Staining method, liquid composition for staining, and kit for staining

Abstract

Provided is a method whereby a biological sample can be stained with a staining solution discharged by an inkjet system. Provided is a staining method characterized by comprising a step for discharging a liquid composition containing water, a surfactant, and at least one selected from a ligand protein having specificity to a target in a biological sample, and an amino acid and its salts from a liquid discharge head of an inkjet system and applying the liquid composition to the biological sample.

Description

Dyeing method, liquid composition for dyeing, and kit for dyeing

[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, a liquid composition for staining, and a kit for staining, 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 salts thereof, a surfactant, and water from an inkjet liquid ejection head onto the biological sample, the liquid composition for use in the staining, and the kit for use in the staining.

[0002] With the recent development of molecular biology, the importance of analyzing substances present in living organisms has increased. 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 for analyzing proteins and chemical substances in biological samples such as cells and tissues. In this case, a method for identifying targets such as proteins and chemical substances in a biological sample is used in which the targets are labeled via proteins (ligand proteins) that have specificity for the targets, such as antibodies, and then detected 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 this requires a place to wash and replace the pipette, which makes the device large, so there is a demand for a more compact device.

[0005] U.S. Patent No. 6,277,623 discloses a method for dispensing droplets of reagent between about 1 pL and about 50 pL onto a biological sample, and also discloses a method for dispensing the droplets by inkjet printing.

[0006] Special Publication No. 2018-517895

[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 using 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 sometimes became 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 are capable of depositing a ligand protein having specificity for a target in a biological sample in a desired droplet volume by inkjet ejection, i.e., capable of depositing in an efficient liquid volume.

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

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

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

[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 comprising the steps 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 salts thereof, a surfactant, and water from an inkjet liquid ejection head and applying the liquid composition to the biological sample.

[0013] This section explains the case where a thermal inkjet inkjet system is used. The liquid ejection units have high accuracy in the size of the ejection port diameter, the heat amount of the heat pulse used for ejection, and the microheaters used therefor, allowing for high reproducibility. A large number of liquid ejection units are densely arranged on the head. Meanwhile, as described above, the high reproducibility of the liquid ejection units allows for a narrow droplet size distribution of the ejected liquid across the multiple liquid ejection units. Furthermore, the head is inexpensive to manufacture and highly versatile. For example, there is a demand for compact ejection devices that allow for the appropriate replacement of heads, and this head is highly applicable to such ejection devices. Therefore, a thermal inkjet ejection device is particularly preferable when compactness and convenience are required for the ejection device.

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

[0015] The reason for the significant decrease in ejection volume is unclear, but the inventors speculate as follows. Proteins are generally known to have a higher-order structure in which the hydrophobic portion is folded. When a protein adsorbs to the heater section via this hydrophobic portion, insoluble matter temporarily precipitates upon heating. Some of the insoluble matter does not re-dissolve in water in time for the next heater cycle. The inventors speculate that the significant decrease in ejection volume is due to only a portion of the heater section being heated. Furthermore, there is concern that the physiological activity of the ligand protein, once insolubilized by heating in the heater section, may be reduced.

[0016] In order to efficiently eject a large amount of liquid, the ejection frequency must be at a certain level or higher. 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 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 combining amino acids and surfactants contributes sufficiently to ensuring a sufficient ejection volume is not clear, but it is presumed as follows. Amino acids interact weakly with proteins in water. As a result, proteins are covered with amino acids, which significantly suppresses the precipitation of insoluble matter in the heated heater. Furthermore, the coexisting surfactant can instantly redissolve any small amount of insoluble matter that accumulates on the heater, keeping the heater clean and preventing clogging due to coagulation of proteins that have accumulated in the heater. It is believed that these factors contribute to ensuring a sufficient ejection volume.

[0019] <Biological Sample> 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, cerebrospinal fluid, sweat, saliva, urine, etc.), hair, excrement, organs, tissues, animals and plants themselves, or samples obtained by fixing and embedding them in paraffin, or dried forms thereof. Further examples include river water, lake water, seawater, water supply and sewage water, soil, etc., which contain biological substances and may contain a target on which the specificity of the imparted ligand protein can function.

