Method for assisting diagnosis of gastrointestinal disease, method for screening diagnostic marker for gastrointestinal disease, and reagent and kit for detecting gastrointestinal disease-related protein

Abstract

This method for assisting diagnosis of gastrointestinal diseases comprises: a step for isolating fecal exosomes from feces obtained from a subject; and a step for detecting gastrointestinal disease-related protein from the isolated fecal exosomes. The gastrointestinal disease-related protein satisfies either condition (a) or (b): (a) the content of the protein with respect to the total amount of the fecal exosomes is 1.5 times or more of the content of the protein with respect to the total amount of the feces; and (b) the protein is detected only from the isolated fecal exosomes and is substantially not detected from the feces.

Description

Method for assisting in the diagnosis of gastrointestinal disease, method for screening diagnostic markers for gastrointestinal disease, and reagent and kit for detecting proteins associated with gastrointestinal disease

[0001] The present invention relates to a method for assisting in the diagnosis of gastrointestinal diseases, a method for screening diagnostic markers for gastrointestinal diseases, and a reagent and kit for detecting proteins associated with gastrointestinal diseases. More particularly, the present invention relates to a method for assisting in the diagnosis of gastrointestinal diseases, a method for screening diagnostic markers for gastrointestinal diseases, and a reagent for detecting proteins associated with gastrointestinal diseases for use in these methods, and a kit containing the same.

[0002] In recent years, many testing methods have been adopted to aid in the diagnosis of various diseases, such as detecting and quantifying target substances as biomarkers for various diseases, including circulating tumor cells, free DNA in the blood, exosomes, and proteins derived from these substances, from liquid samples such as blood and urine samples. These testing methods using liquid samples are useful as minimally invasive testing methods that impose less strain on the body than tissue tests and can be performed multiple times. Therefore, in recent years, they have been expected to be used not only for the diagnosis of tumor-related diseases, but also for selecting treatment methods and follow-up observations. However, the above-mentioned liquid samples generally have very low amounts of biomarkers per total sample volume, and biomarkers derived from various tissues and organs are mixed, making it difficult to detect biomarkers for the target disease with high sensitivity. For example, extracellular vesicles such as exosomes are known to contain marker proteins that are effective in identifying diseases such as cancer. However, as described by Takuya Naito et al., FEBS Letters, Vol. 595, 19, 2021, p. 2522-2532 (Non-Patent Document 1) reports that it is difficult to identify the organ or tissue from which a marker protein contained in extracellular vesicles of a blood sample originates.

[0003] In addition to the above-mentioned liquid samples, minimally invasive testing methods also include methods using feces as a sample, detecting and quantifying marker proteins for various diseases from the sample, and assisting in the diagnosis of the disease. For example, International Publication No. 2013 / 162368 (Patent Document 1) describes a method for screening for colorectal cancer by detecting multiple marker proteins from a homogenized fecal sample.

[0004] International Publication No. 2013 / 162368

[0005] Takuya Naito et al., FEBS Letters, Vol. 595, 19, 2021, p. 2522-2532

[0006] As described above, feces are useful as minimally invasive specimens. However, since feces contain many impurities compared to liquid specimens, there is a problem that the relative content of the target marker protein is low and falls below the detection limit. Furthermore, when detecting a marker protein associated with a gastrointestinal disease, feces also contains many proteins derived from other tissues, particularly the liver and liver diseases, in addition to the target marker protein (Patent Document 1). Therefore, in order to detect such a target marker protein from feces, it is necessary to adopt a specific purification method or a highly sensitive detection method for each marker protein.

[0007] The present invention has been made in consideration of the problems associated with the above-mentioned conventional technologies, and aims to provide a method for assisting in the diagnosis of gastrointestinal diseases, which can efficiently detect gastrointestinal disease-related proteins using feces as a sample, a method for screening diagnostic markers for gastrointestinal diseases, and detection reagents and kits for gastrointestinal disease-related proteins to be used in these methods.

[0008] To achieve the above-mentioned objective, the present inventors conducted extensive research, fractionating exosomes from feces and conducting various analyses of the proteins contained in the fractionated fecal exosomes. As a result, they discovered a novel phenomenon in which gastrointestinal disease-related proteins, including proteins derived from the gastrointestinal tract, are frequently and specifically present (i.e., enriched) in fecal exosomes at a higher concentration than their concentration in the total feces (hereinafter, this phenomenon is sometimes referred to as "enrichment"). They also confirmed that the "enrichment" of gastrointestinal disease-related proteins in fecal exosomes is statistically significant. The phenomenon of gastrointestinal disease-related proteins being enriched in fecal exosomes at a high frequency is surprising, given the difficulty in identifying the source of proteins contained in exosomes and other samples using the above-mentioned blood samples. Therefore, the present inventors have found that by isolating such fecal exosomes from feces and detecting gastrointestinal disease-related proteins "concentrated" in such fecal exosomes, it is possible to very efficiently detect the gastrointestinal disease-related protein of interest without using specific purification methods or highly sensitive detection methods. Furthermore, because gastrointestinal disease-related proteins concentrated in exosomes are detected after isolating them, it is also possible to detect the gastrointestinal disease-related protein of interest by Western blotting or the like, and it has also been found that the presence or absence and amount of the gastrointestinal disease-related protein of interest can be used as an indicator to efficiently diagnose the associated gastrointestinal disease, thereby completing the present invention.

[0009] The present invention, based on these findings, provides the following aspects: [1] A method for assisting in the diagnosis of gastrointestinal diseases, comprising the steps of isolating fecal exosomes from the feces of a subject, and detecting a gastrointestinal disease-associated protein from the isolated fecal exosomes, wherein the gastrointestinal disease-associated protein satisfies either of the following conditions (a) and (b): (a) its content relative to the total amount of the fecal exosomes is 1.5 times or more its content relative to the total amount of the feces, or (b) it is detected only in the isolated fecal exosomes and is not substantially detectable in the feces. [2] The method according to [1], wherein the gastrointestinal disease-associated protein is at least one selected from the group consisting of OLFM4, LGALS3BP, LAMP1, ATP5F1A, DLD, ENPP4, MAPK1, PCYOX1, SMPD3, VILL, and RPS7. [3] The method according to [1] or [2], wherein the gastrointestinal disease is an intestinal disease. [4] The method according to any one of [1] to [3], wherein the gastrointestinal disease is colorectal cancer. [5] The method according to any one of [1] to [4], further comprising a step of quantifying the detected gastrointestinal disease-associated protein. [6] A method for assisting in the diagnosis of a gastrointestinal disease, comprising a step of isolating fecal exosomes from the feces of a subject, and a step of detecting a gastrointestinal disease-associated protein from the separated fecal exosomes, The gastrointestinal disease-related proteins include ZMPSTE24, YWHAG, UGDH, TPM4, TC2N, SUSD2, STOM, ST6GALNAC1, SPTBN1, SLC6A14, SLC46A1, SLC15A4, SERPINF2, SDHB, SDHA, SAFB2, RPS16, RNF128, RNASE3, RAB21, RAB14, PRKG2, PLEC, PLA2G10, and NPEPP. S, MYH14, MT-CYB, MAOA, ITGA3, IGLV7-46, HSD17B4, HARS1, H1-4, GSTA1, GMDS, GFM2, FUT3, FLOT2, DNPEP, CND P2, CLTC, B3GNT7, B2M, ATP9A, ARPC2, AP2A1, AP1B1, AGR2, ACTR2, ABRACL, YWHAH, WDR1, VILL, VAMP8, TUBB4B,<h2 style=";text-align:left;direction:ltr">TSPAN6、TPP1、TPM3、TPM1、TMEM30A、S YPL1、SQOR、SPPL2A、SMPD3、SLC9A3、S LC44A1、SLC2A5、SLC25A5、SLC15A1、S ERPINA7、SCARB2、S100A11、RPS7、RPS 3、RPS23、RPS10、RPL30、RNPEP、RHPN2 、RAB3D、RAB32、RAB2A、PTPN6、PSMB8、 PSMB1、PSMA1、PRKCD、PRKACA、PRDX6、 PRDX5、PRDX1、PPP1CB、PLA2G2A、PKM、 PICALM、PGK1、PGD、PFN1、PCYOX1、PCB P1、P2RX4、NIBAN2、NEDD4L、NCSTN、MY OF、MYO1E、MYO1C、MYO1B、MYH9、MUC4、MUC3A、MGST3、MGLL、MEP1A、LYN、LGAL S4、LGALS3BP、LDHA、LAMP1、IGKV1-39 、IGHM、IGHG2、IDH1、HSPA5、HSP90AB1 、HNRNPA2B1、HLA-DMA、H2BC12、GSTP1 、GRN、GPI、GPD2、GPA33、GP2、GNAI2、GH ITM、GDPD3、GAPDH、FLOT1、F11R、EPCA M、ENPP4、ENO1、EIF5A、EIF4A1、EEF2、 EBP、DSTN、DLD、CYFIP1、CSNK1A1、CPN E3、CLIC1、CHMP2A、CEACAM5、CEACAM1 、CD151、CAPZB、CAPN1、CAP1、C9、ATP1 B1、ATP1A1、ASAH1、ARL1、ARHGDIA、AN XA4、ANXA3、ALDOA、ALDH3B1、ACTN4、A CAA1、XPO1、VPS26A、UQCRC2、UQCRC1、U BA1、TPP2、TMEM33、TMEM205、TMED4、T MED2、TMED10、TM9SF3、TM9SF2、THEMI S2、TARDBP、SYNGR2、SYK、STIP1、STAT 3、STAT1、SSR4、SRSF3、SPCS3、SPAST、 SOD2、SLC3A2、SLC2A3、SLC2A1、SLC16A3、SIRPA、SIL1、SERPINB5、SELENOP、 SEC31A、SEC23A、RPS4X、RPS3A、RPS18 、RPN1、RPL7、RPL6、RPL5、RPL4、RPL21、<h2 style=";text-align:left;direction:ltr">RPL13A、RARS1、QARS1、PTK2B、PTGES 3、PSMD3、PSMD12、PSMD1、PSMC2、PRXL 2A、PRPS1、PRKDC、PFKP、PCK2、PC、PAI CS、OCIAD2、OAF、NT5C2、NIPSNAP2、ND UFB10、MT-CO2、MOB1B、MICAL1、MGST 1、MCCC2、M6PR、LNPEP、LBR、KDELR2、K DELR1、KARS1、ITGAV、IMPDH2、IGKV1- 12、IDH3B、IDH2、HTT、HSPA4、HSD17B1 0、HNRNPUL2、HNRNPU、HNRNPK、HMGCS 2、HLA-B、HK1、HIBCH、HADHA、H1-0、GR TP1、GPD1、GMPPB、GCLM、GBE1、GARS1、 GALNT2、FGR、FCN3、FASN、ERO1A、ENTP D1、EML4、EIF3D、DYNC1H1、DNM2、DLAT 、DHX9、DHRS7、DARS1、CYB5B、CSK、CRY L1、CPT1A、COX5B、CORO1C、COPG1、COP A、CLDN7、CHST5、CCT4、CCT3、C8G、BSG 、B3GNT3、ATP6V0D1、ATP5MG、ARPC3、 ARPC1B、ARHGAP1、APMAP、AKR1C3、ADS 52、ADGRG3、ADGRE5、ACSL1、ACADM、VPS35、TMED9、TMED7、TM9SF4、STXBP6、S LC25A11、SIGMAR1、SF3B3、RPS9、RPS2 4、RPS13、RPLP0、RPL3、RPL18、RACK1、 PYGB、PPP2CB、PPIB、PLTP、PHB2、NOST RIN、NARS1、MAN1A1、LTA4H、JAKMIP3、 ITGA2、HSD17B11、HNRNPC、GOT2、GAN AB、G6PD、ERAP1、EEF1G、DOP1B、DHRS9 、COPB2、ATP5MF、ATP2A2、ATIC、ARCN1 、APRT、APIP、AP1S1、ALDH9A1、ADAM10 、ACSL5、ACLY、TKT、SERPINA6、RPL14、 PARP4、MFGE8、MAPK1、HSP90B1、GPLD1 、DDC、C8B、C7、ATP5F1A、ACAA2、LCP1、 ANXA1、OLFM4、FBLN1、COL6A1、TGFBI、and THBS1. [7] The method according to [6], wherein the gastrointestinal disease-associated protein is at least one selected from the group consisting of OLFM4, LGALS3BP, LAMP1, ATP5F1A, DLD, ENPP4, MAPK1, PCYOX1, SMPD3, VILL, and RPS7. [8] The method according to [6] or [7], wherein the gastrointestinal disease is an intestinal disease. [9] The method according to any one of [6] to [8], wherein the gastrointestinal disease is colorectal cancer.

[10] The method according to any one of [6] to [9], further comprising a step of quantifying the detected gastrointestinal disease-associated protein.

[11] A detection reagent for the gastrointestinal disease-associated protein, for use in the method according to any one of [1] to

[10] .

[12] A kit comprising the detection reagent for the gastrointestinal disease-associated protein, for use in the method according to any one of [1] to

[10] .

[13] A method for screening for diagnostic markers for gastrointestinal diseases, comprising: isolating fecal exosomes from feces; detecting proteins from the separated fecal exosomes; and selecting candidate diagnostic markers for gastrointestinal diseases from the detected proteins using as an indicator whether the detected proteins satisfy either of the following conditions (a) and (b): (a) the content of the detected proteins in the fecal exosomes is 1.5 times or more the content of the detected proteins in the total amount of the fecal exosomes, or (b) the detected proteins are only detected in the separated fecal exosomes and are not substantially detected in the feces.

[14] The screening method according to

[13] , wherein a step of isolating fecal exosomes from the feces of a colorectal cancer patient and a step of detecting proteins from the separated fecal exosomes are carried out for each of a colorectal cancer patient and a healthy subject, and the step of selecting candidate diagnostic markers for gastrointestinal diseases is a step of selecting candidate diagnostic markers for gastrointestinal diseases from the detected proteins using as an indicator whether the detected proteins satisfy either of the following conditions (c) and (d): (c) in a colorectal cancer patient, the condition (a) or (b) is satisfied, or (d) in a healthy subject, the condition (a) or (b) is satisfied.

[15] A step of isolating fecal exosomes from the feces,and a method for immunologically detecting a gastrointestinal disease-associated protein, comprising the step of immunologically detecting the gastrointestinal disease-associated protein from the separated fecal exosomes.

[0010] According to the present invention, gastrointestinal disease-associated proteins can be efficiently detected using feces as a sample. Furthermore, according to the present invention, it is possible to provide a method for assisting the diagnosis of gastrointestinal diseases, a method for screening diagnostic markers for gastrointestinal diseases, and a detection reagent and kit for gastrointestinal disease-associated proteins for use in these methods, which can efficiently detect gastrointestinal disease-associated proteins using feces as a sample.

[0011] 1 is a graph showing the relationship between BLEIA luminescence intensity (CD9 / CD63 luminescence intensity, left axis, line graph) and each fraction (Fraction_1 to 12), and the relationship between the total protein amount (Protein (ng / μL), right axis, bar graph) and each fraction (Fraction_1 to 12), obtained in "(2) Detection of fecal exosomes by BLEIA" in Test Example 1. FIG. 2 is a graph showing the results of Western blotting of Fraction_1 to 12, obtained in "(3) Detection of fecal exosomes by Western blotting" in Test Example 1. FIG. 3 is a graph showing the particle size distribution in Fraction_5 to 9, obtained in "(4) Detection of fecal exosomes by Nanosight" in Test Example 1. 1 is a plot diagram showing the correlation between the quantitative values ​​of proteins in fecal exosomes from healthy subjects and the quantitative values ​​of proteins in whole feces from healthy subjects, obtained in "(1) Proteome analysis by mass spectrometry" in Test Example 2.

