Multi-target circulating tumor cell quality control kit, preparation method and application thereof

Abstract

The application belongs to the technical field of fluorescence detection, and particularly relates to a multi-target circulating tumor cell quality control kit and a preparation method and application thereof. The kit comprises multi-marker quality control microspheres, a fixed storage system and a split packaging unit. The microspheres are 2-6 μm polymer base particles, and the surfaces of the microspheres are covalently coupled with recombinant antigen proteins such as EpCAM, CK8, Vimentin and CD45. Different microsphere subpopulations present various antigen combinations, and are used for simulating the marker characteristics of circulating tumor cells and immune cells. Through cross-linking fixation and freeze-drying protection treatment, the obtained quality control microspheres have good antigen stability and channel recognition performance, and are suitable for multi-platform quality control such as a flow cytometer and a multi-channel immunofluorescence system. The kit can replace traditional quality control cells, and realizes standardized, low-risk and long-term storage multi-target detection quality control.

Description

Technical Field

[0001] This invention belongs to the technical field of fluorescence detection, specifically relating to a multi-target circulating tumor cell quality control kit, its preparation method, and its application. Background Technology

[0002] Circulating tumor cells (CTCs) are tumor cells that detach from primary or metastatic lesions and enter the peripheral blood circulation, reflecting the dynamic process of tumor development and metastasis. In recent years, CTC-based liquid biopsy technology has played an important role in early tumor screening, efficacy evaluation, and recurrence monitoring. CTC detection methods include immunomagnetic bead enrichment, microfluidic chip capture, immunofluorescence staining, and molecular detection. However, due to the extremely low concentration and significant heterogeneity of CTCs in peripheral blood, the detection process requires extremely high standards for sample processing, staining sensitivity, and specificity. Therefore, high-quality quality control materials are urgently needed to validate and evaluate the accuracy and stability of the detection system.

[0003] Currently, most commercially available CTC quality control products are based on single-marker positive cells or single-target staining protocols, which are insufficient to simulate the multiphenotypic characteristics of CTCs in clinical practice. For example, some epithelial CTCs express EpCAM and CK, while some mesenchymal or transitional CTCs show co-expression or attenuated characteristics of epithelial and mesenchymal markers. If the marker coverage of the quality control materials is insufficient, the ability of the multi-target detection platform to identify different cell subtypes cannot be fully verified, potentially leading to false negatives or false positives in the test results.

[0004] Therefore, developing a multi-target CTC quality control kit that can stably express multiple CTC-related biomarkers (such as EpCAM, CK, Vimentin, CD45, etc.) and has long-term preservation capabilities is of great significance for improving the reliability and standardization of detection methods. Summary of the Invention

[0005] To address the above problems, the present invention aims to provide a multi-target circulating tumor antigen quality control kit, comprising:

[0006] a. Multi-marker control microspheres, wherein the quality control microspheres are polymer-based microspheres that have undergone fixation and preservation treatment, and the quality control microsphere population contains microspheres capable of covalently coupling recombinant antigen proteins such as EpCAM, cytokeratin CK8, vimentin, and leukocyte common antigen CD45, wherein different microsphere subpopulations exhibit different combinations of the above-mentioned markers;

[0007] The amino acid sequence of EpCAM is shown in SEQ ID NO:1;

[0008] The amino acid sequence of CK8 is shown in SEQ ID NO:2;

[0009] The amino acid sequence of Vimentin is shown in SEQ ID NO:3;

[0010] The amino acid sequence of CD45 is shown in SEQ ID NO:4;

[0011] b. A fixation and preservation system, wherein the system is a phosphate buffer solution with pH 7.3-7.5 containing 3.8-4.2% (w / v) paraformaldehyde, 0.05-0.08% (v / v) glutaraldehyde, 0.4-0.6% (w / v) trehalose, 4-6% (v / v) glycerol and 0.05-0.1% (v / v) Tween-20;

[0012] c. Packaging unit: The quality control microspheres are vacuum freeze-dried and then packaged into freeze-dried tubes filled with inert gas and sealed, with each tube containing the amount of microspheres required for a single test.

