Kit for separating and recovering after immunocapture of circulating tumor cells, preparation method and application

Abstract

The present application belongs to the technical field of tumor detection and cell separation, and particularly relates to a kit for separating and recovering after immunocapture of circulating tumor cells, a preparation method and application. The kit comprises an immunocapture module, a dissociation and recovery module, a cell protection module and elution and supplement factor solutions. The immunocapture module adopts magnetic microspheres modified by double antibody fusion proteins, can recognize multiple markers such as EpCAM and Vimentin, and realizes efficient capture of heterogeneous CTCs. The dissociation and recovery module uses a low-temperature buffer system and a cleavable cross-linking agent to release cells gently. The cell protection module maintains the integrity and activity of the cell membrane during the separation process. The method of the present application can ensure the capture efficiency while obtaining high-activity CTCs, and is suitable for single-cell sequencing, drug sensitivity analysis, organoid culture and patient-derived cell line construction and other preclinical research.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tumor detection and cell separation, and particularly relates to a kit for separating and recovering circulating tumor cells after immunocapture, a preparation method and application. BACKGROUND

[0002] Circulating tumor cells (CTCs) are rare tumor cells that enter the blood circulation after falling off from primary tumors or metastatic foci, and play a key role in tumor metastasis. Since CTCs are derived from tumor tissues, they carry genetic and phenotypic information of tumors, and have extremely high research and diagnostic value. At present, as a "liquid biopsy" target, CTCs have been widely used in tumor early screening, individualized treatment guidance, efficacy evaluation and recurrence monitoring, etc. However, the number of CTCs in peripheral blood is extremely small (generally only 1-10 per milliliter of blood), and the separation and recovery technology has been an important problem in the field of tumor precision medicine.

[0003] At present, the CTC enrichment method based on immunomagnetic beads is widely used due to its convenient operation and strong targeting. The basic principle is to use antibodies that recognize specific tumor markers (such as EpCAM, CK, Vimentin, etc.) to combine with magnetic microspheres, and capture CTCs through an external magnetic field. However, in the traditional scheme, it is difficult to achieve gentle and efficient cell release after the magnetic beads are combined with CTCs, which can easily lead to cell membrane damage and reduced activity, thereby affecting subsequent single-cell sequencing, organoid culture or drug sensitivity analysis.

[0004] Therefore, it is urgent to develop a kit that can achieve efficient immunocapture and guarantee cell activity recovery, and establish a supporting preparation method and application process to meet the technical needs of CTCs in basic research, preclinical modeling and targeted therapy strategy development, etc. SUMMARY

[0005] In view of the above problems, the purpose of the present application is to provide a kit for separating and recovering circulating tumor cells after immunocapture, which comprises the following components:

[0006] a. An immunocapture solution containing an immunocomplex composed of magnetic microspheres and specific monoclonal antibodies, wherein the surface of the magnetic microspheres is coated with an anti-EpCAM and anti-Vimentin double antibody fusion protein obtained by SELEX screening, and the fusion protein is coupled to carboxyl-modified magnetic microspheres by covalent means;

[0007] b. dissociation recovery solution, including dissociation buffer containing disodium ethylenediaminetetraacetic acid, detergent Triton X-100 and cleavable protein cross-linking agent, for dissociating the binding between cells and magnetic particles under low temperature conditions, and neutralization buffer for adjusting the pH value of cell suspension to physiological level;

[0008] c. cell protection solution, including glycerol, human serum albumin (HSA), anti-apoptotic peptide and antioxidant, for maintaining the integrity and activity of cell membrane during separation process;

[0009] d. elution buffer and supplement factor solution, the former for removing unbound impurities and free antibodies, and the latter containing insulin, growth factors and amino acid mixture for enhancing the subsequent activation and culture ability of recovered cells.

