CN115161277A

CN115161277A - Method for directly separating immune cells

Info

Publication number: CN115161277A
Application number: CN202210991418.8A
Authority: CN
Inventors: 罗绍祥; 牛梦溪; 舒芹
Original assignee: Cusabio Biotech Co ltd
Current assignee: Cusabio Biotech Co ltd
Priority date: 2022-08-18
Filing date: 2022-08-18
Publication date: 2022-10-11

Abstract

The application relates to the field of biotechnology, in particular to a method for directly separating immune cells; the method comprises the following steps: labeling the coupled antigen by using biotin to obtain a modified antigen; coating a solid phase carrier by using avidin, and then adding a modified antigen to carry out biotin labeling and avidin combination to obtain a modified solid phase carrier; adding lymphocytes on the surface of the modified solid phase carrier, and then cleaning to obtain a carrier enriched with B lymphocytes; adding a lysis buffer solution to the surface of the carrier for lysis to obtain B lymphocytes; wherein the biotin label comprises a cleavable functional group, and the lysis buffer is used for cleaving the cleavable functional group; through the combination of biotin labeling and avidin, antigen is combined with specific antibody on the surface of B lymphocyte, so that other unbound cells are conveniently removed, and meanwhile, the cleavable functional group is cleaved through a lysis buffer solution, so that specific B lymphocyte can be obtained, and the separation efficiency of the B lymphocyte is improved.

Description

Method for directly separating immune cells

Technical Field

The application relates to the field of biotechnology, in particular to a method for directly separating immune cells.

Background

Monoclonal antibodies (mabs) are immunoglobulins that are produced by B cells and are specifically targeted to an antigen. Monoclonal antibodies are not only indispensable tools in biochemical, molecular biological and medical research, but their use in clinical therapy has also revolutionized the treatment of various miscellaneous diseases. With the development of monoclonal antibody technology, the current methods for preparing monoclonal antibodies include not only traditional hybridoma technology, but also Phage display technology (Phage display), human antibody transgenic mice (Human antibody-producing mice), and Single B cell antibody technology (Single B cell antibody technology), and these methods have their respective limitations, but have been widely applied to monoclonal antibody screening.

The current monoclonal antibody technology comprises the steps of: a. primary extraction of lymphocytes from peripheral blood or immune organs; b. isolating specific B cells using cell sorting techniques; c. performing single cell culture on the separated B cells; d. identifying the specificity of the antibody secreted by the single B cells using reverse transcription polymerase chain reaction (RT-PCR) and antibody specific primers; e. amplifying specific antibody genes; f. cloning antibody genes into an expression vector and expressing in a bacterial or cellular system; g. the antibody produced by expression is purified and evaluated by ELISA or the like.

Therefore, in the monoclonal technology, the cell sorting technology is the most critical loop in the technical process of the monoclonal antibody, and the sorting process with high specificity and convenient operation can greatly reduce the difficulty and the workload of subsequent experiments. The current common cell sorting methods include flow cytometry (FACS) and magnetic bead sorting (MACS), however, since lymphocytes extracted from peripheral blood or immune organs contain a large amount of T lymphocytes, macrophages and non-specific B lymphocytes, the conventional FACS and MACS techniques are used for cell sorting, although B lymphocytes can be enriched to a certain extent, other immune cells or substances except for B lymphocytes cannot be effectively separated, and thus the problem of low separation efficiency exists, and therefore, how to improve the separation efficiency of B lymphocytes on the basis of B lymphocyte enrichment is a technical problem which needs to be solved at present.

Disclosure of Invention

The application provides a method for directly separating immune cells, which aims to solve the technical problem that the separation efficiency of B lymphocytes is too low on the basis of enriching the B lymphocytes in the prior art.

In a first aspect, the present application provides a method for directly isolating immune cells, the method comprising:

labeling the coupled antigen by using biotin to obtain a modified antigen;

coating a solid phase carrier by using avidin, and then adding the modified antigen to carry out biotin labeling and avidin combination to obtain a modified solid phase carrier;

adding lymphocytes to the surface of the modified solid phase carrier so as to enable the antigen on the modified solid phase carrier to be specifically combined with the B lymphocytes, and then cleaning to obtain a carrier enriched with the B lymphocytes;

adding a lysis buffer solution to the surface of the carrier for lysis to obtain B lymphocytes;

wherein the biotin label comprises a cleavable functional group and the lysis buffer is used to cleave the cleavable functional group.

