Method for rapidly separating igg-positive b cells

Abstract

Provided in the present invention are a micro-droplet screening system and the use thereof. Specifically, by means of the micro-droplet screening system of the present invention, a method for detecting hybridoma cells and splenic B cells of immunized mice is provided, thereby enabling the identification of antibodies on the surface of memory B cells and intracellular IgG in plasma cells without relying on cell viability.

Description

A rapid method for isolating IgG positive B cells

[0001] This application claims priority to the invention patent application No. 2025103322927 filed on March 19, 2025, all contents and elements of which are incorporated herein by reference for all purposes. Technical Field

[0002] This invention belongs to the field of immunology and biotechnology, specifically relating to a method for rapid isolation and screening of IgG positive B cells based on microfluidic technology. Background Technology

[0003] Humoral immunity is the core mechanism of the body's immune defense, which relies on antibodies produced by B cells to recognize, neutralize, and eliminate pathogens.

[0004] Antibody therapy plays an important role in disease prevention, diagnosis and treatment, and works in synergy with cellular immunity to maintain the body's immune balance.

[0005] Plasma cells and memory B cells are the main sources of antibodies. Among them, IgG, as the most abundant and functionally diverse antibody type in the body, plays an important role in long-term immune protection and the development of therapeutic antibodies.

[0006] However, traditional antibody screening techniques suffer from problems such as low efficiency, dependence on cell viability, dependence on cell culture medium, and complex operation.

[0007] Therefore, developing an efficient, rapid, and more versatile method for isolating IgG-positive B cells can not only accelerate the development of antibody drugs but also provide strong technical support for disease diagnosis and personalized treatment, thus possessing significant scientific research value and clinical application prospects. Summary of the Invention

[0008] This invention provides a method for rapid isolation and screening of IgG-positive B cells based on microfluidic technology. This method can efficiently identify surface antibodies on memory B cells and intracellular IgG on plasma cells, and is applicable to antibody drug development, antibody screening for infectious diseases, and personalized immunotherapy.

[0009] In a first aspect of the invention, a screening system for sorting immunoglobulin-positive cell populations is provided, the screening system comprising microdroplets, the microdroplets comprising a cell phase and an antibody phase, the antibody phase comprising antiglobulin antibodies.

[0010] In another preferred embodiment, the immunoglobulin comprises: IgG, IgA, IgM, IgD, IgE, or a combination thereof.

[0011] In another preferred embodiment, the anti-immunoglobulin antibody is a free anti-globulin antibody.

[0012] In another preferred embodiment, the immunoglobulin is IgG.

[0013] In another preferred embodiment, the "antiglobulin antibody" refers to an antibody against immunoglobulins.

[0014] In another preferred embodiment, the antiglobulin antibody is an anti-IgG antibody.

[0015] In another preferred embodiment, the immunoglobulin is an immunoglobulin derived from humans or non-human mammals.

[0016] In another preferred embodiment, the non-human mammals include: mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, sheep, and non-human primates.

[0017] In a second aspect of the invention, a screening system for sorting IgG-positive cell populations is provided, the screening system comprising microdroplets, the microdroplets comprising a cell phase and an antibody phase, the antibody phase comprising anti-IgG antibodies.

[0018] In another preferred embodiment, the IgG positive cell population contains IgG. + B cells and / or IgG + Plasma cells.

[0019] In another preferred embodiment, the diameter of the microdroplets is 10-100 μm, more preferably 50 μm.

[0020] In another preferred embodiment, the microdroplets are an array of microdroplets.

[0021] In another preferred embodiment, the cellular phase comprises a group of cells to be sorted.

[0022] In another preferred embodiment, the cell population comprises hybridoma cells and / or mouse spleen B cells.

[0023] In another preferred embodiment, the cell phase contains a buffer solution suitable for the cell population to be sorted.

[0024] In another preferred embodiment, the buffer solution suitable for the cell population to be sorted comprises a PBS solution.

[0025] In another preferred embodiment, the cell phase comprises: a population of cells to be sorted, and a PBS solution.

[0026] In another preferred embodiment, the cell phase does not contain a culture medium.

[0027] In another preferred embodiment, the total volume of the microdroplets is 100-500 μL, more preferably 200-300 μL, and most preferably 216 μL.

[0028] In another preferred embodiment, the ratio of the cell phase to the antibody phase is 1:2 to 2:1, preferably 1:1.

[0029] In another preferred embodiment, the anti-IgG antibody is a fluorescently labeled anti-IgG antibody.

[0030] In another preferred embodiment, the fluorescent marker is an AF488 fluorescent marker.

[0031] In another preferred embodiment, the fluorescent marker is an AF647 fluorescent marker.

[0032] In another preferred embodiment, the concentration of anti-IgG antibody in the antibody phase is 0.5%-3%, more preferably 1%.

[0033] In another preferred embodiment, the concentration of the anti-IgG antibody refers to a volume concentration v / v.

[0034] In another preferred embodiment, the antibody phase further comprises: a metal ion chelating agent and / or a nonionic detergent.

[0035] In another preferred embodiment, the metal ion chelating agent comprises EDTA.

[0036] In another preferred embodiment, the nonionic detergent is Tween-20.

[0037] In another preferred embodiment, the antibody phase further comprises EDTA and / or Tween-20.

[0038] In another preferred embodiment, the concentration of EDTA in the antibody phase is 0.01 mM-1 mM, more preferably 0.05 mM-0.5 mM, and most preferably 0.1 mM.

[0039] In another preferred embodiment, the concentration of Tween-20 in the antibody phase is 0.01%-0.5%, more preferably 0.1%.

[0040] In another preferred embodiment, the concentration of Tween-20 refers to a volume concentration v / v.

[0041] In another preferred embodiment, the antibody phase comprises anti-IgG antibody, EDTA, and Tween-20, wherein the anti-IgG antibody is a fluorescently labeled anti-IgG antibody.

[0042] In another preferred embodiment, the antibody phase further comprises: an RNase inhibitor.

[0043] In another preferred embodiment, the antibody phase solution and the cell phase solution are the same.

[0044] In another preferred embodiment, the antibody phase solution is a PBS solution.

[0045] In another preferred embodiment, the cell phase includes hybridoma cells and / or mouse spleen B cells.

[0046] In another preferred embodiment, the microdroplet encapsulation rate is: each microdroplet contains 1-3 cells, preferably 1 cell.

[0047] In another preferred embodiment, the screening system does not contain a culture medium.

[0048] In another preferred embodiment, the cells in the cellular phase have a damaged cell membrane barrier.

[0049] In another preferred embodiment, the screening system is prepared by a method comprising the following steps:

[0050] (S1-1) Provide a cell phase containing a population of cells to be sorted;

[0051] (S1-2) Provide an antibody phase comprising anti-IgG antibody;

[0052] (S1-3) The cell phase and the antibody phase are mixed, and the mixture of cell phase and antibody phase is cut with an immiscible oil phase to prepare a screening system containing microdroplets.

[0053] In a third aspect of the invention, the use of the screening system described in the second aspect of the invention is provided for sorting IgG positive cell populations.

[0054] In another preferred embodiment, the IgG-positive cell population includes IgG-positive cells on the cell membrane surface and IgG-positive cells intracellularly.