[0034] FIG. 1 is a graph showing the relationship between BLEIA luminescence intensity (CD9 / CD63 luminescence intensity, left axis, line graph) and each fraction (Fraction_1 to 12), and the relationship between the total protein amount (Protein (ng / μL), right axis, bar graph) and each fraction (Fraction_3 to 8) by BCA protein assay, obtained in "(1) Purification of fecal exosomes from feces of colon cancer patients" in Test Example 3.

[0035] FIG. 1 is a graph showing the results of Western blotting of Fraction_3 to 8, obtained in "(1) Purification of fecal exosomes from feces of colon cancer patients" in Test Example 3. 1 is a graph showing the particle size distribution in Fractions 3 to 8 obtained in "(1) Purification of fecal exosomes from feces of colorectal cancer patients" in Test Example 3. FIG. 2 is a plot showing the correlation between the quantitative values ​​of proteins in fecal exosomes from colorectal cancer patients and the quantitative values ​​of proteins in fecal exosomes from healthy individuals obtained in "(2) Proteome analysis by mass spectrometry" in Test Example 3. FIG. 3 is a diagram showing proteins detected in fecal exosomes from two or more colorectal cancer patient specimens among the gastrointestinal disease-related proteins listed in Tables 10 to 12 obtained in Test Example 5. FIG. 4 is a diagram showing proteins detected in fecal exosomes from two or more colorectal cancer patient specimens among the gastrointestinal disease-related proteins listed in Tables 13 to 15 obtained in Test Example 5.This figure shows proteins detected in fecal exosomes in common across all colon cancer patient specimens (four specimens) from among the gastrointestinal disease-related proteins listed in Tables 10 to 15 obtained in Test Example 5. For five colon cancer-related proteins (LAMP1, ATP5F1A, LGALS3BP, DLD, and ENPP4) obtained in Test Example 6 whose quantitative values ​​in fecal exosomes (fEV) from colon cancer patients were more than twice those in whole feces (feces) from colon cancer patients, (a) a graph comparing the quantitative values ​​(HC) in fecal exosomes from healthy subjects with the quantitative values ​​(CC) in fecal exosomes from colon cancer patients, and (b) a graph comparing the quantitative values ​​(HC) in whole feces from healthy subjects with the quantitative values ​​(CC) in whole feces from colon cancer patients. 1A and 1B are graphs comparing the quantitative values ​​(HC) in fecal exosomes of healthy subjects with the quantitative values ​​(CC) in fecal exosomes of colon cancer patients for five colon cancer-related proteins (MAPK1, PCYOX1, SMPD3, VILL, RPS7) that were not detected in the whole feces (feces) of colon cancer patients but only in the fecal exosomes (fEV) of colon cancer patients obtained in Test Example 6; and (b) the quantitative values ​​(HC) in the whole feces of healthy subjects with the quantitative values ​​(CC) in the whole feces of colon cancer patients.

[0049] Figure 1 shows the results of PLS ​​regression analysis of colon cancer-related proteins obtained in Test Example 7, (a) whose quantitative values ​​in fecal exosomes from healthy subjects were more than twice as high as those in whole feces from healthy subjects, using the quantitative values ​​in fecal exosomes from healthy subjects (white circles, 4 samples) and the quantitative values ​​in fecal exosomes from colon cancer patients (black circles, 4 samples), for each of the following groups: a group with a molecular weight of 25 kDa or less (21 proteins), a group with a molecular weight of 25 to 75 kDa (49 proteins), and a group with a molecular weight of 75 kDa or more (14 proteins). (b) PLS regression analysis of colon cancer-related proteins that were not detected in the whole feces of healthy individuals but only in fecal exosomes from healthy individuals was performed using quantitative values ​​in fecal exosomes from healthy individuals (white circles, 4 samples) and quantitative values ​​in fecal exosomes from colon cancer patients (black circles, 4 samples) for each of the following groups: a group with a molecular weight of 25 kDa or less (92 proteins), a group with a molecular weight of 25 to 75 kDa (220 proteins), and a group with a molecular weight of 75 kDa or more (80 proteins).Regarding the colon cancer-related proteins obtained in Test Example 7 (a), whose quantitative values ​​in fecal exosomes from healthy subjects were at least twice as high as those in whole feces from healthy subjects, the quantitative values ​​in fecal exosomes from healthy subjects (white circles, 4 samples) and the quantitative values ​​in fecal exosomes from colon cancer patients (black circles, 4 samples) were used to identify a group of secreted proteins (18 proteins), a group of membrane proteins (46 proteins), a group of cytoplasmic proteins (56 proteins), and a group of nuclear proteins (56 proteins). (b) Plots showing the results of PLS ​​regression analysis for each group of secreted proteins (secreted protein, 31 proteins), membrane proteins (membrane protein, 232 proteins), cytoplasmic proteins (cytoplasmic protein, 235 proteins), and nuclear proteins (nuclear protein, 92 proteins), using quantitative values ​​in fecal exosomes of healthy individuals (white circles, 4 samples) and quantitative values ​​in fecal exosomes of colorectal cancer patients (black circles, 4 samples) for colon cancer-related proteins that were not detected in the whole feces of healthy individuals but only in fecal exosomes of healthy individuals.

[0049] Figure 1 shows the results of PLS ​​regression analysis of colon cancer-related proteins obtained in Test Example 7, (a) whose quantitative values ​​in fecal exosomes of colon cancer patients were more than twice as high as those in the whole feces of colon cancer patients, using the quantitative values ​​in fecal exosomes of healthy individuals (white circles, 4 samples) and the quantitative values ​​in fecal exosomes of colon cancer patients (black circles, 4 samples), for each of the following groups: a group with a molecular weight of 25 kDa or less (60 proteins), a group with a molecular weight of 25 to 75 kDa (186 proteins), and a group with a molecular weight of 75 kDa or more (64 proteins). (b) Plot diagram showing the results of PLS ​​regression analysis of colon cancer-related proteins that were not detected in the whole feces of colon cancer patients but only in fecal exosomes of colon cancer patients, using quantitative values ​​in fecal exosomes of healthy individuals (white circles, 4 samples) and quantitative values ​​in fecal exosomes of colon cancer patients (black circles, 4 samples) for each of the following groups: a group with a molecular weight of 25 kDa or less (121 proteins), a group with a molecular weight of 25 to 75 kDa (344 proteins), and a group with a molecular weight of 75 kDa or more (156 proteins).Regarding the colon cancer-related proteins obtained in Test Example 7, (a) whose quantitative values ​​in fecal exosomes of colon cancer patients were more than twice as high as those in the whole feces of colon cancer patients, the quantitative values ​​in fecal exosomes of healthy subjects (white circles, 4 specimens) and the quantitative values ​​in fecal exosomes of colon cancer patients (black circles, 4 specimens) were used to identify the secreted protein group (54 proteins), membrane protein group (135 proteins), cytoplasmic protein group (169 proteins), and nuclear protein group (169 proteins). (b) Plots showing the results of PLS ​​regression analysis for each group of secreted proteins (secreted protein, 35 proteins), membrane proteins (membrane protein, 351 proteins), cytoplasmic proteins (cytoplasmic protein, 359 proteins), and nuclear proteins (nuclear protein, 144 proteins), using quantitative values ​​in fecal exosomes of healthy individuals (white circles, 4 samples) and quantitative values ​​in fecal exosomes of colon cancer patients (black circles, 4 samples) for colon cancer-related proteins that were not detected in the whole feces of colon cancer patients but only in fecal exosomes of colon cancer patients. 1 shows (a) a graph comparing the quantitative value (HC) in fecal exosomes of healthy subjects with the quantitative value (CC) in fecal exosomes of colorectal cancer patients for OLFM4 obtained in Test Example 9, and (b) a graph comparing the quantitative value (HC) in whole feces of healthy subjects with the quantitative value (CC) in whole feces of colorectal cancer patients for OLFM4 obtained in Test Example 9.

[0034] FIG. 1 shows the results of Western blot detection of OLFM4, LAMP1, and LGALS3BP in fecal exosomes (HC) of healthy subjects and fecal exosomes (CC) of colorectal cancer patients obtained in Test Example 10.

[0012] The present invention will be described in detail below based on preferred embodiments thereof.

[0013] [Subject] In the present invention, the term "subject" refers to a person from whom feces, which is a sample for the diagnostic support method and diagnostic method of the present invention, is derived, and refers to a subject on whom the diagnostic support method and diagnostic method are performed. Examples of the subject include humans and non-human mammals (mice, rats, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, horses, cows, sheep, goats, pigs, etc.), preferably humans. The subject of the present invention is not particularly limited and may be a healthy person not suffering from a gastrointestinal disease, or a person suffering from a gastrointestinal disease but not yet developing the disease. The subject may also be a patient suffering from a gastrointestinal disease, or a patient already known to have or who has had a gastrointestinal disease in the past, for the purpose of monitoring the progress of the gastrointestinal disease or the prognosis of treatment.

[0014] [Feces] The sample used in the diagnostic support method and diagnostic method of the present invention, the screening method of the present invention, and the detection method of the present invention is "feces." There are no particular limitations on the feces, as long as they contain the fecal exosomes and gastrointestinal disease-related proteins described below. Examples of the feces include feces collected from the subject, such as a human or non-human mammal, and may be collected directly from the large intestine of a human or non-human mammal by endoscopic examination or the like. The feces may be suspended in a suspension solution, if necessary. Examples of the suspension solution include buffers such as phosphate buffer, Tris buffer, Good's buffer, borate buffer, acetate buffer, citrate buffer, glycine buffer, succinate buffer, and phthalate buffer. Furthermore, the feces may contain pH adjusters, stabilizers, preservatives, antiseptics, surfactants, etc., or may be frozen, if necessary.

[0015] [Fecal Exosomes] In the present invention, "fecal exosomes" refer to exosomes contained in or isolated from the feces. In contrast, in this specification, the entire feces prior to exosome isolation (i.e., the sample itself) may be referred to as "whole feces." Exosomes generally refer to intracellular vesicles formed by inward budding of intracellular multivesicular endosomes (MVEs) membranes. In the present invention, "fecal exosomes" refer to extracellular vesicles present in feces, derived from the human or non-human mammal (preferably human) from which the feces was collected. These particles (vesicles) have a particle diameter of 30 to 300 nm and contain at least one exosome marker protein selected from the group consisting of Alix, CD9, CD63, CD81, and Tsg101 on their surface or interior. Furthermore, the "fecal exosomes" of the present invention are preferably vesicles contained in fractions in which the exosome marker protein is detected, preferably fractions with an iodixanol concentration of 30 to 45%, among fractions obtained by density gradient centrifugation at 100,000 x g using 5 to 40% iodixanol as a separation medium, obtained by subjecting a supernatant (e.g., supernatant obtained by centrifuging 3 to 4 times at a centrifugal force of 3,000 to 4,000 x g for 10 to 90 minutes) of the supernatant of a suspension of the feces (e.g., a phosphate buffer suspension) to a centrifugal force of 150,000 x g.

[0016] The particle diameter can be measured, for example, by nanoparticle tracking analysis (e.g., analysis using Nanosight (Malvern Panalytical) described in the Examples) in which the particle diameter in a particle liquid is measured from the speed of its Brownian motion. The particle diameter of the "fecal exosomes" according to the present invention is more preferably 100 to 300 nm.

[0017] Furthermore, the fraction in which the exosome marker protein is detected may be a fraction in which one of the exosome marker proteins is detected, but more preferably a fraction in which two or more of the exosome marker proteins are detected. Examples of methods for detecting these exosome marker proteins include forming an immune complex between each exosome marker protein and a label in which a labeling substance is bound to an antibody (detection antibody) against the exosome marker protein, and then detecting a signal corresponding to the labeling substance. In the present invention, the term "signal" includes color development (color development), quenching, reflected light, luminescence, fluorescence, radiation from a radioisotope, and the like, depending on the labeling substance. This includes signals that can be confirmed with the naked eye as well as signals that can be confirmed using detection methods or devices appropriate for the type of signal.

[0018] In the present invention, the "antibody" may be a polyclonal antibody or a monoclonal antibody. Furthermore, in the present invention, the "antibody" includes not only a complete antibody but also an antibody fragment (e.g., Fab, Fab', F(ab')). ２ , Fv, disulfide-linked Fv, single-chain Fv (scFv), sc(Fv) ２ , diabodies), and polymers thereof. In the present invention, "antibody" includes all immunoglobulin classes (isotypes) and subclasses, and may be a combination of two or more types as appropriate, but is preferably IgG. Furthermore, the origin of such antibodies is not particularly limited, and they may be antibodies derived from humans, antibodies derived from non-human animals (e.g., rabbit antibodies, mouse antibodies, rat antibodies, goat antibodies, sheep antibodies, and camel antibodies), chimeric antibodies, or humanized antibodies. Such antibodies can be prepared as appropriate by conventionally known methods, or commercially available antibodies may be used as appropriate.

[0019] The "labeling substance" can be any substance that is used as a labeling substance in conventionally known immunological detection methods or methods based thereon, without any particular limitation. Examples of such labeling substances include enzymes; luminescent substances such as acridinium derivatives; fluorescent substances such as europium; fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE); １２５Examples of the labeling substance include radioactive substances such as IF; fluorescent dyes such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); particles such as latex particles and gold particles; dinitrophenyl phosphate (DNP); and digoxigenin (DIG), and these may be used alone or in combination of two or more. For example, when an enzyme is used as the labeling substance, various signals can be detected depending on the substrate by adding a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, or the like. Examples of the enzyme include, but are not limited to, peroxidase (POD: horseradish peroxidase (HRP), etc.), alkaline phosphatase (ALP), β-galactosidase (β-gal), glucose oxidase, and luciferase.

[0020] The binding of the labeling substance to the antibody can be performed by a conventionally known method or a method based thereon, as appropriate. The antibody and the labeling substance may be bound directly via an active group or the like, or indirectly via an oligopeptide, a linker, or the like. Alternatively, a biotin-avidin system (e.g., biotinylated antibody is reacted with an avidinated labeling substance, and the interaction between biotin and avidin is utilized to bind the labeling substance to the antibody) may be utilized. Alternatively, the labeling substance may be bound to the antibody, or the antibody may be indirectly bound to the antibody using a secondary antibody or the like bound to a labeling substance, without binding the labeling substance to the antibody. Here, the "secondary antibody" refers to an antibody that exhibits reactivity with an antibody (primary antibody) that directly binds to the substance to be detected. Alternatively, protein G, protein A, or the like bound to a labeling substance may be used instead of the secondary antibody.

[0021] [Gastrointestinal disease-related protein] In the present invention, the "gastrointestinal tract" includes the stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, appendix, colon, rectum), anus, etc. Among these, the "gastrointestinal tract" according to the present invention is preferably at least one selected from the group consisting of the small intestine (duodenum, jejunum, ileum) and large intestine (cecum, appendix, colon, rectum).

[0022] In the present invention, "gastrointestinal disease" includes diseases of the gastrointestinal tract, and is not particularly limited to, for example, gastric diseases such as stomach cancer and gastritis; small intestinal diseases such as small intestinal cancer; and large intestinal diseases such as colon cancer (colon cancer, rectal cancer, colorectal cancer, etc.), colitis, and Crohn's disease, and may be one of these or a combination of two or more of these. Among these, the gastrointestinal disease according to the present invention is preferably an intestinal disease (small intestinal disease, large intestinal disease), more preferably a large intestinal disease, and even more preferably colon cancer.

