Method and kit for detecting cedar allergen-specific IgE antibodies in body fluid samples

Abstract

Disclosed are a method for detecting sugi allergen-specific IgE antibodies in a body fluid sample, which can improve the detection value of sugi allergen-specific IgE antibodies, and a kit for use therein. The method comprises the following steps: a removal step for removing non-IgE antibodies such as IgG antibodies from the body fluid sample; a reaction step for contacting the antibody-removed body fluid sample obtained in the removal step with a reaction well on a substrate having a reaction well, thereby forming a complex between the allergen and the allergen-specific IgE antibody; the reaction well comprising a region immobilizing the sugi allergen and regions immobilizing one or more allergens other than the sugi allergen; and a detection step for detecting the complex between the sugi allergen and the sugi allergen-specific IgE antibody obtained in the reaction step.

Description

Technical Field

[0001] The present invention relates to a method for detecting cedar allergen-specific IgE antibodies in body fluid samples and a kit for detecting cedar allergen-specific IgE antibodies. Background Art

[0002] In recent years, the in vitro detection of specific IgE antibodies in body fluid samples collected from patients has become widely used as a minimally invasive and safe allergy test. This method involves contacting a carrier immobilized with various allergens (allergy-inducing substances) such as cedar, mite, or milk with the body fluid sample, thereby forming a complex between the allergen and the specific IgE antibody. This complex is then detected using an anti-IgE antibody labeled with an enzyme, fluorescent dye, or radioactive substance, thereby detecting specific IgE antibodies in the body fluid sample.

[0003] Under the situation that specific IgE antibody is detected in vitro, there is the specific antibody except that specific IgE antibody that coexists identical with specific IgE antibody allergen specificity in the known body fluid sample, works as the competitive antibody that is combined with allergen with specific IgE antibody competition.That is, when body fluid sample is contacted with allergen immobilized carrier, above-mentioned competitive antibody and allergen form complex, hinder specific IgE antibody and allergenic complex to form, thereby exist and make the sensitivity and the such problem that accuracy of specific IgE antibody detection is reduced.As the solution to this problem, reported importing the part that uses specific absorption above-mentioned competitive antibody, before body fluid sample is contacted with allergen immobilized carrier, removed the method (patent documentation 1,2) of the removal process of above-mentioned competitive antibody in advance from body fluid sample. Patent Document 2 reports that when detecting milk allergen-specific IgE antibodies in body fluid samples using a 96-well microplate immobilized with milk allergen, the introduction of a removal step improves the detection value, allowing the specific IgE antibodies to be measured without being hindered by coexisting competing antibodies.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Laid-Open No. 3-25366

[0007] Patent Document 2: Japanese Patent Application Laid-Open No. 2000-266746 Summary of the Invention

[0008] Problems to be solved by the invention

[0009] The present inventors, in order to expect the effect of improving the detection value brought by removing the competing antibodies coexisting in the body fluid sample, detected the cedar allergen-specific IgE antibody, with reference to the method described in patent documentation 2, implemented the detection of the specific IgE antibody using 96-well microplates. Specifically, as in comparative example 6 described later, the cedar allergen was immobilized on a 96-well microplate, the body fluid sample from which the competing antibodies had been removed was contacted, and the complex of the cedar allergen and the specific IgE antibody was detected. The result was different from the situation of detecting the milk allergen-specific IgE antibody reported in patent documentation 2, and the detection of the cedar allergen-specific IgE antibody implemented by the present inventors did not confirm the improvement of the detection value brought by the removal process. That is, only implementing the removal process could not improve the detection value in the detection of the cedar allergen-specific IgE antibody using 96-well microplates.

[0010] Methods for solving problems

[0011] The present inventors have conducted intensive studies to overcome the above-mentioned problems and have discovered that by using a substrate on which a sugi allergen and one or more allergens other than the sugi allergen are independently immobilized in the same reaction vessel, and contacting the substrate with a body fluid sample from which competing antibodies have been removed, the detection value of sugi allergen-specific IgE antibodies can be improved.

[0012] That is, the present invention is constituted by the following aspects (1) to (8).

