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ctc protein typing kit based on microfluidic technology

A microfluidic technology, protein technology, applied in CTC protein typing kits, medical and biological fields, can solve the problems of damaged cell activity, affecting typing identification, low recovery rate, etc., to reduce steric hindrance, fast automation Detection and typing, the effect of improving recycling efficiency

Active Publication Date: 2018-10-02
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional separation methods have defects such as low recovery rate, damaged cell viability, integrity and surface antigen, which affect the subsequent typing and identification

Method used

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  • ctc protein typing kit based on microfluidic technology
  • ctc protein typing kit based on microfluidic technology
  • ctc protein typing kit based on microfluidic technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The CTC protein typing kit based on microfluidic technology described in this embodiment mainly includes:

[0033] 1. Capture antibody complexes

[0034]The capture antibody complex includes a capture antibody and an aptamer that specifically binds to each marker protein of CTCs, the capture antibody is used to connect the marker protein and the marker antibody, and can specifically bind to the marker protein and the marker antibody, and the capture antibody In order to have high-purity monoclonal antibodies, the reaction characteristics of the selected capture antibodies do not interfere with each other, and the capture antibodies and aptamers are connected by a shearable material. The shearable material includes photosensitive materials, temperature-sensitive materials, and moisture-sensitive materials. Materials, heat-sensitive materials, enzymatic degradation materials, etc. Aptamers are small molecular compounds that can react with specific binders on the cell capt...

Embodiment 2

[0059] Example 2 Using the kit in Example 1 to detect circulating tumor cells in human peripheral blood

[0060] The formula of described various solutions is as follows:

[0061]

[0062] All the antibodies in the list of corresponding marker proteins in Example 1 were used in the capture mixed solution and the chromogenic mixed solution in this example.

[0063] 1. Sample pretreatment

[0064] 1. Preserve the blood sample in the sample preservation tube with preservation solution, centrifuge at 600×g for 5 minutes, discard the supernatant, remove the red blood cells, and add 1mL of LPBS to resuspend.

[0065]2. Take 450uL corresponding magnetic beads for mixing, centrifuge to remove the supernatant, add 1mL PBS to resuspend, use 1mg magnetic beads per 30ug magnetic bead antibody, add magnetic bead antibody mixture to the cell suspension, and react on the mixer for 30min. Centrifuge to remove supernatant, add 10uLPBS to resuspend.

[0066] 2. Detection of microfluidic d...

Embodiment 3

[0095] Example 3: Using the kit in Example 1 to detect tumor cell lines

[0096] 1. Selection of tumor cell lines

[0097] In this example, we added a known number of epithelial cell line MCF-10A, mesenchymal tumor cell line U118, and epithelial-mesenchymal lung cancer cell line PC-9 into the peripheral blood samples of healthy volunteers, using The lymphoblastoid cell line CCRF-HSB-2 was used as a negative control. Those skilled in the art can obtain it by purchasing as long as they know the name of the cell line. The kit in Example 1 was used to test the tumor cell capture efficiency of the present invention.

[0098] 2. Sample testing

[0099] Each get 200 MCF-10A cells, U118 cells and PC-9 cells (determined by cell counter), add in 5mL healthy volunteer peripheral blood samples, mix well, and detect the samples according to the detection process and method described in Example 2 , the capture rate of target cells was calculated by the following formula: capture rate = ...

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Abstract

The invention relates to a microfluidic-technology-based CTC protein typing kit. The kit comprises: (1) capture antibody compounds aiming at different CTCs marker protein, each capture antibody compound being made from a capture antibody in specific binding to corresponding marker protein and an aptamer, wherein the capture antibody and aptamer are connected through a cuttable material which is deoxyribonucleotide double strands having a palindrome structure and two or more than two restriction enzyme sites; and (2) a microfluidic chip, the surface of which is coupled to a cell capture assembly. The cell capture assembly made from a microcolumn is fixed at the surface of the microfluidic chip, and a specific conjugate capable of bonding to the aptamer modifies one end of the microcolumn. Through the microfluidic technology, efficient and fast automatic detection and typing of CTCs is achieved, the kit is simple to operate, false-negative and false-positive results due to man-made operational factors are reduced, and the sensitivity and accuracy of detection are improved.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a CTC protein typing kit based on microfluidic technology, which is suitable for capturing, separating, purifying, and typing identification of circulating tumor cells in blood. Background technique [0002] Circulating tumor cells (CTCs) originate from primary tumors or metastatic tumors, acquire the ability to break away from the basement membrane and enter blood vessels by invading the tissue matrix, and are a general term for various tumor cells present in peripheral blood. According to the difference in the expression of surface antigens, it is mainly divided into epithelial marker positive phenotype (referred to as epithelial cell type), mesenchymal marker positive phenotype (referred to as mesenchymal cell type) and mixed epithelial and mesenchymal phenotype (referred to as epithelial-mesenchymal type). cytoplasmic type), etc. Epithe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/574
CPCG01N33/5304G01N33/5308G01N33/56966G01N33/574G01N33/6854
Inventor 刘志明吴诗扬廖传荣
Owner SUREXAM BIO TECH
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