Dyeing method, liquid composition for dyeing and kit for dyeing

Abstract

To provide a method capable of a biological sample with a stain solution discharged according to an ink jet system.SOLUTION: A dyeing method is provided, including a step of discharging a liquid composition containing: at least one kind selected from a ligand protein having specificity to a target in a biological sample, an amino acid and a salt thereof; a surfactant; and water, from a liquid discharge head of an ink jet system to apply the composition to the biological sample.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a staining method for staining a biological sample, a liquid composition for staining, and a kit for staining. Specifically, the present invention relates to a staining method, which includes a step of ejecting a liquid composition containing a ligand protein having specificity for a target in a biological sample, at least one selected from amino acids and their salts, a surfactant, and water from an inkjet liquid ejection head and applying the liquid composition to the biological sample, a liquid composition used for the staining, and a kit used for the staining. [Background technology]

[0002] With the recent development of molecular biology, the importance of analyzing substances present in the body is increasing. For example, many attempts have been made to identify proteins and chemicals related to recurrence and metastasis from cancer tissue sections.

[0003] Microscopic observation is often used as a means of analyzing proteins and chemical substances in biological samples such as cells and tissues. In this case, a method is used to identify targets such as proteins and chemical substances in a biological sample by labeling the target via a protein (ligand protein) that has specificity for the target, such as an antibody, and detecting the target by color development, luminescence, fluorescence, etc.

[0004] There is a method of applying a liquid composition containing a ligand protein to a target using a pipette dispenser, but since a place for washing and replacing the pipette is required and the apparatus becomes large, there is a demand for a miniaturized apparatus.

[0005] Patent Document 1 discloses a method for dispensing droplets of reagent of about 1 pL to about 50 pL onto a biological sample, and also discloses a method for dispensing the droplets of reagent by inkjet. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2018-517895 Summary of the Invention [Problem to be solved by the invention]

[0007] In Patent Document 1, small droplets of an antibody reagent composition or the like are applied by inkjet to perform immunostaining. The present inventors ejected the antibody reagent composition described in Patent Document 1 by an inkjet method. As a result, particularly when ejecting at a high frequency, i.e., when ejecting at short ejection intervals, the ejected volume of the antibody reagent composition was sometimes significantly smaller than the intended droplet volume.

[0008] An object of the present invention is to provide a staining method, a liquid composition for staining, and a kit for staining, which can dispense a ligand protein having specificity for a target in a biological sample in a desired droplet volume, i.e., an efficient liquid volume, by ejection using an inkjet method. [Means for solving the problem]

[0009] The present invention provides a staining method for staining a biological sample, which comprises a step of ejecting a liquid composition containing a ligand protein having specificity for a target in a biological sample, at least one selected from amino acids and their salts, a surfactant, and water from an inkjet liquid ejection head and applying the liquid composition to the biological sample.

[0010] The present invention also provides a liquid composition for staining a biological sample. More specifically, the liquid composition contains a ligand protein having specificity for a target in a biological sample, at least one selected from amino acids and their salts, a surfactant, and water. The present invention also provides a staining method for staining a kit for staining a biological sample. More specifically, the kit is for staining a biological sample using an inkjet method, and includes a liquid composition containing a ligand protein having specificity for a target in a biological sample, at least one selected from amino acids and their salts, a surfactant, and water. Effect of the Invention

[0011] According to the present invention, by ejecting using an inkjet method, a ligand protein having specificity for a target in a biological sample can be deposited in a desired droplet volume, i.e., in an efficient liquid volume. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described in detail below with reference to preferred embodiments. As a first embodiment, the present invention provides a staining method characterized by comprising a step of ejecting a liquid composition containing a ligand protein having specificity for a target in a biological sample, at least one selected from amino acids and their salts, a surfactant, and water from an inkjet liquid ejection head and applying the liquid composition to the biological sample.

[0013] A case where the inkjet method of the thermal inkjet method is used will be described. As for the liquid ejection unit, the size precision of the aperture of the ejection port, the heat amount of the heat pulse used for ejection, the micro heater used therefor, etc. is high, and it is possible to increase the reproducibility. A large number of liquid ejection units are densely arranged on the head. On the other hand, as described above, since the reproducibility of the liquid ejection unit can be increased, it is possible to narrow the droplet size distribution of the ejected liquid in the entire large number of liquid ejection units. In addition, the head has low manufacturing cost and high versatility. For example, there is a demand for a small ejection device that uses a head by appropriately replacing it, and it is also highly applicable to such an ejection device. Therefore, when miniaturization and convenience of the ejection device are required, the ejection device of the thermal inkjet method is particularly preferable.

[0014] However, according to the studies of the present inventors, when a liquid composition containing a protein is ejected using a thermal inkjet system without adding any additives, it has been confirmed that, although depending on the type and concentration of the protein, the ejection volume is significantly reduced in the high-frequency range of 5 to 50 kHz ejection frequency compared to a composition without added protein.

[0015] The reason for the drastic decrease in ejection volume is unclear, but the inventors speculate as follows. It is known that proteins generally have a higher-order structure in which the hydrophobic portion is folded. When the protein is adsorbed to the heater portion via the hydrophobic portion, insoluble matter is temporarily precipitated by heating. Some of the insoluble matter does not re-dissolve in water in time for the next heater drive. It is presumed that the drastic decrease in ejection volume is observed because only a portion of the heater portion is heated. In addition, there is a concern that the ligand protein, once insolubilized by heating in the heater section, may lose its physiological activity.

[0016] In order to efficiently eject a large amount of liquid, the liquid must be ejected at a high frequency above a certain level. In this embodiment, the driving frequency is preferably 0.1 kHz to 100 kHz, and more preferably 1 kHz to 50 kHz.

[0017] As a result of extensive research, the inventors have found that by adding at least one type selected from amino acids and their salts, and a surfactant to a liquid composition containing a ligand protein, it is possible to maintain the physiological activity of the ligand protein while ensuring a sufficient ejection volume.

