WO2021261483A1 - 腺癌の検出方法及び検査キット - Google Patents

腺癌の検出方法及び検査キット Download PDF

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WO2021261483A1
WO2021261483A1 PCT/JP2021/023578 JP2021023578W WO2021261483A1 WO 2021261483 A1 WO2021261483 A1 WO 2021261483A1 JP 2021023578 W JP2021023578 W JP 2021023578W WO 2021261483 A1 WO2021261483 A1 WO 2021261483A1
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antibody
fragment
terminal
seq
value
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French (fr)
Japanese (ja)
Inventor
駒子 石川
仰 天野
敏文 高尾
宣明 奥村
俊樹 武居
雅光 中里
拡伸 坪内
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Mitsui Chemicals Inc
University of Miyazaki NUC
University of Osaka NUC
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Mitsui Chemicals Inc
Osaka University NUC
University of Miyazaki NUC
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Priority to EP21829606.9A priority Critical patent/EP4169941A4/en
Priority to US18/002,344 priority patent/US20230243835A1/en
Priority to JP2022532492A priority patent/JPWO2021261483A1/ja
Publication of WO2021261483A1 publication Critical patent/WO2021261483A1/ja
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • 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
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57545—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the ovaries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • G01N33/70—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving creatine or creatinine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7023—(Hyper)proliferation
    • G01N2800/7028—Cancer

Definitions

  • the present invention relates to a method for detecting adenocarcinoma and a test kit.
  • Adenocarcinoma is one of the epithelial malignancies that develops from the cells of secretory glandular tissue. It can occur in any organ of the body, including lung adenocarcinoma, liver adenocarcinoma, pancreatic adenocarcinoma, lymph adenocarcinoma, uterine adenocarcinoma, spermatic adenocarcinoma, and gastric adenocarcinoma.
  • lung adenocarcinoma is one of the adenocarcinomas whose initial symptoms are difficult to detect and early detection is difficult.
  • Lung adenocarcinoma has the highest incidence among the histological types of lung cancer, and lung adenocarcinoma accounts for about 40% of male lung cancers, about 70% of females, and about 50% of all lung cancers. Since lung cancer has the highest number of malignant tumor deaths in Japan, there is a need for a technique for early detection of lung adenocarcinoma.
  • the survival rate of lung cancer decreases as the clinical stage progresses.
  • the 5-year survival rates are 82.0%, 66.1%, 54.5% and 46.1%, respectively.
  • 42.8% Japanese lung cancer registry study of 11,663 surgical cases in 2004: demographic and prognosis changes over decade", J.Thorac.Oncol., 6 ( 7), p.1229-1235
  • the median survival time is 22.4 months (Atagi, S., et al. (2012), respectively.
  • Carcinoembryonic antigen (CEA) in serum is used for clinical diagnosis as an existing tumor marker for lung adenocarcinoma.
  • CEA Carcinoembryonic antigen
  • the positive rate of CEA in lung adenocarcinoma cases is only 36.6 to 56.5% (Molina, R., et al. (2016): "Assessment of a Combined Panel of Six Serum Tumor Markers for Lung Cancer". ", Am.J.Respir.Crit.CareMed., 193 (4), doi.org/10.1164/rccm.201404-0603OC, Matsuoka, K., et al.
  • the method for obtaining the protein fragmentation rate (Fn) by the MRM method embodied in the invention described in Patent Document 1 is usually 2 to 2 to pretreatment of the sample until it is subjected to measurement. It takes three days, and it requires frequent manual work. Not only is it prone to errors, but it also has the drawback of being able to quickly inspect multiple samples (hereinafter referred to as "high throughput"). It was found that there is a problem that it is difficult to put it into clinical practical use.
  • the present inventors attempted to detect lung adenocarcinoma by detecting a protein fragment peculiar to lung adenocarcinoma by the sandwich method.
  • the means for solving the above-mentioned problems include the following aspects.
  • ⁇ 1> A method for detecting adenocarcinoma based on a protein fragment of WFDC2 protein in a sample derived from a subject, which is the amount of a protein fragment having the amino acid sequence of SEQ ID NO: 1 obtained by the sandwich method in the sample.
  • the first determination value which is a value obtained by dividing the amount of one fragment by the total WFDC2 protein mass in the sample obtained by the sandwich method or the reference amount defined by the creatinine concentration, with a predetermined threshold value.
  • a method for detecting adenocarcinoma to determine the presence or absence of adenocarcinoma.
  • ⁇ 2> N from the first anti-C-terminal antibody that specifically binds to the C-terminal region of the protein fragment having the amino acid sequence of SEQ ID NO: 1 and the C-terminal region of the protein fragment having the amino acid sequence of SEQ ID NO: 1.
  • the method for detecting adenocarcinoma according to ⁇ 1>, wherein the amount of the protein fragment measured using the anti-N-terminal region antibody that recognizes the terminal side is the amount of the first fragment in the sample.
  • the amount of the second fragment which is the amount of the protein fragment having the amino acid sequence of SEQ ID NO: 2 obtained by the sandwich method in the sample, is divided by the reference amount to obtain the protein fragment.
  • the method for detecting adenocarcinoma according to ⁇ 1> or ⁇ 2> which uses a modified determination value which is the sum of a second determination value which is a value and a value obtained by multiplying the first determination value by 5 to 100.
  • N from the second anti-C-terminal antibody that specifically binds to the C-terminal region of the protein fragment having the amino acid sequence of SEQ ID NO: 2 and the C-terminal region of the protein fragment having the amino acid sequence of SEQ ID NO: 2.
  • the amount of the protein fragment measured using the anti-N-terminal region antibody that recognizes the terminal side is defined as the amount of the second fragment in the sample, and the adenocarcinoma according to ⁇ 3> cited in ⁇ 2>.
  • a third anti-C-terminal antibody that specifically binds to the C-terminal region of the protein fragment having the amino acid sequence of SEQ ID NO: 3 and the N-terminal from the C-terminal region of the protein fragment having the amino acid sequence of SEQ ID NO: 3.
  • the amount of the protein fragment measured using the anti-N-terminal region antibody that recognizes the side is taken as the total WFDC2 protein mass, and ⁇ 2> or ⁇ 3> cited ⁇ 2> is cited in ⁇ 4>.
