KR20200059381A - Monoclonal Antibodies for detecting Foot and Mouth Disease Virus and using the same - Google Patents

Abstract

The present invention relates to a specific monoclonal antibody capable of detecting foot and mouth virus serotype O, A, Asia1, C, SAT1, SAT2 and SAT3, and a composition and kit for the detection of foot and mouth disease virus using the same. Regardless of the foot and mouth virus serotype, rapid and accurate initial preventive measures can be taken to prevent the spread of the disease when the foot and mouth disease breaks out.

Description

Monoclonal Antibodies for detecting Foot and Mouth Disease Virus and using the same}

The present invention is a foot and mouth disease virus (Foot and Mouth Disease Virus; hereinafter, FMDV) serotype O, A, Asia1, C, SAT1, SAT2 and SAT3 monoclonal antibody for simultaneous detection, FMDV serotype O, A using the monoclonal antibody , Asia1, C, SAT1, SAT2 and SAT3 simultaneous detection method and FMDV serotype O, A, Asia1, C, SAT1, SAT2 and SAT3 simultaneous detection kit using the monoclonal antibody.

Foot and Mouth Disease Virus (FMDV) is an RNA virus belonging to Picornaviridae and aphthovirus. Seven serotypes worldwide (O, A, Asia1, C, SAT1, SAT2, and SAT3) and 70 subtypes Viruses are distributed. Individuals infected with FMDV (eg, amphibians) form blisters and crusts between the tongue, mucous membranes, and hoofs, and have a high morbidity rate and are classified as List A diseases in the OIE, and are classified as the first type of infectious disease in Korea.

In Korea, foot-and-mouth disease occurred eight times every two to five years after 2000, causing enormous economic damage (e.g., an estimated 3 trillion won of direct damage if it occurred in Andong in 2010), most recently in December 2014 From May to April 2015, about 185 cases occurred in 7 trials, and 21 foot and mouth diseases occurred between January and March 2016.

Current methods of diagnosing foot and mouth disease virus antigens can be roughly divided into gene detection methods and viral protein antigen detection methods. Gene detection methods include real-time RT-PCR or general RT-PCR, and protein detection methods include Antigen detecting Enzyme Linked Immunosorbent Assay (Ag-ELISA), Rapid kit (immunochromatographic assay), etc. Can be heard.

In the case of real-time RT-PCR, it is the method with the highest sensitivity to detection of foot and mouth virus, and samples of suspicion of infection (blood, saliva, semen, tissues, etc.) are transported to the laboratory and sampled in a special shielded laboratory (Biosafety Level 3 laboratory). Collecting total RNA from, and performing amplification virus specific gene amplification with a fluorescence detection real-time PCR instrument, it is used to confirm whether or not a foot-and-mouth disease is positive, but requires considerable skilled researchers from RNA extraction to the step of operating real-time PCR. , Special laboratories and expensive reagents and equipment are required.

Enzyme immunoassay (ELISA, Enymne Linked Immunosorbent Assay) using an antigen-antibody reaction is a useful method for herd diagnosis because it is possible to diagnose a large amount of samples at the same time as judging the foot-and-mouth disease positive and analyzing the serotype. Currently, the OIE standard antigen ELISA kit (Pirbright, IZSLER), which is used worldwide, including in Korea, is very expensive, equivalent to about 100,000 won per head, and it is difficult to use at the local government's foot and mouth disease diagnosis center because it is not able to supply smoothly. .

Foot-and-mouth disease virus is distinctly divided into seven types of serotypes, and thus has no characteristic of mutually defending serotypes. For proper vaccine selection for rapid initial preventive measures to prevent the spread of disease, serotype differentiation diagnosis is possible up to serotype analysis. Kit development is very urgent.

The present inventors have developed monoclonal antibodies capable of simultaneously detecting serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 of Foot and Mouth Disease Virus (FMDV) with high sensitivity, This is considered to be very useful in the field diagnosis kit for detection of foot and mouth virus and enzyme immunoassay.

Accordingly, it is an object of the present invention to provide antibodies or antigen-binding fragments thereof that specifically bind to FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3.

Another object of the present invention is to provide polynucleotides encoding heavy and light chain variable regions of antibodies specifically binding to FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3.

Another object of the present invention is a polynucleotide encoding a heavy chain variable region and a polynucleotide encoding a light chain variable region of an antibody specifically binding to FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3. It is to provide a recombinant vector containing.

Another object of the present invention is a polynucleotide encoding a heavy chain variable region and a polynucleotide encoding a light chain variable region of an antibody specifically binding to FMDV serotypes O, A, Asia1, SAT1, C, SAT2 and SAT3. It is to provide a host cell transformed with a recombinant vector containing.

Another object of the present invention is to provide a kit for detecting FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 antigens.

Another object of the present invention is to provide a method for simultaneously detecting FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3.

The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds Foot and Mouth Disease Virus (FMDV) serotype O, A, Asia1, C, SAT1, SAT2 and SAT3, and uses thereof will be.

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

An example of the present invention relates to a monoclonal antibody that specifically binds to Foot and Mouth Disease Virus (FMDV) serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3.

Foot-and-mouth disease virus belongs to the genus Picornavirus Aphthovirus, and is the first animal virus to be found as a filterable pathogen, and is infected with cattle such as cattle. The particle of the virus is 22-28 nm in diameter, and the capsid protein composed of 60 molecules of 4 kinds of constituent proteins without an envelope surrounds a single stranded (+) RNA in which the VPg protein is covalently attached to the 5 'end. have.

Foot and mouth virus consists of seven serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3, and no mutual protective immunity is formed between serotypes. For example, even if a suitable protective immunity is formed by inoculating a susceptible animal with a vaccine against foot and mouth virus serotype O, when foot and mouth virus serotype Asia1 invades, it cannot be defended and foot and mouth disease occurs.

As a rapid initial preventive measure to prevent the spread of foot and mouth disease, the development of a diagnostic kit capable of sensitively detecting all of the seven serotypes is a priority in the field of prevention.

) 및 람다(

) 타입을 가진다.A complete antibody is a structure having two full length light chains and two full length heavy chains, each light chain connected by a heavy chain and a disulfide bond. The constant region of the antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and subclasses. Gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant region of the light chain is kappa (

) And lambda (

) Type.

