CN103230784B - 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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- CN103230784B CN103230784B CN201310151726.0A CN201310151726A CN103230784B CN 103230784 B CN103230784 B CN 103230784B CN 201310151726 A CN201310151726 A CN 201310151726A CN 103230784 B CN103230784 B CN 103230784B
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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 composite crystal glue continuous bed medium and preparation method thereof, and this composite crystal glue continuous bed medium quick separating Immunoglobulin IgG and sero-abluminous application from human serum.
(2) background technology
Serum Antibodies and albumin have important application at numerous areas such as biotechnology, clinical medicine and diagnosis, and serum obtains IgG and albuminous main source at present.Existing separation method as saltoutd, the method such as alcohol settling, metal ion affinity chromatography, dye affinity chromatography, Protein A affinity chromatography, amino acid aglucon affinity chromatography has been studied in separation IgG and albumin, portion of techniques obtains application.But, these methods usually run into that such as affinity ligand easily leaks, aglucon enters eluent after being combined with target protein and not easily remove, purification procedures is many, process time is long, IgG and albumin easily lose the problems such as biologically active in separation process.In recent years, mixing mode expanded bed medium also starts for IgG and albuminous adsorbing separation, but because the impact of bed dispersion and the research of efficient separating medium relatively lag behind, to be directly used in from human serum IgG with albuminous a large amount of be separated time, still need and will carry out the work of the system of going deep into.Therefore, study quick separating IgG and albuminous new method from human serum, significant.
The crystal glue chromatography Bio-separation method of nearest appearance, there is the brilliant glue of super large hole for separating medium, can separating bio macromolecular substances from zymotic fluid fast.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 selective little to these two kinds of target proteins of these affinity medias, and what obtain is the mixture of these two kinds of albumen, and be difficult to the two separately to become independently product, 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 IgG of Protein A; but due to Protein A ten points of costlinesses; and there is aglucon and leak and pollute the problem of target protein, scale is separated and is difficult to realize.
(3) summary of the invention
The object of the invention is to provide a kind of composite crystal glue continuous bed medium and preparation and application thereof, and described composite crystal glue continuous bed medium is suitable for immune globulin antibody (IgG) and albumin in quick separating human serum.
The technical solution used in the present invention is:
The invention provides a kind of composite crystal glue continuous bed medium, described composite crystal glue continuous bed pore size of media 0.1 ~ 300 μm, porosity 80 ~ 95%, described composite crystal glue continuous bed medium is with the functional group of the tertiary amino of hydrophobic benzyl-anion exchange formula (I) Suo Shi:
In formula (I), n is positive integer;
Described composite crystal glue continuous bed medium be by immobilized for the tertiary amino-functional group of hydrophobic benzyl-anion exchange in the skeleton of composite crystal glue continuous bed dielectric matrix;
Described composite crystal glue continuous bed dielectric matrix is prepared as follows: added by saturated cellulose grain in the mixed aqueous solution A of hydroxyethyl methacrylate and polyethyleneglycol diacrylate, under the effect of catalyst, through crystallization pore and polymerisation, obtained described composite crystal glue continuous bed dielectric matrix; Described saturated cellulose grain adsorbs saturated in the mixed aqueous solution B of hydroxyethyl methacrylate and polyethyleneglycol diacrylate by cellulose powder, described cellulose powder particle diameter <2mm(preferably 300 ~ 1500 μm); Described catalyst is ammonium persulfate and tetramethyl diethylamine mixing with mass ratio 1:1 ~ 3, and described catalyst quality consumption is 0.5 ~ 1% of hydroxyethyl methacrylate and polyethyleneglycol diacrylate gross mass in mixed aqueous solution A; In described mixed aqueous solution A, hydroxyethyl methacrylate and polyethyleneglycol diacrylate mass ratio are 3:1, and in mixed aqueous solution A, hydroxyethyl methacrylate and polyethyleneglycol diacrylate total mass concentration are 15%; Described saturated cellulose grain quality consumption counts 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, described crystallization pore and the temperature of polymerisation are-15 DEG C, and the reaction time is 20h.
