KR101806522B1 - Pathogenic Escherichia coli-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 novel monoclonal antibody specific to pathogenic Escherichia coli, a hybridoma producing the same, a detection composition containing the same, a detection method and a detection kit. The monoclonal antibody according to the present invention can selectively recognize only pathogenic Escherichia coli in a sample, and thus can be usefully used for the detection of pathogenic Escherichia coli.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel monoclonal antibody specific for pathogenic Escherichia coli, a hybridoma producing the hybridoma, a detection composition containing the same, a detection method and a detection kit Comprising The Antibody And Kit For Detecting The Same}

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

Hemorrhagic Escherichia coli, an aerobic or anaerobic anaerobic Gram negative bacterium that causes hemorrhagic diarrhea and hemolytic uremic syndrome in humans, is known to be the causative organism of the O157: H7 serotype in E. coli. Food poisoning caused by E. coli O157: H7 occurs worldwide in North America, Europe, Asia and Africa after the first occurrence in the United States in 1982. The E. coli O157: H7 is mainly present in the intestines of cattle, Recently, the occurrence of food poisoning through contaminated water, crude oil, vegetables and apple juice as well as ground beef has recently been reported. Therefore, rapid detection of pathogenic E. coli O157: H7 in food Development is urgently required.

The isolation and identification of pathogenic Escherichia coli O157: H7 in food is accomplished by isolating the bacterium from the sample, performing a specific biochemical test and serologically confirming the toxin production ability and several pathogenic factors of the isolate. Performing these standard methods requires some tricky procedures and much time. First, a special enrichment medium and a selective medium are used for the isolation of bacteria from the sample, which makes it difficult to isolate the causative bacteria according to the components of the sample and the competitive action of other bacteria contained in the sample. In particular, pathogenic Escherichia coli O157: H7 can cause food poisoning even with a very small amount (about 100 grains), and it is very difficult to isolate bacteria by the standard separation method because a small number of ordinary samples are present.

On the other hand, after isolation of the bacteria, a characteristic biochemical property test is carried out to identify and confirm the serotype of the bacteria using the antiserum. In this process, the cross-reactivity of the antiserum with other intestinal bacteria and other serogroups of E. coli remains a problem. Therefore, identification of the pathogenic factors of the isolated bacteria is required for confirmation of the final E. coli O157: H7. Among the pathogenic factors, verotoxin is verified by cell culture using verocell, and other pathogenic factors are obtained by enzyme-linked-polymerase chain reaction (PCR) or DNA hybridization using gene probe . It is very useful for the final confirmation of the bacterium because it is possible to identify four pathogenic factors in the recently developed multiplex PCR. However, the multiplex PCR method is difficult to apply directly to the sample, and is troublesome because it has to undergo a DNA extraction process, and there is a problem in retrieving a large number of samples at once.

Therefore, it is urgently required to develop a more rapid and sensitive method for rapidly diagnosing whether or not contamination by pathogenic Escherichia coli is caused.

The present inventors have made an effort to develop a method for detecting pathogenic Escherichia coli. As a result, immunological cells producing the antibody were isolated after immunizing a mouse with the pathogenic Escherichia coli O157: H7 cell antigen, and fused with tumor cells (myeloma cells) to produce a hybridoma that produces a specific antibody against pathogenic Escherichia coli The present inventors completed the present invention by confirming the effect of detecting the pathogenic Escherichia coli specifically, rapidly and easily using the monoclonal antibody produced by the cells.

Accordingly, an object of the present invention is to provide a monoclonal antibody against the pathogenic Escherichia coli outer membrane-binding protein fraction.

Another object of the present invention is to provide a hybridoma cell (Accession No. KCTC 12699BP) that produces the above monoclonal antibody.

It is still another object of the present invention to provide a composition for detecting pathogenic Escherichia coli .

It is still another object of the present invention to provide a method for detecting pathogenic Escherichia coli .

