KR20180001538A - Staphylococcus aureus-Specific Novel Monoclonal Antibody, Hybridoma For Producing The Antibody, Method For Detecting The Same Comprising The Antibody And Kit For Detecting The Same - Google Patents

Abstract

The present invention relates to a Staphylococcus aureus-specific novel monoclonal antibody, hybridoma for producing the same, a detection composition containing the same, and a detection method and a detection kit including the same. The monoclonal antibody according to the present invention can selectively recognize only Staphylococcus aureus in a sample so as to be usefully used for the detection diagnosis of Staphylococcus aureus.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel monoclonal antibody specific to Staphylococcus aureus, a hybridoma producing the hybridoma, a detection composition containing the antibody, a detection method and a detection kit therefor, and a kit for detecting a Staphylococcus aureus-Specific Novel Monoclonal Antibody Detecting The Same Comprising The Antibody And Kit For Detecting The Same}

The present invention relates to a novel monoclonal antibody specific to Staphylococcus aureus, a hybridoma producing the same, a detection composition containing the same, a detection method and a detection kit.

Food poisoning is an acute or chronic disease caused by ingesting foods containing natural poisons or harmful substances, and is accompanied by symptoms such as fever, nausea, vomiting, diarrhea and abdominal pain. Food poisoning can be classified into bacterial food poisoning, chemical food poisoning, natural poisoning food poisoning, and food poisoning by microbial toxic metabolites. There are many kinds of toxic substances belonging to each class. Toxic substances are widely distributed in many foods even though they are not harmful for immediate health. Chronic poisoning is caused by mutagenic and teratogenic allergic reactions It may be the cause.

Most of the food poisoning is bacterial food poisoning caused by bacteria, which includes infectious food poisoning by the pathogenic E. coli and poisonous food poisoning by pathogen - generated toxins.

Infectious food poisoning is caused by ingesting a large amount of living harmful bacteria. Therefore, when the food is heated and eaten, the bacteria are killed, so they are not poisoned. However, poisonous food poisoning remains toxic even if the bacteria die, so the remaining toxin is poisoned when the food is heated.

Staphylococcus aureus is a typical pathogenic bacterium that can cause collective food poisoning in these foods.

Staphylococcus aureus is a gram-positive condition aerobic granuloma forming colonies that are normally colonized by healthy human nose and skin. Approximately 20% to 30% of the population is colonized with Staphylococcus aureus at any given time. Staphylococcus aureus bacteria, also often referred to as "starfish", "starfish aureus" or "s. Aureus", are known to cause mild infections (eg, rashes, Opportunity is considered an infectious agent.

In addition, Staphylococcus aureus infection can cause a variety of diseases or conditions, and non-limiting examples of such diseases or conditions include bacterial infections, soft tissue saliva, eyelid infections, food poisoning, joint infections, skin infections, Lacunar skin syndrome, toxic shock syndrome, pneumonia, osteomyelitis, endocarditis, meningitis and abscess formation.

Therefore, it is necessary to quickly diagnose whether or not the staphylococcus aureus is contaminated.

Currently, there are traditional methods that use various biochemical test methods as identification methods that can be used in hospitals and industries (food companies, food companies and dairy companies), but it is an inefficient method requiring a lot of time and manpower.

Molecular biological methods such as polymerase chain reaction (PCR) have been used as a diagnostic method to quickly identify the infection with Staphylococcus aureus and contamination in foods. However, since the technique and precision are required, It is an unsuitable method for industrial application. In addition, although immunological methods such as enzyme immunoassay (EIA) are widely used, non-specificity appears, resulting in inaccurate test results or additional confirmation tests

Accordingly, it is urgently required to quickly and accurately determine the presence of Staphylococcus aureus, a food poisoning-causing pathogen in food, and to ensure the stability of food.

The present inventors have made an effort to develop a method for detecting Staphylococcus aureus. As a result, after immunizing a mouse with a Staphylococcus aureus cell antigen, the immune cells producing the antibody were isolated and fused with tumor cells (myeloma cells) to produce a specific antibody against Staphylococcus aureus The present inventors have completed the present invention by confirming the effect of specifically detecting the S. pneumoniae by using the monoclonal antibody produced by these cells.

