Cell identification kits and methods of use and their use in identifying mast cells

Abstract

The application provides a cell identification kit and a use method and application thereof in identifying mast cells, and relates to the technical field of cell identification. The cell identification kit comprises CD117 antibody conjugated magnetic microparticles, CD16 antibody conjugated magnetic microparticles and Fc epsilon R1 alpha antibody conjugated magnetic microparticles, and FcR blocking agent and eluent. The cell identification kit can be used to identify one or more of CD117, Fc epsilon R1 alpha and CD16 on the surface of cells, and target cells with a purity of at least 90% can be obtained. The application is applied to identifying whether a cell is CD117 + Fc epsilon R1 alpha + CD16 ‑ mast cells, target cells with a purity higher than 95% can be obtained, the degranulation rate of mast cells is improved, and the technical problem of a low degranulation rate of mast cells obtained by a mast cell identification method in the prior art is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell identification, in particular to a cell identification kit and a use method thereof and application thereof in identifying mast cells. BACKGROUND

[0002] The innate immune cells are the first to encounter the 'invaders', including pathogens and allergens, and are the first cells to discover, respond to and regulate or coordinate the overall immune response to these 'invaders'. Therefore, any method of controlling these responses in specific immune cells in the body will affect the overall immune response. In addition to the above characteristics, mast cells (MCs) have the ability to survive for a longer period of time after activation compared to other types of innate immune cells that can die at the beginning of the contraction phase of the innate response. Mast cells are widely distributed around the microvessels under the skin and internal mucosa. They secrete a variety of cytokines and are involved in immune regulation (activation of T cells (immune cells) and APC cells (antigen-presenting cells)). They express MHC (major histocompatibility complex) molecules, B7 molecules, have APC functions, and also express a large number of IgE Fc receptors and release allergic mediators. Therefore, MCs are considered to be a powerful diagnostic and therapeutic target for allergic diseases as key effector cells that cause allergic reactions in allergic diseases.

[0003] At present, the identification method of mast cells mainly relies on: 1) microscopic observation, mast cells are larger than general lymphocytes, are semi-transparent and round in shape, and are full in overall shape; 2) performing a mast cell degranulation verification experiment to determine the function. At present, there is a lack of specific and comprehensive overall identification means for the surface markers of mast cells. The degranulation rate of mast cells obtained by using the above-mentioned mast cell identification method is low, so there is a need for a mast cell identification product to solve the problem of low degranulation rate of mast cells.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] The first object of the present application is to provide a cell identification kit, which can be used to identify one or more molecular characteristics of CD117, FcεRIα and CD16 on the surface of cells, and can obtain target cells with a purity of at least 90%.

[0006] The second object of the present application is to provide a use method for identifying cells by using the above-mentioned cell identification kit.

[0007] The third object of the present application is to provide a cell identification kit or a method for identifying cells, which can effectively identify and collect CD117 + FcεRIα + CD16 - The third object of the present application is to provide a cell identification kit or a method for identifying cells, which can effectively identify and collect CD117 + FcεRIα + CD16 - The third object of the present application is to provide a cell identification kit or a method for identifying cells, which can effectively identify and collect CD117

[0008] To solve the above technical problems, the present application adopts the following technical solutions:

[0009] According to one aspect of the present application, the present application provides a cell identification kit, comprising antibody-coupled magnetic microparticles, an FcR blocker and an eluent.

[0010] The antibody-coupled magnetic microparticles comprise CD117 antibody-coupled magnetic microparticles, CD16 antibody-coupled magnetic microparticles and FcεRIα antibody-coupled magnetic microparticles; the CD117 antibody-coupled magnetic microparticles are obtained by coupling magnetic microparticles and CD117 antibodies at a mass ratio of 100:1; the CD16 antibody-coupled magnetic microparticles are obtained by coupling magnetic microparticles and CD16 antibodies at a mass ratio of 100:1; and the FcεRIα antibody-coupled magnetic microparticles are obtained by coupling magnetic microparticles and FcεRIα antibodies at a mass ratio of 100:1.

[0011] The eluent contains 50 mM sodium dihydrogen phosphate, 50 mM sodium chloride, 50 mM glycine, 10% w / v bovine serum albumin and 10 U / ml cysteine protease.

[0012] Preferably, the working concentration of the CD117 antibody-coupled magnetic microparticles, the CD16 antibody-coupled magnetic microparticles and the FcεRIα antibody-coupled magnetic microparticles is 200 μg / mL.

[0013] Preferably, the cell identification kit further comprises a flow-through buffer, which is a phosphate buffer with a pH of 7.2 and contains 0.5% w / v bovine serum albumin and 2 mM EDTA.

[0014] Preferably, the cell identification kit further comprises a magnetic microparticle diluent, which contains 2.42 g / L tris-hydroxymethyl aminomethane, 9 g / L NaCl, 10 g / L gelatin, 10 g / L bovine serum albumin, 0.372 g / L EDTA and 0.05% w / v Proclin 300.

[0015] According to another aspect of the present application, the present application also provides a method for identifying cells using the above-mentioned cell identification kit, comprising at least using one of the antibody-conjugated magnetic microparticles to magnetically select the cells to be tested;

[0016] Before the magnetic selection, the cells to be tested are treated with an FcR blocking agent;

[0017] The magnetic selection comprises mixing the cells to be tested with the magnetic microparticles and incubating them to obtain a mixed system containing the magnetic microparticles and the cells to be tested; after fixing the magnetic microparticles in the mixed system containing the magnetic microparticles and the cells to be tested, step (I) or step (II) is performed:

[0018] Step (I): the mixed system is washed with a perfusion buffer, and the collected washing solution is the collection solution;

[0019] Step (II): the mixed system is washed with a perfusion buffer, and then the remaining mixed system is eluted with an elution solution, and the collected elution solution is the collection solution;

[0020] When the target surface molecules of the cells to be selected are negative, step (I) is performed; when the target surface molecules of the cells to be selected are positive, step (II) is performed;

[0021] When a plurality of antibody-conjugated magnetic microparticles are used to magnetically select the cells to be tested, the plurality of antibody-conjugated magnetic microparticles are used in series, and the cells in the collection solution are the cells to be tested for the next magnetic selection; the collection solution after the last magnetic selection is the final collection solution, and the cells collected in the final collection solution are the target cells.

