Method for detecting macadamia nut allergen-specific IgE, in vitro diagnostic agent for diagnosing macadamia nut allergy, kit for detecting macadamia nut allergen-specific IgE, and method for detecting macadamia nut allergen

Abstract

To provide a technology capable of detecting an IgE antibody to be a cause of an onset of macadamia nut allergy with accuracy higher than in conventional ones.SOLUTION: A method for detecting a macadamia nut allergen specific IgE in a target sample includes a contact step for bringing (a) or (b) polypeptide into contact with the target sample, and a detection step for detecting the existence / nonexistence of an IgE specifically combined with the polypeptide after the contact step.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for detecting macadamia nut allergen-specific IgE, an in vitro diagnostic agent for diagnosing macadamia nut allergy, a kit for detecting macadamia nut allergen-specific IgE, and a method for detecting macadamia nut allergen.

Background Art

[0002] Nut allergy is a major causative food of food allergy, causes systemic symptoms, and tends to worsen. Therefore, in clinical practice, when diagnosed with nut allergy, patients are often advised to remove all nuts from their diet. However, removing even nuts that do not induce allergy reduces the quality of life (QOL) of food. For this reason, in order to achieve both a safe diet and an improved food QOL, an accurate diagnosis of the type of nut allergy is necessary.

[0003] As tests for food allergy, generally, crude antigen-specific IgE tests and food challenge tests are performed.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, regarding macadamia nuts, it has been reported that the conventional macadamia nut crude antigen-specific IgE antibody test using macadamia crude antigen has low diagnostic accuracy, and improvement in accuracy is desired (Non-Patent Document 1). In addition, the food challenge test has risks such as causing anaphylactic shock.

[0006] In recent years, in the diagnosis method of nut allergies, a diagnosis method based on the measurement of nut allergen component-specific IgE antibodies (Component Resolved Diagnostics: CRD) has attracted attention. This diagnosis method is known to have more excellent diagnostic accuracy compared to the conventional diagnosis method based on the measurement of nut crude antigen-specific IgE antibodies.

[0007] However, regarding macadamia nuts, although allergen component information is disclosed in the WHO / IUIS ALLERGEN NOMENCLATURE, a diagnosis method for macadamia nut allergy based on the measurement of this allergen component-specific IgE antibody has not been reported (Non-Patent Document 2).

[0008] One aspect of the present invention aims to provide a technique capable of detecting IgE antibodies that cause the onset of macadamia nut allergy with higher accuracy than before.

Means for Solving the Problems

[0009] The inventors of the present invention have conducted intensive studies to achieve the above object. As a result, by using macadamia nut 7S globulin protein as an antigen protein, it is possible to more accurately detect the presence or absence of macadamia nut allergen component-specific IgE antibody (that is, the IgE antibody that causes the onset of macadamia nut allergy) in the blood of a subject than before, and based on the measurement result of the macadamia nut allergen component-specific IgE antibody in the blood of the subject, it is possible to provide an index for more accurately diagnosing whether the subject has macadamia nut allergy than before, and thus the present invention has been completed.

[0010] In order to solve the above problems, a method for detecting macadamia nut allergen-specific IgE according to one aspect of the present invention is a method for detecting macadamia nut allergen-specific IgE in a target sample, comprising a contacting step of contacting a polypeptide of the following (a) or (b) with the target sample, and a detecting step of detecting the presence or absence of IgE specifically bound to the polypeptide after the contacting step: (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) A polypeptide consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having binding affinity for macadamia nut allergen-specific IgE.

[0011] An in vitro diagnostic agent for diagnosing macadamia nut allergy according to one aspect of the present invention comprises, as an antigen, a polypeptide of the following (a) or (b): (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) A polypeptide consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having binding affinity for macadamia nut allergen-specific IgE.

[0012] The kit for detecting macadamia nut allergen-specific IgE according to one aspect of the present invention comprises, as an antigen, the polypeptide of the following (a) or (b): (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) A polypeptide consisting of an amino acid sequence having 95% or more sequence identity with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and having binding affinity for macadamia nut allergen-specific IgE.

[0013] The method for detecting macadamia nut allergen according to one aspect of the present invention is a method for detecting macadamia nut allergen in a target sample, comprising: an antibody or a functional fragment thereof that specifically recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; and a detection step of detecting the presence or absence of an antigen specifically bound to the antibody or the functional fragment thereof after the contact step. [Effect of the Invention]

[0014] According to one aspect of the present invention, the presence or absence of IgE antibodies that cause the onset of macadamia nut allergy in a target sample can be detected with higher accuracy than in the past.

[0015] Also, according to another aspect of the present invention, the presence or absence of macadamia nut allergen in a target sample can be detected with higher accuracy than in the past. [Brief Description of the Drawings]

[0016]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0017] <1. Method for Detecting Macadamia Nut Allergen-Specific IgE> The method for detecting macadamia nut allergen-specific IgE according to one aspect of the present invention is a method for detecting macadamia nut allergen-specific IgE in a target sample, comprising a contacting step of contacting the following polypeptide (a) or (b) with the target sample, and a detecting step of detecting the presence or absence of IgE specifically bound to the polypeptide after the contacting step: (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) A polypeptide consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having binding affinity for macadamia nut allergen-specific IgE. According to the method for detecting macadamia nut allergen-specific IgE of this aspect, it becomes possible to more accurately detect the presence or absence of macadamia nut allergen-specific IgE antibodies in a biological sample collected from a subject, and as a result, an index for accurately diagnosing whether the subject has macadamia nut allergy can be provided.