Accordingly, it is an object of the present invention to provide monoclonal antibodies against Staphylococcus aureus cell wall-binding protein fractions.

Another object of the present invention is to provide a hybridoma cell (Accession No. KCTC 12700BP) that produces the above monoclonal antibody.

It is still another object of the present invention to provide a composition for detecting Staphylococcus aureus .

Still another object of the present invention is to provide a method for detecting Staphylococcus aureus .

It is yet another object of the present invention to provide a Staphylococcus aureus detection kit.

It is still another object of the present invention to provide a pharmaceutical composition for treating or preventing a disease caused by Staphylococcus aureus.

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, the present invention provides a monoclonal antibody against a Staphylococcus aureus cell wall-binding protein fraction produced by a hybridoma of Accession No. KCTC 12700BP and a hybridoma Lt; / RTI >

As used herein, the term "monoclonal antibody" is a term known in the art and refers to a highly specific antibody directed against a single antigenic site.

The antibody of the present invention is a specific antibody to Staphylococcus aureus, and can include an antigen-binding fragment of an antibody molecule as well as a complete antibody form.

A complete antibody is a structure having two full-length light chains and two full-length heavy chains, each light chain linked by a disulfide bond with a heavy chain. The heavy chain constant region has gamma (gamma), mu (mu), alpha (alpha), delta (delta) and epsilon (epsilon) types and subclasses gamma 1 (gamma 1), gamma 2 ), Gamma 4 (gamma 4), alpha 1 (alpha 1) and alpha 2 (alpha 2). The constant region of the light chain has the kappa and lambda types (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WB Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).

An antigen binding fragment of an antibody molecule means a fragment having an antigen binding function and includes Fab, F (ab ') 2, F (ab') 2, Fv and the like. Fabs in the antibody fragment have one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1) of a heavy chain. Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. The F (ab ') 2 antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Recombinant techniques for generating Fv fragments with minimal antibody fragments having only heavy chain variable regions and light chain variable regions are described in PCT International Publication Nos. WO88 / 10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO 88/09344. The double-chain Fv is a non-covalent bond, and the variable region of the heavy chain and the light chain variable region are connected to each other. The single-chain Fv generally has a variable region of the heavy chain and a variable region of the short chain through a peptide linker, Or directly connected at the C-terminus to form a dimer-like structure like the double-stranded Fv. Such an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, a Fab can be obtained by restriction of the whole antibody to papain, and F (ab ') 2 fragment can be obtained by cleavage with pepsin) Can be produced through recombinant DNA technology.

The term "heavy chain ", as used herein, refers to a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and a full length heavy chain comprising three constant region domains CH1, CH2 and CH3 And fragments thereof.

The term "light chain ", as used herein, refers to both the full length light chain and its fragments, including the variable region domain VL and the constant region domain CL comprising an amino acid sequence with sufficient variable region sequence to confer specificity to the antigen do.

Unlike polyclonal antibodies, which typically contain different antibodies directed against different epitopes, monoclonal antibodies are directed against a single determinant on the antigen.

Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and have other advantages that are not contaminated by other immunoglobulins because they are synthesized by fusion culture.

The monoclonal antibody may be prepared by a hybridoma method (see Kohler and Mislstein (1976) European Journal of Immunology 6: 511-519) or a phage antibody library (Clackson et al, Nature, 352: 624 -628, 1991, etc.) techniques. Generally, hybridoma cells that secrete monoclonal antibodies are made by fusing immune cells and cancer cell lines from immunologically appropriate host animals, such as mice injected with antigen proteins. The cell fusion of these two groups is fused using a method known in the art such as polyethylene glycol, and the antibody producing cells are proliferated by a standard culture method. After subcloning by limited dilution to obtain a uniform cell population, hybridoma cells capable of producing an antigen-specific antibody are cultured in vitro or in vivo.

The term " hybridoma " as used herein refers to a cell made by artificially fusing two different kinds of cells. Hybridoma cells are produced by fusing two or more homologous cells or xenogeneic cells using a substance that causes cell fusion such as polyethylene glycol or a virus of a specific species. By forming a hybridoma cell, different functions of different cells can be obtained It can be integrated into one cell.

