KR101431065B1 - Protein marker neural cell adhesion molecule l1-like protein for breast cancer diagnosis, method of detecting the same, and diagnosis kit for breast cancer using antibody against the same - Google Patents

Abstract

The present invention relates to a method for detecting a nerve cell adhesion molecule L1-like protein, which is a protein marker capable of diagnosing breast cancer, in a blood sample through a multiple reaction monitoring method, a method for detecting a breast cancer, , And a method for detecting a neuronal cell adhesion molecule L1-like protein in the blood using an antigen-antibody binding reaction. The method and kit for detecting the neural cell adhesion molecule L1-like protein, which is a protein marker for breast cancer diagnosis of the present invention, in a blood sample through the MRM method or the antigen-antibody binding reaction not only has a high sensitivity and accuracy of diagnosis, And it can be used for early diagnosis of breast cancer because it can diagnose breast cancer very easily.

Description

TECHNICAL FIELD [0001] The present invention relates to a breast cancer diagnostic kit comprising breast cancer cell line, neural cell adhesion molecule L1-like protein, breast cancer diagnostic cell line L1-like protein, FOR BREAST CANCER USING ANTIBODY AGAINST THE SAME}

The present invention relates to breast cancer diagnosis. More particularly, the present invention relates to a neural cell adhesion molecule L1-like protein as a protein marker capable of selectively diagnosing the presence or absence of breast cancer in blood, a method of detecting the same, and a breast cancer diagnostic kit comprising the antibody specifically recognizing the same will be.

Although the cause of breast cancer has not been clearly elucidated, various factors such as female hormone, family history, history, fertility and dietary habits are being discussed. According to a 2005 survey by the National Statistical Office, the incidence of breast cancer in Korean women has risen sharply in recent years, exceeding that of cervical cancer in 1998, accounting for 16.1% of Korean female cancer cases in 2001. In 2002, breast cancer was the most prominent cancer in 2001 (11.1%) compared to 2001, and the number of women who were exposed to female hormone stimulation in the period of physiological changes such as low fertility, short lactation period, early menarche, late menopause , The incidence of breast cancer is rapidly increasing due to the increase in the sensitivity of the mammary gland due to the rapid increase of the mammary gland, the westernization of the diet, and the pollution of the living environment.

The incidence of breast cancer and mortality due to breast cancer is expected to continue for a considerable period of time based on current westernization. Breast cancer usually causes symptoms such as invasion of surrounding tissues or lymph node metastasis due to the growth of cancer cells, but most can be diagnosed by self-examination without any symptoms. Therefore, effective early diagnosis of breast cancer is very important to reduce mortality from breast cancer (Tuli et al., Breast J. 12: 343-348, 2006).

Several methods have been used to diagnose breast cancer. Up to now, 70% of breast cancer patients are diagnosed by self diagnosis. However, such a self-diagnostic method has a disadvantage in that it is very difficult to distinguish malignant tumors from benign tumors. In addition, there are X-ray mammography, ultrasound, fine needle aspiration cytology, magnetic resonance imaging, and so on. X-ray mammography is an excellent way to distinguish whether a lump is benign or malignant by examining the breast by X-ray. It is a method of finding a hiding lump, It is the most effective way to diagnose breast cancer. However, there is a controversy about the fact that mammography like a young woman has developed a lot of mammary glands and that the diagnosis rate is low in Korean women with a small breast and fibroids, and breast cancer may be induced more frequently. Ultrasonography is an alternative to these mammography methods. Although ultrasound is effective in distinguishing between bruising and hard bruising, the ability to distinguish malignant tumors from benign bruises is inferior.

To overcome these drawbacks of conventional diagnostic methods, attempts have been made to diagnose breast cancer by measuring the concentration of tumor markers in the blood of patients (Clinton et al., Biomed Sci . Instrum . 39: 408-414, 2003; Rui et al., Proteomics . 3: 433-439, 2003; Soletormos et al., 48: 229-255, 2001). However, the diagnostic value or prognostic value of these tumor markers has been studied, but there are no markers of breast cancer that have been officially recommended, although they are still limited.

Accordingly, the inventors of the present invention have made extensive efforts to develop a method for using a marker protein that specifically changes the amount of blood in breast cancer patients in the diagnosis of breast cancer. As a result, it has been found that the L1- In addition, the present inventors have found that the amount of the L1-like protein is specifically increased in the blood of patients with breast cancer. In order to efficiently track the neuronal adhesion molecule L1-like protein, The present inventors have completed the present invention by confirming that breast cancer can be diagnosed easily by monitoring the expression level of the peptide shown in SEQ.

Accordingly, it is an object of the present invention to provide a method for easily identifying breast cancer using the change pattern of nerve cell adhesion molecule L1-like protein present in blood according to the onset of breast cancer.

In order to achieve the above object, the present invention provides a nerve cell adhesion molecule L1-like protein having an amino acid sequence of SEQ ID NO: 1 as a protein marker for breast cancer diagnosis.

The present invention also provides a method for selectively detecting the neural cell adhesion molecule L1-like protein in a blood sample through multiple reaction monitoring using a triple tetrahedral mass analyzer.

