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
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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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