  • ⁇ 6> The method for detecting adenocarcinoma according to any one of ⁇ 1> to ⁇ 5>, wherein the reference amount is the total WFDC2 protein mass.
  • ⁇ 7> The method for detecting adenocarcinoma according to any one of ⁇ 1> and ⁇ 4>, wherein the reference amount is the creatinine concentration.
  • ⁇ 8> The method for detecting adenocarcinoma according to any one of ⁇ 1> to ⁇ 7>, wherein the sample is urine of the subject or a sample derived from urine.
  • a test kit for determining the presence or absence of adenocarcinoma by the sandwich method which comprises a first microplate and two antibodies that specifically bind to a protein fragment having the amino acid sequence of SEQ ID NO: 1.
  • the test kit according to ⁇ 9> which is an anti-N-terminal region antibody that recognizes the N-terminal side from the region.
  • ⁇ 11> It further comprises a second microplate and a second anti-C-terminal antibody that specifically binds to the C-terminal region of the protein fragment having the amino acid sequence of SEQ ID NO: 2, the second anti-C-terminal antibody and the anti-N-terminal.
  • the embodiment of the present invention is configured as described above, it is possible to provide a method and a kit for efficiently detecting adenocarcinoma with high sensitivity and specificity in a sandwich method excellent in high throughput.
  • the values not normalized for each of the first judgment value (A), the second judgment value (B), and the modified judgment value (C) are set for each of the patient group and the healthy subject group. It is a plotted graph. It is a graph in which the unnormalized first judgment value and the second judgment value are paired and plotted on a two-dimensional plane when the creatinine concentration is used as a reference amount.
  • the values normalized for each of the first judgment value (A), the second judgment value (B), and the modified judgment value (C) when the total WFDC2 protein mass is used as the reference amount are set for each of the patient group and the healthy subject group, respectively. It is a graph plotted about.
  • the values that are not normalized for each of the first judgment value (A), the second judgment value (B), and the modified judgment value (C) are the values of the patient group and the healthy subject group. It is a graph plotted for each. It is a graph in which the unnormalized first judgment value and the second judgment value are paired and plotted on a two-dimensional plane when the total WFDC2 protein mass is used as a reference amount. It is a graph which plotted the value when the new subject was judged by the correction judgment value when the creatinine concentration was used as a reference amount, for each of a patient and a healthy person group.
  • the method for detecting adenocarcinoma according to the first embodiment is a method for detecting adenocarcinoma based on a protein fragment of WFDC2 protein in a sample derived from a subject, and is a protein having the amino acid sequence of SEQ ID NO: 1 obtained by the sandwich method.
  • the first determination value which is a value obtained by dividing the amount of the first fragment, which is the amount of the fragment in the sample, by the total WFDC2 protein mass in the sample obtained by the sandwich method or the reference amount defined by the creatinine concentration.
  • the presence or absence of adenocarcinoma is determined by comparison with a predetermined threshold.
  • WFDC2 is an abbreviation for "WAP (whey acidic protein) four-disulfide core domain protein 2" and is also known as “human epididymis protein 4" (HE4).
  • the WFDC2 protein is a secretory protein, and it is known that the signal peptide on the N-terminal side is cleaved when it is released extracellularly.
  • the full-length amino acid sequence of the WFDC2 protein released extracellularly typically consists of 94 amino acid residues of SEQ ID NO: 3 shown below.
  • SEQ ID NO: 3 EKTGVCPELQ ADQNCTQECV SDSECADNLK CCSAGCATFC SLPNDKEGSC PQVNINFPQL GLCRDQCQVD SQCPGQMKCC RNGCGKVSCV TPNF
  • either the total WFDC2 protein mass or the creatinine concentration in the sample derived from the subject is measured by the sandwich method, and this is used as the reference amount. Further, the amount of W10409 fragment is measured by the sandwich method, and this is used as the first fragment amount. Then, the first determination value, which is a value obtained by dividing the amount of the first fragment by the reference amount, that is, the ratio of the W10409 fragment to the reference amount is calculated, and the presence or absence of adenocarcinoma is determined by this value. This determination is, for example, positive for adenocarcinoma if it is larger than this threshold value by comparison with a predetermined threshold value.
  • the above-mentioned first determination value is calculated for a sample collected from a plurality of known adenocarcinoma patients (for example, lung adenocarcinoma patient) in advance, and a plurality of healthy subjects (that is, no adenocarcinoma disease is observed).
  • the first judgment value is calculated for the sample collected from the person), the first judgment value is compared between the adenocarcinoma patient and the healthy person, and the sensitivity (that is, the sample of the adenocarcinoma patient is positive).
  • the threshold value can be a value such that the specificity (that is, the ratio at which a healthy person's sample can be determined to be negative) becomes higher.
  • MSE mean square error
  • ROC-AUC Receiveiver Operating Characteristic-Area Under the Curve
  • the threshold value may be a normalized value.
  • the first determination value derived from the sample may be normalized in the same manner as the threshold value, and the normalized threshold value and the normalized first determination value may be compared.
  • the term "threshold value" is a concept including both a non-normalized threshold value and a normalized threshold value.
  • the mean value of the group including the patient group and the healthy person group is 0, and the variance is 1.
  • an antibody against a substance to be detected (antigen) (hereinafter, also referred to as “primary antibody”) is immobilized in a measurement region such as a microplate, magnetic particles, or a sensor chip in advance, and the substance to be detected is subjected to an immune reaction. Then, another antibody (hereinafter, also referred to as “secondary antibody”) that specifically binds to the substance to be detected is bound, and the abundance of the antigen is measured using this secondary antibody. It is a method to do.
  • the method for measuring the abundance of the antigen using the secondary antibody is not particularly limited, and a method may be used in which the secondary antibody is labeled in advance and the abundance of the antigen is measured by the labeling.
  • a method may be used in which a further antibody (hereinafter, also referred to as “tertiary antibody”) that specifically binds to the antibody is labeled in advance and the abundance of the antigen is measured by the labeling.
  • the primary antibody and the secondary antibody are not particularly limited as long as the object of the present embodiment can be achieved, and various antibodies described later and commercially available antibodies can be used.
