Detection kit for capturing CTC by streptavidin coupled antibody

Abstract

The invention belongs to the technical field of biochemical detection, and particularly provides a streptavidin coupled antibody capture CTC detection kit and a preparation method thereof.The kit comprises a biotin-modified EpCAM monoclonal antibody, a biotin-modified Vimentin monoclonal antibody, a pretreatment agent, a separation solution, a stationary solution, a permeation agent, a sealing agent, a fluorescent staining solution, a micro-fluidic chip and other components. Streptavidin is solidified at the bottom layer of the chip channel, and the upper layer is of an asymmetric herring bone structure, so that the binding efficiency of cells and antibodies can be enhanced. The kit realizes efficient capture of different phenotypes of CTCs through multi-antibody composite recognition, performs multi-marker detection in combination with immunofluorescent staining, and has both sensitivity and specificity. The method is standard in technological process, stable in component and suitable for enrichment and analysis of CTC in a clinical sample.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemical tests, and specifically provides a streptavidin-conjugated antibody capture CTC detection kit and a preparation method thereof. Background Art

[0002] Circulating tumor cells (CTC) are tumor cells that shed from primary or metastatic tumors and enter the peripheral blood circulation system. The presence of CTC is considered an important prerequisite for tumor metastasis and has been widely used in the fields of early tumor diagnosis, prognosis evaluation, and treatment response monitoring.

[0003] Currently, most commonly used CTC detection techniques are based on the enrichment of single epithelial cell adhesion molecule (EpCAM) antibodies. However, due to the obvious heterogeneity of CTC phenotypes, some mesenchymal or transformed CTCs may not express EpCAM, resulting in problems such as low capture rate, poor specificity, and high false negative rate in traditional detection methods, making it difficult to meet the detection requirements of multiple types of solid tumors in clinical practice.

[0004] In the prior art, there is a lack of a CTC detection kit that can efficiently enrich heterogeneous CTCs, accurately distinguish tumor cells from normal blood cells, and has good adaptability. Therefore, how to establish a CTC immunodetection system with dual-target recognition ability, stable structure, and high enrichment efficiency is a key problem to be solved urgently. Summary of the Invention

[0005] The present invention aims to solve the problems of low capture efficiency, insufficient specificity, and easy loss of mesenchymal CTCs in the existing detection of circulating tumor cells (CTC), and provides a streptavidin-conjugated antibody capture CTC detection kit, which can efficiently and specifically enrich various types of CTCs in peripheral blood samples, and achieve stable fixation and multiple labeling detection.

[0006] To achieve the above object, the present invention provides the following technical solutions: A streptavidin-conjugated antibody capture CTC detection kit, comprising the following components: a) A pretreatment agent; b) A cell capture agent, the capture agent containing a biotinylated EpCAM monoclonal antibody and a vimentin monoclonal antibody, wherein: The amino acid sequence of the heavy chain variable region of the EpCAM monoclonal antibody is SEQ ID NO:1, and its corresponding coding nucleotide sequence is SEQ ID NO:5; the amino acid sequence of the light chain variable region is SEQ ID NO:2, and the corresponding coding nucleotide sequence is SEQ ID NO:6; The amino acid sequence of the heavy chain variable region of the vimentin monoclonal antibody is SEQ ID NO: 3, and the corresponding coding nucleotide sequence is SEQ ID NO: 7. The amino acid sequence of the light chain variable region is SEQ ID NO: 4, and the corresponding coding nucleotide sequence is SEQ ID NO: 8; c) Separation solution; d) Fixative, permeabilizer and blocking agent; e) The first group of staining mixture, including an antibody labeled with cytokeratin and an antibody labeled with vimentin; f) The second group of staining mixture, including an antibody labeled with CD45 and a nucleic acid dye; g) A microfluidic chip, wherein the bottom layer of the chip is solidified with streptavidin, which can bind the two biotinylated antibodies described above, and the upper layer of the chip is a herringbone structure microchannel array.

[0007] In a preferred technical solution, when the microfluidic chip is connected to a supporting external separation device, its channel structure forms a local rotating liquid flow to enhance the contact between cells and antibodies. The channel length is 20 - 60 mm, the width is 300 - 500 μm, and the depth is 40 - 60 μm, arranged in an asymmetric bifurcated herringbone pattern.

[0008] In a preferred technical solution, the molar ratio of the EpCAM antibody to the Vimentin antibody is between 1:1 and 3:1, and 2 to 10 biotin residues are introduced into each antibody molecule.

[0009] In a preferred technical solution, the pretreatment agent is red blood cell lysate, and the lysate is ammonium chloride buffer with a pH value of 7.2 to 7.4. The main components include ammonium chloride, sodium bicarbonate and EDTA.

[0010] In a preferred technical solution, the separation solution is phosphate buffered saline PBS, or an isotonic washing solution containing 0.5% - 1% bovine serum albumin (BSA) and 1 mM EDTA.

[0011] In a preferred technical solution, the fixative is 4% paraformaldehyde solution, the permeabilizer is 0.1% TritonX - 100 aqueous solution, and the blocking agent is 5% BSA solution or normal goat serum.

