WO2016067637A1 - Ebola virus antibody, and method for producing antibody - Google Patents

Ebola virus antibody, and method for producing antibody Download PDF

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WO2016067637A1
WO2016067637A1 PCT/JP2015/005489 JP2015005489W WO2016067637A1 WO 2016067637 A1 WO2016067637 A1 WO 2016067637A1 JP 2015005489 W JP2015005489 W JP 2015005489W WO 2016067637 A1 WO2016067637 A1 WO 2016067637A1
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antibody
ebola virus
ebola
ostrich
antigen
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French (fr)
Japanese (ja)
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康浩 塚本
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オーストリッチファーマ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A41—WEARING APPAREL
    • A41D—OUTERWEAR; PROTECTIVE GARMENTS; ACCESSORIES
    • A41D13/00—Professional, industrial or sporting protective garments, e.g. surgeons' gowns or garments protecting against blows or punches
    • A—HUMAN NECESSITIES
    • A41—WEARING APPAREL
    • A41D—OUTERWEAR; PROTECTIVE GARMENTS; ACCESSORIES
    • A41D13/00—Professional, industrial or sporting protective garments, e.g. surgeons' gowns or garments protecting against blows or punches
    • A41D13/05—Professional, industrial or sporting protective garments, e.g. surgeons' gowns or garments protecting against blows or punches protecting only a particular body part
    • A41D13/11—Protective face masks, e.g. for surgical use, or for use in foul atmospheres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/12—Aerosols; Foams
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present invention relates to an antibody that recognizes Ebola virus and its production, and particularly relates to an antibody produced by an ostrich.
  • Ebola virus is known as a virus that causes critical clinical symptoms in humans. In addition, it is highly infectious and there is no certified vaccine. For this reason, the biosafety level is designated as the highest level of 4 and can only be handled by facilities that have advanced safety measures.
  • Patent Document 1 it has been carried out to neutralize viruses by using antibodies that bind to viruses. Therefore, it is conceivable to search for a method that can be neutralized with antibodies against Ebola virus.
  • Patent Document 2 discloses that a mammal is immunized with a natural or genetically modified Ebola virus nucleoprotein, and the resulting antibody-producing cells are used as hybridomas to obtain a monoclonal antibody.
  • Patent Document 3 discloses that a monoclonal antibody against a Reston strain of Ebola virus is produced.
  • Ebola virus whose handling is very strictly regulated, is treated as an antigen as it is, an antibody manufacturing site will need a facility that satisfies the requirements of biosafety level 4. However, it is not practical to prepare such a large-scale facility at the antibody production site.
  • Ebola virus which is supposed to pass through the defense mechanism of the living body, is highly effective against polyclonal antibodies that can bind to various epitopes rather than monoclonal antibodies against a single epitope. is there.
  • the present invention has been conceived in view of the above problems, and provides a production method for providing a large amount of antibodies that bind to Ebola virus. More specifically, the method for producing an antibody according to the present invention includes a step of immunizing a female bird using an Ebola virus recombinant protein as an antigen, and a step of obtaining an antibody from the egg yolk of the egg laid by the female bird, To do.
  • an antibody is obtained from an avian egg containing an ostrich, and therefore a large amount of homogeneous antibody can be obtained at a time.
  • a recombinant protein is used as an antigen, antibodies can be produced much more safely than when natural Ebola virus is used.
  • the present invention uses an Ebola virus recombinant protein produced by genetic recombination technology as an antigen.
  • an antigen By using the recombinant protein as an antigen, there is no worry of infection. Therefore, the antibody can be produced without providing particularly high safety equipment.
  • a protein that can be used as an antigen nucleoprotein (NP), glycoprotein (GP) and the like can be suitably used.
  • Ebola virus that can be used is not particularly limited.
  • Ebola viruses such as Zaire Ebola virus, Sudan Ebola virus, Côte d'Irete Ebola virus, Reston Ebola virus can be used. Reston Ebola virus has no record of developing serious clinical symptoms in humans. Therefore, it is recommended to use Zaire strain, Sudan strain, and Côte d'Irium strain.
  • a known method can be used in the step of immunizing female birds.
  • various adjuvants can be used together with the antigen. Immunization may be boosted after the initial immunization.
  • the antibody In the step of recovering antibody (IgY) from eggs obtained from birds after immunization, the antibody can be recovered by a known method.
  • the recovered antibody binds to the Ebola virus recombinant protein. Therefore, it can be expected to bind to natural Ebola virus. This binding neutralizes Ebola virus infection.
  • the obtained antibody can mask Ebola virus as shown in Examples described later. That is, the infection to a cell can be suppressed. Therefore, it can be used as a therapeutic drug (injection drug) for preventing infection. Moreover, it can be used as a spray for sterilization of Ebola virus (preventive spray). In addition, an Ebola virus preventive mask can be obtained by supporting the antibody on the mask.
  • Ebola virus can be neutralized by spraying it on infection prevention clothes and air conditioner filters together with a binder. Moreover, since the obtained antibody binds to Ebola virus, it can be used as a test kit. It can also be used as a spray or spray.
  • a disinfectant can be composed of alcohol, ostrich antibodies, preservatives, stabilizers, and water.
  • alcohol ethyl alcohol, 2-propanol (isopropyl alcohol) or the like can be preferably used.
  • paraoxin benzoates such as methylparaben, ethylparaben, propylparaben, and benzylparaben can be suitably used.
  • disaccharides such as sucrose and trehalose can be used as a stabilizer for stabilizing the antibody in the solvent.
  • the content ratio of the ostrich antibody is 0.01 to 1.0% by mass (preferably 0.02 to 0.5% by mass, more preferably 0.05 to 0.1% by mass).
  • the stabilizer is 0.1 to 10% by mass (preferably 0.2 to 5% by mass, more preferably 0.3 to 2% by mass), the preservative 2 to 8% by mass, and the rest is water or concentration. It can be suitably composed of 60 to 80% or less of alcohol water.
  • “ ⁇ ” represents the following.
  • This disinfectant can also be used by spraying with a sprayer or a sprayer. Therefore, it may be called a spray or a spray.
  • an air conditioner filter that prevents invasion of Ebola virus can be obtained by supporting an ostrich antibody and a binder on a moisturizing nonwoven fabric or the like.
  • the antibody cannot bind to the antigen unless it is in a solvent. Therefore, it can bind to Ebola virus in an environment where moisture exists.
  • a test kit for testing the presence of Ebola virus can be constructed using this ostrich antibody.
  • the materials used in the ELISA method and the sandwich ELISA method introduced in Examples described later can constitute a test kit.
  • the test object of this test kit may be a sample from which a place considered to be contaminated is wiped off or human blood.
  • the ostrich antibody has a high reactivity to the antigen, and the presence of Ebola virus can be examined even in a small amount of sample.
  • Example 1 ⁇ Antigen> As the antigen, a recombinant protein produced in silkworm cells by incorporating the Glyprotein gene (Met-Asp637) of Ebola virus (subtype Sudan, strain Gul) into a baculovirus vector was used. This recombinant protein is hereinafter referred to as “Sudan Ebola protein”.
  • ⁇ Method for producing antibody> Mature female birds (ostrich, chicken, quail) were used. Sudan Ebola protein solution (protein amount 100 ⁇ g) was mixed with 0.2 mL of Freund's complete adjuvant, and each of the five ostriches was immunized for the first time. The antigen was also individually inoculated into 5 chickens and 5 quails. In other words, ostriches, chickens and quails were inoculated with the same amount of antigen (Sudan Ebola protein). After the first immunization, each bird was boosted with a mixture of 50 ⁇ g of antigen and Freund's incomplete adjuvant at 2 and 4 weeks.
