Rare cell capture reagent combinations and methods

Abstract

The present disclosure provides a rare cell capture reagent combination and a capture method thereof, which comprises a substrate, a capture antibody, a blocking solution, an incubation buffer and magnetic beads; the substrate is a compound of formula (I) and its derivatives; the capture antibody includes but is not limited to 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 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 capture method can be widely used for capturing and separating rare cells with low expression of specific antigens. Compared with the traditional secondary antibody capture method, the capture efficiency of the present disclosure is higher, and has higher capture sensitivity while not affecting the specificity.

Description

Technical Field

[0001] This disclosure pertains to the field of biological detection, specifically relating to a reagent combination and method for capturing rare cells. Background Technology

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

[0003] Circulating tumor cells (CTCs) are tumor cells that detach from the primary tumor and invade the bloodstream. Studies have confirmed that CTCs can evade the body's immune system and reside at the primary site or in distant organs, thus forming recurrent and metastatic lesions. The specific mechanism is as follows: When the primary tumor grows to a certain stage, it invades surrounding blood vessels. Tumor cells first attach to the vascular basement membrane through integrin and grow. As the number of tumor cells gradually increases, the amount of matrix metalloproteinases they secrete also gradually increases. By gradually digesting type IV collagen, they break through the basement membrane barrier and enter the bloodstream, becoming known as CTCs. After entering the bloodstream, CTCs circulate throughout the body, leading to recurrence and metastasis. Therefore, CTC detection has important clinical prospects and roles in early auxiliary diagnosis, medication guidance, and efficacy monitoring for patients. However, due to the relatively low number of CTCs in the blood and the high white blood cell background, generally around 10... 7 The number of CTCs in white blood cells is only in the single digits (2-10 / mL), resulting in a low detection rate of CTCs with existing methods, which poses a significant challenge to their clinical application.

[0004] Currently, existing methods for detecting rare cells generally involve first enriching and purifying the cells using a forward capture method based on cell-specific antigens, then labeling the cells with specific detection antibodies, staining with nuclear dyes, and finally detecting them using a fluorescence microscope. This capture method requires target cells to have high levels of target antigen expression. However, in actual clinical applications, the expression levels of the same biomarker (such as HER2, PD-L1, CK, etc.) vary significantly among different patients. When the expression level of the target biomarker is low, conventional forward capture methods often struggle to effectively identify target CTCs, leading to false negatives and delaying treatment progress.

[0005] Therefore, there is an urgent need to develop a new kit and detection method for capturing and detecting rare cells to solve the problems of weak detection signal, low sensitivity, and low detection efficiency of existing detection methods. Summary of the Invention

[0006] The purpose of this 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 linkage of a biotin-labeled substrate to the tyrosine residues of a capturing antibody, thereby generating an activated substrate. This allows the antibody and substrate to be stably covalently bound, indirectly linking and enriching biotin around the target cells. Magnetic beads are then added to achieve highly sensitive capture of rare cells.

[0007] In one aspect, this 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 compounds or derivatives of formula (I), (II), or (III).

[0008]

[0009]

[0010] in:

[0011] A is

[0012] It can be a single bond or a double bond;

[0013] R1 is selected from -(CH2) n1 -, -CH(CH3)-, -C(CH3)2-, cycloalkyl, heterocyclic, 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] R2 is selected from -OH, -NH2, -SH, and -H;

[0018] X is and / or

[0019] n1 is selected from integers between 0 and 10;

[0020] n2 is selected from integers from 1 to 300, preferably, n2 is selected from integers from 1 to 10;

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

[0022] On the other hand, this disclosure provides a kit for rare cell capture that includes the aforementioned reagent combination.

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

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

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

[0026] 1. This disclosure provides a novel reagent combination that 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 to the traditional method of directly applying H2O2 blocking solution to cells, which can easily cause cells to adhere to the centrifuge tube wall when damaged, leading to cell loss, this disclosure uses H2O2 containing 5% FBS for blocking, effectively sealing the damaged cell area and preventing cell adhesion.

[0028] 3. This disclosure provides a novel method for capturing rare cells. Compared with traditional secondary antibody capture methods, the method of this disclosure has higher capture efficiency and higher capture sensitivity, while not affecting specificity. Attached Figure Description

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

[0030] Figure 2 The results of the investigation on the concentration of the blocking solution (0.05-3%) are shown.

[0031] Figure 3 The results of the investigation of serum concentration (0%-10%) during the closed phase are shown.

[0032] Figure 4 The results of the investigation into the concentration of HRP-tagged capture antibodies are shown.

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

[0034] Figure 6 The results of the substrate incubation time investigation are shown.

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

[0036] Figure 8 The results of investigations into different preservatives are shown.

[0037] Figure 9 The results of an investigation into the use of different concentrations of preservatives are shown.

[0038] Figure 10 The results of the cell enrichment and separation effect are shown.

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

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

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

[0042] Figure 14 The results of biotin-XX-tyramine validation are shown.

[0043] Figure 15 The results of biotin-PEG4-tyramine validation are shown.

[0044] Figure 16 The results of the biotin-styreneamide validation are shown.

[0045] Figure 17 The results of biotin-aniline verification are shown. Detailed Implementation

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

[0047] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in this disclosure is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure.

[0048] The terms “comprising” and “having”, and any variations thereof, in this disclosure are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0049] In this disclosure, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects are in an "or" relationship. Furthermore, to better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0050] As used in this disclosure, the terms “enrichment” or “capture” or “separation” refer to the process of separating rare cells from a sample to be tested.

[0051] As used in this disclosure, the term "magnetic bead" refers to tiny particles that possess magnetic properties. Immunomagnetic bead separation of cells is based on the fact that cell surface antigens can bind to specific monoclonal antibodies linked to magnetic beads. In an external magnetic field, cells linked to magnetic beads by antibodies are adsorbed and retained in the magnetic field. Cells without such surface antigens are not magnetic because they cannot bind to specific monoclonal antibodies linked to magnetic beads and therefore do not remain in the magnetic field, thus allowing the cells to be separated.

[0052] As used in this disclosure, the term "rare cells" refers to atypical cells found in biological fluid samples (including blood, pleural fluid, ascites, urine, cerebrospinal fluid, etc.). Studies have shown that the collection and analysis of rare cells are of significant guiding importance for identifying potential therapeutic mechanisms, pathological mechanisms, and developing targeted drugs for diseases.

[0053] As used in this disclosure, the term "peroxidase blocking solution" refers to a reagent used in enzyme detection methods to block endogenous peroxidases. Endogenous peroxidases are widely present in some cells and tissues, including erythrocytes, kidney tissue, and liver tissue. The presence of endogenous peroxidases in tissues or cells can lead to high background noise and even false positive results when using peroxidase methods for sample detection. Therefore, cell or tissue samples should be blocked with an appropriate peroxidase blocking solution before staining to eliminate interference from endogenous peroxidases. Optional peroxidase blocking solutions include, but are not limited to, endogenous peroxidase blocking solution (Beyotime P0100A), strong endogenous peroxidase blocking solution (Beyotime P0100B), or hydrogen peroxide solutions of varying concentrations.

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

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

[0056] In one aspect, this 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 compounds or derivatives of formula (I), (II), or (III).

