KR102241521B1 - Method of detecting antibody produced by foot-and-mouth disease vaccination using an antibody having immune reactivity to FMDV type O - Google Patents

Abstract

An antibody or antigen-binding fragment thereof that exhibits immunoreactivity to foot-and-mouth disease virus type O that specifically binds to foot-and-mouth disease virus structural protein VP1, a composition for detecting foot-and-mouth disease virus type O containing the same, and for detecting foot-and-mouth disease virus type O antibody containing the same It relates to the composition.

Description

{Method of detecting antibody produced by foot-and-mouth disease vaccination using an antibody having immune reactivity to FMDV type O}

It relates to a method for detecting foot-and-mouth disease virus type O, or an antibody thereof, using an antibody or antigen-binding fragment thereof that exhibits immunoreactivity to foot-and-mouth disease virus type O that specifically binds to foot-and-mouth disease virus structural protein VP1.

Foot-and-Mouth Disease Virus (FMDV) is the first type of domestic legal livestock contagious disease that causes high fever and blisters due to 　virus 　 infection in animals of the right-wing animals and causes death in young livestock. In Korea, it occurred 9 times since 2000, and when it occurred in 2010-2011, it caused direct economic damage of about 3 trillion won.

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 the 3C protease, resulting in viral 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 as an important site inducing the generation of neutralizing antibodies (Korean Patent Registration No. 10-1814757).

Accordingly, vaccines have been produced by targeting only structural proteins of foot-and-mouth disease virus, and antibodies generated during vaccination express only antibodies against structural proteins, so a method of detecting neutralizing antibodies targeting structural proteins has been required. Specifically, the empty capsid particle (ECP) antigen expresses only structural proteins constituting the capsid, excluding non-structural proteins, and thus has no infectivity or replication ability, but is a recombinant antigen having excellent antigenicity due to the appearance of foot-and-mouth disease virus. There have been attempts to implement the ECP antigen of foot-and-mouth disease virus for vaccine development, but industrialization is difficult due to a very low yield. The reason for the low yield is that the 3C protease is toxic in the expression host cell, making continuous cultivation difficult. Accordingly, there is a need to maintain the role of 3C protease, but by substituting some amino acids with other amino acids to inactivate toxicity in the expression host cell and increase the expression yield.

Under this background, the present inventors have completed the ECP antigen which is capable of self-assembly by the 3C protease of the structural protein VP1 and the non-structural protein 2A, while reducing the 3C protease activity that affects the growth of host cells. In addition, an antibody that specifically binds to the ECP antigen was selected, and a method for detecting foot-and-mouth disease virus type O or its antibody using the same was completed.

One aspect provides a method of detecting a foot-and-mouth disease virus type O antibody using an antibody or antigen-binding fragment thereof that specifically binds to an epitope consisting of the amino acid sequence of SEQ ID NO: 1.

Another aspect provides a method for detecting foot-and-mouth disease virus type O using an antibody or antigen-binding fragment thereof that specifically binds to an epitope consisting of the amino acid sequence of SEQ ID NO: 1.

One aspect provides a method of detecting a foot-and-mouth disease virus type O antibody using an antibody or antigen-binding fragment thereof that specifically binds to an epitope consisting of the amino acid sequence of SEQ ID NO: 1.

The foot-and-mouth disease virus belongs to the genus Picornavirus family Aphthovirus, and a capsid protein composed of 4 types of constituent proteins without an envelope surrounds the (+) RNA of the single-stranded VPg protein covalently linked at the 5'end. It exists in the form of being. The virus is composed of 7 serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3, and it is known that mutual protective immunity between the serotypes is not formed. Among the vaccines selected as permanent vaccines in Korea, Ariabag from Russia is targeting the O-type Primorsky, but the structural protein full-length amino acid sequence or genomic gene sequence for this has not been known in Korea. Accordingly, various attempts have been made to detect the corresponding neutralizing antibody titer from a specimen inoculated with the vaccine, but the situation is still insufficient.

The method comprises the steps of contacting an antibody or antigen-binding fragment thereof that specifically binds to an epitope consisting of the amino acid sequence of SEQ ID NO: 1 and a sample isolated from the individual; And detecting a complex formed by binding of the sample and the antibody or antigen-binding fragment thereof, wherein the contacting comprises competitive contacting the antibody or antigen-binding fragment thereof with the foot-and-mouth disease virus type O antibody in the sample. It may be to let you do. Here, the foot-and-mouth disease virus O-type antibody in the sample may include not only an antibody against the O-type Jincheonju, but also an antibody against the O-type primoski strain, but is not limited thereto.

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

In one embodiment, the step of contacting may be a competitive contact of the antibody or antigen-binding fragment thereof with the foot-and-mouth disease virus type O antibody in the sample with respect to the antigen, which may be performed simultaneously or sequentially. For example, the contacting may include contacting a sample with a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 10; And contacting the recombinant antigen contacted with the sample with an antibody or antigen-binding fragment thereof that specifically binds to an epitope consisting of the amino acid sequence of SEQ ID NO: 1.

The method includes a substrate comprising a solid support having an antigen immobilized or conjugated thereon; And a reagent including the above-described antibody or antigen-binding fragment thereof. Here, the antigen may be a recombinant antigen comprising the amino acid of SEQ ID NO: 1, or a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 10, and the antibody may be bound to a detection label, but this is a detection condition, detection The enemy can be changed according to the method, detection target, and detection purpose.

The substrate may be a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, and a slide glass made of glass, 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 fluorescent substances include fluorescein. , Eu ³⁺ , Eu ³⁺ chelate, cryptate, FITC, RITC, etc., and the ligand includes biotin derivatives, and the luminescent material includes acridinium ester and isoluminol derivatives, and microparticles The furnace includes colloidal gold and colored latex, and the radioactive isotopes include ⁵⁷ Co, ³ H, ¹²⁵ I and ¹²⁵ I-Bonton Hunter's reagent.

The kit may further include an appropriate amount of a buffer solution, a chromogenic substrate, and the like, as well as an appropriate amount of a buffer solution, as well as a technique known in the art, for immunological detection of the antibody, as well as the above-described substrate and the antibody bound to the detection label. This can be applied without limitation.

According to an embodiment, since the antibody or antigen-binding fragment is in competitive contact with the foot-and-mouth disease virus antibody and the foot-and-mouth disease virus structural protein, when the concentration of the complex formed by the binding of the antibody or antigen-binding fragment is detected high, the antibody It can be judged that the price is low. In addition, when the concentration of the complex is detected low, it can be determined that the antibody price is high.