[0013] As a preferred technical solution, the quality control microspheres include:

[0014] a. Multiantigen microspheres coated with any two or three of the recombinant antigen proteins EpCAM, CK8, and Vimentin;

[0015] b. Single-marker microspheres coated with CD45 recombinant antigen protein;

[0016] The microspheres are mixed in proportion to form a mixed quality control population with multi-antigen recognition characteristics. Different microsphere subpopulations exhibit combination patterns of high EpCAM expression / low Vimentin expression, low EpCAM expression / high Vimentin expression, and co-expression of EpCAM and Vimentin.

[0017] As a preferred technical solution, the microspheres are carboxylated polystyrene microspheres with a particle size of 2-6 μm, and each recombinant antigen protein is covalently coupled to the surface of the microspheres via NHS-EDC chemical cross-linking.

[0018] As a preferred technical solution, the pre-lyophilized quality control microsphere suspension contains 0.2-0.5% (w / v) soluble fluorescently labeled internal reference microspheres with a particle size of 2-4 μm, which are used for channel calibration of the detection platform.

[0019] As a preferred technical solution, after the reconstituted solution of the quality control microspheres is lyophilized, the immunofluorescence signal retention rate of EpCAM and Vimentin antigens is not less than 90% after 12 months of storage at 4°C.

[0020] This invention also provides a method for preparing a multi-target circulating tumor antigen quality control kit, comprising the following steps:

[0021] S1. Antigen protein expression and purification: Recombinant proteins of EpCAM, CK8, Vimentin and CD45 were constructed and expressed, purified by affinity chromatography and their concentrations were adjusted to 0.5-2 mg / mL for each antigen.

[0022] S2. Microsphere activation: Carboxylated polystyrene microspheres were dispersed in MES buffer, and the carboxyl groups on the surface of the microspheres were activated using NHS and EDC.

[0023] S3, Antigen Coupling: Different antigen proteins are added to the microsphere reaction system under activated conditions to prepare microspheres with different antigen combinations in proportion. After reacting at 4℃ for 12-18 h, unreacted sites are blocked.

[0024] S4. Buffering and fixation: The coupled microspheres were stably crosslinked using a PBS system containing 3.8-4.2% paraformaldehyde and 0.05-0.08% glutaraldehyde for 30-35 min.

[0025] S5. Washing and Pre-freezing: The microspheres were washed with PBS, lyophilization protectant was added, and after equilibration, they were pre-frozen.

[0026] S6. Vacuum freeze-drying: Freeze-drying under a vacuum degree not exceeding 50 Pa;

[0027] S7. Uniform dispensing and sealing: The microsphere suspension is kept uniform by magnetic stirring. The number of microspheres in each tube is calibrated by volume control and optical density method. The microspheres are dispensed under nitrogen protection and sealed for storage.

[0028] As a preferred technical solution, after the fixation treatment in step S4, the sample is washed three times with PBS buffer containing 0.1 M glycine and 0.05% Tween-20, each time for 5 min, to block free aldehyde groups and reduce non-specific binding in subsequent staining.

[0029] As a preferred technical solution, step S5 specifically involves: washing the microspheres three times with PBS, adding a lyophilization protectant, equilibrating at 4°C for 30 min, then lowering the temperature to -20°C at a rate of 1-2°C / min and maintaining it for 1 h, and then lowering it to -40°C and maintaining it for 2 h.

[0030] As a preferred technical solution, in step S5, the order of adding the freeze-drying protectant is as follows: add 0.5-1% trehalose and stir evenly, then slowly add 3-6% glycerol, and finally add 0.2-0.4% hydroxyethyl starch and continue stirring for 10 min;

[0031] As a preferred technical solution, in step S6, the freeze drying adopts a two-stage sublimation dehydration process. The temperature of the first stage is controlled at -25℃±2℃, the vacuum degree is 40-50 Pa, and it lasts for 12-15 h. In the second stage, the temperature is slowly raised to -5℃±1℃, the vacuum degree is 20–30 Pa, and it lasts for 8-10 h.