It is still another object of the present invention to provide a pathogenic Escherichia coli detection kit.

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

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 pathogenic Escherichia coli outer membrane-binding protein fraction produced by a hybridoma of Accession No. KCTC 12699BP and a hybridoma cell producing the monoclonal antibody do.

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 against pathogenic Escherichia coli, and may 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, only spleen cells of a mouse were obtained and fused with mouse myeloma cells, and then only hybridoma cells in which pathogenic E. coli antibodies were identified were selected. (Accession No. KCTC 12699BP).

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 a pathogenic Escherichia coli; And (b) fractionating outer membrane-associated proteins from the lysate of (a), and has a size of about 50 kDa.

In addition, as long as the antibody of the present invention specifically recognizes, the pathogenic Escherichia coli strain is not limited. For example, the pathogenic Escherichia coli may be selected from the group consisting of enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli, enteroinvasive E. coli, , Enterohaemorrhagic E. coli) and enterococcal enterococci (EAEC, Enteroadherent E. coli), preferably enterochromic E. coli.

The pathogenic Escherichia coli is selected from the group consisting of enterohemorrhagic Escherichia coli O157: H7, intestinal hemorrhagic Escherichia coli O17: H18, intestinal hemorrhagic Escherichia coli O26: H11, intestinal hemorrhagic Escherichia coli O11: H8, intestinal hemorrhagic Escherichia coli O104: H21, O104: H4, and preferably is enterohemorrhagic Escherichia coli O157: H7. In the present invention, strains of intestinal hemorrhagic Escherichia coli having a deposit number of ATCC 35150 were used.

According to a preferred embodiment of the present invention, the heavy chain of the antibody is of the IgG3 type, and the light chain of the antibody is of the Kappa type.

According to another aspect of the present invention, there is provided a composition for detecting pathogenic Escherichia coli 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, there is provided a pathogenic Escherichia coli detection method 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.

The term "antigen-antibody complex " as used herein means a combination of pathogenic Escherichia coli and an antibody recognizing the presence or absence of pathogenic Escherichia coli in the sample. Preferably, the antigen-antibody complex is a complex of a pathogenic Escherichia coli and a monoclonal antibody according to the present 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.

As used herein, the term "sample" refers to not only food for which the presence or absence of pathogenic Escherichia coli is detected, but also tissues, cells, whole blood, serum, plasma, cerebrospinal fluid, tissue autopsy samples (brain, skin, lymph nodes, But are not limited to, biological samples such as colostrum, joint fluid, saliva, defecation, cell culture supernatant or supernatant. 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.

Since the method of the present invention utilizes the above-described monoclonal antibody, redundant contents thereof are 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 pathogenic Escherichia coli, which comprises said monoclonal antibody.

As long as the monoclonal antibody of the present invention, as well as the pathogenic Escherichia coli can be selectively recognized, the kit of the present invention may contain other antibodies such as a polyclonal antibody, a chimeric antibody, a humanized-antibody, a fragment of a monoclonal antibody Can 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 a pathogenic Escherichia coli comprising the above 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 and may be a powder, A liquid, a granule, or the like.

The diseases caused by the pathogenic Escherichia coli include, for example, infectious diseases caused by infectious diseases caused by pathogenic Escherichia coli infection, preferably food poisoning, hemorrhagic colitis, intestinal hemorrhage, diarrhea, stool, vomiting, fever, Of skin infections.

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 can selectively recognize only pathogenic Escherichia coli in a sample, and thus can be usefully used for the detection of pathogenic Escherichia coli.

