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Method for directly separating CD<4+> and CD<8+> lymphocytes

A technology of lymphocytes and -CD4, applied in the field of biomedicine, can solve the problems of separation failure, change of antibody spatial direction, poor monodispersity of micron magnetic beads, etc., to increase the chance of contact, stabilize the reaction solution, and shorten the separation time.

Inactive Publication Date: 2015-04-15
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current separation technology based on micron-scale immunomagnetic beads has many limitations: 1) The specific surface area of ​​micron-sized magnetic beads is relatively small, which reduces the capture efficiency of magnetic beads; The combination of cells through a multiphase reaction usually takes longer to specifically capture cells in the food matrix; 3) The micron magnetic beads have poor monodispersity and are prone to self-aggregation or self-aggregation in the peripheral blood matrix Precipitation; 4) The traditional immunomagnetic separation technology often directly couples the antibody to the immunomagnetic beads. This process often leads to a greatly reduced activity of the antibody and a change in the spatial direction of the antibody, which increases the inter-antibody interaction. Steric hindrance effect, which reduces the capture efficiency of the antibody 5) Blood viscosity is high and non-CD4 + and CD8 + The blood cell concentration of lymphocytes is high, micron magnetic beads are prone to non-specific adsorption, and it is difficult to achieve CD4 in blood + and CD8 + Specific separation of lymphocytes; 6) Excessive concentration of micron magnetic beads will cause CD4 + and CD8 + The damage of lymphocytes (the magnetic field causes the magnetic beads on the cell surface to attract each other, causing the cells to be squeezed or even ruptured), resulting in the failure of separation; Antibody attached to magnetic bead surface
The distance between the antibody and the surface of the magnetic bead is too close, the nature of the magnetic bead itself and the residual hydrophobic or strong hydrophilic groups on the surface are likely to cause changes in the spatial conformation of the antibody, resulting in a decrease in the biological activity of the antibody

Method used

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  • Method for directly separating CD&lt;4+&gt; and CD&lt;8+&gt; lymphocytes
  • Method for directly separating CD&lt;4+&gt; and CD&lt;8+&gt; lymphocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Multi-Armed Well Star Polymer-CD4 + Antibody complexes were prepared according to the following steps:

[0031] (1) Dissolve 1.0 mg of Dobby Star Polymer Dobby Star polyamide-amine in 2 mL of 0.02 M, pH 6.5 phosphate buffer PBS, add 0.6 mg of N-hydroxysuccinimide NHSS, 0.4 mg ethyl 3-(3-dimethylamino)carbodiimide hydrochloride EDC, stir on a mixer at room temperature, and activate for 15 min;

[0032] (2) Take 2.2 mg mouse anti-human CD4 + The monoclonal antibody was added to the above reaction solution, placed on a mixer at room temperature and stirred for 30 min;

[0033] (3) The above solution was spin-dried under reduced pressure, dissolved in deionized water, and dialyzed in PBS and deionized water for 1 day; after the dialysis, the obtained solution was freeze-dried.

[0034] 2. Multi-Armed Well Star Polymer-CD8 + Antibody complexes were prepared according to the following steps:

[0035] (1) Dissolve 1.0 mg of aminated multi-arm well star polymer in 2 mL ...

Embodiment 2

[0044] Example 2 Enrichment effect experiment

[0045] (1) Take 1 mL of concentration as 10 4 CD4 cells / mL + or CD8 + Centrifuge the cells at 12000 rpm for 5 min in a 1.5 mL sterile centrifuge tube, discard the supernatant, and resuspend with an equal volume of sterile PBS solution.

[0046] (2) Enrichment and capture: respectively set the technical solution group of the present invention (CD4 + or CD8 + Cell antibody and long-chain biotin co-modified multi-armed well star polymer group), CD4 + or CD8 + Cell-specific antibody-modified nanomagnetic bead set, CD4 + or CD8 + Cell-specific antibody-modified micron magnetic bead sets enrich target cells.

[0047] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and the separated CD4 + or CD8 + The immunomagnetic beads of the cells were washed twice with PBST, mixed evenly, and the immunomagnetic bead complex was resuspended with 1 mL sterile PBS solution.

[0048] (4) Capture rate calc...

Embodiment 3

[0060] Example 3 Enrichment capture experiment

[0061] Conventional magnetic stand separation time is 30min, and all the other are with embodiment 2.

[0062] CD4 + Capture efficiency of cell-specific antibody-modified micron magnetic bead sets CD4 + Capture efficiency of cell-specific antibody-modified nanomagnetic bead sets CD4 + Capture efficiency of multi-armed well star polymer sets co-modified with cellular antibodies and long-chain biotin 51.7% 38.1% 89.8% CD8 + Capture efficiency of cell-specific antibody-modified micron magnetic bead sets CD8 + Capture efficiency of cell-specific antibody-modified nanomagnetic bead sets CD8 + Capture efficiency of multi-armed well star polymer sets co-modified with cellular antibodies and long-chain biotin 50.9% 36.9% 88.7%

[0063] The experimental results show that compared with the separation of 3min in Example 2, when the separation time reaches 30min, the capture efficiency of the thre...

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Abstract

The invention discloses a method for directly separating CD<4+> and CD<8+> lymphocytes, lays a better foundation for the subsequent research on the CD<4+> and CD<8+> lymphocytes, and relates to the field of biomedicines. The method comprises steps of: multi-arm well and star-shaped polymer and mouse anti-human CD<4+> or CD<8+> monoclonal antibody covalent coupling, long-chain biotin molecule coating through utilizing a mouse anti-human CD<4+> or CD<8+> monoclonal antibody-modified multi-arm well and star-shaped polymer, peripheral blood sample CD<4+> and CD<8+> lymphocyte acquiring through utilizing a mouse anti-human CD<4+> or CD<8+> monoclonal antibody and long-chain biotin co-modified multi-arm well and star-shaped polymer, peripheral blood long-chain biotinylation multi-arm well and star-shaped polymer identifying and coupling through utilizing streptavidin-modified nano magnetic beads, captured CD<4+> and CD<8+> lymphocyte separating and suspending and the like. A suspension can be directly used for subsequent analysis; and compared with a conventional cell separating method, the method is suitable for magnetically separating complicated peripheral blood sample CD<4+> and CD<8+> lymphocytes, so that the peripheral blood sample CD<4+> and CD<8+> lymphocyte separation efficiency is increased.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to CD4 based on nano magnetic beads + and CD8 + Lymphocyte isolation method. Background technique [0002] T lymphocytes are the most important group of cells in the body's immune system. T lymphocytes are derived from lymphocytes of the bone marrow and perform cellular immune functions. They are not only the main body of direct immune effects, but also produce a variety of cytokines and express adhesion molecules, and play an immune regulatory role through direct or indirect contact with other immune cells. In a normal body, various lymphocyte subsets interact to maintain the normal immune function of the body. When the number and function of different lymphocyte subsets are abnormal, it can lead to immune dysfunction and a series of pathological changes. CD4 + and CD8 + As the most important cell in the lymphocyte subgroup, lymphocytes are closely related to the occurrence and dev...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783
Inventor 许恒毅熊勇华魏华赖卫华
Owner NANCHANG UNIV
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