[0018] (Contacting Step) The contacting step is a step of contacting the polypeptide of (a) or (b) with the target sample. For convenience of explanation, this contacting step may be referred to as the "first contacting step". The polypeptide of (a) or (b) is used as an antigen for detecting macadamia nut allergen-specific IgE in a target sample. Therefore, in the following description, the polypeptide of (a) or (b) is referred to as the "detection antigen" for convenience of explanation.

[0019] The reaction conditions for contacting the detection antigen with the target sample are not particularly limited. When macadamia nut allergen-specific IgE is contained in the target sample, the reaction conditions can be appropriately set such that the macadamia nut allergen-specific IgE specifically binds to the detection antigen and the binding can be maintained.

[0020] The contacting step is exemplified below. For example, the detection antigen immobilized on an arbitrary carrier may be contacted with the target sample. As the method, first, a streptavidin-immobilized plate is prepared by immobilizing 20 μg / mL of streptavidin on a black plate. Next, the biotinylated detection antigen is added to the wells of the plate to immobilize the detection antigen on the plate. Further, the target sample diluted with a 10% FBS solution is added and incubated at room temperature for 2 hours to contact the detection antigen with the target sample.

[0021] (Target sample) The type of the test sample used in the method for detecting macadamia nut allergen-specific IgE of this embodiment is not particularly limited. All samples for which it is desired to detect the presence or absence of macadamia nut allergen-specific IgE are test samples. Such test samples are preferably biological samples collected from a subject and biological samples that may contain macadamia nut allergen-specific IgE. Examples of biological samples that may contain macadamia nut allergen-specific IgE include blood, saliva, sputum, nasal discharge, urine, sweat, tears, tissue fluid, lymph fluid, and the like. Since the amount of IgE present is large compared to other biological samples and it is possible to collect a large amount, it is preferable that the test sample is a sample derived from blood. The sample derived from blood may be whole blood, serum, or plasma.

[0022] The type of the subject from which the test sample is collected is not particularly limited. Examples of the subject include humans and mammals other than humans.

[0023] From the viewpoint of further improving the detection accuracy of macadamia nut allergen-specific IgE, the test sample is preferably a sample in which it has been confirmed that it contains macadamia nut crude antigen-specific IgE. The amount of macadamia nut crude antigen-specific IgE contained in the test sample is not particularly limited, but in the conventional Immunocap method, since macadamia nut crude antigen-specific IgE is determined to be positive at 0.35 kU A / L or more, it is preferable that the test sample is a sample in which it has been confirmed that it contains 0.35 kU A / L or more of macadamia nut crude antigen-specific IgE.

[0024] The method for confirming the amount of macadamia nut crude antigen-specific IgE in a target sample is not particularly limited. For example, the known Immunocap method can be preferably adopted. By detecting the presence or absence of IgE that specifically binds to the polypeptide of (a) or (b) in a sample confirmed to contain macadamia nut crude antigen-specific IgE, the false positive rate can be reduced. As a result, the detection accuracy of macadamia nut allergen-specific IgE can be further enhanced.

[0025] (Macadamia nut allergen-specific IgE) Macadamia nut allergen-specific IgE is an immunoglobulin E that specifically binds to macadamia nut allergens. That is, it is an immunoglobulin E that specifically binds to the allergen component in macadamia nut allergy discovered by the present inventors (a substance that causes an allergic reaction and is also referred to as an antigen), namely, the "macadamia nut 7S globulin protein". Here, "specifically binds" means that macadamia nut allergen-specific IgE binds only to the macadamia nut allergen that is the antigen, i.e., the macadamia nut 7S globulin protein, and does not bind to other proteins. The IgE to be detected by the detection method of macadamia nut allergen-specific IgE in this embodiment may be IgE alone or IgE bound to mast cells or basophils.

[0026] (Macadamia nut 7S globulin protein) The macadamia nut 7S globulin precursor protein is a protein consisting of 666 amino acids and is processed into the macadamia nut 7S globulin protein consisting of 638 amino acids. The macadamia nut 7S globulin protein is cleaved by self-cleavage into multiple polypeptide chains such as MiAMP2 (Macadamia integrifolia antimicrobial protein 2) a, b, c or VLAP (vicilin-like antimicrobial peptides) (Reference 1: Marcus JP, Green JL, Goulter KC, Manners JM. A family of antimicrobial peptides is produced by processing of a 7S globulin protein in Macadamia integrifolia kernels. Plant J 1999;19:699-710.) and Reference 2: Ehlers AM, Rohwer S, Otten HG, Brix B, Le TM, Suer W, Knulst AC. IgEbinding to vicilin-like antimicrobial peptides is associated with systemic reactions to macadamia nut. Clin Transl Allergy 2020;10:55). Therefore, MiAMP2a, b, c or VLAP corresponds to partial peptides of the macadamia nut 7S globulin protein. Also, Mac i 1 described in Non-Patent Document 2 also corresponds to a part of the macadamia nut 7S globulin protein described herein. The macadamia nut 7S globulin protein consists of the amino acid sequence shown in SEQ ID NO: 1. That is, the polypeptide of (a) corresponds to the wild-type macadamia nut 7S globulin protein. In the present specification, the term "protein" is used interchangeably with the term "polypeptide".

