KR20050076377A - Monoclonal antibody specific to human macrophage inhibitory cytokine-1, hybridoma producing the monoclonal antibody and diagnostic kit comprising the monoclonal antibody - Google Patents

Abstract

The present invention provides a macrophage inhibitory cytokine-1 (MIC-1) specific monoclonal antibody, a hybridoma cell line producing the same, a method for detecting a gastric cancer disease marker using the same, and gastric cancer disease comprising the same It is about a diagnostic kit.

Description

Monoclonal antibody specific to human macrophage inhibitory cytokine-1, hybridoma producing the monoclonal antibody and diagnostic kit comprising the monoclonal antibody}

The present invention relates to a monoclonal antibody specific for MIC-1 (Macrophage inhibitory cytokine-1). More specifically, monoclonal antibodies against MIC-1, which are specifically overexpressed in gastric cancer cell lines and found to be involved in cancer cell metastasis, hybridoma cell lines producing MIC-1 monoclonal antibodies, MIC using the monoclonal antibodies The present invention relates to a method for detecting -1 and a diagnostic kit for gastric cancer disease.

Macrophage inhibitory cytokine-1 (MIC-1) was first identified in 1997 as a gene involved in the activation of macrophages by the Breit group in Australia, and was the first branched group member of the TGF-β superfamily. known as placental transforming growth factor-β or prostate-derived factor (Proc Natl Acad Sci US A. 1997 Oct 14; 94 (21): 11514-11519). Like the other TGF-β family, it consists of a total of 308 amino acids, and includes a secretion induction sequence of 29 amino acid residues and a propeptide of 167 amino acid residues. The mature protein is secreted in the form of a 112 amino acid homodimer. Unlike all TGF-β families, the MIC-1 protein is correctly folded into an activated protein with propeptide removed by cleavage of the RXXR moiety. It is characterized by secretion. The secreted protein inhibits the synthesis of TNF-α secreted by macrophages by stimulation of LPS, induces the formation of cartilage and the formation of endochonadal bone in the early stages, and inhibits the proliferation of hematopoietic stem cells. It has been known.

Tissue Northern blots show that most of the MIC-1 transcripts are present in the placenta and MIC-1 is not expressed in resting macrophages, but phorbol myristate acetate, interleukin 1 It is known that expression is induced by activating factors such as tumor necrosis factor alpha.

Recently, MIC-1 has been shown to be specifically overexpressed in gastric cancer cells and involved in cancer cell metastasis (Cancer Res. 2003 Aug 1; 63 (15): 4648-4655) and has high affinity for MIC-1 in gastric cancer tissues. If a specific monoclonal antibody can be obtained, it can be helpful for MIC function research and gastric cancer diagnosis in gastric cancer disease.

Antibodies capable of detecting the MIC-1 protein include antibodies produced by the Breit group that originally identified the gene (polyclonal antibody: 233B3, monoclonal antibody: 13C4H3-J Clin Endocrinol & Metab 85: 4781-4788, 2000), and Although there is an antibody described in US Patent No. 6,465,181, considering the importance of this protein, it is required to develop a highly reliable monoclonal antibody with improved specificity and affinity for MIC-1 protein for cancer cell therapy research.

Under these backgrounds, the present inventors have splenocytes obtained after immunizing TRX-attached purified MIC-1 protein to mice in order to make an antibody having excellent binding and affinity for MIC-1 specifically overexpressing gastric cancer cells. And fusion cell lines of myeloma cells were prepared and succeeded in obtaining stable cell lines secreting antibodies with superior titers. As a result of analyzing each of these characteristics, the obtained antibodies were detected by Western blotting and immunohistochemistry to detect MIC-1 protein and recognize MIC-1 in gastric cancer tissues with high affinity and high specificity. The present invention was completed by confirming the applicability of these antibodies in the MIC-1 cell function analysis study and diagnosis of gastric cancer.

The gist of the invention

The present invention relates to monoclonal antibodies having specificity for human MIC-1 (Macrophage Inhibitory Cytokine-1) produced by hybridoma cells of Accession No. KCTC 10565BP or KCTC 10580BP.

The present invention also relates to hybridoma cells of Accession No. KCTC 10565BP or KCTC 10580BP.

The present invention also relates to a composition for detecting gastric cancer disease markers comprising the monoclonal antibody described above.

The present invention also relates to a gastric cancer disease diagnostic kit comprising the monoclonal antibody described above.

The present invention also relates to a method for detecting gastric cancer disease markers by contacting a monoclonal antibody described above with a biological sample to detect antigen-antibody complex formation.

In one embodiment, the present invention provides a monoclonal antibody having specificity to human Macrophage Inhibitory Cytokine-1 (MIC-1) and a hybridoma secreting the same.