[0020] The biological sample may be immobilized on a solid phase, which makes it possible to easily remove excess liquid composition after application of the liquid composition, for example, by a washing step.

[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 glass slides 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 its target in the biological sample. Examples of surface modifications of the solid phase include the addition of functional groups such as amino groups, carboxyl groups, thiol groups, disulfide groups, and hydroxyl groups, or the addition of compounds having specific amino acid sequences.

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

[0024] <Amino Acid> The term "amino acid" refers to a compound having an amino group and a carboxyl group at the carbon atom at the center, and a derivative thereof. 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 its molecular weight is not limited, and it is not limited to D- or L-form. Preferably, it is 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, which are 20 types of amino acids constituting proteins and peptides constituting living organisms.

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

[0026] Examples of salts include alkali metal salts such as sodium and potassium, and ammonium salts for acidic to neutral amino acids. Furthermore, examples of salts include hydrochlorides, sulfates, phosphates, nitrates, carboxylic acid salts containing acetic acid and fatty acids such as lauric acid, and oxalates for neutral to basic amino acids. Preferred 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 can be used without any particular limitation. Examples include anionic surfactants such as sodium alkyl sulfonate, sodium alkylbenzene sulfonate, sodium dialkyl sulfosuccinate, and sodium alkyl carboxylate; nonionic surfactants such as acetylene glycol surfactants represented by formula (1), polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene alkyl esters, polyoxyethylene polyoxypropylene glycol, 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 the 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 surfactant properties.

[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 Saiinol 465 and 485 (all manufactured by Nissin Chemical Industry Co., Ltd.).

[0030] The surfactant represented by formula (1) is thought to rapidly orient and adsorb to the interface between a heating element, such as a heater, and a liquid composition, thereby suppressing protein adsorption to the heating element. Furthermore, the surfactant represented by formula (1) is thought to rapidly orient and adsorb to proteins that have adhered to the heating element and have exposed hydrophobic surfaces. Due to this action, the surfactant represented by formula (1) is thought to suppress protein deposition on the heating element and contribute to stabilizing ejection. (In the formula (1), x and y satisfy the relationship 1.0≦x+y≦30.0.)

[0031] The amount of surfactant added is preferably 0.01% by mass or more and 3% by mass or less, and more preferably 0.05% by mass or more and 0.40% by mass or less, 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 referred to as inkjet) and applying it to a biological sample.

[0033] Examples of liquid ejection heads include a type that ejects the liquid composition by generating bubbles by causing film boiling in the liquid composition using an electrothermal converter, a type that ejects the liquid composition using an electromechanical 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 electrothermal converter, i.e., a liquid ejection head having a heating element that imparts thermal energy to the liquid composition, is preferred.

[0034] In an inkjet system that ejects a liquid composition from a liquid ejection 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 may accumulate on the heating element. As a result, the action of thermal energy may be difficult to transmit to the liquid composition, which may cause a decrease in ejection volume, ejection failure, etc.

[0035] <Protein having specificity for a 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 thereof include enzymes, antibodies, antigen proteins, receptors, cytokines, hormones, serum proteins, etc.

[0037] The ligand protein may be any known protein other than those described above, or may be a novel protein. From the viewpoints of a low dissociation constant and molecular structural stability, antibodies or antibody fragments are particularly preferred as the ligand protein used for staining in this embodiment.

[0038] <Antibodies, antibody fragments> Antibodies are a general term for the immunoglobulin family induced by the immune system in response to a specific antigen or substance, and are substances 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, etc. can also be used. Antibodies may be either monoclonal or polyclonal.

[0039] An antibody fragment refers to a portion 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) regions alone, light chain variable (VL) regions 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 light chain variable region are linked together.

