Kit for pretreatment of biological samples and method for producing samples

Abstract

The present invention aims to provide a method for producing a sample that can detect target substances in biological samples containing mucin, such as nasal secretions, with high sensitivity. [Solution] The present invention is a method for producing a sample for detecting a target substance in a biological sample containing mucin, such as nasal secretions, comprising the steps of: contacting the biological sample with a lectin; and removing the complex of mucin and the lectin from the biological sample after contact.

Description

Technical Field

[0001] The present invention relates to a method for producing a specimen using a lectin, a method for detecting a target substance to be measured in the specimen obtained by the aforementioned method, and a kit for pretreating a biological sample.

Background Art

[0002] Detection of biomarkers in biological samples is used as a minimally invasive and safe test method for disease diagnosis, prognosis prediction, pharmaceutical efficacy evaluation, and the like. As for detection performance, it is desirable that the target substance to be measured can be detected with high sensitivity, that is, the signal value is high. In particular, when the target substance to be measured is present in a biological sample at a low concentration, pretreatment of the specimen 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 on 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 biological samples such as nasal secretions contain mucin, they attempted to improve the measurement sensitivity by removing mucin from biological samples such as nasal secretions that contain mucin. In other words, the object of the present invention is to provide a method for producing a sample that can detect target substances in biological samples such as nasal secretions that contain mucin with high sensitivity. [Means for solving the problem]

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

[0007] In other words, the present invention is comprised of the following embodiments (1) to (14). (1) A method for producing a sample to detect a target substance in a biological sample containing mucin, The steps of bringing the biological sample into contact with lectin, A step of removing the mucin-lectin complex from the biological sample after contact, Methods that include... (2) The method according to (1), wherein the biological sample is nasal secretions, saliva, or tears. (3) The method according to (1) or (2), wherein the biological sample is a biological sample that has been collected by absorbing or adhering to it using a water-absorbing member or a water-repellent member. (4) The method according to any one of (1) to (3), wherein the substance to be measured is a protein. (5) The method according to (4), wherein the protein is an IgE antibody. (6) The method according to any one of (1) to (5), wherein the lectin is a lectin that binds to N-acetylgalactosamine. (7) The method according to any one of (1) to (6), wherein the lectin is a lectin derived from Dolichos biflorus, Griffonia simplicifolia I, Vicia villosa, Jacalin, or Wisteria floribunda. (8) The method according to any one of (1) to (7), wherein the lectin is a lectin immobilized on a carrier. (9) A specimen prepared by any of the methods described in (1) to (8). (10) A method for detecting a target substance in a biological sample containing mucin, The process of bringing the biological sample into contact with lectin, The process of obtaining a sample by removing the mucin-lectin complex from the biological sample after contact, and A step of detecting the substance to be measured in the sample, Methods that include... (11) A kit for pre-treatment of mucin-containing biological samples, which includes a carrier on which lectins have been immobilized. (12) A kit for detecting a target substance in a mucin-containing biological sample, comprising a carrier on which lectin is immobilized, and a substance that selectively binds to the target substance in the mucin-containing biological sample (excluding mucin). (13) The kit according to (12), wherein the substance that selectively binds is an anti-immunoglobulin antibody. (14) The kit according to (12) or (13), 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 biological samples containing mucin, such as nasal secretions. [Modes for carrying out the invention]

[0009] [1. Method for manufacturing specimens] In this specification, a biological sample refers to a sample such as blood (serum), urine, saliva, nasal secretions, tears, vaginal fluid, or intestinal fluid collected from a subject such as a human, before any pretreatment.

[0010] In the present specification, a subject refers to an individual organism from which a biological sample is collected. A preferred subject is a human.

[0011] In the present specification, pretreatment refers to processing a collected biological sample such as nasal discharge into a state that allows the target substance to be measured, which will be described later, to be detected, and includes at least removing mucin.

[0012] In the present specification, a specimen refers to a product obtained by pretreating a biological sample.

[0013] The present invention relates to a method for producing a specimen for detecting a target substance in a mucin-containing biological sample, the method comprising a step of bringing the biological sample into contact with a lectin, and a step of removing a complex of the mucin and the lectin from the biological sample after the contact.

[0014] [1-1. Mucin] In the present specification, 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.

[0015] The mucin may be any one of the glycoproteins encoded by mucin genes or a mixture thereof, and is not limited to secretory or membrane-bound types, but those encoded by the MUC5B gene and MUC7 gene, which are abundantly contained in saliva, nasal discharge, and tear fluid, are preferred.

