JP7328950B2 - Method for detecting infant atopic dermatitis - Google Patents

Description

The present invention relates to a method for detecting infant atopic dermatitis.

Atopic dermatitis (hereinafter also referred to as “AD”) is an eczematous skin disease that mainly develops in people with atopic predisposition. Typical symptoms of atopic dermatitis are bilaterally occurring chronic and recurrent itching, eruptions, erythema, etc., as well as hypokeratosis, impaired barrier function, dry skin, and the like. Most AD develops in infants and tends to improve with growth, but adult-type and refractory AD are increasing in recent years.

Newborns or infants who have a genetic predisposition to allergies and atopy may develop various allergic diseases (allergic march) with age, such as atopic dermatitis, food allergies, bronchial asthma, and allergic rhinitis. Are known. In this way, when one allergic disease develops, the possibility of developing another allergic disease increases, and the treatment is often long-term. It is said that

Methods for detecting AD using biomarkers include blood peripheral blood eosinophil count, serum total IgE level, LDH (lactate dehydrogenase) level, serum Thymus and Activation-Regulated Chemokine (TARC) and squamous cell carcinoma antigen. It has been proposed to detect 1 (SCCA1 or SerpinB3) and 2 (SCCA2 or SerpinB4) (Non-Patent Documents 1, 2, 3). AD detection by biomarkers is particularly effective in infants who have difficulty in expressing symptoms.

On the other hand, atopic dermatitis biomarkers may have different efficacy depending on the patient's age, eg, pediatric or adult. For example, it has been reported that the sensitivity and specificity of determination of the above serum TARC is lower in children under 2 years of age than in children 2 years of age or older (Non-Patent Document 4). Blood IL-18 has been reported as an effective marker for detecting infant AD (Non-Patent Document 5). In addition, it has been reported that serum SerpinB4 is effective in detecting pediatric and adult AD (Non-Patent Documents 6 and 7). In addition, it has been reported that a decrease in SerpinB12 and an increase in SerpinB3 were observed in the stratum corneum collected from children with AD (Non-Patent Document 8). not

Recently, it has been reported that RNA contained in skin surface lipids (SSL) can be used as a sample for bioanalysis, and that marker genes of the epidermis, sweat glands, hair follicles and sebaceous glands can be detected from SSL (Patent Reference 1). It has also been reported that a marker gene for atopic dermatitis can be detected from SSL (Patent Document 2). On the other hand, there is almost no knowledge about proteins contained in SSL, and it is completely unknown whether protein markers in blood and stratum corneum can be similarly expressed in SSL and have similar accuracy.

International Publication No. 2018/008319 JP 2020-074769 A

Sugawara et al., Allergy (2002) 57:180-181 Ohta et al., Ann Clin Biochem (2012) 49:277-84 Kato et al., Journal of Nissinkai (2018) 128: 2431-2502 Fujisawa et al., Japanese Journal of Pediatric Allergy (2005) 19(5):744-757 Ohnishi et al., Allergology International (2003) 52:123-130 Nagao et al., J Allergy Clin Immunol (2018) 141(5):1934-1936 Okawa et al., Allergology International (2018) 67:124-130 Goleva et.al., J Allergy Clin Immunol (2020) S0091-6749(20):30571-6

The present invention provides a method for detecting infant atopic dermatitis using a skin surface lipid (SSL)-derived protein marker for infant atopic dermatitis detection.

The present inventors found that the expression level of SerpinB4 protein is elevated in SSL collected from infants with atopic dermatitis, and that infant atopic dermatitis can be detected using this as an index.

Accordingly, the present invention provides a method for detecting infant atopic dermatitis in an infant subject, comprising the step of measuring the expression level of SerpinB4 protein in superficial skin lipids collected from the infant subject.
The present invention also provides a test kit for detecting atopic dermatitis in infants, which contains an antibody that recognizes SerpinB4 protein and is used in the method for detecting atopic dermatitis in infants.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to detect infant atopic dermatitis by a simple and non-invasive method.

