WO2018190382A1 - Méthode de détection de cellules cancéreuses pd-l1 positives - Google Patents

Méthode de détection de cellules cancéreuses pd-l1 positives Download PDF

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WO2018190382A1
WO2018190382A1 PCT/JP2018/015274 JP2018015274W WO2018190382A1 WO 2018190382 A1 WO2018190382 A1 WO 2018190382A1 JP 2018015274 W JP2018015274 W JP 2018015274W WO 2018190382 A1 WO2018190382 A1 WO 2018190382A1
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cells
fluorescent dye
antibody
recognizes
cell
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PCT/JP2018/015274
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English (en)
Japanese (ja)
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理美 八木
勝也 遠藤
雅之 樋口
清太 中村
上原　寿茂
泰浩 洪
信之 山本
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日立化成株式会社
公立大学法人和歌山県立医科大学
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Priority to JP2019512558A priority Critical patent/JPWO2018190382A1/ja
Publication of WO2018190382A1 publication Critical patent/WO2018190382A1/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
    • 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
    • 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

Definitions

  • the present invention relates to a method for detecting PD-L1-positive cancer cells.
  • Immuno checkpoint inhibition therapy is known as one of cancer treatment methods.
  • immune checkpoint inhibition therapy immune cells are activated by inhibiting reaction pathways (immune checkpoints) that suppress immune responses.
  • the PD-1 / PD-L1 pathway is known as a reaction pathway that suppresses the immune response, and the development of immune checkpoint inhibitors “pembrolizumab” and “nivolumab” that inhibit this pathway is progressing.
  • pembrolizumab and “nivolumab” that inhibit this pathway is progressing.
  • the efficacy of the inhibitor acting on the PD-1 / PD-L1 pathway is low. Therefore, it is important to confirm the efficacy of the inhibitor before actually administering the inhibitor by detecting PD-L1 in the cancer cells of the patient.
  • Non-patent document 1 As a method for detecting cancer cells expressing PD-L1 (PD-L1-positive cancer cells), a method of collecting a tissue sample from a patient and staining PD-L1 in the sample is known (for example, Non-patent document 1).
  • the conventional method for detecting PD-L1-positive cancer cells requires a large burden on the patient because it requires collecting tumor tissue from the patient.
  • An object of the present invention is to detect PD-L1-positive cancer cells without imposing a heavy burden on a subject.
  • the present invention is a method for detecting PD-L1-positive cancer cells in a blood sample, comprising: (a) collecting cells from the blood sample; and (b) a primary antibody that recognizes PD-L1 in the cells. Contacting and then contacting a secondary antibody that recognizes the primary antibody and labeled with a fluorescent dye, or a cell that is an antibody that recognizes PD-L1 and labeled with a fluorescent dye And (c) irradiating a cell with excitation light of a fluorescent dye to detect fluorescence emitted from the cell.
  • the fluorescent dye may be a first fluorescent dye, and at any stage after step (a) and before step (c), (x1) a cell is contacted with a primary antibody that recognizes a leukocyte marker protein. And then contacting a secondary antibody that recognizes the primary antibody and that is labeled with a second fluorescent dye, or an antibody that recognizes leukocytes and a second fluorescent dye (X2) contacting the cell with a primary antibody that recognizes a marker protein of epithelial cells, and then a secondary antibody that recognizes the primary antibody with a third fluorescent dye.
  • the cells are irradiated with excitation light of the first, second, third and fourth fluorescent dyes, respectively.
  • the fluorescence of the first, second, third, and fourth fluorescent dyes emitted from the cells may be detected, respectively.
  • the primary antibody recognizing PD-L1 or the antibody recognizing PD-L1 may be derived from a 28-8 or SP142 clone.
  • Step (a) may be a step of capturing a cell on a filter by filtering a blood sample with a filter.
  • the leukocyte marker protein may be CD45.
  • the epithelial cell marker protein may be cytokeratin.
  • the first, second, and third fluorescent dyes may be selected from the group consisting of fluorescein, Alexa Fluor (registered trademark) 594, and Alexa Fluor (registered trademark) 647, wherein the fourth fluorescent dye is 4 ', It may be 6-diamidino-2-phenylindole.
  • the PD-L1 positive cancer cell may be derived from lung cancer.
  • the step (x1) may be performed before the step (b), and the step (x2) and the step (x3) may be performed simultaneously after the step (b).
