CN117659205A

CN117659205A - Coupling method for sorting activated magnetic beads and application

Info

Publication number: CN117659205A
Application number: CN202311656536.4A
Authority: CN
Inventors: 邱志宇; 王巧玲; 乔倩; 杨丽微; 陈晓通; 王立燕
Original assignee: Beijing T&l Biotechnology Co ltd
Current assignee: Beijing T&l Biotechnology Co ltd
Priority date: 2023-12-05
Filing date: 2023-12-05
Publication date: 2024-03-08

Abstract

The invention discloses a coupling method of sorting activated magnetic beads and application thereof, and provides a coupling method of magnetic beads. The invention provides an application of a sorting activated magnetic bead obtained by the magnetic bead coupling method in preparing a product. The invention also provides an application of the sorting activated magnetic beads obtained according to the coupling method of the magnetic beads in promoting T cell sorting, activating and/or amplifying. The invention also provides an application of the sorting activated magnetic beads obtained according to the coupling method of the magnetic beads in improving the purity of T cells.

Description

Coupling method for sorting activated magnetic beads and application

Technical Field

The invention belongs to the technical field of biology, relates to a coupling method for sorting activated magnetic beads and application thereof, and particularly relates to CD3/CD28.

Background

The magnetic bead coupling technology is based on biology, and is formed by crossing subjects such as bioinformatics, clinical medicine, materials, and the like, is mainly applied to cell separation, protein and nucleic acid purification, immunodetection, and the like, and brings wide application prospect to the field of magnetic bead coupling antibodies along with the gradual maturation of the magnetic bead coupling technology and the rapid development of the field of cell therapy.

Magnetic beads can be classified into three types according to different structures: 1. core-shell type: the magnetic beads adopt the earliest structure, and the polymer completely coats the magnetic particles in the inner core of the magnetic beads. 2. Sandwich type: the inner core and the outer layer of the magnetic bead are made of polymers, the middle layer is made of magnetic particles, and the process difficulty is higher than that of the core-shell type magnetic bead. 3. Dispersion type: the magnetic particles are randomly distributed in the polymer microsphere, which is a relatively mature process at present.

Compared with agarose beads, the magnetic beads have remarkable advantages as carriers of the whole immune reaction:

1) Unlike agarose bead magnetic bead spongy porous structure, the magnetic bead surface roughness is lower, and the antibody only binds on the magnetic bead surface, can improve the binding efficiency of antibody and magnetic bead, reduces nonspecific binding.

2) The immunomagnetic beads can be enriched rapidly through magnetic separation, and the reaction time is shortened greatly. Since centrifugation is not required when the supernatant is aspirated, the probability of loss of immunomagnetic beads can also be reduced.

3) The nanometer magnetic beads with smaller particle size are colloidal, can be uniformly suspended in the reaction solution, and increase the contact opportunity of the immunomagnetic beads and the sample, thereby accelerating the reaction speed and improving the binding efficiency of the immunomagnetic beads and the sample.

4) The magnetic beads have magnetic responsiveness (also called superparamagnetism), the magnetic beads can be collected through an external magnetic field, the magnetism of the magnetic beads disappears after the magnetic field is removed, and then the magnetic beads are uniformly dispersed in the solution again. The magnetic beads can be endowed with surface functional groups such as hydroxyl, carboxyl, sulfhydryl and amino by copolymerization, surface modification and other methods, and the target molecules can be specifically combined through the functional groups.

Disclosure of Invention

In order to solve the technical problems existing in the prior art, the following technical scheme is provided:

the invention provides a coupling method of magnetic beads, which comprises the following steps:

1) Fully and uniformly mixing magnetic beads, adding MES buffers with different pH values for re-suspension, uniformly mixing, standing on a magnetic frame, discarding supernatant, and repeating the step 1) for a plurality of times;

2) Adding MES buffer with the pH value corresponding to the step 1), re-suspending, adding EDC, and uniformly mixing for 1h;

3) Adding PBS (phosphate buffer solution) for resuspension, uniformly mixing, standing on a magnetic rack, discarding supernatant, and repeating the step 3) for a plurality of times;

4) Adding PBS to resuspend the magnetic beads, adding NHS, and uniformly mixing for 1h;

5) Adding PBS for resuspension, uniformly mixing, standing on a magnetic frame, discarding supernatant, and repeating the step 5) for a plurality of times;

6) Adding PBS (phosphate buffer solution) to resuspend the magnetic beads, adding the CD3 and CD28 monoclonal antibody stock solution, uniformly mixing for 2.5 hours, standing on a magnetic rack, and discarding the supernatant;

7) Adding PBS for resuspension, uniformly mixing, standing on a magnetic frame, discarding supernatant, and repeating the step 7) for a plurality of times;

8) Adding a closed buffer to resuspend the magnetic beads, and uniformly mixing for 1h;

9) Standing on a magnetic rack, and discarding supernatant;

10 Adding a preservation solution buffer for re-suspension, uniformly mixing, standing on a magnetic rack, discarding supernatant, and repeating the step 10) for a plurality of times;

11 Adding a preservation solution buffer to resuspend the magnetic beads to obtain the coupled magnetic beads.

In certain specific embodiments, the magnetic bead material is 1-10 μm in particle size, and in certain specific embodiments, the magnetic bead material is 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm.