[0057]

[0058] in:

[0059] A is

[0060] It can be a single bond or a double bond;

[0061] R1 is selected from -(CH2) n1 -, -CH(CH3)-, -C(CH3)2-, cycloalkyl, heterocyclic, aryl or heteroaryl;

[0062] R2 is selected from -OH, -NH2, -SH, and -H;

[0063] X is and / or

[0064] n1 is selected from integers between 0 and 10;

[0065] n2 is selected from integers from 1 to 300, preferably, n2 is selected from integers from 1 to 10;

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

[0067] Those skilled in the art should also understand that any modification to the compounds or their salts represented by formula (I), (II), or (III) is within the scope of protection of this invention, including but not limited to PEG bridging modification and aminocaproic acid bridging modification. This reagent combination is highly sensitive 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 this disclosure, the cycloalkyl group is selected from...

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

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

[0071] In some preferred embodiments of this disclosure, the biotin-labeled substrate is selected from any one of the compounds shown in Formula 1 to Formula 93 or its derivatives:

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

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

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

[0081] In some preferred embodiments of this disclosure, the capture antibody is selected from one or more of the following antibodies: 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, and anti-CD44.

[0082] In some preferred embodiments of this disclosure, 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.

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

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

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

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

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

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

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

[0090] In some preferred embodiments of this disclosure, the blocking solution is selected from peroxidase blocking solutions; preferably, the peroxidase blocking solution is an H2O2 blocking solution.

[0091] In some preferred embodiments of this disclosure, the H2O2 concentration in the H2O2 blocking solution is 0.1%-0.7%; preferably 0.5%.

[0092] In some preferred embodiments of this disclosure, the H2O2 blocking liquid further contains FBS or BSA.

[0093] In some preferred embodiments of this disclosure, the H2O2 blocking liquid contains FBS.

[0094] In some preferred embodiments of this disclosure, the H2O2 blocking solution contains 5% FBS.

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

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

[0097] In some preferred embodiments of this 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 this disclosure, the buffer solution is any one of PBS solution, hepes buffer, and Hanks' balanced salt solution; preferably, it is PBS solution.

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

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

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

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

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

[0104] In another aspect, this disclosure also provides a kit for rare cell capture comprising the aforementioned reagent combination.

[0105] In some embodiments of this disclosure, the biotin-labeled substrate, capture antibody, blocking solution, incubation buffer, magnetic beads, or preservative are aliquoted into different containers or bags.

[0106] On the other hand, this disclosure also provides a method for capturing rare cells using the aforementioned reagent combination or the aforementioned kit, comprising the following steps:

[0107] (1) Sample collection: Blood samples are collected using anticoagulation blood collection tubes, which contain one or more of the following components: EDTA-dipotassium, EDTA-disodium, sodium citrate, and sodium heparin.

[0108] (2) Red blood cell removal: Sucrose gradient centrifugation and NH4+ were used. + Red blood cells in the sample are removed by one or more methods using red blood cell lysis buffer and red blood cell digestive enzymes; preferably, the sample contains NH4. + Red blood cell lysis buffer;

[0109] (3) Cell fixation: Add 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 sealing: Add the sealing solution to the sample obtained in step (3) and incubate for 15-60 min; preferably 15 min; the sample washing solution is a solution containing FBS or BSA;

[0111] (5) Antibody incubation: Add oxidoreductase-labeled capture antibody to the blocked sample obtained in step (4) and incubate overnight;

[0112] (6) Biotin-labeled substrate incubation: 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: Target cells are sorted using a magnetic separation device;

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

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

[0117] In some preferred embodiments of this 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 this disclosure, the circulating tumor cell sample is selected from one or more of the following: blood, pleural effusion, ascites, saliva, sputum, urine, cerebrospinal fluid, or pericardial effusion of an individual with cancer.

[0119] In some preferred embodiments of this disclosure, the cancer is selected from glioblastoma of the brain, 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, connective tissue proliferative small round cell tumor, ependymoma, Ewing tumor, extraosseous myxoid chondrosarcoma, fibrous dysplasia of bone, fibrous dysplasia of bone, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, and islet cell tumor. Kaposi's sarcoma, renal cell carcinoma, leukemia, liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid adenoma, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, melanoma, renal metastatic carcinoma, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, uterine cancer (one or more of these).

[0120] In some preferred embodiments of this disclosure, the cancer is selected from one or more of breast cancer, lung cancer, stomach 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 or lung adenocarcinoma.

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

[0122] In some preferred embodiments of this disclosure, the cells are rare cells.

[0123] In some preferred embodiments of this disclosure, the cells are tumor cells.

[0124] Example

[0125] The technical solutions of this disclosure are further illustrated below through specific implementation methods. Those skilled in the art should understand that the embodiments described are merely illustrative of this disclosure and should not be construed as specific limitations thereof.

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

[0127] WBC: White blood cells

[0128] Biotin: Biotin

[0129] Streptavidin: Affinity

[0130] HER2: Human Epidermal Growth Factor Receptor 2

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

[0132] Epcam: Epithelial cell adhesion molecule

[0133] HRP: Horseradish peroxidase

[0134] 1. The specific experimental steps for cell detection in this embodiment are as follows:

[0135] (1) Sample sealing: Take cell samples (human breast cancer cells SKBR3 or human leukocytes, etc., with a cell concentration of 3×10⁻⁶). 5 Add blocking buffer (0.1 mL / sample), mix thoroughly, and incubate the cell samples in a 37°C water bath for a period of time. Remove the samples, centrifuge at 500g for 5 min, remove the supernatant and collect the cells. Add 1 mL / sample binding buffer (1% FBS-PBS solution, the same below) to resuspend the cells, centrifuge at 500g for 5 min, remove the supernatant and collect the cells, add 1 mL / sample binding buffer again to resuspend the cells, centrifuge at 500g for 5 min, remove the supernatant and collect the cells, add 100 μL / sample binding buffer to resuspend the cells, and set aside.

[0136] (2) Antibody incubation: Add 100 μL / part of incubation antibody to the cell solution obtained in step (1) according to a certain concentration, incubate at 2-8℃ for 30 min, add binding buffer to resuspend the cells, centrifuge at 500g for 5 min, remove the supernatant and collect the cells, add binding buffer to resuspend the cells again, centrifuge at 500g for 5 min, remove the supernatant and collect the cells to obtain the incubation antibody-cell complex sample, add 100 μL / part of incubation buffer solution 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 (Jackson ImmunoResearch, catalog number 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 500g for 5 min, remove the supernatant and collect the cells, add another 1 mL of binding buffer to resuspend the cells, centrifuge at 500g for 5 min, remove the supernatant and collect the cells to obtain the cell complex sample, and add 100 μL / sample of binding buffer to resuspend the cells.

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

[0139] (5) Cell detection: Samples are taken, DAPI is added to stain the cell nuclei, and finally fluorescence microscopy is used for scanning and analysis.

[0140] 2. The specific experimental steps for cell capture in this embodiment are as follows:

[0141] (1) Sample sealing: Take 1 mL of white blood cell sample per sample (cell concentration of 5 × 10⁻⁶ cells / sample) 7 Add a certain amount of CFSE-prestained human breast cancer cells (SKBR3, etc.) or human leukocytes to a sample (cells / ml). Add blocking buffer, mix thoroughly, and incubate the cell sample in a 37°C water bath for a period of time. Remove the sample, centrifuge at 500g for 5 min, remove the supernatant and collect the cells. Add 1 mL / part binding buffer (1% FBS-PBS solution, the same below) to resuspend the cells, centrifuge at 500g for 5 min, remove the supernatant and collect the cells, add 1 mL / part binding buffer to resuspend the cells, centrifuge at 500g for 5 min, remove the supernatant and collect the cells, add 1 mL / part binding buffer to resuspend the cells.