  • PD-L1-positive cancer cells can be detected easily and in a short time without imposing a heavy burden on the subject by using a blood sample.
  • FIG. 2 is a sectional view taken along line II-II in FIG. 2 is an image of cells fluorescently labeled in Example 1.
  • FIG. 2 is an image of fluorescently labeled cells in Example 2.
  • FIG. 2 is an image of fluorescently labeled cells in Example 2.
  • FIG. 4 is an image of cells fluorescently labeled in Example 3.
  • 4 is an image of cells fluorescently labeled in Example 3.
  • 4 is an image of cells fluorescently labeled in Example 4.
  • the method for detecting PD-L1-positive cancer cells in a blood sample comprises (a) a step of collecting cells from a blood sample, and (b) contacting the cells with a primary antibody that recognizes PD-L1. Next, a step of bringing a secondary antibody that recognizes the primary antibody into contact with a secondary antibody that is labeled with a fluorescent dye, or a cell that is an antibody that recognizes PD-L1 and is labeled with a fluorescent dye A step of bringing the antibody into contact, and (c) a step of irradiating the cell with excitation light of a fluorescent dye to detect fluorescence emitted from the cell.
  • CTC circulating tumor cells
  • CTCs are those in which cancer cells in the lungs, liver, stomach, head and neck, bladder, urothelium, esophagus, biliary tract, breast, ovary, uterus, liver, prostate, or pancreas have entered blood vessels and lymph vessels.
  • PD-L1-positive cancer cells can be detected by using a blood sample of a subject without collecting cancer tissue in these organs.
  • CTC and “cancer cells in a blood sample” are treated as synonymous.
  • blood collected from a subject may be used as it is, or blood diluted with a buffer solution such as phosphate buffered saline (PBS) or other suitable medium may be used.
  • PBS phosphate buffered saline
  • the blood sample may be added with additives that are usually added to blood samples, such as anticoagulants and fixatives.
  • the cells can be collected from the blood sample by, for example, filtering the blood sample with a filter and capturing the cells in the blood sample on the filter.
  • leukocytes have the same diameter as CTC, so that some leukocytes are captured together with CTC on the filter.
  • detection of PD-L1-positive cancer cells can be performed on the filter as it is. That is, all the steps in the present invention can be performed on the cells captured on the filter.
  • Capture means that the liquid containing the cells is filtered through, leaving the cells on the filter.
  • the filter is not particularly limited as long as it can capture CTC present in the blood sample, and a conventionally known filter can be used.
  • the filter may be, for example, a metal or resin filter, and is provided with a substrate and a through-hole provided on the substrate, preferably having a pore diameter of 5 ⁇ m to 15 ⁇ m, more preferably 6 ⁇ m to 12 ⁇ m, and even more preferably 7 ⁇ m to 10 ⁇ m. You may have.
  • the hole diameter of the through hole refers to the maximum value of the diameter of a sphere that can pass through the through hole.
  • contacting a substance with a cell can be performed, for example, by immersing the cell in the substance or a solution of the substance.
  • cleaning liquid with a cell can be performed by filtering these solutions with a filter.
  • the flow rate of the solution is preferably 50 ⁇ L / min to 3000 ⁇ L / min, more preferably 100 ⁇ L / min to 1000 ⁇ L / min, and 200 ⁇ L / min to 600 ⁇ L / min. Further preferred.
  • the cells may be washed.
  • the washing step is performed, for example, by bringing a washing solution containing a known buffer solution such as PBS into contact with the cells.
  • the washing solution may contain additives such as bovine serum albumin (BSA) or ethylenediaminetetraacetic acid (EDTA). Washing is not limited to after step (a), and can be performed appropriately after each step.
  • BSA bovine serum albumin
  • EDTA ethylenediaminetetraacetic acid
  • cells may be immobilized after step (a).
  • the cells can be fixed by contacting the cells with a known fixing agent such as formaldehyde. By fixing the cells, cell spoilage or aggregation can be further reduced.
  • the immobilized cells may then be permeabilized.
  • a cell can be permeabilized by contacting the cell with a known permeabilizing agent.
  • a permeation treatment agent for example, poly (oxyethylene) octylphenyl ether can be used.
  • a secondary antibody that recognizes the primary antibody is contacted with a primary antibody that recognizes PD-L1, and is then labeled with a fluorescent dye (first fluorescent dye). Is contacted (two-step fluorescent labeling).
