Immunoglobulin analysis method

Abstract

To provide a method of analyzing an antigen-specific immunoglobulin in a subject using skin surface lipids that can be easily collected non-invasively.SOLUTION: A method of analyzing an antigen-specific immunoglobulin in a subject is provided, the method comprising measuring the antigen-specific immunoglobulin in skin surface lipids collected from the subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for analyzing antigen-specific immunoglobulins using lipids on the skin surface. [Background technology]

[0002] In order to diagnose the history of infection with many pathogenic microorganisms, including viruses, and to evaluate immunity or susceptibility to infection by microorganisms, immunoglobulin (Ig), a specific antibody produced by infection with the microorganism, is measured.

[0003] When humans are infected with a pathogenic microorganism, the concentration of specific antibodies increases rapidly in the first 2 to 3 weeks. Antigen-specific IgM is usually produced in the early stages of infection, but its production is short-lived and it is only present in the early stages of infection. Therefore, this immunoglobulin is used as a sign to detect early symptoms of infection. Antigen-specific IgG is formed shortly after infection. This IgG antibody has a longer persistence and persists for a long time in many microbial infections. Therefore, antigen-specific IgG antibodies are a sign of infection with the pathogenic microorganism to which they specifically react and immunity to that microorganism, and the absence of this antibody indicates susceptibility to infection (a state in which the body is easily infected by the microorganism).

[0004] In addition, immunoglobulins in the blood are generally involved in allergic reactions. Examples of type I allergic diseases include food allergies, allergic bronchial asthma, and allergic rhinitis. In type I allergies, allergen-specific IgE antibodies are induced in the body by the invasion of allergens, and sensitization occurs when they bind to high-affinity IgE receptors on mast cells. In general, the state of sensitization can be objectively grasped by quantifying the allergen-specific IgE antibody titer in the blood through a blood test. The presence of allergen-specific IgE antibodies indicates sensitization to the allergen in question, and although it is not necessarily the allergen related to the onset of allergy, it is an important indicator for understanding the onset, progression, and treatment course. The immunoglobulin involved in allergic reactions is mainly IgE, but IgG (especially IgG4) and IgA are also said to be involved. Allergen immunotherapy is a treatment that induces immune tolerance by administering minute amounts of allergens to the body, thereby alleviating allergic symptoms associated with exposure to allergens. Since allergen-specific IgG4 increases in the blood early on in the course of treatment, the usefulness of monitoring allergen-specific IgG4 as a way to monitor the course of treatment is being investigated. In addition, immunoglobulins in the blood are known to be involved in type II allergies, type III allergies, autoimmune diseases, collagen diseases, rheumatoid arthritis, etc., and the measurement of specific immunoglobulins against autoantigens produced in the body is considered to be one of the important test items.

[0005] Tests for the presence of antigen-specific immunoglobulins are usually performed by measuring the antigen-specific immunoglobulins in blood (serum) collected by venipuncture. Venipuncture is not a convenient method, is highly invasive, and may not be applicable to children and nervous adults. Venipuncture also requires sterilized needles, syringes, and cotton wool, making it costly and sometimes dangerous or difficult.

[0006] Antigen-specific immunoglobulins are known to exist in body fluids other than blood, such as saliva, tears, and urine. However, from the viewpoint of ease of specimen collection, it is not easy to collect samples, particularly from children and infants. In addition, the amount of secretion of these body fluids and the amount of protein contained therein vary widely between individuals and within a day. These are considered to be issues, and tests or assays using these body fluids have not yet been fully put to practical use.

[0007] On the other hand, it has been reported that sweat contains immunoglobulins such as IgA, IgG, and IgM (Non-Patent Document 1), and that IgM has been detected in antibody-producing cells in human skin (Non-Patent Document 2). However, the presence of quantifiable antigen-specific immunoglobulins in sebum is completely unknown, and no antibody tests using sebum have been conducted to date. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Katchman et al., Proteomics Clin Appl. 12(6):e1800010. (2018) [Non-Patent Document 2] Wilson et al. J Invest Dermatol. 139(12):2477-2487. (2019) Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention relates to providing a method for analyzing antigen-specific immunoglobulins in a subject using lipids on the skin surface, which can be easily collected non-invasively. [Means for solving the problem]

[0010] The inventors conducted research in light of such problems and found that antigen-specific immunoglobulins were detected in lipids on the surface of the skin, and that the concentration and change in the immunoglobulins were correlated with the concentration and change in the immunoglobulins in the blood, and that this can be used to diagnose a history of infection with a pathogenic microorganism and to analyze immunity or susceptibility to infection by the microorganism.

