Prediction method

Abstract

The present invention addresses the problem of constructing a means for predicting the challenge dose of an allergen that can be ingested in a next oral food challenge by a subject that has undergone the oral food challenge of the allergen. The challenge dose of an allergen that can be ingested in a next oral food challenge can be effectively predicted by creating a graph using, as a parameter of the cross-inhibiting antibody ratio of sIgE, the value of sIgE antibody titer / (sIgG1 antibody titer+sIgG4 antibody titer), in addition to the value of the sIgE antigen-binding affinity antibody titer obtained by multiplying the sIgE antibody titer by the value of 1 / IC50 of an sIgE antibody.

Description

Prediction method

[0001] The present invention relates to a method for predicting the amount of allergen that can be loaded in an oral food challenge test. More specifically, the present invention relates to a method for predicting the amount of allergen that a subject who has undergone a first oral food challenge test for an allergen can load in the next oral food challenge test, and a system for predicting the amount of allergen. Furthermore, the present invention relates to a method for predicting the amount of allergen in an oral food challenge test for a subject before undergoing the oral food challenge test.

[0002] Food allergy is defined as "a phenomenon in which an adverse symptom is caused to the living body through an antigen-specific immunological mechanism caused by food" (see, for example, Non-Patent Document 1). Oral immune tolerance (immune tolerance) is normally functioning to prevent the living body from reacting defensively to undigested foreign substances in the food that humans digest and absorb to maintain life. However, it is said that 5-10% of newborn infants develop some kind of food allergy by the age of 1 year. <000001l>

[0003] As a reliable diagnostic method for the presence or absence of allergy onset in infants and children, there is an "oral food challenge test (Oral Food Challenge: OFC)". For example, the Food Allergy Clinical Practice Guidelines 2021 (see Non-Patent Document 1) shows an example of the total load amount in the oral food challenge test. For example, in the case of high-risk subjects such as those who have induced symptoms in small amounts or those with a history of anaphylaxis, an oral food challenge test targeting a low dose is performed. If the result is negative, it is recommended to proceed to an oral food challenge test with a medium dose or a full dose in the next test.

[0004] However, in reality, the mechanisms of allergy development and methods for predicting its onset are not yet fully understood. While hay fever, atopic dermatitis, and food allergies are widely known as specific symptoms in which IgE antibodies are thought to be involved, there are findings that blood IgE levels do not always correlate with allergy symptoms, and that non-IgE-dependent reactions are also involved. Furthermore, antigen-specific antibodies induced by the entry of allergens into the body include not only IgE but also IgA, various IgGs, etc., and there is also the finding that the sum of the effects of these antibodies forms allergy symptoms.

[0005] On the other hand, instead of measuring only the "amount of IgE," the "sum of antigen-antibody binding affinities" (avidity), which represents the "quality of IgE" that the variable region of IgE antibodies exhibits towards allergens, is considered. The value of "1 / IC50" is selected as a parameter indicating the avidity of IgE antibodies towards allergens. Using the value obtained by multiplying the IgE antibody titer of allergen-sensitized infants and children by the value of 1 / IC50 of sIgE antibodies, i.e., the sIgE antigen-binding affinity antibody titer, the presence or absence of anaphylaxis was analyzed, and a significance test of the above parameter was performed. It has been reported that this shows a significantly statistically significant difference between those who developed anaphylaxis and those who did not, compared to using existing parameters (see, for example, Patent Document 1). However, it has not been possible to predict the appropriate loading amount of allergenic food for each test in oral food challenge tests used in the diagnosis of allergy onset.

[0006] International Open Brochure WO2021 / 100719

[0007] Food Allergy Treatment Guidelines 2021, Food Allergy Committee, Japanese Society for Pediatric Allergy, Nutrients 2023, 15, 2770. World Allergy Organ J. 2024, 17:100876. Allergology International Volume 69, Issue 3, July 2020, Pages 370-386.

[0008] Oral food challenge tests are conducted to definitively diagnose food allergies, determine safe intake levels, and confirm tolerance acquisition. As mentioned above, when oral food challenge tests are performed two or more times, if no severe symptoms such as anaphylaxis or allergic symptoms shown in Table 4 below were observed in the previous test, the basic approach is to increase the amount of the allergenic food (allergen) compared to the previous oral food challenge test to determine a sustainable safe intake level. However, the extent to which the amount can be increased is determined in accordance with the above guidelines, and is mainly based on the physician's experience.

[0009] However, in reality, there are a small number of subjects who have experienced severe symptoms such as anaphylaxis with only a slight increase in the food load. On the other hand, there are cases where oral food challenge tests are conducted numerous times, and ultimately the allergic reaction to a particular food is determined to be negative. Even for allergy specialists, it is not easy to find appropriate criteria for determining what amount of the allergenic food should be challenged in the next test. Therefore, there is a need to develop a method that can minimize the possibility of anaphylaxis, conduct oral food challenge tests more efficiently, and determine a safe intake amount.

[0010] The object of the present invention is to develop a means for predicting the amount of allergen that a subject who has undergone an oral food challenge test for an allergen, or a subject who is scheduled to undergo an oral food challenge test, can ingest in the next oral food challenge test (including the first time and / or the first time for subjects who have never undergone an oral food challenge test).

[0011] The present inventors have confirmed that by using the value obtained by multiplying the antigen-specific IgE (sIgE) antibody titer by the value of 1 / IC50 (avidity) of the sIgE antibody, i.e., the value of the sIgE antigen-binding affinity antibody titer, as a parameter and performing ROC (Receiver Operating Characteristic) curve analysis, it is possible to determine the cutoff values ​​for positive and negative food allergy symptom induction, and that it is possible to effectively predict the onset of anaphylaxis in allergy patients for whom there was no effective diagnostic method for the possibility of developing anaphylaxis (see Patent Document 1 above).

[0012] Furthermore, the present inventors have reported on the possibility of calculating the predicted positive value (%) and negative value (%) for food allergy symptom induction by combining the value obtained by multiplying the sIgE antibody titer by the 1 / IC50 value of the sIgE antibody, i.e., the value of the sIgE antigen-binding affinity antibody titer, and the ratio of the sIgE antibody titer to the sIgG4 antibody titer (sIgE cross-inhibitory antibody) as parameters (Non-Patent Literature 2).

[0013] However, the immunoglobulin class switches proposed so far include [IgM → IgG3 → IgG1 → IgG2 → IgG4], [IgM → low affinity IgE, IgA], and [IgG1 → high affinity IgE]. IgG4 appears last in the immunoglobulin class switch process and is usually present in small amounts. Therefore, in some subjects, the antibody titer of sIgG4 may not function adequately as a parameter.

[0014] The inventors continued their search for effective parameters and found that by using the value of "sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer)" as a parameter for the cross-inhibition antibody ratio of sIgE, in addition to the sIgE antigen-binding affinity antibody titer obtained by multiplying the sIgE antibody titer by the 1 / IC50 value of the sIgE antibody, and graphing the results, the amount of allergen that can be ingested in the next oral food challenge test can be effectively predicted. Furthermore, by plotting positive subjects who developed an allergic reaction and negative subjects who did not develop an allergic reaction during the oral food challenge test, and creating graphs for determining negative subjects and positive subjects, it was confirmed that not only allergy specialists but also general practitioners involved in community healthcare can more appropriately predict the amount of allergen to be ingested in the next oral food challenge test. Furthermore, by using the sIgE antigen-binding affinity antibody titer, obtained by multiplying the sIgE antibody titer by the sIgE antibody's 1 / IC50 value, divided by the antigen load (the amount of allergen ingested in the oral food challenge test) (sometimes referred to as "sIgE / sIgE IC50 / antigen load"), and the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) by the antigen load (sometimes referred to as "sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) / antigen load") as parameters for the sIgE cross-inhibition antibody ratio and graphing them, we confirmed that the accuracy of predicting the amount of allergen that can be ingested in the next oral food challenge test can be further improved, thus completing the present invention.

[0015] According to the present invention, a subject who has undergone an oral food challenge test for an allergen can predict the amount of allergen they can ingest in the next oral food challenge test.

[0016] In other words, the present invention is defined by the following: [1] A method for predicting the amount of allergen that a subject who has undergone a primary oral food challenge test for the allergen can be given in a subsequent oral food challenge test. [2] The method according to [1] above, characterized in that the amount of allergen to be given is predicted based on a graph created by measuring antigen-specific sIgE antibody titers, sIgG1 antibody titers, and sIgG4 antibody titers in a sample taken from a subject who has undergone a primary oral food challenge test for the allergen. [3] The method according to [2] above, characterized in that the amount of allergen loading is predicted based on a graph created with the value of the sIgE antigen-binding affinity antibody titer, obtained by multiplying the sIgE antibody titer in a sample taken from a subject who underwent a primary oral food challenge test by the value of 1 / IC50 of the sIgE antibody, as the Y axis, and the value of the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), calculated based on the sIgG1 antibody titer, sIgG4 antibody titer, and sIgE antibody titer in a sample taken from a subject who underwent a primary oral food challenge test, as the Y axis, and the X axis as the value of the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), which is calculated based on the sIgE antibody titer, sIgG4 antibody titer, and sIgE antibody titer in a sample taken from a subject who underwent a primary oral food challenge test. [4] The method according to [2] above, characterized in that the amount of allergen loading is predicted based on a graph created with the value of the sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer in a sample taken from a subject that underwent a primary oral food challenge test by the value of 1 / IC50 of the sIgE antibody, divided by the amount of antigen loaded in the primary oral food challenge test, as the Y axis, and the value of the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), which is calculated based on the sIgG1 antibody titer, sIgG4 antibody titer, and sIgE antibody titer, divided by the amount of antigen loaded, as the X axis.[5] The method described in [3] above, characterized in that the graph is a graph for determining the onset of food allergies, created by dividing the graph into four regions: (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>), based on the orthogonality of the cutoff value <y> determined by ROC analysis based on the value of sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer in a sample taken from a subject who underwent a primary oral food challenge test by the value of 1 / IC50 of the sIgE antibody, and the cutoff value <x> determined by ROC analysis based on the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) calculated based on the sIgG1 antibody titer, sIgG4 antibody titer, and sIgE antibody titer. [6] The method according to [4] above, characterized in that the graph is a graph for determining the onset of food allergies, created by dividing the graph into four regions: (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>), based on the orthogonality of the cutoff value <y> determined by ROC analysis based on the value obtained by dividing the sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer in a sample taken from a subject that underwent a primary oral food challenge test by the value of 1 / IC50 of the sIgE antibody, by the amount of antigen challenged in the primary oral food challenge test, and the cutoff value <x> determined by ROC analysis based on the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), which is calculated based on the sIgE antibody titer, sIgG4 antibody titer, and sIgE antibody titer, by the amount of antigen challenged in the primary oral food challenge test. [7] The method according to [5] or [6] above, characterized in that for each subject that underwent the primary oral food challenge test, two types of graphs are created, a graph for determining negative subjects and a graph for determining positive subjects, by plotting them separately for positive subjects who developed an allergic reaction and negative subjects who did not develop an allergic reaction. [8] The method according to [7] above, characterized in that the graph for determining negative subjects and the graph for determining positive subjects are three types of graphs for determining negative subjects and three types of graphs for determining positive subjects, respectively, created by plotting them separately for subjects who were given a small amount of allergen in the primary oral food challenge test, subjects who were given a moderate amount of allergen, and subjects who were given an amount of allergen that was consumed daily.[9] The method described in [7] above, characterized in that the amount of allergen to be loaded in the next oral food challenge test is predicted as follows: 1) In the graph for determining negative subjects, subjects belonging to the regions (-<x>, -<y>) and (+<x>, -<y>) can be predicted to be loaded at an amount that is 2 to 5 levels higher than the amount ingested in the oral food challenge test, or at an amount equal to or greater than their daily intake; 2) In the graph for determining negative subjects, subjects belonging to the regions (+<x>, +<y>) and (-<x>, +<y>) can be predicted to be loaded at an amount that is 1 to 3 levels higher than the amount ingested in the first oral food challenge test; 3) In the graph for determining positive subjects, subjects belonging to the regions (-<x>, -<y>) and (+<x>, -<y>) can be predicted to be loaded at an amount that is 0.5 to 2 levels lower than the amount ingested in the first oral food challenge test; 4) In the graph for determining positive subjects, subjects belonging to the regions (+<x>, +<y>) and (-<x>, +<y>) can be predicted to ingest a load that is 1 to 5 levels or more lower than the amount ingested in the first oral food challenge test;

