A kit for liver cancer detection based on liquid biopsy

A technology of liquid biopsy and reagent kit, which is applied in the direction of measuring devices, instruments, and biological material analysis, etc. It can solve the problems of weak fluorescence intensity, difficulty in distinguishing, and inconsistency, and achieve the effects of strong fluorescence, good staining effect, and enhanced staining effect

Active Publication Date: 2018-12-18
SHANGHAI YH HEALTH BIOLOGY MEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method can only specifically identify the rare cells through a single monoclonal antibody, which is likely to cause missed detection
At the same time, due to the heterogeneity of cells, the amount of antigen (antibody) expressed by each cell varies, resulting in sometimes very weak fluorescence intensity, which is difficult to distinguish under a fluorescent microscope

Method used

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  • A kit for liver cancer detection based on liquid biopsy
  • A kit for liver cancer detection based on liquid biopsy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 enrichment of target cells in peripheral blood

[0056] (1) Centrifuge peripheral blood to remove plasma protein: 8.5mL peripheral blood was centrifuged at 800g in a horizontal centrifuge for 7min at room temperature, and the supernatant was discarded.

[0057] (2) Add 5-6 mL of PBS buffer solution and 3 mL of lymphocyte separation solution to the plasma in step (1), centrifuge at 450 g in a centrifuge for 7 minutes at room temperature. After centrifugation, it is divided into three layers. The red bottom layer is the erythrocyte layer, the slightly white middle layer is mainly white blood cells and CTC, etc., and the yellow upper layer is plasma. Absorb all the liquid above the erythrocyte layer and remove the bottom erythrocyte layer.

[0058] (3) Add 200 mL of immunomagnetic beads with CD45 antibody coupled to the surface dropwise in step (2), and incubate on a horizontal shaker to obtain a suspension. At room temperature, shake horizontally for 20 minutes...

Embodiment 2

[0060] Example 2 Fluorescent staining of enriched target cells

[0061] (1) Enhanced staining pretreatment: Add 2 μL of staining enhancement solution to about 50 μL of enriched target cells, and let stand at room temperature for 10 min. The staining enhancing solution is a PBS buffer solution of SDS or Triton X-100, and the SDS concentration is 0.1 mg / mL.

[0062] (2) Cell surface staining: Dilute 1 μL each of CD45-Alexa 594 and ASGPR1-Alexa 488 with 198 μL of PBS buffer, add to the cell suspension after pretreatment in step (1), and incubate in the dark for 20 minutes. After incubation, add PBS buffer to wash the cell liquid, centrifuge at 950g for 4min, and remove the supernatant to 100μL.

[0063] (3) Cell fixation: transfer and smear the cells in step (2) onto a glass slide, then add the fixative paraformaldehyde, fix for 10 min, and wash the slide twice with PBS, 5 min each time.

[0064] (4) Cell membrane rupture: After the cells were fixed, 200 μL of cell membrane rup...

Embodiment 3

[0068] Embodiment 3 Fluorescence staining detection of Hep3B human liver cancer cell line

[0069] Hep3B human liver cancer cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were enzymatically digested and 105 Cells, approximately 50 μL, were subjected to cell fluorescence staining and fluorescence microscope examination according to the steps in Example 2. Microscopic examination conditions are as follows: when the excitation light wavelength is 591nm, Alexa594 emits 618nm red light, and the exposure time is 100ms; when the excitation light wavelength is 650nm, CY5 emits 670nm far-red light (invisible to the naked eye, and the microscope scanning is assigned purple). The time is 100ms; when the excitation light wavelength is 499nm, Alexa488 emits 519nm green light, and the exposure time is 100ms; when the excitation light wavelength is 345nm, DAPI emits 455nm blue light, and the exposure time is 10-20ms, the results are as follows figure 1 shown.

[007...

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Abstract

The invention provides a liver cancer detection kit based on liquid biopsy. The liver cancer detection kit comprises staining enhancing liquid and specific antibodies with fluorescent staining labels, wherein the staining enhancing liquid is used for enhancing a staining effect; the specific antibodies include an ASGPR1 antibody, a CD45 antibody and a CPS1 antibody; and the staining enhancing liquid contains a surfactant with a concentration of 0.001mg / mL-1mg / mL. The kit provided by the invention can be used for effectively enriching target cells and determining whether the enriched target cells are derived from early-phase liver cancer patients. Meanwhile, by virtue of double-tumor marker detection, the tumor detection sensitivity is increased, and furthermore, the detection accuracy is guaranteed by virtue of CEP8 detection. Besides, by utilizing the staining enhancing liquid, the staining effect is enhanced, multiple antibodies with the fluorescent staining labels can be integrated with the target cells, and the target cells are stained, so that the staining effect is relatively good, the fluorescence is relatively strong, and borders are clear.

Description

technical field [0001] The invention relates to the field of liquid biopsy, in particular to a kit for detecting liver cancer based on liquid biopsy. Background technique [0002] Liver cancer is a common cancer. Compared with other cancers, its incidence rate ranks fifth in the world, and its mortality rate ranks third. In my country, the morbidity and mortality of liver cancer are higher than the world level. According to statistics, nearly 30 people per 100,000 population are diagnosed with liver cancer each year, and 26 people per 100,000 population die from liver cancer every year, which is only lower than the number of deaths from lung cancer, which ranks first. Therefore, it is particularly important for the early screening of liver cancer and the detection during the development of the disease. In this regard, methods based on circulating tumor cell (CTC) detection have many advantages that traditional detection methods do not have, including non-invasive, fast and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/574
CPCG01N33/57438G01N2800/085
Inventor 段彪陈昌岳张培培张祥林冯丽娜蔡红东
Owner SHANGHAI YH HEALTH BIOLOGY MEDICINE TECH CO LTD
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