JP6873460B2 - How to test a subject for possible pancreatic cancer - Google Patents

Description

The present invention relates to a method for testing the possibility that a subject has pancreatic cancer, a method for distinguishing pancreatic cancer from a pancreatic disease other than pancreatic cancer, and a method for determining the possibility that a subject has pancreatic cancer. Methods for monitoring, reagents for testing the possibility that a subject has pancreatic cancer, reagents for distinguishing pancreatic cancer from pancreatic diseases other than pancreatic cancer, and subjects for pancreatic cancer Concerning reagents for monitoring the likelihood of being affected.

Exosomes are vesicle granules that are present in body fluids in vivo. It is known that various membrane proteins are present on the surface of exosomes, similar to the surface of general cells. In addition, it has been reported that exosomes are secreted from various cells, such as cells of the immune system and various cancer cells, and they function as mediators of cell-cell communication in the living body and are associated with physiological phenomena. Attention is being paid to its association with diseases such as cancer.

In recent years, a method for detecting colorectal cancer by measuring the amount of exosomes expressing specific antigens (CD9, CD63, CD147) on the surface of exosomes (Patent Document 1), in ovarian cancer patients, in blood as the cancer progresses. It has been reported so far that the amount of exosomes is increased (Non-Patent Document 1).

Further, as a method for measuring a component contained in an exosome, an immunoassay method using a first antibody that specifically binds to a first antigen possessed by an exosome and a second antibody that specifically binds to a second antigen possessed by an exosome. Is known (Patent Document 2).
EPS8 (Epidermal Growth Factor Receptor Kinase Substrate 8) is a substrate for epidermal growth factor receptor, a factor that activates Rac, and is high in tissues of patients with pancreatic cancer. It has been reported that it is expressed (Non-Patent Document 2).

International Publication No. 2014/1697969 International Publication No. 2013/094307

Taylor., Et al., Gynecologic Oncol, 100 (2008) pp13-21 T. Welsch., Et al., Cancer Letters, 255 (2007) pp205-218

An object of the present invention is a method and a reagent for testing the possibility that a subject has pancreatic cancer, a method and a reagent for distinguishing pancreatic cancer from a pancreatic disease other than pancreatic cancer, and a subject. To provide methods and reagents for monitoring the likelihood of suffering from pancreatic cancer.

As a result of diligent studies to solve the above problems, the present inventors have suffered from pancreatic cancer by measuring EPS8, particularly EPS8 possessed by exosomes, in a sample collected from the subject. By measuring EPS8 possessed by exosomes in samples collected from patients with pancreatic cancer and patients with pancreatic disease other than pancreatic cancer, it is possible to distinguish between pancreatic cancer and pancreatic disease other than pancreatic cancer. Furthermore, they have found that it is possible to monitor the possibility that a subject has pancreatic cancer, and completed the present invention. That is, the present invention relates to the following [1] to [47].

[1] A method for testing the possibility that a subject has pancreatic cancer, which comprises measuring EPS8 contained in an exosome in a sample collected from a subject.
[2] The method according to [1], which comprises the following steps.
(1) Step of collecting a sample from a subject;
(2) A step of measuring the concentration of EPS8 contained in exosomes in the sample collected in step (1);
(3) If the EPS8 concentration measured in step (2) is higher than the EPS8 concentration of exosomes in a sample collected from a healthy subject, it is highly possible that the subject has pancreatic cancer. If the concentration is the same as or lower than the EPS8 concentration of the exosome in the sample collected from a healthy subject, it is determined that the subject is unlikely to have pancreatic cancer [3]. The method according to [2], wherein in the step (2), the concentration of EPS8 contained in the exosome in the sample collected in the step (1) is measured using the exosome isolated from the sample.
[4] The method according to [2] or [3], wherein the step (2) is performed by an immunological measurement method.
[5] The method according to [4], wherein the immunological measurement method is performed using an antibody that binds to EPS8 or an antibody fragment thereof.
[6] The immunological measurement method uses an antibody or an antibody fragment thereof that binds to EPS8, and an antibody or an antibody fragment thereof that binds to an antigen specifically expressed in an exosome (hereinafter referred to as an exosome-specific antigen). The method according to [4], which is performed.
[7] The method according to [6], wherein the exosome-specific antigen is CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, or LAMP1.
[8] The method according to any one of [1] to [7], wherein the sample is blood.

[9] A method for distinguishing pancreatic cancer from pancreatic disease other than pancreatic cancer in a pancreatic disease patient, which comprises measuring EPS8 possessed by exosomes in a sample collected from a pancreatic disease patient.
[10] The method according to [9], which comprises the following steps.
(1) Step of collecting a sample from a subject;
(2) A step of measuring the concentration of EPS8 contained in exosomes in the sample collected in step (1);
(3) If the EPS8 concentration measured in step (2) is higher than the EPS8 concentration of exosomes in a sample collected from a patient with pancreatic disease other than pancreatic cancer, the subject suffers from pancreatic cancer. If the EPS8 concentration of exosomes is the same as or lower than the EPS8 concentration of exosomes in a sample collected from a patient with pancreatic disease other than pancreatic cancer, the subject suffers from pancreatic disease other than pancreatic cancer. Step [11] In the step (2), the measurement of the concentration of EPS8 contained in the exosome in the sample collected in the step (1) is performed on the exosome isolated from the sample. The method according to [10], which is carried out using the method.
[12] The method according to [10] or [11], wherein the step (2) is performed by an immunological measurement method.
[13] The method according to [12], wherein the immunological measurement method is performed using an antibody that binds to EPS8 or an antibody fragment thereof.
[14] The method according to [12], wherein the immunological measurement method is carried out using an antibody or an antibody fragment thereof that binds to EPS8, and an antibody or an antibody fragment thereof that binds to an exosome-specific antigen.
[15] The method according to [14], wherein the exosome-specific antigen is CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, or LAMP1.
[16] The method according to any one of [9] to [15], wherein the pancreatic disease other than the pancreatic cancer is pancreatitis.
[17] The method according to any one of [9] to [16], wherein the sample is blood.

[18] A method for monitoring the possibility that a subject has pancreatic cancer, which comprises measuring EPS8 contained in an exosome in a sample collected from a subject over time.
[19] The method according to [18], which comprises the following steps.
(1) A step of collecting a sample from a subject over time;
(2) A step of measuring the concentration of EPS8 contained in exosomes in the sample collected in step (1);
(3) If the EPS8 concentration measured in step (2) is maintained at a higher concentration than the EPS8 concentration of exosomes in a sample collected from a healthy subject, the subject suffers from pancreatic cancer. If it is determined that there is a high possibility that the exosome is maintained at the same or lower concentration as the EPS8 concentration of the exosome in the sample collected from a healthy subject, the subject is suffering from pancreatic cancer. The process of determining that the possibility is low.
[20] In the step (2), the concentration of EPS8 contained in the exosome in the sample collected in the step (1) is measured using the exosome isolated from the sample, according to [19]. Method.
[21] The method according to [19] or [20], wherein the step (2) is performed by an immunological measurement method.
[22] The method according to [21], wherein the immunological measurement method is performed using an antibody that binds to EPS8 or an antibody fragment thereof.
[23] The method according to [21], wherein the immunological measurement method is performed using an antibody or an antibody fragment thereof that binds to EPS8, and an antibody or an antibody fragment thereof that binds to an exosome-specific antigen.
[24] The method according to [23], wherein the exosome-specific antigen is CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, or LAMP1.
[25] The method according to any one of [18] to [24], wherein the sample is blood.

[26] A reagent for testing the possibility that a subject has pancreatic cancer, which comprises an EPS8 measuring reagent possessed by an exosome in a sample collected from the subject.
[27] The reagent according to [26], wherein the EPS8 measurement reagent is an immunological measurement reagent.
[28] The reagent according to [27], wherein the immunological measurement reagent contains an antibody or an antibody fragment thereof that binds to EPS8.
[29] The reagent according to [28], wherein the immunological measurement reagent further comprises an antibody or an antibody fragment thereof that binds to an exosome-specific antigen.
[30] The reagent according to [29], wherein the exosome-specific antigen is CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, or LAMP1.
[31] The reagent according to any one of [26] to [30], further comprising an exosome isolation reagent.
[32] The reagent according to any one of [26] to [31], wherein the sample is blood.

[33] the pancreatic disorder in a specimen collected from a patient, exosomes characterized in that it comprises a EPS8 measuring reagent having the reagent for differentiating pancreatic diseases patients other than pancreatic cancer patients and pancreatic cancer.
[34] The reagent according to [33], wherein the EPS8 measurement reagent is an immunological measurement reagent. [35] The reagent according to [34], wherein the immunological measurement reagent contains an antibody or an antibody fragment thereof that binds to EPS8.
[36] The reagent according to [35], wherein the immunological measurement reagent further comprises an antibody or an antibody fragment thereof that binds to an exosome-specific antigen.
[37] The reagent according to [36], wherein the exosome-specific antigen is CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, or LAMP1.
[38] The reagent according to any one of [33] to [37], further comprising an exosome isolation reagent.
[39] The reagent according to any one of [33] to [38], wherein the pancreatic disease other than the pancreatic cancer is pancreatitis.
[40] The reagent according to any one of [33] to [39], wherein the sample is blood.

[41] A reagent for monitoring the possibility that a subject has pancreatic cancer, which comprises an EPS8 measuring reagent possessed by an exosome in a sample collected from the subject over time.
[42] The reagent according to [41], wherein the EPS8 measurement reagent is an immunological measurement reagent.
[43] The reagent according to [42], wherein the immunological measurement reagent contains an antibody or an antibody fragment thereof that binds to EPS8.
[44] The reagent according to [43], wherein the immunological measurement reagent further comprises an antibody or an antibody fragment thereof that binds to an exosome-specific antigen.
[45] The reagent according to [44], wherein the exosome-specific antigen is CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, or LAMP1.
[46] The reagent according to any one of [41] to [45], further comprising an exosome isolation reagent.
[47] The reagent according to any one of [41] to [46], wherein the sample is blood.