[0012] The present invention also provides a preparation method of the streptavidin - conjugated antibody - captured CTC detection kit described above, including the following steps: S1. Antibody expression and purification: Antibodies are prepared according to the nucleotide sequences shown in SEQ ID NO: 5 to SEQ ID NO: 8; S2. Biotinylation: Add the purified antibody to the NHS-biotin reagent at a molar ratio of 1:8 to 1:20, react in a buffer solution for 30 minutes, and then remove the free biotin to obtain the biotinylated antibody; S3. Preparation of the capture agent: Mix the biotinylated EpCAM antibody and the biotinylated Vimentin antibody at a molar ratio of 1:1 to 3:1 to obtain the cell capture agent; S4. Preparation of the microfluidic chip: Use soft lithography technology to construct a microchannel structure with an asymmetric bifurcated herringbone arrangement on the upper layer between PDMS and a glass substrate; S5. Streptavidin immobilization: After plasma treatment of the chip channels, inject streptavidin solution and let it stand for coating to form a high-affinity immobilization interface on the bottom layer, and then rinse with PBS, sterilize with alcohol, and dry for storage; S6. Prepare each functional liquid component, including: red blood cell lysate with a pH of 7.2 to 7.4, separation solution of PBS or containing 0.5% - 1% BSA and 1 mM EDTA, 4% paraformaldehyde fixative, 0.1% TritonX-100 permeabilizer, 5% BSA or goat serum blocking agent, and two staining mixtures containing fluorescently labeled antibodies and nucleic acid dyes; S7. Subpackage, sterilize, and vacuum package the prepared components, assemble them into a complete detection kit in sequence, and finally store them in the warehouse after verifying the capture efficiency and specificity.

[0013] In a preferred technical solution, the S1 step includes: S11. Clone the nucleotide sequences shown in SEQ ID NO:5 to 8 into an expression vector respectively to construct the heavy chain and light chain expression systems of EpCAM and Vimentin antibodies; S12. Transiently transfect the expression vector into CHO-S cells, culture under suspension conditions, collect the expression products, and obtain the required antibodies through chromatography purification.

[0014] In a preferred technical solution, the S4 step includes: S41. Prepare an asymmetric herringbone structure microchannel master mold on a silicon wafer through SU-8 photoresist, and transfer it to a PDMS material to form the upper layer of the chip with a channel length of 20 - 60 mm, a width of 300 - 500 μm, and a depth of 40 - 60 μm; S42. Bond the PDMS layer to the glass substrate, perform plasma treatment, inject streptavidin solution into the channels and let it stand for coating, rinse with PBS and dry, and then package and store for later use.

[0015] Beneficial effects The present invention combines two targeting antibodies, EpCAM and Vimentin, and uses a biotinylation modification and streptavidin immobilization mechanism to firmly immobilize the antibodies on the surface of the microfluidic chip channels, thereby achieving the comprehensive capture of epithelial CTCs and mesenchymal transformed CTCs simultaneously. This strategy significantly expands the detection range and effectively reduces the risk of missed detection of mesenchymal CTCs by traditional methods.

[0016] The herringbone structure microchannel adopted in the present invention has an asymmetric bifurcated arrangement, which can induce a local vortex flow field during the liquid flow process, enhance the interaction between cells and antibodies, and thus improve the overall capture efficiency. Compared with the traditional linear channel structure, this microchannel design provides more cell-interface contact opportunities per unit time, reduces the non-specific adsorption background, and improves the detection accuracy.

[0017] By setting the physical and chemical properties of the red blood cell lysate, isotonic separation solution, fixing solution, permeabilizing agent, blocking solution, etc., the present invention optimizes the retention effect of the morphology and antigenicity of CTCs during the cell processing, avoids cell lysis or antigen masking phenomena, and improves the staining sensitivity and stability.

[0018] In summary, the present invention not only comprehensively improves the capture efficiency, species recognition ability and specificity of CTCs at the technical level, but also enhances the batch producibility and clinical transformation potential of the product through standardized design, with significant technological progress and industrial application value. Brief Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the method steps of Embodiment 1 of the present invention; Figure 2 It is a schematic diagram of the comparative experimental results (total capture rate) of the present invention; Figure 3 It is a schematic diagram of the comparative experimental results of the present invention (CK + cell count); Figure 4 It is a schematic diagram of the comparative experimental results of the present invention (Vim + cell count); Figure 5 It is a schematic diagram of the comparative experimental results of the present invention (CD45 + cell count); Figure 6 It is a schematic diagram of the comparative experimental results of the present invention (white blood cell removal rate). Detailed Embodiments

[0020] To deepen the understanding of the present invention, the following will further elaborate on the present invention in combination with embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation to the protection scope of the present invention.