In addition, the present invention provides a breast cancer diagnostic kit comprising an antibody specific to the nerve cell adhesion molecule L1-like protein marker.

The present invention also provides a method for detecting a neuronal cell adhesion molecule L1-like protein in a blood sample through an antigen-antibody binding reaction using an antibody specific to a neuron cell adhesion molecule L1-like protein having an amino acid sequence of SEQ ID NO: 1 as a breast cancer protein marker ≪ / RTI >

Hereinafter, the present invention will be described in detail.

According to one aspect of the present invention, the present invention provides a nerve cell adhesion molecule L1-like protein having the amino acid sequence of SEQ ID NO: 1 as a protein marker for breast cancer diagnosis.

Neuronal cell adhesion molecule L1-like protein (CHL1) is a protein involved in adhesion between the extracellular matrix and cells, and plays an important role in the development of the nervous system and synaptic plasticity (Wei MH et al, Hum Genet, 103:. .. 355-364, 1998). These proteins are mainly expressed in normal tissues adjacent to the brain or in various types of cancer cell lines and primary tumors (Ross DT et al., Nat Genet 24: 227235, 2000). Recently, it has been reported that the protein acts as an inhibitor of primary tumor growth and is inhibited in various types of cancer growth, and is closely related to the expression and metastasis of cancer (Senchenko VN et al., PLoS One . 6: e15612, 2011).

On the other hand, multiple reaction monitoring (MRM) using triple quadrupole mass-spectrometry is an analytical technique that can selectively detect a specific analyte, detect it, quantify it, and monitor the concentration change . MRM is already used for quantitative analysis of small molecules and is used to diagnose certain genetic diseases. The MRM method is easy to measure multiple peptides at the same time, and has the advantage that the relative concentration difference of protein marker candidates between normal and cancer patients can be confirmed without standard or antibody. In addition, sensitivity and selectivity are excellent, and MRM analysis methods have been introduced for the analysis of complex proteins and peptides in the blood, especially in proteomic analysis using a mass spectrometer (Anderson L. et al., Mol Cell Proteomics , 5: 375-88, 2006; DeSouza, LV et al., Anal . Chem ., 81: 3462-70, 2009).

The present inventors obtained blood samples from 80 breast cancer patients and 80 non-patient control groups in order to select diagnostic markers that can be useful for diagnosis of breast cancer by specifically increasing the amount thereof in blood of breast cancer patients, And quantitatively analyzed by multiple reaction monitoring (MRM) using a mass spectrometer. As a result, it was confirmed that the expression level of neuronal adhesion molecule L1 - like protein was increased in the blood of breast cancer patients compared with the non - patient control group, thereby effectively discriminating breast cancer patients.

In the present invention, it has not been known that L1-like protein binding molecule selected by breast cancer diagnostic marker can be used as a marker for diagnosing the onset and progression of breast cancer. Therefore, the present invention is the first to use the nerve cell adhesion molecule L1-like protein as a diagnostic marker of breast cancer. In addition, since the marker protein can be detected in the blood, it can be used for diagnosis of breast cancer by a simple method without causing inconvenience to the patient because it does not pass through biopsy.

According to another aspect of the present invention, the present invention provides a method for detecting the nerve cell adhesion molecule L1-like protein in a blood sample through multiple reaction monitoring using a triple tetrahedral mass analyzer.

Preferably, the method further comprises

1) making the blood sample of the subject and the protein of the control blood sample into peptide fragments;

2) injecting the peptide fragments into a triple quadrupole mass spectrometer to perform multiple reaction monitoring on a target peptide representing a neuronal cell adhesion molecule L1-like protein;

3) displaying the result of the multiple reaction monitoring as a ratio to the internal standard material; And

4) comparing the detection results of the subject and the control group.

According to one embodiment of the present invention, a blood sample of a subject is collected, and the nerve cell adhesive molecule L1-like protein is detected by the MRM method in the blood sample of the subject and compared with the detection result of the non-patient control group, It is possible to confirm the onset of breast cancer by checking whether the amount of the similar protein is increased.

For the purpose of the present invention, in order to detect a target protein by the MRM method, the selection of a specific peptide representative of each protein and the selection of a parent ion / daughter ion pair targeted for MRM monitoring of each peptide should precede. The target peptide representing the nerve cell adhesion molecule L1-like protein of the present invention has the sequence of SEQ ID NO: 2, and the parent ion / daughter pair of the target peptide is m / z 642.8 and m / z 836.4, respectively. In addition, it is possible to synthesize specific peptides in which some amino acids are replaced by stable isotopes, which are monitored by the MRM method to detect the target protein, and when the MRM method is used as an internal standard material, the absolute amount of the target protein can be measured, Can be derived. In the present invention, any internal standard material commonly used in MRM analysis may be used as the internal standard material, for example, E. coli beta-galactosidase may be used. In order to measure the absolute amount of target protein in the blood, when a specific peptide in which some amino acids are substituted with stable isotopes is synthesized as an internal standard substance, the amino acid substituted with isotopes is preferably lysine or arginine, But is not limited to. It is preferable that the synthesized peptide use peptides separated by 95% or more pure.

