Method for evaluating or predicting food allergy

Abstract

Provided is a method for evaluating or predicting a food allergy.　The present invention provides a method for using, as an index, expression of SCCA in a subject to evaluate or predict the possibility of onset of a food allergy.

Description

Methods for assessing or predicting food allergies

[0001] The present invention provides a method for evaluating or predicting the possibility of developing food allergy using squamous cell carcinoma antigen (hereinafter referred to as "SCCA") in skin stratum corneum cells as an indicator.

[0002] Food allergies are a condition in which the immune system overreacts when a person ingests a particular food. They typically cause symptoms such as itchy skin, rashes, hives, difficulty breathing, nausea, vomiting, and diarrhea, but in severe cases, they can also cause more serious symptoms such as anaphylactic shock.

[0003] Since consuming foods containing allergens causes an allergic reaction and requires avoidance of those foods, it is important to know as early as possible whether one is prone to food allergies. However, many people do not realize they have a food allergy without receiving a food allergy diagnosis. This presents a risk of continuing to consume the food without realizing it. In such cases, people may only receive a diagnosis once their symptoms have worsened, and only then do they realize they have a food allergy. Furthermore, many food allergy patients are infants and young children, and there is also the problem that they often have difficulty complaining about their symptoms at a young age. Therefore, it is desirable to be able to obtain information about the possibility of a food allergy as early as possible.

[0004] Although food allergies are diagnosed by doctors or specialists through patch tests, blood tests, food elimination and reintroduction tests, etc., it would be desirable to have an indicator that allows for easy and objective evaluation or prediction of food allergy tendencies at home. Such evaluation or prediction would be extremely advantageous, especially for people who have difficulty visiting a doctor without an escort.

[0005] Patent No. 6835479

[0006] Clinical Laboratory Tests, Vol. 49, No. 7, July 2005

[0007] An object of the present invention is to provide a method for evaluating or predicting the possibility of developing a food allergy.

[0008] As a result of extensive research, the present inventors have discovered that the possibility of developing a food allergy can be evaluated or predicted by using SCCA expression in a subject as an index, and have completed the following inventions. [1] A method for assisting in the evaluation or prediction of the possibility of developing a food allergy by using the expression level of SCCA in a biological sample collected from a subject as an index. [2] A method for assisting in the evaluation or prediction of the possibility of a subject developing a food allergy by the age of three by using SCCA expression in a biological sample collected from an infant aged 2 months to 3 years as an index. [3] A method for testing a biological sample collected from a subject to determine whether the subject is likely to develop a food allergy, the method comprising measuring SCCA expression in the biological sample. [4] A method for testing a biological sample collected from an infant aged 2 months to 3 years to determine whether the subject is likely to develop a food allergy by the age of three, the method comprising measuring SCCA expression in the biological sample. [5] The method of any one of [1] to [4], comprising a step of comparing the SCCA expression level in the biological sample with a reference value, wherein a higher level of SCCA expression in the biological sample indicates that the subject is likely to develop a food allergy. [6] The method of [5], wherein the reference value is a cutoff value. [7] The method of any one of [1] to [6], wherein the SCCA is SCCA-1. [8] The method of any one of [1] to [7], wherein the biological sample is collected non-invasively by tape stripping. [9] A kit for assessing or predicting the likelihood of developing a food allergy, comprising a medium for collecting a biological sample from a subject and a reagent for measuring SCCA expression in the biological sample collected with the medium.

[10] The kit of [9], wherein the medium is a tape to be applied to the skin of the subject, and the biological sample is collected non-invasively by tape stripping.

[11] A system for assessing or predicting the possibility of developing a food allergy, comprising: a data acquisition unit that acquires data on SCCA expression from a biological sample collected from a subject; an evaluation or prediction unit that refers to the data acquired by the data acquisition unit and, if SCCA expression is high, assesses or predicts that the subject has a high possibility of developing a food allergy; and a display unit that displays the results of the assessment or prediction made by the evaluation or prediction unit.

[0009] By employing the method of the present invention, the possibility of developing a food allergy can be evaluated or predicted.

[0010] Figure 1 shows the mean SCCA-1 values ​​(ng / mg = SCCA amount / total protein amount) in samples collected from each test site of the subject at each measurement time point in Example 1, divided into those with or without the onset of food allergy by age 3 years (t-test). Figure 2 shows the ROC curves for the SCCA-1 values ​​in samples collected from the perioral area (left) and abdominal area (right) of 2-month-old infants in Figure 1 and the presence or absence of the onset of food allergy by age 3 years. Figure 3 shows the mean SCCA-1 values ​​(ng / mg = SCCA amount / total protein amount) in samples collected from each test site of the subject at each measurement time point in Example 1, divided into those with or without the onset of food allergy by age 18 months (t-test). Figure 4 shows the ROC curves for the SCCA-1 values ​​in samples collected from the perioral area (left) of 2-month-old infants and the abdominal area (right) of 4- to 5-month-old infants in Figure 3, and the presence or absence of the onset of food allergy by age 18 months. Figure 5 shows the mean SCCA-1 values ​​(ng / mg = SCCA amount / total protein amount) for samples taken from each test site of the subject at each measurement time point in Example 1, divided into those with or without the onset of food allergies by 8 to 9 months of age (t-test). Figure 6 shows the ROC curves for the SCCA-1 values ​​for samples taken from the abdomen (left) of 2-month-old infants and the abdomen (right) of 4- to 5-month-old infants in Figure 5, and the presence or absence of the onset of food allergies by 8 to 9 months of age. Figure 7 shows the mean SCCA-1 values ​​(ng / mg = SCCA amount / total protein amount) for samples taken from each test site of the subject at each measurement time point in Example 1, divided into those with or without the onset of food allergies by 4 to 5 months of age (t-test). Figure 8 shows the ROC curves for the SCCA-1 values ​​for samples taken from the back (left) and abdomen (right) of 2-month-old infants in Figure 7, and the presence or absence of the onset of food allergies by 4 to 5 months of age. Figure 9 shows the mean SCCA-1 levels (ng / mg = SCCA amount / total protein amount) in samples collected from each test site of the subjects at each measurement time point in Example 1, broken down by the presence or absence of food allergy up to 2 months of age (t-test). Figure 10 shows the age / month and number of subjects who developed food allergy in Example 1, broken down by the type of causative food.Fig. 11 shows ROC curves for the relationship between SCCA-1 values ​​in samples taken from around the mouth (left) and back (right) of 2-month-old infants and the presence or absence of the onset of food allergies by age 3 years in Example 2. Fig. 12 shows ROC curves for the relationship between SCCA-1 values ​​in samples taken from the abdomen (left) and thigh (right) of 2-month-old infants and the presence or absence of the onset of food allergies by age 3 years in Example 2.