Figure 1 shows SDS-PAGE results of the outer membrane-binding protein.
Figure 2 shows the results of ELISA using fractions of various strains (purified protein) and whole cells in order to confirm the specificity of the monoclonal antibody of the present invention.
FIG. 3 shows the result of ELISA using various E. coli strains in order to confirm the specificity of the monoclonal antibody of the present invention.
FIG. 4 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 5 shows the results of Western hybridization to identify the pathogenic Escherichia coli antigen to the monoclonal antibody of the present invention.
FIG. 6 is a graph showing the sensitivity of the monoclonal antibody of the present invention to ELISA plates coated with various concentrations of pathogenic Escherichia coli, followed by using the antibody (2C6) of the present invention or a commercially available pathogenic Escherichia coli antibody (ECcom) And the result of ELISA is shown.

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 present invention as defined by the appended claims. It will be obvious to you.

Experimental Methods and Materials

Isolation of outer membrane fraction

The outer membrane fraction of Gram-negative bacteria was isolated using a method known in the art (Thein et al ., 2010). Briefly, a precipitate of 25 ml of an overnight culture was prepared, 500 μl of 0.2 M Tris-HCl pH 8, 1 M sucrose, 1 mM EDTA was added to the obtained precipitate, and 100 μl lysozyme, Sigma-Aldrich) (5 mg / ml in dH ₂ O) was added and incubated at room temperature for 5 minutes. Then, 2 ml of dH ₂ O was added and incubated at room temperature for 20 minutes to confirm formation of spheroplast Respectively. To the resultant, 3 ml of Tris-HCl pH 8, 2% (w / v) Triton X-100, 10 mM MgCl ₂ and 50 μl DNase I (Applichem) (1 mg / ml in dH ₂ O) The precipitate was obtained by centrifugation at 85,000xg for 30 minutes. The precipitate was washed with 750 μl of buffer (Tris-HCl pH 8, 2% (w / v) Triton X-100, 10 mM MgCl ₂ ) and then resuspended at 85,000 × g for 20 min at 4 ° C. 500 ㎕ dH then washed three times using ₂ O, and stored at -20 ℃. The fraction, Outer Membrane-associated proteins, was isolated and purified and used as an antigen

Prepared antigen-based mouse (Balb / c) immunization

The antigen was emulsified using Freud's complete adjuvant in an amount equivalent to 50 항 of the antigen, and the antigen was firstly injected into the peritoneal cavity of the mice, followed by boosting 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.

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.

To screen for hybridoma cells, the titer was analyzed by ELISA after plating on extended medium (DMEM + 10% FBS + mouse thymus extract) via a limited dilution method.

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: Pathogenic Escherichia coli Escherichia coli ) Preparation of antigen

The present inventors cultured the strain O157: H7 (Accession No. ATCC 35150), which is a pathogenic Escherichia coli, and used it as an antigen. More specifically, after culturing the strain, the culture supernatant was collected and centrifuged to obtain a precipitate. To the obtained precipitate was added 0.2 M Tris-HCl pH 8, 1 M sucrose, 1 mM EDTA, lysozyme (Sigma-Aldrich) (5 mg / ml in dH ₂ O) After incubation, 2 ml of dH ₂ O was added and incubated at room temperature for 20 minutes to confirm formation of spheroplast. To the resultant, 3 ml of Tris-HCl pH 8, 2% (w / v) Triton X-100, 10 mM MgCl ₂ and 50 μl DNase I (Applichem) (1 mg / ml in dH ₂ O) The precipitate was obtained by centrifugation at 85,000xg for 30 minutes. The precipitate was washed with 750 μl of buffer (Tris-HCl pH 8, 2% (w / v) Triton X-100, 10 mM MgCl ₂ ) and then resuspended at 85,000 × g for 20 min at 4 ° C. 500 ㎕ dH then washed three times using ₂ O, and stored at -20 ℃. The fraction, Outer Membrane-associated proteins, was used as an antigen.

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

Example 2: Preparation of hybridoma cells producing monoclonal antibodies against pathogenic Escherichia coli O157: H7

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 the pathogenic Escherichia coli O157: H7 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.

A hybridoma cell that finally produced a monoclonal antibody against the pathogenic Escherichia coli O157: H7 strain was selected and deposited on Nov. 03, 2014 (KCTC 12699BP ).