[0032] This invention also provides an application of the aforementioned multi-target circulating tumor antigen quality control kit in a circulating tumor cell immunofluorescence detection system. The lyophilized quality control microspheres are reconstituted according to the instructions and dropped onto the detection slide or chip. Simultaneously, multi-target immunofluorescence staining of EpCAM, cytokeratin CK8, Vimentin, and CD45 is performed, and multi-channel fluorescence images are acquired to verify the multi-target recognition performance of the detection system.

[0033] This invention also provides an application of the aforementioned multi-target circulating tumor antigen quality control kit in a flow cytometry detection platform. After reconstitution of lyophilized quality control microspheres, immunofluorescence labeling is performed, and the signal distribution of the corresponding biomarkers is detected in FITC, PE, APC, and V450 channels, respectively, to evaluate the instrument channel resolution and the stability of biomarker detection.

[0034] Beneficial effects:

[0035] This invention achieves precise simulation of various common circulating tumor cell and immune cell markers, such as EpCAM, CK8, Vimentin, and CD45, by constructing recombinant protein microspheres with different antigen combinations. Compared to existing quality control materials using fixed cells as carriers, the polymer-based microspheres used in this invention have advantages such as uniform particle size, controllable surface, and high coupling stability. They can stably present different antigen expression patterns without introducing biosafety risks, meeting the target recognition needs of multiple platforms.

[0036] The microspheres described in this invention achieve multi-antigen modification through NHS-EDC covalent cross-linking technology and are further stabilized in a paraformaldehyde / glutaraldehyde cross-linking system. Combined with an optimized lyophilization protection system, the microspheres can be stored for a long time while maintaining antigen structure and fluorescence recognition performance. Experiments show that after 12 months of storage at 4°C, the average fluorescence intensity retention rate of EpCAM and Vimentin is still above 90%, demonstrating excellent stability and batch consistency, effectively ensuring the quality control requirements of immunofluorescence and flow cytometry detection platforms during long-term operation.

[0037] This invention can simulate various combinations of circulating tumor cell phenotypes, and is particularly suitable for verifying the recognition sensitivity of multi-channel detection systems, evaluating antibody screening, and calibrating instrument channel performance. The kit is highly adaptable and scalable, making it particularly suitable for widespread use as a standard quality control material in various in vitro diagnostic applications such as liquid biopsy, CTC detection, automated immunoassay, and microfluidic diagnostics. Attached Figure Description

[0038] Figure 1 This is a schematic flowchart of the preparation method of the reagent kit of the present invention;

[0039] Figure 2 This is a schematic diagram illustrating the retention rate of lyophilized fluorescence intensity in this invention.

[0040] Figure 3 This is a fluorescence image of the positive group four antigen combination microspheres of the present invention;

[0041] Figure 4 This is a fluorescence photograph of the test group microspheres of the present invention. Detailed Implementation

[0042] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0043] Example: Preparation and application validation of multi-target circulating tumor antigen quality control microspheres

[0044] This embodiment provides a method for preparing multi-target circulating tumor antigen quality control microspheres that can be used in flow cytometry and multi-channel immunofluorescence platforms, such as... Figure 1 As shown, the microspheres possess multiple tumor and immune markers such as EpCAM, CK8, Vimentin, and CD45, which can simulate the multi-antigen recognition characteristics of circulating tumor cells on the detection platform. The prepared microspheres exhibit high antigen stability, good lyophilization-reconstitution properties, and platform compatibility, and can be used as quality control standard materials for multi-channel detection systems.

[0045] 1. Raw material preparation:

[0046] 1.1 Microsphere material:

[0047] Carboxylated polystyrene microspheres (Thermo Fisher Scientific) with an average particle size of 3.0 μm and carboxyl groups on their surface were selected and stored in PBS at a suspension concentration of 2.5% at pH 7.4. The microspheres exhibited uniform morphology and stable optical properties, making them suitable for flow cytometry and immunofluorescence platforms.

[0048] 1.2 Antigen protein:

[0049] Procurement of human recombinant antigen proteins separately, including:

[0050] EpCAM (derived from the HEK293 expression system, purity >95%, MW approximately 38 kDa);

[0051] CK8 complex protein (recombinant fusion form);

[0052] Vimentin (full length, with 6×His tags);

[0053] CD45 (extracellular region fused with Fc segment, enhancing coupling efficiency).

