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A Method for Sorting and Enriching Circulating Tumor Cells Relying on Mixed Antibodies

A tumor cell and antibody technology, applied in the field of modern medicine, can solve the problems of delayed disease diagnosis, inappropriate treatment plans, and high mortality of cancer patients

Active Publication Date: 2020-04-14
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinically speaking, the main reason for the high mortality of cancer patients is the delay of disease diagnosis and inappropriate treatment plan

Method used

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  • A Method for Sorting and Enriching Circulating Tumor Cells Relying on Mixed Antibodies
  • A Method for Sorting and Enriching Circulating Tumor Cells Relying on Mixed Antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1, the modification of device or carrier

[0019] Different devices or carriers that rely on the interaction between the device or carrier to link antibodies and cell-specific proteins for capture, first undergo a series of modifications so that the device or carrier can be connected to a large number of antibodies. Then add 10 μg / mL biotin-linked anti-EpCAM and anti-vimentin antibody (1:1 (w / w)) diluted with 1% BSA in PBS, incubate at room temperature for 1 hour, wash, and wash each time All were soaked in PBS solution and placed on a shaker for 5 minutes; finally, blocked with 5% BSA solution prepared in PBS and kept at room temperature for 1 hour to reduce non-specific adhesion of blood cells.

Embodiment 2

[0020] Embodiment 2, blood sample collection and processing

[0021] Healthy people and tumor patients were all completed in the hospital, and were obtained by vacuum blood vessels containing EDTA anticoagulant, placed in a 4°C refrigerator, and processed within 24 hours. The blood was diluted with PBS (1:1 (v / v)), and then the ratio of lymphocyte separation solution and PBS diluted blood solution 2:1 (v / v) was carefully added to the interface of the cell separation solution. After centrifugation at 1500g for 5 minutes, the supernatant was discarded, resuspended in an equal volume of buffer solution and stored in a 4°C refrigerator for later use. patient information such as figure 2 shown.

Embodiment 3

[0022] Example three, identification and counting of circulating tumor cells

[0023] The size, shape and nucleus ratio of the captured cells were preliminarily judged by fluorescence microscopy, and the final identification of circulating tumor cells was completed by means of fluorescence microscopy by further utilizing the method of fluorescent labeling of cytokeratin. Subsequent molecular analysis of CTCs can also be performed according to the PCR method or single-cell sequencing method.

[0024] Such as figure 1 As shown in a and 1b, cancer cells and blood cells can be distinguished. figure 1 c and 1d show that cell capture is more accurate with mixed antibodies corresponding to epithelial and mesenchymal signature molecules, and there are more cancer cells in the blood of patients with advanced breast cancer than patients with early cancer.

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Abstract

The present invention discloses methods for sorting and enriching circulating tumor cells that rely on mixed antibodies. This method is applied to capture and sort CTCs from clinical samples of early and late cancer patients. The method of the present invention has good capturing effect of circulating cancer cells. In the case of 1 mL of blood, patients with advanced breast cancer obtained a capture effect of greater than 5 CTCs, and the capture results of a system modified with mesenchymal-type specific antibodies (anti-vimentin antibodies) were closer to those relying on mixed antibody systems ( The capture results of early cancer patients rely on epithelial-type specific antibodies (anti-EpCAM antibody) modified antibodies to achieve better capture results Close capture results that rely on mixed antibody systems.

Description

technical field [0001] The invention belongs to the modern medical technology, and specifically relates to a cell sorting method and system, in particular to a method for sorting and enriching circulating tumor cells in clinical samples relying on mixed antibodies. Background technique [0002] Cancer is the number one cause of death in developed countries and the second in developing countries. Clinically speaking, the main reason for the high mortality of cancer patients is the delay of disease diagnosis and inappropriate treatment plan. Physiologically, most of the deaths caused by cancer are caused by the migration of tumor cells to other organs in the body through the hematogenous route. In this process, metastatic cancer cells, namely circulating tumor cells (CTCs), play a key role in cancer metastasis. Accumulating evidence indicates that the metastasis of tumor cells from the primary site to other sites occurs early in cancer development. Therefore, on the one han...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N15/10G01N21/64G01N1/34
CPCG01N1/34G01N15/10G01N21/6458G01N21/6486G01N2015/1006G01N2015/103G01N2015/1028G01N2015/1029
Inventor 王本惠兰兰骆严
Owner ZHEJIANG UNIV
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