Bioparticle labeling method, bioparticle analysis method, sorting method, and reagent kit for bioparticle analysis

Abstract

The purpose of the present technology is to provide a technology for effectively labeling a bioparticle in a true-positive case (in a case where an antibody secreted from an antibody-producing cell specifically interacts with an antigen in an antigen-expressing cell) without using an imaging technology or the like.　As a result of intensive studies, the present inventors have found that a true-positive bioparticle can be specifically labeled with or without the occurrence of a FRET reaction by modifying the cell surface of an antigen-expressing cell with one of a donor molecule and an acceptor molecule using a combination of the donor molecule and the acceptor molecule and by using a molecule capable of specifically binding to a target antibody that has been modified with the other of the donor molecule and the acceptor molecule, wherein the donor molecule releases fluorescent energy by excitation light and the acceptor molecule receives the fluorescent energy from the donor molecule and emits fluorescence.

Description

Biological particle labeling method, biological particle analysis method, sorting method, and reagent kit for biological particle analysis

[0001] The present disclosure relates to a biological particle labeling method, a biological particle analysis method, a sorting method, and a reagent kit for biological particle analysis. More specifically, the present disclosure relates to a technique for appropriately labeling even an antigen that is difficult to solubilize and isolate, when an antibody interacts with the antigen.

[0002] Conventionally, screening of antibodies that interact with a target antigen or cells producing such antibodies has been performed.

[0003] For example, Patent Document 1 below discloses a technique for analyzing biological particles in a state contained in a biological particle population. However, this system is based on the premise that the secreted substance captured by the capturing substance is secreted into a medium.

[0004] WO 2022 / 190733

[0005] For example, when it is difficult to solubilize and isolate an antigen, such as when the antigen is present on a cell membrane, two-cell system antibody screening, in which antigen-expressing cells and antibody-producing cells are paired for analysis, is sometimes used to screen for antibody-producing cells that produce an antibody that specifically interacts with the antigen. On the other hand, analysis of target biological particles may not be efficiently performed depending on the proportion of target two-cell pairs formed among all analytes and the physical upper limit of the number of analyzable biological particles. Furthermore, there is a demand for reducing the false positive determination rate and effectively identifying true positive cases (cases where an antibody secreted from an antibody-producing cell specifically interacts with an antigen present in an antigen-expressing cell) without using imaging techniques or the like that are difficult for high-throughput processing.

[0006] Therefore, an object of the present technology is to provide a technique for effectively labeling biological particles in true positive cases (cases where an antibody secreted from an antibody-producing cell specifically interacts with an antigen present in an antigen-expressing cell) without using an imaging technique or the like.

[0007] As a result of diligent research, the inventors have discovered that by using a combination of a donor molecule that emits fluorescence energy upon excitation light and an acceptor molecule that receives fluorescence energy from the donor molecule and emits fluorescence, the cell surface of antigen-expressing cells can be modified with either the donor molecule or the acceptor molecule, and by using a molecule that specifically binds to the modified target antibody, it is possible to specifically label truly positive biological particles based on the presence or absence of a FRET reaction.

[0008] In other words, this technology provides a method for labeling biological particles, comprising: a capture step in which an antibody secreted by an antibody-producing cell captures an antigen expressed on the cell membrane of an antigen-expressing cell, which has a cell surface modified with a first probe containing either a donor molecule that releases energy upon irradiation with excitation light or an acceptor molecule that receives the energy from the donor molecule and emits fluorescence; and a binding step in which an antibody-binding molecule that binds to the antibody, modified with a second probe containing the other of the donor molecule or the acceptor molecule, captures the antibody. In this biological particle labeling method, the first probe may include a lipid linker. In this biological particle labeling method, a crosslinking step may be included after the binding step in which the first probe and the second probe are crosslinked. In this case, the crosslinking may be formed by coordinating bonds, which are created by attaching one of two compounds that can coordinately bond via metal ions to either the first probe or the second probe, and adjusting the concentration of the metal ions in the medium with a chelating agent. In this case, the biological particle labeling method of this technology may include a washing step of washing the chelating agent after the binding step and before the crosslinking step. The biological particle labeling method of this technology may include a pairing step before the capture step, in which a pairing reagent having an antigen-expressing cell binding site that can bind to the antigen-expressing cell and an antibody-producing cell binding site that can bind to the antibody-producing cell is bound to either the antigen-expressing cell or the antibody-producing cell, and then bound to the other. In this case, the pairing reagent may be bound to the antibody-producing cell and then to the antigen-expressing cell. Furthermore, two or more types of pairing reagents may be used. In the biological particle labeling method of this technology, the antibody-producing cell may be assigned an identification code. The biological particle labeling method of this technology may include an isolation step before the capture step, in which a group including the antibody-producing cell and one or more antigen-expressing cells is isolated. In this case, the isolation may be performed by encapsulating the group in a droplet.Furthermore, a droplet destruction step may be included after the binding step to destroy the droplets. Moreover, the isolation may be performed by binding the group to a carrier. In the biological particle labeling method of this technology, when isolation is performed by binding the group to a carrier, a magnetic application step may be included before the isolation step to bind the antigen-expressing cells or the antibody-producing cells to magnetic beads. In this case, a purification step may be included after the isolation step and before the capture step, in which the cells bound to the magnetic beads are separated using magnetism. Next, this technology provides a biological particle analysis method that includes a identification step to identify antibody-producing cells that secrete antibodies that specifically bind to the antigen by irradiating the biological particles labeled by the biological particle labeling method of this technology with excitation light and detecting fluorescence emitted by the interaction between the first probe and the second probe. Furthermore, this technology provides a method for separating the antibody-producing cells identified by the biological particle analysis method of this technology. In this case, the separation may be performed by a flow cytometer.

[0009] Next, the present technology provides a reagent kit for biological particle analysis for identifying antibody-producing cells that secrete antibodies that specifically bind to antigens expressed on the cell membrane of antigen-expressing cells, comprising: a first probe having a modification site that modifies the cell surface of antigen-expressing cells, and containing either a donor molecule that releases energy upon irradiation with excitation light or an acceptor molecule that receives the energy from the donor molecule and emits fluorescence; and an antibody-binding molecule that binds to an antibody, modified by a second probe containing the other of the donor molecule or the acceptor molecule. The biological particle analysis reagent kit of the present technology may further include a pairing reagent comprising an antigen-expressing cell binding site that can bind to the antigen-expressing cells and an antibody-producing cell binding site that can bind to the antibody-producing cells. Furthermore, this technology provides a biological particle analyzer comprising: an irradiation means for irradiating biological particles that have captured the antibody, which is captured by an antibody-producing cell, with excitation light; and an irradiation means for detecting fluorescence emitted by the interaction between the first probe and the second probe, and identifying an antibody-producing cell that secretes an antibody-binding molecule that binds to the antibody, onto an antigen-expressing cell having a cell surface modified with a first probe that includes either a donor molecule that releases energy upon irradiation with excitation light or an acceptor molecule that receives the energy from the donor molecule and emits fluorescence.

[0010] A flowchart of a representative example of the biological particle labeling method of this technology is shown. A flowchart of a modified example of the biological particle labeling method of this technology is shown. An image is shown of the biological particle labeling method of this technology in which the cell surface of an antigen-expressing cell is modified with a first probe containing either a donor molecule or an acceptor molecule. An image is shown of the biological particle labeling method of this technology in which an antibody secreted by an antibody-producing cell captures an antigen expressed on the cell membrane of the antigen-expressing cell. An image is shown of the biological particle labeling method of this technology in which an antibody-binding molecule, modified with a second probe containing either a donor molecule or an acceptor molecule, binds to an antibody secreted by an antibody-producing cell, and labels the biological particle containing the antigen-expressing cell by capturing the antibody. An image is shown of the biological particle labeling method of this technology in which the labeled biological particle containing the antigen-expressing cell emits fluorescence by a FRET reaction. As a modified example of the biological particle labeling method of this technology, an image is shown of the cell surface of an antigen-expressing cell being modified with a first probe to which one of two compounds containing either a donor molecule or an acceptor molecule and capable of coordination bonding via a metal ion is attached. In a modified version of the biological particle labeling method of this technology, an image is shown in which an antibody secreted by an antibody-producing cell captures an antigen expressed on the cell membrane of the antigen-expressing cell. In a modified version of the biological particle labeling method of this technology, an image is shown in which an antibody-binding molecule, which binds to an antibody secreted by an antibody-producing cell, is modified with a second probe to which the other of two compounds that contain the other of a donor molecule or an acceptor molecule and can be coordinately bonded via a metal ion captures the antibody, thereby labeling the biological particle containing the antigen-expressing cell, and by removing the chelating agent, the first probe and the second probe are crosslinked by a coordinate bond, promoting the FRET reaction due to the interaction of the donor molecule and the acceptor molecule, and emitting fluorescence. An image is shown of a biological particle formed by binding an antigen-expressing cell and an antibody-producing cell via a pairing reagent, which has an antigen-expressing cell binding site that can bind to the antigen-expressing cell and an antibody-producing cell binding site that can bind to the antibody-producing cell. An image is shown of the process of pairing using the pairing reagent, where the pairing reagent is first bound to either the antigen-expressing cell or the antibody-producing cell, and then to the other.This document shows an example of a pairing reagent prepared by linking two antibodies, one with a DNA linker that forms an antigen-expressing cell binding site and the other with a DNA linker that forms an antibody-producing cell binding site, using nucleic acid oligomers that hybridize to each DNA linker. It also illustrates the process of adding the pairing reagent when antigen-expressing cells and antibody-producing cells are present simultaneously. Images of biological particles consisting of paired antigen-expressing cells and antibody-producing cells that are acceptable and unacceptable for analysis are shown. Images of the resulting biological particles, where antigen-expressing cells and antibody-producing cells are bound, are shown for both the case using one pairing reagent and the case using two pairing reagents. An image of a biological particle formed when antigen-expressing cells and antibody-producing cells are bound by antibodies secreted from antibody-producing cells is shown. An example of a method for assigning an identification code to biological particles consisting of paired antigen-expressing cells and antibody-producing cells is shown. Finally, an image of a modified version of the method for assigning an identification code to biological particles consisting of paired antigen-expressing cells and antibody-producing cells is shown. This image shows an example of a method for isolating a group containing antibody-producing cells and antigen-expressing cells in the biological particle labeling method of this technology. This image shows a modified version of the method for isolating a group containing antibody-producing cells and antigen-expressing cells in the biological particle labeling method of this technology. This image shows a modified version of the method for isolating a group containing antibody-producing cells and antigen-expressing cells in the biological particle labeling method of this technology. This image shows a method for isolating a group containing antibody-producing cells and antigen-expressing cells that has been encapsulated and isolated in a droplet, after which the target group is separated. This image shows a method for isolating a carrier to which a group containing antibody-producing cells and antigen-expressing cells is bound, using magnetism. This image shows cells to be isolated and cells not to be isolated, used in a modified version of the biological particle isolation method that can be used in the biological particle labeling method of this technology. This image shows a modified version of the biological particle isolation method that can be used in the biological particle labeling method of this technology, in which cells are modified with a labeled antibody that has a labeling site to identify the cells to be isolated.This section shows an example of a carrier having a label-binding molecule that specifically binds to at least a portion of the label site, which can be used in a modified version of the bioparticle isolation method that can be used in the bioparticle labeling method of this technology. This section shows an image of the cells to be isolated binding to the carrier via the label-binding molecule in a modified version of the bioparticle isolation method that can be used in the bioparticle labeling method of this technology. This section shows an image of a dispersion in which cells to be isolated and cells not to be isolated are dispersed when a carrier to which a group including antibody-producing cells and antigen-expressing cells is bound is separated by magnetism in a modified version of the bioparticle isolation method that can be used in the bioparticle labeling method of this technology. This section shows an image of a carrier to which a group including cells to be isolated is bound being separated using a magnet in a modified version of the bioparticle isolation method that can be used in the bioparticle labeling method of this technology. This section shows an example of a carrier having a label-binding molecule that specifically binds to at least a portion of the label site, which can be used in a modified version of the bioparticle isolation method that can be used in the bioparticle labeling method of this technology. This diagram shows a modified version of the bioparticle isolation method that can be used in the bioparticle labeling method of this technology, illustrating how the cells to be isolated bind to the carrier via a label-binding molecule. (Positive selection) This diagram shows a modified version of the bioparticle isolation method that can be used in the bioparticle labeling method of this technology, illustrating how the binding of non-isolated cells to the carrier is inhibited by the label-binding molecule. (Negative selection) This diagram shows an image of analyzing antibody-producing cells possessed by the bioparticles identified by the bioparticle labeling method of this technology. This diagram shows an example of the configuration of the reagent kit for bioparticle analysis of this technology. This diagram shows a modified version of the example configuration of the reagent kit for bioparticle analysis of this technology. This diagram shows a modified version of the example configuration of the reagent kit for bioparticle analysis of this technology.