[0027] The polypeptide of (b) is intended to be a variant of the macadamia nut 7S globulin protein. The polypeptide of (b) consists of an amino acid sequence having 95% or more, preferably 98% or more, more preferably 99% or more identity with the amino acid sequence of SEQ ID NO: 1. The identity of the amino acid sequence can be calculated using known gene information software.

[0028] Further, the polypeptide of (b) consists of an amino acid sequence having mutations in 15 or fewer, preferably 10 or fewer, more preferably 5 or fewer, still more preferably 2 or fewer amino acid residues in the amino acid sequence of SEQ ID NO: 1. The "mutation" is a deletion, substitution, or addition of an amino acid residue.

[0029] Whether the polypeptide of (b) has binding property to macadamia nut allergen-specific IgE can be confirmed by comparing the binding affinity of the polypeptide of (b) to macadamia nut allergen-specific IgE with the binding affinity of the polypeptide of (a) to macadamia nut allergen-specific IgE. When the polypeptide of (b) has a binding affinity equivalent to that of the polypeptide of (a), it can be determined that the polypeptide of (b) has binding property to macadamia nut allergen-specific IgE.

[0030] Also, by confirming the presence or absence of the ability of the polypeptide of (b) to induce a macadamia nut allergic reaction, it is also possible to indirectly confirm that the polypeptide of (b) has binding ability to macadamia nut allergen-specific IgE. For example, a skin test is performed by applying the polypeptide of (b) to the skin of a subject having macadamia nut allergy. If a skin reaction such as swelling occurs in the skin where the polypeptide of (b) is applied, it can be said that the polypeptide of (b) has induced a macadamia nut allergic reaction. Since an allergic reaction occurs as a result of the binding of an antigen and IgE, it can be determined that the polypeptide of (b) has binding ability to macadamia nut allergen-specific IgE based on the presence or absence of the allergic reaction.

[0031] The polypeptide of (a) or (b) can be obtained by a method known in the art (for example, genetic engineering techniques). The method of expressing the polypeptide of (a) or (b) can be appropriately performed by a technique known to those skilled in the art. A vector capable of inducing the expression of the polypeptide can be selected by those skilled in the art according to the type of host cell used. However, in order to facilitate purification, it is preferable that the vector is capable of attaching a tag to the polypeptide of (a) or (b) (for example, pCold-ProS2 vector). Also, the host cell can be appropriately selected by those skilled in the art according to the vector used. The host cell may be derived from a prokaryote such as Escherichia coli (E. coli).

[0032] The method for expressing the polypeptide of (a) is exemplified below. First, the codons of the macadamia nut 7S globulin mRNA sequence (GenBank: AF161883.1) obtained from the NCBI (National Center for Biotechnology Information) website (https: / / www.ncbi.nlm.nih.gov) are optimized and synthesized, and then inserted into the pCold-ProS2 vector. Thereafter, using Escherichia coli (for example, SHuffle T7 Express lysY Competent Escherichia coli), the polypeptide of (a) can be expressed.

[0033] Examples of the method for purifying the polypeptide of (a) include methods using solubility such as salting out and solvent precipitation, methods using differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-PAGE, methods using charge such as ion exchange chromatography, methods using specific affinity such as reverse phase high performance liquid chromatography, methods using differences in hydrophobicity such as reverse phase high performance liquid chromatography, and methods using differences in isoelectric point such as isoelectric focusing.

[0034] The method for purifying the polypeptide of (a) using the His-ProS2 tag is exemplified below. First, the cells are collected by centrifugation and placed in a solution containing, for example, 50 mM Tris-HCl (pH 8.0) containing 200 mM NaCl, 1% Triton, 1 mg / mL lysozyme, and Halt Protease Inhibitor Cocktail, and dissolved using ultrasonic waves. The polypeptide of (a) is purified using a column capable of removing the tag (for example, HisTrap HP column). For example, the ProS2 tag is separated from the His tag using Turbo 3C protease, and the tag and the enzyme are removed by the HisTrap HP column. For the polypeptide of (b), it may be expressed and purified by the same method.

[0035] The polypeptide of (a) or (b) is preferably biotin-labeled, and is preferably immobilized on a streptavidin-immobilized carrier by the interaction between the biotin and the streptavidin. Thereby, in the step of detecting the presence or absence of macadamia nut allergen-specific IgE specifically bound to the polypeptide, methods such as the existing streptavidin solid-phase ELISA method can be used, so that accuracy control becomes easier than using a simple ELISA method.

[0036] Further, the polypeptide of (a) or (b) is preferably biotin-labeled non-specifically with respect to the amino acid sequence. Here, in the present specification, "biotin-labeled non-specifically with respect to the amino acid sequence" means biotin-labeling a target protein regardless of the type of functional group possessed by the amino acid. By biotin-labeling in a non-specific manner, the biotin-labeling can be performed at a uniform frequency over the entire length of the target protein. The macadamia nut 7S globulin protein has a site containing a large number of lysine residues, but by being biotin-labeled non-specifically with respect to the amino acid sequence, it is possible to prevent the antigenicity of a specific site from being lost due to biotin-labeling. As a result, the binding property between the polypeptide and IgE is improved and the antigenicity can be retained, so that highly accurate detection can be achieved.

