CN103305464B - Method for Direct Isolation of CD4+ and CD8+ Lymphocytes - Google Patents

Abstract

The invention discloses a method for directly separating CD<4+> and CD<8+> lymphocytes, lays a better foundation for the subsequent research on the CD<4+> and CD<8+> lymphocytes, and relates to the field of biomedicines. The method comprises steps of: multi-arm well and star-shaped polymer and mouse anti-human CD<4+> or CD<8+> monoclonal antibody covalent coupling, long-chain biotin molecule coating through utilizing a mouse anti-human CD<4+> or CD<8+> monoclonal antibody-modified multi-arm well and star-shaped polymer, peripheral blood sample CD<4+> and CD<8+> lymphocyte acquiring through utilizing a mouse anti-human CD<4+> or CD<8+> monoclonal antibody and long-chain biotin co-modified multi-arm well and star-shaped polymer, peripheral blood long-chain biotinylation multi-arm well and star-shaped polymer identifying and coupling through utilizing streptavidin-modified nano magnetic beads, captured CD<4+> and CD<8+> lymphocyte separating and suspending and the like. A suspension can be directly used for subsequent analysis; and compared with a conventional cell separating method, the method is suitable for magnetically separating complicated peripheral blood sample CD<4+> and CD<8+> lymphocytes, so that the peripheral blood sample CD<4+> and CD<8+> lymphocyte separation efficiency is increased.

Description

Method for Direct Isolation of CD4+ and CD8+ Lymphocytes

technical field

The invention relates to the field of biomedicine, in particular to a method for separating CD4 ⁺ and CD8 ⁺ lymphocytes based on nano magnetic beads.

Background technique

T lymphocytes are the most important group of cells in the body's immune system. T lymphocytes are derived from lymphocytes of the bone marrow and perform cellular immune functions. They are not only the main body of direct immune effects, but also produce a variety of cytokines and express adhesion molecules, and play an immune regulatory role through direct or indirect contact with other immune cells. In a normal body, various lymphocyte subsets interact to maintain the normal immune function of the body. When the number and function of different lymphocyte subsets are abnormal, it can lead to immune dysfunction and a series of pathological changes. Peripheral blood CD4 ⁺ and CD8 ⁺ lymphocytes are the most important lymphocyte subsets, which are closely related to the occurrence and development of tumors and the immune function of the body. Therefore, it is more and more important to study the mechanism of development, differentiation and activation of CD4 ⁺ and CD8 ⁺ T lymphocytes. However, the content of CD4 ⁺ and CD8 ⁺ T lymphocytes in the peripheral blood of normal people is very low, and the existing methods cannot directly separate and collect trace CD4 ⁺ and CD8 ⁺ T lymphocytes in peripheral blood. Therefore, to establish an efficient method for isolating and purifying human peripheral blood CD4 ⁺ and CD8 ⁺ T lymphocytes, and to identify their phenotype and biological function, in order to further study the role of CD4 ⁺ and CD8 ⁺ T lymphocytes in immune response. The role of providing higher purity CD4 ⁺ and CD8 ⁺ T lymphocytes.

Immunomagnetic separation technology is one of the important components of the rapid separation and enrichment technology of peripheral blood cells. This technology can efficiently capture and concentrate target cells in human peripheral blood samples and improve the detection sensitivity of target cells. In recent years, the immunomagnetic separation method (IMS) based on magnetic microbeads connects antibodies to magnetic beads, and then puts the magnetic beads with antibodies into the sample solution to capture, enrich and magnetically separate target cells (see Figure 2A). However, the current separation technology based on micron-scale immunomagnetic beads has many limitations: 1) The specific surface area of micron-sized magnetic beads is relatively small, which reduces the capture efficiency of magnetic beads; The combination of cells through a multiphase reaction usually takes longer to specifically capture cells in the food matrix; 3) The micron magnetic beads have poor monodispersity and are prone to self-aggregation or self-aggregation in the peripheral blood matrix Precipitation; 4) The traditional immunomagnetic separation technology often directly couples the antibody to the immunomagnetic beads. This process often leads to a greatly reduced activity of the antibody and a change in the spatial direction of the antibody, which increases the inter-antibody interaction. Steric hindrance effect, which reduces the capture efficiency of antibodies 5) The blood viscosity is high and the blood cell concentration of non-CD4 ⁺ and CD8 ⁺ lymphocytes is large, micron magnetic beads are prone to non-specific adsorption, and it is difficult to achieve CD4 ⁺ in blood 6) Excessive concentration of micron magnetic beads will cause ^damage to CD4 ⁺ and CD8 ⁺ lymphocytes (the magnetic field causes the magnetic beads on the cell surface to attract each other, causing the cells to be squeezed or even ruptured), resulting in Separation failure; 7) When coupling antibodies to magnetic beads, hydrophobic adsorption or chemical coupling is generally used to couple active antibodies to the surface of magnetic beads. The distance between the antibody and the surface of the magnetic bead is too close, the nature of the magnetic bead itself and the residual hydrophobic or strong hydrophilic groups on the surface may easily cause the change of the spatial conformation of the antibody, resulting in a decrease in the biological activity of the antibody.

