Method for analyzing cells and method for producing target substance

Abstract

Provided is a method for analyzing cells with improved accuracy.　This method for analyzing cells is for analyzing the expression of a target substance in the cells. This method for analyzing cells includes a labeling step, a measurement step, and a determination step. In the labeling step, the cells are labeled, through a chemical or physical action, with fluorescent-labeling resin particles which contain a plurality of fluorescent dye molecules in the resin particles. In the measurement step, a characteristic property of fluorescence emitted from the fluorescent-labeling resin particles that have labeled the cells is measured by flow cytometry. In the determination step, it is determined whether or not the target substance is expressed in the cells from the results of the measurement by flow cytometry. The labeling step includes a first binding step and a second binding step. In the first binding step, an antigen on the cell surface is directly or indirectly bound to a target antibody. In the second binding step, the antigen on the cell surface is bound to the fluorescent-labeling resin particles via the target antibody.

Description

Method for analyzing cells and method for producing target substance

[0001] The present disclosure relates to a method for analyzing cells and a method for producing a target substance.

[0002] Flow cytometry is known as a technique for analyzing the physical and chemical properties of individual cells. In flow cytometry, cells labeled with a fluorescent dye are irradiated with excitation light and the emitted fluorescence is detected for analysis. From the viewpoint of improving the detectability of the fluorescence, techniques are known in which the labeling fluorescent dye is used in the form of fluorescent polymer dots, semiconductor nanoparticles, or the like (Patent Documents 1 to 3).

[0003] JP 2015-512955 A JP 2018-502557 A JP 2004-77389 A

[0004] However, even when fluorescent dyes are used in the form of fluorescent polymer dots, semiconductor nanoparticles, etc., the brightness of the detected fluorescence is insufficient, resulting in a problem of insufficient analytical accuracy.

[0005] The present disclosure has been made in view of the above problems and circumstances, and aims to provide a method for analyzing cells with improved accuracy.

[0006] In order to solve the above-mentioned problems, the present inventors have investigated the causes of the above-mentioned problems. As a result, the present inventors have discovered that in a cell analysis method using flow cytometry, the accuracy of analysis can be improved by labeling cells with fluorescent labeling resin particles, and have arrived at the present disclosure. That is, the above-mentioned problems of the present disclosure can be solved by the following means.

[0007] 1. A method for analyzing the expression of a target substance in a cell, comprising the steps of: labeling the cell with fluorescent labeling resin particles containing a plurality of fluorescent dye molecules in the resin particles by a chemical or physical action; measuring the characteristics of the fluorescence emitted from the fluorescent labeling resin particles that label the cell using flow cytometry; and determining whether the cell expresses the target substance from the measurement results of the flow cytometry, wherein the labeling step comprises a first binding step of directly or indirectly binding an antigen on the cell surface to a target antibody, and a second binding step of binding the antigen on the cell surface to the fluorescent labeling resin particles via the target antibody.

[0008] 2. The method for analyzing cells according to item 1, wherein the average primary particle size of the fluorescent labeling resin particles is within the range of 40 to 200 nm.

[0009] 3. The method for analyzing cells according to item 2, wherein the average primary particle size of the fluorescent labeling resin particles is within the range of 50 to 180 nm.

[0010] 4. The method for analyzing cells according to item 3, wherein the average primary particle size of the fluorescent labeling resin particles is within the range of 60 to 160 nm.

[0011] 5. The method for analyzing cells according to item 1, further comprising the steps of: washing the cells to which the fluorescent labeling resin particles are bound, obtained after the second binding step; and fixing, with a fixative, the cells to which the fluorescent labeling resin particles are bound and which have been retained by the antigen after the washing step.

[0012] 6. The method for analyzing cells according to item 1, comprising the steps of: immobilizing, with a fixative, the cells bound to the target antibody obtained after the first binding step; washing the immobilized cells bound to the target antibody; immobilizing, with a fixative, the cells retained by the antigen and bound to the fluorescent labeling resin particles obtained after the second binding step; and washing the immobilized cells bound to the fluorescent labeling resin particles.

[0013] 7. The method for analyzing cells according to any one of items 1 to 6, wherein the labeling step further comprises a step of masking areas of the cell surface excluding the antigen using a blocking agent.

[0014] 8. The method for analyzing cells according to item 7, wherein the blocking agent comprises a carrier protein.

[0015] 9. The method for analyzing cells according to item 8, wherein the carrier protein is bovine serum albumin or casein.

[0016] 10. The method for analyzing cells according to item 1, wherein the fluorescence emission intensity is measured using the flow cytometry.

[0017] 11. In the flow cytometry measurement results, the mean fluorescence intensity signal value of the reference negative control is 1.0 x 10 ２ Set it to the following: 1.0 x 10 ２ The method for analyzing cells according to claim 1, wherein the cells that are overexpressing the target substance are determined to be the cells that express the target substance.

[0018] 12. A method for producing a target substance by culturing cells, the method comprising the step of adjusting the culture conditions of the cells based on the analysis results obtained by the method for analyzing cells according to claim 1.

[0019] 13. The method for producing a target substance according to item 12, further comprising the step of sorting the cells determined to express the target substance.

[0020] The above-described means of the present disclosure can improve the accuracy of analysis.

[0021] Although the mechanism by which the effects of the present disclosure are expressed or the mechanism of action are not clear, it is speculated as follows: The present disclosure is a method for quantifying proteins exposed on the cell surface, such as cell surface-presenting enzymes.

[0022] In flow cytometry, cells are analyzed by irradiating cells labeled with a fluorescent labeling agent with excitation light and detecting the fluorescence emitted from the fluorescent labeling agent. Therefore, the detected fluorescence must be strong enough for analysis, and the fluorescent labeling agent must have high emission intensity.

[0023] When analyzing the concentration of a specific component on the cell membrane from the intensity of the detected fluorescence, the lower the concentration, the lower the detected fluorescence intensity, making accurate analysis difficult. Also, when cells themselves naturally emit autofluorescence, if the intensity of the fluorescence emitted from the fluorescent labeling agent is low, it is impossible to distinguish whether the detected fluorescence is due to the autofluorescence or the fluorescent labeling agent.

[0024] In this disclosure, fluorescent labeling resin particles are used as the fluorescent labeling agent. The fluorescent labeling resin particles can incorporate multiple fluorescent dye molecules into the resin particles through chemical or physical interactions, allowing the analyte components on the cell membrane to be labeled with a larger number of fluorescent dye molecules. This increases the intensity of the detected fluorescence and is believed to improve the accuracy of the analysis.

[0025] 1 is a flowchart of a method for analyzing cells. FIG. 1 is a flowchart of a labeling step. FIG. 2 is a flowchart of an example of an operating procedure for Pattern 1 in the labeling step. FIG. 3 is a flowchart of an example of an operating procedure for Pattern 2 in the labeling step. FIG. 4 is a graph showing measurement results for a certain cell population, with the horizontal axis representing the intensity of forward scattered light (FS) and the vertical axis representing the intensity of side scattered light (SS). FIG. 5 is a schematic diagram showing an example of a cell sorter. FIG. 6 is a flowchart of a method for producing a target substance. FIG. 7 is a flowchart of a method for producing a target substance with a fractionation step. FIG. 8 is a graph showing the relationship between fluorescence intensity and the number of beads measured in Examples 1 and 2. FIG. 9 is a graph showing the relationship between the concentration of complex B (secondary antibody) and fluorescence intensity. FIG. 10 is a graph showing the relationship between fluorescence intensity and the number of beads measured in Examples 1-7 and 3. FIG. 11 is a graph showing the relationship between fluorescence intensity and the number of beads measured in Examples 4 and 5. FIG. 12 is a graph showing the relationship between fluorescence intensity and the number of beads measured in Examples 5 and 6.

[0026] The cell analysis method of this embodiment is a cell analysis method for analyzing the expression of a target substance in cells. The cell analysis method of the present disclosure includes a labeling step, a measuring step, and a determining step. The labeling step labels cells with fluorescent labeling resin particles containing multiple fluorescent dye molecules in the resin particles through a chemical or physical action. The measuring step uses flow cytometry to measure the characteristics of fluorescence emitted from the fluorescent labeling resin particles that label the cells. The determining step determines whether the cells express the target substance based on the flow cytometry measurement results. The labeling step includes a first binding step and a second binding step. The first binding step directly or indirectly binds an antigen on the cell surface to a target antibody. The second binding step binds the antigen on the cell surface to the fluorescent labeling resin particles via the target antibody. The above features are technical features common to or corresponding to the following embodiments.

[0027] In this embodiment, the average primary particle size of the fluorescent labeling resin particles is preferably within the range of 40 to 200 nm, more preferably within the range of 50 to 180 nm, and even more preferably within the range of 60 to 160 nm, which allows for both the luminescence intensity of the fluorescent labeling resin particles and the indirect binding strength with cells, thereby improving the accuracy of analysis.

[0028] This embodiment preferably includes a step of washing the cells to which the fluorescent labeling resin particles are bound after the second binding step, and a step of fixing the cells to which the fluorescent labeling resin particles are bound and which have been held by the antigen after the washing step with a fixative, which can eliminate nonspecific binding of the fluorescent labeling resin particles and improve the accuracy of the analysis.

[0029] This embodiment preferably includes the steps of immobilizing the target antibody-bound cells obtained after the first binding step with a fixative, washing the immobilized target antibody-bound cells, immobilizing the antigen-retained, fluorescent labeling resin particle-bound cells obtained after the second binding step with a fixative, and washing the immobilized fluorescent labeling resin particle-bound cells, thereby strengthening the indirect binding force of the fluorescent labeling resin particles with the cells and improving the accuracy of the analysis.

[0030] In this embodiment, the labeling step preferably further includes a step of masking the areas of the cell surface other than the antigen using a blocking agent, thereby eliminating non-specific binding of the fluorescent labeling resin particles and improving the accuracy of the analysis.

[0031] In this embodiment, the blocking agent preferably contains a carrier protein. Furthermore, the carrier protein is more preferably bovine serum albumin or casein. This can eliminate nonspecific binding of the fluorescent labeling resin particles and improve the accuracy of the analysis.

[0032] In this embodiment, it is preferable to measure the fluorescence emission intensity using flow cytometry, which can improve the accuracy of analysis when the concentration of the analyte component on the cell membrane is low.

[0033] In this embodiment, in the flow cytometry measurement results, the mean fluorescence intensity signal value of the reference negative control is 1.0 × 10 ２ Set it to the following: 1.0 x 10 ２ It is preferable to determine that cells that exceed the target substance are cells that express the target substance, which makes it possible to exclude fluorescence other than that derived from the fluorescent labeling resin particles from the measurement results, thereby improving the accuracy of the analysis.

[0034] The method for producing a target substance in this embodiment is a method for producing a target substance by culturing cells. The method includes a step of adjusting cell culture conditions based on the analysis results obtained by the above-described cell analysis method. This allows for improved productivity of the target substance.

[0035] In this embodiment, it is preferable to include a step of sorting cells determined to express the target substance, which allows for the selection of highly productive cells and improves the productivity of the target substance.

[0036] One or more embodiments of the present disclosure will be described below with reference to the drawings. However, the scope of the present disclosure is not limited to the disclosed embodiments. In this application, the symbol "to" is used to mean that the numerical values ​​before and after it are included as the lower and upper limits.

[0037] 1. Cell Analysis Method The cell analysis method of this embodiment is a cell analysis method for analyzing the expression of a target substance in cells. The cell analysis method of this embodiment includes a labeling step (labeling step), a measuring step (measurement step), and a determination step (determination step). The labeling step labels cells with fluorescent labeling resin particles containing multiple fluorescent dye molecules in the resin particles through chemical or physical action. The measurement step uses flow cytometry to measure the characteristics of the fluorescence emitted from the fluorescent labeling resin particles that label the cells. The determination step determines whether the cells express the target substance based on the flow cytometry measurement results. The labeling step includes a first binding step and a second binding step. The first binding step directly or indirectly binds an antigen on the cell surface to a target antibody. The second binding step binds the antigen on the cell surface to the fluorescent labeling resin particles via the target antibody.

[0038] FIG. 1 is a flowchart of the cell analysis method according to this embodiment.

[0039] The cell analysis method in this embodiment includes a labeling step (step S1), a measurement step (step S2), and a determination step (step S3). In the labeling step S1, cells are labeled with fluorescently labeled resin particles that contain multiple fluorescent dye molecules in the resin particles through chemical or physical action. In the measurement step S2, flow cytometry is used to measure the characteristics of the fluorescence emitted from the fluorescently labeled resin particles. In the determination step S3, it is determined from the flow cytometry measurement results whether the cells express a target substance.