[0054] All proteins were prepared using a low endotoxin expression system with a purity of no less than 95%. The buffer system consisted of 20 mM Tris, 150 mM NaCl, and pH 7.4.

[0055] 2. Microsphere coupling process:

[0056] 2.1 Microsphere pre-activation:

[0057] Place 10 mL of carboxylated polystyrene microspheres in a centrifuge tube, wash twice with PBS, then resuspend in 0.1 MMES buffer (pH 6.0) to adjust the microsphere concentration to 1%.

[0058] Add 5 mM NHS (N-hydroxysuccinimide) and 10 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) to a final concentration, and react at room temperature in the dark for 30 minutes to activate the carboxyl groups on the surface of the microspheres to form an amide intermediate.

[0059] 2.2 Antigen conjugation:

[0060] The four antigen proteins were pre-prepared to a concentration of 1 mg / mL and then added to microspheres according to the following groups:

[0061] Group A: EpCAM+CK8;

[0062] Group B: Vimentin + EpCAM;

[0063] Group C: EpCAM+CK8+Vimentin;

[0064] Population D: CD45 monoantigen conjugation.

[0065] Add 100 μL of protein solution to each group, ensuring the total protein content does not exceed the loading capacity of 1 mg per milliliter of microspheres. Incubate the reaction system at 4°C with gentle shaking for 12 hours to complete the coupling.

[0066] 2.3 Closure after coupling:

[0067] After protein conjugation, 0.1 M glycine was added to each reaction tube, and the tubes were incubated for 20 minutes to block unreacted active amide sites and prevent subsequent nonspecific binding. The tubes were then washed three times with PBS buffer and centrifuged at 5000 rpm for 5 minutes.

[0068] After washing, the microspheres were resuspended in PBS containing 0.1% BSA and stored at 4°C for later use.

[0069] 3. Microsphere stabilization and freezing intervention:

[0070] 3.1 Fixation process:

[0071] To improve the surface stability of the antigen, a paraformaldehyde / glutaraldehyde crosslinking system was used for surface fixation.

[0072] The coupled microsphere suspension was added to an equal volume of fixative (containing 3.8% paraformaldehyde, 0.07% glutaraldehyde, pH 7.4 PBS), and the mixture was slowly shaken at room temperature for 30 minutes.

[0073] Immediately after the reaction, 0.1 M glycine was added to neutralize the unreacted aldehyde groups, and the mixture was washed three times in 0.05% Tween-20 PBS for 5 minutes each time.

[0074] 3.2 Preparation of lyophilization protection solution:

[0075] The lyophilization protection solution is formulated as follows: Trehalose: 0.8% (w / v); Glycerin: 5% (v / v); Hydroxyethyl starch (HES): 0.3% (w / v); Carrier: PBS, pH 7.4.

[0076] Add trehalose, glycerol and HES sequentially at 4°C, and keep stirring magnetically for 10 minutes to ensure even dissolution.

[0077] 3.3 Pre-freezing procedure:

[0078] The washed microspheres were resuspended in the lyophilization protection solution and the concentration was adjusted to 1×10⁻⁶. 6 Particles / mL. The pre-freezing procedure is as follows:

[0079] Place it on a pre-cooled metal plate and cool it down to -20°C at a rate of 1°C / min, then hold for 60 minutes.

[0080] Continue cooling to -40℃ and maintain for 2 hours to ensure sufficient nucleation and crystallization.

[0081] 4. Vacuum freeze drying and packaging:

[0082] 4.1 Freeze-drying process:

[0083] The pre-frozen samples were placed in a freeze dryer and the following dehydration program was run:

[0084] First stage of sublimation and dehydration: -25℃, vacuum degree 45 Pa, lasting 13 hours;

[0085] The second stage involves a gentle temperature increase: the temperature is raised to -5°C, the vacuum level is 25 Pa, and this process is continued for 9 hours.

[0086] During this process, the heating rate is set not to exceed 5℃ / h to avoid the disintegration of the microsphere structure.

[0087] 4.2 Packaging and Sealing:

[0088] Uniform microsphere powder was obtained after freeze-drying. The unit microsphere quantity was calibrated using the optical density method, and the powder was dispensed into freeze-drying tubes with magnetic stirring. Each tube contained 200 μg of freeze-dried powder, equivalent to approximately 1 × 10⁻⁶ microspheres. 5 Individual antigen quality control microspheres.

