JP2002306164A - Monoclonal antibody recognizing ebola virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a monoclonal antibody specifically recognizing an Ebola virus. SOLUTION: A monoclonal antibody or its fragment recognizing the nucleoprotein of an Ebola virus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a monoclonal antibody that recognizes Ebola virus. More specifically,
The present invention provides a monoclonal antibody that recognizes a nucleoprotein of Ebola virus, a hybridoma that produces the antibody,
And a method for detecting and / or quantifying Ebola virus using the antibody.

[0002]

BACKGROUND OF THE INVENTION Ebola virus infection causes hemorrhagic fever with high mortality (World Health Org).
anization. 1997. Part One: Background informatio
n on the organisms and the disease, p. 1-3.In WHO
recommended guidelines forepidemic preparedness a
nd response: Ebola Haemorrhagic Fever (EHF)). Although the area where the Ebola virus exists is limited, the risk of transmission to humans and animals in other areas is increasing with increasing international traffic and trade. Ebola virus causes secondary human-to-human transmission in healthcare professionals and at home (Dowell, SF et al. 1999. Trans
mission of Ebola hemorrhagic fever: a study of risk
factors in family members, Kikwit, Democratic Rep
ublic of the Congo, 1995. J. Infect. Dis. 179 Supp
l. 1: S87-91; and World Health Organization. 199
7. Part One: Background information on the organi
sms and the disease, p.1-3.In WHO recommended gu
idelines for epidemic preparedness and response: E
bola Haemorrhagic Fever (EHF)), it is important to diagnose in the early stages of infection and call attention to society.

[0003] Based on genetic divergence, Ebola viruses have been classified into four subtypes called Zaire, Sudan, C コ ー ト te d'Ivoire and Reston (Feldman).
n, H. et al. 1999. Classification, Structure, and Repli
cation of Filoviruses, p.1-5. In H.? D. Klenk (e
d.), Marburg and Ebola viruses.Current Topics inM
icrobiology and Immunology. 235. Springer-Verlag,
Berlin Heidelberg; Georges-Courbot, MC et al. 1997.
Isolation and phylogenetic characterization of Ebo
la viruses causing different outbreaks in Gabon.
Emerg. Infect. Dis. 3: 59-62; and Peters, CJ and others 1
999.An introduction to Ebola: Thevirus and the di
sease. J. Infect. Dis. 179 Suppl. 1: iv-xvi). The Zaire, Sudan, and C コ ー ト te d'Ivoire strains show severe clinical symptoms in humans and monkeys, while the Reston strain only causes symptoms in monkeys (Fisher-Hoch, SP et al.
992.Pathogenic potential of filoviruses: Role of
geographic origin ofprimate host and virus strain.
J. Infect. Dis. 166: 753-763; Miller, RK et al. 199
0. Filovirus infections among persons with occupa
tional exposure to nonhuman primates. MMWR. 39: 2
66-267, 273; and Miller, RK et al. 1990. Filovirus i
nfections in animal handlers. MMWR. 39: 221). The onset of Ebola virus infection is acute, and antibody production is often absent at the onset of clinical symptoms (Baiz
e, S. et al. 1999. Defective humoral responses and ext
ensive intravascular apoptosis are associated with
fatal outcome in Ebolavirus-infected patients.Na
t. Med. 5: 423-426; and Ksiazek, TG et al. 1999. Cli
nical virology of Ebola hemorrhagic fever (EHF): vi
rus, virus antigen, and IgG and IgM antibody findi
ngs among EHF patients in Kikwit, Democratic Repub
lic of the Congo, 1995. J. Infect. Dis.179 Suppl.
1: S177-187). On the other hand, the patient's blood and tissues (liver etc.)
Virus is very high (Ksiazek, TG et al. 1
999.Clinical virology of Ebola hemorrhagic fever
(EHF): virus, virus antigen, and IgG and IgM antibo
dy findings among EHF patients in Kikwit, Democrat
ic Republic of the Congo, 1995. J. Infect. Dis. 17
9 Suppl. 1: S177-187). Thus, by detecting viral antigens rather than specific antibodies, a quick and accurate primary screen for Ebola virus infection can be performed (Peters, CJ et al. 1999. An introduction to Ebo
la: The virus and the disease.J. Infect. Dis.179
Suppl. 1: iv-xvi).

