WO2021213315A1 - Kit for detecting mutation expression of braf gene v600e of colorectal cancer patient by means of peripheral blood circulating tumor cells - Google Patents
Kit for detecting mutation expression of braf gene v600e of colorectal cancer patient by means of peripheral blood circulating tumor cells Download PDFInfo
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- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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Definitions
- the invention provides an immunofluorescence kit and a detection method for the expression of BRAF gene V600E mutation in peripheral blood circulating tumor cells of patients with colorectal cancer, and belongs to the technical field of molecular biology.
- Colorectal cancer is one of the common malignant tumors of the digestive tract in China. According to statistics, the incidence and mortality of CRC are among the forefront of all malignant tumors and are on the rise. However, its pathogenic factors and pathogenesis are still not fully understood. It is currently considered to be a complex process involving multiple genes in the regulation, involving abnormal activation of oncogenes (such as RAF, etc.) and inactivation of tumor suppressor genes (such as APC, P53, etc.). Studies have shown that BRAF gene plays an important role in the pathogenesis and treatment of colorectal cancer. However, the relationship between BRAF gene mutation characteristics in colorectal cancer and the relationship between the two and the clinical pathology has not been reported in many studies.
- the BRAF gene is located in the proto-oncogene of human chromosome 7q34, which encodes a serine/threonine specific kinase. It is an activator of the RAF gene and MAPK signal transduction cascade that regulates the serine/threonine kinase. After activating mutations, it can develop in cancer. Plays a carcinogenic effect in. Studies have found that BRAF mutations can inhibit colonic epithelial cell apoptosis and promote cell proliferation, and promote the immortal proliferation, survival and migration of cancer cells. Even in the absence of cytokines and other stimuli, the V600E mutation in BRAF can activate this signaling pathway, leading to immortal cell proliferation and eventually cancer. Based on the mutation of the tumor BRAF gene, the current clinical specimens are mainly tumor tissues, which are derived from surgery or needle biopsy, and it is difficult to achieve multiple or real-time detection.
- Circulating tumor cells are tumor cells that fall off from solid tumors and enter the peripheral blood circulation. Since they were discovered in 1989, there have been a variety of methods for detecting circulating tumor cells in the peripheral blood. Recent studies have shown that its detection has important clinical significance for evaluating the prognosis of cancer patients, especially advanced cancer patients, and selecting appropriate individualized treatments. Because CTC detection has the characteristics of minimally invasive and real-time detection, it is called "liquid biopsy" of tumors.
- Immunofluorescence analysis technology combines immunological methods (antigen-antibody specific binding) and fluorescent labeling technology to study the method of specific protein antigen distribution in cells. Because the fluorescence emitted by fluorescein can be detected under a fluorescence microscope, fluorescein emits bright fluorescence (yellow-green or orange-red) when irradiated by the excitation light, and the cells or tissues where the fluorescence is located can be seen. Quantitative techniques are used to determine the content. In order to carry out cell qualitative and localization analysis of the antigen.
- the specimens for BRAF gene V600E mutation detection in colorectal cancer patients are mainly tumor tissues, which are derived from surgery or needle biopsy, and it is difficult to achieve multiple or real-time detection. Therefore, the detection of circulating tumor cells (CTC) BRAF gene V600E mutation expression is of great value for the prognosis of colorectal cancer and the evaluation of the efficacy of immunotherapy.
- CTC circulating tumor cells
- Shandong First Medical University, Shandong Pharmaceutical Research Institute and Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., Shandong Discovery Biotechnology Co., Ltd. and other units have Research on the industrialization of key technologies for tumor cell detection and identification.
- This project is a major scientific and technological innovation project in Shandong Province. This project will take the Shandong Provincial Pharmaceutical Research Institute on the Jinan Campus of Shandong First Medical University as the core and implement the registrant system.
- Circulating tumor cell detection and identification of core diagnostic technology, and further registration and identification of diagnostic kits to include PD1, PD-L1, ER, PR, Her-2, GPC-3, VEGF, P53, Vimentin, TKI-EGFR, RAS, CK, ALK-D5F3, CD20, ALK/EML4, Beta-catenin, E-Cadherin, EP-CAM, HPV, IDH-1, PSA, PSMA, VEGF, GFAP, Cytokeratin, AE1/AE3, estrogen receptor, pregnancy Hormone receptors, BCA-225, CA 125, CEA, EMA, ERCC1, HPV, Ki-67, P53, TOP2A, etc.
- the identification and diagnosis kits are industrialized and promoted through cooperation with Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., and Shandong Discovery Biotechnology Co., Ltd., which are registered in Jinan.
