WO2021187173A1 - Method for detecting gastrointestinal stromal tumor and detection reagent - Google Patents

Abstract

The present invention addresses the problem of providing a method for easily detecting gastrointestinal stromal tumor (GIST) at a high accuracy and a reagent usable in this method. A method for detecting GIST, said method comprising measuring the amount of azurocidin (AZU1) in a specimen, wherein when the measured value exceeds a preset standard value, then it is deemed that GIST is detected. A reagent, which contains an antibody specifically recognizing AZU1, is used for detecting GIST.

Description

Methods and reagents for detecting gastrointestinal stromal tumors

The present invention relates to a method for detecting a gastrointestinal stromal tumor (hereinafter referred to as "GIST") and a detection reagent for which azurosidin (hereinafter referred to as "AZU1") is to be measured.

GIST is a mesenchymal tumor that develops under the mucosa of the gastrointestinal tract such as the esophagus, stomach, small intestine, large intestine, and rectum.
As an attempt to diagnose GIST by tumor markers present in body fluid samples such as blood, mutations in the KIT gene and PDGFRA gene in blood circulating tumor DNA (ctDNA) (Non-Patent Document 1), blood circulating tumor cells (CTC) ), The expression of anoctamine 1 (ANO1) (Non-Patent Document 2) has been reported.
As a method for measuring a tumor marker in blood, it is possible to collect a test sample only by a blood test. Therefore, it is suitable for test sample collection and repeated measurement according to the course of treatment with the minimum invasion of blood collection only. However, the measurement of gene mutation in ctDNA cannot correspond to the existence of GIST cases without gene mutation, and a tumor marker with higher diagnostic performance is desired, and the measurement of ANO1 expression in CTC is from blood to peripheral blood monocytogenes. Since it has a complicated process of isolating nuclear cells, a simpler measurement method is desired.

By the way, AZU1 is one of the inactive serine proteases also known as heparin-binding protein (HBP) or 37 kDa cationic antibacterial protein (CAP37). Its function is to have a chemical migration effect on monocytes and an antibacterial effect on Gram-negative bacteria. That is, AZU1 is present in Azurophilic granules of neutrophils, and when released from neutrophils that have migrated to the infected site, it induces vascular leakage and edema formation, promotes inflammation, and contributes to biological defense (non-patent). Patent Documents 3 to 7).

Regarding the relationship between AZU1 and disease, a method for diagnosing renal cell carcinoma by isolating extracellular vesicles in body fluid and detecting AZU1 has been reported (Patent Document 1, Non-Patent Document 8).

However, to date, there has been no report on the dynamics of AZU1 in body fluids that can be collected less invasively than biopsy in GIST, and it was unclear whether AZU1 in body fluids could be applied to detect GIST.

International Publication No. 2018/079689 Pamphlet

An object of the present invention is to provide a method for detecting GIST easily and with high accuracy, and a reagent that can be used for the method.

As a result of diligent studies to solve the above problems, the present inventors have found that AZU1 in body fluid shows a significantly higher value in GIST patients than in healthy subjects, and a tumor for AZU1 to detect GIST. We have found that it can be a marker and completed the present invention.

That is, the present invention includes the following aspects.
[1] A method for detecting a gastrointestinal stromal tumor (GIST), which includes measuring the amount of azurosidin (AZU1) in a sample, and when the measured value exceeds a preset reference value, the GIST The method, if detected.
[2] The method according to [1], wherein the amount of AZU1 is measured using an antibody that specifically recognizes AZU1.
[3] In the sample, further detecting a second marker present on the same cell-secreted fine particle as the cell-secreted fine particle in which AZU1 is present, the second marker being any of those listed in Table 1. Or at least one of the methods according to [1] or [2].
[4] The method according to [3], wherein the second marker comprises at least one of CD81, CD63, CD9 and phosphatidylserine.
[5] The method according to [3] or [4], wherein the detection of the second marker is performed using an antibody or receptor that specifically recognizes the second marker.
[6] A reagent for use in the detection of GIST, which comprises an antibody that specifically recognizes AZU1.
[7] The reagent according to [6], further comprising an antibody or receptor that specifically recognizes any of the second markers listed in Table 1.
[8] The reagent according to [7], wherein the second marker comprises at least one of CD81, CD63, CD9 and phosphatidylserine.

INDUSTRIAL APPLICABILITY The present invention provides a method for detecting GIST easily and with high accuracy, and a reagent that can be used for the method.

In Reference Example 8, the figure which shows the screening result of the hybridoma cell culture supernatant by CELISA using the GPI anchor type AZU1 constitutive expression CHO-K1 cell. In Reference Example 9, the figure which shows the screening result of the hybridoma cell culture supernatant by ELISA using the secretory type AZU1. In Reference Example 13, the figure which shows the performance evaluation result of the anti-AZU1 monoclonal antibody as a solid phase antibody by ELISA (conditions 1 to 4) using the cell secretory fine particle. In Reference Example 13, it is a figure which shows the performance evaluation result of the anti-AZU1 monoclonal antibody as a biotin-labeled antibody by ELISA (condition 5-8) using the cell secretory fine particle. The figure which shows the box plot of the concentration of AZU1 contained in a serum sample in a healthy person group and a GIST patient group in Example 1. FIG. FIG. 2 is a diagram showing a box plot of ELISA absorbance using an anti-CD81 antibody as a solid phase antibody and an anti-AZU1 antibody as a biotin-labeled antibody in a healthy subject group and a GIST patient group in Example 2. FIG. 3 is a diagram showing a box plot of ELISA absorbance using an anti-CD9 antibody as a solid phase antibody and an anti-AZU1 antibody as a biotin-labeled antibody in a healthy subject group and a GIST patient group in Example 3. FIG. 6 is a diagram showing a box plot of ELISA absorbance using an anti-CD63 antibody as a solid phase antibody and an anti-AZU1 antibody as a biotin-labeled antibody in a healthy subject group and a GIST patient group in Example 4. FIG. 6 is a diagram showing a box plot of serum AZU1 concentration measured with an AZU1 measuring reagent in a healthy subject group and a GIST patient group in Example 6.

[1] Method for Detecting GIST of the Present Invention The first aspect of the present invention is a method for detecting GIST, which comprises measuring the amount of AZU1 in a sample. This is a method based on the characteristic presence of AZU1 in a biological sample such as GIST blood as compared with a healthy sample. Measurement of the amount of AZU1 in a sample is usually performed in vitro.
According to the method of the present invention, GIST can be detected easily and with high accuracy, as shown in Examples described later.