[0011] Preferred embodiments of the present technology are described below. However, the embodiments shown below are merely examples of typical embodiments of the present technology, and the present technology is not limited to these preferred embodiments, but can be freely modified within the scope of the present technology.

[0012] [Method for Labeling Bioparticles] First, we will specifically explain the bioparticle labeling method of this technology.

[0013] The cell membrane contains membrane proteins involved in intracellular and extracellular signal transduction, such as GPCRs (G protein-coupled receptors) and ion channels. The expression of these membrane proteins can vary depending on the tissue and cell type, making them important targets for drug discovery and diagnostics. Therefore, these membrane proteins are sometimes used as target antigens when designing cell-type-specific antibodies.

[0014] On the other hand, it is generally difficult to solubilize and isolate antigens such as membrane proteins present on cell membranes. Furthermore, even if solubilization is possible, the original three-dimensional structure may be lost during the process, rendering the antigen unsuitable and preventing the creation of the desired antibody.

[0015] Therefore, screening for antibody-producing cells that produce antibodies that specifically interact with such antigens is performed without isolating the antigen or separating the antigen-expressing cells that express it (i.e., while the antigen retains its in vivo structure). More specifically, this is done using a two-cell system, for example, in which antigen-expressing cells and antibody-producing cells that produce antibodies are paired and analyzed. In this case, to improve the accuracy of the screening, it is necessary to distinguish between cases where the antibody produced by the antibody-producing cell specifically interacts with the antigen on the antigen-expressing cell (true positive) and cases where the antibody is conjugated to the antibody-producing cell (false positive).

[0016] As described above, this technology's method for labeling biological particles can effectively label true positive biological particles from false positives without using imaging techniques or other methods. While there are no particular limitations on the antigens expressed on the cell membrane that this technology's biological particle labeling method targets, examples include the membrane proteins mentioned above.

[0017] Herein, in this specification, "biological particle" refers to a particle of biological origin or a particle that functions as a component of a living organism. For example, this includes a single cell, as well as a paired cell group produced by combining an antigen-expressing cell and an antibody-producing cell, as described later. These cells that can constitute biological particles may be, for example, living cells. More specifically, they may be blood cells such as red blood cells and white blood cells, and germ cells such as sperm and fertilized eggs. These cells may be directly collected from a sample such as whole blood, or they may be cultured cells obtained after culturing. Furthermore, the cells constituting the biological particles may be labeled with one or more labeling substances (for example, dyes (particularly fluorescent dyes) and fluorescent dye-labeled antibodies) in addition to the labeling by the biological particle labeling method of this technology.

[0018] Figure 1 shows a flowchart of a typical example of the bioparticle labeling method of this technology. The bioparticle labeling method of this technology consists of at least a supplementation step S101 and a binding step S102. Each step will be described in detail below.

[0019] <Capture Process> In the biological particle labeling method of this technology, the capture process S101 is a process in which antibodies secreted by antibody-producing cells capture antigens expressed on the cell membrane of antigen-expressing cells.

[0020] In this specification, "antibody-producing cell" refers to a cell that produces antibodies. The cells that can be used as antibody-producing cells are not particularly limited, and any cell can be used. Examples of such cells include plasma cells and hybridoma cells. By using the biological particle labeling method of this technology, biological particles containing antibody-producing cells that produce antibodies that specifically interact with an antigen can be identified.

[0021] Furthermore, in this specification, "antigen-expressing cell" refers to a cell that expresses an antigen on its cell membrane. More specifically, in the case of screening using the bioparticle labeling method of this technology, it refers to a cell that expresses the antigen that is the target of the antibody produced by the antibody-producing cell being screened.

[0022] In the biological particle labeling method of this technology, it is preferable that the cell surface of the antigen-expressing cells used is modified with a first probe that includes either a donor molecule that releases energy upon irradiation with excitation light, or an acceptor molecule that receives the energy from the donor molecule and emits fluorescence.

[0023] In this specification, the terms "donor molecule" and "acceptor molecule" refer to a combination of compounds that perform resonance energy transfer via FRET (Fluorescence resonance energy transfer). Specifically, the "donor molecule" is a compound that becomes excited upon irradiation with excitation light and donates energy to the acceptor molecule, while the "acceptor molecule" is a compound that receives the aforementioned energy from the donor molecule and can emit fluorescence.

[0024] The "first probe" used in the biological particle labeling method of this technology contains either the donor molecule or the acceptor molecule. In this way, the first probe containing either the donor molecule or the acceptor molecule modifies the cell surface of the antigen-expressing cell, thereby attaching either the donor molecule or the acceptor molecule to the surface of the antigen-expressing cell.

[0025] Figure 3 shows an image of a two-cell system in which antigen-expressing cell E(201) and antibody-producing cell S(203) are paired for analysis, in which the cell surface of antigen-expressing cell E(201) is modified with a first probe 101 containing either a donor molecule or an acceptor molecule. The antigen-expressing cell E(201) used in this two-cell system expresses the target antigen 202 on its cell membrane, as shown in Figure 3. The state of antigen 202 is not separated from the antigen-expressing cell 201 and can be said to be in a state that retains its in vivo structure. In addition, although Figure 3 shows an example in which the donor molecule 102 is attached to the cell surface of antigen-expressing cell E(201), the design of the first probe may be changed to attach an acceptor molecule to the cell surface of antigen-expressing cell E(201).

[0026] The first probe 101 used in the biological particle labeling method of this technology is equipped with means for modifying the cell surface. Such means are not particularly limited, but any method can be used, such as a lipid linker that can be inserted into the lipids constituting the cell membrane, or an antibody that binds to a specific antigen on the cell membrane.

[0027] Figure 3 shows an example in which a lipid linker 103 is used as a means of modifying the cell surface, but the method is not limited to this. In the example shown in Figure 3, the lipid linker 103 provided on one end of the first probe is inserted (embedded) into the cell membrane of the antigen-expressing cell E (201), and the donor molecule 102 (or acceptor molecule) provided on the other end appears on the surface of the antigen-expressing cell E (201).

[0028] Next, Figure 4 shows an image illustrating how, in the bioparticle labeling method of this technology, antibody 204 secreted by antibody-producing cell 203 captures antigen 202 expressed on the cell membrane of antigen-expressing cell 201. As shown in Figure 4, antibody 204 secreted from antibody-producing cell S (203) is dispersed in the medium of this two-cell system. It can also be confirmed that a portion of the secreted antibody 204 captures antigen 202 expressed on the cell membrane of antigen-expressing cell E (201).

[0029] <Binding Step> In the biological particle labeling method of this technology, binding step S102 is a step in which an antibody-binding molecule 105, which is modified with a second probe 106 containing either a donor molecule or the other of an acceptor molecule, and which binds to an antibody 204, captures the antibody 204 secreted from an antibody-producing cell 203.

[0030] The "second probe" used in this bioparticle labeling method contains a donor molecule or acceptor molecule different from that used in the "first probe." This allows for resonance energy transfer via FRET between the donor molecule or acceptor molecule contained in the first probe, which modifies the surface of antigen-expressing cells.

[0031] The second probe used in the biological particle labeling method of this technology is modified to bind to an antibody-binding molecule that binds to an antibody secreted by an antibody-producing cell. The term "antibody-binding molecule" is not particularly limited as long as it is a molecule capable of binding to an antibody secreted by an antibody-producing cell. The "antibody-binding molecule" may be, for example, an anti-IgG antibody, or an antibody that specifically binds to an antibody secreted by an antibody-producing cell.

[0032] Figure 5 illustrates how, in the bioparticle labeling method of this technology, an antibody-binding molecule 105 that binds to an antibody 204 secreted by an antibody-producing cell S(203), modified with a second probe 106 containing either a donor molecule or the other of an acceptor molecule, captures the antibody 204. In the example in Figure 5, since the first probe 101 contains a donor molecule 102, the second probe contains an acceptor molecule 107. However, if the first probe 101 contains an acceptor molecule, the second probe 106 is designed to contain a donor molecule.

[0033] As mentioned above, the antibodies secreted from antibody-producing cell S(203) are dispersed in the medium of this two-cell system. Therefore, as shown in Figure 5, the antibody-binding molecule captures antibodies 204 that are bound to the surface of antibody-producing cell S(203), antibodies 204 that are suspended in the medium, and antibodies 204 that are capturing antigen 202 expressed on the cell membrane of antigen-expressing cell E(201). Of these, true positivity occurs when there is an antibody 204 that specifically interacts with antigen 202 expressed on the cell membrane of antigen-expressing cell E(201).

[0034] Figure 6 shows an image of the bioparticle labeling method of this technology, in which labeled bioparticles containing antigen-expressing cells 201 emit fluorescence by the FRET reaction. The cell surface of antigen-expressing cells E(201) is modified by the first probe 101, and a donor molecule or acceptor molecule (donor molecule 102 in the example of Figure 6) appears on the cell surface of antigen-expressing cells E(201). Therefore, if an antibody 204 that specifically interacts with antigen 202 expressed on the cell membrane of antigen-expressing cells E(201) is present (true positive), in addition to the donor molecule or acceptor molecule (donor molecule 102 in the example of Figure 6) derived from the first probe 101, as shown in Figure 6, a donor molecule or acceptor molecule (acceptor molecule 106 in the example of Figure 6) derived from the second probe appears on the cell surface of antigen-expressing cells E(201), due to the antibody 204 that captures antigen 202 and the antibody-binding molecule 105 that captures antibody 204.

[0035] When a donor molecule or acceptor molecule derived from the first probe 101 (donor molecule 102 in the example in Figure 6) and a donor molecule or acceptor molecule derived from the second probe 106 (acceptor molecule 107 in the example in Figure 6) are in sufficient proximity on the cell surface of antigen-expressing cell E(201), fluorescence emitted by the interaction (FRET reaction) between the first probe 101 and the second probe 106 can be detected by irradiation with excitation light, as shown in the figure.

[0036] On the other hand, antibodies 204 that are bound to the surface of antibody-producing cells S (203) and captured by antibody-binding molecules 105, or antibodies 204 that are suspended in the medium, do not have a donor molecule or acceptor molecule (donor molecule 102 in the example of Figure 6) derived from the first probe 101 nearby. Therefore, even if excitation light is irradiated, no interaction (FRET reaction) occurs between the first probe 101 and the second probe 106, and fluorescence cannot be detected.

[0037] The bioparticles labeled by this technology can be identified by detecting fluorescence from the FRET reaction if an antibody that specifically interacts with the antigen expressed on the cell membrane of antigen-expressing cell E is present (true positive case). On the other hand, in other cases (false positive case), fluorescence from the FRET reaction is not detected. Therefore, even if there are two different types of cells in the experimental system, it is possible to eliminate false positive cases and identify only true positive cases without using imaging techniques.

[0038] Figure 2 shows a flowchart of a modified example of the bioparticle labeling method of this technology. In addition to the steps shown in the flowchart in Figure 1, the bioparticle labeling method of this technology may optionally include the steps shown in Figure 2. The steps that may optionally be included in the bioparticle labeling method of this technology will be described in detail below.

[0039] <Crosslinking and Washing Processes> In the biological particle labeling method of this technology, the crosslinking process S104 is a process in which the first probe and the second probe are crosslinked after the binding process S102, as shown in Figure 2.

[0040] Here, "crosslinking" refers to the reversible or irreversible bonding of the first probe and the second probe.

[0041] Even if an antibody exists that specifically interacts with the antigen expressed on the cell membrane of an antigen-expressing cell, labeled by the bioparticle labeling method of this technology (in the case of true positivity), if, during irradiation with excitation light, the donor molecule or acceptor molecule derived from the first probe (donor molecule 102 in the example of Figure 6) and the donor molecule or acceptor molecule derived from the second probe (acceptor molecule 107 in the example of Figure 6) are not sufficiently close to each other on the cell surface of the antigen-expressing cell due to fluctuations in the three-dimensional structure of the probes, etc., or fluctuations in their relative positional relationship on the cell surface, then the FRET reaction may not occur, and the desired fluorescence may not be detected.

[0042] In contrast, for example, by cross-linking the first probe and the second probe present on the cell surface of antigen-expressing cells, the positions of the donor molecule and acceptor molecule contained in both probes can be fixed. It is preferable that the fixation is performed by bringing the positions of the donor molecule and the acceptor molecule into close proximity. This reduces fluctuations in the three-dimensional structure and the relative positional relationship on the cell surface. When there is an antibody that specifically interacts with an antigen expressed on the cell membrane of an antigen-expressing cell (in the case of a true positive), the probability of FRET reaction occurrence is improved, and the FRET reaction can be detected with high sensitivity. This can reduce the missed identification of true positives.