- the present invention provides an immunofluorescence kit for the expression of the BRAF gene V600E mutation in circulating tumor cells in the peripheral blood of colorectal cancer patients and a detection method.
- a membrane filtration device is used to separate and obtain circulating tumor cells (CTC) in the peripheral blood of patients with advanced colorectal cancer.
- CTC circulating tumor cells
- An immunofluorescence kit for detecting the BRAF gene V600E mutation expression of circulating tumor cells in the peripheral blood of patients with colorectal cancer including 45mL dilution solution, 1mL decolorization solution, staining solution A 0.5mL, staining solution B 1mL, 200 ⁇ l methanol, 200 ⁇ l 2% PFA, 100 ⁇ l of 10% goat serum, 100 ⁇ l of primary antibody suspension composed of mouse anti-CK, rat anti-CD45 and rabbit anti-BRAF gene V600E mutation, fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, fluorescently labeled goat 100 ⁇ L of anti-rabbit secondary antibody suspension, DAPI mounting plate;
- mice anti-CK, rat anti-CD45 and rabbit anti-BRAF gene V600E mutations in the primary antibody suspension were diluted 1:100, 1:400, and 1:500 respectively, and the total volume was 100 ⁇ L;
- Fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit in the secondary antibody suspension were diluted 1:500.
- the diluent is composed of 1 mmol/L EDTA + 1 mmol moon silicate + 0.1% BSA + 0.2% poloxamer.
- the decolorizing liquid is composed of 95% alcohol and 100% xylene in a volume ratio of 1:1.
- the staining solution A is DAB staining solution
- the staining solution B is hematoxylin staining solution.
- the method for detecting the BRAF gene V600E mutation expression in peripheral blood circulating tumor cells of patients with colorectal cancer for non-diagnostic purposes of the above kit includes the following steps:
- step (7) using immunofluorescence to detect the BRAF gene V600E mutation expression of peripheral blood CTC is as follows:
- the membrane filtration device used in the present invention to separate circulating tumor cells includes a filter, a blood sample container, a waste liquid tank and an iron stand.
- the iron stand is provided with a base, a stand and a bracket.
- the blood sample container is set on the upper part of the iron stand through the bracket.
- Below the blood sample container is a filter, the filter is connected to the waste liquid tank through the infusion set, and the waste liquid tank is arranged on the base.
- the filter includes a filter upper port, a filter membrane, a filter membrane platform and a filter lower port.
- the filter membrane is placed on the filter membrane platform; the upper port of the filter is connected to the blood sample container, and the lower port of the filter is connected to the waste liquid tank through the infusion device.
- the filter membrane is made of hydrophobic material, and the filter holes with a diameter of 8 microns are uniformly spread on it; the diameter of tumor cells is generally greater than 15 microns, and the diameter of blood cells (including red blood cells and white blood cells) is generally less than 8 microns, so when it contains CTC After the peripheral blood is filtered, the blood cells can be filtered because the diameter is smaller than the filter hole, and the CTC is trapped on the filter membrane because the diameter is larger than the filter hole.
- the detection method provided by the present invention can detect the BRAF gene V600E mutation expression of patients with advanced or recurrent colorectal cancer without puncture biopsy to obtain tissue samples, and can realize real-time dynamic detection by using minimally invasive technology.
- the method provided by the present invention has good separation of circulating tumor cells, can avoid the interference of blood cells, can avoid false positive results caused by edge effects that may occur in the staining process, has good stability, reduces cell loss, and improves detection accuracy sex.
- Figure 1 is a schematic diagram of the structure of the membrane filtration device of the present invention.
- FIG. 2 is a schematic cross-sectional view of the structure of the filter of the membrane filtration device of the present invention
- FIG. 3 is a schematic diagram of the structure of the filter membrane of the membrane filtration device of the present invention.
- Figure 4 is an image diagram of circulating tumor cells obtained from peripheral blood of patients with colorectal cancer
- Figure 5 is an immunofluorescence staining image of the BRAF gene V600E mutation of circulating tumor cells in the peripheral blood of patients with advanced colorectal cancer, where A is merge, B is the target gene expression (red), C is CK (green), and D is CD45 (blue).
- Component content Diluent 45 mL Decolorizing liquid (volume ratio of 95% alcohol and 100% xylene 1:1) 1mL Staining solution A (DAB staining solution) 0.5 mL Staining solution B (hematoxylin staining solution) 1mL 10% goat serum (diluted in PBS) 100 ⁇ L A suspension of anti-aliasing 100 ⁇ L Secondary antibody suspensions 100 ⁇ L
- the primary antibody suspension is composed of mouse anti-CK, rat anti-CD45 and rabbit anti-BRAF gene V600E mutations.
