KR20200049387A - Monoclonal antibody specifically binding to OMP28 in Brucella suis, Hybridoma cell line procuding the same and use thereof - Google Patents

Abstract

The present invention relates to a monoclonal antibody specifically bound to OMP28 in Brucella suis, a hybridoma cell line producing the antibody, and a use thereof. More specifically, the present invention relates to a monoclonal antibody specifically bound to OMP28 in Brucella suis, a hybridoma cell line producing the antibody, a composition and a kit for detecting Brucella suis comprising the monoclonal antibody, and a method for detecting the Brucella suis using the monoclonal antibody. According to the present invention, Brucella suis is quickly and accurately analyzed and detected, such that accidents caused by biological weapons can be quickly responded.

Description

Monoclonal antibody specifically binding to OMP28 of Brucella, hybridoma cell line producing the antibody and its use {Monoclonal antibody specifically binding to OMP28 in Brucella suis, Hybridoma cell line procuding the same and use thereof}

The present invention relates to a monoclonal antibody that specifically binds to OMP28 of Brucella, a hybridoma cell line that produces the antibody, and a use thereof, and more specifically, a monoclonal antibody that specifically binds to OMP28 of Brucella. , A hybridoma cell line for producing the antibody, a composition, kit for detecting brucellella comprising the monoclonal antibody, and a method for detecting brucellella using the monoclonal antibody.

A high-risk pathogen is an infectious disease pathogen that can cause serious risks to the public's health when used for the purpose of bioterrorism or leaks to the outside by accidents, etc., as prescribed by Ordinance of the Ministry of Health and Welfare.

As a high-risk pathogen, Yersinia pestis ), Bacillus anthracis , Brucella melitencis , Brucella suis), non jeogyun (Burkholderia mallei), mellitic Oido cis bacteria (Burkholderia pseudomallei ), Clostridium botulinum , Shigella dysenteriae Type 1), Chlamydia psittaci , Coxiella burnetii ), Lactobacillus ( Francisella tularensis ), Typhus ( Rickettsia prowazekii ), Fever rickettsia ( Rickettsia rickettsii ), Coccidioides imitis ( Coccidioides) immitis ) and cholera ( Vibrio cholerae O1O139).

Of these, Brucella melitencis , Brucella suis ) is an aerobic gram-negative bacilli that causes brucellosis, and is known to have been developed as a biochemical weapon in many countries. Globally, the sense of crisis is heightened by IS terrorism and North Korea's practice of chemical and biological warfare training, and civil accidents have occurred in Korea with a large number of unspecified poisons such as pesticides, but harmful substances such as biological agents In the event of a crisis such as terrorism, a system that can promptly analyze harmful substances and make immediate decisions has not been established.

Brucellosis is one of the common infectious diseases caused by bacteria in the genus Brucella, and is designated as a statutory infectious disease. Brucella is also good at spreading air, and it is estimated that inhalation of 10 to 100 cells can cause disease in humans. Livestock such as cows, pigs, and dogs are the main sources of infection, and they can be infected by humans through the urine, milk, and tissues of infected animals, or by ingestion of unpasteurized milk or dairy products or contaminated meat. In humans, besides oral infection through raw milk, dairy products, etc., it is infected by the handling of infected carcasses, and has a characteristic tetanic fever. When a person is infected with this fungus, it has an incubation period of about 3 weeks, and there are systemic symptoms such as irregular fever, fatigue, boredom, and headache, and the fever at this time is called malta fever or mediterranean fever. . The disease has also occurred in Korea, and has spread mainly to oral and contact infections, and to people who have eaten unsterilized dairy products. Brucellosis is a nonspecific fever disorder similar to influenza. Fever, headache, muscle pain, joint pain, back pain, sweating, chills, general weakness, and fatigue are common symptoms. Digestive symptoms such as loss of appetite, nausea, vomiting, diarrhea, and constipation occur in 70% of adults and more rarely in children. Low back pain, lumbar tenderness can occur in 60% of cases due to various osteoarthritis of the spine. When one or both sacr oiliac joints are infected, back pain or hip pain intensifies when stress is applied to the sacroiliac joint on physical examination, and half of the patients are observed with hepatic and splenic zones.

There is no effective treatment for this disease, and it can be prevented by vaccination in livestock, but once an infected livestock is found, it must be slaughtered according to the law. In the case of humans, tetracycline, streptomycin, chloramphenicol, etc. are used for the treatment. The mortality rate is 2% or less, but if left untreated, it causes spondylitis and osteomyelitis.

Therefore, before the Brucella suis is infected with a person, it can be prevented from being infected with a person by detecting the relevant bacteria in a livestock, especially a pig, early, and quickly respond to the occurrence of a bioterrorism using the Brucellus. As a system for securing the safety of drinking water through, development of a method capable of specifically and quickly and accurately detecting brucellella is required.

On the other hand, Korean Patent Publication No. 10-2016-0121673 discloses a protein on a substrate bound with smooth LPS (lipopolysaccharide), which is an antigen derived from B. abortus 1119-3, which causes brucellella disease, and a protein on commarin dendrimer A fluorescence immunoassay kit for diagnosis of brucellosis comprising a condensate of A / G (protein A / G) is disclosed, but only a method for diagnosing brucellosis caused by bovine brassella abortus Therefore, there is still a need for a method for quickly and accurately detecting Brucella suis in pigs.

Accordingly, the present inventors completed the present invention by developing a monoclonal antibody capable of specifically detecting and / or diagnosing Brucella bacteria by specifically binding to OMP28 of Brucella suis .

The present invention is Brucella suis ) The objective is to provide a monoclonal antibody that specifically binds to OMP28 and a hybridoma cell producing the same.

Another object of the present invention is to provide a composition and kit for detecting Brucella suis comprising the aforementioned monoclonal antibody.

