KR20110115292A - Protein marker xage-1d for lung cancer and diagnosis kit for lung cancer using an antibody against the same - Google Patents

Abstract

The present invention relates to a Xage-1d marker for diagnosing lung cancer and a diagnostic kit using the same. More specifically, after confirming that Xage-1d is present in the culture of lung cancer cell line, the amount of Xage-1d in the serum of a real lung cancer patient is normal. By confirming the high sensitivity and excellent sensitivity, Xage-1d can be used as a diagnostic marker for lung cancer, it was confirmed that it can be useful in the lung cancer diagnostic kit.

Description

Protein marker Xage-1d for lung cancer and diagnosis kit for lung cancer using an antibody against the same}

It relates to a lung cancer diagnostic or monitoring kit comprising an antibody specific for Xage-1d and immunochromatography strips and lung cancer diagnostic method using Xage-1d.

According to the Korea Central Cancer Registry released in 2008, 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. By age group, 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). Moreover, 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. Cause of death Statistical yearbook, Statistics Korea).

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).

Various methods have been used to diagnose lung cancer. Until now, the tumor size was measured, the metastasis to lymph nodes, the biopsy of lung tumor tissues and lymph nodes, immunohistochemistry, or chest computed tomography were used. Photography, bronchoscope, etc. (Manser et al ., Curr. Opin. Pulm. Med., 2004, 10, 266-271). However, chest tomography requires measurable lung cancer size of about 0.1 cm or more, which is likely due to the fact that the cancer has already metastasized to other tissues. The method has limitations that make it difficult to observe the tumor at the lung end.

Therefore, there has been a study to develop a lung cancer marker for diagnosing cancer, and as a part of this study, a study for discovering a lung cancer diagnostic marker by examining expression of multiple target genes or proteins has been reported (Hibi et. al ., Am. J. Pathol., 1999, 155: 711-715; Brechot et al . Eur. J. Cancer., 1997, 33: 385-391; Pastor et al ., Eur. Respir. J., 1997, 10: 603-609; Morita et al ., Int. J. Cancer., 1998, 78: 286-292). In addition, there have been reports to discover genes to be used as lung cancer markers using microarray technology. In addition, attempts to diagnose lung cancer by measuring the concentrations of aspartate aminotransferase (AST), alanine transaminase (ALT), total bilirubin or creatinine in the patient's blood (Fishman et al ., Cancer Res. 28,150-154 (1968), Nagamine et al ., Clin. Chem. 29, 379-381 (1983), Muggia et al ., 1, 217-228. (1974). However, despite various studies to discover these tumor markers, the excavated tumor markers are still in limited use, and currently there is no officially recommended lung cancer blood marker.

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 ., Int J Cancer , 2002, 97: 195-204, Lim JH et al ., Int J Cancer , 2005, 116: 200-206. Currently only Xage-1a (formerly Xage-1b) and Xage-1d are registered with NCBI (http://www.ncbi.nlm.nih.gov/gene).

Looking at the conventional Xage-1 study, it has been reported that the study of the Xage-1b of the four types of Xage-1. For example, a study using Xage-1b-stimulated dendritic cells as a tumor vaccine has been published (Clin Exp Immunol. 2008 Sedp; 153 (3): 392-400), and the expression of Xage-1b in patients with non-small cell carcinoma. Although studies have been reported on the relationship (Cancer Immun. 2008 Aug 28; 8:13), no studies on Xage-1d have been reported, and no relationship with lung cancer has been revealed.

Accordingly, 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.

It is an object of the present invention to provide a lung cancer diagnostic or monitoring kit using Xage-1d as a marker and an immunochromatography strip comprising the same.

Another object of the present invention to provide a lung cancer diagnostic method using Xage-1d.

It is an object of the present invention to provide a lung cancer diagnostic or monitoring kit comprising an antibody specific for 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.

In addition, 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. , 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.

Hereinafter, the present invention will be described in detail.

It is an object of the present invention to provide a lung cancer diagnostic or monitoring kit comprising an antibody specific for Xage-1d.

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. In addition, 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 murine 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).

In addition, antibody fragments containing specific binding sites for the Xage-1d protein can be prepared. For example, but not limited to, 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. Alternatively, 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. In addition, the synthetic resins include polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF and nylon.

