CN102553467A - Sulfadimidine affinity membrane and application of sulfadimidine affinity membrane in antibody separation and purification - Google Patents
Sulfadimidine affinity membrane and application of sulfadimidine affinity membrane in antibody separation and purification Download PDFInfo
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- CN102553467A CN102553467A CN201110449444XA CN201110449444A CN102553467A CN 102553467 A CN102553467 A CN 102553467A CN 201110449444X A CN201110449444X A CN 201110449444XA CN 201110449444 A CN201110449444 A CN 201110449444A CN 102553467 A CN102553467 A CN 102553467A
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- sulfamethazine
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Abstract
The invention discloses a sulfadimidine affinity membrane; a preparation method of the sulfadimidine affinity membrane comprises the following steps: (1) taking a cellulose acetate membrane, completely hydrolyzing under an alkaling condition, then adding epoxy chloropropane to perform epoxy activation, and then using deionized water to wash to be neutral after reaction under a suction filter condition, so as to obtain the epoxy activated membrane; (2) dissolving the sulfadimidine into an alkaline buffer solution with pH of 10-12, then dispersing the epoxy activated membrane obtained in the step (1) in the above-mentioned solution, reacting at the temperature of 50-65 DEG C for 24h-36h, then using deionized water to wash under the suction filter condition to make UV absorption of a percolate at 280nm close to zero, and then drying the solution to get the sulfadimidine affinity membrane. The sulfadimidine affinity membrane has good adsorption performance for an antibody, and is particularly suitable for separating and purifying immune globulin IgG.
Description
One, technical field
The present invention relates to field of biomedical materials, particularly relate to the preparation and the application in the antibody separation and purification thereof of sulfamethazine affinity membrane.
Two, background technology
Antibody is in the immune response to antigenic stimulus, the albuminoid that bone-marrow-derived lymphocyte produces.It is can be special with corresponding antigens the globulin that combines, produces various immunological effects (physiological effect).As a kind of special protein molecule, antibody often is used as the reagent of in-vitro diagnosis, the medicine of treatment disease, the aglucon of immunoaffinity chromatography etc., in life science, biotechnology and medical domain, has a wide range of applications.Especially antibody is as the core reagent of various immunoassays, and immunoassay result's sensitivity, specificity played crucial effects.In the research of the new gene outcome close to some and life entity functional relationship, whether have corresponding antibody to detect and the product albumen matter of identifying new gene, be the key that can whole research work be carried out in a deep going way.
The method of separation and purification antibody commonly used has salting out method, caprylic acid-saturated ammonium sulfate method, affinity chromatography, gel-filtration chromatography, hydrophobic chromatography and ion exchange chromatography etc. at present, is not very high antibody but preceding two various methodologies can only obtain purity; Though the affinity chromatography selectivity is better, have steps consuming time such as dressing post, preliminary treatment, and treating capacity is less; Gel permeation chromatography, hydrophobic chromatography and ion-exchange chromatography exist in equally also that treating capacity is little, and the time is long, problems such as dress post.
Since the mid-80, the affinity membrane isolation technics has appearred.It organically combines two kinds of technology; Performance advantage separately; Cover the shortage, not only utilized the Recognition of Biomolecular function, can the high selectively biological product of separating low concentration; And the big characteristics of the permeation flux that utilizes film, can lower operation press with higher flow velocity under target protein is separated and purifying fast; Easy to operate, equipment is simple.Utilize at present affinity membrane can purifying with produce pharmaceutical products such as albumin, monoclonal antibody, interferon, fibrin, trypsase and inhibitor thereof; And demonstrate wide application prospect in fields such as medical test, external circulating therapy technology, receive increasingly extensive concern.The affinity membrane isolation technics combines affinity chromatography and membrane separation technique; Utilize the biological product of Recognition of Biomolecular function separating low concentration; And realize quick separation with the convective mass transfer mode; Have be easy to amplify, characteristics that separating rate is fast, possess the advantage that the affinity chromatography selectivity is good, separation accuracy is high again, be the biological product purifies and separates technology that receives much concern.