Fig. 10 is a boxplot showing the expression level of SerpinB4 protein in SSL derived from a healthy area (face) of a healthy group (HL) and an eruption area (face) of an AD group (AD). The plots of each data are shown in the figure, and the lowest and highest ends of the whiskers are the minimum and maximum values of the data, the first quartile from the bottom of the box, the second quartile (median), The third quartile was displayed (as well as in Figures 2-4 and 7-11 below). ***: P<0.001 (Student's t-test). Fig. 3 is a boxplot showing the expression levels of SerpinB4 protein in SSL derived from the healthy area (face) of a healthy group (HL) of infants, and the eruption area (face) of a mild AD group (Mild) and a moderate AD group (Moderate). . *: P<0.05, ***: P<0.001 (Tukey's test). Fig. 10 is a boxplot showing the expression level of SerpinB4 protein in SSL derived from a healthy part (back) of a healthy group (HL) of infants and a non-erupted part (back) of an AD group (AD). **: P<0.01 (Student's t-test). Boxplots showing the expression levels of SerpinB4 protein in SSL derived from a healthy part (back) of a healthy group (HL) of infants and a non-erupted part (back) of a mild AD group (Mild) and a moderate AD group (Moderate). be. *: P<0.05 (Tukey's test). ROC curve of SerpinB4 protein expression level in SSL derived from healthy area (face) of healthy group (HL) and eruption area (face) of AD group (AD). ROC curve of SerpinB4 protein expression level in SSL from healthy area (back) of healthy group (HL) and non-rash area (back) of AD group (AD). Fig. 3 is a boxplot showing the expression level of SerpinB4 RNA in SSL derived from a healthy area (face) of a healthy group (HL) of infants and an eruption area (face) of an AD group (AD). n. s. : No significant difference (Student's t-test). Fig. 2 is a boxplot showing the expression level of SerpinB4 protein in SSL derived from a healthy area (face) of a healthy adult group (HL) and an eruption area (face) of an AD group (AD). n. s. : No significant difference (Student's t-test). Fig. 3 is a boxplot showing the expression level of IL-18 protein in SSL derived from a healthy part (back) of a healthy group (HL) and a rash (back) of an AD group (AD). n. s. : No significant difference (Student's t-test). Fig. 2 is a boxplot showing the expression level of SerpinB12 protein in SSL derived from a healthy area (face) of a healthy group (HL) and an eruption area (face) of an AD group (AD). n. s. : No significant difference (Student's t-test). Fig. 10 is a boxplot showing the expression level of SerpinB12 protein in SSL derived from a healthy part (back) of a healthy group (HL) and a non-erupted part (back) of an AD group (AD). n. s. : No significant difference (Student's t-test).

All patents, non-patents, and other publications cited herein are hereby incorporated by reference in their entirety.

The names of the proteins disclosed herein follow the Gene Name or Protein Name described in UniProt ([www.uniprot.org/]).

As used herein, "SerpinB4" refers to a protein belonging to the serine protease inhibitor (Serpin) family, also called squamous cell carcinoma antigen 2 (SCCA-2) or leupin. The SerpinB4 protein has been deposited with UniProt as P48594.

As used herein, "skin surface lipids (SSL)" refers to a fat-soluble fraction present on the surface of the skin, and is sometimes called sebum. In general, SSL mainly contains secretions secreted from exocrine glands such as sebaceous glands in the skin, and exists on the skin surface in the form of a thin layer covering the skin surface.

As used herein, the term “skin” is a general term for regions including epidermis, dermis, hair follicles, and tissues such as sweat glands, sebaceous glands, and other glands, unless otherwise specified.

As used herein, "atopic dermatitis" (AD) refers to a disease whose main pathogen is itchy eczema, which repeats exacerbation and remission, and many of its patients are said to have atopic predisposition. there is Atopic predisposition includes: i) family history and medical history (one or more of bronchial asthma, allergic rhinitis/conjunctivitis, atopic dermatitis, and food allergy), or ii) predisposition to easily produce IgE antibodies. , are mentioned.

As used herein, the term "infant" is broadly defined as a concept including "children" before the onset of secondary sexual characteristics, specifically children under the age of 12, preferably from 0 years old to school children. It refers to the age before entering, specifically infants from 0 to 5 years old. Eczema in atopic dermatitis that develops in infants begins on the head or face in infancy and often descends to the trunk or extremities, and after 1 year of age, the eruption on the face decreases, and the eruption on the neck or joints of the extremities. It is characterized by appearing around Regarding the difference between infant atopic dermatitis and adult atopic dermatitis, it has recently been reported that abnormal epidermal keratinization associated with chronic inflammation is observed in adult atopic dermatitis compared to infant atopic dermatitis. (Journal of allergy and clinical immunology, 141(6):2094-2106 (2018)), but the number of reports is small and it is not clear.

The degree of progression (severity) of atopic dermatitis is classified into, for example, no symptoms, slight, mild (mild), moderate (moderate), and severe (severe). The severity should be classified based on the severity evaluation method described in, for example, the guidelines for the treatment of atopic dermatitis (published by the Japanese Dermatological Association, Journal of the Japan Dermatology Association, 128(12): 2431-2502, 2018 (Heisei 30)). can be done. Clinical practice guidelines for atopic dermatitis describe several severity assessment methods, e.g., statistically reliable and valid severity classification methods for assessing overall severity. As, atopic dermatitis severity classification by the Japanese Dermatological Association Atopic Dermatitis Severity Classification Review Committee (Journal of the Japanese Dermatology Association, 111: 2023-2033 (2001), Journal of the Japanese Dermatology Association, 108: 1491-1496 (1998) ), Severity Scoring of Atopic Dermatitis (“SCORAD”; Dermatology, 186:23-31 (1993), Eczema Area and Severity Index (“EASI”; Exp Dermatol, 10:11-18 (2001)), etc. Other severity classification methods described in the clinical practice guidelines for atopic dermatitis include eruption severity evaluation, pruritus evaluation, patient evaluation, and QOL evaluation methods.For example, EASI: The evaluation sites were the head and neck, trunk, upper limbs, and lower limbs, and erythema, edema/infiltration/papules, scratch marks, and lichenification at each evaluation site were evaluated. Calculated based on the percentage (%) of the symptom area, a value from 0 to 72. "Mild" if the EASI score is greater than 0 and less than 6, and "moderate" if the EASI score is greater than or equal to 6 and less than 23. If the EASI score is between 23 and 72, it is classified as "severe".

