WO2020203478A1 - Procédé et système de génération d'informations de condition pathologique, kit d'analyse des chaînes de sucre he4 et he4 - Google Patents

Procédé et système de génération d'informations de condition pathologique, kit d'analyse des chaînes de sucre he4 et he4 Download PDF

Info

Publication number
WO2020203478A1
WO2020203478A1 PCT/JP2020/013080 JP2020013080W WO2020203478A1 WO 2020203478 A1 WO2020203478 A1 WO 2020203478A1 JP 2020013080 W JP2020013080 W JP 2020013080W WO 2020203478 A1 WO2020203478 A1 WO 2020203478A1
Authority
WO
WIPO (PCT)
Prior art keywords
residues
substance
lectin
pathological condition
sugar chain
Prior art date
Application number
PCT/JP2020/013080
Other languages
English (en)
Japanese (ja)
Inventor
智惠 乾
智典 金子
高敏 彼谷
Original Assignee
コニカミノルタ株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by コニカミノルタ株式会社 filed Critical コニカミノルタ株式会社
Priority to JP2021511506A priority Critical patent/JPWO2020203478A1/ja
Publication of WO2020203478A1 publication Critical patent/WO2020203478A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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

Definitions

  • the present invention relates to a pathological condition information generation method, a pathological condition information generation system, a HE4 sugar chain analysis kit, and HE4. More specifically, the present invention relates to a method for generating pathological condition information that enables diagnosis with reduced burden on patients and doctors by evaluating the malignancy of cancer such as ovarian cancer by a simple and non-invasive method.
  • Ovarian cancer is the second most common gynecologic cancer after breast cancer. Early detection of ovarian cancer is difficult because there are few subjective symptoms in the early stages of onset, and symptoms are often already advanced at the time of detection. Therefore, it has a poor prognosis and is a fatal gynecologic malignancies with the highest mortality rate among gynecologic cancers. The majority of ovarian tumors are benign, and distinguishing between malignant and benign is important in determining treatment strategy.
  • tumor marker measurement centered on CA125, transvaginal ultrasonic tomography, MRI (Magnetic Resonance Imaging) examination, and CT (Computed Tomography: Computed Tomography) Imaging method) Inspection is performed as needed.
  • CA125 is the most commonly used tumor marker for the diagnosis of ovarian cancer, but its value has also been increased in benign diseases such as endometriosis, menstruation, pregnancy, and thoracic peritoneal inflammatory diseases. Since it cannot be said that it has sufficient specificity in distinguishing between malignant and benign tumors, the development and clinical application of more useful new markers and analytical methods have been expected.
  • human epididymis protein 4 which is detected at a high concentration in the serum of ovarian cancer patients and whose increase is observed as the stage progresses, is WAP. It is a secretory glycoprotein with a molecular weight of about 25 kDa, which belongs to the (Why Acidic Protein) family and is also called WFDC2 (WAP Four-Disulfide Core Domine Protein 2). It is named because it was found in epididymal epididymal cells. It was attached. Mature human HE4 has a structure in which two WAP domains are linked, but the first or second WAP domain is missing due to splicing, and a plurality of amino acids replaced with 10, 22, or 28 amino acids. Produces an isoform of.
  • HE4 is strongly expressed in serum in ovarian cancer and very high in advanced cancer.
  • HE4 is a highly specific tumor marker that is less likely to increase in gynecological benign diseases such as endometriosis than CA125, which is a mucin glycoprotein.
  • CA125 which is a mucin glycoprotein.
  • HE4 is expressed not only in ovarian cancer but also in lung adenocarcinoma and pulmonary cystic fibrosis, and the size and invasiveness of the tumor and the HE4 iso in which the first WAP domain is replaced with 22 amino acids It has been suggested that it is related to the expression of foam.
  • Increased HE4 levels are also seen in tissues and serum in the case of type I / II endometrial cancer and in urine in the case of transitional cell carcinoma and early / late ovarian cancer.
  • HE4 is expressed in the reproductive tract epithelium, upper respiratory tract, salivary gland canal, and chest of normal men and women, as well as in the distal convoluted tubule, colon, and endometrium. Therefore, even HE4 cannot be said to have sufficient specificity in differentiating between malignant and benign ovarian tumors, and development and clinical application of more useful new markers and analytical methods are still desired. In addition, it is required to develop a method for analyzing HE4 as a more effective tool for detecting a potentially curable early malignant tumor state such as ovarian cancer (see, for example, Patent Document 1).
  • ovarian cancer To distinguish between malignant and benign tumors, there are methods such as pelvic examination, rectal examination, ultrasonography (echo) examination, CT examination, and MRI examination, but for ovarian cancer, benign ovarian examination is performed by imaging examination and examination. Since it is difficult to distinguish between tumors and malignant tumors, it is necessary to confirm the diagnosis by performing pathological examination.
  • Pathological examinations include cytology and histology. Cytology tests the pleural effusion and ascites for cancer cells. The basic method is to collect ascites during surgery, but if ascites is seen before surgery, a needle is pierced through the skin to collect ascites for examination.
  • the present invention has been made in view of the above problems and situations, and the solution problem is to accurately evaluate the morbidity, onset or malignancy of cancer such as ovarian cancer by a rapid, simple and non-invasive method. By doing so, it is possible to provide a pathological condition information generation method, a pathological condition information generation system, a HE4 sugar chain analysis kit, and the like, which enable diagnosis with reduced burden on patients and doctors.
  • the present inventor has identified HE4 having a specific residue at the (non-reducing) end of the sugar chain in the process of examining the sugar chain of HE4 associated with the development of cancer such as ovarian cancer. We have found that it has an important correlation with the cancer of the present invention. That is, the above problem according to the present invention is solved by the following means.
  • a method for generating pathological condition information that generates information on the onset or onset of cancer. Residue of any of four types of residues, ⁇ -N-acetylgalactosamine residue, ⁇ 2,3-sialic acid residue, ⁇ 2,6-sialic acid residue or mannose residue, contained in the test sample.
  • a method for generating pathological condition information which is characterized by having.
  • the four types of residues are subjected to the first step by mass spectrometry or a method of contacting the test sample with a first substance having an affinity for any of the four types of residues.
  • the third step is a step of measuring the total amount of HE4 by mass spectrometry, chromatography, or a method of contacting the test sample with a second substance having an affinity for the polypeptide chain portion of HE4.
  • the method for generating pathological condition information according to any one of the items 1 to 3, wherein the method is characterized by.
  • the second step is a step of calculating the ratio of the amount of HE4 having any one of the four types of residues to the total amount of all the HE4s contained in the test sample.
  • the method for generating pathological condition information according to the fourth item is a step of calculating the ratio of the amount of HE4 having any one of the four types of residues to the total amount of all the HE4s contained in the test sample.
  • the first step is characterized in that the second step is a step of generating information on whether or not the subject has ovarian cancer as information on the morbidity or onset of cancer of the subject.
  • the method for generating pathological condition information according to any one of items 1 to 6.
  • the first step is a method of contacting the test sample with a first substance having an affinity for any of the four residues, and the four residues. This is a step of measuring the amount of HE4 having any residue.
  • the first substance is a lectin having an affinity for HE4 having any of the four types of residues, and is a lectin of any one of ConA, Fujirectin, SSA or MAM.
  • the first substance is immobilized on a support and
  • the third step is a step of measuring the total amount of all HE4 by a method of bringing the second substance having an affinity for the polypeptide chain portion of HE4 into contact with the test sample.
  • the second substance is fixed to the support and
  • the first step is a method of contacting the test sample with a first substance having an affinity for any of the four residues, and the four residues. This is a step of measuring the amount of HE4 having any residue.
  • the first substance is a lectin having an affinity for any of the four residues.
  • the first substance is labeled with a phosphor and In the first step, the intensity of fluorescence emitted from a phosphor labeling the first substance bound to HE4 captured by the second substance is measured, and based on the intensity of the fluorescence, the remaining four types are used.
  • the method for generating pathological condition information according to Item 13, wherein the step is to calculate the amount of HE4 having any residue of the group.
  • the ⁇ -N-acetylgalactosamine residue, the ⁇ 2,3-sialic acid residue, the ⁇ 2,6-sialic acid residue, or the mannose residue is any of four residues.
  • a pathological information generation system that generates information on the onset or onset of cancer. Residue of any of four types of residues, ⁇ -N-acetylgalactosamine residue, ⁇ 2,3-sialic acid residue, ⁇ 2,6-sialic acid residue or mannose residue, contained in the test sample.
  • the first step to obtain information about the amount of HE4 having a group A second step unit that generates information on the morbidity or onset of cancer of the subject according to the test sample based on the amount of HE4 having any of the four residues.
  • a pathological condition information generation system characterized by having.
  • a HE4 sugar chain analysis kit containing HE4 and anti-HE4 antibodies having sugar chains.
  • An HE4 sugar chain analysis kit which is an analysis kit for carrying out the method for generating pathological condition information according to any one of items 1 to 17.
  • HE4 sugar chain analysis kit containing HE4 having sugar chains and anti-HE4 antibodies.
  • the HE4 sugar chain analysis kit according to Item 20 which comprises an anti-HE4 antibody.
  • the HE4 sugar chain analysis kit according to any one of items 20 to 22, wherein the lectin, the anti-HE4 antibody, and the analysis substrate are combined.
  • the HE4 sugar chain analysis kit according to any one of items 20 to 23, wherein the lectin is provided with a fluorescent substance.
  • the HE4 sugar chain analysis kit according to any one of Items 20 to 24, which comprises at least one lectin of Fujirectin or MAM provided with a fluorescent substance.
  • the HE4 sugar chain analysis kit according to any one of items 20 to 25, further comprising a chromatographic apparatus.
  • the HE4 sugar chain analysis kit according to Item 26 which further comprises a labeling reagent for chromatographic measurement.
  • HE4 as a biomarker for cancer testing Any of four types of residues at the (non-reducing) end of the sugar chain: ⁇ -N-acetylgalactosamine residue, ⁇ 2,3-sialic acid residue, ⁇ 2,6-sialic acid residue or mannose residue. HE4 characterized by having a residue of.
  • the means of the present invention it is possible to make a diagnosis with reduced burden on patients and doctors by accurately evaluating the morbidity, onset or malignancy of cancer such as ovarian cancer by a rapid, simple and non-invasive method. It is possible to provide a pathological condition information generation method, a pathological condition information generation system, a HE4 sugar chain analysis kit, and the like.
  • the expression mechanism or action mechanism of HE4 having a specific residue according to the present invention has not been clarified.
  • the pathological condition information generation method, the pathological condition information generation system, the HE4 sugar chain analysis kit, and the like of the present invention can obtain accurate information on whether or not a subject under 50 years old has ovarian cancer. The feature is that it can be done.
  • FIG. 1 Schematic diagram schematically showing the configuration of the SPFS measuring device Partial enlarged view around the sensor chip of the measuring device of FIG.
  • Schematic diagram showing an example of HE4 sugar chain analysis kit The figure which shows the quantification result of HE4 (GalNAcHE4) which has a ⁇ -N-acetylgalactosamine residue at the end of a sugar chain by the lectin column method using WFA as a lectin.
  • the pathological information generation method of the present invention is a pathological information generation method that generates information on the morbidity or onset of cancer, and contains ⁇ -N-acetylgalactosamine residue and ⁇ 2,3-sialic acid residue contained in a test sample.
  • This feature is a technical feature common to or corresponding to each of the following embodiments.
  • the first step is mass spectrometry or a method of bringing the test sample into contact with a first substance having an affinity for any of the four residues. Therefore, it is preferable from the viewpoint of the effect expression and accuracy of the present invention to measure the information regarding the amount of HE4 having any one of the above four types of residues. Further, it is preferable to have a third step of obtaining the total amount of all HE4 contained in the test sample regardless of HE4 having the four kinds of residues from the same viewpoint as described above.