[0018] The reason why the combination of an amino acid and a surfactant contributes sufficiently to ensure a sufficient ejection volume is not clear, but is presumed to be as follows. Amino acids interact weakly with proteins in water. Therefore, proteins are covered with amino acids, which greatly suppresses the precipitation of insoluble matter on the heated heater. Furthermore, the coexisting surfactant can instantly re-dissolve the small amount of insoluble matter that has accumulated on the heater, keeping the heater clean and preventing the proteins that have accumulated on the heater from coagulating and causing clogging. It is believed that the above factors ensure a sufficient discharge volume.

[0019] <Biological samples> The biological sample in this embodiment is not particularly limited as long as it may contain a target on which the specificity of the ligand protein can function. Examples include cultured cells, animal body fluids (e.g., blood, serum, plasma, saliva, sweat, saliva, urine, etc.), hair, excrement, organs, tissues, animals and plants themselves, or samples of these fixed and embedded in paraffin, or dried bodies. In addition, examples include river water, lake water, seawater, water supply and sewage water, soil, etc., which contain substances of biological origin and may contain targets on which the specificity of the ligand protein to be imparted can function.

[0020] The biological sample may be fixed to a solid phase, which makes it possible to easily carry out a step of removing excess liquid composition in a washing step or the like after application of the liquid composition.

[0021] Examples of the solid phase include, but are not limited to, polystyrene resin particles, nylon resin particles, glass particles, glass plates, polystyrene microplates, latex particles, various magnetic particles, metal particles, metal-coated particles, metal plates, metal-coated plates, various porous bodies, various electrodes, etc. Among these, glass plates such as slide glasses are preferably used.

[0022] In this embodiment, a solid phase that has been surface-modified to strengthen the bond between the solid phase and the biological sample is preferably used. The bond between the solid phase and the biological sample is not particularly limited as long as it does not inhibit the specificity of the ligand protein for the target in the biological sample. Examples of surface modification of the solid phase include addition of functional groups such as amino groups, carboxyl groups, thiol groups, disulfide groups, and hydroxyl groups, or addition of compounds having specific amino acid sequences.

[0023] Furthermore, a solid phase that has been subjected to a hydrophilization treatment to suppress non-specific adsorption is also preferably used in this embodiment.

[0024] <Amino acids> The term "amino acid" refers to a compound having an amino group and a carboxyl group at the carbon atom and its derivatives. In this embodiment, the amino acid is not particularly limited, and may be any of hydrophilic amino acids, hydrophobic amino acids, neutral amino acids, acidic amino acids, basic amino acids, branched chain amino acids, aromatic amino acids, sulfur-containing amino acids, etc., and the molecular weight is not limited, and the D-form or L-form is not limited. Preferably, the 20 types of amino acids constituting proteins and peptides constituting living organisms are alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and salts thereof.

[0025] More preferred among the amino acids are arginine, lysine, glycine, proline, glutamic acid, aspartic acid, valine, threonine, alanine, and salts thereof, and among these, particularly preferred are arginine, lysine, glycine, and salts thereof. The amino acids used in the present invention may be of either L- or D-configuration.

[0026] Examples of salts include alkali metal salts such as sodium and potassium, and ammonium salts for acidic to neutral amino acids. Examples of salts include hydrochlorides, sulfates, phosphates, nitrates, carboxylates containing fatty acids such as acetic acid and lauric acid, and oxalates for neutral to basic amino acids. Preferable salts include sodium salts, potassium salts, and ammonium salts for acidic amino acids, sodium salts and potassium salts for neutral amino acids, and hydrochlorides, acetates, and fatty acid salts for basic amino acids.

[0027] <Surfactant> The surfactant is used without any particular limitation. Examples include anionic surfactants such as sodium alkylsulfonate, sodium alkylbenzenesulfonate, sodium dialkylsulfosuccinate, and sodium alkylcarboxylate; nonionic surfactants such as acetylene glycol surfactants represented by formula (1), polyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene alkyl esters, polyoxyethylene polyoxypropylene glycols, sorbitan alkyl esters, polyoxyethylene sorbitan alkyl esters, glycerin alkyl esters, alcohol alkoxylates, and polyoxyethylene hydrogenated castor oil; cationic surfactants such as alkyltrimethylammonium chloride, alkyltrimethylammonium bromide, dialkyldimethylammonium chloride, and alkylbenzyldimethylammonium chloride; and amphoteric surfactants such as alkylbetaine, alkyldimethylamine oxide, and cholate derivatives. Furthermore, polymeric surfactants such as sodium salt of naphthalenesulfonic acid formalin condensate and sodium polyacrylate can be used.

[0028] It is also possible to use a radical polymerizable compound to which an ionic group such as a sulfonic acid group, a carboxylic acid group, or an amino group, or a hydrophilic nonionic group such as a polyoxyethylene group or a polyglyceryl group, has been bonded to impart surface activity.

[0029] As the surfactant, an acetylene glycol surfactant having a structure represented by formula (1) is particularly preferred. Commercially available surfactants represented by formula (1) include Acetylenol E13T, E40, E60, E100, and E200 (all manufactured by Kawaken Fine Chemicals), and Sainylon 465 and 485 (all manufactured by Nissin Chemical Industry Co., Ltd.).

[0030] The surfactant represented by formula (1) is considered to be capable of quickly orienting and adsorbing to the interface between a heating element, such as a heater, and a liquid composition, and thus inhibits the adsorption of proteins to the heating element. The surfactant represented by formula (1) is also considered to be capable of quickly orienting and adsorbing to proteins that are attached to the heating element and have exposed hydrophobic surfaces. Due to this action, the surfactant represented by formula (1) is considered to inhibit the deposition of proteins on the heating element and contribute to stabilizing ejection. [ka] (In the formula (1), x and y satisfy the relationship 1.0≦x+y≦30.0.)

[0031] The amount of the surfactant added is preferably from 0.01% to 3% by mass, and more preferably from 0.05% to 0.40% by mass, based on the total mass of the liquid composition.

[0032] <Inkjet> The method for applying a liquid composition in this embodiment is characterized by having a step of ejecting the liquid from a liquid ejection head of an inkjet system (also simply called inkjet) and applying the liquid to a biological sample.