The term "magnetic beads" refers to microspheres having a very fine particle size that is superparamagnetic and that can aggregate rapidly in a magnetic field and that, after leaving the magnetic field, can be uniformly dispersed to facilitate magnetic separation. Secondly, the particle size is proper and has smaller difference, and the sufficiently strong magnetic responsiveness is ensured without sedimentation. And the kit has abundant surface active groups so as to be coupled with biochemical substances and realize separation from a sample to be detected under the action of an external magnetic field. Compared with the traditional separation method, the magnetic beads are used for separating complex components of the biochemical sample, separation and enrichment can be simultaneously carried out, the separation speed and the enrichment efficiency are effectively improved, and meanwhile, the sensitivity of analysis and detection is greatly improved.

Further, the magnetic beads comprise amino magnetic beads, carboxyl magnetic beads, epoxy magnetic beads, sulfhydryl magnetic beads and uncoated magnetic beads.

In certain specific embodiments, the magnetic beads are carboxyl magnetic beads.

The term "re-suspension" refers to the act of deliberately placing the settled precipitate back into suspension and holding it in suspension while washing is taking place.

The terms "CD3 antibody", "CD3 monoclonal antibody" generally refer to an antibody capable of specifically recognizing the CD3 subunit (e.g., CD3 epsilon, CD3 gamma, CD3 delta, or a complex thereof), which may be a monoclonal antibody capable of recognizing only CD3, or a multi-target antibody capable of recognizing both CD3 and other targets.

The terms "CD28 antibody", "CD28 monoclonal antibody" generally refer to an antibody that specifically recognizes a CD28 subunit (e.g., CD28 epsilon, CD28 gamma, CD28 delta, or a complex thereof), which may be a monoclonal antibody that recognizes only CD28, or a multi-target antibody that recognizes both CD28 and other targets.

In this application, the terms "CD3", "CD28" refer to any natural CD3/CD28 from any vertebrate source, including mammals, such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats). The term encompasses "full length" and unprocessed CD3/CD28 proteins as well as any form of protein or one or more CD3/CD28 chains (polypeptides) (e.g., mature polypeptides) derived from processing in a cell. The term also encompasses naturally occurring variants and isoforms of CD3/CD28, such as splice variants or allelic variants.

Likewise, the terms "CD3 epsilon", "CD28 epsilon", "CD3 gamma", "CD28gamma", "CD3 delta" and/or "CD28 delta" in this application refer to any native CD3/CD28 from any vertebrate source, including mammals, such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats). "CD3 epsilon", "CD28 epsilon", "CD3 gamma", "CD28gamma", "CD3 delta" and/or "CD28 delta" encompass "full length" and unprocessed "CD3 epsilon", "CD28 epsilon", "CD3 gamma", "CD28gamma", "CD3 delta" and/or "CD28 delta" proteins, respectively, and any form of CD3/CD28 chain (polypeptide) (e.g., mature polypeptide) derived from processing in a cell. The term also encompasses naturally occurring variants and isoforms of the CD3/CD28 chain, such as splice variants or allelic variants.

The term "CD28" refers to a protein differentiation cluster 28, which is one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival.

The term "CD3" refers to a multimeric protein complex, historically referred to as a T3 complex, consisting of 4 distinct polypeptide chains. Epsilon (epsilon), gamma (gamma), delta (delta) and zeta (ζ), which are assembled and function as three pairs of dimers (epsilon gamma, epsilon delta, zeta).

Further, the pH value in step 1) of the coupling method is in the range of 4.5-6.5.

In certain specific embodiments, the MES buffer has a pH of 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5. In a preferred embodiment, the MES buffer has a pH of 5.5 to 6.0.

Further, the ratio of MES buffer to added beads used for the resuspension in step 1) was 1mL: 100. Mu.L.

Further, the step 1) is carried out in a way of mixing the materials upside down after adding the MES buffer.

Further, the ratio of MES buffer in step 2) to the magnetic beads added in step 1) was 300. Mu.L: 100. Mu.L.

Further, the ratio of EDC in step 2) to the magnetic beads added in step 1) is 10-50. Mu.L: 100. Mu.L.

In certain specific embodiments, the ratio of EDC to the magnetic beads in step 1) is 10 μl:100 μl,20 μl:100 μl,30 μl:100 μl,40 μl:100 μl,50 μl: 100. Mu.L. In a specific embodiment, the ratio of EDC to the magnetic beads in step 1) is 30 μl: 100. Mu.L.

Further, the method comprises the steps of, mixing uniformly in the step 2) the mode is that the mixture is vibrated and mixed evenly at room temperature.

Further, the ratio of PBS in step 3) to the magnetic beads added in step 1) was 1mL: 100. Mu.L.

Further, the mixing mode in the step 3) is upside down mixing.

Further, the ratio of PBS in step 4) to the magnetic beads added in step 1) was 300. Mu.L: 100. Mu.L.

Further, the ratio of NHS in step 4) to the magnetic beads added in step 1) is 10-100. Mu.L: 100. Mu.L.

In certain specific embodiments, the ratio of NHS to the magnetic beads in step 1) is 10 μl:100 μl,20 μl:100 μl,30 μl:100 μl,40 μl:100 μl,50 μl:100 μl,60 μl:100 μl,70 μl: 100. Mu.L, 80. Mu.L: 100. Mu.L, 90. Mu.L: 100 μL,100 μL: 100. Mu.L. In certain preferred embodiments, the ratio of NHS to magnetic beads in step 1) is 30 to 100. Mu.L: 100 μl, in a certain more preferred embodiment, the ratio of NHS to magnetic beads in step 1) is 30 μl: 100. Mu.L.

Further, the method comprises the steps of, mixing uniformly in the step 4) the mode is that the mixture is vibrated and mixed evenly at room temperature.

Further, the ratio of PBS in step 5) to the magnetic beads added in step 1) is 1mL: 100. Mu.L.

Further, the mixing mode in the step 5) is upside down mixing.

