WO2021213262A1 - Kit de test d'immunofluorescence pour mesurer l'expression de pd-l1 dans des cellules tumorales circulantes dans le sang périphérique d'un patient atteint d'un cancer de l'estomac et procédé de mesure - Google Patents

Kit de test d'immunofluorescence pour mesurer l'expression de pd-l1 dans des cellules tumorales circulantes dans le sang périphérique d'un patient atteint d'un cancer de l'estomac et procédé de mesure Download PDF

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WO2021213262A1
WO2021213262A1 PCT/CN2021/087721 CN2021087721W WO2021213262A1 WO 2021213262 A1 WO2021213262 A1 WO 2021213262A1 CN 2021087721 W CN2021087721 W CN 2021087721W WO 2021213262 A1 WO2021213262 A1 WO 2021213262A1
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peripheral blood
tumor cells
expression
filter
circulating tumor
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PCT/CN2021/087721
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English (en)
Chinese (zh)
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李胜
李�浩
崔凯
夏梅
高德海
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山东第一医科大学(山东省医学科学院)
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Publication of WO2021213262A1 publication Critical patent/WO2021213262A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70532—B7 molecules, e.g. CD80, CD86

Definitions

  • the invention provides a kit and a detection method for detecting the immunofluorescence expression of PD-L1 in peripheral blood circulating tumor cells of patients with gastric cancer, and belongs to the technical field of molecular biology.
  • Gastric cancer is a highly fatal malignant tumor with the fourth highest incidence in the world (950,000 new cases per year) and the third highest among cancer-related deaths in 2012.
  • the 5-year survival rate of gastric cancer patients is less than 30%, and about 50% of gastric cancer patients suffer tumor recurrence or metastasis after curative resection. Tumor recurrence and metastasis are the main causes of death in patients with gastric cancer.
  • 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 immunotherapy with PD-1/PD-L1 as an immune target has brought a new light to the treatment of gastric cancer.
  • immune suppression is closely related to immune escape and the overexpression of PD-L1 in tumor cells.
  • Tumor cells can bind to PD-1 on the surface of immune cells and T cells through PD-L1 on their surface, and conduct inhibitory signals to make T cells
  • the inability to recognize tumor cells and send out attack signals to tumor cells leads to immune escape of tumor cells.
  • CTC circulating tumor cells
  • 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 gastric cancer tissue is positively correlated with tumor aggressiveness.
  • CTC circulating tumor cell
  • 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 PD-L1 test specimens for gastric cancer patients are mainly tumor tissues, which are derived from surgery or needle biopsy, and it is difficult to perform multiple or real-time detections. Therefore, detecting the expression of circulating tumor cells (CTC) PD-L1 is of great value for the prognosis of gastric 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.
  • an immunofluorescence kit and detection method for PD-L1 expression in circulating tumor cells in the peripheral blood of patients with gastric cancer are provided. Circulating tumor cells (CTC) in the peripheral blood of patients with advanced gastric cancer are separated by a membrane filtration device, and CTCs are further detected by immunofluorescence technology. The expression of PD-L1 on the top.
  • An immunofluorescence kit for detecting PD-L1 expression in peripheral blood circulating tumor cells of gastric cancer patients including diluent 45mL, decolorizing solution 1mL, staining solution A 0.5mL, staining solution B 1mL, 200 ⁇ l methanol, 200 ⁇ l 2% PFA, 100 ⁇ l 10% goat Serum, 100 ⁇ l of primary antibody suspension composed of mouse anti-CK, rat anti-CD45 and rabbit anti-PD-L1, composed of fluorescent-labeled goat anti-mouse, fluorescent-labeled goat anti-rat, and fluorescent-labeled goat anti-rabbit 100 ⁇ L of secondary antibody suspension, DAPI mounting plate;
  • mice anti-CK, rat anti-CD45 and rabbit anti-PD-L1 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 are diluted 1:500.
  • the composition of the diluent is: 1 mmol/L EDTA+0.1% BSA+0.1% trehalose+0.2% tyrosine+PBS buffer 150 mmol/L, wherein the percentage is a volume ratio.
  • 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 method for detecting PD-L1 expression of circulating tumor cells in peripheral blood of patients with gastric cancer for non-diagnostic purposes of the kit includes the following steps:
  • the specific method for detecting the expression of PD-L1 of peripheral blood CTC in step (7) 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 expression of PD-L1 in patients with advanced or recurrent gastric 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 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 shows the PD-L1 immunofluorescence staining image of circulating tumor cells in the peripheral blood of patients with advanced gastric cancer.
  • the primary antibody suspension is composed of mouse anti-CK, rat anti-CD45 and rabbit anti-PD-L1.
  • Mouse anti-CK, rat anti-CD45 and rabbit anti-PD-L1 are respectively used with BD wash buffer at 1:100, Dilute 1:500 and 1:400. After dilution, take 10 ⁇ L mouse anti-CK, 50 ⁇ L rat anti-CD45 and 40 ⁇ L rabbit anti-PD-L1 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 respectively 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.
  • Figure 4 is an immunofluorescence staining image of circulating tumor cells in the peripheral blood of patients with advanced hepatocellular carcinoma. According to immunological and morphological findings, it is found that the tumor cells are large in size and have abnormal nucleus-to-cytoplasmic ratios. The immunological features are typical CTCs. The detected circulating tumor cells were confirmed with immunofluorescence to confirm the expression of PD-L1 and compared with the results of PD-L1 in gross specimens of gastric cancer, to observe the differences, mainly for patients with negative expression of PD-L1 in gross specimens and positive expression of circulating tumor cells. Targeted therapy of gastric cancer provides new ideas for targeted therapy of gastric cancer.

