Kit and method for detecting circulating PD-L1 positive cells

Abstract

The present invention provides a kit and method for detecting circulating PD-L1-positive cells, the kit comprising a detection reagent and a PD-L1-positive cell capture reagent; the detection reagent comprising a PD-L1 antibody labeled with a first non-human marker, a leukocyte common antigen antibody labeled with a first fluorescent molecule, a cancer cell antigen antibody labeled with a second fluorescent molecule, an anti-nth non-human marker antibody labeled with a third fluorescent molecule, ... an anti-nth non-human marker antibody labeled with an n-1th non-human marker, and an anti-nth non-human marker antibody labeled with an n-1th non-human marker; wherein n is a positive integer greater than or equal to 1, the first, second, and third fluorescent molecules have different emission wavelengths, and the first to nth non-human markers are different non-human markers. The kit has high sensitivity and a low detection limit for detecting circulating PD-L1-positive cells.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and specifically relates to a kit and method for detecting circulating PD-L1 positive cells. Background Art

[0002] Immunohistochemistry is a classic method for detecting protein expression. It applies the principle of specific binding between antigens and antibodies and uses pathological tissue sections for PD-L1 immunohistochemical staining to evaluate PD-L1 expression in tumor cells or immune cells. Immunohistochemistry is currently the main method for detecting PD-L1 positivity. Clinical trials and practice mainly use immunohistochemistry. In this detection method, in addition to staining technology, the specificity of the antibody is also particularly important.

[0003] Although PD-L1 detection is currently the most important immunotherapy biomarker, its clinical application still has some problems, mainly including:

[0004] (1) In immunohistochemistry, the methodological differences of different detection antibodies and platforms, as well as the different algorithms for calculating expression levels, make it difficult to achieve high consistency, resulting in the lack of a unified clinical diagnostic standard. For example, in the Blueprint study (Hirsch FR, Mcelhinny A, Stanforth D, et al. PD-L1 Immunohistochemistry Assays for Lung Cancer: Results from Phase 1 of the Blueprint PD-L1 IHC Assay Comparison Project [J]. Journal of Thoracic Oncology, 2017, 12 (2): 208-222.), serial sections of 39 paraffin block samples of NSCLC were analyzed for PD-L1 expression levels using the detection methods used in the respective drug clinical trials, and the slides were read by three pathologists. The results showed that the IHC analysis of the three antibodies (28-8, 22C3, SP263) had good consistency in tumor cells, while the SP142 antibody stained relatively few tumor cells. Although the four antibodies all stained immune cells to varying degrees, the consistency was poor. In the Blueprint 2 study (Tsao M, Kerr K, Yatabe Y, et al. PL03.03 Blueprint 2: PD-L1 Immunohistochemistry Comparability Study in Real-Life, Clinical Samples[J]. Journal of Thoracic Oncology, 2017, 12(11): S1606.), the staining comparability of five antibodies (28-8, 22C3, SP142, SP263, and 73-10) was analyzed. The results showed that 28-8, 22C3, and SP263 had consistent staining for tumor cells, SP142 detected fewer positive cells, and 73-10 detected more positive cells. The staining of immune cells was challenging, with poor consistency and unreliable. For cytological samples, the reliability of PD-L1 staining needs further confirmation.

[0005] (2) Biologically, PD-L1 histochemical detection is heterogeneous, because PD-L1 varies in different areas of the same tumor, and there are also differences between primary lesions and metastatic lesions. Therefore, simply taking biopsies from a small number of areas may cause sampling bias, making it difficult to accurately assess the overall situation of the tumor; Ilie et al. (Ilié, ​​M, Szafer-Glusman E, Hofman V, et al. Detection of PD-L1 in circulating tumor cells and white blood cells from patients with advanced non-small-cell lung cancer [J]. Annals of Oncology, 2018.) used SP142 to evaluate PD-L1 staining in surgical resection specimens and lung tissue biopsy specimens of 160 NSCLC patients. There were significant differences in PD-L1 staining, with biopsy specimens and surgical specimens (TC1 / 2 / 3 and / or IC1 / 2 / 3, 26% vs 74%).

[0006] (3) PD-L1 expression is dynamic, fluctuating, up-regulated, or down-regulated over time and in response to treatment. Real-time, multiple-time monitoring is difficult to achieve with tissue biopsy.

[0007] CTCs originate from primary tumor foci and metastatic foci, and can reflect the overall condition of the tumor to a certain extent, reducing the impact of tumor heterogeneity. As a non-invasive blood test method, it can monitor and evaluate the therapeutic effect during the patient's treatment process. Compared with immunohistochemical PD-L1 detection, circulating tumor cells (CTCs) have unique advantages for PD-L1 detection.

[0008] However, there are currently few studies on circulating PD-L1-positive cells, and most technical platforms are based on Epcam single antibody capture. This technical method is prone to miss Epcam-negative PD-L1-positive cells. In addition, due to the existing technical methods, direct detection using fluorescently labeled PD-L1 antibodies is difficult to detect cells with low PD-L1 expression, resulting in low efficiency of PD-L1-positive cell detection using existing methods.

[0009] Therefore, it is urgent to develop a new method for detecting circulating PD-L1-positive cells to solve the problems of weak detection signal and low efficiency of PD-L1-positive cells during the detection process. Summary of the Invention

[0010] The problem to be solved by the present invention is to provide a kit and a detection method for detecting circulating PD-L1-positive cells in a sample to be tested, which has high sensitivity and low detection limit, and can distinguish circulating PD-L1-positive tumor cells from circulating PD-L1-positive immune cells.

[0011] In order to achieve the above objectives, the present invention provides the following solutions:

[0012] In one aspect of the present invention, a kit for detecting circulating PD-L1-positive cells in a sample to be tested is provided, comprising a detection reagent and a PD-L1-positive cell capture reagent; the detection reagent comprises a PD-L1 antibody labeled with a first non-human marker, an antibody to a common leukocyte antigen labeled with a first fluorescent molecule, an antibody to a cancer cell antigen labeled with a second fluorescent molecule, an antibody against an nth non-human marker labeled with a third fluorescent molecule, ... an antibody against an n-2th non-human marker labeled with an n-1th non-human marker, and an antibody against an n-1th non-human marker labeled with an n-1th non-human marker.

[0013] Where n is a positive integer greater than or equal to 1, and the first, second, and third fluorescent molecules have different excitation wavelengths or emission wavelengths to achieve statistical differentiation of different labeled substances. It is understood that when n is less than 1, the kit does not contain... an anti-n-2 non-human marker antibody labeled with an n-1 non-human marker, or an anti-n-1 non-human marker antibody labeled with an n non-human marker.

[0014] The first to nth non-human markers are different non-human markers, so as to form a plurality of pairs of non-human marker-anti-non-human marker antibodies that can specifically bind.

[0015] The circulating PD-L1-positive cells of the present invention include circulating PD-L1-positive tumor cells and circulating PD-L1-positive immune cells.

[0016] In one embodiment, the circulating PD-L1-positive tumor cells are selected from one or more of melanoma cells, non-small cell lung cancer cells, renal cancer cells, prostate cancer cells, pancreatic cancer cells, bile duct cancer cells, esophageal cancer cells, intestinal cancer cells, breast cancer cells, small cell lung cancer cells, gastric cancer cells, liver cancer cells, human placental chorionic villus carcinoma cells, cervical cancer cells, ovarian cancer cells, bladder cancer cells, head and neck tumor cells, skin cancer cells, thymic cancer cells, lymphoma cells, and nasopharyngeal cancer cells.

[0017] In a preferred embodiment, the circulating PD-L1 positive tumor cells are lung cancer cells.

[0018] The kit of the present invention contains n pairs of different non-human markers and anti-non-human marker antibodies with antigen-antibody binding capabilities. By performing multi-stage amplification on the detection process of circulating PD-L1-positive cells, the kit has a low detection limit and high sensitivity, capable of detecting positive cells with low PD-L1 expression. Furthermore, the kit of the present invention also contains detection reagents for detecting white blood cells and cancer cells, and can simultaneously distinguish between circulating PD-L1-positive tumor cells and circulating PD-L1-positive immune cells.

[0019] In a preferred embodiment, 1≤n≤3.

[0020] In one embodiment of the present invention, n=1, and the detection reagent includes a PD-L1 antibody labeled with a first non-human marker, an antibody against a common leukocyte antigen labeled with a first fluorescent molecule, an antibody against a cancer cell antigen labeled with a second fluorescent molecule, and an antibody against the first non-human marker labeled with a third fluorescent molecule. The kit can perform secondary amplification on the detection process of circulating PD-L1-positive cells.

[0021] The PD-L1 antibody labeled with the first non-human marker can be labeled with multiple first non-human marker molecules. Each first non-human marker molecule can bind to a secondary antibody (anti-first non-human marker antibody), and each secondary antibody can be labeled with multiple third fluorescent molecules. Compared to directly detecting the PD-L1 antibody with fluorescent molecules, secondary amplification produces a stronger fluorescent signal. Understandably, if the cost of the kit is not considered, when n is larger, that is, the number of amplification levels is higher, the specificity of the detection is better.

