JP6391477B2 - Antibodies that react with foot-and-mouth disease virus - Google Patents

Description

  The present invention relates to antibodies that react with foot-and-mouth disease virus. More particularly, it relates to antibodies that react with all seven serotypes of foot-and-mouth disease virus (O, A, C, Asia1, SAT1, SAT2, and SAT3).

  Foot and mouth disease virus (FMDV) is one of the infectious diseases of livestock. The foot-and-mouth disease virus is mainly composed of animals belonging to the cetacean (for example, cattle, pigs, deer, sheep, goats, etc.). In Japan, the infectious disease is a domestic animal infectious disease prevention method (formerly known as an infectious disease). ) Is specified. The foot-and-mouth disease virus is not easily inactivated in the surrounding environment and is therefore highly transmissible, significantly reducing the productivity of infected livestock and showing a high mortality rate in infected cubs. Domestic animals infected with foot-and-mouth disease virus due to their particularly high spreadability are slaughtered in Japan as soon as infection is confirmed, based on the Act on Prevention of Livestock Infectious Diseases. In addition, since movement of livestock is restricted in areas and nations where livestock infected with foot-and-mouth disease virus is found, foot-and-mouth disease virus is recognized worldwide as a pathogen that can have a very economic impact on the livestock industry. Yes.

  In Japan, individuals infected with foot-and-mouth disease virus have not been confirmed until 2000 for about a century. However, in 2000 and 2010, individuals infected with foot-and-mouth disease virus were identified. For this reason, the importance of establishing countermeasures against foot-and-mouth disease epidemics is clearly increasing. The most important countermeasure is to determine whether livestock suspected of being infected is infected with the foot-and-mouth disease virus.

  As a general determination method, a gene for foot-and-mouth disease virus is detected by RT-PCR. In this method, if gene amplification is observed, it is determined to be positive (infected with a foot-and-mouth disease virus). However, a method using RT-PCR alone is not preferable in terms of quick and accurate determination. . Therefore, methods for detecting foot-and-mouth disease virus using other antigen detection methods (such as ELISA or immunochromatography) (in combination) have been studied (for example, Non-Patent Document 1 by the present inventors).

  Foot-and-mouth disease viruses are classified into seven serotypes (O, A, C, Asia1, SAT1, SAT2, and SAT3). Non-Patent Document 1 describes antibodies that react with all seven serotypes.

K. Morioka et al., Journal of Clinical Microbiology, Vol.47, No.11, p.3663-3668 (2009)

  In order to detect foot-and-mouth disease with higher accuracy, further development of anti-foot-and-mouth disease virus antibodies is desired.

  As a result of intensive studies to achieve the above object, the present inventors have succeeded in producing a hybridoma that produces anti-foot-and-mouth disease virus antibodies capable of recognizing all seven serotypes of foot-and-mouth disease virus. It came to complete.

That is, the present invention provides the following (1) to (8).
(1) An antibody produced by a hybridoma specified by accession number NITE P-01972.
(2) An antibody-producing hybridoma specified by the accession number NITE P-01972.
(3) A foot-and-mouth disease virus detection kit comprising the antibody according to (1) above.
(4) A foot-and-mouth disease virus serotype O, A, C, Asia1, SAT1, SAT2, and SAT3, which further react with any of the above-mentioned monoclonal antibodies described in (1), further comprising an anti-foot-and-mouth disease virus antibody ( The foot-and-mouth disease virus detection kit as described in 3).
(5) A method for detecting foot-and-mouth disease virus using the antibody according to (1) above.
(6) A step of bringing a sample collected from a subject into contact with the antibody described in (1) above, causing an antigen-antibody reaction between the antigen in the sample and the antibody; and detecting an antigen-antibody reaction product The detection method according to (5) above, comprising a step.
(7) A step of bringing a sample collected from a subject into contact with a primary antibody against foot-and-mouth disease virus immobilized on a base material, and causing an antigen-antibody reaction between the antigen in the sample and the primary antibody; binding to the antigen and the label An anti-foot-and-mouth disease virus secondary antibody contacted to cause an antigen-antibody reaction between the antigen and the secondary antibody; and a step of detecting the label, wherein the primary antibody and the secondary antibody The detection method according to (5) above, wherein at least one is the antibody according to (1) above.
(8) One of the primary antibody and the secondary antibody is the antibody according to (1) above, and the other of the primary antibody and the secondary antibody is serotype O of foot-and-mouth disease virus, The detection method according to (7), which is an anti-foot-and-mouth disease virus antibody different from the antibody according to (1), which reacts with any of A, C, Asia1, SAT1, SAT2, and SAT3.