[0004] An antigen detection system for Ebola virus infection has already been reported and used (Ksiazek, TG et al. 1992. E
nzyme immunosorbent assay for Ebola virus antigens
intissues of infected primates. J. Clin. Microbi
ol. 30: 947-950). However, there is very little information on enzyme-linked immunosorbent assays (ELISAs), for example, no report on the monoclonal antibodies used in this system has been made with respect to the specificity of the molecule.

[0005]

An object of the present invention is to provide a monoclonal antibody that specifically recognizes Ebola virus. Another object of the present invention is to provide a method for specifically detecting Ebola virus in serum and organs by ELISA using the above monoclonal antibody.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have prepared a monoclonal antibody against recombinant Ebola virus nucleoprotein (rNP), and used this antibody for sandwich ELISA.
As a result of trying to detect Ebola virus nucleoprotein in a sample by the method, it was found that this antibody can detect Ebola virus nucleoprotein with high sensitivity. The present invention has been completed based on these findings.

That is, according to the present invention, there is provided a monoclonal antibody or a fragment thereof that recognizes a nucleoprotein of Ebola virus. According to a preferred embodiment of the present invention, a monoclonal antibody or a fragment thereof that recognizes the nucleoprotein of the Ebola virus Zaire strain, the Sudan strain and the Reston strain;
A monoclonal antibody or a fragment thereof that recognizes an epitope consisting of amino acids 631 to 739; a monoclonal antibody or a fragment thereof that recognizes an epitope consisting of 26 amino acids consisting of amino acids 648 to 673 of a nucleoprotein of Ebola virus Zaire strain; Accession number F
A monoclonal antibody produced by a hybridoma having ERM P-18279 or a fragment thereof is provided.

[0008] According to another aspect of the present invention, there is provided a gene encoding the above-described monoclonal antibody of the present invention or a fragment thereof. According to yet another aspect of the present invention,
A hybridoma producing the above-described monoclonal antibody of the present invention is provided. According to a preferred embodiment of the present invention, a hybridoma producing the monoclonal antibody of the present invention, obtained by fusing a mammalian immune cell immunized with an immunogen containing a nucleoprotein of an Ebola virus and a mammalian myeloma cell And accession number FERM P-
A hybridoma having 18279 is provided.

According to still another aspect of the present invention, there is provided a method for producing the monoclonal antibody of the present invention, comprising the steps of culturing the above-described hybridoma of the present invention and collecting the monoclonal antibody produced by the hybridoma. Is done.

According to still another aspect of the present invention, an immunoassay is carried out using the above-described monoclonal antibody of the present invention or a fragment thereof for detecting and / or quantifying a nucleoprotein of Ebola virus. Is provided. According to a preferred embodiment of the present invention, there is provided a method for detecting and / or quantifying a nucleoprotein of Ebola virus, comprising at least the following steps (a) and (b). (A) reacting a sample with a monoclonal antibody or a fragment thereof that recognizes the nucleoprotein of Ebola virus according to the present invention; and (b) an antigen-antibody complex formed in step (1) and a labeled antibody for detection Reacting with

According to yet another aspect of the present invention, a kit for detecting and / or quantifying a nucleoprotein of Ebola virus, comprising a monoclonal antibody recognizing the nucleoprotein of Ebola virus according to the present invention. Is provided.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments and a method of implementing the present invention will be described. 1. The monoclonal antibody of the present invention is characterized in that it recognizes the nucleoprotein of Ebola virus. The type of Ebola virus is not particularly limited, and may be any of a Zaire strain, a Sudan strain, a C コ ー ト te d'Ivoire strain, and a Reston strain. The monoclonal antibody of the present invention may be one that recognizes all of these four strains, or a part of them (for example,
Only the three types, two types or one type among the above four types may be recognized. As the monoclonal antibody of the present invention, a suitable antibody can be appropriately selected and used depending on the purpose. That is, for the purpose of general detection and quantification of Ebola virus, it is preferable to use a monoclonal antibody that recognizes all of the above four strains, and to specifically detect a specific strain. It is preferable to use a monoclonal antibody specific to a specific strain that does not cross-react with other strains.