[0013] (1) A method for detecting cedar allergen-specific IgE antibodies in a body fluid sample, the method comprising the following steps:

[0014] a removal step of removing at least one antibody selected from the group consisting of IgG antibodies, IgM antibodies, IgA antibodies, and IgD antibodies from the body fluid sample;

[0015] a reaction step of contacting the antibody-removed body fluid sample obtained in the removal step with a reaction well of a substrate provided with a reaction well, thereby forming a complex of the allergen and the allergen-specific IgE antibody, wherein the reaction well includes a region where the sugi allergen is immobilized and regions where one or more allergens other than the sugi allergen are immobilized; and

[0016] The detection step is to detect the complex of the cedar allergen and the cedar allergen-specific IgE antibody obtained in the above reaction step.

[0017] (2) The method according to (1), wherein the allergen other than the cedar allergen immobilized on the substrate is at least one allergen selected from the group consisting of shrimp, crab, soybean, peanut, buckwheat, walnut, milk, egg white, wheat, orchard grass, dog, cat, and mite.

[0018] (3) The method according to (1) or (2), wherein the substrate on which the sugi allergen and one or more allergens other than the sugi allergen are immobilized in the same reaction chamber is a microarray.

[0019] (4) The method according to any one of (1) to (3), wherein in the removal step, IgG antibodies are removed from the body fluid sample.

[0020] (5) The method according to any one of (4), wherein the IgG antibodies are removed in the removal step by allowing the IgG antibodies to be adsorbed and removed by a carrier on which protein G is immobilized.

[0021] (6) According to the method according to any one of (1) to (5), the detection of the complex of the allergen and the allergen-specific IgE antibody in the above-mentioned detection step is carried out by reacting an anti-IgE antibody labeled with a fluorescent dye with the above-mentioned complex and detecting the fluorescent dye bound to the complex.

[0022] (7) The method according to any one of (1) to (6), wherein the body fluid sample is blood, serum, or plasma.

[0023] (8) A kit for detecting Sugi allergen-specific IgE antibodies in a body fluid sample, comprising an allergen microarray and a carrier, wherein the allergen microarray has a substrate provided with a reaction tank, the reaction tank comprising an area on which Sugi allergen is immobilized and an area on which one or more allergens other than Sugi allergen are immobilized, and the carrier has a ligand that can selectively bind to at least one antibody selected from IgG antibodies, IgM antibodies, IgA antibodies, and IgD antibodies immobilized thereon.

[0024] (9) The kit according to (8), wherein the carrier on which the ligand capable of selectively binding to the IgG antibody is solid-phased is a carrier on which protein G is solid-phased.

[0025] Effects of the Invention

[0026] By contacting a body fluid sample from which specific IgG, IgM, IgA, or IgD antibodies have been previously removed with a substrate on which a sugi allergen and one or more allergens other than the sugi allergen are independently immobilized in the same reaction chamber, the detection value of sugi allergen-specific IgE antibodies can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a diagram showing an example of a substrate having a reaction chamber used in the method of the present invention.

[0028] Figure 2This is a diagram showing an example of a substrate having partition walls provided on the outer edge of a reaction vessel used in the method of the present invention.

[0029] Figure 3 This is a schematic diagram of a substrate provided with reaction chambers used in Example 1. DETAILED DESCRIPTION

[0030] Examples of body fluid samples used in the present invention include whole blood, plasma, serum, sweat, urine, tears, saliva, sputum / airway secretions, breast milk, amniotic fluid, cerebrospinal fluid, ascites, pleural effusion, joint fluid, semen, and vaginal secretions, but blood (whole blood), plasma, or serum that is highly likely to contain cedar allergen-specific IgE antibodies is preferred.

[0031] The step of removing at least one antibody selected from IgG, IgM, IgA, and IgD antibodies from a body fluid sample can be performed by adsorbing and removing at least one antibody selected from IgG, IgM, IgA, and IgD antibodies (hereinafter, sometimes referred to as "non-IgE antibodies" for convenience). In particular, since IgG antibodies are the most abundant antibodies in a body fluid sample, adsorption and removal of IgG antibodies are preferred to eliminate competition with safflower allergen-specific IgE antibodies. Furthermore, a method that removes at least one of the other three non-IgE antibodies in addition to IgG antibodies is also preferred.

[0032] As a method for removing antibodies by adsorption, chromatography methods such as affinity chromatography and ion exchange chromatography can be cited, but affinity chromatography is preferred due to the short time required. When using affinity chromatography to adsorb non-IgE antibodies in a body fluid sample and remove them, it is sufficient to contact the body fluid sample with a carrier on which a ligand capable of selectively binding to the non-IgE antibodies is immobilized. Specifically, a method in which the body fluid sample is passed through a column filled with the above-mentioned carrier and the body fluid sample is recovered from the carrier by centrifugation or the like can be cited; a method in which the body fluid sample is suspended in the above-mentioned carrier and the suspension is centrifuged to recover the body fluid sample, but a method using a column filled with the above-mentioned carrier is preferred.

