Methods and kits for testing gastrointestinal diseases

Abstract

To provide a novel method for examining gastrointestinal diseases that is less invasive for the test subject. [Solution] A method for testing gastrointestinal diseases, which uses at least one protein selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2 in a fecal mucus sample derived from a test subject as an indicator.

Description

[Technical Field]

[0001] The present invention relates to a method and a kit for testing gastrointestinal diseases. [Background Art]

[0002] Digestive diseases are diseases with a large number of patients, totaling more than 1 million people per year worldwide, and their prevention, diagnosis, and treatment methods have been studied in various institutions. Among digestive diseases, gastrointestinal diseases often require highly invasive testing for diagnosis, and less invasive testing methods are in demand. For example, the allergen-specific lymphocyte stimulation test (ALST), which is used for testing Non-IgE-mediated gastrointestinal food allergy (neonatal / infantile non-IgE-GIFA), a disease that has been increasing in prevalence in recent years, requires a considerable volume of blood samples from neonates and infants. Additionally, a method for diagnosing neonates and infants suspected of having non-IgE-GIFA by measuring interleukins in serum has been proposed (Patent Document 1), but this method also requires blood collection, which is an invasive test for neonates and infants. Furthermore, for definitive diagnosis of neonatal / infantile non-IgE-GIFA, a definitive diagnosis is sometimes made by confirming the recurrence of symptoms after re-administration of the causative food (oral food challenge test [OFC]), but this test is also highly invasive. In addition, non-IgE-GIFA in neonates and infants presents eosinophilic inflammation similar to that observed in allergic diseases such as bronchial asthma and allergic rhinitis. For this reason, the disease may be complicated by eosinophilic gastrointestinal diseases, in which pathological eosinophilic inflammation occurs in the gastrointestinal tract, and analysis of proteins including eosinophil-related molecules is particularly useful. Therefore, development of a less invasive method for testing gastrointestinal diseases is highly desired. [Prior Art Documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2021-63743 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] The present invention aims to provide a novel method for examining gastrointestinal diseases that is less invasive for the subject of the examination. [Means for solving the problem]

[0005] Under these circumstances, the inventors conducted diligent research and found that, among various measurement samples, using a fecal mucus sample allowed for the detection of at least one protein selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2 in greater numbers in gastrointestinal diseases. Based on this novel finding, the inventors completed the present invention after much trial and error regarding the preparation method of the test sample, the measurement method, etc. Accordingly, the present invention provides the following: Item 1. A method for testing gastrointestinal diseases using at least one protein selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2 in a fecal mucus sample derived from a test subject.

[0006] Item 2. A method according to Item 1, wherein the fecal mucus sample is obtained by preparing a fecal smear and extracting components of said fecal smear, or by centrifugation of feces and obtaining the supernatant of said centrifuged sample.

[0007] Item 3. The method described in Item 1, (a) A step of treating a stool smear collected and prepared from the test subject with an extractant, (b) A step of detecting proteins in the extract and measuring the amount of at least one protein selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2. (c) A method in which, if the amount of the measured protein group is more than twice the mean value of the control group and a statistically significant difference (P<0.05, T-test) is observed, it serves as an indicator that the subject has a gastrointestinal disease.

[0008] Item 4. The method according to item 1, wherein the gastrointestinal disease is an eosinophilic gastrointestinal disease.

[0009] Item 5. The method according to Item 1, wherein the gastrointestinal disorder is neonatal or infant food protein-induced gastroenteropathy.

[0010] Item 6. A diagnostic kit for gastrointestinal diseases comprising means for detecting at least one protein selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2, the kit for use in a method comprising the step of measuring at least one protein selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2 in a fecal mucus sample derived from a test subject.

[0011] Item 7. A kit according to item 6, wherein the fecal mucus sample is obtained by preparing a fecal smear and extracting components of said fecal smear, or by centrifugation of feces and obtaining the supernatant of said centrifugation sample.

[0012] Item 8. A kit as described in Item 6, for use in the method described in any one of Items 1 to 5.

[0013] Item 9. The kit according to item 6, wherein the gastrointestinal disease is an eosinophilic gastrointestinal disease.

