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

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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 invention discloses a circulating tumor cell (CTC) sorting and enriching method depending on a mixed antibody. The method is applied to CTCs capturing and sorting of clinical specimens of cancer patients at early and late stages. The method has good circulating tumor cell capturing effect. When the blood is 1 mL, a patient with advanced breast cancer obtains the capturing effect greater than 5 CTCs; meanwhile, a system modified by a mesenchymal type specific antibody (anti-vimentin antibody) has the capturing effect is close to that depending on a mixed antibody system (antibodies of epithelial phenotypic characteristic molecules and antibodies of mesenchymal phenotypic characteristic molecules are combined in a mass ratio of 1 to 1); the patient with early-stage cancer obtains capturing effect on antibodies modified by epithelial special antibodies (anti-EpCAM antibodies) is closer to that depending on the mixed antibody system.

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
CPCG01N1/34G01N15/10G01N21/6458G01N21/6486G01N2015/1006G01N2015/1081G01N2015/1087G01N2015/1093
Inventor 王本惠兰兰骆严
Owner ZHEJIANG UNIV
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