JP2009263318A - Method for production of monoclonal antibody, hybridoma and immunogen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody which recognizes specifically the binding region of glycoproteins. <P>SOLUTION: The immunogen is produced by digesting glycoproteins with protease without performing alkaline hydrolysis. An animal is immunized by the above immunogen, and an antibody-producing cell is collected from the animal. A monoclonal antibody is extracted from the antibody-producing cell or its hybridoma. The obtained antibody is applicable to various uses such as a recognition reagent for the binding region of glycoproteins, a recognition probe of tumor markers or a sugar-chain recognition probe, and has tremendous industrial availableness in various fields such as basic medical science, applied medical science or drug discovery. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a monoclonal antibody, a hybridoma, and an immunogen.

  A sugar chain such as glycosaminoglycan (GAG) is an important substance existing in various tissues or organs such as skin, connective tissue, and brain in a living body. For example, GAGs such as chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and heparin (Hep) are involved in cell growth, differentiation, ontogeny, tissue morphogenesis, etc. (Non-patent documents 1 and 2 etc.). These sugar chains are usually present in vivo in the form of so-called glycoproteins or proteoglycans. The glycoprotein includes the sugar chain, a protein called a core protein, and a binding region, and has a structure in which the sugar chain and the core protein are bound via the binding region (Non-patent Document 3, etc.). .

As described above, glycoproteins, particularly proteoglycans, play an important role as substances that form living tissues or organs or as biologically active substances. For this reason, various antibodies that recognize glycoproteins or proteoglycans have been developed as research tools or for the purpose of application to medicines and the like (Non-patent Document 4, Seikagaku Corporation Catalog, etc.). However, there are still many unclear points about the biological activity of glycoproteins and proteoglycans, and further research is required in each field such as basic medicine, applied medicine, and drug discovery.
Esko, JD, and Selleck, SB (2002) Order out of chaos: assembly of ligand binding sites in heparan sulfate.Annu. Rev. Biochem., 71, 435-471. Sugahara, K., Mikami, T., Uyama, U., Mizuguchi, S., Nomura, K., Kitagawa, H. (2003) Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate. Curr. Opin. Struct Biol., 13, 612-620. "Modulation of neurite formation by chondroitin sulfate / dermatan sulfate"("ExperimentalMedicine" Vol.25, No.7 (extra) pages 990-997, 2007, Shuji Mizumoto, Kazuyuki Sugawara) Sugahara, K., Mikami, T. (2007) Chondroitin / dermatan sulfate in the central nervous system. Curr. Opin. Struct. Biol., 17, 536-545.

  As described above, glycoproteins or proteoglycans include a binding region, and are characterized in that a sugar chain and a core protein are bound by this binding region. Therefore, if an antibody that specifically recognizes this binding region is used, it can be used for studies on the structure and biological activity of glycoproteins or proteoglycans, or for applications related to the biological activity of glycoproteins or proteoglycans. Therefore, it may be useful.

  However, conventionally, there has been no antibody that specifically recognizes (reacts specifically to) a binding region of glycoprotein or proteoglycan. For example, the antibody described in Non-Patent Document 4 is an antibody that recognizes a repetitive disaccharide region in the sugar chain portion of chondroitin sulfate, and is not an antibody that specifically recognizes a binding region.

  Accordingly, an object of the present invention is to provide an antibody that specifically recognizes a binding region of a glycoprotein.

  In order to solve the above problems, the monoclonal antibody of the present invention is a monoclonal antibody that specifically reacts with a binding region between a sugar chain part and a core protein part of a glycoprotein.

  The present invention also provides a hybridoma that produces the antibody of the present invention. Furthermore, the present invention also provides a method for producing an immunogen using a glycoprotein as a raw material, wherein the glycoprotein is digested with a protease and not subjected to alkaline hydrolysis treatment.

  As described above, the monoclonal antibody of the present invention is a monoclonal antibody that specifically reacts with the binding region between the sugar chain portion of the glycoprotein and the core protein portion. That is, according to the monoclonal antibody of the present invention, the binding region of glycoprotein can be specifically recognized. Therefore, the monoclonal antibody of the present invention can be used for various uses such as various products, pharmaceutical compositions, and medicines in both basic research and application.

  According to the present invention, for example, an immunogen produced by the production method of the present invention can be provided. Furthermore, the present invention is a method for immunizing an animal with an immunogen, wherein the immunogen is produced by the production method of the present invention (hereinafter sometimes referred to as “immunogen of the present invention”). An immunization method characterized by the present invention can also be provided.

Furthermore, according to the present invention, for example,
An immunization process in which the animal is immunized with an immunogen;
Extracting a monoclonal antibody from antibody-producing cells of the immunized animal or a hybridoma thereof,
A method for producing a monoclonal antibody comprising:
It is possible to provide a production method characterized in that the immunogen is the immunogen of the present invention.

Furthermore, according to the present invention, for example,
An immunization process in which the animal is immunized with an immunogen;
An antibody-producing cell collection step of collecting antibody-producing cells from the immunized animal,
A method for producing antibody-producing cells, comprising:
It is possible to provide a production method characterized in that the immunogen is the immunogen of the present invention.

Furthermore, according to the present invention, for example,
An immunization process of immunizing an animal with an immunogen;
An antibody-producing cell collection step of collecting antibody-producing cells from the immunized animal;
A fusion step of fusing the antibody-producing cells with other cells,
A method for producing a hybridoma, comprising:
It is possible to provide a production method characterized in that the immunogen is the immunogen of the present invention.

  Furthermore, according to the present invention, for example, the monoclonal antibody of the present invention or the monoclonal antibody produced by the production method of the present invention, a glycoprotein binding region recognition reagent, a tumor marker recognition probe, a sugar chain recognition probe, Products used as sugar chain marker recognition probes, neurite outgrowth inhibitors, neuronal stains, tumor cell stains, or immune tissue stains can be provided.

  Furthermore, according to the present invention, for example, including the monoclonal antibody of the present invention or the monoclonal antibody produced by the production method of the present invention, a genetic disease, inflammation, autoimmune disease, nerve cell adhesion, neurite outgrowth, Growth factor binding, viral infection inhibition, herpes virus infection inhibition, dengue virus infection inhibition, cell growth inhibition, cell death induction, tumor cell growth inhibition, tumor cell invasion inhibition, tumor cell metastasis inhibition, tumor cell migration inhibition, and tumor cell death Pharmaceutical compositions used for applications relating to at least one selected from the group consisting of induction can be provided.

  Furthermore, according to the present invention, for example, including the monoclonal antibody of the present invention or the monoclonal antibody produced by the production method of the present invention, a genetic disease, inflammation, autoimmune disease, nerve cell adhesion, neurite outgrowth, Selected from the group consisting of growth factor binding, viral infection inhibition, herpes virus infection inhibition, dengue virus infection inhibition, and at least one selected from the group consisting of cell proliferation, treatment, diagnosis, symptom reduction and prevention It is possible to provide a medicament used for at least one application.

  Hereinafter, embodiments of the present invention will be described more specifically. However, the present invention is not limited to the following embodiments.

[Monoclonal antibody]
As described above, the monoclonal antibody of the present invention is a monoclonal antibody that specifically reacts with the binding region between the sugar chain portion of the glycoprotein and the core protein portion. Other than this, the monoclonal antibody of the present invention is not particularly limited. For example, it is preferable that the monoclonal antibody does not react with the sugar chain portion and the core protein portion of the glycoprotein. The monoclonal antibody of the present invention reacts only with the binding region and does not react with the sugar chain part and the core protein part. For example, only the glycoprotein can be specifically detected from various biological substances. Alternatively, for example, it is possible to specifically detect a specific glycoprotein among various glycoproteins having different binding region structures. However, the monoclonal antibody of the present invention is not limited thereto, and may be, for example, an antibody that reacts with the sugar chain portion or the core protein portion of the glycoprotein.

  In the present invention, it is clear that an antibody “reacts specifically” or “recognizes specifically” with a specific antigen (immunogen) is reactive with the specific antigen. If it is good. The phrase “specifically reacts” or “specifically recognizes” to the specific antigen includes not only reacting to other antigens at all but also reacting to other antigens. For example, when the monoclonal antibody of the present invention says “specifically reacts with the binding region between the sugar chain part of the glycoprotein and the core protein part”, for example, the monoclonal antibody of the present invention reacts only with the binding region. However, it may have reactivity with other parts of the glycoprotein (sugar chain part, core protein part, etc.). Conventionally, an antibody having reactivity with a binding region of a glycoprotein has not been confirmed, but the monoclonal antibody of the present invention has reactivity with a binding region of a glycoprotein (such as proteoglycan). In the present invention, when an antibody that specifically reacts (recognizes specifically) with the specific antigen also reacts with another antigen, the reactivity with the specific antigen and the reactivity with the other antigen are clearly defined. Preferably they are different. For example, in the case where the monoclonal antibody of the present invention has reactivity to the other part of the glycoprotein (sugar chain part, core protein part, etc.) in addition to the reactivity to the binding region of the glycoprotein, It is preferred that the reactivity towards other parts is clearly different.

  In the present invention, the “binding region” of a glycoprotein refers to all or part of the binding portion between the sugar chain portion of the glycoprotein and the core protein portion and the vicinity thereof. Hereinafter, an example of the structure of the “binding region” of a glycoprotein (eg, proteoglycan) in the present invention will be specifically described. However, the following description is an example, and the structure of the “binding region” is not limited to these. For example, the “binding region” may include both sugar and amino acid, or may include only one of them. In the case of proteoglycans, for example, the binding part between the sugar chain part and the core protein part may contain an oligosaccharide structure different from the repeating disaccharide unit of the sugar chain part (Non-patent Document 3, etc.). The oligosaccharide structure is not particularly limited, and may be, for example, a tetrasaccharide structure of GlcAβ1-3Galβ1-3Galβ1-4Xyl. The tetrasaccharide structure of this binding site is commonly found in GAG sugar chains such as CS, dermatan sulfate (DS), HS, and heparin (Hep). In the present invention, the “binding region” of proteoglycan may or may not include part or all of this oligosaccharide structure (for example, the tetrasaccharide structure). In addition, the “binding region” of proteoglycan may or may not contain a peptide. For example, the “binding region” of proteoglycan may be part or all of the oligosaccharide structure (for example, the tetrasaccharide structure), the peptide, or the oligosaccharide structure (for example, the tetrasaccharide structure). It may be a glycopeptide in which the peptide is bound to a part or all of the (sugar structure). For example, when proteoglycan is thoroughly digested with a sugar chain degrading enzyme such as chondroitinase ABC, a structure in which one repeating disaccharide unit of the sugar chain part is added to the oligosaccharide structure may remain. That is, if the oligosaccharide structure is the tetrasaccharide structure, it becomes a hexasaccharide structure in which a disaccharide unit is added thereto. In the present invention, the “binding region” of proteoglycan may include a structure (for example, the hexasaccharide structure) in which one oligosaccharide structure is added with one repeating disaccharide unit. In addition, a peptide may or may not be added to this structure. In the present invention, the “binding region” of proteoglycan is, for example, a structure in which one monosaccharide is added to the oligosaccharide structure (for example, a pentasaccharide in which one monosaccharide is added to the tetrasaccharide structure). Structure) may or may not be included, and a peptide may or may not be added to this structure. A structure in which one monosaccharide is added to the oligosaccharide structure (for example, a pentasaccharide structure in which one monosaccharide is added to the tetrasaccharide structure) is, for example, a repeating disaccharide of a sugar chain portion in the oligosaccharide structure. It can be prepared by cleaving one monosaccharide (unsaturated uronic acid) by treating a structure with one unit added (for example, the hexasaccharide structure) with mercury acetate or the like. In the present invention, the “binding region” of a glycoprotein preferably includes a characteristic structure different from other parts of the glycoprotein, such as the above-mentioned tetrasaccharide structure, but may not include it. Further, as will be described later, the immunogen of the present invention may contain the entire “binding region” of the glycoprotein, or may contain only a part thereof.