As used herein, "detection of infant atopic dermatitis" includes clarifying the presence (symptoms) or absence (no symptoms) of infant atopic dermatitis, as well as Defining the degree of progression, ie, "mild," "moderate," and "severe," is included. Preferably, each of "no symptoms", "mild" and "moderate" is detected.

As used herein, the term "detection" can also be interchanged with the terms inspection, measurement, determination, evaluation or evaluation support. In addition, the term "judgment" or "evaluation" used herein does not include judgment or evaluation by a doctor.

As shown in the examples below, samples were taken from the faces of healthy infants (healthy infants) and infants suffering from AD (AD patients) (healthy areas in healthy infants, eruption areas (including eruptions) in AD patients). Protein expression analysis in SSL revealed that the expression level of SerpinB4 protein was significantly elevated in children with AD. In addition, when the expression of SerpinB4 protein in SSL collected from the faces of healthy children, children with mild AD, and children with moderate AD was examined, the expression level of SerpinB4 protein increased depending on the severity of AD. Furthermore, when examining the expression of SerpinB4 protein in SSL collected from the backs of healthy children and children with AD (normal areas in healthy children, rash areas (not including eruption) in children with AD), the expression of SerpinB4 protein was found only in rash areas in AD patients. In addition, the expression level of SerpinB4 protein in SSL was elevated also in the non-erupted area.
On the other hand, there was no difference in the expression level of SerpinB4 RNA in SSL between healthy children and children with AD.
In adults, no difference was found in the expression level of SerpinB4 protein in SSL between healthy subjects and AD patients.

Since IL-18 protein in blood and SerpinB12 protein in the stratum corneum are known as AD markers (Non-Patent Documents 5 and 8), the expression of IL-18 protein and SerpinB12 protein in SSL of children with AD was examined. . As a result, as shown in Examples described later, there was no difference in the expression levels of IL-18 protein and SerpinB12 protein in SSL between healthy children and AD patients.

These results indicate that the SerpinB4 protein in SSL is useful as an infant AD marker for detecting infant AD. In addition, SSL that can be collected noninvasively is an important biological sample source for infants, and when SSL is used as a biological sample, known marker proteins such as RNA of SerpinB4 and IL-18 and SerpinB12 are present. Given its unavailability as an infant AD marker, it is unexpected and extremely useful that the SerpinB4 protein in SSL can be used as an infant AD marker.

Accordingly, the present invention provides methods for detecting infant AD. A method for detecting infant AD according to the present invention comprises measuring the expression level of SerpinB4 protein in SSL obtained from an infant subject.

In the present invention, subjects from whom SSL is collected are not particularly limited in terms of sex, race, etc., as long as they are infants. Preferred examples of the subject include infants requiring detection of atopic dermatitis and infants suspected of developing atopic dermatitis.

The site of the skin from which the SSL is collected from the subject may include the skin of the head, face, neck, trunk, extremities, etc., and is not particularly limited. The site from which SSL is collected may or may not be a site where AD symptoms are observed on the skin, and may be, for example, an eruption site or a non-rash site.

Any means used to collect or remove SSL from the skin can be used to collect the SSL from the subject's skin. Preferably, an SSL absorbent material, an SSL adhesive material, or an instrument that scrapes the SSL off the skin, as described below, can be used. The SSL absorbent material or SSL adhesive material is not particularly limited as long as it has affinity for SSL, and examples thereof include polypropylene and pulp. More detailed examples of procedures for collecting SSL from the skin include a method of absorbing SSL into sheet-like materials such as blotting paper and blotting film, a method of adhering SSL to a glass plate, tape, etc., a spatula, a scraper, etc. and a method of scraping off and recovering SSL. In order to improve the adsorptivity of SSL, an SSL absorbent material previously impregnated with a solvent having high fat solubility may be used. On the other hand, if the SSL absorbent material contains a highly water-soluble solvent or moisture, the adsorption of SSL is inhibited, so it is preferable that the content of the highly water-soluble solvent and moisture is small. The SSL absorbent material is preferably used dry.

The SSL collected from the subject may be immediately used in the protein extraction step described below, or may be stored until used in the protein extraction step. When storing, the SSL is preferably stored under low temperature conditions as soon as possible after collection. The temperature condition for storing SSL may be 0° C. or lower, preferably -20±20° C. to -80±20° C., more preferably -20±10° C. to -80±10° C., still more preferably -20° C. ±20°C to -40±20°C, more preferably -20±10°C to -40±10°C, more preferably -20±10°C, still more preferably -20±5°C. The SSL storage period is not particularly limited, but is preferably 12 months or less, for example, 6 hours or more and 12 months or less, more preferably 6 months or less, for example, 1 day or more and 6 months or less, further preferably 3 months or less, such as 3 days or more and 3 months or less.