Further again, composite crystal glue continuous bed medium of the present invention obtains as follows: can be raw material with the monomer of composite crystal 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, by carrying out graft reaction with described composite crystal glue continuous bed dielectric matrix, immobilized in composite crystal glue continuous bed dielectric matrix, namely obtain described composite crystal 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 composite crystal glue continuous bed dielectric matrix volume, described Cu
3+the concentration of deionized water solution is 0.037M, Cu
3+deionized water solution volumetric usage is 3 times of composite crystal glue continuous bed dielectric matrix volume; The temperature of described graft reaction is 40 ~ 55 DEG C, and the reaction time is 0.5 ~ 4h.
Cellulose grain in described composite crystal glue medium matrix is embedded in poly hydroxy ethyl acrylate, has the fine pore of the sub-micron to several microns being suitable for IgG Molecular Adsorption in a large number in particle; And poly hydroxy ethyl acrylate skeleton mainly forms the super large hole that size reaches several microns to hundreds of microns, be convenient to other impurity in serum and foreign protein passes through fast smoothly, 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 μm.
The present invention also provides the application of a kind of described composite crystal glue continuous bed medium in quick separating human serum IgG and albumin, and described is applied as: by the buffer solution (Na of preferred pH7.2 ~ 7.8,20 ~ 50mM of human serum pH7.2 ~ 7.8
2hPO
4/ NaH
2pO
4buffer solution) dilute and make human serum dilution, then with composite crystal glue continuous bed medium for chromatographic column is separated, (NaCl final concentration 50 ~ 100mM, the 150 ~ 200mM of the buffer of pH7.2 ~ 7.8, the eluent of 1M is namely used, the Na of preferred pH7.2 ~ 7.8,20 ~ 50mM respectively with the NaCl eluent of 50 ~ 100mM, 150 ~ 200mM, 1M
2hPO
4/ NaH
2pO
4buffer solution) wash-out, there is the efflux (it doesn't matter for peak shape, as long as there is peak to occur just starting to collect) of eluting peak when collecting the NaCl elution of 50 ~ 100mM, namely obtain human serum albumins through dialysis and freeze drying; There is the efflux of eluting peak when collecting the NaCl elution of 150 ~ 200mM, namely obtain IgG through dialysis and freeze drying.
Further, human serum dilution is made in the phosphate buffer dilution 6 ~ 15 times of described human serum pH7.2 ~ 7.8,20 ~ 50mM.
Further, the application optimum condition of composite crystal glue continuous bed medium of the present invention in quick separating human serum IgG and albumin is as follows: by the phosphate (Na of human serum 20mM, pH7.8
2hPO
4/ NaH
2pO
4) buffer solution dilutes 15 times, with composite crystal glue continuous bed medium for chromatographic column is separated, with the speed loading of 1cm/min, then uses the phosphate (Na of 20mM, pH7.8
2hPO
4/ NaH
2pO
4) wash buffer extremely balance, the eluent wash-out successively of 100mM, 150mM, 1M is respectively again with the NaCl final concentration of the phosphate buffered saline of 20mM, pH7.8, elution flow rate is 1cm/min, the efflux of eluting peak is there is when collecting NaCl final concentration 100mM elution, namely human serum albumins is obtained through dialysis and freeze drying (preferred MWCO50000 dialysis membrane deionized water dialyse 24h, then freeze drying 24h at-60 DEG C); There is the efflux of eluting peak when collecting NaCl final concentration 150mM elution, namely obtain immune globulin antibody IgG through dialysis and freeze drying (preferred MWCO100000 dialysis membrane deionized water dialyse 24h, then freeze drying 24h at-65 DEG C); Be 1cm/min by the flow velocity of composite crystal glue continuous bed dielectric posts in chromatography process.