Contents of the invention

Aiming at the defects of the prior art, the purpose of the present invention is to provide a high capture efficiency, simple and short separation time, specific for CD4 ⁺ and CD8 ⁺ lymphocytes in the complex peripheral blood matrix under low gradient magnetic field (less than 30 T/m) method of rapid separation.

A method for directly isolating CD4 ⁺ and CD8 ⁺ lymphocytes, comprising the steps of:

(1) Dissolve 1.0 mg of aminated multi-arm well star polymer in 2 mL of 0.02 M, pH 6.5 phosphate buffer PBS, add 0.6 mg of N-hydroxysuccinimide NHSS, 0.4 mg of ethyl 3-( 3-Dimethylamino) carbodiimide hydrochloride EDC, placed on a mixer at room temperature, stirred, activated for 15 min; 2.2 mg of mouse anti-human CD4 ⁺ monoclonal antibody was added to the above reaction solution, placed at room temperature for mixing Stir on the instrument for 30 min; spin dry the above solution under reduced pressure, dissolve in deionized water ^, and dialyze in PBS and deionized water for 1 d; (2) Dissolve 1.0 mg of aminated multi-arm well star polymer in 2 mL of 0.02 M, pH 6.5 phosphate buffer PBS, add 0.6 mg of N-hydroxysuccinimide NHSS, 0.4 mg of ethyl 3 -(3-Dimethylamino)carbodiimide hydrochloride EDC, placed on a mixer at room temperature, stirred, and activated for 15 min; 2.2 mg of mouse anti-human CD8 ⁺ monoclonal antibody was added to the above reaction solution, placed at room temperature Stir on a mixer for 30 min; spin dry the above solution under reduced pressure, dissolve in deionized water, and dialyze in PBS and deionized water for 1 day; freeze ^- dry the obtained solution after dialysis Complex; (3) 15 mg long-chain biotin, 3.6 mg NHSS, 2.4 mg EDC were dissolved in 2 mL 0.02 M pH 6.5 PBS buffer; 0.1 mg multi-arm well star polymer-CD4 ⁺ or CD8 ⁺ The antibody complex was added to the above solution, placed on a mixer at room temperature and stirred for 30 min; the above solution was spin-dried under reduced pressure, dissolved in deionized water, and dialyzed in PBS and deionized water for 1 day; after the dialysis, the obtained solution was Freeze-dried to obtain long-chain biotin-multi-armed star polymer-antibody complexes; (4) Enrichment and capture: take 1 mL of the sample solution to be tested, add 0.1 mg long-chain biotin-multi-armed star polymer-CD4 ⁺ Place the antibody complex on a mixer and incubate at room temperature at 30 rpm for 15 min to form a long-chain biotin-multi-armed well star polymer-antibody-CD4 ⁺ cell antigen complex; add 0.1 mg modified streptavidin Hesu nano-magnetic beads were placed on a mixer, and incubated at room temperature for 15 minutes at a speed of 30 rpm, and the conventional magnetic frame was fully separated; after magnetic separation, the cells were resuspended with an equal volume of PBS solution, which were CD4 ⁺ cells ; Wash the separated supernatant with an equal volume of PBS solution, centrifuge at 300rpm/min, 20°C for 10 min, discard the supernatant, and resuspend the cells with an equal volume of PBS solution; add 0.1 mg long-chain biotin-poly Arm well star polymer-CD8 ⁺ antibody complex, placed on a mixer, incubated at room temperature at 30 rpm for 1 Form a long-chain biotin-multi-armed well star polymer-antibody-CD8 ⁺ cell antigen complex for 5 min; add 0.1 mg nano-magnetic beads modified with streptavidin, place it on a mixer, and set it at 30 rpm Incubate at room temperature for another 15 min at a rotating speed, and fully separate with a conventional magnetic frame. After magnetic separation, resuspend the cells with an equal volume of PBS solution to obtain CD8 ⁺ cells; (5) After gently washing with deionized water, resuspend with PBS buffer to obtain cells enriched with CD4 ⁺ or CD8 ⁺ cells The complex is nano magnetic beads-streptavidin-biotin-multi-arm well star polymer-antibody-CD4 ⁺ or CD8 ⁺ cell antigen.

