KR20190026632A - Method for diagnosing pancreatic cancer using methionyl-tRNA synthetase and pancreatic acinar cell-specific marker - Google Patents

Abstract

The present invention relates to a method for diagnosing pancreatic cancer using methionyl-tRNA synthetase and an acinar cell-specific marker. MRS has a higher level of diagnostic accuracy than conventional pancreatic cancer markers such as CEA, particularly allows a significant increase in accuracy of diagnosis of pancreatic cancer when, together with the expression of MRS, an acinar cell-specific marker protein such as chymotrypsin is used additionally as a dual marker, and thus has markedly superior industrial applicability in fields such as in in-vitro diagnostics industry.

Description

TECHNICAL FIELD The present invention relates to a method for diagnosing pancreatic cancer using methionyl-thi ene synthetase and specific cell markers,

The present invention relates to a method for diagnosing pancreatic cancer using a methionyl-tRNA synthetase (MRS) and a precursor cell-specific marker, and more particularly, to a method for diagnosing pancreatic cancer using a methionyl-tRNA synthetase A kit comprising the same, and a method for enhancing the accuracy of diagnosis of pancreatic cancer by using the two proteins as a double marker.

Cancer is a group of diseases that can start from the growth of uncontrolled cells, infiltrate into the surrounding normal tissues or organs, destroy them, create new growth places, and take away the life of the individual. Despite having made remarkable progress in the search for new targets, including regulation of cell cycle or apoptosis, and cancer genes or cancer-suppressing genes, to conquer cancer over the last decade, It is increasing with development.

Among these, pancreatic cancer is the fatal cancer with a 5-year survival rate of 1-4% and a median survival time of 5 months. It has the worst prognosis of cancer of the human body. In the 80-90% of patients, the diagnosis is made in a state in which a radical resection is not possible, which is expected to be cured. Therefore, the prognosis is poor and the treatment depends on chemotherapy. The therapeutic effects of some anticancer drugs including 5-fluorouracil, gemcitabine, and tarceva, which are known to be effective against pancreatic cancer, are extremely disappointing, and the response rate to chemotherapy is only about 15% This suggests that more accurate and quicker diagnostic methods are needed to improve the prognosis of pancreatic cancer patients.

On the other hand, pathological examination refers to a test which uses mainly extracted cells, tissues or organs to elucidate the origins of diseases in a morphological standpoint, and is a method of examining gross findings, optical, electron microscope It is an important test applied to the diagnosis of diseases. These pathological tests include histopathology and cytopathology. On the other hand, there are many differences between histological examination and cytodiagnosis test, and it is known that there is a large difference in the accuracy of prediction between the biopsy and cytology of the well-known cancer markers such as diagnosis sensitivity and specificity. Therefore, even if it is a known cancer marker, it is considered as a separate problem whether the diagnostic efficacy can be practically achieved according to a specific specimen (tissue or cell).

The difficulty in judging atypical cells among the obstacles in the pathological examination of the cells isolated from the body is also a problem. In 1976, Melamed et al. Reported that the inflammatory changes were not inflammatory changes, but they were not enough to diagnose dysplasia, and there have been many controversies about the diagnosis, interpretation, and treatment of atypical cells. Therefore, the bethesda system (TBS) has been established for its improvement and the use of the term atypical cell in TBS can not be diagnosed as inflammatory, (undetermined significance). This is problematic because there may be other views regarding the therapeutic strategy for non-malignant cells. Particularly, there is a problem in that it is significant that diagnosis is made of atypical cells in a tissue or cell level test, or the result is judged only in a tissue sample, although the cancer is actually progressing. Atypism or cellular atypia is often diagnosed when the indeterminate tissue structure or cell type is not clearly distinguishable from inflammatory lesions or neoplasms. Therefore, it is necessary to repeatedly repeat the inspection by other inspection means, and accordingly the time and economic costs are considerably consumed.

Since the pancreas is deeply in the body, it is very difficult to detect when the cancer is present in the pancreas. In the case of pancreatic cancer that can be operated through imaging studies, definitive diagnosis of pancreatic cancer is necessary through biopsy or endoscopic ultrasound cytology before the operation. Even if surgery is not possible, biopsy or cytology is necessary for histologic diagnosis for chemotherapy or radiotherapy. Tumor markers for diagnosis of pancreatic cancer are CEA (reference value: 5.0 ng / mL) and CA 19-9 (reference value: 37 U / mL). However, in the case of CA19-9, there is a problem that the specificity is low as a marker for diagnosing pancreatic cancer. According to one report, CA19-9 was elevated in about 1% of people who underwent physical examinations, and only 2% of cancer-free patients with elevated CA19-9 levels without symptoms were found to have cancer. According to the American Cancer Society guidelines, CA19-9 has been shown to be less sensitive and specific in pancreatic cancer screening.

In addition, biomarkers for diagnosing pancreatic cancer have been actively developed. For example, Korean Patent No. 10-0819122 discloses a method for diagnosing pancreatic cancer by using matrilin, transthyretin, and stratifin as pancreatic cancer markers And Korean Unexamined Patent Application Publication No. 2008-0082372 discloses a technique using various pancreatic cancer markers. Korean Patent Laid-Open Publication No. 2009-0003308 discloses a method for diagnosing pancreatic cancer by detecting the expression level of REG4 protein in a blood sample of an individual. Korean Published Application No. 2012-0009781 discloses a method for diagnosing pancreatic cancer Korean Patent Application Laid-open No. 2007-0119250 discloses an assay method for measuring the expression level of XIST RNA in cancer tissues isolated from an individual in order to provide a new gene LBFL313 differentially expressed in human pancreatic cancer tissue And U.S. Patent Application Publication No. 2011/0294136 discloses a method for diagnosing pancreatic cancer using biomarkers such as keratin 8 protein.

However, the above-mentioned markers have a limitation in that they show a great difference in the diagnostic efficiency and accuracy of each marker, and in particular, they do not depend on the cytology analysis method and are clinically diagnosed as tumor cells or other disease states In the case of atypical cells in which the distinction is not clear, there is no definite marker for determining whether a clear diagnosis of pancreatic cancer is more important. In other words, the previously reported diagnosis of pancreatic cancer markers is poor in sensitivity and specificity in the diagnosis of pancreatic cancer cells, unlike the histology of pancreatic cancer markers.

Therefore, in order to diagnose pancreatic cancer, expensive examination such as CT, endoscopic retrograde cholangiopancreatography (ERCP), ultrasound endoscopy (EUS), and angiography is demanded in addition to the above tumor markers , It is very difficult to diagnose pancreatic cancer accurately. The diagnosis of pancreatic cancer is made through endoscopic ultrasonic micro needle aspiration test in a patient who can not undergo surgery because the pancreatic cancer is a progressive cancer that is not easily resectable at the time of diagnosis and 10-15% . However, endoscopic ultrasound microscopic needle aspiration cytology is difficult to compare with surrounding tissues or cells compared with pancreatic cancer tissues.

Because of this, it is still dependent on pathological diagnosis based on general staining, mainly H & E staining or pap staining, in differentiating the cells of pancreatic cancer cells and other diseases (eg pancreatitis). However, the diagnosis of pancreatic cancer by the above-mentioned conventional staining method may be different according to the experience and the interpretation technique of the medical staff. In particular, when it is judged as atypical cell in the pancreatic cell observation by H & E staining or pap staining, It is very difficult to distinguish between benign disease and other diseases. The diagnosis of the cytopenia is inaccurate, so that it is impossible to appropriately treat a pancreatic cancer patient who can undergo surgery, and conversely, it is difficult to prevent unnecessary surgery. Therefore, accurate diagnosis of cytology is needed to increase the therapeutic effect of pancreatic cancer clinically.

Therefore, the inventors of the present invention conducted a cytological analysis to find a marker capable of diagnosing pancreatic cancer more precisely at the cellular level using pancreatic samples obtained from pancreatic cancer patients. As a result, MRS (methionyl tRNA synthetase) (Atypical cell) was determined by conventional cytopathologic examination using H & E staining or pap staining. In addition, it was confirmed that malignant tumor cells can be clearly distinguished by high expression (MRS) expression and the use of double-specific marker (s), such as chymotrypsin, as a dual marker, may be useful in the diagnosis of pancreatic cancer. Confirming that the accuracy is further increased and completing the present invention .

Accordingly, an object of the present invention is to provide an agent for measuring an expression level of a methionyl-tRNA synthetase (MRS) protein and an agent for measuring an expression level of an acinar cell-specific marker protein And a kit for diagnosing pancreatic cancer comprising the same.

 Another object of the present invention is to provide a method for qualitatively or quantitatively analyzing the expression level of the MRS protein and the secretory cell specific marker protein from a pancreatic sample collected from a potential patient to provide information necessary for diagnosis of pancreatic cancer.

It is yet another object of the present invention to provide a method for improving sensitivity or specificity in cytodiagnosis or histology of pancreatic cancer comprising the steps of:

(a) measuring the level of expression of the precursor cell-specific marker protein and the MRS protein in the pancreas sample collected from a potential patient; And

(b) determining that the precursor cell-specific marker protein is not expressed in the step (a) and that the expression of the MRS protein is increased, the cell is a pancreatic cancer cell.

It is another object of the present invention to provide a method for the cytodiagnosis or histologic examination of pancreatic cancer,

(a) measuring the level of expression of the precursor cell-specific marker protein and the MRS protein in the pancreas sample collected from a potential patient; And

(b) determining that the precursor cell-specific marker protein is not expressed in the step (a) and that the expression of the MRS protein is increased, the pancreatic cancer cell is further characterized in that it is a pancreatic cancer cell A method for providing information necessary for diagnosis of pancreatic cancer.

To achieve these and other advantages and in accordance with the purpose of the present invention,

A composition for measuring the expression level of methionyl-tRNA synthetase (MRS) protein and an agent for measuring the expression level of acinar cell-specific marker protein and a composition for diagnosing pancreatic cancer The present invention provides a kit for diagnosing pancreatic cancer.

In order to achieve another object of the present invention,

There is provided a method for qualitatively or quantitatively analyzing the expression level of the MRS protein and the secretory cell-specific marker protein from a pancreas sample collected from a potential patient to provide information necessary for diagnosis of pancreatic cancer.

In order to accomplish still another object of the present invention, the present invention provides a method for improving sensitivity or specificity in a cytodiagnosis test or biopsy for pancreatic cancer comprising the steps of:

(a) measuring the level of expression of the precursor cell-specific marker protein and the MRS protein in the pancreas sample collected from a potential patient; And

(b) determining that the precursor cell-specific marker protein is not expressed in the step (a) and that the expression of the MRS protein is increased, the cell is a pancreatic cancer cell.

In order to accomplish still another object of the present invention, the present invention provides a method for the cytodiagnosis or histology of pancreatic cancer,

(a) measuring the level of expression of the precursor cell-specific marker protein and the MRS protein in the pancreas sample collected from a potential patient; And

(b) determining that the precursor cell-specific marker protein is not expressed in the step (a) and that the expression of the MRS protein is increased, the pancreatic cancer cell is further characterized in that it is a pancreatic cancer cell To provide information necessary for the diagnosis of pancreatic cancer.

Hereinafter, the present invention will be described in detail.

Throughout the disclosure herein, various aspects or conditions relating to the present invention may be suggested in a range format. The description of the range values in the present specification is intended to include the corresponding boundary values unless otherwise stated, that is, to include all the values from the lower limit value to the upper limit value. It should be understood that the description of a range format is merely for convenience and simplicity and should not be construed as an inflexible limitation to the scope of the present invention. Thus, the description of a range should be considered as disclosing all possible subranges as well as individual numerical values within the range. For example, a description in the range of 1 to 5 may be applied to individual values within the range, such as 1, 2, 2.7, 3, 3.5, 4.3 and 5, as well as from 1 to 3, 1 to 4, 5, 2 to 3, 2 to 4, 3 to 4, and the like. This applies irrespective of the width of the range.

The term " pancreatic cancer " as used herein refers to a malignant tumor or cancer occurring in the pancreas. It refers to a malignant neoplasm having a proliferation rate and having a characteristic of penetrating into peripheral tissues and metastasizing to other organs . The malignant tumor or cancer is distinguished from a benign tumor, which has a slow growth rate and does not metastasize.

More than 90% of cancers in the pancreas occur in pancreatic duct cells, and pancreatic cancer usually refers to pancreatic cancer or pancreatic ductal cancer. Therefore, preferably, the pancreatic cancer in the present invention may be a pancreatic ductal (adeno) carcinoma.

The cause of the pancreatic cancer for the purpose of diagnosis in the present invention is not particularly limited as long as it is cancer of the pancreas due to metastasis or primary cancer. Preferably, it may be targeted to primary cancer.

The term 'normal' in the present invention means a malignant tumor or a non-cancerous condition (Negative for malignancy, malignant tumor cell negative), and includes a complete steady state without any disease, other diseases such as pancreatitis State " and / or " benign ". In the present specification, the positive mark described as 'benign' in the clinical (final) disease state determination is distinguished from the positive mark on the corresponding test indicated as 'positive', and the positive marked as 'positive' Or the result of the test means the possibility of cancer.

About 5% to 10% of pancreatic cancer patients have a genetic predisposition. About 7.8% of pancreatic cancer patients have a family history of pancreatic cancer, compared with 0.6% of pancreatic cancer patients in the general population. Pancreatic cancer is a very poor prognosis with a 5 - year survival rate of less than 5%. The reason for this is that most of the cases are found after cancer has progressed, so surgical resection is possible within 20% of cases, and even if completely resected by visual examination, survival rate is not improved by micro metastasis and response to chemotherapy and radiotherapy It is low.

Currently, pancreatic cancer is diagnosed by computed tomography (CT) or magnetic resonance imaging (MRI), and CT or MRI is used as an imaging technique, And finally diagnose pancreatic cancer by histological examination or cytological examination, which is a pathological method. The histological examination can confirm that cancer is present in a specific area through comparison with surrounding structures or cells. However, in the case of the cytological examination, since the individual cells are extracted and stained, the relationship with the surrounding tissues can not be proved. And cytologic examination are fundamentally different.

Recently, as a clinical sample that can diagnose pancreatic cancer, protein analysis in body fluids such as plasma is measured at the same time to determine the presence or the possibility of pancreatic cancer. However, clinically, serologic methods have only a meaning as a reference in the diagnosis of pancreatic cancer, but they do not provide crucial information for the diagnosis of pancreatic cancer. The most commonly used tumor markers associated with pancreatic cancer are carbohydrate antigen 19-9 (CA19-9) or carcinoembryogenic antigen (CEA). However, CA19-9 is known to be inadequate for the diagnosis of pancreatic cancer because its serum concentration is elevated even in non-cancerous diseases such as hepatitis, cirrhosis and pancreatitis. It is known that CA19-9 is not detectable in pancreatic cell itself, , It can not be said to be pancreatic cancer-specific. Therefore, CA19-9 serum levels are monitored after surgery to determine the prognosis of patients. In addition, CEA does not exhibit sufficient sensitivity, specificity, positive predictive value, and / or negative predictive value for pancreatic cancer, which is a limitation in accurately diagnosing pancreatic cancer.

On the other hand, the present inventors have first found that MRS is expressed in pancreatic cancer (high), and that when using MRS as a pancreatic cancer marker, it is possible to obtain diagnosis results with high accuracy in not only biopsy but also cytodiagnosis . That is, MRS is highly expressed specifically in pancreatic cancer as compared to normal pancreatic cells (ie, non-tumorous pancreatic cells), and MRS is more sensitive to pancreatic cell samples than CEA, which is conventionally used as a pancreatic cancer marker It is possible to distinguish pancreatic cancer cells with highly precise atypical cells which are difficult to diagnose accurately by conventional cell staining methods (for example, H & E staining or pap staining) in the diagnosis of pancreatic cancer. . Especially, the diagnosis accuracy of pancreatic cancer was remarkably elevated when it was diagnosed using double marker as an additional marker specific protein such as chymotrypsin.

Therefore, Methionyl Thieme  Synthetic enzyme ( 메 - tRNA synthetase , MRS) < / RTI > Splenocyte ( acinar  cell specific Marker  A composition for diagnosing pancreatic cancer comprising an agent for measuring the expression level of a protein, and a kit for diagnosing pancreatic cancer comprising the same.

As used herein, the term " diagnosis " means identifying (identifying) the presence or characteristic of a pathological condition. Specifically, in the present invention, the diagnosis may be performed by measuring the expression level or expression level of the MRS protein to confirm the presence or absence of pancreatic cancer.

