Material for identifying specific blood cells, and purification and separation method
Patent Information
- Application Number
- PCT/CN2025/085399
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2025-03-27
- Publication Date
- 2026-10-01
Smart Images
Figure CN2025085399_01102026_PF_FP_ABST
Abstract
Description
Specific hematologic identification materials and purification and separation methods Technical Field This invention discloses a specific hematology-identifying material and a purification and separation method. Specifically, the specific hematology-identifying material is an antibody-modified temperature-responsive polymer. Background Technology In the field of biomolecular separation technology, particularly the separation of specific blood cells or platelets from blood samples, has been a current trend in biomedical research and development. Blood samples typically have a complex composition, including plasma, red blood cells, platelets, white blood cells, and various physiologically active biomolecules or cells. White blood cells are usually separated by centrifugation or using filters containing fibrous materials or anti-biomolecular-adhesion materials such as polyethylene glycol. Known filter materials are generally porous or have surface charge modifications to separate negatively charged platelets and white blood cells. Furthermore, to prevent coagulation or platelet activation during separation, anticoagulants or other agents need to be added to the blood sample. Thirdly, known materials or methods can only roughly separate red blood cells or perform blood sample deleukization procedures for transfusion safety, but cannot achieve precise separation of specific cells in the blood sample. Therefore, the disadvantages of known technologies include complex separation procedures, long processing times, and poor separation recovery rates, all of which constitute technical barriers to the purification and separation of blood components. In conclusion, researching and developing a biomedical material and purification method that can more accurately separate and purify specific blood cells or cells from blood samples to overcome the current technical bottlenecks faced by related industries is an urgent technical issue that needs to be addressed. Summary of the Invention Based on prior art and to meet industry needs, the primary objective of this invention is to provide a specific blood cell identification material that effectively separates and purifies specific blood cells or immune cells, such as B cells, T cells, or natural killer (NK) cells, from a sample. Specifically, the specific blood cell identification material comprises a protein, a temperature-responsive polymer, and an antibody (immunoglobulin). The protein is chemically anchored to the temperature-responsive polymer, and the antibody (immunoglobulin) is non-covalently bound to the protein, thereby constituting the specific blood cell identification material. Specifically, the temperature-responsive polymer of the present invention has reactive functional groups, such as carboxylic acid groups; the carboxylic acid groups can undergo condensation reactions with hydroxyl or amino groups in the protein structure to form chemical covalent bonds of ester or amide bonds, thereby anchoring the protein to the temperature-responsive polymer. Then, the protein combines with antibodies (immunoglobulins) or biotin-labeled antibodies (immunoglobulins) to form a complex, thereby constituting the specific blood cell recognition material described above. Specifically, the protein contains streptavidin, or avidin. Specifically, the temperature-responsive polymer includes acrylic hydrogel, polyacrylic acid, (acrylic acid-N-alkylacrylamide) copolymer, (acrylic acid-N-alkylacrylamide) copolymer network hydrogel, (acrylic acid-alkyl methacrylate) copolymer, (acrylic acid-alkyl methacrylate) copolymer network hydrogel or a combination thereof. Specifically, the antibody (immunoglobulin) is selected from one of the following groups: CD3 antibody, CD16 antibody, CD19 antibody, CD45 antibody, CD56 antibody, anti-CD3 antibody, anti-CD16 antibody, anti-CD19 antibody, anti-CD45 antibody, anti-CD56 antibody, biotin-labeled anti-CD19 antibody, and biotin-labeled anti-CD56 antibody. Preferably, the antibody is a biotin-labeled antibody. More specifically, the chemical bond is selected from one or a combination of the following groups: amide bond, ester bond, acetal bond, ketal bond, ether bond, and disulfide bond. In summary, the specific hematology identification material of this invention is an antibody-modified temperature-responsive polymer. This antibody-modified temperature-responsive polymer is composed of an antibody and a temperature-responsive polymer with protein anchoring. Furthermore, the specific hematology identification material of this invention, due to its complex structure of protein and antibody, also improves its biocompatibility. This invention achieves the effect of identifying different biomolecules through antibodies with biomolecular specificity. Therefore, this invention can be applied to the separation and purification of specific hematology cells or immune cells, such as B cells, T cells, or natural killer (NK) cells, in blood samples and achieve the technical effect of specific identification. In accordance with the aforementioned first objective, the second objective of the present invention is to provide a method for self-sample separation and recovery of B cells, which includes, but is not limited to, the following steps. Step 1: Expose a sample containing B cells to a specific hematology identification material at 30–40°C, thereby causing the B cells to adsorb onto the specific hematology identification material. The specific hematology identification material comprises streptavidin, a temperature-responsive polymer, and an antibody. The streptavidin is anchored to the temperature-responsive polymer via an amide bond. The antibody is a CD19 antibody, CD45 antibody, anti-CD19 antibody, anti-CD45 antibody, biotin-labeled anti-CD19 antibody, or biotin-labeled anti-CD45 antibody. The antibody is non-covalently bound to the streptavidin, thereby constituting the specific hematology identification material. Step 2: Perform the desorption procedure by incubating the specific hematology identification material at 0–10°C for at least 10 minutes or immersing it in a solution containing biotin, thereby desorbing the B cells adsorbed on the specific hematology identification material. Step 3: Perform the recovery procedure by rinsing the specific blood cell identification material with a buffer solution or a solution containing biotin at 0–10°C to recover the detached B cells, thereby achieving the effect of separating and recovering B cells from the sample. In accordance with the aforementioned first objective, the third objective of this invention is to provide a method for separating and recovering NK cells from a sample, specifically