[0011] That is, the present invention relates to the following 1) to 4). 1) A method for analyzing antigen-specific immunoglobulins in a subject, comprising the step of measuring antigen-specific immunoglobulins in lipids on the skin surface collected from the subject. 2) A measurement method for detecting a history of pathogenic microbial infection in a subject, the method comprising a step of measuring pathogenic microbial antigen-specific immunoglobulins in lipids on the skin surface collected from the subject. 3) A measurement method for detecting immunity or susceptibility to infection by pathogenic microorganisms in a subject, the method comprising a step of measuring pathogenic microbial antigen-specific immunoglobulins in lipids on the skin surface collected from the subject. 4) A measurement kit for use in any one of the methods 1) to 3), which contains tools and reagents necessary for collecting and storing lipids on the surface of the skin, and a reagent for measuring the antigen-specific immunoglobulin from lipids on the surface of the skin. Effect of the Invention

[0012] According to the present invention, it is possible to simply and non-invasively diagnose the history of infection with a pathogenic microorganism such as a virus in a subject, determine immunity or susceptibility to infection with the microorganism, for example, determine the effectiveness of a vaccine. [Brief description of the drawings]

[0013] [Figure 1] Correlation between sebum and blood coronavirus antibody concentrations. [Diagram 2] Correlation between changes in COVID-19 antibody concentrations in sebum and blood. [Diagram 3] Response to COVID-19 vaccination (comparison between before and 20 days after vaccination). [Figure 4] Responsiveness to COVID-19 vaccination (comparison before vaccination, 20 days after vaccination, and 100 days after vaccination). [Diagram 5] Correlation between influenza virus A antibody concentrations in sebum and blood. [Figure 6] Response to influenza vaccination (comparison between before and one month after vaccination). [Figure 7] Response to influenza vaccination (comparison between 1.5 months and 4 months after vaccination). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] All patents, non-patent publications, and other publications cited herein are hereby incorporated by reference in their entirety.

[0015] In the present invention, the method for analyzing immunoglobulins in a subject comprises the step of measuring antigen-specific immunoglobulins in lipids on the skin surface collected from the subject.

[0016] In the present invention, the immunoglobulin of the antigen-specific immunoglobulin refers to one or more of the major classes of IgG, IgM, IgA, IgE, and IgD and their subclasses. Subclasses include IgG1, IgG2, IgG3, and IgG4, which are IgG subclasses known in humans. The type of antigen-specific immunoglobulin measured in the present invention varies depending on the antigen, but preferred examples include IgG, IgM, and IgA, and more preferably IgG.

[0017] Antigens for antigen-specific immunoglobulins include antigens derived from pathogenic microorganisms (bacteria, fungi, viruses, etc.), particularly antigens derived from viruses that cause infectious diseases, such as Ebola virus, smallpox virus, Lassa virus, poliovirus, SARS coronavirus (SARS-CoV), MERS coronavirus (MERS-CoV), SARS coronavirus 2 (SARS-CoV-2), avian influenza A (H7N9) virus, hepatitis A virus, rabies virus, monkeypox virus, dengue virus, Japanese encephalitis virus, hepatitis B virus, hepatitis C virus, influenza virus, herpes simplex virus, enterovirus, measles virus, human immunodeficiency virus, rubella virus, adenovirus, rotavirus, norovirus, sapovirus, varicella-zoster virus, coxsackievirus, human parvovirus B19, human herpes virus, influenza virus (types A and B), and human papillomavirus. Among these, the method of the present invention is suitable for analyzing respiratory infectious virus antigens such as SARS-CoV, MERS-CoV, SARS-CoV-2, and influenza virus, and is more suitable for analyzing SARS coronavirus 2 (SARS-CoV-2) antigen-specific immunoglobulins, which are the causative virus of novel coronavirus disease 2019 (COVID-19), or influenza virus antigen-specific immunoglobulins, which are the causative virus of influenza, and is more preferably suitable for analyzing SARS-CoV-2 antigen-specific IgG (anti-SARS-CoV-2 IgG antibody) or influenza virus antigen-specific IgG.