[0022] In the present invention, the non-human marker labeling can be selected from non-human markers commonly used in the art. It can be understood that in the above-mentioned multi-stage amplification, the 1st to nth non-human markers can be n different non-human markers selected from non-human markers commonly used in the art.

[0023] In one embodiment, the non-human marker labeling includes labeling the antibody with biotin, digoxigenin, avidin, horseradish peroxidase (HRP), alkaline phosphatase, and fluorescent protein.

[0024] In one embodiment, the leukocyte common antigen antibody is selected from one or more of CD45, CD11b, CD14, CD68, CD16, CD56, CD57, CD59, CD94, CD3, CD4, CD8, CD80, CD86, CD40, CD21, CD19, CD20, CD18, CD32, CD44, CD55 and CD66b antibodies; the cancer cell antigen antibody is selected from one or more of CK, Epcam, HER2, Trop2, EGFR, CEA, vimentin, MUC1, HER3, C-MET, ALK, claudin18.2 and GPC3 antibodies.

[0025] In one embodiment, in the detection antibodies, the mass ratio of the leukocyte common antigen antibody to the cancer cell antigen antibody is 4:(4-6); preferably, 4:5.

[0026] In a preferred embodiment, the leukocyte common antigen antibody is a CD45 antibody.

[0027] In a preferred embodiment, the cancer cell antigen antibody is a CK antibody.

[0028] In one embodiment, the PD-L1-positive cell capture reagent includes a capture antibody and magnetic beads, wherein the capture antibody is a PD-L1 antibody labeled with an n+1 non-human marker and a tumor-specific protein marker antibody labeled with an n+1 non-human marker, and the magnetic beads are magnetic beads labeled with an antibody against the n+1 non-human marker.

[0029] In the present invention, the first non-human marker and the n+1th non-human marker are different non-human markers to avoid the antibody against the first non-human marker labeled with the third fluorescent molecule in the detection reagent from binding to other non-human markers, resulting in false positives and affecting the accuracy of the detection.

[0030] In one embodiment, the tumor-specific protein marker is selected from one or more of Epcam, HER2, Trop2, EGFR, CK, CEA, MUC1, ALK and C-MET.

[0031] The PD-L1 antibody in the capture reagent and the PD-L1 antibody in the detection reagent are non-competing antibodies.

[0032] In one embodiment, the mass ratio of the PD-L1 antibody in the capture reagent to the PD-L1 antibody in the detection reagent is 1:(3.2-4.8); preferably, 1:4, so as to better capture PD-L1 positive cells while saving antibody usage costs.

[0033] It is understandable that when the cancer cell antigen in the detection reagent and the tumor-specific protein marker in the capture antibody are the same antigen, the leukocyte common antigen antibody and the tumor-specific protein marker antibody are selected from non-competing antibodies.

[0034] In one embodiment, in the capture antibody, the tumor-specific protein marker antibody labeled with the n+1 non-human marker is a mixed antibody, which includes one or more of Epcam, HER2, Trop2, and EGFR labeled with the n+1 non-human marker.

[0035] According to the research of the present invention, in the mixed antibody, the mass ratio of the antibody against EGFR target to the antibody against other targets (Epcam, HER2, Trop2) is 2:(1-2). For example, the capture antibody Epcam:HER2:Trop2:EGFR can be 1:1:1:2, 2:1:1:2, 1:1:2:2, 1:2:1:2, 2:2:1:2, 2:1:2:2, 2:2:2:2. This ratio is conducive to improving the capture efficiency of the mixed antibody, thereby improving the detection limit.

[0036] In a preferred embodiment, it comprises at least two tumor-specific protein marker antibodies labeled with the n+1th non-human marker to avoid missing PD-L1 positive cells.

[0037] In a preferred embodiment, the mixed antibody is an antibody against Epcam, HER2, Trop2 and EGFR, and the mass ratio thereof is 1:1:1:2.

[0038] In a preferred embodiment, the capture antibody is a biotin-labeled mixed antibody and a PD-L1 antibody, and the mass ratio thereof is 5:(0.5-2), for example, 5:0.5, 5:1, 5:1.5, 5:2; more preferably 5:1. In one embodiment, the first, second, and third fluorescent molecules are selected from fluorescent markers that can label proteins, such as iFluor647, iFluor488, iFlour594, iFlour555, iFlour568, iFlour750, FITC, Alexa Fluor488, Alexa Fluor555, Alexa Fluor568, AlexaFluor594, Alexa Fluor750, APC, and Alexa Fluor647, and the first, second, and third fluorescent molecules are fluorescent molecules with different excitation wavelengths or emission wavelengths.

[0039] In a preferred embodiment of the present invention, the first fluorescent molecule is iFluor488, the second fluorescent molecule is iFluor647, and the third fluorescent molecule is Alexa Fluor594.

[0040] In one embodiment, n=1, the first non-human marker is HRP, the n+1th non-human marker is the second non-human marker, the second non-human marker is biotin, and the antibody against the second non-human marker is streptavidin.

[0041] In one embodiment, n=1, the detection process is secondary amplification, and the detection reagent includes an HRP-labeled PD-L1 antibody and an anti-HRP antibody labeled with a third fluorescent molecule.

[0042] In one embodiment, n=2, the detection process is a three-stage amplification, and the detection reagents include a biotin-labeled PD-L1 antibody, an HRP-labeled streptavidin (anti-biotin antibody), and a goat anti-HRP antibody labeled with a third fluorescent molecule.

[0043] In one embodiment, n=3, the detection process is a four-stage amplification, and the detection reagents include HRP-labeled PD-L1 antibody, digoxigenin-labeled anti-HRP antibody, AP-labeled anti-digoxigenin antibody, and anti-digoxigenin antibody labeled with a third fluorescent molecule.

[0044] In one embodiment, n=4, the detection process is a five-stage amplification, and the detection reagents include HRP-labeled PD-L1 antibody, biotin-labeled anti-HRP antibody, digoxigenin-labeled streptavidin, AP-labeled anti-digoxigenin antibody, and anti-AP antibody labeled with a third fluorescent molecule.

[0045] In one embodiment, the detection process is a multi-stage amplification, and the detection reagent includes a PD-L1 antibody labeled with a first non-human marker, ... an anti-n-2 non-human marker antibody labeled with an n-1 non-human marker, an anti-n-1 non-human marker antibody labeled with an n non-human marker, and an anti-n non-human marker antibody labeled with a third fluorescent molecule.

[0046] In one embodiment, the tumor-specific protein markers are Epcam, HER2, Trop2, and EGFR; and the capture reagents include biotin-labeled Epcam, HER2, Trop2, EGFR, and PD-L1 antibodies and streptavidin-labeled magnetic beads.

[0047] In one embodiment, the antibody against the first non-human marker is goat anti-HRP.

[0048] In a preferred embodiment of the present invention, the detection reagent includes an HRP-labeled PD-L1 antibody (i.e., a PD-L1 detection antibody), an iFluor488-labeled CD45 antibody (i.e., a CD45 detection antibody), an iFluor647-labeled CK antibody (i.e., a CK detection antibody), and an Alexa Fluor594-labeled anti-HRP antibody (i.e., a secondary antibody).

[0049] The sample to be tested in the present invention is a body fluid sample, which is selected from a blood sample, a pleural effusion sample, an ascites sample, a cerebrospinal fluid sample, a urine sample and a sputum sample.

[0050] In one embodiment, the HRP-labeled PD-L1 antibody in the detection antibody is used at a concentration of 1-8 μg / ml, for example, 1, 2, 4, 6, or 8 μg / ml; preferably, 4 μg / ml or 8 μg / ml.

[0051] In one embodiment, among the detection antibodies, the CD45 detection antibody is used at a concentration of 2.5-20 μg / ml, for example, 1.5, 5, 10, 15, or 20 μg / ml; preferably, 5 μg / ml.

[0052] In one embodiment, among the detection antibodies, the CK detection antibody is used at a concentration of 2-16 μg / ml, for example, 2, 4, 8, 12, or 16 μg / ml; preferably, 4 μg / ml.

[0053] In one embodiment, the anti-HRP antibody labeled with the third fluorescent molecule is used at a concentration of 1.25-10 μg / ml, for example, 1.25, 2.5, 5, 8, or 10 μg / ml; preferably, 5 μg / ml.

[0054] In one embodiment, the mass ratio of the HRP-labeled PD-L1 antibody to the anti-HRP antibody labeled with the third fluorescent molecule in the kit is (0.5-1.5):(0.5-1.5), for example, it can be 0.5:1, 1:1 or 1:1.5; preferably, it is 1:1.

[0055] In one embodiment, the above-mentioned kit further comprises a sample pre-treatment reagent and a blocking reagent, wherein the pre-treatment reagent comprises a red blood cell removal reagent and a cell fixation reagent.

[0056] Wherein, the blocking reagent is selected from one or more solutions of human IgG, mouse IgG, rabbit IgG and goat IgG.