The glycoprotein that can be recognized by the monoclonal antibody of the present invention is not particularly limited. For example, it is preferably a proteoglycan, and more preferably a proteoglycan having a sugar chain portion formed from glycosaminoglycan (GAG). preferable. Further, for example, the sugar chain portion of the proteoglycan has a repeating disaccharide unit, the binding region has an oligosaccharide structure different from the repeating disaccharide unit, and the monoclonal antibody of the present invention has an oligosaccharide of the binding region. More preferably, it reacts specifically with the sugar structure. As described above, glycoproteins or proteoglycans include a binding region, and are characterized in that a sugar chain and a core protein are bound by this binding region. In the present invention, “glycoprotein” includes proteoglycan, and “proteoglycan” is a kind of glycoprotein. In the present invention, “proteoglycan” is not particularly limited, and is, for example, a glycoprotein in which the sugar chain portion is mainly formed from a repeating structure of disaccharide units. The glycoprotein is more preferably a proteoglycan having a sugar chain portion formed from a chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, keratan sulfate, or a polysaccharide chain or oligosaccharide chain having a similar structure thereto.
The glycoprotein has a sugar chain moiety formed from CS-A, CS-B, CS-C, CS-D, CS-E, CS-K, or CS-H (DS-E). It is particularly preferred that However, these are examples, and the glycoprotein that can be recognized by the monoclonal antibody of the present invention is not limited thereto. Chondroitin sulfate H (CS-H) contains many iE disaccharide units having IdoA as a structural unit. The sulfation pattern of iE disaccharide units is the same as that of E disaccharide units. IdoA, which is a structural unit of CS-H, is common with dermatan sulfate (also called DS or CS-B). For this reason, CS-H can also be considered as a kind of dermatan sulfate. Therefore, in the present invention, chondroitin sulfate H (CS-H) is sometimes referred to as dermatan sulfate E (DS-E).

  The glycoprotein that can be recognized by the monoclonal antibody of the present invention is preferably, for example, a tumor cell-derived glycoprotein. According to this, the monoclonal antibody of this invention can be used for the use regarding a tumor, for example. Although the use regarding the said tumor is not specifically limited, For example, a tumor marker recognition probe, an antitumor agent, etc. are mention | raise | lifted. In the present invention, the “tumor” is not particularly limited, and examples thereof include cancer and sarcoma. The tumor cells are not particularly limited. For example, the tumor cells may be brain tumor, head and neck cancer, neuroblastoma, sinus cancer, pharyngeal cancer, esophageal cancer, lung cancer, stomach cancer, colon cancer, rectal cancer, liver cancer. , Biliary tract cancer, pancreatic cancer, prostate cancer, bladder cancer, testicular cancer, breast cancer, uterine cancer, uterine fibroid, cervical cancer, ovarian cancer, acute leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, malignant lymphoma, erythrocytosis, May be cells derived from focal polycythemia, essential thrombocytosis, myeloma, osteosarcoma, choriocarcinoma, Hodgkin's disease, non-Hodgkin's disease, glioblastoma, astrocytoma, or soft tissue sarcoma preferable.

  The monoclonal antibody of the present invention is particularly preferably deposited at the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation on February 22, 2008, and the accession numbers are NITE P-503 and NITE P-504. , NITE P-505, NITE P-506, NITE P-507, or NITE P-508, monoclonal antibodies produced by hybridomas. However, the monoclonal antibody of the present invention is not limited to these.

[Method for producing monoclonal antibody, etc.]
The method for producing the monoclonal antibody of the present invention is not particularly limited, and for example, it can be produced as follows.

  First, prepare an immunogen. This immunogen may be produced by any method, for example, it can be produced by the production method of the present invention. The method for producing an immunogen of the present invention is not particularly limited except that the glycoprotein is digested with a protease and not subjected to an alkaline hydrolysis treatment. For example, it is as follows. That is, first, a biological tissue containing glycoprotein is dehydrated and degreased by extraction with acetone and air-dried to prepare an acetone powder. The biological tissue is not particularly limited and may be appropriately selected depending on the type of the target glycoprotein. For example, squid cartilage, eel chord, shark cartilage, whale cartilage, horseshoe crab cartilage, pig skin, bovine cartilage, pig Examples include cartilage, chicken cartilage, salmon nasal cartilage, and various fish cartilage. Next, the acetone powder is suspended in water to form a slurry and heated to inactivate the proteolytic enzyme. It is thoroughly digested with protease and purified by an appropriate method such as centrifugation, ethanol precipitation, gel filtration chromatography or the like to obtain an immunogen. The concentration, reaction temperature, reaction time and the like of each reactant are not particularly limited and can be selected as appropriate. Moreover, each process can be changed suitably. For example, when extracting proteoglycan from the living tissue, a method such as hot water extraction may be used without using acetone. In the present invention, “does not perform alkali hydrolysis treatment” means that alkali treatment is not performed under such a strong condition that the protein is hydrolyzed. In the present invention, for example, it is more preferable not to perform the acid hydrolysis treatment in addition to the alkali hydrolysis treatment. The above-mentioned “no acid hydrolysis treatment” means that no acid treatment is performed under such a strong condition that the protein is hydrolyzed. Specifically, in the present invention, for example, the glycoprotein may not be treated with acid or alkali at all. However, as long as the protein is not hydrolyzed, acid treatment, alkali treatment, etc. You may do it. In the present invention, for example, conditions such as acid treatment and alkali treatment are preferably suppressed to such an extent that the binding region of glycoprotein is not decomposed.

  Although the mechanism by which the monoclonal antibody of the present invention can be obtained by the immunogen of the present invention produced by the present invention is not necessarily clear, for example, the alkaline protein is not hydrolyzed so that the glycoprotein binding region is not hydrolyzed. The remaining monoclonal antibody is considered to be able to react specifically with the binding region. In the method for producing an immunogen of the present invention, for example, it is more preferable to further digest the glycoprotein with a glycosylation enzyme. In this way, for example, not only the core protein of the glycoprotein but also the sugar chain is decomposed, and only the binding region remains, so that the specificity for the binding region is further enhanced. However, this description is an example of a mechanism that can be estimated, and does not limit the present invention. In the method for producing an immunogen of the present invention, when the glycoprotein is digested with a glycolytic enzyme, it is further preferable to further include a step of inactivating the glycolytic enzyme by heating. In the present invention, the “glycan degrading enzyme” refers to all enzymes capable of degrading sugar chains. In the present invention, the sugar chain degrading enzyme is not particularly limited, and may be appropriately selected according to the type of sugar chain to be degraded. Examples of the sugar chain degrading enzyme include glycosaminoglycan degrading enzyme. The glycosaminoglycan degrading enzyme is not particularly limited, and examples thereof include various chondroitin sulfate degrading enzymes and heparan sulfate degrading enzymes.

  The immunogen of the present invention preferably includes a structure derived from a glycoprotein binding region. In this case, the immunogen of the present invention may consist only of the structure derived from the binding region, but may contain other structures such as a sugar chain part of a glycoprotein. In addition, the immunogen of the present invention may contain, for example, either the peptide structure derived from the binding region or the oligosaccharide structure, may contain both, but may contain the oligosaccharide structure. More preferred. Moreover, the immunogen of the present invention may contain, for example, all of the oligosaccharide structure, but may contain only a part. For example, when preparing an immunogen using shark cartilage-derived CS-C as a raw material, the shark cartilage is thoroughly digested with a protease and a glycolytic enzyme as described above, and the remaining hexasaccharide peptide is used as the immunogen of the present invention. Also good. The hexasaccharide peptide is further treated with an appropriate method such as mercury acetate treatment, digestion with chondronase AC-I or AC-II to remove terminal sugars, pentasaccharide peptide, tetrasaccharide peptide, etc. After being converted to, it may be used as the immunogen of the present invention. In addition, the production of an immunogen using other glycoproteins such as proteoglycans such as various chondroitin sulfates as a raw material may also be applied in the same manner as the case of the shark cartilage.

  The glycoprotein that is the raw material of the immunogen is not particularly limited, but is preferably a proteoglycan, for example, and more preferably a proteoglycan having a sugar chain portion formed from glycosaminoglycan (GAG). The glycoprotein is more preferably a proteoglycan having a sugar chain portion formed from a chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, keratan sulfate, or a polysaccharide chain or oligosaccharide chain having a similar structure thereto. The glycoprotein has a sugar chain moiety formed from CS-A, CS-B, CS-C, CS-D, CS-E, CS-K, or CS-H (DS-E). It is particularly preferred that

  For example, it is preferable that the glycoprotein used as the raw material of the immunogen is a tumor cell-derived glycoprotein. This is because if an immunogen is produced using this, a monoclonal antibody that reacts specifically with the binding region of the tumor cell-derived glycoprotein can be produced. The tumor cells are not particularly limited, for example, brain tumor, head and neck cancer, neuroblastoma, sinus cancer, pharyngeal cancer, esophageal cancer, lung cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, biliary tract cancer, pancreatic cancer, Prostate cancer, bladder cancer, testicular cancer, breast cancer, uterine cancer, uterine fibroid, cervical cancer, ovarian cancer, acute leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, malignant lymphoma, erythrocytosis, true polycythemia, essential More preferably, the cell is derived from the lesion of thrombocytosis, myeloma, osteosarcoma, choriocarcinoma, Hodgkin's disease, non-Hodgkin's disease, glioblastoma, astrocytoma, or soft tissue sarcoma.

  Next, the animal is immunized with the immunogen. The immunization method is not particularly limited except that the immunogen of the present invention is used. For example, a known immunization method or the like may be referred to as appropriate. The animal is not particularly limited, and for example, a mouse or a rat is preferable.

  Furthermore, antibody-producing cells are collected from the immunized animal. Monoclonal antibodies may be extracted directly from these cells, but it is preferable to extract monoclonal antibodies from the hybridomas after fusing with other cells to form hybridomas. Conditions such as a hybridoma production method and a monoclonal antibody extraction method are not particularly limited, and can be set as appropriate. For example, known hybridoma production methods, monoclonal antibody extraction methods, and the like may be referred to as appropriate.

  As described above, the hybridoma of the present invention can be produced using the immunogen of the present invention to produce the monoclonal antibody of the present invention. The hybridoma of the present invention is not particularly limited. For example, the hybridoma is deposited with the Incorporated Administrative Agency Product Evaluation Technology Organization Patent Microorganism Deposit Center, and the accession numbers are NITE P-503, NITE P-504, NITE P-505, NITE. Hybridomas that are P-506, NITE P-507, or NITE P-508 are particularly preferred. In addition, as for the deposit date of these 6 types of hybridoma, as above-mentioned, all are February 22, 2008.

[Usage]
The application of the monoclonal antibody of the present invention is not particularly limited, and may be used for any application. More specifically, for example, it is as follows.

  First, the monoclonal antibody of the present invention is an extremely useful reagent for basic research on the biosynthesis mechanism of glycoprotein or proteoglycan. As described above, in vivo sugar chains exist in the form of glycoproteins or proteoglycans. On the other hand, when viewed from the protein side, more than 50% of the proteins in the living body usually exist as glycoproteins having a sugar chain covalently bonded thereto or as proteoglycans. For this reason, research on the biosynthetic mechanism of glycoproteins or proteoglycans is very important in biology. Although the detailed mechanism of biosynthesis of glycoprotein or proteoglycan is unknown, for example, it is considered as follows. That is, first, after a simple protein having no sugar chain is synthesized and a binding region oligosaccharide is constructed, it is determined whether it matures into chondroitin sulfate (CS) or heparan sulfate (HS). In that case, for example, the possibility of passing through different compartments of the intracellular Golgi apparatus is considered, and the sulfation modification of the binding region may be involved. The monoclonal antibody of the present invention specifically recognizes a binding region between a sugar chain and a peptide, and the binding region is a starting point for biosynthesis of glycoproteins such as chondroitin sulfate and heparan sulfate. Therefore, the monoclonal antibody of the present invention can be a very useful tool for elucidating the biosynthesis mechanism of glycoprotein or proteoglycan.

  Secondly, in certain molecular diseases (genetic diseases), sugar chain synthesis deficiency may be caused by abnormalities of the sulfation modification. The monoclonal antibody of the present invention can be a sugar chain recognition probe that can be used for diagnosis of such genetic diseases.