[0022] Preferably, the ratio of the number of cells to be tested to the volume of FcR blocking agent is 8×10 4 cells / mL;

[0023] Preferably, the cells to be tested are incubated with the FcR blocking agent at 2-8°C for 30-40 min.

[0024] Preferably, after the cells to be tested are mixed with the magnetic microparticles and incubated, the mixed system is washed with a perfusion buffer, the liquid phase is separated, and the solid phase is resuspended with the perfusion buffer to obtain a suspension containing cells, which is used as the sample to be tested for the first magnetic selection;

[0025] Preferably, the ratio of the number of cells to be tested to the mass of FcεRIα antibody-conjugated magnetic microparticles, CD117 antibody-conjugated magnetic microparticles and CD16 antibody-conjugated magnetic microparticles is independently 1.6×10 3 cells:1μg.

[0026] According to another aspect of the present application, the present application also provides the above-mentioned cell identification kit, or the above-mentioned method for identifying whether the cells are CD117 + FcεRIα+ CD16 - application in mast cells.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] The cell identification kit provided by the present application comprises antibody conjugated magnetic microparticles, FcR blocking agents and eluent. The antibody conjugated magnetic microparticles comprise CD117 antibody conjugated magnetic microparticles, CD16 antibody conjugated magnetic microparticles and FcεRIα antibody conjugated magnetic microparticles, which are used to label CD117 + cells, CD16 + cells and FcεRIα + cells, respectively. The FcR blocking agents are used to avoid non-specific binding of FcεRIα antibodies, CD117 antibodies and CD16 antibodies. The antibody conjugated magnetic microparticles are used in combination with the eluent, so that whether the cell surface has one or more of FcεRIα, CD117 and CD16 molecules can be determined by distinguishing whether the cell is labeled by the specific antibody conjugated magnetic microparticles. The positive cells can be labeled by the antibody conjugated magnetic microparticles and eluted by the eluent, otherwise they are negative cells. The purity of the target cells identified by the kit can be more than 90%. The method for identifying cells provided by the present application is realized by using the above-mentioned cell identification kit, and also has the beneficial effects of the above-mentioned cell identification kit.

[0029] The above-mentioned cell identification kit or the above-mentioned method is used to identify whether the cells are CD117 + FcεRIα + CD16 - mast cells, the purity of CD117 + FcεRIα + CD16 - mast cells in the final collection liquid is higher than 95%, which is 5% higher than the purity of the mast cells captured by using FcεRIα antibody conjugated magnetic microparticles, CD117 antibody conjugated magnetic microparticles or CD16 antibody conjugated magnetic microparticles alone. Moreover, after identification, the degranulation rate of the mast cells is improved, which can be up to 35% and the highest can be up to 38.9%, which produces unexpected technical effects. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the following specific embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0031] Figure 1Figure 3 is a photograph of M2 mature mast cells (test cells) in Example 3 of the present application;

[0032] Figure 2 Figure 4 is a photograph of untreated white blood cells (negative control) in Example 3 of the present application. DETAILED DESCRIPTION

[0033] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0034] Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present application are generally performed according to conventional methods in the art and as described in various general and more specific references that are cited throughout the present specification unless otherwise indicated.

[0035] According to one aspect of the present application, the present application provides a cell identification kit, which comprises antibody conjugated magnetic microparticles, an FcR blocker and an eluent.

[0036] The antibody conjugated magnetic microparticles comprise CD117 antibody conjugated magnetic microparticles, CD16 antibody conjugated magnetic microparticles and FcεRIα antibody conjugated magnetic microparticles; wherein the CD117 antibody conjugated magnetic microparticles are obtained by conjugating magnetic microparticles and CD117 antibodies at a mass ratio of 100:1; the CD16 antibody conjugated magnetic microparticles are obtained by conjugating magnetic microparticles and CD16 antibodies at a mass ratio of 100:1; and the FcεRIα antibody conjugated magnetic microparticles are obtained by conjugating magnetic microparticles and FcεRIα antibodies at a mass ratio of 100:1.

[0037] The eluent contains 50 mM sodium dihydrogen phosphate, 50 mM sodium chloride, 50 mM glycine, 10% w / v bovine serum albumin and 10 U / ml cysteine protease.

[0038] The CD117 antibody conjugated magnetic microparticles are used for labeling cells positive for CD117 on the cell surface (CD117 + cells); the CD16 antibody conjugated magnetic microparticles are used for labeling cells positive for CD16 on the cell surface (CD16 + cells); and the FcεRIα antibody conjugated magnetic microparticles are used for labeling cells positive for FcεRIα on the cell surface (FcεRIα +positive" means that the cell or surface molecule of the cell can specifically bind to the corresponding antibody, and "negative" means that the cell or surface molecule of the cell cannot specifically bind to the corresponding antibody. CD117 + The cell is a cell capable of specifically binding to the CD117 antibody, CD16 + The cell is a cell capable of specifically binding to the CD16 antibody, FcεRIα + The cell is a cell capable of specifically binding to the FcεRIα antibody.