[0040] The cell analysis method of this embodiment can be used to analyze the state, health, viability, proliferation ability, toxicity, etc. of cells. Specifically, it can be applied to biomanufacturing, quality inspection, etc. "Biomanufacturing" refers to the entire process or part of the process of producing a target substance via cells of microorganisms, animals, plants, etc. using genetic technology, and the cell analysis method of this embodiment is used in the production process of producing a target substance using cells. Furthermore, in quality inspection, the cell analysis method of this embodiment can be used to check, for example, whether a solution is contaminated with mold.

[0041] In this embodiment, the term "target substance" refers to a substance produced by a metabolic reaction of cells and a target substance in a production process. In other words, the target substance is included in metabolic products obtained by culturing cells.

[0042] Metabolites containing the target substance can be obtained by disrupting cells after the production process. The components obtained by disrupting cells after the production process include DNA, RNA, proteins, etc. contained in the cells, as well as substances produced by metabolic reactions of the cells, i.e., metabolites. Alternatively, cells after the production process may be used in their intact state without being disrupted.

[0043] Metabolites include primary metabolites and secondary metabolites. Primary metabolites are components produced by biochemical reactions essential for sustaining life. Secondary metabolites are components that are not essential for sustaining life but are produced specifically by each species. Metabolites also include intermediates produced during metabolic reactions.

[0044] In this embodiment, the cells to be used are not particularly limited as long as they are capable of producing a target substance. Examples of the cells include bacteria (Escherichia coli, Bacillus, Streptomyces, Pseudomonas, Rhodococcus, Streptococcus, Staphylococcus, etc.), fungi (e.g., yeasts (Saccharomyces, Schizosaccharomyces, Pichia, Lipomyces, etc.)), filamentous fungi (Aspergillus, etc.)), and the like. genus, etc.), algae (microalgae, including organisms belonging to the cyanobacteria, rhodophyta, glaucophyta, chlorophyta, charophyta, heterokontophyta, dinoflagellates, cryptophyta, haptophyta, euglenopphyta, and chlorarachniophyta phyla), insect cells (Drosophila S2, Spodoptera SF, etc.), plant cells (tobacco-derived (BY-2 cells, etc.), Arabidopsis-derived, rice-derived, soybean-derived, tomato-derived, sesame-derived, and periwinkle-derived cells, etc.), animal cells (hybridoma, COS, CHO, HEK293, 3T3 cells, etc.), etc.

[0045] The cells to be used may be natural cells, or may be mutants that have been treated with a drug such as NTG, ultraviolet light, radiation, etc. Examples of such mutants include those that have improved productivity of a target substance.

[0046] The cells to be used may also be cells into which a nucleic acid for producing a target substance has been introduced. For example, if the target substance is a protein, such a nucleic acid may be a nucleic acid encoding the protein. Methods for introducing a nucleic acid into a host cell include known methods, such as using an expression vector containing the nucleic acid. The expression vector used for introducing the nucleic acid is not particularly limited, and a wide variety of known expression vectors can be used. An appropriate expression vector may be selected, taking into consideration the type of host into which the nucleic acid is introduced. In addition to the nucleic acid, the expression vector may also contain a promoter, enhancer, terminator, polyadenylation signal, selection marker, replication origin, etc. Both autonomously replicating vectors and vectors that are integrated into the genome of the host cell upon introduction and replicated together with the integrated chromosome can be used as expression vectors.

[0047] Methods for constructing expression vectors and introducing the expression vectors into cells are well known and can be carried out with reference to, for example, the descriptions in Sambrook and Russell, Molecular Cloning, A Laboratory Manual 3rd edition, Cold Spring Harbor Laboratory Press (2001), etc. Methods for introducing expression vectors into hosts include, for example, the competent cell method, the protoplast method, the spheroplast method, electroporation, the calcium phosphate method, the lipofection method, the Agrobacterium method, the polyethylene glycol method, the liposome method, the microinjection method, and the lithium acetate method.

[0048] The target substance of the production process is not particularly limited, and examples thereof include fats and oils, fatty acids, vitamins, antibiotics, nucleic acids, amino acids, polysaccharides, organic acids, alcohols, sugars, sugar alcohols, foods, antibodies, enzymes, vaccines, lectins, cytokines, hormones, receptors, ligands, peptides, and proteins. Fatty acids include propionic acid, hydroxypropionic acid, EPA (eicosapentaenoic acid), and DHA (docosahexaenoic acid). Vitamins include riboflavin, thiamine, and ascorbic acid. Organic acids include acetic acid, lactic acid, and succinic acid. Alcohols include ethanol. Sugars include xylose and mannose. Sugar alcohols include xylitol and mannitol.

[0049] Examples of cells that can be used without disruption after producing a target substance include lactic acid bacteria, embryonic stem (ES) cells, pluripotent stem cells such as induced pluripotent stem (iPS) cells, etc. Lactic acid bacteria include not only Lactobacillus and Lactococcus, but also Bifidobacteria, which are considered lactic acid bacteria in a broad sense. Specific examples of lactic acid bacteria include Lactobacillus, Bifidobacterium, Leuconostoc, Lactococcus, Pediococcus, Enterococcus, Streptococcus, Weissella, etc.

[0050] Since fluorescent labeling resin particles have a larger particle size than other fluorescent labeling agents, it is difficult to introduce them into cells without disrupting the cells. Therefore, in this embodiment, the expression state of the target substance is analyzed by observing the components on the cell surface.

[0051] The cell surface component to be observed is not particularly limited, but membrane proteins are preferable. Membrane proteins are proteins embedded in the cell membrane, and their type and structure change depending on the components contained within the cell. Furthermore, the type and structure of membrane proteins change depending on the metabolic reactions of the cell. The type and structure of the membrane protein may be the same as or different from that of the target substance. For example, even if target substance A and membrane protein B are different types, if it is known that the expression level of membrane protein B increases or decreases in accordance with the increase or decrease in the expression level of target substance A, the expression state of target substance A can be analyzed by observing membrane protein B.

[0052] Each step will be described in detail below.

[0053] (1) Labeling Step In the labeling step, cells to be analyzed are labeled with fluorescent labeling resin particles, which contain a plurality of fluorescent dye molecules by chemical or physical action.

[0054] 2 is a flowchart of the labeling process according to this embodiment. The labeling process includes a first binding process (step S12) and a second binding process (step S13). The first binding process S12 binds an antigen on the cell surface to a target antibody. The second binding process S13 binds the antigen on the cell surface to a fluorescently labeled resin particle via the target antibody. In the binding between the antigen on the cell surface and the fluorescently labeled resin particle, a wide variety of antigens can be bound to the fluorescently labeled resin particle by using the target antibody.

[0055] The labeling step may further include a blocking step (step S11). In the blocking step S11, a blocking agent is used to mask areas of the cell surface other than the antigen. In the example shown in FIG. 2, the blocking step S11 is performed before the first binding step S12, but the timing and number of times that the blocking step S11 is performed are not limited.

[0056] (1.1) Fluorescent Labeling Resin Particles In this embodiment, "fluorescent labeling resin particles" refers to resin particles used as a fluorescent labeling agent. Fluorescent labeling resin particles contain multiple fluorescent dye molecules in the resin particles through chemical or physical action. Here, "contained in the resin particles" means that multiple fluorescent dye molecules are fixed to the resin particles through chemical or physical action and are contained in the resin particles. The form of fixation is not limited, and examples of the form include an encapsulation type and an adsorption type.

[0057] Fluorescent labeling resin particles can immobilize a large number of fluorescent dye molecules to the resin particles, resulting in higher brightness than other common fluorescent labeling agents. This high brightness improves the detectability of fluorescence in the measurement process described below. Because of this high brightness, fluorescence with a much higher intensity than the autofluorescence emitted naturally by the cells themselves is detected, making the particles less susceptible to the effects of autofluorescence.

[0058] Furthermore, in the fluorescent labeling resin particles, the fluorescent dye molecules are somewhat covered with resin. Therefore, the fluorescent dye molecules are less susceptible to the effects of light and heat, and deactivation of the dye by light and heat can be suppressed. Furthermore, if the dye is deactivated over time during the measurement process, the measured values ​​of the fluorescence intensity will be unstable. However, by suppressing the deactivation of the dye, the measured values ​​of the fluorescence intensity will be stable, allowing for accurate measurement of the fluorescence intensity.

[0059] In the case of the encapsulated fluorescent labeling resin particles, the fluorescent dye molecules are mainly encapsulated in the resin particles, and the encapsulated fluorescent labeling resin particles are obtained by mixing a resin raw material (such as melamine resin) with the fluorescent dye while allowing the resin to polymerize.

[0060] The encapsulated fluorescent labeling resin particles can immobilize a large number of fluorescent dye molecules, thereby improving the fluorescence intensity. The encapsulated fluorescent labeling resin particles can suppress concentration quenching by selecting more appropriate fluorescent dye molecules, thereby improving the fluorescence intensity and light resistance. In particular, the resin particles preferably contain a curable resin with a dense crosslinked structure, such as a melamine resin.

[0061] In the case of adsorption-type fluorescent labeling resin particles, the fluorescent dye molecules are mainly adsorbed onto the surface of the resin particles. Adsorption-type fluorescent labeling resin particles are obtained by first preparing resin particles using a resin raw material (e.g., melamine resin) and then adding a fluorescent dye to a dispersion of the resin particles.

[0062] When the fluorescent labeling resin particles are of the encapsulation type, the fluorescent dye is confined within the particles by at least the molecular structure of the resin, such as the three-dimensional network structure of a thermosetting resin (e.g., melamine resin).

[0063] It is preferable that an electrostatic interaction, i.e., an ionic bond, occurs between the functional group or moiety of the fluorescent dye molecule and the functional group or moiety of the resin. It is also preferable that a covalent bond is formed between the functional group or moiety of the fluorescent dye molecule and the functional group or moiety of the resin. This allows the fluorescent dye molecule to be more firmly fixed to the resin particle, and prevents the fluorescent dye from leaching out of the particle due to the organic solvent (xylene) used in the tissue staining permeation process.

[0064] When a fluorescent dye molecule has a positively or negatively charged functional group or moiety and a resin molecule has an oppositely charged functional group or moiety, the above-mentioned "electrostatic interaction" occurs. Here, "having a positively or negatively charged functional group or moiety" means that the fluorescent dye or resin has a positively or negatively charged functional group or moiety when dissolved in neutral, acidic, or basic water. Hereinafter, "positively or negatively charged functional group or moiety" will also be simply referred to as "charged functional group or moiety."

[0065] Examples of negatively charged functional groups or moieties include sulfo groups (-SO3-), phosphoryl groups, phenolic hydroxy groups, carboxy groups, etc. Examples of positively charged functional groups or moieties include amino groups (-NH3+), aromatic amino groups, cyclic amino groups (pyridinyl groups, triazolyl groups, etc.), hydrazinyl groups, etc.

[0066] In a resin, the charged functional group or moiety may be introduced into the resin raw material beforehand. Alternatively, the charged functional group or moiety may be formed by a process in which H+ is added to an amino group contained in a melamine resin, urea resin, or the like, resulting in protonation. In a fluorescent dye, the charged functional group or moiety may be present in the fluorescent dye molecule beforehand, or may be introduced by a water-solubilizing treatment.

[0067] The greater the number of charged functional groups or moieties per molecule or per molecular weight in a given unit in the resin and fluorescent dye, the stronger the electrostatic interaction, so it is preferable to adjust the number of charged functional groups or moieties to achieve the desired strength of electrostatic interaction.

[0068] The above-mentioned "covalent bond" includes an amide bond, an ester bond, an ether bond, a C--N bond, and the like.

[0069] The covalent bond can be formed, for example, by the following method: First, a fluorescent dye and a resin raw material (monomer) are selected that have functional groups or sites capable of forming the covalent bond. Next, prior to the resin synthesis reaction, the fluorescent dye and the resin raw material are reacted (pre-step). Next, the resin raw material derivatized in the pre-step is polymerized, if necessary, together with other resin raw materials (comonomers) (polymerization step).

[0070] In the polymerization step, if the fluorescent dye to be added reacts with the resin raw material or the synthesized resin to form a covalent bond, the pre-step does not necessarily have to be carried out.

[0071] When the resin particles for fluorescent labeling are of the adsorption type, electrostatic interaction occurs between the functional groups or moieties on the surface of the resin particles and the functional groups or moieties on the fluorescent dye molecules, and covalent bonds are formed between the functional groups or moieties on the surface of the resin particles and the functional groups or moieties on the fluorescent dye molecules.

[0072] For the adsorption type, the fluorescent dye molecules can be immobilized on the resin particles in the same manner as for the encapsulation type.