The method of the present invention includes up to the stage of detecting GIST, and does not include the final judgment act regarding the diagnosis of GIST. The doctor diagnoses GIST and formulates a treatment policy by referring to the detection results and the like by the method of the present invention.
Usually, the target (test animal) for detecting GIST is a human.

Examples of the sample to be measured in the present invention include blood, urine, saliva, tears, ascites, peritoneal lavage fluid, cerebrospinal fluid, and cell or tissue extract. Blood, urine, saliva and tears are preferred for ease of sample collection. Blood is more preferred given its versatility for other test items. Blood may be used as whole blood or separated into blood components such as serum, plasma, and blood cells, but serum or plasma is preferably used. The dilution ratio of the sample is not particularly limited, but for example, it may be appropriately selected in the range of undiluted to 100-fold dilution according to the type and state of the sample to be used.
The sample usually contains fine particles (cell-secreted fine particles) secreted from cells, which will be described later.

The disease targeted by the present invention is GIST.

AZU1 to be measured in the present invention is a peptide containing at least the sequence from isoleucine at residue 27 to proline at residue 248 of the amino acid sequence of human AZU1 protein disclosed in accession number P20160 of UniPlotKB. , Or a peptide containing an amino acid sequence having 80% or more identity with the above sequence. The identity is preferably 90% or more, more preferably 95% or more. In addition, this peptide may be a peptide consisting of amino acids in which one or several amino acids have been deleted, substituted, inserted or added in the sequence. The number is preferably 2 to 20, more preferably 2 to 10, and even more preferably 2 to 5. In addition, other peptide fragments may be provided on both sides of the sequence.

The AZU1 measured in the present invention may be measured as a soluble protein in body fluid, may be measured on fine particles secreted from cells, or both may be measured. May be good. When measuring AZU1 existing on the fine particles, the one coexisting with the second marker on the fine particles may be measured.
Examples of fine particles secreted by cells include exosomes. Exosomes are membrane vesicles composed of lipid bilayers, usually having a diameter of 50-200 nm. Exosomes are known to contain a large amount of membrane proteins such as tetraspanins and integrins, proteins related to polytope formation, and heat shock proteins. Further, it is known that the lipid bilayer membrane constituting the exosome has phosphatidylserine on the membrane surface. Table 1 shows typical molecules that are abundant in exosomes.

The second marker in the present invention is not particularly limited as long as it is a molecule existing on the cell-secreted fine particles, but preferably refers to at least one included in the group consisting of the protein and phosphatidylserine shown in Table 1 described above. More preferably, it contains at least one of CD81, CD63, CD9 and phosphatidylserine. The protein shown in Table 1 also includes a peptide containing an amino acid sequence having high homology (80% or more, preferably 90% or more, more preferably 95% or more). The second marker is preferably one that is present on the same cell-secreted microparticle as the cell-secreted microparticle in which AZU1 is present.
Since the second marker may be more than one type, the word "second" may be understood to mean "other of AZU1".

In the detection method of the present invention, the method of measuring the amount of AZU1 and the method of measuring (detecting) at least one of the second markers coexisting on the cell secretory fine particles and AZU1 do not interfere with the measurement of the amount of AZU1. There are no particular restrictions as long as it is limited. For example, an immunoassay method using an antibody that specifically recognizes AZU1 and a method using a mass spectrometry method can be mentioned.
Specific examples of the immunoassay method using an antibody that specifically recognizes AZU1 include the following.

[A] A competitive method utilizing an antibody that specifically recognizes AZU1 and a labeled AZU1 that the labeled AZU1 and AZU1 contained in a sample competitively bind to the antibody.
[B] A method using surface plasmon resonance in which a sample is brought into contact with a chip on which an antibody that specifically recognizes AZU1 is immobilized, and a signal depending on the binding between the antibody and AZU1 is detected.
[C] A fluorescent polarization immunoassay method using a fluorescently labeled antibody that specifically recognizes AZU1 and increasing the degree of fluorescence polarization by binding of the antibody to AZU1.

[D] A sandwich method in which two antibodies (one of which is a labeled antibody) that specifically recognizes AZU1 is used to form a three-way complex of the two antibodies and AZU1. At this time, the two antibodies are preferably two types of antibodies having different epitopes.
[E] As a pretreatment, a method of concentrating AZU1 in a sample with an antibody that specifically recognizes AZU1 and then detecting a conjugate of AZU1 and the antibody with a mass spectrometer.
[F] Obtained when a fluorescently labeled antibody that specifically recognizes AZU1 is used, the measurement target is aligned in a flow path after the antibody is bound to AZU1, and individual particles are irradiated with excitation light. A flow cytometry method that counts the complex in which the antibody and the measurement target are bound based on scattered light and fluorescence.

Of these, the methods [d] and [e] are simple and highly versatile, but the method [d] is more preferable in processing a large number of samples because the techniques related to reagents and devices are sufficiently established.

The antibody that specifically recognizes AZU1 is not particularly limited, but by immunizing an animal using the AZU1 protein itself, an oligopeptide consisting of a partial region of AZU1, a polynucleotide encoding the full length or partial region of AZU1, or the like as an immunogen. Obtainable.

If the AZU1 protein itself or an oligopeptide consisting of a partial region of AZU1 is used as an immunogen, its structure may change in the process of preparing the protein or the oligopeptide. Therefore, the obtained antibody may not have high specificity or binding force to the desired antigen, and as a result, AZU1 contained in the sample may not be accurately quantified. On the other hand, when an expression vector containing a polynucleotide encoding the full length or partial region of AZU1 is used as an immunogen, AZU1 is expressed in the body of an immunized animal without undergoing structural change, and therefore, it is expressed against a desired antigen. It is preferable because an antibody having high specificity and binding force (that is, high affinity) can be obtained.

The animal used for immunity is not particularly limited as long as it has an antibody-producing ability, and may be a mammal normally used for immunity such as a mouse, a rat, or a rabbit, or a bird such as a chicken.

The antibody that specifically recognizes AZU1 may be a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferable.

The establishment of hybridoma cells that produce an antibody that specifically recognizes AZU1 may be appropriately selected from the methods for which technology has been established. As an example, selection of hybridoma cells in which B cells are collected from an animal immunized by the method described above, the B cells and myeloma cells are fused electrically or in the presence of polyethylene glycol, and a desired antibody is produced by a HAT medium. By performing the above and cloning the selected hybridoma cells by the limiting dilution method, hybridoma cells that produce a monoclonal antibody that specifically recognizes AZU1 can be established.