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Abstract

La présente invention concerne un kit de test d'immunofluorescence pour mesurer l'expression de PD-L1 dans des cellules tumorales circulantes dans le sang périphérique d'un patient atteint d'un cancer de l'estomac et un procédé de mesure. Un dispositif de filtration à membrane est utilisé pour séparer et acquérir des cellules tumorales circulantes (CTC) à partir du sang périphérique d'un patient atteint d'un cancer de l'estomac de stade avancé et une technologie d'immunofluorescence est en outre utilisée pour mesurer l'expression de PD-L1 dans les CTC. Selon le procédé de mesure de la présente invention, l'expression de PD-L1 chez un patient atteint d'un cancer de l'estomac de stade avancé ou en rechute peut être mesurée sans l'utilisation d'une biopsie par ponction pour prélever un échantillon de tissu. Le procédé selon la présente invention présente une bonne séparation des cellules tumorales circulantes, est capable d'éviter une interférence de cellules sanguines, est capable d'éviter des faux positifs provoqués par des effets de bord éventuellement produits pendant un processus de coloration, et présente une bonne stabilité, une réduction de la perte de cellules et une précision de mesure améliorée. Le marqueur PD-L1 présente une excellente spécificité et une excellente sensibilité lors de l'utilisation, et son application au cancer de l'estomac a été améliorée.
PCT/CN2021/087721 2020-04-20 2021-04-16 Kit de test d'immunofluorescence pour mesurer l'expression de pd-l1 dans des cellules tumorales circulantes dans le sang périphérique d'un patient atteint d'un cancer de l'estomac et procédé de mesure WO2021213262A1 (fr)

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Cited By (1)

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CN115184620A (zh) * 2022-09-14 2022-10-14 山东子峰生物技术有限公司 一种pla2r抗体的量子点荧光检测试纸条、试剂盒及其应用

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CN111562375B (zh) * 2020-04-20 2022-05-06 山东第一医科大学(山东省医学科学院) 一种检测胃癌患者外周血循环肿瘤细胞pd-l1表达的免疫荧光试剂盒及检测方法
CN115792245A (zh) * 2023-02-02 2023-03-14 北京市肿瘤防治研究所 一种用于同时对外周血胃癌循环肿瘤细胞活性和免疫状态进行检测的方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115184620A (zh) * 2022-09-14 2022-10-14 山东子峰生物技术有限公司 一种pla2r抗体的量子点荧光检测试纸条、试剂盒及其应用
CN115184620B (zh) * 2022-09-14 2023-01-24 山东子峰生物技术有限公司 一种pla2r抗体的量子点荧光检测试纸条、试剂盒及其应用

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