As described above, although the gene of the MIC-1 protein was identified before the present invention, and also found that the MIC-1 protein is used as a marker of prostate cancer, breast cancer, colon cancer, rectal cancer and bladder cancer (WO / 0322863, US Patent Appln.No. 20030211531, WO / 181928), and no monoclonal antibodies have been prepared which highly recognize them with high affinity.

To prepare monoclonal antibodies that recognize human MIC-1 proteins with high affinity and specificity for use in diagnosing gastric cancer, the inventors used a SNU-638 cDNA library, a gastric cancer cell line formed by secondary metastasis, as a template. The reaction (PCR) amplified the complete recognition site of the human MIC-1 gene. TRX and His were attached to the N-terminus of amplified human MIC-1 and cloned into pET32a. The produced pET32a-MIC-1 vector was transformed into DE3 strain to immunize mice using MIC-1 obtained from a strain overexpressing recombinant MIC-1, and the splenocytes and myeloma cells extracted therefrom were fused. Hybridoma cell lines that specifically reacted only with the human MIC-1 antigen were selected from the cutting boards. The obtained hybridomas MIC-1-3-14 was December 11, 2003, and MIC-1-26-20-9 was January 12, 2004. KCTC (Korean Eur-dong, Yuseong-gu, Daejeon City) Collection for Type Cultures (KCTC) were deposited under accession numbers KCTC 10565BP and KCTC 10580BP, respectively. Two clones were finally obtained and subclass type identified as clone MIC-1-26-20-9 as IgG1 and another clone MIC-1-3-14 as IgG2a. Antigen reactivity of monoclonal antibodies that specifically respond to human MIC-1 was confirmed using immunohistochemistry and western blotting methods to confirm high affinity specific reactivity with MIC-1 antigen. As can be seen from the results of FIG. 7 showing immunohistochemical results using the monoclonal antibody of the present invention, MIC-1 protein was specifically detected only in advanced cancer and metastatic cancer tissue. In particular, the results of Figure 3 showing the results of Western blotting performed using the digested metastatic cancer cell line, the fusion protein comprising the entire sequence (amino acids 1 to 308) including the secretion induction sequence and propeptide sequence of MIC-1 It shows that the monoclonal antibody prepared for specifically recognizes the MIC-1 protein of gastric cancer cells. As evidenced by these results, the monoclonal antibody of the present invention specifically binds to gastric cancer cells, and thus can be used to diagnose gastric cancer.

On the other hand, US Patent No. discloses that MIC-1 protein can be used as a diagnostic marker of prostate cancer. For 6,465,181, a monoclonal antibody is described using a polypeptide fragment of 20 to 40 amino acids inferred from the sequence of MIC-1 protein as an antigen for use in the diagnosis of prostate cancer or as an inhibitor for the treatment of prostate cancer. However, the monoclonal antibody disclosed in this patent document is clearly different from the monoclonal antibody of the present invention prepared using the entire MIC-1 sequence different from the amino acid sequence of the above-mentioned fragment.

Thus, in a preferred embodiment, the present invention provides monoclonal antibodies having specificity for human MIC-1 produced by hybridoma cells of Accession No. KCTC 10565BP or KCTC 10580BP.

Monoclonal antibodies can be made by fusion methods well known in the art (Kohler et al. European Journal of Immunology 6; 511-519). Generally, hybridoma cells that secrete monoclonal antibodies are made by fusing cancer cell lines with immune cells from immunologically suitable host animals such as mice injected with antigenic proteins. These two populations of cell fusion are fused using methods known in the art, such as polyethylene glycol, and the antibody producing cells are propagated by standard culture methods. After subcloning by limited dilution to obtain a uniform cell population, hybridoma cells capable of producing an antibody specific for the antigen are cultured in large quantities in vitro or in vivo.

Myeloma cells used for cell fusion include p3 / x63-Ag8, p3-U1, NS-1, MPC-11, SP-2 / 0, F0, P3x63 Ag8, V653, S194, and R210 derived from rats. Various cell lines can be used. Specific Examples of the Invention The cell line used is myeloma cell NS-1.

The monoclonal antibodies produced by the hybridoma cells can be used without purification, and can also be used in various conventional methods, for example dialysis, salt precipitation, ion exchange chromatography, size exclusion chromatography, affinity chromatography. It can be used by purifying with high purity using a mathematics or the like.

Various methods commonly used to screen for monoclonals that selectively recognize MIC-1, for example, immunoassay (RIA), enzyme immunosorbent (ELISA), immunofluorescence, Western blotting (Western) blotting and flow cytometry may be used, but are not limited to these. An exemplary method of the present invention is enzyme immunosorbent adsorption (ELISA).

