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A rapid and efficient method for isolating single antigen-specific B cells

A specific, B cell technology, applied in cell dissociation methods, animal cells, vertebrate cells, etc., can solve problems such as affecting the quality and activity of B cells, ranging from hundreds of thousands to millions, and large randomness. , to achieve the effect of shortening the development cycle, strong adaptability, and reducing the impact

Active Publication Date: 2020-02-21
HANGZHOU BIOLYNX TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has many disadvantages: the "abundance" of antigen-specific B cells in spleen cells makes the isolation of antigen-specific clones very random; there must be a myeloma cell
However, this method has the following disadvantages: expensive flow cytometers are required, ranging from hundreds of thousands to millions
However, more labeling reagents require more complex manipulations
Complex operations will inevitably affect the quality and activity of B cells

Method used

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  • A rapid and efficient method for isolating single antigen-specific B cells
  • A rapid and efficient method for isolating single antigen-specific B cells
  • A rapid and efficient method for isolating single antigen-specific B cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment one: if figure 1 As shown, a method for rapidly and efficiently isolating a single antigen-specific B cell provided by the present invention specifically includes the following steps:

[0020] In step 1, the peripheral blood mononuclear cell suspension is obtained by separating whole blood from the peripheral blood of the human or animal infected with the antigen.

[0021] Step 2, preparing activated immunomagnetic beads coupled with an antigen, and the ligand is an artificial antigen corresponding to the antigen.

[0022] Specifically, the steps of preparing activated immunomagnetic beads coupled with antigen mainly include:

[0023] (1) the step of the activation of immunomagnetic beads;

[0024] (2) The step of mixing and incubating the immunomagnetic beads and the ligand;

[0025] Specifically, the ligand is a recombinant protein or polypeptide-BSA (a cross-linked product of polypeptides such as polypeptide-KLH and polypeptide-OVA and a carrier protein...

Embodiment 2

[0034] Example 2. In this example, hydroxyl magnetic beads, rabbit peripheral blood, CRP polypeptide-KLH conjugate as the antigen, and CRP recombinant protein as the ligand are used in this example to specifically illustrate the implementation of the technical solution of the present invention.

[0035] step one:

[0036] (a) Take a 50ml centrifuge tube, add 5-20ml rabbit blood, add 5-25ml PBS, and mix well;

[0037] (b) Take another -50mL centrifuge tube and add 5-20mL lymphocyte separation medium;

[0038] (c) PBS-diluted rabbit blood was evenly added to two centrifuge tubes equipped with lymphocyte separation solution, and the rotation speed was 900-1400 rpm, and the centrifugation was performed for 15-30 minutes;

[0039] (d) Carefully take out the centrifuge tube, slowly suck up the second layer of lymphocytes with a 1ml pipette, and add DPBS to make up to 45ml. Speed ​​900~1400rpm, centrifuge for 5~15min;

[0040] (e) Discard the supernatant and wash once with DPBS; ...

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Abstract

The invention discloses a method for rapidly and efficiently separating an individual antigen specific cell B. The method specifically comprises the following steps: step I, separating peripheral blood whole blood of an antigen-infected person or animal to obtain peripheral blood mononuclear cell suspension; step II, preparing activated immunomagnetic beads of a coupling antigen ligand, wherein the ligand is an artificial antigen of a corresponding antigen; step III, removing a non-specific adsorption cell in the peripheral blood mononuclear cell suspension obtained in step I by using un-coupled un-activated magnetic beads, and separating a mixture obtained in step III by using the activated immunomagnetic beads of the coupling antigen obtained in the step II to obtain the individual antigen specific cell B. The scheme provides a universal method which does not depend on myeloma cell and is low in operation complexity and low in cost and obtains the specific cell B corresponding to the individual antigen, so that the single-cell separation effect is better.

Description

technical field [0001] The present invention relates to a method for isolating antigen-specific B cells. Background technique [0002] B cells account for about 15% of the total number of lymphocytes in the blood. Immunoglobulins (mainly monomeric IgM and IgD) fixed on the surface of B cell membrane are the specific receptors for antigens. When they are sensitized by contact with a certain antigen for the first time, some B cells differentiate and mature into plasma cells, and the plasma cells begin to produce immunoglobulins specific to the antigen and release them into the surrounding tissue fluid, which is the immune system. Antibody. [0003] Methods of isolating, culturing and identifying B cells that produce antibodies that specifically bind a target antigen are of great importance in the field of immunotechnology. In the prior art, the method of obtaining B cells directed against a certain antigen in the immune system and preparing monoclonal antibodies is mainly t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0781
CPCC12N5/0635C12N2509/00
Inventor 李明振徐婷婷其他发明人请求不公开姓名
Owner HANGZHOU BIOLYNX TECH CO LTD
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