KR102614002B1 - Antibody exhibiting immunoreactivity to foot and mouth disease virus type A, composition for detecting foot and mouth disease virus type A antibody comprising the same, and method for detecting foot and mouth disease virus type A antibody using the same - Google Patents

Abstract

It relates to an antibody or antigen-binding fragment thereof showing immunoreactivity to foot-and-mouth disease virus type A, a composition for detecting foot-and-mouth disease virus type A antibody comprising the same, and a method for detecting foot-and-mouth disease virus type A antibody using the same.

Description

Antibody exhibiting immunoreactivity to foot and mouth disease virus type A, composition for detecting foot and mouth disease virus type A antibody comprising the same, and method for detecting foot and mouth disease virus type A antibody using the same {Antibody exhibiting immunoreactivity to foot and mouth disease virus type A, composition for detecting foot and mouth disease virus type A antibody comprising the same, and method for detecting foot and mouth disease virus type A antibody using the same}

It relates to an antibody showing immunoreactivity to foot-and-mouth disease virus type A, a composition containing the same for detecting foot-and-mouth disease virus type A antibody, and a method for detecting foot-and-mouth disease virus type A antibody using the same.

Foot-and-Mouth Disease Virus (FMDV) is a type 1 legal livestock infectious disease in Korea that causes symptoms such as high fever and blisters due to viral infection in artiodactyl animals and causes death in young livestock. In Korea, it has occurred 9 times since 2000, and when it occurred in 2010-2011, it caused a whopping 3 trillion won in direct economic damage.

Foot-and-mouth disease virus consists of four structural proteins, VP4, VP3, VP2, and VP1, and eight non-structural proteins, 2A, 2B, 2C, 3A, 3B, 3C, and 3D. The VP1-2A protein precursor is cleaved and folded by 3C protease, resulting in virus assembly. Among the capsid proteins, VP4 exists inside the capsid and is not exposed, but VP2, VP3, and VP1 are exposed on the capsid surface. In particular, among the exposed structural proteins, the G-H loop contained in VP1 is known to be an important site that induces the production of neutralizing antibodies (Korean Patent No. 10-1814757).

Accordingly, vaccines have been manufactured targeting only the structural proteins of the foot-and-mouth disease virus, and since antibodies produced during vaccination express only antibodies against the structural proteins, there has been a need for an antibody detection method targeting the structural proteins. The Virus Neutralizing Test (VNT) is known as the standard serological test method for structural proteins of foot-and-mouth disease, but this requires a shielded facility for virus culture and requires a test time of at least 72 hours. It has the disadvantage of producing low levels of non-specific positive reactions in certain sera.

Under this background, the present inventors developed a competitive ELISA assay for detecting FMDV type A antibodies that can exhibit significantly superior antibody detection ability even with a simple test operation, and the significantly increased precision, sensitivity, and precision of the ELISA assay compared to existing test methods. By confirming the specificity performance, this application was completed.

One aspect includes a heavy chain variable region comprising HCDR1 consisting of the amino acid sequence of SEQ ID NO: 1, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 3; And a light chain variable region comprising LCDR1 consisting of the amino acid sequence of SEQ ID NO: 4, LCDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and LCDR3 consisting of the amino acid sequence of SEQ ID NO: 6, showing immunoreactivity to foot-and-mouth disease virus type A. To provide an antibody or antigen-binding fragment thereof.

Another aspect is to provide a composition for detecting foot-and-mouth disease virus type A antibody, comprising the antibody or antigen-binding fragment thereof.

Another aspect is to provide a kit for detecting foot-and-mouth disease virus type A antibodies, comprising the composition for detecting foot-and-mouth disease virus type A antibodies.

Another aspect includes immobilizing a recombinant antigen on a solid phase support; Contacting a sample isolated from an individual and the antibody or antigen-binding fragment thereof with the immobilized recombinant antigen; and detecting a complex formed by binding the immobilized recombinant antigen with the antibody or antigen-binding fragment thereof.

Other objects and advantages of the present application will become clearer from the following detailed description together with the appended claims and drawings. Contents not described in this specification can be fully recognized and inferred by a person skilled in the technical field of this application or a similar technical field, so description thereof is omitted.

Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Additionally, the scope of the present application cannot be considered limited by the specific description described below.

One aspect includes a heavy chain variable region comprising HCDR1 consisting of the amino acid sequence of SEQ ID NO: 1, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 3; And a light chain variable region comprising LCDR1 consisting of the amino acid sequence of SEQ ID NO: 4, LCDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and LCDR3 consisting of the amino acid sequence of SEQ ID NO: 6, showing immunoreactivity to foot-and-mouth disease virus type A. Antibodies or antigen-binding fragments thereof are provided.

The antibody or antigen-binding fragment thereof includes a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7; and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.

The Foot-and-Mouth Disease Virus (FMDV) belongs to the Aphthovirus genus of the Picornavirus family and is an animal virus first discovered as a filterable pathogen that infects artiodactyl animals such as cattle. The virus particle is 22 to 28 nm in diameter, has no envelope, and the capsid protein, which is composed of 60 molecules of 4 types of constituent proteins, carries single-stranded (+) RNA with a VPg protein covalently bound to the 5′ end. Surrounding. Foot-and-mouth disease virus consists of seven serotypes: O, A, Asia1, C, SAT1, SAT2, and SAT3, and mutual protective immunity is not formed between serotypes. In other words, even if an appropriate protective immunity is formed by vaccinating a susceptible animal against foot-and-mouth disease virus serotype O, if another foot-and-mouth disease virus serotype A virus invades, the animal is not protected and foot-and-mouth disease occurs. In order to select an appropriate vaccine as a rapid initial preventive measure to prevent the spread of foot-and-mouth disease, there is an urgent need to develop a rapid serotype identification kit that can perform serotype analysis on site. The foot-and-mouth disease virus type A may be one or more selected from the group consisting of Iraq, Cruzeiro, Pocheon, Gimpo, and Argentina, but is not limited thereto.

As used herein, the term “antibody” refers to a specific immunoglobulin directed against an antigenic site. The antibody is a specific antibody against foot-and-mouth disease virus type A, and may be an antibody that specifically binds to the structural protein of foot-and-mouth disease virus type A.

The antibodies may include intact antibodies, antigen-binding fragments of antibody molecules, synthetic antibodies, recombinant antibodies, or antibody hybrids. Meanwhile, sequence information regarding the foot-and-mouth disease virus type A is already known in the art. The antibodies include polyclonal antibodies, monoclonal antibodies, bispecific antibodies, non-human antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fv (scFv), single chain antibodies, Fab fragments, F (ab') fragments. , disulfide-binding Fv (sdFv), and anti-idiotype (anti-Id) antibodies, etc., and may include all immunoglobulin antibodies, and may include epitope-binding fragments of these antibodies. The antibody does not have the structure of an intact antibody with two full-length light chains and two full-length heavy chains, as well as a complete form with two light chains and two heavy chains, but is directed to an antigenic site. It may also include a functional fragment of an antibody molecule that possesses an antigen-binding function and has a specific antigen-binding site, that is, a binding domain.

The complete antibody has a structure of two full-length light chains and two full-length heavy chains, each light chain is connected to the heavy chain by a disulfide bond (SS-bond), and the constant region of the antibody is the heavy chain constant region. It is divided into a and light chain constant regions.

There are five types of heavy chains: γ, δ, α, μ, and ε, and the heavy chain can determine the type of antibody. α and γ are composed of 450 amino acids, and μ and ε are composed of 550 amino acids. The heavy chain has two regions: a variable region and a constant region.

The light chain is of two types, λ and κ, and may consist of approximately 211 to 217 amino acids. Each human antibody has only one chain. The light chain consists of a continuous constant region and a variable region.

As used herein, the term “complementarity determining region (CDR)” refers to the amino acid sequence of the hypervariable region of the heavy and light chains of immunoglobulin. The heavy and light chains may each contain three CDRs (HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3). The CDR may provide key contact residues for the antibody to bind to the antigen or epitope.

The antibody may be a monoclonal antibody.

As used herein, the term “monoclonal antibody” means that the antibody molecule is manufactured to consist of a single molecule. Monoclonal antibodies have the specificity of reacting only to antigens with the same epitope, and can also show affinity only to specific epitopes.

As used herein, the term “antigen-binding fragment” refers to a fragment of the entire structure of an immunoglobulin and a portion of a polypeptide containing a portion to which an antigen can bind. For example, it may be F (ab')2, Fab', Fab, Fv, scFv, or a combination thereof, but is not limited thereto. Among the above antigen-binding fragments, Fab has a structure that includes variable regions of light and heavy chains, a constant region of the light chain, and the first constant region (CH1) of the 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. The F(ab')2 antibody is produced when the cysteine residue in the hinge region of Fab' forms a disulfide bond. Fv is a minimal antibody fragment containing only the heavy chain variable region and the light chain variable region, and recombinant techniques for producing Fv fragments are widely known in the art. In two-chain Fv, the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, while in single-chain Fv (single chain Fv), the variable region of the heavy chain and the variable region of the short chain are generally linked by a covalent bond through a peptide linker. It can be linked or directly linked at the C-terminus to form a dimer-like structure, such as double-chain Fv. The antigen-binding fragment can be obtained using a proteolytic enzyme (for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F (ab')2 fragment can be obtained by digestion with pepsin), It can be produced through genetic recombination technology.

The antibody or antigen-binding fragment thereof may exhibit specific immunoreactivity or virus neutralizing ability against the foot-and-mouth disease virus type A structural proteins VP4, VP2, VP3, VP1, or a combination thereof. The immune reactivity may mean that an immune response occurs due to antigenic stimulation. The immune response may include an increase in cellular or humoral immunity to an antigen. The virus neutralizing ability may mean that the infectivity of the virus is specifically suppressed or lost by a virus neutralizing antibody.

The antibody or antigen-binding fragment thereof specifically binds to the foot-and-mouth disease virus type A antigen, and may bind competitively with the foot-and-mouth disease virus type A antibody and the foot-and-mouth disease virus type A antigen.

The foot-and-mouth disease virus type A antigen may include the amino acid sequence of the structural protein of foot-and-mouth disease virus type A. Additionally, the foot-and-mouth disease virus type A antigen may be a recombinant antigen containing the amino acid sequence of a structural protein of foot-and-mouth disease virus type A, that is, VP4, VP2, VP3, VP1, a portion thereof, or a combination thereof. Additionally, the foot-and-mouth disease virus type A antigen may include the amino acid sequence of the GH loop of VP1, one of the structural proteins of foot-and-mouth disease virus type A. The foot-and-mouth disease virus type A may be one or more selected from the group consisting of Iraq, Cruzeiro, Pocheon, Gimpo, and Argentina, but is not limited thereto.

Specifically, the foot-and-mouth disease virus type A antigen is a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 9, a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 10, a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 11, and an amino acid sequence of SEQ ID NO: 12. It may be a recombinant antigen, or a combination thereof. The recombinant antigen is about 60% or more, for example, about 70% or more, about 80% or more, about 90% or more, about 95% or more of the sequence of SEQ ID NO: 9, 10, 11, 12, or a combination thereof, It may include an amino acid sequence having a sequence identity of about 99% or more, or about 100%. In addition, the recombinant antigen has about 1 or more amino acids, about 2 or more amino acids, about 3 or more amino acids, about 4 or more amino acids, or about 5 amino acids in the sequence of SEQ ID NO: 9, 10, 11, 12, or a combination thereof. The recombinant antigen may have a different sequence of at least about 6 amino acids, or at least about 7 amino acid residues.

The antibody or antigen-binding fragment thereof may be produced from hybridoma cells.