[0016] The state of the mucin of the present invention is not particularly limited, and may be liquid, solid, sol, gel, amorphous, or the like.

[0017] As for the state of mucin molecules in the present invention, the respective mucin molecules may be dispersed, or the mucin molecules may have a structure in which they are aggregated with one another.

[0018] The sugar chain structure of mucin may be an O-linked sugar chain attached to a threonine / serine residue. The proportion of the sugar chain structure per unit molecular weight of mucin is not limited. A higher proportion is preferable in that it improves the binding efficiency between lectin and mucin, so a proportion of 30% to 90% is preferred, and more preferably 50% to 90%.

[0019] [1-2. Biological samples containing mucin] Examples of mucin-containing biological samples used in the present invention include blood (serum), urine, saliva, nasal secretions, tears, vaginal fluid, and intestinal fluid. The biological samples used in the present invention are preferably those containing mucin at high concentrations and that can be collected with minimal invasiveness, such as nasal secretions, saliva, and tears, with nasal secretions being particularly preferred.

[0020] The biological sample of the present invention preferably contains 0.001% to 10% by weight of mucin, and more preferably 0.01% to 1% by weight of mucin.

[0021] The biological sample used in this invention may be in a solution, a solid such as an aggregate or precipitate, or in a dry state.

[0022] Methods for collecting biological samples include dropping them directly into a container, using a suction device, absorbing or adhering them using an absorbent material, and adhering them using a water-repellent material. When an absorbent material is used, the biological sample 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 a water-repellent material is used, the collected biological sample can be recovered and transferred to another container for storage. Examples of water-repellent materials include water-repellent sheets and water-repellent nonwoven fabrics.

[0023] Methods for separating biological samples absorbed or attached to absorbent materials include immersing the absorbent material from which the biological sample has been recovered in an extractant, and separating the sample 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 biological sample is separated.

[0024] 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.

[0025] Examples of methods for storing the above-mentioned biological samples include storing the collected biological sample in a container or storing the biological sample together with the absorbent material from which it was collected. To suppress evaporation and drying of the biological sample, it is preferable to store the container in a sealed container or to place the absorbent material inside a sealed container.

[0026] The ideal storage temperature for biological samples is between 0°C and 37°C to prevent denaturation of the components within the sample, 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 freezing / thawing cycles to prevent deterioration of the biological sample.

[0027] The biological sample may be used in the contact step with the lectin in its undiluted state, or it may be used in the contact step with the lectin after being diluted to any dilution ratio by adding a diluent or separating it from the absorbent material, but the diluted state is preferred. In the case of nasal secretions, a dilution ratio of 2 to 10 times is preferred in order to reduce viscosity and make the sample easier to handle.

[0028] [1-3. Lectins] In this specification, lectin refers to a general term for proteins that have binding activity to monosaccharides or sugar chains.

[0029] In this invention, lectins are used to remove mucin from biological samples and prepare specimens by utilizing their ability to bind to mucin.

[0030] The sugar chain structures recognized by lectins include N-linked sugar chains that bind to asparagine residues and O-linked sugar chains that bind to threonine / serine side chains. In the present invention, lectins that recognize O-linked sugar chains highly expressed in mucins are preferred.

[0031] Examples of O-linked sugar chain structures recognized by lectins include N-acetylglucosamine, N-acetylgalactosamine, fucose, mannose, and xylose, which are sugars bound to threonine / serine. In particular, lectins that recognize N-acetylgalactosamine, which is found in mucin structures, are preferred. Specifically, examples include lectins that recognize structures to which the N-acetylgalactosamine monosaccharide is bound, lectins that recognize structures to which N-acetylglucosamine or galactose is bound to N-acetylgalactosamine, such as Core1, Core2, Core3, Core4, Core5, Core6, Core7, and Core8 structures, and lectins that recognize structures to which sialic acid or fucose is bound to these Core structures.

[0032] The lectins used may be of animal, plant, or microbial origin, but plant-derived lectins, which offer a wide variety of lectins, are preferred, and those derived from leguminous plants, which form a large family, are even more preferred. The lectins used in this invention may be commercially available, or they may be extracted by the inventors themselves from animals, plants, microorganisms, etc.

[0033] Lectins may recognize and bind to the entire sugar chain structure, to recognize and bind to a part of the sugar chain structure, or to recognize and bind to both the sugar chain structure and the amino acid structure. However, lectins that recognize and bind to N-acetylgalactosamine, which is a common structure of O-linked sugar chains contained in mucin, are preferred.

