CN104475041A - A novel method of preparing agarose magnetic microspheres and uses of the agarose magnetic microspheres in separation and purification of an IgG antibody - Google Patents
A novel method of preparing agarose magnetic microspheres and uses of the agarose magnetic microspheres in separation and purification of an IgG antibody Download PDFInfo
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Abstract
The invention relates to a novel method of preparing agarose magnetic microspheres and application of the prepared agarose magnetic microspheres to separation and purification of an IgG antibody by utilization of an immunomagnetic separation technique. In specific, active amino is introduced to surfaces of the agarose magnetic microspheres, glutaraldehyde is adopted as a crosslinking arm to crosslink a ligand, and the ligand is staphylococcus aureus protein A capable of being specifically combined with the IgG antibody, thus preparing immunomagnetic microspheres capable of specifically adsorbing the IgG antibody. In addition, lysate of bacteria generating the IgG antibody by fermentation is separated and purified by utilization of a magnetic separation and purification device, the activity of the prepared antibody can reach 1.5*10<8> g IU/mg, the corresponding activity recovery rate is more than 90%, the purity of the antibody is almost 100% by purification analysis, and each index meets the national standards.
Description
Technical field
The present invention relates to the new method of preparing Agarose Magnetic Microsphere and utilize immunomagnetic isolation technology, by prepared Agarose Magnetic Microsphere for separating of IgG purification antibody, belonging to biological technical field.
Background technology
Antibody refers to the homologous antibody only identifying a certain specific antigen epi-position produced by monospecific polyclonal hybridoma, is through a class distinct antibodies of artificial preparation.Described antibody is the class globulin that B cell produces when being divided into thick liquid cell, be present in body fluid, there is specific binding with corresponding antigens (as pathogen), and play immunological effect in the presence of other immune molecules and cell, can humoral immunity.The high specificity that the physicochemical property height that antibody has is homogeneous, biologically active is single, be combined with antigen, be convenient to artificially process and quality control, also have and be easy to obtain, be easy to carry out the advantages such as large-scale external preparation and purifying.These advantages make it be paid much attention to once coming out, and are widely used in biology and medical research field, have vast potential for future development.
IgG antibody is the one of human immunoglobulin, and be also uniquely can by the immunoglobulin (Ig) of placenta, it be the main antibody composition in serum composition, accounts for about 75% of haemocyanin.The IgG of about 40 ~ 50% is distributed in serum, and remaining is distributed in various tissue.The major function effect of IgG antibody shields in immunity of organism.Most of antiviral, antitoxin and antibacterial antibody all belong to IgG antibody.IgG antibody can tackle the disease such as measles, hepatitis A, and can effectively prevent corresponding infectious diseases.Therefore, neutralizing a toxin, antibacterium, antiviral, in inhibiting tumor cell, IgG antibody plays an important role.
IgG monoclonal antibody mainly utilizes engineering bacteria fermentation to produce, and cording has expression product active in born of the same parents.But its separation and purification process is complicated, needs successively through steps such as clasmatosis, centrifugation, salt precipitation, dissolution precipitation, hydrophobic chromatography, cation and anion exchange chromatography and gel permeation chromatographies.Because above-mentioned separation and purification process steps is many, time-consuming length, cause that product yield is low, cost is high, at present, commercially available IgG monoclonal antibody adopts affinity chromatography to improve purification efficiency and product specificity usually, but also will could obtain higher productive rate through process such as oversalting, hydrophobic or ion-exchange chromatographies before affinity chromatography.In addition, the connection of affinity chromatography medium homospecificity antibody adopts Australia's cyanogen of severe toxicity as coupling agent more, adds the danger of technological process.Therefore, exploitation can simply, the new technology of fast separating and purifying IgG monoclonal antibody become the main flow of this area.
Agarose is that the one extracted from marine alga is linear, water-soluble natural polysaccharide.Due to its rich surface hydroxyl, after chemical activation, can the various aglucon of coupling; Further, agarose has how empty network structure, is convenient to the diffusion of macromolecular substances in agarose, thus increases the effective density of aglucon; There is good hydrophilic characteristics, can make biomolecule be easy near and with part effect, can not biologically active be lost again; The most important is, its surperficial neutral, therefore non-specific adsorption ability is minimum, does not particularly almost have non-specific adsorption to protein.Therefore, agarose is the ideal material of protein separation.