[0010] As a preferred technical solution, the antibody fusion protein consists of the anti-EpCAM antibody variable region shown in SEQ ID NO: 1 and the anti-Vimentin antibody variable region shown in SEQ ID NO: 2, and is connected by triple repeats of flexible connection peptide (GGGGSGGGGSGGGGS), as shown in SEQ ID NO: 3, and the fusion protein is covalently coupled with magnetic microspheres through EDC / NHS activation reaction.

[0011] The heavy chain variable region (VH) of SEQ ID NO: 1 is based on the human IGHV3 family skeleton framework, but multiple mutation sites are introduced in the CDR3 region (CARGRYLDYFDY); in particular, the combination of “RGRY” and “LDYFDY” is different from the common anti-EpCAM antibodies (such as HEA125, C215, etc.), which belongs to non-conservative mutation; in the CDR1 region, the “GFTFSSYAMH” type sequence is replaced by the “GFTFSSY” structure.

[0012] These variations help to form a unique spatial conformation, improve the adaptability of the antibody to different conformation sites of EpCAM protein; enhance the recognition breadth of isomers or splice variants, while avoiding sequence homology with commercially available or literature antibodies; improve the affinity and specificity of the antibody, which is conducive to precise recognition of EpCAM + tumor cells in a complex blood environment.

[0013] The light chain variable region (VL) of SEQ ID NO:2 is based on the human IGKV1 framework, but "YNHNLA" is introduced in the CDR1 region instead of "YSDHLA" or "YSSNLA" commonly found in anti-vimentin antibodies; the CDR3 region "QQYNSYPLT" is also structurally compressed to reduce the redundant glycine sequence; this improvement can improve the recognition ability of different epitopes of Vimentin structure, especially adapt to the conformational change in the EMT state; avoid cross recognition of intermediate filament protein family members, and improve specificity.

[0014] SEQ ID NO:3 is a classic (GGGGSGGGGSGGGGS) flexible linker peptide, but it is first applied to fuse anti-EpCAM and anti-Vimentin variable regions into a single functional module in the present application; compared with common short linker peptides such as (GGGS) and (G4S), the sequence provides a long flexible spacer of 15 amino acids, allowing the two domains to fold independently.

[0015] As a preferred technical solution, the particle size of the magnetic microspheres is 200-300 nanometers, the surface carboxyl density is 50-80 μmol / g, and the free reaction group is treated by three steps, and the non-specific binding site is blocked by BSA to reduce the background adsorption.

[0016] As a preferred technical solution, the specific component concentration of the dissociation buffer is: EDTA 5-10 mM, Triton X-100 0.01-0.05%, the cleavable protein crosslinking agent is a dithiothreitol-sensitive crosslinking agent, the concentration is 1-5 mM, the pH value of the buffer is controlled at 6.8-7.4, and the dissociation reaction is carried out at 4°C, and the duration is 10-20 minutes.

[0017] As a preferred technical solution, the glycerol concentration in the cell protection solution is 5-10%, the HSA concentration is 2-5%, the anti-apoptotic peptide is a Bcl-2 mimetic peptide or a Caspase-3 inhibitory peptide, the concentration is 10-50 μg / mL, and the antioxidant is glutathione or ascorbic acid, the concentration is 100-500 μM, which can effectively inhibit cell apoptosis and oxidative stress response.

[0018] The application also provides a preparation method of an immunocapture solution for the kit for separating and recovering circulating tumor cells after immunocapture.

[0019] S1, obtain the antibody variable region sequences of anti-EpCAM and anti-Vimentin, construct a fusion protein expression vector; the antibody variable region sequence of anti-EpCAM is shown as SEQ ID NO: 1, and the antibody variable region sequence of anti-Vimentin is shown as SEQ ID NO: 2;

[0020] S2, transfect the expression vector into E. coli or CHO cells, induce expression, and purify the fusion protein through affinity chromatography and gel filtration;

[0021] S3, covalently couple the purified fusion protein and carboxyl-modified magnetic microspheres in an EDC / NHS activation system;

[0022] S4, three times of magnetic separation and washing are performed on the coupled magnetic microspheres to remove free proteins, and 1% BSA is used for blocking treatment, so as to obtain a double-antibody functionalized immunocapture module.

