about 800 μ m of average grain diameter) (hydroxyethyl methacrylate quality and polyethyleneglycol diacrylate total mass concentration are 15% in the mixed aqueous solution at the mixed aqueous solution of hydroxyethyl methacrylate and polyethyleneglycol diacrylate, hydroxyethyl methacrylate quality and polyethyleneglycol diacrylate mass ratio are 3:1) in absorption saturated, the saturated cellulose grain of 4.25g is added the mixed aqueous solution of 1.18mL hydroxyethyl methacrylate and polyethyleneglycol diacrylate, and (hydroxyethyl methacrylate quality and polyethyleneglycol diacrylate total mass concentration are 15% in the mixed aqueous solution, mass ratio is 3:1), add the catalyst 0.089g that ammonium persulfate and tetramethyl diethylamine are mixed and made into mass ratio 1:1 again, under-15 ℃ through crystallization pore and polymerisation 20h, prepare the compound brilliant glue continuous bed matrix of poly hydroxy ethyl acrylate matrix and cellulose base, be described compound brilliant glue continuous bed dielectric matrix (diameter 10mm, height 49mm).
(diameter 10mm, height 49mm) is the K of 0.037M in 11.5mL concentration with compound brilliant glue continuous bed dielectric matrix
5[Cu (HIO
6)
2] in the aqueous solution, be the N of 0.5M with 19mL concentration, N, N-trimethyl-ethylene base benzene first aqueous ammonium chloride solution is in 40 ℃ of following graft reaction 4h, obtain the compound brilliant glue continuous bed medium of poly hydroxy ethyl acrylate matrix and cellulose base (being compound brilliant glue continuous bed medium), the sem photograph of its microstructure is seen illustrated in figures 1 and 2.After tested, the mass ratio that the contained cellulose grain of this medium accounts for brilliant gel matrix skeleton is 28%, effective drainage porosity 81%, maximum pore rate 95%, about 0.1～300 μ m of pore size.Be that model protein carries out adsorption chromatography with the bovine serum albumin, the dynamic adsorbance to bovine serum albumin under the flow velocity 1cm/min reaches 1.3mg/mL.
Embodiment 2
Cellulose powder is (commercially available, particle diameter 300～1900 μ m, the about 900 μ m of average grain diameter) (hydroxyethyl methacrylate quality and polyethyleneglycol diacrylate total mass concentration are 15% in the mixed aqueous solution at the mixed aqueous solution of hydroxyethyl methacrylate and polyethyleneglycol diacrylate, mass ratio is 3:1) in absorption saturated, the saturated cellulose grain of 6.5g is added the mixed aqueous solution of 1.1mL hydroxyethyl methacrylate and polyethyleneglycol diacrylate, and (hydroxyethyl methacrylate quality and polyethyleneglycol diacrylate total mass concentration are 15% in the mixed aqueous solution, mass ratio is 3:1), add the catalyst 0.083g that ammonium persulfate and tetramethyl diethylamine are mixed and made into mass ratio 1:1 again, under-15 ℃ through crystallization pore and polymerisation 20h, prepare the compound brilliant glue continuous bed matrix of poly hydroxy ethyl acrylate matrix and cellulose base, be described compound brilliant glue continuous bed dielectric matrix (diameter 10mm, height 50mm).
Be the K of 0.037M in 14.3mL concentration with compound brilliant glue continuous bed dielectric matrix
5[Cu (HIO
6)
2] in the aqueous solution, be the N of 0.5M with 23.8mL concentration, N, N-trimethyl-ethylene base benzene first aqueous ammonium chloride solution is in 55 ℃ of following graft reaction 0.5h, obtain poly hydroxy ethyl acrylate matrix and the compound brilliant glue continuous bed medium of cellulose base of diameter 11mm, height 50mm, the mass ratio that the contained cellulose grain of its matrix accounts for brilliant glue skeleton is 39%, effective drainage porosity 80%, maximum pore rate 90%, about 0.1～200 μ m of pore size.
