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

Abstract

Disclosed are a method for detecting cedar-allergen-specific IgE antibodies in a body fluid sample, and a kit for achieving said method, with which it is possible to improve detection values in the detection of cedar-allergen-specific IgE antibodies. This method includes: a removal step for removing IgG antibodies and other non-IgE antibodies from a body fluid sample; a reaction step for bringing the post-antibody-removal body fluid sample obtained in the removal step into contact with a reaction vessel, which includes a region to which cedar allergens are fixed and a region to which one or more types of allergens other than cedar allergens are fixed, in a substrate provided with the reaction vessel, and forming a complex of the allergens and the allergen-specific IgE antibodies; and a detection step for detecting the complex of the allergens and the cedar-allergen-specific IgE antibodies obtained in the reaction step.

Description

[Technical field]

[0001] The present invention relates to a method for detecting Japanese Cedar allergen-specific IgE antibodies in a body fluid sample and a kit for detecting Japanese Cedar allergen-specific IgE antibodies. [Background technology]

[0002] In recent years, a method for detecting specific IgE antibodies in a body fluid sample collected from a patient outside the body has been widely used as a minimally invasive and safe allergy testing method. In this method, a carrier on which various allergens (allergy-inducing substances), such as cedar, dust mites, or milk, are immobilized is brought into contact with the body fluid sample to form a complex between the allergen and the specific IgE antibody, and the complex is then detected using an anti-IgE antibody labeled with an enzyme, a fluorescent dye, a radioactive substance, or the like, thereby detecting the specific IgE antibody in the body fluid sample.

[0003] When detecting specific IgE antibodies outside the body, it is known that specific antibodies other than the specific IgE antibodies coexisting in a body fluid sample and having the same allergen specificity as the specific IgE antibodies exist, and act as competing antibodies that compete with the specific IgE antibodies to bind to allergens. That is, when a body fluid sample is contacted with an allergen-immobilized carrier, the competing antibodies form a complex with the allergen, inhibiting the formation of a complex between the specific IgE antibody and the allergen, thereby reducing the sensitivity and accuracy of the detection of specific IgE antibodies. As a solution to this problem, a method has been reported in which a removal step is introduced to remove the competing antibodies from the body fluid sample before contacting the body fluid sample with an allergen-immobilized carrier using a ligand that specifically adsorbs the competing antibodies (Patent Documents 1 and 2). Patent Document 2 reports that when a 96-well microplate with immobilized milk allergens is used to detect milk allergen-specific IgE antibodies in a body fluid sample, the detection value is improved by the introduction of a removal step, and the specific IgE antibodies can be measured without interference from the coexisting competing antibodies. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 3-25366 [Patent Document 2] JP 2000-266746 A Summary of the Invention [Problem to be solved by the invention]

[0005] In order to detect Japanese cedar allergen-specific IgE antibodies, the present inventors performed detection of specific IgE antibodies using a 96-well microplate with reference to the method described in Patent Document 2, in hopes of improving the detection value by removing competing antibodies coexisting in a body fluid sample. Specifically, as described in Comparative Example 6 below, Japanese cedar allergen was immobilized on a 96-well microplate, and a body fluid sample from which competing antibodies had been removed in advance was contacted to detect a complex between the Japanese cedar allergen and the specific IgE antibody. As a result, unlike the detection of milk allergen-specific IgE antibodies reported in Patent Document 2, the detection of Japanese cedar allergen-specific IgE antibodies performed by the present inventors did not show an improvement in the detection value due to the removal process. In other words, the detection value could not be improved in the detection of Japanese cedar allergen-specific IgE antibodies using a 96-well microplate by only performing the removal process. [Means for solving the problem]

[0006] As a result of intensive research conducted by the inventors to overcome the above-mentioned problems, they discovered that by using a substrate on which Japanese cedar allergen and one or more allergens other than Japanese cedar allergen are independently immobilized in the same reaction tank, and by contacting the substrate with a body fluid sample from which competing antibodies have been previously removed, it is possible to improve the detection value in the detection of Japanese cedar allergen-specific IgE antibodies.

