WO2021187173A1 - Méthode de détection de tumeur stromale gastro-intestinale et réactif de détection - Google Patents

Méthode de détection de tumeur stromale gastro-intestinale et réactif de détection Download PDF

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WO2021187173A1
WO2021187173A1 PCT/JP2021/008777 JP2021008777W WO2021187173A1 WO 2021187173 A1 WO2021187173 A1 WO 2021187173A1 JP 2021008777 W JP2021008777 W JP 2021008777W WO 2021187173 A1 WO2021187173 A1 WO 2021187173A1
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azu1
antibody
gist
reagent
sample
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PCT/JP2021/008777
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Japanese (ja)
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幸嗣 植田
なおみ 大西
久裕 松原
将之 加野
則久 大竹
康俊 河合
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公益財団法人がん研究会
国立大学法人千葉大学
東ソー株式会社
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Priority to JP2022508218A priority Critical patent/JP7306661B2/ja
Publication of WO2021187173A1 publication Critical patent/WO2021187173A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • 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 gastrointestinal stromal tumor
  • AZU1 a detection reagent for which azurosidin
  • 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.
  • ctDNA blood circulating tumor DNA
  • CTC blood circulating tumor cells
  • ANO1 anoctamine 1
  • 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.
  • 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).
  • Non-Patent Document 8 a method for diagnosing renal cell carcinoma by isolating extracellular vesicles in body fluid and detecting AZU1 has been reported.
  • 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.
  • 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.
  • a method for detecting a gastrointestinal stromal tumor 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.
  • 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.
  • the second marker comprises at least one of CD81, CD63, CD9 and phosphatidylserine.
  • the detection of the second marker is performed using an antibody or receptor that specifically recognizes the second marker.
  • a reagent for use in the detection of GIST which comprises an antibody that specifically recognizes AZU1.
  • the reagent according to [6] further comprising an antibody or receptor that specifically recognizes any of the second markers listed in Table 1.
  • the reagent according to [7], wherein the second marker comprises at least one of CD81, CD63, CD9 and phosphatidylserine.
  • the present invention provides a method for detecting GIST easily and with high accuracy, and a reagent that can be used for the method.
  • 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.
  • 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.
  • the target (test animal) for detecting GIST is a human.
  • sample to be measured in the present invention examples 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.
  • 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.
  • 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.
  • the one coexisting with the second marker on the fine particles may be measured.
  • 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".
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • hybridoma cells that produce an antibody that specifically recognizes AZU1 may be appropriately selected from the methods for which technology has been established.
  • 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.
  • GPI glycosylphosphatidylinositol
  • 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.
  • 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.
  • the antibody can be purified by affinity chromatography using a carrier on which protein A, protein G, protein L or the like is immobilized.
  • 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”).
  • 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.
  • a detection device such as a radioisotope or functional fine particles, and the like.
  • 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.
  • 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.
  • LC / MS liquid chromatography-mass spectrometry
  • MALDI-TOF / MS matrix-assisted laser desorption / ionization time-of-flight mass spectrometry
  • 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.
  • ROC receiver operating characteristic
  • 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.
  • 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.
  • 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.
  • 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].
  • Antibodies and the like 1 Antibodies and the like 1: AZU1 Antibodies, antibodies, etc. that specifically recognize 2: Antibodies or receptors that specifically recognize the second marker
  • 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.
  • 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.
  • 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.
  • 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.
  • the methods shown in [4] to [6] below may be used.
  • 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.
  • 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.
  • 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.
  • 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.
  • the methods shown in the following [7] to [8] may be used.
  • 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.
  • 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.
  • 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.
  • 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].
  • 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).
  • 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.
  • 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.
  • the methods shown in the following [11] to [13] may be used.
  • 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.
  • 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.
  • 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.
  • 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.
  • the amount of antibody or the like 1 to be bound to the carrier is 100 ng to 1000 ⁇ g per reaction system in which 20 ⁇ L 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 ⁇ g.
  • 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.
  • 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.
  • HRP horseradish peroxidase
  • Mouse IgG (negative control, NC) diluted 3 times with TBST and diluted with PBS containing 1% BSA to 2 ⁇ g / mL and a commercially available mouse anti-AZU1 antibody (manufactured by R & D Systems) (positive control, PC).
  • PC mouse anti-AZU1 antibody
  • 50 ⁇ L / well of undiluted hybridoma cell culture supernatant was added, and the mixture was left at room temperature for 1 hour.
  • 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.
  • Example 1 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 ⁇ L / well of a rabbit anti-human AZU1 monoclonal antibody diluted to 2 ⁇ g / mL with PBS was added, and the mixture was allowed to solidify at 4 ° C. overnight.
  • 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 ⁇ L / well of solid phase antibody (anti-CD81 antibody) diluted to 4 ⁇ g / 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.
  • 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.
  • FIG. 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.
  • 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 ⁇ L of the measurement sample (serum sample diluted 10-fold in advance) and 40 ⁇ L 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.
  • FIG. 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.
  • 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.