In another aspect, the present invention provides a composition for detecting gastric cancer disease markers comprising the monoclonal antibody described above.

In another aspect, the present invention provides a method for detecting gastric cancer disease markers by reacting the monoclonal antibody with a biological sample to detect the formation of an antigen-antibody complex.

As used herein, the term "stomach cancer disease marker" refers to a substance that can be expressed in tissues or cells related to gastric cancer and can confirm the onset of gastric cancer by confirming the expression thereof. In the present invention, MIC-1 protein, which is specifically overexpressed in gastric cancer tissues and cells, is used as a diagnostic marker for gastric cancer disease.

As used herein, the term “biological sample” includes tissues, cells, whole blood, serum, plasma, saliva, cerebrospinal fluid, urine and the like. The expression level of MIC-1 can be measured by reacting an antibody of the present invention or the same epitope with an antibody having specificity, with or without manipulation of these biological samples.

As used herein, the term "antigen-antibody complex" refers to a combination of the MIC-1 protein and monoclonal antibody reacted to confirm the expression of MIC-1 in a biological sample.

The formation of the antigen-antibody complexes described above can be performed by colormetric method, electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment and It can be detected by a method selected from the group consisting of scintillation counting method. However, it is not necessarily limited to these and various applications and applications are possible.

"Detection" herein refers to the detection of antigen-antibody complexes using various labels. Specific examples are selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, and radioisotopes. But it is not necessarily limited to these.

Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, β-latamase, and the like. Phosphorus, Eu ³⁺ , Eu ³⁺ chelate or cryptate and the like, and ligands include biotin derivatives and the like, luminescent materials include acridinium esters, isoluminol derivatives and the like, and microparticles include colloids Gold, colored latex, and the like, and radioisotopes include ⁵⁷ Co, ³ H, ¹²⁵ I, ¹²⁵ I-Bolton Hunter reagent, and the like.

Preferably, the antigen-antibody complex can be detected using enzyme immunosorbent adsorption (ELISA). Enzyme immunosorbent methods (ELISA) include direct ELISA using labeled antibodies that recognize antigens attached to a solid support, and indirect ELISAs using labeled secondary antibodies that recognize captured antibodies in a complex of antibodies that recognize antigens attached to a solid support. Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the antibody and the antibody attached to the solid support, followed by reaction with another antibody that recognizes the antigen in the complex of the antibody and the antigen attached to the solid support. Various ELISA methods include indirect sandwich ELISAs using labeled secondary antibodies that recognize the antibody. The monoclonal antibody may have a detection label, and if it does not have a detection label, the monoclonal antibody may be captured and can be identified by treating another antibody having the detection label.

Particularly preferably, the antigen-antibody complex can be detected using a sandwich ELISA.

According to the ELISA detection method, the biological sample is contacted with a monoclonal antibody of the invention coated on a solid support such as a microtiter plate, membrane, test strip, or the like. As a specific example, the wells of a microtiter plate are coated with a monoclonal antibody of the invention and the unoccupied binding site is blocked, for example with BSA, then the wells of the coated plate are incubated with the sample sample and then the antigen-antibody. The presence of the complex can be determined. The presence of the antigen-antibody complex can be confirmed using a monoclonal antibody or polyclonal antibody that specifically binds to an antibody specific for the antigen of the antigen-antibody complex, for example MIC-1. The monoclonal antibody or polyclonal antibody may have a detection label, and in the absence of a detection label, the monoclonal antibody or polyclonal antibody may be identified by treatment with another antibody capable of detecting the monoclonal antibody or polyclonal antibody.

In the sandwich ELISA detection method of the present invention, the antibody that recognizes the antigen bound to the monoclonal antibody (capture antibody) that selectively recognizes MIC-1 is preferably a monoclonal antibody (detection antibody), which is MIC-1. Monoclonal antibodies that selectively recognize but recognize epitopes different from the capture monoclonal antibodies used to complex with the antigen are used.

In another aspect, the present invention provides a gastric cancer disease diagnostic kit comprising the monoclonal antibody described above.

The monoclonal antibody used in the diagnostic kit for detecting the heart disease of the present invention can be used as a fragment of the monoclonal antibody as long as the antibody can selectively recognize MIC-1. Such antibody fragments may include F (ab ') 2, Fab, Fab', Fv fragments, and the like.

The cardiac disease diagnostic kit of the present invention may include a monoclonal antibody or fragment thereof that selectively recognizes MIC-1 and a tool / reagent used for immunological analysis.

Tools / reagents used in immunological assays include suitable carriers, labeling substances capable of producing detectable signals, solubilizers, detergents and the like. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.

Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyesters, Polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated with latex metal, other papers, glass, metals, agarose and combinations thereof.