[0011] As described in Patent Document 1, SCCA in skin stratum corneum cells is known to be associated with the severity of atopic dermatitis. Non-Patent Document 1 also reports that SCCA is associated with bronchitis asthma. However, much remains unknown about the relationship between SCCA and food allergies.

[0012] The present inventors have measured the expression levels of SCCA in biological samples collected from infants and investigated the occurrence of food allergies over a long period of time, and found that the SCCA level is highly correlated with the possibility of developing food allergies.

[0013] Squamous cell carcinoma-associated antigen (SCCA) is an antigen extracted from squamous cell carcinoma cells. It is found at high blood concentrations in squamous cell carcinomas of the cervix, lung, esophagus, and skin, and is often used to diagnose squamous cell carcinoma. Various literature suggests that there is some relationship between allergies and SCCA. However, none of the literature suggests that SCCA can objectively evaluate or predict the possibility of developing food allergies, especially at an early stage such as in infancy.

[0014] SCCA is encoded by two genes, SCCA-1 and SCCA-2, which are arranged in tandem on chromosome 18q21.3. The proteins encoded by these genes, SCCA-1 and SCCA-2, both have a molecular weight of approximately 45,000 and exhibit high homology, with the homology reaching 95% at the nucleic acid level. These SCCAs belong to the ovalbumin-serine protease inhibitor (ov-serpin) family. Ov-serpins have unique characteristics within the serpin superfamily. While serpins are generally secreted and function extracellularly, ov-serpins are protease inhibitors that primarily function intracellularly. The SCCA measured in the present invention may be either SCCA-1 or SCCA-2, with SCCA-1 being preferred.

[0015] Therefore, the present invention provides a method for assessing or predicting the likelihood of a subject developing a food allergy by using SCCA expression in a biological sample collected from an infant or young child as an indicator. The present invention also provides a method for testing a biological sample collected from the subject to determine whether the subject is likely to develop a food allergy. The method of the present invention may comprise measuring SCCA expression in the biological sample.

[0016] As mentioned above, it is important to avoid foods and take appropriate measures as early as possible when it comes to food allergies. However, there are problems in that the presence or absence of a food allergy may not be apparent, and infants, in particular, may not be able to report symptoms themselves. Therefore, the present invention is particularly advantageous for young subjects. Preferably, the subject is between 2 and 5 months of age, e.g., 2 months, 3 months, 4 months, 5 months, 2 months to 3 months, 3 months to 4 months, or 4 months to 5 months. For example, SCCA expression in a biological sample collected from a 2-month-old infant can be used as an indicator to indicate the likelihood that the subject will develop a food allergy by the age of 3. "By the age of 3" refers to any time point, such as 0 months to 3 years, 2 months to 3 years, 2 months, 2 months to 5 months, 4 months to 5 months, 8 months to 3 years, 8 months to 1 year and 6 months, 8 to 9 months, 8 months to 1 year, 9 months to 1 year, 1 year to 1 year and 6 months, or 1 year and 6 months to 3 years.

[0017] As used herein, "assessment" refers to assessing the likelihood that a subject from whom a biological sample was collected will develop a food allergy by the time the biological sample was collected. As used herein, "prediction" refers to predicting the likelihood that a subject from whom a biological sample was collected will develop a food allergy at a time after the time the biological sample was collected. One aspect of prediction is to use a biological sample collected from a 2-month-old infant to predict the likelihood that the subject will develop a food allergy at any time during the period from 4 months to 3 years of age, such as 4 months to 3 years, 4 months to 5 months, 8 months to 3 years, 8 months to 1 year and 6 months, 8 to 9 months, 8 months to 1 year, 9 months to 1 year, 1 year to 1 year and 6 months, or 1 year and 6 months to 3 years. Another aspect of prediction is to use a biological sample collected from an infant or toddler subject aged 2 to 5 months or 4 to 5 months to predict the possibility of the subject developing a food allergy at any time during the period from 8 months to 3 years of age, such as 8 months to 3 years, 8 months to 1 year and 6 months, 8 to 9 months, 8 months to 1 year, 9 months to 1 year, 1 year to 1 year and 6 months, or 1 year and 6 months to 3 years.

[0018] The present invention also provides a method for testing a biological sample collected from a subject to determine whether the subject is likely to develop a food allergy, which may comprise measuring an expression level of SCCA in the biological sample collected from the subject.

[0019] The sites from which the biological sample is collected include the face, back, abdomen, and thighs, with the face, such as around the mouth and / or cheeks, the back, and the abdomen being particularly preferred.

[0020] In one embodiment, the possibility that a subject will develop a food allergy by the age of three is predicted by using the expression of SCCA in a biological sample collected from the perioral or abdominal area of ​​a two-month-old infant subject.

[0021] In one embodiment, the possibility that a subject will develop food allergy by the time the subject is 18 months of age is predicted by using the expression of SCCA in a biological sample collected from the perioral area of ​​a 2-month-old infant subject or from the abdomen of a 4- to 5-month-old infant subject as an indicator.