[0023] The application further provides a circulating tumor cell separation and recovery method, which is based on the kit and comprises the following steps:

[0024] A1, treat the blood sample with a red blood cell lysis buffer, and obtain a mononuclear cell suspension through density gradient centrifugation or filter column treatment;

[0025] A2, add the immunocapture module, incubate at 37 DEG C for 15-30 minutes, and slowly shake and mix to form a complex of circulating tumor cells and double-antibody magnetic beads;

[0026] A3, separate the immunocomplex under an external magnetic field, discard the supernatant, and wash 2-3 times using an elution buffer;

[0027] A4, add a dissociation buffer, incubate at low temperature for 10-20 minutes, centrifuge to remove the magnetic beads, add a neutralization buffer to adjust the pH, and then add a cell protection solution to transfer to a subsequent experimental system.

[0028] As a preferred technical solution, the mass ratio of the magnetic microspheres to the circulating tumor cells is controlled to be 5:1-20:1, a reciprocating shaker is used in the capture process to avoid cell sedimentation, and an automatic magnetic sorting device can be selected for high-throughput processing.

[0029] The application further provides an application of the kit, and the kit is used for separating circulating tumor cells from peripheral blood of a tumor patient and is used for any one of the following researches:

[0030] a. after the circulating tumor cells are recovered, immunofluorescence staining and FACS detection are performed to evaluate the heterogeneity of the circulating tumor cells;

[0031] b. Obtain single CTCs for qPCR, transcriptome sequencing and mutation analysis using single cell sorting technology;

[0032] c. Establish CTC-derived micro-tumor models in organoid culture system for targeted drug screening and drug resistance mechanism research.

[0033] The application also provides another use of the kit for constructing a patient-derived circulating tumor cell line.

[0034] Advantages

[0035] The kit provided by the application realizes the simultaneous recognition and binding of different phenotypic circulating tumor cells such as EpCAM and Vimentin by introducing a diabody fusion protein in the immunocapture module, thereby improving the coverage and capture efficiency of heterogeneous CTCs. The fusion protein uses a flexible linker to connect two variable regions, and is fixed on the surface carboxyl-modified magnetic microspheres through EDC / NHS chemical coupling, so that it can maintain high-affinity binding while reducing non-specific adsorption, thereby ensuring the accuracy and stability of the separation results.

[0036] In terms of cell recovery, the application uses a dissociation buffer containing EDTA, a cleavable protein crosslinking agent and a low-concentration detergent to realize gentle dissociation of the cell-magnetic bead complex under low-temperature conditions, thereby avoiding membrane structure damage caused by mechanical peeling. In combination with a neutralization buffer and a high-osmotic cell protection solution, the cell membrane integrity and metabolic activity can be effectively maintained during the recovery process, thereby providing a high-quality cell source for subsequent single-cell sequencing, drug sensitivity analysis and organoid culture. At the same time, the optimized buffer system reduces the interference of residual chemical reagents on cell function.

[0037] In addition, the matching preparation method and application process of the application are compatible with existing automated magnetic sorting platforms, which are suitable for small-batch scientific experiments and can also be extended to preclinical high-throughput sample processing. The kit has comprehensive advantages in terms of CTC capture efficiency, cell activity maintenance and multi-marker coverage, and can provide stable and repeatable technical support for early tumor screening, efficacy evaluation and transformation research, thereby improving the reliability and applicability of CTC-related detection and functional research. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 It is a preparation method flowchart of the immunocapture solution of the application;

[0039] Figure 2 It is a separation and recovery method flowchart of circulating tumor cells of the application;

[0040] Figure 3 It is a schematic diagram of experimental data (capture efficiency);

[0041] Figure 4 Figure 1 is a schematic diagram of experimental data (residual rate of magnetic beads after separation) ;

[0042] Figure 5 Figure 2 is a schematic diagram of experimental data (recovery rate and recovery cell activity rate) ;

[0043] Figure 6 Figure 3 is a fluorescence photo of a multi-channel fluorescence co-localization experiment. DETAILED DESCRIPTION

[0044] In order to deepen the understanding of the present application, the present application will be further described in detail below in combination with examples, which are only used to explain the present application and do not constitute a limitation on the protection scope of the present application.