[0057] In a preferred embodiment, the blocking reagent is a human IgG solution; the concentration of the human IgG solution is 2.5-20 μg / ml, for example, 2.5, 5, 10, 15, 20 μg / ml; preferably, it is 10 μg / ml.

[0058] In one embodiment, the red blood cell removal reagent is a reagent containing NH 4+ of reagents.

[0059] In one embodiment, the cell fixation reagent is a solution containing one or more of paraformaldehyde, formaldehyde, glutaraldehyde, methanol, and glacial acetic acid.

[0060] In one embodiment, the cell fixation reagent is used at a concentration (mass percentage) of 0.25-4%, for example, 0.25%, 1%, 2%, 3%, or 4%; preferably, 2%.

[0061] In a preferred embodiment, the cell fixation reagent is a solution containing 2% paraformaldehyde.

[0062] In one embodiment, the volume ratio of the red blood cell removal reagent to the sample to be tested is (1:2)-(1:9); preferably, it is 1:3.

[0063] In one embodiment, the volume ratio of the cell fixation reagent to the sample to be tested is (2:1)-(1:5); preferably, it is 1:1.

[0064] In one embodiment, the binding buffer is a solution containing one or more of fetal bovine serum, bovine serum albumin, and goat serum; preferably, it is a solution containing fetal bovine serum.

[0065] Another aspect of the present invention provides a method for preparing the above-mentioned kit, comprising the following steps:

[0066] (1) Preparation of a PD-L1 positive cell capture reagent, which includes preparation of a capture antibody and preparation of magnetic beads;

[0067] (2) a step of preparing a PD-L1 detection antibody, which includes the steps of preparing a PD-L1 antibody labeled with a first non-human marker, an antibody against an nth non-human marker labeled with a third fluorescent molecule, ... an antibody against an n-2th non-human marker labeled with an n-1th non-human marker, and an antibody against an n-1th non-human marker labeled with an n-1th non-human marker;

[0068] (3) Preparation steps of antibodies against common leukocyte antigens and cancer cell antigens;

[0069] (4) Reagent assembly.

[0070] In one embodiment, in the capture antibody preparation step of step (1), the mass ratio of the n+1 non-human marker to the tumor-specific protein marker antibody is 1:(10-120), and can further be 1:(40-120) or 1:(80-100), for example, it can be 1:10, 1:20, 1:40, 1:80, 1:100, 1:120; preferably, it is 1:80.

[0071] In one embodiment, in step (2), the mass ratio of the first non-human marker to the PD-L1 antibody is 1:(0.5-4); for example, it can be 1:0.5, 1:1, 1:2, 1:4; preferably, it is 1:1.

[0072] Another aspect of the present invention provides a method for detecting PD-L1 positive cells in a test sample in vitro using the above kit, comprising the following steps:

[0073] i. (1) n = 1, obtaining a sample to be tested from a subject, adding a PD-L1 antibody labeled with a first non-human marker, an antibody against a common leukocyte antigen labeled with a first fluorescent molecule, and an antibody against a cancer cell antigen labeled with a second fluorescent molecule for incubation; (2) adding an antibody against the first non-human marker labeled with a third fluorescent molecule; (3) adding a nucleic acid dye to separate the cells and performing scanning analysis using a fluorescence microscope; or,

[0074] ii. (1) n = 1, obtain the sample to be tested from the subject, add the capture antibody labeled with the second non-human marker (the PD-L1 antibody labeled with the second non-human marker and the tumor-specific protein marker antibody labeled with the second non-human marker), the PD-L1 antibody labeled with the first non-human marker, the leukocyte common antigen antibody labeled with the first fluorescent molecule, and the cancer cell antigen antibody labeled with the second fluorescent molecule for incubation; (2) add magnetic beads labeled with the second non-human marker and the antibody against the first non-human marker labeled with the third fluorescent molecule, and separate the magnetic beads; (3) add nucleic acid dye to separate the cells and use fluorescence microscope scanning for analysis.

[0075] In one embodiment, the nucleic acid stain is DAPI.

[0076] Specifically, ii. (1) n = 1, a blood sample is obtained from the subject, and capture antibodies (biotin-labeled PD-L1 antibody, biotin-labeled Epcam, HER2, Trop2, EGFR mixed antibody), HRP-labeled PD-L1 antibody, iFluor488-labeled CD45 antibody, and iFluor647-labeled CK antibody are added for incubation; (2) streptavidin-labeled magnetic beads and AlexaFluor594-labeled anti-HRP antibody are added to separate the magnetic beads; (3) DAPI dye is added to separate the cells and then analyzed by fluorescence microscopy scanning.

[0077] In the present invention, in the above step (3), if the fluorescence emitted by the third fluorescent molecule (Alexa Fluor 594) is detected, the detection sample contains circulating PD-L1 positive cells; if both the fluorescence emitted by the third fluorescent molecule (Alexa Fluor 594) and the fluorescence emitted by the first fluorescent molecule (iFluor 488) are detected, the detection sample contains circulating PD-L1 positive immune cells; if both the fluorescence emitted by the third fluorescent molecule (Alexa Fluor 594) and the fluorescence emitted by the second fluorescent molecule (iFluor 647) are detected, the detection sample contains circulating PD-L1 positive tumor cells.

[0078] In the present invention, the above-mentioned kit is used in a method for detecting PD-L1-positive cells in a test sample. The ratio of the concentrations of the various antibodies used, measured by mass concentration (mass / volume of solution), is the mass ratio of the antibodies contained in the above-mentioned kit. For example, in the detection antibodies, the mass ratio of the antibody against the common leukocyte antigen to the antibody against the cancer cell antigen is 4:(4-6); preferably, it is 4:5. When the kit is used to detect PD-L1-positive cells, the ratio of the antibody against the common leukocyte antigen to the antibody against the cancer cell antigen, measured by mass concentration, is 4:(4-6); preferably, it is 4:5.

[0079] The use concentration of the present invention is the concentration of the reagent, antibody or protein in the total solution system when it is finally used.

[0080] Another aspect of the present invention provides a method for detecting circulating PD-L1 positive cells in a sample for non-diagnostic purposes, comprising:

[0081] (1) Obtaining a sample to be tested from a subject, adding a capture antibody, an antibody against a common leukocyte antigen labeled with a first fluorescent molecule, an antibody against a cancer cell antigen labeled with a second fluorescent molecule, and an antibody against PD-L1 labeled with a first non-human marker for incubation;

[0082] (2) adding an antibody against the first non-human marker labeled with the second non-human marker and incubating;

[0083] …

[0084] adding an antibody against the n-2 non-human marker labeled with the n-1 non-human marker for incubation;

[0085] adding an antibody against the n-1 non-human marker labeled with the n non-human marker for incubation;

[0086] (3) adding magnetic beads labeled with the n+1th non-human marker and an antibody against the nth non-human marker labeled with a third fluorescent molecule, and separating the magnetic beads;

[0087] (4) After adding nucleic acid dye to separate cells, use fluorescence microscopy to scan and analyze;

[0088] (5) Result judgment. Wherein, n is a positive integer greater than or equal to 1; the first, second, and third fluorescent molecules have different emission wavelengths, and the first to nth non-human markers are different non-human markers; preferably, n=1.

[0089] According to the research of the present invention, in step (5), if the fluorescence value of the third fluorescent molecule is ≥40, and the fluorescence value of the third fluorescent molecule / the fluorescence value of the first fluorescent molecule is ≥3.0, the PD-L1 positive cells are positive tumor cells; if the fluorescence value of the third fluorescent molecule is ≥40, and the fluorescence value of the third fluorescent molecule / the fluorescence value of the first fluorescent molecule is <3.0, the PD-L1 positive cells are positive immune cells, that is,

[0090] The criteria for determining PD-L1-positive tumor cells are a PD-L1 detection signal fluorescence value ≥40, and the ratio of the PD-L1 detection signal fluorescence value to its CD45 background signal fluorescence value ≥3.0; the criteria for determining PD-L1-positive immune cells are a PD-L1 detection signal fluorescence value ≥40, and the ratio of the PD-L1 detection signal fluorescence value to its CD45 background signal fluorescence value <3.0.

[0091] In one embodiment, the method for detecting circulating PD-L1 positive cells in a non-diagnostic sample comprises the following steps:

[0092] (1) Obtaining a sample to be tested from a subject, adding a capture antibody, an antibody against a common leukocyte antigen labeled with a first fluorescent molecule, an antibody against a cancer cell antigen labeled with a second fluorescent molecule, and an antibody against PD-L1 labeled with a first non-human marker for incubation;

[0093] (2) adding magnetic beads labeled with a second non-human marker and an antibody against the first non-human marker labeled with a third fluorescent molecule, and separating the magnetic beads;

[0094] (3) After adding nucleic acid dye to separate cells, use fluorescence microscopy to scan and analyze;

[0095] (4) Result judgment;

[0096] Wherein, the first, second and third fluorescent molecules have different emission wavelengths;

[0097] In step (4), if the fluorescence value of the third fluorescent molecule is ≥40, and the fluorescence value of the third fluorescent molecule / the fluorescence value of the first fluorescent molecule is ≥3.0, the PD-L1 positive cell is a positive tumor cell;

[0098] If the fluorescence value of the third fluorescent molecule is ≥40, and the fluorescence value of the third fluorescent molecule / the fluorescence value of the first fluorescent molecule is <3.0, the PD-L1 positive cell is a positive immune cell.