According to the present invention, a method and a reagent for testing the possibility that a subject has pancreatic cancer, a method and a reagent for distinguishing pancreatic cancer from a pancreatic disease other than pancreatic cancer, and a method and a reagent for the subject to discriminate between pancreatic diseases and pancreatic diseases. Methods and reagents for monitoring the likelihood of having cancer are provided.

Using exosomes in serum collected from healthy subjects (4 samples), pancreatitis patients (4 samples), and pancreatic cancer patients (4 samples), EPS8 (97 kD) and exosome-specific antigens were used by Western blotting. This is the result of detecting a certain CD63. It is a result of comparing the amount of EPS8 contained in each serum of exosomes in sera collected from healthy subjects (10 samples), pancreatitis patients (10 samples), and pancreatic cancer patients (23 samples). The vertical axis shows the signal ratio of EPS8 / CD63 calculated from the signal of EPS8 and the signal of CD63 obtained by Western blotting. EPS8 (97 kD) was detected by Western blotting using exosomes in serum collected from patients with pancreatic cancer before and after surgery (stage IB: 1 sample, stage IIB: 2 samples, stage III: 2 samples). It is the result of the operation. This is the result of comparing the amount of EPS8 contained in each serum of exosomes in sera collected from patients with recurrent pancreatic cancer (22 samples) and healthy subjects (2 samples). The vertical axis shows the signal ratio of EPS8 / CD9 calculated from the signal of EPS8 and the signal of CD9 obtained by Western blotting.

1. 1. Method for testing the possibility that a subject has pancreatic cancer The method for testing the possibility that a subject has pancreatic cancer according to the present invention is that the method for testing the possibility that a subject has pancreatic cancer is described in a sample collected from a subject. It is a method characterized by measuring the concentration of EPS8 contained in an exosome.

The exosome in the present invention is a membrane vesicle covered with a lipid bilayer membrane having a diameter of 30 to 100 nm secreted from an animal cell.
The sample in the present invention is not particularly limited as long as it is a sample in which EPS8 contained in an exosome can be measured, and examples thereof include blood, urine, saliva, milk, nasal discharge, and cerebrospinal fluid, and blood is preferable. Examples of the blood include whole blood, serum, plasma and the like, and serum is preferable.

In the present invention, EPS8 contained in an exosome means any form of EPS8 such as EPS8 contained in the exosome, EPS8 existing on the membrane surface of the exosome, and EPS8 penetrating the membrane of the exosome. ..
In the present invention, the EPS8 concentration contained in an exosome includes not only the mass or the number of moles per unit volume but also the signal intensity per unit volume.

[Measurement method 1]
In the present invention, the method for testing the possibility that a subject has pancreatic cancer can be carried out by a method including the following steps.
(1) Step of collecting a sample from a subject;
(2) A step of measuring the concentration of EPS8 contained in exosomes in the sample collected in step (1);
(3) If the EPS8 concentration measured in step (2) is higher than the EPS8 concentration of exosomes in a sample collected from a healthy subject, it is highly likely that the subject has pancreatic cancer. A step of determining that the subject is unlikely to have pancreatic cancer if the EPS8 concentration of the exosome in the sample collected from a healthy subject is the same as or lower than that of the exosome.
Hereinafter, each step will be described in detail.

<Process (1)>
Examples of the sample collected from the subject in the step (1) include the above-mentioned sample and the like.

<Process (2)>
The step (2) is a step of measuring the EPS8 concentration of the exosome in the sample collected in the step (1). The EPS8 concentration of exosomes in the sample collected in step (1) can be measured by, for example, an immunological measurement method. As the immunological measurement method, any method can be used as long as the concentration of EPS8 contained in the exosome in the sample can be measured by using an antigen-antibody reaction. For example, an antibody that binds to EPS8 or an antibody fragment thereof. , And a method using an antibody that binds to EPS8 or the antibody fragment thereof and an antibody that binds to an exosome-specific antigen or the antibody fragment thereof.

The antibody that binds to EPS8 in the step (2) is not particularly limited as long as it can measure EPS8, and examples thereof include Purified Mouse Anti-Eps8 (manufactured by Becton Dickinson, Japan, clone: 15 / Eps8). Be done.

Examples of the exosome-specific antigen in the step (2) include CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-I, Rab5, AnnexinV, LAMP1 and the like.

The antibody that binds to the exosome-specific antigen is not particularly limited as long as it is an antibody that can bind to the exosome-specific antigen. Human CD9 (manufactured by Becton Dickinson Japan, clone: ML13), Purified Mouse Anti-Human CD81 (manufactured by Becton Dickinson Japan, clone: JS-81), Purified Mouse Anti-Human CD37 (Beckinson Japan) Manufactured by, clone: MB371), Purified Mouse Anti-Human CD53 (manufactured by Nippon Bekton Dickinson, clone: HI29), Anti-CD82 antibody [C33] (manufactured by Abcum), Purified Mouse Anti-Human CD -Dickinson, clone: WM15), Purified Mouse Anti-Human CD11a (Nippon Becton Dickinson, clone: 27 / CD11a), Purified Mouse Anti-Human CD11b / Mac-1 (Nippon Becton, Dickinson) : ICRF44), Purified Mouse Anti-Human CD11c (manufactured by Becton Dickinson Japan, clone: 3.9), Purified Mouse Anti-Human CD86 (manufactured by Becton Dickinson Japan, clone: 2331 (FUN-1)), Anti -ICAM1 antibody [HM1] (manufactured by Abcam), Purified Mouse Anti-Rab5 (manufactured by Becton Dickinson, Japan, clone: 1 / Rab5), Anti-Annexin V antibody [EPR3979] (manufactured by Abcum), Human Lamp-1 (manufactured by Becton Dickinson, Japan, clone: 25 / Lamp-1) and the like can be mentioned.

The antibody fragment that binds to EPS8 and the antibody fragment that binds to the exosome-specific antigen in the step (2) are not particularly limited as long as they can measure EPS8 and the exosome-specific antigen, respectively. For example, the antibody is treated with papain. Antibody fragments obtained by removing Fc moieties such as Fab, F (ab') ₂ obtained by pepsin treatment, Fab' obtained by pepsin treatment-reduction treatment, antibody fragments from which Fc moieties have been removed by genetic engineering techniques. And so on.

The EPS8 concentration of the exosome in the sample in the step (2) can be measured by sandwich ELISA, the exoscreen method described later, or the like. Of the above two types of antibodies used for measuring the EPS8 concentration of exosomes in sandwich ELISA, an antibody that binds to an exosome-specific antigen or an antibody fragment thereof is used as a solid phase antibody, and an antibody that binds to EPS8 or an antibody fragment thereof is used. It can be used as a labeling antibody. Alternatively, an antibody that binds to EPS8 or the antibody fragment thereof can be used as a solid phase antibody, and an antibody that binds to an exosome-specific antigen or the antibody fragment thereof can be used as a labeling antibody. The preparation of the solid phase antibody and the labeling antibody is not particularly limited and can be carried out according to a known method.

The method for measuring EPS8 contained in an exosome can be any method as long as it can measure EPS8 contained in an exosome, and can be carried out by using a method such as an immunological measurement method.

Specifically, when an antibody that binds to EPS8 or the antibody fragment thereof is used as the antibody for the solid phase, and an antibody that binds to an exosome-specific antigen or the antibody fragment thereof is used as the labeling antibody, the antibody is immobilized on the solid phase. The EPS8-bearing exosome in the sample is bound to the antibody or antibody fragment that binds to EPS8, and then the labeled antibody or antibody fragment that binds to the exosome-specific antigen is added to solidify. A complex of an antibody or antibody fragment thereof that binds to EPS8, an exosome, and a labeled antibody or antibody fragment that binds to an exosome-specific antigen is formed on the phase, and the complex thereof is formed. By measuring the standard, EPS8 contained in the exosome in the sample can be measured.
When an antibody that binds to an exosome-specific antigen or the antibody fragment thereof is used as the antibody for the solid phase, and an antibody that binds to EPS8 or the antibody fragment thereof is used as the labeling antibody, the exosome immobilized on the solid phase is used. The exosome in the sample is bound to the antibody or antibody fragment that binds to a specific antigen, and then the labeled antibody or antibody fragment that binds to EPS8 is added to the exosome-specific antibody or antibody fragment on the solid phase. By forming a complex of an antibody or antibody fragment that binds to an antigen, an exosome, and a labeled antibody that binds to EPS8 or that antibody fragment, and measuring the standard in the formed complex. EPS8 contained in the exosome in the sample can be measured.

In the above, examples of the solid phase include microtiter plates, glass or synthetic resin granules (beads), glass or synthetic resin spheres (balls), latex, magnetic particles, nitrocellulose membrane, and the like. Various membranes, test tubes made of synthetic resin, and the like can be used. As the label, for example, an enzyme, a fluorescent substance, a luminescent substance, a radioisotope, biotin, digoxigenin, a polypeptide containing a tag sequence, metal colloid particles, colored latex particles and the like can be used.

Examples of the enzyme include alkaline phosphatase, peroxidase, galactosidase, glucuronidase, luciferase and the like.
Examples of the fluorescent substance include fluorescein isothiocyanate (FITC) and rhodamine B-isothiocyanate (RITC). Examples of other fluorescent substances include quantum dot (Science, 281, 2016-2018, 1998), phycobili protein such as phycoerythrin, GFP (Green Fluorescent Protein ) , RFP (Redfluorescent Protein), YFP (Reedfluorescent Protein), YFP (YFP). , BFP (Blue fluorescent Protein) and other proteins that emit fluorescence.
Examples of the luminescent substance include aclidinium and its derivatives, ruthenium complex compounds, loffin and the like. As the ruthenium complex compound, those shown in Clin. Chem. 37, 9, 1534-1539, 1991, which emit light electrochemically together with an electron donor, are preferable.
As the radioisotope, ^{e.g., 3 H, 14 C, 35} S, 32 P, 125 I, 131 I and the like.
Examples of the polypeptide containing the tag sequence include FLAG peptide (FLAG tag, Asp Tyr Lys Asp Asp Asp Asp Asp Lys), polyhistidine (His tag, His His His His His), and myc epitope peptide (myc tag, Glu Gln Lys Leu). Ile Ser Glu Glu Asp Leu), hemagglutinin epitope peptide (HA tag, Tyr Pro Tyr Asp Val Pro Asp Tyr Ala) and the like.

