Methods for collecting data for predicting the onset of anaphylaxis

Abstract

A method is provided for predicting the risk of anaphylaxis in infants / children with allergies by measuring the antibody titer of IgE antibodies in samples collected from infants / children with food allergies and the affinity of IgE antibodies for allergens, and predicting the risk of anaphylaxis in infants / children in the early stage by using a judgment criterion prepared based on ROC analysis, thereby predicting the risk of anaphylaxis in infants / children with allergies.

Description

Technical Field

[0001] The present invention relates to a method for collecting data for predicting the possibility of systemic anaphylaxis in infants / children using a critical value determined by ROC analysis based on the degree of affinity (avidity) of IgE antibodies in a sample collected from infants / children suffering from allergies for an allergen as a judgment criterion. Background Art

[0002] Food allergy is defined as "a phenomenon in which food causes adverse symptoms to the body via an antigen-specific immunological mechanism" (for example, see Non-Patent Document 1, etc.). For foods that are digested and absorbed by humans to maintain life, oral immune tolerance usually works to prevent the body from developing a defensive response to undigested foreign matter. However, 5% to 10% of infants and young children in the newborn population will develop certain food allergies within one year of birth, and the pathogenesis or methods of preventing the disease have not yet been fully explained.

[0003] Anaphylaxis is defined as "a hypersensitivity reaction that causes systemic allergic symptoms involving multiple organs (not a mild symptom) due to the invasion of allergens and may be life-threatening" (for example, see Patent Document 2, etc.). Therefore, for patients with allergies, whether anaphylaxis may be induced is a very important concern.

[0004] Currently, various methods for measuring the antibody titer of antigen-specific IgE antibodies, including the ImmunoCAP (Thermo Fisher) method, are used for the diagnosis of allergies. However, for example, in the ImmunoCAP method, when a probability curve showing the possibility of allergy occurrence in a subject is prepared, there are reports that the numerical value of the possibility of anaphylaxis occurring in infants and young children of grades 2 and 3 with relatively low IgE antibody titers is lower than the actual incidence rate (for example, refer to non-patent documents 3, 4, etc.).

[0005] In addition, a method for collecting data for predicting risks has been proposed, in which plasma samples of infants within 6 months of birth are used to quantitatively measure the antibody titers of IgG1 and IgE to allergens in the samples, respectively, and the risk of allergy to the allergens in the infants before the onset of the aforementioned food allergy is predicted based on a judgment standard made based on the degree of immunoglobulin conversion from IgG1 to IgE that has been statistically processed in advance (for example, refer to Patent Document 1, etc.); however, this method makes it difficult to predict whether a systemic allergic reaction will occur.

[0006] In addition, the oral food challenge (OFC), which is considered to be the most reliable diagnostic method for the onset of anaphylaxis in infants and young children with food allergies, is performed in the presence of a doctor, but since the subject may develop anaphylaxis, there is a strong demand for the development of a method for predicting the possibility of anaphylaxis without performing OFC. However, there are currently no reports on effective diagnostic methods, effective biomarkers, etc. for predicting the possibility of anaphylaxis.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: International Publication WO2017 / 195871 Pamphlet

[0010] Non-patent literature

[0011] Non-patent literature 1: Food Allergy Diagnosis and Treatment Guidelines 2011, Food Allergy Research Society

[0012] Non-patent literature 2: Simons FE, Ardusso LR, Bilo MB, EI-Gamal YE, Ledford DK, RingJ, Sanchez-Borges M, Senna GE, Sheikh A, Thong BY, World Allergy Orhan J. 2011; 4:13-37.

[0013] Non-patent document 3: Komata T, Soderstrom L, Borres MP, Tachimoto H, Ebisawa M., J Allergy Clin Immunol. 2007; 119: 1272-1274.

[0014] Non-patent document 4: Furuya K, Nagao M, Sato Y, Ito S, Fujisawa T., Allergy. 2016; 71: 1435-1443. Summary of the invention

[0015] Problems to be solved by the invention

[0016] An object of the present invention is to provide a method for predicting the risk of anaphylaxis in infants and children suffering from allergies.

[0017] Methods for solving problems

[0018] Under the circumstance that the onset of anaphylaxis cannot be effectively predicted using only the antibody titer of IgE antibody as a parameter, the inventors considered whether other parameters are needed in the prediction of the onset of anaphylaxis. That is, in the blood, in addition to IgE antibodies, there are IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, IgM antibodies, IgA antibodies, IgD antibodies, etc. that react to the same allergen, and it is considered that the fact that these antibodies are polyclonal antibodies that recognize multiple epitopes in the antigen needs to be taken into consideration.

[0019] Furthermore, the present inventors have found that when evaluating the binding of the IgE antibody to the allergen, it is not necessary to simply measure the IgE antibody titer obtained by measuring only the constant region of the IgE antibody using a secondary antibody as has been conventionally done, i.e., Figure 1 The "IgE quantity" of the constant part (Fc region) of the IgE antibody shown in the above is measured, while the "sum of the affinity of the antigen and the antibody" (affinity) as the "mass of IgE" shown by the variable part of the IgE antibody for the allergen needs to be taken into consideration. The present inventors selected 1 / IC50 value as a parameter representing the affinity of the IgE antibody for the allergen, and used the numerical value obtained by multiplying the IgE antibody titer of infants / children sensitized to each allergen by 1 / IC50 value and affinity value to analyze the presence or absence of anaphylaxis, and performed a significance test on the above parameters. As a result, it was found that compared with the use of existing parameters, a more significant statistically significant difference was found between those who had anaphylaxis and those who did not have anaphylaxis. In addition, in the ROC (Receiver Operating Characteristic) curve analysis used in the performance evaluation of the test, it was confirmed that the value obtained by multiplying the IgE antibody titer by 1 / IC50 value significantly increased the sensitivity and the area under the curve (AUC) calculated as a usefulness index value based on the specificity compared with the conventional test of only the IgE antibody amount. The present inventors confirmed that the onset of anaphylaxis can be effectively predicted for allergic patients who have not had an effective diagnostic method for the possibility of anaphylaxis, and completed the present invention.

[0020] That is, the present invention is defined by the following matters.

[0021] (1) A method for collecting data for predicting the risk of anaphylaxis in infants / children suffering from food allergies, using as a criterion a cutoff value determined by ROC analysis based on the affinity of IgE antibodies for allergens in samples collected from infants / children suffering from food allergies.

[0022] (2) The method for collecting data described in (1) above is characterized in that the critical value determined by ROC analysis based on the affinity of IgE antibodies for allergens is a critical value determined by ROC analysis based on a numerical value obtained by multiplying the antibody titer of IgE antibodies for allergens by the affinity of IgE antibodies for allergens.

[0023] (3) The method for collecting data described in (2) above is characterized in that the affinity of the IgE antibody for the allergen is quantified as 1 / IC50.

[0024] (4) The method for collecting data described in (3) above, wherein IC50 is a competitive binding inhibition activity value.

[0025] (5) The method for collecting data described in (1) above is characterized in that the critical value determined by ROC analysis based on the affinity of IgE antibodies for allergens is a critical value determined by ROC analysis based on IC50 of IgE antibodies.

[0026] (6) The method for collecting data described in (5) above, wherein IC50 is a competitive binding inhibition activity value.

[0027] (7) The method for collecting data according to any one of (2) to (6) above, characterized in that the antibody titer of IgE antibodies to allergens is measured using a DCP chip.

[0028] (8) The method for collecting data according to any one of (1) to (7) above, wherein the sample is plasma or serum.

[0029] Effects of the Invention

[0030] According to the present invention, the possibility of occurrence of anaphylaxis can be meaningfully predicted for infants and children whose anaphylaxis attacks have been difficult to diagnose in the past. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Shown are the structural features that determine the "quantity" and "quality" of IgE antibodies.

[0032] Figure 2 Schematic diagram showing a DCP chip with immobilized proteins.

[0033] Figure 3The ROC curve shows the results of ROC analysis of 343 infants aged 1 to 6 years old who were judged to be level 2 or above by the ImmunoCAP method, using five different measurement parameters for the heated egg allergen ovomucin (OVM), the measurement parameters being CAP OVM sIgE (UA / mL), DCPOVM sIgE (BUe / mL), DCP OVM sIgE / DCP OVM sIgE IC50 [(BUe / mL) / (nM)], DCP OVM sIgEIC50 (nM), and 1 / DCP OVM sIgE IC50 (nM).

[0034] Figure 4 The ROC curve shows the results of ROC analysis of 256 infants aged 1 to 3 years old who were judged to be level 2 or above by the ImmunoCAP method, using five different parameters for allergy positive / negative persons in heated egg OFC. The parameters were: CAP OVM sIgE (UA / mL), DCP OVM sIgE (BUe / mL), DCP OVM sIgE / DCP OVM sIgEIC50 [(BUe / mL) / (nM)], DCP OVM sIgE IC50 (nM), and 1 / DCP OVM sIgE IC50 (nM).

[0035] Figure 5 The results are shown in which 343 infants aged 1 to 6 years old and judged as grade 2 or above by the ImmunoCAP method were tested for the presence (+) or absence (-) of egg allergy (EA) using five different parameters (A to D) for OFC positive and negative heated eggs [EW (heat)], and the results of detecting significant differences in the presence (+) or absence (-) of anaphylaxis (AN) in those with egg allergy (OFC positive). The parameters are: (A) CAP OVM sIgE (UA / mL), (B) DCP OVM sIgE (BUe / mL), (C) DCP OVM sIgE / DCP OVM sIgE IC50 [(BUe / mL) / (nM)], (D) DCP OVM sIgE IC50 (nM), and (E) 1 / DCP OVM sIgE IC50 (nM).

[0036] Figure 6The results of ROC analysis of 343 infants aged 1 to 6 years old judged as grade 2 or higher by the ImmunoCAP method, based on the OFC-based diagnosis of anaphylaxis, are shown, using five different parameters: CAPOVM sIgE (UA / mL), DCP OVM sIgE (BUe / mL), DCP OVM sIgE / DCP OVM sIgE IC50 [(BUe / mL) / (nM)], DCP OVM sIgE IC50 (nM), and 1 / DCP OVM sIgE IC50 (nM).

[0037] Figure 7 The present invention shows the results of detecting the presence (+) or absence (-) of egg allergy (EA) in 256 infants aged 1 to 3 years and judged as grade 2 or above by the ImmunoCAP method using five different parameters (A to D), and the results of significant differences in detecting the presence (+) or absence (-) of systemic anaphylaxis (AN) based on OFC in those with egg allergy (OFC positive). The parameters are: (A) CAP OVM sIgE (UA / mL), (B) DCP OVM sIgE (BUe / mL), (C) DCP OVM sIgE / DCP OVMsIgE IC50 [(BUe / mL) / (nM)], (D) DCP OVM sIgE IC50 (nM), and (E) 1 / DCP OVM sIgE IC50 (nM).