[0089] The tube is sealed under nitrogen protection, with aluminum foil used for sealing, and then stored at 2–8°C.

[0090] 5. Quality control and performance verification

[0091] 5.1 Antigen Expression Validation (Fluorescent Labeling)

[0092] After reconstitution of each group of microspheres, immunofluorescence labeling was performed with the following antibodies:

[0093] FITC-anti-EpCAM; PE-anti-CK8; APC-anti-Vimentin; V450-anti-CD45.

[0094] The fluorescence intensity distribution and positive rate were detected by flow cytometry (BD FACSCanto II), and the results are shown in Table 1:

[0095] Table 1. Fluorescence intensity distribution and positive rate

[0096]

[0097] The above results indicate that the microsphere antigen expression in each group is accurate, the coupling efficiency is good, and there is no cross-contamination.

[0098] 5.2 Freeze-drying stability test:

[0099] The lyophilized tubes were stored at 4°C, and samples were taken and reconstituted at 0, 3, 6, and 12 months. The fluorescence intensity retention rate of EpCAM and Vimentin (expressed as MFI) was detected using a standard immunofluorescence assay, as shown in Table 2. A schematic diagram is also shown below. Figure 2 As shown:

[0100] Table 2. Retention rate of lyophilized fluorescence intensity

[0101]

[0102] The results show that the quality control microspheres maintained over 90% of the antigen fluorescence intensity after 12 months of storage under normal refrigeration conditions. This indicates that the lyophilization protection system used in this invention effectively stabilizes protein conformation and reduces the risk of antigen denaturation during lyophilization and reconstitution. The antigen coupling method on the microsphere surface exhibits good covalent bond stability, preventing antigen desorption or degradation during storage. Throughout the storage period, the signal decline trends of both EpCAM and Vimentin antigens were stable without significant fluctuations, demonstrating high quality control stability and good reproducibility. The results meet the basic requirements of in vitro diagnostic standards for quality control products with a shelf life of ≥12 months and an antigen retention rate of ≥90%, proving that this kit can be used for long-term quality control.

[0103] 5.3 Platform Compatibility Verification:

[0104] The reconstituted microsphere samples were tested on the following platform:

[0105] Flow cytometry channel (FITC / PE / APC / V450);

[0106] Multichannel immunofluorescence imaging platform;

[0107] Microfluidic chip quantitative detection system.

[0108] All four channels can be detected stably, with a CV value of less than 3% and a signal-to-noise ratio of more than 10:1, which meets the platform's quality control requirements.

[0109] The multi-target antigen quality control microspheres prepared in this embodiment have the following technical advantages:

[0110] It enables multiple combination conjugation of four tumor / immune-related antigens, adapting to multi-channel detection systems;

[0111] Microspheres simulate the behavior of CTC particles, which is beneficial for the calibration of detection systems;

[0112] Employing a precisely controlled freeze-drying protection system and sublimation process, it effectively protects antigen conformation and recognition ability;

[0113] It achieved long-term storage stability (12 months) under normal temperature cold chain transportation and storage conditions;

[0114] It can be widely used in signal comparison, channel verification and batch quality control of platforms such as flow cytometry, microfluidics, biochips and immunoimaging.

[0115] To verify the antigen recognition specificity and detection system compatibility of the multi-target antigen control microspheres described in this invention, a typical control group was designed for system comparison experiments, including a positive control group and a test group. Immunofluorescence staining images of four target antigens—EpCAM, CK8, Vimentin, and CD45—were combined to compare and analyze the antigen recognition effect on the microsphere surface, fluorescence channel matching degree, and non-specific staining level, confirming the discriminative power of this kit. The groups are shown in Table 3, and the fluorescence images are shown in Table 4. Figure 3 , Figure 4 As shown.