In step (2), when measuring the concentration of EPS8 contained in an exosome in a sample without destroying the exosome in the sample, an antibody that binds to EPS8 or an antibody that binds to EPS8 as the antibody fragment thereof. Alternatively, among the antibody fragments, it is preferable to use an antibody or antibody fragment that binds to the EPS8 epitope present on the surface of the exosome.

Examples of the immunological measurement method in the step (2) include the exoscreen method in addition to the sandwich ELISA described above. The exoscreen method is an application of AlphaLISA developed by PerkinElmer. In this method, two types of antibodies having different epitopes are used, one antibody is a biotinylated antibody to which biotin is bound, and the other is an antibody to which AlphaLISA acceptor beads are bound. React. Then, by adding the donor beads to which strep avidin is bound, the biotinylated antibody and the donor beads are bound via strep avidin, and the acceptor beads and the donor beads are adjacent to each other. Singlet oxygen is generated from the donor beads by 680 nm excitation in a state of being adjacent within 200 nm, and when the singlet oxygen reaches the acceptor beads, light of 615 nm is emitted and can be detected as a signal.

Specifically, an antibody that binds to EPS8 or a biotinylated antibody in which biotin is bound to the antibody fragment, and an antibody or antibody fragment that binds to an exosome-specific antigen to which AlphaLISA acceptor beads are bound are contained in a sample. By reacting with EPS8 contained in the exosome, and then adding a donor bead to which strept antibody is bound, a complex of the acceptor bead, the exosome, and the donor bead is formed. The donor beads of the complex are irradiated with excitation light to generate singlet oxygen, and the light generated by the reaction between the generated singlet oxygen and the acceptor beads is measured.
In addition, the exosome in the sample has an antibody that binds to an exosome-specific antigen or a biotinylated antibody in which biotin is bound to the antibody fragment, and an antibody or antibody fragment that binds to EPS8 in which AlphaLISA acceptor beads are bound. By reacting with EPS8 and then adding donor beads to which strept antibody is bound, a complex of the acceptor beads, exosomes, and the donor beads is formed. The donor beads of the complex are irradiated with excitation light to generate singlet oxygen, and the light generated by the reaction between the generated singlet oxygen and the acceptor beads is measured.

<Process (3)>
Step (3) compares the EPS8 concentration of exosomes in the sample collected from the subject obtained in step (2) with the EPS8 concentration of exosomes in the sample collected from healthy subjects. In this step, if the EPS8 concentration of exosomes in a sample collected from a subject is higher than the EPS8 concentration of exosomes in a sample collected from a healthy subject, the subject suffers from pancreatic cancer. If it is determined that there is a high possibility that the exosome has the same or lower EPS8 concentration in the sample collected from a healthy subject, the subject is unlikely to have pancreatic cancer. Is determined.

The EPS8 concentration of exosomes in a sample collected from a healthy subject is a reference value for determining the possibility that the subject has pancreatic cancer, and is a reference value in a sample collected from a plurality of healthy subjects. , The EPS8 concentration of exosomes can be measured, and the EPS8 concentration of exosomes in a sample collected from a healthy person can be set from the statistics.
The sample collected from a healthy person is preferably a sample of the same type as the sample collected from a subject. For example, when the sample collected from a subject is blood, the sample collected from a healthy person also contains blood. It is preferable to use it.
The method for comparing the EPS8 concentration of the exosome in the sample collected from the subject and the EPS8 concentration of the exosome in the sample collected from the healthy subject is not particularly limited, and is a known method (Steel method). , T-test, Wilcoxon test, etc.) can be used. If the analysis shows that the EPS8 concentration of the exosome in the sample collected from the subject is higher than the EPS8 concentration of the exosome in the sample collected from the healthy subject, the subject Is determined to be highly likely to have pancreatic cancer, and if it is shown that the EPS8 concentration of exosomes is the same as or lower than that of exosomes in a sample collected from a healthy subject, the subject is pancreatic cancer. It is determined that the possibility of suffering from is low.

Further, in the step (2) of the measurement method 1, the EPS8 concentration of the exosome in the sample can be measured by using the exosome isolated from the sample collected from the subject. Specifically, a method including the following steps can be mentioned.
[Measurement method 2]
(1A) Step of collecting a sample from a subject;
(2A) Step of isolating exosomes from the sample collected in step (1A);
(3A) A step of measuring the EPS8 concentration of the exosomes isolated in step (2A);
(4A) A step of determining the EPS8 concentration of an exosome in the sample from the EPS8 concentration measured in step (3A);
(5A) If the EPS8 concentration determined in step (4A) is higher than the EPS8 concentration of exosomes in a sample collected from a healthy subject, the subject is likely to have pancreatic cancer. If the concentration is the same as or lower than the EPS8 concentration of the exosome in the sample collected from a healthy subject, it is determined that the subject is unlikely to have pancreatic cancer.
Hereinafter, each step will be described in detail.

<Process (1A)>
Examples of the sample collected from the subject in the step (1A) include the above-mentioned sample and the like.

<Process (2A)>
The method for isolating exosomes in step (2A) is not particularly limited as long as the exosomes in the sample can be isolated. For example, ultracentrifugation method (Trends in Molecular Medicine. 21, 533 (2015)). Examples thereof include a method using an exosome isolation kit EXO-Prep (manufactured by Cosmo Bio Co., Ltd.).

<Process (3A)>
The method for measuring EPS8 contained in an exosome in step (3A) can be carried out by any method as long as it can measure EPS8 contained in an exosome, for example, by using a method such as an immunological measurement method. be able to. Examples of the method for measuring the EPS8 concentration of an exosome by an immunological measurement method include the following aspects.

・ Aspect 1
A method using an antibody that binds to EPS8 or an antibody fragment thereof.
・ Aspect 2
A method using an antibody or an antibody fragment thereof that binds to EPS8, and an antibody or an antibody fragment thereof that binds to an exosome-specific antigen.

Examples of the antibody that binds to EPS8 or the antibody fragment thereof include the above-mentioned antibody that binds to EPS8 or the antibody fragment thereof. Examples of the antibody or the antibody fragment thereof that binds to the exosome-specific antigen include the antibody that binds to the above-mentioned exosome-specific antigen or the antibody fragment thereof.

Examples of the method of the first aspect include a Western blotting method and the like. Examples of the Western blotting method include the methods described in Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996).
As the method of the second aspect, in the case of measuring the isolated exosome without destroying it, the immunological measurement method described in the above-mentioned measurement method 1, for example, the exoscreen method and the like can be mentioned, and the isolated exosome can be used. In the case of breaking and measuring, a sandwich ELISA method or the like can be mentioned. Examples of the exoscreen method include the above-mentioned exoscreen method. When measuring the isolated exosome without destroying it, the antibody that binds to EPS8 or the antibody fragment thereof is the antibody that binds to EPS8 or the antibody fragment that binds to the epitope of EPS8 present on the surface of the exosome. Alternatively, use an antibody fragment.
Examples of the method for destroying the isolated exosome include a method using ultrasonic waves. As the sandwich ELISA method, for example, an antibody that binds to EPS8 or an antibody fragment thereof can be used as a solid phase antibody, and an antibody that binds to an exosome-specific antigen or the antibody fragment can be used as a labeling antibody. The binding antibody or the antibody fragment may be used as a solid phase antibody, and the binding antibody or the antibody fragment may be used as a labeling antibody. Examples of the solid phase and the label include known solid phase and the label, respectively, and for example, the above-mentioned solid phase and the label are used, respectively. Examples of the method for preparing the solid phase antibody and the labeling antibody include known preparation methods.

<Process (4A)>
The step (4A) is a step of determining the EPS8 concentration of the exosome in the sample from the EPS8 concentration measured in the step (3A). From the volume of the sample and the EPS8 concentration measured in the step (3A), the EPS8 concentration of the exosome in the sample can be determined.

<Process (5A)>
In step (5A), the EPS8 concentration of exosomes in the sample collected from the subject determined in step (4A) is compared with the EPS8 concentration of exosomes in the sample collected from a healthy subject. If the EPS8 concentration of exosomes in a sample collected from a subject is higher than the EPS8 concentration of exosomes in a sample collected from a healthy subject, the subject develops pancreatic cancer. If it is determined that the subject is likely to be affected and the EPS8 concentration of the exosome in the sample collected from a healthy subject is the same as or lower than that of the exosome, the subject is likely to have pancreatic cancer. It is judged to be low.

The EPS8 concentration of exosomes in a sample collected from a healthy subject is a reference value for determining the possibility that the subject has pancreatic cancer, and is a reference value in a sample collected from a plurality of healthy subjects. , The EPS8 concentration of exosomes may be measured, and the EPS8 concentration of exosomes in a sample collected from a healthy person may be set from the statistics.
The sample of a healthy person is preferably a sample of the same type as the sample of a subject. For example, when the sample of a subject is blood, it is preferable to use blood as the sample of a healthy person.

As a method for comparing the EPS8 concentration of exosomes in a sample collected from a subject with the EPS8 concentration of exosomes in a sample collected from a healthy subject, the above-mentioned method can be used. If the analysis shows that the EPS8 concentration of the exosome in the sample collected from the subject is higher than the EPS8 concentration of the exosome in the sample collected from the healthy subject, the subject Is determined to be highly likely to have pancreatic cancer, and if it is shown that the EPS8 concentration of exosomes is the same as or lower than that of exosomes in a sample collected from a healthy subject, the subject is pancreatic cancer. It is determined that the possibility of suffering from is low.