[0055] In another preferred embodiment, the IgG positive cell population includes a population of memory B cells that are IgG positive and / or a population of plasma cells that are IgG positive.

[0056] In another preferred embodiment, the sorting rate is 85%-95%, more preferably 95%.

[0057] In another preferred embodiment, it is used to screen for and isolate antigen-specific antibodies, and / or to detect antibody efficacy.

[0058] In another preferred embodiment, the use is for non-diagnostic and non-therapeutic purposes.

[0059] In a fourth aspect of the invention, a sorting system for sorting cells expressing antibodies specific to a target antigen is provided, the sorting system comprising microdroplets, the microdroplets comprising a cell phase, an antibody phase, and an antigen phase;

[0060] The cell phase includes the cell population to be sorted.

[0061] The antibody phase contains antiglobulin antibodies.

[0062] The antigen phase contains the target antigen.

[0063] In another preferred embodiment, the antiglobulin antibody is not fluorescently labeled.

[0064] In another preferred embodiment, the target antigen is a fluorescently labeled target antigen.

[0065] In another preferred embodiment, the antiglobulin antibody is a fluorescently labeled antiglobulin antibody, and the fluorescence of the labeled antiglobulin antibody is different from the fluorescence of the labeled target antigen.

[0066] In another preferred embodiment, the sorting system is prepared by a method comprising the following steps:

[0067] (S1-1) Provide a cell phase containing a population of cells to be sorted;

[0068] (S1-2) Provide an antibody phase comprising an antiglobulin antibody;

[0069] (S1-3) Provide an antigen phase containing a target antigen;

[0070] (S1-4) The cell phase, the antibody phase, and the antigen phase are mixed, and the mixture of the cell phase, antibody phase, and antigen phase is cut using an immiscible oil phase to prepare the sorting system.

[0071] In another preferred embodiment, the "antiglobulin antibody" refers to an antibody against immunoglobulins.

[0072] In another preferred embodiment, the antiglobulin antibody is an anti-IgG antibody.

[0073] In a fifth aspect of the invention, the use of the screening system described in the first or second aspect of the invention, or the sorting system described in the fourth aspect of the invention, is provided for screening antibodies specific to a target antigen.

[0074] In another preferred embodiment, the target antigen comprises: disease-related endogenous antigens and exogenous antigens.

[0075] In another preferred embodiment, the disease-related endogenous antigens include: tumor-related antigens, autoimmune disease and inflammation-related antigens, metabolic and cardiovascular disease-related antigens, or combinations thereof.

[0076] In another preferred embodiment, the exogenous antigen comprises: pathogen proteins, biotoxins, allergens, or combinations thereof.

[0077] In another preferred embodiment, the pathogen protein comprises a pathogen protein derived from an organism selected from the group consisting of viruses, bacteria, fungi, and parasites.

[0078] In a fifth aspect of the invention, a method for sorting immunoglobulin-positive cell populations is provided, the method comprising the following steps:

[0079] (S1) The screening system according to the first aspect of the present invention is provided, the screening system comprising microdroplets;

[0080] (S2) Incubate the microdroplets;

[0081] (S3) Microdroplets with immunoglobulin-positive cells are sorted from the incubated microdroplets to obtain an immunoglobulin-positive cell population.

[0082] In another preferred embodiment, the "antiglobulin antibody" refers to an antibody against immunoglobulins.

[0083] In another preferred embodiment, the antiglobulin antibody is an anti-IgG antibody.

[0084] In a sixth aspect of the invention, a method for sorting IgG-positive cell populations is provided, the method comprising the following steps:

[0085] (S1) Provides the screening system according to the second aspect of the present invention, the screening system comprising microdroplets, the microdroplets comprising a cell phase and an antibody phase, the antibody phase comprising fluorescently labeled anti-IgG antibody;

[0086] (S2) Incubate the microdroplets;

[0087] (S3) Image the incubated microdroplets and sort out the microdroplets with fluorescent signals to obtain IgG positive cell populations.

[0088] In another preferred embodiment, step (S1) includes the following steps:

[0089] (S1-1) A mixture is provided by mixing a cell phase containing the cell population to be sorted with an antibody phase containing anti-IgG antibodies;

[0090] (S1-2) Microdroplets are prepared by cutting the mixture obtained by the immiscible oil phase.

[0091] In another preferred embodiment, step (S1) includes the following steps:

[0092] (S1-1) Provide a cell phase containing a population of cells to be sorted;

[0093] (S1-2) Provide an antibody phase comprising anti-IgG antibody;

[0094] (S1-3) The cell phase and the antibody phase are mixed, and the mixture of cell phase and antibody phase is cut with an immiscible oil phase to prepare a screening system containing microdroplets.

[0095] In another preferred embodiment, the flow of the cell phase, antibody phase, and oil phase is all driven by gas pressure.

[0096] In another preferred embodiment, the cellular phase and the antibody phase are driven by the same pressure.

[0097] In another preferred embodiment, the cell phase and the antibody phase are respectively driven to flow by gas pressure and mixed to provide a mixture.

[0098] In another preferred embodiment, the oil phase is driven to flow by gas pressure, thereby cutting the mixture to prepare a screening system containing microdroplets.

[0099] In another preferred embodiment, the driving pressure between the cell phase and the antibody phase is 1-5 psi, more preferably 3.50 psi.

[0100] In another preferred embodiment, the oil phase drive pressure is 6-20 psi, more preferably 10.00 psi.

[0101] In another preferred embodiment, in step (S2), the incubation temperature is 0-10°C, preferably 4°C.

[0102] In another preferred embodiment, the incubation temperature is 4°C.

[0103] In another preferred embodiment, in step (S2), the incubation time is 2-8 hours, more preferably 2-6 hours, even more preferably 3-6 hours, and most preferably 4 hours.

[0104] In another preferred embodiment, the incubation time is 4 hours.

[0105] In another preferred embodiment, in step (S2), the incubation refers to incubation at 4°C for 4 hours.

[0106] In another preferred embodiment, in step (S2), the microdroplets are incubated, and after incubation, the screening system has one or more of the following characteristics: loss of cell activity, decrease in cell membrane permeability, cell survival, or stability of the microdroplets.

[0107] In another preferred embodiment, in step (S2), the microdroplets are incubated at 4°C for 4 hours, after which the cell activity is lost.

[0108] In another preferred embodiment, in step (S2), the cell membrane permeability decreases after the microdroplets are incubated at 4°C for 4 hours.

[0109] In another preferred embodiment, in step (S2), the cells remain viable after the microdroplets are incubated at 4°C for 4 hours.

[0110] In another preferred embodiment, in step (S2), the microdroplets remain stable after being incubated at 4°C for 4 hours.

[0111] In another preferred embodiment, in step (S3), the imaging is performed by imaging the microdroplets in the screening system using a microscope.

[0112] In another preferred embodiment, the microscope is a fluorescence microscope.

[0113] In another preferred embodiment, the method is a method for non-diagnostic and non-therapeutic purposes.

[0114] In another preferred embodiment, the following steps are also included:

[0115] (S4) Select cells expressing the target antigen-specific antibody from the IgG positive cell population to obtain the target antigen-specific antibody.

[0116] In another preferred embodiment, the method further includes the step of:

[0117] (S5) Verify the structure and / or function of the target antigen-specific antibody.