  • the third step is a step of measuring the total amount of HE4 by mass spectrometry, chromatography, or a method of contacting the test sample with a second substance having an affinity for the polypeptide chain portion of HE4. It is preferable to have.
  • the second step is the ratio of the amount of HE4 having any one of the four residues to the total amount of all HE4 contained in the test sample. Is preferable from the viewpoint of the expression of the effect and the accuracy of the present invention. Further, in the second step, as information on the morbidity or onset of cancer of the subject, whether or not the subject has cancer, the probability that the subject has cancer, and the above. It is preferable that the step is to generate information on the malignancy of the subject's cancer or the stage of the subject's cancer. Further, it is preferable that the second step is a step of generating information on whether or not the subject has ovarian cancer as information on the onset or onset of cancer of the subject.
  • the first step is performed before the third step. Further, in the first step, the first substance having an affinity for one of the four types of residues is brought into contact with the test sample, thereby causing the four types of residues to come into contact with each other. It is a step of measuring the amount of HE4 having any of the residues, and the first substance is a lectin or antibody having an affinity for any of the residues of the four types. Is preferable. Further, it is preferable that the first substance is a lectin having an affinity for HE4 having any of the four types of residues, and is any lectin of ConA, Fujirectin, SSA or MAM.
  • the first substance is immobilized on the support
  • the third step is a method in which the second substance having an affinity for the polypeptide chain portion of HE4 is brought into contact with the test sample. It is a step of measuring the total amount of all the HE4s, and it is the effect of the present invention that the second substance is an antibody having an affinity for any one of the four residues. Preferred from the point of view. Further, it is preferable that the first step is a step by chromatography.
  • the third step is performed before the first step.
  • the second substance is fixed to the support, and the first step includes the first substance having an affinity for any of the four residues and the test sample. It is a step of measuring the amount of HE4 having any one of the four kinds of residues by the method of contacting with, and the first substance is one of the above four kinds of residues.
  • the first substance is labeled with a phosphor, and the first step labels the first substance bound to HE4 captured by the second substance. It is a step of measuring the intensity of the fluorescence emitted from the phosphor and calculating the amount of HE4 having any one of the four types of residues based on the intensity of the fluorescence. It is preferable from the viewpoint of.
  • the third step it is preferable to compare the intensity of the peak obtained by mass spectrometry or chromatography with the intensity of the peak as a predetermined reference from the same viewpoint as above.
  • any one of four types of residues the ⁇ -N-acetylgalactosamine residue, the ⁇ 2,3-sialic acid residue, the ⁇ 2,6-sialic acid residue, or the mannose residue. It is preferable that the residue of is at the (non-reducing) end of the sugar chain of HE4 from the viewpoint of the effect of the present invention. Moreover, it is preferable that the cancer is ovarian cancer.
  • the pathological information generation system of the present invention is a pathological information generation system that generates information on the morbidity or onset of cancer, and contains ⁇ -N-acetylgalactosamine residue and ⁇ 2,3-sialic acid residue contained in a test sample.
  • an embodiment of the present invention is characterized by being a system for implementing the pathological condition information generation method.
  • the HE4 sugar chain analysis kit of the present invention is a HE4 sugar chain analysis kit containing HE4 having a sugar chain and an anti-HE4 antibody, and is an analysis kit for carrying out the above-mentioned pathological condition information generation method of the present invention. And. Further, in the HE4 sugar chain analysis kit containing HE4 having a sugar chain and an anti-HE4 antibody, ⁇ -N-acetylgalactosamine residue, ⁇ 2,3-sialic acid residue, at the (non-reducing) end of the sugar chain, It is characterized by containing a substance having an affinity for HE4 having an ⁇ 2,6-sialic acid residue or a mannose residue and an anti-HE4 antibody.
  • the substance having an affinity for HE4 is a lectin from the viewpoint of accuracy and convenience. Further, it is preferable that the lectin, the anti-HE4 antibody, and the analysis substrate are combined. Further, it is preferable that the lectin is provided with a phosphor. In addition, it is preferable to contain at least one lectin of Fujirectin or MAM equipped with a phosphor. Further, as an embodiment, it is preferable that the configuration is provided with a chromatography device. Further, it is preferable that the configuration includes a labeling reagent for chromatographic measurement.
  • HE4 is HE4 as a biomarker for cancer examination, and ⁇ -N-acetylgalactosamine residue, ⁇ 2,3-sialic acid residue, ⁇ 2,6- at the (non-reducing) end of the sugar chain. It is characterized by having one of four types of residues, a sialic acid residue or a mannose residue.
  • the "detection target sugar chain” (also referred to as “analyte”, “detection target”, “test object”) is such that it is present in a relatively high concentration in blood, for example, when a specific disease develops.
  • the “non-detection target sugar chain” is a sugar chain possessed by impurities (glycoproteins, glycolipids, etc. different from the analysis) present in the sample together with the analysis, and is an obstacle to the detection of the analysis.
  • the "detection method” in the present specification naturally includes not only a method for qualitatively detecting an analyst but also a method for quantitatively detecting and measuring an analyst (a method for quantitatively detecting and measuring an analyst). Further, the detection method is included in the step of the pathological condition information generation method of the present invention.
  • the pathological information generation method of the present invention is a pathological information generation method for generating information on the onset or onset of cancer, and is a ⁇ -N-acetylgalactosamine residue contained in a test sample.
  • detailed explanations of the components and methods will be given in sequence.
  • the substance to be detected according to the present invention is HE4 having a sugar chain, and the sugar chain to be detected is ⁇ -N-acetylgalactosamine residue, ⁇ 2,3-sialic acid residue, ⁇ 2, at the (non-reducing) end of the sugar chain.
  • the sugar chain is a general term for monosaccharides such as glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid, and molecules in which these derivatives are linked in a chain by glycosidic bonds. ..
  • sugar chains are extremely diverse and are deeply involved in various functions of naturally occurring organisms, such as cell-cell communication and regulation of protein functions and interactions. It is becoming clear that this is the case.
  • Biopolymers having sugar chains include, for example, proteoglycans on the cell wall of plant cells that contribute to cell stabilization, glycolipids that affect cell differentiation, proliferation, adhesion, migration, etc., and cell-cell interactions and cells. Examples include glycoproteins involved in recognition.
  • the mechanism by which sugar chains contained in these biopolymers control sophisticated and precise biological reactions while substituting, assisting, amplifying, regulating, or inhibiting each other's functions with the biopolymers is gradually being elucidated. If the relationship between such sugar chains and cell differentiation and proliferation, cell adhesion, immunity, and cell canceration is clarified, this sugar chain engineering will be closely related to medicine, cell engineering, or organ engineering. It can be expected that new developments will be made.
  • lectin is a general term for proteins (sugar chain-binding proteins) that exhibit binding activity to glycoproteins and glycolipid sugar chains that constitute cell membranes. Details of the lectin will be described later.
  • Lectin group (1) Proteins that recognize high mannose-type sugar chains: Lectins such as Artocarpin, CRLL, Conarva, CCA, Heltuba, jacalin, Calsepa, MornigaM, UDA, PSA, LTA, LTL, NPA, HHA, HHL, GNL, GNA, AMA, Succinlylae and ConA. antibody.
  • Lectins such as Artocarpin, CRLL, Conarva, CCA, Heltuba, jacalin, Calsepa, MornigaM, UDA, PSA, LTA, LTL, NPA, HHA, HHL, GNL, GNA, AMA, Succinlylae and ConA. antibody.
  • Protein that recognizes ⁇ 2,6-residues of sialic acid Lectins such as SNA, EBL, BRL, BRA2, TJA-I, PPL, SNA, SAN-I, SSA, RCA120, RCAI and AMA, and anti-sialic acid ⁇ 2,6-residue antibody.
  • Protein that recognizes ⁇ 2,3-residues of sialic acid Lectins such as MALII, MAH and MAM, and anti-sialic acid ⁇ 2,3-residue antibody.
  • Particularly preferably used lectins in the present invention are ⁇ -N-acetylgalactosamine residue (GalNAc ⁇ 1 ⁇ R,) ⁇ 2,3 sialic acid residue and ⁇ 2,6 bound to the (non-reducing) end of the sugar chain of HE4.
  • Lectins having an affinity for sialic acid residues, mannose residues and the like are preferable.
  • lectins having an affinity for ⁇ -N-acetylgalactosamine residues include WFA, TJA-II, ECA, RCA120 and DSA.
  • Examples of lectins having an affinity for ⁇ 2,3 sialic acid residues include MAL and MAM.
  • Examples of the lectin having an affinity for ⁇ 2,6 sialic acid residues include SNA, SSA and TJA-I.
  • Examples of the lectin having an affinity for high mannose (high-mannose) include ConA and UDA.
  • TJA-II is a lectin extracted and purified from the roots of Trichosanthes chinensis, and has a molecular weight of 64 kDa by electrophoresis in a non-reduced state, but is an SS-bound dimer and is an electricity in a reduced state. The molecular weight by electrophoresis shows 32 kDa and 29 kDa.
  • WFA is a lectin extracted and purified from Nodafuji seeds and shows a strong affinity for ⁇ -N-acetylgalactosamine residue (GalNAc ⁇ 1 ⁇ R) itself. It also shows a strong affinity for GalNAc ⁇ 1 ⁇ 4Gal residues and GalNAc ⁇ 1 ⁇ 4GlcNAc residues.
  • the substance having an affinity for HE4 having any of the four residues is ConA, WFA, WFL, SSA or MAM, or Norectin. It is preferably either ConA, WFA or SSA.
  • a lectin is used as a labeling lectin for labeling a detection target (analyte) having a detection target sugar chain.
  • a lectin may be used as a masking sugar chain recognition molecule for masking a sugar chain to be detected contained in a contaminant.
  • the sugar chain recognition molecule used for masking is not limited to lectins, and for example, an antibody that recognizes a sugar chain to be detected as an epitope and specifically binds to it is also included, but is similar to a lectin for labeling.
  • Lectin is preferable from the viewpoint that it is inexpensive, can be obtained in large quantities, has excellent protein stability, and can be stored for a long period of time.
  • Lectin binds to a specific sugar chain according to its type with a high binding ability, but it may also bind to another sugar chain with a low binding ability. Therefore, in the detection method of the present invention, a lectin that binds to a sugar chain to be detected possessed by Anna Reed with a high binding ability and to a sugar chain to be detected not possessed by a contaminant with a weak binding ability is used as a labeling lectin. Applies when used. Then, a sugar chain recognition molecule that binds to a non-detection target sugar chain to which the labeling lectin binds with a weak binding ability is used as a mask sugar chain recognition molecule.
  • Such a mask sugar chain recognition molecule does not substantially bind to the sugar chain to be detected to which the labeling lectin binds with a strong binding ability, or a sugar chain recognition molecule that binds with a weak binding ability (for example, labeling). It needs to be a different type of lectin from the one used for it.
  • test sample containing substance to be detected
  • the test sample (sample) that may contain the detection target substance may be a sample that actually contains the detection target substance, or is actually a test sample (sample) that does not contain the detection target substance.
  • test samples include, for example, biological samples such as blood, serum, plasma, urine, spinal fluid, saliva, cells, tissues, ascites, or organs, or preparations thereof (for example, biopsy specimens). Biological samples can be mentioned.
  • blood, serum or plasma which may contain a cancer antigen / tumor marker, is suitable as a test sample.
  • liquid samples such as blood, serum, plasma, urine, cerebrospinal fluid, saliva, and ascites can be diluted with an appropriate buffer solution before use.
  • solid samples such as cells, tissues, or organs are homogenized with an appropriate buffer solution having a volume of about 2 to 10 times the volume of the solid sample, and the suspension or its supernatant is used as it is. , Or it can be further diluted before use.
  • ⁇ Sign body> in order to detect HE4 (antigen to be detected) having a sugar chain, it is preferable to use a labeled substance complexed with a lectin. Therefore, it is preferable to use a labeled lectin in which a labeled substance and a lectin are complexed (bound).
