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

Abstract

The invention belongs to the technical field of tumor detection and cell separation, and particularly relates to a kit for separating and recycling circulating tumor cells after immunocapture, a preparation method and application. The kit comprises an immunocapture module, a dissociation recovery module, a cell protection module and an elution and supplement factor solution, the immunocapture module adopts magnetic microspheres modified by double-antibody fusion protein and can simultaneously recognize various markers such as EpCAM and Vimentin, and efficient capture of heterogeneity CTCs is achieved; the dissociation and recovery module gently releases cells by using a low-temperature buffer system and a cleavable cross-linking agent; the cell protection module maintains the integrity and activity of cell membranes in the separation process. According to the method, the high-activity CTCs can be obtained while the capture efficiency is guaranteed, and the method is suitable for scientific research and preclinical research such as single cell sequencing, drug sensitivity analysis, organoid culture and patient-derived cell line construction.

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 the primary tumor or metastatic tumor, 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 ensure 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: 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 method; 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; 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; 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.

[0006] 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.

[0007] 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.

[0008] 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 isoforms 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.

[0009] The light chain variable region (VL) of SEQ ID NO:2 is constructed based on a 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 the Vimentin structure, especially to adapt to the conformational change in the EMT state; avoid cross recognition of intermediate filament protein family members and improve specificity.

[0010] 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.

[0011] 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 BSA is used to block the non-specific binding site to reduce the background adsorption.

[0012] 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.

[0013] 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.

[0014] The present application also provides a preparation method of an immunocapture solution for the kit for separating and recovering after immunocapture of circulating tumor cells, comprising the following steps: S1, obtaining the antibody variable region sequences of anti-EpCAM and anti-Vimentin, and constructing 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; S2, transfecting the expression vector into E. coli or CHO cells, inducing expression, and purifying the fusion protein by affinity chromatography and gel filtration; S3, covalently coupling the purified fusion protein with the carboxyl-modified magnetic microspheres in an EDC / NHS activation system; S4, performing three magnetic separation and washing processes on the coupled magnetic microspheres to remove free proteins, and performing blocking treatment using 1% BSA to obtain a double-antibody functionalized immunocapture module.

[0015] The application also provides a circulating tumor cell separation and recovery method based on the kit, comprising the following steps: A1, treating the blood sample with a red blood cell lysis buffer, and performing density gradient centrifugation or filter column treatment to obtain a mononuclear cell suspension; A2, adding the immunocapture module, incubating at 37°C for 15-30 minutes, and slowly shaking to mix, so that the circulating tumor cells form a complex with the double-antibody magnetic beads; A3, separating the immunocomplex under an external magnetic field, discarding the supernatant, and washing 2-3 times using an elution buffer; A4, adding a dissociation buffer, incubating at low temperature for 10-20 minutes, centrifuging to remove the magnetic beads, adding a neutralization buffer to adjust the pH, and adding a cell protection solution to transfer to a subsequent experimental system.

[0016] 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 automated magnetic separation device can be optionally used for high-throughput processing.

[0017] The application also provides an application of the kit, which is used for separating circulating tumor cells from peripheral blood of a tumor patient, and is used for any one of the following studies: a. evaluating the heterogeneity of the recovered circulating tumor cells by immunofluorescence staining and FACS detection; b. obtaining a single CTC by using a single cell sorting technology, and performing qPCR, transcriptome sequencing, and mutation analysis; c. establishing a microtumor model of CTC origin in an organoid culture system, and using the model for targeted drug screening and drug resistance mechanism research.

[0018] The application also provides another application of the kit, which is used for constructing a patient-derived circulating tumor cell line.

[0019] Advantages The kit provided by the application realizes the simultaneous recognition and combination of different phenotypes of circulating tumor cells such as EpCAM and Vimentin by introducing a diabody fusion protein in the immunocapture module, and improves the coverage and capture efficiency of heterogeneous CTCs. The fusion protein uses a flexible connecting peptide 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 combination while reducing non-specific adsorption, and ensure the accuracy and stability of the separation results.

[0020] In terms of cell recovery, the application uses a dissociation buffer containing EDTA, a cleavable protein crosslinking agent and a low concentration of a detergent, which realizes the mild dissociation of the cell-magnetic bead complex under low temperature conditions, avoids the membrane structure damage caused by mechanical peeling. Combined with neutralization buffer and high permeability cell protection liquid, the cell membrane integrity and metabolic activity can be effectively maintained during the recovery process, providing 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.