  • the sign is not particularly limited as long as the object of the present embodiment can be achieved, and a known sign can be used.
  • the label include fluorescent dyes, fluorescent nanoparticles, aggregated nanoparticles, magnetic beads, enzymes / coenzymes, chemiluminescent substances, and radioactive substances.
  • the amount of antigen can be measured by measuring the enzyme substrate reaction and chemical reaction caused by these labels, and the amount of luminescence, absorption, fluorescence, radiation, etc. generated from the label itself.
  • examples of the enzyme / coenzyme include peroxidase (HRP) and alkaline phosphatase (ALP) derived from horseradish peroxidase.
  • HRP peroxidase
  • ALP alkaline phosphatase
  • a calibration curve is prepared using the signal intensity obtained from a biological substance whose concentration is known as a sample, and the signal intensity obtained from the substance to be detected is applied to obtain the concentration. Can be done.
  • the sandwich method known methods have been put into practical use, and any of them may be used.
  • Specific examples of the sandwich method include an ELISA (enzyme-linked immunosorbent assay) method, a CLIA (chemiluminescent immunoassay) method, and an ECLIA (Electro Chemiluminescence Immunoassay) method.
  • the ELISA method is preferable as the sandwich method.
  • sandwich methods may be performed according to the standard method.
  • the primary antibody is fixed in a well of a microplate, a sample is added, and a specific fragment or the like in the sample is bound to the primary antibody.
  • the secondary antibody is bound, and the substrate is reacted with an enzyme such as peroxidase for detection (which may be bound to the secondary antibody later) bound to the secondary antibody, and the color development, luminescence, and fluorescence generated by the reaction are reacted. Etc. are measured.
  • the judgment can usually be made in about 2 to 8 hours, so that the high throughput property is excellent.
  • the ELISA method is preferable from the viewpoint of further improving the quantitativeness and further improving the ROC-AUC.
  • the method for detecting adenocarcinoma in the second embodiment is, in the first embodiment, a first anti-C-terminal antibody that specifically binds to the C-terminal region of the protein fragment having the amino acid sequence of SEQ ID NO: 1 and the above-mentioned SEQ ID NO:.
  • the amount of the protein fragment measured using the anti-N-terminal region antibody that recognizes the N-terminal side from the C-terminal region of the protein fragment having the amino acid sequence of 1 is defined as the amount of the first fragment in the sample.
  • the first anti-C-terminal antibody is an antibody that specifically binds to the C-terminal of the W10409 fragment.
  • the antibody that specifically binds to the C-terminal 4 amino acid residue that is, the C-terminal “PQLG” (proline-glutamine-lysine-glycine) is defined as the first anti-C-terminal antibody.
  • PQLG proline-glutamine-lysine-glycine
  • the number of amino acid residues recognized by the first anti-C-terminal antibody is not particularly limited as long as the first anti-C-terminal antibody can specifically bind to the C-terminal, and can be, for example, 3 to 10.
  • the anti-N-terminal region antibody is an antibody that recognizes the N-terminal side of the C-terminal region to which the first anti-C-terminal antibody binds.
  • the site recognized by the anti-N-terminal region antibody may be a plurality of consecutive amino acid residues on the N-terminal side of the C-terminal region to which the first anti-C-terminal antibody binds, or may not be present. It may have a three-dimensional structure composed of consecutive amino acid residues.
  • the anti-N-terminal region antibody is at least the amino acid residue located on the N-terminal side of this "P" (proline).
  • the W10409 fragment can be detected with high sensitivity in the above-mentioned first embodiment.
  • the W10409 fragment not only the W10409 fragment itself but also the fragment lacking the amino acid residue on the N-terminal side of the site recognized by the anti-N-terminal region antibody binds to the first anti-C-terminal antibody. Any fragment that can be detected can be detected.
  • the method for detecting adenocarcinoma in the third embodiment is the sample of the protein fragment having the amino acid sequence of SEQ ID NO: 2 obtained by the sandwich method in place of the first determination value in the first embodiment or the second embodiment.
  • a correction judgment value which is the sum of the second judgment value which is a value obtained by dividing the amount of the second fragment which is the amount in the medium by the reference amount and the value obtained by multiplying the first judgment value by 5 to 100.
  • the amount of the first fragment which is the amount of the W10409 fragment in the sample
  • the amount of the first fragment is divided by the reference amount to obtain the first determination value.
  • the amount of the second fragment which is the amount of the W14309 fragment in the same sample
  • the sandwich method is measured by the sandwich method, and the amount of the second fragment is divided by the reference amount to calculate the second determination value.
  • the sum of the value obtained by multiplying the amount of the first fragment by a predetermined magnification, specifically 5 to 100, and the second determination value is calculated and used as the correction determination value.
  • the presence or absence of adenocarcinoma is determined by this modified determination value.
  • This determination is, for example, positive for adenocarcinoma if it is larger than this threshold value by comparison with a predetermined threshold value.
  • the above magnification is preferably 7 to 50, more preferably 8 to 40, and particularly preferably 10 to 30 from the viewpoint of better balancing ROC-AUC and MSE.
  • this threshold value as in the first embodiment, any value that can distinguish between positive and negative adenocarcinoma can be selected, but the second determination value is combined with the first determination value. It is desirable to select the threshold value in consideration of the value. For example, for a sample collected from a plurality of known adenocarcinoma patients (for example, lung adenocarcinoma patients) in advance, the above-mentioned first judgment value and second judgment value are calculated, and the correction judgment value is calculated in the same manner as above.
  • the first judgment value and the second judgment value are similarly calculated for the samples collected from a plurality of healthy subjects (that is, those who are not found to have adenocarcinoma), and the correction judgment value is calculated in the same manner as above.
  • the correction judgment values of the adenocarcinoma patient and the healthy subject can be compared, and the sensitivity (that is, the ratio at which the sample of the adenocarcinoma patient can be determined to be positive) and the specificity (that is, the sample of the healthy subject can be determined to be negative).
  • a value such that the ratio) becomes higher can be set as a threshold.
  • a threshold value for example, it is conceivable to select a value that minimizes MSE (mean square error) based on the ROC-AUC value.
