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WO2011129483A1 - Marqueur xage-1d pour le diagnostic du cancer pulmonaire, et trousse de diagnostic utilisant un tel marqueur - Google Patents

Marqueur xage-1d pour le diagnostic du cancer pulmonaire, et trousse de diagnostic utilisant un tel marqueur Download PDF

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Publication number
WO2011129483A1
WO2011129483A1 PCT/KR2010/003710 KR2010003710W WO2011129483A1 WO 2011129483 A1 WO2011129483 A1 WO 2011129483A1 KR 2010003710 W KR2010003710 W KR 2010003710W WO 2011129483 A1 WO2011129483 A1 WO 2011129483A1
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Prior art keywords
xage
lung cancer
antibody
pad
kit
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Korean (ko)
Inventor
박성섭
권기선
하종성
김선영
정봉현
정용원
성문우
윤탁
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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    • G01N33/5752—
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips

Definitions

  • the present invention relates to a lung cancer diagnostic or monitoring kit comprising an antibody specific for Xage-1d, and an immunochromatography strip and a method for diagnosing lung cancer using Xage-1d.
  • lung cancer was the second largest cancer in Korea between 2003 and 2005, accounting for 12.1% of all cancers.
  • the total incidence per 100,000 population was 33.2, and the sex ratio of male and female was 3.83: 1, which occurred more frequently in males.
  • the number of incidence was 2nd among male cancers and 5th among female cancers.
  • the 60s were the highest with 34.3%, followed by the 70s with 31.0% and the 50s with 14.6% (Data released October 15, 2008, Ministry of Health, Welfare and Family Affairs).
  • tumors are rapidly increasing in Korea due to the increase of smoking population and air pollution, and the mortality rate by cancer type is 21.4%, which is the first place, and in males it is 24.9% and 15.1%, respectively.
  • Lung cancer usually causes symptoms such as surrounding tissue involvement or airway obstruction or lymph node metastasis due to the growth of cancer cells, but about 10 to 15% of patients are diagnosed with lung cancer at regular examinations without any symptoms. In addition, since most lung cancers are diagnosed with a significant progression at the time of diagnosis, most cases are difficult to cure. Therefore, it is urgent to diagnose lung cancer early and reduce mortality from lung cancer (Wulfkuhle et al ., Nat Rev. Cancer, 3, 267-275, 2003).
  • the Xage-1 gene was originally identified as a PAGE / GAGE-associated gene located in the X chromosome by EST analysis (Brinkmann U, et al ., Cancer Res 1999, 59: 1445-1448). The expression profile of Xage-1 suggested that it could be used as a cancer / testis (CT) antigen. Transcription of the Xage-1d gene is regulated by methylation of the CpG island of the promoter, and four RNA splicing variants, Xage-1a, b, c and d, have been identified (Zendman AJ et al.
  • the inventors have discovered proteins that increase expression in lung cancer cells, and the relationship between lung cancer of Xage-1d protein and lung cancer cell line, which has no function in splicing variants of Xage1 gene, has been identified.
  • the present invention was completed by confirming high expression amount and sensitivity in the blood of an actual lung cancer patient compared to a normal person.
  • Another object of the present invention to provide a lung cancer diagnostic method using Xage-1d.
  • the present invention also provides an immunochromatography strip for diagnosing lung cancer comprising an antibody specific for Xage-1d.
  • the present invention also provides a method for diagnosing lung cancer using Xage-1d as a protein marker.
  • the present invention also provides a lung cancer prognosis monitoring kit comprising an antibody specific for Xage-1d.
  • the present invention provides a lung cancer prognosis monitoring method comprising an antibody specific for Xage-1d.
  • Xage-1d of the present invention is present not only in the culture of lung cancer cell lines, but also in the blood of lung cancer patients, and more amounts are present in the blood of lung cancer patients compared to normal people, and thus, lung cancer can be sensitively diagnosed with a small amount.
  • Diagnostic or monitoring kits for lung cancer, immunochromatography strips and lung cancer diagnostics can be used as protein markers.
  • 1 is a graph showing the results of enzyme immunoassay confirming the presence of Xage-1d in the culture of lung cancer cell lines.