[0118] In a seventh aspect of the invention, a method for sorting cells expressing antibodies specific to a target antigen is provided, the method comprising the following steps:

[0119] (S1) The screening system according to the first aspect of the present invention is provided, the screening system comprising microdroplets;

[0120] (S2) Incubate the microdroplets;

[0121] (S3) Screening out immunoglobulin-positive cell populations from the incubated microdroplets;

[0122] (S4) Select cells expressing antibodies specific to the target antigen from the immunoglobulin-positive cell population.

[0123] In an eighth aspect of the invention, a method for sorting cells expressing antibodies specific to a target antigen is provided, the method comprising the following steps:

[0124] (S1) A sorting system according to the fourth aspect of the present invention is provided, the sorting system comprising microdroplets;

[0125] (S2) Incubate the microdroplets;

[0126] (S3) Select cells expressing target antigen-specific antibodies from the incubated microdroplets.

[0127] In another preferred embodiment, in step (S3), microdroplets that are positive for both immunoglobulins and target antigens are sorted from the incubated microdroplets to obtain cells expressing antibodies specific to the target antigen.

[0128] In another preferred embodiment, the sorting includes: fluorescent sorting and magnetic sorting.

[0129] In another preferred embodiment, in step (S3), a cell population containing both fluorescent signals labeled with antiglobulin antibodies and fluorescent signals labeled with target antigens is sorted from the incubated microdroplets to obtain cells expressing antibodies specific to the target antigen.

[0130] In a ninth aspect of the invention, an apparatus for sorting IgG-positive cell populations is provided, the apparatus comprising the following modules:

[0131] (Z1) Microdroplet preparation module, the microdroplet preparation module being configured to: provide the screening system according to the second aspect of the present invention, the screening system comprising microdroplets; wherein, a cell phase comprising a cell population to be sorted is mixed with an antibody phase comprising anti-IgG antibody to prepare microdroplets;

[0132] (Z2) Microdroplet incubation module, wherein the microdroplet incubation module is configured to incubate the microdroplets;

[0133] (Z3) Sorting module, the sorting module is configured to: image the incubated microdroplets, sort out the microdroplets with fluorescent signals, and thus obtain IgG positive cell populations.

[0134] In another preferred embodiment, the device further includes the following module: (Z4) a detection and identification module configured to detect the obtained cell population and determine that the obtained cell population is positive for IgG.

[0135] In another preferred embodiment, the microdroplet preparation module further includes:

[0136] (Z1-1) Two-phase mixing module, the two-phase mixing module being configured to: mix a cell phase containing a population of cells to be sorted with an antibody phase containing anti-IgG antibodies, thereby providing a mixture;

[0137] (Z1-2) Microdroplet generation module, wherein the microdroplet generation module is configured to: prepare microdroplets by cutting the mixture obtained by the immiscible oil phase.

[0138] In another preferred embodiment, the microdroplet preparation module further includes:

[0139] (Z1-1) Cell phase providing module, the cell phase providing module being configured to: provide a cell phase, the cell phase comprising a population of cells to be sorted;

[0140] (Z1-2) Antibody phase providing module, the antibody phase providing module being configured to provide an antibody phase containing anti-IgG antibody;

[0141] (Z1-3) Two-phase mixing and microdroplet generation module, wherein the two-phase mixing and microdroplet generation module is configured to: mix the cell phase and the antibody phase, and use an immiscible oil phase to cut the mixture of the cell phase and the antibody phase, thereby preparing a screening system containing microdroplets.

[0142] In another preferred embodiment, the microdroplet incubation module is configured to incubate the microdroplets at 4°C for 4 hours.

[0143] In another preferred embodiment, the diameter of the microdroplets is 10-100 μm, more preferably 50 μm.

[0144] In another preferred embodiment, the volume of the microdroplet is 100-500 μL, more preferably 200-300 μL, and most preferably 216 μL.

[0145] In another preferred embodiment, the ratio of the cell phase to the antibody phase is 1:2 to 2:1, preferably 1:1.

[0146] In another preferred embodiment, the anti-IgG antibody is a fluorescently labeled anti-IgG antibody.

[0147] In another preferred embodiment, the fluorescent marker is an AF647 fluorescent marker.

[0148] In another preferred embodiment, the concentration of anti-IgG antibody in the antibody phase is 0.5%-3%, more preferably 1%.

[0149] In another preferred embodiment, the concentration of the anti-IgG antibody refers to a volume concentration v / v.

[0150] In another preferred embodiment, the antibody phase further comprises EDTA and / or Tween-20.

[0151] In another preferred embodiment, the concentration of EDTA in the antibody phase is 0.01 mM-1 mM, more preferably 0.05 mM-0.5 mM, and most preferably 0.1 mM.

[0152] In another preferred embodiment, the concentration of Tween-20 in the antibody phase is 0.01%-0.5%, more preferably 0.1%.

[0153] In another preferred embodiment, the concentration of Tween-20 refers to a volume concentration v / v.

[0154] In another preferred embodiment, the antibody phase solution is a PBS solution.

[0155] In another preferred embodiment, the cell phase includes hybridoma cells and mouse spleen B cells.

[0156] In another preferred embodiment, the microdroplet encapsulation rate is: each microdroplet contains 1-3 cells, preferably 1 cell.

[0157] In another preferred embodiment, the cell phase solution is a PBS solution.

[0158] In another preferred embodiment, the IgG-positive cell population includes IgG-positive cells on the cell membrane surface and IgG-positive cells intracellularly.

[0159] In another preferred embodiment, the IgG positive cell population includes a population of memory B cells that are IgG positive and / or a population of plasma cells that are IgG positive.

[0160] In another preferred embodiment, the device further includes the following module: (Z4) an antibody screening module configured to screen cell populations containing target antigen-specific antibodies from IgG-positive cell populations, thereby obtaining target antigen-specific antibodies.

[0161] In a tenth aspect of the present invention, an apparatus for sorting and expressing antibodies specific to a target antigen is provided, the apparatus comprising the following modules:

[0162] (Z1) Microdroplet preparation module, the microdroplet preparation module being configured to: provide the sorting system according to the fourth aspect of the present invention, the sorting system comprising microdroplets;

[0163] (Z2) Microdroplet incubation module, wherein the microdroplet incubation module is configured to incubate the microdroplets;

[0164] (Z3) Sorting module, the sorting module being configured to: sort cells expressing target antigen-specific antibodies from the incubated microdroplets.

[0165] In another preferred embodiment, microdroplets that are positive for both immunoglobulins and target antigens are sorted from the incubated microdroplets to obtain cells expressing antibodies specific to the target antigen.

[0166] In another preferred embodiment, cell populations containing both fluorescent signals labeled with antiglobulin antibodies and fluorescent signals labeled with target antigens are sorted from the incubated microdroplets to obtain cells expressing antibodies specific to the target antigen.

[0167] In another preferred embodiment, the sorting refers to sorting using dielectrophoresis.

[0168] In another preferred embodiment, the module (Z3) further includes an antibody acquisition module configured to isolate target antigen-specific antibodies from the cells expressing target antigen-specific antibodies.

[0169] In another preferred embodiment, the device further includes the following modules:

[0170] A verification module is configured to verify the structure and / or function of the target antigen-specific antibody.

[0171] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0172] Figure 1 shows the results of verifying the package buffer system at different time points.