  • the egg yolk antibody (IgY) was purified from the egg yolk of each bird obtained 8 weeks after the first immunization. Specifically, first, the egg yolk of the obtained egg was subjected to 5 times amount of TBS (20 mM Tris-HCl, 0.15 M NaCl, 0.5% NaN 3 ) and the same amount of 10% dextran sulfate / TBS. The mixture was further stirred for 20 minutes.
  • Antibodies obtained from ostrich, chicken and quail eggs are anti-Sudan Ebola ostrich antibody, anti-Sudan Ebola chicken antibody and anti-Sudan Ebola antibody, respectively. These antibodies are polyclonal antibodies, and it is virtually impossible to specify the structure of the antibody paratope (binding to the epitope of the antigen).
  • ⁇ ELISA method> The reactivity of antibodies obtained from each egg yolk to Sudan Ebola protein was verified by ELISA. Specifically, first, 10 ⁇ g of Sudan Ebola protein was separately solidified in each hole of a 96-well ELISA plate (4 hours at room temperature).
  • an anti-Ebola ostrich antibody (a mixture of antibodies from egg yolks obtained from five ostriches each), an anti-Ebola chicken antibody (a mixture of antibodies from egg yolks obtained from five chickens each), and an anti-ebola antibody (5 each)
  • a serial dilution of a mixture of antibodies from egg yolk obtained from quail of feathers) (stock solution is 2 mg / mL) is dropped into each hole, reacted at room temperature for 1 hour, washed, and then HRP-labeled secondary antibody for each antibody is added. The reaction was allowed to proceed for 1 hour at room temperature.
  • Example 2 ⁇ Antigen>
  • a recombinant protein produced in silkworm cells by incorporating the Glyprotein gene (full length lacking the transmembrane region) of Ebola virus (Subtype Zaire) into a baculovirus vector was used as the antigen.
  • This recombinant protein is hereinafter referred to as “Zaire Ebola protein”.
  • ⁇ Antigen production method Mature female birds (ostrich, chicken, quail) were used. Zaire Ebola protein solution (protein amount 100 ⁇ g) was mixed with 0.2 mL of Freund's complete adjuvant, and each of the five ostriches was immunized for the first time. This antigen was also inoculated into 5 chickens and 5 quails. Ostriches, chickens and quails have been vaccinated with the same amount of antigen.
  • each bird was boosted with a mixture of 50 ⁇ g of antigen and Freund's incomplete adjuvant at 2 and 4 weeks.
  • the egg yolk antibody (IgY) was purified from the egg yolk of each bird obtained 8 weeks after the first immunization.
  • Egg yolk antibodies obtained from ostrich, chicken, and quail egg yolk are “anti-Zaire ebola ostrich antibody”, “anti-Zaire ebony chicken antibody”, and “anti-Zaire ebola antibody”. These antibodies are polyclonal antibodies, and it is virtually impossible to specify the structure of the antibody paratope (binding to the epitope of the antigen).
  • ⁇ ELISA method> The reactivity of the obtained egg yolk antibody to Zaire Ebola protein was verified by ELISA. 10 ⁇ g of Zaire Ebola protein was separately solidified in each well of a 96-well ELISA plate (4 hours at room temperature).
  • anti-Zaire ebola ostrich antibody (mixture of antibodies from egg yolk obtained from each of five ostriches)
  • anti-Zaire ebony chicken antibody mixed with antibodies from egg yolk obtained from each of five chicks
  • anti-Zaire ebola Serial dilutions of antibody (mixture of antibodies from egg yolk obtained from 5 quails each) (stock solution is 2 mg / mL) are dropped into each well, reacted at room temperature for 1 hour, washed, and then labeled with HRP for each antibody The secondary antibody was reacted at room temperature for 1 hour.
  • Example 3 Next, the binding of each of the anti-Sudan Ebola ostrich antibody and anti-Zaire Ebola ostrich antibody to each antigen was confirmed by sandwich ELISA.
  • the primary antibody an antibody obtained by immunizing mice with Sudan Ebola protein and Zaire Ebola protein was used. They are called anti-Sudan Ebola mouse antibody and anti-Zaire Ebola mouse antibody, respectively.
  • antibodies obtained by immunizing rabbits with Sudan Ebola protein and Zaire Ebola protein were used as secondary antibodies.
  • the secondary antibody is attached with an HRP label. They are called HRP-labeled anti-Sudan Ebola rabbit antibody and HRP-labeled anti-Zaire Ebola rabbit antibody, respectively.
  • the sandwich ELISA method was performed according to a formula. Briefly, the primary antibody was first immobilized on an ELISA plate, and then the antigen was bound to the primary antibody. Of course, the Sudan Ebola protein was bound to the anti-Sudan Ebola mouse antibody, and the Zaire Ebola protein was bound to the anti-Zaire Ebola mouse antibody. And each ELISA plate was subjected to blocking treatment.
  • Example 2 2 ⁇ g of the anti-Sudan Ebola ostrich antibody solution 50 ⁇ L (concentration 10 mg / ml) prepared in Example 1 was added to the plate on which the Sudan Ebola protein was immobilized on the primary antibody, and reacted at 37 ° C. for 0, 10, 20, and 30 minutes.
  • Example 2 2 ⁇ g of the anti-Zaire Ebola ostrich antibody 50 ⁇ L (concentration 10 mg / ml) prepared in Example 2 was added to the plate on which the Zaire Ebola protein was immobilized on the primary antibody, and reacted at 37 ° C. for 0, 10, 20, and 30 minutes. . Then, after adding a secondary antibody to each plate and making it react for 1 hour, the light absorbency of each plate was measured.
  • FIGS. 1 (a) and 1 (b) Results are shown in FIGS. 1 (a) and 1 (b).
  • FIG. 1 (a) shows the case where the antigen is a Sudan Ebola protein
  • FIG. 1 (b) shows the case of a Zaire Ebola protein.
  • the horizontal axis represents the reaction time (min) of the ostrich antibody and the antigen
  • the vertical axis represents the amount of antigen to which the ostrich antibody did not bind (relative value with the absorbance value at 0 minutes of reaction being 100).
  • each value is represented by an average value of measurement values at 3 wells.
  • the numbers in the graph indicate the antigen amount (%), respectively.
  • the reaction time of 0 min is the absorbance when no ostrich antibody is added. This shows the amount of each antigen immobilized on the primary antibody.
  • the reaction time between the ostrich antibody and each antigen is lengthened, the ostrich antibody binds to each antigen immobilized on the primary antibody. Then, since the secondary antibody masks the antigen to be bound by the ostrich antibody, the number of binding sites is reduced. Therefore, if the absorbance decreases, the amount of antigen not bound to the ostrich antibody decreases.
  • Ebola virus can be masked by the anti-Sudan Ebola ostrich antibody and anti-Zaire Ebola ostrich antibody produced by ostrich.
  • each antibody can suppress infection of virus cells.
  • Ebola virus is known to pass through the immune system unlike other viruses. Therefore, a polyclonal antibody that can bind to various sites is more effective for masking than a monoclonal antibody that binds to a single epitope.
  • Example 4 The following disinfectant was prepared using the anti-Sudan Ebola ostrich antibody prepared in Example 1 and the anti-Zaire Ebola ostrich antibody prepared in Example 2.
  • the “ostrich antibody” represents either an anti-Sudan Ebola ostrich antibody or an anti-Zaire Ebola ostrich antibody.
  • Ostrich antibody solution 0.5% by mass 95% by weight of water Parapen 0.5% by mass Sucrose 4% by mass
  • the ostrich antibody solution is a liquid having a protein concentration of 15 mg / mL.
  • the ostrich antibody corresponds to 0.075% by mass.
  • This disinfectant can suppress the infectious activity of Ebola virus by spraying it on the place where the body fluid of Ebola patient is scattered by spray or sprayer.
  • Example 5 A disinfectant having the following composition was prepared in the same manner as in Example 4.
  • the “ostrich antibody” represents either an anti-Sudan Ebola ostrich antibody or an anti-Zaire Ebola ostrich antibody.