[0014] Item 10. The kit according to item 6, wherein the gastrointestinal disorder is neonatal or infant food protein-induced gastroenteropathy. [Effects of the Invention]

[0015] According to the present invention, a novel method for examining gastrointestinal diseases that is less invasive for the subject of the examination can be provided. [Brief explanation of the drawing]

[0016] [Figure 1] The results for protein content before and after changing the stool smear extraction conditions in Example 1 are shown. The horizontal axis represents the difference in extraction conditions, and the vertical axis represents the number of identified proteins. In Figure 1, the two bar graphs on the left show the measurement results under normal pretreatment conditions for measuring cells, etc., and the two bar graphs on the right show the measurement results after changing the pretreatment conditions. [Figure 2] Figure 2 shows the analysis results of the measured values ​​of various proteins in stool smears in Example 1. The horizontal axis shows the magnitude of the change in expression level (log2 Fold Change). The vertical axis shows the indicator of statistical significance (-log10 P-value). In Figure 2, the upper right region corresponds to the group of proteins that increased in neonatal and infant food protein gastroenteropathy patients compared to the control group. [Figure 3] In Example 2, Eosinophil cationic protein (ECP) was used as the indicator protein, and the results of ROC analysis performed on the patient group and the control group are shown. [Figure 4] Fig. 3 shows a comparison of the ECP / total protein ratio in fecal mucus between groups in Example 3. The ECP / total protein ratio (pg / ng) in the Control group (n=3) and the Non-IgE-GIFA patient group (n=6) is shown in a scatter plot. The horizontal axis represents groups, and the vertical axis represents the ECP / total protein ratio. The figure shows that a high-value range is observed in the patient group. Mode for Carrying Out the Invention

[0017] In one embodiment, the present invention provides a method for testing a gastrointestinal disease, which uses at least one protein selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2 in a fecal mucus sample derived from a test subject as an indicator.

[0018] As used herein, the term "testing" includes testing for the presence or absence of a gastrointestinal disease and testing for the risk of a gastrointestinal disease. Preferably, it refers to testing for the presence or absence of a gastrointestinal disease. The term "testing for risk" includes testing and determining whether there is a possibility of developing a gastrointestinal disease in the future. The term "testing" can also be rephrased as "determination" or "diagnosis".

[0019] Samples, subjects In the testing method of the present invention, a sample collected from a test subject is used. In the present invention, a fecal mucus sample derived from a test subject is used as a measurement target.

[0020] The sample is derived from the test subject of the testing method. The test subject is not particularly limited, and examples thereof include mammals including humans. Examples of non-human mammals include mice, rats, monkeys, dogs, guinea pigs, rabbits, sheep, goats, horses, pigs, and cattle. A preferred test subject for the testing method of the present invention is a human.

[0021] The present invention is characterized in that it targets fecal mucus rather than the entire stool as the object of measurement. Typically, fecal mucus may include intestinal mucosa, which is the shed mucosa of the large and / or small intestines; and mucus from within the intestines. A fecal mucus sample can be obtained, for example, by preparing a fecal smear and extracting the components of the fecal smear. In another embodiment, a fecal mucus sample can also be obtained by centrifugation of stool and obtaining the supernatant of the sample obtained by centrifugation. Therefore, examples of fecal mucus samples include fecal smears and supernatants obtained by centrifugation of stool. A fecal smear is a biological sample obtained by applying stool and mucus to a substrate such as a glass slide and drying it. Examples of substrates to which feces and mucus are applied in the preparation of a fecal smear include polymer compounds such as polypropylene, polystyrene, polyethylene, polytetrafluoroethylene, polyester, polyamide, polyurethane, polycarbonate, polymethyl methacrylate, polyvinyl alcohol, polyetheretherketone, polyimide, polylactic acid, epoxy resin, polyvinyl chloride, polybutadiene, polyphenylene sulfide, polysulfone, polyethersulfone, polyetherimide, polyvinylidene fluoride, polyvinylidene fluoride, polyacetal, polyhydroxybutyrate, polycaprolactone, polychloroprene, polystyrene sulfonic acid, polymethylsiloxane, polyphthalamide, polytrimethylene terephthalate, polypropylene oxide, polythiophene, polypyrrole, polyaniline, and polysulfide; metals such as stainless steel, aluminum, titanium, nickel, silver, copper, molybdenum, and tantalum; and inorganic materials such as ceramics, paper, mica, and gypsum. The shape of the substrate is not limited, but a plate-like form such as a glass slide is preferred. Since the fecal smear is dried, it can be stored for 10 years or more, but it is preferable to use one that has been collected and prepared within the last year. Even if the fecal smear has been stored (for example, stored at room temperature for 1 day or more, 1 week or more, 1 month or more, 1 year or more, 5 years or more, 10 years or more, etc.), it can still be used for proteomics analysis and is therefore useful. In the present invention, the fecal mucus sample may contain fecal-derived components other than fecal mucus (food residue, intestinal bacteria, etc.) to the extent that the effects of the present invention can be obtained.In a typical embodiment of the present invention, the fecal mucus sample preferably contains more fecal mucus than solids such as food residue and intestinal bacteria, and of the total 100% by mass of solids such as food residue and intestinal bacteria and fecal mucus, it is preferable that fecal mucus accounts for 60% by mass or more, preferably 70% by mass or more, and more preferably 80% by mass or more.