[0021] Example 1 (T1) This example provides a streptavidin-conjugated antibody-based CTC capture detection kit, which includes the following components: a) Pretreatment agent: an ammonium chloride buffer solution with a pH adjusted to 7.3, composed of 8.3 g / L ammonium chloride, 1.0 g / L sodium bicarbonate, and 0.05 g / L disodium EDTA, used for red blood cell lysis and sample pretreatment; b) Cell capture agent: contains biotinylated EpCAM monoclonal antibody and vimentin monoclonal antibody. The amino acid sequence of the heavy chain variable region of the EpCAM antibody is SEQ ID NO:1, and the light chain is SEQ ID NO:2; the corresponding coding nucleotide sequences are SEQ ID NO:5 and SEQ ID NO:6. The amino acid sequence of the heavy chain variable region of the vimentin antibody is SEQ ID NO:3, and the light chain is SEQ ID NO:4; the corresponding coding nucleotide sequences are SEQ ID NO:7 and SEQ ID NO:8. The two antibodies are mixed at a molar ratio of 2:1, and each antibody molecule is on average introduced with about 5 biotin residues; c) Separation liquid: phosphate buffer solution (PBS), containing 1% bovine serum albumin (BSA) and 1 mM EDTA, with a pH of 7.4, used for non-specific cell elution; d) Fixative, permeabilizer, and blocking agent: the fixative is a 4% paraformaldehyde solution, the permeabilizer is a 0.1% TritonX-100 aqueous solution, and the blocking agent is a 5% BSA solution, and normal goat serum can be used as an alternative; e) The first group of staining mixture: contains AlexaFluor488-labeled anti-cytokeratin antibody and Cy3-labeled anti-vimentin antibody; f) The second group of staining mixture: contains AlexaFluor647-labeled anti-CD45 antibody and DAPI nucleic acid dye; g) Microfluidic chip: a master mold is made using SU-8 photoresist, and a PDMS channel structure is formed by soft lithography technology and bonded to a glass substrate. The upper channel is an asymmetric bifurcated herringbone structure, with a channel length of 40 mm, a width of 400 μm, and a depth of 50 μm. A 10 μg / mL streptavidin solution is injected into the channel, left to adsorb statically at 4 °C for 1 hour, washed with PBS, sterilized with alcohol, dried, and stored refrigerated.

[0022] The preparation method of this kit is as Figure 1 shown, including the following steps: S1. Antibody Expression and Purification: The nucleotide sequences of SEQ ID NOs: 5 to 8 were inserted into humanized IgG1 and Igκ expression vectors, respectively, and transiently transfected into CHO-S cells for expression. After 7 days, the supernatant was collected and purified by Protein A affinity chromatography and Superdex 200 gel filtration chromatography to obtain highly purified EpCAM and Vimentin antibodies.

[0023] S2. Biotin modification: Each antibody solution was adjusted to 1 mg / mL. NHS-biotin (molar ratio 1:10) was added to HEPES buffer at pH 7.2. After reaction at 4°C for 30 minutes, free biotin was removed by dialysis. The resulting biotinylated antibody had an average of 5–6 biotins introduced per molecule.

[0024] S3. Capture agent preparation: Biotinylated EpCAM antibody and Vimentin antibody were mixed at a molar ratio of 2:1 in PBS buffer to a final concentration of 0.5 mg / mL. After filtration sterilization, the mixture was placed in a light-proof brown bottle and stored at 4°C.

[0025] S4. Microfluidic Chip Fabrication: A silicon wafer master mold with an asymmetric herringbone structure was fabricated using SU-8 photoresist. PDMS was then used to mold the channel, with channel dimensions controlled to 40 mm in length, 400 μm in width, and 50 μm in depth. The PDMS layer was plasma bonded to a glass substrate to form a complete chip.

[0026] S5. Streptavidin curing: Inject 10 μg / mL streptavidin solution into the chip channel and let it stand at 4°C for 1 hour to form a coating layer. Rinse with PBS, sterilize with 75% ethanol, and then dry and package for later use.

[0027] S6. Liquid component preparation: Pretreatment agent: ammonium chloride 8.3g / L, sodium bicarbonate 1.0g / L, disodium EDTA 0.05g / L, pH adjusted to 7.3; Separation buffer: PBS + 1% BSA + 1 mM EDTA, sterilized by 0.22 µm filter; Fixative: 4% paraformaldehyde, freshly prepared for use; Permeabilization agent: 0.1% TritonX-100 aqueous solution; Blocking agent: 5% BSA aqueous solution; Staining solution 1: contains AlexaFluor488-anti-CK antibody and Cy3-anti-Vimentin antibody; Staining solution 2: contains AlexaFluor647-anti-CD45 antibody and DAPI dye.

[0028] S7. Finished Product Assembly and Packaging: Load the above components such as chips, liquid reagents, and antibody solutions into a clean blister tray in sequence, evacuate, fill with nitrogen, and seal with an aluminum foil bag. After attaching a label, store it in a cold chain warehouse.

[0029] Example 2 (T2) The streptavidin-conjugated antibody-based CTC detection kit provided in this example is improved based on Example 1 and is suitable for the detection requirements of samples with a relatively high proportion of mesenchymal CTCs. It specifically includes the following components and preparation steps: a) Pretreatment agent: The formula is the same as that in Example 1, which is ammonium chloride buffer solution with a pH of 7.3, composed of 8.3 g / L of ammonium chloride, 1.0 g / L of sodium bicarbonate, and 0.05 g / L of disodium EDTA, mainly used for red blood cell lysis and retaining the monocyte population.