The method of detecting the neuronal cell adhesion molecule L1-like protein of the present invention using the MRM method is a new diagnostic tool using the blood of a patient. It is not only superior in sensitivity, but also has a higher sensitivity than a conventional immunochemical method using an antigen antibody The selectivity of the blood is very high and the blood can be easily analyzed without using the biopsy, which can be useful for early diagnosis of breast cancer.

In addition, the present invention provides a diagnostic kit for detecting a nerve cell adhesion molecule L1-like protein in a blood sample using the MRM method. This diagnostic kit contains information on the target peptide of the nerve cell adhesion molecule L1-like protein and the parent ion / daughter ion pair of the target peptide necessary for detecting the nerve cell adhesion molecule L1-like protein using the MRM method, Includes tools, reagents and the like commonly used in mass spectrometry.

On the other hand, the neuronal adhesion molecule L1-like protein can be detected not only in the MRM method but also in the blood sample through the antigen-antibody binding reaction. Thus, in accordance with another aspect of the present invention, the present invention provides a method for detecting a neural cell adhesion molecule L1-like protein in a blood sample, comprising an antibody specific to a neuron cell adhesion molecule L1-like protein having the amino acid sequence of SEQ ID NO: Diagnostic reagent and a breast cancer diagnostic kit.

In order to produce an antibody selectively binding to a neuron-binding molecule L1-like protein, the neuronal cell adhesion molecule L1-like protein should be obtained first, and it may be synthesized using the amino acid sequence of SEQ ID NO: 1, Or may be prepared by separating directly from the blood.

For the purpose of the present invention, the antibody may include both a polyclonal antibody and a monoclonal antibody of a nerve cell adhesion molecule L1-like protein, but a monoclonal antibody that can specifically bind to an antigen is preferable.

Polyclonal antibodies can be prepared by injecting an exogenous nerve cell adhesion molecule L1-like protein or fragment thereof into an antigen according to conventional methods known to those skilled in the art. Such external hosts include mammals such as mice, rats, sheep, and rabbits, and immunogens are generally administered by intramuscular, intraperitoneal, or subcutaneous injection with adjuvants to increase antigenicity Administered to immunize an external host. Serum can be collected periodically from the immunized external host to collect serum showing improved titer and specificity for the antigen or isolate and purify the antibody from the serum to prepare a polyclonal antibody specific to the neuron cell adhesion molecule L1-like protein .

Monoclonal antibodies can be prepared using an immortalized cell line generation method (Kohler G. et al., Nature , 256: 495-497, 1975) by fusion known to those skilled in the art. The method is briefly described. First, mice are immunized with pure neuronal cell adhesion molecule L1-like protein or fragments thereof, or their peptides are synthesized and immunized with mice by binding with bovine serum albumin. The antibody-producing B lymphocytes isolated from immunized mice are fused with human or mouse myeloma cells to produce immortalized hybridoma cells. Next, the production of monoclonal antibodies of hybridoma cells was examined by enzyme-linked immunosorbent assay (ELISA), and positive clones were selected. After the cultivation of the positive clones, the antibodies were isolated and purified or injected into the abdominal cavity of rats A plurality of monoclonal antibodies specific for the neuronal cell adhesion molecule L1-like protein can be prepared.

Antibodies used in the detection of the nerve cell adhesion molecule L1-like proteins of the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least antigen binding ability, and includes Fab, F (ab ') 2, F (ab') ₂ and Fv.

The breast cancer diagnostic kit of the present invention includes not only antibodies that selectively recognize the neuronal cell adhesion molecule L1-like protein, but also tools and reagents commonly used in the immunological analysis in the art.

According to one embodiment of the present invention, the breast cancer diagnostic kit comprises an antibody specific for a neuronal cell adhesion molecule L1-like protein; A secondary antibody conjugate conjugated with a marker that develops color by reaction with a substrate; A color developing substrate solution to be colored with the label; Washing liquid; And an enzyme reaction stop solution.

In addition, the breast cancer diagnostic kit of the present invention may further comprise a positive control comprising a neuron cell adhesion molecule L1-like protein standard antigen and a negative control including an antiserum of an animal not infected with the antigen.

The breast cancer diagnostic kit of the present invention can diagnose breast cancer by quantitatively or qualitatively analyzing an antigen against an antibody protein through an antigen-antibody binding reaction. The antigen-antibody binding reaction can be detected by an enzyme immunoassay (ELISA) Methods such as radioimmunoassay (RIA), sandwich assay, Western blotting, immunoprecipitation, immunohistochemical staining, fluorescent immunoassay, enzyme substrate staining, and antigen-antibody aggregation . For example, the diagnostic kit can be provided to perform an ELISA that reacts with a recombinant mAb protein using a 96-well microtiter plate or the like coated with a sample and a control.

As a fixture for the antigen-antibody binding reaction, a well plate synthesized from a nitrocellulose membrane, a PVDF membrane, a polyvinyl resin or a polystyrene resin, and a glass slide glass can be used.