In the present invention, 'MRS' means methionyl-tRNA synthetase. The MRS is an enzyme that mediates aminoacylation reaction between methionine and tRNA. The MRS protein of the present invention is not particularly limited as long as it contains the MRS amino acid sequence known in the art and its specific sequence and its biological origin. For example, in humans, it is encoded in MARS gene, and the sequence information of MRS is known as Genbank (NCBI) accession number such as NM_004990 (mRNA), NP_004981.2, P56192.2 (protein). Preferably, the MRS of the present invention may include a human MRS protein amino acid sequence represented by SEQ ID NO: 1. More preferably, the MRS protein of the present invention may comprise the amino acid sequence of SEQ ID NO: 1. The MRS has two types of isoforms: cytoplasmic methionyl-tRNA synthetase and mitochondrial form (mitochondrial methionyl-tRNA synthetase). The MRS in the present invention may preferably be a cytoplasmic form.

The expression " expression " in the present invention means that a protein or a nucleic acid is produced in a cell.

The term 'protein' as used herein is used interchangeably with 'polypeptide' or 'peptide', for example, a polymer of amino acid residues as commonly found in proteins in nature.

The agent for measuring the expression level of the MRS protein may be an antibody or an aptamer that specifically binds to the MRS protein, although the type thereof is not particularly limited as long as it is known in the art to be capable of measuring the expression level of the protein .

The term " antibody " in the present invention means an immunoglobulin which specifically binds to an antigenic site. More specifically, it refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains connected to each other by a disulfide bond. Each heavy chain consists of a heavy chain variable region (abbreviated as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (called complementarity determining regions (CDRs)) in which more conserved regions, referred to as framework regions (FR), are scattered. Each of VH and VL consists of three CDRs and four FRs arranged from the amino-terminus to the carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The constant region of the antibody may mediate the binding of immunoglobulins to the host tissue or factor, including the various components of the immune system (e. G., Effector cells) and the first component of the traditional complement system (Clq).

The anti-MRS antibody in the present invention is an antibody that specifically binds only to the MRS protein without reacting with other proteins including other aminoacyl thiourea synthetases in addition to MRS. The antibody specifically binding to the MRS protein in the present invention may preferably be an antibody that specifically binds to a protein (MRS) comprising the amino acid sequence represented by SEQ ID NO: 1. The anti-MRS antibody can be prepared by cloning the MRS gene into an expression vector to obtain a protein encoded by the gene, and obtaining an antibody produced by injecting the obtained protein into an animal, such as by an ordinary method in the art can do. The MRS antibody may be prepared through an MRS full length sequence protein, or a fragment of an MRS protein including an MRS antigenic site may be used to produce an MRS protein specific antibody. The specific sequence and form of the antibody of the present invention are not particularly limited and include a polyclonal antibody or a monoclonal antibody. In addition, the type of the immunoglobulin to be provided is not particularly limited, and may be selected from the group consisting of IgG, IgA, IgM, IgE and IgD, preferably an IgG antibody. Furthermore, the antibody of the present invention includes a special antibody such as a humanized antibody, a chimeric antibody and a recombinant antibody as long as it can specifically bind to the MRS protein. Also, as long as it has antigen-antibody binding (reaction), part of the whole antibody is included in the antibody of the present invention, and includes all kinds of immunoglobulin antibodies that specifically bind to MRS. F (ab '), F (ab '), < / RTI > which have antigen binding functions, as well as complete forms of antibodies with two full length light chains and two full length heavy chains, 2, Fv, diabody, scFv, and the like.

Fab (fragment antigen-binding) is an antigen-binding fragment of an antibody, consisting of one variable domain and one constant domain of each of the heavy and light chains. F (ab ') 2 is a fragment produced by hydrolyzing an antibody to pepsin, and two Fabs are linked from a heavy chain hinge to a disulfide bond. F (ab ') is a monomer antibody fragment in which a heavy chain hinge is added to a Fab obtained by reducing disulfide bonds of F (ab') 2 fragments. Fv (variable fragment) is an antibody fragment consisting of only variable regions of heavy and light chains, respectively. A single chain variable fragment (scFv) is a recombinant antibody fragment in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked by a flexible peptide linker. A diabody refers to a fragment of a form in which VH and VL of scFv are linked to a very short linker and can not bind to each other but bind to VL and VH of another scFv of the same type to form a dimer. For the purposes of the invention, the fragment of the antibody is not restricted in structure or form as long as it retains the binding specificity for the human-derived MRS protein.

In the present invention, as long as the anti-MRS antibody (including its functional fragment) is capable of specifically binding to the MRS protein, the site where the antibody interacts with (i.e., binds to) MRS is not particularly limited, Or an antibody or a functional fragment thereof, which specifically binds to an epitope of a region including the amino acid sequence represented by SEQ ID NO: 2 in the MRS. More preferably an antibody or a functional fragment thereof that specifically binds to an epitope comprising the 861th to 900th amino acid regions of the MRS (methionyl-tRNA synthetase) protein represented by SEQ ID NO: 1 .

In one embodiment of the present invention, for the detection (staining) of a high sensitivity of MRS to pancreatic cancer cells, the present inventors obtained an antibody that epitopes the amino acid sequence region represented by SEQ ID NO: 2 in MRS, It has been confirmed that it is possible to provide a high-sensitivity detection capability.

The antibody specifically binding to the epitope of the region including the amino acid sequence represented by SEQ ID NO: 2 is not particularly limited as long as it has the desired specific binding ability,

A light chain complementarity determining region 1 (CDR1) comprising an amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 16; Light chain complementarity determining region 2 (CDR2) comprising the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 18; A light chain variable region (VL) comprising a light chain complementarity determining region 3 (CDR3) comprising an amino acid sequence represented by SEQ ID NO: 8 or SEQ ID NO: 20, and

A heavy chain complementarity determining region 1 (CDR1) comprising an amino acid sequence represented by SEQ ID NO: 10 or SEQ ID NO: 22; A heavy chain complementarity determining region 2 (CDR2) comprising an amino acid sequence represented by SEQ ID NO: 12 or SEQ ID NO: 24; A light chain variable region (VH) comprising a heavy chain complementarity determining region 3 (CDR3) comprising an amino acid sequence represented by SEQ ID NO: 14 or SEQ ID NO: 26.

As a preferred example having the CDR construct, the antibody (including functional fragments thereof) of the present invention may comprise a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 28

And the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 30.

In another preferred example of the CDR construct, the antibody (including functional fragments thereof) of the present invention may comprise a light chain variable region having an amino acid sequence represented by SEQ ID NO: 32 and a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: And may include amino acid sequences.

As a most preferred example, the present invention provides an antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 36 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 37.

As another most preferred example, the present invention provides an antibody comprising a light chain consisting of the amino acid sequence of SEQ ID NO: 38 and a heavy chain consisting of the amino acid sequence of SEQ ID NO: 39.

Detectives (typically antibodies and functional fragments thereof, etc.) in the present invention may be labeled with a detectable moiety, generally for their detection. See, for example, Current Protocols in Immunology, Volumes 1 and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, N. Y., Pubs]. Or various enzyme-substrate labels are available, examples of which include enzymes such as luciferase such as Drosophila luciferase and bacterium luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydropthal Such as peroxidase, alkaline phosphatase, beta -galactosidase, glucoamylase, lysozyme, saccharide oxyde, peroxidase such as chondroitinase, chondrodinase, malate dihydrogenase, urase, horseradish peroxidase (HRPO) (Such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e. G., Free radicals and xanthine oxidases), lactoperoxidase, microperoxy And the like. Techniques for conjugating an enzyme to an antibody are described, for example, in O'Sullivan et al., 1981, Methods for the Preparation of Enzyme-in-vivo Conjugates for Use in Enzyme Immunoassay, in Methods in Enzym. J. Langone & H. Van Vunakis, eds., Academic press, N. Y., 73: 147-166. The label can be conjugated directly or indirectly to the antibody using a variety of known techniques. For example, the antibody can be conjugated to biotin and any label belonging to the three broad categories mentioned above can be conjugated to avidin, or vice versa. Biotin selectively binds to avidin, and thus this label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve an indirect conjugation of the label to the antibody, the antibody may be conjugated to a small hapten (e. G., Digoxin) and one of the different types of labels mentioned above may be conjugated to an anti- Hapten antibody (e. G., An anti-diphoshin antibody). Thus, indirect conjugation of the label to the antibody can be achieved.

In the present invention, the term 'aptamer' refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure as a substance capable of specifically binding to an analyte to be detected in a sample , The presence of the target protein in the sample can be confirmed specifically. Aptamer is prepared by determining the sequence of an oligonucleotide having a selective and high binding ability with respect to a target protein to be identified according to a general method of preparing an aptamer and then synthesizing an oligonucleotide at the 5'end or 3'end of the oligonucleotide But not limited to, -SH, -COOH, -OH, or NH2 to allow binding to the functional groups of the tamer chip.

The term 'acinar cell-specific marker' in the present invention means a marker showing a significant difference between a secretory cell and a non-secretory cell. Specifically, the presence (expression) or abundance (expression amount) of the gene is detected by detecting the presence (expression) or presence (expression) An indicator that can be measured objectively. As such a marker, proteins, DNA, RNA, metabolites and the like are generally used. In the present invention, it is preferable that a precursor cell-specific marker protein is used. As long as it is known in the art as a precursor cell-specific marker protein, the kind thereof is not particularly limited. For example, chymotrypsin, phospholipase A2 group IB (PLA2G1B, phospholipase A2 group IB) and amylase 2A ) May be used.

The above-mentioned 'chymotrypsin' is not particularly limited as long as it is known in the art as chymotrypsin (particularly, human), and its specific amino acid sequence is not particularly limited. For example, Genbank (NCBI) accession number EAW51726.1, EAW51725.1, CAA74031.1, AAH15118.1, CAA74031.1, NP_009203.2, and the like. For example, in one embodiment of the present invention, double-staining was performed with an MRS antibody using an antibody having a marking of a chymotrypsin protein represented by NP_009203.2 (see SEQ ID NO: 3).

The specific amino acid sequence constitution of the 'phospholipase A2 group IB' (PLA2G1B, phospholipase A2 group IB) is not particularly limited as long as it is known in the art as PLA2G1B protein (in particular, human), and for example, Genbank (NCBI) accession numbers AAI06727.1, AAI06726.1, AAH05386.1, NP_000919.1, and the like.

The specific amino acid sequence constitution of the amylase A2 (amylase 2A) is known in the art as amylase A2 (particularly, human). For example, Genbank (NCBI) accession number AAI46998.1 , AAH07060.1, BAD97183.1, AAA51723.1, and the like.

In the present invention, the agent for measuring the expression level of the precursor cell-specific marker protein is not particularly limited as long as it is known to be capable of measuring the expression level of the protein in the art, Binding antibody or aptamer, and a detailed description thereof is based on the anti-MRS antibody and the aptamer described above.

The kit for diagnosing pancreatic cancer according to the present invention may further comprise one or more antibodies or aptamers which selectively recognize each of the proteins, as well as an antibody or aptamer which selectively recognizes the protein or / and the secretory cell- Further, other component compositions, solutions or devices may be included.

In a specific embodiment, the kit may be administered by Western blot, ELISA, radioimmunoassay, radioimmunoprecipitation, Oucheroni immunodiffusion, rocket immunoelectrophoresis, immunostaining, immunoprecipitation assays, complement fixation, FACS, SPR, But are not limited to, diagnostic kits comprising known essential elements and associated elements necessary for performing the assay.

In one embodiment, the kit comprises an antibody specific for the MRS protein. The antibody is a monoclonal antibody, a polyclonal antibody or a recombinant antibody, which has high specificity and affinity for a target marker protein and has substantially no cross reactivity to other proteins. The kit may further comprise an antibody specific for any control protein. The antibody provided in the kit may itself be labeled with a detectable moiety, as described above. The kit may further comprise a separate reagent capable of detecting the bound antibody, for example, a labeled secondary antibody, chromophores, an enzyme (in conjugated form with the antibody) and a substrate or antibody ≪ / RTI > and the like. In addition, the kit of the present invention may include a washing solution or an eluting solution capable of removing surplus chromogenic substrate and unbound protein and retaining only the protein marker bound to the antibody.

The agent that measures the level of expression of the MRS protein may also include an agent that detects the level of expression of the MRS gene (MARS). As an increase in the level of protein expression is accompanied by an increase in the transcript (e. G., MRNA) from the gene encoding the protein, those skilled in the art will recognize that the MRS protein itself, It is obviously understandable that means for directly detecting the related transcripts can be used.

For example, a preparation for detecting MRS mRNA can be used, and if it is a ligand that specifically binds or hybridizes with MRS mRNA, the kind thereof is not particularly limited, but may be, for example, a primer (pair) or a probe.

In addition, the agent for measuring the expression level of acinar cell-specific marker protein includes an agent for detecting the expression level of the gene encoding the secretory cell-specific marker protein. For example, a preparation for detecting an mRNA encoding a precursor cell-specific marker protein can be used. If the ligand specifically binds to or hybridizes with the mRNA, the kind thereof is not particularly limited. For example, ) Or a probe.

The term " primer " refers to a nucleic acid sequence having a short free 3 'hydroxyl group and capable of forming a base pair with a complementary template and having a short nucleic acid sequence serving as a starting point for template strand copying It says. Primers can initiate DNA synthesis in the presence of reagents for polymerization (i. E., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffer solutions and temperatures. The PCR conditions, the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.

The sequence of the primer does not need to have a sequence completely complementary to a part of the nucleotide sequence of the template. It is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, in the present invention, the primer for measuring the expression level of the target mRNA does not need to have a sequence completely complementary to the coding gene sequence of the protein of interest, amplifies a specific region of mRNA or cDNA through DNA synthesis, It is sufficient that it has a length and complementarity that is suitable for the purpose of measuring the amount of the sample. The primer for the amplification reaction is composed of a pair (pair) complementarily binding to a template (or sense) at opposite ends of a specific region of the mRNA to be amplified and an opposite region (antisense), respectively. The primers can be easily designed by those skilled in the art with reference to the mRNA or cDNA base sequence encoding the desired protein.

The term "probe" refers to a fragment of a polynucleotide, such as RNA or DNA having a base pair length of several to several hundreds, which can specifically bind to a specific gene mRNA or cDNA (complementary DNA) And it is labeled so that the presence or expression level of mRNA or cDNA to be bound can be confirmed. For the purpose of the present invention, a probe complementary to a target mRNA can be used for diagnosis by performing hybridization with a sample of a subject to measure the expression amount of a target mRNA. The selection and hybridization conditions of the probes can be appropriately selected according to techniques known in the art.

The primer or probe of the present invention can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. The primer or probe can be chemically synthesized by a method that does not interfere with hybridization with MRS mRNA And can be variously modified according to methods known in the art. Examples of such modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) ) Or charged conjugates (eg, phosphorothioate, phosphorodithioate, etc.), and binding of labeling materials using fluorescence or enzymes.

The diagnostic kit of the present invention may further comprise one or more other component compositions suitable for the assay, as well as primers or probes for recognizing mRNA for each of them in order to measure the expression level of MRS protein and / or secretory cell specific marker protein, Solutions or devices may optionally be included. The kit is not particularly limited as long as it is known in the art as an assay kit for providing a primer (primer pair) or a probe as a component. For example, the kit includes a PCR (polymerase chain reaction), an RNase protection assay, Northern blotting, Southern blotting or kits for DNA microarray chips, and the like.

For example, the diagnostic kit may be a diagnostic kit characterized by comprising essential elements necessary for performing a polymerase reaction. The polymerase chain reaction kit contains the respective primer pairs specific for the marker gene (mRNA). A primer is a nucleotide having a sequence specific to the nucleotide sequence of each marker gene (mRNA), and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length. It may also contain a primer specific for the nucleic acid sequence of the control gene. Other polymerase enzyme reaction kits may be used in combination with test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), DNA polymerases (such as Taq polymerase) DNAse, RNAse inhibitor DEPC-water, sterile water, and the like.

Also,  In order to provide the information necessary for the diagnosis of pancreatic cancer, Splenocyte  Specific Marker  A method for qualitative or quantitative analysis of the expression level of a protein is provided. Specifically,

(a) Pancreas samples taken from potential patients Splenocyte  Specific Marker  Measuring an expression level of the protein and the MRS protein; And

(b) separating the sample from the measurement sample in step (a) Splenocyte  Specific Marker  And determining that the protein is not expressed and the MRS protein is expressed as a pancreatic cancer cell.

The term 'analysis' in the present invention may preferably mean 'measurement', and the qualitative analysis may be a measurement and confirmation of the presence or absence of a target substance, It may be meant to measure and confirm changes in the level of expression (level of expression) or amount. In the present invention, the analysis or measurement can be performed without limitation, including both qualitative and quantitative methods. Therefore, detection of MRS protein includes detection of the presence of MRS protein, or confirmation of an increase (up-regulation) of the amount of protein expression.

Hereinafter, the above method will be described in accordance with each step.

The step (a) is a step of providing a pancreatic sample collected from a potential patient and measuring the expression level of a secretory cell-specific marker protein and MRS protein in the sample.