[0033] In the affinity chromatography, as the ligand immobilized on the carrier, in addition to antibodies such as anti-IgG antibodies, anti-IgM antibodies, anti-IgA antibodies, and anti-IgD antibodies, known ligands such as protein A from Staphylococcus aureus, protein G from type G hemolytic streptococci, protein L from Gram-positive anaerobic cocci, and jacalin from jackfruit can also be used. Among them, when IgG antibodies are excluded, protein G is preferably used because of its high selective binding ability to IgG antibodies, low cost, and easy availability.

[0034] As the material of the carrier used for the above-mentioned affinity chromatography, any one of resin, glass, metal, silicon wafer, etc. can be used, but resin is preferably considered from the viewpoint of the ease of surface treatment and mass production. As the resin that becomes the material of the carrier, for example, agarose, polyacrylate, polymethacrylate, polycarbonate, polystyrene, polyvinyl acetate, polyester, etc. can be mentioned, but agarose with less non-specific adsorption to components in the body fluid sample is preferably used. As the form of the carrier with the above-mentioned ligand immobilization, any one of particles, substrates, etc. can be used, but particles are preferably used in terms of large specific surface area and the amount of immobilization of the above-mentioned ligand.

[0035] The above-mentioned carrier can be purchased from a commercial product such as GE Healthcare and used, or a carrier without a ligand immobilized thereon can be purchased and then immobilized with a desired ligand. For example, as described in the following examples, carriers immobilized with protein G are also commercially available, and therefore such commercially available products can be preferably used.

[0036] In the present invention, in the reaction step, the antibody-removed body fluid sample is brought into contact with a substrate having a reaction reservoir, the reaction reservoir comprising a region immobilizing a sugae allergen and regions immobilizing one or more allergens other than the sugae allergen. The contact conditions may be known conditions for inducing an antigen-antibody reaction between the antibodies in the body fluid sample and the immobilized allergens. For example, the body fluid sample or a dilution thereof can be brought into contact with the immobilized allergens at room temperature to 37°C for approximately 1 to 4 hours.

[0037] An example of the substrate used here is shown in Figure 1 A reaction well 2 is provided in a substrate 1 as an area for contacting a body fluid sample. The reaction well 2 includes two distinct, non-overlapping areas: a sugi allergen-immobilizing area 3 on which a sugi allergen is immobilized, and an allergen-immobilizing area 4 on which one or more allergens other than the sugi allergen are immobilized. Specifically, the substrate used in the present invention allows a body fluid sample to contact both the sugi allergen and the allergen-immobilizing area within the same reaction well.

[0038] Therefore, a substrate consisting of a reaction tank having only a region 3 on which the fir allergen is immobilized and a substrate consisting of a reaction tank having only a region 4 on which allergens other than the fir allergen are immobilized do not correspond to the substrate used in the present invention. In addition, a substrate consisting of a reaction tank having a region 3 for immobilizing the fir allergen and a region 4 for immobilizing allergens other than the fir allergen, which are physically separated from each other by a partition wall, does not correspond to the substrate used in the present invention because it does not allow the body fluid sample to contact both the fir allergen and the allergen other than the fir allergen in the same reaction tank. The partition wall referred to here refers to a convex structure provided on the substrate that physically separates the allergen immobilization regions. For example, a multi-hole plate in which different allergens are immobilized in each well, in which the region for immobilizing the fir allergen and the region for immobilizing the allergen other than the fir allergen are physically separated by a partition wall, does not correspond to the substrate used in the present invention.

[0039] The addition of the antibody-removed body fluid sample to the reaction vessel can be carried out in a manner that allows the body fluid sample to come into contact with all the allergens immobilized in the reaction vessel. The body fluid sample may be diluted with a buffer such as phosphate-buffered saline before addition to the reaction vessel, or may be added to the reaction vessel without dilution.

[0040] In the interior of the reaction tank, as long as the body fluid sample can contact all the allergens immobilized in the reaction tank, a concave structure such as a flow path can be provided. Figure 2 In order to suppress leakage of the body fluid sample to the outside of the reaction tank during the reaction step, a structure such as a partition wall 5 is provided at the outer edge of the reaction tank, thereby physically isolating the reaction tank from the outside.