[0023] In the present invention, the "gastrointestinal disease-related protein" is not particularly limited as long as it is a protein that has already been reported to be associated with the gastrointestinal disease or that may be associated with the gastrointestinal disease, and examples include proteins derived from the gastrointestinal tract whose expression level increases or decreases in association with a gastrointestinal disease, and proteins derived from gastrointestinal cancer, and may be one type or a combination of two or more types.

[0024] More specifically, gastrointestinal disease-related proteins according to the present invention include, for example, proteins listed in Tables 10 to 12 below, namely, ZNF256, ZMPSTE24, YWHAH, YWHAG, WDR1, VILL, VASP, VAMP8, USP5, UGDH, UBE2D3, TUBB4B, TSPAN6, TPP1, TPM4, TPM3, TPM1, TMEM30A, TC2N, SYPL1, SUSD2, SURF4, STOM, ST6GALNAC1, ST13, SQOR, SPTBN1, SPPL2A, SMPD3, SLC9A3, and SLC6A1. 4, SLC46A1, SLC44A1, SLC36A1, SLC2A5, SLC25A5, SLC25A4, SLC16A1, SLC15 A4, SLC15A1, SERPINF2, SERPINA7, SDHB, SDHA, SCARB2, SAFB2, S100A11, RP S7, RPS6KA1, RPS3, RPS23, RPS16, RPS10, RPL35, RPL30, RNPEP, RNF128, RNA SE3, RNASE1, RHPN2, RAB3D, RAB32, RAB2A, RAB21, RAB14, PTPN6, PSMB8, PSMB 1, PSMA1, PRKG2, PRKCD, PRKACA, PRDX6, PRDX5, PRDX2, PRDX1, PPP1CB, PNP, PLEC, PLA2G2A, PLA2G10, PKM, PICALM, PGK1, PGD, PFN1, PCYOX1, PCNT, PCBP 1, PAFAH1B1, P2RX4, OTUB1, NPEPPS, NIBAN2, NEDD4L, NCSTN, MYOF, MYO1E, M YO1C, MYO1B, MYH9, MYH14, MUC4, MUC3A, MT-CYB, MIS12, MIF, MGST3, MGLL, ME P1A, MAOB, MAOA, LYN, LSM2, LGALS4, LGALS3BP, LDHA, LAMP1, ITGA6, ITGA3, ITGA1, IGLV7-46, IGKV1-39, IGHM, IGHG4, IGHG2, IDH1, HTATIP2, HSPA5, HS P90AB1, HSD17B4, HNRNPA2B1, HM13, HLA-DMA, HARS1, H2BC12, H1-4, GSTP1, GSTA1, GSDME, GRN, GPI, GPD2, GPA33, GP2, GNAI2, GMDS, GHITM, GFM2, GDPD3,Examples include GAPDH, FUT3, FLOT2, FLOT1, F11R, EVC2, ETHE1, ESD, ERLIN2, EP-CAM, ENPP4, ENO1, EIF5A, EIF4A1, EEF2, EBP, DSTN, DNPEP, DLD, DDost, CYFIP1, CSRP1, CSNK1A1, CPNE3, CORO1B, CND-P2, CLTC, CLIC1, CHP2, CHMP2A, CEACAM5, CEACAM1, CD151, CASQ1, CAPZB, CAPZA1, CAPN1, CAP1, C9, BLVRB, B3GNT7, B2M, ATP9A, ATP1B3, ATP1B1, ATP1A1, ASAH1, ARPC5, ARPC2, AR-L1, ARHGDI-A, AP2M1, AP2A1, AP1B1, ANXA4, ANXA3, ANO6, ALDOA, ALDH3B1, AGR3, AGR2, ACTR3, ACTR2, ACTN4, ACAA1, ABRACL.、

[0025] Furthermore, examples of gastrointestinal disease-related proteins according to the present invention include proteins listed in Tables 13 to 15 below, namely, YARS1, XRCC6, XRCC5, XPO1, WDR44, VPS35, VPS29, VPS26A, VKORC1L1, VBP1, VAMP7, USP15, USP14, USO1, UQCRC2, UQCRC1, UPF1, UGGT1, UBA1, TUFM, TPP2, TPMT, TOP2B, TMEM33, TMEM205, TMED9, TMED7, TMED5, TMED4, TMED2, TMED10, TM9SF4, TM9SF3, TM9SF2, TM4SF20, TKT, THEMIS2, TEKT2, TBXAS1, TARS1, TARDBP, SY NGR2, SYK, SUCLG2, SUCLG1, SUCLA2, STXBP6, STIP1, STAT3, STAT1, SSR4, SS R1, SRSF3, SRM, SPCS3, SPAST, SORL1, SORD, SOD2, SNX33, SNRPA1, SNRNP70, SND1, SLK, SLC6A6, SLC3A2, SLC35B2, SLC2A3, SLC2A1, SLC25A11, SLC16A3, S LC12A9, SLC11A2, SIRPA, SIL1, SIGMAR1, SHMT2, SHMT1, SF3B3, SERPINB5, S ERPINA6, SEPTIN7, SELENOP, SEC31A, SEC24D, SEC24C, SEC23B, SEC23A, SAC M1L, RUVBL2, RRM2B, RRAGC, RRAGB, RPS9, RPS4X, RPS3A, RPS24, RPS18, RPS1 7, RPS14, RPS13, RPN1, RPLP0, RPL7, RPL6, RPL5, RPL4, RPL3, RPL28, RPL27A, RPL24, RPL21, RPL18A, RPL18, RPL17, RPL14, RPL13A, RPL13, RPL12, RPL10A , RNF149, REEP6, RCC2, RBBP4, RARS1, RACK1, RABGGTA, QARS1, PYGB, PTPRE, P TK2B, PTGES3, PSMF1, PSMD7, PSMD6, PSMD3, PSMD2, PSMD13, PSMD12, PSMD1, PSMC5, PSMC3, PSMC2, PRXL2A, PRPS1, PRPF19, PRMT1, PRKDC, PRKCB, PPP2CB,<h2 style=";text-align:left;direction:ltr">PPIB、POR、PON1、POLR2H、PLTP、PLP2、 PLEKHA1、PLCG2、PITPNA、PIP4P2、PHL DA3、PHB2、PGAP6、PFKP、PDLIM1、PCK2 、PCCA、PCBP2、PC、PARP4、PAK4、PAICS 、PABPC1、PAAF1、PA2G4、OGDH、OCIAD2 、OCIAD1、OAT、OAF、NXF1、NT5C2、NSDH L、NQO1、NPC1、NOSTRIN、NNT、NMT1、NI PSNAP2、NIBAN1、NDUFV1、NDUFB10、NC KAP1L、NARS1、MVD、MT-CO2、MOGS、MOB 1B、MICAL1、MGST2、MGST1、MFGE8、MCC C2、MAT2A、MAPK14、MAPK1、MAN1A1、MA CC1、M6PR、LYPLAL1、LTA4H、LNPEP、LB R、LARS1、KDELR2、KDELR1、KARS1、JAK MIP3、ITGAV、ITGA2、IQGAP2、IMPDH2、 ILF2、IL18R1、IGKV1-12、IDH3B、IDH3 A、IDH2、IDE、IARS1、HTT、HSPH1、HSPA9 、HSPA4、HSP90B1、HSDL2、HSD17B11、H SD17B10、HSD11B2、HNRNPUL2、HNRNPU 、HNRNPK、HNRNPC、HMGCS2、HMGB1、HLA -B、HKDC1、HK2、HK1、HIBCH、HCK、HADH B、HADHA、HADH、H1-5、H1-0、GYS1、GSP T1、GSK3A、GRTP1、GRK2、GPLD1、GPD1、 GOT2、GOLGB1、GNPNAT1、GMPPB、GDI1、 GCLM、GCLC、GBE1、GATD3B、GART、GARS1 、GANAB、GALNT2、GALNT1、G6PD、FGR、FETUB、FCN3、FBXO7、FBXO6、FASN、FARS (1) D、EIF3CL、EIF3B、EIF2S3、EIF2S1、EF R3A、EEF1G、DYNC1H1、DPYSL2、DOP1B、 DNM2、DNAJC13、DNAJB11、DLAT、DHX9、 DHRS9、DHRS7、DDX1、DDC、DARS1、DAD1、CYFIP2, CYB5R3, CYB5B, CSK, CRYZ, CRYL1, CPT2, CPT1A, CPB2, COX5B, CORO7, CORO1C, COPG 1, COPB2, COPA, CMPK1, CLPX, CLINT1, CLDN7, CHST5, CEMIP2, CDCP1, CCT8, CCT7, CCT6A, CC T5, CCT4, CCT3, CCT2, CASP6, C8G, C8B, C7, BUB3, BST2, BSG, BPHL, BLVRA, BAX, BACE1, B3GN T8, B3GNT3, ATP8A1, ATP6V1H, ATP6V1G1, ATP6V1E1, ATP6V1A, ATP6V0D1, ATP6V0A1, ATP5PO , ATP5PB, ATP5MG, ATP5MF, ATP5F1A, ATP2A2, ATP11B, ATIC, ATG3, ASL, ARPC3, ARPC1B, ARH GAP1, ARCN1, APRT, APPL2, APPL1, APMAP, APIP, APEX1, AP1S1, AP1M1, AP1G1, ALOX15, ALDH9 A1, ALDH16A1, AKR1C3, AIMP1, AIFM1, AHSA1, AGPS, AGO2, ADSS2, ADGRG3, ADGRE5, ADAM10, ACSL5, ACSL1, ACOT7, ACOT11, ACLY, ACADS, ACADM, ACAA2, ABRAXAS2, ABCG8, and ABCF1 are also included.

[0026] Furthermore, examples of gastrointestinal disease-related proteins according to the present invention include the proteins listed in Table 16 below, namely, LCP1, ANXA1, OLFM4, FBLN1, COL6A1, TGFBI, and THBS1.

[0027] Other examples of the gastrointestinal disease-related proteins according to the present invention include SLC25A3, ACADM, ACOT7, ACOX1, ACSL5, ADGRE5, ADGRG7, ADH6, AGR2, AHCYL1, AK1, AKAP7, AKR1A1, AKR1C3, ALDH9A1, ALDOA, ANPEP, ANXA13, ANXA2, ANXA3, ANXA7, APIP, APOA4, APOB, APPL2, ARHGDIA, ARPC2, ATIC, ATP10B, ATP11B, ATP5F1A, ATP8B1, BAIAP2L2, C2, C8G, CALM1, CALML4, CAP1, CCT7, CD151, CD177, CD300LF, CD59, CD82, CEACA M1, CEACAM5, CEACAM7, CEL, CHP1, CHP2, CKB, CLCA1, CLDN3, CLDN7, CLTC, CL U, CNDP2, COPA, CORO7, CSNK1G2, CYB5B, CYBRD1, DDAH1, DDOST, DDX3X, DEFA 5, DHRS9, DMBT1, DPEP1, EEF1A1, EIF2S3, EIF3L, ENO1, ENPP7, EPCAM, EPHA2, EPS8L3, EZR, FABP1, FAM151A, FBP1, FBP2, FERMT1, FOLH1, FTH1, FUT3, GAPD H, GBA1, GDA, GFM2, GIPC1, GIPC2, GNPNAT1, GPA33, GPRC5A, GSTP1, H1-0, HA DHB, HLA-DRA, HLA-DRB1, HMGB1, HNRNPA2B1, HNRNPK, HNRNPU, HPGD, HSD17B 11, HSD17B2, HSP90AB1, HSPA1A, HSPA4, HSPA5, HSPA8, HSPA9, IDH2, ILF2, IM PDH2, ITLN1, ITLN2, KCNAB2, KIF20B, KLKB1, KRAS, LASP1, LCT, LGALS3BP, L GALS4, LGALS9, LGALS9B, LNX2, LPCAT3, LSR, LYZ, MEP1A, MEP1B, MINK1, MS4 A12, MTTP, MUC12, MUC13, MUC17, MUC3A, MUC4, MYH14, MYO15B, MYO1A, MYO1D , NAALADL1, NARS1, NCF2, NCKAP1L, NDUFV1, NIPSNAP2, NOS2, NOSTRIN, NOX1,NOX O1, NQO1, OCLN, OTUB1, PA2G4, PAF AH1B1, PARP4, PDC D6, PDC D6IP, PDZK1, PFKL, PFN1, PGA4, PGK1, PKM, PLEC, PLEKHA1, PLS1, PLSCR1, PLTP, POR, PPIA, PPP1CC, PPP2R1A, PROM1, PRPS1, PRSS8, PSMF1, PTPRB, PTPRJ, PYGB, RAB10, RAB1A, RAB21, RAB22A, RAB25, RAC1, RAN, RBP2, REG4, RHOA, RNASE3, RPL13A, RPL18, RPL21, RPL24, RPL27A, RPL28, RPL3, RPL35A, RPL5, RPLP0, RPS10, RPS15A, RPS18, RPS2, RPS3, RPS6KA1, RPS9, RUVBL2, SAFB2, SDHA, SERINC2, SER PIN A4, SER PIN B5, SI, SLC15A1, SLC16A1, SLC23A1, SLC25A3, SLC25A5, SLC26A3, SLC28A2, SLC44A4, SLC9A3, SLPI, SMPDL3B, SOD2, SPINT1, SRI, STK25, STX3, STXB P2, SULT1A1, SYTL2, TAGLN2, TCP1, TFF3, TM4SF20, TM9SF2, TM9SF3, TMBIM1, TMC4, TMED10, TMI GD1, TPM1, TSPAN1, TSPAN8, TUT1, UBB, UBE2V1, UPP1, USH1C, VDAC2, VIL1, VPS26A, VPS35, VPS37B, VTA1, WDR1, XDH, XPNPEP2, YWHA B, YWHA G, YWHA Z, ZG16, AHCYL1, CEA CAM7, GUCY2C, HSP A8, RAB1A, SLC44A4, SULT1A1, VIL1 are mentioned.

[0028] <Method for assisting in the diagnosis of gastrointestinal disease, method for diagnosing gastrointestinal disease> The present invention provides a method for assisting in the diagnosis of gastrointestinal disease (sometimes referred to herein as the "diagnostic assisting method of the present invention"), comprising the steps of isolating fecal exosomes from a subject's feces and detecting a gastrointestinal disease-associated protein from the isolated fecal exosomes. The present invention also provides a method for diagnosing gastrointestinal disease (sometimes referred to herein as the "diagnostic method of the present invention"), comprising the steps of isolating fecal exosomes from a subject's feces and detecting a gastrointestinal disease-associated protein from the isolated fecal exosomes. The present inventors have discovered that because gastrointestinal disease-associated proteins are significantly concentrated in fecal exosomes, the gastrointestinal disease-associated protein can be detected with high efficiency and accuracy by simply isolating fecal exosomes from the feces, and then detecting the gastrointestinal disease-associated protein from the isolated fecal exosomes.

[0029] [Separation Step] The diagnostic support method and diagnostic method of the present invention include a step of separating the fecal exosomes from the feces of the subject (referred to as the "separation step" in the diagnostic support method and diagnostic method of the present invention).

