Specimen manufacturing method and nasal discharge pretreatment kit

Abstract

The present invention aims to provide a method for producing a sample that can detect target substances in nasal secretions with high sensitivity. [Solution] The present invention is a method for producing a sample for detecting a target substance in nasal secretions, comprising the steps of contacting the nasal secretions with an anti-mucin antibody, and removing a complex of mucin and the anti-mucin antibody from the nasal secretions after contact.

Description

Technical Field

[0001] The present invention relates to a method for producing a specimen using an anti-mucin antibody, a method for detecting a target substance to be measured in the specimen obtained by the aforementioned method, and a kit for nasal discharge pretreatment.

Background Art

[0002] Detection of biomarkers in biological samples is used as a minimally invasive and safe testing method for disease diagnosis, prognosis prediction, pharmaceutical efficacy evaluation, and the like. In terms of detection performance, it is desirable that the target substance to be measured can be detected with high sensitivity, that is, a high signal value. In particular, when the target substance to be measured is present in a biological sample at a low concentration, specimen pretreatment is important to enhance detection sensitivity.

[0003] Patent Document 1 describes a method for determining allergic diseases, in which a specimen is brought into contact with an allergen determination chip, and an allergen-recognizing antibody in the specimen captured by the allergen determination chip is detected by an immunoassay using a labeled secondary antibody. Here, a method is described in which saliva, tear fluid, or nasal discharge that has been pressure-filtered with a low protein-adsorptive filter is used as the specimen.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present inventors attempted to detect allergen-specific IgE antibodies using a sample prepared by pretreatment to remove impurities from nasal secretions by membrane filtration, similar to the low-protein-adsorbing filter described in Patent Document 1. However, they found that the measurement sensitivity was low (see Comparative Example 1, described later). Therefore, focusing on the fact that nasal secretions contain mucin, they attempted to improve the measurement sensitivity by removing mucin from the nasal secretions. In other words, the object of the present invention is to provide a method for producing a sample that can detect target substances in nasal secretions with high sensitivity. [Means for solving the problem]

[0006] As a result of diligent research to overcome the above problems, the inventors have found that by detecting the target substance using a sample obtained by removing mucin from nasal secretions, it is possible to detect target substances such as biomarkers contained in nasal secretions with high sensitivity, thereby solving the problems.

[0007] In other words, the present invention is comprised of the following embodiments (1) to (12). (1) A method for producing a sample to detect a target substance in nasal secretions, The steps of contacting the nasal secretion with an anti-mucin antibody, and A step of removing the complex of mucin and the anti-mucin antibody from the nasal discharge after contact, Methods that include... (2) The method according to (1), wherein the nasal discharge is collected by absorbing or adhering it using a water-absorbing member or a water-repellent member. (3) The method according to (1) or (2), wherein the substance to be measured is a protein. (4) The method according to (3), wherein the protein is an IgE antibody. (5) The method according to any one of (1) to (4), wherein the anti-mucin antibody is an anti-mucin antibody that specifically binds to MUC5B or MUC7. (6) The method according to any one of (1) to (5), wherein the anti-mucin antibody is an anti-mucin antibody immobilized on a carrier. (7) A specimen prepared by any of the methods described in (1) to (6). (8) A method for detecting a target substance in nasal secretions, The step of contacting the nasal secretion with an anti-mucin antibody, The process of obtaining a sample by removing the mucin and the anti-mucin antibody complex from the nasal secretions after contact, and A step of detecting the substance to be measured in the sample, Methods that include... (9) A kit for pre-treatment of nasal discharge, containing a carrier on which anti-mucin antibodies are immobilized. (10) A kit for detecting a target substance in nasal secretions, comprising a carrier on which an anti-mucin antibody is immobilized, and a substance that selectively binds to the target substance in nasal secretions. (11) The kit according to (10), wherein the substance that selectively binds is an anti-immunoglobulin antibody. (12) The kit according to (10) or (11), wherein the selectively binding substance is a substance immobilized on a carrier. [Effects of the Invention]

[0008] This invention enables highly sensitive measurement of target substances in nasal secretions. [Modes for carrying out the invention]

[0009] [1. Method for manufacturing specimens] In this specification, a subject refers to an individual organism from which nasal secretions are collected. A preferred subject is a human.