[0037] Examples of the method for biotin-labeling non-specifically with respect to the amino acid sequence include a method of labeling a polypeptide using a photoreactive biotin-labeling reagent. Examples of the photoreactive biotin-labeling reagent include, but are not limited to, EZ-Link TFPA-PEG3-Biotin (manufactured by Thermo Fisher Scientific) used in the examples described later.

[0038] When biotin-labeling the polypeptide of (a) or (b), it is preferable that a biotin molecule is bound to the polypeptide of (a) or (b) via a linker sequence. The presence of a linker sequence between the polypeptide and the biotin molecule can ensure the distance between the biotin-labeled polypeptide and the solid layer surface when the biotin-labeled polypeptide is immobilized on a streptavidin-immobilized carrier. As a result, for the polypeptide, the IgE antibody can access more easily from a direction where it cannot wrap around in the normal ELISA method, so the binding property between the polypeptide and IgE is improved, and more accurate detection can be realized. The linker sequence is not particularly limited, but polyethylene glycol can be preferably used as the linker sequence, such as EZ-Link TFPA-PEG3-Biotin (manufactured by Thermo Fisher Scientific) used in the examples described later.

[0039] (Detection step) The detection step is a step of detecting the presence or absence of macadamia nut allergen-specific IgE specifically bound to the polypeptide after the contact step. For convenience of explanation, this detection step may be referred to as the "first detection step".

[0040] The detection of the binding between the macadamia nut allergen-specific IgE in the test sample and the polypeptide can be carried out by a detection method based on a known antigen-antibody reaction. Such methods can employ, for example, detection methods by ELISA (Enzyme-Linked Immunosorvent Assay), sandwich immunoassay, immunoblotting, immunoprecipitation, immunochromatography, and Immunocap method. All of these are methods of detecting the binding between an antigen and the target IgE by contacting and binding the target IgE to the antigen, allowing an enzyme-labeled secondary antibody to act on the IgE specifically bound to the antigen, adding a substrate of the enzyme (usually a chromogenic or luminescent reagent), and detecting the product of the enzyme reaction.

[0041] In the detection step, it is only necessary to detect the presence or absence of IgE specifically binding to the antigen for detection, but the amount of IgE specifically binding to the antigen for detection may also be quantified. The amount of IgE specifically binding to the antigen for detection can be measured using known methods.

[0042] (Other steps) The method for detecting macadamia nut allergen-specific IgE of this aspect may include steps other than the aforementioned first contact step and first detection step. For example, it may further include a second contact step of contacting the macadamia nut crude antigen with the test sample, and a second detection step of detecting the presence or absence of IgE specifically binding to the macadamia nut crude antigen after the second contact step. By further including the second contact step and the second detection step, the presence or absence of macadamia nut crude antigen-specific IgE in the test sample can be detected.

[0043] The second contact step and the second detection step may be performed before the first contact step or after the first detection step. Regarding the second contact step and the second detection step, except for using the macadamia nut crude antigen as the antigen for detection, they are the same as those described for the first contact step and the first detection step, so the description will not be repeated here. The macadamia nut crude antigen can be prepared by the method described in the examples below.

[0044] Based on the results of the first detection step and the second detection step, it may further include a determination step of determining the presence or absence of macadamia nut allergen-specific IgE in the test sample. In one embodiment, in the determination step, from the result of the first detection step, if the macadamia nut crude antigen-specific IgE in the test sample is contained in an amount equal to or more than a predetermined amount, preferably 0.35 kU A / L or more, and from the result of the second detection step, IgE specifically binding to the polypeptide of (a) or (b) is contained, it may be determined that the test sample contains macadamia nut allergen-specific IgE.

[0045] (Use of the detection result obtained by the detection method) The detection result obtained by performing the detection method of this aspect can be used as one of the diagnostic materials when a doctor makes a diagnosis. In addition, a subject determined to have the above-mentioned macadamia nut allergy or a possibility of having a macadamia nut allergy may, if necessary, receive a diagnosis from a doctor (when the subject is a human) or a veterinarian (when the subject is a mammal other than a human), and then determine a treatment policy according to the degree of the symptoms of the macadamia nut allergy, and receive treatment according to the treatment policy. Here, "treatment" is intended to prevent the onset of macadamia nut allergy and improve the symptoms of macadamia nut allergy. Examples of the treatment method for macadamia nut allergy include, for example, a diet therapy of ingesting a macadamia nut allergen-free diet; an oral immunotherapy using a macadamia nut allergen, etc.

[0046] <2. Kit for detecting macadamia nut allergen-specific IgE> The kit for detecting macadamia nut allergen-specific IgE according to one aspect of the present invention comprises, as an antigen, the polypeptide of the following (a) or (b): (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) A polypeptide consisting of an amino acid sequence having 95% or more sequence identity with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and having binding properties to macadamia nut allergen-specific IgE. According to the detection kit of this aspect, it can be suitably used for the detection method described in the section of <1. Detection method of macadamia nut allergen-specific IgE>.

[0047] The kit for detecting macadamia nut allergen-specific IgE of this aspect preferably further comprises a macadamia nut crude antigen as an antigen (antigen for detection). By further comprising the macadamia nut crude antigen, it becomes possible to detect the presence or absence of macadamia nut allergen-specific IgE in a target sample with higher accuracy based on the detection result of macadamia nut crude antigen-specific IgE and the detection result of the polypeptide-specific IgE of (a) or (b) above.

