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Kit for detecting prostatic cancer based on liquid biopsy

A technology of prostate cancer and liquid biopsy, applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of missed detection, inconsistency, and difficulty in distinguishing, and achieve the effect of high enrichment efficiency

Active Publication Date: 2017-07-21
上海美吉医学检验有限公司
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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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  • Kit for detecting prostatic cancer based on liquid biopsy
  • Kit for detecting prostatic cancer 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 PSMA-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 ruptu...

Embodiment 3

[0068] Example 3 Fluorescence staining detection of LNCaP human prostate cancer cell line

[0069] LNCaP human prostate cancer (purchased from the Cell Bank of the Chinese Academy of Sciences) was enzymatically digested and 10 5Cells, 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.

[0070] ...

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Abstract

The invention provides a kit for detecting prostatic cancer based on liquid biopsy, comprising a staining enhancement solution for enhancing staining effect and specific antibodies with a fluorescent staining marker, wherein the specific antibodies include PSA (prostate specific antigen), CD45 antibody and PSMA (prostate specific membrane antigen); the staining enhancement solution comprises a surfactant having a concentration of 0.001-1 mg / mL. The kit herein is capable of effectively enriching target cells, determining whether the enriched target cells are derived from a patient with early prostatic cancer, and typing CTCs (circulating tumor cells); in addition, double-tumor-marker detection increases detection sensitivity, and detection accuracy is guaranteed through further CEP8 detection; furthermore, the staining effect is enhanced through the staining enhancement solution, each of various antibodies with a fluorescent staining marker can bind with a target cell, the target cells can be stained more effectively, fluorescence is greater, and the boundary is vivid.

Description

technical field [0001] The invention relates to the field of liquid biopsy, in particular to a liquid biopsy-based prostate cancer detection kit. Background technique [0002] Prostate cancer ranks sixth in the incidence of male malignant tumors in China. In 2012, the incidence of prostate cancer in my country's tumor registration areas was 9.92 / 100,000. The onset age of prostate cancer is at a low level before the age of 55, and gradually increases after the age of 55. The incidence rate increases with age, and the peak age is 70-80 years old. Patients with familial hereditary prostate cancer had a slightly earlier age of onset, and 43% of patients were ≤55 years old. [0003] At present, for the early screening of prostate cancer, the method of detecting circulating tumor cells (CTC) based on liquid biopsy has many advantages that traditional detection methods do not have, including non-invasive, fast, and high accuracy. CTC is a kind of tumor cells released into the per...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/58
CPCG01N33/57434G01N33/582
Inventor 张培培陈昌岳段彪张祥林李静蔡红东冯丽娜
Owner 上海美吉医学检验有限公司
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