[0039] CD117 is a tyrosine kinase receptor, belonging to the III subclass family of tyrosine kinase receptors; CD16 is a low-affinity Fc receptor, mainly present on the surface of natural killer cells, polymorphonuclear leukocytes, monocytes and macrophages; FcεRI is a high-affinity IgE receptor on the surface of mast cells, which is a membrane glycoprotein composed of multiple subunits, and FcεRIα is one of the subunits.

[0040] The antibody-coupled magnetic microparticles are used in combination with an eluent, and whether the cell surface has one or more of FcεRIα, CD117 and CD16 molecules can be determined by distinguishing whether the cell is labeled by the antibody-coupled magnetic microparticles. Positive cells can be labeled by antibody-coupled magnetic microparticles and eluted by the eluent; negative cells cannot be labeled by antibody-coupled magnetic microparticles. FcR blockers are used to avoid non-specific binding of FcεRIα antibodies, CD117 antibodies and CD16 antibodies.

[0041] Any one of the antibody-coupled magnetic microparticles in the cell identification kit can be used alone to identify whether the cell is a CD117 + cell, CD117 - cell, CD16 + cell, CD16 - cell, FcεRIα + cell or FcεRIα - cell. By experiment, the CD117 antibody-coupled magnetic microparticles in the cell identification kit are used alone to identify the CD117 + mast cells with a purity of greater than 90%; the FcεRIα antibody-coupled magnetic microparticles in the cell identification kit are used alone to identify the FcεRIα + mast cells with a purity of greater than 90%; the CD16 antibody-coupled magnetic microparticles in the cell identification kit are used alone to identify the CD16 - mast cells with a purity of greater than 90%.

[0042] The cells can be identified by using the three antibody conjugated magnetic particles in the kit in series to identify the CD117 + CD16 + cells, CD117 - CD16 + cells, CD117 + CD16 - cells, CD117 - CD16 - cells, CD117 + FcεRIα + cells, CD117 - FcεRIα + cells, CD117 + FcεRIα - cells, CD117 - FcεRIα - cells, CD16 + FcεRIα + cells, CD16 + FcεRIα - cells, CD16-FcεRIα + cells or CD16-FcεRIα-cells.

[0043] The cells can be identified by using the three antibody conjugated magnetic particles in the kit in series to identify the CD117 + FcεRIα + CD16 + cells, CD117 + FcεRIα + CD16 - cells, CD117 + FcεRIα - CD16 + cells, CD117 - FcεRIα + CD16 + cells, CD117 + FcεRIα-CD16 - cells, CD117 - FcεRIα-CD16 + cells, CD117-FcεRIα + CD16-cells or CD117 - FcεRIα - CD16 - cells. By experiments, the three antibody conjugated magnetic particles in the kit are used in series to identify the CD117 +FcεRIα + CD16 - The purity of the mast cells is higher than 95%. Compared with using FcεRIα antibody conjugated magnetic microspheres, CD117 antibody conjugated magnetic microspheres or CD16 antibody conjugated magnetic microspheres to capture CD117 + FcεRIα + CD16 - The purity of the mast cells is increased by 5%. Meanwhile, the mast cells identified by the mast cell identification method of the present application have improved degranulation rate, which can reach 35% and the highest can reach 38.9%, thus producing unexpected technical effects.

[0044] In some alternative embodiments, the working concentration of the CD117 antibody conjugated magnetic microspheres, the CD16 antibody conjugated magnetic microspheres and the FcεRIα antibody conjugated magnetic microspheres is 200 μg / mL.

[0045] In some alternative embodiments, the cell identification kit further comprises a perfusion buffer, which is a phosphate buffer with pH of 7.2 and contains 0.5% w / v bovine serum albumin and 2 mM EDTA.

[0046] In some alternative embodiments, the cell identification kit further comprises a magnetic microspheres diluent, which contains 2.42 g / L Tris, 9 g / L NaCl, 10 g / L gelatin, 10 g / L bovine serum albumin, 0.372 g / L EDTA and 0.05% w / v Proclin 300.

[0047] In some preferred embodiments, the cell identification kit comprises the above-mentioned antibody conjugated magnetic microspheres, the FcR blocking agent, the above-mentioned eluent, the perfusion buffer and the magnetic microspheres diluent. The perfusion buffer is a phosphate buffer with pH of 7.2 and contains 0.5% w / v bovine serum albumin and 2 mM EDTA; the magnetic microspheres diluent contains 2.42 g / L Tris, 9 g / L NaCl, 10 g / L gelatin, 10 g / L bovine serum albumin, 0.372 g / L EDTA and 0.05% w / v Proclin 300.

[0048] According to another aspect of the present application, the present application further provides a method for identifying cells using the above-mentioned cell identification kit, which comprises treating the cells to be identified with an FcR blocking agent to avoid non-specific binding of the FcεRIα antibody, the CD117 antibody and the CD16 antibody, and then performing magnetic selection on the cells to be identified using at least one of the antibody conjugated magnetic microspheres.

[0049] Before the cells to be tested are treated with the blocking agent, the density of the cells to be tested is preferably adjusted to 8 x 10 4 cells / mL. The ratio of the number of cells to be tested to the volume of the FcR blocking agent is preferably 8 x 10 4 cells / mL.

[0050] Each magnetic micro-particle magnetic selection is performed as follows:

[0051] The cells to be tested are mixed with the magnetic micro-particles and incubated to obtain a mixed system containing the magnetic micro-particles and the cells to be tested. After the magnetic micro-particles in the mixed system containing the magnetic micro-particles and the cells to be tested are fixed, step (I) or step (II) is performed:

[0052] Step (I): The mixed system is washed with the flow-through buffer. The cells not labeled with the magnetic micro-particles in the mixed system enter the flow-through buffer, while the magnetic micro-particles cannot flow with the flow-through buffer because they are fixed, thus achieving the separation of the cells not labeled with the magnetic micro-particles from the magnetic micro-particles. The flow-through buffer after washing is collected as the collection liquid, which contains the cells not labeled with the magnetic micro-particles. When the cells to be screened for the target surface molecule are negative, step (I) is performed. The cells to be screened for the target surface molecule are negative and cannot be labeled with the antibody-coupled magnetic micro-particles, and thus enter the flow-through buffer.