[0007] That is, the present invention is constituted by the following aspects (1) to (8). (1) A method for detecting Japanese cedar allergen-specific IgE antibodies in a body fluid sample, comprising the steps of: a removal step of removing at least one antibody selected from the group consisting of IgG antibody, IgM antibody, IgA antibody, and IgD antibody from the body fluid sample; a reaction step in which the body fluid sample from which the antibodies have been removed, obtained in the removal step, is brought into contact with a reaction chamber of a substrate provided with the reaction chamber, the reaction chamber including an area in which the Japanese cedar allergen is immobilized and an area in which one or more allergens other than the Japanese cedar allergen are immobilized, to form a complex between the allergen and the allergen-specific IgE antibody; and a detection step of detecting the complex between the Japanese cedar allergen and the Japanese cedar allergen-specific IgE antibody obtained in the reaction step; The method includes: (2) The method according to (1), wherein the allergen other than the Japanese 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 dust mite allergens. (3) The method according to (1) or (2), wherein the substrate on which the Japanese cedar allergen and one or more allergens other than the Japanese cedar allergen are immobilized in the same reaction vessel is a microarray. (4) The method according to any one of (1) to (3), wherein in the removing step, IgG antibodies are removed from the body fluid sample. (5) The method according to any one of (4), wherein the removal of the IgG antibody in the removal step is performed by adsorbing the IgG antibody onto a carrier having immobilized protein G. (6) The method according to any one of (1) to (5), wherein the detection of the complex between the allergen and the allergen-specific IgE antibody in the detection step comprises reacting an anti-IgE antibody labeled with a fluorescent dye with the complex and detecting the fluorescent dye bound to the complex. (7) The method according to any one of (1) to (6), wherein the body fluid sample is blood, serum or plasma. (8) A kit for detecting Japanese cedar allergen-specific IgE antibodies in a body fluid sample, comprising an allergen microarray having a substrate provided with a reaction chamber including an area where the Japanese cedar allergen is immobilized and an area where one or more types of allergens other than the Japanese cedar allergen are immobilized, and a carrier having immobilized a ligand capable of selectively binding to at least one type of antibody selected from the group consisting of IgG antibodies, IgM antibodies, IgA antibodies and IgD antibodies. (9) The kit according to (8), wherein the support having a ligand capable of selectively binding to the IgG antibody immobilized thereon is a support having Protein G immobilized thereon. Effect of the Invention

[0008] By contacting a body fluid sample from which specific IgG, IgM, IgA or IgD antibodies have been previously removed with a substrate on which Japanese cedar allergen and one or more allergens other than Japanese cedar allergen are independently immobilized in the same reaction vessel, the detection value can be improved in the detection of Japanese cedar allergen-specific IgE antibodies. [Brief description of the drawings]

[0009] [Figure 1] FIG. 2 is a diagram showing an example of a substrate having a reaction chamber for use in the method of the present invention. [Diagram 2] FIG. 2 is a diagram showing an example of a substrate provided with a partition wall on the outer edge of a reaction vessel, used in the method of the present invention. [Diagram 3] FIG. 2 is a schematic diagram of a substrate provided with a reaction chamber, used in Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The body fluid samples used in the present invention include whole blood, plasma, serum, sweat, urine, tears, saliva, sputum / respiratory tract secretions, breast milk, amniotic fluid, cerebrospinal fluid, ascites, pleural effusion, synovial fluid, semen, vaginal secretions, etc., but blood (whole blood), plasma, or serum is preferred, as they are likely to contain Japanese cedar allergen-specific IgE antibodies.

[0011] The removal step of removing at least one antibody selected from the group consisting of IgG, IgM, IgA and IgD antibodies from a body fluid sample can be carried out by a method of adsorbing and removing at least one antibody selected from the group consisting of IgG, IgM, IgA and IgD antibodies (hereinafter, for convenience, may be referred to as "non-IgE antibody") in the body fluid sample. In particular, since IgG antibodies are the antibody with the highest content in body fluid samples, a form of adsorbing and removing IgG antibodies is preferred in terms of eliminating competition with Japanese cedar allergen-specific IgE antibodies. Note that a form of removing at least one of the other three types of non-IgE antibodies in addition to IgG antibodies is also preferred.