comprising, but not limited to, the following steps. Step 1: Expose a sample containing NK cells to a specific hematology identification material at 30–40°C, thereby causing the NK cells to adsorb onto the specific hematology identification material. The specific hematology identification material comprises streptavidin, a temperature-responsive polymer, and an antibody. The streptavidin is anchored to the temperature-responsive polymer via an amide bond. The antibody is a CD16 antibody, CD56 antibody, anti-CD16 antibody, anti-CD56 antibody, biotin-labeled anti-CD16 antibody, or biotin-labeled anti-CD56 antibody. The antibody is non-covalently bound to the streptavidin, thereby constituting the specific hematology identification material. Step 2: Perform the desorption procedure by incubating the specific hematology identification material at 0-10°C for at least 10 minutes or immersing it in a solution containing biotin, thereby desorbing the NK cells adsorbed on the specific hematology identification material. Step 3: Perform the recovery procedure by rinsing the specific blood cell recognition material with a buffer solution or a solution containing biotin at 0-10°C to recover the detached NK cells, thereby achieving the effect of separating and recovering NK cells from the sample. A fourth objective of this invention is to provide a natural killer cell separation membrane, which has the technical effect of separating and purifying natural killer (NK) cells from biological or blood samples. Specifically, the natural killer cell separation membrane comprises a coating and a substrate, and the coating comprises an antibody-modified polymer. Specifically, the coating comprises multiple antibodies, multiple streptavidins, and a polymer having multiple carboxylic acid groups. The multiple antibodies bind to the multiple streptavidins via biotin, and the multiple streptavidins are chemically coupled to the polymer having multiple carboxylic acid groups, thereby forming the coating; and the coating is coated on the substrate, thereby constituting the natural killer cell separation membrane. More specifically, the polymer of the present invention having multiple carboxylic acid groups is a temperature-responsive polymer; the carboxylic acid groups can undergo a condensation reaction with the hydroxyl or amino groups in the streptavidin structure to form a chemical covalent bond of ester or amide bond, thereby coupling and anchoring the streptavidin to the polymer having multiple carboxylic acid groups, and then binding to the aforementioned multiple antibodies via biotin, thereby forming the coating of the natural killer cell separation membrane. In summary, the specific blood cell identification material of the present invention has the following technical features and effects. (1) The specific blood cell identification material of the present invention is an antibody-modified temperature-responsive polymer. Different antibody-protein complexes can be designed on the temperature-responsive polymer according to the different blood cells or immune cells to be identified, thereby achieving the technical effect of specific blood cell identification. For example, to identify B lymphocytes in a sample, the temperature-responsive polymer material is a biotin-labeled anti-CD19 antibody-streptavidin complex; to identify natural killer cells in a sample, the temperature-responsive polymer material is a biotin-labeled anti-CD56 antibody-streptavidin complex. (2) The specific blood cell identification material of the present invention has temperature-responsive characteristics, and can achieve the technical effect of capturing or releasing specific blood cells or immune cells by utilizing different operating temperatures. Compared with the traditional centrifugation method, the method of the present invention has the technical advantages of simple operation steps and short operation time. (3) The specific blood cell identification material of the present invention has low toxicity and high biocompatibility, and can obtain a high purity specific blood cell or immune cell recovery solution in the process of separating and purifying specific blood cells or immune cells. Attached Figure Description Figure 1 shows the reaction equation for the temperature-responsive hydrogel (AAc-co-NIPAAm hydrogel) of this invention; Figure 2 is a schematic diagram of the synthesis of the specific blood cell recognition material of the present invention; (A) is a schematic diagram of anchoring streptavidin in a temperature-responsive hydrogel; (B) is a schematic diagram of the formation of a biotin-labeled antibody-streptavidin complex; Figure 3 is the Fourier transform infrared spectrum of the specific blood cell identification material of the present invention; Figure 4 is a histogram of the water absorption and hydrogel area difference of the specific blood cell identification material of the present invention; (A) is a histogram of the water absorption of the control group; (B) is a histogram of the water absorption of the experimental group; (C) is a histogram of the water absorption of the control group; (D) is a histogram of the hydrogel area difference of the experimental group. Figure 5 shows the diiodomethane contact angle of the specific blood cell identification material of the present invention; (A) is the diiodomethane contact angle at 4°C; (B) is the diiodomethane contact angle at 37°C. Figure 6 is a water absorption curve of the control group and the experimental group of the specific hematology identification material of the present invention; Figure 7 is a histogram of fibrinogen adhesion of the specific blood cell identification material of the present invention; (A) is a histogram of fibrinogen adhesion at 4°C; (B) is a histogram of fibrinogen adhesion at 37°C. Figure 8 is a histogram of the relative cell survival rate of the specific blood cell identification material of the present invention; (A) is a histogram of the relative cell survival rate of the control group; (B) is a histogram of the relative cell survival rate of the experimental group. Figure 9 is a histogram of the adhesion rate of the (acrylate-butyl methacrylate) copolymer (A1-A4) with streptavidin coupling of the present invention to biotin-labeled antibodies at different pH values. Figure 10 is a histogram of coating density of the natural killer cell separation film (A1-A4, A1E-A4E) of the present invention; Figure 11 is a porosity histogram of the natural killer cell separation membrane (A1-A4, A1E-A4E) of the present invention; Figure 12 is a histogram of the hydration capacity of the natural killer cell separation membrane (A1-A4, A1E-A4E) of the present invention. Detailed Implementation Based on the foregoing description, this invention aims to provide a specific hematology identification material and its application in purifying and separating specific hematology cells and immune cells. Specifically, this specific hematology