The term " potential patient " in the present invention means a patient suspected of having pancreatic cancer, and means a patient suspected of having pancreatic cancer through various tests such as clinical symptoms, hematological examination or imaging test.

That is, the latent patient includes a patient who can be diagnosed as a pancreatic cancer based on an imaging test, and a patient who can not be diagnosed, and means a patient suspected of having a pancreatic cancer in a clinical symptom or hematological test even if the pancreatic cancer diagnosis is impossible. Clinical manifestations of pancreatic cancer include abdominal pain, jaundice, weight loss digestive disorder, and diabetes, but these symptoms are not specific to pancreatic cancer. Hematologic tests may also increase the number of tumor markers such as jaundice, diabetes mellitus, CEA, and CA19-9. Abdominal ultrasonography, abdominal CT, abdominal MRI, and PET-CT can be used for imaging. Pancreatic cancer is suspected when there is a pancreatic mass. These imaging tests may be suspicious for pancreatic cancer but not for pancreatic cancer. The final diagnosis of pancreatic cancer is pathologic, and in patients who can undergo surgery, the disease is confirmed by the tissue obtained after surgery, and in patients whose operation is not possible, it is confirmed by cytology.

 Preferably, the latent patient (suspected patient of pancreatic cancer) of the present invention has general symptoms such as abdominal pain, jaundice, weight loss, digestive disorder, diabetes, and the like which are generally observed in pancreatic cancer, and diagnostic devices such as CT, ultrasound, and MRI The patient may not be able to confirm with pancreatic cancer. More preferably, the latent patient has a need for definitive diagnosis of pancreatic cancer dependent on cytology (cytogenetic) as it is impossible to perform a wide area invasive biopsy or unnecessary patient (i.e., because of the impossibility or unnecessary inspection of pancreatic tissue by surgery) . That is, it may be, but is not limited to, a patient in need of definitive diagnosis of pancreatic cancer by cellular level analysis.

The sample is not particularly limited as long as it is collected from an individual (patient) or a subject to be diagnosed for the presence or absence of pancreatic cancer, but may be preferably a pancreatic tissue or a pancreatic cell. The pancreatic tissue can be obtained from any part of the pancreas including the pancreatic duct, in particular, a suspected lesion. The pancreatic tissue may be one usually obtained by biopsy or surgery in the pancreas. The method for isolating the pancreatic cells is not particularly limited and is understood to include not only a method currently used in the art for separating cells of a human tissue but also a new method to be developed for the same purpose in the future. Preferably, it may be brushing cytology or fine needle aspiration (FNA) or fine-needle aspiration, and most preferably, it may be an endoscopic ultrasonic microneedle inhalation method (EUS-FNA).

In the present invention, the term 'brush cytology' refers to a method of collecting cells by rubbing the pancreas, especially the pancreatic duct surface (particularly, the suspected lesion) using a conventional cytology brush.

The term 'separated by the fine needle aspiration method' in the present invention means a collection method in which cells of a lesion (suspected part of a pancreatic cancer) are aspirated and extracted using a thin needle commonly used for cytodiagnosis .

Preferably, in one embodiment, the pancreatic tissue or pancreatic cell sample essentially comprises pancreatic duct cells. The pancreatic duct is distinct from the pancreatic parenchyma.

The obtained pancreatic cells or tissues may be pretreated according to a conventional sample pretreatment (for example, fixation, centrifugation, slide-on-slide, etc.) methods known in the art.

 In one preferred embodiment, the pancreatic cell or tissue sample may be pretreated by conventional paraffin block or paraffin section preparation methods and provided on a laboratory slide.

In one preferred embodiment, the pancreatic cell or tissue sample may be pre-treated and prepared by a conventional liquid single-layered cell slide preparation method (liquid cell cell slide production method). For example, ThinPrep, SurePath, CellPrep, etc. may be used Based on a liquid-based monolayer attachment method.

The method for diagnosing pancreatic cancer of the present invention may be preferably for analyzing pancreatic cells. Cytology analysis method for directly analyzing pancreatic cells is very different from biopsy, and therefore, there are many differences from pancreatic cancer diagnosis methods reported in the above-mentioned prior art documents in this specification. Because the pancreas cells themselves are used, there is no room for confusion with tumors of other organs.

Previously, histological examination was performed by endoscopically observing a target site or collecting a tissue of a certain area ranging from 1 to 10 ⁹ cells from a tissue suspected of being transformed into cancer, and then performing a cancer diagnosis through a biochemical method such as dyeing do. It is known that such a biopsy is comparatively easy to confirm that cancer is present in a specific area through comparison with surrounding structures or cells. In addition, the expression pattern of the marker at the tissue level is more easily confirmed by comparing the trends with surrounding normal tissues as a whole. However, in the case of the cytopenia test, the diagnosis of the disease at the cellular level is of great significance because it is difficult to prove the relationship with the surrounding tissues since the individual cells are extracted and smoked.

Measuring the expression level of the protein means measuring the expression level (i.e., measuring the presence or absence of expression), or measuring the level of qualitative and quantitative change of the protein. The measurement can be performed without limitation, including both qualitative (analysis) and quantitative methods. The types of qualitative and quantitative methods for measuring protein levels are well known in the art and include the experimental methods described herein. Methods for comparing specific protein levels for each method are well known in the art.

In the present invention, the term 'detection' refers to measuring and confirming the presence (expression) of a desired substance (marker protein, MRS and / or secretory cell-specific marker in the present invention) Expression level) of the test compound. Accordingly, the term " protein detection " in the present invention is meant to include detection of the presence of a target protein or confirmation of an increase (up-regulation) of the expression amount of the protein.

In the present invention, the term " increased expression (or high expression) " of a protein means that an expression is expressed (i.e., an undetectable one is detected) or a relatively overexpressed (i.e., ). The meaning of the opposite term thereof can be understood by one of ordinary skill in the art to have the opposite meaning according to the above definition.

In the present invention, if the MRS protein and the secretory cell-specific marker protein are detected by a method for assaying protein expression levels known in the art, the method is not particularly limited. For example, detection using an antibody specifically binding to the protein Or measured. The antibody specifically binding to the target protein in the present invention is as described above. The method for measuring the protein expression level is not particularly limited as long as it is a method known in the art. For example, Western blotting, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay, Immunoprecipitation assays, complement fixation assays, FACS (Fluorescence activated cell sorter), SPR (surface plasmon resonance immunoassay), immunohistochemistry, immunohistochemistry, immunohistochemistry, resonance or protein chip method. In addition, the measurement method is understood to be the method of measuring the MRS protein and the precursor cell specific marker protein expression level provided by the present invention, and the measurement method thereof in accordance with the description of the kit containing the same.

In the case of conventional cytological examinations, it is often an atypical cell that is not clear whether it is pancreatic cancer or normal cells (including pancreatic cells, for example, which comprehensively refers to non-pancreatic cancer cells) , In which case additional and multiple repetitive re-examinations are required.

Previously reported pancreatic cancer markers have not been effective in cytologic examination due to poor sensitivity and specificity in diagnosis of cell - level, unlike histologic examination. This is well illustrated in one embodiment of the specification of the present invention. CEA, which is one of the most commonly used tumor markers for the diagnosis of pancreatic cancer, was not detected in pancreatic tumor cells, but was weakly expressed, and H & E staining revealed it to be an atypical cell, And that CEA was not detected in pancreatic cancer samples of pancreatic cancer patients. That is, in the case of the previously reported pancreatic cancer markers, it is impossible to definitively diagnose pancreatic cancer or non-coexisting abnormal cells in the atypical cells as a pathological cell diagnostic method using conventional H & E staining, In the case of the MRS according to the present invention, it is possible to clearly determine whether the tumor is a pancreatic cell, which is determined to be atypical. Thus, the MRS exhibits a remarkable effect of diagnosing pancreatic cancer more accurately. That is, the MRS according to the present invention has a high accuracy even when applied to a cytopathological test. Especially, when it is used in combination with a precursor cell-specific marker, the diagnostic accuracy of the false positive by the precursor cell is remarkably reduced, .

Accordingly, the present invention further provides the following steps (i) and (ii) before, simultaneously, or after (i) the measurement of the expression level of the precursor cell-specific marker protein and the MRS protein Thereby further enhancing the diagnostic effect:

(i) pancreatic cells taken from potential patients

(4 ', 6-diamidino-2-phenylindole), methylene blue, acetocarmine, toluidine blue, hematoxylin and Hoechst At least one dyeing solution selected from the group consisting of

Staining the cell with at least one staining solution selected from the group consisting of eosin, crystal violet and orange G staining cytoplasm; And

(ii) determining pancreatic cells as malignant tumor cells, atypical cells or normal cells by staining the cells.

The atypical cells identified in the step (ii) are understood to be undefined and undefined cells, and include, but are not limited to, all of the suspects of malignancy (malignant tumor cell suspicious) .

The above steps (i) and (ii) are cytodiagnosis methods based on a conventional pathological examination of a morphological diagnosis method, which are based on H & E or pap staining used in an embodiment of the present invention. In the present invention, the term " morphological diagnostic pathology " or " morphological examination " refers to examining abnormal morphological changes when normal cells are transformed into cancers.

The specific test item or criterion regarding the abnormal morphological change is not particularly limited as long as it is a kind of morphological change of cancer cells in the art, but preferably cell clustering; Nuclear / cytoplasm ratio (N / C ratio); Shape of nuclear membrane (nuclear membrane irregularity); Aggregation of chromatin; Appearance of nuclear bodies in the nucleus; And the appearance of mitosis. ≪ RTI ID = 0.0 > [0031] < / RTI > The morphological examination can be carried out simultaneously or separately, or simultaneously with the method of providing information necessary for the diagnosis of pancreatic cancer by qualitative or quantitative analysis of expression levels of the MRS protein and secretory cell-specific marker protein provided by the present invention. And may be performed in a sequential manner.

The determination of the pancreatic cell sample as a malignant tumor cell, an atypical cell or a normal cell from the result of the cell staining in the step (ii) in the step (ii) is based on the abnormal morphological change when the normal cell is changed into cancer , And the specific discrimination criteria are well known in the art. At this time, the morphological change of the atypical cell means a malignant tumor cell (cancer cell) or a cell which can not be clearly determined as a normal cell.

In a preferred embodiment of the present invention, the determination of the pancreatic cell sample as a malignant tumor cell, an atypical cell or a normal cell from the cell staining result of the step (i) in the step (ii) It can be done:

Cells are plastified in three dimensions; High nuclear / cytoplasmic ratio (N / c ratio); Appearance of chromatin aggregation; A rough nuclear membrane (the degree of irregularity of the nuclear membrane becomes larger); Emergence of nuclear bodies; And the appearance of mitosis is judged to be malignant tumor cells when two or more morphological abnormalities are selected,

Cells were plated in one layer, and when the nucleus / cytoplasm ratio (N / C ratio) was small and the nuclear membrane was smooth,

If the change in the cell does not reach the malignant cells but can not be determined as normal (including benign), it is judged to be an atypical cell.

If the steps (i) and (ii) are performed before, concurrently with, or after the MRS detection (expression level measurement) step, the cell level test It is characterized by what can be obtained. For example, in the case of performing the steps (i) and (ii) before the MRS detection, in the case of pancreatic cells judged to be malignant tumor cells or normal cells through cell staining, MRS and the expression level (or absence) of the precursor cell-specific marker protein can be more reliably determined to determine whether the cancer is pancreatic cancer or not, thereby making it possible to significantly reduce the diagnosis error and to judge it as an atypical cell through the cell staining , It is possible to make a clear judgment as to whether the tumor is a tumor by analyzing the secretory cell specific marker protein and the MRS expression level (or not) in the subsequent step (a).

The present invention is characterized by the fact that a double-staining method of MRS and a precursor cell-specific marker protein enables accurate diagnosis with high accuracy in examination not only of tissue but also of cell level. In the case of non-malignant cells, there is a need to perform a biopsy again, and a biopsy that requires a large amount of biopsy results in a physical burden on the patient In addition, when considering the problem that it is difficult to obtain a large amount of tissue samples depending on the degree of progression of pancreatic cancer, the present invention which provides accurate diagnosis at the cell level has a great advantage.

In step (b), when the MRS protein is expressed in the measurement sample of step (a) without expressing the precursor cell-specific marker protein, it is determined that the cell is a pancreatic cancer cell.

According to one embodiment of the present invention, MRS was not detected (increased expression) in pancreatic cells determined to be normal cells through H & E staining, but MRS was strongly expressed in tumor cells. That is, it is confirmed that MRS can be used as a pancreatic cancer diagnostic marker. In addition, MRS was detected in the pancreatic cells of patients whose diagnosis was finally confirmed as pancreatic cancer after H & E staining was confirmed to be atypical.

 On the other hand, in the diagnosis of pancreatic cancer, MRS was not expressed in most of the normal cells, but a small number of normal samples showed MRS expression, resulting in false-positive results. This false- Which is caused by the phenomenon of For example, a double staining method using MRS and a dual marker using chymotrypsin as a precursor cell specific marker has been developed. In the case of pancreatic cancer cells, a strong expression signal of MRS is highly prevalent without expression of a leader cell marker (chymotrypsin), and thus, a sample classified as an atypical cell in a conventional Pap smear can be diagnosed as pancreatic cancer and non- .

The step (b) is advantageous in that pancreatic cancer (particularly pancreatic cancer or pancreatic ductal adenocarcinoma) can be detected directly through detection of (high) expression of MRS without comparison with another control group (sample). This is well illustrated in the specification of the present invention.

The level of MRS detection (the level of MRS expression, particularly the level of increase), which is a standard for diagnosis of pancreatic cancer, can be determined by dividing the degree of detection (expression) or the degree of detection (expression) according to the measurement method selected by a person skilled in the art. For example, by measuring the levels of MRS expression in a number of normal subjects and patients, data can be accumulated and analyzed to determine appropriate criteria for diagnosis, such as normal category and pancreatic cancer occurrence category according to the level of MRS detection (expression) .

Also, the step (b) may be performed relatively to the negative control sample (in particular, the normal control sample). Thus, the method may further comprise comparing the MRS protein level detected from the pancreatic sample collected from the potential patient with the negative control sample in step (b) or after step (b). In the present invention, the term normal control group refers to a pancreatic sample collected from a normal human part in a pancreas of a potential patient to be examined (i.e., the same individual as a patient to be examined in the step (a)) or other normal individuals It is meant to include all samples taken from the pancreas. At this time, if the level of MRS protein detected in the pancreas sample collected from the potential patient is higher than the level of the negative control (especially, the normal control), it can be judged that the patient is a pancreatic cancer patient.

Information on such a control group (for example, detection intensity or expression intensity) may be provided in the form of an MRS expression level measurement preparation provided in the present invention described below and a kit containing the same, . Compared to the control group, a higher level of MRS expression in the test sample can be interpreted as a pancreatic cancer patient.

The term " normal pancreatic cell " or " normal control group " in the present invention means a non-tumor (cancer) pancreatic cell. It means a pancreatic cell in a state other than a cancer (cancer) , Benign (benign), and fully healthy pancreatic cells (atherosclerotic pancreatic cells).

The present invention also provides a method for distinguishing atypical cells from cancer cells (malignant tumor cells) and normal cells (non-cancer cells), including steps (a) and (b) described above.

In order to diagnose pancreatic cancer, it is necessary to perform expensive and thorough examination such as computed tomography (CT), endoscopic retrograde cholangiopancreatography (ERCP), ultrasound endoscopy (EUS) and angiography. It is very difficult to diagnose correctly. Ultimately, pathologic methods such as biopsy or cytology should be performed to confirm pancreatic cancer. On the other hand, pancreatic cancer is a case of progressive cancer that is not easily resectable at the time of diagnosis, and in cases where surgery is impossible, only 10-15% of patients can not undergo surgery. Diagnosis of pancreatic cancer is performed through endoscopic ultrasonic micro needle aspiration test . However, there is a limitation in confirming the diagnosis of the cell diagnosis by the endoscopic ultrasonic microneedle aspiration test because it is difficult to compare with the surrounding structures or cells compared with the postoperative pancreatic cancer tissue.

Existing cytopathologic diagnosis depends on general staining such as H & E staining or pap staining to differentiate cells of pancreatic cancer cells and other diseases (for example, pancreatitis). However, the diagnosis of pancreatic cancer by conventional cytopathologic diagnosis based on conventional conventional staining is different according to the experience and interpretation technique of the medical staff. In addition, in the case of these conventional tests, pancreatic cancer or normal cells (non-pancreatic cancer cells (Eg, pancreatitis, etc.). It is often the case that an atypical cell is suspected to be an atypical cell. In such cases, an additional, multiple recurrence is required.

Especially, when it is judged as atypical cell in pancreatic cell observation through H & E staining or pap staining, it is very difficult to distinguish whether it is pancreatic cancer or other benign disease. As the accurate diagnosis of the cell division is delayed, it is impossible to appropriately treat a pancreatic cancer patient who can undergo surgery, and conversely, it is difficult to prevent unnecessary surgery. Therefore, accurate diagnosis of cytology is needed to increase the therapeutic effect of pancreatic cancer clinically.