[0040] <Labeling substance> The ligand protein is preferably modified with a labeling substance. By modifying 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 bonding 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. Physical adsorption methods include mixing and contacting the ligand protein and labeling substance in a solution such as a buffer solution. For example, physical adsorption is effective when the labeling substance is gold colloid or latex. By mixing and contacting the ligand protein and gold colloid in a buffer solution, a gold colloid-labeled ligand protein can be obtained. Chemical binding methods include mixing and contacting the ligand protein and labeling substance with a bivalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide, and reacting the ligand protein with amino groups, carboxyl groups, thiol groups, aldehyde groups, or hydroxyl groups of both the antibody and the labeling substance. Another method involves preparing a derivative by binding a carbodiimide to the labeling substance and then reacting the derivative with the amino group of the ligand protein. For example, chemical binding is effective when the labeling substance is a fluorescent substance, enzyme, or chemiluminescent substance.

[0042] Examples of labeling substances include low molecular weight compounds, microparticles, enzymes, fluorescent dyes, and fluorescent proteins. Specific examples of low molecular weight compounds include biotin, digoxigenin, and dinitrophenyl. Specific examples of microparticles include gold colloids, ferrite particles, latex beads, ferrite-encapsulated latex beads, fluorescent substance-encapsulated latex beads, and agarose beads. Specific examples of enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and luciferase. Specific examples of fluorescent dyes include Cy3, Cy5, Texas Red, fluorescein, indocyanine green, and Alexa dyes (e.g., Alexa 568). 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 Allophycocyanin. Among these, the labeling substance is preferably at least one selected from the group consisting of biotin, enzymes, fluorescent dyes, and fluorescent proteins. That is, the ligand protein is preferably modified with at least one labeling substance selected from the group consisting of biotin, enzymes, fluorescent dyes, and fluorescent proteins. Furthermore, the labeling substance may be a mixture of two or more of the above-listed materials. Furthermore, materials other than those listed above may also be used.

[0043] <Blocking Agent> When staining a biological sample with a ligand protein, a so-called blocking agent can be used to suppress nonspecific adsorption of the ligand protein to non-target regions. By incorporating a blocking agent into the liquid composition along with the ligand protein, favorable staining results can be achieved. Examples of blocking agents include bovine serum albumin, casein, animal serum, gelatin, skim milk, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, phospholipids, and compounds containing these. Among these, the blocking agent is preferably at least one selected from the group consisting of bovine serum albumin and casein. Commercially available blocking agents can also be used. Among these, 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 ejection by an 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, and more preferably 0.001% by mass or more and 0.3% by mass or less, based on the total mass of the liquid composition.

[0044] <Water-Soluble Organic Solvent> The liquid composition contains a ligand protein, at least one amino acid and its salt, a surfactant, and water. Furthermore, a water-soluble organic solvent can be added to the liquid composition as needed to stabilize ejection. Examples of water-soluble organic solvents 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 higher. By incorporating an organic solvent having a boiling point of 180°C or higher, evaporation of liquid components from the ejection port can be effectively suppressed. Two or more selected from these can also be used in combination. The content of the water-soluble organic solvent in the liquid composition is preferably 5% by mass or more and 30% by mass or less, and more preferably 10% by mass or more and 20% by mass or less, based on the total mass of the liquid composition.

[0046] <Second Liquid Composition> The staining method of 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) that has specificity for the ligand protein (first protein) contained in the liquid composition (first liquid composition) described above. Note that "specificity for the ligand protein" refers to a dissociation constant with the ligand protein of 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. Furthermore, 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 a 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 target specificity 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 for applying the second liquid composition can be the inkjet method used for applying the first liquid composition, i.e., a method in which the second liquid composition is ejected from an inkjet liquid ejection head. Alternatively, a contact application method using a gravure roll or blade, or a non-contact application method using a spray or a drop with a pipette can be used. Non-contact application methods are preferred from the viewpoint of not damaging the biological sample.

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

[0050] Therefore, if the ligand protein is modified with an enzyme, the staining method according to this embodiment preferably further comprises the step of applying a substrate for the enzyme to the biological sample to which a liquid composition containing the ligand protein has been applied. Furthermore, if the staining method according to this embodiment comprises the steps of applying a first liquid composition and applying a second liquid composition to the biological sample, and the first protein (ligand protein) contained in the first liquid composition is not modified with an enzyme but the second protein is modified with an enzyme, the staining method according to this embodiment preferably further comprises the step of applying a substrate for the enzyme to the biological sample to which the first and second liquid compositions have been applied. Furthermore, the enzyme substrate may be contained in a third liquid composition.