With the phosphate (Na of the healthy human serum of 0.3mL with 50mM
2HPO
4/ NaH
2PO
4) 15 times of buffer solution (pH=7.8) dilutions, with sample on the 1cm/min flow velocity, carry out chromatography with the compound brilliant glue continuous bed medium of above-mentioned steps preparation, use PBS buffer solution (Na then
2HPO
4/ NaH
2PO
4, pH=7.8,50mM) wash to balance, and use the NaCl eluent (pH=7.8, the preparation of 50mM buffer solution) of 100mM, 200mM, 1M to carry out stepwise elution respectively, the outflow liquid that occurs eluting peak when collecting NaCl final concentration 100mM eluent wash-out, through the MWCO50000 dialysis membrane with the deionized water 24h that dialyses, then at-60 ℃ of following freeze drying 24h, obtain human serum albumins, SDS-PAGE electrophoresis detection purity is about 98%, its adsorption capacity 0.2mg/mL bed; The outflow liquid that occurs eluting peak when collecting NaCl final concentration 200mM eluent wash-out, through the MWCO100000 dialysis membrane with the deionized water 24h that dialyses, then at-65 ℃ of following freeze drying 24h, obtain immune globulin antibody IgG, about 75% through SDS-PAGE electrophoresis detection purity, its adsorption capacity 0.2mg/mL bed.The total protein adsorption capacity reaches the 0.6mg/mL bed.
Embodiment 3
Get the compound brilliant glue continuous bed medium among the embodiment 2, other are operated with embodiment 2, with the phosphate (Na of the healthy human serum of 0.2mL with 20mM
2HPO
4/ NaH
2PO
4) 15 times of buffer solution (pH=6.5) dilutions, carry out compound brilliant glue continuous bed chromatography with the 1cm/min flow velocity, use PBS buffer solution (pH=6.5,20mM) flushing then, and use the NaCl eluent (Na of pH=6.5,20mM of 50mM, 150mM, 1M respectively
2HPO
4/ NaH
2PO
4The buffer solution configuration) carries out stepwise elution, the outflow liquid that occurs eluting peak when collecting the eluent wash-out of NaCl final concentration 50mM, through the MWCO50000 dialysis membrane with the deionized water 24h that dialyses, then at-60 ℃ of following freeze drying 24h, obtain human serum albumins, SDS-PAGE electrophoresis detection purity is about 94%, and it is the 0.21mg/mL bed that the Coomassie brilliant blue method records adsorbance; Collect the eluting peak of the eluent correspondence of NaCl final concentration 150mM, with the deionized water 24h that dialyses, at-65 ℃ of following freeze drying 24h, obtain immune globulin antibody IgG through the MWCO100000 dialysis membrane then, purity is about 82%, and adsorption capacity is the 0.03mg/mL bed.The total protein adsorption capacity is the 0.29mg/mL bed.
Embodiment 4
Get the compound brilliant glue continuous bed medium among the embodiment 2, other are operated with embodiment 2, with phosphate buffer (pH=7.2, the Na of the healthy human serum of 1mL with 20mM
2HPO
4/ NaH
2PO
4) dilute 6 times, carry out compound brilliant glue continuous bed chromatography with the 1cm/min flow velocity, with PBS buffer solution (pH=7.2,20mM, Na
2HPO
4/ NaH
2PO
4) wash to balance, and use 100mM respectively, NaCl eluent (pH=7.2,20mM, the Na of 150mM, 1M
2HPO
4/ NaH
2PO
4The buffer solution preparation) carries out stepwise elution, the outflow liquid that occurs eluting peak when collecting NaCl final concentration 100mM eluent wash-out, through the MWCO50000 dialysis membrane with the deionized water 24h that dialyses, then at-60 ℃ of following freeze drying 24h, obtain human serum albumins, about 40% through SDS-PAGE electrophoresis detection purity, it is the 0.06mg/mL bed that the Coomassie brilliant blue method records adsorbance; The outflow liquid that occurs eluting peak when collecting NaCl final concentration 150mM eluent wash-out, with the deionized water 24h that dialyses, at-65 ℃ of following freeze drying 24h, obtain immune globulin antibody IgG through the MWCO100000 dialysis membrane then, purity is about 74%, and adsorption capacity is the 0.2mg/mL bed.The total protein adsorption capacity is the 1.1mg/mL bed.
