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Method for purification of conjugate of water soluble nano iron oxide and mouse-origin IgG monoclonal antibody

A kind of iron oxide and water-soluble technology, applied in the biological field, can solve the problems of harsh operating conditions, complicated process, low yield, etc., and achieve the effect of low equipment requirements and simple operation

Inactive Publication Date: 2014-03-26
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the traditional method for purification of iron oxide nanoparticle monoclonal antibody conjugates has disadvantages such as harsh operating conditions, complicated process, low yield and difficulty in large-scale production.

Method used

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  • Method for purification of conjugate of water soluble nano iron oxide and mouse-origin IgG monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Synthesis of water-soluble carboxylated iron oxide nanoparticles with amphiphilic polymer as the shell

[0033] At room temperature, 0.1 nmol of FeO(OH), 1.5 mL of oleic acid and 18.75 mL of 1-octadecene were uniformly mixed, and the mixed solution was heated and stirred to 320 °C under nitrogen pressure. When the reaction solution turns from black turbidity to brown transparent, iron carboxylate is formed. When the solution turns black and transparent, ferric oxide (Fe 3 o 4 ) nanocrystal solution formation. To the above ferric oxide (Fe 3 o 4 ) into the nanocrystal solution by adding acetone solution to precipitate iron oxide nanoparticles, and then further purify with chloroform / acetone solution with a volume ratio of 1:10 until iron oxide nanocrystal powder is formed. Take 1 g polystearyl maleic anhydride, 1.2 g 2-(2-aminoethoxy) ethanol and a certain amount of Fe 3 o 4 Crystal powder (Fe 3 o 4 The molar ratio of crystal powder / polymer is 1:10)...

Embodiment 2

[0035] Example 2 This experimental method prepares water-soluble carboxylated iron oxide nanoparticles with a particle size of 8 nm and the coupling and purification process of anti-enrofloxacin monoclonal antibody

[0036] Take 5 mL of carboxylated water-soluble iron oxide nanoparticles with a particle size of 8 nm (concentration: 5 mg / mL) obtained in Example 1 and mix them with an equal volume of 0.05 mol / L borate buffer at pH 6.0; 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide with a molar ratio of iron oxide nanoparticles of 150:1 were reacted at 37°C for 2 hours; Anti-enrofloxacin monoclonal antibody solution with a molar ratio of iron oxide nanoparticles of 10:1, adjusted the pH of the solution to 8.0-9.0 with 1 M NaOH solution, and reacted at room temperature for 3 hours; finally added to the solution with a final concentration of 1% Single-end aminated polyethylene glycol (HO-PEG-NH 3 ), and further adjust the pH to 5.0 with 1 M HCl solu...

Embodiment 3

[0037] Example 3 This experimental method prepares water-soluble carboxylated iron oxide nanoparticles with a particle size of 8 nm and is coupled with anti-clenbuterol monoclonal antibody and purification process

[0038] Take 5 mL of carboxylated water-soluble iron oxide nanoparticles with a particle size of 8 nm (concentration: 5 mg / mL) obtained in Example 1 and mix them with an equal volume of 0.05 mol / L borate buffer at pH 6.0; 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide with a molar ratio of iron oxide nanoparticles of 150:1 were reacted at 37°C for 2 hours; Anti-clenbuterol hydrochloride monoclonal antibody solution with a molar ratio of iron oxide nanoparticles of 10:1, adjust the pH of the solution to 8.0-9.0 with 1 M NaOH solution, and react at room temperature for 3 hours; finally add the final concentration of 1 %Single-end aminated polyethylene glycol (HO-PEG-NH 3 ), and further adjust the pH to 5.0 with 1 M HCl solution. Centrifuge...

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Abstract

The invention discloses a method for large-scale high-efficiency purification of a conjugate of water soluble iron oxide nanoparticles and an IgG monoclonal antibody, and belongs to the field of biotechnology. In allusion to the disadvantages that a conventional iron oxide nanoparticle-antibody conjugate has complex purification process and low recovery rate and difficultly realizes large-scale production, single-terminal aminated polyethylene glycol is adopted to seal residual carboxyl of the iron oxide nanoparticle-antibody conjugate, a surface zeta potential of the conjugate is reduced; by adjusting the pH value of a solution to 4.5-5.0, the net charge content of the conjugate in the solution is further reduced, and the large-scale high-efficiency purification of the iron oxide nanoparticle-antibody conjugate is achieved by an ordinary high-speed centrifugation method. The method simplifies experimental operation procedures of the iron oxide nanoparticle-antibody conjugate, reduces requirements on separation equipment, is suitable for large-batch purification of the iron oxide nanoparticle-antibody conjugate, allows the yield to be more than 90%, and allows optical characteristics and biological activity of the conjugate to have no significant changes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a new method for biological modification of nanometer materials and purification of conjugates. Background technique [0002] Since the monoclonal antibody was first prepared by cell fusion technology in 1975, the basic hybridoma technology based on natural hybridization technology has been developed rapidly. A large number of monoclonal antibodies have been successfully obtained and are widely used in various fields such as immunology, biochemistry, pharmacology, cell biology, microbiology and clinical practice. At present, mouse, rat and human hybridoma cell lines have been prepared, among which the most widely used is still the mouse hybridoma cell line. Therefore, murine IgG monoclonal antibodies naturally become the most important and most widely used monoclonal antibodies. Such antibodies not only have important scientific research value, but also have huge commercial value. H...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/14C07K16/44C07K16/12C07K1/13
Inventor 熊勇华许恒毅罗薇魏华赖卫华黄小林
Owner NANCHANG UNIV
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