[0041] As the processing method for the interior and outer edge of the above-mentioned reaction tank, there are methods of cutting the substrate using a drill or laser, methods of cutting the mold used when molding the substrate and performing injection molding, compression molding, vacuum molding, etc., but considering the stability of quality and the ease of mass production, the above-mentioned method of cutting the mold is more preferred.

[0042] Specific forms of the substrate having the reaction wells include an allergen microarray in which allergens are two-dimensionally immobilized, a microfluidic device in which allergens are immobilized in fine flow channels, and the like. However, an allergen microarray is preferred in which a body fluid sample can easily come into contact with all the allergens immobilized in the same reaction well.

[0043] The cedar allergen and one or more allergens other than the cedar allergen can be immobilized on the reaction vessel by spotting a trace amount of the allergen solution on the surface of the reaction vessel using a spotting device, or by contacting the allergen solution containing each allergen with the surface of the reaction vessel so that they do not mix with each other.

[0044] The immobilized allergen may be bonded to the reaction vessel by physical adsorption or covalent bonding, but covalent bonding is preferred from the viewpoint of suppressing the peeling and elution of the allergen from the substrate during washing of the substrate.

[0045] As the method that allergen is immobilized in reaction tank by covalent bond, the method that the functional groups such as amino, carboxyl, hydroxyl that are present in the surface of allergen react the functional group that is present in reaction tank and covalent bond is formed can be used.In the case that substrate is resin system mentioned later, as the functional group on the surface that is present in reaction tank, amino, carboxyl, isothiocyanate etc. can be enumerated, as the pattern of covalent bond, can be any one in amide bond, thiourea bond, ether bond etc., but consider preferably amide bond from the viewpoint of the easiness and firmness of key formation.Adopt the combination of amide bond for example can be carried out (with reference to following embodiment) by known method by using the succinimide compound such as N-hydroxysuccinimide (NHS) and the carbodiimide compound such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.

[0046] The allergen in the zone of the reaction tank where the immobilization zone of the cedar allergen exists, except the cedar allergen, is preferably at least one selected from shrimp, crab, soybean, peanut, buckwheat, walnut, milk, egg white, wheat, orchard grass, dog, cat and mite.Preferably more than 3 kinds among them, more preferably more than 4 kinds of allergens are immobilized.Each allergen can be purchased from the commercially available products of GREER society products etc., be dissolved in buffers such as phosphate buffered saline and use as allergen solution, or can modulate the solution comprising desired allergen.As the modulation method of allergen solution, for example, the allergen raw material can be supplied to the pulverization process that has used mixing mill etc. or use organic solvents such as ether, acetone, hexane and supply to defatting process, stir in buffers such as phosphate buffered saline and extract the allergen, and this centrifugal supernatant is modulated to suitable allergen concentration and use.

[0047] The material of the substrate used in the present invention may be, for example, any of resin, glass, metal, and silicon wafer. Resin is preferred from the viewpoint of ease of surface treatment and mass productivity.

[0048] Examples of the resin forming the substrate include polyacrylates, polymethacrylates, polycarbonates, polystyrenes, polyvinyl acetates, and polyesters, with polyacrylates and polymethacrylates being preferred. Examples of polymethacrylates include polymethyl methacrylate (PMMA), polyethyl methacrylate (PEMA), and polyalkyl methacrylates (PAMAs) such as polypropyl methacrylate, with PMMA being preferred.

[0049] In the present invention, allergen-specific IgE antibodies can be detected by contacting a body fluid sample from which non-IgE antibodies have been removed with the reaction wells on the substrate, washing the reaction wells to remove unbound matter, and then detecting the complex of allergen and allergen-specific IgE antibodies.

[0050] The method for detecting the complex of allergen and allergen-specific IgE antibody itself is well known, for example, can enumerate, adopt the surface plasmon resonance method using the refractive index difference caused by combination as the detection principle, the quartz crystal microbalance method etc. using the resonance frequency difference as the detection principle, the method using the anti-IgE antibody labeled by fluorescent substance (fluorochrome), the enzyme that generates coloration / luminescent substance, radioisotope element etc. Among them, from the ease of operation and safety consideration, preferably use the method for detecting by the anti-IgE antibody labeled by fluorescent substance. In this case, make the anti-IgE antibody labeled by fluorescent substance react with the above-mentioned complex, after washing, detect the fluorochrome that is combined with the complex.