[0010] In this specification, pretreatment means preparing the collected nasal secretions to be subject to detection of the target substances described below, and includes at least the removal of mucin.

[0011] In this specification, "specimen" refers to a sample obtained by pre-treating nasal secretions.

[0012] The present invention relates to a method for producing a sample for detecting a target substance in nasal secretions, comprising the steps of contacting the nasal secretions with an anti-mucin antibody, and removing a complex of mucin and the anti-mucin antibody from the nasal secretions after contact.

[0013] [1-1. Mucin] As used herein, mucin refers to a mixture of high-molecular-weight glycoproteins secreted from animal epithelial cells and the like. Mucins are encoded by a plurality of mucin genes, and a mucin is any of various glycoproteins encoded by the genes or a mixture of such glycoproteins.

[0014] The mucin may be any one of the glycoproteins encoded by mucin genes or a mixture thereof, and is not limited to a secretory type or a membrane-bound type, but those encoded by the MUC5B gene and MUC7 gene, which are abundantly contained in nasal discharge, are preferred.

[0015] The state of the mucin of the present invention is not particularly limited, and may be liquid, solid, sol, gel, amorphous or the like.

[0016] Regarding the state of mucin molecules in the present invention, each mucin molecule may be dispersed, or the mucin molecules may have a structure aggregated with each other.

[0017] The sugar chain structure possessed by mucin may be an O-linked sugar chain that is bound to a threonine / serine residue. The proportion occupied by the sugar chain structure per molecular weight of mucin is not limited.

[0018] [1-2. Nasal discharge] The nasal discharge used in the present invention preferably contains mucin in an amount of 0.001 wt% to 10 wt%, more preferably 0.01 wt% to 1 wt%.

[0019] The state of the nasal discharge used in the present invention may be a solution, may have solids such as aggregates and precipitates formed therein, or may be in a dried state.

[0020] Methods for collecting nasal discharge include directly dropping it into a container, using a suction device to draw it up, using an absorbent material to absorb or adhere it to the discharge, and using a water-repellent material to adhere it to the discharge. When using an absorbent material, the nasal discharge can be separated from the absorbent material in a later step. Examples of absorbent materials include cotton swabs, tissue paper, towels, absorbent fibers, and absorbent resins, but cotton swabs are preferred because they are easy to handle in the subsequent separation step. When using a water-repellent material, the collected nasal discharge can be collected and transferred to another container for storage. Examples of water-repellent materials include water-repellent sheets and water-repellent nonwoven fabrics.

[0021] Methods for separating nasal discharge absorbed or attached to an absorbent material include immersing the absorbent material from which the nasal discharge has been collected in an extractant, and separating it from the absorbent material without using an extractant by a centrifugal process or the like. In this separation, it is sufficient if some or all of the collected nasal discharge is separated.

[0022] As extractants, you can use aqueous solutions such as water, sodium phosphate buffer, PBS, saline solution, deionized water, Tris-hydrochloride buffer, Tris-maleate buffer, acetate buffer, citrate buffer, carbonate buffer, Good's buffer, and arginine-added aqueous solution, as well as solutions in which an organic solvent is mixed with water, such as acetonitrile-water mixture or ethanol-water mixture.

[0023] Examples of methods for storing the nasal discharge mentioned above include storing the collected nasal discharge in a container or along with the absorbent material in which the nasal discharge was collected. To suppress evaporation and drying of the nasal discharge, it is preferable to store the container in a sealed container or to place the absorbent material inside a sealed container.

[0024] The ideal storage temperature for nasal mucus is between 0°C and 37°C to prevent the denaturation of its components, but between 2°C and 6°C is even more preferable for long-term storage. When storing at temperatures below 0°C, it is desirable to minimize the number of freeze-thaw cycles to prevent deterioration of the nasal mucus.

[0025] Nasal secretions may be used in their undiluted state in the contact step with anti-mucin antibodies, or they may be used in the contact step with anti-mucin antibodies after being diluted to any dilution ratio by adding a diluent or separating them from an absorbent material, but a diluted state is preferred. A dilution ratio of 2 to 10 times is preferred in order to reduce viscosity and make the sample easier to handle.