[0012] Examples of methods for adsorbing and removing antibodies include chromatography such as affinity chromatography and ion exchange chromatography, with affinity chromatography being preferred due to the short time required. To adsorb and remove non-IgE antibodies in a body fluid sample using affinity chromatography, the body fluid sample may be brought into contact with a carrier on which a ligand capable of selectively binding to the non-IgE antibody is immobilized. Specifically, the body fluid sample may be passed through a column packed with the carrier and recovered from the carrier by centrifugation or the like, or the body fluid sample may be suspended in the carrier and the suspension centrifuged to recover the body fluid sample, with the method using a column packed with the carrier being preferred.

[0013] In the above affinity chromatography, examples of the ligand to be immobilized on the carrier include antibodies such as anti-IgG antibodies, anti-IgM antibodies, anti-IgA antibodies, and anti-IgD antibodies, as well as known ligands such as Protein A derived from Staphylococcus aureus, Protein G derived from group G hemolytic streptococci, Protein L derived from Gram-positive anaerobic cocci, and Jacalin derived from jackfruit. Of these, when removing IgG antibodies, it is preferable to use Protein G, since it has high selective binding power for IgG antibodies, is inexpensive, and is easily available.

[0014] The material of the carrier used in the affinity chromatography may be any of resin, glass, metal, silicon wafer, etc., but resin is preferred from the viewpoints of ease of surface treatment and mass production. Examples of resins that can be used as the material of the carrier include agarose, polyacrylic acid ester, polymethacrylic acid ester, polycarbonate, polystyrene, polyvinyl acetate, polyester, etc., but agarose is preferred because it has little non-specific adsorption with components in body fluid samples. The form of the carrier on which the ligand is immobilized may be any of particles, substrates, etc., but particles are preferred because they have a large specific surface area and can increase the amount of the ligand immobilized.

[0015] The carrier may be a commercially available product such as that manufactured by GE Healthcare, or a carrier without immobilized ligand may be purchased and then immobilized with a desired ligand. For example, as described in the following Examples, carriers with immobilized Protein G are commercially available, and such commercially available products can be preferably used.

[0016] In the present invention, in the reaction step, the body fluid sample after antibody removal is contacted with a substrate provided with a reaction chamber including an area where the Japanese cedar allergen is immobilized and an area where one or more allergens other than the Japanese cedar allergen are immobilized. The contact conditions may be well-known conditions under which an antigen-antibody reaction occurs between the antibody in the body fluid sample and each immobilized allergen, and can be carried out, for example, by contacting the body fluid sample or a dilution thereof with each immobilized allergen at room temperature to 37°C for about 1 to 4 hours.

[0017] An example of the substrate used here is shown in Figure 1. Substrate 1 is provided with a reaction chamber 2, which is an area with which a body fluid sample is brought into contact. Reaction chamber 2 contains two distinct areas, namely, a Japanese cedar allergen immobilization area 3 in which a Japanese cedar allergen is immobilized, and an allergen other than Japanese cedar allergen immobilization area 4 in which one or more allergens other than Japanese cedar allergen are immobilized, which do not overlap with each other. In other words, the substrate used in the present invention is one that allows a body fluid sample to come into contact with both Japanese cedar allergen and allergens other than Japanese cedar allergen in the same reaction chamber.

[0018] Therefore, a substrate composed of a reaction vessel having only the region 3 where the Japanese cedar allergen is immobilized, and a substrate composed of a reaction vessel having only the region 4 where an allergen other than the Japanese cedar allergen is immobilized, do not fall under the substrate used in the present invention. In addition, a substrate composed of a reaction vessel in which the immobilization region 3 of the Japanese cedar allergen and the immobilization region 4 of an allergen other than the Japanese cedar allergen are physically separated from each other by a partition wall does not fall under the substrate used in the present invention, since the body fluid sample cannot be brought into contact with both the Japanese cedar allergen and the allergen other than the Japanese cedar allergen in the same reaction vessel. The partition wall here means a convex structure provided on the substrate that physically separates the allergen immobilization regions. For example, a multi-well plate in which different allergens are immobilized in each well does not fall under the substrate used in the present invention, since the immobilization region of the Japanese cedar allergen is physically separated from the immobilization region of the allergen other than the Japanese cedar by a partition wall.

[0019] The body fluid sample after antibody removal may be added to the reaction tank so long as it is capable of contacting all of the allergens immobilized in the reaction tank. The body fluid sample may be diluted with a buffer solution such as phosphate-buffered saline before being added to the reaction tank, or the body fluid sample may be added to the reaction tank without being diluted.