  • Ostrich antibody solution 0.5% by mass Alcohol 65% by mass 30% by weight of water Parapen 0.5% by mass Sucrose 4% by mass
  • the ostrich antibody solution is a liquid having a protein concentration of 15 mg / mL.
  • the ostrich antibody corresponds to 0.075% by mass.
  • Ebola virus is an enveloped virus, so alcohol is effective as a disinfectant.
  • Ostrich antibodies are highly resistant to alcohol and do not denature in alcohol. Therefore, the disinfection can be further enhanced by mixing the alcohol water currently used.
  • the antibody according to the present invention is considered to specifically bind to Ebola virus. Therefore, it can be suitably used for infection prevention / treatment drugs (injection) for Ebola virus, Ebola virus sterilization spray, preventive mask, air conditioner filter and spray, Ebola virus infection prevention and epidemic prevention clothing, etc. it can.

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Abstract

In order to safely disinfect a human body from Ebola virus, the use of an Ebola virus antibody is thought to be effective. However, mass-producing Ebola virus antibodies requires facilities having high safety standards. The present invention provides a method for producing an Ebola virus antibody characterized by including a step for immunizing a female bird with an Ebola virus recombinant protein as the antigen, and a step for obtaining an antibody from the yolk of an egg laid by the female bird. This method uses a recombinant protein, and as a result, there are no concerns about infection. Consequently, it is possible to produce an antibody in a normal breeding facility, and furthermore, the produced antibody is expected to neutralize Ebola virus.

Description

エボラウイルスに対する抗体および抗体の製造方法Antibody against Ebola virus and method for producing antibody
　本発明はエボラウイルスを認識する抗体およびその製造に関するものであり、特にダチョウによって産生された抗体に係るものである。 The present invention relates to an antibody that recognizes Ebola virus and its production, and particularly relates to an antibody produced by an ostrich.
　エボラウイルスは、ヒトに対して危篤的な臨床症状を発生させるウイルスとして知られている。また、感染力も高く、認証されたワクチン等もない。そのため、バイオセーフティーレベルでは最高レベルの４に指定され、高度な安全対策を施した施設でしか扱うことができない。 Ebola virus is known as a virus that causes critical clinical symptoms in humans. In addition, it is highly infectious and there is no certified vaccine. For this reason, the biosafety level is designated as the highest level of 4 and can only be handled by facilities that have advanced safety measures.
　エボラウイルスを予防するためには、身の回りの消毒が重要となる。現在エボラウイルスに対する消毒は、次亜塩素酸ナトリウムやジクロルイソシアヌール酸ナトリウムが利用されている。またエボラウイルスはエンベロープを有するので消毒にはアルコールを利用することができる。しかし、エボラウイルスは感染力が非常に強いので、完全な消毒となると、煮沸やホルマリン燻蒸といった方法が必要とされている。 In order to prevent Ebola virus, disinfection around us is important. Currently, sodium hypochlorite and sodium dichloroisocyanurate are used for disinfection against Ebola virus. Also, since Ebola virus has an envelope, alcohol can be used for disinfection. However, since Ebola virus is very infectious, methods such as boiling and formalin fumigation are required when it is completely disinfected.
　これらの方法は、効果はあると考えられるが、人体に対して直接もしくは間接に接する部分に使用するのは、好ましいとは言えない。人体に対してより安全で、手軽に使え、感染抑制効果を有する消毒法が求められている。 These methods are considered to be effective, but it is not preferable to use them on the part that directly or indirectly contacts the human body. There is a need for a disinfection method that is safer for the human body, easier to use, and has an infection control effect.
　ところで、近年ウイルスに対して結合する抗体を利用することで、ウイルスを無力化することが行われている（特許文献１）。そこで、エボラウイルスに対しても抗体を使って無力化できる方法の模索が考えられる。 Incidentally, in recent years, it has been carried out to neutralize viruses by using antibodies that bind to viruses (Patent Document 1). Therefore, it is conceivable to search for a method that can be neutralized with antibodies against Ebola virus.
　エボラウイルスに対する抗体の産生は、すでに報告がなされている。特許文献２には、天然もしくは遺伝子組み換えでもよいエボラウイルスの核蛋白質を哺乳類に免疫し、得られる抗体産生細胞をハイブリドーマとし、モノクロナール抗体を得る事が開示されている。 The production of antibodies against Ebola virus has already been reported. Patent Document 2 discloses that a mammal is immunized with a natural or genetically modified Ebola virus nucleoprotein, and the resulting antibody-producing cells are used as hybridomas to obtain a monoclonal antibody.
　また、特許文献３では、エボラウイルスのレストン株に対するモノクロナール抗体を産生する点についての開示がある。 Patent Document 3 discloses that a monoclonal antibody against a Reston strain of Ebola virus is produced.
特開２００９－０２３９８５号公報JP 2009-023985 A 特開２００２－３０６１６４号公報JP 2002-306164 A 特開２００４－３１５３９４号公報JP 2004-315394 A
　抗体を消毒に利用しようとする場合、大量の抗体が必要となる。しかし、特許文献２や３のようにハイブリドーマからモノクロナール抗体を産生するのでは、大量の抗体を産生するのに、大規模な設備が必要となる。 When using antibodies for disinfection, a large amount of antibodies are required. However, when a monoclonal antibody is produced from a hybridoma as in Patent Documents 2 and 3, a large-scale facility is required to produce a large amount of antibody.
　また、マウスやウサギといった小動物に抗原を免疫し、その血清よりポリクロナール抗体を得るという方法がある。しかし、これらの動物から得られる血清の量はそれほど多くはない。 There is also a method of immunizing a small animal such as a mouse or rabbit with an antigen and obtaining a polyclonal antibody from the serum. However, the amount of serum obtained from these animals is not very high.
　また、比較的大量に抗体を得る方法としては、鶏に抗原を免疫し、その鶏から得た鶏卵からポリクロナール抗体を得る方法がある。しかし、鶏も個体が異なると免疫力も異なり、均質な抗体を大量に産生するのは無理がある。 Further, as a method for obtaining antibodies in a relatively large amount, there is a method in which a chicken is immunized with an antigen and a polyclonal antibody is obtained from a chicken egg obtained from the chicken. However, different chickens have different immunity, making it impossible to produce large amounts of homogeneous antibodies.
　また、扱いが非常に厳しく規制されているエボラウイルスをそのまま抗原として扱うとすれば、抗体の製造場所もバイオセーフティーレベル４の規定を満足する施設が必要となる。しかし、抗体の製造場所にそのような大規模な設備を準備することは現実的ではない。 Also, if Ebola virus, whose handling is very strictly regulated, is treated as an antigen as it is, an antibody manufacturing site will need a facility that satisfies the requirements of biosafety level 4. However, it is not practical to prepare such a large-scale facility at the antibody production site.
　さらに、他の多くのウイルスと異なり、生体の防御機構をすり抜けるとされているエボラウイルスには、単一のエピトープに対するモノクロナール抗体ではなく、さまざまなエピトープに結合可能なポリクロナール抗体が非常に有効である。 Furthermore, unlike many other viruses, Ebola virus, which is supposed to pass through the defense mechanism of the living body, is highly effective against polyclonal antibodies that can bind to various epitopes rather than monoclonal antibodies against a single epitope. is there.
　本発明は以上のような課題に鑑み想到されたものであり、エボラウイルスに結合する抗体を大量に提供する製造方法を提供するものである。より具体的に本発明に係る抗体の製造方法は、エボラウイルスのリコンビナント蛋白質を抗原として雌性鳥類に免疫する工程と、前記雌性鳥類が産卵した卵の卵黄から抗体を得る工程を含むことを特徴とする。 The present invention has been conceived in view of the above problems, and provides a production method for providing a large amount of antibodies that bind to Ebola virus. More specifically, the method for producing an antibody according to the present invention includes a step of immunizing a female bird using an Ebola virus recombinant protein as an antigen, and a step of obtaining an antibody from the egg yolk of the egg laid by the female bird, To do.