[0048] The polypeptide of (a) or (b) above, and the macadamia nut crude antigen are as described in the section <1. Method for detecting macadamia nut allergen-specific IgE>, so the description will not be repeated here.

[0049] The antigen for detection may be bound to a substrate. Examples of the substrate include plates, membranes, columns, beads, and gold colloids. The substrate may be immobilized with a fluorescent protein or a tag protein, etc. Since a large number of samples can be processed in parallel, the substrate is preferably a microplate (for example, 96 wells).

[0050] (Form of the detection kit) The form of the detection kit of this aspect is not particularly limited. For example, it can be provided in the form of an allergen chip, an ELISA kit, a digital ELISA kit, etc.

[0051] In addition to the polypeptide of (a) or (b) above, the detection kit of this embodiment may also include multiple other allergens as antigens. Thereby, the presence or absence of multiple allergen-specific IgEs including macadamia nut allergen-specific IgE in the test sample can be comprehensively detected. The types of other allergens provided in the detection kit of this embodiment are not particularly limited. For example, by including allergens of nuts such as peanuts, almonds, cashew nuts, pistachios, walnuts, pecan nuts, hazelnuts, and Brazil nuts as antigens, the presence or absence of allergen-specific IgEs against various nuts in the test sample can be comprehensively detected.

[0052] For example, when the detection kit of this embodiment is an allergen chip, it may be a microarray chip on which multiple spots of the polypeptide of (a) or (b) are arranged. Also, it may be a chip on which multiple types of allergens as described above are arranged as different spots for each type.

[0053] The detection kit according to one embodiment of the present invention may further include at least one of a substrate; various reagents (such as secondary antibodies, reporter molecules, and buffers); instruments (such as test tubes and pipettes); an instruction manual for the detection kit; a sample for a control test, etc., depending on the form of the kit. Note that the instruction manual for the detection kit describes, for example, specific procedures related to the detection method described in the section <1. Detection method for macadamia nut allergen-specific IgE>.

[0054] <3. In Vitro Diagnostic Reagent for Macadamia Nut Allergy Diagnosis> An in vitro diagnostic reagent for macadamia nut allergy diagnosis that includes the polypeptide of (a) or (b) below as an antigen is also included in the scope of the present invention: (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) A polypeptide consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having binding activity to Macadamia nut allergen-specific IgE. According to the in vitro diagnostic agent of this embodiment, by using the detection method described in the section of <1. Method for detecting Macadamia nut allergen-specific IgE>, Macadamia nut allergen-specific IgE in a test sample can be detected, so it is possible to diagnose Macadamia nut allergy with higher accuracy.

[0055] The in vitro diagnostic agent of this embodiment preferably further comprises a Macadamia nut crude antigen as an antigen (antigen for detection). By further comprising a Macadamia nut crude antigen, it is possible to diagnose Macadamia nut allergy with higher accuracy based on the detection results of Macadamia nut crude antigen-specific IgE and the detection results of the polypeptide-specific IgE of (a) or (b) above.

[0056] The polypeptide of (a) or (b) above and the Macadamia nut crude antigen are as described in the section of <1. Method for detecting Macadamia nut allergen-specific IgE>, so the description will not be repeated here.

[0057] The in vitro diagnostic agent of this embodiment can also be provided in the form of a kit. The composition of the kit is as described in the section of <2. Kit for detecting Macadamia nut allergen-specific IgE>, so the description will not be repeated here.

[0058] <4. Method for detecting Macadamia nut allergen> The method for detecting macadamia nut allergens according to one aspect of the present invention is a method for detecting macadamia nut allergens in a target sample, comprising a contacting step of contacting an antibody or a functional fragment thereof that specifically recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 with the target sample, and a detecting step of detecting the presence or absence of an antigen specifically bound to the antibody or the functional fragment thereof after the contacting step. According to the method for detecting macadamia nut allergens of this aspect, it is possible to detect the presence or absence of macadamia nut allergens in a target sample with higher accuracy than before.

[0059] (Contacting step) The contacting step is a step of contacting an antibody or a functional fragment thereof that specifically recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 with the target sample. The antibody that specifically recognizes the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is used as an antibody for detecting macadamia nut allergens in a target sample. Therefore, in the following description, the antibody that specifically recognizes the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is referred to as an "antibody for detection" for convenience of explanation.

[0060] The reaction conditions for contacting the antibody for detection with the target sample are not particularly limited, and when macadamia nut allergens are contained in the target sample, reaction conditions can be appropriately set such that the macadamia nut allergens and the antibody for detection specifically bind and the binding can be maintained.

[0061] (Target sample) The type of the target sample to be used in the method for detecting macadamia nut allergens of this aspect is not particularly limited. All samples for which it is desired to detect the presence or absence of macadamia nut allergens are target samples. Such target samples are articles that may be ingested into the human body or absorbed into the body transdermally or through mucous membranes, and more specifically, foods, food ingredients, beverages, pharmaceuticals, quasi-drugs, cosmetics, etc. can be mentioned.

[0062] (An antibody that specifically recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1) The antibody that specifically recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is intended to be an antibody that specifically recognizes at least a part of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 as an epitope and can specifically bind thereto.

[0063] The "detection antibody" used in the method for detecting macadamia nut allergens of this embodiment is intended to be in a form including all classes and subclasses of immunoglobulins, as well as functional fragments of antibodies. The detection antibody is a concept including both polyclonal antibodies and monoclonal antibodies, and in addition, antibodies produced using recombinant DNA technology, as well as functional fragments of such antibodies.