[0022] Gastrointestinal diseases The gastrointestinal diseases that are the subject of examination in this invention are not limited, but examples include allergic gastrointestinal diseases and non-allergic gastrointestinal diseases, with allergic gastrointestinal diseases being preferred. These diseases occur in the gastrointestinal tract, such as the esophagus, stomach, small intestine, and large intestine, but diseases relating to the stomach, small intestine, and large intestine are particularly preferred. Allergic gastrointestinal diseases include neonatal and infant food protein-induced gastrointestinal food allergy (non-IgE-GIFA), specifically Food Protein-Induced Enterocolitis Syndrome (FPIES), Food Protein-Induced Allergic Proctocolitis (FPIAP), and Food Protein-Induced Enteropathy (FPE).

[0023] Furthermore, diseases that can be examined using the present invention include eosinophilic gastrointestinal diseases. It has been pointed out that some neonatal and infant food protein-induced gastroenteropathy and eosinophilic gastrointestinal diseases overlap in their pathophysiology, and examples of eosinophilic gastrointestinal diseases (EGIDs) include eosinophilic esophagitis (EoE) and EGIDs other than esophageal EGIDs. Inflammatory bowel disease (IBD), which is a chronic inflammatory disease of the gastrointestinal tract and involves abnormal immune responses, can also be examined using the present invention. Specifically, examples include ulcerative colitis and Crohn's disease. Food protein-induced gastroenteropathy in neonatal and infants may include food protein-induced gastroenteritis. In one preferred embodiment, the method of the present invention is very useful because it can examine neonatal and infant food protein-induced gastroenteropathy (e.g., food protein-induced gastroenteritis). Neonatal and infant food protein-induced gastrointestinal food allergy (Non-IgE-mediated gastrointestinal food allergy [neonatal and infant non-IgE-GIFA]) is a disease that has been increasing in recent years and is already designated as an intractable disease in Japan as a disease related to eosinophilic gastrointestinal disorders (EGIDs). Although cellular immunity is thought to be involved, the pathogenesis has not yet been clearly elucidated. Neonatal and infant non-IgE-GIFA is suspected when gastrointestinal symptoms such as lethargy, abdominal distension, vomiting, choleretic vomiting, decreased feeding ability, mucous stools, diarrhea, bloody stools, and poor weight gain appear after the start of feeding. If neonatal or infant non-IgE-GIFA is suspected, differential diagnoses should first be ruled out based on the symptoms, and it should be confirmed that the symptoms improve by removing the causative food (elimination test). A comprehensive diagnosis of this disease may be made based on the results of auxiliary diagnostic tests such as blood tests, plain X-rays, contrast tests, endoscopy, ultrasound, food allergen-specific lymphocyte stimulation tests (ALST), eosinophils in fecal mucus, milk-specific IgE antibodies, and intestinal mucosal tissue examinations. Alternatively, a definitive diagnosis may be made by confirming the reappearance of symptoms after reintroducing the causative food (oral food challenge test [OFC]). In this condition, reingesting the causative food for a definitive diagnosis carries a high risk, and the Ministry of Health, Labour and Welfare research group's guidelines for the treatment of neonatal-infant food protein-induced gastroenteropathy suggest that this should be done cautiously, taking into account the facility and the patient's condition. In many cases, the condition is diagnosed with elimination testing and auxiliary diagnosis. Although not mentioned in the guidelines for the treatment of neonatal-infant food protein-induced gastroenteropathy, a patent has been published (JP 2021-63743) for diagnosing patients suspected of having neonatal-infant food protein-induced gastroenteropathy by measuring interleukins in the serum. While this would be useful if it were developed into a diagnostic kit and put into practical use, it requires blood sampling, which is an invasive test for neonates and infants. As mentioned above, it has been difficult to perform non-invasive tests until now, so this invention is particularly useful in the testing of neonatal-infant food protein-induced gastroenteropathy.

[0024] Testing method In a preferred embodiment, the method of the present invention includes a method for measuring at least one (preferably two or more, more preferably three or more, more preferably four or more, more preferably five) selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2 in a fecal mucus sample. In the present invention, "at least one selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2" may also be simply referred to as "ECP, etc."

[0025] In this embodiment, the measurement method for ECP, etc., is not limited, and any method that can be used in the medical technology field to which the present invention belongs may be appropriately adopted. Specific measurement methods for ECP, etc., include, for example, Western blotting, ELISA, mass spectrometry, flow cytometry, chromatography, electrophoresis, and the like.

[0026] In one embodiment, the present invention may include a step of treating a fecal mucus sample (e.g., a fecal smear) collected and prepared from a test subject with an extract. This step can be carried out, for example, by suspending the fecal mucus sample (e.g., a fecal smear) in a buffer and eluting the proteins in the fecal mucus sample into the buffer. Examples of buffers include Tris-buffered saline (TBS), BIS-TRIS buffer, MOPS buffer, PIPES buffer, and TES buffer. A protease inhibitor may also be added to the buffer. Optionally, centrifugation may be performed further during the treatment with the extract.