The multi-arm star polymer is a multi-arm star polymer modified with amino groups at the end, and its molecular weight is 70000 Da. The structure is shown in Figure 1. the

The particle size of the streptavidin-modified nano magnetic beads is 20-50 nm, preferably 30 nm. the

The multi-armed well-star polymer realizes the covalent coupling of the multi-armed well-star polymer and the antibody through the amino group and the carboxyl group of CD4 ⁺ or CD8 ⁺ lymphocyte antibody.

The multi-armed well-star polymer realizes the covalent coupling of the multi-armed well-star polymer and the long-chain biotin through the amino group and the carboxyl group of the long-chain biotin molecule; adding excess long-chain biotin to ensure the closure of the multi-arm well-star polymer exposed amino sites. the

The specific principle is shown in Figure 2B. the

This method is especially suitable for the separation of complex samples, such as human peripheral blood samples. Sample processing can be done according to conventional processing methods. the

Adopt technical scheme of the present invention to have following beneficial effect:

1. The present invention utilizes the cascade amplification effect of the multi-arm well star polymer to exponentially expand the magnetic cell signal, and the separation of the magnetic cell can be realized at a lower magnetic field strength, and in the same time, it is relatively Compared with conventional immunomagnetic bead separation methods, the ability to separate target cells is stronger, and it is especially suitable for the separation of complex samples, such as peripheral blood samples. Aiming at the defects of slow speed and high magnetic field requirements for the separation of target cells in complex matrix samples by purely using 20-50 nm immunomagnetic beads modified by antibodies, multi-arm well star polymers are used to amplify the magnetic signal of nano magnetic beads, thereby improving the The separation efficiency of target cells in complex matrix samples enables the specific and rapid separation of target cells in complex food matrices under low gradient magnetic field (less than 30 T/m).

2. This solution is to couple the antibody molecule to the multi-arm well star polymer, which avoids the shortcomings of the conventional method of coupling the antibody molecule to the surface of the magnetic bead, which leads to the decrease of antibody activity and the large steric hindrance. the

3. The present invention adopts the multi-arm well star polymer, which can make the reaction solution more stable, less prone to precipitation, increases the chance of contact between the antibody and the target cell, and is conducive to improving the capture efficiency; at the same time, the multi-arm well star polymer is connected with A large number of long-chain biotin molecules can be combined with streptavidin-modified nano-magnetic beads, so that a large number of nano-magnetic beads can be combined with the multi-armed well star polymer, realizing the cascade amplification of magnetic cell signals, which is conducive to shortening Separation time of magnetic cells. the

4. After replacing micron-sized magnetic particles with nano-magnetic beads (20-50 nm), due to the small particle size and large specific surface area of nano-magnetic beads, the steric hindrance of binding to cell surface antigens is small, and the coverage efficiency of cell surface magnetic beads is significantly improved. , and the cells covered by magnetic nanoparticles can maintain a normal shape, and the nano-magnetic beads also have good dispersion and stability in complex matrices, so the use of nano-magnetic beads can overcome the above-mentioned defects caused by the use of micron magnetic beads. the

5. In the separation process of the present invention, a multi-armed well-star polymer is introduced, and a large number of long-chain biotin molecules are connected to the multi-arm well-star polymer, which can couple with the matrix solution dispersed in the matrix solution with specificity and high affinity. Linked with streptavidin nano-magnetic beads for recognition, so that a large number of nano-magnetic beads are bound to the multi-arm well star polymer, which greatly increases the number of magnetic beads bound to the surface of the target cells, and realizes rapid separation of captured cells under a magnetic field. target cells. Compared with the traditional cell magnetic separation method, this method is more suitable for magnetic separation of cells in complex matrices because of the addition of more stable nano-magnetic beads in the matrix, improving the separation efficiency of target cells in complex matrix samples . the

6. When coupling antibodies to magnetic beads, hydrophobic adsorption or chemical coupling is generally used to couple active antibodies to the surface of magnetic beads. The distance between the antibody and the surface of the magnetic bead is too close, the nature of the magnetic bead itself and the residual hydrophobic or strong hydrophilic groups on the surface may easily cause the change of the spatial conformation of the antibody, resulting in a decrease in the biological activity of the antibody. However, this experimental protocol introduces a multi-arm well star polymer during the coupling process, which has a certain space size, so that the antibody molecules are kept away from the magnetic beads and the surface of the magnetic beads, avoiding the adverse effects of the properties of the magnetic beads themselves and the surface on the antibody molecules . At the same time, the introduced multi-arm well star polymer will not affect the spatial conformation of the antibody, thereby protecting the biological activity of the antibody molecule. the

