KR100455658B1 - Monoclonal antibody specific for human calcium binding protein s100a2 and fusion cell strain producing same - Google Patents

Abstract

The present invention relates to a monoclonal antibody against human calcium binding protein S100A2 (calcium binding protein; S100A2) overexpressed in specific human tumor tissue and tumor cells constituting the same, and to a fusion cell line producing the same, in particular breast cancer or cervical cancer tissue It relates to a monoclonal antibody derived from a mouse that specifically binds to the calcium binding protein S100A2 overexpressed in and the fusion cell line producing the antibody. Monoclonal antibody to human S100A2 calcium binding protein of the present invention and the fusion cell line producing the same specifically binds to tumor cells expressing human S100A2 protein, so can easily detect cancer tissue through antigen-antibody binding Therefore, it can be useful for the study on the degree of expression and function of human S100A2 protein in tumor cells during tumor development and invasion and for the clinical diagnosis of cancer.

Description

MONOCLONAL ANTIBODY SPECIFIC FOR HUMAN CALCIUM BINDING PROTEIN S100A2 AND FUSION CELL STRAIN PRODUCING SAME}

The present invention relates to a monoclonal antibody against the human calcium binding protein S100A2 (calcium binding protein; S100A2) overexpressed in certain human tumor tissues and tumor cells constituting the same, and a fusion cell line producing the same, specifically breast cancer or cervical cancer tissue It relates to a monoclonal antibody derived from a mouse that specifically binds to the calcium binding protein S100A2 overexpressed in and the fusion cell line producing the antibody.

S100A2 protein is one of 18 S100-based calcium binding proteins that regulate calcium function through binding to intracellular calcium ions and was found in 1996 as one of the genes that constitute the epidermal differentiation complex (EDC). Marenholz, I. et al ., Genomics , 1 (37): 295-302, 1996). The S100A2 gene is very important in the final differentiation of epithelial structures and is located on q21 of human chromosome 1 where EDC, a collection of structural and functionally similar genes, is located. S100A2 calcium binding protein is known to be distributed only in some normal tissues, but has recently been reported to be expressed in certain specific tumors of the human body, such as skin-related tumors, breast cancer, and laryngeal squamous cell carcinoma (Lauriola, L. et al ., Int. J. Cancer , 20 (89): 345-349, 2000; Shrestha, P. et al ., Virchows Arch. , 32: 53-59, 1998; Maelandsmo, GM et al ., Int. J. Cancer , 22 (74): 464-469, 1997).

The human S100A2 gene is known to have a binding site with p53 tumor suppressor protein in the promoter region, and the expression of S100A2 mRNA is induced by p53 activator etoposide. It is known to have the function of tumor suppressor genes regulated by proteins (Tan, M. et al ., FEBS Lett ., 445: 265-268, 1999). However, the increase in the amount of S100A2 protein associated with the activation of epidermal growth factor receptor (EGF-R) in HaCaT cell lines derived from human keratinocytes is associated with the regeneration and tumorigenicity of squamous cells. The possibility of gene involvement is suggested (Stoll, SW et al ., J. Invest. Dermatol ., 111: 1092-1097, 1998). S100A2 protein is specifically of S100A2 calcium-binding protein in the In Vitro results, combined with tropomyosin (tropomyosin) in the presence of calcium ion in the muscle cells, or, but is known to the same kind to form a dimer (homodimer) for cellular function, cell And the exact role and function is not known yet.

Maintaining homeostasis of calcium ions during transformation and invasion of tumor cells is a very important part, and S100A1, S100A2, S100A4, and S100A1, S100A2, S100A6 has been studied in relation to tumor development and metastasis (Ilg, EC et al ., Int. J. Cancer , 4 (68): 325-332, 1997). They are known to play a role in mediating the buffering and signaling of calcium ions during cell adhesion, cell mobility, cell growth and differentiation (Ilg, EC et al ., Int. J. Cancer , 68: 325-332, 1997; Mueller, A. et al ., Histochem. Cell Biol ., 111: 453-459, 1999).

The S100A2 gene used to produce monoclonal antibodies in the present invention is a gene encoding calcium binding protein S100A2, which is known to be regulated by activation of p53 tumor suppressor protein and c-erbB receptor system (EGF-R) protein in animal cells. . Until now, monoclonal antibodies that recognize S100A2 protein and fusion cell lines producing the same have not been reported, and the results of functional analysis and research on S100A2 genes and proteins related to the generation of human constituent cells and tumors are also insignificant.

Accordingly, the present inventors have specifically described a monoclonal derived from a mouse specifically binding to S100A2 calcium binding protein overexpressed in specific human tumor tissues and tumor cells constituting the intracellular functional analysis and clinical tumor diagnosis of S100A2 calcium binding protein The present invention was completed by preparing a fusion cell line producing the antibody and confirming that the anti-S100A2 monoclonal antibody produced therefrom specifically recognized tumor cells.