The antibody that specifically recognizes AZU1 used in the present invention may be selected based on the affinity for glycosylphosphatidylinositol (GPI) anchor type AZU1 derived from the host expression system.

The host is not particularly limited, and may be appropriately selected from microbial cells such as Escherichia coli and yeast, insect cells, and animal cells that are usually used by those skilled in the art for protein expression, but disulfide bonds or sugar chains are added. It is preferable to use a mammalian cell as a host, which can express a protein having a structure similar to that of the natural AZU1 by post-translational modification such as. Examples of mammalian cells include conventionally used human fetal kidney-derived 293T cell lines, monkey kidney-derived COS-7 cell lines, Chinese hamster ovary-derived CHO-K1 cell lines, and cancer cells isolated from humans. Can be mentioned.

Purification of the antibody used in the GIST detection method of the present invention may be carried out by appropriately selecting from the methods for which the technique has been established. As an example, after culturing the antibody-producing hybridoma cells established by the method described above, the culture supernatant is collected, and if necessary, the antibody is concentrated by ammonium sulfate precipitation, followed by ion exchange chromatography and hydrophobic interaction chromatography. Alternatively, the antibody can be purified by affinity chromatography using a carrier on which protein A, protein G, protein L or the like is immobilized.

In the present invention, when the amount of AZU1 present on the same cell-secreted fine particle is measured and the detection of the second marker is performed together, the second marker is specifically recognized in addition to the antibody that recognizes AZU1. It is preferable to further use an antibody or a receptor (hereinafter, also referred to as “antibody or the like”).

In the present invention, the second marker preferably contains at least one of CD81, CD63, CD9 and phosphatidylserine, and the antibody or receptor that specifically recognizes them includes an anti-CD81 antibody, an anti-CD63 antibody, and the like. It is preferably an anti-CD9 antibody or a phosphatidylserine receptor.

The anti-CD81 antibody, anti-CD63 antibody and anti-CD9 antibody used in the present invention can be obtained by the same method as the above-mentioned antibody that recognizes AZU1.

The phosphatidylserine receptor used in the present invention is not particularly limited, and examples thereof include Annexin V, MFG-E8, Tim1, Tim3, and Tim4, and Tim4 having high specificity and binding force to phosphatidylserine (International Publication No. 1). 2016/088689) is preferable. Tim4 may have at least an amino acid sequence of a binding domain (IgV domain) for phosphatidylserine, and includes, for example, the Tim4 protein itself, a peptide consisting of a partial region containing the IgV domain of Tim4, and the IgV domain of Tim4. A fusion protein in which another peptide fragment is bound to a partial region can be used.

The labeled antibody or the like used when performing the binding quantification method by the above-mentioned sandwich method is an enzyme such as peroxidase or alkaline phosphatase, a fluorescent substance, a chemical luminescent substance, a substance that can be detected by a detection device such as a radioisotope or functional fine particles, and the like. Alternatively, it may be labeled with a substance or the like that has a specific binding partner such as avidin to biotin, and the labeling may also be performed by using a method in which the technique is well established.

In the method of the present invention, a method of detecting and quantitatively measuring AZU1 by using a mass spectrometry method will be specifically described below.

The measurement can be performed by liquid chromatography-mass spectrometry (LC / MS), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS), or the like.

In the detection method of the present invention, it is preferable to determine that GIST has been detected when the amount of AZU1 obtained by measurement exceeds the reference value (cutoff value) calculated from the control.
The amount of AZU1 used for the determination may be either a measured value or a converted concentration value. The converted concentration value refers to a value converted from a measured value based on a calibration curve prepared using AZU1 as a standard sample. The concentration of the standard sample may be determined as a value converted from the measured value based on the calibration curve of the standard peptide using mass spectrometry.
The cutoff value can be appropriately set to a measured value showing optimum sensitivity and specificity by measuring a sample collected from a healthy person and a GIST patient, respectively, and using a receiver operating characteristic (ROC) curve.

The method of detecting GIST of the present invention can be applied to the method of treating GIST. That is, according to the present invention, it is a method for treating GIST in a patient.
A method is provided that includes (i) identifying a patient as having a measured value of AZU1 amount exceeding a preset reference value, and (ii) treating the identified patient.
In the identification of the step (i), the amount of AZU1 may be measured by using an antibody that specifically recognizes AZU1 or by using a mass spectrometry method.
Examples of the treatment in the step (ii) include surgical excision, drug therapy, radiation therapy, and the like, but are not particularly limited.

Another aspect of the first aspect of the invention is the use of AZU1 in the manufacture of reagents for detecting GIST.
Another aspect of the invention is the use of AZU1 in the detection of GIST.
Another aspect of the invention is the AZU1 used for the detection of GIST.

[2] Reagent for Detecting GIST of the Present Invention The second aspect of the present invention is a reagent for detecting GIST, which comprises an antibody that specifically recognizes AZU1.

The reagent of the present invention further preferably further contains an antibody or receptor that specifically recognizes the second marker shown in Table 1. The antibody or receptor (antibody or the like) that specifically recognizes the second marker is not particularly limited, but is, for example, an antibody or receptor that specifically recognizes at least one of CD81, CD63, CD9 and phosphatidylserine. Is preferable, and it is more preferable that it is either an anti-CD81 antibody, an anti-CD63 antibody, an anti-CD9 antibody or a phosphatidylserine receptor. The phosphatidylserine receptor is not particularly limited, and examples thereof include Annexin V, MFG-E8, Tim1, Tim3, and Tim4, and Tim4 having high specificity and binding force to phosphatidylserine is preferable. Tim4 may have at least an amino acid sequence of a binding domain (IgV domain) for phosphatidylserine, and includes, for example, the Tim4 protein itself, a peptide consisting of a partial region containing the IgV domain of Tim4, and the IgV domain of Tim4. A fusion protein in which another peptide fragment is bound to a partial region can be used.

The reagent and the measuring method of the present invention will be specifically described below with respect to the three methods of the sandwich method described above. However, the reagent and the measuring method of the present invention are not limited to these three methods.