  For example, when the monoclonal antibody of the present invention specifically recognizes a specific glycoprotein binding region, the specific glycoprotein can be used to distinguish it from other glycoproteins. As described above, when chondroitin sulfate and heparan sulfate are localized in different Golgi compartments in the cell, and the monoclonal antibody of the present invention specifically recognizes the binding region of chondroitin sulfate, the monoclonal antibody of the present invention contains chondroitin sulfate. It can be used as a reagent for detecting intracellular localization.

  The details of the biosynthetic mechanism of proteoglycan are as follows, for example. First, GAG sugar chains such as CS, dermatan sulfate (DS), HS, and heparin (Hep) are composed of repeating disaccharide units. CS and DS are GlcAβ1-3GalNAc or IdoAα1-3GalNAc, and HS and Hep are GlcAβ1-4GlcNAc. Let IdoAα1-4GlcNAc be a constituent disaccharide unit. These disaccharides are variously sulfate-modified and exist in the polysaccharide chain. A unique sequence composed of a combination of these disaccharides having different sulfation modifications is the structural basis of the activity expression of the polysaccharide chain, and has a different sequence for each polysaccharide chain. In this way, these four types of GAG chains have different structures and activities, but there is a common characteristic called GlcAβ1-3Galβ1-3Galβ1-4Xyl at the root (binding region) bound to each core protein. Has a tetrasaccharide structure. The monoclonal antibody of the present invention has a detailed molecular mechanism of biosynthesis in which polysaccharide chains having different structures and activities are constructed on the same oligosaccharide structure (binding region) by specifically recognizing the binding region of proteoglycan. It can be a tool for research.

The structural study of the binding region of proteoglycan is, for example, as follows in more detail. First, the Gal residue in the binding region of CS and DS has a sulfated structure not found in HS and Hep (the following documents (i) to (iv)). As for the hybrid proteoglycan with both CS and DS, when CS and HS were analyzed simultaneously, a modified structure of GlcAβ1-3Gal (4-O-sulfate) β1-3Galβ1-4Xyl was found in CS. There is a study that the sulfation structure of Gal was not found (the following document (v)). This sulfated modified structure also exists in DS, which is a structural isomer of CS (the following document (vi)), but has not been found in Hep, a structural analog of HS (the following document (vii)). . Therefore, the sulfation modification of Gal is considered to be a structure characteristic of CS and DS containing GalNAc instead of GlcNAc in the disaccharide repeat structure. The role played by this sulfated modified structure is not always clear, but may be involved, for example, in proteoglycan biosynthesis sorting, ie, producing CS or DS, or HS or Hep branching. That is, details of the biosynthetic mechanism of proteoglycan are not clear, but first a simple protein without a sugar chain is synthesized, then becomes a glycoprotein with a binding domain oligosaccharide, and then matures into CS, DS, and HS. It is thought that it can be classified into sugar chains having various functions. In the monoclonal antibody of the present invention, it is possible to discriminate slight differences in the structure such as the sulfated structure of the binding region by appropriately selecting an immunogen or the like. Such an antibody can be a useful reagent for elucidating the biosynthesis mechanism of proteoglycan as described above.
(I) Sugahara, K., and Kitagawa, H. (2000) Recent advances in the study of the biosynthesis and functions of sulfated glycosaminoglycans.
Curr. Opin. Struct. Biol., 10 (5), 518-527.
(Ii) Sugahara, K., Yamashina, I., De Waard, P., Van Halbeek, H. and Vliegenthart, JFG (1988) Structural studies on sulfated glycopeptides from the carbohydrate-protein linkage region of chondroitin 4-sulfate proteoglycans of Swarm rat chondrosarcoma. Demonstration of the structure, Gal (4-O-Sulfate) β1-3Galβ1-4Xylβ1-O-Ser.J. Biol. Chem., 263 (21), 10168-10174.
(Iii) Sugahara, K., Ohi, Y., Harada, T., de Waard, P. and Vliegenthart, JFG (1992) Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. I. Six compounds containing 0 or 1 sulfate and / or phosphate residue.J. Biol. Chem., 267 (9), 6027-6035.
(Iv) de Waard, P., Vliegenthart, JFG, Harada, T. and Sugahara, K. (1992) Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. II. Seven compounds containing 2 or 3 sulfate residues. J. Biol. Chem., 267 (9), 6036-6044.
(V) Ueno, M., Yamada, S., Zako, M., Bernfield, M., and Sugahara, K. (2001) Structural characterization of heparan sulfate and chondroitin sulfate of syndecan-1 purified from normal murine mammary gland epithelial Common phosphorylation of xylose and differential sulfation of galactose in the protein linkage region tetrasaccharide sequence.J. Biol. Chem., 276 (31), 29134-29140.
(Vi) Sugahara, K., Masuda, M., Harada, T. and Yamashina, I. (1991) Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin sulfate proteoglycans of whale cartilage. Eur. J. Biochem., 202 (2), 805-811.
(Vii) Sugahara, K., Ohkita, Y., Shibata, Y., Yoshida, K. and Ikegami, A. (1995) Structural studies on the hexasaccharide alditols isolated from the carbohydrate-protein linkage region of dermatan sulphate proteoglycans of bovine aorta. Demonstration of iduronic acid-containing components. J. Biol. Chem., 270 (13), 7204-7212.
(Viii) Sugahara, K. (1998) http://www.glycoforum.gr.jp/science/word/proteoglycan/ PGA06E.html.
(Ix) Sugahara, K., Yamada, S., Yoshida, K., de Waard, P. and Vliegenthart, JFG (1992) A novel sulfated structure in the carbohydrate-protein linkage region isolated from porcine intestinal heparin. J. Biol Chem., 267 (3), 1528-1533.

  The monoclonal antibody of the present invention can be used for various applications other than those described above. For example, it can be used for various products, pharmaceutical compositions or pharmaceuticals as described above. For example, when the monoclonal antibody of the present invention reacts with a binding region of a glycoprotein but does not react with a sugar chain part, it can be used for distinguishing between a glycoprotein containing a binding region and a glycoprotein not containing it. Specifically, for example, when used for a sample containing a sugar chain (food, etc.), the sugar chain contains a sugar chain that does not contain a binding region (that is, an artificial treatment such as an alkali hydrolysis treatment). It is possible to identify whether or not In addition, as described above, the pharmaceutical provided by the present invention includes genetic diseases, inflammation, autoimmune diseases, nerve cell adhesion, neurite outgrowth, growth factor binding, virus infection inhibition, herpes virus infection inhibition, dengue virus infection inhibition. And at least one application selected from the group consisting of treating, diagnosing, alleviating and preventing symptoms related to at least one selected from the group consisting of cell proliferation. The disease relating to the proliferation of the cells is not particularly limited, but for example, brain tumor, head and neck cancer, neuroblastoma, sinus cancer, pharyngeal cancer, esophageal cancer, lung cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, biliary tract cancer , Pancreatic cancer, prostate cancer, bladder cancer, testicular cancer, breast cancer, uterine cancer, uterine fibroid, cervical cancer, ovarian cancer, acute leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, malignant lymphoma, erythrocytosis, true blood At least one disease selected from the group consisting of symptom, essential thrombocytosis, myeloma, osteosarcoma, choriocarcinoma, Hodgkin's disease, non-Hodgkin's disease, glioblastoma, astrocytoma, and soft tissue sarcoma . In addition, the medicament provided by the present invention is preferably used for at least one application selected from the group consisting of, for example, an antitumor agent, an anticancer agent, an anticancer agent, an antimetastatic agent, and a tumor marker detection agent, It is not limited to these. Thus, the present invention has great industrial applicability in various fields such as basic medicine, applied medicine, and drug discovery. Furthermore, the monoclonal antibody of the present invention can be used for any application without being limited thereto.

  Next, examples of the present invention will be described.

[Examples 1 to 11]
The “root” of shark cartilage-derived CS (chondroitin sulfate), that is, the sugar-protein binding region that connects the sugar chain and the core protein, has a characteristic structure in which the hydroxyl group at the 4- or 6-position of Gal is sulfated (References 1 and 2 below, Reference 3 below). That is, GlcA-Gal-Gal (6-O-sulfate) -Xyl and GlcA-Gal (4-O-sulfate) -Gal (6-O-sulfate) -Xyl and GlcA-Gal (6-O-sulfate)- The structure is Gal (6-O-sulfate) -Xyl (Reference Document 4 below).
(Reference 1) Sugahara, K., Ohi, Y., Harada, T., de Waard, P. and Vliegenthart, JFG (1992) Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. I. Six compounds containing 0 or 1 sulfate and / or phosphate residue.J. Biol. Chem., 267 (9), 6027-6035.
(Reference 2) de Waard, P., Vliegenthart, JFG, Harada, T. and Sugahara, K. (1992) Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. II. Seven compounds containing 2 or 3 sulfate residues. J. Biol. Chem., 267 (9), 6036-6044.
(Reference 3) Sugahara, K., and Kitagawa, H. (2000) Recent advances in the study of the biosynthesis and functions of sulfated glycosaminoglycans.
Curr. Opin. Struct. Biol., 10 (5), 518-527.
(Reference 4) Sugahara, K., Masuda, M., Harada, T. and Yamashina, I. (1991) Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin sulfate proteoglycans of whale cartilage.

  In this example, shark cartilage-derived CS (chondroitin sulfate) is thoroughly digested with protease and chondroitinase to decompose the core protein and sugar chain (chondroitin sulfate) portion, leaving a binding region, which is used as an immunogen. A monoclonal antibody was produced. This immunogen has a glycopeptide structure in which a short sugar chain (oligosaccharide) is bound to a peptide. In addition, the reactivity of the monoclonal antibody thus produced was measured, and it was confirmed that the glycoprotein binding region was specifically recognized.

  Hereinafter, the operation of the present embodiment will be specifically described. Unless otherwise indicated, all chemical substances are reagent grade and can be purchased as commercial products. Chondroitinase ABC, chondroitinase AC-I, and chondroitinase AC-II are trade names of Seikagaku Corporation, and actinase E is a trade name of Kaken Pharmaceutical Co., Ltd. Actinase E is sometimes simply referred to as “actinase”, but “actinase E” and “actinase” are the same.

(1. Preparation of immunogen for antibody production)
The shark cartilage was crushed and thoroughly digested with protease to remove the protein component, and the core protein of CS proteoglycan was also degraded to obtain a CS peptide fraction. The thus-prepared CS peptide fraction contains peptides consisting of an average of 5 to 6 amino acids in addition to Ser residues to which CS is bound via Xyl (Reference 1).

Thorough digestion with protease was performed as follows. That is, first, shark cartilage was crushed, dehydrated and degreased by acetone extraction, and air-dried to prepare acetone powder. This acetone powder was thoroughly digested with actinase E (Kaken Pharmaceutical Co., Ltd.) according to the method described in Reference 5 below, and then an unpurified glycosaminoglycan (GAG) -peptide fraction was extracted. Specifically, first, the acetone powder was suspended in water to form a slurry, and heated in a boiling water bath for 30 minutes to inactivate the proteolytic enzyme. Boric acid-NaOH buffer and CaCl ₂ were added to the heated slurry at final concentrations of 0.1 M and 10 mM, respectively. It was digested with protease (actinase E, 2% by weight with respect to the slurry) at 60 ° C. for 24 hours. Immediately after that, that is, 24 hours after the start of digestion, fresh actinase E (1% by mass with respect to the slurry) was added and digestion was continued. After another 24 hours, that is, 48 hours after the start of digestion, fresh actinase E (1% by mass based on the slurry) was added again, and digestion was continued. After a total of 95 hours of digestion, a 50% strength by weight aqueous trichloroacetic acid solution was added until a final concentration of 5% by weight was reached. The mixture was centrifuged, and the resulting precipitate was resuspended in a 5% by mass aqueous trichloroacetic acid solution and centrifuged again. The supernatant thus obtained was collected and excess trichloroacetic acid was removed by extraction with diethyl ether. To the supernatant from which trichloroacetic acid had been removed, an 80% by mass ethanol aqueous solution (containing 5% by mass sodium acetate) was added 4 times in volume, and left at 4 ° C. overnight to precipitate GAG. Thereafter, the precipitate was collected by centrifugation and dried to obtain an unpurified chondroitin sulfate (CS) -peptide fraction.
(Reference 5) Nandini, CD, Itoh, N., and Sugahara, K. (2005) J. Biol. Chem. 280, 4058-4069.