[0034] Examples of lectins that recognize and bind to N-acetylgalactosamine include lectins derived from Dolichos biflorus, Griffonia simplicifolia I, Vicia villosa, Jacalin, or Wisteria floribunda.

[0035] The lectin used may be one type or two or more types. Furthermore, if two or more types are used, the sugar structures recognized by each lectin may be the same or different.

[0036] Alternatively, the mucin removal process may be carried out using a molecule with specific binding activity to mucin, along with the lectin.

[0037] Lectins may be dispersed, aggregated, or immobilized on a carrier. It is preferable to use lectins immobilized on a carrier because mucin bound to the lectin can be easily removed in the removal process described later.

[0038] Examples of carrier materials used for immobilizing lectins 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.

[0039] Examples of carrier shapes used for lectin immobilization include particulate, plate-like, well-like, membrane-like, cylindrical, fragment-like, and sponge-like forms. Preferred carrier shapes are particulate or fragment-like, which can be easily removed from the biological sample as a precipitate after mucin adsorption.

[0040] When the carrier is in particulate form, the particle size is preferably 45 μm to 165 μm, and more preferably 70 μm to 110 μm, for high reaction efficiency.

[0041] You may use commercially available lectins immobilized on a carrier, or you may use lectins that have been immobilized on a carrier through a self-immobilization reaction.

[0042] Lectins can be immobilized on a support by either chemical bonding or physical adsorption. Examples of chemical bonding include bonding via amino acids in the lectin or via linker / spacer molecules. Examples of physical adsorption include intermolecular forces. Stronger chemical bonding is preferred as the method of immobilization on the support. While the type of chemical bonding is not limited, immobilization by amide bonding using amino groups, which are abundant in lectins, is preferred.

[0043] [1-4. Contact process] The present invention provides a method for producing a specimen, which includes a step of contacting the biological sample with a lectin (hereinafter referred to as the "contact step").

[0044] When a biological sample is collected using an absorbent material, the biological sample may be in a state that has already been separated from the absorbent material, or it may be absorbed or attached to the absorbent material, when it comes into contact with lectin. When a biological sample that has been absorbed or attached to an absorbent material comes into contact with lectin, the separation from the absorbent material and contact with the lectin can be achieved by immersing the absorbent material in a liquid containing lectin.

[0045] When a biological sample is collected using a water-repellent material, the biological sample may be in a state that has already been separated from the water-repellent material or in a state that is attached to the water-repellent material when it comes into contact with lectin. When a biological sample attached to a water-repellent material comes into contact with lectin, the separation from the water-repellent material and contact with the lectin can be achieved by immersing the water-repellent material in a liquid containing lectin.

[0046] Examples of methods for contacting a biological sample with lectins include adding lectin or a lectin-immobilized carrier to the biological sample and allowing contact, and, in the case of using lectin immobilized on a particulate carrier, flowing the biological sample through a column packed with the lectin-immobilized carrier. The method of adding lectin or a lectin-immobilized carrier to the biological sample is preferable because it allows for a long contact reaction while preventing evaporation and drying by sealing the container. It is not necessary for the lectin and the biological sample to be in constant contact; it is sufficient for the mucin in the biological sample to bind to the lectin. Contact between the lectin and the biological sample may be carried out by standing or with stirring, but the method of carrying out the contact with stirring is preferred because it provides a higher reaction efficiency.

[0047] The amount of lectin added to the biological sample is not specified, but when nasal secretions are used as the biological sample, the lectin weight 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.

[0048] While there is no specific time requirement for the contact between the lectin and the biological sample, it is preferably one hour or longer, and more preferably until the reversible binding reaction between the lectin and the sugar chain reaches a plateau.

[0049] The temperature at which the lectin and the biological sample are brought into contact should be such that the liquid does not coagulate and the lectin does not denature easily, but it is preferably between 4°C and 37°C, and more preferably between 20°C and 30°C.

[0050] [1-5. Removal process] The present invention provides a method for producing a specimen, which includes a step of removing a mucin-lectin complex from the biological sample after contact (hereinafter referred to as the "removal step").

[0051] Methods for removing mucin-lectin complexes include, when using a carrier on which lectins are immobilized, removing the carrier to which the mucin-lectin complexes have bound after the contact step. When using particulate or fragmentary carriers on which lectins are immobilized, 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, pressurization, 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 is less likely to reduce the amount of the substance to be measured. When using plate-shaped or well-shaped carriers on which lectins are immobilized, the mucin-lectin complexes 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 on which lectins are immobilized, the mucin-lectin complexes bound to the carrier can be removed by crushing the carrier and isolating the sample that seeps out.