[0038] Figure 8 The results of ROC analysis of 256 infants aged 1 to 3 years old judged as grade 2 or higher by the ImmunoCAP method are shown, using five different parameters, CAPOVM sIgE (UA / mL), DCP OVM sIgE (BUe / mL), DCP OVM sIgE / DCP OVM sIgE IC50 [(BUe / mL) / (nM)], DCP OVM sIgE IC50 (nM), and 1 / DCP OVM sIgE IC50 (nM), based on the diagnosis of anaphylaxis based on OFC, between subjects diagnosed as anaphylaxis (AN) onset (+) and subjects diagnosed as AN non-onset (-). DETAILED DESCRIPTION

[0039] The method of collecting data for predicting the risk of anaphylaxis in infants / children with food allergies according to the present invention is not particularly limited as long as it is a method that uses a critical value determined by ROC analysis based on the affinity of IgE antibodies for allergens collected from samples of infants / children with food allergies as a judgment standard; as the critical value determined by ROC analysis based on the affinity of IgE antibodies for allergens, there can be cited a critical value determined by ROC analysis with a value obtained by multiplying the antibody titer of IgE antibodies for allergens by the affinity of IgE antibodies for allergens as a parameter, and a critical value determined by ROC analysis with the IC50 of IgE antibodies as a parameter, and these two critical values ​​can also be used for judgment. In addition, the method of the present invention also includes a case where it is performed non-medically, that is, a method for collecting data non-medically, and also includes a method for collecting data for predicting the risk of anaphylaxis in infants / children with food allergies other than physician behavior. Examples of the food allergy include allergic reactions caused by specific foods that can serve as allergens, and examples of anaphylaxis include hypersensitivity reactions that are particularly severe among allergic reactions and can threaten life.

[0040] The above-mentioned allergens refer to antigens that cause allergen sensitization or induce allergic reactions or even systemic allergic reactions. Specifically, as long as it is any antigen protein or peptide, other sugar chain, nucleic acid, etc. that can induce the production of IgE antibodies in the human body, there is no special limitation. Examples include eggs, milk, meat such as beef, fish such as salmon and tuna, shellfish and mollusks such as shrimp and crab, grains, beans and nuts, fruits, vegetables, brewer's yeast, gelatin, etc., which are food allergens. Among them, the main allergens include: αs1-casein, αs2-casein, β-casein, etc. Milk allergens such as protein, κ-casein, α-lactalbumin, β-lactoglobulin (BLG) which is the main component of whey allergens, ovomucoid (OVM), ovalbumin (OVA), conalbumin or egg white allergens (EW) as a mixture of these, egg yolk allergens and egg allergens mixed with these, wheat allergens such as gliadin and gluten, buckwheat allergens, peanut allergens such as Arah1, sesame allergens such as 11S globulin, and shellfish allergens such as tropomyosin.

[0041] The above-mentioned allergen sensitization refers to a state in which when an allergen enters the body, the immune function that regards it as a foreign body and attempts to eliminate it takes effect, and IgE antibodies that recognize and bind to the allergen are produced. This state is called sensitization to the allergen or the occurrence of allergen sensitization, but allergic reaction does not necessarily occur only through allergen sensitization.

[0042] Examples of the allergic reaction include a reaction in which, when allergens invade again after sensitization to the allergens, IgE antibodies on mast cells bind to the allergens, causing mast cells to release chemical transmitters such as histamine. Symptoms caused by the allergic reaction are called allergic symptoms.

[0043] Examples of the allergic symptoms include: skin symptoms such as itching, urticaria, angioedema, redness, and eczema; eye symptoms such as conjunctival congestion / edema, itching, tearing, and eyelid edema; nasal symptoms such as sneezing, runny nose, and nasal congestion; oral and throat symptoms such as discomfort / swelling of the mouth / lips / tongue, and itchy / swollen throat; digestive system symptoms such as abdominal pain, nausea, vomiting, diarrhea, and bloody stools; respiratory system symptoms such as a feeling of throat constriction, throat edema, hoarseness, coughing, wheezing, dyspnea, and asthma; systemic symptoms such as anaphylactic shock, tachycardia, collapse, impaired consciousness, and decreased blood pressure.

[0044] The sample in the present invention is not particularly limited as long as it is a sample collected from infants / children suffering from allergies, and examples include body fluids such as blood, serum, plasma, saliva, tears, mucus, urine, etc. collected from patients, preferably serum and plasma. When serum is used as a sample, for example, blood can be collected from the patient's brachial vein, and the obtained blood is allowed to stand at 4°C for one night, then centrifuged, and the supernatant is used as serum. Alternatively, a trace amount of 50 to 100 μL of blood obtained by puncturing the ear or fingertips in a minimally invasive manner with a micro-blood collection needle or the like can be collected with a microcapillary tube and used directly.

[0045] The above-mentioned infants / children suffering from food allergies are infants / children aged 1 year and 0 months (1 year) to 12 years old, preferably infants / children aged 1 to 6 years old, and more preferably infants / children aged 1 to 3 years old, and include infants / children who clearly show allergic symptoms due to ingestion of food allergens, or infants / children who are determined to be allergy-positive and suffer from food allergies by known methods. In addition, it also includes infants / children who are obviously sensitized to allergens and may develop allergies but do not show allergic symptoms, and may include infants / children determined to be non-allergy-positive by OFC, which is a previous method for determining food allergies.

[0046] Specific methods for determining whether the infants and children suffer from food allergies include skin prick tests, basophil activation tests, known IgE detection methods, and OFC, and determining whether or not the infants and children suffer from allergies based on the criteria of each method.

[0047] [Determination of IgE antibody titer]

[0048] As a method for quantitatively measuring the antibody titer of the above-mentioned IgE antibody, including the above-mentioned known IgE detection method, as long as it is a method that can detect the IgE antibody for allergens in the sample and quantitatively measure the IgE antibody titer, it is not particularly limited, and preferably the ELISA method using labeled secondary antibodies performed on a carrier can be cited. As the carrier in the ELISA method, commercially available ELISA chips, chips with carbon layers formed on the carrier surface because of the less nonspecific adsorption, chips with chemically modified groups introduced because of the fixation of peptides, or chips activated, preferably DCP chips can be cited. In addition, as the above-mentioned known IgE detection method, DCP method, ImmunoCAP method, Alastat3g, ImmunoFastCheck, Oriton IgE, etc. using the above-mentioned DCP chip can be cited. In addition, when calculating the IgE antibody titer, from the perspective of measuring with higher accuracy within the detection line accuracy range, it is preferred to dilute a blood sample such as serum by 1 to several dozen times, preferably by 1.1 to 10 times, and more preferably by 1.5 to 5 times, and then quantify the IgE antibody titer. In this case, after measuring the IgE antibody titer by the DCP method using a DCP chip, etc., it is necessary to multiply the dilution factor of the blood sample by the dilution factor to finally determine the IgE antibody titer.

[0049] [DCP method]

[0050] In the above-mentioned DCP method, as a method for determining whether the target infant / child suffers from allergies, the determination can be made based on the degree of IgE antibody production in the blood sample of the target infant / child, i.e., the IgE antibody titer. Specifically, although the numerical value varies depending on the allergen, if ovomucin (OVM) is taken as an example, a method can be cited in which the target infant / child is determined to suffer from allergies when the numerical value is above 800BUe / mL, preferably above 1000BUe / mL.

[0051] As the above-mentioned DCP method, there can be cited a method of determining IgE antibodies by ELISA method using a DCP chip, and as the above-mentioned DCP chip, there can be cited: a chip obtained by applying an electrostatic layer treated with an amino compound or its polymer and / or copolymer on the surface of a substrate after a carbon layer is treated with DLC (diamond-like carbon) on the surface of a silicon substrate, or on the surface of a glass slide, and then treating it again with a dicarboxylic acid or a polycarboxylic acid, and then activating it with N-hydroxysuccinimide and / or carbodiimide; or a chip after chemical modification groups are introduced into the carrier surface or the above-mentioned carbon layer; or a chip after further activation treatment, etc.

[0052] As the above-mentioned labeled secondary antibody, there can be cited fluorescent labeled secondary antibodies such as HiLyte Fluor 555, Atto 532, Cy3, Alexa Fluor555, 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. As the above-mentioned secondary antibody, Fab fragments and F(ab')2 fragments of antibodies can also be used. Fab fragments can be prepared by treating antibodies with papain, etc., and F(ab')2 fragments can be prepared by treating antibodies with pepsin, etc.

[0053] [ImmunoCAP method]

[0054] In the above-mentioned ImmunoCAP method, as a method for determining whether the target infant / child suffers from allergies, the following method can be cited: classification is performed, and those with an IgE antibody titer of less than 0.35UA / mL are set as level 0, those with an IgE antibody titer of 0.35-0.69UA / mL are set as level 1, those with an IgE antibody titer of 0.70-3.49UA / mL are set as level 2, those with an IgE antibody titer of 3.50-17.49UA / mL are set as level 3, those with an IgE antibody titer of 17.50-49.99UA / mL are set as level 4, those with an IgE antibody titer of 50.00-99.99UA / mL are set as level 5, and those with an IgE antibody titer of 100UA / mL or more are set as level 6; level 0 is determined to be negative, with no allergen sensitization; level 1 is determined to be a false positive, with allergen sensitization but no allergic symptoms, and levels 2 and above are determined to be positive. The IgE antibody titer unit "UA / mL" is a unit of IgE antibody titer produced against allergens that was originally established by Phadia, the company that developed the ImmunoCAP method. In terms of quantity, it is the same as IU / mL.

[0055] [OFC]

[0056] According to the definition in the "Summary of the Guidelines for the Diagnosis and Treatment of Food Allergies 2016, Chapter 7" (https: / / www.jspaci.jp / allergy_2016 / chap07.html) of the Food Allergy Committee of the Society of Pediatric Allergy, the above OFC refers to a test to confirm the presence or absence of symptoms by taking a food that is known or suspected to cause allergies once or in multiple doses. The total amount of the target food taken in a single dose or in multiple doses is called the total load. Examples of the intake interval and divided method include a plan of gradually increasing the intake amount in 1 to 5 times with an intake interval of 20 to 60 minutes within 1 to 2 hours. For example, in the case of 2 times, 1 / 4 of the total intake amount (first time), 3 / 4 of the total intake amount (second time), or 1 / 3 or 2 / 3 of the total intake amount are taken every 60 minutes; in the case of 3 times, 1 / 8, 3 / 8, 1 / 2 are taken every 30 to 60 minutes; in the case of 5 times, 1 / 16, 1 / 16, 1 / 8, 1 / 4, 1 / 2 are taken every 20 to 40 minutes; in the case of 6 times, 2 / 100, 4 / 100, 8 / 100, 16 / 100, 32 / 100, 38 / 100 are taken, etc. However, for infants and young children, although the presence of a doctor is a prerequisite, since it is a test with a high possibility of systemic allergic reaction, it is preferable to increase the number of times, such as 5 or 6 times or more, and gradually increase the loading amount for the sake of caution.

[0057] Examples of the above-mentioned total load include: in the case of eggs, 1 / 2 egg for a one-year-old child and 1 egg for a child over 2 years old; in the case of milk, 50 ml for a one-year-old child and 200 ml for a child over 2 years old; in the case of wheat, 50 g of noodles and 200 g for a child over 2 years old.

[0058] In the above OFC, the criteria for judging as allergy positive are as follows.