　本発明に係る抗エボラウイルス抗体の製造方法では、ダチョウを含む鳥類の卵から抗体を得るため、一度に大量で均質な抗体を得ることが出来る。また、抗原としてリコンビナント蛋白質を用いるので、天然のエボラウイルスを用いるより遥かに安全に抗体を製造することができる。 In the method for producing an anti-Ebola virus antibody according to the present invention, an antibody is obtained from an avian egg containing an ostrich, and therefore a large amount of homogeneous antibody can be obtained at a time. In addition, since a recombinant protein is used as an antigen, antibodies can be produced much more safely than when natural Ebola virus is used.
エボラウイルス抗原に対するダチョウ抗体の結合性を示すグラフである。It is a graph which shows the binding property of an ostrich antibody with respect to an Ebola virus antigen.
　以下本発明に係る抗体について説明する。なお、以下の説明は本発明の一実施形態を示すものであり、本発明の趣旨を逸脱しない範囲で、以下の実施形態および実施例は改変されてもよい。 Hereinafter, the antibody according to the present invention will be described. The following description shows one embodiment of the present invention, and the following embodiment and examples may be modified without departing from the spirit of the present invention.
　本発明は遺伝子組み換え技術を用いて作製されたエボラウイルスのリコンビナント蛋白質を抗原として用いる。リコンビナント蛋白質を抗原とすることで、感染の心配がない。したがって、特に高度な安全設備を設けることなく抗体を製造することができる。抗原として用いることのできる蛋白質は、核蛋白質（ＮＰ）、糖蛋白質（ＧＰ）などが好適に利用できる。 The present invention uses an Ebola virus recombinant protein produced by genetic recombination technology as an antigen. By using the recombinant protein as an antigen, there is no worry of infection. Therefore, the antibody can be produced without providing particularly high safety equipment. As a protein that can be used as an antigen, nucleoprotein (NP), glycoprotein (GP) and the like can be suitably used.
　また、利用できるエボラウイルスの種類は特に限定されない。ザイールエボラウイルス、スーダンエボラウイルス、コートジボアールエボラウイルス、レストンエボラウイルスといった現在知られているエボラウイルスを用いることができる。なお、レストンエボラウイルスはヒトに重篤な臨床症状を発現させた記録がない。したがって、ザイール株、スーダン株、コートジボアール株を利用するのがよい。 Moreover, the type of Ebola virus that can be used is not particularly limited. Currently known Ebola viruses such as Zaire Ebola virus, Sudan Ebola virus, Côte d'Ivoire Ebola virus, Reston Ebola virus can be used. Reston Ebola virus has no record of developing serious clinical symptoms in humans. Therefore, it is recommended to use Zaire strain, Sudan strain, and Côte d'Ivoire strain.
　雌性鳥類に対して免疫する工程では、公知の方法を利用することができる。免疫の際は、抗原とともに各種アジュバントを利用することができる。また、免疫も初回免疫の後、追加免疫してもよい。 A known method can be used in the step of immunizing female birds. In immunization, various adjuvants can be used together with the antigen. Immunization may be boosted after the initial immunization.
　免疫後の鳥類から得られた卵から抗体（ＩｇＹ）を回収する工程では、公知の方法で抗体を回収することができる。回収された抗体は、エボラウイルスのリコンビナント蛋白質に結合する。したがって、天然のエボラウイルスにも結合することが期待できる。この結合によってエボラウイルスの感染は中和される。 In the step of recovering antibody (IgY) from eggs obtained from birds after immunization, the antibody can be recovered by a known method. The recovered antibody binds to the Ebola virus recombinant protein. Therefore, it can be expected to bind to natural Ebola virus. This binding neutralizes Ebola virus infection.
　得られた抗体は、後述する実施例にも示されるように、エボラウイルスをマスキングすることができる。つまり、細胞への感染を抑制することができる。したがって、感染予防のための治療薬（注射薬）として利用することができる。また、エボラウイルスの殺菌用スプレー剤（予防用スプレー剤）として使用することができる。また、抗体をマスクに担持させることで、エボラウイルスの予防マスクとすることができる。 The obtained antibody can mask Ebola virus as shown in Examples described later. That is, the infection to a cell can be suppressed. Therefore, it can be used as a therapeutic drug (injection drug) for preventing infection. Moreover, it can be used as a spray for sterilization of Ebola virus (preventive spray). In addition, an Ebola virus preventive mask can be obtained by supporting the antibody on the mask.
　また、結合剤とともに感染予防服やエアコンのフィルタに散布することで、エボラウイルスを無力化することができる。また、得られた抗体は、エボラウイルスに結合するので、検査キットとして利用することができる。また、スプレー剤や噴霧剤としても利用することができる。 Moreover, Ebola virus can be neutralized by spraying it on infection prevention clothes and air conditioner filters together with a binder. Moreover, since the obtained antibody binds to Ebola virus, it can be used as a test kit. It can also be used as a spray or spray.
　ダチョウの抗体は、アルコール中で変性することがない。そこで、アルコール、ダチョウ抗体、防腐剤、安定剤、水で消毒剤を構成することができる。アルコールはエチルアルコール、２－プロパノール（イソプロピルアルコール）等が好適に利用できる。 Ostrich antibody is not denatured in alcohol. Thus, a disinfectant can be composed of alcohol, ostrich antibodies, preservatives, stabilizers, and water. As the alcohol, ethyl alcohol, 2-propanol (isopropyl alcohol) or the like can be preferably used.
　また、防腐剤としては、メチルパラベン、エチルパラベン、プロピルパラベン、ベンジルパラベン等の、パラオキシン安息香酸エステル類（パラペン）が好適に利用できる。また、溶媒中の抗体の安定化を図る安定剤には、スクロースやトレハロース等の二糖類を用いることができる。 Moreover, as preservatives, paraoxin benzoates (parapenes) such as methylparaben, ethylparaben, propylparaben, and benzylparaben can be suitably used. Further, disaccharides such as sucrose and trehalose can be used as a stabilizer for stabilizing the antibody in the solvent.
　また、これらの含有比率としては、ダチョウ抗体が０．０１～１．０質量％（好適には、０．０２～０．５質量％、より好適には０．０５～０．１質量％）、安定剤が０．１～１０質量％（好適には０．２～５質量％、より好適には０．３～２質量％）、防腐剤２～８質量％、残りを水若しくは濃度が６０～８０％以下のアルコール水で好適に構成することができる。なお、ここで「～」は以上、以下を表す。この消毒剤は、スプレー器や噴霧器で噴霧することでも使用することが出来る。したがって、スプレー剤や噴霧剤といってもよい。 In addition, the content ratio of the ostrich antibody is 0.01 to 1.0% by mass (preferably 0.02 to 0.5% by mass, more preferably 0.05 to 0.1% by mass). The stabilizer is 0.1 to 10% by mass (preferably 0.2 to 5% by mass, more preferably 0.3 to 2% by mass), the preservative 2 to 8% by mass, and the rest is water or concentration. It can be suitably composed of 60 to 80% or less of alcohol water. Here, “˜” represents the following. This disinfectant can also be used by spraying with a sprayer or a sprayer. Therefore, it may be called a spray or a spray.
　また、ダチョウ抗体を生理食塩水およびブドウ糖などと共に感染者に投与することで、発病および症状の進行を抑制することができる。 In addition, by administering an ostrich antibody together with physiological saline, glucose and the like to an infected person, the onset of disease and progression of symptoms can be suppressed.
　また、ダチョウ抗体をマスクや感染予防服に担持させることで、エボラウイルス用の予防服やマスクからの浸透を防ぐことが出来る。ここで抗体を担持させる方法は、公知の方法を利用することができる。 Also, by carrying the ostrich antibody in a mask or infection prevention clothing, it is possible to prevent penetration from the Ebola virus prevention clothing or mask. Here, a known method can be used as a method for supporting the antibody.