Agarose Magnetic Microsphere, utilize agarose coated magnetic material, form the Agarose Magnetic Microsphere with magnetic core, this Agarose Magnetic Microsphere not only make use of the naturally occurring advantage of agarose, quick separating object can also be realized by the effect in magnetic field, thus reduce operating procedure greatly, shorten the operating time, reduce costs.In recent years, existing report Agarose Magnetic Microsphere being applied to separation and purification field, such as Dong is then raw waits people (with reference to Journal of North westUniversity, Natural Science Edition, Apr.2001, Vo.l 31No.2) utilize reverse microemulsion method coated ferriferrous oxide to prepare Agarose Magnetic Microsphere, and by this Agarose Magnetic Microsphere for separating of urokinase; The people such as Li Jiaxing (Chemical Engineering Journal 172 (2011) 892 – 897) utilize reverse microemulsion method coated ferriferrous oxide, and this Agarose Magnetic Microsphere is used for Adsorption of Radioactive nucleic; Chinese patent CN 1524878A discloses the technology etc. that a kind of Agarose Magnetic Microsphere prepared by reverse microemulsion method is used for purifying gene engineering recombinant interferon.
But, the Agarose Magnetic Microsphere Agarose Magnetic Microsphere preparation method of current application all also exists certain defect, as coated in agarose uneven, coating efficiency is low, magnetisable material becomes that nuclearity is poor, the not balling-up of magnetic ball, reunite serious, specific area is little, separation and purification is considered low and magnetic responsiveness is poor.
Up to now, research Agarose Magnetic Microsphere being applied to antibody does not also have report, the present invention proposes a kind of new method preparing Agarose Magnetic Microsphere, according to the Agarose Magnetic Microsphere that the inventive method obtains, not there is above-mentioned shortcoming, and, also can carry out finishing to the Agarose Magnetic Microsphere of preparation, microballoon is activated.Described finishing such as imports active amino on its surface, and carry out coupling aglucon using glutaraldehyde as crosslinked arm, described aglucon be can with the staphylococcus aureus protein A of IgG antibody generation specific binding, thus obtain the Agarose Magnetic Microsphere that can be used for specific adsorption IgG antibody.By with the use of respective magnetic separation and purification device, eliminate each pre-treatment step in the conventional purification of IgG antibody, achieve " a step separation and purification ", shorten the operating time to greatest extent, reduce product cost.Can realize the fast separating and purifying of IgG antibody according to method of the present invention, purifying rate can reach more than 90%; Prepared antibody activity can reach 1.5 × 10
8g IU/mg, corresponding activity recovery is more than 90%, and indices is all up to state standards.
Summary of the invention
In first aspect, the invention provides a kind of method preparing Agarose Magnetic Microsphere, described method comprises the steps: that (i) preparation is without magnetic core agarose microbeads; (ii) make to be cross-linked without the activation of magnetic core agarose microbeads; (iii) agarose microbeads-ferric ion solutions is prepared; (iv) Agarose Magnetic Microsphere is prepared; V crosslinked knee-joint is entered Agarose Magnetic Microsphere by (); (vi) aglucon is accessed Agarose Magnetic Microsphere.
In second aspect, the invention provides the Agarose Magnetic Microsphere utilizing the method for first aspect to prepare, described Agarose Magnetic Microsphere is with glutaraldehyde cross-linking arm and the aglucon staphylococcus aureus protein A with IgG antibody generation specific binding.
In the third aspect, the invention provides a kind of method of separation and purification IgG antibody, described method comprises the steps:
A) Agarose Magnetic Microsphere of second aspect is mixed with the lysate of expressing IgG antibody cell, make IgG antibody be adsorbed in agarose microbeads;
B) magnetic field is utilized to be separated by the Agarose Magnetic Microsphere being adsorbed with IgG antibody;
C) dissociation solution is used by IgG antibody from wash-out Agarose Magnetic Microsphere, collection IgG antibody.