Mixed aqueous solution A of the present invention and mixed aqueous solution B is mixed aqueous solution, and the amount for ease of distinguishing different step mixed aqueous solution used is different and name, and letter itself does not have implication.
Serum Antibodies provided by the invention and albuminous fast separating process and composite crystal glue medium thereof have following features:
(1) matrix of composite crystal 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 this bi-material, its biocompatibility and biological safety excellent, very stable under serum isolating environment, can reuse, the human serum IgG making to adopt its adsorbing separation to obtain and Albumin products have good security.
(2) composite crystal glue continuous bed medium provided by the invention is different from existing ion-exchange or affine crystal gel medium, in composite crystal glue continuous bed medium of the present invention, cellulose grain is embedded in poly hydroxy ethyl acrylate with porous particle form, define the multilevel pore network simultaneously possessing diffusion mass transfer absorption aperture and convective mass transfer super big hole, be suitable for the large molecule of serum IgG and albuminous adsorption chromatography.
(3) contain the benzyl of amino-type ion-exchange group and certain hydrophobic function in composite crystal glue continuous bed dielectric polymers chain provided by the invention simultaneously, contribute to crystal gel medium to form different competitive multiple spots from the large molecule of IgG and albumin and adsorb, make its through variable concentrations containing salt eluent stepwise elution, be resolved successively, separation purity is high, and separating property is excellent.
(4) chromatography method step provided by the invention is few, simple to operate, and eluent used and buffer solution are all easy to configuration, and separation process is rapid, can realize quick separating, have broad application prospects in the separation of haemocyanin.
(4) detailed description of the invention
Fig. 1 is the scanning electron microscope (SEM) photograph of the inside of composite crystal glue continuous bed medium prepared by embodiment 1.
Fig. 2 is the scanning electron microscope (SEM) photograph on the surface of composite crystal glue continuous bed medium prepared by embodiment 1.
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1
Cellulose powder is (commercially available, particle diameter 300 ~ 1500 μm, average grain diameter about 800 μm) at the mixed aqueous solution of hydroxyethyl methacrylate and polyethyleneglycol diacrylate, (in mixed aqueous solution, hydroxyethyl methacrylate quality and polyethyleneglycol diacrylate total mass concentration are 15%, hydroxyethyl methacrylate quality and polyethyleneglycol diacrylate mass ratio are 3:1) in adsorb saturated, (in mixed aqueous solution, hydroxyethyl methacrylate quality and polyethyleneglycol diacrylate total mass concentration are 15% saturated for 4.25g cellulose grain to be added the mixed aqueous solution of 1.18mL hydroxyethyl methacrylate and polyethyleneglycol diacrylate, mass ratio is 3:1), add the catalyst 0.089g that ammonium persulfate and tetramethyl diethylamine are mixed with mass ratio 1:1 again, through crystallization pore and polymerisation 20h at-15 DEG C, prepare poly hydroxy ethyl acrylate matrix and cellulose base composite crystal glue continuous bed matrix, i.e. described composite crystal glue continuous bed dielectric matrix (diameter 10mm, height 49mm).
Be the K of 0.037M by composite crystal glue continuous bed dielectric matrix (diameter 10mm, height 49mm) in 11.5mL concentration
5[Cu (HIO
6)
2] in the aqueous solution, be the N of 0.5M by 19mL concentration, N, N-trimethyl-ethylene base benzene first aqueous ammonium chloride solution graft reaction 4h at 40 DEG C, obtain poly hydroxy ethyl acrylate matrix and cellulose base composite crystal glue continuous bed medium (i.e. composite crystal glue continuous bed medium), the scanning electron microscope (SEM) photograph of its microstructure is shown in shown in Fig. 1 and Fig. 2.After tested, the mass ratio that the cellulose grain contained by this medium accounts for brilliant gel matrix skeleton is 28%, effective drainage porosity 81%, maximum pore rate 95%, pore size about 0.1 ~ 300 μm.Be that model protein carries out adsorption chromatography with bovine serum albumin, under flow velocity 1cm/min, 1.3mg/mL reached to the dynamic adsorbance of bovine serum albumin.