Another object of the present invention is to provide a method for detecting Brucella suis using the aforementioned monoclonal antibody.

In order to solve the above-described problems, the present invention is produced by the hybridoma cell line B2G12 (Accession No. KCTC18726P) or the hybridoma cell line F4H6 (Accession No. KCTC18727P), Brucella ( Brucella) suis ) provides a monoclonal antibody that specifically binds to OMP28.

The present invention may also provide a hybridoma cell line B2G12 with accession number KCTC18726P or a hybridoma cell line F4H6 with accession number KCTC18727P, which produces a monoclonal antibody that specifically binds to Brucella OMP28.

The present invention also provides a composition and kit for detecting brucellella, comprising a monoclonal antibody that specifically binds to the brucellella OMP28.

The present invention also capture antibody; And detection antibodies; Lateral flow immunoassay kit for detecting Brucella suis , wherein the capture antibody is produced by the hybridoma cell line F4H6 (Accession No. KCTC18727P) and is specific for Brucella suis OMP28. It is a monoclonal antibody that binds to the target, and the detection antibody is produced by the hybridoma cell line B2G12 (Accession No. KCTC18726P), and a side for detecting Brucella, a monoclonal antibody specifically binding to Brucella suis OMP28 Provide a flow analysis kit.

According to one preferred embodiment of the present invention, the lateral flow analysis kit comprises a sample pad, a conjugate pad, a nitrocellulose membran, an absorbent pad, and gold nanoparticles. It may further include.

The present invention also provides a method for detecting brucellella, comprising the step of detecting the formation of an antigen-antibody complex by contacting the monoclonal antibody specifically binding to the brucellella OMP28 with an isolated biological sample.

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

According to another preferred embodiment of the present invention, the formation of the antigen-antibody complex is an enzyme immunoassay (ELISA), Western Blotting, Immunofluorescence, Immunohistochemistry staining, flow cytometry. Flow cytometry, immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assay, immunodiffusion assay, lateral flow immunoassay, complement fixation method Assay) and a protein chip (Protein Chip).

The present invention is also described above, Brucella suis ) Provides a method for detecting brucellella using a lateral flow analysis kit for detection.

The monoclonal antibody according to the present invention is capable of detecting brucellella even in samples containing OMP28 at a concentration of about 1 ng / ml through high specific binding of brucellella to OMP28, a kit for detecting brucellella and a method for detecting brucellella Useful for By using the monoclonal antibodies and kits of the present invention to quickly and accurately analyze and detect Brucella, which corresponds to category B of the US Centers for Disease Control, among biological weapons, it is possible to quickly cope with accidents caused by biological weapons, thereby ensuring the safety of drinking water. It is possible.

1 shows a pBHA vector map for cloning the Brucella OMP28 gene sequence.
FIG. 2 (A) shows a pET21 vector map for expressing Brucella OMP28, and FIG. 2 (B) shows a pET32 vector map for expressing Brucellella OMP28.
Figure 3 shows the results of confirming the recombinant OMP28 gene by Brucellia through electrophoresis.
Figure 4 shows the results of confirming the water-soluble and insoluble protein before and after the induction of overexpression of the Brucella OMP28 protein through electrophoresis.
FIG. 5 shows the results of confirming OMP28 recombinant antigen purified from Brucella OMP28 water-soluble protein obtained after overexpression through electrophoresis.
Figure 6 shows the results of brucellella detection by lateral flow immunoassay using monoclonal antibodies B2G12, D2H4 and F4H6 according to the present invention.

As described above, before the Brucella suis is infected with a human, it can be prevented from being infected with a human by detecting the corresponding bacteria in a livestock, especially a pig, and quickly when a bioterrorism using the Brucella suicide occurs. To cope with this, development of a method capable of specifically and quickly and accurately detecting Brucella suis as a system for securing the safety of drinking water is required.

Accordingly, the present inventors sought to solve the above-mentioned problem by providing a monoclonal antibody capable of specifically detecting and / or diagnosing Brucella bacteria by specifically binding to OMP28 of Brucella bacteria. The monoclonal antibody according to the present invention can detect Brucella bacteria even in samples containing OMP28 protein at a concentration of about 10 ng / ml through high specific binding of Brucella bacteria to the OMP28 protein, thereby detecting Brucella bacteria kit and Brucella bacteria Useful for the method. By using the monoclonal antibodies and kits of the present invention to quickly and accurately analyze and detect Brucella, which corresponds to category B of the US Centers for Disease Control, among biological weapons, it is possible to quickly cope with accidents caused by biological weapons, thereby ensuring the safety of drinking water. It is possible.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art to which the present invention pertains may easily practice. The present invention can be implemented in many different forms and is not limited to the embodiments described herein.

Throughout this specification, when a part “includes” a certain component, it means that the component may further include other components, not to exclude other components, unless otherwise stated.

Throughout this specification, the description of “A and / or B” means “A or B, or A and B”.

As used herein, the terms “about”, “substantially”, and the like are used in or near the numerical values when manufacturing and material tolerances unique to the stated meanings are presented, to aid understanding of the present application Hazards are used to prevent unreasonable abuse by unscrupulous infringers of the disclosures that are either accurate or absolute.

The present invention is produced by the hybridoma cell line B2G12 (Accession No. KCTC18726P) or the hybridoma cell line F4H6 (Accession No. KCTC18727P), a monoclonal antibody specifically binding to Brucella suis OMP28, the monoclonal antibody. It provides a hybridoma cell producing and a method for producing the same.

As used herein, 'Brucellella OMP28' includes the protein itself, its genetically recombined proteins, their artificial variants and mutants, as well as the natural form of the protein and its functional equivalents.