[0033] Examples of the liquid ejection head include a type that ejects the liquid composition by generating film boiling in the liquid composition using an electro-thermal converter to form bubbles, a type that ejects the liquid composition using an electro-mechanical converter, and a type that ejects the liquid composition using static electricity. Among these, from the viewpoint of performing high-speed, high-density printing, a liquid ejection head that uses an electro-thermal converter, i.e., has a heating element that imparts thermal energy to the liquid composition, is preferred.

[0034] In an inkjet method in which a liquid composition is discharged from a liquid discharge head by the action of thermal energy, when a liquid composition containing a protein comes into contact with a heating element, the protein may adhere to the heating element. If this phenomenon continues, the protein accumulates on the heating element. As a result, the action of thermal energy is not easily transmitted to the liquid composition, which may cause a decrease in discharge volume, discharge failure, etc.

[0035] <Proteins with specificity for their target> In this embodiment, the liquid composition contains a ligand protein having specificity for a target. In this specification, a molecule having specificity for a target is referred to as a ligand, and a protein having specificity for a target is referred to as a ligand protein.

[0036] Furthermore, "having specificity for a target" means that the dissociation constant with the target is 1 μM or less. The ligand protein used for staining is not particularly limited as long as it has a dissociation constant with the target of 1 μM or less, and examples of the ligand protein include enzymes, antibodies, antigen proteins, receptors, cytokines, hormones, serum proteins, and the like.

[0037] The ligand protein may be any known protein other than those mentioned above, or may be a novel protein. As the ligand protein used for staining in this embodiment, an antibody or an antibody fragment is particularly preferred from the viewpoints of a low dissociation constant and molecular structural stability.

[0038] <Antibodies and antibody fragments> An antibody is a general term for a family of immunoglobulins induced by the immune system in response to a specific antigen or substance, and is a substance capable of recognizing and binding to a specific target. Antibodies can be obtained from various animal species, such as mice, rabbits, goats, camels, and humans. Humanized antibodies, chimeric antibodies, and the like can also be used. Antibodies may be either monoclonal or polyclonal.

[0039] An antibody fragment is a part of an antibody that can specifically bind to a target molecule. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2, heavy chain variable (VH) domains alone, light chain variable (VL) domains alone, VH and VL complexes, camelized VH domains, peptides containing antibody complementarity determining regions (CDRs), and single chain antibodies (scfv) in which the heavy chain variable region and the light chain variable region are linked together.

[0040] <Labeling substance> The ligand protein is preferably modified with a labeling substance. By being modified with a labeling substance, the location of the target in a biological sample can be confirmed after staining. Here, "modification" refers to physically adsorbing the ligand protein and the labeling substance, chemically binding the ligand protein and the labeling substance, or both.

[0041] The modification of the ligand protein with a labeling substance can be carried out by physical adsorption, chemical binding, or a combination of these methods. The physical adsorption method includes a method in which a ligand protein and a labeling substance are mixed and contacted in a solution such as a buffer solution, etc. For example, when the labeling substance is gold colloid or latex, the physical adsorption method is effective, and a gold colloid-labeled ligand protein can be obtained by mixing and contacting the ligand protein and gold colloid in a buffer solution. Examples of chemical binding methods include a method in which the ligand protein and the labeling substance are mixed and contacted with a bivalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide, and reacted with amino groups, carboxyl groups, thiol groups, aldehyde groups, or hydroxyl groups of both the antibody and the labeling substance. Another method includes a method in which a derivative is prepared by binding a carbodiimide to the labeling substance, and then reacted with an amino group of the ligand protein. For example, when the labeling substance is a fluorescent substance, an enzyme, or a chemiluminescent substance, the chemical binding method is effective.

[0042] Examples of labeling substances include low molecular weight compounds, fine particles, enzymes, fluorescent dyes, and fluorescent proteins. Specific examples of low molecular weight compounds include biotin, digoxigenin, and dinitrophenyl. Specific examples of fine particles include gold colloids, ferrite particles, latex beads, ferrite-containing latex beads, fluorescent substance-containing latex beads, and agarose beads. Specific examples of enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, luciferase, and the like. Specific examples of fluorescent dyes include Cy3, Cy5, Texas Red, fluorescein, indocyanine green, and Alexa dyes (e.g., Alexa568). Rare earth fluorescent complexes such as europium complexes can also be used. Specific examples of fluorescent proteins include GFP (green fluorescent protein) and its derivatives, R-Phycoerythrin, and Allop hycocyanin. Among them, the labeling substance is preferably at least one selected from the group consisting of biotin, an enzyme, a fluorescent dye, and a fluorescent protein. That is, the ligand protein is preferably modified with at least one labeling substance selected from the group consisting of biotin, an enzyme, a fluorescent dye, and a fluorescent protein. The labeling substance may be a mixture of two or more of the above-listed materials, or may be a material other than the above-listed materials.

[0043] <Blocking agent> When staining a biological sample with a ligand protein, a so-called blocking agent can be used to suppress non-specific adsorption of the ligand protein to areas other than the target. By adding a blocking agent to the liquid composition together with the ligand protein, good staining results can be obtained. Examples of blocking agents include bovine serum albumin, casein, animal serum, gelatin, skim milk, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, phospholipids, and compounds containing them. Among them, the blocking agent is preferably at least one selected from the group consisting of bovine serum albumin and casein. In addition, commercially available blocking agents can also be used. Among them, the content of the blocking agent in the liquid composition is preferably 0.1% by mass or more and 10% by mass or less based on the total mass of the liquid composition. When a protein is used as the blocking agent, taking into consideration the ejection by the inkjet method, the content of the total protein including the ligand protein in the liquid composition is preferably 0.001% by mass or more and 3% by mass or less based on the total weight of the liquid composition, and more preferably 0.001% by mass or more and 0.3% by mass or less.