Further, the ratio of PBS in step 6) to the magnetic beads added in step 1) was 600. Mu.L: 100. Mu.L.

Further, the ratio of CD3, CD28 in the step 6) to the magnetic beads added in the step 1) is 200-300 mu L:200-100 μl: 100. Mu.L.

In certain specific embodiments, the CD3 and CD28 are added in an amount of 1-3:1, specifically including 1:1,2:1,3:1. In a preferred embodiment, the CD3 and CD28 are added in an amount of 1:1.

Further, the concentration of CD3 in the step 6) is 1-5mg/mL.

Further, the concentration of CD28 in the step 6) is 1-5mg/mL.

In certain specific embodiments, the CD3 is at a concentration of 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL. In a specific embodiment, the concentration of CD3 is 3mg/mL.

In certain specific embodiments, the CD28 is at a concentration of 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL. In a specific embodiment, the concentration of CD28 is 3mg/mL.

Further, in the step 6), the mixing mode is 2-8 ℃ vibration mixing.

Further, the ratio of PBS in step 7) to the magnetic beads added in step 1) was 1mL: 100. Mu.L.

Further, the mixing mode in the step 7) is upside down mixing.

Further, the ratio of the closed buffer in the step 8) to the magnetic beads added in the step 1) is 1mL: 100. Mu.L.

Further, in the step 8), the mixing mode is 2-8 ℃ vibration mixing.

Further, the ratio of the preservation solution buffer in the step 10) to the magnetic beads added in the step 1) is 1mL: 100. Mu.L.

Further, the mixing mode in the step 10) is upside down mixing.

Further, the ratio of the preservation solution buffer in the step 11) to the magnetic beads added in the step 1) is 200 μl: 200. Mu.L.

Further, the MES buffer comprises the following components: water for injection = 1.59g:50mL, pH was adjusted to 4.5-6.5.

Further, the PBS component is NaCl: KCl: na (Na) ₂ HPO ₄ ：KH ₂ PO ₄ : water for injection = 8g:0.2g:1.44g:0.24g:1L.

Further, the concentration of EDC in the step 2) is 1mg/mL, and the composition of 1mg/mL EDC is EDC: mes=0.01 g:10mL of the EDC was ready to use.

Further, the concentration of NHS in the step 4) is 1mg/mL, and the components of NHS of 1mg/mL are NHS: pbs=0.01 g:10mL, the NHS requires a ready-to-use and ready-to-prepare.

Further, the blocked buffer was composed of 20mM PBS, 1% BSA.

Further, the composition of the preservation solution buffer is 20mM PBS and 5% BSA.

The terms "particle size", "particle size" refer to the average diameter of a particle image, as observed by an electron microscope or optical microscope. The term "particle size" is used in this manner unless otherwise indicated. Unless otherwise indicated, the term "average particle size" means the average particle size of a collection of particles (for particles having an average particle size of at least 500 nm), or the specific surface area (in m) of the particles as determined using the Brunauer-Emmett-Teller method consistent with fully dense particles (for particles having an average particle size of less than 500 nm) ² /g measurement).

The present invention provides a sort-activated magnetic bead obtained according to the coupling method of magnetic beads described above.

The invention provides an application of sorting activated magnetic beads obtained according to the coupling method of the magnetic beads in the preparation of products.

Further, the product comprises a reagent, a kit and chromatographic test paper.

The invention provides the use of a sort-activated magnetic bead obtained according to the coupling method of the magnetic beads described above for promoting T cell sorting, activation and/or expansion.

The invention provides an application of sorting activated magnetic beads obtained according to the coupling method of the magnetic beads in improving the purity of T cells.

In the present application, the term "antibody" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a particular antigen. For example, an antibody may comprise an immunoglobulin of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and include any molecule comprising an antigen binding portion thereof. The term "antibody" includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (e.g., dabs), single chain antibodies (e.g., scFv), and antibody fragments that bind to an antigen (e.g., fab', and (Fab) ₂ Fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, non-glycosylated antibodies, as well as any antigen-binding antibody fragments and derivatives thereof described herein. Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may be composed of a light chain variable region (VL) and a light chain constant region. VH and VL regions can be further distinguished as hypervariable regions called Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved, called Framework Regions (FR). Each VH and VL may be composed of three CDRs and four FR regions, which may be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

The term "monoclonal antibody" refers to the same antibody obtained from a population of substantially homogeneous antibodies, i.e., each antibody comprising the population, but excluding possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are induced against a single antigenic site.

In this application, the term "and/or" is understood to mean either one of the selectable items or both of the selectable items.

In this application, the term "comprising" is generally intended to include the features specifically recited, but does not exclude other elements.

In this application, the term "about" generally means ranging from 0.5% to 10% above or below the specified value, e.g., ranging from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

Drawings

FIG. 1 is a graph showing the separation efficiency of the magnetic beads obtained by the method of example 1

FIG. 2 is a graph showing the activation efficiency of the magnetic beads obtained by the method of example 1;

FIG. 3 is a graph showing amplification factors of magnetic beads obtained by the method of example 1;

FIG. 4 is a graph showing the separation efficiency of the magnetic beads obtained by the method of example 2;

FIG. 5 is a graph showing the activation efficiency of the magnetic beads obtained by the method of example 2;

FIG. 6 is a graph showing amplification factors of magnetic beads obtained by the method of example 2;

FIG. 7 is a graph showing the separation efficiency of the magnetic beads obtained by the method of example 3;

FIG. 8 is a graph showing the activation efficiency of the magnetic beads obtained by the method of example 3;

FIG. 9 is a graph showing amplification factors of magnetic beads obtained by the method of example 3;

FIG. 10 is a graph showing the separation efficiency of the magnetic beads obtained by the method of example 4;