It is very important to determine the tumor status in atypical cells, which are very difficult to distinguish between H & E staining and pap staining, which are commonly used to diagnose cancer. In these atypical cells, MRS (High-grade) expression of the tumor cells (except for the precursor cells) can be judged as tumor cells. In the case of biopsy requiring a relatively large amount of biopsy, the physical burden on the patient is increased in the acquisition of the sample rather than in the cytological examination. In some cases, the operation can not be performed according to the progress of the cancer, The diagnostic method of the present invention, which provides an accurate diagnosis even at the cellular level, has a great advantage.

In the diagnosis of pancreatic cancer, when employing the method of the present invention in which the MRS protein and the precursor cell-specific marker protein are simultaneously used as markers and their detection patterns are analyzed, the sensitivity and specificity , The positive predictive value and / or the negative predictive value are almost 100%.

Accordingly, the present invention relates to a method for screening for pancreatic cancer cytodiagnosis ) In a test or biopsy, a method is provided for improving the sensitivity or specificity, comprising the steps of:

(a) Pancreas samples taken from potential patients Splenocyte  Specific Marker  Measuring an expression level of the protein and the MRS protein; And

(b) In the step (a) Splenocyte  Specific Marker  Expression of MRS protein without protein expression If increased  Determining that the cell is a pancreatic cancer cell.

In the present invention, the term 'sensitivity' refers to the rate at which a final clinical pathological diagnosis is made for a pancreatic cancer sample or a patient through a subject test method (eg, the test method of the present invention).

In the present invention, the term 'specificity' refers to the rate at which a normal determination is made through a subject test method (eg, the test method of the present invention) for a sample or patient whose final clinical pathological diagnosis is normal.

Specifically, at least one selected from the group consisting of the sensitivity, the specificity, the positive predictive value and the negative predictive value is 80% to 100%, preferably 85% to 99%, more preferably 90 to 98% Level). Specific figures for this level are 80%, 81%, 82% and 83%. 98%, 99%, 100%, 85%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% And a range value having a boundary value of two numbers selected from the group consisting of In an embodiment of the present invention, a boundary value of 80% and 95% in the numerical range may be selected, so that all values in the range of 80% to 95% are intended for the present invention It will be apparent to those skilled in the art. In another embodiment, the sensitivity, specificity, positive predictive value and / or negative predictive value are preferably at least 85% (85% to 100%, preferably 87% to 99%, more preferably, 90 to 98%), but is not limited thereto.

It will be apparent to those skilled in the art that the improvement in sensitivity, specificity, positive predictive value, and / or negative predictive value leads to improvement in accuracy. Therefore, the method of the present invention can be understood as a method of improving the accuracy, and it is preferable that the accuracy is in the range of 90% to 100%, and more preferably the accuracy is in the range of 90% to 99%. The specific values of the above levels were 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5% , 97.5%, 98%, 98.5%, 99%, 99.5%, and 100%. In an embodiment of the present invention, a boundary value of 92.5% and 99.5% in the numerical range may be selected, so that all values in the range of 93.5% to 99.5% It will be apparent to those skilled in the art. In yet another embodiment, it is more preferably from 93% to 98%, most preferably from 95% to 98%, but is not limited thereto.

Also,  The invention relates to a method for screening for pancreatic cancer cytodiagnosis ) In the test or biopsy, together with the morphological examination

(a) Pancreas samples taken from potential patients Splenocyte  Specific Marker  Measuring an expression level of the protein and the MRS protein; And

(b) In the step (a) Splenocyte  Specific Marker  Expression of MRS protein without protein expression If increased  The present invention provides a method for providing information necessary for diagnosis of pancreatic cancer, which comprises the further step of determining that the pancreatic cancer cell is a pancreatic cancer cell.

The morphological examination includes all of the other morphological examination methods according to this method, including the tests performed including the steps (i) and (ii) described above as a preferable example. A description thereof will be made by those skilled in the art with reference to the above description.

A detailed description of the steps (a) and (b) is as described above. If the step is performed as an adjuvant (i.e., as an adjuvant therapy), simultaneous, separate, Or in a sequential manner. In addition, the determination method including steps (a) and (b) above can be performed before, simultaneously, or after the morphological inspection.

MRS is more accurate than conventional pancreatic cancer markers such as CEA. Especially, when double-marker markers are used as markers of chimotrypsin, Lt; / RTI >

Brief Description of the Drawings Fig. 1 is a graph showing the MRS binding strength and specificity of the anti-MRS antibodies (1E8, 8A12) of the present invention compared with a known commercially available MRS antibody (Ab50793) using cell extracts of H460 cells treated with si-MRS Results.
2 shows the MRS binding strength and specificity of the anti-MRS antibodies (1E8, 8A12) of the present invention using the cell extracts of the PANC-1 cell line (pancreatic cancer cell line) and the SCK cell line (non-pancreatic cancer cell line) (Ab137105). ≪ / RTI >
FIG. 3 is a graph showing the results of ELISA to confirm the cross-reactivity of 1E8 antibody against other ARS (aminoacyl-tRNA synthetase) and AIMP proteins.
Figure 4 is a graph showing the results of ELISA to confirm the crossactivity of the 8A12 antibody against other ARS (aminoacyl-tRNA synthetase), AIMP protein.
FIG. 5 shows SPR (surface plasmon resonance) test results for confirming antibody affinity of 1E8 antibody against MRS + AIMP3 protein.
Fig. 6 shows the result of SPR (surface plasmon resonance) experiment in which 1E8 antibody was confirmed to be not reactive with AIMP3.
FIG. 7 shows SPR (surface plasmon resonance) test results performed to confirm antibody affinity of 8A12 antibody against MRS + AIMP3 protein.
Fig. 8 shows the result of SPR (surface plasmon resonance) test in which 8A12 antibody was confirmed to be non-reactive with AIMP3.
FIG. 9 shows the results of immunofluorescence staining of PANC-1 cell line (pancreatic cancer cell line) and SCK cell line (non-pancreatic cancer cell line) using the anti-MRS antibody of the invention (1E8, 8A12) and the commercial MRS antibody (Ab137105) .
FIG. 10 shows the results of immunohistochemical staining of Thinprep slides similar to clinical conditions using PANC-1 cell line using Thinprep equipment (Hologic.Inc) used for patient cell sample processing at a clinical site, and immuno-fluorescence of 1E8 antibody and 8A12 antibody of the present invention The result of performing dyeing is shown.
FIG. 11 shows the result of observation of the expression of MRS and the observation of the expression of CEA (each number is the patient code number) as a result of H & E staining of pancreatic cells isolated from a normal patient.
FIG. 12 shows the results of H & E staining of pancreatic cells isolated from pancreatic cancer patients, the observation of the expression of MRS, and the observation of the expression of CEA (each number is the patient code number).
FIG. 13 shows the result of observation of the expression of MRS and the observation of the expression of CEA in the pancreas cytology sample of a patient confirmed to be atypical as a result of H & E staining, and subsequently confirmed as pancreatic cancer (each number is the patient code number).
14 shows the results of observing MRS expression intensity in normal acinar cells in normal pancreatic tissues.
FIG. 15 shows H & E staining results of acinar cells in normal pancreatic tissues.
FIG. 16 shows the results of double detection of MRS and chymotrypsin protein for acinar cells in normal pancreatic tissues.
FIG. 17 shows a pattern pattern in which the MRS was detected very weakly and the relatively high detection of chymotrypsin was found among the representative staining patterns of the precursor cells when the double staining method of the present invention was applied to the pancreatic precursor cell cytology sample .
FIG. 18 shows that, when the double staining method of the present invention was applied to the pancreatic precursor cell cytology sample, the MRS detection was significant as well as the chymotrypsin among the representative staining patterns represented by the precursor cells, but the pattern pattern in which the detection of the chymotrypsin was relatively strong Lt; / RTI >
FIG. 19 shows the results of performing the double staining method of the present invention in a pancreatic cell sample of a pancreas cancer confirmed by papain staining and finally a confirmed pancreatic cancer patient.
20 shows the results of pancreatic cell examination of pancreatic cancer patients in which the double staining method of the present invention was carried out, although the pancreas was classified as atypical cells by conventional cytopathologic examination (pap staining) .

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1: Pancreatic cancer of the present invention Useful antibodies for testing  Production (Obtaining High Specificity Antibodies to MRS)

In vivo, methionyl-tRNA synthetase (MRS) is associated with AIMP3 (Aminoacyl tRNA synthetase complex-interacting multifunctional protein 3) and it is known that this binding state is separated by UV irradiation. Therefore, in order to accurately detect MRS, it is necessary to specifically detect MRS only in a state where MRS binds to AIMP3. However, since there are many similarities in protein structure between AIMP and ARS species, AIMP and ARS species. Thus, for the diagnostic accuracy of the pancreatic cancer assay of the present invention, the present inventors produced a highly sensitive MRS antibody without cross-reactivity to other proteins by the following method.

1-1. MRS-AIMP3 protein production

The MRS-AIMP3 co-purified protein was expressed and purified on E. coli. Specific experimental methods are as follows. The transformants were transformed to express MRS (SEQ ID NO: 1) and AIMP3 (SEQ ID NO: 40, NCBI ref.NM_004280.4) using BL21DE3 strain and cultured in LB medium. Then, single colonies were treated with 5 ml LB containing ampicilin The cells were cultured in a liquid medium so as to have an OD 600 value of 0.6 to 0.8. Then, 1 mM IPTG was added thereto, followed by incubation at 37 ° C for 3 hours, followed by centrifugation for 10 minutes to obtain cells. SDS-PAGE was performed with the cell solution, and expression of the proteins was confirmed using a coomassie stain. Subsequently, the cell solution which induced the overexpression by IPTG was collected and centrifuged to obtain cells. The cells were lysed with 1 ml of DPBS and then lysed with an ultrasonic mill. The MRS-AIMP3 co-purified protein was then separated by centrifugation with the lysed cells.

1-2. Mouse immunization via injection of MRS-AIMP3 protein

To obtain immunized mice necessary for the production of hybridoma cells, the MRS-AIMP3 co-purified protein obtained in Example 1-1 was first injected into the abdominal cavity of four to eight weeks old mice. 10-week old BALB / c mice weighing 25-30 g were purchased from Orient Bio Co. (Sungnam, KyungkiDo, Republic of Korea) and animals were maintained under constant conditions (temperature: 20 ± 2 ° C, humidity: 40-60% 12 hours light / dark cycle). Animal experiments were conducted in accordance with the guidelines of the University Animal Care and Use Committee at Seoul National University. In order to increase the immunity of mice after primary immunization, MRS-AIMP3 co-purified protein of the same dose was injected into mice intraperitoneally two weeks later. One week thereafter, MRS-AIMP3 co-purified protein was injected into the tail vein of mice 3 days before the cell fusion experiment. The immunized mice were anesthetized with ether and blood was collected from the heart with a heparinized syringe. The blood was allowed to stand overnight at 4 ° C and centrifuged to separate the serum. The separated serum was appropriately divided and stored at -80 ° C.

1-3. Production of hybridoma cells

First, myeloma cells were prepared for cell fusion. Myeloma cells were cultured and the cell density was adjusted to 2.5 to 5 x 10 ⁴ cells / ml. Cells were prepared by diluting 1/3 of myeloma cells 24 hours before cell fusion. The mice immunized in Example 1-2 were anesthetized with ether and spleens were collected to separate B cells, followed by washing with SF-DMEM2 (DMEM + 2 x AA) to elute the cells. The cell suspension was collected, placed in a tube, allowed to settle, and the supernatant was transferred to a new tube and centrifuged at 1500 rpm for 5 minutes. The supernatant of the centrifuged splenocytes was removed and tapping followed by filling of SF-DMEM2. B cells and myeloma cells were each centrifuged and washed, and the washing procedure was repeated one more time. The supernatant of the washed myeloma cells was removed, tapped and filled with SF-DMEM2. After removing the supernatant of the washed B cells and tapping, 1 ml of LB (lysis buffer) was treated with RBC (red blood cell), and SF-DMEM2 was filled. Then, B cells and myeloma cells were centrifuged, centrifuged B cells and myeloma cell supernatants were removed, and then tapping and 10 ml of SF-DMEM2 were filled. B cells and myeloma cells were each diluted and counted 100 times in the e-tube to determine the concentration. [B cell concentration (1 × 10 ⁸ , 8 × 10 ⁷ , 5 × 10 ⁷ ), myeloma cell concentration 10 ⁷ , 8 × 10 ⁶ , 5 × 10 ⁶ ). B cells and myeloma cells were determined at a ratio of 10: 1. The determined concentration of B cells and myeloma cells were put into tubes and centrifuged. After removing the supernatant from the centrifuged cells, the supernatant was spun down on the alcohol solution, semi-dried and tapped for 30 seconds to 1 minute. PEG (2 ml) was slowly added thereto for 1 minute while pipetting and reacted. The tube was shaken while inserting SF-DMEM2, followed by centrifugation. After centrifugation, the supernatant was removed and 1 ml of HT medium (HT50 x (HT (sigma) 1 vial + SF-DMEM1 10 ml), FBS 10 ml, SF-DMEM1 (DMEM + 1 x AA) 30 Ml] was dropped dropwise, and gradually increased to 50 ml. The suspension was incubated again in a 37 ° C, 5% CO2 incubator for 3 hours.

1-4. Screening of hybridoma cells producing MRS-specific monoclonal antibodies

Among the fused cell groups prepared in Example 1-3, cells that did not recognize AIMP3 were selected while recognizing MRS, and experiments were conducted as follows to confirm whether or not an antibody was produced.

First, the medium was changed from 8 to 9 days after cell fusion, and cultured in 96 wells and 24 wells were cultured in cDMEM2 until well grown. After replacing the medium, the supernatant of the color-changed wells was collected on days 5 to 7 and filled with cDMEM2, and ELISA test for the binding of MRS and AIMP3 to the antibody produced in each fusion cell was performed. After the ELISA test, wells were selected and cultured in 24 wells. 24 wells and then subjected to ELISA again. Specifically, the concentration of the fusion cell in 24 wells was determined, and the fusion cells were diluted in 15 ml of the culture medium so as to be 0.5 cell / well in a 96 well plate. Fusion cell dilutions were dispensed at 150 각 per well. The wells containing one cell were checked by microscopic examination. The supernatant of the wells to which the cells were grown to some extent was subjected to the first screening by confirming the binding between MRS and AIMP3 with the antibody produced in each fusion cell by ELISA and Western blotting. Fusion cells selected on the basis of the primary screening were transferred to 24 wells, cultured and centrifuged, and the supernatant was collected and subjected to secondary screening by ELISA and Western blotting. (OD value) of the fused cells grown in 24 wells was confirmed by ELISA, and only the fusion cells having an absorbance of 1.0 or more were selected, and the cells were transferred to a 25 T / C culture flask and incubated. After centrifugation, the supernatant was collected and subjected to ELISA and Western blotting And the tertiary screening was carried out. The fusion cells selected on the basis of the tertiary screening were transferred to a 75T / C culture flask, cultured, and the absorbance was confirmed by ELISA to select cells that did not recognize AIMP3 while recognizing MRS. Finally, "1E8" and "8A12" clones Respectively.

1-5. Culturing of hybridoma cells producing MRS-specific monoclonal antibodies and antibody purification

Monoclonal antibodies against MRS can be obtained from the final fusion cells (hybridoma cells " 1E8 " or " 8A12 ") selected in Examples 1-4 above, respectively, by the following two methods.

1) 500 μl of pristane was injected into the abdominal cavity of female mice 7 to 8 weeks of age. Fusion cells cultured in a 75T / C culture flask were collected, centrifuged, and then the supernatant was removed, and the cells were pipetted in a phosphate buffer. 7-10 days after administration of Pristan, the fused cells selected in Example 1-4 were injected into the abdominal cavity of mice at 8 × 10 ⁵ to 4 × 10 ⁷ , respectively. When ascites was filled in the abdominal cavity of the mice 1 to 2 weeks later, the ascites was withdrawn using an 18G injection needle. The ascites was left overnight at 4 ° C and centrifuged the next day to remove lumpy material including yellow fat layer, and only the supernatant was separated. The supernatant was separated and stored at -20 ° C.

Protein A, which is stored in a storage solution (20% ethanol), is filled in an appropriate amount of the column, and 20% ethanol is flowed thereinto. Then, 5-volume volume of binding buffer (20 mM sodium phosphate, pH 7.0) And washed. The aliquots were diluted with a suitable amount of phosphate buffer and loaded onto a Protein A column. (20 mM sodium phosphate, pH 7.0), and eluted with 0.5 ml of 3-Bed Volume elution buffer (0.1 M glycine buffer, pH 3.0 to 2.5). Each fraction was neutralized with 35 μl of neutralization buffer (1 M Tris-HCl, pH 9.0). Through SDS-PAGE, the purity of the fractions was determined and desalted with an Ammersharm GE column.