[0026] [1-3. Anti-mucin antibodies] In this specification, an anti-mucin antibody refers to an antibody that has specific binding activity to mucin. Here, an antibody with specific binding activity to mucin includes antibody fragments that have specific binding activity to mucin, such as the Fab region of the antibody.

[0027] In this invention, anti-mucin antibodies are used to remove mucin from nasal secretions and prepare a sample by utilizing their ability to bind to mucin.

[0028] The anti-mucin antibody used may be either a polyclonal antibody or a monoclonal antibody, but a polyclonal antibody capable of recognizing many mucin structures is preferred. The polyclonal antibody may be any anti-mucin antibody derived from animals commonly used in antibody production, such as mice, rabbits, or goats. The anti-mucin antibody used in this invention may be a commercially available one or one that has been synthesized in-house.

[0029] As for anti-mucin antibodies, those that specifically bind to MUC5B and MUC7, which are abundant in the nasal secretions mentioned above, are preferred.

[0030] The epitopes recognized by anti-mucin antibodies may include the entire structure of the mucin protein, only the amino acid sequence, only the glycan sequence, both, or a partial structure of the mucin protein. When recognizing the glycan structure, anti-mucin antibodies that recognize O-linked glycans highly expressed on mucin are preferred.

[0031] The anti-mucin antibody used may be one type or two or more types. Furthermore, if two or more types are used, the specific sugar structures recognized by each anti-mucin antibody may be the same or different.

[0032] Alternatively, the mucin removal process may be carried out using a molecule that can bind to mucin, along with an anti-mucin antibody.

[0033] The anti-mucin antibodies may be dispersed, aggregated, or immobilized on a carrier. It is preferable to use anti-mucin antibodies immobilized on a carrier because the mucin bound to the anti-mucin antibodies can be easily removed in the removal process described later.

[0034] Examples of carrier materials used for immobilizing anti-mucin antibodies include resins, glass, metals, agarose, silica, silicon wafers, ceramics, cellulose, and nitrocellulose. However, resins, metals, agarose, or silica are preferred because they are easy to mold into particle shapes that increase surface area, and agarose or silica are more preferred because their porous nature can further increase surface area.

[0035] The shapes of carriers used for immobilizing anti-mucin antibodies include particulate, plate-like, well-like, membrane-like, cylindrical, fragment-like, and sponge-like forms. The preferred carrier shape is particulate or fragment-like, which can be easily removed from nasal secretions as a precipitate after adsorbing mucin.

[0036] The particle size of the carrier is preferably 45 μm to 165 μm, and more preferably 70 μm to 110 μm, for high reaction efficiency.

[0037] You may use commercially available anti-mucin antibodies immobilized on a carrier, or you may use anti-mucin antibodies that you have immobilized on a carrier yourself.

[0038] The immobilization of anti-mucin antibodies onto a support may be by chemical bonding or by physical adsorption. Examples of chemical bonding include chemical bonding via amino acids in the anti-mucin antibody or via linker / spacer molecules. Examples of physical adsorption include intermolecular forces. As for the method of immobilization onto the support, stronger chemical bonding is preferred. Although the type of chemical bonding is not limited, immobilization by amide bonding using amino groups, which are abundant in anti-mucin antibodies, is preferred.

[0039] [1-4. Contact process] The present invention provides a method for producing a specimen, which includes a step of contacting the nasal secretion with an anti-mucin antibody (hereinafter referred to as the "contact step").

[0040] When nasal secretions are collected using an absorbent material, the nasal secretions may be in a state that has already been separated from the absorbent material, or they may be absorbed or attached to the absorbent material, when they are brought into contact with anti-mucin antibodies. When bringing nasal secretions that have been absorbed or attached to the absorbent material into contact with anti-mucin antibodies, the separation from the absorbent material and contact with the anti-mucin antibodies can be achieved by immersing the absorbent material in a solution containing anti-mucin antibodies.