[0043] In the cross-linking step of the bioparticle labeling method of the present technology, any cross-linking method that can cross-link the first probe and the second probe can be used. Further, the cross-linking may be reversible or irreversible. As a reversible cross-linking method, for example, there can be mentioned a method in which either one of two types of compounds capable of coordinate bonding via a metal ion is applied to the first probe or the second probe, respectively, and the concentration of the metal ion in the medium is adjusted with a chelating agent; a method of cross-linking the first probe and the second probe with a DNA linker, and the like. Further, as an irreversible cross-linking method, for example, there can be mentioned a method of causing a binding reaction by adding a cross-linking agent (for example, a method using an epoxy-based cross-linking agent, etc.).

[0044] As an example of the bioparticle labeling method including these cross-linking steps, a case where a method of applying either one of two types of compounds capable of coordinate bonding via a metal ion to the first probe or the second probe respectively and adjusting the concentration of the metal ion in the medium with a chelating agent is used as the cross-linking method will be specifically described with reference to the drawings.

[0045] FIG. 7 is a modification of the bioparticle labeling method of the present technology, showing an image of modifying the cell surface of an antigen-expressing cell 201 with the first probe 101 that contains either one of a donor molecule or an acceptor molecule and is provided with either one of two types of compounds capable of coordinate bonding via a metal ion.

[0046] The difference between the modified example of the biological particle labeling method of the present technology shown in FIG. 7 and the example shown in FIG. 3 lies in that one of two compounds capable of coordinate bonding via a metal ion is provided to the first probe 101, the other of the two compounds is provided to the second probe, and a chelating agent is present in a medium, and the metal ion forms a chelate complex with the chelating agent, thereby controlling the concentration of the metal ion to a low level. The first probe 101 and the second probe 103 that can be used in the modified example of the biological particle labeling method of the present technology can adopt the same configuration as that shown in FIG. 3 except that the above compound (described as compounds 108 capable of mutually coordinate bonding to each other in FIG. 7) is provided thereto.

[0047] In the state shown in FIG. 7, since the metal ion forms a chelate complex with a chelating agent, the two types of compounds 108 respectively provided to the first probe 101 and the second probe 106 cannot form a coordinate bond via the metal ion. Therefore, in the state shown in FIG. 7, the first probe 101 and the second probe 106 are not crosslinked.

[0048] Next, FIG. 8 shows an image of an antibody 204 secreted by an antibody-producing cell 203 capturing an antigen 202 expressed on the cell membrane of an antigen-expressing cell 201 in a modified example of the biological particle labeling method of the present technology. Similar to the case shown in FIG. 4, as the capturing step S101, it can be confirmed that the antibody 204 secreted from the antibody-producing cell S (203) is dispersed in the medium of this two-cell system, and a part of the secreted antibody 204 captures the antigen 202 expressed on the cell membrane of the antigen-expressing cell E (201). Furthermore, as the binding step S102, it can be confirmed that the antibody-binding molecule 105 modified with the second probe 106 provided with the other compound 108 corresponding to the compound provided on the first probe 101 captures the antibody 204 that captures the antigen 202, and labels the antigen-expressing cell E (201).

[0049] In the state shown in Figure 8, as in the state shown in Figure 7, the metal ions form chelate complexes with the chelating agent. Therefore, the two compounds 108 applied to the first probe 101 and the second probe 106 cannot form coordinate bonds with each other via the metal ions, and the first probe 101 and the second probe 106 are not crosslinked.

[0050] When crosslinking the first probe 101 and the second probe 106 by applying one of two compounds 108 that can coordinately bond via metal ions to either the first probe 101 or the second probe 106, and adjusting the concentration of the metal ions in the medium with a chelating agent, the adjustment of the metal ion concentration may be performed by washing away the chelating agent. In this case, the biological particle labeling method of this technology includes a washing step S103 after the bonding step S102 and before the crosslinking step S104.

[0051] In the biological particle labeling method of this technology, the washing step S103, as shown in Figure 2, is a step in which the chelating agent is removed from the medium by washing after the binding step S102 and before the crosslinking step S103.

[0052] Figure 9 shows an image illustrating that, as described above, in the binding step S102, when antigen-expressing cells E are labeled with the first probe 101 and the second probe 106, the chelating agent is removed by washing (indicated as [chelating agent -] in Figure 9), causing the first probe 101 and the second probe 106 to be crosslinked by coordination bonds, promoting the FRET reaction through the interaction of donor and acceptor molecules, and resulting in fluorescence emission.

[0053] Here, the crosslinking between the first probe 101 and the second probe 106 is achieved by removing the chelating agent from the medium by washing and replacing it with a medium containing the metal ions necessary for the coordination bond, so that the two compounds 108 attached to the first probe 101 and the second probe 106 present on the cell surface of the antigen-expressing cell E (201) form a coordination bond via the metal ions.

[0054] Furthermore, by performing the washing step S103, in addition to removing the chelating agent from the medium, antibodies 204 secreted from antibody-producing cells S(203) and antibody-binding molecules 105 suspended in the medium can also be removed. This makes it possible to more effectively reduce the probability of detecting false positives.

[0055] When the bioparticle labeling method of this technology includes a crosslinking step, if an antibody that specifically interacts with the antigen expressed on the cell membrane of an antigen-expressing cell exists (in the case of a true positive), as shown in Figure 9, the positions of the donor molecule and acceptor molecule of the first probe 101 and the second probe 106 present on the cell surface of the antigen-expressing cell E(201) can be fixed, and the donor molecule and acceptor molecule can be brought into sufficient proximity. This improves the probability of a FRET reaction occurring, allows for highly sensitive detection of the FRET reaction, and reduces the likelihood of missing a true positive result.

[0056] In the biological particle labeling method of this technology, when a method is used in which one of two compounds that can coordinate bond via a metal ion is attached to either a first probe or a second probe as a crosslinking method, and the concentration of the metal ion in the medium is adjusted with a chelating agent, the chelating agent that can be used is not particularly limited. For example, any chelating agent that can be used for the analysis of biological particles, such as EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), and citric acid, can be used. Similarly, the metal ions that can be used are not particularly limited. For example, any metal ions that can be used as a buffer for biological particles, such as calcium ions and magnesium ions, can be used.

[0057] <Pairing Step> The biological particle labeling method of this technology may include a pairing step S105. As shown in Figure 2, the pairing step S105 is a step in which, before the capture step S101, the pairing reagent is bound to either an antigen-expressing cell or an antibody-producing cell, and then to the other. The pairing step S105 may be performed after the isolation step S106, which will be described later.

[0058] In the pairing step S105, the "pairing reagent" used to bind antigen-expressing cells and antibody-producing cells comprises an antigen-expressing cell binding site that can bind to antigen-expressing cells and an antibody-producing cell binding site that can bind to antibody-producing cells.

[0059] When this technology's bioparticle labeling method can label cells that produce antibodies that specifically interact with the antigen on the cell membrane of antigen-expressing cells (true positive), it is preferable to pre-couple antigen-expressing cells and antibody-producing cells to form a paired cell group in order to efficiently identify the antibody-producing cells that produce such antibodies.

[0060] Figure 10 shows an image of a biological particle formed by binding antigen-expressing cells 201 and antibody-producing cells 203 via a pairing reagent 110, which has an antigen-expressing cell binding site 110-1 that can bind to antigen-expressing cells 201 and an antibody-producing cell binding site 110-2 that can bind to antibody-producing cells 203. <10A> shows the state after the pairing process and before antibodies are secreted from antibody-producing cells S (203), and <10B> shows an image in which antibodies 204 that capture the antigen expressed on the cell membrane of antigen-expressing cells 201 are present (true positive), and antibody-binding molecules 105 capture the antibodies 204. As shown in the example in Figure 10, by keeping antigen-expressing cells 201 and antibody-producing cells 203 as a paired cell group, antibody-producing cells 203 that produce antibodies 204 in the case of true positive can be efficiently identified.

[0061] Furthermore, even when antigen-expressing cells and antibody-producing cells are paired, they can be isolated as a group including both antigen-expressing cells and antibody-producing cells by methods such as encapsulation in droplets or attachment to a carrier, as described later.

[0062] Figure 11 shows an image of the procedure for pairing antigen-expressing cells 201 and antibody-producing cells 203 in the pairing step S105. Here, the pairing reagent 110 that can be used in the pairing step is not particularly limited as long as it has an antigen-expressing cell binding site 110-1 that can bind to antigen-expressing cells 201 and an antibody-producing cell binding site 110-2 that can bind to antibody-producing cells 203, and is capable of binding the target antigen-expressing cells 201 and antibody-producing cells 203.

[0063] As a pairing reagent, for example, the antigen-expressing cell binding site and the antibody-producing cell binding site may be antibodies that capture substances expressed on the cell membrane of antigen-expressing cells or antibody-producing cells as antigens, respectively. In this case, the antibodies that form the antigen-expressing cell binding site and the antibody-producing cell binding site may be, for example, DNA-linked antibodies, and a pairing reagent capable of binding target antigen-expressing cells and antibody-producing cells can be prepared by linking both antibodies using a nucleic acid oligomer having a nucleic acid sequence that hybridizes to the DNA linker attached to the antigen-expressing cell binding site and a nucleic acid sequence that hybridizes to the DNA linker attached to the antibody-producing cell binding site.

[0064] Figure 12 shows an example of a pairing reagent 110 prepared by linking two antibodies: an antibody with a DNA linker 110-3 that forms an antigen-expressing cell binding site, and an antibody with a DNA linker 110-4 that forms an antibody-producing cell binding site, using a nucleic acid oligomer 110-7 that hybridizes to their respective DNA linkers 110-5 and 110-6. Figure 12 confirms that the pairing reagent 110 shows two types of antibodies with DNA linkers (110-3 and 110-4) linked via a nucleic acid oligomer 110-7 that hybridizes to their respective DNA linkers 110-5 or 110-6. As shown in Figure 12, the base sequence at one end of nucleic acid oligomer 110-7 is complementary to the base sequence of DNA linker 110-5 of DNA linker antibody 110-3, and the base sequence at the other end of nucleic acid oligomer 110-7 is complementary to the base sequence of DNA linker 110-6 of DNA linker antibody 110-4. Therefore, the two DNA linker antibodies (110-3 and 110-4) can be linked via the complementary sequences at the ends of nucleic acid oligomer 110-7.

[0065] The pairing step S105 shown in Figure 11 can be performed using any pairing reagent 110, such as the example shown in Figure 12 above. First, <11A> shows an image of adding the pairing reagent 110 to the culture medium of antibody-producing cells S (203). <11B> shows an image of the antibody forming the antibody-producing cell binding site 110-2 of the pairing reagent 110 capturing and binding to the antigen on the cell membrane of the antibody-producing cells S (203). In the case of <11B>, it can be seen that there is an excess of pairing reagent 110 in the medium that is not bound to the antibody-producing cells S (203). Next, <11C> shows an image after the excess pairing reagent 110 that is not bound to the antibody-producing cells S (203) in the medium has been removed by washing with exchange of the medium. <11D> shows the subsequent addition of antigen-expressing cell E (201), while <11E> shows the process of pairing the antigen-expressing cell with the antibody-producing cell by the antibody forming the antigen-expressing cell binding site 110-1 of the pairing reagent 110 capturing and binding to the antigen on the cell membrane of the added antigen-expressing cell E.

[0066] Furthermore, in the final step of pairing <11E>, the binding rate may be low if the cell concentration is low. For this reason, for example, by not mixing the medium and leaving it in a static state, natural cell sedimentation is promoted and a high-density cell state is created, which can improve the pairing binding rate between antigen-expressing cells 201 and antibody-producing cells 203.

[0067] As described above, the time required for the sample to remain still to promote natural cell sedimentation and achieve a high-density cell state can be arbitrarily adjusted according to the medium and cells used, allowing for observation of natural cell sedimentation. For example, any time can be set, such as 60 minutes or more, 75 minutes or more, or 90 minutes or more.

[0068] Furthermore, in order to improve the pairing and binding rate between antigen-expressing cells and antibody-producing cells, methods for adjusting the high-density cell state include not only methods that promote natural cell sedimentation, but also methods that rapidly and forcibly sediment cells by centrifugation. By sedimenting cells by centrifugation, the reaction time can be shortened compared to methods that promote natural cell sedimentation. Shortening the reaction time reduces the decrease in cell viability in the medium and suppresses the subsequent occurrence of cross-contamination.

