WO2021213299A1 - 一种检测前列腺癌患者外周血循环肿瘤细胞pd-l1基因突变的试剂盒及检测方法 - Google Patents
一种检测前列腺癌患者外周血循环肿瘤细胞pd-l1基因突变的试剂盒及检测方法 Download PDFInfo
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- G—PHYSICS
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- 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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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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Definitions
- the invention provides a kit and a detection method for detecting PD-L1 gene mutations in peripheral blood circulating tumor cells of prostate cancer patients, and belongs to the technical field of molecular biology.
- prostate cancer ranks second among male malignancies, and cancer-specific death ranks sixth.
- the difficulty in the treatment of prostate cancer is metastatic prostate cancer, especially castration resistance after endocrine therapy.
- the treatment methods for metastatic prostate cancer after castration resistance are limited, and all conventional treatment methods (radiotherapy, chemotherapy, Endocrine therapy, targeted therapy) have failed.
- the efficacy of PD-1 or PD-L1 immune preparations is mostly related to the immunohistochemical expression level of PD-L1 in tumor tissues, suggesting that the expression level of PD-L1 may be a biomarker for predicting the efficacy of PD-1 immunotherapy; there are also studies It shows that the high expression of PD-L1 in prostate cancer tissue is positively correlated with tumor aggressiveness.
- 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.
- the specimens for PD-L1 detection in prostate 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 cell (CTC) PD-L1 expression is of great value for the prognosis of prostate cancer patients and the evaluation of the efficacy of immunotherapy.
- CTC circulating tumor cell
- 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.
- 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 invention provides a kit for detecting PD-L1 gene mutations in circulating tumor cells in the peripheral blood of patients with prostate cancer and a detection method: a membrane filter device is used to separate circulating tumor cells (CTC) from the peripheral blood of patients with advanced prostate cancer, and further use of immune cells Chemical technology detects the expression of PD-L1 on CTC.
- CTC circulating tumor cells
- a kit for detecting PD-L1 gene mutations in circulating tumor cells in the peripheral blood of patients with prostate cancer including diluent 45mL, decolorizing solution 1mL, staining solution A 0.5mL, staining solution B 1mL, anti-human PD-L1 primary antibody 100 ⁇ L, enzyme-labeled Goat anti-human secondary antibody 100 ⁇ L, 0.1% Triton X-100 100 ⁇ L, 0.3% H 2 O 2 100 ⁇ L, and reagent A 1 mL.
- the diluent is 1 mmol/L EDTA+0.1%BSA+0.3% iron sulfate+0.5% sucrose.
- the decolorizing liquid is composed of 95% alcohol and 100% xylene in a volume ratio of 1:1.
- the staining solution A is a DAB staining solution
- the staining solution B is a hematoxylin staining solution.
- the reagent A is composed of ethanol and paraformaldehyde in a volume ratio of 3:1.
- the method for detecting PD-L1 gene mutations in circulating tumor cells in the peripheral blood of a prostate cancer patient by using the kit for non-diagnostic purposes is characterized in that it comprises the following steps:
- Peripheral blood sample pretreatment Dilute the collected peripheral blood sample 10 times with diluent, add paraformaldehyde to fix the peripheral blood sample for 10 minutes after dilution, and fix the final concentration to 0.25%;
- the specific method for detecting the expression of PD-L1 of peripheral blood CTC in step (5) 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, blood cells can be filtered because the diameter is smaller than the filter hole 10, and the CTC is trapped on the filter membrane 7 because the diameter is larger than the filter hole 10.
- the detection method provided by the present invention can detect the expression of PD-L1 in patients with advanced or recurrent prostate cancer without obtaining tissue specimens by puncture biopsy, 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 the edge effect 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 of circulating tumor cells obtained from peripheral blood of a prostate cancer patient
- 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.
- FIG. 4 is an image of circulating tumor cells obtained from the peripheral blood of a prostate cancer patient.
- the nucleus is large, the shape of the nucleus is irregular, and the ratio of nucleus to cytoplasm is high.
- Reagent A is a mixed solution of ethanol and paraformaldehyde with a volume ratio of 3:1, and after being sealed with a neutral resin, the CTC cells are intact without shrinking or swelling. Other reagents or single reagents will be Failure to achieve this effect will affect the accuracy of the test.
- the detected circulating tumor cells were confirmed by immunohistochemistry to confirm the expression of PD-L1 and compared with the results of PD-L1 in the gross specimens of prostate cancer, to observe the differences, mainly for patients with negative expression of PD-L1 in gross specimens and positive expression of circulating tumor cells , To guide the targeted therapy of prostate cancer and provide new ideas for targeted therapy of prostate cancer.
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Abstract
Description
6×PBS缓冲液 | 60mL |
稀释液(1mmol/LEDTA+0.1%BSA+0.3%硫酸铁+0.5%蔗糖) | 45mL |
脱色液(95%酒精与100%二甲苯体积比1∶1) | 1mL |
染色液A(DAB染色液) | 0.5mL |
染色液B(苏木素染色液) | 1mL |
抗人PD-L1一抗 | 100μL |
酶标羊抗人二抗 | 100μL |
0.1%Triton X-100 | 100μL |
0.3%H 2O 2 | 100μL |
试剂A(乙醇和多聚甲醛体积比3∶1) | 1mL |
Claims (7)
- 一种检测前列腺癌患者外周血循环肿瘤细胞PD-L1基因突变的试剂盒,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、抗人PD-L1一抗100μL、酶标羊抗人二抗100μL、0.1%Triton X-100 100μL、0.3%H 2O 2 100μL、试剂A 1mL。
- 根据权利要求1所述的试剂盒,其特征在于,所述稀释液为1mmol/L EDTA+0.1%BSA+0.3%硫酸铁+0.5%蔗糖。
- 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。
- 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。
- 根据权利要求1所述的试剂盒,其特征在于,所述试剂A为乙醇和多聚甲醛按照体积比3:1组成。
- 一种利用权利要求1-5任一项所述的试剂盒非诊断目的检测前列腺癌患者外周血循环肿瘤细胞基因突变的方法,其特征在于,包括以下步骤:(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前列腺癌患者外周血:采集无法获取组织标本的晚期或复发前列腺癌患者肘正中静脉外周血5ml;(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%;(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;(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,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;(5)运用免疫组化技术检测外周血CTC的PD-L1表达情况。
- 根据权利要求6所述的检测方法,其特征在于,步骤(5)所述检测外周血CTC的PD-L1表达的具体方法如下:(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;(2)滴加100μl 0.1%Triton X-100,室温孵育15min,DI水洗2min×3次;(3)滴加100μl 0.3%H 2O 2,室温孵育10min,PBS洗2min×3次;(4)滴加100μl抗人PD-L1一抗,室温孵育2h或4℃过夜,PBS洗2min×3次;(5)滴加100μl酶标羊抗人二抗,18~26℃温度下孵育20min,PBS洗2min×3次;(6)滴加100μl DAB显色液,18~26℃孵育并随时在显微镜下观察显色情况,观察时间为3~10min;(7)显色完成后,弃掉DAB显色液,流水冲洗5min,苏木素染色5min;(8)盐酸酒精分化8秒,自来水返蓝5min;(9)将返蓝后的CTC采用75%乙醇(1min),95%乙醇(1min),100%乙醇(1min)梯度乙醇脱水,然后加入1mL试剂A,摇动混合均匀后,离心沉淀,将沉淀物采用中性树脂封固;(10)光学显微镜下镜检。
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