identification material is an antibody-modified polymer, more specifically, it is composed of an antibody and a protein-anchored polymer. For example, a biotin-labeled antibody and a streptavidin-anchored polymer constitute the specific hematology identification material of this invention. Another technical feature of this invention is that the polymer material exhibits temperature-responsive properties. Therefore, the specific hematology identification material of this invention achieves the technical effect of capturing or desorbing specific hematology cells and immune cells at different operating temperatures, thereby achieving the technical objective of purifying and separating specific hematology cells and immune cells. Furthermore, due to the good shape plasticity of the polymer material, the specific hematology identification material of this invention can be made into thin layers, granules, or other shapes or sizes, and assembled into separation films or packing beds for specific hematology cells and immune cells, with wide applications in various biomedical devices. The following examples and illustrations specifically illustrate the implementation of this invention. The first embodiment of the present invention provides a specific blood cell identification material, which comprises a protein, a temperature-responsive polymer and an antibody. The protein is chemically anchored to the temperature-responsive polymer, and the antibody is non-covalently bound to the protein, thereby constituting the specific blood cell identification material. In one embodiment, the protein contains streptavidin or avidin. In one embodiment, the temperature-responsive polymer comprises acrylic hydrogel, polyacrylic acid, (acrylic acid-N-alkylacrylamide) copolymer, (acrylic acid-N-alkylacrylamide) copolymer network hydrogel, (acrylic acid-alkyl methacrylate) copolymer, (acrylic acid-alkyl methacrylate) copolymer network hydrogel, or any combination thereof. Preferably, the (acrylic acid-N-alkylacrylamide) copolymer is an (acrylic acid-N-isopropylacrylamide) copolymer; and the (acrylic acid-N-alkylacrylamide) copolymer network hydrogel is an (acrylic acid-N-isopropylacrylamide) copolymer network hydrogel. In one embodiment, the molar ratio of acrylic acid to N-alkylacrylamide in the acrylic acid-N-alkylacrylamide copolymer or the (acrylic acid-N-alkylacrylamide) copolymer network hydrogel is 1 to 20. Preferably, the molar ratio of acrylic acid to N-alkylacrylamide in the acrylic acid-N-alkylacrylamide copolymer or the (acrylic acid-N-alkylacrylamide) copolymer network hydrogel is 5 to 15. In a preferred embodiment, the alkyl group of the (acrylic acid-alkyl methacrylate) copolymer or the (acrylic acid-alkyl methacrylate) copolymer network hydrogel is a linear alkyl group with 1 to 18 carbon atoms, a branched alkyl group with 3 to 18 carbon atoms, or a cyclic alkyl group with 3 to 8 carbon atoms. In a representative embodiment, the (acrylic acid-alkyl methacrylate) copolymer is a (acrylic acid-butyl methacrylate) copolymer. In one embodiment, the antibody is selected from one of the following groups: CD3 antibody, CD16 antibody, CD19 antibody, CD45 antibody, CD56 antibody, anti-CD3 antibody, anti-CD16 antibody, anti-CD19 antibody, anti-CD45 antibody, anti-CD56 antibody, biotin-labeled anti-CD19 antibody, and biotin-labeled anti-CD56 antibody. In a preferred embodiment, the antibody is a biotin-labeled antibody. In one embodiment, the chemical bond is selected from one or a combination of the following groups: amide bond, ester bond, acetal bond, ketal bond, ether bond, and disulfide bond. Preferably, the chemical bond is an amide bond, ester bond, ether bond, or disulfide bond. In one embodiment, its Fourier infrared spectrum has absorption peaks in the following wavenumber range: 1545-1579 cm⁻¹ -1 1627-1654cm -1 and 1710-1740cm -1 . The second embodiment of the present invention provides a method for separating and recovering B cells from a blood sample, which includes the following steps. Step 1: Expose a blood sample containing B cells to a specific hematology identification material at 30–40°C, thereby causing the B cells to adsorb onto the specific hematology identification material. The specific hematology identification material comprises streptavidin, a temperature-responsive polymer, and an antibody. The streptavidin is anchored to the temperature-responsive polymer via an amide bond. The antibody is a CD19 antibody, CD45 antibody, anti-CD19 antibody, anti-CD45 antibody, biotin-labeled anti-CD19 antibody, or biotin-labeled anti-CD45 antibody. The antibody is non-covalently bound to the streptavidin, thereby constituting the specific hematology identification material. Step 2: Perform the desorption procedure by incubating the specific hematology identification material at 0–10°C for at least 10 minutes or immersing it in a solution containing biotin, thereby desorbing the B cells adsorbed on the specific hematology identification material. Step 3: Perform the recovery procedure by rinsing the specific blood cell identification material with a buffer solution or a solution containing biotin at 0–10°C to recover the detached B cells, thereby achieving the purpose of separating and recovering B cells from the blood sample. In one embodiment, the temperature-responsive polymer comprises acrylic hydrogel, polyacrylic acid, (acrylic acid-N-alkylacrylamide) copolymer, (acrylic acid-N-alkylacrylamide) copolymer network hydrogel, (acrylic acid-alkyl methacrylate) copolymer, (acrylic acid-alkyl methacrylate) copolymer network hydrogel, or any combination thereof. Preferably, the (acrylic acid-N-alkylacrylamide) copolymer is an (acrylic acid-N-isopropylacrylamide) copolymer; and the (acrylic acid-N-alkylacrylamide) copolymer network hydrogel is an (acrylic acid-N-isopropylacrylamide) copolymer network hydrogel. In one embodiment, the molar ratio of acrylic acid to N-alkylacrylamide in the acrylic acid-N-alkylacrylamide copolymer or the acrylic acid-N-alkylacrylamide copolymer network hydrogel is 1 to 20. Preferably, the molar ratio of acrylic acid to N-alkylacrylamide in the acrylic acid-N-alkylacrylamide copolymer or the acrylic acid-N-alkylacrylamide copolymer network hydrogel is 5 to 15. In a preferred embodiment, the alkyl group of the (acrylic acid-alkyl methacrylate) copolymer or the (acrylic acid-alkyl methacrylate) copolymer network hydrogel is a linear alkyl group with 1 to 18 carbon atoms, a branched alkyl group with 3 to 18 carbon atoms, or a cyclic alkyl group with 3 to 8 carbon atoms. In a representative embodiment, the (acrylic acid-alkyl methacrylate) copolymer is a (acrylic acid-butyl methacrylate) copolymer. In one embodiment, the Fourier transform infrared spectrum of the specific hematology identification material exhibits absorption peaks in the wavenumber range of 1545-1579 cm⁻¹. -1 1627-1654cm -1 and 1710-1740cm -1 . The third embodiment of the present invention provides a method for