[0020] A concave structure such as a flow channel may be provided inside the reaction vessel as long as the body fluid sample can contact all of the allergens immobilized in the reaction vessel. In addition, as shown in Fig. 2, in order to prevent the body fluid sample from leaking out of the reaction vessel during the reaction step, the reaction vessel may be physically separated from the outside by providing a structure such as a partition wall 5 on the outer edge of the reaction vessel.

[0021] Methods for processing the inside and outer edge of the reaction tank include cutting the substrate using a drill or laser, and cutting the mold used to mold the substrate and then performing injection molding, compression molding, or vacuum molding. From the perspective of quality stability and ease of mass production, the above method of cutting the mold is more preferred.

[0022] Specific examples of the substrate having the above-mentioned reaction chamber include an allergen microarray in which allergens are immobilized in two dimensions, and a microfluidic device in which allergens are immobilized in a fine flow channel. However, an allergen microarray is preferred, which allows a body fluid sample to easily come into contact with all of the allergens immobilized in the same reaction chamber.

[0023] The fixation of Japanese cedar allergen and one or more allergens other than Japanese cedar allergen to the reaction tank can be carried out by a method of spotting a small amount of allergen solution onto the surface of the reaction tank using a spotting device, or by contacting allergen solutions containing each allergen with the reaction tank surface without mixing them with each other.

[0024] The immobilized allergen may be bound to the reaction vessel by physical adsorption or covalent bonding, with covalent bonding being preferred from the viewpoint of preventing the allergen from peeling off or eluting from the substrate during substrate cleaning.

[0025] As a method for immobilizing an allergen on a reaction vessel by a covalent bond, a method can be used in which a functional group such as an amino group, a carboxyl group, or a hydroxyl group present on the surface of the allergen reacts with a functional group present on the surface of the reaction vessel to form a covalent bond. When the substrate is made of a resin as described below, the functional group present on the surface of the reaction vessel can be an amino group, a carboxyl group, an isothiocyanate group, or the like, and the form of the covalent bond can be any of an amide bond, a thiocarbamide bond, an ether bond, or the like, but an amide bond is preferred from the viewpoint of ease of bond formation and strength. Bonding by an amide bond can be performed by a well-known method using, for example, a succinimide compound such as N-hydroxysuccinimide (NHS) and a carbodiimide compound such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (see the following examples).

[0026] The allergens other than the Japanese cedar allergen immobilized in the region of the reaction vessel where the Japanese cedar allergen immobilization region exists are preferably at least one selected from the group consisting of shrimp, crab, soybean, peanut, buckwheat, walnut, milk, egg white, wheat, orchard grass, dog, cat and mite. Of these, it is preferable to immobilize three or more types, more preferably four or more types of allergens. Each allergen may be purchased as a commercially available product such as a GREER product, dissolved in a buffer such as phosphate buffered saline, and then used as an allergen solution, or a solution containing a desired allergen may be prepared. As a method for preparing an allergen solution, for example, the allergen raw material is subjected to a grinding process using a mixer mill or the like, or a degreasing process using an organic solvent such as ether, acetone, or hexane, and stirred in a buffer such as phosphate buffered saline to extract the allergen, and the centrifugal supernatant can be prepared to an appropriate allergen concentration and used.

[0027] The material of the substrate used in the present invention may be, for example, any of resin, glass, metal, and silicon wafer, but resin is preferred from the viewpoints of ease of surface treatment and mass productivity.

[0028] Examples of resins that can be used as the material for the substrate include polyacrylic acid esters, polymethacrylic acid esters, polycarbonates, polystyrenes, polyvinyl acetates, polyesters, etc., with polyacrylic acid esters and polymethacrylic acid esters being preferred. Among these, examples of polymethacrylic acid esters include polyalkyl methacrylates (PAMAs) such as polymethyl methacrylate (PMMA), polyethyl methacrylate (PEMA), and polypropyl methacrylate, with PMMA being preferred.

[0029] In the present invention, detection of Japanese cedar allergen-specific IgE antibodies can be performed by contacting a body fluid sample from which non-IgE antibodies have been removed with a reaction vessel on the above-mentioned substrate, washing the reaction vessel to remove unbound matter, and then detecting a complex between the allergen and the allergen-specific IgE antibody.

