Streptavidin-coupled antibody capture CTC detection kit

Abstract

The present invention belongs to the technical field of biochemical testing, and specifically provides a streptavidin-coupled antibody capture CTC detection kit and a preparation method thereof, wherein the kit includes components such as biotin-modified EpCAM monoclonal antibody and Vimentin monoclonal antibody, a pretreatment agent, a separation liquid, a fixative, a permeabilization agent, a blocking agent, a fluorescent staining liquid and a microfluidic chip. The bottom layer of the chip channel is solidified with streptavidin, and the upper layer is an asymmetric herring bone structure, which can enhance the binding efficiency between cells and antibodies. The kit achieves efficient capture of CTCs of different phenotypes through multi-antibody composite recognition, and combines immunofluorescence staining for multi-marker detection, with both sensitivity and specificity. The method has a standardized process flow and stable components, and is suitable for the enrichment and analysis of CTCs in clinical samples.

Description

Technical Field

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

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

[0003] Currently, commonly used CTC detection technologies are mostly based on the enrichment of a single epithelial cell adhesion molecule (EpCAM) antibody. However, due to the obvious heterogeneity of CTC phenotype, some mesenchymal or transformed CTCs may not express EpCAM, resulting in traditional detection methods having problems such as low capture rate, poor specificity, and high false negative rate, which makes it difficult to meet the clinical needs of detecting multiple types of solid tumors.

[0004] The existing technology lacks a CTC detection kit that can efficiently enrich heterogeneous CTCs, accurately distinguish tumor cells from normal blood cells, and possess good compatibility. Therefore, establishing a CTC immunoassay system with dual-target recognition capabilities, a stable structure, and high enrichment efficiency is a key issue that needs to be addressed. Summary of the Invention

[0005] The present invention aims to address the problems of low capture efficiency, insufficient specificity, and easy loss of interstitial CTCs in existing circulating tumor cell (CTC) detection. A streptavidin-coupled antibody capture CTC detection kit is provided, which can efficiently and specifically enrich multiple 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:

[0007] A streptavidin-coupled antibody capture CTC detection kit, comprising the following components:

[0008] a) pretreatment agent;

[0009] b) a cell capture agent, comprising a biotin-modified EpCAM monoclonal antibody and a vimentin monoclonal antibody, wherein:

[0010] 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;

[0011] 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;

[0012] c) separation liquid;

[0013] d) Fixatives, permeabilizing agents, and blocking agents;

[0014] e) a first staining mixture comprising an antibody for labeling cytokeratin and an antibody for labeling vimentin;

[0015] f) a second staining mixture comprising an antibody labeled with CD45 and a nucleic acid dye;

[0016] 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.

[0017] In a preferred technical solution, 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.

[0018] 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.

[0019] In a preferred technical solution, the pretreatment agent is a red blood cell lysate, and the lysate is an ammonium chloride buffer with a pH value of 7.2 to 7.4, and its main components include ammonium chloride, sodium bicarbonate and EDTA.

[0020] In a preferred technical solution, 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.

[0021] In a preferred technical solution, the fixative is a 4% paraformaldehyde solution, the permeabilizing agent is a 0.1% TritonX-100 aqueous solution, and the blocking agent is a 5% BSA solution or normal goat serum.

[0022] The present invention also provides a method for preparing the streptavidin-coupled antibody capture CTC detection kit, comprising the following steps:

[0023] S1. Antibody expression and purification: Prepare antibodies according to the nucleotide sequences shown in SEQ ID NO: 5 to SEQ ID NO: 8;

[0024] 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;

[0025] 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;

[0026] 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;

[0027] 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.

[0028] 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;

[0029] 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.

[0030] In a preferred technical solution, the step S1 includes:

[0031] 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;

[0032] 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.

[0033] In a preferred technical solution, the S4 step includes:

[0034] 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;

[0035] 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.

[0036] Beneficial effects

[0037] This method combines two targeting antibodies, EpCAM and Vimentin, with biotin modification and streptavidin immobilization to secure the antibodies to the microfluidic chip channel surface, enabling comprehensive capture of both epithelial CTCs and mesenchymal transition CTCs. This strategy significantly expands the detection range and effectively reduces the risk of missing mesenchymal CTCs with traditional methods.