[0044] <Water-soluble organic solvent> The liquid composition contains a ligand protein, at least one selected from amino acids and their salts, a surfactant, and water. Furthermore, a water-soluble organic solvent can be added to the liquid composition as necessary for the purpose of stabilizing the discharge. Examples of the water-soluble organic solvent include alcohols, polyalkylene glycols, glycol ethers, nitrogen-containing compounds, and sulfur-containing compounds. Specific examples include glycerin (290°C), dimethyl sulfoxide (DMSO) (189°C), ethylene glycol (197°C), diethylene glycol (245°C), polyethylene glycol with an average molecular weight of 600 (200°C or higher), propylene glycol (187°C), triethylene glycol (244°C), 1,2-pentanediol (187°C), 1,2-hexanediol (223°C), ethylene glycol monobutyl ether (171°C), diethylene glycol monobutyl ether (180°C), 2-pyrrolidone (245°C), triethanolamine (208°C), and thiodiglycol (282°C). The boiling points are shown in parentheses.

[0045] In this embodiment, it is preferable to use a water-soluble organic solvent having a boiling point of 180° C. or more. By including an organic solvent having a boiling point of 180° C. or more, evaporation of liquid components from the discharge port can be effectively suppressed. Two or more types selected from these can also be used in combination. The content of the water-soluble organic solvent in the liquid composition is preferably from 5% by mass to 30% by mass, and more preferably from 10% by mass to 20% by mass, based on the total mass of the liquid composition.

[0046] <Second liquid composition> The staining method in this embodiment may further include a step of applying a second liquid composition to the biological sample to which the first liquid composition has been applied. The second liquid composition contains a protein (second protein) having specificity for the ligand protein (first protein) contained in the liquid composition (first liquid composition) described above. The term "having specificity for the ligand protein" means that the dissociation constant with the ligand protein is 1 μM or less. For example, if the ligand protein contained in the first liquid composition is a rabbit-derived antibody, a goat-derived anti-rabbit antibody can be used as the ligand protein contained in the second liquid composition. Also, for example, if the ligand protein contained in the first liquid composition is a biotin-labeled rabbit-derived antibody, not only a goat-derived anti-rabbit antibody but also avidin or streptavidin can be used as the ligand protein of the second liquid composition.

[0047] The second protein is preferably modified with the labeling substance described above. By using the second protein modified with this labeling substance, it is not necessary to modify the first protein with a labeling substance, and it is possible to use a first protein whose specificity to a target would be reduced by modification with a labeling substance. The second protein is preferably modified with at least one selected from the group consisting of biotin, an enzyme, a fluorescent dye, and a fluorescent protein.

[0048] The method of applying the second liquid composition may be the inkjet method used for applying the first liquid composition, that is, a method of ejecting the second liquid composition from a liquid ejection head of the inkjet system. In addition, a method of applying the second liquid composition by contact using a gravure roll or a blade, or a method of applying the second liquid composition by dropping using a spray or a pipette may be used. From the viewpoint of not damaging the biological sample, a non-contact method for applying the antibody is preferable.

[0049] <Substrate> When enzymes such as horseradish peroxidase and alkaline phosphatase are used as labeling substances, color can be developed using a color-developing substrate that reacts with the enzyme. It is also possible to use a combination of an enzyme and its color-developing / luminescent substrate. For example, luciferase, horseradish peroxidase, and alkaline phosphatase are examples of enzymes. Color can be developed by combining the following color-developing agents as the substrates. Examples include luciferin, 3,3'-diaminobenzidine (DAB), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 3,3'-(3,3'-dimethoxy-4,4'-biphenylene)bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride] (NBT), etc.

[0050] Therefore, in the case where the ligand protein is modified with an enzyme, the staining method according to the present embodiment preferably further comprises a step of applying an enzyme substrate to the biological sample to which the liquid composition containing the ligand protein has been applied. In addition, in the case where the staining method according to the present embodiment comprises a step of applying a first liquid composition to the biological sample and a step of applying a second liquid composition, and the first protein (ligand protein) contained in the first liquid composition is not modified with an enzyme and the second protein is modified with an enzyme, it is preferable to further comprise a step of applying an enzyme substrate to the biological sample to which the first liquid composition and the second liquid composition have been applied. In addition, the enzyme substrate may be contained in a third liquid composition.

[0051] <Buffer> A buffer solution can be used as a solvent for the liquid composition. Examples of buffer solutions include, but are not limited to, phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer. The use of a buffer solution suppresses pH changes and improves the storage stability of the ligand protein.

[0052] The liquid composition may also contain a salt. Examples of the salt include, but are not limited to, sodium salts such as sodium chloride, potassium salts such as potassium chloride, and magnesium salts such as magnesium chloride. By adjusting the salt concentration, non-specific adsorption other than the binding between a target and a ligand protein having specificity for the target can be suppressed.

[0053] <Cleaning process> It is preferable to include a washing step in which, after providing a ligand protein having specificity for a target to a biological sample, the ligand protein that has not reacted with the biological sample is separated from the sample. By carrying out the washing step, coloring or the like caused by the ligand protein remaining in places other than the target can be suppressed, and the position of the target can be clearly observed after staining.

[0054] <Other> In addition to the above components, the liquid composition may contain various additives, such as antifoaming agents, surfactants, pH adjusters, viscosity adjusters, rust inhibitors, preservatives, antifungal agents, antioxidants, reduction inhibitors, and chelating agents, as necessary. In addition to the above components, the liquid composition may contain water-soluble organic solvents that are solid at 25° C., such as urea or a derivative thereof, polyethylene glycol having an average molecular weight of 1000 or more, trimethylolpropane, and trimethylolethane.

[0055] In addition, as a second embodiment, the present invention provides a liquid composition containing a ligand protein having specificity for a target in a biological sample, at least one selected from amino acids and their salts, a surfactant, and water. In addition, as a third embodiment, the present invention provides a kit for staining a biological sample using an inkjet system, which includes a liquid composition containing a ligand protein having specificity for a target in a biological sample, at least one selected from amino acids and their salts, a surfactant, and water.