[10] The method according to [1] to [6] above, characterized by comprising the following steps (a) to (g). (a) A step of measuring the sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer in samples taken from subjects who underwent a primary oral food challenge test; (b) A step of creating a graph with the sIgE antigen-binding affinity antibody titer, obtained by multiplying the sIgE antibody titer in samples taken from subjects who underwent a primary oral food challenge test by the value of 1 / IC50 of the sIgE antibody, on the Y axis, and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), calculated based on the sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer, on the X axis, or, (b') A process to create a graph with the following parameters: the Y-axis is the value obtained by multiplying the sIgE antibody titer in a sample taken from a subject who underwent a primary oral food challenge test by the 1 / IC50 value of the sIgE antibody, and dividing that value by the antigen load imposed in the primary oral food challenge test; and the X-axis is the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), calculated based on the sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer, by the above antigen load;(c)(b)In the graph created in step (c), the cutoff value <y> by ROC analysis is determined based on the value of the sIgE antigen-binding affinity antibody titer obtained by multiplying the sIgE antibody titer in the sample taken from the subject who underwent the primary oral food challenge test by the value of 1 / IC50 of the sIgE antibody, and the cutoff value <x> by ROC analysis is determined based on the value of the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) in the sample taken from the subject who underwent the primary oral food challenge test, or, (c')(b') In the graph created in step (c')(b'), the cutoff value <y> by ROC analysis is determined based on the value obtained by dividing the sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer in the sample taken from the subject who underwent the primary oral food challenge test by the value of 1 / IC50 of the sIgE antibody, by the antigen loading amount imposed in the primary oral food challenge test, and the cutoff value <x> by ROC analysis is determined based on the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) in the sample taken from the subject who underwent the primary oral food challenge test by the above antigen loading amount; (d) A step in which the graph is divided into four regions, (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>) by the cutoff value <y> and the cutoff value <x> determined in step (c) or (c') by ROC analysis being orthogonal, and a judgment graph is created; (e) A step in which, for each subject that underwent the primary oral food challenge test, positive subjects that developed an allergic reaction and negative subjects that did not develop an allergic reaction are distinguished, and the sIgE antigen-binding affinity antibody titer obtained by multiplying the sIgE antibody titer by the value of 1 / IC50 of the sIgE antibody and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) are plotted on the judgment graph to create a graph for determining negative subjects and a graph for determining positive subjects, or,(e') For each subject who underwent the primary oral food challenge test, a process to distinguish between positive subjects who developed an allergic reaction and negative subjects who did not develop an allergic reaction, and to create a graph for determining negative subjects and a graph for determining positive subjects by plotting the value obtained by dividing the sIgE antigen-binding affinity antibody titer (sIgE antibody titer multiplied by the sIgE antibody 1 / IC50 value) by the amount of antigen loaded in the primary oral food challenge test, and the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) by the above amount of antigen loaded on a determination graph; (f) A step to create three types of graphs for determining negative subjects and three types of graphs for determining positive subjects by further distinguishing and plotting subjects who were loaded with a small amount of allergen, subjects who were loaded with a moderate amount of allergen, and subjects who were loaded with the daily intake amount of allergen in the first oral food challenge test in the graphs for determining negative subjects and the graphs for determining positive subjects created in step (e) or step (e'); (g) A step to make predictions as follows: 1) Subjects belonging to the region of (-<x>, -<y>) and (+<x>, -<y>) in the graph for determining negative subjects can consume an amount that is 2 to 5 steps higher than the amount ingested in the first oral food challenge test, or an amount equal to or greater than the daily intake; 2) In the graph used to determine negative subjects, subjects belonging to the regions (+<x>, +<y>) and (-<x>, +<y>) can consume a load that is 1 to 3 levels higher than the amount consumed in the first oral food challenge test; 3) In the graph used to determine positive subjects, subjects belonging to the regions (-<x>, -<y>) and (+<x>, -<y>) can consume a load that is 0.5 to 2 levels lower or 0.5 to 1 level higher than the amount consumed in the first oral food challenge test; 4) In the graph used to determine positive subjects, subjects belonging to the regions (+<x>, +<y>) and (-<x>, +<y>) can consume a load that is 1 to 5 levels or more lower than the amount consumed in the first oral food challenge test;

[0017] Furthermore, the present invention is defined by the following:

[11] A system for predicting the amount of allergen to be ingested in a subsequent oral food challenge test by a subject who has undergone a primary oral food challenge test for the allergen, comprising: a storage unit for storing the personal data of each subject; a calculation unit that calculates the value of sIgE antigen-binding affinity antibody titer by multiplying the sIgE antibody titer by the 1 / IC50 value of the sIgE antibody based on the sIgE antibody titer in a sample taken from a subject who has undergone a primary oral food challenge test, and calculates the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) based on the sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer; and a graph creation unit that creates a graph with the value of sIgE antigen-binding affinity antibody titer (sIgE antibody titer multiplied by the 1 / IC50 value of the sIgE antibody) as the Y axis and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) as the X axis. The ROC analysis unit determines the cutoff value <y> by ROC analysis based on the sIgE antigen-binding affinity antibody titer obtained by multiplying the sIgE antibody titer by the value of 1 / IC50 of the sIgE antibody, and determines the cutoff value <x> by ROC analysis based on the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), and the graph region division unit divides the graph into four regions: (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>) by orthogonality between the cutoff value <y> determined by ROC analysis and the cutoff value <x> determined by ROC analysis, thereby creating a four-part graph. The system comprises: a plotting unit that, for each subject who underwent a primary oral food challenge test, distinguishes between positive subjects who developed an allergic reaction and negative subjects who did not develop an allergic reaction, and plots the sIgE antigen-binding affinity antibody titer (calculated by multiplying the sIgE antibody titer by the 1 / IC50 value of the sIgE antibody) and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) on the four-part graph to create graphs for determining negative subjects and graphs for determining positive subjects; and a prediction unit that predicts the amount of allergen to be loaded in the next oral food challenge test for each subject.

[12] A system for predicting the amount of allergen to be ingested in a subsequent oral food challenge test by a subject who has undergone a primary oral food challenge test for the allergen, comprising: a storage unit for storing the personal data of each subject; a calculation unit that calculates a value by dividing the sIgE antigen-binding affinity antibody titer, obtained by multiplying the sIgE antibody titer by the 1 / IC50 value of the sIgE antibody, by the amount of antigen loaded in the primary oral food challenge test, based on the sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer, by the amount of antigen loaded in the primary oral food challenge test, based on the sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer, by the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer); A graph creation unit creates a graph on which the Y-axis is the value obtained by dividing the sIgE antigen-binding affinity antibody titer (sIgE antibody titer multiplied by the 1 / IC50 value of the sIgE antibody) by the amount of antigen loaded in the first oral food challenge test, and the X-axis is the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) by the amount of antigen loaded in the first oral food challenge test. An ROC analysis unit determines the cutoff value <y> by ROC analysis based on the value obtained by dividing the sIgE antigen-binding affinity antibody titer (sIgE antibody titer multiplied by the 1 / IC50 value of the sIgE antibody) by the amount of antigen loaded in the first oral food challenge test, and determines the cutoff value <x> by ROC analysis based on the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) by the amount of antigen loaded in the first oral food challenge test. A graph region division unit creates a four-part graph by dividing the graph into four regions: (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>) based on the fact that the cutoff value <y> determined by ROC analysis and the cutoff value <x> determined by ROC analysis are orthogonal.The system comprises: a plotting unit that, for each subject who underwent a primary oral food challenge test, distinguishes between positive subjects who developed an allergic reaction and negative subjects who did not develop an allergic reaction, and plots the value obtained by dividing the sIgE antigen-binding affinity antibody titer (sIgE antibody titer multiplied by the sIgE antibody 1 / IC50 value) by the amount of antigen loaded in the primary oral food challenge test and the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) by the amount of antigen loaded in the primary oral food challenge test on the four-part graph to create a graph for determining negative subjects and a graph for determining positive subjects; and a prediction unit that predicts the amount of allergen loaded in the next oral food challenge test for each subject.

[13] The system according to

[11] or

[12] , characterized in that the plotting section further distinguishes and plots subjects who were loaded with a small amount of allergen, subjects who were loaded with a moderate amount of allergen, and subjects who were loaded with the daily intake amount of allergen in the first oral food challenge test.

[14] The method according to any one of [1] to [7], characterized in that the sample is plasma or serum.

[15] The system according to

[11] or

[12] , characterized in that the sample is plasma or serum.

[16] A method for predicting the amount of allergen that a subject who has not undergone an oral food challenge test for an allergen can be given in an oral food challenge test, using a graph for determining the onset of food allergies created by dividing the sample into four regions: (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>), based on the fact that a cutoff value <y> determined by ROC analysis based on the value of sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer in a sample taken from a subject who has undergone a primary oral food challenge test by the value of 1 / IC50 of the sIgE antibody, and a cutoff value <x> determined by ROC analysis based on the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) calculated based on the sIgG1 antibody titer, sIgG4 antibody titer, and sIgE antibody titer are orthogonal to the value of (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>).

[0018] Furthermore, the present invention may include a method for collecting data to predict the amount of allergen that a subject who has undergone or has not undergone a primary oral food challenge test for an allergen can be given in a subsequent oral food challenge test, and a method for collecting data to predict the amount of allergen to be given based on a graph created by measuring antigen-specific sIgE antibody titers, sIgG1 antibody titers, and sIgG4 antibody titers in a sample taken from a subject who has undergone a primary oral food challenge test for an allergen.

[0019] (a) This graph shows the results of ROC analysis for each of the 311 children, comparing positive and negative cases of food allergy symptom induction in oral food challenge tests, using two parameters: OVM sIgE / OVM sIgE IC50 [(BUe / mL) / (nM)] and OVM sIgE / (OVM sIgG1+OVM sIgG4). (b) This graph shows the results of ROC analysis for each of the 311 children, comparing positive and negative cases of food allergy symptom induction in oral food challenge tests, using two parameters: OVM sIgE / OVM sIgE IC50 / antigen loading [(BUe / mL) / (nM) / (g)] and OVM sIgE / OVM sIgG1+OVM sIgG4 / antigen loading (g). (a) The graph shows four regions divided into (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>) by orthogonally aligning the cutoff values ​​determined by ROC analysis of the OVM sIgE / IC50 value and the cutoff values ​​determined by ROC analysis of the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer). (b) The graph shows four regions divided into (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>) by orthogonally aligning the cutoff values ​​determined by ROC analysis of the OVM sIgE / sIgEIC50 / antigen loading values ​​with the cutoff values ​​determined by ROC analysis of the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) / antigen loading values. The changes in allergy severity on the x and y axes are indicated by arrows. (a) shows a graph for determining negative subjects (n=261) who did not develop an allergic reaction in the first oral food challenge test. The graph plots the sIgE antigen-binding affinity antibody titer (sIgE / IC50), which is calculated by multiplying the sIgE antibody titer by the sIgE antibody 1 / IC50 value, and the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) on a determination graph.(b) shows a graph for determining negative subjects for each subject who underwent the primary oral food challenge test. For negative subjects (n=261) who did not develop an allergic reaction, the sIgE antigen-binding affinity antibody titer, obtained by multiplying the sIgE antibody titer by the sIgE antibody's 1 / IC50 value, further divided by the antigen load, and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) / antigen load are plotted on the determination graph. (a) shows a graph for determining positive subjects for each subject who underwent the primary oral food challenge test. For positive subjects (n=50) who developed an allergic reaction, the sIgE antigen-binding affinity antibody titer, obtained by multiplying the sIgE antibody titer by the sIgE antibody's 1 / IC50 value, and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) are plotted on the determination graph. (b) shows a graph for determining positive subjects, plotting the sIgE antigen-binding affinity antibody titer (calculated by multiplying the sIgE antibody titer by the sIgE antibody 1 / IC50 value) and the antigen loading value (sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) / antigen loading amount) on a determination graph for each subject that tested positive in the primary oral food challenge test and showed an allergic reaction (n=50). (a) The graph for determining negative subjects is shown, further divided into subjects who received a small dose of the allergen (Low-dose), subjects who received a moderate dose of the allergen (Medium-dose), and subjects who received a daily intake amount of the allergen (Full-dose). The graph for determining positive subjects is also shown, further divided into subjects who received a small dose of the allergen (Low-dose), subjects who received a moderate dose of the allergen (Medium-dose), and subjects who received a daily intake amount of the allergen (Full-dose). The sIgE antigen-binding affinity antibody titer, obtained by multiplying the sIgE antibody titer by the sIgE antibody's 1 / IC50 value, and the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) are plotted on the graph.(b) For the graphs above, in both the graph for determining negative subjects (OFC - negative with no symptoms) and the graph for determining positive subjects (OFC - positive with symptoms), the value of the sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer by the value of 1 / IC50 of the sIgE antibody, and then dividing that value by the antigen loading (Y axis), and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), and then dividing that value by the antigen loading (X axis) were plotted. This is a flowchart showing an example of the procedure performed in the system of the present invention. This is a block diagram showing an example of the configuration of the system of the present invention.