It is an object of the present invention to provide a monoclonal antibody against calcium binding protein S100A2 and a fusion cell line producing the same for intracellular function analysis and clinical tumor diagnosis of S100A2 calcium binding protein.

1 is an S100A2 recombinant protein fused to glutathione S-transferase (GST) from E. coli transformed with the expression vector pGEX-4T-1-S100A2 of the present invention containing a human S100A2 gene (about 38 kDa) production followed by staining with Coomassie blue (left panel) and Western blot analysis using anti-GST monoclonal antibody (right panel),

Lane M; Standard molecular weight size labels lanes 1 and 3; GST

Lanes 2 and 4; GST-S100A2 Recombinant Protein

2 is a result of staining with Coomassie blue after separating and purifying pure S100A2 recombinant protein by treating thrombin to a fused recombinant protein to prepare an immunogen,

Lane M; Standard molecular weight size label lane 1; GST-S100A2 Recombinant Protein Lane 2; Isolated and purified S100A2 Recombinant Protein

Figure 3 shows the absorbance at 492 nm wavelength using the indirect enzyme-linked immunosorbent assay (ELISA) method of the antibody to the S100A2 antigen in the serum of the mouse after immunization of the isolated and purified recombinant S100A2 protein with an immunogen Is the measurement result,

Figure 4aIs a result of measuring the absorbance at 492 nm wavelength using an indirect ELISA method to determine the titer of the anti-S100A2 monoclonal antibody of the present invention that specifically reacts only with the antigen in the culture supernatant of the fusion cell line,

Figure 4b is an immunoglobulin subtype kit (Immunoglobulin Isotyping kit, Sigma) to determine the immunoglobulin subtype of the anti-S100A2 monoclonal antibody produced by the fusion cell line of the present invention by fusing splenocytes and parent cells of the immunized mice Using),

Figure 5a is a schematic diagram showing the number and size of amino acids of the S100A1, S100A4 and S100A6 recombinant protein having a high homology with S100A2, which is designed to confirm the specificity of the S100A2 monoclonal antibody of the present invention S100A2,

FIG. 5B shows the results of staining the recombinant proteins GST-S100A1, GST-S100A2, GST-S100A4 and GST-S100A6 of FIG. 5A with Coomassie Blue after SDS-PAGE. FIG.

FIG. 5C is a result of Western blot analysis using anti-GST monoclonal antibody against FIG. 5B ,

Lane M; Standard molecular weight size label lane 1; GST

Lane 2; GST-S100A1 Recombinant Protein Lane 3; GST-S100A2 Recombinant Protein

Lane 4; GST-S100A4 recombinant protein lane 5; GST-S100A6 Recombinant Protein

5D is a result of Western blot analysis to confirm the specificity of the anti-S100A2 monoclonal antibody of the present invention to the GST-S100A2 recombinant protein,

Lane M; Standard molecular weight size label lane 1; GST

Lane 2; GST-S100A1 Recombinant Protein Lane 3; GST-S100A2 Recombinant Protein

Lane 4; GST-S100A4 recombinant protein lane 5; GST-S100A6 Recombinant Protein

Figure 6a is a result of verifying the antigen specificity of the monoclonal antibody of the present invention using immunohistochemical staining in invasive ductal carcinoma tissue removed from human breast cancer patients,

N; Normal tissue cell T; Tumor Cells of Breast Cancer Tissue

S; Epilepsy

Figure 6b is a result of verifying the antigen specificity of the monoclonal antibody of the present invention using immunohistochemical staining in squamous cell carcinoma tissue removed from human cervical cancer patients.

N; Normal tissue cell T; Tumor cells of cervical cancer tissue

S; Epilepsy

In order to achieve the above object, the present invention provides a monoclonal antibody having specificity for the human S100A2 protein, which is one of proteins estimated to be tumor markers of breast cancer and cervical cancer.

The present invention also provides a fusion cell line producing a monoclonal antibody specific for the human S100A2 protein.

In addition, the present invention provides a method for mass production of monoclonal antibodies specific for the human S100A2 protein.

Hereinafter, the present invention will be described in detail.

The present invention provides a monoclonal antibody having specificity for human S100A2 protein, which is one of proteins suspected as tumor markers of breast cancer and cervical cancer, and a fusion cell line producing the same.

In order to prepare monoclonal antibodies that specifically recognize human S100A2 protein and fusion cell lines that produce them, S100A2 protein must first be obtained. S100A2 protein can be synthesized using known amino acid sequences or produced as recombinant protein by genetic engineering methods. For example, using the S100A2 gene listed on the GenBank of the NIH program to prepare an expression vector expressing the S100A2 protein in the form of a recombinant protein; Transforming the expression vector into Escherichia coli to obtain a transformant that produces a S100A2 recombinant protein; The S100A2 recombinant protein may be prepared by culturing the transformant to isolate and purify the human S100A2 recombinant protein. GenBank registration number in the present invention; The human S100A2 gene having the nucleotide sequence set forth in SEQ ID NO: 1 listed as NM-005978 was used.