For example, in the absence or absence of foot-and-mouth disease virus type O antibody in a sample isolated from the individual, the antibody or antigen-binding fragment thereof is present in the sample in a high proportion to the epitope consisting of the amino acid sequence of SEQ ID NO: 1. Combine. Conversely, when the foot-and-mouth disease virus type O antibody is present in the sample, the antibody or antigen-binding fragment thereof binds at a low ratio because binding competition for the epitope occurs. Therefore, when the signal level is weak, the individual can be determined as positive for foot-and-mouth disease virus type O antibody, and when the signal level is strong, the individual can be determined as negative for foot-and-mouth disease virus type O antibody.

The detecting steps include enzyme immunoassay (ELISA), western blotting, immunofluorescence, immunohistochemistry staining, flow cytometry, immunocytochemistry, radioimmunoassay. (RIA), Immunoprecipitation Assay, Immunodiffusion Assay, Complement Fixation Assay, and Protein Chip.It may be to perform any one selected from the group consisting of.

The individual refers to all organisms that can be infected with foot-and-mouth disease virus, and may be cattle, pigs, sheep, goats, deer, camels, and the like.

The sample may be a biological sample. The biological sample may be cells, organs, cell lysates, 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 may be a body fluid, plasma, serum, blood, or a combination thereof.

The sample may further contain a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 10. When the recombinant antigen is additionally included, even if the foot-and-mouth disease virus O-type structural protein is not contained in a sufficient amount in the sample isolated from the individual to be detected, foot-and-mouth disease virus O type can be efficiently detected, or its antibody and Can trigger a competitive reaction. Therefore, as long as the recombinant antigen is included so as to contact the antibody or antigen-binding fragment, it may be additionally included without being limited to conditions such as a method or timing of inclusion in a sample.

The epitope refers to a specific three-dimensional molecular structure region that determines antigen specificity. The epitope comprises an amino acid sequence having sequence identity of 60% or more, for example, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% of the sequence of SEQ ID NO: 1. Can be. In addition, the epitope is a sequence different from one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids, or seven or more amino acid residues in the sequence of SEQ ID NO: 1. It may be an epitope having. In the present specification, the'epitope' may be used interchangeably with an'antigenic determinant'.

The antibody refers to a specific immunoglobulin directed against an antigenic site. The antibody refers to a polypeptide or a combination of polypeptides that specifically binds to an epitope consisting of the amino acid sequence of SEQ ID NO: 1. The form of the antibody includes polyclonal antibodies, monoclonal antibodies, or recombinant antibodies such as ScFv fragments, diabodies, single chain antibodies, and the like, and all immunoglobulin antibodies are included. 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 directed against the antigenic site. A functional fragment of an antibody molecule having a specific antigen-binding site, that is, a binding domain, and retaining an antigen-binding function may also be included.

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

The antigen-binding fragment is a fragment thereof for the entire structure of an immunoglobulin, and refers to a part of a polypeptide including a portion to which an antigen can bind. For example, the antigen-binding fragment can be scFv, (scFv)2, Fv, Fab, Fab', Fv F(ab')2, or a combination thereof.

The antibody or antigen-binding fragment thereof includes a heavy chain variable region including HCDR1 consisting of the amino acid sequence of SEQ ID NO: 2, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 3, and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 4; And LCDR1 consisting of the amino acid sequence of SEQ ID NO: 5, LCDR2 consisting of the amino acid sequence of SEQ ID NO: 6, and LCDR3 consisting of the amino acid sequence of SEQ ID NO: 7 may be included.

The antibody or antigen-binding fragment thereof includes a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 8; And a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 9.

The antibody or antigen-binding fragment thereof may be one that exhibits specific immunoreactivity or viral neutralizing ability against the foot-and-mouth disease virus O-type structural protein VP1 site. The immune reactivity may mean that an immune response occurs in response to antigenic stimulation. The immune response may include an increase in cellular immunity or humoral immunity against an antigen. The virus neutralizing ability may mean that the infectivity of a virus is specifically inhibited or lost by a virus neutralizing antibody.

The antibody or antigen-binding fragment thereof may be produced from hybridoma cells deposited with accession number KCLRF-BP-00465. In addition, the antibody or antigen-binding fragment thereof may be a monoclonal antibody.

Another aspect provides a method for detecting foot-and-mouth disease virus type O using an antibody or antigen-binding fragment thereof that specifically binds to an epitope consisting of the amino acid sequence of SEQ ID NO: 1.

The method comprises the steps of contacting an antibody or antigen-binding fragment thereof that specifically binds to an epitope consisting of the amino acid sequence of SEQ ID NO: 1 and a sample isolated from the individual; And detecting a complex formed by binding of the sample and the antibody or antigen-binding fragment thereof.

The epitope, antibody or antigen-binding fragment thereof, the contact step, the detection step, and the like are as described above.

The detecting step may be to perform a competitive enzyme immunoassay (ELISA). In this case, by comparing the level of the signal by the enzyme or fluorescence labeled on the antibody or antigen-binding fragment thereof, it is possible to distinguish whether the individual is negative or positive for foot-and-mouth disease virus type O.

An antibody or antigen-binding fragment thereof that specifically binds to an epitope consisting of the amino acid sequence of SEQ ID NO: 1 according to one aspect shows an improved expression yield compared to a conventional recombinant antigen.

The composition for detecting foot-and-mouth disease virus comprising the antibody or antigen-binding fragment thereof according to another aspect exhibits improved diagnostic sensitivity compared to the conventional ELISA method for diagnosing foot-and-mouth disease virus.

1 is a diagram showing a strategy for developing an FMDV structural protein antigen.
2 is a diagram showing the results of SDS-PAGE confirming the concentration by quantification of FMDV O type Jincheonju P1 (VP4231) antigen.
3 shows the results of Western blotting of purified FMDV O type Jincheonju ECP antigen.
Figure 4 is a diagram showing the Western blotting results of FMDV O type Jincheonju P1 (VP4231) baculo antigen.
5 is a diagram showing an antibody development process.
6 is a diagram showing the results of titer assay for antisera of mice immunized with baculo FMDV ECP (mouse #1) or P1 (mouse #2) antigen.
7 is a diagram showing the results of Western blotting confirming the epitope of the selected antibody that specifically binds to the FMDV structural protein VP1.
8 is a diagram showing a schematic diagram of an FMDV O type antibody competitive ELISA using an FMDV O type Jincheonju ECP antigen and a selection antibody.