[0020] The present invention provides a method for predicting the amount of allergen that can be ingested. This method is not particularly limited as long as it predicts the amount of allergen that a subject who has undergone a primary oral food challenge test can ingest in a subsequent oral food challenge test. In this specification, the "primary oral food challenge test" already undergone by the "subject who has undergone an oral food challenge test for an allergen" may be referred to as the "first oral food challenge test" or the "previous oral food challenge test." Furthermore, since a subject can undergo multiple oral food challenge tests, for example, in the second and subsequent oral food challenge tests, the "primary oral food challenge test" may be synonymous with the "previous oral food challenge test." In addition, in the method for predicting the amount of allergen load according to the present invention, the amount of allergen actually ingested by the subject in the oral food challenge test may be referred to as the "antigen load."

[0021] Furthermore, the present invention includes a system for predicting the amount of allergen that a subject who has undergone a primary oral food challenge test for an allergen can be given in a subsequent oral food challenge test, as well as a method for collecting data to predict the amount of allergen that a subject who has undergone an oral food challenge test for an allergen in a non-medical manner can be given in a subsequent oral food challenge test, and a method for collecting data to predict the amount of allergen that a subject who has undergone an oral food challenge test for an allergen can be given in a subsequent oral food challenge test, excluding the actions of a physician.

[0022] The above allergens refer to antigens that cause allergen sensitization and / or induce allergic reactions, and even anaphylaxis. Specifically, allergens are not particularly limited as long as they are any antigenic protein or peptide that can induce the production of IgE antibodies in humans, or other sugar chains or nucleic acids, but examples of food allergens include eggs, milk, meat such as beef, fish such as salmon and tuna, crustaceans and mollusks such as shrimp and crab, grains, legumes and nuts, fruits, vegetables, brewer's yeast, gelatin, etc. Among these, αs1-casein, αs2-casein, β-casein, κ-casein, α-lactalbumin, and whey allergens are particularly noteworthy. Examples of major allergens include milk allergens such as β-lactoglobulin (BLG), which is the main component of milk; egg white allergens (EW), egg yolk allergens, and egg allergens that are mixtures thereof, such as ovomucoid (OVM), ovalbumin (OVA), conalbumin, or mixtures thereof; wheat allergens such as gliadin and gluten; buckwheat allergens; peanut allergens such as Arah1 and Arah2; sesame allergens such as 11S globulin; and shellfish allergens such as tropomyosin protein.

[0023] The above-mentioned allergic symptoms include skin symptoms such as itching, hives, angioedema, redness, and eczema; eye symptoms such as conjunctival congestion and edema, itching, tearing, and eyelid edema; nasal symptoms such as sneezing, runny nose, and nasal congestion; oral and pharyngeal symptoms such as discomfort and swelling of the mouth, lips, and tongue, and itching and irritation of the throat; digestive symptoms such as abdominal pain, nausea, vomiting, diarrhea, and bloody stools; respiratory symptoms such as pharyngeal constriction, pharyngeal edema, hoarseness, cough, wheezing, difficulty breathing, and asthma; and systemic symptoms such as anaphylactic shock, tachycardia, collapse, impaired consciousness, and decreased blood pressure. Criteria can be determined by referring to guidelines from organizations that research allergies.

[0024] The subjects of the present invention are not particularly limited as long as they are humans who have undergone a primary oral food challenge test for an allergen. Subjects undergoing a primary oral food challenge test for an allergen may include those who have been confirmed to have an allergy to a specific allergen by known allergy determination methods such as skin prick tests, basophil activation tests, IgE tests, or oral food challenge tests, or those who have not been confirmed to have an allergy. This also includes those who have never been determined by the above-mentioned skin prick tests, basophil activation tests, IgE tests, or oral food challenge tests.

[0025] The oral food challenge test described above is a test conducted in a medical institution under the supervision of a physician, in which test subjects are given a food that causes or may cause allergic symptoms in a single dose or in multiple doses, and the presence or absence of symptoms is checked. It is performed with the aim of identifying the food causing the allergy, determining the amount of the food that can be safely consumed, and diagnosing the acquisition of tolerance.

[0026] In the first oral food challenge test described above, the physician may decide whether to set the intake amount (total load) for each subject to be small, moderate, or equivalent to their daily intake, based on the "Food Allergy Treatment Guidelines 2021, Table 9-6, partially modified" shown in Table 1 below. This decision may be based on the results of previously known allergy assessment methods such as the skin prick test, basophil activation test, IgE test, and oral food challenge test, as well as the presence or absence of eczema, hay fever, and various other allergies.

[0027]

[0028] The specific method for the oral food challenge test described above is to gradually increase the dosage of each allergen while observing clinical symptoms, to the extent that anaphylactic symptoms do not occur. However, for each subject, once it has been determined whether the dosage should be small, moderate, or equal to their daily intake, the dosage can be further subdivided. For example, if the total dosage of the daily intake is set to "1", the small dosage can be subdivided into 0.5 / 100, 1 / 100, 2 / 100, and 4 / 100 of the total dosage; the moderate dosage into 8 / 100, 12.5 / 100, 32 / 100, and 50 / 100 of the total dosage; and the daily intake into 67 / 100 and 100 / 100 of the total dosage, resulting in 10 subdivided levels. There are also examples of subdividing the dosage into 8 or 6 levels instead of 10. One method involves gradually increasing the dosage, dividing each stage into 15-30 minute intervals, and administering foods containing each allergen. The number of stages can be increased or decreased at the discretion of the physician depending on the subject's allergy history, and the dosage for each stage can also be appropriately changed in light of the subject's previous symptoms. However, if signs of severe allergic symptoms appear, for example, if symptoms corresponding to Grade 3 or higher according to the criteria set by the World Allergy Organization (WAO), as shown in Table 2 below, it is strongly desirable to stop the test immediately in the middle of the above stages. Examples of small, moderate, and daily intake amounts include: low-dose group: 10-200 mg of heated egg white powder, moderate-dose group: 300-1800 mg of heated egg white powder, and daily intake: 2000 mg or more of heated egg white powder.

[0029]

[0030] The samples collected from the above subjects are not particularly limited as long as they are collected from the subjects, and include body fluids such as blood, serum, plasma, saliva, tears, nasal secretions, and urine collected from the subjects, but serum and plasma are preferred. When using serum as a sample, for example, blood can be collected from the brachial vein, the obtained blood can be left to stand overnight at 4°C, then centrifuged, and the supernatant can be used as serum. Alternatively, it is also possible to collect a small amount of blood (50-100 μL) obtained by minimally invasively puncturing the earlobe or fingertip with a micro-blood collection needle, etc., and use it directly in a microcapillary tube. Furthermore, while it is ideal to use a sample collected immediately before the oral food challenge test, it is desirable to evaluate using a sample collected within one year before the oral food challenge test, preferably 0-6 months or 3-6 months prior.

[0031] In this invention, the amount of allergen that can be administered in the next oral food challenge test can be predicted by measuring the sIgE antibody (specific immunoglobulin E antibody) titer, sIgG1 antibody (specific immunoglobulin G1 antibody) titer, and sIgG4 antibody (specific immunoglobulin G4 antibody) titer in a sample taken from a subject who has undergone a primary oral food challenge test for the allergen. The sIgE antibody, sIgG1 antibody, and sIgG4 antibody are immunoglobulin E, immunoglobulin G1, and immunoglobulin G4, respectively, which specifically bind to a particular allergen, and the "s" (specific) antibody titer indicates an indicator of the amount of antibody produced against a specific (food) allergen (protein).

[0032] Furthermore, the accuracy of the prediction method of the present invention can be further improved by including the amount of antigen loaded in the first oral food challenge test as a parameter for predicting the amount of allergen that can be loaded in the next oral food challenge test.

[0033] The methods for quantitatively measuring the sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer are not particularly limited as long as they can quantify the concentration of each antibody bound to the allergen in the sample as an antibody titer (Binding Unit: BU) / mL. Preferably, a method can be used that uses a chip that can immobilize various antigens and quantitatively measure the antigen-antibody reaction when antibodies against such immobilized specific antigens are present. The ELISA method using labeled secondary antibodies performed on a carrier is a particularly suitable example. Suitable carriers for the ELISA method include commercially available ELISA chips, chips with a carbon layer formed on the surface of the carrier which are advantageous in that they have less nonspecific adsorption, chips with chemically modified groups introduced or activated chips which are advantageous for immobilizing peptides, and among these, DCP chips are particularly suitable.

[0034] Examples of the DCP chips mentioned above include chips in which an electrostatic layer is applied to the surface of a substrate on which a carbon layer has been DLC (diamond-like carbon) treated on the surface of a silicon substrate, or the surface of a glass slide, and an electrostatic layer treated with an amino group-containing compound or its polymer and / or copolymer is applied, and then further treated with a dicarboxylic acid or polycarboxylic acid, and then activated with N-hydroxysuccinimide and / or carbodiimides, or chips in which chemical modification groups have been introduced to the surface of a support or the carbon layer, or chips that have undergone further activation treatment.

[0035] A specific procedure for activating the above-mentioned chip is as follows: A DCP chip made of a glass substrate is coated with an amino group-containing electrostatic layer, and then a negatively charged carboxyl group is introduced using polyacrylic acid. This substrate is then activated in a chemical crosslinking agent (WSC-HCl, NHS, potassium phosphate buffer) while being shaken at room temperature for 30 minutes under light shielding. After discarding the chemical crosslinking agent, the substrate is washed with MilliQ water while shaking, and then the water is removed using a centrifuge.

[0036] Furthermore, when calculating the IgG1 antibody titer and the IgG4 antibody titer, it is preferable to dilute the blood sample, such as serum, 1 to 50 times, preferably 1.1 to 100 times, before quantifying the antibody titer. Furthermore, since IgE is present in trace amounts, from the viewpoint of measuring with higher accuracy within the accuracy range of the calibration curve, it is preferable to dilute it 1.5 to 2 times before quantifying the IgE antibody titer. It is desirable to measure each antibody titer using the DCP method with a DCP chip, and then multiply by the dilution factor of the blood sample to finally determine each antibody titer.

[0037] Examples of the labeled secondary antibodies include fluorescently labeled secondary antibodies such as HiLyte Fluor 555, Atto 532, Cy3, Alexa Fluor 555, Cy5, FITC, and rhodamine; enzyme-labeled secondary antibodies such as peroxidase and alkaline phosphatase; magnetic bead-labeled secondary antibodies; infrared-labeled secondary antibodies; and labeled anti-human IgE antibodies. The above secondary antibodies may include antibody Fab fragments or F(ab') ２ Fragments can also be used, and the Fab fragment can be treated with an antibody such as papain to produce F(ab'). ２ The fragments can be prepared by treating them with pepsin or the like.

[0038] [Graph Creation] As a method for predicting the amount of the allergen to be administered in the next oral food challenge test according to the present invention, it is preferable to make a prediction based on a graph created with the sIgE antigen-binding affinity antibody titer, which is calculated by multiplying the sIgE antibody titer calculated based on the sIgE antibody titer in a sample taken from a subject who underwent the first oral food challenge test by the value of 1 / IC50 of the sIgE antibody, on the Y axis, and the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), which is calculated based on the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) in a sample taken from a subject who underwent the first oral food challenge test, on the X axis. Note that similar results can be obtained by swapping the parameters of the X and Y axes.

[0039] As a graph in the method for predicting the amount of the allergen to be loaded in the next oral food challenge test of the present invention, the value of the sIgE antigen-binding avidity antibody titer obtained by multiplying the sIgE antibody titer calculated based on the sIgE antibody titer in the sample collected from the subject who received the first oral food challenge test by the value of 1 / IC50 of the sIgE antibody, and further divided by the antigen load amount is used as the Y-axis, and the value of sIgG1 antibody titer, sIgG4 antibody titer, and sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) calculated based on the sIgE antibody titer in the sample collected from the subject who received the first oral food challenge test, and further divided by the antigen load amount is used as the X-axis. It is preferable to predict based on the graph created. Note that similar results can be obtained by exchanging the X-axis and Y-axis parameters.