[0142] (2) Antibody incubation: Add antibody to the cell solution obtained in step (1) at a certain concentration and incubate overnight at 2-8℃. Add binding buffer to resuspend the cells, centrifuge at 500g for 5min, remove the supernatant and collect the cells. Add binding buffer to resuspend the cells again, centrifuge at 500g for 5min, remove the supernatant and collect the cells to obtain the incubated antibody-cell complex sample. Add 1mL / part 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 (Jackson ImmunoResearch, catalog number 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 500g for 5 min, remove the supernatant and collect the cells, add another 1 mL of binding buffer to resuspend the cells, centrifuge at 500g for 5 min, remove the supernatant and collect the cells to obtain the cell complex sample, and add 970 μL / sample of binding buffer to resuspend the cells.

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

[0145] (5) Cell capture and detection: Cells were enriched and separated using a magnetic rack. Simultaneously, DAPI was added to stain the cell nuclei, and finally, fluorescence microscopy was used for scanning analysis.

[0146] Example 1: Investigation of Cell Enclosure Conditions

[0147] To optimize experimental conditions, this embodiment explores the cell blocking conditions. The biotin-labeled substrate is biotinylate (compound of formula 1).

[0148] 1.1 Investigation of Closure Time

[0149] In this embodiment, 3% H2O2 was used as the blocking solution for endogenous peroxidase, and the blocking time was further investigated.

[0150] Using 3% H2O2 as the blocking solution, HRP-labeled HER2 antibody as the incubation antibody (concentration 4 μg / mL), biotin-labeled substrate concentration 0.625 μg / mL, incubation time 30 min, incubation buffer 1% FBS, blocking times in step (1) were 0 min, 5 min, 10 min, 15 min, 30 min, 45 min, and 60 min respectively. After sample blocking, antibody incubation, and substrate incubation as described in the aforementioned cell detection steps (1)-(3), cell complex samples were obtained. 100 μL of binding buffer was added to resuspend the cells, and then streptavidin-FITC (manufacturer: Thermo Fisher Scientific, catalog number: SA1001, final concentration 1:400) was added for staining for 30 min. DAPI was added to stain the cell nuclei, and finally the results were detected and compared using a fluorescence microscope.

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

[0152] Table 1. Experimental setup and results for investigating the closure time.

[0153]

[0154]

[0155] The results of the average fluorescence intensity detection for different incubation times in the negative and positive control groups are shown in the figure. Figure 1 As shown in Table 1, after blocking with H2O2 for 15-60 minutes, the background signal of the negative control group remained low, while the positive test groups all exhibited strong fluorescence detection signals. Furthermore, the difference between the positive and negative test groups was greatest at 15 minutes. Considering both the experimental results and the incubation time cost, a sample blocking time of 15 minutes is optimal.

[0156] 1.2 Investigation of the concentration of the sealing solution

[0157] In this embodiment, H2O2 was used as the blocking solution for endogenous peroxidase, and the sample blocking time was 15 min. The concentration of the H2O2 blocking solution was further investigated.

[0158] Alexa used HRP-labeled HER2 antibody (at a concentration of 4 μg / ml) and pan-Cytokeratin antibody (C11) 647 (manufacturer: Santa Cruz biotechnology, catalog number: SC-8018AF647) was used as the incubation antibody, and 100 μL of each sample was added. The concentration of biotinylated substrate was 0.625 μg / mL, the incubation time was 30 min, and the incubation buffer was 1% FBS. 3%, 2.5%, 2%, 1.5%, 1%, 0.7%, 0.5%, 0.3%, and 0.1% H2O2 solution (containing 5% FBS) were used as blocking solutions, and the blocking time was 15 min. After sample blocking, antibody incubation, and substrate incubation as described in the cell detection steps (1)-(3) above, cell complex samples were obtained. 100 μL of binding buffer was added to resuspend the cells, and then streptavidin-FITC (manufacturer: Thermo Fisher Scientific, catalog number: SA1001, final concentration 1:400) was added for staining for 30 min. DAPI was added to stain the cell nuclei, and finally, the results were detected and compared using a fluorescence microscope.

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

[0160] Table 2. Experimental setup and results for investigating the concentration of blocking solution (0.05-3%)

[0161]

[0162]

[0163] When using a 3% H2O2 blocking solution in this detection system, its blocking effect is too strong and may destroy intracellular antigens such as CK, making the antigens undetectable and affecting the sensitivity of the detection. Conversely, using H2O2 containing 5% FBS for blocking allows the FBS to seal damaged areas of the cells and prevent cell adhesion.

[0164] In the positive test group, when the blocking solution concentration was 0.1-0.7% (see...), Figure 2As shown in Table 2, the CK staining fluorescence values ​​were high (87.51-100.29), and the difference relative to the negative test group was also high (81.61-93.9). Meanwhile, the HER2 staining fluorescence values ​​in the negative control group were low (9.11-21.48). Therefore, blocking solution concentrations within the range of 0.1%-0.7% yielded good detection results. However, when the blocking solution concentration was 0.05%, the HER2 background signal value in the negative test group was 45.23, exceeding 40 (a certain background signal is present under a microscope when the value exceeds 40), indicating a high background level that did not meet the selection criteria. The difference between HER2 staining positive and negative values ​​was greatest at concentrations of 0.5%-0.7%. Considering the difference between CK staining positive and negative values, and to reduce reagent costs, 0.5% was the preferred concentration.

[0165] 1.3 Investigation of serum concentration during closure

[0166] In this experiment, 0.5% H2O2 was selected as the blocking solution and the blocking time was 15 min. The serum concentration during the blocking process was further investigated.

[0167] HRP-labeled HER2 antibody (concentration 4 μg / mL) was used as the incubation antibody, 100 μL / sample was added, biotin-labeled substrate concentration was 0.625 μg / mL, incubation time was 30 min, incubation buffer was 1% FBS, and H2O2 solution containing serum (FBS) concentrations of 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10% was used as blocking solution, blocking time was 15 min respectively. After sample blocking, antibody incubation and substrate incubation as described in the above steps of cell detection (1)-(3), cell complex samples were obtained, 100 μL of binding buffer was added to resuspend the cells, and then streptavidin-FITC (manufacturer: Thermo Fisher Scientific, catalog number: SA1001, final concentration 1:400) was added for staining for 30 min, sample was taken and DAPI was added to stain the cell nuclei, and finally the results were detected and compared using a fluorescence microscope.

[0168] Table 3. Experimental setup and results for investigating serum concentrations (0%-10%) in the blocking solution.

[0169]

[0170]

[0171] Depend on Figure 3As shown in Table 3, during the sealing process, if the solution contains a certain amount of FBS, the detection signal of the test group is stronger, with an average fluorescence intensity value 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 impact on sealing. Therefore, the recommended FBS content is 5%-10%. Considering the detection cost, a 5% FBS concentration is selected for sample sealing.

[0172] Example 2: Investigation of HRP-labeled antibody incubation conditions

[0173] In this embodiment, the concentration conditions of the HRP-labeled antibody were investigated. The biotin-labeled substrate was biotinylate tyramine.