2. Method for Differentiating Pancreatic Cancer from Pancreatic Disease Other Than Pancreatic Cancer in Patients with Pancreatic Disease The method for distinguishing pancreatic cancer from pancreatic disease other than pancreatic cancer in patients with pancreatic disease according to the present invention is a sample collected from a patient with pancreatic disease. It is a method characterized by measuring the concentration of EPS8 contained in exosomes.
The sample in the discrimination method of the present invention is not particularly limited as long as it is a sample in which EPS8 contained in an exosome can be measured, and examples thereof include the above-mentioned samples.
In the discrimination method of the present invention, EPS8 contained in an exosome exists in any form regardless of whether it is contained in the exosome, exists on the membrane surface of the exosome, or penetrates the membrane of the exosome. May be good.
In the discrimination method of the present invention, the EPS8 concentration contained in an exosome includes not only the mass or the number of moles per unit volume but also the signal intensity per unit volume.

[Measurement method 3]
The method of the present invention for distinguishing pancreatic cancer from pancreatic disease other than pancreatic cancer in a pancreatic disease patient can be performed by a method including the following steps.
(1B) Step of collecting a sample from a subject;
(2B) A step of measuring the concentration of EPS8 contained in exosomes in the sample collected in step (1B);
(3B) If the EPS8 concentration measured in step (2B) is higher than the EPS8 concentration of exosomes in a sample collected from a patient with pancreatic disease other than pancreatic cancer, the subject suffers from pancreatic cancer. If the EPS8 concentration of exosomes is the same as or lower than the EPS8 concentration of exosomes in a sample collected from a patient with pancreatic disease other than pancreatic cancer, the subject suffers from pancreatic disease other than pancreatic cancer. The process of determining that there is a high possibility that the disease has occurred.
Hereinafter, each step will be described in detail.

<Process (1B)>
Examples of the sample collected from the subject in the step (1B) include the above-mentioned sample and the like.

<Process (2B)>
In step (2B), the EPS8 concentration of the exosome in the sample collected in step (1B) using the antibody or antibody fragment that binds to EPS8 and the antibody or antibody fragment that binds to an exosome-specific antigen is used. Is the process of measuring.

The antibody that binds to EPS8 in the step (2B) is not particularly limited as long as it is an antibody that can measure EPS8, and examples thereof include the above-mentioned antibody.
Examples of the exosome-specific antigen in the step (2B) include the above-mentioned antigens and the like.

The antibody that binds to the exosome-specific antigen is not particularly limited as long as it can bind to the exosome-specific antigen, and examples thereof include the above-mentioned antibodies.
The antibody fragment that binds to EPS8 and the antibody fragment that binds to the exosome-specific antigen in the step (2B) are not particularly limited as long as they can measure EPS8 and the exosome-specific antigen, respectively. Can be mentioned.
In step (2B), the above two types of antibodies used for measuring the EPS8 concentration of an exosome are an antibody that binds to an exosome-specific antigen or an antibody fragment thereof as a solid phase antibody, and an antibody that binds to EPS8 or the antibody fragment thereof. Can be used as a labeling antibody. Alternatively, an antibody that binds to EPS8 or the antibody fragment thereof can be used as a solid phase antibody, and an antibody that binds to an exosome-specific antigen or the antibody fragment thereof can be used as a labeling antibody. The preparation of the solid phase antibody and the labeling antibody is not particularly limited and can be carried out according to a known method.

The method for measuring EPS8 contained in an exosome can be carried out by any method as long as it can measure EPS8 contained in an exosome, and can be carried out by using, for example, the above-mentioned immunological measuring method or the like. ..
In step (2B), when measuring the concentration of EPS8 contained in an exosome in a sample without destroying the exosome in the sample, an antibody that binds to EPS8 or an antibody that binds to EPS8 as the antibody fragment thereof. Alternatively, among the antibody fragments, it is preferable to use an antibody or antibody fragment that binds to the EPS8 epitope present on the surface of the exosome.
Examples of the above-mentioned immunological measurement method include the above-mentioned sandwich ELISA, exoscreen method and the like.

<Process (3B)>
In step (3B), the EPS8 concentration of exosomes in the sample collected from a pancreatic disease patient obtained in step (2B) and the EPS8 of exosome in a sample collected from a pancreatic disease patient other than pancreatic cancer. It is a step to compare the concentration, and when the EPS8 concentration of the exosome in the sample collected from the pancreatic disease patient is higher than the EPS8 concentration of the exosome in the sample collected from the pancreatic disease patient other than pancreatic cancer. Determines that the pancreatic disease patient is likely to have pancreatic cancer, and if the EPS8 concentration of the exosome in the sample collected from a pancreatic disease patient other than pancreatic cancer is the same as or lower than that of the pancreatic cancer, This is a step of determining that a diseased patient is likely to have a pancreatic disease other than pancreatic cancer.

Examples of pancreatitis other than pancreatic cancer include pancreatitis, and examples of pancreatitis include acute pancreatitis and chronic pancreatitis.

The EPS8 concentration of exosomes in a sample collected from a patient with pancreatic disease other than pancreatic cancer is a criterion for distinguishing whether the examiner has pancreatic cancer or pancreatic disease other than pancreatic cancer. It is a value, and the EPS8 concentration of exosomes in samples collected from multiple patients with pancreatic disease other than pancreatic cancer is measured, and from the statistics, EPS8 possessed by exosomes in samples collected from patients with pancreatic disease other than pancreatic cancer. The concentration can be set.
The sample collected from a patient with a pancreatic disease other than pancreatic cancer is preferably a sample of the same type as the sample collected from the subject. For example, when the sample collected from the subject is blood, other than pancreatic cancer. It is preferable to use blood as a sample collected from a patient with pancreatic disease.
The method for comparing the EPS8 concentration of exosomes in a sample collected from a subject with the EPS8 concentration of exosomes in a sample collected from a patient with a pancreatic disease other than pancreatic cancer is not particularly limited, and is not particularly limited. A method or the like can be used. When the analysis shows that the EPS8 concentration of the exosome in the sample collected from the subject is higher than the EPS8 concentration of the exosome in the sample collected from a patient with a pancreatic disease other than pancreatic cancer. , The subject was determined to have a high possibility of suffering from pancreatic cancer, and it was shown that the EPS8 concentration of exosomes in a sample collected from a patient with pancreatic disease other than pancreatic cancer was the same as or lower than that of the exosome. If so, it is determined that the subject is likely to have a pancreatic disease other than pancreatic cancer.

Further, in the step (2B) of the measurement method 3, the EPS8 concentration of the exosome in the sample can be measured by using the exosome isolated from the sample collected from the subject. Specifically, a method including the following steps can be mentioned.
[Measurement method 4]
(1C) Step of collecting a sample from a patient with pancreatic disease;
(2C) Step of isolating exosomes from the sample collected in step (1C);
(3C) Step of measuring the EPS8 concentration of the exosome isolated in step (2C); (4C) Step of determining the EPS8 concentration of the exosome in the sample from the EPS8 concentration measured in step (3C);
(5C) If the EPS8 concentration determined in step (4C) is higher than the EPS8 concentration of the exosome in the sample collected from a pancreatic disease patient other than pancreatic cancer, the subject suffers from pancreatic cancer. If the EPS8 concentration of exosomes is the same as or lower than the EPS8 concentration of exosomes in a sample collected from a patient with pancreatic disease other than pancreatic cancer, the subject suffers from pancreatic disease other than pancreatic cancer. The process of determining that there is a high possibility that the disease has occurred.

<Process (1C)>
Examples of the sample collected from the subject in the step (1C) include the above-mentioned sample and the like.

<Process (2C)>
The method for isolating exosomes in step (2C) is not particularly limited as long as it can isolate exosomes in a sample, and examples thereof include the above-mentioned methods.

<Process (3C)>
The method for measuring EPS8 contained in an exosome in step (3C) can be carried out by any method as long as it can measure EPS8 contained in an exosome, for example, by using a method such as an immunological measurement method. be able to. Examples of the method for measuring the EPS8 concentration of an exosome by an immunological measurement method include the following aspects.

・ Aspect 1
A method using an antibody that binds to EPS8 or an antibody fragment thereof.
・ Aspect 2
A method using an antibody or an antibody fragment thereof that binds to EPS8, and an antibody or an antibody fragment thereof that binds to an exosome-specific antigen.

Examples of the antibody that binds to EPS8 or the antibody fragment thereof include the above-mentioned antibody that binds to EPS8 or the antibody fragment thereof. Examples of the antibody or the antibody fragment thereof that binds to the exosome-specific antigen include the antibody that binds to the above-mentioned exosome-specific antigen or the antibody fragment thereof.

Examples of the method of the first aspect include a Western blotting method and the like. Examples of the Western blotting method include the above-mentioned methods.
As the method of the second aspect, in the case of measuring the isolated exosome without destroying it, the immunological measurement method described in the above-mentioned measurement method 1, for example, the exoscreen method and the like can be mentioned, and the isolated exosome can be used. In the case of breaking and measuring, a sandwich ELISA method or the like can be mentioned. Examples of the exoscreen method include the above-mentioned exoscreen method.

When measuring the isolated exosome without destroying it, the antibody that binds to EPS8 or the antibody fragment thereof is the antibody that binds to EPS8 or the antibody fragment that binds to the epitope of EPS8 present on the surface of the exosome. Alternatively, use an antibody fragment.
Examples of the method for destroying the isolated exosome include the above-mentioned methods.
As the sandwich ELISA method, for example, even if an antibody that binds to EPS8 or an antibody fragment thereof is used as a solid phase antibody and an antibody that binds to an exosome-specific antigen or the antibody fragment is used as a labeling antibody, it binds to an exosome-specific antigen. The antibody or the antibody fragment thereof may be used as a solid phase antibody, and the antibody that binds to EPS8 or the antibody fragment thereof may be used as a labeling antibody. Examples of the solid phase and the label include known solid phase and the label, respectively, and for example, the above-mentioned solid phase and the label are used, respectively. Examples of the method for preparing the solid phase antibody and the labeling antibody include known preparation methods.