[0053] Step (II): The mixed system is washed with the flow-through buffer as described in step (I), which separates the cells not labeled with the magnetic micro-particles from the mixed system. Then, the remaining mixed system is eluted with the elution liquid, which elutes the cells labeled with the magnetic micro-particles from the magnetic micro-particles into the elution liquid. The elution liquid after elution is collected as the collection liquid, which contains the cells that can be labeled with the magnetic micro-particles. When the cells to be screened for the target surface molecule are positive, step (II) is performed. The cells to be screened for the target surface molecule are positive and can bind to the antibodies on the antibody-coupled magnetic micro-particles, and thus enter the elution liquid after elution with the elution liquid.

[0054] When multiple antibody-coupled magnetic micro-particles are used to magnetically select the cells to be tested, the multiple antibody-coupled magnetic micro-particles are used in series. The multiple antibody-coupled magnetic micro-particles are at least two antibody-coupled magnetic micro-particles. The cells in the collection liquid are the cells to be tested for the next magnetic selection. The collection liquid after the last magnetic selection is the final collection liquid. The cells collected in the final collection liquid are the target cells. The use of multiple antibody-coupled magnetic micro-particles in series allows the cells that have been identified as positive or negative for the target surface molecule in the previous step to enter the screening process for the next surface molecule, thus achieving the identification and screening of the expression characteristics of multiple surface molecules of a cell.

[0055] The mixing and incubation of the test cells with the magnetic microparticles is preferably carried out at 2-8°C for 30 minutes; the ratio of the number of test cells to the mass of FcεRIα antibody-coupled magnetic microparticles, CD117 antibody-coupled magnetic microparticles and CD16 antibody-coupled magnetic microparticles is respectively 1.6 x 10 3 The ratio of the number of test cells to the mass of FcεRIα antibody-coupled magnetic microparticles, CD117 antibody-coupled magnetic microparticles and CD16 antibody-coupled magnetic microparticles is respectively 1.6 x 10

[0056] The fixing of the magnetic microparticles is preferably carried out as follows: the fixing of the magnetic microparticles in the mixture containing the magnetic microparticles and the test cells includes placing the mixture in a magnetic environment so that the magnetic microparticles are adsorbed to the reaction device; a specific embodiment can be, but is not limited to, placing the mixture containing the magnetic microparticles and the test cells in a culture dish, placing the culture dish on a magnetic plane so that the magnetic microparticles are fixed to the bottom of the culture dish; or placing the mixture containing the magnetic microparticles and the test cells in a pre-packed column, and placing the pre-packed column in a magnetic environment, which can cause the magnetic microparticles to be adsorbed to the filler in the pre-packed column, thereby being fixed in the pre-packed column, and the magnetic environment can be provided by a magnetic stand.

[0057] In some preferred embodiments, the target cells are CD117 + FcεRIα + CD16 - The target cells are mast cells, and the FcεRIα antibody-coupled magnetic microparticles, the CD117 antibody-coupled magnetic microparticles and the CD16 antibody-coupled magnetic microparticles are used in series;

[0058] The order of the use of the three kinds of magnetic microparticles in series is selected from one of (A) to (D);

[0059] (A) FcεRIα antibody-coupled magnetic microparticles, CD117 antibody-coupled magnetic microparticles and CD16 antibody-coupled magnetic microparticles in sequence:

[0060] The mixing and incubation of the test cells with the FcεRIα antibody-coupled magnetic microparticles to obtain a mixture containing FcεRIα antibody-coupled magnetic microparticles and test cells; after the fixing of the magnetic microparticles in the mixture containing the magnetic microparticles and the test cells, the cells not labeled by the magnetic microparticles are washed away using a flow-through buffer, and then the cells labeled by the magnetic microparticles are eluted using an eluent, and the cells in the eluent are collected as the test cells for the CD117 antibody-coupled magnetic microparticles; the CD117 antibody-coupled magnetic microparticles are subjected to the same magnetic selection as the FcεRIα antibody-coupled magnetic microparticles, and the cells in the eluent are collected as the test cells for the CD16 antibody-coupled magnetic microparticles;

[0061] The cells to be tested are mixed with CD117 antibody conjugated magnetic microparticles and incubated to obtain a mixed system containing CD117 antibody conjugated magnetic microparticles and the cells to be tested; after fixing the magnetic microparticles in the mixed system containing magnetic microparticles and the cells to be tested, the cells not labeled by the magnetic microparticles are washed away using a flow-through buffer, and the flow-through liquid is collected as the final collection liquid; the cells in the final collection liquid are identified as CD16 + FcεRIα + CD16 - mast cells.

[0062] (B) CD117 antibody conjugated magnetic microparticles, FcεRIα antibody conjugated magnetic microparticles, and CD16 antibody conjugated magnetic microparticles in sequence:

[0063] The cells to be tested are mixed with CD117 antibody conjugated magnetic microparticles and incubated to obtain a mixed system containing CD117 antibody conjugated magnetic microparticles and the cells to be tested; after fixing the magnetic microparticles in the mixed system containing magnetic microparticles and the cells to be tested, the cells not labeled by the magnetic microparticles are washed away using a flow-through buffer, and the flow-through liquid is collected as the final collection liquid; the cells in the final collection liquid are identified as CD16

[0064] The cells to be tested are mixed with CD117 antibody conjugated magnetic microparticles and incubated to obtain a mixed system containing CD117 antibody conjugated magnetic microparticles and the cells to be tested; after fixing the magnetic microparticles in the mixed system containing magnetic microparticles and the cells to be tested, the cells not labeled by the magnetic microparticles are washed away using a flow-through buffer, and the flow-through liquid is collected as the final collection liquid; the cells in the final collection liquid are identified as CD16 + FcεRIα + CD16 - mast cells.