In order to facilitate the isolation and purification of the recombinant protein, glutathione-S-transferase (GST) may be expressed in the form of GST-S100A2 recombinant protein fused with GST affinity (GST affinity). The fusion protein may be separated using chromatography, followed by treatment with thrombin, and the like to cleave GST to readily separate the pure S100A2 recombinant protein. In addition to GST, fusion proteins that can be used in the present invention for the same purpose as described above are histidine-labeled (His-tag), beta-galactosidase (β-galactosidase), luciferase and maltose binding protein. (Maltose binding protein) and the like.

In order to confirm that the protein produced from the E. coli transformant is a GST-S100A2 recombinant protein, Western blot analysis using coomassie blue staining and anti-GST monoclonal antibody showed that the GST- having a molecular weight of about 38.7 kDa. S100A2 recombinant protein was specifically detected to confirm that S100A2 recombinant protein is produced in the form of GST-fusion protein from E. coli transformed with the expression vector of the present invention containing the human S100A2 gene (see FIG. 1 ).

S100A2 recombinant protein (see FIG. 2 ) isolated and purified from E. coli transformants was used for indirect enzyme-linked assays for the selection and analysis of fusion cell lines producing monoclonal antibodies through mouse immunization and cell fusion. It is used as an antigen for immunosorbant assay (ELISA).

Immunized mice required for the development of a fusion cell line were mixed with the human S100A2 recombinant protein isolated above and the same amount of Freund 'complete adjuvant until emulsified, and injected into the abdominal cavity of the mouse. It is obtained by further booster injection to increase the immunity of the mice. At this time, it is preferable to further boost the human S100A2 recombinant protein three times in the abdominal cavity of the mouse without using an auxiliary antigen.

The serum titer of the antibody immunized with the human S100A2 recombinant protein antigen injected was measured to measure antibody titers (see FIG. 3 ) . : 1 ratio, polyethylene glycol is added to the cell fusion reaction, and then culture medium is added and diluted. Next, the fused cells are transferred to a selection medium and cultured, and when the growth of the fusion cell population is clearly confirmed, the culture medium is changed to a selection medium to promote cell proliferation. As the cell fusion parent cell, an SP2 / 0.Ag14 cell line, a P3x653 cell line, or an NS-1 cell line may be used. The culture medium may be RPMI medium, DMEM medium, IMDM medium, F12 medium, or the like. HAT medium may be used as the selection medium.

A fusion cell group that specifically reacts only with the human S100A2 protein antigen among the fusion cell groups growing in the selection medium is selected using an indirect ELISA method. That is, by measuring the absorbance with an ELISA reader, fusion cell lines secreting high-binding antibodies with purified human S100A2 protein were selected (see FIG. 4B ), and limiting dilution was performed so that the selected cells became monoclonal. dilution) is used to establish a cell line consisting of cells that have grown from one cell after dilution of the fusion cells. The fusion cell line of the present invention thus selected was named 'KK0724' and was deposited on February 1, 2001 in the Korea Biotechnology Research Institute Gene Bank (KCTC) (Accession Number: KCTC 0941BP).

Monoclonal antibodies of the invention specific for human S100A2 protein

1) injecting the fusion cell line into the abdominal cavity of a mouse;

2) collecting ascites fluid from the abdominal cavity inflated mice;

3) Mass production of monoclonal antibody protein against human S100A2 protein from ascites fluid by separation.

The monoclonal antibody against human S100A2 secreted from the cell line established in the present invention is a monoclonal antibody isolated and purified from the immunoglobulin G _2b type (IgG _2b ) (see FIG. 4A ) and fused cell line KK0724 of the present invention. In order to determine the specific reactivity of the antibody to the human S100A2 recombinant protein, a reactivity search for S100A1, S100A4 and S100A6 proteins with similar amino acid sequences to the S100A2 protein was performed. As a result, it was confirmed that the monoclonal antibody against the human S100A2 recombinant protein of the present invention is an antibody that specifically reacts only with the human S100A2 recombinant protein (see FIG. 5 ).

The monoclonal antibody of the present invention selectively reacts with the S100A2 protein antigen present in tissues of invasive vascular carcinoma and squamous cell carcinoma from human breast cancer and cervical cancer by immunohistochemical staining only in the cytoplasm of malignant tumor cells. It is strongly expressed in brown and clearly distinguishes tumor cells from normal cells. In particular, the cytoplasm of the cells located in the margins of the infiltrating tumor cell population showed a stronger positive reaction (see FIGS . 6A and 6B ). Therefore, the monoclonal antibody of the present invention can be usefully used not only for determining the concentration of S100A2 protein antigen in a patient's cell but also for studying carcinogenesis and for diagnosing tumors of a specific human body.

