Rare cell capture reagent combination and method

Abstract

The invention provides a rare cell capture reagent combination and a capture method thereof. The reagent combination comprises a substrate, a capture antibody, a confining liquid, an incubation buffer solution and magnetic beads, the substrate is a compound as shown in a formula (I) and a derivative thereof; the capture antibody comprises but is not limited to one or more of an anti-CK antibody, an anti-EpCAM antibody, an anti-PD-L1 antibody, an anti-HER2 antibody, an anti-TROP2 antibody, an anti-c-MET antibody, an anti-Claudin18.2 antibody, an anti-EGFR antibody, an anti-MUC1 antibody, an anti-HER3 antibody, an anti-CD31 antibody, an anti-CD44 antibody and the like; the capture antibody is an oxidoreductase labeled antibody; and the magnetic beads are avidin labeled magnetic beads. The reagent combination and the capturing method can be widely used for capturing and separating rare cells with low expression of specific antigens. Compared with a traditional secondary antibody capturing method, the method disclosed by the invention is higher in capturing efficiency and higher in capturing sensitivity, and meanwhile, the specificity is not influenced.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and particularly relates to a reagent combination and method for capturing rare cells. Background Art

[0002] Rare cells refer to some atypical rare cells in biological fluid samples (including blood, pleural effusion, ascites, urine, cerebrospinal fluid, etc.), and their number is often extremely small. Rare cells mainly include circulating tumor cells (CTCs), circulating epithelial cells, circulating endothelial cells, tumor stem cells, etc. Studies have shown that the collection and analysis of rare cells are of great guiding significance for finding potential treatment mechanisms, pathological mechanisms and targeted drug development of diseases. At present, the main research methods for detecting rare cells in blood include flow sorting technology, morphological separation methods, density gradient centrifugation, membrane filtration and immunomagnetic separation technology. For example, Aria can achieve high-speed cell sorting, but the instantaneous laser of flow sorting will damage the sorted cells, and the activity and function of the cells will be impaired after sorting.

[0003] Among them, circulating tumor cells (CTC) refer to tumor cells that fall off from the primary lesion and invade the blood circulation. Studies have confirmed that CTC can evade the body's immunity and reside in the primary site or distant organs, thereby forming recurrence and metastasis lesions. The specific mechanism is: when the primary tumor grows to a certain stage, it will invade the surrounding blood vessels. Tumor cells first grow by attaching to the basement membrane of the blood vessels through integrins; when the number of tumor cells gradually increases, the matrix metalloproteinases they secrete also gradually increase, and they gradually digest type IV collagen, break through the basement membrane barrier, and enter the blood circulation, which is called CTC; after CTC enters the blood, it will travel throughout the body with the blood circulation, forming recurrence and metastasis. Therefore, the detection of CTC has important clinical prospects and functions for early auxiliary diagnosis, medication guidance, and efficacy monitoring of patients. However, due to the small number of CTCs in the blood and the high white blood cell background, generally about 10 7 The number of CTCs in white blood cells is only in the single digit (2-10 / mL), resulting in a low CTC detection rate using existing methods, which poses a great challenge to its clinical application.

[0004] At present, the existing detection methods of rare cells generally first enrich and purify rare cells through a forward capture method based on cell-specific antigens, then use specific detection antibodies to mark the rare cells, add nuclear dyes and stain them before detecting them under a fluorescence microscope. This capture method requires that the target cells have a high level of target antigen expression. However, in actual clinical applications, the same marker (such as HER2, PD-L1, CK, etc.) has a large difference in expression levels in different individual patients. When the expression level of the detection marker of the target cell is low, it is often difficult to effectively identify the target CTC using conventional forward capture methods, resulting in false negative clinical results and delays in treatment progress.

[0005] Therefore, there is an urgent need to develop a new kit and detection method that can capture and detect rare cells to solve the problems of weak detection signals, low sensitivity, and low detection efficiency of existing detection methods. Summary of the invention

[0006] The purpose of the present disclosure is to provide a reagent combination and method for capturing rare cells, which utilizes an enzyme (e.g., oxidoreductase HRP) to catalyze the covalent attachment of a biotin-labeled substrate to a tyrosine residue of a capture antibody, while generating an activated substrate, so that the antibody and the substrate are stably covalently bound, the biotin is indirectly attached and enriched around the target cells, and then magnetic beads are added, thereby achieving high-sensitivity capture of rare cells.

[0007] In one aspect, the present disclosure provides a reagent combination for rare cell capture, wherein the reagent combination comprises a biotin-labeled substrate and a capture antibody, wherein the biotin-labeled substrate is selected from the compounds or derivatives shown in formula (I), formula (II) or formula (III),

[0008]

[0009]

[0010] in:

[0011] A is

[0012] is a single bond or a double bond;

[0013] R 1 Selected from -(CH 2 ) n1 -、-CH(CH 3 )-、-C(CH 3 ) 2 -, cycloalkyl, heterocyclyl, aryl or heteroaryl;

[0014] Preferably, the cycloalkyl group is selected from

[0015] Preferably, the heterocyclic group is selected from

[0016] Preferably, the aryl group is selected from

[0017] R 2 Selected from -OH, -NH 2 , -SH and -H;

[0018] X is and / or

[0019] n1 is an integer selected from 0 to 10;

[0020] n2 is selected from an integer of 1-300, preferably, n2 is selected from an integer of 1-10;

[0021] n3 is selected from an integer of 1-10.

[0022] In another aspect, the present disclosure provides a rare cell capture kit, which comprises the aforementioned reagent combination.

[0023] In another aspect, the present disclosure provides a method for capturing rare cells based on the aforementioned reagent combination or the aforementioned kit.

[0024] In another aspect, the present disclosure provides a use of the aforementioned reagent combination, the aforementioned kit and / or the aforementioned method in preparing a drug for capturing cells.

[0025] The beneficial effects achieved by the present disclosure are at least as follows:

[0026] 1. The present disclosure provides a new reagent combination, which has high sensitivity and good separation effect, and can be widely used to capture and separate rare cells with low expression of specific antigens.

[0027] 2. Compared with the traditional H 2 O 2 The blocking solution is directly used for the blocking of cells. When the cells are damaged, they are easily adsorbed on the wall of the centrifuge tube, resulting in cell loss. The present invention uses 5% FBS in H 2 O 2 The cells are sealed to effectively seal damaged areas and prevent cell adhesion.

[0028] 3. The present disclosure provides a new rare cell capture method. Compared with the traditional secondary antibody capture method, the present disclosure method has higher capture efficiency and higher capture sensitivity without affecting specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1The results of the closed time exploration are shown.

[0030] Figure 2 Results are shown for a range of blocking buffer concentrations (0.05-3%).

[0031] Figure 3 Results are shown for investigation of serum concentration (0%-10%) at the time of blocking.

[0032] Figure 4 Results of a HRP-labeled capture antibody concentration probe are shown.

[0033] Figure 5 The results of exploring the concentration of substrate used are shown.

[0034] Figure 6 Substrate incubation time exploration results are shown.

[0035] Figure 7 The results of exploring the concentration of FBS used are shown.

[0036] Figure 8 Results from investigations with different preservatives are shown.

[0037] Fig. 9 The results of the investigation of different preservative concentrations are shown.

[0038] Fig.10 The results of cell enrichment and separation validation are shown.

[0039] Fig.11 The validation results of cell lines with different HER2 expression levels are shown.

[0040] Fig.12 The validation results of cell lines with different EpCAM expression levels are shown.

[0041] Fig.13 The validation results of cell lines with different EGFR expression levels are shown.

[0042] Fig.14 Biotin-XX-tyramide validation results are shown.

[0043] Fig.15 Biotin-PEG4-tyramide validation results are shown.

[0044] Fig.16 Biotin-styrylamide validation results are shown.

[0045] Fig.17 Biotin-aniline validation results are shown. DETAILED DESCRIPTION

[0046] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are the terms and routine procedures widely used in the corresponding fields.

[0047] Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meanings as those commonly understood by those skilled in the art to which the present disclosure belongs. The terms used in the specification of the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure.

[0048] The terms "including" and "having" and any variations thereof in the present disclosure are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0049] The "plurality" mentioned in the present disclosure refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects before and after are in an "or" relationship. At the same time, in order to better understand the present disclosure, the definitions and explanations of related terms are provided below.

[0050] The terms "enrichment" or "capture" or "isolation" used in the present disclosure refer to separating rare cells from a sample to be tested.

[0051] The term "magnetic beads" used in the present disclosure refers to tiny particles with magnetism. The immunomagnetic bead method for separating cells is based on the ability of cell surface antigens to bind to specific monoclonal antibodies connected to magnetic beads. In an external magnetic field, cells connected to the magnetic beads through antibodies are adsorbed and retained in the magnetic field, while cells without such surface antigens cannot bind to the specific monoclonal antibodies connected to the magnetic beads and therefore have no magnetism and do not stay in the magnetic field, thereby allowing the cells to be separated.