[Method 1] Two-step sandwich method The reagents used in this method include two types of antibodies and the like (hereinafter referred to as "antibodies and the like 1" and "antibodies and the like 2"). It is preferable that the antibody or the like 1 and the antibody or the like 2 have different binding sites for the substance to be measured. Examples of the combination of the antibody or the like 1 and the antibody or the like 2 include the following three types [a] to [c].
[A] Antibodies and the like 1: Antibodies that specifically recognize AZU1, antibodies and the like 2: Antibodies that specifically recognize AZU1 and are the same as or different from antibody 1 and the like [b] Antibodies and the like 1: AZU1 Antibodies, antibodies, etc. that specifically recognize 2: Antibodies or receptors that specifically recognize the second marker [c] Antibodies, etc. 1: Antibodies or receptors that specifically recognize the second marker, antibodies, etc. 2 : An antibody that specifically recognizes AZU1

The reagent of this method can be prepared by the methods shown in the following [1] to [3].
[1] First, antibody or the like 1 is bound to a B / F separable carrier such as an immunoplate or magnetic particles. The bonding method may be a physical bond using a hydrophobic bond, or a chemical bond using a linker reagent capable of cross-linking between two substances.
[2] After binding the antibody or the like 1 to the carrier, the surface of the carrier is blocked with bovine serum albumin, skim milk, a commercially available blocking agent for immunoassay, or the like to prepare a primary reagent in order to avoid non-specific binding.
[3] Label the other antibody or the like 2 and prepare a solution containing the obtained labeled antibody or the like as a secondary reagent. The substance labeled with 2 such as antibody includes an enzyme such as peroxidase or alkaline phosphatase, a fluorescent substance, a chemical luminescent substance, a substance that can be detected by a detection device such as radioisotope or functional fine particles, or a specific binding such as avidin to biotin. A substance or the like in which a partner exists is preferable. Further, as the solution of the secondary reagent, a buffer solution capable of performing an antigen-antibody reaction satisfactorily, for example, a phosphate buffer solution, a Tris-HCl buffer solution, or the like is preferable.
The reagent of this method produced in this manner may be freeze-dried if necessary.

Next, in order to detect and measure AZU1 using the reagent prepared by this method, the methods shown in [4] to [6] below may be used.
[4] The primary reagent prepared in [2] described above and the sample are brought into contact with each other for a certain period of time at a certain temperature. The reaction conditions may be a temperature range of 4 ° C. to 40 ° C. for 5 minutes to 180 minutes.
[5] The unreacted substance is removed by B / F separation, and then the secondary reagent prepared in [3] is brought into contact with the secondary reagent for a certain period of time at a certain temperature to form a sandwich complex. The reaction conditions may be a temperature range of 4 ° C. to 40 ° C. for 5 minutes to 180 minutes.
[6] Unreacted substances are removed by B / F separation, labeled substances such as labeled antibodies are quantified, and the AZU1 concentration in the sample is quantified by a calibration curve prepared using a known concentration of AZU1 as a standard.

[Method 2] 1-step sandwich method The reagent used in this method contains an antibody or the like 1 and an antibody or the like 2 in the same manner as in the above-mentioned method 1.

In order to prepare a reagent of this method, an antibody or the like 1 is bound to a carrier by the method shown in [1] to [2] of Method 1 to perform a blocking treatment, and the antibody or the like is immobilized on the carrier. , A buffer solution containing labeled antibody or the like 2 may be further added.
The reagent of this method produced in this manner may be freeze-dried if necessary.

Next, in order to detect and measure AZU1 using the reagent prepared by this method, the methods shown in the following [7] to [8] may be used.
[7] The reagent prepared by the above method and the sample are brought into contact with each other for a certain period of time at a certain temperature to form a sandwich complex. The reaction conditions may be a temperature range of 4 ° C. to 40 ° C. for 5 minutes to 180 minutes.
[8] Unreacted substances are removed by B / F separation, labeled substances such as labeled antibodies are quantified, and the AZU1 concentration in the sample is quantified by a calibration curve prepared using a known concentration of AZU1 as a standard.

[Method 3] Homogeneous sandwich method The reagent used in this method contains an antibody or the like 1 and an antibody or the like 2 in the same manner as in the above-mentioned methods 1 and 2, and further contains a labeling substance coated with streptavidin and excited by excitation light. include. As the streptavidin-coated labeling substance, for example, AlphaScreen streptavidin donor beads (manufactured by PerkinElmer) can be preferably used.

The reagent of this method can be prepared by the methods shown in the following [9] to [10].
[9] First, antibody or the like 1 is labeled with biotin. The biotin labeling may be carried out by a conventionally known method, and examples thereof include a method using a Biotin labeling Kit-NH2 labeling kit (manufactured by Dojin Chemical Research Institute).
[10] The other antibody or the like 2 is labeled with a substance that emits a signal by singlet oxygen. This singlet oxygen is generated when the streptavidin-coated labeling substance is excited. The signal is preferably a fluorescent signal. As the signal generating substance, for example, AlphaLISA acceptor beads (manufactured by PerkinElmer) can be preferably used. The method of binding the antibody or the like 2 to the signal generating substance is not particularly limited, and examples thereof include reductive amination cross-linking to the aldehyde group on the surface of the signal generating substance using sodium cyanoborohydride.

Next, in order to detect and measure AZU1 using the reagent prepared by this method, the methods shown in the following [11] to [13] may be used.
[11] The biotin-labeled antibody or the like prepared in [9] described above, the signal-generating substance-labeled antibody or the like 2 prepared in [10], and the sample are brought into contact with each other for a certain period of time under constant temperature and light-shielding conditions. Form a sandwich complex. The reaction conditions may be a temperature range of 4 ° C. to 40 ° C. for 5 minutes to 180 minutes.
[12] Subsequently, a streptavidin-coated labeling substance is added and contacted for a certain period of time under constant temperature and light-shielding conditions to bind the biotin-labeled antibody and the streptavidin-coated labeling substance. The reaction conditions may be a temperature range of 4 ° C. to 40 ° C. for 5 minutes to 180 minutes.
[13] Using an analyzer, the signal emitted from the signal-generating substance-labeled antibody or the like 21 when irradiated with excitation light is quantified, and the AZU1 concentration in the sample is determined by a calibration curve prepared using a known concentration of AZU1 as a standard. Quantify. As the analyzer, for example, EnSpire (manufactured by PerkinElmer) can be preferably used.

The amount of the reagent component contained in the reagent of the present invention may be appropriately set according to various conditions such as the amount of the sample, the type of the sample, the type of the reagent, and the measurement method. For example, when 20 μL of serum or plasma is used as a sample and the amount of AZU1 is measured by the sandwich method, the amount of antibody or the like 1 to be bound to the carrier is 100 ng to 1000 μg per reaction system in which 20 μL of the sample is reacted with an antibody or the like. The amount of the labeled antibody or the like 2 may be 2 ng to 20 μg.