  • magnification so that the MSE is minimized, multiply this by the amount of the first fragment, and further add the amount of the second fragment to obtain the correction determination value and the threshold value. Further, it is also preferable to adjust the above-mentioned magnification so that the ROC-AUC is maximized, multiply this by the first fragment amount, and further add the second fragment amount to obtain the correction determination value and the threshold value. Further, it is also preferable to adjust the above-mentioned magnification in consideration of the balance between MSE and ROC-AUC, multiply this by the amount of the first fragment, and further add the amount of the second fragment to obtain the correction determination value and the threshold value. ..
  • the presence or absence of adenocarcinoma can be determined with higher sensitivity and specificity by using not only the amount of W10409 fragment in the sample but also the amount of W14309 fragment as an index. It becomes possible to judge.
  • the method for detecting adenocarcinoma in the fourth embodiment is the second anti-C-terminal that specifically binds to the C-terminal region of the protein fragment having the amino acid sequence of SEQ ID NO: 2 in the third embodiment based on the second embodiment.
  • the amount of the protein fragment measured using the antibody and the anti-N-terminal region antibody that recognizes the N-terminal side of the C-terminal region of the protein fragment having the amino acid sequence of SEQ ID NO: 2 is the amount of the protein fragment in the sample. The amount is two fragments.
  • the second anti-C-terminal antibody is an antibody that specifically binds to the C-terminal of the W14309 fragment.
  • an antibody that specifically binds to the C-terminal 5 amino acid residue that is, the C-terminal "QVNIN" (glutamine-valine-asparagine-isoleucine-asparagine) is a second anti-C-terminal antibody.
  • QVNIN glutamine-valine-asparagine-isoleucine-asparagine
  • the number of amino acid residues recognized by the second anti-C-terminal antibody is not particularly limited as long as the second anti-C-terminal antibody can specifically bind to the C-terminal, and can be, for example, 3 to 10.
  • the anti-N-terminal region antibody is an antibody that recognizes the N-terminal side of the C-terminal region to which the second anti-C-terminal antibody binds.
  • the site recognized by the anti-N-terminal region antibody may be a plurality of consecutive amino acid residues on the N-terminal side of the C-terminal region to which the second anti-C-terminal antibody binds, or may not be present. It may have a three-dimensional structure composed of consecutive amino acid residues.
  • the anti-N-terminal region antibody is at least the amino acid residue located on the N-terminal side of this "Q" (glutamine). It is desirable to make it a part of the site that recognizes the group. However, if the second anti-C-terminal antibody and the antigen recognition site are in close proximity, binding may compete, so it is desirable that the antigen recognition sites are as far apart as possible.
  • the anti-N-terminal region antibody for detecting the W14309 fragment may be a different antibody from the anti-N-terminal region antibody for detecting the W10409 fragment, but it is desirable to use the same antibody.
  • the W14309 fragment can be detected with high sensitivity in the above-mentioned third embodiment.
  • the W14309 fragment not only the W14309 fragment itself but also the fragment lacking the amino acid residue on the N-terminal side of the site recognized by the anti-N-terminal region antibody binds to the second anti-C-terminal antibody. Any fragment that can be detected can be detected.
  • the method for detecting adenocarcinoma in the fifth embodiment is the C-terminal region of the protein fragment having the amino acid sequence of SEQ ID NO: 3 in the fourth embodiment based on the second embodiment or the third embodiment based on the second embodiment.
  • a protein fragment measured using a third anti-C-terminal antibody that specifically binds and an anti-N-terminal region antibody that recognizes the N-terminal side of the C-terminal region of the protein fragment having the amino acid sequence of SEQ ID NO: 3. Is the total WFDC2 protein mass.
  • the third anti-C-terminal antibody is an antibody that specifically binds to the C-terminal of a protein fragment having the amino acid sequence of SEQ ID NO: 3.
  • the antibody that specifically binds to the C-terminal 5 amino acid residue that is, the C-terminal “VTPNF” (valine-threonine-proline-asparagin-phenylalanine) is a third anti-C-terminal antibody.
  • VTPNF valine-threonine-proline-asparagin-phenylalanine
  • the number of amino acid residues recognized by the third anti-C-terminal antibody is not particularly limited as long as the third anti-C-terminal antibody can specifically bind to the C-terminal, and can be, for example, 3 to 10.
  • the anti-N-terminal region antibody is an antibody that recognizes the N-terminal side of the C-terminal region to which the third anti-C-terminal antibody binds.
  • the site recognized by the anti-N-terminal region antibody may be a plurality of consecutive amino acid residues on the N-terminal side of the C-terminal region to which the third anti-C-terminal antibody binds, or may not be present. It may have a three-dimensional structure composed of consecutive amino acid residues.
  • the anti-N-terminal region antibody is at least the amino acid residue located on the N-terminal side of this "V" (valine).
  • the anti-N-terminal region antibody for detecting the protein fragment having the amino acid sequence of SEQ ID NO: 3 is the anti-N-terminal region antibody for detecting the W10409 fragment and the anti-N-terminal region antibody for detecting the W14309 fragment. It may be different from any of the region antibodies, or it may be the same as any of these, but it is desirable to use the same antibody as both of these. In that case, the anti-N-terminal region antibody should be a part of the site that recognizes the amino acid residue located on the N-terminal side of the site to which the second anti-C-terminal antibody that binds to the C-terminal of the W14309 fragment binds. Is desirable.
  • the protein fragment having the amino acid sequence of SEQ ID NO: 3 is sensitively obtained in the above-mentioned second embodiment and fourth embodiment. Can be detected.
  • the protein fragment itself having the amino acid sequence of SEQ ID NO: 3 but also the fragment lacking the amino acid residue on the N-terminal side of the site recognized by the anti-N-terminal region antibody may be used. Any fragment to which the third anti-C-terminal antibody can bind can be detected.
  • the method for detecting adenocarcinoma in the sixth embodiment is any of the first to fifth embodiments, in which the reference amount is the total WFDC2 protein mass.
  • the total WFDC2 protein mass is one in which no nick is contained at any position and one in which the disulfide bond between the fragments separated by the nick is maintained although the nick is contained as described above. And include.