The marker of the secondary antibody is preferably a conventional coloring agent that undergoes a color development reaction, and is preferably selected from the group consisting of HRP (horseradish peroxidase), alkaline phosphatase, coloid gold, FITC (poly-L-lysine- (Fluorescein) and dye (dye) can be used as the labeling agent, for example, cyanate, cyanate, and RITC (rhodamine-B-isothiocyanate).

The coloring substrate for inducing color development is preferably used in accordance with a labeling substance that undergoes a color reaction. TMB (3,3 ', 5,5'-tetramethylbenzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o-phenylenediamine), and the like. At this time, it is more preferable that the chromogenic substrate is provided in a dissolved state in a buffer solution (0.1 M NaAc, pH 5.5). The chromogenic substrate such as TMB is decomposed by HRP used as a marker of the secondary antibody conjugate to generate a chromophore, and the degree of deposition of the chromophore is visualized to determine the presence or absence of the neuron cell adhesion molecule L1-like protein .

The washing solution preferably comprises a phosphate buffer solution, NaCl and Tween 20, and more preferably a buffer solution (PBST) composed of 0.02 M phosphate buffer solution, 0.13 M NaCl, and 0.05% Tween 20. After the antigen-antibody binding reaction, the washing solution is allowed to react with the secondary antibody to the antigen-antibody conjugate, and then an appropriate amount is added to the solid body and washed three to six times. The reaction stop solution may preferably be a sulfuric acid solution (H ₂ SO ₄ ).

In addition, another embodiment of the present invention is a method for detecting a neural cell adhesion molecule L1-like in a blood sample through an antigen-antibody binding reaction using an antibody specific to a neuron cell adhesion molecule L1-like protein having an amino acid sequence of SEQ ID NO: 1 as a breast cancer protein marker A method for detecting a protein is provided.

The detection method involves immobilizing proteins in the blood or separating them by electrophoresis (SDS-PAGE), transferring them to a PVDF membrane, and bringing them into contact with an antibody selectively recognizing a neuronal cell adhesion molecule L1-like protein, Lt; RTI ID = 0.0 > L1 < / RTI > like protein. Examples of the antigen-antibody binding reaction include enzyme immunoassay (ELISA), radioactive immunoassay (RIA), sandwich assay, Western blotting, immunoprecipitation, immunohistochemical staining, fluorescent immunoassay, enzyme substrate staining, And the like. As the sample, serum, plasma or blood can be used, and plasma is most preferable.

According to a preferred embodiment of the present invention,

1) coating or fixing the protein of the subject's blood sample and the control blood sample to the fixture;

2) performing an antigen-antibody binding reaction by adding an antibody specific to a nerve cell adhesion molecule L1-like protein to the fixative;

3) detecting an antigen-antibody binding reaction product generated through the antigen-antibody binding reaction using a secondary antibody conjugate and a chromogenic substrate solution; And

4) comparing the detection results for the blood sample of the subject and the control blood sample.

The detection method will be described in detail. First, the plasma protein is separated from the blood sample according to the molecular weight. The neuron-binding molecule L1-like protein has a molecular weight of 135 kDa. Therefore, electrophoresis is carried out using 12% polyacrylamide gel, and proteins isolated therefrom are transferred and fixed to a fixture such as a PVDF membrane. Then, an antigen-antibody binding reaction is performed by adding a specific antibody to the immobilized protein antigen to the nerve cell adhesion molecule L1-like protein. If there is a nerve cell adhesion molecule L1-like protein in the blood sample, an antigen-antibody binding reaction occurs when a nerve cell adhesion molecule L1-like protein-specific antibody is added to the protein-immobilized membrane. To measure the degree of binding of the nerve cell adhesion molecule L1-like protein to the antibody, a step of binding with a secondary antibody having affinity to the nerve cell adhesion molecule L1-like protein antibody, such as anti-human IgG-HRP, The horseradish peroxidase (HRP) conjugated to the secondary antibody reacted with the enhanced ECL (enhanced chemiluminescence) to determine whether or not the color reaction occurred. And whether or not the amount thereof is increased compared to the control group.

On the other hand, a biological microchip capable of detecting an antigen for the antibody protein by immobilizing an antibody specific to a neuron-binding molecule L1-like protein on a biological microchip and reacting with a blood sample separated from the subject, and An automated microarray system has the advantage of being able to analyze large volumes of samples in a single analysis. Accordingly, the present invention provides a biochip in which an antibody specifically binding to a nerve cell adhesion molecule L1-like protein is integrated in a solid substrate. Examples of the solid substrate of the biochip include plastic, glass, metal, silicon and the like.

As described above, the method of detecting the neural cell adhesion molecule L1-like protein, which is a marker protein for breast cancer diagnosis, of the present invention through blood MRM method is a new diagnostic tool using blood of a patient, The present inventors have found that the present invention can be used for early diagnosis of breast cancer because the selectivity to the protein to be detected is very high and the blood can be easily analyzed without using the biopsy, The use of antibody-like antibodies in breast cancer diagnostic kits and diagnostic methods is also relatively easy to collect blood samples, so unlike conventional breast cancer diagnostic methods for biopsy, it is very easy to diagnose breast cancer without burdening patients. In addition to the diagnostic accuracy and Higher degrees can be useful in the early diagnosis of breast cancer.

