Preparation method of CD4 + T cell sorting magnetic beads

Abstract

The invention provides a preparation method of CD4 + T cell sorting magnetic beads. The preparation method comprises the following specific steps: preparing a ferroferric oxide magnetic core; the magnetic core of the ferroferric oxide is coated with glucan; activating hydroxyl on the surface of the glucan by using 1, 4-butanediol diglycidyl ether; preparing an immunomagnetic bead with a CD4 antibody; carrying out CD4 + T cell sorting; preparing a flow cytometry sample; and verifying the hydroxyl activation effect of the 1, 4-butanediol diglycidyl ether. Due to the fact that the coating on the surface of the magnetic nucleus can cause magnetism reduction, a magnetic field stronger than that of a common magnet can be provided when the Miltenyi cell manual sorting set is adopted to sort cells, it is guaranteed that immunomagnetic beads can be fully magnetically attracted in the cell sorting process, water serves as a solvent, the process from magnetic nucleus preparation to activation is simple and continuous, and the efficiency is high. The complex degree of the activation step and the toxicity of the activated organic matter are greatly reduced, finally, reactants are removed through water dialysis, large-scale production can be achieved, and after the CD4 antibody is coupled, the CD4 + T cells in the human peripheral blood mononuclear cells are extremely high in purity.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and in particular to a method for preparing CD4+T cell sorting magnetic beads. Background Art

[0002] CD4 cells refer to T lymphocytes with CD4+T molecules on their surface. They are an important immune cell in the human immune system. Since the HIV virus attacks CD4+T cells, their detection results play an important role in judging the effectiveness of HIV treatment and the patient's immune function.

[0003] Immunomagnetic bead sorting is a cell separation method widely used in clinical immunotherapy. Immunomagnetic beads are functionalized magnetic nanoparticles with immune function characteristics formed by coupling functional groups (such as amino, carboxyl, hydroxyl, epoxy, cyano, streptavidin and other functional groups) on the surface of magnetic nanoparticles with biological antibodies. The principle of immunomagnetic bead sorting is to use specific antibodies coupled with functional groups on the surface of magnetic beads to specifically bind to antigens on the cell surface and to sort cells that match the antibodies. After adding immunomagnetic beads to a suspension containing target cells, an external magnetic field is applied. The target cells connected to the antibodies on the surface of the magnetic beads are adsorbed and retained in the magnetic field, while cells without such surface antigens cannot bind to the specific antibodies connected to the magnetic beads and thus do not stay in the magnetic field, achieving the purpose of separating the target cells. The equipment required for immunomagnetic bead cell sorting is simple, the requirements for technical personnel are not high, the cells obtained by sorting are highly sensitive, pure, and have good cell activity and recovery rate, which has little impact on downstream applications and has a wide application prospect.

[0004] Methods for preparing the magnetic cores of immunomagnetic beads include physical and chemical methods. Mechanical ball milling is the predominant physical method. Ball milling involves grinding micron or submicron particles for a long period of time and then dispersing them in an oil-based medium. This method produces particles with a relatively wide size distribution and is also time-consuming. Chemical methods include coprecipitation, pyrolysis, microemulsion, sol-gel, sonochemical, laser decomposition, and electrochemical deposition. Coprecipitation has the advantages of a simple reaction principle, relatively inexpensive equipment and raw materials, and suitability for mass production, making it the most widely studied method. The composition and shape of the magnetic core of immunomagnetic beads determine its suitability for specific applications. For example, composite particles containing superparamagnetic iron oxide are commonly used for small-scale affinity separations, particularly for cell separation, followed by flow cytometry analysis or fluorescence-activated cell sorting. The temperature of the coprecipitation process significantly affects the superparamagnetism of the magnetic cores of immunomagnetic beads. High temperatures can enhance the superparamagnetism of the cores and mitigate the effects of the reduced superparamagnetism caused by the formation of a magnetic dead layer during the coating step, which isolates the cores from magnetic attraction.

[0005] 1. After the magnetic core of the immunomagnetic beads is prepared, the surface of the magnetic core needs to be coated to reduce the toxicity of the magnetic core to cells, and activated functional groups are added to the surface of the coating layer to achieve antibody coupling and cell sorting. Since small-particle magnetic cores have the least effect on cell activity, polymers are usually used to coat the surface of the prepared small-particle magnetic cores. The surface coating materials of the magnetic core usually include polymers with hydroxyl groups such as dextran, chitosan, and agarose. Although hydroxyl groups do not react spontaneously to functional groups on biological molecules, they can be activated through many known reaction mechanisms and thus used for covalent coupling of antibodies.

[0006] 2. Traditional hydroxyl activation methods involve dissolving substances such as N,N'-disuccinimidyl carbonate, imidazole carbamate, and cyanogen bromide in organic solvents such as dioxane, acetonitrile, and tetrahydrofuran. Activation is then performed by adding magnetic cores bearing hydroxyl groups. The activated reactants are then washed with organic solvents to remove the activated reactants, and finally washed with ultrapure water for antibody conjugation. These activating substances and organic solvents are highly toxic and can have serious health risks for experimenters. Furthermore, the experimental steps from activation to washing are complex. Therefore, a method for preparing magnetic beads for CD4+ T cell sorting is proposed. Summary of the Invention

[0007] (1) Technical problems solved

[0008] In view of the shortcomings of the existing technology, the present invention provides a method for preparing CD4+ T cell sorting magnetic beads, which solves the technical problems of complex washing steps and reducing the toxicity of activated organic matter.