Described isolation and purification method also can comprise step
D) Agarose Magnetic Microsphere is reclaimed.
In fourth aspect, the invention provides a kind of Magnetic Isolation purification devices for implementing method described in the third aspect, described Magnetic Isolation purification devices comprises the magnet (1) being positioned over liquid trap (3) sidewall, and with liquid separatnig valve (2).
Specifically comprise:
Prepare a method for Agarose Magnetic Microsphere, described method comprises the steps: that (i) preparation is without magnetic core agarose microbeads; (ii) make to be cross-linked without the activation of magnetic core agarose microbeads; (iii) agarose microbeads-ferric ion solutions is prepared; (iv) Agarose Magnetic Microsphere is prepared; V crosslinked knee-joint is entered Agarose Magnetic Microsphere by (); (vi) aglucon is accessed Agarose Magnetic Microsphere.
Described step (i), for being dissolved in ultra-pure water by agarose heating, prepares the agarose aqueous phase W of 0.1wt%-20wt%; Mixed with aqueous phase W by the oil phase O comprising emulsifying agent and organic reagent subsequently, obtain W/O emulsion, in the oil phase of described W/O emulsion, emulsifying agent is 0.1wt%-1.5wt%, and aqueous phase compares 1:1-1:1000 with oil phase volume; Under 40 DEG C of-90 DEG C of constant temperature to W/O emulsion heating, and under 200-1000rpm mechanical agitation, reaction 1-5h; Subsequently, make W/O emulsion be quickly cooled to 15-40 DEG C, obtain W/O emulsion droplets, thus form the agarose microbeads without magnetic core.
Described organic solvent is the combination of atoleine, benzinum, olive oil, cottonseed oil, soybean oil, sunflower seed oil, alkanes or above-mentioned organic solvent.
Described emulsifying agent is the oil emulsifier of glycerin ether polymer (PO-500), NOFABLE SO-992 (Arlace 183), Cremophor RH40, sorbitan trioleate (Span 85), dehydrated sorbitol mono-fatty acid ester (Span80), sorbitan tristearate (Span 65) or lipophilic-hydrophilic block polymer.
Described step (ii) is: take microballoon according to agarose microbeads and sodium borohydride mass ratio 1000:1-10:1, add the sodium hydroxide solution of 0.1mol/L-2mol/L, make the agarose microbeads-concentration of sodium borohydride mixture in sodium hydroxide solution be 0.01-10g/ml, under constant temperature, constant speed stirs; Subsequently, add the mixed solution of epoxychloropropane and dimethyl sulfoxide (DMSO) in aforesaid liquid, under constant temperature, constant speed stirs, and the volume ratio of described epoxychloropropane and dimethyl sulfoxide (DMSO) is 1:1-1:100.
Described step (iii) is: solution step (ii) obtained carries out with ferrous ion-ferric ion mixed solution or mixes, and to make in iron ion mixed solution agarose microbeads concentration range at 0.01-2g/ml.Under 30-60 DEG C of constant temperature, 200-600rpm constant speed, soaks 10-120h, thus it is crosslinked that agarose microbeads and iron ion are activated.
In the mixed solution of described ferrous ion and ferric ion, the mol ratio of ferrous ion and ferric ion is 1:1-10:1; The mixed solution of described ferrous ion and ferric ion is frerrous chloride-ferric chloride solution, ferrous nitrate-iron nitrate solution.
Described step (iv) is: in the agarose microbeads-ferric ion solutions of step (iii), add ammoniacal liquor, make the volume ratio 1:1-100:1 of agarose microbeads-ferric ion solutions and ammoniacal liquor; Under 40-80 DEG C of constant temperature, in the nitrogen environment of 200-1000rpm constant speed, reaction 0.5-3h, preparation has the Agarose Magnetic Microsphere of tri-iron tetroxide magnetic core; Be separated the Agarose Magnetic Microsphere obtained with magnetic field, save backup after washing.
Described step (v) is: in ammoniacal liquor, add Agarose Magnetic Microsphere prepared by step (iv), 2-4 hour is reacted under room temperature, add the glutaraldehyde water solution of 2-20% after abundant drip washing again, under room temperature, react 1-3 hour, thoroughly clean excessive glutaraldehyde with ultra-pure water.