[0018] In the present invention, "skin surface lipids (SSL)" refers to the fat-soluble fraction present on the surface of the skin, and is also called sebum. Generally, 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. Unless otherwise specified, the term "skin" is a general term for an area including tissues such as the epidermis, dermis, hair follicles, sweat glands, sebaceous glands, and other glands on the body surface.

[0019] The subject from which the SSL is obtained may be any organism having an SSL on its skin, including mammals, including humans and non-human mammals, but preferably humans. Preferably, the subject is a human or non-human mammal in need of or desire for detection of antigen-specific immunoglobulins.

[0020] The site of the skin from which the SSL is collected is not particularly limited and may be any site of the skin on the head, face, neck, trunk, limbs, etc., with sites that secrete a lot of sebum, such as the skin of the face, being preferred.

[0021] Any means used for recovering or removing SSL from the skin can be used to collect SSL from the skin of a subject. Preferably, an SSL absorbent material, an SSL adhesive material, or an instrument for scraping SSL from 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 an affinity for SSL, and examples thereof include polypropylene, pulp, and the like. More detailed examples of procedures for collecting SSL from the skin include a method of absorbing SSL into a sheet-like material such as oil blotting paper or oil blotting film, a method of adhering SSL to a glass plate, tape, or the like, and a method of scraping SSL off and collecting it with a spatula, scraper, or the like. In order to improve the adsorption of SSL, an SSL absorbent material that has been previously impregnated with a highly lipid-soluble solvent 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 highly water-soluble solvent or moisture is low. It is preferable to use the SSL absorbent material in a dry state.

[0022] The SSL collected from the subject may be immediately used for the isolation of immunoglobulins, which will be described later, or may be stored until the isolation of immunoglobulins. The SSL may be stored in a refrigerator, at a low temperature below freezing, or at room temperature. For example, an SSL absorbent material or an SSL adhesive material containing an SSL may be stored at room temperature, in a refrigerator, or at a low temperature.

[0023] Immunoglobulins can be extracted from the collected SSL using methods commonly used for extracting or purifying proteins from biological samples, such as extraction methods using water, phosphate-buffered saline solution (aqueous solution, phosphate-buffered saline solution, etc.), or solutions containing surfactants such as Triton X-100 or Tween 20, or extraction methods using commercially available protein extraction reagents or kits such as M-PER buffer (Thermo Fisher Scientific), MPEX PTS Reagent (GL science), QIAzol Lysis Reagent (Qiagen), or EasyPep (trademark) Mini MS Sample Prep Kit (ThermoFisher Scientific).

[0024] The antigen-specific immunoglobulins derived from the separated SSL can be measured by using an immunological measurement method, in which an antigen for the immunoglobulin to be measured is contacted with a sample, the immunoglobulin bound to the antigen is detected, and the level is measured. Examples of the immunological measurement method include immunochromatography, radioimmunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), indirect fluorescent antibody assay (Indirect Fluorescence assay), luminescent immunoassay (Luminescent immunoassay), physicochemical measurement methods (TIA, LAPIA, PCIA), Western blotting, electrochemiluminescence immunoassay (ECLIA), chemiluminescence immunoassay (CLIA), chemiluminescence enzyme immunoassay (CLEIA), ultrasensitive immunoassay (Simoa Immunoassays), etc., and the ELISA method is preferred. In the ELISA method, for example, a sample is added to a plate on which an antigen has been immobilized and reacted to allow the antigen-specific immunoglobulin in the sample to bind to the antigen, and then a labeled antibody that binds to the immunoglobulin is reacted to form an antigen-antigen-specific immunoglobulin-labeled antibody complex, and finally the signal derived from the labeled substance is measured using a radiation measuring device, a fluorescence detector, etc. The antigens used in the measurement may be inactivated microorganisms, proteins derived from microorganisms, or peptide fragments thereof, as long as they bind to antigen-specific immunoglobulin. For example, SARS-CoV-2 antigens include the nucleocapsid protein (N protein), spike protein (S protein), and S protein receptor binding domain of SARS-CoV-2, and influenza virus antigens include the nucleoprotein (NP) of type A or type B viruses, hemagglutinin (HA) on the particle surface, and neuraminidase (NA), which is a glycoprotein, and these may be genetically engineered recombinant forms.