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

Example 1: Development of FMDV ECP antigen and FMDV structural protein P1 antigen

1-1. FMDV ECP gene synthesis and cloning

Among the vaccines currently selected as the domestic regular vaccine strain for Foot-and-Mouth Disease Virus (FMDV), Ariabag from Russia is targeting the O-type primosky strain. On the other hand, with respect to the O-type primoski strain, the full length amino acid sequence or genomic gene sequence of the structural protein has not yet been known, and only the sequence of VP1, which is a part of the structural protein, is disclosed. Under this technical background, in this example, a recombinant antigen was prepared for the structural protein of the Jincheonju foot-and-mouth disease virus, specifically O-type Jincheonju foot-and-mouth disease virus, which has been isolated in Korea.

For the production of a recombinant ECP (empty capsid particle) antigen against FMDV type O Jincheonju, known genetic information, NCBI Accession No. With reference to KX1625590, P1-2A-FS3C (C142T), which is involved in the structural protein, was synthesized as a DNA template. During gene synthesis, codon-optimization was performed in Spodoptera frugiperda , which is an expression host cell of baculovirus, to attempt high-efficiency production of heterologous proteins.

1-2. FMDV structural protein P1 gene synthesis and cloning

The ECP antigen prepared above expresses P1-2A and 3C protease that form structural proteins together. Cysteine (C), which is the 142nd amino acid of 3C protease, is substituted with threonine (T), and HIV- 1 By positioning the ribosomal frameshift (FS), it was attempted to solve the problem of low expression rate due to intracellular toxicity. However, there is still a problem that takes a lot of time in the purification process. In addition, when intact P1-2A is expressed in mammalian cells, effective antigenicity is shown through FMDV antigen ELISA analysis, and in particular, it has been reported that it effectively binds to integrin αvβ6, a major receptor for FMDV. Accordingly, as shown in FIG. 1, in addition to the ECP antigen with improved expression yield, it was intended to develop an FMDV structural protein P1 antigen that exhibits effective antigenicity.

To clone the full length of P1 (VP4, VP2, VP3, VP1) containing the recombinant structural protein for FMDV type O Jincheonju into baculovirus expression vector, after designing the FMDV O type structural protein P1, to synthesize the gene. I did. PCR was performed using the synthesized gene as a template to obtain amplified DNA of FMDV O type Jincheonju (KX162590) P1. Oligonucleotide primer sequences for performing PCR are shown in Table 1 below.

Abbreviation Primer sequence (5'-3') direction Jincheon P1 F 5'- GGA TCC gga gcc ggg caa tcc-3' Forward direction Jincheon P1 R 5'- GTC GAC caa gga ctg ctt tac-3' Reverse

1-3. Preparation of expression vectors of FMDV ECP and FMDV P1

The FMDV ECP antigen and P1 full-length protein were intended to be cloned, cultured, and purified using a baculovirus expression system. In order to express the antigens P1-2A-FS3C (C142T) and P1 (VP4231) consisting of the amino acid sequence of SEQ ID NO: 10 including the recombinant FMDV structural protein, each of the synthesized genes was cloned into a baculovirus expression vector. I did. Through this, the expressed and purified antigen candidates were checked for reactivity and used as immunogens when developing coating materials and antibodies for ELISA plates.

For the expression of FMDV O type Jincheonju P1-2A-FS3C (C142T) and P1 (VP4231) antigens, the following procedures were performed, respectively. The expression vector pFastbac vector (Invitrogen, Cat No.10360-014, USA) was prepared, and the primer linker site of FMDV O type Jincheonju P1-2A-FS3C (C142T) or P1 (VP4231) was BamHI (NEB, Cat No. #R0136S, USA) and SalI (NEB, Cat No. #R0138S, USA) were cut and electrophoresed on an agarose gel. This was extracted using a gene clean kit (Gene Clean Kit, MP biochemical, Cat No. #1101-400, France) FMDV O type Jincheonju P1-2A-FS3C (C142T) or P1 (VP4231). After mixing the vector for insertion of FMDV O type Jincheonju P1-2A-FS3C (C142T) or P1 (VP4231) treated with BamHI and SalI and the pFastbac vector treated with BamHI and SalI, T4 DNA ligase (Roche, 10481220001, Germany) at 16° C. overnight, and then transformed into soluble cells Top10F'.

Transformation is heat-shock method. After taking out water-soluble cells stored at -70°C and thawing them on ice, add 5uL of ligate and mix for 3 seconds, and then on ice for 30 minutes. Let it politicize. The tube containing the water-soluble cells mixed with the reactants was allowed to stand in a water bath at 42° C. for 90 seconds, and then immediately put on ice. 1 mL of LB liquid medium was added to the tube, and after reacting at 37° C. for 1 hour, 100 μL of bacterial solution was plated on an LB agar plate containing ampicillin as a selection marker, and cultured at 37° C. overnight. Colonies grown on LB agar plates were inoculated into liquid LB medium. When the cells were grown, DNA was extracted using the Wizard Plus SV Minipreps Kit (Promega, Cat No.#A1460, USA), and the extracted DNA was confirmed through sequencing.

1-4. Selection of Recombinant Bacmid of FMDV ECP and FMDV P1

To make pFastbac FMDV O type Jincheonju P1-2A-FS3C(C142T) and pFastbac FMDV O type Jincheonju P1 (VP4231), respectively, FMDV O type Jincheonju P1-2A-FS3C(C142T) or P1( VP4231) Plasmid DNA was mixed in an appropriate amount and left to stand on ice for 10 minutes. After heat-shock for 90 seconds in a 42° C. water bath, it was left standing again for 5 minutes on ice. After adding 1 mL of LB broth, shaking incubation was performed at 37°C for 4 hours. After spreading on LB agar plates to which antibiotics were added, they were incubated at 37°C for 48 hours. After selecting and culturing only white colonies, a small amount of the plasmid was extracted (mini-prep). PCR was performed using M13 forward and reverse primers to confirm the recombinant bacmid.