Protein extraction from collected SSL includes methods commonly used for protein extraction or purification from biological samples, such as water, phosphate-buffered saline solutions, or surfactants such as Triton X-100 and Tween 20. Solution extraction method, or M-PER buffer (Thermo Fisher Scientific), MPEX PTS Reagent (GL science), QIAzol Lysis Reagent (Qiagen), EasyPep ^TM Mini MS Prep Kit (ThermoFish er Scientific) and other commercially available protein extraction reagents A kit-based extraction method can be used.

In measuring the expression level of SerpinB4 protein by the method of the present invention, the amount or activity of SerpinB4 protein itself may be measured, or an antibody against SerpinB4 may be used. Alternatively, molecules that interact with SerpinB4 protein, such as other proteins, sugars, lipids, fatty acids, phosphorylated products thereof, alkylated products, sugar adducts thereof, etc., and the amount or activity of any of the above complexes may be measured. The expression level of SerpinB4 protein to be calculated may be a value based on the absolute amount of SerpinB4 protein in SSL or a value relative to other standard substances or total protein in SSL. Values are relative to total protein.

Techniques for measuring the expression level of SerpinB4 protein in the method of the present invention include Western blotting, protein chip analysis, immunoassays (e.g., ELISA), chromatography, mass spectrometry (e.g., LC-MS/MS, MALDI- TOF/MS), 1-hybrid method (PNAS, 100:12271-12276 (2003)), 2-hybrid method (Biol Reprod, 58:302-311 (1998)), etc. can be used. For example, the expression level of SerpinB4 protein can be measured by contacting an antibody against SerpinB4 protein with an SSL-derived protein sample and detecting protein in the sample bound to the antibody. For example, according to Western blotting, after using the above antibody as a primary antibody, an antibody that binds to the primary antibody labeled with a radioisotope, fluorescent substance, enzyme, etc. is used as a secondary antibody, and the primary antibody is Labeling is performed, and signals derived from these labeling substances are measured with a radiometer, a fluorescence detector, or the like. The primary antibody described above may be a polyclonal antibody or a monoclonal antibody. Commercially available products can be used for these antibodies. Alternatively, it can be produced according to a known method. Specifically, a polyclonal antibody is obtained by immunizing a non-human animal such as a rabbit using a protein expressed in Escherichia coli or the like and purified according to a conventional method, or by synthesizing a partial polypeptide of the protein according to a conventional method, It can be obtained from the serum of the immunized animal according to a conventional method. On the other hand, monoclonal antibodies are obtained by immunizing a non-human animal such as a mouse with a protein expressed in Escherichia coli or the like and purified according to a conventional method or a partial polypeptide of the protein, and fusing the obtained spleen cells with myeloma cells. It can be obtained from prepared hybridoma cells. Monoclonal antibodies may also be generated using phage display (Current Opinion in Biotechnology, 9(1):102-108 (1998)).

Thus, the expression level of SerpinB4 in SSL collected from a subject is measured, and infant AD is detected based on the expression level. In one example, the detection is performed by comparing the measured expression level of SerpinB4 to a reference value. More specifically, by comparing the expression level of SerpinB4 in SSL in a subject to a reference value, the presence or absence of, or the degree of progression of, infantile AD in the subject can be detected.

The "reference value" can be arbitrarily set according to the purpose of detection. An example of a "reference value" is the expression level of SerpinB4 protein in SSL in healthy children. For example, as the expression level of healthy children, a statistical value (eg, average value, etc.) of the expression level of SerpinB4 protein in SSL measured from a population of healthy children can be used. Depending on the purpose of detection, the expression level of SerpinB4 protein in SSL in children with mild or moderate AD may be used as a "reference value".

In one embodiment, the presence or absence of infantile AD is detected by comparing the expression level of SerpinB4 protein in SSL in a subject to a reference value based on a healthy population as described above. In one example, it is detected whether the expression level of SerpinB4 protein in SSL in a subject is higher than the reference value based on the healthy population described above. If the subject's expression level is now higher than the reference value, the subject can be detected as having infantile AD.

In another embodiment, the expression level of SerpinB4 protein in SSL in a subject is compared to the reference value based on the above-mentioned healthy child population, and the reference value based on the population of children with mild or moderate AD. is detected. In one example, it is detected whether the expression level of SerpinB4 protein in SSL in a subject is higher than respective reference values. For example, if the expression level of SerpinB4 protein in SSL in a subject is higher than the reference value based on the healthy population and equal to or higher than the reference value based on the moderate AD patient population, the subject is detected as having moderate AD. obtain. Alternatively, if the expression level of SerpinB4 protein in SSL in a subject is higher than a reference value based on a healthy population but lower than a reference value based on a moderate AD population, the subject can be detected as having mild AD.