The reagent of the present invention can also be used for measurement by a conventional method, and can also be used for measurement using an automatic immunodiagnosis device. In particular, measurement using an automatic immunodiagnosis device can be performed without being affected by intrinsic measurement obstructing factors and competing enzymes contained in the sample, and AZU1 in the sample can be quantified in a short time. ,preferable.

Another aspect of the second aspect of the invention is the use of an antibody that specifically recognizes AZU1 in the manufacture of reagents for detecting GIST. It is also the use of an antibody that specifically recognizes AZU1 and an antibody or receptor that specifically recognizes any of the second markers listed in Table 1 in the manufacture of reagents for detecting GIST. ..
Another aspect of the invention is the use of antibodies that specifically recognize AZU1 in the detection of GIST. Also, an antibody that specifically recognizes AZU1 and an antibody or receptor that specifically recognizes any of the second markers listed in Table 1 is used in the detection of GIST.
Another aspect of the invention is an antibody that specifically recognizes AZU1 used for the detection of GIST. It is also an antibody that specifically recognizes AZU1 and is used in combination with an antibody or receptor that specifically recognizes any of the second markers listed in Table 1 for the detection of GIST.

Reference examples and examples are shown below to specifically explain the present invention, but these reference examples and examples show an example of the present invention, and the present invention is limited to the reference examples and the examples. It's not a thing.
Antibodies that specifically recognize AZU1 were obtained according to Reference Examples 1 to 13 described below.

[Reference Example 1] Preparation of GPI-anchored AZU1 expression plasmid The region encoding the sequence from isoleucine at residue 27 to proline at residue 248 of the amino acid sequence of human AZU1 protein was amplified by the PCR method. The above-mentioned PCR amplification product was added to a plasmid pFLAG1 (manufactured by Sigma-Aldrich) containing a coding region of a FLAG tag peptide consisting of the amino acid sequence DYKDDDDK (SEQ ID NO: 1) and a coding region of a signal peptide of a GPI anchor derived from a placenta-type alkaline phosphatase. Can express GPI-anchored AZU1 to which FLAG tag peptide is added to the N-terminal side and GPI-anchored signal peptide is added to the C-terminal side. Peptide was prepared.

[Reference Example 2] Preparation of secretory AZU1 expression plasmid A BNC peptide (Japanese Patent Laid-Open No. 2009-240300) consisting of a coding region of a FLAG tag peptide and a C-terminal 7 amino acid sequence CKVLRRH (SEQ ID NO: 2) of BNP (cerebral sodium diuretic peptide). The PCR amplification product of the AZU1 coding region prepared in Reference Example 1 was inserted into the plasmid pFLAG1 (manufactured by Sigma-Aldrich) containing the coding region of No. 1) using an In-fusion HD cloning kit (manufactured by Takara Bio), and the N-terminal was inserted. A plasmid capable of expressing secretory AZU1 to which a FLAG tag peptide was added to the side and a BNC peptide was added to the C-terminal side was prepared.

[Reference Example 3] Preparation of GPI-anchored AZU1 constitutive expression CHO-K1 cells 10% of the GPI-anchored AZU1 expression plasmid prepared in Reference Example 1 was introduced into a Chinese hamster ovary-derived CHO-K1 cell line according to a conventional method. _{The cells were cultured in a 5% CO 2} incubator using FBS-added Ham's F12 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at 37 ° C. for 24 hours. After culturing, an antibiotic G418 solution (manufactured by Thermo Fisher Scientific) was added to 250 μg / mL, and the cells were further cultured for 3 weeks. CHO-K1 cells constitutively expressing GPI-anchored AZU1 were acquired by a cell sorter using an anti-FLAG antibody.

[Reference Example 4] Preparation of secretory AZU1 transiently expressed 293T cells After gene transfer of the secretory AZU1 expression plasmid prepared in Reference Example 2 into a human fetal kidney-derived 293T cell line according to a conventional method, 10% FBS-added D- AZU1 was transiently expressed by culturing at 37 ° C. in _{a 5% CO 2} incubator using MEM medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[Reference Example 5] Recovery of secretory AZU1 solution After culturing the secretory AZU1 transiently expressed 293T cells prepared in Reference Example 4 for 72 hours, the culture solution was centrifuged, and the supernatant was recovered as a secretory AZU1 solution. ..

[Reference Example 6] Recovery of cell-secreted fine particles by ultracentrifugal method 293T cell line, secretory AZU1 transiently expressed 293T cell prepared in Reference Example 4, human renal cancer-derived ACHN cell line, and AZU1-FLAG-expressing ACHN cell ( International Publication No. 2018/079689 pamphlet and Int J Cancer. 2018 Feb 1; 142 (3): 607-617) were ultra-filtered with AMICON ULTRA-15, 100 KDa cutoff (manufactured by Merck Millipore), and 10% of FBS was obtained. _{After culturing in a 5% CO 2} incubator at 37 ° C. for 48 hours using the added D-MEM medium, cell-secreted fine particles were recovered by the following method.

[1] 60 mL of the culture solution was centrifuged at 2000 × g at 4 ° C. for 30 minutes, and the supernatant was collected.
[2] The above centrifugal supernatant was centrifuged at 16000 × g at 4 ° C. for 30 minutes, and the supernatant was collected.
[3] The above-mentioned centrifugal supernatant was centrifuged at 4 ° C. and 100,000 × g for 16 hours to remove the supernatant, and the precipitate was collected.
[4] PBS was added to the above ultracentrifugal precipitate, and the mixture was ultracentrifuged at 4 ° C. and 100,000 × g for 16 hours to remove the supernatant and recover the precipitate.
[5] 200 μL of PBS was added to the above ultracentrifugal precipitate, suspended by pipetting, and cell-secreted fine particles were collected.

[Reference Example 7] Immunity and Cell Fusion To 4 Balb / c mice, 40 μg / animal of the GPI-anchored AZU1 expression plasmid constructed in Reference Example 1 was administered every 7 days for a total of 6 times, and then the spleen. Cells were harvested. According to a conventional method, the collected spleen cells and mouse myeloma Sp2 / 0 cell line are fused in the presence of polyethylene glycol, and cultured in GIT medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with HAT (manufactured by Sigma-Aldrich) for about 10 days. Hybridoma cells were selected by the above.