[0030] The method for separating fecal exosomes from the feces is not particularly limited as long as it can separate the fecal exosomes according to the present invention, and any conventionally known method or method equivalent thereto can be appropriately adopted. Examples of such separation methods include the density gradient centrifugation described above, as well as density gradient centrifugation using a separation medium other than iodixanol (e.g., a method using sucrose: bio-protocol 10(08):e3584, DOI:10.21769 / BioProtoc.3584,2020); ultracentrifugation; immunological methods of capturing exosomes with antibodies against the exosome marker protein (e.g., immunoprecipitation using beads on which the antibodies are immobilized); elution using a size exclusion chromatography column; precipitation using polymers such as polyethylene glycol; and size exclusion using an ultrafiltration membrane, and two or more of these methods may be combined. Among these methods for separating fecal exosomes according to the present invention, density gradient centrifugation using iodixanol as a separation medium is preferred, given its ease and tendency to separate highly purified fecal exosomes from feces. Furthermore, a method in which a crude product obtained by separation using a separation method other than density gradient centrifugation (e.g., centrifugation, filtration, etc.) is first subjected to density gradient centrifugation, which can further improve the degree of purification of the separated fecal exosomes. Combining purification methods in this way can further increase the degree of purification (purity) of the fecal exosomes when separating fecal exosomes from feces containing a large amount of impurities.

[0031] After the separation step, the presence and purity of the separated fecal exosomes may be confirmed by, for example, confirming the expression of exosome marker proteins by Western blotting or the above-mentioned detection methods, measuring particle size, observing using an electron microscope, or a combination thereof. Therefore, the diagnostic auxiliary method and diagnostic method of the present invention may further include a confirmation step of confirming the presence and purity of such fecal exosomes.

[0032] [Detection step] The diagnostic support method and diagnostic method of the present invention include a step of detecting the gastrointestinal disease-related protein from the fecal exosomes separated in the separation step (referred to as the "detection step" in the diagnostic support method and diagnostic method of the present invention).

[0033] The gastrointestinal disease-related proteins detected in the diagnostic support method and diagnostic method of the present invention preferably include the above-mentioned gastrointestinal disease-related proteins, and may be one or more of these. Furthermore, the gastrointestinal disease-related proteins detected in the diagnostic support method and diagnostic method of the present invention are preferably proteins concentrated in fecal exosomes, and their content relative to the total amount of fecal exosomes is greater than their content relative to the total amount of the feces (whole feces), or they are preferably proteins that are detected only in the fecal exosomes and are not substantially detected in the feces. More specifically, the gastrointestinal disease-related proteins used in the diagnostic support method and diagnostic method of the present invention are gastrointestinal disease-related proteins that satisfy either of the following conditions (a) and (b): (a) their content relative to the total amount of the fecal exosomes is 1.5 times or more their content relative to the total amount of the feces; or (b) they are detected only in the separated fecal exosomes and are not substantially detected in the feces.

[0034] Here, the content of a gastrointestinal disease-associated protein relative to the total amount of fecal exosomes may be 1.5 times or more, more preferably 2 times or more, the content relative to the total amount of the feces (whole feces). The upper limit of this factor is not particularly limited. Furthermore, a gastrointestinal disease-associated protein being "substantially undetectable" in feces refers to a content below the detection limit in the total amount of the feces. Methods for detecting a gastrointestinal disease-associated protein from feces or fecal exosomes and methods for quantifying a gastrointestinal disease-associated protein are described below. For example, when the following immunological detection method is used as the detection method of the present invention, each content can be calculated from the amount of protein quantified based on the signal measurement value and the amount of sample used for the measurement (i.e., the total amount of whole feces or the total amount of fecal exosomes, the same applies hereinafter). The term "substantially undetectable" refers to a signal not being detected in whole feces by a detection method corresponding to the following labeling substance when the same sample amounts of whole feces and fecal exosomes are subjected to the same measurement. Furthermore, for example, when the detection method according to the present invention uses the MS / MS analysis or LC / MS / MS analysis described below, each content can be determined from the absolute quantitative value quantified based on the analytical results and the amount of sample used for the measurement, but when comparing the content relative to the total amount of fecal exosomes with the content relative to the total amount of whole feces, the abundance ratio (ratio of relative quantitative values) can be replaced with the content ratio. Furthermore, when MS / MS analysis or LC / MS / MS analysis is used, "substantially not detected" means that when the same sample amounts of whole feces and fecal exosomes are used for measurement, the spectrum is not observed in whole feces or is below the detection limit (usually three times the standard deviation).

[0035] Such gastrointestinal disease-associated proteins include the gastrointestinal disease-associated proteins described above. Among these, at least one protein selected from the group consisting of the proteins listed in Tables 10 to 15 above is more preferred, from the viewpoint of being significantly concentrated in fecal exosomes and being particularly concentrated in fecal exosomes from gastrointestinal disease patients (preferably colorectal cancer patients).

[0036] Among them, the proteins described in FIG. 9, namely, ZMPSTE24, YWHA G, UGDH, TPM4, TC2N, SUS D2, ST OM, ST6GALNAC1, SPTBN1, SLC6A14, SLC46A1, SLC15A4, SER PINF2, SDHB, SDHA, SAFB2, RPS16, RNF128, RNASE3, RAB21, RAB14, PRKG2, PLEC, PLA2G10, NPEPPS, MYH14, MT - CYB, MAOA, ITGA3, IGLV7 - 46, HSD17B4, HARS1, H1 - 4, GST A1, GMDS, GFM2, FUT3, FLOT2, DNPEP, CND P2, CLTC, B3GNT7, B2M, ATP9A, ARPC2, AP2A1, AP1B1, AGR2, ACTR2, ABRACL, YWHAH, WDR1, VILL, VAMP8, TUBB4B, TSPAN6, TPP1, TPM3, TPM1, TMEM30A, SYPL1, SQOR, SPPL2A, SMPD3, SLC9A3, SLC44A1, SLC2A5, SLC25A5, SLC15A1, SER PINA7, SCARB2, S100A11, RPS7, RPS3, RPS23, RPS10, RPL3, RNP EP, RHPN2, RAB3D, RAB32, RAB2A, PTPN6, PSMB8, PSMB1, PSMA1, PRKCD, PRKACA, PRDX6, PRDX5, PRDX1, PPP1CB, PLA2G2A, PKM, PICALM, PGK1, PGD, PFN1, PCYOX1, PCBP1, P2RX4, NIBAN2, NEDD4L, NCSTN, MYOF, MYO1E, MYO1C, MYO1B, MYH9, MUC4, MUC3A, MGST3, MGLL, MEP1A, LYN, LGALS4, LGALS3BP, LDHA, LAMP1, IGKV1 - 39, IGHM, IGHG2, IDH1, HSP A5, HSP90AB1, HNRNPA2B1, HLA - DMA, H2BC12, GSTP1, GRN, GPI, GPD2, GPA33, GP2, GNAI2, GHITM, GDPD3, GAPDH, FLOT1, F11R, EP CAM, ENPP4, ENO1, EIF5A, EIF4A1, EEF2, EBP, DSTN, DLD, CYFIP1, CSNK1A1, CPNE3, CLIC1, CHMP2A, CEACAM5, CEACAM1, CD151,CAPZB, CAPN1, CAP1, C9, ATP1B1, ATP1A1, ASAH1, ARL1, ARHGDIA, ANXA4, ANXA3, ALDOA, ALDH3B1, ACTN4, and ACAA1; proteins listed in FIG. 10, namely, XPO1, VPS26A, UQCRC2, UQCRC1, UBA1, TPP2, TMEM33, TMEM205, TMED4, TMED2, TMED10, TM9SF3, TM9SF2, THEMIS2, TARDBP, SYNGR2, SYK, STIP1, STAT3, STAT1, SSR4, SRSF 3, SPCS3, SPAST, SOD2, SLC3A2, SLC2A3, SLC2A1, SLC16A3, SIRPA, SIL1, SER PINB5, SELENOP, SEC31A, SEC23A, RPS4X, RPS3A, RPS18, RPN1, RPL7, RPL6, R PL5, RPL4, RPL21, RPL13A, RARS1, QARS1, PTK2B, PTGES3, PSMD3, PSMD12, PS MD1, PSMC2, PRXL2A, PRPS1, PRKDC, PFKP, PCK2, PC, PAICS, OCIAD2, OAF, NT5C 2, NIPSNAP2, NDUFB10, MT-CO2, MOB1B, MICAL1, MGST1, MCCC2, M6PR, LNPEP, LBR, KDELR2, KDELR1, KARS1, ITGAV, IMPDH2, IGKV1-12, IDH3B, IDH2, HTT, H SPA4, HSD17B10, HNRNPUL2, HNRNPU, HNRNPK, HMGCS2, HLA-B, HK1, HIBCH, HA DHA, H1-0, GRTP1, GPD1, GMPPB, GCLM, GBE1, GARS1, GALNT2, FGR, FCN3, FASN, ERO1A, ENTPD1, ​​EML4, EIF3D, DYNC1H1, DNM2, DLAT, DHX9, DHRS7, DARS1, CYB 5B, CSK, CRYL1, CPT1A, COX5B, CORO1C, COPG1, COPA, CLDN7, CHST5, CCT4, CC T3, C8G, BSG, B3GNT3, ATP6V0D1, ATP5MG, ARPC3, ARPC1B, ARHGAP1, APMAP, A KR1C3, ADSS2, ADGRG3, ADGRE5, ACSL1, ACADM, VPS35, TMED9, TMED7, TM9SF4,STXBP6, SLC25A11, SIGMAR1, SF3B3, RPS9, RPS24, RPS13, RPLP0, RPL3, RPL18, RACK1, PYGB, PPP2CB, PPIB, PLTP, PHB2, NOSTRIN, NAR S1, MAN1A1, LTA4H, JAKMIP3, ITGA2, HSD17B11, HNRNPC, GOT2, GANAB, G6PD, ERAP1, EEF1G, DOP1B, DHRS9, COPB2, ATP5MF, ATP2A2, ATI C, ARCN1, APRT, APIP, AP1S1, ALDH9A1, ADAM10, ACSL5, ACLY, TKT, SERPINA6, RPL14, PARP4, MFGE8, MAPK1, HSP90B1, GPLD1, DDC, C8B, C7, ATP5F1A, and ACAA2; and at least one selected from the group consisting of the proteins listed in Table 16, i.e., LCP1, ANXA1, OLFM4, FBLN1, COL6A1, TGFBI, and THBS1.

[0037] The present invention also provides, as a diagnostic auxiliary method and a diagnostic method of the present invention, a method comprising the separation step and the detection step, wherein the gastrointestinal disease-related protein is selected from the group consisting of the proteins listed in Figure 9, the proteins listed in Figure 10, and the proteins listed in Table 16.

[0038] Among the above, particularly preferred gastrointestinal disease-related proteins to be detected by the diagnostic aid method and diagnostic method of the present invention are the proteins shown in FIG. 11, namely, YWHAH, WDR1, VILL, VAMP8, TUBB4B, TSPAN6, TPP1, TPM3, TPM1, TMEM30A, SYPL1, SQOR, SPPL2A, SMPD3, SLC9A3, SLC44A1, SLC2A5, SLC25A5, SLC15A1, SERPINA7, SCARB2, S100A11, RPS7, RPS3, RPS23, RPS 10, RPL30, RNPEP, RHPN2, RAB3D, RAB32, RAB2A, PTPN6, PSMB8, PSMB1, PS MA1, PRKCD, PRKACA, PRDX6, PRDX5, PRDX1, PPP1CB, PLA2G2A, PKM, PICAL M, PGK1, PGD, PFN1, PCYOX1, PCBP1, P2RX4, NIBAN2, NEDD4L, NCSTN, MYOF , MYO1E, MYO1C, MYO1B, MYH9, MUC4, MUC3A, MGST3, MGLL, MEP1A, LYN, LGA LS4, LGALS3BP, LDHA, LAMP1, IGKV1-39, IGHM, IGHG2, IDH1, HSPA5, HSP 90AB1, HNRNPA2B1, HLA-DMA, H2BC12, GSTP1, GRN, GPI, GPD2, GPA33, GP2 , GNAI2, GHITM, GDPD3, GAPDH, FLOT1, F11R, EPCAM, ENPP4, ENO1, EIF5A, EIF4A1, EEF2, EBP, DSTN, DLD, CYFIP1, CSNK1A1, CPNE3, CLIC1, CHMP2A, CEACAM5, CEACAM1, CD151, CAPZB, CAPN1, CAP1, C9, ATP1B1, ATP1A1, ASAH1, ARL1, ARHGDIA, ANXA4, ANXA3, ALDOA, ALDH3B1, ACTN4, ACAA1, TKT, SERPINA6, RPL14, PARP4, MFGE8, MAPK1, HSP90B1, GPLD1, DDC, C8B, C7, ATP5F1A, and ACAA2; and at least one selected from the group consisting of OLFM4, LCP1, and ANXA1.

[0039] Furthermore, the gastrointestinal disease-related protein is more preferably at least one selected from the group consisting of OLFM4, LGALS3BP, LAMP1, ATP5F1A, DLD, ENPP4, MAPK1, PCYOX1, SMPD3, VILL, and RPS7.

[0040] The method for detecting the gastrointestinal disease-associated protein (sometimes simply referred to as a "detection method" herein) is not particularly limited as long as it is capable of detecting the type of gastrointestinal disease-associated protein to be detected, and a conventionally known method or a method similar thereto can be appropriately adopted. Examples include immunological detection methods using antibodies against gastrointestinal disease-associated proteins; MS / MS analysis; LC / MS / MS analysis; and Western blotting, and two or more of these methods may be appropriately combined. Among these, immunological detection methods are preferred as the detection method according to the present invention.

[0041] The immunological detection method may be a conventionally known method, a method based on the method, or a combination thereof, as appropriate. Examples of such immunological detection methods include a method in which an immune complex is formed between each gastrointestinal disease-related protein and a label obtained by binding a label to an antibody (detection antibody) against the protein, and a signal corresponding to the label is detected. Examples of such immunological detection methods include, but are not limited to, EIA (enzyme-linked immunosorbent assay) using an enzyme as the label, ELISA (an embodiment of EIA), CLIA (chemiluminescent immunoassay) using a chemiluminescent compound as the label, IRMA (radioimmunoassay) or RIA (radioimmunoassay) using a radioisotope as the label, and combinations thereof (e.g., CLEIA (chemiluminescent enzyme immunoassay) and BLEIA (bioluminescent enzyme immunoassay)); immunochromatography; and immunoagglutination methods such as immunonephelometry and immunoturbidimetry (latex agglutination, gold colloid agglutination, etc.). Furthermore, the detection method according to the present invention may be a non-competitive detection method or a competitive detection method.

[0042] As the detection method according to the present invention, sandwich methods such as ELISA, CLIA, IRMA, CLEIA, and BLEIA are preferred because they tend to have higher sensitivity. In the sandwich method, a detection target substance (a gastrointestinal disease-related protein in this invention) is captured by a capture antibody (capturer) immobilized (immobilized) on a solid phase, which is then recognized by a detection antibody (labeled) bound to a labeling substance to form a capture antibody-detection target substance-detection antibody complex, and after B / F separation (washing), the complex is detected based on a signal corresponding to the type of labeling substance. Examples of such sandwich methods include a two-step forward sandwich method (a method in which a capture antibody reacts with a target substance and then the target substance bound to the capture antibody reacts with the detection antibody), a reverse sandwich method (a method in which a detection antibody reacts with a target substance in advance and the resulting complex is reacted with the capture antibody), and a one-step method (a method in which the target substance, capture antibody, and detection antibody react simultaneously in one step), any of which can be employed. Furthermore, as in immunochromatography, a detection antibody may be used to recognize a target substance, and while B / F separation is being performed, the target substance may be captured by a capture antibody, and a signal corresponding to the type of labeling substance may be detected.