separating and recovering NK cells from a blood sample, specifically comprising the following steps. Step 1: Expose a blood sample containing NK cells to a specific hematology identification material at 30–40°C, thereby causing the NK cells to adsorb onto the specific hematology identification material. The specific hematology identification material comprises streptavidin, a temperature-responsive polymer, and an antibody. The streptavidin is anchored to the temperature-responsive polymer via an amide bond. The antibody is a CD16 antibody, CD56 antibody, anti-CD16 antibody, anti-CD56 antibody, biotin-labeled anti-CD16 antibody, or biotin-labeled anti-CD56 antibody. The antibody is non-covalently bound to the streptavidin, thereby constituting the specific hematology identification material. Step 2: Perform the desorption procedure by incubating the specific hematology identification material at 0-10°C for at least 10 minutes or immersing it in a solution containing biotin, thereby desorbing the NK cells adsorbed on the specific hematology identification material. Step 3: Perform the recovery procedure by rinsing the specific blood cell identification material with a buffer solution or a solution containing biotin at 0-10°C to recover the detached NK cells, thereby achieving the purpose of separating and recovering NK cells from the blood sample. In one embodiment, the temperature-responsive polymer comprises acrylic hydrogel, polyacrylic acid, (acrylic acid-N-alkylacrylamide) copolymer, (acrylic acid-N-alkylacrylamide) copolymer network hydrogel, (acrylic acid-alkyl methacrylate) copolymer, (acrylic acid-alkyl methacrylate) copolymer network hydrogel, or any combination thereof. Preferably, the (acrylic acid-N-alkylacrylamide) copolymer is an (acrylic acid-N-isopropylacrylamide) copolymer; and the (acrylic acid-N-alkylacrylamide) copolymer network hydrogel is an (acrylic acid-N-isopropylacrylamide) copolymer network hydrogel. In one embodiment, the molar ratio of acrylic acid to N-alkylacrylamide in the (acrylic acid-N-alkylacrylamide) copolymer or the (acrylic acid-N-alkylacrylamide) copolymer network hydrogel is 1 to 20. Preferably, the molar ratio of acrylic acid to N-alkylacrylamide in the (acrylic acid-N-alkylacrylamide) copolymer or the (acrylic acid-N-alkylacrylamide) copolymer network hydrogel is 5 to 15. In a preferred embodiment, the alkyl group of the (acrylic acid-alkyl methacrylate) copolymer or the (acrylic acid-alkyl methacrylate) copolymer network hydrogel is a linear alkyl group with 1 to 18 carbon atoms, a branched alkyl group with 3 to 18 carbon atoms, or a cyclic alkyl group with 3 to 8 carbon atoms. In a representative embodiment, the (acrylic acid-alkyl methacrylate) copolymer is a (acrylic acid-butyl methacrylate) copolymer. In one embodiment, the Fourier transform infrared spectrum of the specific hematology identification material exhibits absorption peaks in the wavenumber range of 1545-1579 cm⁻¹. -1 1627-1654cm -1and 1710-1740cm -1 . A fourth embodiment of the present invention provides a natural killer cell separation membrane, comprising a coating and a substrate. Specifically, the coating comprises a plurality of antibodies, a plurality of streptavidins, and a polymer having a plurality of carboxylic acid groups. The plurality of antibodies bind to the plurality of streptavidins via biotin, and the plurality of streptavidins are chemically coupled to the polymer having a plurality of carboxylic acid groups, thereby forming the coating; and the coating is coated on the substrate, thereby constituting the natural killer cell separation membrane. In one embodiment, the polymer having multiple carboxylic acid groups comprises acrylic hydrogel, polyacrylic acid, (acrylic acid-N-alkylacrylamide) copolymer, (acrylic acid-N-alkylacrylamide) copolymer network hydrogel, (acrylic acid-alkyl methacrylate) copolymer, (acrylic acid-alkyl methacrylate) copolymer network hydrogel, or any combination thereof. Preferably, the (acrylic acid-N-alkylacrylamide) copolymer is an (acrylic acid-N-isopropylacrylamide) copolymer; and the (acrylic acid-N-alkylacrylamide) copolymer network hydrogel is an (acrylic acid-N-isopropylacrylamide) copolymer network hydrogel. In a preferred embodiment, the alkyl group of the (acrylic acid-alkyl methacrylate) copolymer or the (acrylic acid-alkyl methacrylate) copolymer network hydrogel is a linear alkyl group with 1 to 18 carbon atoms, a branched alkyl group with 3 to 18 carbon atoms, or a cyclic alkyl group with 3 to 8 carbon atoms. In a representative embodiment, the (acrylic acid-alkyl methacrylate) copolymer is a (acrylic acid-butyl methacrylate) copolymer. In one embodiment, the molar ratio of acrylic acid to N-alkylacrylamide in the acrylic acid-N-alkylacrylamide copolymer or the (acrylic acid-N-alkylacrylamide) copolymer network hydrogel is 1 to 20. Preferably, the molar ratio of acrylic acid to N-alkylacrylamide in the acrylic acid-N-alkylacrylamide copolymer or the (acrylic acid-N-alkylacrylamide) copolymer network hydrogel is 5 to 15. In one embodiment, the antibody is selected from one of the following groups: CD3 antibody, CD16 antibody, CD19 antibody, CD45 antibody, CD56 antibody, anti-CD3 antibody, anti-CD16 antibody, anti-CD19 antibody, anti-CD45 antibody, anti-CD56 antibody, biotin-labeled anti-CD19 antibody, and biotin-labeled anti-CD56 antibody. In a preferred embodiment, the antibody is a biotin-labeled antibody. In one embodiment, the chemical bond is selected from one or a combination of the following groups: amide bond, ester bond, acetal bond, ketal bond, ether bond, and disulfide bond. Preferably, the chemical bond is an amide bond or an ester bond. In one embodiment, the Fourier transform infrared spectrum of the coating of the natural killer cell separation membrane has absorption peaks in the wavenumber range of 1545-1579 cm⁻¹. -1 1627-1654cm -1 1710-1740cm -1 and 3000-3600cm -1 . In one embodiment, the substrate comprises polyethylene, polypropylene, polystyrene, polytetrafluoroethylene, polyvinylidene fluoride, ceramic, or filament. The following examples illustrate in detail the preparation method, structural analysis, surface properties, biocompatibility, and technical efficacy of the specific blood cell identification material of the present invention in separating and recovering specific blood cells and immune cells. Example: A general preparation method for specific hematology identification materials Referring to the reaction formula shown in Figure 1, acrylic monomers (AAc) and N-isopropylacrylamide monomers (NIPAAm) in different molar ratios are added to deionized water and stirred at 25°C. A crosslinking agent (NMBA) is added and stirring continues. Then, an initiator (Ammonium persulfate, APS / Tetramethylethylenediamine N,N,N',N'tetramethylethylenediamine, TEMED) is added and stirred. The polymerization reaction is carried out at 60°C. After the reaction is completed, the mixture is washed with deionized water to