CN107356744B - Method for sorting and/or enriching circulating tumor cells and kit thereof - Google Patents

Abstract

The invention provides a method for sorting and/or enriching circulating tumor cells, which utilizes the extremely high and stable affinity between a biotin-labeled anti-Her 2 monoclonal antibody, a biotin-labeled anti-EpCAM monoclonal antibody, a biotin-labeled anti-Trop 2 monoclonal antibody and an avidin-coated magnetic bead, and provides an immunomagnetic bead method for sorting and/or enriching the circulating tumor cells. The invention also provides a kit for sorting and/or enriching the tumor cells. The method of the invention can sensitively capture the tumor cells, is suitable for the broad-spectrum screening of the tumor cells, overcomes the defect of steric effect of common magnetic bead screening, and achieves the effect of efficiently capturing the tumor cells.

Description

Method for sorting and/or enriching circulating tumor cells and kit thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a method for sorting and/or enriching circulating tumor cells and a kit for sorting and/or enriching circulating tumor cells.

Background

Circulating Tumor Cells (CTCs) are tumor cells that fall off into the blood circulation, and a large number of experiments have confirmed that CTC detection is helpful for monitoring tumor recurrence and metastasis, judging patient prognosis, guiding postoperative adjuvant therapy, and the like. CTCs, however, are found in very small numbers in peripheral blood, usually only a few tumor cells in about 1 million white blood cells and 500 million red blood cells. Therefore, to increase the detection rate of circulating tumor cells, it is often necessary to enrich the circulating tumor cells prior to detection. Enrichment of circulating tumor cells can be achieved by using specific markers for tumor cells for isolation (immuno-isolation) or by exploiting differences in cell morphological characteristics such as cell volume and density.

The commonly used cell enrichment techniques mainly include immunomagnetic bead sorting, density gradient centrifugation, cell filtration and the like. Immunomagnetic bead sorting is a method for separating and enriching circulating tumor cells based on antigen-antibody combination, and is the most commonly used method at present (such as CN201110005376.8, a method for separating and enriching target cells from tissues, detection of circulating tumor cells and clinical application thereof in breast cancer patients, Maderliang and the like, journal of clinical oncology, 11 th of 2010, 15 th of 2010, 11 th of 2010, detection of circulating tumor cells, Lishuai and the like, medical review, 16 th of 3 th of 2010, 6 th of 16 th of 2010) and the like. The antibody immunomagnetic beads combine specific monoclonal antibodies with magnetic beads, cell surface antigens can be combined with the specific monoclonal antibodies connected with the magnetic beads, cells connected with the magnetic beads through the antibodies are adsorbed and retained in the magnetic field in an external magnetic field, and cells without corresponding surface antigens do not have magnetism because of being incapable of being combined with the specific monoclonal antibodies connected with the magnetic beads, and do not stay in the magnetic field, so that the cells are separated.

However, current direct coupling of antibodies to magnetic beads typically employs the NHS-activated ester method, i.e., activated carboxylated magnetic beads are reacted with primary amino groups of the antibody. Wherein, the stronger the electronegativity of the amino group, the higher the coupling efficiency. However, charged amino acids often play an important role in the structural stability of antibodies and in the binding process of antibodies to antigens. The cross-linking of these key amino acids with magnetic beads can seriously affect the stability of the antibody structure and the activity of the antibody. In addition, in the method of magnetic bead-antibody direct coupling, the magnetic bead is directly crosslinked with amino acid on the antibody, and the steric hindrance of the magnetic bead can affect the structure of the antibody, so that the exposure of a hydrophobic region of the antibody is easily caused, the focusing of the antibody is easily caused to lose activity, and the stability of the product is affected. Moreover, the Circulating Tumor Cells (CTC) in peripheral blood are rare in number, and have the characteristics of heterogeneity, easy aggregation and the like. The method also has a plurality of problems in the aspects of sensitivity, specificity, repeatability and the like.