Optionally, the cleavable functional group comprises at least one of disulfide bond, dadps, dde, and PC.

Optionally, the biotin label comprises at least one of biotin-SS-NHS ester, sulfo-NHS ester-SS-biotin, and NHS ester-SS-polyethylene glycol-biotin.

Optionally, the lysis buffer comprises a reducing agent.

Optionally, the reducing agent comprises at least one of glutathione, dithiothreitol, and β -mercaptoethanol.

Optionally, the concentration of the glutathione is 45 mmol/L-55 mmol/L.

Optionally, the molar ratio of the biotin label to the antigen is 1.

Optionally, the mass ratio of the modified antigen to the avidin is 1.

Optionally, the avidin comprises at least one of ovalbumin, streptavidin, and neutravidin.

Optionally, the cracking time is more than or equal to 15min.

Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:

according to the method for directly separating immune cells provided by the embodiment of the application, firstly, the combination of the biotin label and the avidin is the strongest non-covalent action at present, so that the antigen and the solid-phase carrier can be firmly combined by the combination of the biotin label and the avidin, and then the antigen and the specific antibody on the surface of the B lymphocyte are combined, so that the target cells can be fixed on the surface of the solid-phase carrier, and other cells which are not combined are conveniently removed.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious for those skilled in the art to obtain other drawings without inventive labor.

Fig. 1 is a schematic flow chart of a method provided in an embodiment of the present application;

FIG. 2 is a graph showing the results of PCT positivity in the culture medium provided in the examples of the present application;

FIG. 3 is a graph showing the results of the cell viability rate in the medium provided in the examples of the present application;

wherein P is <0.001, ns is irrelevant.

Detailed Description

The present invention will be specifically explained below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, 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. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

The inventive thinking of the application is that:

although the hybridoma technology and the phage display technology are widely applied to the production of monoclonal antibodies, the disadvantages that are difficult to overcome still exist and restrict the production process of the antibodies, such as low cell fusion efficiency of the hybridoma technology, loss of natural homologous pairing of heavy chains and light chains caused by the phage display technology and the like. In order to overcome the technical limitations, continuous development and improvement of monoclonal antibodies are of great significance, the cell sorting technology is the most critical loop in the technical process of monoclonal antibodies in the steps of the existing monoclonal antibody technology, and the sorting process with high specificity and convenient operation can greatly reduce the difficulty and workload of subsequent experiments. Common cell sorting methods today include flow cytometric sorting (FACS) and magnetic bead sorting (MACS).

In the flow cytometry sorting, a fluorescent pigment marked on the cell surface is excited by laser with a specific wavelength by utilizing the sorting function of a flow cytometer, and a target cell is sorted out under the action of a high-voltage electric field and finally flows into a collection container. Flow cytometric sorting is a highly accurate and automated method, but the disadvantages of this method are: 1) The equipment is expensive, the market price is high, and great economic pressure is exerted on the early investment and the subsequent development of scientific research; 2) The cells need to be tested by pressure such as laser irradiation, high-voltage electric field, airflow and the like in the sorting process, and the subsequent culture of the cells is not challenged; 3) Usually, the cells are labeled by fluorescent antibodies in advance in flow sorting, the molecular weight of fluorescent protein is large, and the fluorescent protein on the surfaces of the sorted cells is difficult to remove, so that certain restrictions are brought to the development of subsequent experiments; 4) In the flow-type cell sorting process, in order to reduce the mechanical damage to the cells, the sorting flow rate cannot be set too high, if the total amount of the sorted cells is more than 10^9, the time is dozens of hours, and the cost of the machine and the activity of the cells are not little examined.