- Mouse anti-CK, rat anti-CD45 and rabbit anti-BRAF gene V600E mutations are respectively used with BD wash buffer at 1:100 , 1:500 and 1:400 dilution, after dilution, take 10 ⁇ L mouse anti-CK, 50 ⁇ L rat anti-CD45 and 40 ⁇ L rabbit anti-BRAF gene V600E mutation to form the primary antibody suspension;
- the secondary antibody suspension is composed of fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit. They are commercially available Alexa Fluor 546 goat Anti-mouse, Alexa Fluor 488 goat Anti -rat and Alexa Fluor 647 goat Anti-rabbit, take the same amount of the above three fluorescently labeled secondary antibodies, dilute them with BD wash buffer at 1:500 and mix to obtain the secondary antibody suspension.
- the membrane filtration device is composed of a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5, and an iron stand 1;
- the diameter of tumor cells is generally greater than 15 microns, and the diameter of blood cells (including red blood cells and white blood cells) is generally less than 8 microns. Therefore, after the peripheral blood containing CTC is filtered, the blood cells can be filtered because the diameter is smaller than the filter hole 10, and the CTC is larger than the diameter.
- the filter hole 10 is trapped on the filter membrane 7. What needs to be explained here is that the diluent plays the role of de-adhesion and dispersion. The laurate and poloxamer are used together to ensure that blood cells and CTC do not adhere and are fully dispersed in the diluent to effectively pass through the filter membrane. 7 was detained.
- Figure 5 is an immunofluorescence staining image of circulating tumor cells in the peripheral blood of a patient with advanced colorectal cancer. According to immunological and morphological findings, it is found that the tumor cells are large in size, have abnormal nucleus-to-cytoplasm ratio, and immunologically show typical CTCs.
- the detected circulating tumor cells were confirmed by immunofluorescence to confirm the BRAF gene V600E mutation and compared with the results of the BRAF gene V600E mutation gene of colorectal cancer specimens, to observe the differences, mainly for patients with negative BRAF gene V600E mutation in the gross specimens and positive expression of circulating tumor cells , To guide the targeted therapy of colorectal cancer and provide new ideas for targeted therapy of colorectal cancer.
- the detected circulating tumor cells were confirmed by immunofluorescence to confirm the expression of BRAF gene V600E mutation and compared with the results of BRAF gene V600E mutation in colorectal cancer specimens. Observe the differences, mainly for the negative expression of BRAF gene V600E mutation in gross specimens and positive expression of circulating tumor cells Of patients, guide the targeted therapy of colorectal cancer, and provide new ideas for targeted therapy of colorectal cancer.
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Abstract
Description
组分Component | 含量content |
稀释液Diluent | 45 mL 45 mL |
脱色液(95%酒精与100%二甲苯体积比1∶1)Decolorizing liquid (volume ratio of 95% alcohol and 100% xylene 1:1) | 1mL1mL |
染色液A(DAB染色液)Staining solution A (DAB staining solution) | 0.5 mL 0.5 mL |
染色液B(苏木素染色液)Staining solution B (hematoxylin staining solution) |
1mL |
10%山羊血清(PBS稀释)10% goat serum (diluted in PBS) | 100μL100μL |
一抗混悬液 A suspension of anti-aliasing | 100μL100μL |
二抗混悬液 Secondary antibody suspensions | 100μL100μL |
Claims (6)
- 一种检测结直肠癌患者外周血循环肿瘤细胞BRAF基因V600E突变表达的免疫荧光试剂盒,其特征在于,包括稀释液45mL,脱色液1mL,染色液A 0.5mL,染色液B 1mL,200μl甲醇、200μl 2%PFA,100μl 10%山羊血清,小鼠抗CK、大鼠抗CD45和兔抗BRAF基因V600E突变组成的一抗混悬液100μl,荧光标记的羊抗小鼠、荧光标记的羊抗大鼠、荧光标记的羊抗兔组成的二抗混悬液100μL,DAPI封片剂;An immunofluorescence kit for detecting the BRAF gene V600E mutation expression of circulating tumor cells in the peripheral blood of patients with colorectal cancer, which is characterized in that it includes a dilution solution of 45mL, a decolorizing solution of 1mL, staining solution A 0.5mL, staining solution B 1mL, 200μl methanol, 200μl 2% PFA, 100μl 10% goat serum, 100μl of primary antibody suspension composed of mouse anti-CK, rat anti-CD45 and rabbit anti-BRAF gene V600E mutation, fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat , 100μL of secondary antibody suspension composed of fluorescently labeled goat anti-rabbit, DAPI mounting plate;一抗混悬液中小鼠抗CK、大鼠抗CD45和兔抗BRAF基因V600E突变分别按1:100、1:400和1:500稀释,总体积为100μL;The mouse anti-CK, rat anti-CD45 and rabbit anti-BRAF gene V600E mutations in the primary antibody suspension were diluted 1:100, 1:400, and 1:500 respectively, and the total volume was 100 μL;二抗混悬液中荧光标记的羊抗小鼠、荧光标记的羊抗大鼠、荧光标记的羊抗兔按1:500稀释。Fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit in the secondary antibody suspension were diluted 1:500.