Assay systems for use in the detection methods and diagnostic kits of the invention include, but are not limited to, ELISA plates, dip-stick devices, immunochromatography test strips and radiation split immunoassay devices, and flow-through through) devices and the like.

Hereinafter, the present invention will be described in detail through examples. The following examples illustrate the invention and are not intended to limit the scope of the invention.

Example 1 Preparation of cDNA Expression Vector of MIC-1

Using the SNU-638 cDNA library, a gastric cancer cell line formed by secondary metastasis, as a template, a forward primer described in SEQ ID NO: 1 with an EcoRI recognition site inserted therein and a reverse primer described with SEQ ID NO: 2 with an XhoI recognition site inserted therein. , The complete recognition site of the human MIC-1 gene (PCR) by PCR (PCR) in a reaction condition of 1 minute at 94 ° C, 1 minute at 55 ° C, 30 minutes at 72 ° C (SEQ ID NO: 3; SEQ ID NO: 4 reverse) was amplified.

The cDNA amplified above was digested using EcoR1 and XhoI restriction enzymes, and inserted into pBluescript KS plasmid (Clontech, palo, CA, USA) and subjected to sequencing to confirm that the cloning was correct. After confirming the nucleotide sequence, TRX and His were attached to the N-terminus of the MIC-1 protein by inserting cICNA of MIC-1 cut with EcoRI and XhoI into pET32a plasmid (Novagen, Inc) digested with restriction enzymes of EcoRI and XhoI. PET32a-MIC-1 vector was prepared.

To express the recombinant MIC-1 gene, a gene corresponding to the amino acid sequence of Nos. 197 to 308 described in SEQ ID NO: 5, the MIC-1 recognition site of the main protein, was amplified using pfu polymerase (Stratagene). Specifically, after preparing the oligonucleotides represented by SEQ ID NO: 6 and SEQ ID NO: 7, the polymerization chain reaction (PCR) is repeated 25 times in a cycle of 1 minute at 94 ℃, 1 minute at 55 ℃, 2 minutes at 72 ℃ To amplify the gene. The PCR product corresponding to the main protein was digested with EcoRI and XhoI, and then inserted into the pFLAG-CMV-1 (Sigma, St. Louis, MI, USA) vector digested with EcoRI and XhoI, and the N- of the MIC-1 protein. PFLAG-MIC-1 recombinant vector was prepared having FLAG attached to the end. The FLAG is characterized by consisting of an amino acid sequence set forth in SEQ ID NO: 8, commonly referred to as a FLAG tag. Such FLAG sequences are very rare in protein sequences of eukaryotic cells, including humans. Therefore, when using an antibody that fuses the FLAG tag with the MIC-1 protein and binds the FLAG-tag, only the MIC-1 protein is recognized, isolated and purified. It is easy.

Example 2 Preparation of Transgenic Cell Line and Expression Analysis of Recombinant MIC-1

PFLAG-MIC-1 vector prepared in Example 1 was transduced into human embryonic kidney cell line 293 using a calcium phosphate kit (Clon Tech). Specifically, the human embryonic kidney cell line 293 to express recombinant MIC-1 was prepared at 1 × 10 6 / ml in a 150 mm culture vessel using 10% DMEM medium the day before transduction, and cultured at 37 ° C. and 5% CO 2 for 24 hours. . Three hours before transduction, the cells were exchanged with fresh 10% FBS-added DMEM medium, and then 40 µg / dish of pFLAG-MIC-1 vector was precipitated in 2 X HBS (HEPEs Buffered Saline) with 2 M CaCl2 and slowly Added. After 6 hours of addition, fresh 10% FBS was added to DMEM medium, and MIC-1 recombinant protein with FLAG attached to the MIC-1 protein N-terminus was forced to express for 48 hours.

After 3 days of transduction, the kidney cell line 293, for which MIC-1 was forcibly expressed, was washed three times or more with PBS (phosphate buffered saline), followed by 10 ml of lysis buffer solution (50 mM Tri-HCl, 150 mM NaCl, 1 mM). EDTA, 1% Triton X-100, pH 7.4) was added, and the cells were completely lysed for 30 minutes in a 4 ° C stirrer, followed by centrifugation at 4 ° C and 12,000 rpm for 10 minutes to remove insoluble protein. The supernatant containing the MIC-1 recombinant protein with FLAG attached to the N-terminus, except the precipitated cell debris, was activated by washing at least five times with TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). Passed through an anti-FLAG M2 affinity column (Sigma). TBS was passed three times or more to remove the unbound protein from the column, and then 1.5 ml eppen containing 50 μl of Tris-HCl (1 M pH 8.0) using 100 mM glycine buffer (pH 3.0). Fractions were separated by 1 ml in a dope tube. The separated MIC-1 protein was placed in an activated dialysis membrane and dialyzed at least 4 times at 4 ° C. using 500 ml of PBS (phosphate buffered saline). MIC-1 protein purified by Micro BCA Protein Assay Reagent Kit (PIERCE) was measured and the concentration was 100 μl and stored at -70 ° C.