The Chinese patent application with the application number of CN201410497998.0 discloses an antibody immunomagnetic bead method, and discloses the application of coupling immunomagnetic beads by combining three monoclonal antibodies of anti-HLA-G, EpCAM and CK8/18 in tumor cell sorting. However, this method uses a nonselective aldehyde coupling method. The aldehyde group can react with both amino group and carboxyl group. This method is often used in protein-protein coupling reactions. The protein and the poly-crosslinking between the proteins can be removed by methods such as ultra-high speed centrifugation/molecular sieve and ultrafiltration. Because the reaction process of the method is uncontrollable and the self-crosslinking of the antibody cannot be effectively removed, the method cannot be generally adopted for coupling the antibody and the magnetic beads. The uncontrollable reaction process can cause the coupling efficiency of the antibody and the magnetic beads to be low, and the difference between batches can be large. Therefore, in practical production applications, it is difficult to produce a stable product by this method. In addition, the direct coupling of the antibody and the magnetic bead can cause the magnetic bead to have stronger steric hindrance effect on the antibody, the influence on the activity of the antibody after the coupling is larger, and the capture efficiency of the tumor cells is easily influenced. The optimized combination ratio of the three antibodies has no repeatability.

Based on this, there is a need for a highly reproducible and efficient method for enriching circulating tumor cells that can be used in practical production applications.

Disclosure of Invention

Therefore, in order to solve the defects of the prior art, the invention aims to provide a method for sorting and/or enriching circulating tumor cells, which utilizes the extremely high and stable affinity between biotin-labeled anti-Her 2, EpCAM and Trop2 antibodies and avidin-coated magnetic beads, and provides an immunomagnetic bead method for sorting and/or enriching circulating tumor cells. The method provided by the invention greatly improves the capture efficiency of the circulating tumor cells. It is another object of the present invention to provide a kit for tumor cell sorting and/or enrichment.

In one aspect, the present invention provides a method of sorting and/or enriching for circulating tumor cells, the method comprising the steps of:

1) labeling an anti-Her 2 (human epidermal growth factor receptor 2) monoclonal antibody, an anti-EpCAM (epithelial cell adhesion molecule) monoclonal antibody, and an anti-Trop 2 (human trophoblast cell surface antigen 2) monoclonal antibody with biotin, and mixing them;

wherein, the mass percentage range of the mixed three monoclonal antibodies is as follows:

20-40% of biotin-labeled anti-Her 2 monoclonal antibody;

10-25% of biotin-labeled anti-EpCAM monoclonal antibody;

40-60% of biotin-labeled anti-Trop 2 monoclonal antibody;

2) adding the mixture obtained in the step 1) into cell fluid to be sorted;

wherein the volume ratio of the mixture to the cell sap is 1-5: 1000;

3) adding streptavidin coupled magnetic beads, incubating for 20 minutes at 4 ℃, and collecting the magnetic beads to obtain the product;

wherein the volume ratio of the cell sap to be sorted to the magnetic beads is 1000: 10-30.

Preferably, in step 1), the mass percentage range of the three monoclonal antibodies after mixing is:

35% of a biotin-labeled anti-Her 2 monoclonal antibody;

20% of biotin-labeled anti-EpCAM monoclonal antibody;

45% of biotin-labeled anti-Trop 2 monoclonal antibody;

preferably, in the step 2), the volume ratio of the mixture to the cell sap is 2-3: 1000;

preferably, in the step 3), the volume ratio of the cell liquid to be sorted to the magnetic beads is 1000: 20. Preferably, the tumor according to the present invention is selected from breast cancer, esophageal cancer, non-small cell lung cancer, cervical cancer, colon cancer, stomach cancer, uterine cancer and/or nasopharyngeal cancer.

Preferably, the Anti-EpCAM monoclonal Antibody is selected from EBA-1 (product name: Ep-CAM Antibody (EBA-1), product number sc-66020), AUA1 (product name: Ep-CAM Antibody (AUA1), product number sc-53277) or C-10 (product name: Ep-CAM Antibody (C-10), product number sc-25308), both from Santa Cruz Biotechnology, the Anti-Ephems 2 monoclonal Antibody is selected from 24D2 (product name: Neu Antibody (24D2), product number sc-23864), 0.N.211 (product name Neu Antibody (0.N.211), product number sc-71667) or 3B5 (product name: Neu Antibody (3B5), product number Neu Antibody (0.N.211), product number sc-3668), all from Santa Cruz Biotechnology, product name: Anti-Antibody is selected from product name: Neu Antibody (product name: Neu Antibody) (product number Sc-35162), product number Anti-Antibody from Santa Cruz Biotechnology, product name: Santa Antibody (product number Sc-36162), product number Clont-3646), product code 563243), B-9 (product name TROP-2 antibodyy (B-9), product code sc-376746) or F-5 (product name TROP-2 antibodyy (F-5), product code sc-376181), wherein 162-46 is from BD Bioscience, and B-9, F-5 are from Santa Cruz Biotechnology, Inc.).