[0022] In one embodiment, the possibility of a subject developing food allergy by 8 to 9 months of age is predicted by using the expression of SCCA in a biological sample collected from the abdomen of a subject aged 2 to 5 months, or an infant aged 2 months or 4 to 5 months, as an indicator.

[0023] In one embodiment, the possibility of a subject developing food allergy at 4 to 5 months of age is predicted by using the expression of SCCA in a biological sample collected from the back or abdomen of a 2-month-old infant subject as an indicator.

[0024] In one embodiment, the expression of SCCA in a biological sample collected from the abdomen of a 1 year and 6 month old infant subject is used as an indicator to assess the possibility that the subject will have developed a food allergy by the time the biological sample is collected, at which time the subject is 1 year and 6 months old.

[0025] In one embodiment, the possibility that a subject will develop a food allergy by the time the subject is 4 to 5 months old, at which time the biological sample is collected, is assessed by using the expression of SCCA in a biological sample collected from the cheek, perioral area, or back of the subject.

[0026] In one embodiment, the expression of SCCA in a biological sample collected from the back of a two-month-old infant subject is used as an indicator to assess the possibility that the subject will develop a food allergy by the time the biological sample is collected, at the age of two months.

[0027] The type of food allergy to be evaluated or predicted is not limited, but may include eggs; grains such as wheat; nuts such as peanuts and almonds; fruits such as apples and mandarins; seafood such as shellfish and seafood; legumes such as soybeans; and dairy products such as powdered milk, milk, and cheese. In one embodiment, the possibility of developing a food allergy to powdered milk in an infant aged 2 to 5 months, or 2 months or 4 to 5 months, is evaluated or predicted. In another embodiment, the possibility of developing a food allergy to eggs, powdered milk, milk, soybeans, peanuts, miso ramen, wheat, apples, yams, peaches, mandarins, bananas, tomatoes, Alaska pollock, sesame, walnuts, and / or salmon roe is evaluated or predicted in an infant aged 8 to 3 years, or 8 to 9 months, 1 year and 6 months, or 3 years.

[0028] The SCCA expression assay of the present invention can be performed quantitatively or qualitatively using any method capable of measuring SCCA. Specifically, various immunoassay methods using SCCA-specific antibodies, such as enzyme-labeled ELISA, radioactive labeled RIA, immunoturbidimetry, Western blotting, latex agglutination, hemagglutination, immunochromatography, resonance plasmon resonance, SAW (surface acoustic wave) devices, and electronic device assays, are available. Immunoassay methods include competitive assays and sandwich assays. Alternatively, the expression level of SCCA can be measured by measuring the intracellular expression level of the gene encoding it. In this case, SCCA expression is preferably determined by measuring the amount of intracellular mRNA encoding SCCA. Extraction of mRNA and quantitative or qualitative measurement of its amount are also well known in the art and can be performed using various well-known methods, such as PCR, 3SR, NASBA, and TMA. Alternatively, SCCA expression may be qualitatively determined by in situ hybridization or by measuring its biological activity.

[0029] Although biological samples can be collected by any method, non-invasive collection by tape stripping is preferred from the viewpoint of simplicity. Tape stripping is a method of collecting stratum corneum samples by applying a piece of adhesive tape to the surface of the skin, peeling it off, and allowing the stratum corneum to adhere to the peeled adhesive tape. Tape stripping allows for measurement of SCCA expression simply by collecting a piece of stratum corneum with a single piece of tape, enabling a method for understanding the pathology of food allergies using SCCA as an indicator. A preferred method of tape stripping involves first removing sebum, dirt, etc. from the surface of the skin, lightly placing a piece of adhesive tape cut to an appropriate size (e.g., 2 x 6 cm) on the skin surface, applying uniform pressure to the entire tape to flatten it, and then peeling off the adhesive tape with uniform pressure. Methods for removing sebum, dirt, etc. from the surface of the skin include wiping with tissue paper, washing with soap, and purifying with water or ethanol. The adhesive tape may be commercially available cellophane tape, for example, Scotch Superstrength Mailing Tape (manufactured by 3M Corporation) or cellophane tape (Cellotape (registered trademark); Nichiban Co., Ltd.). SCCA-1 in a stratum corneum sample attached to adhesive tape can be isolated and extracted from the tape by immersing the tape piece in an appropriate extraction solution, for example, Tris-buffer (pH 8.0) (0.1 M Tris-HCl, 0.14 M NaCl, 0.1% Tween-20), and extracting the stratum corneum.

[0030] A high SCCA expression level in a biological sample indicates that the subject is likely to develop food allergies. However, the absolute value of the SCCA expression level will vary depending on the SCCA measurement system used. Therefore, in order to assign the subject to an appropriate probability of developing food allergy according to the SCCA expression level, a reference value that can be used as a judgment standard for the SCCA measurement system used can be determined.

[0031] Thus, in one embodiment, the method of the present invention may include a step of comparing the SCCA expression level in the biological sample with a reference value. If the SCCA expression level in the biological sample is higher than the reference value, this indicates that the subject is likely to develop a food allergy. For example, if the SCCA expression level in a biological sample collected from an infant aged 2 months to 3 years is higher than the reference value, this may indicate that the subject is likely to develop a food allergy by the age of 2 months to 3 years.