[0021] In addition, the matching preparation method and application process of the application are compatible with the existing automatic magnetic sorting platform, which is suitable for small batch scientific experiments, and can also be extended to preclinical high-throughput sample processing. The kit has comprehensive advantages in CTC capture efficiency, cell activity maintenance and multi-marker coverage, and can provide stable and repeatable technical support for early screening, efficacy evaluation and transformation research of tumors, so as to improve the reliability and applicability of CTC related detection and functional research. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The figure is a preparation method flowchart of the immunocapture liquid of the application; Figure 2 The figure is a separation and recovery method flowchart of circulating tumor cells of the application; Figure 3 The figure is an experimental data (capture efficiency) schematic diagram; Figure 4 The figure is an experimental data (residual rate of magnetic beads after separation) schematic diagram; Figure 5 The figure is an experimental data (recovery rate and recovery cell activity rate) schematic diagram; Figure 6 The figure is a fluorescence photo of a multi-channel fluorescence co-localization experiment. DETAILED DESCRIPTION

[0023] In order to deepen the understanding of the application, the application will be further described in combination with the embodiments below, and the embodiments are only used to explain the application and do not constitute a limitation on the protection scope of the application.

[0024] Embodiment: The 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 immune capture.

[0025] The kit comprises the following core components: a. Immune capture solution: using carboxyl-modified magnetic microspheres (particle size 250 nm, surface carboxyl density 65 μmol / g) as the basic material, the surface is covalently coupled with anti-EpCAM and anti-Vimentin double antibody fusion protein. The fusion protein is formed by connecting the anti-EpCAM antibody variable region shown in SEQ ID NO: 1 with the anti-Vimentin antibody variable region 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.

[0026] b. Dissociation and recovery solution: comprising two parts: 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; 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.

[0027] c. Cell protection solution: formula is 8% glycerol, 3% human serum albumin (HSA), 30 μg / mL Bcl-2 mimic peptide, 250 μM glutathione, which can effectively protect the CTC cell membrane and activity after release, and ensure the subsequent analysis, culture and modeling application.

[0028] d. Elution buffer: the elution buffer uses PBS base solution plus 0.05% Tween-20, which can buffer impurities and non-specific protein adsorption; e. Supplement factor solution contains insulin 10 μg / mL, EGF 20 ng / mL and amino acid mixture 1×, which is used to activate the metabolic state of the recovered CTCs.

[0029] As shown in Figure 1 The preparation method of the immune capture solution comprises: S1. Obtain anti-EpCAM and anti-Vimentin antibody variable region sequences, construct a double-antibody fusion protein expression vector; S2. Transfect the constructed vector into a CHO cell line, and after IPTG induction expression, purify the expression product using Protein A column chromatography and Superdex200 gel filtration chromatography; S3. After activation of the carboxyl-modified magnetic microspheres with the EDC / NHS system, slowly add the fusion protein and stir at 4°C for 4 hours to form stable covalent bonds; S4. Remove the unbound protein using magnetic separation three times, and block with 1% BSA, adjust the final concentration to 1 mg / mL, and store in PBS solution at 4°C for standby.

[0030] As shown in Figure 2 , the separation and recovery method of circulating tumor cells includes the following steps: A1. Collect peripheral blood samples from tumor patients (7.5 mL per sample), treat with red blood cell lysis buffer, and extract peripheral mononuclear cells (PBMC) using density gradient centrifugation; A2. Add the prepared immunocapture solution (magnetic beads: cell mass ratio 10:1) to the treated PBMC, incubate at 37°C for 30 minutes, and gently shake during the incubation period to promote the complexation of CTCs with magnetic beads; 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; A4. Add dissociation buffer, incubate at 4°C for 15 minutes with gentle mixing, remove the 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.

[0031] Performance verification and actual application: The verification experiment uses human triple-negative breast cancer cell line MDA-MB-468 as a CTC simulation model. This cell line 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.

[0032] For easy observation before and after separation, key components in the experiment are labeled with three colors of fluorescence: The surface of the magnetic microspheres is labeled with FITC (green), which is used to track the capture particles; EpCAM is labeled with Alexa Fluor 405 (blue); Vimentin is labeled with Alexa Fluor 594 (red); The enrichment state of the three-color fusion particles before capture and the subsiding state after separation are observed.

