A T cell sorting magnetic microsphere and its preparation method and application

Abstract

The present invention relates to a T cell sorting magnetic microsphere and its preparation method and application, specifically a magnetic microsphere for T cell sorting, wherein the magnetic microsphere comprises magnetic nanoparticles and specific antibodies, wherein the specific antibodies are selected from CD8 antibodies and / or CD4 antibodies; wherein the magnetic nanoparticles are surface-modified with carboxyl groups; the CD8 antibody is selected from one or more CD8 monoclonal antibodies with clone numbers QA18A37, HIT8a, S21010A, SK1, and RPA-T8; and the CD4 antibody is selected from one or more CD4 monoclonal antibodies with clone numbers SK3, HIT4b, HIT4a, RPA-T4, and OKT4. The magnetic microspheres provided by the present invention have excellent T cell sorting effect.

Description

Technical Field

[0001] The present invention relates to the technical field of magnetic microspheres, and in particular to a T cell sorting magnetic microsphere, a preparation method and an application thereof. Background Art

[0002] T cells, or T lymphocytes, are a key cell type in the immune system, primarily responsible for cell-mediated immune responses. They play a crucial role in identifying and eliminating pathogens and tumor cells, as well as in immune regulation. T cells can be further divided into various subpopulations based on the molecules on their surface, such as helper T cells (Th cells), cytotoxic T cells (CTLs), and regulatory T cells (Tregs). Each subpopulation has unique functions and biological characteristics.

[0003] T cell sorting technology involves isolating distinct T cell subsets from a mixed cell population using specific methods. This technology has broad applications in immunology research, cell therapy, and vaccine development. Precise T cell sorting allows researchers to delve deeper into the functions of specific T cell subsets, potentially providing new strategies for disease diagnosis and treatment.

[0004] Currently, T cell sorting primarily relies on flow cytometry and magnetic bead sorting techniques. Flow cytometry uses fluorescently labeled antibodies to identify specific T cell surface markers, followed by rapid analysis and sorting using a flow cytometer. Magnetic bead sorting, on the other hand, uses magnetic beads labeled with specific antibodies to bind to target T cells. Key strategies include CD4 / CD8 sorting or CD3 sorting, using a magnetic field to separate cells. Each of these methods has advantages, but also limitations.

[0005] Although magnetic bead separation technology is favored for its ease of use and high separation efficiency, it still faces several challenges in practical application. Antibody selection is crucial, as the affinity and specificity of antibodies directly impact T cell separation efficiency and purity. Improper antibody selection can lead to nonspecific binding, reduce separation accuracy, and even affect cell viability and function.

[0006] Given the above issues, it is particularly necessary to develop new magnetic beads for T cell sorting and their sorting methods, especially to select antibodies on the magnetic beads that are suitable for T cell sorting. Such magnetic beads should have high affinity and high specificity to ensure efficient and accurate T cell sorting. Summary of the Invention

[0007] In view of the above-mentioned defects of the prior art, the present invention aims to provide a magnetic microsphere with high purity and high T cell recovery rate and a preparation method thereof.

[0008] Solutions for solving problems

[0009] The present invention provides a magnetic microsphere for T cell sorting, wherein the magnetic microsphere comprises magnetic nanoparticles and specific antibodies, wherein the specific antibodies are selected from CD8 antibodies and / or CD4 antibodies;

[0010] Wherein, the magnetic nanoparticles are magnetic nanoparticles with carboxyl groups modified on the surface;

[0011] The CD8 antibody is selected from one or more CD8 monoclonal antibodies with clone numbers of QA18A37, HIT8a, S21010A, SK1 and RPA-T8;

[0012] The CD4 antibody is selected from one or more CD4 monoclonal antibodies with clone numbers of SK3, HIT4b, HIT4a, RPA-T4 and OKT4.

[0013] Preferably, the CD8 antibody is selected from the CD8 monoclonal antibody with clone number SK1.

[0014] Preferably, the CD4 antibody is selected from the CD4 monoclonal antibody with clone number HIT4a.

[0015] Preferably, the mass ratio of the magnetic nanoparticles to the specific antibody is 1:0.1-1, preferably 1:0.1-0.5, and more preferably 1:0.3.

[0016] Preferably, the particle size of the magnetic microspheres is 40-60 nm.