[0073] The particle size of the fluorescent labeling resin particles is not particularly limited as long as it is suitable for the intended use, such as immunostaining of tissue sections. The particle size is preferably within the range of 40 to 200 nm, more preferably within the range of 50 to 180 nm, and even more preferably within the range of 60 to 160 nm. The coefficient of variation, which indicates the variation in particle size, is not particularly limited, but is preferably 20% or less, and preferably within the range of 5 to 15%. Fluorescent labeling resin particles that satisfy the above particle size conditions can be obtained by the manufacturing method described below.

[0074] Although it is not clear, it is speculated that by setting the particle size within the range of 40 to 200 nm, it is possible to achieve both the luminescence intensity of the fluorescent labeling resin particles and the indirect binding strength with cells, thereby improving the accuracy of the analysis. Furthermore, it is speculated that a particle size within the range of 50 to 180 nm is preferable from the viewpoint of noise reduction, such as suppressing nonspecific adsorption. In addition, it is speculated that a particle size within the range of 60 to 160 nm is even more preferable from the viewpoint of improving the labeling binding efficiency. From the above, it is speculated that by setting the particle size of the fluorescent labeling resin particles within this range, the average intensity signal value will gradually improve.

[0075] The particle size of the fluorescent labeling resin particles can be measured by the following method. An electron micrograph of the fluorescent labeling resin particles is taken using a scanning electron microscope (SEM). The cross-sectional area of ​​the fluorescent labeling resin particles is measured from the electron micrograph. When the measured value is taken as the area of ​​a circle equivalent to the cross-sectional area, the diameter of the circle (diameter equivalent to the circle area) is taken as the particle size.

[0076] In a population of fluorescent labeling resin particles, the average particle size and coefficient of variation can be measured by the following method. The particle sizes of a sufficient number of fluorescent labeling resin particles (for example, 1,000 particles) for measurement are measured by the above method. The average particle size is the arithmetic mean value of the measured particle sizes. The coefficient of variation is calculated by the following formula (i): Formula (i): Coefficient of variation [%] = 100 × standard deviation of particle size ÷ average particle size

[0077] The fluorescent labeling resin particles have sufficient initial luminescence intensity at the start of excitation light irradiation and can maintain a certain luminescence intensity even after irradiation with excitation light for a predetermined period of time, i.e., they have excellent light resistance. The initial luminescence intensity and luminescence intensity retention vary depending on the manufacturing conditions of the fluorescent labeling resin particles, the selection of constituent materials, etc. Furthermore, the initial luminescence intensity and luminescence intensity retention vary depending on the evaluation method. As an example, when fluorescent labeling resin particles are manufactured by the method shown in the Examples below and light resistance is evaluated after 10 minutes of irradiation with excitation light, the luminescence intensity retention of the fluorescent labeling resin particles is preferably 60% or more, and more preferably 75% or more.

[0078] (1.1.1) Fluorescent Dye The fluorescent dye is not particularly limited. In particular, from the viewpoint of luminescence intensity, the fluorescent dye preferably satisfies the following condition (1) or condition (2), and more preferably satisfies both conditions (1) and (2). Condition (1): The peak of luminescence intensity, which depends on the concentration of the fluorescent dye molecule in the aqueous solution, is in the range of 30 to 80 μM. Condition (2): The luminescence intensity of the fluorescent dye molecule in the aqueous solution at a concentration of 100 μM is 80% or more of the peak luminescence intensity.

[0079] Whether or not condition (1) is met can be determined by the following method. First, multiple serial dilutions are prepared using an aqueous solution of the fluorescent dye alone. Next, the luminescence intensity of each dilution is measured under the same conditions and compared. The determination can be made by determining whether the concentration of the dilution that shows the luminescence intensity peak p1 is within the range of 30 to 80 μM.

[0080] Whether or not condition (2) is satisfied can be determined by preparing a diluted solution of 100 μM concentration during the measurement related to the above condition (1) and determining whether or not the luminescence intensity is 80% or more of the luminescence intensity of the above peak p1.

[0081] In condition (2), the luminescence intensity at a concentration of 100 μM is more preferably 85% or more, and even more preferably 90% or more, of the luminescence intensity of the peak p1.

[0082] The method for measuring the emission intensity is not particularly limited, and can be measured using a general fluorometer under general measurement conditions. The wavelength of the irradiated excitation light is the maximum excitation wavelength of the fluorescent dye or a wavelength close to it. The emission wavelength to be measured is the maximum emission wavelength of the fluorescent dye or a wavelength close to it. It is preferable to adjust the slit widths of the excitation light and emission light within an appropriate range. The maximum excitation wavelength and maximum emission wavelength can be measured by a general method, and if the fluorescent dye is a commercially available product, they may be considered to be the wavelengths listed in the catalog.

[0083] Generally, the greater the Stokes shift of a fluorescent dye, i.e., the difference between the maximum excitation wavelength and the maximum emission wavelength, the smaller the overlap (autoabsorption) between the absorption spectrum and the emission spectrum. As a result, concentration quenching is suppressed, improving the emission intensity and quantitative sensitivity. The magnitude of the Stokes shift is not particularly limited, but it is preferable that the Stokes shift is 25 nm or more. A Stokes shift of 25 nm or more makes it easier to satisfy the above conditions (1) and (2).

[0084] Fluorescent dyes are more likely to satisfy conditions (1) and (2) by undergoing a water-solubilizing treatment. That is, even fluorescent dyes that do not satisfy conditions (1) and (2) may be made to satisfy conditions (1) and (2) by undergoing a water-solubilizing treatment. Furthermore, fluorescent dyes that satisfy conditions (1) and (2) even without undergoing a water-solubilizing treatment may be subjected to a water-solubilizing treatment. In this case, the satisfaction of conditions (1) and (2) is maintained.

[0085] The water-solubilizing treatment is preferably performed on the fluorescent dye to be immobilized in the encapsulated fluorescent labeling resin particles, which improves the dispersibility and luminescence intensity of the fluorescent dye, thereby obtaining fluorescent labeling resin particles with high light resistance and luminescence intensity.

[0086] A fluorescent dye having a Stokes shift of less than 25 nm may be subjected to a water-solubilizing treatment to widen the Stokes shift to 25 nm or more.

[0087] In this embodiment, the term "water-solubilizing treatment" refers to a treatment for making a fluorescent dye water-soluble, i.e., a treatment for improving the solubility of the fluorescent dye in water. The water-solubilizing treatment is not particularly limited. Examples of the water-solubilizing treatment include a method of treating a fluorescent dye with an acid or an aldehyde. Examples of acids that can be used include concentrated sulfuric acid, concentrated hydrochloric acid, acetic acid, and formic acid. Examples of aldehydes that can be used include formaldehyde and acetaldehyde. From the viewpoint of the effectiveness of the water-solubilizing treatment, an acid treatment is preferred.

[0088] The compound used in the water-solubilizing treatment, for example, the acid used in the acid treatment, is appropriately selected depending on the fluorescent dye to be treated. For example, concentrated hydrochloric acid is suitable for "HiLyte Fluor (registered trademark)" (manufactured by Anaspec Co., Ltd.), but concentrated sulfuric acid is likely to cause quenching. Concentrated sulfuric acid is suitable for "Dylight (registered trademark) 594" (manufactured by Thermo Fisher Scientific Co., Ltd.), but concentrated hydrochloric acid is likely to cause discoloration.

[0089] In determining conditions (1) and (2), the measured value of the luminous intensity may be a relative value expressed in an arbitrary unit au (arbitrary unit) as long as the measurement conditions of the objects to be compared are the same. Alternatively, the measured value of the luminous intensity may be an absolute value, such as the luminance expressed by the following formula (ii):

[0090] Equation (ii): Brightness = molar extinction coefficient (ε) × quantum yield (φ) × 1 / 1000

[0091] In the conditions (1) and (2), the luminescence intensity of the peak p1 is not particularly limited. The luminescence intensity of the peak p1 is preferably 100,000 or more in terms of brightness represented by the formula (ii). A brightness of 100,000 or more allows for the production of fluorescent labeling resin particles with high luminescence intensity.

[0092] Examples of commonly available or producible fluorescent dyes include rhodamine-based dye molecules, squarylium-based dye molecules, cyanine-based dye molecules, aromatic hydrocarbon-based dye molecules, oxazine-based dye molecules, carbopyronine-based dye molecules, pyrromethene-based dye molecules, etc. Examples of commercially available fluorescent dyes include "Alexa Fluor" (registered trademark, manufactured by Invitrogen Corporation), "BODIPY" (registered trademark, manufactured by Invitrogen Corporation), "Cy" (registered trademark, manufactured by GE Healthcare Corporation), "DY-based dye molecules (registered trademark, manufactured by DYOMICS Corporation), "HiLyte" (registered trademark, manufactured by Anaspec Corporation), "DyLight" (registered trademark, manufactured by Thermo Scientific Corporation), "ATTO" (registered trademark, manufactured by ATTO-TEC Corporation), "MFP" (registered trademark, manufactured by Mobitec Corporation), and near-infrared dye tricarbocyanine-based dye molecules. These dye molecules are collectively named based on the main structure (skeleton) of the compound or a registered trademark, and the range of fluorescent dyes belonging to each group can be adequately understood by those skilled in the art without excessive trial and error.

[0093] Examples of rhodamine dye molecules include 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine (ROX), 5-carboxy-X-rhodamine (5-ROX), Texas Red, Spectrum Red, LD700 PERCHLORATE, CAL Fluor Red 610, and CAL Fluor Red 615.

[0094] Examples of squarylium-based dye molecules include SRfluor 680-Carboxylate, 1,3-Bis[4-(dimethylamino)-2-hydroxyphenyl]-2,4-dihydroxycyclobutenediylidene dihydroxide, bis, 1,3-Bis[4-(dimethylamino)phenyl]-2,4-dihydroxycyclobutenediylidene dihydroxide, bis, 2-(4-(Diethylamino)-2-hydroxyphenyl)-4-(4-(diethylium)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-1-enolate, 2-(4-(Dibutylamino)-2-hydroxyphenyl)-4-(4-(dibutylium)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-1-enolate, 2-(8-Hydroxy-1,1,7,7-tetramethyl-1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinolin-9-yl)-4-(8-hydroxy-1,1,7,7-tetramethyl- 2,3,6,7-tetrahydro-1H-pyrido[3,2,1-ij]quinolinium-9(5H)-ylidene)-3-oxocyclobut- 1-enolate, and the like.

[0095] Examples of cyanine dye molecules include 1-butyl-2-[5-(1-butyl-1,3-dihydro-3,3-dimethyl-2H-indol-2-ylidene)-penta-1,3-dienyl]-3,3-dimethyl-3eiti-indolium hexafluorophosphate, 1-butyl-2-[5-(1-butyl-3,3-dimethyl-1,3-dihydro-indol-2-ylidene)-3-chloro-penta-1,3-dienyl]-3,3-dimethyl-3H-indolium hexafluorophosphate, 3-ethyl-2-[5-(3-ethyl-3H-benzothiazol-2-ylidene)-penta-1,3-dienyl]-benzothiazol-3-ium iodide, and the like.

[0096] As aromatic hydrocarbon-based pigment molecules, there are N,N-Bis-(2,6-diisopropylphenyl)-1,6,7,12-(4-tert-butylphenoxy)-perylene-3,4,9,10-tetracarboxylic acid diimide, N,N'-Bis(2,6-diisopropylphenyl)-1,6,7,12-tetraphenoxyperylene-3,4:9,10-tetracarboxydiimide, N,N'-Bis(2,6-diisopropylphenyl)perylene-3,4,9,10-bis(dicarimide), 16,N,N'-Bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylic diimide, 4,4'-[(8,16-Dihydro-8,16-dioxodibenzo[a,j]perylene-2,10-diyldioxy]dibutyric acid, 2,10-Dihydroxy-dibenzo[a,j]perylene-8,16-dione, 2,10-Bis(3-aminopropoxy)dibenzo[a,j]perylene-8,16-dione, 3,3'-[(8,16-Dihydro-8,16-dioxodibenzo[a,j]perylen-2,10-diyldioxy]dipropylamine, 17-BIS(Octyloxy)Anthra[9,1,2-cde-]Benzol[RST]Pentaphene-5-10-Dione, Octadecanoic acid, 5,10-dihydro-5,10-dioxoanthra[9,1,2-cde]benzol[rst]pentaphene-16,17-dylester, Dihydroxydibenzanthrone, Benzenesulfonic acid, 4,4',4'',4''' - [[2,9-bis[2,6-bis(1-methylethyl)phenyl]-1,2,3,8,9,10-hexahydro-1,3,8,10-tetraoxoanthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-5,6,12,13-tetrayl]tetrak is(oxy)]tetrakis-, Benzeneethanaminium, 4,4',4'',4'''-[[2,9-bis[2,6-bis(1-methyleth yl)phenyl]-1,2,3,8,9,10-hexahydro-1,3,8,10-tetraoxoanthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-5,6,12,13-tetrayl]tetrakis(oxy)]tetrakis[N,N,N-trimethyl-] and the like.