　また、ダチョウ抗体と結合剤を保湿性のある不織布などに担持させることでエボラウイルスの侵入を防ぐエアコンフィルタとすることができる。抗体は溶媒中でなければ、抗原に結合できない。したがって、水分が存在する環境下で好適にエボラウイルスに結合することができる。 In addition, an air conditioner filter that prevents invasion of Ebola virus can be obtained by supporting an ostrich antibody and a binder on a moisturizing nonwoven fabric or the like. The antibody cannot bind to the antigen unless it is in a solvent. Therefore, it can bind to Ebola virus in an environment where moisture exists.
　また、本ダチョウ抗体を用いてエボラウイルスの存在を検査する検査キットを構成することができる。たとえば、後述する実施例において紹介するＥＬＩＳＡ法およびサンドイッチＥＬＩＳＡ法に用いた材料は検査キットを構成することができる。この検査キットの検査対象は、汚染されていると考えられる場所をふき取ったサンプルであってもよいし、ヒトの血液であってもよい。ダチョウ抗体は、抗原に対する反応性が高く、微量のサンプルであっても、エボラウイルスの存在を調べることができる。 Also, a test kit for testing the presence of Ebola virus can be constructed using this ostrich antibody. For example, the materials used in the ELISA method and the sandwich ELISA method introduced in Examples described later can constitute a test kit. The test object of this test kit may be a sample from which a place considered to be contaminated is wiped off or human blood. The ostrich antibody has a high reactivity to the antigen, and the presence of Ebola virus can be examined even in a small amount of sample.
　（実施例１）
　＜抗原＞
　抗原は、バキュロウイルスベクターにＥｂｏｌａ　ｖｉｒｕｓ（ｓｕｂｔｙｐｅ　Ｓｕｄａｎ，ｓｔｒａｉｎ　Ｇｕｌｕ）のＧｌｙｐｒｏｔｅｉｎ遺伝子（Ｍｅｔ－Ａｓｐ６３７）を組み込み、カイコ細胞で作製したリコンビナント蛋白質を用いた。このリコンビナント蛋白質を以後「スーダンエボラ蛋白質」と呼ぶ。 (Example 1)
<Antigen>
As the antigen, a recombinant protein produced in silkworm cells by incorporating the Glyprotein gene (Met-Asp637) of Ebola virus (subtype Sudan, strain Gul) into a baculovirus vector was used. This recombinant protein is hereinafter referred to as “Sudan Ebola protein”.
　＜抗体の製造方法＞
　成熟したメス鳥（ダチョウ、ニワトリ、ウズラ）を用いた。スーダンエボラ蛋白質液（蛋白量１００μｇ）をフロイントの完全アジュバント０．２ｍＬと混和し、５羽のダチョウそれぞれに初回免疫した。また、この抗原を個別に５羽のニワトリ、５羽のウズラにも接種した。つまり、ダチョウもニワトリもウズラも同量の抗原（スーダンエボラ蛋白質）を接種したことになる。初回免疫後、２週目と４週目に５０μｇの抗原とフロイントの不完全アジュバントの混和液を、各鳥に追加免疫した。 <Method for producing antibody>
Mature female birds (ostrich, chicken, quail) were used. Sudan Ebola protein solution (protein amount 100 μg) was mixed with 0.2 mL of Freund's complete adjuvant, and each of the five ostriches was immunized for the first time. The antigen was also individually inoculated into 5 chickens and 5 quails. In other words, ostriches, chickens and quails were inoculated with the same amount of antigen (Sudan Ebola protein). After the first immunization, each bird was boosted with a mixture of 50 μg of antigen and Freund's incomplete adjuvant at 2 and 4 weeks.
　初回免疫後８週目に得られた各鳥からの卵の卵黄より卵黄抗体（ＩｇＹ）を精製した。具体的には、まず、得られた卵の卵黄に５倍量のＴＢＳ（２０ｍＭのＴｒｉｓ－ＨＣｌ、０．１５ＭのＮａＣｌ、０．５％ＮａＮ３）と同量の１０％デキストラン硫酸／ＴＢＳを加え２０分攪拌した。 The egg yolk antibody (IgY) was purified from the egg yolk of each bird obtained 8 weeks after the first immunization. Specifically, first, the egg yolk of the obtained egg was subjected to 5 times amount of TBS (20 mM Tris-HCl, 0.15 M NaCl, 0.5% NaN 3 ) and the same amount of 10% dextran sulfate / TBS. The mixture was further stirred for 20 minutes.
　次に１ＭのＣａＣｌ２／ＴＢＳを卵黄と同量加え攪拌し、１２時間静置した。その後、１５０００ｒｐｍで２０分遠心し上清を回収した。そして、最終濃度が４０％になるように硫酸アンモニウムを加え４℃で１２時間静置した。 Next, 1M CaCl 2 / TBS was added in the same amount as egg yolk, stirred and allowed to stand for 12 hours. Subsequently, the supernatant was collected by centrifugation at 15000 rpm for 20 minutes. And ammonium sulfate was added so that a final concentration might be 40%, and it left still at 4 degreeC for 12 hours.
　１２時間の静置後、１５０００ｒｐｍで２０分遠心し、沈殿物を回収した。最後に、卵黄と同量のＴＢＳに再懸濁し、ＴＢＳにて透析した。以上の方法で、各卵から純度９０％の抗体（ＩｇＹ）が回収できた。ダチョウ、ニワトリ、ウズラの各卵から得られた抗体は、それぞれ抗スーダンエボラダチョウ抗体、抗スーダンエボラニワトリ抗体、抗スーダンエボラウズラ抗体である。なお、これらの抗体は、ポリクロナール抗体であり、抗体のパラトープ（抗原のエピトープに結合する）部分の構造を特定するのは、実質的に不可能である。 After standing for 12 hours, the precipitate was collected by centrifugation at 15000 rpm for 20 minutes. Finally, it was resuspended in the same amount of TBS as egg yolk and dialyzed with TBS. By the above method, 90% purity antibody (IgY) could be recovered from each egg. Antibodies obtained from ostrich, chicken and quail eggs are anti-Sudan Ebola ostrich antibody, anti-Sudan Ebola chicken antibody and anti-Sudan Ebola antibody, respectively. These antibodies are polyclonal antibodies, and it is virtually impossible to specify the structure of the antibody paratope (binding to the epitope of the antigen).
　＜ＥＬＩＳＡ法＞
　各卵黄から得られた抗体のスーダンエボラ蛋白質に対する反応性はＥＬＩＳＡにより検証した。具体的には、まず９６穴ＥＬＩＳＡプレートの各穴にスーダンエボラ蛋白質をそれぞれ１０μｇを別々に固層化した（室温で４時間）。 <ELISA method>
The reactivity of antibodies obtained from each egg yolk to Sudan Ebola protein was verified by ELISA. Specifically, first, 10 μg of Sudan Ebola protein was separately solidified in each hole of a 96-well ELISA plate (4 hours at room temperature).
　その後、抗エボラダチョウ抗体（各５羽のダチョウから得た卵黄からの抗体の混合物）、抗エボラニワトリ抗体（各５羽のニワトリから得た卵黄からの抗体の混合物）、抗エボラウズラ抗体（各５羽のウズラから得た卵黄からの抗体の混合物）の段階希釈液（原液は２ｍｇ／ｍＬ）を各穴に滴下し、室温で１時間反応させ、洗浄後、各抗体に対するＨＲＰ標識２次抗体を室温で１時間反応させた。 Thereafter, an anti-Ebola ostrich antibody (a mixture of antibodies from egg yolks obtained from five ostriches each), an anti-Ebola chicken antibody (a mixture of antibodies from egg yolks obtained from five chickens each), and an anti-ebola antibody (5 each) A serial dilution of a mixture of antibodies from egg yolk obtained from quail of feathers) (stock solution is 2 mg / mL) is dropped into each hole, reacted at room temperature for 1 hour, washed, and then HRP-labeled secondary antibody for each antibody is added. The reaction was allowed to proceed for 1 hour at room temperature.