  • an antibody that recognizes PD-L1 and is labeled with a fluorescent dye (first fluorescent dye) is brought into contact with cells (one-step fluorescent labeling).
  • PD-L1 fluorescent labeling may be performed in two steps or one step.
  • the primary antibody that recognizes PD-L1 or the antibody that recognizes PD-L1 and is labeled with a fluorescent dye is, for example, 28-8, SP142, E1L3N (registered trademark), And a clone selected from the group consisting of EPR1161 (2), or a polyclonal antibody (for example, catalog number: 4059 from Prosci).
  • a fluorescent dye for example, 28-8, SP142, E1L3N (registered trademark)
  • a clone selected from the group consisting of EPR1161 (2), or a polyclonal antibody for example, catalog number: 4059 from Prosci.
  • the primary antibody that recognizes PD-L1 or the antibody that recognizes PD-L1 is preferably derived from 28-8 or SP142.
  • the antibodies derived from these clones are all anti-PD-L1 rabbit monoclonal antibodies.
  • the fluorescent dye is not particularly limited as long as it is a fluorescent dye usually used for fluorescent labeling of antibodies.
  • the first fluorescent dye is, for example, Alexa Fluor (registered trademark) 647 or Cy (registered trademark) 5.
  • step (c) the cells are irradiated with excitation light of a fluorescent dye to detect fluorescence emitted from the cells.
  • a cell in which fluorescence due to the fluorescent dye (first fluorescent dye) is detected (positive) is identified as a PD-L1-positive cancer cell.
  • the detected PD-L1-positive cancer cells can then be analyzed for DNA, RNA or protein.
  • sequencer, next-generation sequencer, DNA chip, microarray, comparative genomic hybridization, fluorescence in situ hybridization, digital PCR, quantitative reverse transcription PCR, ELISA, Western plotting, TOF -MS, MALDI-MS, Raman spectroscopy, chromatography, X-ray crystallography, two-dimensional electrophoresis, nuclear magnetic resonance spectroscopy, flow cytometer (FCM), etc. can be used for analysis.
  • step (b) an antibody recognizing PD-L1 binds to PD-L1-negative cells, and fluorescence indicating PD-L1 may be observed from PD-L1-negative cells (false positive). ). From the viewpoint of reducing such false positives and obtaining a more reliable detection result, the following steps (x1) to (x3) are performed at any stage after step (a) and before step (c). Further, it is preferable to carry out.
  • a cell is contacted with a primary antibody that recognizes a marker protein of leukocytes, and then a secondary antibody that recognizes the primary antibody and is labeled with a second fluorescent dye is contacted (Two-step fluorescent labeling).
  • a secondary antibody that recognizes the primary antibody and is labeled with a second fluorescent dye is contacted.
  • cells are contacted with an antibody that recognizes leukocytes and is labeled with a second fluorescent dye (one-step fluorescent labeling).
  • leukocytes are fluorescently labeled.
  • the fluorescent labeling of leukocytes may be performed in either two steps or one step as described above.
  • Leukocyte marker protein is, for example, CD45 expressed in all hematopoietic stem cells.
  • a primary antibody that recognizes a leukocyte marker protein, a secondary antibody that is labeled with a second fluorescent dye, and an antibody that recognizes a leukocyte marker protein and is labeled with a second fluorescent dye It is not limited, A polyclonal antibody or a monoclonal antibody may be sufficient.
  • the animal from which the antibody is derived is not particularly limited as long as the animal from which the primary antibody is derived is different from the animal from which the secondary antibody is derived.
  • the second fluorescent dye is not particularly limited as long as it is a fluorescent dye usually used for fluorescent labeling of antibodies.
  • the second fluorescent dye is, for example, Alexa Fluor (registered trademark) 594 or Texas Red (registered trademark).
  • the second fluorescent dye is a fluorescent dye different from the first, third and fourth fluorescent dyes. Each fluorescent dye is distinguishable because it has a different fluorescence wavelength.
  • the first, second, and third fluorescent dyes are selected from the group consisting of fluorescein, Alexa Fluor 594, and Alexa Fluor 647, and the fourth fluorescent dye is 4 ', 6-diamidino-2-phenyl. Indole (DAPI).