[0174] Using 0.5% H2O2 (containing 5% FBS) as the blocking solution, the blocking time was 15 min. The biotin-labeled substrate concentration was 0.625 μg / mL, the incubation time was 30 min, the incubation buffer was 1% FBS, and HRP-labeled HER2 antibody was used as the incubation antibody (concentrations of 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, and 0.5 μg / ml were used). After sample blocking, antibody incubation, and substrate incubation as described in the cell detection (1)-(3) above, cell complex samples were obtained. 100 μL of binding buffer was added to resuspend the cells, and then streptavidin-FITC (manufacturer: Thermo Fisher Scientific, catalog number: SA1001, final concentration 1:400) was added for staining for 30 min. DAPI was added to the sample to stain the cell nuclei, and finally the results were detected and compared using a fluorescence microscope.

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

[0176] The secondary antibody test group (Table 4, AE group) replaced the substrate incubation in step (3) with incubation using secondary antibody (Biotin-SP Goat Anti-HRP antibody, Jackson Immuno Research, catalog number 123-065-021). The specific steps were as follows: 100 μL / part of secondary antibody (5 μg / ml) solution was added and thoroughly mixed, and then incubated at 37℃ for 30 min. Cell complex samples were obtained, and the cells were resuspended in 100 μL of binding buffer. Then, streptavidin-FITC (manufacturer: Thermo Fisher Scientific, catalog number: SA1001, final concentration 1:400) was added for staining for 30 min. DAPI was added to stain the cell nuclei, and finally, the results were compared using a fluorescence microscope. The results are shown in Table 4 and 2019. Figure 4 .

[0177] Table 4. Experimental setup and results for investigating labeled antibody concentrations.

[0178]

[0179] Depend on Figure 4 As shown in Table 4, when the concentration of HRP-labeled HER2 antibody was 1-8 μg / mL, the substrate test group exhibited a strong detection signal (118.15-143.02); the secondary antibody test group also showed a strong detection signal (72.40-93.67) when the concentration of HER2 antibody was 4-8 μg / mL; and the negative control group showed very low average fluorescence intensity (5.43-8.79). Therefore, the recommended concentration of HRP-labeled antibody is 1-8 μg / mL. Considering both cost and the stability of the detection results, and given that the positive test group showed the strongest detection signal at a concentration of 4 μg / mL, a concentration of 4 μg / mL was chosen for HRP incubation.

[0180] Example 3: Investigation of substrate incubation conditions

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

[0182] 3.1 Investigation of Substrate Concentration

[0183] To further improve reaction efficiency, this embodiment further explores the substrate concentration conditions.

[0184] Using 0.5% H2O2 (containing 5% FBS) as the blocking solution, the blocking time was 15 min. HRP-labeled HER2 antibody was used as the incubation antibody (concentration of 4 μg / mL), and the incubation buffer was 1% FBS. Biotin-labeled substrates were incubated at concentrations of 0.3125 μg / mL, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, and 5 μg / mL for 30 min. After sample blocking, antibody incubation, and substrate incubation as described in the cell detection (1)-(3) above, cell complex samples were obtained. Then, streptavidin-FITC (manufacturer: Thermo Fisher Scientific, catalog number: SA1001, final concentration 1:400) was added for staining for 30 min. DAPI was added to the sample to stain the cell nuclei. Finally, the results were compared using a fluorescence microscope.

[0185] The control group (AE) was tested using negative WBC cells. The positive test group (FJ) was tested using positive SKBR3 cells. The experimental setup and results for investigating the concentration of biotin-labeled substrates are shown in Table 5. Figure 5 .

[0186] Table 5. Experimental setup and results for investigating the concentration of biotin-labeled substrates.

[0187] The results show that ( Figure 5 As shown in Table 5, when the biotin-labeled substrate concentration was 0.3125 μg / mL-5 μg / mL, the detection signal of SKBR3 in positive cells was greater than the background signal of leukocytes in the negative control group, meeting the detection requirements. However, when the concentration was 2.5 μg / mL-5 μg / mL, the background signal of leukocytes was high; and when the concentration was 0.3125 μg / mL, the detection signal in the test group was too low. Therefore, considering the stability of the detection results, and given that the difference between the positive test group and the negative control group was the largest at 1.25 μg / mL, 1.25 μg / mL was preferred.

[0188] 3.2 Investigation of substrate incubation time

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

[0190] 0.5% H2O2 (containing 5% FBS) was used as the blocking solution for 15 min. HRP-labeled HER2 antibody was used as the incubation antibody (concentration of 4 μg / mL). Biotin-labeled substrate was used at a concentration of 1.25 μg / mL. The incubation buffer was 1% FBS. The substrate was incubated for 0 min, 5 min, 10 min, 15 min, 30 min, and 60 min, respectively. After sample blocking, antibody incubation, and substrate incubation as described in the cell detection (1)-(3) above, cell complex samples were obtained. Then, streptavidin-FITC (manufacturer: Thermo Fisher Scientific, catalog number: SA1001, final concentration 1:400) was added for staining for 30 min. DAPI was added to the sample to stain the cell nuclei. Finally, the results were compared using a fluorescence microscope.

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

[0192] Table 6. Experimental setup and results for investigating substrate incubation time.

[0193]

[0194] The results show that ( Figure 6(Table 6) When the substrate was incubated for 15-60 min, the positive cells in the test group all showed high detection signals of SKBR3, while the background signal in the negative control group was low. Therefore, the substrate incubation time was selected as 15-60 min to consider the stability of the detection results. At the same time, the difference between the positive and negative test groups was the largest when the incubation time was 30 min. Therefore, the substrate incubation time is preferably 30 min.

[0195] 3.3 Investigation of Incubation Buffer

[0196] 3.3.1 Investigation into the concentration of FBS

[0197] Further investigation is needed into the appropriate concentration of the incubation buffer FBS.

[0198] 0.5% H2O2 was used as the blocking solution for 15 min. HRP-labeled HER2 antibody was used as the incubation antibody (concentration 4 μg / mL), and biotin-labeled substrate was used at a concentration of 1.25 μg / mL for 30 min. The incubation buffers were 0% FBS, 1% FBS, 2% FBS, 3% FBS, 4% FBS, 5% FBS, 6% FBS, 7% FBS, 8% FBS, 9% FBS, and 10% FBS. After sample blocking, antibody incubation, and substrate incubation as described in the cell detection (1)-(3) above, cell complex samples were obtained. Streptavidin-FITC (manufacturer: Thermo Fisher Scientific, catalog number: SA1001, final concentration 1:400) was added for staining for 30 min. DAPI was then added to stain the cell nuclei. Finally, fluorescence microscopy was used to detect and compare the results. The experimental results are shown in Table 7 and 8. Figure 7 .

[0199] Table 7. Experimental setup and results for exploring incubation buffer.

[0200]

[0201]

[0202] Depend on Figure 7As shown in Table 7, if the FBS content in the solution exceeds 5% during substrate incubation, it will affect substrate binding. Within the FBS content range of 0%-5%, the average fluorescence intensity value is greater than 100 (high detection signal). When the content is 1%-2%, the average fluorescence intensity is close to that of 0% FBS, indicating that FBS in this concentration range has the least impact on substrate binding. Therefore, an FBS content of 0%-2% is recommended. However, a certain amount of FBS has a protective effect on cells. When using 1X-PBS or 1% FBS for substrate incubation, the fluorescence staining signal is strong, and the impact on substrate binding is relatively small. Therefore, considering all factors, a 1% FBS solution is chosen for substrate incubation.