  Next, this unpurified chondroitin sulfate (CS) -peptide fraction is thoroughly digested with chondroitinase ABC derived from bacteria to decompose and remove the disaccharide repeat region of the polysaccharide chain, and the glycopeptide structure in the binding region Left. This was subjected to gel filtration chromatography to recover the glycopeptide fraction. The oligosaccharide chain contained in this fraction has a hexasaccharide skeleton (ΔHexA-GalNAc-GlcA-Gal-Gal-Xyl, where ΔHexA indicates unsaturated uronic acid generated by elimination reaction by chondroitinase) Since terminal unsaturated uronic acid is known to have strong antigenicity, it was chemically removed with mercury acetate to obtain a pentasaccharide peptide. Specifically, the glycopeptide fraction containing the oligosaccharide chain (hexaglycopeptide) was added to a 130 mM sodium acetate buffer (pH 5.0) containing 10 mM mercury acetate and allowed to react at room temperature for 5 hours and a half. (Ludwigs U, Elgavish A, Esko JD, Meezan E, Roden L. “Reaction of unsaturated uronic acid residues with mercuric salts. Cleavage of the hyaluronic acid disaccharide 2-acetamido-2-deoxy-3-O- (beta-D -gluco-4-enepyranosyluronic acid) -D-glucose. "Biochem J. 1987 Aug 1; 245 (3): 795-804.). After the reaction, the fraction containing the target pentasaccharide peptide was collected by gel filtration chromatography. The structures of the glycopeptide fraction (unsaturated hexasaccharide peptide fraction) immediately after digestion with chondroitinase ABC and the pentasaccharide peptide fraction from which uronic acid has been removed are analytical values (MS, NMR, etc.). And confirmed. The pentasaccharide peptide fraction thus obtained was covalently bound to a carrier protein KLH (keyhole limpet hemocyanin) to obtain an immunogen. Specifically, in a phosphate buffer (pH 7.4) physiological saline containing 0.3% glutaraldehyde, mixing was performed so that the mass ratio of the pentasaccharide peptide fraction and KLH was 1: 100 at room temperature. The target immunogen was obtained by reacting overnight. BalbC mice were immunized with this immunogen.

(2. Mouse immunization and blood collection)
According to a conventional method, mice were boosted and the antibody titer in the blood was measured. More specifically, it is as follows.

(2-1. Preparation)
Mouse (use 4 BALB / c 週 6 weeks old), FCA, FIA, PBS (-), 1ml syringe, 26G x 1/2 needle, ultrasonic homogenizer, mouse retainer, alcohol cotton, nipper, hematocrit capillary A micro syringe and a micro tube were prepared. In addition, necessary reagents and instruments such as the immunogen were prepared. Mice were purchased from Nippon Claire Co., Ltd. and used one week after acclimatization. Bad mice were removed from the subject.

(2-2. Immunization)
The mice were immunized by the following procedure. That is, first, the immunogen (the pentasaccharide peptide fraction) was dissolved in phosphate buffered saline to obtain a 250 mg / L solution. An equal volume of adjuvant was added to this immunogen solution, placed in a 1 ml syringe, and emulsified with an ultrasonic homogenizer to give an injection for immunization. The mixing and emulsification were performed while cooling in order not to impair the activity of the immunogen. As the adjuvant, FCA (Freund's complete adjuvant) was used for the first immunization, and FIA (Freund's incomplete adjuvant) was used for the second and subsequent immunizations. Next, a 26G × 1/2 injection needle was attached to the 1 ml syringe, and the injection solution for immunization was dispersedly administered subcutaneously to the back of the mouse (after disinfection with alcohol cotton). The standard dose of the immunogen was 25 μg / animal / dose, and the liquid volume was 200 μl / animal. Immunization was performed 4 times, 0w, 2w, 4w, and 6w. “0w” refers to the time of the first immunization, and 2w, 4w, and 6w indicate that the immunization was performed 2 weeks, 4 weeks, and 6 weeks after the initial immunization, respectively.

(2-3. Blood collection)
The mouse that had been immunized 4 times was placed in a restraint device, and the tail was lightly rubbed with alcohol cotton to make the blood vessels angry. Next, a 26 G × 1/2 injection needle with the needle tip removed with a nipper was inserted into the tail vein of the mouse. The blood that emerged was collected with a hematocrit capillary (2 / animal). The blood was centrifuged at 8,000 rpm for 20 minutes at room temperature after 1 hour incubation (about 37 ° C.). After centrifugation, serum was collected with a microsyringe and transferred into a microtube. It was stored in a −20 ° C. freezer until use. Blood collection was performed at 0 w, 5 w, and 7 w, respectively. As described above, “0w” indicates the time of the first immunization administration, and 5w and 7w indicate 5 weeks and 7 weeks after the initial immunization administration, respectively.

(2-4. Measurement of antibody titer by ELISA using plastic plate)
The antibody titer of each serum collected from the mice was measured according to a conventional method by ELISA using a plastic plate (trade name Maxisorp plate, Nunc). That is, first, the unsaturated hexasaccharide peptide fraction derived from the shark cartilage chondroitin sulfate binding region was directly applied to a plastic plate (the Maxisorp plate) and immobilized. The wells were blocked with 1% bovine serum albumin (BSA), incubated with 5,000-fold diluted serum (including primary antibody) with 0.1% BSA / PBS for 1 hour at 37 ° C, followed by They were treated with goat anti-mouse IgG / IgM labeled with alkaline phosphatase as the secondary antibody. The antibody titer was evaluated by adding paranitrophenyl phosphate as a substrate and measuring the absorption of 415 nm of the released paranitrophenol.

(3. Production of hybridoma)
The mouse spleen (one mouse) that showed a particularly high antibody titer by the ELISA method was fused with myeloma cells according to a conventional method to prepare a hybridoma, which was then screened by the same ELISA method as described above. Three types of positive cell fractions (4E1, 3F11, 1B5) that react with saturated hexasaccharide peptides were obtained. The hybridoma was produced as follows. In addition, 3 days before mouse sacrifice, the above-mentioned injection solution for immunization was used, and 10 μg / mouse · times of immunogen was injected into the mouse for the final boost.

(3-1. Preparation)
Mouse (Three days after the final boost), Beaker (300ml, 1 piece), Sterile petri dish (1 piece), Tweezers (Large / Small) , Stainless bat (2), sterile gauze, stainless mesh (several), spoon (1), vortex mixer, sterile pipette (1, 2, 5, 10 ml), mouse myeloma (SP2), PBS (-) 10% FBS RPMI1640 medium (with HAT), 10% FBS RPMI1640 medium (without HAT), RPMI1640 medium (without serum), sterile 50 ml centrifuge tube, PEG, sterile pipette tip (~ 200 μl), 96-well culture plate, octapipette, A cryotube (2 ml), a suction bottle set, a hemocytometer, and other necessary items were prepared.

(3-2. Cell fusion and hybridoma culture)
Cell fusion and hybridoma culture were performed as follows. That is, first, a mouse 3 days after the final boost was dislocated from the cervical spine, sacrificed, and then disinfected with ethanol. Next, the whole body of the mouse was immersed in ethanol for disinfection, the back muscle was cut with scissors, and the spleen was removed. The extracted spleen was placed on a stainless steel mesh in a sterilized petri dish, an appropriate amount of PBS was added, several holes were opened in the spleen using barbed tweezers, and the mixture was crushed using a spoon. The stainless mesh and sterilized petri dish were washed with PBS, and PBS containing splenocytes was collected and transferred to a 50 ml centrifuge tube. The centrifuge tube from which PBS was collected was centrifuged at 1,300 rpm for 5 minutes at room temperature, and then the supernatant was discarded, fresh PBS was added, pipetted, and centrifuged again under the same conditions and washed.

  Meanwhile, the prepared spleen cells and myeloma cells were mixed at a ratio of 5: 1, PBS was added, and the mixture was centrifuged at 1,300 rpm for 5 minutes at room temperature. After discarding the supernatant, cell fusion was performed by the 50% PEG method to obtain a hybridoma. To this hybridoma, 50 ml of 10% FBS RPMI1640 medium (with HAT) was added and suspended. Using 2 ml of sterile pipette, 100 μl of the hybridoma suspension was seeded in each well of five 96-well culture plates prepared in advance. Five days later, 100 μl of 10% FBS RPMI1640 medium (without HAT) was added to each well using an octapipette. Thereafter, the observation of the size of the colony and the color of the medium was continued, and after 7 days after the addition of the medium, it was determined that the growth of the colony reached a sufficient level for screening.

(4. Screening and cloning)
Further cloning was performed to obtain clones from the above three cell fractions, and clones were obtained only from 4E1, and a total of 11 clones (A4, A8, A9, B6, B11, C6, C11, D6, D12, F4, H4) was obtained. These hybridomas and monoclonal antibodies were obtained from Example 1 (A4), Example 2 (A8), Example 3 (A9), Example 4 (B6), Example 5 (B11), and Example 6 (C6), respectively. Example 7 (C11), Example 8 (D6), Example 9 (D12), Example 10 (F4), and Example 11 (H4). Specifically, screening and cloning of hybridomas were performed as follows.

(4-1. Preparation)
ELISA equipment, equipment, reagents, 96-well culture plate (with cells), octapipette, sterile pipette tip (up to 200 μl), pipette tip disposal container and sterile pipette (1, 2, 5, 10 ml), 10% FBS RPMI1640 medium (With HAT), 10% FBS RPMI1640 medium (without HAT), RPMI1640 medium (without serum), sterile 50 ml centrifuge tube, 96-well culture plate, 24-well culture plate, cell banker, cryotube (2 ml), suction bottle set, hemocytometer 25 cm ² culture flask, PBS (−), and other necessary items were prepared. As the “96-well culture plate (with cells)”, the 96-well culture plate containing the hybridoma colonies cultured in the above-mentioned “3. Production of hybridoma” was used as it was.

(4-2. Screening)
Hybridomas were screened by the same ELISA method as in “2-4. Measurement of antibody titer using plastic plate” described above. That is, first, the unsaturated hexasaccharide peptide fraction (antigen) was immobilized (immobilized) on a plastic plate (96-well ELISA plate). The wells were blocked with 1% bovine serum albumin (BSA). On the other hand, using an octapipette, the culture supernatant containing the antibody was extracted from the “96 well culture plate (with cells)” containing the hybridoma. 50 μl of this culture supernatant was dispensed into each well of the plastic plate (96 well ELISA plate) in which the antigen was immobilized and blocked with BSA. This was incubated at 37 ° C. for 1 hour and then treated with alkaline phosphatase-conjugated goat anti-mouse Ig (G + M). Enzyme activity was detected using p-nitrophenyl phosphate and absorbance was measured at 415 nm.

(4-3. Cloning)
Hybridomas in wells judged positive in the screening after cell fusion were collected in a 24-well culture plate in which 1 ml of 10% FBS RPMI1640 medium (with HAT) was dispensed per well. After culturing for several days, 11 types of hybridomas were selected and 1st cloning was performed. That is, first, the hybridoma was seeded on a new 96-well culture plate prepared in advance by limiting dilution method so that there were 4 per well. As a solvent for limiting dilution, 10% FBS RPMI1640 medium (with HAT) was used, and 100 μl per well was seeded. In addition, 0.5 96-well culture plates were used for one type of hybridoma (one for two types). Five days after seeding, an octapipette was used, and 100% of 10% FBS RPMI1640 medium (without HAT) was dispensed per well of each plate. After culturing for several days, colonies were confirmed with the naked eye, and screening was performed when the color of the medium became light yellow. The screening method is the same as described above.

  Meanwhile, 1 ml of 10% FBSRPMI1640 medium (with HAT) was dispensed into a new 24-well culture plate prepared in advance. 11 kinds of hybridomas in positive wells obtained by the screening after the 1st cloning were added to each. After culturing for several days, when the number of cells increased to some extent, 2nd cloning was performed. That is, first, the hybridoma was seeded on a new 96-well culture plate prepared in advance by a limiting dilution method so that there was one per well. As a solvent for limiting dilution, 10% FBS RPMI1640 medium (with HAT) was used, and 100 μl per well was seeded. In addition, 0.5 96-well culture plates were used for one type of hybridoma (one for two types). Further, using an octapipette, 100 μl of 10% FBS RPMI1640 medium (without HAT) was dispensed per well of each plate. After culturing for about 10 days, colonies were confirmed with the naked eye, and screening was performed when the color of the medium became light yellow. The screening method is the same as described above.