The monoclonal antibody that specifically binds to the Brucellella OMP28 of the present invention can be prepared by a fusion method well known in the art (Kohler and Milstein (1976) European Jounral of Immunology 6). : 511-519). One of the two cell populations that are fused to make a 'hybridoma' that secretes monoclonal antibodies uses cells from an immunologically compatible host animal, such as a mouse injected with the Brucella OMP28 protein, and the other population As a cancer or myeloma cell line is used. These two populations of cells are fused by methods known in the art, such as polyethylene glycol, and then antibody-producing cells are propagated by standard tissue culture methods. After obtaining a homogeneous cell population by subcloning by a limited dilution technique, a hybridoma cell line capable of producing an antibody specifically binding to Brucella OMP28 is enzyme-linked immunosorbent assay. , ELISA) or Western blot analysis, and cultured in vitro or in vivo according to standard techniques. The in vivo bulk culture means that the hybridoma cell line is injected into the abdominal cavity of a mouse to induce high concentration of antibody production, and then is separated from ascites.

The monoclonal antibody produced by the hybridoma cell line may be used without purification, or purified by high purity (eg, 95% or more) according to a method well known in the art. As such a purification technique, for example, gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity antigen column chromatography, or the like may be used to separate from the culture medium or ascites.

Among the three hybridomas producing monoclonal antibodies according to the present invention, the two finally selected hybridomas were designated as B2G12 and F4H6, respectively, and as of October 25, 2018, Korea Research Institute of Bioscience and Biotechnology (Korean Collection for Korean Collection for Type Cultures, KCTC) deposited at the Biological Resource Center (BRC), the hybridoma cell line B2G12 was deposited with accession number KCTC18726P, and the hybridoma cell line F4H6 was deposited with accession number KCTC18727P. In the present invention, the monoclonal antibodies produced by the 'hybridoma cell line B2G12' and the 'hybridoma cell line F4H6' were named 'B2G12' and 'F4H6', respectively.

The advantage of using monoclonal antibodies to detect antigens is that they have a specific interaction by recognizing a single epitope. As described above, various approaches can be used to map epitopes involved in the interaction between antigen and antibody. For example, a fragment containing an epitope sequence is determined by immunoblotting and screening a fragment generated by digesting a bio-panning, antigenic polypeptide with a protease using a phage display peptide library, activated membrane or solid phase such as polyethylene pin The screening of the peptide array immobilized on, a competitive ELISA assay using a soluble peptide for the importance of each amino acid sequence residue in the epitope sequence, and the like.

The monoclonal antibody according to the present invention may be selected from IgG, IgA, IgM, IgE, and IgD isotypes, but may not be limited thereto.

According to an embodiment of the present invention, the IgG isotype may be selected from IgG1, IgG2, IgG3 and IgG4, but may not be limited thereto.

The term 'monoclonal antibody' as used herein refers to a highly specific antibody directed against a single antigenic site as a term known in the art. Typically, monoclonal antibodies are directed against a single determinant on an antigen, unlike polyclonal antibodies that contain different antibodies directed against different epitopes (antigen determinants). Monoclonal antibodies have the advantage of improving selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and also have the advantage of not being contaminated by other immunoglobulins because they are synthesized by hybridoma culture. In the present specification, an antibody against the Brucella OMP28 protein, which includes not only the entire antibody form but also a functional fragment of the antibody molecule. The whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected by a heavy chain and a disulfide bond. The heavy chain constant regions have gamma (γ), mu (μ), alpha (α), delta (δ), epsilon (ε) types, and subclasses gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3) ), Gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant regions of the light chain are of kappa (κ) and lambda (λ) 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)).

A functional fragment of an antibody molecule refers to a fragment having an antigen-binding function, and includes Fab, F (ab '), F (ab') ₂ , and Fv. Among antibody fragments, Fab has 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, and has one antigen-binding site. Fab 'differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. In the F (ab ') 2 antibody, the cysteine residue of the hinge region of Fab' forms a disulfide bond. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. Recombination techniques for generating Fv fragments are described in International Patent Publications WO 88/10649, WO 88/106630, WO88 / 07085, WO 88/07086 and WO 88 / 09344. dsFv (bi-chain Fv) is a non-covalent heavy chain variable region and a light chain variable region are connected, and scFv (single-chain Fv) is usually a covalent bond between the variable region of the heavy chain and the variable region of the single chain through a peptide linker or C-terminal Since it is directly connected to, it can form a dimer-like structure like dsFv.

Such antibody fragments can be obtained using proteolytic enzymes (e.g., restriction digestion of the entire antibody with papain yields Fab and cleavage with pepsin yields F (ab ') ₂ fragment), preferably It can be produced through genetic recombination technology. The antibody in the present invention is preferably in Fab form or in whole antibody form. In addition, the heavy chain constant region can be selected from any of the isotypes of gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). The light chain constant region can be of kappa or lambda type.

Further, the antibody of the present invention may be a chimeric antibody or a humanized antibody or a CDR-grafted antibody (CDR-grafted antibody) to further reduce the immunogenicity of the chimeric antibody.

Chimeric antibody refers to an antibody variable domain derived from a non-human animal (eg, mouse, rabbit, poultry, etc.), and the antibody constant domain refers to an antibody derived from human. do. Such chimeric antibodies may be produced by methods such as gene recombination known in the art.

Humanized antibodies (CDR-grafted antibodies) or human-derived antibodies (CDR-grafted antibodies) of animal-derived monoclonal antibodies in order to maintain the high affinity and specificity of animal-derived monoclonal antibodies and to reduce immunogenicity in the human body Refers to an antibody produced by implanting a complementarity determining region (CDR) into a human antibody, and selects the degree of humanization by considering the affinity for the antigen and the degree of immunity in the human body.

The present invention also provides a composition and / or kit for the detection of Brucella suis comprising a monoclonal antibody that specifically binds to Brucella OMP28 .