The hybridoma cells can be produced using methods known in the art. Specifically, the hybridoma cells are created by immunizing an animal with the immunogenic foot-and-mouth disease virus type A or the foot-and-mouth disease virus type A antigen, and fusing B cells, which are antibody producing cells derived from the immunized animal, with myeloma cells to create hybridoma cells. It can be manufactured by selecting a hybridoma that produces a monoclonal antibody that specifically binds to the foot-and-mouth disease virus type A or the foot-and-mouth disease virus type A antigen. The immunized animal may be not only the mouse used in the examples, but also animals such as goats, sheep, guinea pigs, rats, or rabbits. As a method for immunizing the immunized animal, methods already known in the art can be used. After immunizing the immunized animal, the spleen or lymph nodes can be removed and the B cells contained in these tissues can be fused with myeloma cells in the presence of a fusion promoter according to a cell fusion method already known in the art. The fusion promoter 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 and Sp2/0-Ag14, and rat-derived cells such as AG1 and AG2 can be used, but are not limited to these. In addition, hybridomas that produce monoclonal antibodies that specifically bind to the foot-and-mouth disease virus type A or the foot-and-mouth disease virus type A antigen are cultured in a selective medium such as HAT medium in which only hybridomas are viable, for example. And, the antibody activity in the hybridoma culture supernatant can be measured and selected using a method such as ELISA. Finally, hybridomas that produce monoclonal antibodies that specifically bind to foot-and-mouth disease virus type A or the foot-and-mouth disease virus type A antigen are, for example, specific to the foot-and-mouth disease virus type A or the foot-and-mouth disease virus type A antigen. Hybridomas that produce binding monoclonal antibodies can be selected by repeating cloning using methods such as limiting dilution. Meanwhile, the monoclonal antibody may be of the IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgM, IgE, IgA1, IgA5, or IgD type.

The antibody or antigen-binding fragment thereof may be bound or conjugated to a detection label. The detection label may include an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, or a radioactive isotope. Enzymes used as detection labels include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, and β-latamase, and the fluorescent substance is fluorescein. Phosphorus, Eu ³⁺ , Eu ³⁺ chelate, cryptate, FITC, RITC, etc. are included. Ligands include biotin derivatives, and luminescent substances include acridinium ester and isoluminol derivatives. Particles include colloidal gold and colored latex, and radioactive isotopes include ⁵⁷ Co, ³ H, ¹²⁵ I, and ¹²⁵ I-Bonton Hunter reagent.

The antibody or antigen-binding fragment thereof may include a variant of the amino acid sequence shown in the attached SEQ ID NO to the extent that it can specifically recognize the foot-and-mouth disease virus type A or the recombinant foot-and-mouth disease virus type A antigen. For example, changes can be made to the amino acid sequence of an antibody to improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody. These amino acid mutations are made based on the relative similarity of amino acid side chain substitutions, such as properties 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 can be said to be biologically equivalent in function. On the other hand, amino acid exchanges in proteins that do not overall alter the activity of the molecule are 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/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Considering the mutation having the biological equivalent activity, the antibody or antigen-binding fragment thereof can be interpreted as including a sequence showing substantial identity with the sequence shown in SEQ ID NO: The above substantial identity is at least about 60% when the amino acid sequence of the above-mentioned sequence number and any other sequence are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. % homology, at least about 70% homology, at least about 80% homology, or at least about 90% homology. Alignment methods for comparison of sequences are known in the art. For example, sequence analysis programs such as blastp, blastx, tblastn, and tblastx are available on the Internet through the NCBI Basic Local Alignment Search Tool (BLAST).

Another aspect provides a composition for detecting foot-and-mouth disease virus type A antibody comprising the antibody or antigen-binding fragment thereof.

The antibody refers to an antibody that binds to physiologically active substances such as viruses, toxins, enzymes, etc. and inhibits their pathogenicity or biological activity. In particular, neutralizing antibodies, which are antibodies that inhibit the proliferation of viruses, exist in the serum of patients infected with a certain type of virus, and their quantification can be used for diagnosis or epidemiological investigation.

The foot-and-mouth disease virus type A antibody refers to an antibody formed within an individual and may include neutralizing antibodies or non-neutralizing antibodies.

In the composition for detecting antibodies according to one embodiment, the formed antigen-antibody complex may be confirmed using enzyme-linked immunosorbent assay (ELISA). The enzyme immunosorbent method includes blocking ELISA, which uses the principle that antibodies against various disease-causing agents present in biological samples react with antigens attached to a solid support, thereby preventing the reaction of the labeled antibodies with the antigens; Competitive ELISA, which uses the principle that labeled antibodies and antibodies against disease-causing agents present in a biological sample simultaneously compete to react with an antigen attached to a solid support; Direct ELISA using labeled antibodies that recognize antigens attached to a solid support; Indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of an antibody that recognizes an antigen attached to a solid support; Direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of an antibody and antigen attached to a solid support; and indirect sandwich ELISA, which uses a labeled secondary antibody that recognizes the antibody after reacting it with another antibody that recognizes the antigen in a complex of an antibody and an antigen attached to a solid support. In addition, the antibody may be bound or conjugated to a detection label, and if the antibody is not bound to the detection label, the antigen formed by processing another antibody capable of capturing the antibody and having a detection label - Antibody complexes can be identified.

In addition, the formation of the antigen-antibody complex can be performed using colorimetric methods, electrochemical methods, fluorimetric methods, luminometry, particle counting methods, and visual assessment methods. , or it can be detected by scintillation counting method.

Since the antibody or antigen-binding fragment thereof can react competitively with foot-and-mouth disease virus type A antibodies for foot-and-mouth disease virus type A antigens, it can be used to detect or evaluate the level of foot-and-mouth disease virus type A antibodies. For this purpose, the composition for detecting antibodies may further include not only the above-described antibody or antigen-binding fragment thereof, but also foot-and-mouth disease virus type A antigen. Specifically, the foot-and-mouth disease virus type A antigen is a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 9, a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 10, a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 11, and an amino acid sequence of SEQ ID NO: 12. It may be a recombinant antigen, or a combination thereof. The recombinant antigen is about 60% or more, for example, about 70% or more, about 80% or more, about 90% or more, about 95% or more of the sequence of SEQ ID NO: 9, 10, 11, 12, or a combination thereof, It may include an amino acid sequence having a sequence identity of about 99% or more, or about 100%. In addition, the recombinant antigen has about 1 or more amino acids, about 2 or more amino acids, about 3 or more amino acids, about 4 or more amino acids, or about 5 amino acids in the sequence of SEQ ID NO: 9, 10, 11, 12, or a combination thereof. The recombinant antigen may have a different sequence of at least about 6 amino acids, or at least about 7 amino acid residues.

Any terms or elements mentioned in the composition that are the same as those mentioned in the description of the antibody or antigen-binding fragment thereof are understood to be the same as previously mentioned in the description of the antibody or antigen-binding fragment thereof.

Another aspect provides a kit for detecting foot-and-mouth disease virus type A antibody, including the composition for detecting the antibody.

The kit may be for a competitive ELISA method, and specifically, a substrate comprising a solid-phase support on which foot-and-mouth disease virus type A antigen is immobilized, coated, or conjugated; And it may include a reagent containing the above-described antibody or antigen-binding fragment thereof. Here, the foot-and-mouth disease virus type A antigen may be a recombinant antigen as described above, and the antibody or antigen-binding fragment thereof may be bound to a detection label, but this is determined by the detection conditions, detection method, detection target, and detection. The enemy can be changed depending on the purpose, etc.

The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide, but is not limited thereto.

The detection label may include an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, or a radioactive isotope. Enzymes used as detection labels include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, and β-latamase, and the fluorescent substance is fluorescein. Phosphorus, Eu ³⁺ , Eu ³⁺ chelate, cryptate, FITC, RITC, etc. are included. Ligands include biotin derivatives, and luminescent substances include acridinium ester and isoluminol derivatives. Particles include colloidal gold and colored latex, and radioactive isotopes include ⁵⁷ Co, ³ H, ¹²⁵ I, and ¹²⁵ I-Bonton Hunter reagent.

For immunological detection of antibodies, the kit may further include not only the above-mentioned substrate and an antibody or antigen-binding fragment thereof bound to a detection label, but also an appropriate amount of buffer solution, a chromogenic substrate, etc., Techniques known in the art may be applied without limitation.

According to one embodiment, the antibody or antigen-binding fragment thereof is in competitive contact with the foot-and-mouth disease virus type A antibody and the foot-and-mouth disease virus type A antigen, so that the antibody or antigen-binding fragment thereof is in competitive contact with the foot-and-mouth disease virus type A antigen. If a high concentration of the formed complex is detected, the antibody titer can be determined to be low. Additionally, when the concentration of the complex is detected to be low, it can be determined that the antibody titer is high. For example, when there is no or almost no foot-and-mouth disease virus type A antibody in a sample isolated from an individual, the antibody or antigen-binding fragment thereof binds to the foot-and-mouth disease virus type A antigen immobilized on the solid support at a high rate. Conversely, when foot-and-mouth disease virus type A antibody is present in the sample, competition for binding to the foot-and-mouth disease virus type A antigen immobilized on the solid support occurs, so the antibody or antigen-binding fragment thereof binds at a low rate. Therefore, when the signal appears weak, the individual can be determined to be positive for foot-and-mouth disease virus type A antibodies, and when the signal appears strong, the individual can be determined to be negative for foot-and-mouth disease virus type A antibodies.

The sample or specimen may be a biological sample. The biological sample may be a cell, organ, cell lysate, whole blood, blood, serum, plasma, lymph, extracellular fluid, body fluid, urine, feces, tissue, bone marrow, saliva, sputum, cerebrospinal fluid, or a combination thereof. Specifically, the sample or specimen may be body fluid, plasma, serum, blood, or a combination thereof. Additionally, the sample or specimen may be isolated from an individual. The object refers to any living organism that can be infected with the foot-and-mouth disease virus, and may be an artiodactyl animal including cattle, pigs, sheep, goats, deer, camels, etc.

Any terms or elements mentioned in the kit that are the same as those mentioned in the description of the composition or the antibody or antigen-binding fragment thereof are the same as those previously mentioned in the description of the composition or the antibody or antigen-binding fragment thereof. It is understood that

Another aspect includes immobilizing an antigen on a solid phase support; Contacting a sample isolated from an individual and the antibody or antigen-binding fragment thereof with the immobilized antigen; and detecting a complex formed by binding the immobilized antigen with the antibody or antigen-binding fragment thereof.

The foot-and-mouth disease virus type A antibody refers to an antibody formed within an individual and may include neutralizing antibodies or non-neutralizing antibodies. The neutralizing antibody refers to an antibody that binds to physiologically active substances such as viruses, toxins, enzymes, etc. and inhibits their pathogenicity or biological activity. In particular, neutralizing antibodies, which are antibodies that inhibit the proliferation of viruses, exist in the serum of patients infected with a certain type of virus, and their quantification can be used for diagnosis or epidemiological investigation. Therefore, the method can be used to measure neutralizing antibody titer in individuals who have been vaccinated against foot-and-mouth disease.

The antigen immobilized on the solid support may be the same as the foot-and-mouth disease virus type A antigen described above. Specifically, the antigen immobilized on the solid support is a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 9, a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 10, a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 11, and an amino acid sequence of SEQ ID NO: 12. It may be a recombinant antigen consisting of, or a combination thereof.

In one embodiment, the contacting step may be a competitive contact between the antibody or antigen-binding fragment thereof and the foot-and-mouth disease virus type A antibody in the sample with respect to the antigen, which may be performed simultaneously or sequentially. . For example, the contacting step may include contacting the antigen with a sample; And it may include contacting the antibody or antigen-binding fragment thereof with the antigen in contact with the sample.

The method includes a substrate comprising a solid-phase support on which an antigen is immobilized, coated, or conjugated; and a reagent containing the above-described antibody or antigen-binding fragment thereof. Here, the antigen may be the above-described foot-and-mouth disease virus type A antigen, and the antibody or antigen-binding fragment thereof may be bound to a detection label, but this may be related to detection conditions, detection method, detection target, and detection purpose, etc. Depending on the enemy, it can be changed. The substrate and the detection label are as described in the kit.