[0069] In this context, cross-contamination occurs when antibodies secreted from antibody-producing cells capture antigens from antigen-expressing cells other than the antigen-expressing cell with which the antibody-producing cell was paired. When such cross-contamination occurs, the antibody-producing cell that produces the target antibody cannot be properly identified.

[0070] When cells are settled by centrifugation to adjust the high-density cell state, the conditions can be arbitrarily adjusted according to the medium and cells used. For example, any conditions used when washing unwanted components from a dispersion medium containing cells can be adopted. Specifically, conditions such as 200 × g, 500 × g for centrifugal force, and 5 minutes, 3 minutes, or 1 minute for centrifugation time may be adopted.

[0071] Figure 11 shows an example of a preferred procedure for the pairing step, while Figure 13 shows an image of the case where the pairing reagent 110 is added in the pairing step S105 while antigen-expressing cell E (201) and antibody-producing cell S (203) are present simultaneously. In this case, as shown in Figure 13, one binding site (110- or 110-2) of the pairing reagent 110 binds to the antigen on the cell membrane of antigen-expressing cell E (201) and antibody-producing cell S (203), respectively, blocking the binding sites of antigen-expressing cell E (201) and antibody-producing cell S (203). As a result, the other binding site of the pairing reagent 110 cannot capture the antigen of the other cell, and the pairing binding rate may be significantly reduced.

[0072] Next, Figure 14 shows an image illustrating the cases in which biological particles consisting of paired antigen-expressing cells 201 and antibody-producing cells 203 can be analyzed using the biological particle labeling method of this technology, and whether they are acceptable or unacceptable as analytes.

[0073] <14A> shows an image of what is acceptable for analysis. In the group of cells forming the biological particles shown in <14A>, although there are multiple antigen-expressing cells E (201), it can be confirmed that there is only one antibody-producing cell S (203). In other words, when the biological particles shown in <14A> are labeled using this technology, if the antibody produced by antibody-producing cell 203 specifically interacts with the antigen on antigen-expressing cell 203 (true positive), the antibody-producing cell that produces that antibody can be identified.

[0074] On the other hand, <14B> illustrates a case where the analysis is unacceptable. It can be confirmed that the group of cells forming the biological particles shown in <14B> contains multiple antibody-producing cells S (203). In this case, even if the antibody produced by the antibody-producing cells 203 specifically interacts with the antigen on the antigen-expressing cells 201 (true positive) as a result of labeling the biological particles shown in <14B> using this technology, it is not possible to identify which antibody-producing cell 203 contained in the biological particles is producing the target antibody.

[0075] Based on the above, in the pairing step of the biological particle labeling method of this technology, it is preferable that the pairing reagent be bound to each cell in the order shown in Figure 11, by first binding the pairing reagent to the antibody-producing cell 203 and then to the antigen-expressing cell 201. This makes it possible to avoid the inclusion of multiple antibody-producing cells in the group of cells that form the biological particles.

[0076] Furthermore, by setting the ratio of antigen-expressing cells to antibody-producing cells to a number greater than 1 beforehand, it is possible to avoid the inclusion of multiple antibody-producing cells in the biological particles formed by pairing. The ratio of antigen-expressing cells to antibody-producing cells can be set within any range greater than 1.

[0077] Next, the binding ability of a pairing reagent to antigen-expressing cells via its antigen-expressing cell binding site does not necessarily coincide with its binding ability to antibody-producing cells via its antibody-producing cell binding site. Furthermore, the binding ability changes depending on the medium used and the conditions of the cells. For this reason, the pairing binding rate, which represents the proportion of target biological particles relative to the total number of antibody-producing cells used, may be low. In other words, improving the pairing binding rate can increase the proportion of biological particles available for analysis, thereby improving analytical efficiency.

[0078] For example, by using two or more pairing reagents, it is expected that the pairing and binding rate between antibody-producing cells and antigen-expressing cells can be improved, thereby enhancing the efficiency of subsequent analysis.

[0079] Figure 15 shows images of the biological particles formed when one type of pairing reagent 110 is used and when two types of pairing reagents (111 and 112) are used, in which antigen-expressing cells 201 and antibody-producing cells 203 are bound. <15A> is the biological particle when one type of pairing reagent 110 is used, and <15B> is the biological particle when two types of pairing reagents (111 and 112) are used.

[0080] When two or more pairing reagents are used, as shown in <15B>, the antigen-expressing cell binding site and the antibody-producing cell binding site of each pairing reagent (111 and 112) target different substances expressed on the cell membrane of antigen-expressing cell E (201) or antibody-producing cell S (203). That is, the antigen-expressing cell binding sites of pairing reagent 1 (111) and pairing reagent 2 (112) shown in <15B> each target different substances, and the antibody-producing cell binding sites of pairing reagent 1 (111) and pairing reagent 2 (112) also target different substances.

[0081] As shown in <15B>, by using multiple pairing reagents that target different substances expressed on the cell membrane of antigen-expressing cell E (201) or antibody-producing cell S (203), even if a pairing reagent fails to exhibit sufficient binding ability due to the medium used or the conditions of the cells, its binding ability can be reinforced by binding with other pairing reagents, thereby improving the pairing binding rate. This can increase the proportion of biological particles subjected to analysis and improve analytical efficiency.

[0082] Furthermore, pairing antigen-expressing cells with antibody-producing cells using two or more pairing reagents is not limited to analysis using the bioparticle labeling method of this technology, but can also be suitably used as a pairing method in the analysis of other two-cell systems, independently of the bioparticle labeling method of this technology.

[0083] Furthermore, in the example shown in Figure 15, for the sake of explanation, it is depicted that antigen-expressing cell E (201) and antibody-producing cell S (203) form one bond each with one pairing reagent. However, as shown in the example in Figure 10, each pairing reagent may form multiple bonds.

[0084] In the pairing step of the biological particle labeling method of this technology, an example was shown in which DNA linker-equipped antibodies are linked to each other via nucleic acid oligomers as the pairing reagent. However, as mentioned above, the pairing reagents that can be used in this technology are not limited to this. In addition to the above, other pairing reagents that can be used in this technology include compounds that react selectively with only specific chemical groups without interfering with other molecules in the body.

[0085] One example of such a system is one using Trans-Cyclooctene (TCO) and Tetrazine (Tz). In this case, compounds to which N-Hydroxysuccinimide active ester (NHS) is conjugated to TCO and Tz, respectively, are used as pairing reagents. Each of these pairing reagents forms a covalent bond with a lysine residue in any protein on the cell surface via NHS. Therefore, the cell surface of the antigen-expressing cell is modified by one pairing reagent, and the cell surface of the antibody-producing cell is modified by the other pairing reagent. Subsequently, the antigen-expressing cell and the antibody-producing cell can be efficiently paired by an inverse electron-demand Diels-Alder (IEDDA) reaction between TCO and Tz.

[0086] In the pairing process, when using the above-mentioned pairing reagent, after pairing antigen-expressing cells with antibody-producing cells, the unreacted TCO remaining on the cell surface may be treated with tetrazine acid (TzA) to stop the IEDDA reaction between TCO and Tz. This can prevent excessive aggregation of cells in subsequent steps.

[0087] In the pairing step of the biological particle labeling method of this technology, the pairing and binding rate between antigen-expressing cells and antibody-producing cells can be improved by combining the methods described above.

[0088] <Modification of the Pairing Process> In the above, the pairing process is defined as the process of binding a pairing reagent to either an antigen-expressing cell or an antibody-producing cell, and then to the other, and an example of adding the pairing reagent to a medium is described. On the other hand, as shown in Figure 16, there are cases where the antibody 204 secreted by the antibody-producing cell S (203) remains on the surface of the antibody-producing cell S (203), and the antibody 204 captures the antigen 202 on the cell membrane of the antigen-expressing cell E (201), causing the antigen-expressing cell E (201) and the antibody-producing cell S (203) to bind via the antibody 204. In this case, the antibody 204 secreted by the antibody-producing cell S (203) plays the role of the pairing reagent.

[0089] One possible method for promoting the binding of antibody-producing cells 201 and antibody-producing cells 203 by antibodies 204 secreted from antibody-producing cells 203 is to adjust the cell density by centrifugation without adding a specific pairing reagent. In this case, for example, by separating biological particles in heteropair as a pretreatment, it is possible to efficiently identify candidates for which antibodies that capture the antigen expressed on the cell membrane of antigen-expressing cells E are present (true positive), and to efficiently perform subsequent labeling, identification, and analysis.

[0090] For example, in the biological particle labeling method of this technology, when performing the pairing step of this modified example, if the amount of antibody secreted from antibody-producing cells is large and the amount of antigen expressed on antigen-expressing cells is also large, or if the binding affinity between the antibody-producing secreted antibody and the antigen on the cell membrane of the antigen-expressing cell is strong, then pre-treatment separation can streamline subsequent labeling, identification, and analysis. Furthermore, even when such circumstances do not exist, pre-treatment separation can streamline subsequent labeling, identification, and analysis, thereby improving the throughput of cell identification.

[0091] In this case, the conditions for sedimentation by centrifugation can be the same as those used when centrifugation is performed to settle cells in order to adjust the high-density cell state when using the aforementioned pairing reagent.

[0092] <Identification Code> Antibody-producing cells used in the biological particle labeling method of this technology may be assigned an identification code. By assigning an identification code to antibody-producing cells, these cells can be efficiently subjected to single-cell analysis. Examples of such identification codes include those using nucleic acid sequences. Thus, when antibody-producing cells identified by labeling with the biological particle labeling method of this technology are assigned an identification code using a nucleic acid sequence, it is possible to seamlessly perform analysis such as NGS (next generation sequencing) after separating the cells.

[0093] The method for assigning an identification code to antibody-producing cells is not particularly limited, and any method can be used. For example, one method is to assign an identification code to a cell by encapsulating a single cell (antibody-producing cell) and a barcoding bead (or gel bead) having an identification code using a nucleic acid sequence or the like in a microspace such as a well or emulsion. Another method is to capture a cell (antibody-producing cell) and assign an identification code using a substrate equipped with an identification code such as a nucleic acid sequence and a capture part for capturing cells on its surface.

[0094] Furthermore, the identification code used can be designed by arbitrarily combining cell identifiers (cell bars) to identify the cells from which the nucleic acids to be analyzed originate, and molecular identifiers (molecular bars, UMIs) to identify individual mRNAs, according to the conditions and objectives of subsequent analysis of antibody-producing cells.

[0095] Figure 17 shows an example of a method for assigning an identification code 301 to biological particles consisting of paired antigen-expressing cells 201 and antibody-producing cells 203. In the example shown in Figure 17, the antibody-producing cell S (203) and antigen-expressing cell E (201) are paired first, and then the identification code 301 is assigned.

[0096] <17A> is an image of a biological particle in which an antibody-producing cell S (203) and an antigen-expressing cell E (201) are bound via a pairing reagent. <17B> shows an image of assigning an identification code 301 to the biological particle. In the example shown in <17B>, an example is shown in which an antibody-producing cell 203 of the paired biological particle is captured using a substrate 303 equipped with an identification code 301 such as a nucleic acid sequence and a capture part 302 for capturing cells on its surface, and the identification code 301 is assigned to the antibody-producing cell 203, but other methods of assigning an identification code may be used. Although not explicitly stated, it is preferable to remove biological particles that are not captured on the substrate 303 in <17B> (biological particles that are not assigned the identification code 301) by washing. Furthermore, in the region on the substrate 303 where the identification code 301 is assigned, it is preferable to adjust the concentration of biological particles in the dispersion medium to a low level in order to prevent captured biological particles from overlapping and being assigned the same identification code 301.

[0097] <17C> shows an image in which antibody 204 that captures the antigen expressed on the cell membrane of antigen-expressing cell E (201) is present (true positive), and antibody-binding molecule 105 captures antibody 204. <17D> shows an image of the biological particles shown in <17C> separated from the substrate 303. The true positive biological particles shown in <17D> are assigned an identification code 301 as shown in the figure, and after being separated using a flow cytometer or the like, antibody-producing cell S (203) can be efficiently analyzed by NGS (next generation sequencing) or the like. In this case, by NGS analysis, for example, the sequence of antibody 204 that captures the antigen expressed on the cell membrane of antigen-expressing cell E (201) can be identified.

[0098] Next, Figure 18 shows an image of a modified method for assigning an identification code 301 to biological particles consisting of paired antigen-expressing cells 201 and antibody-producing cells 203. In the example shown in Figure 18, the identification code 301 is assigned to the antibody-producing cell S (203), and then the antibody-producing cell S (203) is paired with the antigen-expressing cell E (201).