[0051] <Buffer Solution> 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 solution, glycine buffer solution, Good's buffer solution, Tris buffer solution, and ammonia buffer solution. Use of a buffer solution suppresses changes in pH and improves the storage stability of the ligand protein.

[0052] The liquid composition may also contain a salt. Examples 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. Adjusting the salt concentration can suppress nonspecific adsorption other than the binding between a target and a ligand protein having specificity for that target.

[0053] <Washing step> After adding a ligand protein specific to a target to a biological sample, it is preferable to include a washing step in which unreacted ligand protein is separated from the biological sample. By performing the washing step, color development due to the ligand protein remaining in places other than the target can be suppressed, and the position of the target can be clearly identified by observation after staining.

[0054] <Others> In addition to the above components, the liquid composition may contain various additives, such as an antifoaming agent, a surfactant, a pH adjuster, a viscosity adjuster, a rust inhibitor, a preservative, an antifungal agent, an antioxidant, an antireducing agent, a chelating agent, etc. In addition to the above components, the liquid composition may also contain a water-soluble organic solvent that is solid at 25°C, such as urea or a derivative thereof, polyethylene glycol having an average molecular weight of 1000 or more, trimethylolpropane, or 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 salts thereof, 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 salts thereof, a surfactant, and water.

[0056] In the kit, a liquid composition containing a ligand protein having specificity for a 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 either individually or mixed together. The kit may also include an additional diluent, a blocking agent, a positive control, a negative control, instructions, etc. Examples of positive controls 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.

[0057] The present embodiment will be described in more detail below using examples and comparative examples. The present invention is not limited to 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 Posicon slide (PS-17001, Pathology Research 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 50 mM Tris-base aqueous solution and a 150 mM sodium chloride aqueous solution were mixed, adjusted to pH 7.6 with hydrochloric acid, and then pressure-filtered through a 0.22 μm pore size sterile filter to prepare a buffer solution. This buffer solution will be referred to as TBS buffer hereafter. 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, manufactured by Dako) was used as the ligand protein 1. HER2 is human epidermal growth factor receptor 2.

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

[0062] The ligand protein 3 used was a FITC-labeled F(ab')2 fragment obtained by digesting a polyclonal rabbit anti-human HER2 antibody (A0485, manufactured by Dako) with pepsin. F(ab')2 fragments were prepared according to the following protocol. First, the antibody was dialyzed against 0.1 M sodium acetate buffer (pH 3.75). Then, pepsin-immobilized agarose derived from porcine gastric mucosa (Sigma) was added, and the reaction was allowed to proceed at 37°C for 3 hours. The reaction was then terminated by adding 0.5 M Tris buffer (pH 8.0) to adjust the pH to 7.0. The pepsin-immobilized agarose fragment was removed by centrifugation. The solution was then passed through a Protein A-immobilized agarose column (Thermo Fisher Scientific). Dialysis against TBS buffer at room temperature for 2 hours yielded 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, manufactured by 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 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 anti-human HER2 antibody labeled with horseradish peroxidase.

[0065] A polyclonal rabbit-derived anti-human HER2 antibody (A0485, manufactured by 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-derived anti-human HER2 antibody.

[0066] As ligand protein 7, a FITC-labeled polyclonal rabbit anti-human HER2 antibody (A0485, manufactured by 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, manufactured by Dako) was labeled with R-Phycoerythrin using 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 is.

[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 proteins other than ligand proteins, BSA (bovine serum albumin, Sigma) was used.

[0075] <Surfactants> 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 is a compound where x + y = 4. (c) Tween 80 (Tokyo Chemical Industry Co., Ltd.) A polyoxyethylene sorbitan alkyl ester-based structure, and is not a surfactant represented by formula (1). (d) BIO-SOFT N91-8 (Stepan) An alcohol alkoxylate-based structure, and is not a surfactant represented by formula (1). (e) Triton-X100 (Merck) A polyoxyethylene alkyl phenyl ether-based structure, and is not a surfactant represented by formula (1). (f) SDS: sodium dodecyl sulfate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) is not a surfactant represented by formula (1). (g) CTAB: cetyltrimethylammonium bromide (manufactured by Tokyo Chemical Industry Co., Ltd.) is an alkyltrimethylammonium bromide-based structure and is not a surfactant represented by formula (1). (h) CHAPS: 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonic acid (manufactured by Dojindo Laboratories) is an amphoteric surfactant of a cholate derivative and is not a surfactant represented by formula (1).