[0052] When lectins that are not immobilized on a carrier are used, the mucin-lectin complex can be removed after the contact step by removing the aggregates formed by the binding of mucin and lectin to each other. Methods for removing these aggregates include removing the precipitated aggregates and isolating the supernatant to use as a sample, or removing the aggregates 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 aggregates is more preferable because it is less likely to reduce the amount of the substance to be measured.

[0053] Furthermore, the removal of mucin by removing the mucin-lectin 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.

[0054] [1-6. Target substances to be detected] In this specification, the substance to be measured refers to a substance such as a biomarker that is to be detected in a biological sample such as nasal secretions.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] [2. Method for detecting the target substance] The present invention provides a method for detecting a target substance in a biological sample containing mucin, comprising the steps of: contacting the biological sample with lectin; removing the mucin-lectin complex from the biological sample after contact to obtain a sample; and detecting the target substance in the sample.

[0059] [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.

[0060] [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.

[0061] [3. Kit Contents] The present invention's method for producing a specimen using lectins can be carried out using a biological sample pretreatment kit containing mucin, which includes a carrier on which the lectins are immobilized.

[0062] A lectin-immobilized carrier can be used as a device to bring a biological sample into contact with the carrier. There are no particular restrictions on the specific configuration of the kit, but at a minimum, it should be a device that can remove mucin from the biological sample as a complex of mucin and the lectin after the lectin-immobilized carrier has been brought into contact with the biological sample.

[0063] Configurations that allow a lectin-immobilized carrier to come into contact with a biological sample include, for example, strips, filters, tubes, etc., for which lectins are bound to and immobilized on strips, filters, tubes, etc., and then a biological sample is added to the strips, etc., to bring them into contact, or for which a lectin-immobilized carrier is mounted or immobilized on strips, filters, tubes, etc., and a biological sample is added to them to bring them into contact. The immobilization method for immobilizing the lectin or lectin-immobilized carrier on the above-mentioned strips, 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.

[0064] The configuration for removing the mucin-lectin 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 lectin-immobilized carrier and collecting the supernatant, and a pipette.

[0065] The kit may also be configured to include a substance that selectively binds to the target substance (excluding mucin), thereby providing a kit for detecting the target substance in a biological sample. This substance is preferably an anti-immunoglobulin antibody. This substance may be immobilized on a carrier or included in the kit in an unimmobilized state. If the substance is immobilized on a carrier, the immobilized portion can be used as the detection area for the target substance. If it is unimmobilized, it can be added separately when using the kit; this can be done by adding the substance to a biological sample from which mucin has been removed and bringing it into contact with the sample. The method for detecting the complex of the selectively binding substance and the target substance is not particularly limited, and known detection methods such as fluorescence and chemiluminescence can be used. [Examples]

[0066] Examples are shown below, but the present invention is not limited to these examples.

[0067] [Examples 1 and 2] Examples 1 and 2 were carried out using the same procedure, except that the nasal secretions used were different.

[0068] (1) Preparation of lectin-immobilized carriers Lectin derived from Griffonia simplicifolia I (EY Laboratories) was dissolved at a concentration of 1 mg / mL in a coupling solution (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3). 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 lectin-immobilized Sepharose particles, a type of lectin immobilized on a carrier, was obtained.

[0069] (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. 20 μL of the collected nasal secretions were mixed with 40 μL of the lectin-immobilized Sepharose particle suspension prepared in (1), and the mixture was stirred for 1 hour. After the lectin-immobilized Sepharose particles had settled, the supernatant was collected to obtain a nasal secretion sample from which the lectin-immobilized Sepharose particles had been removed.

[0070] (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.

[0071] (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.

[0072] 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.

[0073] (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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] (6) Contact of the sample with the allergen-immobilized microarray (reaction step) The sample used was a nasal secretion sample prepared using the lectin 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.

[0078] (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.

[0079] (Fluorescence intensity measurement conditions) Equipment: 3D-Gene® Scanner 3000 (manufactured by Toray Industries) Excitation light wavelength: 635 nm Detection wavelength: 670nm PMT:33

[0080] The results are shown in Tables 1 and 2.

[0081] [Comparative Examples 1 and 2] Comparative Examples 1 and 2 were performed using the same procedure, except that the nasal secretions used were different.