  • a labeling substance such as a fluorescent dye (fluorescent substance), an enzyme / coenzyme, a chemiluminescent substance, and a radioactive substance can be used.
  • the fluorescent dye (fluorescent body) include a fluorescein family fluorescent dye (Integrated DNA Technologies), a polyhalofluoresane family fluorescent dye (Applied Biosystems Japan Co., Ltd.), and a hexachlorofluoresane family fluorescent dye ().
  • Fluorophores of the Kumarin family Invitrogen Co., Ltd.
  • Fluorophores of the Rhodamine family GE Healthcare Biosciences Co., Ltd.
  • Fluorophores of the cyanine family Indocarbocyanine Family fluorescent dyes, Oxazine family fluorescent dyes, Thiadine family fluorescent dyes, Squaline family fluorescent dyes, chelated lanthanide family fluorescent dyes, BODIPY® family fluorescent dyes (Invitrogen Co., Ltd.
  • Naphthalene sulfonic acid family fluorescent dyes Naphthalene sulfonic acid family fluorescent dyes, pyrene family fluorescent dyes, triphenylmethane family fluorescent dyes, Alexa Fluor (registered trademark) dye series (Invitrogen Co., Ltd.) and other organic fluorescent dyes. ..
  • rare earth complex fluorescent dyes such as Eu and Tb (for example, ATBTA-Eu3 +), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and red fluorescence.
  • BFP blue fluorescent protein
  • CFP cyan fluorescent protein
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • Fluorescent proteins typified by protein (DsRed) or Allophycocyanin (APC; LyoFlogen®), fluorescent fine particles such as latex and silica, and the like are also mentioned as fluorescent dyes.
  • the maximum fluorescence wavelength in the near infrared region such as Cy5 or Alexa Fluor 647 is set in order to minimize the influence of absorption by iron derived from blood cell components in blood. It is desirable to use a fluorescent dye that has. Radioisotopes (32P, 14C, 125I, 3H, 131I, etc.) can be mentioned as radioactive substances.
  • a detection target analyte: detection target antigen
  • a labeling lectin a labeling lectin
  • lectin a mask sugar chain recognition molecule
  • the former has a substance to be detected such as a cancer antigen / tumor marker, impurities (glycoproteins and glycolipids other than the antigen to be detected) that are easily contained in the sample, and the type of sugar chain possessed by each.
  • a labeling lectin that easily binds to the sugar chain to be detected and a mask sugar chain recognition molecule (lectin) that is easily bound to the non-detection target sugar chain of the latter that may bind to the lectin at the same time are appropriately selected and combined. It can also be used.
  • HE4 antibody a molecule that specifically recognizes and binds HE4 (analyte: substance to be detected) and does not interfere with sugar chain recognition by lectins can be used as an antigen-capturing substance (ligand).
  • the antigen-capturing substance is not particularly limited as long as it is an appropriate substance corresponding to HE4, and for example, an antibody, an aptamer, a synthetic peptide, an enzyme such as PSA, trypsin, or proteinase K that binds to HE4 can be used.
  • a monoclonal antibody against the antigen to be detected is suitable.
  • an antibody monoclonal antibody or the like
  • an antigen-capturing substance For example, when HE4 is used as an antigen, an anti-human-HE4 antibody may be used.
  • antibody is used to include not only a complete antibody but also an arbitrary antibody fragment or derivative, and in addition to the complete antibody, Fab, F (ab') 2, CDR, humanized antibody, multifunctional antibody, etc.
  • Various antibodies such as single chain antibody (ScFv) are also included.
  • a support for immobilizing an antigen-capturing substance (ligand) (capturing an object to be detected therein) can be used depending on the measurement system.
  • the support include insoluble polysaccharides such as agarose and cellulose, synthetic resins such as silicon resin, polystyrene resin, polyacrylamide resin, nylon resin and polycarbonate resin, and insoluble supports such as glass. ..
  • These supports are used in the form of beads (mainly spherical), plates (mainly flat), and the like.
  • the beads a column filled with magnetic beads, resin beads, or the like can be used.
  • a multi-well plate 96-well multi-well plate, etc.
  • a biosensor chip, or the like can be used.
  • a flat support such as a plate may be called a substrate.
  • the binding between the antigen-capturing substance (ligand) and the support can be performed by a commonly used method such as chemical binding or physical adsorption. As all of these supports, commercially available ones can be preferably used.
  • the pathological information generation method of the present invention is a pathological information generation method that generates information on the onset or onset of cancer, and is contained in a test sample, ⁇ -N-. Obtain information on the amount of HE4 having one of four residues: acetylgalactosamine residue, ⁇ 2,3-sialic acid residue, ⁇ 2,6-sialic acid residue or mannose residue.
  • preferred embodiments of each basic step will be described.
  • (A) First step In the first step, 4 of ⁇ -N-acetylgalactosamine residue, ⁇ 2,3-sialic acid residue, ⁇ 2,6-sialic acid residue or mannose residue contained in the test sample. It is a step of obtaining information on the amount of HE4 having any of the residues of the species.
  • the four types of residues are subjected to mass spectrometry or a method of contacting the test sample with a first substance having an affinity for any of the four types of residues. From the viewpoint of accuracy, convenience, efficiency and the like, it is preferable that the step is to measure information on the amount of HE4 having any of the residues.
  • mass spectrometry examples include high-speed liquid chromatograph mass spectrometry (LC-MS), high-speed liquid chromatogram tandem mass spectrometry (LC-MS / MS), gas chromatogram mass spectrometry (GC-MS), and gas chromatogram mass spectrometry.
  • LC-MS high-speed liquid chromatograph mass spectrometry
  • LC-MS / MS high-speed liquid chromatogram tandem mass spectrometry
  • GC-MS gas chromatogram mass spectrometry
  • gas chromatogram mass spectrometry examples of mass spectrometry
  • gas chromatogram mass spectrometry examples include gas chromatogram mass spectrometry.
  • Analytical methods GC-MS / MS
  • CE-MS capillary electroswimming mass spectrometry (CE-MS), ICP mass spectrometry (ICP-MS) and the like can be used.
  • chromatography including a lectin column
  • ICP-MS ICP mass spectrometry
  • the first substance is a lectin or antibody having an affinity for any one of the four types of residues. Further, it is preferable that the first substance is a lectin having an affinity for HE4 having any of the four types of residues, and is any lectin of ConA, Fujirectin, SSA or MAM. It should be noted that any of four types of residues: the ⁇ -N-acetylgalactosamine residue, the ⁇ 2,3-sialic acid residue, the ⁇ 2,6-sialic acid residue, or the mannose residue. Is preferably at the (non-reducing) end of the sugar chain of HE4.
  • the second step is information on the morbidity or onset of cancer of the subject according to the test sample, based on the amount of HE4 having any of the above four types of residues. Is the process of producing.
  • the second step has any residue of the four types of residues with respect to the total amount of all the HE4s contained in the test sample, regardless of HE4 having the four types of residues. It is also preferable that the step is a step of calculating the ratio of the amount of HE4.
  • the second step as information on the morbidity or onset of cancer of the subject, whether or not the subject has cancer, the probability that the subject has cancer, and the subject. It is also preferable that it is a step of generating information on the malignancy of the examiner's cancer or the stage of the examinee's cancer. Further, it is also preferable that the second step is a step of generating information on whether or not the subject has ovarian cancer as information on the morbidity or onset of cancer of the subject.
  • the method for generating pathological condition information of the present invention further includes a third step of obtaining the total amount of all HE4 contained in the test sample regardless of HE4 having the four types of residues. This is preferable from the viewpoint of accuracy. It is preferable that the third step is a step of measuring the total amount of HE4 by mass spectrometry or a method of contacting the test sample with a second substance having an affinity for the polypeptide chain portion of HE4.
  • the mass spectrometry method for example, the above-mentioned various mass spectrometry methods can be used.
  • chromatography or immunoreaction using an antibody having an affinity for the polypeptide chain portion of HE4 is used.
  • Blotting colloidal gold method, latex agglutination method, radioimmunoassay method, capillary electrophoresis method, enzyme-activated luminescence method, etc.
  • the third step is a step of measuring the total amount of all the HE4s by a method of bringing the second substance having an affinity for the polypeptide chain portion of HE4 into contact with the test sample.
  • the two substances are antibodies that have an affinity for any of the above four residues
  • the method for detecting the sugar chain of HE4 is the separation method by liquid chromatography, and HE4 having a sugar chain.
  • This is a step of contacting a test sample containing the above-mentioned sugar chain with a carrier having a function of capturing the sugar chain, undergoing a step of capturing the sugar chain on the carrier, and then releasing the sugar chain from the carrier that captured the sugar chain. Is preferable.
  • the third step is a step of measuring the total amount of all the HE4s by mass spectrometry, and the sugar chains released from the HE4 having the sugar chains are separated by chromatography to distribute the sugar chain structure. It is preferable to include a step of obtaining the above-mentioned step and a step of comparing the relative intensity of the peak in the sugar chain structure distribution with the relative intensity of the corresponding peak in the reference.
  • the second substance is fixed to the support
  • the first step comprises the first substance having an affinity for any one of the four residues.
  • This is a step of measuring the amount of HE4 having any one of the four types of residues by a method of contacting the test sample, wherein the first substance is one of the four types of residues.
  • the first substance is a lectin having an affinity for any of the residues of the above, the first substance is labeled with a phosphor, and the first step is the first step which is bound to HE4 captured by the second substance.
  • the first step is performed before the third step from the viewpoint of accuracy, convenience, efficiency and the like.
  • the third step is performed before the first step.
  • the first step comprises a first substance having an affinity for any of the four residues. It is a step of measuring the amount of HE4 having any one of the four types of residues by the method of contacting with the test sample, and the first substance is the step of measuring the amount of the four types of residues. It is preferably a lectin or antibody having an affinity for any of the residues. Further, it is preferable that the first substance is a lectin having an affinity for HE4 having any of the four types of residues, and is any lectin of ConA, Fujirectin, SSA or MAM.
  • the first substance is immobilized on a support (for example, a carrier of a lectin column), and the third step involves a second substance having an affinity for the polypeptide chain portion of HE4 and the test sample. It is a step of measuring the total amount of all the HE4s by the method of contacting with, and the second substance is an antibody having an affinity for any residue of the four kinds of residues. Is preferable from the viewpoint of exhibiting the effect of the present invention. Further, it is preferable that the first step is a step by chromatography.
  • the third step is performed before the first step
  • the second substance is fixed to the support
  • the first step is any of the four types of residues.
  • This is a step of measuring the amount of HE4 having any one of the four types of residues by a method of contacting the first substance having an affinity with the residue with the test sample.
  • the first substance is a lectin having an affinity for any of the four residues, and the first substance is labeled with a phosphor.
  • the intensity of fluorescence emitted from the phosphor labeling the first substance bound to HE4 captured by the second substance was measured, and based on the intensity of the fluorescence, any one of the four residues. From the viewpoint of accuracy and the like, it is preferable that the step is to calculate the amount of HE4 having the residue of.
  • the third step is a step of measuring the total amount of all the HE4s by mass spectrometry, and the sugar chain structure distribution of all the HE4s obtained by the mass spectrometry and the sugar chain structure. From the same viewpoint as described above, it is preferable to further include a step of comparing the relative intensity of the peak in the distribution with the relative intensity of the corresponding peak in the reference.
  • (1.2) Method for detecting HE4 sugar chain The method for detecting HE4 sugar chain according to the present invention is, as described above, in the first step, the mass analysis method or the residue of any one of the above four types of residues.
  • the lectin and HE4 may be included as a method for contacting the test sample with the first substance having an affinity for any one of the four residues.
  • a method of contacting a sample to detect and measure the amount of HE4 having an affinity for lectin will be described in detail. More specifically, the following analysis methods (A) and (B) can be mentioned.