[0116] Table 3 Grouping of Discrimination Experiment

[0117]

[0118] Microsphere preparation process and antigen conjugation:

[0119] The microsphere preparation process followed the aforementioned scheme, using 3 μm carboxylated polystyrene microspheres as a carrier, and covalently linking the antigen protein to the surface via EDC / NHS chemical coupling. Key parameters were controlled as follows:

[0120] Dosage for each antigen: 10–30 μg / mg microspheres;

[0121] Reaction system: MES buffer, pH 6.0;

[0122] Coupling time: Slowly rotate at 4℃ for 12–16 hours;

[0123] Immobilized crosslinking: 3.8% paraformaldehyde + 0.07% glutaraldehyde, treated at room temperature for 30 minutes;

[0124] Lyophilization protectant: 0.5% trehalose + 5% glycerol + 0.3% HES;

[0125] Freeze-drying program: -25℃, 45 Pa, 13 hours for initial sublimation; -5℃, 25 Pa, 9 hours for final dehydration;

[0126] After lyophilization, the samples were aliquoted into lyophilized tubes, and each tube was rehydrated and used for one test.

[0127] Commercially available fluorescently labeled antibodies were selected, corresponding to the following targets and channels, as shown in Table 4:

[0128] Table 4 Fluorescent dyes and channels

[0129]

[0130] The reconstituted microspheres were incubated in a staining system containing an antibody mixture at 4°C in the dark for 30 minutes. After washing three times with PBS, the microspheres were added to a glass slide and sealed. Multi-channel imaging was performed using a Nikon Eclipse Ti2 multi-channel fluorescence microscope.

[0131] In the positive control group images, almost all microspheres showed clear and well-defined fluorescence signals, corresponding to: green fluorescence in the FITC (EpCAM) channel; yellow fluorescence in the PE (CK8) channel; red fluorescence in the APC (Vimentin) channel; and sky blue fluorescence in the V450 (CD45) channel. The four-channel signals achieved high co-localization on a single microsphere, and there was no obvious channel leakage, offset, or cross-contamination, indicating that the microsphere-conjugated antigen was stable and the detection system had good recognition consistency.

[0132] In the test group, the mixed population composed of triantigen-coated microspheres and monoantigen microspheres exhibited two types of fluorescence characteristics: the multiantigen microspheres showed sky-blue fluorescence in the V450 (CD45) channel; the Vimentin-coated microspheres showed red fluorescence in the APC channel; and the EpCAM-coated microspheres showed green fluorescence in the FITC channel. Furthermore, the triantigen-coated microspheres and monoantigen microspheres did not emit light when excited in each other's channels, demonstrating good anti-interference properties, and the different fluorescence images after repeated exposures showed good distinguishability.

[0133] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A multi-target circulating tumor antigen quality control kit, characterized in that, include: a. Multi-marker control microspheres, wherein the quality control microspheres are polymer-based microspheres that have undergone fixation and preservation treatment, and the quality control microsphere population contains microspheres capable of covalently coupling recombinant antigen proteins such as EpCAM, cytokeratin CK8, vimentin, and leukocyte common antigen CD45, wherein different microsphere subpopulations exhibit different combinations of the above-mentioned markers; The amino acid sequence of EpCAM is shown in SEQ ID NO:1; The amino acid sequence of CK8 is shown in SEQ ID NO:2; The amino acid sequence of Vimentin is shown in SEQ ID NO:3; The amino acid sequence of CD45 is shown in SEQ ID NO:4; The quality control microspheres include: a. Multiantigen microspheres coated with any two or three of the recombinant antigen proteins EpCAM, CK8, and Vimentin; b. Single-marker microspheres coated with CD45 recombinant antigen protein; The microspheres are mixed in proportion to form a mixed quality control population with multi-antigen recognition characteristics. Different microsphere subpopulations exhibit combination patterns of high EpCAM expression / low Vimentin expression, low EpCAM expression / high Vimentin expression, and co-expression of EpCAM and Vimentin, respectively. b. A fixation and preservation system, wherein the system is a phosphate buffer solution with pH 7.3-7.5 containing 3.8-4.2% (w / v) paraformaldehyde, 0.05-0.08% (v / v) glutaraldehyde, 0.4-0.6% (w / v) trehalose, 4-6% (v / v) glycerol and 0.05-0.1% (v / v) Tween-20; c. Packaging unit: The quality control microspheres are vacuum freeze-dried and then packaged into freeze-dried tubes filled with inert gas and sealed, with each tube containing the amount of microspheres required for a single test.