[0173] Figure 2 shows that anti-IgG antibodies can specifically bind to hybridoma cells.

[0174] Figure 3 shows that the anti-IgG antibody can specifically bind to mouse spleen B cells.

[0175] Figure 4 shows the recognition of membrane antibodies and intracellular antibodies by anti-IgG antibodies.

[0176] Figure 5 shows the characteristics analysis of antibody heavy chain, light chain, and heavy-light chain pairings. Specifically, Figure 5A shows the distribution of the heavy chain variable region (IGHV) gene family used in the screened antibodies; Figure 5B shows the distribution of the light chain variable region (IGKV / IGLV) gene family used in the screened antibodies; and Figure 5C shows the distribution of heavy chain–light chain (VH–VL) pairings used in the screened antibodies.

[0177] Figure 6 shows the in vitro expression verification of intact IgG antibodies secreted by cell clones in HEK293 cells.

[0178] Figure 7 shows the binding activity of the antibodies obtained from screening and ELISA validation to the target antigen. Representative antibodies G2 and H2 (both with the same light chain gene composition, IGHV1 / IGKV4) that were successfully expressed in vitro were selected, and their binding ability to the target antigen Gn protein was detected by ELISA. Detailed Implementation

[0179] Through extensive and in-depth research, the inventors unexpectedly discovered a microdroplet screening system for the first time. Based on this system, high-throughput, high-enrichment, and high-resolution rapid sorting of memory B cell surface antibodies can be achieved without relying on culture media, while also efficiently sorting intracellular IgG from plasma cells. This sorting method is independent of cell viability, has high sample throughput, short sorting time, high recovery rate, and is not limited by sample volume. This invention was developed based on this discovery.

[0180] Specifically, this invention provides a novel microfluidic screening technology that, by detecting hybridoma cells and B cells from the spleen of immunized mice, can identify surface antibodies on memory B cells and intracellular IgG on plasma cells without depending on cell viability, significantly reducing screening time. This will provide a new paradigm for antibody screening against infectious diseases.

[0181] This invention uses IgG as an example, sorting IgG-positive cell populations to screen for antibodies specific to the target antigen. It should be noted that those skilled in the art can select other types of immunoglobulins according to actual needs, thereby screening for antibodies specific to the target antigen by sorting cell populations positive for any immunoglobulin.

[0182] In a specific embodiment, the anti-immunoglobulin antibody of the present invention is a free anti-globulin antibody. In a specific embodiment, the anti-immunoglobulin antibody is not immobilized on microspheres, chips, or carriers.

[0183] the term

[0184] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0185] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0186] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0187] Where a numerical range is provided, unless the context clearly indicates otherwise, it should be understood that every intermediate integer of the value, every tenth of every intermediate integer of the value, any intermediate value between the upper and lower limits of the range, and any other intermediate value within the specified range are included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered within the scope of this invention, but are subject to any express exclusions within the specified range. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0188] As used herein, the terms "encapsulation system," "microdroplet system," "screening system," and "encapsulation buffer system" are interchangeable and all refer to the microfluidic system described in this invention.

[0189] In this invention, in order to distinguish the oil generated by encapsulating droplets, the oil generated by encapsulating droplets is called the oil phase, and the two phases composed of cells and antibodies are collectively referred to as the aqueous phase, which can also be called the reagent phase.

[0190] In this invention, the "buffer solution suitable for the cell population to be sorted" refers to a liquid environment that can stabilize the pH, not harm the cells, and allow the cells to survive / function normally. In specific embodiments, the buffer solution suitable for the cell population to be sorted does not include culture medium. In specific embodiments, the buffer solution suitable for the cell population to be sorted includes PBS solution, Hanks solution, etc.

[0191] Cell sorting

[0192] Cell sorting is a technique for isolating specific cell types from a cell population, playing a crucial role in life science research and clinical applications. Its core objective is to obtain highly pure, viable target cells to meet the needs of subsequent experiments or treatments.

[0193] Cell sorting is based on various differences in cell characteristics. Physical characteristics, such as cell size and density, can be separated using methods like centrifugation. Differences in surface antigen expression among different cells form the basis for immunomagnetic bead sorting and flow cytometry.

[0194] During immunomagnetic bead sorting, the magnetic beads are linked to specific antibodies against the surface antigens of the target cells. Under an external magnetic field, the target cells carrying the magnetic beads are separated.

[0195] Flow cytometry is more advanced. Cells are prepared as single-cell suspensions and passed in a single file through a laser-irradiated area while encased in sheath fluid. Cells generate different light signals due to their inherent characteristics. These signals are detected and analyzed, and the target cells are charged and deflected under an electric field for collection. However, flow cytometry is generally not suitable for intracellular protein detection. Insufficient cell permeabilization prevents large antibody molecules from entering the cells, resulting in a low positive rate. Conversely, excessive antibody addition leads to high background noise and insufficient differentiation between positive and negative populations, which has always been a bottleneck issue.

[0196] Cell sorting has wide applications in many fields. In basic research, it helps scientists obtain specific cell types to explore cell function and developmental mechanisms. In tumor research, it allows the isolation of tumor cells and immune cells from tumor tissue to study tumor development and immune escape mechanisms. In clinical applications, hematopoietic stem cell sorting is of great significance for hematopoietic stem cell transplantation to treat blood diseases; circulating tumor cell sorting helps in the early diagnosis and monitoring of tumors.

[0197] Traditional methods for screening IgG-positive populations in memory B cells and plasma cells often have many limitations, such as dependence on cell viability, single isolation target, long time consumption, high cost, high sample requirements, and strong equipment dependence and complexity.

[0198] Specifically, traditional methods (such as flow cytometry FACS and magnetic bead sorting MACS) require cells to remain viable to ensure labeling efficiency. However, prolonged sample storage or fixation treatment can lead to cell death, affecting the separation effect. Therefore, the inability to handle frozen, fixed, or long-term stored samples limits the flexibility of clinical and research scenarios.

[0199] Flow cytometry typically targets only surface antibodies (such as CD27+IgG+ on memory B cells), while IgG in plasma cells is expressed intracellularly, requiring an additional membrane permeation step, which is complex and may damage the cells. Magnetic bead sorting relies on specific surface markers and cannot simultaneously separate IgG-positive populations in memory B cells and plasma cells. Therefore, neither method can efficiently and simultaneously analyze surface antibodies (memory B cells) and intracellular antibodies (plasma cells), requiring multiple sorting steps and resulting in low efficiency.

[0200] Traditional methods require multiple rounds of labeling, sorting, and validation (such as FACS sorting combined with ELISA validation), which takes hours to days and relies on expensive equipment (such as flow cytometers). This makes it difficult for traditional methods to meet the needs of rapid antibody screening (e.g., emergency research and development during infectious disease outbreaks).

[0201] Flow cytometry or magnetic bead sorting requires a large number of starting cells (10^6 to 10^7), making it unsuitable for small samples (such as clinical biopsy samples). Furthermore, flow cytometry requires specialized operators and complex data analysis, while magnetic bead sorting is susceptible to non-specific binding interference. These characteristics limit the application of these methods in resource-constrained scenarios (such as personalized medicine), and the high technical barriers also make it difficult to popularize them in primary care laboratories or clinical institutions.