[0027] In one embodiment, the method of the present invention may include a step of detecting proteins in a fecal mucus sample or an extract that has undergone the above-mentioned processing step, and measuring the abundance of at least one protein selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2. The measurement method for ECP, etc., is as described above.

[0028] Furthermore, in one embodiment, in the method of the present invention, if the amount of the measured protein group exceeds a predetermined reference value, the measurement result may be used to determine that the subject has a gastrointestinal disease. In this embodiment, the reference value can be set, for example, by the following method. For example, in one embodiment, according to the present invention, if the mass of ECP etc. in the same amount (same mass) of sample being measured is greater than the mass of ECP etc. in a sample of a subject that does not suffer from a gastrointestinal disease, which is used as a comparison, for example, if it is more than 1 times the mass of ECP etc. in the sample of a subject that does not suffer from a gastrointestinal disease, preferably 1.5 times or more, more preferably 2.0 times or more, then it can be determined that the subject being measured has a gastrointestinal disease. Here, if there are multiple samples of subjects that do not suffer from a gastrointestinal disease that are used as a comparison, then "the mass of ECP etc. in the sample of a subject that does not suffer from a gastrointestinal disease that is used as a comparison" can be read as the average value of the mass of ECP etc. in the sample of a subject that does not suffer from a gastrointestinal disease that is used as a comparison. Similarly, if there are multiple samples being measured, then "the mass of ECP etc. in the sample being measured" can be read as the average value of the mass.

[0029] Furthermore, in one embodiment, if there is a statistically significant difference between the mass of ECP etc. in a sample of a subject not suffering from a gastrointestinal disease and the mass of ECP etc. in the same amount (same mass) of sample being measured (for example, if the P value is less than 0.05, or if the P value is less than 0.01, etc.), it can be determined that the subject being measured has a gastrointestinal disease.

[0030] Furthermore, in one embodiment, if the mass of ECP, etc., in the sample to be measured is "larger" and "statistically significant" compared to the mass of ECP, etc., in the same amount (same mass) of sample of a subject without gastrointestinal disease, according to the above criteria, it can be determined that the subject has a gastrointestinal disease. In the present invention, the same amount (same mass) is not limited to measuring exactly the same amount (same mass) of samples, but also includes cases where ECP, etc., is measured in different amounts of samples, and the measured values ​​are converted to values ​​that would be obtained if the sample amounts were the same.

[0031] Furthermore, as described above, in these embodiments, if the mass of at least one protein among Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2 is "larger" and / or "statistically significant" compared to the mass of the same protein in the same amount (same mass) of sample of a subject without gastrointestinal disease, according to the above criteria, it can be determined that the subject has a gastrointestinal disease. In another embodiment, a subject may be determined to have a gastrointestinal disease if, for at least two (e.g., at least three, at least four, or all five) of the proteins Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2, the mass of the proteins in the sample being measured is "larger" and / or "statistically significant" compared to the mass of the proteins in the same amount (same mass) of sample of a subject without a gastrointestinal disease, according to the above criteria.