[Reference Example 8] Screening of anti-AZU1 antibody-producing cells (1)
Hybridoma cells producing anti-AZU1 antibody were screened by the cell enzyme immunoassay (CELISA) shown below using the GPI-anchored AZU1 constitutive expression CHO-K1 cells prepared in Reference Example 3.

^{[1] 5 × 10 4} cells / well of GPI-anchored AZU1 constitutive expression CHO-K1 cells were added to a 96-well microplate (manufactured by Falcon), and 10% FBS-added Ham's F12 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). ) Was cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours.
[2] Mouse IgG (negative control, NC) diluted 3 times with PBS and diluted to 2 μg / mL with PBS containing 1% BSA and commercially available mouse anti-AZU1 antibody (manufactured by R & D Systems) (positive control, PC). In addition, 50 μL / well of undiluted hybridoma cell culture supernatant was added, and the mixture was left at room temperature for 1 hour.
[3] Washed three times with PBS, and added 50 μL / well of horseradish peroxidase (HRP) -labeled anti-mouse immunoglobulin G-Fc antibody (manufactured by Sigma-Aldrich) diluted 20000 times with PBS containing 1% BSA. It was left at room temperature for 1 hour.

[4] Washing was performed 3 times with PBS, 50 μL / well of TMB Microwellell Peroxidase Substrate (manufactured by SeraCare Life Sciences) was added, and the mixture was left at room temperature for 10 minutes.
[5] 50 μL / well of a 1 mol / L phosphoric acid aqueous solution was added to terminate the reaction.
[6] The absorbance at 450 nm was measured with an absorption plate reader.
The measurement results are shown in FIG. High signals were detected in 6 hybridoma cell culture supernatants (1-2, 1-7, 1-8, 1-13, 1-14 and 1-15).

[Reference Example 9] Screening of anti-AZU1 antibody-producing cells (2)
Hybridomas producing anti-AZU1 antibody were screened by the sandwich ELISA shown below using the secretory AZU1 solution prepared in Reference Example 5.

[1] To a Maxisorp 96-well microplate (manufactured by Thermo Fisher Scientific), 50 μL / well of anti-FLAG antibody diluted to 2 μg / mL with a carbonate buffer (pH 9.8) was added, and the mixture was allowed to stand overnight at 4 ° C. to solidify. It became compatible.
[2] Washing was performed 3 times with TBS (TBST) containing 0.05% Tween 20, 200 μL / well of SuperBlock (PBS) (manufactured by Thermo Fisher Scientific) was added, and the mixture was left at room temperature for 1 hour.
[3] Washing was performed 3 times with TBST, 50 μL / well of the secretory AZU1 prepared in Reference Example 5 diluted 5-fold with PBS containing 1% BSA was added, and the mixture was left at room temperature for 2 hours.

[4] Mouse IgG (negative control, NC) diluted 3 times with TBST and diluted with PBS containing 1% BSA to 2 μg / mL and a commercially available mouse anti-AZU1 antibody (manufactured by R & D Systems) (positive control, PC). In addition, 50 μL / well of undiluted hybridoma cell culture supernatant was added, and the mixture was left at room temperature for 1 hour.
[5] Washed three times with TBST, and added 50 μL / well of horseradish peroxidase (HRP) -labeled anti-mouse immunoglobulin G-Fc antibody (manufactured by Sigma-Aldrich) diluted 20000 times with PBS containing 1% BSA. It was left at room temperature for 1 hour.
[6] Washing was performed 3 times by TBST, 50 μL / well of SureBlue Reserve TMB (manufactured by SeraCare Life Sciences) was added, and the mixture was left at room temperature for 10 minutes.

[7] 50 μL / well of a 1 mol / L phosphoric acid aqueous solution was added to terminate the reaction.
[8] The absorbance at 450 nm was measured with an absorption plate reader.
The measurement results are shown in FIG. High signals were detected in 7 hybridoma cell culture supernatants (1-2, 1-5, 1-7, 1-8, 1-13, 1-14 and 1-15).

[Reference Example 10] Cloning of anti-AZU1 antibody-producing cells Three hybridoma cells (1-2, 1-8 and 1-14) selected based on the results of Reference Example 8 and Reference Example 9 were subjected to a limiting dilution method. Cloning was performed, HT (Sigma-Aldrich) -added GIT medium was acclimated to HT-free GIT medium, and finally three anti-AZU1 antibody-producing cell lines (clones 1-2, 1-8 and 1-14) were cultured. ) Was established.

[Reference Example 11] Preparation of anti-AZU1 monoclonal antibody From the culture supernatants of the three anti-AZU1 antibody-producing cells (clones 1-2, 1-8 and 1-14) established in Reference Example 10, using a Protein G column. , The monoclonal antibody was purified according to a conventional method. After dialysis of each purified antibody with PBS, the absorbance at 280 nm was measured to quantify the protein concentration.

[Reference Example 12] Preparation of biotin-labeled antibody A commercially available anti-CD63 antibody (manufactured by Frontier Laboratory), a commercially available anti-AZU1 antibody (manufactured by R & D Systems), and three anti-AZU1 antibodies prepared in Reference Example 11 (clone 1-2, 1-8 and 1-14) were biotin-labeled using the Biotin labeling Kit-NH ₂ labeling kit (manufactured by Dojin Chemical Laboratory), respectively, according to the protocol described in the product instruction manual.

[Reference Example 13] Performance evaluation of anti-AZU1 monoclonal antibody (1)
Secretory microparticles derived from 293T, 293T-AZU1, ACHN, and ACHN-AZU1 cells prepared in Reference Example 6 were measured by eight sandwich ELISAs shown in Table 2.

The specific method is shown below.
[1] To a Maxisorp 96-well microplate (manufactured by Thermo Fisher Scientific), 50 μL / well of the solid phase antibody shown in Table 2 diluted with a carbonate buffer (pH 9.8) to 4 μg / mL was added, and the temperature was 1 at 4 ° C. It was left to solidify overnight.
[2] Washing was performed 3 times with PBS, 300 μL / well of SuperBlock (PBS) (manufactured by Thermo Fisher Scientific) was added, and the mixture was left at room temperature for 1 hour.
[3] Washing was performed 3 times with PBS, 50 μL / well of ultracentrifugal precipitate diluted 10-fold with PBS containing 1% BSA was added, and the mixture was left at room temperature for 2 hours.