The monoclonal antibodies of the present invention include, for example, 631 of the nucleoprotein of Ebola virus Zaire strain.
And those recognizing an epitope consisting of 26 amino acids consisting of amino acids 648 to 673 of the nucleoprotein of the Ebola virus Zaire strain. Can be An example of such a monoclonal antibody includes the monoclonal antibody 3-3D described in the examples of the present specification. Monoclonal antibody 3-3
The hybridoma producing D is deposited under accession number FERMP
Deposited on March 28, 2001 at the National Institute of Advanced Industrial Science and Technology, Institute of Biotechnology, Industrial Technology Research Institute, Patent Microorganisms Depositary Center (1-3-3 Higashi, Tsukuba, Ibaraki, Japan) .

In the present specification, the term "antibody" is used to include not only a full-length antibody but also a fragment of the antibody. That is, according to the present invention, a fragment of a monoclonal antibody that recognizes a nucleoprotein of Ebola virus is provided. Antibody fragments are preferably functional fragments, and include, for example, F (ab ′) ₂ and Fab ′. F
(Ab ') ₂ , Fab' is produced by treating immunoglobulin with a protease (for example, pepsin or papain), and is present between two H chains in the hinge region. Antibody fragments produced by digestion before and after disulfide bonds. Furthermore, when a fragment of an antibody is referred to in the present specification, it includes a protein containing an antigen-binding site derived from a gene encoding the antibody.

For example, when IgG1 is treated with papain, it is cleaved upstream of a disulfide bond existing between two H chains in the hinge region to form VL (light chain variable region) and CL.
(L chain constant region) and an H chain fragment consisting of VH (H chain variable region) and CHγ1 (γ1 region in the H chain constant region) are linked by a disulfide bond at the C-terminal region. Two antibody fragments can be produced. Each of these two homologous antibody fragments is called Fab '. When IgG is treated with pepsin, it is cleaved downstream of a disulfide bond existing between two H chains in the hinge region to produce an antibody fragment slightly larger than the two Fab's connected by the hinge region. Can be. This antibody fragment is
(Ab ') ₂ .

The monoclonal antibody of the present invention can be used as an immobilized antibody immobilized on an insoluble carrier such as a solid phase carrier, or as a labeled antibody labeled with a labeling substance. All such immobilized antibodies and labeled antibodies are within the scope of the present invention.

The term "immobilized antibody" refers to an antibody that has been carried on an insoluble carrier by physical adsorption or chemical bonding. These immobilized antibodies are used to detect, quantify, separate or purify antigens (ie, Ebola virus nucleoprotein) contained in samples (eg, body fluid samples such as plasma, culture supernatants, centrifugal supernatants, etc.). Can be used. Examples of insoluble carriers that can be used to immobilize antibodies include (1) plastics made of polystyrene resin, polycarbonate resin, silicone resin, nylon resin, and the like, and water-insoluble substances such as glass. Plates, test tubes or tubes with internal volume, beads, balls, filters,
Alternatively, affinity chromatography such as a membrane carrier and (2) a cellulose carrier, an agarose carrier, a polyacrylamide carrier, a dextran carrier, a polystyrene carrier, a polyvinyl alcohol carrier, a polyamino acid carrier or a porous silica carrier. Examples include insoluble carriers used for chromatography.