[0045] Embodiment:

[0046] This embodiment aims to specifically illustrate the composition structure, preparation method and actual application process of a kit for separating and recovering circulating tumor cells (CTCs) after immunocapture.

[0047] The kit comprises the following core components:

[0048] a. Immunocapture liquid: carboxyl-modified magnetic microspheres (particle size of 250 nm, surface carboxyl density of 65 μmol / g) are used as the basic material, and the surface is covalently coupled with a double-antibody fusion protein of anti-EpCAM and anti-Vimentin. The fusion protein is formed by connecting the variable region of the anti-EpCAM antibody shown in SEQ ID NO: 1 with the variable region of the anti-Vimentin antibody shown in SEQ ID NO: 2, and the connection region is a triple repeat flexible connection peptide (GGGGSGGGGSGGGGS). The fusion protein is covalently combined with the magnetic microspheres through EDC / NHS coupling chemical reaction, and finally forms magnetic immunoparticles with double recognition ability, which has good heterogeneity CTC recognition ability.

[0049] b. Dissociation and recovery liquid: comprising two parts:

[0050] Dissociation buffer: containing 5 mM EDTA, 0.02% Triton X-100 and 3 mM dithiothreitol-sensitive protein crosslinking agent (SPDP), pH adjusted to 7.2, and incubated at 4°C for 10-15 minutes to gently destroy the antibody-antigen binding site, realize the release of CTC from the surface of the magnetic microspheres;

[0051] Neutralization buffer: Tris-HCl buffer (pH=7.4) containing 1% BSA and appropriate amount of HEPES, used to quickly terminate the dissociation reaction and adjust the physiological environment of the cells.

[0052] c. Cell protection solution: the formula is 8% glycerol, 3% human serum albumin (HSA), 30 μg / mL Bcl-2 mimic peptide, and 250 μM glutathione, which can effectively protect the cell membrane and activity of CTCs after release, ensuring subsequent analysis, culture and modeling applications.

[0053] d. Elution buffer: the elution buffer uses PBS base solution plus 0.05% Tween-20 to buffer impurities and non-specific protein adsorption;

[0054] e. Supplement factor solution contains insulin 10 μg / mL, EGF 20 ng / mL and amino acid mixture 1x, which is used to activate the metabolic state of recovered CTCs.

[0055] As shown in Figure 1 , the preparation method of the immune capture solution comprises:

[0056] S1. Obtain the variable region sequences of anti-EpCAM and anti-Vimentin antibodies, and construct a double-antibody fusion protein expression vector;

[0057] S2. Transfect the constructed vector into a CHO cell line, and after IPTG induction expression, purify the expression product by Protein A column chromatography and Superdex200 gel filtration chromatography;

[0058] S3. After activating the carboxyl modified magnetic microspheres with EDC / NHS system, slowly add the fusion protein and stir at 4°C for 4 hours to form stable covalent connection;

[0059] S4. Remove the unbound protein using three times of magnetic separation, and block with 1% BSA. The final concentration is adjusted to 1 mg / mL, and the solution is stored in PBS at 4°C for standby.

[0060] As shown in Figure 2 , the separation and recovery method of circulating tumor cells comprises the following steps:

[0061] A1. Collect peripheral blood samples of tumor patients (7.5 mL per sample), and extract peripheral mononuclear cells (PBMCs) using density gradient centrifugation after treatment with red blood cell lysis buffer;

[0062] A2. Add the prepared immune capture solution (magnetic beads: cell mass ratio 10:1) to the treated PBMCs, incubate at 37°C for 30 minutes in a water bath, and slowly shake during the period to promote the complexation of CTCs and magnetic beads;

[0063] A3. Complete the separation of the complex under the action of an external magnetic field, discard the supernatant, and wash 3 times with elution buffer to remove non-specific binding components;

[0064] A4. Add dissociation buffer, incubate at 4°C for 15 minutes, remove magnetic beads by magnetic separation; the recovered supernatant is the released CTCs, followed by the addition of neutralization buffer and cell protection solution for physiological recovery.

