WO2018190382A1 - Method for detecting pd-l1-positive cancer cells - Google Patents

Abstract

This PD-L1-positive cancer cell detection method comprises: (a) a step for collecting cells from a blood sample; (b) a step for bringing the cells into contact with a primary antibody capable of recognizing PD-L1 and further bringing the cells into contact with a secondary antibody which is capable of recognizing the primary antibody and which is labeled with a fluorescent dye, or a step for bringing the cells into contact with an antibody capable of recognizing PD-L1 and labeled with a fluorescent dye; and (c) a step for irradiating the cells with excitation light for the fluorescent dye so as to detect fluorescence emitted from the cells. This method enables detecting a PD-L1-positive cancer cell without imposing too much burden on a test subject.

Description

Method for detecting PD-L1-positive cancer cells

The present invention relates to a method for detecting PD-L1-positive cancer cells.

“Immune checkpoint inhibition therapy” is known as one of cancer treatment methods. In 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. When the patient's cancer cells do not have PD-L1, 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.

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. A step of contacting a labeled secondary antibody, or a step of contacting a cell with an antibody that recognizes an epithelial cell marker protein and labeled with a third fluorescent dye, and (x3) a cell The nucleus of the fourth And a step of labeling with a fluorescent dye in any order. In step (c), 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).

According to the method of the present invention, 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.

It is a perspective view which shows one Embodiment of a cell capture | acquisition cartridge. 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 according to the present invention 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. In the blood of cancer patients, there are cases where cancer cells circulating in the body through blood vessels and lymph vessels are called circulating tumor cells (hereinafter also referred to as “CTC”). 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. . Therefore, PD-L1-positive cancer cells (PD-L1-positive CTC) can be detected by using a blood sample of a subject without collecting cancer tissue in these organs. In this specification, “CTC” and “cancer cells in a blood sample” are treated as synonymous.

As a blood sample, 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. The blood sample may be added with additives that are usually added to blood samples, such as anticoagulants and fixatives.

In step (a), 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. Among cells contained in blood, leukocytes have the same diameter as CTC, so that some leukocytes are captured together with CTC on the filter. When cells in a blood sample are collected by a 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 μm to 15 μm, more preferably 6 μm to 12 μm, and even more preferably 7 μm to 10 μm. 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.

In this specification, “contacting” a substance with a cell can be performed, for example, by immersing the cell in the substance or a solution of the substance. When using a filter for collection | recovery of a cell, contacting a reaction liquid or a washing | cleaning liquid with a cell can be performed by filtering these solutions with a filter. From the viewpoint of minimizing damage to cells during filtration, the flow rate of the solution is preferably 50 μL / min to 3000 μL / min, more preferably 100 μL / min to 1000 μL / min, and 200 μL / min to 600 μL / min. Further preferred.

After the step (a), 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.

Furthermore, 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. As the permeation treatment agent, for example, poly (oxyethylene) octylphenyl ether can be used.

In the step (b), 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). Alternatively, 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). Through this step, PD-L1 is fluorescently labeled. As described above, 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 (first 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). From the viewpoint of detecting PD-L1-positive cancer cells with higher sensitivity, the primary antibody that recognizes PD-L1 or the antibody that recognizes PD-L1 is preferably derived from 28-8 or SP142. In addition, by using an antibody derived from 28-8 or an antibody derived from SP142, non-specific binding of the antibody can be reduced, and thus a more reliable detection result with fewer false positives. Obtainable. The antibodies derived from these clones are all anti-PD-L1 rabbit monoclonal antibodies.

The fluorescent dye (first 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.

Finally, in 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. For example, 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.

In addition to PD-L1-positive cancer cells, there are many other cells such as PD-L1-negative cancer cells and leukocytes in the blood sample. Therefore, in 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.

In the step (x1), 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). Alternatively, cells are contacted with an antibody that recognizes leukocytes and is labeled with a second fluorescent dye (one-step fluorescent labeling). By this step, 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. Preferably, 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).

In 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). Alternatively, 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). By this step, CTC is fluorescently labeled. CTC fluorescent labeling may be performed in either two steps or one step as described above.

Examples of epithelial cell marker proteins 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 Without being particularly limited, it 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.

In step (x3), 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). ).

When these optional steps (x1) to (x3) are performed, in 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

When detecting PD-L1-positive cancer cells in a blood sample by the above method, for example, the cartridge shown in FIGS. 1 and 2 can be used. Hereinafter, 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. Further, 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.

First, 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. Next, 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.

Further, after the cells are captured on the filter 105, 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.

Similarly, a 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. Alternatively, 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)).

Finally, 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. As described in the above embodiment, 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. As cell lines, NCI-H820 (high expression of PD-L1), NCI-H441 (expressed in PD-L1), A549 (low expression of PD-L1), and NCI-H23 differed in the expression level of PD-L1, respectively. Four types of blood samples were prepared using four types (PD-L1 negative). As a blood collection tube, a blood collection tube containing EDTA-2K (ethylenediaminetetraacetic acid dipotassium salt) manufactured by Becton Dickinson & Company was used.

Within one hour after blood collection, PD-L1-positive cancer cells in the above four blood samples were detected using a CTC capture device as follows. 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 μm) having a large number of through-holes having a major axis of 100 μm and a minor axis of 8 μm. This corresponds to the cartridge 100.

First, 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 μL / 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 μL / 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 μL / 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 μL / 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 μL / min, and the reaction solution in the cartridge was discharged.