[0202] Therefore, rapid sorting systems and methods for IgG-positive populations in memory B cells and plasma cells with high retention, high recovery rate, high sample throughput, and unlimited sample size have significant research prospects and market value.

[0203] The microfluidic system of the present invention

[0204] The microfluidic system of this invention, which is also the screening system of this invention, is independent of cell viability: This invention overcomes the limitations of traditional methods on cell viability, simplifies the droplet encapsulation of buffer solution, and can encapsulate cells in immobilized or frozen samples with oil droplets, as long as the cells to be tested still have a cell membrane as a physical barrier, especially since frozen cells do not require a thawing step. Therefore, it is suitable for long-term preservation of samples or clinical pathological archives, expanding its application scenarios.

[0205] Simultaneous separation of IgG-positive populations of memory B cells and plasma cells: Simultaneous identification of surface markers of memory B cells and intracellular IgG of plasma cells achieves "two birds with one stone" sorting, thus simplifying the operation process and avoiding cell loss caused by multi-step sorting.

[0206] Rapid and efficient (screening time reduced by more than 50%): By integrating sample processing, labeling, and detection steps through a microfluidic chip, the entire process can be completed within 1 hour, significantly improving screening efficiency. Therefore, it meets the needs of rapid development of infectious disease antibodies (such as the enrichment of IgG-positive cells in the early stages of screening neutralizing antibodies for viral variants).

[0207] Low sample consumption and high sensitivity: Microfluidic chips can process minute samples (down to 10^3 cells) and improve detection sensitivity through signal amplification techniques (such as nanoscale fluorescent labeling). Therefore, they are suitable for precious clinical samples (such as tumor-infiltrating B cells) or small animal models (such as mouse spleen B cells).

[0208] Lightweight and Low-Cost Equipment: This invention utilizes microfluidic chips in conjunction with portable detection systems, reducing equipment costs. Furthermore, it can be freely combined with other microfluidic chips with sorting capabilities, eliminating the need for expensive flow cytometers and lowering the technical barrier. In addition, single-indicator sorting can be achieved through methods such as conjugating anti-IgG antibodies with magnetic beads for magnetic enrichment, thus facilitating downstream antibody discovery processes (e.g., adding antigen proteins as a second indicator to directly obtain antigen-specific IgG cells, enabling more rapid discovery of monoclonal antibodies). Therefore, it promotes the widespread adoption of this technology in primary healthcare institutions and resource-limited areas.

[0209] Microdroplets obtained by replacing the oil used in the screening system of the present invention with other materials, or microdroplets obtained by further encapsulating a single layer of oil droplets, all belong to the screening system of the present invention.

[0210] In the microfluidic system of this invention, the antibody type (anti-IgG antibody), reagent components (PBS), microdroplet size (50 μm), and the concentration of each substance in each phase are all designed and screened through extensive experiments. The microfluidic system of this invention can achieve high-throughput, high-enrichment, and high-resolution rapid sorting of memory B cell surface antibodies without relying on culture medium, and can also efficiently sort intracellular IgG in plasma cells.

[0211] The method of the present invention

[0212] The method of the present invention refers to the method for sorting IgG positive cell populations, the method comprising the following steps:

[0213] (S1) A cell phase containing the cell population to be sorted is mixed with an antibody phase containing 1% anti-IgG antibody in a 1:1 ratio to prepare a microdroplet array, wherein the solutions of the cell phase and the antibody phase are both PBS solutions.

[0214] (S2) Incubate the microdroplets at 4°C for 4 hours;

[0215] (S3) Image the incubated microdroplets and sort out the microdroplets with fluorescent signals to obtain IgG positive cell populations.

[0216] In the method of the present invention, the microdroplet with fluorescence signal refers to the microdroplet whose fluorescence signal is significantly higher than the background fluorescence signal under a fluorescence microscope. The term "significantly higher" means that the microdroplet with fluorescence signal is visible to the naked eye compared to the background fluorescence signal (fluorescence noise), or the microdroplet with fluorescence intensity is obtained by analysis using software for quantitative fluorescence analysis.

[0217] The imaging results of this invention can more accurately identify positive cells by integrating localization information; furthermore, by increasing the number of detection antibodies, double-positive cell screening or even multi-indicator screening can be achieved. In addition to cell identification, detection indicators can be added to achieve simultaneous identification and detection. For example, after anti-IgG labels IgG positive cells, an apoptosis indicator can be added to detect the proportion of apoptotic IgG positive cells.

[0218] The method of this invention addresses the activity dependence, low efficiency, and equipment barriers of traditional methods, providing innovative solutions for the following scenarios: For example, infectious disease emergency response: rapid screening of neutralizing antibodies (e.g., Ebola); tumor immunotherapy: efficient isolation of tumor-specific IgG+ B cells from patient samples; autoimmune disease research: analysis of antibody secretion profiles of plasma cells and memory B cells under pathological conditions; vaccine development evaluation: direct detection of the ratio of memory B cells to plasma cells and antibody characteristics after vaccine immunization; application methods in infectious disease antibody screening, tumor immunotherapy, and autoimmune disease research; specific application procedures in vaccine development evaluation, etc.

[0219] Specifically, the method of the present invention can be further used for the isolation of monoclonal antibodies, such as the isolation of IgG cells from the spleen of mice or rabbits after immunization, which can then be used for the preparation of hybridoma cells; the addition of antigens such as PE-labeled antigens can be used to screen antigen-specific IgG cells, thereby rapidly isolating antigen-specific antibodies; vaccine efficacy can be evaluated by detecting the proportion of antigen-specific IgG cells in peripheral blood to detect antibody efficacy; other target protein antibodies can be used to replace the anti-IgG antibody in the present invention, such as IgE antibodies for detecting allergic reactions, etc. Theoretically, the method of the present invention can complete the detection of any target protein.

[0220] In the method of this invention, the driving pressures of the cell phase, antibody phase, and oil phase affect the formation of microdroplets and the state of the cells. Excessively high overall pressures can affect the stability of the droplet formation process and increase cell damage; excessively low pressures can prolong droplet formation time, affect cell viability, and increase the risk of microsphere aggregation and blockage. The relative flow rates caused by the difference in driving pressure between the cell phase and the reagent phase affect the proportion of each phase in the droplet, influencing the encapsulation rate of cells and microspheres, as well as the reaction efficiency within the droplet.

[0221] The method of the present invention also includes a method for sorting cells expressing target antigen-specific antibodies. In a specific embodiment, the droplet contains both fluorescently labeled anti-IgG antibodies and fluorescently labeled target antigens, using different fluorescence for the two labels. If the cells are positive, the anti-IgG antibody binds to the "constant region" of IgG, while the target antigen binds to the "variable region" of IgG. Through these two fluorescence signals, cells that simultaneously highly express IgG antibodies and exhibit target antigen specificity are sorted. In a specific embodiment, an IgG-positive cell population can be screened first, followed by screening for antigen-specific cells.

[0222] The device of the present invention

[0223] The apparatus of the present invention refers to a set of devices for sorting IgG positive cell populations corresponding to the method of the present invention, the device comprising a microdroplet preparation module, a microdroplet incubation module, and a sorting module.

