JP7693173B2 - 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 - Google Patents

Description

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 allergens.

Nut allergies are the main cause of food allergies, and they tend to cause systemic symptoms and become severe. Therefore, in clinical practice, when a patient is diagnosed with a nut allergy, they are often advised to eliminate all nuts from their diet. However, eliminating even types of nuts that do not cause allergies reduces the patient's dietary QOL (quality of life). For this reason, in order to achieve both a safe diet and improved dietary QOL, it is necessary to accurately diagnose which types of nuts the patient is allergic to.

Food allergy tests generally involve crude antigen-specific IgE testing and food challenge testing.

Ekbote A, Hayman G, Bansal A: Macadamia nut allergy: potentially misleading specific IgE results. Allergy 2010;65:1345 Information on macadamia nut allergen components disclosed in the WHO/IUIS ALLERGEN NOMENCLATURE [searched June 24, 2021], Internet <URL: http://www.allergen.org/viewallergen.php?aid=1016>

However, when it comes to macadamia nuts, it has been reported that the diagnostic accuracy of the conventional macadamia nut crude antigen-specific IgE antibody test using macadamia nut crude antigen is not high, and there is a need to improve the accuracy (Non-Patent Document 1). In addition, food challenge tests have the risk of causing anaphylactic shock.

In recent years, component resolved diagnostics (CRD) based on the measurement of nut allergen component-specific IgE antibodies has been attracting attention as a method for diagnosing nut allergies. This diagnostic method is known to have superior diagnostic accuracy compared to conventional diagnostic methods based on the measurement of nut crude antigen-specific IgE antibodies.

However, with regard to macadamia nuts, although information on allergen components is disclosed in the WHO/IUIS ALLERGEN NOMENCLATURE, no method for diagnosing macadamia nut allergy based on measuring IgE antibodies specific to these allergen components has been reported (Non-Patent Document 2).

One aspect of the present invention aims to provide a technology that enables more accurate detection of IgE antibodies, which are the cause of macadamia nut allergies, than ever before.

The present inventors have conducted extensive research to achieve the above object. As a result, they have discovered that it is possible to detect the presence or absence of macadamia nut allergen component-specific IgE antibodies (i.e., IgE antibodies that cause the onset of macadamia nut allergy) in the blood of a subject with greater accuracy than before by using macadamia nut 7S globulin protein as an antigen protein, and that it is possible to provide an indicator for diagnosing whether or not a subject has a macadamia nut allergy with greater accuracy than before based on the measurement results of macadamia nut allergen component-specific IgE antibodies in the subject's blood, thereby completing the present invention.

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

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

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

A method for detecting a macadamia nut allergen according to one embodiment of the present invention is a method for detecting a macadamia nut allergen in a target sample, and includes an antibody or a functional fragment thereof that specifically recognizes a polypeptide having the amino acid sequence shown in SEQ ID NO:1, and a detection step for detecting the presence or absence of an antigen that specifically binds to the antibody or the functional fragment thereof after the contact step.

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 more accurately than ever before.

In addition, according to another aspect of the present invention, the presence or absence of macadamia nut allergens in a target sample can be detected with greater accuracy than in the past.

FIG. 1 shows the results of an example and is a flow chart for classifying test subjects. FIG. 1 shows the results of an example, and shows the results of evaluating macadamia nut crude antigen sIgE and macadamia nut 7S globulin protein sIgE in the serum of participants. FIG. 1 shows the results of an example, in which macadamia nut crude antigen sIgE and macadamia nut 7S globulin protein sIgE were evaluated for samples in which the macadamia nut crude antigen sIgE in the serum of participants was 0.35 kU _A /L or higher. FIG. 1 shows the results of an example, comparing the measured values of macadamia nut 7S globulin protein sIgE and macadamia nut crude antigen sIgE in the serum of participants.