Such hybridoma cells can be produced by fusing cancer cells that normally proliferate even in vitro, and large cells having a specific differentiation trait extracted from a living body. A typical hybridoma is a lymphocyte hybridoma, which is a hybridoma cell in which a myeloma cell and a B cell are fused, a T cell that is a tumor cell, a hybridoma cell that is a tumor cell thereof, a T cell that produces a lymphocaine (a physiologically active substance) Hybridoma cells that are tumor cells thereof, etc. It is obvious that the hybridoma cell line of the present invention can be fused and cultured under optimal conditions for effectively producing the monoclonal antibody of the present invention according to the selection of a person skilled in the art.

In a preferred embodiment of the present invention, mouse spleen cells were obtained and fused with mouse myeloma cells, followed by selection of only hybridoma cells identified as Staphylococcus aureus antibody. November 3, 2014 (accession number KCTC 12700BP).

The monoclonal antibody produced by the hybridoma cells of the present invention may be used as a cell culture solution without purification. However, in order to obtain the best results, the monoclonal antibody produced by the hybridoma cells of the present invention may be purified in high purity (for example, 95% or more). Such purification techniques may include, for example, gel electrophoresis, dialysis, salt precipitation, chromatography, and the like.

According to a preferred embodiment of the present invention, the antigen recognized by the antibody of the present invention comprises (a) dissolving Staphylococcus aureus; And (b) fractionating cell wall-associated proteins from the lysate of (a), and has a size of about 75 kDa.

In addition, the strain of Staphylococcus aureus is not limited as long as the antibody of the present invention specifically recognizes it. Preferably, the strain of Staphylococcus aureus is a strain of Staphylococcus aureus with accession number ATCC 25923.

According to a preferred embodiment of the present invention, the heavy chain of the antibody is of the IgG2b type and the light chain of the antibody is of the lambda type.

According to another aspect of the present invention, there is provided a composition for detecting Staphylococcus aureus comprising the above monoclonal antibody.

Since the composition of the present invention includes the above-mentioned monoclonal antibody, the duplication thereof is omitted in order to avoid the excessive complexity of the present specification.

According to another aspect of the present invention, the present invention provides a method for detecting Staphylococcus aureus , comprising contacting said monoclonal antibody with a specimen sample to confirm formation of an antigen-antibody complex.

According to a preferred embodiment of the present invention, confirmation of the formation of the antigen-antibody complex can be confirmed by enzyme immunoassay (ELISA), Western blotting, immunofluorescence, immunohistochemistry staining, (RIA), Immunoprecipitation Assay, Immunodiffusion Assay, Complement Fixation Assay, and Protein Chip, which are used in the present invention. Lt; / RTI > group.

As used herein, the term "antigen-antibody complex" means a conjugate of Staphylococcus aureus and an antibody recognizing the presence or absence of Staphylococcus aureus in a sample. Preferably the antigen-antibody complex is a complex of Staphylococcus aureus and a monoclonal antibody according to the invention.

The confirmation of the formation of the antigen-antibody complex can be confirmed by enzyme immunoassay (ELISA), Western blotting, immunofluorescence, immunohistochemistry staining, flow cytometry, (RIA), Immunoprecipitation Assay, Immunodiffusion Assay, Complement Fixation Assay, Protein Chip, or Kit, and the like, but are not limited thereto . The enzyme immunoassay (ELISA) includes direct ELISA using a labeled antibody recognizing an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody recognizing a capture antibody in a complex of an antibody recognizing an antigen attached to a solid support ELISA, a direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of an antibody and an antigen attached to a solid support, a method of reacting with another antibody recognizing an antigen in a complex of an antibody and an antigen attached to a solid support And an indirect sandwich ELISA using a labeled secondary antibody that recognizes this antibody.

Labels that enable qualitative or quantitative measurement of the formation of antigen-antibody complexes include, but are not necessarily limited to, enzymes, minerals, ligands, emitters, microparticles, redox molecules, and radioisotopes . Such enzymes include, but are not limited to,? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, Such as GDPase, RNase, glucose oxidase and luciferase, phosphofructokutase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decarboxylase, But are not limited to.