  The antibody according to the present invention can react with all seven serotypes of foot-and-mouth disease virus with high sensitivity.

It is a figure which shows the result of indirect sandwich ELISA in the Example of this invention. It is a figure which shows the result of MSD-ELISA in the Example of this invention. It is a figure which shows the result of MSD-ELISA in the Example of this invention. It is a figure which shows the result of MSD-ELISA in the Example of this invention.

[1. antibody〕
The antibody according to the present invention is an antibody (named “16D6”) produced by a hybridoma specified by the accession number NITE P-01972. The 16D6 antibody is a monoclonal antibody capable of recognizing all seven serotypes of foot-and-mouth disease virus (O, A, C, Asia1, SAT1, SAT2, and SAT3). The 16D6 antibody is produced by culturing a hybridoma specified by the accession number NITE P-01972, for example, in a medium containing 10% fetal serum or in a serum-free medium under conditions of 37 ° C. and 5% CO _2. The 16D6 antibody can be produced by recovery and purification according to a known method.

[2. Hybridoma)
The present invention also provides a hybridoma for producing an antibody identified by accession number NITE P-01972. The hybridoma according to the present invention is a foot-and-mouth disease virus A22 / IRQ / 24/64 strain isolated from cattle (reference: ARROWSMITH, AEM (1975). Variation among strains of type A foot-and-mouth disease virus in the Eastern Mediterranean region). 1964-1972. Journal of Hygiene 75, 387-397) As an antigen, a hybridoma (anti-foot-and-mouth disease virus hybridoma) prepared by immunizing a mouse and fusing the spleen cell of the immunized mouse with a myeloma cell-derived strain of the mouse 16D6 (accession number NITE P-01972)). The hybridoma 16D6 is deposited with the Patent Microorganism Depositary Center of the National Institute for Product Evaluation Technology (Kazusa-Kamazu City, Chiba Pref., 2-5-5, Room 8122).

[3. Foot-and-mouth disease virus detection kit]
The present invention further provides a foot-and-mouth disease virus detection kit comprising 16D6 antibody. The foot-and-mouth disease virus detection kit which concerns on this invention can be a kit using ELISA method, an immunochromatography method, or an immunodiffusion measuring method etc., for example.

  The foot-and-mouth disease virus detection kit according to the present invention may further contain an anti-foot-and-mouth disease virus antibody different from the 16D6 antibody that reacts with any of serotypes O, A, C, Asia1, SAT1, SAT2, and SAT3 of foot-and-mouth disease virus. Good. Such anti-foot-and-mouth disease virus antibodies can be monoclonal antibodies. Examples of such anti-foot-and-mouth disease virus antibodies include 1H5 antibody described in Non-Patent Document 1. As shown in Examples below, foot-and-mouth disease virus can be detected with higher sensitivity by using 16D6 antibody in combination with other anti-foot-and-mouth disease virus antibodies that react with all seven serotypes. Note that “react” means that an antibody reacts with a foot-and-mouth disease virus (antigen) by an antigen-antibody reaction and can also be expressed as “bind” or “recognize”.

  In addition, the foot-and-mouth disease virus detection kit according to the present invention may include an anti-foot-and-mouth disease virus antibody that specifically reacts with any of serotypes O, A, C, Asia1, SAT1, SAT2, and SAT3 of foot-and-mouth disease virus. Such anti-foot-and-mouth disease virus antibodies can be monoclonal antibodies. Examples of such anti-foot-and-mouth disease virus antibodies include 16C6 antibody (serotype A specific), 70C4 antibody (serotype O specific), and 65H6 antibody (serotype O specific) described in Non-Patent Document 1. And 12C7 antibody (serotype Asia1 specific) and 13F1 antibody (serotype C specific) described in JP2013-49645A. By comparing the reactivity with these antibodies, the serotype of the foot-and-mouth disease virus contained in the specimen of the subject can be determined by a single test.

  In addition, the foot-and-mouth disease virus detection kit according to the present invention includes an anti-foot-and-mouth disease virus antibody that specifically reacts with any two or more of serotypes O, A, C, Asia1, SAT1, SAT2, and SAT3 of foot-and-mouth disease virus. Also good.