  • (Judgment) analysis method hereinafter referred to as analysis method (A)
  • the analysis method (A) is (A) As the lectin of the present invention, for example, by contacting a lectin having an affinity for a ⁇ -N-acetylgalactosamine residue with a sample having a possibility of containing HE4, HE4 having an affinity for the lectin and the above-mentioned lectin A step of separating HE4 having no affinity for lectin (hereinafter, also referred to as “separation step (a)”), and (b) a step of determining the amount of HE4 having an affinity for lectin in the sample (hereinafter, also referred to as “separation step (a)”). , Also referred to as "judgment stage (b)”) including.
  • the method for separating HE4 having an affinity for lectin and HE4 having no affinity for lectin in the separation step (a) is not particularly limited as long as it is a method utilizing the affinity between lectin and HE4.
  • a lectin is bound to a carrier, and a sample that may contain HE4 is brought into contact with the lectin bound to the carrier (hereinafter, also referred to as "lectin affinity column” or simply “lectin column”) to bind to the lectin. This can be done by separating the HE4 that has been formed and the HE4 that does not bind to the lectin.
  • the carrier is not limited as long as it can bind lectins, and for example, cepharose, cellulose, agarose, dextran, polyacrylate, polystyrene, polyacrylamide, polymethacrylicamide, styrene and divinylbenzene.
  • Copolymers, polyamides, polyesters, polycarbonates, polyethylene oxides, hydroxypropylmethylcellulose, polyvinyl chlorides, polymethylacrylates, polystyrenes and polystyrene copolymers, polyvinyl alcohols, polyacrylic acids, collagens, calcium alginates, latexs, polysulfones, silica, zirconia, Acrylate, polystyrene and ceramics can be mentioned.
  • the shape of the carrier is not particularly limited, but a carrier having a shape such as particulate beads, a plate, or a gel can be used.
  • a carrier having a shape such as particulate beads, a plate, or a gel
  • the carrier preferably has a gel shape.
  • the lectin affinity column can be prepared according to a conventional method.
  • CNBr-activated Sepharose 4B can be used and coupled according to a protocol recommended by the manufacturer to prepare a lectin column.
  • the amount of lectin bound to the Sepharose gel is preferably 2 to 10 mg / mL.
  • the method of separating HE4 having an affinity for the lectin from HE4 having no affinity for the lectin using the lectin affinity column can be carried out according to a usual method for separating glycoproteins using the lectin affinity column.
  • the lectin affinity column is equilibrated with buffer before introducing a sample that may contain HE4.
  • As the equilibration buffer for example, 0.1% bovine serum albumin (BSA) -containing phosphate buffer or 0.1% bovine serum albumin (BSA) -containing Tris-hydrochloric acid buffer can be used.
  • a sample that may contain HE4 is added and allowed to stand for a period of time to bring the lectin into contact with HE4.
  • the contact time is not particularly limited and can be appropriately determined depending on the type of lectin and the affinity of HE4, but in consideration of the binding speed and efficiency, it is usually 15 to 30 minutes. ..
  • the temperature at which the lectin and HE4 are brought into contact with each other is not particularly limited and can be appropriately determined depending on the type of lectin and the affinity with HE4, but it is possible to prevent freezing of the column or to use a protein having no affinity for the lectin. Considering the prevention of formation of non-specific bonds, the temperature can be 0 ° C.
  • the temperature at which TJA-II and HE4 are brought into contact with each other is not particularly limited, but it is usually preferable to bring them into contact at 4 ° C. to 10 ° C.
  • the temperature at which WFA and HE4 are brought into contact with each other is not particularly limited, but it is usually preferable to bring them into contact at 4 ° C. to 10 ° C.
  • the unbound fraction to the lectin can be obtained by adding a wash buffer to the column and collecting the pass-through fraction.
  • the washing buffer is not limited as long as it is a buffer that allows the unbound component to flow out without dissociating the bond between the lectin and HE4, but for example, the buffer used for the equilibration. Can be used.
  • the volume of the washing buffer solution can be appropriately determined depending on the type of lectin and the affinity with HE4, but is preferably 3 to 7 times, more preferably about 5 times, the column volume.
  • the bound fraction to the lectin can be obtained by adding an elution buffer to the column and collecting the elution fraction.
  • the elution buffer contains a hapten sugar, which allows HE4 bound to the lectin to be separated from the lectin.
  • the hapten sugar can be appropriately selected depending on the specificity of the lectin, but in the case of TJA-II, lactose or the like can be used as the hapten sugar, for example, 10 mM lactose, 0.1% bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the eluted fraction can be recovered using the contained phosphate buffer.
  • GalNAc or the like can be used as a hapten sugar, and for example, an eluted fraction can be recovered using a phosphate buffer solution containing 10 mM GalNAc and 0.1% bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the volume of the elution buffer can be appropriately selected, but is preferably 3 to 7 times, more preferably about 5 times, the column volume.
  • the elution temperature is also not particularly limited, but can be 0 to 40 ° C., preferably 2 to 25 ° C., more preferably 4 to 20 ° C.
  • the temperature at which HE4 is eluted from TJA-II is not particularly limited, but it is preferably eluted at room temperature.
  • the temperature at which HE4 is eluted from WFA is also not particularly limited, but it is preferably eluted at room temperature.
  • the determination of the amount of HE4 having an affinity for lectin in the determination step (b) is (1) Judgment by measurement of the amount of HE4 having an affinity for the fractionated lectin, (2) Measurement of the amount of HE4 in the sample before fractionation and determination by measurement of HE4 having an affinity for the fractionated lectin, or (3) Measurement of the amount of HE4 in the sample before fractionation and the fractionated lectin Judgment by measurement of the amount of HE4 having no affinity for the above can be mentioned.
  • the determination (1) by measuring the amount of HE4 having an affinity for the separated lectin can be performed by quantitatively or semi-quantitatively measuring the amount of HE4 in the fraction bound to the lectin. That is, the determination is made by measuring the absolute amount of HE4 having an affinity for the lectin contained in the blood of the patient.
  • the determination (2) by measuring the amount of HE4 in the sample before fractionation and measuring the amount of HE4 having an affinity for the separated lectin is determined by the amount of HE4 in the sample before fractionation (or the binding image to the lectin). This can be done by comparing the amount of HE4 in the fraction bound to the lectin and the amount of HE4 in the fraction bound to the lectin. Specifically, the amount of HE4 in the sample bound to the lectin (or the total amount of the amount of HE4 in the fraction bound to the lectin and the amount of HE4 in the fraction not bound to the lectin) in the sample before sorting).
  • the determination (3) by measuring the amount of HE4 in the sample before fractionation and measuring the amount of HE4 having no affinity for the fractionated lectin is determined by the amount of HE4 in the sample before fractionation (or binding to the lectin). This can be done by comparing the amount of HE4 in the fraction and the non-binding fraction to the lectin) and the amount of HE4 in the non-binding fraction to the lectin.
  • HE4 bound to the lectin is separated from the lectin and the amount thereof is measured.
  • the amount of HE4 bound to the lectin is not bound to the amount of HE4 in the sample before fractionation.
  • the amount of HE4 bound to the lectin is not measured, so that the amount of HE4 bound to the lectin can be determined without separation.
  • the method for measuring HE4 for determining the amount of HE4 having an affinity for lectin is particularly limited as long as it is a method capable of quantitatively or semi-quantitatively determining HE4.
  • a total HE4 measuring method or a free HE4 measuring method can be used.
  • the total HE4 measurement method or the free HE4 measurement method is an immunological method using an antibody or a fragment thereof according to a conventional method (for example, enzyme immunoassay, latex agglutination immunoassay, chemoluminescence immunoassay, fluorescent antibody method, etc. It can be measured by radioimmunoassay, immunoprecipitation, immunohistological staining, Western blot, etc.), and a commercially available PSA measurement kit can also be used.
  • the lectin affinity column using MAM or WFA that can be used in the method for analyzing HE4 of the present invention can recover about 100% (at least 97% or more) of HE4 in the sample. It turned out to be possible.
  • the analysis method (B) is a method for detecting HE4 having an affinity for lectins directly with lectins, and specific examples thereof include lectin blots by electrophoresis and lectin blots by dot blots. Both lectin blots can be performed according to a conventional method, but in the case of a lectin blot by electrophoresis, a sample that may contain HE4 is electrophoresed, HE4 is transferred to a nitrocellulose membrane or a PVDF membrane, and used as a sample membrane. Use.
  • a sample that may contain HE4 is adsorbed on a nitrocellulose membrane, a PVDF membrane, or the like by a dot blotting device, and used as a sample membrane.
  • the sample membrane is blocked with a blocking buffer and contacted in a solution containing a biotin-labeled lectin, such as a biotin-labeled WFA or biotin-labeled TJA-II.
  • a color-developing enzyme or a luciferase for example, avidin labeled with HRP, and the color-developing solution or the luminescent solution is contacted, and the obtained signal is detected.
  • immunoblot and enzyme-linked immunosorbent assay can be partially modified.
  • the monoclonal antibody or polyclonal antibody against HE4 is immobilized on a nitrocellulose membrane or the like, an ELISA plate or the like, and blocking is performed with a blocking buffer.
  • a sample that may contain HE4 is contacted with a nitrocellulose membrane, ELISA plate, etc., and then with a biotin-labeled lectin, such as biotin-labeled WFA or biotin-labeled TJA-II.
  • a coloring enzyme or a luciferase for example, avidin labeled with HRP can be bound, and the signal can be detected by the coloring solution or the luminescent solution.
  • the test sample used in the method for analyzing HE4 of the present invention can be used, but a biopsy sample of blood, serum, plasma, ascites or ovary is preferable, and blood, serum, or plasma is particularly preferable. Is preferable.
  • HE4 having ⁇ -N-acetylgalactosamine residue and / or ⁇ 2,3 sialic acid residue and ⁇ 2,6 sialic acid residue in the blood, serum, or plasma of a healthy person and having these sugar chains in cancer patients This is because the content or ratio of HE4 is different.
  • Liquid samples such as urine, blood, serum, plasma, spinal fluid, saliva, and ascites are prepared in the analysis method (A) or analysis method (B) with an appropriate buffer solution according to each analysis method. It can be diluted and used. Further, a solid sample such as a cell, tissue, or organ is homogenized with an appropriate buffer solution having a volume of about 2 to 10 times the volume of the solid sample, and the suspension or its supernatant is prepared as described above. In the analysis method (A) or the analysis method (B), it can be used as it is or after being further diluted.
  • the suspension of the liquid sample or the solid sample or the supernatant thereof can be diluted with an appropriate buffer solution before use.
  • the dilution ratio is not particularly limited as long as the binding between the lectin and HE4 is not inhibited, but is preferably 2 to 400 times, more preferably 2 to 300 times, and most preferably 4 to 200 times.
  • the volume of the sample applied to the lectin affinity column is preferably 40% or less, more preferably 30% or less, and most preferably 20% or less of the bed volume of the column.
  • 400 ⁇ L or less is preferable, 300 ⁇ L or less is more preferable, and 200 ⁇ L or less is most preferable.
  • the sandwich-type assay according to the present invention is a sandwich-type assay for quantifying HE4 as a substance to be detected by using a labeled lectin and a capture substance immobilized on the measurement region, and is non-specific to the measurement region. Includes a treatment that suppresses the binding of the labeled lectin to the same sugar chain as the substance to be detected, which is contained in the contaminants adsorbed.
  • the "measurement region” is a region (space) in which the intensity of fluorescence emitted from the measurement region is measured, which will be described in detail later, and is an immobilized capture substance, a support supporting the capture substance, and a support. Furthermore, it refers to the area including the surface of the member on which they are placed.
  • the treatment for suppressing the binding of the labeled lectin to the same sugar chain as the substance to be detected, which is contained in the contaminants non-specifically adsorbed in the measurement region, is not particularly limited, and the type of the contaminants (glycoprotein or not? , Glycolipid), detection target substance, capture substance, labeled lectin, measurement system, etc. can be modified.