1. Method for detecting macadamia nut allergen-specific IgE
A method for detecting macadamia nut allergen-specific IgE according to one embodiment of the present invention is a method for detecting macadamia nut allergen-specific IgE in a target sample, comprising the steps of: contacting a polypeptide of the following (a) or (b) with the target sample; and detecting, after the contacting step, the presence or absence of IgE specifically bound to the polypeptide:
(a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1;
(b) a polypeptide having an amino acid sequence with a sequence identity of 95% or more to the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 and having binding ability to macadamia nut allergen-specific IgE. According to the method for detecting macadamia nut allergen-specific IgE of this embodiment, it is possible to detect the presence or absence of macadamia nut allergen-specific IgE antibody in a biological sample collected from a subject with higher accuracy than conventional methods, and as a result, it is possible to provide an indicator for accurately diagnosing whether or not the subject has a macadamia nut allergy.

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

The reaction conditions for contacting the detection antigen with the target sample are not particularly limited, and when the target sample contains macadamia nut allergen-specific IgE, the reaction conditions can be appropriately set so that the macadamia nut allergen-specific IgE specifically binds to the detection antigen and the binding can be maintained.

The contact step is exemplified below. For example, the detection antigen may be immobilized on any carrier and then brought into contact with the target sample. The method involves first immobilizing 20 μg/mL streptavidin on a black plate to create a streptavidin-immobilized plate. Next, biotinylated detection antigen is added to the wells of the plate to immobilize the detection antigen on the plate. The target sample diluted with 10% FBS solution is then added and incubated at room temperature for 2 hours to bring the detection antigen into contact with the target sample.

(Target sample)
The type of target 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 target samples. Such target samples are, for example, biological samples collected from subjects, and are preferably 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 mucus, urine, sweat, tears, tissue fluid, lymph, etc. The target sample is preferably a sample derived from blood, since it has a higher amount of IgE than other biological samples and can be collected in large quantities. The sample derived from blood may be whole blood, serum, or plasma.

The type of subject from which the target sample is collected is not particularly limited. Subjects may be, for example, humans or non-human mammals.

From the viewpoint of further improving the detection accuracy of macadamia nut allergen-specific IgE, the target sample is preferably a sample confirmed to contain macadamia nut crude antigen-specific IgE. Although the amount of macadamia nut crude antigen-specific IgE contained in the target sample is not particularly limited, in the conventional Immunocap method, macadamia nut crude antigen-specific IgE of 0.35 kU _A / L or more is judged to be positive, so the target sample is preferably a sample confirmed to contain macadamia nut crude antigen-specific IgE of 0.35 kU _A / L or more.

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 suitably adopted. By detecting the presence or absence of IgE that specifically binds to the polypeptide (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 improved.

(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 "macadamia nut 7S globulin protein", which is an allergen component (a substance that induces an allergic reaction and is also called an antigen) in macadamia nut allergy discovered by the present inventors. Here, "specifically bind" means that macadamia nut allergen-specific IgE binds only to the macadamia nut allergen, i.e., the macadamia nut 7S globulin protein, which is an antigen, and does not bind to other proteins. The IgE to be detected by the method for detecting macadamia nut allergen-specific IgE of this embodiment may be IgE alone, or IgE bound to mast cells or basophils.

(Macadamia nut 7S globulin protein)
The macadamia nut 7S globulin precursor protein is a protein consisting of 666 amino acids, which is processed to produce the macadamia nut 7S globulin protein consisting of 638 amino acids. Macadamia nut 7S globulin protein is split into multiple polypeptide chains such as MiAMP2 (Macadamia integrifolia antimicrobial protein 2) a, b, c or VLAP (vicilin-like antimicrobial peptides) by autocleavage (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 a partial peptide of the macadamia nut 7S globulin protein. In addition, Mac i 1 described in Non-Patent Document 2 also corresponds to a part of the macadamia nut 7S globulin protein described in this specification. 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 this specification, the term "protein" is used interchangeably with the term "polypeptide".

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

The polypeptide (b) has an amino acid sequence having mutations in 15 or less, preferably 10 or less, more preferably 5 or less, and even more preferably 2 or less amino acid residues in the amino acid sequence of SEQ ID NO: 1. The "mutations" are deletions, substitutions, or additions of amino acid residues.

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

In addition, by confirming whether or not the polypeptide (b) induces a macadamia nut allergy reaction, it is possible to indirectly confirm that the polypeptide (b) has binding ability to macadamia nut allergen-specific IgE. For example, if a skin test is performed in which the polypeptide (b) is applied to the skin of a subject with macadamia nut allergy, and a skin reaction such as swelling occurs in the area where the polypeptide (b) is applied, it can be said that the polypeptide (b) has induced a macadamia nut allergy reaction. Since an allergic reaction occurs as a result of the binding between an antigen and IgE, it can be determined that the polypeptide (b) has binding ability to macadamia nut allergen-specific IgE based on the presence or absence of an allergic reaction.