  • Figure 2 is a graph showing the results of enzyme immunoassay confirming the presence of Xage-1d in the blood.
  • Figure 3 is a graph showing the results of absorbance measurements by biotin-peptide and absorbance by peptide competition.
  • the anti-Xage-1d antibody may be prepared by injecting Xage-1d protein or commercially available, and Xage-1d (X antigen family member 1 subtype d) may be human Xage-1d. It is preferable to have the Xage-1d amino acid sequence set forth in SEQ ID NO: 1.
  • the antibodies include polyclonal antibodies, monoclonal antibodies, fragments capable of binding epitopes, and the like.
  • Polyclonal antibodies can be produced by conventional methods of injecting the Xage-1d protein into an animal and collecting blood from the animal to obtain a serum comprising the antibody.
  • Such polyclonal antibodies can be purified by any method known in the art and can be made from any animal species host such as goat, rabbit, rat, rat, chicken, sheep, monkey, horse, pig, cow, dog, and the like. It is possible to use a rabbit as a host, but is not limited thereto.
  • Monoclonal antibodies can be prepared using any technique that provides for the production of antibody molecules through the culture of continuous cell lines. Such techniques include, but are not limited to, hybridoma technology, human and rat B-cell hybridoma technology, and EBV-hybridoma technology (Kohler G et al ., Nature 256: 495-497, 1975). Kozbor D et al. , J Immunol Methods 81: 31-42, 1985; Cote RJ et al. , Proc Natl Acad Sci 80: 2026-2030, 1983; and Cole SP et al ., Mol Cell Biol 62: 109- 120, 1984).
  • antibody fragments containing specific binding sites for the Xage-1d protein can be prepared.
  • F (ab ') 2 fragments can be prepared by digesting antibody molecules with pepsin, and Fab fragments can be prepared by reducing the disulfide bridges of F (ab') 2 fragments.
  • the Fab expression library can be made smaller to quickly and easily identify monoclonal Fab fragments with the desired specificity (Huse WD et al. , Science 254: 1275-1281, 1989).
  • the antibody can be bound to a solid substrate to facilitate subsequent steps such as washing or separation of the complex.
  • Solid substrates include synthetic resins, nitrocellulose, glass substrates, metal substrates, glass fibers, microspheres and microbeads.
  • the synthetic resins include polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF and nylon.
  • Xage-1a is an amino acid consisting of 81 amino acids
  • Xage-1d is composed of 69 amino acids
  • Table 1 two kinds of transcription variants of Xage1, Xage-1a and Xage-1d protein sequences
  • Xage-1d in order to clarify the relationship between Xage-1d and lung cancer, in order to compare the expression of Xage-1d in a culture of lung cancer cell line, first, after culturing three kinds of lung cancer cell lines, As a result of confirming the expression of -1d, there was a difference according to the cell line, but the expression of Xage-1d in the lung cancer cell line was confirmed. This trend was confirmed to increase as the duration of the culture of lung cancer cell line (see Fig. 1).
  • ELISA using GFGFRRQGEDNT peptide and its biotin-bound biotin-peptide to quantify the amount of Xage-1d present in serum results in Xage in the patient's blood compared to normal The expression level and sensitivity of -1d was confirmed to be high (see FIG. 3 and Table 3).
  • Xage-1d protein is present not only in the culture of lung cancer cell lines, but also in the serum of actual lung cancer patients, and confirmed that Xage-1d expression and sensitivity are higher in lung cancer patients compared to normal individuals.
  • Kits containing antibodies specific for 1d may be usefully used for diagnosing or monitoring lung cancer.
  • lung cancer diagnostic or monitoring kit of the present invention preferably comprises an Xage-1d specific primer as an essential component, but is not limited thereto.
  • reagents necessary for PCR amplification such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus ( Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs.
  • Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above.
  • the anti-Xage-1d antibody is preferably bound to a chromophore, a chromophore, or a fluorescent molecule, but is not limited thereto.
  • the anti-Xage-1d antibody has biotin or biotin having essentially the same binding action as biotin to avidin or streptavidin. It is preferable to use the one bound to the ligand, which is a derivative, and the visualization conjugate bound to the ligand-specific binding molecule to which the chromophore, chromophore or fluorescent molecule is bound is preferably used. It doesn't work.