The term "labeled antibody" refers to an antibody labeled with a labeling substance, and these labeled antibodies are composed of an antigen (for example, a body fluid sample such as plasma, a culture supernatant or a centrifugal supernatant) contained in a sample. That is, it can be used for detecting or quantifying Ebola virus nucleoprotein). The labeling substance that can be used in the present invention is not particularly limited as long as the substance can be detected by binding to the antibody by physical or chemical bonding or the like. Specific examples of the labeling substance include an enzyme, a fluorescent substance, a chemiluminescent substance, biotin, avidin or a radioisotope, and more specifically, peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucosidase, and the like. Enzymes such as -s-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase, fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates , Fluorescent substances such as dansyl chloride or tetramethylrhodamine isothiocyanate, ³ H, ¹⁴ C, ¹²⁵
Radioisotopes such as I or ¹³¹ I, biotin, avidin, or chemiluminescent substances. The binding method between the labeling substance and the antibody is a glutaraldehyde method, a maleimide method,
A known method such as a pyridyl disulfide method or a periodic acid method can be used.

Here, a radioisotope and a fluorescent substance alone can give a detectable signal, but enzymes, chemiluminescent substances, biotin and avidin alone cannot give a detectable signal. Further reaction with one or more other substances produces a detectable signal. For example, in the case of an enzyme, at least a substrate is required, and various substrates are used depending on the method for measuring the enzyme activity (colorimetric method, fluorescent method, bioluminescent method, chemiluminescent method, etc.). In the case of biotin, it is common to react at least avidin or enzyme-modified avidin. If necessary, various coloring substances depending on the substrate may be used.

[0020] 2. Produces the monoclonal antibody of the invention
The present invention also relates to a hybridoma that produces a monoclonal antibody that recognizes the nucleoprotein of Ebola virus described above. The monoclonal antibody of the present invention can be produced using the hybridoma. Hereinafter, a method for producing a hybridoma that produces a monoclonal antibody that recognizes the nucleoprotein of the Ebola virus of the present invention will be described.

First, antibody-producing cells are prepared in an animal by immunizing a mammal with a nucleoprotein of Ebola virus. The type of mammal is not particularly limited,
In general, mice, rats, cows, rabbits, goats, sheep and the like can be mentioned, preferably rodents such as mice, rats and rabbits, and more preferably mice. Examples of mice include A / J strain, BALB / C strain, DBA
/ 2 system, C57BL / 6 system, C3H / He system, S
JL strain, NZB strain, and CBA / JNCrj strain mice can be mentioned. BALB / C strain mice are preferred because a myeloma-derived cell line of the same strain has been established at the time of hybridoma production.

The nucleoprotein (NP) of Ebola virus used for immunization may be a natural NP, a recombinant NP, a chemically synthesized NP, or a fragment thereof. Preferably, a recombinant NP is used. Prior to immunization, NPs may be mixed with an adjuvant to enhance the immune response. Examples of adjuvants include water-in-oil emulsions (eg, incomplete Freund's adjuvant), water-in-oil-in-water emulsions, oil-in-water emulsions, liposomes, aluminum hydroxide gels, silica adjuvants, powdered bentonite, and tapioca adjuvants. , BCG, Pr
Bacteria such as opionobacterium acnes, cell walls and trehalose dicholate (TDM)
Lipopolysaccharide (LPS) and lipid A fractions, which are endotoxins of Gram-negative bacteria; β-glucan (polysaccharide); muramyl dipeptide (MDP); bestatin; synthetic compounds such as levamisole; Proteins or peptide substances derived from biological components such as hormones, thymic hormone humoral factors and tuftsin; and mixtures thereof (eg, complete Freund's adjuvant). These adjuvants can be administered by route of administration,
It is effective in enhancing or suppressing the immune response depending on the administration timing and the like. Furthermore, depending on the type of adjuvant, there are differences in the production of antibodies to blood against antigen, induction of cellular immunity, immunoglobulin class, and the like. Therefore, it is preferable to select an adjuvant appropriately according to the intended immune response. Adjuvant treatment methods are known in the art.