[0017] The present invention provides a method for preparing the magnetic microspheres described in any one of the above items, the method comprising the following steps:

[0018] (1) Activating magnetic nanoparticles using an activation reagent;

[0019] (2) coupling the activated magnetic nanoparticles obtained in step (1) with specific antibodies;

[0020] (3) blocking the conjugate obtained in step (2), thereby obtaining the magnetic microspheres;

[0021] The mass ratio of the activated magnetic nanoparticles to the specific antibody is 1:0.1-1, preferably 1:0.1-0.5, and more preferably 1:0.3.

[0022] Preferably, the components and concentrations of the activation reagent are 0.1-0.5 g / L EDC, 0.1-0.5 g / L NHS, 3-5 g / L MES, and 20-35 g / L NaCl, respectively;

[0023] The coupling is performed using a coupling buffer, wherein the coupling buffer contains components and their concentrations are 5-10 g / L NaCl, 0.1-1 g / L KCl, 2-5 g / L Na2HPO4·12H2O, and 0.1-1 g / L KH2PO4, respectively;

[0024] The blocking is performed using a blocking buffer, wherein the components and concentrations of the blocking buffer are 5-10 g / L Tris, 5-15 g / L BSA, 0.1-2 mL / L PC300, and 1-5 mL / L ethanolamine, respectively.

[0025] The present invention provides use of any of the magnetic microspheres described above or the magnetic microspheres obtained by the preparation method described above in preparing a T cell sorting product.

[0026] The present invention provides a method for sorting T cells, comprising:

[0027] (1) contacting the magnetic microspheres described in any one of the above items or the magnetic microspheres obtained by the above preparation method with cells to be sorted and incubating them;

[0028] (2) placing the mixture after incubation in step (1) in a separation column, adding a magnetic field, and removing cells that are not bound to the magnetic microspheres;

[0029] (3) Remove the magnetic field and collect the target cells.

[0030] Preferably, every 10 to 30 μL of the magnetic microspheres and every 10 7 The cells to be sorted are mixed.

[0031] Preferably, collecting the target cells comprises removing the cells bound to the magnetic beads from the separation column using an air plug.

[0032] Preferably, the cells to be sorted are PBMCs.

[0033] Effects of the Invention

[0034] The present invention selects magnetic nanoparticles with carboxylic acid-rich surfaces and specific antibodies (such as CD4 or CD8 antibodies) through chemical covalent coupling to firmly bind the two, thereby obtaining the magnetic microspheres of the present invention. When used in T cell sorting, they have the advantages of high recovery rate and high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the percentage of CD8+ in human PBMCs before sorting;

[0036] Figure 2Schematic diagram of the sorting results of CD8 sorting magnetic beads (CD8 antibody clone number is QA18A37) prepared in Example 1;

[0037] Figure 3 This is a schematic diagram of the sorting results of the CD8 sorting magnetic beads (CD8 antibody clone number is HIT8a) prepared in Example 2;

[0038] Figure 4 Schematic diagram of the sorting results of CD8 sorting magnetic beads (CD8 antibody clone number is S21010A) prepared in Example 3;

[0039] Figure 5 This is a schematic diagram of the sorting results of the CD8 sorting magnetic beads (CD8 antibody clone number is SK1) prepared in Example 4;

[0040] Figure 6 This is a schematic diagram of the sorting results of CD8 sorting magnetic beads (CD8 antibody clone number is RPA-T8) prepared in Example 5;

[0041] Figure 7 This is a schematic diagram of the percentage of CD4+ in human PBMCs before sorting;

[0042] Figure 8 This is a schematic diagram of the sorting results of the CD4 sorting magnetic beads (CD4 antibody clone number is SK3) prepared in Example 6;

[0043] Figure 9 This is a schematic diagram of the sorting results of the CD4 sorting magnetic beads (CD4 antibody clone number is HIT4b) prepared in Example 7;

[0044] Figure 10 This is a schematic diagram of the sorting results of the CD4 sorting magnetic beads (CD4 antibody clone number is HIT4a) prepared in Example 8;

[0045] Figure 11 This is a schematic diagram of the sorting results of CD4 sorting magnetic beads (CD4 antibody clone number is RPA-T4) prepared in Example 9;

[0046] Figure 12 This is a schematic diagram of the sorting results of CD4 sorting magnetic beads (CD4 antibody clone number is OKT4) prepared in Example 10;