Methods for diagnosing cancer using the monoclonal antibodies of the present invention include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, The diagnosis can be made by confirming whether the protein is expressed in a biological sample of the subject by a method such as Western blot or immunoblot analysis on a polyacrylamide gel.

The present invention also provides a protein chip capable of detecting an antigen for the antibody protein by immobilizing the monoclonal antibody on a biological microchip and then reacting with a biological sample of the subject. For example, samples can be analyzed in large quantities using known biological microchips and automated microarray systems in addition to ELISA methods.

In addition, the present invention provides a diagnostic kit comprising the monoclonal antibody protein for diagnosing certain human tumors, for example, cancers such as breast cancer or cervical cancer.

The diagnostic kit can diagnose various cancers by quantitatively or qualitatively analyzing the antigen to the antibody protein through the monoclonal antigen-antibody binding reaction of the present invention, and the antigen-antibody binding reaction is conventional ELISA, RIA, sandwich. It can be measured by a method of measurement, Western blot on polyacrylamide gel or by immunoblot analysis. For example, the diagnostic kit may be provided to perform ELISA using a 96 well microtiter plate coated with the recombinant monoclonal antibody protein on the surface thereof. Substrates to which the antibody protein can be coated include a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, and a slide glass made of glass. This can be used. Such a diagnostic kit may further include a secondary antibody, a coloring substrate solution, etc., in which a suitable buffer solution, a standard antibody, a chromophore or a fluorescent material is bound, in addition to a biological microchip in which a monoclonal antibody protein is immobilized on a surface thereof. Peroxidase, alkaline phosphatase may be used as a chromophore bound to the antibody, and the fluorescent material may be FITC, RITC, or the like. As the coloring substrate solution, ABTS [2, 2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o-Phenylenediamine), TMB (Tetramethyl Benzidine) and the like can be used.

In addition, the diagnostic kit may use an automated analysis method using a biological microchip for the diagnosis of various cancers. For example, the diagnostic kit may be configured to perform an ELISA using a protein chip such as slide glass coated on the surface of the recombinant monoclonal antibody protein.

Hereinafter, the present invention will be described in detail by way of examples.

The following examples are merely illustrative of the present invention, but the content of the present invention is not limited by the examples.

Example 1 Preparation of Expression Vector Producing Human S100A2 Recombinant Protein

In order to obtain the full nucleotide sequence of the human S100A2 gene, cDNA obtained by reverse transcription of mRNA extracted from human breast cancer cell line MDA-MB231 (ATCC, HTB-26) was used as a template, and SEQ ID NO: 2 and SEQ ID NO: The entire open reading frame (ORF) was amplified by polymerase chain reaction (PCR) using the described S100A2 gene specific primer pairs. The primer pair is a protein coding end region and a protein coding region of the first to 18th protein coding sequence of the human S100A2 gene ( GenBank Accession No .; NM-005978) described in SEQ ID NO: 1 listed on GenBank of the NIH program. It was designed to specifically recognize the 18th base sequence of (see Table 2 ). The amplified PCR product was digested with restriction enzymes Bam HI and Eco RI, and DNA fragments consisting of 300 nucleotides were isolated and purified. The DNA fragment was cloned into pGEX-4T-1 vector (Novagen) treated with the same restriction enzymes Bam HI and Eco RI to produce the recombinant protein glutathione-S-transferase (GST) -S100A2. An expression vector pGEX-4T-1-S100A2 was prepared.

The pGEX-4T-1 vector is a vector capable of expressing a target protein in the form of a fusion protein by fusion of GST to a protein to be expressed. When the S100A2 expression vector is transformed into E. coli DH5α and induces expression, GST-fused S100A2 protein GST-S100A2 can be produced.

Example 2 Isolation and Purification of Human S100A2 Recombinant Protein

E. coli DH5α was transformed with the expression vector pGEX-4T-1-S100A2 containing the human S100A2 gene and shaken and cultured at 37 ° C. in LB medium containing 50 μg / ml of ampicillin. Only converted colonies were selected. In order to induce the production of human S100A2 protein from the selected E. coli transformants, 1 mM IPTG was added to the culture medium during the logarithmic growth of the cells. The culture supernatant obtained by centrifuging the culture solution and then crushed by ultrasound was used for glutathione Sepharose R ^R 4B (Glutathione Sepharose R ^R 4B, Korea Amersham-Pharmacia Biotech) having affinity for GST. Recombinant protein in the form of GST-S100A2 was purified by GST affinity chromatography. 10 μl of the purified sample was electrophoresed with 10% SDS-PAGE and stained with Coomassie Blue to confirm that GST-fused recombinant protein was produced from E. coli transformants ( FIG. 1 , left panel).