The magnetic bead sorting is to combine the specific antibody coated on the magnetic bead with the cell surface antigen, and to retain the specific cell in the magnetic field under the action of the magnetic field, so that the cell incapable of being combined with the specific antibody will leave the magnetic field with the mobile phase to separate specific cell. Compared with a flow sorting method, the magnetic bead sorting technology has the advantages of low degree of mechanization, small cell damage and low equipment cost, but still cannot completely replace the flow sorting. Its limitations are: 1) The mechanical degree of magnetic bead sorting is low, although the damage to cells is less than that of flow sorting, if the sorted cells need to be subjected to single cell culture, more manpower is needed for subsequent experiments, and the experiment precision is lower than that of flow sorting; 2) Although the cost of the magnetic bead sorting equipment is lower than that of a flow type sorter, the equipment such as a widely-used magnetic rack still needs tens of thousands to hundreds of thousands, and the material consumption is expensive; 3) If the forward screening is used, cells combined with the magnetic bead specific antibodies are finally obtained, and the magnetic beads are difficult to separate from the cells subsequently, so that the cells are easy to aggregate or precipitate due to too large magnetic beads, and the cells enter the cells along with endocytosis due to too small magnetic beads, so that the biological activity of the cells is influenced; 4) If a reverse screening is used, cells which cannot bind to the specific antibodies on the magnetic beads are finally obtained, so that the influence of the magnetic beads on the activity of the cells can be avoided, but more antibodies are consumed, and the uniformity of the obtained cells is slightly poor.

Other techniques for isolating lymphocytes from peripheral blood have also been disclosed, for example, according to step 1: whole blood sample pretreatment, ficoll method separation, MNC collection and washing, MNC counting, MNC freezing, cell recovery, MNC counting and cell expansion; and 2, step: adding specific antibody labeled by biotin and coupled with streptavidin microvesicle to make target cell labeled by microvesicle, and separating target cell by centrifugation; and step 3: a method for automatically extracting immune cells from adult peripheral blood using an AXP fully automated cell separation apparatus; and 4, step 4: capturing a specific antibody secreted by target lymphocyte B cells by using magnetic particle microspheres of an immobilized antigen, separating the magnetic particle microspheres by using magnetic force, determining a positive hole, improving the internal and external drug effects of the monoclonal antibody through antibody engineering, evaluating the internal drug properties of the monoclonal antibody, and obtaining a pre-clinical candidate antibody. However, the above method has a complicated procedure or a low separation efficiency, and thus it is impossible to obtain a high-purity target cell with high activity in a short time.

Therefore, if the B lymphocytes secreting specific antibodies can be efficiently separated and enriched from immune organ cells or peripheral blood cells, the workload of single B cell culture can be reduced, and the production efficiency can be improved.

In order to solve the technical problems, the embodiment of the invention provides the following general ideas:

in one embodiment of the present application, as shown in fig. 1, there is provided a method of directly isolating immune cells, the method comprising:

s1, labeling a coupling antigen by using biotin to obtain a modified antigen;

s2, coating the solid phase carrier by using avidin, and then adding the modified antigen to combine biotin labeling and avidin to obtain a modified solid phase carrier;

s3, adding lymphocytes to the surface of the modified solid phase carrier to ensure that the antigens on the modified solid phase carrier are specifically combined with the B lymphocytes, and then cleaning to obtain a carrier enriched with the B lymphocytes;

s4, adding a lysis buffer solution to the surface of the carrier for lysis to obtain B lymphocytes;

In some alternative embodiments, the cleavable functional group comprises at least one of disulfide bond, dadps, dde, and PC, wherein the cleavable functional group can be a disulfide bond, dadps, dde, or PC.

In the embodiment of the application, the types of the cleavable functional groups are controlled, and the cleavable functional groups can be cleaved by most of the lysis buffer solution, so that the B lymphocytes with specific antibodies are dissociated from the surface of the solid-phase carrier, a suspension of the B lymphocytes specifically combined with antigens is obtained, and the separation efficiency of the B lymphocytes is improved.

In some alternative embodiments, the biotin label comprises at least one of biotin-SS-NHS ester, sulfo-NHS ester-SS-biotin, and NHS ester-SS-polyethylene glycol-biotin.

In the embodiment of the present application, the type of the biotin label can be controlled to include most of the possible biotin and labels of the cleavable functional group, so that the method of the present application has universality.