In an embodiment of the present invention, two kinds of transcription variants of Xage1, Xage-1a and Xage-1d protein sequences, were compared. Xage-1a is an amino acid consisting of 81 amino acids, it can be seen that Xage-1d is composed of 69 amino acids, and comparing the two amino acid sequences, the two proteins have a common 32 amino acid sequence from the amino acid N terminus Yes, it was confirmed (see Table 1).

In addition, in an embodiment of the present invention, 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).

In addition, in an embodiment of the present invention, in order to examine the relationship between Xage-1d and actual lung cancer, the blood of lung cancer patients, the presence of Xage-1d present in the blood using the blood of normal patients as a control Compared. As a result, it was confirmed that the expression of Xage-1d was more present in the blood of lung cancer patients than in normal blood, and these results were analyzed by the sensitivity and absorbance of Xage-1d for lung cancer. Showed high sensitivity to lung cancer (see Table 2).

In addition, in an embodiment of the present invention, 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 blood of the patient compared to the normal person. The expression level and sensitivity of -1d was confirmed to be high (see FIG. 3 and Table 3).

Thus, in an embodiment of the present invention, 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.

In addition, 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. When Xage-1d detection using Xage-1d specific primers is applied to PCR amplification, optionally, reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus ( Tth), Thermusfiliformis, 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.

In lung cancer diagnostic or monitoring kit comprising an antibody specific for Xage-1d of the present invention, the anti-Xage-1d antibody is preferably bound to a chromophore, a chromophore, or a fluorescent molecule, but is not limited thereto.

In addition, in a lung cancer diagnostic or monitoring kit comprising an antibody specific for Xage-1d of the present invention, 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 HRP (horseradish peroxidase) or basic dephosphatase (alkaline phosphatase), the color development material is preferably colloidal gold (coloid gold), the fluorescent molecule is a FITC (poly L-lysine-fluorescein isothiocyanate ) And RITC (rhodamine-B-isothiocyanate), but is not limited thereto.

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.

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.

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) 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.

In addition, the present invention provides an immunochromatography strip for diagnosing lung cancer using an antibody specific for Xage-1d.

In an embodiment of the present invention, 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.

Specifically, the immunochromatography strip preferably has the following configuration, but is not limited thereto:

1) a support;

2) a sample pad attached to the upper surface of the support, attaching a test sample to be analyzed;

3) a conjugate pad linked to the sample pad and containing a first antibody conjugate that specifically binds to the Xage-1d protein contained in the sample to be tested;

4) a 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. A signal detection pad; And

5) An absorbent pad located downstream of the signal detecting pad, which absorbs the test sample after the signal detecting reaction is completed.

In the above configuration, the coloring agent of step 3) is preferably colloidal gold particles (gold particles) or horseradish peroxidase (HRP), but is not limited thereto.

In the above configuration, the signal detection pad of step 4) is preferably made of a nitrocellulose film, but is not limited thereto.

In the above configuration, 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.

In the above configuration, 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.

In the above configuration, 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.

In the immunochromatography strip according to the present invention, plasma, which is a test sample, is first supplied to the immunochromatography strip through the sample pad. 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. If necessary, 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. To this end, 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.

In particular, when the secondary antibody-signal complex is used as the detection means, 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 (β- 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.

In addition, 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.

In addition, as a method for searching for the amount of Xage-1d from the subject, it is preferable to use 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.

In addition, 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. To provide.

In addition, the present invention provides a lung cancer diagnostic method using Xage-1d as a protein marker.

In the embodiment of the present invention, 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.

Specifically, the lung cancer diagnostic method of the present invention preferably includes the following steps, but is not limited thereto:

1) measuring the expression level of Xage-1d in a blood sample derived from the subject;

2) comparing the expression level of Xage-1d in step 1) with the expression level of Xage-1d in the normal blood sample; And

3) Determining that there is a high risk of lung cancer when the amount of Xage-1d expression is increased compared to the normal control group.

In the method, 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.

In the method, 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.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

< Example  1> Cell Culture

To observe protein secretion in cell lines, lung cancer cell lines A549 (lung carcinoma), H460 (lung adenocarcinoma) (Zhu et al., British J. Cancer, 94, 1936-1941 (2006)), non-small cell cancer cell line HOP92 (Zhong et. al., J. Biol. Chem. 284, 23225-23233. (2009)) and 293T (human embryonic kidney cell line) as a control (Senechal et. al., Human Mol. Genetics 14, 1613- 1620, (2005)) cells were cultured. The A549, H460, and HOP92 cell lines were maintained at 5% CO ₂ in humidity by adding 10% fetal bovine serum (FBS, Invitrogen) to RPMI1640 medium (Invitrogen). The cells were cultured in a 37 ^° C. incubator, and 293T cell lines were cultured 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.