[0065] Performance verification and actual application:

[0066] The verification experiment selected human triple-negative breast cancer cell line MDA-MB-468 as a CTC simulation model. This cell line simultaneously expresses EpCAM (epithelial cell adhesion molecule) and Vimentin (mesenchymal marker), which can be used to verify the recognition effect of the double-antibody fusion magnetic beads on heterogeneous CTCs.

[0067] For easy observation before and after separation, key ingredients were labeled with three colors of fluorescence in the experiment:

[0068] Magnetic microspheres were labeled with FITC (green) on the surface for tracking the capture particles;

[0069] EpCAM was labeled with Alexa Fluor 405 (blue);

[0070] Vimentin was labeled with Alexa Fluor 594 (red);

[0071] The observation object was the enrichment state of the three-color fusion particles before capture and the subsidence after separation.

[0072] The experiment had a total of 4 groups, as shown in Table 1 below:

[0073] Table 1: Experimental grouping

[0074]

[0075] Experimental procedure:

[0076] Blood sample preparation: 1×10 4 MDA-MB-468 cells were added to 7.5 mL of peripheral blood from healthy volunteers, red blood cells were removed using red blood cell lysis buffer, and then PBMCs were separated by density gradient centrifugation;

[0077] Capture incubation: Add the corresponding magnetic capture solution (magnetic beads: cell mass ratio of 10:1) to the PBMCs, incubate at 37°C for 30 minutes in a water bath, and gently shake during the incubation;

[0078] Magnetic separation and washing: Use an external magnetic field to settle the complex, discard the supernatant, and then wash three times with PBS-Tween elution buffer to remove non-specific binding components;

[0079] Dissociation and recovery: add dissociation buffer of the kit to the complex, incubate at 4℃ for 15 minutes, remove the microspheres by magnetic force, and recover the released CTCs;

[0080] Cell protection treatment: add the recovered CTCs to the neutralization buffer and the cell protection solution to restore the cell osmotic pressure and physiological activity;

[0081] Detection and analysis: use a flow cytometer to count the capture and recovery efficiency; and use AO / PI double staining to evaluate the cell activity.

[0082] The experimental results are shown in Table 2.

[0083] Table 2: Experimental result data

[0084]

[0085] Data analysis:

[0086] The double-antibody fusion magnetic beads constructed in the application perform much better than the control groups (C1: EpCAM magnetic bead group; C2: Vimentin magnetic bead group) in the separation and recovery of circulating tumor cells (CTCs), and the specific analysis is as follows:

[0087] 1. Capture efficiency comparison:

[0088] After adding the same number of simulated CTC cells (10,000), the number of captured cells in the T1 group of the application is 9,460±175, and the capture efficiency is as high as 94.6±1.7%, which is significantly better than 70.5±2.1% of the C1 group and 68.2±1.9% of the C2 group, as shown in Table 2. Figure 3 This result shows that the double-antibody fusion strategy significantly improves the recognition breadth and specificity: the fusion protein used in the application is composed of anti-EpCAM and anti-Vimentin variable regions, and the spatial conformation is stabilized by a flexible linker (GGGGSGGGGSGGGGS), so that the magnetic beads can recognize both epithelial CTCs and mesenchymal CTCs, significantly improving the capture ability of heterogeneous cell populations;

[0089] In comparison, the C1 group only recognizes epithelial CTCs expressing EpCAM, and the C2 group only recognizes mesenchymal CTCs expressing Vimentin, both of which have the problem of insufficient recognition of part of the CTC subpopulation, resulting in a significant decrease in the overall capture rate.