[0038] The herringbone microchannels employed in this study feature an asymmetric bifurcated arrangement, which induces localized vortexes during liquid flow, enhancing the interaction between cells and antibodies and thus improving overall capture efficiency. Compared to traditional linear channel structures, this microchannel design provides more cell-interface contact opportunities per unit time, reduces nonspecific adsorption background, and improves detection accuracy.

[0039] By setting the physical and chemical properties of the formulas such as red blood cell lysis solution, isotonic separation solution, fixative, permeabilization agent and blocking solution, the present invention optimizes the retention of CTC morphology and antigenicity during cell processing, avoids cell lysis or antigen masking, and improves staining sensitivity and stability.

[0040] In summary, the present invention not only achieves a comprehensive improvement in CTC capture efficiency, type identification ability and specificity at the technical level, but also enhances the product's mass manufacturability and clinical transformation potential through standardized design, with significant technological progress and industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a schematic diagram of the method steps of Example 1 of the present invention;

[0042] Figure 2Schematic diagram of comparative experimental results (total capture rate) of the present invention;

[0043] Figure 3 The comparative experimental results of the present invention (CK + Cell number) schematic diagram;

[0044] Figure 4 The comparative experimental results of the present invention (Vim + Cell number) schematic diagram;

[0045] Figure 5 The comparative experimental results of the present invention (CD45 + Cell number) schematic diagram;

[0046] Figure 6 Schematic diagram of comparative experimental results (leukocyte removal rate) of the present invention. DETAILED DESCRIPTION

[0047] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the examples. The examples are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0048] Example 1 (T1)

[0049] This example provides a streptavidin-coupled antibody capture CTC detection kit, comprising the following components:

[0050] a) Pretreatment agent: ammonium chloride buffer, 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;

[0051] b) Cell capture agent: contains biotin-modified 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 encoding 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 encoding nucleotide sequences are SEQ ID NO: 7 and SEQ ID NO: 8. The two antibodies are mixed in a molar ratio of 2:1, and an average of about 5 biotin residues are introduced into each antibody molecule;

[0052] c) Separation buffer: phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 1 mM EDTA, pH 7.4, used for eluting nonspecific cells;

[0053] d) Fixative, permeabilizing agent, and blocking agent: The fixative is 4% paraformaldehyde solution, the permeabilizing agent is 0.1% Triton X-100 aqueous solution, and the blocking agent is 5% BSA solution. Normal goat serum can be used as an alternative.

[0054] e) The first staining mixture: containing AlexaFluor488-labeled anti-cytokeratin antibody and Cy3-labeled anti-vimentin antibody;

[0055] f) The second staining mixture: containing AlexaFluor647-labeled anti-CD45 antibody and DAPI nucleic acid dye;

[0056] g) Microfluidic Chip: A master mold was prepared using SU-8 photoresist, and a PDMS channel structure was formed by soft lithography and bonded to a glass substrate. The upper channel was an asymmetric bifurcated herringbone structure with a length of 40 mm, a width of 400 μm, and a depth of 50 μm. A 10 μg / mL streptavidin solution was injected into the channel and allowed to adsorb at 4°C for 1 hour. The sample was then washed with PBS, sterilized with alcohol, and dried before being refrigerated.

[0057] The preparation method of the kit is as follows Figure 1 As shown, the following steps are included:

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] S6. Liquid component preparation:

[0064] Pretreatment agent: ammonium chloride 8.3g / L, sodium bicarbonate 1.0g / L, disodium EDTA 0.05g / L, pH adjusted to 7.3;

[0065] Separation buffer: PBS + 1% BSA + 1 mM EDTA, sterilized by 0.22 µm filter;

[0066] Fixative: 4% paraformaldehyde, freshly prepared for use;

[0067] Permeabilization agent: 0.1% TritonX-100 aqueous solution;

[0068] Blocking agent: 5% BSA aqueous solution;

[0069] Staining solution 1: contains AlexaFluor488-anti-CK antibody and Cy3-anti-Vimentin antibody;

[0070] Staining solution 2: contains AlexaFluor647-anti-CD45 antibody and DAPI dye.