Example 3: Expression Analysis of Recombinant MIC-1 in Escherichia Coli Cells

The DE3 strain was transformed into the pET32a-MIC-1 vector prepared in Example 1 using a heat shock method, and then plated in a solid medium containing ampicillin and incubated in a 37 ° C. incubator for 24 hours. Single clones transformed with pET32a-MIC-1 were selected. The selected monoclonal strains were inoculated in 5 ml of sterile LB-ampicillin culture solution, incubated in shaker for 24 hours, and then inoculated in 250 ml of LB-ampicillin culture solution and cultured in large capacity. When the OD600 value is 0.6 to 1.0 after the start of large-scale incubation, isopropyl-1β-D-thiogalactopyranoside (IPTG), which promotes the T7 promoter in the pET32 vector, is 1 mM. It was added at a concentration and further incubated for about 3 hours to obtain a large amount of strains that induced forced overexpression of MIC-1 having TRX and His attached to the N-terminus. The obtained strain was isolated and purified only MIC-1 according to the protein purification procedure provided by His-bind kits (Novagen, Inc). Purified recombinant MIC-1 was placed in an activated dialysis membrane and dialyzed at least 3 times at 4 ° C. using 500 ml of TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4), followed by Micro BCA Protein Assay. After the concentration was measured using Reagent Kit (PIERCE), 100 μl fractions were stored and frozen at -70 ° C.

Example 4: Antigen Immunization of Mice

To obtain immunized mice for the development of hybridoma cell lines, 50 μg recombinant pET32a-MIC-1 fusion protein and the same amount of adjuvant (MPL-TDM Adjuvant) were mixed and bathed for 30 minutes at 40 to 45 ° C. Four to six week old Balb / c mice were injected intraperitoneally. Two weeks later, 25 μg of pET32a-MIC-1 fusion protein and the same amount of adjuvant (MPL + TDM Adjuvant) were mixed well and boosted into the abdominal cavity of mice. After 4-5 days, a small amount of blood was drawn from the mouse ocular vein to confirm the titer, and the final injection of pET32a-MIC-1 protein mixed with the adjuvant antigen was performed intraperitoneally 3 days before the cell fusion experiment.

Example 5: Preparation of hybridomas by cell fusion

To carry out the cell fusion required in the production of hybridoma cells, collected 10 ⁶ splenocytes obtained by scanning the prepared immunogenic mice 10 ⁷ dogs and myeloma cells (myeloma cell, NS-1) in 50 ml test tubes. NS-1 was used as the parent cell of the cell fusion. These blasts were always maintained at a maximum density of 5-10 ⁵ / ml in DMEM medium containing 10% FBS. After anesthetizing the immunized mice with ether in Example 4, take out the spleen located on the left side of the body, grind it finely into a mesh to make a suspension, and centrifuge the splenocytes obtained at this time in a 15 ml centrifuge tube. This method was repeated twice to thoroughly wash the spleen cells. After resuspending each of the splenocytes and NS-1 cells by 10 ml, the cells were counted, and the splenocytes and NS-1 were mixed in a 50 ml centrifuge tube so as to have a ratio of 10: 1, and precipitated again by centrifugation. Disperse the centrifuged precipitate with a light tap with a finger, hold for 1 minute at 37 ° C, and then add 1 ml of 45% polyethylene glycol (PES) fusogen in Hans buffer (HBSS). It was put in for 1 minute and shaken slightly for another 1 minute. Then, 9 ml of culture medium (DMEM) was added over 1 minute, and DMEM was added slowly while shaking until it became 50 ml. After centrifugation of the suspension again, the cell precipitate was resuspended in a separation medium (HAT medium) at about 1 to 2 × 10 ⁵ / ml, aliquoted in a 96 well microtiter plate at 0.2 ml, and then in a 37 ° C. wet carbon dioxide incubator. Incubated.