  • the present invention will be described mainly according to an embodiment in the case where the contaminant is HE4 having the same sugar chain as the substance to be detected, but the sugar having the same sugar chain as the substance to be detected. Even in the case of a substance other than a protein, for example, a glycolipid, the present invention can be carried out so that the desired action and effect can be obtained by applying the description.
  • the contaminants non-specifically adsorbed on the measurement region are glycoproteins having the same sugar chain as the substance to be detected.
  • the treatment for suppressing the binding of the labeled lectin to the sugar chain is a sugar protein in which a substance exhibiting an action of cleaving a disulfide bond (referred to as a “SS bond cleaving agent” in the present specification) is a contaminant. It is preferable that the treatment releases a domain containing the same sugar chain as the substance to be detected by contacting with.
  • the substance (SS bond cleaving agent) that exhibits the action of cleaving the disulfide bond of the contaminant is not particularly limited, but for example, the reducing agent, that is, the crosslinked structure of —S—S—is divided into two, ⁇ SH and HS—.
  • the reducing agent that is, the crosslinked structure of —S—S—is divided into two, ⁇ SH and HS—.
  • a compound having an action of reducing to a sulfhydryl group (thiol group) is preferable.
  • the reducing agent is not particularly limited, and can be selected from various reducing agents used for cleaving the disulfide bond of the protein.
  • a reducing agent include lower oxidates such as sulfite, hyposulfate, phosphorous acid and hypophosphorous acid, alkali hydroxide, and thiol salt as a nucleophile.
  • lower oxidates such as sulfite, hyposulfate, phosphorous acid and hypophosphorous acid
  • alkali hydroxide thiol salt as a nucleophile.
  • Hydrochloride tris (2-carboxyethyl) phosphine and the like can be used, and among these, sodium metabisulfite is particularly preferable.
  • a disulfide reductase disulfide reductase
  • disulfide reductase can be used in addition to the above compounds.
  • the analysis (measurement) method using column chromatography includes (a) a step of purifying HE4 from a sample derived from a sample, (b) a step of preparing a HE4 derivative from HE4 purified in the above step (a), and ( c) The step of labeling the HE4 derivative obtained in the step (b) and (d) the labeled HE4 derivative obtained in the step (c) of the step (c) using high performance liquid chromatography, chromatography such as capillary electroswimming.
  • step (1) includes a step of removing albumin in a serum sample using protein A agarose (PIERCE) or the like, a step of purifying HE4 protein using beads or the like equipped with an anti-HE4 antibody, and the like. Be done.
  • step (b) there is a step of cleaving the sugar chain and HE4 protein using HE4 protein proteinase K, thermolysin, etc. recovered in step (a).
  • step (c) there is a method of labeling the HE4 derivative prepared in step (b) with a compound that binds to an aldehyde group of a sugar chain such as BOA, PA, 2-AA, 2-AB. It is also preferable to attach the lectin affinity column (also referred to as “lectin column”) to a high performance liquid chromatography device for measurement.
  • lectin affinity column also referred to as “lectin column”
  • Mass spectrometry> It includes a step of analyzing a HE4 derivative prepared in the same manner as in the steps (a) to (b) of the column chromatography method by mass spectrometry.
  • mass spectrometry method LC-MS, MALDI-ToF-MS, GC-MS and the like can be used.
  • mass spectrometry the following method can be carried out.
  • mass spectrometry after ionizing the sample to be measured, various generated ions are sent to a mass spectrometer, and the ions are generated for each mass-to-charge ratio m / z, which is the ratio of the mass number m and the valence z of the ion. Measure the strength.
  • the mass spectrum obtained as a result consists of the measured ionic strength peaks (ion peaks) for each mass-to-charge ratio m / z value.
  • Mass spectrometry of the ionized sample itself in this way is called MS1.
  • MS1 Mass spectrometry of the ionized sample itself in this way.
  • MSn + 1 mass spectrometry of the dissociated ion species by dissociating the parent ion in n stages.
  • the parent ion is dissociated in multiple stages (1st stage, 2nd stage, ..., N stages) and the mass number of the ion species generated in each stage is analyzed (MS2, MS3, ... , MSn + 1).
  • SPFS surface plasmon-field enhanced fluorescence spectroscopy
  • ATR total reflection attenuation
  • the measuring member for SPFS is generally used for forming a sandwich-type immune complex, a sensor chip in which a field (measurement region) for forming a sandwich-type immune complex and performing fluorescence measurement by SPFS is formed, and the like.
  • the sensor chip basically includes a transparent support for introducing excitation light into the back surface of the metal thin film, a metal thin film formed on the transparent support for generating surface plasmon resonance, and the metal thin film.
  • the phosphor formed above including the reaction layer for capturing the analyze on the sensor surface, and optionally, formed between the metal thin film and the reaction layer, is too close to the metal thin film. It may include a spacer layer to prevent the metal dimming of the fluorescence caused by the above.
  • the site where the reaction layer is formed corresponds to the measurement area.
  • a reaction layer may be formed over the entire bottom surface of the flow path or well to serve as a measurement region, or a reaction layer may be formed only on a part of the bottom surface (with desired patterning if necessary) to serve as a measurement region.
  • the area of the measurement area can be adjusted while considering the irradiation area of the excitation light generally irradiated as the laser light. For example, if the spot diameter of the excitation light is about 1 mm ⁇ , the assay area is usually designed to have an area of at least several mm square.
  • a measuring member is constructed by loading a "top plate” with a liquid feed inlet and a liquid feed discharge port at a position corresponding to the hole of the flow cell and fixing them in close contact with each other. To do.
  • the surface of the sensor chip at the position corresponding to the hole of the flow cell forms the bottom surface of the flow path, and the measurement region is formed there.
  • liquids can be sent into the flow path from the liquid feed inlet and discharged from the liquid feed discharge port by using a liquid feed means including a pump or a tube, if necessary. It is also possible to perform reciprocating type and circulation type liquid feeding. Conditions such as liquid feed rate and liquid feed (circulation) time include the amount of sample, the concentration of allite in the sample, the size of the flow path or well, the mode of the reaction layer (density of immobilized ligand, etc.), and the pump It can be adjusted as appropriate while considering the performance and the like.
  • the SPFS system is a "well type” in which various solutions are stored in a space wider than the above flow path
  • a "well member” having a through hole for forming a well is provided on the sensor chip.
  • the measuring member is constructed by loading and fixing.
  • various liquids can be added to and removed from the well using, for example, a pipette-shaped member.
  • the flow cell can be made of, for example, a sheet of polydimethylsiloxane (PDMS).
  • the top plate is made of a transparent material so that the fluorescence emitted from the measurement region can be measured, and can be made of, for example, a plate-shaped polymethyl methacrylate (PMMA).
  • PMMA polymethyl methacrylate
  • the flow cell and the top plate can be made of plastic having a desired shape by molding or photolithography.
  • the means for closely contacting and fixing the flow cell or well member on the sensor chip is not particularly limited, and generally, pressure may be physically applied from above and below, and if necessary, a transparent support. Adhesives, matching oils, transparent adhesive sheets, etc. having the same light refractive index as above may be used.
  • the measuring method according to the present invention can be carried out using a general measuring device for SPFS.
  • the SPFS measuring member is basically removable, and a light source and excitation for irradiating excitation light (preferably laser light) having an appropriate wavelength according to the phosphor to be used.
  • a prism for incident light on the back surface of the metal thin film of the sensor chip at a predetermined angle receives the light reflected by the metal thin film and measures its intensity.
  • a light receiver a lens for condensing fluorescence emitted from a phosphor, a detector for measuring the intensity of the fluorescence, and only light having a predetermined wavelength is transmitted from excitation light and fluorescence to cut other light. It is equipped with various filters for this purpose.
  • FIG. 1 is a schematic view schematically showing an outline of a quantitative measurement device for explaining the method for quantitative measurement of Anna Reed of the present invention
  • FIG. 2 is a partially enlarged view of the quantitative measurement device of FIG.
  • the quantitative measurement device 10 of the present invention is formed on a prism-shaped dielectric member 12 having a substantially trapezoidal vertical cross-sectional shape and a horizontal upper surface 12a of the dielectric member 12.
  • a sensor chip 16 having a metal film 14 is provided, and the sensor chip 16 is loaded in the sensor chip loading unit 18 of the quantitative measurement device 10.
  • a light source 20 is arranged on the side of one side surface 12b below the dielectric member 12, and the incident light 22 from the light source 20 is emitted from the outer lower side of the dielectric member 12. , It is incident on the side surface 12b of the dielectric member 12 and is irradiated toward the metal film 14 formed on the upper surface 12a of the dielectric member 12 via the dielectric member 12.
  • a polarizing filter for converting the laser light emitted from the light source 20 into P-polarized light that efficiently generates surface plasmons on the metal film 14 can be provided between the light source 20 and the dielectric member 12. ..
  • a light receiving means 26 for receiving the metal film reflected light 24 in which the incident light 22 is reflected by the metal film 14 is provided on the side of the other side surface 12c below the dielectric member 12. ing.
  • the light source 20 is provided with incident angle adjusting means (not shown) that allows the incident light 22 emitted from the light source 20 to appropriately change the incident angle ⁇ 1 with respect to the metal film 14.
  • the light receiving means 26 is also provided with a movable means (not shown), and even when the reflection angle of the metal film reflected light 24 changes, the metal film reflected light 24 is reliably received in synchronization with the light source 20. It is configured to do.
  • the sensor chip 16, the light source 20, and the light receiving means 26 constitute an SPR measuring unit 28 that measures SPR (Surface plasmon resonance) of the quantitative measuring device 10 of the present invention. Further, above the sensor chip 16, a photodetecting means 32 for receiving the fluorescence 30 emitted by being excited by a fluorescent substance as described later is provided.
  • a cut filter, a condenser lens, or the like may be provided between the sensor chip 16 and the light detection means 32, for example.
  • the sensor chip 16, the light source 20, and the photodetector 32 constitute an SPFS measurement unit 34 that performs SPFS measurement of the quantitative measurement device 10 of the present invention.
  • the light receiving means 26 and the light detecting means 32 are each connected to the quantitative calculation means 40, and the amount of light of the metal film reflected light 24 received by the light receiving means 26 and the amount of light of the fluorescence 30 received by the light detecting means 32. Is configured to be transmitted to the quantitative calculation means 40.
  • the fine flow path 36 is formed on the upper surface 14a of the metal film 14.
  • a sensor unit 38 on which a molecule (ligand) that specifically binds to the detection target antigen (analyte) is immobilized is provided in a part of the microchannel 36.
  • the pathological information generation system of the present invention is a pathological information generation system that generates information on the onset or onset of cancer, and contains ⁇ -N-acetylgalactosamine residues, which are contained in a test sample.
  • a second step unit that generates information on the morbidity or onset of cancer of the subject according to the test sample, based on the amount of HE4 having any of the four residues. It is characterized by having.
  • the system is for carrying out the above-mentioned pathological condition information generation method of the present invention.
  • the HE4 sugar chain analysis kit of the present invention is the HE4 sugar chain analysis kit of the present invention, which is a HE4 sugar chain analysis kit containing HE4 having a sugar chain and an anti-HE4 antibody. It is an analysis kit for carrying out the above-mentioned method for generating pathological condition information.
  • ⁇ -N-acetylgalactosamine residue, ⁇ 2,3-sialic acid residue, at the (non-reducing) end of the sugar chain It is characterized by containing a substance having an affinity for HE4 having an ⁇ 2,6-sialic acid residue or a mannose residue and an anti-HE4 antibody.
  • the substance having an affinity for HE4 is any lectin selected from the whole of the lectin groups (1) to (4) from the viewpoint of exhibiting the effect of the present invention. Further, it is preferable that the substance having an affinity for HE4 is two or more kinds of lectins selected from a plurality of groups among the lectins groups (1) to (4).