The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. The luminescent material includes, but is not limited to, acridinium ester, luciferin, luciferase, and the like. The fine particles include, but are not limited to, colloidal gold, colored latex and the like. The redox molecules include ferrocene, ruthenium complex compounds, Biology hydrogen, quinone, Ti ions, Cs ions, diimide, 1,4-benzoquinone, _{hydroquinone, K 4 W (CN) 8} , [Os (bpy) 3] 2 ⁺ , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^4-, and the like. The radioisotope compartments include ^{3 H, 14 C, 32 P} , 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re , and are not limited to .

According to a preferred embodiment of the present invention, the sample is any one selected from the group consisting of tissue, cell, whole blood, serum, plasma, cerebrospinal fluid, colostrum, joint fluid, saliva, feces, cell culture supernatant and food.

The term "sample" used in the present specification is intended to include not only foods for which the detection of Staphylococcus aureus is to be detected but also tissues, cells, whole blood, serum, plasma, cerebrospinal fluid, , Spinal cord, etc.), colostrum, joint fluid, saliva, defecation, cell culture supernatant or supernatant, and the like. The biological sample can be collected from various animals such as a human, a pig, a hamster, a blue fox, an emu, a deer, a dog, a guinea pig, a horse, a Lercus marcus monkey, an ostrich, a rabbit and a rat.

The method of the present invention uses the above-described monoclonal antibody, and redundant description thereof is omitted in order to avoid the excessive complexity of the present specification.

According to another aspect of the present invention, the present invention provides a detection kit for detecting Staphylococcus aureus , which comprises said monoclonal antibody.

As long as the monoclonal antibody of the present invention, as well as Staphylococcus aureus, can be selectively recognized, the kit of the present invention may contain other antibodies such as polyclonal antibodies, chimeric antibodies, humanized antibodies, monoclonal antibodies Antibody fragments can also be used together.

In addition, the kit of the present invention may include tools and reagents used for immunological analysis. Such tools and reagents include a carrier, a labeling substance capable of generating a detectable signal, a solubilizer, a cleaning agent, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring the enzyme activity and a reaction terminator. Such carriers include, but are not limited to, soluble carriers, such as physiologically acceptable buffers known in the art, such as PBS; Insoluble carrier such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal , Agarose, and combinations thereof.

Assay systems for use in the kits of the invention include, but are not limited to, ELISA plates, dip-stick devices, immunochromatographic test strips, split-split immune assay devices, flow-through devices, etc. do.

Since the kits of the present invention include the above-described monoclonal antibodies, duplicate descriptions thereof are omitted in order to avoid the excessive complexity of the present specification.

According to another aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing a disease caused by Staphylococcus aureus, which comprises the monoclonal antibody as an active ingredient.

In addition, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier or ingredient.

In addition, the pharmaceutical composition may also include antibiotic drugs such as streptomycin, penicillin, and vancomycin, preferably antibiotic drugs (e.g., ).

The pharmaceutical composition is suitably adjusted depending on the purpose of use, the intended use, the patient's condition, age, sex, body weight, prescription drug and disease, and is usually in the range of 0.1 to 100 mg / kg Includes monoclonal antibodies in body weight.

The pharmaceutical composition may be used for parenteral or oral use, and parenteral routes may include intravenous, intra-muscular, intra-dermal, subcutaneous, intraperitoneal, intraperitoneal, peritoneal, topical, intra-nasal administration, or inhalation spray.

The composition may further comprise at least one additive selected from the group consisting of pharmaceutically acceptable excipients, disintegrants, binders, and glidants. The additive is not particularly limited, Capsules, liquid preparations or granules.

The diseases caused by Staphylococcus aureus include, for example, infectious diseases caused by infection with Staphylococcus aureus infection, preferably food poisoning, bacteremia, soft tissue salts, eyelid infections, joint infections, Skin infection, thermal skin syndrome, toxic shock syndrome, pneumonia, osteomyelitis, endocarditis, meningitis or abscess formation.