According to one embodiment, the antibody or antigen-binding fragment thereof makes competitive contact with the foot-and-mouth disease virus type A antibody and the antigen immobilized on the solid-phase support (e.g., the foot-and-mouth disease virus type A antigen). If the concentration of the complex formed by the binding of the antigen is detected to be high, the antibody titer can be determined to be low. Additionally, when the concentration of the complex is detected to be low, it can be determined that the antibody titer is high. For example, when there is no or almost no foot-and-mouth disease virus type A antibody in a sample isolated from an individual, the antibody or antigen-binding fragment thereof binds to the antigen at a high rate. Conversely, when foot-and-mouth disease virus type A antibodies are present in the sample, competition for binding to the antigen occurs, so the antibody or antigen-binding fragment thereof binds at a low rate. Therefore, when the signal appears weak, the individual can be determined to be positive for foot-and-mouth disease virus type A antibodies, and when the signal appears strong, the individual can be determined to be negative for foot-and-mouth disease virus type A antibodies.

The detection step includes enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence, immunohistochemistry staining, flow cytometry, immunocytochemistry, and radioimmunoassay. (RIA), immunoprecipitation assay (Immunoprecipitation Assay), immunodiffusion assay (Immunodiffusion assay), complement fixation assay (Complement Fixation Assay), and protein chip (Protein Chip). .

Any of the terms or elements mentioned in the above method that are the same as those mentioned in the description of the kit, the composition, or the antibody or antigen-binding fragment thereof are the same as those previously mentioned in the description of the kit, the composition, or the antibody or antigen-binding fragment thereof. It is understood to be the same as mentioned in the explanation.

According to an antibody or antigen-binding fragment, composition, kit, or method thereof according to one aspect, the diagnostic or detection sensitivity performance is significantly improved compared to the existing foot-and-mouth disease virus type A diagnosis method or the foot-and-mouth disease virus type A antibody detection method.

Figure 1 is a diagram showing the results of comparative analysis of amino acid-based homology of structural protein P1 (VP4, VP2, VP3, VP1) of A22 Iraqi strain, A24 Cruzeiro strain, type A Pocheon strain, and type A Gimpo strain. am.
Figure 2 is a diagram showing SDS-PAGE results confirming purified FMDV type A P1 (VP4231) recombinant antigen.
Figure 3 is a diagram showing the development process of a monoclonal antibody raw material (Detector) showing immunoreactivity to FMDV type A.
Figure 4 is a diagram showing the results of an antibody titer test for the antiserum of mice immunized with a peptide (VP1 GH loop, FMDV type A Iraqi strain) antigen.
Figure 5 is a schematic diagram showing the process of selecting hybridoma cell lines.
Figure 6 is a schematic diagram showing the testing principle of the competitive ELISA assay for detecting FMDV type A antibodies.

Hereinafter, the present invention will be described in more detail through experimental examples and examples. However, these experimental examples and examples are for illustrative purposes only and the scope of the present invention is not limited to these experimental examples and examples.

In addition, unless there is a special definition in this specification, all scientific and technical terms used in this specification may have the same meaning as commonly understood by a person skilled in the art in the technical field to which the present invention pertains.

Example 1: Development of P1 recombinant antigen containing FMDV structural protein

In this example, recombinant antigens were developed targeting the A22 Iraqi strain and A24 Cruzeiro strain, which are used as Foot-and-Mouth Disease Virus (FMDV) type A vaccines, and additionally, In preparation for type A, recombinant antigens targeting type A Pocheonju and Gimpoju were developed.

Specifically, recombinant antigens were developed targeting the structural proteins (VP4, VP2, VP3, and VP1) of FMDV type A. Among FMDV structural proteins, the RGD motif present in the GH loop of VP1 is known to be a major immunogenic site for inducing neutralizing antibodies. Therefore, peptide synthesis in the GH loop region was performed for each virus strain.

Figure 1 is a diagram showing the results of comparative analysis of amino acid-based homology of structural protein P1 (VP4, VP2, VP3, VP1) of A22 Iraqi strain, A24 Cruzeiro strain, type A Pocheon strain, and type A Gimpo strain. am.

For the production of recombinant antigens against FMDV type A structural protein, A22-Iraq (AY593763), A24-Cruzeiro (AY593768), type A Pocheon (KC588943), and type A Gimpo (A/GP/ Genetic information from (SKR/2018, provided by the Korea Quarantine Service) was obtained, and during gene synthesis, codon optimization was performed on Spodoptera frugiperda, the expression host of the baculovirus, to enable the production of heterologous proteins with high efficiency.

Using the synthesized gene, the full-length P1 (VP4, VP2, VP3, VP1) containing the FMDV structural protein was cloned into a baculovirus expression vector, then expressed and purified to determine the reactivity of the obtained antigen candidates, through which , and finally, FMDV type A P1 (VP4231) recombinant antigen containing FMDV structural protein was obtained. The obtained FMDV type A P1 (VP4231) recombinant antigen was used as a raw material for plate coating in a competitive ELISA assay for detecting FMDV type A antibodies, and a monoclonal antibody raw material (detector) used in a competitive ELISA assay for detecting FMDV type A antibodies was developed. It was used as an immunogen to

1-1. Preparation of FMDV type A P1 (VP4231) recombinant antigen expression vector

In this example, in order to prepare FMDV type A P1 (VP4231) recombinant antigen, a vector expressing FMDV type A P1 (VP4231) was prepared. Specifically, in order to manufacture a total of four types of FMDV A type P1 (VP4231) recombinant antigens containing structural proteins of A22 Iraqi strain, A24 Cruzeiro strain, A type Pocheon strain, and A type Gimpo strain, respectively, a total of four types were used. An expression vector was prepared. The four types of expression vectors above differ only in the gene sequence of FMDV type A P1 (VP4231) they contain, and other than that, the sequences constituting the vector and the vector production method are all the same.

Specifically, to prepare the expression vector, pFastbac vector (Invitrogen, Cat No.10360-014, USA) was prepared, and the primer linker site of FMDV type A P1 (VP4231) was linked to BamHI (NEB). , Cat No. #R0136S, USA) and SalI (NEB, Cat No. #R0138S, USA) and electrophoresed on an agarose gel. The FMDV type A P1 (VP4231) was extracted using a gene clean kit (MP biochemical, Cat No. #1101-400, France). Afterwards, the extracted FMDV type A P1 (VP4231) was treated with BamHI and SalI to generate an insert (FMDV type A P1 (VP4231)-Insert), and the insert was mixed with the pFastbac vector treated with BamHI and SalI. After this, the cells were ligated overnight at about 16°C using T ₄ DNA ligase (Roche, 10481220001, Germany) and then transformed into competent cells (Top10F').

The transformation was performed through heat-shock. Specifically, soluble cells stored at about -70°C were taken out and thawed on ice, then about 5 uL of ligate was added and mixed for about 3 seconds, and then left on ice for about 30 minutes. The tube containing the soluble cells mixed with the reactants was placed in a constant temperature water bath at approximately 42°C for approximately 90 seconds and then immediately placed on ice. About 1 mL of LB liquid medium was added to the tube, reacted at about 37°C for about 1 hour to obtain a bacterial solution, and about 100 uL of the bacterial solution was plated on an LB agar plate containing ampicillin as a selection marker and incubated at about 37°C. Cultured overnight. Colonies grown on LB agar plates were inoculated into liquid LB medium and cultured. DNA was extracted from the cells in the culture medium obtained after culturing using the Wizard Plus SV Minipreps Kit (Promega, Cat No.#A1460, USA), and the extracted DNA was confirmed through sequencing.

1-2. Preparation of FMDV type A P1 (VP4231) recombinant bacmid.

In this example, to create pFastbac FMDV type A P1 (VP4231) recombinant bacmid, FMDV type A P1 (VP4231) plasmid DNA was inserted into DH10bac competent cells and cultured, and then the recombinant bacmid was extracted.

Specifically, an appropriate amount of FMDV type A P1 (VP4231) plasmid DNA was mixed with DH10bac competent cells and left on ice for about 10 minutes. After being heat-shocked in a water bath at about 42°C for about 90 seconds, it was placed on ice again for about 5 minutes. After adding about 1 mL of LB broth, shaking incubation was performed at about 37°C for about 4 hours. After being plated on LB agar plates to which antibiotics were added, the cells were cultured at about 37°C for about 48 hours. After selecting and culturing only white colonies, a small amount of recombinant bacmid was extracted (mini-prep). PCR was performed using M13 forward and reverse primers to confirm the recombinant bacmid.

1-3. Transfection and culture of recombinant bacmid

In this example, cell pectin reagent (Invitrogen, Cat No. 10362-100, USA) was used to transfect the FMDV type A P1 (VP4231) recombinant bacmid prepared above.

Specifically, about 2X10 ⁶ sf9 cells were dispensed into a 6-well culture plate, left for about 1 hour, and then washed twice with medium without antibiotics. About 10 uL of the FMDV type A P1 (VP4231) recombinant bacmid prepared above was mixed with about 100 uL of antibiotic-free medium, and about 8 uL of cell pectin reagent was mixed with about 100 uL of antibiotic-free medium. . The medium containing the bacmid and the medium containing the cell pectin reagent were mixed, left to stand at room temperature for about 25 minutes, and then about 800 uL of medium without antibiotics was added. The mixture containing the complex of the FMDV type A P1 (VP4231) recombinant bacmid and the cell pectin was spotted on the sf9 cell plate. After culturing at about 27°C for about 5 hours, the supernatant was removed and cultured for about 4 days in medium supplemented with about 2% FBS. After confirming the cytopathic effect (CPE), which is an indicator of virus proliferation, it was subcultured and used.

1-4. Extraction and purification of FMDV type A P1 (VP4231) recombinant antigen

In this example, FMDV type A P1 (VP4231) expressed in the cells transfected in Example 1-3 was extracted, and affinity chromatography using the histidine-tag contained in the extracted FMDV type A P1 (VP4231) was performed. Through graphy, the FMDV type A P1 (VP4231) recombinant antigen was purified.

Specifically, the transfected cells of Example 1-3 were cultured to express FMDV type A P1 (VP4231), and then the cell culture was disrupted with an ultrasonic disruptor, and the supernatant was recovered through centrifugation. The recovered supernatant was passed through a Ni-NTA-coupled resin column to allow the target antigen to bind to the resin, and then gradient elution was performed for each concentration of the imidazole solution. For each eluted concentration fraction, the eluate was selected and recovered into a fraction tube containing the target protein through SDS-PAGE. The recovered purified antigen was quantified using the BCA quantitative method to confirm the concentration.

Figure 2 is a diagram showing SDS-PAGE results confirming purified FMDV type A P1 (VP4231) recombinant antigen.

As a result, as shown in Figure 2, a total of four types of FMDV A type P1 containing the structural proteins of A22 Iraq strain, A24 Cruzeiro strain, A type Pocheon strain, and A type Gimpo strain, respectively, prepared in Example 1. (VP4231) It was confirmed that each recombinant antigen showed a distinct band in the SDS-PAGE results, and through this, it was confirmed that each FMDV type A P1 (VP4231) recombinant antigen was purified to a high level of purity.

Through this example, as shown in Table 1, a total of four types of FMDV A, each containing structural proteins of A22 Iraqi strain, A24 Cruzeiro strain, type A Pocheon strain, and type A Gimpo strain, and having high purity. Type P1 (VP4231) recombinant antigen was obtained.

Recombinant antigen name derived cell line order FMDV Type A P1 (VP4231) Iraq P1 A22 Iraq Province SEQ ID NO: 9 Cruzeiro P1 A24 Cruzeiroju SEQ ID NO: 10 Pocheon P1 Type A Pocheonju SEQ ID NO: 11 Gimpo P1 Type A, Kim Po-ju SEQ ID NO: 12

Example 2: Development of monoclonal antibody raw material (Detector) showing immunoreactivity to FMDV type A

2-1. Monoclonal antibody raw material (Detector) development process

Figure 3 is a diagram showing the development process of a monoclonal antibody raw material (Detector) showing immunoreactivity to FMDV type A.