[0071] S7. Finished product assembly and packaging: The above-mentioned chips, liquid reagents, antibody solutions and other components are placed in a clean blister tray in order, vacuumed, filled with nitrogen and sealed in aluminum foil bags, labeled and put into cold chain storage.

[0072] Example 2 (T2)

[0073] The streptavidin-conjugated antibody capture CTC detection kit provided in this example is improved on the basis of Example 1 and is suitable for detecting samples with a high proportion of interstitial CTCs. It specifically includes the following components and preparation steps:

[0074] a) Pretreatment agent: The formulation is the same as that of Example 1, i.e., ammonium chloride buffer, pH 7.3, composed of 8.3 g / L ammonium chloride, 1.0 g / L sodium bicarbonate, and 0.05 g / L disodium EDTA, mainly used for lysis of red blood cells and retention of monocyte populations.

[0075] b) Cell capture agent: The antibody composition is the same as in Example 1, consisting of EpCAM antibodies (SEQ ID NOs: 1 / 5, 2 / 6) and Vimentin antibodies (SEQ ID NOs: 3 / 7, 4 / 8). However, in this example, the amount of Vimentin antibody was increased, with a molar ratio of EpCAM to Vimentin of 1:2. This is used to preferentially enrich for CTC subpopulations expressing vimentin during epithelial-mesenchymal transition (EMT). The number of biotin residues introduced per molecule of each antibody was controlled between 3 and 5 to minimize steric interference.

[0076] c) Separation buffer: It is a low-protein PBS washing buffer containing 0.5% BSA and 1mM EDTA, suitable for rapid elution of non-specifically bound cells.

[0077] d) Fixative, permeabilizing agent and blocking agent:

[0078] Fixative: freshly prepared 4% paraformaldehyde aqueous solution;

[0079] Permeabilization agent: 0.1% TritonX-100;

[0080] Blocking agent: normal goat serum, diluted to 5% concentration, used to suppress background fluorescence signals.

[0081] e) The first staining mixture contains FITC-labeled anti-cytokeratin (CK) antibody and Cy3-labeled anti-vimentin antibody to facilitate the differentiation of CTC types.

[0082] f) The second staining mixture contains AlexaFluor647-labeled anti-CD45 antibody and DAPI nucleic acid dye, which are used for leukocyte identification and cell nucleus imaging, respectively.

[0083] g) Microfluidic Chip: The chip body was constructed using the same PDMS-glass material as in Example 1. Channel design parameters were adjusted to: length: 60 mm; width: 300 μm; depth: 40 μm. The branch spacing density of the upper herring bone channel was increased to create more localized rotating flow, enhancing cell sedimentation and antibody binding. The bottom of the chip was plasma-treated and then coated with 10 μg / mL streptavidin. The chip was incubated at 4°C for 60 minutes and then washed with PBS for later use.

[0084] The preparation steps are as follows:

[0085] S1. Antibody expression and purification:

[0086] Sequences represented by SEQ ID NOs: 5-8 were constructed into heavy and light chain expression vectors, respectively, and transfected into CHO-K1 cells for transient expression. The culture supernatant was collected after 7 days of culture. EpCAM and Vimentin antibodies were purified by Protein A affinity chromatography and Superdex 200 gel filtration, respectively, and the concentration was adjusted to 1 mg / mL.

[0087] S2, biotin modification:

[0088] NHS-biotin solution was added to the antibody solution at a molar ratio of 1:12, maintained at pH 7.2, and reacted at 4°C for 30 minutes. Free biotin was removed using a dialysis bag (molecular weight cutoff 7 kDa). The reaction efficiency was controlled at 3–5 biotin molecules per molecule using the HABA colorimetric assay.

[0089] S3. Antibody compounding:

[0090] EpCAM and Vimentin antibodies were mixed at a molar ratio of 1:2 and adjusted to a final concentration of 0.8 mg / mL using PBS buffer. The mixture was sterilized by 0.22 µm filtration and then aliquoted.