[0097] Examples of oxazine dye molecules include Cresyl violet, Oxazine 170, EVO blue 30, and Nile Blue.

[0098] Examples of carbopyronin dye molecules include CARBOPYRONIN 149.

[0099] Specific examples of dipyrromethene (pyrromethene) dye molecules include PYRROMETHENE 650 and the like.

[0100] Examples of Alexa Fluor dye molecules mainly having a rhodamine skeleton include Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750 (all manufactured by Invitrogen Corporation).

[0101] Examples of BODIPY dye molecules mainly having a dipyrromethene skeleton include BODIPY FL, BODIPY TMR, BODIPY TR, BODIPY 493 / 503, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, and BODIPY 650 / 665 (all manufactured by Invitrogen Corporation).

[0102] Examples of Cy-based dye molecules include Cy3.5, Cy5, and Cy5.5 (all manufactured by GE Healthcare Corporation).

[0103] Examples of DY dye molecules mainly having a rhodamine skeleton include DY-590, DY-610, DY-615, DY-630, DY-631, DY-632, DY-633, and DY-634 (all manufactured by DYOMICS Corporation).

[0104] Examples of HiLyte dye molecules include HiLyte594 and HiLyteFluor TR (both manufactured by Anaspec, Inc.).

[0105] Examples of DyLight dye molecules mainly having a rhodamine skeleton include DyLight 594 and DyLight 633 (both manufactured by Thermo Scientific Co., Ltd.).

[0106] Examples of ATTO dye molecules mainly having a rhodamine skeleton include ATTO590, ATTO610, ATTO620, ATTO633, and ATTO655 (all manufactured by ATTO-TEC Corporation).

[0107] Examples of MFP dye molecules include MFP590 and MFP631 (both manufactured by Mobitec Corporation).

[0108] Other dyes include C-Phycocyanin, Phycocyanin, APC (Allophycocyanin), APC-XL, NorthernLights 637 (all manufactured by R&D Systems Co., Ltd.), and near-infrared dye Indocyanine Green.

[0109] In this embodiment, the fluorescent dye molecules (compounds) can be selected from the above-mentioned known fluorescent dye molecules (compounds) or their water-solubilized products. It is preferable to select a compound that satisfies the above-mentioned conditions (1) and (2), and it is more preferable to select a compound that satisfies the above-mentioned conditions related to the Stokes shift or brightness. The selection can be made by a person skilled in the art without excessive trial and error by following the above-mentioned method for determining the conditions.

[0110] The emission wavelength of the fluorescent dye can be selected depending on the application.

[0111] Specific examples of fluorescent dyes that can be used in this embodiment are as follows, but the present disclosure is not limited to embodiments that use these fluorescent dyes.

[0112] Examples of fluorescent dyes include 5-carboxy-X-rhodamine (5-ROX), Spectrum Red, Alexa Fluor 594, ATTO 590, DY-590, CAL Fluor Red 610, and DyLight 594. These can be used either with or without water-solubilization treatment.

[0113] Examples of fluorescent dyes that can be used after being subjected to a water-solubilizing treatment include BODIPY 576 / 589, PYRROMETHENE 650, Cresyl violet, DY-610, and Texas Red.

[0114] The above fluorescent dyes are summarized in Table I below.

[0115]

[0116] (1.1.2) Resin The resin may be a thermosetting resin or a thermoplastic resin. It is preferable that the resin is one from which the fluorescent dye is not easily eluted during a permeation process using an organic solvent such as xylene. In other words, it is preferable that the resin be capable of immobilizing the fluorescent dye inside a dense cross-linked structure. An example of such a resin is melamine resin.

[0117] Examples of monomers constituting the thermosetting resin include melamine, urea, guanamines (benzoguanamine, acetoguanamine, etc.), phenols (phenol, cresol, xylenol, etc.), xylene, and derivatives thereof. These monomers may be used alone or in combination of two or more. Comonomers other than the above-mentioned monomers may also be used. Comonomers may be used alone or in combination of two or more.

[0118] Examples of the thermosetting resin include melamine-formaldehyde resin, urea-formaldehyde resin, benzoguanamine-formaldehyde resin, phenol-formaldehyde resin, and metaxylene-formaldehyde resin.

[0119] As raw materials for thermosetting resins, not only the above-mentioned monomers themselves but also prepolymers may be used. Prepolymers are obtained by preliminarily reacting a monomer with a crosslinking agent such as formaldehyde. For example, in the production of melamine-formaldehyde resins, methylolmelamine, which is prepared by condensing melamine and formaldehyde under alkaline conditions, is generally used as the prepolymer. The prepolymer may be alkyl-etherified. Examples of alkyl-etherification include methylation to improve stability in water and butylation to improve solubility in organic solvents.

[0120] In thermosetting resins, at least a portion of the hydrogen atoms contained in the structural units may be replaced with a charged substituent or a substituent capable of forming a covalent bond. Such thermosetting resins can be synthesized by using, as raw materials, monomers in which at least one hydrogen atom has been replaced with the above-described substituent (derivatized) by known methods. Melamine resins, urea resins, benzoguanamine resins, etc., typically naturally have cations generated from amino groups or moieties derived therefrom. Phenol resins, xylene resins, etc. typically naturally have anions generated from hydroxy groups or moieties derived therefrom.

[0121] Thermosetting resins can be synthesized by known methods. For example, melamine-formaldehyde resins can be synthesized by heating pre-prepared methylolmelamine, optionally with the addition of a reaction accelerator such as an acid, to cause polycondensation.

[0122] Examples of monomers constituting thermoplastic resins include styrene, (meth)acrylic acid, its alkyl esters, acrylonitrile, and derivatives thereof. These monomers are monofunctional monomers having one group involved in the polymerization reaction per molecule. In the above example, the group involved in the polymerization reaction is a vinyl group. These monomers may be used alone or in combination of two or more. Comonomers other than the above monomers may also be used. Comonomers may be used alone or in combination of two or more.

[0123] Examples of thermoplastic resins include polystyrene, styrene-based resins composed of styrene and other monomers, and polymethyl methacrylate. Examples of thermoplastic resins include (meth)acrylic acid, acrylic resins composed of alkyl esters of (meth)acrylic acid and other monomers, and polyacrylonitrile. Examples of thermoplastic resins include acrylonitrile-styrene copolymers (AS resins), acrylonitrile-styrene-methyl acrylate copolymers (ASA resins), and acrylonitrile-based resins composed of acrylonitrile and other monomers.

[0124] The thermoplastic resin may contain a structural unit formed from a polyfunctional monomer having two or more groups involved in a polymerization reaction in one molecule, i.e., a crosslinking site. An example of the polyfunctional monomer is divinylbenzene. In divinylbenzene, the group involved in the polymerization reaction is a vinyl group. An example of a thermoplastic resin containing a crosslinking site is a crosslinked product of polymethyl methacrylate.

[0125] At least a portion of the hydrogen atoms contained in the structural units of a thermoplastic resin may be replaced with a charged substituent or a substituent capable of forming a covalent bond. An example of such a thermoplastic resin is 4-aminostyrene. 4-aminostyrene can be synthesized by using, as a raw material, a monomer in which at least one hydrogen atom has been replaced with the above-described substituent (a derivative).

[0126] The thermosetting resin and thermoplastic resin may have functional groups for surface modification of the fluorescent labeling resin particles. For example, by using a monomer such as glycidyl methacrylate having an epoxy group as a raw material, fluorescent labeling resin particles with the epoxy group oriented on the surface can be produced. The epoxy group can be converted to an amino group by reacting with excess aqueous ammonia. The formed amino group can be bound to cells via a linker molecule according to known methods.

[0127] (1.1.3) Method for producing fluorescently labeled resin particles: Fluorescently labeled resin particles can be produced using a fluorescent dye in accordance with the known polymerization process (1) for various resins. Furthermore, a biorelevant binding substance may be linked to the resulting fluorescently labeled resin particles in the modification process (2). The term "biorelevant binding substance" used herein refers to a substance used to indirectly bind antigens on the cell surface to the fluorescently labeled resin particles.

[0128] [Thermosetting Resin - Encapsulating Type] Encapsulating type fluorescent labeling resin particles using a thermosetting resin can basically be produced using emulsion polymerization. Among these, it is preferable to produce them by the following polymerization process using a surfactant and a polymerization reaction accelerator. In the following polymerization process, substantially all of the fluorescent dye is immobilized in a state where it is encapsulated in the resin particles, but some of the fluorescent dye may be immobilized in a state where it is bound to or attached to the surface of the resin particles.

[0129] There are no limitations on the chemical or physical mechanism by which the fluorescent dye is immobilized in the resin particles when the fluorescent dye is encapsulated. In the polymerization process described below, fluorescent labeling resin particles with excellent luminescence intensity, light resistance, etc. can be obtained without a prior derivatization step for covalently bonding the resin raw material and the fluorescent dye or for introducing a charged substituent into the resin raw material. However, a derivatization step may be performed if necessary.

[0130] (a) Polymerization step: The polymerization step is a step in which a reaction mixture containing a fluorescent dye and a resin raw material is heated to promote a polymerization reaction of the resin, thereby producing resin particles encapsulating the fluorescent dye. The reaction mixture preferably contains a surfactant and a polymerization reaction accelerator. The resin raw material includes a monomer, oligomer, or prepolymer.

[0131] The order of addition of the components contained in the reaction mixture is not particularly limited. For example, the order of addition may be such that a surfactant is added to an aqueous solution of the fluorescent dye, followed by addition of the resin raw material to the resulting mixture, and finally addition of a polymerization reaction accelerator to the resulting mixture. Alternatively, the order of addition may be such that a resin raw material is added to an aqueous solution of the surfactant, followed by addition of a polymerization reaction accelerator to the resulting mixture to allow the synthesis reaction of the resin particles to proceed, while the aqueous solution of the fluorescent dye is added. The concentration of the aqueous solution of the fluorescent dye used in the polymerization step is preferably relatively high, for example, in the range of 250 to 450 μM.

[0132] The polymerization reaction conditions (temperature, time, etc.) can be set as needed, taking into consideration the type of resin, the composition of the raw material mixture, etc. In the synthesis of thermosetting resins such as melamine resin, the reaction temperature is preferably within the range of 70 to 200°C, and the reaction time is preferably within the range of 20 to 120 minutes. It is appropriate that the reaction temperature is a temperature at which the performance of the fluorescent dye does not deteriorate, i.e., within the heat-resistant temperature range. Heating may be carried out in multiple stages; for example, the reaction may be carried out at a relatively low temperature for a certain period of time, followed by heating and reaction at a relatively high temperature for a certain period of time.

[0133] After the polymerization reaction is complete, impurities such as excess resin raw material, fluorescent dye, and surfactant are removed from the reaction solution, and the resulting fluorescently labeled resin particles are recovered and purified. For example, the reaction solution is centrifuged, the supernatant containing impurities is removed, and then ultrapure water is added and the particles are redispersed and washed by ultrasonic irradiation. These procedures are preferably repeated multiple times until no absorption or fluorescence from the resin or fluorescent dye is observed in the supernatant.

[0134] (Surfactant) Known emulsifiers for emulsion polymerization can be used as the surfactant. Surfactants can be classified as anionic (negative ion), nonionic (nonionic), or cationic (cationic). When synthesizing a thermosetting resin having a positively charged substituent or moiety, i.e., a cationic type, it is preferable to use an anionic or nonionic surfactant. Conversely, when synthesizing a thermosetting resin having a negatively charged substituent or moiety, i.e., an anionic type, it is preferable to use a cationic or nonionic surfactant.

[0135] Examples of anionic surfactants include the "Neopelex" series (sodium dodecylbenzenesulfonate, manufactured by Kao Corporation). Examples of nonionic surfactants include the "Emulgen" series (polyoxyethylene alkyl ether compounds, manufactured by Kao Corporation). Other examples of nonionic surfactants include polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA). Examples of cationic surfactants include dodecyltrimethylammonium bromide.

[0136] By adjusting the amount of surfactant added, the particle size of the resin particles can be adjusted and the particle size coefficient of variation can be small, i.e., fluorescent labeling resin particles with a uniform particle size can be produced. The amount of surfactant added is preferably within the range of 10 to 60 parts by mass per 100 parts by mass of the resin raw material, and is preferably within the range of 0.1 to 3.0% by mass of the entire raw material mixture. Increasing the amount of surfactant added tends to reduce the particle size, while decreasing the amount of surfactant added tends to increase the particle size.