  • step (x2) the cell is contacted with a primary antibody that recognizes a marker protein of epithelial cells, and then a secondary antibody that recognizes the primary antibody and is labeled with a third fluorescent dye is contacted (Two-step fluorescent labeling).
  • a secondary antibody that recognizes the primary antibody and is labeled with a third fluorescent dye is contacted.
  • cells are contacted with an antibody that recognizes a marker protein of epithelial cells and labeled with a third fluorescent dye (one-step fluorescent labeling).
  • CTC is fluorescently labeled.
  • CTC fluorescent labeling may be performed in either two steps or one step as described above.
  • epithelial cell marker proteins examples include cytokeratin, epithelial cell adhesion molecule (EpCAM), CD146, and CD176, with cytokeratin being preferred. Since CTC is derived from epithelial cells, it has a marker protein for these epithelial cells.
  • the third fluorescent dye is not particularly limited as long as it is a fluorescent dye usually used for fluorescent labeling of antibodies.
  • the third fluorescent dye is, for example, fluorescein such as fluorescein isothiocyanate (FITC) or Alexa Fluor (registered trademark) 488.
  • a primary antibody that recognizes a marker protein of epithelial cells, a secondary antibody that is labeled with a third fluorescent dye, and an antibody that recognizes a marker protein of epithelial cells and is labeled with a third fluorescent dye may be a polyclonal antibody or a monoclonal antibody.
  • the animal from which the antibody is derived is not particularly limited as long as the animal from which the primary antibody is derived is different from the animal from which the secondary antibody is derived.
  • the cell nucleus is labeled with a fourth fluorescent dye.
  • the fourth fluorescent dye for labeling the nucleus is not particularly limited as long as it is a fluorescent dye capable of binding to a nucleic acid, and a fluorescent dye usually used for fluorescently labeling a nucleus can be used.
  • Examples of the fourth fluorescent dye include DAPI and 2 ′-(4-ethoxyphenyl) -5- (4-methyl-1-piperazinyl) -2,5′-bi-1H-benzimidazole trihydrochloride (Hoechst 33342). ).
  • step (c) the cells are irradiated with excitation light of the first, second, third and fourth fluorescent dyes, respectively. Fluorescence of the first, second, third and fourth fluorescent dyes emitted from is detected respectively. PD-L1-positive cancer cells are labeled with the first, third, and fourth fluorescent dyes, but are not labeled with the second fluorescent dye. Therefore, cells in which the fluorescence of the second fluorescent dye is not detected (negative) and the fluorescence of the first, third, and fourth fluorescent dyes are detected (positive) are identified as PD-L1-positive cancer cells.
  • the cartridge shown in FIGS. 1 and 2 can be used.
  • a method for detecting PD-L1-positive cancer cells in a blood sample using a cartridge according to an embodiment of the present invention will be described. Unless otherwise stated, the details of each step and the order of the steps are as described in the above embodiment.
  • a CTC capturing cartridge (cartridge) 100 shown in FIGS. 1 and 2 has a housing having an inlet 130 to which an inflow pipe 125 into which liquid flows is connected and an outlet 140 to which an outflow pipe 135 from which liquid flows out is connected.
  • a body 120 and a filter 105 are provided.
  • the filter 105 is fixed by a casing 120 including an upper member 110 and a lower member 115.
  • the blood sample, the cleaning liquid, and other reaction liquids are introduced into the housing 120 through the inflow pipe 125, and are discharged to the outside through the filter 105 through the outflow pipe 135.
  • Such a liquid flow can be created, for example, by connecting a pump upstream of the inflow pipe 125 or downstream of the outflow pipe 135.
  • a cock may be provided upstream of the inflow pipe 125 and / or downstream of the outflow pipe 135 to control the flow of the liquid.
  • a blood sample is introduced into the cartridge 100 from the inflow tube 125, and the blood sample is filtered by the filter 105 (step (a)).
  • CTC and some white blood cells in the blood sample cannot pass through the through hole 106 of the filter 105 and remain on the surface of the filter 105.
  • Other components in the blood sample pass through the through hole 106 and are discharged out of the cartridge 100.
  • the filter 105 may be cleaned by passing a cleaning solution through the filter 105.
  • the filter 105 can be appropriately washed after the following steps.
  • a reaction solution containing a fixing agent and then a permeabilizing agent is optionally introduced into the cartridge 100, and held in the cartridge 100 for a predetermined time.