1, SEQ ID NO: 1 2 shows a MRM chromatogram obtained by quantitative analysis of a peptide of SEQ ID NO: 2, which is one of the proposed neural cell adhesion molecule L1-like tryptic peptides, by a multi-reaction monitoring method using a triple quadrupole mass spectrometer.
FIGS. 2a and 2b show the results obtained by measuring the concentration of the antigen measured by the multiple reaction monitoring method for 80 breast cancer patients and 80 non-patient control groups (for the coliform beta-galactosidase used as an internal standard) Ratio).
a: box chart; And
b: ROC curve.
3A and 3B are graphs showing the effect of the nerve cell adhesion molecule L1-like protein on the concentration of the antigen measured by the multi-response monitoring method for 37 first stage breast cancer patients and 80 non-patient control strains (E. coli beta-galactosidase In the case of the first embodiment.
a: box chart; And
b: ROC curve.
FIG. 4 is a graph showing the results of immunoblotting with an anti-nerve cell adhesion molecule L1-like protein antibody after analyzing plasma of a cancer patient group by electrophoresis.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these examples.

Example 1. Detection of Neuronal Cell Adhesion Molecule L1-like Protein for Diagnosis of Breast Cancer Using Multiple Reaction Monitoring (MRM)

The inventors of the present invention conducted a multi-reaction monitoring method using a triple quadrupole mass spectrometer in order to confirm whether the neuron-binding molecule L1-like protein shown in SEQ ID NO: 1 can be used as a marker for the selective diagnosis of breast cancer in blood (Anderson L. et al., Mol Cell Proteomics, 5: 375-88, 2006). MRM is a method that is mainly used for quantification using a mass spectrometer, and is a method of obtaining information by observing specific daughter ions generated from a mother ion of interest. For example, if only one of the several parent ions with m / z 1000 has daughter ions at m / z 500, then the parent ion with m / z 1000 is selected and fragmented to track these ions and m It is MRM to irradiate daughter ions at / z 500.

1.1 Preparation of sample

To confirm the diagnostic efficiency of breast cancer cell adhesion molecule L1-like protein, the protein expression level in blood samples obtained from 80 patients with breast cancer and 80 patients with non-patient control group was examined and statistically processed to determine the breast cancer diagnosis Efficiency was confirmed. 200 μg of each protein sample was prepared from blood obtained from 160 individuals. Each sample was treated with 10 mM dithiothreitol and reacted for 1 hour to allow the binding of the cystine to the thiol residue of amino acids in the protein that could interfere with the process of making the protein into a peptide fragment I cut it off. The cyanol bond was treated with 60 mM iodoacetamide and reacted for 1 hour in the absence of light to prevent recombination during the cyanobacterial period. To the prepared protein sample, 20 쨉 l of 4 쨉 g trypsin (Promega, USA) corresponding to 1/50 of 200 쨉 g of total protein was added and treated at 37 째 C for 16 hours to separate into a plurality of peptide fragments. The resulting peptide fragments were prepared as final samples for mass spectrometry analysis by removing salts using a C18 cartridge.

1.2 Triple Drama  Multiple reactions using a mass spectrometer monitoring  Perform

For MRM analysis, a pair of parent and daughter ions should be selected first to select a peptide that can represent a specific protein and to detect the daughter ion, that is, the target peptide, generated through fragmentation from the peptide. A peptide representative of the protein was synthesized (JPT Peptide Technologies Gmbh, Germany) in order to confirm the retention time and the tandem mass spectrometry spectrum in the column of the neuron adhesion molecule L1-like protein shown in SEQ ID NO: 1. As a result, the peptide shown in SEQ ID NO: 2 for monitoring the neuronal adhesion molecule L1-like protein was synthesized, and the parent ion / daughter ion pair was selected from the tandem mass spectrum of the synthesized peptide.

Target protein Target peptide
MRM transition (m / z) MRM ratio (cancer / normal) Mother ion Daughter ion Breast cancer 1st stage breast cancer Neuronal cell adhesion molecule L1-like protein GDLYFANVEEK 642.8 836.4 1.43 1.4 Coli beta-galactosidase GDFQFNISR 542.3 636.3 Internal reference material

The final samples prepared in Example 1.1 were subjected to reverse phase resin chromatography to separate plasma peptide fragments and the MRM spectra of each peptide was obtained using a triple quadrupole mass spectrometer (instrument: 5500 Qtrap, AB Sciex, USA). At this time, reverse phase resin chromatography was carried out on a HALO ( ^TM) C18 column (Eksigent, USA) column using a gradient of acetonitrile concentration of 5% to 40% for 45 minutes. The quantitative information was confirmed by calculating the peak area of the MRM chromatogram of the target peptide with a MultiQuant ^TM computer quantitative analysis program (AB Sciex, USA) (FIG. 1). At this time, the quantitative value of each target peptide was expressed as a ratio with respect to the peak area of MRM chromatogram of E. coli beta-galactosidase (Table 1) put into the internal standard material. By determining the ratio of MRM chromatograms of each peptide, differences in the amount of protein expression between the breast cancer patient and the non-patient control group could be confirmed.

