Method for determining immune activity and device for determining immune activity

Abstract

Disclosed is a method for determining immune activity of a subject, the method comprising a determination step for determining immune activity of the subject on the basis of the amount of at least one biomarker in urine collected from the subject.

Description

Method for determining immune activity, and apparatus for determining immune activity

[0001] The present invention relates to a method for determining immunoactivity and an apparatus for determining immunoactivity.

[0002] The immune system is a biological defense mechanism that prevents foreign substances from entering the body and detects and eliminates foreign substances that have entered the body, playing a very important role in maintaining health. In human immune activity, the activity of immune cells, especially plasmacytoid dendritic cells (pDCs, also called "plasmacytoid dendritic cells"), is known to be important, and it is known that the activity of pDCs in subjects such as humans can be determined, for example, by measuring the number of pDCs that produce IFN-α using the subject's blood (Non-Patent Literature 1).

[0003] Clemence Ngo et al., “The role of plasmacytoid dendritic cells(pDCs) in immunity during viral infections and beyond” Cell Mol. Immunol., volume 21, p1008-1035 (2024).

[0004] As mentioned above, while there are methods for determining immune activity using blood samples, these require blood collection and measurement equipment, making them relatively invasive, not simple, and costly, and therefore not suitable for everyday use. On the other hand, there is a need for a simple method to determine immune activity for purposes such as maintaining health.

[0005] Therefore, the present invention aims to provide a simple method for determining immune activity. The present invention also aims to provide a device for simply determining immune activity.

[0006] As a result of diligent research, the inventors have found a correlation between pDC activity and the amount of a specific biomarker in urine. This invention is based on this novel finding.

[0007] In other words, the present invention relates to the following inventions: [1] A method for determining the immune activity of a subject, comprising a determination step of determining the immune activity of the subject based on the amount of at least one biomarker in the urine collected from the subject. [2] The method according to [1], wherein the biomarker comprises a protein and / or miRNA.[3] The above proteins include IgA, RAB21, Nectin 4, PD-L2, SARP-2, LIRA5, RWDD1, CI061, Caspase 4, DAB2, Adrenomedullin, IMP2L, SOD3, LG3BP, IL-6 soluble receptor α chain, MYO6, Gelsolin, NALD2, FCGRN, TCP2L, RAC1, AIF1L, F AAA, PSME2, ​​RAB5B, SH3G2, NCAM-L1, MAG, Radixin, Arylsulfatase A, DHR11, Glyoxalase I, BLVRB, BMPR1A, I5P1, ASB9, Cathepsin H, MXRA8:ECD, STK4, LACB2, Ceruloplasmin, CHSP1, FLRT3:ECD, LTB4DH, NPTX2, GLO2, RHG30, VILI, GNPTG, RAB1B, 14-3-3 Protein β / α, 14-3-3 Protein γ, Type III Collagen, SCG3, BGAL, 14-3-3 Protein ε, Calcifosin, DHPR, NRP1, Glutaminilcyclase, FCN2, Cdc42, SDCB2, ACY3 GSTA2, RB11B, Testican 2, IF1AY, MITD1, RAB8B, RCN1, Lysosomal acid phosphatase, Thioredoxin reductase 1, IL-17 soluble receptor, PROSC, RB11A, AN32B, Paraoxonase 2, SPT46, SIA10, UBE2N / UBE2V2 complex, Albumin, IKB The method according to [2], wherein at least one selected from the group consisting of ε, GALNS, VATC2, NCF-2, NUBP1, SDHB, CREB-binding protein, SHPS1, ARL5B, PHS, C1GLC, EphB6, PAFAH β subunit, proteasome α5 subunit, RBP-III, 14-3-3 protein ζ / δ, MTL26, RNase 2, PHIPL, UBE2K, SAP, cargranulin A, TIMD3, ferritin, ferritin light chain, PPIC, S100A12, PACN2, ALG2, annexin V, IF5A2, HSP 70, and hCG.[4] The above protein is at least one selected from the group consisting of adrenomedullin, IMP2L, IgA, TCP2L, NALD2, caspase 4, NCF-2, CHSP1, RHG30, NPTX2, lysosomal acid phosphatase, ARL5B, AN32B, SDHB, ceruloplasmin, NUBP1, STK4, albumin, S100A12, CREB-binding protein, cargranulin A, SPT46, paraoxonase 2, proteasome α5 subunit, RNase 2, annexin V, PAFAH β subunit, IF5A2, IKB ε, ALG2, and hCG, RAB21, Nectin 4, CI061, SARP-2, LIRA5, NCAM-L1, 14-3-3 protein ε, MYO6, RAB1B, PD-L2, RAB5B, LTB4DH, Gelsolin, SOD3, SH3G2, BMPR1A, DHR11, VILI, RWDD1, IL-17 soluble receptor, LG3BP, MAG, FAAA, PSME2, ​​FCGRN, DAB2, Arylsulfatase A, AIF1L, Glyoxalase I, SHPS1, IL-6 soluble receptor α chain, BLVRB, DHPR, MXRA8:ECD, 14-3-3 protein γ, RCN1, I5P1, P HIPL, FLRT3:ECD, SCG3, Type III collagen, Glutaminyl cyclase, VATC2, ASB9, Cdc42, 14-3-3 protein β / α, Cathepsin H, Calcifosin, GLO2, RAC1, Testican 2, Radixin, FCN2, BGAL, SDCB2, RAB8B, UBE2K, PHS, LACB2, PROSC, IF1AY, GNPTG, ACY3, SIA10, Thioredoxin reductase 1, UBE2N / UBE2V2 complex, MITD1, RB11B, NRP1, EphB6, GALNS, GSTA2, RB11A, C1GLC, HSP The method according to [2] or [3], wherein at least one protein is selected from the group consisting of 70, 14-3-3 protein ζ / δ, SAP, ferritin light chain, ferritin, TIMD3, PACN2, MTL26, RBP-III, and PPIC. [5] The method according to any one of [2] to [4], wherein the above proteins are one to four types. [6] The method according to any one of [2] to [5], wherein the above proteins are two to four types.[7] The method according to any one of [2] to [6], wherein the protein is one of two types: AN32B, SDHB, SPT46, or STK4 and RAB21, one of two types: TCP2L and radixin, one of two types: caspase 4 and DHR11, one of two types: IgA and SARP-2, one of two types: IgA and RWDD1, or one of two types: IgA and HSP70. [8] The above proteins include four types: SOD3, SARP-2, AN32B, and RAB21; four types: SOD3, SARP-2, AN32B, and PHIPL; four types: SOD3, IgA, SARP-2, and RAB1B; four types: S100A12, SOD3, IgA, and SARP-2; four types: SARP-2, IgA, SOD3, and MTL26; four types: IgA, SARP-2, SOD3, and SHPS1; and IgA, SARP-2, and RNase. The method according to any one of [2] to [6], wherein the four types are 2 and SOD3, the four types are IgA, SARP-2, ferritin light chain and SOD3, the four types are MYO6, SOD3, IgA and SARP-2, the four types are SHPS1, SARP-2, adrenomedullin and IgA, or the four types are caspase 4, DHR11, Cdc42 and IgA.[9] The above miRNAs are hsa-miR-151a-5p, hsa-miR-99b-5p, hsa-miR-200b-3p, hsa-miR-99 a-5p, hsa-miR-125b-5p, ENST00000531977.1, hsa-let-7c-5p, ENST000005165 07.1, hsa-miR-30c-5p, hsa-miR-200c-3p, hsa-miR-204-5p, hsa-miR-103a-3p , hsa-miR-125a-5p, hsa-miR-29a-3p, ENST00000568314.1, ENST00000563103. The method according to any one of [2] to [8], wherein at least one selected from the group consisting of hsa-miR-30b-5p, hsa-miR-151a-3p, hsa-miR-92a-3p, hsa-miR-182-5p, hsa-miR-22-3p, hsa-miR-100-5p, hsa-miR-23b-3p, ENST00000658801.1, hsa-miR-9-5p, ENST00000655322.1, hsa-miR-141-3p, hsa-miR-191-5p, ENST00000630360.1, and hsa-miR-30c-2-3p.

[10] The method according to any one of [2] to [9], wherein the miRNA is at least one selected from the group consisting of hsa-miR-151a-5p, hsa-miR-99b-5p, and hsa-miR-200b-3p.

[11] The method according to any one of [2] to

[10] , wherein the miRNA is one to eight types.

[12] The method according to any one of [2] to

[11] , wherein the miRNA is two to eight types.

[13] The method according to any one of [2] to

[12] , wherein the miRNA is three to eight types.

[14] The method according to any one of [2] to

[13] , wherein the miRNA is four to eight types.

[15] The method according to any one of [2] to

[12] , wherein the miRNA is one of two types: hsa-miR-29a-3p and hsa-miR-200c-3p, one of two types: hsa-miR-30b-5p and hsa-miR-22-3p, one of two types: hsa-miR-141-3p and hsa-miR-30b-5p, or one of two types: hsa-miR-182-5p and hsa-miR-29a-3p.

[16] The above miRNAs are three types: hsa-miR-141-3p, hsa-miR-30b-5p and hsa-miR-22-3p; three types: hsa-miR-141-3p, hsa-miR-30b-5p and hsa-miR-103a-3p; three types: hsa-miR-182-5p, hsa-miR-29a-3p and hsa-miR-200c-3p; hsa-miR-141-3p, hsa-miR-30b-5p and hs The method according to any one of [2] to

[13] , wherein the three types are a-miR-200c-3p, the three types are hsa-miR-29a-3p, hsa-miR-200c-3p and hsa-miR-22-3p, the three types are hsa-miR-182-5p, hsa-miR-30b-5p and hsa-miR-22-3p, or the three types are hsa-miR-30c-2-3p, hsa-miR-200c-3p and hsa-miR-22-3p.

[17] The above miRNAs are four types: hsa-miR-141-3p, hsa-miR-30c-5p, hsa-miR-200c-3p and hsa-miR-22-3p; four types: hsa-miR-30b-5p, hsa-miR-29a-3p, hsa-miR-200c-3p and hsa-miR-22-3p; four types: hsa-miR-141-3p, hsa-miR-30b-5p, hsa-miR-200c-3p and hsa-miR-22-3p; and hsa-miR-14 The method according to any one of [2] to

[14] , wherein the four types are 1-3p, hsa-miR-30b-5p, hsa-miR-200c-3p and hsa-miR-103a-3p, the four types are hsa-miR-141-3p, hsa-miR-30b-5p, hsa-miR-103a-3p and hsa-miR-22-3p, or the four types are hsa-miR-182-5p, hsa-miR-29a-3p, hsa-miR-200c-3p and hsa-miR-103a-3p.

[18] The method according to any one of [2] to

[14] , wherein the miRNA is one of eight types: hsa-miR-200c-3p, hsa-miR-141-3p, hsa-miR-103a-3p, hsa-miR-30c-5p, hsa-miR-29a-3p, hsa-miR-30b-5p, hsa-miR-182-5p, and hsa-miR-22-3p.

[19] The method according to any one of [1] to

[18] , wherein the biomarker is IgA.

[20] The method according to any one of [1] to

[19] , wherein the determination step is at least one of the following (A) to (D). (A): Determining that the immune activity of the subject is high if the value of a parameter based on the amount of the above biomarker is higher than a predetermined threshold. (B): Determining that the immune activity of the subject is low if the value of a parameter based on the amount of the above biomarker is lower than a predetermined threshold. (C): Determining that the immune activity of the subject is low if the value of a parameter based on the amount of the above biomarker is higher than a predetermined threshold. (D): Determining that the immune activity of the subject is high if the value of a parameter based on the amount of the above biomarker is lower than a predetermined threshold.

[21] The method according to any one of [1] to

[20] , wherein the immune activity is the activity of plasmacytoid dendritic cells.

[22] A method for supporting the improvement or maintenance of immune activity, comprising: a determination step of determining immune activity by the method according to any one of [1] to

[21] ; and a determination result presentation step of presenting a report showing the determination result obtained in the determination step.

[23] The method according to

[22] , further comprising: a specification step of identifying an action for improving or maintaining immune activity in accordance with the determination result obtained in the determination step; and a specification result presentation step of presenting a report showing the result identified in the specification step.

[24] A reagent or reagent kit for use in the method of any one of [1] to

[21] , comprising a substance capable of specifically detecting the above-mentioned biomarker in urine.

[25] A method for evaluating the immunostimulatory activity of a test substance, comprising an evaluation step of evaluating the immunostimulatory activity of the test substance based on the amount of at least one biomarker in urine collected from a subject who has ingested the test substance.

[26] The method according to

[25] , wherein the above biomarker comprises a protein and / or miRNA.

[27] The above protein is IgA, RAB21, Nectin 4, PD-L2, SARP-2, LIRA5, RWDD1, CI061, Caspase 4, DAB2, Adrenomedullin, IMP2L, SOD3, LG3BP, IL-6 soluble receptor α chain, MYO6, Gelsolin, NALD2, FCGRN, TCP2L, RAC1, AIF1L, F AAA, PSME2, ​​RAB5B, SH3G2, NCAM-L1, MAG, Radixin, Arylsulfatase A, DHR11, Glyoxalase I, BLVRB, BMPR1A, I5P1, ASB9, Cathepsin H, MXRA8:ECD, STK4, LACB2, Ceruloplasmin, CHSP1, FLRT3:ECD, LTB4DH, NPTX2, GLO2, RHG30, VILI, GNPTG, RAB1B, 14-3-3 Protein β / α, 14-3-3 Protein γ, Type III Collagen, SCG3, BGAL, 14-3-3 Protein ε, Calcifosin, DHPR, NRP1, Glutaminilcyclase, FCN2, Cdc42, SDCB2, ACY3 GSTA2, RB11B, Testican 2, IF1AY, MITD1, RAB8B, RCN1, Lysosomal acid phosphatase, Thioredoxin reductase 1, IL-17 soluble receptor, PROSC, RB11A, AN32B, Paraoxonase 2, SPT46, SIA10, UBE2N / UBE2V2 complex, Albumin, IKB The method according to

[26] , wherein at least one selected from the group consisting of ε, GALNS, VATC2, NCF-2, NUBP1, SDHB, CREB-binding protein, SHPS1, ARL5B, PHS, C1GLC, EphB6, PAFAH β subunit, proteasome α5 subunit, RBP-III, 14-3-3 protein ζ / δ, MTL26, RNase 2, PHIPL, UBE2K, SAP, cargranulin A, TIMD3, ferritin, ferritin light chain, PPIC, S100A12, PACN2, ALG2, annexin V, IF5A2, HSP 70, and hCG.

[28] The above protein is at least one selected from the group consisting of adrenomedullin, IMP2L, IgA, TCP2L, NALD2, caspase 4, NCF-2, CHSP1, RHG30, NPTX2, lysosomal acid phosphatase, ARL5B, AN32B, SDHB, ceruloplasmin, NUBP1, STK4, albumin, S100A12, CREB-binding protein, cargranulin A, SPT46, paraoxonase 2, proteasome α5 subunit, RNase 2, annexin V, PAFAH β subunit, IF5A2, IKB ε, ALG2, and hCG, RAB21, Nectin 4, CI061, SARP-2, LIRA5, NCAM-L1, 14-3-3 protein ε, MYO6, RAB1B, PD-L2, RAB5B, LTB4DH, Gelsolin, SOD3, SH3G2, BMPR1A, DHR11, VILI, RWDD1, IL-17 soluble receptor, LG3BP, MAG, FAAA, PSME2, ​​FCGRN, DAB2, Arylsulfatase A, AIF1L, Glyoxalase I, SHPS1, IL-6 soluble receptor α chain, BLVRB, DHPR, MXRA8:ECD, 14-3-3 protein γ, RCN1, I5P1, P HIPL, FLRT3:ECD, SCG3, Type III collagen, Glutaminyl cyclase, VATC2, ASB9, Cdc42, 14-3-3 protein β / α, Cathepsin H, Calcifosin, GLO2, RAC1, Testican 2, Radixin, FCN2, BGAL, SDCB2, RAB8B, UBE2K, PHS, LACB2, PROSC, IF1AY, GNPTG, ACY3, SIA10, Thioredoxin reductase 1, UBE2N / UBE2V2 complex, MITD1, RB11B, NRP1, EphB6, GALNS, GSTA2, RB11A, C1GLC, HSP 70. The method according to

[26] or

[27] , wherein at least one selected from the group consisting of 14-3-3 protein ζ / δ, SAP, ferritin light chain, ferritin, TIMD3, PACN2, MTL26, RBP-III, and PPIC.

[29] The method according to any one of

[26] to

[28] , wherein one to four of the above proteins.

[30] The method according to any one of

[26] to

[29] , wherein the above proteins are two or more and four or less.

[31] The method according to any one of

[26] to

[30] , wherein the above proteins are two types of AN32B, SDHB, SPT46 or STK4 and RAB21, two types of TCP2L and radixin, two types of caspase 4 and DHR11, two types of IgA and SARP-2, two types of IgA and RWDD1, or two types of IgA and HSP70.

[32] The above proteins include four types: SOD3, SARP-2, AN32B, and RAB21; four types: SOD3, SARP-2, AN32B, and PHIPL; four types: SOD3, IgA, SARP-2, and RAB1B; four types: S100A12, SOD3, IgA, and SARP-2; four types: SARP-2, IgA, SOD3, and MTL26; four types: IgA, SARP-2, SOD3, and SHPS1; and IgA, SARP-2, and RNase. The method according to any one of

[26] to

[30] , wherein the four types are 2 and SOD3, the four types are IgA, SARP-2, ferritin light chain and SOD3, the four types are MYO6, SOD3, IgA and SARP-2, the four types are SHPS1, SARP-2, adrenomedullin and IgA, or the four types are caspase 4, DHR11, Cdc42 and IgA.

[33] The above miRNAs are hsa-miR-151a-5p, hsa-miR-99b-5p, hsa-miR-200b-3p, hsa-miR-99 a-5p, hsa-miR-125b-5p, ENST00000531977.1, hsa-let-7c-5p, ENST000005165 07.1, hsa-miR-30c-5p, hsa-miR-200c-3p, hsa-miR-204-5p, hsa-miR-103a-3p , hsa-miR-125a-5p, hsa-miR-29a-3p, ENST00000568314.1, ENST00000563103.1 The method according to any one of

[26] to

[32] , wherein at least one selected from the group consisting of hsa-miR-30b-5p, hsa-miR-151a-3p, hsa-miR-92a-3p, hsa-miR-182-5p, hsa-miR-22-3p, hsa-miR-100-5p, hsa-miR-23b-3p, ENST00000658801.1, hsa-miR-9-5p, ENST00000655322.1, hsa-miR-141-3p, hsa-miR-191-5p, ENST00000630360.1, and hsa-miR-30c-2-3p.

[34] The method according to any one of

[26] to

[33] , wherein the miRNA is at least one selected from the group consisting of hsa-miR-151a-5p, hsa-miR-99b-5p, and hsa-miR-200b-3p.

[35] The method according to any one of

[26] to

[34] , wherein the miRNA is one to eight types.

[36] The method according to any one of

[26] to

[35] , wherein the miRNA is two to eight types.

[37] The method according to any one of

[26] to

[36] , wherein the miRNA is three to eight types.

[38] The method according to any one of

[26] to

[37] , wherein the miRNA is four to eight types.

[39] The method according to any one of

[26] to

[36] , wherein the miRNA is one of two types: hsa-miR-29a-3p and hsa-miR-200c-3p, one of two types: hsa-miR-30b-5p and hsa-miR-22-3p, one of two types: hsa-miR-141-3p and hsa-miR-30b-5p, or one of two types: hsa-miR-182-5p and hsa-miR-29a-3p.

[40] The above miRNAs are three types: hsa-miR-141-3p, hsa-miR-30b-5p and hsa-miR-22-3p; three types: hsa-miR-141-3p, hsa-miR-30b-5p and hsa-miR-103a-3p; three types: hsa-miR-182-5p, hsa-miR-29a-3p and hsa-miR-200c-3p; hsa-miR-141-3p, hsa-miR-30b-5p and hs The method according to any one of

[26] to

[37] , wherein the three types are a-miR-200c-3p, the three types are hsa-miR-29a-3p, hsa-miR-200c-3p and hsa-miR-22-3p, the three types are hsa-miR-182-5p, hsa-miR-30b-5p and hsa-miR-22-3p, or the three types are hsa-miR-30c-2-3p, hsa-miR-200c-3p and hsa-miR-22-3p.