[0137] (Polymerization Reaction Accelerator) A polymerization reaction accelerator promotes the polycondensation reaction of a thermosetting resin such as a melamine resin, and also imparts a proton (H+) to functional groups such as amino groups contained in the resin or fluorescent dye, thereby charging them and facilitating electrostatic interactions. Although the reaction of a thermosetting resin proceeds by heating alone, the addition of a polymerization reaction accelerator allows the reaction to proceed at a lower temperature, so it can be added within a range that allows the reaction and the above-mentioned function to be controlled. Examples of polymerization reaction accelerators include acids such as formic acid, acetic acid, sulfuric acid, paratoluenesulfonic acid, and dodecylbenzenesulfonic acid. When the fluorescent dye is a compound having a carboxy group or a sulfo group, the fluorescent dye can also donate a proton in the same way as the above-mentioned acids.

[0138] (b) Modification step The modification step, which is performed as needed, is a step for linking a biologically relevant binding substance or the like to the surface of the fluorescent labeling resin particle depending on the intended use of the fluorescent labeling resin particle. Various methods for linking a fluorescent label to a biologically relevant binding substance or the like are known, and any known method can be used.

[0139] For example, reactions occurring between reactive functional groups such as carboxyl groups, amino groups, aldehyde groups, thiol groups, and maleimide groups can be utilized. This allows one reactive functional group present on the surface of a fluorescent label to be bonded to another reactive functional group present in the molecule of a biologically relevant binding substance. Furthermore, if these functional groups cannot be bonded directly to each other, they can be bonded via a "linker molecule" that has specific functional groups at both ends of the molecule. These reactions can be carried out by adding the necessary reagents and allowing the required time to elapse.

[0140] Specifically, amino groups are introduced by reacting a silane coupling agent with fluorescent labeling resin particles having hydroxyl groups on their surfaces. Examples of silane coupling agents include aminopropyltrimethoxysilane. Meanwhile, thiol groups are introduced by reacting streptavidin with a thiol group introduction reagent. Examples of thiol group introduction reagents include N-succimidyl S-acetylthioacetate. Finally, fluorescent labeling resin particles and streptavidin are linked by reacting them with a PEG (polyethylene glycol)-based linker molecule having maleimide groups at both ends. Maleimide groups are reactive with both amino and thiol groups.

[0141] For example, when synthesizing an acrylic resin using glycidyl methacrylate as a raw material monomer, epoxy groups derived from the monomer are oriented on the surface of the fluorescent labeling resin particles. By adding ammonia water to the fluorescent labeling resin particles, the epoxy groups oriented on the surface are converted to amino groups, and the desired biologically relevant binding substances can be linked to the amino groups.

[0142] [Thermosetting Resin - Adsorption Type] The method for producing adsorption-type fluorescent labeling resin particles using a thermosetting resin is as follows. First, fluorescent dye-free thermosetting resin particles are prepared using the same method as the polymerization step (a) for the aforementioned encapsulated fluorescent labeling resin particles, except that no fluorescent dye is added as a raw material. Next, a dispersion of the resulting resin particles is mixed with an aqueous solution of the fluorescent dye, and the fluorescent dye is adsorbed onto the surface of the resin particles. In this case, the thermosetting resin and fluorescent dye of the resin particles preferably have substituents or moieties with opposite charges, and are adsorbed by electrostatic interaction.

[0143] [Thermoplastic resin - encapsulated type] Thermoplastic resins can be synthesized according to known methods such as radical polymerization, ionic polymerization (anionic polymerization, etc.). The encapsulated fluorescent labeling resin particles using a thermoplastic resin can also be produced in accordance with these methods. For example, the encapsulated fluorescent labeling resin particles using a thermoplastic resin are preferably produced by a polymerization step according to a soap-free emulsion polymerization method.

[0144] In this case, the polymerization step (a) is, for example, a step of heating a reaction mixture containing a fluorescent dye, a resin raw material, and a polymerization initiator to promote a resin polymerization reaction and generate resin particles encapsulating the fluorescent dye. Examples of polymerization initiators include benzoyl peroxide and azobisisobutyronitrile. The type of polymerization initiator and polymerization reaction conditions (temperature, time, etc.) can be set as needed, taking into account the type of resin, etc. In the synthesis of a thermoplastic resin, the reaction temperature is preferably within the range of 20 to 150°C, and the reaction time is preferably within the range of 10 to 240 minutes.

[0145] [Thermoplastic Resin - Adsorption Type] The method for producing adsorption-type fluorescent labeling resin particles using a thermoplastic resin is as follows. First, fluorescent dye-free thermoplastic resin particles are prepared using the same method as the polymerization step (1) for the above-described encapsulated fluorescent labeling resin particles, except that no fluorescent dye is added as a raw material. Next, a dispersion of the obtained resin particles is mixed with an aqueous solution of the fluorescent dye, and the fluorescent dye is adsorbed onto the surface of the resin particles. In this case, the thermoplastic resin and fluorescent dye of the resin particles preferably have substituents or moieties with opposite charges, and are adsorbed by electrostatic interaction.

[0146] (1.2) Binding Step The first binding step and second binding step will be described together. In this embodiment, the "first binding step" refers to a step of directly or indirectly binding an antigen on the cell surface to a target antibody. For example, when a primary antibody and a secondary antibody are sequentially bound to an antigen on the cell surface, the primary antibody binds directly to the antigen, and the secondary antibody binds indirectly to the antigen. In this embodiment, both are included in the first binding step. The "second binding step" refers to a step of binding an antigen on the cell surface to a fluorescently labeled resin particle via a target antibody.

[0147] "Immunocytochemical staining" is a method for detecting intracellular antigens using the specificity of antibodies. By using a fluorescent labeling agent in immuneocytochemical staining, the presence of antigens can be detected by fluorescence. In this embodiment, the above-mentioned fluorescent labeling resin particles are used as the fluorescent labeling agent. Furthermore, as mentioned above, the antigen does not necessarily have to be the target substance, as long as it is a substance whose expression level increases or decreases in accordance with an increase or decrease in the expression level of the target substance.

[0148] Immunocytostaining can be performed by a direct method or an indirect method, and the labeling step according to this embodiment may be performed by either the direct method or the indirect method.

[0149] In the direct method (primary antibody method), a fluorescent labeling agent is directly bound to a primary antibody that specifically binds to an antigen to be detected. In this embodiment, in the case of the direct method, the step of binding the antigen to be detected and the primary antibody is referred to as the first binding step, and the step of binding the primary antibody bound to the antigen and fluorescent labeling resin particles is referred to as the second binding step.

[0150] In the indirect method (secondary antibody method), a secondary antibody bound to a fluorescent labeling agent is bound to a primary antibody that specifically binds to an antigen to be detected. In this embodiment, in the case of the indirect method, the step of binding the antigen to be detected and the primary antibody is referred to as the first binding step, and the step of binding the primary antibody bound to the antigen and the secondary antibody bound to the fluorescent labeling resin particles is referred to as the second binding step.

[0151] Furthermore, in immunocytostaining, binding of biotin with avidin or streptavidin may be used. This binding is very strong, and one molecule of avidin or streptavidin can bind to four molecules of biotin. Although streptavidin will not be described below, it has the same functions as avidin.

[0152] For example, a complex of an antigen bound to a primary antibody, a complex of a secondary antibody bound to biotin, and a complex of avidin bound to fluorescent labeling resin particles are generated. These complexes can be bound by binding the primary antibody to the secondary antibody and then binding biotin to avidin. By using this method, even if it is difficult to directly bind the secondary antibody to the fluorescent labeling resin particles, indirect binding is possible.

[0153] The combination of biotin and avidin may also be reversed. A complex of an antigen bound to a primary antibody, a complex of a secondary antibody bound to avidin, and a complex of biotin bound to fluorescently labeled resin particles are generated, respectively. These complexes can be bound by binding the primary antibody to the secondary antibody and then binding avidin to biotin. By using this method, even if direct binding of a secondary antibody to fluorescently labeled resin particles is difficult, indirect binding is possible. In addition, because one molecule of avidin can bind to four molecules of biotin, many fluorescently labeled resin particles can be bound to the secondary antibody, thereby increasing the fluorescence emission intensity per antigen molecule.

[0154] The primary antibody may be selected depending on the antigen to be detected, so as to specifically bind to the antigen. For example, if the antigen to be detected is HER2, an anti-HER2 monoclonal antibody may be used as the primary antibody. Such a primary antibody (monoclonal antibody) may be produced by a general method using an immunized animal such as a mouse, rabbit, cow, goat, sheep, dog, or chicken.

[0155] The secondary antibody may be selected depending on the selected primary antibody, so long as it binds to the primary antibody. For example, if the primary antibody is a rabbit anti-HER2 monoclonal antibody, an anti-rabbit IgG antibody can be used as the secondary antibody. Such secondary antibodies can also be produced by a common method.

[0156] The method for producing a complex in which a secondary antibody is bound to a resin particle for fluorescent labeling is not particularly limited and may be a known method. For example, amidation by the reaction of an amine with a carboxylic acid, sulfidation by the reaction of a maleimide with a thiol, imination by the reaction of an aldehyde with an amine, or amination by the reaction of an epoxy with an amine can be used. The functional groups involved in these reactions may be functional groups that are pre-existing on the surface of the resin particles, i.e., functional groups derived from the raw material monomers of the resin. Furthermore, the functional groups involved in these reactions may be functional groups obtained by converting functional groups present on the surface of the resin particles using a known method, or functional groups introduced onto the surface of the resin particles by surface modification, etc. An appropriate linker molecule may be used, if necessary, to bind the resin particle for fluorescent labeling to the secondary antibody.

[0157] (1.3) Blocking step: In the blocking step, areas of the cell surface other than the antigen are masked, thereby preventing the fluorescent labeling resin particles from binding to components other than the antigen on the cell surface (non-specific binding), thereby improving the accuracy of the analysis.

[0158] In immunocytostaining, an antibody specifically binds to an antigen epitope. This antibody-antigen binding involves forces such as hydrophobic interactions, ionic interactions, hydrogen bonds, and intermolecular interactions. However, these forces may also result in nonspecific binding between the antibody and a component other than the antigen, in addition to the antibody-antigen binding. If the antibody forms a nonspecific bond with a component other than the antigen, the amount and location of the antigen cannot be accurately detected. Therefore, it is preferable to use a blocking agent to mask components other than the antigen and prevent forces such as hydrophobic interactions, ionic interactions, hydrogen bonds, and intermolecular interactions from occurring between the antibody and the antibody.

[0159] The blocking agent is not particularly limited. Examples of polymer compounds contained in the blocking agent include casein, skim milk, albumin, gelatin, and polyethylene glycol. Albumin includes bovine serum albumin. Examples of low molecular weight compounds contained in the blocking agent include phospholipids, ethylenediamine, and acetonitrile.

[0160] In particular, it is preferable that the blocking agent contains a carrier protein. A "carrier protein" is a protein that has the function of becoming a complete antigen with immunogenicity when bound to a hapten. A "hapten" is a substance that does not exhibit immunogenicity on its own due to its small molecular weight. Blocking with a carrier protein makes it less likely for nonspecific binding with antibodies to occur.

[0161] From the viewpoint of blocking effect, the carrier protein is preferably bovine serum albumin or casein. The immunogenic complete antigen generated by binding these to the hapten is less likely to bind to the resin particles for fluorescent labeling, and thus has a high blocking effect.

[0162] (1.4) An example of the procedure in the labeling step An example of the procedure in the labeling step will be described below. In pattern 1, after the second binding reaction, the cells are first washed and then fixed. Fixation is performed only after the second binding reaction. In pattern 2, after each binding reaction, the cells are first fixed and then washed. Fixation is performed after each binding reaction. In the example shown below, an indirect method (secondary antibody method) is used.

[0163] The procedure for the blocking step is not particularly limited, but in the example shown below, a blocking agent may be added to the washing solution, the diluted solution of the target antibody, the diluted solution of the resin particles for fluorescent labeling, etc. For example, by adding a blocking agent to the washing solution, washing and blocking of the cells can be performed simultaneously.

[0164] (1.4.1) Pattern 1: Fixation after Washing Figure 3 is a flowchart showing an example of the operation procedure for Pattern 1 in the labeling step. The cell population to be analyzed is first washed with a washing solution and then centrifuged (Step S101). Then, the antigen on the surface of each cell is bound to the primary antibody (Step S102). The cell population to which the primary antibody has bound is washed with a washing solution and then centrifuged (Step S103). Then, the primary antibody bound to each cell is bound to the secondary antibody (Step S104).

[0165] The cell population bound to the secondary antibody is washed with a washing solution and then centrifuged (step S105).Then, the secondary antibody bound to each cell is bound to the fluorescent labeling resin particles (step S106).The cell population bound to the fluorescent labeling resin particles is washed with a washing solution and then centrifuged (step S107).Then, the cell population bound to the fluorescent labeling resin particles is fixed with a fixative (step S108), and the labeling process is completed.