In the above embodiments, for example, if the SerpinB4 protein expression level in SSL in a subject is preferably 110% or more, more preferably 120% or more, and still more preferably 150% or more relative to the reference value, then in the subject SerpinB4 protein expression levels in SSL can be judged to be "higher" than the reference value. Alternatively, reference values such as the mean +2SD, mean +SD, mean +1/2SD, or mean +1/3SD of the SerpinB4 protein expression level in SSL of a healthy child population or AD (for example, mild, moderate, etc.) patient population As such, it can be determined whether the SerpinB4 protein expression level in SSL in a subject is higher than a reference value.

Another example of a "reference value" is a cutoff value determined based on the expression levels of SerpinB4 protein in SSL measured from infant populations including healthy children and children with AD. The cutoff value can be determined by various statistical analysis methods. For example, a cutoff value based on ROC curve (Receiver Operating Characteristic curve) analysis is exemplified. The ROC curve shows the expression level of SerpinB4 protein in SSL measured from the infant population, the probability (%) of a positive result in positive patients (True Position Fraction (TPF), sensitivity) and negative in negative patients. It can be created by obtaining the probability (%) (specificity) that the result is obtained and plotting the sensitivity against [100-specificity] (false positive rate (FPF: False Position Fraction)). Which point of the ROC curve is to be adopted as the cutoff value may be determined according to the severity of the disease, the positioning of the examination, and various other conditions. Generally, in order to increase both sensitivity and specificity (close to 100%), the cutoff value is the true positive rate (sensitivity) on the vertical axis (Y axis) and the false positive rate on the horizontal axis (X axis). At the point on the ROC curve where the expression level at the point closest to (0, 100), or ["true positive (sensitivity)" - "false positive (100 - specificity)"] is maximized ( Youden index).
Therefore, in yet another embodiment of the present invention, the degree of progression of infant AD is detected by comparing the expression level of SerpinB4 protein in SSL in a subject with a reference value based on the cutoff value described above. . In one example, it is detected whether the expression level of SerpinB4 protein in SSL in a subject is higher than a reference value based on the cutoff value described above. If the subject's expression level is now higher than the reference value, the subject can be detected as having infantile AD.

The present invention also provides a test kit for detecting infant AD, which can be used in the method for detecting infant AD according to the present invention described above. In one embodiment, the kit of the invention comprises reagents or instruments for measuring the expression level of SerpinB4 protein. For example, the kit of the present invention may comprise reagents for quantifying SerpinB4 protein (eg, reagents for immunoassay, etc.). Preferably, the kit of the invention contains an antibody that recognizes SerpinB4 protein. Antibodies included in the kit can be obtained from commercial products or by known methods, as described above. In addition to the above antibodies, the kit may contain labeling reagents, buffers, chromogenic substrates, secondary antibodies, blocking agents, equipment necessary for testing, and control reagents used as positive and negative controls. good.
Preferably, the kit of the present invention further comprises an indicator or guidance for evaluating the expression level of SerpinB4 protein. For example, the kit of the invention can include guidance describing a reference level of SerpinB4 protein expression for AD detection.
Further, the kits of the present invention may include an SSL collection device (e.g., the SSL absorbent material or SSL adhesive material described above), reagents for extracting proteins from biological samples, preservatives and preservatives for the sample collection device after biological sample collection. A container or the like may be further provided.

Further disclosed herein are the following materials, methods of manufacture, uses, methods, etc., as exemplary embodiments of the present invention. However, the invention is not limited to these embodiments.

[1] A method for detecting infant atopic dermatitis in an infant subject, comprising the step of measuring the expression level of SerpinB4 protein in surface lipids collected from the infant subject.
[2] Preferably, the method of [1] further comprises comparing the measured expression level of the SerpinB4 protein with a reference value to detect the presence or absence of infantile atopic dermatitis or the degree of progression thereof. the method of.
[3] Preferably, the detection of the degree of progression of infantile atopic dermatitis is detection of mild or moderate atopic dermatitis.
[4] Preferably, the method according to any one of [1] to [3], wherein the infant is 0 to 5 years old.
[5] Preferably, the method according to any one of [1] to [4], further comprising collecting superficial skin lipids from the subject.
[6] A test kit for detecting infant atopic dermatitis used in the method according to any one of [1] to [5], containing an antibody that recognizes SerpinB4 protein.
[7] Use of SerpinB4 protein in epidermal lipids collected from infant subjects for the detection of infantile atopic dermatitis.
[8] Preferably, the use of [7] for detecting the presence or absence of infantile atopic dermatitis or the degree of progression thereof.
[9] Preferably, the use according to [8], wherein the detection of the degree of progression of infantile atopic dermatitis is detection of mild or moderate atopic dermatitis.
[10] The use according to any one of [7] to [9], wherein preferably the infant is 0 to 5 years old.