[0059] (A) Within minutes or hours after the onset of allergic load

[0060] Skin symptoms—pruritus, urticaria, erythema, angioedema; and

[0061] Mucosal symptoms—conjunctival congestion, runny nose, sneezing, pharyngeal discomfort; one or more of the following

[0062] (B) Within minutes or hours after the onset of allergic load

[0063] Respiratory symptoms—cough, wheezing;

[0064] Digestive symptoms—abdominal pain, vomiting, diarrhea; and

[0065] Systemic symptoms - pallor, low blood pressure, decreased level of consciousness; more than one of the following

[0066] (C) Within minutes or hours after the onset of allergic load

[0067] Reduce systolic blood pressure by more than 30% for the child's age

[0068] When one or more of the above (A), (B), and (C) are satisfied, the patient is determined to be positive for the specific allergen.

[0069] In addition, the criteria for judging the occurrence of anaphylaxis are as follows.

[0070] “1 Skin or mucous membrane symptoms (e.g., generalized urticaria, itching, flushing, swelling of lips / tongue / uvula) occur within minutes or hours after the onset of allergen loading and one or more of the following A) or B) is met.

[0071] A) Respiratory disorders (e.g., dyspnea, expiratory wheezing, inspiratory wheezing, decreased PEF (peak expiratory flow), hypoxia)

[0072] B) Decreased blood pressure or related organ failure (e.g., hypotension, collapse, syncope, incontinence)

[0073] 2 Two or more of the following symptoms C) to F) appear within minutes or hours after the onset of allergen exposure

[0074] C) Skin or mucous membrane symptoms (e.g., generalized urticaria, itching, flushing, swelling of lips / tongue / uvula)

[0075] D) Respiratory disorders (e.g., dyspnea, expiratory wheezing, inspiratory wheezing, decreased PEF (peak expiratory flow), hypoxia)

[0076] E) Decreased blood pressure or related organ failure (e.g., hypotension, collapse, syncope, incontinence)

[0077] F) Persistent gastrointestinal symptoms (e.g. colic, vomiting)"

[0078] The judgment criteria used in the present invention for predicting the risk of anaphylaxis in infants / children suffering from food allergies are not particularly limited as long as they are criteria made using the antibody titer of IgE antibodies for allergens and the affinity of IgE antibodies for allergens, but it is preferred to use a value obtained by multiplying the antibody titer of IgE antibodies by the affinity of IgE antibodies (1 / IC50).

[0079] [Calculation of IgE antibody affinity for allergens]

[0080] As the value of the affinity of the IgE antibody for the allergen in the present invention, the value of the IgE antibody quantified as IC50 or 1 / IC50 can be cited. The IC50 value is sometimes also referred to as the competitive binding inhibition activity value, and can be obtained by calculating the value representing the allergen concentration that inhibits the antigen-antibody binding reaction by 50%, for example, as the competitive binding inhibition activity between the solid-phase allergen and the soluble allergen. According to this calculation method, it is preferable that the IC50 value can be evaluated even if there are IgG, IgA, IgD and / or IgM antibodies other than IgE that recognize the same antigen.

[0081] The method for calculating the IC50 value of the above-mentioned IgE antibody is not particularly limited as long as it is based on a method that can perform a competitive binding assay for the IgE antibody, and examples thereof include a measurement method based on a biochemical assay such as ELISA. For monoclonal antibodies and purified IgE antibodies, an affinity measurement method based on Biacore, which can measure the degree of interaction using a surface plasmon resonance technique, and isothermal titration calorimetry can also be mentioned.

[0082] As a method for calculating the IC50 value by the above-mentioned ELISA, there can be cited a method of measuring the affinity of IgE antibodies for allergens using a protein modifier as a chaotropic agent that reduces the intermolecular attraction between antigen and antibody, or a method of measuring by competitive ELISA. Since the chaotropic agent may reduce the intermolecular attraction between not only the antibody but also the antibody and the antigen, and since the antigen-antibody binding inhibition effect varies even if the concentration of the chaotropic agent is the same, depending on the dilution factor of the serum used as the sample, it cannot be said that the reproducibility is high. On the other hand, the determination of the IC50 value by the competitive ELISA using the competitive binding inhibition reaction method with the addition of various concentrations of allergens is highly reproducible because the IC50 value varies less depending on the dilution factor of the sample, and the IC50 values ​​of different allergens can be compared with each other, and the total binding activity of the antibody to the allergen can be quantitatively compared, so it is preferred in this regard. Therefore, it was confirmed that when calculating the IC50 value, even in the case of diluting a blood sample such as plasma or serum, it is not necessary to multiply the dilution factor of the sample. An example of the specific procedure of ELISA using the competitive binding inhibition reaction method is shown below.

[0083] (Preliminary reaction)

[0084] To a sample containing IgE antibodies that bind to antigen-specific allergens, the allergens are added at known concentrations that are adjusted in stages as competitive binding inhibitors, and the mixture is allowed to react for a certain period of time. The allergens at known concentrations that are adjusted in stages are adjusted in stages from a concentration of 0 (the antigen that serves as a competitive binding inhibitor is not present in the sample) to a concentration sufficient for the allergen to bind to all IgE antibodies within a certain period of time.

[0085] As a certain time in the above-mentioned pre-stage reaction, for example, 15 minutes to 2 hours, preferably 30 minutes to 1 hour can be cited. In addition, as the above-mentioned known concentration that has been modulated in stages, a concentration appropriately selected by a person skilled in the art according to the amount of antibodies present in the sample, etc., can be used. For example, as an example of a staged allergen concentration (final concentration) added to serum or plasma for competitive inhibition, a combination of 0nM, 0.1nM, 1.0nM, 10nM, 100nM, 1000nM, or a combination of 0nM, 0.1nM, 1.0nM, 10nM, 100nM, 200nM, etc. can be cited, but it is not limited to these concentrations. In addition, for allergens with a clear molecular weight, it is preferred to perform ELISA using a competitive binding inhibition reaction method, but for allergens with an unknown molecular weight and the allergen concentration cannot be adjusted by molar concentration, the above-mentioned staged allergen concentration can be adjusted by units such as mg / mL and μg / mL.

[0086] (Competitive binding inhibition reaction)

[0087] The solution after the above-mentioned previous stage reaction is provided to a carrier on which the allergen is immobilized, and the free IgE antibody (primary antibody) in the solution after the above-mentioned previous stage reaction is reacted by binding with the immobilized allergen, and then after the primary antibody is removed and washed, it is reacted with the labeled secondary antibody, and the binding amount of the bound IgE antibody is calculated.

[0088] The above-mentioned allergen concentration that inhibits the antigen-antibody binding reaction by 50%, namely "IC50", can be expressed as the antigen concentration when the above-mentioned labeling amount is 50% (half maximal inhibitory concentration: 50% inhibitory concentration) when the labeling amount of the labeled secondary antibody detected in the solution with a concentration of 0 in the absence of the competing allergen in the above-mentioned previous stage reaction is set to 100%.

[0089] As described above, the affinity of IgE antibodies for allergens can be expressed based on the IC50 value, using "1 / IC50" which is the reciprocal of the 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 IgE primary antibody present in the solution after the previous stage reaction is greater, so the amount of antibody bound to the solidified allergen becomes greater, and the IC50 value is greater. On the other hand, when the affinity of the IgE antibody in the sample is higher, the amount of primary antibody present in the solution after the previous stage reaction is less, so the amount of IgE antibody bound to the solidified allergen becomes less, and the IC50 value is smaller. Therefore, in order to set the affinity to a lower value when the affinity of the IgE antibody is lower and to set the affinity to a higher value when the affinity of the IgE antibody is higher, the reciprocal of the IC50 value is sometimes used for convenience. In particular, when evaluating the IgE antibody titer by multiplying the antigen affinity (affinity) of IgE, the reciprocal of the IC50 value is used.

[0090] As the labeled secondary antibody for calculating affinity, there can be cited fluorescent labeled secondary antibodies such as HiLyte Fluor 555, Atto532, 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, etc. In addition, as the above-mentioned secondary antibody, Fab fragments and F(ab')2 fragments of antibodies can also be used. Fab fragments can be obtained by treating antibodies with papain, etc., and F(ab')2 fragments can be obtained by treating antibodies with pepsin, etc.

[0091] Examples of the carrier with high binding sensitivity to the above-mentioned allergen immobilized thereon include commercially available ELISA chips, chips having a carbon layer formed on the carrier surface due to their advantage in terms of less nonspecific adsorption, chips having chemical modification groups introduced therein to facilitate peptide fixation, and chips subjected to activation treatment. Among these, the DCP chip is preferred.

[0092] As the above-mentioned chip with allergens immobilized thereon and the quantitative measurement method of IgE antibodies using the same, known chips and methods can be used, for example, a method that can include using the chips described in Japanese Patent Publication Nos. 2006-267058, 2006-267063 and 2015-169616, i.e., the above-mentioned DCP chip.

[0093] [ROC analysis]

[0094] The criterion prepared according to the antibody titer of IgE antibody and the affinity of IgE antibody for allergen in the present invention is not particularly limited as long as it is a criterion that can predict the risk of anaphylaxis in infants / children. Preferably, prediction is performed using a critical value determined by ROC analysis using "a value obtained by multiplying the antibody titer of IgE antibody by the affinity of IgE antibody for allergen" as a parameter.

[0095] The ROC is a term translated as receiver operating characteristic or receiver action characteristic, and is a method for comparing the predictive ability of an independent variable with respect to an output quantity as a binary variable or for setting a critical value of an independent variable with respect to an output quantity.

[0096] In the present invention, as a method for comparing the predictive ability of the independent variable to the output as a binary variable, for example, the following method can be cited: if the setting of the threshold value of anaphylaxis is changed in each of n different parameters, the true positive rate and false positive rate for anaphylaxis-positive persons will change accordingly. When the true positive rate and the false positive rate are plotted with the true positive rate as the vertical axis and the false positive rate as the horizontal axis to prepare n types of ROC curves, the larger the area under the curve (AUC), the better the parameter is, and the higher the predictive ability is evaluated.

[0097] As a method for preparing the above-mentioned ROC curve, the following method can be cited: when a certain parameter is selected, for example, when an arbitrary threshold value is set for the anaphylaxis positive value in the subject classification, if the case where the anaphylaxis positive subject is correctly classified as Y is represented as TY, the case where the anaphylaxis positive subject is incorrectly classified as Y is represented as FY, the case where the anaphylaxis negative subject is correctly classified as N is represented as TN, and the case where the anaphylaxis negative subject is incorrectly classified as N is represented as FN, the true positive rate (sensitivity) when the positive subject is correctly classified as positive is set to TY / (TY+FN), and the false positive rate (1-specificity) when the negative subject is classified as positive is set to FY / (FY+TN), then for a certain threshold value, (true positive rate, false positive rate), that is, (sensitivity, 1-specificity) can be plotted, and further, the ROC curve can be prepared by setting various threshold values ​​and performing a large number of plots.

[0098] The method for setting the critical value in the above-mentioned ROC analysis is not particularly limited as long as it is a method that can improve the predictive ability. An example of a method is to determine the maximum value of sensitivity-(1-specificity) as the critical value. The setting of the critical value can also be determined by a known order using the ROC analysis equipment or commercially available software shown below.