[0052] The term "rare cells" used in this disclosure refers to some atypical cells in biological fluid samples (including blood, pleural effusion, ascites, urine, cerebrospinal fluid, etc.). Studies have shown that the collection and analysis of rare cells has important guiding significance for finding potential treatment mechanisms, pathological mechanisms and targeted drug development of diseases.

[0053] The term "peroxidase blocking solution" used in the present disclosure is a reagent for blocking endogenous peroxide in enzyme detection methods. Endogenous peroxidase is widely present in some cells and tissues, including red blood cells, kidney and liver tissues, etc. Due to the presence of endogenous peroxidase in tissues or cells, a higher background may occur when the method for peroxidase is used to detect samples, or even false positive results may occur. Therefore, cells or tissue samples, etc. should be sealed with appropriate peroxidase blocking solutions before staining to eliminate the interference of endogenous peroxidase. Optional peroxidase blocking solutions include but are not limited to endogenous peroxidase blocking solutions (Biyuntian P0100A), endogenous peroxidase strong blocking solutions (Biyuntian P0100B) or hydrogen peroxide solutions of varying concentrations.

[0054] The terms "recovery rate" and "capture efficiency" used in the present disclosure refer to the ratio of the number of recovered cells to the number of actually added cells in a simulation experiment of target cell enrichment technology.

[0055] The term "effective recovery rate" used in the present disclosure refers to an evaluation index of the target cell enrichment technology, that is, the minimum value that the ratio of the number of recovered cells to the number of actually added cells should reach. According to the effective recovery rate set by the target cell enrichment technology or the enrichment object, the effective recovery rate can be selected from 10%-98%, more specifically, it can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. The effective recovery rate of the present disclosure can be selected from 65%-90%, preferably, the effective recovery rate is 80%. When the recovery rate is greater than or equal to the effective recovery rate set, it can be considered that the technical solution can be effectively applied to the detection of clinical samples.

[0056] In one aspect, the present disclosure provides a reagent combination for rare cell capture, wherein the reagent combination comprises a biotin-labeled substrate and a capture antibody, wherein the biotin-labeled substrate is selected from the compounds or derivatives shown in formula (I), formula (II) or formula (III),

[0057]

[0058] in:

[0059] A is

[0060] is a single bond or a double bond;

[0061] R 1 Selected from -(CH 2 ) n1 -、-CH(CH 3 )-、-C(CH 3 ) 2-, cycloalkyl, heterocyclyl, aryl or heteroaryl;

[0062] R 2 Selected from -OH, -NH 2 , -SH and -H;

[0063] X is and / or

[0064] n1 is an integer selected from 0 to 10;

[0065] n2 is selected from an integer of 1-300, preferably, n2 is selected from an integer of 1-10;

[0066] n3 is selected from an integer of 1-10.

[0067] Those skilled in the art should also understand that any modification of the compound represented by formula (I), formula (II) or formula (III) or its salt is within the scope of protection of the present invention, including but not limited to PEG bridging modification and aminocaproic acid bridging modification. The reagent combination has high sensitivity and can be widely used to capture rare cells with low expression of specific antigens, such as circulating tumor cell samples with low expression of tumor markers.

[0068] In some preferred embodiments of the present disclosure, the cycloalkyl group is selected from

[0069] In some preferred embodiments of the present disclosure, the heterocyclic group is selected from

[0070] In some preferred embodiments of the present disclosure, the aryl group is selected from

[0071] In some preferred embodiments of the present disclosure, the biotin-labeled substrate is selected from a compound or a derivative thereof represented by any one of Formulas 1 to 93:

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079] In some more preferred embodiments of the present disclosure, the biotin-labeled substrate is selected from the compounds shown in Formula 1, Formula 2, Formula 85, Formula 87 or Formula 90 or their derivatives.

[0080] In some preferred embodiments of the present disclosure, the reagent combination further comprises a blocking solution, an incubation buffer and magnetic beads.

[0081] In some preferred embodiments of the present disclosure, the capture antibody is selected from one or more of anti-CK, anti-EpCAM, anti-PD-L1, anti-HER2, anti-TROP2, anti-c-MET, anti-Claudin18.2, anti-EGFR, anti-MUC1, anti-HER3, anti-CD31, anti-CD44 and the like antibodies.

[0082] In some preferred embodiments of the present disclosure, the capture antibody is a labeled antibody, preferably an oxidoreductase labeled antibody; more preferably, the oxidoreductase is selected from any one of a dehydrogenase, an oxidase, a peroxidase, and an oxygenase; preferably, the oxidoreductase is selected from any one of horseradish peroxidase, catalase, and superoxide dismutase; preferably, the oxidoreductase is horseradish peroxidase.

[0083] In some preferred embodiments of the present disclosure, the magnetic beads are labeled magnetic beads, preferably avidin-labeled magnetic beads.

[0084] In some preferred embodiments of the present disclosure, the avidin is streptavidin or neutravidin; preferably streptavidin.

[0085] In some preferred embodiments of the present disclosure, the concentration of the substrate is 0.1-5 μg / mL.

[0086] In some preferred embodiments of the present disclosure, the concentration of the substrate is 0.625-1.25 μg / mL.

[0087] In some preferred embodiments of the present disclosure, the concentration of the substrate is 1.25 μg / mL.

[0088] In some preferred embodiments of the present disclosure, the concentration of the capture antibody is 1-8 μg / mL.

[0089] In some preferred embodiments of the present disclosure, the concentration of the capture antibody is 4 μg / mL.

[0090] In some preferred embodiments of the present disclosure, the blocking solution is selected from a peroxidase blocking solution; preferably, the peroxidase blocking solution is H 2 O 2 Blocking solution.

[0091] In some preferred embodiments of the present disclosure, the H 2 O 2 H in the blocking solution 2 O 2 The concentration is 0.1%-0.7%; preferably 0.5%.

[0092] In some preferred embodiments of the present disclosure, the H 2 O 2 The blocking solution also contains FBS or BSA.

[0093] In some preferred embodiments of the present disclosure, the H 2 O 2 The blocking buffer contains FBS.

[0094] In some preferred embodiments of the present disclosure, the H 2 O 2 The blocking solution contained 5% FBS.

[0095] In some preferred embodiments of the present disclosure, the incubation buffer is a buffer solution containing FBS or BSA.

[0096] In some preferred embodiments of the present disclosure, the incubation buffer is a buffer solution containing FBS.

[0097] In some preferred embodiments of the present disclosure, the incubation buffer is a buffer solution containing 1%-5% FBS; preferably, it is a buffer solution containing 1% FBS.

[0098] In some preferred embodiments of the present disclosure, the buffer solution is any one of PBS solution, hepes buffer, and Hanks' balanced salt solution; preferably PBS solution.

[0099] In some preferred embodiments of the present disclosure, an optional preservative is also included. The purpose of adding the preservative is to extend the storage time of the incubation buffer. Without adding the preservative, the reagent can be prepared and used immediately without affecting the capture and detection effect of rare cells.

[0100] In some preferred embodiments of the present disclosure, the preservative is selected from isothiazolinone preservatives.

[0101] In some preferred embodiments of the present disclosure, the active ingredient of the isothiazolinone preservative is selected from one or more of 2-methyl-4-isothiazoline-3-one, 5-chloro-2-methyl-4-isothiazoline-3-one, 1,2-benzisothiazolin-3-one, and 2-n-octyl-4-isothiazoline-3-one.

[0102] In some preferred embodiments of the present disclosure, the preservative is krovin-300M or proclin-300; preferably krovin-300M.

[0103] In some preferred embodiments of the present disclosure, the concentration of the preservative krovin-300M is 0.05%-0.1%, preferably 0.08%.

[0104] In another aspect, the present disclosure also provides a kit for capturing rare cells, comprising the aforementioned reagent combination.

[0105] In some embodiments of the present disclosure, the biotin-labeled substrate, capture antibody, blocking solution, incubation buffer, magnetic beads or preservatives are packaged in different containers or packaging bags.