[0099] Specifically, as shown in <18A>, first, an identification code 301 is assigned to the antibody-producing cells S (203). In <18A>, an example is shown in which antibody-producing cells S (203) are captured using a substrate 303 having an identification code 301 such as a nucleic acid sequence and a capture unit 302 for capturing cells on its surface, and the identification code 301 is assigned to the cells. However, other methods for assigning the identification code may also be used. In the step of <18A>, it is preferable to remove antibody-producing cells that are not captured on the substrate 303 (antibody-producing cells that are not assigned the identification code) by washing. Furthermore, in the region on the substrate 303 where the identification code 301 is assigned, it is preferable to adjust the concentration of antibody-producing cells in the dispersion medium to a low level in order to prevent captured antibody-producing cells from being assigned the same identification code 301 repeatedly.

[0100] Next, as shown in <18B>, antibody-producing cells S (203) and antigen-expressing cells E (201) are paired on a substrate 303 equipped with an identification code 301. To improve the pairing binding rate between antibody-producing cells 203 and antigen-expressing cells 201, it is preferable that the number of antigen-expressing cells E (203) added in step <18B> is greater than the number of captured antibody-producing cells S (203) on the substrate 303. In addition, to suppress the occurrence of cross-contamination, unbound antigen-expressing cells 203 may be removed by washing after the pairing reaction. By performing the above procedure, <18B> will be substantially the same as the state shown in <17B>. The subsequent steps <18C> and <18D> are substantially the same as <17C> and <17D>, and similar conditions can be used and similar effects can be expected.

[0101] <Isolation Step and Droplet Breaking Step> The biological particle labeling method of this technology may include an isolation step S106. As shown in Figure 2, the isolation step S106 is a step in which a group including antibody-producing cells and one or more antigen-expressing cells is isolated before the capture step S101.

[0102] By isolating a group of antibody-producing cells and one or more antigen-expressing cells, the secretion of antibodies from the antibody-producing cells and the capture of antigens expressed on the cell membrane of the antigen-expressing cells by those antibodies occur within the isolated space. This prevents contamination by antibodies secreted from other antibody-producing cells, thus effectively suppressing cross-contamination.

[0103] In the isolation process, the method for isolating a group containing antibody-producing cells and one or more antigen-expressing cells is not particularly limited, and any method can be used. Examples include a closed-system isolation method, such as encapsulating the group containing antibody-producing cells and one or more antigen-expressing cells in a droplet (emulsion), and an open-system or semi-open-system isolation method, such as binding the group containing antibody-producing cells and one or more antigen-expressing cells to a carrier.

[0104] Figure 19 shows an image illustrating an example of a method for isolating a group containing antibody-producing cells 203 and antigen-expressing cells 201 in the biological particle labeling method of this technology. In the example shown in Figure 19, a group containing antibody-producing cells 203 and one or more antigen-expressing cells 203 is isolated by encapsulating it in a droplet 310.

[0105] <19A> is an image of biological particles in which antibody-producing cells S (203) and antigen-expressing cells E (201) have been bound via pairing reagent 110. <19B> shows that biological particles in which antibody-producing cells S (203) and antigen-expressing cells E (201) have been bound via pairing reagent 110 are detected from the sample flowing through the microchannel 311 by arbitrary means such as laser irradiation, and only the target biological particles are selectively encapsulated in droplets 310 (emulsion) and isolated. Note that <19B> is just one example of a means to encapsulate and isolate the target group in droplets 310, and is not limited to this, as long as it is a means that can isolate the target group.

[0106] <19C> shows a state in which a biological particle containing an antibody-producing cell S (203) and an antigen-expressing cell E (201) is isolated within a droplet 310 isolated in <19B>. It can also be confirmed that an antibody-binding molecule 105 modified with a second probe 106 for labeling the biological particle using the biological particle labeling method of this technology is also enclosed within the droplet.

[0107] <19D> shows that within the droplet 310 forming the isolation space, the antibody 204 produced by the antibody-producing cell 203 specifically interacts (true positive) with the antigen expressed on the cell membrane of the antigen-expressing cell 201, and the antibody 204 is captured by the antibody-binding molecule 105 modified with the second probe 106, thereby confirming that the biological particle is labeled. In this way, by performing the capture step S101 and the binding step S102 within the droplet 310, contamination by antibodies secreted from other antibody-producing cells can be prevented, and thus the occurrence of cross-contamination can be effectively suppressed.

[0108] Next, Figure 20 shows a modified version of the isolation method shown in Figure 19, which involves encapsulating a group containing antibody-producing cells 203 and one or more antigen-expressing cells 201 in a droplet 310. Specifically, in the isolation method shown in Figure 19, an example is shown in which biological particles formed using antibody-producing cells S (203) to which identification code 301 has been assigned are used as biological particles formed by linking antibody-producing cells S (203) and antigen-expressing cells E (201) via the pairing reagent 110 shown in <19A>. The isolation method shown in Figure 20 is the same as the isolation method in Figure 19, except that biological particles formed using antibody-producing cells S (203) to which identification code 301 has been assigned are used as biological particles. Furthermore, the same conditions can be adopted for each step.

[0109] In the example of the isolation method shown in Figure 20, by using biological particles formed with antibody-producing cells S(203) to which identification code 301 has been assigned, as mentioned above, after separating the antibody-producing cells S(203), analysis by NGS or the like can be performed seamlessly.

[0110] Figure 21 shows a modified example of the method for isolating a group containing antibody-producing cells 203 and one or more antigen-expressing cells 201 by encapsulating them in a droplet 310, as shown in Figure 19 or Figure 20. Specifically, this method involves preparing a droplet 310 containing a group containing antibody-producing cells 203 and one or more antigen-expressing cells 201 by fusing droplets 310 each containing antibody-producing cells S (203) and antigen-expressing cells E (201), without performing a pairing step or preparing biological particles by linking antibody-producing cells S and antigen-expressing cells E via a pairing reagent.

[0111] Figure 21A shows an image of a dispersion containing antibody-producing cells S (203) and antigen-expressing cells E (201) in a dispersion medium. In the isolation method shown in Figure 21, first, without performing a pairing step, the antibody-producing cells S (203) and antigen-expressing cells E (201) are enclosed in a dispersion without preparing biological particles by binding them via a pairing reagent. From the viewpoint of avoiding the inclusion of multiple antibody-producing cells S (203) in the final droplet 310, it is preferable that the dispersion contains more antigen-expressing cells E (201) than antibody-producing cells S (203).

[0112] <21B> shows that antibody-producing cells S (203) and antigen-expressing cells E (201) contained in the dispersion are detected individually by the same method as shown in <19B>, and each is sealed in a droplet 310 and isolated.

[0113] <21C> illustrates the image of two droplets 310 colliding and fusing by controlling the movement speed of a droplet 310 containing antibody-producing cells S (203) along a channel to be faster than the movement speed of a droplet 310 containing antigen-expressing cells E (201) that follows along the channel. It can be confirmed that the droplet 310 obtained by collision and fusion contains a group consisting of antibody-producing cells S (203) and antigen-expressing cells E (201). Furthermore, by preparing the dispersion to contain more antigen-expressing cells E (201) than antibody-producing cells S (203), the probability of a droplet 310 containing antigen-expressing cells E (201) following a droplet 310 containing antibody-producing cells S (203) is increased.

[0114] Furthermore, although not explicitly shown in the figure, the droplet 310 also contains an antibody-binding molecule modified with a second probe for labeling biological particles using the biological particle labeling method of this technology. As a result, when an antibody produced by an antibody-producing cell S (203) specifically interacts with an antigen expressed on the cell membrane of an antigen-expressing cell E (201) within the droplet (true positive), the antibody is captured by the antibody-binding molecule modified with the second probe, thereby suitably labeling the biological particle.

[0115] Furthermore, even when using the isolation method shown in Figure 21, the pairing reagent may be encapsulated within the droplet 310 formed by collision fusion, and the antibody-producing cell S (203) and antigen-expressing cell E (201) may be paired within the droplet 310.

[0116] In the biological particle labeling method of this technology, when a closed-system isolation method is used as the isolation step, in which a group containing antibody-producing cells 203 and one or more antigen-expressing cells 201 is isolated by encapsulating them in a droplet 310, as shown in Figures 19 to 21, a droplet destruction step may be included to wash away components contained in the droplet 310 that are no longer needed in subsequent steps.

[0117] As shown in Figure 2, the droplet breaking step S107 is a step that breaks the droplets after the bonding step S102. If the specific step S110 described later is performed, the droplet breaking step S107 is performed before the specific step S110. In the droplet breaking step, by breaking the droplets and replacing the medium, components contained in the droplets that are unnecessary in subsequent steps can be washed away.

[0118] In particular, when crosslinking a first probe and a second probe to improve the probability of the FRET reaction occurring is performed by adjusting the concentration of the metal ions in the medium with a chelating agent, thereby enabling coordinate bonding via the metal ions, it becomes necessary to wash the chelating agent in the droplet to promote coordinate bonding. In this case, the droplet breaking step breaks the droplet and replaces the medium, thereby washing and removing the chelating agent contained in the droplet, and enabling crosslinking of the first probe and the second probe by coordinate bonding.

[0119] Furthermore, the droplet disruption process, by exchanging the medium, removes not only the chelating agent from the medium but also antibodies secreted from antibody-producing cells S and antibody-binding molecules suspended in the medium. This more effectively reduces the probability of detecting false positives and improves the efficiency of subsequent analysis.

[0120] Figure 22 shows an image illustrating the process in the biological particle labeling method of this technology, where a group containing antibody-producing cells 203 and antigen-expressing cells 201, which have been encapsulated and isolated in a droplet 310, is separated from the target group after the droplet 310 has been destroyed.

[0121] <22A> shows an image, similar to that shown in <20D>, of biological particles formed by the binding of antibody-producing cells S (203) and antigen-expressing cells E (201) within a droplet 310, which are labeled by the biological particle labeling method of this technology. Note that <22A> is merely an example, and shows an example in which biological particles formed using antibody-producing cells S (203) to which identification code 301 has been assigned are included in the droplet 310, but is not limited to this.

[0122] In <22B>, it can be confirmed that the droplet 310 covering the biological particles has been destroyed. This also removes the chelating agent, the antibodies 204 secreted from antibody-producing cells S (203) that are suspended in the medium, and the antibody-binding molecules 105.

[0123] <22C> indicates that, using a method similar to that shown in <20B>, etc., the target biological particles are detected, and only the target biological particles are selectively sealed in droplet 310, re-isolated, and separated. The separated biological particles can then be subjected to analysis by NGS or the like.

[0124] In addition, in the biological particle labeling method of this technology, the isolation step may be performed by binding a group containing antibody-producing cells and one or more antigen-expressing cells to a carrier. In this case, isolation can be performed in an open or semi-open system. When isolation is performed in an open or semi-open system, unlike the sealed system isolation method using droplets described above, the droplets can be destroyed in a droplet destruction step, and chelating agents, antibodies secreted from antibody-producing cells S suspended in the medium, and antibody-binding molecules can also be removed without exchanging the medium.

[0125] The carrier that can be used in the isolation process is not particularly limited, and any carrier capable of supporting biological components such as cells can be used. For example, the carrier may hold the biological components on its outer surface or on its inner surface. The shape of the carrier is also not particularly limited and may have any shape, such as spherical, elliptical, or rod-shaped. Furthermore, the material constituting the carrier is not particularly limited, and any material that can be used for the analysis of biological particles, such as agarose gel, sodium alginate, or extracellular matrix (collagen, fibronectin, laminin, elastin, etc.), can be suitably used.

[0126] <Magnetic Imprinting and Purification Process> Using the biological particle labeling method of this technology, the labeled biological particles are identified and separated by detecting the fluorescence emitted by the interaction between a first probe and a second probe upon irradiation with excitation light, for example, using a flow cytometer. This identifies the biological particles containing antibody-producing cells that are truly positive.

[0127] On the other hand, in order to improve the number of processes per unit time for the series of events from identification to sorting, it is desirable to remove free antigen-expressing cells and other cells that are not bound to antibody-producing cells and are not to be analyzed beforehand. In particular, when antigen-expressing cells are introduced in excess of antibody-producing cells in order to increase the rate of generation of biological particles paired with antibody-producing cells, the number of free cells increases. As a result, the proportion of free cells that are not to be analyzed processed increases relative to the total number of processes per unit time for the series of events from identification to sorting, reducing processing efficiency and hindering the achievement of high-speed processing (high throughput).

[0128] Furthermore, when this technology includes a method for labeling biological particles, and the isolation is performed by binding a group containing antibody-producing cells and antigen-expressing cells to a carrier, an excess of carrier may be added to the expected group, resulting in the generation of free carrier that does not bind to the group. Since this free carrier is not the target of analysis, it is desirable to remove it before processing in order to reduce unnecessary consumption of the processing unit.