In some alternative embodiments, the lysis buffer comprises a reducing agent.

In the embodiment of the application, the lysis buffer solution is controlled to comprise a reducing agent, the reducible functional group in the cleavable functional group is removed in a reduction mode, and the reduction mode is safer and more efficient than the modes between other lysis buffer solutions and the cleavable functional group, so that the biotin label can be completely lysed, the separation speed of the antigen and the solid-phase carrier can be increased, and the separation efficiency of the B lymphocyte can be improved.

In some alternative embodiments, the reducing agent comprises at least one of glutathione, dithiothreitol, and β -mercaptoethanol, wherein the reducing agent can be glutathione, dithiothreitol, or β -mercaptoethanol.

In the embodiment of the application, the type of the reducing agent can be controlled to include most of the types of the biological reducing agent, so that the method has universality.

In some optional embodiments, the concentration of glutathione is 45mmol/L to 55mmol/L, wherein the concentration of glutathione may be 45mmol/L, or 50mmol/L, or 55mmol/L.

In the embodiment of the application, the positive effect of controlling the concentration of glutathione to be 45 mmol/L-55 mmol/L is that in the concentration range, the content of glutathione serving as a reducing agent is sufficient, and the glutathione and a reducing functional group in a cleavable functional group are ensured to be subjected to reduction reaction and cracked, so that an antigen is dissociated from a solid phase carrier, and then B lymphocytes specifically combined with the antigen are separated, and the separation efficiency of the B lymphocytes is improved.

In some alternative embodiments, the molar ratio of the biotin label to the antigen is 1.

In the embodiment of the application, the positive effect of controlling the molar ratio of the biotin label to the antigen is that in the range, the biotin label can be coupled with sufficient antigen, so that the tight combination between the subsequent B lymphocyte with the specific antibody and the antigen is ensured, the subsequent separation between the specifically combined B lymphocyte and the uncombined cell is facilitated, and the separation efficiency of the B lymphocyte is improved.

In some alternative embodiments, the mass ratio of the modified antigen to the avidin is 1.

In the embodiment of the application, the positive effect of controlling the mass ratio of the modified antigen and the avidin is that in the range of the mass ratio, the full combination between the biotin label on the modified antigen and the avidin can be ensured, and the subsequent separation effect on the B lymphocyte can be ensured.

In some alternative embodiments, the avidin comprises at least one of ovalbumin, streptavidin, and neutravidin.

In the embodiments of the present application, the specific type of avidin can be controlled to cover most avidin bound to the biotin label, so that the method of the present application has universality.

In some alternative embodiments, the time for the lysis is ≧ 15min.

In the embodiment of the application, the positive effect of controlling the lysis time to be more than or equal to 15min is that in the time range, the action time of the lysis buffer solution on the cleavable functional groups can be ensured, so that the lysis can be fully performed, the antigen and the solid-phase carrier can be fully separated, and the B lymphocyte and the solid-phase carrier can be completely separated.

Example 1

As shown in fig. 1, a method for directly isolating immune cells, comprising:

s1, using biotin to mark a coupled antigen to obtain a modified antigen, and specifically comprising the following steps:

(1) Dissolving biotin with the concentration of 20mg/mL in a PBS solution, then mixing the biotin and an antigen according to the molecular molar ratio of 1 ^TM Sulfo-NHS-SS-biotin；

(2) Free biotin and other reagents were removed with an ultrafiltration tube, and the antibody was preserved with an antibody preservation solution.