[0099] Based on the kit and its use method described in the present invention, the present invention introduces the fluorescence threshold and the ratio of the PD-L1 detection signal to the background signal for joint judgment to achieve the statistical distinction between PD-L1 positive immune cells and PD-L1 positive tumor cells.

[0100] In another aspect of the present invention, a use of the above-mentioned kit in preparing a reagent for detecting circulating PD-L1-positive cells in a sample to be tested is provided, wherein the circulating PD-L1-positive cells include circulating PD-L1-positive tumor cells and circulating PD-L1-positive immune cells; and the sample to be tested is a body fluid sample, including a blood sample, a pleural effusion sample, an ascites sample, a cerebrospinal fluid sample, a urine sample, and a sputum sample.

[0101] Beneficial effects of the present invention:

[0102] 1. The kit and detection system described in the present invention perform multi-stage amplification of the detection process of circulating PD-L1-positive cells, so that the kit has a lower detection limit and higher sensitivity, and can detect positive cells with low PD-L1 expression.

[0103] 2. The kit described in the present invention can detect and separate PD-L1-positive cells while distinguishing and counting circulating PD-L1-positive tumor cells and circulating PD-L1-positive immune cells, and the judgment criteria are explored.

[0104] 3. Based on the detection scheme of the present invention, the present invention targets the characteristics of circulating tumor cells and conducts creative exploration of the selection of common leukocyte antigens and cancer cell antigens in the detection kit, the selection of capture reagents, the antibody concentration, the ratio between different detection antibodies, the ratio between the detection antibody and capture antibody concentrations, and the ratio between different capture antibodies. As a result, the detection system of the present invention can achieve a specificity of 99.7% for PD-L1-positive cells, a detection limit as low as 1 cell, and a capture efficiency of over 80% for different types of lung cancer cell lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 The results of the antibody biotin labeling process investigation in Example 1 are shown.

[0106] Figure 2 The results of the HRP labeling process investigation in Example 1 are shown.

[0107] Figure 3 The comparison results of PD-L1 primary, secondary and tertiary amplification detection are shown.

[0108] Figure 4 The recovery rates of red blood cells treated with lysis buffer at different volume ratios are shown.

[0109] Figure 5 The results of the sample fixed condition exploration are shown ( Figure 5 A in the figure represents the effect of different fixation conditions on cell integrity; Figure 5 (B) The effect of different fixation conditions on CK binding in tumor cells.

[0110] Figure 6 The results of flow cytometry analysis of the concentration of PD-L1 antibodies are shown.

[0111] Figure 7 The results of the cell enrichment and separation method to explore the concentration of PD-L1 antibody are shown.

[0112] Figure 8 Shown is a comparison of cell enrichment and separation methods using anti-HRP fluorescent secondary antibody concentrations.

[0113] Figure 9 The results of flow cytometry analysis of the concentration of CK detection antibodies are shown.

[0114] Figure 10 The results of the cell enrichment and separation method to explore the concentration of CK detection antibodies are shown.

[0115] Figure 11 The results of flow cytometry analysis of the concentration of CD45 detection antibodies are shown.

[0116] Figure 12 The results of the cell enrichment and separation method exploring the concentration of CD45 detection antibody used are shown.

[0117] Figure 13 The comparison results of the best concentration of blocking agents for cell enrichment and separation methods are shown ( Figure 13 A in the figure is the white blood cell background comparison; Figure 13 B in the figure is a comparison of positive signals of tumor cells).

[0118] Figure 14 The results of the capture antibody ratio exploration are shown.

[0119] Figure 15 Shown are the recovery results of different cell lines captured by the capture antibodies.

[0120] Figure 16 The results of the PD-L1 positive cell detection of the present invention are compared with those of other detection platforms.

[0121] Figure 17 The method for determining PD-L1 positive tumor cells and immune cells is shown ( Figure 17A in the figure is the threshold for judging PD-L1-positive tumor cells; Figure 17 B in the figure is the threshold for judging PD-L1 positive immune cells). DETAILED DESCRIPTION

[0122] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0123] In this article, the term "enrichment" refers to the separation of circulating PD-L1-positive cells from the sample to be tested.

[0124] As used herein, the term "streptavidin" may refer to a protein or peptide that can bind to biotin, and may include: native egg-white avidin, recombinant avidin, a deglycosylated form of avidin, bacterial streptavidin, recombinant streptavidin, truncated streptavidin, and / or any derivatives thereof.

[0125] As used herein, the term "common leukocyte antigens" refers to a class of membrane proteins expressed on all leukocytes.

[0126] As used herein, the term "cancer cell antigen" refers to a class of proteins expressed on cancer cells.

[0127] As used herein, the term "magnetic beads" refers to tiny particles that have magnetic properties.

[0128] Herein, the term "PD-L1 positive cells" refers to cells expressing PD-L1.

[0129] As used herein, the term "PD-L1-positive tumor cells" refers to tumor cells expressing PD-L1.

[0130] Herein, the term "PD-L1 positive immune cells" refers to immune cells expressing PD-L1.

[0131] Herein, the terms "secondary antibody", "cancer cell detection antibody (CK detection antibody)" and "leukocyte detection antibody (CD45 detection antibody)" are all antibodies with fluorescent labels.

[0132] Herein, the term "PD-L1 detection antibody" refers to a PD-L1 antibody labeled with a first non-human marker.

[0133] In this article, the term "LiquidBiopsy system" refers to the automated cell enrichment and separation system with registration number Yuezhu Xiebei 20170142, which originated from Zhuhai Lizhu Shengmei Medical Diagnostic Technology Co., Ltd.

[0134] Example

[0135] Example 1: Research on non-human marker labeling process

[0136] 1.1 Study on biotin labeling process

[0137] 1.1.1 Biotin labeling

[0138] Using the Japanese Dojindo Chemical Biotin Labeling Kit (Cat. No. LK03, components: ultrafiltration column, WS buffer, labeling reaction solution, activated biotin), pipette the WS buffer into a centrifuge tube, add EGFR (tumor marker) antibody and mix thoroughly, then apply the mixture to the ultrafiltration column and centrifuge. After washing with WS buffer, add the labeling reaction solution to the ultrafiltration column and simultaneously add activated biotin at varying molar ratios (antibody:biotin = 1:10, 1:20, 1:40, 1:80, 1:100, 1:120). Incubate at 37°C for 10 min. Wash twice with WS buffer. Add 200 μl of PBS solution and transfer to a 0.5 ml tube to obtain the biotin-labeled EGFR antibody.

[0139] 1.1.2 Marker Detection

[0140] HCC827 cells (human non-small cell lung cancer cells) were diluted to 300,000 cells / ml in binding buffer (Shengmei Biodiagnostic Technology Co., Ltd., registration number: Yuezhu Xiebei 20160072). Biotin-labeled EGFR antibodies were incubated with HCC827 cells, followed by the addition of streptavidin-FITC (Streptavidin, Solarbio SF068), mixing and continued incubation. Cells were harvested, washed, and assayed for binding activity of the EGFR-biotin antibody to HCC82 cells. An EGFR-biotin antibody from Capricobio was used as a control (antibody:biotin = 1:80).

[0141] The results showed that ( Figure 1 ), the binding activity of EGFR antibodies after labeling at a ratio of 1:(10-120) is relatively high, and at a ratio of 1:80, the activity is higher than that of Capricobio's EGFR-biotin (control) antibody. Therefore, the preferred ratio for biotin labeling of EGFR-biotin antibodies is 1:80.

[0142] 1.2 Research on antibody HRp labeling process

[0143] 1.2.1 HRP labeling

[0144] The antibody was labeled using the HRP labeling kit (Cat. No. BF06095-1000; composition: ultrafiltration tube, labeling reaction solution, activated HRP) from Beijing Biolong Immunotechnology Co., Ltd. The specific steps were to add activated HRP in different mass ratios (HRP:PD-L1 antibody = 1:0.5, 1:1, 1:2, 1:4) and incubate, then add PBS and transfer to the reagent tube to obtain the HRP-labeled PD-L1 antibody. 1.2.2 Marker Detection

[0145] NCI-H820 cells (adherent human papillary lung adenocarcinoma cells) were diluted to 300,000 cells / ml using binding buffer (Shengmei Biodiagnostic Technology Co., Ltd.). HRP-labeled PD-L1 antibody was incubated with NCI-H820 cells. Goat anti-HRP IgG-Alexa Fluor 488 (fluorescent secondary antibody, Jackson ImmunoResearch) was then added, mixed, and incubated for further analysis. After washing with binding buffer, flow cytometry was performed to compare the binding activity of the PD-L1 HRP-labeled antibody to NCI-H820 cells.