[0129] There are no particular restrictions on the method for removing free cells beforehand, but possible methods include strainer treatment, weak centrifugation or removal of the supernatant by natural sedimentation, and selective separation of target cells by attaching magnetic beads to the target cells. Depending on the target to be isolated and the environmental conditions, the above methods can be used individually or in combination. For example, when the difference in sedimentation rates between the carrier and free cells is small, or when the carrier is easily reversibly adsorbed to a plastic surface, removing the supernatant by natural sedimentation at room temperature or above may be effective. In addition, other appropriate methods can be selected depending on the conditions.

[0130] When the biological particle labeling method of this technology includes an isolation step, and this isolation is performed by binding a group including antibody-producing cells and antigen-expressing cells to a carrier, among the methods for removing the free cells etc. mentioned above in advance, a separation method using magnetic beads is preferably used from the viewpoint of improving the utilization rate of biological particles including antibody-producing cells and reducing the burden on cells by speeding up the operation.

[0131] In other words, when the biological particle labeling method of this technology includes an isolation step, and the isolation is performed by binding a group including antibody-producing cells and antigen-expressing cells to a carrier, a magnetic application step S108 may be included before the isolation step S106, as shown in Figure 2. Here, the "magnetic application step" refers to the step of binding the antigen-expressing cells or the antibody-producing cells to magnetic beads.

[0132] Furthermore, if the biological particle labeling method of this technology includes a magnetic application step, a purification step S109 may be included after the isolation step S106 and before the capture step S101, as shown in Figure 2. Here, the "purification step" refers to a step of separating the cells bound to the magnetic beads using magnetism.

[0133] Figure 23 shows an image illustrating the bioparticle labeling method of this technology, in which antibody-producing cells or antigen-expressing cells (collectively referred to as cell 321 in Figure 23) are bound to magnetic beads 320, and then the carrier to which the group containing the antibody-producing cells and antigen-expressing cells is bound is separated by magnetism.

[0134] Figure 23 (1) shows an image of adding magnetic beads 320 to a dispersion containing antibody-producing cells and antigen-expressing cells (collectively referred to as cell 321). The magnetic beads 320 contain, for example, an antibody that specifically binds to a substance expressed on the cell surface of antibody-producing cells and / or antigen-expressing cells as an antigen. This allows the magnetic beads 320 to be attached to the antibody-producing cells and / or antigen-expressing cells (collectively referred to as cell 321). Figure 23 (2) shows a dispersion containing cells 322 to which the magnetic beads have been attached in this manner. In this bioparticle labeling method of the present technology, from the viewpoint of selectively separating bioparticles including antibody-producing cells, the magnetic beads 320 may be designed to attach specifically to antibody-producing cells.

[0135] Figure 23 (3) shows the state after adding carrier 150 to the dispersion shown in Figure 23 (2). Figure 23 (4) shows that cells 322 bound to carrier 150 and isolated in the dispersion, and free cells 322 that cannot bind to the carrier and float in the dispersion, have been generated. It can also be confirmed that free carrier 150 that is not bound to cells 322 has been generated in the dispersion. In this state, the free cells 322 can be removed from the dispersion by straining the dispersion (Figure 23 (5a)). It can be confirmed that the remaining dispersion after straining (Figure 23 (5b)) contains carrier 150 bound to cells 322 and free carrier 150 that is not bound to cells 322.

[0136] From the dispersion shown in (5b) in Figure 23, the carrier 150 bound to the cells 322 with magnetic beads attached can be distinguished and identified from the free carrier 150 not bound to the cells 322 by using a magnet 323 as shown in (6) of the same figure. Subsequently, as shown in (7) of the same figure, the carrier 150 bound to the cells 322 with magnetic beads can be separated by a method such as decanting. Since this separation does not use a column or the like, physical damage to the cells 322 can be minimized. The separated carrier 150 bound to the cells 322 with magnetic beads attached can be resuspended as shown in (8) of the same figure to complete the purification of the target cells.

[0137] Furthermore, in the cell purification using magnetic beads as described above, the free cells removed by strainer treatment and the free carrier separated by methods such as decanting may be reused and used in other experiments.

[0138] When isolating a group of antibody-producing cells and antigen-expressing cells by binding them to a carrier, purifying the target cells using magnetic beads as described above removes free cells and carriers. This increases the proportion of biological particles to be analyzed relative to the total number of processes per unit time in the subsequent series of events, from identification by fluorescence detection to sorting, thereby improving processing efficiency.

[0139] Furthermore, the purification of target cells using magnetic beads can be utilized not only for analysis using the bioparticle labeling method of this technology, but also independently as a generation method for the analysis of other two-cell systems.

[0140] <Modifications of the Method for Isolating Biological Particles> When the biological particle labeling method of this technology includes an isolation step, as described above, the method for isolating a group of cells to be isolated, such as antibody-producing cells, is not particularly limited, and any method can be used. Here, as a method for isolating a group consisting of one or more cells by binding them to a carrier, when binding target cells to a carrier from a group that includes target cells and non-target cells, a carrier equipped with an antibody that specifically binds to an antigen on the cell surface of the target cells is generally used. This makes it possible to bind only the group containing the target cells to the carrier.

[0141] On the other hand, if there are few biomarker antigens on the cell surface, for example, if the antibody used for fluorescent labeling to identify target cells and the antibody used to bind to the carrier are designed to recognize the same target antigen, the two antibodies will compete, and the antibody binding efficiency will decrease. As a result, problems such as a decrease in the rate of cell binding to the carrier may occur.

[0142] As a way to resolve the above-mentioned problems, for example, the cells to be isolated can be modified with a labeled antibody having a labeling site that identifies those cells, and binding to the carrier can be performed via a label-binding molecule that specifically binds to at least a portion of the labeling site. This avoids competition between the labeled antibody and the antibody used to bind to the carrier. As a result, even when isolating cells with few biomarker antigens on their cell surface, a decrease in the binding rate of the cells to be isolated to the carrier can be suppressed. In this case, the cells to be isolated and the carrier bind as a positive selection.

[0143] Conversely, if cells not to be isolated are modified with a labeled antibody having a labeling site that identifies those cells, binding to the carrier is inhibited by the binding of a label-binding molecule to the labeling site of the cells not to be isolated. This allows the antibody on the carrier surface to suitably capture only the cells to be isolated, and suppresses a decrease in the binding rate of the cells to be isolated to the carrier. In this case, even when isolating cells with few biomarker antigens on their cell surface, the cells to be isolated and the carrier can be suitably isolated as a negative selection.

[0144] Furthermore, in the modified method for isolating biological particles, which addresses the above-mentioned problems, a positive selection or negative selection is made according to the characteristics of the cells to be isolated (combination of antigen expression levels). By designing the labeled antibody, labeling site, and label-binding molecule described later, even cells with few biomarker antigens on their cell surface can be effectively bound to the carrier and isolated.

[0145] Here, the "labeling site" refers to a site that can identify a cell and specify a labeled cell. The method of identifying cells is not particularly limited, and any method can be used. For example, one method is to identify labeled cells by labeling them with a fluorescent dye that absorbs light of a specific wavelength and emits light of a different wavelength, or by labeling them with a specific compound and then identifying the labeled cells using another compound that specifically binds to that compound.

[0146] In a method for identifying labeled cells by labeling with a fluorescent dye, the labeling site is the fluorescent dye. In a method for identifying labeled cells by labeling with a specific compound and then using another compound that specifically binds to that compound, the labeling site is the specific compound.

[0147] Here, the fluorescent dye that can be used as the labeling site is not particularly limited, and any fluorescent dye that can be used in methods for analyzing biological particles can be suitably used. Examples of such fluorescent dyes include phycoerythrin (PE), allophycocyanin, cy3, cy5, cy7, and the like.

[0148] Furthermore, combinations of a specific compound with another compound that specifically binds to it include, for example, antigen-antibody combinations, or biotin-avidin or streptavidin combinations.

[0149] Furthermore, "labeled antibody" refers to an antibody equipped with the labeling site described above. Here, as mentioned above, the antibody related to the labeled antibody can be designed to target any antigen according to the characteristics of the cells to be isolated (combination of antigen expression levels). Examples of such antibodies include anti-CD138 antibody targeting CD138, a maturation marker of Plasma cell; anti-CD38 antibody targeting CD38; anti-CD59 antibody targeting CD59; and anti-B220 antibody targeting B220.

[0150] A "labeled molecule" refers to a compound that can specifically bind to at least a portion of a labeled site. Examples of labeled molecules include antibodies, Fab, Fv, scFv, VHH, affibody, aptamers, and peptide aptamers, which recognize the labeled site as an antigen.

[0151] The carrier used in the method for isolating biological particles according to this modified example is not particularly limited, as long as it has a label-binding molecule on its surface that specifically binds to at least a portion of the labeled site, and any carrier capable of supporting biological components such as cells can be used, similar to the carrier described above. These carriers may be open systems or semi-open systems.

[0152] Furthermore, the cells to be isolated in the method for isolating biological particles according to this modified example are not particularly limited, but for example, when identifying antibody-producing cells that secrete antibodies that specifically interact with antigens expressed on the cell membrane of antigen-expressing cells, it is preferable to use antibody-producing cells. This allows antibody-producing cells to be effectively isolated from other antibody-producing cells. In this case, the antibody-producing cells are modified with the labeled antibody. The isolation may also be performed by isolating a group including the antibody-producing cells and one or more antigen-expressing cells.

[0153] Furthermore, the biological particle isolation method according to this modified example is not limited to its use as an isolation means in the biological particle labeling method of this technology, but can also be suitably used independently of the biological particle labeling method of this technology as an isolation method in other biological particle labeling or analysis methods.

[0154] Next, the method for isolating biological particles according to this modified example will be explained in more detail using diagrams.

[0155] <Separation by magnetism using a modified method for isolating biological particles> First, we will explain a method for separating a group of cells to be isolated by magnetism using the biological particle isolating method described in this modified version.

[0156] Figure 24 shows images of cells to be isolated and cells not to be isolated, used in the method for isolating biological particles according to this modified example. <24A> shows a cell 331 (Target cell) to be isolated, and <24B> shows an image of a cell 332 (Non-target cell) not to be isolated. It can be seen that the cell surface of the cells to be isolated has an identification antigen 333 (Target antigen) to which a labeled antibody equipped with a labeling site for identifying the cells to be isolated specifically binds, while the surface of the cells not to be isolated does not have the above-mentioned identification antigen 333. As mentioned above, the cells to be isolated are not particularly limited, but may include antibody-producing cells, etc.

[0157] Figure 25 shows an image of how the labeled antibody 334 modifies cells in the method for isolating biological particles according to this modified example. As shown in <25A>, the labeled antibody 334 specifically binds to the identification antigen 333 present on the cell surface of the cell 331 to be isolated. On the other hand, as shown in <25B>, the labeling antibody 334 does not bind to the surface of the non-isolated cell 332 because the identification antigen 333 is not present there. In the example shown in Figure 25, biotin is used as the labeling site 335 of the labeled antibody 334 that identifies the cell 331 to be isolated, but as mentioned above, the compounds that can be used as the labeling site are not limited to this.

[0158] Figure 26 shows an example of a carrier 150 having a label-binding molecule 151 that specifically binds to at least a portion of the label site 335, which can be used in the bioparticle isolation method according to this modified example. In the example shown in Figure 26, magnetic beads are used as the carrier 150 to separate a group containing cells to be isolated by magnetism, but any carrier suitable for the subsequent analysis of bioparticles can be used. Furthermore, it can be confirmed that the surface of the carrier 150 shown in Figure 26 contains compounds that can bind to biotin, which is the label site for identifying cells to be isolated as shown in Figure 25 (biotin-binding molecules / biotin-recognizing antibodies or streptavidin, etc.), as the label-binding molecule 151.

[0159] Figure 27 shows an image of how the cells to be isolated 331 bind to the carrier 150 via the label-binding molecule 151 in the bioparticle isolation method according to this modified example. As shown in <27A>, it can be confirmed that in the cells to be isolated 331, the label-binding molecule 151 (biotin-binding molecules) provided on the carrier 150 is specifically bound to the label site 335 (biotin) of the labeled antibody 334 that binds to the identification antigen 333 on the cell surface. As a result, the cells to be isolated 331 can suitably bind to the carrier 150 relating to magnetic beads. On the other hand, the cells 332 not to be isolated, as shown in <27B>, are not modified with the labeled antibody 334, as shown in <25B>, and therefore cannot bind to the carrier 150.

