JP2013049645A - Antibody responding to foot-and-mouth disease virus, method of detecting foot-and-mouth disease virus using the antibody, and strip containing the antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antibody that does not show cross reactivity with foot-and-mouth disease viruses of other serum types.SOLUTION: The antibody binds only to a foot-and-mouth disease virus of a specific serum type, such as C and Asia1. Then the antibody does not respond to the foot-and-mouth disease virus of the specific serum type.

Description

  The present invention relates to antibodies that specifically react only with a specific serotype of foot-and-mouth disease virus, and uses thereof.

  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 (eg, cattle, pigs, deer, sheep, goats, etc.). In Japan, the infectious disease is designated as a legal infectious disease in the Domestic Animal Infectious Disease Prevention Law. . 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. Due to its particularly high transmission, livestock infected with foot-and-mouth disease virus will be killed as soon as infection is confirmed. 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.

  Foot-and-mouth disease viruses are classified into seven serotypes (O, A, C, Asia1, SAT1, SAT2, and SAT3). Of these, serotypes SAT1, SAT2 and SAT3 have been confirmed to exist only in Africa. Therefore, in other regions, only serotypes O, A, C, and Asia1 have been confirmed.

  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 the suspected livestock is infected with the foot-and-mouth disease virus and, if so, whether the serotype is one of the above four. It is.

  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 foot-and-mouth disease virus), but serotype needs to be further tested by other methods. Therefore, a method using RT-PCR alone is not preferable in that the serotype is quickly determined. Thus, a method for determining the serotype of foot-and-mouth disease virus using ELISA has been studied (for example, Non-Patent Document 1 by the present inventors).

JOUNAL OF CLINICAL Microbiology, NOV. 2009, p.3663-3668

  However, at present, only two types of antibodies (antibodies specific to serotype O or A) are found that specifically bind to one of the foot-and-mouth disease viruses of a specific serotype and do not bind to the foot-and-mouth disease virus of other serotypes. It has not been. Therefore, at present, serotypes of foot-and-mouth disease viruses other than serotypes O and A cannot be detected. For this reason, in one step, it is impossible to determine the presence or absence of serotype and the presence of infection against foot-and-mouth disease virus.

  That is, there is a need for an antibody that specifically recognizes only serotype C or Asia1 foot-and-mouth disease virus. However, when an antibody that specifically recognizes serotype Asia1 described in Non-Patent Document 1 is taken as an example, the antibody shows cross-reactivity with other serotypes. Thus, it is not easy to obtain an antibody that recognizes only a single serotype of foot-and-mouth disease virus without showing cross-reactivity.

  In view of the above problems, an object of the present invention is to provide an antibody that specifically recognizes only serotype C or Asia1 foot-and-mouth disease virus.

  In order to solve the above problems, the antibody of the present invention specifically binds to serotype C of foot-and-mouth disease virus and does not specifically bind to serotypes O, A and Asia1 of foot-and-mouth disease virus.

  In order to solve the above problems, the antibody of the present invention specifically binds to foot-and-mouth disease virus serotype Asia1, and does not specifically bind to foot-and-mouth disease virus serotypes O, A and C.

  All of the above antibodies are preferably monoclonal antibodies.

  All of the above antibodies are preferably polyclonal antibodies.

  Preferably, the antibody is an antibody produced by hybridoma anti-foot-and-mouth disease virus serotype Asia1 hybridoma 12C7 (self-deposited strain).

  The antibody is preferably an antibody produced by a hybridoma anti-foot-and-mouth disease virus serotype C hybridoma 13F1 (self-deposited strain).

  In order to solve the above-described problems, the detection strip of the present invention includes a membrane on which any one of the above-described antibodies is applied.

  In the strip, both of the above antibodies, an antibody that specifically binds to serotype O of foot-and-mouth disease virus, does not specifically bind to serotypes A, C and Asia1 of foot-and-mouth disease virus, and specific to serotype A of foot-and-mouth disease virus It is preferable that an antibody that specifically binds to and does not specifically bind to serotypes O, C, and Asia1 of foot-and-mouth disease virus is applied.