The present invention also capture antibody; And detection antibodies; Lateral flow immunoassay kit for detecting Brucella suis , wherein the capture antibody is produced by the hybridoma cell line F4H6 (Accession No. KCTC18727P) and is specific for Brucella suis OMP28. and enemy monoclonal antibody that binds to the detection antibody hybridoma cell line B2G12 (Accession No KCTC18726P) on the side for the Brucella bacteria detection monoclonal antibody specifically binding to Brucella bacteria (Brucella suis) OMP28, produced by Provide a flow analysis kit.

In the present invention, the lateral flow analysis kit may further include a sample pad, a conjugate pad, a nitrocellulose membran, an absorbent pad, and gold nanoparticles. have.

Specifically, the lateral flow analysis kit may include a structure in which a sample pad, a conjugate pad, a nitrocellulose membran, and an absorbent pad are sequentially connected.

Specifically, the lateral flow analysis strip sensor includes a structure in which a sample pad, a conjugate pad, a membrane and an absorption pad are sequentially connected, and the conjugate pad is a nanoparticle connected to a nanoparticle and a detection antibody that binds to the antigen- A detection antibody conjugate is included, and a test line, an antigen line, and a control line are sequentially formed from the conjugate pad to the absorption pad on the membrane, and a capture antibody binding to the antigen is immobilized on the test line. The antigen line is immobilized on the antigen line, and the secondary antibody binding to the detection antibody may be immobilized on the control line.

In the kit of the present invention, the "sample pad" means a pad capable of diffusion flow by receiving a sample to be analyzed, and is composed of a material having a porosity sufficient to receive and contain a sample to be analyzed. Examples of such porous materials include fibrous paper, microporous membranes made of cellulose materials, cellulose derivatives such as cellulose and cellulose acetate, nitrocellulose, glass fibers, naturally occurring cotton, textiles such as nylon, or porous gels, etc. , But is not limited to this.

In the kit of the present invention, the “conjugate pad” accommodates a sample that is diffused and moved from a sample pad, and at the same time, a pad containing a “nanoparticle-detecting antibody conjugate” in which a detection antibody binding to the nanoparticle and an antigen is connected to be. The conjugate pad, like the sample pad, is made of a material capable of diffusion flow.

The nanoparticles of “nanoparticle-detecting antibody conjugate” contained in the conjugate pad refer to nanoparticles that act as detectable labels. The nanoparticles are preferably nanoparticles of metal, and examples of the metal include gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iridium (Ir), and rhodium (Rh), Precious metals of ruthenium (Ru); Titanium (Ti), zirconium (Zr), hafnium (Hf), vanadium (V), niobium (Nb), tantalum (Ta), chromium (Cr), molybdenum (Mo), tungsten (W), ruthenium (Ru), a transition metal of osmium (Os); Metals such as iron (Fe), nickel (Ni), and cobalt (Co); Magnesium oxide (MgO), titanium dioxide (TiO ₂ ), vanadium pentoxide (V ₂ O ₅ ), metal oxides of zinc oxide (ZnO), and the like, but are not limited thereto. The nanoparticles are most preferably gold (Au) nanoparticles.

The detection antibody refers to an antibody that specifically binds to an antigen to be analyzed, and if it possesses binding specificity, it also means to include a fragment of the antibody.

The detection antibody may be a monoclonal antibody or a polyclonal antibody, and most preferably, a monoclonal antibody. In the present invention, monoclonal antibody B2G12 was used as a detection antibody.

The binding of the nanoparticles to the detection antibody includes, but is not limited to, ionic bonds, covalent bonds, metal bonds, coordination bonds, hydrogen bonds, and van der Waals bonds.

In the kit of the present invention, the “membrane” may be made of various materials through which the sample material can pass. Natural, synthetic, or synthetically modified naturally occurring substances, such as polysaccharides (such as cellulose materials, paper, cellulose derivatives such as cellulose acetate and nitrocellulose); Polyether sulfone; Polyethylene; nylon; Polyvinylidene fluoride (PVDF); Polyester; Polypropylene; Silica; Inorganic materials uniformly dispersed in the porous polymer matrix together with polymers such as vinyl chloride, vinyl chloride-propylene copolymer and vinyl chloride-vinyl acetate copolymer, such as inactivated alumina, diatomaceous earth, MgSO ₄ , or other inorganic fines matter; Naturally occurring (eg cotton) and synthetic (eg nylon or rayon) fabrics; Porous gels such as silica gel, agarose, dextran and gelatin; Polymer films, such as polyacrylamide; And the like.

Preferably the membrane comprises a polymeric material such as nitrocellulose, polyethersulfone, polyethylene, nylon, polyvinylidene fluoride, polyester and polypropylene.

In the kit of the present invention, the "absorption pad" may be located adjacent to or near the end of the membrane. Absorption pads generally accept fluid samples that travel through the entire membrane. The absorbent pad can help promote capillary action through the membrane and diffusion flow of the fluid.

In the kit of the present invention, the sample pad, conjugate pad, membrane and absorbent pad described above are sequentially connected on the same support. The support may be formed of any material as long as it can support and transport the sample pad, conjugate pad, membrane, and absorbent pad described above, but generally, the fluid of the sample diffusing through the membrane does not leak through the support. It is preferably liquid impermeable. Glass, for example; Polymer materials include, but are not limited to, polystyrene, polypropylene, polyester, polybutadiene, polyvinylchloride, polyamide, polycarbonate, epoxide, methacrylate, and polymelamine.

In the kit of the present invention, a test line, an antigen line, and a control line are sequentially formed on the membrane in the direction from the conjugate pad to the absorption pad.

In the kit of the present invention, a capture antibody binding to the antigen is fixed to the "test line". In the present invention, monoclonal antibody F4H6 was used as a capture antibody.