  A plurality of antibodies other than the 16D6 antibody described above may be included in the foot-and-mouth disease virus detection kit according to the present invention. Further, the 16D6 antibody and the antibody other than the 16D6 antibody may be used as mixed or may be used without being mixed.

  Further, the 16D6 antibody and / or an antibody other than the 16D6 antibody described above may be bound to a label. Examples of the label include enzymes, enzyme substrates, radioisotopes, luminescent substances, fluorescent substances, biotin, and colored substances. Examples of the enzyme include peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase and glucose-6-phosphate dehydrogenase. These enzymes and antibodies can be bound by a known method using a crosslinking agent such as a maleimide compound and an N-hydroxysuccinimide ester compound. As the enzyme substrate, a known substance can be used according to the enzyme to be used. For example, when peroxidase is used as an enzyme, OPD (orthophenylenediamine) and TMB (tetramethylbenzidine) are used. When alkaline phosphatase is used as an enzyme, nitroblue tetrazolium (NBT) and 5-bromo- A mixed substrate of 4-chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) or the like can be used.

Examples of the radioisotope include those used in usual radioimmunoassay such as ¹²⁵ I, ³ H or ¹⁴ C. Radiolabeling of the antibody can be performed using a known method. Examples of the fluorescent dye include those used in usual fluorescent antibody methods such as fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate and phycoerythrin. Examples of the luminescent substance include isoluminol, acridine ester and lucigenin. At this time, a known method can be used as the labeling method. Examples of the coloring substance include colored latex particles and gold colloid.

  Further, the 16D6 antibody and / or an antibody other than the 16D6 antibody described above may be immobilized on a substrate. In this case, the base material is also a component of the foot-and-mouth disease virus detection kit. As a base material, well-known things, such as a nitrocellulose membrane, a silica membrane, a glass filter, a polystyrene particle, a polystyrene plate, or a silica magnetic particle, can be used. In one example, the foot-and-mouth disease virus detection kit according to the present invention includes an antibody other than the 16D6 antibody immobilized on the substrate and the labeled 16D6 antibody. In another example, the foot-and-mouth disease virus detection kit according to the present invention includes an antibody other than 16D6 antibody immobilized on a substrate and a labeled 16D6 antibody.

  The foot-and-mouth disease virus detection kit which concerns on this invention may further contain the reagent for detecting a label | marker. In addition, the foot-and-mouth disease virus detection kit according to the present invention is a member (secondary antibody, coloring reagent, blocking reagent, etc.) necessary for performing an immune reaction such as ELISA, immunochromatography, Western blotting, etc., plate (96 Well plate, etc.) and tubes may be included. Further, a secondary antibody for detecting the 16D6 antibody, a label enzyme substrate bound to the secondary antibody, and the like may be provided.

  Moreover, you may provide the 1 or more containers (for example, vial, tube, ampoule, bottle, etc.) for storing the component which comprises the detection kit which concerns on this invention. Moreover, you may further provide the use manual in which the detail was described about the detection method used in the detection kit which concerns on this invention. The detection method will be described later [4. The detection method demonstrated by the detection method of foot-and-mouth disease virus] is mentioned.

[4. (Food and foot disease virus detection method)
The present invention further provides a method for detecting foot-and-mouth disease virus using the 16D6 antibody. Examples of subjects include cattle, pigs, deer, sheep, goats, buffalos, wild boars, and antelopes. Samples collected from the subject include blood, blister fluid, feces, urine, throat swab, nasal swab, nasal aspirate, bullous epithelial emulsion, lesion wiping fluid, various tissues and organs, and the like. By detecting the foot-and-mouth disease virus in the sample using the detection method according to the present invention, it is possible to quickly and easily and reliably diagnose whether or not the subject from which the sample is derived is infected with the foot-and-mouth disease virus. .

  The detection method according to the present invention can be based on, for example, an ELISA method (for example, a sandwich ELISA method), an immunochromatography method, an immunodiffusion measurement method, or the like.

  One aspect of the detection method according to the present invention is a step of bringing a sample collected from a subject into contact with a 16D6 antibody, and causing an antigen-antibody reaction between the antigen in the sample and the 16D6 antibody; Detecting.