obtain a temperature-responsive hydrogel [(acrylic acid-N-isopropylacrylamide) copolymer network hydrogel (AAc-co-NIPAAm hydrogel)]. Alternatively, acrylic monomers (AA) and butyl methacrylate monomers (BMA) in different molar ratios are added to ethanol and stirred. An initiator is added and the polymerization reaction is carried out at 60°C. After the reaction is completed, the mixture is washed with deionized water to obtain a (acrylic acid-butyl methacrylate) copolymer (poly(AA-co-BMA)). Both are stored at 4°C for later use.Please refer to the schematic diagrams in Figures 2(A) and 2(B). The (acrylate-N-isopropylacrylamide) copolymer network hydrogel or (acrylate-butyl methacrylate) copolymer is a molecule with carboxylate functional groups. This (acrylate-N-isopropylacrylamide) copolymer network hydrogel or (acrylate-butyl methacrylate) copolymer is added to MES (sodium 2-(N-morpholino)ethanesulfonic acid) at 25°C. The solution of amino acids (MES) was stirred for 50 minutes, then (1-ethyl-3-(-3-dimethylamino)propyl)-carbodiimide (EDC) was added, and the mixture was stirred at 25°C for 1 hour. Next, NHS (N-hydroxysuccinimide, NHS) was added, and the mixture was stirred at 25°C for 1 hour to generate an activated NHS ester. After adding streptavidin, a grafting reaction was carried out for 1 hour, allowing the amino groups in the streptavidin structure to form stable amide bonds with the carboxylic acid groups of the (acrylate-N-isopropylacrylamide) copolymer hydrogel or (acrylate-butyl methacrylate) copolymer. A streptavidin-anchored (acrylate-N-isopropylacrylamide) copolymer hydrogel or streptavidin-anchored (acrylate-butyl methacrylate) copolymer is prepared by bonding. Then, a biotinylated antibody is added, and the mixture is stirred at 25°C for 1 hour to allow the biotinylated antibody to bind with the streptavidin on the streptavidin-anchored (acrylate-N-isopropylacrylamide) copolymer or the streptavidin-anchored (acrylate-butyl methacrylate) copolymer, forming a biotinylated antibody-streptavidin complex via non-covalent bonding. Finally, the mixture is washed three times with PBS buffer to obtain the specific hematology recognition material. Specifically, this specific hematology recognition material is a polymer material anchored to a biotinylated antibody-streptavidin complex; more specifically, the biotinylated antibody is a biotinylated anti-CD19 antibody or a biotinylated anti-CD56 antibody. In the following description, the polymer material codes A-5NI and A-5NI* of this invention represent, respectively, a hydrogel of (acrylate-N-isopropylacrylamide) copolymer network anchored by a streptavidin complex of a biotin-labeled antibody (anti-CD19 or anti-CD56) and a hydrogel of (acrylate-N-isopropylacrylamide) copolymer network anchored by a streptavidin complex of a biotin-labeled antibody (anti-CD19 or anti-CD56); wherein, the polymer material code without an asterisk (*) indicates a hydrogel without biotin labeling. The control group for antibodies, and the polymer materials marked with * are the experimental groups containing biotin-labeled antibodies; based on the total mole percentage (100%) of the reacting monomers, A-5NI and A-5NI* have a mole percentage of 95% acrylic acid and 5% N-isopropylacrylamide; A-10NI and A-10NI* have a mole percentage of 90% acrylic acid and 10% N-isopropylacrylamide. The meanings of other polymer material codes such as A-15NI and A-15NI* are defined and explained according to the above. Example: Structural analysis of specific hematology identification materials The structural analysis of the specific blood cell identification material of the present invention was performed using Fourier transform infrared spectroscopy to analyze the functional groups. The specific spectra and analysis results are shown in Figure 3 and Table 1. Table 1 Example: Analysis of water absorption and hydrocolloid area difference of specific hematology identification materials In this example, the water absorption and hydrogel area difference of the control group and the experimental group were measured at 4℃ and 37℃, respectively. The specific experimental results are shown in Figures 4 and 6. According to the experimental results, the specific blood cell identification material of the present invention has the technical characteristics of high critical solution temperature (UCST) and low critical solution temperature (LCST), and has different folding structures at different operating temperatures, which can achieve the technical effect of adsorbing specific blood cells or immune cells at 37℃ or desorbing them at 4℃. Example: Surface analysis of the specific blood cell identification material of the present invention In this example, the diiodomethane contact angle of the control group and the experimental group was measured at 4℃ and 37℃, respectively. The specific experimental results are shown in Figure 5. According to the results, the specific hematology identification material of this invention exhibits different diiodomethane contact angles at different temperatures. Specifically, at 4℃, the surface of the specific hematology identification material has a larger contact angle, which is beneficial for cell detachment; at 37℃, its surface has a smaller contact angle, which is beneficial for cell adsorption. Therefore, the surface of the specific hematology identification material of this invention has temperature-responsive characteristics and is a temperature-responsive polymer material. Example: Fibrinogen adhesion assay for specific blood cell recognition materials This example uses ELISA to measure and analyze the adhesion of fibrinogen to the surfaces of the control and experimental groups at different temperatures. The experimental results are shown in Figure 7. The specific hematology identification material of this invention exhibits a higher fibrinogen adhesion at 37°C than at 4°C, thus demonstrating that the surface of the specific hematology identification material of this invention has a temperature-responsive technical feature for the adsorption of biomolecules. Example: Biocompatibility test of the specific blood cell identification material of the present invention This example uses mouse fibroblast cell line (L929) as the experimental subject. The experimental results show that the average relative survival rate of cells in the control group is 75%, while the average relative survival rate of cells in the experimental group is increased to 82%. Based on this, the specific blood cell identification material of the present invention improves the relative survival rate of cells, indicating better biocompatibility. Example: Adsorption / desorption experiment of specific blood cells or immune cells The blood samples used in this example include, but are not limited to, whole blood (WB) or white blood cell concentrate (WBC) samples. Specifically, the white blood cell concentrate sample can be separated from the whole blood sample after a specific