[0099] ROC analysis can also be performed using known commercially available analysis equipment, such as GraphPad Prismver.5.4 (GraphPad) and JMP14 (jmp.Statistical Discovery. TM ) and other software.

[0100] Hereinafter, the present invention will be described in more detail by way of examples, but the technical scope of the present invention is not limited to these examples.

[0101] Example

[0102] [Example 1]

[0103] (sample)

[0104] The studies in this example were approved by the Ethics Committee of the University of Tokushima (#175-4) and were conducted between August 2012 and January 2019 at the University of Tokushima, the Health Insurance Naruto Hospital of the University of Tokushima-related hospitals, and the National Hospital Organization Mie Hospital.

[0105] The study was conducted on infants and young children aged 1 year 0 months to 6 years who were confirmed to be sensitized or allergic to egg allergens, or suspected to be allergic to eggs, with full informed consent from both parents or one parent. In addition, OFC was conducted in accordance with the Food Allergy Committee of the Japanese Society of Child Allergy, the Food Allergy Diagnosis and Treatment Guidelines 2016, and the Kyowa Planning Guidelines.

[0106] The selection criteria for the test infants and young children are as follows.

[0107] (1) Infants and young children who have been confirmed to be sensitized to egg allergens and have been on an egg-free diet for more than 6 months before OFC.

[0108] (However, this does not apply to infants and young children who have received heated egg OFC for diagnostic purposes within 3 months before OFC.)

[0109] (2) Infants and young children who have been confirmed to be sensitized to egg allergens and whose ovomucoid-specific IgE levels have been measured by the ImmunoCAP method or the DCP method in a blood test immediately before or within 4 months of OFC.

[0110] Based on this criterion, 472 people were selected.

[0111] (Allergen Selection)

[0112] Ovalbumin, a component of egg, is known to lose its allergen activity when heated. Therefore, in this study, by ingesting heated egg powder in OFC, ovomucoid (OVM), which does not lose its allergen activity even when heated, was used as an indicator to examine whether infants and young children with egg allergy had systemic anaphylaxis.

[0113] For the above 472 test infants, serum collected from all subjects from 4 weeks before OFC to the day of OFC implementation was stored at less than -30°C and not frozen and thawed until analysis. Before OFC, the production of IgE antibodies against ovomucoid (OVM) was measured by blood test using the commonly used ImmunoCAP method (Phadia), and it was divided into levels 0 to 6. In the present invention, since the purpose is to verify the usefulness of IgE antibody affinity measurement in the diagnosis of anaphylaxis, the negative level 0 and false positive level 1, whose IgE antibody titer is low and affinity is difficult to measure, were excluded, and OFC was performed on 343 infants and young children of level 2 or above who were judged to be sensitized to the allergen of ovomucoid and whose affinity value can calculate the allergen concentration that inhibits the antigen-antibody binding reaction by 50%.

[0114] (Implementation of OFC)

[0115] In the above-mentioned ImmunoCAP method, samples diagnosed with egg allergy were used for affinity determination by conducting OFC on 343 infants and young children of grade 2 or above who were determined to be positive for allergen sensitization to OVM. OFC was implemented based on the methods described in Furuya K et al., Allergy. 2016; 71: 1435-1443 and the Food Allergy Committee of the Japanese Society of Child Allergy, Food Allergy Diagnosis and Treatment Guidelines 2016, and Kyowa Planning Guidelines.

[0116] Specifically, the test infants and young children were divided into a 1-year-old group and a 2- to 6-year-old group according to the age-determined intake. The total egg load in the OFC for 1-year-old children was 6.5 g of heated egg powder (Kewpie Co., Ltd.) (equivalent to 1 / 2 egg). The total egg load in the OFC for 2- to 6-year-old children was 13 g of heated egg powder (equivalent to one egg).

[0117] The total load was divided into 6 stages: 2 / 100, 4 / 100, 8 / 100, 16 / 100, 32 / 100, and 38 / 100. The test was conducted by gradually increasing the load and letting the test infants and young children take heated egg powder 6 times at intervals of 15 to 30 minutes. That is, 1-year-old children took heated egg powder 6 times in the order of 0.13 g, 0.26 g, 0.52 g, 1.04 g, 2.08 g, and 2.47 g at intervals of 15 to 30 minutes. Children aged 2 to 6 years took heated egg powder 6 times in the order of 0.26 g, 0.52 g, 1.04 g, 2.08 g, 4.16 g, and 4.94 g at intervals of 15 to 30 minutes.

[0118] In order to prevent the egg from being detected and to correct the difference in the amount of intake per intake, the heated egg powder was provided to each infant as a mixed powder with pumpkin or sweet potato powder added.

[0119] If the infants and young children who ingested the heated egg powder met the following criteria, they were determined to be positive for egg allergy in OFC. If they did not meet the following criteria, they were determined to be negative for egg allergy.

[0120] (Criteria for determining positive allergy test)

[0121] (A) Within a few minutes or hours after the start of heating the egg powder load,

[0122] Skin symptoms—pruritus, urticaria, erythema, angioedema; and

[0123] Mucosal symptoms—conjunctival congestion, runny nose, sneezing, pharyngeal discomfort; one or more of the following

[0124] (B) within a few minutes or hours after the start of heating the egg powder load,

[0125] Respiratory symptoms—cough, wheezing;

[0126] Digestive symptoms—abdominal pain, vomiting, diarrhea; and

[0127] Systemic symptoms - pallor, low blood pressure, decreased level of consciousness; more than one of the following

[0128] (C) Within a few minutes or hours after the start of the egg powder load heating

[0129] Reduce systolic blood pressure by more than 30% for the child's age

[0130] When one or more of the above (A), (B), and (C) are met, the egg allergy is determined to be positive.

[0131] (Criteria for determining the onset of anaphylaxis)

[0132] According to the World Allergy Organization Anaphylaxis Guidelines (World Allergy Orhan J. 2011; 4: 13-37), anaphylaxis is considered to have occurred when any one or more of the following criteria 1 to 3 are met.

[0133] 1. Skin or mucous membrane symptoms (e.g., generalized urticaria, itching, flushing, swelling of lips / tongue / uvula) occur within minutes or hours after the start of the heated egg powder load and one or more of the following A) or B) is met.

[0134] A) Respiratory disorders (e.g., dyspnea, expiratory wheezing, inspiratory wheezing, decreased PEF (peak expiratory flow), hypoxia)

[0135] B) Decreased blood pressure or related organ failure (e.g., hypotension, collapse, syncope, incontinence)

[0136] 2 Two or more of the following symptoms C) to F) appear within a few minutes or hours after the start of the egg powder heating load

[0137] C) Skin or mucous membrane symptoms (e.g., generalized urticaria, itching, flushing, swelling of lips / tongue / uvula)

[0138] D) Respiratory disorders (e.g., dyspnea, expiratory wheezing, inspiratory wheezing, decreased PEF (peak expiratory flow), hypoxia)

[0139] E) Decreased blood pressure or related organ failure (e.g., hypotension, collapse, syncope, incontinence)

[0140] F) Persistent gastrointestinal symptoms (e.g. colic, vomiting)

[0141] 3. When a decrease in systolic blood pressure corresponding to age occurs within a few minutes or hours after the start of the heated egg powder load, or when systolic blood pressure decreases by more than 30%.

[0142] Infants and young children who were judged as positive in the above OFC were diagnosed as having egg allergy, and infants and young children who were judged as negative in the OFC were diagnosed as not having egg allergy. In addition, among the test infants and young children diagnosed as having egg allergy, infants and young children who showed systemic anaphylaxis were called systemic anaphylaxis (AN+), and infants and young children who did not show systemic anaphylaxis were called non-systemic anaphylaxis (AN-). In addition, for the test infants and young children, the level of classification by blood test using the ImmunoCAP method performed before the implementation of OFC was also recorded. The results are shown in Table 1.

[0143] [Table 1]

[0144] Patient background: Level 2-6, 1-6 years old (343 cases)

[0145]

[0146] (M: boy, F: girl. The upper part (%) indicates the ratio to the total 343 cases, and the lower part (%) indicates the ratio to the 204 cases with egg allergy.)

[0147] (Result 1)

[0148] As is clear from Table 1, among the 343 infants aged 1 to 6 years who underwent OFC using heated egg powder, 204 were determined to be OFC positive and egg allergic. On the other hand, 139 were determined to be OFC negative and not egg allergic. Furthermore, among the 204 infants determined to be egg allergic, 38 were determined to have anaphylaxis, and 166 were determined not to have anaphylaxis.

[0149] Therefore, among infants aged 1 to 6 years who underwent OFC, 59.5% were determined to be allergic to egg, and 40.5% were determined to be non-egg allergic. It should be noted that in the ImmunoCAP method performed in the previous stage, the male-female ratio of infants and young children who were determined to be positive for ovomucoid sensitization and classified as grades 2 to 6 was 66.5% for boys and 33.5% for girls, with boys showing significantly higher values. However, in the definitive diagnosis of egg allergy performed by OFC, no significant difference was seen in the male-female ratio of those with egg allergy, those without egg allergy, those with egg allergy and AN+, and those with egg allergy and AN-. In addition, among infants and young children determined to be allergic to egg, 18.6% were AN+ infants and young children who showed severe symptoms. Although it was confirmed that they were allergic to egg, the allergic symptoms of infants and young children with AN- were not a hypersensitivity state that could endanger their lives.

[0150] (Result 2)

[0151] Table 2 shows only the data of the above-mentioned infants and young children aged 1 to 3 years, whose allergy diagnosis is difficult in daily diagnosis and treatment due to their underdeveloped immune system.

[0152] [Table 2]

[0153] Patient background: Level 2-6, 1-3 years old (256 cases)

[0154]

[0155] (AN+: with systemic allergic reaction, AN-: without systemic allergic reaction, M: boy, F: girl. The upper part (%) shows the proportion relative to the total 256 cases investigated, and the lower part (%) shows the proportion relative to the 147 cases diagnosed with egg allergy.)

[0156] As is clear from Table 2, among the 256 infants aged 1 to 3 years who underwent OFC using heated egg powder, 147 were determined to be OFC positive and egg allergic. On the other hand, 109 were determined to be OFC negative and not egg allergic. Furthermore, among the 147 infants determined to be egg allergic, 28 were determined to have anaphylaxis, and 119 were determined not to have anaphylaxis.

[0157] Therefore, among infants and young children aged 1 to 3 years who underwent OFC, 57.4% were determined to be allergic to egg, and 42.6% were determined to be non-egg allergic. In addition, among infants and young children determined to be allergic to egg, 19.0% of them were AN+ with severe symptoms. Although it was confirmed that they were allergic to egg, the allergic symptoms of infants and young children with AN- were not a hypersensitivity state that could endanger their lives. In summary, it was confirmed that among infants and young children aged 1 to 3 years who were confirmed to be allergic to egg, the proportion of infants and young children who developed systemic anaphylaxis among infants and young children with immature immune systems was not significantly different from that of infants and young children aged 1 to 6 years.