[0032] Furthermore, the mass of ECP, etc., in a sample of a positive control subject suffering from a gastrointestinal disease can be measured in advance, and the presence or absence of a gastrointestinal disease can be determined by comparing it with the mass of ECP, etc., in the same amount (same mass) of sample to be measured. For example, if the mass of ECP, etc., in the sample to be measured is about the same as or greater than the mass of ECP, etc., in the same amount (same mass) of sample of a positive control subject suffering from a gastrointestinal disease (e.g., 0.7 times or more, 0.8 times or more, 0.9 times or more, 1.0 times or more, etc.), it can be determined that the subject has a gastrointestinal disease. In this embodiment, it can also be determined that the subject has a gastrointestinal disease if the mass of ECP, etc., in the sample to be measured is statistically significant to be about the same as or greater than the mass of ECP, etc., in the same amount (same mass) of sample of a positive control subject suffering from a gastrointestinal disease (e.g., 0.7 times or more, 0.8 times or more, 0.9 times or more, 1.0 times or more). For example, when a t-test is performed with the alternative hypothesis that the mass of ECP etc. in the sample to be measured is approximately the same as (e.g., 0.7 times or more, 0.8 times or more, 0.9 times or more, 1.0 times or more) or greater than the mass of ECP etc. in the same amount (same mass) of a positive control subject suffering from a gastrointestinal disease, if the p-value is smaller than a predetermined significance level (e.g., 0.05, 0.01, etc.), it can be determined that the subject to be measured has a gastrointestinal disease. In these embodiments as well, if there are multiple samples of positive control subjects suffering from gastrointestinal diseases to be used for comparison, "mass of ECP etc. in the samples of positive control subjects suffering from gastrointestinal diseases to be used for comparison" can be read as the average mass of ECP etc. in the samples of positive control subjects suffering from gastrointestinal diseases to be used for comparison. Similarly, in these embodiments as well, if there are multiple samples to be measured, "mass of ECP etc. in the sample to be measured" can be read as the average mass. Furthermore, the mass of ECP, etc., in a sample of a negative control that does not have a gastrointestinal disease can be measured in advance, and the presence or absence of a gastrointestinal disease can be determined by comparing it with the mass of ECP, etc., in the same amount (same mass) of sample to be measured.For example, if the mass of ECP etc. in the sample being measured is significantly greater than the mass of ECP etc. in the same amount (same mass) of a negative control subject who does not have a gastrointestinal disease (e.g., 2.0 times or more, 5.0 times or more, 10 times or more, etc.), it can be determined that the subject has a gastrointestinal disease. In this embodiment, it can also be determined that the subject has a gastrointestinal disease if the mass of ECP etc. in the sample being measured is significantly greater than the mass of ECP etc. in the same amount (same mass) of a negative control subject who does not have a gastrointestinal disease (e.g., 2.0 times or more, 5.0 times or more, 10 times or more) or more, and this difference is statistically significant. For example, if a statistical test (e.g., t-test, Mann-Whitney U test, etc.) is performed with the alternative hypothesis that the mass of ECP etc. in the sample being measured is significantly greater (e.g., 2.0 times or more, 5.0 times or more, 10 times or more) than the mass of ECP etc. in the same amount (same mass) of a negative control sample that does not have a gastrointestinal disease, and the p-value is smaller than a predetermined significance level (e.g., 0.05, 0.01, etc.), then it can be determined that the subject being measured has a gastrointestinal disease. Statistical tests such as the t-test and Mann-Whitney U test can be used. In these embodiments as well, if there are multiple samples of negative controls that do not have a gastrointestinal disease for comparison, the mean or median mass of ECP etc. in the negative control samples that do not have a gastrointestinal disease can be used as the "mass of ECP etc. in the negative control samples that do not have a gastrointestinal disease for comparison." Similarly, in these embodiments as well, if there are multiple samples being measured, the mean or median mass can be used as the "mass of ECP etc. in the sample being measured."

[0033] Furthermore, in one embodiment of the present invention, it is preferable to use the mass ratio of ECP to total protein in the sample [ECP / total protein] as an indicator. Specifically, if the [ECP / total protein] (pg / ng) in the target sample is greater than a predetermined value (for example, 0.01 (pg / ng) or more, 0.02 (pg / ng) or more, 0.03 (pg / ng) or more, 0.05 (pg / ng) or more, 0.07 (pg / ng) or more, 0.10 (pg / ng) or more, etc.), it can be determined that the subject of measurement has a gastrointestinal disease.

[0034] According to the present invention, gastrointestinal diseases can be easily and non-invasively examined by using Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and / or Hemoglobin subunit gamma-2 in a fecal mucus sample as indicators. Such effects were not predictable from the prior art. Specifically, there have been no previous reports that Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2 can be used as indicators for the examination of gastrointestinal diseases. In particular, regarding Eosinophil cationic protein, if the entire stool sample is used instead of a fecal mucus sample, the Eosinophil cationic protein is degraded and difficult to detect. Therefore, it was not predictable that Eosinophil cationic protein could be used as an indicator. As described above, the effects of the present invention are not predictable from the prior art.

[0035] Kits, etc. In one embodiment, the present invention provides a gastrointestinal disease testing kit comprising means for detecting at least one protein selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2, for use in a method comprising the step of measuring at least one protein selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2 in a fecal mucus sample derived from a test subject. Details regarding the method of use of the kit, fecal mucus sample, gastrointestinal disease, etc., are as described above. The kit may also include means for collecting, preparing, and storing the fecal mucus sample, means for detecting ECP, etc., from the fecal mucus sample, etc.

[0036] Means for collecting, preparing, and storing fecal mucus samples include, for example, a substrate (for making a fecal smear), a centrifuge tube (for centrifuging the feces), a dropper (for obtaining the supernatant of the centrifuged sample), a buffer solution (for treating the fecal smear with an extract), and a container for storing the fecal mucus sample. Means for detecting ECP, etc., from the fecal mucus sample include antibodies that specifically bind to ECP, etc. These means may be used individually or in combination of two or more. Furthermore, the kit of the present invention may include instructions describing how to use the kit, that is, the gastrointestinal disease testing method of the present invention.

[0037] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the examples, EPX refers to eosinophil peroxidase. [Examples]

[0038] Example 1 <patient> This study included 41 neonatal and infant patients suspected of having non-IgE-GIFA or eosinophilic gastrointestinal disease based on their symptoms, who subsequently received a stool smear and were diagnosed with the disease. Stool smears were collected between 2014 and 2024. The diagnosis of neonatal and infant non-IgE-GIFA was made in accordance with the Neonatal and Infant Food Protein Gastroenteropathy Treatment Guidelines (2019) and the Child and Adult Eosinophilic Gastrointestinal Disease Treatment Guidelines (2020).