[0009] (2) Technical solution

[0010] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0011] A method for preparing CD4+ T cell sorting magnetic beads, the specific steps are as follows:

[0012] (1) Preparation of ferroferric oxide magnetic core;

[0013] (2) ferroferric oxide magnetic core coated with dextran;

[0014] (3) 1,4-Butanediol diglycidyl ether activates the surface hydroxyl groups of dextran;

[0015] (4) Preparation of immunomagnetic beads with CD4 antibodies;

[0016] (5) CD4+ T cell sorting;

[0017] (6) Flow cytometry sample preparation;

[0018] (7) Verification of the effect of activating hydroxyl groups by 1,4-butanediol diglycidyl ether.

[0019] Preferably, the preparation of the ferroferric oxide magnetic core (the magnetic core is prepared according to the coprecipitation chemical formula Fe2++2Fe3++8OH-=Fe3O4+4H2O)

[0020] 1. Dissolve 2.16g of ferric chloride hexahydrate and 1.11g of ferrous sulfate heptahydrate in 30mL of ultrapure water to ensure that Fe 2+ / Fe 3+ The molar ratio is 0.5, and then 0.6 mol / L NaOH solution and 0.1 mol / L ethylenediaminetetraacetic acid (EDTA) solution are prepared.

[0021] 2. Raise the water temperature of the ultrasonic cleaning machine to 80 degrees, add 100ml of 0.6mol / LNaOH solution, 10ml of 0.1mol / LEDTA solution and 20ml of anhydrous ethanol to a 250mL three-necked flask. After the temperature of the mixed solution rises to 80 degrees, turn on the ultrasonic cleaning machine, turn on the electric stirrer, and stir at a rate of 300 revolutions per minute.

[0022] 3. Put Fe 2+ / Fe 3+ The mixed iron salt solution was added to the constant pressure dropping funnel and gradually dripped into the three-necked flask at a rate of 1 drop per second.

[0023] 4. After the mixed iron salt solution is dripped, add 2.5g of citric acid monohydrate, keep the water temperature in the ultrasonic cleaning machine constant at 80 degrees, the electric stirrer speed remains unchanged at 300 revolutions per minute, and continue ultrasonication for 5 hours.

[0024] Preferably, the ferroferric oxide magnetic core is coated with dextran

[0025] 1. Add the magnetic core to a 500mL three-necked flask, add 160mL of ultrapure water, set the water temperature of the ultrasonic cleaning machine to 25 degrees, turn on the ultrasonic cleaning machine, turn on the electric stirrer, and stir at a rate of 300 revolutions per minute for 10 minutes.

[0026] After 10 minutes, weigh 0.5 g of dextran (Bome Bio, molecular weight 40,000), dissolve it in 5 ml of ultrapure water, and drop it into the three-necked flask. Keep the electric stirrer speed at 300 rpm and continue ultrasonication at 25 degrees for 5 hours.

[0027] (3) Activation of the hydroxyl groups on the surface of dextran by 1,4-butanediol diglycidyl ether

[0028] 1. Add the magnetic core to a 1000mL three-necked flask, add 320mL of ultrapure water, set the water temperature of the ultrasonic cleaning machine to 40 degrees, turn on the ultrasonic cleaning machine, turn on the electric stirrer, and stir at a rate of 300 revolutions per minute for 10 minutes.

[0029] After 10 minutes, weigh 80 mg of sodium borohydride, dissolve it in 4 ml of 0.6 mol / L NaOH solution, and add it dropwise to the three-necked flask. Then add 10 ml of 1,4-butanediol diglycidyl ether dropwise. Maintain the electric stirrer at 300 rpm and continue ultrasonication at 40°C for 5 hours.

[0030] 3. After the ultrasound, add the prepared epoxy-functionalized magnetic beads into a dialysis bag with a molecular weight of 300,000 and dialyze with ultrapure water for 12 hours, changing the water every 2 hours to filter out the remaining unreacted substances.

[0031] Preferably, preparation of immunomagnetic beads with CD4 antibodies

[0032] 1. Place the Miltenyi LS Column on the Miltenyi Manual Cell Sorting Kit, add 3 ml of the prepared epoxy-functionalized magnetic beads, and wash three times with 3 ml of ultrapure water.

[0033] 2. Remove the LS Column from the manual cell sorting kit, add 3 ml of ultrapure water to the LS Column, transfer the washed magnetic beads to a centrifuge tube using a push rod, and add 0.4 mg of CD4 antibody.

[0034] 3. Place the centrifuge tube on a rotary mixer and rotate and mix at 37 degrees for 1 hour. After 1 hour, place the centrifuge tube in a refrigerator and store it at 4 degrees.

[0035] Preferably, CD4+ T cell sorting

[0036] 1. Resuspend human peripheral blood mononuclear cells (hPBMCs) in a certain volume of cell buffer (1000 ml 1× PBS containing 0.5% bovine serum albumin and 2 mmol EDTA) to 1.25×10 8 Take 80 μl of the resuspended cell solution, add 20 μl of immunomagnetic beads, and incubate at 4°C for 20 minutes.

[0037] 2. Add 1-2 mL of cell buffer, centrifuge at 400 g for 10 minutes, discard the supernatant, and then add 1-2 mL of cell buffer to resuspend the cells. Repeat the centrifugation step once, and finally add 500 μL of cell buffer to resuspend the cells.