  In the antigen-antibody reaction step, the sample and the antibody may be incubated. The step of detecting the antigen-antibody reaction product can be performed using a known method such as detecting the antibody labeled in advance.

  In one embodiment of the detection method according to the present invention, a sandwich ELISA method is used, and a sample collected from a subject is contacted with an anti-foot-and-mouth disease virus primary antibody immobilized on a substrate, and an antigen in the sample is contacted. An antigen-antibody reaction between the antigen and the primary antibody; an antigen-antibody-and-mouth disease virus secondary antibody bound to the antigen and a label; and the antigen and the secondary antibody react with each other; and the label And at least one of the primary antibody and the secondary antibody is a 16D6 antibody.

  In the detection method according to the present invention, both the primary antibody and the secondary antibody can be prepared using the 16D6 antibody, but one of the primary antibody and the secondary antibody is prepared using the 16D6 antibody. Can be prepared using an anti-foot-and-mouth disease virus antibody separate from the 16D6 antibody, the other of which reacts with any of the seven serotypes of foot-and-mouth disease virus. Examples of the anti-foot-and-mouth disease virus antibody different from the 16D6 antibody include the 1H5 antibody described above. The combination of the 16D6 antibody and the 1H5 antibody can further improve the detection sensitivity of foot-and-mouth disease virus. Furthermore, it is preferable to use 16D6 antibody as the primary antibody immobilized on the substrate and 1H5 antibody as the secondary antibody. The detection sensitivity of foot-and-mouth disease virus can be further improved.

  Examples will be shown below, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention. Moreover, all the literatures described in this specification are used as reference.

[Production of monoclonal antibodies]
IB-RS-2 cells were inoculated with foot-and-mouth disease virus A22 / IRQ / 24/64 strain, and rotated and cultured overnight. The collected culture was centrifuged at 1500 G for 10 minutes, and the supernatant was collected. The supernatant was mixed with an equal volume of saturated ammonium sulfate solution and stirred overnight at 4 ° C. to precipitate the virus. The precipitated virus was spun down at 5000 G for 30 minutes, and the supernatant was removed and suspended in an appropriate amount of PBS. Again, centrifugation was performed at 5000 G for 30 minutes, and the supernatant was removed and suspended in 1 mL of PBS. A sucrose density gradient (15-45%) virus suspension was added, centrifuged at 20000 rpm for 2 hours, and the desired band was collected to purify 146S complete particles.

  8-12 week old female BALB / c mice were inoculated intraperitoneally with complete particles of the foot-and-mouth disease virus A22 / IRQ / 24/64 strain purified as described above. For the first immunization, use an equivalent mixture of Freund's complete adjuvant (manufactured by Yatron) and purified virus solution, and for additional inoculation, use an equivalent mixture of Freund's complete adjuvant (manufactured by Yatron) and purified virus solution. To micelles. After final immunization, mouse spleen cells and mouse myeloma cells P3U1 were fused using polyethylene glycol 4000 (Merck), and HAT medium (0.1 mM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine, and Cultured in 96-well plates using RPMI-1640 medium (Nissui) containing 20% FCS. After 2 weeks, the cells were cultured using an HT selection medium (RPMI-1640 medium (Nissui) containing 0.1 mM hypoxanthine, 16 μM thymidine, and 20% FCS).

  By culturing as described above, hybridoma colonies were formed, and hybridomas producing the desired monoclonal antibody were screened by ELISA and immunostaining using foot-and-mouth disease virus-infected cells. Details of the screening are as follows.

  In ELISA, a rabbit anti-foot-and-mouth disease virus antibody was adsorbed to a solid phase and reacted with complete particles of A22 / IRQ / 24/64 strain. Next, after removing the virus, the culture supernatant of the hybridoma was added and allowed to react. Then, after removing the culture supernatant, a mouse anti-IgG antibody labeled with HRP was added and reacted. After discarding the labeled antibody, it was reacted with a chromogenic substrate, and the absorbance was measured.

  In immunostaining using foot-and-mouth disease virus-infected cells, IB-SR-2 cells were seeded on a plate having a plurality of wells, and then foot-and-mouth disease virus was inoculated into serotypes. The culture solution was discarded at an appropriate time after infection (so that the infected cells were not peeled off), and the infected cells were fixed using cooled acetone. Each plate was stored at −80 ° C. until used at each time point of screening. In other words, as many infected cells as necessary for seven serotypes were prepared for one of the hybridomas to be screened. The culture supernatant of the hybridoma to be screened was added to the preserved plate and incubated. After discarding the supernatant, an anti-mouse IgG antibody labeled with HRP was added and reacted. The labeled antibody was discarded and reacted with a chromogenic substrate, and the presence or absence of the antibody was determined under a microscope.