1-5. Transfection and culture of FMDV ECP and FMDV P1 (VP4231)

The production of baculo antigens of FMDV O-type Jincheonju P1-2A-FS3C (C142T) and FMDV O-type Jincheonju P1 (VP4231) was performed using a cell fectin reagent (Invitrogen, Cat No. 10362-100, USA). Was used for transfection. ^{2X10 6} sf9 cells were dispensed into a 6-well culture plate, allowed to stand for 1 hour, and washed twice with a medium containing no antibiotics. 10 uL of bacmid was mixed with 100 uL of antibiotic-free medium, and about 8 uL of cell pectin reagent was mixed with 100 uL of antibiotic-free medium. The bacmid-mixed medium and the cell-pectin reagent-containing medium were mixed, allowed to stand at room temperature for 25 minutes, and then 800 uL of an antibiotic-free medium was added. The mixed recombinant bacmid complex was instilled on the above sf9 cell plate. After incubation at 27° C. for 5 hours, the supernatant was removed, and cultured for 4 days in a medium to which 2% FBS was added. After confirming the cytopathic effect (CPE), which is an index of viral proliferation, it was used after subculture.

1-6. Purification of FMDV ECP and FMDV P1 (VP4231) antigen

In order to purify the recombinant antigen from the cultured transfected cells, in the case of FMDV O-type Jincheonju P1-2A-FS3C (C142T), FMDV O-type Jincheonju P1-2A-FS3C (C142T) baculo culture was obtained and centrifuged. The pellet recovered through separation (3000 rpm, 10 minutes) was mixed with PBS and released, and then the cells were lysed through a freeze-thaw process. Then, the supernatant was recovered through centrifugation (12,000 rpm, 10 minutes). The recovered supernatant was subjected to gradient fractionation with 30% sucrose (w/v in PBS), centrifuged at 100,000xg for 16 hours, and then only the uppermost layer was taken. The recovered purified antigen was quantified through the BCA quantification method to confirm the concentration.

In the case of FMDV O type Jincheonju P1 (VP4231), affinity chromatography using histidine-tag included in the recombinant antigen was performed. The antigen culture was disrupted with an ultrasonic disruptor, and the supernatant was recovered through centrifugation. The recovered supernatant was passed through a Ni-NTA-conjugated 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 portion of the eluted concentration, the eluate was selected and recovered in a fraction tube containing the target protein through SDS-PAGE. The recovered purified antigen was quantified through the BCA quantification method to confirm the concentration. As a result, in the case of the P1-2A-FS3C(C142T) gene recombinant antigen, since the four antigen fragments VP1, VP2, VP3 and VP4 are in the form of empty capsid particles, a distinct antigen band can be identified as a result of SDS-PAGE. There wasn't. In contrast, as shown in FIG. 2, it was confirmed that the FMDV O type Jincheonju P1 (VP4231) recombinant antigen was purified by confirming a distinct band in the SDS-PAGE result.

1-7. Confirmation of expression of FMDV ECP antigen

Western blotting was performed to confirm the expression of the FMDV O type Jincheonju ECP antigen purified in Example 1-6. 3 shows the results of Western blotting of purified FMDV O type Jincheonju ECP antigen. As a result, as shown in FIG. 3, since a reactive band appeared at about 24 kDa corresponding to the size of VP1 using a VP1-specific antibody, expression of the ECP antigen can be confirmed through this. In particular, in the ECP antigen expressed with 3C protease wild-type (WT), the band signal reacting with the VP1 antibody was insignificant, but in the ECP antigen expressed with 3C (C142T), a band of the size of VP1 was shown as a definite signal. This means that the expression yield was improved through the mutation of the 3C protease.

1-8. Confirmation of expression of FMDV P1 (VP4231) antigen

Western blotting was performed to confirm the expression of the purified FMDV O type Jincheonju P1 (VP4231) antigen. Figure 4 is a diagram showing the Western blotting results of FMDV O type Jincheonju P1 (VP4231) baculo antigen. As a result, as shown in FIG. 4, since a reactive band appeared at about 150 kDa corresponding to the size of P1 using an antibody specific to the structural protein P1, through this, FMDV O type Jincheonju P1 (VP4231) baculo antigen The expression of can be confirmed.

Example 2: Confirmation of the performance of FMDV ECP and FMDV P1 (VP4231) antigens

After coating the purified antigen on a microplate, antigenicity was verified using an indirect ELISA using negative and positive serum for FMDV O type antibody as a sample.

After the sample diluted 1/10 times was injected into the plate, it was reacted at 37° C. for 1 hour. The unreacted sample was removed through washing, and the secondary antibody-HRP was injected, followed by reaction at 37° C. for 30 minutes. At this time, bovine serum was reacted with anti-Bovine IgG-HRP, and pig serum was reacted with anti-Pig IgG-HRP. After removing the remaining unreacted HRP conjugate through washing, the TMB substrate was reacted at room temperature for 15 minutes, the reaction was stopped, and the absorbance was measured. Table 2 shows the results. As a result, as shown in Table 2, the difference between the negative and positive response signals of bovine serum and pig serum was confirmed, and the level of the signal difference was equal to or higher than that of the Prionics plate. Through these results, the antigenicity of the ECP and P1 (VP4231) antigens prepared in Example 1 can be confirmed.

Secondary antibody-HRP Specimen plate JC ECP JC P1 Prionics Anti-Bovine　IgG-HRP Negative serum Negative bovine serum 0.0580 0.0580 0.0590 manisa
Vaccination
Bovine serum Positive bovine serum 1 1.6350 1.5220 0.8000 Benign bovine serum 51 1.3240 1.2060 0.8360 Positive bovine serum 58 0.1980 0.1170 0.1540 Anti-Pig　IgG-HRP Negative serum Negative pig serum 0.0930 0.1060 0.0810 Negative pig serum (5A-5) 0.0630 0.0650 0.0570 Primorsky
Vaccination
Pig serum 2-125 0.3240 0.3840 0.4960 3-213 0.3080 0.2720 0.4840 4-225 0.4560 0.3830 0.4630 FMDvirus
Attack vaccination
Pig serum DPE-4 0.2460 0.2930 0.4940 DPE 11 0.3540 0.4180 0.3570 DPE 23 0.4620 0.4100 0.3470 DPE 27 0.5640 0.4730 0.3910

Among the two antigens prepared in Example 1, the expression and purification yield of the ECP antigen was higher than that of the FMDV O type Jincheonju P1 (VP4231) antigen, so the FMDV O type Jincheonju ECP was used as a plate coating material for the ELISA kit The antigen was selected.