  In order to solve the above problems, the method of the present invention specifically detects a foot-and-mouth disease virus for a serotype using any of the above-described antibodies.

  In order to solve the above problems, the method of the present invention specifically detects foot-and-mouth disease virus for serotypes using the strip for detection.

  The present invention has an effect that the foot-and-mouth disease virus of serotype C and serotype Asia1 can be detected separately from other serotype viruses.

It is a figure which shows the difference in the antigen specificity of the monoclonal antibody of this invention, and the conventional monoclonal antibody. It is a figure which illustrates the lateral flow assay which concerns on this invention typically. It is a figure which shows the result of having detected foot-and-mouth disease virus using the lateral flow assay which concerns on this invention.

  Details of the present invention will be described below.

  The antibody of the present invention specifically binds to serotype C of foot-and-mouth disease virus and does not specifically bind to serotypes O, A and Asia1 of foot-and-mouth disease virus, or specifically binds to serotype Asia1 of foot-and-mouth disease virus. An antibody that does not specifically bind to serotypes O, A and C of foot-and-mouth disease virus. Therefore, if these antibodies according to the present invention are used, the foot-and-mouth disease virus of serotype Asia1 or C can be detected without cross-reacting with other serotypes.

  Any of the antibodies of the present invention can be a monoclonal antibody or a polyclonal antibody. However, from the viewpoint of less possibility of showing more specific reactivity and cross-reactivity, it is preferable that all of the antibodies of the present invention are monoclonal antibodies.

  The antibody of the present invention is preferably produced by the hybridoma anti-foot-and-mouth disease virus serotype Asia1 hybridoma 12C7 (self-deposited strain). The antibody of the present invention is preferably produced by hybridoma anti-foot-and-mouth disease virus serotype C. hybridoma 13F1 (self-deposited strain).

  Here, all these hybridomas are related to the deposit of microorganisms related to the patent application filed by the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center, assuming that the possibility that a pathogen of BSL3 or higher is attached cannot be denied. Since it falls under the provisions of Article 4, Paragraph 1, Item 1 of the Regulations, the Center has been informed that a deposit certificate cannot be issued (date of notification of deposit receipt non-delivery notice: August 2011) 15th). Therefore, the applicant himself stores these hybridomas as self-deposited strains.

  In addition, as described above, foot-and-mouth disease virus is a pathogen that directly causes a disease designated as a legal infectious disease in the livestock infectious disease prevention method. Furthermore, these hybridomas and the antibodies produced from them cannot completely rule out the possibility that the foot-and-mouth disease virus is attached. Therefore, at present, these hybridomas and antibodies cannot be taken out of the facility of the National Institute of Agricultural and Food Industry Research (Applicant), which is approved for handling by the Ministry of Agriculture, Forestry and Fisheries.

  As described above, the antibodies and hybridomas according to the present invention are distributed only to third parties deemed appropriate based on public safety and legal regulations, and cannot be taken out of the facility. .

  The antibody of the present invention is used in a method for detecting a serotype Asia1 or C foot-and-mouth disease virus that is an antigen by being applied to or attached to an appropriate substrate (for example, a membrane) or the like. obtain. The method is a variety of known methods (for example, immunochromatography) using an antigen-antibody reaction.

  Therefore, the antibody of the present invention can be used by being applied on a membrane. That is, the present invention can be a detection strip provided with the above membrane in order to detect foot-and-mouth disease virus serotype-specifically. Such a detection strip is used in a lateral flow assay for serotype-specific detection of foot-and-mouth disease virus bound to a labeled compound (eg, colloidal gold). The details may be referred to an example described later.

  When the antibody of the present invention is applied on a membrane, it specifically binds only to two antibodies of the present invention, an antibody that specifically binds only to serotype O foot-and-mouth disease virus, and only to serotype A foot-and-mouth disease virus It is particularly preferred that all of the antibodies to be applied are applied on the membrane. A one-step assay can simultaneously determine whether the pathogen contained in the subject of the assay is a foot-and-mouth disease virus or one of the four serotypes.