[0041] When nasal secretions are collected using a water-repellent material, the nasal secretions may be in a state that has already been separated from the water-repellent material or in a state that is still attached to the water-repellent material when they are brought into contact with the anti-mucin antibody. When bringing nasal secretions that are attached to the water-repellent material into contact with the anti-mucin antibody, the separation from the water-repellent material and contact with the anti-mucin antibody can be achieved by immersing the water-repellent material in a solution containing the anti-mucin antibody.

[0042] Examples of methods for contacting nasal secretions with anti-mucin antibodies include adding anti-mucin antibodies or an anti-mucin antibody immobilized carrier to the nasal secretions and allowing contact, and, in the case of using anti-mucin antibodies immobilized on particulate carriers, flowing nasal secretions through a column packed with the carrier immobilized with anti-mucin antibodies. The method of adding anti-mucin antibodies or an anti-mucin antibody immobilized carrier to nasal secretions is preferable because the contact reaction can be carried out for a long time while preventing evaporation and drying by sealing the container. It is not necessary for the anti-mucin antibodies and nasal secretions to be in constant contact; it is sufficient for the mucin in the nasal secretions to bind to the anti-mucin antibodies. Contact between anti-mucin antibodies and nasal secretions may be carried out by standing or with stirring, but the method with stirring is preferred because it has a higher reaction efficiency.

[0043] The amount of anti-mucin antibody added to nasal secretions is not specified, but the weight of anti-mucin antibody per volume of directly collected nasal secretions is preferably 0.01 mg / mL or more and 1 mg / mL or less, and more preferably 0.05 mg / mL or more and 0.3 mg / mL or less.

[0044] While there is no specific time requirement for the contact between the anti-mucin antibody and nasal secretion, it is preferably one hour or longer, and more preferably until the reversible binding reaction between the anti-mucin antibody and mucin reaches a plateau.

[0045] The temperature at which the anti-mucin antibody is brought into contact with the nasal secretion should be such that the liquid does not coagulate and the anti-mucin antibody does not easily denature, but it is preferably between 4°C and 37°C, and more preferably between 20°C and 30°C.

[0046] [1-5. Removal process] The present invention's method for producing a specimen includes a step of removing the complex of mucin and the anti-mucin antibody from the nasal secretion after contact (hereinafter referred to as the "removal step").

[0047] Methods for removing mucin-anti-mucin antibody complexes include, when using a carrier immobilized with anti-mucin antibodies, removing the carrier to which the mucin-anti-mucin antibody complex has bound after the contact step. When using particulate or fragmentary carriers immobilized with anti-mucin antibodies, the precipitated carrier can be removed and the supernatant isolated and used as a sample, or the carrier can be removed by membrane filtration using centrifugation, pressure, or aspiration, and the flow-through isolated and used as a sample. However, the method of removing the precipitated carrier is more preferable because it has a lower possibility of reducing the amount of substance to be measured. When using plate-shaped or well-shaped carriers immobilized with anti-mucin antibodies, the mucin-anti-mucin antibody complex bound to the carrier can be removed by isolating the sample on top of the carrier. When using easily crushable carriers such as membranes or sponges immobilized with anti-mucin antibodies, the mucin-anti-mucin antibody complex bound to the carrier can be removed by crushing the carrier and isolating the sample that seeps out.

[0048] When using anti-mucin antibodies that are not immobilized on a carrier, the mucin-anti-mucin antibody complex can be removed after the contact step by removing the agglutinations formed by the binding of mucin and anti-mucin antibodies to each other. Methods for removing these agglutinations include removing the precipitated agglutinations and isolating the supernatant to use as a sample, or removing the agglutinations by membrane filtration using centrifugation, pressurization, or aspiration, and isolating the flow-through to use as a sample. However, the method of removing the precipitated agglutinations is more preferable because it has a lower possibility of reducing the amount of the target substance.

[0049] Furthermore, the removal of mucin by removing the mucin-anti-mucin antibody complex only needs to be to the extent that it does not affect the detection step of the next target substance, and does not necessarily need to be completely removed. The removal efficiency that does not affect the detection step is preferably such that the ratio of remaining mucin to the mucin before removal is 10% by weight or less, and more preferably 1% by weight or less.

