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Enrichment detection method for epithelial-mesenchymal hybrid and PD-L1 positive circulating tumor cell

A PD-L1 and detection method technology, applied in the field of enrichment and detection of circulating tumor cell subtypes, can solve the problems of large and small blood consumption for detection

Active Publication Date: 2019-10-29
杭州华得森生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the large amount of blood used for the detection of existing CTCs enrichment detection technology, it is limited to the enrichment detection of epithelial or mesenchymal CTCs of a single group, and cannot enrich the detection of mixed epithelial-mesenchymal CTCs and different types of CTCs PD-L1 immune Due to the technical limitations of phenotype, the purpose of the present invention is to provide an epithelial-mesenchymal mixture through the combination of epithelial-mesenchymal specific capture and detection antibody markers, specific immune checkpoint antibody markers and capture staining enhancement fluid combined with microfluidic technology. The enrichment detection method of PD-L1 positive circulating tumor cells, specifically a small blood volume, simple, efficient, sensitive and specific enrichment detection of epithelial type, mesenchymal type, mixed epithelial-mesenchymal type CTCs and epithelial type PD -L1-positive phenotype, mixed epithelial-mesenchymal PD-L1-positive phenotype, and mesenchymal PD-L1-positive phenotype CTCs

Method used

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  • Enrichment detection method for epithelial-mesenchymal hybrid and PD-L1 positive circulating tumor cell
  • Enrichment detection method for epithelial-mesenchymal hybrid and PD-L1 positive circulating tumor cell
  • Enrichment detection method for epithelial-mesenchymal hybrid and PD-L1 positive circulating tumor cell

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Type detection of circulating tumor cells in lung cancer and detection of PD-L1 expression

[0068] Materials: The microfluidic carrier uses a microfluidic chip, and the microfluidic pump system uses a circulating tumor cell analyzer (WY-C3000, produced by Hangzhou Watson Biotechnology Co., Ltd.)

[0069] 1. Preparation of biotin-labeled epithelial-mesenchymal mixed antibody microfluidic immune capture carrier

[0070] 1.1. Use a pipette to draw 300 μL streptavidin solution with a concentration of 5 μg / mL, add it to the microfluidic chip, incubate at room temperature 25°C for 4 hours, and then place it in a drying room with an ambient humidity below 30% to dry for 24 hours until There is no liquid residue in the microfluidic chip.

[0071] 1.2. Draw 300 μL of 95% ethanol by volume and inject it into the microfluidic chip, and then immediately wash it twice with 1×PBS buffer.

[0072] 1.3. Mix 200 μL of biotin-labeled anti-EpCAM (epithelial cell adhesion mole...

Embodiment 2

[0100] Example 2 Comparison experiment of CTCs enrichment detection efficiency

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Abstract

The invention discloses an enrichment detection method for epithelial-mesenchymal hybrid and PD-L1 positive circulating tumor cells, comprising the steps of: preparation of a biotin-labeled epithelial-mesenchymal hybrid antibody (biotin-labeled EpCAM antibody and biotin-labeled anti-CSV antibody); separation and enrichment of epithelial-mesomixed hybrid CTCs; immunodetection of fluorescein-labeledepithelial-mesenchymal hybrid antibody (fluorescein-labeled anti-PanCK antibody, fluorescein-labeled anti-Vimentin antibody, fluorescein-labeled antibody CD45 antibody) to different subtypes of CTCs;and detection of PD-L1 phenotype of captured different subtypes of CTCs by PD-L1 primary antibody and fluorescein-labeled PD-L1 secondary antibody. The method of the invention can not only enrichmentdetect epithelial-mesenchymal hybrid CTCs in a simple, efficient and specific manner, but also can enrichment detect epithelial and mesenchymal CTCs.

Description

technical field [0001] The invention relates to the field of enrichment of circulating tumor cell subtypes and a detection method thereof, in particular to an enrichment detection method of epithelial-mesenchymal mixed type and PD-L1 positive circulating tumor cells. Background technique [0002] Circulating Tumor Cells (CTCs) are tumor cells that are released from solid tumors or metastases into the peripheral blood circulation spontaneously or due to diagnostic and therapeutic procedures, and are a general term for various types of tumor cells that exist in peripheral blood. CTCs are mainly composed of epithelial CTCs, mesenchymal CTCs, and epithelial-mesenchymal mixed (epithelial-mesenchymal transition and mesenchymal-epithelial transition) CTCs subgroups, as well as CTCs PD-L1 positive or negative subtypes of the above subgroups. types of heterogeneous populations. [0003] Metastatic spread of tumors is a major cause of cancer progression and associated death. CTCs pl...

Claims

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Application Information

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IPC IPC(8): G01N33/533G01N33/536G01N1/40G01N33/574
CPCG01N33/533G01N33/536G01N1/40G01N33/574
Inventor 崔莹张开山于杰
Owner 杭州华得森生物技术有限公司
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