[0030] b) Cell capture agent: The antibodies are composed in the same way as in Example 1, both composed of EpCAM antibodies (SEQ ID NO:1 / 5, SEQ ID NO:2 / 6) and Vimentin antibodies (SEQ ID NO:3 / 7, SEQ ID NO:4 / 8). However, in this example, the dosage of Vimentin antibody is increased, and the molar ratio of EpCAM to Vimentin is 1:2, which is preferentially used to enrich the CTC subset expressing vimentin during epithelial-mesenchymal transition (EMT). The number of biotin residues introduced per molecule of each antibody is controlled between 3 and 5 to reduce steric hindrance interference.

[0031] c) Separation solution: It is a low-protein PBS washing solution containing 0.5% BSA and 1 mM EDTA, suitable for quickly eluting non-specifically bound cells.

[0032] d) Fixative, permeabilizer, and blocking agent: Fixative: Prepare a freshly prepared 4% paraformaldehyde aqueous solution; Permeabilizer: 0.1% TritonX-100; Blocking agent: Normal goat serum, diluted at a concentration of 5%, used to inhibit background fluorescence signals.

[0033] e) The first group of staining mixture: Contains FITC-labeled anti-cytokeratin antibody (CK) and Cy3-labeled anti-Vimentin antibody, which is convenient for distinguishing CTC types.

[0034] f) The second group of staining mixture: Contains AlexaFluor647-labeled anti-CD45 antibody and DAPI nucleic acid dye, which are respectively used for white blood cell identification and cell nucleus imaging.

[0035] g) Microfluidic chip: The chip body is made of the same PDMS-glass material as in Example 1, and the channel design parameters are adjusted as follows: length: 60 mm; width: 300 μm; depth: 40 μm; the bifurcation spacing density of the upper herringbone channel is increased to form more local rotational flows, enhancing the probability of cell sedimentation and antibody binding. After the bottom of the chip is treated with plasma, it is coated with 10 μg / mL streptavidin, left standing at 4 °C for 60 min, and then washed with PBS for standby.

[0036] The preparation method steps are as follows: S1. Antibody expression and purification: The sequences shown in SEQ ID NO:5-8 are respectively constructed into heavy chain and light chain expression vectors, transfected into CHO-K1 cells for transient expression, and the culture supernatant is collected after 7 days of culture. The EpCAM antibody and Vimentin antibody are purified by ProteinA affinity chromatography and Superdex200 gel filtration respectively, and the concentration is adjusted to 1 mg / mL.

[0037] S2. Biotin modification: The NHS-biotin solution is added to the antibody solution at a molar ratio of 1:12, the pH is maintained at 7.2, and the reaction is carried out at 4 °C for 30 minutes. Free biotin is removed through a dialysis bag (molecular weight cut-off 7 kDa), and the reaction efficiency is detected by the HABA colorimetric method and controlled at 3-5 biotins / molecule.

[0038] S3. Antibody compounding: The EpCAM and Vimentin antibodies are mixed at a molar ratio of 1:2, adjusted to a final concentration of 0.8 mg / mL, in a PBS buffer system, filtered and sterilized through a 0.22 µm filter, and then aliquoted.

[0039] S4. Microfluidic chip construction and coating: A high-density asymmetric herringbone channel master mold is constructed by SU-8 lithography technology, and the PDMS is molded by replication and plasma bonded with the glass substrate layer to form a chip, and the channel geometric parameters are 60 mm × 300 μm × 40 μm. Subsequently, 10 μg / mL streptavidin solution is injected into the chip, adsorbed at 4 °C for 1 hour, rinsed with PBS, sterilized with ethanol, and then stored in vacuum after drying.

[0040] S5. Reagent liquid preparation: Red blood cell lysate: the same as in Example 1; Separation liquid: PBS + 0.5% BSA + 1 mM EDTA; The fixing solution, permeabilizing agent, blocking agent and staining solution all refer to the foregoing ratios and concentrations, and are prepared and used immediately; All liquids are sterilized through a 0.22 µm filter membrane and stored at 4 °C.

[0041] S6. Component assembly: Assemble components such as chips, capture agents, lysis solutions, elution solutions, and staining mixtures into a blister tray in sequence. After encapsulating with an aluminum foil bag, evacuate and fill with nitrogen, then seal and package, and attach batch and expiration date labels.

[0042] Example 3 (T3) This example provides a streptavidin-conjugated antibody capture CTC detection kit dedicated to detecting mesenchymal CTCs. A capture system is constructed using a single biotinylated Vimentin antibody, supplemented with a high-density herringbone structure microfluidic chip to enhance the recognition and enrichment ability of Vimentin + / CD45 - type circulating tumor cells.

[0043] This kit includes the following components: a) Pretreatment agent: Ammonium chloride buffer solution, with components of ammonium chloride 8.3 g / L, sodium bicarbonate 1.0 g / L, disodium EDTA 0.05 g / L, pH adjusted to 7.3, mainly used for lysing red blood cells and retaining the monocyte population in peripheral blood.