  • the HE4 sugar chain analysis kit of the present invention preferably has a configuration in which the lectin, the anti-HE4 antibody, and the analysis substrate are combined from the viewpoint of performing accurate measurement. Further, it is preferable that the lectin is provided with a phosphor. In addition, it preferably contains at least one lectin of WFA or MAM equipped with a phosphor.
  • the configuration is provided with a chromatography device from the viewpoint of accuracy and convenience. Further, it is preferable that the configuration is provided with a labeling reagent for chromatographic measurement.
  • FIG. 3 shows a schematic diagram of an example of the HE4 sugar chain analysis kit of the present invention.
  • the HE4 sugar chain analysis kit 50 of the present invention at least the sample holding well 51, the sample collecting well 52, the drug well 53, and the sample chip 54 are connected and integrated as shown in FIG. , It is possible to detect while sending a liquid such as an injected sample.
  • the detection cartridge disclosed in International Publication No. 2017/130369 can be referred to.
  • the drug recovery well 53 has an affinity for HE4 having a ⁇ -N-acetylgalactosamine residue, an ⁇ 2,3-sialic acid residue, an ⁇ 2,6-sialic acid residue, or a high mannose residue at the end of the sugar chain.
  • a sexual substance or a substance having an affinity for a HE4 peptide such as a HE4 antibody can be retained in the well. Both the substance having an affinity for the HE4 sugar chain and the HE4 antibody may be provided in the drug well.
  • the HE4 sugar chain analysis kit of the present invention can be preferably applied as a kit for carrying out the method for detecting the HE4 sugar chain of the present invention.
  • the pathological information generation method of the present invention is a pathological information generation method that generates information on the onset or onset of cancer, and is a ⁇ -N-acetylgalactosamine residue, ⁇ 2, contained in a test sample.
  • the pathological information generation method of the present invention is a cancer test method for detecting HE4 sugar chains in order to diagnose or predict the onset of cancer, and quantification of HE4 sugar chains having sugar chains having an affinity for a specific lectin. It is characterized in that a subject evaluates whether or not a patient has ovarian cancer based on the value or the ratio of the quantified value of the sugar chain to the quantified value of all HE4. It is also characterized by assessing the malignancy or stage of cancer in ovarian cancer patients.
  • the possibility of ovarian cancer and the degree of malignancy can be differentiated.
  • the patient has ovarian cancer. It is possible to determine whether or not there is. More specifically, the patient has an ovarian ovary when the lectin-affinitive HE4 present in the patient's sample is significantly higher than the lectin-affinitive HE4 present in the healthy subject's sample. It can be determined that it is cancer.
  • Example 1 Quantification of GalNAcHE4 by the lectin column method
  • a WFA column using WFA as a lectin was prepared, and a patient diagnosed with ovarian cancer and a healthy person who was a non-cancer sample,
  • the amount of HE4 (abbreviated as "GalNAcHE4") having a ⁇ -N-acetylgalactosamine residue at the end of the sugar chain was measured by the HE4 sugar chain analysis method according to the present invention.
  • the total HE4 amount of the serum sample before sorting by the WFA column, the WFA unbound fraction, and the WFA bound fraction was measured using ARCHITEC T i1000SR (manufactured by Abbott).
  • the HE4 recovery rate from the WFA column was always 97% to 100%.
  • Table I shows the total He4 amount before sorting, the WFA binding ratio (GalNAcHE4 ratio), and the grade of ovarian cancer patients.
  • the WFA binding rate was calculated by the following formula.
  • WFA binding rate (HE4 amount in WFA-bound fraction / (HE4 amount in WFA-bound fraction + HE4 amount in WFA non-bonded fraction) x 100%
  • the WFA binding rate indicates the terminal GalNAc sugar chain HE4 with respect to the total HE4 in the sample.
  • total HE4 represents the concentration of HE4 protein including all sugar chain structures.
  • Example 2 Quantification of HE4 having various sugar chains by the lectin column method (culture of cancer cells) Ovarian cancer cells (cell number JCRB1046, cell name OVSAHO) purchased from the JCRB cell bank purchase were cultured. Add OVSAHO cell suspension to RPMI 1640 Medium 11875-093 20 mL to a concentration of 2.7-4.2 ⁇ 10 5 cells / mL and add to Nunc cell culture treated EasyFlask at 36.7 ° C. , CO 25% concentration environment for 3 days. The cell culture supernatant was collected to prepare an ovarian cancer cell culture supernatant.
  • HE4 having a sugar chain of ⁇ -N-acetylgalactosamine residue has changed. It can also be seen that the content ratio (secretion amount) of ⁇ 2,3-sialic acid and ⁇ 2,6-sialic acid increases.
  • HE4 having a sugar chain of ⁇ -N-acetylgalactosamine residue is cancerous or non-cancerous because the change in the content ratio is relatively remarkable compared to HE4 having a sugar chain of other species of residues. It is especially useful in differentiating cancers.
  • Example 3 Measurement by surface plasmon excitation enhanced fluorescence spectroscopy
  • SPFS surface plasmon excitation enhanced fluorescence spectroscopy
  • a chromium film was first formed on one side of the plasma-cleaned substrate by a sputtering method, and then a gold film was formed on the surface thereof by a sputtering method.
  • the thickness of the chromium film was 1 to 3 nm, and the thickness of the gold film was 44 to 52 nm.
  • the substrate thus obtained was immersed in 10 mL of an ethanol solution of 10-carboxy-1-decanthyl adjusted to 25 mg / mL for 24 hours, and a self-assembled monolayer (Self-Assembled) was placed on the surface of the gold film.
  • Monolayer; SAM was formed.
  • the substrate was removed from the ethanol solution, washed sequentially with ethanol and isopropanol, and then dried using an air gun.
  • CMD carboxymethyl dextran
  • NHS N-hydroxysuccinimide
  • WSC water-soluble carbodiimide
  • a sheet-shaped silicon rubber spacer having a thickness of 0.5 mm and having a hole of 2 mm ⁇ 14 mm is placed.
  • the substrate is provided so that the surface is inside the flow path (however, the silicon rubber spacer is in a state where it does not come into contact with the liquid feed), and the thickness is 2 mm so as to cover the substrate from the outside of the flow path.
  • a polymethyl methacrylate plate was placed and crimped, and the flow path and the polymethyl methacrylate plate were fixed with screws.
  • Ultrapure water was circulated as a liquid feed for 10 minutes, and then PBS was circulated for 20 minutes at room temperature and a flow rate of 500 ⁇ L / min by a perista pump. Subsequently, 5 mL of PBS containing 50 mM of NHS and 100 mM of WSC was sent and circulated for 20 minutes, and then 2.5 mL of an anti-HE4 monoclonal antibody (3C24; manufactured by hytest) was circulated for 30 minutes. By doing so, the primary antibody was immobilized on the immobilized layer having a three-dimensional structure. Finally, a plasmon sensor was prepared by performing nonspecific adsorption prevention treatment by circulating the solution in PBS buffered saline containing 1% bovine serum albumin [BSA] for 30 minutes.
  • BSA bovine serum albumin
  • Serum samples derived from patients suspected of having cancer and serum samples derived from patients who received a definitive diagnosis of cancer were diluted 3-54 times with a phosphate buffer solution to prepare serum samples.
  • Second washing step Tris-buffered saline containing 0.05% by mass of Tween® 20 was circulated for 10 minutes.
  • Blank measurement step With the flow path filled with Tris-buffered physiological saline, the amount of photons was adjusted by an optical filter with a laser beam having a wavelength of 635 nm, and the metal thin film was irradiated from the back surface of the plasmon excitation sensor through a prism.
  • a CCD image sensor (Texas Instrument Co., Ltd.) equipped with an objective lens with a magnification of 20 times installed at the top of the measurement area measured the intensity of light that passed through a filter that cuts wavelengths other than fluorescent components. ..
  • First reaction step The prepared serum sample was circulated and sent to the flow path in which the anti-HE4 antibody-immobilized region was formed for 30 minutes.
  • Second washing step Tris-buffered saline containing 0.05% by mass of Tween® 20 Second reaction step: The prepared labeled MAM solution was circulated for 10 minutes.
  • Tris-buffered saline containing 0.05% by mass of Tween (registered trademark) 20 was circulated for 10 minutes.
  • Signal measurement step With the flow path filled with Tris-buffered saline, the amount of photons was adjusted with an optical filter, and the metal thin film was irradiated from the back surface of the plasmon excitation sensor through a prism.
  • a CCD image sensor (Texas Instrument Co., Ltd.) equipped with an objective lens with a magnification of 20 times installed at the top of the measurement area measured the intensity of light that passed through a filter that cuts wavelengths other than fluorescent components. (Signal value). This measured value is applied to a sugar chain (sia2,3-sugar chain) having an ⁇ 2,3-sialic acid residue (hereinafter, abbreviated as “sia2,3”) existing on HE4 bound to the HE4 antibody.
  • Fluorescence emitted by a phosphor labeled with the bound MAM which is obtained by subtracting the amount of fluorescent light obtained in the blank measurement step from the amount of fluorescent light obtained in the signal measurement step and multiplying the value obtained by diluting the sample by the magnification of sia2.
  • a quantitative value of the amount of 3-sugar chain (relative fluorescence intensity value; unit: pW) was used.
  • the numerical values shown in the columns of Examples (sia2,3-HE4) in Tables III and IV are the fluorescence intensity values.
  • ROC curve calculation method The ROC curves obtained from the results shown in Tables III and IV are shown in FIGS. 5, 6 (A, B, C) and 7 (A, B, C).
  • cancer sample and non-cancer sample including healthy person and endometriosis patient
  • cancer patient under 50 years old and non-cancer sample healthy person and endometriosis patient
  • ROC curves obtained by comparing grade 1 cancer specimens and non-cancer specimens are shown in FIGS. 5 to 7, respectively.
  • Table V summarizes the area under the curve (ROC-AUC) and the like of the ROC curve determined based on the figure.
  • the ROC curve line (also referred to as “receiver operating characteristic curve”) shown here is used when the false positive rate is plotted on the horizontal axis and the sensitivity is plotted on the vertical axis, and the threshold value is used as a parameter to change from large to small. It is a curve to be drawn. At first, it is difficult to capture both positive and negative persons, so both take small values, but the sensitivity gradually increases, and the false positive rate increases later. Ultimately, both sensitivity and false positives will be 100%. If the biomarker is competent, the ROC curve deviates to the upper left from a 45 degree tilt. That is, the area under the curve (ROC-AUC) of the ROC curve represents the performance of the biomarker.
  • ROC-AUC area under the curve
  • the area under the ROC curve based on the evaluation result of ⁇ 2,3-sialic acid HE4 is the largest as compared with total HE4 and ROMA. It can be seen that such ⁇ 2,3-sialic acid is an excellent biomarker.
  • the area under the curve (ROC-AUC) of the ROC curve exceeded 0.7, and the high sensitivity and specificity at the optimum threshold value of the biomarker of the present invention. It shows that the diagnostic performance is high.
  • FIG. 8 shows the relationship between the amount of HE4 protein having an ⁇ 2,3-sialic acid residue terminal and the grade of ovarian cancer (healthy subjects, G1, G2, and G3) obtained from the measurement results of Example 3. Shown.
  • the Y-axis is plotted as a logarithm of the ⁇ S value, which is a quantitative value of ⁇ 2,3-sialic acid. From the obtained results, it can be seen that the higher the grade of ovarian cancer, the higher the amount of HE4 having the ⁇ 2,3-sialic acid residue terminal.
  • the sugar chain structure content of the HE4 protein has changed, and it can be seen that the amount of HE4 having a sugar chain having an ⁇ 2,3-sialic acid residue correlates with the grade of ovarian cancer in the diagnosis of ovarian cancer.
  • HE4 with can be a biomarker for ovarian cancer. Therefore, according to the present invention, it is possible to provide a pathological condition information generation method and a pathological condition information generation method system that generate information on the morbidity or onset of cancer including ovarian cancer. That is, since HE4 expressed in association with ovarian cancer can be effectively detected by a simple and inexpensive method, early detection, diagnosis and treatment of ovarian cancer become possible. Further, according to the method of the present invention, ovarian cancer can be detected minimally invasively using patient blood, so that ovarian cancer can be detected easily and quickly.
  • the present invention can be used in the medical field.
  • Quantitative measurement device Dielectric member 12a Top surface 12b Side surface 12c Side surface 14 Metal film 14a Top surface 16 Sensor chip 18 Sensor chip loading unit 20 Light source 22 Incident light 24 Metal film reflected light 26 Light receiving means 28 SPR measuring unit 30 Fluorescence 32 Light detection means 34 SPFS measurement unit 36 Microchannel 38 Sensor unit 40 Quantitative calculation means 50 HE4 Sugar chain analysis kit 51 Specimen holding well 52 Specimen collection well 53 Drug well 54 Specimen chip