[0039] <Comparison subjects> For comparison, we included 26 patients who were suspected of having neonatal or infant food protein-induced enteropathy or eosinophilic gastrointestinal disease based on their symptoms, had stool smears prepared, and were later diagnosed with a different disease, or were hospitalized for other reasons and confirmed not to have the disease in question. The procedure for preparing stool smears was the same for patients hospitalized for other reasons.

[0040] <Creating a fecal smear> The mucous portion of the stool surface from the patient or control group was collected on a glass slide and smeared onto it. Immediately after smearing, it was dried with air at room temperature, and after drying, it was stored at room temperature until extraction.

[0041] <Extraction of fecal smear> Fecal smears were suspended in Tris-buffered saline (TBS) containing a protease inhibitor (Roche, cOmplete® ULTRA tablets, Min, EASYpack protease inhibitor cocktail), and this was used as the extract. The same amount of TBS per slide was used for suspension, and the extracts were measured for absorbance along with a standard substance. The concentration was adjusted to ensure uniform protein concentration in the extracts of each sample. The extracts were collected in tubes and cooled with ice. The ice-cooled tubes were pipetted and then centrifuged at 4°C at 15000g for 20 minutes. The supernatant was collected, and 12.5% ​​trichloroacetic acid was added to the supernatant to precipitate the protein. The protein was washed with 80% propanol and ethanol. The washed protein was collected in a Tris-HCl 0.024% LMNG CaCl solution. The protein was digested with Trypsin to form peptide fragments, which were then subjected to reductive alkylation and desalted. The desalted samples were dried using a centrifugal evaporator and redissolved in 0.02% DMNG-0.1% TFA.

[0042] As an example of existing extraction conditions, the protein is suspended in 100 mM Tris-HCl (pH 8.0) and 20 mM NaCl (containing 4% SDS), and extracted by sonication for 10 minutes. The protein concentration in this protein extract is measured and adjusted. This extract is then used to prepare peptides using the SP3 method, digested with Trypsin, and converted into peptide fragments. These peptide fragments are subjected to reductive alkylation and then desalted. The desalted sample is dried using a centrifugal evaporator and redissolved in 0.02% DMNG-0.1% TFA. Figure 1 shows the results of protein quantity before and after changing the stool smear extraction conditions in Example 1. The horizontal axis represents the difference in extraction conditions, and the vertical axis represents the number of identified proteins. In Figure 1, the two bar graphs on the left show the measurement results under existing pretreatment conditions for measuring cells, etc., and the two bar graphs on the right show the measurement results after changing the pretreatment conditions. As shown in Figure 1, the number of identified proteins increased by approximately three times with the extraction conditions of the present invention compared to the existing extraction conditions.

[0043] <Peptide measurement and data analysis> This peptide was measured by LC-MS / MS. The raw data files obtained by LC-MS / MS were imported into DIA-NN. DIA-NN is software specifically designed for the analysis of data-independent acquisition (DIA) proteomics data, utilizing deep neural networks to achieve highly accurate peptide and protein identification and quantification. For data analysis, a protein sequence database in FASTA format, widely used in proteome analysis, was used. In this analysis, human-derived FASTA files provided by Prosit (https: / / www.proteomicsdb.org / prosit) were used. These FASTA files contain protein sequences included in the human proteome and were used for identification and quantitative analysis of LCMS / MS data. The spectral library was generated directly from the FASTA files using DIA-NN's library-free mode. DIA-NN generated a report containing the identification results and quantitative information for each peptide. This report file output from DIA-NN was loaded into Perseus. Perseus is software for statistical analysis and visualization of proteomics data and is suitable for post-processing of data obtained from DIA-NN. The loaded quantitative data was log2 transformed, data normalization and handling of missing values ​​were performed, and outstanding proteins were identified using t-tests. Figure 2 shows the group of proteins increased in neonatal and infant food protein gastroenteropathy patients using a Volcano Plot. The horizontal axis of Figure 2 shows the magnitude of the change in expression level (log2Fold Change). Specifically, for each protein detected in the patient-derived sample, it shows the base-2 logarithm (log2) of the ratio of the mass of the protein in the patient-derived sample to the mass of the protein in the comparison subject-derived sample (log2{[mass of the protein in the patient-derived sample] / [mass of the protein in the comparison subject-derived sample]}). The vertical axis is an indicator of statistical significance (-log). 10This shows the P-value. Specifically, for each protein detected in the patient-derived sample, it is the common logarithm (log) of the P-value calculated by a T-test between the mass of the protein in the patient-derived sample and the mass of the protein in the control sample. 10 The number obtained by multiplying () by -1 (-log 10 The p-value is shown. As shown in Figure 2, the amounts of Eosinophil cationic protein (RNASE3), Eosinophil peroxidase (EPX), Hemoglobin subunit gamma-1 (HBG1), Hemoglobin subunit beta (HBB), and Hemoglobin subunit gamma-2 (HBG2) were significantly higher in patient-derived samples than in control group samples.