[0038] 3. Add the resuspended cells to the Miltenyi MS Column. After the cell suspension is dripped, add 500 μL of cell buffer to wash the cell sorting column MS Column. Repeat this step twice.

[0039] 4. Add 1 mL of cell buffer, push out the magnetically labeled cells using the plunger, and store in a refrigerator at 4°C.

[0040] Preferably, flow cytometry sample preparation

[0041] 1. Place the magnetically labeled cells in a centrifuge and centrifuge at 500g for 6 minutes. Discard the supernatant of the cell buffer. Resuspend and wash the cells in 1ml of 1× PBS, centrifuge again at 500g for 6 minutes, and discard the supernatant.

[0042] 2. Resuspend the cells in 1 ml of 1× PBS, centrifuge at 500 g for 6 minutes, and resuspend the cells in 100 μL of cell buffer.

[0043] 3. Add 5 μL of Tonbo TM FITCAnti-HumanCD4 was used for fluorescent labeling and incubated in the dark at 4°C for 15-20 minutes.

[0044] 4. After incubation, add 1 ml of 1× PBS to each tube, mix thoroughly, and centrifuge at 500 g for 6 minutes. Discard the supernatant. Resuspend the cells in 1 ml of 1× PBS and repeat the centrifugation step once. Finally, resuspend the cells in 400 μL of 1× PBS and analyze them on a flow cytometer.

[0045] 5. Repeat (5) and (6) to perform a blank experiment without adding immunomagnetic beads. Add the resuspended cells to the Miltenyi MS Column. Since cells without immunomagnetic beads will not be magnetically attracted to the MS Column, collect the cell fluid that flows out to prepare flow cytometry samples.

[0046] Preferably, the effect of 1,4-butanediol diglycidyl ether activation of hydroxyl groups is verified

[0047] 1. Add 3 ml of epoxy-functionalized immunomagnetic beads to a Miltenyi LS Column and wash three times with 3 ml of ultrapure water. After three washes, add 3 ml of ultrapure water and push out the washed epoxy-IONPs with a pusher and transfer to a 10 ml centrifuge tube.

[0048] 2. Add 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, and 1 mg of CD4 antibody, place the centrifuge tube on a vertical mixer, and then place it in a constant temperature shaker and rotate and mix at 37°C for 1 hour.

[0049] 3. Add 300 μl of CD4 antibody-coupled epoxy-functionalized immunomagnetic beads to the Miltenyi LS Column. After the supernatant flows out, add 300 μl of ultrapure water to wash once. After washing, add 300 μl of ultrapure water and push out and collect with a push rod.

[0050] 4. Prepare BCA working solution according to the instructions in the Micro BCA Protein Assay Kit (Shanghai Sangon Biotech Co., Ltd.). Add 100 μl of BCA working solution and 100 μl of a mixture of CD4 antibody-conjugated epoxy-functionalized immunomagnetic beads to the ELISA plate. Place the plate in a thermostatic shaker and incubate at 37°C for 30 minutes. After incubation, cool the plate to room temperature and place it in an ELISA analyzer. Set the primary wavelength to 570 nm and the secondary wavelength to 595 nm. Record the absorbance reading.

[0051] 5. Repeat the steps in (7) for the magnetic core coated with dextran but not activated with 1,4-butanediol diglycidyl ether, record the absorbance readings, and finally detect the CD4 antibody conjugation content using the fitted curve.

[0052] (3) Beneficial effects

[0053] 1. Under high temperature conditions of 80 degrees, by adding a specific ratio of ethylenediaminetetraacetic acid (EDTA) and citric acid monohydrate, a negatively charged chelate is formed on the surface of ferroferric oxide to prevent the magnetic core from settling.

[0054] 2. Select dextran with a molecular weight of 40,000 to coat the magnetic core, so that the dextran magnetic beads have good stability and texture. The low molecular weight dextran has a low degree of side chain, which can prevent the cross-linking of dextran on the surface of the magnetic core from causing sedimentation, while providing sufficient hydroxyl functional groups for activation.

[0055] 3. 1,4-Butanediol diglycidyl ether was selected as the activating organic compound and water as the activating medium, which reduced the toxicity of traditional activating organic compounds and organic solvents on the experimenters and simplified the activation reaction steps.

[0056] 4. From the preparation of magnetic cores to the coating of magnetic cores with dextran and then to the activation with 1,4-butanediol diglycidyl ether, the reaction volume is doubled each time, which can make the magnetic beads more dispersed and facilitate the reaction.

[0057] Because the coating on the surface of the magnetic core can reduce magnetism, using the Miltenyi Biotec manual cell sorting kit for cell sorting can provide a stronger magnetic field than ordinary magnets, ensuring that the immunomagnetic beads can be fully magnetically attracted during cell sorting. This invention uses water as the solvent and 1,4-butanediol diglycidyl ether as the activator. The process from magnetic core preparation to activation is simple and continuous, greatly reducing the complexity of the activation step and the toxicity of the activated organic matter. Finally, the reactants are dialyzed with water. This series of simple steps is conducive to large-scale production. After coupling with CD4 antibodies, the CD4+ T cells in human peripheral blood mononuclear cells (hPBMCs) are shown to be extremely pure. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings.