Description of drawings

Fig. 1 Schematic diagram of the structure of multi-arm well star polymer. the

Fig. 2 Operation flow chart of the conventional magnetic separation technology (A) and the magnetic separation technology (B) involved in the present invention. the

Detailed ways

In order to make the present invention clearer, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention. the

Long-chain biotin was purchased from Thermo Fisher Scientific, USA, carboxylated long-chain biotin (EZ-Link Sulfo-NHS-LC-Biotin, molecular weight 556.59). the

Nanomagnetic beads (30 nm) modified with streptavidin were purchased from Ocean NanoTech, USA. the

The multi-arm star polymer is a multi-arm star polyamide-amine with a molecular weight of 70,000 Da, which was purchased from Weihai Chenyuan Chemical New Material Co., Ltd. the

Conventional magnetic stand separation magnetic field strength is less than 30T/m. the

N-Hydroxysuccinimide NHSS, Ethyl 3-(3-dimethylamino)carbodiimide hydrochloride EDC, etc. are all conventional reagents and will not be repeated here. the

0.1%PBST preparation method: Dissolve 8.0 g NaCl, 0.2 g KCl, 0.24 g KH ₂ PO ₄ , 1.44 g Na ₂ HPO ₄ in 800 mL of distilled water, adjust the pH to 7.4 with 5 M NaOH, and then dilute to 1000 mL. 0.01 M PBS was obtained. Add Tween 20 at a volume ratio of 1/1000 (V/V) to obtain 0.1% PBST.

Example 1

1. The multi-arm well star polymer-CD4 ⁺ antibody complex is prepared according to the following steps:

(1) Dissolve 1.0 mg of Dobby Star Polymer Dobby Star polyamide-amine in 2 mL of 0.02 M, pH 6.5 phosphate buffer PBS, add 0.6 mg of N-hydroxysuccinimide NHSS, 0.4 mg ethyl 3-(3-dimethylamino)carbodiimide hydrochloride EDC, stir on a mixer at room temperature, activate for 15 min;

(2) Take 2.2 mg of mouse anti-human CD4 ⁺ monoclonal antibody and add it to the above reaction solution, place it on a mixer at room temperature and stir for 30 min;

(3) The above solution was spin-dried under reduced pressure, dissolved in deionized water, and dialyzed in PBS and deionized water for 1 day; after the dialysis, the obtained solution was freeze-dried.

2. The multi-arm well star polymer-CD8 ⁺ antibody complex is prepared according to the following steps:

(1) Dissolve 1.0 mg of aminated multi-arm well star polymer in 2 mL of 0.02 M, pH 6.5 phosphate buffer PBS, add 0.6 mg of N-hydroxysuccinimide NHSS, 0.4 mg of ethyl 3-( 3-Dimethylamino) carbodiimide hydrochloride EDC, stirred on a mixer at room temperature, and activated for 15 min;

(2) Take 2.2 mg mouse anti-human CD8 ⁺ monoclonal antibody and add it to the above reaction solution, place it on a mixer at room temperature and stir for 30 min;

(3) The above solution was spin-dried under reduced pressure, dissolved in deionized water, and dialyzed in PBS and deionized water for 1 day; after the dialysis, the obtained solution was freeze-dried.

3. The long-chain biotin-multi-armed well star polymer-antibody complex is prepared according to the following steps: 

(1) Dissolve 15 mg long-chain biotin, 3.6 mg NHSS, and 2.4 mg EDC in 2 mL 0.02 M pH 6.5 PBS buffer;

(2) Add 0.1 mg of multi-arm well star polymer-CD4 ⁺ or CD8 ⁺ antibody complex to the above solution, and place it on a mixer at room temperature and stir for 30 min;

(3) The above solution was spin-dried under reduced pressure, dissolved in deionized water, and dialyzed in PBS and deionized water for 1 day; after the dialysis, the obtained solution was freeze-dried.