[0051] Another embodiment of the present invention is a kit for detecting sugi allergen-specific IgE antibodies in a body fluid sample, the kit comprising an allergen microarray and a carrier as components. The allergen microarray comprises a substrate provided with reaction wells, the reaction wells comprising regions immobilizing a sugi allergen and regions immobilizing one or more allergens other than the sugi allergen, and the carrier immobilizing a ligand that selectively binds to at least one antibody selected from IgG, IgM, IgA, and IgD antibodies (for example, a carrier immobilizing protein G as a ligand that selectively binds to IgG). This substrate, similar to the substrate used in the method for detecting sugi allergen-specific IgE antibodies of the present invention, contains regions immobilized within the reaction wells for the sugi allergen and one or more allergens other than the sugi allergen. A carrier on which a ligand (eg, protein G) capable of selectively binding to these antibodies is immobilized is used in the step of removing the antibodies (eg, IgG antibodies) from a body fluid sample in the method of detecting sugi allergen-specific IgE antibodies of the present invention.

[0052] The test kit for detecting sprue allergen-specific IgE antibodies in body fluid samples of the present invention may include, as components other than those mentioned above, anti-IgE antibodies labeled with fluorescent substances (fluorochromes), enzymes that generate color-emitting / luminescent substances, radioactive isotope elements, etc., standard samples, diluents, washing solutions, reaction stop solutions, etc. Each of the above-mentioned components may be in the form of a suspension, solution, or freeze-dried product. Furthermore, the kit may include the apparatus and software required for the assay / analysis of the kit, a computer equipped with the software, instructions for use of the kit, and instructions explaining the procedures.

[0053] Example

[0054] Examples are shown below, but the present invention is not limited to these examples.

[0055] Example 1

[0056] Detection of cedar allergen-specific IgE antibodies in IgG-depleted human serum samples using microarrays immobilized with cedar, shrimp, crab, soybean, peanut, and buckwheat allergens

[0057] (1) Preparation of NHS-esterified PMMA substrate

[0058] Will Figure 3 A polymethyl methacrylate (PMMA) substrate (75 mm × 25 mm × 1.0 mm) equipped with a reaction tank (12.5 mm × 10 mm × 0.15 mm) as shown was immersed in a 10N sodium hydroxide aqueous solution at 70°C for 15 hours. The substrate was then washed in pure water, 0.1N HCl aqueous solution, and pure water, in that order. This hydrolyzed the PMMA side chains on the substrate surface, generating carboxyl groups.

[0059] 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 0.1N sodium hydroxide). The hydrolyzed PMMA substrate was immersed in this mixed solution and stirred for 1 hour using a micro stirrer to obtain a PMMA substrate esterified with NHS.

[0060] (2) Preparation of allergen solution

[0061] Sugi allergen was obtained from Hayashibara Co., Ltd. as a 0.2 mg / mL phosphate-buffered saline solution and used directly in the subsequent steps. Shrimp, crab, soybean, peanut, and buckwheat allergens were obtained from GREER Co., Ltd. as freeze-dried powders. Each freeze-dried powder was dissolved in pure water to a protein concentration of 1.0 mg / mL and used as the allergen solution in the subsequent steps.

[0062] (3) Preparation of allergen-immobilized microarray

[0063] The allergen solutions of cedar, shrimp, crab, soybean, peanut, and buckwheat prepared in (2) were spotted in one reaction tank of the NHS-esterified PMMA resin substrate prepared above using a spotting robot (GTMASStamp-2, manufactured by Japan Rayza Electronics Co., Ltd.). Next, the substrate was placed in a sealed plastic container and incubated overnight at 37°C and 100% humidity to immobilize the allergens on the surface of the reaction tank. After incubation, the substrate was washed with phosphate-buffered saline (0.05% Tween 20 (trade name)). By operating as described above, a microarray in which the allergens of cedar, shrimp, crab, soybean, peanut, and buckwheat were immobilized in the same reaction tank was obtained.

[0064] (4) Removal of IgG antibodies from human serum (removal step)

[0065] 50 μL of a suspension of a protein G-immobilized carrier (Protein G Sepharose 4 Fast Flow) (GE Healthcare Chemical Science) was dispensed into a spin column (MoBiTec), and the liquid component of the suspension was separated from the carrier by centrifugation (1500 G, 1 minute), and the carrier was dried on the column.