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

[0077] <Preparation of first liquid composition> First liquid compositions A-1 to A-49 were prepared by mixing the components so as to obtain the contents shown in Tables 1-1, 1-2, 1-3, and 1-4. The numbers in the tables indicate the solid content (% by mass).

[0078]

[0079]

[0080]

[0081]

[0082] <Preparation of second liquid composition> 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 content. Second liquid composition B-1 was used as supplied by the manufacturer, and this is noted in Table 2.

[0083]

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

[0085]

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

[0087] <Ligand Protein> A monoclonal mouse-derived anti-human Ki-67 antibody (M7240, manufactured by Dako) was used as the 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 telogen phase. HRP-labeled goat-derived anti-mouse IgG (Funakoshi Co., Ltd.) was prepared as the ligand protein 16.

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

[0089] <Surfactant> The following surfactant (a) was used: (a) A-E100: Acetylenol E100 (manufactured by Kawaken Fine Chemicals)

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

[0091] <Preparation of First Liquid Composition> The first liquid composition A-50 was prepared with TBS buffer to contain 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 0.1% by mass of surfactant A-E100.

[0092] <Preparation of Second Liquid Composition> The second liquid composition B-7 was prepared with TBS buffer to contain 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 0.1% by mass of surfactant A-E100.

[0093] (Evaluation of Discharge Volume) A thermal jet liquid discharge head having a nozzle diameter of 3 μm was prepared, and a tank connected to the head was filled with BC-345Bk ink (manufactured by Canon). The discharge head was driven by a controller electrically connected to the liquid discharge head, and liquid was discharged from the discharge port at a frequency of 15 kHz and a voltage of 13 volts. The discharged liquid was observed with an ultra-high speed camera, and the volume was calculated by the ligament method, which was set as 100%. Subsequently, the liquid compositions of Liquids A-1 to A-50, Liquids B-1 to B-7, and Liquids C-1 and C-2 were discharged under the same conditions, and the discharge volumes of the discharged 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 and C-2.

[0095]

[0096] <Evaluation of staining gradation> Cells were stained using each liquid composition. After observation, staining 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, evaluation was performed according to the following criteria. A: The staining was clear and graded according to the expression levels of HER2 "0", "1+", "2+", and "3+", making it easy to judge. B: The staining was graded and graded according to the expression level of HER2, making it possible to judge. C: HER2 "0", "2+", and "3+" stained graded, but the difference between "0" and "1+" was unclear, making it difficult to judge whether it was negative or weakly positive. D: HER2 "0" and "3+" stained graded, but the difference between "0", "1+", and "2+" was unclear, making it difficult to judge whether it was 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, evaluation was performed according to the following criteria. A: It was easy to determine the staining of Ki67 "-" and "+". E: It was difficult to determine the staining of Ki67 "-" and "+". The evaluation results of staining gradation are shown in Tables 5-1, 5-2, 5-3, 5-4, and 5-5.

[0097]

[0098]

[0099]

[0100]

[0101]

[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 application of liquid, 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 allowed to stand at room temperature for 5 minutes. This procedure was repeated twice. b) The specimens were immersed in a container containing 99.5% ethanol (Kishida Chemical Co., Ltd.) and allowed to stand at room temperature for 3 minutes. This procedure was repeated twice. c) The specimens were immersed in a container containing 95% ethanol and allowed to stand at room temperature for 5 minutes. This procedure was repeated twice. d) The specimens were 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, they 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 procedure 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 added dropwise to 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 procedure was repeated twice. The subsequent steps were performed using different methods for each Example.

