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Specific method for detecting breast cancer circulating tumor cells by adopting HER2 antibody immunofluorescence method

A tumor cell, immunofluorescence technology, applied in the field of molecular biology testing, to avoid false negative results, accurate results, and increase the difficulty of work

Pending Publication Date: 2022-01-11
SHANGHAI PERMED BIOMEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] With the increase in the mortality rate of female breast cancer year by year, breast cancer has become an important cause of female death, and there are still some deficiencies in the detection and prognosis of breast cancer in the existing technology
In addition, since the course of breast cancer is a long-term process, there may be heterogeneity in the HER-2 receptor

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  • Specific method for detecting breast cancer circulating tumor cells by adopting HER2 antibody immunofluorescence method

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Experimental program
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Embodiment 1

[0027] A specific method for detecting circulating tumor cells in breast cancer by using HER2 antibody immunofluorescence method, comprising the following steps: S1: collecting 8ml of peripheral blood from patients with breast cancer, mixing the collected blood sample with lysate at a ratio of 1:8, and mixing at room temperature 15min. Centrifuge at 200RCF for 5 minutes, aspirate the supernatant and keep the cells. Add 2mL of 1% FPBS to the centrifuge tube, mix well and centrifuge at 200RCF for 5 minutes, remove the supernatant; add 1ml of FPBS and mix well, add the mixed cells into the treated culture dish, and incubate at 37°C for 45min After culturing, put the petri dish in a 4°C refrigerator and let it stand for 10 minutes; suck off the FPBS, add 4% formaldehyde to the petri dish and put it at 4°C for 10 minutes. Remove formaldehyde by suction, add 1mL of methanol, and place at -20°C for 10min. Aspirate off the methanol and wash three times with 2 mL of PBS each time.

...

Embodiment 2

[0045] A specific method for detecting circulating tumor cells in breast cancer by using HER2 antibody immunofluorescence method, comprising the following steps: S1: collecting 9ml of peripheral blood from patients with breast cancer, mixing the collected blood sample with lysate at a ratio of 1:8, and mixing at room temperature 15min. Centrifuge at 200RCF for 5 minutes, aspirate the supernatant and keep the cells. Add 2mL of 1% FPBS to the centrifuge tube, mix well and centrifuge at 200RCF for 5 minutes, remove the supernatant; add 1ml of FPBS and mix well, add the mixed cells into the treated culture dish, and incubate at 37°C for 45min After culturing, put the petri dish in a 4°C refrigerator and let it stand for 10 minutes; suck off the FPBS, add 4% formaldehyde to the petri dish and put it at 4°C for 10 minutes. Remove formaldehyde by suction, add 1mL of methanol, and place at -20°C for 10min. Aspirate off the methanol and wash three times with 2 mL of PBS each time.

...

Embodiment 3

[0063] A specific method for detecting circulating tumor cells in breast cancer by using HER2 antibody immunofluorescence method, comprising the following steps: S1: collecting 10 ml of peripheral blood from patients with breast cancer, mixing the collected blood sample with lysate at a ratio of 1:9, and mixing at room temperature 15min. Centrifuge at 200RCF for 5 minutes, aspirate the supernatant and keep the cells. Add 2mL of 1% FPBS to the centrifuge tube, mix well and centrifuge at 200RCF for 5 minutes, remove the supernatant; add 1ml of FPBS and mix well, add the mixed cells into the treated culture dish, and incubate at 37°C for 45min After culturing, put the petri dish in a 4°C refrigerator and let it stand for 10 minutes; suck off the FPBS, add 4% formaldehyde to the petri dish and put it at 4°C for 10 minutes. Remove formaldehyde by suction, add 1mL of methanol, and place at -20°C for 10min. Aspirate off the methanol and wash three times with 2 mL of PBS each time. ...

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Abstract

The invention discloses a specificity method for detecting breast cancer circulating tumor cells by adopting an HER2 antibody immunofluorescence method. The method comprises the following steps that S1, peripheral blood of a breast cancer patient is collected, and all nucleated cells in the blood are flatly laid and enriched on a nanometer substrate to be fixed and separated through a specially-made nanometer substrate membrane; s2, after the cells are subjected to fluorescence labeling, CTCs are detected through a high-content scanner, and counting and protein analysis are carried out as shown in the figure 1; s3, if yes, the breast cancer circulating tumor cells are cultured and if not, the steps S1 to S2 are repeated; S4, an HER2 antibody is added into the screened breast cancer circulating tumor cells for co-incubation; and S5, the HER2 expression condition of the breast cancer circulating tumor cells is detected. The method disclosed by the invention is simple to operate and high in detection accuracy, can avoid false negative and other conditions, and improves the checking accuracy of breast cancer.

Description

[technical field] [0001] The invention relates to the technical field of molecular biology testing, in particular to a specific method for detecting breast cancer CTCs by using HER2 antibody immunofluorescence method. [Background technique] [0002] Circulating tumor cells (CTCs) refer to tumor cells that are shed from the primary tumor and invade the blood circulation. The vast majority of tumor cells entering the circulation die within a short period of time. Only a very small number of tumor cells with high vitality and high metastatic potential survive in the circulation system, aggregate with each other to form tiny tumor thrombi, and develop into metastases under certain conditions. CTCs detection is regarded as a liquid biopsy with real-time monitoring function. It is one of the few new tumor markers that have been researched and applied in the past 30 years. By detecting the number of CTCs in the blood, individualized treatment, curative effect evaluation and monito...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/574
CPCG01N33/57488G01N33/57415G01N33/56966G01N33/74G01N2333/71
Inventor 罗静尹红玲吴浪
Owner SHANGHAI PERMED BIOMEDICAL CO LTD
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