Embodiment 5
Get the compound brilliant glue continuous bed medium among the embodiment 2, other are operated with embodiment 2, with phosphate buffer (pH=7.8, the Na of the healthy human serum of 0.2mL with 20mM
2HPO
4/ NaH
2PO
4) dilute 15 times, carry out compound brilliant glue continuous bed chromatography with the 1cm/min flow velocity, use PBS buffer solution (pH=7.8,20mM, Na then
2HPO
4/ NaH
2PO
4) wash to balance, and use 100mM respectively, NaCl eluent (pH=7.8,20mM, the Na of 150mM, 1M
2HPO
4/ NaH
2PO
4The buffer solution configuration) carries out stepwise elution, the outflow liquid that occurs eluting peak when collecting NaCl final concentration 100mM eluent wash-out, through the MWCO50000 dialysis membrane with the deionized water 24h that dialyses, then at-60 ℃ of following freeze drying 24h, obtain human serum albumins, about 98% through SDS-PAGE electrophoresis detection purity, recording adsorbance in conjunction with the Coomassie brilliant blue method is the 0.2mg/mL bed; The outflow liquid that occurs eluting peak when collecting NaCl final concentration 200mM eluent wash-out, the MWCO100000 dialysis membrane at-65 ℃ of following freeze drying 24h, obtains immune globulin antibody IgG then with the deionized water 24h that dialyses, purity is about 84%, and adsorption capacity is the 0.04mg/mL bed.The total protein adsorption capacity is the 0.3mg/mL bed.
Claims (6)
1. compound brilliant glue continuous bed medium, it is characterized in that described compound brilliant glue continuous bed pore size of media 0.1～300 μ m, porosity 80～95%, described compound brilliant glue continuous bed medium has the functional group of hydrophobic benzyl shown in the formula (I)-anion exchange uncle amino:
N is positive integer in the formula (I);
Described compound brilliant glue continuous bed medium is that hydrophobic benzyl-anion exchange uncle amino functional group is immobilized in the skeleton of compound brilliant glue continuous bed dielectric matrix;
Described compound brilliant glue continuous bed dielectric matrix prepares as follows: saturated cellulose grain is added among the mixed aqueous solution A of hydroxyethyl methacrylate and polyethyleneglycol diacrylate, under the effect of catalyst, through crystallization pore and polymerisation, make described compound brilliant glue continuous bed dielectric matrix; Described saturated cellulose grain be cellulose powder is adsorbed in the mixed aqueous solution B of hydroxyethyl methacrylate and polyethyleneglycol diacrylate saturated, described cellulose powder particle diameter＜2mm; Described catalyst is ammonium persulfate with the tetramethyl diethylamine with the mixing of mass ratio 1:1～3, described catalyst quality consumption be among the mixed aqueous solution A hydroxyethyl methacrylate and polyethyleneglycol diacrylate gross mass 0.5～1%; Hydroxyethyl methacrylate and polyethyleneglycol diacrylate mass ratio are 3:1 among the described mixed aqueous solution A, and hydroxyethyl methacrylate and polyethyleneglycol diacrylate total mass concentration are 15% among the mixed aqueous solution A; Described saturated cellulose grain quality consumption is counted 3.6～6.4g/ml with the volumetric usage of mixed aqueous solution A; Described mixed aqueous solution B forms identical with mixed aqueous solution A.
2. compound brilliant glue continuous bed medium according to claim 1 is characterized in that 15 ℃ of the temperature Wei – of described crystallization pore and polymerisation, and the reaction time is 20h.