[0166] From the viewpoint of analytical accuracy, it is preferable that the labeling be performed accurately. That is, it is preferable that the fluorescent labeling resin particles bind to the antigen to be detected without leakage, and that they do not form nonspecific bonds with substances other than the antigen.

[0167] In Pattern 1, the cell population bound to the fluorescent labeling resin particles is washed with a washing solution and then fixed. The washing weakens the binding force (nonspecific binding) between substances other than the antigen and the fluorescent labeling resin particles, thereby eliminating the nonspecific binding. Furthermore, the fluorescent labeling resin particles have a relatively large particle size and are easily detached from the antigen. Therefore, the cells are then fixed using a fixative, i.e., by strengthening the binding force between the secondary antibody and the fluorescent labeling resin particles, the fluorescent labeling resin particles held by the antigen are less likely to detach.

[0168] (1.4.2) Pattern 2: Washing after Fixation Figure 4 is a flowchart showing an example of the operation procedure for Pattern 2 in the labeling step. The cell population to be analyzed is first washed with a washing solution and then centrifuged (step S111). Then, the antigen on the surface of each cell is bound to the primary antibody (step S112). The cell population to which the primary antibody has bound is washed with a washing solution and then centrifuged (step S113). Then, the primary antibody bound to each cell is bound to the secondary antibody (step S114).

[0169] The cell population bound to the secondary antibody is fixed with a fixative (step S115). The cell population bound to the fixed secondary antibody is washed with a washing solution and then centrifuged (step S116). The secondary antibody bound to each cell is then bound to the fluorescent labeling resin particles (step S117). The cell population bound to the fluorescent labeling resin particles is fixed with a fixative (step S118). The cell population bound to the fixed fluorescent labeling resin particles is washed with a washing solution and then centrifuged (step S119), completing the labeling process.

[0170] In Pattern 2, the cell population bound to the secondary antibody is first immobilized and then washed. Next, the cell population bound to the fluorescent labeling resin particles is first immobilized and then washed. That is, in Pattern 2, immobilization is performed first and then washing is performed for each binding step. This makes it possible to strengthen the binding strength between the primary antibody and the secondary antibody, and between the secondary antibody and the fluorescent labeling resin particles, and makes it difficult for the fluorescent labeling resin particles held by the antigen to be released.

[0171] In addition, in pattern 2, the cell population to which the primary antibody is bound is not fixed, i.e., the fixation process is not performed between step S112 and step S113, but the fixation process may be performed if necessary.

[0172] In this embodiment, either Pattern 1 or Pattern 2 may be used. Pattern 1 can more easily eliminate non-specific binding than Pattern 2, and Pattern 2 can make it harder for fluorescent labeling resin particles held by antigens to come off than Pattern 1. For example, if non-specific binding is likely to occur, it is preferable to select Pattern 1, and if fluorescent labeling resin particles held by antigens are likely to come off, it is preferable to select Pattern 2.

[0173] (2) Measurement step: In the measurement step, the characteristics of the fluorescence emitted from the fluorescent labeling resin particles are measured using flow cytometry for the cells labeled in the labeling step. The measurement items are not particularly limited, and examples thereof include the fluorescence emission intensity and the decay time of the fluorescence emission.

[0174] In flow cytometry, a fluid containing labeled cells is flowed at high speed through a tube equipped with an observation window, and the cells passing through the observation window are irradiated with laser light to measure the characteristics of the light emitted from the cells. The cells are analyzed based on the obtained measurement results. In this embodiment, the cells are irradiated with excitation light, and the characteristics of the fluorescence emitted from the cells are measured.

[0175] The light source for irradiating the laser light is not particularly limited as long as it is a light source that can irradiate excitation light, and multiple light sources may be used. The light irradiated from the light source may be continuous light or pulsed light. Examples of the light source include a laser light source and a semiconductor laser light source.

[0176] Fluorescence emitted from cells upon irradiation with excitation light is detected by a photosensor (photodetector), such as a PMT (photomultiplier), an APD (avalanche photodiode), or a PD (photodetector).

[0177] In the measurement step, optical properties other than fluorescence properties may be measured for the cells labeled in the labeling step. For example, the cells may be irradiated with light in a specific wavelength range, and the properties of the light transmitted through or reflected by the cells may be measured. Below, forward and side scatter gating, which extracts the cell type to be measured by measuring the scattered light from the cells, will be described.

[0178] (Forward and side scatter gating) When light is irradiated onto a cell, the irradiated light is refracted by the cell, generating forward scattered light (FS) and side scattered light (SS). The intensity of the forward scattered light can be used to evaluate the size of the cell, and the intensity of the side scattered light can be used to evaluate the granularity and complexity of the cell.

[0179] Figure 5 is a graph showing the measurement results for a certain cell population, with the horizontal axis representing the intensity of forward scattered light (FS) and the vertical axis representing the intensity of side scattered light (SS). In Figure 5, there are two main areas (areas PA and PB) where the plots are concentrated, and it can be determined that this cell population contains two or more cell types with different shapes. By selecting an area that is thought to correspond to the cells to be measured and extracting the plots present in that area, it is possible to eliminate the measurement data for cells that are not to be measured.

[0180] In other words, when a sample contains a mixture of multiple cell types or foreign bodies with significantly different shapes, forward and side scatter gating can be used to extract the cell type of interest, resulting in more accurate cell analysis.

[0181] (3) Determination Step In the determination step, it is determined whether or not the cells express the target substance based on the measurement results obtained in the measurement step.

[0182] The method for determining whether a cell expresses a target substance is not particularly limited. For example, cells with a known expression level of the target substance are separately prepared, and the cell surface components (membrane proteins, etc.) of these cells are analyzed. Based on the analysis results, a target antibody is prepared that targets the cell surface component as an antigen, and the cells are labeled with fluorescently labeled resin particles. Fluorescence emitted from the fluorescently labeled resin particles of the labeled cells is measured using flow cytometry. A calibration curve is created by plotting the expression level of the target substance versus the measured fluorescence intensity. Using the created calibration curve, it is possible to determine whether the target substance is expressed from the fluorescence intensity, and also to predict the expression level.

[0183] It is not necessary to prepare cells with known expression levels of the target substance. For example, the fluorescence emitted from fluorescently labeled resin particles is measured for labeled cells using flow cytometry. The cells are then disrupted to extract the target substance and measure its expression level. A calibration curve is created by plotting the measured expression level of the target substance against the measured fluorescence intensity.

[0184] Furthermore, the measurement results obtained in the measurement step may contain fluorescence other than that derived from the fluorescent labeling resin particles, such as the autofluorescence described above. From the viewpoint of analytical accuracy, it is preferable to remove fluorescence other than that derived from the fluorescent labeling resin particles. Examples of methods for removing this include the following: When the mean fluorescence intensity signal value of the reference negative control is 1.0 × 10 ２ Set the average fluorescence intensity signal value to 1.0 x 10 ２ Cells that exceed the target substance are determined to be cells expressing the target substance. The reference mean fluorescence intensity signal value is not limited to the above values ​​and is preferably set appropriately. Note that the "negative control" here specifically refers to the measurement results using an unstained (unlabeled) sample or an isotype control. An isotype control is a measurement using an antibody raised against an antigen that is not present in the cells to be analyzed, and the cells to be analyzed are not labeled.

[0185] Alternatively, a machine learning model may be used to determine whether a target substance is expressed or to predict the amount of expression.

[0186] (3.1) Machine Learning Machine learning may be either supervised learning or unsupervised learning. "Supervised learning" refers to a learning method that learns the "relationship between input and output" from training data with correct answer labels. "Unsupervised learning" refers to a learning method that learns the "structure of a data group" from training data without correct answer labels.

[0187] Machine learning may be reinforcement learning, deep learning, or deep reinforcement learning. "Reinforcement learning" refers to a learning method that learns "optimal action sequences" through trial and error. "Deep learning" refers to a learning method that learns the features contained in data in a stepwise and deeper manner (at a deeper level) from a large amount of data. "Deep reinforcement learning" refers to a learning method that combines reinforcement learning and deep learning.

[0188] Common analytical methods (algorithms) can be applied to machine learning. For example, predictive models constructed by analytical methods such as linear regression, random forest, decision tree, support vector machine (SVM), support vector regression (SVR), neural network, and discriminant analysis can be applied to machine learning. Examples of linear regression include multiple regression analysis, partial least squares (PLS) regression, LASSO regression, Ridge regression, and principal component regression (PCR). A trained model is constructed by combining the results of multiple predictive models.

[0189] The prediction model is constructed based on data linking the expression level of the target substance with measurement data on the fluorescence characteristics. The prediction model can be constructed by performing machine learning using the features in the measurement data on the fluorescence characteristics as explanatory variables and the expression level of the target substance as the objective variable.

[0190] When referring to a trained model, the data obtained in the measurement step is applied to the trained model. The prediction result may be obtained as, for example, classification, regression, clustering, anomaly detection (outlier detection), etc.

[0191] 2. Cell Analysis Devices Devices that perform flow cytometry include flow cytometers, and examples of such devices include cell sorters and cell analyzers. Cell sorters analyze cells using measurement results, and then sort and recover the cells based on the analysis results. Cell sorters are also called FACS (Fluorescence Activated Cell Sorting). On the other hand, cell analyzers only analyze cells using measurement results. The cell analysis method in this embodiment can be performed using either a cell sorter or a cell analyzer.

[0192] 6 is a schematic diagram showing an example of a cell sorter used in this embodiment. The cell sorter 10 includes a flow cell 11, a light source 12, a first detector 13, a second detector 14, a controller 15, a deflection electrode plate 16, a collection container 17, a waste container 18, etc.

[0193] Cells labeled with fluorescent labeling resin particles are discharged from the flow cell 11. The discharged cells are individually irradiated with light from a light source 12. Within the photomultiplier tube, the scattered light is detected by a first detector 13, and the fluorescent light is detected by a second detector 14, and the scattered light intensity and fluorescent intensity are measured. The obtained optical signal is converted into digital data and processed by an analytical computer. Cells that are not considered to be measurement targets are extracted based on the scattered light intensity, and cells that are not considered to be measurement targets are removed. In addition, the expression state of the target substance is analyzed based on the fluorescence intensity.

[0194] Thereafter, an electric charge is applied to the cells using a controller 15. The path of the cells is controlled using a deflection electrode plate 16 so that highly productive cells are sorted based on the analysis results. The cells to be sorted are separated into a sorting container 17, and the cells to be discarded are separated into a waste container 18. Note that the cells sorted into the waste container 18 do not necessarily need to be discarded, and the culture may be continued until the desired expression level is exhibited.

[0195] 3. Target substance production method The cell analysis method of this embodiment is preferably used in a target substance production method, and it is preferable to adjust the cell culture conditions based on the analysis results.

[0196] Flow cytometry allows analysis of target components contained within the cell membrane and cells without disrupting the cells. Therefore, if analysis reveals that the expression of the target substance is insufficient, the cells can be continued to be cultured until the expression of the target substance becomes sufficient, at which point they can be harvested.

[0197] FIG. 7 is a flowchart of the method for producing a target substance in this embodiment.

[0198] The method for producing a target substance in this embodiment includes a culture step (step S201), an analysis step (step S202), a determination step (step S203), and an adjustment step (step S205). In the culture step S201, cells are cultured to produce the target substance. In the analysis step S202, the aforementioned analysis method is used to determine whether the cultured cells express the target substance. In the determination step S203, it is determined whether the expression level of the target substance in the entire cultured cell population meets a reference value. In the adjustment step S205, the culture conditions for the entire cell population are adjusted.

[0199] If the reference value is met in the determination step S203 (YES), the production of the target substance, i.e., the culture of the entire cell population, is terminated and the target substance is recovered. If the reference value is not met in the determination step S203 (NO), the culture conditions of the entire cell population are adjusted (adjustment step S205), and the production of the target substance, i.e., the culture, is continued.

[0200] In this method, cells are not separated individually but are treated as a whole cell population, so a cell analyzer can be used.

[0201] 8 is a flowchart of a method for producing a target substance that includes a separation step. In this case, the target substance production method includes a separation step (step S204) in addition to the above steps. In the determination step S203, it is determined whether the expression level of the target substance in the cultured single cell satisfies a reference value. In the adjustment step S205, the culture conditions of cells whose expression level of the target substance does not satisfy the reference value are adjusted. In the separation step S204, cells whose expression level of the target substance satisfies the reference value are separated.

[0202] If the standard value is met in the determination step S203 (YES), the cells are separated into individual cells (separation step S204). The separated cells are terminated for production of the target substance, i.e., culture, and the target substance is recovered. If the standard value is not met in the determination step S203 (NO), the cell culture conditions are adjusted (adjustment step S205), and production of the target substance, i.e., culture, is continued.

