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High-specificity and high-purity tumor cell sorting method based on double-antibody and cell density

A tumor cell, high-specificity technology, applied in the field of high-specificity and high-purity tumor cell sorting, can solve the problems of purity and sensitivity reduction, and achieve the effects of improving screening purity, enhancing capture efficiency, and increasing density

Inactive Publication Date: 2016-09-21
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method can only remove a large number of red blood cells in the blood sample. Because the density of CTCs and white blood cells has an intersection, during the centrifugal separation process, CTCs may co-sediment with white blood cells, resulting in reduced purity and sensitivity.

Method used

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  • High-specificity and high-purity tumor cell sorting method based on double-antibody and cell density
  • High-specificity and high-purity tumor cell sorting method based on double-antibody and cell density
  • High-specificity and high-purity tumor cell sorting method based on double-antibody and cell density

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 SiO 2 Comparison of the preparation of @Gel and the capture efficiency of tumor cells before and after encapsulating gelatin

[0049] (1) SiO 2 Preparation of @Gel

[0050] Weigh 0.1 g of SiO with a size of 50 µm and a density of 1.2 g / mL 2 After the microspheres were centrifuged and washed with absolute ethanol for 3 times, they were transferred into a conical flask, added with 50 mL of absolute ethanol, and placed on a heating and stirring table; after the temperature rose to 60°C, 0.2 mL of 3-aminopropyl Triethoxysilane (APS), after stirring for 3h on SiO 2 Amino groups were introduced into the surface of microspheres to obtain amino-modified SiO 2 Microspheres (NH 2 -SiO 2 ); NH 2 -SiO 2 After centrifugation and washing with absolute ethanol and deionized water for 3 times, resuspend in 50 mL of deionized water, add 0.2 mL of 50% glutaraldehyde solution dropwise, stir at room temperature for 10 h, and introduce aldehyde groups on the surface of the...

Embodiment 2

[0055] Example 2 Selection of tumor cell surface markers

[0056]CTCs will undergo a complex EMT process during the transfer process. During this process, EpCAM on the surface of many CTCs will be low or not expressed, and the CD146 antigen will be highly expressed on the cell surface. Therefore, in order to improve the capture efficiency of CTCs, the antibody anti -CD146 and anti-EpCAM work together to capture CTCs in the peripheral blood of cancer patients.

[0057] This example studies the expression of CD146 and EpCAM on the surface of breast cancer cell MCF-7 and colorectal cancer cell HCT116. The expression of corresponding antigens in tumor cells was marked with fluorescein-grafted antibodies PE-anti-CD146 and APC-anti-EpCAM, and the cells were observed and counted by confocal microscopy and flow cytometry, and RT was performed on the two tumor cells -PCR sequencing, specific methods and experimental results are as follows:

[0058] (1) Confocal microscope observation...

Embodiment 3

[0064] Example 3 SiO 2 Modification of @Gel surface antibody

[0065] (1) Use PBS to prepare 0.1M MES (2-morpholinoethanesulfonic acid) solution, and use MES solution to prepare 4mg / mL EDC (N-(3-dimethylaminopropyl-N'-ethylcar-bodiimide)) and 6mg / mL -NHS (N-hydroxysuc-cinimide) solution, the 100µL quantity is 1.5×10 5 SiO 2 The @Gel suspension was stirred with 100 µL of the above EDC / NHS solution at room temperature for 40 minutes to activate the carboxyl groups on the surface of the gelatin, and washed by centrifugation with PBS for 3 times.

[0066] (2) Use PBS to prepare a streptavidin (SA) solution with a concentration of 50 µg / mL, immerse the microspheres treated in the previous step in 100 µL SA solution, react at room temperature for 60 min, and centrifuge and wash with PBS three times.

[0067] (3) Use PBS to prepare a biotinylated anti-EpCAM solution of 10 µg / mL and a biotinylated anti-CD146 solution of 10 µg / mL, and immerse the microspheres treated in the previou...

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Abstract

The invention discloses a high-specificity and high-purity tumor cell sorting method based on a double-antibody and cell density. The method comprises the following steps of chemically bonding a layer of gelatin with the thickness of 20 to 100nm on the surface of a hydroxyl microsphere with the grain size of 20 to 100mum and the density of 1.16 to 3g / mL, and obtaining the hydroxyl microsphere coated with the gelatin; modifying the antibodies of anti-EpCAM and anti-CD146 on the surface of the hydroxyl microsphere coated with the gelatin; uniformly mixing a blood sample of a patient and the hydroxyl microsphere with the modified antibodies, incubating to obtain a mixed sample, dropwise adding to an upper layer of 1.15 to 1.20g / mL of cell separation fluid, and after centrifuge separation, and deposing the microsphere acquiring a tumor cell to the bottom part of the cell separation fluid; using a gelatinase on the microsphere acquiring the tumor cell to degrade the gelatin on the surface of the microsphere, and releasing the tumor cell. The high-specificity and high-purity tumor cell sorting method based on the double-antibody and the cell density provided by the invention is higher in sorting efficiency and purity, and the sorted tumor cell can be released and cultured.

Description

technical field [0001] The present invention relates to a method for sorting tumor cells, in particular to a method for sorting tumor cells with high specificity and high purity based on double antibodies and cell density. Background technique [0002] Circulating Tumor Cells (CTCs) are tumor cells that break away from the primary tumor tissue and enter the peripheral blood of tumor patients. They have important clinical research value for the early diagnosis and treatment evaluation of tumors. In view of the fact that most of the deaths from cancer in patients with solid tumors are due to tumor metastasis, and the detection of CTCs in the blood circulation system will indicate the possibility of distant metastasis of tumors, early diagnosis of circulating tumor cells in human tumor biopsy and play a key role in judging disease progression. However, the content of CTCs in the blood circulation system is extremely low. There are only a few to hundreds of CTCs per milliliter ...

Claims

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Application Information

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IPC IPC(8): C12N5/09
CPCC12N5/0694C12N2509/00
Inventor 赵兴中黄琴琴蔡博
Owner WUHAN UNIV
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