1.25 mL of PBS solution containing formaldehyde in an amount of 0.5% to 4% by mass was introduced into the cartridge at a flow rate of 400 μL / min and reacted at room temperature for 10 minutes to immobilize the cells. 1.40 mL of the washing solution was introduced into the cartridge at a flow rate of 400 μL / min, and the reaction solution in the cartridge was discharged.

By introducing 1.25 mL of a PBS solution containing 0.05 mass% to 0.1 mass% of Triton X-100 (manufactured by Sigma Aldrich) into the cartridge at a flow rate of 400 μL / min and reacting at room temperature for 10 minutes. The cells were permeabilized. 1.40 mL of the washing solution was introduced into the cartridge at a flow rate of 400 μL / 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 μL / 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 μL / 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 μL / 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 μL / min, and the reaction solution in the cartridge was discharged.

1.25 mL of 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 μL / 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 μL / 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.

The results are shown in FIG. In the figure, H820 means NCI-H820, H441 means NCI-H441, and H23 means NCI-H23 (hereinafter the same). In experiments using NCI-H820 and NCI-H441, which are PD-L1-positive cancer cell lines, the cell nucleus (DAPI), cytokeratin (FITC), and PD-L1 (Alexa Fluor 647) are positive, and , A fluorescent image of cells negative for CD45 (Alexa Fluor 594) was obtained. In an experiment using 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. On the other hand, in each experiment, a leukocyte fluorescence image in which cell nuclei and CD45 were positive and cytokeratin and PD-L1 were negative was also obtained (not shown). There were no false positives due to nonspecific binding of antibodies. Note that “positive” means that fluorescence was detected, and “negative” means that fluorescence was not detected.

(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.

The results are shown in 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. The same as Example 1 except that the anti-human PD-L1 rabbit monoclonal antibody was changed to an antibody derived from E1L3N (registered trademark), an antibody derived from EPR1161 (2), and a polyclonal antibody (manufactured by Prosci, catalog number: 4059). The fluorescence of the cells was observed by this method. The results are shown in FIGS.

As shown in FIG. 6 and FIG. 7, in the experiments using any of the antibodies, fluorescence images of cells that were positive for cell nucleus, cytokeratin, and PD-L1, and negative for CD45 were obtained. The fluorescence intensity of PD-L1 was high in the experiment using the antibody derived from E1L3N and low in the experiment using the polyclonal antibody. In an experiment using an antibody derived from EPR1161 (2), the fluorescence of PD-L1 was only slightly confirmed.

As shown in FIG. 7, in the experiment using any of the antibodies, cell nuclei and white blood cells that are positive for CD45 and negative for cytokeratin and PD-L1 are observed. On the other hand, FIG. 7 also 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. .

The results are shown in FIG. When Alexa Fluor 680 and Alexa Fluor 700 were used, the fluorescence intensity of PD-L1 was low.

DESCRIPTION OF SYMBOLS 100 ... CTC capture cartridge, 105 ... Filter, 106 ... Through hole, 110 ... Upper member, 115 ... Lower member, 120 ... Housing, 125 ... Inflow pipe, 130 ... Inlet, 135 ... Outlet, 140 ... Outlet

Claims (9)

1. A method for detecting PD-L1-positive cancer cells in a blood sample, comprising:
   (A) collecting cells from a blood sample;
   (B) contacting a cell with a primary antibody that recognizes PD-L1, and then contacting a secondary antibody that recognizes the primary antibody and labeled with a fluorescent dye, or the cell, Contacting an antibody that recognizes PD-L1 and labeled with a fluorescent dye;
   (C) irradiating a cell with excitation light of a fluorescent dye, and detecting fluorescence emitted from the cell.
2. The fluorescent dye is a first fluorescent dye;
   At any stage after step (a) and before step (c),
   (X1) contacting a cell with a primary antibody that recognizes a leukocyte marker protein, and then contacting a secondary antibody that recognizes the primary antibody and labeled with a second fluorescent dye, or Contacting the cell with an antibody that recognizes leukocytes and is labeled with a second fluorescent dye;
   (X2) contacting a cell with a primary antibody that recognizes a marker protein of epithelial cells, and then a secondary antibody that recognizes the primary antibody and labeled with a third fluorescent dye, Or contacting a cell with an antibody that recognizes a marker protein of epithelial cells and labeled with a third fluorescent dye;
   (X3) labeling the cell nucleus with a fourth fluorescent dye;
   Further in any order,
   In the step (c), the cells are irradiated with excitation light of the first, second, third and fourth fluorescent dyes, respectively, and the first, second, third and fourth fluorescence emitted from the cells. The method according to claim 1, wherein fluorescence of each dye is detected.
3. The method according to claim 2, wherein the primary antibody that recognizes PD-L1 or the antibody that recognizes PD-L1 is derived from a clone of 28-8 or SP142.
4. The method according to claim 2 or 3, wherein step (a) is a step of filtering a blood sample with a filter to capture cells on the filter.
5. The method according to any one of claims 2 to 4, wherein the leukocyte marker protein is CD45.
6. The method according to any one of claims 2 to 5, wherein the marker protein of epithelial cells is cytokeratin.
7. The first, second, and third fluorescent dyes are selected from the group consisting of fluorescein, Alexa Fluor (registered trademark) 594, and Alexa Fluor (registered trademark) 647, and the fourth fluorescent dye is 4 ', 6- The method according to any one of claims 2 to 6, which is diamidino-2-phenylindole.
8. The method according to any one of claims 2 to 7, wherein the PD-L1-positive cancer cells are derived from lung cancer.
9. Performing step (x1) before step (b),
   The method according to any one of claims 2 to 8, wherein the step (x2) and the step (x3) are simultaneously performed after the step (b).

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