[41] The above miRNAs are four types: hsa-miR-141-3p, hsa-miR-30c-5p, hsa-miR-200c-3p and hsa-miR-22-3p; four types: hsa-miR-30b-5p, hsa-miR-29a-3p, hsa-miR-200c-3p and hsa-miR-22-3p; four types: hsa-miR-141-3p, hsa-miR-30b-5p, hsa-miR-200c-3p and hsa-miR-22-3p; and hsa-miR-14 The method according to any one of

[26] to

[38] , wherein the four types are 1-3p, hsa-miR-30b-5p, hsa-miR-200c-3p and hsa-miR-103a-3p, the four types are hsa-miR-141-3p, hsa-miR-30b-5p, hsa-miR-103a-3p and hsa-miR-22-3p, or the four types are hsa-miR-182-5p, hsa-miR-29a-3p, hsa-miR-200c-3p and hsa-miR-103a-3p.

[42] The method according to any one of

[26] to

[38] , wherein the miRNA is one of eight types: hsa-miR-200c-3p, hsa-miR-141-3p, hsa-miR-103a-3p, hsa-miR-30c-5p, hsa-miR-29a-3p, hsa-miR-30b-5p, hsa-miR-182-5p, and hsa-miR-22-3p.

[43] The method according to any one of

[25] to

[42] , wherein the biomarker is IgA.

[44] An apparatus for determining immune activity, comprising a computer including a processor and a memory under the control of the processor, wherein the memory contains a computer program for causing the computer to perform: A) a determination step of determining the immune activity of a subject based on the amount of at least one biomarker in urine collected from the subject; and B) an output step of outputting the determination result of the immune activity of the subject.

[45] The apparatus according to

[44] , wherein the biomarker comprises a protein and / or miRNA.

[46] The above proteins include IgA, RAB21, Nectin 4, PD-L2, SARP-2, LIRA5, RWDD1, CI061, Caspase 4, DAB2, Adrenomedullin, IMP2L, SOD3, LG3BP, IL-6 soluble receptor α chain, MYO6, Gelsolin, NALD2, FCGRN, TCP2L, RAC1, AIF1L, F AAA, PSME2, ​​RAB5B, SH3G2, NCAM-L1, MAG, Radixin, Arylsulfatase A, DHR11, Glyoxalase I, BLVRB, BMPR1A, I5P1, ASB9, Cathepsin H, MXRA8:ECD, STK4, LACB2, Ceruloplasmin, CHSP1, FLRT3:ECD, LTB4DH, NPTX2, GLO2, RHG30, VILI, GNPTG, RAB1B, 14-3-3 Protein β / α, 14-3-3 Protein γ, Type III Collagen, SCG3, BGAL, 14-3-3 Protein ε, Calcifosin, DHPR, NRP1, Glutaminilcyclase, FCN2, Cdc42, SDCB2, ACY3 GSTA2, RB11B, Testican 2, IF1AY, MITD1, RAB8B, RCN1, Lysosomal acid phosphatase, Thioredoxin reductase 1, IL-17 soluble receptor, PROSC, RB11A, AN32B, Paraoxonase 2, SPT46, SIA10, UBE2N / UBE2V2 complex, Albumin, IKB The apparatus according to

[45] , wherein at least one selected from the group consisting of ε, GALNS, VATC2, NCF-2, NUBP1, SDHB, CREB-binding protein, SHPS1, ARL5B, PHS, C1GLC, EphB6, PAFAH β subunit, proteasome α5 subunit, RBP-III, 14-3-3 protein ζ / δ, MTL26, RNase 2, PHIPL, UBE2K, SAP, cargranulin A, TIMD3, ferritin, ferritin light chain, PPIC, S100A12, PACN2, ALG2, annexin V, IF5A2, HSP 70, and hCG.

[47] The above protein is at least one selected from the group consisting of adrenomedullin, IMP2L, IgA, TCP2L, NALD2, caspase 4, NCF-2, CHSP1, RHG30, NPTX2, lysosomal acid phosphatase, ARL5B, AN32B, SDHB, ceruloplasmin, NUBP1, STK4, albumin, S100A12, CREB-binding protein, cargranulin A, SPT46, paraoxonase 2, proteasome α5 subunit, RNase 2, annexin V, PAFAH β subunit, IF5A2, IKB ε, ALG2, and hCG, RAB21, Nectin 4, CI061, SARP-2, LIRA5, NCAM-L1, 14-3-3 protein ε, MYO6, RAB1B, PD-L2, RAB5B, LTB4DH, Gelsolin, SOD3, SH3G2, BMPR1A, DHR11, VILI, RWDD1, IL-17 soluble receptor, LG3BP, MAG, FAAA, PSME2, ​​FCGRN, DAB2, Arylsulfatase A, AIF1L, Glyoxalase I, SHPS1, IL-6 soluble receptor α chain, BLVRB, DHPR, MXRA8:ECD, 14-3-3 protein γ, RCN1, I5P1, P HIPL, FLRT3:ECD, SCG3, Type III collagen, Glutaminyl cyclase, VATC2, ASB9, Cdc42, 14-3-3 protein β / α, Cathepsin H, Calcifosin, GLO2, RAC1, Testican 2, Radixin, FCN2, BGAL, SDCB2, RAB8B, UBE2K, PHS, LACB2, PROSC, IF1AY, GNPTG, ACY3, SIA10, Thioredoxin reductase 1, UBE2N / UBE2V2 complex, MITD1, RB11B, NRP1, EphB6, GALNS, GSTA2, RB11A, C1GLC, HSP 70. The apparatus according to

[45] or

[46] , wherein the protein is at least one selected from the group consisting of 14-3-3 protein ζ / δ, SAP, ferritin light chain, ferritin, TIMD3, PACN2, MTL26, RBP-III, and PPIC.

[48] The apparatus according to any one of

[45] to

[47] , wherein the protein is one to four types.

[49] The apparatus according to any one of

[45] to

[48] , wherein the above proteins are two or more and four or less.

[50] The apparatus according to any one of

[45] to

[49] , wherein the above proteins are two types of AN32B, SDHB, SPT46 or STK4 and RAB21, two types of TCP2L and radixin, two types of caspase 4 and DHR11, two types of IgA and SARP-2, two types of IgA and RWDD1, or two types of IgA and HSP70.

[51] The above proteins include four types: SOD3, SARP-2, AN32B, and RAB21; four types: SOD3, SARP-2, AN32B, and PHIPL; four types: SOD3, IgA, SARP-2, and RAB1B; four types: S100A12, SOD3, IgA, and SARP-2; four types: SARP-2, IgA, SOD3, and MTL26; four types: IgA, SARP-2, SOD3, and SHPS1; and IgA, SARP-2, and RNase. The apparatus according to any one of

[45] to

[49] , wherein the apparatus consists of four types: 2 and SOD3, four types: IgA, SARP-2, ferritin light chain and SOD3, four types: MYO6, SOD3, IgA and SARP-2, four types: SHPS1, SARP-2, adrenomedullin and IgA, or four types: caspase 4, DHR11, Cdc42 and IgA.

[52] The above miRNAs are hsa-miR-151a-5p, hsa-miR-99b-5p, hsa-miR-200b-3p, hsa-miR-99 a-5p, hsa-miR-125b-5p, ENST00000531977.1, hsa-let-7c-5p, ENST000005165 07.1, hsa-miR-30c-5p, hsa-miR-200c-3p, hsa-miR-204-5p, hsa-miR-103a-3p , hsa-miR-125a-5p, hsa-miR-29a-3p, ENST00000568314.1, ENST00000563103.1 The apparatus according to any one of

[45] to

[51] , wherein at least one selected from the group consisting of hsa-miR-30b-5p, hsa-miR-151a-3p, hsa-miR-92a-3p, hsa-miR-182-5p, hsa-miR-22-3p, hsa-miR-100-5p, hsa-miR-23b-3p, ENST00000658801.1, hsa-miR-9-5p, ENST00000655322.1, hsa-miR-141-3p, hsa-miR-191-5p, ENST00000630360.1, and hsa-miR-30c-2-3p.

[53] The apparatus according to any one of

[45] to

[52] , wherein the miRNA is at least one selected from the group consisting of hsa-miR-151a-5p, hsa-miR-99b-5p, and hsa-miR-200b-3p.

[54] The apparatus according to any one of

[45] to

[53] , wherein the miRNA is one to eight types.

[55] The apparatus according to any one of

[45] to

[54] , wherein the miRNA is two to eight types.

[56] The apparatus according to any one of

[45] to

[55] , wherein the miRNA is three to eight types.

[57] The apparatus according to any one of

[45] to

[56] , wherein the miRNA is four to eight types.

[58] The apparatus according to any one of

[45] to

[55] , wherein the miRNA is one of two types: hsa-miR-29a-3p and hsa-miR-200c-3p, one of two types: hsa-miR-30b-5p and hsa-miR-22-3p, one of two types: hsa-miR-141-3p and hsa-miR-30b-5p, or one of two types: hsa-miR-182-5p and hsa-miR-29a-3p.

[59] The above miRNAs are three types: hsa-miR-141-3p, hsa-miR-30b-5p and hsa-miR-22-3p; three types: hsa-miR-141-3p, hsa-miR-30b-5p and hsa-miR-103a-3p; three types: hsa-miR-182-5p, hsa-miR-29a-3p and hsa-miR-200c-3p; hsa-miR-141-3p, hsa-miR-30b-5p and hs The apparatus according to any one of

[45] to

[56] , which includes three types of a-miR-200c-3p, three types of hsa-miR-29a-3p, hsa-miR-200c-3p and hsa-miR-22-3p, three types of hsa-miR-182-5p, hsa-miR-30b-5p and hsa-miR-22-3p, or three types of hsa-miR-30c-2-3p, hsa-miR-200c-3p and hsa-miR-22-3p.

[60] The above miRNAs are four types: hsa-miR-141-3p, hsa-miR-30c-5p, hsa-miR-200c-3p and hsa-miR-22-3p; four types: hsa-miR-30b-5p, hsa-miR-29a-3p, hsa-miR-200c-3p and hsa-miR-22-3p; four types: hsa-miR-141-3p, hsa-miR-30b-5p, hsa-miR-200c-3p and hsa-miR-22-3p; and hsa-miR-14 The apparatus according to any one of

[45] to

[57] , wherein the four types are 1-3p, hsa-miR-30b-5p, hsa-miR-200c-3p and hsa-miR-103a-3p, the four types are hsa-miR-141-3p, hsa-miR-30b-5p, hsa-miR-103a-3p and hsa-miR-22-3p, or the four types are hsa-miR-182-5p, hsa-miR-29a-3p, hsa-miR-200c-3p and hsa-miR-103a-3p.

[61] The apparatus according to any one of

[45] to

[57] , wherein the miRNA is one of eight types: hsa-miR-200c-3p, hsa-miR-141-3p, hsa-miR-103a-3p, hsa-miR-30c-5p, hsa-miR-29a-3p, hsa-miR-30b-5p, hsa-miR-182-5p, and hsa-miR-22-3p.

[62] The apparatus according to any one of

[45] to

[61] , wherein the biomarker is IgA.

[63] The apparatus according to any one of

[44] to

[62] , wherein the determination step is at least one of the following (A) to (D). (A): Determining that the immune activity of the subject is high if the value of a parameter based on the amount of the above biomarker is higher than a predetermined threshold. (B): Determining that the immune activity of the subject is low if the value of a parameter based on the amount of the above biomarker is lower than a predetermined threshold. (C): Determining that the immune activity of the subject is low if the value of a parameter based on the amount of the above biomarker is higher than a predetermined threshold. (D): Determining that the immune activity of the subject is high if the value of a parameter based on the amount of the above biomarker is lower than a predetermined threshold.

[64] A) A determination step of determining the immune activity of the subject based on the amount of at least one biomarker in the urine collected from the subject. B) An output step of outputting the determination result of the immune activity of the subject.

[65] The computer program according to

[64] , wherein the biomarker comprises a protein and / or miRNA.

[66] The above proteins include IgA, RAB21, Nectin 4, PD-L2, SARP-2, LIRA5, RWDD1, CI061, Caspase 4, DAB2, Adrenomedullin, IMP2L, SOD3, LG3BP, IL-6 soluble receptor α chain, MYO6, Gelsolin, NALD2, FCGRN, TCP2L, RAC1, AIF1L, F AAA, PSME2, ​​RAB5B, SH3G2, NCAM-L1, MAG, Radixin, Arylsulfatase A, DHR11, Glyoxalase I, BLVRB, BMPR1A, I5P1, ASB9, Cathepsin H, MXRA8:ECD, STK4, LACB2, Ceruloplasmin, CHSP1, FLRT3:ECD, LTB4DH, NPTX2, GLO2, RHG30, VILI, GNPTG, RAB1B, 14-3-3 Protein β / α, 14-3-3 Protein γ, Type III Collagen, SCG3, BGAL, 14-3-3 Protein ε, Calcifosin, DHPR, NRP1, Glutaminilcyclase, FCN2, Cdc42, SDCB2, ACY3 GSTA2, RB11B, Testican 2, IF1AY, MITD1, RAB8B, RCN1, Lysosomal acid phosphatase, Thioredoxin reductase 1, IL-17 soluble receptor, PROSC, RB11A, AN32B, Paraoxonase 2, SPT46, SIA10, UBE2N / UBE2V2 complex, Albumin, IKB The computer program according to

[65] , which is at least one selected from the group consisting of ε, GALNS, VATC2, NCF-2, NUBP1, SDHB, CREB-binding protein, SHPS1, ARL5B, PHS, C1GLC, EphB6, PAFAH β subunit, proteasome α5 subunit, RBP-III, 14-3-3 protein ζ / δ, MTL26, RNase 2, PHIPL, UBE2K, SAP, cargranulin A, TIMD3, ferritin, ferritin light chain, PPIC, S100A12, PACN2, ALG2, annexin V, IF5A2, HSP 70, and hCG.

[67] The above protein is at least one selected from the group consisting of adrenomedullin, IMP2L, IgA, TCP2L, NALD2, caspase 4, NCF-2, CHSP1, RHG30, NPTX2, lysosomal acid phosphatase, ARL5B, AN32B, SDHB, ceruloplasmin, NUBP1, STK4, albumin, S100A12, CREB-binding protein, cargranulin A, SPT46, paraoxonase 2, proteasome α5 subunit, RNase 2, annexin V, PAFAH β subunit, IF5A2, IKB ε, ALG2, and hCG, RAB21, Nectin 4, CI061, SARP-2, LIRA5, NCAM-L1, 14-3-3 protein ε, MYO6, RAB1B, PD-L2, RAB5B, LTB4DH, Gelsolin, SOD3, SH3G2, BMPR1A, DHR11, VILI, RWDD1, IL-17 soluble receptor, LG3BP, MAG, FAAA, PSME2, ​​FCGRN, DAB2, Arylsulfatase A, AIF1L, Glyoxalase I, SHPS1, IL-6 soluble receptor α chain, BLVRB, DHPR, MXRA8:ECD, 14-3-3 protein γ, RCN1, I5P1, P HIPL, FLRT3:ECD, SCG3, Type III collagen, Glutaminyl cyclase, VATC2, ASB9, Cdc42, 14-3-3 protein β / α, Cathepsin H, Calcifosin, GLO2, RAC1, Testican 2, Radixin, FCN2, BGAL, SDCB2, RAB8B, UBE2K, PHS, LACB2, PROSC, IF1AY, GNPTG, ACY3, SIA10, Thioredoxin reductase 1, UBE2N / UBE2V2 complex, MITD1, RB11B, NRP1, EphB6, GALNS, GSTA2, RB11A, C1GLC, HSP 70. A computer program according to

[65] or

[66] , wherein the computer program is at least one selected from the group consisting of 14-3-3 protein ζ / δ, SAP, ferritin light chain, ferritin, TIMD3, PACN2, MTL26, RBP-III, and PPIC.

[68] A computer program according to any one of

[65] to

[67] , wherein the above proteins are one to four types.

[69] A computer program according to any one of

[65] to

[68] , wherein the above proteins are two or more but not more than four types.

[70] A computer program according to any one of

[65] to

[69] , wherein the above proteins are two types of AN32B, SDHB, SPT46 or STK4 and RAB21, two types of TCP2L and radixin, two types of caspase 4 and DHR11, two types of IgA and SARP-2, two types of IgA and RWDD1, or two types of IgA and HSP70.

[71] The above proteins include four types: SOD3, SARP-2, AN32B, and RAB21; four types: SOD3, SARP-2, AN32B, and PHIPL; four types: SOD3, IgA, SARP-2, and RAB1B; four types: S100A12, SOD3, IgA, and SARP-2; four types: SARP-2, IgA, SOD3, and MTL26; four types: IgA, SARP-2, SOD3, and SHPS1; and IgA, SARP-2, and RNase. A computer program according to any one of

[65] to

[69] , comprising four types of 2 and SOD3, four types of IgA, SARP-2, ferritin light chain and SOD3, four types of MYO6, SOD3, IgA and SARP-2, four types of SHPS1, SARP-2, adrenomedullin and IgA, or four types of caspase 4, DHR11, Cdc42 and IgA.

[72] The above miRNAs are hsa-miR-151a-5p, hsa-miR-99b-5p, hsa-miR-200b-3p, hsa-miR-99a -5p, hsa-miR-125b-5p, ENST00000531977.1, hsa-let-7c-5p, ENST00000516507 .. 1, hsa-miR-30c-5p, hsa-miR-200c-3p, hsa-miR-204-5p, hsa-miR-103a-3p, hsa -miR-125a-5p, hsa-miR-29a-3p, ENST00000568314.1, ENST00000563103.1, hsa A computer program according to any one of

[65] to

[71] , which is at least one selected from the group consisting of -miR-30b-5p, hsa-miR-151a-3p, hsa-miR-92a-3p, hsa-miR-182-5p, hsa-miR-22-3p, hsa-miR-100-5p, hsa-miR-23b-3p, ENST00000658801.1, hsa-miR-9-5p, ENST00000655322.1, hsa-miR-141-3p, hsa-miR-191-5p, ENST00000630360.1, and hsa-miR-30c-2-3p.

[73] A computer program according to any one of

[65] to

[72] , wherein the miRNA is at least one selected from the group consisting of hsa-miR-151a-5p, hsa-miR-99b-5p, and hsa-miR-200b-3p.

[74] A computer program according to any one of

[65] to

[73] , wherein the miRNA is one to eight types.

[75] A computer program according to any one of

[65] to

[74] , wherein the miRNA is two to eight types.

[76] A computer program according to any one of

[65] to

[75] , wherein the miRNA is three to eight types.

[77] A computer program according to any one of

[65] to

[76] , wherein the miRNA is four to eight types.

[78] A computer program according to any one of

[65] to

[75] , wherein the miRNAs are two types: hsa-miR-29a-3p and hsa-miR-200c-3p, two types: hsa-miR-30b-5p and hsa-miR-22-3p, two types: hsa-miR-141-3p and hsa-miR-30b-5p, or two types: hsa-miR-182-5p and hsa-miR-29a-3p.

[79] The above miRNAs are three types: hsa-miR-141-3p, hsa-miR-30b-5p and hsa-miR-22-3p, three types: hsa-miR-141-3p, hsa-miR-30b-5p and hsa-miR-103a-3p, three types: hsa-miR-182-5p, hsa-miR-29a-3p and hsa-miR-200c-3p, hsa-miR-141-3p, hsa-miR-30b-5p and hsa-miR A computer program as described in any of

[65] to

[76] , which is one of the following types: -200c-3p, hsa-miR-29a-3p, hsa-miR-200c-3p and hsa-miR-22-3p, one of the following types: hsa-miR-182-5p, hsa-miR-30b-5p and hsa-miR-22-3p, or one of the following types: hsa-miR-30c-2-3p, hsa-miR-200c-3p and hsa-miR-22-3p.