[0158] [Example 2]

[0159] [Determination of OVM-specific IgE antibody titer using the DCP method]

[0160] For the above 343 infants aged 1 to 6 years, serum samples collected before OFC were used as antigenic proteins, and OVM (manufactured by Sigma) with a molecular weight known as a clear allergen contained in eggs was immobilized on a DCP chip as an antigen, and the amount of IgE antibody bound to OVM was measured as the OVM-specific IgE antibody titer. It should be noted that when quantifying the IgE antibody titer, a fluorescence intensity calibration curve based on a known concentration of human standard IgE antibody (75 / 502) purchased from the National Institute for Biological Standards and Control (Hertfordshire, England) was prepared, and the OVM-specific IgE antibody titer (BUe / mL) was measured using a DCP chip.

[0161] [Manufacturing of OVM-cured DCP chips]

[0162] (Chip activation)

[0163] The DCP chip composed of a glass substrate was provided with an amino group-containing electrostatic layer and a carboxyl group with negative charge from polyacrylic acid was introduced into the substrate. The substrate was activated in a chemical crosslinking agent (100mM WSC·HCl, 100mM NHS, 0.1M potassium phosphate buffer (pH6.0)) at room temperature under light shielding while shaking for 30 minutes. After the chemical crosslinking agent after the reaction was discarded, the substrate was washed twice for 1 minute with MilliQ water while shaking, and then immediately centrifuged using a desktop centrifuge (Allegra). TM X-22R Centrifuge, manufactured by Beckman Coulter, Inc.) to remove moisture and prepare an activated chip.

[0164] (Coupling reaction of OVM)

[0165] OVM (manufactured by Sigma) as an antigen protein was dissolved in a solution added with 5% to 30% DMSO or 5% to 30% PEG300 at a concentration of 0.2 to 2.0 mg / mL to obtain an antigen protein solution. The obtained antigen protein solution was dispensed into a flat-bottomed 384-well plate (manufactured by Corning), and a 4 nL spot was formed on the above-mentioned activation chip by a microarray production device (OmniGridAccent, manufactured by DIGILAB), and then dried at 15°C to 30°C for 1 to 18 hours to solidify the OVM antigen protein. Figure 2 A schematic diagram of a DCP chip with OVM cured is shown.

[0166] (Blocking reaction of unreacted active groups)

[0167] For the above-mentioned chip that solidifies OVM as antigen protein, add blocking reagent Blockmaster (JSR company) to the reaction well groove (hole), leave it to stand in refrigeration (4 ℃) under light shielding, and react overnight. After removing the above-mentioned blocking reagent by suction with an aspirator (VARIABLE SPEED PUMP, manufactured by BIORAD), move it to the reaction plate again, add 10mL cleaning solution (50mM TTBS), shake for 5 minutes, and then remove the cleaning solution by suction with an aspirator. After washing 3 times in the same way, wash it 3 times with pure water (MilliQ water). Use a centrifuge (Allegra (trademark), X-22R Centrifuge (manufactured by BECKMAN COULTER)) to perform centrifugal water droplet removal operation (1 minute at 2000rpm) to remove water droplets on the chip surface to obtain an OVM solidified DCP chip.

[0168] (Primary Antibody Reaction)

[0169] The serum samples of each tested infant were appropriately diluted with sample diluent (20 mM phosphate buffer, pH 7.4 / 0.3 M KCl / 0.05% Tween 20) to prepare serum dilution solution (primary antibody solution), 10 μL of the diluted sample was added to the reaction well, and the solution was allowed to stand at 37°C for 2 hours in the dark.

[0170] (Reaction with secondary antibody)

[0171] After removing the diluted sample obtained by the above operation by suction with an aspirator (VARIABLE SPEED PUMP, manufactured by BIORAD), move the chip to a cleaning box, add 10 mL of cleaning solution (TTBS: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.95% Tween 20), and then use Double-Shaker NR3 to repeat the cleaning operation 3 times in 5 minutes for cleaning, and then add purified water (MilliQ water) and wash 3 times in 1 minute. Use the above centrifuge to remove centrifugal droplets (at 2000 rpm for 1 minute) to remove the droplets on the surface of the chip. Next, prepare a fluorescently labeled secondary antibody (HiLyteFluor (trademark) or 555connected antibody human IgE (HyTest) (diluent, IMMUNO SHOTPlatimun / 1% BSA, final dilution concentration 10μg / mL)) as a secondary antibody solution. 10 μL of the secondary antibody solution was dispensed into each reaction well on the slide glass, and the mixture was left to stand at 37° C. for 2 hours in the dark.

[0172] (Detection of primary antibody captured by antigen)

[0173] After removing the above-mentioned diluted secondary antibody solution by suction with an aspirator, place the chip in a cleaning box and use Double-Shaker NR3 (manufactured by TAITEC) to repeat the cleaning operation 3 times in 5 minutes. Then, add purified water (MilliQ water) and rinse 3 times in 1 minute, remove water droplets and dry with the above-mentioned centrifuge. Use a fluorescence scanner (3D GeneScanner, manufactured by Toray) to measure the fluorescence intensity (Ex: 532nm, Em: 570nm), and digitize the fluorescence intensity of the spots obtained from each chip. As the titer of IgE antibodies that bind to the antigen through antigen-antibody reaction, the measurement unit is expressed in binding units (BUe), and is measured and expressed based on the fluorescence intensity detection line of each standard antibody of known concentration solidified on the chip. The following expression is 1BUe=2.4ng.

[0174] [Example 3]

[0175] [Affinity measurement of OVM-specific IgE antibodies]

[0176] The affinity of OVM-specific IgE antibodies to OVM was measured using serum samples collected before OFC and OVM (manufactured by Sigma) as an antigen protein from the 343 infants aged 1 to 6 years.

[0177] Use OVM to cure the DCP chip and perform the following reactions.

[0178] (Primary Antibody Reaction)

[0179] The serum samples of each tested infant are appropriately diluted with a sample diluent (20mM phosphate buffer, pH7.4 / 0.3M KCl / 0.05% Tween20), for example, diluted 2.5 times, to prepare a serum dilution solution (primary antibody solution), and the antibody titer is measured. Next, the antibody titer of the obtained dilution is readjusted so that the competitive binding inhibition activity (affinity) dependent on the amount of soluble antigen added is within the detectable range. Specifically, it is adjusted so that the lower limit value of the fluorescence intensity represented by OVM IgE> the measurement limit value (blank + 5SD), and the upper limit value of the fluorescence intensity is within the range of 5000 or less. In addition, 6 OVM solutions adjusted to 6 levels of concentration of 0, 0.2, 2.0, 20, 100 and 200nM with the above-mentioned sample diluent were prepared as competitive binding inhibitors. As for the IC50 value, when the antigen-antibody reaction between the IgE antibody contained in the measuring sample and the OVM antigen immobilized on the chip is carried out, the pre-stage reaction is to add various concentrations of soluble OVM, that is, the above 6 kinds of OVM solutions are added to the above serum dilution solution (6μL) in advance (a total of 12μL), and react for 30 minutes at 25°C. After the reaction, 10μL of each pre-stage reaction solution is collected, added to the OVM immobilized DCP chip, and left to stand at 37°C for 2 hours in the dark. It should be noted that the blank + 5SD above represents the lower limit of the detection limit. Specifically, the fluorescence intensity derived from the non-specific binding of the fluorescent-labeled secondary antibody contained in the reaction solution to the antigen under the condition of only buffer without sample serum is set as the blank value. As for the fluorescence intensity, the measurement is repeated 5 times and the standard deviation (SD) value of the fluorescence intensity is calculated, and the case of the fluorescence intensity above the blank + 5SD (lower limit of detection limit) is set as the fluorescence intensity of the IgE derived from the sample.

[0180] (Reaction with secondary antibody)

[0181] After removing the serum dilution solution reacted on the DCP chip by suction with an aspirator (VARIABLE SPEED PUMP, manufactured by BIORAD), the chip was moved to a cleaning box, 10 mL of cleaning solution (50 mM TTBS) was added, and then the Double-Shaker NR3 was used to repeat the cleaning operation 3 times in 5 minutes for cleaning, and then pure water (MilliQ water) was added to clean 3 times in 1 minute. The water droplets on the surface of the chip were removed by centrifugal removal of water droplets (at 2000 rpm for 1 minute) using the above-mentioned centrifuge. Next, a fluorescently labeled secondary antibody (HiLyte Fluor (trademark) or 555 connected antibody human IgE (HyTest) (diluent, IMMUNO SHOT Platimun / 1% BSA, final dilution concentration 10 μg / mL)) was prepared. 10 μL of the secondary antibody solution was dispensed into each reaction well slot on the slide and allowed to stand at 37°C for 2 hours in the dark.

[0182] Then, after suction removal with an aspirator, the chip is moved to a cleaning box, 10 mL of cleaning solution (50 mM TTBS) is added, and the cleaning operation is repeated 3 times in 5 minutes using Double-Shaker NR3. Then, pure water (MilliQ water) is added and washed 3 times in 1 minute. The water droplets on the surface of the chip are removed by centrifugal water drop removal (at 2000 rpm for 1 minute) using the above-mentioned centrifuge. The residual fluorescence amount is measured with a fluorescence scanner (3D Gene Scanner, manufactured by Toray) for fluorescence intensity (Ex: 532 nm, Em: 570 nm), thereby quantifying the fluorescence intensity of the spots obtained from each chip. For each tested infant / child, when the fluorescence intensity of the solution without OVM, that is, the concentration of the competitive binding inhibitor is 0, is set to 100%, the OVM concentration representing a fluorescence intensity of 50% is expressed as the "IC50 value".

[0183] [ROC analysis]

[0184] [Reference Example 1]

[0185] ROC analysis was performed on infants and young children aged 1 to 6 years (343 cases) who underwent OFC in [Example 1]. Based on the results that OFC-positive was diagnosed as egg allergy and OFC-negative was diagnosed as no egg allergy, ROC analysis was performed on the following five parameters using the OVM-specific IgE titer and affinity of OVM-specific IgE measured by the DCP method and the OVM-specific IgE titer measured by the ImmunoCAP method. The results are shown in Figure 3Subsequently, ROC analysis was performed using GraphPad Prism ver.5.4 (GraphPad), JMP14 (jmp.Statistical Discovery. TM )conduct.

[0186] (1) [DCP OVM sIgE / DCP OVMs IgE IC50 [(BUe / mL) / (nM)]] was obtained by multiplying the OVM-specific IgE titer measured by the DCP method by 1 / IC50 value;

[0187] (2) IC50 value of OVM-specific IgE measured by the DCP method (DCPOVM sIgE IC50 (nM));

[0188] (3) 1 / IC50 value of OVM-specific IgE measured by the DCP method (1 / DCP OVMs IgE IC50 (nM));

[0189] (4) OVM-specific IgE titer measured by the DCP method (DCP OVMs IgE (BUe / mL));

[0190] (5) OVM-specific IgE titer measured by the ImmunoCAP method (CAP OVMs IgE (UA / mL));

[0191] In addition, 343 infants aged 1 to 6 years were selected as subjects, and ROC analysis was performed for each of the five parameters for subjects diagnosed as positive allergy diagnoses and subjects diagnosed as negative allergy diagnoses based on OFC for allergy diagnosis of OVM, and AUC values, cutoff values, sensitivity, and specificity were calculated. The results are shown in Table 3.

[0192] [Table 3]

[0193]

[0194] ①, ②, ③, ④, ⑤ and Figure 3 The parameters recorded are consistent.