2) Hybridoma cells obtained in the above Example 1-4 were acclimated in serum-free medium (Thermo) supplemented with GlutaMAX (Gibco) (final 5 mM) and 1x Choleserol lipid concentrate (Gibco) Breydoma cells are also named 34-8F2). The cells were then cultured at a maximum culture volume of 860 mL using Cellstack-5 (Corning, Corning, NY). GlutaMAX (Gibco) (final 5 mM) and 1x Choleserol lipid concentrate (Gibco) were added to serum-free medium (Thermo) and initial cell concentration was inoculated at 1.4-2.0 × 10 ⁵ cells / mL. After 4 to 5 days of inoculation, the cells were centrifuged at 2000 rpm for 10 minutes, and the supernatant was recovered. After confirming the pH of the supernatant, the pH was adjusted to 7.6 using a 20X binding solution (1M Potassium phosphate dibasic) (pH 9.0). And then filtered using a 0.22um filter to obtain a neutralized antibody culture.

The obtained antibody culture was purified through a protein A column. 10 column volumes of distilled water was flowed through the protein A column and an equal volume of 1X binding solution (50 mM Potassium phosphate dibasic) (pH 9.0) was flowed through. Then, the obtained antibody culture solution was flowed to bind the antibody to protein A, followed by washing with 1X binding solution (50 mM Potassium phosphate dibasic) (pH 9.0). Then, two column volumes of elution solution (0.2 M citric acid) (pH 3.0) were flown through to elute antibody bound to protein A to obtain an eluate. After neutralization with 1 M Tris, antibody concentration was determined by measuring absorbance at 280 nm.

Thereafter, the GE PD-10 column was equilibrated with 25 ml of physiological saline and centrifuged (1000 g, 2 minutes). Then, 2.5 ml of the antibody eluate obtained from the protein A column was placed in the GE PD-10 column, centrifuged (1000 g, 2 minutes), and the antibody-exchanged antibody was collected with physiological saline. The antibody concentration was determined by measuring the absorbance at 280 nm, and divided and stored at -80 캜.

1-6. Sequence analysis and cloning of antibodies

Cloning and sequencing of the 1E8, 8A12 clone expressing antibodies, respectively, were performed by YBIO inc. And Abclon Inc. Korea. Briefly, cDNA was synthesized by first extracting RNA from 1E8 or 8A12 hybridoma cells. PCR was then performed using primers specific for VL, CL, VH and CH1, respectively. The PCR product of the expected size was purified on agarose gel and sequenced to confirm the sequence and confirmed the CDR region by Kabat numbering. The results of identification of the antigen binding site of the 1E8 antibody are shown in Table 1, and the results of confirming the sequence of the antigen binding site of the 8A12 antibody are shown in Table 2. Fab was synthesized with the confirmed sequence and confirmed by ELISA that MRS shows high binding ability.

Also, it was confirmed that the sequence identified above was consistent with the result of protein sequence analysis (mass spectrometry result) of the antibody obtained through multiple purification after the hybridoma cells were injected into mouse abdominal cavity in Example 1-5.

The resulting 1E8 Fab or 8A12 Fab sequence was cloned into mouse IgG heavy chain (pFUSE-mIgG2a-Fc, InvivoGen) and mouse light chain sequence vector (pFUSE2-CLIg-mK, InvivoGen), respectively. The vector was then co-transformed into freestyle 293F cells using PEI (Polysciences, 23966-2), allowing the light and heavy chains of the antibody to coexpress simultaneously in the cells. Transfected 293F cells were cultured at 37 ° C and 8% CO ₂ for 7 days. Cells were then harvested and centrifuged to obtain supernatants. After confirming the pH of the supernatant, the pH of the supernatant was adjusted to 7.6 using the prepared 20X binding solution (1M Potassium phosphate dibasic) (pH 9.0). The supernatant was then filtered with a 0.22 [mu] m filter to obtain a neutralized antibody culture. Antibodies were obtained from the antibody culture in the manner described in 2) of Example 1-5. The total antibody of 1E8 IgG thus obtained was confirmed to consist of a light chain consisting of the amino acid sequence of SEQ ID NO: 36 and a heavy chain consisting of the amino acid sequence of SEQ ID NO: 37. It was also confirmed that the whole antibody of 8A12 IgG was composed of the light chain consisting of the amino acid sequence of SEQ ID NO: 38 and the heavy chain consisting of the amino acid sequence of SEQ ID NO: 39.

1-7. Confirmation of binding specificity of antibody against MRS - Western blotting experiment

Western blotting experiments were performed as follows to confirm the MRS binding ability of the 1E8 antibody and 8A12 antibody obtained in the above example. H460 cells were cultured in DMEM (Hyclone, GE lifesciences) medium containing 10% FBS (Fetal bovine serum, Hyclone, GE lifesciences) and 1% penicillin (Hyclone, GE lifesciences). Each cell was cultured under conditions of 5% CO ₂ and 37 ° C. The cultured H460 cells were treated with si-MRS for 72 hours. The H460 cells were then harvested, lysed and then Western blotted with H460 cell lysate. The experiment was repeated twice. As a primary antibody, 1E8 antibody or 8A12 antibody was diluted to 1: 5000 (0.2 μg / ml), and MRS antibody (Abcam, Ab50793) distributed in the market was used for the comparison of binding ability. (Tublin) was used.

As shown in FIG. 1, conventional MRS antibodies circulating in the market failed to detect MRS in the si-MRS-treated group, and in the non-treated group of si-MRS, the 8A12 antibody of the present invention and 1E8 (Ability to bind to MRS) at a significantly lower level than the antibody. On the other hand, the 1E8 antibody and the 8A12 antibody of the present invention were remarkably superior to the conventional MRS antibody in MRS specific binding ability and sensitivity, and the 8A12 antibody showed excellent binding ability and sensitivity.

In addition, PANC-1 pancreatic cancer cell line and SCK cell line (non-pancreatic cancer cell line) were further used to confirm the MRS binding ability of 1E8 antibody and 8A12 antibody. As a control, commercially available MRS antibody (Abcam, Ab137105) was used (antibodies were diluted to 1: 1000 and used at 0.137 / / ml), except for the treatment of si-MRS, Western blot experiment.

Experimental Results As shown in FIG. 2, the 1E8 antibody and 8A12 antibody of the present invention specifically detected MRS, but the existing commercial antibody Ab137105 antibody exhibited a large number of nonspecific bands under the same conditions, indicating that the selective detection ability was very poor. MRS was not detected in SCK cell line (non-pancreatic cancer cell line) under this experimental condition.

1-8. Determine cross-reactivity to other proteins - ELISA

The following experiment was carried out to confirm whether the 8A12 antibody obtained in the above example had cross activity against other ARS (aminoacyl-tRNA synthetase) proteins.

(His-MRS, MRS full) and other ARS proteins (DX2 tag free, 34S-DX2, 34S-AIMP2, His-CRS, His) were added to 96 well plate (Corning 3690 flat bottom, -AIMP1, His-GRS, His-WRS, and His-KRS) at a concentration of 1 μg / ml. 1E8 antibody or 8A12 antibody was added to a 96-well plate coated with each of the ARS proteins at a concentration of 500 ng / ml and reacted for 1 hour. Then, HRP-conjugated anti-mouse IgG secondary antibody was added and reacted for 1 hour. Absorbance was measured at 450 nm by ELISA. TMB (3,3 ', 5,5'-Tetramethylbenzidine) was used as the substrate.

As a result, as shown in Fig. 3, the 1E8 antibody reacted only with MRS and did not react with other ARS proteins and AIMP proteins. Also, as shown in Fig. 4, the 8A12 antibody reacts only with MRS and does not react with other ARS proteins and AIMP proteins. As a result, it was confirmed that the 1E8 antibody and the 8A12 antibody had no cross-reactivity to other ARS protein and AIMP protein and specifically detected only MRS.

1-9. Confirm antibody affinity using surface plasmon resonance

SPR (surface plasmon resonance) experiments were conducted using MRS-AIMP3 co-purified protein (hereinafter referred to as MRS + AIMP3 protein) and AIMP3 protein in order to confirm the MRS specific affinity of 1E8 antibody and 8A12 antibody Respectively. MRS + AIMP3 or AIMP3 protein was coated on a CM5 chip and the degree of binding reaction with protein was measured while flowing 1E8 antibody or 8A12 antibody at various concentrations. The analytes and buffers were injected at a flow rate of 30 μl / min for 8 minutes and washed for 20 minutes.

As a result, as shown in FIG. 5 and FIG. 6, it was confirmed that the 1E8 antibody binds to the MRS + AIMP3 protein but does not bind to the AIMP3 protein. It was also confirmed that the 1E8 antibody had a KD value of 5.42 nM for MRS

As shown in FIGS. 7 and 8, it was confirmed that the 8A12 antibody binds to the MRS + AIMP3 protein but not the AIMP3 protein. It was also confirmed that the 8A12 antibody had a KD value of 1.56 nM for MRS.

1-10. Confirmation of binding site of MRS antibody

The following experiment was conducted to confirm the binding site (epitope, main binding site) of the 1E8 antibody or 8A12 antibody.

First, several MRS fragments with different lengths and positions were prepared by considering the GST, catalytic domain, and tRNA binding domain regions of the entire MRS protein. To the pcDNA3 vector (EV), MRS whole protein or each MRS fragment (MRS fragment) . The positions of the respective MRS fragments are 1 to 266aa fragment, 267 to 597aa fragment, 1 to 598aa fragment, 598 to 900aa fragment, 660 to 860aa fragment, 660 to 900 fragment, 730 to 900 fragment , And so on. At this time, the Myc protein was bound to the N-terminal of each peptide, and the Myc protein was used as a control. Then, 2 μg of cloned vector DNA was transfected into H460 cells using Turbofect (Thermo) according to the manufacturer's instructions. After 24 hours, cells were obtained and subjected to Western blotting. At this time, 1E8 antibody and 8A12 antibody were diluted to 1: 5000 (0.2 μg / mL) as a primary antibody.

Through the above experiment, the epitope of the 1E8 antibody and the 8A12 antibody was found in the region of at least 598-900 aa among the MRS proteins of SEQ ID NO: 1.

Thus, the 811-840aa fragment, 821-850aa fragment, 831-860aa fragment, 841-870aa fragment, 846-875aa fragment, 851-880aa fragment, 856-885aa fragment, 861-890aa fragment, 866-895aa fragment, 871-900aa A variety of small fragment peptide fragments including fragments were prepared and each peptide was coated on a 96 well ELISA plate at 300 ng / well and ELISA was performed according to a conventional protocol. As a primary antibody, 1E8 antibody or 8A12 antibody was diluted (1x PBST-Tween 0.05%) to a concentration of 10 nM and HRP-conjugated Goat anti-mouse IgG (Thermo) was diluted 1: 10000 with a secondary antibody (1xPBST-Tween0. 05%), and absorbance was measured at 450 nm.

As a result, it was confirmed that the 1E8 antibody and the 8A12 antibody specifically recognize the 861-900 aa region (AQKADKNEVA AEVAKLLDLK KQLAVAEGKP PEAPKGKKKK, SEQ ID NO: 2) as an epitope among the MRS proteins. These results suggest that other binding molecules (such as other antibodies and functional fragments thereof) that recognize the 861-900 aa region as an epitope will also be able to distinguish between MRS-specific binding and MRS.

Hereinafter, the present inventors have applied an antibody having excellent MRS detection capability to the pancreatic cancer test method newly devised by the present inventors.

1-11. Pancreatic cancer cell staining using the anti-MRS antibody of the present invention and the effect of the anti-MRS antibody against commercial antibodies

MRS fluorescence staining was performed on PANC-1 pancreatic cancer cell line and SCK cell line (non-pancreatic cancer cell line) using 1E8 antibody or 8A12 antibody, respectively. At this time, a commercially available MRS antibody (Abcam, Ab137105) was used as a control. Specifically, dyeing was carried out by the following procedure. Each target cell prepared on the slide was treated with PBS containing 0.2% tween 20 to increase permeability and then blocked with 2% goat serum for 1 hour. As a primary antibody, the antibodies (1E8 antibody or 8A12 antibody, manufactured by Oncotag) or Ab137105 antibody (Abcam) of the present invention were treated at 1 ug / ml for 1 hour at 37 ° C and washed three times with 0.05% TBST . Alexa-488-conjugated secondary antibodies (Molecular probes, Cat. No. A11001) were diluted 1: 100 as a secondary antibody with a fluorescent substance bound thereto and treated for 1 hour in a dark place at room temperature. And washed three times with TBST (500 μl). The mounting solution with DAPI (ProLong Gold antifade regent with DAPI / Molecular probes, Cat. No. P36931) was treated with 20 μl of tissue on the slide, covered with a coverslip, and observed with a confocal laser microscope and a fluorescence microscope .

As shown in FIG. 9, the commercially available Ab137105 antibody, which was confirmed to have low MRS specificity in Example 1-7, nonspecifically stained both PANC-1 pancreatic cancer cell and SCK cell (non-pancreatic cancer cell line), but the 1E8 antibody of the present invention And 8A12 antibodies were able to specifically stain only MRS.

Thinprep slides were prepared using PANC-1 cell line similar to the clinical conditions (see Example 2 below), using the Thinprep equipment (Hologic.Inc) used for patient cell sample processing at the clinical site, and the 1E8 antibody And the 8A12 antibody.

As a result, as shown in FIG. 10, it was confirmed that the 1E8 antibody and 8A12 antibody of the present invention can be used together with a sample providing method (Thinprep slide, etc.) commonly used in a clinical field.

Example  2: Cellular ( cytodiagnosis ) In the test method , Identification of pancreatic cancer cell-specific MRS expression detection (staining method) and its effect

Experimental Method

1) Pancreas cells as a specimen were obtained according to a conventional endoscopic ultrasonic fine-needle aspiration (EUS-FNA). Ultrasonography was performed using an ultrasound system equipped with a linear array echo endoscope (EUS) (GF-UCT140 or GF-UCT180, company: Olympus, Japan) Respectively. Pancreatic cells were obtained by introducing a needle for fine needle aspiration (product name: 22G Echo-ultra ^TM , company: Cook Medical, Cork, Ireland) into an ultrasonically targeted mass.

Then, the collected pancreatic cells were treated with a conventional method using a Cellular Automated Cell Block System (Hologic) (Antonio Ieni et al., Cell-block procedure in endoscopic ultrasound-guided-fine-needle-aspiration of gastrointestinal solid neoplastic lesions, World J Gastrointest Endosc 2015 August 25; 7 (11): 1014-1022) as cell-based paraffin sections. Also, the pancreatic cell sample can be provided on a ThinPrep slide by a conventional method using ThinPrep (Hologic.Inc) or by direct smear (de Luna R et al., Comparison of ThinPrep and conventional preparations in a pancreatic fine-needle aspiration biopsy, Diagnostic Cytopathology, 2004 Feb; 30 (2): 71-6.). The results of these cell samples were compared by the following test methods.

2) Conventional pathologic cytology by conventional cytologic examination method can be obtained by the result of staining using H & E staining method which is commonly used so far. The H & E staining was carried out using hematoxylin and eosin according to conventional protocols (see detailed protocol in Example 3 below). It can also be reduced by staining with Pap staining method. The Pap staining was performed using hematoxylin, OG-6 (Orange G-6), and eosin azure according to conventional protocols (see detailed protocol in Example 3 below). When a paraffin slice sample is used, paraffin removal and hydration are performed by a conventional method, and the dye materials are treated.

Cells are stained with a single layer on the slide. When the nucleus / cytoplasm ratio (N / C ratio) is small and the nuclear membrane is smooth, the cell is judged as benign (normal) The ratio is high, chromatin aggregation is seen, the nuclear membrane is coarse, and the nucleolus and mitosis appear, and it is judged to be a malignant tumor cell. If the cell change does not reach the malignant cell but it can not be judged as benign, (atypical cell).

3) Clinical end result was determined by imaging (abdominal ultrasonography, abdominal computed tomography, abdominal magnetic resonance imaging, endoscopic retrograde ganglionectomy, positron emission tomography) and pathological examination (cytology, biopsy) Based on the results, the doctor made a final judgment.

4) We developed immunohistochemistry (especially, immunofluorescence staining) for measuring MRS expression in pancreatic cancer cells and normal pancreatic cells (including non-cancerous benign pancreatic cells) as described below. Specifically, when the paraffin section was used, the following treatments were carried out.