Preferably, in step 1), the three biotin-labeled monoclonal antibodies are prepared by a method comprising the steps of:

a. purifying the antibody solution by using an ultrafiltration column;

b. adding NHS-PEG4-Biotin solution, and reacting for 0.5-4 h at room temperature;

wherein, the molar concentration ratio of the NHS-PEG4-Biotin to the antibody is 10-50: 1;

preferably at room temperature for 0.5-3 h; more preferably at room temperature for 1 h;

c. separating and purifying to remove free biotin, and obtaining a biotin-labeled monoclonal antibody solution.

Preferably, the separation and purification is performed using sephadex.

Preferably, the step a is accomplished by a method comprising the steps of:

preparing a marking reaction solution;

the marking solution comprises the following components:

NaCl：10-300mmol/L；KCl：1-5mmol/L；Na₂HPO₄：5-50mmol/L；KH₂PO₄：1-5mmol/L；

preferably, the labeling reaction solution comprises the following components:

NaCl：137mmol/L；KCl：2.7mmol/L；Na₂HPO₄：10mmol/L；KH₂PO₄：2mmol/L；

adding a labeled reaction solution into the ultrafiltration column, adding the monoclonal antibody, uniformly mixing, centrifuging, and removing a filtrate;

wherein the volume mass ratio of the labeling solution to the monoclonal antibody is 1:1-50 (mu l/mu g);

preferably, repeating the step for 2-3 times;

thirdly, uniformly mixing the residual liquid in the ultrafiltration column, standing at room temperature, reversely inverting the ultrafiltration column, placing the ultrafiltration column in a new ultrafiltration tube, centrifugally collecting filtrate, uniformly mixing PBS in the ultrafiltration column, and standing; inverting the ultrafiltration column, and centrifuging to collect filtrate;

in a preferred embodiment, the biotin-labeled monoclonal antibody is prepared by a method comprising the steps of:

adding 200 μ L of labeled reaction solution (NaCl137 mmol/L; KCl2.7 mmol/L; Na)₂HPO₄10mmol/L；KH₂PO₄2mmol/L), adding 500 mu g of monoclonal antibody, and mixing uniformly; centrifuging at 4 deg.C for 2min at 3500g, and removing filtrate; adding 100 μ L of labeled reaction solution (NaCl137 mmol/L; KCl2.7 mmol/L; Na)₂HPO₄10mmol/L；KH₂PO₄2mmol/L), mixing, and centrifuging at 5000g for 2min at 4 deg.C; repeating the steps of adding 100 mul of labeled reaction solution into the ultrafiltration column, mixing uniformly, and centrifuging for 2 times at the temperature of 4 ℃ for 2min at 5000 g;

mixing the residual liquid in the ultrafiltration column, and standing at room temperature for 1 min; inverting the ultrafiltration column, placing in a new ultrafiltration tube, centrifuging at 4 deg.C for 2min at 1000g, and collecting filtrate; mixing 50 μ l PBS in ultrafiltration column, and standing for 1 min; inverting the ultrafiltration column, centrifuging at 4 deg.C for 2min at 3500g, and collecting filtrate; mixing the above filtrates, and standing at 4 deg.C. Using a labeling reaction solution (NaCl137 mmol/L; KCl2.7 mmol/L; Na)₂HPO₄10mmol/L；KH₂PO₄2mmol/L) was adjusted to 2mg/ml (0.25 ml).

The filtrate after ultrafiltration was added with NHS-PEG4-Biotin solution (0.1-0.5mmol/L) and reacted at room temperature for 1 h. Separating and purifying the glucan gel to remove free biotin; obtaining the biotin-labeled monoclonal antibody solution.

Preferably, in step 2), the biotin-labeled anti-Her 2 monoclonal antibody, the biotin-labeled anti-EpCAM monoclonal antibody and the biotin-labeled anti-Trop 2 monoclonal antibody are used in the cell fluid at final concentrations of 300 μ g/ml to 600 μ g/ml, respectively.

Preferably, the biotin-labeled anti-Her 2 monoclonal antibody, the biotin-labeled anti-EpCAM monoclonal antibody and the biotin-labeled anti-Trop 2 monoclonal antibody are used at a final concentration of 500 μ g/ml in the cell fluid, respectively.