[80] The above miRNAs are four types: hsa-miR-141-3p, hsa-miR-30c-5p, hsa-miR-200c-3p and hsa-miR-22-3p, four types: hsa-miR-30b-5p, hsa-miR-29a-3p, hsa-miR-200c-3p and hsa-miR-22-3p, four types: hsa-miR-141-3p, hsa-miR-30b-5p, hsa-miR-200c-3p and hsa-miR-22-3p, hsa-miR-141-3p, A computer program as described in any of

[65] to

[77] , which is one of four types: hsa-miR-30b-5p, hsa-miR-200c-3p and hsa-miR-103a-3p; one of four types: hsa-miR-141-3p, hsa-miR-30b-5p, hsa-miR-103a-3p and hsa-miR-22-3p; or one of four types: hsa-miR-182-5p, hsa-miR-29a-3p, hsa-miR-200c-3p and hsa-miR-103a-3p.

[81] A computer program according to any one of

[65] to

[77] , wherein the miRNA is one of eight types: hsa-miR-200c-3p, hsa-miR-141-3p, hsa-miR-103a-3p, hsa-miR-30c-5p, hsa-miR-29a-3p, hsa-miR-30b-5p, hsa-miR-182-5p, and hsa-miR-22-3p.

[82] A computer program according to any one of

[64] to

[81] , wherein the biomarker is IgA.

[83] A computer program according to any one of

[64] to

[82] , wherein the determination step is at least one of the following (A) to (D).(A): If the value of the parameter based on the amount of the above biomarker is higher than a predetermined threshold, it is determined that the immune activity of the subject is high. (B): If the value of the parameter based on the amount of the above biomarker is lower than a predetermined threshold, it is determined that the immune activity of the subject is low. (C): If the value of the parameter based on the amount of the above biomarker is higher than a predetermined threshold, it is determined that the immune activity of the subject is low. (D): If the value of the parameter based on the amount of the above biomarker is lower than a predetermined threshold, it is determined that the immune activity of the subject is high.

[84] A method for screening a biomarker in a biological sample for determining immune activity, comprising: a first measurement step of measuring the immune activity of a subject; a second measurement step of measuring the amount of at least one biomarker in a biological sample taken from a subject; and a determination step of determining whether the biomarker is a useful biomarker for determining immune activity based on the correlation between the immune activity of the subject and the amount of the above biomarker.

[85] The method according to

[84] , wherein the immune activity is the activity of plasmacytoid dendritic cells.

[86] An immunostimulatory composition containing an immunostimulatory substance to be administered to a subject that was not determined to have high immune activity or low immune activity by the method of

[20] or

[21] .

[87] A method to assist in determining the immune activity of a subject, comprising a determination step of determining the immune activity of the subject based on the amount of at least one biomarker in the urine collected from the subject.

[88] An immunostimulatory method comprising administering an immunostimulatory composition containing an immunostimulatory substance to a subject that was determined to have low immune activity by the method of any one of [1] to

[21] and

[87] .

[89] An immunostimulatory substance for use in immunostimulation, for use in immunostimulation of a subject that was determined to have low immune activity by the method of any one of [1] to

[21] and

[87] .

[90] Use of an immunostimulatory substance for immunostimulation of a subject that was determined to have low immune activity by the method of any one of [1] to

[21] and

[87] .

[91] Use of a substance having immunostimulatory activity to produce an immunostimulatory composition for immunostimulating a subject whose immunoactivity has been determined to be low by any of the methods in [1] to

[21] and

[87] .

[92] A method for determining the immunoactivity of a subject using the amount of at least one biomarker in urine collected from the subject as an indicator for determining the immunoactivity of the subject.

[93] A method for collecting data for determining the immunoactivity of a subject, comprising a determination step of determining the immunoactivity of the subject based on the amount of at least one biomarker in urine collected from the subject.

[94] A method for determining the disease risk of a subject, comprising a determination step of determining the disease risk of the subject based on the amount of at least one biomarker in urine collected from the subject.

[95] A method for determining the disease risk of a subject, comprising a determination step of determining the disease risk of the subject based on the amount of at least one biomarker in urine collected from the subject.

[96] The method according to

[94] or

[95] , wherein the subject is a subject whose immunoactivity has been determined to be low by any of the methods in [9] to

[21] .

[97] The method according to any one of

[94] to

[96] , wherein the disease is at least one selected from the group consisting of cancer, endocrine disorders, metabolic disorders, infectious diseases, cardiovascular diseases, and neurodegenerative diseases.

[98] The method according to any one of

[94] to

[97] , wherein the infectious disease is at least one selected from the group consisting of bacterial, viral, and parasitic infections.

[0008] The present invention provides a simple method for determining immune activity. In particular, because it is a non-invasive method utilizing a urine sample, it can be widely used. The present invention also provides an apparatus for simply determining immune activity.

[0009] This is a schematic diagram of an immunoactivity determination device according to one embodiment. This is a schematic diagram showing the hardware configuration of the immunoactivity determination device according to one embodiment. This is a flowchart of the immunoactivity determination method according to one embodiment. This is a graph showing the results of comparing the relative amount of IgA in the urine of participants with high immunoactivity and the relative amount of IgA in the urine of participants with low immunoactivity in Test Example 2. This is a figure showing the results of predicting immunoactivity using IgA in urine as an indicator in Test Example 3.

[0010] The following describes in detail embodiments for carrying out the present invention. However, the present invention is not limited to the following embodiments.

[0011] [Method for determining immune activity] The method for determining the immune activity of a target according to this embodiment includes a determination step of determining the immune activity of the target based on the amount of at least one biomarker in the urine collected from the target.

[0012] Preferably, the amount of at least one biomarker in the urine collected from the subject is positively or negatively correlated with immune activity, and the level of immune activity of the subject can be determined based on the amount of at least one biomarker in the urine collected from the subject. This allows for a simple and non-invasive determination of immune activity.

[0013] The subjects may include mammals, for example, specifically humans, monkeys, mice, and rats, but are not limited to these.

[0014] In this specification, "immune activity" means the strength of the immune response ability of the innate and / or adaptive immune systems against foreign substances, and is also called immune function. From the viewpoint of representing the body's immune state, immune activity is preferably the activity of the innate immune system, and for example, it is preferably the activity of immune cells involved in the innate immune system. Immune cells of the innate immune system include, for example, eosinophils, neutrophils, basophils, macrophages, dendritic cells, and natural killer cells (NK cells), but among these, it is preferably the activity of dendritic cells, which are central to the immune system in capturing pathogens that have invaded the body and activating other immune cells, and it is particularly preferably the activity of plasmacytoid dendritic cells (pDCs).

[0015] Dendritic cells are a type of immune cell that functions as an antigen-presenting cell. Plasmacytoid dendritic cells, also known as plasmacytoid dendritic cells (pDCs), are a type of dendritic cell that constitutes the innate immune system. pDCs are activated in response to viruses that have entered the body, or by stimulation such as TLR7 / 8 ligands and TLR9 ligands that recognize CpG DNA, and produce interferons such as type I interferon and type III interferon. Type I interferons exhibit inhibitory activity against the proliferation of viruses and other pathogens. Interferon-alpha (IFN-α) and interferon-beta (IFN-β) are well-known representative type I interferons. These type I interferons, as well as type III interferon, interferon-λ (IFN-λ), are thought to have immune-activating effects. The activity of pDCs may be at least one IFN production-promoting activity (IFN production-promoting ability) selected from the group consisting of IFN-α, IFN-β, and IFN-λ, or it may be IFN-α production-promoting activity.

[0016] Immune activity can be measured by the quantity of various immune cells such as dendritic cells, eosinophils, neutrophils, basophils, macrophages, and natural killer cells (NK cells), or by measuring the expression levels of surface markers or production factors that serve as indicators of activation or inactivation. When immune activity is pDC activity, the number of pDCs producing IFN-α relative to the number of pDCs in peripheral blood mononuclear cells (PBMCs) is measured. ＋ pDC activity can be measured by the ratio of pDCs. A higher ratio indicates higher pDC activity. In addition, the expression level of surface markers that serve as indicators of pDC activation can be measured. Examples of surface markers that serve as indicators of pDC activation include CD86, HLA-DR, CD80, CD40, CD123, and CD303.

[0017] In this specification, "immunostimulatory activity" means the strength with which a substance enhances the ability of the innate and / or adaptive immune systems to respond to foreign substances, and is also called immune activation ability. The immunostimulatory activity is preferably activating activity of the innate immune system, and for example, it is preferably activating activity of immune cells involved in the innate immune system. Immune cells of the innate immune system include eosinophils, neutrophils, basophils, macrophages, dendritic cells, NK cells, etc., but among these, it is preferably activating activity of dendritic cells, which are central to the immune system in capturing pathogens that have entered the body and activating other immune cells, and it is particularly preferably activating activity of pDCs.

[0018] The immunostimulatory activity of a test substance can be evaluated by measuring changes in the expression levels of surface markers or production factors that serve as indicators of the activation of various immune cells. If the immunostimulatory activity is the activating activity of pDCs, then the number of pDCs in PBMCs that produce IFN-α (IFN-α ＋ An increase in the proportion of pDCs can indicate that the test substance has immunostimulatory activity. Furthermore, changes in the expression levels of surface markers that serve as indicators of pDC activation can be measured. Examples of surface markers that serve as indicators of pDC activation include CD86, HLA-DR, CD80, CD123, and CD303.

[0019] Since the above determination step is performed based on the amount of at least one biomarker in the urine collected from the subject, the method for determining the immune activity of a subject according to this embodiment may further include a measurement step for measuring the amount of the biomarker.

[0020] In this specification, "urinary biomarkers" are biomolecules present in urine that correlate with immune activity. Urinary biomarkers may be, for example, proteins, nucleic acids (e.g., DNA, mRNA, and miRNA), sugars, and lipids, and may preferably include proteins and / or miRNA, and more preferably proteins and / or miRNA.

[0021] When a biomarker in urine contains a protein, for example, such a biomarker may be IgA (immunoglobulin A), RAB21 (Ras-related in brain 21), Nectin 4, PD-L2 (Programmed cell death ligand 2), SARP-2 (Secretized frizzled-related protein 1), LIRA5 (Leukocyte immunoglobulin-like receptor subfamily A member 5), RWDD1 (RWD domain-containing protein) 1; RWD domain-containing protein 1), CI061 (Protein FAM189A2), caspase 4, DAB2 (Disabled homolog 2), adrenomedullin, IMP2L (Mitochondrial inner membrane protease subunit 2), SOD3 (Superoxide dismutase 3), LG3BP (Galectin-3-binding protein), IL-6 soluble receptor α chain, MYO6 (Unconventional myosin-VI (non-classical myosin VI), gelsolin, NALD2 (N-acetylated-alpha-linked acid dipeptidase 2), FCGRN (IgG receptor FcRn large subunit p51), TCP2L (T-complex protein 10A homolog 2), RAC1 (Ras-related C3 botulinum toxin substitute) 1; Ras-related C3 botulinum toxin substrate 1), AIF1L (Allograft inflammation factor 1-like;Allograft inflammatory factor 1-like protein), FAAA (Fumarylacetase), PSME2 (Proteasome activator complex subunit 2), RAB5B (Ras-related protein Rab-5B), SH3G2 (Endophyllin-A1), NCAM-L1 (Neural cell adhesion molecule L1), MAG (Myelin-associated protein Glycoprotein (myelin-related glycoprotein), Radixin, Arylsulfatase A, DHR11 (Dehydrogenase / reductase SDR family member 11), Glyoxalase I, BLVRB (Flavin reductase (NADPH)), BMPR1A (Bone morphogenetic protein receptor type-1A), I5P1 (Type I inositol 1,4,5-trisphosphate) 5-phosphatase; type I inositol 1,4,5-triphosphate-5-phosphatase), ASB9 (Ankyrin repeat and SOCS box protein 9), cathepsin H, MXRA8:ECD (Matrix-remodeling-associated protein 8: Extracellular domain), STK4 (Serine / threonine-protein kinase 4), LACB2 (Endoribonuclease) LACTB2 (endoribonuclease LACTB2), ceruloplasmin, CHSP1 (Calcium-regulated heat-stable protein 1)Calcium-regulated thermostable protein 1), FLRT3:ECD (Leucine-rich repeat transmembrane protein FLRT3; Extracellular domain), LTB4DH (Prostaglandin reductase 1), NPTX2 (Neuronal pentraxin-2), GLO2 (Hydroxyacyl glutathione hydrolase, mitochondrial), RHG30 (Rho GTPase-activating protein 30 (Rho GTPase activating protein 30), VILI (Villin-1), GNPTG (N-acetylglucosamine-1-phosphotransferase subunit Gamma), RAB1B (Ras-related protein) Rab-1B (Ras-related protein Rab-1B), 14-3-3 protein β / α, 14-3-3 protein γ, type III collagen, SCG3 (Secretogranin-3), BGAL (β-galactosidase), 14-3-3 protein ε, calcifosin, DHPR (Dihydropteridine reductase), NRP1 (Neuropilin-1), glutaminylcyclase, FCN2 (Ficolin-2), Cdc42 (Cell division control protein) 42; Cell division regulatory protein 42), SDCB2 (Syntenin-2), ACY3 (Aspartoacylase-2), GSTA2 (Glutathione S-transferase A2), RB11B (Ras-related protein Rab-11B;Ras-related protein Rab-11B), Testican 2, IF1AY (Eukaryotic translation initiation factor 1A, Y-chromosome), MITD1 (MIT domain-containing protein 1), RAB8B (Ras-related protein Rab-8B), RCN1 (Reticulocalbin-1), Lysosomal acid phosphatase, Thioredoxin reductase 1, IL-17 soluble receptor, PROSC (Proline synthase) co-transcribed bacterial homolog protein (proline synthase co-transcribed bacterial homolog protein), RB11A (Ras-related protein Rab-11A), AN32B (acidic leucine-rich nuclear phosphoprotein 32 family member B), paraoxonase 2, SPT46 (spermatogenesis-associated protein 46), SIA10 (Type 2 lactosamine alpha-2,3-sialyltransferase (type 2 lactosamine α-2,3-sialyltransferase), UBE2N / UBE2V2 complex, albumin, IKB ε, GALNS (N-acetylgalactosamine-6-sulfatase), VATC2 (V-type proton ATPase subunit C2), NCF-2 (neutrophil cytoplasmic factor 2), NUBP1 (nucleotide-binding protein 1)Nucleotide-binding protein 1), SDHB (Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial), CREB-binding protein, SHPS1 (Tyrosine-protein phosphatase non-receptor type substrate 1), ARL5B (ADP-ribosylation factor-like protein 5B), PHS (Pterin-4-alpha-carbonolamine) dehydrase (pterin-4α-carbinolamine dehydrogenase), C1GLC (C1GALT1-specific chaperone 1), EphB6 (Ephrin type-B receptor 6), PAFAH β subunit (Platelet-activating factor acetylhydrolase IB subunit β), proteasome α5 subunit, RBP-III (retinol-binding protein) 5 (Retinol-binding protein 5), 14-3-3 protein ζ / δ, MTL26 (Methyltransferase-like protein 26), RNase 2 (Non-secretory ribonuclease), PHIPL (Phytanoyl-CoA hydroxylase-interacting protein-like protein), UBE2K (Ubiquitin-conjugating enzyme E2 K), SAP (Serum amyloid) P-component (serum amyloid P component), cargranulin A, TIMD3 (Hepatitis A virus cellular receptor 2;Hepatitis A virus cell receptor 2), ferritin, ferritin light chain, PPIC (Peptidyl-prolyl c-s-trans isomerase C), S100A12 (S100 calcium-binding protein A12), PACN2 (Protein kinase C and casein kinase substrate in neurons protein 2), ALG2 (Alpha-1,3-mannosyltransferase ALG2) α-1,3-mannosyltransferase ALG2), Annexin V, IF5A2 (Eukaryotic translation initiation factor 5A-2), HSP 70 (Heat shock 70 kDa protein 1A), and hCG (Human chorionic gonadotropin), Tenascin, CHM2B (Charged Multivesical Body Protein 2B), or CgA (Chromogranin A; contains chromogranin A, etc., preferably containing IgA.

[0022] If the biomarker in the urine contains protein, the protein may be, for example, IgA, RAB21, Nectin 4, PD-L2, SARP-2, LIRA5, RWDD1, caspase 4, DAB2, adrenomedullin, IMP2L, SOD3, LG3BP, IL-6 soluble receptor α chain, MYO6, Gelsolin, NALD2, FCGRN, or TCP2L. RAC1, AIF1L, FAAA, PSME2, ​​RAB5B, SH3G2, NCAM-L1, MAG, Radixin, Arylsulfatase A, DHR11, Glyoxalase I, BLVRB, BMPR1A, I5P1, ASB9, Cathepsin H, MXRA8:ECD, STK4, LACB2, Ceruloplasmin, CHSP1, FLRT3 : ECD, LTB4DH, NPTX2, GLO2, RHG30, VILI, GNPTG, RAB1B, 14-3-3 protein β / α, 14-3-3 protein γ, type III collagen, SCG3, BGAL, 14-3-3 protein ε, calcifosin, DHPR, NRP1, glutaminylcyclase, FCN2, Cdc42, SDCB2, ACY3, GSTA2, RB11B, Testican 2, IF1AY, MITD1, RAB8B, RCN1, Lysosomal acid phosphatase, Thioredoxin reductase 1, IL-17 soluble receptor, PROSC, RB11A, AN32B, Paraoxonase 2, SPT46, SIA10, UBE2N / UBE2V2 complex, Albumin, IKB It may also be at least one selected from the group consisting of ε, GALNS, VATC2, NCF-2, NUBP1, SDHB, CREB-binding protein, SHPS1, ARL5B, PHS, C1GLC, EphB6, PAFAH β subunit, proteasome α5 subunit, RBP-III, 14-3-3 protein ζ / δ, MTL26, RNase 2, PHIPL, UBE2K, SAP, cargranulin A, TIMD3, ferritin, ferritin light chain, PPIC, S100A12, PACN2, ALG2, annexin V, IF5A2, HSP 70, and hCG.

[0023] If the biomarker in the urine contains protein, the protein may be, for example, adrenomedullin, IMP2L, IgA, TCP2L, NALD2, caspase 4, NCF-2, CHSP1, RHG30, NPTX2, lysosomal acid phosphatase, ARL5B, AN32B, SDHB, ceruloplasmin, NUBP1, STK4, albumin, S100A12, CREB-binding protein, cargranulin A, SPT46, paraoxonase 2, proteasome α5 subunit, RNase 2, annexin V, PAFAH β subunit, IF5A2, IKB At least one selected from the group consisting of ε, ALG2, and hCG, and RAB21, Nectin 4, CI061, SARP-2, LIRA5, NCAM-L1, 14-3-3 protein ε, MYO6, RAB1B, PD-L2, RAB5B, LTB4DH, Gelsolin, SOD3, SH3G2, BMPR1A, DHR11, VILI, RWDD1, IL-17 soluble receptor, LG3BP, MAG, FAAA, PSME2, ​​FCGRN, DAB2, arylsulfatase A, AIF1L, glyoxalase I, SHPS1, IL-6 soluble receptor α chain, BLVRB, DHPR, MXRA8:ECD, 14-3-3 Protein γ, RCN1, I5P1, PHIPL, FLRT3:ECD, SCG3, Type III collagen, Glutaminyl cyclase, VATC2, ASB9, Cdc42, 14-3-3 protein β / α, Cathepsin H, Calcifosin, GLO2, RAC1, Testican 2, Radixin, FCN2, BGAL, SDCB2, RAB8B, UBE2K, PHS, LACB2, PROSC, IF1AY, GNPTG, ACY3, SIA10, Thioredoxin reductase 1, UBE2N / UBE2V2 complex, MITD1, RB11B, NRP1, EphB6, GALNS, GSTA2, RB11A, C1GLC, HSP 70, at least one selected from the group consisting of 14-3-3 protein ζ / δ, SAP, ferritin light chain, ferritin, TIMD3, PACN2, MTL26, RBP-III, and PPIC.In other words, it may be at least one protein selected from the 31 proteins that were present in relatively large amounts in the group with high immune activity, as analyzed in the examples described later, and at least one protein selected from the 84 proteins that were present in relatively large amounts in the group with low immune activity.