[0040] [Calculation of the avidity of IgE antibody to allergen] As the value of the (antigen-specific IgE avidity) of the sIgE antibody to the allergen set on the Y-axis, the value quantified as the 1 / IC50 value of the IgE antibody can be cited as the avidity. The value of IC50 is a value that may also be called a competitive binding inhibition activity value. For example, as the competitive binding inhibition activity between the immobilized allergen and the soluble allergen, it can be obtained by calculating the value representing the concentration of the allergen that inhibits the antigen-antibody binding reaction by 50%. According to this calculation method, it is preferable in that the values of IC50 and 1 / IC50 can be evaluated even when IgG, IgA, IgD, and / or IgM antibodies other than IgE that recognize the same antigen are present.

[0041] As an example of a method for calculating IC50 values ​​using the above-mentioned ELISA, a competitive ELISA measurement method can be cited. Measuring IC50 values ​​by competitive ELISA using a competitive binding inhibition reaction method, in which various concentrations of allergens are added, is preferable because the IC50 value does not change much with the dilution ratio of the sample, it is possible to compare IC50 values ​​between different allergens, and the sum of the antigen-binding activities of antibodies against allergens can be quantitatively compared. When calculating IC50 values, it has been confirmed that it is not necessary to multiply by the dilution ratio of the sample even when blood samples such as plasma and serum are diluted. An example of a specific procedure using ELISA with a competitive binding inhibition reaction method is shown below.

[0042] A sample containing IgE antibodies that bind to antigen-specific allergens is treated with a known concentration of allergen, prepared stepwise from a concentration of 0 (no antigen present in the sample as a competitive binding inhibitor) to a concentration sufficient to bind the allergen to all IgE antibodies within a certain time. The sample is then allowed to react for a certain period of time.

[0043] The specified time in the above preliminary reaction can be, for example, 15 minutes to 2 hours, preferably 30 minutes to 1 hour. Furthermore, the known concentrations prepared in stages can be those appropriately determined by a person skilled in the art based on the antibody titer present in the sample. For example, examples of stepwise allergen concentrations (final concentrations) used for competitive inhibition added to serum or plasma include combinations of 0 nM, 0.1 nM, 1.0 nM, 10 nM, 100 nM, and 1000 nM, or combinations of 0 nM, 0.1 nM, 1.0 nM, 10 nM, 100 nM, and 200 nM, but are not limited to these concentrations. While competitive binding inhibition reaction ELISA is preferably performed for allergens with a clearly defined molecular weight, for allergens whose molecular weight is unknown and whose concentration cannot be adjusted in molar concentration, the above stepwise allergen concentrations can be adjusted in units such as mg / mL and μg / mL.

[0044] The solution after the above-mentioned preliminary reaction is subjected to a carrier on which an allergen is immobilized, and an antigen-antibody reaction is carried out until it reaches an equilibrium state by a method such as binding the IgE antibody (primary antibody) free in the solution after the above-mentioned preliminary reaction to the immobilized allergen. After that, after the removal and washing process of the primary antibody, it is further reacted with a labeled secondary antibody, and the binding amount of the IgE antibody bound to the immobilized allergen is calculated.

[0045] The concentration of the allergen that inhibits the antigen-antibody binding reaction by 50%, that is, the "IC50", in the above-mentioned preliminary reaction, for a solution with a concentration of 0 of the competing allergen, when the labeled amount of the detected labeled secondary antibody is taken as 100%, is expressed as the antigen concentration (half maximal inhibitory concentration: 50% inhibitory concentration) at which the labeled amount becomes 50%.

[0046] The avidity of the IgE antibody for the allergen can be expressed as "1 / IC50", which is the reciprocal of the IC50 value, based on the above IC50 value. In the ELISA using the above-mentioned competitive binding inhibition reaction method, when the affinity of the IgE antibody in the sample is lower, the amount of free primary IgE antibody present in the solution after the preliminary reaction is larger, so the antibody titer binding to the immobilized allergen increases and the IC50 value becomes larger. On the other hand, when the affinity of the IgE antibody in the sample is higher, the amount of free primary IgE antibody present in the solution after the preliminary reaction is smaller, so the IgE antibody titer binding to the immobilized allergen decreases and the IC50 value becomes smaller. Therefore, in order to set a lower value for avidity when the affinity of the IgE antibody is lower and a higher value for avidity when the affinity of the IgE antibody is higher, the reciprocal of the IC50 value may be used for convenience. Especially when evaluating the IgE antibody titer by multiplying it by the antigen-binding affinity (avidity) of IgE, the reciprocal of the IC50 value can be used.

[0047] Examples of labeled secondary antibodies used in avidity calculation include fluorescently labeled secondary antibodies such as HiLyte Fluor 555, Atto 532, Cy3, Alexa Fluor 555, Cy5, FITC, and rhodamine; enzyme-labeled secondary antibodies such as peroxidase and alkaline phosphatase; magnetic bead-labeled secondary antibodies; and infrared-labeled secondary antibodies. Furthermore, the above secondary antibodies may also include antibody Fab fragments or F(ab') ２ Fragments can also be used, and the Fab fragment can be treated with an antibody such as papain to produce F(ab'). ２ The fragments can be prepared by treating them with pepsin or the like.

[0048] Examples of highly sensitive carriers on which the allergen is immobilized include commercially available ELISA chips, chips with a carbon layer formed on the surface of the carrier which are advantageous in that they have low nonspecific adsorption, and chips into which chemically modified groups have been introduced or which have undergone activation treatment which are advantageous for immobilizing peptides. Among these, the DCP chip is preferred.

[0049] [Calculation of the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer)] In the graph created in the present invention, the value of sIgG1 antibody titer + sIgG4 antibody titer can be used as a parameter as a cross-inhibitory antibody against sIgE antibody, and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) can be calculated from the above sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer measured from samples taken from subjects who underwent a primary oral food challenge test for allergens.

[0050] (Calculation of the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) / antigen loading) In the graph created in the present invention, it is more preferable to use the value of sIgG1 antibody titer + sIgG4 antibody titer / antigen loading as a parameter for cross-inhibitory antibodies against sIgE antibodies, and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) / antigen loading can be calculated from the sIgE antibody titer, sIgG1 antibody titer, sIgG4 antibody titer, and antigen loading amount that were imposed on the subject who underwent the first oral food challenge test, measured from a sample taken from the subject who underwent the first oral food challenge test for the allergen.

[0051] As shown in the graph above, the cutoff values ​​for positive and negative food allergy symptom induction determined by ROC analysis based on the sIgE antigen-binding affinity antibody titer, which is calculated by multiplying the sIgE antibody titer in samples taken from subjects who underwent a primary oral food challenge test by the 1 / IC50 value of the sIgE antibody, and the cutoff values ​​for positive and negative food allergy symptom induction determined by ROC analysis based on the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) in samples taken from subjects who underwent an oral food challenge test, are orthogonal to each other. This results in a graph divided into four regions: (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>). This graph can be used to determine the onset of food allergies. The Y-axis and X-axis can also be swapped.

[0052] The graph described above can also be used as a judgment graph for determining the onset of food allergies, divided into four regions: (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>), by orthogonality between <y>, which is the cutoff value for positive and negative induction of food allergy symptoms determined by ROC analysis based on the value of the sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer in the sample taken from a subject who underwent a primary oral food challenge test by the value of 1 / IC50 of the sIgE antibody, and then dividing that value by the antigen load, and <x>, which is the cutoff value for positive and negative induction of food allergy symptoms determined by ROC analysis based on the value of the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) in the sample taken from a subject who underwent an oral food challenge test, and then dividing that value by the antigen load.

[0053] The above-mentioned ROC is a term translated as receiver operating characteristic or receiver operating characteristic, and is a method used to compare the predictive power of an independent variable to a binary outcome variable, or to set a cutoff for the independent variable to the outcome.

[0054] In the present invention, a method for comparing the predictive power of independent variables to the outcome, which is the binary variable, is to change the threshold setting for anaphylaxis positivity for each of n different parameters. The true positive rate and false positive rate for anaphylaxis positivity change accordingly. When the true positive rate and false positive rate are plotted with the true positive rate on the vertical axis and the false positive rate on the horizontal axis, and n types of ROC curves are created, a parameter is evaluated as being superior and having high predictive power if the area under the curve (AUC) is large.

[0055] As for the method of creating the ROC curve described above, if a certain parameter is selected, for example, if an arbitrary threshold is set for the anaphylaxis positive value in the classification of subjects, then TY is shown when anaphylaxis positive subjects are correctly classified as Y, FY is shown when anaphylaxis positive subjects are incorrectly classified as Y, TN is shown when anaphylaxis negative subjects are correctly classified as N, and FN is shown when anaphylaxis negative subjects are incorrectly classified as N. The true positive rate (sensitivity) when positive subjects are correctly classified as positive is TY / (TY+FN), and the false positive rate (1-specificity) when negative subjects are classified as positive is FY / (FY+TN). Then, (true positive rate, false positive rate), i.e., (sensitivity, 1-specificity), can be plotted for a certain threshold. Furthermore, an ROC curve can be created by setting various thresholds and plotting many of them.

[0056] In the above ROC analysis, the method for setting the cutoff values ​​for positive and negative food allergy symptom induction is not particularly limited as long as it can improve predictive ability. However, one example is determining the sensitivity value that maximizes sensitivity - (1 - specificity) as the cutoff value. Such cutoff values ​​can be determined using the ROC analyzer shown below by known procedures, or they can be determined using commercially available software.

[0057] ROC analysis can also be performed using commercially available analytical instruments, such as GraphPad Prism ver. 5.4 (GraphPad Inc.), JMP14 (jmp.Statistical Discovery.™), and JMP Pro17.

[0058] For the above-mentioned determination graph, it is preferable to create graphs for determining negative subjects and graphs for determining positive subjects by plotting the sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer in the sample by the 1 / IC50 value of the sIgE antibody, and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) for each subject who underwent the primary oral food challenge test, and distinguishing between positive subjects who developed an allergic reaction and negative subjects who did not develop an allergic reaction.

[0059] To distinguish between subjects that exhibited an allergic reaction (positive) and those that did not (negative), a positive result for allergy to a food allergen (antigen) was determined if one or more of the WAO's criteria for positive allergic symptoms shown in Table 2 above were met. If none of the criteria were met, the allergy to egg white was determined to be negative. Antigens can include food allergens such as eggs, milk, beef and other meats, salmon, tuna and other fish, crustaceans and mollusks such as shrimp and crab, grains, legumes and nuts, fruits, vegetables, brewer's yeast, and gelatin.

[0060] Furthermore, for the above-mentioned judgment graph, when plotting the sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer in the sample by the 1 / IC50 value of the sIgE antibody, and then dividing that value by the antigen loading amount, and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) / antigen loading amount for each subject that underwent the primary oral food challenge test, it is preferable to create two types of graphs for determining negative subjects and positive subjects by distinguishing between positive subjects who developed an allergic reaction and negative subjects who did not develop an allergic reaction.

[0061] The graphs for determining negative and positive subjects described above can also be further divided into three types of graphs each, resulting in a total of six types of graphs, by plotting subjects who ingested a small amount of allergen, a moderate amount of allergen, and a daily intake amount of allergen in the first oral food challenge test. Using these six types of graphs, it is immediately clear whether the subjects plotted on each graph ingested a small amount, a moderate amount, or a daily intake amount of allergen in the first oral food challenge test, thus allowing for a more rapid prediction of the next food challenge dose.

[0062] The following criteria can be used as examples of a method for predicting the amount of allergen loaded in the next oral food challenge test according to the present invention.

[0063] If the subject is a negative subject who has shown an allergic reaction, the amount of allergen that can be administered in the next oral food challenge test can be predicted using the negative subject determination graph. Hereinafter, "stage" refers to the same meaning as the "stage" exemplified by subdividing the (total) load of daily intake into 10 stages, for example, small amounts into 0.5 / 100, 1 / 100, 2 / 100, 4 / 100 of the (total) load, moderate amounts into 8 / 100, 12.5 / 100, 32 / 100, 50 / 100 of the (total) load, and daily intake into 67 / 100 and 100 / 100 of the (total) load. The specific number of stages that can be increased or decreased can be determined by taking into account medical history, the subject's age, family allergy history, etc. The following is an example of 10 subdivided stages. (a) If the data for the subject is plotted in the (-<x>, -<y>) or (+<x>, -<y>) region of the graph for determining negative subjects, it can be predicted that the amount of allergen that can be ingested in the next oral food challenge test can be increased from the maximum load administered in the first oral food challenge test, and that the amount can be two, three, four, or five levels higher, or equivalent to or greater than the usual intake. This allows for the determination of the amount of allergen that can be sustainably ingested, which is one of the objectives of the test. (b) If the data for the subject is plotted in the (+<x>, +<y>) or (-<x>, +<y>) region of the graph for determining negative subjects, it can be predicted that the amount of allergen that can be ingested in the next oral food challenge test can be one to two levels, or one to three levels higher, than the actual load administered in the first oral food challenge test.