[0203] In this method, cells are handled individually, so a cell sorter can be used.

[0204] 9 is a flowchart of a method for producing a target substance when a separation step is included. In this case, the method for producing a target substance includes an analysis step S202, a determination step S203, a separation step S204, an adjustment step S205, and a discarding step S206 between a pre-culture step S211 and a main culture step S212. In the discarding step S206, cells that do not satisfy the standard value in the expression level of the target substance are discarded without proceeding to main culture.

[0205] Typically, cell culture is performed by pre-culture and main culture, scaling up from a small scale to a large scale. There is an upper limit to the number of cells that can be cultured in a culture tank such as a reactor. Therefore, it is preferable to culture only highly productive cells and remove as many low-productivity cells as possible.

[0206] In the determination step S203, if the standard value is met (YES), the cells are sorted into individual cells. The sorted cells have their culture conditions adjusted based on the analysis results (adjustment step S205) and are then subjected to main culture (main culture step S212). Note that the main culture conditions do not necessarily need to be adjusted based on the analysis results, and the main culture conditions may be determined in advance. In the determination step S203, if the standard value is not met (NO), the cells are not subjected to main culture and are discarded (discarding step S206).

[0207] In this method, cells are handled individually, so a cell sorter can be used.

[0208] In the production of a target substance, analysis may be performed multiple times. For example, in the method shown in Figure 9, analysis may be performed during or after the main culture.

[0209] (1) Culturing step In the culturing step, cells are cultured. Finally, metabolic products produced by the culture are collected as products. The metabolic products include the target substance.

[0210] The medium used to culture cells is not particularly limited as long as it allows the cells to grow and produce the target substance. When the cells are microorganisms, synthetic or natural media containing nutrient sources such as carbon sources, nitrogen sources, and inorganic salts can be used. Examples of carbon sources include glucose, sucrose, fructose, maltose, glycerin, dextrin, starch, oligosaccharides, molasses, malt extract, and organic acids. Nitrogen sources include organic nitrogen sources such as various peptones, yeast extract, corn steep liquor, soybean flour, bran extract, meat extract, casein, amino acids, and urea. Nitrogen sources include inorganic nitrogen sources such as nitrates and ammonium salts. Inorganic salts include sodium salts, potassium salts, magnesium salts, iron salts, and other metal salts. Other nutrient sources include vitamins, amino acids, and nucleic acids.

[0211] The culture is preferably liquid culture, and various general cell culture methods can be used. The liquid culture is preferably aerobic culture, and the medium is preferably agitated. An example of such a culture is aerobic agitation culture. In aerobic agitation culture, the medium may be agitated only by aeration, such as by airlift.

[0212] The oxygen concentration in aerobic culture is preferably, for example, within the range of 5 to 50% of the saturation concentration. The liquid culture method may be any of batch culture, fed-batch culture, and continuous culture. Culture conditions (temperature, pH, culture time, etc.) can be set appropriately depending on the growth characteristics of the cells to be cultured. The culture temperature is usually within the range of 10 to 40°C, preferably within the range of 30 to 37°C. The pH is usually within the range of 4 to 8, preferably within the range of 5 to 7. The culture time is usually within the range of 1 to 500 hours.

[0213] (2) Sorting step: In the sorting step, cells determined to express a target substance are sorted. Alternatively, only cells whose expression level of the target substance meets a predetermined standard value may be sorted. Cell sorting can be performed using the cell sorter described above.

[0214] The present disclosure will be specifically described below using examples, but the present disclosure is not limited thereto. In the examples, the terms "parts" and "%" are used, but unless otherwise specified, they represent "parts by mass" or "% by mass." In the following examples, unless otherwise specified, operations were performed at room temperature (25°C).

[0215] Example I In Example I, fluorescent labeling resin particles having an average primary particle size of 130 nm as a fluorescent labeling agent were compared with a fluorescent dye.

[0216] (1) Preparation of Resin Particles for Fluorescent Labeling Resin particles for fluorescent labeling were prepared by encapsulating a fluorescent dye in melamine resin particles.

[0217] A surfactant solution with a concentration of 0.5% by volume was prepared by adding the surfactant "Neopelex (registered trademark) G-15" (manufactured by Kao Corporation) to 22 mL of water. "Neopelex (registered trademark) G-15" contained sodium dodecylbenzenesulfonate as an active ingredient, and the amount of the active ingredient was 16% by mass.

[0218] To the resulting surfactant solution, 5-carboxy-X-rhodamine (5-ROX) was added as a fluorescent dye. The amount added was such that the concentration of the resulting mixture was 380 μM. The resulting mixture was heated to 70°C while stirring on a hot stirrer. Note that, although an example using 5-ROX as the fluorescent dye will be described below, results comparable to those obtained with the 5-ROX example were also obtained when the fluorescent dyes listed in Table I above were used instead of 5-ROX.

[0219] To the heated mixture, 0.6 g of a melamine resin raw material "Nikarac (registered trademark) MX-730" (manufactured by Sanwa Chemical Co., Ltd.) was added. In addition, 680 μL of a polymerization reaction accelerator (dodecylbenzenesulfonic acid) with a concentration of 10.0% by volume was added to the heated mixture.

[0220] The resulting mixture was heated and stirred at 70°C for 50 minutes. The mixture was then heated to 90°C and heated and stirred for 20 minutes to allow the reaction to occur. This produced a dispersion of fluorescent labeling resin particles in which the fluorescent dye was encapsulated in the melamine resin particles. The resulting dispersion was centrifuged at 20,000 G for 15 minutes to recover the particles, which were then washed and purified.

[0221] Details of 5-ROX used as the fluorescent dye are as follows: Stokes shift: 29 nm Emission intensity peak: 106 a.u. at a concentration of 55 μM (equivalent to 102043 in brightness) Emission intensity at a concentration of 100 μM: 89 a.u. (equivalent to 85677 in brightness) Details of fluorescent dyes other than 5-ROX are as shown in Table I above.

[0222] (2) Preparation of Fluorescent Dye Alexa Fluor (registered trademark) 488 (manufactured by Invitrogen Corporation) was prepared as a fluorescent dye for comparison with the fluorescently labeled resin particles.

[0223] (3) Labeling of the sample to be analyzed: A group of 10 μm cell-mimicking beads was prepared as a sample to be analyzed. This group of beads was a mixed group of two types of beads, including beads A bound (modified) with a mouse anti-human CD5 antibody and beads A not bound (modified) with an antibody. In other words, the antibody-bound beads A were beads to which part of the first binding step had already been performed.

[0224] A complex B in which biotin and a secondary antibody were bound was prepared.

[0225] A complex C1 in which the above-mentioned fluorescent labeling resin particles and streptavidin were bound, and a complex C2 in which the above-mentioned fluorescent dye and streptavidin were bound were prepared.

[0226] In Example 1 (Example), the primary antibody of bead A was bound to the secondary antibody of complex B. Then, the biotin of complex B was bound to the streptavidin of complex C1, and the cell-mimicking beads were labeled with fluorescent labeling resin particles. That is, part of the first binding step and the second binding step were carried out in order to obtain beads D1 labeled with fluorescent labeling resin particles. The detailed procedure is shown below.

[0227] The labeling process used the same procedure as in Pattern 1 ( FIG. 3 ), except that the immobilization step (S108) was not performed. The blocking agent, "Antibody Diluent with Background-Reducing Components (Dako Corporation: S3022)," is a solution containing casein as a carrier protein and adjusted to an appropriate pH and ionic strength.

[0228] A group of 10 μm cell-mimicking beads, including beads A and beads a, was washed with a washing solution containing Antibody Diluent with Background-Reducing Components (Dako: S3022). The washed beads were then centrifuged at 4°C for 5 minutes. The separated beads were reacted with a diluted solution of Complex B containing Antibody Diluent with Background-Reducing Components (Dako: S3022), allowing Complex B to bind to the beads.

[0229] The resulting bead group was washed with a washing solution containing Antibody Diluent with Background-Reducing Components (Dako: S3022). Then, the beads were centrifuged at 4°C for 5 minutes. The separated bead group was reacted with a diluted solution (concentration: 0.05 nM) of Complex C1 containing Antibody Diluent with Background-Reducing Components (Dako: S3022), and Complex C1 was bound to the beads. The resulting bead group was washed with a washing solution containing Antibody Diluent with Background-Reducing Components (Dako: S3022). Then, the beads were centrifuged at 4°C for 5 minutes. The washing and centrifugation were repeated under the same conditions, and the resulting bead group was diluted with Flow Buffer to prepare a sample for flow cytometry.

[0230] Here, if sufficient amounts of complex B and complex C1 are not added, not all of the antigen to be detected will be labeled, and only a portion of the antigen will be labeled. In order to change the concentration of the labeled antigen, the concentration of the diluted solution of complex B was changed as follows. The same amount was added in all cases. The lower the concentration of the diluted solution of complex B, the lower the concentration of the labeled antigen. Note that in Example 1-1, no diluted solution of complex B was added.

[0231] Example 1-1: 0 μg / mL Example 1-2: 1.0 × 10 －４ μg / mL Example 1-3: 1.0×10 －３ μg / mL Example 1-4: 1.0×10 －２ μg / mL Example 1-5: 1.0×10 －１ μg / mL Example 1-6: 1.0 μg / mL Example 1-7: 2.0 μg / mL

[0232] In Example 2 (Comparative Example), the primary antibody of bead A was bound to the secondary antibody of complex B. Then, the biotin of complex B was bound to the streptavidin of complex C2, and the cell-mimetic beads were labeled with a fluorescent dye. This resulted in fluorescent dye-labeled beads D2. The labeling process was performed in the same manner as in Example 1.

[0233] In Example 2, the amount of Complex B added was changed in the same manner as in Example 1. Example 2-1: 0 μg / mL Example 2-2: 1.0 × 10 －４ μg / mL Example 2-3: 1.0×10 －３ μg / mL Example 2-4: 1.0×10 －２ μg / mL Example 2-5: 1.0×10 －１ μg / mL Example 2-6: 1.0 μg / mL Example 2-7: 2.0 μg / mL

[0234] (4) Measurement of Fluorescence The characteristics of the fluorescence emitted from the labeled bead group were measured using flow cytometry.

[0235] The labeled beads were dispersed in sheath fluid, and the resulting dispersion was then injected into the flow cell of a flow cytometer. The beads were aligned in a laminar flow and then ejected from the flow cell. Each ejected bead was irradiated with a laser beam from a light source, and the intensity of the fluorescence emitted from each bead was measured.

[0236] FIG. 10 is a graph showing the relationship between the fluorescence intensity measured in Examples 1 and 2 and the number of beads. The horizontal axis of the graph represents the measured fluorescence intensity, and the vertical axis of the graph represents the number of beads (count number) for which the fluorescence intensity was measured. Note that fluorescence not derived from the fluorescent labeling agent was also detected in the measurement. In the graph of FIG. 10, the fluorescence intensity is plotted at a predetermined intensity (1.0×10 ２ Fluorescence below [au] was determined not to be derived from the fluorescent labeling agent, and data on fluorescence not derived from the fluorescent labeling agent was excluded.

[0237] In Example 1 (Example) of FIG. 10, Example 1-1 (0 μg / mL) and Example 1-2 (1.0 × 10 －４ In Example 1-2, the number of counts at higher fluorescence intensities (signal detection) increased compared to Example 1-1, and Example 1-1 and Example 1-2 showed different graph shapes. That is, in Example 1, the number of counts at concentrations of 0 μg / mL and 1.0 × 10 －４ It was found that it was possible to distinguish between μg / mL and μg / mL.

[0238] In Example 2 (Comparative Example) of FIG. 10, Example 2-1 (0 μg / mL) and Example 2-2 (1.0 × 10 －４ μg / mL) and Example 2-3 (1.0 × 10 －３ In Example 2-2 and Example 2-3, the shape of the graph was almost the same as in Example 2-1 (no signal was detected), and in Example 2, the concentration was 0 μg / mL and 1.0 × 10 －４ μg / mL and 1.0 × 10 －３ It was found that it was not possible to distinguish between the 1.0 × 10 μg / mL and the 1.0 × 10 μg / mL. －３ μg / mL) and Example 2-4 (1.0 × 10 －２ In Example 2-4, the number of counts at higher fluorescence intensities (signal detection) increased compared to Example 2-3, and Example 2-3 and Example 2-4 showed different graph shapes. That is, in Example 2, the concentration of 1.0 × 10 －３ μg / mL and 1.0 × 10 －２ It was found that it was possible to distinguish between μg / mL and μg / mL.

[0239] 11 is a graph showing the relationship between the concentration of complex B (secondary antibody) and fluorescence intensity. The horizontal axis of the graph represents the concentration of complex B (secondary antibody) [μg / mL], and the vertical axis of the graph represents the mean fluorescence intensity signal value [au]. The mean fluorescence intensity signal value is the arithmetic mean value calculated by dividing the sum of the fluorescence intensities of the beads by the number of beads.