[0064] The "functional fragment of an antibody" refers to a region of a part of the aforementioned antibody that has antigen-binding ability (synonymous with a binding fragment). The natural antibody is not particularly limited and can be derived from any species including humans, mice, rats, goats, rabbits, camels, llamas, cows, chickens, sharks, and fish. Antibodies produced using recombinant DNA technology include, but are not particularly limited to, chimeric antibodies such as humanized antibodies and primatized antibodies obtained by genetically modifying natural antibodies, synthetic antibodies, recombinant antibodies, mutagenized antibodies, and grafted antibodies (for example, antibodies conjugated or fused with other proteins and radiolabels), and antibodies obtained by performing the same modifications as those for genetically modifying natural antibodies on antibodies already produced using recombinant DNA technology are also included. Specific examples of the functional fragment of an antibody include, for example, F(ab’)2, Fab’, Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv), and dAb (single domain antibody) (George et al, Exp. Opin. Ther. Patents, Vol.6, No.5, p.441-456, 1996).

[0065] Furthermore, in the present invention, the binding fragment also includes, as the concept of the binding fragment, an antibody fragment into which mutations have been introduced within a range that maintains reactivity with the target protein. The aforementioned introduction of mutations is performed using known techniques such as gene modification techniques, which are appropriately selected by those skilled in the art.

[0066] Since it can recognize various epitopes of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the detection antibody is preferably a polyclonal antibody. Based on the information of the amino acid sequence shown in SEQ ID NO: 1, those skilled in the art can easily determine an appropriate amino acid sequence as an antigen for producing an antibody that specifically recognizes the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0067] (Detection step) The detection step is a step of detecting the presence or absence of an antigen specifically bound to the detection antibody after the contact step. Here, the "antigen" is a polypeptide specifically recognized by the detection antibody, that is, at least a part of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. Detection of the binding between the antigen in the target sample and the detection antibody can be performed by a detection method based on a known antigen-antibody reaction. The detection method based on the antigen-antibody reaction is as described in the section of <1. Detection method for macadamia nut allergen-specific IgE>, so the description will not be repeated here.

[0068] In the detection step, it is only necessary to detect the presence or absence of the antigen specifically bound to the detection antibody, but the amount of the antigen specifically bound to the detection antibody may also be measured. The amount of the antigen specifically bound to the detection antibody can be measured using known methods.

[0069] (Utilization of the detection result obtained by the detection method) The detection result obtained by performing the detection method of this aspect can be used as one of the judgment materials when conducting quality control at the manufacturing sites of foods, food ingredients, beverages, pharmaceuticals, quasi-drugs, cosmetics, etc. Further, according to the detection result, a treatment for removing macadamia nut allergen from the target sample may be performed. For example, by using an affinity column in which an antibody that specifically recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is immobilized on a carrier and passing the target sample through the affinity column, the macadamia nut allergen can be removed from the target sample.

[0070] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0071] 〔Summary〕 The method for detecting macadamia nut allergen-specific IgE according to Aspect 1 of the present invention is a method for detecting macadamia nut allergen-specific IgE in a target sample, comprising a contact step of contacting a polypeptide of the following (a) or (b) with the target sample, and a detection step of detecting the presence or absence of IgE specifically bound to the polypeptide after the contact step: (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) a polypeptide consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having binding affinity for macadamia nut allergen-specific IgE.

[0072] The method for detecting macadamia nut allergen-specific IgE according to Aspect 2 of the present invention is preferably a sample in which, in the above Aspect 1, the target sample has been confirmed to contain macadamia nut crude antigen-specific IgE.

[0073] The method for detecting macadamia nut allergen-specific IgE according to Aspect 3 of the present invention is preferably a sample derived from blood in the above Aspect 1 or 2.

[0074] In the method for detecting macadamia nut allergen-specific IgE according to Aspect 4 of the present invention, in any one of the above Aspects 1 to 3, the polypeptide of (a) or (b) is biotin-labeled, and it is preferably immobilized on a streptavidin-immobilized carrier by the interaction between the biotin and the streptavidin.

[0075] In the method for detecting macadamia nut allergen-specific IgE according to Aspect 5 of the present invention, in the above Aspect 4, the polypeptide of (a) or (b) is preferably biotin-labeled in a non-sequence-specific manner.

[0076] The in vitro diagnostic agent for diagnosing macadamia nut allergy according to Aspect 6 of the present invention has a configuration comprising the polypeptide of the following (a) or (b) as an antigen: (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) a polypeptide consisting of an amino acid sequence having 95% or more sequence identity with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and having binding affinity for macadamia nut allergen-specific IgE.

[0077] The kit for detecting macadamia nut allergen-specific IgE according to Aspect 7 of the present invention has a configuration comprising the polypeptide of the following (a) or (b) as an antigen: (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) a polypeptide consisting of an amino acid sequence having 95% or more sequence identity with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and having binding affinity for macadamia nut allergen-specific IgE.

[0078] The method for detecting macadamia nut allergen according to Aspect 8 of the present invention is a method for detecting macadamia nut allergen in a test sample, comprising a contacting step of contacting an antibody or a functional fragment thereof that specifically recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 with the test sample, and a detecting step of detecting the presence or absence of an antigen specifically bound to the antibody or the functional fragment thereof after the contacting step.

Example

[0079] One embodiment of the present invention will be described below.