① Removal of paraffin: Paraffin is dissolved in an oven at 60 ° C

② Hydration: Washing with xylene three times for 5 minutes, washing with 100% ethanol for 2 minutes, washing with 95% ethanol for 2 minutes, washing with 90% ethanol for 2 minutes, washing with 70% ethanol for 2 minutes, DW for 2 min, PBS for 5 min

(3) Increase in permeabilization: treatment with 0.2% Triton X-100 for 30 minutes and washing with PBS for 5 minutes

④ Pretreatment: Blocking with 2% goat serum for 1 hour,

(5) Treatment with primary antibody: An anti-MRS antibody (typically using the antibody 8A12 of the present invention, produced by Oncotag) was diluted 1: 300, treated overnight at 4 ° C, washed three times with PBS for 5 minutes each

⑥ Color development: Alexa-488-conjugated secondary antibodies (Molecular probes, Cat. No. A11001) were diluted to 1: 200 ~ 1: 300 as a secondary antibody with a fluorescent substance attached. , Washed three times with PBS for 5 min each

(7) A mounting solution with DAPI (ProLong Gold antifade regent with DAPI / Molecular probes, Cat. No. P36931) was treated with 20 μl of tissue on the slide and covered with a coverslip.

In the case of the thinprep slide samples, the samples were processed by a method including the above steps 3 to 7 without removing the paraffin. The sample prepared by the above method was observed with a confocal laser microscope and a fluorescence microscope.

The cells in which the MRS staining intensity was increased more than 2 times on the basis of the negative control sample in the above specimens were judged to be pancreatic cancer cells and these results were compared with pathological findings and clinical final diagnosis results of the specimens, (Sensitivity and specificity).

Experiment result

Specific experimental results are shown in Tables 3, 4 and 5 below. As a result of applying the MRS staining method of pancreatic cancer according to the present invention to 14 benign pancreatic cell samples and 94 pancreatic cancer malignancy cell samples, the conventional cytopathological test using H & E staining showed sensitivity The sensitivity of the MRS staining of the present invention was 92.6%, and compared with that of the present invention, the purity of the pancreatic cancer cell sample, which was inevitably classified as atypia by the cytopathological examination method, It was possible to distinguish whether These results indicate that the staining method of the present invention using MRS as a marker in pancreatic cancer has a high discriminating ability in diagnosis at the cellular level. As shown in Example 3 described below, the conventional pancreatic cancer marker Were clearly different from those that were difficult to demonstrate efficacy in cell-level diagnostics (ie, cytodiagnosis).

Comparison Details: Compared to the result of the final clinical pathological diagnosis, the result of the conventional cytologic examination (conventional pathologic cytology) and the test result by the MRS immunostaining of the present invention Conventional
과과학 cytology Final clinicopathological diagnosis MRS immunostaining Positive Negative Malignancy
(n = 43) Malignancy (n = 43) 42 One Benign (n = 0) 0 0 Suspicious of malignancy (n = 29) Malignancy (n = 28) 26 2 Benign (n = 1) One 0 Atypical
(n = 21) Malignancy (n = 18) 17 One Benign (n = 3) 2 One Negative for malignancy (n = 15) Malignancy (n = 5) 2 3 Benign (n = 10) 2 8

Comparison of final cytopathological diagnosis results and existing cytological examination results (The positive cytology test and the suspicious malignancy judgment in the existing cytological test results of Table 3 were classified as the final positive, and the Atypia judgment and the negative for malignancy judgment Are classified as final negative) Final clinicopathological diagnosis Malignancy Benign Conventional
과과학 cytology Positive 71 One Negative 23 13 Sensitivity: 75.5% Specificity: 92.9% Accuracy = 77.9% PPV: 98.6% NPV: 36.1%

PPV: positive predictive value, NPV: negative predictive value

Comparison Summary: Compared to the final clinical pathological diagnosis result, the test result of MRS immunostaining of the present invention was compared Final clinicopathological diagnosis Malignancy Benign MRS immunostaining Positive 87 5 Negative 7 9 Sensitivity: 92.6% Specificity: 64.3% Accuracy = 88.1% PPV: 94.6% NPV: 56.3%

PPV: positive predictive value, NPV: negative predictive value

Example  3: At the cellular level, commercial pancreatic cancer Marker CEA and  The pancreatic cancer of the MRS of the present invention Discrimination ability  compare

Experimental Method

1) Obtaining pancreatic cell samples for patient and cytodiagnosis

In 26 suspected cases of pancreatic cancer, 26 pancreatic cell specimens were collected and obtained by endoscopic ultrasound-assisted cell resection (fine needle aspiration). The study was approved by the Research Ethics Committee of the Gangnam Severance Hospital. Pancreatic cell samples from the above patients were obtained through endoscopic ultrasonic fine-needle aspiration (EUS-FNA) in the same manner as in Example 2 above. The pancreatic cells thus obtained were prepared by a conventional method using a Cellular Automated Cell Block System (Hologic) in a state of paraffin sections (Cellent paraffin sections) or Thinprep method (see Example 2).

Twenty - six patients with suspected pancreatic cancer were followed - up for pancreatic cancer (13 cases) and normal pancreas (13 cases). Of the 13 patients who were diagnosed as pancreatic cancer, 7 were classified as pancreatic cancer cells by histologic examination by pathologist. The remaining 6 patients were classified as atypical cells by histologic examination. And finally diagnosed as pancreatic cancer.

At the time of pancreatic cell harvesting, the patient was informed by papers, and pancreatic cancer cells, atypical cells, and normal cells were histologically confirmed by a pathologist (see judgment criteria of Example 2). Representative diagnostic examples of each type of normal cells, tumor cells, and atypical cells in the 26 samples obtained are shown in the drawings of each experiment.

2) Conventional cytogenetic method

H & E staining: Paraffin slices were treated with Xylene three times for 5 minutes, 100% ethanol for 2 minutes, 95% ethanol for 2 minutes, 90% ethanol for 2 minutes, 70% ethanol for 2 minutes, Minute treatment, respectively. Paraffin removal and hydration were performed. Hematoxylin was reacted at room temperature for 30 seconds and washed with tap water for 10 minutes. Eosin was reacted at room temperature for 1 minute and washed with tap water for 10 minutes. Dehydration and clearing were carried out for 1 minute in 70% ethanol, 1 minute in 90% ethanol, 1 minute in 95% ethanol, 1 minute in 100% ethanol, and 3 times in 5 minutes for Xylene. The mounting solution was dropped onto the slide tissue, the cells were covered with a coverslide, and the sample was observed under an optical microscope.

pap staining: Papanicolaou stain was performed using a Varistain 24-4 stainer from Thermo Scientific according to the protocol embedded in the instrument. The protocol is shown in Table 6 below.

reagent program 1 program 2 One water 10 minutes 10 minutes 2 Hema. 1 minute 30 seconds 1 minute 30 seconds 3 water 30 seconds 30 seconds 4 water 30 seconds 30 seconds 5 0.5% HCl. 7 seconds 7 seconds 6 0.5% Amm. 5 seconds 5 seconds 7 water 30 seconds 30 seconds 8 50% alc. 30 seconds 30 seconds 9 70% alc. 30 seconds 30 seconds 10 80% alc. 30 seconds 30 seconds 11 95% alc. 30 seconds 30 seconds 12 MV 6 1 minute 10 seconds 13 95% alc. 30 seconds 30 seconds 14 95% alc. 30 seconds 30 seconds 15 EA 50 1 minute 10 seconds 16 95% alc. 30 seconds 30 seconds 17 95% alc. 30 seconds 30 seconds 18 95% alc. 30 seconds 30 seconds 19 99% alc. 30 seconds 30 seconds 20 99% + xyl. 20 seconds 20 seconds 21 xyl. 30 seconds 30 seconds 22 xyl. 30 seconds 30 seconds 23 xyl. 30 seconds 30 seconds 24 xyl. 30 seconds 30 seconds 25 end.

3) Discrimination criteria from cytodiagnosis by morphological pathologic examination by conventional staining method

The most definitive evidence for the morphologic diagnosis of malignant tumors is invasive growth in the surrounding normal tissue, but unlike histology, which is easy to demonstrate the relationship between cancer and surrounding tissues, We can not prove the relationship with the surrounding organization because we extracted it and spoke. Therefore, atypia of individual cells is assessed as a secondary measure. Atypia is defined as the nucleus of the cell is enlarged and the cytoplasm is decreased, and the nucleus to cytoplasmic ratio (N / c ratio ), Chromatin is not uniformly distributed in the nucleus, and clumping occurs partially, nucleolus appears in the nucleus, and mitosis appears. When these atypical findings are all present and the severity is significant, a cytological examination can be used to diagnose malignant tumors. However, if these findings are partially present or if they are mild, they should be classified as atypical cells. These lesions may be partially seen in benign lesions such as severe inflammation. Conversely, if none of these findings are visible, normal cells can be determined. Of course, it should be presumed that the cells can be observed at the site where the cells were collected.

4) Comparative staining using CEA, a known commercial pancreatic cancer marker, and MRS of the present invention

Immunostaining for MRS was performed in the same manner as described in Example 2 above. For immunohistochemistry for CEA, the same procedure as in Example 2 was performed except that Carcinoembryonic Antigen (CEA) antibody (Dako, Cat. No. M7072) was used as the primary antibody.

5) Statistical analysis

Experimental results were expressed as mean ± standard deviation based on at least 3 independent experiments. Data were analyzed using Student's t test for statistical significance. The P-values of less than 0.05 were considered to be significant.

Experiment result

3-1. Immunostaining of normal cells

Cells isolated from the pancreas of normal or pancreatic cancer patients by the EUS-FNA method were analyzed by H & E staining, and normal cells were subjected to immunofluorescence staining using MRS or CEA antibody. The results are shown in Fig.

As shown in Fig. 11, the pancreatic cells obtained from four patients did not show any tumor findings in H & E staining and were judged to be normal cells. In the normal pancreas cells, it was confirmed that neither MRS nor CEA stained .

3-2. Immunostaining of tumor cells

Cells isolated from the pancreas of pancreatic cancer patients were analyzed by H & E staining. Immunofluorescent staining was performed using MRS or CEA antibody as the tumor cells. The results are shown in FIG.

As shown in Fig. 12, pancreatic cells obtained from four patients were judged to be tumor cells in H & E staining. MRS staining was stronger in all four pancreatic cancer tumor cells, which was statistically significant (p = 0.019) as compared with MRS staining of normal cells. However, CEA staining was weakly stained only in two patient samples, which was not statistically significant (p = 0.187) compared with MRS staining of normal cells.

3-3. Immunostaining of atypical cells

MRS or CEA staining was performed using pancreatic cells of a patient who was finally identified as a tumor by following up the prognosis of the patient as an atypical cell, which is difficult to clearly determine whether it is a tumor cell alone by H & E staining. The results are shown in Fig.

As shown in FIG. 13, it can be seen that the division of pancreatic cells classified into atypical cells through H & E staining is not clear whether the tumor is a tumor or a normal cell. On the other hand, the patients included in the atypical cell group are all those who have been diagnosed as pancreatic cancer in the future. MRS staining was strongly observed in all four atypical pancreatic cells, which was statistically significant (p = 0.020) compared with MRS staining of normal cells. However, no CEA staining was observed in all four atypical pancreatic cells.

The results of Example 3 above indicate that pancreatic cancer cells can be diagnosed with much higher accuracy than other tumor markers by performing H & E staining and MRS staining in pancreatic cells isolated from suspected pancreatic cancer patients. In addition, conventional tumor markers (eg, CEA) in atypical cells, which can not be clearly distinguished from tumor cells or normal cells by H & E staining, can not be clearly distinguished from tumors, but MRS Is a diagnostic marker for pancreatic cancer that is very significant in that these atypical pancreatic cells can also be clearly distinguished from tumor cells by a fairly high degree of accuracy.

Example  4: Establishment of accurate pancreatic cancer discrimination method _MRS double staining method

4-1. Find the cause of false positive results

As shown in Example 3, the MRS was proved to be able to diagnose pancreatic cancer with higher accuracy than the existing pancreatic cancer markers such as CEA. However, since the experiments in the above-described embodiments were performed on samples having all the determinations, It is necessary to confirm the degree of accuracy and diagnosis in blind (unknown) state after tissue collection from patients suspected of having pancreatic cancer. In addition, as shown in Example 2 above, MRS showed a very good sensitivity in the diagnosis of pancreatic cancer, but the specificity of MRS alone was slightly lower than the sensitivity in the case of pancreatic cancer. In this study, we examined the ability of MRS to discriminate pancreatic cancer from pancreatic specimens obtained from new patients. Among the 10 samples with negative cytology (non-pancreatic cancer), three samples showed MRS expression Respectively. In other words, it was confirmed that the normal pancreatic cell sample had a high MRS expression level, and therefore, there was a problem that the result of false positives (which can be regarded as the occurrence of pancreatic cancer, which is not pancreatic cancer) is included.

As a result, the cause of the false positive judgment was determined by performing MRS staining after discriminating cancer tissues and normal tissues with H & E stain for all the specimens. As a result, as shown in FIG. 14, acinar cell, pancreatic parenchymal cells) were found to be highly expressed in MRS.

4-2. Establish strategy to improve MRS diagnostic accuracy

Example 4-1. As shown in the results of the item, since the MRS is expressed at a high level in the normal acinar cell, it alone contributes to the false positive rate of the pancreatic cancer judgment (that is, when the MRS alone is used, (MRS) and acinar cell specific marker proteins were used to reduce the false positive rate. The present inventors confirmed the staining pattern in the precursor cells using chymotrypsin, for example, among the acinar cell-specific marker candidates.

FIG. 16 shows the result of verifying the double staining strategy of the present invention by the ICC (Immunohistochemistry) method. The tissue of FIG. 16 is a normal tissue as shown in the H & E staining image of FIG. FIG. 16 shows an enlarged view of the acinar cell and the surrounding cells when the double staining of the present invention is performed on this normal tissue. MRS and chymotrypsin were both strongly expressed in acinar cells, and the color was strongly overlapped, and MRS was not detected in other parts of normal tissues (FIG. 16). Based on this staining pattern, cell and tissue regions in which a precursor cell marker (typically chymotrypsin) is detected at high intensity can be excluded from the process of pancreatic cancer discrimination.

4-3. Establish double staining method

Paraffin slices were prepared by first dissolving paraffin in a 60 ° C oven, washing 3 times for 5 minutes with xylene, 2 minutes for 100% ethanol, 2 minutes for 95% ethanol, 2 minutes for 90% Ethanol for 2 minutes and DW for 2 minutes. Paraffin removal and hydration were performed.

For double staining of MRS and chymotrypsin, a specific experimental protocol was established to detect MRS as green (Alexa Fluor 488) and chymotrypsin as red (Texas Red). First, 0.2% Triton X-100 was treated to increase cell permeability. blocking solution, 2% goat serum for 1 hour. The pancreatic cells were then incubated overnight at 4 ° C with the primary antibody, Methionyl tRNA synthetase antibody (1: 100) and Anti-Chymotrypsin antibody (1: 100 dilution, abcam, catalog no. Ab155400). After dipping 30 times or more in 1X TBST (counting 1 cycle), rinse 3 times or more and dilute the secondary antibody (1: 200 ~ 1: 300 diluted with Alexa Fluor 488 and Texas Red) / ThermoFisher Scientific, catalog no. A -11001 / SANTA CRUZ BIOTECHNOLOGY, INC., Catalog no. Sc-278) were treated in the dark at room temperature for 1 hour. After dipping 30 times or more in 1X TBST PBS (3 times), the mounting solution (ProLong Gold antifade regent with DAPI, Molecular Probes, Cat. No. P36931) with DAPI was dropped on the slide The cells were covered with a coverslide and then the sample was observed under a fluorescence microscope.

The criteria for discriminating pancreatic cancer were determined as shown in Table 7 below. When chymotrypsin was not detected (expressed) and the MRS protein was detected (expressed), it was judged to be a pancreatic cancer cell. That is, in the case of MRS (+) and ACT (-) of the present invention, the pancreatic cancer is classified as positive, and in the remaining cases, it is classified as negative.

no. MRS Chymotrypsin
(ACT) Reading One + + acinar cell 2 + - 가소 셀 3 - + acinar cell 4 - - fibrotic cell or others

Example  5: Cytopathology ( cytodiagnosis ), The pancreatic cancer of the present invention double staining method Discrimination ability  evaluation

Pancreatic cancer was identified by applying the double staining method of the present invention to 26 new pancreatic cell samples obtained from different samples from those used in the above-mentioned examples. These pancreatic cancer cells were collected by the EUS-FNA method as described above. In performing the double staining method of the present invention, pathological diagnosis results or final clinical diagnosis proceeded in a blind state.

5-1. Normal pancreas In acinar cells, Immunofluorescent staining  Confirm aspect

 It was confirmed how the normal acinar cell group obtained by the EUS-FNA method from the pancreas was actually stained with the double staining method of the present invention. The results are shown in Fig. 17 and Fig.