Described step (vi) is: at the NaHCO containing staphylococcus aureus protein A
3-Na
2cO
3the Agarose Magnetic Microsphere with crosslinked arm obtained in step (v) is added in buffer solution, clean with ultrapure water after reaction, add ethanolamine solutions and close unreacted epoxide group; Add bovine serum albumin subsequently, close unreacted aldehyde radical, reduce non-specific adsorption, finally clean with a large amount of ultra-pure waters, obtain Agarose Magnetic Microsphere product.
The Agarose Magnetic Microsphere utilizing above-mentioned method to prepare, is characterized in that, described Agarose Magnetic Microsphere is with glutaraldehyde cross-linking arm and the aglucon staphylococcus aureus protein A with IgG antibody generation specific binding.
A method for separation and purification IgG antibody, described method comprises the steps:
A) Agarose Magnetic Microsphere as claimed in claim 11 is mixed with the lysate of expressing IgG antibody cell, make IgG antibody be adsorbed in agarose microbeads;
B) magnetic field is utilized to be separated by the Agarose Magnetic Microsphere being adsorbed with IgG antibody;
C) dissociation solution is used by IgG antibody from wash-out Agarose Magnetic Microsphere, collection IgG antibody.
Described dissociation solution is glycine-hydrochloride buffer.
Described method also comprises step
D) Agarose Magnetic Microsphere is reclaimed.
It is characterized in that, described steps d) use PBS to clean, and the Agarose Magnetic Microsphere after cleaning is stored in 20% ethanol water.
Carrying out, step is a) front, fully cleans balance with PBS buffer solution to described immune magnetic microsphere.
Described IgG antibody is IgG monoclonal antibody.
Described separation and purification is carried out in Magnetic Isolation purification devices, and described Magnetic Isolation purification devices comprises the magnet (1) being positioned over liquid trap (3) sidewall, and with liquid separatnig valve (2).
Beneficial effect
The Agarose Magnetic Microsphere that according to a first aspect of the present invention prepared by method has good spherical in shape, and spheroid is mellow and full, caking of not easily reuniting, good dispersion, even particle size distribution, specific area is large, magnetic core productive rate reaches more than 90%, can reuse, average more than 20 times of service life.
Method carries out separation and purification to IgG antibody and/or IgG monoclonal antibody according to a second aspect of the present invention, can save clasmatosis, a series of pre-treatment such as centrifugal, realize the object of " a step separation and purification ".The antibody purification rate of recovery can reach more than 90%, and antibody purity is close to 100%, and Antibody dynamics absorption carrying capacity >27mg/mL, through active testing, indices is all up to state standards.
Magnetic Isolation purification devices according to a third aspect of the present invention, the built-in strong magnet of this device, magnetic field intensity is large, can place conical flask withdrawal liquid down, and middle liquid separatnig valve controls.Like this be arranged so that easy and simple to handle saving time.
Accompanying drawing illustrates:
Fig. 1 is Magnetic Isolation purification devices of the present invention, and wherein, in figure, numbering corresponds to (1) magnet; (2) liquid separatnig valve; (3) liquid trap; (4) Agarose Magnetic Microsphere; (5) reactant liquor; (6) liquid is collected.
Fig. 2 is the optical microscope photograph of Agarose Magnetic Microsphere prepared according to the methods of the invention.
Fig. 3 is the SDS-PAGE gel electrophoresis analysis to the IgG monoclonal antibody obtained according to the inventive method separation and purification.Wherein, swimming lane A and B is respectively the IgG monoclonal antibody utilizing Agarose Magnetic Microsphere separation and purification of the present invention to obtain; Swimming lane C is the IgG antibody standard items with test sample comparable sodium; Swimming lane D is the IgG antibody standard items with the Marker comparable sodium of swimming lane E; Swimming lane E is and Marker#SM0661.
Fig. 4 is the high performance liquid chromatography test result of the IgG monoclonal antibody utilizing Agarose Magnetic Microsphere separation and purification of the present invention to obtain.
Fig. 5 is the adsorption percentage of Agarose Magnetic Microsphere reuses the relation of number of times result with it.