Example 6: Selection of Hybridoma Cells Producing Monoclonal Antibodies

The hybridoma cell group prepared in Example 5 was screened by an enzyme immunosorbent adsorption (ELISA) assay using TRX and TRX-MIC-1 antigens to select hybridoma cells that specifically react with MIC-1 antigen. . 50 microliters (2 ug / ml) of TRX or TRX-MIC-1 antigen was added to the microtiter plate, respectively, per well to attach to the surface of the plate. The culture medium of hybridoma cells was reacted for 1 hour by adding 50 ul to each well, and then washed with phosphate buffer-Tween 20 (PBST) solution sufficiently to remove the unreacted culture. To this was added goat anti-mouse IgG-horseradish peroxidase (goat anti-mouse IgG-HRP), reacted at room temperature for 1 hour, and then sufficiently washed with PBST solution. Subsequently, the substrate solution (OPD) of peroxidase was added and reacted, and the reaction degree was measured by absorbance at 490 nm. As a result, hybridoma cell lines that secrete antibodies with high specificity to MIC-1 antigen were selected, and hybridoma cell lines that specifically reacted to MIC-1 antigen were selected through repeated experiments. Hybridoma cell lines producing monoclonal antibodies by limiting dilution to monoclonal were selected. Two clones, MIC-1-3-14 and MIC-1-26-20-9, were finally obtained. This hybridoma MIC-1-3-14 is December 11, 2003, and MIC-1-26-20-9 is January 12, 2004. KCTC (Korean Collection), 52 Eoeun-dong, Yuseong-gu, Daejeon, Korea for Type Cultures (KCTC) were deposited with accession numbers KCTC 10565BP and 10580BP, respectively.

In addition, these clones were cloned and frozen, and the supernatants were assayed for titer by enzyme immunosorbent adsorption, and subclass type was identified. One clone (MIC-1-26-20-9) turned out to be IgG1 and another clone (MIC-1-3-14) turned out to be IgG2a and showed a high specificity for MIC-1. . These two clones were injected into mice to produce ascites fluid.

Example 7: Mass Production of Monoclonal Antibodies

Mice (Balb / c) were injected intraperitoneally with 0.5 ml of Incomplete Freund Adjuvant to mass produce monoclonal antibodies from the continually secreting antibody obtained in Example 6. After one week, each of the hybridomas was injected 2 × 10 ⁶ per horse in the abdominal cavity of the mice, and ascites fluid was collected from the mice in which the abdominal cavity was inflated after 7 to 10 days. The high concentration of hybridoma cells contained in this ascites solution was removed by centrifugation at 10,000 rpm, the obtained supernatant was taken and stored at -70 ° C until some were purified, and some of the Protein A & G agarose column was prepared. Purification was carried out using (Fig. 6).

Example 8 Detection of MIC-1 Proteins by Immunohistochemical Staining

The antibody purified in Example 7 was subjected to immunohistochemical staining to investigate the expression level of MIC-1 in gastric cancer tissues. Paraffin was removed from the paraffin sections of gastric cancer tissues obtained from the patient by using xylene, hydrated with alcohol, and then placed in 10 mM citrate buffer (pH 6.0) to project microwaves three times for 5 minutes. Next, working in the Vector kit (Cat No. PK 6102) to prevent the binding of nonspecific proteins after inhibiting the internally generated peroxidase activity by treating methanol containing 3% hydrogen peroxide for 6 minutes. ) Solution was blocked for 30 minutes. MIC-1 IgG2a antibody diluted 50 × 1 in 1 × PBS was reacted at room temperature for 2 hours, and then biotinylated anti-mouse Ig Ab (Vector kit) was reacted at room temperature for 30 minutes. Next, after reacting ABC Elite kit (Vector kit) for 30 minutes at room temperature, diaminobenzidine tetrahydrochloride substrate (Vector kit) for 2 minutes to confirm the expression of MIC-1 in gastric cancer tissue (Fig. 7 ).

As a result, the expression of MIC-1 protein in gastric cancer tissues was found to increase as the metastasis of cancer progresses.

Example 9 Sandwich ELISA Using Monoclonal Antibody Attached with Horseradish Peroxidase (HRP)

Horseradish peroxidase was attached to each of the two monoclonal antibodies purified in Example 6. Briefly, peroxidase was dissolved in 0.1M phosphate (pH6.5) containing 1.25% glutaraldehyde and incubated overnight at 4 ° C. Peroxidase was dialyzed in 0.1M carbonate (pH9.0) buffer to remove glutaraldehyde, and purified monoclonal antibody was dialyzed in 0.1M sodium carbonate buffer (pH9.2) and 3 mg peroxidase. And incubated overnight at 4 ° C. The buffer composition of the mixture was changed to 0.1 M phosphate buffer through dialysis, purified through Sephadex G-25 column, measured at absorbance 280 and 403, and the dialysis fractions were collected by dialysis again to obtain peroxidase attachment. Only the antibody was isolated. In order to measure the activity of the isolated antibody, 100 μl (4 μg / ml) of polyclonal and monoclonal antibodies against MIC-1 were coated on a microtiter plate, followed by dilution of the TRX-MIC-1 antigen per well. Each 100 μl was added and reacted at room temperature for 2 hours. Unreacted antigens were washed out, diluted with peroxidase-attached IgG2a antibody at 1:50 and IgG1 antibody at 1: 100, and 100 μl per well was added. The polyclonal antibody was treated with IgG1, reacted at room temperature for 1 hour, and then reacted with the substrate OPD of peroxidase, and the reaction degree was measured at 490 nm using an ELISA Reader (FIG. 8). The degree of response of polyclonal and monoclonal antibodies to recombinant MIC-1 protein was less than 1 ng / ml at all. something to do.