Example 3: Development of FMDV recombinant antibody

3-1. Antibody development process

5 is a diagram showing an antibody development process. As shown in FIG. 5, in order to obtain an immunized mouse required for the development of a hybridoma cell line, an immunogen and a complete Freund's adjuvant were mixed at a ratio of 1:1 to obtain a magnetic 6-week-old Balb/c mouse. It was injected intraperitoneally. In the same way, three additional immunizations were performed at weekly intervals. One week after the last 4th immunization, the same antigen used as an immunogen was injected into the tail vein of the mouse 3 times at 1 day intervals to boost it. Before performing cell fusion, blood was collected from the tail of the immunized mouse to obtain serum, and then the immunity was confirmed by confirming that the antibody titer against the immunogen was increased through the ELISA method. By confirming the antibody titer, a mouse having a sufficient amount of antibody was selected to perform a cell fusion process. The spleen of the immunized mouse was excised to separate the cells, and one splenocyte and five myeloma cells were obtained to prepare hybridoma cells, and the cells were cultured.

In order to select cells that specifically respond only to FMDV among the hybridoma cell groups, ELISA was performed using a microplate coated with an antigen. As a result of performing ELISA, hybridoma cell lines secreting an antibody having a specific high binding ability with an antigen were selected.

3-2. Immunogen used for antibody development

As the immunogen used for antibody development, in addition to the recombinant ECP antigen and the baculo antigen of the P1 antigen prepared in Example 1, a total of four types of immunogens, purified virus, vaccine, and peptide were used to attempt antibody development. . Detailed information on the immunogen is shown in Table 3 below.

Target Immunogen type Cell line source ECP Baculo Jincheon Bio Note P1 Baculo Jincheon Bio Note Native Purified virus
(BEI inactivation) Boeun Quarantine Headquarters O-type structural protein vaccine Campos Biogenesis Bago O-type structural protein vaccine Primoskey FGBI ARRIAH VP1 G-H loop Peptide Primoskey Peptron

3-3. Titer assay in immunized mice

Antisera obtained by collecting a small amount of blood from the eyeballs of immunized mice for a certain period of time was tested for immunity by performing a titer assay using an indirect ELISA test method using a microplate coated with an immunogen. 6 is a diagram showing the results of an anti-serum titer assay of mice immunized with baculo FMDV ECP (mouse #1) or P1 (mouse #2) antigen. As a result, as shown in Figure 6, it can be confirmed that the immunization was successful in the mouse immunized with the baculo ECP antigen or the P1 antigen. Thus, the spleen of the mouse was excised and fused with myeloma cells to prepare a hybridoma cell line secreting an antibody exhibiting immunoreactivity to FMDV O type.

3-4. Hybridoma cell line selection

After culturing the prepared hybridoma cell line for a certain period of time, hybridoma cell lines secreting an antibody having a specific high binding ability with an antigen were selected using an indirect ELISA method. As shown in Table 4 below, there are a total of 375 antibodies developed to date, of which 349 antibodies were purified to perform a screening test of an antibody raw material suitable for application of an ELISA diagnostic method. As a result of performing ELISA using the culture supernatant of the hybridoma cell line, compared to antibodies developed using other types of immunogens, when the baculo ECP antigen or P1 antigen was used as an immunogen, a total of 200 clones of hybridoma cell lines Since the culture supernatant showed high binding ability, it was selected to produce antibodies.

Immunogen Antibody development Target Immunogen type Strain manufacturer Clone ECP or P1 Baculo Jincheon Bio Note 200 Native Purified virus (BEI inactivation) Boeun Quarantine Headquarters 135 O-type structural protein vaccine Campos Biogenesis Bago 14 O-type structural protein vaccine Primoskey FGBI ARRIAH 13 VP1 G-H loop Peptide Primoskey Peptron 13

3-5. Selection of antibodies exhibiting immunoreactivity to FMDV type O

In order to select an antibody that can be used as a detector among the 349 developed antibodies as shown in Table 4, all of the candidate antibody raw materials were combined with horseradish peroxidase (HRP). Thereafter, the JC ECP or P1 antigen-coated plate developed in Example 1 was reacted with the detector using negative and positive sera of cattle and pigs as samples. As a result of selecting a clone with discrimination power for negative and positive samples, it was decided to apply the raw material for the clone 8P#10 antibody detector (Detector #2) to ELISA.

3-6. Identification of the epitope of the selected antibody

To identify the epitope for the selected detector clone 8P#10 antibody, it was expressed as VP0 (VP4+VP2), VP3 and VP1 recombinant proteins, and the reactivity of the clone 8P#10 antibody with each protein was confirmed. Western blotting was performed to do this. 7 is a diagram showing the results of Western blotting confirming the epitope of the selected antibody that specifically binds to the FMDV structural protein VP1. As a result, as shown in Fig. 7, it was confirmed that the clone 8P#10 antibody exhibited reactivity in the VP1 recombinant protein.

Example 4: Development of a competitive ELISA using FMDV type O antibody

4-1. Development process of competitive ELISA using FMDV type O antibody

As shown in Figure 8, using the FMDV O type Jincheonju ECP antigen prepared in Example 1 and the antibody selected in Example 3, an FMDV O type antibody competitive ELISA was developed. Specifically, an antibody produced from hybridoma cells deposited with accession number KCLRF-BP-00465 as the FMDV O type antibody was used.

To a microplate coated with FMDV type O Jincheonju ECP antigen, 25 uL of the sample and 100 uL of the antibody anti-FMDV type O-HRP conjugate mixture as a detector enzyme conjugate were simultaneously injected and reacted at 37° C. for 90 minutes. In the case of negative specimens, since there is no anti-FMDV antibody to react with the antigen on the plate surface, all of the detectors injected together bind to the antigen. In the case of a positive sample, a competition reaction with the detector injected together is induced, so that the number of detectors binding to the antigen of the plate is relatively small. Therefore, in the case of a positive sample, the TMB color development and color development after stopping the reaction are weak. On the other hand, in the case of a negative sample, the color development is dark. According to the difference in the degree of color development, negative and positive can be distinguished for the FMDV O type antibody in the sample.

4-2. Verification of competitive ELISA using FMDV type O antibody: performance comparison with existing ELISA kit

Specimens with low detection ability in the previously developed ELISA using antigens and antibodies based on the manisa cell line were tested by the Jincheonju-based ELISA assay developed in the present invention, and the performance was compared. It is shown in Table 5 below. As a result, as shown in Table 5, compared to the existing Manisa-based ELISA kit, Jincheonju-based competitive ELISA using the antibody of Example 3 showed excellent sensitivity to positive individuals, In particular, the sensitivity to the samples vaccinated with Primoski vaccine was remarkable. In addition, the PI value of the DPI-6 specimen at the initial stage of bovine challenge vaccination was also significantly improved.