[0195] The AUC values ​​of OVM-specific IgE titers measured by the CAP OVM sIgE method (UA / mL) and the DCP OVM sIgE method (BUe / mL) were almost the same. Among the five parameters studied, the AUC value of "DCPOVM sIgE / DCP OVM sIgE IC50", which is the multiplication of IgE antibody titer by OVM affinity, was the highest, at 0.79, and the critical value for confirming the diagnosis of allergy at this time was 4459.00 (BUe / mL) / (nM). Therefore, when the value of DCP OVM sIgE / DCP OVM sIgE IC50, which is the multiplication of IgE antibody titer by OVM affinity, is above the critical value, i.e., 4459.00 (BUe / mL) / (nM), it can be judged that the risk of egg allergy is high, and when it is less than this critical value, it can be judged that the risk of egg allergy is low. Therefore, it is presumed that it would be beneficial to improve the quality of life of patients if the dietary restrictions that have been imposed on all patients suspected of having egg allergy were only imposed on patients with higher risk and were lifted for patients with lower risk. Figure 3 As shown in the lower part, the AUC value of DCP OVM sIgE / DCP OVM sIgE IC50 (quantity × mass) obtained by multiplying the IgE quantitative value by the OVM affinity shows a P value indicating a significant difference compared with individual parameters such as the IC50 value or the OVM affinity, indicating that the risk of egg allergy and the absence of egg allergy can be correctly determined. It should be noted that in this specification, "OVM sIgE" refers to IgE specific to ovomucoid.

[0196] [Reference Example 2]

[0197] Among the subjects (1 to 6 years old) shown in [Example 1], 256 subjects (1 to 3 years old) whose immune system was underdeveloped and whose allergies were considered difficult to diagnose in daily medical treatment were selected as the subjects. Based on the confirmed diagnosis of allergy for OVM based on OFC, ROC analysis was performed on the above five parameters for subjects diagnosed as allergy-positive confirmed subjects and subjects diagnosed as allergy-negative confirmed subjects. The results are shown in Figure 4 .

[0198] In addition, 256 infants aged 1 to 3 years were selected as subjects, and ROC analysis was performed for each of the five parameters for subjects diagnosed as positive allergy diagnoses and subjects diagnosed as negative allergy diagnoses based on OFC for allergy diagnosis of OVM, and AUC values, cutoff values, sensitivity, and specificity were calculated. The results are shown in Table 4.

[0199] [Table 4]

[0200]

[0201] ①, ②, ③, ④, ⑤ and Figure 4 The parameters recorded are consistent.

[0202] (result)

[0203] In subjects (1 to 3 years old) who are considered difficult to diagnose allergies, the same five parameters were used for the study as in the case of subjects (1 to 6 years old). It was confirmed that in subjects (1 to 3 years old), the AUC values ​​of OVM-specific IgE values ​​(BUe / mL) measured by the ImmunoCAP method (UA / mL) and the DCP method were approximately the same as those in the case of subjects (1 to 6 years old). Among the five parameters studied, the AUC value of DCP OVM sIgE / DCP OVM sIgE IC50, which is obtained by multiplying the DCP OVM sIgE value by the affinity, was the highest, at 0.79, compared with the other parameters. At this time, the critical value for confirming the diagnosis of allergies was 1556.70 (BUe / mL) / (nM), which is 35% of the critical value of the subjects (1 to 6 years old), which is a low value. In addition, as Figure 4 As shown in the lower part, similarly to the subjects (1 to 6 years old), in the subjects (1 to 3 years old), the AUC value of DCP OVMsIgE / DCP OVM sIgE IC50 (quantity × mass), which is obtained by multiplying the IgE quantitative value by the OVM affinity, is a P value indicating a significant difference compared with individual parameters such as the IC50 value or OVM affinity, indicating that the risk of egg allergy and the lack of egg allergy can be correctly determined.

[0204] [Example 4]

[0205] Among the 343 infants aged 1 to 6 years who were positive for egg allergen sensitization shown in Table 1, the diagnosis of anaphylaxis risk based on OFC was as follows: Figure 5 As shown, the diagnostic accuracy and usefulness of the five parameters (A) CAP OVM sIgE (UA / mL), (B) DCPOVM sIgE (BUe / mL), (C) DCP OVM sIgE / DCP OVM sIgE IC50 [(BUe / mL) / (nM)], (D) DCP OVM sIgE IC50 (nM), and (E) 1 / DCP OVM sIgE IC50 (nM) were evaluated, and a significant difference test was performed between the groups. Figure 5 The statistical data are shown in Table 5 below.

[0206] (result)

[0207] according to Figure 5As is clear from Table 5, the conventional allergen-specific IgE quantitative method (A) CAP OVM sIgE (UA / mL) and (B) DCP OVM sIgE (BUe / mL) showed significant differences in the diagnosis of egg allergy (EA+) and egg allergy-free (EA-), and the diagnosis was made possible. However, no significant difference was detected between the two groups in the diagnosis of "hypersensitivity state, anaphylaxis that can threaten life" [(AN+) and (AN-)]. However, when (C) DCP OVM sIgE / DCP OVM sIgE IC50 [(BUe / mL) / (nM)] was used as a parameter, there was a significant difference between (AN+) and (AN-) in egg allergy-positive subjects (P=0.0041), indicating that identification was possible. It should be noted that (D) DCP OVM sIgE IC50 (nM) and (E) 1 / DCP OVM sIgE IC50 (nM) values ​​also showed relatively good values ​​of 0.0183 and 0.0184, respectively. Thus, by using OVM affinity, the diagnostic accuracy of anaphylaxis risk diagnosis is improved, and the effect is most significant when the parameter DCP OVM sIgE / DCP OVM sIgE IC50 [(BUe / mL) / (nM)].

[0208] [Table 5]

[0209]

[0210] ROC analysis was performed on the above five parameters for the diagnosis of anaphylaxis to OVM based on OFC results in subjects aged 1 to 6 years (343 subjects). The results are shown in Figure 6 .

[0211] In addition, for 343 infants aged 1 to 6 years who underwent the ROC analysis, the AUC value, cutoff value, sensitivity, and specificity were calculated for subjects diagnosed with anaphylaxis and subjects diagnosed with no anaphylaxis based on OFC diagnosis of anaphylaxis onset for OVM using the five parameters described above. The results are shown in Table 6.

[0212] [Table 6]

[0213]

[0214] ①, ②, ③, ④, ⑤ and Figure 6 The parameters recorded are consistent.

[0215] In the ROC analysis, if DCP OVM sIgE / DCP OVM sIgE IC50 [(BUe / mL) / (nM)] obtained by multiplying the affinity of OVM is used as a parameter, the AUC is 0.76, which is the highest compared to other parameters. Since it is within 0.7 to 0.8, the diagnostic accuracy for the onset of egg anaphylaxis is judged to be good. The critical value of this parameter is 8960.10 [(BUe / mL) / (nM)], which is about twice the risk value of egg allergy 4459.00 [(BUe / mL) / (nM)]. In the case of a value exceeding this value, it can be inferred that the risk of onset of egg anaphylaxis is increased. In addition, if Figure 6 As shown in the lower part, in the subjects (1 to 6 years old), the AUC value of DCP OVMsIgE / DCP OVM sIgE IC50 (quantity × mass) obtained by multiplying the IgE quantitative value by the OVM affinity showed a significant difference in P value compared with the previous IgE quantitative value [(UA / mL) / mL] single parameter, indicating that the risk of egg allergy and the lack of egg allergy can be correctly determined.

[0216] [Example 5]

[0217] In 256 infants and young children aged 1 to 3 years whose immune systems are immature and whose allergy diagnosis is difficult as shown in Table 2, the diagnostic accuracy and usefulness of OFC-based diagnosis of the risk of anaphylaxis were evaluated using five parameters: (A) CAP OVM sIgE (UA / mL), (B) DCP OVM sIgE (BUe / mL), (C) DCP OVM sIgE / DCP OVM sIgE IC50 [(BUe / mL) / (nM)], (D) DCP OVM sIgE IC50 (nM), and (E) 1 / DCP OVM sIgE IC50 (nM). A significant difference test was performed between the groups, and the results are shown in the table. Figure 7 In addition, Figure 7 The statistical data are shown in Table 7 below.

[0218] (result)

[0219] according to Figure 7It is clear that the diagnosis of egg allergy was significantly different between those with (EA+) and those without (EA-) in (A) CAP OVM sIgE (UA / mL) and (B) DCP OVM sIgE (BUe / mL), but no significant difference was observed between the two groups in the diagnosis of anaphylaxis risk, as in the case of 1 to 6 years old. However, when (C) DCPOVM sIgE / DCP OVM sIgE IC50 [(BUe / mL) / (nM)] was used as a parameter, there was a significant difference between (AN+) and (AN-) in egg allergy-positive subjects (P=0.0077), indicating that identification was possible. In addition, the antigen affinity parameters (D) DCPOVM sIgE IC50 (nM) and (E) 1 / DCP OVM sIgE IC50 (nM) had P values ​​of 0.0092 and 0.0093, respectively, showing good values, indicating that it is useful for predicting the onset of anaphylaxis.

[0220] [Table 7]

[0221]

[0222] The above ROC analysis was performed on 256 infants aged 1 to 3 years, and the AUC value, cutoff value, sensitivity, and specificity were calculated for subjects diagnosed with anaphylaxis and subjects diagnosed with no anaphylaxis, respectively, based on OFC-based anaphylaxis diagnosis. The results are shown in Figure 8 and Table 8.

[0223] [Table 8]

[0224]

[0225] ①, ②, ③, ④, ④ and Figure 8 The parameters recorded are consistent.

[0226] The critical value of DCP OVM sIgE / DCP OVM sIgE IC50 [(BUe / mL) / (nM)] in the ROC analysis between anaphylactic patients and non-anaphylactic patients in infants aged 1 to 3 years, in which anaphylactic episodes were difficult to diagnose in the past, was 1225.80, as shown in Table 8, which was 13.7% of the critical value (8960.10) in infants aged 1 to 6 years, and was low, clearly indicating that anaphylaxis is prone to occur in infants aged 1 to 3 years. Regarding the above parameter (quantity × quality) obtained by multiplying the OVM affinity by the DCP OVM sIgE value, as shown in Table 8, Figure 8As shown in Table 8, the AUC value shown by the ROC curve was 0.77, the highest among the five groups studied, and the sensitivity at this time showed the highest value of 1.00, indicating that it is useful for diagnosing the risk of systemic allergic reaction. Figure 7 In (C) DCP OVM sIgE / DCP OVM sIgE IC50[(BUe / mL) / (nM)] shown in Table 7, the P value between the EA(+)(AN-) group and the EA(+)(AN+) group was P=0.0077, showing a significant difference (P<0.008). In addition, in (D) DCP OVM sIgE IC50(nM) and (E) 1 / DCP OVM sIgE IC50(nM), the P values ​​between the EA(+)(AN-) group and the EA(+)(AN+) group were P=0.0092 and P=0.0093, respectively, both of which were relatively good results. In addition, as Figure 8 As shown in the lower part of the figure, in the subjects (1 to 3 years old), the AUC value of DCP OVM sIgE / DCP OVMsIgE IC50 (quantity × mass) obtained by multiplying the IgE quantitative value by the OVM affinity showed a significant difference compared with the previous single parameters of IgE antibody titer [(UA / mL) / mL] or (BUe / mL), showing that it is possible to correctly judge the risk of systemic anaphylaxis to eggs and the situation without systemic anaphylaxis to eggs. In summary, it can be inferred that when the critical value of DCP OVM sIgE / DCP OVM sIgE IC50 [(BUe / mL) / (nM)] in the diagnosis of the risk of systemic anaphylaxis in 1 to 3 years old is 1225.80 or more, it can be judged that the risk of systemic anaphylaxis to eggs is high.