The concentration of the neuron-binding molecule L1-like protein measured by the above method is shown graphically. Specifically, the nerve cell adhesion molecule L1-like protein shown in SEQ ID NO: 1 was quantitatively analyzed in 80 breast cancer patients and 80 non-patient control groups, and as a result, as shown in Fig. 2A, And 1.43 times more in the breast cancer group than in the control group. As shown in FIG. 2B, the area under the curve (AUC) of the marker protein was 0.75. In addition, the sensitivity was 56.25% at a specificity of 80%, indicating that the assay had higher sensitivity and accuracy than conventional breast cancer markers.

In addition, when the neuron adhesion molecule L1-like protein was compared between 37 first stage breast cancer patients and 80 non-patient control groups, as shown in the box chart in FIG. 3A, the marker proteins were compared with the non- 1 in breast cancer patients. As shown in FIG. 3B, the area under the curve (AUC) of the marker protein was 0.79. In addition, the sensitivity was 54.05% at a specificity of 80%, indicating that the above assay is very effective in the diagnosis of early breast cancer patients.

For reference, the ROC graph mainly shows the accuracy of the test with a graph of all sensitivity / specificity pairs obtained by continuously changing the decision threshold over the entire range of observed data (Zweig et al . , Clin . Chem . 39: 561-577,1993).

Example  2. Immunity blotting  Nerve cell adhesion molecule L1  Detection of similar proteins

The neuronal adhesion molecule L1-like protein shown in SEQ ID NO: 1 can also be used for the selective diagnosis of breast cancer in blood through immunoblotting.

Specifically, blood is taken from 93 cancer patients and the amount of protein in blood is quantified. Each 10 μg of the prepared plasma protein sample was mixed with a buffer solution containing 1% SDS, followed by electrophoresis using 12% SDS polyacrylamide gel. The separated serum proteins were transferred to a PVDF membrane (Bio-Rad, USA) in a solution containing 25 mM Tris-HCl, 190 mM glycine and 10% methanol, The reaction was carried out in TBST (Tris-buffered saline Tween 20) containing skimmed milk powder for 1 hour. Then, the PVDF membrane was reacted with a fibronectin antibody (Biohit Oy, Finland) at 4 ° C for 16 hours. After washing with TBST three times, mouse IgG-HRP (Amersham Bioscience, USA) binding to mouse immunoglobulin and 25 Lt; 0 > C for 1 hour. Thereafter, the PVDF membrane was reacted with ECL (enhanced chemiluminescence, Amersham Bioscience, USA) and exposed to X-ray film to measure the concentration of the neuronal cell adhesion molecule L1-like protein antigen.

As a result, immunoblot analysis results were obtained as shown in FIG. 4, and the detection of the neuronal cell adhesion molecule L1-like protein through an antigen-antibody reaction was confirmed.