[0050] [1-6. Target substances to be detected] In this specification, the substance to be measured refers to a substance in nasal secretions that is a biomarker or the like that is to be detected.

[0051] Examples of substances to be measured include biologically derived components such as nucleic acids, proteins, peptides, and amino acids, as well as ingested components such as drugs, food, and food residues. Examples of nucleic acids include cell-free DNA, genomic DNA, messenger RNA, microRNA, and artificially synthesized nucleic acids. Examples of proteins include antibodies, antigens, cytokines, and allergens. An example of an antibody is IgE antibody. Among IgE antibodies, allergen-specific IgE antibodies that can be used in allergy testing are particularly preferred. Examples of amino acids include glutamic acid and leucine, which contain free amino acids. Examples of drugs include hallucinogens, psychostimulants, analgesics, inhibitors, abuse inhalation antigens, hypnotics, and alcohol. Furthermore, the substances to be measured may be pre-labeled with fluorescent substances or the like. The method for producing a sample of the present invention is particularly suitable when the substance to be measured is protein, and among them, IgE antibody is particularly suitable.

[0052] The detection of the target substance in this invention can be performed by known methods. For example, a method can be described that includes the steps of contacting a sample containing the target substance with a carrier on which the target substance and a substance that selectively binds to it (hereinafter referred to as "selective binding substance") are immobilized to form a complex with the selective binding substance, and detecting the formed complex. The method for detecting the complex is not particularly limited and can be performed by known methods such as fluorescence or chemiluminescence. If the target substance is labeled with a fluorescent substance or the like, the label may be used to detect the complex, or the complex may be labeled with another fluorescent substance or the like before detection. A specific method for detecting the complex is to use a labeled secondary antibody that specifically binds to the complex. As the selective binding substance, a substance immobilized on a carrier is preferred.

[0053] Examples of selective binding substances include antibodies that specifically bind to the target substance as an antigen. When the target substance is a nucleic acid, a complementary nucleic acid that specifically binds to that nucleic acid can be used as the selective binding substance. When the target substance is an allergen, an allergen-specific antibody that specifically binds to that antigen can be used as the selective binding substance. When the target substance is an antibody, an anti-immunoglobulin antibody can be used as the selective binding substance. When the target substance is an IgE antibody, an anti-IgE antibody can be used as the selective binding substance.

[0054] [2. Method for detecting the target substance] The present invention provides a method for detecting a target substance in nasal secretions, comprising the steps of: contacting the nasal secretions with an anti-mucin antibody; removing the complex of mucin and the anti-mucin antibody from the nasal secretions after contact to obtain a sample; and detecting the target substance in the sample.

[0055] [2-1. Contact and Removal Processes] The contact step and removal step in the detection method of the present invention are performed by the contact step and removal step in the method for producing a sample of the present invention.

[0056] [2-2. Detection Process] The detection method of the present invention includes the step of detecting the target substance contained in a sample obtained by the manufacturing method of the present invention. The method for detecting the target substance is as described above.

[0057] [3. Kit Contents] [3-1. Kit containing a carrier on which anti-mucin antibodies are immobilized] The present invention's method for producing a sample using an anti-mucin antibody can be carried out using a nasal secretion pretreatment kit that includes a carrier on which the anti-mucin antibody is immobilized. There are no particular restrictions on the specific configuration of the kit, but at a minimum, it should be configured to remove mucin derived from nasal secretions as a complex of mucin and the anti-mucin antibody after contacting the carrier on which the anti-mucin antibody is immobilized with the nasal secretions.

[0058] Configurations that allow a carrier immobilized with anti-mucin antibodies to come into contact with nasal secretions include, for example, a method in which anti-mucin antibodies are bound to and immobilized on a strip, filter, tube, etc., and then nasal secretions are added to the strip, etc. to bring them into contact, or a method in which an anti-mucin antibody immobilized carrier is mounted or immobilized on a strip, filter, tube, etc., and then nasal secretions are added to it to bring it into contact. The immobilization method for immobilizing the anti-mucin antibody or the anti-mucin antibody immobilized carrier on the above-mentioned strip, etc., may be any method such as chemical bonding or physical adsorption, but from the viewpoint of more stable immobilization, a chemical bonding method is preferred.