[0056] In the kit, the liquid composition containing the ligand protein having specificity for the target, at least one selected from amino acids and their salts, a surfactant, and water may be mixed or separate, and may be contained in a container separately or mixed together. The kit may also include an additional diluent, a blocking agent, a positive control, a negative control, instructions, and the like. Examples of the positive control include tissue sections and liquid samples that clearly contain a measurable target. Furthermore, the kit may also include a cartridge container that can be attached to an inkjet ejection head. EXAMPLES

[0057] The present embodiment will be described in more detail below using examples and comparative examples. The present invention is not limited by the following examples without departing from the gist of the present invention. In the following description of the examples, "parts" are by mass unless otherwise specified.

[0058] <Biological sample 1> A HER2-IHC positive control slide (PS-17001, Pathology Institute) was used as biological sample 1. Four types of cultured cell lines were attached to this commercially available slide according to the expression level of HER2 protein: negative (0), weakly positive (1+), moderately positive (2+), and strongly positive (3+).

[0059] <Preparation of Buffer Solution> A 50mM Tris·base solution and a 150mM sodium chloride solution were mixed, adjusted to pH 7.6 with hydrochloric acid, and then pressure filtered through a 0.22μm pore size sterile filter to prepare a buffer solution. This buffer solution is hereafter referred to as TBS buffer. Various ligand proteins, various surfactants, and solid solvents were dissolved in this buffer solution before preparation.

[0060] <Ligand protein> Polyclonal rabbit anti-human HER2 antibody (A0485, Dako) was used as the ligand protein 1. HER2 is human epidermal growth factor receptor 2.

[0061] As ligand protein 2, a monoclonal rabbit anti-human HER2 antibody (4290, Cell Signaling Technology) was used.

[0062] As the ligand protein 3, a polyclonal rabbit-derived anti-human HER2 antibody (A0485, Dako) was digested with pepsin to obtain F(ab′)2 labeled with FITC. F(ab′)2 was prepared according to the following protocol. First, the antibody was dialyzed against 0.1 M sodium acetate buffer (pH 3.75). Then, porcine gastric mucosa-derived pepsin-immobilized agarose (Sigma product) was added and reacted at 37°C for 3 hours. Then, 0.5 M Tris buffer (pH 8.0) was added to adjust the pH to 7.0 to stop the reaction. The pepsin-immobilized agarose was removed by centrifugation. Furthermore, the solution was passed through a Protein A-immobilized agarose column (Thermo Fisher Scientific). Dialysis was performed against TBS buffer at room temperature for 2 hours to obtain an F(ab′)2 solution. Furthermore, FITC-labeled F(ab')2 was prepared using Fluorescein Labeling Kit-NH2 (trade name, Dojindo Laboratories) according to the manufacturer's protocol.

[0063] A polyclonal rabbit anti-human HER2 antibody (A0485, Dako) labeled with biotin was used as the ligand protein 4. Biotin labeling was performed using Biotin Labeling Kit-NH2 (trade name, Dojindo Laboratories) according to the manufacturer's protocol to prepare a biotin-labeled polyclonal rabbit anti-human HER2 antibody.

[0064] A polyclonal rabbit-derived anti-human HER2 antibody (A0485, manufactured by Dako) labeled with horseradish peroxidase was used as the ligand protein 5. Enzyme labeling was performed using Peroxidase Labeling Kit-NH2 (trade name, Dojindo Laboratories) according to the manufacturer's protocol to prepare a polyclonal rabbit-derived anti-human HER2 antibody labeled with horseradish peroxidase.

[0065] A polyclonal rabbit anti-human HER2 antibody (A0485, Dako) labeled with alkaline phosphatase was used as the ligand protein 6. Enzyme labeling was performed using Alkaline Phosphatase Labeling Kit-NH2 (trade name, Dojindo Laboratories) according to the manufacturer's protocol to prepare an alkaline phosphatase-labeled polyclonal rabbit anti-human HER2 antibody.

[0066] As ligand protein 7, a FITC-labeled polyclonal rabbit anti-human HER2 antibody (A0485, Dako) was prepared using Fluorescein Labeling Kit-NH2 (trade name, Dojindo Laboratories) according to the manufacturer's protocol.

[0067] As ligand protein 8, a polyclonal rabbit anti-human HER2 antibody (A0485, Dako) was used with the R-Phycoerythrin Labeling Kit-NH2 (trade name, Dojindo Laboratories) according to the manufacturer's protocol to prepare an R-phycoerythrin-labeled polyclonal rabbit anti-human HER2 antibody.

[0068] As the ligand protein 9, Dual Link System-HRP (HRP-labeled polymer reagent) (K4063, Dako) was prepared and used as it was.

[0069] As the ligand protein 10, FITC-labeled streptavidin (Funakoshi Co., Ltd.) was prepared.

[0070] As the ligand protein 11, biotin-labeled goat anti-rabbit IgG (Funakoshi Co., Ltd.) was prepared.

[0071] As the ligand protein 12, HRP-labeled goat anti-rabbit IgG (Funakoshi Co., Ltd.) was prepared.

[0072] As the ligand protein 13, FITC-labeled goat anti-rabbit IgG (Funakoshi Co., Ltd.) was prepared.

[0073] As the ligand protein 14, R-Phycoerythrin-labeled goat anti-rabbit IgG (Funakoshi Co., Ltd.) was prepared.

[0074] <Proteins other than ligand proteins (blocking agents)> As a protein other than the ligand protein, BSA (bovine serum albumin, Sigma) was used.

[0075] <Surfactant> The following surfactants (a) to (h) were used. (a) A-E100: Acetylenol E100 (Kawaken Fine Chemicals) In formula (1), this is a compound where x+y=10. (b) A-E40: Acetylenol E100 (Kawaken Fine Chemicals) In formula (1), this compound has x+y=4. (c) Tween 80 (Tokyo Chemical Industry Co., Ltd.) It has a polyoxyethylene sorbitan alkyl ester structure and is not a surfactant represented by formula (1). (d) BIО-SОFT N91-8 (manufactured by Stepan) It has an alcohol alkoxylate structure and is not a surfactant represented by formula (1). (e) Triton-X100 (Merck) It has a polyoxyethylene alkylphenyl ether structure and is not a surfactant represented by formula (1). (f) SDS: sodium dodecyl sulfate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) It is not a surfactant represented by formula (1). (g) CTAB: cetyltrimethylammonium bromide (Tokyo Chemical Industry Co., Ltd.) This has an alkyltrimethylammonium bromide structure and is not a surfactant represented by formula (1). (h) CHAPS: 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonic acid (manufactured by Dojindo Laboratories) It is an amphoteric surfactant derived from a cholate salt, and is not a surfactant represented by formula (1).