< Experimental Example  1> Lung cancer cell line in culture Xage -1d detection

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.

As a result, as shown in Table 1, Xage-1a is an amino acid consisting of 81 amino acids, it was confirmed that Xage-1d is composed of 69 amino acids, and 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).

In addition, in order to confirm the presence or absence of Xage-1d confirming the difference of the amino acid sequence in the culture of lung cancer cell line, cultured lung cancer cell line A549, lung adenocarcinoma cell line H460, arsenic lung cancer line HOP92 and 293T cells cultured in the above example After that, 100 µl of the cell culture was taken to confirm the presence of Xage-1d.

Specifically, an enzyme-linked immunosorbent assay (ELISA) 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 μg per well was dissolved in and coated in a 96-well plate (Nunc # 439454) for 24 hours. To inhibit nonspecific binding, 200 μl 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. After 2 hours, the blocking solution was discarded from the wells and the wells were washed 5 times with washing solution (PBS containing 0.05% Thimerosal, 0.05% Tween-20), and 100 μl of the cell line culture medium Was added to the wells. As a control, the same amount of 293T human embryonic kidney cell line culture cultured for 3 days was used. The coated plate was stored at room temperature for 2 to 3 hours to induce the reaction, and then washed 5 times with 200 μl 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 μl per well, followed by 2 at room temperature. It was reacted for 3 hours. After the reaction, the mixture was washed five times with 200 μl of washing solution per well, and then HRP (horse radish peroxidase) conjugated anti-rabbit-goat antibody diluted 1: 5,000 to 10,000 in PBS was added to each well at room temperature. After reacting for 1 hour, the mixture was washed 5 times with a washing solution. To develop peroxides, 50 μl of TMB substrate solution (GenDEPOT) was added to each well for 10 minutes at room temperature, and then 50 μl of stop solution (GenDEPOT) was added to stop the reaction. Absorbance was measured.

As a result, as shown in FIG. 1, the presence of Xage-1d was confirmed in the three types of lung cancer cell lines used in the experiment. When comparing the lung cancer cell lines, 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)

Protein Sequences by Transcription Variants of Xage-1 Xage1 variant types Reference sequence number order Number of amino acids Xage-1a
(Even with Xage-1b
Known) NP_001091061
NP_001091063
NP_001091066
NP_065144
NP_001091073 MESPKKKNQQ LKVGILHLGS RQKKIRIQLR SQ CATWKVIC KSCISQTPGI NLDLGSGVKV KIIPKEEHCK MPEAGEEQPQ V 81 Xage-1d NP_001091062
NP_001091065
NP_001091067
NP_597673
NP_001091074 MESPKKKNQQ LKVGILHLGS RQKKIRIQLR SQ VLGREMRD MEGDLQELHQ SNTGDKSGFG FRRQGEDNT 69

* Both proteins contain an underlined amino acid in common at the amino terminus.

< Experimental Example  2> In lung cancer patients blood Xage -1d detection

In order to check whether Xage-1d detected in the culture of lung cancer cell line is present in the blood of lung cancer patients, 90 blood of lung cancer patients and 31 normal bloods provided by the National Cancer Center were used in the blood of Xage-1d. The amounts were compared through ELISA experiments. 100 μl of erythrocyte and platelet-free serum was analyzed using the enzyme immunoassay experiment.

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 μg per well and coated in a 96-well plate (Nunc # 439454) for 24 hours. To inhibit nonspecific binding, 200 μl of blocking solution per well [0.1% BSA (Bovine serum albumin), PBS containing 0.02% Thimerosal] was treated at room temperature for 2 hours. After 2 hours, the blocking solution was discarded from the wells, and the wells were washed 5 times with 200 μl of washing solution [PBS containing 0.05% Thimerosal, 0.05% Tween-20], and 100 μl of lung cancer patients were added to each well. . As a control, the blood of normal people was used in the same amount. The coated plate was stored at room temperature for 2 to 3 hours to induce the reaction, and then washed 5 times with 200 μl 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 μl per well, followed by 2 at room temperature. It was reacted for 3 hours. After the reaction, the cells were washed with 200 μl of washing solution per well, and then HRP (horse radish peroxidase) conjugated anti-rabbit-chlorine antibody was diluted 1: 5,000 to 10,000 in PBS and added to each well for 1 hour at room temperature. After the reaction, the mixture was washed five times with a washing solution. To develop peroxides, 50 μl of TMB substrate solution (GenDEPOT) was added to each well for 10 minutes at room temperature, and then 50 μl of stop solution (GenDEPOT) was added to stop the reaction. Absorbance was measured.