[0064] If the subject is a positive subject who has shown an allergic reaction, the amount of allergen that can be administered in the next oral food challenge test can be predicted using the positive subject determination graph. Specifically, the number of steps that can be increased or decreased can be determined by taking into account the severity of the allergic reaction that occurred in the first oral food challenge test, medical history, the subject's age, family allergy history, etc. (c) If the subject's data is plotted in the (-<x>, -<y>) or (+<x>, -<y>) region of the positive subject determination graph, the amount of allergen that can be administered in the next oral food challenge test can be predicted to be 0.5 steps, 1 step, or 2 steps less than the actual amount administered in the first oral food challenge test. (d) If the data in question is plotted in the (+<x>, +<y>) or (-<x>, +<y>) region of the graph for determining positive subjects, it can be predicted that the amount of allergen that can be administered in the next oral food challenge test will be one, two, three, four, or five levels less than the actual amount administered in the first oral food challenge test.

[0065] The prediction system of the present invention includes a storage unit that stores personal data for each subject, a calculation unit that calculates the value of sIgE antigen-binding affinity antibody titer by multiplying the sIgE antibody titer by the value of 1 / IC50 of the sIgE antibody based on the sIgE antibody titer in a sample taken from a subject that underwent a primary oral food challenge test, and calculates the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) based on the sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer, and a graph creation unit that creates a graph with the avidity value of sIgE antibody on the Y axis and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) on the X axis, The ROC analysis unit determines the cutoff value <y> for positive and negative food allergy symptom induction by ROC analysis based on the sIgE antigen-binding affinity antibody titer obtained by multiplying the sIgE antibody titer by the 1 / IC50 value of the sIgE antibody, and determines the cutoff value <x> for positive and negative food allergy symptom induction by ROC analysis based on the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), and the graph region division unit divides the graph into four regions: (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>) based on the orthogonality of the cutoff value <y> and the cutoff value <x> determined by ROC analysis. The system comprises a plotting unit that, for each subject who underwent a primary oral food challenge test, distinguishes between negative subjects who developed an allergic reaction and positive subjects who did not develop an allergic reaction, and plots the sIgE antigen-binding affinity antibody titer (calculated by multiplying the sIgE antibody titer by the sIgE antibody's 1 / IC50 value) and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) on a determination graph to create graphs for determining negative subjects and graphs for determining positive subjects; and a prediction unit that predicts the amount of allergen to be loaded in the next oral food challenge test for each subject. The system is not particularly limited as long as it is for predicting the amount of allergen to be loaded in the next oral food challenge test for subjects who have undergone a primary oral food challenge test for an allergen. Furthermore, the plotting unit can also further distinguish and plot subjects who were loaded with a small amount of allergen, subjects who were loaded with a moderate amount of allergen, and subjects who were loaded with the daily intake amount of allergen in the primary oral food challenge test.

[0066] Furthermore, if the subject is negative and did not develop an allergic reaction, the amount of allergen that can be administered in the next oral food challenge test can be predicted using the graphs in Figures 3-1 and 4. This is because even if the subject is negative and did not develop an allergic reaction, there is still a possibility of false negatives (individuals who would actually develop an allergic reaction but were judged negative because the amount of allergen administered was too small to trigger a reaction).

[0067] The judgment method for the graphs in Figures 3-1, 3-2, and Figure 4 is described below. (a) If the data of the subject is plotted in the (-<x>, -<y>) or (+<x>, -<y>) region of the graph for determining negative subjects, there is a high possibility that the subject is truly negative. However, it can be predicted that the amount of allergen that can be administered in the next oral food challenge test can be increased from the maximum dose ingested in the first oral food challenge test, and that the subject can ingest an amount two, three, four, or five levels higher, or even exceed their daily intake. The extent to which the dose can be increased or decreased can be determined by taking into account the subject's age, family allergy history, etc. (b) If the data for the subject is plotted in the (+<x>, +<y>) or (-<x>, +<y>) region of the graph for determining negative subjects, there is a possibility that the subject is a false negative, and it can be predicted that the amount of allergen that can be administered in the next oral food challenge test will be one to two levels, or one to three levels, higher than the actual amount administered in the first oral food challenge test. (c) If the data for the subject is plotted in the (-<x>, -<y>) or (+<x>, -<y>) region of the graph for determining positive subjects, it can be predicted that the amount of allergen that can be administered in the next oral food challenge test will be 0.5 levels, one level, or two levels lower than the actual amount administered in the first oral food challenge test. (d) If the data in question is plotted in the (+<x>, +<y>) or (-<x>, +<y>) region of the graph for determining positive subjects, it can be predicted that the amount of allergen that can be administered in the next oral food challenge test will be one, two, three, four, or five levels less than the actual amount administered in the first oral food challenge test. By making this prediction (determination), the amount of allergen that can be administered in the next oral food challenge test can be determined as the safe intake amount (allergen load that will not cause an anaphylactic reaction).

[0068] The memory unit can store personal data for each subject, including their name, gender, age, height, weight, allergy history, preliminary test data, the amount of food load in the first oral food challenge test, and sIgE antibody titers, sIgG1 antibody titers, and sIgG4 antibody titers in samples taken from each subject who underwent the first oral food challenge test. It can also store calculation results data from the calculation unit, and for this purpose, memory such as RAM or ROM is used.

[0069] In the above-mentioned calculation unit, based on signals stored in the above-mentioned storage unit and input via I / O circuits, etc., the calculation means performs calculations such as multiplying the sIgE antibody titer by the 1 / IC50 value of the sIgE antibody, and calculating the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), based on the data of the sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer of subjects who have undergone a primary oral food challenge test.

[0070] In the graph creation unit described above, a graph is created with the sIgE antigen-binding affinity antibody titer, calculated by multiplying the sIgE antibody titer (calculated in the calculation unit and input via an I / O circuit, etc.) by the sIgE antibody's 1 / IC50 value, as the Y-axis, and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) as the X-axis.

[0071] In the ROC analysis unit described above, a calculation means for performing ROC analysis is used to determine the cutoff value <y> by ROC analysis based on the value of the sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer by the value of 1 / IC50 of the sIgE antibody, and the cutoff value <x> is determined by ROC analysis based on the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer).

[0072] In the graph region division section described above, the cutoff value <y> determined in the ROC analysis section and the cutoff value <x> determined by the ROC analysis are orthogonal to each other, resulting in the graph being divided into four regions: (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>).

[0073] In the plotting section described above, for each subject who underwent the primary oral food challenge test, positive subjects who developed an allergic reaction and negative subjects who did not develop an allergic reaction are distinguished. The sIgE antigen-binding affinity antibody titer, obtained by multiplying the sIgE antibody titer by the sIgE antibody's 1 / IC50 value, and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) are plotted on a judgment graph, thereby creating a graph for determining negative subjects and a graph for determining positive subjects.

[0074] In the plotting section described above, by further distinguishing and plotting subjects who ingested a small amount of allergen in the first oral food challenge test, subjects who ingested a moderate amount of allergen, and subjects who ingested their daily intake of allergen, three types of graphs for determining negative results and three types of graphs for determining positive results can be created.

[0075] The prediction unit described above can predict the amount of allergen to be loaded in the next oral food challenge test for each subject.

[0076] In the processing of the above-mentioned memory unit, calculation unit, graph creation unit, ROC analysis unit, graph region division unit, plotting unit, and prediction unit, the value of the sIgE antigen-binding affinity antibody titer obtained by multiplying the sIgE antibody titer by the value of 1 / IC50 of the sIgE antibody can be replaced with the value obtained by dividing the sIgE antigen-binding affinity antibody titer obtained by multiplying the sIgE antibody titer by the value of 1 / IC50 of the sIgE antibody by the antigen loading, and / or the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) can be replaced with the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) by the antigen loading, and a prediction system can be constructed. By including the amount of allergen actually ingested by each subject as the antigen loading in the formula, more precise analysis can be performed. Furthermore, by using such a system, data for predicting the amount of allergen that can be ingested in an oral food challenge test can be efficiently collected.

[0077] The present invention will be specifically described below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0078] [Example 1] [Conducting the first oral food challenge test]

[0079] The study was conducted according to procedures approved by the Ethics Committee of the National Hospital Organization Mie Hospital. It was conducted as an exploratory, retrospective, single-center cohort study at the Allergy Center of the National Hospital Organization Mie Hospital, identifying cohorts at a past point in time and examining the prevalence in each cohort towards the present. This study involved 311 children suspected of having cooked egg (HE) allergy, with full informed consent obtained from both or one parent. Oral food challenge tests were performed according to the Japanese Guidelines for food allergy 2020 (Non-Patent Literature 4), and the severity of symptoms was evaluated according to the WAO consensus on the definition of severity of food allergy symptoms (see Table 2 above) (Non-Patent Literature 3).

[0080] (Subjects) The breakdown of the 311 children mentioned above is as follows: (1) Children aged 1 to 17 years (0 to 17 years old, 67.2% male, median age 3 years) who were suspected of having heated egg (HE) allergy and underwent an oral food challenge test with heated egg white (HEW) (corresponding to the first oral food challenge test) at the National Hospital Organization Mie Hospital between 2021 and 2023. (2) Patients with severe atopic dermatitis; poorly controlled asthma (baseline FEV1 (forced expiratory volume in one second) less than 80% of the predicted value) (in accordance with the Japanese Guidelines for the Treatment and Management of Pediatric Bronchial Asthma (Allergol Int. 2017;66:190-204)); pollen-food allergy syndrome; and poorly controlled food protein-induced enteritis syndrome were not included in the 311 children mentioned above.

[0081] (Selection of allergens) Ovalbumin, one of the components of chicken eggs, is known to lose its allergenic activity when heated. Therefore, in this study, we proceeded with the investigation using ovomucoid (OVM), which does not lose allergenic activity even when heated, as the indicator.

[0082] (Preliminary Study) For the 311 children mentioned above, serum samples collected from all subjects up to six months prior to the study were stored at below -30°C without freezing or thawing until analysis. Before conducting the oral food challenge test, the level of IgE antibody production against egg white (Immuno CAP HEW level) was measured by blood tests using the commonly used ImmunoCAP method (Phadia), and allergists confirmed the findings based on the subjects' medical history and blood immunological test results. The findings for the 311 subjects, including ovomucoid (OVM)-specific IgE (sIgE) levels, are shown in Table 3 below.

[0083]

[0084] (Oral Food Challenge Test) Based on the above findings, the allergist carefully selected the total loading dose of heated egg white or heated egg white powder (manufactured by Kewpie Corporation) for each subject in the oral food challenge test. The amount of heated egg white or heated egg white powder administered to each subject in the first oral food challenge test was then classified into three groups: a small intake group (78 subjects), a moderate intake group (103 subjects), or a daily intake group (130 subjects).

[0085] For oral food challenge tests for ovomucoid, the load amounts of whole eggs were as follows: low intake group: 1 / 32 to 1 / 25 of a cooked whole egg; moderate intake group: 1 / 8 to 1 / 2 of a cooked whole egg; and daily intake group: 2 / 3 to 1 cooked whole egg. The moderate intake group's 1 / 8 to 1 / 2 of a cooked whole egg was adjusted as appropriate based on age, weight, etc. When using cooked egg white powder (manufactured by Kewpie Corporation), the (total) load amounts were: low intake group: 10 to 200 mg of cooked egg white powder; moderate intake group: 300 to 1800 mg of cooked egg white powder; and daily intake group: 2000 mg or more of cooked egg white powder.

[0086] (Loading Amount) When implementing the test, the total daily intake loading amount is set to "1". Small amounts can be divided into 10 stages: 0.5 / 100, 1 / 100, 2 / 100, and 4 / 100 of the total loading amount; moderate amounts into 8 / 100, 12.5 / 100, 32 / 100, and 50 / 100 of the total loading amount; and the daily intake into 10 stages: 67 / 100 and 100 / 100 of the total loading amount. When implementing the test, the loading amount was gradually increased in multiple sessions at 15-30 minute intervals for each stage, and the test was conducted by having subjects ingest a predetermined amount of whole egg or heated egg white powder.