Embodiment 2
Cellulose powder is (commercially available, particle diameter 300 ~ 1900 μm, average grain diameter about 900 μm) at the mixed aqueous solution of hydroxyethyl methacrylate and polyethyleneglycol diacrylate, (in mixed aqueous solution, hydroxyethyl methacrylate quality and polyethyleneglycol diacrylate total mass concentration are 15%, mass ratio is 3:1) in absorption saturated, (in mixed aqueous solution, hydroxyethyl methacrylate quality and polyethyleneglycol diacrylate total mass concentration are 15% saturated for 6.5g cellulose grain to be added the mixed aqueous solution of 1.1mL hydroxyethyl methacrylate and polyethyleneglycol diacrylate, mass ratio is 3:1), add the catalyst 0.083g that ammonium persulfate and tetramethyl diethylamine are mixed with mass ratio 1:1 again, through crystallization pore and polymerisation 20h at-15 DEG C, prepare poly hydroxy ethyl acrylate matrix and cellulose base composite crystal glue continuous bed matrix, i.e. described composite crystal glue continuous bed dielectric matrix (diameter 10mm, height 50mm).
Be the K of 0.037M by composite crystal glue continuous bed dielectric matrix in 14.3mL concentration
5[Cu (HIO
6)
2] in the aqueous solution, be the N of 0.5M by 23.8mL concentration, N, N-trimethyl-ethylene base benzene first aqueous ammonium chloride solution graft reaction 0.5h at 55 DEG C, obtain poly hydroxy ethyl acrylate matrix and the cellulose base composite crystal glue continuous bed medium of diameter 11mm, highly 50mm, the mass ratio that the cellulose grain contained by its matrix accounts for brilliant glue skeleton is 39%, effective drainage porosity 80%, maximum pore rate 90%, pore size about 0.1 ~ 200 μm.
By the phosphate (Na of 0.3mL Healthy Human Serum 50mM
2hPO
4/ NaH
2pO
4) buffer solution (pH=7.8) dilutes 15 times, with 1cm/min flow velocity loading, the composite crystal glue continuous bed medium prepared by above-mentioned steps carries out chromatography, then uses PBS buffer solution (Na
2hPO
4/ NaH
2pO
4, pH=7.8,50mM) rinse to balance, and use the NaCl eluent of 100mM, 200mM, 1M (pH=7.8,50mM buffer) to carry out stepwise elution respectively, the efflux of eluting peak is there is when collecting NaCl final concentration 100mM elution, to dialyse 24h through MWCO50000 dialysis membrane deionized water, then freeze drying 24h at-60 DEG C, obtain human serum albumins, SDS-PAGE electrophoresis detection purity about 98%, its adsorption capacity 0.2mg/mL bed; The efflux of eluting peak is there is when collecting NaCl final concentration 200mM elution, to dialyse 24h through MWCO100000 dialysis membrane deionized water, then freeze drying 24h at-65 DEG C, obtain immune globulin antibody IgG, through SDS-PAGE electrophoresis detection purity about 75%, its adsorption capacity 0.2mg/mL bed.Total protein adsorption capacity reaches 0.6mg/mL bed.