The term "monoclonal antibody" means that the antibody molecule is made up of a single molecule. Monoclonal antibodies have the specificity of responding only to antigens with the same epitope, and also exhibit affinity for specific epitopes.

The monoclonal antibody may be produced in hybridoma cells with accession number KCTC 18621P.

The hybridoma cells can be prepared using methods known in the art. Specifically, the hybridoma cells are immunized with FMDV serotype A (IRAQ), an immunogen, to the animal, and the antibody-producing cells derived from the immunized animal are fused with myeloma cells to prepare hybridomas. , Among them, can be prepared by selecting a hybridoma that produces monoclonal antibodies that bind to foot and mouth virus serotypes O, A, Asia1, C, SAT1, SAT2, and SAT3.

The immunized animal can use animals such as goats, sheep, mormots, rats, or rabbits, as well as the mice used in the examples.

As the method for immunizing the immunized animal, a method already known in the art can be used. For example, when immunizing a mouse, 1 to 100 μg of the immunogen is emulsified with an equal amount of physiological saline and / or antigen adjuvant such as Freund's adjuvant at one time, and subcutaneously in the abdomen of the animal to be immunized. Alternatively, it may be performed by inoculating 2 to 6 times every 2 to 5 weeks in the abdominal cavity. After immunization of immunized animals, spleens or lymph nodes are removed 3 to 5 days after the final immunization, and according to a cell fusion method known in the art, B cells contained in their tissues in the presence of a fusion promoter are myeloma. It fuses with the cells. The fusion accelerator may be, for example, a material such as polyethylene glycol (PEG).

The myeloma cells are, for example, P3U1, NS-1, P3x63. Mouse-derived cells such as Ag 8.653, Sp2 / 0-Ag14, and rat-derived cells such as AG1 and AG2 can be used, but are not limited thereto.

In addition, in the cell fusion method known in the art, for example, B cells and myeloma cells are mixed at a ratio of 1: 1 to 10: 1, whereby PEG having a molecular weight of 1,000 to 6,000 is used at a concentration of 10 to 80%. In addition, it may be performed by incubating for 1 to 10 minutes at 30 to 37 ° C.

In addition, the hybridomas that produce monoclonal antibodies specifically binding to the FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 are, for example, a HAT medium in which only hybridomas can survive. It can be cultured in a selective medium such as, and the antibody activity in the hybridoma culture supernatant can be selected by measuring using a method such as ELISA.

Finally, hybridomas that produce monoclonal antibodies that specifically bind to FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3, for example, FMDV serotypes O, A, Asia1, Hybridomas that produce monoclonal antibodies that specifically bind to C, SAT1, SAT2, and SAT3 can be selected by repeating cloning by methods such as limiting dilution.

On the other hand, the monoclonal antibody may be of the IgG ₁ , IgG _2a , IgG _2b , IgG ₃ , IgA or IgM type, for example, IgG ₁ type.

또는

형일 수 있다.In addition, the light chain constant region of the antibody

or

It can be older brother.

Another example of the present invention is a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 2 and at least one heavy chain CDR (amino acid sequence) selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 5, SEQ ID NO: 6 and An antibody specifically binding to FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 comprising a light chain variable region comprising at least one light chain CDR amino acid sequence selected from the group consisting of SEQ ID NO: 7 or Antigen-binding fragments.

The antibody or antigen-binding fragment thereof comprises amino acid sequences of heavy chain complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) represented by SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. Comprising a heavy chain variable region; And a light chain variable region comprising the light chain complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3) amino acid sequences represented by SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; It may be included.

In addition, the heavy chain variable region may consist of the amino acid sequence of SEQ ID NO: 4, and the light chain variable region may consist of the amino acid sequence of SEQ ID NO: 8.

The term “antibody” is a specific antibody that responds to all FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3, and all FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 It specifically binds and includes the antigen-binding fragment of the antibody molecule as well as the complete antibody form. The description of the complete antibody is as mentioned above.

The term "heavy chain" comprises a variable region domain V _H and three constant region domains C _H1 , C _H2 and C _H3 comprising an amino acid sequence having a variable region sequence sufficient to confer specificity to the antigen. It is construed to include both the full-length heavy chain and fragments thereof.

The term "light chain" encompasses both the full-length light chain comprising the variable region domain V _L and the constant region domain C _L and a fragment thereof, comprising an amino acid sequence having sufficient variable region sequences to confer antigenic specificity. It is interpreted as meaning.

The term "complementarity determining region" (CDR) refers to the amino acid sequence of the hypervariable regions of the heavy and light chains of immunoglobulins. The heavy chain and light chain may each include three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs can provide a major contact residue for the antibody to bind to an antigen or epitope.

The term "antigen-binding fragment" is a fragment of the entire structure of an immunoglobulin and refers to a portion of a polypeptide comprising a portion to which an antigen can bind. For example, F (ab ') ₂ , Fab', Fab, Fv or scFv, but is not limited thereto. Among the antigen-binding 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 (C _H1 ) 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 C _H1 domain. The F (ab ') ₂ antibody is produced by cysteine residues in the hinge region of Fab' forming disulfide bonds. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and recombinant techniques for generating Fv fragments are well known in the art. The double-chain Fv (two-chain Fv) is a non-covalently linked heavy chain variable region and a light chain variable region, and the single-chain Fv (single-chain Fv) generally shares the variable region of the heavy chain and the variable region of the single chain through a peptide linker. It can be linked by a bond or directly linked at the C-terminus to form a dimer-like structure such as double-chain Fv. The antigen-binding fragments can be obtained using proteolytic enzymes (e.g., digestion of the entire antibody with papain to obtain Fab and digestion with pepsin to obtain F (ab ') ₂ fragment), It can be produced through genetic recombination technology.

Such antibodies are monoclonal antibodies, bispecific antibodies, non-human antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F (ab ') fragments, disulfide-binding Fvs (sdFV) and anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of the antibodies.

The antibody may be a humanized antibody or a human antibody. The humanized antibody of a non-human, e.g., mouse, is a chimeric immunoglobulin, a chain of immunoglobulins or fragments thereof, e.g., Fv, Fab, Fab ', F, comprising a minimal sequence derived from the immunoglobulin of the mouse (ab ') ₂ or other antigen-binding subsequence of the antibody.

Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues introduced from a non-human source. Humanization can be performed essentially by substituting the CDRs or CDR sequences of rodent animals for sequences that correspond to human antibodies. Thus, such humanized antibodies are chimeric antibodies, and regions less than the variable regions of substantially intact human antibodies can be replaced by corresponding sequences from non-human species. For example, a humanized antibody may be a humanized antibody in which some CDR residues and possibly some framework (FR) residues have been replaced with residues from similar sites in the antibodies of rodent animals.

The human antibody refers to an antibody in which the sequences of the variable and constant regions of the heavy and light chains are derived from humans, and the human antibody is a variety of techniques well known to those skilled in the art, including phage display libraries, for example, genetic recombination techniques and cell engineering. It can be created using technology.

The term "chimeric" means that the antibody or antigen-binding site comprises sequences from two different species.

Antibodies or antigen-binding fragments thereof that specifically bind to all of the FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3, FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 Variants of the amino acid sequence set forth in the attached SEQ ID NO can be included within a specific recognizable range.

For example, the amino acid sequence of an antibody can be altered to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion and / or substitution of amino acid sequence residues of the antibody. These amino acid variations are based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, and size. For example, arginine, lysine and histidine are all positively charged residues, alanine, glycine and serine have similar sizes, and phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on the above considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan, and tyrosine are biologically equivalent functions.

On the other hand, amino acid exchange in proteins that do not entirely change the activity of the molecule is known in the art. The most common exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. If considering the variation having the biologically equivalent activity, the antibody specifically binding to the FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 or an antigen-binding fragment thereof is substantially identical to the sequence set forth in SEQ ID NO: (Substantial identity) can also be interpreted as including the sequence.

The substantial identity of the amino acid sequence of the above-described sequence number and any other sequence is aligned so as to correspond as much as possible, and when the aligned sequence is analyzed using an algorithm commonly used in the art, at least 60%. It may be a sequence showing the homology of, at least 70% homology, at least 80% homology or at least 90% homology.

Alignment methods for comparison of sequences are known in the art. For example, through the NCBI Basic Local Alignment Search Tool (BLAST), sequence analysis programs such as blastp, blastx, tblastn and tblastx can be used on the Internet.

Another example of the invention relates to polynucleotides encoding heavy and / or light chain variable regions of antibodies that specifically bind to FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3. .

In one aspect, the polynucleotide encoding the heavy chain variable region comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4 of an antibody specifically binding to FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3. It may be a polynucleotide encoding.

The polynucleotide encoding the heavy chain variable region may be composed of the nucleotide sequence of SEQ ID NO: 12.

In one aspect, the polynucleotide encoding the light chain variable region comprises a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8 of an antibody specifically binding to FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3. It may be a polynucleotide encoding.

The polynucleotide encoding the light chain variable region may be composed of the nucleotide sequence of SEQ ID NO: 16.

The term "polynucleotide" is a polymer of deoxyribonucleotides or ribonucleotides present in single-stranded or double-stranded form. It encompasses RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless otherwise specified.

The polynucleotide is not only the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region or light chain variable region of the antibody specifically binding to the FMDV serotype O, A, Asia1, C, SAT1, SAT2 and SAT3, but also the sequence thereof. Complementary sequences are also included.

The complementary sequence includes not only perfectly complementary sequences, but also substantially complementary sequences, which are stringent conditions known in the art, for example, the Foot and Mouth Disease Virus; FMDV) to be hybridized with the nucleotide sequence of the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region or light chain variable region of an antibody specifically binding to serotype O, A, Asia1, C, SAT1, SAT2 and SAT3 Means a sequence that can.

In addition, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region and / or light chain variable region may be modified. Such modifications include addition, deletion or non-conservative substitutions or conservative substitutions of nucleotides.

The polynucleotide encoding the amino acid sequence of the heavy chain variable region and / or the light chain variable region of the antibody specifically binding to FMDV serotype O, A, Asia1, C, SAT1, SAT2 and SAT3 is substantially relative to the nucleotide sequence. It is interpreted as including nucleotide sequences showing identity.

The substantial identity is at least 80% homology when aligning the nucleotide sequence with any other sequence to the maximal correspondence and analyzing the aligned sequence using an algorithm commonly used in the art. It may be a sequence that exhibits at least 90% homology or at least 95% homology.

Another object of the present invention is a polynucleotide encoding a heavy chain variable region and a polynucleotide encoding a light chain variable region of an antibody specifically binding to FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3. It is to provide a recombinant vector containing.

The light chain variable region may be a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8, and the heavy chain variable region may be a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4.

The polynucleotide encoding the light chain variable region may consist of the nucleotide sequence of SEQ ID NO: 16, and the polynucleotide encoding the heavy chain variable region may consist of the nucleotide sequence of SEQ ID NO: 12.

gt4

B,

-Charon,

z1 및 M13 등) 또는 바이러스 (예를 들명, SV40 등)를 조작하여 제작될 수 있다. The term "vector" refers to a means for expressing a gene of interest in a host cell. For example, viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retroviral vectors and adeno-associated virus vectors. Vectors that can be used as the recombinant vector are plasmids often used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14) , pGEX series, pET series and pUC19, etc.), phage (e.g.,

gt4

B,

-Charon,

z1 and M13, etc.) or viruses (eg, SV40, etc.).

The polynucleotide encoding the amino acid sequence of the heavy chain variable region and the light chain variable region in the recombinant vector may be operably linked to a promoter. The term “operatively linked” refers to a functional binding between a nucleotide expression control sequence (eg, promoter sequence) and another nucleotide sequence. Thus, the regulatory sequence can thereby regulate the transcription and / or translation of the other nucleotide sequence.

The recombinant vector can typically be constructed as a vector for cloning or for expression. The expression vector can be used in the art, conventional ones used to express foreign proteins in plants, animals or microorganisms. The recombinant vector can be constructed through various methods known in the art.

프로모터, trp 프로모터, lac 프로모터, tac 프로모터, T7 프로모터 등), 해독의 개시를 위한 라이보좀 결합 자리 및 전사/해독 종결 서열을 포함하는 것이 일반적이다. 진핵 세포를 숙주로 하는 경우에는, 벡터에 포함되는 진핵 세포에서 작동하는 복제원점은 f1 복제원점, SV40 복제원점, pMB1 복제원점, 아데노 복제원점, AAV 복제원점 및 BBV 복제원점 등을 포함하나, 이에 한정되는 것은 아니다. The recombinant vector can be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vector used is an expression vector and a prokaryotic cell is a host, a strong promoter (eg, p _L) that can proceed transcription

It is common to include a promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription / detox termination sequence. When eukaryotic cells are used as hosts, replication origins that operate on eukaryotic cells included in vectors include f1 replication origin, SV40 replication origin, pMB1 replication origin, adeno replication origin, AAV replication origin, and BBV replication origin, etc. It is not limited.