[0087] A child who ingested heated egg white powder was determined to have a positive allergy to OVM if they met one or more of the WAO's criteria for positive allergy symptoms shown in Table 2 above. If a child did not meet any of the criteria, they were determined to have a negative allergy to egg white.

[0088] (Results) Of the 311 children who underwent the oral food challenge test with the heated egg white powder described above, 50 were confirmed as positive subjects who developed an allergic reaction. On the other hand, 261 were confirmed as negative subjects who did not develop an allergic reaction. Children who were determined to be positive in the oral food challenge test were considered positive subjects who developed an allergic reaction, and children who were determined to be negative subjects who did not develop an allergic reaction were considered negative subjects for further study.

[0089] [Example 2] [Measurement of OVM-specific antibody titers by DCP method] For the above 311 children, antibody titers for sIgE, sIgG4, and sIgG1 that bind to ovomucoid (OVM) were calculated using a high-density carboxylated protein microarray (commonly known as DCP chip) (manufactured by Americ-Applied Enzyme Medical Research Institute Co., Ltd.) using serum samples collected before the first oral food challenge test. The DCP chip has been confirmed to correlate with the ImmunoCAP system (manufactured by Thermo Fisher Diagnostics).

[0090] [Fabrication of OVM-immobilized DCP chips] (Chip activation) A DCP chip made of a glass substrate was coated with an amino group-containing electrostatic layer, and then negatively charged carboxyl groups were introduced using polyacrylic acid. The substrate was activated in a chemical crosslinking agent (100 mM WSC·HCl, 100 mM NHS, 0.1 M potassium phosphate buffer (pH 6.0)) in a light-shielded environment at room temperature for 30 minutes while shaking. After discarding the chemical crosslinking agent after the reaction, the substrate was washed twice for 1 minute each time with MilliQ water while shaking, and then immediately subjected to a benchtop centrifuge (Allegra ＴＭ Moisture was removed using an X-22R Centrifger (manufactured by Beckman Coulter) to prepare an activated tip.

[0091] (OVM Coupling Reaction) An antigen protein solution was prepared by dissolving OVM (Sigma-America) as the antigen protein at a concentration of 0.2–2.0 mg / mL in a solution containing 5–30% DMSO or 5–30% PEG300. The prepared antigen protein solution was dispensed into a 384-well flat-bottom plate (Corning), and 4 nL spots were made onto the activation chip using a microarray production system (OmniGridAccent, DIGILAB). The OVM antigen protein was then immobilized by drying at 15°C–30°C for 1–18 hours. The microarray production system is not limited to OmniGridAccent; any system with similar performance can be used. For example, a microarray production system (AD6020) ＴＭ It is also possible to spot 6 nL of material onto the activation chip using the Aspirate Dispense System (manufactured by BioDot), and after spotting, the same procedure as described above is performed.

[0092] (Blocking reaction of unreacted active groups) Blockmaster (JSR Corporation), a blocking reagent, was added to the wells of the chip immobilized with OVM as the antigen protein, and the reaction was allowed to proceed overnight under light-shielding and refrigeration (4°C). After removing the blocking reagent by aspirator (VARIABLE SPEED PUMP, BIORAD), the chip was transferred back to the reaction plate, 10 mL of washing solution (50 mM TTBS) was added, and the chip was agitated for 5 minutes before the washing solution was aspirated and removed. After washing three times in the same manner, the chip was washed three more times with purified water (MilliQ water). The chip surface was centrifuged using a centrifuge (Allegra®, X-22R Centrifuge (BECKMAN COULTER)) to remove water droplets (2000 rpm for 1 minute) and an OVM-immobilized DCP chip was prepared.

[0093] (Primary Antibody Reaction) Serum samples from each test infant were appropriately diluted with sample diluent (20 mM phosphor buffer, pH 7.4 / 0.3 M KCl / 0.05% Tween 20) to prepare serum dilution solutions (primary antibody solutions). 10 μL of these diluted samples were added to the reaction well tank and allowed to stand for 2 hours at 37°C under light protection.

[0094] (Reaction with secondary antibody) After removing the diluted sample obtained in the above procedure by aspirator (VARIABLE SPEED PUMP, BIORAD), the tip was transferred to a washing case, 10 mL of washing solution (TTBS: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.95% Tween 20) was added, and the washing process was repeated three times for 5 minutes using a Double-Shaker NR3. Then, purified water (MilliQ water) was added and washed three times for 1 minute. The water droplets on the tip surface were removed by centrifugation (2000 rpm for 1 minute) using the above centrifuge. Next, a fluorescently labeled secondary antibody (HiLyte Fluor® or 555-conjugated anti-human IgE (HyTest)) (dilution, IMMUNO SHOT Platinum / 1% BSA, final dilution concentration 10 μg / mL) was prepared as the secondary antibody solution. 10 μL of this secondary antibody solution was dispensed into each reaction well on the slide and allowed to stand for 2 hours at 37°C under light protection.

[0095] (Detection of primary antibody captured by antigen) After removing the diluted secondary antibody solution using an aspirator, the tip was placed in a washing case and washed three times for 5 minutes each using a Double-Shaker NR3 (TAITEC). Then, purified water (MilliQ water) was added and rinsed three times for 1 minute each, and the water droplets were removed using the centrifuge and dried. The fluorescence intensity was measured (Ex: 532 nm, Em: 570 nm) using a fluorescence scanner (3D Gene Scanner, Toray Industries), or any other device with similar performance, not limited to the 3D Gene Scanner, such as an InnoScan710AL-Dx (Innopsys), and the fluorescence intensity of the spots obtained from each tip was quantified. The unit of measurement is expressed as the IgE antibody titer bound to the antigen by the antigen-antibody reaction, in units of Binding Unit (BUe). This was measured and expressed from a calibration curve based on the fluorescence intensity of each standard antibody at known concentrations immobilized on a chip. The following notation is based on 1 BUe = 2.4 ng.

[0096] For the quantification of IgE antibody titers, a fluorescence intensity calibration curve was created using human standard IgE antibodies (75 / 502) of known concentrations purchased from the National Institute for Biological Standards and Control (Hertfordshire, England), and the OVM-specific IgE antibody titer (BUe / mL) was measured using a DCP chip.

[0097] For the quantification of IgG1 antibody titers, a fluorescence intensity calibration curve was created using human standard IgG1 antibody of known concentration (Human IgG1 catalog number: PHP010) purchased from Bio-Rad, and the OVM-specific IgG1 antibody titer (BUe / mL) was measured using a DCP chip.

[0098] For the quantification of IgG4 antibody titers, a fluorescence intensity calibration curve was created using human standard IgG4 antibody of known concentration (Human IgG4 catalog number: 5254-3004) purchased from Bio-Rad, and the OVM-specific IgG4 antibody titer (BUe / mL) was measured using a DCP chip.

[0099] [Example 3] [Measurement of Avidity of OVM-Specific IgE Antibodies] For the 311 children mentioned above, serum samples were collected before the first oral food challenge test, and OVM (manufactured by Sigma-A) was used as the antigen protein. The sIgE antibody against OVM, which is OVM-specific IgE antibody, and the sIgE antigen-binding affinity antibody titer, which is calculated by multiplying the sIgE antibody titer by the 1 / IC50 value of the sIgE antibody, were calculated.

[0100] The following reaction was carried out using the aforementioned OVM-immobilized DCP chip.

[0101] (Primary Antibody Reaction) Serum samples from each test infant were appropriately diluted with sample diluent (20 mM phosphorate buffer, pH 7.4 / 0.3 M KCl / 0.05% Tween 20), for example, 2.5 times, to prepare serum dilution solutions (primary antibody solutions), and the antibody titer was measured. Next, the antibody titer of the obtained dilution was readjusted so that the competitive antigen-binding inhibitory activity (avidity) dependent on the amount of added soluble antigen was within a detectable range. Specifically, the lower limit of the fluorescence intensity shown by OVMIgE was adjusted so that > ​​the detection limit (blank + 5 SD), and the upper limit of the fluorescence intensity was within a range of 5,000 or less. In addition, six types of OVM solutions were prepared as competitive binding inhibitors by adjusting them with the above sample diluent to six different concentrations: 0, 0.2, 2.0, 20, 100, and 200 nM. To measure the IC50 value, a preliminary reaction was performed by adding 6 μL each of the six types of soluble OVM solutions (6 μL each) to the serum dilution solution (6 μL) (total 12 μL) and reacting them at 25°C for 30 minutes. After the reaction, 10 μL of each preliminary reaction solution was taken and added to the OVM-immobilized DCP tip, and left to stand at 37°C for 2 hours under light shielding. The blank + 5 SD indicates the lower limit of detection. Specifically, the blank value was the fluorescence intensity derived from the non-specific binding of the fluorescently labeled secondary antibody in the reaction solution to the antigen under conditions of buffer only and no sample serum. This fluorescence intensity was measured five times to calculate the standard deviation (SD) value of the fluorescence intensity, and the fluorescence intensity showing a value of blank + 5 SD (lower limit of detection) or higher was considered to be the fluorescence intensity of IgE derived from the sample.

[0102] (Reaction with secondary antibody) After removing the serum dilution solution, which had reacted on the DCP tip, using an aspirator (VARIABLESPEED PUMP, BIORAD), the tip was transferred to a washing case. 10 mL of washing solution (50 mM TTBS) was added, and the tip was washed using a Double-Shaker NR3 for 5 minutes three times. Further washing was performed with purified water (MilliQ water) for 1 minute three times. The tip surface was then centrifugated to remove water droplets (2000 rpm for 1 minute) using the above centrifuge. Next, a fluorescently labeled secondary antibody (HiLyte Fluor® or 555-conjugated anti-human IgE (HyTest)) was prepared (dilution: IMMUNO SHOT Platinum / 1% BSA, final dilution concentration 10 μg / mL). 10 μL of this secondary antibody solution was dispensed into each reaction well on the slide and allowed to stand for 2 hours at 37°C under light protection.

[0103] After removing the residue with an aspirator, the tips were transferred to a washing case, 10 mL of washing solution (50 mM TTBS) was added, and the washing process was repeated three times for 5 minutes each using a Double-Shaker NR3. Then, purified water (MilliQ water) was added and washed three times for 1 minute each. The tips were centrifuged using the above centrifuge to remove water droplets (2000 rpm for 1 minute) to remove water droplets from the tip surface. The remaining fluorescence amount was measured using a fluorescence scanner (3D Gene Scanner, Toray Industries) and an InnoScan 710AL-Dx (Innopsys), and the fluorescence intensity of the spots obtained from each tip was quantified (Ex: 532 nm, Em: 570 nm). For each infant and child tested, the OVM concentration that resulted in a fluorescence intensity of 50% was defined as the "IC50" value, with the fluorescence intensity of a solution without OVM (i.e., when the concentration of the competitive binding inhibitor) was 0 being set to 100%.

[0104] [Example 4] [ROC Analysis] Based on the sIgG1 antibody titer, sIgG antibody titer, and sIgE antibody titer data of each of the 311 children mentioned above, ROC analysis was performed on two parameters: OVM sIgE / OVM sIgE IC50 [(BUe / mL) / (nM)] and OVM sIgE / OVM sIgG1 + OVM sIgG4. The results are shown in Figure 1(a). The cutoff value, sensitivity at the cutoff value, specificity, AUC value, lower limit 95%, and upper limit 95% are shown in Table 4-1. From here on, ROC analysis was performed using GraphPad Prism ver. 5.4 (GraphPad Inc.) and JMP14 (SAS Institute, Cary, NC).

[0105]

[0106] Similarly, based on the sIgG1 antibody titers, sIgG antibody titers, and sIgE antibody titers of each of the 311 children mentioned above, ROC analysis was performed on two parameters: OVM sIgE / OVM sIgEIC50 / antigen loading [(BUe / mL) / (nM) / (g)] and OVM sIgE / OVM sIgG1+(DCP)OVM sIgG4 / antigen loading. The results are shown in Figure 1(b). The cutoff value, sensitivity, specificity, AUC value, lower limit 95%, and upper limit 95% are shown in Table 4-2.

[0107]

[0108] Compared to Table 4-1, the data in Table 4-2 showed increases in AUC (0.81–0.82), specificity (0.71–0.83), lower limit of 95% (0.74–0.76), and upper limit of 95% (0.86–0.87). Although there was a slight decrease in sensitivity (0.70–0.84), overall, it can be said that this led to an increase in diagnostic accuracy from a statistical standpoint.