  • a fixing agent and a permeation treatment agent may be reacted with each other.
  • reaction solution containing a primary antibody that recognizes PD-L1 and then a secondary antibody that recognizes the primary antibody and is labeled with a fluorescent dye (first fluorescent dye) are applied to the cells.
  • the contained reaction solution is reacted with the cells captured on the filter 105.
  • a reaction solution containing an antibody that recognizes PD-L1 and is labeled with a fluorescent dye (first fluorescent dye) is allowed to react with the cells captured on the filter 105 (step (step ( b)).
  • the cartridge 100 is irradiated with excitation light of a fluorescent dye using a fluorescence microscope to detect fluorescence emitted from the cells captured on the filter 105 (step (c)).
  • the fluorescence is detected by, for example, observing the cartridge 100 from the upper surface in the vertical direction of the cartridge 100 and processing the fluorescence observation image.
  • the steps (x1) to (x3) can optionally be further performed.
  • Example 1 The non-small cell lung cancer cell line contained in the culture flask was cultured at 37 ° C. in a carbon dioxide incubator. Trypsin-EDTA with a concentration of 0.25% was added to the culture flask, and the cultured cells attached to the flask were detached from the flask. The detached cells were counted using a hemocytometer and a phase contrast microscope. A blood sample in which the blood of a lung cancer patient was sprinkled was prepared by adding 100 cells to the blood of a healthy person collected in a blood collection tube.
  • NCI-H820 high expression of PD-L1
  • NCI-H441 expressed in PD-L1
  • A549 low expression of PD-L1
  • NCI-H23 differed in the expression level of PD-L1, respectively.
  • Four types of blood samples were prepared using four types (PD-L1 negative).
  • a blood collection tube a blood collection tube containing EDTA-2K (ethylenediaminetetraacetic acid dipotassium salt) manufactured by Becton Dickinson & Company was used.
  • the CTC capture device includes a reservoir for introducing a blood sample and other reaction solution, and a CTC capture cartridge.
  • the CTC capture cartridge (hereinafter also referred to as cartridge) includes a thin-film metal filter (membrane area 6 mm ⁇ 6 mm, film thickness 18 ⁇ m) having a large number of through-holes having a major axis of 100 ⁇ m and a minor axis of 8 ⁇ m. This corresponds to the cartridge 100.
  • the cartridge was filled with a PBS solution containing 0.5% BSA and 2 mM EDTA (hereinafter referred to as “cleaning solution”). 7 mL of the washing solution was placed in the reservoir, and 3 mL of the blood sample was added under the washing solution so that the blood sample and the washing solution were layered.
  • the CTC capture device was activated, the blood sample and the washing solution in the reservoir were introduced into the cartridge at a flow rate of 200 ⁇ L / min, and the cells in the blood sample were captured on the filter.
  • a washing solution was introduced into the cartridge to wash away blood components remaining on the filter.
  • a reaction solution containing 1.25 mL of anti-human CD45 mouse monoclonal antibody (clone: 2D1) was introduced into the cartridge at a flow rate of 200 ⁇ L / min and reacted at room temperature for 30 minutes.
  • 1.40 mL of the washing solution was introduced into the cartridge at a flow rate of 400 ⁇ L / min, and the reaction solution in the cartridge was discharged.
  • a reaction solution containing 1.25 mL of Alexa Fluor 594-labeled anti-mouse IgG goat polyclonal antibody was introduced into the cartridge at a flow rate of 400 ⁇ L / min and reacted at room temperature for 30 minutes.
  • 1.40 mL of the washing solution was introduced into the cartridge at a flow rate of 400 ⁇ L / min, and the reaction solution in the cartridge was discharged.
  • a reaction solution containing 1.25 mL of anti-human PD-L1 rabbit monoclonal antibody (clone: 28-8) was introduced into the cartridge at a flow rate of 200 ⁇ L / min and reacted at room temperature for 60 minutes. 1.40 mL of the washing solution was introduced into the cartridge at a flow rate of 400 ⁇ L / min, and the reaction solution in the cartridge was discharged.
  • a reaction solution containing 1.25 mL of Alexa Fluor 647-labeled anti-rabbit IgG goat polyclonal antibody was introduced into the cartridge at a flow rate of 400 ⁇ L / min and reacted at room temperature for 30 minutes. 1.40 mL of the washing solution was introduced into the cartridge at a flow rate of 400 ⁇ L / min, and the reaction solution in the cartridge was discharged.