3. compound brilliant glue continuous bed medium as claimed in claim 1 is characterized in that described compound brilliant glue continuous bed medium makes as follows: can be raw material with the monomer of compound brilliant glue continuous bed dielectric matrix generation graft reaction, with Cu
3+Deionized water solution is catalyst, and is immobilized in compound brilliant glue continuous bed dielectric matrix by carrying out graft reaction with described compound brilliant glue continuous bed dielectric matrix, namely obtains described compound brilliant glue continuous bed medium; The monomer of described graft reaction is N, N, N-trimethyl-ethylene base benzene first ammonium chloride; The monomer of described graft reaction adds with the form of 0.5mol/L monomer solution, and the volumetric usage of described monomer solution is 5 times of compound brilliant glue continuous bed dielectric matrix volume, described Cu
3+The concentration of deionized water solution is 0.037M, Cu
3+The deionized water solution volumetric usage is 3 times of compound brilliant glue continuous bed dielectric matrix volume; The temperature of described graft reaction is 40～55 ℃, and the reaction time is 0.5～4h.
4. the application of the described compound brilliant glue continuous bed medium of claim 1 in quick separation of human serum IgG and albumin, it is characterized in that described being applied as: human serum is made the human serum dilution with the buffer solution dilution of pH7.2～7.8, be that chromatographic column separates with compound brilliant glue continuous bed medium then, respectively with the NaCl eluent wash-out of 50～100mM, 150～200mM, 1M, occur the outflow liquid of eluting peak when collecting the NaCl eluent wash-out of 50～100mM, namely get human serum albumins through dialysis and freeze drying; Occur the outflow liquid of eluting peak when collecting the NaCl eluent wash-out of 150～200mM, namely get IgG through dialysis and freeze drying.
5. as application as described in the claim 4, it is characterized in that described human serum is with 6～15 times of the phosphate buffer dilutions of pH7.2～7.8,20～50mM.
6. as application as described in the claim 4, it is characterized in that described being applied as: with human serum 20mM, 15 times of the phosphate buffer dilutions of pH7.8, be that chromatographic column separates with compound brilliant glue continuous bed medium, with sample on the speed of 1cm/min, use 20mM then, the phosphate buffer flushing of pH7.8, use 20mM again, the NaCl final concentration of the phosphate buffer preparation of pH7.8 is respectively 100mM, 150mM, the eluent of 1M is wash-out successively, elution flow rate is 1cm/min, occur the outflow liquid of eluting peak when collecting NaCl final concentration 100mM eluent wash-out, namely get human serum albumins through dialysis and freeze drying; Occur the outflow liquid of eluting peak when collecting NaCl final concentration 150mM eluent wash-out, namely get immune globulin antibody IgG through dialysis and freeze drying.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593340A (en) * | 2015-01-15 | 2015-05-06 | 浙江工业大学 | Method for separating lactoperoxidase from bovine whey |
| CN104607162A (en) * | 2015-01-15 | 2015-05-13 | 浙江工业大学 | Cation exchange chimeric cryogel separation medium and preparation method thereof |
| CN106674443A (en) * | 2016-12-23 | 2017-05-17 | 浙江工业大学 | Glucan-poly(hydroxyethyl methacrylate) based continuous bed cryogel separation medium and preparation method thereof |
| CN113087960A (en) * | 2021-05-19 | 2021-07-09 | 石河子大学 | Porous crystal glue and preparation method thereof |
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| WO2004096127A2 (en) * | 2003-04-25 | 2004-11-11 | Kos Life Sciences, Inc. | Formation of strong superporous hydrogels |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593340A (en) * | 2015-01-15 | 2015-05-06 | 浙江工业大学 | Method for separating lactoperoxidase from bovine whey |
| CN104607162A (en) * | 2015-01-15 | 2015-05-13 | 浙江工业大学 | Cation exchange chimeric cryogel separation medium and preparation method thereof |
| CN106674443A (en) * | 2016-12-23 | 2017-05-17 | 浙江工业大学 | Glucan-poly(hydroxyethyl methacrylate) based continuous bed cryogel separation medium and preparation method thereof |
| CN113087960A (en) * | 2021-05-19 | 2021-07-09 | 石河子大学 | Porous crystal glue and preparation method thereof |
| CN113087960B (en) * | 2021-05-19 | 2022-07-08 | 石河子大学 | Porous crystal glue and preparation method thereof |
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|---|---|
| CN103230784B (en) | 2015-01-28 |
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