[Preparation of monoclonal antibodies (13F1 and 12C7)]
(Production of hybridoma)
Asia1 Shamir and C / PHI / 7 / U strains of foot-and-mouth disease virus were inoculated into IB-SR-2 cells and rotated overnight. The collected culture solution was centrifuged at 4000 G for 30 minutes, and then the supernatant was collected. The supernatant was mixed with an equal volume of saturated ammonium sulfide solution and stirred overnight at 4 ° C. to precipitate the virus. The precipitated virus was precipitated by centrifugation at 4000 G for 30 minutes, and the supernatant was discarded and then suspended using an appropriate amount of PBS. The mixture was centrifuged again at 170000 G (32000 rpm) for 2 hours, the supernatant was removed, and the virus was suspended using 1 ml of PBS. By placing the virus suspension on a sucrose density gradient (15-45%), centrifuging at 280000 G (41000 rpm) for 1.5 hours, and recovering the target band that appeared in place, the 146S Full particles were purified.

  8-12 week old female BALB / c mice were inoculated intraperitoneally with complete particles of Asia1 Shamir strain and C / PHI / 7/84 strain of foot-and-mouth disease virus purified as described above. In the first immunization, an equivalent mixture of Freund's complete adjuvant (manufactured by Yatron) and purified virus solution is used, and for booster immunization, an equivalent mixture of Freund's complete adjuvant (manufactured by Yatron) and purified virus solution is micelles using a connecting needle. To produce an emulsion. After the final immunization, mouse spleen cells and mouse myeloma cells P3U1 were fused using polyethylene glycol 4000 (manufactured by Merck), and HAT medium (containing 0.1 mM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine). 20% FCS RPMI-1640 medium (Nissui)) was cultured in a 96-well plate. After 2 weeks, the cells were cultured in an HT selection medium (20% FCS RPMI-1640 medium (Nissui) containing 0.1 mM hypoxanthine 16 μM thymidine).

  By culturing as described above, colonies were formed in the hybridomas, and hybridomas producing the target monoclonal antibody were screened by immunostaining using ELISA and 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 serotype foot-and-mouth disease virus complete particles (either C / PHI / 7/84 strain or Asia1 Shamir 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 divided into serotypes and inoculated. 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, infected cells were prepared for one of the hybridomas to be screened as much as necessary for the four serotypes. 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 further screened by immunostaining using foot-and-mouth disease virus-infected cells to confirm that the results of screening by ELISA are not nonspecific reactions. did.

  By cloning the hybridoma selected by screening according to a standard method, two types of hybridomas stably producing the target monoclonal antibody were established. Each of the hybridomas was named anti-foot-and-mouth disease virus serotype Asia1 hybridoma 12C7 (self-deposited strain) and anti-foot-and-mouth disease virus serotype C hybridoma 13F1 (self-deposited strain). Further, each of the monoclonal antibodies produced by these hybridomas is classified into 12C7 (monoclonal antibody specifically recognizing serotype Asia1 foot-and-mouth disease virus) and 13F1 (monoclonal antibody specifically recognizing serotype C foot-and-mouth disease virus). ).

(Determining the properties of 12C7 and 13F1)
Antigen characteristics and neutralizing activity of the obtained monoclonal antibody were examined by (1) ELISA method, (2) virus neutralization test, and (3) immunostaining of foot-and-mouth disease infected cells. (1) to (3) were performed using the following materials and procedures.

(1) ELISA method The procedure was the same as ELISA in screening. As a monoclonal antibody to be compared, an antibody (7C2) showing cross-reactivity with serotype O and serotype Asia1 foot-and-mouth disease viruses was used. Similarly for the following (2) and (3), 7C2 was used as a comparison target. For 7C2, JOUNAL OF CLINICAL Microbiology, NOV. 2009, p.3663-3668 may be referred to.