  • a reaction solution containing FITC-labeled anti-human cytokeratin mouse monoclonal antibody (clone: mixture of CK3, 6H5, AE1, and AE3) and DAPI is introduced into the cartridge at 400 ⁇ L / min and reacted at room temperature for 30 minutes. I let you. 3.00 mL of the cleaning solution was introduced into the cartridge at a flow rate of 400 ⁇ L / min, and the reaction solution in the cartridge was discharged. The cartridge was then removed from the CTC capture device.
  • the cartridge was set on a fluorescence microscope. Fluorescent mirror units (FITC, Alexa Fluor 594, Alexa Fluor 647, and DAPI) were each excited using a fluorescent mirror unit. The fluorescence emitted from each fluorescent dye was photographed, and the resulting images were synthesized.
  • Fluorescent mirror units FITC, Alexa Fluor 594, Alexa Fluor 647, and DAPI
  • H820 means NCI-H820
  • H441 means NCI-H441
  • H23 means NCI-H23 (hereinafter the same).
  • NCI-H820 and NCI-H441 which are PD-L1-positive cancer cell lines
  • the cell nucleus (DAPI), cytokeratin (FITC), and PD-L1 are positive
  • FITC cytokeratin
  • PD-L1 Alexa Fluor 647
  • a fluorescent image of cells negative for CD45 Alexa Fluor 594
  • NCI-H23 which is a PD-L1-negative cancer cell line
  • fluorescence images of cells positive for cell nuclei and cytokeratin and negative for CD45 and PD-L1 were obtained.
  • Example 2 NCI-H820 was used as a cell line. Cell fluorescence was observed in the same manner as in Example 1 except that the clone of the anti-human PD-L1 rabbit monoclonal antibody was changed to SP142.
  • FIGS. As shown in these figures, fluorescence images of cells positive for cell nuclei, cytokeratin, and PD-L1, and negative for CD45 were obtained. In the lower right of FIG. 5, leukocytes that are positive for cell nuclei and CD45, and negative for cytokeratin and PD-L1 are seen. There were no false positives due to nonspecific binding of antibodies.
  • Example 3 NCI-H820 was used as a cell line.
  • FIG. 7 shows that non-specific binding of the antibody was observed in the experiment using any antibody.
  • Example 4 Cell fluorescence was observed in the same manner as in Example 1 except that the labeling dye of the secondary antibody (anti-rabbit IgG goat polyclonal antibody) against PD-L1 was changed to Alexa Fluor 680 and Alexa Fluor (registered trademark) 700. .

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Abstract

Cette méthode de détection de cellules cancéreuses PD-L1 positives comprend : (a) une étape de collecte de cellules à partir d'un échantillon sanguin ; (b) une étape destinée à mettre les cellules en contact avec un anticorps primaire capable de reconnaître PD-L1 et à mettre en outre les cellules en contact avec un anticorps secondaire qui est capable de reconnaître l'anticorps primaire et qui est marqué avec un colorant fluorescent, ou une étape destinée à mettre en contact des cellules avec un anticorps capable de reconnaître PD-L1 et marqué par un colorant fluorescent ; et (c) une étape consistant à soumettre à un rayonnement les cellules avec une lumière d'excitation pour le colorant fluorescent de sorte à détecter la fluorescence émise par les cellules. Ce procédé permet de détecter une cellule cancéreuse PD-L1 positive sans imposer de charge trop importante à un sujet faisant l'objet du test.
PCT/JP2018/015274 2017-04-13 2018-04-11 Méthode de détection de cellules cancéreuses pd-l1 positives WO2018190382A1 (fr)

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JP2016086736A (ja) * 2014-11-05 2016-05-23 日立化成株式会社 血中希少細胞含有液の製造方法
WO2018029858A1 (fr) * 2016-08-12 2018-02-15 日立化成株式会社 Procédé de détection de cellules cancéreuses circulantes dans le sang, et procédé de prétraitement destiné à détecter des cellules cancéreuses circulantes dans le sang
KR20180048215A (ko) * 2016-11-02 2018-05-10 주식회사 싸이토젠 혈중 순환 암세포를 이용한 pd-l1 타겟 면역치료법을 위한 암 환자 선별 방법

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