[0032] Regarding the reference value, for example, in one aspect of prediction, the reference value is a cutoff value of the SCCA-1 or SCCA-2 value per total protein in a stratum corneum sample collected by tape stripping from a specific time point and site of an infant subject. The cutoff value can be determined by a logistic regression model using an ROC cutoff value, with the presence or absence of food allergy at each time point as the objective variable and the SCCA expression level as the explanatory variable. In this case, a method that maximizes the Youden Index ("sensitivity - (1 - specificity)") can be used. Furthermore, regarding the reference value, for example, in one aspect of prediction, the reference value may be set as follows based on the average SCCA-1 or SCCA-2 values ​​at each time point and site in a food allergy-developing group and a non-developing group:

[0033] In one example, the value of SCCA-1 or SCCA-2 in a skin stratum corneum sample collected by tape stripping from around the mouth of a 2-month-old infant subject is, for example, about 500 ng / mg or more, about 600 ng / mg or more, about 700 ng / mg or more, about 750 ng / mg or more, about 760 ng / mg or more, about 800 ng / mg or more, about 825.2 ng / mg or more, A level of about 900 ng / mg, about 1000 ng / mg, about 1100 ng / mg, about 1200 ng / mg, about 1500 ng / mg, about 1700 ng / mg, about 1900 ng / mg, about 2000 ng / mg, about 2300 ng / mg, about 2400 ng / mg, or about 2500 ng / mg or higher indicates that the subject may develop a food allergy by the age of 3.

[0034] In one example, if the SCCA-1 level in a skin stratum corneum sample collected by tape stripping from around the mouth of a 2-month-old infant is higher than the cutoff value of 761 ng / mg of total protein, the subject is at risk of developing food allergies by the age of 3.

[0035] In one example, if the SCCA-1 level in a skin stratum corneum sample collected by tape stripping from around the mouth of a 2-month-old infant is equal to or higher than the cutoff value of 825.2 ng / mg of total protein, this indicates that the subject is likely to develop food allergies by the age of 3.

[0036] In one example, the SCCA-1 or SCCA-2 value in a stratum corneum sample collected by tape stripping from the abdomen of a 2-month-old infant subject is, for example, about 50 ng / mg or more, about 60 ng / mg or more, about 64 ng / mg or more, about 65 ng / mg or more, about 70 ng / mg or more, about 80 ng / mg or more, about 90 ng / mg, about 100 ng / mg or more, about 110 ng / mg or more, or about 120 ng / mg or more per total protein. , about 130 ng / mg or more, about 140 ng / mg or more, about 150 ng / mg or more, about 160 ng / mg or more, about 170 ng / mg or more, about 180 ng / mg or more, about 190 ng / mg or more, about 200 ng / mg, about 230 ng / mg or more, about 250 ng / mg or more, or about 260 ng / mg, or about 270 ng / mg or more indicates that the subject is at risk of developing a food allergy by the age of 3.

[0037] In one example, if the SCCA-1 level in a skin stratum corneum sample collected by tape stripping from the abdomen of a 2-month-old infant is higher than the cutoff value of 65 ng / mg of total protein, the subject is at risk of developing food allergies by the age of 3.

[0038] In one example, if the SCCA-1 level in a skin stratum corneum sample collected by tape stripping from the abdomen of a 2-month-old infant is equal to or higher than the cutoff value of 89.5 ng / mg of total protein, this indicates that the subject is likely to develop food allergies by the age of 3.

[0039] In one example, if the SCCA-1 or SCCA-2 level per total protein in a skin stratum corneum sample collected by tape stripping from the thigh of a 2-month-old infant subject is, for example, about 60 ng / mg or more, about 70 ng / mg or more, about 80 ng / mg or more, about 90 ng / mg or more, about 100 ng / mg or more, about 110 ng / mg or more, about 120 ng / mg or more, about 130 ng / mg or more, about 140 ng / mg or more, about 150 ng / mg or more, about 160 ng / mg or more, about 170 ng / mg or more, about 180 ng / mg or more, about 190 ng / mg or more, about 200 ng / mg or more, about 205 ng / mg or more, or about 210 ng / mg or more, this indicates that the subject is likely to develop a food allergy by the age of 3.

[0040] In one example, if the SCCA-1 level in a skin stratum corneum sample collected by tape stripping from the thigh of a 2-month-old infant is equal to or higher than the cutoff value of 208.8 ng / mg of total protein, this indicates that the subject is likely to develop food allergies by the age of 3.

[0041] In one example, if the SCCA-1 or SCCA-2 value per total protein in a stratum corneum sample collected by tape stripping from around the mouth of a 2-month-old infant is, for example, about 500 ng / mg or more, about 600 ng / mg or more, about 700 ng / mg or more, about 750 ng / mg or more, about 760 ng / mg or more, about 800 ng / mg or more, or about 900 ng / mg or more, this indicates that the subject is likely to develop a food allergy by the time the subject reaches 18 months of age.

[0042] In one example, if the SCCA-1 level in a skin stratum corneum sample collected by tape stripping from around the mouth of a 2-month-old infant is higher than the cutoff value of 761 ng / mg of total protein, it indicates that the subject is likely to develop food allergies by the age of 18 months.

[0043] In one example, if the SCCA-1 or SCCA-2 value per total protein in a skin stratum corneum sample collected by tape stripping from the abdomen of a 4- to 5-month-old infant subject is, for example, about 200 ng / mg or more, about 250 ng / mg or more, about 300 ng / mg or more, about 350 ng / mg or more, about 360 ng / mg or more, about 400 ng / mg or more, or about 500 ng / mg or more, this indicates that the subject is likely to develop a food allergy by the time the subject reaches 18 months of age.

[0044] In one example, if the SCCA-1 level in a skin stratum corneum sample collected by tape stripping from the abdomen of a 4- to 5-month-old infant is higher than the cutoff value of 366 ng / mg of total protein, it indicates that the subject is likely to develop food allergy by the age of 18 months.

[0045] In one example, if the SCCA-1 or SCCA-2 level in a skin stratum corneum sample collected by tape stripping from the abdomen of a 2-month-old infant subject is, for example, about 50 ng / mg or more, about 60 ng / mg or more, about 70 ng / mg or more, about 80 ng / mg or more, about 85 ng / mg or more, about 86 ng / mg or more, or about 90 ng / mg or more per total protein, this indicates that the subject is likely to develop a food allergy by 8 to 9 months of age.