[0240] In Example 1 (Example) of FIG. 11, Example 1-1 (0 μg / mL) and Example 1-2 (1.0 × 10 －４ On the other hand, in Example 2 (Comparative Example) in FIG. 11 , there is a difference in the mean fluorescence intensity signal value between Example 2-1 (0 μg / mL) and Example 2-2 (1.0 × 10 －４ μg / mL) and Example 2-3 (1.0 × 10 －３ It can be seen that there is no difference in the mean fluorescence intensity signal value between Example 2-3 (1.0 × 10 －３ μg / mL) and Example 2-4 (1.0 × 10 －２ It can be seen that there is a difference in the mean fluorescence intensity signal value between the 100 μg / mL and 100 μg / mL.

[0241] 10 and 11 show that the use of fluorescent labeling resin particles as a fluorescent labeling agent provides higher detectability at low concentrations than the use of fluorescent dyes. Furthermore, the use of fluorescent labeling resin particles allows detection at concentrations approximately 1 / 100 of the lower limit of the detectable concentration using fluorescent dyes.

[0242] Example II In Example II, bovine serum albumin (BSA) and casein were compared as carrier proteins for blocking agents, and fluorescent labeling resin particles were used as the fluorescent labeling agent.

[0243] (1) Preparation of Fluorescent Labeling Resin Particles Fluorescent labeling resin particles were prepared in the same manner as in Example I.

[0244] (2) Labeling of Samples to be Analyzed In Example 3, beads labeled with fluorescent labeling resin particles were obtained using the same procedures as in Examples 1-7, except that the blocking agent was changed to a solution prepared with phosphate buffered saline containing bovine serum albumin (BSA), called a "PBS / BSA solution."

[0245] (3) Measurement of Fluorescence The characteristics of the fluorescence emitted from the labeled bead group were measured using flow cytometry.

[0246] Figure 12 is a graph showing the relationship between the fluorescence intensity measured in Example 3 and Examples 1-7 and the number of beads. The horizontal axis of the graph represents the measured fluorescence intensity, and the vertical axis of the graph represents the number of beads (count number) for which the fluorescence intensity was measured. Note that the graph of Figure 12 also reflects data on fluorescence not derived from the fluorescent labeling agent, which was excluded from the graph of Figure 10, and peaks are observed in the region of low fluorescence intensity.

[0247] In Figure 12, Example 3 and Examples 1-7 were compared, focusing on the peaks in the region of high fluorescence intensity. The mean fluorescence intensity signal value (MFI) at the peak of the region of high fluorescence intensity in Example 3 was 63,912 [au]. The mean fluorescence intensity signal value (MFI) at the peak of the region of high fluorescence intensity in Examples 1-7 was 209,816 [au]. In other words, it can be seen that the fluorescence intensity in Example 3 was improved by about 3.3 times compared to Examples 1-7.

[0248] Example A: In Example A, a fluorescent dye was compared with fluorescent labeling resin particles. The primary particle sizes of the fluorescent labeling resin particles were compared: 35 nm, 40 nm, 50 nm, 60 nm, 80 nm, 130 nm, 160 nm, 180 nm, 200 nm, and 210 nm.

[0249] (1) Preparation of Fluorescently Labeled Resin Particles Fluorescently labeled resin particles were prepared using the same procedure as in Example I, and the primary particle size was 130 nm. By gradually increasing the amount of surfactant using the procedure in Example I, fluorescently labeled resin particles with primary particle sizes of 80 nm, 60 nm, 50 nm, 40 nm, and 35 nm were obtained. Furthermore, by gradually decreasing the amount of surfactant using the procedure in Example I, fluorescently labeled resin particles with primary particle sizes of 160 nm, 180 nm, and 200 nm were obtained.

[0250] (2) Labeling of Samples to be Analyzed In Example A, beads labeled with fluorescent labeling resin particles were obtained using the same procedure as in Example 1-7 above. Also, beads labeled with a fluorescent dye were obtained using the same procedure as in Example 2-7 above.

[0251] (3) Measurement of Fluorescence: The characteristics of the fluorescence emitted from the labeled beads were measured using flow cytometry. Using the same method as in Example II, the mean fluorescence intensity (MFI) signal values ​​at the peak of the high fluorescence intensity region in each measurement were compared.

[0252] The mean fluorescence intensity signal values ​​(MFI) at the peak of the region with high light intensity in each measurement were arranged in descending order. At this time, the ranking of the mean fluorescence intensity signal value in each measurement, that is, the mean fluorescence intensity signal value in each measurement, was relatively evaluated according to the following criteria. Note that although Examples A-1 and A-10 were evaluated as D, when the mean fluorescence intensity signal value was confirmed, there was no practical problem, and there were also no practical problems for Examples A to C. Example A-11 was evaluated as E, and when the mean fluorescence intensity signal value was confirmed, there was a practical problem.

[0253] A: The MFI ranking is in the top 40% or more. B: The MFI ranking is in the top 40% or more but less than 60%. C: The MFI ranking is in the top 60% or more but less than 80%. D: The MFI ranking is in the top 80% or more but less than 95%. E: The MFI ranking is not in the top 95% or less and is the lowest of all measurements.

[0254]

[0255] Example III In Example III, a comparison was made between the presence and absence of immobilization treatment in a series of steps in the labeling process. Fluorescent labeling resin particles were used as the fluorescent labeling agent.

[0256] (1) Preparation of Fluorescent Labeling Resin Particles Fluorescent labeling resin particles were prepared in the same manner as in Example I.

[0257] (2) Labeling of Sample to be Analyzed In Example 4, beads labeled with fluorescent labeling resin particles were obtained using the same procedure as in Examples 1-4.

[0258] In Example 5, beads labeled with fluorescent labeling resin particles were obtained by the same procedure as in Example 4, except that the fixation treatment (S108) in the above-mentioned Pattern 1 ( FIG. 3 ) was performed. In the fixation treatment (S108), a "4% Paraformaldehyde Phosphate Buffer Solution" was used as the fixative, and the beads labeled with fluorescent labeling resin particles were reacted with the fixative for 10 minutes.

[0259] (3) Measurement of Fluorescence The characteristics of the fluorescence emitted from the labeled bead group were measured using flow cytometry.

[0260] Figure 13 is a graph showing the relationship between the fluorescence intensity and the number of beads measured in Examples 4 and 5. The horizontal axis of the graph represents the measured fluorescence intensity, and the vertical axis of the graph represents the number of beads (count number) for which the fluorescence intensity was measured. Note that the graph of Figure 13 also reflects data on fluorescence not derived from the fluorescent labeling agent, which was excluded from the graph of Figure 10, and a peak is observed in the region of low fluorescence intensity.

[0261] In Figure 13, Example 4 and Example 5 were compared, focusing on the peaks in the region of high fluorescence intensity. The mean fluorescence intensity signal value (MFI) at the peak of the region of high fluorescence intensity in Example 4 was 810 [au]. The mean fluorescence intensity signal value (MFI) at the peak of the region of high fluorescence intensity in Example 5 was 1335 [au]. In other words, it can be seen that the fluorescence intensity in Example 5 was improved by about 1.6 times compared to Example 4.

[0262] Example IV In Example IV, Pattern 1 (FIG. 3) and Pattern 2 (FIG. 4) were compared in the labeling procedure. That is, fixation before washing was compared with fixation after washing. Fluorescent labeling resin particles were used as the fluorescent labeling agent.

[0263] (1) Preparation of Fluorescent Labeling Resin Particles Fluorescent labeling resin particles were prepared in the same manner as in Example I.

[0264] (2) Labeling of Sample to be Analyzed In Example 6, beads labeled with fluorescent labeling resin particles were obtained using the same procedure as in Example 5, except that in the series of steps in the labeling process, Pattern 1 (FIG. 3) was changed to Pattern 2 (FIG. 4).

[0265] (3) Measurement of Fluorescence The characteristics of the fluorescence emitted from the labeled bead group were measured using flow cytometry.

[0266] Figure 14 is a graph showing the relationship between the fluorescence intensity and the number of beads measured in Examples 5 and 6. The horizontal axis of the graph represents the measured fluorescence intensity, and the vertical axis of the graph represents the number of beads (count number) for which the fluorescence intensity was measured. Note that the graph of Figure 14 also reflects data on fluorescence not derived from the fluorescent labeling agent, which was excluded from the graph of Figure 10, and peaks are observed in the low fluorescence intensity region.

[0267] In Figure 14, Example 5 and Example 6 were compared, focusing on the peaks in the region of high fluorescence intensity. The mean fluorescence intensity signal value (MFI) at the peak of the region of high fluorescence intensity in Example 5 was 1335 [au]. The mean fluorescence intensity signal value (MFI) at the peak of the region of high fluorescence intensity in Example 6 was 13454 [au]. In other words, it can be seen that the fluorescence intensity in Example 6 was improved by about 10 times compared to Example 5.

[0268] A comparison between Example 1 and Example 2 in Example I shows that the cell analysis method in this embodiment can sufficiently analyze even low concentrations of the analyte, and can improve the accuracy of the analysis.

[0269] A comparison of Example 3 and Examples 1 to 7 in Example II shows that the inclusion of casein as a carrier protein in the blocking agent improves the intensity of the detected fluorescence, thereby improving the accuracy of the analysis.

[0270] A comparison of Example 4 and Example 5 in Example III shows that immobilizing the labeled cells (beads) with a fixative solution improves the intensity of the detected fluorescence, thereby improving the accuracy of the analysis.

[0271] A comparison of Example 5 and Example 6 in Example IV shows that fixation before washing improves the intensity of detected fluorescence, thereby improving the accuracy of the analysis.

[0272] By using the present disclosure, it is possible to improve the accuracy of a cell analysis method using flow cytometry. Based on the analysis results with improved accuracy, it is possible to adjust the cell culture conditions and improve productivity.

[0273] 10 Cell sorter 11 Flow cell 12 Light source 13 First detector 14 Second detector 15 Controller 16 Deflection electrode plate 17 Fractionation container 18 Disposal container

Claims

1. A method for analyzing the expression of a target substance in a cell, comprising the steps of: labeling the cell with fluorescent labeling resin particles containing a plurality of fluorescent dye molecules in the resin particles by chemical or physical action; measuring the characteristics of the fluorescence emitted from the fluorescent labeling resin particles that label the cell using flow cytometry; and determining whether the cell expresses the target substance from the flow cytometry measurement results, wherein the labeling step comprises a first binding step of directly or indirectly binding an antigen on the cell surface to a target antibody, and a second binding step of binding the antigen on the cell surface to the fluorescent labeling resin particles via the target antibody.

2. The method for analyzing cells according to claim 1, wherein the average primary particle size of the fluorescent labeling resin particles is within the range of 40 to 200 nm.

3. The method for analyzing cells according to claim 2, wherein the average primary particle size of the fluorescent labeling resin particles is within the range of 50 to 180 nm.

4. The method for analyzing cells according to claim 3, wherein the average primary particle size of the fluorescent labeling resin particles is within the range of 60 to 160 nm.

5. A method for analyzing cells as described in claim 1, comprising a step of washing the cells bound with the fluorescent labeling resin particles obtained after the second binding step, and a step of fixing the cells bound with the fluorescent labeling resin particles that are retained on the antigen after washing with a fixative.

6. A method for analyzing cells according to claim 1, comprising the steps of: immobilizing, with a fixative, the cells bound to the target antibody obtained after the first binding step; washing the immobilized cells bound to the target antibody; immobilizing, with a fixative, the cells bound to the fluorescent labeling resin particles held by the antigen obtained after the second binding step; and washing the immobilized cells bound to the fluorescent labeling resin particles.

7. A method for analyzing cells according to any one of claims 1 to 6, wherein the labeling step further comprises a step of masking areas of the cell surface other than the antigen using a blocking agent.

8. The method for analyzing cells according to claim 7, wherein the blocking agent comprises a carrier protein.

9. The method for analyzing cells according to claim 8, wherein the carrier protein is bovine serum albumin or casein.

10. The method for analyzing cells according to claim 1, wherein the fluorescence intensity is measured using flow cytometry.

11. In the flow cytometry measurement results, the mean fluorescence intensity signal value of the reference negative control is 1.0 x 10 ２ Set it to the following: 1.0 x 10 ２ The method for analyzing cells according to claim 1 , wherein the cells that are overexpressing the target substance are determined to be the cells that express the target substance.

12. A method for producing a target substance by culturing cells, comprising the step of adjusting the culture conditions of the cells based on the analysis results obtained by the cell analysis method described in claim 1.

13. A method for producing a target substance according to claim 12, further comprising the step of isolating the cells determined to express the target substance.

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