As shown in Figure 3, a hybridoma cell line was developed from the immunized mouse, and hybridoma cell lines secreting antibodies with specific high binding affinity to the antigen were selected, and then the selected hybridoma cell lines were The antibody secreted was separated to obtain monoclonal antibody raw material (detector).

2-2. Immunogen used to develop monoclonal antibody raw material (Detector)

In this example, immunogens to be used in the development of monoclonal antibody raw materials (Detectors) were selected.

Immunogens used in the development of monoclonal antibody raw materials (Detectors) include, in addition to the recombinant baculo antigen (FMDV type A P1 (VP4231)) prepared in Example 1, purified viruses, vaccines, and a total of peptides. An attempt was made to develop a monoclonal antibody raw material (detector) using four types of immunogens. Detailed information about the immunogens is shown in Table 2.

target Immunogen type cell line source VP1 G-H loop peptide Iraq Peptron VP1 G-H loop peptide Cruzeiro Peptron VP1 G-H loop peptide Pocheon Peptron Native Culture concentrate
(BEI deactivated) Iraq (A22) Quarantine Headquarters Native Culture medium SGUC
(BEI deactivated) Iraq (A22) Quarantine Headquarters P1 baculo Iraq bionote P1 baculo Cruzeiro bionote P1 baculo Pocheon bionote

2-3. Preparation of hybridoma cell lines from immunized mice

In this example, immunized mice and hybridoma cell lines were prepared to develop monoclonal antibody raw materials (Detectors).

Specifically, in order to obtain immunized mice required for the development of hybridoma cell lines, the immunogen of Example 2-2 and complete Freund's adjuvant were mixed 1:1 and incubated with magnetic Balb mice about 6 weeks old. /c was injected into the mouse's abdominal cavity. Additional immunizations were performed three times at one-week intervals using the same method. One week after the last 4th immunization, the same antigen used as an immunogen was boosted by injecting the mouse into the tail vein of the mouse about 3 times at intervals of about 1 day. Before performing cell fusion, blood was collected from the eye of the immunized mouse to obtain serum, and then immunity was confirmed by confirming that the antibody titer against the immunogen increased through ELISA method.

Figure 4 is a diagram showing the results of an antibody titer test for the antiserum of mice immunized with a peptide (VP1 G-H loop, FMDV type A Iraqi strain) antigen.

As a result, as shown in Figure 4, it was confirmed that the mice immunized with the peptide (VP1 G-H loop, FMDV type A Iraqi strain) antigen were successfully immunized.

The spleen of the immunized mouse was removed, the cells were separated to obtain one spleen cell and five myeloma cells, and then the spleen cell and myeloma cell were fused to produce a high-quality antibody that secretes antibodies showing immunoreactivity to FMDV type A. Bridoma cell lines were prepared.

2-4. Selection of hybridoma cell lines and acquisition of candidate monoclonal antibody raw materials (Detectors)

In this example, hybridoma cell lines were selected and candidate monoclonal antibody raw material (Detector) was obtained.

Figure 5 is a schematic diagram showing the process of selecting hybridoma cell lines.

Specifically, as shown in Figure 5, after culturing the prepared hybridoma cell line for a certain period of time, hybridoma cell lines secreting antibodies with specific high binding affinity to the antigen were cultured using an indirect ELISA method. Selected.

In addition, candidate monoclonal antibody raw material (Detector) was obtained using the selected hybridoma cell line. Specifically, after the hybridoma cell line was injected into the abdominal cavity of a mouse, ascites fluid was collected from the mouse with a swollen abdominal cavity, and the antibody was isolated from the ascites fluid and purified to obtain a candidate monoclonal antibody raw material (detector).

As a result, a total of 12 types of candidate monoclonal antibody raw materials (Detectors) were obtained (Detector 1 to Detector 12). In addition, in order to visually confirm the degree of reaction between the FMDV type A P1 (VP4231) recombinant antigen and the monoclonal antibody raw material (Detector), an enzyme was conjugated to each obtained monoclonal antibody raw material (Detector) to form a monoclonal antibody raw material (Detector)-enzyme. Conjugates were prepared.

2-5. Selection of antigen raw materials and monoclonal antibody raw materials (Detectors)

In this example, among the FMDV type A P1 (VP4231) recombinant antigen of Table 1 and the candidate monoclonal antibody raw material (Detector) obtained in Example 2-4, antigen raw materials and monoclonal antibodies available for detection of FMDV type A antibody Raw materials (Detector) were selected.

Specifically, the vaccine was applied to plates coated with the recombinant antigens in Table 1, Iraq P1, Cruzeiro P1, Pocheon P1, Gimpo P1, and combinations thereof. Inoculation specimen or vaccination specimen, i.e., New Zealand bovine serum, Canadian pig serum, Merial-inoculated bovine serum, AriaVac Plus-inoculated pig serum, or Bio-Atogen-inoculated pig serum, and the candidate monoclones obtained in Examples 2-4 above. Antibody raw material (Detector)-enzyme conjugate was injected and reacted. Afterwards, the signal intensity generated by the reaction between the recombinant antigens coated on the plate and the injected candidate monoclonal antibody raw materials (Detectors) was analyzed. The degree of reaction between the recombinant antigens coated on the plate and the injected candidate monoclonal antibody raw material (detector) may vary depending on the presence or absence of anti-FMDV type A antibody in the specimen, and the case in which the difference in the degree of reaction is most evident is selected. did. In Table 3, Ag 1 represents Iraq P1 (Iraq P1), Ag 2 represents Cruzeiro P1 (Cruzeiro P1), Ag 3 represents Pocheon P1 (Pocheon P1), and Ag 4 represents Gimpo P1 (Gimpo P1). indicates. In addition, NC 1 in Table 3 represents New Zealand bovine serum, NC 2 represents Canadian pig serum, PC 1 represents Merial-inoculated bovine serum, PC 2 represents AriaVac Plus-inoculated pig serum, and PC 3 represents bio Atogene-inoculated pig serum is shown.

As a result, as shown in Table 3, when Iraq P1, Cruzeiro P1, and Pocheon P1 of Table 1 were used as a capture recombinant antigen to be coated on the plate, the difference between negative and positive signals was most clearly seen. was confirmed, and when Detector 1 (29B91), which was obtained by immunizing Iraqi strains among the candidate monoclonal antibody raw materials (Detectors) obtained in Example 2-4, was used as a monoclonal antibody raw material (Detector), negative and positive results were obtained. It was confirmed that the signal difference appeared most clearly.

As a result of sequence analysis of the monoclonal antibody raw material Detector 1 (29B91), the monoclonal antibody raw material Detector 1 (29B91) consists of HCDR1 consisting of the amino acid sequence of SEQ ID NO: 1, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and amino acid of SEQ ID NO: 3. A heavy chain variable region comprising HCDR3 consisting of the sequence; and a light chain variable region including LCDR1 consisting of the amino acid sequence of SEQ ID NO: 4, LCDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and LCDR3 consisting of the amino acid sequence of SEQ ID NO: 6. In addition, the monoclonal antibody raw material Detector 1 (29B91) includes a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7; and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.

FMDV type A P1 (VP4231) recombinant antigen
candidate monoclonal
antibody raw materials sample Ag 1 … Ag 4 Ag1+2 … Ag 3+4 Ag 1+2+3 … Ag 2+3+4 Ag 1+2+3+4 Detector 1 NC 1 2.039 1.313 2.01 2.257 2.894 1.44 2.09 2.243 2.234 2.111 NC 2 2.119 1.464 2.022 2.262 2.817 1.558 2.174 2.25 2.134 2.035 PC 1 0.037 0.007 0.025 0.042 0.039 0.025 0.01 0.153 0.725 0.012 PC 2 0.361 0.375 0.295 1.205 0.893 0.415 0.086 0.338 1.016 0.374 PC 3 0.044 0.034 0.075 0.378 0.088 0.063 0.009 0.143 0.697 0.01 .
.
. Detector 12 NC 1 1.991 3.507 3.088 2.743 2.341 2.587 2.433 2.788 2.379 2.46 NC 2 1.898 3.46 3.152 2.704 2.245 2.448 2.446 2.729 2.068 2.394 PC 1 0.039 0.009 0.038 0.007 0.02 0.01 0.019 0.017 0.094 0.122 PC 2 0.396 1.17 0.701 0.666 0.628 0.91 0.68 1.25 0.843 0.796 PC 3 0.664 2.879 1.577 1.309 1.233 1.516 1.626 2.001 0.696 0.834

Example 3: Development of a competitive ELISA assay for detecting FMDV type A antibodies

In this example, the developed FMDV type A P1 (VP4231) recombinant antigen (Iraq P1, Cruzeiro P1, Pocheon P1) and monoclonal antibody raw material (Detector 1, 29B91) were used to detect a competitive FMDV type A antibody. An ELISA assay was developed.

Figure 6 is a schematic diagram showing the testing principle of the competitive ELISA assay for detecting FMDV type A antibodies.

Specifically, as shown in Figure 6, about 25 uL of sample and monoclonal antibody raw material (Detector 1, 29B91)-enzyme (HRP) conjugate (FMDV Type A Ab) were placed on a microplate coated with FMDV Type A P1 (VP4231) recombinant antigen. -HRP conjugate) mixture is simultaneously injected with about 100 uL and reacted at about 37°C for about 90 minutes. In the case of a negative sample, since there is no anti-FMDV type A antibody in the sample to react with the recombinant antigen on the plate surface, all monoclonal antibody raw materials (detectors) injected with the sample can bind to the recombinant antigen on the plate surface. there is. In the case of a positive specimen, a competition reaction is induced between the anti-FMDV type A antibody in the specimen and the monoclonal antibody raw material (Detector) injected together with the specimen, so that the monoclonal antibody raw material (Detector) that binds to the recombinant antigen on the plate is relatively small. It decreases. Therefore, in the case of a positive sample, the color development of TMB becomes weak after the reaction stops. On the other hand, in the case of negative samples, the color is dark. Depending on the difference in the degree of color development, the sample can be classified as negative or positive for FMDV type A antibodies.

The competitive ELISA assay for detecting FMDV type A antibodies developed in this way can show significantly improved diagnostic or detection sensitivity performance compared to existing foot-and-mouth disease virus type A diagnosis methods or foot-and-mouth disease virus type A antibody detection methods. This is described in detail in Example 5 below.

Example 4: Setup of sample types for competitive ELISA assay for detection of FMDV type A antibodies

In this example, in order to confirm the type of sample applicable to the competitive ELISA assay for detecting FMDV type A antibodies, serum and plasma samples were obtained from the same individual, and the results were compared.

Specifically, Table 4 shows sample information used. In the case of negative samples, it is difficult to obtain serum and plasma samples for livestock species that are proven to be negative for FMDV type A antibodies in Korea, so among pre-vaccinated individuals (pigs), negative type O antibodies were confirmed through ELISA test and Type A antibodies from Prionics were confirmed. A sample obtained from an individual that was found to be negative as a result of the ELISA kit was used as a negative sample.

Sample number Specimen type Specimen details anticoagulant standard One bovine serum Cattle vaccinated with O/A vaccine (Merial bivalent) Do not process positivity 2 bovine plasma Heparin positivity 3 pig serum O/A vaccine (Bago bivalent) 2nd dose pig Do not process positivity 4 pig plasma Heparin positivity 5 pig serum Pig serum before vaccination
(FMD SP O type ELISA negative,
Prionics-FMD SP type A ELISA negative) Do not process voice 6 pig plasma Heparin voice

As a result, as shown in Table 5, it was confirmed that the difference in sample types between serum and plasma did not affect the results of competitive ELISA analysis for detecting FMDV type A antibodies. Because of this, it was confirmed that both serum and plasma samples can be applied to the competitive ELISA assay for detecting FMDV type A antibodies.