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Sustainable Development (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention aborde le problème consistant à fournir un procédé de génération d'informations d'état pathologique, de manière à pouvoir évaluer de manière aisée et non invasive l'acquisition, l'apparition, le grade malin, etc. du cancer tel que le cancer de l'ovaire et, par conséquent, de pouvoir diagnostiquer le cancer tout en réduisant les effets néfastes tant pour un patient que pour un médecin; un système pour générer des informations de condition pathologiqu ; et un kit d'analyse de la chaîne de sucre HE4.　La présente invention porte sur un procédé pour générer des informations d'état pathologique, ledit procédé étant destiné à générer des informations concernant l'acquisition ou l'apparition d'un cancer, est caractérisé en ce qu'il comprend une première étape consistant à acquérir des informations relatives à la quantité de HE4 qui a l'un quelconque des quatre types de résidus, c'est-à-dire, un résidu de β-N-acétylgalactosamine, un résidu d'acide α2,3-sialique, un résidu d'acide α2,6-sialique et un résidu de mannose, contenu dans un échantillon d'essai; et une seconde étape pour générer des informations concernant l'acquisition ou l'apparition d'un cancer chez le sujet, qui utilise l'échantillon d'essai, en fonction de la quantité de HE4 qui comprend l'un quelconque des quatre types de résidus décrit ci-dessus
PCT/JP2020/013080 2019-04-02 2020-03-24 Procédé et système de génération d'informations de condition pathologique, kit d'analyse des chaînes de sucre he4 et he4 WO2020203478A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2021511506A JPWO2020203478A1 (fr) 2019-04-02 2020-03-24