[0024] If the biomarker in the urine contains a protein, the protein may be, for example, one of the following (1) or (2): (1) at least one selected from the group consisting of adrenomedullin, IMP2L, NALD2, caspase 4, NPTX2, AN32B, SDHB, ceruloplasmin, and STK4, and RAB21, nectin 4, CI061, SARP-2, LIRA5, NCAM-L1, 14-3-3 protein ε, MYO6, RAB1B, PD-L2, RAB5B, LTB4DH, Gelsolin, SOD3, SH3G2, BMPR1A, DHR11, VILI, RWDD1, IL-17 soluble receptor, LG3BP, MAG, FAAA, PSME2, ​​FCGRN, DAB2, arylsulfatase A, AIF1L, glyoxalase I, SHPS1, IL-6 soluble receptor α chain, B LVRB, DHPR, MXRA8:ECD, 14-3-3 protein γ, RCN1, I5P1, PHIPL, FLRT3:ECD, SCG3, Type III collagen, Glutaminyl cyclase, VATC2, ASB9, Cdc42, 14-3-3 protein β / α, Cathepsin H, Calcifosin, GLO2, RAC1, Testican 2, Rad At least one selected from the group consisting of Ixin, FCN2, BGAL, SDCB2, RAB8B, UBE2K, PHS, LACB2, PROSC, IF1AY, GNPTG, ACY3, SIA10, Thioredoxin reductase 1, UBE2N / UBE2V2 complex, MITD1, RB11B, NRP1, EphB6, GALNS, GSTA2, RB11A, C1GLC, HSP 70, 14-3-3 protein ζ / δ, SAP, ferritin light chain, ferritin, TIMD3, PACN2, MTL26, RBP-III, and PPIC.(2) Adrenomedullin, IMP2L, IgA, TCP2L, NALD2, Caspase 4, NCF-2, CHSP1, RHG30, NPTX2, Lysosomal Acid Phosphatase, ARL5B, AN32B, SDHB, Ceruloplasmin, NUBP1, STK4, Albumin, S100A12, CREB-binding protein, Cargranulin A, SPT46, Paraoxonase 2, Proteasome α5 subunit, RNase 2, Annexin V, PAFAH β subunit, IF5A2, IKB At least one selected from the group consisting of ε, ALG2, and hCG, and RAB21, Nectin 4, CI061, SARP-2, LIRA5, NCAM-L1, 14-3-3 protein ε, MYO6, RAB1B, RAB5B, LTB4DH, Gelsolin, SOD3, BMPR1A, DHR11, VILI, RWDD1, IL-17 soluble receptor, LG3BP, MAG, FAAA, PSME2, ​​FCGRN, DAB2, aryl sulfa At least one selected from the group consisting of Tase A, AIF1L, Glyoxalase I, IL-6 soluble receptor α chain, BLVRB, DHPR, MXRA8:ECD, Glutaminil cyclase, VATC2, ASB9, Cdc42, Cathepsin H, Calcifosin, GLO2, RAC1, Radixin, FCN2, BGAL, RAB8B, PHS, LACB2, IF1AY, GNPTG, SIA10, UBE2N / UBE2V2 complex, MITD1, RB11B, GSTA2, and HSP 70.

[0025] If the biomarker in the urine contains a protein, the protein may be, for example, one of two combinations shown in Tables 5 to 7, such as two types of AN32B, SDHB, SPT46 or STK4 and RAB21, two types of TCP2L and radixin, two types of caspase 4 and DHR11, two types of IgA and SARP-2, two types of IgA and RWDD1, or two types of IgA and HSP70.

[0026] If the biomarker in the urine contains protein, the protein may be, for example, one of the four combinations shown in Table 8: SOD3, SARP-2, AN32B, and RAB21; SOD3, SARP-2, AN32B, and PHIPL; SOD3, IgA, SARP-2, and RAB1B; S100A12, SOD3, IgA, and SARP-2; SARP-2, IgA, SOD3, and MTL26; IgA, SARP-2, SOD3, and SHPS1; IgA, SARP-2, and RNase It may also be four types of 2 and SOD3, four types of IgA, SARP-2, ferritin light chain and SOD3, four types of MYO6, SOD3, IgA and SARP-2, four types of SHPS1, SARP-2, adrenomedullin and IgA, or four types of caspase 4, DHR11, Cdc42 and IgA.

[0027] If the biomarker in the urine contains miRNA, the biomarker may be, for example, hsa-let-7c-5p, hsa-miR-99a-5p, hsa-miR-30c-5p, ENST00000516507.1, ENST00000698785.1, hsa-miR-125b-5p, ENST00000531977.1, ENST00000663798.1, hsa-miR-30b-5p, ENST00000433588.1, EN It includes ST00000643616.1, ENST00000622359.1, ENST00000629295.1, etc., preferably including hsa-let-7c-5p, hsa-miR-99a-5p, hsa-miR-30c-5p, ENST00000516507.1, hsa-miR-125b-5p, ENST00000531977.1, hsa-miR-30b-5p, etc., and is particularly preferably including hsa-let-7c-5p.

[0028] If the biomarker in the urine contains miRNA, the miRNA may be, for example, hsa-miR-151a-5p, hsa-miR-99b-5p, hsa-miR-200b-3p, hsa-miR-99a-5p, hsa-miR-125b-5p, ENST00000531977.1, hsa-l et-7c-5p, ENST00000516507.1, hsa-miR-30c-5p, hsa-miR-200c-3p, hsa-miR-2 04-5p, hsa-miR-103a-3p, hsa-miR-125a-5p, hsa-miR-29a-3p, ENST0000056831 4.1, ENST00000563103.1, hsa-miR-30b-5p, hsa-miR-151a-3p, hsa-miR-92a-3p , hsa-miR-182-5p, hsa-miR-22-3p, hsa-miR-100-5p, hsa-miR-23b-3p, ENST000 It may be at least one selected from the group consisting of 00658801.1, hsa-miR-9-5p, ENST00000655322.1, hsa-miR-141-3p, hsa-miR-191-5p, ENST00000630360.1, and hsa-miR-30c-2-3p.

[0029] If the biomarker in the urine contains miRNA, the miRNA may be at least one selected from the group consisting of, for example, hsa-miR-151a-5p, hsa-miR-99b-5p, and hsa-miR-200b-3p.

[0030] If the biomarker in the urine contains miRNA, the miRNA may be, for example, two types: hsa-miR-29a-3p and hsa-miR-200c-3p, two types: hsa-miR-30b-5p and hsa-miR-22-3p, two types: hsa-miR-141-3p and hsa-miR-30b-5p, or two types: hsa-miR-182-5p and hsa-miR-29a-3p.

[0031] If the biomarker in the urine contains miRNA, the miRNA may be, for example, three types: hsa-miR-141-3p, hsa-miR-30b-5p and hsa-miR-22-3p; three types: hsa-miR-141-3p, hsa-miR-30b-5p and hsa-miR-103a-3p; three types: hsa-miR-182-5p, hsa-miR-29a-3p and hsa-miR-200c-3p; or hsa-miR-141- It may be one of three types: 3p, hsa-miR-30b-5p and hsa-miR-200c-3p; one of three types: hsa-miR-29a-3p, hsa-miR-200c-3p and hsa-miR-22-3p; one of three types: hsa-miR-182-5p, hsa-miR-30b-5p and hsa-miR-22-3p; or one of three types: hsa-miR-30c-2-3p, hsa-miR-200c-3p and hsa-miR-22-3p.

[0032] If the biomarker in the urine contains miRNA, the miRNA may be, for example, four types: hsa-miR-141-3p, hsa-miR-30c-5p, hsa-miR-200c-3p and hsa-miR-22-3p, four types: hsa-miR-30b-5p, hsa-miR-29a-3p, hsa-miR-200c-3p and hsa-miR-22-3p, hsa-miR-141-3p, hsa-miR-30b-5p, hsa-miR-200c-3p and hsa-miR It may be four types of -22-3p, four types of hsa-miR-141-3p, hsa-miR-30b-5p, hsa-miR-200c-3p and hsa-miR-103a-3p, four types of hsa-miR-141-3p, hsa-miR-30b-5p, hsa-miR-103a-3p and hsa-miR-22-3p, or four types of hsa-miR-182-5p, hsa-miR-29a-3p, hsa-miR-200c-3p and hsa-miR-103a-3p.

[0033] If the biomarker in the urine contains miRNA, the miRNA may be one of eight types, for example, hsa-miR-200c-3p, hsa-miR-141-3p, hsa-miR-103a-3p, hsa-miR-30c-5p, hsa-miR-29a-3p, hsa-miR-30b-5p, hsa-miR-182-5p, and hsa-miR-22-3p.

[0034] The biomarkers in the urine may be those whose AUC (Area Under the Curve) of the ROC (Receiver Operating Characteristic) curve used to distinguish between a group with high immune activity and a group with low immune activity is greater than 0.5 and less than or equal to 1.0, 0.6 to less than or equal to 1.0, 0.65 to less than or equal to 1.0, 0.70 to less than or equal to 1.0, 0.75 to less than or equal to 1.0, 0.80 to less than or equal to 1.0, or 0.85 to less than or equal to 1.0.

[0035] The biomarkers in the urine used in the determination process may be one or more, two or more, three or more, four or more, or five or more, and may be ten or fewer. Furthermore, the biomarkers in the urine may be a combination of different molecular species, for example, a combination of protein and miRNA.

[0036] If the biomarker in the urine used in the determination step contains protein, the protein may be, for example, one or more types, two or more types, or three or more types, and may be 10 or fewer types, 9 or fewer types, 8 or fewer types, 7 or fewer types, 6 or fewer types, 5 or fewer types, or 4 or fewer types. If the biomarker in the urine used in the determination step contains protein, the protein may be, for example, one to four types, or two to four types.

[0037] If the biomarker in the urine used in the determination step contains miRNA, the miRNA may be, for example, one or more types, two or more types, or three or more types, and may be 10 or fewer types, 9 or fewer types, 8 or fewer types, 7 or fewer types, 6 or fewer types, 5 or fewer types, or 4 or fewer types. If the biomarker in the urine used in the determination step contains miRNA, the miRNA may be, for example, one to eight types, two to eight types, three to eight types, or four to eight types.

[0038] The "amount" of the biomarker in urine is not particularly limited as long as it is a value serving as an index of the abundance of the biomarker in urine. For example, it may be the signal intensity (e.g., fluorescence intensity, etc.) in the measurement by a predetermined measurement method, or a value such as the concentration of the biomarker in urine. The concentration may be, for example, mass percentage concentration, molar concentration, molality, or volume percentage concentration, etc. In this specification, the measured value of the amount of the biomarker in urine means the measured value of the value serving as an index of the abundance of these biomarkers in urine.

[0039] The "value of the parameter based on the amount of the biomarker in urine" means a value calculated according to a predetermined parameter based on the measured value of the amount of the biomarker in urine. Here, the parameter based on the amount of the biomarker in urine may be a discriminant using the amount of the biomarker in urine. Here, the discriminant may be the measured value of the amount of the biomarker in urine itself, or may be an equation for calculating by substituting the measured value of the amount of the biomarker in urine. Therefore, the value of the parameter based on the amount of the biomarker in urine may be the measured value of the amount of the biomarker in urine itself, or may be a value calculated based on the measured value of the amount of the biomarker in urine. The discriminant may be created by analyzing the correlation between the data of the "measured value of the amount of the biomarker in urine" obtained from a plurality of subjects and the data of "immunoreactivity" using a commercially available analysis system, a machine learning system, etc.

[0040] The measured value of the amount of the biomarker in urine may be a value normalized by the mass of the internal standard substance. Thereby, accurate determination can be performed. The internal standard substance is preferably a substance that exists in urine at a constant concentration. By normalizing the amount of the biomarker by the amount of the internal standard substance, for example, a value excluding the influence of the amount of urine volume, etc. can be obtained. The normalization may be, for example, dividing the amount of the biomarker in urine by the amount of the internal standard substance in urine.

[0041] Examples of the internal standard substance include total protein, albumin, creatinine, and the like. The amount of the biomarker in urine may be the concentration of the biomarker in urine per 1 mg / mL of the concentration of total protein in urine, or may be the concentration of the biomarker in the urine per 1 g of creatinine in urine.

[0042] When the biomarker is a protein, examples of the measurement of the amount of the biomarker include the BCA method, pyrogallol red method, immunoturbidimetry, enzyme method, Western blotting method, enzyme-linked immunosorbent assay method (ELISA method), DNA chip method using an aptamer (microarray method), amino acid sequencing method, and mass spectrometry method, and the like. When the biomarker is a nucleic acid, examples of the measurement of the amount of the biomarker include next-generation sequencing, PCR method, and Southern blotting method, and the like. When the biomarker is a sugar, examples of the measurement of the amount of the biomarker include the enzyme-linked immunosorbent assay method (ELISA method), high-performance liquid chromatography method (HPLC method), gas chromatography method (GC method), and mass spectrometry method, and the like. When the biomarker is a lipid, examples of the measurement of the amount of the biomarker include the enzyme-linked immunosorbent assay method (ELISA method), high-performance liquid chromatography method (HPLC method), gas chromatography method (GC method), and mass spectrometry method, and the like.

[0043] When there are multiple types of biomarkers to be measured, the measurement of the biomarker amounts may be performed using a known measurement method that can measure the amounts of multiple biomarkers at once, from the viewpoint of efficiently measuring their amounts. For example, if the biomarkers include proteins, they can be measured using a DNA chip method (microarray method) that uses the same number of aptamers as the number of proteins to be measured. More specifically, the protein measurement service provided by SomaLogic can also be used. Protein measurement in the protein measurement service provided by SomaLogic is performed using a method called the SomaScan® assay. The SomaScan® assay comprehensively measures proteins using 7,288 specific SOMAmers (single-stranded DNA aptamers) for 6,596 types of proteins, and the relative measurement value (relative amount) of protein abundance can be obtained as relative fluorescence units (RFU). It is also possible to obtain measurement values ​​that are standardized based on plasma samples from a healthy population in the United States.

[0044] Furthermore, for example, by using the value of the amount of protein in urine (e.g., the amount of albumin in urine) obtained by the DNA chip method described above and the value of the protein concentration in urine obtained by a measurement method other than the DNA chip method described above, a conversion formula can be created from the value obtained by the DNA chip method to the value of the protein concentration in urine. According to this conversion formula, the concentration (absolute value) of the amount of protein in urine obtained by the DNA chip method described above (e.g., the amount of IgA in urine) can be used to determine the concentration (absolute value) of the protein in urine. The determined concentration (absolute value) of the protein in urine may be normalized, for example, by the internal standard substance described above.

[0045] When the biomarker is IgA, it is preferable to measure the amount of IgA in urine using methods such as Western blotting, enzyme-linked immunosorbent assay (ELISA), nephelometry, or immunoturbidimetry.

[0046] The total protein concentration in urine is preferably measured by the pyrogallol red method. The albumin concentration in urine is preferably measured by immunoturbidimetry. The creatinine concentration in urine is preferably measured by the enzymatic method.

[0047] When there are multiple types of biomarkers to be measured, the measurement of the amounts of these biomarkers may be performed by a known measurement method that can measure the amounts of multiple biomarkers at once, from the viewpoint of efficiently measuring their amounts. For example, if the biomarkers include miRNA, they can be measured by next-generation sequencing as described in the examples below.

[0048] When measuring the amount of biomarkers in urine, the obtained urine may be used directly for measurement, or it may be pre-treated before measurement. Examples of pre-treatment include removal of precipitates and concentration.

[0049] The determination step may be based on the amount of at least one biomarker in the urine collected from the subject. For example, it may be a step in which the subject's immune activity is determined to be high if the value of a parameter based on the amount of the biomarker is higher than a predetermined threshold, or it may be a step in which the subject's immune activity is determined to be high if the value of a parameter based on the amount of the biomarker is lower than a predetermined threshold. Conversely, the determination step may be a step in which the subject's immune activity is determined to be low if the value of a parameter based on the amount of the biomarker is lower than a predetermined threshold, or it may be a step in which the subject's immune activity is determined to be low if the value of a parameter based on the amount of the biomarker is higher than a predetermined threshold. In other words, the determination step may include at least one of the following (A) to (D). (A) If the value of the parameter based on the amount of the above biomarker is higher than a predetermined threshold, it is determined that the immune activity of the above subject is high. (B) If the value of the parameter based on the amount of the above biomarker is lower than a predetermined threshold, it is determined that the immune activity of the above subject is low. (C) If the value of the parameter based on the amount of the above biomarker is higher than a predetermined threshold, it is determined that the immune activity of the above subject is low. (D) If the value of the parameter based on the amount of the above biomarker is lower than a predetermined threshold, it is determined that the immune activity of the above subject is high.

[0050] The threshold may be a value that allows determination of whether the target's immune activity is high or low by comparing it to a parameter value based on the amount of biomarkers in the target's urine. Conversely, the threshold may be a value that allows determination of whether the target's immune activity is low or low by comparing it to a parameter value based on the amount of biomarkers in the target's urine. Different threshold values ​​can be used depending on the type of biomarker and the discriminant formula used for determination. To calculate the threshold, for example, urine samples are obtained from targets that have been previously determined to have high immune activity and targets that have been previously determined to have low immune activity, and data on the amount of biomarkers in the target's urine is obtained. The "immune activity" data and the "amount of biomarkers in urine" data obtained from multiple targets are calculated using a parameter (discriminant formula), and a value that can distinguish between targets with high immune activity and targets with low immune activity is obtained, and this value is set as the predetermined threshold.

[0051] In calculating the threshold, for example, at least one biomarker in urine screened using the method described in [Screening Method for Biomarkers] below is selected, and the correlation between the data on the "amount of biomarker in urine" and the "immune activity" obtained from multiple subjects is analyzed using a commercially available analysis system or machine learning system. Through this analysis, a discriminant formula for distinguishing between subjects with high and low immune activity based on the amount of biomarker in urine, and a threshold for distinguishing between subjects with high and low immune activity in that discriminant formula can be calculated and used. For example, in a machine learning system, a discriminant formula for predicting immune activity is created using the measured amount of biomarker as a training sample, the measured amount of biomarker in urine collected from a subject is substituted into the discriminant formula, and the immune activity of the subject is predicted from the obtained result. By repeating this, a discriminant formula with higher accuracy in predicting immune activity and a threshold for distinguishing between subjects with high and low immune activity in that discriminant formula can be calculated.

[0052] The threshold may be set according to the purpose, such as whether to prioritize a high positive rate, a low false positive rate, or a balance between the positive and false positive rates. The threshold may also be set considering the age and gender of the target group.

[0053] Specifically, for example, the threshold may be calculated such that the discriminant accuracy, determined by the following formula, is 70% or higher, 75% or higher, 80% or higher, 85% or higher, or 90% or higher: Discriminant accuracy (%) = {(Number of accurately classified samples) / (Total number of samples)} × 100

[0054] When the biomarker is IgA, the threshold for determining whether the target immune activity is high is, for example, an IgA concentration [mg / mL] that is greater than 0.01031 and 0.01033 or less, greater than 0.00985 and 0.01021 or less, or greater than 0.0056 and 0.00569 or less, more preferably greater than 0.00959 and 0.00985 or less, or greater than 0.00569 and 0.00595 or less, more preferably greater than 0.00892 and 0.00959 or less, or greater than 0.00595 and 0.00600 or less, and even more preferably greater than 0.00858 and 0.0892 or less.

[0055] Furthermore, when the biomarker is IgA, the threshold for determining whether the target immune activity is high may be, for example, in the range of relative IgA amount [RFU] greater than 8314 and 8329 or less, greater than 7943 and 8235 or less, or greater than 4517 and 4587 or less, preferably in the range of greater than 7732 and 7943 or greater than 4587 and 4800 or less, more preferably in the range of greater than 7197 and 7732 or greater than 4800 and 4839 or less, and even more preferably in the range of greater than 6923 and 7197 or less. The relative IgA amount [RFU] is the amount of IgA that was relatively quantified by using SomaScan® Assay (SomaLogic service) as performed in the examples described later.