[0203] 3.3.2 Investigation into the effects of different preservatives

[0204] Preservatives are beneficial for the preservation of incubation buffer, but since oxidoreductases are introduced into the system, different types of preservatives may have different effects on the experimental results. In order to minimize the impact on the capture and detection of rare cells, this embodiment further screens for the addition of preservatives.

[0205] Using 0.5% H2O2 (containing 5% FBS) as the blocking solution, the blocking time was 15 min. HRP-labeled HER2 antibody was used as the labeling antibody (concentration of 4 μg / mL), and biotin-labeled substrate was used at a concentration of 1.25 μg / mL. The incubation time was 30 min, and the incubation buffer was PBS buffer containing 1% FBS. In step (2), the preservatives listed in Table 8 were added to the incubation buffer. After sample blocking, antibody incubation, and substrate incubation as described in the cell detection (1)-(3) above, cell complex samples were obtained. Then, streptavidin-FITC (manufacturer: Thermo Fisher Scientific, catalog number: SA1001, final concentration 1:400) was added for staining for 30 min. DAPI was added to the sample to stain the cell nuclei. Finally, the results were compared using a fluorescence microscope.

[0206] The control group received no preservatives. The experimental results are shown in Table 8. Figure 8 .

[0207] Table 8 Experimental setup and results for preservative investigation

[0208]

[0209] The results show that ( Figure 8(Table 8) Using 0.1% Krovin-600, 0.1% Krovin-500, and 0.02% NaN3 as preservatives in the substrate incubation solution all affected 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) was used as a preservative, a fluorescence signal meeting the detection requirements was obtained. Among these, 0.1% Krovin-300M had the least impact on substrate staining. Therefore, 0.1% Krovin-300M was chosen as the preservative for subsequent substrate incubation solutions.

[0210] 3.3.3 Investigation into the concentration of preservatives used

[0211] Further investigation is needed into the concentration of preservatives used.

[0212] Using 0.5% H2O2 (containing 5% FBS) as the blocking solution, the blocking time was 15 min. HRP-labeled HER2 antibody was used as the labeling antibody (concentration of 4 μg / mL). Biotin-labeled substrate was used at a concentration of 1.25 μg / mL. The incubation time was 30 min. The incubation buffer was 1% FBS. In step (2), the preservatives listed in Table 9 were added to the incubation buffer (according to the product instructions, the recommended concentration is 1:1000-1:2000. Therefore, this example compared the effect of 0.05%-0.1% concentration on the detection results). After sample blocking, antibody incubation, and substrate incubation as described in the cell detection steps (1)-(3) of the above example, cell complex samples were obtained. Then, streptavidin-FITC (manufacturer: Thermo Fisher Scientific, catalog number: SA1001, final concentration of 1:400) was added for staining for 30 min. DAPI was added to the sample to stain the cell nucleus. Finally, the results were compared using a fluorescence microscope.

[0213] The control group contained no preservatives. The test results are shown in Table 9 and... Figure 9 .

[0214] Table 9. Experimental setup and results for investigating the concentration of preservatives used.

[0215]

[0216] Depend on Figure 9 As shown in Table 9, when the concentration of krovin-300M was 0.05%-0.08%, the average fluorescence intensity value was basically the same as that of the control group, indicating that the preservative had little effect on cell capture and detection within this concentration range. Considering the preservative effect of the preservative (the higher the concentration, the better the preservative effect), 0.08% of krovin-300M is preferred.

[0217] Example 4: Cell enrichment and isolation

[0218] Cell enrichment and separation is based on a magnetic separation technique, where magnetic separation refers to the technique of treating substances with a magnetic field. Suitable magnetic separation methods include magnetic rack separation and chip separation (such as Liquidbiopsy). This embodiment demonstrates a magnetic rack separation method, and the specific experimental procedure is as follows:

[0219] Using 0.5% H2O2 (containing 5% FBS) as the blocking solution, the blocking time was 15 min. HRP-labeled HER2 antibody was used as the incubation antibody (concentration 4 μg / mL), and biotin-labeled substrate was used at a concentration of 1.25 μg / mL for 30 min. The incubation buffer was 1% FBS. After sample blocking, antibody incubation, and substrate incubation as described in the cell detection examples (1)-(3) above, cell complex samples were obtained. Then, streptavidin-FITC (manufacturer: Thermo Fisher Scientific, catalog number: SA1001, final concentration 1:400) was added for staining for 30 min. During sample separation, the centrifuge tube was placed on a magnetic rack and allowed to stand for 6-8 minutes. Uncaptured leukocyte solution was aspirated with a pipette, and the cells were resuspended with 5 ml / part binding buffer. The centrifuge tube was placed on a magnetic rack and allowed to stand for 6-8 minutes. Uncaptured leukocyte solution was aspirated with a pipette. The cells were resuspended and enriched by adding 20 μL of binding buffer, dropped onto a glass slide, and stained with DAPI to stain the cell nuclei. Finally, the cells were scanned and analyzed using a fluorescence microscope.

[0220] Example 5: Verification of Rare Cell Capture Effect

[0221] Based on the above embodiments, the separation effect of the rare cell capture kit was verified. The biotin-labeled substrate was biotinylate tyramine.

[0222] Specifically as follows:

[0223] Using 0.5% H2O2 (containing 5% FBS) as the blocking solution, the blocking time was 15 min. HRP-labeled HER2 antibody was used as the incubation antibody (concentration 4 μg / mL), and biotin-labeled substrate was used at a concentration of 1.25 μg / mL. The incubation time was 30 min, and the incubation buffer was 1% FBS. Following the steps described in Example (1)-(5) of the above embodiment, the cell capture samples were obtained after sample blocking, antibody incubation, substrate incubation, and magnetic bead incubation. DAPI staining was added, and the results were compared using a fluorescence microscope. The difference was that the cell samples in step (1) were replaced with 1 mL / sample of leukocytes (cell concentration 5 × 10⁻⁶) of SKBR3 cells added at concentrations of 5, 25, and 100 cells respectively. 7(samples / mL); in step (2), HRP-labeled HER2 antibody was added along with fluorescently labeled CK detection antibody (manufacturer: Santa Cruz biotechnology, catalog number: SC-8018AF647). The experimental results for each sample are shown in Table 10 and... Figure 10 .

[0224] Table 10 Experimental setup and results for verifying cell enrichment and isolation effects.

[0225]

[0226] Depend on Figure 10 As shown in Table 10, the number of tumor cells recovered from samples A, B, and C were 5, 25, and 96, respectively, with recovery rates of 100%, 100%, and 96%, all meeting the acceptable recovery rates. Furthermore, the CK staining fluorescence signal was very strong. In summary, the scheme described in this disclosure can be used for the efficient detection of rare cells and possesses high capture efficiency.