Three, summary of the invention
First technical problem that the present invention will solve provides a kind of sulfamethazine affinity membrane; Described sulfamethazine affinity membrane antagonist (especially Immunoglobulin IgG) has the specific adsorption effect; Gained antibody has higher purity and activity, is applicable to extensive separation and purification antibody.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
A kind of sulfamethazine affinity membrane, the preparation method of described sulfamethazine comprises the steps:
(1) get CAM, abundant hydrolysis under alkali condition earlier adds epoxychloropropane then and carries out the epoxy activation, and reaction finishes the back and under the suction filtration condition, is washed till neutrality with deionized water, obtains the film after the epoxy activation;
(2) sulfamethazine is dissolved in the alkaline buffer solution of pH 10～12; Then the film after the epoxy activation of step (1) acquisition is dispersed in the above-mentioned solution; At 50～65 ℃ of reaction 24h～36h; Under the suction filtration condition, be washed till the uv absorption of filter liquor and approach 0, promptly get the sulfamethazine affinity membrane after the drying at the 280nm place with deionized water.
It is basement membrane that the present invention selects the film of cellulose acetate for use, and this is that activation method is easier, so it is more suitable for as basement membrane of the present invention with respect to nitrocellulose filter and cellulose mixture film etc. because the CAM non-specific adsorption is lower.Cellulose acetate film of the present invention uses the commercial goods.
The present invention needs processings that be hydrolyzed of first Dichlorodiphenyl Acetate tunica fibrosa, with the modification density of aglucon on the raising film in preparation affinity membrane process.The present invention recommends the hydrolysis of said CAM in 1.5～2mol/LNaOH solution, to carry out, and hydrolysis temperature is 60～80 ℃, and hydrolysis time is 1.5～2.5h.
Among the present invention, for improving the epoxy activation effect, preferred epoxy activation temperature is 40～60 ℃, and preferred epoxy soak time is 4～6h.
The adding volume of the preferred said epoxychloropropane of the present invention is 15%～20% of a hydrolysis afterreaction system volume.
Among the present invention, the adding quality optimization of said sulfamethazine be after the epoxy activation film quality 10%～20%.
Among the present invention, the alkaline buffer solution of described pH 10～12 is preferably PBS cushioning liquid or the carbonate buffer solution of pH 10～12.
The present invention as basement membrane, through to the design of reactions step and the control of reaction condition, has obtained to have the sulfamethazine affinity membrane that better aglucon is modified density with cellulose acetate film.
Second technical problem that the present invention will solve is that described sulfamethazine affinity membrane is applied to the application in the antibody separation and purification, to obtain having higher degree and active antibody.
Concrete; The device sketch map of described antibody separation and purification is as shown in Figure 1; Described separation and purification can be carried out according to following steps: get the sulfamethazine affinity membrane and pack in the film cup, will with the ascites after the cushioning liquid dilution with peristaltic pump pack into let in the film cup aglucon on itself and the affinity membrane fully adsorb after, pump into deionized water with peristaltic pump and be close to 0 until the uv absorption of filter liquor at the 280nm place; And then pump into elution buffer with peristaltic pump; Collect the material that wash-out comes out, the eluent that obtains is the antibody after the separation and purification from ascites, detects its purity with SDS-PAGE.
Further, because the intrinsic property of described sulfamethazine affinity membrane, separation and purification of the present invention is preferably carried out under acid condition, preferably under the condition of pH5.5～7.5, carries out, and more preferably under the condition of pH5.5～6.0, carries out.In the separation and purification process, utilize the pH value of the buffer solution hierarchy of control, for example the PBS buffer solution.
Further, contain NaCl in the described elution buffer, wherein the concentration of NaCl is 1.0～3.0mol/L, adopts the eluting salt of high concentration to avoid pickling to take off the active influence of antagonist.Preferred elution buffer is the PBS buffer solution of pH5.5～6.0 of containing 1.5～2.0mol/L NaCl.
Sulfamethazine affinity membrane of the present invention has absorption property preferably for antibody, is particularly suited for the separation and purification Immunoglobulin IgG.
Compared with prior art, beneficial effect of the present invention is:
(1) the present invention is that basement membrane sulfamethazine aglucon in epoxy activation and coupling makes the sulfamethazine affinity membrane with the CAM, and method is simple for this, and the sulfamethazine affinity membrane that makes has good aglucon modification density;
(2) sulfamethazine affinity membrane of the present invention is used for separation and purification antibody and has higher selectivity, and gained antibody has higher purity and activity; And isolation and purification method of the present invention is simple, and operating condition is gentle, need not adorn post and handle, and treats that the mode that adopts film to separate after the adsorption equilibrium removes the foreign protein that is not combined, and can obtain purity greater than 90% antibody.