[0025] In the present invention, analysis of antigen-specific immunoglobulins in a subject includes various analyses, such as qualitative analysis, quantitative analysis, and correlation analysis, using the antigen-specific immunoglobulins in the measured SSL. As shown in the Examples below, quantifiable IgA, IgD, IgE, IgM, and IgG (IgG1, IgG2, IgG3, IgG4) are detected in the SSL. Quantifiable anti-SARS-CoV-2 IgG antibodies are detected in the SSL of subjects vaccinated with the novel coronavirus (SARS-CoV-2) vaccine, and quantifiable anti-influenza virus A IgG antibodies are detected in the SSL of subjects vaccinated with the influenza virus vaccine. Therefore, the analysis of antigen-specific immunoglobulins in a subject of the present invention includes quantitative analysis that quantifies antigen-specific immunoglobulins in a subject.

[0026] Furthermore, a significant positive correlation is observed between the immunoglobulin concentration in SSL and the immunoglobulin concentration in blood samples, and a significant correlation is also observed between the change in the immunoglobulin concentration in SSL and the change in the immunoglobulin concentration in blood samples. That is, the amount of the immunoglobulin in blood or the change therein can be estimated from the amount of antigen-specific immunoglobulin in SSL or the change therein, and therefore, the analysis of antigen-specific immunoglobulin in a subject of the present invention includes a correlation analysis with the amount of antigen-specific immunoglobulin in blood in the subject or the change therein.

[0027] In such quantitative analysis using antigen-specific immunoglobulins in SSL, the measured value of the antigen-specific immunoglobulin amount in SSL may be used, but from the viewpoint of correcting for variations in the amount of SSL collected from each subject, it is preferable to correct the amount of antigen-specific immunoglobulins in SSL by various methods such as the weight of SSL, the volume of extract, or the amount of a specific component in SSL. For example, it is preferable to use a correction value obtained by dividing the amount of antigen-specific immunoglobulins by the total amount of immunoglobulins, the amount of internal standard protein, the total amount of protein, or the total amount of lipids in the SSL, and among these, it is more preferable to use a correction value obtained by dividing by the total amount of immunoglobulins in the SSL. Here, the total immunoglobulin amount refers to the total amount of immunoglobulins of the same isotype as the antigen-specific immunoglobulin to be measured, for example, when the antigen-specific immunoglobulin to be measured is IgG, it is the total IgG amount in the SSL. In addition, as the internal standard protein, it is preferable to use a protein that exhibits smaller expression variations between subjects in lipids on the sebum surface.

[0028] Analysis of antigen-specific immunoglobulins in such a subject makes it possible to detect a history of infection with a pathogenic microorganism such as a virus in the subject, and to detect immunity or susceptibility to infection with the microorganism. Thus, the present invention provides a measurement method for detecting a history of infection with a pathogenic microorganism in a subject, the method comprising a step of measuring pathogenic microorganism antigen-specific immunoglobulins in SSL, and a measurement method for detecting immunity or susceptibility to infection with a pathogenic microorganism in a subject, the method comprising a step of measuring pathogenic microorganism antigen-specific immunoglobulins in SSL. Here, detecting a history of infection with a pathogenic microorganism includes detecting whether a subject has ever been infected with a pathogenic microorganism, and detecting immunity or susceptibility to infection with a pathogenic microorganism includes, for example, detecting the effectiveness of vaccination against the pathogenic microorganism or the durability of the vaccine effect. The term "detection" means to clarify whether or not a subject has been infected with a pathogenic microorganism, or whether or not the subject has immunity to or susceptibility to a pathogenic microorganism, and can be expressed in other terms such as examination, measurement, judgment, evaluation support, or prediction. In the present invention, the terms "detection," "examination," "measurement," "judgment," or "evaluation" do not include diagnosis by a physician.