[0043] The capture antibody and detection antibody are antibodies against the target substance. Examples of such antibodies are as described above, and can be selected appropriately depending on the target substance, i.e., the gastrointestinal disease-related protein. The labeling substance, the binding between the detection antibody and the labeling substance, and the signal are the same as those described above for fecal exosomes. The solid phase can be any suitable solid phase typically used in immunological detection methods or methods similar thereto. For example, particles such as magnetic particles or latex particles, plates such as plastic plates, and fibrous materials such as nitrocellulose fibers can be used. Immobilization of the antibody on the solid phase can be performed using conventionally known methods or methods similar thereto. The antibody can be directly immobilized on the solid phase by known methods such as physical adsorption, chemical binding, or a combination of these. Alternatively, the antibody can be indirectly immobilized using the secondary antibody, protein G, protein A, biotin-avidin, or the like.

[0044] This allows the presence or absence of a gastrointestinal disease-associated protein in the feces or fecal exosomes to be detected as the presence or absence of a signal derived from the labeling substance. Furthermore, in the diagnostic support method and diagnostic method of the present invention, it is also preferable to quantify the gastrointestinal disease-associated protein from the measured value of the detected signal. Quantification of the gastrointestinal disease-associated protein can generally be performed by comparison with measurements using a standard substance (e.g., a standard protein). In this case, the amount of the gastrointestinal disease-associated protein in the feces or fecal exosomes can be determined, for example, by determining where the obtained measured value is located on a standard curve created based on measurements using a standard substance of known concentration. For example, when MS / MS analysis or LC / MS / MS analysis is used as the detection method of the present invention, the quantification of the gastrointestinal disease-associated protein can be, for example, an absolute quantitative value determined based on the analysis results, or a relative quantitative value (i.e., content) obtained from the absolute quantitative value and the amount of sample used in the analysis, which can be used as a value corresponding to the quantitative value.

[0045] [Diagnostic Assistance, Diagnosis (Assessment Step)] The diagnostic assistance methods and diagnostic methods of the present invention detect gastrointestinal disease-associated proteins concentrated in fecal exosomes, thereby enabling highly sensitive detection of the target protein (gastrointestinal disease-associated protein) at a content higher than that in total feces, or at a sufficient content even when the content in total feces was below the detection limit, and can quantitate the target protein as needed. Therefore, the detection and / or quantitation results obtained by the diagnostic assistance methods and diagnostic methods of the present invention can be used as indicators for diagnosing the presence or absence of a gastrointestinal disease-associated protein and / or the amount of the gastrointestinal disease-associated protein, depending on the type of gastrointestinal disease (preferably intestinal disease, more preferably colorectal cancer), to determine the presence or absence, likelihood of morbidity, severity, or the quality of follow-up of the gastrointestinal disease. Thus, the present invention provides such diagnostic methods or methods for assisting diagnosis, and the method of obtaining data regarding the presence or absence of a gastrointestinal disease-associated protein and / or its amount using the diagnostic assistance methods of the present invention can also be described as a method of collecting such data for a physician's diagnosis, a method of presenting such data to a physician, or a method of testing for a gastrointestinal disease.

[0046] Furthermore, the diagnostic support method and diagnostic method of the present invention may include, for example, a determination step of determining the severity of gastrointestinal disease in the subject based on the presence or absence of a gastrointestinal disease-related protein detected in the detection step and / or by comparing the amount of the gastrointestinal disease-related protein with a reference amount.

[0047] In the assessment step, the severity of the gastrointestinal disease is determined. Determining the "severity" includes determining the type and severity of the gastrointestinal disease, determining whether or not a subject has a gastrointestinal disease, and selecting subjects predicted to be at a high risk of developing a gastrointestinal disease. It also includes distinguishing between healthy subjects and subjects suffering from diseases other than gastrointestinal disease. The "reference amount" to be compared is not particularly limited and can be appropriately determined depending on the type of gastrointestinal disease or gastrointestinal disease-associated protein, the purpose of the assessment, the subject, etc. For example, a person skilled in the art can set it as a so-called cutoff value based on the above-mentioned detection method and statistical analysis method. Furthermore, the degree of "higher" or "lower" than the reference amount can be appropriately determined based on statistical analysis methods depending on the type of gastrointestinal disease or gastrointestinal disease-associated protein, the purpose of the assessment, the subject, etc.

[0048] <Screening Method> The present invention also provides a method for screening for diagnostic markers for gastrointestinal diseases (sometimes referred to herein as the "screening method of the present invention"), comprising the steps of: isolating fecal exosomes from feces; detecting proteins from the isolated fecal exosomes; and selecting candidate diagnostic markers for gastrointestinal diseases from the detected proteins based on whether the detected proteins satisfy either of the following conditions (a) or (b): (a) their content relative to the total amount of the fecal exosomes is 1.5 times or more their content relative to the total amount of the feces; or (b) they are detected only in the isolated fecal exosomes and are not substantially detected in the feces. The present inventors have found that, because gastrointestinal disease-related proteins are present in fecal exosomes at a high frequency and significantly enriched and can be detected with high sensitivity, as described above, proteins enriched in fecal exosomes can be efficiently screened as candidates for gastrointestinal disease-related proteins, i.e., diagnostic markers useful for diagnosing gastrointestinal diseases.

[0049] [Separation Step] The screening method of the present invention includes a step of separating fecal exosomes from feces (referred to as the "separation step" in the screening method of the present invention). For example, feces from a healthy subject and feces from a patient known to have a gastrointestinal disease, such as feces from a healthy subject known not to have colon cancer and feces from a patient known to have colon cancer, are used. By comparing the two, it becomes possible to screen for gastrointestinal disease-related proteins specific to the gastrointestinal disease. The method for separating fecal exosomes from the feces, including preferred embodiments thereof, is the same as the method described as the separation step in the diagnostic support method and diagnostic method of the present invention.

[0050] [Detection step] The screening method of the present invention includes a step of detecting proteins from the fecal exosomes separated in the separation step (referred to as the "detection step" in the screening method of the present invention). The method for detecting the proteins, including preferred embodiments thereof, is the same as the method described as the detection step in the diagnostic auxiliary method and diagnostic method of the present invention.

[0051] However, in the screening method of the present invention, the target proteins to be detected are not limited to the gastrointestinal disease-related proteins, but may be any proteins. Examples of such protein detection methods that target all proteins include comprehensive protein detection methods using proteome analysis by MS / MS analysis or LC / MS / MS analysis. For the proteome analysis, for example, analysis software such as Scaffold DIA (manufactured by Proteome Software) and DIA-NN (Demichev et al., DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput, Nat. Methods, 2020, DOI: 10.1038 / s41592-019-0638-x) can be used.

[0052] In the screening methods of the present invention, it is also preferable to quantify the amount of each protein in the feces or fecal exosomes. Such quantification methods, including preferred embodiments thereof, are the same as those described in the detection step of the diagnostic support method and diagnostic method of the present invention. For example, the relative quantitative value calculated by proteomic analysis using Scaffold DIA or DIA-NN can be used as the content ratio relative to the amount of sample subjected to each analysis (i.e., the total amount of all feces or the total amount of fecal exosomes), and this can be used in the selection step described below.

[0053] [Selection step] The screening method of the present invention includes a step of selecting candidate diagnostic markers for gastrointestinal diseases from the proteins detected in the detection step (referred to as the "selection step" in the screening method of the present invention) using as an indicator either (a) that the protein has a content relative to the total amount of the fecal exosomes that is 1.5 times or more (preferably 2 times or more) the content relative to the total amount of the feces, or (b) that the protein is detected only in the separated fecal exosomes and is not substantially detected in the feces.

[0054] The content of the protein relative to the total amount of fecal exosomes or the total amount of feces, and the method for confirming whether or not the protein is detected in fecal exosomes or feces, are the same as those described in the detection step of the diagnostic auxiliary method and diagnostic method of the present invention, including their preferred embodiments, except that the gastrointestinal disease-related protein is replaced with a protein that is not limited to this.

[0055] In the screening method of the present invention, it is more preferable that the detection step involves, for example, performing the separation step and the detection step using the feces of the above-mentioned healthy subjects and the feces of colon cancer patients as the feces, and selecting candidate diagnostic markers for gastrointestinal diseases from the detected proteins using as an index whether the detected proteins satisfy one of the following conditions (c) and (d): (c) in colon cancer patients, either of conditions (a) and (b) is satisfied; (d) in healthy subjects, either of conditions (a) and (b) is satisfied;

[0056] Furthermore, the detection step may satisfy at least one of the above conditions (c) and (d) (preferably either one, more preferably (c)), and further satisfy the following conditions (e) and (f): (e) the Ratio (%) calculated by the following formula: Ratio = (content of fecal exosomes from colorectal cancer patients relative to the total amount of fecal exosomes) / (content of fecal exosomes from healthy subjects relative to the total amount of fecal exosomes) × 100 is 150% or more, (f) in proteome analysis by DIA-NN, the exosomes are not substantially detected in fecal exosomes from healthy subjects, and the content of the exosomes relative to the total amount of fecal exosomes from colorectal cancer patients is 9.0 × 10 ３ It is more preferable to select a candidate diagnostic marker for a gastrointestinal disease using as an index whether or not the candidate satisfies any of the above conditions.

[0057] In condition (e), the ratio may be 150% or more, more preferably 200% or more, and even more preferably 300% or more. There is no particular upper limit to the ratio, but it is preferably 11,000% or less, and more preferably 7,000% or less. For example, the ratio may be 150 to 7,000%, or 200 to 7,000%.

[0058] Further, under condition (f), the content of fecal exosomes of the colon cancer patient relative to the total amount of fecal exosomes is 9.0 × 10 ３ It is fine if it is 5.0 x 10 ４ More preferably, it is 1.0 × 10 or more. ５ The upper limit is not particularly limited, but is more preferably 5.0 × 10 ６ Preferably, it is 3.0 × 10 or less. ６ For example, the content is preferably 9.0×10 ３ ~5.0 x 10 ６ , 5.0 × 10 ４ ~5.0 x 10 ６ , 1.0×10 ５ ~3.0 x 10 ６Furthermore, the number of healthy subjects and colon cancer patients (i.e., the number of specimens) is preferably 3 or more (e.g., 3 to 4), and each of the above contents is preferably an average value thereof.

[0059] The proteins selected in the selection step are frequently gastrointestinal disease-associated proteins, and can therefore be used as candidate diagnostic markers for gastrointestinal diseases. The screening method of the present invention preferably further comprises a step of detecting the gastrointestinal disease-associated proteins from the selected proteins (candidate diagnostic markers). Methods for detecting the gastrointestinal disease-associated proteins from the proteins can be appropriately selected from known methods or methods based thereon, including, for example, immunological detection methods using antibodies against the gastrointestinal disease-associated proteins; methods combining MS / MS analysis or LC / MS / MS analysis with proteome analysis; and Western blotting, and two or more of these methods may be combined.

[0060] Furthermore, the candidate diagnostic markers for gastrointestinal diseases selected in the selection step can be evaluated for their usability as diagnostic markers for the gastrointestinal diseases, for example, by analyzing increases or decreases in expression levels associated with gastrointestinal diseases by comparing healthy individuals with patients with gastrointestinal diseases, analyzing whether the protein is derived from gastrointestinal cancer, etc. Proteins (gastrointestinal disease-related proteins) evaluated in this manner include, but are not limited to, the gastrointestinal disease-related proteins listed as preferred detection targets in the detection step of the diagnostic auxiliary method and diagnostic method of the present invention.

[0061] <Immunological Detection Method> The present invention also provides a method for immunologically detecting a gastrointestinal disease-associated protein (sometimes referred to herein as the "detection method of the present invention"), comprising the steps of: isolating fecal exosomes from feces; and immunologically detecting a gastrointestinal disease-associated protein from the isolated fecal exosomes. As described above, gastrointestinal disease-associated proteins are significantly concentrated in fecal exosomes. Therefore, even if a gastrointestinal disease-associated protein is difficult to detect immunologically due to its low concentration in whole feces or the presence of many impurities, the step of isolating fecal exosomes enables the gastrointestinal disease-associated protein to be immunologically detected with high sensitivity.

[0062] [Separation Step] The detection method of the present invention includes a step of separating fecal exosomes from feces (referred to as the "separation step" in the detection method of the present invention). The feces are as described above, and are not particularly limited as long as they can contain the gastrointestinal disease-related protein that is the detection target substance. The method for separating fecal exosomes from the feces, including preferred embodiments thereof, is the same as the method described as the separation step in the diagnostic auxiliary method and diagnostic method of the present invention.

[0063] [Detection step] The detection method of the present invention includes a step of immunologically detecting a gastrointestinal disease-associated protein from the fecal exosomes separated in the separation step (referred to as the "detection step" in the detection method of the present invention). In this detection step, the method for detecting the gastrointestinal disease-associated protein is an immunological detection method among the detection methods described in the diagnostic support method and diagnostic method of the present invention. The immunological detection method is the same as the method described in the diagnostic support method and diagnostic method of the present invention, including preferred embodiments thereof.

[0064] <Detection Reagent and Kit for Gastrointestinal Disease-Associated Proteins> The present invention provides a detection reagent for a gastrointestinal disease-associated protein (sometimes simply referred to herein as "the reagent of the present invention") as a reagent for use in the diagnostic auxiliary method and diagnostic method of the present invention, the screening method of the present invention, and / or the detection method of the present invention. The present invention also provides a kit containing the reagent of the present invention (sometimes simply referred to herein as "the kit of the present invention") as a kit for use in the diagnostic auxiliary method and diagnostic method of the present invention, the screening method of the present invention, and / or the detection method of the present invention.

[0065] The reagent of the present invention includes reagents for use in the detection method of the gastrointestinal disease-associated protein, depending on the method, and is not particularly limited. When the detection method is an immunological detection method, examples of the reagent include an antibody against the gastrointestinal disease-associated protein; the labeling substance or an antibody bound to the labeling substance; a secondary antibody, protein G, or protein A bound to the labeling substance; the solid phase or an antibody bound to the solid phase. Each of the reagents may consist of one or more of these, or may be a composition containing one or more of these. The composition may further contain other components. Examples of the other components include, but are not limited to, sterilized water, physiological saline, surfactants, preservatives, etc., and may be one or a combination of two or more of these.

[0066] In addition to the reagents of the present invention, the kits of the present invention may further include, as needed, for example, a fecal suspension solution, a reagent for separating fecal exosomes (e.g., a separation medium for density gradient centrifugation), a reagent for detecting fecal exosomes (e.g., an antibody against an exosome marker protein or a labeled form in which the antibody is bound to a labeling substance), reagents for use in various immunological detection methods (e.g., a substrate solution, a reaction solution, and a reaction stop solution when the labeling substance is an enzyme), a control, or a combination thereof. Furthermore, the kits of the present invention may further include instructions for use of the kit.

[0067] The present invention will be described in more detail below based on test examples, but the present invention is not limited to the following test examples. In these test examples, "%" indicating concentration means "weight / volume % (w / v %)" unless otherwise specified.

[0068] Test Example 1: Purification of fecal exosomes from healthy human feces (1) Fractionation by density gradient centrifugation 2-7 g of feces (pooled sample: a mixture of feces from 20 healthy individuals) was weighed into a tube, suspended in 25 mL of phosphate buffer containing protease inhibitor (Roche), and then allowed to stand on ice for 30 minutes. After resuspending, the sample was centrifuged at 3,000 × g for 10 minutes, and supernatant 1 was collected. The precipitate was again suspended in 25 mL of phosphate buffer containing protease inhibitor (Roche), and then centrifuged at 3,000 × g for 10 minutes. Supernatant 2 was then collected and mixed with the previously collected supernatant 1. The mixed supernatant was then centrifuged at 3,000 × g for 30 minutes, and the resulting supernatant was filtered through a filter paper (Advantec), and the filtrate was centrifuged at 40,000 × g for 90 minutes. After centrifugation, the supernatant was filtered through a 0.45 μm filter (Whatman). The filtrate was then centrifuged at 150,000 × g for 150 minutes to obtain crude fecal exosomes as a precipitate.