The polypeptide (a) or (b) can be obtained by a method known in the art (e.g., genetic engineering). The method for expressing the polypeptide (a) or (b) can be appropriately performed by a method known to those skilled in the art. A vector capable of inducing expression of the polypeptide can be selected by those skilled in the art depending on the type of host cell used, but in order to facilitate purification, it is preferable that the vector is capable of adding a tag to the polypeptide (a) or (b) (e.g., pCold-ProS2 vector). In addition, a host cell can be appropriately selected by those skilled in the art depending on the vector used. The host cell may be derived from a prokaryote such as Escherichia coli (E. coli).

The following is an example of a method for expressing the polypeptide (a). 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. Then, the polypeptide (a) can be expressed using Escherichia coli (e.g., SHuffle T7 Express lysY Competent Escherichia coli).

Methods for purifying the polypeptide of (a) include, for example, methods that utilize solubility such as salting out and solvent precipitation, methods that utilize differences in molecular weight such as dialysis, ultrafiltration, gel filtration and SDS-PAGE, methods that utilize charge such as ion exchange chromatography, methods that utilize specific affinity such as reversed-phase high performance liquid chromatography, methods that utilize differences in hydrophobicity such as reversed-phase high performance liquid chromatography, and methods that utilize differences in isoelectric point such as isoelectric focusing.

The following is an example of a purification method using the His-ProS2 tag of the polypeptide (a). First, the cells are collected by centrifugation, and then placed in a solution containing, for example, 50 mM Tris-HCl (pH 8.0) containing 200 mM NaCl and 1% Triton, 1 mg/mL lysozyme, and Halt Protease Inhibitor Cocktail, and then dissolved using ultrasound. The polypeptide (a) is purified using a column capable of removing the tag (for example, a HisTrap HP column). For example, the ProS2 tag is separated from the His tag using Turbo 3C protease, and the tag and enzyme are removed using a HisTrap HP column. The polypeptide (b) may also be expressed and purified in a similar manner.

The polypeptide (a) or (b) is preferably labeled with biotin, and is preferably immobilized on a streptavidin-immobilized carrier by the interaction between the biotin and the streptavidin. This allows the use of existing methods such as streptavidin immobilization ELISA in the process of detecting the presence or absence of macadamia nut allergen-specific IgE that has specifically bound to the polypeptide, making it easier to control accuracy than when using a simple ELISA method.

In addition, the polypeptide of (a) or (b) is preferably non-specifically biotin-labeled with respect to the amino acid sequence. Here, in this specification, "non-specifically biotin-labeled with respect to the amino acid sequence" refers to biotin-labeling of a target protein regardless of the type of functional group that the amino acid has. By biotin-labeling in a non-specific manner, it is possible to biotin-label the target protein at a uniform frequency over the entire length. Macadamia nut 7S globulin protein has a site containing many lysine residues, but by non-specifically biotin-labeling 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 between the polypeptide and IgE is improved and antigenicity can be maintained, thereby realizing highly accurate detection.

As a method for non-specifically labeling with biotin based on an amino acid sequence, for example, a method of labeling a polypeptide using a photoreactive biotin labeling reagent can be mentioned. An example of a photoreactive biotin labeling reagent is EZ-Link TFPA-PEG3-Biotin (manufactured by Thermo Fisher Scientific) used in the examples described below, but is not limited thereto.

When the polypeptide (a) or (b) is labeled with biotin, it is preferable that a biotin molecule is bound to the polypeptide (a) or (b) via a linker sequence. By providing a linker sequence between the polypeptide and the biotin molecule, when the biotin-labeled polypeptide is immobilized on a streptavidin-immobilized carrier, the distance between the polypeptide and the immobilization surface can be secured. As a result, the IgE antibody can easily access the polypeptide from a direction that the IgE antibody cannot reach in a normal ELISA method, improving the binding between the polypeptide and IgE and enabling more accurate detection. The linker sequence is not particularly limited, but polyethylene glycol can be suitably used as the linker sequence, as in EZ-Link TFPA-PEG3-Biotin (manufactured by Thermo Fisher Scientific) used in the examples described below.