Eleven types of positive-well hybridomas obtained by the screening after the 2nd cloning were collected in 24-well culture plates in which 1 ml of 10% FBS RPMI1640 medium (with HAT) was dispensed per well. After culturing for several days and a certain amount of cells increased, each cell was transferred to two 25 cm ² culture flasks. Each culture flask was previously filled with 5 ml of 10% FBS RPMI1640 medium (with HAT). In each cell, one of the two 25 cm ² culture culture flasks, one is the hybridomas was performed conditioned culture as can be cultured in a 10% FBS RPMI1640 medium (without HAT). The remaining one was frozen stock. Acclimatized hybridomas can be cultured in 10% FBS RPMI1640 medium, and when they approached confluence, they were frozen and stocked in three cryotubes containing a cell cryopreservation solution (trade name Cell Banker). Three cryotubes were used for each hybridoma. At this point, clones were established (11 clones). As described above, these 11 types of clones were named A4, A8, A9, B6, B11, C6, C11, D6, D12, F4, and H4, respectively. These hybridomas and monoclonal antibodies produced thereby were obtained in Example 1 (A4), Example 2 (A8), Example 3 (A9), Example 4 (B6), Example 5 (B11), Example 6 (C6), Example 7 (C11), Example 8 (D6), Example 9 (D12), Example 10 (F4), and Example 11 (H4) are used.

(5. Ascites collection)
Among the 11 types of clones described above, Example 1 (A4), Example 2 (A8), Example 4 (B6), Example 5 (B11), Example 6 (C6), and Example 8 (D6) 6 types of hybridomas were inoculated intraperitoneally into mice and ascites was collected. The method for collecting ascites is as follows.

(5-1. Preparation)
Mouse (BALB / c ♂ 6 weeks old), pristane, 2.5ml syringe, 5ml syringe, 25G x 5/8 needle, 18G x 11/2 needle, 15ml centrifuge tube (or 50ml centrifuge tube), EDTA 2K An aqueous solution (80 mg / ml), alcohol cotton and other necessary items were prepared.

(5-2. Ascites collection)
Mice bred for 1 week were administered 0.5 ml of pristane per mouse in a 2.5 ml syringe with a 25G × 5/8 needle. After 1 week or more after the administration of pristane, Example 1 (A4), Example 2 (A8), Example 4 (B6), Example 5 (B11), Example 6 (C6), and Example PBS (-) in which any hybridoma of 8 (D6) was suspended was administered at 1 ml per mouse in a 2.5 ml syringe with a 25 G x 5/8 needle. The number of cells administered per mouse was about 5 × 10 ⁶ cells. After cell administration, the progress was observed, and ascites was collected about 10 days later. Specifically, an 18G × 11/2 injection needle was inserted into the abdomen of the mouse, and ascites was collected by dropping into a 15 ml centrifuge tube (or 50 ml centrifuge tube) containing EDTA 2K solution (2 mg per 1 ml of ascites). If possible, ascitic fluid was collected again (2-3 times) after several days. The collected ascites was centrifuged at 3,500 rpm for 10 minutes at 4 ° C. and stored frozen in a centrifuge tube. The number of days until ascites collection may vary slightly depending on the nature of the hybridoma, the nature of the mouse, the breeding conditions, and the like.

(6. Properties of monoclonal antibodies)
The culture supernatant produced by the three cell fractions (4E1, 3F11, 1B5) is not only the unsaturated hexasaccharide peptide fraction derived from the binding region, but also GalNAc-GlcA-Gal- prepared from it by mercury acetate treatment. Unsaturation containing ΔHexA-Gal-Gal-Xyl skeleton prepared by treating pentasaccharide peptide fraction containing Gal-Xyl skeleton and unsaturated hexasaccharide peptide with a mixture of chondroitinase AC-I and AC-II It also reacted with the tetrasaccharide peptide fraction. However, these three types of antibodies did not react with the unsaturated hexasaccharide peptide fraction derived from the CS binding region prepared in the same manner as in the case of shark cartilage from whale cartilage. The specific method of mercury acetate treatment is as described above. In addition, the method for preparing the unsaturated tetrasaccharide peptide fraction is specifically about 5 nmU of each enzyme (chondroitinase AC-I and AC) with respect to about 1 nmol substrate (the unsaturated hexasaccharide peptide fraction). -II mixture solution), and incubated in 50 mM Tris-HCl buffer (pH 7.3) at 37 ° C. for 1 hour. The desired unsaturated tetrasaccharide peptide fraction was recovered by gel filtration chromatography. The difference in structure between the shark cartilage-derived CS binding region and the whale cartilage-derived CS binding region is described in detail in, for example, Reference 6 and Reference 7 below.
(Reference 6) Kazuyuki Sugawara, http://www.glycoforum.gr.jp/science/word/proteoglycan/PGA06J.html
(Reference 7) Sugahara, K., Ohkita, Y., Shibata, Y., Yoshida, K. and Ikegami, A. (1995) Structural studies on the hexasaccharide alditols isolated from the carbohydrate-protein linkage region of dermatan sulphate proteoglycans of bovine aorta. Demonstration of iduronic acid-containing components.

  The mechanism by which the three types of monoclonal antibodies specifically recognize the shark cartilage-derived CS binding region and not the whale cartilage-derived CS binding region is not necessarily clear, but is considered as follows, for example. That is, first, since the CS binding region of whale cartilage has a structure (GlcA-Gal (4-O-sulfate) -Gal-Xyl) different from the sulfation modification structure of the shark binding region (see the above-mentioned reference). 8) The three types of monoclonal antibodies may recognize differences in galactose sulfate structure or peptide sequences. In addition, CS, like HS, can undergo phosphorylation modification at position 2 in Xyl of the binding region, and thus there is a possibility that phosphorylation modification is involved in the antibody recognition mechanism. However, these are examples of mechanisms that can be estimated, and do not limit the present invention.

Furthermore, Example 1 (A4), Example 2 (A8), Example 3 (A9), Example 4 (B6), Example 5 (B11), Example 6 (from the cell fraction 4E1) For the 11 monoclonal antibodies of C6), Example 7 (C11), Example 8 (D6), Example 9 (D12), Example 10 (F4), and Example 11 (H4), shark cartilage-derived CS The reactivity to the binding region and the whale cartilage-derived CS binding region was tested by ELISA as described above. That is, first, a hexasaccharide peptide fraction derived from a shark cartilage CS binding region, a pentasaccharide peptide fraction, a tetrasaccharide peptide fraction, or a hexasaccharide peptide fraction or pentasaccharide peptide fraction prepared from a whale cartilage CS binding region. The minute was immobilized on a Maxisorp plate. Next, any one of the 11 culture supernatant clones of Examples 1 to 11 was added. Further, goat anti-mouse IgG / IgE antibody (alkaline phosphatase labeled) was added, paranitrophenyl phosphate as a substrate was added, and the mixture was reacted at 37 ° C. for 8 minutes. The amount of released paranitrophenol was detected by measuring absorbance at a wavelength of 415 nm, and the reactivity between each monoclonal antibody and each glycopeptide fraction was evaluated. The results are shown in the bar graph of FIG. In the figure, the vertical axis represents the absorbance (relative value) at a wavelength of 415 nm, that is, an index of the reactivity of the monoclonal antibody. “A4” indicates the measurement result of the monoclonal antibody of Example 1. “A8” indicates the measurement result of the monoclonal antibody of Example 2. “A9” indicates the measurement result of the monoclonal antibody of Example 3. “B6” indicates the measurement result of the monoclonal antibody of Example 4. “B11” indicates the measurement result of the monoclonal antibody of Example 5. “C6” indicates the measurement result of the monoclonal antibody of Example 6. “C11” indicates the measurement result of the monoclonal antibody of Example 7. “D6” indicates the measurement result of the monoclonal antibody of Example 8. “D12” indicates the measurement result of the monoclonal antibody of Example 9. “F4” indicates the measurement result of the monoclonal antibody of Example 10. “H4” indicates the measurement result of the monoclonal antibody of Example 11. Blank indicates the measurement result when the primary antibody (monoclonal antibody) was not added. In the monoclonal antibody and Blank of each Example, each bar indicates the absorbance in the following cases (1) to (6) in order from the left side.
(1) When reacted with shark cartilage-derived unsaturated hexasaccharide peptide fraction (hexa lk re peptide (sh.))
(2) When reacted with a shark cartilage-derived pentasaccharide peptide fraction (penta lk re peptide (sh.))
(3) When reacted with shark cartilage-derived tetrasaccharide peptide fraction (tetra lk re peptide (sh.))
(4) When reacted with an unsaturated hexasaccharide peptide fraction derived from whale cartilage (hexa lk re peptide (wh.))
(5) When reacted with a whale cartilage-derived pentasaccharide peptide fraction (penta lk re peptide (sh.))
(6) When no antigen is added (no Antigen)

  As shown in the figure, the 11 clones of Examples 1 to 11 all reacted with the hexasaccharide peptide fraction, the pentasaccharide peptide fraction, and the tetrasaccharide peptide fraction derived from the shark binding region, as in the case of the original 4E1. However, it did not react at all with the CS-derived hexasaccharide peptide fraction or pentasaccharide peptide fraction of whale cartilage. As for the shark cartilage-derived peptide fraction, the reactivity decreased when it was cut from pentasaccharide to tetrasaccharide, so it can be judged that the cut GalNAc was recognized. The tetrasaccharide peptide fraction also showed significant reactivity, although the reactivity was weaker than the hexasaccharide peptide fraction and the pentasaccharide peptide fraction. From this, it was confirmed that the clones of Examples 1 to 11 specifically recognize not only the GalNAc structure of the shark cartilage CS-derived binding region but also the structure of other parts. Although not necessarily clear, there is a possibility that not only the sugar chain modification structure of the binding region as described above but also the difference in the amino acid sequence of the peptide is recognized. Thus, the monoclonal antibodies of Examples 1 to 11 were antibodies that showed very specific reactivity to the shark cartilage CS-derived binding region. The reactivity of the 11 clones was very similar. In addition, since it did not react with the well which did not add the glycopeptide of an antigen, it was confirmed that there is no non-specific adsorption.

Among the 11 types of clones described above, Example 1 (A4), Example 2 (A8), Example 4 (B6), Example 5 (B11), Example 6 (C6), and Example 8 (D6) 6 types of hybridomas were inoculated intraperitoneally into mice and ascites was collected. The method for collecting ascites is as described above. The reactivity of the obtained ascites was further confirmed by ELISA. The ELISA method was performed in the same manner as in FIG. The results are shown in the bar graph of FIG. In the figure, the vertical axis represents the absorbance (relative value) at a wavelength of 415 nm, that is, an index of the reactivity of the monoclonal antibody. “A4” indicates the measurement result of the monoclonal antibody of Example 1. “A8” indicates the measurement result of the monoclonal antibody of Example 2. “B6” indicates the measurement result of the monoclonal antibody of Example 4. “B11” indicates the measurement result of the monoclonal antibody of Example 5. “C6” indicates the measurement result of the monoclonal antibody of Example 6. “D6” indicates the measurement result of the monoclonal antibody of Example 8. “No primary Ab” indicates the measurement result when the primary antibody (monoclonal antibody) was not added. In the monoclonal antibody and “no primary Ab” of each Example, each bar indicates the absorbance in the following cases (1) to (4) in order from the left side.
(1) When reacted with shark cartilage-derived unsaturated hexasaccharide peptide fraction (shark hexa)
(2) When reacted with shark cartilage-derived tetrasaccharide peptide fraction (shark tetra)
(3) When reacted with an unsaturated hexasaccharide peptide fraction derived from whale cartilage (whale hexa)
(4) When no antigen is added (no GAGs)

  As shown in the figure, the monoclonal antibody obtained from ascites also showed the same reactivity as the monoclonal antibody obtained from the hybridoma culture supernatant. That is, it showed reactivity with tetrasaccharide, pentasaccharide and hexasaccharide peptide fractions derived from shark CS, but did not show reactivity with the glycopeptide fraction derived from CS-derived binding region of whale cartilage. It did not react with wells (no GAGs) to which no glycopeptide of the antigen was added, and nonspecific adsorption was not observed even with blanks (no primary Ab) without primary antibodies. The reactivity of the six types of ascites was very similar.