As a result, as shown in FIG. 2, the absorbance measured in 31 normal samples was 0.353 in average and 0.448 in average, whereas the mean value of the measured values in 90 patient samples was 1.256, minimum was 0.124, and maximum was 2.365. High absorbance was shown in comparison with normal patients (FIG. 2).

Seven cases of lung cancer patients showed lower values than normal samples, but the sensitivity and specificity of the absorbance values based on the highest values of normal people were 92.2% and 100%, respectively. That is, despite the very high specificity, the sensitivity was also very high, it can be seen that the Xage-1d present in the blood is very sensitive to lung cancer (Table 2).

Sensitivity and specificity when the absorbance value is set to 0.448, the maximum value of a normal sample. normal patient Samples (121 total) 31 90

Absorbance 0.448 standard
≤ 0.448 31 7 ＞ 0.448 0 83 responsiveness
(Sensitivity) Sensitivity = (83/90) × 100 = 92.2 (%) Specificity
(Specificity) Specificity = (31/31) × 100 = 100 (%)

< Experimental Example  3> in serum Xage Quantification curve analysis of -1d protein

In the ELISA test method, 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.

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. After washing the well with a washing solution, 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.

As a result, as shown in FIG. 3A, 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.

Therefore, if the amount of biotin-peptide is fixed and the biotin-unbound peptide (GFGFRRQGEDNT) is added at various concentrations, then competitive binding to the coated antibody changes the absorbance according to the concentration of the added peptide. This method was used to draw a quantification curve to calculate the concentration of Xage-1d in serum. 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 that is not coated with the washing solution, add the biotin-bind peptide to the biotin-peptide fixed at 4 ng according to the concentration, mix well, add to the plate, and react at room temperature for 2 to 3 hours. I was. After washing the plate five times with a washing solution, STR-HRP (Pierce Co., Ltd.), in which HRP is bound to biotin-specific streptavidin, was added at a dilution of 1: 10,000, bound for 2-3 hours, and then well again. After washing with a washing solution, 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.

As a result, a quantification curve such as B of FIG. 3 having a reduced absorbance in inverse proportion to the amount of peptide added was completed (B of FIG. 3).

< Experimental Example  4> Biotin In serum using peptides Xage -1d Protein Quantification

In order to quantify Xage-1d protein in serum, absorbance was measured using serum instead of peptide in Experimental Example 3, and the measured absorbance was substituted into B of FIG. 3 to calculate the concentration of Xage-1d. Antibodies specific for the peptide (GFGFRRQGEDNT) were dissolved in the coating solution and coated on 96-well plates, and then nonspecific binding was inhibited with the blocking solution. After washing the antibody or blocking solution not coated with the washing solution, the serum was dissolved in PBS by concentration and added to 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. After washing the well with a washing solution, 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.

As a result, as shown in Table 3, the average value of the sample of the normal person was 0.867 ng / ㎖ while the mean of the patient's blood was 22.342 ng / ㎖ 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).

Comparison of sensitivity and specificity based on peak and mean volume of normal blood.
Samples (108 total) normal patient 33 75 Max (ng / ml) 1.306 90.949 Minimum amount (ng / ml) 0.607 0.812 Average (ng / ml) 0.867 22.342 1.306 ng / ml standard
(Max of normal person)


 1.306 33 8 > 1.306 0 67 responsiveness
(Sensitivity) Sensitivity = (67/75) × 100 = 89.3 Specificity
(Specificity) Specificity = (33/33) × 100 = 100 Based on 0.867 ng / ml
(Average of normal people)


 0.867 16 One > 0.867 17 74 responsiveness
(Sensitivity) Sensitivity = (74/75) × 100 = 98.7 Specificity
(Specificity) Specificity = (16/33) × 100 = 48.5

As described above, the 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. 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.