Preferably, the biotinylated anti-Her 2 monoclonal antibody is biotinylated 24D2, the biotinylated anti-EpCAM monoclonal antibody is biotinylated EBA-1, and the biotinylated anti-Trop 2 monoclonal antibody is biotinylated 162-46(24D2, EBA-1, 162-46 are all from Santa Cruz Biotechnology).

In another aspect, the invention provides a kit for sorting and/or enriching circulating tumor cells, the kit comprising a mixture of a biotin-labeled anti-Her 2 monoclonal antibody, a biotin-labeled anti-EpCAM monoclonal antibody, and a biotin-labeled anti-Trop 2 monoclonal antibody.

Preferably, the mass percentage ranges of the three monoclonal antibodies are as follows:

20-40% of biotin-labeled anti-Her 2 monoclonal antibody;

10-25% of biotin-labeled anti-EpCAM monoclonal antibody;

40-60% of biotin-labeled anti-Trop 2 monoclonal antibody;

preferably, the mass percentage ranges of the three monoclonal antibodies are as follows:

35% of a biotin-labeled anti-Her 2 monoclonal antibody;

20% of biotin-labeled anti-EpCAM monoclonal antibody;

45% of biotin-labeled anti-Trop 2 monoclonal antibody;

preferably, the kit of the present invention further comprises streptavidin-labeled magnetic beads (source company: BD Bioscience, cat # 557812).

In a further aspect, the invention also provides the use of the methods and kits of the invention for sorting and/or enriching circulating tumor cells.

Compared with the prior art, the technical scheme of the invention has the following advantages:

1) the invention relates to a method for sorting circulating tumor cells by using three monoclonal antibodies coupled by biotin, which adopts a mixture of anti-EpCAM (EpCAM), anti-Her 2 and anti-Trop 2 monoclonal antibodies, can sensitively capture tumor cells, is suitable for broad-spectrum screening of the tumor cells, overcomes the defect of steric effect of common magnetic bead screening, and achieves the effect of efficiently capturing the tumor cells.

2) It is known to those skilled in the art that, due to the inconsistent binding efficiency of various antibodies to tumor cell antigens, the combination of three antibodies for tumor cell enrichment directly affects the capturing effect of tumor cells, and the combination of three antibodies also affects the binding efficiency of each other. However, the inventors of the present invention have unexpectedly found that the coverage of tumor cells can be maximized within a suitable mass percentage range and at a reasonable final concentration by using the three monoclonal antibodies of the present invention, thereby greatly improving the efficiency and saving the cost.

3) The invention adopts the immunomagnetic bead compound consisting of the biotin-labeled monoclonal antibody and the avidin-modified magnetic bead to replace the method of directly crosslinking the antibody and the magnetic bead in the prior art, effectively ensures the activity of the immunomagnetic bead and improves the capture efficiency of tumor cells. The adding ratio of the antibody to the magnetic beads is 4:1 to 1:1, so that the antibody used as a core reagent is greatly saved.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The anti-EpCAM monoclonal antibodies of the invention are EBA-1, AUA1 and C-10, all purchased from SANTA CRUZBEICHNOGY; the anti-Her 2 monoclonal antibodies were 24D2, 0.N.211, and 3B5, all purchased from SANTA CRUZBETECHNOLOGY; the anti-Trop 2 monoclonal antibodies were 162-46, B-9 and F-5, with 162-46 being purchased from BD Bioscience and B-9 or F-5 being purchased from SANTA CRUZ BIOTECHNOLOGY. Magnetic beads of the present invention were purchased from BD Bioscience, cat #: 557812.

example 1 sorting of tumor cells in combination with three monoclonal antibodies coupled to biotin

1. Preparation of Biotin-labeled monoclonal antibodies

1.1 preparation of Biotin-labeled anti-EpCAM monoclonal antibodies

200. mu.l of a labeling reaction solution (NaCl137 mmol/L; KCl2.7 mmol/L; Na) was added to an ultrafiltration column (Millipore, cat # UFC501096)₂HPO₄10mmol/L；KH₂PO₄2mmol/L), 500. mu.g of anti-EpCAM monoclonal antibody (EBA-1) was added and mixed well. Centrifuging at 4 deg.C for 2min at 3500g, and removing filtrate; adding 100 μ L of labeled reaction solution (NaCl137 mmol/L; KCl 2.7mmol/L; Na)₂HPO₄10mmol/L；KH₂PO₄2mmol/L), mixing, and centrifuging at 5000g for 2min at 4 deg.C; repeating the steps of adding 100 mul of labeled reaction solution into the ultrafiltration column, mixing uniformly, and centrifuging for 2 times at the temperature of 4 ℃ for 2min at 5000 g;