By detecting the MIC-1 protein as a diagnostic marker for gastric cancer using a monoclonal antibody specific for human MIC-1 produced according to the present invention, the occurrence and metastasis of gastric cancer can be known and gastric cancer comprising such monoclonal antibody. Disease marker detection compositions and gastric cancer disease diagnostic kits may be provided.

Figure 1a is an electrophoresis result showing the expression of MIC-1 after transforming Escherichia coli with pET32a-MIC-1, Figure 1b is the result of electrophoresis after purification and dialysis of the expressed protein using an affinity column to be.

Figure 2 is an enzyme immunosorbent assay (ELISA) that examines the degree of reaction of the antibody produced in the fusion cell line obtained after injecting the human MIC-1 recombinant protein fused with TRX to the recombinant antigen expressed from pET32a-MIC-1 (ELISA) ) Is the result.

Figure 3 shows the results of electrophoresis of the digested gastric cancer cell line and Western blotting of the MIC-1-3-14 clone using the culture supernatant. Figure 3a is a result showing that FLAG Ab recognizes pFLAG-MIC-1 as a control and Figure 3b is a result showing the recognition of MIC-1 protein in gastric cancer cell lines using the culture supernatant of the fusion cell line of the present invention.

FIG. 4 shows the results of sub-cloning two clones that specifically recognize MIC-1 among three clones found to secrete MIC-1 specific monoclonal antibodies in FIG. 2. Enzyme immunosorbent adsorption (ELISA) analysis of the degree of reaction of each antibody produced from a total of 57 clones against TRX and the recombinant antigen expressed from pET32a-MIC-1.

5 is a result of determining the isotype by selecting 13 clones from 27 clones found to secrete MIC-1 specific monoclonal antibodies in FIG. MIC-1-3-14 clones secrete antibodies in the IgG2a isotype form and MIC-1-26-20-9 clones show the antibodies in the IgG1 isotype form.

FIG. 6 shows that the MIC-1-3-14 clone and the MIC-1-26-20-9 clone, which produce monoclonal antibodies against human MIC-1, were harvested after mass culture in the abdominal cavity of mice and were collected from affinity columns (Protein). The result was separated and purified using A & G agarose column.

7 shows the results of immunohistochemical staining of gastric cancer tissues using monoclonal antibodies against purified human MIC-1.

Figure 8 shows the results of antibody antibody analysis by sandwich enzyme immunosorbent (sandwich ELISA) by attaching a peroxidase to a monoclonal antibody against human MIC-1.