Sample number standard Specimen type PI value result One positivity bovine serum 77.8 positivity 2 bovine plasma 73.5 positivity 3 positivity pig serum 64.2 positivity 4 pig plasma 63.4 positivity 5 voice pig serum 17.6 - 6 pig plasma 20.8 -

Example 5: Performance evaluation of competitive ELISA assay for detecting FMDV type A antibodies

5-1. Basis for cut-off setting

In this example, when samples were classified into negative and positive through a competitive ELISA assay for detecting FMDV type A antibodies, the PI value with the best agreement rate was set as the cut-off value. Table 6 shows information on the samples used in the competitive ELISA assay for detecting FMDV type A antibodies. VNT in Table 6 refers to the Virus Neutralizing Test.

Liver tumor Specimen details
Number of samples note cow voice
(VNT voice) ① FMD clean country serum 1,257 specificity positivity
(VNT positive) ② Vaccination serum 311 responsiveness pig voice
(VNT voice) ③ FMD clean country serum 1,192 specificity ④ Vaccination serum ⑤ Vaccination or infection serum positivity
(VNT positive) ⑥ Vaccination serum 1,343 responsiveness ⑦ Vaccination and infection serum

As a result, as shown in Tables 7 and 8, PI 50 was set for bovine serum and PI 30 for pig serum was set as the standard for judging negative and positive.

division Number of samples Cut-off standard (PI value) - small 30 40 50 60 70 Specificity (%) 1257 85.76%
(1078/1257) 95.47%
(1200/1257) 98.73%
(1241/1257) 99.36%
(1249/1257) 99.76%
(1254/1257) responsiveness(%) 311 96.14%
(299/311) 93.89%
(292/311) 90.35%
(281/311) 83.92%
(261/311) 78.14%
(243/311) accuracy(%) 1568 87.82%
(1377/1568) 95.15%
(1492/1568) 97.07%
(1522/1568) 96.30%
(1510/1568) 95.47%
(1497/1568)

division Number of samples Cut-off criteria (PI value) - Pig 30 40 50 60 70 Specificity (%) 1192 97.73%
(1165/1192) 98.99%
(1180/1192) 99.66%
(1188/1192) 99.83%
(1190/1192) 100%
(1192/1192) responsiveness(%) 1343 87.10%
(1170/1343) 79.37%
(1066/1343) 72.45%
(973/1343) 61.28%
(823/1343) 50.19%
(467/1343) accuracy(%) 2535 92.11%
(2335/2535) 88.60%
(2246/2535) 85.25%
(2161/2535) 79.41%
(2013/2535) 73.61%
(1866/2535)

5-2. Analytical performance evaluation

5-2-1. Analytical sensitivity performance evaluation (Limit of Detection, LOD)

In this example, in order to confirm the detection limit of the competitive ELISA assay for detecting FMDV type A antibodies, vaccination sera with confirmed neutralizing antibody titers against type A were applied at 2 points each for each livestock species (pig, cow), The test was performed by diluting each sample in negative control serum. Table 9 shows serum panel information for LOD analysis.

sample Liver tumor vaccine information Period after vaccination VNT results One cow O/A vaccine (Merial bivalent) 2 weeks after 2 doses 90 2 cow O/A vaccine (ARRIAH bivalent) 4 weeks after 2 doses >256 3 pig O/A vaccine (Bago bivalent) 4 weeks after 2 doses >256 4 pig O/A vaccine (Bago bivalent) 6 weeks after 2 doses >256

As a result, as shown in Table 10, when using the competitive ELISA assay for detecting FMDV type A antibodies, it is possible to detect FMDV type A antibodies from bovine samples up to about 5.6 to 32 times the titer based on the neutralizing antibody test, and pig samples It was confirmed that detection of FMDV type A antibodies was possible up to a titer of approximately 2 times that of the neutralizing antibody test. In particular, when using a competitive ELISA assay for detecting FMDV type A antibodies in bovine samples, when comparing the detection limit with the case using Prionics' analysis method, it was confirmed that the detection ability of FMDV type A antibodies was significantly superior.

sample VNT results Based on dilution factor
VNT Titer dilution factor Prionics
bionote
PI value result PI value result One 90 45 2 59.1 Pos. 80.3 Pos. 22.5 4 52.7 Pos. 69.4 Pos. 11.3 8 43.5 - 55.9 Pos. 5.6 16 31.5 - 50.1 Pos. 2.8 32 25.0 - 35.2 - 1.4 64 25.7 - 26.2 - 0.7 128 26.1 - 23.8 - 0.4 256 24.1 - 25.2 - 2 >256 256 2 59.0 Pos. 82.4 Pos. 128 4 52.2 Pos. 72.8 Pos. 64 8 43.9 - 61.4 Pos. 32 16 33.8 - 53.8 Pos. 16 32 29.1 - 40.4 - 8 64 32.6 - 29 - 4 128 22.0 - 25.5 - 2 256 20.4 - 26.2 - 3 >256 256 2 85.8 Pos. 97.6 Pos. 128 4 85.3 Pos. 94.2 Pos. 64 8 84.1 Pos. 89.8 Pos. 32 16 80.1 Pos. 83.1 Pos. 16 32 77.9 Pos. 74.1 Pos. 8 64 71.7 Pos. 64.4 Pos. 4 128 65.2 Pos. 53.2 Pos. 2 256 50.1 Pos. 38.2 Pos. 4 >256 256 2 87.4 Pos. 98.3 Pos. 128 4 87.8 Pos. 96.7 Pos. 64 8 86.0 Pos. 94.3 Pos. 32 16 82.9 Pos. 90.7 Pos. 16 32 82.0 Pos. 84.6 Pos. 8 64 78.3 Pos. 74.4 Pos. 4 128 71.1 Pos. 63.8 Pos. 2 256 57.4 Pos. 47.4 Pos.

5-2-2. Analytical specificity performance evaluation

In this example, to evaluate the analytical specificity performance of the competitive ELISA assay for detecting FMDV type A antibodies, when using the competitive ELISA assay for detecting FMDV type A antibodies, cross-reactivity between FMD serotypes and cross-over between FMD-like diseases The presence of reactivity was assessed.

(1) Confirmation of cross-reactivity between FMD serotypes

As a result of evaluating whether there was cross-reactivity between FMD serotypes other than FMDV type A using a competitive ELISA assay for detecting FMDV type A antibodies with respect to bovine serum, the IAEA FMD Control panel, as shown in Table 11, FMDV type A When using a competitive ELISA assay for antibody detection, only FMDV type A antiserum was determined to be positive.

serotype PI value result Type O 37.9 - Type A 72.3 positivity Type Asia1 32.6 - SAT1 28.3 - SAT2 2.4 - SAT3 20.2 -

In addition, as a result of evaluating whether there was cross-reactivity between FMD serotypes other than FMDV type A using a competitive ELISA assay for detecting FMDV type A antibodies for the Pirbright FMDV Reference serum panel, as shown in Table 12, FMDV type A When using a competitive ELISA assay for antibody detection, only FMDV type A antiserum was determined to be positive.

serotype Cat.No PI value result Type A A IRN 1996-WP 69 positivity A IRN 1996-SP 86 positivity Type O O SKR 1/2000-WP 19 voice O SKR 1/2000-SP 39 voice Type O O1 Manisa 09/97-WP 14 voice O1 Manisa 09/97-SP 21 voice Type Asia1 Asia1 Shamir-WP 2 20 voice Asia1 Shamir-SP 39 voice Type C C Noville 09/97-WP 42 voice C Noville 09/97-SP 28 voice

In addition, cross-reactivity between FMDV serotypes other than FMDV type A was confirmed by inoculation with FMDV type O single vaccine (O1 Campos, O1 Priomorskiy, O1 Manisa+O 3039) and using pig antiserum infected with Boeunju, the outbreak strain in 2017. As a result of the evaluation, as shown in Table 13, the specificity of the competitive ELISA assay for detecting FMDV type A antibodies was confirmed to be 100% for all vaccine sera and infection sera.

Livestock sample details Number of samples Specificity (%) O ₁ Campos vaccine 3 100 Porcine O ₁ Priomorskiy vaccine 3 100 O ₁ Manisa + O 3039 Vaccine 3 100 Bo Eun-ju (2017-O type) infection serum 8 100

(2) Confirmation of cross-reactivity between FMD-like diseases

As shown in Table 14, the cross-reactivity test results for blistering diseases similar to FMD showed that when using a competitive ELISA assay for detecting FMDV type A antibodies, there was a positive reaction only to BVD sera from FMDV type A-infected cattle. , negative results were confirmed for all other similar diseases.

disease PI value result Infectious Bovine Rinotracheitis 4 voice Bovine Viral Diarrhea 87 positivity Vesicular Exanthema of Swine virus -One voice Swine Vesicular Disease -9 voice Seneca Valley virus -One voice

5-2-3. Analytical precision performance evaluation

In this example, in order to evaluate the analytical precision performance of the competitive ELISA assay for detecting FMDV type A antibodies, evaluation of accuracy and reproducibility performance was performed.

(1) Day-to-day reproducibility

When using the competitive ELISA assay for detecting FMDV type A antibodies, one tester performed the test on each panel at one location for 10 days, 3 replicates per sample, and deviations in the results were checked. As a result, as shown in Table 15, it was confirmed that the difference between days of the competitive ELISA assay for detecting FMDV type A antibodies satisfied the acceptable range.

between dates panel 1 panel 2 panel 3 panel 4 Allowable range Ave. 1.092 1.224 0.410 0.363 Std. Deviation 0.06 0.06 0.02 0.06 CV (%) 5.9% 4.5% 5.6% 15.3% CV ≤ 20%

(2) Lot-to-lot reproducibility (Batch-to-batch)

When using a competitive ELISA assay for detecting FMDV type A antibodies, 3 lots of kits were tested on each panel by one tester at one location for 5 days, and deviations in the results were checked. As a result, as shown in Table 16, it was confirmed that the lot-to-lot variation of the competitive ELISA assay for detecting FMDV type A antibodies satisfied the acceptable range.

Between lots panel 1 panel 2 panel 3 panel 4 Allowable range Ave. 1.097 1.213 0.408 0.375 Std. Deviation 0.066 0.059 0.022 0.052 CV (%) 6.0% 4.8% 5.4% 13.9% CV ≤ 20%

(3) Between-site reproducibility

When using the competitive ELISA assay for detecting FMDV type A antibodies, one tester performed the test with one lot for each panel at three locations for five days, and any deviation in the results was checked. As a result, as shown in Table 17, it was confirmed that the site-to-site variation of the competitive ELISA assay for detecting FMDV type A antibodies met the acceptable range.

between places panel 1 panel 2 panel 3 panel 4 Allowable range Ave. 1.097 1.240 0.409 0.365 Std. Deviation 0.068 0.058 0.021 0.052 CV (%) 6.2% 4.7% 5.2% 14.3% CV ≤ 20%

(4) Between-operators

When using the competitive ELISA assay for detecting FMDV type A antibodies, three testers performed the test in one lot at one location for 5 days, and deviations in the results were checked. As a result, as shown in Table 18, it was confirmed that the inter-person variation of the competitive ELISA assay for detecting FMDV type A antibodies satisfied the acceptable range.

between people panel 1 panel 2 panel 3 panel 4 Allowable range Ave. 1.089 1.223 0.412 0.372 Std. Deviation 0.065 0.061 0.022 0.055 CV (%) 5.9% 5.0% 5.5% 14.7% CV ≤ 20%

5-3. Clinical performance evaluation

5-3-1. Sensitivity performance evaluation

In this example, in order to evaluate the clinical sensitivity performance of the competitive ELISA assay for detecting FMDV type A antibodies, the degree of agreement with the OIE standard test method, the Virus Neutralizing Test (VNT), was evaluated, and the subject was vaccinated against the virus. The time when antibodies can be detected after vaccination and the time when antibodies can be detected after virus challenge in vaccinated subjects were evaluated.

(1) Sensitivity evaluation of type A positive samples (evaluation of consistency with neutralization test (VNT))

Serum (vaccination serum) and O/A vaccine (Arial bivalent, O1 Primorskiy + A Zabaikalskiy) of the group vaccinated with type A monovalent (A Zabaikalskiy) vaccine and O/A vaccine (Arial bivalent, O1 Primorskiy + A Zabaikalskiy) A neutralization test and a competitive ELISA assay for detection of FMDV type A antibodies were performed on sera from pigs (139 points) infected with foot-and-mouth disease virus.