[0046] In one example, if the SCCA-1 level in a skin stratum corneum sample collected by tape stripping from the abdomen of a 2-month-old infant is higher than the cutoff value of 86 ng / mg of total protein, this indicates that the subject is likely to develop food allergies by 8 to 9 months of age.

[0047] In one example, if the SCCA-1 or SCCA-2 level in a skin stratum corneum sample collected by tape stripping from the abdomen of a 4- to 5-month-old infant subject is, for example, about 200 ng / mg or more, about 250 ng / mg or more, about 300 ng / mg or more, about 350 ng / mg or more, about 360 ng / mg or more, about 400 ng / mg or more, or about 500 ng / mg or more per total protein, this indicates that the subject is likely to develop a food allergy by the time the subject is 8 to 9 months of age.

[0048] In one example, if the SCCA-1 level in a skin stratum corneum sample collected by tape stripping from the abdomen of a 4- to 5-month-old infant is higher than the cutoff value of 366 ng / mg of total protein, this indicates that the subject is likely to develop food allergies by 8 to 9 months of age.

[0049] In one example, if the SCCA-1 or SCCA-2 value per total protein in a stratum corneum sample collected by tape stripping from the back of a 2-month-old infant subject is, for example, about 60 ng / mg or more, about 70 ng / mg or more, about 80 ng / mg or more, about 90 ng / mg or more, about 91 ng / mg or more, about 92 ng / mg or more, or about 95 ng / mg or more, this indicates that the subject is likely to develop a food allergy by the time the subject is 4 to 5 months of age.

[0050] In one example, if the SCCA-1 level in a skin stratum corneum sample collected by tape stripping from the back of a 2-month-old infant is higher than the cutoff value of 92 ng / mg of total protein, this indicates that the subject is likely to develop food allergies by 4 to 5 months of age.

[0051] In one example, if the SCCA-1 or SCCA-2 value per total protein in a stratum corneum sample collected by tape stripping from the back of a 2-month-old infant subject is, for example, about 100 ng / mg or more, about 110 ng / mg or more, about 120 ng / mg or more, about 130 ng / mg or more, about 140 ng / mg or more, about 150 ng / mg or more, about 200 ng / mg or more, about 220 ng / mg or more, about 230 ng / mg, about 240 ng / mg, or about 250 ng / mg or more, this indicates that the subject is likely to develop a food allergy by the age of 3.

[0052] In one example, if the SCCA-1 level in a skin stratum corneum sample collected by tape stripping from the back of a 2-month-old infant is equal to or higher than the cutoff value of 102.1 ng / mg of total protein, this indicates that the subject is likely to develop food allergies by the age of 3.

[0053] In one example, if the SCCA-1 or SCCA-2 level in a skin stratum corneum sample collected by tape stripping from the abdomen of a 2-month-old infant subject is, for example, about 400 ng / mg or more, about 450 ng / mg or more, about 460 ng / mg or more, about 470 ng / mg or more, about 473 ng / mg or more, about 475 ng / mg or more, or about 500 ng / mg or more per total protein, this indicates that the subject is likely to develop a food allergy by the time the subject is 4 to 5 months of age.

[0054] In one example, if the SCCA-1 level in a skin stratum corneum sample collected by tape stripping from the abdomen of a 2-month-old infant is higher than the cutoff value of 474 ng / mg of total protein, it indicates that the subject is likely to develop food allergies by 4 to 5 months of age.

[0055] Regarding the reference value, for example, in one aspect of the evaluation, the reference value can be set as follows based on the average value of the SCCA-1 or SCCA-2 values ​​at each time point and site in the food allergy-developed group and the non-developed group.

[0056] In one example, if the SCCA-1 or SCCA-2 value per total protein in a skin stratum corneum sample collected by tape stripping from the abdomen of a 18-month-old infant or toddler subject is, for example, about 107 ng / mg or more, about 120 ng / mg or more, about 150 ng / mg or more, about 170 ng / mg or more, about 180 ng / mg or more, about 190 ng / mg or more, or about 192 ng / mg or more, this indicates that the subject is likely to develop a food allergy by the time the subject is 18 months of age.

[0057] In one example, if the SCCA-1 or SCCA-2 level in a skin stratum corneum sample collected by tape stripping from the cheek of a 4-5 month old infant is, for example, about 1086 ng / mg or more, about 1100 ng / mg or more, about 1500 ng / mg or more, about 2000 ng / mg or more, about 3000 ng / mg or more, about 4000 ng / mg or more, or about 4017 ng / mg or more per total protein, this indicates that the subject is likely to develop a food allergy by the time the subject is 4-5 months old.

[0058] In one example, if the SCCA-1 or SCCA-2 value in a skin stratum corneum sample collected by tape stripping from around the mouth of a 4- to 5-month-old infant subject is, for example, about 1083 ng / mg or more, about 1100 ng / mg or more, about 1500 ng / mg or more, about 2000 ng / mg or more, about 2500 ng / mg or more, about 3000 ng / mg or more, or about 3287 ng / mg or more per total protein, this indicates that the subject is likely to develop a food allergy by the time the subject is 4 to 5 months of age.

[0059] In one example, if the SCCA-1 or SCCA-2 value per total protein in a skin stratum corneum sample collected by tape stripping from the back of a 4-5 month old infant or toddler subject is, for example, about 219 ng / mg or more, about 250 ng / mg or more, about 300 ng / mg or more, about 500 ng / mg or more, about 700 ng / mg or more, about 800 ng / mg or more, or about 819 ng / mg or more, this indicates that the subject is likely to develop a food allergy by the time the subject is 4-5 months old.