[0065] (C) CD16 antibody conjugated magnetic microparticles, FcεRIα antibody conjugated magnetic microparticles, and CD117 antibody conjugated magnetic microparticles in sequence:

[0066] The cells to be tested are mixed with CD117 antibody conjugated magnetic microparticles and incubated to obtain a mixed system containing CD117 antibody conjugated magnetic microparticles and the cells to be tested; after fixing the magnetic microparticles in the mixed system containing magnetic microparticles and the cells to be tested, the cells not labeled by the magnetic microparticles are washed away using a flow-through buffer, and the flow-through liquid is collected as the final collection liquid; the cells in the final collection liquid are identified as CD16

[0067] The cells to be tested are mixed with the FcεRIα antibody conjugated magnetic microparticles and incubated to obtain a mixed system containing FcεRIα antibody conjugated magnetic microparticles and the cells to be tested; after the magnetic microparticles in the mixed system containing magnetic microparticles and the cells to be tested are fixed, the cells not labeled by the magnetic microparticles are removed by using a flow-through buffer, and then the cells labeled by the magnetic microparticles are eluted by using an eluent, and the cells in the eluent are collected as the cells to be tested for the CD117 antibody conjugated magnetic microparticles; the CD117 antibody conjugated magnetic microparticles are magnetically selected in the same way as the FcεRIα antibody conjugated magnetic microparticles, and the eluent is collected as the final collection, and the cells in the final collection are identified as CD117 + FcεRIα + CD16- mast cells.

[0068] (D) CD16 antibody conjugated magnetic microparticles, CD117 antibody conjugated magnetic microparticles and FcεRIα antibody conjugated magnetic microparticles in sequence;

[0069] The cells to be tested are mixed with the CD16 antibody conjugated magnetic microparticles and incubated to obtain a mixed system containing CD16 antibody conjugated magnetic microparticles and the cells to be tested; after the magnetic microparticles in the mixed system containing magnetic microparticles and the cells to be tested are fixed, the cells not labeled by the magnetic microparticles are removed by using a flow-through buffer, and the cells in the flow-through are collected as the cells to be tested for the CD117 antibody conjugated magnetic microparticles;

[0070] The cells to be tested are mixed with the CD117 antibody conjugated magnetic microparticles and incubated to obtain a mixed system containing CD117 antibody conjugated magnetic microparticles and the cells to be tested; after the magnetic microparticles in the mixed system containing magnetic microparticles and the cells to be tested are fixed, the cells not labeled by the magnetic microparticles are removed by using a flow-through buffer, and then the cells labeled by the magnetic microparticles are eluted by using an eluent, and the cells in the eluent are collected as the cells to be tested for the FcεRIα antibody conjugated magnetic microparticles; the FcεRIα antibody conjugated magnetic microparticles are magnetically selected in the same way as the CD117 antibody conjugated magnetic microparticles, and the eluent is collected as the final collection, and the cells in the final collection are identified as CD117 + FcεRIα + CD16 - Mast cells.

[0071] According to another aspect of the present application, the present application also provides application of the above-mentioned cell identification kit or the above-mentioned method for identifying cells to identify whether the cells are CD117 + FcεRIα + CD16 - Mast cells. The above-mentioned cell identification kit or the above-mentioned method for identifying cells is used to identify whether the cells to be tested are CD117 + FcεRIα + CD16 - Mast cells, and CD117 +FcεRIα + CD16 - The mast cells are identified, and the degranulation rate of the mast cells is improved to 35% or even 38.9%, which achieves an unexpected technical effect.

[0072] Preparation of antibody-coupled magnetic bead working solution

[0073] 1. Antibody-coupled magnetic beads:

[0074] 1.1 Preparation of antibodies:

[0075] First antibody: FcεRIα antibody (anti-FceR1 alpha Monoclonal Antibody)

[0076] Second antibody: CD117 antibody (anti-c-Kit(Ab81) Mouse mAb)

[0077] Third antibody: CD16 antibody (anti-CD16(3G8) Mouse mAb)

[0078] 1.2 Preparation of solutions:

[0079] The magnetic bead washing solution contains 1.2114 g / L Tris, 58.44 g / L NaCl, 0.186 g / L EDTA-2Na and 0.005% Tween20.

[0080] The magnetic bead coupling solution contains 2.42 g / L Tris.

[0081] The magnetic bead blocking solution contains 1.21 g / L Tris, 9 g / L NaCl, 1 g / L bovine serum albumin and 0.05% w / v Proclin300.

[0082] The magnetic bead aging solution contains 2.42 g / L Tris, 9 g / L NaCl, 0.186 g / L EDTA-2Na, 10 g / L bovine serum albumin, 0.05% w / v Proclin300 and 10 g / L gelatin.

[0083] The magnetic bead dilution solution contains 2.42 g / L Tris, 9 g / L NaCl, 10 g / L gelatin, 10 g / L bovine serum albumin, 0.372 g / L EDTA and 0.05% w / v Proclin300.

[0084] All solutions are equilibrated at room temperature for 30 minutes before use, and are shaken well.