As a result, as shown in Table 19, the detection rate of the neutralization test method was confirmed to be about 87.1%. Compared to the neutralization test method, the sensitivity of the competitive ELISA assay for detecting FMDV type A antibodies was confirmed to be approximately 73.6% (89/121), and the specificity was approximately 94.4% (17/18).

N=139 (Porcine)
VNT
positivity voice Sum SP-A ELISA positivity 89 One 90 voice 32 17 49 Sum 121 18 139

(2) Evaluation of antibody detection period after virus attack vaccination

As a result of examining the blood serum collected by days (Days Post Injection, DPI) after inoculation with type A Yeoncheonju virus in SPF pigs, the time when antibody detection was possible was confirmed. As shown in Table 20, the neutralization test method showed that the challenge inoculation It was confirmed that the competitive ELISA assay for detecting FMDV type A antibodies was confirmed to be positive approximately 9 to 10 days after challenge.

SPF Pig
DPI
Specimen name method of inspection One 2 3 4 6 7 8 9 10 11 12 13 14 18 5 VNT <16 32 90 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 ELISA 14 4 -26 -26 -12 4 One 31 58 57 53 85 83 6 VNT <16 <16 90 >256 >256 >256 >256 >256 >256 >256 ELISA 35 4 -16 24 20 21 41 48 68 92

As a result of examining the blood serum collected by Days Post Injection (DPI) after inoculation with type A Yeoncheonju virus in non-SPF pigs, the time when antibody detection was possible was confirmed. As shown in Table 21, neutralization test was performed. It was confirmed that the test was positive starting from about 5 to 9 days after the challenge vaccination and from about 4 days after the challenge vaccination using the competitive ELISA assay for detecting FMDV type A antibodies.

pig
DPI
Specimen name method of inspection One 2 3 4 5 9 12 15 GA-21 VNT <16 90 16 <16 22 45 64 45 ELISA 7 19 12 76 77 70 61 62 GA-22 VNT 16 181 <16 16 16 90 256 181 ELISA 15 32 16 40 44 74 50 51 GA-23 VNT 16 181 <16 16 16 90 256 181 ELISA 15 32 16 40 44 74 50 51 GA-24 VNT <16 128 <16 16 45 64 128 128 ELISA 9 7 5 45 46 74 72 74

Through this example, it was confirmed that when using a competitive ELISA assay for detecting FMDV type A antibodies on a type A Yeoncheonju infection sample, positive detection was possible from about 4 to 10 days after infection.

(3) Evaluation of antibody detection time after virus challenge in vaccinated subjects

After inoculating SPF pigs with the Merial vaccine, they were challenged with type A Yeoncheonju virus 7 days later, 21 days later, and 35 days later, respectively, and the serum collected according to the days after the virus challenge vaccination (Days Post Contact, DPC) was tested. Thus, the time when antibody detection was possible was confirmed.

As a result, as shown in Table 22, the competitive ELISA assay for detecting FMDV type A antibodies was determined to be positive from about 5 to 7 days after the virus challenge in the sample inoculated with the virus challenge 7 days after vaccination. Confirmed. In addition, in samples that received a viral challenge 21 days after vaccination, positive results were obtained approximately 4 to 6 days after vaccination, and in all samples that received a viral challenge 35 days after vaccination, antibodies caused by the vaccine were detected. It was confirmed that the test was positive from the time of virus attack.

After receiving Merial vaccine
attack inoculation DPC
Specimen name vaccine information method of inspection One 2 3 4 5 6 7 8 9 10 11 12 16 7dpv VNT >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 ELISA -4 -9 -14 -6 48 49 44 43 62 59 17 7dpv VNT >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 ELISA 14 16 6 23 39 59 57 52 51 65 59 18 7dpv VNT >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 ELISA -10 -9 0 36 52 73 80 75 78 81 71 19 7dpv VNT >256 >256 ELISA -29 -22 12 21dpv VNT >256 181 181 >256 >256 >256 >256 >256 >256 >256 >256 >256 ELISA 16 11 10 16 60 68 82 87 90 91 92 92 13 21dpv VNT >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 ELISA 32 19 9 One 12 40 69 83 90 91 92 14 21dpv VNT >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 ELISA 0 8 4 22 54 75 87 90 89 92 89 15 21dpv VNT >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 ELISA 19 27 21 33 57 67 84 83 79 82 79 One 35dpv VNT >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 ELISA 65 69 54 53 61 81 92 96 96 97 96 97 2 35dpv VNT >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 ELISA 59 63 50 56 62 62 73 88 91 91 3 35dpv VNT >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 ELISA 32 29 32 48 82 89 93 96 97 97 95 4 35dpv VNT >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 ELISA 52 58 58 55 33 78 88 90 91 91 93 92

After inoculating SPF pigs with the Eastern vaccine, they were challenged with type A Yeoncheonju virus 7 days and 28 days later, respectively. Antibodies can be detected by testing serum collected according to the days after the virus challenge (Days Post Contact, DPC). The timing was confirmed.

As a result, as shown in Table 23, it was confirmed that the competitive ELISA assay for detecting FMDV type A antibodies was determined to be positive about 1 to 3 days after the virus challenge in the sample inoculated with the virus challenge 7 days after vaccination. did. In addition, it was confirmed that most of the samples that were vaccinated against the virus 28 days after vaccination were tested positive approximately 1 day after the vaccination. Some individuals tested positive approximately 3 days after being vaccinated against the virus, which is understood to be due to differences in immunity levels among individuals.

After receiving the Eastern vaccine
attack inoculation DPC
Specimen name vaccine information method of inspection One 3 4 5 9 12 15 16 7dpv VNT 45 90 90 90 45 45 45 ELISA 24 34 61 48 52 31 7dpv VNT 16 <16 22 32 <16 32 32 ELISA 36 44 64 40 60 32 7dpv VNT <16 <16 <16 <16 32 181 181 ELISA 15 35 49 48 69 36 7dpv VNT 22 32 16 32 22 45 32 ELISA 25 52 76 59 74 37 7dpv VNT 22 22 16 32 22 32 32 ELISA 22 33 65 65 80 39 7dpv VNT 22 16 16 22 22 <16 16 ELISA 29 39 68 57 84 40 7dpv VNT 90 128 128 128 90 90 ELISA 47 59 66 61 69 41 7dpv VNT 64 90 90 128 32 90 90 ELISA 42 61 72 63 74 One 28dpv VNT 64 90 32 45 90 45 64 ELISA 61 68 72 66 74 2 28dpv VNT 128 128 181 128 64 90 ELISA 70 78 81 68 81 4 28dpv VNT 64 45 45 45 64 64 64 ELISA 64 70 86 81 83 5 28dpv VNT 64 90 45 64 32 128 256 ELISA 67 74 84 77 88 6 28dpv VNT 64 128 32 90 90 128 90 ELISA 58 62 84 78 67 8 28dpv VNT 22 32 32 32 32 22 45 ELISA 60 71 83 74 81 9 28dpv VNT <16 <16 16 16 <16 16 22 ELISA 30 33 78 67 57 10 28dpv VNT 22 <16 22 32 32 22 ELISA 45 48 49 48 73 11 28dpv VNT 64 45 90 64 256 128 128 ELISA 27 30 64 53 94 12 28dpv VNT 22 64 32 45 32 22 ELISA 70 67 47 49 84

Through this example, it was confirmed that the competitive ELISA assay for detecting FMDV type A antibodies showed relatively higher sensitivity for the Oriental vaccine (A zabaikalsky) strain compared to the Merial vaccine strain.

5-3-2. Specificity performance evaluation

In this example, to evaluate the clinical specificity performance of the competitive ELISA assay for detecting FMDV type A antibodies, samples obtained from clean countries, which are non-vaccinated samples of foot-and-mouth disease vaccine, and type A-negative domestic samples, which are type O monovalent vaccine-vaccinated samples, A competitive ELISA assay for detecting FMDV type A antibodies was performed on field samples, and the specificity performance for detecting FMDV type A antibodies was evaluated.

(1) Evaluation of specificity of samples from non-vaccinated/foot-and-mouth disease-free countries

The specificity performance of the competitive ELISA assay for detecting FMDV type A antibodies was evaluated using bovine serum and porcine serum samples obtained from clean countries (New Zealand and Canada), which were not vaccinated with foot-and-mouth disease vaccine.

As a result, as shown in Table 24, it was confirmed that the competitive ELISA assay for detecting FMDV type A antibodies showed a specificity performance of 100% (1,100/1,100) for a total of 1,100 negative samples.

Liver tumor Sample type Introduction country Introduction Number of samples FMDV type A antibody negative test rate (%) cow negative serum New Zealand Invetus 600 100 (600/600) pig negative serum Canada VIDO-Intervac 500 100 (500/500) Sum 1,100 100 (1100/1100)

(2) Type O monovalent vaccination / Type A negative domestic outdoor sample specificity evaluation

To determine whether specific detection of foot-and-mouth disease type A antibodies was possible, the specificity performance of a competitive ELISA assay for detecting FMDV type A antibodies was evaluated using type A-negative domestic field samples, which were type O monovalent vaccination samples.

As a result, as shown in Table 25, the competitive ELISA assay for detecting FMDV type A antibodies showed a negative determination of foot-and-mouth disease type A antibodies for about 99.3% of the samples in which all foot-and-mouth disease type O antibody titers were found to be positive; It was confirmed that the specificity performance was (143/144).

Liver tumor vaccine type Number of samples Negative test rate (%) Positive test rate (%) FMD Type A FMD Type O bionote Prionics pig Aria bag 43 100% (43/43) 100% (43/43) Atogene 66 98.5% (65/66) 100% (66/66) Merial 17 100% (17/17) 100% (17/17) subtotal 126 99.2% (125/126) 100% (126/126) cow Merial 18 100% (18/18) 100% (18/18) subtotal 18 100% (18/18) 100% (18/18) Sum
144 99.3% (143/144) 100% (144/144)

5-3-3. Domestic outdoor application evaluation results

In this example, in order to evaluate the clinical sensitivity performance of the competitive ELISA assay for detecting FMDV type A antibodies, a comparative evaluation was performed with the Prionics kit test for each vaccine strain using field O+A vaccination field samples collected in Korea. carried out.

As a result, as shown in Table 26, it was confirmed that the competitive ELISA assay for detecting FMDV type A antibodies showed a positive determination rate for type A antibodies of about 87.4% (76/87) for bovine samples, and about 87.4% (76/87) for pig samples. It was confirmed that the positive test rate for type A antibodies was 43.9% (279/635). In particular, it was confirmed that the competitive ELISA assay for detecting FMDV type A antibodies showed better sensitivity performance compared to the Prionics kit test.

