WO2014057528A1 - 肺扁平上皮癌の検出方法 - Google Patents
肺扁平上皮癌の検出方法 Download PDFInfo
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- WO2014057528A1 WO2014057528A1 PCT/JP2012/076082 JP2012076082W WO2014057528A1 WO 2014057528 A1 WO2014057528 A1 WO 2014057528A1 JP 2012076082 W JP2012076082 W JP 2012076082W WO 2014057528 A1 WO2014057528 A1 WO 2014057528A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
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- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
Definitions
- the present invention relates to a method for detecting lung squamous cell carcinoma. More specifically, the present invention relates to a method for detecting lung squamous cell carcinoma by measuring desmoglein 3 in a blood sample.
- lung cancer is the most mortal cancer in both men and women.
- the death rate of lung cancer in Japan has increased since 1950.
- the number of lung cancer deaths in 1998 was 50,871 and death of all malignant tumors.
- early detection and treatment of lung cancer can significantly improve 5-year survival. If the disease is detected early and surgical resection is possible, the 5-year survival rate increases to 40% (see Patent Document 1).
- lung cancer The basic histological types of lung cancer are adenocarcinoma, squamous cell carcinoma (squamous cell carcinoma), adenosquamous carcinoma, large cell carcinoma (large cell carcinoma), small cell carcinoma (small cell ⁇ carcinoma) Consists of.
- the former four are collectively referred to as non-small cell lung cancer because there is no significant difference in prognosis or treatment policy.
- Non-small cell lung cancer accounts for 80 to 85% of all lung cancer cases.
- Non-small cell lung cancer is characterized by slow progression compared to small cell cancer and inadequate response to chemotherapy and radiation therapy. Therefore, surgical excision is the primary option when the tumor is localized, but the outcome is significantly inferior to other carcinomas such as gastric cancer that corresponds to the same stage in the TNM classification.
- pharmacological treatment an effective treatment method leading to complete remission has not been established. Therefore, early detection is important, and a simple, rapid and sensitive inspection method is required.
- Serum biomarkers for lung cancer have been developed to achieve early detection of lung cancer and improve clinical management. Nevertheless, its clinical usefulness is limited.
- CEA carcinoembryonic antigen
- CYFRA21-1 cytokeratin 19 fragment
- they are clinically effective for monitoring the disease state and evaluating the response to treatment.
- they are not suitable for use in clinical diagnosis. This is because they are known to be associated with other diseases such as smoking and pneumonia and other types of cancer, and moreover, early stage lung cancer cannot be detected (See Patent Document 2).
- SCC squamous cell carcinoma related antigen
- SLX sialyl Lewisx-i antigen
- Non-patent Document 2 the positive rate for early stage cancer is still low, and the development of a blood diagnostic marker that ensures non-small cell lung cancer is desired.
- lung squamous cell carcinoma is known to have a different reactivity to anticancer agents from other non-small cell lung cancers, and the development of blood diagnostic markers that specifically detect lung squamous cell carcinoma is desired. (See Non-Patent Document 3).
- Desmoglein 3 (Desmoglein 3), a member of the cadherin family involved in cell adhesion, is a membrane protein molecule that is particularly highly expressed in lung squamous cell carcinoma tissue, and is useful as a diagnostic marker for lung squamous cell carcinoma (For example, see Patent Document 3 and Non-Patent Document 3).
- autoantibodies against desmoglein 3 are known, and autoantibodies are detected in the blood even in healthy individuals. From this, it is easily expected that even if desmoglein 3 is present in the blood, it will be neutralized and inactivated by autoantibodies.
- the autoantibody When the autoantibody is overexpressed, it reacts with desmoglein 3 which is involved in the adhesion between normal cells of the skin and inhibits the adhesion, resulting in a pathological condition of an autoimmune disease called pemphigus.
- the autoantibodies are slightly detected in the blood even in healthy individuals who do not overexpress the autoantibodies and in patients other than pemphigus.
- An object of the present invention is to provide a method capable of easily and rapidly detecting lung squamous cell carcinoma. Moreover, it is providing the detection method which improves the detection power of lung squamous cell carcinoma more. Furthermore, another object of the present invention is to provide a method for detecting blood desmoglein 3 with higher sensitivity in the detection of lung squamous cell carcinoma.
- desmoglein 3 is present in a specific high concentration in the blood of lung squamous cell carcinoma patients despite being a cadherin family protein. I found out. In addition, it has been found that blood desmoglein 3 shows an expression pattern different from existing lung cancer markers, can detect lung squamous cell carcinoma patients that cannot be detected with existing lung cancer markers, and in combination with existing lung cancer markers, It has been found that the detection power of lung squamous cell carcinoma is further improved.
- the present invention includes the following matters.
- a method for detecting squamous cell carcinoma of the lung by determination comprising the following steps: (1) a step of measuring the content of desmoglein 3 in a blood sample collected from a subject, and (2) The content of desmoglein 3 obtained by the above measurement is compared with the content of desmoglein 3 in the blood sample collected from healthy subjects, and the content of desmoglein 3 in the blood sample collected from the subject Estimating the presence of squamous cell carcinoma of the lung in the subject when the amount is greater.
- HCDR1 represented by the amino acid sequence of SEQ ID NO: 4
- HCDR2 represented by the amino acid sequence of SEQ ID NO: 5
- SEQ ID NO: 6 DF366m having HCDR3 represented by the amino acid sequence
- LCDR1 represented by the amino acid sequence of SEQ ID NO: 7
- LCDR2 represented by the amino acid sequence of SEQ ID NO: 8
- LCDR3 represented by the amino acid sequence of SEQ ID NO: 9.
- HCDR1 represented by the amino acid sequence of SEQ ID NO: 17, HCDR2 represented by the amino acid sequence of SEQ ID NO: 18, HCDR3 represented by the amino acid sequence of SEQ ID NO: 19, and SEQ ID NO: : LCDR1 represented by the amino acid sequence of 20, LCDR2 represented by the amino acid sequence of SEQ ID NO: 21, and DF151 antibody having LCDR3 represented by the amino acid sequence of SEQ ID NO: 22, or MAB1720 antibody (R & D systems The method according to [3].
- the specimen is contacted with the DF366m antibody immobilized on the carrier, and then the DF151 antibody or MAB1720 antibody (R & D systems) is contacted. [3] or [4 ] Method.
- the DF366m antibody is a complete antibody comprising the H chain represented by the amino acid sequence of SEQ ID NO: 2 and the L chain represented by SEQ ID NO: 3, or the amino acid sequence of SEQ ID NO: 10 as VH
- a kit for diagnosing squamous cell carcinoma of the lung containing the following reagents used in sandwich immunoassay: (1) HCDR1 represented by the amino acid sequence of SEQ ID NO: 4, HCDR2 represented by the amino acid sequence of SEQ ID NO: 5, HCDR3 represented by the amino acid sequence of SEQ ID NO: 6 as a solid-phased antibody, sequence DF366m antibody having LCDR1 represented by the amino acid sequence of No. 7; LCDR2 represented by the amino acid sequence of SEQ ID No. 8; and LCDR3 represented by the amino acid sequence of SEQ ID No.
- HCDR1 represented by the amino acid sequence of SEQ ID NO: 17, HCDR2 represented by the amino acid sequence of SEQ ID NO: 18, and HCDR3 represented by the amino acid sequence of SEQ ID NO: 19 as the detection antibody DF151 antibody or MAB1720 antibody (R & D systems) having LCDR1 represented by the amino acid sequence of SEQ ID NO: 20, LCDR2 represented by the amino acid sequence of SEQ ID NO: 21, and LCDR3 represented by the amino acid sequence of SEQ ID NO: 22. Company).
- the DF366m antibody is a complete antibody comprising an H chain represented by the amino acid sequence of SEQ ID NO: 2 and an L chain represented by SEQ ID NO: 3, or the amino acid sequence of SEQ ID NO: 10 as VH
- a simple and quick method for detecting lung squamous cell carcinoma can be provided.
- a lung squamous cell carcinoma patient that cannot be detected by an existing lung cancer marker can be detected, and further, the detection power of the lung squamous cell carcinoma is further improved by combining with an existing lung cancer marker.
- FIG. 1 is a graph showing measurement results of desmoglein 3 concentration in blood samples of healthy subjects, pancreatic cancer patients, lung adenocarcinoma patients, and lung squamous cell carcinoma patients (10 cases each).
- FIG. 2 is a graph showing the measurement results of desmoglein 3 concentration in blood samples of lung squamous cell carcinoma patients (40 cases).
- FIG. 3 is a graph showing the correlation between desmoglein 3 and SCC concentrations in blood samples of lung squamous cell carcinoma patients (40 cases).
- FIG. 4 is a graph showing the correlation between desmoglein 3 and CYFRA21-1 concentrations in blood samples of lung squamous cell carcinoma patients (40 cases).
- FIG. 5 is a graph showing the correlation between SCC and CYFRA21-1 concentrations in blood samples of lung squamous cell carcinoma patients (40 cases).
- FIG. 6 is a graph showing the results of measuring the amount of desmoglein 3 in a buffer solution by SPFS.
- “sandwich immunoassay” refers to two different antibodies (solid phase antibody and detection antibody) that recognize different epitopes on the antigen to be detected, Means a method for detecting the presence and abundance.
- “desmoglein 3” means “human desmoglein 3 protein” represented by SEQ ID NO: 1.
- “Blood” refers to whole blood, whole blood that has been anticoagulated if necessary, plasma obtained by centrifuging whole blood that has been anticoagulated and removing blood cell components, or coagulated whole blood to precipitate It means serum obtained by removing substances (blood clots).
- Blood specimen means a blood-derived sample that has been subjected to a treatment such as centrifugation, dilution, or mixing with a reagent as necessary in addition to the blood.
- Subject means a human.
- the expression “X to Y” indicating a numerical range means X or more and Y or less.
- the present invention is a method for detecting lung squamous cell carcinoma of a subject by measuring the amount of desmoglein 3 in the blood.
- the detection method of the present invention the diagnostic kit, the anti-desmoglein 3 monoclonal antibody and the sandwich immunoassay according to the present invention will be described in detail.
- the present invention detects lung squamous cell carcinoma by measuring using desmoglein 3 in a blood sample collected from a subject.
- the blood concentration of desmoglein 3 specifically increases in patients with lung squamous cell carcinoma, and thus is an effective diagnostic marker for lung squamous cell carcinoma, and can be used for diagnosis or support of lung squamous cell carcinoma.
- it is only necessary to measure the concentration in the blood, so there is no need to perform a tissue biopsy that is a heavy burden on the subject, and since the subsequent analysis work is easy, It can be detected simply and quickly.
- the content of desmoglein 3 in a sample can be measured by any method as long as it can specifically detect desmoglein 3 protein in a blood sample with the required sensitivity. It is measured by an immunoassay from the viewpoint of convenience.
- the immunological measurement method is preferably a sandwich type immunological measurement method from the viewpoint of improving detection sensitivity and specificity.