(2) Virus neutralization test IB-SR-2 cells were used as cells for infection. In accordance with a conventional method, cells were inoculated after incubating a hybridoma culture supernatant or a growth medium alone and a mixture of virus solutions. It dye | stained on the 5th day after infection, and it was evaluated whether the proliferation of the virus was suppressed by mixing and incubation of the virus liquid with a culture supernatant.

(3) Immunostaining of foot-and-mouth disease infected cells The procedure was the same as immunostaining in screening.

  The results of (1) to (3) are shown in FIG. As shown in FIG. 1, all of the monoclonal antibodies obtained as described above specifically recognize only foot-and-mouth disease virus serotype C or serotype Asia1. 12C7 also showed neutralizing activity against serotype Asia1 of foot-and-mouth disease virus.

[Lateral flow assay]
(Antibodies used)
The following six antibodies are used in this assay. In the following description, the types of antibodies correspond to the following numbers.
1. Monoclonal antibody specifically recognizing serotype O foot-and-mouth disease virus (70C4)
2. Monoclonal antibody specifically recognizing serotype A foot-and-mouth disease virus (16C6)
3. Monoclonal antibody specifically recognizing serotype C foot-and-mouth disease virus (13F1)
4). Monoclonal antibody specifically recognizing foot-and-mouth disease virus of serotype Asia1 (12C7)
5. Monoclonal antibody specifically recognizing foot-and-mouth disease virus of all serotypes (1H5)
6). Anti-mouse IgG monoclonal antibody (anti-mouse IgG)
For antibodies 1, 2 and 5, refer to JOUNAL OF CLINICAL Microbiology, NOV. 2009, p.3663-3668.

(Purification of monoclonal antibody)
The monoclonal antibody used was applied to a protein G affinity column (MAb Trap Kit; GH 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 Amicon ultra Centrifugal filter units (Millipore, product code: UFC805024). Those substituted with 5 mM phosphate buffer (PB) (pH 7.5) were prepared for each of antibodies 1 to 6, and those substituted with 5 mM PB (pH 8.0) were prepared for antibody 5.

(Preparation of antibody-coated membrane)
The concentration of each of antibodies 1-6 was adjusted to about 1500 μg / ml using 5 mM PB (pH 7.5) containing 3% ethanol. The antibodies 1 to 6 having adjusted concentrations were applied on a nitrocellulose membrane (Millipore, HF180XSS) using an immunoliner 200 (System Biotics) with a linear shape having a width of about 1 mm and an interval of 5 mm. As shown in FIG. 2, the antibodies 1 to 6 were applied in order from the upstream (from bottom to top in FIG. 2). The converted antibody concentration was 0.8 μl / cm.

(Assembly of detection strip)
The membrane was dried at 50 ° C. for 30 minutes and then immersed in blocking buffer (50 mM borate buffer (pH 8.5) containing 0.5% casein) at room temperature for 30 minutes. Then, it was immersed in a washing buffer (50 mM Tris-HCl (pH 7.4) containing 0.5% sucrose) at room temperature for 30 minutes, and then air-dried at room temperature.

  Using a backing sheet (ARcare 7815: Adhesives Research), a sample pad (Standard 14 (glass fiber), Whatman) is pasted on the upstream side of the dried membrane, and an absorbent pad (CF6 (glass fiber and A mixture with cotton), Whatman) was applied. In this way, the detection strip having the configuration shown in FIG. 2 was assembled. The detection strip was stored in a sealed container containing silica gel under conditions of humidity of 40% or less.

(Labeling monoclonal antibodies with colloidal gold)
The concentration of antibody 5 was adjusted to 20 μg / 100 μl using 5 mM PB (pH 8.0). After sufficiently sonicating colloidal gold (diameter 40-50 nm) (Wine Red Chemical Co., WRGH2 (OD525 = 12)), the pH was adjusted to 8.0 using 1 mM K ₂ CO ₃ . Next, the antibody solution and the gold colloid solution were mixed at a ratio of 1: 8 in the silicon coating tube. 1% bovine serum albumin (BSA) and 0.05% PEG were added and mixed, followed by centrifugation at 10,000 rpm at room temperature for 30 minutes. After removing the centrifugation supernatant and sonicating, it was suspended using a solution (solution A) in which 0.5% BSA and 0.05% PEG were added to PBS. After centrifugation again at 10000 rpm for 30 minutes at room temperature, the supernatant was removed, suspended by sonication, and adjusted to OD525 = 2.0 using solution A. After adding an equal volume of 15 mM Tris-HCl (pH 8.2) containing 20% sucrose, 100 μl of the suspension containing colloidal gold-labeled antibody was dispensed into 2 ml tubes. The mouth of each tube was covered with parafilm and then several holes were made in the parafilm and frozen at -80 ° C for 1 hour. After vacuum drying overnight using a vacuum dryer, the parafilm was peeled off, the tube cap was closed and stored at 4 ° C.