&Lt; 110 > Biomedieng Co., Ltd. <120> PROTEIN MARKER NEURAL CELL ADHESION MOLECULE L1-LIKE PROTEIN FOR          BREAST CANCER DIAGNOSIS, METHOD OF DETECTING THE SAME, AND          DIAGNOSIS KIT FOR BREAST CANCER USING ANTIBODY AGAINST THE SAME <130> SDP50345 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1208 <212> PRT <213> Homo sapiens <400> 1 Met Glu Pro Leu Leu Leu Gly Arg Gly Leu Ile Val Tyr Leu Met Phe   1 5 10 15 Leu Leu Leu Lys Phe Ser Lys Ala Ile Glu Ile Pro Ser Ser Val Gln              20 25 30 Gln Val Pro Thr Ile Ile Lys Gln Ser Lys Val Gln Val Ala Phe Pro          35 40 45 Phe Asp Glu Tyr Phe Gln Ile Glu Cys Glu Ala Lys Gly Asn Pro Glu      50 55 60 Pro Thr Phe Ser Trp Thr Lys Asp Gly Asn Pro Phe Tyr Phe Thr Asp  65 70 75 80 His Arg Ile Ile Pro Ser Asn Asn Ser Gly Thr Phe Arg Ile Pro Asn                  85 90 95 Glu Gly His Ile Ser His Phe Gln Gly Lys Tyr Arg Cys Phe Ala Ser             100 105 110 Asn Lys Leu Gly Ile Ala Met Ser Glu Glu Ile Glu Phe Ile Val Pro         115 120 125 Ser Val Pro Lys Phe Pro Lys Glu Lys Ile Asp Pro Leu Glu Val Glu     130 135 140 Glu Gly Asp Pro Ile Val Leu Pro Cys Asn Pro Pro Lys Gly Leu Pro 145 150 155 160 Pro Leu His Ile Tyr Trp Met Asn Ile Glu Leu Glu His Ile Glu Gln                 165 170 175 Asp Glu Arg Val Tyr Met Ser Gln Lys Gly Asp Leu Tyr Phe Ala Asn             180 185 190 Val Glu Glu Lys Asp Ser Arg Asn Asp Tyr Cys Cys Phe Ala Ala Phe         195 200 205 Pro Arg Leu Arg Thr Ile Val Gln Lys Met Pro Met Lys Leu Thr Val     210 215 220 Asn Ser Ser Asn Ser Ile Lys Gln Arg Lys Pro Lys Leu Leu Leu Pro 225 230 235 240 Pro Thr Glu Ser Gly Ser Glu Ser Ser Ile Thr Ile Leu Lys Gly Glu                 245 250 255 Ile Leu Leu Leu Glu Cys Phe Ala Glu Gly Leu Pro Thr Pro Gln Val             260 265 270 Asp Trp Asn Lys Ile Gly Gly Asp Leu Pro Lys Gly Arg Glu Thr Lys         275 280 285 Glu Asn Tyr Gly Lys Thr Leu Lys Ile Glu Asn Val Ser Tyr Gln Asp     290 295 300 Lys Gly Asn Tyr Arg Cys Thr Ala Ser Asn Phe Leu Gly Thr Ala Thr 305 310 315 320 His Asp Phe His Val Ile Val Glu Glu Pro Pro Arg Trp Thr Lys Lys                 325 330 335 Pro Gln Ser Ala Val Tyr Ser Thr Gly Ser Asn Gly Ile Leu Leu Cys             340 345 350 Glu Ala Glu Gly Glu Pro Gln Pro Thr Ile Lys Trp Arg Val Asn Gly         355 360 365 Ser Pro Val Asp Asn His Pro Phe Ala Gly Asp Val Val Phe Pro Arg     370 375 380 Glu Ile Ser Phe Thr Asn Leu Gln Pro Asn His Thr Ala Val Tyr Gln 385 390 395 400 Cys Glu Ala Ser Asn Val Gly Thr Ile Leu Ala Asn Ala Asn Ile                 405 410 415 Asp Val Val Asp Val Arg Pro Leu Ile Gln Thr Lys Asp Gly Glu Asn             420 425 430 Tyr Ala Thr Val Val Gly Tyr Ser Ala Phe Leu His Cys Glu Phe Phe         435 440 445 Ala Ser Pro Glu Ala Val Val Ser Trp Gln Lys Val Glu Glu Val Lys     450 455 460 Pro Leu Glu Gly Arg Arg Tyr His Ile Tyr Glu Asn Gly Thr Leu Gln 465 470 475 480 Ile Asn Arg Thr Thr Glu Glu Asp Ala Gly Ser Tyr Ser Cys Trp Val                 485 490 495 Glu Asn Ala Ile Gly Lys Thr Ala Val Thr Ala Asn Leu Asp Ile Arg             500 505 510 Asn Ala Thr Lys Leu Arg Val Ser Pro Lys Asn Pro Arg Ile Pro Lys         515 520 525 Leu His Met Leu Glu Leu His Cys Glu Ser Lys Cys Asp Ser His Leu     530 535 540 Lys His Ser Leu Lys Leu Ser Trp Ser Lys Asp Gly Glu Ala Phe Glu 545 550 555 560 Ile Asn Gly Thr Glu Asp Gly                 565 570 575 Thr Ile Ser Asn Val Thr Leu Glu Asp Gln Gly Ile Tyr Cys Cys Ser             580 585 590 Ala His Thr Ala Leu Asp Ser Ala Ala Asp Ile Thr Gln Val Thr Val         595 600 605 Leu Asp Val Pro Asp Pro Pro Glu Asn Leu His Leu Ser Glu Arg Gln     610 615 620 Asn Arg Ser Val Arg Leu Thr Trp Glu Ala Gly Ala Asp His Asn Ser 625 630 635 640 Asn Ile Ser Glu Tyr Ile Val Glu Phe Glu Gly Asn Lys Glu Glu Pro                 645 650 655 Gly Arg Trp Glu Glu Leu Thr Arg Val Gln Gly Lys Lys Thr Thr Val             660 665 670 Ile Leu Pro Leu Ala Pro Phe Val Arg Tyr Gln Phe Arg Val Ile Ala         675 680 685 Val Asn Glu Val Gly Arg Ser Gln Pro Ser Gln Pro Ser Asp His His     690 695 700 Glu Thr Pro Pro Ala Ala Pro Asp Arg Asn Pro Gln Asn Ile Arg Val 705 710 715 720 Gln Ala Ser Gln Pro Lys Glu Met Ile Ile Lys Trp Glu Pro Leu Lys                 725 730 735 Ser Met Glu Gln Asn Gly Pro Gly Leu Glu Tyr Arg Val Thr Trp Lys             740 745 750 Pro Gln Gly Ala Pro Val Glu Trp Glu Glu Glu Thr Val Thr Asn His         755 760 765 Thr Leu Arg Val Met Thr Pro Ala Val Tyr Ala Pro Tyr Asp Val Lys     770 775 780 Val Gln Ala Ile Asn Gln Leu Gly Ser Gly Pro Asp Pro Gln Ser Val 785 790 795 800 Thr Leu Tyr Ser Gly Glu Asp Tyr Pro Asp Thr Ala Pro Val Ile His                 805 810 815 Gly Val Asp Val Ile Asn Ser Thr Leu Val Lys Val Thr Trp Ser Thr             820 825 830 Val Pro Lys Asp Arg Val His Gly Arg Leu Lys Gly Tyr Gln Ile Asn         835 840 845 Trp Trp Lys Thr Lys Ser Leu Leu Asp Gly Arg Thr His Pro Lys Glu     850 855 860 Val Asn Ile Leu Arg Phe Ser Gly Gln Arg Asn Ser Gly Met Val Pro 865 870 875 880 Ser Leu Asp Ala Phe Ser Glu Phe His Leu Thr Val Leu Ala Tyr Asn                 885 890 895 Ser Lys Gly Ala Gly Pro Glu Ser Glu Pro Tyr Ile Phe Gln Thr Pro             900 905 910 Glu Gly Val Pro Glu Gln Pro Thr Phe Leu Lys Val Ile Lys Val Asp         915 920 925 Lys Asp Thr Ala Thr Leu Ser Trp Gly Leu Pro Lys Lys Leu Asn Gly     930 935 940 Asn Leu Thr Gly Tyr Leu Leu Gln Tyr Gln Ile Ile Asn Asp Thr Tyr 945 950 955 960 Glu Ile Gly Glu Leu Asn Asp Ile Asn Ile Thr Thr Pro Ser Lys Pro                 965 970 975 Ser Trp His Leu Ser Asn Leu Asn Ala Thr Thr Lys Tyr Lys Phe Tyr             980 985 990 Leu Arg Ala Cys Thr Ser Gln Gly Cys Gly Lys Pro Ile Thr Glu Glu         995 1000 1005 Ser Ser Thr Leu Gly Gly Gly Ser Lys Gly Ile Gly Lys Ile Ser Gly    1010 1015 1020 Val Asn Leu Thr Gln Lys Thr His Pro Ile Glu Val Phe Glu Pro Gly 1025 1030 1035 1040 Ala Glu His Ile Val Arg Leu Met Thr Lys Asn Trp Gly Asp Asn Asp                1045 1050 1055 Ser Ile Phe Gln Asp Val Ile Glu Thr Arg Gly Arg Glu Tyr Ala Gly            1060 1065 1070 Leu Tyr Asp Asp Ile Ser Thr Gln Gly Trp Phe Ile Gly Leu Met Cys        1075 1080 1085 Ala Ile Ala Leu Leu Thr Leu Leu Leu Leu Thr Val Cys Phe Val Lys    1090 1095 1100 Arg Asn Arg Gly Gly Lys Tyr Ser Val Lys Glu Lys Glu Asp Leu His 1105 1110 1115 1120 Pro Asp Pro Glu Ile Gln Ser Val Lys Asp Glu Thr Phe Gly Glu Tyr                1125 1130 1135 Ser Asp Ser Asp Glu Lys Pro Leu Lys Gly Ser Leu Arg Ser Leu Asn            1140 1145 1150 Arg Asp Met Gln Pro Thr Glu Ser Ala Asp Ser Leu Val Glu Tyr Gly        1155 1160 1165 Glu Gly Asp His Gly Leu Phe Ser Glu Asp Gly Ser Phe Ile Gly Ala    1170 1175 1180 Tyr Ala Gly Ser Lys Glu Lys Gly Ser Val Glu Ser Asn Gly Ser Ser 1185 1190 1195 1200 Thr Ala Thr Phe Pro Leu Arg Ala                1205 <210> 2 <211> 11 <212> PRT <213> Homo sapiens <400> 2 Gly Asp Leu Tyr Phe Ala Asn Val Glu Glu Lys   1 5 10 <210> 3 <211> 9 <212> PRT <213> Escherichia coli <400> 3 Gly Asp Phe Gln Phe Asn Ile Ser Arg   1 5