[0029] The measurement kit of the present invention is a kit for carrying out the above-mentioned method for analyzing antigen-specific immunoglobulins in a subject, the measurement method for detecting a history of infection with a pathogenic microorganism in a subject, and the measurement method for detecting immunity or susceptibility to infection with a pathogenic microorganism in a subject. The kit of the present invention contains tools and reagents necessary for collecting and storing SSL, and reagents for measuring antigen-specific immunoglobulins from SSL. Examples of tools and reagents necessary for collecting and storing SSL include oil-removing films for collecting SSL, reagents for storing collected SSL, storage containers, etc. Examples of reagents for measuring antigen-specific immunoglobulins include reagents for extracting and purifying immunoglobulins from collected SSL, reagents for immunological measurements including antigens that bind to antigen-specific immunoglobulins, as well as labeling reagents, buffer solutions, color-developing substrates, secondary antibodies, blocking agents, control reagents used as positive and negative controls, tools necessary for testing, and indicators or guidance for detecting antigen-specific immunoglobulins.

[0030] In relation to the above-described embodiment, the present invention further discloses the following aspects. 1) A method for analyzing antigen-specific immunoglobulins in a subject, comprising the step of measuring antigen-specific immunoglobulins in lipids on the skin surface collected from the subject. 2) The method according to 1), wherein the measurement comprises extracting and isolating antigen-specific immunoglobulins in the lipids on the skin surface. 3) The method according to 1) or 2), wherein the antigen-specific immunoglobulin is an infectious disease virus antigen-specific immunoglobulin. 4) The method according to 3), wherein the infectious disease viral antigen is an antigen derived from a virus selected from Ebola virus, smallpox virus, Lassa virus, poliovirus, SARS coronavirus (SARS-CoV), MERS coronavirus (MERS-CoV), SARS coronavirus 2 (SARS-CoV-2), avian influenza A (H7N9) virus, hepatitis A virus, rabies virus, monkeypox virus, dengue virus, Japanese encephalitis virus, hepatitis B virus, hepatitis C virus, influenza virus, herpes simplex virus, enterovirus, measles virus, human immunodeficiency virus, rubella virus, adenovirus, rotavirus, norovirus, sapovirus, varicella-zoster virus, coxsackievirus, human parvovirus B19, human herpes virus, influenza virus (types A and B), and human papillomavirus. 5) The method according to 1) or 2), wherein the antigen-specific immunoglobulin is a respiratory infectious virus antigen-specific immunoglobulin. 6) The method according to 1) or 2), wherein the antigen-specific immunoglobulin is a SARS-CoV-2 antigen-specific immunoglobulin or an influenza virus antigen-specific immunoglobulin. 7) The method according to 1) or 2), wherein the antigen-specific immunoglobulin is a SARS-CoV-2 antigen-specific immunoglobulin. 8) The method according to 1) or 2), wherein the antigen-specific immunoglobulin is an influenza virus antigen-specific immunoglobulin. 9) The method according to 1) or 2), wherein the antigen-specific immunoglobulin is at least one selected from the group consisting of IgA, IgM, IgE, IgD, IgG1, IgG2, IgG3 and IgG4. 10) The method according to 1) or 2), wherein the antigen-specific immunoglobulin is IgG. 11) The method according to 1) or 2), wherein the antigen-specific immunoglobulin is SARS-CoV-2 antigen-specific IgG or influenza virus antigen-specific IgG. 12) The method according to 1) or 2), wherein the antigen-specific immunoglobulin is SARS-CoV-2 antigen-specific IgG. 13) The method according to 1) or 2), wherein the antigen-specific immunoglobulin is influenza virus antigen-specific IgG. 14) Any of the methods 1) to 13), wherein the analysis is a quantitative analysis that quantifies antigen-specific immunoglobulin in the subject. 15) Any of the methods 1) to 14), in which the analysis is a correlation analysis that estimates the amount of antigen-specific immunoglobulin in the blood based on the amount of the antigen-specific immunoglobulin in lipids on the skin surface. 16) The method of 14) or 15), wherein in the analysis, a corrected value obtained by dividing the antigen-specific immunoglobulin amount in the lipids on the skin surface by the total immunoglobulin amount, the amount of internal standard protein, the total protein amount, or the total lipid amount in the lipids on the skin surface is used. 17) A measurement method for detecting a history of pathogenic microbial infection in a subject, the method comprising a step of measuring pathogenic microbial antigen-specific immunoglobulins in lipids on the skin surface collected from the subject. 18) A measurement method for detecting immunity or susceptibility to infection by a pathogenic microorganism in a subject, the method comprising a step of measuring pathogenic microorganism antigen-specific immunoglobulins in lipids on the skin surface collected from the subject. 19) The method according to 18), wherein the detection of immunity or susceptibility to a pathogenic microorganism is the detection of the effectiveness of a vaccine against the pathogenic microorganism or the persistence of the effectiveness of the vaccine. 20) A measurement kit for use in any of the methods 1) to 19), comprising tools and reagents necessary for collecting and preserving lipids on the surface of the skin, and a reagent for measuring the antigen-specific immunoglobulin from lipids on the surface of the skin. EXAMPLES