[0047] Figure 13 This is a schematic diagram of the percentage of CD3+ in human PBMCs in three samples before sorting;

[0048] Figure 14 This is a schematic diagram of the sorting results of using CD8 sorting magnetic beads in combination with CD4 sorting magnetic beads in sample 1;

[0049] Figure 15 This is a schematic diagram of the sorting results of using CD8 sorting magnetic beads in combination with CD4 sorting magnetic beads in sample 2;

[0050] Figure 16 Schematic diagram of the sorting results when CD8 sorting magnetic beads and CD4 sorting magnetic beads were used together in sample 3. DETAILED DESCRIPTION

[0051] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0052] The present invention provides a magnetic microsphere for T cell sorting, wherein the magnetic microsphere comprises magnetic nanoparticles and specific antibodies, wherein the specific antibodies are selected from CD8 antibodies and / or CD4 antibodies;

[0053] Wherein, the magnetic nanoparticles are magnetic nanoparticles with carboxyl groups modified on the surface;

[0054] The CD8 antibody is selected from one or more CD8 monoclonal antibodies with clone numbers of QA18A37, HIT8a, S21010A, SK1 and RPA-T8;

[0055] The CD4 antibody is selected from one or more CD4 monoclonal antibodies with clone numbers of SK3, HIT4b, HIT4a, RPA-T4 and OKT4.

[0056] In the present application, the magnetic nanoparticles are conventional commercial products, for example, they can be obtained from Suzhou Weidu Biotechnology Co., Ltd., Miltenyi Biotec Co., Ltd., etc.

[0057] In the present application, the CD8 antibody and CD4 antibody are both conventional commercially available products. For example, CD8 antibody can be commercially available from Biolegend, Thermo Fisher Scientific, etc., and CD4 antibody can be commercially available from Beijing Kuangbo Bio, Thermo Fisher Scientific, Biolegend, etc.

[0058] In certain embodiments, the CD8 antibody is selected from the CD8 monoclonal antibody with clone number SK1.

[0059] In the present application, the CD8 monoclonal antibody with clone number QA18A37 was purchased from Biolegend with catalog number 303802; the CD8 monoclonal antibody with clone number HIT8a was purchased from Biolegend with catalog number 300902; the CD8 monoclonal antibody with clone number S21010A was purchased from Biolegend with catalog number 388702; the CD8 monoclonal antibody with clone number SK1 was purchased from Biolegend with catalog number 344702; and the CD8 monoclonal antibody with clone number RPA-T8 was purchased from Biolegend with catalog number 301002.

[0060] In certain embodiments, the CD4 antibody is selected from the CD4 monoclonal antibody with clone number HIT4a.

[0061] In this application, the CD4 monoclonal antibody with clone number SK3 was purchased from Beijing Kuangbo Biotechnology with the catalog number A1141; the CD4 monoclonal antibody with clone number HIT4b was purchased from Beijing Kuangbo Biotechnology with the catalog number A7661; the CD4 monoclonal antibody with clone number HIT4a was purchased from Beijing Kuangbo Biotechnology with the catalog number A6081; the CD4 monoclonal antibody with clone number RPA-T4 was purchased from Beijing Kuangbo Biotechnology with the catalog number A7601; and the CD4 monoclonal antibody with clone number OKT4 was purchased from Biolegend with the catalog number 317402.

[0062] In certain embodiments, the mass ratio of the magnetic nanoparticles to the specific antibody is 1:0.1-1, for example, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, etc.

[0063] In certain embodiments, the mass ratio of the magnetic nanoparticles to the specific antibody is 1:0.1-0.5.

[0064] In certain embodiments, the mass ratio of the magnetic nanoparticles to the specific antibody is 1:0.3.

[0065] In certain embodiments, the magnetic microspheres have a particle size of 40 to 60 nm, for example, 42 nm, 44 nm, 46 nm, 48 nm, 50 nm, 52 nm, 54 nm, 56 nm, 58 nm, and 60 nm. In this application, the nanomagnetic beads are small in size and require a strong magnetic field to be attracted. Therefore, a separation column capable of amplifying the magnetic field is required for use with the magnetic separator. Other strong magnetic field devices may also be used instead.