Liver tumor Type O+A vaccine Number of samples Number of vaccinations Positive test rate (%)
FMD Type A
FMD Type O bionote Prionics bionote cow Merial 72 - 90.3% (65/72) 81.9% (59/72) 100% (72/72) Aria Bag Plus 15 - 73.3% (11/15) 73.3% (11/15) 86.7% (13/15) subtotal 87 - 87.4% (76/87) 80.5% (70/87) 97.7% (85/87) pig Merial 30 Primary 16.7% (5/30) 3.3% (1/30) 33.3% (10/30) bioatogen 45 Primary 77.8% (35/45) 66.7% (30/45) 68.9% (31/45) 40 Secondary 70.0% (28/40) 70.0% (28/40) 67.5% (27/40) Aria Bag Plus 168 Primary 17.3% (29/168) 8.9% (15/168) 26.8% (45/168) 342 Secondary 50.9% (174/342) 29.2% (100/342) 58.5% (200/342) 10 3rd 80% (8/10) 30% (3/10) 90.0% (9/10) subtotal 635 43.9% (279/635) 27.9% (177/635) 50.7% (322/635) Sum
722 49.2% (355/722) 34.2% (247/722) 56.4% (407/722)

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

<110> REPUBLIC OF KOREA(Animal and Plant Quarantine Agency) BIONOTE, INC. <120> Antibody exhibiting immunoreactivity to foot and mouth disease virus type A, composition for detecting foot and mouth disease virus type A antibody comprising the same, and method for detecting foot and mouth disease virus type A antibody using the same <130> PN136867KR < 160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 1 Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Asn 1 5 10 < 210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 2 Asn Ile Tyr Pro Ser Asp Ser Ser Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210 > 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 3 Asp His Tyr Tyr Gly Ser Cys Pro Phe Ala Tyr 1 5 10 <210> 4 <211> 10 <212 > PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 4 Arg Ala Ser Ser Gly Val Ser Tyr Met Phe 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220 > <223> LCDR2 <400> 5 Asp Thr Ser Asn Leu Ala Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 6 Gln Gln Trp Ser Phe Tyr Pro Arg Thr 1 5 <210> 7 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region <400> 7 Glu Val Lys Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Ser Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp His Tyr Tyr Gly Ser Cys Pro Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Pro 115 120 <210> 8 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region <400> 8 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Val Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Val Thr Cys Arg Ala Ser Ser Gly Val Ser Tyr Met 20 25 30 Phe Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Tyr Pro Arg Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro 100 105 110 Thr Val Ser 115 <210> 9 <211> 738 <212> PRT <213> Artificial Sequence <220> <223> Iraq P1 <400> 9 Met Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser 1 5 10 15 Gly Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln 20 25 30 Asn Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser 35 40 45 Asn Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln 50 55 60 Asn Asn Asp Trp Phe Ser Lys Leu Ala Ser Ser Ala Phe Ser Gly Leu 65 70 75 80 Phe Gly Ala Leu Leu Ala Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu 85 90 95 Glu Asp Arg Ile Leu Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr 100 105 110 Gln Ser Ser Val Gly Val Thr Tyr Gly Tyr Ser Thr Gln Glu Asp His 115 120 125 Val Ser Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg Val Val Gln Ala 130 135 140 Glu Arg Phe Phe Lys Lys His Leu Phe Asp Trp Thr Pro Asp Lys Ala 145 150 155 160 Phe Gly His Leu Glu Lys Leu Glu Leu Pro Thr Asp His Lys Gly Val 165 170 175 Tyr Gly His Leu Val Asp Ser Phe Ala Tyr Met Arg Asn Gly Trp Asp 180 185 190 Val Glu Val Ser Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu 195 200 205 Val Ala Met Val Pro Glu Trp Lys Glu Phe Thr Pro Arg Glu Lys Tyr 210 215 220 Gln Leu Thr Leu Phe Pro His Gln Phe Ile Ser Pro Arg Thr Asn Met 225 230 235 240 Thr Ala His Ile Val Val Pro Tyr Leu Gly Val Asn Arg Tyr Asp Gln 245 250 255 Tyr Lys Lys His Lys Pro Trp Thr Leu Val Val Met Val Val Ser Pro 260 265 270 Leu Thr Thr Asn Thr Val Ser Ala Gly Gln Ile Lys Val Tyr Ala Asn 275 280 285 Ile Ala Pro Thr His Val His Val Ala Gly Glu Leu Pro Ser Lys Glu 290 295 300 Gly Ile Val Pro Val Ala Cys Ser Asp Gly Tyr Gly Gly Leu Val Thr 305 310 315 320 Thr Asp Pro Lys Thr Ala Asp Pro Val Tyr Gly Met Val Tyr Asn Pro 325 330 335 Pro Arg Thr Asn Tyr Pro Gly Arg Phe Thr Asn Leu Leu Asp Val Ala 340 345 350 Glu Ala Cys Pro Thr Phe Leu Cys Phe Asp Glu Gly Lys Pro Tyr Val 355 360 365 Val Thr Arg Thr Asp Glu Gln Arg Leu Leu Ala Lys Phe Asp Val Ser 370 375 380 Leu Ala Ala Lys His Met Ser Asn Thr Tyr Leu Ser Gly Ile Ala Gln 385 390 395 400 Tyr Tyr Ala Gln Tyr Ser Gly Thr Ile Asn Leu His Phe Met Phe Thr 405 410 415 Gly Ser Thr Asp Ser Lys Ala Arg Tyr Met Val Ala Tyr Val Pro Pro 420 425 430 Gly Val Glu Thr Pro Pro Asp Thr Pro Glu Lys Ala Ala His Cys Ile 435 440 445 His Ala Glu Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile 450 455 460 Pro Tyr Val Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Val Ala 465 470 475 480 Glu Thr Thr Asn Val Gln Gly Trp Val Cys Ile Tyr Gln Ile Thr His 485 490 495 Gly Lys Ala Glu Gln Asp Thr Leu Val Val Ser Val Ser Ala Gly Lys 500 505 510 Asp Phe Glu Leu Arg Leu Pro Ile Asp Pro Arg Ser Gln Thr Thr 515 520 525 Thr Gly Glu Ser Ala Asp Pro Val Thr Thr Thr Val Glu Asn Tyr Gly 530 535 540 Gly Glu Thr Gln Val Gln Arg Arg Gln His Thr Asp Val Thr Phe Ile 545 550 555 560 Met Asp Arg Phe Val Lys Ile Gln Asn Leu Asn Pro Thr His Val Ile 565 570 575 Asp Leu Met Gln Thr His Gln His Gly Leu Val Gly Ala Leu Leu Arg 580 585 590 Ala Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Ile Val Val Arg His Asp 595 600 605 Gly Asn Leu Thr Trp Val Pro Asn Gly Ala Pro Glu Ala Ala Leu Ser 610 615 620 Asn Thr Gly Asn Pro Thr Ala Tyr Leu Lys Ala Pro Phe Thr Arg Leu 625 630 635 640 Ala Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ala Thr Val Tyr Asn 645 650 655 Gly Thr Ser Lys Tyr Ser Ala Gly Gly Thr Gly Arg Arg Gly Asp Leu 660 665 670 Gly Pro Leu Ala Ala Arg Val Ala Ala Gln Leu Pro Ala Ser Phe Asn 675 680 685 Phe Gly Ala Ile Gln Ala Thr Thr Ile His Glu Leu Leu Val Arg Met 690 695 700 Lys Arg Ala Glu Leu Tyr Cys Pro Arg Pro Leu Leu Ala Val Glu Val 705 710 715 720 Ser Ser Gln Asp Arg His Lys Gln Lys Ile Ile Ala Pro Ala Lys Gln 725 730 735 Leu Leu <210> 10 <211> 738 <212> PRT <213> Artificial Sequence <220> <223> Cruzeiro P1 <400> 10 Met Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser 1 5 10 15 Gly Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln 20 25 30 Asn Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser 35 40 45 Asn Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln 50 55 60 Asn Asn Asp Trp Phe Ser Lys Leu Ala Ser Ala Phe Thr Gly Leu 65 70 75 80 Phe Gly Ala Leu Leu Ala Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu 85 90 95 Glu Asp Arg Ile Leu Thr Arg Asn Gly His Thr Thr Ser Thr Thr 100 105 110 Gln Ser Ser Val Gly Val Thr His Gly Tyr Ser Thr Glu Glu Asp His 115 120 125 Val Ala Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg Val Val Gln Ala 130 135 140 Glu Arg Phe Tyr Lys Lys Tyr Leu Phe Asp Trp Thr Thr Asp Lys Ala 145 150 155 160 Phe Gly His Leu Glu Lys Leu Glu Leu Pro Ser Asp His His Gly Val 165 170 175 Phe Gly His Leu Val Asp Ser Tyr Ala Tyr Met Arg Asn Gly Trp Asp 180 185 190 Val Glu Val Ser Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu 195 200 205 Val Ala Met Val Pro Glu Trp Lys Glu Phe Asp Thr Arg Glu Lys Tyr 210 215 220 Gln Leu Thr Leu Phe Pro His Gln Phe Ile Ser Pro Arg Thr Asn Met 225 230 235 240 Thr Ala His Ile Thr Val Pro Tyr Leu Gly Val Asn Arg Tyr Asp Gln 245 250 255 Tyr Lys Lys His Lys Pro Trp Thr Leu Val Val Met Val Val Ser Pro 260 265 270 Leu Thr Val Asn Asn Thr Ser Ala Ala Gln Ile Lys Val Tyr Ala Asn 275 280 285 Ile Ala Pro Thr Tyr Val His Val Ala Gly Glu Leu Pro Ser Lys Glu 290 295 300 Gly Ile Phe Pro Val Ala Cys Ala Asp Gly Tyr Gly Gly Leu Val Thr 305 310 315 320 Thr Asp Pro Lys Thr Ala Asp Pro Ala Tyr Gly Lys Val Tyr Asn Pro 325 330 335 Pro Arg Thr Asn Tyr Pro Gly Arg Phe Thr Asn Leu Leu Asp Val Ala 340 345 350 Glu Ala Cys Pro Thr Phe Leu Cys Phe Asp Asp Gly Lys Pro Tyr Val 355 360 365 Thr Thr Arg Thr Asp Asp Thr Arg Leu Leu Ala Lys Phe Asp Leu Ser 370 375 380 Leu Ala Ala Lys His Met Ser Asn Thr Tyr Leu Ser Gly Ile Ala Gln 385 390 395 400 Tyr Tyr Thr Gln Tyr Ser Gly Thr Ile Asn Leu His Phe Met Phe Thr 405 410 415 Gly Ser Thr Asp Ser Lys Ala Arg Tyr Met Val Ala Tyr Ile Pro Pro Pro 420 425 430 Gly Val Glu Thr Pro Pro Asp Thr Pro Glu Arg Ala Ala His Cys Ile 435 440 445 His Ala Glu Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile 450 455 460 Pro Tyr Val Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Thr Ala 465 470 475 480 Glu Thr Ile Asn Val Gln Gly Trp Val Cys Ile Tyr Gln Ile Thr His 485 490 495 Gly Lys Ala Glu Asn Asp Thr Leu Val Val Ser Val Ser Ala Gly Lys 500 505 510 Asp Phe Glu Leu Arg Leu Pro Ile Asp Pro Arg Gln Gln Thr Thr Ala 515 520 525 Thr Gly Glu Ser Ala Asp Pro Val Thr Thr Thr Val Glu Asn Tyr Gly 530 535 540 Gly Glu Thr Gln Ile Gln Arg Arg His His Thr Asp Ile Gly Phe Ile 545 550 555 560 Met Asp Arg Phe Val Lys Ile Gln Ser Leu Ser Pro Thr His Val Ile 565 570 575 Asp Leu Met Gln Thr His Gln His Gly Leu Val Gly Ala Leu Leu Arg 580 585 590 Ala Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Ile Val Val Arg His Glu 595 600 605 Gly Asn Leu Thr Trp Val Pro Asn Gly Ala Pro Glu Ser Ala Leu Leu 610 615 620 Asn Thr Ser Asn Pro Thr Ala Tyr Asn Lys Ala Pro Phe Thr Arg Leu 625 630 635 640 Ala Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ala Thr Val Tyr Asn 645 650 655 Gly Thr Ser Lys Tyr Ala Val Gly Gly Ser Gly Arg Arg Gly Asp Met 660 665 670 Gly Ser Leu Ala Ala Arg Val Val Lys Gln Leu Pro Ala Ser Phe Asn 675 680 685 Tyr Gly Ala Ile Lys Ala Asp Ala Ile His Glu Leu Leu Val Arg Met 690 695 700 Lys Arg Ala Glu Leu Tyr Cys Pro Arg Pro Leu Leu Ala Ile Glu Val 705 710 715 720 Ser Ser Gln Asp Arg His Lys Gln Lys Ile Ile Ala Pro Ala Lys Gln 725 730 735 Leu Leu <210> 11 <211> 737 <212> PRT <213> Artificial Sequence <220> <223> Pocheon P1 <400> 11 Met Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser 1 5 10 15 Gly Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln 20 25 30 Asn Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser 35 40 45 Asn Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln 50 55 60 Asn Asn Asp Trp Phe Ser Lys Leu Ala Ser Ser Ala Phe Thr Gly Leu 65 70 75 80 Phe Gly Ala Leu Leu Ala Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu 85 90 95 Glu Asp Arg Ile Leu Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr 100 105 110 Gln Ser Ser Val Gly Val Thr Tyr Gly Tyr Ser Thr Gly Glu Asp His 115 120 125 Val Ser Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg Val Val Gln Ala 130 135 140 Glu Arg Phe Phe Lys Lys His Leu Phe Asp Trp Thr Thr Asp Lys Pro 145 150 155 160 Phe Gly His Val Glu Lys Leu Glu Leu Pro Thr Asp His Lys Gly Val 165 170 175 Tyr Gly Gln Leu Val Asp Ser Phe Ala Tyr Met Arg Asn Gly Trp Asp 180 185 190 Val Glu Val Ser Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu 195 200 205 Val Ala Met Val Pro Glu Phe Lys Glu Phe Thr Arg Glu Lys Tyr 210 215 220 Gln Leu Thr Leu Phe Pro His Gln Phe Ile Ser Pro Arg Thr Asn Met 225 230 235 240 Thr Ala His Ile Thr Val Pro Tyr Leu Gly Val Asn Arg Tyr Asp Gln 245 250 255 Tyr Asn Lys His Lys Pro Trp Thr Leu Val Val Met Val Val Ser Pro 260 265 270 Leu Thr Thr Ser Ser Ile Gly Ala Ser Gln Ile Lys Val Tyr Thr Asn 275 280 285 Ile Ala Pro Thr His Val His Val Ala Gly Glu Leu Pro Ser Lys Glu 290 295 300 Gly Ile Val Pro Val Ala Cys Ser Asp Gly Tyr Gly Gly Leu Val Thr 305 310 315 320 Thr Asp Pro Lys Thr Ala Asp Pro Ala Tyr Gly Met Val Tyr Asn Pro 325 330 335 Pro Arg Thr Asn Tyr Pro Gly Arg Phe Thr Asn Leu Leu Asp Val Ala 340 345 350 Glu Ala Cys Pro Thr Phe Leu Cys Phe Asp Asp Gly Lys Pro Tyr Val 355 360 365 Val Thr Arg Thr Asp Glu Gln Arg Leu Leu Ala Lys Phe Asp Leu Ser 370 375 380 Leu Ala Ala Lys His Met Ser Asn Thr Leu Ser Gly Ile Ala Gln 385 390 395 400 Tyr Tyr Ala Gln Tyr Ser Gly Thr Ile Asn Leu His Phe Met Phe Thr 405 410 415 Gly Ser Thr Asp Ser Lys Ala Arg Tyr Met Val Ala Tyr Val Pro Pro 420 425 430 Gly Val Thr Thr Pro Pro Asp Thr Pro Glu Arg Ala Ala His Cys Ile 435 440 445 His Ala Glu Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile 450 455 460 Pro Tyr Val Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Val Ala 465 470 475 480 Asp Thr Thr Asn Val Gln Gly Trp Val Cys Ile Tyr Gln Ile Thr His 485 490 495 Gly Lys Ala Glu Gln Asp Thr Leu Val Val Ser Val Ser Ala Gly Lys 500 505 510 Asp Phe Glu Leu Arg Leu Pro Ile Asp Pro Arg Ala Gln Thr Thr Ala 515 520 525 Thr Gly Glu Ser Ala Asp Pro Val Thr Thr Thr Val Glu Asn Tyr Gly 530 535 540 Gly Glu Thr Gln Val Gln Arg Arg His His Thr Asp Val Ser Phe Ile 545 550 555 560 Met Asp Arg Phe Val Gln Ile Lys Pro Val Ser Pro Thr His Val Ile 565 570 575 Asp Leu Met Gln Thr His Gln His Gly Leu Val Gly Ala Met Leu Arg 580 585 590 Ala Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Ile Val Val Asn His Thr 595 600 605 Gly Arg Leu Thr Trp Val Pro Asn Gly Ala Pro Glu Ala Ala Leu Asp 610 615 620 Asn Thr Ser Asn Pro Thr Ala Tyr His Lys Ala Pro Phe Thr Arg Leu 625 630 635 640 Ala Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ala Thr Val Tyr Asn 645 650 655 Gly Thr Ser Arg Tyr Ser Ala Pro Ala Thr Arg Arg Gly Asp Leu Gly 660 665 670 Ser Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala Ser Phe Asn Tyr 675 680 685 Gly Ala Val Arg Ala Thr Glu Ile Gln Glu Leu Leu Val Arg Met Lys 690 695 700 Arg Ala Glu Leu Tyr Cys Pro Arg Pro Leu Leu Ala Val Glu Val Thr 705 710 715 720 Ser Gln Asp Arg His Lys Gln Lys Ile Ile Ala Pro Ala Lys Gln Leu 725 730 735 Leu <210> 12 <211> 737 <212> PRT <213> Artificial Sequence <220> <223> Gimpo P1 <400> 12 Met Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser 1 5 10 15 Gly Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln 20 25 30 Asn Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser 35 40 45 Asn Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln 50 55 60 Asn Asn Asp Trp Phe Ser Lys Leu Ala Ser Ser Ala Phe Thr Gly Leu 65 70 75 80 Phe Gly Ala Leu Leu Ala Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu 85 90 95 Glu Asp Arg Ile Leu Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr 100 105 110 Gln Ser Ser Val Gly Val Thr Cys Gly Tyr Ser Thr Gly Glu Asp His 115 120 125 Val Ser Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg Val Val Gln Ala 130 135 140 Glu Arg Phe Phe Lys Lys Tyr Leu Phe Asp Trp Thr Thr Asp Lys Pro 145 150 155 160 Phe Gly His Val Val Lys Leu Glu Leu Pro Thr Glu His Lys Gly Val 165 170 175 Tyr Gly Gln Leu Val Glu Ser Phe Ala Tyr Met Arg Asn Gly Trp Asp 180 185 190 Val Glu Val Ser Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu 195 200 205 Val Ala Met Ile Pro Glu Phe Lys Glu Phe Thr Gln Arg Glu Lys Tyr 210 215 220 Gln Leu Thr Leu Phe Pro His Gln Phe Ile Ser Pro Arg Thr Asn Met 225 230 235 240 Thr Ala His Ile Thr Val Pro Tyr Leu Gly Val Asn Arg Tyr Asp Gln 245 250 255 Tyr Lys Lys His Lys Pro Trp Thr Leu Val Val Met Val Val Ser Pro 260 265 270 Leu Thr Thr Ser Ser Ile Gly Ala Thr Gln Ile Lys Val Tyr Ala Asn 275 280 285 Ile Ala Pro Thr Tyr Val His Val Ala Gly Glu Leu Pro Ser Lys Glu 290 295 300 Gly Ile Val Pro Val Ala Cys Ser Asp Gly Tyr Gly Gly Leu Val Thr 305 310 315 320 Thr Asp Pro Lys Thr Ala Asp Pro Ala Tyr Gly Met Val Tyr Asn Pro 325 330 335 Pro Arg Thr Asn Tyr Pro Gly Arg Phe Thr Asn Leu Leu Asp Val Ala 340 345 350 Glu Ala Cys Pro Thr Phe Leu Cys Phe Asp Asp Gly Lys Pro Tyr Ile 355 360 365 Val Thr Arg Thr Asp Glu Gln Arg Leu Leu Ala Lys Phe Asp Leu Ser 370 375 380 Leu Ala Ala Lys His Met Ser Asn Thr Tyr Leu Ser Gly Leu Ala Gln 385 390 395 400 Tyr Tyr Ala Gln Tyr Ser Gly Thr Ile Asn Leu His Phe Met Phe Thr 405 410 415 Gly Ser Thr Asp Ser Lys Ala Arg Tyr Met Val Ala Tyr Val Pro Pro 420 425 430 Gly Val Glu Thr Pro Pro Asp Thr Pro Glu Lys Ala Ala His Cys Ile 435 440 445 His Ala Glu Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile 450 455 460 Pro Tyr Val Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Glu Ala 465 470 475 480 Glu Thr Thr Asn Val Gln Gly Trp Val Cys Ile Tyr Gln Ile Thr His 485 490 495 Gly Lys Ala Glu Gln Asp Thr Leu Val Val Ser Val Ser Ala Gly Lys 500 505 510 Asp Phe Glu Leu Arg Leu Pro Ile Asp Pro Arg Ala Gln Thr Thr 515 520 525 Thr Gly Glu Ser Ala Asp Pro Val Thr Thr Thr Val Glu Asn Tyr Gly 530 535 540 Gly Glu Thr Gln Val Gln Arg Arg Tyr His Thr Asp Val Gly Phe Leu 545 550 555 560 Met Asp Arg Phe Val Gln Ile Lys Pro Val Gly Ser Thr His Val Ile 565 570 575 Asp Leu Met Gln Thr His Gln His Gly Leu Val Gly Ala Met Leu Arg 580 585 590 Ala Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Ile Val Val Asn His Thr 595 600 605 Gly Asn Leu Thr Trp Val Pro Asn Gly Ala Pro Glu Ala Ala Leu Gln 610 615 620 Asn Thr Ser Asn Pro Thr Ala Tyr His Lys Ala Pro Phe Thr Arg Leu 625 630 635 640 Ala Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ala Thr Val Tyr Ser 645 650 655 Gly Thr Ser Lys Tyr Ser Ala Pro Gln Asn Arg Arg Gly Asp Ser Gly 660 665 670 Pro Leu Val Val Lys Pro Ala Thr Gln Leu Pro Ala Ser Phe Asn Phe 675 680 685 Gly Ala Ile Arg Ala Thr Glu Ile Arg Glu Leu Leu Val Arg Met Lys 690 695 700 Arg Ala Glu Leu Tyr Cys Pro Arg Pro Leu Leu Ala Val Glu Val Ser 705 710 715 720 Ser Gln Asp Arg His Lys Gln Lys Ile Ile Ala Pro Ala Lys Gln Leu 725 730 735Leu