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

The binding between the macadamia nut allergen-specific IgE in the subject sample and the polypeptide can be detected by a detection method based on a known antigen-antibody reaction. For example, such methods include ELISA (Enzyme-Linked Immunosorbent Assay), sandwich immunoassay, immunoblot, immunoprecipitation, immunochromatography, and immunocap. All of these methods detect the binding between the antigen and the subject's IgE by contacting the antigen with the subject's IgE to bind it, allowing an enzyme-labeled secondary antibody to act on the IgE that has specifically bound to the antigen, and adding a substrate for the enzyme (usually a color-developing or luminescent reagent) to detect the product of the enzyme reaction.

In the detection step, it is sufficient to detect the presence or absence of IgE specifically bound to the detection antigen, but the amount of IgE specifically bound to the detection antigen may also be quantified. The amount of IgE specifically bound to the detection antigen can be measured using a known method.

(Other processes)
The method for detecting macadamia nut allergen-specific IgE of this embodiment may include steps other than the first contact step and the first detection step described above. For example, it may further include a second contact step of contacting a macadamia nut crude antigen with a target sample, and a second detection step of detecting the presence or absence of IgE specifically bound 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 target sample can be detected.

The second contact step and the second detection step may be performed before the first contact step or after the first detection step. The second contact step and the second detection step are the same as those described for the first contact step and the first detection step, except that the macadamia nut crude antigen is used as the detection antigen, and therefore the description will not be repeated here. The macadamia nut crude antigen can be prepared by the method described in the Examples below.

The method may further include a step of determining the presence or absence of macadamia nut allergen-specific IgE in the target sample based on the results of the first and second detection steps. In one embodiment, in the determination step, if the result of the first detection step indicates that the target sample contains a predetermined amount or more of macadamia nut crude antigen-specific IgE, preferably 0.35 kU _A /L or more, and the result of the second detection step indicates that the target sample contains IgE that specifically binds to the polypeptide (a) or (b), then the target sample is determined to contain macadamia nut allergen-specific IgE.

(Use of detection results obtained by detection methods)
The detection result obtained by carrying out the detection method of this embodiment can be used as one of the diagnostic materials when a doctor makes a diagnosis. In addition, the subject who is judged to have the above-mentioned macadamia nut allergy or to have the possibility of having macadamia nut allergy may be diagnosed by a doctor (if the subject is a human) or a veterinarian (if the subject is a mammal other than a human) as necessary, and then a treatment plan according to the degree of symptoms of macadamia nut allergy may be determined and treatment according to the treatment plan. 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 dietary therapy of ingesting macadamia nut allergen-free diet; oral immunotherapy using macadamia nut allergen, etc.

<2. Macadamia nut allergen-specific IgE detection kit>
A kit for detecting macadamia nut allergen-specific IgE according to one embodiment of the present invention comprises the following polypeptide (a) or (b) as an antigen:
(a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1;
(b) a polypeptide having an amino acid sequence with a sequence identity of 95% or more to the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 and having binding ability to macadamia nut allergen-specific IgE. The detection kit of this embodiment can be suitably used in the detection method described in Section <1. Method for detecting macadamia nut allergen-specific IgE>.

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

The polypeptide (a) or (b) and the macadamia nut crude antigen are as described in Section 1. Method for detecting macadamia nut allergen-specific IgE, so the description will not be repeated here.

The antigen for detection may be bound to a substrate. Examples of substrates include plates, membranes, columns, beads, and gold colloids. A fluorescent protein or a tag protein may be immobilized on the substrate. Since multiple samples can be processed in parallel, the substrate is preferably a microwell plate (e.g., 96 wells).

(Form of detection kit)
The form of the detection kit of this embodiment is not particularly limited, and may be provided in the form of, for example, an allergen chip, an ELISA kit, a digital ELISA kit, or the like.