[0044] Example 2 <patient> This study included 40 neonates and infants diagnosed with neonatal non-IgE GIFA. Fecal smears were collected from these subjects between 2014 and 2024. The diagnosis of neonatal and infant non-IgE-GIFA was made in accordance with the Neonatal and Infant Food Protein Gastroenteropathy Treatment Guidelines (2019) and the Child and Adult Eosinophilic Gastrointestinal Disease Treatment Guidelines (2020).

[0045] <Comparison subjects> For comparison, we included 19 newborns and infants who were hospitalized for reasons other than gastrointestinal disease (unrelated hospitalization) and did not have the disease in question. The procedure for preparing stool smears was the same for patients hospitalized for unrelated reasons.

[0046] <Creating a fecal smear> The mucous portion of the stool surface from the patient or control group was collected on a glass slide and smeared onto it. Immediately after smearing, it was dried with air at room temperature, and after drying, it was stored at room temperature until extraction.

[0047] <Extraction of fecal smear> Fecal smears were suspended in Tris-buffered saline (TBS) containing a protease inhibitor (Roche, cOmplete® ULTRA tablets, Min, EASYpack protease inhibitor cocktail), and this was used as the extract. The same amount of TBS per slide was used for suspension, and the extracts were measured for absorbance along with a standard substance. The concentration was adjusted to ensure uniform protein concentration in the extracts of each sample. The extracts were collected in tubes and cooled with ice. The ice-cooled tubes were pipetted and then centrifuged at 4°C at 15000g for 20 minutes. The supernatant was collected, and 12.5% ​​trichloroacetic acid was added to the supernatant to precipitate the protein. The protein was washed with 80% propanol and ethanol. The washed protein was collected in a Tris-HCl 0.024% LMNG CaCl solution. The protein was digested with Trypsin to form peptide fragments, which were then subjected to reductive alkylation and desalted. The desalted samples were dried using a centrifugal evaporator and redissolved in 0.02% DMNG-0.1% TFA.

[0048] <Peptide measurement and data analysis> This peptide was measured by LC-MS / MS. The raw data files obtained by LC-MS / MS were imported into DIA-NN. DIA-NN is software specifically designed for the analysis of data-independent acquisition (DIA) proteomics data, utilizing deep neural networks to achieve highly accurate peptide and protein identification and quantification. For data analysis, a protein sequence database in FASTA format, widely used in proteome analysis, was used. In this analysis, human-derived FASTA files provided by Prosit (https: / / www.proteomicsdb.org / prosit) were used. These FASTA files contain protein sequences included in the human proteome and were used for identification and quantitative analysis of LCMS / MS data. The spectral library was generated directly from the FASTA files using DIA-NN's library-free mode. DIA-NN generated a report containing the identification results and quantitative information for each peptide. This report file output from DIA-NN was loaded into Perseus. Perseus is software for statistical analysis and visualization of proteomics data and is suitable for post-processing of data obtained from DIA-NN. Eosinophil cationic protein (ECP) was used as the indicator protein, and ROC analysis was performed on patient and control groups. The ROC analysis was performed using the Graphpad prism 10 analysis software. The results are shown in Figure 3. High discriminative ability (AUC approximately 0.98) was confirmed. Considering clinical application and prioritizing the avoidance of false positives, a cutoff was set under the condition that all control groups in this dataset were negative (Intensity 15465800). Subjects with ECP levels above the cutoff value were judged positive, and subjects below the cutoff value were judged negative. As a result, 38 out of 40 patients tested positive, and 2 tested negative. In contrast, all 19 control patients tested negative (Table 1). Similarly, high discriminative ability has been confirmed when using eosinophil peroxidase (EPX) as an indicator.

[0049]

Table 1

[0050] From the above results, it was shown that gastrointestinal diseases can be discriminated by using the amount of ECP in fecal mucus samples as an index.