[0227] Example 6: Capture and Comparative Validation of Low-Expression Protein Biomarker Samples

[0228] The kit described in this disclosure can be used for the efficient detection of rare cells. This embodiment further compares the capture sensitivity and specificity of this method with the secondary antibody capture method when facing rare cell samples with low expression levels of protein biomarkers. The biotin-labeled substrate is biotinylate (compound of formula 1). Details are as follows:

[0229] 6.1. Validation of HER2 expression in different cell lines

[0230] 0.5% H2O2 (containing 5% FBS) was used as the blocking solution for 15 min. HRP-labeled HER2 antibody was used as the incubation antibody (concentration 4 μg / mL), and biotin-labeled substrate was used at a concentration of 1.25 μg / mL for 30 min. The incubation buffer was 1% FBS. 500 cells / sample of SKBR3 (human breast cancer cells), ZR-75-1 (human breast cancer cells), MCF7 (human breast cancer cells), A549 (human lung adenocarcinoma cells), and WBC cells were added as cell samples. The cell samples were pre-stained with CFSE (eBbioscience CFSE, catalog number: 65-0850-84) beforehand. After sample blocking, antibody incubation, substrate incubation, and magnetic bead incubation as described in the cell capture steps (1)-(5) of the above example, the test samples were obtained. After DAPI staining, the results were detected and compared using a fluorescence microscope. The difference is that in step (2), HRP-labeled HER2 antibody is added along with fluorescently labeled CK detection antibody (manufacturer: Santa Cruz biotechnology, catalog number: SC-8018AF647).

[0231] Step (3) of the secondary antibody detection group is to replace substrate incubation with secondary antibody incubation. The specific steps are as follows: 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℃ for 30 min. Then add magnetic beads for incubation.

[0232] Table 11 Experimental setup and results for detecting low-expression protein biomarkers

[0233]

[0234] The results show that ( Figure 11 (Table 11) In cell lines with low expression of protein markers, the biotin-labeled substrate capture system showed significantly higher capture efficiency than the secondary antibody capture system, and both were negative in non-expressing cell lines (WBCs). Therefore, the biotin-labeled substrate capture system offers higher capture sensitivity without compromising specificity.

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

[0236] 0.5% H2O2 (containing 5% FBS) was used as the blocking solution for 15 min. HRP-labeled EpCAM antibody was used as the incubation antibody (concentration 4 μg / mL), and biotin-labeled substrate was used at a concentration of 1.25 μg / mL for 30 min. The incubation buffer was 1% FBS. 500 cells / sample 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), and WBC cells were added as cell samples. The cell samples were pre-stained with CFSE (eBbioscience CFSE, catalog number: 65-0850-84) beforehand. After sample blocking, antibody incubation, substrate incubation, and magnetic bead incubation as described in the cell capture steps (1)-(5) of the above example, the test samples were obtained. After DAPI staining, the results were detected and compared using a fluorescence microscope. The difference is that in step (2), HRP-labeled EpCAM antibody is added along with fluorescently labeled CK detection antibody (manufacturer: Santa Cruz biotechnology, catalog number: SC-8018AF647).

[0237] Step (3) of the secondary antibody detection group is to replace substrate incubation with secondary antibody incubation. The specific steps are as follows: 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℃ for 30 min. Then add magnetic beads for incubation.

[0238] Table 12 Experimental setup and results for the detection of low-expression protein biomarkers.

[0239]

[0240] The results show that ( Figure 12 (Table 12) In cell lines with low expression of protein markers, the biotin-labeled substrate capture system showed significantly higher capture efficiency than the secondary antibody capture system, and both were negative in non-expressing cell lines (WBCs). Therefore, the biotin-labeled substrate capture system offers higher capture sensitivity without compromising specificity.

[0241] 6.3. Validation of EGFR-expressing cell lines

[0242] 0.5% H2O2 (containing 5% FBS) was used as the blocking solution for 15 min. HRP-labeled EGFR antibody was used as the incubation antibody (concentration 4 μg / mL), and biotin-labeled substrate was used at a concentration of 1.25 μg / mL for 30 min. The incubation buffer was 1% FBS. 500 cells / sample 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), and WBC cells were added as cell samples. The cell samples were pre-stained with CFSE (eBbioscience CFSE, catalog number: 65-0850-84) beforehand. After sample blocking, antibody incubation, substrate incubation, and magnetic bead incubation as described in the cell capture steps (1)-(5) of the above example, the test samples were obtained. After DAPI staining, the results were detected and compared using a fluorescence microscope. The difference is that in step (2), HRP-labeled EGFR antibody is added along with fluorescently labeled CK detection antibody (manufacturer: Santa Cruz biotechnology, catalog number: SC-8018AF647).

[0243] Step (3) of the secondary antibody detection group is to replace substrate incubation with secondary antibody incubation. The specific steps are as follows: 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℃ for 30 min. Then add magnetic beads for incubation.

[0244] Table 13 Experimental setup and results for the detection of low-expression protein biomarkers.

[0245]

[0246] The results show that ( Figure 13 (Table 13) In cell lines with low expression of protein markers, the biotin-labeled substrate capture system showed significantly higher capture efficiency than the secondary antibody capture system, and both were negative in non-expressing cell lines (WBCs). Therefore, the biotin-labeled substrate capture system offers higher capture sensitivity without compromising specificity.

[0247] Example 7: Verification using other substrates

[0248] 7.1. Biotin-XX-tyramine (Compound 85, Manufacturer: APExBIO, Catalog No.: A8012) Test

[0249] Using 0.5% H2O2 (containing 5% FBS) as the blocking solution, the blocking time was 15 min. HRP-labeled HER2 antibody was used as the labeling antibody (concentration of 4 μg / mL), and biotin-labeled substrate (concentration of 0.625 μg / mL-10 μg / mL) was used. The incubation time was 30 min, and the incubation buffer was 1% FBS (with 0.08% krovin-300M preservative added). After sample blocking, antibody incubation, and substrate incubation as described in the above-mentioned cell detection examples (1)-(3), cell complex samples were obtained. Then, streptavidin-FITC (manufacturer: Thermo Fisher Scientific, catalog number: SA1001, final concentration 1:400) was added for staining for 30 min. DAPI was added to the sample to stain the cell nuclei. Finally, the results were detected and compared using a fluorescence microscope.

[0250] Table 14. Verification results of biotin-XX-tyramine test.

[0251]

[0252] The results show that ( Figure 14 (Table 14) When the concentration of biotin-labeled substrate was 0.625 μg / mL-10 μg / mL, the detection signal of SKBR3 in positive cells was greater than the background signal of leukocytes in the negative control group, which met the detection requirements.

[0253] 7.2. Biotin-PEG4-tyramine (Compound 87, Manufacturer: CONFLUOR, Catalog No.: BBBP-1-100mg) Testing and Verification

[0254] Using 0.5% H2O2 (containing 5% FBS) as the blocking solution, the blocking time was 15 min. HRP-labeled HER2 antibody was used as the labeling antibody (concentration of 4 μg / mL), and biotin-labeled substrate (concentration of 0.625 μg / mL-10 μg / mL) was used. The incubation time was 30 min, and the incubation buffer was 1% FBS (with 0.08% krovin-300M preservative added). After sample blocking, antibody incubation, and substrate incubation as described in the above-mentioned cell detection examples (1)-(3), cell complex samples were obtained. Then, streptavidin-FITC (manufacturer: Thermo Fisher Scientific, catalog number: SA1001, final concentration 1:400) was added for staining for 30 min. DAPI was added to the sample to stain the cell nuclei. Finally, the results were detected and compared using a fluorescence microscope.

[0255] Table 15. Verification results of biotin-PEG4-tyramine test.