4. Enrichment and capture: Take 1 mL of the sample solution to be tested, add 0.1 mg of long-chain biotin-multi-armed well star polymer-CD4 ⁺ antibody complex, place it on a mixer, and incubate at room temperature for 15 min at a speed of 30 rpm Form a long-chain biotin-multi-armed well star polymer-antibody-CD4 ⁺ cell antigen complex; add 0.1 mg nano-magnetic beads modified with streptavidin, place on a mixer, and re-mix at a speed of 30 rpm Incubate at room temperature for 15 min, and insert the centrifuge tube into a conventional magnetic stand for sufficient separation. After magnetic separation, resuspend the cells with an equal volume of PBS solution, which are CD4 ⁺ cells. Wash the separated supernatant with an equal volume of PBS solution, centrifuge (300 rpm/min, 20°C) for 10 min, discard the supernatant, and resuspend the cells with an equal volume of PBS solution. Add 0.1 mg long-chain biotin-multi-armed well star polymer-CD8 ⁺ antibody complex, place it on a mixer, and incubate at room temperature for 15 min at a speed of 30 rpm to form long-chain biotin-multi-arm well star polymer- Antibody-CD8 ⁺ cell antigen complex; add 0.1 mg nano-magnetic beads modified with streptavidin, place on a mixer, incubate at room temperature for 15 min at a speed of 30 rpm, insert the centrifuge tube into a conventional magnetic stand for full separate. After magnetic separation, resuspend the cells with an equal volume of PBS solution, which are CD8 ⁺ cells.

5. After gently washing with deionized water, mix and resuspend with PBS buffer to obtain a complex enriched with CD4 ⁺ or CD8 ⁺ cells, that is, nano-magnetic beads-streptavidin-biotin-multi-armed well star polymerization Object - Antibody - CD4 ⁺ or CD8 ⁺ cell antigen.

Example 2 Enrichment effect experiment

(1) Take 1 mL of CD4 ⁺ or CD8 ⁺ cells with a concentration of 10 ⁴ cells/mL in a 1.5 mL sterile centrifuge tube, centrifuge at 12,000 rpm for 5 min, discard the supernatant, and resuspend with an equal volume of sterile PBS solution.

(2) Enrichment and capture: Set up the technical solution group of the present invention (multi-arm well star polymer group co-modified with CD4 ⁺ or CD8 ⁺ cell antibody and long-chain biotin), CD4 ⁺ or CD8 ⁺ cell-specific antibody modified Nano-magnetic beads group, CD4 ⁺ or CD8 ⁺ cell-specific antibody-modified micro-magnetic beads set enrich target cells.

(3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and wash the separated immunomagnetic beads with CD4 ⁺ or CD8 ⁺ cells twice with PBST, mix well, and rinse with 1 mL of sterile PBS solution to resuspend the immunomagnetic bead complex.

(4) Capture rate calculation: after serial dilution of the enriched target cell resuspension in each group, the flow cytometer (Flow Cytometer) was used to detect the number, and the capture efficiency of target cells was calculated by the capture efficiency formula, and each experiment was repeated three times. The formula for calculating the capture efficiency of each group is as follows: (total number of target cells that are enriched and adsorbed/total number of all cells) × 100%. the

The schemes for each group to enrich and capture target cells are as follows:

a. The technical solution group of the present invention (multi-armed well-star polymer group co-modified with CD4 ⁺ or CD8 ⁺ cell antibody and long-chain biotin) enrichment and capture of target cells is as in Example 1, specifically as follows:

Add 0.1 mg of CD4 ⁺ cell antibody and biotin co-modified multi-armed well star polymer, that is, biotin-multi-armed well star polymer-antibody complex, into a centrifuge tube containing target cells, place it on a mixer, and Incubate at room temperature for 15 min at 30 rpm. Then, 0.1 mg of nano-magnetic beads modified with streptavidin were added, placed on a mixer, and incubated at room temperature for 15 min at a speed of 30 rpm. Finally, insert the centrifuge tube into a conventional magnetic stand to separate fully. After magnetic separation, resuspend the cells with an equal volume of PBS solution, which are CD4 ⁺ cells. Wash the separated supernatant with an equal volume of PBS solution, centrifuge (300 rpm/min, 20°C) for 10 min, discard the supernatant, and resuspend the cells with an equal volume of PBS solution. Add 0.1 mg long-chain biotin-multi-armed well star polymer-CD8 ⁺ antibody complex, place it on a mixer, and incubate at room temperature for 15 min at a speed of 30 rpm to form long-chain biotin-multi-arm well star polymer- Antibody-CD8 ⁺ cell antigen complex; add 0.1 mg nano-magnetic beads modified with streptavidin, place on a mixer, incubate at room temperature for 15 min at a speed of 30 rpm, insert the centrifuge tube into a conventional magnetic stand for full separate. After magnetic separation, resuspend the cells with an equal volume of PBS solution, which are CD8 ⁺ cells.