[0030] The method of detecting a complex between an allergen and an allergen-specific IgE antibody is well known, and examples of such methods include surface plasmon resonance, which uses the difference in refractive index caused by binding as its detection principle, quartz crystal microbalance, which uses the difference in resonance frequency as its detection principle, and methods using anti-IgE antibodies labeled with fluorescent substances (fluorescent dyes) or enzymes that generate color- or luminescent substances, radioisotopes, etc. Among these, detection methods using anti-IgE antibodies labeled with fluorescent substances are preferred from the viewpoint of ease of handling and safety. In this case, anti-IgE antibodies labeled with fluorescent substances are reacted with the complex, and after washing, the fluorescent dye bound to the complex is detected.

[0031] Another embodiment of the present invention is a kit for detecting Japanese Cedar allergen-specific IgE antibodies in a body fluid sample, which comprises an allergen microarray having a substrate provided with a reaction chamber including an area where Japanese Cedar allergen is immobilized and an area where one or more allergens other than Japanese Cedar allergen are immobilized, and a carrier having a ligand immobilized that can selectively bind to at least one antibody selected from the group consisting of IgG antibodies, IgM antibodies, IgA antibodies, and IgD antibodies (for example, a carrier having protein G immobilized as a ligand that can selectively bind to IgG antibodies). The substrate is the same as the substrate used in the above-mentioned method for detecting Japanese Cedar allergen-specific IgE antibodies of the present invention, and contains areas in the reaction chamber where Japanese Cedar allergen and one or more allergens other than Japanese Cedar allergen are immobilized. The carrier having a ligand (for example, protein G) that can selectively bind to these antibodies immobilized is used in the removal step of removing the antibodies (for example, IgG antibodies) from the body fluid sample in the above-mentioned method for detecting Japanese Cedar allergen-specific IgE antibodies of the present invention.

[0032] The kit for detecting Japanese cedar allergen-specific IgE antibodies in body fluid samples of the present invention can contain, in addition to the above-mentioned components, fluorescent substances (fluorescent dyes) or enzymes that generate color- and luminescent substances, anti-IgE antibodies labeled with radioisotopes, standard samples, diluents, washing solutions, reaction stop solutions, etc. Each of the above-mentioned component reagents can be in the form of a suspension, solution, or freeze-dried product. In addition, the kit can contain devices and software required for measurement and analysis, a computer with the software installed, and specifications explaining the method of use and protocol of the kit. EXAMPLES

[0033] Examples are shown below, but the present invention is not limited to these examples.

[0034] Example 1 Detection of Japanese cedar allergen-specific IgE antibodies in human serum samples from which IgG antibodies have been removed, using a microarray immobilizing Japanese cedar, shrimp, crab, soybean, peanut, and buckwheat allergens. (1) Preparation of NHS-esterified PMMA substrate A polymethyl methacrylate (PMMA) substrate (75 mm x 25 mm x 1.0 mm) equipped with a reaction tank (12.5 mm x 10 mm x 0.15 mm) as shown in Figure 3 was immersed in a 10N sodium hydroxide aqueous solution at 70°C for 15 hours. It was then washed with pure water, a 0.1N HCl aqueous solution, and pure water in that order. The side chains of PMMA on the substrate surface were hydrolyzed to generate carboxyl groups.

[0035] 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 0.1 N sodium hydroxide). The PMMA substrate after hydrolysis was immersed in the mixed solution and stirred with a micro stirrer for 1 hour to obtain an NHS-esterified PMMA substrate.

[0036] (2) Preparation of allergen solution Japanese cedar allergen was obtained from Hayashibara Co., Ltd. as a 0.2 mg / mL solution in phosphate buffered saline and used as such in the subsequent steps. Shrimp, crab, soybean, peanut, and buckwheat allergens were obtained from Greer Co., Ltd. as freeze-dried powders, and 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.

[0037] (3) Preparation of allergen-immobilized microarrays The cedar, shrimp, crab, soybean, peanut, and buckwheat allergen solutions prepared in (2) were spotted into one of the reaction chambers of the NHS-esterified PMMA resin substrate prepared above using a spotting robot (Nihon Laser Electronics Co., Ltd., GTMAS Stamp-2). The substrate was then placed in a sealed plastic container and incubated overnight at 37°C and 100% humidity to immobilize each allergen on the reaction chamber surface. After incubation, the substrate was washed with phosphate-buffered saline (0.05% Tween 20 (trade name)). In this manner, a microarray was obtained in which the cedar, shrimp, crab, soybean, peanut, and buckwheat allergens were immobilized in the same reaction chamber.