  • the amount of the W10409 fragment or the W14309 fragment as the substance to be measured in the sample can be standardized and grasped as the ratio of the mutated WFDC2 protein to the entire WFDC2 protein. ..
  • the method for detecting adenocarcinoma in the seventh embodiment is any of the first to fourth embodiments, in which the reference amount is the creatinine concentration.
  • Creatinine is said to be produced almost constantly in one day, and all of it is excreted in urine. Therefore, it is used in clinical tests to correct the concentration error due to the difference in urine volume of substances to be measured in urine such as protein. Therefore, the creatinine concentration has significance as a reference substance in standardizing the amount of the W10409 fragment or the W14309 fragment as the measurement target substance in the sample.
  • the method for detecting adenocarcinoma in the eighth embodiment is, in any one of the first to seventh embodiments, the sample from which the subject's urine or urine is derived. Since the WFDC2 protein is excreted in urine and there are few extra proteins such as serum albumin, it is desirable that the sample is the urine of the subject or a sample derived from urine.
  • the sample derived from urine refers to a sample obtained by diluting the urine itself collected from the subject or adding an appropriate reagent for the convenience of measurement.
  • the test kit of the ninth embodiment is a test kit for determining the presence or absence of adenocarcinoma by the sandwich method, and specifically binds to the first microplate and the protein fragment having the amino acid sequence of SEQ ID NO: 1 2
  • a test kit comprising a seed antibody and one of the two antibodies immobilized on a first microplate.
  • the microplate used as the first microplate consists of a flat plate with a large number of dents, and each dent is used as a test tube or a petri dish.
  • a 96-hole type is widely used as the microplate.
  • the first microplate in this embodiment contains two antibodies that specifically bind to a protein fragment having the amino acid sequence of SEQ ID NO: 1, and one of the two or more antibodies is immobilized on the microplate A. Has been done. Immobilization is preferably performed on all indentations, but is not limited to this. Further, it is preferable that the antibody to be immobilized is only one of the above-mentioned two antibodies.
  • the two types of antibodies contained in the test kit of the ninth embodiment are antibodies that are less likely to compete with each other.
  • the site recognized by the two antibodies is not particularly limited as long as it can quantify the amino acid sequence of SEQ ID NO: 1.
  • the two kinds of antibodies may be a polyclonal antibody or a monoclonal antibody.
  • the unimmobilized antibody is included in the kit in a container.
  • the non-immobilized antibody may be labeled with horseradish peroxidase (HRP) or the like for quantification.
  • the test kit of the ninth embodiment may further contain materials commonly used in the sandwich method, in addition to the above-mentioned two types of antibodies and the first microplate.
  • Other materials included in the kit of the ninth embodiment include, for example, one of the two antibodies, a tertiary antibody capable of specifically binding to an antibody not particularly immobilized, a buffer solution, a washing solution, a coloring solution, and the like. Can be mentioned.
  • Other materials included in the test kit of the ninth embodiment are not limited thereto.
  • the tertiary antibody may be labeled with horseradish peroxidase (HRP) or the like.
  • test kit of the ninth embodiment can be used for the adenocarcinoma detection method described in the above-described embodiment.
  • inspection kit of the ninth embodiment By using the inspection kit of the ninth embodiment, the throughput of the above-described embodiment can be further improved.
  • the test kit of the tenth embodiment is the kit described in the ninth embodiment, in which the two antibodies specifically bind to the C-terminal region of the protein fragment having the amino acid sequence of SEQ ID NO: 1. It is an anti-C-terminal antibody and an anti-N-terminal region antibody that recognizes the N-terminal side of the C-terminal region of the protein fragment having the amino acid sequence of SEQ ID NO: 1.
  • the first anti-C-terminal antibody and the anti-N-terminal region antibody are the same as in the second embodiment.
  • the test kit of the eleventh embodiment is the test kit according to the tenth embodiment, and further specifically binds to the second microplate and the C-terminal region of the protein fragment having the amino acid sequence of SEQ ID NO: 2.
  • the second anti-C-terminal antibody and one of the anti-N-terminal region antibody are immobilized on the second microplate.
  • one of the second anti-C-terminal antibody and the anti-N-terminal region antibody is immobilized.
  • the microplate used as the second microplate is the same as in the ninth embodiment.
  • the second anti-C-terminal antibody and the anti-N-terminal region antibody are the same as those in the fourth embodiment.
  • SEQ ID NO: 4 (standard peptide 1): TGAEKTGVCP ELQADQNCTQ ECVSDSECAD NLKCCSAGCA TFCSLPNDKE GSSPQVNINF PQLG
  • SEQ ID NO: 5 (standard peptide 2): TGAEKTGVCP ELQADQNCTQ ECVSDSECAD NLKCCSAGCA TFCSLPNDKE GSSPQVNIN
  • the sulfhydryl group of the 50th cysteine of SEQ ID NO: 1 and SEQ ID NO: 2 forms a disulfide bond with the sulfhydryl group of the 80th cysteine of SEQ ID NO: 3. Therefore, in SEQ ID NO: 1 and SEQ ID NO: 2 in which the 80th cysteine residue does not exist, a disulfide bond is formed with the sulfhydryl group of another cysteine, and the proteins of SEQ ID NO: 1 and SEQ ID NO: 2 existing in the living body. It may have a three-dimensional structure different from that of the fragment. If the three-dimensional structure changes, the antibody may not be recognized. Therefore, amino acids are substituted as described above.
  • the standard peptide 3 differs from the full-length WFDC2 protein shown in SEQ ID NO: 3 only in that the three amino acid residues T (threonine) -G (glycine) -A (alanine) are added to the N-terminal.
  • SEQ ID NO: 6 standard peptide 3: TGAEKTGVCP ELQADQNCTQ ECVSDSECAD NLKCCSAGCA TFCSLPNDKE GSCPQVNINF PQLGLCRDQC QVDSQCPGQM KCCRNGCGKV SCVTPNF
  • the first anti-C-terminal antibody as the C-terminal recognition antibody of the W10409 fragment shown in SEQ ID NO: 1 is the "new edition anti-peptide antibody experimental protocol" (Shinobu Oumi, Kunio Tsujimura). , Supervised by Masaki Inagaki, Shujunsha, published on September 6, 2004).
