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Reagent for detection of ROR1 protein of circulating tumor cells in peripheral blood and application thereof

A tumor cell and peripheral blood technology, applied in the field of protein detection, can solve problems such as lack, achieve the effect of improving accuracy and reliability, and overcoming limitations

Active Publication Date: 2014-05-14
益杰立科(上海)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims to provide a reagent for detecting ROR1 protein expressed by tumor cells in circulating blood and its application, so as to solve the existing technical problems of lack of simple, sensitive and reliable early diagnosis of cancer and monitoring of cancer metastasis

Method used

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  • Reagent for detection of ROR1 protein of circulating tumor cells in peripheral blood and application thereof
  • Reagent for detection of ROR1 protein of circulating tumor cells in peripheral blood and application thereof
  • Reagent for detection of ROR1 protein of circulating tumor cells in peripheral blood and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1. Detection of ROR1 on circulating tumor cells - immunofluorescent staining

[0037] Circulating tumor cell ROR1 protein detection reagents include: fixative solution, buffer solution, fluorescently coupled anti-ROR1 antibody, fluorescently coupled anti-CD45 antibody mixture, DAPI fluorescent dye and mounting solution. ROR1 antibody is prepared from the recombinant protein obtained from CHO cells transfected with the extracellular fragment of human ROR1 (the sequence of the recombinant protein of ROR1 antibody is as follows). After incubating the enriched circulating tumor cells of lung cancer fixed on the glass slide with PBS at room temperature for 5 minutes, the PBS was discarded by suction. Add 1:200 Alexa Flour 488-coupled anti-ROR1 antibody and 1:400 Alexa Flour 594-coupled anti-CD45 antibody mixture (0.5% BSA-PBS), and incubate at room temperature for 30 minutes in the dark. Aspirate the antibody. Wash slides 3 times with 0.5% BSA-PBS for 3 minutes eac...

Embodiment 2

[0045] Example 2. Flow cytometry is used to detect circulating tumor cells expressing ROR1 in the blood of cancer patients

[0046] Circulating tumor cells from cancer patients were incubated with fluorescently labeled ROR1 antibody or control antibody (Isotype IgG) in incubation solution for 20 minutes at 4°C. The incubation solution contained 3% FBS and 1M HEPES in phosphate buffer (pH 7.4). After washing twice with phosphate buffer, the cells were measured by flow cytometry. The results were analyzed with FlowJo software and figure 2 shown in . The picture shows the detection results of a representative number of circulating cells. On the left, cells stained with a fluorescently labeled isotype control antibody (blue); on the right, cells stained with a fluorescently labeled ROR1 antibody (red).

Embodiment 3

[0047] Example 3. Detection of circulating tumor cells in lung cancer patients: comparison between ROR1 kit and traditional methods

[0048] A comparative study was carried out on 25 patients diagnosed as primary lung cancer in clinical cases. Tumor cells enriched from each lung cancer patient were suspended and equally divided into two parts. One part uses the existing technology to detect the keratin (CK) expression of circulating tumor cells; the other part uses the technology of the present invention to detect the ROR1 protein of circulating tumor cells. After acetone fixation, the former was stained with FITC-conjugated anti-CK antibody mixture (anti-CK18, etc.) and DyLight 549-conjugated anti-CD45 antibody, and the nuclei were stained with DAPI; the latter was detected with the ROR1 protein reagent described in Example 1. Circulating tumor cells were positive for CK or ROR1, negative for CD45, and positive for nuclear staining. As shown in Table 1, the number of circul...

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Abstract

The invention discloses a reagent for detection of an ROR1 protein in circulating blood and an application thereof, and particularly discloses the reagent for the detection of the ROR1 protein of circulating tumor cells in peripheral blood. The reagent includes an ROR1 antibody, a buffer solution, a marker, a nuclear dye and a lymphocyte identification antibody. A characteristic that ROR1 is only expressed in cancer cells and is not expressed in normal adult tissue cells is utilized, and the reagent is applied to detect the expressed ROR1 protein of the circulating tumor cells or tumor stem cells in the circulating blood, wherein the circulating tumor cells or the tumor stem cells fall off from a primary tumor nidus into the blood; limitations of a traditional method are overcome, and the accuracy and the reliability of the detection of the circulating tumor cells are greatly improved. The reagent can be used for helping detect the tumor cells in the circulating blood of a tumor patient, and is a potential breakthrough point for tumor disease diagnosis, evaluation of therapeutic effects and cancer metastasis monitoring.

Description

technical field [0001] The invention relates to the technical field of protein detection, in particular to a reagent for detecting the ROR1 protein of circulating tumor cells in peripheral blood and its application. Background technique [0002] In the process of tumor growth, tumor cells will fall off from the primary lesion and enter the circulatory system to become "circulating tumor cells" and serve as a tumor-specific test indicator. Tumor metastasis is when circulating tumor cells enter the blood circulation from the primary tumor lesion, attach to distant tissues and begin to grow and develop into new tumors. A large number of clinical data show that circulating tumor cells are the main cause of death in cancer patients. The recently developed isolation method for circulating tumor cells is very important for the early diagnosis of tumors. Importantly, the quantification of circulating tumor cells can be used to predict the outcome of tumor metastasis. In addition,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/53C12Q1/68
CPCG01N33/57488G01N33/6893G01N2500/00
Inventor 张宝弘林平
Owner 益杰立科(上海)生物科技有限公司
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