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A kind of preparation method of separating memory B cells in vitro

A B cell and memory technology, which is applied in the field of preparation of memory B cells in vitro, can solve the problems of poor purity, low harvest rate, and affecting B cell function, and achieve high purity, high harvest rate, and good specificity Effect

Active Publication Date: 2016-08-24
NANCHANG UNIV
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Problems solved by technology

[0004] However, most of the methods currently used to isolate memory B lymphocytes have obvious disadvantages: affecting B cell function, poor purity, non-specific binding, and low harvest rate

Method used

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  • A kind of preparation method of separating memory B cells in vitro
  • A kind of preparation method of separating memory B cells in vitro
  • A kind of preparation method of separating memory B cells in vitro

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Embodiment Construction

[0017] 1. Screening of blood-positive patients with RSV infection and blood collection: Since almost 100% of the population had been infected with RSV in childhood, we randomly selected volunteers, drew 3mL of fresh blood from each person, and set the centrifugation Centrifuge at 2000 rpm for 5 min, at which time all the cells gather at the bottom of the tube. Carefully pipette up into a fresh EP tube, number and use the Human RSV Elisa Kit to screen the volunteers with high positive RSV in vivo.

[0018] 2. Separation of blood mononuclear cells: Take 20mL of fresh blood from a positive respiratory syncytial virus patient and use heparin sodium as an anticoagulant, then add an equal volume of buffer system Hanks solution to mix with anticoagulated whole blood samples Set the centrifuge speed at 2000rpm and centrifuge for 20min in the lymphocyte separation medium, carefully take out the centrifuge tube, the cloudy layer in the solution in the tube is the peripheral blood mononu...

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Abstract

The invention provides establishment of an in-vitro memory B cell separating method, which can finally realize separation and purification of a memory B cell by utilizing characteristics of general lymphocytes and memory B cells in plasma. The method comprises the following steps: separating general lymphocytes, namely peripheral blood monouclear cells, by utilizing a lymphocyte separation liquid according to different densities of lymphocytes and erythrocyte in plasma; separating and purifying to obtain a relatively pure memory B cell by utilizing a magnetic activated cell sorting method according to specific recognition proteins CD19 and CD27 of memory B cells in the peripheral blood monouclear cells. The establishment has the advantages that compared with other in-vitro memory B lymphocyte separating methods, the memory B lymphocyte sorted by utilizing the magnetic activated cell sorting method does not influence normal growth and functions of B lymphocytes, is good in specificity, high in purity and high in yield.

Description

technical field [0001] The invention relates to a preparation method for separating memory B cells in vitro. 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. A small number of B cells stimulated by antigens develop into memory B cells, which have a long life span and maintain specificity, and the progeny cells generated by their proliferation also maintain this specificity. When they come into contact with the same specific antigen again, they can be quickly activated and become specific B...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0781
Inventor 陈廷涛辛洪波魏强
Owner NANCHANG UNIV
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