In the kit of the present invention, the antigen is fixed to the “antigen line”. Preferably, the antigen immobilized on the antigen line is immobilized via a capture antibody immobilized on the membrane. Rather than immobilizing the antigen directly on the membrane in the antigen line, the antigen can be exposed in an appropriate direction so that the binding with the nanoparticle-detecting antibody is facilitated by binding to the capture antibody and consequently detecting the antigen of the kit. Performance can be improved.

In the kit of the present invention, a secondary antibody binding to the detection antibody is immobilized on the “control line”. The secondary antibody of the control line binds to the nanoparticle-detecting agent or nanoparticle-detecting antibody-antigen complex and generates a detection signal for it.

In this specification, the term “antigen” refers to a substance to be analyzed for concentration or presence, for example, Brucella suis ) or OMP28 thereof.

The monoclonal antibody used in the kit for detecting brucellella of the present invention can also use a fragment of the monoclonal antibody as long as the antibody can selectively recognize brucellella OMP28. Such antibody fragments may include F (ab ') ₂ , Fab, Fab', Fv fragments, and the like.

The kit for detecting brucellosis may be prepared in the form of a protein chip to which the monoclonal antibody of the present invention is attached. The kit for detecting brucellella of the present invention may include a monoclonal antibody or fragment thereof that selectively recognizes brucellella OMP28, and a tool / reagent used for immunological analysis.

Tools / reagents used in the immunological analysis include suitable carriers, labeling substances capable of generating detectable signals, solubilizers, detergents, and the like. In addition, when the labeling substance is an enzyme, a substrate capable of measuring enzyme activity and a reaction stopper may be included.

Suitable carriers include, but are not limited to, soluble carriers, such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, Polymers such as polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharide, magnetic fine particles plated with metal in latex, other paper, glass, metal, agarose, and combinations thereof.

The assay system for use in the kit for detecting brucellella of the present invention is, but is not limited to, ELISA plates, dip-stick devices, immunochromatographic test strips and radiation splitting immunoassay devices, and flowthrough Devices and the like.

The invention also is contacted with the biological sample to remove the above-mentioned monoclonal antibody antigen- Brucella comprising the step of detecting the antibody complex formation bacteria (Brucella suis ) detection method.

In the method for detecting brucellella of the present invention, the biological sample may be selected from the group consisting of tissue, cells, whole blood, serum, plasma, cerebrospinal fluid, colostrum, joint fluid, saliva, feces, cell culture supernatant and food, but brucellella Any sample to check for infection or presence can be used without limitation.

In the method for detecting brucellella of the present invention, the term 'antigen-antibody complex' refers to a combination of brucellella OMP28 and monoclonal antibodies that recognize it, to confirm the presence of brucellella in the biological sample.

The antigen-antibody complex can be detected using a detection label. For example, it may be selected from enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, or radioisotopes, and is not particularly limited thereto.

Such enzymes include, for example, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, Hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphenolpyruvate decarboxylase, β-ratama And others, but are not limited to this. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoerytherin, phycocyanin, allopicocyanine, o-phthalaldehyde, fluorescamine, 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. Microparticles include, but are not limited to, colloidal gold, gold nanoparticles, and colored latex. Redox molecules include ferrocene, ruthenium complex, biogen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K ₄ W (CN) ₈ , [Os (bpy) ₃ ] ²⁺ _{, [RU (bpy) 3]} 2 + or ^{_{[MO (CN) 8] 4}} - and the like, without limitation. Radioisotopes include, but are not limited to, ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I or ¹⁸⁶ Re.

Formation of the antigen-antibody complex is a colorimetric method, an electrochemical method, a fluorimetric method, a luminometry, a particle counting method, a visual assessment, or It can be detected using a scintillation counting method or the like. For example, enzyme immunoassay (ELISA), Western Blotting, Immunofluorescence, Immunohistochemistry staining, Flow cytometry, Immunocytochemistry, Radioimmunoassay (RIA) , Immunoprecipitation Assay, Immunodiffusion assay, Lateral flow immunoassay, Complement Fixation Assay, and Protein Chip have.

More specifically, it can be detected through immunoprecipitation and immunoblotting, which are useful for recovering a small amount of the target protein.

Most specifically, the antigen-antibody complex can be detected using an enzyme-linked immunosorbent assay (ELISA). The ELISA is a direct ELISA using a labeled antibody recognizing an antigen attached to a solid support, an indirect ELISA using a labeled antibody recognizing a capture antibody in a complex of antibodies recognizing an antigen attached to a solid support, to a solid support Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the attached antibody and antigen, reacted with another antibody that recognizes the antigen in the complex of the antibody and the antigen attached to the solid support, and then recognizes this antibody And various ELISA methods, such as indirect sandwich ELISA using labeled secondary antibodies.

According to the ELISA detection method, a biological sample is contacted with a monoclonal antibody of the present invention coated on a solid support, such as a microtiter plate, membrane, test strip, or the like. As a specific example, the wells of a microtiter plate are coated with a monoclonal antibody of the present invention and a nonoccupied binding site is blocked with, for example, BSA, and then the wells of the coated plate are incubated with a sample sample, The presence of the antigen-antibody complex can then be determined. The presence of the antigen-antibody complex can be confirmed using an antibody specific for the antigen of the antigen-antibody complex, for example, a monoclonal antibody or polyclonal antibody that specifically binds to Brucella OMP28. The monoclonal antibody or polyclonal antibody may have a detection label, and when it does not have a detection label, it may be confirmed by treating with another antibody capable of detecting these monoclonal antibodies or polyclonal antibodies.

As a specific example, a capture monoclonal antibody attached to a support is reacted with a biological sample, and then a monoclonal antibody that specifically binds to Brucellia OMP28 is bound to measure a detection label signal or generate a detectable signal in these complexes. Brucellella can be detected by adding a secondary antibody having a label capable of measuring a detection label signal therefrom.

According to one embodiment of the present invention, Brucella suis may be detected using the above-described lateral flow analysis kit.