[0227] (summary)

[0228] To date, in allergy diagnosis, e.g. Figure 1 As shown, only the amount of allergen-specific IgE in the blood was measured, and the antigen affinity, which is the quality of the allergen-specific IgE, was not considered. Since the reaction exhibited by the antibody is achieved according to the "quantity" and "quality" of the antibody, the biological reaction of the allergen-specific IgE should be evaluated in combination with its "quantity" and "quality". As the "quality" of the antibody, the usefulness of the antigen affinity was studied in the present invention.

[0229] The affinity of IgE for allergens can be measured by the competitive binding inhibition activity (IC50 value) between solidified allergens and soluble allergens, and is expressed as 1 / IC50 value. This method has the following advantages: it can be evaluated even if there are IgG, IgA, IgD and IgM antibodies other than IgE that recognize the same antigen, and in addition, it can also evaluate the affinity difference between allergens and the affinity difference between different allergens using the same parameter. In the present invention, it has been confirmed that it is difficult to diagnose the risk of anaphylaxis by measuring the affinity (1 / IC50 value) of polyclonal IgE antibodies for allergens in human serum using the conventional allergen-specific IgE quantitative method, but by using DCP OVM sIgE / DCP OVM sIgE IC50 [(BUe / mL) / (nM)] obtained by multiplying the antibody titer to DCP OVM sIgE (BUe / mL) by the affinity as a parameter, the risk of anaphylaxis can be predicted and diagnosed. In addition, the following fact was newly discovered: in the diagnosis of the risk of anaphylaxis in patients aged 1 to 6 years and 1 to 3 years who were allergic to egg, even the DCPOVM sIgE IC50 (nM) value and 1 / DCP OVM sIgE IC50 (nM) value, which are individual parameters of affinity for allergens, had a smaller degree of significant difference than DCP OVM sIgE / DCP OVM sIgE IC50 [(BUe / mL) / (nM)] obtained by multiplying the antibody titer (BUe / mL) to DCP OVM sIgE by affinity, but they had a significant difference and could distinguish the presence or absence of anaphylaxis.

[0230] [Example 6]

[0231] In order to verify the feasibility (effectiveness) of the critical value of the judgment criterion [(BUe / mL) / IC50] prepared based on the IC50 values ​​for egg allergy and systemic anaphylaxis to egg calculated from the previous ROC analysis, the IgE antibody titer, and the affinity of the IgE antibody for the allergen, the inventors commissioned the Osaka Prefecture Takatsuki Hospital, which is a hospital in a different region from the hospital from which the previous data (Tables 1 to 8, etc.) were obtained, and with the approval of the Hospital Ethics Committee of Takatsuki Hospital (Ethics Committee Approval No.: 2019-92), allergic patients who underwent OFC were recruited from February 20, 2020, and blood was collected with the consent of the patients and their families, and the OVM-specific IgE value was measured and its affinity value was calculated.

[0232] As the first phase of the measurement, among the 19 patients with egg allergy collected in about four months from February 20, 2020 to June 17, 2020, 15 cases of heated egg OFC were intervened by OVM. The other four patients with egg allergy were excluded from this study because it was determined that the allergy was caused by ovalbumin, which is an allergen of raw eggs. For the 15 cases that underwent heated egg OFC, the validity of the critical values ​​of IC50 and [(BUe / mL) / IC50] values ​​for egg allergy and systemic anaphylaxis to egg recorded in the patent was verified.

[0233] Of the 15 cases that could be treated with heated egg OFC at the Department of Pediatrics at Takatsuki Hospital, 2 were aged 1 to 3 years, and the other 13 were aged 8 to 15 years. In this study, it was decided to use the cutoff value of 1 to 6 years for the 13 cases aged 8 to 15 years to study the effectiveness.

[0234] [Table 9]

[0235]

[0236] (Critical value for diagnosis of egg allergy)

[0237] The contents of the numerical values ​​shown in the above Table 9 are as follows.

[0238] [1] Among infants and young children aged 1 to 3 years and 1 to 6 years, based on the definitive diagnosis of allergy to OVM based on OFC, the critical values ​​of the process of ROC analysis were extracted from Tables 3 and 4 as follows for subjects diagnosed as positive for egg allergy and subjects diagnosed as negative for egg allergy.

[0239] (1) The cut-off value for allergy-presence or absence of OVM-specific IgE IC50 value (OVM sIgE IC50 (nM)) measured by the DCP method;

[0240] (That is, if the level is less than 1.13 nM in children aged 1 to 3 years and less than 1.30 nM in children aged 1 to 6 years, it can be determined as positive for allergy.)

[0241] (2) The cutoff value for the presence or absence of allergy was obtained by multiplying the OVM-specific IgE titer measured by the DCP method by 1 / IC50: [OVM sIgE / OVMs IgE IC50 [(BUe / mL) / (nM)]];

[0242] (That is, when the value is 1556.70 (BUe / mL) / (nM) in the case of 1 to 3 years old and 4459.00 (BUe / mL) / (nM) or more in the case of 1 to 6 years old, it can be determined as positive for allergy.)

[0243] (Critical value for diagnosis of systemic allergic reaction to egg)

[0244] [2] In infants aged 1 to 3 years and 1 to 6 years, based on the diagnosis of egg anaphylaxis based on OFC, the critical values ​​of the results of ROC analysis were extracted from Tables 6 and 8 as follows for subjects diagnosed as anaphylaxis and subjects diagnosed as non-anaphylaxis. They are respectively expressed as:

[0245] (3) The critical value of the IC50 value of OVM-specific IgE (OVM sIgE IC50 (nM)) measured by the DCP method for the occurrence of systemic anaphylaxis (i.e., less than 2.15 nM for children aged 1 to 3 years and less than 1.03 nM for children aged 1 to 6 years, which can be judged as the possibility of systemic anaphylaxis).

[0246] (4) The critical value for the presence or absence of anaphylaxis is obtained by multiplying the OVM-specific IgE titer measured by the DCP method by 1 / IC50: [OVM sIgE / OVMs IgE IC50 [(BUe / mL) / (nM)]];

[0247] (That is, when it is 1225.80 (BUe / mL) / (nM) or more in the case of 1 to 3 years old, and when it is 8960.00 (BUe / mL) / (nM) or more in the case of 1 to 6 years old, it can be determined that there is a possibility of systemic anaphylaxis.).

[0248] Regarding the cases that underwent OFC at Takatsuki Hospital, the results of three parameters obtained from 2 cases aged 1 to 3 years and 13 cases aged 8 to 15 years: DCP OVM-sIgE, DCP OVM-sIgE IC50, and DCP OVM-sIgE / DCP OVM-sIgEIC50 are shown in Table 10 below.

[0249] [Table 10]

[0250]

[0251] Table 10 (A) is a group in which the results of the heated egg OFC were judged to be negative for egg allergy (egg allergy negative-anaphylaxis negative group), and 2 infants aged 1 to 3 years and 8 children aged 8 to 15 years belong to this group. (B) is a group in which the results of the heated egg OFC were judged to be positive for egg allergy and no anaphylaxis occurred (egg allergy positive-anaphylaxis negative group). (C) is a group in which the results of the heated egg OFC were judged to be positive for egg allergy and anaphylaxis occurred (egg anaphylaxis group).

[0252] (Egg allergy negative-anaphylaxis negative group)

[0253] For the two infants aged 1 to 3 years who were judged to be negative for egg allergy as a result of the heated egg OFC, the IC50 values ​​of OVM-specific IgE (OVM sIgE IC50 (nM)) measured by the DCP method were 4.39 nM and 194.71 nM, which were above the critical value of 1.13 nM for the IC50 value of OVM-specific IgE for 1 to 3 years old, and thus were confirmed to fall within the range of negative for allergy. In addition, the values ​​obtained by multiplying the OVM-specific IgE titer measured by the DCP method by the value of 1 / IC50 were 757.12 (BUe / mL) / (nM) and 11.82 (BUe / mL) / (nM), which were lower than the critical value of 1556.70 for the IC50 of OVM sIgE / OVMs IgE for 1 to 3 years old, and thus were confirmed to fall within the range of negative for allergy. In fact, no signs of egg allergy were confirmed in these two infants.

[0254] It should be noted that, in the two cases, even with regard to the presence or absence of anaphylaxis, the values ​​were higher than the critical value of 2.15 nM for the OVM-specific IgE IC50 value (OVM sIgE IC50 (nM)) for 1 to 3 years old. In addition, the values ​​were lower than the critical value of 1225.80 (BUe / mL) / (nM) for the presence or absence of anaphylaxis using the OVM sIgE / OVMs IgE IC50 parameter for 1 to 3 years old, and the possibility of anaphylaxis was considered to be very low. So far, no reports of anaphylaxis due to egg ingestion have been received for these two cases.

[0255] Therefore, it was confirmed that in the case of 1 to 3 years old, the critical value for the presence or absence of systemic anaphylaxis determined by ROC analysis using the IC50 value of OVM-specific IgE as a parameter, and the critical value for the presence or absence of systemic anaphylaxis determined by ROC analysis using the value obtained by multiplying the OVM-specific IgE titer by 1 / IC50 value as a parameter can be effectively utilized.

[0256] For the six children aged 8 to 15 years, except for THG-23 and THG-44, the IC50 values ​​of OVM-specific IgE measured by the DCP method (OVM sIgE IC50 (nM)) were in the range of 1.73nM to 16.03nM, which was above the critical value of 1.30nM for the IC50 values ​​of OVM-specific IgE for 1 to 6 years old regarding the presence or absence of allergies, so it was confirmed that they fell into the negative range of allergies. In addition, since the value obtained by multiplying the OVM-specific IgE titer measured by the DCP method by the value of 1 / IC50 was 170.93 (BUe / mL) / (nM) to 1082.41 (BUe / mL) / (nM), that is, less than the critical value of 4459.00 (BUe / mL) / (nM) for the IC50 values ​​of OVM sIgE / OVMs IgE for 1 to 6 years old regarding the presence or absence of allergies, it was confirmed that they fell into the negative range of allergies.

[0257] In addition, for the 7 children aged 8 to 15 years, except for one THG-44, all showed values ​​higher than the critical value of 1.03nM for the presence or absence of anaphylaxis using the IC50 value of OVM-specific IgE as a parameter for 1 to 6 years old, and showed values ​​lower than the critical value of 8960.00 (BUe / mL) / (nM) for the presence or absence of anaphylaxis using the OVM sIgE / OVMs IgE IC50 as a parameter for 1 to 6 years old. These 6 children aged 8 to 15 years showed no signs of anaphylaxis at all, confirming that the onset of anaphylaxis can be predicted using the critical value for 1 to 6 years old.