  • SEQ ID NO: 7 the synthetic peptide shown in SEQ ID NO: 7 below is used, in which CGGG (cysteine-glycine-glycine-glycine) is attached to the N-terminal side of PQLG (proline-glutamine-lysine-glycine) which is the C-terminal of the W10409 fragment. board.
  • This synthetic peptide was administered intravenously to chickens at weekly intervals. After the 4th administration, the antibody titer in chicken serum was measured by the ELISA method. As a result, it was found that the antibody titer was sufficiently increased, and the antiserum was collected one week after the day of the final immunization.
  • the synthetic peptide of SEQ ID NO: 7 was immobilized on an agarose carrier, and the antibody was purified from antiserum using this as an affinity column.
  • the antibody thus obtained was subclass IgY, and this antibody was used as the first anti-C-terminal antibody.
  • the second anti-C-terminal antibody as the C-terminal recognition antibody of the W14309 fragment shown in SEQ ID NO: 2 is the "new anti-peptide antibody experiment" shown in (1-3). It was made according to the method described in "Protocol”.
  • the immunogen used was the synthetic peptide shown in SEQ ID NO: 8 below, in which CGGG was added to the N-terminal side of QVNIN (glutamine-valine-asparagine-isoleucine-asparagine), which is the C-terminal of the W14309 fragment.
  • This synthetic peptide was administered intravenously to rabbits at weekly intervals. After the 4th administration, the antibody titer in rabbit serum was measured by the ELISA method. As a result, it was found that the antibody titer was sufficiently increased, and the antiserum was collected one week after the day of the final immunization.
  • the synthetic peptide of SEQ ID NO: 8 was immobilized on an agarose carrier, and the antibody was purified from antiserum using this as an affinity column.
  • the antibody thus obtained was a subclass IgG, and this antibody was used as a second anti-C-terminal antibody.
  • This synthetic peptide was administered intravenously to chickens at weekly intervals. After the 4th administration, the antibody titer in chicken serum was measured by the ELISA method. As a result, it was found that the antibody titer was sufficiently increased, and the antiserum was collected one week after the day of the final immunization.
  • the synthetic peptide of SEQ ID NO: 9 was immobilized on an agarose carrier, and the antibody was purified from antiserum using this as an affinity column.
  • the antibody thus obtained was subclass IgY, and this antibody was used as a third anti-C-terminal antibody.
  • Anti-N-terminal region antibody As the anti-N-terminal region antibody, a commercially available anti-HE4 antibody (Product No.ab200828, Abcam) was used. This antibody has an amino acid sequence on the N-terminal side of QVNIN, which is the amino acid sequence recognized by the second anti-C-terminal antibody, or a three-dimensional structure composed of several amino acids in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. Is presumed to recognize.
  • the standard peptide 4 shown in SEQ ID NO: 10 below was synthesized as a peptide on the C-terminal side of the standard peptide 1 (SEQ ID NO: 1) and the standard peptide 2 (SEQ ID NO: 2). .. The method for synthesizing the standard peptide 4 was based on the standard peptide 1 and the standard peptide 2.
  • the commercially available anti-HE4 antibody has an amino acid on the N-terminal side. It was found to recognize a sequence or a three-dimensional structure composed of several amino acids.
  • Example 1 when the creatinine concentration in the urine of the sample is used as a reference amount and the first fragment amount, which is the W10409 fragment amount, is used as an index as a detection model for lung adenocarcinoma, the second fragment amount, which is the W14309 fragment amount, is used as an index. In addition, the case where the combined amount of the first fragment amount and the second fragment amount was used as an index was examined.
  • the ELISA plate was washed 3 times with PBS-Tween, and then 200 ⁇ L of Blocking One (Product No. 03953-66, Nacalai Tesque) diluted 1: 5 with pure water was added per well for 2 hours at 25 ° C. After incubation, washing was performed three times with Tween-added PBS (hereinafter referred to as "PBS-Tween"). Then, 100 ⁇ L of a sample diluted 1: 3 with BSA-PBS was added per well, incubated at 25 ° C. for 2 hours, and washed with PBS-Tween three times.
  • PBS-Tween Tween-added PBS
  • the first anti-C-terminal antibody diluted 1: 5000 with BSA-PBS was added at 100 ⁇ L per well, incubated at 25 ° C. for 1.5 hours, and then washed with PBS-Tween. It was carried out three times. After that, a horseradish peroxidase (HRP) -labeled antibody (Peroxidase Affinity pure Donkey Anti-Chicken IgY (IgG) (H + L), Product No.703-035-155, Jacson IRL), which is an anti-chicken IgY antibody, was used as BSA-. It was diluted 1: 5000 with PBS, 100 ⁇ L was added per well, incubated at 25 ° C.
  • HRP horseradish peroxidase
  • TMB Substrate Ultra Solution (Product No. ES022-100ML, Merc) was added per well as a substrate. After the enzymatic reaction at 25 ° C. for 5 to 10 minutes, the reaction was stopped by adding sulfuric acid, and the absorbance at 450 nm was measured using a microplate reader (iMark microplate reader, BIO-RAD). The measured absorbance was applied to a calibration curve prepared in advance using the standard peptide 1 to calculate the amount of the first fragment.
  • the first judgment value was obtained by dividing the amount of the first fragment obtained for each sample by the urinary creatinine concentration. Similarly, the amount of the second fragment obtained for each sample was divided by the urinary creatinine concentration to obtain the second judgment value. Further, for each sample, the correction judgment value (10 times) was obtained by adding the second judgment value to the value obtained by multiplying the first judgment value by 10. Further, the magnification was changed to 20 times and 30 times to obtain a correction determination value (20 times) and a correction determination value (30 times).
  • the values normalized in this way are shown in FIG. 2 (A) for the first judgment value, in FIG. 2 (B) for the second judgment value, and in FIG. 2 (C) for the correction judgment value (10 times). Plotd on the graph as follows.
  • non-normalized first judgment value is shown in FIG. 3 (A)
  • the non-normalized second judgment value is shown in FIG. 3 (B)
  • the non-normalized correction judgment value (10 times) is shown in FIG. 3 ( Plots were made on the graph as shown in C).