Example 2

Example 2 is compared to example 1, with example 2 differing from example 1 in that:

s2, coating the solid phase carrier with avidin, and then adding the modified antigen to carry out biotin labeling and avidin combination to obtain the modified solid phase carrier, wherein the method specifically comprises the following steps:

(1) Adding 0.8% glutaraldehyde solution into a polystyrene cell culture dish, coating for 2h at 37 ℃, then absorbing and discarding the glutaraldehyde solution, washing the culture dish with sterile water for 3 times, 3min each time, and removing the residual glutaraldehyde solution to obtain a solid phase carrier;

(2) Adding 2 mu g/mL streptavidin solution into a cell culture dish, coating overnight at 37 ℃, then absorbing and discarding the solution, washing the culture dish for 3 times with sterile PBST solution, 3min each time, and removing the redundant streptavidin solution;

(3) Adding 2 mu g/mL biotin-antigen complex into a cell culture dish, coating for 1h at 37 ℃, then sucking away the solution, washing the culture dish for 3 times with sterile PBS (phosphate buffer solution) for 3min each time, and removing redundant antigen;

(4) Adding a BSA solution with the mass concentration of 2% into a cell culture dish, sealing for 1h at 37 ℃, then sucking away the solution, washing the culture dish for 1 time by using sterile PBS, putting the culture dish back into a 37 ℃ incubator, and drying for 1 h-2 h.

Example 3

Example 3 is compared with example 2, which differs from example 2 in that:

s3, adding lymphocytes to the surface of the modified solid phase carrier to enable the antigen on the modified solid phase carrier to be specifically combined with the B lymphocytes, and then cleaning to obtain the carrier enriched with the B lymphocytes, wherein the specific steps are as follows:

(1) Adding 1mL of spleen cells or peripheral blood cells into a centrifuge tube with the volume of 15mL, adding erythrocyte lysate to split the centrifuge tube and fill the centrifuge tube, turning the centrifuge tube upside down for 10min until the liquid is red, centrifuging at 1500rpm for 5min, discarding supernatant, resuspending the cells with 5mL of DMEM solution, centrifuging at 1500rpm for 5min, absorbing the supernatant again, and resuspending the cells with 5mL of DMEM solution to obtain the splenocytes or peripheral blood cells without erythrocytes;

(2) Adding 5mL of lymphocyte separation liquid into a 15mL centrifuge tube, slowly and carefully dropwise adding the cell suspension obtained in the previous step on the surface of the lymphocyte separation liquid, centrifuging at 400G for 20min, dividing the centrifuged cells into four layers, and sequentially adding the following components: DMEM, lymphocytes, separating medium and red blood cells. Sucking the second layer of lymphocytes into a new centrifuge tube carefully, dispersing the cells with 5mL of DMEM (DMEM), centrifuging at 1500rpm for 5min, sucking the supernatant, and suspending the cells with 5mL of DMEM containing 10% FBS (FBS) to obtain lymphocyte suspension;

(3) Adding the collected lymphocyte suspension into the prepared antigen-coated cell culture dish, and then putting the cell culture dish into the cell culture dishCO at 37 ℃ and 5% by volume ₂ Incubating in an incubator for 1h, taking out the culture dish every 20min, and gently shaking;

(4) The supernatant in the culture dish was aspirated, and the culture dish was washed 10 times with DMEM medium containing FBS at a mass concentration of 2% for 1min each time to remove unbound cells.

Example 4

Example 4 is compared with example 3, and example 4 differs from example 3 in that:

s4, adding a lysis buffer solution to the surface of the carrier for lysis to obtain B lymphocytes, and the specific steps comprise:

(1) Adding lysis buffer, processing at room temperature for 15min, and shaking the culture dish for several times to help the cut cells to dissociate, wherein the formula of the lysis buffer comprises: 50mM of glutaminone, 75mM of NaCl,10mM of EDTA, 2% by mass BSA,0.075N NaOH;

(2) And transferring the dissociated cell suspension into a centrifuge tube, washing a culture dish for 1 time by using a DMEM (DMEM) medium containing 2% FBS, transferring the solution into the centrifuge tube, centrifuging at 1500rpm for 5min, sucking and discarding supernatant, and re-suspending cells by using 1mL of PBS solution containing 1% FBS to obtain the target cell suspension.

Example 5

Example 5 is compared to example 4, with example 5 differing from example 4 in that:

detecting cells of the target cell suspension, which comprises the following steps:

(1) Marking the cells in the separated target cell suspension as a 'separated sample', marking the lymphocyte suspension before cell separation as an 'unseparated sample', mixing 10 mu L of each of the two sample cells with trypan blue solution of the same volume, counting by using a cell counter, and calculating the cell activity in the sample;

(2) Irradiating CD40L-NIH-3T3 cells by using Gamma acell1000, counting the treated cells, and preparing an IMDM culture medium of B lymphocytes: FBS with the mass concentration of 10 percent, penicillin-Streptomyces (10000U/mL) with the mass concentration of 1 percent1. Mu.g/L of CPG-ODN,10 ⁸ CD40L-NIH-3T3 cells after irradiation.