[0256]

[0257]

[0258] The results show that ( Figure 15 (Table 15) When the concentration of biotin-labeled substrate was 0.625 μg / mL-10 μg / mL, the detection signal of SKBR3 in positive cells was greater than the background signal of leukocytes in the negative control group, which met the detection requirements.

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

[0260] Using 0.5% H2O2 (containing 5% FBS) as the blocking solution, the blocking time was 15 min. HRP-labeled HER2 antibody was used as the labeling antibody (concentration of 4 μg / mL), and biotin-labeled substrate (concentration of 0.625 μg / mL-10 μg / mL) was used. The incubation time was 30 min, and the incubation buffer was 1% FBS (with 0.08% krovin-300M preservative added). After sample blocking, antibody incubation, and substrate incubation as described in the above-mentioned cell detection examples (1)-(3), cell complex samples were obtained. Then, streptavidin-FITC (manufacturer: Thermo Fisher Scientific, catalog number: SA1001, final concentration 1:400) was added for staining for 30 min. DAPI was added to the sample to stain the cell nucleus. Finally, the results were detected and compared using a fluorescence microscope.

[0261] Table 16. Verification Results of Biotin-Styreneamide Test

[0262]

[0263] The results show that ( Figure 16 (Table 16) When the concentration of biotin-labeled substrate was 0.625 μg / mL-10 μg / mL, the detection signal of SKBR3 in positive cells was greater than the background signal of leukocytes in the negative control group, which met the detection requirements.

[0264] 7.4. Biotin-aniline (Compound of Formula 2, Manufacturer: MedMol, CAS No.: 769933-15-5) Testing and Verification

[0265] Using 0.5% H2O2 (containing 5% FBS) as the blocking solution, the blocking time was 15 min. HRP-labeled HER2 antibody was used as the labeling antibody (concentration of 4 μg / mL), and biotin-labeled substrate (concentration of 0.625 μg / mL-10 μg / mL) was used. The incubation time was 30 min, and the incubation buffer was 1% FBS (with 0.08% krovin-300M preservative added). After sample blocking, antibody incubation, and substrate incubation as described in the above-mentioned cell detection examples (1)-(3), cell complex samples were obtained. Then, streptavidin-FITC (manufacturer: Thermo Fisher Scientific, catalog number: SA1001, final concentration 1:400) was added for staining for 30 min. DAPI was added to the sample to stain the cell nucleus. Finally, the results were detected and compared using a fluorescence microscope.

[0266] Table 17. Verification Results of Biotin-Aniline Test

[0267]

[0268]

[0269] The results show that ( Figure 17 (Table 17) When the concentration of biotin-labeled substrate was 0.625 μg / mL-10 μg / mL, the detection signal of SKBR3 in positive cells was greater than the background signal of leukocytes in the negative control group, which met the detection requirements.

[0270] Example 8: Validation Tests for Capture of Other Substrates

[0271] Using 0.5% H2O2 (containing 5% FBS) as the blocking solution, the blocking time was 15 min. HRP-labeled HER2 antibody was used as the incubation antibody (concentration of 4 μg / mL), and biotin-labeled substrate was used at a concentration of 1.25 μg / mL. The incubation time was 30 min, and the incubation buffer was 1% FBS (with 0.08% krovin-300M preservative added). After sample blocking, antibody incubation, substrate incubation, and magnetic bead incubation as described in the cell capture (1)-(5) steps of the above example, the test samples were obtained. After adding DAPI staining, the results were detected and compared using a fluorescence microscope. The difference is that in step (3), the substrate was selected from (biotin-styreneamide, biotin-XX-tyramine, biotin-PEG-tyramine, biotin-aniline); in step (2), HRP-labeled HER2 antibody was added along with fluorescently labeled CK detection antibody (manufacturer: Santa Cruz biotechnology, catalog number: SC-8018AF647). The test results of each sample are shown in Table 18. The results showed that when the concentration was 1.25 μg / ml, the recoveries of the four biotin substrates all exceeded 90%, with biotin-styreneamide, biotin-XX-tyramine, and biotin-PEG-tyramine having higher recoveries than biotin-aniline.

[0272] Table 18 Validation results of capture tests on different substrates

[0273]

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

[0275] Example 9: Comparison Test with Different Substrates

[0276] As shown in Examples 6 and 8, the recovery rates of SKBR3 in positive cells using Formulas 1, 2, 85, 87, and 90 at 1.25 μg / ml were 98%, 90%, 99%, 99%, and 97%, respectively. However, the minimum concentration required for effective recovery varied among the different substrates. This example studies the recovery rates of different substrates at different concentrations.

[0277] Experimental methods:

[0278] Using 0.5% H2O2 (containing 5% FBS) as the blocking solution, the blocking time was 15 min. HRP-labeled HER2 antibody was used as the incubation antibody (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). After cell capture (1)-(5) as described in the above steps, the sample was blocked, incubated with antibody, incubated with substrate, and incubated with magnetic beads to obtain the test sample. After adding DAPI staining, the results were detected and compared using a fluorescence microscope. The difference lies in the substrate selection: in step (3), the substrate is chosen from (biotin-tyramine, biotin-styreneamide, biotin-XX-tyramine, biotin-PEG-tyramine, biotin-aniline), with concentrations of 1.0 μg / ml, 0.7 μg / ml, 0.5 μg / ml, 0.3 μg / ml, and 0.1 μg / ml, respectively; in step (2), HRP-labeled HER2 antibody is added along with fluorescently labeled CK detection antibody (manufacturer: Santa Cruz biotechnology, catalog number: SC-8018AF647). The experimental results for each sample are shown in Table 19. The results indicate that the recovery rate of biotin-styreneamide is high and more stable when the concentration is between 0.1 μg / ml and 0.5 μg / ml. Therefore, considering cost and performance stability, biotin-styreneamide is preferred at lower concentrations.

[0279] Table 19 Comparison of recoveries of different substrates

[0280]

[0281]

[0282] In summary, the kit described in this disclosure can be used for the efficient detection of rare cells. Furthermore, the detection system of this disclosure 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 embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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 embodiments described above are merely illustrative of several implementations of this disclosure, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this disclosure, and these all fall within the protection scope of this disclosure. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. A reagent combination for rare cell capture, wherein, The reagent combination comprises a biotin-labeled substrate, a capture antibody, a peroxidase blocking solution, and magnetic beads, the capture antibody is an oxidoreductase-labeled antibody, the oxidoreductase is horseradish peroxidase, and the capture antibody is selected from one or more of anti-EpCAM, anti-HER2, and anti-EGFR antibodies; The biotin-labeled substrate is selected from any one of the compounds shown in formula 1, formula 2, formula 85, formula 87, and biotin-styryl amide: The biotin-styryl amide is biotin-styryl amide with a product number of 45300 produced by AAT.

2. The reagent combination according to claim 1, wherein, The reagent combination further comprises an incubation buffer.

3. The combination of agents according to claim 1 or 2, wherein, The magnetic beads are labeled magnetic beads.

4. The combination of agents according to claim 3, wherein, The magnetic beads are avidin-labeled magnetic beads.

5. The combination of agents according to claim 4, wherein, The avidin is streptavidin or neutral avidin.

6. The combination of agents according to claim 5, wherein, The avidin is streptavidin.

7. The combination of agents according to claim 1 or 2, wherein, The concentration of the substrate is 0.1-5 μg / mL.

8. The combination of agents according to claim 7, wherein, The concentration of the substrate is 0.625-1.25 μg / mL.

9. The combination of agents according to claim 1 or 2, wherein, The concentration of the capture antibody is 1-8 μg / mL.