[0106] In another aspect, the present disclosure further provides a method for capturing rare cells using the aforementioned reagent combination or the aforementioned kit, comprising the following steps:

[0107] (1) Sample collection: Collect blood samples using anticoagulant blood collection tubes containing one or more components selected from the group consisting of EDTA-dipotassium, EDTA-disodium, sodium citrate, and heparin sodium;

[0108] (2) Removal of red blood cells: using sucrose gradient centrifugation, NH4 + One or more methods of red blood cell lysis solution and red blood cell digestion enzyme are used to remove red blood cells from the sample; preferably containing NH4 + Red blood cell lysis buffer;

[0109] (3) Cell fixation: adding a fixative to the sample obtained in step (2), wherein the fixative contains one or more of paraformaldehyde, formaldehyde, glutaraldehyde, methanol, ethanol, and acetone; preferably paraformaldehyde;

[0110] (4) Sample blocking: adding blocking solution to the sample obtained in step (3) and incubating for 15-60 min, preferably 15 min; the sample cleaning solution is a solution containing FBS or BSA;

[0111] (5) Antibody incubation: adding the capture antibody labeled with redox enzyme to the blocked sample obtained in step (4) and incubating overnight;

[0112] (6) Incubation with biotin-labeled substrate: Add biotin-labeled substrate to the sample obtained in step (5) and incubate for 15-60 min, preferably 30 min;

[0113] (7) Magnetic bead incubation: Add avidin-labeled magnetic beads to the sample obtained in step (6) and incubate for 15-60 min;

[0114] (8) Cell separation: Use a magnetic separation device to sort target cells;

[0115] (9) Cell detection: Scan and analyze the fluorescence signal of the sample.

[0116] In some preferred embodiments of the present disclosure, the sample is a body fluid sample.

[0117] In some preferred embodiments of the present disclosure, the rare cells are one or more of circulating tumor cells, circulating epithelial cells, circulating endothelial cells, and tumor stem cells.

[0118] In some preferred embodiments of the present disclosure, the circulating tumor cell sample is selected from one or more of blood, pleural effusion, peritoneal effusion, saliva, sputum, urine, cerebrospinal fluid or pericardial effusion of an individual with cancer.

[0119] In some preferred embodiments of the present disclosure, the cancer is selected from brain astroblastoma, pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal chromophobe cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, fibrous dysplasia, fibrous dysplasia, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, pancreatic islet cell tumor , Kaposi sarcoma, kidney cancer, leukemia, liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid cancer, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, melanoma, metastatic renal cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, uterine cancer.

[0120] In some preferred embodiments of the present disclosure, the cancer is selected from one or more of breast cancer, lung cancer, gastric cancer, skin cancer, liver cancer, pancreatic cancer, kidney cancer, prostate cancer, cervical cancer, uterine cancer, ovarian cancer, nasopharyngeal cancer, head and neck cancer, esophageal cancer, pharyngeal cancer, glioblastoma, thymic cancer, and thyroid cancer; preferably breast cancer and lung adenocarcinoma.

[0121] In another aspect, the present disclosure also provides a use of the aforementioned reagent combination, the aforementioned kit and / or the aforementioned method in preparing a drug for capturing cells.

[0122] In some preferred embodiments of the present disclosure, the cell is a rare cell.

[0123] In some preferred embodiments of the present disclosure, the cell is a tumor cell.

[0124] Example

[0125] The technical solution of the present disclosure is further described below by way of specific implementation. It should be clear to those skilled in the art that the embodiments are only used to help understand the present disclosure and should not be regarded as specific limitations of the present disclosure.

[0126] Wherein, the meanings of the abbreviations in the following embodiments are as follows:

[0127] WBC: White blood cells

[0128] Biotin: Biotin

[0129] Streptavidin: Avidin

[0130] HER2: human epidermal growth factor receptor 2

[0131] CK: Cytokeratin, a tumor cell epithelial marker

[0132] Epcam: epithelial cell adhesion molecule

[0133] HRP: Horseradish peroxidase

[0134] 1. The specific experimental steps of cell detection in the embodiment of the present disclosure are as follows:

[0135] (1) Sample blocking: Take a cell sample (human breast cancer cell SKBR3 or human leukocyte sample, etc., with a cell concentration of 3×10 5 / mL, 0.1mL / portion), add blocking solution, mix thoroughly, and incubate the cell sample in a 37°C water bath for a period of time; take out the sample, centrifuge at 500g for 5min, remove the supernatant to collect the cells, add 1mL / portion binding buffer (1% FBS-PBS solution, the same below) to resuspend the cells, centrifuge at 500g for 5min, remove the supernatant to collect the cells, add 1mL / portion binding buffer to resuspend the cells again, centrifuge at 500g for 5min, remove the supernatant to collect the cells, add 100μL / portion binding buffer to resuspend the cells for use.

[0136] (2) Antibody incubation: 100 μL / portion of incubation antibody is added to the cell solution obtained in step (1) according to a certain working concentration, incubated at 2-8°C for 30 min, the cells are resuspended in binding buffer, centrifuged at 500 g for 5 min, the supernatant is removed and the cells are collected, the cells are resuspended in binding buffer again, centrifuged at 500 g for 5 min, the supernatant is removed and the cells are collected to obtain an incubation antibody-cell complex sample, and 100 μL / portion of incubation buffer solution is added to resuspend the cells for later use.

[0137] (3) Substrate incubation: Add 100 μL of biotin-labeled substrate (in the secondary antibody detection method, biotin-labeled secondary antibody Biotin-SP Goat Anti-HRP antibody is used instead (Jackson ImmunoResearch, Catalog No. 123-065-021)) to the cell solution obtained in step (2) according to a certain concentration, mix thoroughly, and incubate at 37°C for a period of time. Add 1 mL of binding buffer to resuspend the cells, centrifuge at 500 g for 5 min, remove the supernatant to collect the cells, add 1 mL of binding buffer to resuspend the cells, centrifuge at 500 g for 5 min, remove the supernatant to collect the cells, obtain a cell complex sample, and add 100 μL / portion of binding buffer to resuspend the cells.

[0138] (4) Fluorescent antibody incubation: Add 100 μL / portion of streptavidin-FITC (manufacturer: ThermoFisher, catalog number: SA1001, final concentration 1:400) to the cell complex sample obtained in step (3) to stain the cells, and incubate at 2-8°C for 30 min. Centrifuge at 500 g for 5 min, remove the supernatant to collect the cells, then add 1 mL / portion of binding buffer to resuspend the cells, centrifuge at 500 g for 5 min, remove the supernatant to collect the cells, and add 100 μL / portion of binding buffer.

[0139] (5) Cell detection: Take samples, add DAPI to stain the cell nucleus, and finally use fluorescence microscopy to scan and analyze.

[0140] 2. The specific experimental steps of cell capture in the disclosed embodiment are as follows:

[0141] (1) Sample sealing: Take 1 mL / portion of white blood cell sample (cell concentration is 5×10 7 / ml), add a certain amount of human breast cancer cells SKBR3 or human leukocyte samples prestained with CFSE, add blocking solution, mix well, and incubate the cell sample in a 37°C water bath for a period of time; take out the sample, centrifuge at 500g for 5min, remove the supernatant to collect the cells, add 1mL / portion of binding buffer (1% FBS-PBS solution, the same below) to resuspend the cells, centrifuge at 500g for 5min, remove the supernatant to collect the cells, add 1mL / portion of binding buffer to resuspend the cells, centrifuge at 500g for 5min, remove the supernatant to collect the cells, and add 1mL / portion of binding buffer to resuspend the cells.

[0142] (2) Antibody incubation: Add antibodies at a certain concentration and incubate in the cell solution obtained in step (1), incubate at 2-8°C overnight, add binding buffer to resuspend the cells, centrifuge at 500g for 5 min, remove the supernatant to collect the cells, add binding buffer to resuspend the cells, centrifuge at 500g for 5 min, remove the supernatant to collect the cells, obtain an incubated antibody-cell complex sample, add 1 mL / portion of incubation buffer solution to resuspend the cells for later use.

[0143] (3) Substrate incubation: Add biotin-labeled substrate (in the secondary antibody capture method, biotin-labeled secondary antibody Biotin-SP Goat Anti-HRP antibody is used instead (Jackson ImmunoResearch, Catalog No. 123-065-021)) to the cell solution obtained in step (2) at a certain concentration, mix thoroughly, and incubate at 37°C for a period of time. Add 1 ml of binding buffer to resuspend the cells, centrifuge at 500 g for 5 min, remove the supernatant to collect the cells, add 1 ml of binding buffer to resuspend the cells, centrifuge at 500 g for 5 min, remove the supernatant to collect the cells, obtain a cell complex sample, and add 970 μL / portion of binding buffer to resuspend the cells.

[0144] (4) Magnetic bead incubation: Add 30 μL / portion of magnetic beads (BDbiosciences, catalog number 557812) to the cell complex sample obtained in step (3) and incubate at 2-8°C for 60 min. Centrifuge at 500 g for 5 min, remove the supernatant to collect the cells, add 1 mL / portion of binding buffer to resuspend the cells, centrifuge at 500 g for 5 min, remove the supernatant to collect the cells, and add 1 mL / portion of binding buffer.

[0145] (5) Cell capture and detection: Use a magnetic rack for enrichment and separation. At the same time, add DAPI to stain the cell nucleus, and finally use a fluorescence microscope for scanning and analysis.

[0146] Example 1: Investigation of cell blocking conditions

[0147] In order to optimize the experimental conditions, the cell blocking conditions were explored in this example. Wherein, the biotin-labeled substrate is biotin tyrosinamide (compound of formula 1).