[0056] Furthermore, when the biomarker is IgA, the threshold for determining whether the target immune activity is high is preferably, for example, that the amount of IgA in the urine per gram of creatinine in the urine (mg) [mg / g] is within the range of greater than 0.38 and less than or equal to 0.41, greater than 0.43 and less than or equal to 0.45, or greater than 0.80 and less than or equal to 0.82.

[0057] If the biomarker contains adremedullin, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on SomaScan assay measurements within the ranges of 500 to 2500, 1000 to 2000, or 1400 to 1550.

[0058] If the biomarker contains IMP2L, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 10 to 200, 50 to 150, or 100 to 110.

[0059] If the biomarker contains TCP2L, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 80 to 300, 100 to 250, or 180 to 210.

[0060] If the biomarker contains NALD2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 10 to 150, 30 to 100, or 40 to 50.

[0061] If the biomarker contains caspase 4, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 100 to 350, 150 to 300, 200 to 250, or 220 to 223.

[0062] If the biomarker contains NCF-2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 1000 to 2000, 1200 to 1500, 1300 to 1450, or 1370 to 1440.

[0063] If the biomarker contains CHSP1, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 800 to 1500, 850 to 1200, 950 to 1100, or 980 to 1080.

[0064] If the biomarker contains RHG30, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 300 to 600, 350 to 500, 400 to 450, or 410 to 415.

[0065] If the biomarker contains NPTX2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 50 to 500, 100 to 250, 150 to 200, or 170 to 185.

[0066] If the biomarker contains lysosomal acid phosphatase, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 2500 to 4500, 3000 to 4000, or 3600 to 3610.

[0067] If the biomarker contains ARL5B, the threshold for determining whether the target immune activity is high may be, for example, within the range of relative volume [RFU] based on SomaScan® assay measurements of 2500 to 5000, 3500 to 4500, 3800 to 4000, or 3830 to 3850.

[0068] If the biomarker contains AN32B, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 500 to 2000, 700 to 1500, or 800 to 1100.

[0069] If the biomarker contains SDHB, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 100 to 1000, 200 to 500, or 300 to 450.

[0070] If the biomarker contains ceruloplasmin, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 300 to 600, 400 to 550, or 420 to 480.

[0071] If the biomarker contains NUBP1, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 700 to 1500, 800 to 1000, or 880 to 900.

[0072] If the biomarker contains STK4, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 200 to 500, 300 to 400, or 280 to 350.

[0073] If the biomarker contains albumin, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 140,000 to 180,000, 150,000 to 170,000, or 155,000 to 169,000.

[0074] If the biomarker contains S100A12, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 800 to 2000, 900 to 1500, or 940 to 1100.

[0075] If the biomarker contains a CREB-binding protein, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 2000 to 4000, 2500 to 3500, or 3100 to 3200.

[0076] If the biomarker contains cargranulin A, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 40 to 800, 500 to 700, or 590 to 610.

[0077] If the biomarker contains SPT46, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 200 to 500, 300 to 400, or 310 to 360.

[0078] If the biomarker contains paraoxonase 2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 300 to 600, 400 to 500, or 460 to 540.

[0079] If the biomarker contains the proteasome α5 subunit, the threshold for determining whether the target immune activity is high may be, for example, a relative quantity [RFU] based on the SomaScan® assay measurement range of 100 to 500, 200 to 400, or 300 to 330.

[0080] If the biomarker contains RNase 2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 2000 to 4000, 2500 to 3500, or 3000 to 3200.

[0081] If the biomarker contains annexin V, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement within the range of 400 to 700, 500 to 600, or 570 to 590.

[0082] If the biomarker contains the PAFAH β subunit, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 500 to 900, 600 to 800, or 700 to 720.

[0083] If the biomarker contains IF5A2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 100 to 400, 200 to 300, or 220 to 240.

[0084] If the biomarker includes IKB ε, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 1 to 40, 5 to 20, or 10 to 15.

[0085] If the biomarker includes ALG2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 40 to 80, 50 to 70, 60 to 65, or 60 to 61.

[0086] If the biomarker contains hCG, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 500 to 900, 600 to 800, or 620 to 700.

[0087] If the biomarker contains RAB21, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 600 to 1000, 700 to 900, or 760 to 820.

[0088] If the biomarker contains nectin 4, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 200 to 500, 300 to 450, or 400 to 415.

[0089] If the biomarker contains CI061, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 200 to 500, 300 to 400, or 375 to 385.

[0090] If the biomarker contains SARP-2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 300 to 700, 400 to 600, or 485 to 510.

[0091] If the biomarker contains LIRA5, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 1700 to 2000, 1800 to 2100, or 1900 to 2060.

[0092] If the biomarker contains NCAM-L1, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement within the range of 150 to 300, 170 to 250, or 180 to 190.

[0093] When the biomarker contains 14-3-3 protein ε, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 300 to 700, 400 to 600, or 450 to 500.

[0094] If the biomarker contains MYO6, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 100 to 400, 200 to 300, or 240 to 265.

[0095] If the biomarker includes RAB1B, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 50 to 300, 100 to 150, or 125 to 135.

[0096] When the biomarker contains PD-L2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 900 to 1500, 1000 to 1300, or 1140 to 1200.

[0097] If the biomarker contains RAB5B, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 1500 to 1900, 1600 to 1800, or 1700 to 1750.

[0098] If the biomarker contains LTB4DH, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 3900 to 4500, 4000 to 4600, or 3900 to 4600.

[0099] If the biomarker contains gelsolin, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 9,000 to 11,000, 10,000 to 10,500, or 10,200 to 10,300.

[0100] If the biomarker contains SOD3, the threshold for determining whether the target immune activity is high may be, for example, within the range of relative volume [RFU] based on SomaScan® assay measurements of 17,000 to 21,000, 18,000 to 20,000, 19,000 to 19,750, or 19,400 to 19,600.

[0101] When the biomarker contains SH3G2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 50 to 200, 100 to 150, or 120 to 135.

[0102] If the biomarker contains BMPR1A, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 100 to 300, 150 to 200, or 170 to 185.

[0103] If the biomarker contains DHR11, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 400 to 700, 500 to 600, or 530 to 560.

[0104] If the biomarker includes VILI, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 200 to 500, 300 to 400, or 350 to 370.

[0105] If the biomarker contains RWDD1, the threshold for determining whether the target immune activity is high may be, for example, a relative quantity [RFU] based on the SomaScan® assay measurement range of 200 to 550, 300 to 450, or 340 to 390.

[0106] If the biomarker includes the IL-17 soluble receptor, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 350 to 900, 450 to 800, or 550 to 700.

[0107] If the biomarker contains LG3BP, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 700 to 1100, 800 to 1000, or 860 to 930.

[0108] If the biomarker contains MAG, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 500 to 800, 600 to 700, or 640 to 670.

[0109] If the biomarker contains FAAA, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 400 to 800, 500 to 700, or 590 to 610.

[0110] If the biomarker contains PSME2, ​​the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 300 to 700, 400 to 600, or 470 to 490.

[0111] If the biomarker contains FCGRN, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 400 to 900, 500 to 800, or 580 to 690.

[0112] If the biomarker contains DAB2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 300 to 700, 400 to 600, or 490 to 510.

[0113] If the biomarker contains arylsulfatase A, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 500 to 1000, 600 to 900, or 680 to 780.

[0114] If the biomarker contains AIF1L, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 900 to 1400, 1000 to 1300, or 1100 to 1200.

[0115] When the biomarker contains glyoxalase I, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 600 to 1200, 700 to 1100, or 810 to 940.

[0116] If the biomarker contains SHPS1, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 1800 to 2300, 1900 to 2200, or 1970 to 2070.

[0117] When the biomarker contains the IL-6 soluble receptor α chain, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 2300 to 2800, 2400 to 2700, or 2560 to 2570.

[0118] When the biomarker contains BLVRB, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 10 to 500, 100 to 400, or 240 to 260.

[0119] If the biomarker includes DHPR, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 800 to 1400, 900 to 1300, or 990 to 1150.

[0120] If the biomarker includes MXRA8:ECD, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 11,500 to 13,500, 12,000 to 13,700, or 12,700 to 13,850.

[0121] When the biomarker contains 14-3-3 protein γ, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 10 to 400, 50 to 300, or 90 to 150.

[0122] If the biomarker contains RCN1, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 10 to 400, 50 to 300, or 160 to 170.

[0123] If the biomarker contains I5P1, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 10 to 400, 50 to 300, or 180 to 190.

[0124] If the biomarker includes PHIPL, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement within the range of 50 to 500, 100 to 350, or 210 to 220.

[0125] If the biomarker includes FLRT3:ECD, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 100 to 600, 200 to 500, or 330 to 350.

[0126] If the biomarker contains SCG3, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 100 to 600, 200 to 500, or 320 to 350.

[0127] When the biomarker contains type III collagen, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 200 to 600, 300 to 500, or 390 to 405.

[0128] If the biomarker contains glutaminilcyclase, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 200 to 700, 300 to 600, or 460 to 500.

[0129] If the biomarker contains VATC2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 200 to 700, 300 to 600, or 440 to 470.

[0130] If the biomarker contains ASB9, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 10 to 500, 100 to 350, or 210 to 230.

[0131] If the biomarker contains Cdc42, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 300 to 700, 400 to 600, or 520 to 530.

[0132] When the biomarker contains 14-3-3 protein β / α, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 600 to 1000, 700 to 900, or 790 to 805.

[0133] If the biomarker contains cathepsin H, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 800 to 1200, 900 to 1300, or 1040 to 1130.

[0134] If the biomarker contains calcifosin, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 1000 to 1500, 1100 to 1400, or 1280 to 1300.

[0135] If the biomarker contains GLO2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 10 to 400, 100 to 300, or 200 to 230.

[0136] If the biomarker contains RAC1, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 1200 to 1700, 1300 to 1600, or 1400 to 1460.

[0137] If the biomarker contains Testican 2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 5500 to 6100, 5600 to 6000, or 5700 to 5880.

[0138] If the biomarker contains radixin, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 9200 to 11200, 9300 to 11100, or 9400 to 11000.

[0139] If the biomarker contains FCN2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 5200 to 5800, 5300 to 5700, or 5400 to 5600.

[0140] When the biomarker includes BGAL, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement within the range of 0.1 to 10, 1 to 5, or 3 to 3.5.

[0141] When the biomarker contains SDCB2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 10 to 400, 100 to 300, or 200 to 230.

[0142] If the biomarker contains RAB8B, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 1 to 300, 10 to 200, or 120 to 135.

[0143] If the biomarker contains UBE2K, the threshold for determining whether the target immune activity is high may be, for example, within the range of relative volume [RFU] of 100 to 600, 200 to 500, or 370 to 405 based on the SomaScan® assay measurement.

[0144] If the biomarker includes PHS, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 1 to 300, 10 to 200, or 80 to 100.

[0145] If the biomarker contains LACB2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 1 to 400, 100 to 300, or 190 to 200.

[0146] If the biomarker contains PROSC, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 10 to 500, 100 to 400, or 240 to 270.

[0147] If the biomarker includes IF1AY, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 10 to 400, 100 to 300, or 200 to 210.

[0148] If the biomarker includes GNPTG, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 500 to 1000, 600 to 900, or 720 to 780.

[0149] If the biomarker contains ACY3, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 700 to 1300, 800 to 1200, or 960 to 1110.

[0150] If the biomarker contains SIA10, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 300 to 800, 400 to 700, or 500 to 560.

[0151] If the biomarker contains thioredoxin reductase 1, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 800 to 1300, 900 to 1200, or 1000 to 1050.

[0152] When the biomarker contains the UBE2N / UBE2V2 complex, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 1000 to 1500, 1100 to 1400, or 1200 to 1225.

[0153] If the biomarker contains MITD1, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 600 to 1100, 700 to 1000, or 860 to 890.

[0154] If the biomarker contains RB11B, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 600 to 1000, 700 to 900, or 800 to 820.

[0155] If the biomarker contains NRP1, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 3200 to 3700, 3300 to 3600, or 3400 to 3490.

[0156] If the biomarker contains EphB6, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 9100 to 11000, 9200 to 10900, or 9300 to 10730.

[0157] When the biomarker includes GALNS, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 4500 to 6300, 4600 to 6200, or 4700 to 6050.

[0158] If the biomarker contains GSTA2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 8500 to 9000, 8600 to 8900, or 8700 to 8790.

[0159] If the biomarker contains RB11A, the threshold for determining whether the target immune activity is high may be, for example, a relative quantity [RFU] based on the SomaScan® assay measurement range of 1 to 300, 10 to 200, or 100 to 110.

[0160] When the biomarker contains C1GLC, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 10 to 500, 100 to 400, or 210 to 255.

[0161] If the biomarker contains HSP 70, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 10 to 500, 100 to 400, or 190 to 220.

[0162] When the biomarker contains the 14-3-3 protein ζ / δ, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 5100 to 5700, 5200 to 5600, or 5300 to 5490.

[0163] If the biomarker includes SAP, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 6100 to 6800, 6200 to 6700, or 6360 to 6600.

[0164] If the biomarker contains ferritin light chains, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 6200 to 8200, 6300 to 8000, or 6420 to 7880.

[0165] If the biomarker includes ferritin, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 6600 to 8800, 6700 to 8500, or 6890 to 8350.

[0166] If the biomarker contains TIMD3, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 100 to 600, 200 to 500, or 320 to 350.

[0167] If the biomarker contains PACN2, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 100 to 600, 200 to 500, or 340 to 370.

[0168] If the biomarker contains MTL26, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 10 to 500, 100 to 400, or 210 to 230.

[0169] When the biomarker contains RBP-III, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 1000 to 1500, 1100 to 1600, or 1230 to 1500.

[0170] If the biomarker includes PPIC, the threshold for determining whether the target immune activity is high may be, for example, a relative amount [RFU] based on the SomaScan® assay measurement range of 5600 to 6300, 5700 to 6200, or 5800 to 6100.

[0171] The threshold may be set, for example, based on the ROC curve used to distinguish between a group with high immune activity and a group with low immune activity. As a method for setting the threshold based on the ROC curve, for example, the point at which the distance from the upper left corner of the ROC curve is minimized may be used as the threshold, or the point (Youden Index) furthest from the diagonal line where the area under the ROC curve is 0.5 may be used as the threshold.

[0172] The determination step may also be based on the amount of at least one biomarker in the urine collected from the subject. For example, it may be a step of calculating the standard score of the parameter value based on the amount of the biomarker when multiple subjects are considered as a population, and making a determination based on the standard score. The standard score used for determination may differ depending on the type of biomarker. For example, the determination step may be a step of determining that the subject has high immune activity if the standard score of the parameter value based on the amount of the biomarker is 50 or higher, 51 or higher, 52 or higher, 53 or higher, 54 or higher, 55 or higher, 56 or higher, 57 or higher, 58 or higher, 59 or higher, 60 or higher, 61 or higher, 62 or higher, 63 or higher, 64 or higher, or 65 or higher. The determination step may also be a step of determining that the subject has high immune activity if the standard score of the parameter value based on the amount of the biomarker is less than 50, 49 or lower, 48 or lower, 47 or lower, 46 or lower, 45 or lower, 44 or lower, 43 or lower, 42 or lower, 41 or lower, or 40 or lower. The determination step may be a step in which the immune activity of the subject is determined to be low if the standard score of the parameter value based on the amount of the biomarker is less than 50, 49 or less, 48 ​​or less, 47 or less, 46 or less, 45 or less, 44 or less, 43 or less, 42 or less, 41 or less, or 40 or less. The determination step may be a step in which the immune activity of the subject is determined to be low if the standard score of the parameter value based on the amount of the biomarker is 50 or more, 51 or more, 52 or more, 53 or more, 54 or more, 55 or more, 56 or more, 57 or more, 58 or more, 59 or more, 60 or more, 61 or more, 62 or more, 63 or more, 64 or more, or 65 or more. The standard score may be calculated taking into account the age and sex of the subject, and in that case, the age and sex of the population when calculating the standard score may also be taken into account.

[0173] [Method to support improvement or maintenance of immune activity] The method to support improvement or maintenance of immune activity according to this embodiment comprises a determination step of determining immune activity using the method for determining the target immune activity according to this embodiment, and a determination result presentation step of presenting a report showing the determination result obtained in the determination step. The determination step is as described above.

[0174] The method for supporting the improvement or maintenance of immune activity according to this embodiment includes the above-described steps, thereby enabling the subject to recognize their own immune activity and increasing their awareness of taking actions to improve or maintain their immune activity. This makes it possible to support the improvement or maintenance of the subject's immune activity.

[0175] The judgment result presentation step presents a report showing the judgment result obtained in the judgment step. If the judgment step is a step in which the immune activity of the subject is determined to be high when the value of the parameter based on the amount of the biomarker is higher or lower than a predetermined threshold, the report may indicate that the immune activity of the subject is high. If the judgment step is a step in which the immune activity of the subject is determined to be low when the value of the parameter based on the amount of the biomarker is lower or higher than a predetermined threshold, the report may indicate that the immune activity of the subject is low. Furthermore, if the judgment step is a step in which the standard score of the parameter value based on the amount of the biomarker is calculated for a population of multiple subjects and the judgment is made based on the standard score, the report may show the standard score, and may indicate that the immune activity of the subject is high or low based on the standard score.

[0176] The method for supporting the improvement or maintenance of immune activity according to this embodiment may further include: an identification step that identifies an action for improving or maintaining immune activity (hereinafter also referred to as "immune care action") based on the determination result obtained in the determination step; and an identification result presentation step that presents a report showing the results identified in the identification step.

[0177] The report showing the judgment results obtained in the judgment process and / or the results identified in the specific process is not limited in form as long as it shows the results, but may be, for example, a printed document showing these results, or an electronic screen showing these results.

[0178] The method for supporting the improvement or maintenance of immune activity according to this embodiment includes the above-mentioned specific step and specific result presentation step, so that the actions that the subject should take to improve or maintain their immune activity become clear, and thus the improvement or maintenance of the subject's immune activity can be supported more specifically.

[0179] If, in a specific process, the assessment process determines that "immune activity is low," immune care actions may include, for example, regularly measuring immune activity, taking supplements or foods / beverages known to have immunostimulatory activity (especially pDC activating activity), reviewing lifestyle habits, exercise habits, sleep habits, and dietary habits.

[0180] If, in a specific process, the determination process determines that "immune activity is high," immune care actions may include, for example, maintaining healthy lifestyle habits, taking supplements or foods known to have immune-boosting activity (especially pDC-boosting activity) as needed, maintaining appropriate exercise habits, maintaining appropriate sleep habits, and maintaining appropriate dietary habits.

[0181] Foods and beverages known to have immune-boosting activity include, for example, bacteria such as lactic acid bacteria, bifidobacteria, and acetic acid bacteria or compositions containing them, vitamins, lactoferrin, oligosaccharides, dietary fiber, fermented foods, and other substances that improve the intestinal environment.

[0182] If the subject is one whose immune activity has already been determined by the method for determining the immune activity of a subject according to this embodiment, the identification step may be a step in which, in addition to the determination result from the determination step, actions for improving or maintaining immune activity in accordance with the determination result from the previous determination step are identified based on the determination result from the above determination step.

[0183] [Apparatus for Determining Immune Activity] The apparatus for determining immune activity according to this embodiment (hereinafter also referred to as the "immune activity determination apparatus") comprises a computer including a processor and memory under the control of the processor. The memory contains a computer program that causes the computer to perform the following steps: A) a determination step of determining the immune activity of a subject based on the amount of at least one biomarker in urine collected from the subject, and B) an output step of outputting the determination result of the immune activity of the subject.

[0184] Figure 1 is a schematic diagram of an immunoactivity determination device according to one embodiment. The immunoactivity determination device 10 shown in Figure 1 includes a device 20 (measuring device 20) for measuring the amount of the above-mentioned biomarker and a computer system 30 connected to the measuring device 20.