The detection kit of this embodiment may contain multiple types of other allergens as antigens in addition to the polypeptide (a) or (b). This allows comprehensive detection of the presence or absence of multiple types of allergen-specific IgE, including macadamia nut allergen-specific IgE, in a target sample. The types of other allergens contained in the detection kit of this embodiment are not particularly limited. For example, by containing nut allergens such as peanuts, almonds, cashew nuts, pistachios, walnuts, pecan nuts, hazel nuts, and Brazil nuts as antigens, the presence or absence of allergen-specific IgE to various nuts in a target sample can be comprehensively detected.

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 (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.

Depending on the form of the kit, the detection kit according to one embodiment of the present invention may further include at least one of the following, if necessary: a substrate; various reagents (secondary antibodies, reporter molecules, buffers, etc.); equipment (test tubes, pipettes, etc.); instructions for use of the detection kit; and samples for control tests. The instructions for use of the detection kit include, for example, specific procedures for the detection method described in Section <1. Method for detecting macadamia nut allergen-specific IgE>.

<3. In vitro diagnostics for macadamia nut allergy>
The scope of the present invention also includes an in vitro diagnostic agent for diagnosing macadamia nut allergy, comprising the following polypeptide (a) or (b) as an antigen:
(a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1;
(b) a polypeptide having an amino acid sequence with a sequence identity of 95% or more to the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 and having binding ability to macadamia nut allergen-specific IgE. According to the in vitro diagnostic agent of this embodiment, the detection method described in Section <1. Method for detecting macadamia nut allergen-specific IgE> is used to detect macadamia nut allergen-specific IgE in a subject sample, so that macadamia nut allergy can be diagnosed with higher accuracy.

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

The polypeptide (a) or (b) and the macadamia nut crude antigen are as described in Section 1. Method for detecting macadamia nut allergen-specific IgE, so the description will not be repeated here.

The in vitro diagnostic agent of this embodiment can also be provided in the form of a kit. The configuration of the kit is as explained in Section 2. Kit for detecting macadamia nut allergen-specific IgE, so the explanation will not be repeated here.

4. Method for detecting macadamia nut allergens
A method for detecting a macadamia nut allergen according to one embodiment of the present invention is a method for detecting a macadamia nut allergen in a target sample, comprising a contacting step of contacting the target sample with 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 that specifically binds to the antibody or the functional fragment thereof after the contacting step. According to the method for detecting a macadamia nut allergen of this embodiment, it is possible to detect the presence or absence of a macadamia nut allergen in a target sample more accurately than before.

(Contact process)
The contacting step is a step of contacting the target sample with an antibody or a functional fragment thereof that specifically recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. 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 explanation, the antibody that specifically recognizes the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is referred to as a "detection antibody" for convenience of explanation.

The reaction conditions for contacting the detection antibody with the target sample are not particularly limited, and if the target sample contains a macadamia nut allergen, the reaction conditions can be appropriately set so that the macadamia nut allergen specifically binds to the detection antibody and the binding can be maintained.

(Target sample)
The type of target sample that is used in the detection method of macadamia nut allergen of this embodiment is not particularly limited.All samples that are desired to detect the presence or absence of macadamia nut allergen are target samples.Such target samples are articles that may be ingested into the human body or absorbed into the human body through the skin or mucous membrane, and more specifically, can include food, foodstuffs, beverages, medicines, quasi-drugs, cosmetics, etc.

(An antibody that specifically recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1)
An antibody that specifically recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is intended to mean an antibody that specifically recognizes at least a portion of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 as an epitope and can specifically bind to it.

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

"Functional fragments of antibodies" refer to those that have a partial region of the aforementioned antibody and have antigen-binding ability (synonymous with binding fragments). Natural antibodies can be derived from any species, including, but not limited to, humans, mice, rats, goats, rabbits, camels, llamas, cows, chickens, sharks, and fish. Antibodies produced using recombinant technology include, but are not limited to, chimeric antibodies such as humanized antibodies and primatized antibodies obtained by genetically modifying natural antibodies, synthetic antibodies, recombinant antibodies, mutated antibodies, and grafted antibodies (e.g., antibodies conjugated or fused with other proteins and radioactive labels), and also include antibodies that have been modified in the same way as when genetically modifying natural antibodies as described above, for antibodies already produced using recombinant technology. Specific examples of functional antibody fragments include 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).