[0085] 1.3 Preparation of antibody-coupled magnetic bead working solution:

[0086] Take streptavidin magnetic beads, and remove the supernatant by magnetic separation. Add the magnetic bead washing solution to wash. Remove the supernatant by magnetic separation. The FcεRIα antibody, CD117 antibody, and CD16 antibody are all coupled with biotin molecules, and are mixed and coated according to the ratio of magnetic bead mass: antibody mass = 100: 1, and constant temperature shaking for 20 hours. After the completion of the coating reaction, the supernatant is removed by magnetic separation, and the magnetic bead washing solution is added for washing. The supernatant is removed by magnetic separation, and FcεRIα antibody-coupled magnetic beads, CD117 antibody-coupled magnetic beads, and CD16 antibody-coupled magnetic beads are obtained.

[0087] Add the magnetic bead blocking solution to the constant temperature shaker for 5 hours. After the reaction is completed, magnetic separation is performed to remove the supernatant. The magnetic bead washing solution is added for washing. Magnetic separation is performed to remove the supernatant, the magnetic bead aging solution is added, and the reaction is performed in the constant temperature shaker for 20 hours. After the reaction is completed, magnetic separation is performed to remove the supernatant. The magnetic bead washing solution is added for washing. Magnetic separation is performed to remove the supernatant, and the FcεRIα antibody-coupled magnetic beads, CD117 antibody-coupled magnetic beads, and CD16 antibody-coupled magnetic beads are respectively resuspended with the magnetic bead dilution solution, and are respectively transferred to 50 mL centrifuge tubes to prepare antibody-coupled working solutions with a concentration of 200 μg / mL. The FcεRIα antibody-coupled magnetic beads are denoted as magnetic beads a, the CD117 antibody-coupled magnetic beads are denoted as magnetic beads b, and the CD16 antibody-coupled magnetic beads are denoted as magnetic beads c.

[0088] Example 2: Verification of the ability to capture cells

[0089] 2.1 Buffer configuration:

[0090] Perfusion buffer: phosphate buffer containing 0.5% bovine serum albumin, 2 mM EDTA, pH 7.2.

[0091] Elution solution: containing 50 mM sodium dihydrogen phosphate, 50 mM sodium chloride, 50 mM glycine, 10% bovine serum albumin, 10 U / ml cysteine protease.

[0092] 2.2 Sample preparation:

[0093] The human mast cells are centrifuged, and the supernatant is removed and suspended with the above-mentioned phosphate buffer (perfusion buffer). The cell clumps are removed by a nylon mesh filter. The mast cells are washed once with the above-mentioned phosphate buffer, resuspended with the above-mentioned phosphate buffer, and adjusted to 8 x 10 4 cells / mL.

[0094] 2.3 Magnetic labeling:

[0095] 3.2 x 10 4one mast cell (i.e. 0.4 mL), the ratio of the number of mast cells to the volume of FcR blocker was equal to 8 x 103cells / mL. The FcR blocker was added to avoid non-specific binding of the FcεRIα antibody, the CD117 antibody, and the CD16 antibody. Then 20 μg of magnetic beads a, b, and c (i.e. the ratio of the number of mast cells to the mass of the antibody-coupled magnetic beads was equal to 1.6 x 103cells: 1 μg) prepared in Example 1 were added to the three centrifuge tubes, respectively, and incubated at 2-8°C for 30 minutes. The reacted mast cells were washed with 5 mL of the above phosphate buffer and centrifuged at 300 g for 10 minutes to remove the supernatant. The cells were suspended with 500 μl of the above phosphate buffer. 4 cells / mL. The FcR blocker was added to avoid non-specific binding of the FcεRIα antibody, the CD117 antibody, and the CD16 antibody. Then 20 μg of magnetic beads a, b, and c (i.e. the ratio of the number of mast cells to the mass of the antibody-coupled magnetic beads was equal to 1.6 x 103cells: 1 μg) prepared in Example 1 were added to the three centrifuge tubes, respectively, and incubated at 2-8°C for 30 minutes. The reacted mast cells were washed with 5 mL of the above phosphate buffer and centrifuged at 300 g for 10 minutes to remove the supernatant. The cells were suspended with 500 μl of the above phosphate buffer.

[0096] 2.4 Magnetic separation:

[0097] The pre-packed column was installed in a magnetic frame (a magnetic environment was provided to adsorb and fix the magnetic beads), and the pre-packed column was washed with 500 μl of the above phosphate buffer. The three cell suspensions prepared in the above step were added to the corresponding pre-packed columns, respectively, and the pre-packed columns were flushed with 1500 μl of the above phosphate buffer to remove the substances not bound to the FcεRIα antibody, the CD117 antibody, and the CD16 antibody, respectively. The effluent 1 of the FcεRIα antibody-coupled magnetic beads, the effluent 2 of the CD117 antibody-coupled magnetic beads, and the effluent 3 of the CD16 antibody-coupled magnetic beads were collected, respectively. The collection tube was placed under the magnetic frame, and 1 mL of eluent (50 mM sodium phosphate monobasic, 50 mM sodium chloride, 50 mM glycine, 10% bovine serum albumin, and 10 U / ml cysteine protease) was added to the column. The plunger was pushed into the column, and immediately the mast cells bound to the FcεRIα antibody-coupled magnetic beads, the CD117 antibody-coupled magnetic beads, and the CD16 antibody-coupled magnetic beads were eluted to separate them from the magnetic beads, and the eluent 1 of the FcεRIα antibody-coupled magnetic beads, the eluent 2 of the CD117 antibody-coupled magnetic beads, and the eluent 3 of the CD16 antibody-coupled magnetic beads were collected. All experiments were repeated three times.

[0098] The SA magnetic beads (streptavidin magnetic beads) were used to collect the corresponding flow-through buffer and eluent according to the above experimental process.