EXAMPLES The present invention will be described in more detail below based on examples, but the present invention is not limited to these.

Example 1 Identification of AD-related proteins in infant SSL and expression analysis of SerpinB4 protein 1) Subjects and SSL collection Healthy children (6 months to 5 years old male and female) 23 (healthy group) and children with atopic dermatitis (AD patients ) (6 months to 5 years old male and female) 16 subjects (AD group) were used as subjects. In the recruitment of children with AD, children with AD who fulfilled the UKWP criteria (The UK Working Party; Br J Dermatol, 131:406-416 (1994)) as judged by their parents were collected, and the informed consent of the parents was obtained. We selected patients who were For selected children with AD, a dermatologist observed and interviewed the skin of the whole body, and diagnosed AD based on the clinical guidelines for atopic dermatitis (Journal of the Japanese Skin Society, 128(12):2431-2502, 2018). . Based on the Eczema Area and Severity Index (EASI; Exp Dermatol, 10:11-18 (2001)), among AD children diagnosed with AD, symptoms such as mild or more AD-like eczema and dryness on the face were selected. Infants with symptoms were selected and used as subjects. The 16 AD groups selected included 9 mild (mild AD group) and 7 moderate (moderate AD group) based on EASI scores.

From each part of each subject's entire face (including the rash for AD patients) and the entire back (not including the rash for AD patients), using an oil removing film (5 x 8 cm, polypropylene, 3M company) Sebum was collected. The blotting films were transferred to glass vials and stored at −80° C. for approximately one month before being used for protein extraction.

2) Protein preparation The blotting film of 1) above was cut into an appropriate size, and a protein precipitate was obtained using QIAzol Lysis Reagent (Qiagen) according to the attached protocol. From the resulting protein precipitate, using MPEX PTS Reagent (GL science), the protein was dissolved with a lysate according to the attached protocol, and then digested with trypsin. The obtained digestive fluid was dried under reduced pressure (35° C.) and then dissolved in an aqueous solution containing 0.1% (v/v) formic acid and 2% (v/v) acetonitrile to prepare a peptide solution. Peptide concentrations in solutions were measured using a microplate reader (Corona Electric) according to the protocol of Pierce ^™ Quantitative Fluorometric Peptide Assay (ThermoFisher Scientific). The concentration of the peptide solution was adjusted to a constant value, and the protein quantification value was calculated by LC-MS/MS analysis. Peptide solutions from 1 back sample from a healthy child and 1 face sample from an AD child were excluded from the LC-MS/MS analysis because the required peptide amounts were not obtained.

3) LC-MS/MS Analysis and Data Analysis The sample peptide solution obtained in 2) above was subjected to LC-MS/MS analysis under the conditions shown in Table 1 below.

For analysis of spectral data obtained by LC-MS/MS analysis, Proteome Discoverer ver. 2.2 (ThermoFisher Scientific) was used. Human-derived proteins were identified using Mascot database search (Matrix Science), setting Swiss Prot as the reference database and Homo sapiens as Taxonomy. In the search, Enzyme is Trypsin, Missed cleavage is 2, Dynamic modifications are Oxidation (M), Acetyl (N-term), Acetyl (Protein N-term), Static Modifications are Carbamidomethyl (C). set to Peptides satisfying a false discovery rate (FDR) of p<0.01 were searched. The identified proteins were subjected to precursor ion-based Label Free Quantification (LFQ). The protein abundance was calculated from the peak intensity of the precursor ion derived from the peptide, and if the peak intensity was below the detection limit, it was taken as a missing value. To correct experimental bias, the protein abundance was normalized by the Total peptide amount method, and the protein abundance ratio was calculated by the Summed Abundance Based method. ANOVA (Individual Based, t-test) was used to calculate the p-value indicating the significance of the existence difference between groups. Among the identified human-derived proteins, proteins with a false discovery rate (FDR) of 0.1 or higher were excluded from the analysis. Prism8 ver. 3.0 was used. A Log ₂ (Abundance+1) value was calculated by converting the value obtained by dividing the protein abundance before normalization by the total sum of all human-derived detected protein abundances into base 2 logarithmic values, and used as each protein quantification value.

4) Expression analysis (eruption area)
First, from the analysis of human-derived proteins contained in SSL collected from the face (including the eruption area in the AD group), the protein abundance that was not a missing value was calculated in 75% or more of the subjects in either the healthy group or the AD group. 533 kinds of proteins were extracted as analysis targets. We identified 116 proteins whose abundance ratio increased by 1.5 times or more (p<0.05) in the AD group compared to the healthy group, including SerpinB4 protein. Plots of quantitative values (Log ₂ (Abundance+1)) of SerpinB4 protein in face-derived SSL of each subject in the healthy group and AD group are shown in FIG. It was shown that the expression level of SerpinB4 protein in SSL collected from the eruption area (face) of the AD group was statistically significantly increased compared to the healthy group (face) (Student's t -test, P<0.001).