b. The CD4 ⁺ or CD8 ⁺ cell-specific antibody-modified nano-magnetic bead group enrichment and capture target cell scheme is as follows:

Add 0.1 mg of prepared CD4 ⁺ cell-specific antibody-modified nano-magnetic beads into a centrifuge tube containing target cells, place on a mixer, and incubate at room temperature for 15 min at a speed of 30 rpm. Finally, insert the centrifuge tube into a conventional magnetic stand for separation for 3 min. After magnetic separation, resuspend the cells with an equal volume of PBS solution, which are CD4 ⁺ cells. Wash the separated supernatant with an equal volume of PBS solution, centrifuge (300 rpm/min, 20°C) for 10 min, discard the supernatant, and resuspend the cells with an equal volume of PBS solution. Add 0.1 mg of CD8 ⁺ cell-specific antibody-modified nano-magnetic beads, place them on a mixer, incubate at room temperature for 15 min at a speed of 30 rpm, and insert the centrifuge tube into a conventional magnetic stand for sufficient separation. After magnetic separation, resuspend the cells with an equal volume of PBS solution, which are CD8 ⁺ cells.

Preparation of the CD4 ⁺ or CD8 ⁺ cell-specific antibody-modified nano-magnetic beads: (1) Take 10 mg nano-magnetic beads (30 nm, not coupled to streptavidin) with absolute ethanol, 1 M NaOH, Wash once each with 1 M HCl, wash three times with PBS (0.02 M, pH 4.0), and resuspend in sterile PBS. Add 0.4 mg of NHSS and 0.35 mg of EDC, place on a mixer to keep the magnetic beads suspended, and activate at 37°C for 2 h. (2) Collect the magnetic beads on the magnetic stand. After washing three times with PBS (0.02 M, pH 4.0), the magnetic beads were resuspended in sterile PBS, and 80 μg of CD4 ⁺ or CD8 ⁺ cell-specific antibody was added to each mg of magnetic beads. Coupling was performed on a mixer at 37°C for 2 h. (3) Add ethanolamine to block at room temperature for 2 h. The magnetic beads were recovered on the magnetic rack, washed three times with PBS, and the immunomagnetic beads were resuspended in 10 ml PBS (containing 0.05% NaN ₃ , 0.5% BSA, pH 7.4) and stored in a refrigerator at 4°C for later use.

c. The CD4 ⁺ or CD8 ⁺ cell-specific antibody-modified micron magnetic bead group enrichment and capture target cell scheme is as follows:

Add 0.1 mg of prepared CD4 ⁺ cell-specific antibody-modified micron magnetic beads into a centrifuge tube containing target cells, place it on a mixer, and incubate at room temperature for 15 min at a speed of 30 rpm. Finally, insert the centrifuge tube into a conventional magnetic stand for separation for 3 min. After magnetic separation, resuspend the cells with an equal volume of PBS solution, which are CD4 ⁺ cells. Wash the separated supernatant with an equal volume of PBS solution, centrifuge (300 rpm/min, 20°C) for 10 min, discard the supernatant, and resuspend the cells with an equal volume of PBS solution. Add 0.1 mg CD8 ⁺ cell-specific antibody-modified micron magnetic beads, place on a mixer, incubate at room temperature for 15 min at a speed of 30 rpm, and insert the centrifuge tube into a conventional magnetic stand for sufficient separation. After magnetic separation, resuspend the cells with an equal volume of PBS solution, which are CD8 ⁺ cells.

The CD4 ⁺ or CD8 ⁺ cell-specific antibody-modified micron magnetic beads were prepared: (1) Take 10 mg of micron magnetic beads (1150 nm, without coupling streptavidin) with absolute ethanol, 1 M NaOH, Wash once each with 1 M HCl, wash three times with PBS (0.02 M, pH 4.0), and resuspend in sterile PBS. Add 0.4 mg of NHSS and 0.35 mg of EDC, place on a mixer to keep the magnetic beads suspended, and activate at 37°C for 2 h. (2) Collect the magnetic beads on the magnetic stand. After washing three times with PBS (0.02 M, pH 4.0), the magnetic beads were resuspended in sterile PBS, and 80 μg of CD4 ⁺ or CD8 ⁺ cell-specific antibody was added to each mg of magnetic beads. Coupling was performed on a mixer at 37°C for 2 h. (3) Add ethanolamine to block at room temperature for 2 h. The magnetic beads were recovered on the magnetic rack, washed three times with PBS, and the immunomagnetic beads were resuspended in 10 ml PBS (containing 0.05% NaN ₃ , 0.5% BSA, pH 7.4) and stored in a refrigerator at 4°C for later use.