Detailed description of the invention
The present invention is elaborated below for following aspect.
Prepared by Agarose Magnetic Microsphere
The method preparing Agarose Magnetic Microsphere of the present invention comprises the steps: that (i) preparation is without magnetic core agarose microbeads; (ii) make to be cross-linked without the activation of magnetic core agarose microbeads; (iii) agarose microbeads-ferric ion solutions is prepared; (iv) Agarose Magnetic Microsphere is prepared; V crosslinked knee-joint is entered Agarose Magnetic Microsphere by (); (vi) aglucon is accessed Agarose Magnetic Microsphere.
In one embodiment, described step (i) is: be dissolved in ultra-pure water by agarose heating, the agarose aqueous phase W of preparation 0.1wt%-20wt%; Mixed with aqueous phase W by the oil phase O comprising emulsifying agent and organic reagent subsequently, obtain W/O emulsion, in the oil phase of described W/O emulsion, emulsifying agent is 0.1wt%-1.5wt%, and aqueous phase compares 1:1-1:1000 with oil phase volume; Under 40 DEG C of-90 DEG C of constant temperature to W/O emulsion heating, and under 200-1000rpm mechanical agitation, reaction 1-5h; Subsequently, make W/O emulsion be quickly cooled to 15-40 DEG C, obtain W/O emulsion droplets, thus form the agarose microbeads without magnetic core.
Oil phase described in step (i) and aqueous phase can not be miscible, and described oil phase is one or more organic solvents being dissolved with one or more emulsifying agents.Described organic solvent must immiscible with water, there is certain viscosity and for liquid under room temperature, preferably there is the organic reagent of higher boiling, low volatility, as, atoleine, benzinum, olive oil, cottonseed oil, soybean oil, sunflower seed oil or some alkanes.The organic solvent of oil phase also can be the mixing of above-mentioned multiple organic solvent.One or more emulsifying agents comprised in oil phase must be able to be dissolved in described oil phase, described emulsifying agent is glycerin ether polymer (PO-500), NOFABLE SO-992 (Arlace 183) such as, the oil emulsifier of Cremophor RH40, sorbitan trioleate (Span 85), dehydrated sorbitol mono-fatty acid ester (Span 80), sorbitan tristearate (Span 65) or lipophilic-hydrophilic block polymer.
In one embodiment, described step (ii) is: be that 1000:1-10:1 takes microballoon according to the mass ratio without magnetic core agarose microbeads and sodium borohydride, add the sodium hydroxide solution of 0.1mol/L-2mol/L, make the agarose microbeads-concentration of sodium borohydride mixture in sodium hydroxide solution be 0.01-10g/ml, under constant temperature, constant speed stirs; Subsequently, add the mixed solution (volume ratio of epoxychloropropane and dimethyl sulfoxide (DMSO) is 1:1-1:100) of epoxychloropropane and dimethyl sulfoxide (DMSO) in aforesaid liquid, under constant temperature, constant speed stirs.
In one embodiment, described step (iii) is: solution step (ii) obtained carries out with ferrous ion-ferric ion mixed solution or mixes, and to make in iron ion mixed solution microballoon concentration range at 0.01-2g/ml.Under 30-60 DEG C of constant temperature, 200-600rpm constant speed, soaks 10-120h, thus it is crosslinked that agarose microbeads and iron ion are activated.
In the mixed solution of described ferrous ion and ferric ion, the mol ratio of ferrous ion and ferric ion is 1:1-10:1.The mixed solution of described ferrous ion and ferric ion can be frerrous chloride-ferric chloride solution, ferrous nitrate-iron nitrate solution etc.
In one embodiment, described step (iv) is: in the microballoon-ferric ion solutions of step (iii), add ammoniacal liquor, make the volume ratio 1:1-100:1 of microballoon-ferric ion solutions and ammoniacal liquor; Under 40-80 DEG C of constant temperature, in the nitrogen environment of 200-1000rpm constant speed, reaction 0.5-3h, preparation has the Agarose Magnetic Microsphere of tri-iron tetroxide magnetic core, is separated the Agarose Magnetic Microsphere obtained, saves backup after washing with magnetic field.