[0066] The present invention provides a method for preparing the magnetic microspheres described in any one of the above items, the method comprising the following steps:

[0067] (1) Activating magnetic nanoparticles using an activation reagent;

[0068] (2) coupling the activated magnetic nanoparticles obtained in step (1) with specific antibodies;

[0069] (3) blocking the conjugate obtained in step (2), thereby obtaining the magnetic microspheres;

[0070] The mass ratio of the activated magnetic nanoparticles to the specific antibody is 1:0.1 to 1, for example, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, etc.

[0071] In certain embodiments, the mass ratio of the magnetic nanoparticles to the specific antibody is 1:0.1-0.5.

[0072] In certain embodiments, the mass ratio of the magnetic nanoparticles to the specific antibody is 1:0.3.

[0073] In certain embodiments, the activation reagent contains components and their concentrations are 0.1-0.5 g / L EDC, 0.1-0.5 g / L NHS, 3-5 g / L MES, and 20-35 g / L NaCl, respectively.

[0074] In certain embodiments, the concentration of the EDC in the activation reagent is 0.1-0.4 g / L, for example, 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, etc.

[0075] In certain embodiments, the concentration of NHS in the activation reagent is 0.1-0.4 g / L, for example, 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, etc.

[0076] In certain embodiments, the concentration of MES in the activation reagent is 3.5-5 g / L, for example, 3.8 g / L, 4 g / L, 4.2 g / L, 4.4 g / L, 4.6 g / L, 4.8 g / L, 5 g / L, etc.

[0077] In certain embodiments, the concentration of NaCl in the activation reagent is 26-30 g / L, for example, 26 g / L, 26.5 g / L, 27 g / L, 27.5 g / L, 28 g / L, 28.5 g / L, 29 g / L, 29.5 g / L, 30 g / L, etc.

[0078] In certain embodiments, the coupling is performed using a coupling buffer, wherein the components and concentrations of the coupling buffer are 5-10 g / L NaCl, 0.1-1 g / L KCl, 2-5 g / L Na2HPO4·12H2O, and 0.1-1 g / L KH2PO4, respectively.

[0079] In certain embodiments, the concentration of NaCl in the coupling buffer is 6-10 g / L, for example, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, etc.

[0080] In certain embodiments, the concentration of KCl in the coupling buffer is 0.1-0.5 g / L, for example, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, etc.

[0081] In certain embodiments, the concentration of Na2HPO4·12H2O in the coupling buffer is 2-4 g / L, for example, 2.2 g / L, 2.4 g / L, 2.6 g / L, 2.8 g / L, 3 g / L, 3.2 g / L, 3.4 g / L, 3.6 g / L, 3.8 g / L, etc.

[0082] In certain embodiments, the concentration of KH2PO4 in the coupling buffer is 0.1-0.5 g / L, for example, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, etc.

[0083] In certain embodiments, the blocking is performed using a blocking buffer comprising components and their concentrations of 5-10 g / L Tris, 5-15 g / L BSA, 0.1-2 mL / L PC300, and 1-5 mL / L ethanolamine, respectively.

[0084] In certain embodiments, the concentration of Tris in the blocking buffer is 5-8 g / L, for example, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, etc.

[0085] In certain embodiments, the concentration of BSA in the blocking buffer is 8-12 g / L, for example, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, 10.5 g / L, 11 g / L, 11.5 g / L, 12 g / L, etc.

[0086] In certain embodiments, the concentration of PC300 in the blocking buffer is 0.5-2 mL / L, for example, 1 mL / L, 1.5 mL / L, 2 mL / L, etc.

[0087] In certain embodiments, the concentration of ethanolamine in the blocking buffer is 2-5 mL / L, for example, 2 mL / L, 2.5 mL / L, 3 mL / L, 3.5 mL / L, 4 mL / L, 4.5 mL / L, etc.

[0088] The present invention provides use of any of the magnetic microspheres described above or the magnetic microspheres obtained by the preparation method described above in preparing a T cell sorting product.

[0089] The present invention provides a method for sorting T cells, comprising:

[0090] (1) contacting the magnetic microspheres described in any one of the above items or the magnetic microspheres obtained by the above preparation method with cells to be sorted and incubating them;

[0091] (2) placing the mixture after incubation in step (1) in a separation column, adding a magnetic field, and removing cells that are not bound to the magnetic microspheres;

[0092] (3) Remove the magnetic field and collect the target cells.