Four, description of drawings
Fig. 1 is the installation drawing of embodiment 1 this separation method of gained; Wherein, 1: magnetic stirring apparatus, 2: micro-filtration, 3: level pad, 4: ascites solution, 5: elution buffer, 6: peristaltic pump, 7: Pressure gauge, 8: fraction collector, 9: UV-detector;
Fig. 2 is the adsorption isotherm of embodiment 1 gained affinity membrane;
Fig. 3 is embodiment 2 gained adsorption effect figure, wherein 1=albumen marker; 2=standard I gG; 3=ascites; 4=antibody behind the purifying from ascites.
Five, the specific embodiment
With specific embodiment technical scheme of the present invention is further specified below, but protection scope of the present invention is not limited thereto:
Embodiment 1: the preparation of sulfamethazine affinity membrane
(1) (promise of Hangzhou section, Φ=10cm) be dispersed in the NaOH solution that 100mL concentration is 2mol/L is in 70 ℃ of (175rpm) hydrolysis 1.5h in the water-bath percussion table with 10 CAMs.After being cooled to 40 ℃, add volume fraction and be 20% epoxychloropropane solution, in 40 ℃ of reaction 4h, reaction finishes the back and under the suction filtration condition, is washed till neutrality with deionized water, promptly gets the film after the epoxy activation.
(2) the 1.0g sulfamethazine is dissolved in pH10.5, in the PBS cushioning liquid of 20mmol/L, the film 10 (about 5g) after the activation of step (1) gained epoxy is scattered in this cushioning liquid, in 55 ℃ of reaction water-bath concussion (175rpm) 36h.Under the suction filtration condition, be washed till the uv absorption of filter liquor at the 280nm place with deionized water then and approach 0, promptly get the sulfamethazine affinity membrane after the drying, it is as shown in the table that it modifies density parameter:
| Affinity membrane | Epoxide group | Sulfamethazine |
| Modify density (umol/cm 2) | 1.01 | 0.73 |
Taking by weighing 100mg (being equivalent to 1/4 film) after affinity membrane shredded respectively is dispersed in the solution of 6 different antibodies IgG of 5mL content (0.0615,0.123,0.1845,0.246,0.3075,0.369mg/mL); The room temperature vibration is adsorbed to balance; The concentration of antibody in the solution when measuring balance; Obtain this affinity membrane adsorption isotherm with equilibrium concentration and adsorption capacity mapping, as shown in Figure 2.
Embodiment 2: the application of sulfamethazine affinity membrane in the antibody separation and purification
Get 10～15 of the sulfamethazine affinity membranes that embodiment 1 makes; With in its film cup of packing into (like Fig. 1); 10ml ascites is diluted to 100mL with the PBS cushioning liquid of pH 5.5,20mmol/L; And then after pumping into the aglucon that lets in the film cup on itself and the film and fully adsorb with the flow velocity of 2ml/min with peristaltic pump, pump into deionized water with identical flow velocity and be close to 0 until the uv absorption of filter liquor at the 280nm place.Pump into elution buffer (the PBS+1.5mol/L NaCl of PH5.5 20mmol/L) with identical flow velocity then.Collect the material that wash-out comes out, behind the centrifugal 15min of ultra-filtration centrifuge tube 3000rpm, it is sub-packed in the pipe of 1.5mL freezing.
Detect antibody purity with SDS-PAGE, the result is as shown in Figure 3,1 expression albumen marker, 2 expression standard antibody, 3 expression ascites, antibody behind the 4 expression purifying.What band 4 was represented is the antibody with this method separation and purification, thereby can find out that ascites has obtained good purifying.
The elisa method is surveyed tiring all 10 of antibody indirectly
-7More than, biologically active is good.
Claims (10)
1. sulfamethazine affinity membrane, it is characterized in that: the preparation method of said sulfamethazine affinity membrane comprises the steps:
(1) get CAM, abundant hydrolysis under alkali condition earlier adds epoxychloropropane then and carries out the epoxy activation, and reaction finishes the back and under the suction filtration condition, is washed till neutrality with deionized water, obtains the film after the epoxy activation;
(2) sulfamethazine is dissolved in the alkaline buffer solution of pH 10～12; Then the film after the epoxy activation of step (1) acquisition is dispersed in the above-mentioned solution; At 50～65 ℃ of reaction 24h～36h; Under the suction filtration condition, be washed till the uv absorption of filter liquor and approach 0, promptly get the sulfamethazine affinity membrane after the drying at the 280nm place with deionized water.