As shown in Fig. 17 and Fig. 18, chimotrypsin was strongly detected (expressed) in red cells, and the red color was very strong. Specifically, as shown in FIG. 17, there was a pattern in which MRS was very weakly detected, a relatively strong detection of chymotrypsin was found, and a pattern in which MRS was detected with chymotrypsin as shown in FIG. However, even in the case of the staining pattern shown in Fig. 18, the red color representing the chymotrypsin appears to have a high intensity in the merge image.

5-2. Existing cytopathologic examination confirmed pancreatic cancer, and eventually confirmed pancreatic cancer For patients  Staining aspect

The cells isolated from the pancreas were analyzed by pap staging and the staining pattern of the cell samples judged to be tumor cells was evaluated by the double staining method of the present invention.

As shown in FIG. 19, the cytopenic samples used in this experiment (cells determined to be tumor cells by papa staining) show little red color and very strong green color, that is, chymotrypsin is almost And the expression of MRS was very strongly expressed. Thus, it was confirmed that it can be judged as pancreatic cancer.

5-3. Existing cell Pathologically,  By atypical cells Classified and confirmed  However, finally, the pancreatic cancer confirmed For patients  Staining aspect

The cell staining (cell sample) staining pattern of patients with pancreatic cancer confirmed by conventional pap staining was evaluated by the double staining method of the present invention, although it was atypical cells.

As a result, as shown in FIG. 20, the expression intensity of MRS was very high, and the detection strength of chymotrypsin was relatively low, confirming that it was a cancer cell (see merge image). As described above, the double staining method of the present invention confirmed that cancer can be judged also on non-specific cells.

5-4. Result synthesis

The results of the double-staining of the 26 cell samples were compared with the results of the final clinical diagnosis. The results are shown in Table 8 below. As a result of the double staining of the present invention, it was classified as positive for pancreatic cancer in the case of MRS (+) and dorsal cell marker (-), and as MRS (+) and deficient cell marker (+ -) and a precursor cell marker (-), or MRS (-) and precursor cell marker (+)).

Compared with the results of the final clinicopathological diagnosis, the test results of MRS and chymotrypsin double staining of the present invention were compared Final clinicopathological diagnosis Malignancy Benign The staining method of the present invention Positive 23 0 Negative 0 3 Sensitivity: 23/23 = 100% Specificity: 3/3 = 100% PPV: 23/23 = 100% NPV = 3/3 = 100%

PPV: positive predictive value, NPV: negative predictive value

As shown in Table 8, the pancreatic cancer determination by the double-staining method of the MRS of the present invention and the precursor cell markers (typically, chymotrypsin) was 100% in sensitivity, 100% in specificity, 100% in positive predictive value (PPV) (NPV) was 100%.

In addition, as shown in Example 5 above, the double staining method of the present invention can not be confirmed or undetected by conventional cytopathological judgment methods (pap-staining or H & E stainig based morphological judgment method) including atypical cells It was confirmed that the presence or absence of malignant tumor cells can be clearly discriminated against the pancreatic cell (particularly, Atypia), and the diagnostic efficiency in the cytology can be remarkably increased.

As described above, the present invention relates to a method for diagnosing pancreatic cancer using a methionyl-thi ene synthase and a secretory cell-specific marker. MRS is more accurate than conventional pancreatic cancer markers such as CEA. Especially, when double-marker markers are used as markers of chimotrypsin, Is remarkably elevated, so that it is highly industrially applicable in fields such as an in vitro diagnostic industry.

<110> Oncotag Diagnostics Co., Ltd. <120> Method for diagnosing pancreatic cancer using methionyl-tRNA          synthetase and pancreatic acinar cell-specific marker <130> NP17-0068P <150> KR 10-2017-0113372 <151> 2017-09-05 <160> 72 <170> KoPatentin 3.0 <210> 1 <211> 900 <212> PRT <213> Artificial Sequence <220> The amino acid sequence of MRS (methionyl-tRNA synthetase) <400> 1 Met Arg Leu Phe Val Ser Asp Gly Val Pro Gly Cys Leu Pro Val Leu   1 5 10 15 Ala Ala Ala Gly Arg Ala Arg Gly Arg Ala Glu Val Leu Ile Ser Thr              20 25 30 Val Gly Pro Glu Asp Cys Val Val Pro Phe Leu Thr Arg Pro Lys Val          35 40 45 Pro Val Leu Gln Leu Asp Ser Gly Asn Tyr Leu Phe Ser Thr Ser Ala      50 55 60 Ile Cys Arg Tyr Phe Leu Leu Ser Gly Trp Glu Gln Asp Asp Leu  65 70 75 80 Thr Asn Gln Trp Leu Glu Trp Glu Ala Thr Glu Leu Gln Pro Ala Leu                  85 90 95 Ser Ala Leu Tyr Tyr Leu Val Val Gln Gly Lys Lys Gly Glu Asp             100 105 110 Val Leu Gly Ser Val Arg Arg Ala Leu Thr His Ile Asp His Ser Leu         115 120 125 Ser Arg Gln Asn Cys Pro Phe Leu Ala Gly Glu Thr Glu Ser Leu Ala     130 135 140 Asp Ile Val Leu Trp Gly Ala Leu Tyr Pro Leu Leu Gln Asp Pro Ala 145 150 155 160 Tyr Leu Pro Glu Glu Leu Ser Ala Leu His Ser Trp Phe Gln Thr Leu                 165 170 175 Ser Thr Gln Glu Pro Cys Gln Arg Ala Ala Glu Thr Val Leu Lys Gln             180 185 190 Gln Gly Val Leu Ala Leu Arg Pro Tyr Leu Gln Lys Gln Pro Gln Pro         195 200 205 Ser Pro Ala Glu Gly Arg Ala Val Thr Asn Glu Pro Glu Glu Glu Glu     210 215 220 Leu Ala Thr Leu Ser Glu Glu Glu Ile Ala Met Ala Val Thr Ala Trp 225 230 235 240 Glu Lys Gly Leu Glu Ser Leu Pro Pro Leu Arg Pro Gln Gln Asn Pro                 245 250 255 Val Leu Pro Val Ala Gly Glu Arg Asn Val Leu Ile Thr Ser Ala Leu             260 265 270 Pro Tyr Val Asn As Val Val Pro His Leu Gly Asn Ile Gly Cys Val         275 280 285 Leu Ser Ala Asp Val Phe Ala Arg Tyr Ser Arg Leu Arg Gln Trp Asn     290 295 300 Thr Leu Tyr Leu Cys Gly Thr Asp Glu Tyr Gly Thr Ala Thr Glu Thr 305 310 315 320 Lys Ala Leu Glu Glu Gly Leu Thr Pro Gln Glu Ile Cys Asp Lys Tyr                 325 330 335 His Ile Ile His Ala Asp Ile Tyr Arg Trp Phe Asn Ile Ser Phe Asp             340 345 350 Ile Phe Gly Arg Thr Thr Thr Pro Gln Gln Thr Lys Ile Thr Gln Asp         355 360 365 Ile Phe Gln Gln Leu Leu Lys Arg Gly Phe Val Leu Gln Asp Thr Val     370 375 380 Glu Gln Leu Arg Cys Glu His Cys Ala Arg Phe Leu Ala Asp Arg Phe 385 390 395 400 Val Glu Gly Val Cys Pro Phe Cys Gly Tyr Glu Glu Ala Arg Gly Asp                 405 410 415 Gln Cys Asp Lys Cys Gly Lys Leu Ile Asn Ala Val Glu Leu Lys Lys             420 425 430 Pro Gln Cys Lys Val Cys Arg Ser Cys Pro Val Val Gln Ser Ser Gln         435 440 445 His Leu Phe Leu Asp Leu Pro Lys Leu Glu Lys Arg Leu Glu Glu Trp     450 455 460 Leu Gly Arg Thr Leu Pro Gly Ser Asp Trp Thr Pro Asn Ala Gln Phe 465 470 475 480 Ile Thr Arg Ser Trp Leu Arg Asp Gly Leu Lys Pro Arg Cys Ile Thr                 485 490 495 Arg Asp Leu Lys Trp Gly Thr Pro Val Leu Glu Gly Phe Glu Asp             500 505 510 Lys Val Phe Tyr Val Trp Phe Asp Ala Thr Ile Gly Tyr Leu Ser Ile         515 520 525 Thr Ala Asn Tyr Thr Asp Gln Trp Glu Arg Trp Trp Lys Asn Pro Glu     530 535 540 Gln Val Asp Leu Tyr Gln Phe Met Ala Lys Asp Asn Val Pro Phe His 545 550 555 560 Ser Leu Val Phe Pro Cys Ser Ala Leu Gly Ala Glu Asp Asn Tyr Thr                 565 570 575 Leu Val Ser His Leu Ile Ala Thr Glu Tyr Leu Asn Tyr Glu Asp Gly             580 585 590 Lys Phe Ser Lys Ser Arg Gly Val Gly Val Phe Gly Asp Met Ala Gln         595 600 605 Asp Thr Gly Ile Pro Ala Asp Ile Trp Arg Phe Tyr Leu Leu Tyr Ile     610 615 620 Arg Pro Glu Gly Gln Asp Ser Ala Phe Ser Trp Thr Asp Leu Leu Leu 625 630 635 640 Lys Asn Asn Ser Glu Leu Leu Asn Asn Leu Gly Asn Phe Ile Asn Arg                 645 650 655 Ala Gly Met Phe Val Ser Lys Phe Phe Gly Gly Tyr Val Pro Glu Met             660 665 670 Val Leu Thr Pro Asp Asp Gln Arg Leu Leu Ala His Val Thr Leu Glu         675 680 685 Leu Gln His Tyr His Gln Leu Leu Glu Lys Val Arg Ile Arg Asp Ala     690 695 700 Leu Arg Ser Ile Leu Thr Ile Ser Arg His Gly Asn Gln Tyr Ile Gln 705 710 715 720 Val Asn Glu Pro Trp Lys Arg Ile Lys Gly Ser Glu Ala Asp Arg Gln                 725 730 735 Arg Ala Gly Thr Val Thr Gly Leu Ala Val Asn Ile Ala Ala Leu Leu             740 745 750 Ser Val Met Leu Gln Pro Tyr Met Pro Thr Val Ser Ala Thr Ile Gln         755 760 765 Ala Gln Leu Gln Leu Pro Pro Ala Cys Ser Ile Leu Leu Thr Asn     770 775 780 Phe Leu Cys Thr Leu Pro Ala Gly His Gln Ile Gly Thr Val Ser Pro 785 790 795 800 Leu Phe Gln Lys Leu Glu Asn Asp Gln Ile Glu Ser Leu Arg Gln Arg                 805 810 815 Phe Gly Gly Gly Gln Ala Lys Thr Ser Pro Lys Pro Ala Val Val Glu             820 825 830 Thr Val Thr Thr Ala Lys Pro Gln Gln Ile Gln Ala Leu Met Asp Glu         835 840 845 Val Thr Lys Gln Gly Asn Ile Val Arg Glu Leu Lys Ala Gln Lys Ala     850 855 860 Asp Lys Asn Glu Val Ala Ala Glu Val Ala Lys Leu Leu Asp Leu Lys 865 870 875 880 Lys Gln Leu Ala Val Ala Glu Gly Lys Pro Pro Glu Ala Pro Lys Gly                 885 890 895 Lys Lys Lys Lys             900 <210> 2 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> antibody epitope (main binding site) <400> 2 Ala Gln Lys Ala Asp Lys Asn Glu Val Ala Ala Glu Val Ala Lys Leu   1 5 10 15 Leu Asp Leu Lys Lys Gln Leu Ala Val Ala Glu Gly Lys Pro Pro Glu              20 25 30 Ala Pro Lys Gys Lys Lys Lys Lys          35 40 <210> 3 <211> 268 <212> PRT <213> Artificial Sequence <220> <223> human chymotrypsin <400> 3 Met Leu Gly Ile Thr Val Leu Ala Ala Leu Ala Cys Ala Ser Ser   1 5 10 15 Cys Gly Val Pro Ser Phe Pro Pro Asn Leu Ser Ala Arg Val Val Gly              20 25 30 Gly Glu Asp Ala Arg Pro His Ser Trp Pro Trp Gln Ile Ser Leu Gln          35 40 45 Tyr Leu Lys Asn Asp Thr Trp Arg His Thr Cys Gly Gly Thr Leu Ile      50 55 60 Ala Ser Asn Phe Val Leu Thr Ala Ala His Cys Ile Ser Asn Thr Arg  65 70 75 80 Thr Tyr Arg Val Ala Val Gly Lys Asn Asn Leu Glu Val Glu Asp Glu                  85 90 95 Glu Gly Ser Leu Phe Val Gly Val Asp Thr Ile His Val His Lys Arg             100 105 110 Trp Asn Ala Leu Leu Leu Arg Asn Asp Ile Ala Leu Ile Lys Leu Ala         115 120 125 Glu His Val Glu Leu Ser Asp Thr Ile Gln Val Ala Cys Leu Pro Glu     130 135 140 Lys Asp Ser Leu Leu Pro Lys Asp Tyr Pro Cys Tyr Val Thr Gly Trp 145 150 155 160 Gly Arg Leu Trp Thr Asn Gly Pro Ile Ala Asp Lys Leu Gln Gln Gly                 165 170 175 Leu Gln Pro Val Val Asp His Ala Thr Cys Ser Arg Ile Asp Trp Trp             180 185 190 Gly Phe Arg Val Lys Lys Thr Met Val Cys Ala Gly Gly Asp Gly Val         195 200 205 Ile Ser Ala Cys Asn Gly Asp Ser Gly Gly Pro Leu Asn Cys Gln Leu     210 215 220 Glu Asn Gly Ser Trp Glu Val Phe Gly Ile Val Ser Phe Gly Ser Arg 225 230 235 240 Arg Gly Cys Asn Thr Arg Lys Lys Pro Val Val Tyr Thr Arg Val Ser                 245 250 255 Ala Tyr Ile Asp Trp Ile Asn Glu Lys Met Gln Leu             260 265 <210> 4 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VL CDR1 <400> 4 Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu   1 5 10 15 Ala     <210> 5 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VL CDR1 <400> 5 aagtccagtc agagcctttt atatagtagc aatcaaaaga actacttggc c 51 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VL CDR2 <400> 6 Trp Ala Ser Thr Arg Glu Ser   1 5 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VL CDR2 <400> 7 tgggcatcca ctagggaatc t 21 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VL CDR3 <400> 8 Gln Gln Tyr Tyr Ser Tyr Pro Thr   1 5 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VL CDR3 <400> 9 cagcaatatt atagctatcc gacg 24 <210> 10 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VH CDR1 <400> 10 Ser Asp Tyr Ala Trp Asn   1 5 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VH CDR1 <400> 11 agtgattatg cctggaac 18 <210> 12 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VH CDR2 <400> 12 Tyr Ile Ser Tyr Ser Gly Arg Thr Ser Tyr Lys Ser Ser Leu Lys Ser   1 5 10 15 <210> 13 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VH CDR2 <400> 13 tacataagct acagtggtcg cactagctac aaatcatctc tcaaaagt 48 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VH CDR3 <400> 14 Asp Tyr Gly Asn Phe Val Gly Tyr Phe Asp Val   1 5 10 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VH CDR3 <400> 15 gactatggta acttcgtagg ttacttcgat gtc 33 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VL CDR1 <400> 16 Lys Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser   1 5 10 <210> 17 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VL CDR1 <400> 17 aaggcgagtc aggacattaa tagctattta agc 33 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VL CDR2 <400> 18 Arg Ala Asn Arg Leu Val Asp   1 5 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VL CDR2 <400> 19 cgtgcaaaca gattggtaga t 21 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VL CDR3 <400> 20 Leu Gln Tyr Asp Glu Phe Pro Arg Thr   1 5 <210> 21 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VL CDR3 <400> 21 ctacagtatg atgagtttcc tcggacg 27 <210> 22 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VH CDR1 <400> 22 Ser Glu Tyr Ala Trp Thr   1 5 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VH CDR1 <400> 23 agtgagtatg cctggacc 18 <210> 24 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VH CDR2 <400> 24 Tyr Ile Asn Tyr Asn Gly Asn Thr Asn Leu Asn Pro Ser Leu Lys Ser   1 5 10 15 <210> 25 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VH CDR2 <400> 25 tacataaact acaatggcaa cactaactta aatccatctc tcaaaagt 48 <210> 26 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VH CDR3 <400> 26 Ser Leu Trp Pro Arg Gly Trp Phe Ala Tyr   1 5 10 <210> 27 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VH CDR3 <400> 27 tcactttggc ccaggggctg gtttgcttac 30 <210> 28 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VL <400> 28 Asp Ile Val Met Thr Gln Ser Ser Ser Leu Ala Val Ser Val Gly   1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser              20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln          35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Ser Tyr Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 29 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VL <400> 29 gacattgtga tgacccagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 60 atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc 120 tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 180 gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagaatt cactctcacc 240 atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatagctat 300 ccgacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 30 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VH <400> 30 Asp Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Asn Pro Ser Gln   1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp              20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp          35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Arg Thr Ser Tyr Lys Ser Ser Leu      50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe  65 70 75 80 Leu Glu Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys                  85 90 95 Ala Arg Asp Tyr Gly Asn Phe Val Gly Tyr Phe Asp Val Trp Gly Ala             100 105 110 Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 31 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VH <400> 31 gatgtgaagc ttcaggagtc gggacctggc ctggtgaatc cttctcagtc tctgtccctc 60 acctgcactg tcactggcta ttcaatcacc agtgattatg cctggaactg gatccggcag 120 tttccaggaa acaaactgga gtggatgggc tacataagct acagtggtcg cactagctac 180 aaatcatctc tcaaaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc 240 ctggagttga attctgtgac tactgaggac acagccacat attactgtgc aagagactat 300 ggtaacttcg taggttactt cgatgtctgg ggcgcaggga ccacggtcac cgtctcctca 360                                                                          360 <210> 32 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VL <400> 32 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly   1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr              20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Met          35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr  65 70 75 80 Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Arg                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 33 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VL <400> 33 gacattctga tgacccagtc tccatcttcc atgtatgcat ctctaggaga gagagtcact 60 atcacttgca aggcgagtca ggacattaat agctatttaa gctggttcca gcagaaacca 120 gggaaatctc ctaagaccct gatgtatcgt gcaaacagat tggtagatgg ggtcccatca 180 aggttcagtg gcagtggatc tggccaagat tattctctca ccatcagcag cctggaatat 240 gaagatatgg gaatttatta ttgtctacag tatgatgagt ttcctcggac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 <210> 34 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VH <400> 34 Asp Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln   1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Glu              20 25 30 Tyr Ala Trp Thr Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp          35 40 45 Met Gly Tyr Ile Asn Tyr Asn Gly Asn Thr Asn Leu Asn Pro Ser Leu      50 55 60 Lys Ser Arg Ile Ser Ile Ile Arg Asp Thr Ser Lys Asn Gln Phe Phe  65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys                  85 90 95 Ala Arg Ser Leu Trp Pro Arg Gly Trp Phe Ala Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ala         115 <210> 35 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VH <400> 35 gatgtgaagc ttcaggagtc gggacctggc ctggtgaaac cttctcagtc tctgtccctc 60 acctgcactg tcactggcta ttcaatcacc agtgagtatg cctggacctg gatccggcag 120 tttccaggaa acaaactgga atggatgggc tacataaact acaatggcaa cactaactta 180 aatccatctc tcaaaagtcg aatctctatc attcgagaca catccaagaa ccagttcttc 240 ctgcagttga attctgtgac aactgaggac acagccacat attactgtgc aagatcactt 300 tggcccaggg gctggtttgc ttactggggc caagggactc tggtcactgt ctctgca 357 <210> 36 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> 1E8 light chain <400> 36 Asp Ile Val Met Thr Gln Ser Ser Ser Leu Ala Val Ser Val Gly   1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser              20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln          35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Ser Tyr Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln         115 120 125 Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr     130 135 140 Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 145 150 155 160 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr                 165 170 175 Tyr Ser Met Ser Arg Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg             180 185 190 His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro         195 200 205 Ile Val Lys Ser Phe Asn Arg Asn Glu Cys     210 215 <210> 37 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> 1E8 Heavy chain <400> 37 Asp Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Asn Pro Ser Gln   1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp              20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp          35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Arg Thr Ser Tyr Lys Ser Ser Leu      50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe  65 70 75 80 Leu Glu Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys                  85 90 95 Ala Arg Asp Tyr Gly Asn Phe Val Gly Tyr Phe Asp Val Trp Gly Ala             100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr Ser Ser Val Ser Val         115 120 125 Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr     130 135 140 Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr 145 150 155 160 Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val                 165 170 175 Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser             180 185 190 Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala         195 200 205 Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile     210 215 220 Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile                 245 250 255 Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp             260 265 270 Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His         275 280 285 Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg     290 295 300 Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys 305 310 315 320 Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu                 325 330 335 Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr             340 345 350 Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu         355 360 365 Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp     370 375 380 Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu                 405 410 415 Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His             420 425 430 Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro         435 440 445 Gly Lys     450 <210> 38 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> 8A12 light chain <400> 38 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly   1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr              20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Met          35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr  65 70 75 80 Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Arg                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ala Asp Ala Ala Pro             100 105 110 Thr Val Ser Ile Phe Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly         115 120 125 Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn     130 135 140 Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn 145 150 155 160 Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Arg                 165 170 175 Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr             180 185 190 Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe         195 200 205 Asn Arg Asn Glu Cys     210 <210> 39 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> 8A12 Heavy chain <400> 39 Asp Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln   1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Glu              20 25 30 Tyr Ala Trp Thr Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp          35 40 45 Met Gly Tyr Ile Asn Tyr Asn Gly Asn Thr Asn Leu Asn Pro Ser Leu      50 55 60 Lys Ser Arg Ile Ser Ile Ile Arg Asp Thr Ser Lys Asn Gln Phe Phe  65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys                  85 90 95 Ala Arg Ser Leu Trp Pro Arg Gly Trp Phe Ala Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Ala Pro Ser Val Tyr         115 120 125 Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu     130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp 145 150 155 160 Ser Ser Ser Ser Ser Val Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Val Ser                 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser             180 185 190 Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser         195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys     210 215 220 Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser                 245 250 255 Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp             260 265 270 Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr         275 280 285 Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val     290 295 300 Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu 305 310 315 320 Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg                 325 330 335 Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val             340 345 350 Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr         355 360 365 Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr     370 375 380 Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys                 405 410 415 Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu             420 425 430 Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly         435 440 445 Lys     <210> 40 <211> 174 <212> PRT <213> Artificial Sequence <220> <223> Aminoacyl tRNA synthetase complex-interacting          multifunctional protein 3) <400> 40 Met Ala Ala Ala Glu Leu Ser Leu Leu Glu Lys Ser Leu Gly Leu   1 5 10 15 Ser Lys Gly Asn Lys Tyr Ser Ala Gln Gly Glu Arg Gln Ile Pro Val              20 25 30 Leu Gln Thr Asn Asn Gly Pro Ser Leu Thr Gly Leu Thr Thr Ile Ala          35 40 45 Ala His Leu Val Lys Gln Ala Asn Lys Glu Tyr Leu Leu Gly Ser Thr      50 55 60 Ala Glu Glu Lys Ala Ile Val Gln Gln Trp Leu Glu Tyr Arg Val Thr  65 70 75 80 Gln Val Asp Gly His Ser Ser Lys Asn Asp Ile His Thr Leu Leu Lys                  85 90 95 Asp Leu Asn Ser Tyr Leu Glu Asp Lys Val Tyr Leu Thr Gly Tyr Asn             100 105 110 Phe Thr Leu Ala Asp Ile Leu Leu Tyr Tyr Gly Leu His Arg Phe Ile         115 120 125 Val Asp Leu Thr Val Gln Glu Lys Glu Lys Tyr Leu Asn Val Ser Arg     130 135 140 Trp Phe Cys His Ile Gln His Tyr Pro Gly Ile Arg Gln His Leu Ser 145 150 155 160 Ser Val Val Phe Ile Lys Asn Arg Leu Tyr Thr Asn Ser His                 165 170 <210> 41 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VH FR1 <400> 41 Asp Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Asn Pro Ser Gln   1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr              20 25 30 <210> 42 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VH FR1 <400> 42 gatgtgaagc ttcaggagtc gggacctggc ctggtgaatc cttctcagtc tctgtccctc 60 acctgcactg tcactggcta ttcaatcacc 90 <210> 43 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VH FR2 <400> 43 Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly   1 5 10 <210> 44 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VH FR2 <400> 44 tggatccggc agtttccagg aaacaaactg gagtggatgg gc 42 <210> 45 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VH FR3 <400> 45 Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Glu   1 5 10 15 Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg              20 25 30 <210> 46 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VH FR3 <400> 46 cgaatctcta tcactcgaga cacatccaag aaccagttct tcctggagtt gaattctgtg 60 actactgagg acacagccac atattactgt gcaaga 96 <210> 47 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VH FR4 <400> 47 Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser   1 5 10 <210> 48 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VH FR4 <400> 48 tggggcgcag ggaccacggt caccgtctcc tca 33 <210> 49 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VL FR1 <400> 49 Asp Ile Val Met Thr Gln Ser Ser Ser Leu Ala Val Ser Val Gly   1 5 10 15 Glu Lys Val Thr Met Ser Cys              20 <210> 50 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VL FR1 <400> 50 gacattgtga tgacccagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 60 atgagctgc 69 <210> 51 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VL FR2 <400> 51 Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr   1 5 10 15 <210> 52 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VL FR2 <400> 52 tggtaccagc agaaaccagg gcagtctcct aaactgctga tttac 45 <210> 53 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VL FR3 <400> 53 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Glu Phe Thr   1 5 10 15 Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys              20 25 30 <210> 54 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VL FR3 <400> 54 ggggtccctg atcgcttcac aggcagtgga tctgggacag aattcactct caccatcagc 60 agtgtgaagg ctgaagacct ggcagtttat tactgt 96 <210> 55 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 1E8 VL FR4 <400> 55 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys   1 5 10 <210> 56 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 1E8 VL FR4 <400> 56 ttcggtggag gcaccaagct ggaaatcaaa 30 <210> 57 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VH FR1 <400> 57 Asp Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln   1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr              20 25 30 <210> 58 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VH FR1 <400> 58 gatgtgaagc ttcaggagtc gggacctggc ctggtgaaac cttctcagtc tctgtccctc 60 acctgcactg tcactggcta ttcaatcacc 90 <210> 59 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VH FR2 <400> 59 Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly   1 5 10 <210> 60 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VH FR2 <400> 60 tggatccggc agtttccagg aaacaaactg gaatggatgg gc 42 <210> 61 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VH FR3 <400> 61 Arg Ile Ser Ile Ile Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln   1 5 10 15 Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg              20 25 30 <210> 62 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VH FR3 <400> 62 cgaatctcta tcattcgaga cacatccaag aaccagttct tcctgcagtt gaattctgtg 60 acaactgagg acacagccac atattactgt gcaaga 96 <210> 63 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VH FR4 <400> 63 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala   1 5 10 <210> 64 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VH FR4 <400> 64 tggggccaag ggactctggt cactgtctct gca 33 <210> 65 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VL FR1 <400> 65 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly   1 5 10 15 Glu Arg Val Thr Ile Thr Cys              20 <210> 66 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VL FR1 <400> 66 gacattctga tgacccagtc tccatcttcc atgtatgcat ctctaggaga gagagtcact 60 atcacttgc 69 <210> 67 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VL FR2 <400> 67 Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Met Tyr   1 5 10 15 <210> 68 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VL FR2 <400> 68 tggttccagc agaaaccagg gaaatctcct aagaccctga tgtat 45 <210> 69 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VL FR3 <400> 69 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser   1 5 10 15 Leu Thr Ile Ser Ser Leu Glu Tyr Glu Asp Met Gly Ile Tyr Tyr Cys              20 25 30 <210> 70 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VL FR3 <400> 70 ggggtcccat caaggttcag tggcagtgga tctggccaag attattctct caccatcagc 60 agcctggaat atgaagatat gggaatttat tattgt 96 <210> 71 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 8A12 VL FR4 <400> 71 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys   1 5 10 <210> 72 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 8A12 VL FR4 <400> 72 ttcggtggag gcaccaagct ggaaatcaaa 30