In addition, a promoter derived from the genome of a mammalian cell (eg, a metallothionine promoter) or a promoter derived from a mammalian virus (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, Cytomegalovirus promoter and HSV's tk promoter) can be used and generally have a polyadenylation sequence as the transcription termination sequence.

Meanwhile, the vector capable of expressing the heavy chain variable region and the light chain variable region of the antibody is a vector system in which the heavy chain variable region and the light chain variable region are simultaneously expressed in one vector, or the heavy chain variable region and the light chain variable region are separate vectors, respectively. Both expression systems are possible. In the latter case, both vectors can be introduced into the host cell through co-transfomation and targeted transformation.

Another example of the present invention is a polynucleotide encoding a light chain variable region of an antibody specifically binding to FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 and a polynucleotide encoding a heavy chain variable region It relates to a host cell transformed with a recombinant vector comprising a.

The host cell comprises a polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4; And it may be transformed with a recombinant vector comprising a polynucleotide encoding a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.

That is, the host cell comprises a polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4; And a polynucleotide encoding a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8 on the genome of a host cell, or a recombinant vector containing the polynucleotide sequence.

The polynucleotide encoding the heavy chain variable region may be composed of the nucleotide sequence of SEQ ID NO: 12.

The polynucleotide encoding the light chain variable region may be composed of the nucleotide sequence of SEQ ID NO: 16.

As the host cell capable of continuously cloning or expressing the recombinant vector while being stable, any host cell known in the art can be used. For example, E. coli JM109, E. coli BL21, E Bacillus strains such as coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcesons And various intestinal bacteria and strains such as various Pseudomonas species, and when transformed into eukaryotic cells, as host cells, yeast ( Saccharomyce cerevisiae ), insect cells, plant cells and animal cells, such as Sp2 / 0, CHO ( Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN and MDCK cell lines can be used.

For transport of the polynucleotide or a recombinant vector containing the same into a host cell, a transport method well known in the art can be used. For the transport method, for example, when the host cell is a prokaryotic cell, a CaCl ₂ method or an electroporation method can be used. When the host cell is a eukaryotic cell, a micro-injection method, a calcium phosphate precipitation method, an electroporation method, Liposomal-mediated transfection and gene bombardment may be used, but are not limited thereto.

When using microorganisms such as E. coli , productivity is higher than that of animal cells, but due to glycosylation problems, it is not suitable for producing antibodies in the form of intact Ig, but antigen-binding fragments such as Fab and Fv It can be used for production.

The method for selecting the transformed host cell can be easily performed according to a method well known in the art using a phenotype expressed by a selection label. For example, when the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

Another example of the present invention is to produce a monoclonal antibody specifically binding to Foot and Mouth Disease Virus (FMDV) serotype O, A, Asia1, C, SAT1, SAT2 and SAT3. It relates to hybridoma cells.

The hybridoma cells may be hybridoma cells having accession number KCTC 18621P.

The hybridoma cells were deposited with the Korean Biotechnology Research Institute KCTC (Korean Collection for Type Cultures) with accession number KCTC 18621P on September 27, 2017.

On the other hand, the deposited hybridoma cells are stored according to the provisions of the Budapest Treaty on the Deposit of Microorganisms and can be distributed to the general public by referring to the accession number. That is, the deposits are for a period of at least 5 years after the most recent request for deposit sample management, in some cases for a period of at least 30 years after the deposit date, or during the compulsory period of any patent promulgating the disclosure of the deposit, Maintain the survival of deposits and keep them with all care to avoid contamination.

The depositor is obliged to replace the deposit if the depository is unable to sell the sample upon request due to the condition of the deposit. All restrictions restricting the public's availability of the deposits of interest will be completely eliminated if the patent issuing the deposit is granted.

The method for producing the hybridoma cells is as described above.

Another example of the present invention includes a monoclonal antibody that specifically binds to Foot and Mouth Disease Virus (FMDV) serotype O, A, Asia1, C, SAT1, SAT2 and SAT3. FMDV serotype O, A, Asia1, C, SAT1, SAT2 and SAT3.

The kit may include an enzyme binding agent (conjugate), but is not limited thereto.

The enzyme binding agent (conjugate) may be a monoclonal antibody specifically binding to FMDV serotype O, A, Asia1, C, SAT1, SAT2, and SAT3 and an enzyme covalently bound.

The monoclonal antibody is a heavy chain comprising the heavy chain CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3) amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. Variable region; And a light chain variable region comprising the light chain complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3) amino acid sequences represented by SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; It may be included.

In addition, the monoclonal antibody may include a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4, a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.

In addition, the monoclonal antibody may be a monoclonal antibody produced by hybridoma cells having accession number KCTC 18621P.

The monoclonal antibody is a heavy chain comprising the heavy chain CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3) amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. Variable region; And a light chain variable region comprising the light chain complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3) amino acid sequences represented by SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; It may be included.

In addition, the monoclonal antibody may include a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4, a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.

In addition, the monoclonal antibody may be a monoclonal antibody produced by hybridoma cells having accession number KCTC 18621P.

The kit may be used for enzyme immunoassay, but is not limited thereto.

Enzyme immunoassay of the present invention is a diagnostic method that enables the examination of various diseases using an antigen-antibody reaction in general. Antigens to be tested from a number of samples using a specific antibody (conjugate) in which a specific antibody and an enzyme are combined. It is a kind of immunoassay that can detect.

The kit of the present invention uses FMDV serotypes O, A, Asia1, C, SAT1, SAT2, and SAT3 with high affinity to react with monoclonal antibodies to FMDV serum from various samples derived from animal individuals infected with foot and mouth disease virus Types O, A, Asia1, C, SAT1, SAT2 and SAT3 infections are all specifically diagnosed.

Another example of the present invention relates to a method for simultaneously detecting Foot and Mouth Disease Virus (FMDV) serotype O, A, Asia1, C, SAT1, SAT2 and SAT3.