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2019-070727 2019-04-02
JP2019070727 2019-04-02

Publications (1)

Publication Number Publication Date
WO2020203478A1 true WO2020203478A1 (fr) 2020-10-08

Family

ID=72669109

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2020/013080 WO2020203478A1 (fr) 2019-04-02 2020-03-24 Procédé et système de génération d'informations de condition pathologique, kit d'analyse des chaînes de sucre he4 et he4

Country Status (2)

Country Link
JP (1) JPWO2020203478A1 (fr)
WO (1) WO2020203478A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009195240A (ja) * 2001-08-29 2009-09-03 Pacific Northwest Research Inst 癌腫の診断
WO2010090264A1 (fr) * 2009-02-04 2010-08-12 国立大学法人東京工業大学 Procédé d'analyse de l'aps, et procédé pour différencier un cancer de la prostate d'une prostatomégalie employant le procédé d'analyse
JP2014507153A (ja) * 2011-02-17 2014-03-27 フジレビオ ダイアグノスティックス インコーポレイテッド ＨＥ４ａの測定のための組成物および使用方法
US20150031561A1 (en) * 2011-02-24 2015-01-29 Vermillion, Inc. Biomarker panels, diagnostic methods and test kits for ovarian cancer
WO2015097863A1 (fr) * 2013-12-27 2015-07-02 コニカミノルタ株式会社 Méthode d'analyse d'informations pour réaliser un diagnostic et son kit
JP2018520353A (ja) * 2015-07-03 2018-07-26 トゥルン イリオピスト 婦人科疾患、特に卵巣上皮がんの診断法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009195240A (ja) * 2001-08-29 2009-09-03 Pacific Northwest Research Inst 癌腫の診断
WO2010090264A1 (fr) * 2009-02-04 2010-08-12 国立大学法人東京工業大学 Procédé d'analyse de l'aps, et procédé pour différencier un cancer de la prostate d'une prostatomégalie employant le procédé d'analyse
JP2014507153A (ja) * 2011-02-17 2014-03-27 フジレビオ ダイアグノスティックス インコーポレイテッド ＨＥ４ａの測定のための組成物および使用方法
US20150031561A1 (en) * 2011-02-24 2015-01-29 Vermillion, Inc. Biomarker panels, diagnostic methods and test kits for ovarian cancer
WO2015097863A1 (fr) * 2013-12-27 2015-07-02 コニカミノルタ株式会社 Méthode d'analyse d'informations pour réaliser un diagnostic et son kit
JP2018520353A (ja) * 2015-07-03 2018-07-26 トゥルン イリオピスト 婦人科疾患、特に卵巣上皮がんの診断法

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
COOLING, L. L. W. ET AL.: "Trends in Glycoscience and Glycotechnology", GLYCOSPHINGOLIPIDS, vol. 9, no. 46, 1997, pages 191 - 209 *
DRAPKIN, R. ET AL.: "Human epididymis protein 4 (HE4) is a secreted glycoprotein that is overexpressed by serous and endometrioid ovarian carcinomas", CANCER RESEARCH, vol. 65, no. 6, 15 March 2005 (2005-03-15), pages 2162 - 2169, XP002600809, DOI: 10.1158/0008-5472.CAN-04-3924 *
HATAKEYAMA, SHINGO, BASIC KNOWLEDGE OF SUGAR CHAINS, 29 March 2017 (2017-03-29), Retrieved from the Internet <URL:http://web.archive.org/web/20170329165338/http://www.med.hirosaki-u.ac.jp/~uro/html/Research-Publications/Glyco_info_Hatakeyama.pdf> [retrieved on 20200615] *
HUA, L. ET AL.: "Expression and biochemical characterization of recombinant human epididymis protein 4", PROTEIN EXPRESSION AND PURIFICATION, vol. 102, 2014, pages 52 - 62, XP055745448 *
INUI, CHIE ET AL.: "Development of Novel Glycoprotein Biomarker for Ovarian Cancer Diagnosis with Surface Plasmon field-enhanced Fluorescence Spectroscopy (SPFS", KONICA MINOLTA TECHNOLOGY REPORT, vol. 17, January 2020 (2020-01-01), pages 76 - 80, XP029053855 *
NARIMATSU, M. K. ET AL., FUCOSYLTRANSFERASE FAMILY, 15 May 2001 (2001-05-15), Retrieved from the Internet <URL:https://www.glycoforum.gr.jp/glycoword/glycoprotein/GPB02J.html> [retrieved on 20200612] *
ZHUANG, H. Y. ET AL.: "Co-expression of Lewis y antigen with human epididymis protein 4 in ovarian epithelial carcinoma", PLOS ONE, vol. 8, no. 7, 2013, pages e68994, XP055745449 *
ZHUANG, H. Y. ET AL.: "Overexpression of Lewis y antigen promotes human epididymis protein 4- mediated invasion and metastasis of ovarian cancer cells", BIOCHIMIE, vol. 105, 2014, pages 91 - 98, XP029053855, DOI: 10.1016/j.biochi.2014.06.022 *

Also Published As

Publication number Publication date
JPWO2020203478A1 (fr) 2020-10-08

Similar Documents

Publication Publication Date Title
US11988663B2 (en) Compositions and methods for the detection and molecular profiling of membrane bound vesicles
JP6520940B2 (ja) 標識化レクチンを用いるサンドイッチ型アッセイおよびそのためのキット
JP6260541B2 (ja) 夾雑物の影響を低減する免疫測定法
EP1934607A1 (fr) Analyse immunohistochimique multiplexee in situ
US11162940B2 (en) Method for suppressing nonspecific signals from contaminants in an immunoassay using surface plasmon-field enhanced fluorescence spectroscopy (SPFS)
EP3550304B1 (fr) Procédé d&#39;estimation du score de gleason du cancer de la prostate, procédé d&#39;estimation de classification des stades pathologiques, et procédé d&#39;acquisition d&#39;informations auxiliaires, sur la base de la teneur en antigène prostatique spécifique dans un échantillon
JP6323463B2 (ja) 診断用情報の分析方法およびそのためのキット
JP5991032B2 (ja) レクチンを用いたアナライトの検出方法
JP6729595B2 (ja) 前立腺癌の病理組織診断結果（グリーソンスコア）の推定方法
WO2020203478A1 (fr) Procédé et système de génération d&#39;informations de condition pathologique, kit d&#39;analyse des chaînes de sucre he4 et he4
JP6414078B2 (ja) 診断用情報の分析方法およびそのためのキット
JP5221825B1 (ja) 肺扁平上皮癌の検出方法
WO2022230991A1 (fr) Procédé de détection d&#39;une lésion néoplasique orale, réactif d&#39;essai, kit d&#39;essai et composition thérapeutique
JP7412344B2 (ja) 補助情報の取得方法
WO2021111820A1 (fr) Substance standard pour la quantification de psa, son procédé de préparation, solution standard pour la quantification de psa et procédé de quantification de psa
US20230324376A1 (en) Compositions and methods for the detection and molecular profiling of membrane bound vesicles with nanoparticles

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20784422

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021511506

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20784422

Country of ref document: EP

Kind code of ref document: A1