Although one embodiment of the present invention has been described above, the spirit of the present invention is not limited to the embodiments presented herein, and those skilled in the art to understand the spirit of the present invention may add elements within the scope of the same spirit. However, other embodiments may be easily proposed by changing, deleting, adding, or the like, but this will also be considered to be within the scope of the present invention.

Brucellella  Development of immunogen for the production of monoclonal antibodies

1-1. Brucellella OMP28  Gene synthesis

The brucellella OMP28 gene sequence is as shown in Table 1 below, and the biosynthesis was commissioned to obtain a gene synthesis to obtain a cloned brucellella OMP28 plasmid DNA in the pBHA vector (FIG. 1). In the case of gene synthesis, for cloning with a protein expression vector, HindIII (AAGCTT) restriction enzyme was added to the 5 'end and XhoI ( CTCGAG ) restriction enzyme was added to the 3' end.

Gene name Sequence Sequence number Brucella OMP28 AAGCTT AAC ACT CGT GCT AGC AAT TTT CTC GCA GCC TCA TTT TCC ACA ATC ATG CTC GTC GGC GCT TTC AGC CTG CCC GCT TTC GCA CAG GAG AAT CAG ATG ACG ACG CAG CCC GCG CGC ATC GCC GTC ACC GGG GAA GGC ATG TCG AC CCC GAT ATG GCC ATT CTC AAT CTC TCG GTG CTA CGC CAG GCA AAG ACC GCG CGC GAA GCC ATG ACC GCG AAT AAT GAA GCC ATG ACA AAA GTG CTC GAT GCC ATG AAG AAG GCC GGC ATC GAA GAT CGC GAT CTC CAG ATC AAT ATC CAG CCG ATT TAT GTC TAT CCT GAC GAC AAG AAC AAC CTG AAA GAG CCT ACC ATC ACC GGC TAT TCT GTA TCC ACC AGT CTC ACG GTT CGC GTG CGC GAA CTG GCC AAT GTT GGA AAA ATT TTG GAT GAA TTC GTC GTT AAT CAG GGC GGT GAT TTG AAC CTG GTC AAT GAT AAT CCC TCC GCC GTG ATC AAC GAG GCG CGC AAG CGC GCA GTG GCC AAT GCC ATT GCC AAG GCG AAG ACG CTT GCC GAC GCT GCA GGC GTG GGG CTT GG CGT GTC CG AGT GAA CTG AGC CGC CCG CCC ATG CCG ATG CCA ATT GCG CGC GGA CAG TTC AGA ACC ATG CTA GCA GCC GCA CCG GAC AAT TCC GTG CCG ATT GCC GCA GGC GAA AAC AGC TAT AAC GTA TCG GTC AAT GTC GTT TTT GAA ATC AAG CTCG AG One

1-2. Genetically modified plasmid production

The protein expression vector is a pET21b vector (Merck) containing 6 histidines (Fig. 2 (A)) and Trx (Thioredoxin), known as a solubility enhancer, to increase the water solubility of the antigen, and 6 histidines are fused. The pET32 vector (Figure 2 (B)) was used. Purification of Brucella OMP28, pET21b, and pET32b plasmid DNA using Nucleogen's Plasmid Purification Mini Kit, followed by TAKARA's HindIII, XhoI restriction enzyme and reaction buffer, followed by cleavage at 37 ° C for 3 hours Did. After the restriction enzyme cleavage reaction was completed, the cellulase OMP28 and the protein expression vectors pET21b and pET32b were subjected to 1% agarose gel electrophoresis, and compared to the DNA size markers, the gene product at 747bp for Brucella OMP28, 5443bp for pET21b vector, and 5900bp for pET32b vector. After cutting, DNA was purely separated using a gel extraction kit of Nucleogen.

After purification, the DNA of the Brucella OMP28, pET21b, and pET32b vectors was subjected to DNA quantification, and the molar concentrations of the Brucella OMP28 and pET21b vectors, and the Brucella OMP28 and pET32b vector DNA were 5: 1, respectively, using NEB's T4 DNA ligase, 25 The reaction was carried out for 2 hours or more at ℃. After the ligation was completed, the reactant was transformed into E.coli (DH5α) and spread on an LB plate medium containing antibiotic ampicillin and incubated for 16 hours in an incubator at 37 ° C. The resulting single colonies were grown in LB liquid medium to purify plasmid DNA. The purely isolated Brucella OMP28 gene recombinant DNA was again digested with HindIII and XhoI restriction enzymes to confirm that pET21, pET32 vector and Brucella OMP28 were separated (FIG. 3).

1-3. Brucellella OMP28  Protein overexpression of gene

Cloning was performed on the expression vector pMAL (NEB) vector containing maltose binding protein (MBP) to improve the purity and yield of the Brucella OMP28 recombinant antigen. After confirming the OMP28-pMAL vector recombinant gene clone, the Brucella OMP28 recombinant gene plasmid DNA was purified purely, and the purified Brucella OMP28 recombinant DNA was BL21 (DE3), a receiving strain for recombinant protein expression, to induce overexpression of the protein. Transformed.

For transformation, after dissolving a cell (competent cell) capable of reacting with the antibody of BL21 (DE3), 1 ul of the Brucellella OMP28 recombinant DNA was added to the BL21 (DE3) cell and mixed well. Cells were placed in a heat block set to 42 ° C., subjected to heat shock for 90 seconds, and then taken out. The cells were plated on LB plate medium containing antibiotic ampicillin and cultured for more than 16 hours in a 37 ° C. incubator. The inoculated single colony was inoculated into LB medium containing antibiotics and cultured, followed by inoculation of the culture medium sufficiently grown in LB medium to 1% in new LB medium, followed by cultivation, and the Brucella OMP28 recombinant protein was expressed in BL21 (DE3) strain. In order to induce the cells, when the value of OD 600 grew to 0.8 level, IPTG (final concentration 0.5 mM) was added and further cultured in a 25 ° C. incubator for 3 hours to induce overexpression of Brucella OMP28 protein.