[0185] The computer system 30 includes a computer main unit 300, an input unit 301, and a display unit 302 that displays sample information, judgment results, etc. The computer system 30 receives information on the amount of at least one biomarker in the urine collected from the subject from the measuring device 20. The processor of the computer system 30 then executes the computer program installed on the hard disk 313 based on the information on the amount of the biomarker. The computer system 30 may be a separate device from the measuring device 20, as shown in Figure 1, or it may be a device that encloses the measuring device 20. In the latter case, the computer system 30 may itself be the immunoactivity determination device 10. The computer program may be installed on a commercially available measuring device. Furthermore, the immunoactivity determination device 10 only needs to be able to receive information on the amount of at least one biomarker in the urine collected from the subject from the measuring device 20, and does not need to include the measuring device 20. Moreover, the immunoactivity determination device 10 may include the measuring device 20, in which case the measuring device 20 may be a small device held by the subject.

[0186] As shown in Figure 2, the computer main unit 300 includes a CPU (Central Processing Unit) 310, a ROM (Read Only Memory) 311, a RAM (Random Access Memory) 312, a hard disk 313, an input / output interface 314, a reader 315, a communication interface 316, and an image output interface 317. The CPU 310, ROM 311, RAM 312, hard disk 313, input / output interface 314, reader 315, communication interface 316, and image output interface 317 are connected via a bus 318 for data communication. The measuring device 20 is also connected to the computer system 30 via the communication interface 316 for communication.

[0187] The CPU 310 is a processor capable of executing computer programs stored in the ROM 311 or hard disk 313 and computer programs loaded into the RAM 312. The CPU 310 determines the immune activity of a subject based on the amount of at least one biomarker in the urine collected from the subject. The CPU 310 reads threshold values ​​or deviation values, or information for calculating these values, stored in the ROM 311 or hard disk 313 as needed, and determines the immune activity of the subject. The CPU 310 outputs the result of the determination of the subject's immune activity and displays it on the display unit 302.

[0188] ROM 311 is a memory module composed of a mask ROM, PROM, EPROM, EEPROM, etc. As mentioned above, ROM 311 stores computer programs executed by the CPU 310 and data used to execute said computer programs. ROM 311 may also store data used in the immunoactivity determination method performed by the immunoactivity determination device 10 described later, such as threshold values ​​or deviation values, or information for calculating these values.

[0189] The RAM 312 is composed of SRAM, DRAM, etc. The RAM 312 is used to read computer programs recorded in the ROM 311 and the hard disk 313. The RAM 312 is also used as a workspace for the CPU 310 when executing these computer programs.

[0190] The hard disk 313 has computer programs such as an operating system and application programs (the computer programs mentioned above) installed on it, as well as data used to execute the computer programs, which are to be executed by the CPU 310. The hard disk 313 may also contain data used in the immunoactivity determination method performed by the immunoactivity determination device 10 described later, such as information for threshold calculation or information for standard score calculation.

[0191] The reading device 315 is composed of a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, etc. The reading device 315 can read computer programs or data recorded on the portable recording medium 40.

[0192] The input / output interface 314 is composed of, for example, a serial interface such as USB, IEEE 1394, or RS-232C, a parallel interface such as SCSI, IDE, or IEEE 1284, and an analog interface consisting of a D / A converter, an A / D converter, etc. An input unit 301 such as a keyboard or mouse is connected to the input / output interface 314. The operator can input various commands to the computer main unit 300 using the input unit 301.

[0193] The communication interface 316 is, for example, an Ethernet® interface. The computer main unit 300 can also transmit judgment result data to a printer, as well as to the target's smartphone, computer, and small device such as the measuring device 20, via the communication interface 316.

[0194] The image output interface 317 is connected to a display unit 302, which is composed of an LCD, CRT, or the like. This allows the display unit 302 to output a video signal corresponding to the image data provided by the CPU 310. The display unit 302 displays an image (screen) according to the input video signal.

[0195] The immunoactivity determination method performed by the immunoactivity determination device 10 will now be described. The immunoactivity determination method performed by the immunoactivity determination device 10 involves a computer program stored in memory (ROM 311, etc.) which performs the following steps: A) a determination step of determining the immune activity of a subject based on the amount of at least one type of biomarker in the urine collected from the subject; and B) an output step of outputting the determination result of the immune activity of the subject.

[0196] The specific details of the determination step may be as described in [Method for Determining Immune Activity]. The determination step may be performed based on the amount of at least one biomarker in the urine collected from the subject. For example, it may be a step in which the immune activity of the subject is determined to be high if the value of the parameter based on the amount of the biomarker is higher than a predetermined threshold, or it may be a step in which the immune activity of the subject is determined to be high if the value of the parameter based on the amount of the biomarker is lower than a predetermined threshold. Conversely, the determination step may be a step in which the immune activity of the subject is determined to be low if the value of the parameter based on the amount of the biomarker is lower than a predetermined threshold, or it may be a step in which the immune activity of the subject is determined to be low if the value of the parameter based on the amount of the biomarker is higher than a predetermined threshold. In other words, the determination step may include at least one of (A) to (D) above. The determination step may also be performed based on the amount of at least one biomarker in the urine collected from the subject. For example, it may be a step in which the standard score of the parameter value based on the amount of the biomarker is calculated when multiple subjects are used as a population, and the determination is made based on the standard score.

[0197] Figure 3 is a flowchart showing an example of the immunoactivity determination method. The immunoactivity determination method performed by the immunoactivity determination device 10 allows for the determination of immunoactivity in a simple and non-invasive manner.

[0198] [Step S1: Information Acquisition from Target Information Database (DB)] First, information on the amount of at least one biomarker in the urine collected from the subject, measured by the measuring device 20, is acquired from the target information database. In step S1, personal information of the subject, such as age and gender, may also be acquired.

[0199] [Step S2: Information Acquisition from Threshold Calculation Information DB] Next, information necessary for threshold calculation is acquired from the threshold calculation information DB. Examples of information necessary for threshold calculation include information on the amount of at least one biomarker in the urine of a group of people of the same age or generation as the subject, a group of people of the same sex as the subject, a group of people with similar genetic characteristics, physical characteristics or lifestyle habits to the subject, information on immune activity, and background information of the group (age, sex, lifestyle habits, genetic information, etc.).

[0200] [Threshold Calculation Step S3] Next, the threshold is calculated based on the information necessary for threshold calculation. The threshold calculation is as described above.

[0201] [High / Low Determination Step S4] Next, the amount of at least one biomarker in the urine collected from the subject is compared with a threshold value to determine whether the immune activity is high or low. Step S4 may be performed without going through steps S2 and S3, for example by reading a threshold value stored in ROM 311 or hard disk 313.

[0202] [Step S5: Comparison with Previous Assessment] If the subject has already had its immune activity level assessed, the previous assessment of immune activity level is compared with the current assessment of immune activity level. If the subject has never had its immune activity level assessed, step S5 is skipped, and the process proceeds from step S4 to step S6.

[0203] [Step S6: Confirmation of the presence / details of immune care behaviors] Next, obtain information on the presence / details of immune care behaviors that the subjects have responded to. The details of the immune care behaviors may be as described above.

[0204] [Result Report Content Generation Step S7] Next, based on the results from steps S4 to S6, a report summarizing those results is generated (output step). For example, the report may include the judgment result from step S4, the comparison result with the previous judgment result from step S5, and the immune care actions corresponding to the judgment result from step S6. If steps S10 to S11 have been completed, based on the results from steps S10 to S11, a report summarizing those results is generated.

[0205] [Result Report Output Step S8] Next, the report generated in step S7 is output.

[0206] [Step S9: Information Acquisition from the Standard Score Calculation Information Database] Following step S1, information necessary for calculating the standard score is acquired from the standard score calculation information database. Examples of information necessary for calculating the standard score include information on the amount of at least one biomarker in the urine of a group of people of the same age or generation as the subject, a group of people of the same sex as the subject, a group of people with similar genetic characteristics, physical characteristics or lifestyle habits to the subject, information on immune activity, and background information of the group (age, sex, lifestyle habits, genetic information, etc.).

[0207] [Standard Score Calculation Step S10] Following Step S9, the standard score is calculated based on the information necessary for calculating the standard score. The calculation of the standard score is as described above.

[0208] [Comparison with previous standard score step S11] After step S10, if the subject has already had a standard score for immune activity calculated, the previous standard score for immune activity is compared with the current standard score for immune activity. If the subject has never had its level of immune activity determined, step S11 is skipped and the process proceeds from step S10 to step S6.

[0209] [Computer Program] The computer program according to this embodiment is a computer program that causes a computer to perform the following steps: A) a determination step of determining the immune activity of a subject based on the amount of at least one biomarker in the urine collected from the subject; and B) an output step of outputting the determination result of the immune activity of the subject.

[0210] The computer program according to this embodiment causes the computer to execute steps A) and B) above, and therefore can determine immune activity. Accordingly, the computer program according to this embodiment is a computer program for determining immune activity. By having the computer execute the computer program according to this embodiment, the computer operates as an immune activity determination device.

[0211] The computer program according to this embodiment is provided, for example, by being recorded on a computer-readable recording medium. The recording medium may be a non-temporary recording medium. Examples of recording media include flexible disks, CDs, DVDs, ROMs, semiconductor memory, and the like.

[0212] [Reagent or Reagent Kit] The reagent or reagent kit according to this embodiment is a reagent or reagent kit for use in a method for determining the target immunoactivity according to this embodiment, and contains a substance capable of specifically detecting the above-mentioned biomarker in urine.

[0213] As described above, the immune activity of the subject can be determined based on the amount of at least one biomarker in the urine collected from the subject. Therefore, a substance capable of specifically detecting the above biomarker in urine can be used in the novel application of a method for determining the immune activity of a subject according to this embodiment.

[0214] Substances capable of specifically detecting the above-mentioned biomarkers in urine may include, for example, antibodies, aptamers, or complexes thereof with dyes that bind to them.

[0215] When the biological marker in urine is IgA, examples of substances that can specifically detect IgA in urine include anti-IgA antibodies and IgA-specific aptamers. When the biological markers in urine are ARL5B, SOD3, BGAL, SARP-2, Tenascin, DHR11, CHM2B, GNPTG, CgA, and RAB21, examples of substances that can specifically detect ARL5B, SOD3, BGAL, SARP-2, Tenascin, DHR11, CHM2B, GNPTG, CgA, and RAB21 in urine include specific antibodies and specific aptamers against them. When the biological marker in urine is hsa-let-7c-5p, examples of substances that can specifically detect hsa-let-7c-5p in urine include specific primers, target probes, and aptamers against hsa-let-7c-5p. If the biomarkers in the urine are hsa-miR-99a-5p, hsa-miR-30c-5p, ENST00000516507.1, ENST00000698785.1, hsa-miR-125b-5p, ENST00000531977.1, ENST00000663798.1, hsa-miR-30b-5p, ENST00000433588.1, ENST00000643616.1, ENST00000622359.1, and ENST00000629295.1, then the urinary biomarkers are hsa-miR-99a-5p and hsa-m Substances that can specifically detect iR-30c-5p, ENST00000516507.1, ENST00000698785.1, hsa-miR-125b-5p, ENST00000531977.1, ENST00000663798.1, hsa-miR-30b-5p, ENST00000433588.1, ENST00000643616.1, ENST00000622359.1, and ENST00000629295.1 include, for example, specific primers, target probes, and aptamers for these substances.If the biomarkers in urine are hsa-miR-99a-5p, hsa-miR-30c-5p, ENST00000516507.1, hsa-miR-125b-5p, ENST00000531977.1, and hsa-miR-30b-5p, then examples of substances that can specifically detect hsa-miR-99a-5p, hsa-miR-30c-5p, ENST00000516507.1, hsa-miR-125b-5p, ENST00000531977.1, and hsa-miR-30b-5p in urine include specific primers, target probes, and aptamers for these markers.

[0216] The reagent or reagent kit according to this embodiment may further contain, in addition to a substance capable of specifically detecting biomarkers in urine, a buffer solution, a protease inhibitor, a nuclease inhibitor, and the like.

[0217] [Method for evaluating the immunostimulatory activity of a test substance] The method for evaluating the immunostimulatory activity of a test substance according to this embodiment includes an evaluation step of evaluating the immunostimulatory activity of the test substance based on the amount of at least one biomarker in the urine collected from a subject who ingested the test substance.

[0218] The method for evaluating the immunostimulatory activity of a test substance according to this embodiment may further include a measurement step of measuring the amount of at least one biomarker in the urine collected from a subject who ingested the test substance. The measurement step is as described above. Here, in order to perform a more accurate evaluation, the number of subjects may be multiple (for example, two or more, five or more, ten or more, fifteen or more, twenty or more, etc.), and in the case of multiple subjects, the amount of at least one biomarker in the urine of the subjects may be the average value of all subjects, and this may be called "the amount of at least one biomarker in the urine of the target group".

[0219] The test substance is not particularly limited as long as it is a substance that is expected to have immunostimulatory activity. Immunostimulatory activity is the activity that enhances immune activity, and immunostimulatory activity and immune activity are as described above.

[0220] As described above, the amount of at least one biomarker in the urine collected from a subject is positively or negatively correlated with immune activity. Therefore, the immune activity of the subject can be determined based on the amount of at least one biomarker in the urine collected from the subject. Accordingly, the immunostimulatory activity of the test substance can be evaluated by assessing the increase or decrease in the amount of at least one biomarker in the urine collected from a subject that has ingested the test substance. Evaluating the increase or decrease in the amount of at least one biomarker in the urine collected from a subject that has ingested the test substance may also be done, for example, by evaluating the increase or decrease based on the value of a parameter based on the amount of at least one biomarker in the urine collected from the subject before ingestion of the test substance. The value of the parameter based on the amount of biomarker is as described above.

[0221] The duration and amount of intake of the test substance can be adjusted as appropriate depending on the type of test substance. For example, the duration of intake of the test substance may be one day or more, two days or more, three days or more, one week or more, two weeks or more, three weeks or more, four weeks or more, one month or more, two months or more, three months or more, four months or more, twelve months or less, ten months or less, eight months or less, or six months or less. For example, the amount of test substance taken per dose may be 10 mg or more, 100 mg or more, 500 mg or more, 1 g or more, 5 g or more, 10 g or more, 100 g or less, 80 g or less, 60 g or less, or 15 g or less. The amount of the test substance ingested per dose may be 10 mg to 100 g, 10 mg to 80 g, 10 mg to 15 g, 100 mg to 80 g, 100 mg to 15 g, 500 mg to 100 g, 500 mg to 80 g, or 500 mg to 15 g.

[0222] The evaluation step may include, for example, comparing the value of a parameter based on the amount of at least one biomarker in urine collected from a subject before ingestion of the test substance (value of the parameter based on the amount of biomarker before ingestion) with the value of a parameter based on the amount of at least one biomarker in urine collected from a subject after ingestion of the test substance (value of the parameter based on the amount of biomarker after ingestion). In this case, the method for evaluating the immunostimulatory activity of the test substance according to this embodiment may further include a first measurement step of measuring the amount of biomarker before ingestion and a second measurement step of measuring the amount of biomarker after ingestion. The measurement of the amount of biomarker may be as described above.

[0223] Furthermore, the evaluation process may include, for example, comparing the parameter value based on the amount of biomarkers before ingestion with the parameter value based on the amount of biomarkers after ingestion, and evaluating that the ingested test substance has immunostimulatory activity if the parameter value based on the amount of biomarkers after ingestion is higher, or comparing the parameter value based on the amount of biomarkers before ingestion with the parameter value based on the amount of biomarkers after ingestion, and evaluating that the ingested test substance has immunostimulatory activity if the parameter value based on the amount of biomarkers after ingestion is lower.

[0224] The evaluation process may include, for example, preparing subjects (or control groups) that ingest the test substance and subjects (or control groups) that do not ingest the test substance, and comparing the value of a parameter based on the amount of at least one biomarker in the urine collected from the subjects (or control groups) that ingested the test substance with the value of a parameter based on the amount of at least one biomarker in the urine collected from the subjects (or control groups) that did not ingest the test substance.

[0225] Furthermore, the evaluation process may include comparing the parameter value based on the amount of biomarkers in urine collected from subjects (or control groups) that ingested the test substance with the parameter value based on the amount of biomarkers in urine collected from subjects (or control groups) that did not ingest the test substance, and evaluating that the ingested test substance has immunostimulatory activity if the parameter value based on the amount of biomarkers in urine collected from subjects (or control groups) that ingested the test substance is higher. The evaluation process may also include comparing the parameter value based on the amount of biomarkers in urine collected from subjects (or control groups) that ingested the test substance with the parameter value based on the amount of biomarkers in urine collected from subjects (or control groups) that did not ingest the test substance, and evaluating that the ingested test substance has immunostimulatory activity if the parameter value based on the amount of biomarkers in urine collected from subjects (or control groups) that ingested the test substance is lower.

[0226] The method for evaluating the immunostimulatory activity of a test substance according to this embodiment can be used as a screening method for substances having immunostimulatory activity.

[0227] [Biomarker Screening Method] The biomarker screening method according to this embodiment is a method for screening biomarkers in a biological sample for determining immune activity, and includes a first measurement step of measuring the immune activity of a target, a second measurement step of measuring the amount of at least one biomarker in a biological sample taken from the target, and a determination step of determining whether or not the biomarker is a biomarker useful for determining immune activity based on the correlation between the immune activity of the target and the amount of the biomarker.

[0228] Examples of biological samples include urine, blood, saliva, and feces, with urine being preferred.

[0229] The first measurement step is not particularly limited, as long as it can measure an indicator of the target's immune activity by a conventionally known method. For example, the first measurement step may be a step of separating peripheral blood mononuclear cells (PBMCs) from blood collected from the target, stimulating the PBMCs with TLR7 / 8 ligand (R848), and a step of determining the number of pDCs in the PBMCs that produce IFN-α (IFN-α ＋ The method may also include a step of measuring the proportion of pDCs. The specific procedure is as described in the examples below. The method may also include a step of measuring the quantity of various immune cells such as dendritic cells, eosinophils, neutrophils, basophils, macrophages, and natural killer cells (NK cells), or the expression level of surface markers or production factors that serve as indicators of activation or inactivation.

[0230] The biomarkers measured in the second measurement step may be, for example, proteins, nucleic acids (e.g., DNA, mRNA, and miRNA), sugars, and lipids. The number of types of biomarkers measured in the second measurement step is not particularly limited and may be, for example, one or more, five or more, ten or more, fifty or more, one hundred or more, five hundred or more, one thousand or more, five thousand or more, or seven thousand or more.

[0231] The second measurement step may be the same as the measurement step described in [Method for Determining Immune Activity], but with "in urine" replaced by "in a biological sample." The second measurement step can be carried out by a method known to those skilled in the art. In particular, if there are multiple types of biomarkers to be measured in the second measurement step, the second measurement step may be carried out by a known measurement method that can measure the amounts of multiple biomarkers at once, from the viewpoint of efficient screening. For example, if the biomarkers include proteins, they can be measured by a DNA chip method (microarray method) using the same number of aptamers as the type of protein to be measured. More specifically, the protein measurement service provided by SomaLogic can also be used. Furthermore, if the biological sample is urine, as described above, the concentration (absolute value) of protein in the urine may be calculated from the value obtained by the said method, and the concentration of protein in the urine may be further normalized by the amount of internal standard substance.

[0232] The determination step determines whether a biomarker is a useful biomarker for determining immune activity, based on the correlation between the immune activity of the target obtained in the first measurement step and the amount of at least one biomarker in the biological sample taken from the target obtained in the second measurement step. The immune activity of the target obtained in the first measurement step only needs to be an indicator of the immune activity of the target obtained in the first measurement step, and the number of pDCs in PBMCs separated from the blood taken from the target is the number of pDCs that produce IFN-α (IFN-α ＋ The ratio of the number of pDCs may also be acceptable.

[0233] In the determination step, when evaluating the correlation between the immune activity of the target obtained in the first measurement step and the amount of at least one biomarker in the biological sample taken from the target obtained in the second measurement step, the correlation may be calculated and evaluated using data from all targets, or the correlation may be calculated and evaluated using data from some targets based on their immune activity. When calculating and evaluating the correlation using data from some targets based on their immune activity, for example, the correlation may be calculated and evaluated using data from the top 25% of targets and the bottom 25% of targets based on their immune activity.