[4] The biotin-labeled antibody shown in Table 2 was washed 3 times with PBS, diluted with PBS containing 1% BSA to 2 μg / mL, and left at room temperature for 1 hour.
[5] Washing was performed 3 times with PBS, 50 μL / well of Streptavidin-PolyHRP40 (manufactured by Stereospecific Detection technologies) diluted 50,000 times with PBS containing 1% BSA was added, and the mixture was left at room temperature for 30 minutes.
[6] Washing was performed 3 times with PBS, 50 μL / well of SureBlue Reserve TMB (manufactured by SeraCare Life Sciences) was added, and the mixture was left at room temperature for 10 minutes.

[7] 50 μL / well of a 1 mol / L phosphoric acid aqueous solution was added to terminate the reaction.
[8] The absorbance at 450 nm was measured with an absorption plate reader.

The measurement results are shown in FIGS. 3 and 4 (NC is a negative control). It was shown that by using the anti-AZU1 antibody as either a solid phase antibody or a biotin-labeled antibody, cell-secreted fine particles containing AZU1 can be detected. In particular, clone 1-14 was shown to have high detection sensitivity.

All of the sample collections and measurements in Examples 1 to 4 described below were informed by the sample provider with the approval of the Institutional Review Board of Chiba University Graduate School of Medicine and The Cancer Institute Ariake Hospital.・ It was done with the consent of the informed consent.
[Example 1] AZU1 measurement of serum samples of healthy subjects and GIST patients (1)
Table 3 shows the serum samples of healthy subjects and GIST patients used in this example.

The concentration of AZU1 contained in the above serum sample was measured using a commercially available AZU1 ELISA kit (manufactured by Sino Biological, product number SEK10660). The AZU1 measured by this example is both AZU1 existing as a soluble protein and AZU1 existing on cell-secreted fine particles.
The specific method is shown below.
[1] To a Maxisorp 96-well microplate (manufactured by Thermo Fisher Scientific), 100 μL / well of a rabbit anti-human AZU1 monoclonal antibody diluted to 2 μg / mL with PBS was added, and the mixture was allowed to solidify at 4 ° C. overnight.
[2] Washing was performed 3 times with TBS containing 0.05% Tween 20, 300 μL / well of TBS containing 2% BSA and 0.05% Tween 20 was added, and the mixture was left at room temperature for 1 hour.
[3] Wash three times with TBS containing 0.05% Tween 20 and with TBS containing 0.1% BSA, 0.05% Tween 20 and 0.05 mg / mL atrophic inhibitor HBR1 (manufactured by Scantibodies Laboratory). A serum sample diluted 10-fold or a standard sample having a known concentration prepared by adding recombinant human AZU1 to the above-mentioned diluted solution was added at 100 μL / well and left at room temperature for 2 hours.
[4] HRP-labeled rabbit anti-human AZU1 washed 3 times with TBS containing 0.05% Tween 20 and diluted to 0.5 μg / mL with TBS containing 0.5% BSA and 0.05% Tween 20. 100 μL / well of the polyclonal antibody was added, and the mixture was left at room temperature for 1 hour.
[5] Washing was performed three times with TBS containing 0.05% Tween 20, 200 μL / well of SureBlue Reserve TMB (manufactured by SeraCare Life Sciences) was added, and the mixture was left at room temperature for 10 minutes.
[6] 50 μL / well of a 1 mol / L sulfuric acid aqueous solution was added to terminate the reaction.
[7] The absorbance at 450 nm was measured with an absorption plate reader.
[8] A calibration curve was prepared based on the standard sample, and the AZU1 concentration in the sample was calculated.

A box plot of the AZU1 concentration contained in the serum sample is shown in FIG. Table 4 shows the minimum, 25th percentile, median, 75th percentile, maximum value, AZU1 concentration range in the 95% confidence interval, and the p value of the Mann-Whitney U test in the healthy and GIST patient groups. .. The GIST patient group tended to have a higher value than the healthy subject group. The p value was less than 0.001 which was smaller than the significance level (0.05), and a statistically significant difference was observed.

[Example 2] AZU1 measurement of serum samples of healthy subjects and GIST patients (2)
Table 5 shows the serum samples of healthy subjects and GIST patients used in this example.

The amount of AZU1 present on the cell-secreted fine particles expressing the CD81 molecule contained in the above serum sample was measured by a commercially available anti-CD81 antibody (manufactured by Frontier Laboratory) and an anti-AZU1 antibody (clone) obtained by Reference Examples 1 to 13. It was measured by sandwich ELISA using 1-14).
The specific method is shown below.
[1] 50 μL / well of solid phase antibody (anti-CD81 antibody) diluted to 4 μg / mL with carbonate buffer (pH 9.8) was added to a Maxisorp 96-well microplate (manufactured by Thermo Fisher Scientific) at 4 ° C. It was left to solidify overnight.
[2] Washing was performed 3 times with PBS, 300 μL / well of SuperBlock (PBS) (manufactured by Thermo Fisher Scientific) was added, and the mixture was left at room temperature for 1 hour.
[3] Wash with PBS three times, add 50 μL / well of serum sample diluted 5-fold with PBS containing 1% BSA and 0.05 mg / mL heterophilic inhibitor HBR1 (manufactured by Scantibodies Laboratory), and add at room temperature. It was left for 2 hours.

[4] Washing was performed 3 times with PBS, 50 μL / well of a biotin-labeled detection antibody (anti-AZU1 antibody) diluted with PBS containing 1% BSA to 2 μg / mL was added, and the mixture was left at room temperature for 1 hour.
[5] Washing was performed 3 times with PBS, 50 μL / well of Streptavidin-PolyHRP40 (manufactured by Stereospecific Detection technologies) diluted 50,000 times with PBS containing 1% BSA was added, and the mixture was left at room temperature for 1 hour.
[6] Washing was performed 3 times with PBS, 50 μL / well of SureBlue Reserve TMB (manufactured by SeraCare Life Sciences) was added, and the mixture was left at room temperature for 10 minutes.

[7] 50 μL / well of a 1 mol / L phosphoric acid aqueous solution was added to terminate the reaction.
[8] The absorbance at 450 nm was measured with an absorption plate reader.

FIG. 6 shows a box plot of the amount of AZU1 present on the cell-secreted fine particles expressing the CD81 molecule contained in the serum sample. Table 6 shows the minimum, 25th, median, 75th percentile, maximum absorbance, absorbance range in the 95% confidence interval, and the Mann-Whitney U-test p-value for the healthy and GIST patient groups. The GIST patient group tended to have a higher value than the healthy subject group. The p value was less than 0.001 which was smaller than the significance level (0.05), and a statistically significant difference was observed.