  Hybridomas in which antibodies are present in the culture supernatant are identified by ELISA, and in order to confirm that the result of screening by ELISA is not a non-specific reaction, again by immunostaining with foot-and-mouth disease virus-infected cells. Screened.

  By cloning the hybridoma selected by screening according to a standard method, one type of hybridoma stably producing the target monoclonal antibody was established. This hybridoma was named anti-foot-and-mouth disease virus hybridoma 16D6. The hybridoma was deposited with the Patent Microorganism Depositary Center for Product Evaluation Technology, an independent administrative agency (Accession Number NITE P-01972). The monoclonal antibody produced by this hybridoma (monoclonal antibody that reacts with foot-and-mouth disease virus of all seven serotypes) was named 16D6.

  The 16D6 antibody was examined using the swine vesicular disease virus (SVDV) strain JI and UKG 27/72 in order to examine the cross-reactivity with other viruses. (No OD <0.01 for SVDV when OD> 0.5 for all types of foot-and-mouth disease virus).

  The monoclonal antibody-producing hybridoma cloned by the manipulation method using CellTram / vario (Eppendorf) was cultured using BriClone Hybridoma Cloning Medium (QD Bioscience).

[Indirect sandwich ELISA for comparison of monoclonal antibody performance]
To confirm the performance of monoclonal antibody 16D6, an indirect sandwich ELISA was performed. For comparison, a known monoclonal antibody 1H5 that reacts with foot-and-mouth disease virus of all seven serotypes was used. For the 1H5 antibody, Non-Patent Document 1 may be referred to.

  As a capture antibody, anti-foot-and-mouth disease virus rabbit antibody of each serotype was diluted to × 800 with 0.05M carbonate buffer (pH 9.6), and 50 μL each was immobilized on a 96-well plate (Immulon II HB Thermo) at 4 ° C. overnight. Turned into. After washing three times with 300 μL / well of 0.002M PBS, 50 μL of the sample containing foot-and-mouth disease virus (antigen) was added to each well and reacted at 37 ° C. for 1 hour while shaking at 100 rpm. At this time, as a negative sample, solution A in which 0.05% (v / v) Tween 20 was added to 0.01 M PBS (pH 7.4) was used. After washing in the same manner, 5% skim milk was added to solution A to prepare solution B, which was blocked at 37 ° C. for 30 minutes. After discarding the B solution, monoclonal antibodies 1H5 and 16D6, each of which was 5-fold serially diluted with the A solution from 1 μg to 0.000064 μg, were added without washing. After reacting at 37 ° C. for 1 hour, 50 μL of peroxidase-labeled anti-mouse immunoglobulin goat antibody diluted to 3000 × B solution was added and reacted at room temperature for 45 minutes. After washing 8 times, 50 μL of chromogenic substrate TMB was added and reacted at room temperature for 15 minutes in the dark. The reaction was stopped with 50 μL of 1.25 M sulfuric acid and a wavelength of 450 nm was measured using an ELISA plate reader.

  In addition, O / JPN / 2000 strain as serotype O, A / IRN / 1/2011 strain and A22 / IRQ / 24/64 strain as serotype A, Asia1 Shamir (ISR 3/89) strain as serotype Asia1, and serum C / PHI / 7/84 strain as type C, SAT1 / KEN / 117/2009 strain as serotype SAT1, SAT2 / SAU / 6/2000 strain as serotype SAT2, SAT3 / ZIM / 3/83 strain as serotype SAT3 Was used.

  The results are shown in FIG. As shown in FIG. 3, the 16D6 antibody was more reactive than the 1H5 antibody in 7 out of 8 strains. In particular, the A / IRN / 1/2011 strain, SAT1 / KEN / 117/2009 strain, SAT2 / SAU / 6/2000 strain, and SAT3 / ZIM / 3/83 strain, which have relatively low reactivity with the 1H5 antibody, are prominent. The reactivity is high. Thus, it was found that the 16D6 antibody exhibits a very excellent reactivity as compared with the 1H5 antibody.