[0031] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0032] Example 1 Quantitative Analysis of Immunoglobulin in Sebum Immunoglobulins in sebum samples from a healthy subject (male, 39 years old) were quantified. Sebum was collected from the entire face using an oil blotting film (5 x 8 cm, polypropylene, 3M). The oil-blotting film was then transferred to a tube and stored in a freezer. In the laboratory, the oil-blotting film was immersed in 500 μL of PBS solution containing 0.1% Triton X-100. After soaking in water and shaking for 15 minutes at room temperature, the supernatant was centrifuged (15,000 g, 4°C, 5 minutes) to remove the residue, and the supernatant was used as the immunoglobulin extract for the following analysis. The immunoglobulin in the extract was quantified by Funakoshi Co., Ltd. using a Human Ig Isotype Array (QAH-ISO-1-2, RayBiotech).

[0033] <Result> The immunoglobulin concentrations of IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3, and IgG4 in the sebum samples are shown in Table 1. This indicates that quantifiable IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3, and IgG4 were detected in the sebum samples.

[0034] [Table 1]

[0035] Example 2 Quantification of anti-SARS-CoV-2 IgG antibodies in sebum The antibody titers against the novel coronavirus (SARS-CoV-2) were measured in blood and sebum samples from 35 healthy subjects (men and women, aged 24 to 60 years). The subjects had received two doses of the novel coronavirus vaccine before participating in the study, and were scheduled to receive a third dose of the novel coronavirus vaccine. Blood and sebum samples were collected in the morning on the day before or on the day of the third dose, and 19 or 20 days after vaccination. In addition, blood and sebum samples were similarly collected from 15 of the subjects 99 or 100 days after vaccination. Anti-SARS-CoV-2 IgG antibody titers in the blood were measured using the antibody testing service provided by iGenomics Japan, Inc. Following the instructions provided with the testing service, subjects collected blood from their fingertips using a simple blood collection kit (lancet) at home, soaked the blood on filter paper, dried it, and then mailed it to Igenomics Japan, Inc. The analytical values ​​of anti-SARS-CoV-2 IgG antibodies in the blood notified by Igenomics Japan, Inc. were used for analysis. In addition, the subjects collected sebum from the entire face using an oil blotting film (5 x 8 cm, made of polypropylene, 3M) at home, transferred the oil blotting film to a tube, and stored in a freezer. In the laboratory, the oil blotting film was immersed in 550 μL of PBS solution containing 0.1% Triton X-100, shaken and mixed at room temperature for 15 minutes, and then centrifuged (15,000 g, 4 ° C, 5 minutes) to remove residues, and the supernatant was used as an antibody extract for subsequent analysis. The anti-SARS-CoV-2 IgG antibody concentration in the extract was quantified using the same Anti-SARS-CoV-2 QuantiVac ELISA (IgG) kit (EI 2606, EUROIMMUN) used in the blood antibody titer measurement method at iGenomics Japan. In addition, an IgG ELISA kit (EI7200-1, AssayPro) was used to quantify the total IgG antibody concentration in the antibody extract, a CBQCA assay kit (MP 06667, Molecular Probes) was used to quantify total protein, and a Lipid Quantification Kit (STA-617, Cell Biolabs) was used to quantify total lipids.

[0036] <Result> The anti-SARS-CoV-2 IgG antibody concentrations in the sebum samples from the subjects ranged from 0.45 to 11.38 RU / mL, indicating that quantifiable anti-SARS-CoV-2 IgG antibodies could be detected in sebum samples. When the correlation between the antibody concentration in the sebum samples and the antibody titer in the blood samples was analyzed, a significant positive correlation was found (Figure 1-(1), Spearman's rank correlation coefficient, ρ = 0.658, p < 0.001, n = 84). When the antibody concentration in the sebum samples was corrected by dividing it by the total IgG antibody concentration, total protein concentration, and total lipid concentration, and the correlation between each corrected value and the antibody titer in the blood samples was analyzed, a significant correlation was found for all of them, and in particular, a high correlation was shown with the corrected value divided by the total IgG antibody concentration and the total protein concentration (Figure 1-(2) to (4)).