Furthermore, in the present invention, the concept of a binding fragment also includes an antibody fragment that has been mutated to the extent that it maintains its reactivity with the target protein. The above-mentioned mutations are introduced using known techniques such as gene modification techniques that can be appropriately selected by those skilled in the art.

The detection antibody is preferably a polyclonal antibody, since it can recognize various epitopes of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. Based on the information of the amino acid sequence shown in SEQ ID NO: 1, a person 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.

(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. The 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 explained in the section <1. Method for detecting macadamia nut allergen-specific IgE>, so the explanation will not be repeated here.

In the detection step, it is sufficient to detect the presence or absence of an antigen that has specifically bound to the detection antibody, but the amount of the antigen that has specifically bound to the detection antibody may also be measured. The amount of the antigen that has specifically bound to the detection antibody can be measured using a known method.

(Use of detection results obtained by detection methods)
The detection results obtained by carrying out the detection method of this embodiment can be used as one of the judgment materials when performing quality control at the manufacturing site of food, ingredients, beverages, medicines, quasi-drugs, cosmetics, etc. In addition, depending on the detection results, a process for removing macadamia nut allergens from the target sample may be performed. For example, 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 fixed to a carrier is used, and the target sample is passed through the affinity column to remove macadamia nut allergens from the target sample.

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

〔summary〕
A 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 contacting step of contacting the target sample with a polypeptide of (a) or (b) below, and a detection 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 a sequence identity of 95% or more to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and having binding ability to macadamia nut allergen-specific IgE.

In the method for detecting macadamia nut allergen-specific IgE according to aspect 2 of the present invention, in the above-mentioned aspect 1, the target sample is preferably a sample that has been confirmed to contain macadamia nut crude antigen-specific IgE.

In the method for detecting macadamia nut allergen-specific IgE according to aspect 3 of the present invention, in the above-mentioned aspect 1 or 2, it is preferable that the target sample is a sample derived from blood.

In the method for detecting macadamia nut allergen-specific IgE according to aspect 4 of the present invention, in any one of aspects 1 to 3, it is preferable that the polypeptide (a) or (b) is labeled with biotin and immobilized on a streptavidin-immobilized carrier by the interaction between the biotin and the streptavidin.

In the method for detecting macadamia nut allergen-specific IgE according to aspect 5 of the present invention, in the above-mentioned aspect 4, it is preferable that the polypeptide (a) or (b) is non-specifically labeled with biotin in terms of the amino acid sequence.

The in vitro diagnostic agent for diagnosing macadamia nut allergies according to aspect 6 of the present invention is configured to include the following polypeptide (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 a sequence identity of 95% or more with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and having binding ability to macadamia nut allergen-specific IgE.

The kit for detecting macadamia nut allergen-specific IgE according to aspect 7 of the present invention is configured to include the following polypeptide (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 a sequence identity of 95% or more with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and having binding ability to macadamia nut allergen-specific IgE.

The method for detecting a macadamia nut allergen according to aspect 8 of the present invention is a method for detecting a macadamia nut allergen in a target sample, and includes a contacting step of contacting the target sample with 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 that specifically binds to the antibody or the functional fragment thereof after the contacting step.

One embodiment of the present invention is described below.

(Participant)
A total of 86 participants were provided from the University of Tokyo Hospital, Aichi Children's Health and Medical Center, National Hospital Organization Mie Hospital, National Center for Child Health and Development, Yamaguchi University Hospital, and other institutions. Ethics approval was obtained from the ethics committees of all hospitals where the recruited participants were affiliated. Informed consent was obtained from all children and/or parents.

(Criteria for patients included in the study)
Eighty-six people under the age of 20 were included in the study, and the patient group was classified according to Figure 1. The patient group was made up of patients with a confirmed medical history of immediate reactions to nuts, including peanuts and/or macadamia nuts (n=18) who had a definite medical history of macadamia nut allergy. If there was no history of immediate reactions, an oral food challenge (OFC) was performed, and OFC-positive patients (n=27) were added to the patient group, and OFC-negative patients (n=41) were included in the control group.

(OFC test)
The OFC test was performed according to the Japanese Pediatric Guideline for Food Allergy, and was performed according to the method determined by each hospital. Macadamia nuts were administered at 3.5 to 10 g for all schedules. After testing the oral intake dose for immunity, the results were judged as positive or negative based on whether or not an allergic reaction was observed.