[0059] The configuration for removing the mucin-anti-mucin antibody complex may include, but is not limited to, a developing solution for adding to the sample and allowing it to flow down, a tube for centrifugation, a tube for settling the carrier on which the anti-mucin antibody is immobilized and collecting the supernatant, a pipette, etc.

[0060] The kit may also be configured to include the aforementioned selective binding substance, thereby providing a kit for detecting target substances in nasal secretions. A preferred selective binding substance is an anti-immunoglobulin antibody. The selective binding substance may be immobilized on a carrier or included in the kit in an unimmobilized state. If the selective binding substance is immobilized on a carrier, the immobilized portion can be used as the detection area for the target substance. On the other hand, if it is unimmobilized, it is sufficient to add it separately when using the kit, by adding the substance to nasal secretions from which mucin has been removed and bringing them into contact. The method for detecting the complex of the selective binding substance and the target substance is not particularly limited, and known detection methods such as fluorescence and chemiluminescence can be used. [Examples]

[0061] Examples are shown below, but the present invention is not limited to these examples.

[0062] [Example 1] (1) Preparation of anti-mucin antibody immobilized carriers MUC5B Polyclonal antibody (Proteintech Japan) was dissolved at a concentration of 1 mg / mL in a coupling solution (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) as an anti-mucin antibody. 100 μL of NHS-activated Sepharose 4 Fast Flow (Cytiva) was added to 100 μL of this solution, and the mixture was allowed to stand at room temperature (25°C) for 2 hours. Then, 500 μL of 0.1 M Tris-HCl (pH 8.0) was added to form a suspension, and the solvent was removed by centrifugation. Finally, a washing step was performed (three times with 300 μL of 0.1 M Tris-HCl (pH 8.0) and three times with 300 μL of 0.1 M acetate buffer (pH 4.5)), after which 200 μL of 20 wt% ethanol solution was added to dilute the mixture, and a suspension of anti-mucin antibody-immobilized Sepharose particles, a type of anti-mucin antibody immobilized on a carrier, was obtained.

[0063] (2) Obtaining a nasal secretion sample Nasal secretions were absorbed onto a Mediswab flocked cotton swab (Heiwa Medic Co., Ltd. NA01 PP for nasopharyngeal use), the tip of the swab was cut off, and the secretions were collected by centrifugation in a tube. 40 μL of the anti-mucin antibody-immobilized Sepharose particle suspension prepared in (1) was added to 20 μL of the collected nasal secretions, and a stirring reaction was carried out for 1 hour. After the anti-mucin antibody-immobilized Sepharose particles were allowed to precipitate, the supernatant was collected to obtain a nasal secretion sample from which the anti-mucin antibody-immobilized Sepharose particles had been removed.

[0064] (3) Preparation of crude allergen extract solution Dog, cat, soy, wheat, and timothy grass allergens were purchased from GREER. Sugar allergen was purchased from SBP. Dust mite and egg white allergens were purchased from Torii Pharmaceutical Co., Ltd. Ovomucoid allergen was purchased from LSBio. Each allergen was diluted with 20 mM phosphate buffer to a final concentration of 1 mg / mL to obtain an allergen extract.

[0065] (4) Fabrication of NHS esterified PMMA substrates A polymethyl methacrylate (PMMA) resin substrate (manufactured by Kuraray, external dimensions 75 mm x 25 mm x 1 mm, average molecular weight 30,000) was used as the substrate. This substrate was immersed in 10 N sodium hydroxide at 70°C for 14 hours, then washed three times with pure water and dried. In this way, the side chains of the PMMA on the substrate surface were hydrolyzed to generate carboxyl groups.

[0066] Next, 100 mg of N-hydroxysuccinimide (NHS) and 350 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were dissolved in 400 mL of 2-morpholinoethanesulfonic acid monohydrate (MES) buffer (adjusted to pH 5.0 with 1N hydrochloric acid). The PMMA substrate, after hydrolysis, was immersed in this mixed solution and stirred with a micro-stirrer for 1 hour to obtain an NHS-esterified PMMA substrate.