[0059] Figure 1 The particle size microstructure of the magnetic core in the preparation method of CD4+T cell sorting magnetic beads of the present invention Figure 1 ;

[0060] Figure 2 The particle size microstructure of the magnetic core in the preparation method of CD4+T cell sorting magnetic beads of the present invention Figure 2 ;

[0061] Figure 3 This is a hydrodynamic particle size diagram of immunomagnetic beads prepared using Bomei Bio's 40,000 molecular weight dextran in a method for preparing CD4+ T cell sorting magnetic beads of the present invention;

[0062] Figure 4 This is a diagram showing the results of sorting human peripheral blood mononuclear cells using immunomagnetic beads without CD4 antibody coupling in a method for preparing CD4+ T cell sorting magnetic beads of the present invention;

[0063] Figure 5 This is a diagram showing the results of sorting human peripheral blood mononuclear cells by adding CD4 antibody-coupled immunomagnetic beads in a method for preparing CD4+ T cell sorting magnetic beads of the present invention;

[0064] Figure 6 This is a hydrodynamic particle size diagram of the immunomagnetic beads prepared after the rotation speed is increased to 600 revolutions per minute in a method for preparing CD4+ T cell sorting magnetic beads of the present invention;

[0065] Figure 7 This is a hydrodynamic particle size diagram of immunomagnetic beads prepared using Yisheng Bio's 40,000 molecular weight dextran in a method for preparing CD4+ T cell sorting magnetic beads of the present invention;

[0066] Figure 8 This is a hydrodynamic particle size diagram of immunomagnetic beads prepared using 40,000 molecular weight dextran from a Chinese medicine chemical reagent in a method for preparing CD4+ T cell sorting magnetic beads of the present invention;

[0067] Figure 9 This is a hydrodynamic particle size diagram of immunomagnetic beads prepared using 20,000 molecular weight dextran from a Chinese medicine chemical reagent in a method for preparing CD4+ T cell sorting magnetic beads of the present invention;

[0068] Figure 10This is a diagram showing the results of human peripheral blood mononuclear cell sorting using immunomagnetic beads prepared with 20,000 molecular weight dextran from a Chinese medicine chemical reagent and coupled with CD4 antibodies in a method for preparing CD4+ T cell sorting magnetic beads of the present invention;

[0069] Figure 11 This is a diagram showing the effects of dextran 40000 and 1,4-butanediol diglycidyl ether coating on the saturation magnetization value of magnetic cores in a method for preparing CD4+ T cell sorting magnetic beads of the present invention;

[0070] Figure 12 This is a curve diagram fitted according to the instructions of the micro BCA protein assay kit in the preparation method of CD4+ T cell sorting magnetic beads of the present invention;

[0071] Figure 13 This is a diagram showing the coupling results of different amounts of CD4 antibodies before and after activation of the magnetic beads with 1,4-butanediol diglycidyl ether in a method for preparing CD4+ T cell sorting magnetic beads of the present invention. DETAILED DESCRIPTION

[0072] The embodiment of the present application provides a method for preparing CD4+ T cell sorting magnetic beads, which solves the problems of complex washing steps and reducing the toxicity of activated organic matter in the prior art. Water is used as a solvent and 1,4-butanediol diglycidyl ether is used as an activator. The process from preparation of magnetic cores to activation is simple and continuous, which greatly reduces the complexity of the activation step and the toxicity of the activated organic matter. Finally, the reactants are dialyzed with water. This series of simple steps is conducive to large-scale production and coupled with CD4 antibodies.

[0073] Example 1

[0074] The technical solution in the embodiment of the present application is to solve the problem of complex washing steps and reduce the toxicity of activated organic matter. The overall idea is as follows:

[0075] In response to the problems existing in the prior art, the present invention provides a method for preparing CD4+ T cell sorting magnetic beads, the specific steps of which are as follows:

[0076] (1) Preparation of ferroferric oxide magnetic core;

[0077] (2) ferroferric oxide magnetic core coated with dextran;

[0078] (3) 1,4-Butanediol diglycidyl ether activates the surface hydroxyl groups of dextran;

[0079] (6) Preparation of immunomagnetic beads with CD4 antibodies;

[0080] (7) CD4+ T cell sorting;

[0081] (6) Flow cytometry sample preparation;

[0082] (7) Verification of the effect of activating hydroxyl groups by 1,4-butanediol diglycidyl ether.

[0083] (1) Preparation of ferroferric oxide magnetic core (prepared according to the coprecipitation chemical formula Fe2++2Fe3++8OH-=Fe3O4+4H2O)

[0084] 1. Dissolve 2.16g of ferric chloride hexahydrate and 1.11g of ferrous sulfate heptahydrate in 30mL of ultrapure water to ensure that Fe 2+ / Fe 3+ The molar ratio is 0.5, and then 0.6 mol / L NaOH solution and 0.1 mol / L ethylenediaminetetraacetic acid (EDTA) solution are prepared.

[0085] 2. Raise the water temperature of the ultrasonic cleaning machine to 80 degrees, add 100ml of 0.6mol / LNaOH solution, 10ml of 0.1mol / LEDTA solution and 20ml of anhydrous ethanol to a 250mL three-necked flask. After the temperature of the mixed solution rises to 80 degrees, turn on the ultrasonic cleaning machine, turn on the electric stirrer, and stir at a rate of 300 revolutions per minute.

[0086] 5. Put Fe 2+ / Fe 3+ The mixed iron salt solution was added to the constant pressure dropping funnel and gradually dripped into the three-necked flask at a rate of 1 drop per second.