10. The combination of agents according to claim 9, wherein, The concentration of the capture antibody is 4 μg / mL.

11. The combination of agents of claim 7, wherein, The concentration of the substrate is 1.25 μg / mL.

12. The reagent combination of claim 1 or 2, wherein the peroxidase blocking solution is an H2O2 blocking solution.

13. The combination of agents according to claim 12, wherein, The H2O2 blocking solution further contains FBS or BSA.

14. The combination of agents according to claim 13, wherein, The H2O2 blocking solution contains FBS.

15. The combination of agents according to claim 14, wherein, The H2O2 blocking solution contains 5% FBS.

16. The combination of agents of claim 12, wherein, The concentration of H2O2 in the H2O2 blocking solution is 0.1%-0.7%.

17. The combination of agents according to claim 16, wherein, The concentration of H2O2 in the H2O2 blocking solution is 0.5%.

18. The combination of agents of claim 2, wherein, The incubation buffer is a buffer solution containing FBS or BSA.

19. The combination of agents of claim 18, wherein, The incubation buffer is a buffer solution containing FBS.

20. The combination of agents of claim 19, wherein, The incubation buffer is a buffer solution containing 1%-5% FBS.

21. The combination of agents of claim 20, wherein, The incubation buffer is a buffer solution containing 1% FBS.

22. The combination of agents of claim 18, wherein, The buffer solution is any one of a PBS solution, a hepes buffer, and a Hanks' balanced salt solution.

23. The combination of agents of claim 22, wherein, The buffer solution is a PBS solution.

24. The combination of agents of claim 1 or 2, wherein, Further comprising an optional preservative.

25. The combination of agents of claim 24, wherein, The preservative is selected from isothiazolinone preservatives.

26. The combination of reagents according to claim 25, wherein, The active ingredient of the isothiazolinone preservative is selected from one or more of 2-methyl-4-isothiazolin-3-one, 5-chloro-2-methyl-4-isothiazolin-3-one, 1,2-benzisothiazolin-3-one, and 2-n-octyl-4-isothiazolin-3-one.

27. The combination of agents of claim 24, wherein, The preservative is krovin-300M or proclin-300.

28. The combination of agents of claim 27, wherein, The preservative is krovin-300M.

29. The combination of agents of claim 28, wherein, The concentration of the preservative krovin-300M is 0.05%-0.1%.

30. The combination of reagents according to claim 29, wherein, The concentration of the preservative krovin-300M is 0.08%.

31. A kit for capturing rare cells, comprising the reagent combination of any one of claims 1-30.

32. Use of the reagent combination of any one of claims 1-30 in the preparation of a kit for capturing rare cells.

33. The use of claim 32, wherein, The method for capturing rare cells comprises the following steps: The method for capturing rare cells comprises the following steps: (1) sample collection: blood sample is collected by using an anticoagulant blood collection tube containing one or more components of EDTA-dipotassium, EDTA-disodium, sodium citrate, heparin sodium; (2) Red blood cell removal: removal of red blood cells from the sample using one or more of sucrose gradient centrifugation, red blood cell lysis solution, red blood cell digestion enzyme + red blood cell lysis solution, red blood cell digestion enzyme (3) cell fixation: a fixing solution containing one or more of paraformaldehyde, formaldehyde, glutaraldehyde, methanol, ethanol, acetone is added to the sample obtained in step (2); (4) sample blocking: blocking solution is added to the sample obtained in step (3) and incubated for 15-60 min; the sample washing solution is a solution containing FBS or BSA; (5) antibody incubation: redox enzyme-labeled capture antibody is added to the blocked sample obtained in step (4) and incubated overnight; (6) biotin-labeled substrate incubation: biotin-labeled substrate is added to the sample obtained in step (5) and incubated for 15-60 min; (7) magnetic bead incubation: avidin-labeled magnetic beads are added to the sample obtained in step (6) and incubated for 15-60 min; (8) cell separation: the target cells are sorted using a magnetic separation device; (9) cell detection: the fluorescence signal of the sample is scanned and analyzed.

34. The use of claim 33, wherein, In step (2), NH4 + Red blood cell lysing solution removes red blood cells from the sample.

35. Use according to claim 33 or 34, wherein, In step (3), the fixing solution is a fixing solution containing paraformaldehyde.

36. The use of claim 33 or 34, wherein, In step (4), the incubation time is 15 min.

37. The use of claim 33 or 34, wherein, In step (6), the incubation time is 30 min.

38. The use of claim 33 or 34, wherein, The sample is a body fluid sample.

39. The use of claim 32 or 33, wherein, The rare cells are one or more of circulating tumor cells, circulating epithelial cells, circulating endothelial cells, tumor stem cells.

40. The use of claim 39, wherein, 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.

41. The use of claim 40, wherein, The cancer is selected from one or more of adrenal gland tumor, bladder cancer, bone cancer, breast cancer, carotid body tumor, cervical cancer, chordoma, clear cell carcinoma, colon cancer, desmoplastic small round cell tumor, ependymal cell tumor, Ewing's tumor, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, kidney cancer, leukemia, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, pediatric cancer, peripheral nerve sheath tumor, prostate cancer, rhabdoid tumor, squamous cell carcinoma, testicular cancer, thymic carcinoma, thymoma, uterine cancer.

42. The use of claim 40, wherein, The cancer is brain astrocytoma, metastatic brain tumor or meningioma.

43. The use of claim 40, wherein, The cancer is astrocytoma.

44. The use of claim 40, wherein, The cancer is oropharyngeal cancer.

45. The use of claim 40, wherein, The cancer is brain and spinal cord cancer.

46. The use of claim 40, wherein, The cancer is fibrous dysplasia of bone, fibrous dysplasia of bone, multiple myeloma or myelodysplastic syndrome.

47. The use of claim 40, wherein, The cancer is alveolar soft part sarcoma, chondrosarcoma, extraskeletal myxoid chondrosarcoma, Kaposi's sarcoma, liposarcoma, rhabdomyosarcoma, soft tissue sarcoma or synovial sarcoma.

48. The use of claim 40, wherein, The cancer is malignant lipomatous tumor.

49. The use of claim 40, wherein, The cancer is renal chromophobe carcinoma or renal metastatic carcinoma.

50. The use of claim 40, wherein, The cancer is hepatocellular carcinoma.

51. The use of claim 40, wherein, The cancer is thyroid metastatic carcinoma.

52. The use of claim 40, wherein, The cancer is multiple endocrine neoplasia.

53. The use of claim 40, wherein, The cancer is islet cell tumor.

54. The use of claim 40, wherein, The cancer is pheochromocytoma, pituitary tumor, papillary thyroid carcinoma or parathyroid tumor.

55. The use of claim 40, wherein, The cancer is an AIDS-related cancer.

56. The use of claim 40, wherein, The cancer is a colorectal cancer.

57. The use of claim 40, wherein, The cancer is a sarcoma.

58. The use of claim 40, wherein, The cancer is a skin cancer.

59. The use of claim 40, wherein, 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, head and neck cancer, esophageal cancer, glioblastoma, thymus cancer, thyroid cancer.

60. The use of claim 59, wherein, The cancer is breast cancer or lung cancer.

61. The use of claim 59, wherein, The cancer is nasopharyngeal cancer or oropharyngeal cancer.

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