[0148] 1.1 Closed time exploration

[0149] This example uses 3% H 2 O 2 As an endogenous peroxidase blocking solution, the blocking time was further explored.

[0150] Use 3% H 2 O 2 As blocking solution, HRP-labeled HER2 antibody was used as incubation antibody (using concentration 4 μg / mL), biotin-labeled substrate was used at a concentration of 0.625 μg / mL, incubation time was 30 min, incubation buffer was 1% FBS, the blocking time in step (1) was 0 min, 5 min, 10 min, 15 min, 30 min, 45 min, 60 min, respectively, and cell complex samples were obtained after sample blocking, antibody incubation, and substrate incubation according to the aforementioned cell detection steps (1)-(3), 100 μL of binding buffer was added to resuspend the cells, and streptavidin-FITC (manufacturer: ThermoFisher, product number: SA1001, using a final concentration of 1:400) was added for staining for 30 min, and DAPI was added to stain the cell nucleus, and finally the results were detected and compared using a fluorescence microscope.

[0151] The negative control group (AG) was tested using negative cells WBC. The positive test group (HN) was tested using positive cells SKBR3. The experimental settings and test results of each experimental group are shown in Table 1.

[0152] Table 1 Experimental settings and results of closure time exploration

[0153]

[0154]

[0155] The average fluorescence intensity detection results of the negative and positive control groups at different incubation times are shown in Figure 1 and Table 1, using H 2 O 2 After blocking for 15-60 minutes, the background signal of the negative control group is still low, while the positive test group has a strong fluorescence detection signal, and at 15 minutes, the difference between the positive test group and the negative test group is the largest. Considering the comprehensive experimental results and the incubation time cost, the sample blocking time is preferably 15 minutes.

[0156] 1.2 Blocking solution concentration study

[0157] This embodiment uses H 2 O 2 As the endogenous peroxidase blocking solution, the sample blocking time was 15min. 2 O 2 The concentration of the blocking solution was further investigated.

[0158] Alexa Fluor 500 was used to detect HER2 with HRP-labeled antibody (4 μg / ml) and pan-Cytokeratin antibody (C11). 647 (manufacturer: Santa Cruz biotechnology, catalog number: SC-8018AF647) was used as the incubation antibody, 100 μL / portion was added, the biotin-labeled substrate was used at a concentration of 0.625 μg / mL, the incubation time was 30 min, the incubation buffer was 1% FBS, and 3%, 2.5%, 2%, 1.5%, 1%, 0.7%, 0.5%, 0.3%, and 0.1% H 2 O 2 Solution (containing 5% FBS) was used as blocking solution, and the blocking time was 15 minutes. After sample blocking, antibody incubation, and substrate incubation as described in the above cell detection steps (1)-(3), the cell complex sample was obtained. 100 μL of binding buffer was added to resuspend the cells, and then streptavidin-FITC (manufacturer: ThermoFisher, product number: SA1001, final concentration 1:400) was added for staining for 30 minutes. The sample was taken and DAPI was added to stain the cell nucleus. Finally, a fluorescence microscope was used to detect and compare the results.

[0159] The negative control group (Table 2, AJ) was tested using negative cells WBC. The positive test group (Table 2, KT) was tested using positive cells SKBR3. The experimental settings and test results of each experimental group are shown in Table 2.

[0160] Table 2 Blocking solution (0.05-3%) concentration exploration experiment settings and results

[0161]

[0162]

[0163] H 2 O 2 When the blocking solution is used at a concentration of 3% in this detection system, its blocking effect is too strong and will destroy intracellular antigens such as CK, making the antigen undetectable and affecting the sensitivity of the detection. 2 O 2 FBS can block damaged areas of cells and prevent cell adhesion.

[0164] In the positive test group, the blocking solution was used at a concentration of 0.1-0.7% (see Figure 2 and Table 2), the CK staining fluorescence value is high (87.51-100.29), and the difference relative to the negative test group is also high (81.61-93.9); at the same time, the HER2 staining fluorescence value in the negative control group is low (9.11-21.48), so the blocking solution concentration in the range of 0.1%-0.7% has good detection results. At the same time, when the blocking solution concentration is 0.05%, the HER2 background signal value in the negative test group is 45.23, which exceeds 40 (there is a certain background signal under the microscope when it exceeds 40), and the background is high, which does not meet the selection criteria. When the concentration is 0.5%-0.7%, the difference between HER2 staining positive and negative is the largest. At the same time, considering the difference between CK staining positive and negative, and in order to reduce the cost of reagents, 0.5% is preferred after comprehensive consideration.

[0165] 1.3 Investigation of serum concentration during closure

[0166] In this experiment, the sample blocking solution was 0.5% HO. 2 O 2 The sample blocking time was 15 minutes, and the serum concentration during blocking was further investigated.

[0167] HRP-labeled HER2 antibody (using concentration 4 μg / mL) was used as the incubation antibody, 100 μL / portion was added, the biotin-labeled substrate was used at a concentration of 0.625 μg / mL, the incubation time was 30 min, the incubation buffer was 1% FBS, and HRP-labeled HER2 antibody (using concentration 4 μg / mL) was added at 100 μL / portion, the biotin-labeled substrate was used at a concentration of 0.625 μg / mL, the incubation time was 30 min, the incubation buffer was 1% FBS, and the ... 2 O 2 The solution was used as a blocking solution, and the blocking time was 15 minutes. After sample blocking, antibody incubation, and substrate incubation according to the above-mentioned step embodiment cell detection (1)-(3), a cell complex sample was obtained. 100 μL of binding buffer was added to resuspend the cells, and then streptavidin-FITC (manufacturer: ThermoFisher, product number: SA1001, using a final concentration of 1:400) was added for staining for 30 minutes. The sample was taken and DAPI was added to stain the cell nucleus. Finally, a fluorescence microscope was used to detect and compare the results.

[0168] Table 3 Blocking solution serum concentration (0%-10%) Experimental settings and results

[0169]

[0170]

[0171] Depend on Figure 3 As shown in Table 3, during the blocking process of the sample, if the solution contains a certain amount of FBS, the detection signal of the test group is strong, and the average fluorescence intensity value is greater than 100; when the content is 5%-10%, the average fluorescence intensity values ​​are close, indicating that FBS in this concentration range has the least effect on blocking, so the FBS content is recommended to be 5%-10%. Considering the detection cost issue comprehensively, the FBS concentration of 5% is selected when the sample is blocked.

[0172] Example 2: Study on incubation conditions of HRP-labeled antibodies

[0173] In this embodiment, the concentration conditions of HRP labeled antibody are explored. Wherein, the biotin labeled substrate is biotin tyrosinamide.

[0174] Use 0.5% HO 2 O 2 (containing 5% FBS) was used as blocking solution, the blocking time was 15 minutes, the biotin-labeled substrate was used at a concentration of 0.625 μg / mL, the incubation time was 30 minutes, the incubation buffer was 1% FBS, and HRP-labeled HER2 antibody was used as the incubation antibody (the concentrations used were 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, and 0.5 μg / ml, respectively). After sample blocking, antibody incubation, and substrate incubation as described in the aforementioned cell detection (1)-(3), the cell complex sample was obtained, 100 μL of binding buffer was added to resuspend the cells, and streptavidin-FITC (manufacturer: ThermoFisher, product number: SA1001, final concentration 1:400) was added for staining for 30 minutes. The sample was taken and DAPI was added to stain the cell nucleus. Finally, a fluorescence microscope was used to detect and compare the results.

[0175] The negative control group (Table 4, LP group) was tested using negative cells WBC. The positive (substrate) test group (Table 4, FK group) was tested using positive cells SKBR3.

[0176] In the secondary antibody test group (Table 4, AE group), the substrate incubation in step (3) was replaced with secondary antibody (Biotin-SP Goat Anti-HRP antibody, Jackson ImmunoResearch, catalog number 123-065-021) incubation. The specific steps were as follows: 100 μL / portion of secondary antibody (5 μg / ml) solution was added and mixed thoroughly, and then incubated at 37°C for 30 min. The cell complex sample was obtained, 100 μL of binding buffer was added to resuspend the cells, and then streptavidin-FITC (manufacturer: ThermoFisher, catalog number: SA1001, using a final concentration of 1:400) was added for staining for 30 min. The sample was then added with DAPI to stain the cell nucleus, and the results were compared using a fluorescence microscope. The results are shown in Tables 4 and Figure 4 .