centrifugation procedure. Blood is primarily composed of blood cells and plasma. Blood cells make up about 45% of the blood, including red blood cells, white blood cells, and platelets, with a ratio of approximately 600:1:45. Plasma makes up about 55% of the blood, consisting of 90% water and 10% chemical components. Under normal circumstances, the number of white blood cells in a healthy adult is 4 × 10⁻⁶. 9 Up to 11×10 9 / per liter of blood. White blood cells can be further subdivided by their percentage: 54-62% neutrophils, 1-6% eosinophils, <1% basophils, 2-10% monocytes, and 25-33% lymphocytes. Lymphocytes, often functioning as immune recognition cells, can be classified into three main groups based on their biological function and the expression of cell surface antigens: T lymphocytes, B lymphocytes, and natural killer (NK) cells. NK cells account for approximately 5-10% of human lymphocytes. This example further demonstrates the effectiveness of the aforementioned specific blood cell recognition material in recovering a very small number of specific functional immune cells, such as B lymphocytes or natural killer cells, from blood samples or thick white blood cell saturates. The general procedure of this experiment includes, but is not limited to, the following: (1) At 37°C, 1 ml of blood sample is placed in contact with 10 mm diameter test material (water gel placed in a 24-well plate) for 2 hours to adsorb specific blood cells or immune cells; (2) Remove the unadsorbed blood sample; (3) Rinse the surface of the adsorbed test material 3 times with buffer (DPBS, 1 ml); (4) Maintain at 4°C for 4 hours to desorb cells and collect the cell desorption solution; (5) Perform cell staining using glutaraldehyde and DAPI; (6) Store the above test material in buffer (DPBS) and control at 4°C; (7) Perform subsequent qualitative and quantitative analysis of the adsorption and desorption of specific blood cells or immune cells using a conjugate laser scanning microscope (CLSM) or flow cytometer. In the leukocyte adsorption-desorption experiment, according to paragraph
[0061] The relevant steps were performed. Whole blood samples were used, and the test materials included a control group and an experimental group [biotin-labeled anti-CD56 antibody streptavidin complex-anchored acrylic hydrogel, (acrylate-N-isopropylacrylamide) copolymer hydrogel, and N-isopropylacrylamide hydrogel]; the control group was sulfobetaine (SBMA). The experimental results are shown in Table 2, and the p-values of the data in Table 2 are less than 0.05. Table 2 1. Desorption rate (%) = [(White blood cell adhesion amount at 37℃ - White blood cell desorption amount at 4℃) / White blood cell adhesion amount at 37℃] × 100% According to the experimental data in Table 2, the amount of white blood cells adhering to the surface at 37℃ in the experimental group was significantly higher than that in the control group. This indicates that the acrylate hydrogel (AAc*), (acrylate-N-isopropylacrylamide) copolymer hydrogel or copolymer anchored by the biotin-labeled anti-CD56 antibody (anti-CD56) streptavidin complex has a better white blood cell capture effect, which can improve the ability to extract white blood cells from blood samples. This also indicates an improved specificity for capturing white blood cells from blood samples. On the other hand, the white blood cell desorption rate data also show that the (acrylate-N-isopropylacrylamide) copolymer hydrogel or copolymer anchored by the biotin-labeled anti-CD56 antibody (anti-CD56) streptavidin complex can release the white blood cells originally adhering to the surface more completely at low temperatures than the control group. Among them, when the test material is A-15NI*, the white blood cell desorption rate reaches 90.2%. Accordingly, the biotin-labeled antibody streptavidin complex-anchored acrylate hydrogel (AAc*), (acrylate-N-isopropylacrylamide) copolymer hydrogel or copolymer disclosed in this invention has better leukocyte capture specificity and recovery effect when applied in the field of separating and purifying leukocytes from blood samples. In the B cell adsorption-desorption experiment, according to paragraph
[0061] The relevant steps were performed. The blood sample used was a thickened leukocyte solution, and the test materials used included a control group and an experimental group [biotin-labeled anti-CD19 antibody (anti-CD19)-streptavidin complex-anchored acrylic hydrogel, (acrylate-N-isopropylacrylamide) copolymer hydrogel, N-isopropylacrylamide hydrogel]. Specifically, the B cell desorption procedure in this example included recovering the desorbed B cells with DPBS and recovering the desorbed B cells with a 1 mM or 10 mM biotin solution. The experimental results are shown in Table 3, and the p-values of the data in Table 3 are less than 0.05. Table 3 2. B cell detachment rate (%) = [Amount of B cells detached from the surface at 4℃ / Amount of B cells attached to the surface at 37℃] × 100% According to the experimental data in Table 3, the amount of B cells attached to the surface at 37°C in the experimental group was significantly higher than that in the control group. This indicates that the acrylic hydrogel (AAc*), (acrylate-N-isopropylacrylamide) copolymer network hydrogel or copolymer anchored with biotin-labeled anti-CD19 antibody (anti-CD19)-streptavidin complex has a better B cell capture effect, which can improve the ability to extract B cells from blood samples. This also indicates an improved specificity for capturing B cells from blood samples. On the other hand, the use of different desorption procedure buffers has a significant impact on the desorption rate of B cells. When detached B cells were recovered with DPBS at 4°C, the detachment rate of B cells in the experimental group (A-15NI*) reached 51%, higher than the 34% in the control group. When detached B cells were recovered with 1mM or 10mM biotin solutions at 4°C, the detachment rates of B cells in the experimental group (A-15NI*) reached 74% and 76% respectively, significantly higher than the 34% in the control group. Therefore, the biotin-labeled anti-CD19 antibody-streptavidin complex-anchored acrylate hydrogel (AAc*), (acrylate-N-isopropylacrylamide) copolymer hydrogel, or copolymer disclosed in this invention exhibits better B cell capture specificity and recovery efficiency when applied in the field of separating and purifying B cells from blood samples. In the natural killer (NK) cell adsorption-desorption experiment, according to paragraph