[0224] Specifically, the present invention provides an apparatus for sorting IgG positive cell populations, the apparatus comprising the following modules:

[0225] (Z1) Microdroplet preparation module, the microdroplet preparation module being configured to: provide the screening system according to the first aspect of the present invention, the screening system comprising microdroplets; mix a cell phase containing a cell population to be sorted with an antibody phase containing anti-IgG antibodies to prepare microdroplets;

[0226] (Z2) Microdroplet incubation module, wherein the microdroplet incubation module is configured to incubate the microdroplets;

[0227] (Z3) Sorting module, the sorting module is configured to: image the incubated microdroplets, sort out the microdroplets with fluorescent signals, and thus obtain IgG positive cell populations.

[0228] In another preferred embodiment, the microdroplet incubation module is configured to incubate the microdroplets at 4°C for 4 hours.

[0229] In another preferred embodiment, the device further includes the following module: (Z4) a detection and identification module configured to detect the obtained cell population and determine that the obtained cell population is positive for IgG.

[0230] In another preferred embodiment, the diameter of the microdroplets is 10-100 μm, more preferably 50 μm.

[0231] In another preferred embodiment, the volume of the microdroplet is 100-500 μL, more preferably 200-300 μL, and most preferably 216 μL.

[0232] In another preferred embodiment, the ratio of the cell phase to the antibody phase is 1:2 to 2:1, preferably 1:1.

[0233] In another preferred embodiment, the anti-IgG antibody is a fluorescently labeled anti-IgG antibody.

[0234] In another preferred embodiment, the fluorescent marker is an AF647 fluorescent marker.

[0235] In another preferred embodiment, the concentration of anti-IgG antibody in the antibody phase is 0.5%-3%, more preferably 1%.

[0236] In another preferred embodiment, the concentration of the anti-IgG antibody refers to a volume concentration v / v.

[0237] In another preferred embodiment, the antibody phase further comprises: a metal ion chelating agent and / or a nonionic detergent.

[0238] In another preferred embodiment, the metal ion chelating agent comprises EDTA.

[0239] In another preferred embodiment, the nonionic detergent is Tween-20.

[0240] In another preferred embodiment, the antibody phase further comprises EDTA and / or Tween-20.

[0241] In another preferred embodiment, the concentration of EDTA in the antibody phase is 0.01 mM-1 mM, more preferably 0.05 mM-0.5 mM, and most preferably 0.1 mM.

[0242] In another preferred embodiment, the concentration of Tween-20 in the antibody phase is 0.01%-0.5%, more preferably 0.1%.

[0243] In another preferred embodiment, the concentration of Tween-20 refers to a volume concentration v / v.

[0244] In another preferred embodiment, the antibody phase further comprises: an RNase inhibitor.

[0245] In another preferred embodiment, the antibody phase solution is a PBS solution.

[0246] In another preferred embodiment, the cell phase includes hybridoma cells and mouse spleen B cells.

[0247] In another preferred embodiment, the microdroplet encapsulation rate is: each microdroplet contains 1-3 cells, preferably 1 cell.

[0248] In another preferred embodiment, the cell phase solution is a PBS solution.

[0249] In another preferred embodiment, the IgG-positive cell population includes IgG-positive cells on the cell membrane surface and IgG-positive cells intracellularly.

[0250] In another preferred embodiment, the IgG positive cell population includes a population of memory B cells that are IgG positive and / or a population of plasma cells that are IgG positive.

[0251] In a specific implementation, the device includes a cell sorting module containing a miniature chip configured to actuate magnetic beads upon electrolysis of a positive single droplet. These magnetic beads are simultaneously coupled with magnetic beads containing both antibody heavy and light chain mRNA. The device can be used in point-of-care research to analyze patient BCR repertoire profiles, specifically for rapidly acquiring sufficient BCR profile information; particularly suitable for bedside placement and diagnostic applications; and especially applicable to large-scale clinical cohort studies. In another preferred embodiment, single-cell target gene combinatorial sequencing or single-cell transcript sequencing can also be achieved through customization of magnetic beads containing other target genes.

[0252] In a specific implementation, the device includes an antibody DNA module, which contains a PCR module (microchip). Specifically, before the microdroplet preparation module, the cell phase is pretreated so that the cells contain microspheres (containing barcodes) containing reagents required for reverse transcription and PCR amplification. The generated droplets are subjected to a PCR reaction, resulting in the generation of an antibody DNA sequence in each microdroplet (the antibody heavy and light chains are synthesized into a linear DNA sequence using fusion PCR). The generated droplets undergo PCR in each chamber of a microplate. Subsequently, all the DNA is collected using conventional methods for subsequent sequencing or antibody expression. The device is used to obtain antibodies, and the generated linear DNA contains paired heavy and light chain sequences.

[0253] The main advantages of this invention include:

[0254] (a) The microfluidic system of the present invention has simple components and can simultaneously separate IgG positive populations of memory B cells and plasma cells, achieving efficient and simultaneous sorting of two antigen-specific IgG positive cells (memory B cells and plasma cells).

[0255] (b) Based on the microfluidic system of the present invention, antibody detection independent of cell activity can be achieved through anti-IgG antibody labeling strategies and other methods of designing and using specific binding cell target proteins.

[0256] (c) The microfluidic system of the present invention can perform rapid screening and high-throughput analysis, and can complete sample processing to result output within 1 hour, significantly shortening the screening time; and can process multiple samples at a time, making it suitable for large-scale antibody screening.

[0257] (d) The antibody detection method of the present invention has the advantages of low sample consumption and high antibody detection sensitivity. Specifically, it requires only a small amount of sample (as low as 10^3 cells) and is supplemented by signal amplification technology (such as nanoscale fluorescent labeling) to improve detection sensitivity.

[0258] (e) The antibody detection method of the present invention is simple to operate, uses lightweight equipment, and is low in cost. Specifically, the microfluidic chip used can be paired with portable detection devices, reducing the equipment cost and operational threshold of antibody detection.

[0259] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0260] Example 1: Experimental method.

[0261] First, the encapsulation system was constructed. Frozen mouse spleen cells / human peripheral blood total B cells were thawed, and 3 mL of PBS was added. After centrifugation at 300g for 3 min, the supernatant was discarded. The cells were resuspended in 1 mL of PBS, and 18 μL of the cell suspension and 2 μL of 0.4% trypan blue solution were used to determine cell viability and count the cells. Then, two-phase reagents were prepared according to the system shown in Table 1. The base solution for both phases was PBS, and the final total volume was 216 μL. The prepared cell phase and antibody phase were encapsulated 1:1. A 50 μm droplet generation chip (microdroplet array) was used to generate uniform microdroplets with a diameter of about 50 μm. The droplets contained cells and reagent components (i.e., antibody phase components). The obtained microdroplets were placed at 4 degrees Celsius and the incubation time was optimized. Finally, the reaction was observed under a microscope.

[0262] Table 1 Formulations of two-phase solvents

[0263] Secondly, a mouse infection model was constructed. Overnight cultured KP strain (Klebsiella pneumoniae) was washed with PBS and centrifuged at 8000 rpm for 10 min at 4°C. The supernatant was collected, 1 mL of paraformaldehyde was added, and the mixture was fixed at room temperature for 30 min. The mixture was centrifuged again at 8000 rpm at 4°C, the supernatant was discarded, and the sample was washed with PBS. This step was repeated once. 1 mL of PBS was added to the fixed sample for resuspending. When using aluminum adjuvant, 50 μL of aluminum adjuvant was added to 500 μL of fixed bacteria and vortexed at room temperature for 30 min to ensure thorough mixing. The bacteria prepared with aluminum adjuvant were injected intraperitoneally into C57BL / 6 genetic background mice, with each mouse receiving 200 μL of the mixed reagent.