[0090] Therefore, the high capture rate of the T1 group is due to its double-target synergistic recognition mechanism, which can fully enrich the heterogeneous CTC population in blood samples.

[0091] 2. Recovery rate comparison:

[0092] After the completion of the immunocapture, the T1 group recovered 8,960 ± 165 cells after treatment with the mild dissociation solution, with a recovery rate of 89.6 ± 1.6%, while the recovery rates of the C1 and C2 groups were only 53.2 ± 1.8% and 49.5 ± 1.6%, respectively, with a significant difference, as shown in Figure 4 .

[0093] The reason for the significant improvement in the recovery rate of the present application is that:

[0094] A dissociation system with reductive sensitivity is used: EDTA, non-ionic surfactant Triton X-100, and a breakable disulfide bond cross-linking agent (SPDP) are introduced into the recovery solution, which can gently destroy the antigen-antibody binding interface at a low temperature of 4°C, and release the cells without damaging the cell membrane structure;

[0095] The control group lacks a specific dissociation system and usually relies on mechanical shaking, PBS flushing, or warm conditions to induce antibody detachment, which has low release efficiency and is prone to cause cell damage or adhesion residues, thereby causing a decrease in the recovery rate.

[0096] Therefore, the high recovery rate of the T1 group is directly due to the biocompatible and targeted dissociation mechanism.

[0097] 3. Cell activity retention rate:

[0098] In terms of the activity of the recovered CTCs, the T1 group reached 93.1 ± 1.3%, which was much higher than that of the C1 (78.4 ± 2.0%) and C2 (75.2 ± 2.3%), as shown in Figure 5 . This result indicates that:

[0099] The key role of the cell protection solution of the present application: 8% glycerol is used to adjust the osmotic pressure, 3% human serum albumin (HSA) is used to maintain a stable external protein environment, and Bcl-2 mimetic peptide and glutathione are introduced to synergistically inhibit the apoptosis process, effectively protecting the cell membrane integrity and mitochondrial function;

[0100] On the contrary, the control group did not use any form of physiological protection solution, and the cells were exposed to high shear force or stress conditions during the release process, resulting in a significant decrease in the activity.

[0101] It can be seen that the good state of the recovered cells is a key condition for ensuring subsequent single-cell sequencing, culture modeling, and functional research, and the present application provides protection for this by constructing a whole-process protection system.

[0102] 4. Magnetic bead residual rate:

[0103] The residual rate of the magnetic beads of the T1 group is only 3.1 ± 0.4 %, which is far lower than that of the C1 (8.9 ± 0.7 %) and the C2 (9.5 ± 0.8 %). This shows that, in the design of the magnetic particles, the characteristics of a higher carboxyl density (65 μmol / g) and a smaller particle size (250 nm) are combined, so that the particles have higher stability and controllability after immunocapture;

[0104] The EDTA and the surfactant in the dissociation system can cooperatively strip the antibodies on the surface of the magnetic beads, and at the same time, the particle aggregation and cell adhesion can be avoided, so that the magnetic bead removal efficiency is improved.

[0105] The synergistic effect of the structure and the cleaning system effectively solves the technical pain points of the difficulty in removing the magnetic beads and the interference with subsequent detection in the traditional magnetic bead separation method.

[0106] According to the comprehensive analysis of the experimental data, the double-antibody fusion magnetic beads combined with the mild dissociation and the cell protection system form an optimized scheme from CTC enrichment, separation, recovery to state maintenance, which is significantly better than the existing single-antibody magnetic bead system in the four key indicators of capture rate, recovery rate, activity retention rate and magnetic bead removal rate, and has wide clinical application potential and research and conversion value.

[0107] Multi-channel fluorescence co-localization experiment:

[0108] In order to verify the binding state between the magnetic microspheres and the circulating tumor cells and the separation effect after release, a three-color fluorescence labeling system is used to dynamically track the co-focal imaging.