[0066] 20 μL of human serum as a body fluid sample was added dropwise to the protein G-immobilized carrier column prepared as described above, and the column was allowed to permeate. The column was then allowed to stand at room temperature for 30 minutes to allow the IgG antibodies in the sample to adsorb to the protein G. The column was placed in a 1.5 mL Eppendorf tube, and the human serum was transferred from the column to a 1.5 mL Eppendorf tube by centrifugation (1500 g, 1 minute) to separate it from the carrier, thereby obtaining a human serum sample from which the IgG antibodies had been removed.

[0067] (5) Contact of human serum with allergen-immobilized microarray (reaction step)

[0068] The IgG antibody-depleted human serum sample obtained in (4) was diluted 3-fold with phosphate-buffered saline. 50 μL of this dilution was added dropwise to the reaction chamber of the substrate prepared in (3). The reaction was sealed with a gap cover glass (manufactured by Matsunami Glass Industries, Ltd.: 24 mm × 25 mm, gap size 20 μm). After reacting at 37°C for 2 hours, the gap cover glass was removed and the substrate was washed with phosphate-buffered saline (0.05% Tween 20 (trade name)).

[0069] (6) Detection of Sugi Allergen-Specific IgE Antibodies (Detection Step)

[0070] A 1.0 mg / mL solution of Dylight-650 dye-labeled anti-human IgE goat polyclonal antibody (manufactured by Novus Biologicals) was diluted 1000-fold with phosphate-buffered saline (0.05% Tween 20 (trade name)) containing 1% by weight of bovine serum albumin. 50 μL of the resulting dilution was added dropwise to the reaction chamber of the substrate that had been in contact with human serum in (5). The reaction was allowed to proceed at room temperature for 1 hour with a gap cover glass. The gap cover glass was then removed, and the substrate was washed with phosphate-buffered saline (0.05% Tween 20 (trade name)).

[0071] The substrate was placed on a "3D Gene (registered trademark) Scanner" (Toray Co., Ltd.), and fluorescence detection of the sugi allergen-immobilized area was performed with the excitation light set to 635 nm, laser output 100%, and PMT 30. The fluorescence intensity is shown in Table 1. The detected fluorescence originates from the fluorescent dye contained in the labeled antibody bound to the complex of sugi allergen and sugi allergen-specific IgE antibody. A higher amount of sugi allergen-specific IgE antibody bound to the allergen-immobilized microarray indicates a higher detection value.

[0072] Comparative Example 1

[0073] As a control experiment for Example 1, detection of cedar allergen-specific IgE antibodies in human serum samples was carried out in the same manner as in Example 1, except that the step of removing IgG antibodies from the serum samples in (4) of Example 1 was omitted. The results of the fluorescence intensity measurement are shown in Table 1.

[0074] Example 2

[0075] Detection of cedar allergen-specific IgE antibodies in IgG-depleted human serum samples using microarrays immobilized with cedar, walnut, milk, egg white, and wheat allergens

[0076] Detection of cedar allergen-specific IgE antibodies in human serum samples was carried out in the same manner as in Example 1, except that walnut, milk, egg white, and wheat allergens (GREER) were used as allergens other than the cedar allergen in (2) and (3) of Example 1. The results of the fluorescence intensity measurement are shown in Table 1.

[0077] Comparative Example 2

[0078] As a control experiment for Example 2, detection of cedar allergen-specific IgE antibodies in human serum samples was carried out in the same manner as in Example 2, except that the step of removing IgG antibodies from the serum samples was not performed. The results of the fluorescence intensity measurement are shown in Table 1.

[0079] Example 3

[0080] Detection of cedar allergen-specific IgE antibodies in IgG-depleted human serum samples using microarrays immobilized with cedar, orchardgrass, dog, cat, and mite allergens

[0081] Detection of cedar allergen-specific IgE antibodies in human serum samples was carried out in the same manner as in Example 1, except that orchardgrass, dog, cat, and mite allergens (GREER) were used as allergens in addition to the cedar allergen in (2) and (3) of Example 1. The results of the fluorescence intensity measurement are shown in Table 1.

[0082] Comparative Example 3

[0083] As a control experiment for Example 3, detection of cedar allergen-specific IgE antibodies in human serum samples was carried out in the same manner as in Example 3, except that the step of removing IgG antibodies from serum samples was not performed. The results of the fluorescence intensity measurement are shown in Table 1.

[0084] Comparative Example 4

[0085] Detection of Sugi Allergen-Specific IgE Antibodies in IgG-Depleted Human Serum Samples Using a Sugi Allergen-Immobilized Microarray

[0086] Detection of cedar allergen-specific IgE antibodies in human serum samples was carried out in the same manner as in Example 1, except that no allergen other than cedar allergen was used in (2) and (3) of Example 1. The results of the fluorescence intensity measurement are shown in Table 1.