Western blot analysis was performed using an anti-GST monoclonal antibody to confirm that the protein produced from the E. coli transformant was a GST-S100A2 recombinant protein. First, the proteins separated by 10% SDS-PAGE were transferred to the membrane filter by an electrical method, and then the membrane was washed with 3% bovine serum albumin solution for 1 to 2 hours to reduce the nonspecific reactions of other proteins transferred to the membrane filter. Blocked. Anti GST monoclonal antibody (Sigma) was added thereto and reacted at room temperature for 2 hours. The membrane filter was washed with a phosphate buffer solution containing 0.5% Tween 20, and then fluorescently colored using ECL solution (Amersham Co., Ltd.), and confirmed by photosensitive X-ray film (Kodak). 1 , right panel).

The results of the SDS-PAGE gel and Western blot analysis are shown in FIG. 1 . In Figure 1 lane M is the standard molecular weight size label, lanes 1 and 3 are GST, lanes 2 and 4 represent the GST-S100A2 recombinant protein of the present invention, the left panel is the result of staining with Coomassie blue and the right panel Is the result of Western blot analysis using anti-GST monoclonal antibody.

As shown in FIG. 1 , the GST protein has a molecular weight of 28 kDa while the GST-S100A2 recombinant protein has a molecular weight of about 38.7 kDa, and the GST and GST-S100A2 recombinant proteins were analyzed in Western blot analysis using an anti-GST monoclonal antibody. Specifically detected. Therefore, the S100A2 recombinant protein was purified and purified from E. coli transformed with the expression vector pGEX-4T-1-S100A2 of the present invention containing the human S100A2 gene, and the purified and purified S100A2 recombinant protein was GST-fusion protein. It was confirmed that it is produced in the form.

FIG. 2 illustrates that 1 M CaCl ₂ was added to obtain a final concentration of 2.5 mM GST-S100A2 fusion protein from the GST-S100A2 fusion protein protein isolated above, and then 0.02% thrombin was added at room temperature. Pure S100A2 protein obtained by treatment for 24 hours was extracted on the gel by electrophoresis on SDS-PAGE gel. Specifically, the SDS-PAGE gel from which the GST-S100A2 recombinant protein was separated was stained with 25 mM CuCl ₂ solution, and the GST-S100A2 recombinant protein which was not bound to Cu ²⁺ ions was cut and extracted on the gel by electrophoresis. The recombinant protein was concentrated by dialysis with phosphate buffer. The concentration of the protein measured by the Bradford method in the concentrated final sample was 1.2 mg / ml.

In this example, the isolated and purified S100A2 recombinant protein was used as an antigen for enzyme-linked immunosorbent for screening and analyzing fusion cell lines producing mouse immunization and monoclonal antibody.

Example 3 Immunization of Mice

In order to obtain immunized mice for the production of fusion cell lines producing S100A2 monoclonal antibody, human S100A2 recombinant protein prepared in Example 2 was dissolved in phosphate buffer at a concentration of 10 μg / 100 μl and then the same volume of Freund Freund's complete adjuvant (FCA) was mixed well until emulsified and injected intraperitoneally of 6-8 week old Balb / c mice (prepared from the Institute of Biotechnology). Two weeks later, the same amount of human S100A2 recombinant protein as first immunized was mixed with the same amount of incomplete adjuvant (FIA) and injected into the same site of mice. After 4 to 5 days, a small amount of blood was drawn from the blood vessels of the eye region of the mouse, and the titer of the antibody was confirmed by ELISA as shown in FIG. 3 , and the titer was confirmed even in diluted serum more than 1,000-fold. 3 to 4 days before the cell fusion experiment, 10 μg of human S100A2 recombinant protein was dissolved in 0.85% physiological saline and injected into the vein and abdominal cavity of the mouse to double the immunity of the mouse and to be used for cell fusion. Mice were secured.

Example 4 Cell Fusion

In Example 3, the immunized mice were anesthetized with ether, and spleens located on the left side of the body were extracted, and finely crushed with a tissue homogenizer, followed by Hans' buffered saline solution, HBSS) was used to make a suspension, and the splenocytes obtained at this time were placed in a 15 ml centrifuge tube and centrifuged. The procedure was repeated twice to fully wash the spleen cells with HBSS.

On the other hand, as a parent cell of cell fusion, the SP2 / 0.Ag14 cell line (ATCC CRL-1581) was cultured in RPMI medium containing 10% fetal bovine serum (FBS) while maintaining a maximum density of about 5x10 ⁵ / ml. It was. The parental cells SP2 / 0.Ag14 were also washed by centrifugation twice using HBSS.