[0258] For one child aged 8 to 15 years (THG-44), the IC50 value of OVM-specific IgE (OVM sIgEIC50 (nM)) measured by the DCP method was 0.93 nM, which was lower than the critical value of 1.30 nM for the IC50 value of OVM-specific IgE for 1 to 6 years old, which was related to the presence or absence of allergy, so it fell into the range of allergy-positive. In addition, since it was lower than the critical value of 1.03 nM for the IC50 value of OVM-specific IgE for 1 to 6 years old, which was related to the presence or absence of systemic allergic reaction, it was estimated that there was a possibility of systemic allergic reaction.

[0259] In the case of THG-44, the value obtained by multiplying the OVM-specific IgE titer measured by the DCP method by 1 / IC50 was 15133.54 (BUe / mL) / (nM), which is higher than the critical value of 1556.70 for the presence or absence of allergy in OVM sIgE / OVMs IgE IC50 for 1 to 6 years old, and falls into the range of positive allergy. In addition, since it is higher than the critical value of 8960.10 (BUe / mL) / (nM) for the presence or absence of systemic anaphylaxis in OVM sIgE / OVMsIgE IC50 for 1 to 6 years old, it can be predicted that the possibility of systemic anaphylaxis is very high. Therefore, for this child, although allergic symptoms caused by egg ingestion have not been confirmed, it is believed that systemic anaphylaxis may occur in the future, and dietary guidance related to eggs is being carried out under the supervision of a doctor, and the progress is being observed.

[0260] For one child aged 8 to 15 years (THG-23), the IC50 value of OVM-specific IgE (OVMsIgE IC50 (nM)) measured by the DCP method was 1.25 nM, which was lower than the critical value of 1.30 nM for the IC50 value of OVM-specific IgE for 1 to 6 years old, and therefore fell into the range of allergy-positive. However, it was higher than the critical value of 1.03 nM for the IC50 value of OVM-specific IgE for 1 to 6 years old, which was higher than the critical value of 1.03 nM for the IC50 value of OVM-specific IgE for 1 to 6 years old, and it was estimated that the possibility of anaphylaxis was low.

[0261] In the case of THG-23, the value obtained by multiplying the OVM-specific IgE titer measured by the DCP method by the value of 1 / IC50 was 942.36 (BUe / mL) / (nM), which is lower than the critical value of 4459.00 (BUe / mL) / (nM) for the presence or absence of allergies in OVM sIgE / OVMs IgE IC50 for children aged 1 to 6 years, so it falls into the negative range of allergies. In addition, it is lower than the critical value of 8960.10 (BUe / mL) / (nM) for the presence or absence of systemic allergic reactions in OVM sIgE / OVMs IgE IC50 for children aged 1 to 6 years. When the two critical values ​​of 1 to 6 years are applied, it can be said that the child is less likely to have a systemic allergic reaction. As for this child, since no allergic symptoms have been shown even after ingesting eggs, the progress is still under observation.

[0262] (Summary of the egg allergy negative group)

[0263] In the egg allergy negative group (Group (A)), the critical value for the presence or absence of anaphylaxis using the IC50 value of OVM-specific IgE and the critical value for the presence or absence of anaphylaxis using the value obtained by multiplying the OVM-specific IgE titer by 1 / IC50 were confirmed. In addition, it was confirmed that the critical value calculated based on the data of 1 to 6 years old could also be applied to children aged 8 to 15 years.

[0264] (Egg allergy positive-anaphylaxis negative group)

[0265] For two children aged 8 to 15 years (THG-31 and 47) who were judged to be positive for egg allergy as a result of heated egg OFC, the OVM-specific IgE IC50 values ​​(OVM sIgE IC50 (nM)) measured by the DCP method were 1.05nM and 0.48nM. Since the IC50 values ​​of OVM-specific IgE for 1 to 6 years old are below the critical value of 1.30nM for the presence or absence of allergy, it was confirmed that they fell into the range of positive allergy.

[0266] The values ​​obtained by multiplying the OVM-specific IgE titer measured by the DCP method by 1 / IC50 were 1465.64 (BUe / mL) / (nM) and 3897.79 (BUe / mL) / (nM), respectively, which are lower than the critical value of 4459.00 (BUe / mL) / (nM) for the IC50 of OVM sIgE / OVMs IgE for 1 to 6 years old, which indicates the presence or absence of allergy, and fall into the negative range of allergy. Although these two children have mild symptoms of egg allergy, it is highly likely that they are in the transition period from positive to negative egg allergy, and their progress is being monitored.

[0267] In addition, since these children had a much lower value than the critical value of 8960.10 (BUe / mL) / (nM) for the presence or absence of anaphylaxis in OVM sIgE / OVMs IgE IC50 for 1 to 6 years old, it was predicted that they were less likely to have anaphylaxis, and in fact, they did not show symptoms of anaphylaxis. However, in the case of THG-47, the IC50 value of IgE was lower than the critical value for the presence or absence of anaphylaxis, and further observation is needed.

[0268] For one child aged 8 to 15 years (THG-21), the IC50 value of OVM-specific IgE measured by the DCP method (OVMsIgE IC50 (nM)) was 1.74 nM, which was above the critical value of 1.30 nM for the IC50 value of OVM-specific IgE for children aged 1 to 6 years, and therefore fell within the range of negative allergy. The value obtained by multiplying the OVM-specific IgE titer measured by the DCP method by the value of 1 / IC50 was 4897.47 (BUe / mL) / (nM), which was higher than the critical value of 4459.00 (BUe / mL) / (nM) for the IC50 value of OVM sIgE / OVMs IgE for children aged 1 to 6 years, and thus fell within the range of positive allergy.

[0269] In the case of THG-21, the IC50 value was higher than the critical value of 1.03nM for the presence or absence of anaphylaxis using the IC50 value of OVM-specific IgE for children aged 1 to 6 years, and the value obtained by multiplying the OVM-specific IgE titer by 1 / IC50 was lower than the critical value of 8960.10 (BUe / mL) / (nM) for the presence or absence of anaphylaxis using the OVM sIgE / OVMs IgE IC50 for children aged 1 to 6 years. In the case of applying the two critical values ​​for children aged 1 to 6 years, it can be said that the possibility of anaphylaxis is low. In fact, the child had mild symptoms and had no history of anaphylaxis. Therefore, it was confirmed that the critical value calculated based on the data for children aged 1 to 6 years can also be used for children aged 8 to 15 years.

[0270] For one child aged 8 to 15 years (THG-13), the IC50 value of OVM-specific IgE measured by the DCP method (OVMsIgE IC50 (nM)) was 2.7 nM, which was above the critical value of 1.30 nM for the IC50 value of OVM-specific IgE for children aged 1 to 6 years, and therefore fell within the range of negative allergy. In addition, the value obtained by multiplying the OVM-specific IgE titer measured by the DCP method by the value of 1 / IC50 was 317.17 (BUe / mL) / (nM), which was much lower than the critical value of 4459.00 (BUe / mL) / (nM) for the IC50 of OVM sIgE / OVMs IgE for children aged 1 to 6 years, and thus fell within the range of negative allergy.

[0271] In case THG-13, the IC50 value of OVM-specific IgE for 1 to 6 years old is above the critical value of 1.03nM for the presence or absence of systemic allergic reaction, so the possibility of systemic allergic reaction is low. In addition, since it is significantly lower than the critical value of 8960.10 (BUe / mL) / (nM) for the presence or absence of systemic allergic reaction of OVMsIgE / OVMs IgE IC50 for 1 to 6 years old, it can be predicted that the possibility of systemic allergic reaction is low. Although the child currently has allergic symptoms, there is no history of systemic allergic reaction. The OVM antibody titer itself is very low, and the trend of allergic symptoms becoming milder has been observed, so the progress is under observation.

[0272] (Egg systemic allergic reaction group)

[0273] This is a study on 10-year-old children who had anaphylaxis. The IC50 value of OVM-specific IgE measured by the DCP method (OVM sIgE IC50 (nM)) was 0.26 nM, which is lower than the critical value of 1.03 nM for the IC50 value of OVM-specific IgE for 1 to 6 years old, which is consistent with the occurrence of anaphylaxis. In addition, the value obtained by multiplying the OVM-specific IgE titer measured by the DCP method by 1 / IC50 is 81174.34 (BUe / mL) / (nM), which is significantly higher than the critical value of 8960.10 (BUe / mL) / (nM) for the IC50 value of OVM sIgE for 1 to 6 years old / OVMs IgE, which is consistent with the actual occurrence of anaphylaxis.

[0274] Therefore, it was confirmed that the critical value for the presence or absence of anaphylaxis using the IC50 value of OVM-specific IgE for children aged 1 to 6 years and the critical value for the presence or absence of anaphylaxis using the OVMsIgE value / OVMsIgE IC50 for children aged 1 to 6 years can also be applied to the 10-year-old children.

[0275] (Summarize)

[0276] It was confirmed that the cutoff value calculated based on the antibody titer of IgE antibodies and the affinity of IgE antibodies for allergens and determined by ROC analysis can predict the risk of anaphylaxis in infants and children. In addition, it was confirmed that the cutoff value for the presence or absence of anaphylaxis using OVM sIgE / OVMs IgE IC50 calculated based on data of children aged 1 to 6 years and the cutoff value for the presence or absence of anaphylaxis using IgE IC50 as a parameter, or both, can be applied to children aged 8 to 15 years. It should be noted that according to the present invention, infants and children who have not yet had a systemic allergic reaction can receive dietary guidance from doctors, etc., for example, when the OVM sIgE value / OVMs IgE IC50 [(BUe / mL) / (nM)]] obtained by multiplying the OVM-specific IgE titer by 1 / IC50 or the IgE IC50 (nM) critical value for the presence or absence of a systemic allergic reaction, which is clearly exceeded, which is good.

Claims

1. A method for collecting data, wherein the method comprises collecting critical value data determined by ROC analysis based on the affinity of IgE antibodies for allergens in samples collected from infants / children suffering from food allergies, wherein: The affinity of IgE antibodies for allergens is numerically expressed as 1 / IC50, and the critical value determined by ROC analysis based on the affinity of IgE antibodies for allergens is a critical value determined by ROC analysis using as a parameter a value obtained by multiplying the antibody titer of IgE antibodies for allergens by the affinity of IgE antibodies for allergens.

2. The method for collecting data according to claim 1, characterized in that: IC50 is the competitive binding inhibition activity value.

3. The method for collecting data according to claim 1, characterized in that: The antibody titer of IgE antibodies against allergens is measured using 1) a chip obtained by treating a carbon layer on a silicon substrate surface with diamond-like carbon and introducing chemical modification groups into the carbon layer, 2) a chip obtained by applying an electrostatic layer treated with an amino compound or its polymer and / or copolymer on the surface of a glass slide and then treating it again with a dicarboxylic acid or a polycarboxylic acid and activating it with N-hydroxysuccinimide and / or carbodiimide, or 3) a chip obtained by introducing chemical modification groups onto the surface of a carrier.

4. The method for collecting data according to claim 1, characterized in that: The sample is plasma or serum.

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