FIG. 11 is a graph showing the activation efficiency of the magnetic beads obtained by the method of example 4;

FIG. 12 is a graph showing amplification factors of magnetic beads obtained by the method of example 4;

FIG. 13 is a graph showing the separation efficiency of the magnetic beads obtained by the method of example 5;

FIG. 14 is a graph showing the activation efficiency of the magnetic beads obtained by the method of example 5;

FIG. 15 is a graph showing amplification factors of magnetic beads obtained by the method of example 5;

FIG. 16 is a graph showing the separation efficiency of magnetic beads obtained by the method of example 6;

FIG. 17 is a graph showing the activation efficiency of the magnetic beads obtained by the method of example 6;

FIG. 18 is a graph showing the amplification factors of magnetic beads obtained by the method of example 6.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

In order to solve the problems of T cell sorting, activation and amplification, the invention adopts a magnetic bead coupling antibody technology to chemically couple CD3 and CD28 antibodies with magnetic beads. Through co-incubation of CD3/CD28 immunomagnetic beads (hereinafter called immunomagnetic beads) and cells, the immunomagnetic beads specifically bind to T cells, and the T cells are separated and purified by a magnetic separation technology, so that the purity of the T cells is improved. The immune magnetic beads are provided with a first messenger CD3 and a second messenger CD28 for activating T cells, so that the T cells can be quickly activated, and the expansion of the T cells is promoted.

Example 1

1. Experimental materials

Carboxyl magnetic beads (nude beads), MES buffer, CB buffer, blocking solution buffer, preservation solution buffer, anti-human CD3 monoclonal antibody, anti-human CD28 monoclonal antibody, EDC, NHS,

2. Reagent preparation

1) MES buffer (molecular weight: 213.25): MES1.59g is weighed and dissolved in 50mL of water for injection, and the pH value is adjusted to be 4.5-6.5 by NaOH.

2) 1mg/mL EDC:0.01g of EDC dry powder was dissolved in 10mL of 0.15M MES (as prepared).

3) 1mg/mL NHS:0.01g of NHS dry powder was dissolved in 10mL of 20mM PBS (Ready to use).

4)PBS：NaCl 8g、KCl 0.2g、Na ₂ HPO ₄ 1.44g、KH ₂ PO ₄ 0.24g, dissolved in 1L of water for injection.

5) Sealing liquid: 20mM PBS+1% BSA.

6) Preservation solution: 20mM PBS+5%o BSA.

3. Experimental method

(1) Magnetic bead coupling

1) And taking out the carboxyl magnetic beads from the refrigerator at the temperature of 2-8 ℃ and fully and uniformly mixing the carboxyl magnetic beads for 1min by a vortex oscillator.

2) The carboxyl magnetic beads are placed into a biological safety cabinet, after being fully and evenly mixed up and down, 100 mu L of carboxyl magnetic beads are taken out, a sterile 1.5mL Ep tube is placed, 1mL MESbuffer with different pH values is added for resuspension according to the following experimental scheme, and the details are shown in the table 1, and the mixture is evenly mixed up and down.

TABLE 1

	Group 1	Group 2	Group 3	Group 4	Group 5
						MES pH value	4.5	5.0	5.5	6.0	6.5

3) The Ep tube was placed on a magnetic rack and allowed to stand for 1min, after which the supernatant was discarded.

4) The tube was removed from the magnet frame, resuspended in 1mL MES buffer at the appropriate pH, and mixed upside down.

5) Repeating step 3) and step 4);

6) The Ep tube was placed on a magnetic stand for 1min, the supernatant was discarded, the supernatant was removed as much as possible, and 300. Mu.L of MES buffer at the corresponding pH was added to resuspend the beads.

7) 30. Mu.L of 1mg/mL EDC was added and mixed with shaking at room temperature for 1h.

8) 1mL PBS was added for resuspension, the Ep tube was allowed to stand on a magnetic rack for 1min, and the supernatant was discarded.

9) The tube was removed from the magnet holder, resuspended in 1mL PBS, mixed upside down, the Ep tube was placed on the magnet holder, allowed to stand for 1min, and the supernatant was discarded.

10 Repeating step 9) 2 times.

11 300. Mu.L PBS was added to the beads, 30. Mu.L of 1mg/mL NHS was added thereto, and the mixture was stirred and mixed at room temperature for 1 hour.

12 Add 1mL PBS to resuspend, put Ep tube on a magnetic rack, stand for 1min and discard supernatant.

13 The tube was removed from the magnet holder, resuspended in 1mL PBS, mixed upside down, the Ep tube was allowed to stand on the magnet holder for 1min and the supernatant was discarded.

14 Step 13) was repeated 2 times and the beads were resuspended by adding 1mL of PBS.

15 The Ep tube was removed from the magnet holder, 600. Mu.L of PBS was added to resuspend the beads, followed by 200. Mu.L of CD3, CD28 monoclonal antibody stock at a concentration of 1mg/mL each.

16 Upside down mixing, shaking and mixing for 2.5 hours at the temperature of 2-8 ℃.

17 Placing the Ep tube on a magnetic rack, standing for 1min, and discarding the supernatant.

18 The tube was removed from the magnet holder, resuspended in 1mL PBS and mixed upside down.

19 Repeating step 17), step 18);

20 Placing the Ep tube on a magnetic rack, standing for 1min, discarding the supernatant, sucking the supernatant as much as possible, and re-suspending the magnetic beads by 1mL of a closed buffer.

21 2-8 ℃ and evenly mixing for 1h.