[0076] <Substrate> The following substrates were used: (a) Liquid DAB+ (product name K3465, manufactured by Dako) (b) BCIP-NBT solution kit (Nacalai Tesque, Inc.)

[0077] <Preparation of First Liquid Composition> As the first liquid composition, A-1 to A-49 were prepared by mixing each component so as to have the content shown in Table 1-1, Table 1-2, Table 1-3, and Table 1-4. The numbers in the tables indicate the solid content (mass%).

[0078] [Table 1-1]

[0079] [Table 1-2]

[0080] [Table 1-3]

[0081] [Table 1-4]

[0082] <Preparation of second liquid composition> As second liquid compositions, B-1 to B-6 were prepared by mixing the components so as to obtain the contents shown in Table 2. The numbers in the table indicate the solid contents. As for the second liquid composition B-1, the one supplied by the manufacturer was used as it was, and this is noted in Table 2.

[0083] [Table 2]

[0084] <Preparation of third liquid composition> As the third liquid composition, C-1 to C-3 were prepared. C-1 and C-2 were prepared by preparing each substrate according to the manufacturer's protocol, and then mixing each component to the content shown in Table 3. The numbers in the table indicate the solid content. As C-3, the same one prepared as the second liquid composition, B-2, was used. Note that neither C-1 nor C-2 contained a ligand protein.

[0085] [Table 3]

[0086] <Biological sample 2> A Ki67-IHC positive control slide (PS-17004, Pathology Research Institute) was used as biological sample 2. Two types of samples with different levels of Ki67 protein expression, a negative "-" porcine liver tissue and a strongly positive "+" cell line embedded sample, were attached to this commercially available slide.

[0087] <Ligand protein> Monoclonal mouse anti-human Ki-67 antibody (M7240, Dako) was used as ligand protein 15. Ki-67 is a protein expressed by a gene present on the long arm of chromosome 10, and is used as a cell proliferation marker because it is expressed in all cell nuclei except those in the resting phase. As the ligand protein 16, HRP-labeled goat anti-mouse IgG (Funakoshi Co., Ltd.) was prepared.

[0088] <Proteins other than ligand proteins (blocking agents)> As a protein other than the ligand protein, BSA (bovine serum albumin, Sigma) was used.

[0089] <Surfactant> The surfactants shown in (a) below were used. (a) A-E100: Acetylenol E100 (Kawaken Fine Chemicals)

[0090] <Substrate> The following substrates were used: (a) Liquid DAB+ (product name K3465, manufactured by Dako) (b) BCIP-NBT solution kit (Nacalai Tesque, Inc.)

[0091] <Preparation of First Liquid Composition> The first liquid composition A-50 contains 0.001% by mass of ligand protein 15, 0.5% by mass of BSA, 1% by mass of lysine, 10% by mass of glycerin, and surfactant A-E100. was adjusted to a concentration of 0.1% by mass with TBS buffer.

[0092] <Preparation of second liquid composition> The second liquid composition B-7 contains 0.001% by mass of ligand protein 16, 0.5% by mass of BSA, 1% by mass of lysine, 10% by mass of glycerin, and surfactant A-E100. was adjusted to a concentration of 0.1% by mass with TBS buffer.

[0093] (Discharge volume evaluation) A liquid ejection head using a thermal jet method with a nozzle diameter of 3 μm was prepared, and a tank connected to it was filled with BC-345Bk ink (Canon).The ejection head was driven by a controller electrically connected to the liquid ejection head, and liquid was ejected from the ejection port at a frequency of 15 kHz and a voltage of 13 volts.The ejected liquid was observed with an ultra-high speed camera, and the volume was calculated by the ligament method, which was set as 100%. Next, the liquid compositions of Liquids A-1 to A-50, Liquids B-1 to B-7, and Liquids C-1 and C-2 were ejected under the same conditions, and the ejection volumes of the ejected liquids were evaluated according to the following criteria. (Evaluation Criteria) A: 95% or more B: 90% or more but less than 95% C: 70% or more but less than 90% D: 40% or more but less than 70% E: Less than 40%

[0094] Table 4 shows the evaluation results of the ejection volumes of the liquid compositions A-1 to A-50, B-1 to B-7, and C-1 to C-2.

[0095] [Table 4]

[0096] <Evaluation of dye gradation> Each liquid composition was used to stain cells. As a result of the observation, the dye gradation was evaluated according to the following criteria. The evaluation results are shown in Tables 5-1, 5-2, 5-3, and 5-4. When biological sample 1 was used, the evaluation was performed according to the following criteria. A: HER2 expression levels of "0", "1+", "2+", and "3+" were stained clearly in stages, making it easy to judge. B: Staining was performed in stages according to the amount of HER2 expression. C: HER2 stains in a graded sequence from "0" to "2+" to "3+", but the difference between "0" and "1+" is unclear, making it difficult to determine whether it was negative or weakly positive. D: HER2 "0" and "3+" stain in a gradual manner, but the difference between "0", "1+", and "2+" is unclear, making it difficult to determine whether it is negative, weakly positive, or moderately positive. E: The difference between HER2 "0", "1+", "2+", and "3+" was unclear and could not be determined at all. When biological sample 2 was used, the evaluation was performed according to the following criteria. A: It was easy to determine whether Ki67 was "-" or "+" staining. E: It was difficult to determine whether Ki67 was “-” or “+” staining. The evaluation results of dye gradation are shown in Tables 5-1, 5-2, 5-3, 5-4, and 5-5.

[0097] [Table 5-1]

[0098] [Table 5-2]

[0099] [Table 5-3]

[0100] [Table 5-4]

[0101] [Table 5-5]

[0102] The deparaffinization process for biological samples 1 and 2, the antigen activation process for biological samples 1 and 2, the blocking process for biological samples 1 and 2, the permeabilization process for biological sample 2, the liquid application, and the observation were carried out as follows.