  • FIG. 3 (A) the degree of overlap in the first determination value of FIG. 3 (A) is shown in FIG. 3 (B). It can be seen that it is smaller than the degree of overlap in the second determination value. Further, it can be seen that the degree of overlap in the correction determination value of FIG. 3C is smaller than that of both the first determination value of FIG. 3A and the second determination value of FIG. 3B.
  • ROC- when the first judgment value, the second judgment value, the correction judgment value (10 times), the correction judgment value (20 times), and the correction judgment value (30 times) in the case of normalization are used as inspection indexes.
  • the AUC was calculated, it was 0.83, 0.62, 0.84, 0.87 and 0.83, respectively. From this ROC-AUC value, it can be said that the first determination value based on the W10409 fragment amount is superior to the second determination value based on the W14309 fragment amount as a test index for lung adenocarcinoma. Further, the correction judgment value obtained by combining the first judgment value and the second judgment value is further excellent as an inspection index as compared with the case where the first judgment value and the second judgment value are used alone. Can be said.
  • the ROC-AUC when not normalized has the same value as above.
  • FIG. 4 shows a graph in which the unnormalized first judgment value and the second judgment value are paired and plotted on a two-dimensional plane for each sample.
  • the circular symbol in the graph indicates a healthy person's sample, and the square symbol indicates a patient's sample.
  • neither the first judgment value nor the second judgment value shows a prominent value in the sample of a healthy person, but either the first judgment value or the second judgment value is shown in the patient sample.
  • the frequency of mutations in which the W10409 fragment occurs is higher than the frequency of mutations in which the W14309 fragment occurs, it can be explained that the first determination value is superior to the second determination value as a test index. Then, it is not possible to determine that the mutation that causes the W14309 fragment is positive with the first determination value, but it is presumed that this mutation can be made positive without being missed to some extent by adding the second determination value. Will be done.
  • N is the total number of samples
  • t is the threshold value
  • x i is the individual judgment value in the patient group
  • x j is the individual judgment value in the healthy subject group.
  • the MSE was 0.0269, which was the minimum when the threshold value was -0.21.
  • the MSE was 0.0948, which was the minimum when the threshold value was 0.62.
  • the MSE was 0.00681, which was the minimum when the threshold value was ⁇ 0.106.
  • the MSE was 0.0835 when the threshold value was ⁇ 0.079.
  • the MSE was 0.01258 when the threshold value was 0.08405.
  • the MSE For the first judgment value that was not normalized, the MSE was 0.000245, which was the minimum when the threshold value was 0.141. Regarding the second determination value not normalized, the MSE was 0.02, which was the minimum when the threshold value was 0.485. Further, regarding the normalized correction determination value (10 times), the MSE was 0.0069, which was the minimum when the threshold value was 2.1. Further, for the correction judgment value (20 times) that was not normalized, the MSE was 0.03 when the threshold value was 3.668. Further, for the unnormalized correction determination value (30 times), the MSE was 0.087 when the threshold value was 5.192.
  • ROC-AUC, threshold value and MSE for each of the above-mentioned normalized judgment values are shown in Table 1 below, and the ROC-AUC, threshold value and MSE for each of the above-mentioned non-normalized judgment values are shown in Table 2 below.
  • the judgment model using the first judgment value based on the detection of the W10409 fragment has an extremely high ROC-AUC and a smaller MSE than the judgment model using the second judgment value based on the detection of the W14309 fragment. Therefore, it was found to be an excellent method for detecting lung adenocarcinoma.
  • the judgment model that uses the modified judgment value that takes into account the second judgment value while placing more emphasis on the first judgment value has almost the same ROC-AUC as the judgment model with the first judgment value alone. There was. In addition, MSE was remarkably small in each of the correction determination values. Therefore, it was found that the judgment model using the modified judgment value is superior to the judgment model using the first judgment value alone as a method for detecting lung adenocarcinoma.
  • the method for detecting lung adenocarcinoma is as follows. That is, urine is collected from a subject whose patient is unknown whether or not the patient has lung adenocarcinoma, and the creatinine concentration in the urine is measured according to the above (2-1) using this urine as a sample. Measure the amount of fragments. Then, if the first determination value calculated according to (2-4) above and the value normalized in the same manner is ⁇ 0.21 or more shown in Table 1 above, it can be determined to be positive for lung adenocarcinoma. can. When not normalized, the value before normalization is used as the threshold value, and if it is 0.141 or more shown in Table 2 above, it can be determined to be positive for lung adenocarcinoma.
  • the detection method of lung adenocarcinoma is as follows when the detection of the W14309 fragment is taken into consideration. That is, urine is collected from a subject whose patient is unknown whether or not the patient has lung adenocarcinoma, and the creatinine concentration in the urine is measured according to the above (2-1) using this urine as a sample. The amount of the fragment is measured, and the amount of the second fragment is further measured according to (2-3) above. Then, the first judgment value and the second judgment value are calculated according to the above (2-4), the correction judgment value is further calculated from these, and the values normalized in the same manner are shown in Table 1 above.
  • the value before normalization is used as the threshold value, and if it is 2.1 or more shown in Table 2 above, it can be determined to be positive for lung adenocarcinoma.
  • the case of the correction determination value of 10 times has been described as an example, but the same applies to the correction determination values of other magnifications. This also applies to Example 2 described later.
  • the ELISA method is adopted for measuring the amount of the first fragment and the amount of the second fragment, and it is completed within 8 hours from the addition of the sample derived from the subject until each judgment value is obtained.
  • a general-purpose automated device it is possible to measure a large number of samples at the same time in a short time, and it is excellent in high throughput. The same applies to Example 2 described later.
  • Example 2 when the total WFDC2 protein mass in the urine of the sample is used as a reference amount and the first fragment amount, which is the W10409 fragment amount, is used as an index as a detection model for lung adenocarcinoma, the second fragment amount, which is the W14309 fragment amount, is used as an index. Was used as an index, and the case where the combined amount of the first fragment amount and the second fragment amount was used as an index was examined.