22 Placing the Ep tube on a magnetic rack, standing for 1min, and discarding the supernatant.

23 The tube was removed from the magnet rack, resuspended in 1mL buffer, and mixed upside down.

24 Repeating step 22), step 23) 2 times;

25 Placing the Ep tube on a magnetic rack, standing for 1min, discarding the supernatant, sucking as completely as possible, and re-suspending the magnetic beads with 200 mu L of preservation solution buffer.

26 If the sealing film is not used for the next step, the pipe orifice is wound, and the pipe orifice is put into 4 ℃ for preservation.

(2) Magnetic bead Activity test

1) Human PBMC cells were resuspended in PBS buffer containing 1% HSA, sampled, counted and labeled CD3 for flow detection.

2) According to the CD3 positive rate of PBMC cells, the CD3+ T cell density was adjusted to 1X 10 with 1% HSA in PBS buffer ⁷ cells/mL。

3) The beads were resuspended (vortexing for no less than 30s, or mixing upside down for 5 min).

4) Transfer to centrifuge tubes according to the relationship between the number of magnetic beads and the incubation ratio of CD3+ T cells 1:1.

5) To the centrifuge tube, l mL of 1% HSA in PBS buffer was added, and the beads were resuspended, blown or vortexed for washing.

6) Placing the centrifuge tube with the resuspended magnetic beads on a magnetic rack (special for the magnetic beads), standing for 1min, discarding the supernatant, and taking care not to suck the magnetic beads.

7) And adding PBMC with the adjusted CD3+ T cell density into the washed magnetic beads according to a proportion, and uniformly mixing.

8) The centrifuge tube is placed on a sample mixer with the rotating speed of 15-30 rpm, and incubated for 30min at room temperature.

9) The centrifuge tube was placed on a magnetic rack (special for magnetic beads), allowed to stand for 1min, and the supernatant was collected and counted and flow tested, labeled CD3.

10 Resuspension of the mixture of magnetic beads and cells with complete medium containing 200IU/mL rh IL-2 and adjustment of the cell density to 1X 10 ⁶ cells/mL。

11 Placing the above cell suspension in a cell culture container at 37deg.C, 5% CO ₂ Incubators were incubated for 48h, samples were counted after removal of the beads and activation efficiencies (cd69+cd25+ percentages) were tested.

12 Observing the cell state and periodically replenishing the liquid when the cell density exceeds 2.5X10) ⁶ When cells/mL or the culture medium turns yellow, the cell density is adjusted to 1X 10 ⁶ cells/mL. Cells were harvested by culturing the cells for 14 days.

4. Experimental results

1) Sorting efficiency

As shown in FIG. 1, as the pH of the MES solution gradually increased, the sorting efficiency was highest when the MES pH was 6.0, indicating that the optimum pH for EDC to react with the carboxyl groups on the beads was 6.0. The specific component separation efficiencies are shown in table 2.

TABLE 2 sorting efficiency

Group 1	Group 2	Group 3	Group 4	Group 5
					20.54	40.82	85.08	98.22	90.31
30.58	48.93	90.58	94.54	85.37

2) Activation efficiency

As shown in FIG. 2, there was no significant difference in activation efficiency between groups 1-5, indicating that the pH of the MES had no effect on activation efficiency. The specific activation efficiencies are shown in table 3.

TABLE 3 activation efficiency

Group 1	Group 2	Group 3	Group 4	Group 5
					80.2	79.1	82.6	81.7	82.5
84.39	82.54	88.49	83.49	87.43

3) Amplification factor

As shown in FIG. 3, the amplification factors of group 3 and group 4 are higher than those of other groups, which indicates that the pH value of MES is between 5.5 and 6.0, and the amplification factors are all acceptable. Specific amplification factors are shown in Table 4.

TABLE 4 amplification factors

In summary, in example 1, the reaction of EDC and carboxyl groups on the magnetic beads was optimized by pH values of different MES, and analysis was performed by 3 indexes of sorting efficiency, activation efficiency, amplification factor, and when experimental data were compared, it was found that when the pH value of MES was 6.0, each index data was superior, so that it was possible to draw conclusions: when the pH value of MES is 6.0, the optimal condition is adopted in the experiment.

Example 2

1. Experimental materials

2. Reagent preparation

The reagent was prepared as in example 1.

3. Experimental method

(1) Magnetic bead coupling

1) And taking out the carboxyl magnetic beads from the refrigerator at the temperature of 2-8 ℃ according to an experimental scheme, and fully and uniformly mixing for 1min by a vortex oscillator.

2) The carboxyl magnetic beads are put into a biological safety cabinet, after being fully and evenly mixed up and down, 100 mu L of carboxyl magnetic beads are taken out, an aseptic 1.5mL Ep tube is put into the biological safety cabinet, and 1mL MES buffer is added for re-suspension, and the biological safety cabinet is evenly mixed up and down.

4) The tube was removed from the magnet rack, resuspended in 1mL MES buffer, and mixed upside down.

5) Repeating step 3) and step 4);

6) The Ep tube was placed on a magnetic rack for 1min, the supernatant was discarded, and the beads were resuspended in 300. Mu.L MES buffer.

7) According to the experimental scheme shown in Table 5, EDC with different volumes was added and mixed by shaking at room temperature for 1h.

TABLE 5

	Group 1	Group 2	Group 3	Group 4
					EDC addition amount	10μL	30μL	50μL	100μL

10 Repeating step 9) 2 times.

19 Repeating step 17), step 18);

21 2-8 ℃ and evenly mixing for 1h.

24 Repeating step 22), step 23) 2 times;

(2) Magnetic bead Activity test

The procedure was as for the magnetic bead activity test of example 1.