<Process (4C)>
The step (4C) is a step of determining the EPS8 concentration of the exosome in the sample from the EPS8 concentration measured in the step (3C). From the volume of the sample and the EPS8 concentration measured in the step (3C), the EPS8 concentration of the exosome in the sample can be determined.

<Process (5C)>
The step (5C) includes the EPS8 concentration of the exosome in the sample collected from the pancreatic disease patient determined in the step (4C) and the exosome in the sample collected from the pancreatic disease patient other than pancreatic cancer. This is a step of comparing the EPS8 concentration with the EPS8 concentration of the exosome in the sample collected from the pancreatic disease patient , which is higher than the EPS8 concentration of the exosome in the sample collected from the pancreatic disease patient other than pancreatic cancer. If the patient with pancreatic disease is determined to have a high possibility of suffering from pancreatic cancer, and the EPS8 concentration of the exosome in the sample collected from the patient with pancreatic disease other than pancreatic cancer is the same as or lower than that of the exosome. It is determined that the pancreatic disease patient is likely to have a pancreatic disease other than pancreatic cancer.

The EPS8 concentration of exosomes in a sample collected from a patient with pancreatic disease other than pancreatic cancer is used to distinguish whether the patient with pancreatic cancer has pancreatic cancer or a pancreatic disease other than pancreatic cancer. It is a reference value, and the EPS8 concentration of exosomes in samples collected from multiple patients with pancreatic disease other than pancreatic cancer is measured, and from the statistics, exosomes have in samples collected from patients with pancreatic disease other than pancreatic cancer. The EPS8 concentration can be set.
In this case, the sample of the pancreatic disease patient other than pancreatic cancer used when setting the reference value is preferably a sample of the same type as the sample of the pancreatic disease patient who is the subject, for example, the subject. When the sample of a pancreatic disease patient is blood, it is preferable to use blood as a sample of a pancreatic disease patient other than pancreatic cancer.
As a method for comparing the EPS8 concentration of the exosome of the subject with the EPS8 concentration of the exosome of a patient with a pancreatic disease other than pancreatic cancer, the above-mentioned method can be used. When the analysis shows that the EPS8 concentration of the exosome in the sample collected from the subject is higher than the EPS8 concentration of the exosome in the sample collected from a patient with a pancreatic disease other than pancreatic cancer. , The subject was determined to have a high possibility of suffering from pancreatic cancer, and it was shown that the EPS8 concentration of exosomes in a sample collected from a patient with pancreatic disease other than pancreatic cancer was the same as or lower than that of the exosome. If so, it is determined that the subject is likely to be suffering from a pancreatic disease patient other than pancreatic cancer.

3. 3. Method for monitoring the possibility that a subject has pancreatic cancer The method for monitoring the possibility that a subject has pancreatic cancer according to the present invention is a sample collected from a subject over time. It is a method characterized by measuring the concentration of EPS8 contained in an exosome.

The sample in the present invention is not particularly limited as long as it is a sample in which EPS8 contained in an exosome can be measured, and examples thereof include the above-mentioned samples.
In the present invention, EPS8 contained in an exosome may be contained in the exosome, present on the surface of the membrane of the exosome, penetrate the membrane of the exosome, or exist in any form.
In the present invention, the EPS8 concentration contained in an exosome includes not only the mass or the number of moles per unit volume but also the signal intensity per unit volume.

[Measurement method 5]
In the present invention, the method of monitoring the possibility that the subject has pancreatic cancer can be carried out by a method including the following steps.
(1D) Step of collecting a sample from a subject over time;
(2D) A step of measuring the concentration of EPS8 contained in exosomes in the sample collected in step (1D);
(3D) If the EPS8 concentration measured in step (2D) is maintained at a higher concentration than the EPS8 concentration of exosomes in a sample collected from a healthy subject, the subject suffers from pancreatic cancer. If it is determined that there is a high possibility that the exosome is maintained at the same or lower concentration as the EPS8 concentration of the exosome in the sample collected from a healthy subject, the subject is suffering from pancreatic cancer. The process of determining that the possibility is low.

<Process (1D)>
Examples of the sample collected from the subject in the step (1D) include the above-mentioned sample and the like.

<Process (2D)>
In the step (2D), the EPS8 concentration of the exosome in the sample collected in the step (1D) using the antibody or the antibody fragment that binds to EPS8 and the antibody or the antibody fragment that binds to the exosome-specific antigen is used. Is the process of measuring.
The antibody that binds to EPS8 in the step (2D) is not particularly limited as long as it is an antibody that can measure EPS8, and examples thereof include the above-mentioned antibody.
Examples of the exosome-specific antigen in the step (2D) include the above-mentioned antigens and the like.

The antibody that binds to the exosome-specific antigen is not particularly limited as long as it can bind to the exosome-specific antigen, and examples thereof include the above-mentioned antibodies.
The antibody fragment that binds to EPS8 and the antibody fragment that binds to the exosome-specific antigen in the step (2D) are not particularly limited as long as they can measure EPS8 and the exosome-specific antigen, respectively. Can be mentioned.
In the step (2D), the above two types of antibodies used for measuring the EPS8 concentration of an exosome are an antibody that binds to an exosome-specific antigen or an antibody fragment thereof as a solid phase antibody, and an antibody that binds to EPS8 or the antibody fragment thereof. Can be used as a labeling antibody. Alternatively, an antibody that binds to EPS8 or the antibody fragment thereof can be used as a solid phase antibody, and an antibody that binds to an exosome-specific antigen or the antibody fragment thereof can be used as a labeling antibody. The preparation of the solid phase antibody and the labeling antibody is not particularly limited and can be carried out according to a known method.
The method for measuring EPS8 contained in an exosome can be carried out by any method as long as it can measure EPS8 contained in an exosome, and can be carried out by using, for example, the above-mentioned immunological measuring method or the like. ..
In the step (2D), when measuring the concentration of EPS8 contained in an exosome in a sample without destroying the exosome in the sample, an antibody that binds to EPS8 or an antibody that binds to EPS8 as the antibody fragment thereof. Alternatively, among the antibody fragments, it is preferable to use an antibody or antibody fragment that binds to the EPS8 epitope present on the surface of the exosome.
Examples of the above-mentioned immunological measurement method include the above-mentioned sandwich ELISA, exoscreen method and the like.

<Process (3D)>
In the step (3D), the EPS8 concentration of the exosome in the sample collected from the subject obtained in the step (2D) is compared with the EPS8 concentration of the exosome in the sample collected from a healthy subject. In this step, if the EPS8 concentration of exosomes in a sample collected from a subject is higher than the EPS8 concentration of exosomes in a sample collected from a healthy subject, the subject suffers from pancreatic cancer. If the concentration of EPS8 in the sample collected from a healthy subject is the same as or lower than the EPS8 concentration of exosomes, it is considered that the subject is unlikely to have pancreatic cancer. judge.

The EPS8 concentration of exosomes in a sample collected from a healthy subject is a reference value for determining the possibility that the examiner has pancreatic cancer, and is a reference value in a sample collected from a plurality of healthy subjects. The EPS8 concentration of exosomes can be measured, and the EPS8 concentration of exosomes in a sample collected from a healthy person can be set from the statistics.
The sample collected from a healthy person is preferably a sample of the same type as the sample collected from a subject. For example, when the sample collected from a subject is blood, the sample collected from a healthy person also contains blood. It is preferable to use it.
The method for comparing the EPS8 concentration of exosomes in a sample collected from a subject over time with the EPS8 concentration of exosomes in a sample collected from a healthy subject is not particularly limited, and for example, the above-mentioned method or the like is used. Can be used. According to the analysis, if the EPS8 concentration of the exosome in the sample collected from the subject is maintained at a higher concentration than the EPS8 concentration of the exosome in the sample collected from the healthy subject, the test is performed. If a person is determined to have a high possibility of suffering from pancreatic cancer and is maintained at the same or lower concentration of EPS8 possessed by exosomes in a sample collected from a healthy person, the subject is considered to have the same or lower concentration. It is determined that the possibility of having pancreatic cancer is low.

Further, in the step (2D) of the measurement method 5, the EPS8 concentration of the exosome in the sample can be measured by using the exosome isolated from the sample collected from the subject. Specifically, a method including the following steps can be mentioned.
[Measurement method 6]
(1E) Step of collecting a sample from a subject over time;
(2E) A step of isolating exosomes from the sample collected in step (1E);
(3E) A step of measuring the EPS8 concentration of the exosomes isolated in step (2E);
(4E) A step of determining the EPS8 concentration of an exosome in the sample from the EPS8 concentration measured in step (3E);
(5E) If the EPS8 concentration determined in step (4E) is maintained at a higher concentration than the EPS8 concentration of exosomes in a sample collected from a healthy subject, the subject suffers from pancreatic cancer. If it is determined that there is a high possibility that the exosome is maintained at the same or lower concentration as the EPS8 concentration of the exosome in the sample collected from a healthy subject, the subject is suffering from pancreatic cancer. The process of determining that the possibility is low.

<Process (1E)>
Examples of the sample collected from the subject in the step (1E) include the above-mentioned sample and the like.

<Process (2E)>
The method for isolating exosomes in step (2E) is not particularly limited as long as it can isolate exosomes in a sample, and examples thereof include the above-mentioned methods.

<Process (3E)>
The method for measuring EPS8 contained in an exosome in step (3E) can be carried out by any method as long as it can measure EPS8 contained in an exosome, for example, by using a method such as an immunological measurement method. be able to. Examples of the method for measuring the EPS8 concentration of an exosome by an immunological measurement method include the following aspects.

・ Aspect 1
A method using an antibody that binds to EPS8 or an antibody fragment thereof.
・ Aspect 2
A method using an antibody or an antibody fragment thereof that binds to EPS8, and an antibody or an antibody fragment thereof that binds to an exosome-specific antigen.