[0099] 1.5 Cell counting:

[0100] The mast cells in the effluent and the eluent were counted by a cell counter, respectively. The number of mast cells in the eluent of magnetic beads a and magnetic beads b was ≥ 90% of the total number, i.e. the FcεRIα antibody-coupled magnetic beads and the CD117 antibody-coupled magnetic beads were considered to be successfully prepared. The number of mast cells in the flow-through of magnetic beads c was ≥ 90% of the total number, i.e. the CD16 antibody-coupled magnetic beads were considered to be successfully prepared.

[0101] 1.6 Result analysis

[0102] Through the binding of the cells after culture with FcεRIα antibody conjugated magnetic beads and CD117 antibody conjugated magnetic beads, the mast cells can be efficiently captured, other cells are removed, and the purity of the obtained mast cells is improved; through the mixing of the cells after culture with CD16 antibody conjugated magnetic beads, the neutrophils and eosinophils and other non-specifically bound cells in the cells after culture except the mast cells can be better removed. The three methods can all obtain a mast cell suspension with a purity greater than 90%.

[0103] Table 1 cell counting statistics (pieces)

[0104]

[0105] Example 3 Antibody conjugated magnetic beads used in series

[0106] 1. Capture

[0107] The mature mast cells were used as the cells to be tested, and the blood leukocytes were used as the negative cells. Through four combinations, combination 1 (magnetic beads a, magnetic beads b, magnetic beads c used in series in order), combination 2 (magnetic beads b, magnetic beads a, magnetic beads c used in series in order), combination 3 (magnetic beads c, magnetic beads a, magnetic beads b used in series in order), and combination 4 (magnetic beads c, magnetic beads b, magnetic beads a used in series in order). The liquid collection was performed according to the process in Example 2, the target collection liquid of magnetic beads a and magnetic beads b was the elution liquid, and the target collection liquid of magnetic beads c was the flow-through liquid. The target liquid after the above step of capture was used as the sample for the next step of capture, and after 3 times of capture, the final target liquid, i.e., the final cell to be tested. The negative cells were operated synchronously. The experiments were repeated three times.

[0108] If magnetic beads a, magnetic beads c, and magnetic beads b, or magnetic beads b, magnetic beads c, and magnetic beads a are used in series in order, the flow-through buffer is collected between the collection of the elution liquid on both sides, the operation is complex, and the loss of the amount of cells collected is caused due to the existence of non-specific binding, so the method of using magnetic beads a, magnetic beads c, and magnetic beads b, or magnetic beads b, magnetic beads c, and magnetic beads a in series is not adopted.

[0109] 2. Cell counting and photographing

[0110] The cells to be tested and the negative cells were photographed and counted, and the photographing results are shown in FIGS. 1-4, and the counting results are shown in Table 2. Figure 1 and Figure 2

[0111] 3. Result analysis

[0112] ​The results of the capture and separation from different combinations show that the purity of mast cells under the four combinations is higher than 95%, which is 5% higher than the purity of mast cells captured by using one of the FcεRIα antibody coupled magnetic beads, CD117 antibody coupled magnetic beads or CD16 antibody coupled magnetic beads alone. The negative cells cannot be combined with the FcεRIα antibody coupled magnetic beads and the CD117 antibody coupled magnetic beads, resulting in loss of the cells in the process of flowing through the liquid flow, and the number of cells in the eluent is extremely small and can be ignored.

[0113] Table 2 cell count statistics

[0114]

[0115] Example 4: Identification of mast cell degranulation function

[0116] 1. Degranulation stimulation experiment

[0117] The amplification experiment was carried out according to the method of Example 3. The mast cell solutions obtained by the four combinations were centrifuged, resuspended with cell culture solution, and the mast cells not treated by the above magnetic beads obtained by observation under a microscope were used as a control group, and the density of the mast cells was adjusted to 1 x 10 6 cells / mL. 5 x 10 4 cells were added to each well of a 96-well plate. The working concentration of 10% of the dust mite patient positive serum was added to each well, and incubated overnight at 37°C in 5% carbon dioxide. The working concentration of 2% of Triton X-100 lysis solution and the working concentration of 4 μg / mL of IgE antibody were added, and incubated at 37°C in 5% carbon dioxide for 1 hour. A 2.5 mg / mL hexokinase substrate solution was prepared with citric acid buffer (pH 4.5, 0.05 mol / L), and a working concentration of 0.09 mg / mL was added. After incubation, the liquid in each well was centrifuged at 300g for 5 minutes, and 20 uL of supernatant was transferred to a substrate enzyme-labeled plate well, and mixed well. Incubate in a 37°C incubator for 2 hours, add 200 μL of stop solution to each well. Measure the OD value at 405 nm, and calculate the degranulation rate.

[0118] 2. Cell degranulation rate statistics

[0119] Table 3 degranulation rate statistics

[0120]

[0121] 3. Results analysis

[0122] The degranulation rates of the four combinations are all greater than or equal to 35%, and greater than the control group (using mast cells not treated by the magnetic beads by observing under a microscope), proving that the purity of the purified mast cells is improved, and the degranulation function of the mast cells is normal. The degranulation efficiency of combination 2 (CD117 antibody coupled magnetic beads, FcεRIα antibody coupled magnetic beads, CD16 antibody coupled magnetic beads) is the highest, reaching 38.9%, which is increased by 53.15% compared with the control group, and unexpected technical effects are produced. The degranulation efficiency of combination 1 (FcεRIα antibody coupled magnetic beads, CD117 antibody coupled magnetic beads, CD16 antibody coupled magnetic beads) is the lowest, being 37.9%, which is increased by 49.21% compared with the control group, and unexpected technical effects are also produced.