[0264] Enrichment of mouse-specific B cells. Mice (the resulting mouse infection model) were euthanized after immunization, and their spleens were removed. The spleens were placed in 5 mL of PBS, ground, and filtered through a 70 μm filter. They were then centrifuged at 300 g for 5 min, and the supernatant was discarded. Magnetic beads were added to the resulting spleen tissue cells for enrichment, and the cells were centrifuged at 300 g for 5 min, and the supernatant was discarded. Finally, the remaining cells were counted, cryopreserved, and aliquoted.

[0265] Example 2: Optimization of microdroplet incubation time.

[0266] By constructing 293T cells expressing GFP fluorescent protein, the cells were encapsulated using the microfluidic method described in Example 1, and the cell viability was observed at 2 hours and 4 hours.

[0267] The results are shown in Figure 1.

[0268] The results showed that after the obtained microdroplets were incubated at 4°C for 2 hours, the fluorescent protein expression in 293T cells was obvious, indicating that the cells had high activity and intact cell membranes.

[0269] After incubating the resulting microdroplets at 4°C for 4 hours, the expression of fluorescent proteins in 293T cells significantly decreased, indicating low cell viability and cell membrane damage. At this point, the cell membranes within the microdroplets had largely perforated, and the stability of the generated microdroplets remained relatively good. This demonstrates that the buffering system efficiently and stably perforates cell membranes without affecting the stability of the generated droplets.

[0270] Example 3: Verification of the system of the present invention.

[0271] Microscopic observation was performed by incubating anti-IgG antibodies with negative cells (CTRL) and hybridoma cells (8D3), respectively, to verify that the anti-IgG antibodies could specifically bind to hybridoma cells. The negative cells were 293T cells.

[0272] The results are shown in Figure 2.

[0273] The results showed that in the CTRL group, almost no binding of anti-IgG antibodies to negative cells was observed after incubation; however, in the 8D3 group, the binding of anti-IgG antibodies to hybridoma cells was observed in the microdroplets encapsulating the cells after incubation. This indicates that anti-IgG antibodies can specifically bind to hybridoma cells, suggesting that under these encapsulation conditions, specific antibodies can effectively perform their functions.

[0274] In addition, L / D live / dead cell staining results showed that under fluorescence microscopy, the L / D live / dead dye could pass through the cell membrane of dead cells with damaged cell membranes and enter the cell to produce fluorescence, while the cell membrane of live cells remained intact and could not pass through. This proves that the buffer system can play a role and can destroy the hybridoma cell membrane.

[0275] By enriching immunized mouse spleen B cells, anti-IgG antibodies were incubated with splenic B cells to verify that anti-IgG antibodies can specifically bind to mouse spleen B cells.

[0276] The results are shown in Figure 3.

[0277] The results showed that the anti-IgG antibody could bind to mouse spleen B cells without affecting its function. This indicates that the microdroplet system has good screening effect and adaptability.

[0278] Anti-IgG antibodies were encapsulated and incubated with mouse spleen cells. After 4 hours, the cells were observed under a microscope to verify that the anti-IgG antibodies could specifically recognize membrane antibodies and intracellular antibodies.

[0279] The results are shown in Figure 4.

[0280] The results showed that the encapsulation system could recognize both B cell surface antibodies (i.e., membrane antibodies) and antibodies present inside B cells (i.e., intracellular antibodies), demonstrating the comprehensiveness of the encapsulation system in screening specific antibodies.

[0281] Example 4: A comparative example of the sorting method of the present invention.

[0282] The efficiency comparison between the sorting method of the present invention and traditional flow cytometry cell sorting and magnetic bead sorting is shown in Table 2.

[0283] Table 2 Comparison of Sorting Efficiency

[0284] Specifically, flow cytometry can be used to efficiently separate single antigen-specific B lymphocytes from spleen cells for the preparation of monoclonal antibodies in hybridomas. The sorting purity is usually >95%, making it suitable for sorting cells with complex phenotypes (multiple marker combinations) and low abundance. However, flow cytometry sorting is relatively time-consuming, requiring approximately 1-2 hours per sample on average after sample preparation, loading, and sorting.

[0285] In addition, when flow cytometry is used to sort IgG positive cells after membrane perforation, the recovery rate is generally 60-80% (positive selection); when using fluorescently labeled IgM polyclonal antibodies and pre-labeled screening antigens to further negatively screen the spleen cells obtained above, the flow cytometry technique is used to remove B cells that do not express IgG molecules on the surface and retain the target lymphocytes that bind to the pre-labeled screening antigens, the recovery rate is only 10%-30% (negative selection).

[0286] Magnetic bead sorting (MACS) is typically used for large sample (generally starting cell number 10^8 / mL) high-throughput sorting, which can be completed rapidly within 30 minutes. Purity is approximately 85-95% (positive sorting) or higher (negative sorting). Recovery rate is typically >80% (positive sorting).

[0287] However, it is often not suitable for small samples. In addition, there are no IgG direct sorting products for magnetic bead sorting. Generally, B cells are separated by surface protein CD19 or plasma cells are separated by CD138.

[0288] Example 5: Improvement and application of the device of the present invention.

[0289] By adding a cell sorting module, BCR profiles can be obtained.

[0290] Specifically, the complete device is formed by connecting another cell sorting module (small chip).

[0291] This device is mainly used for point-of-care research in hospitals, and its main application is the analysis of patients' BCR repertoire spectra.

[0292] The cell sorting module (miniature chip) is designed to inject magnetic beads (which can simultaneously couple antibody heavy and light chain mRNA) when a positive single droplet is energized.

[0293] Other target genes can also be customized using magnetic beads to achieve combined sequencing of single-cell target genes; or single-cell transcript sequencing.

[0294] Antibodies can be obtained by adding antibody DNA modules.

[0295] Specifically, by connecting another PCR module (small chip), a complete device is formed.

[0296] Before generating microdroplets, the cells are first treated so that microspheres (containing barcodes) containing reagents required for reverse transcription and PCR amplification are introduced into the cells;

[0297] The generated microdroplets are mainly generated through PCR reaction, which produces antibody DNA sequences in each microdroplet (the antibody heavy chain and light chain are synthesized into a linear DNA using fusion PCR).

[0298] Each PCR reaction is performed in each chamber of a microplate, and the DNA inside is collected using conventional methods for subsequent sequencing or antibody expression.

[0299] The resulting linear DNA contains paired heavy and light chain sequences.

[0300] Example 6: Validation of the effectiveness of screening specific antibodies against Simbnia virus Gn protein (as a representative viral surface antigen) in low-activity frozen samples.

[0301] To further verify the practical application effect of the antibody screening platform described in this invention in complex, low-quality biological samples, peripheral blood total B cell samples from Simbnia patients, frozen for two years with cell viability below 50%, were used as starting materials. Simbnia virus Gn protein (as a representative viral surface antigen) was used as the target antigen. The screening platform of this invention was used to obtain 31 paired monoclonal antibodies, and their structural characteristics and functional activities were analyzed. The specific screening method is as follows: the antibody phase simultaneously contained fluorescently labeled anti-IgG antibody (anti-IgG antibody-AF488) and fluorescently labeled Gn protein (Gn protein-AF647). The anti-IgG antibody binds to the "constant region" of IgG, and the Gn protein binds to the "variable region" of IgG. Cells that simultaneously highly express IgG antibodies and can bind to the Gn-specific antigen were sorted using the two fluorescence signals.