[0234] By analyzing the correlation between the immune activity of the target obtained in the first measurement step and the amount of the biomarker obtained in the second measurement step, if a correlation is found between the two, it can be determined that the biomarker is a useful biomarker for determining immune activity.

[0235] [Immunostimulating Composition] The immunostimulating composition according to this embodiment may be an immunostimulating composition containing a substance having immunostimulating activity, which is administered to a subject whose immune activity is determined to be low by the method for determining the immune activity of the subject according to this embodiment.

[0236] Furthermore, the immunostimulatory composition according to this embodiment may be an immunostimulatory composition containing a substance having immunostimulatory activity, which is administered to a subject that was not determined to have high immune activity by the method for determining the immune activity of the subject according to this embodiment. The substance having immunostimulatory activity is not particularly limited and examples include low molecular weight compounds, proteins, glycoproteins, nucleic acids, bacteria such as lactic acid bacteria, bifidobacteria and acetic acid bacteria or compositions containing them, vitamins, lactoferrin, and substances that have an effect of improving the intestinal environment, such as oligosaccharides, dietary fiber and fermented foods.

[0237] The content of the substance having immunostimulatory activity in the immunostimulatory composition according to this embodiment may be, for example, 0.0001% by mass or more, 0.001% by mass or more, 0.01% by mass or more, 0.1% by mass or more, 1% by mass or more, 3% by mass or more, 5% by mass or more, or 10% by mass or more, relative to the total amount of the immunostimulatory composition, and may be 90% by mass or less, 85% by mass or less, 80% by mass or less, 75% by mass or less, 70% by mass or less, or 65% by mass or less. Furthermore, the content of the substance having immunostimulatory activity in the immunostimulatory composition according to this embodiment may be, for example, 0.0001% by mass or more and 90.0% by mass or less, 0.001% by mass or more and 85.0% by mass or less, 0.1% by mass or more and 80.0% by mass or less, 1% by mass or more and 75.0% by mass or less, 5% by mass or more and 70.0% by mass or less, or 10% by mass or more and 65.0% by mass or less, relative to the total amount of the immunostimulatory composition.

[0238] The immunostimulatory composition according to this embodiment may be a food or beverage, a pharmaceutical product, or a quasi-drug. Food and beverages, pharmaceuticals, or quasi-drugs can each be manufactured in accordance with conventional methods. The content of the substance having immunostimulatory activity in the food or beverage, pharmaceutical, or quasi-drug is not particularly limited and can be freely set according to the purpose.

[0239] If the immunostimulatory composition according to this embodiment is a food or beverage, pharmaceutical, or quasi-drug, the food or beverage, pharmaceutical, or quasi-drug may contain, in addition to the substance having immunostimulatory activity, components that can be commonly used in food or beverages, pharmaceuticals, or quasi-drugs. The food or beverage, pharmaceutical, or quasi-drug according to one embodiment may contain bases, carriers, additives, etc., that are commonly used in food or beverages, pharmaceuticals, or quasi-drugs. Examples of additives include excipients, oils, powders, buffers, solubilizers, antioxidants, surfactants, thickeners, preservatives, pH adjusters, chelating agents, stabilizers, irritation reducers, antiseptics, pigments, colorants, fragrances, gloss enhancers, gelling agents, alcohols, water-soluble polymers, film-forming agents, resins, etc. The base, carrier, and the various additives described above can be used individually or in combination as needed.

[0240] When the immune-boosting composition according to this embodiment is a food or beverage, such food or beverage may be a health food, functional food, nutritional composition, nutritional supplement, supplement, health food, food for specified health uses, food with nutritional function claims, or food with functional claims. Such food compositions can be labeled, for example, with claims such as supporting the maintenance of immune function in healthy people (immune care), or for people concerned about a decline in immune function, or for suppressing the decline in immune function. The immune-boosting composition according to this embodiment can also be used as a food additive. When the immune-boosting composition according to this embodiment is administered to animals other than humans, the food or beverage is used as animal feed.

[0241] Examples of such food and beverages include seasonings, processed meat products, processed agricultural products, beverages (lactic acid bacteria beverages, soft drinks, alcoholic beverages, carbonated beverages, milk beverages, fruit juices, tea, coffee, nutritional drinks, etc.), powdered beverages (powdered juice, powdered soup, powdered milk, etc.), concentrated beverages, confectionery (candy (throat lozenges), cookies, biscuits, gum, gummies, chewable tablets, tablets, chocolate, etc.), bread, cereals, etc. In the case of Foods for Specified Health Uses, Foods with Nutrient Function Claims, Foods with Function Claims, etc., they may also be in the form of capsules, granules, powders, syrups, lozenges, etc.

[0242] If the immunostimulatory composition according to this embodiment is a pharmaceutical or quasi-drug, the dosage form of the pharmaceutical or quasi-drug may be, for example, a liquid, suspension, capsule, granule, pill, powder, tablet, syrup, lozenge, etc. Possible indications for use as a pharmaceutical include allergies, obesity, and heart failure.

[0243] The immunostimulatory composition according to this embodiment is preferably ingested into the body. The administration method may be oral or parenteral, but oral administration is preferred. The immunostimulatory composition according to this embodiment may be ingested only once or multiple times, and if sustained immune stimulation is desired, it is preferable to ingest it continuously or intermittently over a certain period of time. The food composition according to one embodiment may be ingested, for example, 1 to 5 times a day, once every 2 days, once every 3 days, once every 4 days, or once a week over a period of 1 day or more, 1 week or more, 2 weeks or more, 1 month or more, 3 months or more, 6 months or more, 1 year or more, 3 years or more, 5 years or more, or 10 years or more.

[0244] The immunostimulatory composition according to this embodiment may be manufactured, for example, by a method that includes a step of screening for substances having immunostimulatory activity using the screening method according to this embodiment.

[0245] [Method, substance having immunostimulatory activity, and use thereof] As described above, in the method for determining the immune activity of a subject according to this embodiment, the level of the subject's immune activity can be determined based on the amount of at least one biomarker collected from the subject, so the subject's immune activity can be determined mechanically. Therefore, the determination result can also be used as an aid in the overall determination of the subject's immune activity.

[0246] In other words, the present invention provides a method for assisting in the determination of the immune activity of a subject, comprising a determination step of determining the immune activity of the subject based on the amount of at least one biomarker in the urine collected from the subject. The present invention also provides a method for using the amount of at least one biomarker in the urine collected from the subject as an indicator for determining the immune activity of the subject. The present invention further provides a method for collecting data for determining the immune activity of a subject, comprising a determination step of determining the immune activity of the subject based on the amount of at least one biomarker in the urine collected from the subject.

[0247] The present invention also provides an immunostimulation method, which includes administering an immunostimulatory composition containing a substance having immunostimulatory activity to a subject that has been determined to have low immune activity by a method for assisting in determining the immune activity of a subject or a method for determining the immune activity of a subject.

[0248] The present invention also provides a substance having immunostimulatory activity for use in immunostimulation, which is used for immunostimulation of a target whose immune activity has been determined to be low by a method for assisting in determining the immune activity of the target or by a method for determining the immune activity of the target.

[0249] The present invention also provides the use of a substance having immunostimulatory activity for immunostimulation of a target whose immune activity has been determined to be low by a method for assisting in determining the immune activity of a target or by a method for determining the immune activity of a target.

[0250] The present invention also provides the use of a substance having immunostimulatory activity for producing an immunostimulatory composition for immunostimulating a target whose immune activity has been determined to be low by a method for assisting in determining the immune activity of a target or by a method for determining the immune activity of a target.

[0251] [Method for Determining the Risk of Disease Involving a Target] The method for determining the risk of disease in a target according to this embodiment may include a determination step of determining the risk of disease in the target based on the amount of at least one biomarker in the urine collected from the target. The biomarker may be as described above, and preferably includes miRNA. In particular, the miRNA described above shows a significant difference between targets with high immune activity and targets with low immune activity, and targets genes involved in the development of specific diseases. Since immune activity can be determined based on the amount of the biomarker described above, the method for determining the risk of disease in a target according to this embodiment can, for example, determine the risk of disease involuntarily developing in conjunction with a decrease in immune activity.

[0252] The determination step in the method for determining the disease risk of a subject according to this embodiment may be performed based on the amount of at least one biomarker in the urine collected from the subject. For example, the determination step may be a step in which the subject is determined to have a high risk of disease if the value of the parameter based on the amount of the biomarker is higher than a predetermined threshold, or a step in which the subject is determined to have a high risk of disease if the value of the parameter based on the amount of the biomarker is lower than a predetermined threshold. Conversely, the determination step may be a step in which the subject is determined to have a low risk of disease if the value of the parameter based on the amount of the biomarker is lower than a predetermined threshold, or a step in which the subject is determined to have a low risk of disease if the value of the parameter based on the amount of the biomarker is higher than a predetermined threshold. In other words, the determination step may include at least one of the following (E) to (H). (E): If the value of the parameter based on the amount of the above biomarker is higher than a predetermined threshold, the subject is determined to have a high risk of developing the disease. (F): If the value of the parameter based on the amount of the above biomarker is lower than a predetermined threshold, the subject is determined to have a low risk of developing the disease. (G): If the value of the parameter based on the amount of the above biomarker is higher than a predetermined threshold, the subject is determined to have a low risk of developing the disease. (H): If the value of the parameter based on the amount of the above biomarker is lower than a predetermined threshold, the subject is determined to have a high risk of developing the disease.

[0253] The target is not particularly limited, but it may be a target that has been determined to have low immune activity by the method for determining the immune activity of the target according to this embodiment.

[0254] The disease is not particularly limited, but may be at least one selected from the group consisting of cancer, endocrine disorders, metabolic disorders, infectious diseases, cardiovascular diseases, and neurodegenerative diseases. The infectious disease may be at least one selected from the group consisting of bacterial, viral, and parasitic infections.

[0255] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0256] (Isolation of Peripheral Blood Mononuclear Cells (PBMCs)) Blood samples were collected from 223 participants aged 51-55 years (average age 53.0 years, 90 males, 133 females) of the Wakayama Health Promotion Study. The collected blood was gathered in blood collection tubes for mononuclear cell isolation, centrifuged at 1800 x g for 20 minutes, and then mixed by inversion about 10 times. Within 24 hours, the mononuclear cell layer was transferred to a tube containing PBS, centrifuged, and the supernatant was removed. The cell aggregates were suspended in cell / tissue cryopreservation solution CELLBANKER2 (registered trademark) (manufactured by Takara Bio Inc.) and stored at -80°C until use.

[0257] (Collection of Urine Samples) Urine samples collected by the participants of the Wakayama Health Promotion Study were stored at -80°C until measurement.

[0258] (Test Example 1: Measurement of Immunoactive Activity Using pDC Activity as an Indicator) Frozen peripheral blood mononuclear cells were thawed and resuspended in RPMI 1640 medium (+ 10% FBS, 1% penicillin / streptomycin) (Sigma-Aldrich). Then, centrifugation was performed, the supernatant was removed, and the cells were washed with phosphate-buffered saline. After that, the cells were resuspended in RPMI 1640 medium and passed through a 45 μm filter. Next, centrifugation was performed, the supernatant was removed, and the peripheral blood mononuclear cells were measured at 1.5 × 10⁶ ６ Cells were suspended in RPMI 1640 medium to a concentration of cells / mL and seeded into 12-well plates (IWAKI). Cells were either left unstimulated or stimulated with the TLR7 / 8 ligand R848 (final concentration 10 μM) (BioLegend), and then incubated at 37°C and 5% CO2. ２The cells were incubated for 4 hours. Two hours after stimulation, Brefeldin A (BioLegend) was added to retain the cytokines within the cells. After incubation, the cells were harvested and washed with PBS. The cells were stained with Zombie Red Fixable Viability Kit (BioLegend, 423109) for 15 minutes, and then washed with FACS buffer (PBS + 0.5% BSA). After treating the cells with Fc block (BD Pharmaingen, 564220) for 5 minutes, antibodies against cell surface markers were applied (FITC anti-human Lineage Cocktail (CD3, CD14, CD19, CD20, CD56) (BioLegend, 348701), PE / Cyanine7 anti-human CD11c antibody (BioLegend, 301608), APC-H7 mouse anti-human HLA-DR (BD, 561358), PerCP / Cyanine5.5 anti-human CD123). Cells were stained with antibody (BioLegend, 306016) and Brilliant Violet 421 (registered trademark) anti-human CD303 (BDCA-2) antibody (BioLegend, 354212) for 30 minutes and washed twice with FACS buffer. The cells were treated with Fixation / Permeabilization solution (BD, 554714) for 20 minutes, washed twice with FACS buffer, and washed three times with Permeabilization buffer (BD, 554714). Cells were stained with PE mouse anti-human IFN-α [2b] (BD, 560097), an antibody against intracellular cytokines (IFN-α), for 20 minutes, and then washed twice with Permeabilization buffer. The cells were resuspended in FACS buffer and analyzed using a Navios EX flow cytokineter (Beckman Coulter). Analysis was performed using Kaluza (Beckman Coulter).After removing dead cells, the population of lineage-HLA-DR+ CD11c- CD123+ CD303+ was analyzed as pDC. Gate determination was performed using fluorescence minus one and isotype controls, and the ratio of the number of pDCs producing IFN-α (IFN-α. ＋ pDC) to the number of pDCs in PBMC was used as an activity index of pDC to measure immune activity.

[0259] (Test Example 2 Analysis of Urinary Markers (Proteins)) Among the 223 participants whose immune activity (pDC activity) was measured, participants with a pDC ratio in PBMC above the median of the whole were selected, and participants with a history of COVID-19 infection were excluded. Also, arranged in descending order of immune activity (ratio of the number of IFN-α ＋ pDC to the number of pDCs), participants with a ratio of the number of IFN-α ＋ pDC to the number of pDCs less than 1% and those determined as outliers were excluded, and then 20 participants each were selected from those included in the upper 25% or lower 25% (upper 25%: 8 males and 12 females (participants with high immune activity), lower 25%: 10 males and 10 females (participants with low immune activity)). The frozen urine of the 40 selected participants was thawed, and the total protein concentration was measured by the BCA method. After diluting to a total protein concentration of 200 μg / mL, it was subjected to SomaScan (registered trademark) Assay (service of SomaLogic), and more than 7000 proteins including IgA and albumin were relatively quantified. The amount of IgA relatively quantified by SomaScan (registered trademark) Assay (service of SomaLogic) was defined as the IgA relative amount. Also, the total protein concentration in urine was measured by the pyrogallol red method, the albumin concentration in urine was measured by the immunoturbidimetry method, and the creatinine concentration in urine was measured by the enzymatic method. A conversion coefficient was obtained from the albumin measurement value by SomaScan (registered trademark) Assay, the total protein concentration by the pyrogallol red method, and the albumin measurement value by the immunoturbidimetry method, and the IgA concentration in urine was calculated using the conversion coefficient. Using the calculated values of the IgA concentration in urine and the creatinine concentration in urine, the mass (mg) of IgA in urine per 1 g of creatinine in urine (ratio of the mass (mg) of IgA in urine to the creatinine concentration (g) in urine; IgA / creatinine ratio) was calculated.

[0260] Figure 4 shows a comparison of the relative IgA levels of participants with high immune activity and those with low immune activity.

[0261] As shown in Figure 4, relative IgA levels were significantly higher in participants with high immune activity. This suggests a correlation between immune activity (particularly pDC activity) and the amount of IgA in urine.

[0262] (Test Example 3: Prediction of immune activity using urinary IgA as an indicator) IFN-α in relation to the number of pDCs in PBMCs ＋ A threshold for distinguishing between high and low levels of immune activity was set using the proportion of pDCs, the relative amount of IgA measured by SomaScan® Assay, the calculated IgA concentration, or the IgA / creatinine ratio. This threshold was set so that the discrimination accuracy was 70% or higher. The discrimination accuracy was calculated using the following formula: Discrimination accuracy (%) = {(Number of accurately classified samples) / (Total number of samples)} × 100

[0263] When the threshold was set within the range of IgA concentration [mg / mL] greater than 0.00858 and 0.0892 or less, or relative IgA amount [RFU] greater than 6923 and 7197 or less, the discriminant accuracy was 77.5%. The results when the threshold was set at a relative IgA amount of 7000 RFU are shown in Table 1 and Figure 5. The discriminant accuracy is calculated as {(14 + 17) / 40} × 100 = 77.5%.

[0264] Similarly, when the threshold was set within the range of IgA concentration [mg / mL] greater than 0.00892 and 0.00959 or less, or greater than 0.00595 and 0.00600 or less, or relative IgA amount [RFU] greater than 7197 and 7732 or less, or greater than 4800 and 4839 or less, the discriminant accuracy was 75%.

[0265] Similarly, when the threshold was set within the range of IgA concentration [mg / mL] greater than 0.00959 and 0.00985 or greater than 0.00569 and 0.00595 or greater, or relative IgA amount [RFU] greater than 7732 and 7943 or greater than 4587 and 4800 or greater, the discriminant accuracy was 72.5%.

[0266] Similarly, when thresholds were set within the ranges of IgA concentration [mg / mL] greater than 0.01031 and 0.01033 or less, greater than 0.00985 and 0.01021 or less, or greater than 0.0056 and 0.00569 or less, or relative IgA amount [RFU] greater than 8314 and 8329 or less, greater than 7943 and 8235 or less, or greater than 4517 and 4587 or less, the discriminant accuracy was 70%.

[0267] Similarly, when the threshold was set within the range of IgA / creatinine ratio greater than 0.38 and less than or equal to 0.41, greater than or equal to 0.43 and less than or equal to 0.45, or greater than or equal to 0.80 and less than or equal to 0.82, the discriminant accuracy was 70%.

[0268] From these results, it was found that the level of immune activity can be easily and non-invasively determined using urinary IgA as an indicator.

[0269] (Test Example 4: Analysis of Urinary Markers (Proteins) (2)) As described in Test Example 2, the samples were subjected to SomaScan® Assay (a service provided by SomaLogic Inc.) to perform relative quantification of more than 7,000 types of proteins, including IgA and albumin. Based on the relative amounts of over 7,000 proteins quantified using SomaScan® Assay (a service provided by SomaLogic), proteins were extracted that met the following criteria: the ratio of the average relative amounts between the high-immune-activity group and the low-immune-activity group was 1.5 or higher (average relative amount of the high-immune-activity group / average relative amount of the low-immune-activity group ≥ 1.5, or average relative amount of the low-immune-activity group / average relative amount of the high-immune-activity group ≥ 1.5), and the p-value when comparing immune activity between the high-immune-activity group and the low-immune-activity group was less than 0.05 (student t-test). As a result, 115 proteins (120 SOMAmers) were identified.

[0270] (Test Example 5: Prediction of Immune Activity Using Urinary Markers (Proteins) as Indicators (1)) In order to evaluate the effectiveness of assessing the amount of each of the 115 types of proteins listed above in distinguishing between a group with high immune activity and a group with low immune activity, the AUC (Area Under the Curve) of the ROC (Receiver Operating Characteristic) curve was calculated. The results are shown in Table 2.

[0271] The ROC curve is a curve created by plotting (1 - specificity) against sensitivity. An AUC value of 1 indicates perfect discriminative ability, while an AUC value of 0.5 indicates random discriminative ability. An AUC value greater than 0.55 and less than or equal to 1 indicates discriminative ability, with values ​​closer to 1 indicating higher discriminative ability.

[0272]

[0273] As shown in Table 2, one protein with an AUC of ≥ 0.85, thirteen proteins with an AUC of ≥ 0.80, fifty proteins with an AUC of ≥ 0.75, eighty-seven proteins with an AUC of ≥ 0.70, eleven proteins with an AUC of ≥ 0.65, and eleven five proteins with an AUC of ≥ 0.55 were identified. These results demonstrate that by evaluating the amount of each of the 115 proteins mentioned above, it is possible to distinguish between groups with high and low immune activity. In particular, it was shown that evaluating the amount of proteins with an AUC between 0.6 and 1 is effective in distinguishing between groups with high and low immune activity.