[0146] The results showed that the PD-L1 antibody and HRP had detectable fluorescent signals after labeling at a mass ratio of 1:(0.5-4). The detection signal after 1:1 labeling was higher than that of 1:2, 1:4 and 1:0.5. Therefore, the HRP labeling ratio is preferably 1:1.

[0147] Example 2 PD-L1 multi-stage amplification and fluorescence channel exploration

[0148] Dilute NCI-H820 cells to 300,000 cells / ml using binding buffer, and take 100 μl of cell fluid per aliquot.

[0149] 2.1 First-stage amplification detection (n=0)

[0150] iFluor488 fluorescently labeled PD-L1 antibody, iFluor594 fluorescently labeled PD-L1 antibody, and iFluor647 fluorescently labeled PD-L1 antibody were diluted and incubated with NCI-H820 cell fluid respectively. The cells were collected, washed, and then detected.

[0151] 2.2 Secondary Amplification Detection (n=1)

[0152] HRP-labeled PD-L1 antibody was co-incubated with NCI-H820 cells; then iFluor488 fluorescently labeled goat anti-HRP antibody (secondary antibody), iFluor594 fluorescently labeled goat anti-HRP antibody (secondary antibody), and iFluor647 fluorescently labeled goat anti-HRP antibody (secondary antibody) were added and co-incubated, and the cells were collected, washed, and then detected.

[0153] 2.3 Three-stage amplification detection (n=2)

[0154] Biotin-labeled PD-L1 antibody was co-incubated with NCI-H820 cells; after the cells were collected and washed, HRP-labeled streptavidin was added and incubated for further treatment; then iFluor488 fluorescently labeled goat anti-HRP antibody, iFluor594 fluorescently labeled goat anti-HRP antibody, and iFluor647 fluorescently labeled goat anti-HRP antibody were added and incubated together, and the cells were collected, washed, and then detected.

[0155] Take the liquid droplets obtained from the first, second and third level amplification tests respectively, load them onto glass slides and bake them dry, add DAPI reagent and scan them using a Leica fluorescence microscope. The results are as follows: Figure 3 shown.

[0156] The results showed that ( Figure 3 ), the detection signal using the secondary and tertiary amplification methods is stronger than that using the primary amplification method. When using ifluor594 fluorescein and ifluor647 fluorescein, the secondary amplification detection signal already has a strong fluorescent signal that meets the detection requirements. Therefore, the secondary amplification method is preferably used for PD-L1 detection.

[0157] Example 3 Investigation of red blood cell removal conditions

[0158] NCI-H820 cells were labeled with CFSE fluorescent dye; the labeled NCI-H820 cells were added to the blood; 1x red blood cell lysis buffer was used at a volume ratio (blood volume: 1x red blood cell lysis buffer) of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 (corresponding to Figure 4 1 ml, 1.5 ml, 2 ml, 2.5 ml, 3 ml, 3.5 ml, 4 ml, 4.5 ml) of the solution was added to the blood supplemented with NCI-H820 cells. After lysis, the cells were collected and resuspended, and the red blood cell integrity was detected by flow cytometry, and the red blood cell recovery rate was calculated.

[0159] The results showed that there was no significant difference in the number of red blood cells after the red blood cells were treated with different ratios of lysis buffer, and the recovery rate of intact cells ( Figure 4) showed no significant difference, indicating no significant difference in tumor cell integrity, and the lysis effect was good (fewer residual cell particles). Therefore, the dosage of 1x red blood cell lysis buffer can be used based on a blood volume: lysis buffer volume ratio of 1:2-1:9, with 1:2 or 1:3 being preferred.

[0160] Example 4 Study on sample fixation conditions

[0161] NCI-H820 cells were lysed according to the lysis conditions described in Example 3 above; after resuspending the cells, 4% PFA (paraformaldehyde) was added at a volume ratio of 1:1 (fixative reagent: test sample) and fixed at room temperature for 15 minutes, 30 minutes, and 60 minutes, and 2% PFA was added at room temperature for 15 minutes, 30 minutes, and 60 minutes, respectively. The cell fluid was washed with binding buffer and collected; CK detection antibody and CD45 detection antibody were added to the cell fluid and incubated overnight (incubation for 12-20 hours). After the cells were collected, they were detected using a flow cytometer.

[0162] The results showed that 2% PFA fixation at room temperature for 30 min and 1% PFA fixation at room temperature for 60 min retained a larger proportion of cells ( Figure 5 A) in the figure indicates that the cell integrity is well preserved and the CK staining signal value is also high ( Figure 5 Therefore, it is recommended that samples be fixed with 2% PFA for 30 min at room temperature or 1% PFA for 60 min at room temperature.

[0163] Example 5 Exploration of the concentration of detection antibodies and corresponding secondary antibodies

[0164] 5.1. Exploration of the Concentration of PD-L1 Detection Antibodies

[0165] Flow cytometry and cell enrichment separation methods were used to explore the optimal concentration of PD-L1 detection antibodies in the secondary amplification detection scheme.

[0166] 5.1.1 Flow cytometry

[0167] NCI-H820 cells were diluted to 300,000 cells / ml using binding buffer, and 100 μl of cell fluid was taken per portion; NCI-H820 cells were incubated with different concentrations of PD-L1 detection antibody (HRP-labeled PD-L1 antibody, the concentrations were 16 μg / ml, 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.0625 μg / ml, 0.03125 μg / ml, 0.016 μg / ml); Alexa Fluor 488-labeled goat anti-HRP fluorescent secondary antibody was added and incubation continued. After collecting the cells, washing, and then using a flow cytometer for mixed detection.

[0168] 5.1.2 Cell Enrichment and Separation Methods

[0169] 500 freshly prepared NCI-H820 cells were added to 8 ml of blood; the NCI-H820 cell sample was lysed at a ratio of 1:3 according to the lysis conditions described in Example 3 above; FBS was added for resuspending and then PFA was added for fixation at room temperature for 1 hour; the cells were collected and washed, and then biotin-labeled capture antibodies (including PD-L1, Epcam, HER2, Trop2, and EGFR antibodies, using a mass concentration ratio of 2:1:1:1:1), iFluor647-labeled CK detection antibody, iFluor488-labeled CD45 detection antibody, and HRP-labeled PD-L1 detection antibody were added and incubated overnight; the cells were collected and washed, and then streptavidin-labeled magnetic beads and Alexa Fluor594-labeled goat anti-HRP secondary antibody were added for incubation; the cells were collected, washed, resuspended, and DAPI dye was added. After enrichment using the LiquidBiopsy system, the cells were molecularly enriched and separated using a fluorescence microscope, and the PD-L1 detection signal fluorescence value of the NCI-H820 cells was counted. The concentrations of PD-L1 detection antibodies used in the experimental groups were 8μg / ml, 4μg / ml, 2μg / ml, and 1μg / ml, respectively.

[0170] The results showed that in the flow cytometry test results, when the PD-L1 detection antibody was used at a concentration of 1-15μg / ml, the PD-L1 detection antibody and tumor cells could be well recognized and bound, obtaining a fluorescent signal that met the detection requirements. At 4μg / ml, it basically reached saturation ( Figure 6 ), so the recommended concentration of PD-L1 detection antibody is 4μg / ml. In the cell enrichment and separation method test, when other detection antibodies and capture antibodies are present in the detection system, the PD-L1 detection antibody can also be well recognized and bound to tumor cells when the concentration is 1-8μg / ml, obtaining a fluorescent signal that meets the detection requirements ( Figure 7 ), 4μg / ml and 8μg / ml, the PD-L1 detection signal fluorescence value of NCI-H820 cells is higher, and at 4μg / ml and 8μg / ml, the fluorescence values ​​of the two are close. Therefore, in the detection system of the present invention, the use of the PD-L1 detection antibody at a concentration of 1-8μg / ml can obtain good detection results. For economic considerations, the recommended concentration of the PD-L1 detection antibody is 4μg / ml.

[0171] 5.2 Study on the concentration of anti-HRP fluorescent secondary antibody

[0172] PD-L1-positive NCI-H820 cells and PD-L1-negative A549 cells were tested. 500 freshly prepared NCI-H820 cells and 500 freshly prepared A549 cells were added to 8 ml of blood. The procedure was as described in Example 5.1.2. The concentrations of the fluorescent goat anti-HRP secondary antibody used in the experimental groups were 10 μg / ml, 8 μg / ml, 5 μg / ml, and 2.5 μg / ml, respectively. The fluorescence values ​​of the PD-L1 detection signals from the enriched and isolated NCI-H820 and A549 cells were calculated.

[0173] The results showed that ( Figure 8 ) When the goat anti-HRP fluorescent secondary antibody is used at a concentration of 2.5-10 μg / ml, PD-L1-positive NCI-H820 cells exhibit a high fluorescent signal (mean fluorescence intensity 60-120), while PD-L1-negative A549 cells exhibit a low signal (mean fluorescence intensity 20-30). This concentration range produces good detection results. Overall, the recommended concentration of goat anti-HRP fluorescent secondary antibody is 5 μg / ml.