[0087] 6. After the mixed iron salt solution is dripped, add 2.5g of citric acid monohydrate, keep the water temperature in the ultrasonic cleaning machine constant at 80 degrees, the electric stirrer speed remains unchanged at 300 revolutions per minute, and continue ultrasonication for 5 hours.

[0088] (2) Ferroferric oxide magnetic core coated with dextran

[0089] 1. Add the magnetic core to a 500mL three-necked flask, add 160mL of ultrapure water, set the water temperature of the ultrasonic cleaning machine to 25 degrees, turn on the ultrasonic cleaning machine, turn on the electric stirrer, and stir at a rate of 300 revolutions per minute for 10 minutes.

[0090] After 10 minutes, weigh 0.5 g of dextran (Bome Bio, molecular weight 40,000), dissolve it in 5 ml of ultrapure water, and drop it into the three-necked flask. Keep the electric stirrer speed at 300 rpm and continue ultrasonication at 25 degrees for 5 hours.

[0091] (3) Activation of the hydroxyl groups on the surface of dextran by 1,4-butanediol diglycidyl ether

[0092] 1. Add the magnetic core to a 1000mL three-necked flask, add 320mL of ultrapure water, set the water temperature of the ultrasonic cleaning machine to 40 degrees, turn on the ultrasonic cleaning machine, turn on the electric stirrer, and stir at a rate of 300 revolutions per minute for 10 minutes.

[0093] After 10 minutes, weigh 80 mg of sodium borohydride, dissolve it in 4 ml of 0.6 mol / L NaOH solution, and add it dropwise to the three-necked flask. Then add 10 ml of 1,4-butanediol diglycidyl ether dropwise. Maintain the electric stirrer at 300 rpm and continue ultrasonication at 40°C for 5 hours.

[0094] 3. After the ultrasound, add the prepared epoxy-functionalized magnetic beads into a dialysis bag with a molecular weight of 300,000 and dialyze with ultrapure water for 12 hours, changing the water every 2 hours to filter out the remaining unreacted substances.

[0095] (4) Preparation of immunomagnetic beads with CD4 antibodies

[0096] 1. Place the Miltenyi LS Column on the Miltenyi Manual Cell Sorting Kit, add 3 ml of the prepared epoxy-functionalized magnetic beads, and wash three times with 3 ml of ultrapure water.

[0097] 2. Remove the LS Column from the manual cell sorting kit, add 3 ml of ultrapure water to the LS Column, transfer the washed magnetic beads to a centrifuge tube using a push rod, and add 0.4 mg of CD4 antibody.

[0098] 3. Place the centrifuge tube on a rotary mixer and rotate and mix at 37 degrees for 1 hour. After 1 hour, place the centrifuge tube in a refrigerator and store it at 4 degrees.

[0099] (5) CD4+ T cell sorting

[0100] 1. Resuspend human peripheral blood mononuclear cells (hPBMCs) in a certain volume of cell buffer (1000 ml 1× PBS containing 0.5% bovine serum albumin and 2 mmol EDTA) to 1.25×10 8 Take 80 μl of the resuspended cell solution, add 20 μl of immunomagnetic beads, and incubate at 4°C for 20 minutes.

[0101] 2. Add 1-2 mL of cell buffer, centrifuge at 400 g for 10 minutes, discard the supernatant, and then add 1-2 mL of cell buffer to resuspend the cells. Repeat the centrifugation step once, and finally add 500 μL of cell buffer to resuspend the cells.

[0102] 3. Add the resuspended cells to the Miltenyi MS Column. After the cell suspension is dripped, add 500 μL of cell buffer to wash the cell sorting column MS Column. Repeat this step twice.

[0103] 4. Add 1 mL of cell buffer, push out the magnetically labeled cells using the plunger, and store in a refrigerator at 4°C.

[0104] (6) Flow cytometry sample preparation

[0105] 1. Place the magnetically labeled cells in a centrifuge and centrifuge at 500g for 6 minutes. Discard the supernatant of the cell buffer. Resuspend and wash the cells in 1ml of 1× PBS, centrifuge again at 500g for 6 minutes, and discard the supernatant.

[0106] 2. Resuspend the cells in 1 ml of 1× PBS, centrifuge at 500 g for 6 minutes, and resuspend the cells in 100 μL of cell buffer.

[0107] 3. Add 5 μL of Tonbo TM FITCAnti-HumanCD4 was used for fluorescent labeling and incubated in the dark at 4°C for 15-20 minutes.

[0108] 4. After incubation, add 1 ml of 1× PBS to each tube, mix thoroughly, and centrifuge at 500 g for 6 minutes. Discard the supernatant. Resuspend the cells in 1 ml of 1× PBS and repeat the centrifugation step once. Finally, resuspend the cells in 400 μL of 1× PBS and analyze them on a flow cytometer.

[0109] 5. Repeat (5) and (6) to perform a blank experiment without adding immunomagnetic beads. Add the resuspended cells to the Miltenyi MS Column. Since cells without immunomagnetic beads will not be magnetically attracted to the MS Column, collect the cell fluid that flows out to prepare flow cytometry samples.