mixing the residual liquid in the ultrafiltration column, and standing at room temperature for 1 min. Inverting the ultrafiltration column, placing in a new ultrafiltration tube, centrifuging at 4 deg.C for 2min at 1000g, and collecting filtrate. 50 μ l PBS was put on the ultrafiltration column and mixed well, and left to stand for 1 min. Inverting the ultrafiltration column, centrifuging at 4 deg.C for 2min at 3500g, and collecting filtrate. Mixing the above filtrates, and standing at 4 deg.C. Using a labeling reaction solution (NaCl137 mmol/L; KCl 2.7mmol/L; Na)₂HPO₄10mmol/L；KH₂PO₄2mmol/L) was adjusted to 2mg/ml (0.25 ml).

The filtrate after ultrafiltration was added with NHS-PEG4-Biotin solution (0.1-0.5mmol/L) and reacted at room temperature for 1 h. And separating and purifying the dextran gel (removing free biotin) to obtain a biotin-labeled monoclonal antibody solution A.

1.2 preparation of Biotin-labeled monoclonal anti-Her 2 antibodies

200. mu.l of a labeling reaction solution (NaCl137 mmol/L; KCl2.7 mmol/L; Na) was added to an ultrafiltration column (Millipore, cat # UFC501096)₂HPO₄10mmol/L；KH₂PO₄2mmol/L), 500. mu.g of anti-Her 2mg were addedAnd (3) mixing the monoclonal antibody (24D 2). Centrifuging at 4 deg.C for 2min at 3500g, and removing filtrate; adding 100 μ L of labeled reaction solution (NaCl137 mmol/L; KCl 2.7mmol/L; Na)₂HPO₄10mmol/L；KH₂PO₄2mmol/L), mixing, and centrifuging at 5000g for 2min at 4 deg.C; the above was repeated and 100. mu.l of the labeled reaction solution (NaCl137 mmol/L; KCl 2.7mmol/L; Na)₂HPO₄10mmol/L；KH₂PO₄2mmol/L), mixing, centrifuging at 4 deg.C for 2 times at 5000g for 2 min;

mixing the residual liquid in the ultrafiltration column, and standing at room temperature for 1 min. Inverting the ultrafiltration column, placing in a new ultrafiltration tube, centrifuging at 4 deg.C for 2min at 1000g, and collecting filtrate. 50 μ l PBS was put on the ultrafiltration column and mixed well, and left to stand for 1 min. Inverting the ultrafiltration column, centrifuging at 4 deg.C for 2min at 3500g, and collecting filtrate. Mixing the above filtrates, and standing at 4 deg.C. Using a labeling reaction solution (NaCl137 mmol/L; KCl 2.7mmol/L; Na)₂HPO₄10mmol/L；KH₂PO₄2mmol/L) was adjusted to 2mg/ml (0.25 ml).

The filtrate after ultrafiltration was added with NHS-PEG4-Biotin solution (0.1-0.5mmol/L) and reacted at room temperature for 1 h. And separating and purifying the dextran gel (removing free biotin) to obtain a biotin-labeled monoclonal antibody solution B.

1.3 preparation of Biotin-labeled monoclonal antibody anti-Trop 2antibody

200. mu.l of a labeling reaction solution (NaCl137 mmol/L; KCl2.7 mmol/L; Na) was added to an ultrafiltration column (Millipore, cat # UFC501096)₂HPO₄10mmol/L；KH₂PO₄2mmol/L), adding 500 μ g of anti-Trop 2 monoclonal antibody (162-46), and mixing. Centrifuging at 4 deg.C for 2min at 3500g, and removing filtrate; adding 100 μ L of labeled reaction solution (NaCl137 mmol/L; KCl 2.7mmol/L; Na)₂HPO₄10mmol/L；KH₂PO₄2mmol/L), mixing, and centrifuging at 5000g for 2min at 4 deg.C; the above was repeated and 100. mu.l of the labeled reaction solution (NaCl137 mmol/L; KCl 2.7mmol/L; Na)₂HPO₄10mmol/L；KH₂PO₄2mmol/L), mixing, centrifuging at 4 deg.C for 2mi at 5000gn, 2 times;