  Furthermore, using the monoclonal antibodies of Examples 1 to 11, cancer cell staining, tumor tissue staining, and cancer cell metastasis-inhibiting effects could be confirmed for human and mouse cells, respectively.

[Examples 12 to 17]
In this example, a hybridoma was prepared according to a conventional method using spleen cells obtained by immunizing a mouse using chondroitin sulfate E type extracted and purified from squid cartilage containing as much as 60% E disaccharide unit. did. In addition, the hybridoma was injected into the abdominal cavity of the mouse to produce ascites. And the reactivity of the monoclonal antibody which the said hybridoma produces was confirmed.

  Hereinafter, the operation of the present embodiment will be specifically described. Unless otherwise indicated, all chemical substances are reagent grade and can be purchased as commercial products.

(1. Preparation of immunogen for antibody production)
In accordance with the methods of References 8 and 9 below, squid skull cartilage is thoroughly digested with actinase E, a proteolytic enzyme, and then polysaccharide (CS-E, ie, chondroitin sulfate E) is precipitated with ethanol, and ion using Dowex 1 column. The main fraction purified by exchange chromatography and eluted with 3.0M NaCl was used as the immunogen.
(Reference 8) Kawai, Y., Seno, N., Anno, K. (1966) The Journal of Biochemistry, Vol. 60, No. 3, 317-321
(Reference 9) Nandini, CD, Mikami, T., Ohta, M., Itoh, N., Akiyama-Nambu, F., and Sugahara, K. (2004) J. Biol. Chem. 279, 50799-50809

More specifically, the method for preparing the immunogen (squid cartilage-derived CS-E) is as follows. That is, first, other tissues were removed from the squid crab cartilage ( Ommastrephes sloani pacificus ), 1.5 volumes of water was added, and homogenized with a homogenizer (trade name Waring blender). The obtained suspension was heated in a boiling water bath for 1 hour, cooled, 2% by weight NaOH aqueous solution was added, and the mixture was stirred at 4-5 ° C. for 24 hours to extract GAG. The obtained extract was centrifuged, adjusted to pH 7.8 by adding acetic acid, 5 mg of actinase E [EC class 3.4.4] was added per gram of protein, and digested by incubation at 40 ° C. for 24 hours. Thereafter, the half amount of actinase E was added, and the mixture was further digested by further incubation for 24 hours. After digestion, 10% by mass concentration of trichloroacetic acid was added, and after centrifugation and dialysis, the resulting clear solution was concentrated under reduced pressure. The mucopolysaccharide mixture thus obtained was precipitated by adding 3 volumes of ethanol (containing 1% by weight sodium acetate). The precipitated GAG mixture, Dowex 1-Cl ^- were separated by column (Dow Chemical Company trade name). Separation was performed by gradually increasing the NaCl concentration to elute each component in turn. The fraction obtained by elution with 3.0M NaCl was characterized, and heparan sulfate (HS) was removed by the so-called Shively and Conrad method in which a newly prepared aqueous nitrite solution (pH 1.5) was added. Thereafter, 0.5 M Na ₂ CO ₃ aqueous solution was added to neutralize nitrous acid, and CS-E and HS were separated through a Sephadex G-50 column (trade name, 56 × 1 cm). As the eluent, 50 mM pyridine acetate buffer (pH 5.0) was used, and the elution rate was 0.6 ml / min. In this way, the hydrophobic peptide was removed from the CS-E fraction excluding HS through a C18 cartridge (trade name, cartridge containing C18-bound silica gel). The CS-E preparation thus obtained was used as an immunogen.

The CS-E preparation was purified by anion exchange chromatography according to the description in Reference 10 below, and then a biotinylated CS-E fraction was prepared. This biotinylated CS-E fraction was used to monitor the antibody production status.
(Reference 10) Deepa, SS, Umehara, Y., Higashiyama, S., Itoh N., and Sugahara, K. (2002) J. Biol. Chem. 277, 43707-43716

[Preparation of biotinylated CS-E]
The CS-E preparation was dissolved in 100 mM MES (N-morpholinoethanesulfonic acid) buffer (pH 5.5) at a concentration of 2 mg / ml. On the other hand, a DMSO (dimethyl sulfoxide) solution of 50 mM biotin-LC-hydrazide (Pierce) was newly prepared, and the CS-E solution was mixed therewith. At this time, the mass ratio of CS-H or CS-E and biotin-LC-hydrazide was set to 20: 1. To this mixture, 0.5M EDAC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) hydrochloride aqueous solution dissolved in the same buffer (100 mM MES (morpholinoethanesulfonic acid) aqueous solution (pH 5.5)) as above. Was added. At this time, the mass ratio of CS-E and EDAC was set to 8: 1. When this solution was gently stirred at room temperature overnight, a labeling reaction occurred. The reaction mixture was dialyzed against phosphate buffered saline (PBS) at room temperature for 24 hours. The dialysis was performed using a Spectra / Pormolecular porous membrane (trade name of Spectrum Medical Industries, Inc., Laguna Hills, Canada) having a molecular weight cut-off value of 3,500 Da. Hexuronic acid in each preparation was quantified by the usual carbazole method using GlcUA as a standard substance. The binding ratio of biotin to CS-E (mol / mol) is the so-called protocol of Green that utilizes the formation of a complex between HABA dye (2- (4'-hydroxyazobenzene-2-carboxylic acid) and avidin. Specifically, 60 μl of 10 mM HABA aqueous solution was added to 1.94 ml of 100 mM sodium phosphate aqueous solution (containing 150 mM NaCl, pH 7.2) containing 1 mg of avidin. The absorbance of the solution was measured at 500 nm, and 10 μl of biotinylated CS-E aqueous solution (1.7 mg / ml) or 10 μl of CS-H aqueous solution (2.1 mg / ml) was added to 90 μl of the above reagent solution. A decrease in absorbance was observed as a result of substitution of HABA with HABA, and as a result, when the binding ratio (mol / mol) of biotin to CS-E was quantified, about 4.2% of hexuronic acid groups were labeled with biotin. It was.

(2. Mouse immunization and blood collection)
Mice were immunized and blood collected as follows.

(2-1. Preparation)
Mouse (use 4 BALB / c 週 6 weeks old), FCA, FIA, PBS (-), 1ml syringe, 26G x 1/2 needle, ultrasonic homogenizer, mouse retainer, alcohol cotton, nipper, hematocrit capillary A micro syringe and a micro tube were prepared. In addition, necessary reagents and instruments such as the immunogen were prepared. Mice were purchased from CLEA Japan and used one week after acclimatization. Bad mice were removed from the subject.

(2-2. Immunization)
The mice were immunized by the following procedure. That is, first, an immunogen (the squid cartilage-derived CS-E) was dissolved in phosphate buffered saline to obtain a 2000 mg / L solution. An equal volume of adjuvant was added to this immunogen solution, placed in a 1 ml syringe, and emulsified with an ultrasonic homogenizer to give an injection for immunization. The mixing and emulsification were performed while cooling in order not to impair the activity of the immunogen. As the adjuvant, FCA (Freund's complete adjuvant) was used for the first immunization, and FIA (Freund's incomplete adjuvant) was used for the second and subsequent immunizations. Next, a 26G × 1/2 injection needle was attached to the 1 ml syringe, and the injection solution for immunization was dispersedly administered subcutaneously to the back of the mouse (after disinfection with alcohol cotton). The standard dose of the immunogen was 200 μg / animal / dose, and the liquid volume was 200 μl / animal. Immunization was performed 4 times, 0w, 2w, 4w, and 6w. “0w” refers to the time of the first immunization, and 2w, 4w, and 6w indicate that the immunization was performed 2 weeks, 4 weeks, and 6 weeks after the initial immunization, respectively.

(2-3. Blood collection)
The mouse that had been immunized 4 times was placed in a restraint device, and the tail was lightly rubbed with alcohol cotton to make the blood vessels angry. Next, a 26 G × 1/2 injection needle with the needle tip removed with a nipper was inserted into the tail vein of the mouse. The blood that emerged was collected with a hematocrit capillary (2 / animal). The blood was centrifuged at 8,000 rpm for 20 minutes at room temperature after 1 hour incubation (about 37 ° C.). After centrifugation, serum was collected with a microsyringe and transferred into a microtube. It was stored in a −20 ° C. freezer until use. Blood collection was performed at 0 w, 5 w, and 7 w, respectively. As described above, “0w” indicates the time of the first immunization administration, and 5w and 7w indicate 5 weeks and 7 weeks after the initial immunization administration, respectively.

(2-4. Antibody titer measurement by ELISA using streptavidin plate)
The antibody titer of each serum collected from the mouse was measured according to a conventional method by ELISA using a streptavidin plate. That is, first, the biotinylated CS-E fraction was immobilized on the streptavidin plate (96 well ELISA plate) as described above. The wells were blocked with 1% bovine serum albumin (BSA), incubated with 5,000-fold diluted serum (including primary antibody) with 0.1% BSA / PBS for 1 hour at 37 ° C, followed by Alkaline phosphatase-conjugated goat anti-mouse Ig (G + M) was treated. Enzyme activity was detected using p-nitrophenyl phosphate and absorbance was measured at 415 nm.

(3. Production of hybridoma)
One mouse having a particularly high antibody titer as determined by the ELISA method was selected, and the spleen cells were fused with myeloma cells to produce a hybridoma. In addition, 3 days before mouse sacrifice, the above-mentioned injection solution for immunization was used, and 100 μg / mouse · times of immunogen was injected into the mouse for the final boost.

(3-1. Preparation)
Mouse (Three days after the final boost), Beaker (300ml, 1 piece), Sterile petri dish (1 piece), Tweezers (Large / Small) , Stainless bat (2), sterile gauze, stainless mesh (several), spoon (1), vortex mixer, sterile pipette (1, 2, 5, 10 ml), mouse myeloma (SP2), PBS (-) 10% FBS RPMI1640 medium (with HAT), 10% FBS RPMI1640 medium (without HAT), RPMI1640 medium (without serum), sterile 50 ml centrifuge tube, PEG, sterile pipette tip (~ 200 μl), 96-well culture plate, octapipette, A cryotube (2 ml), a suction bottle set, a hemocytometer, and other necessary items were prepared.

(3-2. Cell fusion and hybridoma culture)
Cell fusion and hybridoma culture were performed as follows. That is, first, a mouse 3 days after the final boost was dislocated from the cervical spine, sacrificed, and then disinfected with ethanol. Next, the whole body of the mouse was immersed in ethanol for disinfection, the back muscle was cut with scissors, and the spleen was removed. The extracted spleen was placed on a stainless steel mesh in a sterilized petri dish, an appropriate amount of PBS was added, several holes were opened in the spleen using barbed tweezers, and the mixture was crushed using a spoon. The stainless mesh and sterilized petri dish were washed with PBS, and PBS containing splenocytes was collected and transferred to a 50 ml centrifuge tube. The centrifuge tube from which PBS was collected was centrifuged at 1,300 rpm for 5 minutes at room temperature, and then the supernatant was discarded, fresh PBS was added, pipetted, and centrifuged again under the same conditions and washed.

  Meanwhile, the prepared spleen cells and myeloma cells were mixed at a ratio of 5: 1, PBS was added, and the mixture was centrifuged at 1,300 rpm for 5 minutes at room temperature. After discarding the supernatant, cell fusion was performed by the 50% PEG method to obtain a hybridoma. To this hybridoma, 50 ml of 10% FBS RPMI1640 medium (with HAT) was added and suspended. Using 2 ml of sterile pipette, 100 μl of the hybridoma suspension was seeded in each well of five 96-well culture plates prepared in advance. Five days later, 100 μl of 10% FBS RPMI1640 medium (without HAT) was added to each well using an octapipette. Thereafter, the observation of the size of the colony and the color of the medium was continued, and after 7 days after the addition of the medium, it was determined that the growth of the colony reached a sufficient level for screening.

(4. Screening and cloning)
Hybridoma screening and cloning were performed as follows.