Prionics Existing　ELISA (based on manisa) Jincheonju 　-based ELISA PI result PI result PI result Bovine field serum Bovine serum 4 95.0 Pos. 99.8 Pos. 99.0 Pos. Bovine field serum Bovine serum 47 67.6 Pos. 69.9 Pos. 62.3 Pos. Bovine challenge serum 2910 DPI-6 59.8 Pos. 19.1 - 38.9 - Primosky vaccine field specimen 2-125 63.4 Pos. 41.3 - 67.3 Pos. Pig vaccination serum DPE 23 77.0 Pos. 46.4 - 52.9 Pos. Primoski　Vaccination pig serum Pig pooling 1X 52.4 Pos. 54.1 Pos. 65.6 Pos. Pig pooling 2X 37.5 - 40.0 - 54.9 Pos. Specificity Pig 5A-5 19.0 - -43.9 - 21.7 - New Zealand cattle 4-83 14.4 - 10.1 - 28.3 -

4-3. Verification of competitive ELISA using FMDV type O antibody: performance comparison with Prionics' ELISA kit

By examining 150 samples of vaccinated pig serum and 150 samples of vaccinated bovine serum obtained from domestic farms, we tried to confirm the sensitivity performance of competitive ELISA using FMDV type O antibody. The negative and positive test results for the specimens were based on the results of the trial by simultaneously testing Prionics' Priocheck FMDV O type antibody ELISA kit, which tests the titer of vaccination antibodies in the existing field. As a result, as shown in Table 6, the sensitivity to vaccinated pig serum was 97.3% (144/145), and the judgment match rate was 96.7% (145/150) based on the results of Prionics' ELISA test. The sensitivity to vaccination bovine serum was 100% (139/139), and the concordance rate was 98.0% (147/150).

Vaccination pig serum Prionics positivity voice Sum FMDV type O antibody positivity 144 One 145 voice 4 One 5 Sum 148 2 150 Vaccination bovine serum Prionics positivity voice Sum FMDV type O antibody Yang 7 stars 139 3 142 voice 0 8 8 Sum 139 11 150

In addition, 43 bovine-negative specimens and 48 pig-negative specimens obtained from New Zealand, a clean country for foot-and-mouth disease, were tested to confirm the specificity performance of a competitive ELISA using FMDV O type antibody. As a result, the performance of cow specificity 100% (43/43) and pig specificity 100% (48/48) was confirmed. 100% of the test results for the specimen were consistent with Prionics' ELISA results.

Bovine negative specimen Prionics positivity voice Sum FMDV type O antibody positivity 0 0 0 voice 0 43 43 Sum 0 43 43 Pig negative sample Prionics positivity voice Sum FMDV type O antibody positivity 0 0 0 voice 0 48 48 Sum 0 48 48

4-4. Verification of competitive ELISA using FMDV type O antibody: performance comparison with neutralization assay

Porcine serum collected 8 weeks after vaccination was examined, and the sensitivity performance of the competitive ELISA diagnostic method using FMDV O type antibody was confirmed through comparison of the developed competitive ELISA method, the Prionics ELISA method, and the determination result of the neutralization test method. I wanted to.

As a result, as shown in Table 8 below, the positive test rates for the three vaccines Manisa, Campos, and Primoski FMDV of the three kits were 100% as the result of the existing VNT neutralization test , The method of the present invention showed a sensitivity performance of Manissa 147% (28/28), Campos 104% (28/28), and Primoski 158% (30/30). On the other hand, Prionics' ELISA method showed a sensitivity performance of 132% (25/28), Campos 104% (28/28), and 116% (22/30) of Manis, compared with the positivity of the present invention. It showed equivalent or similar sensitivity performance.

VNT Bio Note Prionics Sample group Number of samples Number of positive judgments Positive rate
(standard) Number of positive judgments Positive rate Number of positive judgments Positive rate O manisa 28 19 100% 28 147% 25 132% O campos 28 27 100% 28 104% 28 104% O primorsky 30 19 100% 30 158% 22 116%

The above results show that the competitive ELISA method using an antibody that specifically binds to the ECP antigen P1-2A-FS3C (C142T) can diagnose foot-and-mouth disease virus with high sensitivity compared to the conventional method for diagnosis of foot-and-mouth disease virus. will be.

