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Reagent kit for detecting non-small cell lung cancer on basis of liquid biopsy

A technology of non-small cell lung cancer and liquid biopsy, which is applied in the direction of measuring devices, preparation of test samples, instruments, etc., can solve the problems of weak fluorescence intensity, difficult to distinguish, missed detection, etc., and achieve simple methods, easy to distinguish, and enrichment high efficiency effect

Active Publication Date: 2017-07-28
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 on the surface of 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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  • Reagent kit for detecting non-small cell lung cancer on basis of liquid biopsy
  • Reagent kit for detecting non-small cell lung cancer on basis of liquid biopsy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[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 horizonta...

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 SDS (sodium dodecylsulfonate) or Triton X-100 in PBS buffer solution, and the concentration of SDS is 0.1 mg / mL.

[0062] (2) Cell surface staining: Dilute 1 μL each of CD45-Alexa 594, EGFR-Alexa 488, and Folate-CY5 with 197 μL of PBS buffer, add to the cell suspension after pretreatment in step (1), and then avoid Incubate with light for 20min. 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 buffer, 5 min each time.

[0064] (4) Fluorescent in situ hybridi...

Embodiment 3

[0066] Example 3 Fluorescence staining detection of Calu-1 human lung cancer cell line

[0067] Calu-1 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were enzymatically digested and 10 5Cells, approximately 50 μL, were subjected to fluorescent staining and fluorescent microscope examination on the cell surface 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.

[0068] The ...

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Abstract

The invention provides a reagent kit for detecting non-small cell lung cancer on the basis of liquid biopsy. The reagent kid comprises staining enhancement solution for enhancing staining effects and specific ligands with fluorescent staining markers. The specific ligands comprise EGFR (epidermal growth factor receptor) antibodies, CD45 antibodies and folate ligands; the staining enhancement solution comprises surfactants with the concentration of 0.001-1 mg / mL. The reagent kit has the advantages that target cells can be effectively enriched by the reagent kit, and whether the enriched target cells come from early-stage patients suffering from the non-small cell lung cancer or not can be verified; the CTC (circulating tumor cell) detection sensitivity can be improved by means of double-tumor-marker detection, and the detection accuracy further can be guaranteed by means of CEP8 detection; the staining effects can be enhanced by the staining enhancement solution, the diversified ligands with the fluorescent staining markers can be combined with the surfaces of the target cells, accordingly, the surfaces of the target cells can be stained, the good staining effects can be realized, fluorescence is intense, and boundaries are clear.

Description

technical field [0001] The invention relates to the field of liquid biopsy, in particular to a liquid biopsy-based detection kit for non-small cell lung cancer. Background technique [0002] Lung cancer is one of the most common malignant tumors in the world, and its morbidity and mortality both rank first in my country. Lung cancer includes non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), wherein non-small cell lung cancer includes squamous cell carcinoma (squamous cell carcinoma), adenocarcinoma, and large cell carcinoma. Compared with SCLC, although NSCLC cells grow and divide slower, and spread and metastasize relatively late, but because it accounts for about 80% of all lung cancers, and about 75% of patients are found in the middle and advanced stages, the 5-year survival rate is very low. Therefore, there are many studies on NSCLC, and more people pay attention to it. Based on the above characteristics of NSCLC, its early screening and diagnosis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N1/30
CPCG01N1/30G01N21/6428G01N2021/6439
Inventor 张培培段彪陈昌岳张祥林冯丽娜蔡红东
Owner 上海美吉医学检验有限公司
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