[0109] (Creation of a graph for determination 1) The Y-axis represents the sIgE antigen-binding affinity antibody titer (sIgE / IC50), which is obtained by multiplying the sIgE antibody titer against OVM by the sIgE antibody's 1 / IC50 value, and the X-axis represents the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer). The cutoff value for positive and negative food allergy symptom induction determined by ROC analysis of the above OVMsIgE / IC50 value is <y>, i.e., 5.76 (BUe / mL) / (nM) (a straight line extended horizontally to the right from the Y-axis on the above graph). Based on the ROC analysis of the above sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), four regions were defined by the orthogonality of the two cutoff values: <x>, which is 0.74 (a straight line drawn vertically upward from the X-axis on the graph above), and the cutoff value for positive and negative induction of food allergy symptoms, i.e., 0.74. These four regions are (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>) on the graph above (Figure 2(a)).

[0110] (Creation of a graph for judgment 2) In order to investigate whether the predictive ability can be further improved, the value of the sIgE antigen-binding affinity antibody titer (sIgE / IC50), which is obtained by multiplying the sIgE antibody titer against OVM by the value of 1 / IC50 of the sIgE antibody, is divided by the antigen loading (sIgE / sIgE In a graph where the Y-axis represents IC50 / antigen loading and the X-axis represents the value obtained by dividing sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) by the antigen loading, the cutoff value for positive and negative food allergy symptom induction determined by ROC analysis of the Y-axis value <y>, i.e., 5.82 (BUe / mL) / (nM) / (g) (a straight line extended horizontally to the right from the Y-axis on the graph above), and the cutoff value for positive and negative food allergy symptom induction determined by ROC analysis of the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) / antigen loading, i.e., the cutoff value for positive and negative food allergy symptom induction determined by ROC analysis of the value, i.e., We attempted to plot the four regions formed by the orthogonality of the two cutoff values, -1.30 (a straight line drawn vertically upward from the X-axis on the graph above), namely (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>) on the graph above (Figure 2(b)).

[0111] For each subject who underwent the primary oral food challenge test, when plotting the sIgE antigen-binding affinity antibody titer (calculated by multiplying the sIgE antibody titer in the sample by the 1 / IC50 value of the sIgE antibody) and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) on a judgment graph, a graph for determining negative subjects (Figure 3-1(a)) and a graph for determining positive subjects (Figure 3-2(a)) were created by distinguishing between positive subjects who developed an allergic reaction and negative subjects who did not develop an allergic reaction.

[0112] Furthermore, when plotting the values ​​of sIgE / sIgE IC50 / antigen loading and sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) / antigen loading on a graph for determination, we created a graph for determining negative subjects (Figure 3-1(b)) and a graph for determining positive subjects (Figure 3-2(b)) by distinguishing between positive subjects who developed an allergic reaction and negative subjects who did not develop an allergic reaction.

[0113] Furthermore, by further distinguishing and plotting the graphs for determining negative subjects (Figure 3-1(a)) and positive subjects (Figure 3-2(a)) into subjects who ingested a small amount of the allergen, subjects who ingested a moderate amount of the allergen, and subjects who ingested a daily intake of the allergen, six types of determination graphs (a) to (f) in Figure 4(a) were created.

[0114] (Prediction of next load 1) If the subject is a negative subject that does not produce an allergic reaction, the amount of allergen that can be ingested in the next oral food challenge test was predicted using the graphs (a) to (c) in Figure 4(a).

[0115] (a) If the data for a subject is plotted in the (-<x>, -<y>) or (+<x>, -<y>) region of the negative subject determination graph in Figure 4(a) above, it was predicted that the amount of allergen that could be ingested in the next oral food challenge test could be increased from the maximum load administered in the first oral food challenge test, and that the subject could ingest an amount two, three, four, or five levels higher, or even exceed the daily intake. The degree of increase was determined taking into account the subject's age, family allergy history, etc. In Figure 4(a) above, 12 subjects in graph (a), 54 subjects in graph (b), and 88 subjects in graph (c) met this determination. (b) If the data for the subject is plotted in the (+<x>, +<y>) or (-<x>, +<y>) region of the negative subject determination graph in Figure 4(a) above, it was predicted that the amount of allergen that could be ingested in the next oral food challenge test would be one or three levels higher than the actual amount ingested in the first oral food challenge test. In Figure 4(a) above, 37 people in graph (a), 31 people in graph (b), and 39 people in graph (c) fell into this category.

[0116] If the subject was a positive subject that had shown an allergic reaction, the amount of allergen that could be ingested in the next oral food challenge test was predicted using the graphs (d) to (f) in Figure 4(a) above.

[0117] (c) If the data for the subject is plotted in the (-<x>, -<y>) or (+<x>, -<y>) region of the graph for determining positive subjects in Figure 4(a) above, it was predicted that the amount of allergen that could be ingested in the next oral food challenge test would be 0.5 or 2 levels less than the actual amount administered in the first oral food challenge test. The degree of increase was determined by taking into account the degree of the positive reaction that occurred in the first oral food challenge test, the subject's age, family allergy history, etc. In Figure 4(a) above, there was 1 person in graph (d), 5 people in graph (e), and 1 person in graph (f). (d) If the data for a subject is plotted in the (+<x>, +<y>) or (-<x>, +<y>) region of the graph for determining positive subjects in Figure 4(a) above, it was predicted that the amount of allergen that could be ingested in the next oral food challenge test should be one, two, three, four, or five levels less than the actual amount ingested in the first oral food challenge test. The degree of reduction was determined by taking into account the degree of the positive reaction that occurred in the first oral food challenge test, the subject's age, family allergy history, etc. In Figure 4(a) above, 28 subjects were in graph (d), 13 subjects in graph (e), and 2 subjects in graph (f).

[0118] (Prediction of next load amount 2) If the subject is a negative subject that does not produce an allergic reaction, the amount of allergen that can be ingested in the next oral food challenge test was predicted using the graphs (a) to (c) in Figure 4(b).

[0119] (a) If the data for a subject is plotted in the (-<x>, -<y>) or (+<x>, -<y>) region of the negative subject determination graph in Figure 4(b) above, it was predicted that the amount of allergen that could be ingested in the next oral food challenge test could be increased from the maximum load administered in the first oral food challenge test, and that the subject could ingest an amount two, three, four, or five levels higher, or even exceed the daily intake. The degree of increase was determined by taking into account the subject's age, family allergy history, etc. In Figure 4(b) above, 12 subjects in graph (a), 78 subjects in graph (b), and 127 subjects in graph (c) met this determination. (b) If the data for the subject is plotted in the (+<x>, +<y>) or (-<x>, +<y>) region of the negative subject determination graph in Figure 4(b) above, it was predicted that the amount of allergen that could be ingested in the next oral food challenge test would be one or three levels higher than the actual amount ingested in the first oral food challenge test. In Figure 4(b) above, 37 people in graph (a), 7 people in graph (b), and 0 people in graph (c) fell into this category.

[0120] If the subject was a positive subject that had shown an allergic reaction, the amount of allergen that could be ingested in the next oral food challenge test was predicted using the graphs (d) to (f) in Figure 4(b) above.

[0121] (c) If the data for the subject is plotted in the (-<x>, -<y>) or (+<x>, -<y>) region of the graph for determining positive subjects in Figure 4(b) above, it was predicted that the amount of allergen that could be ingested in the next oral food challenge test could be reduced by 0.5 or 2 levels from the actual amount administered in the first oral food challenge test. The degree of increase in levels was determined by taking into account the degree of the positive reaction that occurred in the first oral food challenge test, the subject's age, family allergy history, etc. In Figure 4(b) above, there was 1 person in graph (d), 11 people in graph (e), and 3 people in graph (f). (d) If the data for the subject was plotted in the (+<x>, +<y>) or (-<x>, +<y>) region of the graph for determining positive subjects in Figure 4(b) above, it was predicted that the amount of allergen that could be ingested in the next oral food challenge test should be one, two, three, four, or five levels less than the actual amount ingested in the first oral food challenge test. The degree of reduction was determined by taking into account the degree of the positive reaction that occurred in the first oral food challenge test, the subject's age, family allergy history, etc. In Figure 4(b) above, 28 subjects in graph (d), 7 subjects in graph (e), and 0 subjects in graph (f).

[0122] The diagnostic significance of each region in the four-part diagram illustrated in Figure 4(b) of the present invention is determined as follows, but it is believed that including the antigen loading amount as a parameter will more accurately indicate the significance of each region and thus accurately demonstrate the diagnostic performance. <x> , + <y>): Area where allergies occur, (- <x> , + <y>): Mild symptom area close to the allergy onset area (+ <x> , - <y>): Mild allergy symptom area, close to the area of ​​no allergy symptoms (- <x> , - <y>): Areas where no allergy symptoms developed. For example, plotted in Figure 4(a)(c) for subjects who underwent the test, (+ <x> ,+ <y>)and(- <x> ,+ <y>The 39 individuals who belonged to the allergy-causing region were replotted in the format shown in Figure 4(b) with the antigen load from the oral food challenge test added, as shown in Figure 4(b)(c) (- <x> ,- <y>) and (+ <x> ,- <y>These data clearly confirm that the individuals were reclassified into the 127-person allergy-free zone and that oral food challenge tests are not necessarily required for them.

[0123] (Necessity of oral food challenge tests and estimation of antigen load) The four regions in Figures 2(a) and (b) indicate that a means can be constructed to predict the amount of allergen that a subject who has undergone an oral food challenge test for an allergen can ingest in the next oral food challenge test. This means using the four quadrants of Figure 2, created by setting positive and negative cutoff values ​​for the test based on the immunological parameters of the patient's blood and the load during the challenge test.

[0124] As we have seen, for subjects who have undergone oral food challenge tests, plotting the data with the antigen load as a parameter slightly improves predictive accuracy. On the other hand, when using graphs created without including the antigen load as a parameter, the judgment tends to be stricter, so the possibility of an anaphylactic reaction due to an excessively high allergen load in the next test is lower. In cases where more careful judgment is required, such as with infants and young children, it may be useful to use graphs that do not include the antigen load as a parameter.

[0125] Furthermore, by using Figure 2(a), which was created using values ​​excluding the antigen loading parameter, and Figure 4(a), which uses eggs as an example, it was found that even for subjects who have never undergone an oral food challenge test or are about to undergo one, it is possible to roughly determine whether an oral food challenge test is necessary and estimate the antigen loading amount at the time of the test. The criteria for determining whether an oral food challenge test is necessary and for estimating the antigen loading amount based on Figure 2(a) are shown in Table 5 below. Until now, the antigen loading amount in the first oral food challenge test has been determined mainly based on the physician's experience, but by measuring the antigen-specific sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer of the subject, it is possible to determine whether an oral food challenge test is necessary and to determine the antigen loading amount more objectively.

[0126]

[0127] Currently, there are no clear criteria for determining whether or not an oral food challenge test is necessary and for setting the appropriate antigen load. These criteria rely solely on patient information, physician experience, and allergen sensitization information. However, the ability to predict the outcome in patients before an oral food challenge test through statistical analysis represents a significant advance in the field of allergy diagnosis.

[0128] This invention is useful in the field of diagnosis and treatment of food allergies.

