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Liquid-phase cellular immunity identification method and application thereof

A technology of cellular immunity and identification method, which is applied to the liquid-phase cellular immunity identification method and its application field, and can solve the problems of large changes in cells, troublesome, and unfavorable practical application.

Inactive Publication Date: 2019-05-14
宜昌美光硅谷生命科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as precision medicine needs to be analyzed at the gene level, it is not enough to obtain the count of circulating tumor cells. It is also necessary to separate and extract circulating tumor cells and conduct gene analysis including a small number or single cells
In the existing scientific research and clinical applications, basically, for the obtained circulating tumor cells, only one of immunofluorescence identification or gene analysis can be selected, or the cells are centrifuged onto a glass slide, dried and stained, and then removed from the glass slide. Try to collect target cells on the chip for gene analysis. In this way, not only the cells change greatly and the collected cells are incomplete, which greatly affects the reliability of the gene analysis results, but also the operation is very complicated and cumbersome, which is not conducive to scientific research and clinical application. practical application of
Cells in a liquid phase environment, it is easy to operate whole cells or complete single cells, but there is a lack of liquid phases in the market to identify circulating tumor cells and then use them for counting and subsequent cell analysis including genetic analysis of single cells or a small number of pure cells technology

Method used

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  • Liquid-phase cellular immunity identification method and application thereof
  • Liquid-phase cellular immunity identification method and application thereof
  • Liquid-phase cellular immunity identification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Magnetic bead labeling and liquid phase immunostaining identification of breast cancer tumor cell lines

[0040] 1. Magnetic bead labeling method for breast cancer cell lines

[0041] 1. Put the blood sample into the centrifuge and centrifuge at 1000rpm for 5 minutes. After the centrifugation, take out the blood sample carefully and avoid shaking.

[0042] 2. Use a pipette gun to discard the supernatant, that is, the plasma, and add the same volume of cell washing solution as the blood sample, and gently invert and mix 4-5 times.

[0043] 3. Put the blood sample into the centrifuge and centrifuge at 1900rpm for 10 minutes. After the centrifugation, take out the blood sample carefully and avoid shaking.

[0044] 4. Use a Pasteur pipette to discard the supernatant.

[0045] 5. Add cell washing solution to restore to the original volume of the blood sample.

[0046] 6. Take EpCAM magnetic beads, blow and mix with a 20ul pipette gun, take 4ul magnetic beads into ...

Embodiment 2

[0056] Example 2 Liquid phase immunofluorescence identification of circulating tumor cells in the blood of cancer patients

[0057] 1. Liquid phase immunofluorescence identification method of circulating tumor cells in the blood of cancer patients

[0058] 1. Immunofluorescence Staining

[0059] (1) Take out the culture plate from the magnetic trap, absorb 100ul of 4% paraformaldehyde fixation solution and drop it in the center of the release tank, and fix it at room temperature for 10min.

[0060] (2) Antibody preparation on ice: take 20ul Anti-cytokeratin (CAM5.2), 2ul CD45 Ab-1 (Bra55 / 2), 0.25ul Alexa in each well 568goat anti-mouse IgG1 and 0.25ul Alexa 488anti-mouse IgG2a was added to a 1.5mL EP tube, and the volume was supplemented to 100ul with 1xPBS.

[0061] (3) Discard the fixative, add 100ul antibody dropwise to the center of the release tank, and incubate at room temperature for 30min.

[0062] (4) Add 100ul DAPI dropwise to the center of the release tank, an...

Embodiment 3

[0066] Example 3 Analysis of single-cell gene phenotype after liquid-phase immunostaining identification of breast cancer tumor cell lines

[0067] 1. Single cell genotype analysis method

[0068] 1. Pre-amplification

[0069]

[0070]

[0071] Reaction conditions: 50°C for 15min→(95°C for 15sec, 60°C for 4min)*18 cycles→5-fold dilution of cDNA product.

[0072] 2. Microfluidic dynamic array

[0073] (1) The chip is filled with the lubricant of the IFC controller;

[0074] (2) After the sample mixture is mixed, add it to the sample inlet

[0075] 2*TaqMan Master Mix 2.5ul

[0076] DA sample loading reagent 0.25ul

[0077] Preamplified cDNA 2.25ul

[0078] Total 5ul

[0079] (3) Add the reaction mixture to the analysis inlet after mixing

[0080] 20*TaqMan gene expression assay mix 2.5ul

[0081] DA Assay loading reagent 2.5ul

[0082] Total 5ul

[0083] (4) Load the chip and mix it into the IFC controller;

[0084] (5) qRT-PCR reaction of the chip

[0085] (...

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Abstract

The invention discloses a liquid-phase cellular immunity identification method and application thereof. The liquid-phase cellular immunity identification method comprises the steps of: S1, centrifuging a blood sample, absorbing blood plasma, adding cell buffer solution, and uniformly mixing cells, so that a blood cell sample is obtained; S2, taking a tumour cell identification antibody magnetic bead, adding into the blood cell sample, slowly rotating on a table, and hatching at room temperature; S3, after instantaneously centrifuging the hatched blood cell sample, transferring into a cell culture plate, putting the cell culture plate in a magnetic trap instrument, and performing separation and extracting living circulating tumour cells; S4, performing fluorescent staining or fluorescent amplification staining and reagent solution exchange on the living circulating tumour cells, so that the circulating tumour cells are subjected to marker staining in a liquid state; and S5, in the liquid state, performing immunofluorescence identification on the stained circulating tumour cells. By means of the liquid-phase cellular immunity identification method and application thereof in the invention, after separated and extracted living target cells are subjected to cellular qualitative fluorescence immunoidentification, a mobile complete single cell is used for all kinds of subsequent cellanalysis.

Description

technical field [0001] The invention relates to the field of molecular cell biology; in particular, it relates to a liquid-phase cell immune identification method and its application. Background technique [0002] In recent years, blood circulating tumor cells, as the most important part of liquid biopsy, have become a hotspot in clinical research due to their clinical effects in early screening, diagnosis, treatment, and curative effect tracking and monitoring of different tumors. It is an effective technology for non-invasive tumor diagnosis and real-time curative effect detection, and its clinical application value is extremely significant. However, as precision medicine needs to be analyzed at the gene level, it is not enough to only obtain the count of circulating tumor cells. It is also necessary to separate and extract circulating tumor cells and conduct gene analysis including a small number or single cells. In the existing scientific research and clinical applicati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/533C12Q1/6869
Inventor 邓亚光许新华孙长胜覃程陈李宴清邓伟平
Owner 宜昌美光硅谷生命科技股份有限公司