- 根据权利要求1所述的试剂盒,其特征在于,所述稀释液是由1mmol/L EDTA+1mmol月硅酸盐+0.1%BSA+0.2%泊洛沙姆组成。The kit according to claim 1, wherein the diluent is composed of 1 mmol/L EDTA + 1 mmol moon silicate + 0.1% BSA + 0.2% poloxamer.
- 根据权利要求1所述的试剂盒,其特征在于,所述脱色液是由95%酒精与100%二甲苯按体积比1:1组成。The kit according to claim 1, wherein the decolorizing solution is composed of 95% alcohol and 100% xylene in a volume ratio of 1:1.
- 根据权利要求1所述的试剂盒,其特征在于,所述染色液A为DAB染色液;所述染色液B为苏木素染色液。The kit according to claim 1, wherein the staining solution A is a DAB staining solution; the staining solution B is a hematoxylin staining solution.
- 一种利用权利要求1-4任一项所述的试剂盒非诊断目的检测结直肠癌患者外周血循环肿瘤细胞BRAF基因V600E突变表达的方法,其特征在于,包括以下步骤:A method for detecting the BRAF gene V600E mutation expression of circulating tumor cells in the peripheral blood of patients with colorectal cancer for non-diagnostic purposes using the kit according to any one of claims 1 to 4, characterized in that it comprises the following steps:(1)利用膜过滤装置分离获取无法获得组织标本的晚期或复发结直肠癌患者外周血:采集无法获取组织标本的晚期或复发结直肠癌患者肘正中静脉外周血5ml;(1) Use membrane filtration device to separate and obtain peripheral blood of patients with advanced or recurrent colorectal cancer who cannot obtain tissue samples: Collect 5ml of peripheral blood from the median cubital vein of patients with advanced or recurrent colorectal cancer who cannot obtain tissue samples;(2)外周血样预处理:将采集的外周血样采用稀释液进行10倍稀释,稀释后加多聚甲醛固定外周血样10分钟,固定终浓度为0.25%;(2) Pretreatment of peripheral blood samples: Dilute the collected peripheral blood samples 10 times with diluent, add paraformaldehyde to fix the peripheral blood samples for 10 minutes after dilution, and fix the final concentration to 0.25%;(3)利用膜过滤分离肿瘤细胞装置过滤外周血样,分离获得外周血CTC:将预处理的外周血样加入到膜过滤分离肿瘤细胞装置的血样容器中,使其依靠重力自然过滤;(3) Use membrane filtration device to separate tumor cells to filter peripheral blood samples to separate and obtain peripheral blood CTC: add the pretreated peripheral blood sample to the blood sample container of the membrane filtration device to separate tumor cells so that it can be filtered naturally by gravity;(4)过滤结束后,从膜过滤分离肿瘤细胞装置中取下滤器,将循环肿瘤细胞染色液A液0.5ml加入到滤器中,染色3min,PBS缓冲液冲洗干净;滤液过滤完全后加入染色液B液1ml,染色2min,纯水1ml冲洗2次;(4) After filtering, remove the filter from the device for separating tumor cells by membrane filtration, add 0.5 ml of circulating tumor cell staining solution A to the filter, stain for 3 minutes, and rinse with PBS buffer; add staining solution after filtering the filtrate 1ml B solution, stain for 2min, rinse 2 times with 1ml pure water;(5)向滤器中加入200μl 2%PFA,室温固定5min,完成后0.5ml PBS漂洗3次,每次2min;(5) Add 200μl 2% PFA to the filter, fix at room temperature for 5 minutes, and rinse with 0.5ml PBS for 3 times, 2 minutes each time;(6)再向滤器中加入200μl预冷的甲醇,4℃固定15min,取下滤膜,放置在载玻片上,干燥后在显微镜下观察,确定是否存在CTC;(6) Add 200μl of pre-cooled methanol to the filter, fix it at 4°C for 15 minutes, remove the filter membrane, place it on a glass slide, and observe under a microscope after drying to determine whether there is CTC;(7)运用免疫荧光方法检测外周血CTC的BRAF基因V600E突变表达情况。(7) Use immunofluorescence method to detect the expression of BRAF gene V600E mutation in peripheral blood CTC.