The suspended splenocytes and blast cells were resuspended in HBSS to 10 ml each, and the cells in each suspension were counted. Centrifugation of the two suspensions was carried out by centrifugation of 50 ml of mice to have 10 ⁸ spleen cells and 10 ⁷ blast cells. The mixture was mixed in a tube and centrifuged again to precipitate. Dissolve the precipitate in the centrifuge tube with your finger and disperse it, and let stand for 1 minute at 37 ℃, 1 ml of fusogen of 45% polyethylene glycol (PEG) -5% DMSO in HBSS for 1 minute Slowly applied over and gently shaken again for 1 minute. Thereafter, 9 ml of culture medium (RPMI 1640, Sigma) was slowly added over 3 minutes, and the culture medium (RPMI 1640) was slowly added while gently shaking again to finally bring the suspension volume to 50 ml. After centrifugation of the suspension again, the cell precipitates were resuspended in a selection medium (HAT medium, Sigma) at about 1 × 2 × 10 ⁵ / ml to secure a fusion cell population. 0.2 ml of each 96-well micro-titer plate was dispensed and maintained in a humidified carbon dioxide cell incubator maintained at 37 ° C.

Example 5 Selection of Fusion Cell Lines Producing Monoclonal Antibodies

Enzymatic binding using the human S100A2 recombinant protein isolated and purified from the E. coli transformant of Example 2 as an antigen to select fusion cells that specifically react with human S100A2 recombinant protein antigen from the fusion cell group prepared in Example 4 Immunosorbent assay was performed.

After the phosphate buffer in which the human S100A2 recombinant protein obtained in Example 2 was dissolved was added to a micro-titer plate, 50 μl (2 μg / μl) of each well was attached to the protein surface, Unreacted antigen was removed by washing with phosphate buffer-Tween 20 (PBST) solution. 50 μl of the culture solution of the fusion cells obtained in Example 4 was added to the plate and allowed to react for 1 hour, followed by sufficient washing with phosphate buffer-Tween 20 solution to remove unreacted culture. Goat anti-mouse IgG-horseradish peroxidase (HRP; Sigma) was added thereto and reacted at room temperature for 1 hour, followed by sufficient washing with PBST. Each well was washed with the peroxidase substrate solution (OPD), and the reaction was determined by measuring the absorbance at 492 nm with an ELISA reader.

From the above results, fusion cells secreting antibodies having a specific high binding capacity to human S100A2 recombinant protein antigen were first selected, and then the above-described procedure was repeated several times on the selected cells, thereby providing high binding capacity only to the human S100A2 recombinant protein antigen. And specifically responsive fusion cell populations. In order to separate monoclonal cells from the fusion cell population, restriction dilution was performed to establish a fusion cell line derived from one cell producing a good monoclonal antibody, and the titer of the antibody secreted from the supernatant of the established cell line was enzyme-linked immunosorbent. Analyzes were performed by assay (ELISA) ( FIG. 4A ). The fusion cell line clone finally obtained therefrom was named KK0724, and only the clone was purely freeze-dried and preserved. The fusion cell line KK0724, which secretes antibodies with high specific binding capacity to human S100A2 recombinant protein antigen, was deposited on February 1, 2001 with the Korea Biotechnology Research Institute Gene Bank (KCTC) (Accession No .: KCTC 0941BP).

After culturing the fusion cell line KK0724, the supernatant was used to measure the titer of the antibody by enzyme immunoassay, and the immunoglobulin subtype was determined using an immunoglobulin isotyping kit (Sigma). Was identified as IgG _2b ( FIG. 4B ) and the monoclonal antibody (subtype: IgG _2b ) isolated from fusion cell line KK0724 was named S100A2 # 7-4.

The fusion cell lines obtained in this example and the immunoglobulins they produce are shown in Table 1 below.

Fusion cell line Absorbance (492 nm) Subtypes of Immunoglobulins (Name) KK0724 1.120 IgG _2b (S100A2 # 7-4)

Example 6 Mass Production of Monoclonal Antibodies

In order to mass produce monoclonal antibodies against human S100A2 protein from the fusion cell line established in Example 5, 0.5 ml of pristane per mouse (Balb / c) was first injected intraperitoneally. After one week, 5 × 10 ⁶ fusion cell lines KK0724 were injected per mouse, and ascites fluid was collected from the abdominal cavity inflated mice. Since the ascites solution contained fusion cells grown at high concentration, the collected ascites solution was centrifuged at 10,000 rpm to precipitate the fusion cells, and only the supernatant was taken. The supernatant was stored at minus 70 ° C. until the antibody protein was purified.

In order to purify monoclonal antibody against human S100A2 recombinant protein from ascites liquid, saturated ammonium sulfate solution was added in equal amounts to the supernatant liquid from which the protein was precipitated. The precipitate isolated therefrom was dissolved in phosphate buffer, dialyzed in phosphate buffer for 24 to 42 hours and a single protein against human S100A2 protein using Protein G agarose affinity column (Biorad). After the cloned antibody was isolated, the amount of the antibody protein was quantified by a Bradford assay and stored at minus 70 ° C.

Example 7 Identification of Antigen-Antibody Reaction of Monoclonal Antibody of the Present Invention with S100A2 Protein

In Example 6, Western blot analysis was performed to determine specific reactivity of the human S100A2 recombinant protein of the purified and isolated monoclonal antibody from the fusion cell line KK0724.