[0093] In certain embodiments, the magnetic microspheres are mixed with each 10 to 30 μL (e.g., 12 μL, 14 μL, 16 μL, 18 μL, 20 μL, 22 μL, 24 μL, 26 μL, 28 μL, 30 μL) of each 10 μL. 7 The cells to be sorted are mixed.

[0094] In certain embodiments, the density of the magnetic microspheres is 1×10 11 ~30×10 11 indivual.

[0095] In certain embodiments, the density of the magnetic microspheres coupled to the CD8 antibody is 1×10 11 ~10×10 11 indivual.

[0096] In certain embodiments, the density of the magnetic microspheres coupled to CD8 antibodies is 6.8×10 11 indivual.

[0097] In certain embodiments, the density of the magnetic microspheres coupled to CD4 antibodies is 10×10 11 ~30×10 11 indivual.

[0098] In certain embodiments, the density of the magnetic microspheres coupled to CD4 antibodies is 26×10 11 indivual.

[0099] In certain embodiments, collecting the target cells comprises removing the magnetic bead-bound cells from the separation column using an air plug.

[0100] In certain embodiments, the cells to be sorted are PBMCs.

[0101] As used herein, the term "sorting" generally refers to the process of separating target cells (e.g., T cells) from a mixture (e.g., blood, lymphocytes, PBMCs, etc.). This separation does not necessarily require 100% separation; for example, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of the target cells in the mixture may be separated. The separated target cells also do not necessarily require 100% purity; for example, they may be substantially pure, for example, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or more of the cell purity may be achieved.

[0102] As used herein, the term "CD4" generally refers to the CD4 receptor, also known as the "Cluster of Differentiation 4 receptors." In the biomedical field, CD4 is a glycoprotein molecule found on the surface of immune cells (e.g., helper T cells, monocytes, macrophages, and dendritic cells). It was discovered in the late 1970s and was known as Ieu-3 and T4 before 1984. The CD4 receptor is typically one of the surface markers of helper T cells and is an important receptor for helper T cells to function. When antigen-presenting cells (e.g., macrophages, dendritic cells, and B cells themselves) degrade foreign pathogens, the antigens are bound to the major histocompatibility complex (MHC) and presented to helper T cells (i.e., they bind to the CD4 receptor on the helper T cell surface).

[0103] As used herein, the term "CD8" generally refers to the CD8 (cluster of differentiation 8) receptor, a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR). Like the TCR, CD8 typically binds to major histocompatibility complex (MHC) molecules but is specific for MHC class I proteins. There are two CD8 isoforms, α and β, encoded by separate genes. In humans, both genes are located at position 2p12 on chromosome 2. CD8 is primarily expressed on the surface of cytotoxic T cells but can also be found on natural killer cells, cortical thymocytes, and dendritic cells. The CD8 molecule can serve as a marker for cytotoxic T cell populations.

[0104] As used herein, the term "PBMC" generally refers to peripheral blood mononuclear cells (PBMCs), which are any peripheral blood cells with a round or nearly round nucleus. These cells may include lymphocytes (T cells, B cells, NK cells) and monocytes. Red blood cells and platelets do not fall into this category due to their lack of nuclei; granulocytes (including neutrophils, basophils, and eosinophils) do not fall into this category due to their multilobed nuclei. In certain embodiments, PBMCs can be extracted from whole blood using Ficoll (a hydrophilic polysaccharide that separates blood layers) and gradient centrifugation. For example, blood can be separated into a top layer of plasma, followed by a layer of PBMCs and a bottom layer of polymorphonuclear cells (such as neutrophils and eosinophils) and red blood cells.

[0105] The following are the reagents used in the examples of the present invention:

[0106] The activation buffer formulation was: MES 4.265 g / L, NaCl 28 g / L, pH 4.0;

[0107] The coupling buffer formula is: NaCl 8 g / L, KCl 0.2 g / L, Na2HPO4·12H2O 3.63 g / L, and KH2PO4 0.24 g / L, pH 7.0;

[0108] The blocking solution formula is: Tris 6.6 g / L, ethanolamine 3 mL / L, BSA 10 g / L, PC300 1 mL / L, pH 7.4;

[0109] The formula of magnetic bead storage solution is: Tris 6.6g / L, BSA 0.5g / L, Tween 20 0.5mL / L, PC300 1mL / L, BND10 0.5mI / L, pH 7.4.