[0087] Comparative Example 5

[0088] As a control experiment for Comparative Example 4, detection of cedar allergen-specific IgE antibodies in human serum samples was carried out in the same manner as in Comparative Example 4, except that the step of removing IgG antibodies from the serum samples was not performed. The results of the fluorescence intensity measurement are shown in Table 1.

[0089] Table 1

[0090] Sugi allergen immobilization microarray

[0091]

[0092] When using human serum from which IgG antibodies had been removed in Examples 1 to 3, the fluorescence intensity detected was higher than when using human serum from which IgG antibodies had not been removed in Comparative Examples 1 to 3. This is presumably due to the removal of IgG antibodies from the human serum, which act as inhibitors against the formation of complexes between sugi allergen and sugi allergen-specific IgE antibodies, and the resulting improvement in the efficiency of binding of sugi allergen-specific IgE antibodies to the sugi allergen immobilized on the microarray.

[0093] On the other hand, Comparative Example 4, which used a substrate immobilized with only sugi allergen and human serum from which IgG antibodies had been removed, did not show a higher value than Comparative Example 5, which used human serum from which IgG antibodies had not been removed. This indicates that even if IgG antibodies were removed from human serum, the binding efficiency of sugi allergen-specific IgE antibodies to sugi allergen was not improved.

[0094] Improvement in the binding efficiency of sugi allergen-specific IgE antibodies due to the removal of IgG antibodies was observed only in Examples 1, 2, and 3 using substrates immobilized with sugi allergen and one or more allergens other than sugi allergen. Therefore, it is speculated that when human serum is brought into contact with the reaction vessel, the immobilized allergens other than sugi allergen coexisting with the sugi allergen in the reaction vessel synergize with the removal of IgG antibodies to improve the binding efficiency of sugi allergen-specific IgE antibodies to the sugi allergen.

[0095] It was found that in order to detect sugi allergen-specific IgE antibodies without being hindered by competition with specific IgG antibodies, it is necessary to use a reaction vessel in which sugi allergen and allergens other than sugi allergen are co-immobilized.

[0096] Comparative Example 6

[0097] Detection of Sugi Allergen-Specific IgE Antibodies in IgG-Depleted Human Serum Samples Using a Sugi Allergen-Immobilized 96-Well Microplate

[0098] (1) Preparation of cedar allergen solution

[0099] Sugi allergen was obtained from Hayashibara Co., Ltd. as a 0.2 mg / mL phosphate-buffered saline solution and diluted 10-fold with phosphate-buffered saline to prepare a sugi allergen solution with a final concentration of 0.02 mg / mL.

[0100] (2) Preparation of 96-well microplates with immobilized cedar allergen

[0101] 50 μL of the cedar allergen solution prepared in (1) was added to each well of a polystyrene 96-well microplate (manufactured by Watson), and the plate was left to stand at 4°C for 16 hours. The solution in the well was discarded, and 200 μL of phosphate-buffered saline containing 1% by weight bovine serum albumin was added to the same well, and the plate was left to stand at room temperature for 2.5 hours. The well was then washed with phosphate-buffered saline (0.05% Tween 20 (trade name)). As described above, a microplate with cedar allergen immobilized in each well was obtained.

[0102] (3) Contact of human serum with the cedar allergen-immobilized microplate

[0103] In the same manner as in Example 1 (4), a human serum sample from which IgG antibodies had been removed was prepared.

[0104] The human serum sample was diluted threefold with phosphate-buffered saline containing 1% by weight bovine serum albumin. 50 μL of this dilution was added to the wells of a microplate immobilized with sugae allergen and allowed to stand at 37°C for 2 hours. The wells were then washed with phosphate-buffered saline (0.05% Tween 20 (trade name)).

[0105] (4) Detection of cedar allergen-specific IgE antibodies

[0106] The horseradish peroxidase-labeled anti-human IgE goat polyclonal antibody (SouthernBiotech) 1.0 mg / mL solution was diluted 5000 times with phosphate-buffered saline (0.05% Tween20 (trade name)) containing 1% by weight of bovine serum albumin, and 50 μL of the dilution was added to the hole in which the cedar allergen was immobilized and allowed to stand at 37°C for 1 hour. The same hole was washed with phosphate-buffered saline (0.05% Tween20 (trade name)). 50 μL of TMB OneComponent HRP Microwell Substrate (Cosmobio) was added to the same hole and allowed to stand at room temperature for 15 minutes. 50 μL of 1 mol / mL sulfuric acid aqueous solution was added to the same hole, and the absorbance at a wavelength of 450 nm was measured using an enzyme reader (Morekiura Debisu). The results are shown in Table 2.