- sandwich immunoassay sandwich ELISA is preferable from the viewpoint of simplicity, and SPFS is preferable from the viewpoint of detection sensitivity.
- the antibody used in the sandwich immunoassay is preferably a combination of specific antibodies described later.
- the blood sample to be used may be collected from the subject by a known method, but peripheral blood collected from the vein of the subject's arm is preferable.
- the collected blood sample may be appropriately processed by a known method and used for measurement, but it is preferable to use serum or plasma after fractionation.
- the determination of the presence of lung squamous cell carcinoma in the subject includes the following steps: (1) a step of measuring the content of desmoglein 3 in a blood sample collected from a subject; (2) The content of desmoglein 3 obtained by the above measurement is compared with the content of desmoglein 3 in the blood sample collected from healthy subjects, and desmoglein 3 in the blood sample collected from the subject Estimating the presence of squamous cell carcinoma of the lung in a subject with a high content of.
- the content of desmoglein 3 in blood samples collected from healthy subjects may be measured simultaneously as a control sample for each measurement, but desmoglein in blood samples collected from a certain number of healthy subjects 3
- the content can be measured in advance by the same method, and can be used as a comparison target as a value of a known healthy person.
- the content of desmoglein 3 in the blood sample collected from the subject When comparing the content of desmoglein 3 in a blood sample collected from a subject and the content of desmoglein 3 in a blood sample collected from a healthy subject, the content of desmoglein 3 in the blood sample collected from the subject When the content is high (the concentration is high), the presence of lung squamous cell carcinoma in the subject can be estimated.
- the determination of “high content” is made by setting an appropriate cut-off value and determining that the content is high when the desmoglein 3 concentration in the blood sample is equal to or higher than that value.
- the cut-off value may be appropriately adjusted according to the detection limit value of the detection method, the purpose of determination, etc. (screening, definitive diagnosis, etc.). For example, since the healthy person's desmoglein 3 concentration in FIG. 2 is 5 ng / mL or less, the false negative can be made zero by setting the cutoff value to 5 ng / mL or more. On the other hand, when the cut-off value is set to less than 5 ng / mL, a false positive that determines that a healthy person is positive occurs, but the positive rate for estimating the presence of lung squamous cell carcinoma is improved.
- the presence of squamous cell carcinoma of the lung in the subject may be estimated when a statistically significant difference is observed with respect to the desmoglein 3 concentration in the blood sample of a certain number of healthy subjects.
- the present invention combines the determination based on the content of desmoglein 3 in a blood sample collected from a subject and the determination based on the expression level of an existing lung cancer marker obtained from the sample of the same subject.
- a method for detecting epithelial cancer is included.
- the determination of the content of desmoglein 3 in the blood sample collected from the subject is performed by the method described in the method for detecting lung squamous cell carcinoma.
- any marker may be used as long as it significantly varies in the blood of lung cancer patients compared to healthy individuals.
- Examples thereof include SCC, CYFRA, CEA, SLX and the like.
- at least one selected from the group consisting of SCC and CYFRA is preferable from the viewpoint of improving the detection ability of lung squamous cell carcinoma.
- These lung cancer markers are highly specific for lung squamous cell carcinoma (cancer and chemotherapy, 28, 2089-2093, 2001) and have a different expression tendency from desmoglein 3, Undetectable lung squamous cell carcinomas can be detected from each other, and combining them is preferable because the detection power is improved.
- ⁇ Detection of desmoglein 3 by sandwich immunoassay> it is preferable to measure the desmoglein 3 protein contained in the test sample by a sandwich immunoassay using an anti-desmoglein 3 antibody.
- sandwich immunoassay sandwich ELISA or surface plasmon-field enhanced fluorescence spectroscopy (hereinafter referred to as “SPFS”) is more preferable.
- the sandwich immunoassay method of the present invention may be performed based on a known method, but is preferably performed by the following steps.
- the support (carrier) used for immobilizing the anti-desmoglein 3 antibody include synthesis of insoluble polysaccharides such as agarose and cellulose, silicone resin, polystyrene resin, polyacrylamide resin, nylon resin, and polycarbonate resin. Examples thereof include an insoluble support such as a resin or glass.
- These supports are used in the form of beads, plates and the like. In the case of beads, a column packed with these can be used. In the case of a plate, a multiwell plate (96-well multiwell plate or the like), a biosensor chip, or the like can be used.
- the binding between the anti-desmoglein 3 antibody and the support can be performed by a commonly used method such as chemical bonding or physical adsorption. All of these supports can be used commercially.
- Blocking can be performed using, for example, calf serum albumin (BSA), gelatin, albumin or the like diluted with a buffer. Blocking can usually be performed by incubating at 4 ° C. to 37 ° C. for about 1 to 24 hours.
- BSA calf serum albumin
- Blocking can usually be performed by incubating at 4 ° C. to 37 ° C. for about 1 to 24 hours.
- the test sample is appropriately diluted with a buffer solution, blood, protein-containing solution or the like as necessary.
- a buffer solution for example, phosphate buffer solution, Tris buffer solution, citrate buffer solution, borate buffer solution, carbonate buffer solution can be used.
- blood for example, bovine serum and the like
- protein-containing solution for example, a BSA-containing buffer or the like is preferably used.
- Contact can be usually carried out by incubating at 4 ° C. to 37 ° C. for about 1 to 24 hours.
- a control sample can be appropriately prepared in addition to the test sample for detecting the desmoglein 3 content.
- the control sample include a negative control sample not containing desmoglein 3 and a positive control sample containing desmoglein 3 standard. In this case, by comparing the results obtained with the test sample with the results obtained with the negative control sample without desmoglein 3 and with the positive control sample with the desmoglein 3 standard, The presence or absence of desmoglein 3 in the test sample can be confirmed.
- Binding of desmoglein 3 bound to the immobilized antibody and the detection antibody is performed by bringing the detection antibody into contact with desmoglein 3.
- the binding between desmoglein 3 and the detection antibody is usually performed in a buffer.
- the buffer solution for example, phosphate buffer solution, Tris buffer solution, citrate buffer solution, borate buffer solution, carbonate buffer solution and the like can be used.
- Contact can be usually carried out by incubating at 4 ° C. to 37 ° C. for about 1 to 24 hours.
- the detection antibody is an anti-desmoglein 3 antibody labeled with a labeling substance.
- the labeling of the anti-desmoglein 3 antibody can be performed by a known method.
- labeling substances known to those skilled in the art such as fluorescent dyes, enzymes / coenzymes, chemiluminescent substances, radioactive substances, and the like can be used.
- fluorescent dyes include fluorescein family fluorescent dyes (Integrated DNA Technologies), polyhalofluorescein family fluorescent dyes (Applied Biosystems Japan Co., Ltd.), and hexachlorofluorescein family fluorescent dyes (applied).
- rare earth complex fluorescent dyes such as Eu and Tb (for example, ATBTA-Eu 3+ ), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP),
- BFP blue fluorescent protein
- CFP cyan fluorescent protein
- GFP green fluorescent protein
- YFP yellow fluorescent protein
- fluorescent dyes include fluorescent proteins typified by red fluorescent protein (DsRed) or Allophycocyanin (APC; LyoFlogen (registered trademark)), and fluorescent fine particles such as latex and silica.
- the maximum fluorescence wavelength is set in the near-infrared region, such as Cy5 and Alexa Fluor 647. It is desirable to use a fluorescent dye having the same.
- the radioactive substance a radioisotope (such as 32 P, 14 C, 125 I , 3 H, 131 I) can be mentioned.
- biotin When biotin is used as the labeling substance, it is preferable to further add avidin to which an enzyme such as alkaline phosphatase is bound after adding the biotin-labeled antibody.
- an enzyme such as alkaline phosphatase is bound after adding the biotin-labeled antibody.
- known methods such as the glutaraldehyde method, the maleimide method, the pyridyl disulfide method, and the periodic acid method can be used.
- one or more types of primary antibodies specifically recognizing desmoglein 3 protein, and a secondary antibody specifically recognizing the primary antibody 1 can be mentioned.
- a secondary antibody that can bind only to the primary antibody, It reacts with the complex of the desmoglein 3 and the primary antibody.
- secondary antibodies that can only bind to the primary antibody include, for example, a class (IgM, IgD, IgG, IgE or IgA) specific to the primary antibody, or an isotype (IgG1, IgG2, IgG3).
- an antibody that specifically binds to the constant region of IgG4 can be mentioned.
- Such an anti-desmoglein 3 antibody can be isolated using a known hybridoma technique, and an antibody gene encoding a specific anti-desmoglein 3 antibody is assigned to a constant region of a desired class or isotype. It can also be isolated as a recombinant antibody linked in-frame to the encoding antibody gene.
- a method of detecting desmoglein 3 in a test sample by qualitatively or quantitatively detecting the bound secondary antibody can be mentioned. In this case, it is appropriate that the secondary antibody is labeled with the labeling substance.
- the labeled substance can be detected by a method known to those skilled in the art suitable for each labeled substance. For example, when detecting a detection antibody labeled with a radioactive substance, it can be detected by liquid scintillation or RIA. When detecting a detection antibody labeled with a fluorescent dye, it can be detected with a luminometer, an SPSF measuring instrument or the like. When detecting a detection antibody labeled with an enzyme, a substrate corresponding to the labeled enzyme is added, and then detected by measuring a chemical change of the substrate by the enzyme, for example, color development, fluorescence, chemiluminescence, etc. be able to.
- the solvent used for washing is not particularly limited as long as it does not inhibit the sandwich immunoassay process, but a buffer solution is usually used.
- a buffer solution for example, phosphate buffer solution, Tris buffer solution, citrate buffer solution, borate buffer solution, carbonate buffer solution and the like can be used.
- a buffer solution containing a surfactant can be used.
- a phosphate buffer solution (pH 7.4) containing 0.02% polyoxyethylene sorbitan monolaurate (Tween-20, trade name) are preferably used.
- sandwich ELISA is preferably used in the present invention.
- Sandwich ELISA is a method in which an enzyme is used as a labeling substance in the sandwich immunoassay.
- enzyme known ones may be used, but luciferase, peroxidase, myeloperoxidase, alkaline phosphatase, ⁇ -galatatosidase, ⁇ -glucosidase, horseradish peroxidase, glucoamylase, lysozyme, saccharide oxidase, micropenoleoxidase, etc. are used. be able to.
- a known substrate may be used as the enzyme substrate, but a substrate that develops color as a result of the enzyme reaction (hereinafter referred to as “chromogenic substrate”), a substrate that emits fluorescence (hereinafter referred to as “fluorescent substrate”), and a substrate that emits chemiluminescence. (Hereinafter referred to as “chemiluminescent substrate”) and the like can be preferably used.
- chromogenic substrates examples include 3,3′-diaminobenzidine (DAB), 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,2-phenylenediamine Min (ortho-phenylenediamine), o-phenylenediamine dihydrochloride (OPD), 3,3 ′, 5,5′-tetramethylbenzidine (TMB) and the like.