The present invention is a foot and mouth disease virus (Foot and Mouth Disease Virus; hereinafter, FMDV) serotype O, A, Asia1, C, SAT1, SAT2 and SAT3 monoclonal antibody for simultaneous detection, FMDV serotype O, A using the monoclonal antibody , Asia1, C, SAT1, SAT2 and SAT3 simultaneous detection method and FMDV serotype O, A, Asia1, C, SAT1, SAT2 and SAT3 simultaneous detection kit using the monoclonal antibody Since it can detect all of the sentences, it is possible to perform quicker early preventive measures to prevent the spread of foot and mouth disease.

1 is a schematic diagram showing the principle of an enzyme immunoassay method for screening hybridoma cultures according to an embodiment of the present invention.
2A to 2E are results of capture / detector matching test using 7 monoclonal antibodies that respond to all FMDV serotypes according to an embodiment of the present invention.
3 is a result of the reactivity of 7 monoclonal antibodies FMDV serotype of monoclonal antibody 1E58 according to an embodiment of the present invention.
Figure 4 is a schematic diagram showing the principle of an enzyme immunoassay for foot and mouth virus antigen diagnosis using monoclonal antibody 1E58 according to an embodiment of the present invention.
5 is a reaction optimization result of the enzyme immunoassay for foot and mouth virus antigen diagnosis using monoclonal antibody 1E58 according to an embodiment of the present invention.
Figure 6 is a result of comparing the sensitivity of the enzyme immunoassay for foot and mouth virus antigen diagnostic method using monoclonal antibody 1E58 according to an embodiment of the present invention compared to other commercialization kits.
7 is a result confirming the specificity of the enzyme immunoassay for foot and mouth virus antigen diagnosis using monoclonal antibody 1E58 according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Preparation of hybridomas, production of monoclonal antibodies and evaluation of performance

1-1. Immunogen preparation

FMDV A (IRAQ) was inoculated into BHK21 cells, and FMDV A (IRAQ) amplified through incubation for 24 hours at 37 degrees was collected. Then, after treating with binary Ethylenimine (BEI) at a concentration of 0.003N, shaking and incubating for 24 hours at 37 ° C, and then neutralizing by adding 0.002N of sodium thiosulfate. To the inactivated culture supernatant, PEG6000 (Poly Ethylene Glycol; PEG) and sodium chloride (NaCl) were added 7.5% and 2.3% respectively, followed by stirring incubation at 4 degrees for 24 hours. The supernatant treated with PEG6000 was centrifuged at 11,000 RPM for 20 minutes, and the pellet was again released with TN buffer (pH 7.6). The supernatant was centrifuged again at 11,000 RPM for 20 minutes, and ultracentrifugation (30,000 rpm, 4 hours) was performed by sugar density gradient centrifugation to obtain a high purity FMDV Asia1 fraction.

1-2. Immunization

Immunization was performed on experimental animals (balb / c mice) using the inactivated FMDV A (IRAQ) obtained in 1-1. Specifically, 200 μl of inactivated FMDV A (IRAQ) as an antigen was prepared at a concentration of 0.25 mg / ml, and mixed with 200 ul of Freund's Adjuvant (FA) to perform 1-3 primary immunization in the mouse (Orient Bio Co., Ltd.) intraperitoneally every two weeks. Did. One week after the third immunization for fusion immunoassay, indirect ELISA was performed using mouse serum. For final boosting, 100ul of inactivated FMDV A (IRAQ) was immunized intraperitoneally and used for cell fusion 4 days later.

1-3. Cell fusion and hybridoma production

The spleen located on the left side of the immunized mouse body was removed and suspended in Dulbecco's modified Eagle's medium (DMEM) medium. The extracted spleen was ground with a mesh to separate cells, and mixed with culture medium DMEM to prepare a spleen cell suspension. Then, the suspension was centrifuged at 1,200 rpm for 5 minutes to remove the supernatant, and splenocytes and bone marrow cell lines (SP2 / 0 Ag14 (ATCC, USA)) were mixed at a ratio of 1: 5 cells, The cells were precipitated by centrifugation at 1,200 rpm for 5 minutes to remove the supernatant. After slowly dispersing the centrifuged cells, 1.0 ml of polyethylene glycol 1500 (PEG1500, Roche) was treated, maintained at 37 ° C. for 1 minute, and then 1 ml of DMEM was added. Then, 10 ml of DMEM was slowly added for 1 minute, reacted in water at 37 ° C. for 5 minutes, adjusted to 50.0 ml, and centrifuged at 1,200 rpm for 5 minutes. 1 x 10 ⁵ to the cell precipitate in the separation medium (HAT medium) The cells were resuspended at a concentration of 2 X 10 ⁵ cells / ml, dispensed 0.1 ml in 96-well plates, and then cultured in a 37 ° C carbon dioxide incubator to prepare hybridoma cells.

1-4. Screening of hybridoma cells

In order to select hybridoma cells that specifically respond to FMDV serotypes O, A, Asia1, C, SAT1, SAT2, and SAT3 among the prepared hybridoma cell groups, an ELISA analysis method was used as shown in FIG. 1. .

Specifically, the target inactivation FMDV serotypes O, A, Asia1, C, SAT1, SAT2, and SAT3 were attached to the plate surface by adding 100 ul (0.5 ug / ml) each per well of a 96-well microplate, and reacting. Antigens that had not been removed were removed by washing. Then, 0.1% casein-PBS blocking solution was dispensed at 100 ul per well and reacted at 37 ° C. for 2 hours, followed by phosphate buffer solution-Tween 20 (0.05% (v / v) Tween 20). Phosphate buffer solution, pH 7.4); It was washed three times with PBS-T).

Then, 100 ul of each hybridoma cell culture was added to each well to react at room temperature for 1 hour, followed by washing three times with PBS-T solution to remove unreacted culture. Goat anti-mouse IgG-horseradish peroxidase (Goat anti-mouse IgG-HRP) was added thereto, reacted at room temperature for 1 hour, and then washed 3 times with PBS-T solution. After washing, peroxidase substrate solution (TMB) was added to react, 0.5 M sulfuric acid was dispensed into each well of the plate 50 ul, and absorbance was measured at 450 nm using a Sunrise ELISA reader (Tecan). Hybridoma cell lines secreting antibodies highly reactive to FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 were selected.