Embodiment 3
Composite crystal glue continuous bed medium in Example 2, other operate with embodiment 2, by the phosphate (Na of 0.2mL Healthy Human Serum 20mM
2hPO
4/ NaH
2pO
4) buffer solution (pH=6.5) dilutes 15 times, carry out composite crystal glue continuous bed chromatography with 1cm/min flow velocity, then use PBS buffer solution (pH=6.5,20mM) to rinse, and use the NaCl eluent (Na of pH=6.5,20mM of 50mM, 150mM, 1M respectively
2hPO
4/ NaH
2pO
4buffer solution configures) carry out stepwise elution, the efflux of eluting peak is there is when collecting the elution of NaCl final concentration 50mM, to dialyse 24h through MWCO50000 dialysis membrane deionized water, then freeze drying 24h at-60 DEG C, obtain human serum albumins, SDS-PAGE electrophoresis detection purity about 94%, it is 0.21mg/mL bed that Coomassie Brilliant Blue records adsorbance; Collect the eluting peak that the eluent of NaCl final concentration 150mM is corresponding, to dialyse 24h, then freeze drying 24h at-65 DEG C through MWCO100000 dialysis membrane deionized water, obtain immune globulin antibody IgG, purity about 82%, adsorption capacity is 0.03mg/mL bed.Total protein adsorption capacity is 0.29mg/mL bed.
Embodiment 4
Composite crystal glue continuous bed medium in Example 2, other operate with embodiment 2, by phosphate buffer (pH=7.2, Na of 1mL Healthy Human Serum 20mM
2hPO
4/ NaH
2pO
4) dilution 6 times, carry out composite crystal glue continuous bed chromatography with 1cm/min flow velocity, with PBS buffer solution (pH=7.2,20mM, Na
2hPO
4/ NaH
2pO
4) rinse to balance, and use NaCl eluent (pH=7.2,20mM, Na of 100mM, 150mM, 1M respectively
2hPO
4/ NaH
2pO
4buffer) carry out stepwise elution, the efflux of eluting peak is there is when collecting NaCl final concentration 100mM elution, to dialyse 24h through MWCO50000 dialysis membrane deionized water, then freeze drying 24h at-60 DEG C, obtain human serum albumins, through SDS-PAGE electrophoresis detection purity about 40%, it is 0.06mg/mL bed that Coomassie Brilliant Blue records adsorbance; The efflux of eluting peak is there is when collecting NaCl final concentration 150mM elution, to dialyse 24h, then freeze drying 24h at-65 DEG C through MWCO100000 dialysis membrane deionized water, obtain immune globulin antibody IgG, purity about 74%, adsorption capacity is 0.2mg/mL bed.Total protein adsorption capacity is 1.1mg/mL bed.
Embodiment 5
Composite crystal glue continuous bed medium in Example 2, other operate with embodiment 2, by phosphate buffer (pH=7.8, Na of 0.2mL Healthy Human Serum 20mM
2hPO
4/ NaH
2pO
4) dilution 15 times, carry out composite crystal glue continuous bed chromatography with 1cm/min flow velocity, then use PBS buffer solution (pH=7.8,20mM, Na
2hPO
4/ NaH
2pO
4) rinse to balance, and use NaCl eluent (pH=7.8,20mM, Na of 100mM, 150mM, 1M respectively
2hPO
4/ NaH
2pO
4buffer solution configures) carry out stepwise elution, the efflux of eluting peak is there is when collecting NaCl final concentration 100mM elution, to dialyse 24h through MWCO50000 dialysis membrane deionized water, then freeze drying 24h at-60 DEG C, obtain human serum albumins, through SDS-PAGE electrophoresis detection purity about 98%, recording adsorbance in conjunction with Coomassie Brilliant Blue is 0.2mg/mL bed; The efflux of eluting peak is there is when collecting NaCl final concentration 200mM elution, MWCO100000 dialysis membrane deionized water is dialysed 24h, then freeze drying 24h at-65 DEG C, obtains immune globulin antibody IgG, purity about 84%, adsorption capacity is 0.04mg/mL bed.Total protein adsorption capacity is 0.3mg/mL bed.