[0082] As a control experiment for Examples 1 and 2, in Example 1(2), instead of collecting the supernatant after contacting nasal secretions with a suspension of lectin-immobilized Sepharose particles (corresponding to lectins immobilized on a carrier), 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.

[0083] The results are shown in Tables 1 and 2.

[0084] [Example 3] Experiments were conducted using different types of lectins than those used in Examples 1 and 2. Except for using a lectin derived from Dolichos biflorus (Sigma Aldrich) instead of a lectin derived from Griffonia simplicifolia I in Example 1(1), allergen-specific IgE in the samples was measured and evaluated in the same manner as in Example 1.

[0085] The results are shown in Table 2.

[0086] [Example 4] Experiments were conducted using different types of lectins than those used in Examples 1 and 2. Except for using a lectin derived from Jacalin (Thermo Fisher Scientific) instead of the lectin derived from Griffonia simplicifolia I in Example 1(1), allergen-specific IgE in the samples was measured and evaluated in the same manner as in Example 1.

[0087] The results are shown in Table 2.

[0088] [Table 1]

[0089] [Result 1: Comparison of Example 1 and Comparative Example 1] Using a sample obtained by removing mucin from nasal secretions with lectin-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). One possible explanation for this result is 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.

[0090] [Table 2]

[0091] [Result 2: Comparison of Examples 2-4 and Comparative Example 2] In addition to lectins derived from Griffonia simplicifolia I (Example 2), mucin was removed from nasal secretions using lectins derived from Dolichos biflorus (Example 3) and Jacalin (Example 4). Allergen-specific IgE antibodies in these samples were measured using a microarray method. The results showed improved signal values ​​for positive items compared to samples obtained by removing impurities from nasal secretions using membrane filtration (Comparative Example 2). This suggests that, compared to Comparative Example 2, the loss of allergen-specific IgE antibodies was reduced and detection was possible even when using different types of lectins in Examples 3 and 4, in addition to Example 2.

[0092] [Example 5] 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.

[0093] The results are shown in Table 3.

[0094] [Comparative Example 3] As a control experiment for Example 5, instead of the procedure in Example 5 in which rhinorrhea was brought into contact with a suspension of lectin-immobilized Sepharose particles (corresponding to lectins 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 rhinorrhea was placed on the membrane and quickly centrifuged (2000G, 1 min)). The albumin in the membrane-filtered sample was measured and evaluated in the same manner as in Example 5.

[0095] The results are shown in Table 3.

[0096] [Table 3]

[0097] [Result 3: Comparison of Example 5 and Comparative Example 3] When albumin in a sample obtained by removing mucin from nasal secretions using lectin-immobilized Sepharose particles (Example 5) 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 3). 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 3, albumin is significantly lost due to adsorption to the membrane, whereas in Example 5, albumin loss is reduced, allowing for detection.

Claims

1. A method for producing a sample to detect a target substance in a biological sample containing mucin, The steps of bringing the biological sample into contact with lectin, A step of removing the mucin-lectin complex from the biological sample after contact, Methods that include...

2. The method according to claim 1, wherein the biological sample is nasal secretions, saliva, or tears.

3. The method according to claim 2, wherein the biological sample is a biological sample collected by absorbing or adhering it using a water-absorbing member or a water-repellent member.

4. The method according to any one of claims 1 to 3, wherein the substance to be measured is a protein.

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

6. The method according to any one of claims 1 to 3, wherein the lectin is a lectin that binds to N-acetylgalactosamine.

7. The method according to any one of claims 1 to 3, wherein the lectin is a lectin derived from Dolichos bifloras, Griffonia simplicifolia I, Vicia villosa, Jacalin, or Wisteria floribunda.

8. The method according to any one of claims 1 to 3, wherein the lectin is a lectin immobilized on a carrier.

9. A specimen manufactured by the method described in any one of claims 1 to 3.

10. A method for detecting a target substance in a biological sample containing mucin, The process of bringing the biological sample into contact with lectin, The process of obtaining a sample by removing the mucin-lectin complex from the biological sample after contact, and A step of detecting the substance to be measured in the sample, Methods that include...

11. A kit for pre-treatment of mucin-containing biological samples, containing a lectin-immobilized carrier.

12. A kit for detecting target substances in mucin-containing biological samples, comprising a lectin-immobilized carrier and a substance that selectively binds to target substances (excluding mucin) in mucin-containing biological samples.

13. The kit according to claim 12, wherein the substance that selectively binds is an anti-immunoglobulin antibody.

14. The kit according to claim 12 or 13, wherein the selectively binding substance is a substance immobilized on a carrier.

Images