Examples of the antibody that binds to EPS8 or the antibody fragment thereof include the above-mentioned antibody that binds to EPS8 or the antibody fragment thereof. Examples of the antibody or the antibody fragment thereof that binds to the exosome-specific antigen include the antibody that binds to the above-mentioned exosome-specific antigen or the antibody fragment thereof.

Examples of the method of the first aspect include a Western blotting method and the like. Examples of the Western blotting method include the above-mentioned methods.

As the method of the second aspect, in the case of measuring the isolated exosome without destroying it, the immunological measurement method described in the above-mentioned measurement method 1, for example, the exoscreen method and the like can be mentioned, and the isolated exosome can be used. In the case of breaking and measuring, a sandwich ELISA method or the like can be mentioned. Examples of the exoscreen method include the above-mentioned exoscreen method.
When measuring the isolated exosome without destroying it, the antibody that binds to EPS8 or the antibody fragment thereof is the antibody that binds to EPS8 or the antibody fragment that binds to the epitope of EPS8 present on the surface of the exosome. Alternatively, use an antibody fragment.
Examples of the method for destroying the isolated exosome include the above-mentioned methods. As the sandwich ELISA method, for example, even if an antibody that binds to EPS8 or an antibody fragment thereof is used as a solid phase antibody and an antibody that binds to an exosome-specific antigen or the antibody fragment is used as a labeling antibody, it binds to an exosome-specific antigen. The antibody or the antibody fragment thereof may be used as a solid phase antibody, and the antibody that binds to EPS8 or the antibody fragment thereof may be used as a labeling antibody. Examples of the solid phase and the label include known solid phase and the label, respectively, and for example, the above-mentioned solid phase and the label are used, respectively. Examples of the method for preparing the solid phase antibody and the labeling antibody include known preparation methods.

<Process (4E)>
The step (4E) is a step of determining the EPS8 concentration of the exosome in the sample from the EPS8 concentration measured in the step (3E). From the volume of the sample and the EPS8 concentration measured in the step (3E), the EPS8 concentration of the exosome in the sample can be determined.

<Process (5E)>
In step (5E), the EPS8 concentration of exosomes in the sample collected from the subject determined in step (4E) is compared with the EPS8 concentration of exosomes in the sample collected from a healthy subject. If the EPS8 concentration of the exosome in the sample collected from the subject is maintained at a higher concentration than the EPS8 concentration of the exosome in the sample collected from the healthy subject, the subject is concerned. If it is determined that the examiner is likely to have pancreatic cancer and the concentration is maintained at the same or lower than the EPS8 concentration of the exosome in the sample collected from a healthy person, the test is performed. A person is determined to be less likely to have pancreatic cancer.

The EPS8 concentration of exosomes in a sample collected from a healthy subject is a reference value for determining the possibility that the examiner has pancreatic cancer, and is a reference value in a sample collected from a plurality of healthy subjects. The EPS8 concentration of exosomes can be measured, and the EPS8 concentration of exosomes in a sample collected from a healthy person can be set from the statistics.
The sample of a healthy person is preferably a sample of the same type as the sample of a subject. For example, when the sample of a subject is blood, it is preferable to use blood as the sample of a healthy person.
As a method for comparing the EPS8 concentration of exosomes in a sample collected from a subject with the EPS8 concentration of exosomes in a sample collected from a healthy subject, the above-mentioned method can be used. According to the analysis, if the EPS8 concentration of the exosome in the sample collected from the subject is maintained at a higher concentration than the EPS8 concentration of the exosome in the sample collected from the healthy subject, the test is performed. If a person is determined to have a high possibility of suffering from pancreatic cancer and is maintained at the same or lower concentration of EPS8 possessed by exosomes in a sample collected from a healthy person, the subject is considered to have the same or lower concentration. It is determined that the possibility of having pancreatic cancer is low.

As will be described later in Examples, according to the present invention, the therapeutic effect when surgery is performed by measuring the EPS8 concentration of exosomes in a sample collected from a pancreatic cancer patient before and after surgery. , The condition of pancreatic cancer patients such as shrinkage of cancerous part can be monitored.
Further, according to the present invention, recurrence of pancreatic cancer can be predicted by measuring the EPS8 concentration of exosomes in a sample collected from a patient with pancreatic cancer being treated.
Further, according to the present invention, the possibility that the subject has pancreatic cancer can be monitored by measuring the EPS8 concentration of the exosome in the sample collected from the subject.

4. Reagent for testing the possibility that a subject has pancreatic cancer The reagent of the present invention for testing the possibility that a subject has pancreatic cancer is an EPS8 measurement reagent possessed by Exosome. It is a reagent characterized by containing, and can be used in the method of the present invention for testing the possibility that a subject has pancreatic cancer.

Further, in the reagent of the present invention for testing the possibility that the subject has pancreatic cancer, the EPS8 concentration of the exosome in the sample collected from the subject is further used in a healthy subject. If it is higher than the EPS8 concentration of exosomes in the sample collected, the subject is likely to have pancreatic cancer, and the EPS8 concentration of exosomes in the sample collected from healthy subjects If the same or lower, a criteria table may be included that describes the criteria that the subject is less likely to have pancreatic cancer.

The EPS8 measurement reagent possessed by an exosome in the reagent of the present invention for testing the possibility that a subject has pancreatic cancer is a reagent used in the above-mentioned method for measuring EPS8 possessed by an exosome. The EPS8 measuring reagent contained in the exosome is not particularly limited as long as it is a reagent capable of measuring EPS8 contained in the exosome in the sample, and examples thereof include an immunological measuring reagent. Examples of the immunological measurement reagent include reagents based on the above-mentioned immunological measurement method.
Specific embodiments of the EPS8 measurement reagent contained in the exosome based on the immunological measurement method are shown below.

・ Measuring reagent 1
A reagent containing an antibody that binds to EPS8 or an antibody fragment thereof.
・ Measuring reagent 2
A reagent containing an antibody or antibody fragment thereof that binds to EPS8 to which the label is bound.
・ Measuring reagent 3
A reagent containing an antibody or an antibody fragment thereof that binds to EPS8 and an antibody or an antibody fragment thereof that binds to an exosome-specific antigen.
・ Measuring reagent 4
A reagent containing an antibody or an antibody fragment thereof that binds to EPS8 immobilized on a solid phase and an antibody or an antibody fragment thereof that binds to an exosome-specific antigen to which a label is bound.
・ Measuring reagent 5
A reagent containing an antibody or an antibody fragment thereof that binds to an exosome-specific antigen immobilized on a solid phase and an antibody or an antibody fragment thereof that binds to EPS8 to which a label is bound.
・ Measuring reagent 6
A reagent containing a first antibody or antibody fragment thereof immobilized on a solid phase and binding to EPS8, and a second antibody or antibody fragment thereof bound to EPS8 to which a label is bound.
The antibody or antibody fragment that binds to EPS8 and the antibody or antibody fragment that binds to an exosome-specific antigen include the antibody or antibody fragment that binds to EPS8 or an antibody that binds to an exosome-specific antigen, respectively. Alternatively, the antibody fragment and the like can be mentioned.

Examples of the label in the label-bound antibody that binds to EPS8 or the antibody fragment thereof, and the label-bound antibody that binds to the exosome-specific antigen or the antibody fragment include the above-mentioned labels and the like. The label-bound antibody or antibody fragment thereof that binds to EPS8 and the antibody that binds to the exosome-specific antigen to which the label is bound or the antibody fragment thereof are prepared, for example, according to a known method for preparing a labeled antibody. Can be done.
Examples of the solid phase in the solid phase-immobilized antibody or antibody fragment thereof that binds to EPS8 and the antibody or antibody fragment that binds to an exosome-specific antigen immobilized on the solid phase are described above. Equality can be mentioned. Immobilization of an antibody or antibody fragment that binds to EPS8 on a solid phase and immobilization of an antibody or antibody fragment that binds to an exosome-specific antigen on a solid phase can be performed, for example, according to a known method.
In the measurement reagent 6, the epitope to which the first antibody or the antibody fragment that binds to EPS8 binds and the epitope to which the second antibody or the antibody fragment that binds to EPS8 binds may be different or the same, but they are different. Is preferable.
The measurement reagents 1 to 6 may further contain an exosome isolation reagent. The exosome isolation reagent is not particularly limited as long as it is a reagent capable of isolating exosomes from a sample, and examples thereof include the above-mentioned exosome isolation kit.

5. Reagent for Differentiating Pancreatic Cancer from Pancreatic Disease Other Than Pancreatic Cancer in Patients with Pancreatic Disease The reagent of the present invention for distinguishing pancreatic cancer from pancreatic disease other than pancreatic cancer in patients with pancreatic disease is Exome. a reagent which is characterized by containing a EPS8 measurement reagent having, of the present invention, the pancreatic disorder patients, can be used in a method for differentiating pancreatic diseases other than pancreatic cancer and pancreatic cancer.

Further, in the reagent of the present invention for distinguishing pancreatic cancer from pancreatic disease other than pancreatic cancer in a pancreatic disease patient, the EPS8 concentration contained in the exosome in the sample collected from the pancreatic disease patient is further added. If the EPS8 concentration of the exosomes in the sample of a pancreatic disease patient other than pancreatic cancer is higher than the EPS8 concentration, the pancreatic disease patient is likely to have pancreatic cancer, and the sample of a pancreatic disease patient other than pancreatic cancer. If the EPS8 concentration of the exosome is the same as or lower than that of the exosome, a reference table containing the criteria that the pancreatic disease patient is likely to have a pancreatic disease other than pancreatic cancer may be included. ..