(4-1. Preparation)
ELISA equipment, equipment, reagents, 96-well culture plate (with cells), octapipette, sterile pipette tip (up to 200 μl), pipette tip disposal container and sterile pipette (1, 2, 5, 10 ml), 10% FBS RPMI1640 medium (With HAT), 10% FBS RPMI1640 medium (without HAT), RPMI1640 medium (without serum), sterile 50 ml centrifuge tube, 96-well culture plate, 24-well culture plate, cell banker, cryotube (2 ml), suction bottle set, hemocytometer 25 cm ² culture flask, PBS (−), and other necessary items were prepared. As the “96-well culture plate (with cells)”, the 96-well culture plate containing the hybridoma colonies cultured in the above-mentioned “3. Production of hybridoma” was used as it was.

(4-2. Screening)
Hybridomas were screened by the same ELISA method as described above in “2-4. Antibody titer measurement using streptavidin plate”. That is, first, as described above, the biotinylated CS-E fraction (antigen) was immobilized (immobilized) on a streptavidin plate (96-well ELISA plate). The wells were blocked with 1% bovine serum albumin (BSA). On the other hand, using an octapipette, the culture supernatant containing the antibody was extracted from the “96 well culture plate (with cells)” containing the hybridoma. 50 μl of this culture supernatant was dispensed into each well of the streptavidin plate (96 well ELISA plate) in which the antigen was immobilized and blocked with BSA. This was incubated at 37 ° C. for 1 hour and then treated with alkaline phosphatase-conjugated goat anti-mouse Ig (G + M). Enzyme activity was detected using p-nitrophenyl phosphate and absorbance was measured at 415 nm.

(4-3. Cloning)
Hybridomas in wells judged positive in the screening after cell fusion were collected in a 24-well culture plate in which 1 ml of 10% FBS RPMI1640 medium (with HAT) was dispensed per well. After culturing for several days, 6 types of hybridomas were selected and 1st cloning was performed. That is, first, the hybridoma was seeded on a new 96-well culture plate prepared in advance by limiting dilution method so that there were 4 per well. As a solvent for limiting dilution, 10% FBS RPMI1640 medium (with HAT) was used, and 100 μl per well was seeded. In addition, 0.5 96-well culture plates were used for one type of hybridoma (one for two types). Five days after seeding, an octapipette was used, and 100% of 10% FBS RPMI1640 medium (without HAT) was dispensed per well of each plate. After culturing for several days, colonies were confirmed with the naked eye, and screening was performed when the color of the medium became light yellow. The screening method is the same as described above.

  Meanwhile, 1 ml of 10% FBS RPMI1640 medium (with HAT) was dispensed into a new 24-well culture plate prepared in advance. Six kinds of hybridomas in positive wells obtained by the screening after the 1st cloning were added thereto. After culturing for several days, when the number of cells increased to some extent, 2nd cloning was performed. That is, first, the hybridoma was seeded on a new 96-well culture plate prepared in advance by a limiting dilution method so that there was one per well. As a solvent for limiting dilution, 10% FBS RPMI1640 medium (with HAT) was used, and 100 μl per well was seeded. In addition, 0.5 96-well culture plates were used for one type of hybridoma (one for two types). Further, using an octapipette, 100 μl of 10% FBS RPMI1640 medium (without HAT) was dispensed per well of each plate. After culturing for about 10 days, colonies were confirmed with the naked eye, and screening was performed when the color of the medium became light yellow. The screening method is the same as described above.

Six types of positive well hybridomas obtained by the screening after the 2nd cloning were collected in 24-well culture plates in which 1 ml of 10% FBS RPMI1640 medium (with HAT) was dispensed per well. After culturing for several days and a certain amount of cells increased, each cell was transferred to two 25 cm ² culture flasks. Each culture flask was previously filled with 5 ml of 10% FBS RPMI1640 medium (with HAT). In each cell, one of the ^two 25 cm ² culture flasks was conditioned so that the hybridoma could be cultured in 10% FBS RPMI1640 medium (without HAT). The remaining one was frozen stock. Acclimatized hybridomas can be cultured in 10% FBS RPMI1640 medium, and when they approached confluence, they were frozen and stocked in three cryotubes containing a cell cryopreservation solution (trade name Cell Banker). Three cryotubes were used for each hybridoma. At this point, clones were established (6 clones). These clones were named 1C7 (C3), 1C7 (H10), 1C7 (B7), 2A2 (A8), 1C7 (D4), and 2A2 (F9), respectively. Each clone was cultured to the number of cells (5 × 10 ⁶ cells / mouse) that could be administered to mice. The collected hybridomas were suspended in PBS (−) so that they could be administered to mice.

The six clones (hybridomas) and the monoclonal antibodies produced thereby are referred to as Examples 12 to 17, respectively. The hybridomas produced according to Examples 12 to 17 are deposited at the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation. The deposit date is February 22, 2008. Table 1 below shows the relationship between the example number, the hybridoma name, and the accession number.

[Table 1]
Example number Hybridoma name Accession number
Example 12 1C7 (C3) NITE P-503
Example 13 1C7 (H10) NITE P-504
Example 14 1C7 (B7) NITE P-505
Example 15 2A2 (A8) NITE P-506
Example 16 1C7 (D4) NITE P-507
Example 17 2A2 (F9) NITE P-508

(5. Ascites collection)
Ascites was collected as follows using the 6 clones.

(5-1. Preparation)
Mouse (BALB / c ♂ 6 weeks old), pristane, 2.5ml syringe, 5ml syringe, 25G x 5/8 needle, 18G x 11/2 needle, 15ml centrifuge tube (or 50ml centrifuge tube), EDTA 2K An aqueous solution (80 mg / ml), alcohol cotton and other necessary items were prepared.

(5-2. Ascites collection)
Mice bred for 1 week were administered 0.5 ml of pristane per mouse in a 2.5 ml syringe with a 25G × 5/8 needle. One week or more after the administration of pristane, PBS (−) in which the hybridoma of any of Examples 18 to 23 was suspended was added to each mouse in a 2.5 ml syringe with a 25 G × 5/8 injection needle. 1 ml was administered. The number of cells administered per mouse was about 5 × 10 ⁶ cells. After cell administration, the progress was observed, and ascites was collected about 10 days later. Specifically, an 18G × 11/2 injection needle was inserted into the abdomen of the mouse, and ascites was collected by dropping into a 15 ml centrifuge tube (or 50 ml centrifuge tube) containing EDTA 2K solution (2 mg per 1 ml of ascites). If possible, ascitic fluid was collected again (2-3 times) after several days. The collected ascites was centrifuged at 3,500 rpm for 10 minutes at 4 ° C. and stored frozen in a centrifuge tube. The number of days until ascites collection may vary slightly depending on the nature of the hybridoma, the nature of the mouse, the breeding conditions, and the like.

(6. Reactivity of monoclonal antibodies)
Using the ascites containing the monoclonal antibody, the reactivity of the monoclonal antibodies of Examples 12 to 17 with CS-E and CS-H (DS-E) was confirmed. In addition, various commercially available chondroitin sulfates (CS-A from whale cartilage, CS-C and CS-D from shark cartilage), dermatan sulfate from pig skin (also called CS-B), and heparin from pig small intestine The reactivity with was also confirmed.

As the antigen CS-E, biotinylated CS-E prepared as described above was used. On the other hand, CS-H is disclosed in Reference Document 9 (Nandini, CD, Mikami, T., Ohta, M., Itoh, N., Akiyama-Nambu, F., and Sugahara, K. (2004) J. Biol. Chem. 279, 50799-50809) and isolated and purified from Japanese eel ( Eptatretus burgeri ) notochord. That is, first, CS-E was extracted from the notochord with acetone, and then thoroughly digested with (actinase E). The protein was removed by adding 20% aqueous trichloroacetic acid to precipitate. Thereafter, 2 volumes of ethanol (containing 2.5 mass% calcium acetate and 0.25 M acetic acid) were added to the GAG aqueous solution to precipitate GAG. The precipitated GAG mixture, Dowex 1-Cl ^- were separated by column (Dow Chemical Company trade name). Separation was performed by gradually increasing the NaCl concentration to elute each component in turn. The fraction obtained by elution with 2.0M NaCl was characterized, and heparan sulfate (HS) was removed by the so-called Shively and Conrad method in which a newly prepared aqueous nitrite solution (pH 1.5) was added. Thereafter, 0.5 M Na ₂ CO ₃ aqueous solution was added to neutralize nitrous acid, and CS-H and HS were separated through a Sephadex G-50 column (trade name, 56 × 1 cm). As the eluent, 50 mM pyridine acetate buffer (pH 5.0) was used, and the elution rate was 0.6 ml / min. Further, the hydrophobic peptide was removed through a C18 cartridge (trade name, cartridge containing C18-bonded silica gel) to obtain the intended CS-H preparation. In addition, the CS-H preparation was biotinylated by the same method as CS-E and used as an antigen for confirming the reactivity of the monoclonal antibody. Furthermore, CS-A, CS-B, CS-C, and CS-D were used as commercially available products in the same manner. Commercially available heparin purified from porcine small intestine was also used after biotinylation.

  The reactivity of the monoclonal antibodies of Examples 12 to 17 with the respective GAGs (CS-A, CS-B, CS-C, CS-D, CS-E, CS-H and heparin) was determined by the same ELISA as described above. Confirmed by law. That is, first, the various biotinylated GAG samples were immobilized on a plastic plate coated with streptavidin. Thereto was added a diluted solution obtained by diluting ascites (antiserum) containing any of the monoclonal antibodies of Examples 12 to 17 with 0.1% BSA / PBS for reaction. A secondary antibody was added thereto for color development, the bound primary antibody was detected, and the antibody titer was measured. More specific operations of the ELISA method are as described above. The reaction time (incubation time) was 60 minutes.

  The bar graphs of FIGS. 3A to 3F show the reactivity of the monoclonal antibodies of Examples 1 to 6 confirmed by the ELISA method with the respective GAGs. FIG. 3A is a graph showing the reactivity of the monoclonal antibody of Example 12 (1C7 (C3)). FIG. 3B is a graph showing the reactivity of the monoclonal antibody of Example 13 (1C7 (H10)). FIG. 3C is a graph showing the reactivity of the monoclonal antibody of Example 14 (1C7 (B7)). FIG. 3D is a graph showing the reactivity of the monoclonal antibody of Example 15 (2A2 (A8)). FIG. 3E is a graph showing the reactivity of the monoclonal antibody of Example 16 (1C7 (D4)). FIG. 3F is a graph showing the reactivity of the monoclonal antibody of Example 17 (2A2 (F9)). In each figure, the vertical axis represents the absorbance (relative value) at a wavelength of 415 nm, that is, an index of the reactivity (antibody titer) of the monoclonal antibody. Each bar in each figure shows the absorbance when each GAG is reacted with a monoclonal antibody. The rightmost “Background” indicates the absorbance when no GAG is added.

  As shown, the three types of hybridomas (1C7 (C3), 1C7 (H10), 1C7 (B7)) of Examples 12 to 14 are CS-E (containing a large amount of E disaccharide units) and CS-H (iE). Reacted with both). It also reacted with heparin with a very high degree of sulfation derived from porcine small intestine. On the other hand, the three types of hybridomas (2A2 (A8), 1C7 (D4), 2A2 (F9)) of Examples 15 to 17 have particularly high reactivity with CS-H, and are reactive with CS-E and heparin. It was weak. In addition, any of the monoclonal antibodies of Examples 12 to 17 hardly reacted with CS-A, CS-B, CS-C, and CS-D, and were not coated with a sugar chain (Background) There was no non-specific binding to.

  The E disaccharide unit is a structure that is abundant in chondroitin sulfate E (CS-E), and is represented by [GlcA-GalNAc (4-O-sulfate, 6-O-sulfate)] with GlcA as a structural unit. Is done. The iE disaccharide unit is a structure that is abundant in chondroitin sulfate H (CS-H), with IdoA as a constituent unit, and represented by [IdoA-GalNAc (4-O-sulfate, 6-O-sulfate)] It has the same sulfation pattern as the E disaccharide unit (Non-patent Document 3, etc.). Both the E disaccharide unit and the iE disaccharide unit are structures important for biological activity. In particular, the E disaccharide unit has been confirmed to be related to tumor metastasis and the like from the test results of metastasis of mouse lung cancer cells to the lung. For this reason, research and development relating to antibodies that recognize E disaccharide units or iE disaccharide units are also underway (JP 2007-256217 A, etc.).