To this end, first, a GST fusion recombinant protein of S100A1, S100A4 and S100A6 proteins having high homology with human S100A2 protein of the present invention was prepared ( FIG. 5A). The base sequences of the template cDNA and primer pairs used to amplify the respective genes as shown in Examples 1 and 2 to demonstrate the repeatability of the preparation of S100A1, S100A4 and S100A6 recombinant proteins are summarized in Table 2 . , The treated restriction enzyme was used Bam HI and Eco RI as in S100A2.

Forward primer sequence cDNA derived Estimated RT-PCR Product Backward primer sequence S100A2 SEQ ID NO: 1 HepG2, Hepatocellular Sarcoma Cell Line 301 bp SEQ ID NO: 2 S100A2 SEQ ID NO: 3 MDA-MB231, Human Breast Cancer Cell Line 310 bp SEQ ID NO: 4 S100A4 SEQ ID NO: 5 Human fetal liver tissue 322 bp SEQ ID NO: 6 S100A6 SEQ ID NO: 7 Human fetal liver tissue 289 bp SEQ ID NO: 8

While the S100A2 recombinant protein of the present invention consists of 97 amino acids and has a molecular weight of 38.7 kDa, the S100A1 protein fused to GST consists of 94 amino acids and has a molecular weight of about 38.3 kDa. In addition, the S100A4 and S100A6 proteins consist of 101 and 90 amino acids, respectively, and have molecular weights of 39.1 kDa and 37.9 kDa.

Expression vectors pGEX-4T-1-S100A1, pGEX-4T-1-S100A2, pGEX-4T-1-S100A4 and pGEX-4T-1, respectively, producing the S100A1, S100A2, S100A4 and S100A6 recombinant proteins prepared as described above. E. coli was transformed with -S100A6. Cell culture fluid of Escherichia coli which induced expression of recombinant protein and cell culture fluid of Escherichia coli transformed with pGEX-4T-1 vector expressing only GST as a control group were electrophoresed on a 10% SDS-PAGE gel and then electrophoresed to a membrane filter. Moved in the usual way. Membranes were blocked for 1 to 2 hours with 3% bovine serum albumin solution to reduce nonspecific responses exhibited by other proteins transferred to the membrane filter. To identify monoclonal antibodies that specifically react to human S100A2 recombinant protein, monoclonal antibody against S100A2 recombinant protein produced from fusion cell line KK0724 in Example 5 was added as a primary antibody and reacted at room temperature for 2 hours. . The membrane filter was washed three times with a phosphate buffer solution containing 0.5% bovine serum albumin, followed by goat anti-mouse immunoglobulin G (Goat-anti-mouse-) conjugated with horseradish peroxidase with a secondary antibody. IgG-HRP; ICN Inc., USA) was added and reacted at room temperature. The membrane filter was washed again with phosphate buffer solution containing 0.5% Tween 20, and then color development was induced using ECL (Enhanced chemiluminescence) western blotting solution (Amersham- Pharmacia Biotech Co., Ltd., Korea).

FIG. 5B shows the results of staining recombinant proteins GST-S100A1, GST-S100A2, GST-S100A4 and GST-S100A6 with Coomassie Blue after SDS-PAGE, lane M for standard molecular weight size label, lane 1 for GST, Lanes 2, 3, 4, and 5 represent GST-S100A1, GST-S100A2, GST-S100A4, and GST-S100A6 recombinant proteins isolated and purified from each E. coli transformed with the expression vector. Figure 5c is the result of Western blot analysis using an anti-GST monoclonal antibody (anti-GST IgG-HRP conjugate antibody, Novagen) as a specific antibody of the recombinant protein, Figure 5d is from the fusion cell line KK0724 of the present invention Western blot analysis was performed with the obtained anti-S100A2 monoclonal antibody.

From the results of FIG. 5 , it was confirmed that the monoclonal antibodies directed against the human S100A2 recombinant protein of the present invention are antibodies that specifically react only with the human S100A2 recombinant protein.

<Example 8> Confirmation of immunohistochemical staining of human breast cancer tissue and cervical cancer tissue of monoclonal antibody against human S100A2 recombinant protein

As another method of verifying the specificity of the monoclonal antibody against human S100A2 of the present invention, tissues of breast cancer and cervical cancer patients were confirmed by immunohistochemical staining using monoclonal antibodies.

First, tissues extracted from tumor patients were fixed in 10% neutral formalin buffer and embedded in dissolved paraffin. Paraffin-embedded tissue was thinly sliced with a microtome, and then the sections were fixed on a glass slide to remove paraffin with xylene and histoclear solvent. The sections were washed with 1 M Tris buffer and then monoclonal antibody against human S100A2 of the present invention diluted 200-fold with 1 M Tris buffer with primary antibody for 20 minutes at 40 ° C. The unreacted antibody was completely washed with 1 M Tris buffer solution, and then removed, and then avidin-horseradish peroxidase solution (Immunotech Co., Ltd.) at 10 ° C. Treatment was for minutes. After washing and removing the second antibody that did not react again, the fragment was reacted with 3-amine-9-ethylcarbazole solution containing 0.05% H ₂ O ₂ , a peroxidase substrate solution. Cross-stained with Meyer's hematoxylin and the slides were sutured and checked by light microscopy.