[0177] Table 4 Experimental setup and results of labeling antibody concentration exploration

[0178]

[0179] Depend on Figure 4 As shown in Table 4, when the HRP-labeled HER2 antibody is used at a concentration of 1-8 μg / mL, the substrate test group has a strong detection signal (118.15-143.02); the secondary antibody test group has a strong detection signal (72.40-93.67) when the HER2 antibody is used at a concentration of 4 μg / mL-8 μg / mL; in the negative control group, the detection signal values ​​of the average fluorescence intensity are very low (5.43-8.79). Therefore, the recommended concentration of the HRP-labeled antibody is 1 μg / mL-8 μg / mL, based on comprehensive considerations of cost and stability of the test results, and the detection signal of the positive test group is the strongest when the concentration is 4 μg / mL. Therefore, the concentration of the HRP incubation antibody is selected to be 4 μg / mL.

[0180] Example 3: Investigation of substrate incubation conditions

[0181] This example explores substrate incubation conditions, wherein the biotin-labeled substrate is biotin tyrosinamide.

[0182] 3.1 Study on the concentration of substrate used

[0183] In order to further improve the reaction efficiency, this example further explores the substrate concentration conditions.

[0184] Use 0.5% HO 2 O 2(containing 5% FBS) was used as blocking solution for 15 minutes, HRP-labeled HER2 antibody was used as incubation antibody (using concentration 4 μg / mL), incubation buffer was 1% FBS, biotin-labeled substrate was used at concentrations of 0.3125 μg / mL, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, and 5 μg / mL, respectively, and incubation time was 30 minutes. After sample blocking, antibody incubation, and substrate incubation as described in the above-mentioned cell detection (1)-(3), cell complex samples were obtained, and then streptavidin-FITC (manufacturer: ThermoFisher, product number: SA1001, using a final concentration of 1:400) was added for staining for 30 minutes. The sample was taken and DAPI was added to stain the cell nucleus. Finally, fluorescence microscopy was used to detect and compare the results.

[0185] The control group (AE) was tested using negative cells WBC. The positive test group (FJ) was tested using positive cells SKBR3. The experimental settings and results of the biotin-labeled substrate concentration exploration are shown in Tables 5 and Figure 5 .

[0186] Table 5 Experimental setup and results of biotin-labeled substrate concentration exploration

[0187] The results show that ( Figure 5 , Table 5), when the concentration of biotin-labeled substrate is 0.3125μg / mL-5μg / mL, the detection signal of positive cell SKBR3 is greater than the background signal of leukocytes in the negative control group, which meets the detection requirements, but when the concentration is 2.5μg / mL-5μg / mL, the background signal of leukocytes is high; when the concentration is 0.3125μg / mL, the detection signal of the test group is too low. Therefore, the concentration of biotin-labeled substrate is selected to be 0.625μg / mL-1.25μg / mL, considering the stability of the test results, and at 1.25μg / mL, the difference between the positive test group and the negative control group is the largest, therefore, it is preferred to use 1.25μg / mL.

[0188] 3.2 Study on substrate incubation time

[0189] After determining the concentration range of the substrate, this example further explored the substrate incubation time.

[0190] Use 0.5% HO 2 O 2(containing 5% FBS) was used as blocking solution, the blocking time was 15 minutes, HRP-labeled HER2 antibody was used as incubation antibody (using concentration 4 μg / mL), biotin-labeled substrate was used at a concentration of 1.25 μg / mL, the incubation buffer was 1% FBS, and the substrate was incubated for 0 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, and 60 minutes, respectively. After sample blocking, antibody incubation, and substrate incubation as described in the aforementioned cell detection (1)-(3), the cell complex sample was obtained, and then streptavidin-FITC (manufacturer: ThermoFisher, product number: SA1001, using a final concentration of 1:400) was added for staining for 30 minutes. The sample was taken and DAPI was added to stain the cell nucleus. Finally, a fluorescence microscope was used to detect and compare the results.

[0191] The control group (AF) was tested using negative cells WBC. The positive test group (GL) was tested using positive cells SKBR3. The test results are shown in Figure 6 and Table 6.

[0192] Table 6 Experimental setup and results of substrate incubation time exploration

[0193]

[0194] The results show that ( Figure 6 , Table 6), when the substrate was incubated for 15min-60min, the positive cells SKBR3 in the test group all had a higher detection signal, and the background signal of the negative control group was lower. Therefore, the substrate incubation time was selected to be 15-60min, considering the stability of the test results. At the same time, when the incubation time was 30min, the difference between the positive test group and the negative test group was the largest, so the substrate incubation time was preferably 30min.

[0195] 3.3 Incubation buffer exploration

[0196] 3.3.1 Study on FBS concentration

[0197] The concentration of FBS used in the incubation buffer was further explored.

[0198] Use 0.5% HO 2 O 2As blocking solution, the blocking time was 15 minutes, HRP-labeled HER2 antibody was used as incubation antibody (using concentration 4 μg / mL), biotin-labeled substrate was used at a concentration of 1.25 μg / mL, incubation time was 30 minutes, incubation buffer was 0% FBS, 1% FBS, 2% FBS, 3% FBS, 4% FBS, 5% FBS, 6% FBS, 7% FBS, 8% FBS, 9% FBS, 10% FBS, and the cell complex sample was obtained after sample blocking, antibody incubation, and substrate incubation as described in the above cell detection (1)-(3), and then streptavidin-FITC (manufacturer: ThermoFisher, product number: SA1001, using a final concentration of 1:400) was added for staining for 30 minutes, and then DAPI was added to the sample to stain the cell nucleus, and finally the results were compared using a fluorescence microscope. The experimental results are shown in Tables 7 and Figure 7 .

[0199] Table 7 Incubation buffer exploration experiment settings and results

[0200]

[0201]

[0202] Depend on Figure 7 As shown in Table 7, during the incubation of the substrate, if the FBS content in the solution exceeds 5%, it will affect the binding of the substrate. When the FBS content is in the range of 0%-5%, the average fluorescence intensity value is greater than 100 (the detection signal is higher); when the content is 1%-2%, the average fluorescence intensity is close to the average fluorescence intensity value of 0%, indicating that FBS in this concentration range has the least effect on substrate binding, so the FBS content is recommended to be 0%-2%. However, a certain amount of FBS has a certain protective effect on cells. When 1X-PBS or 1% FBS is used for substrate incubation, the fluorescent staining signal is stronger and the effect on substrate binding is also smaller. Therefore, considering comprehensively, 1% FBS solution is selected as the substrate incubation solution.

[0203] 3.3.2 Study on the effects of different preservatives

[0204] Preservatives are beneficial to the preservation of incubation buffer, but since oxidoreductase is introduced into the system, different types of preservatives may have different effects on the test results. In order to minimize the impact on the capture and detection of rare cells, this example further screened the addition of preservatives.

[0205] Use 0.5% HO 2 O 2(containing 5% FBS) was used as blocking solution, the blocking time was 15 minutes, HRP-labeled HER2 antibody was used as labeling antibody (using concentration 4 μg / mL), biotin-labeled substrate was used at a concentration of 1.25 μg / mL, the incubation time was 30 minutes, the incubation buffer was PBS buffer containing 1% FBS, in step (2), the preservatives described in Table 8 below were added to the incubation buffer, and the cell complex sample was obtained after sample blocking, antibody incubation, and substrate incubation as described in the above-mentioned cell detection (1)-(3), and then streptavidin-FITC (manufacturer: ThermoFisher, product number: SA1001, using a final concentration of 1:400) was added for staining for 30 minutes, and then DAPI was added to the sample to stain the cell nucleus, and finally the results were detected and compared using a fluorescence microscope.

[0206] The control group did not add any preservatives. The test results are shown in Table 8 and Figure 8 .

[0207] Table 8 Experimental setup and results of preservative exploration

[0208]

[0209] The results show that ( Figure 8 , Table 8), the substrate incubation solution used 0.1% krovin-600, 0.1% krovin-500, 0.02% NaN 3 As preservatives, all of them will affect the detection signal. When 0.1% krovin-300M (manufacturer: Xibao Biotechnology, catalog number: ACN0037B-500ml) or 0.05% proclin-300 (manufacturer: Sigma, catalog number: 48912-U) is used as a preservative, a fluorescent signal that meets the detection requirements can be obtained, among which 0.1% krovin-300M has the least effect on substrate staining. Therefore, 0.1% krovin-300M is selected as a preservative in the subsequent substrate incubation solution.

[0210] 3.3.3 Study on the concentration of preservatives used

[0211] The concentration of preservatives used was further explored.

[0212] Use 0.5% HO 2 O 2(containing 5% FBS) was used as blocking solution, the blocking time was 15 minutes, HRP-labeled HER2 antibody was used as labeling antibody (using concentration 4 μg / mL), biotin-labeled substrate was used at a concentration of 1.25 μg / mL, the incubation time was 30 minutes, and the incubation buffer was 1% FBS. In step (2), the preservatives described in Table 9 below were added to the incubation buffer (according to the product manual, the recommended concentration is 1:1000-1:2000, so this embodiment compares the effect of 0.05%-0.1% concentration on the detection results). According to the cell detection (1)-(3) of the above step embodiment, after sample blocking, antibody incubation, and substrate incubation, a cell complex sample was obtained, and then streptavidin-FITC (manufacturer: ThermoFisher, product number: SA1001, using a final concentration of 1:400) was added for staining for 30 minutes, and then DAPI was added to stain the cell nucleus, and finally the results were detected and compared using a fluorescence microscope.