[0091] S4. Microfluidic chip construction and coating:

[0092] A high-density asymmetric herring bone channel master mold was constructed using SU-8 photolithography. PDMS was then overmolded and plasma-bonded to a glass substrate to form a chip with channel geometry of 60 mm × 300 μm × 40 μm. A 10 μg / mL streptavidin solution was then injected into the chip, adsorbed at 4°C for 1 hour, rinsed with PBS, sterilized with ethanol, and then vacuum-dried for storage.

[0093] S5. Preparation of reagent liquid:

[0094] Red blood cell lysis solution: same as in Example 1;

[0095] Separation buffer: PBS + 0.5% BSA + 1 mM EDTA;

[0096] Fixative, permeabilizing agent, blocking agent and staining solution should all be prepared fresh for use according to the above ratios and concentrations;

[0097] All liquids were sterilized by passing through a 0.22 μm filter and stored at 4°C.

[0098] S6. Component assembly:

[0099] The chip, capture agent, lysate, eluent, staining mixture and other components are assembled into blister trays in order, sealed in aluminum foil bags, vacuum-packed and nitrogen-filled, and sealed with batch and expiration date labels.

[0100] Example 3 (T3)

[0101] This example provides a CTC detection kit specifically for detecting interstitial CTCs by capturing them with a streptavidin-coupled antibody. The capture system is constructed using a single biotinylated Vimentin antibody and supplemented with a high-density herringbone structure microfluidic chip to enhance the capture of Vimentin. + / CD45 - Ability to identify and enrich circulating tumor cells.

[0102] This kit contains the following components:

[0103] a) Pretreatment agent: ammonium chloride buffer, composed of 8.3 g / L ammonium chloride, 1.0 g / L sodium bicarbonate, and 0.05 g / L disodium EDTA, with a pH adjusted to 7.3. It is mainly used to lyse red blood cells and preserve the mononuclear cell population in peripheral blood.

[0104] b) Cell capture agent: Contains only the biotinylated Vimentin monoclonal antibody, whose heavy chain variable region amino acid sequence is SEQ ID NO: 3 and light chain variable region is SEQ ID NO: 4, with corresponding nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 8. The antibody is modified with an average of four biotin residues per molecule. The solution is prepared at a concentration of 1 mg / mL in PBS, sterile-filtered through 0.22 μm, and stored at 4°C.

[0105] c) Separation buffer: Phosphate buffer containing 1% BSA and 1 mM EDTA, used to remove unbound cells and reduce nonspecific background.

[0106] d) Fixative, permeabilizing agent and blocking agent:

[0107] Fixative: 4% paraformaldehyde solution;

[0108] Permeabilization agent: 0.1% TritonX-100 aqueous solution;

[0109] Blocking agent: 5% BSA solution, stored at 4°C in the dark after preparation;

[0110] e) The first staining mixture: Cy3-labeled anti-Vimentin antibody staining solution, used for marking interstitial CTCs;

[0111] f) The second staining mixture contains AlexaFluor 647-labeled anti-CD45 antibody and DAPI dye, which is used to distinguish leukocyte background from cell nuclear localization;

[0112] g) Microfluidic chip: The upper channel layer features a herringbone structure with a high-density bifurcation design. The channel width is compressed to 280 μm, the length is 40 mm, and the depth is 40 μm. The channel corner radius is reduced to enhance the ability of cells to settle as the fluid rotates. The bottom layer is plasma-treated and then injected with 10 μg / mL streptavidin. The solution is allowed to adsorb for 60 minutes at 4°C, rinsed with PBS, and then packaged.

[0113] Preparation method

[0114] S1. Antibody expression and purification:

[0115] The heavy and light chain coding sequences of the Vimentin antibody shown in SEQ ID NO:7 and SEQ ID NO:8 were constructed into the pFUSE and pCAG expression vectors, respectively, and transiently co-transfected into CHO-S cells. After 7 days of culture, the supernatant was collected and purified by Protein A affinity chromatography and then Superdex200 gel filtration to a final concentration of 1 mg / mL.

[0116] S2, biotin modification:

[0117] NHS-biotin was added to the purified antibody solution at a molar ratio of 1:10. The reaction was allowed to proceed for 30 minutes, and free biotin was removed by dialysis. The HABA colorimetric assay was used to confirm the incorporation of approximately four biotin residues per antibody molecule.