The catch rate of each group is as follows: CD4 ⁺ cell-specific antibody-modified micron magnetic bead set capture rate Capture efficiency of CD4 ⁺ cell-specific antibody-modified magnetic nanobeads Capture efficiency of CD4 ⁺ cell antibody and long-chain biotin co-modified multi-armed well star polymer group 52.1% 20.8% 87.6% CD8 ⁺ cell-specific antibody-modified micron magnetic bead set capture rate Capture efficiency of CD8 ⁺ cell-specific antibody-modified magnetic nanobeads Capture efficiency of CD8 ⁺ cell antibody and long-chain biotin co-modified multi-armed well star polymer group 49.8% 19.5% 88.3%

The experimental results showed that the capture efficiency of the CD4 ⁺ or CD8 ⁺ cell-specific antibody-modified micron magnetic beads group was significantly higher than that of the nano magnetic bead group, which indicated that compared with the nano magnetic bead group, due to the large volume and strong magnetic properties of the micron magnetic beads , and more target cells can be isolated and enriched in a short time. However, the capture efficiency of the technical solution group of the present invention is far greater than that of the CD4 ⁺ or CD8 ⁺ cell-specific antibody-modified micron magnetic bead group, which shows that the technical solution of the present invention can increase the target cell surface nanomagnetic The coverage of the beads has greatly improved the magnetic properties, thereby realizing the efficient separation and enrichment of CD4 ⁺ or CD8 ⁺ cells in a short time (3min).

Example 3 Enrichment capture experiment

Conventional magnetic stand separation time is 30min, and all the other are with embodiment 2.

The catch rate of each group is as follows: CD4 ⁺ cell-specific antibody-modified micron magnetic bead set capture rate Capture efficiency of CD4 ⁺ cell-specific antibody-modified magnetic nanobeads Capture efficiency of CD4 ⁺ cell antibody and long-chain biotin co-modified multi-armed well star polymer group 51.7% 38.1% 89.8% CD8 ⁺ cell-specific antibody-modified micron magnetic bead set capture rate Capture efficiency of CD8 ⁺ cell-specific antibody-modified magnetic nanobeads Capture efficiency of CD8 ⁺ cell antibody and long-chain biotin co-modified multi-armed well star polymer group 50.9% 36.9% 88.7%

The experimental results show that compared with the separation of 3 minutes in Example 2, when the separation time reaches 30 minutes, the capture efficiency of the three groups has been improved, especially the capture efficiency of the CD4 ⁺ or CD8 ⁺ cell-specific antibody-modified nano magnetic beads group. Most notably, this shows that the capture efficiency of the nanomagnetic bead set can be greatly improved by extending the time, but it is still lower than that of multi-armed wells co-modified with CD4 ⁺ or CD8 ⁺ cell antibodies and long-chain biotin for a short separation time (3 min). Capture efficiency of star polymer groups. This shows that the technical scheme of the present invention can efficiently separate and enrich CD4 ⁺ or CD8 ⁺ cells in a short time (3 minutes).

Example 4 Nano-magnetic Beads Enrichment of CD4 ⁺ and CD8 ⁺ Lymphocytes in the Peripheral Blood of Healthy Volunteers

Take 20 mL of sterile EDTA-anticoagulated peripheral blood from healthy volunteers, separate peripheral blood mononuclear cells by density gradient centrifugation, and resuspend the cells in 2 mL of PBS solution. Take the above PBS cell resuspension in a centrifuge tube, centrifuge (300 rpm/min, 20°C) for 10 min, discard the supernatant, and resuspend the cells, the number of cells per 80 μL PBS is 10 ⁷ , and the number of cells per 10 ⁷ Add 0.1 mg of long-chain biotin-multi-arm well star polymer-mouse anti-human CD4 ⁺ antibody complex, mix thoroughly, and incubate at 4-8°C for 15 min. Wash the separated cell suspension with an equal volume of PBS solution, centrifuge (300rpm/min, 20°C) for 10 min, discard the supernatant, and resuspend the cells with an equal volume of PBS solution. Add 0.1 mg long-chain biotin-multi-armed well star polymer-CD8 ⁺ antibody complex, place it on a mixer, and incubate at room temperature for 15 min at a speed of 30 rpm to form long-chain biotin-multi-arm well star polymer- Antibody-CD8 ⁺ cell antigen complex; add 0.1 mg nano-magnetic beads modified with streptavidin, place on a mixer, incubate at room temperature for 15 min at a speed of 30 rpm, insert the centrifuge tube into a conventional magnetic stand for full separate. After magnetic separation, pour the supernatant into a sterile centrifuge tube, and wash the separated immunomagnetic beads with CD4 ⁺ or CD8 ⁺ cells twice with PBST, mix well, and resuspend with 1 mL of sterile PBS solution Immunomagnetic beads. The capture rate is obtained as in Example 2, and the rest are the same as in Example 2. The results are shown in Table 1, indicating that this protocol can efficiently enrich CD4 ⁺ or CD8 ⁺ cells in isolated samples.