[0044] b) Cell capture agent: Only contains biotinylated Vimentin monoclonal antibody, the amino acid sequence of its heavy chain variable region is SEQ ID NO:3, the light chain is SEQ ID NO:4, and the corresponding nucleotide sequences are SEQ ID NO:7 and SEQ ID NO:8. Each molecule of the antibody is modified with 4 biotin residues on average, the solution concentration is 1 mg / mL, formulated in PBS, filtered through a 0.22 μm filter for sterilization, and stored at 4°C.

[0045] c) Separation solution: Phosphate buffer solution containing 1% BSA and 1 mM EDTA, used to remove unbound cells and reduce non-specific background.

[0046] d) Fixative, permeabilizer, and blocking agent: Fixative: 4% paraformaldehyde solution; Permeabilizer: 0.1% TritonX-100 aqueous solution; Blocking agent: 5% BSA solution, formulated and stored in the dark at 4°C; e) First group of staining mixture: Cy3-labeled anti-Vimentin antibody staining solution, used for mesenchymal CTC labeling; f) Second group of staining mixture: Contains AlexaFluor647-labeled anti-CD45 antibody and DAPI dye, used to distinguish leukocyte background and nuclear localization; g) Microfluidic chip: The upper layer of the channel has a herringbone structure, designed with a high bifurcation density. The channel width is compressed to 280 μm, the length is 40 mm, and the depth is 40 μm. The radius of the channel corner is reduced to enhance the ability of cells to rotate and deposit with the fluid. The bottom layer is treated with plasma and then 10 μg / mL streptavidin is injected, left to stand and adsorb for 60 minutes at 4°C, rinsed with PBS and then encapsulated.

[0047] Preparation method S1. Antibody expression and purification: The heavy and light chain coding sequences of the Vimentin antibody shown in SEQ ID NO:7 and SEQ ID NO:8 are respectively constructed into pFUSE and pCAG expression vectors, transiently co-transfected in CHO-S cells, and the supernatant is collected after 7 days of culture. First, it is subjected to Protein A affinity chromatography, and then purified by Superdex200 gel filtration. The final concentration is adjusted to 1 mg / mL.

[0048] S2. Biotin modification: NHS-biotin is added to the purified antibody solution at a molar ratio of 1:10, reacted for 30 minutes, and free biotin is removed by dialysis. The HABA colorimetric method is used to confirm that about 4 biotin residues are introduced per molecule of antibody.

[0049] S3. Microfluidic chip preparation: By SU-8 photolithography for mold making, PDMS is used for mold replication and plasma bonded with the glass substrate to form a channel structure. The upper channel has a length of 40 mm, a width of 280 μm, and a depth of 40 μm. The bifurcation spacing of the herringbone structure is half of the standard spacing, improving the flow disturbance and cell sedimentation ability. 10 μg / mL streptavidin solution is injected into the channel, adsorbed for 1 hour, washed with PBS, and dried for standby.

[0050] S4. Solution preparation and dispensing: All liquid components are prepared according to the above ratios, filtered and sterilized with 0.22 μm, and stored at 4°C. Both the anti-CD45 antibody and the Vimentin antibody are fluorescently labeled, and the DAPI staining solution is purchased from a ready-made kit. All components are loaded into a blister tray in the order of operation, vacuum encapsulated and then stored in the warehouse.

[0051] Comparative example 1 (C1) This comparative example provides a CTC detection kit that only uses a single EpCAM antibody as a capture agent. Its chip structure and solution preparation method are basically the same as those in Example 1 of the present invention, but do not contain the Vimentin antibody. The specific composition is as follows: a) Pretreatment agent: It is ammonium chloride lysis solution, with the same formulation as in Example 1, consisting of 8.3 g / L ammonium chloride, 1.0 g / L sodium bicarbonate, and 0.05 g / L disodium EDTA, and the pH is adjusted to 7.3.

[0052] b) Cell capture agent: It only contains biotinylated EpCAM monoclonal antibody, and the amino acid sequences of the heavy chain and light chain variable regions are SEQ ID NO:1 and SEQ ID NO:2 respectively, and the corresponding nucleotide sequences are SEQ ID NO:5 and SEQ ID NO:6. The antibody is modified with NHS-biotin, introducing about 5 biotin residues per molecule, with a final concentration of 1 mg / mL, and stored in PBS buffer.

[0053] c) Separation liquid: PBS buffer, containing 1% BSA and 1 mM EDTA.

[0054] d) Fixative, permeabilizer, blocking agent: They are all the same as in Example 1, which are 4% paraformaldehyde solution, 0.1% Triton X-100 aqueous solution, and 5% BSA blocking solution respectively.

[0055] e) Staining mixture: The first group of staining solution only contains FITC-labeled anti-cytokeratin (CK) antibody; The second group of staining solution contains Alexa Fluor 647-labeled anti-CD45 antibody and DAPI dye.

[0056] f) Microfluidic chip: It is the same as in Example 1. The upper channel has a length of 40 mm, a width of 400 μm, and a depth of 50 μm, and the structure is a standard asymmetric herringbone bifurcation arrangement. After the bottom layer is treated with plasma, a 10 μg / mL streptavidin solution is injected, adsorbed for 1 hour, washed with PBS, dried, and then encapsulated.