[0110] (7) Verification of the effect of 1,4-butanediol diglycidyl ether on the activation of hydroxyl groups

[0111] 1. Add 3 ml of epoxy-functionalized immunomagnetic beads to a Miltenyi LS Column and wash three times with 3 ml of ultrapure water. After three washes, add 3 ml of ultrapure water and push out the washed epoxy-IONPs with a pusher and transfer to a 10 ml centrifuge tube.

[0112] 2. Add 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, and 1 mg of CD4 antibody, place the centrifuge tube on a vertical mixer, and then place it in a constant temperature shaker and rotate and mix at 37°C for 1 hour.

[0113] 3. Add 300 μl of CD4 antibody-coupled epoxy-functionalized immunomagnetic beads to the Miltenyi LS Column. After the supernatant flows out, add 300 μl of ultrapure water to wash once. After washing, add 300 μl of ultrapure water and push out and collect with a push rod.

[0114] 4. Prepare BCA working solution according to the instructions in the Micro BCA Protein Assay Kit (Shanghai Sangon Biotech Co., Ltd.). Add 100 μl of BCA working solution and 100 μl of a mixture of CD4 antibody-conjugated epoxy-functionalized immunomagnetic beads to the ELISA plate. Place the plate in a thermostatic shaker and incubate at 37°C for 30 minutes. After incubation, cool the plate to room temperature and place it in an ELISA analyzer. Set the primary wavelength to 570 nm and the secondary wavelength to 595 nm. Record the absorbance reading.

[0115] 5. Repeat the steps in (7) for the magnetic core coated with dextran but not activated with 1,4-butanediol diglycidyl ether, record the absorbance readings, and finally detect the CD4 antibody conjugation content using the fitted curve.

[0116] Example 2

[0117] Based on Example 1, in order to ensure that the device maintains a stable shape when folded or unfolded, the overall concept of this embodiment is as follows:

[0118] Figure 1-2 As shown, after the preparation step (1) of the ferroferric oxide magnetic core is completed, the synthesized magnetic core is characterized by transmission electron microscopy, and it can be observed that the particle size of the magnetic core is about 10 nm, which is conducive to the coupling of biomolecules.

[0119] Figure 3 As shown in the figure, after the activation of the surface hydroxyl groups of dextran by (3) 1,4-butanediol diglycidyl ether, the particle size of the prepared magnetic beads increased, the hydrodynamic particle size was 80.81 nm, and the polydispersity index (PDI) was 0.143, indicating that the particle size distribution of the magnetic beads was relatively uniform.

[0120] like Figure 4-5 As shown, after CD4 antibodies were conjugated to epoxy groups to prepare immunomagnetic beads, flow cytometric analysis revealed that even without the addition of CD4 antibodies, 58.6% of CD4+ T cells were present after MS column and flow cytometry sample preparation. After adding the immunomagnetic beads, the purity of CD4+ T cells detected by flow cytometry reached 95.7%, demonstrating that the prepared immunomagnetic beads can effectively isolate CD4+ T cells from human peripheral blood mononuclear cells with high purity.

[0121] like Figure 6As shown in the figure, the effects of changing experimental parameters on epoxy-functionalized immunomagnetic beads:

[0122] Change the speed

[0123] To verify the effect of increased shear force on the hydrodynamic particle size of immunomagnetic beads after increasing the rotational speed, the rotational speed of the electric stirrer in (1)(2)(3) was increased from 300 rpm to 600 rpm, while other parameters remained unchanged. At this time, the particle size distribution of the magnetic beads was 81.36 nm, and the polydispersity index (PDI) was 0.278. The results showed that increasing the rotational speed had little effect on the particle size, but increasing the shear force would reduce the uniformity of the particle size distribution. In addition, large-sized magnetic beads have poor biocompatibility, which would reduce the viability of the sorted cells.

[0124] like Figure 7 As shown, different brands of dextran

[0125] After replacing the 40,000 molecular weight dextran with dextran produced by Yisheng Bio and Sinopharm Chemical Reagent, while keeping other parameters unchanged, the magnetic bead particle size results are as follows:

[0126] After adding Yisheng Bio's 40,000 molecular weight dextran to coat the magnetic core and then activating it with 1,4-butanediol diglycidyl ether, the magnetic bead particle size distribution was 84.37 nm and the polydispersity index (PDI) was 0.177.

[0127] like Figure 8 As shown in the figure, after adding 40,000 molecular weight dextran from Sinopharm Chemical Reagent to coat the magnetic core and then activating it with 1,4-butanediol diglycidyl ether, the particle size distribution of the magnetic beads was 87.53 nm and the polydispersity index (PDI) was 0.16.

[0128] Although the particle size of the magnetic beads prepared using two different brands of 40,000 molecular weight dextran is still small, the magnetic beads prepared using 40,000 molecular weight dextran from Bomei Biotechnology have the smallest particle size and polydispersity coefficient. This may be related to the impurities contained in dextran prepared from different brands. In general, epoxy-functionalized magnetic beads prepared using Bomei Biotechnology's dextran are a good choice for downstream antibody coupling.

[0129] 3. Dextran molecular weight

[0130] After adding 20,000 molecular weight dextran from Sinopharm Chemical Reagent to coat the magnetic cores, keeping other parameters unchanged and then activating with 1,4-butanediol diglycidyl ether, the magnetic beads had a particle size distribution of 81.83 nm and a polydispersity index (PDI) of 0.156. After flow cytometric sorting, the purity of CD4+ T cells was 91.3%, indicating that the hydroxyl functional groups on the side chains of the 20,000 molecular weight dextran are less than those on the 40,000 molecular weight dextran, which will affect the purity of the final sorting. Figure 9 and 10 shown.