[0110] The method of the present invention is described below by means of specific examples. It should be understood that these examples are used to illustrate the basic principles, main features and advantages of the present invention, and the present invention is not limited to the scope of the following examples. The implementation conditions adopted in the examples can be further adjusted according to specific requirements. The implementation conditions not specified are generally the conditions in routine experiments. Example 1

[0111] The preparation method of antibody-coupled magnetic beads is as follows:

[0112] (1) Place the separation column in the magnetic separator, rinse the separation column with 3 mL of activation buffer, take 1 mg of nanomagnetic beads (Suzhou Weidu Biotechnology Co., Ltd., product number CSM0050CA) into the separation column, and wash the nanomagnetic beads three times with 3 mL of activation buffer. After the liquid has flowed out, remove the separation column from the magnetic separator and place it in a 10 mL EP tube. Add 4 mL of activation buffer to the separation column. At this time, the nanomagnetic beads flow into the EP tube with the buffer.

[0113] (2) Add 0.5 mg of NHS to the washed nanomagnetic beads, mix well, and then add 0.5 mg of EDC. After mixing, place in a rotary incubator and activate at room temperature for 30 minutes.

[0114] (3) Place the separation column in a magnetic separator and rinse the separation column with 3 mL of coupling buffer. Inject the activated nanomagnetic beads into the separation column and wash the nanomagnetic beads three times with 3 mL of coupling buffer. After the liquid has flowed out, remove the separation column from the magnetic separator and place it in a 10 mL EP tube. Add 4 mL of coupling buffer to the separation column. The activated nanomagnetic beads are collected in the EP tube. Add 0.3 mg of CD8 monoclonal antibody (clone number QA18A37) to the magnetic beads, mix well, and place in a rotary incubator for coupling at room temperature for 2 hours.

[0115] (4) Place the separation column in a magnetic separator and rinse the separation column with 3 mL of blocking solution. Pour the coupled nanomagnetic beads into the separation column. After the liquid has flowed out, remove the separation column from the magnetic separator and place it in a 10 mL EP tube. Add 4 mL of blocking solution to the separation column. The coupled nanomagnetic beads are collected in the EP tube. After mixing, place the tube in a rotary incubator and block for 30 minutes at room temperature.

[0116] (5) Place the separation column in a magnetic separator and rinse the separation column with 3 mL of magnetic bead preservation solution. Inject the sealed nanomagnetic beads into the separation column and wash the nanomagnetic beads three times with 3 mL of magnetic bead preservation solution. After the liquid has flowed out, remove the separation column from the magnetic separator and place it in a 10 mL EP tube. Add 2 mL of magnetic bead preservation solution to the separation column to obtain magnetic beads coupled to CD8 (clone number QA18A37) antibodies. The antibody magnetic beads should be stored at 2-8°C.

[0117] The preparation methods of the magnetic beads of Examples 2 to 5 are basically the same as those of Example 1, except that the clone numbers of the CD8 antibodies are different from those in Example 1. The specific antibodies coupled to the magnetic beads prepared in Examples 2 to 5 are shown in Table 1.

[0118] The preparation method of the magnetic beads coupled with CD4 antibody is basically the same as that of Example 1, except that the CD8 antibody in Example 1 is replaced by CD4 antibody. The specific antibodies coupled with the magnetic beads prepared in Examples 6 to 10 are shown in Table 2.

[0119] Test Case

[0120] The magnetic beads for sorting T cells were prepared using the above Examples 1 to 10, and the operation steps are as follows:

[0121] (1) Resuspend human peripheral blood mononuclear cells (PBMC) in MACS buffer (purchased from Miltenyi Biotec), take samples and count them, and take 1×10 7 Transfer 100 cells to a 1.5 mL EP tube and centrifuge at 1500 rpm for 5 min.

[0122] (2) Discard the supernatant and resuspend the cell pellet in 100 μL MACS buffer, then add 20 μL CD4 sorting magnetic beads (magnetic bead concentration is 2.6*10 12 / mL) or CD8 sorting magnetic beads (magnetic bead concentration is 6.8*10 11 After mixing evenly, place the mixture in a refrigerator at 2-8°C for 15 min.

[0123] (3) Place the LS column on the MACS separator and rinse twice with 1 mL of MACS buffer.