Since the composition of the present invention includes the above-mentioned monoclonal antibody, the duplication thereof is omitted in order to avoid the excessive complexity of the present specification.

The monoclonal antibody according to the present invention is capable of selectively recognizing only Staphylococcus aureus in the sample, and thus can be usefully used for diagnosis of Staphylococcus aureus .

Figure 1 shows SDS-PAGE results of cell wall-binding proteins.
Fig. 2 shows the results of ELISA using fractions of various strains (purified proteins) in order to confirm the specificity of the monoclonal antibody of the present invention.
FIG. 3 shows the result of ELISA using whole cells of various strains to confirm the specificity of the monoclonal antibody of the present invention.
FIG. 4 shows the results of ELISA using Staphylococcus aureus isolated from various foods and humans to confirm the detection of Staphylococcus aureus in a sample using the monoclonal antibody of the present invention.
FIG. 5 shows the result of Western blotting using crude protein extracts of various strains in order to confirm the strain specificity of the monoclonal antibody of the present invention.
Figure 6 shows the results of Western hybridization in order to identify Staphylococcus aureus antigen for the monoclonal antibody of the present invention.
FIG. 7 is a graph showing the sensitivity of the monoclonal antibody of the present invention. To confirm the sensitivity of the monoclonal antibody of the present invention, various concentrations of Staphylococcus aureus were coated on an ELISA plate and the antibody (7D6) of the present invention or the commercially available Staphylococcus aure ELISA using anti-mouse antibody (SA com).

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Experimental Methods and Materials

Separation of cell wall fraction

The cell wall fraction of Gram-positive bacteria was isolated using a method known in the art (Jonquieres et al ., 1999). Briefly, pellets of 1 ml exponentional phase cells were washed once with PBS. And then washed once with TS buffer (10 mM Tris HCl pH 6.9, 10 mM MgCl ₂ , 0.5 M sucrose). After addition of lysis solution (TS buffer containing 60 mg / ml mutanolysin, 250 mg / ml RNAseA, 2 mM serine protease inhibitor AEBSF), the cells were incubated at 30 ° C for 1 hour. The supernatant was settled with 16% TCA and stored.

Prepared antigen-based mouse (Balb / c) immunization

The antigen was emulsified using Freud ' s Complete adjuvant in an amount equivalent to 50 쨉 g of the antigen, and the antigen was firstly injected into the peritoneal cavity of the mouse, followed by incomplete adjuvant And boosted. Splenocytes were obtained from mice after boosting 4 times.

In addition, mouse serum was obtained, and antibody titer was analyzed by ELISA using whole cells.

Fabrication of hybridoma cells

Splenocytes obtained from immunized mice were fused with myeloma cells (SP2 / 0-Ag 14) using PEG (50%) to prepare hybridoma cells. Thereafter, unfused cells were removed using a medium supplemented with HAT.

* Hybridoma cells were plated on expanded medium (DMEM + 10% FBS + mouse thymus extract) via a limited dilution method and analyzed for titer by ELISA.

Mass production of monoclonal antibodies using cell culture

Clones producing monoclonal antibodies were cultured in a specific medium (DMEM) and the concentrations and titers of the monoclonal antibodies in the supernatants were analyzed.

Mass production of monoclonal antibody using in vivo model (multiple liquid)

To mass-produce monoclonal antibodies, 250 μl Incomplete adjuvant was intraperitoneally injected into 8-week old mice (Balb / c). After 2 weeks, 5x106 cells were injected and ascites were obtained 7-10 days later.

Pure Isolation of Monoclonal Antibody (MAb)

The monoclonal antibody was purified by mass spectrometry using protein A or protein G affinity chromatography. The monoclonal antibody was purified by mass spectrometry using a multiple liquid or hybridoma cell culture supernatant.

Analysis of specificity of MAbs by Indirect ELISA

A variety of whole cell cultures (Overnight culture) were resuspended in carbonate coating buffer and then added to 96-well ELISA plates and stored overnight at refrigeration temperature. Reacted with Ab diluted to various concentrations and reacted with AP- or HRP-conjugated anti-Rabbit Ab. Thereafter, a soluble substrate was added to confirm the enzyme reaction, and the absorbance was measured at an appropriate wavelength.