[0038] (4) Removal of IgG antibodies from human serum (removal step) A 50 μL portion of the Protein G immobilized carrier suspension (Protein G Sepharose 4 Fast Flow) (GE Healthcare Life Sciences) was dispensed into a spin column (MoBiTec), and the liquid components of the suspension were separated and removed from the carrier by centrifugation (1500 G, 1 minute), and the carrier was dried on the column.

[0039] 20 μL of human serum as a body fluid sample was dropped onto the Protein G immobilized carrier column prepared as described above, allowed to permeate, and left to stand at room temperature for 30 minutes to allow the IgG antibodies in the sample to be adsorbed by 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), separated from the carrier, and a human serum sample from which the IgG antibodies had been removed was obtained.

[0040] (5) Contact of human serum with the allergen-immobilized microarray (reaction step) The human serum sample from which the IgG antibodies were removed, obtained in (4), was diluted 3-fold with phosphate buffered saline, and 50 μL of the diluted solution was dropped into the reaction vessel of the substrate prepared in (3), and the vessel was covered and sealed with a gap cover glass (manufactured by Matsunami Glass Industry Co., 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)).

[0041] (6) Detection of Japanese cedar allergen-specific IgE antibodies (detection step) A 1.0 mg / mL solution of Dylight-650 dye-labeled anti-human IgE goat polyclonal antibody (Novus biologicals) was diluted 1000-fold with phosphate buffered saline (0.05% Tween 20 (trade name)) containing 1% by weight of bovine serum albumin, and 50 μL of the diluted solution was dropped into the reaction vessel of the substrate that had been contacted with human serum in (5), covered with a gap cover glass, and reacted at room temperature for 1 hour. Thereafter, the gap cover glass was removed, and the substrate was washed with phosphate buffered saline (0.05% Tween 20 (trade name)).

[0042] The substrate was placed in a "3D Gene" (registered trademark) Scanner (Toray Industries, Inc.), and the areas where the Japanese cedar allergen was fixed were subjected to fluorescence detection with excitation light at 635 nm, laser output at 100%, and PMT at 30. The fluorescence intensity is shown in Table 1. The fluorescence detected here originates from the fluorescent dye contained in the labeled antibody bound to the complex of the Japanese cedar allergen and the Japanese cedar allergen-specific IgE antibody, and the greater the amount of Japanese cedar allergen-specific IgE antibody bound to the allergen-immobilized microarray, the higher the detection value.

[0043] Comparative Example 1 As a control experiment for Example 1, detection of Japanese 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 serum samples was not carried out in (4) of Example 1. The measurement results of fluorescence intensity are shown in Table 1.

[0044] Example 2 Detection of Japanese cedar allergen-specific IgE antibodies in human serum samples from which IgG antibodies have been removed, using microarrays immobilizing Japanese cedar, walnut, milk, egg white, and wheat allergens In (2) and (3) of Example 1, detection of Cryptomeria japonica 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 Cryptomeria japonica allergen. The measurement results of fluorescence intensity are shown in Table 1.

[0045] Comparative Example 2 As a control experiment for Example 2, detection of Japanese 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 serum samples in Example 2 was not carried out. The measurement results of fluorescence intensity are shown in Table 1.

[0046] Example 3 Detection of Japanese cedar allergen-specific IgE antibodies in human serum samples from which IgG antibodies have been removed, using microarrays immobilizing Japanese cedar, orchard grass, dog, cat, and mite allergens In (2) and (3) of Example 1, detection of Japanese cedar allergen-specific IgE antibodies in human serum samples was carried out in the same manner as in Example 1, except that orchard grass, dog, cat and mite allergens (GREER) were used as allergens other than Japanese cedar allergen. The measurement results of fluorescence intensity are shown in Table 1.

[0047] Comparative Example 3 As a control experiment for Example 3, detection of Japanese 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 in Example 3 was not carried out. The measurement results of fluorescence intensity are shown in Table 1.