The EPS8 measuring reagent contained in the exosome in the reagent for distinguishing pancreatic cancer from a pancreatic disease other than pancreatic cancer in a pancreatic disease patient of the present invention is a reagent used in the above-mentioned method for measuring EPS8 possessed by an exosome. is there. The EPS8 measuring reagent contained in the exosome is not particularly limited as long as it is a reagent capable of measuring EPS8 contained in the exosome in the sample, and examples thereof include an immunological measuring reagent. Examples of the immunological measurement reagent include reagents based on the above-mentioned immunological measurement method.
Specific embodiments of the EPS8 measurement reagent contained in the exosome based on the immunological measurement method are shown below.

・ Measuring reagent 7
A reagent containing an antibody that binds to EPS8 or an antibody fragment thereof.
・ Measuring reagent 8
A reagent containing an antibody or antibody fragment thereof that binds to EPS8 to which the label is bound.
・ Measuring reagent 9
A reagent containing an antibody or an antibody fragment thereof that binds to EPS8 and an antibody or an antibody fragment thereof that binds to an exosome-specific antigen.
-Measuring reagent 10
A reagent containing an antibody or an antibody fragment thereof that binds to EPS8 immobilized on a solid phase and an antibody or an antibody fragment thereof that binds to an exosome-specific antigen to which a label is bound.
-Measuring reagent 11
A reagent containing an antibody or an antibody fragment thereof that binds to an exosome-specific antigen immobilized on a solid phase and an antibody or an antibody fragment thereof that binds to EPS8 to which a label is bound.
-Measuring reagent 12
A reagent containing a first antibody or antibody fragment thereof immobilized on a solid phase and binding to EPS8, and a second antibody or antibody fragment thereof bound to EPS8 to which a label is bound.

The antibody or antibody fragment that binds to EPS8 and the antibody or antibody fragment that binds to an exosome-specific antigen include the antibody or antibody fragment that binds to EPS8 or an antibody that binds to an exosome-specific antigen, respectively. Alternatively, the antibody fragment and the like can be mentioned.
Examples of the label in the label-bound antibody that binds to EPS8 or the antibody fragment thereof, and the label-bound antibody that binds to the exosome-specific antigen or the antibody fragment include the above-mentioned labels and the like. The label-bound antibody or antibody fragment thereof that binds to EPS8 and the antibody that binds to the exosome-specific antigen to which the label is bound or the antibody fragment thereof are prepared, for example, according to a known method for preparing a labeled antibody. Can be done.
Examples of the solid phase in the solid phase-immobilized antibody or antibody fragment thereof that binds to EPS8 and the antibody or antibody fragment that binds to an exosome-specific antigen immobilized on the solid phase are described above. Equality can be mentioned. Immobilization of an antibody or antibody fragment that binds to EPS8 on a solid phase and immobilization of an antibody or antibody fragment that binds to an exosome-specific antigen on a solid phase can be performed, for example, according to a known method.
In the measurement reagent 12, the epitope to which the first antibody or the antibody fragment that binds to EPS8 binds and the epitope to which the second antibody or the antibody fragment that binds to EPS8 binds may be different or the same, but they are different. Is preferable.
The measurement reagents 7 to 12 may further contain exosome isolation reagents. The exosome isolation reagent is not particularly limited as long as it is a reagent capable of isolating exosomes from a sample, and examples thereof include the above-mentioned exosome isolation kit.

6. Reagent for monitoring the possibility that a subject has pancreatic cancer The reagent of the present invention for monitoring the possibility that a subject has pancreatic cancer is an EPS8 measurement reagent possessed by Exosome. It is a reagent characterized by containing, and can be used in the method of the present invention for monitoring the possibility that a subject has pancreatic cancer.

In addition, the reagent of the present invention for monitoring the possibility that a subject has pancreatic cancer further includes the EPS8 concentration of exosomes in a sample collected from the subject over time. If the concentration is maintained higher than the EPS8 concentration of the exosomes in the healthy subject's sample, the subject is likely to have pancreatic cancer, and the exosomes in the healthy subject's sample have. A criteria table may be included that describes the criteria that the subject is less likely to have pancreatic cancer if the concentration is maintained at the same or lower than the EPS8 concentration.

The EPS8 measurement reagent possessed by an exosome in the reagent of the present invention for monitoring the possibility that a subject has pancreatic cancer is a reagent used in the above-mentioned method for measuring EPS8 possessed by an exosome. The EPS8 measuring reagent contained in the exosome is not particularly limited as long as it is a reagent capable of measuring EPS8 contained in the exosome in the sample, and examples thereof include an immunological measuring reagent. Examples of the immunological measurement reagent include reagents based on the above-mentioned immunological measurement method.
Specific embodiments of the EPS8 measurement reagent contained in the exosome based on the immunological measurement method are shown below.

-Measuring reagent 13
A reagent containing an antibody that binds to EPS8 or an antibody fragment thereof.
-Measuring reagent 14
A reagent containing an antibody or antibody fragment thereof that binds to EPS8 to which the label is bound.
-Measuring reagent 15
A reagent containing an antibody or an antibody fragment thereof that binds to EPS8 and an antibody or an antibody fragment thereof that binds to an exosome-specific antigen.
-Measuring reagent 16
A reagent containing an antibody or an antibody fragment thereof that binds to EPS8 immobilized on a solid phase and an antibody or an antibody fragment thereof that binds to an exosome-specific antigen to which a label is bound.
・ Measuring reagent 17
A reagent containing an antibody or an antibody fragment thereof that binds to an exosome-specific antigen immobilized on a solid phase and an antibody or an antibody fragment thereof that binds to EPS8 to which a label is bound.
-Measuring reagent 18
A reagent containing a first antibody or antibody fragment thereof immobilized on a solid phase and binding to EPS8, and a second antibody or antibody fragment thereof bound to EPS8 to which a label is bound.

The antibody or antibody fragment that binds to EPS8 and the antibody or antibody fragment that binds to an exosome-specific antigen include the antibody or antibody fragment that binds to EPS8 or an antibody that binds to an exosome-specific antigen, respectively. Alternatively, the antibody fragment and the like can be mentioned.
Examples of the label in the label-bound antibody that binds to EPS8 or the antibody fragment thereof, and the label-bound antibody that binds to the exosome-specific antigen or the antibody fragment include the above-mentioned labels and the like. The label-bound antibody or antibody fragment thereof that binds to EPS8 and the antibody that binds to the exosome-specific antigen to which the label is bound or the antibody fragment thereof are prepared, for example, according to a known method for preparing a labeled antibody. Can be done.
Examples of the solid phase in the solid phase-immobilized antibody or antibody fragment thereof that binds to EPS8 and the antibody or antibody fragment that binds to an exosome-specific antigen immobilized on the solid phase are described above. Equality can be mentioned. Immobilization of an antibody or antibody fragment that binds to EPS8 on a solid phase and immobilization of an antibody or antibody fragment that binds to an exosome-specific antigen on a solid phase can be performed, for example, according to a known method.
In the measurement reagent 18, the epitope to which the first antibody or the antibody fragment that binds to EPS8 binds and the epitope to which the second antibody or the antibody fragment that binds to EPS8 binds may be different or the same, but they are different. Is preferable.
The measurement reagents 13 to 18 may further contain exosome isolation reagents. The exosome isolation reagent is not particularly limited as long as it is a reagent capable of isolating exosomes from a sample, and examples thereof include the above-mentioned exosome isolation kit.

Further, in each of the above inventions, the "concentration of EPS8 possessed by an exosome" may be not only a protein level concentration, that is, an EPS8 concentration but also a gene level concentration. Examples of the method for measuring the “EPS8 concentration in an exosome” at the gene level include a primer set capable of amplifying the EPS8 gene or a measurement method using a probe that specifically hybridizes to the mRNA of the EPS8 gene.

(Primer set)
The primer set capable of amplifying the EPS8 gene is not particularly limited as long as it can amplify the mRNA of the EPS8 gene by RT-PCR or the like.
For example, the mRNA of the EPS8 gene in the sample can be detected by performing RT-PCR or the like using this primer set using the tissue of the subject as the sample.

(probe)
The probe is not particularly limited as long as it specifically hybridizes to the mRNA of the EPS8 gene. The probe may be immobilized on a carrier to form a DNA microarray or the like. The step of measuring the "concentration of EPS8 contained in exosomes" can be performed by RT-PCR using the above-mentioned primer set, quantitative RT-PCR, or the like. Alternatively, the expression level of the EPS8 gene may be measured by DNA microarray analysis using the above-mentioned probe, Northern blotting, or the like. Here, the measured expression level of the EPS8 gene may be converted into a quantitative value by using a fragment of each gene having a known concentration that is serially diluted as a reference. Examples of the carrier include the above-mentioned carriers and the like.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to this Example.

[Example 1]
Detection of EPS8 contained in exosomes in each serum Serum collected from healthy subjects (4 samples), serum collected from patients with pancreatitis (4 samples), and serum collected from patients with pancreatic cancer (4 samples: 2 types of stage IIA, After ultracentrifugation of 1 type of stage IIB and 1 type of stage III at 100,000 × g or more (4 ° C) for 70 minutes with a swing rotor, the supernatant is discarded, and PBS is added to precipitate the precipitate. The fraction was defined as an exosome fraction. This exosome fraction was lysed with PBS (total volume 100 μL). An exosome fraction solution equivalent to 100 μL of serum per well contains the solution and 1/3 volume of 4 × sample buffer (4% sodium dodecyl sulfate, 10% sucrose, 0.01% bromophenol blue, 10% 2-mercaptoethanol). , 0.125 mol / L Tris buffer [pH 6.8]) was added and mixed, and the mixture was kept warm at 95 ° C. for 5 minutes and then ice-cooled. The treatment solution was applied to a well of 4-15% Mini-Protian TGX gel (manufactured by Bio-Rad), and SDS-PAGE was performed. After transferring the gel after SDS-PAGE to a PVDF membrane, the membrane was blocked with Nacalai Blocking One solution [manufactured by Nacalai Tesque] at room temperature (25 ° C.) for 1 hour. Next, a primary antibody reaction was carried out at room temperature (25 ° C.) for 1 hour (Purified Mouse Anti-EPS8 (15 / EPS8) mouse monoclonal [manufactured by Becton Dickinson, Japan] 1: 2000). Subsequently, a secondary antibody reaction was carried out at room temperature (25 ° C.) for 1 hour (Mouse IgG HRP [GE Healthcare] 1: 5000). Finally, a color reaction (room temperature [25 ° C] for 5 minutes) was performed using ImmunoStar LD (manufactured by Wako Pure Chemical Industries, Ltd.), blotting images were taken, and EPS8 signals contained in exosomes in each serum were detected. .. The result of the blotting image is shown in FIG.