[0109] In the experiment, the magnetic microspheres are pre-labeled with FITC fluorescence, which is a green channel; the EpCAM marker is labeled with AlexaFluor 405, which is a blue channel; and the Vimentin marker is labeled with Alexa Fluor 568, which is a red channel.

[0110] The experimental steps are as follows:

[0111] Cell preparation: MDA-MB-468 cells are cultured, and a single-cell suspension is prepared after TrypLE enzymolysis, and the concentration is adjusted to 1 × 10 6

[0112] Simulation environment construction: 7.5 mL of healthy human whole blood samples are taken, and the above MDA-MB-468 cell suspension (doping concentration of about 100 per sample) is added, and after mixing, a CTC background blood is simulated;

[0113] ​Capture and labeling: add three-color labeled immunocapture solution to the sample, incubate in a 37℃ constant temperature water bath for 30 minutes, and mix slowly at the same time to promote the binding of magnetic microspheres and CTC; then separate in a magnetic sorting frame, discard the unbound components, and wash twice with elution buffer;

[0114] Capture state observation: take about 20 μL of the captured complex, drop it onto the center of a pre-cooled glass slide, cover it with a cover glass, fix it with methanol, and dye the nucleus with DAPI, collect green (magnetic beads), blue (EpCAM), and red (Vimentin) channel images under a laser confocal microscope, and perform co-localization analysis;

[0115] Dissociation treatment: add the remaining capture product to the dissociation recovery solution, incubate at 4℃ with gentle shaking for 15 minutes, remove the magnetic beads by magnetic separation, transfer the supernatant, and process again;

[0116] Separation state observation: repeat imaging and analyze the degree of three-channel overlap under the same laser confocal system.

[0117] The results show that, as shown in Figure 6 the fluorescence of the single target channel, the double target channel, the target microsphere double channel, and the three-channel can be clearly displayed before separation, and the recognition degree is high, which indicates that the double target capture can be effectively performed; after the treatment of the kit and the separation method of the application, the single target channel (EpCAM channel) and the double target channel (EpCAM channel + Vimentin channel) are obviously colored, while the target microsphere double channel and the three-channel only show the fluorescence of the microspheres, and almost do not contain target fluorescence, which indicates that the separation effect is good.

[0118] The fluorescence experiment fully verifies that the dissociation recovery solution in the application can realize the effective separation between CTC and magnetic microspheres on the premise of maintaining the integrity of cell surface markers, and provides a stable and high-quality cell sample source for subsequent flow detection, drug sensitivity analysis, and single-cell sequencing.

[0119] The kit is suitable for the separation and recovery of circulating tumor cells in the peripheral blood of breast cancer, lung cancer, prostate cancer, colorectal cancer and other solid tumors, and is suitable for flow cytometry, single-cell sequencing, organoid culture, PDX modeling and other downstream application platforms, and has good universality, sensitivity and cell activity maintenance ability.

[0120] This embodiment fully verifies the applicability and advantages of the kit in circulating tumor cell recognition coverage, separation efficiency, activity maintenance ability, and subsequent functional research, solves the technical problems of insufficient heterogeneity recognition and cell activity destruction in the release process in the traditional CTC capture scheme, and provides a reliable cell source basis for individualized tumor treatment.