[0057] Comparative Example 2 (C2) This comparative example provides a CTC detection kit that immobilizes antibodies by physical adsorption. Although its capture agent contains a combination of EpCAM antibody and Vimentin antibody, it does not use a streptavidin-coated chip and biotinylated antibody to construct a high-affinity coupling system, and only immobilizes the antibody on the bottom layer of the chip by direct adsorption. The specific composition is as follows: a) Pretreatment agent: It is ammonium chloride lysis solution, with the same formulation as in Example 1, consisting of 8.3 g / L ammonium chloride, 1.0 g / L sodium bicarbonate, and 0.05 g / L disodium EDTA, and the pH is adjusted to 7.3.

[0058] b) Cell capture agent: Comprising unbiotinylated EpCAM antibody and Vimentin antibody, the amino acid sequences of the heavy and light chain variable regions are SEQ ID NO:1 - 4 respectively, and the corresponding coding nucleotide sequences are SEQ ID NO:5 - 8. The two antibodies are mixed at a molar ratio of 1:1, with a final concentration of 0.5 mg / mL for each antibody, and are directly formulated in PBS buffer without any biotinylation treatment.

[0059] c) Separation solution: PBS buffer containing 1% BSA and 1 mM EDTA.

[0060] d) Fixative, permeabilizer, blocking agent: Are the same as in Example 1, which are 4% paraformaldehyde solution, 0.1% Triton X - 100 aqueous solution and 5% BSA blocking solution respectively.

[0061] e) Staining mixture: The first group of staining solution contains FITC - labeled anti - cytokeratin antibody (CK) and Cy3 - labeled anti - Vimentin antibody; The second group of staining solution contains Alexa Fluor 647 - labeled anti - CD45 antibody and DAPI dye.

[0062] f) Microfluidic chip: The chip structure is the same as in Example 1. The upper - layer channel has a length of 40 mm, a width of 400 μm, and a depth of 50 μm, and the structure is a standard asymmetric herringbone bifurcation arrangement. Only after plasma treatment, the antibody mixture is directly injected into the bottom layer of the chip channel, incubated at 37 °C for 1 hour to form a physical adsorption coating, and there is no streptavidin coating step. Subsequently, it is rinsed with PBS and dried and encapsulated.

[0063] Comparative Example 3 (C3) This comparative example provides a CTC detection kit that only uses cytokeratin (CK) labeling in the staining detection step. Its capture step is the same as in Example 1, both using the dual - antibody capture strategy of EpCAM and Vimentin antibodies, but only CK and CD45 staining are performed in the staining stage, without including Vimentin staining label. The specific composition is as follows: a) Pretreatment agent: Ammonium chloride lysis solution, with the same formula as in Example 1, consisting of ammonium chloride 8.3 g / L, sodium bicarbonate 1.0 g / L, and disodium EDTA 0.05 g / L, and the pH is adjusted to 7.3.

[0064] b) Cell capture agent: It contains biotinylated EpCAM monoclonal antibody and Vimentin monoclonal antibody. The amino acid sequences of the heavy and light chain variable regions are SEQ ID NO:1 - 4 respectively, and the corresponding nucleotide sequences are SEQ ID NO:5 - 8. The two antibodies are mixed at a molar ratio of 1:1, with a final concentration of 1 mg / mL, modified with NHS-biotin, introducing about 5 biotin residues per molecule, and stored in PBS buffer.

[0065] c) Separation solution: PBS buffer containing 1% BSA and 1 mM EDTA.

[0066] d) Fixative, permeabilizer, blocking agent: They are the same as those in Example 1, which are 4% paraformaldehyde solution, 0.1% Triton X - 100 aqueous solution and 5% BSA blocking solution respectively.

[0067] e) Staining mixture: The first group of staining solution only contains FITC-labeled anti-cytokeratin (CK) antibody; The second group of staining solution contains Alexa Fluor 647-labeled anti-CD45 antibody and DAPI dye; There is no staining channel set for detecting Vimentin.

[0068] f) Microfluidic chip: It is the same as that in Example 1. The upper channel has a length of 40 mm, a width of 400 μm, and a depth of 50 μm. The structure is an asymmetric herringbone bifurcation arrangement. After the bottom layer is treated with plasma, 10 μg / mL streptavidin solution is injected, left to adsorb for 1 hour, washed with PBS, dried, and then encapsulated.

[0069] Comparative experiment: To systematically evaluate the overall performance of the embodiments of the present invention (T1, T2, T3) and three comparative example schemes (C1, C2, C3) in the enrichment and detection of circulating tumor cells (CTC), the following comparative experiment was designed.

[0070] 1. Experimental materials and grouping Positive model cells: Epithelial MCF-7 cells (EpCAM + / Vim - ).

[0071] Mesenchymal MDA-MB-231 cells (EpCAM - / Vim + ).

[0072] Blood matrix: Peripheral blood of healthy volunteers, collected after ethical approval.

[0073] Simulated sample preparation: Add 20 MCF-7 cells and 20 MDA-MB-231 cells to 3 mL of whole blood respectively. After gentle mixing, immediately proceed to the subsequent process.