[0048] Comparative Example 4 Detection of Japanese cedar allergen-specific IgE antibodies in human serum samples from which IgG antibodies have been removed, using a microarray immobilizing Japanese cedar allergen Except for not using any allergens other than the Japanese cedar allergen in (2) and (3) of Example 1, detection of Japanese cedar allergen-specific IgE antibodies in human serum samples was carried out in the same manner as in Example 1. The measurement results of fluorescence intensity are shown in Table 1.

[0049] Comparative Example 5 comparison As a control experiment for Example 4, comparison In Example 4, the procedure was repeated except that the step of removing IgG antibodies from the serum sample was not performed. comparison Detection of Japanese cedar allergen-specific IgE antibodies in human serum samples was carried out in the same manner as in Example 4. The measurement results of fluorescence intensity are shown in Table 1.

[0050] [Table 1]

[0051] When human serum from which IgG antibodies had been removed in Examples 1 to 3 was used, the fluorescence intensity detection values ​​were higher than those from which human serum from which IgG antibodies had not been removed in the corresponding Comparative Examples 1 to 3. It is presumed that the removal of IgG antibodies, which are inhibitory substances that block the formation of complexes between Japanese cedar allergens and Japanese cedar allergen-specific IgE antibodies, from human serum improved the binding efficiency of Japanese cedar allergens immobilized on the microarray.

[0052] On the other hand, in Comparative Example 4, which used a substrate on which only the Japanese cedar allergen was immobilized and human serum from which IgG antibodies had been removed, no higher values ​​were observed compared to Comparative Example 5, which used human serum from which IgG antibodies had not been removed. It was found that removing IgG antibodies from human serum did not improve the binding efficiency of Japanese cedar allergen-specific IgE antibodies to the Japanese cedar allergen.

[0053] Since an improvement in the binding efficiency of the cedar allergen-specific IgE antibody due to removal of IgG antibodies was observed only in Examples 1, 2, and 3, which used substrates on which the cedar allergen and one or more allergens other than the cedar allergen were immobilized, it is presumed that when human serum was contacted with the reaction vessel, the immobilized allergens other than the cedar allergen that coexisted with the cedar allergen in the reaction vessel cooperated with the removal of IgG antibodies to improve the binding efficiency of the cedar allergen-specific IgE antibody to the cedar allergen.

[0054] It was found that in order to detect Japanese cedar allergen-specific IgE antibodies without being interfered with by competition with specific IgG antibodies, it is necessary to use a reaction vessel in which Japanese cedar allergen and allergens other than Japanese cedar allergen are immobilized in the coexistence.

[0055] Comparative Example 6 Detection of Japanese cedar allergen-specific IgE antibodies in human serum samples from which IgG antibodies have been removed using a Japanese cedar allergen-immobilized 96-well microplate (1) Preparation of Japanese cedar allergen solution Cryptomeria japonica allergen was obtained from Hayashibara Co., Ltd. as a 0.2 mg / mL solution in phosphate buffered saline, and was diluted 10-fold with phosphate buffered saline to prepare a Cryptomeria japonica allergen solution with a final concentration of 0.02 mg / mL.

[0056] (2) Preparation of a 96-well microplate immobilizing Japanese cedar allergen 50 μL of the Japanese Cedar allergen solution prepared in (1) was added per well to a polystyrene 96-well microplate (WATSON), and the plate was left to stand at 4°C for 16 hours. The solution in the wells 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 same well was washed with phosphate-buffered saline (0.05% Tween 20 (trade name)). In this way, a microplate was obtained in which Japanese Cedar allergen was immobilized in each well.

[0057] (3) Contact of human serum with Japanese cedar allergen-immobilized microplates A human serum sample from which IgG antibodies had been removed was prepared in the same manner as in Example 1(4).

[0058] This human serum sample was diluted 3-fold with phosphate buffered saline containing 1% by weight of bovine serum albumin, and the diluted solution was added to the wells of the microplate on which the Japanese cedar allergen was immobilized at 50 μL per well and allowed to stand for 2 hours at 37° C. The same wells were washed with phosphate buffered saline (0.05% Tween 20 (trade name)).

