Method for high-efficiency purification of quantum dot-IgG monoclonal antibody conjugate

A monoclonal antibody and quantum dot technology, applied in the biological field, can solve problems such as harsh operating conditions, complicated process, and low yield, and achieve the effect of low equipment requirements, simple operation, and high-efficiency separation

Active Publication Date: 2014-03-26
江西昌大业力生物技术有限公司
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  • Description
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AI Technical Summary

Problems solved by technology

However, the traditional purification method of quantum dot 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 high-efficiency purification of quantum dot-IgG monoclonal antibody conjugate

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Embodiment 1

[0031] Embodiment 1 water-soluble quantum dot anti-aflatoxin B 1 Monoclonal antibody conjugates and purification process

[0032] Take 5 mL of commercial carboxylated water-soluble quantum dots (concentration: 50 nmol / L) and mix with an equal volume of 0.05 mol / L borate buffer at pH 6.0; respectively add 1- Ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide were reacted at 37°C for 2 hours; anti-aflatoxin was added with a molar ratio of 10:1 to quantum dots 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 Monoclonal antibody against water-soluble...

Embodiment 2

[0033] Example 2 Water-soluble quantum dot anti-ochratoxin monoclonal antibody conjugate and purification process

[0034] Take 5 mL of commercial carboxylated water-soluble quantum dots (concentration: 50 nmol / L) and mix with an equal volume of 0.05 mol / L borate buffer at pH 6.0; respectively add 1 -Ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide, reacted at 37°C for 2 hours; Toxin monoclonal antibody solution, after adjusting the pH of the solution to 8.0 with 1 M NaOH solution, 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. 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 The water-soluble quantum dot anti-ochratoxin monoclonal antibody conjugate without free anti-ochratoxin monoclonal antibody was obtained by dissolving in 0.05 mol / L phosphate buffer (pH 7.0-7.5). After the water-...

Embodiment 3

[0035] Example 3 Water-soluble carboxyl quantum dot anti-zearalenone monoclonal antibody conjugate and purification process

[0036] Take 5 mL of commercial carboxylated water-soluble quantum dots (concentration: 50 nmol / L) and mix with an equal volume of 0.05 mol / L borate buffer at pH 5.5; respectively add 1- Ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide were reacted at 37°C for 2 hours; the molar ratio of quantum dots was 3:1 by adding anti-zeb For enone monoclonal antibody solution, adjust the pH of the solution to 8.0-9.0 with 1 M NaOH solution, 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 The water-soluble quantum dot anti-zearalenone monoclonal antibody conjugate without free anti-zearalenone monoclonal antibody w...

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Abstract

The invention discloses a new method suitable for large-scale high-efficiency purification of a water soluble quantum dot-IgG monoclonal antibody conjugate, and belongs to the field of biotechnology. In allusion to the disadvantages that a conventional quantum dot-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 quantum dot-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 quantum dot-antibody conjugate is achieved by an ordinary high-speed centrifugation method. The method simplifies experimental operation procedures of the quantum dot-antibody conjugate, reduces requirements on separation equipment, is suitable for large-batch purification of the quantum dot-IgG monoclonal antibody conjugate, allows the yield to be more than 85%, and allows fluorescence 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, 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 have huge commercial value. However, mouse-derived IgG monoclonal antibodies do not have optical and electrical pro...

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 熊勇华许恒毅罗薇许杨魏华徐威
Owner 江西昌大业力生物技术有限公司
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