mixing the residual liquid in the ultrafiltration column, and standing at room temperature for 1 min. Inverting the ultrafiltration column, placing in a new ultrafiltration tube, centrifuging at 4 deg.C for 2min at 1000g, and collecting filtrate. 50 μ l PBS was put on the ultrafiltration column and mixed well, and left to stand for 1 min. Inverting the ultrafiltration column, centrifuging at 4 deg.C for 2min at 3500g, and collecting filtrate. Mixing the above filtrates, and standing at 4 deg.C. Using a labeling reaction solution (NaCl137 mmol/L; KCl 2.7mmol/L; Na)₂HPO₄10mmol/L；KH₂PO₄2mmol/L) was adjusted to 2mg/ml (0.25 ml).

The filtrate after ultrafiltration was added with NHS-PEG4-Biotin solution (0.1-0.5mmol/L) and reacted at room temperature for 1 h. And separating and purifying the dextran gel (removing free biotin) to obtain a biotin-labeled monoclonal antibody solution C.

2. Detection of antibody activity by flow cytometry

The SKBR3 cell is a Her2 receptor positive cell line and can be specifically combined with a Her2 antibody; MCF-7 cells are Epcam receptor positive cell lines and can be specifically combined with Epcam antibodies; the H1650 cell is a Trop2 receptor positive cell line and can be specifically combined with a Trop2 antibody; in order to verify the activity of the Her 2antibody, the Epcam antibody and the Trop 2antibody, monoclonal antibody solutions A, B and C obtained in the preparation process of the biotin-labeled monoclonal antibody are respectively incubated with SKBR3 cells, MCF-7 cells and H1650 cells, and binding signals are detected by a flow cytometry method.

2.1 preparation of a stationary liquid: the cell preservation solution A (250. mu.l) and the cell preservation solution B (250. mu.l) were mixed and then left at room temperature for 15min in the dark for use.

2.2SKBR3 cells, MCF-7 cells, H1650 cells collection: the cells were digested with 2ml of 0.25% trypsin, stopped by adding 5ml of medium, centrifuged at 1000rpm for 5min, the supernatant was discarded, and 500ul of PBS was added to resuspend the cells.

2.3SKBR3 cells, MCF-7 cells and H1650 cells: 500. mu.l of the cell suspension and 500. mu.l of the fixation solution were mixed well and left at room temperature for 1 hour in the dark. Centrifuging at 700g for 5min, discarding supernatant, adding 1ml binding buffer to resuspend cells for cell counting, and diluting cells to 2.8 × 10 with binding buffer⁵One per ml.

2.4 antibody dilution: antibody gradient dilutions were performed with binding buffer.

2.5 antibody incubation: the diluted antibodies were mixed at 100 ul/tube with 100. mu.l of cell suspension (about 2.8 ten thousand) and incubated with shaking at 4 ℃ for 30 min.

2.6 cell washing: each tube was added with 800ul PBST, centrifuged at 700g for 5min, 900. mu.l of supernatant was aspirated, 900. mu.l PBST was added, centrifuged at 700g for 5min, and 900. mu.l of supernatant was aspirated.

2.7 incubation with secondary antibody: 100ul of secondary antibody was added to each tube and incubated at 4 ℃ with shaking in the dark for 30 min.

2.8 repeat step 2.6 for cell washing.

2.9, detection on a computer: resuspend cells and detect with flow cytometry.

2.9 data analysis: the data was processed with softmax5.3 software.

Delta F% ((experimental group fluorescence value-negative group fluorescence value)/negative group fluorescence value 100%). The percentage of absolute fluorescence values relative to negative values is indicated.

3. Tumor cell sorting and/or enrichment

The laboratory cultured breast cancer cells (MCF-7, from the Chinese academy of sciences cell Bank), lung cancer cells (H1650, from the Chinese academy of sciences cell Bank), and colon cancer cells (SW480, from the Chinese academy of sciences cell Bank). Cells were diluted to 50000/ml and 4.8. mu.l were added per 8ml of blood for a total of 240/tube.

