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Method for purification of conjugate of water soluble nano silver particles and mouse-origin IgG monoclonal antibody

A nano-silver particle and water-soluble technology, applied in chemical instruments and methods, peptide preparation methods, organic chemistry, etc., can solve the problems of harsh operating conditions, complicated processes, and low yields, and achieve low equipment requirements and simple operations Effect

Active Publication Date: 2014-03-26
HUNAN ANSON MEDICAL POLYMER MATERIALS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional methods for purification of monoclonal antibody conjugates with silver nanoparticles have disadvantages such as harsh operating conditions, complicated procedures, low yields, and difficulty in large-scale production.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Synthesis of water-soluble carboxylated silver nanoparticles with an amphiphilic polymer as the shell

[0031] The concentration is 98% concentrated sulfuric acid (H 2 SO 4 ) and a concentration of 30% hydrogen peroxide (H 2 o 2 ) in a volume ratio (1:3) and mix them evenly, and heat them on an electric stove until boiling. Take a certain amount of quartz and silicon wafers and slowly add them to the boiling solution, react for 20 minutes, and rinse with a large amount of distilled water. The rinsed quartz and silicon wafers were immersed in an aqueous solution of polydiallyldimethylammonium chloride (PDDA, 1.0 mg / mL) and reacted for 20 min. The above reaction solution was added to 10 mM Ti(SO 4 ) 2 Aqueous solution (0.1 M H 2 SO 4 ), react for 5 min. The reaction matrix was then transferred to a phosphate buffer (pH 4.0) for a few seconds and then transferred to another phosphate buffer (pH 4.0) for 5 min. Finally, rinse with copious amounts of dist...

Embodiment 2

[0033] Embodiment 2 water-soluble nano-silver particles resist aflatoxin B 1 Monoclonal antibody conjugates and purification process

[0034] Take 5 mL of commercial carboxylated water-soluble silver nanoparticles (concentration: 50 nmol / L) and mix with an equal volume of 0.05 mol / L borate buffer at pH 6.0; 1-Ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide were reacted at 37°C for 2 hours; the molar ratio of 10:1 to the nano-silver particles was added Aflatoxin B 1 Monoclonal antibody solution, after adjusting the pH of the solution to 7.5 with 1 M NaOH solution, react at room temperature for 3 hours; finally add 2% glucosamine to the solution, and further adjust the pH to 4.5 with 1 M HCl solution. 18,000 rpm (about 29,000 g) centrifuge at 4°C for 30 min, discard the supernatant, and precipitate with 25% glycerol, 0.01% NaN 3 0.05 mol / L phosphate buffer (pH 7.0-7.5) was dissolved to obtain free anti-aflatoxin B 1 Water-soluble silver nanoparticles...

Embodiment 3

[0035] Example 3 Water-soluble nano-silver particle anti-ochratoxin monoclonal antibody conjugate and purification process

[0036] Take 5 mL of commercial carboxylated water-soluble silver nanoparticles (concentration: 50 nmol / L) and mix with an equal volume of 0.05 mol / L borate buffer at pH 6.0; 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide were reacted at 37°C for 2 hours; Anti-ochratoxin monoclonal antibody solution, adjust the pH of the solution to 8.0 with 1 M NaOH solution, and react at room temperature for 3 hours; finally add 2.5% glucosamine to the solution, and further adjust the pH to 4.5 with 1 M HCl solution. Centrifuge at 18,000 rpm (about 29,000 g) at 4°C for 30 min, discard the supernatant, and use 25% glycerol, 0.01% NaN for precipitation 3 0.05 mol / L phosphate buffer (pH 7.0-7.5) was dissolved to obtain the water-soluble nano-silver particle anti-ochratoxin monoclonal antibody conjugate without free anti-ochratoxin monoclonal an...

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Abstract

The invention discloses a new method for large-scale high-efficiency purification of a conjugate of water soluble nano silver particles and a mouse-origin IgG monoclonal antibody, and belongs to the field of biotechnology. In allusion to the disadvantages that a conventional nano silver particle-antibody conjugate has complex purification process and low recovery rate and difficultly realizes large-scale production, glucosamine is adopted to seal residual carboxyl of the nano silver particle-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 nano silver particle-antibody conjugate is achieved by an ordinary high-speed centrifugation method. The method simplifies experimental operation procedures of the nano silver particle-antibody conjugate, reduces requirements on separation equipment, is suitable for large-batch purification of the conjugate of the nano silver particles and the mouse-origin IgG monoclonal antibody, 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 relates to a new method for biological modification of nanomaterials and purification of conjugates, in particular to a new method for highly efficient purification of carboxylated water-soluble nano-silver particle mouse-derived IgG monoclonal antibody conjugates. Background technique [0002] Since the monoclonal antibody was first prepared by cell fusion technology in 1975, hybridoma technology has developed rapidly. A large number of monoclonal antibodies have been successfully prepared and widely used in immunology, biochemistry, pharmacology, cell biology, microbiology and clinical fields. 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 ...

Claims

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Application Information

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IPC IPC(8): C07K16/14C07K16/44C07K16/12C07K1/13
Inventor 许恒毅熊勇华罗薇魏华赖卫华黄小林
Owner HUNAN ANSON MEDICAL POLYMER MATERIALS
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