  • the color development enzyme is preferably horseradish peroxidase (HRP) or basic phosphatase (alkaline phosphatase), the color development material is preferably colloidal gold (colloid gold), the fluorescent molecule is FITC (poly L-lysine-fluorescein isothiocyanate ) And RITC (rhodamine-B-isothiocyanate), but is not limited thereto.
  • HRP horseradish peroxidase
  • basic phosphatase alkaline phosphatase
  • the color development material is preferably colloidal gold (colloid gold)
  • the fluorescent molecule is FITC (poly L-lysine-fluorescein isothiocyanate )
  • RITC rhodamine-B-isothiocyanate
  • Lung cancer diagnostic or monitoring kit of the present invention can diagnose lung cancer by quantitatively or qualitatively analyzing the binding reaction through the antigen-antibody binding reaction, or protein-ligand binding reaction, the binding reaction is a conventional enzyme immunoassay (ELISA) ), Radioimmunoassay (RIA), sandwich assay, western blot, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate coloration, and antigen-antibody aggregation. Can be measured.
  • ELISA enzyme immunoassay
  • RIA Radioimmunoassay
  • sandwich assay Western blot
  • immunoprecipitation immunohistochemical staining
  • fluorescence immunoassay enzyme substrate coloration
  • antigen-antibody aggregation can be measured.
  • the lung cancer diagnosis or monitoring kit of the present invention may be used as a support, a well plate synthesized with a nitrocellulose membrane, a PVDF membrane, a polyvinyl resin or a polystyrene resin, a slide glass made of glass, or the like. have.
  • the Lung cancer diagnostic or monitoring kit of the present invention is a label
  • the chromophore, a chromophoric substance or a fluorescent molecule is preferably a conventional chromophore that the color reaction, HRP (horseradish peroxidase), basic dephosphorase (alkaline phosphatase), colloidal gold (coloid)
  • HRP horseradish peroxidase
  • basic dephosphorase alkaline phosphatase
  • colloidal gold colloidal gold
  • Fluorescent materials such as gold, poly L-lysine-fluorescein isothiocyanate (FITC), and rhodamine-B-isothiocyanate (RITC) and dyes may be used.
  • Lung cancer diagnostic or monitoring kit of the present invention preferably comprises a phosphate buffer, NaCl and Tween 20 as a wash solution, but is not limited thereto.
  • the present invention provides an immunochromatography strip for diagnosing lung cancer using an antibody specific for Xage-1d.
  • the relationship between Xage-1d and lung cancer was confirmed in lung cancer cell lines and actual lung cancer patients. Since the expression level and sensitivity of Xage-1d were significantly higher in lung cancer, antibodies specific for Xage-1d were identified. Immunochromatographic strips can be usefully used for diagnosing lung cancer.
  • the immunochromatographic strip according to the present invention preferably comprises an adhesive plastic support, and a sample pad, a conjugate pad, a signal detection pad, and an absorption pad attached to the adhesive plastic support, but are not limited thereto.
  • the immunochromatography strip preferably has the following configuration, but is not limited thereto:
  • test line interlocked with the conjugate pad, in which a second antibody binding to the conjugate is linearly immobilized, and a control line in which an anti-first antibody immunoglobulin is immobilized.
  • An absorbent pad located downstream of the signal detecting pad, which absorbs the test sample after the signal detecting reaction is completed.
  • the coloring agent of step 3) is preferably colloidal gold particles (gold particles) or horseradish peroxidase (HRP), but is not limited thereto.
  • the signal detection pad of step 4) is preferably made of a nitrocellulose film, but is not limited thereto.
  • the first antibody of step 4) is preferably a monoclonal or polyclonal antibody specifically binding to the Xage-1d protein, but is not limited thereto.
  • the second antibody of step 4) is preferably a monoclonal or polyclonal antibody that specifically binds to the primary antibody-conjugate, but is not limited thereto.
  • the absorbent pad of step 5) preferably includes a porous support and an absorbent dispersed in pores of the porous support or adsorbed or coated on the fiber yarn of the porous support. It is preferable to further include a porous film layer attached to the upper surface, but is not limited thereto.