[0037] Furthermore, the change in antibody concentration in sebum and blood samples within the same individual between each time point (before vaccination on the day before or on the day of vaccination, 19 or 20 days after vaccination, and 99 or 100 days after vaccination) was calculated, and a significant positive correlation was observed (Figure 2-(1), Spearman's rank correlation coefficient, ρ = 0.614, p < 0.001, n = 64). Positive correlations were also observed with each correction value, and in particular, a high correlation was observed with the correction value divided by the total IgG antibody concentration. These results suggest that the anti-SARS-CoV-2 IgG antibody concentration in sebum samples showed a positive correlation with the concentration in blood samples, and in particular, the correction method of dividing by the total IgG antibody concentration showed the highest correlation (Figures 2-(2) to (4)).

[0038] Next, the antibody concentrations in the sebum and blood samples were analyzed for changes at each time point (two time points, the day before or on the day of the third vaccination (D0), 19 or 20 days after vaccination (D20) (35 patients), and three time points including 99 or 100 days after vaccination (D100) (15 patients)). The anti-SARS-CoV-2 IgG antibody concentration in the blood samples was significantly increased on D20 compared to D0, and the anti-SARS-CoV-2 IgG antibody concentration in the sebum samples was also significantly increased on D20 compared to D0 (Figure 3-(1) and (2), Paired t-test, p<0.001, blood samples n=34, sebum samples n=35). Similarly, significant increases were observed in the antibody concentrations in the sebum samples when corrected for total IgG antibody concentration, total protein concentration, and total lipid concentration (Figures 3-(3), (4), and (5)).

[0039] Furthermore, a comparison of D20 and D100 confirmed a significant decrease in the anti-SARS-CoV-2 IgG antibody concentration in the blood samples, and a similar significant decrease was observed in the anti-SARS-CoV-2 IgG antibody concentration in the sebum samples corrected for the total IgG antibody concentration (Figure 4-(1) to (5)). This suggests that by quantifying the antigen-specific antibody (anti-SARS-CoV-2 IgG antibody) concentration in sebum, it is possible to understand the changes in antibody titers due to vaccination (COVID-19 vaccine).

[0040] Example 3 Quantification of anti-influenza virus A IgG antibodies in sebum The amount of anti-influenza virus A IgG antibodies in blood and sebum samples from four healthy subjects (ID01-ID04) (male and female, aged 24 to 60 years old) was evaluated. Blood and sebum samples were collected from two of the subjects (ID01, ID02) before influenza vaccination and approximately one month after vaccination. Blood and sebum samples were collected from the other two subjects (ID03, ID04) approximately 1.5 months and approximately 4 months after influenza vaccination. Blood was collected by standard methods via puncture of the inside of the forearm, and serum was separated by centrifugation (1,500 × g, 4°C, 15 minutes). The amount of anti-influenza virus A IgG antibody in the serum was evaluated using an Anti-Influenza virus A IgG ELISA kit (ab108745, Abcam). Sebum was collected from the entire face using an oil blotting film (5×8 cm, polypropylene, 3M). The oil blotting film was transferred to a tube and stored in a freezer. In the laboratory, the oil blotting film was immersed in 550 μL of PBS solution containing 0.1% Triton X-100, and shaken and mixed at room temperature for 15 minutes. The supernatant was centrifuged (15,000 g, 4° C., 5 minutes) to remove residues, and the supernatant was used as an antibody extract. The amount of anti-influenza virus A IgG antibody in the extract was evaluated using an Anti-Influenza virus A IgG ELISA kit (30114050, TECAN). The amount of anti-influenza virus A IgG antibody in the serum and sebum samples was calculated as a relative value when the amount of antibody in the attached Positive Control solution was set to 1.