2. sulfamethazine affinity membrane as claimed in claim 1 is characterized in that: the hydrolysis of said CAM is carried out in 1.5～2mol/L NaOH solution, and hydrolysis temperature is 60～80 ℃, and hydrolysis time is 1.5～2.5h.
3. sulfamethazine affinity membrane as claimed in claim 2 is characterized in that: the epoxy activation temperature is 40～60 ℃, and the epoxy soak time is 4～6h.
4. sulfamethazine affinity membrane as claimed in claim 3 is characterized in that: the adding volume of said epoxychloropropane is 15%～20% of a hydrolysis afterreaction system volume.
5. like the described sulfamethazine affinity membrane of one of claim 1～4, it is characterized in that: the adding quality of said sulfamethazine be after the epoxy activation film quality 10%～20%.
6. like the described sulfamethazine affinity membrane of one of claim 1～4, it is characterized in that: the alkaline buffer solution of described pH 10～12 is PBS cushioning liquid or the carbonate buffer solution of pH 10～12.
7. the application of sulfamethazine affinity membrane as claimed in claim 1 in the antibody separation and purification is characterized in that: carry out under the condition of the described pH5.5 of being applied in～7.5.
8. the application of sulfamethazine affinity membrane as claimed in claim 7 in the antibody separation and purification is characterized in that: carry out under the condition of the described pH of being applied in 5.5～6.0.
9. like claim 7 or 8 application of described sulfamethazine affinity membrane in the antibody separation and purification, it is characterized in that: described antibody is Immunoglobulin IgG.
10. the application of sulfamethazine affinity membrane as claimed in claim 7 in the antibody separation and purification; It is characterized in that: described separation and purification is carried out according to following steps: get the sulfamethazine affinity membrane and pack in the film cup; To pack into peristaltic pump with the ascites after the dilution of the PBS cushioning liquid of pH5.5～6.0 lets in the film cup aglucon on itself and the sulfamethazine affinity membrane fully adsorb; Pump into deionized water with peristaltic pump then and be close to 0 until the uv absorption of filter liquor at the 280nm place; Pump into the PBS buffer solution elution of pH5.5～6.0 of containing 1.5～2.0mol/L NaCl again with peristaltic pump; Collect eluent, promptly get the antibody after the separation and purification from ascites.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112657347A (en) * | 2020-12-16 | 2021-04-16 | 杭州科百特科技有限公司 | Regenerated cellulose ultrafiltration membrane and application and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5006287A (en) * | 1988-01-14 | 1991-04-09 | The Standard Oil Company | Affinity membranes having pendant hydroxy groups and processes for the preparation and use thereof |
| CN1548224A (en) * | 2003-05-21 | 2004-11-24 | 中国科学院化学研究所 | Affinity chromatographic stuffing with sulfadimidine as ligand |
| CN101596422A (en) * | 2009-07-07 | 2009-12-09 | 浙江大学 | With amino acid is the preparation method of the polyvinylidene fluoride affinity membrane of aglucon |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5006287A (en) * | 1988-01-14 | 1991-04-09 | The Standard Oil Company | Affinity membranes having pendant hydroxy groups and processes for the preparation and use thereof |
| CN1548224A (en) * | 2003-05-21 | 2004-11-24 | 中国科学院化学研究所 | Affinity chromatographic stuffing with sulfadimidine as ligand |
| CN101596422A (en) * | 2009-07-07 | 2009-12-09 | 浙江大学 | With amino acid is the preparation method of the polyvinylidene fluoride affinity membrane of aglucon |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112657347A (en) * | 2020-12-16 | 2021-04-16 | 杭州科百特科技有限公司 | Regenerated cellulose ultrafiltration membrane and application and preparation method thereof |
| CN112657347B (en) * | 2020-12-16 | 2022-11-29 | 杭州科百特科技有限公司 | Regenerated cellulose ultrafiltration membrane and application and preparation method thereof |
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Address after: No.60, Beijing Middle Road, Yantai Development Zone, Yantai area, China (Shandong) pilot Free Trade Zone, Yantai City, Shandong Province 264006 Patentee after: Yantai maibairui international biomedical Co.,Ltd. Address before: No.60, Beijing Middle Road, Yantai Development Zone, Yantai area, China (Shandong) pilot Free Trade Zone, Yantai City, Shandong Province 264006 Patentee before: MABPLEX INTERNATIONAL, Ltd. |
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