The selected hybridoma cell line was diluted by limiting dilution to perform cloning, and then, the hybridoma cell line secreting the target antibody was selected in the above manner, and the results are shown in Table 1.

No Clone name Isotype One 8C23 IgG1, k 2 8E72 IgG1, k 3 9D6 IgG1, k 4 10C59 IgG1, k 5 1E58 IgG1, k 6 9G21 IgG1, k 7 10D2 IgG1, k

As can be seen in Table 1, seven monoclonal antibodies were selected that respond to all of the inactivated FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3.

1-5. Multiple production and purification

FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 were filtered using 0.4 kinds of ascitic fluids produced using seven hybridomas that specifically respond to protein G column (GE, Sweden), and eluted only with a monoclonal antibody using an elution buffer (100 mM glycin HClr, pH 2.8) and then exchanged with PBS (pH 7.0) through dialysis (MWCO 12,000 to 14,000). The purified monoclonal antibody was stored at -70 ° C.

1-6. FMDV serotype O, A, Asia1, SAT1, SAT2 and SAT3 specific monoclonal antibody screening (cross-reactivity evaluation)

ELISA and immunochromatographic assays were used to select monoclonal antibodies that specifically respond to FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3.

In the case of ELISA, specifically, in order to confirm the cross-reactivity with the target inactivated FMDV O, A, Asia1, the inactivated FMDV O, A, Asia1 were each 100 ul (0.5 ug / 0.5 well per well of a 96-well microplate) ml) to attach to the plate surface, and the unreacted antigen was washed and removed. 0.1% casein-PBS blocking solution was dispensed at 100 ul per well, and then reacted at 37 ° C. for 2 hours, followed by washing three times with PBS-T solution.

Then, 100 ul of each hybridoma cell culture was added to each well to react at room temperature for 1 hour, followed by washing three times with PBS-T solution to remove unreacted culture. Goat anti-mouse IgG-horseradish peroxidase (Goat anti-mouse IgG-HRP) was added thereto, reacted at room temperature for 1 hour, and then washed 3 times with PBS-T solution. After washing, the substrate solution (TMB) of peroxidase was added to react, and 50 M of sulfuric acid was dispensed into each well of the plate, and absorbance was measured at 450 nm using a Sunrise ELISA reader (Tecan). Reactivity and cross-reactivity were measured, and the results are shown in Table 2.

No. Clone Name ELISA screening plate (O.D) O A Asia1 One 8C23 2.01 1.99 1.50 2 8E72 2.02 1.98 2.00 3 9D6 1.86 2.06 1.30 4 10C59 2.02 1.88 1.81 5 1E58 2.02 2.13 1.93 6 9G21 1.77 2.08 1.23 7 10D2 2.25 2.13 1.97

In the case of immunochromatography, specifically, in order to confirm the cross-reactivity with the targeted inactivated FMDV serotypes O, A, Asia1, C, SAT1, SAT2, and SAT3, 7 monoclonal antibodies in Table 1 were each 2 mg / A nitrofibrin membrane immobilized at a concentration of ml was prepared, and the seven monoclonal antibodies were fused to gold nanoparticles of about 40 nm to prepare seven monoclonal antibody-gold nanoparticle condensers to inactivate FMDV serotypes. Capture antibodies and detector antibodies that detect all O, A, Asia1, C, SAT1, SAT2, and SAT3 were selected, and the reactivity of the antibodies was analyzed, and the results are shown in FIGS. 2 and 3. .

2 and 3, it was confirmed that 1E58 monoclonal antibody responds to FMDV serotypes O, A, Asia1, C, SAT1, SAT2, and SAT3.

Example 2. FMDV serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 Optimization of enzyme immunoassay reaction for foot and mouth virus antigen diagnosis using monoclonal antibody 1E58 that can be detected

2-1. Monoclonal antibody immobilization

1E58 monoclonal antibody was attached to the plate surface by adding 100 ul (1-8 ug / ml) per well of a 96-well microplate, and unreacted monoclonal antibody was washed and removed. 0.1% casein-PBS blocking solution was dispensed at 100 ul per well, and then reacted at 37 ° C. for 2 hours, followed by washing three times with PBS-T solution.

2-2. Preparation of enzyme-monoclonal antibody 1E58 conjugate

HRP coupling was performed according to the user manual using a commercialized HRP conjugation kit (Roche, cat. No. 11 829 696 001).

2-3. Specimen dilution buffer

The sample dilution buffer for diluting the FMDV culture prepared at the quarantine headquarters was prepared with 0.1M carbonate, 1% BSA, 0.1% Tween 20, 0.05% NaCl, 0.1% sodium azide, and pH 9.6. .

2-4. Enzyme immunoassay reaction optimization test

1E58 monoclonal antibody was coated with 1 ~ 8ug / ml of 96-well microplate and foot-and-mouth disease virus serotype O culture (cal1 ~ cal4) was added to each well 100 ul, and reacted at room temperature for 1 hour, followed by PBS-T The unreacted culture was removed by washing with the solution 3 times. Here, enzyme-monoclonal antibody 1E58 conjugate was added at various concentrations (0.2 ug / ml to 0.8 ug / ml) to react at room temperature for 1 hour, and then washed 3 times with PBS-T solution. After washing, the substrate solution (TMB) of peroxidase was added to react, and 50 M of sulfuric acid was dispensed into each well of the plate, and absorbance was measured at 450 nm using a Sunrise ELISA reader (Tecan). By measuring the reactivity, the results are shown in Fig. 5 and Table 3.

coating
conc. 1 ug / ml 2 ug / ml 4 ug / ml 8 ug / ml detector
conc.
(ug / ml) 0.8 0.6 0.4 0.2 0.8 0.6 0.4 0.2 0.8 0.6 0.4 0.2 0.8 0.6 0.4 0.2 O.D O.D O.D O.D O.D O.D O.D O.D O.D O.D O.D O.D O.D O.D O.D O.D cal1 3.21 2.98 2.43 1.81 4.00 3.68 3.87 3.12 4.00 4.00 3.15 3.47 4.00 3.27 3.18 2.35 cal2 1.97 1.55 1.40 1.06 3.67 3.42 2.49 1.80 3.75 3.47 3.08 2.09 4.00 1.84 1.85 1.24 cal3 1.17 0.83 0.77 0.54 2.08 1.89 1.37 0.98 2.65 2.15 1.78 1.14 1.31 1.13 0.96 0.62 cal4 1.07 0.74 0.65 0.50 1.05 0.87 0.56 0.62 1.46 1.11 0.88 0.63 0.59 0.62 0.39 0.27

5 and Table 3, the coating concentration was determined to be 4 ug / ml, and the concentration of the enzyme-monoclonal antibody 1E58 conjugate was 0.8 ug / ml.