Immunization of a mammal is performed according to a method known in the art. For example, the antigen, NP, is injected subcutaneously, intradermally, intravenously, or intraperitoneally into a mammal. Since the immune response varies depending on the type and strain of mammal to be immunized, the immunization schedule is appropriately set according to the animal used. The antigen administration is repeated several times after the first immunization. Booster immunizations can be performed, for example, four weeks, six weeks, and six months after the initial immunization.

After immunization, blood is collected from the mammal, and the obtained blood is assayed for the presence of NP binding activity, thereby confirming that antibodies to NP are produced in the body of the mammal. Assay methods include enzyme immunoassay (ELISA), radioimmunoassay (RIA),
Known methods such as a fluorescent antibody method and the like can be mentioned.

After confirming the production of the NP-binding antibody, a boost (additional injection of an immunogen) can be performed to bring the immune cells capable of producing the specific antibody into a state suitable for cell fusion. The amount of NP administered in the boost is not particularly limited, but is preferably about 4 to 5 times the amount of NP immunized first. Boosting can generally be performed using an emulsion of NP and incomplete Freund's adjuvant. The administration route is appropriately selected from subcutaneous, intradermal, intravenous, intraperitoneal or the like.

After the final immunization, spleen cells are excised from the immunized mammal and fused with a cell line derived from myeloma. For cell fusion, it is preferable to use a cell line having a high proliferation ability, and it is preferable that the cell line derived from myeloma be compatible with the mammal from which the spleen cells to be fused are derived. Cell lines derived from mouse myeloma include P3X63-Ag8.
653, Sp2 / O-Ag14, FO · 1, S194 /
5. XX0 BU. l, P3 / NS1 / 1-Ag4-1 and the like. Cell fusion is performed according to methods known in the art. Examples of the cell fusion method include a polyethylene glycol method, a method using Sendai virus, a method using electric current, and the like. The resulting fused cells can be grown according to conditions known in the art. The desired fused cells are selected based on the binding ability of the produced antibody.

The binding ability of the antibody produced from the fused cells is as follows:
Assay can be performed based on methods known in the art. In the present invention, in order to obtain a fusion cell that produces an antibody having specific and high binding ability to NP, a cell line of interest is cloned using selection based on the binding ability to NP. The binding ability of the antibody can be determined by the ELISA method, the RIA method,
Assay can be performed using a method such as a fluorescent antibody method. The ELISA method is preferred because it is simple and has high sensitivity.

The cloning of the fused cells can be carried out using a method known in the art. Examples of the cloning method include a limiting dilution method and a soft agar method, and the limiting dilution method is preferable because the operation is easy and the reproducibility is high.
In order to select useful cells efficiently from many fusion cells obtained by cell fusion, cell selection is preferably performed from an early stage of cloning. In this way, a fusion cell line that produces an antibody having the desired binding ability can be finally selected.

By culturing the monoclonal antibody-producing cell line selected as described above in a large amount, a large amount of a monoclonal antibody specific to NP can be produced. Methods for mass culture of monoclonal antibody producing cell lines include in vivo and in vitro culture. As an example of in vivo large-scale culture, there is a method in which a fused cell is injected into a peritoneal cavity of a mammal to proliferate the antibody, and an antibody is produced in ascites. For in vitro culture, the fused cells are cultured in a medium and the antibodies are produced in the medium.

The monoclonal antibody of the present invention can be purified from the ascites fluid or the culture supernatant obtained by mass culture using a method known in the art. For purification, for example, DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation,
PEG fractionation, ethanol fractionation and the like are used in appropriate combination. The antibodies of the present invention preferably have about 90%
, Preferably about 95%, more preferably about 98%.