- DAB 3,3′-diaminobenzidine
- ABTS 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
- ABTS 1,2-phenylenediamine Min (ortho-phenylenediamine), o-phenylenediamine dihydrochloride (OPD), 3,3 ′, 5,5′-tetramethylbenzidine (TMB) and the like.
- fluorescent substrate examples include AttoPhos (registered trademark), SPECTROFLUOR (registered trademark), 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP), QuantaBlu (registered trademark), and the like.
- chemiluminescent substrate examples include luminol compounds (eg, luminol), dioxetane compounds (eg, AMPPD (registered trademark), CSPD (registered trademark), CDP-Star (registered trademark)) and the like.
- luminol compounds eg, luminol
- dioxetane compounds eg, AMPPD (registered trademark), CSPD (registered trademark), CDP-Star (registered trademark)
- AMPPD registered trademark
- CSPD registered trademark
- CDP-Star registered trademark
- Solid-phased (captured) anti-desmoglein 3 antibody diluted with sodium bicarbonate buffer is added to the multiwell plate and incubated overnight at 4 ° C to immobilize on the plate.
- blocking is performed by adding 1% BSA-PBS (-) (blocking solution) and incubating at room temperature for 2 hours. Subsequently, after removing the blocking solution and washing with PBS, a test sample is added and incubated at room temperature for 1 hour to allow desmoglein 3 to bind to the immobilized antibody.
- BSA-PBS blocking solution
- SPFS Surface Plasmon-field enhanced fluorescence spectroscopy
- ATR total reflection attenuation
- the evanescent wave transmitted through the metal thin film is several tens of times higher due to resonance with surface plasmons. It is a method of efficiently exciting a phosphor for labeling an analyte (analyte) captured in the vicinity of a metal thin film and measuring the fluorescence signal by utilizing the fact that it is enhanced several hundred times. Since such SPFS is extremely sensitive compared to general fluorescent labeling methods, it can be quantified even when only a very small amount of analyte is present in the sample.
- the measurement member for SPFS is generally used to form a sandwich-type immune complex, a sensor chip on which a field (measurement region) for measuring fluorescence by SPFS is formed and a sandwich-type immune complex is formed.
- a structure in which various solutions used can be held on the measurement area and laminated with members for constructing channels or wells. Take.
- 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 for generating surface plasmon resonance formed on the transparent support, and the metal thin film. Including a reaction layer formed on the sensor surface for capturing the analyte on the sensor surface, and if necessary, the phosphor formed between the metal thin film and the reaction layer is too close to the metal thin film. A spacer layer may be included to prevent the metal quenching of fluorescence caused by.
- the part where the reaction layer is formed corresponds to the measurement region.
- a reaction layer may be formed over the entire bottom surface of the flow path or the well to be a measurement region, or a reaction layer may be formed only on a part of the bottom surface (with a desired patterning if necessary) to be a measurement region.
- the area of the measurement region can be adjusted in consideration of the irradiation area of excitation light generally irradiated as 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 “flow cell” with holes to form the channel is loaded on the sensor chip.
- a measurement member is constructed by loading a “top plate” with a liquid feed inlet and a liquid feed outlet at a position corresponding to the hole of the flow cell and fixing them in close contact with each other. To do.
- the sensor chip surface at a position corresponding to the hole of the flow cell forms the bottom surface of the flow path, and a measurement region is formed there.
- various liquids can be fed into the flow channel from the liquid feed inlet and discharged from the liquid feed outlet using a liquid feeding means including a pump and a tube.
- reciprocating and circulating liquid feeding can also be performed.
- Conditions such as the feeding speed and feeding (circulation) time are as follows: the amount of the sample, the concentration of the analyte in the sample, the size of the channel or well, the mode of the reaction layer (density of the immobilized ligand, etc.), the pump It can be adjusted as appropriate in consideration of performance and the like.
- the SPFS system is a “well type” in which various solutions are stored in a space wider than the flow path
- a “well member” having a through hole for forming a well is formed on the sensor chip.
- a measuring member is constructed by loading and fixing.
- various liquids can be added to the well and removed using, for example, a pipette-shaped member.
- the flow cell can be made of, for example, a sheet-like polydimethylsiloxane (PDMS).
- PDMS polydimethylsiloxane
- the top plate is made of a material having transparency so that fluorescence emitted from the measurement region can be measured.
- the top plate can be made of 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 fixing the flow cell or well member in close contact with the sensor chip is not particularly limited.
- the pressure may be physically applied from above and below, and if necessary, a transparent support.
- Adhesives having the same optical refractive index, matching oil, transparent adhesive sheets, and the like may be used.
- the immunological measurement method according to the present invention can be carried out using a general SPFS measurement apparatus.
- the measurement device for SPFS basically has an SFPFS measurement member that can be attached and detached, and a light source and excitation for irradiating excitation light (preferably laser light) with an appropriate wavelength according to the phosphor used.
- Prism for making light incident on the back of the metal thin film of the sensor chip at a predetermined angle (when the transparent support uses a sensor chip with a flat substrate), receiving the light reflected by the metal thin film and measuring its intensity Light receiver, lens for condensing fluorescence emitted from the phosphor, detector for measuring the intensity of the fluorescence, excitation light and only light having a predetermined wavelength is transmitted from the fluorescence and cut the other light It is equipped with various filters for doing so.
- various documents such as JP 2010-145272 A can be referred to.
- the SPFS measurement method includes the following steps 1 and 2 for desmoglein 3 in a blood sample: (Step 1) forming a sandwich-type immune complex comprising an immobilized anti-desmoglein 3 antibody, desmoglein 3 and a fluorescently labeled anti-desmoglein 3 antibody, and (step 2) formed A step of measuring the fluorescence intensity emitted from the phosphor contained in the sandwich-type immune complex by SPFS (surface plasmon excitation enhanced fluorescence spectroscopy).
- step 1 The step of forming the sandwich-type immune complex shown as step 1 is not particularly limited as long as the sandwich-type immune complex can be formed before moving to step 2, but for example, the following step 1a And embodiments comprising 1b are common: (Step 1a) reacting the immobilized anti-desmoglein 3 antibody with desmoglein 3 in the sample to form a complex, and (step 1b) the formed immune complex and the fluorescent labeling Reacting with the anti-desmoglein 3 antibody thus formed to form the sandwich immune complex.
- a process may be provided.
- step 2 in the same manner as general SPFS, the metal thin film is irradiated with excitation light, and evanescent waves enhanced by surface plasmon resonance occurring in the metal thin film are generated, thereby forming a sandwich-type immune complex. What is necessary is just to measure the intensity
- the fluorescence intensity generated by irradiating the metal thin film with excitation light in the same manner as in the step 2 in the state where the sandwich immune complex is not formed ( It is preferable that the signal value is corrected (subtracted or removed) by the noise value.
- the flow path or well is usually filled with an aqueous solvent (for example, phosphate buffer) containing neither analyte nor fluorescently labeled antibody.
- an aqueous solvent for example, phosphate buffer
- it may be in a state filled with a solvent other than the aqueous solvent or air.
- the desmoglein 3 in the analyzed sample The concentration can be quantified.
- the concentration of desmoglein 3 in the sample thus measured can be used as reference data when diagnosing various diseases or symptoms in which the desmoglein 3 functions as a biomarker.
- an anti-human desmoglein 3 antibody is used.
- the anti-human desmoglein 3 antibody used in the measurement according to the present invention is not particularly limited as long as it is an anti-human desmoglein 3 antibody capable of achieving the effects of the present invention.
- Specific examples of the antibody include DF120, DF122, DF148, DF151, DF153, DF168, DF331, DF364, DF366, DF151c, DF364c, DF366c, DF366m, and YB-DF366c.
- anti-human desmoglein 3 antibodies The amino acid sequence of some of these anti-human desmoglein 3 antibodies is disclosed in Patent Document 3.
- Commercially available anti-human desmoglein 3 antibodies other than those described above can also be used.
- Preferred examples of commercially available antibodies include MAB1720 from R & D Systems, D219-3 from Medical and Biological Laboratories.
- an immobilized antibody hereinafter also referred to as “capture antibody”
- a detection antibody is used.
- the immobilized antibody is immobilized on a support (carrier) and specifically captures desmoglein 3 in a specimen.
- the detection antibody is an antibody modified with a substance used for detection (hereinafter referred to as “labeling substance”), and binds to desmoglein 3 captured by the immobilized antibody to label desmoglein 3 with a labeling substance. To do. By measuring the presence and abundance of this labeling substance, the presence and abundance of desmoglein 3 in the specimen can be measured.
- blood desmoglein 3 can be detected with higher sensitivity by using the following specific anti-human desmoglein 3 antibody as a solid-phased antibody and a detection antibody.
- a DF366m antibody As a combination of antibodies used in the sandwich immunoassay method according to the present invention, it is more preferable to use a DF366m antibody as a solid-phased antibody and a DF151 antibody or MAB1720 antibody as a detection antibody. It is more preferable to use the DF366m antibody as the immobilized antibody and the DF151 antibody as the detection antibody from the viewpoint of improving detection sensitivity.
- the DF366m antibody consists of HCDR1 represented by the amino acid sequence of SEQ ID NO: 4, HCDR2 represented by the amino acid sequence of SEQ ID NO: 5, HCDR3 represented by the amino acid sequence of SEQ ID NO: 6, and amino acid sequence of SEQ ID NO: 7.
- a complete antibody (hereinafter referred to as “DF366m [I] antibody”) comprising an H chain and an L chain represented by SEQ ID NO: 3, and an antibody having a part of the amino acids of the complete antibody (hereinafter referred to as “DF366 [P ] Antibody ”).
- Each of the CDRs of the DF366m antibody is the same as the corresponding CDR of the DF366 antibody (see Patent Document 3).
- the DF366m [P] antibody is an antibody having a part of amino acids of the DF366m [I] antibody, and includes the following group of antibodies.
- DF366m [CDR] antibody an antibody in which at least all of the CDR regions are the same as the DF366m [I] antibody
- DF366m [CDR] antibody The amino acid sequence described in SEQ ID NO: 4 as CDR1 (the HCDR1 sequence of the DF366m antibody), the amino acid sequence described in SEQ ID NO: 5 as the CDR2 (the HCDR2 sequence of DF366m antibody), and the SEQ ID NO: 6 described as the CDR3
- An antibody comprising an L chain having the sequence (LCDR3 sequence of DF366m antibody).
- DF366m [V] antibody an antibody having at least a variable region (V region) identical to the DF366m [I] antibody (hereinafter referred to as “DF366m [V] antibody”), An H chain having the amino acid sequence (SEQ ID NO: 10 VH sequence of the DF366m antibody) as VH and an L chain having the amino acid sequence (SEQ ID NO: 11 VL sequence of the DF366m antibody) as VL Including antibodies.