[0160] Figure 28 shows an image of a dispersion in which cells to be isolated 331 and cells not to be isolated 332, as shown in Figure 27, are dispersed in the bioparticle isolation method according to this modified example. Figure 29 shows an image of separating the carrier 150 to which the group containing cells to be isolated 331 is bound from the dispersion shown in Figure 28 using a magnet 323. As shown in Figure 29, only cells to be isolated, such as antibody-producing cells, bound to the carrier 150 relating to magnetic beads are attracted to the magnet 323.

[0161] Subsequently, as shown in Figure 30, non-isolated cells 332 can be removed by decanting or other methods, and the isolation target cells 331 bound to the carrier 150 related to the magnetic beads can be separated. The separated isolation target cells 331 can be purified by resuspending them.

[0162] ≪Positive selection and negative selection using a modified method for isolating biological particles≫ In the separation method using the modified method for isolating biological particles described above, a carrier related to magnetic beads was used for magnetic separation. However, the modified method for isolating biological particles can use any carrier that is suitable for the subsequent analysis of the biological particles. Furthermore, positive selection can be performed by using cells modified with labeled antibodies as the cells to be isolated, and negative selection can be performed by using cells modified with labeled antibodies as the cells not to be isolated.

[0163] Figure 31 shows modified form 150-1 of a carrier having a label-binding molecule that specifically binds to at least a portion of the label site, which can be used in the bioparticle isolation method according to this modified form. For example, when bound to streptavidin, the surface of carrier 150-1 is biotinylated (in this case, it becomes a Biotinylated carrier, and the carrier modification molecule 336 becomes biotin). Also, when bound to an antigen, the surface of carrier 150-1 is modified with an antibody that specifically binds to the antigen (in this case, it becomes an Antibody-conjugated carrier, and the carrier modification molecule 336 becomes the antibody). Note that although carrier 150-1 shown in Figure 31 is an open-system carrier as an example, the carriers that can be used in the bioparticle isolation method according to this modified form are not limited to this, as described above, and any carrier capable of supporting biological components such as cells can be used.

[0164] Figure 32 shows an example of positive selection in the bioparticle isolation method according to this modified example, illustrating how target cells 331 modified with a labeled antibody 334 having a labeling site 335 bind to carrier 15-1 (Biotinylated carrier) via a label-binding molecule 151 that specifically binds to at least a portion of the labeling site 335. In the example shown in Figure 32, the binding of target cells 331 to carrier 150-1 is performed via another carrier 150 having the label-binding molecule 151. More specifically, as shown in Figure 32, this other carrier 150 having the label-binding molecule 151 binds to carrier 150-1 (Biotinylated carrier) via a label-binding molecule 151 located on a different side from the label-binding molecule 151 that binds to the labeling site 335 of the labeled antibody 334 that modifies the surface of target cells 331.

[0165] The label-binding molecule 151 binds to the label site 335 on the labeled antibody 334, rather than to the antigen on the surface of the cells 331 to be isolated. This avoids competition between the labeled antibody 334 and the label-binding molecule 151 on the surface of the carrier 150. Furthermore, as shown in Figure 32, positive selection is achieved by specifically binding the cells to be isolated to the carrier 150-1 via the label-binding molecule 151 positioned on the opposite side from the label-binding molecule 151 that binds to the labeled antibody 334.

[0166] On the other hand, Figure 33 shows an example of negative selection in the bioparticle isolation method according to this modified example, in which non-target cells 332 modified with a labeled antibody 334 having a labeling site 335 have their binding to the carrier 150-1 inhibited by the label-binding molecule 151. As shown in Figure 33, the labeled antibody 334 having a labeling site 335 binds to the antigen on the surface of the non-target cells 332, and another carrier 150 having a label-binding molecule 151 that binds to the labeling site 335 covers the non-target cells 332, so that the non-target cells 332 cannot bind to the carrier 150-1 (Antibody-conjugated carrier). This allows the antibody (carrier-modified molecule 336) on the surface of the carrier 150-1 to suitably capture only the cells 331 to be sequestered, and suppresses a decrease in the binding rate of the cells 331 to the carrier 150-1.

[0167] It should be noted that the above is merely an example of positive selection and negative selection. By arbitrarily designing the target to be modified with the labeled antibody and the binding method to the carrier, a group containing the cells to be isolated can be bound to the carrier and appropriately isolated.

[0168] Furthermore, in the separation method using the bioparticle isolation method described above, magnetic beads were used as a carrier for separation by magnetism, but the separation of the carrier is not limited to this. For example, the carrier bound to the target cells may be separated using a device capable of separating specific bioparticles, such as a flow cytometer.

[0169] <Other Processes> The biological particle labeling method of this technology may be combined with any other process that can be performed in sample preparation, in accordance with the analysis to be performed, as long as the desired effect is not significantly impaired.

[0170] [Method for analyzing biological particles] Next, this technology provides a method for analyzing biological particles using biological particles labeled by the biological particle labeling method of this technology. Specifically, the biological particle analysis method of this technology includes a selection step (S110 in Figure 2) in which an antibody-producing cell that secretes an antibody that specifically binds to the antigen is identified by detecting fluorescence emitted by the interaction between the first probe and the second probe when the biological particles labeled by the biological particle labeling method of this technology are irradiated with excitation light.

[0171] When antibodies produced by antibody-producing cells specifically interact with antigens expressed on the cell membrane of antigen-expressing cells (true positive), fluorescence is emitted due to the interaction (FRET reaction) between donor or acceptor molecules derived from the first probe and donor or acceptor molecules derived from the second probe upon irradiation of biological particles with excitation light. By detecting this fluorescence, antibody-producing cells that secrete antibodies that specifically bind to the antigen can be identified.

[0172] The aforementioned identification can be performed using any device capable of irradiating biological particles with excitation light and detecting their fluorescence. For example, a flow cytometer can suitably perform the above identification.

[0173] [Separation Method] This technology provides a method for separating biological particles that include antibody-producing cells that produce antibodies that specifically interact with antigens expressed on the cell membrane of antigen-expressing cells, as identified by the biological particle analysis method of this technology. This separation can be performed using any device capable of separating the identified biological particles. For example, a flow cytometer can suitably perform the above separation.

[0174] Figure 34 shows an image of analyzing antibody-producing cells after separating biological particles containing target antibody-producing cells using a flow cytometer or the like. <34A> shows an image of sorting the separated cells as single cells on a plate as a pretreatment for NGS. At this time, the cells may be cultured if necessary. <34B> shows an image of analyzing the sequence of antibodies in the antibody-producing cells that specifically interact with antigens expressed on the cell membrane of antigen-expressing cells using NGS. <34C> is an image of antibody-producing cells created based on the sequence of antibodies identified by NGS.

[0175] Note that the analysis shown in Figure 34 is merely an example, and antibody-producing cells identified and isolated using this technology can be subjected to various analyses depending on the purpose.

[0176] [Cell Identification Kit] This technology realizes the biological particle labeling method of this technology and provides a reagent kit for biological particle analysis to identify antibody-producing cells that secrete antibodies that specifically bind to antigens expressed on the cell membrane of antigen-expressing cells.

[0177] As shown in Figure 35, the reagent kit 100 for biological particle analysis of this technology comprises at least a first probe 101 having a modification site 103 that modifies the cell surface of antigen-expressing cells 201, and which includes at least one of a donor molecule that releases energy upon irradiation with excitation light and an acceptor molecule (102 in Figure 35) that receives the energy from the donor molecule and emits fluorescence; and an antibody-binding molecule 105 that binds to an antibody 204, which is modified with a second probe 106 that includes the other of the donor molecule or the acceptor molecule (107 in Figure 35).

[0178] The above-mentioned elements of the reagent kit 100 for biological particle analysis of this technology each have the functions described herein. Specifically, by labeling biological particles with the reagent kit 100 for biological particle analysis of this technology, when an antibody 204 that specifically interacts with the antigen 202 expressed on the cell membrane of antigen-expressing cells 201 is present (true positive case) upon irradiation of the biological particles with excitation light, fluorescence emitted by the interaction (FRET reaction) between one of the donor molecule or acceptor molecule 102 of the first probe 101 and the other of the donor molecule or acceptor molecule 107 of the second probe 106 is detected. By detecting or not detecting this fluorescence, it is possible to identify antibody-producing cells 203 that secrete antibodies 204 that specifically bind to the antigen 202 expressed on the cell membrane of antigen-expressing cells 201, without using imaging techniques or the like, while excluding false positive cases.

[0179] Figure 36 shows a modified example of the configuration of the reagent kit for biological particle analysis of this technology. In addition to the configuration shown in Figure 35, the reagent kit 100 for biological particle analysis of this technology may optionally include elements having the functions described herein. For example, as shown in the example in Figure 36, it may include a pairing reagent 110 having an antigen-expressing cell binding site that can bind to antigen-expressing cells 201 and an antibody-producing cell binding site that can bind to antibody-producing cells 203. Furthermore, if the first probe 101 and the second probe of the reagent kit 100 for biological particle analysis are each coated with one of two compounds that can coordinately bond via metal ions, it may also include a chelating agent 120 for adjusting the concentration of the metal ions in the medium.

[0180] Figure 37 shows another modified example of the configuration of the reagent kit for biological particle analysis of this technology. The reagent kit 100 for biological particle analysis according to this modified example further comprises, in addition to the configuration shown in Figure 36, a labeled antibody 140 having a labeling site 141 for identifying cells, and a carrier 150 having a label-binding molecule 151 that specifically binds to at least a portion of the labeling site 141. By having these configurations, the reagent kit 100 for biological particle analysis according to this modified example can suitably bind to the carrier 150 even when, for example, antibody-producing cells 203 have few antigens that serve as biomarkers on their cell surface. This allows for the suitable isolation of groups containing antibody-producing cells 203.

[0181] The reagent kit for biological particle analysis shown in Figure 37 is an example of a configuration in which the labeled antibody 140 modifies antibody-producing cells 203, which are the target of isolation, and a group including antibody-producing cells 203 can be suitably isolated by positive selection. However, the kit may also be designed to modify cells other than antibody-producing cells 203 with the labeled antibody 140, and a group including antibody-producing cells 203 can be isolated by negative selection.

[0182] Furthermore, as shown in Figure 37, this technology can also be implemented as a reagent kit 130 for isolating biological particles, separate from the reagent kit 100 for analyzing biological particles. In this case, the reagent kit 130 for isolating biological particles can be used independently of the biological particle labeling method of this technology, as a kit for implementing isolation methods in other biological particle labeling and analysis methods.

[0183] The reagent kit 130 for isolating biological particles comprises, for example, a labeled antibody 140 having a labeling site 141 for identifying cells, and a carrier 150 having a label-binding molecule 151 that specifically binds to at least a portion of the labeling site 141, as shown in Figure 37.

[0184] The reagent kits for biological particle analysis and biological particle isolation of this technology may optionally include other elements that provide the functions described herein, and may have other configurations depending on the analysis being performed, as long as they do not significantly impair the desired effect.

[0185] [Biological Particle Analyzer] This technology realizes the biological particle labeling method of this technology and provides a biological particle analyzer for identifying antibody-producing cells that secrete antibodies that specifically bind to antigens expressed on the cell membrane of antigen-expressing cells.

[0186] The biological particle analyzer of this technology comprises: an irradiation means for irradiating biological particles that have captured the antibody, which is captured by an antibody-producing cell, with excitation light; an irradiation means for detecting fluorescence emitted by the interaction between the first probe and the second probe and identifying antibody-producing cells that secrete antibodies that bind to the antibody, which are modified by a second probe that contains the other of the donor molecule or the acceptor molecule; and an identification means for identifying antibody-producing cells that secrete antibodies that bind to the antibody and specifically bind to the antigen. The first probe has a cell surface modified with a first probe that contains either a donor molecule that releases energy upon irradiation with excitation light, or an acceptor molecule that receives the energy from the donor molecule and emits fluorescence.

[0187] The bioparticle analyzer of this technology includes an irradiation means for irradiating bioparticles labeled by the bioparticle labeling method of this technology with excitation light, and a specific means for detecting fluorescence emitted by the interaction (FRET reaction) between one of the donor molecules or acceptor molecules of the first probe and the other of the donor molecules or acceptor molecules of the second probe. These means are not particularly limited, and any means capable of realizing these functions may be used.

[0188] Furthermore, the biological particle analyzer of this technology may optionally be equipped with other means capable of realizing the functions described herein, and may be equipped with other means in accordance with the analysis to be performed, as long as the desired effect is not significantly impaired.