Claims (21)

An agent for measuring the expression level of methionyl-tRNA synthetase (MRS) protein and an agent for measuring the expression level of acinar cell-specific marker protein.
The method of claim 1, wherein the precursor cell-specific marker protein is at least one selected from the group consisting of chymotrypsin, phospholipase A2 group IB (PLA2G1B, phospholipase A2 group IB) and amylase A2 (amylase 2A) Wherein the composition is for the diagnosis of pancreatic cancer.
The composition for diagnosing pancreatic cancer according to claim 1, wherein the MRS protein comprises the amino acid sequence of SEQ ID NO: 1.
The composition for diagnosing pancreatic cancer according to claim 1, wherein the agent is an antibody or an aptamer that specifically binds to each of the MRS protein or the secretory cell-specific marker protein.
5. The antibody or the functional fragment thereof according to claim 4, wherein the antibody specifically binding to the MRS protein specifically binds to an epitope of a region including the amino acid sequence represented by SEQ ID NO: 2 in MRS Lt; / RTI &gt; fragment.
6. The antibody of claim 5, wherein the antibody
A light chain variable region (VL) comprising an amino acid sequence represented by SEQ ID NO: 28; And
A heavy chain variable region (VH) comprising an amino acid sequence represented by SEQ ID NO: 30; or
A light chain variable region (VL) comprising an amino acid sequence represented by SEQ ID NO: 32; And
A heavy chain variable region (VH) comprising an amino acid sequence represented by SEQ ID NO: 34.
The composition according to claim 1, wherein the agent is a primer or a probe that specifically binds to each mRNA encoding an MRS protein or a secretory cell-specific marker protein.
An agent for measuring the expression level of the methionyl thiourea synthetase protein and an agent for measuring the expression level of the precursor cell specific marker protein.
A method for qualitatively or quantitatively analyzing the expression level of methionyl thiolne synthase protein and secretory cell specific marker protein from a pancreas sample collected from a potential patient to provide information necessary for diagnosis of pancreatic cancer.
10. The method of claim 9, wherein the precursor cell-specific marker protein is at least one selected from the group consisting of chymotrypsin, phospholipase A2 group IB (PLA2G1B, phospholipase A2 group IB) and amylase A2 (amylase 2A) Lt; / RTI &gt;
10. The method of claim 9,
(a) measuring the level of expression of the precursor cell-specific marker protein and the MRS protein in the pancreas sample collected from a potential patient; And
(b) determining that the precursor cell-specific marker protein is not expressed in the measurement sample of step (a) and that the MRS protein is expressed, thereby being a pancreatic cancer cell.
12. The method according to any one of claims 9 to 11, wherein the sample is a pancreatic cell.
13. The method of claim 12, wherein the pancreatic cells are separated by fine needle aspiration.
12. The method of claim 11, wherein said method further comprises the following steps, simultaneously or in addition, before measuring the level of expression of the precursor cell-specific marker protein and MRS protein:
(i) pancreatic cells taken from potential patients
(4 ', 6-diamidino-2-phenylindole), methylene blue, acetocarmine, toluidine blue, hematoxylin and Hoechst At least one dyeing solution selected from the group consisting of
Staining the cell with at least one staining solution selected from the group consisting of eosin, crystal violet and orange G staining cytoplasm; And
(ii) determining pancreatic cells as malignant tumor cells, atypical cells or normal cells by staining the cells.
15. The method of claim 14, wherein step (ii)
From the cell staining results of step (i)
Cells are plastified in three dimensions; High nuclear / cytoplasm ratio (N / c ratio); Appearance of chromatin aggregation; Rough-shaped nucleus; Emergence of nuclear bodies; And the appearance of mitosis is judged to be malignant tumor cells when two or more morphological abnormalities are selected,
Cells were plated in one layer, and when the nucleus / cytoplasm ratio (N / C ratio) was small and the nuclear membrane was smooth,
If the change of the cell is not found in malignant tumor cells but can not be judged as normal, it is judged as atypical cell
. &Lt; / RTI &gt;
12. The method of claim 11, wherein the protein expression level is determined by Western blotting, enzyme immunoassay (ELISA), radioimmunoassay, radioimmunoprecipitation, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, Characterized in that the method comprises using any one of an assay method, a complement fixation assay, a fluorescence activated cell sorter (FACS), a surface plasmon resonance (SPR) or a protein chip method.
A method for improving sensitivity or specificity in cytodiagnosis or histology of pancreatic cancer, comprising the steps of:
(a) measuring the level of expression of the precursor cell-specific marker protein and the MRS protein in the pancreas sample collected from a potential patient; And
(b) determining that the precursor cell-specific marker protein is not expressed in the step (a) and that the expression of the MRS protein is increased, the cell is a pancreatic cancer cell.
18. The method of claim 17, wherein the method is characterized by a sensitivity or specificity of at least 80%.
In the cytodiagnosis or histologic examination of pancreatic cancer,
(a) measuring the level of expression of the precursor cell-specific marker protein and the MRS protein in the pancreas sample collected from a potential patient; And
(b) determining that the precursor cell-specific marker protein is not expressed in the step (a) and that the expression of the MRS protein is increased, the pancreatic cancer cell is further characterized in that it is a pancreatic cancer cell A method of providing information necessary for the diagnosis of pancreatic cancer.
20. The method according to claim 19, wherein the discrimination method is performed simultaneously, separately or sequentially with morphological examination.
20. The method of claim 19,
Cell clustering; Nuclear / cytoplasm ratio (N / C ratio); Shape of nucleus; Aggregation of chromatin; Appearance of nuclear bodies in the nucleus; And the appearance of mitosis. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt;

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