[0107] Comparative Example 7

[0108] As a control experiment for Comparative Example 6, the sugi allergen-specific IgE antibodies in human serum were detected using a sugi allergen-immobilized 96-well microplate in the same manner as in Comparative Example 6, except that IgG antibodies were not removed from the serum samples in (3) of Comparative Example 6. The results are shown in Table 2.

[0109] Table 2. 96-well microplate with immobilized cedar allergen

[0110] IgG removal process Absorbance measurement value Comparative Example 6 have 3.23 Comparative Example 7 none 3.40

[0111] Table 2 shows the results of detection of sugi allergen-specific IgE antibodies using a sugi allergen-immobilized 96-well microplate, performed in Comparative Examples 6 and 7. The absorbance detected here originates from TMB substrate oxidized by horseradish peroxidase and contained in the labeled antibody bound to the complex of sugi allergen and sugi allergen-specific IgE antibody. A higher amount of sugi allergen-specific IgE antibody bound to the sugi allergen-immobilized 96-well microplate indicates a higher detection value.

[0112] Similar to Comparative Examples 4 and 5, which used microarrays immobilizing only the sugi allergen, in Comparative Examples 6 and 7, which used 96-well microplates immobilizing only the sugi allergen, Comparative Example 6, which used human serum from which IgG antibodies had been removed, did not show a higher value than Comparative Example 7, which used human serum from which IgG antibodies had not been removed. This indicates that even when a 96-well microplate was used, the binding efficiency of sugi allergen-specific IgE antibodies to sugi allergen was not improved.

[0113] Explanation of symbols

[0114] 1 substrate

[0115] 2 reaction tanks

[0116] 3 Immobilization area of cedar allergen

[0117] 4 Immobilization area of allergens other than cedar allergen

[0118] 5 next door.

Claims

1. A method for detecting cedar allergen-specific IgE antibodies in a body fluid sample, the method comprising the following steps: Removal process, removing IgG antibodies from body fluid samples; a reaction step of contacting the antibody-removed body fluid sample obtained in the removal step with a reaction well of a substrate provided with a reaction well, thereby forming a complex of the allergen and the allergen-specific IgE antibody, the reaction well comprising a region where the sugi allergen is immobilized and a region where an allergen other than the sugi allergen is immobilized; and a detection step of detecting the complex of the cedar allergen and the cedar allergen-specific IgE antibody obtained in the reaction step, The allergens other than the cedar allergen immobilized on the substrate are at least four allergens selected from the group consisting of shrimp, crab, soybean, peanut, buckwheat, walnut, milk, egg white, wheat, orchard grass, dog, cat, and mite. 2 . The method according to claim 1 , wherein the substrate on which the sugi allergen and allergens other than the sugi allergen are immobilized in the same reaction chamber is a microarray. 3 . The method according to claim 1 , wherein the IgG antibodies are removed in the removal step by allowing the IgG antibodies to be adsorbed and removed by a carrier on which protein G is immobilized.

4. The method according to claim 1 or 2, wherein the detection of the complex of the allergen and the allergen-specific IgE antibody in the detection step is performed by reacting an anti-IgE antibody labeled with a fluorescent dye with the complex and detecting the fluorescent dye bound to the complex. The method according to claim 1 or 2, wherein the body fluid sample is blood, serum or plasma.

6. A kit for detecting cedar allergen-specific IgE antibodies in body fluid samples, comprising an allergen microarray and a carrier, The allergen microarray comprises a substrate provided with reaction chambers, wherein the reaction chambers include a region where a sugi allergen is immobilized and a region where an allergen other than the sugi allergen is immobilized. The carrier solid-phases a ligand that can selectively bind to an IgG antibody. The allergens other than the cedar allergen immobilized on the substrate are at least four allergens selected from the group consisting of shrimp, crab, soybean, peanut, buckwheat, walnut, milk, egg white, wheat, orchard grass, dog, cat, and mite. 7 . The kit according to claim 6 , wherein the carrier on which the ligand capable of selectively binding to the IgG antibody is immobilized is a carrier on which protein G is immobilized.

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