The overexpressed BL21 (DE3) cells were harvested by centrifugation, the harvested cells were suspended with lysis buffer, decomposed through ultrasonic crushing, and the cell extract was centrifuged to take the supernatant to separate the soluble protein, and the supernatant After taking the remaining pellets again, sufficiently dissolve the pellets with 8M urea (Urea) until all the pellets were released, and centrifuging to obtain a supernatant to separate insoluble proteins.

Before and after inducing overexpression of the recombinant OMP28 recombinant protein, the water-soluble protein and the insoluble protein were mixed with 5 times concentrated SDS sample loading buffer, boiled in boiling water for 5 minutes, and then the sample was subjected to electrophoresis by SDS-PAGE. As a result of staining with Coomassie Blue, as shown in FIG. 4, it was confirmed that the Brucella OMP28 recombinant protein was overexpressed in the form of a water-soluble protein more than an insoluble protein with a size of about 70 kDa.

1-4. Brucellella OMP28  Antigen purification

The affinity chromatography was performed to isolate purely the Brucella OMP28 recombinant protein cloned into the pMAL vector. For affinity chromatography, amylose resin to which maltose was bound was used. First, the amylose resin was sufficiently washed and equilibrated with a buffer solution, and then overexpressed in the amylose resin, the brucellella OMP28 water-soluble protein solution obtained by crushing was flowed to adsorb the brucellella OMP28 protein to the resin. The washing solution was sufficiently flowed to remove non-specific proteins from the column other than Brucellella OMP28 protein. After equilibrating with the buffer solution again, 10 mM maltose solution was flowed to elute the Brucellia OMP28 antigen adsorbed on the column. The purified Brucellia OMP28 recombinant protein was confirmed by staining with Coomassie Blue after SDS-PAGE electrophoresis. As a result of the verification, it was confirmed that the recombinant antigen was purified from the Brucellella OMP28 in a more pure manner than the antigen purified by the previous pET32 vector. (Figure 5).

Brucellella OMP28  That specifically binds to proteins Monoclonal antibody  production

2-1. Mouse Immunity

To obtain immunized mice for antibody production, the previously purified Brucellia OMP28 recombinant antigen was diluted to a concentration of 1 mg / ml, and then the same volume of FCA (Freund's complete adjuvant, Sigma) was mixed with the antigen until emulsification. After the emulsion was prepared, it was injected into the soles of BALB / c mice 6-8 weeks of age. After the first immunization, 3 additional immunizations were performed every 2 weeks, and an additional emulsion was prepared by mixing FIA (Freund's incomplete adjuvant, Sigma) and Brucellia OMP28 antigen instead of FCA and preparing an emulsion.

2-2. Cell fusion

After the final immunization, the mice were further raised for 1 week and sacrificed using CO ₂ gas. The sacrificed mice were fixed and surgical lymph nodes and tweezers were used to extract the popliteal lymphnode. The extracted lymph nodes were washed with sterile RPMI 1640 medium, grinded, filtered through a 40 μm mesh (SPL) to remove fat, and lymph node cells were recovered. The collected lymph node cells were precipitated by centrifugation at 1,200 rpm for 3 minutes, washed twice with RPMI 1640 medium, and resuspended in 20 ml of the same medium to release well.

The collected lymph node cells and cultured SP2 / 0 myeloma cells were stained with tryptophan blue solution (Sigma) and observed under a microscope to measure living cells. After mixing each cell so that the ratio of SP2 / 0 myeloma cells and lymph node cells was 1: 5, the cells were precipitated by centrifugation at 1,200 rpm for 3 minutes. The supernatant was removed by inhalation and the cells containing the precipitated cells were lightly beaten to mix the cells well. After 1 ml of 50% (w / v) PEG 1500 (Invitrogen) solution was slowly added for 1 minute, 1 ml of RPMI 1640 medium was added for 30 seconds, 3 ml for 30 seconds, and 17 ml was slowly added for 1 minute, followed by 10 ml. Added. The cells were precipitated by centrifugation at 1,200 rpm for 5 minutes, the supernatant was removed by inhalation, and the precipitated cells were suspended in RPMI 1640 medium containing HAT (hypoxanthine, aminopterin, thymidine), and then 4 96 well plates (SPL) Each well was dispensed at 100 μl per well, and 200 μl per well was added to RPMI 1640 medium containing HAT, and cultured in the presence of 37 ° C. and 5% CO ₂ to continuously observe cell proliferation and condition.

2-3. Monoclonal antibody  Selection of fusion cells to produce

In order to select fusion cells that specifically responded only to the Brucella OMP28 antigen, the purified Brucella OMP28 protein was selected by ELISA using the antigen. The Brucella OMP28 protein was diluted in 50 mM sodium hydrogen carbonate buffer (pH 9.6) to a concentration of 100 µg / ml, then dispensed 100 µl in a 96-well immunoplate (SPL) and coated overnight at 4 ° C. for 16 hours. After attaching to the plate surface, the unattached antigen was removed by washing with PBS-0.05% Tween 20 solution. After culturing the fusion cell line in each well, 100 µl of the supernatant obtained was added and reacted for 1 hour, followed by washing with PBS-0.05% Tween 20 solution sufficiently to remove the unreacted culture solution, and recognize IgG of mice obtained from goats The antibody and HRP (horseradish peroxidase, Sigma) 100 µl of the complex was added, reacted at room temperature for 1 hour, and then sufficiently washed with PBS-0.05% Tween 20 solution. After putting TRP solution of substrate solution of HRP into each well after washing, after about 10 minutes, the reaction was stopped with 0.16 MH ₂ SO ₄ solution, and the absorbance at a wavelength of 450 nm was measured using an ELISA reader. Thus, 49 cell lines were obtained.