[0129] 1. System for predicting the amount of allergens 2. Memory unit 3. Calculation unit 4. Graph creation unit 5. ROC analysis unit 6. Graph region division unit 7. Plotting unit 8. Prediction unit< / y> < / x> < / y> < / x> < / y> < / x> < / y> < / x> < / y> < / x> < / y> < / x> < / y> < / x> < / y> < / x>

Claims

A method for predicting the amount of allergen that a subject who has undergone a primary oral food challenge test for that allergen can be given in a subsequent oral food challenge test. The method according to claim 1, characterized in that the amount of allergen load is predicted based on a graph created by measuring antigen-specific sIgE antibody titers, sIgG1 antibody titers, and sIgG4 antibody titers in samples collected from subjects who underwent a primary oral food challenge test for the allergen. The method according to claim 2, characterized in that the amount of allergen loading is predicted based on a graph created with the value of sIgE antigen-binding affinity antibody titer, obtained by multiplying the sIgE antibody titer in a sample taken from a subject who underwent a primary oral food challenge test by the value of 1 / IC50 of the sIgE antibody, as the Y axis, and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), calculated based on the sIgG1 antibody titer, sIgG4 antibody titer, and sIgE antibody titer in a sample taken from a subject who underwent a primary oral food challenge test, as the Y axis, and the X axis as the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), which is calculated based on the sIgE antibody titer, sIgG1 antibody titer, and sIgE antibody titer in a sample taken from a subject who underwent a primary oral food challenge test. The method according to claim 2, characterized in that the amount of allergen loading is predicted based on a graph created with the value of the sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer in a sample taken from a subject that underwent a primary oral food challenge test by the value of 1 / IC50 of the sIgE antibody, divided by the amount of antigen loaded in the primary oral food challenge test, as the Y axis, and the value of the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), calculated based on the sIgG1 antibody titer, sIgG4 antibody titer, and sIgE antibody titer, divided by the amount of antigen loaded, as the X axis. The method according to claim 3, characterized in that the graph is a graph for determining the onset of food allergies, created by dividing the graph into four regions: (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>), based on the orthogonality of the cutoff value <y> determined by ROC analysis based on the sIgE antigen-binding affinity antibody titer obtained by multiplying the sIgE antibody titer in a sample taken from a subject who underwent a primary oral food challenge test by the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) calculated based on the sIgG1 antibody titer, sIgG4 antibody titer, and sIgE antibody titer. The method according to claim 4, characterized in that the graph is a graph for determining the onset of food allergies, created by dividing the graph into four regions: (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>), based on the orthogonality of the cutoff value <y> determined by ROC analysis based on the value obtained by dividing the sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer in a sample taken from a subject who underwent a primary oral food challenge test by the value of 1 / IC50 of the sIgE antibody, by the amount of antigen challenged in the primary oral food challenge test, and the cutoff value <x> determined by ROC analysis based on the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), which is calculated based on the sIgE antibody titer, sIgG4 antibody titer, and sIgE antibody titer, by the amount of antigen challenged in the primary oral food challenge test. The method according to claim 5 or 6, characterized in that the graph is created by plotting, for each subject who underwent the primary oral food challenge test, positive subjects who developed an allergic reaction and negative subjects who did not develop an allergic reaction, thereby creating two types of graphs: a graph for determining negative subjects and a graph for determining positive subjects. The method according to claim 7, characterized in that the graphs for determining negative subjects and the graphs for determining positive subjects are three types of graphs for determining negative subjects and three types of graphs for determining positive subjects, respectively, created by plotting separately the subjects who were given a small amount of allergen, the subjects who were given a moderate amount of allergen, and the subjects who were given the daily intake amount of allergen in the first oral food challenge test. The method according to claim 7, characterized in that the amount of allergen loaded in the next oral food challenge test is predicted as follows: 1) to 4). 1) In the graph used to determine negative subjects, subjects belonging to the regions (-<x>, -<y>) and (+<x>, -<y>) can be predicted to have consumed an amount that is 2 to 5 levels higher than the amount ingested in the oral food challenge test, or an amount equal to or greater than their daily intake; 2) In the graph for determining negative subjects, subjects belonging to the regions (+<x>, +<y>) and (-<x>, +<y>) can be predicted to consume a load that is 1 to 3 levels higher than the amount ingested in the first oral food challenge test; 3) In the graph for determining positive cases, individuals belonging to the regions (-<x>, -<y>) and (+<x>, -<y>) can be predicted to consume a load 0.5 to 2 levels lower than the amount ingested in the first oral food challenge test; 4) In the graph for determining positive cases, individuals belonging to the regions (+<x>, +<y>) and (-<x>, +<y>) can be predicted to consume a load 1 to 5 levels or more lower than the amount ingested in the first oral food challenge test; The method according to any one of claims 1 to 6, characterized by comprising the following steps (a) to (g). (a) A step of measuring the sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer in a sample taken from a subject who underwent a primary oral food challenge test; (b) A process of creating a graph with the sIgE antigen-binding affinity antibody titer, obtained by multiplying the sIgE antibody titer in the sample taken from a subject who underwent a primary oral food challenge test by the 1 / IC50 value of the sIgE antibody, on the Y axis, and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), calculated based on the sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer, on the X axis. Or, (b') A process to create a graph with the following parameters: the Y-axis is the value obtained by multiplying the sIgE antibody titer in a sample taken from a subject who underwent a primary oral food challenge test by the 1 / IC50 value of the sIgE antibody, and dividing that value by the antigen load imposed in the primary oral food challenge test; and the X-axis is the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), calculated based on the sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer, by the above antigen load; (c)(b)In the graph created in step (c), the cutoff value <y> by ROC analysis is determined based on the value of sIgE antigen-binding affinity antibody titer obtained by multiplying the sIgE antibody titer in the sample taken from the subject who underwent the primary oral food challenge test by the value of 1 / IC50 of the sIgE antibody, and the cutoff value <x> by ROC analysis is determined based on the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) in the sample taken from the subject who underwent the primary oral food challenge test. Or, (c')(b') In the graph created in step (c')(b'), the cutoff value <y> by ROC analysis is determined based on the value obtained by dividing the sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer in the sample taken from the subject who underwent the primary oral food challenge test by the value of 1 / IC50 of the sIgE antibody, by the antigen loading amount imposed in the primary oral food challenge test, and the cutoff value <x> by ROC analysis is determined based on the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) in the sample taken from the subject who underwent the primary oral food challenge test by the above antigen loading amount; (d) A process in which the cutoff value <y> determined in step (c) or step (c') by ROC analysis and the cutoff value <x> determined by ROC analysis are orthogonal, thereby dividing the graph into four regions: (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>), and creating a judgment graph; (e) For each subject who underwent the primary oral food challenge test, a process to distinguish between positive subjects who developed an allergic reaction and negative subjects who did not develop an allergic reaction, and to create a graph for determining negative subjects and a graph for determining positive subjects by plotting the value of the sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer by the value of 1 / IC50 of the sIgE antibody, and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) on a determination graph. Or, (e') For each subject who underwent the primary oral food challenge test, a process to distinguish between positive subjects who developed an allergic reaction and negative subjects who did not develop an allergic reaction, and to create a graph for determining negative subjects and a graph for determining positive subjects by plotting the value obtained by dividing the sIgE antigen-binding affinity antibody titer (sIgE antibody titer multiplied by the sIgE antibody 1 / IC50 value) by the amount of antigen loaded in the primary oral food challenge test, and the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) by the above amount of antigen loaded on a determination graph; (f) A step to create three types of graphs for determining negative subjects and three types of graphs for determining positive subjects by further distinguishing and plotting subjects who were given a small amount of allergen in the first oral food challenge test, subjects who were given a moderate amount of allergen, and subjects who were given the daily intake amount of allergen; (g) The process of making predictions as follows: 1) In the graph used to determine negative subjects, subjects belonging to the regions (-<x>, -<y>) and (+<x>, -<y>) can consume an amount that is 2 to 5 levels higher than the amount ingested in the first oral food challenge test, or an amount equivalent to or greater than their daily intake; 2) In the graph used to determine negative subjects, subjects belonging to the regions (+<x>, +<y>) and (-<x>, +<y>) can consume a load that is 1 to 3 levels higher than the amount ingested in the first oral food challenge test; 3) In the graph for determining positive cases, subjects belonging to the regions (-<x>, -<y>) and (+<x>, -<y>) can consume an amount that is 0.5 to 2 steps lower or 0.5 to 1 step higher than the amount ingested in the first oral food challenge test; 4) In the graph used to determine positive cases, subjects belonging to the regions (+<x>, +<y>) and (-<x>, +<y>) can consume a load that is 1 to 5 levels or more lower than the amount ingested in the first oral food challenge test; A system for predicting the amount of allergen to be ingested in a subsequent oral food challenge test by a subject who has undergone a primary oral food challenge test for that allergen, A storage unit that stores the personal data of each target, A calculation unit calculates the sIgE antigen-binding affinity antibody titer by multiplying the sIgE antibody titer by the 1 / IC50 value of the sIgE antibody, based on the sIgE antibody titer in samples taken from subjects who underwent a primary oral food challenge test, and then calculates the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) based on the sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer. A graph creation unit creates a graph where the sIgE antigen-binding affinity antibody titer, obtained by multiplying the sIgE antibody titer by the 1 / IC50 value of the sIgE antibody, is on the Y axis, and the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) is on the X axis. Based on the sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer by the 1 / IC50 value of the sIgE antibody, a cutoff value <y> is determined by ROC analysis, and based on the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), a cutoff value <x> is determined by ROC analysis, comprising: an ROC analysis unit; A graph region division unit creates a four-part graph by dividing the graph into four regions: (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>) based on the fact that the cutoff value <y> determined by ROC analysis and the cutoff value <x> determined by ROC analysis are orthogonal. For each subject who underwent the primary oral food challenge test, the system distinguishes between positive subjects who developed an allergic reaction and negative subjects who did not develop an allergic reaction. The system then plots the sIgE antigen-binding affinity antibody titer (sIgE antibody titer multiplied by the sIgE antibody's 1 / IC50 value) and the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) on the aforementioned four-part graph to create graphs for determining negative subjects and graphs for determining positive subjects. The system comprises a prediction unit that predicts the amount of allergen to be loaded in the next oral food challenge test for each subject. A system for predicting the amount of allergen to be ingested in a subsequent oral food challenge test by a subject who has undergone a primary oral food challenge test for that allergen, A storage unit that stores the personal data of each target, A calculation unit calculates the value of the sIgE antigen-binding affinity antibody titer, obtained by multiplying the sIgE antibody titer by the 1 / IC50 value of the sIgE antibody, based on the sIgE antibody titer in a sample taken from a subject who underwent a primary oral food challenge test, by dividing the value by the amount of antigen loaded in the primary oral food challenge test. Based on the sIgE antibody titer, sIgG1 antibody titer, and sIgG4 antibody titer, a calculation unit calculates the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) by the amount of antigen loaded in the primary oral food challenge test. A graph creation unit creates a graph in which the sIgE antigen-binding affinity antibody titer, obtained by multiplying the sIgE antibody titer by the sIgE antibody's 1 / IC50 value, is divided by the antigen load imposed in the primary oral food challenge test, and the Y-axis is the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) by the antigen load imposed in the primary oral food challenge test, and the X-axis is the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) by the antigen load imposed in the primary oral food challenge test. Based on the value obtained by dividing the sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer by the 1 / IC50 value of the sIgE antibody, by the amount of antigen loaded in the primary oral food challenge test, a cutoff value <y> is determined by ROC analysis, and based on the value obtained by dividing the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) by the amount of antigen loaded in the primary oral food challenge test, a cutoff value <x> is determined by ROC analysis, in the ROC analysis unit, A graph region division unit creates a four-part graph by dividing the graph into four regions: (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>) based on the fact that the cutoff value <y> determined by ROC analysis and the cutoff value <x> determined by ROC analysis are orthogonal. For each subject who underwent the primary oral food challenge test, a graph for determining negative subjects and a graph for determining positive subjects are created by plotting the following values ​​on the four-part graph: the sIgE antigen-binding affinity antibody titer, obtained by multiplying the sIgE antibody titer by the sIgE antibody 1 / IC50 value, divided by the antigen load administered in the primary oral food challenge test; and the sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer), divided by the antigen load administered in the primary oral food challenge test. The system comprises a prediction unit that predicts the amount of allergen to be loaded in the next oral food challenge test for each subject. The system according to claim 11 or 12, characterized in that the plotting section further distinguishes and plots subjects who were given a small amount of allergen, subjects who were given a moderate amount of allergen, and subjects who were given a daily intake amount of allergen in the first oral food challenge test. The method according to any one of claims 1 to 7, characterized in that the sample is plasma or serum. The system according to claim 11 or 12, characterized in that the sample is plasma or serum. A method for predicting the amount of allergen that a subject who has not undergone an oral food challenge test for an allergen can be given in an oral food challenge test, using a graph for determining the onset of food allergies created by dividing the sample taken from a subject who has undergone a primary oral food challenge test into four regions: (+<x>, +<y>), (-<x>, +<y>), (+<x>, -<y>), and (-<x>, -<y>), based on the orthogonality of the cutoff value <y> determined by ROC analysis based on the value of sIgE antigen-binding affinity antibody titer, which is obtained by multiplying the sIgE antibody titer in a sample taken from a subject who has undergone a primary oral food challenge test by the value of 1 / IC50 of the sIgE antibody, and the cutoff value <x> determined by ROC analysis based on the value of sIgE antibody titer / (sIgG1 antibody titer + sIgG4 antibody titer) calculated based on the sIgG1 antibody titer, sIgG4 antibody titer, and sIgE antibody titer.

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