Claims (6)
1. a composite crystal glue continuous bed medium, it is characterized in that described composite crystal glue continuous bed pore size of media 0.1 ~ 300 μm, porosity 80 ~ 95%, described composite crystal glue continuous bed medium is with the functional group of the tertiary amino of hydrophobic benzyl-anion exchange formula (I) Suo Shi:
In formula (I), n is positive integer;
Described composite crystal glue continuous bed medium be by immobilized for the tertiary amino-functional group of hydrophobic benzyl-anion exchange in the skeleton of composite crystal glue continuous bed dielectric matrix;
Described composite crystal glue continuous bed dielectric matrix is prepared as follows: added by saturated cellulose grain in the mixed aqueous solution A of hydroxyethyl methacrylate and polyethyleneglycol diacrylate, under the effect of catalyst, through crystallization pore and polymerisation, obtained described composite crystal glue continuous bed dielectric matrix; Described saturated cellulose grain adsorbs saturated in the mixed aqueous solution B of hydroxyethyl methacrylate and polyethyleneglycol diacrylate by cellulose powder, described cellulose powder particle diameter < 2mm; Described catalyst is ammonium persulfate and tetramethyl diethylamine mixing with mass ratio 1:1 ~ 3, and described catalyst quality consumption is 0.5 ~ 1% of hydroxyethyl methacrylate and polyethyleneglycol diacrylate gross mass in mixed aqueous solution A; In described mixed aqueous solution A, hydroxyethyl methacrylate and polyethyleneglycol diacrylate mass ratio are 3:1, and in mixed aqueous solution A, hydroxyethyl methacrylate and polyethyleneglycol diacrylate total mass concentration are 15%; Described saturated cellulose grain quality consumption counts 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. composite crystal glue continuous bed medium as claimed in claim 1, it is characterized in that the temperature Wei – 15 DEG C of described crystallization pore and polymerisation, the reaction time is 20h.
3. composite crystal glue continuous bed medium as claimed in claim 1, is characterized in that described composite crystal glue continuous bed medium obtains as follows: can be raw material with the monomer of composite crystal glue continuous bed dielectric matrix generation graft reaction, with Cu
3+deionized water solution is catalyst, by carrying out graft reaction with described composite crystal glue continuous bed dielectric matrix, immobilized in composite crystal glue continuous bed dielectric matrix, namely obtains described composite crystal 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 composite crystal glue continuous bed dielectric matrix volume, described Cu
3+the concentration of deionized water solution is 0.037M, Cu
3+deionized water solution volumetric usage is 3 times of composite crystal glue continuous bed dielectric matrix volume; The temperature of described graft reaction is 40 ~ 55 DEG C, and the reaction time is 0.5 ~ 4h.
4. the application of composite crystal glue continuous bed medium in quick separating human serum IgG and albumin described in a claim 1, it is characterized in that described being applied as: human serum dilution is made in the dilution of the buffer solution of human serum pH7.2 ~ 7.8, then with composite crystal glue continuous bed medium for chromatographic column is separated, respectively with the NaCl elution of 50 ~ 100mM, 150 ~ 200mM, 1M, there is the efflux of eluting peak when collecting the NaCl elution of 50 ~ 100mM, namely obtain human serum albumins through dialysis and freeze drying; There is the efflux of eluting peak when collecting the NaCl elution of 150 ~ 200mM, namely obtain IgG through dialysis and freeze drying.
5. apply as claimed in claim 4, it is characterized in that the phosphate buffer dilution 6 ~ 15 times of described human serum pH7.2 ~ 7.8,20 ~ 50mM.
6. apply as claimed in claim 4, be applied as described in it is characterized in that: by human serum 20mM, the phosphate buffer of pH7.8 dilutes 15 times, with composite crystal glue continuous bed medium for chromatographic column is separated, with the speed loading of 1cm/min, then 20mM is used, the phosphate buffer of pH7.8 rinses, use 20mM again, the NaCl final concentration of the phosphate buffered saline of pH7.8 is respectively 100mM, 150mM, the eluent wash-out successively of 1M, elution flow rate is 1cm/min, the efflux of eluting peak is there is when collecting NaCl final concentration 100mM elution, namely human serum albumins is obtained through dialysis and freeze drying, there is the efflux of eluting peak when collecting NaCl final concentration 150mM elution, namely obtain immune globulin antibody IgG through dialysis and freeze drying.
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