  • the absorbent is preferably selected from the group consisting of calcium chloride, magnesium chloride, diatomaceous earth, bentonite, dolomite, gypsum, silica gel and mixtures thereof, but is not limited thereto.
  • the immunochromatography strip according to the present invention is characterized in that a test sample is determined as a positive sample of lung cancer when colored lines appear in the control line and the detection line on the immunochromatography strip.
  • plasma which is a test sample
  • the sample pad may further have a function of filtering in order to further improve the selectivity for the analyte or to minimize the influence of the interference material which may be included in the test sample.
  • an auxiliary pad may be further provided upstream of the sample pad containing a substance that can increase the reaction between the analyte and the conjugate or eliminate the influence by the interference.
  • Blood introduced through the sample pad is transferred to a conjugate pad located upstream of the sample pad through chromatographic movement.
  • the conjugate pad contains a conjugate that specifically binds to the Xage-1d protein contained in blood.
  • the conjugate is labeled with gold particles, latex particles, fluorescent materials, enzymes and the like.
  • the test sample passing through the conjugate pad moves to the signal detection pad.
  • the signal detection pad includes a detection line for detecting whether an analyte is present in the test sample, and a control line for confirming whether the assay kit is normally operated regardless of the presence or absence of the analyte.
  • the detection line is coated with a substance (or signal detection material) that selectively and specifically binds to a binding product between the analyte and the conjugate contained in the conjugate pad, and the control line is coated with the conjugate pad. It is preferable that the material specifically binding to the conjugate contained in the coating is not limited thereto.
  • the signal detection pad is composed of a porous membrane pad, it may be made of nitrocellulose, cellulose, polyethylene, polyethersulfone, nylon and the like.
  • the present invention also provides a biosensor for diagnosing or monitoring lung cancer comprising an Xage-1d specific antibody.
  • the biosensor is characterized in that the capacitance and the amount of the microelectromagnetic field and its amount of change by a biosensor of a single or multi-channel biosensor radiated from biological tissues such as cells, tissues, organs, etc. Since the biosensor detects the amount of change, it can be used as a means for diagnosing or monitoring lung cancer.
  • the biosensor includes a lung cancer diagnostic substrate to which an antibody capable of specifically binding to Xage-1d is attached, and a detection means for detecting lung cancer specific antigen bound to an antibody on an electrical substrate, wherein the antigen is bound to an antibody on a substrate.
  • the primary antibody-gold conjugate that can specifically bind to or the complex of a secondary antibody-signal material that specifically binds to the primary antibody and the primary antibody that can specifically bind to the antigen bound to the antibody of the electrical substrate. It is preferable to use.
  • the signal substance is not particularly limited thereto, but the fluorescent substance (for example, Cy-3, Cy-5, FITC, GFP (green fluorescent protein), RFP) (red fluorescent protein), Texas Red, etc.), luminescent materials, radioisotopes, enzymes (e.g. horse raddish peroxidase), alkaline phosphatase, beta galactosidase ( ⁇ - galactosidase), luciferase, etc.), and the like, and in the case of using the enzyme as a signal material, the kit for lung cancer diagnosis may further include a coloring reagent causing a color reaction by an electric enzyme.
  • the fluorescent substance for example, Cy-3, Cy-5, FITC, GFP (green fluorescent protein), RFP) (red fluorescent protein), Texas Red, etc.
  • luminescent materials for example, radioisotopes, enzymes (e.g. horse raddish peroxidase), alkaline phosphatase, beta galactosidase ( ⁇ - gal
  • biomolecules that specifically bind to the Xage-1d protein provide a biochip for monitoring, diagnosing and screening breast cancer integrated in a solid substrate.
  • the biomolecule is preferably an antibody or aptamer, but is not limited thereto.
  • the solid substrate is preferably selected from the group consisting of plastic, glass, metal and silicon, but is not limited thereto.