- DF366m [Fab] an antibody in which at least the Fab region is identical to the DF366m [I] antibody (hereinafter referred to as “DF366m [Fab]”), The V region described in (2) above, and the H chain having the amino acid sequence of SEQ ID NO: 12 (CH1 sequence of DF366m antibody) as CH1, and the amino acid sequence of SEQ ID NO: 13 as CL (CL of DF366m antibody CL An antibody comprising an L chain having a sequence).
- an H chain having the amino acid sequence of SEQ ID NO: 14 (a chimeric antibody of VH having the amino acid sequence of SEQ ID NO: 10 and CH of mouse IgG1), SEQ ID NO: :
- An antibody containing an L chain having an amino acid sequence of 3 (corresponding to DF366 (natural type) described in Patent Document 3).
- the antibody used in the present invention is preferably a DF366m [V] antibody, DF366m [Fab] antibody or DF366m [I] antibody from the viewpoint of improving detection sensitivity, and the DF366m [Fab] antibody or DF366m [I] antibody. It is more preferable that the antibody is DF366m [I] antibody.
- the DF151 antibody has HCDR1 represented by the amino acid sequence of SEQ ID NO: 17, HCDR2 represented by the amino acid sequence of SEQ ID NO: 18, HCDR3 represented by the amino acid sequence of SEQ ID NO: 19, and amino acid sequence of SEQ ID NO: 20.
- a complete antibody (hereinafter referred to as “DF151 [I] antibody”) comprising the H chain and the L chain represented by SEQ ID NO: 16, and an antibody having a part of the amino acids of the complete antibody (hereinafter referred to as “DF151 [P ] Antibody ”)).
- the DF151 [P] antibody is an antibody having a part of the amino acids of the DF151 [I] antibody, and includes the following group of antibodies.
- DF151 [CDR] antibody an antibody in which at least all of the CDR regions are the same as the DF151 [I] antibody
- DF151 [CDR] antibody The amino acid sequence described in SEQ ID NO: 17 as CDR1 (HCDR1 sequence of the DF151 antibody), the amino acid sequence described in SEQ ID NO: 18 as the CDR2 (HCDR2 sequence of DF151 antibody), and the SEQ ID NO: 19 as CDR3
- An antibody comprising an L chain having a sequence (LCDR3 sequence of DF151 antibody).
- DF151 [V] antibody an antibody having at least a variable region (V region) identical to the DF151 [I] antibody (hereinafter referred to as “DF151 [V] antibody”), An H chain having the amino acid sequence described in SEQ ID NO: 23 (the VH sequence of the DF151 antibody) as VH, and an L chain having the amino acid sequence described in SEQ ID NO: 24 (the VL sequence of the DF151 antibody) as VL Including antibodies.
- DF151 [Fab] antibody an antibody in which at least the Fab region is the same as the DF151 [I] antibody (hereinafter referred to as “DF151 [Fab] antibody”), The V region described in (2) above, and an H chain having the amino acid sequence of SEQ ID NO: 25 (CH1 sequence of DF151 antibody) as CH1, and the amino acid sequence of SEQ ID NO: 3 as CL (CL of DF151 antibody An antibody comprising an L chain having a sequence).
- the antibody used in the present invention is preferably a DF151 [V] antibody, DF151 [Fab] antibody or DF151 [I] antibody, from the viewpoint of improving detection sensitivity, and is preferably a DF151 [Fab] antibody or DF151 [I] antibody. More preferably, it is DF151 [I] antibody.
- the MAB1720 antibody is a mouse anti-human desmoglein 3 monoclonal antibody (subclass: IgG 2b ) produced by Clone # 216519, and can be purchased from R & D systems.
- the antibody used in the present invention is not limited to a full-length antibody molecule (complete antibody), and is within the range that does not impair the effects of the present invention, and on the condition that it has the amino acid sequence of each CDR defined above. It may be a molecular antibody, a multimer, a chimeric antibody or a humanized antibody.
- the low molecular weight antibody include Fab, Fab ′, F (ab ′) 2 , Fv, scFv (single chain Fv), Diabody, sc (Fv) 2 (single chain (Fv) 2 ) and the like. it can.
- multimers eg, dimers, trimers, tetramers, polymers
- a chimeric antibody or a humanized antibody can be prepared by a known technique.
- the antibody used in the present invention is preferably a complete antibody, F (ab ′) 2 or Fab, more preferably a complete antibody or F (ab ′) 2 from the viewpoint of improving detection sensitivity. More preferably, it is an antibody.
- the region used for immobilization to the sensor chip or binding of the labeling substance preferably a constant that does not participate in binding to desmoglein 3. It is appropriate to use an antibody having a sufficiently long amino acid sequence having a region), and such an antibody can be selected appropriately by those skilled in the art.
- amino acid substitutions, deletions, additions and / or insertions may be made as long as the effects of the present invention are not impaired. That is, among the amino acids constituting the antibody used in the present invention, one having a mutated amino acid sequence and an antibody functionally equivalent to the antibody to be used in the present invention and the effect to be achieved by the present invention Are also included in the antibodies of the present invention.
- the number of amino acids to be mutated is usually 50 amino acids or less, preferably 30 amino acids or less, and more preferably 10 amino acids or less (for example, 5 amino acids or less).
- the amino acid substitution is preferably a conservative amino acid substitution.
- Each of the antibodies used in the present invention can be prepared by a known method. For example, by introducing a nucleic acid having a sequence encoding the amino acid of the target antibody into an expression plasmid, introducing the nucleic acid into an appropriate expression cell, and culturing the expression cell in an appropriate medium, It can be obtained (see, for example, Patent Document 3).
- a sample to be subjected to the method of the present invention is a blood sample collected from a subject.
- the blood can be whole blood, whole blood anti-coagulated if necessary, whole blood that has been anti-coagulated, centrifuged to remove blood cell components, or whole blood clotted to precipitate (blood Serum obtained by removing (ii) may be used.
- the blood sample includes a blood-derived sample that has been subjected to a treatment such as centrifugation, dilution, or mixing with a reagent as necessary. Of these, serum or plasma is preferred.
- the present invention includes a lung squamous cell carcinoma diagnostic kit for detecting lung squamous cell carcinoma by detecting desmoglein 3 in a specimen.
- the lung squamous cell carcinoma diagnostic kit contains the following reagents used in a sandwich immunoassay: (1) DF366m antibody as a solid-phased antibody, and (2) One antibody selected from the group consisting of DF151 antibody and MAB1720 antibody (R & D systems) as a detection antibody.
- the lung squamous cell carcinoma diagnostic kit may further contain substances or instruments that can be used for immobilization of antibodies, detection of antibodies, and the like.
- a carrier such as a microtiter plate or a sensor chip for SPFS
- a solid phase immobilization liquid such as a carbonate buffer, or a blocking liquid containing gelatin or albumin
- antibody detection for example, a labeled substance, a labeled secondary antibody, a substrate necessary for detection of the label, a carrier, a washing buffer, a sample diluent, an enzyme substrate, a reaction stop solution, and purified.
- desmoglein 3 protein as a standard substance, instructions for use and the like may be included.
- the contents of the instruction manual usually include a method for detecting squamous cell carcinoma of the lung according to the present invention and a method for detecting desmoglein 3 in a blood sample by a sandwich immunological method.
- the lung squamous cell carcinoma diagnostic kit can be configured according to the sandwich immunoassay employed. Furthermore, in order to combine the determination based on the expression level of at least one selected from the group consisting of SCC and CYFRA other than existing lung cancer markers other than desmoglein 3, a sandwich immunoassay is used to combine other lung cancer markers. Reagents for use and detection (for example, other immobilized antibodies and detection antibodies for lung cancer markers) may be included.
- ELISA has the feature that an inexpensive reagent can be used, an expensive machine is not required for measurement, and a large number of specimens can be processed simultaneously, particularly when a multiwell plate is used. Therefore, it is preferable for the purpose of processing multiple specimens such as primary screening of specimens, for example, health examinations.
- the SPFS method has a demerit that requires a dedicated and expensive machine as compared with the plate ELISA method in terms of the number of samples processed.
- it has a very high detection sensitivity and allows more detailed analysis. Therefore, a use purpose requiring high accuracy, for example, a definite diagnosis of a specimen that becomes false positive in the primary screening, and a use for basic research purposes are preferable. Therefore, SPFS is not sufficient to detect the target object, and it is usually required to greatly reduce the cut-off value of the inspection value as compared with, for example, the ELISA method.
- Example 1 ⁇ Selection test of appropriate antibody using standard antigen> The following antibodies were used.
- the antibodies (1) to (3) were each prepared from the culture supernatant of an expression cell into which a hybridoma or a plasmid had been prepared by the method described in Patent Document 3.
- a soluble human DSG3 / mouse IgG2aFc fusion protein in which a human DSG3 extracellular region (Met1-Leu616) represented by SEQ ID NO: 1 and a mouse IgG2a constant region were combined was prepared and used.
- the fusion protein was produced in the same manner as described in Example 3 of Patent Document 3.
- the obtained antigen protein was diluted with PBS ( ⁇ ) to prepare an antigen solution of 0 to 25 ng / mL.
- Each solid phase antibody was diluted to 5 ⁇ g / mL in 100 mM sodium bicarbonate buffer (pH 9.6), and this solution was added to a polystyrene ELISA plate (Maxi-soup plate (trade name), NUNC) at 50 ⁇ L / mL. Each antibody was immobilized by adding wells and incubating at 4 ° C. for 12 hours.
- PBS ( ⁇ ) containing 1% bovine serum albumin (hereinafter referred to as “BSA”) (hereinafter referred to as “1% BSA-PBS ( ⁇ )”) is prepared as a blocking solution, and antibodies are prepared from the previous plate. After removing the solution, 100 ⁇ L / well of this blocking solution was added and incubated at 37 ° C. for 1 hour for blocking.
- BSA bovine serum albumin
- biotinylated antibody solution was removed, washed 3 times with 0.05% Tween20-PBS, and then prepared with 12.5 ng / mL streptavidin-conjugated horseradish peroxidase (Thermo'scientific Inc., hereinafter referred to as “streptavidin-HRP”). .) was added at 50 ⁇ L / well and incubated at room temperature for 30 minutes.
- the results are shown in Table 1.
- the numerical values in the table indicate the minimum antigen solution concentration (hereinafter referred to as “detection limit concentration”) with an S / N ratio exceeding 2, and the unit of the numerical values is pg / mL.
- the S / N ratio was obtained by dividing the luminescence intensity at each antigen solution concentration (1.6, 8, 40, 200, 1,000, 5,000, 25,000 pg / mL) by the luminescence intensity in the blank antigen solution (0 pg / mL) containing no antigen. Is.
- the antibody combination to be used was the most preferable combination when DF366m was used as the immobilized antibody and DF151 was used as the detection antibody, showing a particularly high S / N ratio.
- Table 2 shows the S / N ratio at each sample concentration for antibody combinations with a detection limit concentration of 1,000 pg / mL or less.