Fifteen AD patients, except one who was excluded from the LC-MS/MS analysis, were divided into a mild AD group (9) and a moderate AD group (6). FIG. 2 shows plots of quantitative values (Log ₂ (Abundance+1)) of SerpinB4 protein in face-derived SSL of each subject in the healthy group, mild AD group, and moderate AD group. The expression level of SerpinB4 protein in SSL collected from the eruption area (face) of the mild AD group and moderate AD group was statistically significantly increased compared to the healthy group (face). (Tukey's test, P<0.05 or P<0.001).

5) Expression analysis (non-rash area)
Next, from the analysis of SSL-derived proteins collected from the back without eruption, 894 proteins for which non-missing protein abundance was calculated in 75% or more of the subjects in either the healthy group or the AD group were analyzed. extracted as We identified 135 proteins whose abundance ratio increased by 1.5 times or more (p<0.05) in the AD group compared to the healthy group, including SerpinB4 protein. FIG. 3 shows plots of quantitative values (Log ₂ (Abundance+1)) of SerpinB4 protein in SSL derived from the back of each subject in the healthy group and the AD group. It was shown that the expression level of SerpinB4 protein in SSL collected from the non-erupted area (back) of the AD group was statistically significantly increased compared to the healthy group (back) (Student's t-test, P<0.01).

16 AD patients were divided into a mild AD group (9 people) and a moderate AD group (7 people), and SerpinB4 protein in the back-derived SSL of each subject in the healthy group, mild AD group, and moderate AD group was quantified. A plot of the values (Log ₂ (Abundance+1)) is shown in FIG. The expression level of serpinB4 protein in SSL collected from the non-erupted part (back) of the mild AD group and the moderate AD group was statistically significantly increased compared to the healthy group (back). (Tukey's test, P<0.05).

6) ROC analysis SerpinB4 protein quantitative value (Log ₂ (Abundance + 1)) in SSL collected from the face (eruption area for AD group) and back (non-eruption area for AD group) of each subject in the healthy group and AD group was used. Then, ROC curves were created (FIGS. 5 and 6). The SerpinB4 protein in SSL collected from the face (AD group is the eruption area) had an area under the ROC curve of 0.86 and a significant P value of 0.0002. Effectiveness in detecting atopic dermatitis was demonstrated. The detection accuracy of AD with a cutoff value of 7.76 by Youden index was 93.33% sensitivity and 65.22% specificity (Fig. 5). On the other hand, for SerpinB4 protein in SSL collected from the back (AD group, non-erupted area), the area under the ROC curve was 0.80, and the P value was significant at 0.0016. Effectiveness in detecting infantile atopic dermatitis was demonstrated even when the expression level was used as an index. The detection accuracy of AD with a cutoff value of 8.05 by Youden index was 87.50% sensitivity and 72.73% specificity (Fig. 6).

Comparative Example 1 Expression analysis of AD-related RNA in infant SSL 1) RNA preparation and sequencing From the nucleic acid-containing fraction, the subject's SSL-derived RNA was extracted. Based on the extracted RNA, reverse transcription was performed at 42° C. for 90 minutes using SuperScript VILO cDNA Synthesis kit (Life Technologies Japan Co., Ltd.) to synthesize cDNA. Random primers attached to the kit were used as primers for the reverse transcription reaction. A library containing DNA derived from the 20802 gene was prepared from the resulting cDNA by multiplex PCR. Multiplex PCR was performed using Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan Co., Ltd.) [99 ° C., 2 minutes → (99 ° C., 15 seconds → 62 ° C., 16 minutes) × 20 cycles → 4 ° C., Hold ]. The resulting PCR product was purified with Ampure XP (Beckman Coulter, Inc.) and then subjected to buffer reconstitution, primer sequence digestion, adapter ligation and purification, and amplification to prepare a library. The prepared library was loaded into the Ion 540 Chip and sequenced using the Ion S5/XL system (Life Technologies Japan).

2) Data analysis
i) Data used The expression level data (read count value) of subject-derived RNA measured in 1) above was corrected using DESeq2. Log ₂ (Normalized count+1) was calculated from the corrected count value (Normalized count value) and used for RNA expression analysis.
ii) RNA Expression Analysis Plots of SerpinB4 RNA expression levels (Log ₂ (Normalized count+1)) for each subject in the healthy group and AD group are shown in FIG. SerpinB4 RNA expression level was not significantly elevated in the AD group compared to the healthy group. That is, from Example 1 and this example, it was found that the SerpinB4 RNA expression level in SSL was not significantly elevated in the AD group, while the SerpinB4 protein expression level was significantly elevated in the AD group. , showed that the expression of SerpinB4 in SSL is discordant between protein and RNA.