[0131]

[0132] Table 1

[0133] like Figure 11 As shown in the results of vibrating sample magnetometer characterization, the saturation magnetization values of the co-precipitation prepared magnetic core and the functionalized magnetic beads are 68.21 emu / g and 4.64 emu / g, respectively. Dextran 40000 and 1,4-butanediol diglycidyl ether polymer molecules successfully form a magnetic dead layer on the surface of the magnetic core, suppressing the surface magnetic moment. However, the superparamagnetic properties of the magnetic core itself are not altered.

[0134] like Figure 12 As shown, according to the curve fitted in the instructions of the micro BCA protein assay kit, the formula of BSA concentration and absorbance can be obtained by linear fitting: Y=0.0043x+0.00967, where Y is absorbance and x is BSA concentration (μg / mL).

[0135] like Figure 13 As shown in the figure, with the increase in the amount of CD4 antibody added, the CD4 protein coupling content of both unactivated and epoxy-activated magnetic beads showed an upward trend. The reason for the increase in CD4 protein coupling content in unactivated magnetic beads is that proteins themselves typically contain a hydrophobic core structure and a predominantly hydrophilic surface. Therefore, their interaction with the hydrophobic dextran surface inevitably involves significant conformational changes, resulting in passive adsorption. The reason for the increase in CD4 protein coupling content in epoxy-activated magnetic beads is that the epoxy groups covalently couple to the amino functional groups in the protein through a ring-opening reaction. This is a stronger chemical force than adsorption, and the reaction time is shorter than passive adsorption, resulting in a higher antibody coupling content.

[0136] Beneficial effects:

[0137] 1. Under high temperature conditions of 80 degrees, by adding a specific ratio of ethylenediaminetetraacetic acid (EDTA) and citric acid monohydrate, a negatively charged chelate is formed on the surface of ferroferric oxide to prevent the magnetic core from settling.

[0138] 2. Select dextran with a molecular weight of 40,000 to coat the magnetic core, so that the dextran magnetic beads have good stability and texture. The low molecular weight dextran has a low degree of side chain, which can prevent the cross-linking of dextran on the surface of the magnetic core from causing sedimentation, while providing sufficient hydroxyl functional groups for activation.

[0139] 3. Selecting 1,4-butanediol diglycidyl ether as the activated organic matter and water as the activation medium reduces the toxicity of traditional activated organic matter and organic solvents on the experimenters and simplifies the activation reaction steps.

[0140] 4. From the preparation of magnetic cores to the coating of magnetic cores with dextran and then to the activation with 1,4-butanediol diglycidyl ether, the reaction volume is doubled each time, which can make the magnetic beads more dispersed and facilitate the reaction.

[0141] Since the coating on the surface of the magnetic core will reduce the magnetism, the use of Miltenyi cell sorting kit to sort cells can provide a stronger magnetic field than ordinary magnets, ensuring that the immunomagnetic beads can be fully magnetically attracted during cell sorting.

[0142] Finally, it should be noted that the above embodiments are merely examples for the purpose of illustrating the present invention and are not intended to limit the embodiments. Those skilled in the art will readily appreciate that other variations or modifications based on the above description are possible. It is not necessary and impossible to provide an exhaustive list of all embodiments. However, obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing CD4+ T cell sorting magnetic beads, characterized in that: The specific steps are as follows: (1) Preparation of ferroferric oxide magnetic core; (2) ferroferric oxide magnetic core coated with dextran; (3) 1,4-Butanediol diglycidyl ether activates the surface hydroxyl groups of dextran; (4) Preparation of immunomagnetic beads with CD4 antibodies; (5) CD4+ T cell sorting; (6) Flow cytometry sample preparation; (7) Verification of the effect of activating hydroxyl groups by 1,4-butanediol diglycidyl ether.

2. The method for preparing CD4+ T cell sorting magnetic beads according to claim 1, characterized in that: (1) Preparation of ferroferric oxide magnetic core (according to the coprecipitation chemical formula Fe 2+ +2Fe 3+ +8OH-=Fe3O4+4H2O to prepare the magnetic core) a solution preparation Dissolve 2.16g of ferric chloride hexahydrate (FeCl3·6H2O) and 1.11g of ferrous sulfate heptahydrate (FeSO4·7H2O) in 30mL of ultrapure water (Fe 2+ / Fe 3+ Molar ratio = 0.5), prepare 0.6mol / L NaOH solution and 0.1mol / L EDTA solution; b. Reaction system construction In an 80°C ultrasonic cleaner, add 100 mL of 0.6 mol / L NaOH, 10 mL of 0.1 mol / L EDTA, and 20 mL of anhydrous ethanol to a 250 mL three-necked flask. After the temperature of the mixed solution rises to 80°C, turn on the ultrasonic cleaner and the electric stirrer and stir at 300 rpm. c Iron salt solution dropwise Fe 2+ / Fe 3+ The mixed iron salt solution was dripped into the three-necked flask at a rate of 1 drop / second through a constant pressure dropping funnel to gradually mix; Surface modification After the mixed iron salt solution was added, 2.5 g of citric acid monohydrate was added, and the mixture was kept under ultrasonication at 80° C. and stirred at 300 rpm for 5 hours.