- the detection limit concentration is 1,000 pg / mL
- DF151 The S / N ratio was high in the order of the combination of (solid phase) and D219-3 (detection), and it was revealed that this combination was preferable in this order (Table 2).
- surface shows S / N ratio in a detection limit density
- Example 2 ⁇ Detection of blood-added desmoglein 3 standard> Among the antibody combinations shown in Table 2, for the combination having a detection limit concentration of 1,000 pg / mL or less, a sample obtained by adding desmoglein 3 standard to human serum was measured.
- human desmoglein 3 was diluted with human serum (Kohjin-Bio, catalog number 12181201) to prepare an antigen solution of 0 to 25 ng / mL. Otherwise, the same procedure as in Example 1 was performed. The combination of DF151 (solid phase) and D219-3 (detection), which had the lowest S / N ratio, and the same antibody combination, DF151 (solid phase) and DF151 (detection), were not tested. It was.
- the detection limit concentration can be lowered particularly when DF366m is used as the immobilized antibody and DF151 or MAB1720 is used as the detection antibody, which is a preferable combination.
- Example 3 ⁇ Detection of desmoglein 3 in patient blood (serum and plasma) samples by sandwich ELISA> Detection of desmoglein 3 in blood samples of cancer patients was performed as follows.
- test solution Human blood samples (serum or plasma) obtained from the blood bank (healthy subjects, pancreatic cancer patients, lung adenocarcinoma patients and lung squamous cell carcinoma patients, 10 cases each) were diluted 10 times with bovine pool serum (Kohjin Bio). A test solution was obtained.
- DF366m was diluted to 5 ⁇ g / mL with 100mM sodium bicarbonate buffer (pH 9.6), and this solution was added to a polystyrene ELISA plate (Maxi-soup plate (trade name), NUNC) at 50 ⁇ L / well. It was immobilized by incubating overnight at 4 ° C.
- 1% BSA-PBS (-) (blocking solution) was added at 100 ⁇ L / well and incubated at room temperature for 2 hours for blocking.
- test solution 50 ⁇ L / well of the test solution was added and incubated at room temperature for 1 hour. Then, the test solution was removed, and 0.05% Tween20 After washing 3 times with PBS, 50 ⁇ L / well of 1 ⁇ g / mL biotinylated DF-151 antibody solution (1% BSA-PBS ( ⁇ )) was added and incubated at room temperature for 1 hour.
- Example 4 ⁇ Detection of desmoglein 3 in blood (serum) specimens of lung squamous cell carcinoma patients>
- blood samples from 30 patients with squamous cell carcinoma of the lung were obtained from the blood bank, and additional experiments were conducted. The same procedure as in Example 2 was performed except that the stock solution was used as the test solution. The obtained results were evaluated together with the results of 10 healthy subjects and 10 lung squamous cell carcinoma patients measured in Example 2.
- the result is shown in figure 2.
- the blood desmoglein 3 concentration in 10 healthy subjects was about 0 to 4 ng / mL.
- the cut-off value for judgment of lung squamous cell carcinoma was set to 1 ng / mL.
- high concentration of desmoglein 3 higher than the cut-off value could be detected in 20 of 40 cases (positive rate 50%).
- Example 5 ⁇ Comparison with other lung cancer markers> Forty blood samples from lung squamous cell carcinoma patients of Example 3, characteristics were compared with other lung cancer markers. The data of desmoglein 3 used the results of Example 3. SCC and CYFRA21-1 (hereinafter referred to as “CYFRA”) were selected and examined as other lung cancer markers.
- SCC was measured using an SCC measurement kit CanAg SCC EIA (trade name, FUJIREBIO Diagnostics, Inc.) according to the attached instructions, and the cut-off value was set to 1.5 ng / mL.
- the measurement of CYFRA was carried out using a CYFRA measurement kit CYFRA21-1 EIA (trade name, FUJIREBIO Diagnostics, Inc) according to the attached instructions, and the cutoff value was set to 3.5 ng / mL.
- the reference values are 1.5 ng / mL and 2.0 ng / mL (latest medical supplement, “Lung Cancer”, (latest medical company) 2012).
- the detection power of lung squamous cell carcinoma was improved by using the measurement results of the amounts of desmoglein 3, SCC and CYFRA in the blood in combination. That is, the detection rates when desmoglein 3 (when the cutoff value is 1 ng / mL), SCC, and CYFRA alone are used (20/40), (4/40), (6/40), respectively.
- the detection rate when desmoglein 3 and SCC were combined was (21/40), and the detection rate when desmoglein 3 and CYFRA were combined was (20/40).
- the detection rate when the two were combined was (21/40). At this time, the false positive rate of desmoglein 3 was (3/10: 30%).
- the detection rates when SCC and CYFRA were used alone were (13/40), (4/40), and (6/40), respectively.
- combining the three The detection rate was (17/40).
- the false positive rate of desmoglein 3 at this time is (1/10: 10%), and when combined with SCC and / or CYFRA, it is possible to maintain high detection power while reducing the false positive rate in desmoglein 3. Met.
- the specimen that showed a high desmoglein 3 value with the antibody combination (DF366m + biotinylated DF-151) in Example 3 also showed a high desmoglein 3 value when measured with the other antibody combinations used in Example 1.
- specimens that showed a low value of desmoglein 3 also showed a low value. That is, even when measured with a combination of antibodies other than DF366m + biotinylated DF-151, it was possible to diagnose lung squamous cell carcinoma patients with lung squamous cell carcinoma that cannot be determined as cancer with existing lung cancer markers.
- Example 6 ⁇ Detection of desmograin 3 standard in buffer solution by SPFS (1)> Desmoglein 3 in the buffer was measured by SPFS. DF366m described in Example 1 was used as the immobilized antibody, and DF151 described in Example 1 was used as the detection antibody.
- the transparent support provided with the metal thin film obtained by the above process was immersed in 10 mL of an ethanol solution of 11-amino-1-undecanthiol adjusted to 1 mM for 24 hours to form SAM on the surface of the gold thin film.
- the transparent support was taken out from the ethanol solution, washed with ethanol and isopropanol, and then dried using an air gun.
- the transparent support provided with the SAM obtained by the above process was prepared by adding 1 mg / mL of carboxymethyldextran (CMD) having a molecular weight of 500,000 to 1,000,000 and 0.5 mM of N-hydroxysuccinimide (NHS).
- CMD carboxymethyldextran
- NHS N-hydroxysuccinimide
- MES buffered saline MES
- EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
- WSC water-soluble carbodiimide
- the transparent support provided with the solid phase layer obtained in the above step was immersed in MES containing NHS 50 mM and WSC 100 mM for 1 hour, and then a DF366m antibody solution (2.5 ⁇ g / mL) The monoclonal antibody was immobilized on CMD by immersing in CMD for 30 minutes.
- non-specific adsorption prevention treatment was performed by circulating and feeding PBS containing 1% by mass bovine serum albumin (BSA) and 1M aminoethanol for 30 minutes.
- BSA bovine serum albumin
- a PDMS sheet having a thickness of 10 mm and a width of 5 mm and having a thickness of 0.5 mm was placed on the sensor chip obtained as described above, and a silicone rubber spacer was arranged around the PDMS sheet.
- a PMMA top plate in which holes for introducing and discharging liquids were formed at positions corresponding to the holes of the PDMS sheet was placed.
- a laminate of these sensor chip, PDMS sheet and silicone rubber spacer, and PMMA top plate was pressure-bonded at the outer periphery and fixed with screws to produce an SPFS measurement member.
- Tris-buffered saline (TBS) containing 0.05% by mass of Tween 20 was fed, circulated for 10 minutes to wash the flow path, and then 2 ⁇ g / mL of AlexaFluor647-labeled DF151 prepared in the above step (2).
- the containing PBS solution was fed and circulated for 5 minutes.
- TBS Tris-buffered saline
- PMT photomultiplier tube
- the concentration of the desmoglein 3 PBS solution to be delivered is 10,000 pg / mL, 5,000 pg / mL, 1,000 pg / mL, 200 pg / mL, 50 pg / mL, 10 pg / mL, 3.16 pg / mL, 1 pg / mL,
- the amount of fluorescence was measured by the same procedure as above except that the amount was changed to 0.316 pg / mL, 0.1 pg / mL, and 0.031 pg / mL, and those measured values were defined as “signal” (S) at each concentration.
- Example 1 when various combinations of antibodies were changed as in Example 1, it was confirmed that the same plot as in FIG. 6 was obtained. Furthermore, when comparing the S / N ratio in the 200-1000 pg / mL concentration range, DF366 (solid phase), MAB1720 (detection), and DF151 (solid phase) were used when DF366m was used as the immobilized antibody and DF151 was used as the detection antibody. ) And MAB1720 (detection), MAB1720 (solid phase) and DF151 (detection), DF151 (solid phase) and D219-3 (detection) in the order of combination, and this order is obtained in Example 1. The order was almost the same. That is, the S / N ratio at a concentration of 1000 pg / mL was 140.0, 92.2, 60.8, 34.5, and 24.4 in the above order.
- Example 7 ⁇ Detection of blood-added desmoglein 3 standard by SPFS> The same procedure as in Example 6 was performed except that the sample solution of Example 2 was used as the sample.
- Sequence number 2 Artificial sequence DF366 VH-Murine IgG2a CH (DF366 H chain variable region-mouse IgG2a H chain constant region).
- Sequence number 3 Artificial sequence DF366 VL-Murine IgG2a CL (kappa) (DF366 L chain variable region-mouse IgG2a L chain ( ⁇ chain) constant region).