[0213] The control group did not add any preservatives. The test results are shown in Table 9 and Fig. 9 .

[0214] Table 9 Experimental settings and results of exploring the concentration of preservatives

[0215]

[0216] Depend on Fig. 9 As can be seen from Table 9, when the concentration of krovin-300M is 0.05%-0.08%, the average fluorescence intensity value is basically no different from that of the control group, indicating that within this concentration range, the preservative has little effect on cell capture and detection. Taking into account the preservative effect of the preservative (the higher the concentration, the better the preservative effect), krovin-300M is preferably used at 0.08%.

[0217] Example 4: Cell Enrichment and Separation

[0218] Cell enrichment and separation is a technical solution based on magnetic separation, wherein magnetic separation refers to the technology of subjecting a substance to a magnetic field. The optional magnetic separation solutions include magnetic frame separation, chip separation (such as Liquidbiopsy), etc.; the solution illustrated in this embodiment is magnetic frame separation, and the specific experimental process is as follows:

[0219] Use 0.5% HO 2 O 2(containing 5% FBS) as blocking solution, the blocking time is 15 minutes, HRP-labeled HER2 antibody as incubation antibody (using concentration 4 μg / mL), biotin-labeled substrate using concentration 1.25 μg / mL, incubation time 30 minutes, incubation buffer is 1% FBS, according to the above steps of cell detection (1)-(3) described in the embodiment, after sample blocking, antibody incubation, substrate incubation, a cell complex sample is obtained. Then streptavidin-FITC (manufacturer: ThermoFisher, item number: SA1001, using a final concentration of 1:400) is added for staining for 30 minutes. During the sample separation process, the centrifuge tube is placed on a magnetic stand, allowed to stand for 6-8 minutes, the uncaptured leukocyte solution is aspirated with a pipette, and then 5 ml / portion of binding buffer is added to resuspend the cells, the centrifuge tube is placed on a magnetic stand, allowed to stand for 6-8 minutes, and the uncaptured leukocyte solution is aspirated with a pipette. Add 20 μL of binding buffer to resuspend the enriched cells, drop them onto a glass slide, add DAPI to stain the cell nuclei, and finally scan and analyze them using a fluorescence microscope.

[0220] Example 5: Verification of rare cell capture effect

[0221] In combination with the above examples, the separation effect of the above rare cell capture kit is verified. Wherein, the biotin-labeled substrate is biotin tyrosinamide.

[0222] The details are as follows:

[0223] Use 0.5% HO 2 O 2 (containing 5% FBS) as blocking solution, blocking time is 15min, HRP-labeled HER2 antibody as incubation antibody (using concentration 4μg / mL), biotin-labeled substrate using concentration 1.25μg / mL, incubation time 30min, incubation buffer is 1% FBS, according to the above step example cell capture (1)-(5) after sample blocking, antibody incubation, substrate incubation, magnetic bead incubation to obtain the test sample, after adding DAPI staining, fluorescence microscope detection comparison results. The difference is that the cell sample in step (1) is replaced by 1mL / portion of white blood cells (cell concentration is 5×10 7 pcs / mL); in step (2), HRP-labeled HER2 antibody was added and fluorescent molecule-labeled CK detection antibody (manufacturer: Santa Cruz biotechnology, catalog number: SC-8018AF647) was added. The test results of each sample are shown in Table 10 and Fig.10 .

[0224] Table 10 Experimental settings and results for verification of cell enrichment and separation effects

[0225]

[0226] Depend on Fig.10 As shown in Table 10, the number of tumor cells recovered in samples A, B, and C are 5, 25, and 96, respectively, and the recovery rates are 100%, 100%, and 96%, respectively. The recovery rates are all qualified, and the CK staining fluorescence signal is very strong. In summary, the scheme disclosed in the present invention can be used for efficient detection of rare cells and has a high capture efficiency.

[0227] Example 6: Capture comparison and verification of low expression protein marker samples

[0228] The kit disclosed in the present invention can be used for efficient detection of rare cells. This example further compares the capture sensitivity and specificity of this scheme and the secondary antibody capture method when facing rare cell samples with low expression protein markers. Among them, the biotin-labeled substrate is biotin tyrosinamide (compound of formula 1). The details are as follows:

[0229] 6.1. Capture and validation of cell lines with different HER2 expression

[0230] Use 0.5% HO 2 O 2 (containing 5% FBS) as blocking solution, the blocking time is 15min, HRP-labeled HER2 antibody as incubation antibody (using concentration 4μg / ml), biotin-labeled substrate using concentration 1.25μg / mL, incubation time 30min, incubation buffer is 1% FBS, 500 / portion of SKBR3 (human breast cancer cells), ZR-75-1 (human breast cancer cells), MCF7 (human breast cancer cells), A549 (human lung adenocarcinoma cells), WBC cells are added as cell samples, and the added cell samples are pre-stained with CFSE (eBbioscience CFSE, catalog number: 65-0850-84) in advance. According to the above step cell capture example (1)-(5), after sample blocking, antibody incubation, substrate incubation, and magnetic bead incubation, the test sample is obtained, and after adding DAPI staining, the results are detected and compared using a fluorescence microscope. The difference is that in step (2), a fluorescent molecule-labeled CK detection antibody (manufacturer: Santa Cruz biotechnology, catalog number: SC-8018AF647) is added at the same time as the HRP-labeled HER2 antibody.

[0231] Secondary antibody detection group step (3) Substrate incubation was replaced by secondary antibody incubation, specifically: add 5 μg / mL secondary antibody solution (Biotin-SP Goat Anti-HRP antibody, Jackson ImmunoResearch, catalog number 123-065-021), mix thoroughly, and incubate at 37°C for 30 min. Then add magnetic beads for incubation.

[0232] Table 11 Experimental settings and results of low expression protein marker detection

[0233]

[0234] The results show that ( Fig.11 , Table 11), in the cell lines with low protein marker expression, the capture efficiency of the biotin-labeled substrate capture system is much higher than that of the secondary antibody capture system, and in the cell lines without expression (WBC), both are negative. Therefore, the use of the biotin-labeled substrate capture system has higher capture sensitivity without affecting specificity.

[0235] 6.2. Validation of EpCAM Capture in Cell Lines with Different Expressions

[0236] Use 0.5% HO 2 O 2 (containing 5% FBS) as blocking solution, blocking time is 15min, HRP-labeled EpCAM antibody as incubation antibody (using concentration 4μg / ml), biotin-labeled substrate using concentration 1.25μg / mL, incubation time 30min, incubation buffer is 1% FBS, 500 / portion of MCF-7 (human breast cancer cells), SKBR3 (human breast cancer cells), MDA-MB-231 (human breast cancer cells), Calu-1 (human lung adenocarcinoma cells), WBC cells are added as cell samples, and the added cell samples are pre-stained with CFSE (eBbioscience CFSE, catalog number: 65-0850-84) in advance. According to the above step cell capture example (1)-(5), after sample blocking, antibody incubation, substrate incubation, and magnetic bead incubation, the test sample is obtained, and after adding DAPI staining, the results are detected and compared using a fluorescence microscope. The difference is that in step (2), a fluorescent molecule-labeled CK detection antibody (manufacturer: Santa Cruz biotechnology, catalog number: SC-8018AF647) is added at the same time as the HRP-labeled EpCAM antibody.

[0237] Secondary antibody detection group step (3) Substrate incubation was replaced by secondary antibody incubation, specifically: add 5 μg / mL secondary antibody solution (Biotin-SP Goat Anti-HRP antibody, Jackson ImmunoResearch, catalog number 123-065-021), mix thoroughly, and incubate at 37°C for 30 min. Then add magnetic beads for incubation.

[0238] Table 12 Experimental settings and results of low expression protein marker detection

[0239]

[0240] The results show that ( Fig.12 , Table 12), in the cell lines with low protein marker expression, the capture efficiency of the biotin-labeled substrate capture system is much higher than that of the secondary antibody capture system, and in the cell lines without expression (WBC), both are negative. Therefore, the use of the biotin-labeled substrate capture system has a higher capture sensitivity without affecting the specificity.