[0041] <Result> When we analyzed the correlation between the amounts of anti-influenza virus A IgG antibodies in serum and sebum, a significant positive correlation was found (Figure 5, Spearman's rank correlation coefficient, ρ = 0.952, p < 0.01, n = 8). Among the subjects whose blood and sebum were collected before and about one month after influenza vaccination, in ID01, the amount of antibodies in the serum increased after vaccination, and similarly, the amount of antibodies in the sebum also increased (Fig. 6 (1) and (2)). On the other hand, in ID02, no significant increase in the amount of antibodies in the serum was observed after vaccination, and similarly, the amount of antibodies in the sebum showed almost no change (Fig. 6 (1) and (2)). Furthermore, among subjects whose blood and sebum were collected approximately 1.5 months and 4 months after influenza vaccination, in ID03, the amount of antibodies in the serum decreased from approximately 1.5 months to approximately 4 months after vaccination, and the amount of antibodies in the sebum also decreased (Figures 7(1) and (2)). On the other hand, in ID04, no significant decrease in the amount of antibodies in the serum was observed from approximately 1.5 months to approximately 4 months after vaccination, and similarly, the amount of antibodies in the sebum showed almost no change (Figures 7(1) and (2)). These results suggest that influenza virus antigen-specific immunoglobulins can be detected in sebum, that the amount of antibodies in the blood correlates with the amount of antibodies in sebum, and that by quantifying the amount of antibodies in sebum, it is possible to understand changes in antibody titers due to vaccination.

Claims

1. A method for analyzing antigen-specific immunoglobulins in a subject, comprising a step of measuring antigen-specific immunoglobulins in lipids on the skin surface collected from the subject.

2. The method of claim 1 , wherein said measuring comprises extracting and isolating antigen-specific immunoglobulins in said lipids on the skin surface.

3. The method according to claim 1 or 2, wherein the antigen-specific immunoglobulin is an infectious disease virus antigen-specific immunoglobulin.

4. The method according to claim 1 or 2, wherein the antigen-specific immunoglobulin is a respiratory infectious virus antigen-specific immunoglobulin.

5. The method according to claim 1 or 2, wherein the antigen-specific immunoglobulin is a SARS-CoV-2 antigen-specific immunoglobulin or an influenza virus antigen-specific immunoglobulin.

6. The method of claim 1 or 2, wherein the antigen-specific immunoglobulin is a SARS-CoV-2 antigen-specific immunoglobulin.

7. The method according to claim 1 or 2, wherein the antigen-specific immunoglobulin is an influenza virus antigen-specific immunoglobulin.

8. The method according to claim 1 or 2, wherein the antigen-specific immunoglobulin is at least one selected from the group consisting of IgA, IgM, IgE, IgD, IgG1, IgG2, IgG3 and IgG4.

9. The method according to claim 1 or 2, wherein the antigen-specific immunoglobulin is SARS-CoV-2 antigen-specific IgG or influenza virus antigen-specific IgG.

10. The method of claim 1 or 2, wherein the antigen-specific immunoglobulin is a SARS-CoV-2 antigen-specific IgG.

11. The method according to claim 1 or 2, wherein the antigen-specific immunoglobulin is an influenza virus antigen-specific IgG.

12. The method of claim 1, wherein the analysis is a quantitative analysis that quantifies antigen-specific immunoglobulins in the subject.

13. The method according to claim 1, wherein the analysis is a correlation analysis in which the amount of antigen-specific immunoglobulin in blood is estimated based on the amount of said immunoglobulin in lipids on the surface of the skin.

14. The method according to claim 12 or 13, wherein in the analysis, a corrected value obtained by dividing the amount of antigen-specific immunoglobulin in the lipids on the skin surface by the total amount of immunoglobulin, the amount of internal standard protein, the total amount of protein or the total amount of lipid in the lipids on the skin surface is used.

15. A measurement method for detecting a history of infection with a pathogenic microorganism in a subject, the method comprising a step of measuring pathogenic microorganism antigen-specific immunoglobulins in lipids on the skin surface collected from the subject.

16. A measurement method for detecting immunity or susceptibility to infection by pathogenic microorganisms in a subject, the method comprising a step of measuring pathogenic microbial antigen-specific immunoglobulins in lipids on the skin surface collected from the subject.

17. The method according to claim 16, wherein the detection of immunity or susceptibility to a pathogenic microorganism is the detection of the efficacy of a vaccine against the pathogenic microorganism or the persistence of the efficacy of the vaccine.

18. 17. A measuring kit for use in the method according to claim 1, 15 or 16, comprising tools and reagents necessary for collecting and storing lipids on the surface of the skin, and reagents for measuring the antigen-specific immunoglobulins from lipids on the surface of the skin.

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