  • HRP Horseradish peroxidase
  • Peroxidase Affinity pure Donkey Anti-Rabbit IgG, Product No.711-035-152, Jacson IRL that recognizes the above-mentioned N-terminal region antibody is diluted 1: 5000 with BSA-PBS. 100 ⁇ L was added per well, and the mixture was incubated at 25 ° C. for 1 hour, and washed with PBS-Tween 5 times. Then, 100 ⁇ L of TMB Substrate Ultra Solution (Product No. ES022-100ML, Merck) was added per well as a substrate. After the enzymatic reaction at 25 ° C.
  • TMB Substrate Ultra Solution Product No. ES022-100ML, Merck
  • the reaction was stopped by adding sulfuric acid, and the absorbance at 450 nm was measured using a microplate reader (iMark microplate reader, BIO-RAD). The measured absorbance was applied to a calibration curve prepared in advance using the standard peptide 3, and the total WFDC2 protein mass was calculated.
  • the first judgment value was obtained by dividing the amount of the first fragment obtained for each sample by the total WFDC2 protein mass. Similarly, the second fragment amount obtained for each sample was divided by the total WFDC2 protein mass to obtain a second determination value. Further, for each sample, the correction judgment value was obtained by adding the second judgment value to the value obtained by multiplying the first judgment value by 10.
  • the first judgment value, the second judgment value, and the correction judgment value obtained above are the average values and standard deviations of the combined group of the patient group and the healthy subject group, and are the same as in (2-4) above. Normalization was done. The values normalized in this way are shown in the graph as shown in FIG. 5 (A) for the first judgment value, FIG. 5 (B) for the second judgment value, and FIG. 5 (C) for the correction judgment value. Plotted.
  • the non-normalized first judgment value is shown in FIG. 6 (A)
  • the non-normalized second judgment value is shown in FIG. 6 (B)
  • the non-normalized correction judgment value is shown in FIG. 6 (C). It was plotted on the graph as follows.
  • the ROC-AUC when the first judgment value, the second judgment value and the correction judgment value were used as inspection indexes were 0.76, 0.72 and 0.76, respectively. From this ROC-AUC value, it can be said that the first determination value based on the W10409 fragment amount is slightly superior to the second determination value based on the W14309 fragment amount as a test index for lung adenocarcinoma. Further, it can be said that the correction determination value obtained by combining the first determination value and the second determination value is equivalent to the first determination value from the ROC-AUC value.
  • the ROC-AUC in the non-normalized case has the same value as above.
  • FIG. 4 shows a graph in which the unnormalized first judgment value and the second judgment value are paired and plotted on a two-dimensional plane for each sample.
  • the circular symbol in the graph indicates a healthy person's sample, and the square symbol indicates a patient's sample.
  • neither the first judgment value nor the second judgment value shows a prominent value in the sample of a healthy person, but either the first judgment value or the second judgment value is shown in the patient sample. May show a prominent value for the sample. That is, in a lung adenocarcinoma patient, when a mutation occurs in the WFDC2 protein, it is presumed that either the mutation resulting in the W10409 fragment occurs or the mutation resulting in the W14309 fragment occurs. Since the frequency of mutations in which the W10409 fragment occurs is higher than the frequency of mutations in which the W14309 fragment occurs, it is presumed that the first determination value is somewhat superior to the second determination value as a test index. ..
  • the MSE was 0.0713, which was the minimum when the threshold value was -0.31.
  • the MSE was 0.047, which was the minimum when the threshold value was ⁇ 0.31.
  • the MSE was 0.0581, which was the minimum when the threshold value was set to ⁇ 0.34.
  • the MSE was 0.0004, which was the minimum when the threshold value was 0.051.
  • the MSE was 0.004, which was the minimum when the threshold value was 0.157.
  • the MSE was 0.045, which was the minimum when the threshold value was 0.68.
  • ROC-AUC, threshold value and MSE for each of the above-mentioned normalized judgment values are shown in Table 3 below, and the ROC-AUC, threshold value and MSE for each of the above-mentioned non-normalized judgment values are shown in Table 4 below.
  • the ROC-AUC is higher in the former and the lung adenocarcinoma. It can be seen that it is an excellent detection method.
  • the judgment model that uses the modified judgment value that takes into account the second judgment value while placing more emphasis on the first judgment value has the same ROC-AUC as the judgment model of the first judgment value alone, but MSE. I was able to make it smaller. Since ROC-AUC itself has a certain height, it can be seen that it is useful as a determination model for lung adenocarcinoma.
  • the method for detecting lung adenocarcinoma is as follows. That is, urine is collected from a subject whose patient is unknown whether or not it is a lung adenocarcinoma patient, and the creatinine concentration in the urine is measured according to the above (3-1) using this urine as a sample, and the first according to the above (3-2). Measure the amount of fragments. Then, if the first determination value calculated according to (3-4) above and the value normalized in the same manner is ⁇ 0.31 or more shown in Table 3 above, it can be determined to be positive for lung adenocarcinoma. can. When not normalized, if it is 0.051 or more shown in 4 above, it can be determined to be positive for lung adenocarcinoma.
  • the detection method of lung adenocarcinoma is as follows when the detection of the W14309 fragment is taken into consideration. That is, urine is collected from a subject whose patient is unknown whether or not the patient has lung adenocarcinoma, and the creatinine concentration in the urine is measured according to the above (3-1) using this urine as a sample. The amount of the fragment is measured, and the amount of the second fragment is further measured according to (3-3) above. Then, the first judgment value and the second judgment value are calculated according to the above (3-4), the correction judgment value is further calculated from these, and the values normalized in the same manner are shown in Table 3 above.
  • the model using the creatinine concentration as the reference amount is the total WFDC2 protein mass even if it is based on the same first fragment amount and second fragment amount. It was shown that it is superior to the model based on the above as a method for detecting lung adenocarcinoma.
  • Example 3 In Example 3, it was verified whether or not lung adenocarcinoma could be detected by using the threshold value 2.1 of the correction determination value (10 times) obtained in Example 1.
  • the two patients diagnosed with lung adenocarcinoma were patients diagnosed with lung adenocarcinoma by surgery, transbronchial biopsy, lymph node biopsy or cytology.
  • the 10 patients who were determined not to have lung adenocarcinoma were healthy subjects. The result is shown in FIG.

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