In one embodiment, described step (v) is: in ammoniacal liquor, add Agarose Magnetic Microsphere prepared by step (iv), 2-4 hour is reacted under room temperature, the glutaraldehyde water solution of 2-20% is added again after abundant drip washing, react 1-3 hour under room temperature, thoroughly clean excessive glutaraldehyde with ultra-pure water.
In one embodiment, described step (vi) is: at the NaHCO containing staphylococcus aureus protein A
3-Na
2cO
3the Agarose Magnetic Microsphere with crosslinked arm obtained in step (v) is added in buffer solution, clean with ultrapure water after reaction, add ethanolamine solutions and close unreacted epoxide group; Add bovine serum albumin subsequently, close unreacted aldehyde radical, reduce non-specific adsorption, finally clean with a large amount of ultra-pure waters, the product obtained is Agarose Magnetic Microsphere of the present invention.
Utilize immune magnetic technology separation IgG purification antibody
Fully balance is cleaned to following the immune magnetic microsphere prepared according to method of the present invention with PBS buffer solution, add the cell pyrolysis liquid of the PBS buffer solution dilution containing IgG antibody, 300rpm slowly shakes 0.5-3 hour, liquid is moved into (as Fig. 1) in Magnetic Isolation purification devices after reaction, leave standstill 3h, make the Agarose Magnetic Microsphere being adsorbed with IgG antibody be attracted to the sidewall of knockout, open control valve, discard reactant liquor.Subsequently, PBS buffer solution is added in knockout, described Agarose Magnetic Microsphere is rinsed balance, remove magnetic field, the Agarose Magnetic Microsphere buffer solution being adsorbed with IgG antibody is taken out from knockout, add the glycine-hydrochloride buffer of pH2.8,300rpm slowly shakes reaction 0.5-1 hour, makes IgG antibody from desorption Agarose Magnetic Microsphere.Subsequently, reactant liquor is moved in Magnetic Isolation purification devices, Agarose Magnetic Microsphere is separated with IgG antibody, collect dissociation solution, use PBS buffer solution for cleaning immediately, obtain the IgG antibody of separation and purification.Reclaim Agarose Magnetic Microsphere by removing magnetic field, the microballoon after cleaning can be kept in the ethanol water of 20%, reuses.
Embodiment
The medicine source of employing in embodiment: agarose: Shanghai Hui Shi biochemical reagents Co., Ltd; Atoleine: Xilong Chemical Co., Ltd;
Benzinum: Xilong Chemical Co., Ltd; Glutaraldehyde: Xilong Chemical Co., Ltd;
Staphylococcus aureus protein A: breathe out medicine bio-pharmaceuticals; IgG antibody standard items: breathe out medicine bio-pharmaceuticals; Express the lysate of IgG antibody cell: breathe out medicine bio-pharmaceuticals.
Embodiment 1: the preparation of Agarose Magnetic Microsphere
I () preparation is without magnetic core agarose microbeads:
2.0g agarose is added 100ml ultra-pure water, and 100 DEG C of heating, prepare agarose aqueous phase W.Subsequently, in atoleine 180ml, benzinum 40ml, add 8.0g emulsifying agent Span 80, fully oil phase O is prepared in dispersion.By oil phase O 80 DEG C of heated at constant temperature, with 400rpm mechanical agitation, rapidly aqueous phase is moved into, reaction 3h.Ice-water bath is cooled to rapidly 15 DEG C.
(ii) make to be cross-linked without the activation of magnetic core agarose microbeads:
Get microballoon 5g, add the NaOH7.5ml of 0.8mol/L, sodium borohydride 0.01g, 25 DEG C, 400rpm shakes 30min.Dimethyl sulfoxide (DMSO) 7.5ml is added, epoxychloropropane 3.75ml in the solution of preparation like this.30 DEG C of constant temperature 400rpm react 20h.Wash subsequently, remove unreacted reagent.
(iii) agarose-ferric ion solutions is prepared
Take 4.32gFeCl
3and 15.92gFeCl
2, be dissolved in 200ml deoxidized water, preparation ferric ion solutions.Be dissolved in by 30g microballoon in above-mentioned ferric ion solutions, 45 DEG C, 90h is soaked in 400rpm concussion.
