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Capturing method and capturing kit for circulating tumor cells

A tumor cell and capture reagent technology, applied in the field of biomedicine, can solve problems such as insufficient sensitivity, low CTC content, lack of epidermal antigen expression capture, etc., to achieve the effect of improving capture efficiency and detection rate

Inactive Publication Date: 2019-06-07
BEIJING USCI MEDICAL LAB CO LTD
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  • Abstract
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Problems solved by technology

However, the CellSearch system itself also has unsolvable defects: first, the detection sensitivity is not high enough
Studies have confirmed that not all tumor cells express EpCAM and its expression varies greatly on the surface of different cells, so it is difficult for EpCAM to be used as a general recognition antigen
At the same time, some CTCs cannot be captured due to the lack of epidermal antigen expression due to the EMT process.
Once breast cancer cells undergo EMT transformation, they will have stronger activity than cancer cells that have not undergone EMT, and are prone to metastasize to the brain, liver, lung, and bone. In addition, the EMT process will also lead to stagnation of cancer cell proliferation and damage chemotherapy sensitivity. Therefore, breast cancer cells undergoing EMT transformation have a higher survival rate and greater risk in the blood
However, due to the EMT transformation of the cells, the epithelial markers on the cell surface will be lost, so they cannot be captured by the immunomagnetic beads bound to the EpCAM antibody, resulting in the loss of important clinical information
In addition, some patients with metastatic cancer cannot be captured by the CellSearch system because the CTC content in their blood is too low, so this test has a high false negative rate.
The detection of the Cellsearch system also requires specific instruments, reagents and consumables, all of which are expensive, which limits its further large-scale clinical application
[0007] In addition, the content of CTC in peripheral blood is extremely low, and it is generally believed that there are about 10 5 ~10 7 There is only one CTC in every monocyte, and the cellsearch method only uses the EpCAM antibody for one positive enrichment during the enrichment process of CTC, which will lead to a large number of non-specific enrichment of leukocytes, which is useful for the subsequent staining and identification of CTC. cause great disturbance

Method used

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  • Capturing method and capturing kit for circulating tumor cells
  • Capturing method and capturing kit for circulating tumor cells

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Embodiment approach

[0029] According to a typical embodiment of the present invention, the capture method further includes: S4, detecting the number of captured cells by immunofluorescent staining; preferably, S4 specifically includes: S41, fixing the isolated circulating tumor cells and labeling the nuclei with DAPI fluorescent dye ; S42, cells were labeled with CD45 green fluorescent antibody; S43, cells were labeled with CKmix, EpCAM, vimentin red fluorescent antibody; S44, the results were analyzed under a three-color fluorescence microscope, DAPI+, CD45-, CKmix+ or EpCAM+ or vimentin+ cells Defined as CTCs.

[0030] According to a typical implementation of the present invention, the capture method includes:

[0031] 1. Isolation of Monocytes

[0032] 1) Collect 8 mL of peripheral blood from breast cancer patients with EDTA tubes.

[0033] 2) Dilute 8 mL of blood by 1 time with PBS.

[0034] 3) Add 15mL of Ficoll lymphocyte separation medium to a 50mL centrifuge tube, and carefully add the...

Embodiment 1

[0062] EpCAM alone captures peripheral blood CTCs and EpCAM+Vimentin captures peripheral blood CTCs capture efficiency verification.

[0063] Specific steps are as follows:

[0064] 1) Collect 8 mL of peripheral blood from a healthy person in an EDTA tube, and mix it with the same number of MCF-7 cells and MDA-MB-231 cells.

[0065] 2) Dilute one time with PBS.

[0066] 3) Add 15mL of Ficoll lymphocyte separation medium to a 50mL centrifuge tube, and carefully add the diluted blood to the upper layer of the separation medium.

[0067] 4) Centrifuge at 400g room temperature for 30min.

[0068] 5) If stratification occurs after centrifugation, take the buffy coat layer in the middle, which is the cell layer, and transfer it to a new 15mL centrifuge tube.

[0069] 6) Wash twice with PBS.

[0070] 7) Add 250 μL of CD45 magnetic beads, and incubate at 4° C. for 30 min on a rotary mixer.

[0071] 8) Stick the cell magnetic bead mixture on the magnetic stand.

[0072] 9) When t...

Embodiment 2

[0092] CD45 magnetic beads were not used to remove leukocytes, and EpCAM alone captured peripheral blood CTCs and EpCAM+Vimentin captured peripheral blood CTCs to verify the capture efficiency.

[0093] Specific steps are as follows:

[0094] 1) Collect 8 mL of peripheral blood from a healthy person in an EDTA tube, and mix it with the same number of MCF-7 cells and MDA-MB-231 cells.

[0095] 2) Dilute one time with PBS.

[0096] 3) Add 15mL of Ficoll lymphocyte separation medium to a 50mL centrifuge tube, and carefully add the diluted blood to the upper layer of the separation medium.

[0097] 4) Centrifuge at 400g room temperature for 30min.

[0098] 5) If stratification occurs after centrifugation, take the buffy coat layer in the middle, which is the cell layer, and transfer it to a new 15mL centrifuge tube.

[0099] 6) Wash twice with PBS.

[0100] 7) Only 1 μg EpCAM antibody was added to one group, and 1 μg EpCAM antibody and 1 μg Vimentin antibody were added to the ...

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Abstract

The invention discloses a capturing method and a capturing kit for circulating tumor cells. The capturing method includes the following steps: S1, using lymphocyte separation solution to separate cells from peripheral blood to obtain albuginear cells; and S2, using an EpCAM antibody and a Vimentin antibody to capture circulating tumor cells in the albuginear cells. According to the technical scheme of the invention, by adding an interstitial marker (Vimentin) antibody and mixing the Vimentin antibody with the EpCAM antibody to capture CTCs (Circulating Tumor Cells), both conventional epithelial CTCs and more dangerous CTCs with EMT (Epithelial-Mesenchymal Transition) can be captured, the capture efficiency can be greatly improved, and the detection rate of CTCs can be improved.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method and a kit for capturing circulating tumor cells. Background technique [0002] Circulating Tumor Cells (CTCs) are tumor cells detached from tumor lesions and disseminated into the blood. They are an important cause of postoperative recurrence and distant metastasis in patients with malignant tumors, and are also an important factor leading to the death of cancer patients. A large number of experiments have confirmed that the detection of CTCs is helpful for early diagnosis of tumors, judgment of patient prognosis, evaluation of the efficacy of anticancer drugs, and formulation of individualized treatment plans. [0003] Since metastasis is the main cause of cancer-related death, CTCs are regarded as the seeds of metastasis. Studies based on human tumor cell lines and mouse models have shown that tumor cells undergoing epithelial-mesenchymal transition (EMT) play a k...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/574
Inventor 骆靖华王燕刘霞方楠王建伟伍启熹刘倩刘珂弟唐宇
Owner BEIJING USCI MEDICAL LAB CO LTD
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