3. The method for preparing CD4+ T cell sorting magnetic beads according to claim 1, characterized in that: The (2) ferroferric oxide magnetic core coated with dextran aMagnetic core dispersion Transfer the magnetic core to a 500 mL three-necked flask, add 160 mL of ultrapure water, set the water temperature of the ultrasonic cleaning machine to 25 degrees, turn on the ultrasonic cleaning machine, turn on the electric stirrer, and stir at a rate of 300 rpm for 10 minutes; bGlucan coating After 10 minutes, 0.5 g of dextran (molecular weight 40,000) was dissolved in 5 mL of ultrapure water and added dropwise to the three-necked flask. Ultrasonication was performed at 25° C. for 5 hours while the electric stirrer kept rotating at 300 rpm.

4. The method for preparing CD4+ T cell sorting magnetic beads according to claim 1, wherein: The (3) 1,4-butanediol diglycidyl ether activates the surface hydroxyl groups of dextran a Magnetic core redispersion The magnetic core was transferred to a 1000 mL three-necked flask, 320 mL of ultrapure water was added, and ultrasonication was performed at 40 °C for 10 min with stirring at 300 rpm. b Epoxy functionalization 80 mg of sodium borohydride dissolved in 4 mL of 0.6 mol / L NaOH and 10 mL of 1,4-butanediol diglycidyl ether were added dropwise, and ultrasonicated at 40 °C for 5 h with stirring at 300 rpm; Purification After the ultrasonication, the prepared epoxy-functionalized magnetic beads were added to a dialysis bag with a molecular weight of 300,000 and dialyzed with ultrapure water for 12 h, with the water changed every 2 h to filter out the remaining unreacted substances.

5. The method for preparing CD4+ T cell sorting magnetic beads according to claim 1, characterized in that: (4) Preparation of immunomagnetic beads with CD4 antibodies, a Magnetic bead washing Wash the epoxy magnetic beads three times with a Miltenyi LS Column and 3 mL of ultrapure water; bAntibody conjugation Transfer the magnetic beads to a centrifuge tube, add 0.4 mg of CD4 antibody, place the centrifuge tube on a rotary mixer, rotate and mix at 37°C for 1 hour, and store in a refrigerator at 4°C.

6. The method for preparing CD4+ T cell sorting magnetic beads according to claim 1, characterized in that: (5) CD4+ T cell sorting, a cell incubation hPBMCs were resuspended in cell buffer to a volume of 1.25 × 10 8 / mL, 80 μl of cell solution was incubated with 20 μL of immunomagnetic beads at 4°C for 20 minutes; b. Wash by centrifugation, add 1-2 mL of buffer, centrifuge at 400 g for 10 minutes, discard the supernatant, and then add 1-2 mL of cell buffer to resuspend the cells. Repeat the wash twice and finally resuspend in 500 μL of buffer; c Magnetic separation The cell suspension was passed through a Miltenyi MS Column, washed twice with 500 μL of buffer, and the magnetic cells were eluted with 1 mL of buffer and stored at 4°C. dBlank control Repeat step (5) without adding magnetic beads and collect the effluent cells for flow cytometry.

7. The method for preparing CD4+ T cell sorting magnetic beads according to claim 1, characterized in that: (6) flow cytometry sample preparation, a. Cell washing The magnetically labeled cells were placed in a centrifuge and centrifuged at 500 g for 6 minutes. The supernatant of the cell buffer was discarded, and the cells were washed twice with PBS and finally suspended in 100 μL of buffer. bFluorescent labeling Add 5 μL FITC Anti-Human CD4 and incubate at 4°C in the dark for 15-20 minutes; cSample processing Wash twice with PBS, resuspend in 400 μL PBS, and detect on the instrument.

8. The method for preparing CD4+ T cell sorting magnetic beads according to claim 1, characterized in that: Verification of the effect of activating hydroxyl groups by (7) 1,4-butanediol diglycidyl ether: a. Add 3 ml of epoxy-functionalized immunomagnetic beads to a Miltenyi LS Column and wash three times with 3 ml of ultrapure water. After three washes, add 3 ml of ultrapure water and push out the washed epoxy-IONPs with a push rod and transfer to a 10 ml centrifuge tube. b. Add 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, and 1 mg of CD4 antibody, place the centrifuge tube on a vertical mixer, and then place it in a constant temperature shaker and rotate and mix at 37°C for 1 hour; c. Add 300 μl of CD4 antibody-coupled epoxy-functionalized immunomagnetic beads to the Miltenyi LS Column. After the supernatant flows out, add 300 μl of ultrapure water to wash once. After washing, add 300 μl of ultrapure water and push out and collect with a push rod. d. Prepare BCA working solution according to the instructions of the Micro BCA Protein Assay Kit (Shanghai Sangon Biotech Co., Ltd.). Add 100 μl of BCA working solution and 100 μl of CD4 antibody-coupled epoxy-functionalized immunomagnetic beads mixture to the ELISA plate. Place the ELISA plate in a constant temperature shaker and incubate at 37°C for 30 min. After incubation, remove the plate and cool it to room temperature. Place the plate in an ELISA analyzer and set the main wavelength to 570 nm and the secondary wavelength to 595 nm. Record the absorbance readings. e Repeat the steps in (7) for the experiment with the magnetic core coated with dextran but not activated with 1,4-butanediol diglycidyl ether, record the absorbance values read, and finally detect the CD4 antibody coupling content by fitting the curve.

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