[0080] (Participants) All 86 participants were supplied by the University of Tokyo Hospital, Aichi Children's Comprehensive Medical Center, Mie University Hospital of the National Hospital Organization, National Center for Child Health and Development, Yamaguchi University Hospital and other institutions. Ethical approval was obtained from the ethics committees of all hospitals to which the participants belonged. Informed consent was also obtained from all children and / or parents.

[0081] (Test subject criteria) Eighty-six people under 20 years old were targeted, and the patient group was classified according to Figure 1. The medical history of immediate reactions to nuts containing peanuts and / or macadamia nuts was confirmed, and patients with a definite medical history of macadamia nut allergy (n = 18) were used as the patient group. In the case of no medical history of immediate reaction, an oral food challenge (OFC) was performed, and OFC-positive subjects (n = 27) were added to the patient group, while OFC-negative subjects (n = 41) were used as the control group.

[0082] (OFC test) The OFC test was performed in a defined manner at each hospital according to the Japanese Pediatric Guidelines for Food Allergy. Macadamia nuts were administered at 3.5 - 10 g throughout the schedule. After testing the immunity of the oral intake amount, positive or negative was determined based on whether an allergic reaction was observed or not.

[0083] (Preparation of macadamia nut 7S globulin protein) The sequence (GenBank: AF161883.1, SEQ ID NO: 2) of the polynucleotide (coding region) encoding the macadamia nut 7S globulin protein was obtained from the NCBI (National Center for Biotechnology Information) website (https: / / www.ncbi.nlm.nih.gov). The encoded sequence was codon-optimized and synthesized (Integrated DNA Technologies). This sequence was inserted into the pCold-ProS2 vector (Takara Bio Inc.), and the macadamia nut 7S globulin protein was expressed using SHuffle T7 Express lysY Competent Escherichia coli (New England Biolabs) as competent cells. The amino acid sequence of the macadamia nut 7S globulin protein is shown in SEQ ID NO: 1.

[0084] Cells were collected by centrifugation and then placed in a solution containing 50 mM Tris-HCl (pH 8.0), 1 mg / mL lysozyme (Merck), Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific), 200 mM NaCl, and 1% Triton, and lysed using ultrasonic waves. The recombinant protein with His-ProS2 tag was purified using a HisTrap HP column (GE Healthcare). The ProS2 tag was separated from the His tag using Turbo 3C protease (Accelagen). The tag and the enzyme were removed by the HisTrap HP column. The concentration of the purified protein was measured using Pierce BCA Protein Assay (Thermo Fisher Scientific). This protein was biotinylated using EZ-Link TFPA-PEG3-Biotin (Thermo Fisher Scientific).

[0085] (Preparation of Macadamia Nut Crude Antigen) The preparation of macadamia nut crude antigen was commissioned to Thermo Fisher Scientific, Inc.

[0086] (Measurement of Macadamia Nut Crude Antigen-Specific IgE) The amount of macadamia nut crude antigen-specific IgE in serum was measured by ImmunoCAP (registered trademark) by commissioning Thermo Fisher Scientific, Inc.

[0087] (Measurement of Macadamia Nut 7S Globulin Protein-Specific IgE) The amount of macadamia nut 7S globulin protein-specific IgE (macadamia nut 7S globulin protein sIgE) in serum was measured by the streptavidin solid-phase ELISA method. First, Black 384-well plates (manufactured by Greiner Bio-One) were coated with 20 μg / mL streptavidin (manufactured by Merck) overnight at 4°C. Then, the washed plates were blocked with phosphate buffered saline containing 10% FBS to prepare streptavidin solid-phase plates.

[0088] 50 μg / mL biotinylated recombinant macadamia nut 7S globulin protein was added to the wells. Human serum samples were diluted with 10% FBS solution and then added to the wells and incubated at room temperature for 2 hours. IgE bound to the biotinylated recombinant macadamia nut 7S globulin protein was detected by sequentially reacting with anti-human IgE rabbit antibody (manufactured by Dako) and horseradish peroxidase-conjugated anti-rabbit antibody (manufactured by Cell Signaling Technology). The luminescence signal of Western Lightning ECL Pro (manufactured by PerkinElmer) was measured by a GloMax luminometer (manufactured by Promega).

[0089] (Statistical Analysis) Statistical analysis was performed using JMP 15 (manufactured by SAS Institute Inc.) and Prism 8 (manufactured by GraphPad Software). The Mann-Whitney U test and Pearson's chi-square test were used to compare continuous and categorical data between groups. The diagnostic performance was evaluated by the area under the ROC curve (ROC: Receiver operating characteristic curve).

[0090] 〔Example 1〕 The clinical and serological characteristics of children suspected of having macadamia nut allergy are summarized in Table 1. As shown in Table 1, there was no significant difference in the total IgE level and the number of patients with allergic complications between the patient group and the control group. On the other hand, the amount of macadamia nut crude antigen-specific IgE (macadamia nut crude antigen sIgE) was significantly increased in the patient group compared with the control group (p < 0.01).

[0091]

Table 1

[0092] The amount of macadamia nut 7S globulin protein sIgE in the serum samples of the patient group and the control group was analyzed by streptavidin immobilized ELISA, and the amount of macadamia nut crude antigen sIgE was analyzed by the Immunocap method, and the results were compared. In addition, ROC analysis of the amount of macadamia nut crude antigen sIgE and macadamia nut 7S globulin protein sIgE detected in the patient group and the control group was performed, and the respective AUCs (AUC: Area under the curve) were compared.