[0103] (1) Deparaffinization of Biological Samples 1 and 2 a) Biological samples 1 and 2 were immersed in a container containing a xylene substitute, Clear Plus (Pharma Co., Ltd.), and left to stand at room temperature for 5 minutes. This operation was repeated twice. b) The specimen was immersed in a container containing 99.5% ethanol (Kishida Chemical Co., Ltd.) and left to stand at room temperature for 3 minutes. This procedure was repeated twice. c) The specimen was immersed in a container containing 95% ethanol and left to stand at room temperature for 5 minutes. This procedure was repeated twice. d) The specimen was immersed in a container containing pure water and allowed to stand at room temperature for 1 minute.

[0104] (2) Antigen retrieval treatment of biological samples 1 and 2 a) Biological samples 1 and 2 were immersed in Target Retrieval Solution, pH 9.0 (Dako) heated to 96-98°C and heated for 40 minutes. After heating, the samples were allowed to cool to room temperature. b) Biological samples 1 and 2 were immersed in a container containing TBS buffer and allowed to stand at room temperature for 3 minutes. This operation was repeated twice.

[0105] (3) Blocking treatment of biological samples 1 and 2 a) 100 μL of peroxidase blocking reagent (Peroxidase-Blocking Solution Dako REAL (Dako)) was dropped onto biological samples 1 and 2, and the samples were allowed to stand in a humidified box for 5 minutes. b) Biological samples 1 and 2 were immersed in a container containing TBS buffer and allowed to stand at room temperature for 3 minutes. This operation was repeated twice. The subsequent steps were carried out in different ways for each Example.

[0106] (4) Permeabilization of Biological Sample 2 The biological sample 2 was immersed in TBS buffer containing 0.2% by mass of a surfactant (e) and allowed to stand at room temperature for 15 minutes. The following steps were carried out in common for Examples 1 to 52 and Comparative Examples 1 to 4.

[0107] (1) Applying a first liquid composition to biological samples 1 and 2 a) In biological sample 1, 1 cm x 1 cm CT images were placed around four types of cultured cell lines with different levels of HER2 protein expression, and in biological sample 2, 1 cm x 1 cm CT images were placed around two types of cultured cell lines with different levels of Ki-67 protein expression. 2 The frame was made using a PapPen (Liquid Blocker, manufactured by Daido Sangyo Co., Ltd.). b) As shown in Tables 5-1 to 5-5, the first liquid composition was ejected by inkjet and dropped onto biological samples 1 and 2. Specifically, the first liquid composition was filled into an ink tank for an inkjet printer (product name: TS-203, manufactured by Canon Inc.) and attached to a print head. The ejection volume of one droplet was measured in advance by ejection volume evaluation, and then the ejection volume of one droplet was measured by 1 cm 2 The first liquid composition was dropped into the frame made with a poultice pen under conditions set so that the amount of drop was 30 μL per drop. After the dropping, the mixture was left to stand in a humid box for 60 minutes. c) After a predetermined time had elapsed, the biological samples 1 and 2 were immersed in a container containing TBS buffer and left to stand at room temperature for 3 minutes. This operation was also repeated twice.

[0108] (2) Application of second liquid composition a) As shown in Tables 5-1 to 5-5, the second liquid composition was applied to biological samples 1 and 2 using a dab pen to form a frame. The ejection volume of one droplet was measured in advance by the ejection volume evaluation, and then the ejection volume was measured by 1 cm 2 The second liquid composition was dropped under conditions set so that 50 μL per drop was added. After dropping, the mixture was allowed to stand in a humidified box for 30 minutes. In Tables 5-1 to 5-5, "unused" means that the above operation was not carried out. b) Biological samples 1 and 2 were immersed in a container containing TBS buffer and allowed to stand at room temperature for 3 minutes. This procedure was repeated twice.

[0109] (3) Application of the third liquid composition a) As shown in Tables 5-1 to 5-5, the third liquid composition was applied to a biological sample 1 using a dab pen within a frame prepared by measuring the discharge volume of one droplet in advance by the discharge volume evaluation, and then the third liquid composition was applied to a frame within 1 cm 2The third liquid composition was dropped under conditions set so that 50 μL per drop was added, and the mixture was allowed to stand for 5 minutes in a humidified box. In Tables 5-1 to 5-5, "unused" means that the above procedure was not carried out. b) The biological sample 1 was immersed in a container containing water that had been pressure-filtered through a sterile filter with a pore size of 0.22 μm, and allowed to stand at room temperature for 3 minutes. This operation was also repeated twice.

[0110] (4) Observation A microscope BZ-X810 (Keyence Corporation) was used for Examples 1, 2, 5, 6, 9 to 45, and 47, and Comparative Examples 1 to 4. Images were acquired using a 40x objective lens compatible with bright field observation. In Examples 3, 4, 7, 8, 46, and 48 to 52, a 40x objective lens compatible with fluorescence observation and a BZ-X filter GFPOP-87763 (Keyence Corporation, excitation wavelength 470 nm, detection wavelength 525 nm), which is a fluorescence filter unit, were used to obtain a fluorescence image of the biological sample 1. Next, a phase contrast image of the biological sample 1 was obtained using an objective lens compatible with phase contrast observation and a condenser for phase contrast observation. Next, a green substitute color was applied to the obtained fluorescence image, and a gray substitute color was applied to the phase contrast image. After that, the two images were superimposed by an additive synthesis method to generate one synthetic image. In Example 8, a fluorescent image of the biological sample 1 was acquired using a 40x objective lens compatible with fluorescent observation and a fluorescent filter unit, BZ-X filter TRITCOP-87764 (Keyence Corporation, excitation wavelength 545 nm, detection wavelength 605 nm). Next, a phase contrast image of the biological sample 1 was acquired using an objective lens compatible with phase contrast observation and a condenser for phase contrast observation. Next, an orange substitute color was applied to the acquired fluorescent image, and a gray substitute color was applied to the phase contrast image. After that, the two images were superimposed by additive synthesis to generate one composite image.