[0241] 6.3. Capture validation of cell lines with different EGFR expression

[0242] Use 0.5% HO 2 O 2 (containing 5% FBS) as blocking solution, the blocking time is 15min, HRP-labeled EGFR antibody as incubation antibody (using concentration 4μg / mL), biotin-labeled substrate using concentration 1.25μg / mL, incubation time 30min, incubation buffer is 1% FBS, 500 / portion of HCC827 (human lung adenocarcinoma cells), MDA-MB-231 (human breast cancer cells), MCF-7 (human breast cancer cells), T-47D (human lung adenocarcinoma cells), WBC cells are added as cell samples, and the added cell samples are pre-stained with CFSE (eBbioscience CFSE, catalog number: 65-0850-84) in advance. According to the above step cell capture example (1)-(5), after sample blocking, antibody incubation, substrate incubation, and magnetic bead incubation, the test sample is obtained, and after adding DAPI staining, the results are detected and compared using a fluorescence microscope. The difference is that in step (2), a fluorescent molecule-labeled CK detection antibody (manufacturer: Santa Cruz biotechnology, catalog number: SC-8018AF647) is added at the same time as the HRP-labeled EGFR antibody.

[0243] Secondary antibody detection group step (3) Substrate incubation was replaced by secondary antibody incubation, specifically: add 5 μg / mL secondary antibody solution (Biotin-SP Goat Anti-HRP antibody, Jackson ImmunoResearch, catalog number 123-065-021), mix thoroughly, and incubate at 37°C for 30 min. Then add magnetic beads for incubation.

[0244] Table 13 Experimental settings and results of low expression protein marker detection

[0245]

[0246] The results show that ( Fig.13 , Table 13), in the cell lines with low protein marker expression, the capture efficiency of the biotin-labeled substrate capture system is much higher than that of the secondary antibody capture system, and in the cell lines without expression (WBC), both are negative. Therefore, the use of the biotin-labeled substrate capture system has a higher capture sensitivity without affecting the specificity.

[0247] Example 7: Other substrate testing and verification

[0248] 7.1. Biotin-XX-tyramine (compound of formula 85, manufacturer: APExBIO, catalog number: A8012) test

[0249] Use 0.5% HO 2 O 2 (containing 5% FBS) was used as blocking solution for 15 minutes, HRP-labeled HER2 antibody was used as labeling antibody (using concentration 4 μg / mL), biotin-labeled substrate (using concentration 0.625 μg / mL-10 μg / mL), incubation time was 30 minutes, incubation buffer was 1% FBS (added with 0.08% krovin-300M preservative), and cell complex samples were obtained after sample blocking, antibody incubation, and substrate incubation according to the above-mentioned steps of cell detection (1)-(3), and then streptavidin-FITC (manufacturer: ThermoFisher, product number: SA1001, using a final concentration of 1:400) was added for staining for 30 minutes, and then DAPI was added to stain the cell nucleus, and finally the results were detected and compared using a fluorescence microscope.

[0250] Table 14 Biotin-XX-Tyramide Test Verification Results

[0251]

[0252] The results show that ( Fig.14, Table 14), when the biotin-labeled substrate was used at a concentration of 0.625 μg / mL-10 μg / mL, the detection signal of the positive cell SKBR3 was greater than the white blood cell background signal in the negative control group, meeting the detection requirements.

[0253] 7.2. Biotin-PEG4-tyramine (Formula 87 compound, manufacturer: CONFLUOR, product number: BBBP-1-100mg) test verification

[0254] Use 0.5% HO 2 O 2 (containing 5% FBS) was used as blocking solution for 15 minutes, HRP-labeled HER2 antibody was used as labeling antibody (using concentration 4 μg / mL), biotin-labeled substrate (using concentration 0.625 μg / mL-10 μg / mL), incubation time was 30 minutes, incubation buffer was 1% FBS (added with 0.08% krovin-300M preservative), and cell complex samples were obtained after sample blocking, antibody incubation, and substrate incubation according to the above-mentioned steps of cell detection (1)-(3), and then streptavidin-FITC (manufacturer: ThermoFisher, product number: SA1001, using a final concentration of 1:400) was added for staining for 30 minutes, and then DAPI was added to stain the cell nucleus, and finally the results were detected and compared using a fluorescence microscope.

[0255] Table 15 Biotin-PEG4-Tyramide Test Verification Results

[0256]

[0257]

[0258] The results show that ( Fig.15 , Table 15), when the biotin-labeled substrate was used at a concentration of 0.625 μg / mL-10 μg / mL, the detection signal of the positive cell SKBR3 was greater than the white blood cell background signal in the negative control group, meeting the detection requirements.

[0259] 7.3. Biotin-styrylamide (Formula 90 compound, manufacturer: AAT, catalog number: 45300) test verification

[0260] Use 0.5% HO 2 O 2(containing 5% FBS) was used as blocking solution for 15 minutes, HRP-labeled HER2 antibody was used as labeling antibody (using concentration 4 μg / mL), biotin-labeled substrate (using concentration 0.625 μg / mL-10 μg / mL), incubation time was 30 minutes, incubation buffer was 1% FBS (added with 0.08% krovin-300M preservative), and cell complex samples were obtained after sample blocking, antibody incubation, and substrate incubation according to the above-mentioned steps of cell detection (1)-(3), and then streptavidin-FITC (manufacturer: ThermoFisher, product number: SA1001, using a final concentration of 1:400) was added for staining for 30 minutes, and then DAPI was added to stain the cell nucleus, and finally the results were detected and compared using a fluorescence microscope.

[0261] Table 16 Biotin-styrylamide test verification results

[0262]

[0263] The results show that ( Fig.16 , Table 16), when the biotin-labeled substrate was used at a concentration of 0.625 μg / mL-10 μg / mL, the detection signal of the positive cell SKBR3 was greater than the white blood cell background signal in the negative control group, meeting the detection requirements.

[0264] 7.4. Biotin-aniline (Formula 2 compound, manufacturer: MedMol, Cas number: 769933-15-5) test verification

[0265] Use 0.5% HO 2 O 2 (containing 5% FBS) was used as blocking solution for 15 minutes, HRP-labeled HER2 antibody was used as labeling antibody (using concentration 4 μg / mL), biotin-labeled substrate (using concentration 0.625 μg / mL-10 μg / mL), incubation time was 30 minutes, incubation buffer was 1% FBS (added with 0.08% krovin-300M preservative), and cell complex samples were obtained after sample blocking, antibody incubation, and substrate incubation according to the above-mentioned steps of cell detection (1)-(3), and then streptavidin-FITC (manufacturer: ThermoFisher, product number: SA1001, using a final concentration of 1:400) was added for staining for 30 minutes, and then DAPI was added to stain the cell nucleus, and finally the results were detected and compared using a fluorescence microscope.

[0266] Table 17 Biotin-aniline test verification results

[0267]

[0268]

[0269] The results show that ( Fig.17 , Table 17), when the biotin-labeled substrate was used at a concentration of 0.625 μg / mL-10 μg / mL, the detection signal of the positive cell SKBR3 was greater than the white blood cell background signal in the negative control group, meeting the detection requirements.

[0270] Example 8: Other Substrate Capture Validation Tests

[0271] Use 0.5% HO 2 O 2 (containing 5% FBS) as blocking solution, the blocking time is 15min, HRP-labeled HER2 antibody as incubation antibody (using concentration 4μg / mL), biotin-labeled substrate using concentration 1.25μg / mL, incubation time 30min, incubation buffer is 1% FBS (added with 0.08% krovin-300M preservative), according to the above step example cell capture (1)-(5) after sample blocking, antibody incubation, substrate incubation, magnetic bead incubation to obtain the test sample, add DAPI staining and use fluorescence microscope to detect and compare the results. The difference is that in step (3), the substrate is selected from (biotin-styrylamide, biotin-XX-tyramide, biotin-PEG-tyramide, biotin-aniline); in step (2), the HRP-labeled HER2 antibody is added and the fluorescent molecule-labeled CK detection antibody (manufacturer: Santa Cruz biotechnology, item number: SC-8018AF647) is added. The test results of each sample are shown in Table 18. The results showed that when the concentration was 1.25 μg / ml, the recovery rates of the four biotin substrates were all over 90%, and the recovery rates of biotin-styrylamide, biotin-XX-tyramide and biotin-PEG-tyramide were higher than that of biotin-aniline.

[0272] Table 18 Capture test verification results of different substrates

[0273]

[0274] The results showed (Table 18) that when the biotin-labeled substrates Formula 2, Formula 85, Formula 87 and Formula 90 were used at a concentration of 1.25 μg / mL, the recovery rates of positive cells SKBR3 were 90%, 99%, 99% and 97%, respectively.

[0275] Example 9: Comparative test of different substrates

[0276] As can be seen from Examples 6 and 8, the positive cell SKBR3 recovery rates of Formula 1, Formula 2, Formula 85, Formula 87 and Formula 90 at 1.25 μg / ml were 98%, 90%, 99%, 99% and 97%, respectively, but the minimum concentrations of different substrates to achieve effective recovery rates were different. This example studies the recovery rates of different substrates at different concentrations.