[0060] In one example, if the SCCA-1 or SCCA-2 level in a skin stratum corneum sample collected by tape stripping from the back of a 2-month-old infant subject is, for example, about 145 ng / mg or more, about 200 ng / mg or more, about 300 ng / mg or more, about 400 ng / mg or more, about 500 ng / mg or more, about 600 ng / mg or more, or about 619 ng / mg or more per total protein, this indicates that the subject is likely to develop a food allergy by the age of 2 months.

[0061] The method of the present invention uses the expression level of SCCA as an index, and therefore does not require the involvement of a physician or medical professional. Thus, one embodiment of the present invention is a method for assisting in the evaluation or prediction of the possibility of developing a food allergy without requiring a diagnosis by a physician or medical professional.

[0062] The present invention also provides a kit for assessing or predicting the likelihood of developing a food allergy. The kit of the present invention includes a medium for collecting a biological sample from a subject and a reagent for measuring SCCA expression in the biological sample collected using the medium. If the SCCA expression in the biological sample measured using the kit is higher than a reference value, it indicates that the subject is at risk of developing a food allergy. The age, collection site, reference value, and timing of assessment or prediction of the subject's biological sample, as well as the type of food allergy, of the subject are as described above. The medium preferably includes a tape as described above for application to the subject's skin, and the biological sample is collected non-invasively by tape stripping. The reagent is not limited as long as it is used to measure SCCA expression, and examples include reagents necessary for measuring SCCA expression using the above-described method. Examples include an anti-SCCA-1 monoclonal antibody, an anti-SCCA-2 monoclonal antibody, an anti-SCCA-1 polyclonal antibody, an anti-SCCA-2 polyclonal antibody, or a reagent for measuring any of these antibodies, which are necessary for an ELISA assay to measure SCCA levels.

[0063] The present invention also provides a system for assessing or predicting the likelihood of developing a food allergy, comprising: a data acquisition unit that acquires data on SCCA expression from a biological sample collected from a subject; an evaluation or prediction unit that refers to the data acquired by the data acquisition unit and assesses or predicts that the subject is likely to develop a food allergy if SCCA expression is high; and a display unit that displays the results of the evaluation or prediction by the evaluation or prediction unit. The age, collection site, reference value, and timing of evaluation or prediction of the subject from whom the biological sample is collected, as well as the type of food allergy, are as described above.

[0064] The present invention will be described in more detail below with reference to specific examples, although the present invention is not limited thereto.

[0065] 1. Measurement and observation items (1) Subjects 117 2-month-old infants (55 females, 62 males) (at the start of measurements) (2) Measurement sites Cheeks, around the mouth, back, abdomen, and front of the thighs of the subjects (3) Measurement time points SCCA-1 expression levels and food allergy status were measured over time using the following methods at 2 months, 4-5 months, 8-9 months, 1 year and 6 months, and 3 years of age.

[0066] 2. Measurement of SCCA-1 Expression Levels (1) Collection and Measurement of Stratum Corneum Tapes Stratum corneum samples were collected from each test site (perioral, cheek, back, abdomen, and anterior thigh; for 1 year 6 months and 3 years old, only the back, abdomen, and anterior thigh) using Cellotape (registered trademark) (2 cm x 6 cm). The obtained stratum corneum samples were immersed in a protein extract (0.1 M Tris-HCl (pH 8.0), 0.14 M NaCl, 0.1% Tween 20) and extracted using a Bioruptor. After centrifugation, the supernatant was used for ELISA. (2) Immunological SCCA-1 Measurement Method Recombinant human SCCA-1 was obtained by inserting SCCA-1 cDNA into the pQE30 vector. The SCCA-1 assay method described here involved purifying the recombinant with Ni-Agarose, then further purifying it with Mono Q chromatography, and using it as the standard for the ELISA assay. A rabbit anti-SCCA-1 polyclonal antibody (manufactured by Shiseido) was immobilized on a 96-well plate as the primary antibody. After blocking with ImmunoBlock (DS Pharma Biomedical, Osaka, Japan), 100 μl of stratum corneum extract was added to each well, and the plate was incubated for 1 hour at 37°C. A monoclonal anti-SCCA1 antibody (Santa Cruz) was used as the secondary antibody, and the incubation was continued for 1 hour at 37°C. Then, horseradish peroxidase-conjugated anti-mouse F(ab')2 (GE Healthcare) was added, and color development was carried out using a TMB peroxidase EIA substrate kit (Bio-Rad). The reaction was stopped with 1 M H2SO4 and measured at 450 nm.

[0067] 3. Doctor's findings Even experienced doctors can have difficulty determining the severity of food allergies, and it is extremely difficult for ordinary people without medical experience to determine the severity. In this example, an experienced doctor examined the food allergy at the above measurement period, comprehensively assessed the following items, classified the presence or absence of food allergies, and identified the foods that were the allergic causes: - Allergic symptoms (skin, mouth, eyes, respiratory tract, digestive tract) appear when exposed to a specific food - Food allergies have been diagnosed by a family doctor

[0068] 4. Creation of ROC curves The SCCA-1 values ​​at each test site at each measurement time point and the presence or absence of onset of food allergy up to age 3 were plotted on a plane and connected with a line to create an ROC curve. The cutoff value was determined using a logistic regression model with the presence or absence of onset of food allergy at age 3 as the objective variable and the SCCA expression value as the explanatory variable. In this case, a method was used that maximized the Youden Index ("sensitivity - (1 - specificity)")

[0069] 5. Results Figures 1 to 9 show the SCCA-1 levels in samples collected from each test site at each measurement time point, divided into those with and without the onset of food allergy up to that point. It can be seen that there is a correlation between the SCCA-1 levels and the onset of food allergy in infants.