Claims (23)

1) making the blood sample of the subject and the protein of the control blood sample into peptide fragments;
2) The peptide fragment was injected into a triple quadrupole mass spectrometer to have a sequence of SEQ ID NO: 2 representing the neuronal adhesion molecule L1-like protein, and the mother and daughter ions of the target peptide were m / z 642.8 and m / z Performing multiple reaction monitoring on a target peptide that is 836.4;
3) displaying the result of the multiple reaction monitoring as a ratio to the internal standard material; And
4) comparing the results of the detection of the subject and the control group. The multi-reaction monitoring using the triple quadrupole mass spectrometer can be used to measure the adhesion of the nerve cell having the sequence of SEQ ID NO: 1 A method for selectively detecting a molecular L1-like protein. delete delete [Claim 3] The method according to claim 1, wherein the target peptide represented by E. coli beta-galactosidase has the sequence of SEQ ID NO: 3 and the parent and daughter ions are m / z 542.3 and m / z 636.3. The method according to claim 1, wherein the sample is plasma or serum. delete delete A target peptide of a nerve cell adhesion molecule L1-like protein necessary for detecting a neuron adhesion molecule L1-like protein in a blood sample using the detection method of the above-mentioned 1, and information about a mother ion / daughter ion pair of the target peptide Breast cancer diagnostic kit. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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