(iv) Agarose Magnetic Microsphere is prepared
Microballoon after above-mentioned for 15g immersion is added 150ml deoxidized water.At 60 DEG C, add 40ml ammoniacal liquor, stir and pass into nitrogen, reaction 3h.Agarose Magnetic Microsphere is separated by externally-applied magnetic field, successively adopts absolute ethyl alcohol, water washing.Finally, microballoon is kept in 20% ethanolic solution, 4 DEG C of preservations.
V crosslinked knee-joint is entered Agarose Magnetic Microsphere by ()
Get the above-mentioned Agarose Magnetic Microsphere of 3g and add 6ml ammoniacal liquor, react 4 hours under room temperature.Make the thorough ammonia solution of epoxide group.By hydrolysis after Agarose Magnetic Microsphere clean, add the glutaraldehyde water solution of 20ml volume fraction 15%, under room temperature react 3 hours, thoroughly clean up, obtain the Agarose Magnetic Microsphere with active aldehyde radical, place 20% ethanol water in 4 DEG C of preservations.
(vi) aglucon is accessed Agarose Magnetic Microsphere
Get and activate and connect the magnetic microsphere 2ml of crosslinked arm, add the NaHCO that 10ml contains 30mg staphylococcus aureus protein A
3-Na
2cO
3buffer solution, 4 DEG C of reactions are spent the night, clean with ultrapure water, add 5mL 1mol/L ethanolamine solutions, react 4 hours at 37 DEG C.To close unreacted epoxide group, then add the bovine serum albumin reaction of 5ml 1mg/ml, close unreacted aldehyde radical, reduce non-specific adsorption, finally clean with a large amount of ultra-pure waters, for subsequent use.
The Magnetic Isolation purifying of embodiment 2:IgG monoclonal antibody
Get the Agarose Magnetic Microsphere 1ml prepared, after PBS buffer solution fully cleans balance, add the lysate 10ml being diluted to certain density expression IgG monoclonal antibody cell with buffer solution, slow concussion reaction response 1 hour, liquid is moved in Magnetic Isolation purification devices, absorption fixed magnetic microballoon, shift out separator and discard reactant liquor, then 10mlPBS buffer solution for cleaning is used 3 times, add the glycine-hydrochloride buffer of 4ml 0.1M, the slow concussion reaction 1 hour antibody for Dissociative, continue to move in Magnetic Isolation purification devices, absorption magnetic microsphere, collect dissociation solution.
Embodiment 3: the wash-out of magnetic microsphere reclaims
Collect after dissociation solution, adsorb in magnetic ball to embodiment 2 and add PBS buffer solution and fully clean for several times, equilibrium magnetism microballoon, discards cleaning fluid, and the microballoon after cleaning is placed on 4 DEG C of preservations in the ethanol water of 20%, reusable more than 20 times.
The mensuration of embodiment 4:IgG antibody activity
The immunocompetence of IgG antibody after employing ELISA method mensuration purifying, the indices of the antibody after purifying is all up to state standards, the Antibody dynamics absorption carrying capacity >27mg/mL of direct purification zymophyte lysate.The average activity rate of recovery is 92%, and average specific activity is 1.5 × 108g IU/mg.Through SDS-PAGE gel electrophoresis and high performance liquid chromatography purity analysis known, purity about 99.7%, indices is all up to state standards, and according to reusing number of times for allowing the standard used more than 50%, microballoon access times are greatly about about 20 times by analysis.
Claims (10)
1. prepare a method for Agarose Magnetic Microsphere, described method comprises the steps: that (i) preparation is without magnetic core agarose microbeads; (ii) make to be cross-linked without the activation of magnetic core agarose microbeads; (iii) agarose microbeads-ferric ion solutions is prepared; (iv) Agarose Magnetic Microsphere is prepared; V crosslinked knee-joint is entered Agarose Magnetic Microsphere by (); (vi) aglucon is accessed Agarose Magnetic Microsphere.