Example 3. Foot and mouth disease virus using monoclonal antibody 1E58 Evaluation of the sensitivity and specificity of foot and mouth virus in the enzyme immunoassay for antigen diagnosis

3-1. Enzyme immunoassay sensitivity and specificity evaluation test

1E58 monoclonal antibody coated with 4ug / ml 96-well microplates for foot-and-mouth disease virus culture medium and specificity test samples (salt, swine, novel, pig, small, small, and host cell (BHK21) culture) 50 ul each After mixing 50 ul of the sample dilution buffer and adding 100 ul to each well, the mixture was reacted at room temperature for 1 hour, and then washed 3 times with PBS-T solution to remove unreacted culture. The enzyme-monoclonal antibody 1E58 conjugate was added thereto at a concentration of 0.3 ug / ml, reacted at room temperature for 1 hour, and then washed three times with PBS-T solution. After washing, the substrate solution (TMB) of peroxidase was added to react, and 50 M of sulfuric acid was dispensed into each well of the plate, and absorbance was measured at 450 nm using a Sunrise ELISA reader (Tecan). By measuring the reactivity, the results are shown in Fig. 6 and Table 4.

Foot and mouth disease virus
Serotype Strain people O.D (450nm) Remark 1E58
Enzyme immunoassay Commercialization of other companies
Enzyme immunoassay O Primorsky 4.000 1.353 Manisa 4.000 0.616 Taiwan97 4.000 0.499 Campos 3.957 0.630 BFS 3.763 0.468 liquor 4.000 0.449 Jincheon 4.000 0.708 Hapcheon 3.994 0.621 Gimje 4.000 0.876 Boeun 1st 3.643 0.224 Jeongeup 3.861 0.413 A Malaysia97 4.000 2.218 Iran05 4.000 2.986 A22iraq 3.793 1.563 Pocheon 3.874 0.595 Yeoncheon 3.495 0.616 Asia1 MOG 3.847 0.994 CAM 4.000 0.724 Shamir 4.000 0.471 SAT SAT1 2.219 3.220 SAT2 1.949 1.191 SAT3 3.600 0.000 C C3 3.755 1.320

As can be seen in Figure 6 and Table 4, in all viruses except the SAT1 virus culture medium, the reactivity was higher than the third-party kit. In the case of the SAT3 virus culture solution, there was no reaction result of the third-party kit, and thus it was omitted in FIG. 6.

Enzyme immunoassay assays for the measurement of foot and mouth antigens tested are listed in Table 5 below.

No. Serotype Strain Topotype / Lineage Year Titer One A Pocheon Asia / Sea-97 2010 4.14 × 10 ^ 6 2 A Yeoncheonju Asia / Sea-97 2017 1.50 × 10 ^ 6 3 A Malaysia97 Asia / Sea-97 2.00 × 10 ^ 6 4 A Iran05 Asia / Iran-05 6.32 × 10 ^ 5 5 A A22 Iraq Asia / G-IV 2006 1.12 × 10 ^ 7 6 Asia1 MOG / 05 G-V 2005 1.50 × 10 ^ 7 7 Asia1 CAM / 9/80 G-V 2006 8.43 × 10 ^ 6 8 Asia1 Shamir G-V 2009 1.50 × 10 ^ 6 9 C C3 / Resende / BRA / 55 Euro-SA 2006 1.12 × 10 ^ 6 10 O Yang-Ju SEA / Mya-98 2010 6.32 × 10 ^ 6 11 O Jincheon SEA / Mya-98 2014 1.50 × 10 ^ 6 12 O O / Hapcheon / KOR / 2014 SEA / Mya-98 2014 1.12 × 10 ^ 6 13 O Gimje SEA / Mya-98 2016 1.50 × 10 ^ 6 14 O Boeun 1st ME-SA / ind-2001d 2017 1.12 × 10 ^ 6 15 O Jeongeup ME-SA / ind-2001d 2017 3.56 × 10 ^ 6 16 O O1 Manisa ME-SA Vaccine 1.12 × 10 ^ 6 17 O O1 / BFS / 860 / UK / 67 Euro-SA 2001 1.50 × 10 ^ 6 18 O Yunlin / Taiwan / 97 Cathay 2.00 × 10 ^ 6 19 O Primorsky SEA / Mya-98 2014 3.56 × 10 ^ 5 20 O Campos Euro-SA 2.00 × 10 ^ 8 21 SAT1 SAT1 / BOT / 1/68 WZ (III) 2006 2.00 × 10 ^ 7 22 SAT2 SAT2 / ZIM / 5/81 WZ (II) 2006 6.32 × 10 ^ 6 23 SAT3 SAT3 / ZIM / 4/81 WZ (I) 2006 8.43 × 10 ^ 6

In addition, as can be seen in Figure 7 and Table 6, it was confirmed that the enzyme immunoassay using 1E58 did not react with bovine, pig saliva and tissues (snow, foot), cell culture fluid (BHK21), and the like.

Foot and mouth disease virus
Negative sample 1E58 Enzyme immunoassay
OD (450nm) Foot and mouth disease virus
Negative sample 1E58 Enzyme immunoassay
OD (450nm) PC (Positive Control) 1.09 Snow Epithelium 1 0.047 NC (Negative Control) 0.032 Snowy Epithelium 2 0.089 Money Snow Epithelium 1 0.062 Snowy Epithelium 3 0.060 Money Snow Epithelium 2 0.056 Snowy Epithelium 4 0.087 Money Snow Epithelium 3 0.077 Snow Epithelium 5 0.066 Money Snow Epithelium 4 0.080 Right Epithelium 1 0.060 Money Snow Epithelium 5 0.073 Right Epithelium 2 0.052 Don's epithelium 1 0.071 Right Epithelium 3 0.048 Don's epithelium 2 0.052 Right Epithelium 4 0.051 Don's epithelium 3 0.050 Right Epithelium 5 0.051 Don's epithelium 4 0.051 Right saliva 1 0.045 Don's epithelium 5 0.055 Right saliva 2 0.048 Money Salvation 1 0.047 Right saliva 3 0.053 Money salvage 2 0.044 Right saliva 4 0.049 Money salvation 3 0.047 Right saliva 5 0.062 Money salvation 4 0.049 BHK21 cell culture 0.053 Money Saliva 5 0.046