[0189] Furthermore, this technology can take the following configurations: [1] A method for labeling biological particles, comprising: a capture step in which an antibody secreted by an antibody-producing cell captures an antigen expressed on the cell membrane of an antigen-expressing cell, which has a cell surface modified with a first probe containing either a donor molecule that releases energy upon irradiation with excitation light or an acceptor molecule that receives the energy from the donor molecule and emits fluorescence; and a binding step in which an antibody-binding molecule that binds to the antibody, modified with a second probe containing the other of the donor molecule or the acceptor molecule, captures the antibody; and creating labeled biological particles. [2] The method for labeling biological particles according to [1], wherein the first probe comprises a lipid linker. [3] The method for labeling biological particles according to [1] or [2], further comprising a crosslinking step in which the first probe and the second probe are crosslinked after the binding step. [4] The method for labeling biological particles according to [3], wherein the crosslinking is formed by attaching one of two compounds that can coordinately bond to the first probe or the second probe via a metal ion, and adjusting the concentration of the metal ion in the medium with a chelating agent, and the particles are bound by the coordination bond. [5] The method for labeling biological particles according to [4], further comprising a washing step of washing the chelating agent after the binding step and before the crosslinking step. [6] The method for labeling biological particles according to any one of [1] to [5], further comprising a pairing step of binding a pairing reagent, which has an antigen-expressing cell binding site that can bind to the antigen-expressing cell and an antibody-producing cell binding site that can bind to the antibody-producing cell, to either the antigen-expressing cell or the antibody-producing cell, and then to the other, before the capture step. [7] The method for labeling biological particles according to [6], wherein the pairing reagent is bound to the antibody-producing cell and then to the antigen-expressing cell. [8] The method for labeling biological particles according to [6] or [7], wherein two or more pairing reagents are used. [9] A method for labeling biological particles according to any one of [1] to [8], wherein the ratio of the number of antigen-expressing cells to the antibody-producing cells is greater than 1.

[10] A method for labeling biological particles according to any one of [1] to [9], wherein the antibody-producing cells are assigned an identification code.

[11] A method for labeling biological particles according to any one of [1] to

[10] , comprising an isolation step of isolating a group comprising the antibody-producing cells and one or more antigen-expressing cells before the capture step.

[12] The method for labeling biological particles according to

[11] , wherein the isolation is performed by encapsulating the group in a droplet.

[13] The method for labeling biological particles according to

[12] , comprising a droplet-breaking step of breaking the droplet after the binding step.

[14] The method for labeling biological particles according to

[11] , wherein the isolation is performed by binding the group to a carrier.

[15] The method for labeling biological particles according to

[14] , wherein the antibody-producing cells are modified with a labeled antibody having a label site that identifies the antibody-producing cells, and the binding to the carrier is performed via a label-binding molecule that specifically binds to at least a part of the label site.

[16] The method for labeling biological particles according to

[14] , wherein cells other than the antibody-producing cells are modified with a labeled antibody having a label site that identifies the antibody-producing cells, and the binding of cells other than the antibody-producing cells to the carrier is inhibited by a label-binding molecule that specifically binds to at least a part of the label site.

[17] The method for labeling biological particles according to

[15] or

[16] , wherein the labeling site comprises a fluorescent dye.

[18] The method for labeling biological particles according to any one of

[15] to

[17] , wherein the labeling binding molecule is an antibody.

[19] The method for labeling biological particles according to any one of

[15] to

[18] , wherein the labeling antibody is an anti-CD138 antibody.

[20] The method for labeling biological particles according to any one of

[15] to

[19] , wherein the carrier is an open system and binding to the carrier is performed on the surface of the carrier.

[21] The method for labeling biological particles according to any one of

[14] to

[20] , further comprising a magnetic application step of binding the antigen-expressing cells or the antibody-producing cells to magnetic beads before the isolation step.

[22] The method for labeling biological particles according to

[21] , further comprising a purification step of separating the cells bound to the magnetic beads using magnetism after the isolation step and before the capture step.

[23] The method for labeling biological particles according to any one of [1] to

[22] , wherein the antibody-binding molecule is an anti-IgG antibody.

[24] A biological particle analysis method comprising: a selection step of identifying antibody-producing cells that secrete antibodies that specifically bind to the antigen by detecting fluorescence emitted by interaction between the first probe and the second probe upon irradiation of the biological particles labeled by the biological particle labeling method described in any of [1] to

[23] with excitation light.

[25] A method for separating antibody-producing cells identified by the biological particle analysis method described in

[24] .

[26] The separation method described in

[25] , wherein the separation is performed by a flow cytometer.

[27] A biological particle analysis reagent kit for identifying antibody-producing cells that secrete antibodies that specifically bind to an antigen expressed on the cell membrane of an antigen-expressing cell, comprising: a first probe having a modification site that modifies the cell surface of an antigen-expressing cell, and containing either a donor molecule that releases energy upon irradiation with excitation light or an acceptor molecule that receives the energy from the donor molecule and emits fluorescence; and an antibody-binding molecule that binds to an antibody, modified by a second probe containing the other of the donor molecule or the acceptor molecule.

[28] The reagent kit for biological particle analysis according to

[27] , further comprising a pairing reagent having an antigen-expressing cell binding site that can bind to the antigen-expressing cell and an antibody-producing cell binding site that can bind to the antibody-producing cell.

[29] The reagent kit for biological particle analysis according to

[27] or

[28] , further comprising a labeled antibody having a labeling site for identifying cells and a carrier having a label-binding molecule that specifically binds to at least a part of the labeling site.

[30] A biological particle analyzer comprising: an irradiation means for irradiating a biological particle that has captured the antibody, which is obtained by an antibody-producing cell, with excitation light; an irradiation means for irradiating an antigen-expressing cell having a cell surface modified with a first probe that includes either a donor molecule that releases energy upon irradiation with excitation light or an acceptor molecule that receives the energy from the donor molecule and emits fluorescence; an irradiation means for irradiating a biological particle that has captured the antibody, which is modified with a second probe that includes the other of the donor molecule or the acceptor molecule, with excitation light; and an identification means for detecting fluorescence emitted by the interaction between the first probe and the second probe, and identifying an antibody-producing cell that secretes an antibody that specifically binds to the antigen.

[31] A biological particle in which an antigen-expressing cell and an antibody-producing cell are bound by forming two or more bindings via two or more pairing reagents, each having an antigen-expressing cell binding site that can bind to the antigen-expressing cell and an antibody-producing cell binding site that can bind to the antibody-producing cell.

[32] A method for isolating cells, comprising: a magnetic application step of binding antigen-expressing cells or antibody-producing cells to magnetic beads; an isolation step of isolating a group including the antibody-producing cells and one or more antigen-expressing cells by binding them to a carrier; and a purification step of separating the cells bound to the magnetic beads using magnetism.

[33] A method for isolating biological particles, wherein a group consisting of one or more cells is isolated by binding them to a carrier, the cells being modified with a labeled antibody having a labeling site for identifying the cells, and the binding to the carrier being carried out via a label-binding molecule that specifically binds to at least a part of the labeling site, or inhibited by the binding of the label-binding molecule.

[34] The method for isolating biological particles according to

[33] , wherein the cells are antibody-producing cells.

[35] The method for isolating biological particles according to

[33] or

[34] , wherein the labeling site includes a fluorescent dye.

[36] The method for isolating biological particles according to any one of

[33] to

[35] , wherein the label-binding molecule is an antibody.

[37] The isolation method according to any one of

[33] to

[36] , wherein the labeled antibody is an anti-CD138 antibody.

[38] The isolation method according to any one of

[33] to

[37] , wherein the carrier is an open system and binding to the carrier is performed on the surface of the carrier.

[39] A reagent kit for isolating biological particles, comprising a labeled antibody having a labeling site for identifying cells, and a carrier having a label-binding molecule that specifically binds to at least a portion of the labeling site.

[0190] 100 Reagent kit for biological particle analysis 101 First probe 102 Donor molecule or acceptor molecule of the first probe 103 Modification site (lipid linker) that modifies the cell surface of antigen-expressing cells 105 Antibody-binding molecule 106 Second probe 107 Donor molecule or acceptor molecule of the second probe 108 Compounds that can coordinate with each other 110 Pairing reagent 110-1 Antigen-producing cell binding site 110-2 Antibody-expressing cell binding site 110-3 Antibody with DNA linker that forms antigen-expressing cell binding site 110-4 Antibody with DNA linker that forms antibody-expressing cell binding site 110-5, 110-6 DNA linker 110-7 Nucleic acid oligomer 111 Pairing reagent 1 112 Pairing reagent 2 120 Chelating agent 130 Reagent kit for biological particle isolation 140 Labeled antibody 141 Labeling site 150, 150-1 Carrier 151 Label-binding molecule 201 Antigen-expressing cell 202 Antigen 203 Antibody-producing cell 204 Antibody 301 Identification code 302 Capture site 303 Substrate 310 Droplet 311 Microchannel 320 Magnetic bead 321 Cell 322 Cell to which magnetic bead is bound 323 Magnet 331 Target cell 332 Non-target cell 333 Identification antigen 334 Labeled antibody 335 Labeling site 336 Carrier-modified molecule

Claims

For antigen-expressing cells having a cell surface modified with a first probe containing either a donor molecule that releases energy upon irradiation with excitation light, or an acceptor molecule that receives the energy from the donor molecule and emits fluorescence, A capture step in which antibodies secreted by antibody-producing cells capture antigens expressed on the cell membrane of the antigen-expressing cells, A method for labeling biological particles, comprising creating labeled biological particles by a binding step in which an antibody-binding molecule, which binds to the antibody and is modified with a second probe containing the other of the donor molecule or the acceptor molecule, captures the antibody. 　 The method for labeling biological particles according to claim 1, wherein the first probe comprises a lipid linker. 　 The method for labeling biological particles according to claim 1, further comprising a crosslinking step of crosslinking the first probe and the second probe after the bonding step. 　 The method for labeling biological particles according to claim 3, wherein the crosslinking is formed by attaching one of two compounds that can coordinately bond to the first probe or the second probe via a metal ion, and adjusting the concentration of the metal ion in the medium with a chelating agent, and is bonded by the coordination bond. 　 The method for labeling biological particles according to claim 4, further comprising a washing step of washing the chelating agent after the bonding step and before the crosslinking step. 　 Before the aforementioned capture process, A method for labeling biological particles according to claim 1, comprising a pairing step of binding a pairing reagent, which has an antigen-expressing cell binding site that can bind to the antigen-expressing cell and an antibody-producing cell binding site that can bind to the antibody-producing cell, to either the antigen-expressing cell or the antibody-producing cell, and then binding the reagent to the other. 　 The method for labeling biological particles according to claim 6, wherein the pairing reagent is bound to the antibody-producing cells and then to the antigen-expressing cells. 　 The method for labeling biological particles according to claim 6, wherein two or more of the pairing reagents are used. 　 The method for labeling biological particles according to claim 1, wherein the antibody-producing cells are assigned an identification code. 　 Before the aforementioned capture process, The method for labeling biological particles according to claim 1, comprising an isolation step of isolating a group comprising the antibody-producing cells and one or more antigen-expressing cells. 　 The method for labeling biological particles according to claim 10, wherein the isolation is performed by encapsulating the group in a droplet. 　 The method for labeling biological particles according to claim 11, further comprising a droplet destruction step of destroying the droplet after the bonding step. 　 The method for labeling biological particles according to claim 10, wherein the isolation is performed by binding the group to a carrier. 　 Before the aforementioned isolation process, The method for labeling biological particles according to claim 13, comprising a magnetic application step of binding the antigen-expressing cells or the antibody-producing cells with magnetic beads. 　 After the isolation step and before the capture step, A method for labeling biological particles according to claim 14, comprising a purification step of separating cells bound to the magnetic beads using magnetism. 　 A method for analyzing biological particles, comprising: a selection step of identifying antibody-producing cells that secrete antibodies that specifically bind to the antigen by irradiating the biological particles labeled by the biological particle labeling method described in claim 1 with excitation light, and detecting fluorescence emitted by the interaction between the first probe and the second probe. 　 A method for separating antibody-producing cells identified by the biological particle analysis method described in claim 16. 　 The separation method according to claim 17, wherein the separation is performed by a flow cytometer. 　 A first probe comprising either a donor molecule that releases energy upon irradiation with excitation light, or an acceptor molecule that receives the energy from the donor molecule and emits fluorescence, and having a modification site that modifies the cell surface of an antigen-expressing cell, The system comprises an antibody-binding molecule that binds to an antibody, modified with a second probe containing the other of the donor molecule or the acceptor molecule, A reagent kit for biological particle analysis to identify antibody-producing cells that secrete antibodies that specifically bind to antigens expressed on the cell membrane of the aforementioned antigen-expressing cells. 　 Furthermore, the reagent kit for biological particle analysis according to claim 19 comprises a pairing reagent having an antigen-expressing cell binding site that can bind to the antigen-expressing cell and an antibody-producing cell binding site that can bind to the antibody-producing cell.

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