- 根据权利要求5所述的检测方法,其特征在于,所述的步骤(7)运用免疫荧光方法检测外周血CTC的BRAF基因V600E突变表达的具体方法如下:The detection method according to claim 5, characterized in that, in the step (7), the specific method for detecting the V600E mutation expression of the BRAF gene in peripheral blood CTC by using an immunofluorescence method is as follows:(1)脱色:将带有CTC的滤膜从载玻片上取下,置于脱色液中浸泡4-6小时,脱去CTC染色液,PBS洗2min×3次;(1) Decolorization: Remove the filter membrane with CTC from the glass slide, soak it in the decolorization solution for 4-6 hours, remove the CTC staining solution, and wash with PBS for 2min×3 times;(2)封闭:向滤膜上滴加100μl 10%山羊血清,室温放置30min,完成后吸去多余的血清(注:羊血清用PBS稀释);(2) Blocking: Add 100μl 10% goat serum to the filter membrane, leave it at room temperature for 30 minutes, and suck off the excess serum after completion (Note: Goat serum is diluted with PBS);(3)一抗孵育:向滤膜上滴加100μl小鼠抗CK、大鼠抗CD45和兔抗BRAF基因V600E突变组成的一抗混悬液,37℃孵育1h或4℃过夜,完成后PBS洗3min×3次;(3) Primary antibody incubation: Add 100μl mouse anti-CK, rat anti-CD45 and rabbit anti-BRAF gene V600E mutations to the filter membrane, and incubate at 37℃ for 1h or 4℃ overnight. After completion, PBS Wash 3min×3 times;(4)二抗孵育:向滤膜上滴加100μl荧光标记的羊抗小鼠、荧光标记的羊抗大鼠、荧光标记的羊抗兔组成的二抗混悬液,室温孵育30min,完成后PBS洗2min×3次;(4) Secondary antibody incubation: drop 100μl of the secondary antibody suspension composed of fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit on the filter membrane, and incubate at room temperature for 30 minutes. Wash with PBS for 2min×3 times;(5)使用含DAPI的封片剂封片,阅片,采图;(5) Use DAPI-containing mounting tablets to mount, read and take pictures;(6)采照完成后,脱片后进行瑞氏吉姆萨染色,与IF结果进行对比。(6) After taking the photos, take off the pieces and perform Wright's Giemsa staining to compare with the IF results.
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CN105588943A (en) * | 2016-01-28 | 2016-05-18 | 山东省肿瘤防治研究院 | Detection method for peripheral blood CTC (Circulating Tumor Cell) Her-2 gene of stomach cancer patient |
WO2019232485A1 (en) * | 2018-05-31 | 2019-12-05 | Nvigen, Inc. | Accurate blood test to predict cancer incidence, recurrence, guide and monitor treatment intervention |
CN110716041A (en) * | 2019-10-23 | 2020-01-21 | 郑州大学 | Serum protein marker, kit and detection method for early screening and diagnosis of gastric cancer |
CN111521801A (en) * | 2020-04-22 | 2020-08-11 | 山东第一医科大学(山东省医学科学院) | Kit for detecting BRAF gene V600E mutation expression of colorectal cancer patient through peripheral blood circulation tumor cells |
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CN105588943A (en) * | 2016-01-28 | 2016-05-18 | 山东省肿瘤防治研究院 | Detection method for peripheral blood CTC (Circulating Tumor Cell) Her-2 gene of stomach cancer patient |
WO2019232485A1 (en) * | 2018-05-31 | 2019-12-05 | Nvigen, Inc. | Accurate blood test to predict cancer incidence, recurrence, guide and monitor treatment intervention |
CN110716041A (en) * | 2019-10-23 | 2020-01-21 | 郑州大学 | Serum protein marker, kit and detection method for early screening and diagnosis of gastric cancer |
CN111521801A (en) * | 2020-04-22 | 2020-08-11 | 山东第一医科大学(山东省医学科学院) | Kit for detecting BRAF gene V600E mutation expression of colorectal cancer patient through peripheral blood circulation tumor cells |
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