  • a high throughput screening (HTS) system which includes a fluorescence method performed by detecting a fluorescence by attaching a fluorescent substance to a detector or Radiation method performed by attaching a radioisotope to a detector to detect radiation; It is preferable to use a surface plasmon resonance (SPR) method for measuring the plasmon resonance change of the surface in real time without labeling the detector or a surface plasmon resonance imaging (SPRI) method for imaging and confirming the SPR system.
  • SPR surface plasmon resonance
  • SPRI surface plasmon resonance imaging
  • lung cancer diagnosis or monitoring method in which a sample is reacted with a plate having various kinds of antibodies including an antibody capable of specifically binding to Xage-1d, and then the bound protein is analyzed by mass spectrometry.
  • the present invention provides a lung cancer diagnostic method using Xage-1d as a protein marker.
  • the relationship between the Xage-1d protein and lung cancer was confirmed in lung cancer cell lines and actual lung cancer patients, and the expression level and sensitivity of Xage-1d were significantly higher in lung cancer, so Xage-1d was used for the diagnosis of lung cancer. It can be useful.
  • the lung cancer diagnostic method of the present invention preferably includes the following steps, but is not limited thereto:
  • step 2) comparing the expression level of Xage-1d in step 1) with the expression level of Xage-1d in the normal blood sample;
  • the subject of step 1) is a vertebrate, including human, preferably a mammal, more preferably human, ape, bovine, pig, rat, rabbit, guinea peak, hamster, dog or Cats can be used, but are not limited thereto.
  • the expression level of Xage-1d in step 1) is Western blotting, Enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining, immunoprecipitation ( It is preferable to measure by any one selected from the group consisting of immunoprecipitation) and immunofluorescence, but is not limited thereto.
  • lung cancer cell lines A549 lung carcinoma
  • H460 lung adenocarcinoma
  • non-small cell carcinoma HOP92 Zhong et. al., J. Biol. Chem. 284, 23225-23233.
  • 293T human embryonic kidney cell line
  • A549, H460 and HOP92 cell lines were cultured in a humidified 5% CO 2 37 o C incubator with 10% fetal bovine serum (FBS, Invitrogen) added to RPMI1640 medium (Invitrogen). The culture was incubated in Dulbecco's modification of Eagle's medium (DMEM, Invitrogen) with 10% FBS. All cells were divided into 1 ⁇ 10 5 cells in a 60 mm culture dish, and after 2 days and 3 days, the medium was taken and used in the experimental example.
  • FBS fetal bovine serum
  • DMEM Dulbecco's modification of Eagle's medium
  • Xage1 has four types of a, b, c, and d. There are transcription variants by RNA splicing, and two kinds of Xage1 transcription variants Xage-1a and Xage-1d protein sequences were compared.
  • Xage-1a is an amino acid consisting of 81 amino acids
  • Xage-1d is composed of 69 amino acids
  • comparing the two amino acid sequences, the two proteins are amino acid N terminal From the results, it was confirmed that 32 amino acid sequences were included in common (see Table 1).
  • an enzyme-linked immunosorbent assay was performed.
  • Antibodies specific for xage-1d include antibody AB27477 (Abcam), which binds to GFGFRRQGEDNT at the carboxyl terminus, with a coating solution [1.59 g Na 2 CO 3 , 2.93 g NaHCO 3 , 2 ml 10% NaN 3 , pH 9.5 based on 1 L]. 1 ⁇ g per well was dissolved in and coated in a 96-well plate (Nunc # 439454) for 24 hours. To inhibit nonspecific binding, 200 ⁇ l of blocking solution per well [PBS containing 0.1% BSA (Bovine serum albumin) and 0.02% Thimerosal] was treated at room temperature for 2 hours.
  • BSA Bovine serum albumin
  • washing solution PBS containing 0.05% Thimerosal, 0.05% Tween-20
  • 100 ⁇ l of the cell line culture medium was added to the wells.
  • washing solution PBS containing 0.05% Thimerosal, 0.05% Tween-20
  • 100 ⁇ l of the cell line culture medium was added to the wells.
  • the coated plate was stored at room temperature for 2 to 3 hours to induce the reaction, and then washed 5 times with 200 ⁇ l of washing solution.
  • the primary antibody for detecting Xage-1d was diluted in PBS at 1: 2,000 in LS-B318 (Lifespan) rabbit antibody, which is known to specifically recognize the amino terminal, and added to 100 ⁇ l per well, followed by 2 at room temperature.
