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Method for carrying cell sorting by using gold magnetism particles

A technology of gold magnetic particles and cells, applied in biochemical equipment and methods, microorganisms, tissue culture, etc., can solve the problems of loss of antibody activity, not involving cell sorting, and complex fixation methods.

Active Publication Date: 2008-04-23
XIAN GOLDMAG NANOBIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of these magnetic particles used for cell separation are organic / inorganic magnetic composite particles, that is, with magnetic materials as the core, organic small molecules, natural polymers, organic synthetic polymers, etc. as the shell, these magnetic particles are used for cell separation. However, there are still some defects in this method: for example, antibody molecules are fixed by covalent modification on the surface, and the immobilization method is complicated and easily causes the loss of antibody activity; the number of antibody molecules immobilized by magnetic particles per unit mass is small, and the dissociation of magnetic particles and cells in the subsequent step of cell sorting is complicated and difficult. high cost, expensive
[0004] The preparation methods and structural composition of assembled magnetic composite particles and core / shell superparamagnetic composite particles have been disclosed in Chinese patents ZL 03153486.4 and ZL 03124061.5 respectively, but their applications mainly include molecular or cell labeling and related detection , not related to the content of cell sorting

Method used

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  • Method for carrying cell sorting by using gold magnetism particles
  • Method for carrying cell sorting by using gold magnetism particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Monoclonal antibody against cell surface antigen (CD3 monoclonal antibody) is directly immobilized on the surface of gold magnetic particles.

[0085] Take 1 mg of gold magnetic particles, put them in a centrifuge tube, add 500 μl of 0.02M Tris-HCl pH7.4, shake by hand, magnetically separate for about 2 minutes, discard the supernatant, and dissolve 75 mg of CD3 monoclonal antibody in 500 μl of 0.02M Tris - In HCl pH7.4, take 400 μl and add it to the centrifuge tube containing the pretreated gold magnetic particles, and reserve 100 μl for UV detection; Under the conditions of 37°C and 180r / min, fully react for 30min. After the reaction is completed, magnetically separate, take 100μl of the supernatant for ultraviolet detection, and discard the rest. Wash the gold magnetic particles to wash away excess antibody molecules, add 500 μl of 1×PBS containing 5% Tween and shake well, magnetically separate and discard the supernatant, repeat this step twice, and use 400 μl of 5%...

Embodiment 2

[0088] The gold magnetic particles immobilized the monoclonal antibody (anti-CD3 * monoclonal antibody).

[0089] Take 1 mg of gold magnetic particles, put them in a centrifuge tube, add 500 μl of 0.02M Tris-HCl pH7.4, shake well, magnetically separate for 2 minutes, discard the supernatant, and dissolve 75 μg of goat anti-mouse IgG antibody solution in 500 μl of 0.02M Tris-HCl pH7.4, and then added to the centrifuge tube containing the pretreated gold magnetic particles; put the centrifuge tube containing the gold magnetic particles and antibody in a shaker, at 37°C, 180r / min After fully reacting for 30 min, after the reaction was completed, magnetic separation was performed, and the supernatant was discarded; the gold magnetic particles were washed once by adding 500 μl of 1×PBS containing 0.05% Tween 20, and then washed with 400 μl of 0.01M Tris- HCl was added to the gold magnetic particles, then placed in a shaker, fully reacted at 37°C and 180r / min for 30min, and washed ...

Embodiment 3

[0091] Separation of CD3+ cells by gold magnetic particles indirectly immobilized monoclonal antibody molecules by secondary antibody

[0092] Preparation of cell samples: Count Raji (CD3-) and Jurkat (CD3+) cells separately, mix them at a ratio of 4:1, centrifuge at 1200r / min at 4°C for 5min, discard the supernatant, collect the precipitate, and use sorting buffer 1 ×PBS containing 1% BSA+2mM EDTA resuspended to a certain volume.

[0093] Combination of cells and gold magnetic particles: cell samples and gold magnetic particles 6 Add 200 μg of gold magnetic particles indirectly immobilized with monoclonal antibody molecules by the secondary antibody to each target cell, mix well, incubate at room temperature for 30 minutes, and gently shake once every 5 minutes to prevent the cells from sinking to the bottom of the tube, so that the cell surface antigen and monoclonal antibody are fully Combined to fix cells on the surface of gold magnetic particles;

[0094] Magnetic separ...

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Abstract

The present invention is technological scheme of magnetic metal particle process for separating out target cell from cell mixture. The process includes the following steps: 1. fixing monoclonal antibody molecule onto the surface of magnetic metal particle; 2. incubating the magnetic metal particle with fixed monoclonal antibody molecule and the cell mixture to be separated for 15-60 min for combining the cell surface antigen to the monoclonal antibody and combining the cell onto the surface of magnetic metal particle; 3. magnetically separating the target cell from other cells in a magnetic separator; and 4. washing the magnetic metal particle to eliminate non-specifically combined cells. The process has the advantages of short time, low cost, low cell toxicity, no influence on cell activity, etc.

Description

technical field [0001] The invention relates to a method for separating desired target cells from a mixed sample containing different cells by using gold magnetic particles. Background technique [0002] With the continuous development of life sciences, the basic research of cell biology and immunology, as well as the diagnosis and treatment of clinical diseases all need to separate purified cells from mixed samples containing different cells. The existing cell separation technologies mainly include: Ficoll density gradient Centrifugation, centrifugal elution, two-phase separation, these methods are based on the characteristics of physical properties such as cell size, density, surface charge, etc., and it is difficult to obtain ideal purity and recovery rate. Although flow cytometry sorting can obtain high High purity and recovery rate require expensive instruments, and the operation process is complicated and time-consuming. CN1376779 discloses a method for separating feta...

Claims

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Application Information

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IPC IPC(8): C12N5/00
Inventor 崔亚丽陈超刘蓓高婧
Owner XIAN GOLDMAG NANOBIOTECH
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