2. The multi-target circulating tumor antigen quality control kit according to claim 1, characterized in that, The microspheres are carboxylated polystyrene microspheres with a particle size of 2-6 μm, and each recombinant antigen protein is covalently coupled to the surface of the microspheres via NHS-EDC chemical cross-linking.

3. The multi-target circulating tumor antigen quality control kit according to claim 2, characterized in that, The pre-lyophilized quality control microsphere suspension contains 0.2-0.5% (w / v) soluble fluorescently labeled internal reference microspheres with a particle size of 2-4 μm, which are used for channel calibration of the detection platform.

4. The multi-target circulating tumor antigen quality control kit according to claim 3, characterized in that, After the reconstituted solution of the quality control microspheres was lyophilized, it was stored at 4°C for 12 months, and the immunofluorescence signal retention rate of EpCAM and Vimentin antigens was not less than 90%.

5. A method for preparing a multi-target circulating tumor antigen quality control kit, used to prepare the multi-target circulating tumor antigen quality control kit as described in claim 1, characterized in that, Includes the following steps: S1. Antigen protein expression and purification: Recombinant proteins of EpCAM, CK8, Vimentin and CD45 were constructed and expressed, purified by affinity chromatography and their concentrations were adjusted to 0.5-2 mg / mL for each antigen. S2. Microsphere activation: Carboxylated polystyrene microspheres were dispersed in MES buffer, and the carboxyl groups on the surface of the microspheres were activated using NHS and EDC. S3, Antigen Coupling: Different antigen proteins are added to the microsphere reaction system under activated conditions to prepare microspheres with different antigen combinations in proportion. After reacting at 4℃ for 12-18 h, unreacted sites are blocked. S4. Buffering and fixation: The coupled microspheres were stably crosslinked using a PBS system containing 3.8-4.2% paraformaldehyde and 0.05-0.08% glutaraldehyde for 30-35 min. S5. Washing and Pre-freezing: The microspheres were washed with PBS, lyophilization protectant was added, and after equilibration, they were pre-frozen. S6. Vacuum freeze-drying: Freeze-drying under a vacuum degree not exceeding 50 Pa; S7. Uniform dispensing and sealing: The microsphere suspension is kept uniform by magnetic stirring. The number of microspheres in each tube is calibrated by volume control and optical density method. The microspheres are dispensed under nitrogen protection and sealed for storage.

6. The preparation method according to claim 5, characterized in that, After fixation in step S4, the sample was washed three times with PBS buffer containing 0.1 M glycine and 0.05% Tween-20 for 5 min each time to block free aldehyde groups and reduce non-specific binding in subsequent staining.

7. The preparation method according to claim 6, characterized in that, Step S5 is as follows: after washing the microspheres three times with PBS, add the lyophilization protectant, equilibrate at 4℃ for 30 min, then lower the temperature to -20℃ at a rate of 1-2℃ / min and maintain for 1 h, then lower the temperature to -40℃ and maintain for 2 h.

8. The preparation method according to claim 7, characterized in that, In step S5, the order of adding the freeze-drying protectant is as follows: add 0.5-1% trehalose and stir evenly, then slowly add 3-6% glycerol, and finally add 0.2-0.4% hydroxyethyl starch and continue stirring for 10 min.

9. The preparation method according to claim 8, characterized in that, In step S6, the freeze-drying process adopts a two-stage sublimation dehydration process. The temperature of the first stage is controlled at -25℃±2℃, the vacuum degree is 40-50 Pa, and it lasts for 12-15 h. In the second stage, the temperature is slowly raised to -5℃±1℃, the vacuum degree is 20–30 Pa, and it lasts for 8-10 h.

10. The application of a multi-target circulating tumor antigen quality control kit as described in any one of claims 1-4 on a flow cytometry detection platform, characterized in that, After rehydration of the lyophilized quality control microspheres, they were labeled with immunofluorescence, and the signal distribution of the corresponding biomarkers was detected in the FITC, PE, APC, and V450 channels to evaluate the instrument channel resolution and the stability of biomarker detection.

Images