Table 1 Separation effect of CD4 ⁺ and CD8 ⁺ lymphocytes in peripheral blood Experimental group number Capture efficiency of CD4 ⁺ cell antibody and long-chain biotin co-modified multi-armed well star polymer group Capture efficiency of CD8 ⁺ cell antibody and long-chain biotin co-modified multi-armed well star polymer group Example 4 83.6% 82.3%

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention within.

Claims (3)

1. direct separation of C D4 ⁺and CD8 ⁺lymphocytic method, is characterized in that comprising the following steps:

(1) 1.0 mg multi-arm well star polymer dissolution are often got in 2 mL 0.02 M, pH 6.5 phosphoric acid buffer PBS, adds 0.6 mg N-hydroxysuccinimide NHSS, 0.4 mg ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC, room temperature is placed on blending instrument and stirs, and activates 15 min; Get 2.2 mg mouse-anti people CD4 ⁺monoclonal antibody adds in above-mentioned reaction solution, and room temperature is placed on blending instrument and stirs 30 min; Above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to be obtained multi-arm well star polymkeric substance-CD4 ⁺antibody complex; (2) 1.0 mg arm well star polymer dissolution are often got in 2 mL 0.02 M, pH 6.5 phosphoric acid buffer PBS, adds 0.6 mg N-hydroxysuccinimide NHSS, 0.4 mg ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC, room temperature is placed on blending instrument and stirs, and activates 15 min; Get 2.2 mg mouse-anti people CD8 ⁺monoclonal antibody adds in above-mentioned reaction solution, and room temperature is placed on blending instrument and stirs 30 min; Above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize multi-arm well star polymkeric substance-CD8 that will obtain ⁺antibody complex; (3) often get 15 mg long-chain biological elements, 3.6 mg NHSS, 2.4 mg EDC are dissolved in 2 mL 0.02 M pH 6.5 PBS damping fluids; By 0.1 mg multi-arm well star polymkeric substance-CD4 ⁺or CD8 ⁺antibody complex joins in above-mentioned solution, and room temperature is placed on blending instrument and stirs 30 min; Above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to be obtained long-chain biological element-multi-arm well star polymer-antibody complex; (4) enrichment is caught: get testing sample solution 1mL, adds 0.1 mg long-chain biological element-multi-arm well star polymkeric substance-CD4 ⁺antibody complex, is placed on blending instrument, forms long-chain biological element-multi-arm well star polymer-antibody-CD4 with rotating speed incubated at room 15 min of 30 rpm ⁺cell antigen mixture; Add the nanometer magnetic bead that 0.1 mg is modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again, conventional magnetic frame is fully separated; The described nanometer magnetic bead particle diameter being modified with Streptavidin is 20-50 nm; After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD4 ⁺cell; Washed by isopyknic PBS solution by supernatant liquor after being separated, centrifugal 300 rpm/min, 20 DEG C of 10 min, supernatant discarded, with isopyknic PBS solution re-suspended cell; Add 0.1 mg long-chain biological element-multi-arm well star polymkeric substance-CD8 ⁺antibody complex, is placed on blending instrument, forms long-chain biological element-multi-arm well star polymer-antibody-CD8 with rotating speed incubated at room 15 min of 30 rpm ⁺cell antigen mixture; Add the nanometer magnetic bead that 0.1 mg is modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again, conventional magnetic frame is fully separated; After Magneto separate, with isopyknic PBS solution re-suspended cell, be CD8 ⁺cell; (5), after deionized water cleans gently, mix resuspended namely obtaining with PBS damping fluid and be enriched with CD4 ⁺or CD8 ⁺the mixture of cell and nanometer magnetic bead-Streptavidin-vitamin H-multi-arm well star polymer-antibody-CD4 ⁺or CD8 ⁺cell antigen.

2. method according to claim 1, it is characterized in that described multi-arm well star polymkeric substance is multi-arm well star polyamide-amide, its molecular weight is 70000 Da.

3. method according to claim 1, is characterized in that the described nanometer magnetic bead particle diameter having modified Streptavidin is 30 nm.