[0074] Detection system: T1, T2, and T3 correspond to Examples 1, 2, and 3 respectively; C1, C2, and C3 correspond to three comparative examples of only EpCAM capture, physical adsorption fixation, and absence of Vimentin staining respectively.

[0075] Six simulated samples are detected in parallel for each system.

[0076] 2. Operation process Pretreatment: Add ammonium chloride lysis solution to each group to remove red blood cells and obtain mononuclear cell suspension.

[0077] Chip enrichment: Constant flow rate of 1 mL / h -1 Inject into the microfluidic chip.

[0078] Elution and fixation: Wash unbound cells with PBS → Fix with 4% paraformaldehyde → Permeabilize with 0.1% Triton X-100 → Block with 5% BSA.

[0079] Fluorescent staining: The staining protocol is four-color immunofluorescent labeling, labeling CK, Vimentin, CD45, and cell nucleus (DAPI) respectively; The Vimentin antibody step is absent in C1 and C3.

[0080] Microscopic counting: Confocal microscope scans each well, record the number of cells of CK + Vim + / CK + Vim - / Vim + CK -  and CD45 - Record the number of cells of CD45 + Residual white blood cells.

[0081] 3. Evaluation indicators Total capture rate = (number of CTCs detected in the chip / total number of added CTCs) × 100%.

[0082] EpCAM type detection rate = (number of detected MCF-7 / number of added MCF-7) × 100%.

[0083] Vimentin type detection rate = (number of detected MDA-MB-231 / number of added MDA-MB-231) × 100%.

[0084] Leukocyte removal rate = (number of loaded WBCs - number of residual WBCs) / number of loaded WBCs.

[0085] Background noise: number of misjudged cells in fluorescence images.

[0086] The experimental results are shown in Table 1: Table 1 Comparative experimental results

[0087] Data analysis As Figure 2 shown, Examples T1, T2, and T3 all showed relatively high total CTC capture rates, which were 80.5%, 72.4%, and 76.1% respectively, significantly higher than those of Comparative Examples C1 (38.7%), C2 (43.5%), and C4 (35.8%). This indicates that the dual-antibody capture system (EpCAM and Vimentin) adopted in the solution of the present invention has significant cell enrichment ability, especially for CTC subgroups with strong phenotypic heterogeneity, and has better recognition and binding effects.

[0088] In terms of biomarker recognition, as Figure 3 shown, the number of cytokeratin-positive (CK + ) cells in Group T1 was 47, and the number of vimentin-positive (Vim + ) cells was 39, both being the highest among all groups, indicating that this solution has advantages in capturing epithelial and mesenchymal CTCs. Although the total CTC capture rate of Comparative Example C3 was close to that of T1 (79.8%), the number of its Vim + cells was 0, indicating that this solution did not include Vimentin staining, resulting in the inability to recognize mesenchymal CTC cells and serious missed detection. Taking the number of Vim + cells in T1 as a reference, the missed detection rate of mesenchymal CTCs in C3 reached 100%, indicating that when relying solely on EpCAM as a screening biomarker, it is very easy to miss tumor cells after phenotypic transformation.

[0089] In addition, as Figure 4 shown, in Comparative Example C1, only the EpCAM antibody was used, and its total capture rate was 38.7%. The number of CK + cells was only 22, and the number of Vim + cells was 0, further verifying the importance of jointly using epithelial and mesenchymal biomarkers in the present invention; although Group C2 had the Vimentin antibody, it was not biotinylated and could not bind to the streptavidin at the bottom layer of the chip, resulting in low antibody immobilization efficiency and ultimately no significant improvement in the capture efficiency. The number of CTC-positive cells was similar to that of C1, and the synergistic effect of the dual-antibody system was not fully exerted.

[0090] As Figure 5As shown, Example T1 also exhibits good specificity, with the CD45 + white blood cell count maintained within 9, significantly lower than that of most comparative groups (such as the CD45 + cells in C2 reaching as high as 19), demonstrating the background noise control ability after optimizing the blocking agent and washing buffer system, effectively reducing non-specific binding and improving the signal-to-noise ratio.

[0091] In terms of white blood cell removal ability, as Figure 6 shown, Examples T1 to T3 all show relatively high white blood cell removal rates, reaching 93.1%, 91.8% and 91.8% respectively, much higher than those of Comparative Examples C1 (74.3%), C2 (82.4%) and C3 (76.6%), indicating that using the streptavidin immobilization system and optimizing the blocking agent scheme can effectively reduce the residual CD45 + background cells and improve the specificity and signal-to-noise ratio of CTC detection.

[0092] In summary, Example T1 performs excellently in terms of capture efficiency, comprehensive cell recognition and non-specific binding control, especially in recognizing CK + / Vim + double-positive CTCs, which is significantly superior to the existing technical solutions. By jointly using biotinylated double antibodies and streptavidin functional chip structure, the efficient enrichment and accurate detection of CTCs with different phenotypes are effectively achieved, verifying the reliability and technical advantages of the present invention in practical applications.