Analysis of specificity of MAb using Western blot

The protein (overnight culture protein, cell wall fraction) used as an antigen was separated by SDS-PAGE. Protein was transferred to PVDF membrane and reacted with prepared MAb. Then, insoluble substrate was added to confirm enzyme reaction.

A comparative test for the specificity of commercially available MAb and MAb

ELISA and Western blotting were performed on the bacteria to be tested. At this time, the MAbs already developed and the novel MAb of the present invention were simultaneously applied to confirm the specificity of the MAb.

Example 1: Staphylococcus aureus ( Staphylococcus aureus ) Preparation of antigen

The present inventors cultured Staphylococcus aureus strain (Accession No. ATCC 25923) and used it as an antigen. More specifically, after culturing the strain, the culture supernatant was collected and centrifuged to obtain a precipitate. The resulting precipitate was washed once with PBS once with TS buffer (10 mM Tris HCl pH 6.9, 10 mM MgCl ₂ , 0.5 M sucrose). After lysis buffer (TS buffer containing 60 mg / ml mythanosine, 250 mg / ml RNAse A, 2 mM serine protease inhibitor AEBSF) was added and incubated at 30 ° C for 60 minutes, The supernatant was obtained by centrifugation, and the precipitate was stored in 16% TCA and used as an antigen.

In addition, qualitative and quantitative determination of the purified protein was confirmed using SDS-PAGE (Fig. 1).

Example 2: Preparation of hybridoma cells producing monoclonal antibodies against Staphylococcus aureus

2-1. Obtain antibody-producing cells

The present inventors immunized mice (Balb / c) with the antigen obtained in Example 1 to induce mouse immunity against Staphylococcus aureus strain (Accession No. ATCC 25923), and then immunized cells were obtained.

Briefly, the antigen was emulsified using Freud's complete adjuvant in an amount equivalent to 50 쨉 g of the antigen obtained in Example 1, and the mice were injected with the antigen in the peritoneal cavity first, then injected with an incomplete adjuvant every two weeks . Splenocytes were obtained from mice after boosting 4 times.

In addition, mouse serum was obtained, and antibody titer was analyzed by ELISA using whole cells.

2-2. Production and mass production of hybridoma cells

The present inventors prepared hybridoma cells by fusing spleen cells obtained from the immunized mouse of Example 2-1 with myeloma cells (SP2 / 0-Ag 14) using PEG (50%). Thereafter, unfused cells were removed using a medium supplemented with HAT.

2-3. Screening of hybridoma cells and monoclonal antibody isolation

We plated on expanded medium (DMEM + 10% FBS + mouse thymus extract) via a limited dilution method to select hybridoma cells and then analyzed the titer by ELISA.

In addition, clones producing monoclonal antibodies were cultured in a specific medium (DMEM), and the concentrations and titers of the monoclonal antibodies in the supernatants were analyzed.

Finally, a hybridoma cell producing a monoclonal antibody to the selected Staphylococcus aureus strain was selected and deposited on Nov. 03, 2014 in the Korean Cell Line Research Foundation (Accession No. KCTC 12700BP).

On the other hand, in order to mass-produce the monoclonal antibody, the present inventors intraperitoneally injected 250 μl of an incomplete adjuvant into an 8-week old mouse (Balb / c). After 2 weeks, 5x10 < ⁶ > cells were injected and ascites was obtained 7-10 days later.

The monoclonal antibody was purified by mass spectrometry using protein A or protein G affinity chromatography. The monoclonal antibody was purified by mass spectrometry using a multiple liquid or hybridoma cell culture supernatant.

Example 3: Isotype confirmation of monoclonal antibody against Staphylococcus aureus and identification of monoclonal antibody specificity

3-1. Isotype confirmation of monoclonal antibody

The present inventors confirmed the isotype of the monoclonal antibody against Staphylococcus aureus produced by the hybridoma of the accession number KCTC 12700BP, and the results are shown in Table 1 below.

As shown in Table 1, the heavy chain of the monoclonal antibody of the present invention was of the IgG2a type and the light chain was of the lambda type.