[0033] The experiment includes four groups, and the details are shown in Table 1. Table 1: Experimental grouping

[0034] Experimental procedure: Blood sample preparation: 1x10 4 MDA-MB-468 cells, red blood cells are removed using red blood cell lysis buffer, and then PBMCs are separated by density gradient centrifugation; Capture incubation: Add the corresponding magnetic capture solution (magnetic beads: cell mass ratio is 10:1) to the PBMCs, incubate at 37 DEG C in a water bath for 30 minutes, and gently shake during the incubation; 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; Dissociation and recovery: Add the dissociation buffer of the kit to the complex, incubate at 4 DEG C for 15 minutes, remove the microspheres by magnetic force, and recover the released CTCs; Cell protection treatment: Add the recovered CTCs to the neutralization buffer and cell protection solution to restore the cell osmotic pressure and physiological activity; Detection and analysis: Use a flow cytometer to count the capture and recovery efficiency; and use AO / PI double staining method to evaluate the cell activity.

[0035] The experimental results are shown in Table 2. Table 2: Experimental results

[0036] Data analysis: The performance of the double-antibody fusion magnetic beads constructed in the application in the separation and recovery of circulating tumor cells (CTCs) is much better than that of the control groups (C1: EpCAM magnetic bead group; C2: Vimentin magnetic bead group) using single-antibody magnetic beads. The specific analysis is as follows: 1. Capture efficiency comparison: 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 Figure 3The results show that the dual antibody fusion strategy significantly improves the recognition breadth and specificity. The fusion protein used in the application is composed of two antibody variable regions of anti-EpCAM and anti-Vimentin, and the spatial conformation is stabilized by a flexible connecting peptide (GGGGSGGGGSGGGGS), so that the magnetic beads can recognize both epithelial CTCs and mesenchymal CTCs, and significantly improve the capture ability of heterogeneous cell populations. 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.

[0037] Therefore, the high capture rate of the T1 group is due to its dual target point synergistic recognition mechanism, which can fully enrich the heterogeneous CTC population in the blood sample.

[0038] 2. Recovery rate comparison: After completing the immunocapture, the T1 group recovers 8,960±165 cells after treatment with a mild dissociation recovery solution, with a recovery rate of 89.6±1.6%, while the recovery rates of the C1 and C2 groups are only 53.2±1.8% and 49.5±1.6%, respectively, with a significant difference, as shown in Figure 4 .

[0039] The reason for the significant improvement in the recovery rate of the application is: The use of a dissociation system with reductive sensitivity: EDTA, non-ionic surfactant Triton X-100, and breakable disulfide bond cross-linking agent (SPDP) are introduced into the recovery solution, which can gently destroy the antigen-antibody binding interface at low temperature of 4℃, release the cells without damaging the cell membrane structure; 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 easy to cause cell damage or adhesion residue, resulting in a decrease in recovery rate.

[0040] Therefore, the high recovery rate of the T1 group is directly due to the biocompatible and target-breaking mild dissociation mechanism.

[0041] 3. Cell activity retention rate: In terms of cell activity after recovery, the T1 group reaches 93.1±1.3%, which is much higher than that of C1 (78.4±2.0%) and C2 (75.2±2.3%), as shown in Figure 5 . The results show that: The key role of the cell protection solution of the application: 8% glycerol is used to regulate 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 integrity of the cell membrane and the function of mitochondria. 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 viability.

[0042] It can be seen that the good state of the recovered cells is a key condition to ensure subsequent single-cell sequencing, culture modeling and functional research, and the application provides protection through the construction of a whole-process protection system.

[0043] 4. Magnetic bead residual rate: The magnetic bead residual rate of the T1 group was only 3.1±0.4%, which was much lower than that of C1 (8.9±0.7%) and C2 (9.5±0.8%). This indicates that in the design of the magnetic particles of the application, the combination of a relatively high carboxyl group density (65 μmol / g) and a relatively small particle size (250 nm) characteristics makes the particles have higher stability and controllability after immunocapture; EDTA and surfactant in the dissociation system can synergistically strip the antibodies on the surface of the magnetic beads, while avoiding particle aggregation and cell attachment, thereby improving the efficiency of magnetic bead removal.

[0044] The synergistic effect of this structure and the cleaning system effectively solves the technical pain points of difficulty in removing magnetic beads and interference with subsequent detection in traditional magnetic bead separation methods.