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Abstract
Description
(1)被検体から採取した血液検体中のデスモグレイン3の含有量を測定する工程、および、
(2)前記測定により得られたデスモグレイン3の含有量と、健常者から採取した血液検体中のデスモグレイン3の含有量を比較し、被検体から採取した血液検体中のデスモグレイン3の含有量の方が多いことをもって、被検体における肺扁平上皮癌の存在を推定する工程。
(1)固相化抗体としての、配列番号:4のアミノ酸配列で表されるHCDR1、配列番号:5のアミノ酸配列で表されるHCDR2、配列番号:6のアミノ酸配列で表されるHCDR3、配列番号:7のアミノ酸配列で表されるLCDR1、配列番号:8のアミノ酸配列で表されるLCDR2、および配列番号:9のアミノ酸配列で表されるLCDR3を有するDF366m抗体、および、
(2)検出抗体としての、検出抗体として、配列番号:17のアミノ酸配列で表されるHCDR1、配列番号:18のアミノ酸配列で表されるHCDR2、配列番号:19のアミノ酸配列で表されるHCDR3、配列番号:20のアミノ酸配列で表されるLCDR1、配列番号:21のアミノ酸配列で表されるLCDR2、および配列番号:22のアミノ酸配列で表されるLCDR3を有するDF151抗体またはMAB1720抗体(R&D systems社)。
(3)SCCおよびCYFRAからなる群から選ばれる少なくとも1つの肺癌マーカーを、サンドイッチ型免疫学的測定法を用いて検出するための試薬。
本発明は、被検体から採取した血液検体中のデスモグレイン3を用いて測定することにより、肺扁平上皮癌を検出する。血液中のデスモグレイン3濃度は、肺扁平上皮癌患者で特異的に上昇するため、肺扁平上皮癌の有効な診断マーカーであり、肺扁平上皮癌の診断または診断の支援に用いることができる。また、本発明によれば、血液中の濃度を測定すればよいため、被験者の負担の大きい組織生検をする必要が無く、また、その後の解析作業も容易であるため、肺扁平上皮癌を簡便かつ迅速に検出することができる。
(1)被検体から採取した血液検体中のデスモグレイン3の含有量を測定する工程、
(2)前記測定により得られたデスモグレイン3の含有量と、健常者から採取した血液検体中のデスモグレイン3の含有量を比較し、被検体から採取した血液検体中のデスモグレイン3の方の含有量が多いことをもって、被検体における肺扁平上皮癌の存在を推定する工程。
本発明は、被験者から採取した血液検体中のデスモグレイン3の含有量に基づく前記判定と、同一被検体の検体から得られた、既存の肺癌マーカーの発現量に基づく判定を組み合わせた、肺扁平上皮癌の検出方法を含む。
本発明では、被検試料に含まれるデスモグレイン3タンパク質を、抗デスモグレイン3抗体を用いたサンドイッチ型免疫学的測定法により測定することが好ましい。サンドイッチ型免疫学的測定法としては、サンドイッチELISAまたは表面プラズモン励起増強蛍光分光法(Surface Plasmon-field enhanced Fluorescence Spectroscopy、以下「SPFS」という)がより好ましい。
抗デスモグレイン3抗体を固定するために用いられる支持体(担体)としては、例えば、アガロース、セルロースなどの不溶性の多糖類、シリコン樹脂、ポリスチレン樹脂 、ポリアクリルアミド樹脂、ナイロン樹脂、ポリカーボネイト樹脂などの合成樹脂や、ガラスなどの不溶性の支持体を挙げることができる。これらの支持体は、ビーズ、プレートなどの形状で用いられる。ビーズの場合、これらが充填されたカラムなどが使用できる。プレートの場合、マルチウェルプレート(96穴マルチウェルプレート等)、バイオセンサーチップなどが使用できる。抗デスモグレイン3抗体と支持体との結合は、化学結合や物理的な吸着などの通常用いられる方法により行うことができる。これらの支持体はすべて市販のものが好適に使用できる。
試料中のデスモグレイン3の支持体に対する非特異的な結合を防ぐため、固相のブロッキングを行うことが好ましい。ブロッキングは、例えば、緩衝液で希釈した仔牛血清アルブミン(BSA)、ゼラチン、アルブミンなどを用いて行うことができる。ブロッキングは、通常、4℃~37℃で、1時間~24時間程度インキュベートすることで行うことができる。
被験試料を支持体に固相化された抗体に接触させることで、デスモグレイン3と固相化抗体を結合させる。被験試料は、必要により、緩衝液、血液、タンパク含有溶液等で適宜希釈して用いる。緩衝液としては、例えば、リン酸緩衝液、Tris緩衝液、クエン酸緩衝液、ホウ酸塩緩衝液、炭酸塩緩衝液を用いることができる。また、血液としては、例えばウシ血清等、タンパク含有溶液としては、例えばBSA含有緩衝液等が好適に用いられる。接触は通常、4℃~37℃で、1時間~24時間程度インキュベートすることで行うことができる。
固相化抗体と結合したデスモグレイン3と検出抗体との結合は、検出抗体をデスモグレイン3に接触させることで行う。デスモグレイン3と検出抗体の結合は、通常、緩衝液中で行われる。緩衝液としては、例えば、リン酸緩衝液、Tris緩衝液、クエン酸緩衝液、ホウ酸塩緩衝液、炭酸塩緩衝液等を用いることができる。接触は通常、4℃~37℃で、1時間~24時間程度インキュベートすることで行うことができる。
標識物質の検出は、それぞれの標識物質に適した当業者に公知の方法により行うことができる。例えば、放射性物質により標識された検出抗体を検出する場合には、液体シンチレーシヨンやRIA法により検出することができる。蛍光色素により標識された検出抗体を検出する場合には、ルミノメーター、SPSF測定器等により検出することができる。酵素で標識された検出抗体を検出する場合は、標識された酵素に対応した基質を加えた後に、酵素による基質の化学的変化、例えば、発色、蛍光、化学発光等を測定することにより検出することができる。
サンドイッチ型免疫学的測定法のなかでも、本発明ではサンドイッチELISAが好適に用いられる。サンドイッチELISAは、前記サンドイッチ型免疫学的測定法において、標識物質に酵素を用いる方法である。
本発明のサンドイッチELISAの測定方法として、担体(支持体)としてマルチプレートを用いた以下の方法が好適に挙げられる。
本発明のサンドイッチ型免疫学的測定方法として、SPFSが好適に用いられる。SPFSは、誘電体部材上に形成された金属薄膜に全反射減衰(ATR)が生じる角度で励起光を照射したときに、金属薄膜を透過したエバネッセント波が表面プラズモンとの共鳴により数十倍~数百倍に増強されることを利用して、金属薄膜近傍に捕捉されたアナライト(分析対象物)を標識する蛍光体を効率的に励起させ、その蛍光シグナルを測定する方法である。このようなSPFSは、一般的な蛍光標識法などに比べて極めて感度が高いため、サンプル中にアナライトがごく微量しか存在しない場合であってもそれを定量することができる。
SPFS用測定部材は、一般的に、サンドイッチ型免疫複合体を形成してSPFSによる蛍光測定を行うための場(測定領域)が形成されているセンサーチップと、サンドイッチ型免疫複合体の形成などに用いられる各種の溶液(アナライトを含む試料、標識リガンド溶液、その他の反応試薬等)を測定領域上に保持することのできる、流路またはウェルを構築するための部材とを積層化した構成をとる。
本発明に係る免疫学的測定法は、一般的なSPFS用測定装置を使用して実施することができる。SPFS用測定装置は、基本的に、SFPFS用測定部材が着脱可能となっており、使用する蛍光体に応じた適切な波長の励起光(好適にはレーザー光)を照射するための光源、励起光をセンサーチップの金属薄膜の裏面に所定の角度で入射させるためのプリズム(透明支持体が平面基板状のセンサーチップを使用する場合)、金属薄膜で反射した光を受光しその強度を測定する受光器、蛍光体から発せられる蛍光を集光するためのレンズおよびその蛍光の強度を測定するための検出器、励起光および蛍光から所定の波長を有する光のみを透過させそれ以外の光をカットするための各種のフィルタなどを備える。より具体的な態様は、たとえば特開2010-145272号公報など、各種の文献を参照することができる。
本発明に係るSPFSの測定法は、血液検体中のデスモグレイン3を対象として、下記工程1および2を行うことを含む:
(工程1)固相化された抗デスモグレイン3抗体、デスモグレイン3、および蛍光標識化された抗デスモグレイン3抗体を含むサンドイッチ型免疫複合体を形成する工程、および
(工程2)形成されたサンドイッチ型免疫複合体に含まれる蛍光体から発せられる蛍光強度をSPFS(表面プラズモン励起増強蛍光分光法)により測定する工程。
(工程1a)前記固相化された抗デスモグレイン3抗体と試料中のデスモグレイン3とを反応させて複合体を形成する工程、および
(工程1b)形成された免疫複合体と前記蛍光標識化された抗デスモグレイン3抗体とを反応させて前記サンドイッチ型免疫複合体を形成する工程。
〔免疫学的測定法に用いる抗体〕
本発明に係る免疫学的測定法では、抗ヒトデスモグレイン3抗体を用いる。本発明に係る測定に用いる抗ヒトデスモグレイン3抗体は、本発明の効果を達成できる抗ヒトデスモグレイン3抗体であれば特に限定はされないが、例えば、特許文献3に記載の方法で得られた抗体を挙げることができ、具体的には、例えば、DF120、DF122、DF148、DF151、DF153、DF168、DF331、DF364、DF366、DF151c、DF364c、DF366c、DF366m、YB-DF366c等を挙げることができる。これらの抗ヒトデスモグレイン3抗体のうちのいくつかはアミノ酸配列は特許文献3に開示されている。また、上記以外の市販の抗ヒトデスモグレイン3抗体を用いることもできる。市販の抗体としては、例えば、R&D Systems社のMAB1720、(株)医学生物学研究所のD219-3等が好適に挙げられる。
本発明に係るサンドイッチ型免疫学的測定法では、固相化抗体(以下「捕捉抗体」ともいう。)および検出抗体を用いる。固相化抗体は、支持体(担体)に固定され、検体中のデスモグレイン3を特異的に捕捉するものである。検出抗体は、検出に用いる物質(以下「標識物質」という。)で修飾された抗体であり、前記固相化抗体で捕捉されたデスモグレイン3に結合することでデスモグレイン3を標識物質により標識するものである。この標識物質の存在および存在量を測定することにより、検体中のデスモグレイン3の存在および存在量を測定することができる。本発明では、以下の特定の抗ヒトデスモグレイン3抗体を固相化抗体および検出抗体として用いることで、より高感度で血中デスモグレイン3を検出することができる。
本発明に係るサンドイッチ型免疫学的測定方法では、後述するDF366m抗体、DF151抗体およびMAB1720抗体からなる群から選ばれる2つの抗体を使用することが好ましい。
DF366m抗体は、配列番号:4のアミノ酸配列で表されるHCDR1、配列番号:5のアミノ酸配列で表されるHCDR2、配列番号:6のアミノ酸配列で表されるHCDR3、配列番号:7のアミノ酸配列で表されるLCDR1、配列番号:8のアミノ酸配列で表されるLCDR2、および配列番号:9のアミノ酸配列で表されるLCDR3を有するモノクローナル抗体であって、配列番号:2のアミノ酸配列で表されるH鎖および配列番号:3で表されるL鎖を含む完全抗体(以下「DF366m[I]抗体」という。)、ならびに、当該完全抗体のアミノ酸の一部を有する抗体(以下「DF366[P]抗体」という。)が包含される。なお、DF366m抗体の上記各CDRは、DF366抗体のそれぞれ対応するCDRと同一である(特許文献3参照)。
DF366m[P]抗体は、DF366m[I]抗体のアミノ酸の一部を有する抗体であり、以下の一群の抗体を含む。
CDR1として配列番号:4に記載のアミノ酸配列(DF366m抗体のHCDR1の配列)、CDR2として配列番号:5に記載のアミノ酸配列(DF366m抗体のHCDR2の配列)、および、CDR3として配列番号:6に記載のアミノ酸配列(DF366m抗体のHCDR3の配列)を有するH鎖、ならびに、