Korea Cell Line Research Foundation KCLRFBP00465 20190123

<110> REPUBLIC OF KOREA (Animal and Plant Quarantine Agency) BIONOTE, INC. <120> Method of detecting antibody produced by foot-and-mouth disease vaccination using an antibody having immune reactivity to FMDV type O <130> PN125522 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 737 <212> PRT <213> Foot-and-mouth disease virus <400> 1 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 Ile Thr His Gly Tyr Ala Thr Ala Glu Asp Phe 115 120 125 Val Ser Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg Val Ile Gln Ala 130 135 140 Glu Arg Phe Phe Lys Thr His Leu Phe Asp Trp Val Thr Ser Asp Pro 145 150 155 160 Phe Gly Arg Cys Tyr Leu Leu Glu Leu Pro Thr Asp His Lys Gly Val 165 170 175 Tyr Gly Ser Leu Thr Asp Ser Tyr Ala Tyr Met Arg Asn Gly Trp Asp 180 185 190 Val Glu Val Thr Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu 195 200 205 Val Ala Met Val Pro Glu Leu Cys Ser Ile Gly Gln Arg Glu Leu Phe 210 215 220 Gln Leu Thr Leu Phe Pro His Gln Phe Ile Asn Pro Arg Thr Asn Met 225 230 235 240 Thr Ala His Ile Lys Val Pro Phe Val Gly Val Asn Arg Tyr Asp Gln 245 250 255 Tyr Lys Val His Lys Pro Trp Thr Leu Val Val Met Val Val Ala Pro 260 265 270 Leu Thr Val Asn Thr Glu Gly Ala Pro Gln Ile Lys Val Tyr Ala Asn 275 280 285 Ile Ala Pro Thr Asn Val His Val Ala Gly Glu Phe Pro Ser Lys Glu 290 295 300 Gly Ile Phe 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 Lys Val Phe Asn Pro 325 330 335 Pro Arg Asn Met Leu Pro Gly Arg Phe Thr Asn Leu Leu Asp Val Ala 340 345 350 Glu Ala Cys Pro Thr Phe Leu His Phe Asp Gly Gly Val Pro Tyr Val 355 360 365 Thr Thr Lys Thr Asp Ser Asp Arg Val Leu Thr Gln Phe Asp Leu Ser 370 375 380 Leu Ala Ala Lys His Met Ser Asn Thr Phe Leu Ala Gly Leu 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 Pro Thr Asp Ala Lys Ala Arg Tyr Met Ile Ala Tyr Ala Pro Pro 420 425 430 Gly Met Glu Pro Pro Lys Thr Pro Glu Ala Ala Ala His Cys Ile His 435 440 445 Ala Glu Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile Pro 450 455 460 Tyr Leu Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Ala Ala Glu 465 470 475 480 Thr Thr Asn Val Gln Gly Trp Val Cys Leu Phe Gln Ile Thr His Gly 485 490 495 Lys Ala Glu Gly Asp Ala Leu Val Val Leu Ala Ser Ala Gly Lys Asp 500 505 510 Phe Glu Leu Arg Leu Pro Val Asp Ala Arg Gln Gln Thr Thr Ser Thr 515 520 525 Gly Glu Ser Ala Asp Pro Val Thr Ala Thr Val Glu Asn Tyr Gly Gly 530 535 540 Glu Thr Gln Ile Gln Arg Arg His His Thr Asp Val Ser Phe Ile Leu 545 550 555 560 Asp Arg Phe Val Lys Val Thr Pro Lys Asp Ser Thr Asn Val Leu Asp 565 570 575 Leu Met Gln Thr Pro Pro His Thr Leu Val Gly Ala Leu Leu Arg Ala 580 585 590 Ala Thr Tyr Tyr Phe Ala Asp Leu Glu Val Ala Val Lys His Glu Gly 595 600 605 Asp Leu Thr Trp Val Pro Asn Gly Ala Pro Glu Ala Ala Leu Asp Asn 610 615 620 Thr Thr Asn Pro Thr Ala Tyr His Lys Ala Pro Leu Thr Arg Leu Ala 625 630 635 640 Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ala Thr Val Tyr Asn Gly 645 650 655 Asn Cys Lys Tyr Thr Gly Gly Ser Leu Pro Asn Val Arg Gly Asp Leu 660 665 670 Gln Val Leu Ala Pro Lys Ala Ala Arg Pro Leu Pro Thr Ser Phe Asn 675 680 685 Tyr Gly Ala Ile Lys Ala Thr Arg Val Thr Glu Leu Leu Tyr Arg Met 690 695 700 Lys Arg Ala Glu Thr Tyr Cys Pro Arg Pro Leu Leu Ala Val His Pro 705 710 715 720 Ser Ala Ala Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln Ser 725 730 735 Leu <210> 2 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> 8P#10 antibody HCDR1 <400> 2 Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr Leu Ile Ala 1 5 10 <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 8P#10 antibody HCDR2 <400> 3 Met Leu Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Asp <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> 8P#10 antibody HCDR3 <400> 4 Glu Thr Thr Ile Ile Gly Thr Arg Trp Tyr Phe Asp Val 1 5 10 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 8P#10 antibody LCDR1 <400> 5 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 8P#10 antibody LCDR2 <400> 6 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 8P#10 antibody LCDR3 <400> 7 Gln Asn Asp Tyr Ser Tyr Pro Phe Thr 1 5 <210> 8 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> 8P#10 antibody VH <400> 8 Glu Val Gln Leu Gln Glu Ser Gly Thr Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30 Leu Ile Ala Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Leu Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Lys Ala Ala Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Thr Thr Ile Ile Gly Thr Arg Trp Tyr Phe Asp Val Trp 100 105 110 Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 9 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> 8P#10 antibody VL <400> 9 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 10 <211> 985 <212> PRT <213> Foot-and-mouth disease virus <400> 10 Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser Gly 1 5 10 15 Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln Asn 20 25 30 Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser Asn 35 40 45 Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln Asn 50 55 60 Asn Asp Trp Phe Ser Lys Leu Ala Ser Ser Ala Phe Ser Gly Leu Phe 65 70 75 80 Gly Ala Leu Leu Ala Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu Glu 85 90 95 Asp Arg Ile Leu Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr Gln 100 105 110 Ser Ser Val Gly Ile Thr His Gly Tyr Ala Thr Ala Glu Asp Phe Val 115 120 125 Ser Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg Val Ile Gln Ala Glu 130 135 140 Arg Phe Phe Lys Thr His Leu Phe Asp Trp Val Thr Ser Asp Pro Phe 145 150 155 160 Gly Arg Cys Tyr Leu Leu Glu Leu Pro Thr Asp His Lys Gly Val Tyr 165 170 175 Gly Ser Leu Thr Asp Ser Tyr Ala Tyr Met Arg Asn Gly Trp Asp Val 180 185 190 Glu Val Thr Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu Val 195 200 205 Ala Met Val Pro Glu Leu Cys Ser Ile Gly Gln Arg Glu Leu Phe Gln 210 215 220 Leu Thr Leu Phe Pro His Gln Phe Ile Asn Pro Arg Thr Asn Met Thr 225 230 235 240 Ala His Ile Lys Val Pro Phe Val Gly Val Asn Arg Tyr Asp Gln Tyr 245 250 255 Lys Val His Lys Pro Trp Thr Leu Val Val Met Val Val Ala Pro Leu 260 265 270 Thr Val Asn Thr Glu Gly Ala Pro Gln Ile Lys Val Tyr Ala Asn Ile 275 280 285 Ala Pro Thr Asn Val His Val Ala Gly Glu Phe Pro Ser Lys Glu Gly 290 295 300 Ile Phe Pro Val Ala Cys Ser Asp Gly Tyr Gly Gly Leu Val Thr Thr 305 310 315 320 Asp Pro Lys Thr Ala Asp Pro Val Tyr Gly Lys Val Phe Asn Pro Pro 325 330 335 Arg Asn Met Leu Pro Gly Arg Phe Thr Asn Leu Leu Asp Val Ala Glu 340 345 350 Ala Cys Pro Thr Phe Leu His Phe Asp Gly Gly Val Pro Tyr Val Thr 355 360 365 Thr Lys Thr Asp Ser Asp Arg Val Leu Thr Gln Phe Asp Leu Ser Leu 370 375 380 Ala Ala Lys His Met Ser Asn Thr Phe Leu Ala Gly Leu Ala Gln Tyr 385 390 395 400 Tyr Thr Gln Tyr Ser Gly Thr Ile Asn Leu His Phe Met Phe Thr Gly 405 410 415 Pro Thr Asp Ala Lys Ala Arg Tyr Met Ile Ala Tyr Ala Pro Pro Gly 420 425 430 Met Glu Pro Pro Lys Thr Pro Glu Ala Ala Ala His Cys Ile His Ala 435 440 445 Glu Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile Pro Tyr 450 455 460 Leu Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Ala Ala Glu Thr 465 470 475 480 Thr Asn Val Gln Gly Trp Val Cys Leu Phe Gln Ile Thr His Gly Lys 485 490 495 Ala Glu Gly Asp Ala Leu Val Val Leu Ala Ser Ala Gly Lys Asp Phe 500 505 510 Glu Leu Arg Leu Pro Val Asp Ala Arg Gln Gln Thr Thr Ser Thr Gly 515 520 525 Glu Ser Ala Asp Pro Val Thr Ala Thr Val Glu Asn Tyr Gly Gly Glu 530 535 540 Thr Gln Ile Gln Arg Arg His His Thr Asp Val Ser Phe Ile Leu Asp 545 550 555 560 Arg Phe Val Lys Val Thr Pro Lys Asp Ser Thr Asn Val Leu Asp Leu 565 570 575 Met Gln Thr Pro Pro His Thr Leu Val Gly Ala Leu Leu Arg Ala Ala 580 585 590 Thr Tyr Tyr Phe Ala Asp Leu Glu Val Ala Val Lys His Glu Gly Asp 595 600 605 Leu Thr Trp Val Pro Asn Gly Ala Pro Glu Ala Ala Leu Asp Asn Thr 610 615 620 Thr Asn Pro Thr Ala Tyr His Lys Ala Pro Leu Thr Arg Leu Ala Leu 625 630 635 640 Pro Tyr Thr Ala Pro His Arg Val Leu Ala Thr Val Tyr Asn Gly Asn 645 650 655 Cys Lys Tyr Thr Gly Gly Ser Leu Pro Asn Val Arg Gly Asp Leu Gln 660 665 670 Val Leu Ala Pro Lys Ala Ala Arg Pro Leu Pro Thr Ser Phe Asn Tyr 675 680 685 Gly Ala Ile Lys Ala Thr Arg Val Thr Glu Leu Leu Tyr Arg Met Lys 690 695 700 Arg Ala Glu Thr Tyr Cys Pro Arg Pro Leu Leu Ala Val His Pro Ser 705 710 715 720 Ala Ala Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln Ser Leu 725 730 735 Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly 740 745 750 Pro Leu Phe Phe Phe Arg Glu Asp Leu Ala Phe Leu Gln Gly Lys Ala 755 760 765 Arg Glu Phe Ser Ser Gly Ala Pro Pro Thr Asp Leu Gln Lys Met Val 770 775 780 Met Gly Asn Thr Lys Pro Val Glu Leu Ile Leu Asp Gly Lys Thr Val 785 790 795 800 Ala Ile Cys Cys Ala Thr Gly Val Phe Gly Thr Ala Tyr Leu Val Pro 805 810 815 Arg His Leu Phe Ala Glu Lys Tyr Asp Lys Ile Met Leu Asp Gly Arg 820 825 830 Ala Met Thr Asp Ser Asp Tyr Arg Val Phe Glu Phe Glu Ile Lys Val 835 840 845 Lys Gly Gln Asp Met Leu Ser Asp Ala Ala Leu Met Val Leu His Arg 850 855 860 Gly Asn Arg Val Arg Asp Ile Thr Lys His Phe Arg Asp Thr Ala Arg 865 870 875 880 Met Lys Lys Gly Thr Pro Val Val Gly Val Ile Asn Asn Ala Asp Val 885 890 895 Gly Arg Leu Ile Phe Ser Gly Glu Ala Leu Thr Tyr Lys Asp Ile Val 900 905 910 Val Thr Met Asp Gly Asp Thr Met Pro Gly Leu Phe Ala Tyr Lys Ala 915 920 925 Ala Thr Lys Ala Gly Tyr Cys Gly Gly Ala Val Leu Ala Lys Asp Gly 930 935 940 Ala Asp Thr Phe Ile Val Gly Thr His Ser Ala Gly Gly Asn Gly Val 945 950 955 960 Gly Tyr Cys Ser Cys Val Ser Arg Ser Met Leu Gln Lys Met Lys Ala 965 970 975 His Ile Asp Pro Glu Pro His His Glu 980 985