[0121] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above-mentioned embodiments, and the above-mentioned embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A kit for the separation and recovery of circulating tumor cells after immune capture, characterized in that, It includes the following components: a. Immune capture fluid: contains an immune complex composed of magnetic microspheres and specific monoclonal antibodies; The magnetic microspheres are coated with a dual antibody fusion protein of anti-EpCAM and anti-Vimentin obtained by SELEX screening; the fusion protein is covalently coupled to the carboxyl-modified magnetic microspheres; the antibody fusion protein consists of the variable region of the anti-EpCAM antibody shown in SEQ ID NO:1 and the variable region of the anti-Vimentin antibody shown in SEQ ID NO:2, and is linked by a flexible linker peptide GGGGSGGGGSGGGGS, as shown in SEQ ID NO:3; the fusion protein and the magnetic microspheres are covalently coupled through an EDC / NHS activation reaction. b. Dissociation recovery solution: comprising dissociation buffer and neutralization buffer, wherein the dissociation buffer contains disodium ethylenediaminetetraacetate, detergent Triton X-100 and cleavable protein crosslinking agent; c. Cell protection solution: including glycerol, human serum albumin (HSA), anti-apoptotic peptides, and antioxidants; d. Elution buffer and supplemental factor solution: The elution buffer is used to remove unbound impurities and free antibodies; the supplemental factor solution contains a mixture of insulin, growth factors and amino acids.

2. The kit for isolating and recovering circulating tumor cells after immune capture according to claim 1, characterized in that, The magnetic microspheres have a particle size of 200–300 nm and a surface carboxyl group density of 50–80 μmol / g. They undergo a three-step process to remove free reactive groups and use BSA to block non-specific binding sites to reduce background adsorption.

3. The kit for isolating and recovering circulating tumor cells after immune capture according to claim 1, characterized in that, The specific component concentrations of the dissociation buffer are as follows: EDTA 5-10 mM; Triton X-100 0.01~0.05%; The cleavable protein cross-linking agent is a dithiothreitol-sensitive cross-linking agent with a concentration of 1–5 mM; The pH of the buffer solution was controlled at 6.8–7.4, and the dissociation reaction was carried out at 4°C for 10–20 minutes.

4. The kit for isolating and recovering circulating tumor cells after immune capture according to claim 1, characterized in that, The cell protection solution contains 5–10% glycerol, 2–5% HSA, and the anti-apoptotic peptide is a Bcl-2 mimic peptide or a Caspase-3 inhibitory peptide at a concentration of 10–50 μg / mL. The antioxidant is glutathione or ascorbic acid at a concentration of 100–500 μM.

5. A method for preparing an immune capture solution, used in the kit for the separation and recovery of circulating tumor cells after immune capture as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Obtain the variable region sequences of the anti-EpCAM and anti-Vimentin antibodies and construct a fusion protein expression vector; the variable region sequence of the anti-EpCAM antibody is shown in SEQ ID NO:1, and the variable region sequence of the anti-Vimentin antibody is shown in SEQ ID NO:

2. S2. Transfect the expression vector into E. coli or CHO cells, induce expression, and purify the fusion protein by affinity chromatography and gel filtration. S3. The purified fusion protein and carboxyl-modified magnetic microspheres were covalently coupled in an EDC / NHS activation system. S4. The coupled magnetic microspheres were subjected to three magnetic separation and washing processes to remove free proteins, and then blocked with 1% BSA to obtain a dual-antibody functionalized immune capture module.

6. A method for isolating and recovering circulating tumor cells, implemented based on the kit described in any one of claims 1-4, characterized in that, Includes the following steps: A1. Blood samples were treated with red blood cell lysis buffer and then subjected to density gradient centrifugation or column filtration to obtain a mononuclear cell suspension. A2. Add the immune capture module, incubate at 37°C for 15–30 minutes, and slowly shake to mix, so that the circulating tumor cells form a complex with the dual antibody magnetic beads. A3. Separate immune complexes under an external magnetic field, discard the supernatant, and wash 2–3 times with elution buffer; A4. Add dissociation buffer, incubate at low temperature for 10–20 minutes, centrifuge to remove magnetic beads, add neutralization buffer to adjust pH, add cell protection solution, and transfer to subsequent experimental systems.

7. The method for isolating and recovering circulating tumor cells according to claim 6, characterized in that, The mass ratio of the magnetic microspheres to circulating tumor cells is controlled at 5:1 to 20:1, and a reciprocating shaker is used during the capture process to avoid cell sedimentation.

8. An application of the reagent kit according to claim 1, characterized in that, This kit is used to construct patient-derived circulating tumor cell lines.

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