[0069] Next, the crude fecal exosomes obtained above were subjected to density gradient centrifugation. First, OptiPrep (60% iodixanol solution, manufactured by Abbott Diagnostics Technologies) and 8-26% sucrose solution were mixed so that the iodixanol concentration was 5-40%, and then layered in a 1.4 cm diameter centrifuge tube in four layers, starting from the bottom: 3 mL of 40% iodixanol solution, 3 mL of 20% iodixanol solution, 3 mL of 10% iodixanol solution, and 2.5 mL of 5% iodixanol solution. The resulting crude fecal exosomes (precipitate) was suspended in 500 μL of phosphate buffer and placed on top to form a total of five layers. This was then centrifuged at 100,000 × g for 18 hours (density gradient centrifugation). After centrifugation, 1 mL of each sample was collected from the top surface at a depth of 0.742 cm, and these were used as purified fecal exosome fractions (Fraction 1-12).

[0070] Each fecal exosome purified fraction was directly used for the detection of fecal exosomes by BLEIA described below. Each fecal exosome purified fraction (1 mL) was further diluted with 12 mL of phosphate buffer, centrifuged at 150,000 × g for 120 minutes, the supernatant was removed, and the mixture was suspended in 100 μL of phosphate buffer to obtain purified fecal exosomes for each fraction.

[0071] (2) Detection of fecal exosomes by BLEIA Each fecal exosome purified fraction was analyzed by BLEIA, which is based on bioluminescence, using a fully automated analyzer, BLEIA. ＴＭFecal exosomes were detected using a ELISA kit (manufactured by Eiken Chemical Co., Ltd.) with a ELISA kit (manufactured by Eiken Chemical Co., Ltd.). Specifically, each fecal exosome purified fraction obtained in (1) above was used as a sample. For the primary reaction, 50 μL of the sample was mixed with magnetic particles immobilized with an anti-human CD9 antibody (manufactured by Cosmo Bio Co., Ltd., SHI-EXO-M01) and a biotinylated anti-human CD63 antibody (manufactured by Cosmo Bio Co., Ltd., SHI-EXO-M02-B) and allowed to react at 37 ° C for 15 minutes. Subsequently, impurities not captured by the magnetic particles were washed to remove them, and 80 μL of Tris buffer containing streptavidin and biotinylated luciferase was added, followed by a secondary reaction at 37 ° C for an additional 15 minutes. After the secondary reaction, washing was again performed, and 100 μL of luminescent substrate solution (manufactured by Eiken Chemical Co., Ltd.) was added to detect the luminescent signal.

[0072] The total protein content of each fraction was quantified by BCA protein assay. Specifically, BCA Working Reagent (Thermo Fisher Scientific) was added to each fecal exosome purified fraction solution obtained in (1) above, and the mixture was incubated at 37°C for 30 minutes, then cooled to room temperature. The color (purple) was measured by absorbance at 562 nm. The total protein content (ng / μL) in each fecal exosome purified fraction was calculated using a calibration curve created from the absorbance measured in the same manner for standard proteins of known concentrations.

[0073] Figure 1 shows the relationship between BLEIA luminescence intensity (CD9 / CD63 luminescence intensity, left axis, line graph) and each fraction (Fraction_1-12), as well as the relationship between the total protein amount (Protein (ng / μL), right axis, bar graph) and each fraction (Fraction_1-12). As shown in Figure 1, of the obtained purified fecal exosome fractions, Fractions_6-7 showed particularly strong luminescence intensities corresponding to CD9 and CD63, which are surface marker proteins of fecal exosomes, relative to the total protein amount. Therefore, it was confirmed that in the purified fecal exosome fractions obtained in (1) above, fecal exosomes were abundant in Fractions_6-7, and that the purity of fecal exosomes in these fractions was also high. Fractions 6 to 7 are fractions with an iodixanol concentration of 30 to 45% in the density gradient centrifugation.

[0074] (3) Detection of fecal exosomes by Western blot. Fecal exosomes were detected by Western blot for each fraction of purified fecal exosome solution. Specifically, 17.5 μL of each purified fecal exosome solution obtained in (1) above was mixed with SDS and DTT and then heated at 95°C for 10 minutes to prepare a Western blot sample. 20 μL of the Western blot sample was electrophoresed on a 10-20% acrylamide gel, transferred to a PVDF membrane, and reacted overnight with anti-Alix antibody (rabbit-derived, Proteintech), anti-CD63 antibody (rabbit-derived, Proteintech), or anti-CD9 antibody (rabbit-derived, Proteintech). Next, the sections were reacted with HRP-labeled anti-rabbit antibody for 1 hour, and then colored with ECL prime (Cytiva). Specific bands corresponding to each antibody were detected using iBright Imaging Systems (Thermo Fisher Scientific).

[0075] The results of Western blotting are shown in Figure 2. As shown in Figure 2, bands for Alix, CD63, and CD9, which are marker proteins on the surface or encapsulated in fecal exosomes, were confirmed in Fractions 6 to 7 of the purified fecal exosome solution obtained. Therefore, analysis of the purified fecal exosome solution by Western blotting also confirmed that fecal exosomes were contained at high purity in Fractions 6 to 7 obtained in (1) above.

[0076] (4) Detection of fecal exosomes using Nanosight Particle analysis was performed on the purified fecal exosome solution of each fraction using Nanosight (Malvern Panalytical). Specifically, each purified fecal exosome solution obtained in (1) above was diluted 350 to 1000 times with phosphate buffer, and the number of particles (particles) with a particle size of up to 1000 nm was measured while this was being pumped into Nanosight (Malvern Panalytical) using a syringe pump, and the concentration (particles / mL) was calculated. Measurements were performed in triplicate, and the average value was used.

[0077] A graph showing the particle size distribution in Fractions 5 to 9 is shown in Figure 3. As shown in Figure 3, it was confirmed that, of the purified fecal exosome solution obtained in (1), Fractions 6 to 7 contained a high concentration of particles with particle diameters of 100 to 300 nm, which correspond to fecal exosomes. Therefore, analysis of the purified fecal exosome solution using Nanosight also confirmed that fecal exosomes were contained at a high purity in Fractions 6 to 7 obtained in (1) above.

[0078] Test Example 2: Comparison of fecal exosomes and whole feces in healthy individuals (1) Proteome analysis by mass spectrometry Proteome analysis by mass spectrometry was performed using the purified fecal exosome solution of Fractions 6 to 7 obtained in Test Example 1 above as a fecal exosome sample from a healthy individual, and the feces (pooled specimen) used in Test Example 1 above as a whole feces sample from a healthy individual. Each sample was processed, measured, and analyzed by a contractor (Promega Corporation, DIA Proteome Analysis Contract Service) according to the following procedure.

[0079] After reductive alkylation, 200 ng of each sample was added to 500 ng of Trypsin / Lys-C Mix (Promega Corporation) and digested overnight at 37 ° C. After digestion, the sample was desalted using a reversed-phase spin column (GL Tip SDD, GL Sciences) to prepare the analytical sample. 200 ng of the analytical sample was separated using an UltiMate 3000 RSLCnano LC System (Thermo Fisher Scientific), and then subjected to MS / MS analysis (overlapping window DIA) using a connected Q Exactive HF X (Thermo Fisher Scientific). The obtained data was analyzed using Scaffold DIA (Proteome Software) to identify proteins (including peptides, the same applies hereinafter) and calculate quantitative values. Identification and calculation of quantitative values ​​were performed only for proteins whose Peptide FDR (False Discovery Rate, the same applies hereinafter) and Protein FDR were both 1% or less. Quantitative values ​​were calculated as relative quantitative values ​​(contents) relative to the amount of sample used for analysis using Scaffold DIA software (Proteome Software) (unitless).

[0080] The numbers of proteins identified in healthy subject fecal exosomes and healthy subject whole feces are shown in Table 1 below. As shown in Table 1, a greater number of proteins were identified in fecal exosomes. Here, 271 proteins were common to healthy subject fecal exosomes and healthy subject whole feces, and of these, 89 proteins had a high content (quantitative value) in healthy subject fecal exosomes, more than twice the content in healthy subject whole feces. Note that because the same amount of sample was provided for all MS / MS analysis, each quantitative value was interpreted as the content in each sample. Furthermore, because the detection limit (three times the standard deviation) was common to all samples, proteins present at concentrations lower than this detection limit were not detected. Figure 4 shows a plot illustrating the correlation between the quantitative values ​​of proteins in fecal exosomes and whole feces of healthy individuals for proteins (271 types) common to both fecal exosomes and whole feces of healthy individuals. However, no correlation was observed between the quantitative values ​​of proteins in fecal exosomes and whole feces of healthy individuals.

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[0082] (2) Enrichment Analysis Among the proteins identified in (1) above and common to fecal exosomes and whole feces of healthy subjects, 89 proteins (quantitative value) whose content in fecal exosomes of healthy subjects was more than twice (200%) that of whole feces of healthy subjects, or 287 proteins (detected only in fecal exosomes of healthy subjects but not in whole feces of healthy subjects) were analyzed using The Database for Annotation, Visualization and Integrated Discovery (DAVID) (https: / / david.ncifcrf.gov / , DAVID) to investigate whether proteins derived from the digestive tract (gastrointestinal tract or its cancer) were significantly enriched. Enrichment analysis was performed using the DAVID Knowledgebase v2022q4 (Dec. 2021). Tissues or organs with a p-value (P value) of less than 0.05 and an FDR of less than 5% were extracted. Among these, the analysis results for tissues or organs belonging to the digestive tract are shown in Table 2 below, along with the corresponding proteins (i.e., digestive tract-derived proteins). As shown in Table 2, proteins whose content in healthy subject fecal exosomes was more than twice the content in healthy subject whole feces, or proteins that were not detected in healthy subject whole feces but only in healthy subject fecal exosomes, contained significantly higher amounts of proteins derived from the digestive tract, such as colon (colon), colon adenocarcinoma (colon cancer), and small intestine (small intestine), and it was confirmed that these proteins were significantly enriched in fecal exosomes. In addition, other tissues or organs from which the above-mentioned enriched proteins originate, such as the liver, were also cited as tissues or organs that met the above conditions (p-value and FDR). However, when a similar analysis was also performed on proteins that were not detected in fecal exosomes from healthy individuals but were detected only in the whole feces of healthy individuals, the liver had a high count of 76, which met the above conditions (p-value and FDR), confirming that liver-derived proteins cannot generally be said to be significantly enriched only in fecal exosomes.On the other hand, in an analysis of proteins that were not detected in fecal exosomes from healthy individuals but were detected only in the whole feces of healthy individuals, no tissues or organs belonging to the digestive tract were identified as meeting the above conditions (p-value and FDR), confirming that proteins derived from the digestive tract were significantly enriched only in fecal exosomes.

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[0084] Test Example 3 Comparison of fecal exosomes from healthy individuals and fecal exosomes from colorectal cancer patients 1 (1) Purification of exosomes from feces of colorectal cancer patients Test Example 1 confirmed that fecal exosomes can be purified with high purity from feces by the method described in the same test example. Therefore, the feces of the colorectal cancer patient were fractionated by density gradient centrifugation in the same manner as in Test Example 1, except that feces collected from a colorectal cancer patient (one specimen) was used as the feces, and fecal exosomes were detected by BLEIA, Western blotting, and Nanosight.

[0085] Figure 5 shows the relationship between BLEIA luminescence intensity (CD9 / CD63 luminescence intensity, left axis, line graph) and each fraction (Fraction_1-12), as well as the relationship between total protein amount (Protein (ng / μL), right axis, bar graph) and each fraction (Fraction_3-8) by BCA protein assay. Figure 6 also shows the results of Western blotting of purified fecal exosome solutions from Fractions_3-8. Figure 7 also shows a graph showing the particle size distribution of purified fecal exosome solutions from Fractions_3-8. As shown in Figures 5 to 7, it was confirmed that fecal exosomes were contained in high purity in Fractions_6-7 of the purified fecal exosome fractions. Therefore, the purified fecal exosome solutions from Fractions_6-7 were used as fecal exosome samples from colorectal cancer patients.

[0086] (2) Proteome Analysis by Mass Spectrometry A fecal exosome sample from a healthy subject (purified fecal exosome solution of Fractions 6-7) was obtained in the same manner as in Test Example 1, except that a different feces sample (pooled sample: a mixture of feces from 20 healthy subjects other than those in Test Example 1) was used as the sample. Proteome analysis by mass spectrometry was performed on this sample and the fecal exosome sample from a colon cancer patient obtained in (1) above. Each sample was processed, measured, and analyzed by a contractor (Promega Corporation, DIA Proteome Analysis Contract Service) according to the following procedure.

[0087] After reductive alkylation, 200 ng of each sample was added with 500 ng of Trypsin / Lys-C Mix (Promega Corporation) and digested overnight at 37 ° C. After digestion, the sample was desalted using a reversed-phase spin column (GL Tip SDD, GL Sciences) to prepare the analytical sample. 200 ng of the analytical sample was separated using an UltiMate 3000 RSLCnano LC System (Thermo Fisher Scientific), and then subjected to MS / MS analysis (DIA MS) using a connected Q Exactive HF X (Thermo Fisher Scientific). The obtained data was analyzed using DIA-NN (Demichev et al., DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput, Nat. Methods, 2020, DOI: 10.1038 / s41592-019-0638-x) to identify proteins and calculate quantitative values. Note that only proteins with precursor FDR and protein FDR of 1% or less were identified and quantitative values ​​were calculated. The quantitative value was calculated by DIA-NN as a relative quantitative value (i.e., content in each sample) relative to the amount of sample used for analysis (unitless).

[0088] The numbers of proteins identified in fecal exosomes from healthy individuals and fecal exosomes from colorectal cancer patients are shown in Table 3 below. The above analysis identified 1,200 proteins in fecal exosomes from colorectal cancer patients, far more than the 457 proteins identified in fecal exosomes from healthy individuals. Figure 8 shows a plot of the proteins (442 proteins) common to both fecal exosomes from healthy individuals and those from colorectal cancer patients, showing the correlation between the quantitative values ​​of proteins in fecal exosomes from colorectal cancer patients and those in healthy individuals. In Figure 8, the correlation coefficient (r) between the quantitative values ​​of proteins in fecal exosomes from colorectal cancer patients and those in healthy individuals was 0.2736, indicating no correlation between the two.

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[0090] (3) Enrichment Analysis To investigate whether the 758 proteins identified in (2) above and detected only in fecal exosomes from colon cancer patients were significantly enriched in proteins derived from the digestive tract (gastrointestinal tract or its cancer), enrichment analysis was performed in the same manner as in (2) of Test Example 2. Tissues or organs with a p-value (P value) of less than 0.05 and an FDR of less than 5% were extracted. It was confirmed that, similar to the fecal exosomes from healthy individuals, the fecal exosomes from colon cancer patients also showed significant enrichment of proteins derived from the digestive tract, such as colon (large intestine), small intestine, and colon adenocarcinoma (colon cancer).

[0091] Test Example 4 Comparison of Fecal Exosomes with Whole Feces in Healthy Subjects and Cancer Patients (1) Purification of Exosomes from Feces of Healthy Subjects and Feces of Colon Cancer Patients Healthy subject fecal exosome samples (purified fecal exosome liquid of Fractions 6-7) were obtained in the same manner as Test Example 1, except that four fecal specimens (healthy subjects 1-4) collected from four healthy subjects were used as the healthy subject feces. Furthermore, colon cancer patient fecal exosome samples (purified fecal exosome liquid of Fractions 6-7) were obtained in the same manner as Test Example 3(1), except that four fecal specimens (colon cancer subjects 1-4) collected from four colon cancer patients were used as the feces.