(3) Diluting the two cells in (2) to 10 mL with 200mL B lymphocyte IMDM medium ⁴ Mixing the cell suspension, inoculating into 10 96-well plates, adding 200 μ L of cell suspension into each well, placing the cells at 37 deg.C and 5% CO ₂ After culturing for 11-13 days in the incubator, collecting the culture medium supernatant for detection, detecting the titer of the PCT antibody in the culture medium, and calculating the proportion of positive cells, wherein the detection results are shown in Table 1.

TABLE 1

In table 1, the unpaired test is adopted, and the specific results are shown in fig. 2 and fig. 3, it is known that, by adopting the separation method of the present application, reducible biotin and antigen-labeled B lymphocytes are utilized to enable the B lymphocytes to be tightly bound to the solid phase carrier of the cell culture dish, which facilitates the subsequent separation of unbound cells, lysis buffer is utilized to cut biotin, and the reaction between the cleavable functional group in the biotin and the lysis buffer is utilized to enable the antigen-labeled B lymphocytes to be dissociated from the solid phase carrier, so that B cells with higher activity and better specificity can be obtained.

One or more technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

(1) According to the method provided by the embodiment of the application, the antigen and the solid-phase carrier are firmly combined in a biotin labeling and avidin combining mode, and then the antigen is combined with the specific antibody on the surface of the B lymphocyte, so that the target cell can be fixed on the surface of the solid-phase carrier, other cells which are not combined are conveniently removed, meanwhile, the cleavable functional group of the biomarker is cleaved through a lysis buffer solution, the antigen coupled with the biotin is released, the B lymphocyte is also released along with the release of the antigen, so that the specific B lymphocyte can be obtained, the effective separation of the B lymphocyte is realized, and the content of the specific B lymphocyte is improved.

(2) The solid phase carrier may be from 1.27 x 10 in the methods provided in the examples of the present application ⁹ Per m ² ～6.37*10 ⁹ Per m ² Enriching the specific B lymphocytes in the original cells.

(3) The method provided by the embodiment of the application can obtain the target cells with high activity and high purity in a very short time, the whole operation time on the cells can be finished within 1.5-2 h, and the damage on the cell activity is reduced.

(4) The method provided by the embodiment of the application can enable experimenters to more simply and efficiently separate and obtain target cells such as immune cells, hematopoietic stem cells and the like from human peripheral blood and animal immune organs.

It is noted that, in this document, relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrases "comprising 8230; \8230;" comprises 8230; "does not exclude the presence of additional like elements in a process, method, article, or apparatus that comprises the element.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and these ranges or values should be understood to encompass values close to these ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for directly isolating immune cells, the method comprising:

labeling the coupled antigen by using biotin to obtain a modified antigen;

2. The method of claim 1, wherein the cleavable functional group comprises at least one of a disulfide bond, dadps, dde, and PC.

3. The method of claim 2, wherein the biotin label comprises at least one of biotin-SS-NHS ester, sulfo-NHS ester-SS-biotin, and NHS ester-SS-polyethylene glycol-biotin.

4. The method of claim 1, wherein the lysis buffer comprises a reducing agent.

5. The method of claim 4, wherein the reducing agent comprises at least one of glutathione, dithiothreitol, and β -mercaptoethanol.

6. The method according to claim 5, wherein the concentration of glutathione is 45mmol/L to 55mmol/L.

7. The method according to claim 1, wherein the molar ratio of the biotin label to the antigen is 1.

8. The method according to claim 1, wherein the mass ratio of the modified antigen to the avidin is 1.

9. The method of claim 1 or 8, wherein the avidin comprises at least one of ovalbumin, streptavidin, and neutravidin.

10. The method of claim 1, wherein the time for the lysis is 15min or more.