* Donal epithelium: pig tongue epithelial tissue, pig epithelium: pig foot epithelial tissue, pig saliva: pig saliva (spit)

* PC: inactivated foot and mouth virus serotype O, NC: sample dilution buffer

* BHK21 cell culture medium: foot and mouth virus host cell culture medium

Korea Research Institute of Bioscience and Biotechnology Biological Resource Center KCTC18621P 20170926

<110> MEDIAN Diagnostics Inc.          REPUBLIC OF KOREA (Animal and Plant Quarantine Agency) <120> Monoclonal Antibodies for detecting Foot and Mouth Disease Virus          and using the same <130> PN180409 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR 1_1E58 <400> 1 Gly His Thr Phe Thr Thr Tyr Thr   1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR 2_1E58 <400> 2 Phe Asn Pro Asn Asn Gly Gly Ala   1 5 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> HCDR 3_1E58 <400> 3 Ala Tyr Gly Asn Tyr Val Asn Phe Asp Tyr   1 5 10 <210> 4 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 1E58 Heavy chain V-region <400> 4 Glu Phe Gln Leu Gln Gln Ser Arg Pro Glu Leu Ala Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly His Thr Phe Thr Thr Tyr              20 25 30 Thr Met His Trp Val Lys Gln Ser His Val Lys Ser Leu Glu Trp Ile          35 40 45 Gly Cys Phe Asn Pro Asn Asn Gly Gly Ala Thr Tyr Asn Gln Lys Phe      50 55 60 Lys Gly Lys Ala Thr Leu Thr Met Asp Lys Ser Ser Asn Thr Ala Tyr  65 70 75 80 Val Glu Val Arg Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys                  85 90 95 Ala Tyr Gly Asn Tyr Val Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr             100 105 110 Leu Thr Val Ser Ser         115 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> LCDR 1_1E58 <400> 5 Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr   1 5 10 <210> 6 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> LCDR 2_1E58 <400> 6 Leu Val Ser   One <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR 3_1E58 <400> 7 Gln His Ile Arg Glu Leu Thr   1 5 <210> 8 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> 1E58 Light chain V-region <400> 8 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly   1 5 10 15 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser              20 25 30 Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro          35 40 45 Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala      50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His  65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg                  85 90 95 Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys Asn             100 105 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> HCDR 1_1E58 <400> 9 gggcacacat tcactacata cacc 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> HCDR 2_1E58 <400> 10 tttaatccta ataatggtgg tgct 24 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> HCDR 3_1E58 <400> 11 gcctatggta actacgtaaa ctttgactac 30 <210> 12 <211> 352 <212> DNA <213> Artificial Sequence <220> <223> 1E58 Heavy chain V-region <400> 12 gaattccagc tgcaacagtc tagacctgag ttggcgaagc ctggggcttc agtgaagata 60 tcctgcaaga cttctgggca cacattcact acatacacca tgcactgggt gaagcagagc 120 catgtaaaga gccttgagtg gattggatgt tttaatccta ataatggtgg tgctacttac 180 aaccagaagt tcaagggcaa ggccacactg actatggaca agtcctccaa tacagcctac 240 gtggaggtcc gcagcctgac atctgatgat tctgcagtct attactgtgc ctatggtaac 300 tacgtaaact ttgactactg gggccaaggc accactctca cagtctcatc ag 352 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> LCDR 1_1E58 <400> 13 aaaagtgtca gtacatctgg ctatagttat 30 <210> 14 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> LCDR 2_1E58 <400> 14 cttgtatcc 9 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> LCDR 3_1E58 <400> 15 cagcacatta gggagcttac acg 23 <210> 16 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> 1E58 Light chain V-region <400> 16 gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60 atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120 caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180 ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300 tcggaggggg gaccaagctg gaaataaaac 330

Claims (13)

Foot and Mouth Disease Virus (FMDV) serotype O, A, Asia1, C, SAT1, monoclonal antibody that specifically binds to SAT2 and SAT3. The monoclonal antibody of claim 1, wherein the monoclonal antibody is of the IgG1 type. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is produced in accession number KCTC18621P hybridoma cells. A heavy chain variable region comprising the heavy chain complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3) amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; And
A light chain variable region comprising the light chain complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3) amino acid sequences represented by SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively;
Foot and Mouth Disease Virus (FMDV) detection antibody or antigen-binding fragment thereof comprising a. The antibody or antigen-binding fragment thereof according to claim 4, wherein the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 4, and the light chain variable region consists of the amino acid sequence of SEQ ID NO: 8. A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4; And a polynucleotide encoding a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8. The recombinant vector of claim 6, wherein the polynucleotide encoding the heavy chain variable region consists of the nucleotide sequence of SEQ ID NO: 12, and the polynucleotide encoding the light chain variable region consists of the nucleotide sequence of SEQ ID NO: 16. A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4; And a polynucleotide encoding a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8. Hybridoma cells with accession number KCTC18621P producing monoclonal antibodies specifically binding to Foot and Mouth Disease Virus (FMDV) serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 . FMDV serotypes O, A, Asia1 containing monoclonal antibodies specifically binding to Foot and Mouth Disease Virus (FMDV) serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 , C, SAT1, SAT2 and SAT3 antigen detection kits. The method of claim 10, wherein the monoclonal antibody,
A heavy chain variable region comprising the heavy chain complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3) amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; And
Light chain variable region comprising the light chain CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3) amino acid sequences represented by SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively; FMDV serotype O, A, Asia1, C, SAT1, SAT2 and SAT3 antigen detection kit. The method of claim 10, wherein the monoclonal antibody comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4, light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8, FMDV serotype O, A, Asia1, C , SAT1, SAT2 and SAT3 antigen detection kits. The kit according to claim 10, wherein the monoclonal antibody is a monoclonal antibody produced by hybridoma cells having accession number KCTC18621P, FMDV serotype O, A, Asia1, C, SAT1, SAT2 and SAT3 antigen detection kit. .

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