(Evaluation of lateral flow assay)
FMD virus serotype O (O / JPN / 2000 strain), serotype A (A15 / TAI / 1/60 strain), serotype C (C / PHI / 7/84 strain), serotype Asia1 (Asia1 Shamir strain) ) Was adjusted to 10 ⁶ TCID ₅₀ respectively. 100 μl of 5mMPB (pH 7.5) was used to dissolve the freeze-dried gold colloid-labeled antibody, and then 100 μl of virus dilution was added to each tube.

  As shown in FIG. 2, the virus-gold colloid-labeled antibody solution in each tube was dropped onto the sample pads of the four detection strips, and the solution was allowed to penetrate into the absorption pad. In all of the strips for detection, a red line appeared at the position where the antibodies 1 to 6 were applied on the membrane according to the serotype of the dropped virus. The result is shown in FIG.

  As shown in FIG. 3, a red line appeared at the position where antibodies 1, 5 and 6 were applied on the detection strip to which serotype O foot-and-mouth disease virus was dropped. In addition, a red line appeared at the position where antibodies 2, 5 and 6 were applied on the detection strip to which serotype A foot-and-mouth disease virus was dropped. In the detection strip to which serotype C foot-and-mouth disease virus was dropped, a red line appeared at the position where antibodies 3, 5 and 6 were applied. A red line appeared at the position where antibodies 4, 5 and 6 were applied on the detection strip to which the foot-and-mouth disease virus of serotype Asia1 was dropped. As described above, by using the strip for detection according to the present invention, the serotype of the foot-and-mouth disease virus to be detected can be determined quickly and accurately.

  INDUSTRIAL APPLICABILITY The present invention can be used in a technique that can detect foot-and-mouth disease viruses that infect livestock by dividing them into serotypes.

Claims (10)

  An antibody that specifically binds to serotype C of foot-and-mouth disease virus and does not specifically bind to serotypes O, A and Asia1 of foot-and-mouth disease virus.   An antibody that specifically binds to serotype Asia1 of foot-and-mouth disease virus and does not specifically bind to serotypes O, A and C of foot-and-mouth disease virus.   The antibody according to claim 1 or 2, which is a monoclonal antibody.   The antibody according to claim 1 or 2, which is a polyclonal antibody.   The antibody according to claim 1, which is produced by hybridoma anti-foot-and-mouth disease virus serotype C hybridoma 13F1 (self-deposited strain).   The antibody according to claim 2, which is produced by hybridoma anti-foot-and-mouth disease virus serotype Asia1 hybridoma 12C7 (self-deposited strain).   A detection strip provided with a membrane coated with the antibody according to claim 1 or the antibody according to claim 2.   The antibody according to claim 1, the antibody according to claim 2, an antibody that specifically binds to serotype O of foot-and-mouth disease virus and does not specifically bind to serotypes A, C and Asia1 of foot-and-mouth disease virus, and foot-and-mouth disease virus The detection strip according to claim 7, which is coated with an antibody that specifically binds to serotype A and does not specifically bind to serotypes O, C, and Asia1 of foot-and-mouth disease virus.   A method for specifically detecting serotypes of foot-and-mouth disease virus using the antibody according to claim 1 or the antibody according to claim 2.   9. A method for specifically detecting foot-and-mouth disease virus for serotypes using the strip for detection according to claim 7 or 8.