<110> Jinan Red Ginsen Oriental Medicine Cluster Corporation <120> Composition for the prevention and treatment of lipid-related          cardiovascular disease or obesity containing the extracts of          Dipsacus asperoides as active ingredient <130> 10p-04-07 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> UCP1 forward primer <400> 1 gggcccttgt aaacaacaaa 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> UCP1 reward primer <400> 2 gccacaaacc ctttgaaaaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> UCP2 forward primer <400> 3 agctgggtac tcgggattct 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> UCP2 reward primer <400> 4 catggagagg ctcagaaagg 20

Claims (22)

Lung cancer diagnostic kit comprising an antibody specific for Xage-1d described in SEQ ID NO: 1.
According to claim 1, wherein the anti-Xage-1d antibody lung cancer diagnostic kit, characterized in that coupled to the chromophore, chromophores or fluorescent molecules.
The kit for diagnosing lung cancer according to claim 2, wherein the color developing enzyme is horseradish peroxidase (HRP) or basic dephosphatase (alkaline phosphatase).
The kit for diagnosing lung cancer according to claim 2, wherein the coloring material is colloidal gold.
The kit for diagnosing lung cancer according to claim 2, wherein the fluorescent molecule is poly L-lysine-fluorescein isothiocyanate (FITC) or rhodamine-B-isothiocyanate (RITC).
The kit for diagnosing lung cancer according to claim 1, wherein the anti-Xage-1d antibody is bound to a ligand.
7. The kit for diagnosing lung cancer according to claim 6, wherein the ligand is biotin or a biotin derivative having essentially the same binding action as biotin to avidin or streptavidin.
7. The kit for diagnosing lung cancer according to claim 6, further comprising a visualization conjugate in which a chromophore, a chromophore, or a fluorescent molecule bound to a ligand specific binding molecule is bound.
7. The kit for diagnosing lung cancer according to claim 6, wherein the chromatase is horseradish peroxidase (HRP) or basic dephosphatase (alkaline phosphatase).
7. The lung cancer diagnostic kit of claim 6, wherein the coloring material is colloidal gold.
The kit for diagnosing lung cancer according to claim 4, wherein the fluorescent molecule is poly L-lysine-fluorescein isothiocyanate (FITC) or rhodamine-B-isothiocyanate (RITC).
1) a support;
2) a sample pad attached to the upper surface of the support, attaching a test sample to be analyzed;
3) a conjugate pad linked to the sample pad and containing a first antibody conjugate that specifically binds to the Xage-1d protein contained in the sample to be tested;
4) a 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. A signal detection pad; And
5) An immunochromatography strip for diagnosing lung cancer, comprising an absorbent pad located downstream of the signal detecting pad, the absorbing test sample having completed the signal detecting reaction.
13. The immunochromatographic strip of claim 12, wherein the color developer of step 3) is a colloidal gold particle or horseradish peroxidase (HRP).
13. The immunochromatography strip according to claim 12, wherein the signal detection pad of step 4) is any one selected from the group consisting of nitrocellulose, cellulose, polyethylene, polyethersulfone and nylon.
13. The immunochromatographic strip of claim 12, wherein the first antibody of step 4) is a mono or monoclonal or polyclonal antibody that specifically binds to the Xage-1d protein.
The immunochromatographic strip of claim 12, wherein the second antibody of step 4) is a monoclonal or polyclonal antibody that specifically binds to a primary antibody-conjugate.
13. The immunochromatography according to claim 12, wherein the absorbent pad of step 5) comprises a porous support and an absorbent dispersed in a pore of the porous support or adsorbed or coated on the fiber yarn of the porous support. strip.
The immunochromatographic strip according to claim 12, wherein when a colored line appears in the control line and the detection line on the immunochromatography strip, the test sample is determined as a positive sample of lung cancer.
1) measuring the expression level of Xage-1d in a blood sample derived from the subject;
2) comparing the expression level of Xage-1d in step 1) with the expression level of Xage-1d in the normal blood sample; And
3) Protein detection method for providing information of lung cancer diagnosis comprising the step of determining that there is a high risk of lung cancer when the amount of Xage-1d expression compared to the normal control.
20. The method of claim 19, wherein the expression level of Xage-1d in step 1) is Western blotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining, immunoprecipitation Protein detection method, characterized in that selected from the group consisting of (immunoprecipitation) and immunofluorescence (immunofluorescence).
A lung cancer prognosis monitoring kit comprising an antibody specific for Xage-1d set forth in SEQ ID NO: 1.
A lung cancer prognosis monitoring method comprising an antibody specific for Xage-1d described in SEQ ID NO: 1.

Images