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 Identification of Isotype of Monoclonal Antibody against Pathogenic Escherichia coli O157: H7 and Identification of Monoclonal Antibody Specificity

3-1. Isotype confirmation of monoclonal antibody

The present inventors confirmed the isotype of the monoclonal antibody against the pathogenic Escherichia coli O157: H7 produced by the hybridoma of accession number KCTC 12699BP, and the results are shown in Table 1 below.

As shown in Table 1, it was confirmed that the heavy chain of the monoclonal antibody of the present invention was IgG3 type and the light chain was kappa type.

3-2. Identification of monoclonal antibody specificity

(FIG. 2A, purified protein) and whole cells (FIG. 2B) to identify the specificity of the mAb to the pathogenic E. coli O157: H7 produced by the hybridoma of accession number KCTC 12699BP, , And the results are shown in Fig.

As shown in Fig. 2, the monoclonal antibody of the present invention was confirmed to react only with the pathogenic Escherichia coli O157: H7 among the fractions of various strains and whole cells.

In addition, as shown in Fig. 3, it was confirmed that the monoclonal antibody of the present invention reacts only with pathogenic Escherichia coli O157: H7 strains among various strains.

Example 4: Detection using E. coli O157: H7 monoclonal antibody

The present inventors conducted Western blotting using crude protein extracts of various strains in order to confirm the strain specificity of the monoclonal antibody against the pathogenic Escherichia coli O157: H7 produced by the hybridoma of the accession number KCTC 12699BP And the results are shown in Fig.

As shown in Fig. 4, it was confirmed that the monoclonal antibody of the present invention reacts only with pathogenic Escherichia coli O157: H7 among the crude protein extracts of various strains.

Meanwhile, the present inventors performed Western hybridization in order to identify the pathogenic E. coli O157: H7 antigen to the monoclonal antibody of the present invention, and the results are shown in FIG.

As shown in FIG. 5, it was confirmed that the antibody of the present invention specifically reacted with an antigen having a size of about 50 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 the pathogenic E. coli O157: H7 and then incubated with an antibody of the present invention or a commercially available pathogenic Escherichia coli O157: H7 antibody , And the results are shown in FIG.

As shown in FIG. 6, the detection limit of the antibody of the present invention was confirmed to be 10 ³ CFU / ml or less, which is about 10 times higher than that of a commercially available antibody as a control.

Korea Biotechnology Research Institute KCTC12699BP 20141103

Claims (11)

Monoclonal antibody 2C6, which is produced by hybridoma cells deposited with accession number KCTC 12699BP and recognizes intestinal hemorrhagic Escherichia coli O157: H7 Outer Membrane-associated proteins with a molecular mass of 50 kDa as antigen. Monoclonal antibody 2C6, which is produced by hybridoma cells deposited with accession number KCTC 12699BP and recognizes the intestinal hemorrhagic E. coli O157: H7 Outer Membrane-associated proteins with a molecular weight of 50 kDa, And contacting the sample with a sample to confirm the formation of an antigen-antibody complex. The method of detecting Enterohaemorrhagic E. coli O157: H7. delete delete delete delete delete 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. (O7: H7 Outer Membrane-associated proteins), which are produced by hybridoma cells deposited with accession number KCTC 12699BP and have a molecular weight of 50 kDa, as antigens, to form an antigen-antibody complex A detection kit for detecting Enterohaemorrhagic E. coli O157: H7, comprising the monoclonal antibody 2C6 . (O7: H7 Outer Membrane-associated proteins), which are produced by hybridoma cells deposited with accession number KCTC 12699BP and have a molecular weight of 50 kDa, as antigens, to form an antigen-antibody complex Wherein the composition comprises a monoclonal antibody 2C6, wherein the antibody is selected from the group consisting of: < RTI ID = 0.0 > O157: < / RTI > H7 for detecting Enterohaemorrhagic E. coli .

Images