Comparative Example 2 Expression analysis of SerpinB4 protein in adult SSL 1) Subjects and SSL collection 18 healthy subjects (20 to 59 years old, male) (healthy group) and atopic dermatitis patients (AD patients) (20 to 59 years old, 26 males (AD group) were used as subjects. Informed consent was obtained from the subjects. Subjects in the AD group were evaluated on the day of the test by a dermatologist comprehensively evaluating the severity of their faces in five stages of "slight", "mild", "moderate", "severe" and "most severe". , were AD patients diagnosed with mild and moderate atopic dermatitis. Sebum was collected from the entire face of each subject (including the eruption area for AD patients) using an oil removing film (5×8 cm, polypropylene, 3M). The blotting films were transferred to vials and stored at −80° C. for approximately one month until used for protein extraction.

2) Protein preparation Instead of MPEX PTS Reagent (GL science), EasyPep ^™ Mini MS Sample Prep Kit (ThermoFisher Scientific) was used to obtain a peptide solution according to the attached protocol, and the procedure was the same as in Example 1. A peptide solution was prepared in and the peptide concentration was measured.

3) LC-MS/MS analysis and data analysis Protein analysis and data analysis were performed under the same conditions and procedures as in Example 1.

4) Results Among the identified proteins, proteins with a false discovery rate (FDR) of 0.1 or higher were excluded from the analysis. 1075 kinds of proteins for which protein abundance was calculated without a missing value in 75% or more of the subjects in either the healthy group or the AD group were extracted as analysis targets. One AD patient with many missing values for protein abundance was excluded from the analysis. We obtained 205 proteins whose abundance ratio increased by 1.5 times or more (p<0.05) in the AD group compared to the healthy group, but SerpinB4 protein was not included among them. FIG. 8 shows plots of quantitative values (Log ₂ (Abundance+1)) of SerpinB4 protein for each subject in the healthy group and AD group. Previous reports have reported that serum SerpinB4 protein levels are elevated in children and adult AD patients (Non-Patent Document 7). On the other hand, from the results of Example 1 and this example, it was clarified that the expression level of SerpinB4 protein in SSL increases in infant AD but does not increase in adult AD, and does not necessarily coincide with changes in blood. .

Comparative Example 3 Expression analysis of known AD-related proteins in infant SSL SerpinB12 protein in the stratum corneum has been reported to decrease compared to healthy children (Non-Patent Documents 5 and 8). In this example, the expression of IL-18 protein and SerpinB12 protein in infant SSL collected in Example 1 was analyzed.
FIG. 9 shows a plot of quantitative values (Log ₂ (Abundance+1)) of IL-18 protein in SSL collected from the back of each subject in the healthy group and AD group (non-erupted area in AD group). No significant difference in IL-18 protein expression level was observed between the healthy group and the AD group. No IL-18 protein was identified in the face (the eruption area in the AD group).
In addition, a plot of the quantitative value (Log ₂ (Abundance+1)) of SerpinB12 protein in SSL collected from the face (eruption area for AD group) and back (non-eruption area for AD group) of each subject in the healthy group and AD group was performed. Shown in FIGS. 10 and 11. FIG. No significant difference was observed between the healthy group and the AD group at any site.

Whether or not various proteins are expressed in SSL and their expression behavior have not yet been fully elucidated. For example, as shown in Comparative Example 1, the expression level of the protein contained in SSL does not necessarily match the expression level of the RNA that encodes it. These facts mean that it is difficult to predict the expression behavior of various proteins in SSL. Moreover, the results of this experiment revealed that the expression behavior of proteins in SSL does not necessarily match that in blood or in the stratum corneum. As shown in FIGS. 9 to 11, IL-18 protein and SerpinB12 protein, which have been reported to be related to AD, do not show any relationship to AD in SSL, unlike in blood or stratum corneum. Previous reports have not clearly shown whether SerpinB4 protein in the stratum corneum of infants is related to AD (Non-Patent Document 8). Further, conventionally, the SerpinB4 protein in blood has been reported as a marker for pediatric and adult AD (Non-Patent Document 6), but as shown in Comparative Example 2, the SerpinB4 protein in SSL does not show a relationship with adult AD. These results of this experiment indicate that the expression of SerpinB4 protein in SSL and its relationship with AD cannot be inferred from conventional knowledge.
Based on the above-described conventional knowledge about proteins in SSL and the results of the present experiment, the technology provided by the present invention, which uses SerpinB4 protein in SSL as an infant AD marker, is completely unexpected and difficult to find. indicates that there is

Claims (4)

A method of measuring protein for detecting infant atopic dermatitis in an infant subject, comprising the step of measuring the expression level of SerpinB4 protein in superficial skin lipids collected from a site of the skin of an infant subject that does not contain an eruption. There is
The infant is an infant aged 0 to 5 years,
How . The method is a method of measuring a protein to detect the presence or absence of infant atopic dermatitis, or the degree of progress thereof, and the method includes measuring the expression level of the SerpinB4 protein as a reference value 2. The method of claim 1, further comprising comparing to . 3. The method according to claim 2, wherein the detection of the degree of progression of infantile atopic dermatitis is detection of mild or moderate atopic dermatitis. A test kit for detecting infant atopic dermatitis used in the method according to any one of claims 1 to 3 , containing an antibody that recognizes SerpinB4 protein.