  As described above, the monoclonal antibodies of Examples 12 to 17 were prepared using CS-E as an immunogen, but also showed reactivity to CS-H and heparin. On the other hand, there was no reactivity to commercially available CS-A, CS-B, CS-C, and CS-D. This indicates that the monoclonal antibodies of Examples 12 to 17 recognize the tetrasaccharide structure (GlcAβ1-3Galβ1-3Galβ1-4Xyl) of the binding region, which is a structure common to CS-E, CS-H and heparin. To suggest. Since CS-E, CS-H and heparin used in Fig. 3 were digested thoroughly with actinase E without acid hydrolysis treatment or alkaline hydrolysis treatment, the binding region was different from commercially available CS-A and CS-C. It is because it is thought that it remains. On the other hand, commercially available CS-A, CS-B, CS-C, and CS-D were not reacted with the monoclonal antibodies of Examples 12 to 17 because the binding region was degraded by alkaline hydrolysis treatment. Conceivable. However, these considerations are examples of a presumable reaction mechanism, and do not limit the present invention.

  Furthermore, the monoclonal antibody of Examples 12 to 17 and the above-described method were used in the same manner as in FIG. 3 except that the dilution rate of ascites (antiserum) was variously changed to 10 times, 50 times, 100 times, 400 times and 1600 times. Reactivity with various GAGs was confirmed. The line graphs of FIGS. 4A to 4F show the reactivity with CS-E and CS-H. FIG. 4A is a graph showing the reactivity of the monoclonal antibody of Example 12 (1C7 (C3)). FIG. 4B is a graph showing the reactivity of the monoclonal antibody of Example 13 (1C7 (H10)). FIG. 4C is a graph showing the reactivity of the monoclonal antibody of Example 14 (1C7 (B7)). FIG. 4D is a graph showing the reactivity of the monoclonal antibody of Example 15 (2A2 (A8)). FIG. 4E is a graph showing the reactivity of the monoclonal antibody of Example 16 (1C7 (D4)). FIG. 4F is a graph showing the reactivity of the monoclonal antibody of Example 17 (2A2 (F9)). In each figure, the vertical axis represents the absorbance (relative value) at a wavelength of 415 nm, that is, an index of the reactivity (antibody titer) of the monoclonal antibody. The numerical value (Dilution) on the horizontal axis indicates the dilution rate of ascites (antiserum). The polygonal lines “CS-E” and “CS-H” in each figure indicate the absorbance when CS-E and CS-H are reacted with a monoclonal antibody, respectively. “Background” indicates the absorbance when no GAG was added. As shown, each of the monoclonal antibodies of Examples 12 to 14 showed high reactivity against both CS-E and CS-H. On the other hand, each monoclonal antibody of Examples 15 to 17 reacted specifically with CS-H, and the reactivity with CS-E was very weak. These agreed with the results of FIG. In addition, each monoclonal antibody of Examples 12 to 17 maintains the aforementioned reactivity (antibody titer) and reaction specificity for CS-E and CS-H even when the dilution rate of ascites (antiserum) is increased. It was.

  Furthermore, the same CS-E, CS-H and heparin as used in FIG. 3 were thoroughly digested with chondroitinase ABC to obtain a hexasaccharide peptide, and the reactivity was confirmed by the ELISA method similar to FIG. Sex did not disappear. This hexasaccharide peptide is composed of the tetrasaccharide structure (GlcAβ1-3Galβ1-3Galβ1-4Xyl), a disaccharide unit unique to each GAG sugar chain (for example, E disaccharide unit in CS-E, iE disaccharide in CS-H) Unit) has one added hexasaccharide unit, and a peptide is further bound to the hexasaccharide unit. Furthermore, the hexasaccharide peptide was digested with a mixed solution of chondroitinase AC-I and AC-II to obtain a tetrasaccharide peptide in which a tetrasaccharide peptide was bound to the tetrasaccharide structure. The reactivity to the monoclonal antibody did not disappear. The specific operation of enzyme digestion is the same as described above.

  FIG. 5 shows the results of the reactivity confirmation test using CS-E. In the figure, the vertical axis represents the absorbance (relative value) at a wavelength of 415 nm, that is, an index of the reactivity (antibody titer) of the monoclonal antibody. Each bar shows the absorbance when CS-E or its digest is reacted with a monoclonal antibody. In FIG. 5, CS-E was derived from the squid cartilage and commercially available. In the figure, each bar shows the absorbance when each CS-E or its digest (biotinylated) was reacted with the monoclonal antibody of the example. That is, “CS-E (S)” indicates the absorbance of CS-E prepared from squid cartilage. “CS-E (S) + ABC” indicates the absorbance of the CS-E prepared from squid cartilage and thoroughly digested with chondroitinase ABC. “CS-E (S) + ACI + AC2” is the above-mentioned CS-E prepared from squid cartilage, thoroughly digested with chondroitinase ABC, and then thoroughly digested with a mixture of chondroitinase AC-I and AC-II The absorbance is shown. “CS-E (C)” indicates the absorbance of commercially available CS-E. “CS-E (C) + ABC” indicates the absorbance of the commercially available CS-E thoroughly digested with chondroitinase ABC. “CS-E (C) + ACI + AC2” is the absorbance of the commercially available CS-E after thorough digestion with chondroitinase ABC and further thorough digestion with a mixture of chondroitinase AC-I and AC-II. Indicates. The “Background” on the rightmost side indicates the absorbance when no antigen (GAG) is added. The three bars in each antigen are, in order from the left, the absorbance when reacted with the monoclonal antibody of Example 13 (1C7 (H10)) and when reacted with the monoclonal antibody of Example 17 (2A2 (F9)). And the absorbance when no primary antibody was added (Blank). As shown in the figure, even if squid cartilage-derived (self-prepared) CS-E and commercially available CS-E were thoroughly digested with chondroitinase ABC, Example 13 (1C7 (H10)) and Example 17 (2A2 ( The reactivity with the monoclonal antibody of F9) was almost unchanged. Further, when the digestion was thoroughly performed with a mixed solution of chondroitinase AC-I and AC-II, the reactivity was slightly lowered, but the reactivity still remained. In addition, non-specific binding to well (Background) not coated with a sugar chain was not observed. Similarly, heparin was confirmed to remain reactive even after enzymatic digestion. For CS-H, either thorough digestion with chondroitinase ABC, further thorough digestion with a mixture of chondroitinase AC-I and AC-II, or further thorough digestion with chondroitinase B However, the reactivity was lower than that without enzyme digestion, but the reactivity remained.

  As shown in FIG. 5, since the reactivity was not lost even after enzymatic digestion of the GAG sugar chain, the monoclonal antibodies of Examples 12 to 17 had the tetrasaccharide structure (GlcAβ1-3Galβ1-3Galβ1-4Xyl). It was suggested that they were aware. However, as described above, the hexasaccharide peptide includes not only the tetrasaccharide structure but also a disaccharide unit unique to each GAG sugar chain. When the disaccharide unit was thoroughly digested with a mixed solution of chondroitinase AC-I and AC-II, the reactivity slightly decreased. From this, it is considered that the monoclonal antibodies of Examples 12 to 17 recognize not only the tetrasaccharide structure but also the disaccharide unit. This is also suggested by the fact that in FIG. 3, the reactivity to CS-E, CS-H and heparin is different. However, these considerations are examples of a presumable reaction mechanism, and do not limit the present invention.

  Furthermore, inhibition of cancer cell metastasis was performed using the monoclonal antibodies of Examples 13 to 27, and a strong inhibitory effect could be confirmed.

  As described above, according to the present invention, it is possible to provide a monoclonal antibody that specifically reacts with a binding region between a sugar chain part and a core protein part of a glycoprotein. That is, according to the monoclonal antibody of the present invention, the binding region of glycoprotein can be specifically recognized. For this reason, the monoclonal antibody of the present invention can be used for various uses such as various products, pharmaceutical compositions, and pharmaceuticals as described above in both basic research and application. For example, in the above-described biosynthetic mechanism research of glycoproteins, there is a possibility that it can be used as a unique reagent for basic research. It is also considered applicable to serodiagnosis of diseases using antibodies, for example, diagnosis of various diseases including genetic diseases, inflammation, and autoimmune diseases. Thus, the present invention has great industrial applicability in various fields such as basic medicine, applied medicine, and drug discovery. Furthermore, the application of the present invention is not limited to the above, and various applications are possible, and any application may be used.

FIG. 1 is a graph showing the reactivity of the shark cartilage-derived chondroitin sulfate binding region and the whale cartilage-derived chondroitin sulfate binding region in the monoclonal antibody of the example of the present invention. FIG. 2 is a graph showing the reactivity of the monoclonal antibody collected from mouse ascites with the binding region of shark cartilage-derived chondroitin sulfate and the binding region of whale cartilage-derived chondroitin sulfate in the example of FIG. FIG. 3A is a graph showing the reactivity of the monoclonal antibody of Example of the present invention with various glycosaminoglycans (GAG). FIG. 3B is a graph showing the reactivity of a monoclonal antibody of another example with various glycosaminoglycans (GAG). FIG. 3C is a graph showing the reactivity of a monoclonal antibody of another example with various glycosaminoglycans (GAG). FIG. 3D is a graph showing the reactivity of a monoclonal antibody of another example with various glycosaminoglycans (GAG). FIG. 3E is a graph showing the reactivity of a monoclonal antibody of another example with various glycosaminoglycans (GAG). FIG. 3F is a graph showing the reactivity of the monoclonal antibody of another example with various glycosaminoglycans (GAG). FIG. 4A is a graph showing the reactivity of the monoclonal antibody of Example of the present invention with chondroitin sulfate E (CS-E) and chondroitin sulfate H (CS-H). FIG. 4B is a graph showing the reactivity of the monoclonal antibody of another example with chondroitin sulfate E (CS-E) and chondroitin sulfate H (CS-H). FIG. 4C is a graph showing the reactivity of the monoclonal antibody of another example with chondroitin sulfate E (CS-E) and chondroitin sulfate H (CS-H). FIG. 4D is a graph showing the reactivity of the monoclonal antibody of another example with chondroitin sulfate E (CS-E) and chondroitin sulfate H (CS-H). FIG. 4E is a graph showing the reactivity of the monoclonal antibody of another example with chondroitin sulfate E (CS-E) and chondroitin sulfate H (CS-H). FIG. 4F is a graph showing the reactivity of the monoclonal antibody of another example with chondroitin sulfate E (CS-E) and chondroitin sulfate H (CS-H). FIG. 5 is a graph showing reactivity in the reaction of the monoclonal antibody of Example with chondroitin sulfate E (CS-E) when chondroitin sulfate E (CS-E) is digested with chondroitinase and when it is not digested. It is.

Claims (10)

  A monoclonal antibody that reacts specifically with the binding region between the sugar chain portion of the glycoprotein and the core protein portion.   The monoclonal antibody according to claim 1, wherein the glycoprotein is a proteoglycan.   The monoclonal antibody according to claim 1 or 2, which does not react with a sugar chain part and a core protein part of the glycoprotein.   The glycoprotein is a proteoglycan having a sugar chain portion formed from a chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, keratan sulfate, a polysaccharide chain or an oligosaccharide chain having a similar structure thereto. The monoclonal antibody as described in any one of.   5. The product according to claim 1, which is produced by a hybridoma whose accession number is NITE P-503, NITE P-504, NITE P-505, NITE P-506, NITE P-507, or NITE P-508. Monoclonal antibodies.   A hybridoma producing the monoclonal antibody according to any one of claims 1 to 5.   The hybridoma according to claim 6, wherein the accession number is NITE P-503, NITE P-504, NITE P-505, NITE P-506, NITE P-507, or NITE P-508.   A method for producing an immunogen using a glycoprotein as a raw material, wherein the glycoprotein is digested with a protease and not subjected to an alkaline hydrolysis treatment.   The production method according to claim 8, wherein the glycoprotein is a proteoglycan.   10. The proteoglycan having a sugar chain portion formed from a chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, keratan sulfate, a polysaccharide chain or an oligosaccharide chain having a similar structure thereto. The manufacturing method as described.

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