<110> Korea Reserarch Institute of Bioscience and Biotechnology <120> Monoclonal antibody specific to human macrophage inhibitory cytokine-1, hybridoma producing the monoclonal antibody and diagnostic kit comprising the monoclonal antibody <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for the amplification of MIC-1 <400> 1 gggggtggac ccatcctcta 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for the amplification of MIC-1 <400> 2 cgcgcagctg taaacggtag 20 <210> 3 <211> 927 <212> DNA <213> Homo sapiens <220> <221> gene (222) (1) .. (927) <223> Human MIC-1 (macrophase inhibitory cytokime-1) <400> 3 atgcccgggc aagaactcag gacgctgaat ggctctcaga tgctcctggt gttgctggtg 60 ctctcgtggc tgccgcatgg gggcgccctg tctctggccg aggcgagccg cgcaagtttc 120 ccgggaccct cagagttgca caccgaagac tccagattcc gagagttgcg gaaacgctac 180 gaggacctgc taaccaggct gcgggccaac cagagctggg aagattcgaa caccgacctc 240 gtcccggccc ctgcagtccg gatactcacg ccagaagtgc ggctgggatc cggcggccac 300 ctgcacctgc gtatctctcg ggccgccctt cccgaggggc tccccgaggc ctcccgcctt 360 caccgggctc tgttccggct gtccccgacg gcgtcaaggt cgtgggacgt gacacgacct 420 ctgcggcgtc agctcagcct tgcaagaccc caggcgcccg cgctgcacct gcgactgtcg 480 ccgccgccgt cgcagtcgga ccaactgctg gcagaatctt cgtccgcacg gccccagctg 540 gagttgcact tgcggccgca agccgccagg gggcgccgca gagcgcgtgc gcgcaacggg 600 gaccactgtc cgctcgggcc cgggcgttgc tgccgtctgc acacggtccg cgcgtcgctg 660 gaagacctgg gctgggccga ttgggtgctg tcgccacggg aggtgcaagt gaccatgtgc 720 atcggcgcgt gcccgagcca gttccgggcg gcaaacatgc acgcgcagat caagacgagc 780 ctgcaccgcc tgaagcccga cacggtgcca gcgccctgct gcgtgcccgc cagctacaat 840 cccatggtgc tcattcaaaa gaccgacacc ggggtgtcgc tccagaccta tgatgacttg 900 ttagccaaag actgccactg catatga 927 <210> 4 <211> 927 <212> DNA <213> Homo sapiens <220> <221> gene (222) (1) .. (927) <223> Human MIC-1 (macrophase inhibitory cytokime-1) <400> 4 tcatatgcag tggcagtctt tggctaacaa gtcatcatag gtctggagcg acaccccggt 60 gtcggtcttt tgaatgagca ccatgggatt gtagctggcg ggcacgcagc agggcgctgg 120 caccgtgtcg ggcttcaggc ggtgcaggct cgtcttgatc tgcgcgtgca tgtttgccgc 180 ccggaactgg ctcgggcacg cgccgatgca catggtcact tgcacctccc gtggcgacag 240 cacccaatcg gcccagccca ggtcttccag cgacgcgcgg accgtgtgca gacggcagca 300 acgcccgggc ccgagcggac agtggtcccc gttgcgcgca cgcgctctgc ggcgccccct 360 ggcggcttgc ggccgcaagt gcaactccag ctggggccgt gcggacgaag attctgccag 420 cagttggtcc gactgcgacg gcggcggcga cagtcgcagg tgcagcgcgg gcgcctgggg 480 tcttgcaagg ctgagctgac gccgcagagg tcgtgtcacg tcccacgacc ttgacgccgt 540 cggggacagc cggaacagag cccggtgaag gcgggaggcc tcggggagcc cctcgggaag 600 ggcggcccga gagatacgca ggtgcaggtg gccgccggat cccagccgca cttctggcgt 660 gagtatccgg actgcagggg ccgggacgag gtcggtgttc gaatcttccc agctctggtt 720 ggcccgcagc ctggttagca ggtcctcgta gcgtttccgc aactctcgga atctggagtc 780 ttcggtgtgc aactctgagg gtcccgggaa acttgcgcgg ctcgcctcgg ccagagacag 840 ggcgccccca tgcggcagcc acgagagcac cagcaacacc aggagcatct gagagccatt 900 cagcgtcctg agttcttgcc cgggcat 927 <210> 5 <211> 336 <212> DNA <213> Homo sapiens <220> <221> gene (222) (1) .. (336) <223> Human MIC-1 (macrophase inhibitory cytokime-1) <400> 5 gcgcgcaacg gggaccactg tccgctcggg cccgggcgtt gctgccgtct gcacacggtc 60 cgcgcgtcgc tggaagacct gggctgggcc gattgggtgc tgtcgccacg ggaggtgcaa 120 gtgaccatgt gcatcggcgc gtgcccgagc cagttccggg cggcaaacat gcacgcgcag 180 atcaagacga gcctgcaccg cctgaagccc gacacggtgc cagcgccctg ctgcgtgccc 240 gccagctaca atcccatggt gctcattcaa aagaccgaca ccggggtgtc gctccagacc 300 tatgatgact tgttagccaa agactgccac tgcata 336 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer for the amplification of MIC-1 <400> 6 cggaattcaa tggcgcgcaa cggg 24 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer for the amplification of MIC-1 <400> 7 ccctcgagtc atatgcagtc tttggc 26 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> flag <400> 8 Met Asp Tyr Lys Asp Asp Asp Asp Lys 1 5

Claims (9)

A monoclonal antibody having specificity for human Macrophage Inhibitory Cytokine-1 (MIC-1) produced by hybridoma cells of Accession No. KCTC 10565BP. Hybridoma cells of Accession No. KCTC 10565BP. Monoclonal antibody having specificity for human MIC-1 produced by hybridoma cells of Accession No. KCTC 10580BP. Hybridoma cells of Accession No. KCTC 10580BP. Composition for the detection of gastric cancer disease markers comprising the monoclonal antibody of claim 1 or claim 3. Gastric cancer disease diagnostic kit comprising a monoclonal antibody of claim 1 or 3. A method of detecting gastric cancer disease markers by contacting a monoclonal antibody of claim 1 or 3 with a biological sample to detect antigen-antibody complex formation. 8. The method of claim 7, wherein the antigen-antibody complex formation is detected by ELISA. The method of claim 8, wherein antigen-antibody complex formation is detected by sandwich ELISA.