[Foot-and-mouth disease virus antigen detection ELISA using monoclonal antibodies]
(Purification of antibodies)
The monoclonal antibody used was applied to a protein G affinity column (MAb Trap Kit; GE Healthcare 17-1128-01) to purify only the IgG fraction for use in the assay.

(Replacement of buffer in antibody suspension)
The buffer in the antibody suspension containing the IgG fraction obtained by antibody purification was replaced with 0.01 M PBS using Amicon ultra Centrifugal filter units (Millipore, product code: UFC805024).

(Preparation of peroxidase-labeled monoclonal antibody)
Using a Peroxidase Labeling Kit-SH (Dojin Chemical Co., Ltd., product code: LK09), a monoclonal antibody (16D6 or 1H5) for antigen detection against each antigen was labeled with peroxidase.

(MSD (monoclonal antibody based sandwich direct) -ELISA method)
Monoclonal antibody (16D6 or 1H5) as a capture antibody is diluted to 0.05 μg / 50 μL with 0.05 M carbonate buffer (pH 9.6), and 50 μL is dispensed into each well of a 96-well plate (Immulon II HB Thermo). And solidified overnight at 4 ° C. After washing 4 times with a washing solution (0.002 M PBS, 300 μL / well), a sample containing foot-and-mouth disease virus antigen was added to each well by 50 μL, and reacted at 37 ° C. for 1 hour while shaking at 100 rpm. At this time, as a negative sample, solution A in which 0.05% (v / v) Tween 20 was added to 0.01 M PBS (pH 7.4) was used. After washing in the same manner as after the solid phase, blocking was performed at room temperature for 20 minutes using solution B (solution obtained by adding 8% skim milk to solution A). After discarding solution B, without washing, 50 μL of peroxidase-labeled monoclonal antibody diluted 500 times with solution B was added to each well and allowed to react at 37 ° C. for 45 minutes. After washing 8 times with the above washing solution, 50 μL each of chromogenic substrate in which 30 mg OPD and 3% (w / v) hydrogen peroxide were added to 50 mL of 0.05 M phosphate citrate buffer (pH 5.0) And allowed to react for 15 minutes at room temperature in the dark. The reaction was stopped with 50 μL of 1.25 M sulfuric acid and wavelengths of 492 nm and 690 nm were measured using an ELISA plate reader.

  The results are shown in FIGS. As shown in FIGS. 2 to 4, when the 16D6 antibody and the 1H5 antibody were combined, higher reactivity was observed. In particular, when 1H5 antibody was used as a capture antibody and 16D6 antibody was used as an antigen detection antibody, higher reactivity was observed.

  The present invention can be used for detection of foot-and-mouth disease virus.

  NITE P-01972

Claims (8)

  An antibody produced by a hybridoma identified by accession number NITE P-01972.   The hybridoma for antibody production specified by accession number NITE P-01972.   The foot-and-mouth disease virus detection kit containing the antibody of Claim 1.   The foot-and-mouth disease of claim 3, further comprising an anti-foot-and-mouth disease virus antibody different from the antibody of claim 1 that reacts with any of serotypes O, A, C, Asia1, SAT1, SAT2, and SAT3 of the foot-and-mouth disease virus. Virus detection kit.   A method for detecting foot-and-mouth disease virus using the antibody according to claim 1. A step of bringing a sample collected from a subject into contact with the antibody according to claim 1 and causing an antigen-antibody reaction between the antigen in the sample and the antibody; and a step of detecting an antigen-antibody reaction product, The detection method according to claim 5. A step of bringing a sample collected from a subject into contact with a primary antibody against foot-and-mouth disease virus immobilized on a base material, and causing an antigen-antibody reaction between the antigen in the sample and the primary antibody;
Contacting the antigen with an anti-foot-and-mouth disease virus secondary antibody bound to a label, causing the antigen to react with the secondary antibody; and detecting the label;
The detection method according to claim 5, wherein at least one of the primary antibody and the secondary antibody is the antibody according to claim 1. One of the primary antibody and the secondary antibody is the antibody of claim 1,
The antibody according to claim 1 or 2, wherein the other of the primary antibody and the secondary antibody reacts with any of serotypes O, A, C, Asia1, SAT1, SAT2, and SAT3 of foot-and-mouth disease virus. The detection method according to claim 7, which is another anti-foot-and-mouth disease virus antibody.