[0070] As shown in Figure 1, there was a significant correlation between SCCA-1 levels in perioral and abdominal samples of infants at 2 months of age and the development of food allergies by age 3 years. Figure 2 shows the ROC curves for SCCA-1 levels in samples taken from the perioral (left) and abdominal (right) areas of 2-month-old infants and the presence or absence of food allergies by age 3 years. When the cutoff value for SCCA-1 in samples taken from the perioral area of ​​2-month-old infants was set at 761 ng / mg (SCCA amount / total protein amount), the sensitivity was 0.6563 and the specificity was 0.5395. When the cutoff value for SCCA-1 in samples taken from the abdominal area of ​​2-month-old infants was set at 65 ng / mg (SCCA amount / total protein amount), the sensitivity was 0.8333 and the specificity was 0.4143.

[0071] As shown in Figure 3, there was a significant correlation between SCCA-1 levels in the perioral area of ​​infants at 2 months of age and in the abdominal area of ​​infants aged 4-5 months and the onset of food allergies by 18 months of age. Figure 4 shows ROC curves for SCCA-1 levels in samples taken from the perioral area of ​​2-month-old infants (left) and the abdominal area of ​​4-5-month-old infants (right) and the presence or absence of food allergy by 18 months of age. When the cutoff value for SCCA-1 in samples taken from the perioral area of ​​2-month-old infants was set to 761 ng / mg (SCCA amount / total protein amount), the sensitivity was 0.6667 and the specificity was 0.5432. When the cutoff value for SCCA-1 in samples taken from the abdominal area of ​​infants aged 4-5 months of age was set to 366 ng / mg (SCCA amount / total protein amount), the sensitivity was 0.2069 and the specificity was 0.9481.

[0072] Furthermore, as shown in Figure 3, there was a significant correlation between abdominal SCCA-1 levels in infants at 1 year and 6 months of age and the onset of food allergies up to 1 year and 6 months of age. In the group without food allergies, the mean abdominal SCCA-1 level at 1 year and 6 months of age was 107 ng / mg, while in the group with food allergies, it was 192 ng / mg.

[0073] As shown in Figure 5, there was a significant correlation between SCCA-1 levels in the backs of infants at 2 months and in the abdomens of infants at 4 to 5 months and the onset of food allergies by 8 to 9 months. Figure 6 shows the ROC curves for SCCA-1 levels in samples taken from the abdomens of 2-month-old infants (left) and 4- to 5-month-old infants (right) in Figure 5 and the presence or absence of food allergies by 8 to 9 months. When the cutoff value for SCCA-1 in samples taken from the abdomens of 2-month-old infants was set to 86 ng / mg (SCCA amount / total protein amount), the sensitivity was 0.7895 and the specificity was 0.5172. When the cutoff value for SCCA-1 in samples taken from the abdomens of 4- to 5-month-old infants was set to 366 ng / mg (SCCA amount / total protein amount), the sensitivity was 0.2500 and the specificity was 0.9432.

[0074] As shown in Figure 7, there was a significant correlation between SCCA-1 levels in the back or abdomen of infants at 2 months of age and the onset of food allergies by 4 to 5 months of age. Figure 8 shows the ROC curves for SCCA-1 levels in samples taken from the back (left) and abdomen (right) of 2-month-old infants in Figure 7 and the presence or absence of food allergy onset by 4 to 5 months of age. When the cutoff value for SCCA-1 in samples taken from the back of 2-month-old infants was set to 92 ng / mg (SCCA amount / total protein amount), the sensitivity was 1.000 and the specificity was 0.4545. When the cutoff value for SCCA-1 in samples taken from the abdomen of 2-month-old infants was set to 474 ng / mg (SCCA amount / total protein amount), the sensitivity was 0.5000 and the specificity was 0.9709.

[0075] Furthermore, as shown in Figure 7, there was a significant correlation between the SCCA-1 levels in the cheeks, perioral areas, or backs of infants at 4 to 5 months of age and the onset of food allergies by 4 to 5 months of age. In the food allergy-free group, the mean SCCA-1 levels in the cheeks, perioral areas, or backs at 1 year and 6 months of age were 1086 ng / mg, 1083 ng / mg, and 219 ng / mg, respectively, while in the food allergy-developing group, the mean values ​​were 4017 ng / mg, 3287 ng / mg, and 819 ng / mg.

[0076] Furthermore, as shown in Figure 9, there was a significant correlation between the SCCA-1 levels on the backs of infants at 2 months of age and the onset of food allergies up to 2 months of age. The mean SCCA-1 levels on the backs of infants at 2 months of age in the group without food allergies were 145 ng / mg, while in the group with food allergies the mean was 619 ng / mg.

[0077] Figure 10 shows the age / month of subjects who developed food allergies and the type of food allergy. Infants aged 2 months (2M) or 4 to 5 months (4-5M) often only consume powdered milk, so powdered milk accounts for 100%. However, as they grow, opportunities to consume a variety of foods increase, and the types of foods that can cause allergies also increase at the ages of 8 to 9 months (8-9M), 1 year and 6 months (1Y6M), and 3 years (3Y).

[0078] Since Example 1 suggested that it is possible to evaluate or predict the onset of food allergy up to the age of three at two months of age, subjects were selected under stricter conditions and data analysis was performed. 1. Measurement and observation items (1) Subjects 91 infants and toddlers aged 2 months (at the start of measurements) (after the following exclusions) (2) Measurement sites Perioral, back, abdomen, and thighs of subjects (3) Measurement time At two months and three years of age, the expression level of SCCA-1 and the state of food allergy were measured using the following methods.

[0079] 2. Measurement Method The expression level of SCCA-1 was measured using the same method as in Example 1, but the food allergy state was measured under stricter conditions than in Example 1. The food allergy state was diagnosed by an experienced pediatrician. Patients who exhibited one or more clinical symptoms of rash, runny nose, sneezing, cough, wheezing, vomiting, and diarrhea due to allergen foods and whose sensitization was confirmed by blood tests or skin prick tests were diagnosed as having food allergies. To obtain more accurate results, cases in which clinical symptoms were observed but blood tests or skin prick tests were not performed, and cases in which clinical symptoms could not be confirmed because food allergens were eliminated first were excluded from the analysis. An ROC curve was created using the same method as in Example 1, and a cutoff value was determined from the ROC curve using the Youden Index method. This cutoff value was used as the basis for calculating the odds ratio. Univariate analysis was performed using JMP Pro version 15.0.0 (SAS Institute Inc., Cary, NC, USA) with the SCCA-1 cutoff value in samples collected from each measurement site at 2 months of age as the explanatory variable, to calculate odds ratios for the development of food allergy by age 3 years.