10ml of the fixing solution was added to 8ml of the blood sample, and the mixture was fixed for 24 hours or more. The blood sample was centrifuged at 700g for 10min and the supernatant was removed. Adding 45ml erythrocyte lysate, and rotating and cracking for 15min at room temperature. Centrifuge at 500g for 5min and remove supernatant. Add binding buffer 10ml heavy suspension cells, washing cells once, 300g centrifugation for 5 min. Removing supernatant, adding appropriate amount of washing binding buffer solution, and diluting to 1 ml. A, B, C mixture of three biotin-labeled antibodies, FITC-labeled anti-CK antibody and APC-labeled anti-CD 45 antibody are added in proportion, wherein the final concentration of the mixture of the biotin-labeled anti-Her 2antibody, the biotin-labeled anti-EpCAM antibody and the biotin-labeled anti-Trop 2antibody in the cell fluid is 500 mug/ml, the cell is incubated at 4 ℃ for 30min in a rotating mode, 10ml of washing buffer is added to wash the cells for 1 time, and the supernatant is removed after 300g of centrifugation. Adding 1ml of mixed solution consisting of streptavidin-labeled magnetic beads and FITC (FITC-activated carbon) reinforcing agent, wherein the volume ratio of the cell fluid to be sorted to the magnetic beads is 1000:20, 4 ℃ rotary incubation for 20 min. Tumor cell capture was performed.

3.1 analysis of cell staining results

The staining of the cells was observed under a microscope using a fluorescent antibody, and the number of tumor cells was counted to calculate the recovery rate.

Analysis of results of microscopic scanning by Leica DM 6000M: the DAPI stained cell nucleus presents blue fluorescence to identify nucleated cells, the leucocytes express CD45, the tumor cells do not express CD45, the marked CD45 antibody can be used for identifying the leucocytes, and CK is mainly distributed in epithelial cells, so that cells of CK8+ CD 45-are tumor cells, and cells of CK8-CD45+ are leucocytes.

Example 2 selection of three antibodies

The biotin antibody preparation method and the method for detecting antibody activity by flow cytometry as described in example 1, wherein the Delta F% values of the biotin-labeled anti-Her 2 monoclonal antibody, the biotin-labeled anti-EpCAM monoclonal antibody, and the biotin-labeled anti-Trop 2 monoclonal antibody are shown in tables 1-3 below:

TABLE 1 Her 2antibody binding SKBR3 cell Delta F% statistics

TABLE 2 Epcam antibody binding to MCF-7 cells Delta F% statistics

TABLE 3 Trop 2antibody binding H1650 cells Delta F% statistics

As can be seen from tables 1, 2 and 3, the Her 2antibody with the clone number 24D2 was most effective against the Her2 monoclonal antibody. Epcam antibodies with clone number EBA-1 work best for Epcam monoclonal antibodies. For the Trop2 monoclonal antibody, the Trop 2antibody with clone number 162-46 works best.

EXAMPLE 3 determination of the mixing ratio of three different monoclonal antibodies

The mixing ratios of the biotin-labeled anti-Her 2 monoclonal antibody (24D2), the biotin-labeled anti-EpCAM monoclonal antibody (EBA-1), the biotin-labeled anti-Trop 2 monoclonal antibody (162-46) are shown in Table 4 below, as described in example 1:

table 4:

and (4) conclusion: the recovery rate of the tumor cells of the groups 1, 4 and 5 is more than 70 percent, wherein the effect of the group 1 is the best, and the recovery rates of the tumor cells of the groups 2 and 3 are low and are not in accordance with the requirements.

EXAMPLE 4 determination of the amount of antibody mixture used

The experimental protocol was as described in example 1, wherein the mixed mass percentages of the biotin-labeled anti-Her 2 monoclonal antibody (24D2), the biotin-labeled anti-EpCAM monoclonal antibody (EBA-1), and the biotin-labeled anti-Trop 2 monoclonal antibody (162-46) were 35%, 20%, and 45%, respectively. The final concentrations of the above antibody mixtures used in the cell fluids are shown in Table 5 below

TABLE 5 determination of the amount of antibody mixture used

And (4) conclusion: the recovery rate of the tumor cells of the groups 2, 3 and 4 is more than 80%, wherein the group 3 has the best effect, the recovery rate of the tumor cells reaches 94%, and the recovery rates of the tumor cells of the groups 1, 5 and 6 are low and do not meet the requirement.

Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.