[0067] (5) Fabrication of allergen-immobilized microarrays The nine allergen extracts prepared in (3) were spotted onto the same NHS esterified PMMA resin substrate prepared above using a spotting robot (Nippon Laser Electronics Co., Ltd., GTMAS Stamp-2). Several allergen-immobilized areas were present on the substrate after spotting, with each immobilized area measuring 0.007 mm². 2 That was the case.

[0068] Here, we measure allergen-specific IgE antibodies contained in samples using nine types of allergen proteins. To ensure that each sample comes into contact with all types of allergen proteins, we configured a set of immobilized allergen protein sections for each sample.

[0069] Next, in the incubation process, the substrate was placed in a plastic container with a small amount of water, sealed, and reacted overnight at 37°C and 100% humidity to immobilize each allergen extract onto the substrate surface. Subsequently, it was washed three times with Phosphate Buffered Saline with Tween® 20 (PBS-T) pH 7.4 (hereinafter referred to as "PBST"), the washing solution was removed by centrifugation (1000G, 5 minutes), and the substrate surface was dried to obtain an allergen-immobilized microarray for specific antibody detection.

[0070] Finally, the allergen-immobilized microarrays were blocked by immersion overnight at 4°C in Phosphate Buffered Saline (PBS) pH 7.4 (hereinafter referred to as "PBS") containing 1% by weight bovine serum albumin (BSA) (Sigma-Aldrich), followed by three washes with PBST.

[0071] (6) Contact of the sample with the allergen-immobilized microarray (reaction step) The sample used was a nasal secretion sample prepared using the anti-mucin antibody described in (2). This sample was negative for dog, cat, dust mite, soy, egg white, wheat, and ovomucoid, and positive for cedar and timothy grass. The nasal secretion sample was brought into contact with the allergen protein immobilized portion of the allergen-immobilized microarray described in (5). Specifically, a solution prepared by diluting 5 μL of the sample three-fold with PBS containing 1 wt% BSA was dropped onto the allergen-immobilized portion of the carrier after the above treatment, reacted at 37°C for 2 hours, and then washed three times with PBST.

[0072] (7) Detection of allergen-specific IgE antibodies (detection step) Next, 15 μL of DyLight-650 dye-labeled anti-human IgE goat polyclonal antibody (Novus biologicals), diluted 1000-fold with PBST containing 1 wt% BSA, was added to the substrate and reacted for 1 hour at room temperature under light-shielding conditions. After washing three times with PBST and drying, the fluorescence intensity was measured using a scanner (Toray Industries, Inc., "3D-Gene® Scanner 3000") under the following conditions. The fluorescence detected here originates from the fluorescent dye contained in the labeled antibody bound to the complex of allergen extract and allergen-specific IgE antibody; a higher amount of allergen-specific IgE antibody bound to the allergen-immobilized microarray results in a higher detection value. Fluorescence intensity was obtained at the pixel level within the spot, and the median of these fluorescence intensities was evaluated as the overall fluorescence intensity.

[0073] (Fluorescence intensity measurement conditions) Equipment: 3D-Gene® Scanner 3000 (manufactured by Toray Industries) Excitation light wavelength: 635 nm Detection wavelength: 670nm PMT:33

[0074] The results are shown in Table 1.

[0075] [Comparative Example 1] As a control experiment for Example 1, instead of performing the procedure in Example 1(2) of contacting nasal secretions with a suspension of anti-mucin antibody-immobilized Sepharose particles (corresponding to anti-mucin antibodies immobilized on a carrier) and collecting the supernatant, impurities were removed by membrane filtration using a spin-empty column (BioRad, #7326204) (specifically, the tip of the cotton swab used to collect nasal secretions was placed on the membrane and quickly centrifuged (2000G, 1 min)). The allergen-specific IgE in the membrane-filtered sample was measured and evaluated in the same manner as in Example 1.

[0076] The results are shown in Table 1.