　十分な洗浄後、ペルオキシダーゼ用発色キット（Ｓ－Ｂｉｏ　ＳＵＭＩＬＯＮ）を用いてプレートリーダーにて吸光度（４５０ｎｍ）を測定した。免疫前の各鳥種の卵黄抗体の２倍以上の吸光度値を示す最大希釈倍率をＥＬＩＳＡ値として示した。結果を表１に示す。表１中の「ダチョウ」、「ニワトリ」、「ウズラ」は、それぞれ「抗スーダンエボラダチョウ抗体」、「抗スーダンエボラニワトリ抗体」、「抗スーダンエボラウズラ抗体」である。抗原の「Ｅｂｏｌａ　ｖｉｒｕｓ　（ｓｕｂｔｙｐｅ　Ｓｕｄａｎ，ｓｔｒａｉｎ　Ｇｕｌｕ）　Ｇｌｙｐｒｏｔｅｉｎ」はスーダンエボラ蛋白質を表す。 After sufficient washing, absorbance (450 nm) was measured with a plate reader using a peroxidase coloring kit (S-Bio SUMILON). The maximum dilution factor showing the absorbance value of 2 times or more of the yolk antibody of each bird species before immunization was shown as the ELISA value. The results are shown in Table 1. In Table 1, "ostrich", "chicken", and "quail" are "anti-Sudan Ebola ostrich antibody", "anti-Sudan Ebola chick antibody", and "anti-Sudan Ebola antibody", respectively. The antigen “Ebola virus (subtype Sudan, strain Gulu) Glyprotein” represents a Sudan Ebola protein.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　スーダンエボラ蛋白質を免疫することにより、ダチョウ、ニワトリ、ウズラに高感度の卵黄抗体が作製されることが判明した。特に、各鳥種類には同量の抗原を免疫したのにもかかわらず、巨大なダチョウが最も反応性が高い抗体が産生されていた。これは、ダチョウを使うことで、少量の抗原でも高感度の抗体が産生できることを示している。 It was found that immunization with Sudan Ebola protein produced highly sensitive egg yolk antibodies against ostriches, chickens and quails. In particular, despite the immunization of the same amount of antigen for each bird species, giant ostriches produced the most reactive antibodies. This shows that by using ostriches, highly sensitive antibodies can be produced even with a small amount of antigen.
　（実施例２）
　＜抗原＞
　抗原は、バキュロウイルスベクターにＥｂｏｌａ　ｖｉｒｕｓ　（Ｓｕｂｔｙｐｅ　Ｚａｉｒｅ）のＧｌｙｐｒｏｔｅｉｎ遺伝子（膜貫通領域欠く全長）を組み込み、カイコ細胞で作製したリコンビナント蛋白質を用いた。このリコンビナント蛋白質を以後「ザイールエボラ蛋白質」と呼ぶ。 (Example 2)
<Antigen>
As the antigen, a recombinant protein produced in silkworm cells by incorporating the Glyprotein gene (full length lacking the transmembrane region) of Ebola virus (Subtype Zaire) into a baculovirus vector was used. This recombinant protein is hereinafter referred to as “Zaire Ebola protein”.
　＜抗原製造方法＞
　成熟したメス鳥（ダチョウ、ニワトリ、ウズラ）を用いた。ザイールエボラ蛋白質液（蛋白量１００μｇ）をフロイントの完全アジュバント０．２ｍＬと混和し、５羽のダチョウそれぞれに初回免疫した。この抗原を、５羽のニワトリ、５羽のウズラにも接種した。ダチョウもニワトリもウズラも同量の抗原を接種したことになる。 <Antigen production method>
Mature female birds (ostrich, chicken, quail) were used. Zaire Ebola protein solution (protein amount 100 μg) was mixed with 0.2 mL of Freund's complete adjuvant, and each of the five ostriches was immunized for the first time. This antigen was also inoculated into 5 chickens and 5 quails. Ostriches, chickens and quails have been vaccinated with the same amount of antigen.
　初回免疫後、２週目と４週目に５０μｇの抗原とフロイントの不完全アジュバントの混和液を、各鳥に追加免疫した。初回免疫後８週目に得られた各鳥からの卵の卵黄より卵黄抗体（ＩｇＹ）を精製した。ダチョウ、ニワトリ、ウズラの各卵黄から得た卵黄抗体は、「抗ザイールエボラダチョウ抗体」、「抗ザイールエボラニワトリ抗体」、「抗ザイールエボラウズラ抗体」である。なお、これらの抗体は、ポリクロナール抗体であり、抗体のパラトープ（抗原のエピトープに結合する）部分の構造を特定するのは、実質的に不可能
である。 After the first immunization, each bird was boosted with a mixture of 50 μg of antigen and Freund's incomplete adjuvant at 2 and 4 weeks. The egg yolk antibody (IgY) was purified from the egg yolk of each bird obtained 8 weeks after the first immunization. Egg yolk antibodies obtained from ostrich, chicken, and quail egg yolk are “anti-Zaire ebola ostrich antibody”, “anti-Zaire ebony chicken antibody”, and “anti-Zaire ebola antibody”. These antibodies are polyclonal antibodies, and it is virtually impossible to specify the structure of the antibody paratope (binding to the epitope of the antigen).
　＜ＥＬＩＳＡ法＞
　得られた卵黄抗体のザイールエボラ蛋白質に対する反応性をＥＬＩＳＡにより検証した。９６穴ＥＬＩＳＡプレートの各穴にザイールエボラ蛋白質をそれぞれ１０μｇを別々に固層化した（室温で４時間）。その後、抗ザイールエボラダチョウ抗体（各５羽のダチョウから得た卵黄からの抗体の混合物）、抗ザイールエボラニワトリ抗体（各５羽のニワトリから得た卵黄からの抗体の混合物）、抗ザイールエボラウズラ抗体（各５羽のウズラから得た卵黄からの抗体の混合物）の段階希釈液（原液は２ｍｇ／ｍＬ）を各穴に滴下し、室温で１時間反応させ、洗浄後、各抗体に対するＨＲＰ標識２次抗体を室温で１時間反応させた。 <ELISA method>
The reactivity of the obtained egg yolk antibody to Zaire Ebola protein was verified by ELISA. 10 μg of Zaire Ebola protein was separately solidified in each well of a 96-well ELISA plate (4 hours at room temperature). Thereafter, anti-Zaire ebola ostrich antibody (mixture of antibodies from egg yolk obtained from each of five ostriches), anti-Zaire ebony chicken antibody (mixture of antibodies from egg yolk obtained from each of five chicks), anti-Zaire ebola Serial dilutions of antibody (mixture of antibodies from egg yolk obtained from 5 quails each) (stock solution is 2 mg / mL) are dropped into each well, reacted at room temperature for 1 hour, washed, and then labeled with HRP for each antibody The secondary antibody was reacted at room temperature for 1 hour.