[0118] S3. Microfluidic chip preparation:

[0119] The channel structure was formed by SU-8 photolithography, PDMS overmolding, and plasma bonding to a glass substrate. The upper channel was 40 mm long, 280 μm wide, and 40 μm deep. The herringbone bifurcation spacing was half the standard spacing, enhancing flow perturbation and cell sedimentation. A 10 μg / mL streptavidin solution was injected into the channel, adsorbed for 1 hour, then washed with PBS and dried for later use.

[0120] S4, liquid preparation and packaging:

[0121] All liquid components were prepared according to the above ratios, sterilized by 0.22 μm filtration, and stored at 4°C. Anti-CD45 and Vimentin antibodies were fluorescently labeled, and DAPI stain was purchased from a pre-existing kit. All components were loaded into blister trays according to the order in which they were prepared, vacuum-sealed, and stored.

[0122] Comparative Example 1 (C1)

[0123] This comparative example provides a CTC detection kit using only a single EpCAM antibody as a capture agent. The chip structure and liquid preparation method thereof are basically based on Example 1 of the present invention, but without vimentin antibody. The specific composition is as follows:

[0124] a) Pretreatment agent: ammonium chloride lysis solution, the formula of which is the same as that 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.

[0125] b) Cell capture agent: Contains only a biotinylated EpCAM monoclonal antibody, whose heavy and light chain variable region amino acid sequences 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 approximately five biotin residues per molecule, at a final concentration of 1 mg / mL, and stored in PBS buffer.

[0126] c) Separation Buffer: PBS buffer containing 1% BSA and 1 mM EDTA.

[0127] d) Fixative, permeabilizing agent, and blocking agent: all were the same as those in Example 1, namely, 4% paraformaldehyde solution, 0.1% Triton X-100 aqueous solution, and 5% BSA blocking solution, respectively.

[0128] e) Staining mixture:

[0129] The first set of staining solutions contained only FITC-labeled anti-cytokeratin (CK) antibodies;

[0130] The second staining solution contains Alexa Fluor 647-labeled anti-CD45 antibody and DAPI dye.

[0131] f) Microfluidic chip: Similar to Example 1, the upper channel had a length of 40 mm, a width of 400 μm, and a depth of 50 μm, with a standard asymmetric herringbone bifurcated arrangement. The bottom layer was plasma-treated and then injected with a 10 μg / mL streptavidin solution for 1 hour. The solution was then washed with PBS, dried, and packaged.

[0132] Comparative Example 2 (C2)

[0133] This comparative example provides a CTC detection kit that uses physical adsorption to immobilize antibodies. Although the capture agent comprises a combination of EpCAM antibodies and Vimentin antibodies, a high-affinity coupling system is not constructed using a streptavidin-coated chip and a biotin-modified antibody. Instead, the antibodies are fixed to the chip substrate by direct adsorption. The specific composition is as follows:

[0134] a) Pretreatment agent: ammonium chloride lysis solution, the formula of which is the same as that 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.

[0135] b) Cell capture agent: Contains unbiotinylated EpCAM and Vimentin antibodies, with the heavy and light chain variable region amino acid sequences of SEQ ID NOs: 1-4, respectively, and the corresponding encoding nucleotide sequences of SEQ ID NOs: 5-8. The two antibodies were mixed in a 1:1 molar ratio to a final concentration of 0.5 mg / mL each in PBS buffer without any biotin labeling.

[0136] c) Separation Buffer: PBS buffer containing 1% BSA and 1 mM EDTA.

[0137] d) Fixative, permeabilizing agent, and blocking agent: all were the same as those in Example 1, namely, 4% paraformaldehyde solution, 0.1% Triton X-100 aqueous solution, and 5% BSA blocking solution, respectively.

[0138] e) Staining mixture:

[0139] The first set of staining solution contains FITC-labeled anti-cytokeratin antibody (CK) and Cy3-labeled anti-Vimentin antibody;

[0140] The second staining solution contains Alexa Fluor 647-labeled anti-CD45 antibody and DAPI dye.