[0077] [Table 1]

[0078] [Result 1: Comparison of Example 1 and Comparative Example 1] Using a sample obtained by removing mucin from nasal secretions with anti-mucin antibody-immobilized Sepharose particles (Example 1), allergen-specific IgE antibodies in the sample were measured by microarray. The results showed improved signal values ​​for positive items compared to a sample obtained by removing impurities from nasal secretions by membrane filtration (Comparative Example 1). This suggests that in Comparative Example 1, allergen-specific IgE antibodies of the target substance were significantly lost due to adsorption to the membrane, while in Example 1, the loss of allergen-specific IgE antibodies was reduced, allowing for detection.

[0079] [Example 2] The target substance for measurement was albumin in the sample. In (3) of Example 1, instead of the crude allergen extract solution, an anti-human albumin rabbit monoclonal antibody (Thermo Fisher Scientific) was used and diluted in 20 mM phosphate buffer to obtain an anti-albumin antibody solution with a final concentration of 0.2 mg / mL. The anti-albumin antibody-immobilized microarray was prepared and the sample was brought into contact with it in the same manner as in (5) and (6) of Example 1. Subsequently, in (7), instead of the DyLight-650 dye-labeled anti-human IgE goat polyclonal antibody, the DyLight-650 dye-labeled anti-human albumin rabbit monoclonal antibody was used, and fluorescence derived from the fluorescent dye contained in the labeled antibody bound to the anti-albumin antibody-albumin complex was detected. The remaining steps were the same as in Example 1, and the albumin in the sample was measured and evaluated.

[0080] The results are shown in Table 2.

[0081] [Comparative Example 2] As a control experiment for Example 2, instead of the procedure in Example 2 in which nasal secretions were brought into contact with a suspension of anti-mucin antibody-immobilized Sepharose particles (corresponding to anti-mucin antibodies immobilized on a carrier) and the supernatant was collected, impurities were removed by membrane filtration using a spin-empty column (specifically, the tip of the cotton swab used to collect nasal secretions was placed on the membrane and quickly centrifuged (2000G, 1 min)). The albumin content in the membrane-filtered sample was measured and evaluated in the same manner as in Example 2.

[0082] The results are shown in Table 2.

[0083] [Table 2]

[0084] [Result 2: Comparison between Example 2 and Comparative Example 2] When albumin in a sample obtained by removing mucin from nasal secretions using anti-mucin antibody-immobilized Sepharose particles (Example 2) was measured by microarray, the signal value was improved compared to a sample obtained by removing impurities from nasal secretions by membrane filtration (Comparative Example 2). This result suggests that, similar to the case where the target substance is allergen-specific IgE antibody, when the target substance is albumin, in Comparative Example 2, albumin is significantly lost due to adsorption to the membrane, while in Example 2, albumin loss is reduced, allowing for detection.

Claims

1. A method for producing a sample to detect a target substance in nasal secretions, The steps of contacting the nasal secretion with an anti-mucin antibody, and A step of removing the complex of mucin and the anti-mucin antibody from the nasal discharge after contact, Methods that include...

2. The method according to claim 1, wherein the nasal discharge is collected by absorbing or adhering it using a water-absorbing member or a water-repellent member.

3. The method according to claim 1 or 2, wherein the substance to be measured is a protein.

4. The method according to claim 3, wherein the protein is an IgE antibody.

5. The method according to claim 1 or 2, wherein the anti-mucin antibody is an anti-mucin antibody that specifically binds to MUC5B or MUC7.

6. The method according to claim 1 or 2, wherein the anti-mucin antibody is an anti-mucin antibody immobilized on a carrier.

7. A specimen manufactured by the method described in claim 1 or 2.

8. A method for detecting a target substance in nasal secretions, The step of contacting the nasal secretion with an anti-mucin antibody, The process of obtaining a sample by removing the mucin and the anti-mucin antibody complex from the nasal secretions after contact, and A step of detecting the substance to be measured in the sample, Methods that include...

9. A kit for pre-treatment of nasal discharge, containing a carrier immobilized with anti-mucin antibodies.

10. A kit for detecting target substances in nasal secretions, comprising a carrier on which anti-mucin antibodies are immobilized, and a substance that selectively binds to the target substance in nasal secretions.

11. The kit according to claim 10, wherein the substance that selectively binds is an anti-immunoglobulin antibody.

12. The kit according to claim 10 or 11, wherein the selectively binding substance is a substance immobilized on a carrier.

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