CDR1として配列番号:7に記載のアミノ酸配列(DF366m抗体のLCDR1の配列)、CDR2として配列番号:8に記載のアミノ酸配列(DF366m抗体のLCDR2の配列)、CDR3として配列番号:9に記載のアミノ酸配列(DF366m抗体のLCDR3の配列)を有するL鎖
を含む抗体。
VHとして配列番号:10に記載のアミノ酸配列(DF366m抗体のVHの配列)を有するH鎖、および、VLとして配列番号:11に記載のアミノ酸配列(DF366m抗体のVLの配列)を有するL鎖を含む抗体。
前記(2)に記載のV領域、ならびにCH1として配列番号:12のアミノ酸配列(DF366m抗体のCH1の配列)を有するH鎖、および、CLとして配列番号:13のアミノ酸配列(DF366m抗体のCLの配列)を有するL鎖を含む抗体。
DF151抗体は、配列番号:17のアミノ酸配列で表されるHCDR1、配列番号:18のアミノ酸配列で表されるHCDR2、配列番号:19のアミノ酸配列で表されるHCDR3、配列番号:20のアミノ酸配列で表されるLCDR1、配列番号:21のアミノ酸配列で表されるLCDR2、および配列番号:22のアミノ酸配列で表されるLCDR3を有するモノクローナル抗体であって、配列番号:15のアミノ酸配列で表されるH鎖および配列番号:16で表されるL鎖を含む完全抗体(以下「DF151[I]抗体」という。)、ならびに、当該完全抗体のアミノ酸の一部を有する抗体(以下「DF151[P]抗体」という。)が包含される。
DF151[P]抗体は、DF151[I]抗体のアミノ酸の一部を有する抗体であり、以下の一群の抗体を含む。
CDR1として配列番号:17に記載のアミノ酸配列(DF151抗体のHCDR1の配列)、CDR2として配列番号:18に記載のアミノ酸配列(DF151抗体のHCDR2の配列)、および、CDR3として配列番号:19に記載のアミノ酸配列(DF151抗体のHCDR3の配列)を有するH鎖、ならびに、
CDR1として配列番号:20に記載のアミノ酸配列(DF151抗体のLCDR1の配列)、CDR2として配列番号:21に記載のアミノ酸配列(DF151抗体のLCDR2の配列)、CDR3として配列番号:22に記載のアミノ酸配列(DF151抗体のLCDR3の配列)を有するL鎖を含む抗体。
VHとして配列番号:23に記載のアミノ酸配列(DF151抗体のVHの配列)を有するH鎖、および、VLとして配列番号:24に記載のアミノ酸配列(DF151抗体のVLの配列)を有するL鎖を含む抗体。
前記(2)に記載のV領域、ならびにCH1として配列番号:25のアミノ酸配列(DF151抗体のCH1の配列)を有するH鎖、および、CLとして配列番号:3のアミノ酸配列(DF151抗体のCLの配列)を有するL鎖を含む抗体。
MAB1720抗体は、Clone#216519により産生されるマウス抗ヒトデスモグレイン3モノクローナル抗体(サブクラス:IgG2b)であり、R&D systems社より購入することが可能である。
本発明で使用される抗体は、抗体の全長分子(完全抗体)に限られず、本発明の効果を損ねない範囲において、かつ、前記に規定した各CDRのアミノ酸配列を有することを条件として、低分子化抗体、多量体、あるいはキメラ抗体またはヒト化抗体であってもよい。低分子化抗体としては、例えば、Fab、Fab’、F(ab')2、Fv、scFv(single chain Fv)、Diabody、sc(Fv)2(single chain (Fv)2)などを挙げることができる。また、これら抗体の多量体(例えば、ダイマー、トリマー、テトラマー、ポリマー)も、本発明の抗体に含まれる。さらに、公知の手法によってキメラ抗体またはヒト化抗体を作製することもできる。
本発明に用いる抗体のアミノ酸配列は、本発明の効果を損ねない範囲において、アミノ酸の置換、欠失、付加および/または挿入がされていてもよい。すなわち、本発明に用いる抗体を構成するアミノ酸のうち、一もしくは複数のアミノ酸が変異したアミノ酸配列を有し、前記本発明に用いる抗体と、本発明の達成するべき効果において機能的に同等な抗体もまた本発明の抗体に含まれる。このような変異体における、変異するアミノ酸数は、通常、50アミノ酸以内であり、好ましくは30アミノ酸以内であり、さらに好ましくは10アミノ酸以内(例えば、5アミノ酸以内)である。前記アミノ酸置換は保存的アミノ酸置換であることが好ましい。
本発明に用いる前記各抗体は、公知の方法により、作製することができる。例えば、目的とする抗体のアミノ酸をコードする配列を有する核酸を発現プラスミドに導入し、これを適切な発現細胞に導入し、該発現細胞を適切な培地中で培養することにより、目的の抗体を得ることができる(例えば特許文献3参照)。
本発明の方法の対象となる検体は、被検体より採取した血液検体である。血液は、全血、必要に応じて抗凝固処理した全血、抗凝固処理をした全血を遠心分離して血球成分を除去して得られる血漿、または全血を凝固させて沈殿物(血餅)を除去して得られる血清等であってよい。また、血液検体は、前記血液に加え、必要に応じてこれらを遠心分離、希釈、試薬との混合等の処理をした血液由来試料を含む。これらのなかでも、血清または血漿が好ましい。
本発明は、検体中のデスモグレイン3を検出することによって肺扁平上皮癌を検出する肺扁平上皮癌診断用キットを含む。該肺扁平上皮癌診断用キットは、サンドイッチ型免疫学的測定法に用いる下記試薬を含有する:
(1)固相化抗体としてのDF366m抗体、および、
(2)検出抗体としての、DF151抗体およびMAB1720抗体(R&D systems社)からなる群から選ばれる1つの抗体。
上記サンドイッチ型免疫学的測定法のうち、血液検体の測定では、ELISA法とSPFS法が汎用される。
<標準抗原を用いた適切な抗体の選択試験>
抗体は以下のものを用いた。(1)~(3)の抗体は、いずれも特許文献3に記載の方法で作成した、ハイブリドーマまたはプラスミドを導入した発現細胞の培養上清より調製した。
(2)DF151(DF151[I]抗体)
(3)D219-3((株)医学生物学研究所)
(4)MAB1720(R&D systems社、カタログ番号MAB1720)
<ビオチン標識抗体の調製>
モノクローナル抗体のビオチン化は、以下のように行った。
ヒトデスモグレイン3として、配列番号:1で表されるヒトDSG3細胞外領域(Met1-Leu616)とマウスIgG2a定常領域を結合させた可溶型ヒトDSG3/マウスIgG2aFc融合タンパク質を作製して使用した。融合タンパクの作製は、特許文献3の実施例3に記載の方法と同様に行った。得られた抗原タンパクをPBS(-)で希釈し、0~25ng/mLの抗原溶液を作製した。
各固相抗体を、100mM 炭酸水素ナトリウム緩衝液(pH9.6)中に5μg/mLに希釈し、この溶液をポリスチレン製ELISAプレート(Maxi-soup plate (商品名)、NUNC社)に各50μL/ウェルずつ添加し、4℃で12時間インキュベートすることで、各抗体を固相化した。
結果を表1に示す。表中の数値は、S/N比が2を超える最小の抗原溶液濃度(以下「検出限界濃度」という。)を示し、数値の単位はpg/mLである。S/N比は各抗原溶液濃度(1.6、8、40、200、1,000、5,000、25,000pg/mL)における発光強度を、抗原を含まないブランク抗原溶液(0pg/mL)における発光強度で除したものである。
<血液添加デスモグレイン3標準品の検出>
表2に示す抗体の組合せのうち、検出限界濃度が1,000pg/mL以下であった組合せについて、デスモグレイン3標準品をヒト血清に添加した検体の測定をおこなった。
結果を表3に示す。表中の数値は検出限界濃度であり、数値の単位はpg/mLである。本発明の抗体の組合せを用いれば、血液中のデスモグレイン3量であっても高感度で測定できることが明らかとなった。
<患者血液(血清および血漿)検体中のデスモグレイン3のサンドイッチELISAによる検出>
癌患者の血液検体中のデスモグレイン3の検出を以下の通り行った。
血液バンクより入手したヒト血液検体(血清または血漿)(健常者、膵癌患者、肺腺癌患者および肺扁平上皮癌患者、各10例)を、ウシプール血清(コージンバイオ社)で10倍に希釈し、被験溶液とした。
DF366mを100mM炭酸水素ナトリウム緩衝液(pH9.6)で5μg/mLに希釈し、この溶液をポリスチレン製ELISAプレート(Maxi-soup plate (商品名)、NUNC社)に各50μL/ウェルずつ添加し、4℃で終夜インキュベートすることで固相化した。
結果を図1に示す。肺扁平上皮癌患者の1検体において、健常者と比較して有意に高いデスモグレイン3の存在を検出することができた。健常者、膵癌患者および肺腺癌患者の検体中では、デスモグレイン3の高い上昇は認められなかった。したがって、肺扁平上皮癌と他の癌を区別することができ、血中デスモグレイン3量は、肺扁平上皮癌の特異的診断マーカーとして用いることができることが示唆された。
<肺扁平上皮癌患者血液(血清)検体中のデスモグレイン3の検出>
さらに、血液バンクより、肺扁平上皮癌患者30名の血液検体を入手し、追加実験を行った。被験溶液として原液を用いた他は実施例2と同様に行った。得られた結果は、実施例2で測定した健常者10例および肺扁平上皮癌患者10例の結果と合わせて評価した。
<他の肺癌マーカーとの比較>
実施例3の肺扁平上皮癌患者血液検体40例について、他の肺癌マーカーとの特性の比較検討を行った。デスモグレイン3のデータは実施例3の結果を用いた。他の肺癌マーカーとしてSCCおよびCYFRA21-1(以下「CYFRA」という。)を選択し検討を行った。
SCCの測定は、はSCC測定キットCanAg SCC EIA(商品名、FUJIREBIO Diagnostics,Inc)を用いて、添付指示書にしたがって実施し、カットオフ値は、1.5ng/mLに設定した。CYFRAの測定は、CYFRA測定キットCYFRA21-1 EIA(商品名、FUJIREBIO Diagnostics,Inc)を用いて、添付指示書にしたがって実施し、カットオフ値は3.5ng/mLに設定した。
図3~図5に結果を示す。本来、血中濃度としてマイナスの値は通常想定できないが、定量限界に近い濃度領域ではノイズ値がシグナル値を超える場合も多々あるために、マイナス値が算出されており、図3~図5においては、算出値をそのまま記載している。
<緩衝液中のデスモグレイン3標準品のSPFSによる検出(1)>
緩衝液中のデスモグレイン3をSPFSにより測定した。固相化抗体として、実施例1に記載のDF366mを、検出抗体として実施例1に記載のDF151を用いた。
厚さ1mmのガラス製の透明支持体「S-LAL 10」((株)オハラ、屈折率(nd):1.72)をプラズマ洗浄した後、該支持体の片面にクロム薄膜をスパッタリング法により形成し、さらにその表面に金薄膜をスパッタリング法により形成した。クロム薄膜の厚さは1~3nm以下、金薄膜の厚さは42~47nmであった。
DF151抗体溶液(2.5μg/mL)と、AlexaFluor647標識キット(Invitrogen社)とを用いて、当該キットの所定の手順に従い、AlexaFluor647標識化DF151を作製した。その後、分子量カットフィルタ(日本ミリポア(株))を用いて未反応物を除去し、AlexaFluor647標識化DF151抗体を精製し、下記アッセイの実施まで4℃で保存した。
前記工程(1)で作製した測定部材の流路に、デスモグレイン3を100pg/mL(=0.1ng/mL)含むPBS溶液0.1mLを送液し、25分間循環させた。
結果を図6に示す。縦軸は「シグナル」-「ノイズ」(S-N)の値(単位:a.u.)、横軸はデスモグレイン3の濃度(単位:pg/mL)である。このプロットに基づき、上記測定系によるデスモグレイン3の検出限界濃度は5pg/mLであると判定でき、サンドイッチELISAによる測定と比べて、約40倍の検出感度が得られることが明らかとなった。すなわち、SPFS法によれば、ELISA法で必要な検体量の1/40の検体量で同等の評価が行えるという効果が得られることになる。
[実施例7]