[0124] (4) Add 1 mL of MACS buffer to the incubated sample, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0125] (5) Add 1 mL of MACS buffer to resuspend the sample and then add the sample to the separation column.

[0126] (6) Collect the effluent in a 15 mL centrifuge tube, wash the column three times with 2 mL of MACS buffer, and collect the effluent (unlabeled cells).

[0127] (7) After all the MACS buffer has flowed out, remove the separation column from the MACS separator and place it in another new 15 mL centrifuge tube. Add 2 mL of MACS buffer to the separation column and push the piston of the LS separation column to directly flush out the magnetically labeled cells.

[0128] (8) Place the 15 mL centrifuge tube containing the collected solution into a horizontal centrifuge and centrifuge at 1500 rpm for 5 minutes.

[0129] (9) After centrifugation, discard the supernatant, take 1 mL of 1× DPBS solution to resuspend the cells, count them and perform flow cytometry.

[0130] The test results are as follows (the same samples were used to test the sorting effects of the magnetic beads prepared in Examples 1-10):

[0131] The results of CD8 magnetic beads separation of CD8+ T cells prepared in Examples 1 to 5 are shown in Table 1 and Figures 2 to 6 Before sorting, CD8+ T cells accounted for 17.92% of PBMCs. The flow cytometry results are shown in Figure 1 .

[0132] Table 1

[0133]

[0134] From Table 1 and Figures 2 to 6 As can be seen, the separation performance of different CD8 antibody clones after coupling to magnetic beads varied significantly. Among the five clones, SK1 showed the best separation performance after coupling to magnetic beads, with a purity of 92.11% and a CD8+ recovery rate of 95.33%. The separation purity and CD8+ recovery rates of the remaining clones were poor.

[0135] The results of CD4 magnetic beads separation of CD4+ T cells prepared in Examples 6 to 10 are shown in Table 2 and Figures 8-12 Before sorting, CD4+ T cells accounted for 24.25% of PBMCs. The flow cytometry results are shown in Figure 7 The CD4 antibodies with clone numbers SK3, HIT4b, HIT4a, and RPA-T4 in the CD4 magnetic beads were purchased from Beijing Kuangbo Biotechnology, and the CD4 antibody with clone number 317402 was purchased from Biolegend.

[0136] Table 2

[0137]

[0138] From Table 2 and Figures 8-12 Similar to the CD8 antibody, the separation performance of different CD4 antibody clones after coupling to magnetic beads also showed significant differences. Among the five clones, HIT4a showed the best separation performance after coupling to magnetic beads, with a purity of 93.75% and a CD4+ recovery rate of 90.55%. RPA-T4 performed second best after coupling to magnetic beads, while the separation performance of the remaining three clones was poor.

[0139] Furthermore, the present invention combines the most effective CD8 separation magnetic beads (CD8 antibody clone number SK1) with the most effective CD4 separation magnetic beads (CD4 antibody clone number HIT4a) to isolate T cells. The specific experimental steps are as follows:

[0140] (1) Three samples (donor, different from the samples in the above verification experiment) were selected, and human peripheral blood mononuclear cells (PBMC) were resuspended in MACS buffer, sampled and counted, and 1×10 7Transfer 100 cells to a 1.5 mL EP tube and centrifuge at 1500 rpm for 5 min.

[0141] (2) Discard the supernatant and resuspend the cell pellet in 100 μL MACS buffer. Then add 20 μL CD4 and 20 μL CD8 sorting magnetic beads, mix well and place in a refrigerator at 2-8°C for 15 minutes.

[0142] (3) Place the LS column on the MACS separator and rinse twice with 1 mL of MACS buffer.

[0143] (4) Add 1 mL of MACS buffer to the incubated sample, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0144] (5) Add 1 mL of MACS Buffer to resuspend the sample, and then add the sample to the separation column.

[0145] (6) Collect the effluent in a 15 mL centrifuge tube, wash the column three times with 2 mL of MACS buffer, and collect the effluent (unlabeled cells).

[0146] (7) After all the MACS buffer has flowed out, remove the separation column from the MACS separator and place it in another new 15 mL centrifuge tube. Add 2 mL of MACS buffer to the separation column and push the piston of the LS separation column to directly flush out the magnetically labeled cells.

