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A novel method of preparing agarose magnetic microspheres and uses of the agarose magnetic microspheres in separation and purification of an IgG antibody

A technology of agarose microspheres and magnetic microspheres, applied in other chemical processes, chemical instruments and methods, inorganic chemistry, etc., can solve the problems of small specific surface area, low separation and purification, low coating efficiency, etc., and achieve particle size distribution. Uniform, not easy to agglomerate and agglomerate, easy to operate and save time

Inactive Publication Date: 2015-04-01
HARBIN INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the preparation methods of agarose magnetic microspheres currently used all have certain defects, such as uneven coating of agarose, low coating efficiency, poor nucleation of magnetic substances, non-balling of magnetic balls, and serious agglomeration. , small specific surface area, low separation and purification filter and poor magnetic response
[0008] Up to now, the research of applying agarose magnetic microspheres to antibodies has not been reported. The present invention proposes a new method for preparing agarose magnetic microspheres. The agarose magnetic microspheres obtained according to the method of the present invention do not have the above-mentioned disadvantages. Moreover, the surface modification of the prepared agarose magnetic microspheres can also be carried out, so that the microspheres can be activated

Method used

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  • A novel method of preparing agarose magnetic microspheres and uses of the agarose magnetic microspheres in separation and purification of an IgG antibody
  • A novel method of preparing agarose magnetic microspheres and uses of the agarose magnetic microspheres in separation and purification of an IgG antibody
  • A novel method of preparing agarose magnetic microspheres and uses of the agarose magnetic microspheres in separation and purification of an IgG antibody

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Experimental program
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Effect test

Embodiment 1

[0067] Embodiment 1: Preparation of agarose magnetic microspheres

[0068] (i) Preparation of non-magnetic core agarose microspheres:

[0069] Add 2.0 g of agarose to 100 ml of ultrapure water and heat at 100°C to prepare the agarose aqueous phase W. Subsequently, 8.0 g of emulsifier Span 80 was added to 180 ml of liquid paraffin and 40 ml of petroleum ether, and fully dispersed to prepare oil phase O. The oil phase O was heated at a constant temperature of 80°C, mechanically stirred at 400rpm, and the water phase was quickly transferred in, and reacted for 3h. The ice-water bath was cooled rapidly to 15°C.

[0070] (ii) Activation and cross-linking of non-magnetic core agarose microspheres:

[0071]Take 5g of microspheres, add 7.5ml of 0.8mol / L NaOH, 0.01g of sodium borohydride, shake at 25°C and 400rpm for 30min. To the solution thus prepared were added 7.5 ml of dimethyl sulfoxide and 3.75 ml of epichlorohydrin. React at a constant temperature of 30°C and 400rpm for 20...

Embodiment 2

[0080] Embodiment 2: Magnetic separation and purification of IgG monoclonal antibody

[0081] Take 1ml of the prepared agarose magnetic microspheres, after fully washing and balancing with PBS buffer, add 10ml of lysate of cells expressing IgG monoclonal antibody diluted to a certain concentration with buffer, react with slow shaking for 1 hour, and transfer the liquid to Into the magnetic separation and purification device, adsorb and fix the magnetic microspheres, remove the separation device and discard the reaction solution, then wash with 10ml PBS buffer solution for 3 times, add 4ml 0.1M glycine-hydrochloric acid buffer solution, and slowly shake for 1 hour for dissociation The adsorbed antibodies are then transferred to the magnetic separation and purification device, the magnetic microspheres are adsorbed, and the dissociation solution is collected.

Embodiment 3

[0082] Embodiment 3: the elution recovery of magnetic microsphere

[0083] After collecting the dissociation solution, add PBS buffer solution to the adsorption magnetic balls of Example 2 and wash them several times, balance the magnetic microspheres, discard the cleaning solution, and store the washed microspheres in 20% ethanol aqueous solution at 4°C , can be reused more than 20 times.

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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 [0001] The invention relates to a new method for preparing agarose magnetic microspheres and using the prepared agarose magnetic microspheres to separate and purify IgG antibodies by using immunomagnetic separation technology, belonging to the field of biotechnology. Background technique [0002] Antibody refers to a homologous antibody produced by a single clonal hybridoma cell that only recognizes a specific epitope, and is a special type of antibody that has been artificially prepared. The antibody is a type of globulin produced when B cells differentiate into plasma cells, exists in body fluids, specifically binds to corresponding antigens (such as pathogens), and exerts immune effects with the participation of other immune molecules and cells. Can mediate humoral immunity. Antibodies have highly uniform physical and chemical properties, single biological activity, strong specificity in binding to antigens, are convenient for manual handling and quality ...

Claims

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Application Information

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IPC IPC(8): B01J20/24B01J20/28B01J20/30G01N33/531
CPCB01J20/24B01J20/28009B01J20/3085B01J2220/4825G01N33/53
Inventor 赵九蓬马丽华李垚李娜李会成梁秋波曹喜生
Owner HARBIN INST OF TECH
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