<血液添加デスモグレイン3標準品のSPFSによる検出>
検体として、実施例2の検体溶液を用いたほかは、実施例6と同様に実施した。
実施例6と同様の結果が得られ、血中のデスモグレイン3の検出限界濃度は5pg/mLと判定された。すなわち、本発明に係る検体の組合せを用いれば、血液検体中のデスモグレイン3をSPFSにより高感度で検出できることが明らかとなり、他の測定方法、例えばELISA法と比較して遥かに少ない検体量で検出することができ、また、肺扁平上皮癌の判定のカットオフ値をELISA法に比べてさらに下げられることが示された。
Claims (11)
- 以下の工程を含む判定により、肺扁平上皮癌を検出する方法:
(1)被検体から採取した血液検体中のデスモグレイン3の含有量を測定する工程、および、
(2)前記測定により得られたデスモグレイン3の含有量と、健常者から採取した血液検体中のデスモグレイン3の含有量を比較し、被検体から採取した血液検体中のデスモグレイン3の含有量の方が多いことをもって、被検体における肺扁平上皮癌の存在を推定する工程。 - さらに、同一被検体の検体から得られた、既存の肺癌マーカーである、SCCおよびCYFRAからなる群から選ばれる少なくとも1つの発現量に基づく判定を組み合わせた、請求項1に記載の方法。
- 前記測定がサンドイッチ型免疫学的測定法である、請求項1または2に記載の方法。
- 前記サンドイッチ型免疫学的測定法において、
固相化抗体として、配列番号:4のアミノ酸配列で表されるHCDR1、配列番号:5のアミノ酸配列で表されるHCDR2、配列番号:6のアミノ酸配列で表されるHCDR3、配列番号:7のアミノ酸配列で表されるLCDR1、配列番号:8のアミノ酸配列で表されるLCDR2、および配列番号:9のアミノ酸配列で表されるLCDR3を有するDF366m抗体を使用し、
検出抗体として、配列番号:17のアミノ酸配列で表されるHCDR1、配列番号:18のアミノ酸配列で表されるHCDR2、配列番号:19のアミノ酸配列で表されるHCDR3、配列番号:20のアミノ酸配列で表されるLCDR1、配列番号:21のアミノ酸配列で表されるLCDR2、および配列番号:22のアミノ酸配列で表されるLCDR3を有するDF151抗体、またはMAB1720抗体(R&D systems社)を使用する、請求項3に記載の方法。 - 前記サンドイッチ型免疫学的測定法において、担体に固相化されたDF366m抗体に検体を接触させ、その後、DF151抗体またはMAB1720抗体(R&D systems社)を接触させる、請求項3または4に記載の方法。
- 前記DF366m抗体が、配列番号:2のアミノ酸配列で表されるH鎖および配列番号:3で表されるL鎖を含む完全抗体、またはVHとして配列番号:10に記載のアミノ酸配列を有するH鎖およびVLとして配列番号:11に記載のアミノ酸配列を有するL鎖を含む抗体であり、前記DF151抗体が、配列番号:15のアミノ酸配列で表されるH鎖および配列番号:16で表されるL鎖を含む完全抗体、またはVHとして配列番号:23に記載のアミノ酸配列を有するH鎖およびVLとして配列番号:24に記載のアミノ酸配列を有するL鎖を含む抗体である、請求項4または5に記載の方法。
- 前記サンドイッチ型免疫学的測定法が、サンドイッチELISA法である、請求項3~6のいずれか一項に記載の方法。
- 前記サンドイッチ型免疫学的測定法が、SPFS法である、請求項3~6のいずれか一項に記載の方法。
- サンドイッチ型免疫学的測定法に用いる下記試薬を含有する、肺扁平上皮癌診断用キット:
(1)固相化抗体としての、配列番号:4のアミノ酸配列で表されるHCDR1、配列番号:5のアミノ酸配列で表されるHCDR2、配列番号:6のアミノ酸配列で表されるHCDR3、配列番号:7のアミノ酸配列で表されるLCDR1、配列番号:8のアミノ酸配列で表されるLCDR2、および配列番号:9のアミノ酸配列で表されるLCDR3を有するDF366m抗体、および、
(2)検出抗体としての、検出抗体として、配列番号:17のアミノ酸配列で表されるHCDR1、配列番号:18のアミノ酸配列で表されるHCDR2、配列番号:19のアミノ酸配列で表されるHCDR3、配列番号:20のアミノ酸配列で表されるLCDR1、配列番号:21のアミノ酸配列で表されるLCDR2、および配列番号:22のアミノ酸配列で表されるLCDR3を有するDF151抗体またはMAB1720抗体(R&D systems社)。 - 前記DF366m抗体が、配列番号:2のアミノ酸配列で表されるH鎖および配列番号:3で表されるL鎖を含む完全抗体、またはVHとして配列番号:10に記載のアミノ酸配列を有するH鎖およびVLとして配列番号:11に記載のアミノ酸配列を有するL鎖を含む抗体であり、前記DF151抗体が、配列番号:15のアミノ酸配列で表されるH鎖および配列番号:16で表されるL鎖を含む完全抗体、またはVHとして配列番号:23に記載のアミノ酸配列を有するH鎖およびVLとして配列番号:24に記載のアミノ酸配列を有するL鎖を含む抗体である、請求項9に記載の肺扁平上皮癌診断用キット。
- さらに、下記試薬を含有する、請求項9または10の肺扁平上皮癌診断用キット:
(3)SCCおよびCYFRAからなる群から選ばれる少なくとも1つの肺癌マーカーを、サンドイッチ型免疫学的測定法を用いて検出するための試薬。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004529849A (ja) | 2000-09-01 | 2004-09-30 | インターナショナル バイオイムン システムズ,インコーポレーテッド | 扁平上皮癌に対する特異的モノクローナル抗体の同定と開発 |
JP2007186508A (ja) * | 1995-03-07 | 2007-07-26 | President & Fellows Of Harvard College | 自己免疫疾患に関連する自己及び非自己抗原の同定 |
WO2008020586A1 (en) | 2006-08-14 | 2008-02-21 | Forerunner Pharma Research Co., Ltd. | Diagnosis and treatment of cancer using anti-desmoglein-3 antibody |
JP2010145272A (ja) | 2008-12-19 | 2010-07-01 | Konica Minolta Holdings Inc | プラズモン励起センサを用いたアッセイ法 |
JP2012051822A (ja) | 2010-08-31 | 2012-03-15 | Institute Of Physical & Chemical Research | 肺癌診断用ポリペプチド、肺癌の検出方法、および治療効果の評価方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101669031A (zh) * | 2007-02-27 | 2010-03-10 | 森托科隆股份公司 | 使用与细胞外标记物结合的试剂组多重检测肿瘤细胞 |
WO2010144808A2 (en) * | 2009-06-12 | 2010-12-16 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Identification of dsg-3 as a biomarker for the detection of metastasis in lymph nodes |
-
2012
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- 2012-10-09 WO PCT/JP2012/076082 patent/WO2014057528A1/ja active Application Filing
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007186508A (ja) * | 1995-03-07 | 2007-07-26 | President & Fellows Of Harvard College | 自己免疫疾患に関連する自己及び非自己抗原の同定 |
JP2004529849A (ja) | 2000-09-01 | 2004-09-30 | インターナショナル バイオイムン システムズ,インコーポレーテッド | 扁平上皮癌に対する特異的モノクローナル抗体の同定と開発 |
WO2008020586A1 (en) | 2006-08-14 | 2008-02-21 | Forerunner Pharma Research Co., Ltd. | Diagnosis and treatment of cancer using anti-desmoglein-3 antibody |
JP2010145272A (ja) | 2008-12-19 | 2010-07-01 | Konica Minolta Holdings Inc | プラズモン励起センサを用いたアッセイ法 |
JP2012051822A (ja) | 2010-08-31 | 2012-03-15 | Institute Of Physical & Chemical Research | 肺癌診断用ポリペプチド、肺癌の検出方法、および治療効果の評価方法 |
Non-Patent Citations (11)
Title |
---|
"Health, Labour and Welfare Statistics Association, Trend of National Health", JOURNAL OF HEALTH AND WELFARE STATISTICS, vol. 47, 2000, pages 52 - 53 |
"Practical Method for Reading Tumor Markers", LUNG CANCER. THE JAPANESE JOURNAL OF CLINICAL AND EXPERIMENTAL MEDICINE, vol. 78, 2001, pages 35 - 40 |
"The Medical Frontline", 2012, article "Lung Cancer" |
CEMILE DILARA SAVCI-HEIJINK: "The Role of Desmoglein-3 in the Diagnosis of Squamous Cell Carcinoma of the Lung", THE AMERICAN JOURNAL OF PATHOLOGY, vol. 174, no. 5, 2009, pages 1629 - 1637, XP055248103 * |
DALBADIE-MCFARLAND, G. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 79, 1982, pages 6409 - 6413 |
MARK, D. F. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 5662 - 5666 |
OURNAL OF CANCER AND CHEMOTHERAPY, vol. 28, 2001, pages 2089 - 2093 |
SAVCI-HEIJINK ET AL., THE AMERICAN JOURNAL OF PATHOLOGY, vol. 174, no. 5, 2009, pages 1629 - 1637 |
See also references of EP2908133A4 |
WANG, A ET AL., SCIENCE, vol. 224, pages 1431 - 1433 |
ZOLLER, M. J.; SMITH, M., NUCLEICACIDS RESEARCH, vol. 10, 1982, pages 6487 - 6500 |
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