[0092] (2) Proteome analysis by mass spectrometry The feces used in (1) above were designated as the whole feces samples from healthy individuals and the whole feces samples from colorectal cancer patients, respectively. Proteome analysis by mass spectrometry was performed on these samples, as well as on the fecal exosome samples from healthy individuals and the fecal exosome samples from colorectal cancer patients obtained in (1) above, in the same manner as in (2) of Test Example 3.

[0093] The number of proteins identified in each healthy subject fecal exosome and in the whole feces of healthy subjects is shown in Table 4 below, and the number of proteins identified in each colon cancer patient fecal exosome and in the whole feces of colon cancer patients is shown in Table 5 below. Note that the number of proteins in the whole feces of colon cancer patients was not identified for the colon cancer 4 specimen. As in Test Example 2 (1) and Test Example 3 (2), it was confirmed that the number of proteins identified in each fecal exosome was greater than the number of proteins identified in the whole feces by the same analysis for both specimens.

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[0096] (3) Enrichment Analysis In a total of seven specimens (healthy subjects 1-4 and colon cancer 1-3), proteins identified in (2) above and common to each fecal exosome and each whole feces were identified for proteins whose content (quantitative value) in fecal exosomes was more than twice (200%) that of the whole feces, or proteins identified in (2) above but not in the whole feces but only in fecal exosomes in a total of seven specimens (healthy subjects 1-4 and colon cancer 1-3). To investigate whether proteins derived from the digestive tract (gastrointestinal tract or its cancer) were significantly enriched (enriched), enrichment analysis was performed in the same manner as in (2) of Test Example 2. Tissues or organs with a p-value (P value) of less than 0.05 and an FDR of less than 5% were extracted, and the analysis results of tissues or organs belonging to the digestive tract, together with the corresponding proteins (i.e., digestive tract-derived proteins), are shown in Tables 6 to 9 below. As shown in Tables 6 to 9, proteins whose content in fecal exosomes was more than twice that in total feces (Tables 6 and 8) or that were not detected in total feces but only in fecal exosomes (Tables 7 and 9) were found to contain significantly higher amounts of gastrointestinal proteins in both healthy subjects and colon cancer patient samples, as in Test Example 2 (2) and Test Example 3 (3), confirming that these proteins were significantly enriched in fecal exosomes. In this analysis, as in Test Example 2 (2), other tissues or organs that met the above conditions (p-value and FDR) included the liver, etc. However, when a similar analysis was performed on proteins that were not detected in fecal exosomes but were detected only in whole feces, no tissues or organs belonging to the digestive tract that met the above conditions (p-value and FDR) were identified, whereas the liver had a high Count of 90 or more, which met the above conditions (p-value and FDR), confirming that liver-derived proteins cannot generally be said to be significantly enriched in fecal exosomes alone. Similar analyses were also performed on proteins derived from tissues or organs other than the digestive tract and liver, but none of them met the above conditions (p-value and FDR). In this analysis, the only tissue or organ in which significant protein enrichment was confirmed in fecal exosomes alone across all samples was the digestive tract.

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[0101] <Test Example 5> Comparison of fecal exosomes from healthy individuals and fecal exosomes from colorectal cancer patients 2 First, the pathology data of "The Human Protein Atlas Database (https: / / www.proteinatlas.org / )," a database of protein staining profiles and staining levels in human tumor tissues based on immunohistochemistry, was downloaded (June 16, 2023), and colorectal cancer data was obtained from them. From the obtained data, the ratio (n / N) of the total number of proteins (n) with a staining level of High, Medium, or Low divided by the total number of stains (N) was calculated, and those with a ratio greater than 0.5 were selected as colorectal cancer-related proteins. 9,723 colorectal cancer-related proteins were selected from the database.

[0102] Next, proteins identical to the above 9723 proteins were extracted from the proteins identified in the fecal exosomes of each healthy subject and the fecal exosomes of colon cancer patients in Test Example 4 (2). From these proteins, proteins common to both the fecal exosomes of healthy subjects and the whole feces of healthy subjects, whose content in the fecal exosomes of healthy subjects was more than twice (200% or more) the content (quantitative value) in the whole feces of healthy subjects, and proteins detected only in the fecal exosomes of healthy subjects, were selected. Proteins: Proteins that were common to both fecal exosomes of colorectal cancer patients and whole feces of colorectal cancer patients, and whose content in fecal exosomes of colorectal cancer patients was at least twice (200% or more) that in whole feces of colorectal cancer patients; and proteins that were not detected in whole feces of colorectal cancer patients but only in fecal exosomes of colorectal cancer patients. For at least one of these proteins, proteome analysis by mass spectrometry was performed in the same manner as in Test Example 3 (2), and quantitative values ​​(contents) were calculated.

[0103] For the analyzed gastrointestinal disease-related proteins, the ratio (Ratio: Quantitative value in fecal exosomes of colon cancer patients / Quantitative value in fecal exosomes of healthy individuals × 100 (%)) of the average quantitative value in fecal exosomes of healthy individuals (average of healthy individuals 1 to 4; quantitative values ​​in specimens in which the protein was not identified were calculated as 0) to the average quantitative value in fecal exosomes of colon cancer patients (average of colon cancer individuals 1 to 3; quantitative values ​​in specimens in which the protein was not identified were calculated as 0) was calculated. Tables 10 to 12 show a list of proteins with this ratio (Ratio) of 150% (1.5 times) or higher, as well as the average quantitative values ​​and ratios in each of these fecal exosomes. Tables 13 to 15 also show a list of analyzed gastrointestinal disease-related proteins that were not detected in fecal exosomes from healthy individuals (samples 1 to 4) and were detected only in fecal exosomes from at least one of colorectal cancer patients (colorectal cancer 1 to 3), as well as the average quantitative values ​​for each of these fecal exosomes (average values ​​for colorectal cancer 1 to 3; the quantitative values ​​for samples in which the protein was not identified were calculated as 0).

[0104] Furthermore, of the gastrointestinal disease-associated proteins listed in Tables 10 to 12, proteins that were detected in fecal exosomes in common in two or more colon cancer patient specimens are shown in Figure 9, and of the gastrointestinal disease-associated proteins listed in Tables 13 to 15, proteins that were detected in fecal exosomes in common in two or more colon cancer patient specimens are shown in Figure 10. Furthermore, of the gastrointestinal disease-associated proteins listed in Tables 10 to 15, proteins that were detected in common in fecal exosomes in all colon cancer patient specimens (four specimens) are shown in Figure 11.

[0105] In both healthy individuals and colorectal cancer patients, gastrointestinal disease-related proteins were enriched in fecal exosomes (i.e., the quantitative value (content) in each fecal exosome was more than twice the quantitative value (content) in each whole feces, or the protein was not detected in each whole feces but was detected only in each fecal exosome). However, as shown in Tables 10 to 12, many gastrointestinal disease-related proteins were enriched in fecal exosomes from colorectal cancer patients, and the content was significantly higher than in fecal exosomes from healthy individuals (i.e., the ratio was 150% or higher). Furthermore, as shown in Tables 13 to 15, more gastrointestinal disease-related proteins were enriched and detected in fecal exosomes from colorectal cancer patients than in fecal exosomes from healthy individuals (i.e., the protein was not detected in fecal exosomes from healthy individuals but was enriched only in fecal exosomes from colorectal cancer patients). These findings confirmed that gastrointestinal disease-related proteins (e.g., colon cancer-related proteins) are enriched in fecal exosomes (particularly fecal exosomes from patients with gastrointestinal diseases (e.g., colon cancer patients)). Furthermore, gastrointestinal disease-related proteins differ depending on the severity of the disease and individual differences, so they do not necessarily need to be common to all patients. However, as shown in Figures 9 to 11, it was confirmed that at least many gastrointestinal disease-related proteins are enriched in fecal exosomes from colon cancer patients in common across samples.

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[0112] Test Example 6 Comparison of colon cancer-related proteins in fecal exosomes and in whole feces 1 From the proteins shown in Figure 11 obtained in Test Example 5 above, proteins whose quantitative value (content) in fecal exosomes (fEV) of colon cancer patients was more than twice that in whole feces (feces) of colon cancer patients were selected, and for five of these proteins (LAMP1, ATP5F1A, LGALS3BP, DLD, ENPP4) as an example, the quantitative values ​​(average values) in fecal exosomes of healthy subjects and fecal exosomes of colon cancer patients, as well as the quantitative values ​​(average values) in whole feces of healthy subjects and whole feces of colon cancer patients were compared. Figure 12 shows (a) a graph comparing the quantitative values ​​(HC) in fecal exosomes from healthy subjects with the quantitative values ​​(CC) in fecal exosomes from colorectal cancer patients, and (b) a graph comparing the quantitative values ​​(HC) in whole feces from healthy subjects with the quantitative values ​​(CC) in whole feces from colorectal cancer patients.

[0113] Furthermore, from the proteins shown in Figure 11 obtained in Test Example 5 above, proteins that were not detected in the whole feces (feces) of colorectal cancer patients but were detected only in the fecal exosomes (fEV) of colorectal cancer patients were selected, and for five of these (MAPK1, PCYOX1, SMPD3, VILL, RPS7), the quantitative values ​​(mean values) in the fecal exosomes of healthy individuals were compared with the quantitative values ​​(mean values) in the fecal exosomes of colorectal cancer patients, and the quantitative values ​​(mean values) in the whole feces of healthy individuals were compared with the quantitative values ​​(mean values) in the whole feces of colorectal cancer patients. Figure 13 shows (a) a graph comparing the quantitative values ​​(HC) in the fecal exosomes of healthy individuals with the quantitative values ​​(CC) in the fecal exosomes of colorectal cancer patients, and (b) a graph comparing the quantitative values ​​(HC) in the whole feces of healthy individuals with the quantitative values ​​(CC) in the whole feces of colorectal cancer patients. In Figures 12 and 13, "*" indicates that p<0.05 in the Mann-Whitney U test, and "ns" indicates that there was no significant difference.

[0114] As shown in Figures 12 and 13 , the quantitative values ​​of colon cancer-related proteins enriched in fecal exosomes from colon cancer patients, i.e., proteins whose quantitative value (content) in fecal exosomes from colon cancer patients was more than twice that in whole feces from colon cancer patients, or proteins that were not detected in whole feces from colon cancer patients but were detected only in fecal exosomes from colon cancer patients, were significantly different between colon cancer patients and healthy individuals, confirming that detecting these colon cancer-related proteins in fecal exosomes, for example, can be used as an indicator to detect colon cancer patients ( Figures 12 and 13 (a)). On the other hand, when the quantitative values ​​of these proteins in whole feces were compared, there was no significant difference between colon cancer patients and healthy individuals, confirming that it is difficult to distinguish between the two groups based on whole feces ( Figures 12 and 13 (b)).

[0115] <Test Example 7> PLS regression (partial least squares regression) analysis Pathology data from "The Human Protein Atlas database (https: / / www.proteinatlas.org / )" was downloaded (June 16, 2023), and colorectal cancer data was obtained from them. From the obtained data, the ratio (n / N) of the total number of proteins (n) with a staining level of High, Medium, or Low divided by the total number of stains (N) was calculated, and proteins with a ratio greater than 0.5 were selected as colorectal cancer-related proteins. 9,723 colorectal cancer-related proteins were selected from the database.

[0116] Next, proteins corresponding to the above 9723 proteins were extracted from the proteins identified in the fecal exosomes of each healthy subject and the fecal exosomes of colon cancer patients in Test Example 4 (2). Furthermore, for proteins whose quantitative values ​​(content) in the fecal exosomes of healthy subjects were more than twice that of the quantitative values ​​in the whole feces of healthy subjects, the quantitative values ​​in the fecal exosomes of healthy subjects (4 samples) and the quantitative values ​​in the fecal exosomes of colon cancer patients (4 samples) were used to perform PLS regression analysis using JMP software (JMP 17.0.0, JMP Statistical Discovery LLC) with the NIPALS method selected for each group. A plot showing the results is shown in Figure 14(a). In addition, PLS regression analysis was also performed on colon cancer-related proteins that were not detected in the whole feces of healthy individuals, but were detected only in fecal exosomes of healthy individuals. The plots showing the results are shown in Figure 14(b).

[0117] Furthermore, for proteins whose quantitative values ​​in fecal exosomes from healthy individuals were more than twice as high as those in whole feces from healthy individuals among the 9,723 proteins listed above, quantitative values ​​in fecal exosomes from healthy individuals (4 samples) and quantitative values ​​in fecal exosomes from colorectal cancer patients (4 samples) were used to perform PLS regression analysis on each of the following groups: secreted protein group (secreted protein, 18 proteins), membrane protein group (membrane protein, 46 proteins), cytoplasmic protein group (cytoplasmic protein, 56 proteins), and nuclear protein group (nuclear protein, 23 proteins) using JMP software and the NIPALS method. A plot showing the results is shown in Figure 15(a). In addition, PLS regression analysis was also performed on colon cancer-related proteins that were not detected in the whole feces of healthy individuals, but were detected only in fecal exosomes of healthy individuals. The plots showing the results are shown in Figure 15(b).

[0118] Furthermore, for colon cancer-related proteins whose quantitative values ​​in fecal exosomes from colon cancer patients were more than twice as high as those in whole feces from colon cancer patients, quantitative values ​​in fecal exosomes from healthy individuals (four samples) and fecal exosomes from colon cancer patients (four samples) were used. PLS regression analysis was performed using JMP software with the NIPALS method selected for each group: molecular weights of 25 kDa or less (60 proteins), molecular weights of 25-75 kDa (186 proteins), and molecular weights of 75 kDa or more (64 proteins). A plot showing the results is shown in Figure 16(a). A similar PLS regression analysis was also performed on colon cancer-related proteins that were not detected in whole feces from colon cancer patients but were only detected in fecal exosomes from colon cancer patients. A plot showing the results is shown in Figure 16(b).

[0119] Furthermore, for colon cancer-related proteins whose quantitative values ​​in fecal exosomes from colon cancer patients were more than twice as high as those in the whole feces of colon cancer patients among the proteins matching the above 9723 proteins, quantitative values ​​in fecal exosomes from healthy individuals (4 samples) and quantitative values ​​in fecal exosomes from colon cancer patients (4 samples) were used to perform PLS regression analysis on each of the following groups: secreted protein group (secreted protein, 54 proteins), membrane protein group (membrane protein, 135 proteins), cytoplasmic protein group (cytoplasmic protein, 169 proteins), and nuclear protein group (nuclear protein, 77 proteins) using JMP software and selecting the NIPALS method. A plot showing the results is shown in Figure 17(a). In addition, PLS regression analysis was also performed on colon cancer-related proteins that were not detected in the whole feces of colon cancer patients but were detected only in fecal exosomes of colon cancer patients. The plots showing the results are shown in Figure 17(b).

[0120] As shown in Figures 14 to 17 , the distribution trend of the quantitative values ​​in each fecal exosome of colon cancer-associated proteins that were concentrated in each fecal exosome, i.e., proteins whose quantitative value (content) in each fecal exosome was twice or more that in each whole feces, or proteins that were not detected in each whole feces but were detected only in each fecal exosome, showed that regardless of differences in protein molecular weight ( Figures 14 and 16 ) or protein localization ( Figures 15 and 17 ), the fecal exosomes were separated into two groups, that is, between colon cancer patients and healthy subjects, and it was confirmed that colon cancer patients can be detected using the quantitative values ​​of these colon cancer-associated proteins as indicators.