[0061] The relevant steps were performed as described. The blood sample used was a thick leukocyte solution, and the test materials used included a control group and an experimental group [biotin-labeled anti-CD56 antibody-streptavidin complex-anchored (acrylate-N-isopropylacrylamide) copolymer hydrogel]. Specifically, the natural killer (NK) cell desorption procedure of this example included recovering desorbed natural killer (NK) cells with DPBS buffer and recovering desorbed natural killer (NK) cells with 1 mM and 10 mM biotin solutions. The experimental results are shown in Tables 4 and 5, with p-values less than 0.05 for the data in Tables 4 and 5. Table 4 3. NK cell capture rate (%) = [NK cell adhesion amount on surface at 37℃ / Theoretical NK cell adhesion amount on surface at 37℃] × 100% According to the experimental data in Table 4, the amount of NK cells attached to the surface at 37℃ in the experimental group was significantly higher than that in the control group. When the test materials were A-10NI*, A-15NI*, and A-20NI*, the NK cell capture rate increased significantly to over 70%, which was significantly better than that in the control group. This indicates that the acrylic hydrogel (AAc*), (acrylic acid-N-isopropylacrylamide) copolymer hydrogel or copolymer anchored with biotin-labeled anti-CD56 antibody (anti-CD56)-streptavidin complex has a better NK cell capture effect and can improve the ability to extract NK cells from blood samples. This also indicates an improved specificity for capturing NK cells from blood samples. Table 5 4. NK cell detachment rate (%) = [Amount of NK cells detached from the surface at 4℃ / Amount of NK cells attached to the surface at 37℃] × 100% According to the experimental data in Table 5, the NK cell detachment rate of the experimental groups was greater than 70%. When the test material was A-15NI*, the NK cell detachment rate was as high as 93%, indicating that the effect of detachment and recovery of NK cells was better than that of the control group. Combining the experimental data in Tables 4 and 5, it can be seen that the acrylate hydrogel (AAc*), (acrylate-N-isopropylacrylamide) copolymer hydrogel or copolymer anchored with biotin-labeled anti-CD56 antibody (anti-CD56)-streptavidin complex disclosed in this invention has better NK cell capture specificity and recovery effect when applied to the separation and purification of NK cells from blood samples. Example: Analysis of cell viability after adsorption and detachment The testing method of this example is as follows: The test samples of this invention were loaded into 24-well plates, washed three times with DPBS, and 1 ml of concentrated white blood cell solution was added to each well. The plates were then placed in a 37°C oven for 2 hours for white blood cell adhesion. After adhesion, 1 ml of the sample was transferred to a 2 ml centrifuge tube, and the hydrogel was transferred to a new 24-well plate. The plate was then washed three times with DPBS to remove any white blood cells or other impurities not adhered to the hydrogel surface. Conversely, for desorption, the DPBS-washed hydrogel was transferred to a new 24-well plate, and 1 ml of 10 mM excess biotin solution was added to each well. The plate was then placed in a 4°C refrigerator for 4 hours to perform the white blood cell desorption experiment. Finally, the amount of white blood cells adsorbed / desorbed and the viability were analyzed using the white blood cell-specific antibody CD45 and the 7-aminoactinomycin DViability Dye (7-AAD) dye 7AAD, respectively. The experimental results are shown in Table 6, and the p-value of the data in Table 6 is less than 0.05. Experimental results confirmed that the survival rate of leukocytes desorbed from the test material (experimental group) of the present invention at 4°C was significantly higher than that of the control group. Therefore, it is demonstrated that the specific blood cell identification material of the present invention has better cell compatibility and can improve the purity and recovery rate of leukocyte recovery solution. Table 6 Example: Preparation steps of natural killer cell separation membrane A non-covalent affinity reaction was carried out between a streptavidin-coupled (acrylate-butyl methacrylate) copolymer and a biotin-labeled anti-CD56 antibody. The experimental results are shown in Figure 9. Based on this, the biotin-labeled anti-CD56 antibody exhibited the best adhesion rate, i.e., the best affinity effect, to the streptavidin-coupled (acrylate-butyl methacrylate) copolymer of the present invention at pH 4.5 (designations A1-4.5, A2-4.5, A3-4.5, A4-4.5). Designations A1-A4 and A1-E-A4-E represent film coatings composed of streptavidin-coupled (acrylate-butyl methacrylate) copolymers and film coatings composed of antibody-containing (acrylate-butyl methacrylate) copolymers synthesized in different molar ratios, respectively. The natural killer cell separation membrane was constructed by coating a streptavidin-coupled copolymer and an anti-CD56 antibody-modified copolymer onto a porous polypropylene meltblown fiber membrane using an immersion coating method. The coating density of the membrane was between 0.15 and 0.28 mg / cm³. 2 The specific data is shown in Figure 10. Surface elemental analysis of the coating was performed using XPS, and the results are shown in Table 7. Table 7 Example: Characteristic Analysis of Natural Killer Cell Separation Membranes Referring to Figure 11, after the coating process, the natural killer separation membrane of the present invention does not affect the porosity of the original porous polypropylene meltblown fiber membrane. Referring to Figure 12, the natural killer separation membrane of the present invention with anti-CD56 antibody modified coatings (A1-E to A4-E) has a higher hydration capacity than the coatings without antibody modification (A1 to A4), indicating that it has better cell affinity. Example: The technological benefits of natural killer cell separation membranes The test samples used in this example included polypropylene films [blank group (Virgin)], polypropylene films with an antibody-modified coating (A1), polypropylene films with an antibody-modified coating (A1-E), polypropylene films with SBMA (sulfobetaine) as the coating, and polyethylene cell culture dishes (TCPS). The SBMA coating and the polyethylene cell culture dishes (TCPS) served as the control group, while the polypropylene films with an antibody-modified coating (A1) served as the experimental control group. The testing method followed the aforementioned steps, and the experimental results are shown in Tables 8, 9, and 10. Table 8 Table 9 Table 10 According to the data in Tables 8 to 10, the polypropylene film with the antibody-modified coating (A1-E) exhibited the highest natural killer cell adhesion at 37°C. In contrast, the adhesion levels of natural killer cells in the control and general groups were relatively low, indicating a lack of specificity in capturing natural killer cells. Subsequently, natural killer cell desorption experiments were conducted at 4°C using DPBS and biotin, respectively. The results confirmed that the polypropylene film with the antibody-modified coating (A1-E) achieved a desorption rate of over 97.5%, enabling nearly 100% recovery of natural killer cells from blood samples, demonstrating a breakthrough in current natural killer cell isolation technology.