[0093] The above shows and describes 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 by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A streptavidin-coupled antibody capture CTC detection kit, characterized in that: Includes the following components: a) pretreatment agent; b) a cell capture agent, comprising a biotin-modified EpCAM monoclonal antibody and a vimentin monoclonal antibody, wherein: The amino acid sequence of the heavy chain variable region of the EpCAM monoclonal antibody is SEQ ID NO: 1, and the corresponding encoding nucleotide sequence is SEQ ID NO: 5; the amino acid sequence of the light chain variable region is SEQ ID NO: 2, and the corresponding encoding nucleotide sequence is SEQ ID NO: 6; The amino acid sequence of the heavy chain variable region of the vimentin monoclonal antibody is SEQ ID NO: 3, and the corresponding encoding nucleotide sequence is SEQ ID NO: 7; the amino acid sequence of the light chain variable region is SEQ ID NO: 4, and the corresponding encoding nucleotide sequence is SEQ ID NO: 8; c) separation liquid; d) Fixatives, permeabilizing agents, and blocking agents; e) a first staining mixture comprising an antibody for labeling cytokeratin and an antibody for labeling vimentin; f) a second staining mixture comprising an antibody labeled with CD45 and a nucleic acid dye; g) A microfluidic chip, wherein the bottom layer of the chip is immobilized with streptavidin capable of binding to the two biotin-modified antibodies, and the upper layer of the chip is a microchannel array of herring bone structure.

2. The kit according to claim 1, wherein When the microfluidic chip is connected to a matching external separation device, its channel structure forms a local rotating liquid flow to enhance the contact between cells and antibodies. The channel has a length of 20-60 mm, a width of 300-500 μm, and a depth of 40-60 μm, and is arranged in an asymmetric forked herringbone pattern.

3. The kit according to claim 1 or 2, characterized in that The molar ratio of the EpCAM antibody to the Vimentin antibody is between 1:1 and 3:1, and 2 to 10 biotin residues are introduced into each antibody molecule.

4. The kit according to claim 3, wherein The pretreatment agent is a red blood cell lysis solution, which is an ammonium chloride buffer with a pH value of 7.2 to 7.

4. The main components include ammonium chloride, sodium bicarbonate and EDTA.

5. The kit according to claim 4, characterized in that The separation solution is phosphate buffered saline (PBS), or an isotonic washing solution containing 0.5% to 1% bovine serum albumin (BSA) and 1 mM EDTA.

6. The kit according to claim 1, wherein The fixing solution is 4% paraformaldehyde solution, the permeabilizing agent is 0.1% TritonX-100 aqueous solution, and the blocking agent is 5% BSA solution or normal goat serum.

7. The method for preparing a CTC detection kit for capturing CTCs by a streptavidin-coupled antibody according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1. Antibody expression and purification: Prepare antibodies according to the nucleotide sequences shown in SEQ ID NO: 5 to SEQ ID NO: 8; S2. Biotin modification: Add NHS-biotin reagent to the purified antibody at a molar ratio of 1:8 to 1:20, react in buffer for 30 minutes, and then remove free biotin to prepare the biotin-modified antibody; S3. Capture agent preparation: biotinylated EpCAM antibody and biotinylated Vimentin antibody are mixed in a molar ratio of 1:1 to 3:1 to prepare a cell capture agent; S4. Microfluidic chip preparation: Soft lithography was used to construct a microchannel structure with an asymmetric bifurcated herringbone arrangement on the upper layer between PDMS and a glass substrate; S5, Streptavidin curing: After plasma treatment of the chip channel, inject streptavidin solution and let it sit for coating to form a bottom high-affinity curing interface. The chip is then rinsed with PBS, sterilized with alcohol, and dried for storage. S6. Prepare the functional liquid components, including: red blood cell lysis buffer with a pH of 7.2 to 7.4, PBS or separation buffer containing 0.5% to 1% BSA and 1 mM EDTA, 4% paraformaldehyde fixative, 0.1% Triton X-100 permeabilizer, 5% BSA or goat serum blocking agent, and two sets of staining mixtures containing fluorescent-labeled antibodies and nucleic acid dyes; S7. The prepared components are packaged, sterilized and vacuum-packed, and assembled into a complete detection kit in sequence. Finally, the kit is put into storage after the capture efficiency and specificity are verified.

8. The preparation method according to claim 7, characterized in that The S1 step includes: S11. Cloning the nucleotide sequences shown in SEQ ID NOs: 5 to 8 into expression vectors, respectively, to construct heavy chain and light chain expression systems for EpCAM and Vimentin antibodies; S12. Transiently transfect the expression vector into CHO-S cells, culture them under suspension conditions, collect the expression products, and purify them by chromatography to obtain the desired antibody.

9. The preparation method according to claim 7, characterized in that The S4 step includes: S41, prepare an asymmetric herring bone structure microchannel master mold on a silicon wafer using SU-8 photoresist, and transfer it to PDMS material to form a chip upper layer with a channel length of 20–60 mm, a width of 300–500 μm, and a depth of 40–60 μm; S42. After bonding the PDMS layer to the glass substrate, perform plasma treatment, inject streptavidin solution into the channel and let it sit for coating, rinse with PBS, dry, and then package and store for later use.

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