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Abstract

Le problème à résoudre par la présente invention concerne la fourniture d'une méthode permettant de détecter facilement une tumeur stromale gastro-intestinale (GIST) avec une grande précision et un réactif pouvant être utilisé dans cette méthode. L'invention concerne un procédé de détection de GIST, ledit procédé consiste à mesurer la quantité d'azurocidine (AZU1) dans un échantillon ; lorsque la valeur mesurée dépasse une valeur standard prédéfinie, on considère alors que la GIST est détectée. Un réactif contenant un anticorps reconnaissant de manière spécifique AZU1 est utilisé pour la détection de la GIST.
PCT/JP2021/008777 2020-03-19 2021-03-05 Méthode de détection de tumeur stromale gastro-intestinale et réactif de détection WO2021187173A1 (fr)

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Citations (6)

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Publication number Priority date Publication date Assignee Title
JP2012088220A (ja) * 2010-10-21 2012-05-10 Shinya Watanabe Ｇｉｓｔ検出用マーカー
JP2012088221A (ja) * 2010-10-21 2012-05-10 Shinya Watanabe Ｇｉｓｔの診断方法
JP2013128434A (ja) * 2011-12-20 2013-07-04 Sapporo Medical Univ 消化管間質腫瘍マーカー
US20170153240A1 (en) * 2015-11-30 2017-06-01 Sungkwang Medical Foundation Composition, kit, and method for diagnosing and treating ovarian cancer
WO2018079689A1 (fr) * 2016-10-28 2018-05-03 公益財団法人がん研究会 Biomarqueur, procédé de recherche d'un gène associé à une maladie, et marqueur du cancer du rein
WO2018217087A1 (fr) * 2017-05-23 2018-11-29 Stichting Het Nederlands Kanker Instituut-Antoni van Leeuwenhoek Ziekenhuis Nouveaux biomarqueurs protéiques basés sur les selles pour le dépistage du cancer colorectal

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Publication number Priority date Publication date Assignee Title
JP2012088220A (ja) * 2010-10-21 2012-05-10 Shinya Watanabe Ｇｉｓｔ検出用マーカー
JP2012088221A (ja) * 2010-10-21 2012-05-10 Shinya Watanabe Ｇｉｓｔの診断方法
JP2013128434A (ja) * 2011-12-20 2013-07-04 Sapporo Medical Univ 消化管間質腫瘍マーカー
US20170153240A1 (en) * 2015-11-30 2017-06-01 Sungkwang Medical Foundation Composition, kit, and method for diagnosing and treating ovarian cancer
WO2018079689A1 (fr) * 2016-10-28 2018-05-03 公益財団法人がん研究会 Biomarqueur, procédé de recherche d'un gène associé à une maladie, et marqueur du cancer du rein
WO2018217087A1 (fr) * 2017-05-23 2018-11-29 Stichting Het Nederlands Kanker Instituut-Antoni van Leeuwenhoek Ziekenhuis Nouveaux biomarqueurs protéiques basés sur les selles pour le dépistage du cancer colorectal

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