[0045] Based on the analysis of the above experimental data, the double-antibody fusion magnetic beads combined with mild dissociation and cell protection system proposed by the application form a whole-process optimization scheme from CTC enrichment, separation, recovery to state maintenance, which is significantly better than the existing single-antibody magnetic bead system in terms of capture rate, recovery rate, activity maintenance rate and magnetic bead removal rate, and has wide clinical application potential and research and conversion value.

[0046] Multi-channel fluorescence co-localization experiment: 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 was used to dynamically track the co-focal imaging.

[0047] In the experiment, the magnetic microspheres were pre-labeled with FITC fluorescence, showing a green channel; the EpCAM marker was labeled with AlexaFluor 405, showing a blue channel; and the Vimentin marker was labeled with Alexa Fluor 568, showing a red channel.

[0048] The experimental steps are as follows: Cell preparation: MDA-MB-468 cells were cultured and prepared into single-cell suspensions after digestion with TrypLE enzyme. The concentration was adjusted to 1×10⁻⁶ cells after cell counting. 6 cells / mL; Simulation environment construction: Take 7.5 mL of whole blood sample from a healthy person, add the above MDA-MB-468 cell suspension (doping concentration of about 100 cells / sample), mix well, and then simulate the blood background of CTCs; Capture and labeling: Add tricolor labeled immunocapture solution to the sample and incubate in a 37°C constant temperature water bath for 30 minutes while slowly rotating to mix and promote the binding of magnetic microspheres to CTCs; then place in a magnetic sorting rack for separation, discard unbound components, and wash twice with elution buffer; Observation of the captured state: Take about 20 µL of the captured complex and drop it into the center of a pre-cooled glass slide. After covering with a coverslip, fix it with methanol and stain the nucleus with DAPI. Acquire three-channel images (green (magnetic beads), blue (EpCAM), and red (Vimentin)) under a laser confocal microscope and perform colocalization analysis. Dissociation process: The remaining captured products were added to the dissociation recovery solution, gently shaken and incubated at 4°C for 15 minutes, and then magnetically separated to remove the magnetic beads. The supernatant was then transferred and prepared into slides again. Separation state observation: Repeated imaging and analysis of the degree of overlap of the three channels under the same laser confocal system.

[0049] The results show that: Figure 6 As shown, before separation, the fluorescence of the single-target channel, dual-target channel, dual-target microsphere channel, and triple-target channel can all be clearly displayed with high recognition, indicating that dual-target capture can be effectively performed. After processing with the kit and separation method of this invention, the single-target channel (EpCAM channel) and dual-target channel (EpCAM channel + Vimentin channel) are clearly colored, while the dual-target microsphere channel and triple-target channel only show the fluorescence of the microspheres and contain almost no target fluorescence, indicating that the separation effect is good.

[0050] This fluorescence experiment fully verifies that the dissociation recovery solution in this invention can effectively separate CTCs from magnetic microspheres while maintaining the integrity of cell surface marker expression, providing a stable and high-quality source of cell samples for subsequent flow cytometry detection, drug sensitivity analysis, and single-cell sequencing.

[0051] This kit is suitable for the isolation and recovery of circulating tumor cells from peripheral blood of various solid tumors such as breast cancer, lung cancer, prostate cancer, and colorectal cancer. It is also compatible with various downstream application platforms such as flow cytometry, single-cell sequencing, organoid culture, and PDX modeling, and has good versatility, sensitivity, and cell viability preservation capabilities.

[0052] The present embodiment fully verifies the applicability and advantages of the kit in the identification coverage of circulating tumor cells, separation efficiency, activity maintenance ability and subsequent functional study, solves the technical problems of insufficient heterogeneous identification 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.

[0053] 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 examples, and the above examples 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 surface of the magnetic microspheres is coated with a fusion protein of anti-EpCAM and anti-Vimentin obtained by SELEX screening; the fusion protein is covalently coupled to the carboxyl-modified magnetic microspheres. 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 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 is covalently coupled to the magnetic microspheres through an EDC / NHS activation reaction.

3. 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.

4. 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.

5. 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.

6. 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 1-5, 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.

7. A method for isolating and recovering circulating tumor cells, implemented based on the kit described in any one of claims 1-5, 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.

8. The method for isolating and recovering circulating tumor cells according to claim 7, 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.

9. 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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