[0141] f) Microfluidic chip: The chip structure was identical to that of Example 1. The upper channel had a length of 40 mm, a width of 400 μm, and a depth of 50 μm, with a standard asymmetric herringbone bifurcation arrangement. The bottom channel layer of the chip was plasma-treated only, then directly injected with the antibody mixture and incubated at 37°C for 1 hour to form a physical adsorption coating. No streptavidin coating was performed. The chip was then rinsed with PBS, dried, and packaged.

[0142] Comparative Example 3 (C3)

[0143] This comparative example provides a CTC detection kit that uses only cytokeratin (CK) markers in the staining detection step. The capture step is the same as that of Example 1, both using a dual capture strategy of EpCAM and Vimentin antibodies. However, only CK and CD45 staining are performed in the staining stage, and Vimentin staining markers are not included. The specific composition is as follows:

[0144] a) Pretreatment agent: ammonium chloride lysis solution, the formula of which is the same as that 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.

[0145] b) Cell capture agent: Contains biotin-modified EpCAM monoclonal antibody and Vimentin monoclonal antibody, with the heavy and light chain variable region amino acid sequences of SEQ ID NOs: 1-4, and the corresponding nucleotide sequences of SEQ ID NOs: 5-8, respectively. The two antibodies were mixed in a 1:1 molar ratio to a final concentration of 1 mg / mL. They were modified with NHS-biotin to introduce approximately five biotin residues per molecule and stored in PBS buffer.

[0146] c) Separation Buffer: PBS buffer containing 1% BSA and 1 mM EDTA.

[0147] d) Fixative, permeabilizing agent, and blocking agent: all were the same as those in Example 1, namely, 4% paraformaldehyde solution, 0.1% Triton X-100 aqueous solution, and 5% BSA blocking solution, respectively.

[0148] e) Staining mixture:

[0149] The first set of staining solutions contained only FITC-labeled anti-cytokeratin (CK) antibodies;

[0150] The second staining solution contained Alexa Fluor 647-labeled anti-CD45 antibody and DAPI dye;

[0151] No staining channel was set for detecting Vimentin.

[0152] f) Microfluidic chip: Similar to Example 1, the upper channel had a length of 40 mm, a width of 400 μm, and a depth of 50 μm. The structure was an asymmetric herringbone bifurcated arrangement. The bottom layer was plasma-treated and then injected with a 10 μg / mL streptavidin solution. The solution was allowed to adsorb for 1 hour, washed with PBS, dried, and then encapsulated.

[0153] Comparative experiment:

[0154] To systematically evaluate the overall performance of the embodiments of the present invention (T1, T2, and T3) and three comparative examples (C1, C2, and C3) in circulating tumor cell (CTC) enrichment and detection, the following comparative experiments were designed.

[0155] 1. Experimental Materials and Grouping

[0156] Positive model cells:

[0157] Epithelial MCF-7 cells (EpCAM + / Vim - ).

[0158] Mesenchymal MDA-MB-231 cells (EpCAM - / Vim + ).

[0159] Blood matrix: Peripheral blood from healthy volunteers, collected after ethical approval.

[0160] Simulated sample preparation: 20 MCF-7 cells and 20 MDA-MB-231 cells were added to 3 mL of whole blood, respectively. After gentle mixing, the cells were immediately transferred to the subsequent steps.

[0161] Detection system:

[0162] T1, T2, and T3 correspond to embodiments 1, 2, and 3;

[0163] C1, C2, and C3 correspond to the three comparative examples of EpCAM capture only, physical adsorption fixation, and lack of Vimentin staining, respectively.

[0164] Six simulated samples were tested in parallel in each system.

[0165] 2. Operational Process

[0166] Pretreatment: Ammonium chloride lysis buffer was added to each group to remove red blood cells and obtain mononuclear cell suspension.

[0167] Chip enrichment: constant flow rate 1 mL h -1 Inject into the microfluidic chip.

[0168] Elution and fixation: PBS elution of unbound cells → 4% paraformaldehyde fixation → 0.1% Triton X-100 permeabilization → 5% BSA blocking.

[0169] Fluorescent staining: The staining scheme is a four-color immunofluorescence labeling, which labels CK, Vimentin, CD45 and cell nucleus (DAPI) respectively; C1 and C3 lack the Vimentin antibody link.