[0123] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method of using a cell identification kit, characterized in that, The kit is used to sort mast cells with high degranulation rates as target cells. The cell identification kit includes antibody-conjugated magnetic microparticles, an FcR blocking agent, and an elution buffer. The antibody-coupled magnetic microparticles include CD117 antibody-coupled magnetic microparticles, CD16 antibody-coupled magnetic microparticles, and FcεRIα antibody-coupled magnetic microparticles. The CD117 antibody-conjugated magnetic microparticles are obtained by conjugating magnetic microparticles and CD117 antibody at a mass ratio of 100:

1. The CD16 antibody-conjugated magnetic microparticles are obtained by conjugating magnetic microparticles and CD16 antibody at a mass ratio of 100:

1. The FcεRIα antibody-conjugated magnetic microparticles are obtained by conjugating magnetic microparticles and FcεRIα antibody at a mass ratio of 100:

1. The eluent contains 50 mM sodium dihydrogen phosphate, 50 mM sodium chloride, 50 mM glycine, 10% w / v bovine serum albumin and 10 U / ml cysteine ​​protease. The target cells were CD117 cells. + FcεRIα + CD16 - Mast cells; the method includes magnetic separation of test cells by using FcεRIα antibody-conjugated magnetic microparticles, CD117 antibody-conjugated magnetic microparticles and CD16 antibody-conjugated magnetic microparticles in series. The cells to be tested were treated with an FcR inhibitor before magnetic separation; The magnetic separation includes mixing and incubating the test cells with magnetic microparticles to obtain a mixed system containing magnetic microparticles and test cells; after immobilizing the magnetic microparticles in the mixed system containing magnetic microparticles and test cells, step (I) or step (II) is performed: Step (I): Wash the mixture with flow buffer and collect the washed flow buffer as the collection solution; Step (II): Wash the mixture with flow buffer, then elute the remaining mixture with elution buffer, and collect the eluent as the collection solution; When using CD16 antibody-conjugated magnetic microparticles for magnetic separation, perform step (I); when using CD117 antibody-conjugated magnetic microparticles or FcεRIα antibody-conjugated magnetic microparticles for magnetic separation, perform step (II). The cells in the collection solution are the cells to be tested in the next magnetic separation; the collection solution of the last magnetic separation is the final collection solution, and the cells collected in the final collection solution are the target cells; The order in which the three magnetic particles are used in series is selected from one of (A) to (D); (A) FcεRIα antibody-conjugated magnetic microparticles, CD117 antibody-conjugated magnetic microparticles, and CD16 antibody-conjugated magnetic microparticles in sequence; (B) CD117 antibody-conjugated magnetic microparticles, FcεRIα antibody-conjugated magnetic microparticles, and CD16 antibody-conjugated magnetic microparticles in sequence; (C) CD16 antibody-conjugated magnetic microparticles, FcεRIα antibody-conjugated magnetic microparticles, and CD117 antibody-conjugated magnetic microparticles in sequence; (D) CD16 antibody-conjugated magnetic microparticles, CD117 antibody-conjugated magnetic microparticles, and FcεRIα antibody-conjugated magnetic microparticles in sequence.

2. The method of use according to claim 1, characterized in that, The working concentrations of the CD117 antibody-conjugated magnetic microparticles, the CD16 antibody-conjugated magnetic microparticles, and the FcεRIα antibody-conjugated magnetic microparticles are 200 μg / mL.

3. The method of use according to claim 1, characterized in that, The flow buffer is a phosphate buffer with a pH of 7.2, and contains 0.5% w / v bovine serum albumin and 2 mM EDTA.

4. The method of use according to claim 1, characterized in that, The cell identification kit also includes a magnetic microparticle diluent; the magnetic microparticle diluent contains 2.42 g / L tris(hydroxymethyl)aminomethane, 9 g / L NaCl, 10 g / L gelatin, 10 g / L bovine serum albumin, 0.372 g / L EDTA and 0.05% w / v Proclin 300.

5. The method of use according to claim 1, characterized in that, The cell identification kit includes antibody-conjugated magnetic microparticles, FcR blocking agent, elution buffer, flow buffer, and magnetic microparticle diluent; the flow buffer is a phosphate buffer with a pH of 7.2 and contains 0.5% w / v bovine serum albumin and 2 mM EDTA; the magnetic microparticle diluent contains 2.42 g / L tris(hydroxymethyl)aminomethane, 9 g / L NaCl, 10 g / L gelatin, 10 g / L bovine serum albumin, 0.372 g / L EDTA, and 0.05% w / v Proclin 300.

6. The method of use according to any one of claims 1 to 5, characterized in that, The ratio of the number of cells to be tested to the volume of FcR inhibitor was 8 × 10⁻⁶. 4 cells / mL.

7. The method of use according to any one of claims 1 to 5, characterized in that, The number of cells to be tested and the FcR inhibitor were incubated at 2-8°C for 30-40 minutes.

8. The method of use according to any one of claims 1 to 5, characterized in that, After mixing and incubating the cells to be tested with magnetic microparticles, the mixture was washed with flow buffer, the liquid phase was separated, and the solid phase was resuspended with flow buffer to obtain a suspension containing cells, which was used as the sample to be tested for the first magnetic separation.

9. The method of use according to any one of claims 1 to 5, characterized in that, The number of cells to be tested was independently determined by the mass ratio of FcεRIα antibody-conjugated magnetic microparticles, CD117 antibody-conjugated magnetic microparticles, and CD16 antibody-conjugated magnetic microparticles to 1.6 × 10⁻⁶. 3 cells: 1 μg.

10. The method of use according to any one of claims 1 to 9 for identifying whether cells are CD117 + FcεRIα + CD16 - Application in mast cells.

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