4. Experimental results

1) Sorting efficiency

As shown in fig. 4, the sorting efficiency gradually increased as the addition amount of EDC gradually increased. The specific component separation efficiencies are shown in table 6.

TABLE 6 sorting efficiency

Group 1	Group 2	Group 3	Group 4
				33.3	45.71	54.15	60.42
31.88	51.74	56.12	63.19

2) Activation efficiency

As shown in fig. 5, there was no significant difference in the activation efficiencies of groups 1 to 3, and the activation efficiency of group 4 was decreased. It was revealed that the EDC addition amount of 1mg/mL was 10. Mu.L to 50. Mu.L, and had no effect on the activation efficiency. The specific results are shown in Table 7.

TABLE 7 activation efficiency

Group 1	Group 2	Group 3	Group 4
				77	73.3	79.5	68.15
80.6	82.6	77.4	64.42

3) Amplification factor

As shown in FIG. 6, the amplification factors of group 2 and group 3 were higher than those of group 1 and group 4, indicating that the EDC addition amount of 1mg/mL was 10. Mu.L to 50. Mu.L. Specific amplification factors are shown in Table 8.

TABLE 8 amplification factors

In summary, in example 2, the coupling reaction of CD3/CD28 antibody and magnetic beads was performed by different EDC addition amounts, and the experimental data of comprehensive sorting efficiency, activation efficiency and amplification factor found that when 30 μL to 50 μL of EDC was added to 1mg/mL, the result was good, and the cost factor was combined, so that it was possible to draw conclusions: 30. Mu.L of EDC was added at 1mg/mL, which was the optimal condition for the experiment.

Example 3

1. Experimental materials

2. Reagent preparation

The reagent was prepared as in example 1.

3. Experimental method

5) Repeating step 3) and step 4);

10 Repeating step 9) 2 times.

11 300. Mu.L of PBS was used to resuspend the beads, NHS was added as shown in Table 9 and mixed by shaking at room temperature for 1h.

TABLE 9

	Group 1	Group 2	Group 3	Group 4
					NHS addition amount	10μL	30μL	50μL	100μL

14 Step 13) was repeated 2 times and the beads were resuspended by adding 1 mL of PBS.

15 The Ep tube was removed from the magnet holder, 600. Mu.L of PBS was added to resuspend the beads, followed by 200. Mu.L of CD3, CD28 monoclonal antibody stock at a concentration of 1 mg/mL each.

16 Upside down mixing, shaking and mixing at 2-8 ℃ for 2.5-h.

17 Placing the Ep tube on a magnetic rack, standing for 1 min, and discarding the supernatant.

18 The tube was removed from the magnet holder, resuspended in 1 mL PBS and mixed upside down.

19 Repeating step 17), step 18);

20 Placing the Ep tube on a magnetic rack, standing for 1 min, discarding the supernatant, sucking the supernatant as much as possible, and sealing the buffer to resuspend the magnetic beads by 1 mL.

21 2-8 ℃ and evenly mixing by shaking 1-h.

22 Placing the Ep tube on a magnetic rack, standing for 1 min, and discarding the supernatant.

23 The tube is removed from the magnetic frame, added with 1 mL preservation solution buffer for resuspension, and mixed upside down.

24 Repeating step 22), step 23) 2 times;

25 Placing the Ep tube on a magnetic rack, standing for 1 min, discarding the supernatant, sucking as completely as possible, and re-suspending the magnetic beads with 200 mu L of preservation solution buffer.

(2) Magnetic bead Activity test

The procedure was as for the magnetic bead activity test of example 1.

4. Experimental results

1) Sorting efficiency

As shown in fig. 7, as the addition amount of NHS gradually increases, the sorting efficiency gradually increases. The specific sorting efficiencies of the respective groups are shown in table 10.

TABLE 10 sorting efficiency

Group 1	Group 2	Group 3	Group 4
				44.32	58.67	67.97	62.05
46.32	54.67	59.97	60.05

2) Activation efficiency

As shown in FIG. 8, there was no significant difference in activation efficiency between groups 1 and 4, indicating that the addition of NHS at 1mg/mL was between 10. Mu.L and 100. Mu.L without affecting activation efficiency. The specific activation efficiencies of each group are shown in table 11.

TABLE 11 activation efficiency

Group 1	Group 2	Group 3	Group 4
				77.7	84.7	83.4	78.15
70.43	82.8	78.4	72.42

3) Amplification factor

As shown in FIG. 9, the amplification factors of group 1 were low, and there was no significant difference in the amplification factors of group 2 to group 4, indicating that the NHS addition amount of 1mg/mL was 30. Mu.L to 100. Mu.L. Specific amplification factors for each group are shown in Table 12.

TABLE 12 amplification factors

In summary, in example 3, the coupling reaction of CD3/CD28 antibody and magnetic beads was performed by different NHS addition amounts, and the experimental data of comprehensive sorting efficiency, activation efficiency and amplification factor found that when NHS was added at 30. Mu.L to 100. Mu.L per 1mg/mL, the results were good and the cost factor was combined, so that it can be concluded that: when 30. Mu.L of NHS was added at 1mg/mL, the conditions were optimized for this experiment.

Example 4

1. Experimental materials

2. Reagent preparation

The reagent was prepared as in example 1.

3. Experimental method

(1) Magnetic bead coupling

5) Repeating step 3) and step 4);

10 Repeating step 9) 2 times.

15 Ep tube was removed from the magnet holder, 600. Mu.L PBS was added to resuspend the beads, and CD3/CD28 monoclonal antibody stock solutions at different concentrations were added as shown in Table 13.