[0051] Example 3 ECP quantification and total protein correction analysis by ELISA <ECP Measurement Method> Fecal smears and fecal mucus sample extracts were prepared from feces of patient group and control group by the same method as in Examples 1 and 2. Eosinophil cationic protein (ECP) in the fecal mucus sample extract was quantified using a Human Eosinophil Cationic Protein ELISA Kit (#SK00128-06, manufactured by Aviscera Bioscience). A standard curve was prepared using the standard ECP protein included with the kit, and the ECP concentration (pg / mL) of each sample was calculated by absorbance measurement. The obtained concentration was converted based on an extract volume of 220 μL, and the total amount of ECP per slide (ng / slide) was calculated. <Measurement of Total Protein Amount> The total amount of protein in the extract was measured by the BCA (Bicinchoninic Acid) protein quantification method. Specifically, using a commercially available BCA Protein Assay Kit, a standard curve was prepared with bovine serum albumin (BSA) as the standard protein, and the total protein concentration in the extract was calculated. From the obtained total protein concentration, the total amount of protein per slide (μg / slide) was calculated based on an extract volume of 220 μL. <Normalization Processing> Fecal smears were prepared with a uniform application area of 1482 mm 2 , and the application amount was also standardized. The ECP amount was calculated using the following index. ECP / total protein (pg / ng) ECP_total (value calculated by converting the ELISA measurement value (pg / mL) to 220 μL) is divided by the total protein amount measured by the BCA method. <Comparison between groups> We analyzed the data for the control group (n=3) and the non-IgE-GIFA patient group (n=6). ECP / Total Protein (pg / ng) (Figure 4) Control group Median 0.006709 Average value: 0.01024 Standard deviation 0.006624 patient group Median 0.03099 Average value: 0.1038 Standard deviation 0.1635 A comparison using the Mann-Whitney test (two-tailed) yielded a p-value of 0.0952. The Hodges-Lehmann estimated difference was 0.01899 pg / ng. The ROC analysis revealed an AUC of 0.8889. <Consideration> The core of this invention lies in its ability to non-invasively diagnose gastrointestinal diseases, particularly neonatal and infant food protein-induced gastroenteropathy, using fecal mucus samples. Traditionally, the diagnosis of this disease has required elimination tests, oral food challenge tests, blood tests, and endoscopy, which have been highly invasive methods for neonates and infants. Furthermore, when the entire stool sample is used, ECP is easily degraded, making stable detection difficult. In contrast, this invention focuses on fecal mucus rather than the entire stool. Fecal mucus contains many components derived from the intestinal mucosa and more directly reflects information about the site of inflammation. As a result, the amount of ECP in fecal mucus showed an increasing trend in the disease group and demonstrated high discriminative ability (AUC = 0.8889) in ROC analysis. Furthermore, in this embodiment, considering the variability in sample volume and extraction efficiency specific to fecal smears, the amount of ECP was corrected by the total protein amount (measured by the BCA method). As a result, the ECP / total protein ratio showed higher discriminative ability compared to the absolute amount of ECP alone. However, total protein correction is not an essential component of the present invention, but rather an example of a preferred embodiment for further stabilizing the diagnostic method using fecal mucus samples. The technical significance of the present invention lies in the fact that it enables non-invasive diagnosis of gastrointestinal diseases in neonates and infants by using ECP in fecal mucus samples as an indicator. This diagnostic approach using fecal mucus is difficult to predict with conventional techniques that rely on whole-stool measurement or serum testing, and therefore the present invention has significant technical importance.

Claims

1. A method for testing gastrointestinal diseases, using at least one protein selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2 in a fecal mucus sample derived from a test subject as an indicator.

2. A method according to claim 1, wherein the fecal mucus sample is obtained by preparing a fecal smear and extracting components of the fecal smear, or by centrifugation of feces and obtaining the supernatant of the centrifuged sample.

3. The method according to claim 1, (a) A step of treating a stool smear collected and prepared from the test subject with an extractant, (b) A step of detecting proteins in the extract and measuring the amount of at least one protein selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2. (c) A method in which, if the amount of the measured protein group is more than twice the mean value of the control group and a statistically significant difference (P<0.05, T-test) is observed, it serves as an indicator that the subject has a gastrointestinal disease.

4. The method according to claim 1, wherein the gastrointestinal disease is an eosinophilic gastrointestinal disease.

5. The method according to claim 1, wherein the gastrointestinal disorder is neonatal or infant food protein-induced gastroenteropathy.

6. A diagnostic kit for gastrointestinal diseases, comprising means for detecting at least one protein selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2, the kit for use in a method comprising the step of measuring at least one protein selected from the group consisting of Eosinophil cationic protein, Eosinophil peroxidase, Hemoglobin subunit gamma-1, Hemoglobin subunit beta, and Hemoglobin subunit gamma-2 in a fecal mucus sample derived from a test subject.

7. A kit according to claim 6, wherein the fecal mucus sample is obtained by preparing a fecal smear and extracting components of the fecal smear, or by centrifugation of feces and obtaining the supernatant of the centrifuged sample.

8. A kit according to claim 6, for use in the method according to any one of claims 1 to 5.

9. The kit according to claim 6, wherein the gastrointestinal disease is an eosinophilic gastrointestinal disease.

10. The kit according to claim 6, wherein the gastrointestinal disorder is neonatal or infant food protein-induced gastroenteropathy.

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