[Example 3] AZU1 measurement of serum samples of healthy subjects and GIST patients (3)
Table 7 shows the healthy person serum sample and the GIST patient serum sample used in this example.

The amount of AZU1 present on the cell-secreted fine particles expressing the CD9 molecule contained in the above serum sample was measured by a commercially available anti-CD9 antibody (manufactured by Frontier Laboratory) and an anti-AZU1 antibody (clone) obtained by Reference Examples 1 to 13. It was measured by sandwich ELISA using 1-14). The specific method is the same as in Example 2 except that an anti-CD9 antibody is used as the solid phase antibody.
A box plot of the amount of AZU1 present on the cell secretory microparticles expressing the CD9 molecule contained in the serum sample is shown in FIG. Table 8 shows the minimum, 25th, median, 75th percentile, maximum absorbance, absorbance range in the 95% confidence interval, and the Mann-Whitney U-test p-value for the healthy and GIST patient groups. The GIST patient group tended to have a higher value than the healthy subject group. The p value was less than 0.001 which was smaller than the significance level (0.05), and a statistically significant difference was observed.

[Example 4] AZU1 measurement of serum samples of healthy subjects and GIST patients (4)
The healthy person serum sample and the GIST patient serum sample used in this example are the same as in Example 3.
The amount of AZU1 present on the cell-secreted fine particles expressing the CD63 molecule contained in the above serum sample was measured by a commercially available anti-CD63 antibody (manufactured by Frontier Laboratory) and an anti-AZU1 antibody (clone) obtained by Reference Examples 1 to 13. It was measured by sandwich ELISA using 1-14). The specific method is the same as that of Example 2 except that the anti-CD63 antibody is used as the solid phase antibody.
A box plot of the amount of AZU1 present on the cell secretory microparticles expressing the CD63 molecule contained in the serum sample is shown in FIG. Table 9 shows the minimum, 25th, median, 75th percentile, maximum absorbance, absorbance range in the 95% confidence interval, and the Mann-Whitney U-test p-value for the healthy and GIST patient groups. The GIST patient group tended to have a higher value than the healthy subject group. The p value was less than 0.001 which was smaller than the significance level (0.05), and a statistically significant difference was observed.

[Example 5] Preparation of AZU1 measurement reagent A container having two compartments was prepared, and the anti-AZU1 monoclonal antibody (clone 1-2) prepared in Reference Example 11 was bound to one compartment and then blocked. A solution containing the particles was dispensed into the other compartment, and a solution containing the anti-AZU1 monoclonal antibody (clone 1-14) prepared in Reference Example 11 labeled with alkaline phosphatase was dispensed. The solution dispensed into the container was freeze-dried, and the container was covered with an aluminum seal to prepare an AZU1 measurement reagent. The AZU1 measured by the measuring reagent prepared in this example is both AZU1 existing as a soluble protein and AZU1 existing on the cell-secreted fine particles.

[Example 6] AZU1 measurement of serum samples of healthy subjects and GIST patients (5)
Table 10 shows the healthy person serum sample and the GIST patient serum sample used in this example.
The healthy person serum sample in this example was collected at the Health Management Center of the Tokyo Research Center of Tosoh Corporation with the approval of the Institutional Review Board of the Bioscience Division of Tosoh Corporation and the consent of the sample provider. be. In addition, the collection and measurement of GIST patient serum samples in this example were carried out with the approval of the Institutional Review Board of the Graduate School of Medicine, Chiba University, and with the consent of the sample provider through informed consent. It is a thing.

The measurement of AZU1 contained in the sample was carried out by a two-step sandwich method in which the AZU1 measuring reagent prepared in Example 5 was set in a fully automatic chemiluminescent enzyme immunoassay device AIA-CL2400 (manufactured by Tosoh).
The specific method is shown below.
[1] To the freeze-dried magnetic particles of the measurement reagent, 10 μL of the measurement sample (serum sample diluted 10-fold in advance) and 40 μL of the diluted solution were added, and the primary antigen-antibody reaction was carried out for 5 minutes.
[2] The reaction solution was removed, and washing with a washing solution (first B / F separation) was performed.
[3] The lyophilized alkaline phosphatase-labeled antibody of the measurement reagent was dissolved in a diluted solution, 50 μL of which was added to the magnetic particles, and the secondary antigen-antibody reaction was carried out for 3 minutes.
[4] The reaction solution was removed, and washing with a washing solution (second B / F separation) was performed.
[5] A luminescent substrate was added to the magnetic particles, and the luminescence intensity was measured.
[6] A calibration curve was prepared based on a standard sample having a known concentration, and the AZU1 concentration in the sample was calculated.

A box plot of the AZU1 concentration contained in the serum sample is shown in FIG. Table 11 shows the minimum, 25th percentile, median, 75th percentile, maximum value, AZU1 concentration range in the 95% confidence interval, and the p value of the Mann-Whitney U test in the healthy and GIST patient groups. .. The GIST patient group tended to have a higher value than the healthy subject group. The p value was less than 0.001 which was smaller than the significance level (0.05), and a statistically significant difference was observed.

INDUSTRIAL APPLICABILITY The present invention provides a method for detecting GIST easily and with high accuracy, and a reagent that can be used for the method. These are very useful industrially because they can be suitably used for GIST screening, therapeutic effect determination, and postoperative follow-up.

Claims (8)

1. A method for detecting gastrointestinal stromal tumors (GIST)
   In the sample, including measuring the amount of azurosidin (AZU1),
   A method in which GIST is detected when the measured value exceeds a preset reference value.
2. The method according to claim 1, wherein the amount of AZU1 is measured using an antibody that specifically recognizes AZU1.
3. In the sample, further detection of a second marker present on the same cell secretory microparticles as the cell secretory microparticles in which AZU1 is present comprises further detection.
   The method of claim 1 or 2, wherein the second marker is at least one of those listed in Table 1.
   

   

   
   

   
4. The method of claim 3, wherein the second marker comprises at least one of CD81, CD63, CD9 and phosphatidylserine.
5. The method according to claim 3 or 4, wherein the detection of the second marker is performed using an antibody or receptor that specifically recognizes the second marker.
6. A reagent for use in the detection of GIST, which contains an antibody that specifically recognizes AZU1.
7. The reagent of claim 6, further comprising an antibody or receptor that specifically recognizes any of the second markers listed in Table 1.
   [Table 2-1]
   

   

   
   [Table 2-2]
   

   
8. The reagent according to claim 7, wherein the second marker contains at least one of CD81, CD63, CD9 and phosphatidylserine.

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