[0174] 5.3 Study on the concentration of cancer cell antigen antibodies

[0175] Flow cytometry and cell enrichment separation methods were used to test and explore the optimal concentration of cancer cell antigen detection antibodies (CK antibodies were selected in this example) in the secondary amplification detection scheme.

[0176] 5.3.1 Flow cytometry

[0177] NCI-H820 cells were diluted to 300,000 cells / ml using binding buffer, and 100 μl of cell fluid was taken per portion; NCI-H820 cells were mixed and incubated with different concentrations of iFluor647-labeled CK detection antibodies (the concentrations used were 16 μg / ml, 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.0625 μg / ml, 0.03125 μg / ml, and 0.016 μg / ml), the cells were collected, washed, and then detected using a flow cytometer.

[0178] 5.3.1 Cell Enrichment and Separation Methods

[0179] 500 freshly prepared NCI-H820 cells were added to 8 ml of blood; the steps were as described in Example 5.1.2. The experimental groups set the CK detection antibody concentrations to 2 μg / ml, 4 μg / ml, 8 μg / ml, and 16 μg / ml, respectively, and the CK detection signal fluorescence values ​​of the enriched and isolated NCI-H820 cells were calculated.

[0180] The results showed that when the concentration of CK detection antibody was 2-16 μg / ml, CK antibody and tumor cells could be well recognized and combined, and the flow cytometry signal value was greater than 2.5 million, with a strong fluorescence signal ( Figure 9 ), At the same time, in the cell enrichment and separation method, when other detection antibodies and capture antibodies are present in the detection system, CK antibodies and tumor cells can also be well recognized and combined, and a fluorescent signal that meets the detection requirements can be obtained ( Figure 10 ). Therefore, in the detection system of the present invention, the use concentration of cancer cell antigen detection antibody is 2-16 μg / ml to obtain good detection results. Based on the comprehensive consideration of economy and detection efficiency, the optimal concentration of CK detection antibody is 4 μg / ml.

[0181] 5.4 Study on the concentration of leukocyte common antigen antibodies

[0182] Flow cytometry and cell enrichment separation methods were used to test and explore the optimal concentration of the leukocyte common antigen detection antibody (CD45 antibody was selected in this example) in the secondary amplification detection scheme.

[0183] 5.4.1 Flow cytometry

[0184] WBCs were diluted to 300,000 / ml using binding buffer, and 100 μl of cell fluid was taken per portion; WBCs were mixed and incubated with different concentrations of iFluor488-labeled CD45 detection antibodies (20 μg / ml, 10 μg / ml, 5 μg / ml, 2.5 μg / ml, 1.25 μg / ml, 0.625 μg / ml, 0.3125 μg / ml, 0.16 μg / ml, 0.08 μg / ml, 0.04 μg / ml, 0.02 μg / ml), the cells were collected, washed, and then detected using a flow cytometer.

[0185] 5.4.2 Cell Enrichment and Separation Methods

[0186] 500 freshly prepared NCI-H820 cells were added to 8 ml of blood; the steps were as described in Example 5.1.2. The concentrations of CD45 detection antibody in the experimental groups were set at 2.5 μg / ml, 5 μg / ml, 10 μg / ml, and 20 μg / ml, respectively. The fluorescence values ​​of the CD45 detection signals of the enriched and separated leukocytes were calculated.

[0187] When the concentration of CD45 detection antibody was 2.5-20 μg / ml, CD45 antibody and tumor cells could be well recognized and bound, and the flow cytometry signal value was greater than 400,000, with a strong fluorescence signal ( Figure 11), At the same time, in the cell enrichment and separation method, when other detection antibodies and capture antibodies are present in the detection system, CD45 antibodies and tumor cells can also be well recognized and combined, and a fluorescent signal that meets the detection requirements can be obtained ( Figure 12 ). Therefore, in the detection system of the present invention, the use concentration of leukocyte common antigen detection antibody is 2.5-20μg / ml to obtain good detection results. Based on the comprehensive consideration of economy and detection efficiency, the optimal concentration of CK detection antibody is 5μg / ml.

[0188] Example 6 Sample Sealing Condition Test

[0189] Lung cancer patient samples were obtained as described in Example 5.1.2. After lysing and fixing the samples, cells were collected and washed. Human IgG was then added to the experimental groups at concentrations of 0, 2.5, 5, 10, 15, and 20 μg / mL and incubated at room temperature. The background signal value of leukocyte CD45 and the positive signal value of tumor cells after enrichment and separation were calculated.

[0190] The results showed that ( Figure 13 ), when blocked with human IgG, it can reduce white blood cells ( Figure 13 B) of the background signal fluorescence value, while increasing the positive signal of tumor cells ( Figure 13 The fluorescence values ​​in A) are shown in Table 1. At a concentration of 10 μg / ml-15 μg / ml, the fluorescence value of the white blood cell background signal is lowest, while the fluorescence value of the tumor cell positive signal is highest. Therefore, human IgG is recommended for sample blocking at a concentration of 10 μg / ml-15 μg / ml, preferably 10 μg / ml.

[0191] Example 7: Exploration of Capture Antibody Ratio

[0192] 7.1 Exploration of different capture antibody ratios

[0193] Take 100 μl of NCI-H820 cells, and the steps are as described in Example 5.1.2. In the experimental group, PD-L1 capture antibody and capture antibodies against other targets (Epcam: HER2: Trop2: EGFR are 1:1:1:1, 2:1:1:1, 1:2:1:1, 1:1:2:1, 1:1:1:2, 2:2:1:1, 2:1:2:1, 2:1:1:2, 1:1:2:2, 1:2:2:1, 1:2:1:2, 2:2:2:2, respectively) are added to 100 μl / portion of binding buffer at different mass ratios, and detected by flow cytometry.

[0194] The results showed that when the mass ratio of EGFR antibody to capture antibodies against other targets (Epcam, HER2, Trop2) in the capture antibody was 2:(1-2), the capture efficiency was higher. Therefore, the ratio of EGFR antibody to Epcam, HER2, Trop2 antibody in the capture antibody was selected as 2:1:1:1 ( Figure 14 ).

[0195] 7.2 Capture effect verification

[0196] 500 NCI-H1975 (human non-small cell lung adenocarcinoma cells), Calu-1 (human lung cancer cells), NCI-H441 (human lung adenocarcinoma cells), A431 (human skin squamous cell carcinoma cells), HepG2 (human liver cancer cells), HCC827 (human non-small cell lung adenocarcinoma cells), PC-3 (human prostate cancer cells), SW480 (human colorectal cancer cells), HGC-27 cells (human gastric cancer cells), NCI-H820 (human papillary lung adenocarcinoma cells), NCI-H1650 (human non-small cell lung adenocarcinoma cells), and MDA-MB-231 (human breast cancer cells) were added to 8 mL / portion of blood, respectively. The steps were as described in Example 5.1.2. Capture antibodies including only HER2, Trop2, and Epcam were added at the same concentration as controls, and the separated cells were analyzed by scanning using a fluorescence microscope.

[0197] The results showed that when using capture antibodies targeting HER2, Epcam, and Trop2, the capture efficiency of the lung cancer cell line H1650 cells could reach 96.4%, but the capture efficiency for other lung cancer cell lines was lower. After adding EGFR antibodies and PD-L1 antibodies in proportion, the capture efficiency of all cell lines was greater than 80% ( Figure 15 ).

[0198] Example 8 Comparison of the PD-L1 Detection System of the Present Invention with the Conventional PD-L1 Detection System

[0199] 8 ml of blood was collected from patients with PD-L1 tissue-positive lung cancer, and the steps were as described in Example 5.1.2. The fluorescence value of the PD-L1 detection signal of the enriched and separated PD-L1-positive tumor cells was calculated.

[0200] At the same time, the results were compared with other technology platforms in the literature (Control 1: Fankhauser CD, Curioni Fontecedro A, Beyer J, et al. Frequent expression of PD-L1 in testicular germ cell tumors[J]. 2015.; Control 2: Guibert N, Delaunay M, Lusque A, et al. PD-L1 expression incirculating tumor cells of advanced non-small cell lung cancer patients treated with nivolumab[J]. Lung Cancer, 2018, 120: 108-112.; Control 3: Yue C, Jiang Y, Li P, et al. Dynamic change of PD-L1 expression on circulating tumor cells in advanced solid tumor patients undergoing PD-1blockade therapy[J]. Oncoimmunology, 2018, 7(7): e1438111.).

[0201] The results showed that ( Figure 16 ) The PD-L1-positive cells detected by this method from PD-L1-positive lung cancer patients showed stronger detection signals and better imaging quality than those of the other three control platforms. Therefore, the PD-L1 detection method developed in this case has higher detection specificity and sensitivity than other technology platforms.