[0302] (1) The use of heavy and light chain genes for antibody screening showed a significant non-randomness.

[0303] Genetic analysis of the 31 obtained antibodies revealed a clear preference for the use of heavy chain variable region gene families. The IGHV3 family was the most used, followed by the IGHV1 family, while the IGHV4 and IGHV5 families accounted for a relatively low proportion and did not exhibit a uniform distribution characteristic of random V(D)J rearrangements (Figure 5A).

[0304] Regarding the light chain, the antibody is mainly composed of the κ chain, primarily concentrated in the IGKV1, IGKV3, and IGKV4 families, while the λ chain is used at a significantly lower rate (Figure 5B).

[0305] The above results indicate that even under low-activity cryopreservation conditions, antibodies obtained through the antibody screening platform of this invention still exhibit significant antigen-driven selection characteristics at both the heavy and light chain levels.

[0306] (2) Heavy chain – light chain pairing displays non-random combination features related to functional display.

[0307] After joint analysis of the pairing relationship between the heavy and light chains of the antibodies, it was found that the pairing between different heavy and light chains did not occur randomly. Among them, IGHV3–IGKV1, IGHV3–IGKV3 and IGHV1–IGKV4 were the most frequently occurring pairing combinations (Figure 5C).

[0308] The results showed that the antibodies obtained by screening exhibited obvious non-random distribution characteristics in the heavy chain, light chain and their pairing levels, suggesting that the antibodies originated from antigen-driven functional B cell clonal expansion. This indicates that the antibody screening platform of the present invention can effectively enrich antibody molecules derived from functional B cell clonal expansion during the screening process, rather than non-specific antibodies generated by random rearrangement.

[0309] (3) In vitro functional verification confirmed that the antibodies obtained from screening had antigen-binding activity.

[0310] The 31 antibodies obtained through the antibody screening platform of this invention were analyzed for in vitro expression. Specifically, some B cell clones obtained by screening were expressed in vitro in HEK293 cells, and the antibodies in the culture supernatant were enriched by Protein A / G and Western blot analysis was performed under non-denaturing (Native) conditions.

[0311] The results are shown in Figure 6. Lane 1 is the protein marker lane; lanes 2–8 are the antibody lanes expressed by different B cell clones, respectively; lane 9 represents the negative control. The band at 150–170 kDa is the main IgG monomer band, and clear signals were observed in almost all clones in this region. The band intensity varied among different clones, reflecting differences in secretion efficiency. The band at 200–250 kDa is the IgG dimer or multimer band, which is more common under non-denaturing conditions and may be related to IgG molecule aggregation or Fc–Fc interactions. Overall, the detected antibodies all exhibited consistent IgG characteristic bands, while no obvious signal was detected in the negative control. This indicates that the selected antibodies can stably secrete structurally intact IgG molecules in the mammalian cell system, demonstrating that this platform can stably obtain antibody molecules suitable for in vitro expression and subsequent functional studies and development.

[0312] The binding affinity of the above antibodies to the target antigen Gn protein was detected using an ELISA method. The G2 and H2 antibodies share the same light chain gene composition (IGHV1 / IGKV4).

[0313] As shown in Figure 7, both G2 and H2 antibodies were able to bind to the target antigen with significant specificity, and their OD450 values ​​were significantly higher than those of the negative control group, which consisted of HEK293 cells transfected with an empty vector.

[0314] The in vitro functional validation results further demonstrate that the antibodies obtained through the antibody screening platform of this invention not only have consistency and selectivity at the structural level, but also have clear antigen-binding activity at the functional level.

[0315] In summary, even under conditions of low-activity cryopreserved samples, the antibody screening platform described in this invention can still:

[0316] 1) Achieve antigen-driven selective antibody enrichment;

[0317] 2) Obtain antibody molecules in which the heavy chain, light chain, and their pairings all exhibit non-random characteristics;

[0318] 3) Screening yields functional antibodies with actual antigen-binding activity.

[0319] The above results fully verify the effectiveness, stability and practicality of the antibody screening platform of the present invention under complex sample conditions from multiple aspects such as structural characteristics, expression ability and functional activity, and further confirm the effectiveness of the aforementioned antibody screening platform mechanism.

[0320] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A screening system for sorting immunoglobulin-positive cell populations, characterized in that, The screening system comprises microdroplets, each microdroplet containing a cellular phase and an antibody phase, the antibody phase containing antiglobulin antibodies.

2. A screening system for sorting IgG-positive cell populations, characterized in that, The screening system comprises microdroplets, each microdroplet containing a cell phase and an antibody phase, the antibody phase containing anti-IgG antibodies.

3. The screening system as described in claim 2, characterized in that, The cell phase comprises: a population of cells to be sorted; and the antibody phase comprises anti-IgG antibody, EDTA, and Tween-20; The cell phase solution is a PBS solution, and the antibody phase solution is a PBS solution.

4. The use of the screening system as described in claim 2, characterized in that, Used to sort IgG positive cell populations.

5. The use as described in claim 4, characterized in that, The IgG-positive cell population includes both IgG-positive cells on the cell membrane surface and IgG-positive cells intracellularly.

6. A sorting system for sorting cells expressing antibodies specific to a target antigen, characterized in that, The sorting system comprises microdroplets, which contain cellular phase, antibody phase, and antigen phase. The cell phase includes the cell population to be sorted. The antibody phase contains antiglobulin antibodies. The antigen phase contains the target antigen.

7. The use of the screening system according to claim 1 or 2 or the sorting system according to claim 6, characterized in that, Used to screen for antibodies specific to the target antigen.

8. A method for sorting IgG positive cell populations, characterized in that, The method includes the following steps: (S1) Provides the screening system of claim 2, the screening system comprising microdroplets, the microdroplets comprising a cell phase and an antibody phase, the antibody phase comprising fluorescently labeled anti-IgG antibody; (S2) Incubate the microdroplets; (S3) Image the incubated microdroplets and sort out the microdroplets with fluorescent signals to obtain IgG positive cell populations.

9. A method for sorting cells expressing antibodies specific to a target antigen, characterized in that, The method includes the following steps: (S1) The sorting system of claim 6 is provided, wherein the sorting system comprises microdroplets; (S2) Incubate the microdroplets; (S3) Select cells expressing target antigen-specific antibodies from the incubated microdroplets.

10. An apparatus for sorting IgG positive cell populations, characterized in that, The device includes the following modules: (Z1) Microdroplet preparation module, the microdroplet preparation module being configured to: provide the screening system of claim 2, the screening system comprising microdroplets; wherein, a cell phase comprising a cell population to be sorted is mixed with an antibody phase comprising anti-IgG antibody to prepare microdroplets; (Z2) Microdroplet incubation module, wherein the microdroplet incubation module is configured to incubate the microdroplets; (Z3) Sorting module, the sorting module is configured to: image the incubated microdroplets, sort out the microdroplets with fluorescent signals, and thus obtain IgG positive cell populations.

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