　十分な洗浄後、ペルオキシダーゼ用発色キット（Ｓ－Ｂｉｏ　ＳＵＭＩＬＯＮ）を用いてプレートリーダーにて吸光度（４５０ｎｍ）を測定した。免疫前の各鳥種の卵黄抗体の２倍以上の吸光度値を示す最大希釈倍率をＥＬＩＳＡ値として表２に示した。なお、表２中「ダチョウ」、「ニワトリ」、「ウズラ」は、それぞれ「抗ザイールエボラダチョウ抗体」、「抗ザイールエボラニワトリ抗体」、「抗ザイールエボラウズラ抗体」である。また、抗原の「Ｅｂｏｌａ　ｖｉｒｕｓ　（Ｚａｉｒｅ種）Ｇｌｙｐｒｏｔｅｉｎ」は、ザイールエボラ蛋白質である。 After sufficient washing, absorbance (450 nm) was measured with a plate reader using a peroxidase coloring kit (S-Bio SUMILON). Table 2 shows the maximum dilution factor showing an absorbance value of 2 times or more of the yolk antibody of each bird species before immunization as an ELISA value. In Table 2, “ostrich”, “chicken”, and “quail” are “anti-Zaire ebola ostrich antibody”, “anti-Zeil ebony chicken antibody”, and “anti-Zeil ebola antibody”, respectively. The antigen “Ebola virus (Zaire species) Glyprotein” is a Zaire Ebola protein.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　ザイールエボラ蛋白質を免疫することにより、ダチョウ、ニワトリ、ウズラに高感度の卵黄抗体が作製されることが判明した。特に、各鳥種類には同量の抗原を免疫したのにもかかわらず、巨大なダチョウが最も反応性が高い抗体が産生されている（少量の抗原でも高感度の抗体が産生）。 It was found that highly sensitive egg yolk antibodies were produced against ostriches, chickens and quails by immunizing Zaire Ebola protein. In particular, despite the immunization with the same amount of antigen for each type of bird, the giant ostrich produces the most reactive antibody (a small amount of antigen produces a highly sensitive antibody).
　（実施例３）
　次に抗スーダンエボラダチョウ抗体と、抗ザイールエボラダチョウ抗体の各抗原に対する結合性をサンドイッチＥＬＩＳＡ法により確認した。一次抗体は、スーダンエボラ蛋白質およびザイールエボラ蛋白質をマウスに免疫することで得られた抗体を用いた。それぞれ抗スーダンエボラマウス抗体、抗ザイールエボラマウス抗体と呼ぶ。 (Example 3)
Next, the binding of each of the anti-Sudan Ebola ostrich antibody and anti-Zaire Ebola ostrich antibody to each antigen was confirmed by sandwich ELISA. As the primary antibody, an antibody obtained by immunizing mice with Sudan Ebola protein and Zaire Ebola protein was used. They are called anti-Sudan Ebola mouse antibody and anti-Zaire Ebola mouse antibody, respectively.
　また、２次抗体として、同様にスーダンエボラ蛋白質およびザイールエボラ蛋白質をウサギに免疫することで得られた抗体を用いた。なお、２次抗体には、ＨＲＰ標識が取り付けてある。それぞれＨＲＰ標識抗スーダンエボラウサギ抗体、ＨＲＰ標識抗ザイールエボラウサギ抗体と呼ぶ。 Similarly, antibodies obtained by immunizing rabbits with Sudan Ebola protein and Zaire Ebola protein were used as secondary antibodies. The secondary antibody is attached with an HRP label. They are called HRP-labeled anti-Sudan Ebola rabbit antibody and HRP-labeled anti-Zaire Ebola rabbit antibody, respectively.
　サンドイッチＥＬＩＳＡ法は定式によって行った。簡単に手順を示すと、まず、ＥＬＩＳＡプレート上に、一次抗体を固定し、次に抗原を一次抗体に結合させた。もちろん、抗スーダンエボラマウス抗体には、スーダンエボラ蛋白質を結合させ、抗ザイールエボラマウス抗体にはザイールエボラ蛋白質を結合させた。そして、各ＥＬＩＳＡプレートにブロッキング処理を施した。 The sandwich ELISA method was performed according to a formula. Briefly, the primary antibody was first immobilized on an ELISA plate, and then the antigen was bound to the primary antibody. Of course, the Sudan Ebola protein was bound to the anti-Sudan Ebola mouse antibody, and the Zaire Ebola protein was bound to the anti-Zaire Ebola mouse antibody. And each ELISA plate was subjected to blocking treatment.
　実施例１で作成した抗スーダンエボラダチョウ抗体液５０μＬ（濃度１０ｍｇ／ｍｌ）をスーダンエボラ蛋白質が一次抗体に固定されたプレートに２μｇ加え、３７℃で０、１０、２０、３０分間反応させた。 2 μg of the anti-Sudan Ebola ostrich antibody solution 50 μL (concentration 10 mg / ml) prepared in Example 1 was added to the plate on which the Sudan Ebola protein was immobilized on the primary antibody, and reacted at 37 ° C. for 0, 10, 20, and 30 minutes.
　また、実施例２で作成した抗ザイールエボラダチョウ抗体５０μＬ（濃度１０ｍｇ／ｍｌ）をザイールエボラ蛋白質が一次抗体に固定されたプレートに２μｇ加え、３７℃で０、１０、２０、３０分間反応させた。その後、各プレートに２次抗体を加え１時間反応させた後、各プレートの吸光度を測定した。 Further, 2 μg of the anti-Zaire Ebola ostrich antibody 50 μL (concentration 10 mg / ml) prepared in Example 2 was added to the plate on which the Zaire Ebola protein was immobilized on the primary antibody, and reacted at 37 ° C. for 0, 10, 20, and 30 minutes. . Then, after adding a secondary antibody to each plate and making it react for 1 hour, the light absorbency of each plate was measured.
　結果を図１（ａ）（ｂ）に示す。図１を参照して、図１（ａ）は、抗原がスーダンエボラ蛋白質である場合を示し、図１（ｂ）は、ザイールエボラ蛋白質の場合を示す。 Results are shown in FIGS. 1 (a) and 1 (b). Referring to FIG. 1, FIG. 1 (a) shows the case where the antigen is a Sudan Ebola protein, and FIG. 1 (b) shows the case of a Zaire Ebola protein.
　それぞれのグラフでは横軸がダチョウ抗体と抗原の反応時間（ｍｉｎ）であり、縦軸は、ダチョウ抗体が結合しなかった抗原量（反応０分時の吸光度の値を１００とする相対値で表記した。）である。なお、値はそれぞれ３ｗｅｌｌでの測定値の平均値で表している。また、グラフの中の数字はそれぞれ、抗原量の数値（％）を示したものである。 In each graph, the horizontal axis represents the reaction time (min) of the ostrich antibody and the antigen, and the vertical axis represents the amount of antigen to which the ostrich antibody did not bind (relative value with the absorbance value at 0 minutes of reaction being 100). ). In addition, each value is represented by an average value of measurement values at 3 wells. In addition, the numbers in the graph indicate the antigen amount (%), respectively.
　反応時間０ｍｉｎとは、ダチョウ抗体を入れない状態の吸光度である。これは、１次抗体に固定された各抗原量を示している。ダチョウ抗体と各抗原との反応時間を長くしていくと、１次抗体に固定されている各抗原にダチョウ抗体が結合する。すると、２次抗体は結合する抗原をダチョウ抗体にマスキングされるため、結合場所が少なくなる。したがって、吸光度が減少すれば、ダチョウ抗体に結合していない抗原量は減少する。 The reaction time of 0 min is the absorbance when no ostrich antibody is added. This shows the amount of each antigen immobilized on the primary antibody. When the reaction time between the ostrich antibody and each antigen is lengthened, the ostrich antibody binds to each antigen immobilized on the primary antibody. Then, since the secondary antibody masks the antigen to be bound by the ostrich antibody, the number of binding sites is reduced. Therefore, if the absorbance decreases, the amount of antigen not bound to the ostrich antibody decreases.
　図１（ａ）、（ｂ）を見ると、スーダンエボラ蛋白質の場合も、ザイールエボラ蛋白質の場合も、時間の経過と共に、抗原量が減少しているのがわかる。結合時間が３０分になると、残存している抗原は５％以下になった。 Referring to FIGS. 1 (a) and 1 (b), it can be seen that the amount of antigen decreases with time in both cases of Sudan Ebola protein and Zaire Ebola protein. When the binding time was 30 minutes, the remaining antigen was 5% or less.