Claims (6)

Contacting a sample isolated from the individual with an antibody or antigen-binding fragment thereof that specifically binds to an epitope consisting of the amino acid sequence of SEQ ID NO: 1; And
And detecting a complex formed by binding of the sample and the antibody or antigen-binding fragment thereof,
The contacting step is to competitively contact the antibody or antigen-binding fragment thereof with a foot-and-mouth disease virus type O antibody,
The antibody or antigen-binding fragment thereof includes a heavy chain variable region including HCDR1 consisting of the amino acid sequence of SEQ ID NO: 2, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 3, and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 4; And
A light chain variable region comprising LCDR1 consisting of the amino acid sequence of SEQ ID NO: 5, LCDR2 consisting of the amino acid sequence of SEQ ID NO: 6, and LCDR3 consisting of the amino acid sequence of SEQ ID NO: 7 to detect foot-and-mouth disease virus type O antibody Way.
delete The method according to claim 1, wherein the antibody or antigen-binding fragment thereof is a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 8; And
The method comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 9.
The method of claim 1, wherein the antibody or antigen-binding fragment thereof is produced from hybridoma cells deposited with accession number KCLRF-BP-00465.
The method of claim 1, wherein the detecting step comprises enzyme immunoassay (ELISA), western blotting, immunofluorescence, immunohistochemistry staining, flow cytometry, and immunocytochemistry. Method, radioimmunoassay (RIA), immunoprecipitation assay (Immunoprecipitation Assay), immunodiffusion assay (Immunodiffusion assay), complement fixation assay (Complement Fixation Assay) and protein chip (Protein Chip). That, how.
The method of claim 1, wherein the contacting step,
Contacting the sample with a recombinant antigen consisting of the amino acid sequence of SEQ ID NO: 10; And
The method comprising the step of contacting the recombinant antigen contacted with the sample and an antibody or antigen-binding fragment thereof that specifically binds an epitope consisting of the amino acid sequence of SEQ ID NO: 1.

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