TABLE 13

	Group 1	Group 2	Group 3	Group 4
					CD3/CD28 monoclonal antibody stock solution concentration	0.5mg/mL	1mg/mL	3mg/mL	5mg/mL
CD3 monoclonal antibody stock solution addition amount	200μL	200μL	200μL	200μL
					CD28 monoclonal antibody stock solution addition amount	200μL	200μL	200μL	200μL

19 Repeating step 17), step 18);

21 2-8 ℃ and evenly mixing for 1h.

24 Repeating step 22), step 23) 2 times;

(2) Magnetic bead Activity test

The procedure was as for the magnetic bead activity test of example 1.

4. Experimental results

1) Sorting efficiency

As shown in FIG. 10, the separation efficiencies of groups 2 to 4 were not significantly different except that the separation efficiency of group 1 was poor, indicating that the separation efficiency was between 1 and 5mg/mL for the antibody concentration. The specific sorting efficiency is shown in Table 14.

TABLE 14 sorting efficiency

Group 1	Group 2	Group 3	Group 4
				42.36	53.03	62.66	66.42
32.72	58.87	57.6	68.19

2) Activation efficiency

As shown in fig. 11, there was no significant difference in activation efficiency between groups 1 to 4, indicating that the antibody concentration had no effect on activation efficiency. The specific activation efficiencies are shown in table 15.

TABLE 15 activation efficiency

Group 1	Group 2	Group 3	Group 4
				87.28	88.07	87.3	85.17
86.76	88.09	88.44	86.42

3) Amplification factor

As shown in FIG. 12, the amplification factors of groups 2 to 4 were higher than that of group 1, and there was no obvious difference between groups 2 to 4, indicating that the concentration of the antibody was between 1 and 5 mg/mL. Specific amplification factors are shown in Table 16.

TABLE 16 amplification factors

In summary, in example 4, by coupling different antibody concentrations with magnetic beads, and integrating experimental data of sorting efficiency, activation efficiency and amplification factor, it was found that when the concentration of CD3 and CD28 antibodies is not less than 1mg/mL, there is no obvious difference between each index, and the cost factor of the antibodies is combined, so that it can be concluded that: the optimal conditions for this experiment were found to be when the CD3 and CD28 antibody concentrations were equal to 1 mg/mL.

Example 5

1. Experimental materials

2. Reagent preparation

The reagent was prepared as in example 1.

3. Experimental method

(1) Magnetic bead coupling

5) Repeating step 3) and step 4);

10 Repeating step 9) 2 times.

15 Ep tube was removed from the magnet holder, 600. Mu.L PBS was added to resuspend the beads, and CD3/CD28 monoclonal antibody stock was added in different ratios as shown in Table 17.

TABLE 17

	Group 1	Group 2	Group 3	Group 4
					CD3/CD28 monoclonal antibody stock solution concentration	1mg/mL	1mg/mL	1mg/mL	1mg/mL
CD3: CD28 monoclonal antibody stock solution addition proportion	0.5:1	1:1	2:1	3:1
					CD3 monoclonal antibody stock solution addition amount	133μL	200μL	267μL	300μL
CD28 monoclonal antibody stock solution addition amount	267μL	200μL	133μL	100μL

19 Repeating step 17), step 18);

21 2-8 ℃ and evenly mixing for 1h.

24 Repeating step 22), step 23) 2 times;

(2) Magnetic bead Activity test

The procedure was as for the magnetic bead activity test of example 1.

4. Experimental results

1) Sorting efficiency: as shown in fig. 13, there was no significant difference in the separation efficiency between groups 1 to 4, indicating that the antibody ratio relationship did not affect the separation efficiency. The specific sorting efficiency is shown in table 18.

TABLE 18 sorting efficiency

Group 1	Group 2	Group 3	Group 4
				42.36	53.03	62.66	66.42
32.72	58.87	57.6	68.19

2) Activation efficiency: as shown in fig. 14, the activation efficiency of group 1 was slightly less than that of the other 3 groups of experiments, and there was no obvious difference in activation efficiency between groups 2 and 4, indicating that the ratio of CD3 and CD28 antibodies was 1-3:1 without influence on the activation efficiency. The specific activation efficiencies are shown in table 19.

TABLE 19 activation efficiency

Group 1	Group 2	Group 3	Group 4
				87.28	88.07	87.3	85.17
86.76	88.09	88.44	86.42

3) Amplification factor: as shown in FIG. 15, the amplification factors of groups 2 to 4 are higher than that of group 1, and no obvious difference exists between groups 2 to 4, which indicates that the ratio of CD3 and CD28 antibodies is 1 to 3:1, and the amplification factors are not influenced. Specific amplification factors are shown in Table 20.

TABLE 20 amplification factors

In summary, in example 5, the magnetic beads were coupled according to the ratio of different CD3 and CD28 antibodies, and the experimental data of comprehensive sorting efficiency, activation efficiency and amplification factor found that when the ratio of CD3 to CD28 is 1-3: 1, each index is better.

Example 6

1. Experimental materials

2. Reagent preparation

The reagent was prepared as in example 1.

3. Experimental method

Alignment according to different coupling modes

Group 1: coupling was performed according to patent application publication number CN110568176a, a method for directional coupling of antibodies and magnetic beads.

Group 2: coupling was performed according to the optimization protocol herein:

5) Repeating step 3) and step 4);

10 Repeating step 9) 2 times.

19 Repeating step 17), step 18);

21 2-8 ℃ and evenly mixing for 1h.

24 Repeating step 22), step 23) 2 times;

(2) Magnetic bead Activity test

The procedure was as for the magnetic bead activity test of example 1.

4. Experimental results

1) Sorting efficiency

As shown in fig. 16, the group 2 sorting efficiency was higher than the group 1, demonstrating that optimizing the coupling method herein can increase the sorting efficiency of CD3/CD28 sorting activated magnetic beads. The specific sorting efficiency is shown in table 21.