  • FIG. 1 the presence of Xage-1d was confirmed in the three types of lung cancer cell lines used in the experiment.
  • the amount of Aage or adenocarcinoma cell line H460 was higher than that of A549 or HOP92. It can be seen that this exists (Fig. 1).
  • Antibodies specific for xage-1d include antibody AB27477 (Abcam), which binds to GFGFRRQGEDNT at the carboxyl terminus, with a coating solution [1.59 g Na 2 CO 3 , 2.93 g NaHCO 3 , 2 ml 10% NaN 3 , pH 9.5 based on 1 L]. It was dissolved in 1 ⁇ g per well and coated in a 96-well plate (Nunc # 439454) for 24 hours. To inhibit nonspecific binding, 200 ⁇ l of blocking solution per well [0.1% BSA (Bovine serum albumin), PBS containing 0.02% Thimerosal] was treated at room temperature for 2 hours.
  • BSA Bovine serum albumin
  • the blocking solution was discarded from the wells, and the wells were washed 5 times with 200 ⁇ l of washing solution [PBS containing 0.05% Thimerosal, 0.05% Tween-20], and 100 ⁇ l of lung cancer patients were added to each well. .
  • washing solution [PBS containing 0.05% Thimerosal, 0.05% Tween-20]
  • 100 ⁇ l of lung cancer patients were added to each well. .
  • the coated plate was stored at room temperature for 2 to 3 hours to induce the reaction, and then washed 5 times with 200 ⁇ l of washing solution.
  • the primary antibody for detecting Xage-1d was diluted in PBS at 1: 2,000 in LS-B318 (Lifespan) rabbit antibody, which is known to specifically recognize the amino terminal, and added to 100 ⁇ l per well, followed by 2 at room temperature.
  • peptide competition enzyme immunoassay was performed to quantitatively measure the expression level of Xage-1d in serum, while avoiding the reaction between antibodies that can be induced by the use of various kinds of antibodies. That is, HRP-bound streptavidin (Peptron) using a peptide (GFGFRRQGEDNT, Abcam) corresponding to the carboxyl terminus of Xage-1d and a biotin-synthesized biotin-GFGFRRQGEDNT (Peptron) STR-HRP) to induce color development.
  • HRP-bound streptavidin Peptron
  • GFGFRRQGEDNT a peptide
  • Abcam biotin-synthesized biotin-GFGFRRQGEDNT
  • Antibody (AB27477) that specifically binds to the carboxyl terminus of Xage-1d was dissolved in a coating solution and coated on a 96-well plate, and then nonspecific binding was inhibited with a blocking solution. After washing the antibody or blocking solution not coated with the washing solution, the biotin-peptide (biotin-GFGFRRQGEDNT) was dissolved in PBS by concentration in the plate and then reacted at room temperature for 2 to 3 hours. After washing the plate 5 times with washing solution, each well was added STR-HRP (Pierce Co., Ltd.) with HRP conjugated to streptavidin specific for biotin, diluted 1: 10,000, and then bound again for 2 to 3 hours.
  • STR-HRP Pieris Co., Ltd.
  • the reaction was performed at room temperature by adding TMB substrate solution (GenDEPOT) to each well for peroxidase color development, and then stopping the reaction by adding a stop solution (GenDEPOT) again at 450 nm wavelength. Absorbance was measured.
  • biotin-peptide (biotin-GFGFRRQGEDNT) specifically binds to the coated antibody and is colored by STR-HRP, and the absorbance increases in proportion to the amount of the bio-peptide. It could be confirmed (A of FIG. 3). Based on these results, the amount of biotin-peptide to be used for antigen competitive enzyme immunoassay was determined to be 4 ng / ml.
  • the reaction was performed at room temperature by adding TMB substrate solution (GenDEPOT) to each well for peroxidase color development, and then stop the reaction by adding a stop solution (GenDEPOT) and absorbance at 450 nm wavelength was measured.
  • TMB substrate solution GenDEPOT
  • GenDEPOT stop solution
  • each well was added STR-HRP (Pierce Co., Ltd.) with HRP conjugated to streptavidin specific for biotin, diluted 1: 10,000, and then bound again for 2 to 3 hours.