3-2. Identification of monoclonal antibody specificity

To confirm the specificity of the monoclonal antibody against Staphylococcus aureus produced by the hybridoma of accession number KCTC 12700BP, the present inventors conducted an ELISA using fractions (purified protein) and whole cells of various strains And the results are shown in FIGS. 2 and 3. FIG.

As shown in Fig. 2, the monoclonal antibody of the present invention was confirmed to react only with the fraction of Staphylococcus aureus among the fractions of various strains.

Further, as shown in Fig. 3, the monoclonal antibody of the present invention can be used in a variety of strains (Fig. 3A shows comparative confirmation with gram negative bacteria, Fig. 3B shows the reaction in Staphylococcus aureus, Of the total cells of Staphylococcus aureus strains.

Example 4: Detection of Staphylococcus aureus using a monoclonal antibody

The present inventors have conducted extensive studies to confirm the detection of Staphylococcus aureus in a sample using a monoclonal antibody against Staphylococcus aureus produced by a hybridoma of accession number KCTC 12700BP, ELISA was performed using Staphylococcus aureus and the results are shown in Fig.

As shown in Fig. 4, the monoclonal antibody of the present invention was found to react with Staphylococcus aureus isolated from various foods (Fig. 4a) and human (Fig. 4b).

In order to confirm the strain specificity of the monoclonal antibody against Staphylococcus aureus produced by the hybridomas of the accession number KCTC 12700BP, the present inventors also used crude protein extracts of various strains to obtain Western And the results are shown in Fig.

As shown in Fig. 5, it was confirmed that the monoclonal antibody of the present invention reacts only with Staphylococcus aureus among protein crude extracts of various strains.

On the other hand, the present inventors conducted Western hybridization in order to identify Staphylococcus aureus antigen against the monoclonal antibody of the present invention, and the results are shown in FIG.

As shown in FIG. 6, it was confirmed that the antibody of the present invention specifically reacted with an antigen having a size of about 75 kDa.

Further, in order to confirm the sensitivity of the monoclonal antibody of the present invention, the inventors of the present invention coated an ELISA plate with various concentrations of Staphylococcus aureus, and then, using the antibody of the present invention or commercially available Staphylococcus aureus ELISA was performed using the antibody, and the results are shown in Fig.

As shown in FIG. 7, the detection limit of the antibody of the present invention was confirmed to be 10 ² CFU / ml or less, which is about 100 times higher than that of a commercially available antibody as a control.

Korea Biotechnology Research Institute KCTC12700BP 20141103

Claims (6)

Monoclonal antibody 7D6, produced by a hybridoma deposited with accession number KCTC 12700BP and recognizing a cell wall-associated protein as an antigen, having a molecular weight of 75 kDa. A monoclonal antibody 7D6, produced by a hybridoma deposited with accession number KCTC 12700BP and recognizing a Staphylococcus aureus cell wall-associated protein having a molecular weight of 75 kDa as an antigen, And contacting the sample with a sample to confirm the formation of an antigen-antibody complex. ≪ Desc / Clms Page number 19 > The method according to claim 2, wherein the formation of the antigen-antibody complex is confirmed by an enzyme immunoassay (ELISA), Western blotting, immunofluorescence, immunohistochemical staining, flow cytometry, , Immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assay, immunodiffusion assay, complement fixation assay, and protein chip. RTI ID = 0.0 > 1, < / RTI > The method according to claim 2, wherein the specimen is at least one selected from the group consisting of tissue, cell, whole blood, serum, plasma, cerebrospinal fluid, colostrum, joint fluid, saliva, feces, cell culture supernatant and food. A cell wall-associated protein, produced by a hybridoma deposited with accession number KCTC 12700BP and having a molecular weight of 75 kDa, is recognized as an antigen and forms an antigen-antibody complex A detection kit for detecting Staphylococcus aureus , comprising monoclonal antibody 7D6. A cell wall-associated protein, produced by a hybridoma deposited with accession number KCTC 12700BP and having a molecular weight of 75 kDa, is recognized as an antigen and forms an antigen-antibody complex A composition for detecting Staphylococcus aureus , comprising monoclonal antibody 7D6.

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