TABLE 21 sorting efficiency

Group 1	Group 2
		48.27	68.83
53.34	64.53

2) Activation efficiency

As shown in fig. 17, there was no significant difference in the activation efficiency of group 1, group 2, indicating that the different coupling processes had no effect on the activation efficiency of the cells. The specific activation efficiencies are shown in table 22.

Table 22 activation efficiency

Group 1	Group 2
		88.45	89.58
83.58	85.83

3) Amplification factor

As shown in fig. 18, the expansion ratio of group 2 was higher than that of group 1, indicating that optimizing the coupling method herein can increase the expansion ratio of T cells. Specific amplification factors are shown in Table 23.

TABLE 23 amplification factors

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In summary, example 6 shows that the optimized magnetic bead antibody coupling process is more suitable for coupling CD3/CD28 antibodies according to the experimental data of comprehensive sorting efficiency, activation efficiency and amplification factors by comparing the conventional and optimized magnetic bead antibody coupling processes.

Claims

1. A method of coupling magnetic beads, the method comprising:

9) Standing on a magnetic rack, and discarding supernatant;

2. The coupling process according to claim 1, wherein the pH in step 1) of the coupling process is in the range of 4.5 to 6.5;

preferably, the ratio of MES buffer to added beads used for the resuspension in step 1) is 1mL: 100. Mu.L;

preferably, in the step 1), after the MES buffer is added, the mixing mode is up-down reverse mixing;

preferably, the ratio of MES buffer in step 2) to the magnetic beads added in step 1) is 300. Mu.L: 100. Mu.L;

preferably, the ratio of EDC in step 2) to the magnetic beads added in step 1) is 10-50. Mu.L: 100. Mu.L;

preferably, the method comprises the steps of, mixing uniformly in the step 2) the mode is that the mixture is vibrated and mixed evenly at room temperature;

preferably, the ratio of PBS in step 3) to the magnetic beads added in step 1) is 1mL: 100. Mu.L;

preferably, the mixing mode in the step 3) is upside down mixing;

preferably, the ratio of PBS in step 4) to the magnetic beads added in step 1) is 300. Mu.L: 100. Mu.L;

Preferably, the ratio of NHS in step 4) to the magnetic beads added in step 1) is 10-100. Mu.L: 100. Mu.L;

preferably, the method comprises the steps of, mixing uniformly in the step 4) the mode is that the mixture is vibrated and mixed evenly at room temperature;

preferably, the ratio of PBS in step 5) to the magnetic beads added in step 1) is 1mL: 100. Mu.L;

preferably, the mixing mode in the step 5) is upside down mixing;

preferably, the ratio of PBS in step 6) to the magnetic beads added in step 1) is 600. Mu.L: 100. Mu.L;

preferably, the ratio of CD3, CD28 in step 6) to the magnetic beads added in step 1) is 200-300. Mu.L: 200-100 μl: 100. Mu.L;

preferably, the concentration of CD3 in the step 6) is 1-5mg/mL;

preferably, the concentration of CD28 in step 6) is 1-5mg/mL;

preferably, in the step 6), the mixing mode is 2-8 ℃ shaking mixing;

preferably, the ratio of PBS in step 7) to the magnetic beads added in step 1) is 1mL: 100. Mu.L;

preferably, the mixing mode in the step 7) is upside down mixing;

preferably, the ratio of the closed buffer in the step 8) to the magnetic beads added in the step 1) is 1mL: 100. Mu.L;

preferably, in the step 8), the mixing mode is 2-8 ℃ shaking mixing;

preferably, the ratio of the buffer in the step 10) to the magnetic beads added in the step 1) is 1mL: 100. Mu.L;

Preferably, the mixing mode in the step 10) is upside down mixing;

preferably, the ratio of the preservation solution buffer in the step 11) to the magnetic beads added in the step 1) is 200 μl: 200. Mu.L.

3. The coupling method according to claim 1, wherein the MES buffer component is MES: water for injection = 1.59g:50mL, pH was adjusted to 4.5-6.5.

4. The coupling method according to claim 1, wherein the PBS component is NaCl: KCl: na (Na) ₂ HPO ₄ ：KH ₂ PO ₄ : water for injection = 8g:0.2g:1.44g:0.24g:1L.

5. The coupling process according to claim 1, wherein the concentration of EDC in step 2) is 1mg/mL, and the composition of 1mg/mL EDC is EDC: mes=0.01 g:10mL, the EDC is prepared in a need of being used and matched;

preferably, the concentration of NHS in the step 4) is 1mg/mL, and the components of NHS of 1mg/mL are NHS: pbs=0.01 g:10mL, the NHS requires a ready-to-use and ready-to-prepare.

6. The coupling method according to claim 1, wherein the blocked buffer is composed of 20mM PBS, 1% BSA;

preferably, the composition of the preservation solution buffer is 20mM PBS, 5% BSA.

7. A sort activated magnetic bead obtained according to the method of coupling of magnetic beads according to any one of claims 1 to 6.

8. Use of a sort-activated magnetic bead obtained according to the method of coupling of a magnetic bead according to any one of claims 1-6 for the preparation of a product;

preferably, the product comprises a reagent, a kit and a chromatographic test paper.

9. Use of a sort-activated magnetic bead obtained according to the method of coupling of magnetic beads according to any one of claims 1 to 6 for promoting T cell sorting, activation and/or expansion.

10. Use of a sort-activated magnetic bead obtained according to the method of coupling of magnetic beads according to any one of claims 1 to 6 for increasing the purity of T cells.