[0093] The results are shown in Figure 2. The ROC curve graph shown in Figure 2 has sensitivity on the vertical axis and (1 - specificity) on the horizontal axis. For the ROC of the macadamia nut crude antigen sIgE measurement value being 0.748, the ROC of the macadamia nut 7S globulin protein sIgE measurement value was 0.812, which was an increase compared to the ROC of the macadamia nut crude antigen sIgE measurement value. Also, the AUC (0.834) based on the ROC analysis of macadamia nut 7S globulin protein sIgE showed a significant increase compared to the AUC (0.660) based on the ROC analysis of macadamia nut crude antigen sIgE (p < 0.01).

[0094] Next, the results when targeting the patient group and control group where the amount of macadamia nut crude antigen sIgE is 0.35 kU A / L or more are shown in Figure 3. The ROC curve graph shown in Figure 3 has sensitivity on the vertical axis and (1 - specificity) on the horizontal axis. Also, in the results of the macadamia nut 7S globulin protein sIgE measurement value, the samples with negative measurement values were enclosed by a dotted line. When targeting the patient group and control group where the amount of macadamia nut crude antigen sIgE is 0.35 kU A / L or more, the ROC of the macadamia nut 7S globulin protein sIgE measurement value was 0.885. Also, the AUC (0.823) based on the ROC analysis of macadamia nut 7S globulin protein sIgE showed that the difference between the control group and the patient group became larger compared to the AUC (0.586) based on the ROC analysis of macadamia nut crude antigen sIgE (p < 0.01).

[0095] The results of comparing the measurement values of macadamia nut 7S globulin protein sIgE and macadamia nut crude antigen sIgE in serum are shown in Figure 4. From the results in Figure 4, it was shown that the 25 samples in the control group with positive measurement values of macadamia nut crude antigen sIgE were negative in the measurement values of macadamia nut 7S globulin protein sIgE.

[0096] 〔Results〕 According to the detection method according to one aspect of the present invention, by detecting specific IgE against macadamia nut 7S globulin protein in the serum of a subject, it has been clarified that the presence or absence of IgE antibodies that cause the onset of macadamia nut allergy in a target sample can be detected with higher accuracy than before. And by using specific IgE against macadamia nut 7S globulin protein in serum as an index, compared with the method of measuring the conventional specific IgE value of macadamia nut crude antigen, it has been clarified that it is possible to provide an index for more accurately diagnosing whether a subject has macadamia nut allergy.

[0097] Furthermore, by using specific IgE against macadamia nut 7S globulin protein in serum as an index, it has been clarified that cases of the negative group that are false positives can be identified with high accuracy from among the cases determined to be positive for macadamia nut allergy in the diagnosis based on the conventional specific IgE value of macadamia nut crude antigen. From these results, it has been shown that it is possible to provide an index that can more accurately diagnose whether a subject has macadamia nut allergy than before by detecting specific IgE against macadamia nut 7S globulin protein in the serum of the subject.

Industrial Applicability

[0098] According to one aspect of the present invention, the presence or absence of IgE antibodies that cause the onset of macadamia nut allergy in a target sample can be detected with higher accuracy than before. Therefore, it can be suitably used in the medical field. Also, according to another aspect of the present invention, the presence or absence of macadamia nut allergens in a target sample can be detected with higher accuracy than before. Therefore, it can be suitably used in the manufacturing field of foods and the like.

Claims

1. A method for assisting in the diagnosis of macadamia nut allergy, comprising: a contacting step of contacting a polypeptide of the following (a) or (b) with a test sample; a detecting step of detecting the presence or absence of IgE specifically bound to the polypeptide after the contacting step; A method for assisting in the diagnosis of macadamia nut allergy, characterized by comprising: (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) a polypeptide consisting of an amino acid sequence having 95% or more sequence identity with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and having binding activity to macadamia nut allergen-specific IgE.

2. The method for assisting in the diagnosis of macadamia nut allergy according to claim 1, wherein the test sample is a sample confirmed to contain macadamia nut crude antigen-specific IgE.

3. The method for assisting in the diagnosis of macadamia nut allergy according to claim 1 or 2, wherein the test sample is a sample derived from blood.

4. The polypeptide of (a) or (b) is biotin-labeled, and is immobilized on a streptavidin-immobilized carrier by the interaction between the biotin and the streptavidin. The method for assisting in the diagnosis of macadamia nut allergy according to any one of claims 1 to 3.

5. The method for assisting in the diagnosis of macadamia nut allergy according to claim 4, wherein the polypeptide of (a) or (b) is biotin-labeled non-specifically with respect to the amino acid sequence.

6. An in vitro diagnostic agent for diagnosing macadamia nut allergy, characterized by comprising a polypeptide of the following (a) or (b) as an antigen: (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) A polypeptide consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having binding activity to macadamia nut allergen-specific IgE. **Claim 7** A method for detecting an allergen involved in the onset of macadamia nut allergy in a target sample, comprising: A contact step of contacting an antibody or a functional fragment thereof that specifically recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 with the target sample; A detection step of detecting the presence or absence of an antigen, which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, specifically bound to the antibody or the functional fragment thereof after the contact step; A method for detecting an allergen involved in the onset of macadamia nut allergy, characterized by comprising the above steps.

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