Claims (13)

1. A method of sorting and/or enriching for circulating tumor cells, the method comprising the steps of:

1) labeling an anti-Her 2 (human epidermal growth factor receptor 2) monoclonal antibody, an anti-EpCAM (epithelial cell adhesion molecule) monoclonal antibody, and an anti-Trop 2 (human trophoblast cell surface antigen 2) monoclonal antibody with biotin, and mixing them;

wherein, the mass percentage range of the mixed three monoclonal antibodies is as follows:

35% of a biotin-labeled anti-Her 2 monoclonal antibody;

20% of biotin-labeled anti-EpCAM monoclonal antibody;

45% of biotin-labeled anti-Trop 2 monoclonal antibody;

2) adding the mixture obtained in the step 1) into cell fluid to be sorted;

wherein the volume ratio of the mixture to the cell sap is 1-5: 1000;

the final concentrations of the mixture of biotin-labeled anti-Her 2 monoclonal antibody, biotin-labeled anti-EpCAM monoclonal antibody and biotin-labeled anti-Trop 2 monoclonal antibody used in the cell fluid were 500 μ g/ml respectively;

3) adding streptavidin coupled magnetic beads, incubating for 20 minutes at 4 ℃, and collecting the magnetic beads to obtain the product;

wherein the volume ratio of the cell sap to be sorted to the magnetic beads is 1000: 10-30;

wherein the tumor is selected from breast cancer, non-small cell lung cancer and/or colon cancer.

2. The method according to claim 1, wherein in step 2), the volume ratio of the mixture to the cell sap is 2-3: 1000.

3. The method of claim 1, wherein in step 3), the volume ratio of the cell fluid to be sorted to the magnetic beads is 1000: 20.

4. The method according to claim 1, wherein, in step 1), the three biotin-labeled monoclonal antibodies are prepared by a method comprising the steps of:

a. purifying the antibody solution by using an ultrafiltration column;

b. adding NHS-PEG4-Biotin solution, and reacting for 0.5-4 h at room temperature;

wherein, the molar concentration ratio of the NHS-PEG4-Biotin to the antibody is 10-50: 1;

c. separating and purifying to remove free biotin, and obtaining a biotin-labeled monoclonal antibody solution.

5. The method as claimed in claim 4, wherein in step b, NHS-PEG4-Biotin solution is added and reacted at room temperature for 0.5-3 h.

6. The method as claimed in claim 4, wherein in step b, NHS-PEG4-Biotin solution is added and reacted for 1h at room temperature.

7. The method according to claim 4, wherein in step c, the separation and purification is performed using sephadex.

8. The method of claim 4, wherein step a is accomplished by a method comprising the steps of:

preparing a marking reaction solution;

the labeling reaction solution comprises the following components:

NaCl：10-300mmol/L；KCl：1-5mmol/L；Na₂HPO₄：5-50mmol/L；KH₂PO₄：1-5mmol/L；

adding a labeled reaction solution into the ultrafiltration column, adding the monoclonal antibody, uniformly mixing, centrifuging, and removing a filtrate;

wherein the volume mass ratio of the labeling solution to the monoclonal antibody is 1:1-50 (mu l/mu g);

thirdly, uniformly mixing the residual liquid in the ultrafiltration column, standing at room temperature, reversely inverting the ultrafiltration column, placing the ultrafiltration column in a new ultrafiltration tube, centrifugally collecting filtrate, uniformly mixing PBS in the ultrafiltration column, and standing; inverting the ultrafiltration column, centrifuging and collecting filtrate.

9. The method according to claim 4, wherein, in step (r), the labeling reaction solution comprises the following components:

NaCl：137mmol/L；KCl：2.7mmol/L；Na₂HPO₄：10mmol/L；KH₂PO₄：2mmol/L。

10. the method of claim 4, wherein in step (II), the step (III) is repeated 2 to 3 times.

11. A kit for sorting and/or enriching circulating tumor cells, the kit comprising a mixture of a biotin-labeled anti-Her 2 monoclonal antibody, a biotin-labeled anti-EpCAM monoclonal antibody, and a biotin-labeled anti-Trop 2 monoclonal antibody;

wherein, the mass percentage ranges of the three monoclonal antibodies are as follows:

35% of a biotin-labeled anti-Her 2 monoclonal antibody;

20% of biotin-labeled anti-EpCAM monoclonal antibody;

45% of biotin-labeled anti-Trop 2 monoclonal antibody.

12. The kit of claim 11, further comprising streptavidin-labeled magnetic beads.

13. Use of the method according to any one of claims 1-10 or the kit according to claim 11 or 12 for sorting and/or enriching circulating tumor cells;

wherein the tumor is selected from breast cancer, non-small cell lung cancer and/or colon cancer.