[0080] 3. Results After the above exclusions, 91 infants were included in the analysis. Of these, 32 showed clinical symptoms induced by ingestion of allergen foods by the age of 3 years, of which 28 had atopic dermatitis, and 16 were confirmed to have been sensitized by blood tests or skin prick tests. Therefore, 16 were diagnosed with food allergy by the age of 3 years, and 75 were diagnosed as not having food allergy.

[0081] Table 1 shows the mean SCCA-1 levels (ng / mg = SCCA amount / total protein amount) in samples taken from each test site at 2 months of age, divided by the presence or absence of food allergy by the age of 3 years (t-test).

[0082] Analysis under strict conditions in Example 2 showed a correlation between SCCA-1 values ​​at all measurement sites in infants at 2 months of age and the onset of food allergies by age 3, as shown in Table 1. Figure 11 shows ROC curves for the relationship between SCCA-1 values ​​in samples collected from the perioral region (left) and back (right) of 2-month-old infants and the presence or absence of food allergy by age 3. When the cutoff value for SCCA-1 in samples collected from the perioral region of 2-month-old infants was set to 825.2 ng / mg (SCCA amount / total protein amount), the sensitivity was 0.81 and the specificity was 0.57. When the cutoff value for SCCA-1 in samples collected from the back of 2-month-old infants was set to 102.1 ng / mg (SCCA amount / total protein amount), the sensitivity was 0.69 and the specificity was 0.55. Figure 12 shows ROC curves for the relationship between SCCA-1 levels in samples collected from the abdomen (left) and thigh (right) of 2-month-old infants and the presence or absence of food allergy by age 3. When the cutoff value for SCCA-1 in samples collected from the abdomen of 2-month-old infants was set at 89.5 ng / mg (SCCA amount / total protein amount), the sensitivity was 0.87 and the specificity was 0.51. When the cutoff value for SCCA-1 in samples collected from the thigh of 2-month-old infants was set at 208.8 ng / mg (SCCA amount / total protein amount), the sensitivity was 0.25 and the specificity was 0.95.

[0083] The results of the univariate analysis are shown in Table 2.

[0084] As shown in Table 2, the odds ratio was far greater than 1 for all SCCA-1 values ​​at 2 months of age, whether measured around the mouth, back, abdomen, or thigh. In particular, it was shown that when SCCA-1 values ​​at 2 months of age were equal to or greater than the respective cutoff values ​​in the perioral, abdominal, or thigh regions, there was a high possibility of the development of food allergy by the age of 3. These results demonstrate that even with analysis under more strict conditions, the SCCA-1 value at 2 months of age can be used as an index for assessing or predicting the possibility of developing food allergy by the age of 3.

[0085] As mentioned above, since it is important to avoid foods at an early stage in the treatment of food allergies, it is extremely advantageous to be able to evaluate or predict the possibility of onset at an early stage. Furthermore, SCCA can be collected by a very simple and non-invasive procedure, such as tape stripping. With the development of a simple measurement kit, SCCA measurement can be performed at home, not just in a hospital or laboratory. Therefore, it is extremely advantageous to be able to easily evaluate or predict the possibility of disease at home, especially for infants and young children who are unable to report symptoms themselves.

[0086] By using SCCA as an indicator, the present invention makes it possible to provide a method for evaluating or predicting the possibility of developing a food allergy using a simple and objective indicator.

Claims

1. A method for assisting in the evaluation or prediction of the likelihood of developing food allergy, using the expression level of SCCA in a biological sample collected from a subject as an indicator.

2. A method for assisting in the evaluation or prediction of the likelihood that a subject, who is an infant aged 2 months to 3 years, will develop food allergy by the age of 3 years, using the expression of SCCA in a biological sample collected from the subject as an indicator.

3. A method for examining a biological sample collected from a subject to determine whether the subject is likely to develop food allergy, the method comprising measuring the expression of SCCA in the biological sample.

4. A method for examining a biological sample collected from a subject, who is an infant aged 2 months to 3 years, to determine whether the subject is likely to develop food allergy by the age of 3 years, the method comprising measuring the expression of SCCA in the biological sample.

5. The method according to any one of claims 1 to 4, comprising a step of comparing the expression value of SCCA in the biological sample with a reference value, and indicating that the subject is likely to develop food allergy when the expression of SCCA in the biological sample is higher than the reference value.

6. The method according to claim 5, wherein the reference value is a cut-off value.

7. The method according to any one of claims 1 to 4, wherein the SCCA is SCCA-1.

8. The method according to any one of claims 1 to 4, wherein the biological sample is collected non-invasively by tape stripping.

9. A kit for evaluating or predicting the likelihood of developing food allergy, comprising a medium for collecting a biological sample from a subject and a reagent for measuring the expression of SCCA in the biological sample collected by the medium.

10. The kit according to claim 9, wherein the medium is a tape for attaching to the skin of the subject, and the biological sample is collected non-invasively by tape stripping.

11. A system for evaluating or predicting the likelihood of developing food allergy, comprising: a data acquisition unit that acquires data related to SCCA expression from a biological sample collected from a subject; an evaluation or prediction unit that refers to the data acquired by the data acquisition unit and evaluates or predicts that the likelihood of the subject developing food allergy is high when the SCCA expression is high; and a display unit that displays the result evaluated or predicted by the evaluation or prediction unit.

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