  • the reaction was performed at room temperature by adding TMB substrate solution (GenDEPOT) to each well for peroxidase color development, and then stopping the reaction by adding a stop solution (GenDEPOT) again at 450 nm wavelength. Absorbance was measured.
  • Xage-1d present in blood was quantified by calculating the molecular weight ratio of peptide and Xage-1d protein to the absorbance value.
  • the mean value of the sample of the normal person was 0.867 ng / ml while the mean of the patient's blood was 22.342 ng / ml that is more than 25 times. Based on the maximum amount of normal subjects who were completely excluded from normal subjects, the patient's blood showed 8 low values, and the sensitivity of lung cancer patients was 89.3. 3).
  • Xage-1d of the present invention is present not only in the culture of lung cancer cell lines, but also in the blood of lung cancer patients, and is present in more blood in lung cancer patients compared to normal people, and is sensitive to small amounts. Since lung cancer can be diagnosed, using Xage-1d as a protein marker for diagnosing lung cancer, it can be used for diagnosis kits, immunochromatography strips and lung cancer diagnosis.

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Abstract

La présente invention concerne un marqueur de Xage-1D pour le diagnostic du cancer pulmonaire, et une trousse de diagnostic utilisant un tel marqueur. Plus précisément, suite à la confirmation de la présence de Xage-1D dans un fluide de culture d'une lignée de cellules cancéreuses du poumon, on vérifie que la quantité de Xage-1D dans le sérum d'un patient actuel atteint de cancer pulmonaire est supérieure à celle d'une personne normale, et que la sensibilité est excellente, confirmant ainsi que le gène Xage-1D peut être utilisé comme marqueur de diagnostic du cancer pulmonaire et peut être utilisé dans la trousse de diagnostic pour le cancer pulmonaire.
PCT/KR2010/003710 2010-04-15 2010-06-10 Marqueur xage-1d pour le diagnostic du cancer pulmonaire, et trousse de diagnostic utilisant un tel marqueur Ceased WO2011129483A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113474342A (zh) * 2019-02-28 2021-10-01 国立大学法人东京大学 检测癌症的荧光探针
CN113614536A (zh) * 2018-12-12 2021-11-05 盛捷宁克斯私人有限公司 非小细胞肺癌的生物标志物的检测

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080292633A1 (en) * 1999-06-30 2008-11-27 Corixa Corporation Compositions and methods for the therapy and diagnosis of lung cancer
US7608394B2 (en) * 2004-03-26 2009-10-27 Sequenom, Inc. Methods and compositions for phenotype identification based on nucleic acid methylation
US20090324651A1 (en) * 2005-10-04 2009-12-31 Ludwig Institute For Cancer Research Methods for Stimulating an Immune Response Using Bacterial Antigen Delivery System

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080292633A1 (en) * 1999-06-30 2008-11-27 Corixa Corporation Compositions and methods for the therapy and diagnosis of lung cancer
US7608394B2 (en) * 2004-03-26 2009-10-27 Sequenom, Inc. Methods and compositions for phenotype identification based on nucleic acid methylation
US20090324651A1 (en) * 2005-10-04 2009-12-31 Ludwig Institute For Cancer Research Methods for Stimulating an Immune Response Using Bacterial Antigen Delivery System

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EGLAND KA. ET AL.: "Characterization of overlapping XAGE-1 transcripts encoding a cancer testis antigen expressed in lung, breast, and other types of cancers.", MOL. CANCER THER., vol. 1, 2002, pages 441 - 450 *
SATO S. ET AL.: "Identification of XAGE-1 isoforms: predominant expression of XAGE-1b in testis and tumors", CANCER IMMUNITY., vol. 7, 2007, pages 5 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113614536A (zh) * 2018-12-12 2021-11-05 盛捷宁克斯私人有限公司 非小细胞肺癌的生物标志物的检测
CN113474342A (zh) * 2019-02-28 2021-10-01 国立大学法人东京大学 检测癌症的荧光探针

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KR101130842B1 (ko) 2012-03-29

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