[0274] (Test Example 6: Prediction of Immune Activity Using Urinary Markers (Proteins) as Indicators (2)) The effectiveness of evaluating the amount of each of the 115 proteins listed above in distinguishing between a group with high immune activity and a group with low immune activity was investigated. Tables 3 and 4 show the highest discriminative accuracy and threshold examples for each protein. The discriminative accuracy was calculated using the following formula: Discriminative Accuracy (%) = {(Number of accurately classified samples) / (Total number of samples)} × 100

[0275]

[0276]

[0277] As shown in Tables 3 and 4, one protein was identified with a discrimination accuracy of 85% or higher, seven proteins with an accuracy of 80% or higher, 44 proteins with an accuracy of 75% or higher, 96 proteins with an accuracy of 70% or higher, 114 proteins with an accuracy of 65% or higher, and 115 proteins with an accuracy of 60% or higher. These results also demonstrate that evaluating the amount of each of the above 115 proteins is effective in distinguishing between a population with high immune activity and a population with low immune activity.

[0278] (Test Example 7: Prediction of Immune Activity Using Urinary Markers (Proteins) as Indicators (3)) Using the ratio of the relative amount of one factor from among the proteins that were abundant in the group with high immune activity (31 types) to the relative amount of one factor from among the proteins that were abundant in the group with low immune activity (84 types) (relative amount of proteins abundant in the group with high immune activity / relative amount of proteins abundant in the group with low immune activity), an ROC curve was drawn to distinguish between the group with high immune activity and the group with low immune activity, and the AUC was calculated.

[0279] As a result, six protein combinations were identified with an AUC ≥ 0.90, 174 with an AUC ≥ 0.85, 847 with an AUC ≥ 0.80, 1901 with an AUC ≥ 0.75, 2510 with an AUC ≥ 0.70, and 2604 with an AUC > 0.63. Table 5 shows the protein combinations with an AUC ≥ 0.85.

[0280]

[0281] For nine proteins that are abundant in the highly immune-active population, the AUC (Amount Underlying Consumption) was ≥ 0.70 in all combinations with the 84 proteins that are abundant in the less immune-active population. Table 6 shows the protein combinations that result in the lowest AUC for each of these nine proteins. For 53 proteins that are abundant in the less immune-active population, the AUC was ≥ 0.70 in all combinations with the 31 proteins that are abundant in the highly immune-active population. Table 7 shows the protein combinations that result in the lowest AUC for each of these 53 proteins.

[0282]

[0283]

[0284] (Test Example 8: Prediction of Immune Activity Using Urinary Markers (Proteins) as Indicators (4)) From 120 protein measurement data including 115 types of proteins, we searched for combinations of four factors suitable for distinguishing between a group with high immune activity and a group with low immune activity. For the identified combinations of four factors, we created a model using XG-BOOST and calculated the AUC. One combination was identified with an AUC of ≥ 0.95, 261 combinations with an AUC of ≥ 0.90, 446 combinations with an AUC of ≥ 0.85, and 458 combinations with an AUC of ≥ 0.80. The results are shown in Table 8.

[0285]

[0286] (Example 9: Analysis of urinary markers (miRNA)) Of the 223 participants whose immune activity (pDC activity) was measured, participants whose proportion of pDCs in PBMCs was equal to or greater than the overall median were selected, and participants with a history of COVID-19 infection were excluded. In addition, immune activity (IFN-α relative to the number of pDCs) was analyzed.＋ The items are sorted in descending order of the ratio of pDCs, and IFN-α is the ratio of pDCs. ＋ After excluding participants with a pDC count of less than 1% and those identified as outliers, 20 participants each were selected from the top 25% and bottom 25% (top 25%: 8 males, 12 females (participants with high immune activity); bottom 25%: 10 males, 10 females (participants with low immune activity)). The frozen urine of the 40 selected participants was thawed, and comprehensive analysis of over 70,000 types of exosome miRNAs in the urine was performed using next-generation sequencing as described below. From the more than 70,000 miRNAs analyzed, we selected miRNAs that met the following criteria: the ratio of the mean expression levels between the high-immunity and low-immunity populations was 1.0 or greater (mean expression level of high-immunity population / mean expression level of low-immunity population ≥ 1.0 or mean expression level of low-immunity population / mean expression level of high-immunity population ≥ 1.0), and the corrected p-value when comparing the high-immunity and low-immunity populations was less than 0.1 (Deseq2 analysis pipeline). The mean expression levels between the high-immunity and low-immunity populations are calculated using the Deseq2 analysis pipeline after estimating the size factor and calculating the normalized count. Furthermore, the ratio of the mean expression levels between the high-immunity and low-immunity populations is a value obtained by further log2 transformation using the ratio of normalized count values, and the actual mean ratio is 2.0 or greater.

[0287] <Exosome miRNA Extraction from Urine Samples> Exosome miRNA extraction was performed using QIAGEN's "miRCURY Exosome Cell / Urine / CSF Kit" (product number 76743), following the steps outlined in the product manual. 1 mL of urine from each sample was centrifuged at 3,000 x g or higher for 10 minutes to separate the cell residue. The supernatant was transferred to a new tube, taking care not to disturb the cell residue pellet. 0.4 times the volume of Exosome Precipitation Solution (Urine) was added to the supernatant and thoroughly mixed by vortexing for 10 seconds. After incubation at 4°C for 60 minutes, the mixture was centrifuged again at 10,000 x g or higher for 30 minutes at 20°C. After centrifugation, the supernatant was carefully and completely removed, and the resulting pellet (exosomes) was suspended in 100 μL of resuspension buffer. The resulting exosome fraction was then purified of miRNA from the exosomes using QIAGEN's "miRNeasy Serum / Plasma kit" (product number 217184) according to the product manual. The total amount of miRNA extracted from each sample was quantified using Qubit.

[0288] <miRNA Sequencing Library Construction> The urinary miRNA sequencing library was prepared using the "QIAseq miRNA Library Kit" (product number 331905) from QIAGEN, following the steps outlined in the product manual. 5 μL of input miRNA was used for each sample. Adapter ligation was performed to both ends of the miRNA, and reverse transcription was carried out using primers that recognize the RNA 3' adapter to synthesize single-stranded cDNA. Using the obtained single-stranded cDNA as a template, PCR amplification was performed for 22 cycles using UDI primers. After PCR, cleanup was performed using magnetic beads, and each library was quality-evaluated and quantified using an Agilent 2100 BioAnalyzer. Equal volumes of the libraries from each sample were mixed, and only small RNAs were size-sorted using BluePippin to obtain the final sequencing library.

[0289] <Urinary miRNA Sequencing Analysis> miRNA sequencing was performed using Illumina NovaSeq 6000 (PE150). The raw sequence data obtained was converted to FASTQ format using bcl2fastq2 Conversion Software v2.20. Subsequently, miRNAs with significantly different expression levels were selected following the steps below. Finally, the top 30 differentially expressed miRNA candidates were selected. The raw FASTQ data was quality-checked using FastQC. Then, adapter trimming and removal of low-quality reads were performed using seqtk (ver 1.3-r106). Next, mapping and UMI deduplication were performed using CLC Genomics Workbench (ver 20.0.4) and Biomedical Genomics Analysis Plugin (ver 20.2). Subsequently, read annotation and count statistics were performed using Python (ver 3.6.8). For mapping, miRBase (ver 22.1) and Ensemble Homo sapiens GRCh38 (ver 110) were used as reference genome sequences to obtain gene symbols, RNA types, transcript lengths, and read counts. Differential expression analysis was performed using DESeq2 (ver 1.48.1) with miRNA read counts. During the analysis, the selection criteria for differentially expressed miRNAs were: corrected p-value < 0.1, |LFC| > 1, and basemean > 100.

[0290] (Test Example 10: Prediction of immune activity using differentially expressed miRNAs as indicators) The top 30 differentially expressed miRNAs were selected from the candidate groups. The 30 candidate factors are specifically: hsa-miR-99a-5p, hsa-miR-125b-5p, ENST00000531977.1, hsa-let-7c-5p, ENST00000516507.1, hsa-miR-30c-5p, hsa-miR-200c-3p, hsa-miR-151a-5p, hsa-miR-204-5p, hsa-miR-99b-5p, hsa-miR-103a-3p, hsa-miR-125a-5p, hsa-miR-29a-3p, ENST00000568314.1, ENST00000 563103.1, hsa-miR-30b-5p, hsa-miR-151a-3p, hsa-miR-200b-3p, hsa-mi R-92a-3p, hsa-miR-182-5p, hsa-miR-22-3p, hsa-miR-100-5p, hsa-miR-23 b-3p, ENST00000658801.1, hsa-miR-9-5p, ENST00000655322.1, hsa-miR- 141-3p, hsa-miR-191-5p, ENST00000630360.1, and hsa-miR-30c-2-3p. These miRNA candidate factors showed low expression levels in groups with high immune activity.

[0291] Furthermore, the TMP value (Transscripts per million (corrected value of the measured amount)) of the above miRNA candidate factors was used as the expression level, and an Xgboost model was constructed to calculate the predicted rate of immune activity (AUC). As a result, in the case of single factors, the miRNAs that had an AUC > 0.7 were hsa-miR-151a-5p (AUC = 0.722), hsa-miR-99b-5p (AUC = 0.714), and hsa-miR-200b-3p (AUC = 0.702). In the case of combinations of multiple factors, the combinations of miRNAs that had an AUC ≥ 0.7 are shown in Tables 9 to 11.

[0292]

[0293]

[0294]

[0295] (Test Example 11: Prediction of Disease Risk Using Differential Expression miRNAs as Indicators) Using MIENTURNET (MicroRNA ENrichment TURNED NETwork), the target genes of the top 30 miRNAs were identified. Furthermore, human diseases associated with these identified target genes were analyzed by referring to the KEGG database using Clusterprofiler (ver 4.16.0) to identify disease categories with enrichment p-values ​​< 0.05. The target genes of the top 30 miRNAs are shown in Table 12, the correspondence between the miRNAs and disease categories is shown in Table 13, and the correspondence between the target genes and disease categories is shown in Table 14.

[0296]

[0297]

[0298]

[0299] As shown in Tables 12-14, the top 30 miRNAs target multiple genes involved in the development of the specific diseases mentioned above. Therefore, it is suggested that the risk of developing these specific diseases can be determined based on the amount of these miRNAs.

[0300] We acknowledge SomaLogic Operating Co., Inc. as the provider of the proteomic data measured using the modified aptamer-based SomaScan (registered trade mark) Assay. SomaScan (registered trade mark), SOMAmer (registered trademark) and SomaSignal (trade mark) are trademarks of SomaLogic Operating Co., Inc.

[0301] It is clear to those skilled in the art that various modifications or alterations can be conceived within the scope of the claims, and these will naturally fall within the technical scope of the present invention. Furthermore, the components of the above embodiments may be combined in any way without departing from the spirit of the invention.

[0302] This application is based on the Japanese application (Japanese Patent Application No. 2024-229166) filed on December 25, 2024, the contents of which are incorporated by reference in this application. Furthermore, all publications, patent gazettes, and published patent gazettes cited herein are incorporated herein by reference in their entirety.

[0303] 10...Immunoactivation determination device, 20...Device for measuring the amount of biomarkers (measuring device), 30...Computer system, 40...Recording medium, 300...Computer main unit, 301...Input unit, 302...Display unit, 310...CPU, 311...ROM, 312...RAM, 313...Hard disk, 314...Input / output interface, 315...Reading device, 316...Communication interface, 317...Image output interface, 318...Bus.

Claims

1. A method for determining the immune activity of a subject, comprising a determination step of determining the immune activity of the subject based on the amount of at least one biomarker in the urine collected from the subject.

2. The method according to claim 1, wherein the biomarker comprises a protein and / or miRNA.

3. The aforementioned proteins include IgA, RAB21, Nectin 4, PD-L2, SARP-2, LIRA5, RWDD1, CI061, Caspase 4, DAB2, Adrenomedullin, IMP2L, SOD3, LG3BP, IL-6 soluble receptor α chain, MYO6, Gelsolin, NALD2, FCGRN, TCP2L, RAC1, AIF1L, F AAA, PSME2, ​​RAB5B, SH3G2, NCAM-L1, MAG, Radixin, Arylsulfatase A, DHR11, Glyoxalase I, BLVRB, BMPR1A, I5P1, ASB9, Cathepsin H, MXRA8:ECD, STK4, LACB2, Ceruloplasmin, CHSP1, FLRT3:ECD, LTB4DH, NPTX2, GLO2, RHG30, VILI, GNPTG, RAB1B, 14-3-3 Protein β / α, 14-3-3 Protein γ, Type III Collagen, SCG3, BGAL, 14-3-3 Protein ε, Calcifosin, DHPR, NRP1, Glutaminilcyclase, FCN2, Cdc42, SDCB2, ACY3 GSTA2, RB11B, Testican 2, IF1AY, MITD1, RAB8B, RCN1, Lysosomal acid phosphatase, Thioredoxin reductase 1, IL-17 soluble receptor, PROSC, RB11A, AN32B, Paraoxonase 2, SPT46, SIA10, UBE2N / UBE2V2 complex, Albumin, IKB The method according to claim 2, wherein the protein is at least one selected from the group consisting of ε, GALNS, VATC2, NCF-2, NUBP1, SDHB, CREB-binding protein, SHPS1, ARL5B, PHS, C1GLC, EphB6, PAFAH β subunit, proteasome α5 subunit, RBP-III, 14-3-3 protein ζ / δ, MTL26, RNase 2, PHIPL, UBE2K, SAP, cargranulin A, TIMD3, ferritin, ferritin light chain, PPIC, S100A12, PACN2, ALG2, annexin V, IF5A2, HSP 70, and hCG.

4. The protein is at least one selected from the group consisting of adrenomedullin, IMP2L, IgA, TCP2L, NALD2, caspase 4, NCF-2, CHSP1, RHG30, NPTX2, lysosomal acid phosphatase, ARL5B, AN32B, SDHB, ceruloplasmin, NUBP1, STK4, albumin, S100A12, CREB-binding protein, cargranulin A, SPT46, paraoxonase 2, proteasome α5 subunit, RNase 2, annexin V, PAFAH β subunit, IF5A2, IKB ε, ALG2, and hCG, and RAB21, Nectin 4, CI061, SARP-2, LIRA5, NCAM-L1, 14-3-3 protein ε, MYO6, RAB1B, PD-L2, RAB5B, LTB4DH, Gelsolin, SOD3, SH3G2, BMPR1A, DHR11, VILI, RWDD1, IL-17 soluble receptor, LG3BP, MAG, FAAA, PSME2, ​​FCGRN, DAB2, Arylsulfatase A, AIF1L, Glyoxalase I, SHPS1, IL-6 soluble receptor α chain, BLVRB, DHPR, MXRA8:ECD, 14-3-3 protein γ, RCN1, I5P1, P HIPL, FLRT3:ECD, SCG3, Type III collagen, Glutaminyl cyclase, VATC2, ASB9, Cdc42, 14-3-3 protein β / α, Cathepsin H, Calcifosin, GLO2, RAC1, Testican 2, Radixin, FCN2, BGAL, SDCB2, RAB8B, UBE2K, PHS, LACB2, PROSC, IF1AY, GNPTG, ACY3, SIA10, Thioredoxin reductase 1, UBE2N / UBE2V2 complex, MITD1, RB11B, NRP1, EphB6, GALNS, GSTA2, RB11A, C1GLC, HSP The method according to claim 2, wherein the selected protein is at least one selected from the group consisting of 70,14-3-3 protein ζ / δ, SAP, ferritin light chain, ferritin, TIMD3, PACN2, MTL26, RBP-III, and PPIC.

5. The method according to claim 3, wherein the protein is one to four types.

6. The method according to claim 4, wherein the protein is two or more types and not more than four types.

7. The miRNA is hsa-miR-151a-5p, hsa-miR-99b-5p, hsa-miR-200b-3p, hsa-miR-9 9a-5p, hsa-miR-125b-5p, ENST00000531977.1, hsa-let-7c-5p, ENST0000051 6507.1, hsa-miR-30c-5p, hsa-miR-200c-3p, hsa-miR-204-5p, hsa-miR-103a -3p, hsa-miR-125a-5p, hsa-miR-29a-3p, ENST00000568314.1, ENST00000563 The method according to claim 2, wherein at least one selected from the group consisting of 103.1, hsa-miR-30b-5p, hsa-miR-151a-3p, hsa-miR-92a-3p, hsa-miR-182-5p, hsa-miR-22-3p, hsa-miR-100-5p, hsa-miR-23b-3p, ENST00000658801.1, hsa-miR-9-5p, ENST00000655322.1, hsa-miR-141-3p, hsa-miR-191-5p, ENST00000630360.1, and hsa-miR-30c-2-3p.

8. The method according to claim 2, wherein the miRNA is at least one selected from the group consisting of hsa-miR-151a-5p, hsa-miR-99b-5p, and hsa-miR-200b-3p.

9. The method according to claim 7, wherein the miRNAs are one to four types.

10. The method according to claim 7, wherein the miRNAs are two or more but four or fewer.

11. The method according to claim 7, wherein the miRNAs are three or more but four or fewer.

12. The method according to claim 1, wherein the biomarker includes IgA.

13. The method according to claim 1, wherein the determination step includes at least one of the following (A) to (D): (A) Determining that the immune activity of the target is high when the value of the parameter based on the amount of the biomarker is higher than a predetermined threshold; (B) Determining that the immune activity of the target is low when the value of the parameter based on the amount of the biomarker is lower than a predetermined threshold; (C) Determining that the immune activity of the target is low when the value of the parameter based on the amount of the biomarker is higher than a predetermined threshold; (D) Determining that the immune activity of the target is high when the value of the parameter based on the amount of the biomarker is lower than a predetermined threshold.

14. The method according to claim 1, wherein the immune activity is the activity of plasmacytoid dendritic cells.

15. A method for supporting the improvement or maintenance of immune activity, comprising: a determination step of determining immune activity by the method described in any one of claims 1 to 14; and a determination result presentation step of presenting a report showing the determination result obtained in the determination step.

16. The method according to claim 15, further comprising: a determination step of identifying an action for improving or maintaining immune activity in accordance with the determination result obtained in the determination step; and a determination result presentation step of presenting a report showing the result identified in the determination step.

17. A reagent or reagent kit for use in the method according to any one of claims 1 to 14, comprising a substance capable of specifically detecting the biomarker in urine.

18. A method for evaluating the immunostimulatory activity of a test substance, comprising an evaluation step of evaluating the immunostimulatory activity of the test substance based on the amount of at least one biomarker in the urine collected from a subject who has ingested the test substance.

19. The method according to claim 18, wherein the biomarker comprises IgA.

20. An apparatus for determining immune activity, comprising a computer including a processor and memory under the control of the processor, wherein the memory contains a computer program for causing the computer to perform: A) a determination step of determining the immune activity of a subject based on the amount of at least one biomarker in urine collected from the subject; and B) an output step of outputting the determination result of the immune activity of the subject.

21. The apparatus according to claim 20, wherein the biomarker includes IgA.

22. A) A determination step of determining the immune activity of a subject based on the amount of at least one biomarker in the urine collected from the subject; and B) an output step of outputting the determination result of the immune activity of the subject. A computer program for causing a computer to perform these steps.

23. The computer program according to claim 22, wherein the biomarker comprises IgA.

24. A method for screening biomarkers in a biological sample for determining immune activity, comprising: a first measurement step of measuring the immune activity of a target; a second measurement step of measuring the amount of at least one biomarker in a biological sample taken from a target; and a determination step of determining whether the biomarker is a useful biomarker for determining immune activity based on the correlation between the immune activity of the target and the amount of the biomarker.

25. The method according to claim 24, wherein the immune activity is the activity of plasmacytoid dendritic cells.

26. An immunostimulatory composition containing a substance having immunostimulatory activity, to be administered to a subject that was not determined to have high immune activity or low immune activity by the method described in claim 13.

27. A method for determining the disease risk of a subject, comprising a determination step of determining the disease risk of the subject based on the amount of at least one biomarker in the urine collected from the subject.

28. The method according to claim 27, wherein the subject is determined to have low immune activity by the method according to claim 8.

29. The method according to claim 27 or 28, wherein the disease is at least one selected from the group consisting of cancer, endocrine disorders, metabolic disorders, infectious diseases, cardiovascular diseases, and neurodegenerative diseases.

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