(Preparation of Macadamia Nut 7S Globulin Protein)
The sequence of the polynucleotide (coding region) encoding the macadamia nut 7S globulin protein (GenBank: AF161883.1, SEQ ID NO: 2) was obtained from the NCBI (National Center for Biotechnology Information) website (https://www.ncbi.nlm.nih.gov). The encoded sequence was synthesized with codon optimization (Integrated DNA Technologies). This sequence was inserted into pCold-ProS2 vector (Takara Bio Inc.), and 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 macadamia nut 7S globulin protein is shown in SEQ ID NO:1.

Cells were harvested by centrifugation and then sonicated in a solution containing 50 mM Tris-HCl (pH 8.0) containing 200 mM NaCl and 1% Triton, 1 mg/mL lysozyme (Merck), and Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). The His-ProS2 tagged recombinant protein 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 enzyme were removed using a HisTrap HP column. The concentration of the purified protein was measured using a Pierce BCA Protein Assay (Thermo Fisher Scientific). The protein was biotinylated using EZ-Link TFPA-PEG3-Biotin (Thermo Fisher Scientific).

(Preparation of Macadamia Nut Crude Antigen)
Preparation of the macadamia nut crude antigen was outsourced to Thermo Fisher Scientific.

(Measurement of macadamia nut crude antigen-specific IgE)
The amount of macadamia nut crude antigen-specific IgE in the serum was measured by ImmunoCAP (registered trademark) at Thermo Fisher Scientific.

(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 streptavidin immobilization ELISA. First, in an environment of 4 ° C., Black 384-well plates (Greiner Bio-One) were coated overnight with 20 μg / mL streptavidin (Merck). Then, the washed plate was blocked with phosphate buffer salt containing 10% FBS to prepare a streptavidin immobilized plate.

50 μg/mL of biotinylated recombinant macadamia nut 7S globulin protein was added to the wells. Human serum samples were diluted with 10% FBS solution, 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 sequential reaction with anti-human IgE rabbit antibody (Dako) followed by horseradish peroxidase-conjugated anti-rabbit antibody (Cell Signaling Technology). Luminescence signals of Western Lightning ECL Pro (PerkinElmer) were measured using a GloMax luminometer (Promega).

(statistical analysis)
Statistical analysis was performed using JMP 15 (SAS Institute Inc.) and Prism 8 (GraphPad Software). Mann-Whitney U test and Pearson's chi-square test were used to compare continuous and categorical data between groups. Diagnostic performance was evaluated by the area under the receiver operating characteristic curve (ROC).

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 people who developed allergic complications between the patient group and the control group. On the other hand, the level of macadamia nut crude antigen-specific IgE (macadamia nut crude antigen sIgE) was significantly increased in the patient group compared to the control group (p<0.01).

The amount of macadamia nut 7S globulin protein sIgE in serum samples from the patient and control groups was analyzed by the streptavidin immobilization ELISA method, and the amount of macadamia nut crude antigen sIgE was analyzed by the Immunocap method, and the results were compared. In addition, ROC analysis was performed on the amount of macadamia nut crude antigen sIgE and the amount of macadamia nut 7S globulin protein sIgE detected in the patient and control groups, and the AUC (AUC: Area under the curve) of each was compared.

The results are shown in Figure 2. In the ROC curve graph shown in Figure 2, the vertical axis represents sensitivity and the horizontal axis represents (1-specificity). The ROC based on macadamia nut crude antigen sIgE measurements was 0.748, while the ROC based on macadamia nut 7S globulin protein sIgE measurements was 0.812, which was increased compared to the ROC based on macadamia nut crude antigen sIgE measurements. In addition, the AUC based on the ROC analysis of macadamia nut 7S globulin protein sIgE (0.834) showed a significant increase compared to the AUC based on the ROC analysis of macadamia nut crude antigen sIgE (0.660) (p<0.01).