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

[0107] (1) Application of the first liquid composition to biological samples 1 and 2 a) In biological sample 1, a 1 cm liquid composition was applied to surround four types of cultured cell lines with different levels of HER2 protein expression, and in biological sample 2, a 1 cm liquid composition was applied to surround two types of cultured cell lines with different levels of Ki-67 protein expression. ２ The frames were made using a pap pen (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 the print head. The ejection volume of one droplet was measured in advance in an ejection volume evaluation, and then the volume of the droplet was measured by 1 cm. ２ The first liquid composition was dropped into the frame made with a sachet pen under conditions set to 30 μL per drop. After dropping, the sample was allowed to stand in a humidified 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 allowed to stand at room temperature for 3 minutes. This procedure was repeated twice.

[0108] (2) Application of the 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 dap pen within a frame prepared by measuring the ejection volume of one droplet in advance using an ejection volume evaluation. ２The second liquid composition was added dropwise under conditions set so that 50 μL per sample was added. After addition, the sample was allowed to stand for 30 minutes in a humidified box. In Tables 5-1 to 5-5, "Unused" indicates that the above procedure was not performed. 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 also performed 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 the biological sample 1 using a dap pen within a frame prepared by measuring the ejection volume of one droplet in advance by the ejection volume evaluation, and then the third liquid composition was applied to the biological sample 1 using a dap pen within a frame prepared by the ... ２ The third liquid composition was added dropwise under conditions set so that 50 μL per sample was obtained, and the sample was allowed to stand in a humidified box for 5 minutes. In Tables 5-1 to 5-5, "Unused" indicates that the above procedure was not performed. b) 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 the sample was allowed to stand at room temperature for 3 minutes. This procedure was also performed twice.

[0110] (4) Observation A BZ-X810 microscope (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) fluorescence filter unit were used to acquire fluorescence images of the biological sample 1. Next, a phase-contrast image of the biological sample 1 was acquired using an objective lens compatible with phase-contrast observation and a phase-contrast observation condenser. Next, a green substitute color was applied to the acquired fluorescence image, and a gray substitute color was applied to the phase-contrast image. The two images were then superimposed using additive synthesis to generate a single composite image. In Example 8, a fluorescence image of the biological sample 1 was acquired using a 40x objective lens compatible with fluorescence observation and a BZ-X filter TRITCOP-87764 (Keyence Corporation, excitation wavelength 545 nm, detection wavelength 605 nm), which is a fluorescence filter unit. Next, a phase-contrast image of the biological sample 1 was acquired using an objective lens compatible with phase-contrast observation and a phase-contrast observation condenser. Next, an orange substitute color was applied to the acquired fluorescence image, and a gray substitute color was applied to the phase-contrast image. The two images were then superimposed using an additive synthesis method to generate a single composite image.

[0111] The present invention is not limited to the above-described embodiments, and various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the following claims are appended to apprise the public of the scope of the present invention.

[0112] This application claims priority based on Japanese Patent Application No. 2023-146445 filed on September 8, 2023 and Japanese Patent Application No. 2024-139231 filed on August 20, 2024, the entire contents of which are incorporated herein by reference.

Claims

1. A staining method comprising the steps 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.

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

3. The staining method according to claim 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.

4. The staining method according to any one of claims 1 to 3, wherein the ligand protein is modified with an enzyme, and the method further comprises a step of applying a substrate for the enzyme to the biological sample to which the liquid composition has been applied.

5. A dyeing method according to any one of claims 1 to 4, wherein the liquid composition further contains at least one selected from the group consisting of bovine serum albumin and casein.

6. A dyeing method according to any one of claims 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.

7. The dyeing method according to any one of claims 1 to 6, characterized in that the surfactant has a structure represented by the following formula (1): (In the formula (1), x and y satisfy the relationship 1.0≦x+y≦30.0.) 8. A dyeing method according to any one of claims 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.

9. The dyeing method according to any one of claims 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.

10. The dyeing 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 any one of claims 1 to 10, wherein the liquid composition contains a water-soluble organic solvent.

12. A staining method according to any one of claims 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.

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 or 13, wherein the second protein is modified with an enzyme, and further comprising a step of applying a chromogenic substrate for the enzyme to the biological sample to which the second liquid composition has been applied.

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

16. A kit for staining a biological sample using an inkjet method, comprising 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.

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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