[0059] (4) Detection of Japanese cedar allergen-specific IgE antibodies A 1.0 mg / mL solution of horseradish peroxidase-labeled anti-human IgE goat polyclonal antibody (manufactured by SouthernBiotech) was diluted 5000-fold with phosphate buffered saline (0.05% Tween20 (trade name)) containing 1% by weight of bovine serum albumin, and 50 μL of the diluted solution was added to the well in which the Japanese cedar allergen was immobilized and left to stand at 37°C for 1 hour. The same well was washed with phosphate buffered saline (0.05% Tween20 (trade name)). 50 μL of TMB One Component HRP Microwell Substrate (Cosmo Bio Co., Ltd.) was added to the same well and left to stand at room temperature for 15 minutes. 50 μL of 1 mol / mL sulfuric acid aqueous solution was added to the same well, and the absorbance at 450 nm wavelength was measured using a plate reader (manufactured by Molecular Devices Co., Ltd.). The results are shown in Table 2.

[0060] Comparative Example 7 As a control experiment for Comparative Example 6, detection of Japanese Cedar allergen-specific IgE antibodies in human serum was carried out using a Japanese Cedar 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.

[0061] [Table 2]

[0062] Table 2 shows the results of detection of Japanese Cedar allergen-specific IgE antibodies using the Japanese Cedar allergen-immobilized 96-well microplate carried out in Comparative Examples 6 and 7. The absorbance detected here is derived from the TMB substrate oxidized by horseradish peroxidase contained in the labeled antibody bound to the complex of Japanese Cedar allergen and Japanese Cedar allergen-specific IgE antibodies, and the greater the amount of Japanese Cedar allergen-specific IgE antibodies bound to the Japanese Cedar allergen-immobilized 96-well microplate, the higher the detection value.

[0063] As in Comparative Examples 4 and 5, which used microarrays on which only the Japanese cedar allergen was immobilized, Comparative Examples 6 and 7, which used 96-well microplates on which only the Japanese cedar allergen was immobilized, 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. It was also found that when a 96-well microplate was used, the binding efficiency of the Japanese cedar allergen-specific IgE antibody to the Japanese cedar allergen did not improve even if the IgG antibody was removed from the human serum. [Explanation of symbols]

[0064] 1 Board 2. Reactor 3. Immobilization region of Japanese cedar allergen 4. Immobilization area of ​​allergens other than Japanese cedar allergen 5 Bulkhead

Claims

1. A method for detecting Japanese cedar allergen-specific IgE antibodies in a body fluid sample, comprising the steps of: a removing step of removing IgG antibodies from the body fluid sample; a reaction step in which the body fluid sample from which the antibodies have been removed, obtained in the removal step, is brought into contact with a reaction chamber of a substrate provided with the reaction chamber, the reaction chamber including a region in which the Japanese cedar allergen is immobilized and a region in which one or more allergens other than the Japanese cedar allergen are immobilized, to form a complex between the allergen and the allergen-specific IgE antibody; and a detection step of detecting the complex between the Japanese cedar allergen and the Japanese cedar allergen-specific IgE antibody obtained in the reaction step; The method includes:

2. The method according to claim 1, wherein the allergen other than the Japanese 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 dust mite allergens.

3. 3. The method according to claim 1, wherein the substrate on which the Japanese cedar allergen and one or more allergens other than the Japanese cedar allergen are immobilized in the same reaction vessel is a microarray.

4. The method according to any one of claims 1 to 3, wherein the removal of the IgG antibody in the removal step is performed by adsorbing the IgG antibody onto a carrier on which Protein G is immobilized.

5. The method according to any one of claims 1 to 4, wherein the detection of the complex between the allergen and the allergen-specific IgE antibody in the detection step comprises reacting an anti-IgE antibody labeled with a fluorescent dye with the complex and detecting the fluorescent dye bound to the complex.

6. The method according to any one of claims 1 to 5, wherein the body fluid sample is blood, serum or plasma.

7. A kit for detecting Japanese cedar allergen-specific IgE antibodies in a body fluid sample, comprising an allergen microarray having a substrate provided with reaction chambers including an area where Japanese cedar allergen is immobilized and an area where one or more types of allergens other than Japanese cedar allergen are immobilized, and a carrier having immobilized thereon a ligand capable of selectively binding to IgG antibodies.

8. The kit according to claim 7, wherein the support having a ligand capable of selectively binding to an IgG antibody immobilized thereon is a support having Protein G immobilized thereon.

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