[0277] Experimental methods:

[0278] Use 0.5% HO 2 O 2 (containing 5% FBS) was used as the blocking solution for 15 min, HRP-labeled HER2 antibody was used as the incubation antibody (using a concentration of 4 μg / mL), biotin-labeled substrates were used at different concentrations, the incubation time was 30 min, the incubation buffer was 1% FBS (with 0.08% krovin-300M preservative added), and the test samples were obtained after sample blocking, antibody incubation, substrate incubation, and magnetic bead incubation as described in the above-mentioned step embodiment cell capture (1)-(5), and DAPI staining was added and the results were compared using a fluorescence microscope. The difference is that in step (3), the substrate is selected from (biotin-tyramine, biotin-styramide, biotin-XX-tyramine, biotin-PEG-tyramine, biotin-aniline), and the concentrations used are 1.0μg / ml, 0.7μg / ml, 0.5μg / ml, 0.3μg / ml, and 0.1μg / ml, respectively; in step (2), the HRP-labeled HER2 antibody is added and the fluorescent molecule-labeled CK detection antibody (manufacturer: Santa Cruz biotechnology, item number: SC-8018AF647) is added. The test results of each sample are shown in Table 19. The results show that when the concentration is 0.1μg / ml-0.5μg / ml, the recovery rate of biotin-styramide is higher, and the recovery rate of biotin-styramide is more stable. Therefore, at a lower concentration, biotin-styramide is preferred in terms of cost and performance stability.

[0279] Table 19 Comparison of recovery rates of different substrates

[0280]

[0281]

[0282] In summary, the kit disclosed in the present invention can be used for efficient detection of rare cells. Furthermore, the detection system disclosed in the present invention can also be used for the detection of other similar substrates, and compared with the secondary antibody capture system, it has higher capture sensitivity and capture efficiency without affecting specificity.

[0283] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0284] The above-mentioned embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure shall be subject to the attached claims.

Claims

1. A reagent combination for rare cell capture, wherein: The reagent combination comprises a biotin-labeled substrate and a capture antibody, wherein the biotin-labeled substrate is selected from the compounds or derivatives represented by formula (I), formula (II) or formula (III), in: A is is a single bond or a double bond; R1 is selected from -(CH2) n1 -, -CH(CH3)-, -C(CH3)2-, cycloalkyl, heterocyclyl, aryl or heteroaryl; Preferably, the cycloalkyl group is selected from Preferably, the heterocyclic group is selected from Preferably, the aryl group is selected from R2 is selected from -OH, -NH2, -SH and -H; X is and / or n1 is an integer selected from 0 to 10; n2 is selected from an integer of 1-300, preferably, n2 is selected from an integer of 1-10; n3 is selected from an integer of 1-10.

2. The reagent combination according to claim 1, wherein The biotin-labeled substrate is selected from a compound or a derivative thereof represented by any one of Formulas 1 to 93: More preferably, the biotin-labeled substrate is selected from the compounds shown in Formula 1, Formula 2, Formula 85, Formula 87 or Formula 90 or their derivatives.

3. The reagent combination according to claim 1 or 2, wherein The reagent combination also includes a blocking solution, an incubation buffer and magnetic beads; Preferably, the capture antibody is selected from one or more of anti-CK, anti-EpCAM, anti-PD-L1, anti-HER2, anti-TROP2, anti-c-MET, anti-Claudin18.2, anti-EGFR, anti-MUC1, anti-HER3, anti-CD31, anti-CD44 and the like antibodies; Preferably, the capture antibody is a labeled antibody, preferably an oxidoreductase labeled antibody; more preferably, the oxidoreductase is selected from any one of dehydrogenase, oxidase, peroxidase, and oxygenase; preferably, the oxidoreductase is selected from any one of horseradish peroxidase, catalase, and superoxide dismutase; preferably, the oxidoreductase is horseradish peroxidase; Preferably, the magnetic beads are labeled magnetic beads, preferably avidin-labeled magnetic beads; Preferably, the avidin is streptavidin or neutravidin; preferably streptavidin.

4. The reagent combination according to any one of claims 1 to 3, wherein: The concentration of the substrate is 0.1-5 μg / mL; Preferably, the concentration of the substrate is 0.625-1.25 μg / mL; Preferably, the concentration of the capture antibody is 1-8 μg / mL; Preferably, the concentration of the capture antibody is 4 μg / mL; More preferably, the concentration of the substrate is 1.25 μg / mL.

5. The reagent combination according to any one of claims 1 to 4, wherein: The blocking solution is selected from peroxidase blocking solution; Preferably, the peroxidase blocking solution is H2O2 blocking solution; Preferably, the H2O2 blocking solution further contains FBS or BSA; Preferably, the H2O2 blocking solution contains FBS; Preferably, the H2O2 blocking solution contains 5% FBS; Preferably, the concentration of H2O2 in the H2O2 sealing solution is 0.1%-0.7%, preferably 0.5%.

6. The reagent combination according to any one of claims 1 to 5, wherein: The incubation buffer is a buffer solution containing FBS or BSA; Preferably, the incubation buffer is a buffer solution containing FBS; Preferably, the incubation buffer is a buffer solution containing 1%-5% FBS; preferably a buffer solution containing 1% FBS; Preferably, the buffer solution is any one of PBS solution, HEPES buffer, and Hanks' balanced salt solution; preferably PBS solution.

7. The reagent combination according to any one of claims 1 to 6, characterized in that: Also included is an optional preservative; Preferably, the preservative is selected from isothiazolinone preservatives; Preferably, the active ingredient of the isothiazolinone preservative is selected from one or more of 2-methyl-4-isothiazoline-3-one, 5-chloro-2-methyl-4-isothiazoline-3-one, 1,2-benzisothiazolin-3-one, and 2-n-octyl-4-isothiazoline-3-one; Preferably, the preservative is krovin-300M or proclin-300; preferably krovin-300M; Preferably, the concentration of the preservative krovin-300M is 0.05%-0.1%; preferably 0.08%.

8. A kit for capturing rare cells, comprising the reagent combination according to claims 1-7.

9. A method for capturing rare cells based on the reagent combination according to any one of claims 1 to 7 or the kit according to claim 8, comprising the following steps: (1) Sample collection: Collect blood samples using anticoagulant blood collection tubes containing one or more components selected from the group consisting of EDTA-dipotassium, EDTA-disodium, sodium citrate, and heparin sodium; (2) Removal of red blood cells: using sucrose gradient centrifugation, NH4 + One or more methods of red blood cell lysis solution and red blood cell digestion enzyme are used to remove red blood cells from the sample; preferably containing NH4 + Red blood cell lysis buffer; (3) Cell fixation: adding a fixative to the sample obtained in step (2), wherein the fixative contains one or more of paraformaldehyde, formaldehyde, glutaraldehyde, methanol, ethanol, and acetone; preferably paraformaldehyde; (4) Sample blocking: adding blocking solution to the sample obtained in step (3) and incubating for 15-60 min, preferably 15 min; the sample cleaning solution is a solution containing FBS or BSA; (5) Antibody incubation: adding the capture antibody labeled with redox enzyme to the blocked sample obtained in step (4) and incubating overnight; (6) Incubation with biotin-labeled substrate: Add biotin-labeled substrate to the sample obtained in step (5) and incubate for 15-60 min, preferably 30 min; (7) Magnetic bead incubation: Add avidin-labeled magnetic beads to the sample obtained in step (6) and incubate for 15-60 min; (8) Cell separation: Use a magnetic separation device to sort target cells; (9) Cell detection: Scan and analyze the fluorescence signal of the sample; Preferably, the sample is a body fluid sample; Preferably, the rare cells are one or more of circulating tumor cells, circulating epithelial cells, circulating endothelial cells, and tumor stem cells; Preferably, the circulating tumor cell sample is selected from one or more of blood, pleural effusion, peritoneal effusion, saliva, sputum, urine, cerebrospinal fluid or pericardial effusion of an individual with cancer; Preferably, the cancer is selected from the group consisting of glioblastoma, cerebral astrocytoma, pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, fibrous dysplasia, fibrous dysplasia, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, Kaposi sarcoma , renal cancer, leukemia, liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, melanoma, metastatic renal cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, uterine cancer; Preferably, the cancer is selected from one or more of breast cancer, lung cancer, gastric cancer, skin cancer, liver cancer, pancreatic cancer, kidney cancer, prostate cancer, cervical cancer, uterine cancer, ovarian cancer, nasopharyngeal cancer, head and neck cancer, esophageal cancer, pharyngeal cancer, glioblastoma, thymic cancer, and thyroid cancer; preferably breast cancer and lung adenocarcinoma.

10. Use of the reagent combination according to any one of claims 1 to 7, the kit according to claim 8 and / or the method according to claim 9 in preparing a drug for capturing cells; Preferably, the cell is a rare cell; Preferably, the cell is a tumor cell.

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