[0111] The present invention includes the following methods and compositions. (Method 1) a ligand protein having specificity for a target in a biological sample; At least one selected from amino acids and salts thereof; Surfactant and water A staining method comprising the steps of: discharging a liquid composition containing the above-mentioned formula (1) from an inkjet liquid discharge head and applying the liquid composition to the biological sample. (Method 2) The staining method according to method 1, wherein the ligand protein comprises an antibody or an antibody fragment. (Method 3) The staining method according to Method 1 or 2, wherein the ligand protein is modified with at least one selected from the group consisting of biotin, an enzyme, a fluorescent dye, and a fluorescent protein. (Method 4) 4. The staining method according to any one of methods 1 to 3, wherein the ligand protein is modified with an enzyme, and the staining method further comprises a step of applying a substrate for the enzyme to the biological sample to which the liquid composition has been applied. (Method 5) The dyeing method according to any one of Methods 1 to 4, wherein the liquid composition further contains at least one selected from the group consisting of bovine serum albumin and casein. (Method 6) The staining method according to any one of Methods 1 to 5, wherein the content of the ligand protein in the liquid composition is 0.001% by mass or more and 3% by mass or less, based on the total mass of the liquid composition. (Method 7) The dyeing method according to any one of Methods 1 to 6, wherein the surfactant has a structure represented by the following formula (1): [ka] (In the formula (1), x and y satisfy the relationship 1.0≦x+y≦30.0.) (Method 8) The staining method according to any one of Methods 1 to 7, wherein the content of the ligand protein in the liquid composition is 0.001% by mass or more and 0.3% by mass or less, based on the total mass of the liquid composition. (Method 9) 9. The dyeing method according to any one of methods 1 to 8, wherein the amino acid comprises at least one selected from the group consisting of arginine, lysine, glycine, proline, glutamic acid, aspartic acid, valine, threonine, and alanine. (Method 10) 10. The staining method according to any one of methods 1 to 9, wherein the amino acid comprises at least one selected from the group consisting of arginine, lysine, and glycine. (Method 11) 11. The method according to any one of Methods 1 to 10, wherein the liquid composition contains a water-soluble organic solvent. (Method 12) 12. The staining method according to any one of methods 1 to 11, further comprising the step of applying a second liquid composition containing a second protein having specificity for the ligand protein to the biological sample to which the liquid composition has been applied. (Method 13) 13. The staining method according to claim 12, wherein the second protein is modified with at least one selected from the group consisting of biotin, an enzyme, a fluorescent dye, and a fluorescent protein. (Method 14) The staining method according to method 12 or 13, wherein the second protein is modified with an enzyme, and further comprises a step of applying a chromogenic substrate for the enzyme to the biological sample to which the second liquid composition has been applied. (Configuration 1) a ligand protein having specificity for a target in a biological sample; At least one selected from amino acids and salts thereof; Surfactants, and water A liquid composition comprising: (Configuration 2) a ligand protein having specificity for a target in a biological sample; At least one selected from amino acids and salts thereof; Surfactants, and water The liquid composition includes A kit for staining biological samples using the inkjet method. (Configuration 3) The kit according to configuration 2 further includes a cartridge container that can be attached to an inkjet ejection head.

Claims

1. a ligand protein having specificity for a target in a biological sample; At least one selected from amino acids and salts thereof; Surfactant and water A staining method comprising the steps of: discharging a liquid composition containing the above-mentioned formula (1) from an inkjet liquid discharge head and applying the liquid composition to the biological sample.

2. The staining method according to claim 1 , wherein the ligand protein comprises an antibody or an antibody fragment.

3. The staining method according to claim 1 , wherein the ligand protein is modified with at least one selected from the group consisting of biotin, an enzyme, a fluorescent dye, and a fluorescent protein.

4. The staining method according to claim 1 , wherein the ligand protein is modified with an enzyme, and the method further comprises the step of applying a substrate for the enzyme to the biological sample to which the liquid composition has been applied.

5. The dyeing method according to claim 1, wherein the liquid composition further contains at least one selected from the group consisting of bovine serum albumin and casein.

6. The staining method according to claim 1 , wherein the content of the ligand protein in the liquid composition is from 0.001% by mass to 3% by mass, based on the total mass of the liquid composition.

7. The dyeing method according to claim 1, wherein the surfactant has a structure represented by the following formula (1): 【Chemistry 1】 (In the formula (1), x and y satisfy the relationship 1.0≦x+y≦30.0.)

8. The staining method according to claim 1 , wherein the content of the ligand protein in the liquid composition is 0.001% by mass or more and 0.3% by mass or less, based on the total mass of the liquid composition.

9. 2. The method according to claim 1, wherein the amino acid comprises at least one selected from the group consisting of arginine, lysine, glycine, proline, glutamic acid, aspartic acid, valine, threonine, and alanine.

10. 2. The method according to claim 1, wherein the amino acid comprises at least one selected from the group consisting of arginine, lysine, and glycine.

11. The dyeing method according to claim 1 , wherein the liquid composition contains a water-soluble organic solvent.

12. The staining method according to claim 1, further comprising the step of applying a second liquid composition containing a second protein having specificity for the ligand protein to the biological sample to which the liquid composition has been applied.

13. The staining method according to claim 12, wherein the second protein is modified with at least one selected from the group consisting of biotin, an enzyme, a fluorescent dye, and a fluorescent protein.

14. The staining method according to claim 12, wherein the second protein is modified with an enzyme, and the method further comprises the step of applying a chromogenic substrate for the enzyme to the biological sample to which the second liquid composition has been applied.

15. a ligand protein having specificity for a target in a biological sample; At least one selected from amino acids and salts thereof; Surfactants, and water A liquid composition comprising:

16. a ligand protein having specificity for a target in a biological sample; At least one selected from amino acids and salts thereof; Surfactants, and water The liquid composition includes A kit for staining biological samples using the inkjet method.

17. The kit according to claim 16, further comprising a cartridge container that can be attached to an ink-jet ejection head.

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