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Separation method of micro circulating tumor cells

A technology of tumor cells and separation methods, which is applied in the field of separation of circulating tumor cells based on nano-magnetic beads, can solve the problems of CTCs damage, changes, and poor monodispersity of micron magnetic beads, so as to increase the chance of contact, improve the separation efficiency, shorten the The effect of separation time

Inactive Publication Date: 2013-09-04
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 whole blood matrix; 3) Micron magnetic beads have poor monodispersity and are prone to self-aggregation in the whole blood matrix Or form a precipitate; 4) The traditional immunomagnetic separation technology often directly couples specific substances to immunomagnetic beads. Changes occur, thereby increasing the steric hindrance effect between specific substances and reducing the capture efficiency of specific substances; 5) The blood viscosity is high and the blood cell concentration of non-CTCs is large, and micron magnetic beads are prone to non-specific adsorption, which is difficult Realize the specific separation of CTCs in blood; 6) Excessive concentration of micron magnetic beads will cause damage to CTCs (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 failure of separation; (7) ) When coupling antibodies to magnetic beads, hydrophobic adsorption or chemical coupling is generally used to couple active antibodies to the surface of magnetic beads
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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  • Separation method of micro circulating tumor cells
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  • Separation method of micro circulating tumor cells

Examples

Experimental program
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Effect test

Embodiment 1

[0039] 1. The dendrimer-anti-HER2 antibody complex is prepared according to the following steps:

[0040] (1) Dissolve 10.5 mg of aminated polyamide-amine dendrimer PAMAM G4 in 2 mL, 0.02 M, pH 6.5 PBS, add 0.6 mg NHSS, 0.4 mg EDC, and place on a mixer at room temperature to stir , activated for 15 min;

[0041] (2) Take 0.8 mg of anti-HER2 antibody and add it to the above reaction solution, place it on a mixer at room temperature and stir for 30 min;

[0042] (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.

[0043] 2. The long-chain biotin-dendrimer-anti-HER2 antibody complex is prepared according to the following steps:

[0044] (1) Dissolve 22.5 mg long-chain biotin, 5.4 mg NHSS, and 3.6 mg EDC in 3 mL 0.02 M pH 6.5 PBS buffer;

[0045] (2) Add 8.0 mg of the dendrimer-anti-HER2 antibody complex to the above solution, p...

Embodiment 2

[0049] Example 2 Enrichment effect experiment

[0050] (1) Take 1 mL of concentration as 10 4 Cells / mL SK-BR-3 cells were placed in a 1.5 mL sterile centrifuge tube, centrifuged at 12,000 rpm for 5 min, the supernatant was discarded, and resuspended with an equal volume of sterile PBS solution.

[0051] (2) Enrichment and capture: Set up the technical solution group of the present invention (dendrimer group co-modified with SK-BR-3 cell antibody and long-chain biotin), and nano magnetic beads modified with SK-BR-3 cell-specific antibody Group, SK-BR-3 cell-specific antibody-modified micron magnetic beads group enriched target cells.

[0052] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and wash the separated immunomagnetic beads with SK-BR-3 cells twice with PBST, mix well, and wash with 1 mL sterile PBS solution to resuspend the immunomagnetic bead complex.

[0053] (4) Capture rate calculation: after serial dilution of the enriched ...

Embodiment 3

[0066] Example 3 Enrichment capture experiment

[0067] Conventional magnetic stand separation time is 30 min, all the other are with embodiment 2.

[0068] The catch rate of each group is as follows:

[0069] Capture rate of SK-BR-3 cell-specific antibody modified micron magnetic bead set Capture rate of SK-BR-3 cell-specific antibody-modified nano-magnetic bead set Capture efficiency of dendrimers co-modified with antibody and long-chain biotin in SK-BR-3 cells 51.2% 37.9% 91.2%

[0070] The experimental results show that compared with the separation of 3 min in Example 2, when the separation time reaches 30 min, the capture efficiency of the three groups has been improved, especially the capture of the SK-BR-3 cell-specific antibody-modified nano magnetic beads group The efficiency improvement is the most obvious, which shows that the capture efficiency of the nano-magnetic bead group can be greatly improved by extending the time, but it is stil...

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Abstract

The invention discloses a method for separating and enriching micro circulating tumor cells (circulating tumor cells, CTCs) in whole blood for further providing a basis for subsequent study of tumor cells, and relates to the biomedical field. The method comprises the following steps of: covalent coupling of dendrimers and matters (special matters) specifically combined with target cells; further coating long-chain biotin molecules by dendrimers modified by the specific matters; capturing CTCs in a whole blood sample by dendrimers co-modified by the special matters and the long-chain biotin; identifying and coupling long-chain biotin dendrimers in the whole blood by streptavidin modified nanomagnetic beads; and separating and resuspending the captured CTCs. The resuspension can be directly used for subsequent analysis. Compared with conventional cell separation methods, the method is more suitable for magnetic separation of CTCs in complex whole blood samples, so that the magnetic separation time is shortened, and the CTCs separation efficiency in the whole blood sample is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for separating circulating tumor cells based on nano magnetic beads. Background technique [0002] Circulating tumor cells (CTCs) refer to tumor cells that enter the peripheral blood of the human body. Foreign studies have found that the appearance of CTCs in the peripheral blood of tumor patients is often earlier than that of visible solid tumors. Therefore, the detection of CTCs has been widely used in the early diagnosis of tumor patients in vitro. However, the number of CTCs in the peripheral blood of cancer patients is very rare in the early stage, and the existing methods cannot directly detect trace CTCs in peripheral blood. Therefore, realizing the high-efficiency enrichment of CTCs in human peripheral blood to meet the sensitivity requirements of the detection method is not only for the early detection of tumors, but also for the rapid evaluation of chemotherapy d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09
Inventor 许恒毅
Owner NANCHANG UNIV
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