2. the method for claim 1, is characterized in that, described step (i), for being dissolved in ultra-pure water by agarose heating, prepares the agarose aqueous phase W of 0.1wt%-20wt%; Mixed with aqueous phase W by the oil phase O comprising emulsifying agent and organic reagent subsequently, obtain W/O emulsion, in the oil phase of described W/O emulsion, emulsifying agent is 0.1wt%-1.5wt%, and aqueous phase compares 1:1-1:1000 with oil phase volume; Under 40 DEG C of-90 DEG C of constant temperature to W/O emulsion heating, and under 200-1000rpm mechanical agitation, reaction 1-5h; Subsequently, make W/O emulsion be quickly cooled to 15-40 DEG C, obtain W/O emulsion droplets, thus form the agarose microbeads without magnetic core.
3. method as claimed in claim 2, it is characterized in that, described organic solvent is the combination of atoleine, benzinum, olive oil, cottonseed oil, soybean oil, sunflower seed oil, alkanes or above-mentioned organic solvent.
4. the method for claim 1, it is characterized in that, described step (ii) is: take microballoon according to agarose microbeads and sodium borohydride mass ratio 1000:1-10:1, add the sodium hydroxide solution of 0.1mol/L-2mol/L, make the agarose microbeads-concentration of sodium borohydride mixture in sodium hydroxide solution be 0.01-10g/ml, under constant temperature, constant speed stirs; Subsequently, add the mixed solution of epoxychloropropane and dimethyl sulfoxide (DMSO) in aforesaid liquid, under constant temperature, constant speed stirs, and the volume ratio of described epoxychloropropane and dimethyl sulfoxide (DMSO) is 1:1-1:100.
5. the method for claim 1, it is characterized in that, described step (iii) is: solution step (ii) obtained carries out with ferrous ion-ferric ion mixed solution or mixes, and to make in iron ion mixed solution agarose microbeads concentration range at 0.01-2g/ml.Under 30-60 DEG C of constant temperature, 200-600rpm constant speed, soaks 10-120h, thus it is crosslinked that agarose microbeads and iron ion are activated.
6. the method for claim 1, it is characterized in that, described step (iv) is: in the agarose microbeads-ferric ion solutions of step (iii), add ammoniacal liquor, make the volume ratio 1:1-100:1 of agarose microbeads-ferric ion solutions and ammoniacal liquor; Under 40-80 DEG C of constant temperature, in the nitrogen environment of 200-1000rpm constant speed, reaction 0.5-3h, preparation has the Agarose Magnetic Microsphere of tri-iron tetroxide magnetic core; Be separated the Agarose Magnetic Microsphere obtained with magnetic field, save backup after washing.
7. the method for claim 1, it is characterized in that, described step (v) is: in ammoniacal liquor, add Agarose Magnetic Microsphere prepared by step (iv), 2-4 hour is reacted under room temperature, the glutaraldehyde water solution of 2-20% is added again after abundant drip washing, react 1-3 hour under room temperature, thoroughly clean excessive glutaraldehyde with ultra-pure water.
8. the method as described in claim 1-7, is characterized in that, described step (vi) is: at the NaHCO containing staphylococcus aureus protein A
3-Na
2cO
3the Agarose Magnetic Microsphere with crosslinked arm obtained in step (v) is added in buffer solution, clean with ultrapure water after reaction, add ethanolamine solutions and close unreacted epoxide group; Add bovine serum albumin subsequently, close unreacted aldehyde radical, reduce non-specific adsorption, finally clean with a large amount of ultra-pure waters, obtain Agarose Magnetic Microsphere product.
9. the Agarose Magnetic Microsphere utilizing the method described in claim 1-8 to prepare, is characterized in that, described Agarose Magnetic Microsphere is with glutaraldehyde cross-linking arm and the aglucon staphylococcus aureus protein A with IgG antibody generation specific binding.
10. a method for separation and purification IgG antibody, described method comprises the steps:
A) Agarose Magnetic Microsphere as claimed in claim 9 is mixed with the lysate of expressing IgG antibody cell, make IgG antibody be adsorbed in agarose microbeads;
B) magnetic field is utilized to be separated by the Agarose Magnetic Microsphere being adsorbed with IgG antibody;
C) dissociation solution is used by IgG antibody from wash-out Agarose Magnetic Microsphere, collection IgG antibody.
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