In addition, according to the above method, the expression level of CD63 contained in exosomes in each serum was evaluated by Western blotting using Purified Mouse Anti-Human CD63 (manufactured by Becton Dickinson, Japan, clone: H5C6). The result is shown in FIG.
As is clear from FIG. 1, it was found that EPS8 contained in exosomes isolated from the serum of the pancreatic cancer patient was highly expressed in the pancreatic cancer patient.

[Example 2]
Comparison of EPS8 concentration of exosomes in each serum using serum (10 samples) collected from healthy subjects, serum (10 samples) collected from pancreatitis patients, and serum (23 samples) collected from pancreatic cancer patients. Therefore, according to the method of Example 1, the signals of EPS8 and CD63 contained in the exosomes in each serum were detected. The signal ratio of EPS8 / CD63 is shown in FIG.
As is clear from FIG. 2, it was found that the EPS8 concentration of exosomes in the serum collected from pancreatic cancer patients was higher than the EPS8 concentration of exosomes in the serum collected from healthy subjects. Further, it was found that the EPS8 concentration of exosomes in the serum collected from pancreatic cancer patients was higher than the EPS8 concentration of exosomes in the serum collected from pancreatitis patients.

[Example 3]
Pancreatic cancer patients (5 cases) were evaluated by Western blotting according to the method of Example 1 in order to compare the amount of EPS8 contained in exosomes in each serum before and after surgery. The result of the blotting image is shown in FIG.
As is clear from FIG. 3, it was found that the amount of EPS8 contained in exosomes in the patient's serum after surgery was reduced.

[Example 4]
In order to compare the amount of EPS8 contained in the exosomes in the serum of patients with recurrent pancreatic cancer or the exosomes in the serum of healthy subjects, signals were detected by Western blotting according to the method of Example 1. At the same time, biotinylated anti-CD9 antibody (1000-fold dilution) obtained by biotinlating anti-CD9 antibody (8A12) (manufactured by Cosmobio) and HRP-binding streptavidin (manufactured by CST) (2000-fold dilution) were used. The amount of CD9 contained in exosomes in the serum of horseradish peroxidase was detected by Western blotting. The signal ratio of EPS8 / CD9 calculated from the signal of EPS8 and the signal of CD9 is shown in FIG.
As is clear from FIG. 4, the amount of EPS8 contained in exosomes in the serum of patients with recurrent pancreatic cancer is higher than the amount of EPS8 contained in exosomes in the serum of healthy subjects. found.

The method and reagent of the present invention for testing the possibility that a subject has pancreatic cancer, the method and reagent for distinguishing pancreatic cancer from a pancreatic disease other than pancreatic cancer, and the subject Methods and reagents for monitoring the likelihood of having pancreatic cancer are useful in clinical diagnosis.

Claims (23)

A measurement step of measuring EPS8 (Epidermal Growth Factor Receptor Kinase Substrate 8: Epidermal Growth Factor Receptor Kinase Substrate 8) possessed by exosomes at a protein level in a blood sample collected from a subject is included.
The measurement step is performed by an immunological measurement method using an antibody that binds to EPS8 or the antibody fragment thereof, and an antibody that binds to an antigen specifically expressed in exosomes (hereinafter referred to as an exosome-specific antigen) or the antibody fragment. and wherein the dividing method of testing a potentially affected subject is pancreatic cancer. Is EPS8 concentration measured in the previous SL measurement step, in the blood sample collected from a healthy person, it is likely suffering該被examiner pancreatic cancer is higher than EPS8 concentration with the exosomes The present invention according to claim 1, wherein if the EPS8 concentration of the exosome in the blood sample collected from a healthy subject is the same as or lower than that of the exosome, the subject is unlikely to have pancreatic cancer. the method of. The method according to claim 1 or 2, wherein in the measurement step, the concentration of EPS8 contained in the exosome at the protein level in the collected blood sample is measured using the exosome isolated from the blood sample. .. The exosome-specific antigen is CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, or LAMP1, according to any one of claims 1 to 3. The method described. Including a measurement step of measuring EPS8 (Epidermal Growth Factor Receptor Kinase Substrate 8: Epidermal Growth Factor Receptor Kinase Substrate 8) contained in exosomes at a protein level in a blood sample collected from a patient with pancreatic disease.
The measurement step is performed by an immunological measurement method using an antibody that binds to EPS8 or the antibody fragment thereof, and an antibody that binds to an antigen specifically expressed in exosomes (hereinafter referred to as an exosome-specific antigen) or the antibody fragment. and wherein the dividing, in pancreatic disease patients, method for differentiating pancreatic diseases other than pancreatic cancer and pancreatic cancer. Is EPS8 concentration measured in the previous SL measurement step, in the blood sample collected from pancreatic disease patients than pancreatic cancer,該被examiner is higher than EPS8 concentration with the exosomes can suffering from pancreatic cancer If the EPS8 concentration of exosomes is the same as or lower than the EPS8 concentration of exosomes in blood samples collected from patients with pancreatic disease other than pancreatic cancer, the subject suffers from pancreatic disease other than pancreatic cancer. The method of claim 5 , indicating that there is a high probability of being present. The method according to claim 5 or 6 , wherein in the measurement step, the concentration of EPS8 contained in the exosome at the protein level in the collected blood sample is measured using the exosome isolated from the blood sample. .. The exosome-specific antigen is CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, or LAMP1, according to any one of claims 5 to 7. The method described. The method according to any one of claims 5 to 8 , wherein the pancreatic disease other than pancreatic cancer is pancreatitis. A measurement step of measuring EPS8 (Epidermal Growth Factor Receptor Kinase Substrate 8: Epidermal Growth Factor Receptor Kinase Substrate 8) possessed by exosomes at a protein level in a blood sample collected from a subject over time is included.
The measurement step is performed by an immunological measurement method using an antibody that binds to EPS8 or the antibody fragment thereof, and an antibody that binds to an antigen specifically expressed in exosomes (hereinafter referred to as an exosome-specific antigen) or the antibody fragment. and wherein the dividing method of monitoring potentially affected subject is pancreatic cancer. Before Symbol measurement step EPS8 concentrations measured at the, in the blood sample collected from a healthy person, suffering該被examiner pancreatic cancer if it is maintained at a concentration higher than EPS8 concentration with the exosomes The subject may have pancreatic cancer if it indicates a high probability and is maintained at the same or lower EPS8 concentration of exosomes in blood samples collected from healthy subjects. The method of claim 10 , which shows that the sex is low. The method according to claim 10 or 11, wherein in the measurement step, the concentration of EPS8 contained in the exosome at the protein level in the collected blood sample is measured using the exosome isolated from the blood sample. .. Any one of claims 10 to 12, wherein the exosome-specific antigen is CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, or LAMP1. The method described in the section. Contains an immunological measurement reagent for measuring EPS8 (Epidermal Growth Factor Receptor Kinase Substrate 8: Epidermal Growth Factor Receptor Kinase Substrate 8) possessed by exosomes at a protein level in a blood sample collected from a subject.
The immunological measurement reagent contains an antibody or an antibody fragment thereof that binds to EPS8, and an antibody or an antibody fragment thereof that binds to an antigen specifically expressed in an exosome (hereinafter referred to as an exosome-specific antigen).
Testing the possibility that a subject has pancreatic cancer, which is used in the method for testing the possibility of having pancreatic cancer according to any one of claims 1 to 4. Reagents to do. The reagent according to claim 14 , wherein the exosome-specific antigen is CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, or LAMP1. The reagent according to claim 14 or 15 , further comprising an exosome isolation reagent. Contains an immunological measurement reagent for measuring EPS8 (Epidermal Growth Factor Receptor Kinase Substrate 8: Epidermal Growth Factor Receptor Kinase Substrate 8) possessed by exosomes at a protein level in a blood sample collected from a subject.
The immunological measurement reagent contains an antibody or an antibody fragment thereof that binds to EPS8, and an antibody or an antibody fragment thereof that binds to an antigen specifically expressed in an exosome (hereinafter referred to as an exosome-specific antigen).
A pancreatic cancer patient and a pancreatic disease patient other than pancreatic cancer, which is used in a method for distinguishing between pancreatic cancer and a pancreatic disease other than pancreatic cancer according to any one of claims 5 to 9. A reagent for differentiating. The reagent according to claim 17 , wherein the exosome-specific antigen is CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, or LAMP1. The reagent according to claim 17 or 18 , further comprising an exosome isolation reagent. The reagent according to any one of claims 17 to 19 , wherein the pancreatic disease other than pancreatic cancer is pancreatitis. Contains an immunological measurement reagent for measuring EPS8 (Epidermal Growth Factor Receptor Kinase Substrate 8: Epidermal Growth Factor Receptor Kinase Substrate 8) possessed by exosomes at a protein level in a blood sample collected from a subject over time.
The immunological measurement reagent contains an antibody or an antibody fragment thereof that binds to EPS8, and an antibody or an antibody fragment thereof that binds to an antigen specifically expressed in an exosome (hereinafter referred to as an exosome-specific antigen).
Monitoring the possibility that a subject has pancreatic cancer, which is used in the method for monitoring the possibility of having pancreatic cancer according to any one of claims 10 to 13. Reagents to do. The reagent according to claim 21 , wherein the exosome-specific antigen is CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, or LAMP1. The reagent according to claim 21 or 22 , further comprising an exosome isolation reagent.

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