Claims
1. A specific blood cell identification material, characterized in that, Its composition includes a protein, a temperature-responsive polymer, and an antibody. The protein is chemically anchored to the temperature-responsive polymer, and the antibody is non-covalently bound to the protein, thereby constituting the specific blood cell recognition material.
2. The specific blood cell identification material as described in claim 1, characterized in that, This protein contains streptavidin or avidin.
3. The specific blood cell identification material as described in claim 1, characterized in that, The temperature-responsive polymer includes acrylic hydrogel, polyacrylic acid, (acrylic acid-N-alkylacrylamide) copolymer, (acrylic acid-N-alkylacrylamide) copolymer network hydrogel, (acrylic acid-alkyl methacrylate) copolymer, (acrylic acid-alkyl methacrylate) copolymer network hydrogel, or any combination thereof.
4. The specific blood cell identification material as described in claim 3, characterized in that, The molar ratio of acrylic acid to N-alkylacrylamide in the acrylic acid-N-alkylacrylamide copolymer or the acrylic acid-N-alkylacrylamide copolymer network hydrogel is 1 to 20.
5. The specific blood cell identification material as described in claim 1, characterized in that, The antibody is selected from one of the following groups: CD3 antibody, CD16 antibody, CD19 antibody, CD45 antibody, CD56 antibody, anti-CD3 antibody, anti-CD16 antibody, anti-CD19 antibody, anti-CD45 antibody, anti-CD56 antibody, biotin-labeled anti-CD19 antibody, and biotin-labeled anti-CD56 antibody.
6. The specific blood cell identification material as described in claim 1, characterized in that, The chemical bond is selected from one or a combination of the following groups: amide bond, ester bond, acetal bond, ketal bond, ether bond, and disulfide bond.
7. The specific blood cell identification material as described in claim 1, characterized in that, Its Fourier transform infrared spectrum shows absorption peaks in the following wavenumber range: 1545-1579 cm⁻¹ -1 1627-1654cm -1 and 1710-1740cm -1 .
8. A method for separating and recovering B cells from a sample, characterized in that, It includes the following steps: A sample containing B cells is exposed to a specific hematology identification material at 30–40°C, thereby causing the B cells to adsorb onto the specific hematology identification material. The specific hematology identification material comprises streptavidin, a temperature-responsive polymer, and an antibody. The streptavidin is anchored to the temperature-responsive polymer via an amide bond. The antibody is a CD19 antibody, a CD45 antibody, an anti-CD19 antibody, an anti-CD45 antibody, a biotin-labeled anti-CD19 antibody, or a biotin-labeled anti-CD45 antibody. The antibody is non-covalently bound to the streptavidin, thereby constituting the specific hematology identification material. Perform a desorption procedure by incubating the specific hematology identification material at 0–10°C for at least 10 minutes or immersing it in a solution containing biotin, thereby desorbing B cells adsorbed on the specific hematology identification material. and The recovery procedure involves rinsing the specific hematology identification material with a solution at 0–10°C to recover the detached B cells, thereby achieving the goal of separating and recovering B cells from the sample.
9. The method for separating and recovering B cells from a sample as described in claim 8, characterized in that, The temperature-responsive polymer includes acrylic hydrogel, polyacrylic acid, (acrylic acid-N-alkylacrylamide) copolymer, (acrylic acid-N-alkylacrylamide) copolymer network hydrogel, (acrylic acid-alkyl methacrylate) copolymer, (acrylic acid-alkyl methacrylate) copolymer network hydrogel, or any combination thereof.
10. The method for separating and recovering B cells from a sample as described in claim 9, characterized in that, The molar ratio of acrylic acid to N-alkylacrylamide in the acrylic acid-N-alkylacrylamide copolymer or the acrylic acid-N-alkylacrylamide copolymer network hydrogel is 1 to 20.
11. The method for separating and recovering B cells from a sample as described in claim 8, characterized in that, The Fourier transform infrared spectrum of this specific hematology identification material exhibits absorption peaks in the following wavenumber range: 1545-1579 cm⁻¹ -1 1627-1654cm -1 and 1710-1740cm -1 .
12. A method for separating and recovering NK cells from a sample, characterized in that, It includes the following steps: A sample containing NK cells is exposed to a specific hematology identification material at 30–40°C, thereby causing the NK cells to adsorb onto the specific hematology identification material. The specific hematology identification material comprises streptavidin, a temperature-responsive polymer, and an antibody. The streptavidin is anchored to the temperature-responsive polymer via an amide bond. The antibody is a CD16 antibody, a CD56 antibody, an anti-CD16 antibody, an anti-CD56 antibody, a biotin-labeled anti-CD16 antibody, or a biotin-labeled anti-CD56 antibody. The antibody is non-covalently bound to the streptavidin, thereby constituting the specific hematology identification material. Perform the desorption procedure by incubating the specific hematocyte recognition material at 0–10°C for at least 10 minutes or immersing it in a solution containing biotin, thereby detaching NK cells adsorbed on the specific hematocyte recognition material; and The recovery procedure involves rinsing the specific hematology identification material with a solution at 0–10°C to recover the detached NK cells, thereby achieving the goal of separating and recovering NK cells from the sample.
13. The method for self-sample separation and recovery of NK cells according to claim 12, characterized in that, The temperature-responsive polymer includes acrylic hydrogel, polyacrylic acid, (acrylic acid-N-alkylacrylamide) copolymer, (acrylic acid-N-alkylacrylamide) copolymer network hydrogel, (acrylic acid-alkyl methacrylate) copolymer, (acrylic acid-alkyl methacrylate) copolymer network hydrogel, or any combination thereof.
14. The method for separating and recovering NK cells from a sample as described in claim 13, characterized in that, The molar ratio of acrylic acid to N-alkylacrylamide in the acrylic acid-N-alkylacrylamide copolymer or the acrylic acid-N-alkylacrylamide copolymer network hydrogel is 1 to 20.
15. The method for self-sample separation and recovery of NK cells as described in claim 12, characterized in that, The Fourier transform infrared spectrum of this specific hematology identification material exhibits absorption peaks in the following wavenumber range: 1545-1579 cm⁻¹ -1 1627-1654cm -1 and 1710-1740cm -1 .