　以上のことより、ダチョウで作製した抗スーダンエボラダチョウ抗体および、抗ザイールエボラダチョウ抗体により、エボラウイルスのマスキングが可能となる。これによって、各抗体は、ウイルスの細胞への感染が抑制できることがわかった。 From the above, Ebola virus can be masked by the anti-Sudan Ebola ostrich antibody and anti-Zaire Ebola ostrich antibody produced by ostrich. Thus, it was found that each antibody can suppress infection of virus cells.
　特に、エボラウイルスは他のウイルスと異なり、免疫系をすり抜けることが知られている。したがって、単一のエピトープに結合するモノクロナール抗体よりも、さまざまな部位に結合することの出来るポリクロナール抗体がマスキングには有効である。 In particular, Ebola virus is known to pass through the immune system unlike other viruses. Therefore, a polyclonal antibody that can bind to various sites is more effective for masking than a monoclonal antibody that binds to a single epitope.
　（実施例４）
　実施例１で作製した抗スーダンエボラダチョウ抗体と、実施例２で作製した抗ザイールエボラダチョウ抗体を用いて、以下のような消毒剤を作製した。なお、「ダチョウ抗体」は、抗スーダンエボラダチョウ抗体若しくは抗ザイールエボラダチョウ抗体のいずれかを表す。
　ダチョウ抗体溶液　　　０．５質量％
　水　　　　　　　　　　９５質量％
　パラペン　　　　　　　０．５質量％
　スクロース　　　　　　４質量％
　なお、ダチョウ抗体溶液は１５ｍｇ／ｍＬのタンパク濃度の液体である。ダチョウ抗体は０．０７５質量％に相当する。 Example 4
The following disinfectant was prepared using the anti-Sudan Ebola ostrich antibody prepared in Example 1 and the anti-Zaire Ebola ostrich antibody prepared in Example 2. The “ostrich antibody” represents either an anti-Sudan Ebola ostrich antibody or an anti-Zaire Ebola ostrich antibody.
Ostrich antibody solution 0.5% by mass
95% by weight of water
Parapen 0.5% by mass
Sucrose 4% by mass
The ostrich antibody solution is a liquid having a protein concentration of 15 mg / mL. The ostrich antibody corresponds to 0.075% by mass.
　この消毒剤は、スプレー若しくは噴霧器でエボラ患者の体液が飛散したと考えられる箇所に噴霧することで、エボラウイルスの感染活性を抑制することが出来る。 こ の This disinfectant can suppress the infectious activity of Ebola virus by spraying it on the place where the body fluid of Ebola patient is scattered by spray or sprayer.
　現在エボラウイルスの消毒には、アルコール水や次亜塩素酸ナトリウム等が用いられている。次亜塩素酸ナトリウム等は、消毒性は高いものの、腐食性も強く、人体や食器などに直接用いることはできない。一方、宗教上の理由で消毒にアルコールを使えない場合も世界には多い。このような場合に本実施例のようなアルコールを用いないエボラウイルス用消毒薬は大変有用である。 Currently, alcoholic water and sodium hypochlorite are used to disinfect Ebola virus. Sodium hypochlorite and the like are highly disinfecting, but are highly corrosive and cannot be used directly on the human body or tableware. On the other hand, there are many cases in the world where alcohol cannot be used for disinfection for religious reasons. In such a case, the Ebola virus disinfectant that does not use alcohol as in this example is very useful.
　また、エボラウイルスの場合は、さまざまな部位に結合する抗体を含むポリクロナール抗体が大変有用である。 In the case of Ebola virus, polyclonal antibodies containing antibodies that bind to various sites are very useful.
　（実施例５）
　実施例４と同様に以下の組成の消毒剤を作製した。なお、「ダチョウ抗体」は、抗スーダンエボラダチョウ抗体若しくは抗ザイールエボラダチョウ抗体のいずれかを表す。
　ダチョウ抗体溶液　　０．５質量％
　アルコール　　　　　６５質量％
　水　　　　　　　　　３０質量％
　パラペン　　　　　　０．５質量％
　スクロース　　　　　４質量％
　なお、ダチョウ抗体溶液は１５ｍｇ／ｍＬのタンパク濃度の液体である。ダチョウ抗体は０．０７５質量％に相当する。 (Example 5)
A disinfectant having the following composition was prepared in the same manner as in Example 4. The “ostrich antibody” represents either an anti-Sudan Ebola ostrich antibody or an anti-Zaire Ebola ostrich antibody.
Ostrich antibody solution 0.5% by mass
Alcohol 65% by mass
30% by weight of water
Parapen 0.5% by mass
Sucrose 4% by mass
The ostrich antibody solution is a liquid having a protein concentration of 15 mg / mL. The ostrich antibody corresponds to 0.075% by mass.
　エボラウイルスは、エンベロープを有するウイルスなので、アルコールは消毒剤として有効である。ダチョウ抗体は、アルコールに対する耐性が強く、アルコール中でも変性しない。したがって、現在使用されているアルコール水中に混ぜて使用することで、一層消毒性を高めることができる。 Ebola virus is an enveloped virus, so alcohol is effective as a disinfectant. Ostrich antibodies are highly resistant to alcohol and do not denature in alcohol. Therefore, the disinfection can be further enhanced by mixing the alcohol water currently used.
　また、エボラウイルスの場合は、さまざまな部位に結合する抗体を含むポリクロナール抗体が大変有用である。 In the case of Ebola virus, polyclonal antibodies containing antibodies that bind to various sites are very useful.
　本発明に係る抗体は、エボラウイルスに特異的に結合すると考えられる。したがって、対エボラウイルスのための感染予防・治療薬（注射薬）、エボラウイルスの殺菌用スプレー剤、予防用マスク、エアコンフィルタおよび噴霧剤およびエボラウイルス感染予防防疫服等に好適に利用することができる。 The antibody according to the present invention is considered to specifically bind to Ebola virus. Therefore, it can be suitably used for infection prevention / treatment drugs (injection) for Ebola virus, Ebola virus sterilization spray, preventive mask, air conditioner filter and spray, Ebola virus infection prevention and epidemic prevention clothing, etc. it can.

Claims (9)

  1. 　エボラウイルスのリコンビナント蛋白質を抗原として雌性鳥類に免疫する工程と、前記雌性鳥類が産卵した卵の卵黄から抗体を得る工程を含むことを特徴とする抗エボラウイルス抗体の製造方法。 A method for producing an anti-Ebola virus antibody, comprising a step of immunizing a female bird using an Ebola virus recombinant protein as an antigen, and a step of obtaining an antibody from the egg yolk laid by the female bird.
  2. 　前記雌性鳥類がダチョウであることを特徴とする請求項１に記載された抗エボラウイルス抗体の製造方法。 The method for producing an anti-Ebola virus antibody according to claim 1, wherein the female bird is an ostrich.
  3. 　ダチョウの黄卵抗体であって、エボラウイルスのグリプロテイン遺伝子から作製したリコンビナント蛋白質に結合する抗体。 An ostrich yolk antibody that binds to a recombinant protein prepared from the Ebola virus glycoprotein gene.
  4. 　請求項３の抗体と、防腐剤と、安定剤と、水からなるエボラウイルス用消毒剤。 A disinfectant for Ebola virus comprising the antibody of claim 3, an antiseptic, a stabilizer, and water.
  5. 　請求項３の抗体を有するマスク。 A mask having the antibody of claim 3.
  6. 　請求項３の抗体を有するエアコンフィルタ。 An air conditioner filter comprising the antibody of claim 3.
  7. 　請求項３の抗体を表面に担持された感染予防服。 Infection prevention clothes carrying the antibody of claim 3 on the surface.
  8. 　請求項３の抗体を用いた検査キット。 A test kit using the antibody of claim 3.
  9. 　請求項３の抗体と生理食塩水を含むエボラウイルス用治療剤。 A therapeutic agent for Ebola virus comprising the antibody of claim 3 and physiological saline.
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