[0202] Example 9: Exploration of the method for determining PD-L1 positive cells

[0203] 500 PD-L1 positive cells NCI-H820 and 500 PD-L1 negative cells A549 were added to 8 ml of blood respectively; the steps were as described in Example 5.1.2, and the enriched and separated cells were scanned and analyzed using a fluorescence microscope. The PD-L1 signal fluorescence value and its CD45 signal fluorescence value of each image of tumor cells, and the PD-L1 signal fluorescence value and its CD45 signal fluorescence value of each image of white blood cells were counted, and the ratio was calculated at the same time. The results showed that when the ratio was 3.0, PD-L1 positive cells NCI-H820 and PD-L1 negative cells A549 could be well distinguished ( Figure 17A in the figure), the sensitivity and specificity were both 98%, but there was overlap between PD-L1 positive immune cells and PD-L1 negative immune cells in leukocytes. When the fluorescence value was set to 40, PD-L1 positive immune cells and PD-L1 negative immune cells were well distinguished ( Figure 17 B), the sensitivity and specificity were both above 99% ( Figure 17 ).

[0204] Example 10 Performance of the PD-L1 detection system of the present invention

[0205] The PD-L1 detection kit developed in this case was used to perform accuracy and precision tests.

[0206] 10.1 Accuracy

[0207] The recovery rate of cell addition test was tested by taking 1ml / portion of white blood cells (cell concentration 50 million / ml (calculated based on the white blood cell content of normal people, white blood cells 4-10×10 6 / ml, that is, the white blood cell content in 8mL of blood is 3.2-8×10 7 Therefore, 5×10 white blood cells were selected. 7 25, 100, 500, and 1000 NCI-H820 cells were added, along with 18 μl of capture antibody, 5 μl of CK detection antibody, 5 μl of CD45 detection antibody, and 2 μl of PD-L1 detection antibody (final concentration 4 μg / ml), and incubated overnight at 2-8°C. The next day, the sample tubes were centrifuged at 500 g for 5 minutes to collect the cells, and 1 ml of binding buffer was added to resuspend the cells. The sample tubes were centrifuged at 500 g for 5 minutes to collect the cells, and 1 ml of binding buffer, 30 μl of magnetic beads, and 1.7 μl of goat anti-HRP fluorescent secondary antibody were added, and the cells were incubated at 2-8°C for 60 minutes. Place the sample tube in a centrifuge and centrifuge at 500g for 5 minutes to collect the cells. Add 1 ml of binding buffer to resuspend the cells. Place the sample tube in a centrifuge and centrifuge at 500g for 5 minutes to collect the cells. Resuspend the cells in 1.1 ml of binding buffer and add 5 μl of DAPI nucleic acid dye. Cells were enriched using the LiquidBiopsy system and analyzed by fluorescence microscopy. Repeat the experiment four times for each cell concentration. Calculate the recovery rate. R is the recovery rate; M is the actual number of recovered cells; N is the number of cell lines added.

[0208] Table 1 PD-L1 positive cell accuracy test results

[0209]

[0210] The results showed (Table 1) that the recovery rates were all qualified (within the range of 70%-130%).

[0211] 10.2 Precision Test

[0212] The cell addition test method was used to detect the presence of 10 cells in 10 groups. 100 NCI-H820 cells were added to 5×10 7 in white blood cells (calculated based on the white blood cell count of a normal person, white blood cells 4-10×10 6 / mL, that is, the white blood cell content in 8ml of blood is 3.2-8×10 7 Therefore, 5×10 white blood cells were selected. 7 ), add 18 μl capture antibody, 5 μl CK detection antibody, 5 μl CD45 detection antibody, 2 μl PD-L1 detection antibody (final concentration 4 μg / ml), and incubate overnight at 2-8°C. The next day, place the sample tube in a centrifuge and centrifuge at 500g for 5 minutes to collect the cells. Then add 1 ml binding buffer to resuspend the cells. Place the sample tube in a centrifuge and centrifuge at 500g for 5 minutes to collect the cells. Add 1 ml binding buffer, 30 μl magnetic beads, and 1.7 μl goat anti-HRP fluorescent secondary antibody, and incubate at 2-8°C for 60 minutes. Place the sample tube in a centrifuge and centrifuge at 500g for 5 minutes to collect the cells. Add 1 ml of binding buffer to resuspend the cells. Place the sample tube in a centrifuge and centrifuge at 500g for 5 minutes to collect the cells. Use 1.1 ml of binding buffer to resuspend the cells. Add 5 μl of DAPI nucleic acid dye and use the LiquidBiopsy system for cell enrichment. Scan and analyze the enriched and separated cells using a fluorescence microscope. Repeat the experiment 10 times and calculate the mean M, standard deviation SD, and coefficient of variation CV.

[0213] M is the mean; SD is the standard deviation.

[0214] Table 2 PD-L1 positive cell precision test results

[0215]

[0216] The results (Table 2) show that the recovery rate for each test group was acceptable (81%-96%), and the CV for the ten test groups was 6%, which is considered normal (less than 20%). Therefore, the PD-L1 detection reagent developed in this case study has high accuracy and precision.

[0217] 10.3 Detection Limit Test

[0218] The cell addition test method was used to detect the presence of 0 or 5 NCI-H820 cells in 5×10 7 in white blood cells (calculated based on the white blood cell count of a normal person, white blood cells 4-10×106 / mL, that is, the white blood cell content in 8ml of blood is 3.2-8×10 7 Therefore, 5×10 white blood cells were selected. 7 ), add 18 μl capture antibody, 5 μl CK detection antibody, 5 μl CD45 detection antibody, 2 μl PD-L1 detection antibody (final concentration 4 μg / ml), and incubate overnight at 2-8°C. The next day, place the sample tube in a centrifuge and centrifuge at 500g for 5 minutes to collect the cells. Then add 1 ml binding buffer to resuspend the cells. Place the sample tube in a centrifuge and centrifuge at 500g for 5 minutes to collect the cells. Add 1 ml binding buffer, 30 μl magnetic beads, and 1.7 μl goat anti-HRP fluorescent secondary antibody, and incubate at 2-8°C for 60 minutes. Place the sample tube in a centrifuge and centrifuge at 500g for 5 minutes to collect the cells. Then add 1ml of binding buffer to resuspend the cells. Place the sample tube in a centrifuge and centrifuge at 500g for 5 minutes to collect the cells. Use 1.1ml of binding buffer to resuspend the cells. Add 5μl of DAPI nucleic acid dye and use the LiquidBiopsy system to enrich the cells. Scan and analyze the enriched and separated cells using a fluorescence microscope. Repeat the experiment 6 times. The detection limit is calculated according to the analytical sensitivity performance evaluation method in the "Clinical Laboratory Methodology Evaluation" and the calculation formula is LoD = LoB + C β SD S , C β =1.645 / [1-1 / (4×f)]).

[0219] Table 3 Detection limit test results of PD-L1 positive cells

[0220]

[0221] The results show (Table 3) that the results of adding 0 NCI-H820 cells are all 0. After calculation:

[0222] SD S =0.52, f(degrees of freedom)=6×(6-1)=30, C β =1.645 / [1-1 / (4×f)]=1.66;

[0223] LoD=LoB+C β SD S =0+1.66×0.52=0.86<1.

[0224] 10.4 Specificity Test

[0225] The cell addition test method was used to detect the presence of 500 PD-L1 negative cells (A549) in 5×10 7 in white blood cells (calculated based on the white blood cell count of a normal person, white blood cells 4-10×106 cells / mL, that is, the cell content in 8 ml of blood is 3.2-8×10 7 Therefore, 5×10 white blood cells were selected. 7 ), add 18μl capture antibody, 5μl CK detection antibody, 5μl CD45 detection antibody, and 2μl PD-L1 detection antibody (final concentration 4μg / ml), and incubate overnight at 2-8°C. The next day, place the sample tube in a centrifuge and centrifuge at 500g for 5 minutes to collect the cells. Then add 1ml binding buffer to resuspend the cells. Place the sample tube in a centrifuge and centrifuge at 500g for 5 minutes to collect the cells. Add 1ml binding buffer, 30μl magnetic beads, and 1.7μl goat anti-HRP fluorescent secondary antibody. Incubate at 2-8°C for 60 minutes. Place the sample tube in a centrifuge and centrifuge at 500g for 5 minutes to collect the cells. Then add 1ml binding buffer to resuspend the cells. Place the sample tube in a centrifuge and centrifuge at 500g for 5 minutes to collect the cells. Resuspend the cells in 1.1ml binding buffer and add 5μl DAPI nucleic acid dye. Cell enrichment is performed using the LiquidBiopsy system. The enriched and separated cells are scanned and analyzed using a fluorescence microscope. Repeat the experiment three times.

[0226] The results showed that out of a total of 1563 A549 cells, 1558 were PD-L1 negative, with a specificity of 99.7%.

[0227] 10.5 Breast cancer, gastric cancer, and skin squamous cell carcinoma tests

[0228] Add 500 MDA-MB-231 (human breast cancer) cells, 500 HGC-27 (human gastric cancer) cells, and 500 A431 (human skin squamous cell carcinoma) cells to the blood at 8 mL / aliquot. Follow the same steps as in Example 5.1.2. Analyze the enriched and isolated cells using a fluorescence microscope to count the number of circulating PD-L1-positive tumor cells.