Next, the results of the case where the patient group and the control group were subjects whose macadamia nut crude antigen sIgE amount was 0.35 kU _A / L or more are shown in FIG. 3. In the graph of the ROC curve shown in FIG. 3, the vertical axis represents sensitivity and the horizontal axis represents (1-specificity). In addition, in the results of the macadamia nut 7S globulin protein sIgE measurement, samples whose measurement values were found to be negative are surrounded by a dotted line. In the case where the patient group and the control group were subjects whose macadamia nut crude antigen sIgE amount was 0.35 kU _A / L or more, the ROC based on the macadamia nut 7S globulin protein sIgE measurement value was 0.885. In addition, the AUC (0.823) based on the ROC analysis of the macadamia nut 7S globulin protein sIgE showed a larger difference between the control group and the patient group compared to the AUC (0.586) based on the ROC analysis of the macadamia nut crude antigen sIgE (p<0.01).

Figure 4 shows the results of comparing serum macadamia nut 7S globulin protein sIgE with macadamia nut crude antigen sIgE. The results in Figure 4 show that the 25 control samples with positive macadamia nut crude antigen sIgE measurements were negative for macadamia nut 7S globulin protein sIgE.

〔result〕
According to the detection method of one aspect of the present invention, it has been revealed that the presence or absence of IgE antibodies that cause the onset of macadamia nut allergy in a target sample can be detected more accurately than in the past by detecting macadamia nut 7S globulin protein-specific IgE in the serum of a subject. It has also been revealed that by using macadamia nut 7S globulin protein-specific IgE in serum as an indicator, it is possible to provide an indicator for more accurately diagnosing whether or not a subject has macadamia nut allergy, compared to the conventional method of measuring macadamia nut crude antigen-specific IgE levels.

Furthermore, it was revealed that by using macadamia nut 7S globulin protein-specific IgE in serum as an indicator, it is possible to distinguish with high accuracy false positive negative cases from those cases that were determined to be positive for macadamia nut allergy in a conventional diagnosis based on macadamia nut crude antigen-specific IgE values. These results show that by detecting macadamia nut 7S globulin protein-specific IgE in the serum of a subject, it is possible to provide an indicator that can more accurately diagnose whether or not a subject has a macadamia nut allergy than before.

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 more accurately than in the past. This makes it suitable for use in the medical field. According to another aspect of the present invention, the presence or absence of macadamia nut allergens in a target sample can be detected more accurately than in the past. This makes it suitable for use in the field of food manufacturing, etc.

Claims (7)

A method for supporting diagnosis of macadamia nut allergy, comprising:
A contacting step of contacting a subject sample with a polypeptide of the following (a) or (b):
a detection step, after the contacting step, of detecting the presence or absence of IgE specifically bound to the polypeptide;
A method for supporting diagnosis of macadamia nut allergy , comprising:
(a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1;
(b) a polypeptide having an amino acid sequence that has a sequence identity of 95% or more with a polypeptide having the amino acid sequence shown in SEQ ID NO:1 and has binding ability to macadamia nut allergen-specific IgE. The method for supporting diagnosis of macadamia nut allergy according to claim 1 , wherein the target sample is a sample confirmed to contain macadamia nut crude antigen-specific IgE. The method for supporting diagnosis of macadamia nut allergy according to claim 1 or 2, characterized in that the target sample is a sample derived from blood. the polypeptide (a) or (b) is biotin-labeled;
The method for supporting diagnosis of macadamia nut allergy according to any one of claims 1 to 3, wherein the biotin is immobilized on a streptavidin-immobilized carrier by interaction between the biotin and the streptavidin. The method for supporting diagnosis of macadamia nut allergy according to claim 4, wherein the polypeptide (a) or (b) is non-specifically labeled with biotin in an amino acid sequence manner. An in vitro diagnostic agent for diagnosing macadamia nut allergies, comprising the following polypeptide (a) or (b) as an antigen:
(a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1;
(b) a polypeptide having an amino acid sequence that has a sequence identity of 95% or more with a polypeptide having the amino acid sequence shown in SEQ ID NO:1 and has binding ability to macadamia nut allergen-specific IgE. A method for detecting an allergen involved in the development of macadamia nut allergy in a subject sample, comprising:
a contacting step of contacting the target sample with an antibody or a functional fragment thereof that specifically recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1;
a detection step of detecting the presence or absence of an antigen , which is a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, that specifically binds to the antibody or a functional fragment thereof after the contacting step;
A method for detecting an allergen involved in the onset of macadamia nut allergy , comprising:

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