As a result, it was confirmed that a much larger amount of S100A2 protein antigen was present in tumor cells (T) of breast cancer tissues of FIG. 6A and tumor cells (T) of cervical cancer tissues of FIG. 6B compared to normal tissue cells (N). .

As described above, the S100A2 monoclonal antibody of the present invention, which specifically responds to the human S100A2 protein, binds specifically to tumor cells in cancer tissues excised from tumor patients such as breast cancer or cervical cancer and thus is antigen-antibody. Western blot analysis using immunosorbent binding or immunohistochemical staining can be applied in actual clinical diagnosis and laboratory. Therefore, using the S100A2 monoclonal antibody of the present invention, it is possible to clearly determine the extent and distribution of the S100A2 protein in various normal tissues and corresponding tumor tissues of the human body, and to confirm clinically accurate lesions. It can be used as an accurate and useful marker for diagnosing tumors.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Monoclonal antibody specific for human calcium binding protein S100A2 and fusion cell strain producing same <130> FPD / 200104-0051 <160> 9 <170> KopatentIn 1.71 <210> 1 < 211> 428 <212> DNA <213> Homo sapiens S100A2 <400> 1 ctgggtctgt ctctgccacc tggtctgcca cagatccatg atgtgcagtt ctctggagca 60 ggcgctggct gtgctggtca ctaccttcca caagtactcc tgccaagagg gcgacaagtt 120 caagctgagt aagggggaaa tgaaggaact tctgcacaag gagctgccca gctttgtggg 180 ggagaaagtg gatgaggagg ggctgaagaa gctgatgggc agcctggatg agaacagtga 240 ccagcaggtg gacttccagg agtatgctgt tttcctggca ctcatcactg tcatgtgcaa 300 tgacttcttc cagggctgcc cagaccgacc ctgaagcaga actcttgact tcctgccatg 360 gatctcttgg gcccaggact gttgatgcct ttgagttttg tattcaataa actttttttg 420 tctgttga 428 <210> 2 <211> 26 <212> ia l Sequence <220> <223> forward primer of S100A2 <400> 2 cgggatccat gtgcagttct ctggag 26 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of S100A2 <400> 3 cggaattctc agggtcggtc tgggca 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer of S100A1 <400> 4 cgggatccat gggctctgag ctggag 26 <210> 5 <211> 26 <212 > DNA <213> Artificial Sequence <220> <223> reverse primer of S100A1 <400> 5 cggaattctc aactgttctc ccagaa 26 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer of S100A4 <400> 6 cgggatccat ggcgtgccct ctggag 26 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse p rimer of S100A4 <400> 7 cggaattctc atttcttcct gggctg 26 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer of S100A6 <400> 8 cgggatccat ggcatgcccc tggatcagg 29 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of S100A6 <400> 9 cggaattctc agcccttgag ggcttcat 28

Claims (10)

Monoclonal antibody that specifically binds to the human calcium binding protein S100A2 and is produced by the fusion cell line KK0724 (Accession Number: KCTC 0941BP). The monoclonal antibody of claim 1, wherein the subclass type of antibody is IgG _2b . The monoclonal antibody of claim 1, wherein S100A2 is a protein expressed in large quantities in breast and cervical cancer tissues. Fusion cell line KK0724 (Accession Number: KCTC 0941BP), which produces the monoclonal antibody of claim 1. 1) injecting the fusion cell line of claim 4 into the abdominal cavity of a mouse; 2) collecting ascites fluid from the abdominal cavity inflated mice; 3) A method of mass-producing a monoclonal antibody against human S100A2 of claim 1, comprising separating the monoclonal antibody protein against human S100A2 protein from the ascites fluid. The diagnostic kit for diagnosing cancer by using the monoclonal antibody against human S100A2 of claim 1 as a component and measuring the expression level of S100A2 protein in a biological sample through an antigen-antibody binding reaction. The method of claim 6, wherein the antigen-antibody binding reaction is subjected to an enzyme linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), a sandwich assay, a western blot on a polyacrylamide gel, or A diagnostic kit characterized by the method of immunoblot analysis. The diagnostic kit of claim 6, wherein the cancer is a skin-related tumor, breast cancer, squamous cell carcinoma of the larynx, or cervical cancer. The diagnostic kit of claim 6, wherein the monoclonal antibody is provided in the form of a protein chip immobilized on a micro substrate. 7. The diagnostic kit of claim 6, further comprising a secondary antibody and a chromogenic substrate labeled with a suitable buffer, standard antibody, chromophore or fluorescent substance.