[0229] Table 4 Test results of breast cancer, gastric cancer and skin squamous cell carcinoma cells

[0230] Group MDA-MB-231 HGC-27 A431 Recycling volume (units) 480 475 465 Recovery rate (%) 96％ 95％ 93％

[0231] The results showed that the detection efficiency of circulating PD-L1-positive tumor cells in human breast cancer cell samples was 96%, in human gastric cancer cell samples was 95%, and in human skin cancer samples was 93%. Therefore, the detection method and kit provided in this case also have high detection efficiency for PD-L1-positive cells in other cancer types.

Claims

1. A kit for detecting circulating PD-L1-positive cells in a sample to be tested, the kit comprising a detection reagent and a PD-L1-positive cell capture reagent; the detection reagent comprising a PD-L1 antibody labeled with a first non-human marker, an antibody to a common leukocyte antigen labeled with a first fluorescent molecule, an antibody to a cancer cell antigen labeled with a second fluorescent molecule, and an antibody against the first non-human marker labeled with a third fluorescent molecule; in, The first, second and third fluorescent molecules have different excitation wavelengths or emission wavelengths; The leukocyte common antigen antibody is selected from one or more of CD45, CD11b, CD14, CD68, CD16, CD56, CD57, CD59, CD94, CD3, CD4, CD8, CD80, CD86, CD40, CD21, CD19, CD20, CD18, CD32, CD44, CD55 and CD66b antibodies; The cancer cell antigen antibody is selected from one or more of CK, Epcam, HER2, Trop2, EGFR and CEA antibodies; The first non-human marker is selected from one or more of HRP and alkaline phosphatase markers; The mass ratio of the PD-L1 antibody in the capture reagent to the PD-L1 antibody in the detection reagent is 1:(3.2-4.8); The PD-L1 positive cell capture reagent includes a capture antibody and magnetic beads, wherein the capture antibody is a PD-L1 antibody labeled with a second non-human marker and a tumor-specific protein marker antibody labeled with a second non-human marker; The magnetic beads are magnetic beads labeled with an antibody against the second non-human marker; the tumor-specific protein marker antibody labeled with the second non-human marker is a mixed antibody, which includes Epcam, HER2, Trop2 and EGFR antibodies labeled with the second non-human marker; wherein the mass ratio of Epcam antibody: HER2 antibody: Trop2 antibody: EGFR antibody is (1-2): (1-2): (1-2): 2; the mass ratio of the tumor-specific protein marker antibody labeled with the second non-human marker and the PD-L1 antibody labeled with the second non-human marker is 5: (0.5-2); The PD-L1 antibody in the capture reagent and the PD-L1 antibody in the detection reagent are non-competing antibodies; The first and second non-human markers are different non-human markers.

2. The kit according to claim 1, wherein The circulating PD-L1-positive cells include circulating PD-L1-positive tumor cells and circulating PD-L1-positive immune cells; The circulating PD-L1-positive tumor cells are selected from one or more of melanoma cells, non-small cell lung cancer cells, renal cancer cells, prostate cancer cells, pancreatic cancer cells, bile duct cancer cells, esophageal cancer cells, intestinal cancer cells, breast cancer cells, small cell lung cancer cells, gastric cancer cells, liver cancer cells, human placental chorionic villus cells, cervical cancer cells, ovarian cancer cells, bladder cancer cells, head and neck tumor cells, skin cancer cells, thymic cancer cells, lymphoma cells, and nasopharyngeal cancer cells; The sample to be tested is a body fluid sample, which is selected from a blood sample, a pleural effusion sample, an ascites sample, a cerebrospinal fluid sample, a urine sample and a sputum sample.

3. The kit according to claim 1 or 2, wherein The first, second and third fluorescent molecules are selected from iFluor647, iFluor488, iFlour594, iFlour555, iFlour568, iFlour750, FITC, Alexa Fluor488, AlexaFluor555, Alexa Fluor568, Alexa Fluor594, Alexa Fluor750, APC and Alexa Fluor647, and the first, second and third fluorescent molecules are fluorescent molecules with different excitation wavelengths or emission wavelengths.

4. The kit according to claim 1 or 2, wherein The antibody against the common leukocyte antigen is CD45 antibody; the antibody against the cancer cell antigen is CK antibody.

5. The kit according to claim 1 or 2, wherein The mass ratio of Epcam, HER2, Trop2 and EGFR antibodies in the capture antibody is 1:1:1:2, 2:1:1:2, 1:1:2:2, 1:2:1:2, 2:2:1:2, 2:1:2:2 or 2:2:2:

2.

6. The kit according to claim 1 or 2, wherein The detection reagents include HRP-labeled PD-L1 antibody, iFluor488-labeled CD45 antibody, iFluor647-labeled CK antibody, and Alexa Fluor594-labeled anti-HRP antibody.

7. The kit according to claim 1 or 2, wherein The kit includes HRP-labeled PD-L1 antibody, iFluor488-labeled CD45 antibody, iFluor647-labeled CK antibody, Alexa Fluor594-labeled anti-HRP antibody, biotin-labeled Epcam, HER2, Trop2, EGFR, PD-L1 antibodies and streptavidin-labeled magnetic beads.

8. The kit according to claim 7, wherein The concentration of the HRP-labeled PD-L1 antibody in the kit is 1-8 μg / ml, the concentration of the Alexa Fluor 594-labeled anti-HRP antibody is 1.25-10 μg / ml, the concentration of the CD45 antibody is 2.5-20 μg / ml, and the concentration of the CK detection antibody is 2-16 μg / ml.

9. The kit according to claim 1 or 2, wherein The sample pre-treatment reagent and the blocking reagent are also included, wherein the pre-treatment reagent includes a red blood cell removal reagent and a cell fixation reagent; the blocking reagent is selected from one or more solutions of human IgG, mouse IgG, rabbit IgG, and goat IgG; The red blood cell removal reagent contains NH4 + Reagent; the cell fixation reagent is a solution containing one or more of paraformaldehyde, formaldehyde, glutaraldehyde, methanol, and glacial acetic acid.

10. The kit according to claim 9, wherein The volume ratio of the red blood cell removal reagent to the sample to be tested is (1:2)-(1:9).

11. The kit according to claim 10, wherein The volume ratio of the red blood cell removal reagent to the sample to be tested is 1:

3.

12. The kit according to claim 9, wherein The volume ratio of the cell fixation reagent to the sample to be tested is (2:1)-(1:5).

13. The kit according to claim 12, wherein The volume ratio of the cell fixation reagent to the sample to be tested is 1:

1.

14. The kit according to claim 9, wherein The cell fixation reagent is used at a concentration of 0.25%-4%.

15. The kit according to claim 14, wherein The cell fixation reagent is used at a concentration of 2%.

16. The kit according to claim 9, wherein The blocking reagent is used at a concentration of 2.5-20 μg / ml.

17. The kit according to claim 16, wherein The blocking reagent is human IgG.

18. The method for preparing the kit according to any one of claims 1 to 17, wherein: The following steps are involved: (1) Preparation steps of PD-L1 positive cell capture reagent, which includes capture antibody preparation and magnetic bead preparation; (2) a step of preparing a PD-L1 detection antibody, which includes the steps of preparing a PD-L1 antibody labeled with a first non-human marker and an antibody against the first non-human marker labeled with a third fluorescent molecule; (3) Preparation steps of antibodies against common leukocyte antigens and cancer cell antigens; (4) Reagent assembly.

19. A method for detecting circulating PD-L1 positive cells in a sample using the kit of claim 1 for non-diagnostic purposes, the method comprising the following steps: (1) Obtain the sample to be tested from the subject, add the capture antibody, the leukocyte common antigen antibody labeled with the first fluorescent molecule, the cancer cell antigen antibody labeled with the second fluorescent molecule, and the PD-L1 antibody labeled with the first non-human marker for incubation; (2) adding magnetic beads labeled with a second non-human marker and an antibody against the first non-human marker labeled with a third fluorescent molecule, and separating the magnetic beads; (3) After adding nucleic acid dye to separate cells, use fluorescence microscopy to scan and analyze; (4) Result judgment; Wherein, the first, second and third fluorescent molecules have different emission wavelengths; In step (4), if the fluorescence value of the third fluorescent molecule is ≥40, and the fluorescence value of the third fluorescent molecule / the fluorescence value of the first fluorescent molecule is ≥3.0, the PD-L1 positive cell is a positive tumor cell; If the fluorescence value of the third fluorescent molecule is ≥40, and the fluorescence value of the third fluorescent molecule / the fluorescence value of the first fluorescent molecule is <3.0, the PD-L1 positive cell is a positive immune cell.

20. Use of the kit according to any one of claims 1 to 17, or the kit prepared by the method according to claim 18, in preparing a reagent or kit for detecting circulating PD-L1-positive cells in a test sample, wherein the circulating PD-L1-positive cells include circulating PD-L1-positive tumor cells and circulating PD-L1-positive immune cells.

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