Claims (12)

A heavy chain variable region comprising HCDR1 consisting of the amino acid sequence of SEQ ID NO: 1, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 3; and
An antibody showing immunoreactivity to foot-and-mouth disease virus type A, comprising a light chain variable region comprising LCDR1 having the amino acid sequence of SEQ ID NO: 4, LCDR2 having the amino acid sequence of SEQ ID NO: 5, and LCDR3 having the amino acid sequence of SEQ ID NO: 6. or as an antigen-binding fragment thereof,
The antibody or antigen-binding fragment thereof specifically binds to a foot-and-mouth disease virus type A antigen. The method according to claim 1, wherein the antibody or antigen-binding fragment thereof includes a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7; and
An antibody or antigen-binding fragment thereof comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8. delete The method of claim 1, wherein the foot-and-mouth disease virus type A antigen is a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 9, a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 10, a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 11, and an amino acid of SEQ ID NO: 12. An antibody or antigen-binding fragment thereof, which is a recombinant antigen consisting of a sequence, or a combination thereof. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is produced from hybridoma cells. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is bound or conjugated to a detection label. A composition for detecting foot-and-mouth disease virus type A antibody, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1, 2, and 4 to 7. A kit for detecting foot-and-mouth disease virus type A antibodies, comprising the composition for detecting foot-and-mouth disease virus type A antibodies of claim 8. Immobilizing foot-and-mouth disease virus type A antigen on a solid support;
Contacting a sample isolated from an individual and the antibody or antigen-binding fragment thereof of claim 1 with the immobilized antigen; and
Comprising a step of detecting a complex formed by binding of the immobilized antigen and the antibody or antigen-binding fragment thereof,
The foot-and-mouth disease virus type A antigen is a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 9, a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 10, a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 11, and a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 12. , or a combination thereof, a method for detecting foot-and-mouth disease virus type A antibodies. delete The method of claim 10, wherein the method is performed using competitive enzyme-linked immunosorbent assay (ELISA).