The well absorbance wells of the well plates were selected, diluted in a new 96-well plate by limiting dilution, and further cultured for 5-7 days, and ELISA was performed to isolate and establish cells producing Brucella OMP28 antibody. The final selected clones were B2G12, D2H4, and F4H6, and ascites were made by injecting into the abdominal cavity of the cell line BALB / c mice that secrete monoclonal antibodies, and monoclonal antibodies were purified from the produced ascites.

The results of the ELISA test are shown in Table 2 below.

Confirmation of antigen-antibody response through Lateral flow immunoassay

In this example, the test for detecting brucellosis by lateral flow immunoassay was performed using the three monoclonal antibodies finally selected in Examples 2-3.

The strip test for detecting brucellella consists of four pads: a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. As a marker, 20 nm gold nanoparticles were used. A complex in which gold nanoparticles and antibodies were combined was used as a detector, and the Brucellella OMP28 purified protein was diluted to a concentration of 10 ng / ml and 1 ng / ml, respectively, and injected into a sample pad to capture the antibody. Combined to confirm the detection signal.

The detection antibody and the capture antibody were tested in combination as shown in Table 3 below.

Test 1 Test 2 Test 3 Test 4 Capture antibody B2G12 F4H6 F4H6 D2H4 Detection antibody D2H4 B2G12 D2H4 B2G12

As a result, it was confirmed that the combination of monoclonal antibodies in Test 2 was most effective in detecting Brucella (Fig. 6).

Korea Research Institute of Bioscience and Biotechnology KCTC18726P 20181025 Korea Research Institute of Bioscience and Biotechnology KCTC18727P 20181025

<110> KOREA WATER RESOURCES CORPORATION <120> Monoclonal antibody specifically binding to OMP28 in Brucella          suis, Hybridoma cell line procuding the same and use thereof <130> 1064374 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 759 <212> DNA <213> Artificial Sequence <220> <223> OMP28 gene <400> 1 aagcttaaca ctcgtgctag caattttctc gcagcctcat tttccacaat catgctcgtc 60 ggcgctttca gcctgcccgc tttcgcacag gagaatcaga tgacgacgca gcccgcgcgc 120 atcgccgtca ccggggaagg catgatgacg gcctcgcccg atatggccat tctcaatctc 180 tcggtgctac gccaggcaaa gaccgcgcgc gaagccatga ccgcgaataa tgaagccatg 240 acaaaagtgc tcgatgccat gaagaaggcc ggcatcgaag atcgcgatct ccagacaggc 300 ggcatcaata tccagccgat ttatgtctat cctgacgaca agaacaacct gaaagagcct 360 accatcaccg gctattctgt atccaccagt ctcacggttc gcgtgcgcga actggccaat 420 gttggaaaaa ttttggatga atccgtcacg ctcggtgtta atcagggcgg tgatttgaac 480 ctggtcaatg ataatccctc cgccgtgatc aacgaggcgc gcaagcgcgc agtggccaat 540 gccattgcca aggcgaagac gcttgccgac gctgcaggcg tggggcttgg ccgtgtggtg 600 gaaatcagtg aactgagccg cccgcccatg ccgatgccaa ttgcgcgcgg acagttcaga 660 accatgctag cagccgcacc ggacaattcc gtgccgattg ccgcaggcga aaacagctat 720 aacgtatcgg tcaatgtcgt ttttgaaatc aagctcgag 759

Claims (12)

Brucella , produced by the hybridoma cell line B2G12 (Accession No. KCTC18726P) suis ) A monoclonal antibody that specifically binds OMP28. Brucella , produced by the hybridoma cell line F4H6 (Accession No. KCTC18727P) suis ) A monoclonal antibody that specifically binds OMP28. A hybridoma cell line B2G12 with accession number KCTC18726P, which produces the monoclonal antibody of claim 1. The hybridoma cell line F4H6 with accession number KCTC18727P producing the monoclonal antibody of claim 2. A composition for detecting Brucella suis , comprising the monoclonal antibody according to claim 1 or 2. A kit for detecting Brucella suis , comprising the monoclonal antibody according to claim 1 or 2. Capture antibody; And
Detection antibody; Brucella containing ( Brucella suis ) Lateral flow immunoassay kit for detection,
The capture antibody is produced by the hybridoma cell line F4H6 (Accession No. KCTC18727P), Brucella ( Brucella suis ) is a monoclonal antibody that specifically binds to OMP28,
The detection antibody is produced by the hybridoma cell line B2G12 (Accession No. KCTC18726P), Brucella ( Brucella suis ) A kit for the detection of brucellella, a monoclonal antibody that specifically binds to OMP28. The method of claim 7, wherein the lateral flow analysis kit further comprises a sample pad, a conjugate pad, a nitrocellulose membran, an absorbent pad, and gold nanoparticles. Kit for detecting brucellella, characterized in that. Claim 1 or claim 2 is brought into contact with the biological sample to remove the monoclonal antibody antigen- Brucella bacteria comprising the step of detecting the antibody complex formation (Brucella suis ) detection method. 10. The method of claim 9, wherein the biological sample is selected from the group consisting of tissue, cells, whole blood, serum, plasma, cerebrospinal fluid, colostrum, joint fluid, saliva, feces, cell culture supernatant, and food. The method of claim 9, wherein the formation of the antigen-antibody complex is enzymatic immunoassay (ELISA), Western Blotting, Immunofluorescence, Immunohistochemistry staining, Flow cytometry. , Immunocytochemistry, Radioimmunoassay (RIA), Immunoprecipitation Assay, Immunodiffusion assay, Lateral flow immunoassay, Complement Fixation Assay and Protein Chip (Protein Chip) brucellella detection method characterized in that it is identified by any one selected from the group consisting of. A method for detecting Brucella suis using the lateral flow analysis kit according to claim 7 or 8.

Images