Method for detecting CTC (circulating tumor cell) surface marker molecule GPC3 (glypican-3)

A technology of molecular phosphatidylinositol and surface markers, applied in the field of molecular biology, can solve problems such as large reactions and incompleteness, and achieve high sensitivity, simple detection methods, and good specificity

Inactive Publication Date: 2017-05-10
SHANGHAI PERMED BIOMEDICAL CO LTD
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  • Abstract
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  • Claims
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Problems solved by technology

[0006] The purpose of the present invention is to solve the above-mentioned deficiencies and provide a detection method for circulating tumor cell surface marker molecule Glypican-3, which is simple, fast, economical, high in sensitivity, good in specificity, and Solve the problem that the detection of GPC3 in the existing technology requires a large amount of samples and the reaction is not thorough

Method used

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Embodiment Construction

[0018] Below in conjunction with specific embodiment, the present invention is further described as follows:

[0019] The present invention provides a method for detecting Glypican-3, a marker molecule on the surface of circulating tumor cells, which specifically includes the following steps:

[0020] 1) Whole blood processing:

[0021] a. Add 200 μL of 1× red blood cell lysate to 2 mL of whole blood, place at room temperature for 15 min, and shake evenly during the period.

[0022] b. Centrifuge at 200RCF for 5 minutes, aspirate the supernatant and keep the cells.

[0023] c. Add 2 mL of 1% FPBS (V(FBS):V(PBS)=1:99) to the centrifuge tube, mix well, centrifuge at 200RCF for 5 minutes, and remove the supernatant.

[0024] 2) Cell full enrichment:

[0025] a. Add 1mL FPBS and mix well, then transfer to the treated petri dish, and place the petri dish in a shaker at 37°C for 45min.

[0026] b. After culturing, place the petri dish in a refrigerator at 4°C for 1 min.

[0027...

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Abstract

The invention belongs to the field of molecular biology, and particularly discloses a method for detecting a CTC (circulating tumor cell) surface marker molecule GPC3 (glypican-3). The method comprises the following steps: performing erythrocyte splitting on blood, spreading and enriching all of left karyotes (mainly comprising CTCs and lymphocytes) on a nano-substrate for fixation by virtue of a nanotechnology, marking all the cells by virtue of a nucleus fluorescent dye DAPI, performing positive selection by virtue of a tumor immune fluorescent marker, i.e. a cytokeratin antibody anti-CK, and since a CTC surface has a specific expression of the GPC3, incubating all the cells by virtue of a GPC3 primary antibody, performing incubation by virtue of a secondary antibody marked with an FITC fluorescence group, and finally performing scanning by virtue of a high-throughput technology. Compared with the prior art, the detection method has the advantages of convenience, rapidness, economy, high sensitivity and specificity and capability of overcoming the defects of large sample amount requirement and incomplete reaction of GPC3 detection in the prior art.

Description

[0001] [technical field] [0002] The invention belongs to the field of molecular biology, in particular to a detection method of circulating tumor cell surface marker molecule Glypican-3. [0003] [Background technique] [0004] Circulating tumor cells (CTCs) are tumor cells that come off from the primary tumor and enter the peripheral blood. They will choose a suitable microenvironment along with the blood flow, plant in distant organs, and form metastases. At present, peripheral blood CTC detection technology can only realize the qualitative and relatively quantitative detection of CTCs in peripheral blood, and cannot track which organ the CTCs in the blood come from. CTCs are tumor cells that have fallen off from the primary tumor. Its surface contains marker molecules specifically expressed on the primary tumor. For example, glypican-3 (GPC3) is a heparin-like sulfate proteoglycan polymerized on the surface of the cell membrane, which can be anchored on the cell surface t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/533
CPCG01N33/533G01N33/57496
Inventor 张群祝琳
Owner SHANGHAI PERMED BIOMEDICAL CO LTD
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