[0170] Microscopic counting: Scan each well with a confocal microscope and record CK + Vim + / CK + Vim - / Vim + CK - and CD45 - The number of cells; record CD45 + Residual white blood cells.

[0171] 3. Evaluation Metrics

[0172] Total capture rate = (number of CTCs detected in the chip / total number of CTCs added) × 100%.

[0173] EpCAM type detection rate = (number of MCF-7 detected / number of MCF-7 added) × 100%.

[0174] Vimentin type detection rate = (number of MDA-MB-231 detected / number of MDA-MB-231 added) × 100%.

[0175] Leukocyte removal rate = (number of WBCs loaded - number of residual WBCs) / number of WBCs loaded.

[0176] Background noise: misidentification of cell numbers in fluorescence images.

[0177] The experimental results are shown in Table 1:

[0178] Table 1 Comparative experimental results

[0179]

[0180] Data Analysis

[0181] like Figure 2 As shown, Examples T1, T2, and T3 all showed relatively high total CTC capture rates of 80.5%, 72.4%, and 76.1%, respectively, which were significantly higher than those of Comparative Examples C1 (38.7%), C2 (43.5%), and C4 (35.8%), indicating that the dual antibody capture system (EpCAM and Vimentin) used in the present invention has significant cell enrichment capabilities, especially for CTC subpopulations with strong phenotypic heterogeneity, with better recognition and binding effects.

[0182] In terms of marker identification, Figure 3 As shown, cytokeratin was positive in T1 group (CK + ) The number of cells was 47, and the number of cells positive for vimentin (Vim + ) cells were 39, the highest in all groups, indicating that this approach has advantages in capturing both epithelial and mesenchymal CTCs. In contrast, although the total CTC capture rate of C3 was close to that of T1 (79.8%), its Vim + The cell count is 0, indicating that the protocol does not include Vimentin staining, resulting in the inability to identify interstitial CTC cells and a serious missed detection phenomenon. + Taking the cell number as a reference, the missed detection rate of interstitial CTCs in C3 reached 100%, indicating that it is very easy to miss tumor cells after phenotypic conversion when relying solely on EpCAM as a screening marker.

[0183] In addition, if Figure 4 As shown, in comparative example C1, only EpCAM antibody was used, and the total capture rate was 38.7%. + There are only 22 cells in Vim +The number of CTC-positive cells in group C2 was 0, which further verified the importance of the combined use of epithelial and mesenchymal markers in the present invention. Although group C2 had Vimentin antibodies, it was not biotin-modified and could not bind to the streptavidin at the bottom of the chip, resulting in low antibody fixation efficiency. The final capture efficiency was not significantly improved, and the number of CTC-positive cells was similar to that of C1, failing to fully exert the synergistic effect of the dual antibody system.

[0184] like Figure 5 As shown, Example T1 also showed good specificity, CD45 + The white blood cell count was maintained within 9, which was significantly lower than that of most control groups (such as CD45 + The optimized blocking agent and washing buffer system can effectively reduce nonspecific binding and improve the signal-to-noise ratio.

[0185] In terms of leukocyte removal capacity, Figure 6 As shown in the figure, Examples T1 to T3 all showed high leukocyte removal rates, reaching 93.1%, 91.8% and 91.8% respectively, which were much higher than those of Comparative Examples C1 (74.3%), C2 (82.4%) and C3 (76.6%), indicating that the use of streptavidin fixation system and optimized blocking agent solution can effectively reduce CD45 + Background cells remain, improving the specificity and signal-to-noise ratio of CTC detection.

[0186] In summary, Example T1 performed well in terms of capture efficiency, comprehensive cell recognition, and non-specific binding control, especially in recognizing CK + / Vim + The results show that the dual-positive CTC detection method is significantly superior to existing technology solutions. By combining the biotinylated double antibody and streptavidin functional chip structure, efficient enrichment and accurate detection of CTCs of different phenotypes are effectively achieved, verifying the reliability and technical advantages of the present invention in practical applications.

[0187] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention 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 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 and sterilized with alcohol before drying and 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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