[0121] Test Example 8 Comparison of fecal exosomes from healthy individuals and fecal exosomes from colorectal cancer patients 3 First, in addition to the 9723 proteins selected in Test Example 5, 155 proteins that have previously been reported as biomarkers or potential biomarkers for colorectal cancer were selected.

[0122] Next, from the proteins identified in the fecal exosomes of each healthy subject and the fecal exosomes of colon cancer patients in Test Example 4 (2), 57 proteins that matched the above 155 proteins were extracted, and from these, proteins that were common to both the fecal exosomes of healthy subjects and the whole feces of healthy subjects and whose content in the fecal exosomes of healthy subjects was more than twice (200% or more) the content (quantitative value) in the whole feces of healthy subjects; proteins that were not detected in the whole feces of healthy subjects but only in the fecal exosomes of healthy subjects were selected. proteins that were common to both fecal exosomes of colorectal cancer patients and whole feces of colorectal cancer patients and whose content in fecal exosomes of colorectal cancer patients was at least twice (200% or more) that of whole feces of colorectal cancer patients; and proteins that were not detected in whole feces of colorectal cancer patients but were detected only in fecal exosomes of colorectal cancer patients; proteome analysis by mass spectrometry was performed in the same manner as in Test Example 3 (2) to calculate quantitative values ​​(contents).

[0123] For the colon cancer-related proteins analyzed, the ratio (Ratio: Quantitative value in fecal exosomes of colon cancer patients / Quantitative value in fecal exosomes of healthy individuals × 100 (%)) of the average quantitative value in fecal exosomes of healthy individuals (average of healthy individuals 1 to 4; quantitative values ​​in specimens in which the protein was not identified were calculated as 0) to the average quantitative value in fecal exosomes of colon cancer patients (average of colon cancer individuals 1 to 3; quantitative values ​​in specimens in which the protein was not identified were calculated as 0) was calculated. Table 16 below shows a list of proteins with this ratio (Ratio) of 150% (1.5 times) or higher, as well as the average quantitative values ​​and ratios in each of these fecal exosomes. Furthermore, for the colon cancer-related proteins analyzed, proteins that were not detected in fecal exosomes from healthy individuals (samples 1 to 4) and were detected only in fecal exosomes from at least one colon cancer patient (colon cancer 1 to 3) and the average quantitative values ​​for each of these fecal exosomes (average values ​​for colon cancer 1 to 3; quantitative values ​​for samples in which the protein was not identified were calculated as 0) are listed in Table 16 below. In Table 16, LCP1, ANXA1, and OLFM4 were detected in fecal exosomes in all colon cancer patient samples (4 samples), while FBLN1, COL6A1, TGFBI, and THBS1 were detected in fecal exosomes in two or three colon cancer patient samples.

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[0125] In both healthy individuals and colorectal cancer patients, colon cancer-associated proteins were enriched in fecal exosomes (i.e., the quantitative value (content) in each fecal exosome was more than twice the quantitative value (content) in each whole feces, or the protein was not detected in each whole feces but was detected only in each fecal exosome). However, as shown in Table 16, many colon cancer-associated proteins were enriched in fecal exosomes from colorectal cancer patients, with the content being significantly higher than in fecal exosomes from healthy individuals (i.e., the ratio was 150% or higher). Furthermore, as shown in Table 16, there were colon cancer-associated proteins in fecal exosomes from colorectal cancer patients that were not detected in fecal exosomes from healthy individuals but were enriched and detected only in fecal exosomes from colorectal cancer patients. These results confirmed that gastrointestinal disease-associated proteins (e.g., colon cancer-associated proteins) are enriched in fecal exosomes (particularly fecal exosomes from patients with gastrointestinal diseases (e.g., colon cancer patients)).

[0126] Test Example 9: Comparison of colon cancer-associated proteins in fecal exosomes and in whole feces 2

[0111] Of the seven proteins listed in Table 16 obtained in Test Example 8, OLFM4 was used as an example. The quantitative values ​​(mean values) in fecal exosomes from healthy individuals and fecal exosomes from colon cancer patients were compared, as well as the quantitative values ​​(mean values) in whole feces from healthy individuals and fecal exosomes from colon cancer patients. Figure 18 shows a graph comparing the quantitative values ​​(HC) in fecal exosomes from healthy individuals and fecal exosomes from colon cancer patients (a: fecal exosomes (fEV)), and a graph comparing the quantitative values ​​(HC) in whole feces from healthy individuals and fecal exosomes from colon cancer patients (b: whole feces (feces)), respectively.

[0127] As shown in Figure 18, the quantitative value of colon cancer-associated proteins enriched in fecal exosomes from colon cancer patients, i.e., the quantitative value of OLFM4 in fecal exosomes, whose quantitative value (content) in fecal exosomes from colon cancer patients was more than twice that in whole feces from colon cancer patients, was significantly different between colon cancer patients and healthy individuals, confirming that detecting such colon cancer-associated proteins in fecal exosomes, for example, can be used as an indicator to detect colon cancer patients (Figure 18(a)). On the other hand, when the quantitative value of such proteins in whole feces was compared, there was no significant difference between colon cancer patients and healthy individuals, confirming that it is difficult to distinguish between the two from whole feces (Figure 18(b)).

[0128] Test Example 10: Detection of colon cancer-related proteins by Western blotting Of the proteins shown in FIG. 11 obtained in Test Example 5 above and shown in Table 16 obtained in Test Example 9 above, three proteins (OLFM4, LAMP1, and LGALS3BP) were detected from fecal exosomes by Western blotting.

[0129] Specifically, first, the protein concentrations of the fecal exosome samples from healthy individuals (HC) and colon cancer patients (CC) obtained in (1) of Test Example 4 were measured by the BCA method. From the obtained protein concentrations, an aliquot of each fecal exosome sample was taken so as to contain 2.5 μg of protein, mixed with purified water, SDS, and DTT, and then heated at 95°C for 10 minutes to prepare samples for Western blotting. 20 μL of the prepared sample was electrophoresed on a 10-20% acrylamide gel, then transferred to a PVDF membrane and reacted overnight with anti-OLFM4 antibody (rabbit-derived, Cell Signaling Technology), anti-LAMP1 antibody (rabbit-derived, Cell Signaling Technology), or anti-LGALS3BP antibody (rabbit-derived, Proteintech) in a refrigerator. The sample was then reacted with an HRP-labeled anti-rabbit antibody for 1 hour, after which it was developed with ECL prime (Cytiva). Specific bands corresponding to each antibody were detected using iBright Imaging Systems (Thermo Fisher Scientific).

[0130] The results of Western blot are shown in Figure 19. As shown in Figure 19, for all colon cancer-associated proteins, stronger bands were detected in fecal exosomes from colon cancer patients (CC) than in fecal exosomes from healthy individuals (HC). This confirmed that colon cancer patients can also be detected by Western blot by isolating fecal exosomes and then detecting colon cancer-associated proteins.

[0131] According to the present invention, gastrointestinal disease-associated proteins can be efficiently detected using feces as a sample. Furthermore, according to the present invention, it is possible to provide a method for assisting the diagnosis of gastrointestinal diseases, a method for screening diagnostic markers for gastrointestinal diseases, and a detection reagent and kit for gastrointestinal disease-associated proteins for use in these methods, which can efficiently detect gastrointestinal disease-associated proteins using feces as a sample.

Claims

1. A method for assisting in the diagnosis of a gastrointestinal disease, comprising the steps of: isolating fecal exosomes from the stool of a subject; and detecting a gastrointestinal disease-associated protein from the isolated fecal exosomes, wherein the gastrointestinal disease-associated protein satisfies either of the following conditions (a) and (b): (a) its content in the total amount of the fecal exosomes is 1.5 times or more its content in the total amount of the stool; and (b) it is detected only in the isolated fecal exosomes and is not substantially detectable in the stool.

2. The method of claim 1, wherein the gastrointestinal disease-related protein is at least one selected from the group consisting of OLFM4, LGALS3BP, LAMP1, ATP5F1A, DLD, ENPP4, MAPK1, PCYOX1, SMPD3, VILL, and RPS7.

3. The method of claim 1, wherein the gastrointestinal disease is an intestinal disease.

4. The method of claim 1, wherein the gastrointestinal disease is colon cancer.

5. The method of claim 1, further comprising a step of quantifying the detected gastrointestinal disease-related protein.

6. A method for assisting in the diagnosis of a gastrointestinal disease, comprising: isolating fecal exosomes from the feces of a subject; and detecting a gastrointestinal disease-related protein from the isolated fecal exosomes, The gastrointestinal disease-related protein is ZMPSTE24, YWHAG, UGDH, TPM4, TC2N, SUSD2, STOM, ST6GALNAC1, SPTBN1, SLC6A14, SLC46A1, SLC15A4, SERPINF2, SDHB, SDHA, SAFB2, RPS16, RNF128, RNASE3, RAB21, RAB14, PRKG2, PLEC, PLA2G10, NPEPPS, MYH14, MT-CYB, MAOA, ITGA3, IGLV7-46, HSD17B4, HAR S1, H1-4, GSTA1, GMDS, GFM2, FUT3, FLOT2, DNPEP, CNDP2, CLTC, B3GNT7, B2M, ATP9A, ARPC2, AP2A1, AP1B1, AGR2, ACTR2, ABRACL, YWHAH, WD R1, VILL, VAMP8, TUBB4B, TSPAN6, TPP1, TPM3, TPM1, TMEM30A, SYPL1, SQOR, SPPL2A, SMPD3, SLC9A3, SLC44A1, SLC2A5, SLC25A5, SLC15A1, S ERPINA7, SCARB2, S100A11, RPS7, RPS3, RPS23, RPS10, RPL30, RNPEP, RHPN2, RAB3D, RAB32, RAB2A, PTPN6, PSMB8, PSMB1, PSMA1, PRKCD, PR KACA, PRDX6, PRDX5, PRDX1, PPP1CB, PLA2G2A, PKM, PICALM, PGK1, PGD, PFN1, PCYOX1, PCBP1, P2RX4, NIBAN2, NEDD4L, NCSTN, MYOF, MYO1E, M YO1C, MYO1B, MYH9, MUC4, MUC3A, MGST3, MGLL, MEP1A, LYN, LGALS4, LGALS3BP, LDHA, LAMP1, IGKV1-39, IGHM, IGHG2, IDH1, HSPA5, HSP90AB 1, HNRNPA2B1, HLA-DMA, H2BC12, GSTP1, GRN, GPI, GPD2, GPA33, GP2, GNAI2, GHITM, GDPD3, GAPDH, FLOT1, F11R, EPCAM, ENPP4, ENO1, EIF5A,<h2 style=";text-align:left;direction:ltr">EIF4A1、EEF2、EBP、DSTN、DLD、CYFIP1 、CSNK1A1、CPNE3、CLIC1、CHMP2A、CEA CAM5、CEACAM1、CD151、CAPZB、CAPN1、 CAP1、C9、ATP1B1、ATP1A1、ASAH1、ARL 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 69, 64, 65, 59, 67, 59, 69, 65, 59 ... CRC2、UQCRC1、UBA1、TPP2、TMEM33、TM EM205、TMED4、TMED2、TMED10、TM9SF3、 TM9SF2、THEMIS2、TARDBP、SYNGR2、SY K、STIP1、STAT3、STAT1、SSR4、SRSF3、 I'm sorry, I'm sorry, I'm sorry, I'm sorry, I'm sorry, I'm sorry B5、SELENOP、SEC31A、SEC23A、RPS4X、RPS3A、RPS18、RPN1、RPL7、RPL6、RPL5 、RPL4、RPL21、RPL13A、RARS1、QARS1、 PTK2B、PTGES3、PSMD3、PSMD12、PSMD1、 PSMC2、PRXL2A、PRPS1、PRKDC、PFKP、P CK2、PC、PAICS、OCIAD2、OAF、NT5C2、N IPSNAP2、NDUFB10、MT-CO2、MOB1B、MI CAL1、MGST1、MCCC2、M6PR、LNPEP、LBR、 KDELR2、KDELR1、KARS1、ITGAV、IMPDH 2、IGKV1-12、IDH3B、IDH2、HTT、HSPA4 、HSD17B10、HNRNPUL2、HNRNPU、HNRNP K、HMGCS2、HLA-B、HK1、HIBCH、HADHA、H 1-0、GRTP1、GPD1、GMPPB、GCLM、GBE1、 GARS1、GALNT2、FGR、FCN3、FASN、ERO1 A、ENTPD1、EML4、EIF3D、DYNC1H1、DNM 2、DLAT、DHX9、DHRS7、DARS1、CYB5B、CS K、CRYL1、CPT1A、COX5B、CORO1C、COPG 1、COPA、CLDN7、CHST5、CCT4、CCT3、C8 G、BSG、B3GNT3、ATP6V0D1、ATP5MG、AR PC3、ARPC1B、ARHGAP1、APMAP、AKR1C3、ADSS2, ADGRG3, ADGRE5, ACSL1, ACADM, VPS35, TMED9, TMED7, TM9SF4, S TXBP6, SLC25A11, SIGMAR1, SF3B3, RPS9, RPS24, RPS13, RPLP0, RPL3, RP L18, RACK1, PYGB, PPP2CB, PPIB, PLTP, PHB2, NOSTRIN, NARS1, MAN1A1, LTA4H, JAKMIP3, ITGA2, HSD17B11, HNRNPC, GOT2, GANAB, G6PD, ERAP1, E EF1G, DOP1B, DHRS9, COPB2, ATP5MF, ATP2A2, ATIC, ARCN1, APRT, APIP, AP1S1, ALDH9A1, ADAM10, ACSL5, ACLY, TKT, SERPINA6, RPL14, PARP4, MFGE8, MAPK1, HSP90B1, GPLD1, DDC, C8B, C7, ATP5F1A, ACAA2, LCP1, ANXA1, OLFM4, FBLN1, COL6A1, TGFBI, and at least one selected from the group consisting of THBS1.

7. The method of claim 6, wherein the gastrointestinal disease-related protein is at least one selected from the group consisting of OLFM4, LGALS3BP, LAMP1, ATP5F1A, DLD, ENPP4, MAPK1, PCYOX1, SMPD3, VILL, and RPS7.

8. The method of claim 6, wherein the gastrointestinal disease is an intestinal disease.

9. The method of claim 6, wherein the gastrointestinal disease is colon cancer.

10. The method of claim 6, further comprising a step of quantifying the detected gastrointestinal disease-related protein.

11. A detection reagent for the gastrointestinal disease-related protein, for use in the method according to any one of claims 1 to 10.

12. A kit comprising a detection reagent for detecting the gastrointestinal disease-related protein for use in the method according to any one of claims 1 to 10.

13. A method for screening diagnostic markers for gastrointestinal diseases, comprising: isolating fecal exosomes from stool; detecting proteins from the separated fecal exosomes; and selecting candidate diagnostic markers for gastrointestinal diseases from the detected proteins using as an indicator whether the detected proteins satisfy any one of the following conditions (a) and (b): (a) the content in the total amount of the fecal exosomes is 1.5 times or more the content in the total amount of the feces; or (b) the proteins are detected only in the separated fecal exosomes and are not substantially detected in the feces.

14. The screening method according to claim 13, comprising carrying out a step of isolating fecal exosomes from the stool of colorectal cancer patients and a step of detecting proteins from the separated fecal exosomes in each of colorectal cancer patients and healthy individuals, and selecting the candidate diagnostic marker for gastrointestinal disease from the detected proteins using as an index whether the detected proteins satisfy any of the following conditions (c) and (d): (c) in colorectal cancer patients, either of conditions (a) and (b) is satisfied; (d) in healthy individuals, either of conditions (a) and (b) is satisfied.

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