[0147] (8) Place the 15 mL centrifuge tube containing the collected solution into a horizontal centrifuge and centrifuge at 1500 rpm for 5 minutes.

[0148] (9) After centrifugation, discard the supernatant, take 1 mL of 1× DPBS solution to resuspend the cells, count them and perform flow cytometry.

[0149] Before sorting, the proportion of CD3+ T cells in PBMC in the three samples was shown in Figure 2. Figure 13 The results of using CD8 sorting magnetic beads in combination with CD4 sorting magnetic beads to sort CD3+ T cells are shown in Table 3 and Figures 14 to 16 .

[0150] Table 3

[0151]

[0152] From Table 3 and Figures 14 to 16It can be seen that in different samples, the combined use of CD8 sorting magnetic beads and CD4 sorting magnetic beads showed good sorting effects, with sorting purities greater than 90% and target cell recovery rates greater than 90% (currently there are no industry standard requirements for the purity and recovery rate of cell sorting, and the sorting data achieved by similar products of Miltenyi Biotec are mainly used as a reference, generally with a purity greater than 90% and a recovery rate greater than 70%). Therefore, the combined use of the CD8 sorting magnetic beads and CD4 sorting magnetic beads provided by the present invention can more efficiently and accurately sort CD3+ T cells.

[0153] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A magnetic microsphere for T cell sorting, characterized by: The magnetic microspheres include magnetic nanoparticles and specific antibodies, and the specific antibodies are selected from CD8 antibodies and / or CD4 antibodies; The magnetic nanoparticles are produced by Suzhou Weidu Biotechnology Co., Ltd. with a product number of CSM0050CA, and the surface of the magnetic nanoparticles is modified with carboxyl groups. The CD8 antibody is selected from the CD8 monoclonal antibody with clone number SK1; The CD4 antibody is selected from the CD4 monoclonal antibody with clone number HIT4a; the mass ratio of the magnetic nanoparticles to the specific antibody is 1:0.1~1.

2. The magnetic microspheres according to claim 1, characterized in that: The mass ratio of the magnetic nanoparticles to the specific antibody is 1:0.

3.

3. The magnetic microspheres according to claim 1, characterized in that: The particle size of the magnetic microspheres is 40-60 nm.

4. The method for preparing magnetic microspheres according to any one of claims 1 to 3, wherein: The method comprises the following steps: (1) Activating magnetic nanoparticles using an activation reagent; (2) coupling the activated magnetic nanoparticles obtained in step (1) with specific antibodies; (3) blocking the conjugate obtained in step (2), thereby obtaining the magnetic microspheres; Wherein, the mass ratio of the activated magnetic nanoparticles to the specific antibody is 1:0.1~1.

5. The preparation method according to claim 4, characterized in that: The activation reagent contains components and their concentrations are 0.1-0.5 g / L EDC, 0.1-0.5 g / L NHS, 3-5 g / L MES and 20-35 g / L NaCl respectively; The coupling is performed using a coupling buffer, wherein the coupling buffer contains components and their concentrations are 5-10 g / L NaCl, 0.1-1 g / L KCl, 2-5 g / L Na2HPO4·12H2O, and 0.1-1 g / L KH2PO4, respectively; The blocking is performed using a blocking buffer, wherein the components and concentrations of the blocking buffer are 5-10 g / L Tris, 5-15 g / L BSA, 0.1-2 mL / L PC300, and 1-5 mL / L ethanolamine, respectively.

6. Use of the magnetic microspheres according to any one of claims 1 to 3 or the magnetic microspheres obtained by the preparation method according to claim 4 or 5 in preparing a T cell sorting product.

7. A method for sorting T cells, characterized in that: The method comprises: (1) contacting the magnetic microspheres according to any one of claims 1 to 3 or the magnetic microspheres obtained by the preparation method according to claim 4 or 5 with cells to be sorted and incubating; (2) placing the mixture after incubation in step (1) in a separation column, adding a magnetic field, and removing cells that are not bound to the magnetic microspheres; (3) Remove the magnetic field and collect the target cells.

8. The method according to claim 7, wherein: Each 10~30 μL of the magnetic microspheres and each 10 7 Mixing the cells to be sorted; and / or, collecting the target cells comprises removing the cells bound to the magnetic beads from the separation column using an air plug; And / or, the cells to be sorted are PBMCs.

Images