[0031] 3. Using the monoclonal antibody of the present invention
Detection and / or quantification of Ebola virus nucleoprotein The present invention further comprises performing an immunoassay using the monoclonal antibody of the present invention or a fragment thereof,
It also relates to a method for the detection and / or quantification of Ebola virus nucleoprotein. This method preferably includes at least the following steps (a) and (b). (A) reacting the monoclonal antibody of the present invention or a fragment thereof with a sample; and (b) reacting the antigen-antibody complex formed in step (1) with a labeled antibody for detection;

The detection and / or quantification method of the present invention may be any method as long as it is an assay using an antibody, that is, an immunoassay. For example, an enzyme immunoassay (ELIS)
A), fluorescence immunoassay, radioimmunoassay (RIA), luminescence immunoassay, enzyme antibody method, fluorescent antibody method, immunoturbidimetry,
Latex agglutination, latex turbidimetry, red blood cell agglutination, particle agglutination, western blot, and the like.

Samples to be subjected to the detection and / or quantification method of the present invention include blood, serum, plasma, lymphocyte culture supernatant,
It is not particularly limited as long as it is a biological sample such as urine, cerebrospinal fluid, saliva, sweat, ascites, amniotic fluid, or a cell or organ extract, which may contain a nucleoprotein of Ebola virus. When the detection and / or quantification method of the present invention is performed by an immunoassay using a labeled antibody such as an enzyme immunoassay, a fluorescence immunoassay, a radioimmunoassay, or a luminescence immunoassay, a sandwich method or a competition method In the case of the sandwich method, at least one of the immobilized antibody and the labeled antibody may be the monoclonal antibody of the present invention.

As the solid phase carrier, those described herein above as specific examples of the insoluble carrier in relation to the immobilized antibody can be used. As the labeling substance, those described hereinabove in connection with the labeled antibody can be used.

The method of measurement is known in the art (Clinical Pathology Special Edition, Special Issue No. 53, Immunoassay for Clinical Examinations-Techniques and Applications-edited by The Japanese Society of Clinical Pathology, Clinical Pathology Publishing Association, 1983, Eiji Ishikawa et al. "Enzyme Immunoassay", 3rd Edition, Medical Shoin, 1987, Tsunehiro Kitagawa et al., "Enzyme Immunoassay for Protein Nucleic Acid Enzyme Separate Volume No. 31," Kyoritsu Shuppan,
1987). For example, the sample is reacted with the immobilized antibody and the labeled antibody at the same time, or the labeled antibody is reacted after washing to form a complex of the immobilized antibody-antigen-labeled antibody. Then, the unbound labeled antibody is separated by washing, and the amount of antigen in the sample can be measured from the amount of the bound labeled antibody. Specifically, in the case of enzyme immunoassay (ELISA), a labeling enzyme is reacted with a substrate under the optimal conditions, and the amount of the reaction product is measured by an optical method or the like. In the case of the fluorescence immunoassay, the fluorescence intensity by the fluorescent substance label is measured, and in the case of the radioimmunoassay, the radiation dose by the radioactive substance label is measured. In the case of the luminescence immunoassay, the amount of luminescence by the luminescence reaction system is measured.

The detection and / or quantification method of the present invention may be performed by using an immunoturbidimetry, a latex agglutination reaction, a latex turbidimetry, an erythrocyte agglutination reaction, a particle agglutination reaction, or the like to determine the formation of an immunocomplex aggregate and the transmitted light or scattering thereof. When the light is measured by an optical method or when the measurement is performed by a visual measurement method, a phosphate buffer, a glycine buffer, a Tris buffer, a Good buffer, or the like can be used as a solvent. And a non-specific reaction inhibitor.

When the antibody is sensitized to a solid support, the solid support may be polystyrene, styrene-butadiene copolymer, (meth) acrylate polymer, latex, gelatin, liposome, microcapsule. , Red blood cells, silica, alumina, carbon black,
Particles made of a material such as a metal compound, a metal, a ceramic or a magnetic material can be used.

As a method of this sensitization, a physical adsorption method,
Known methods such as a chemical bonding method or a combination of these methods can be used. The operation of the measurement can be performed by a known method.For example, when the measurement is performed by an optical method, the sample is reacted with the antibody or the sample and the antibody sensitized to the solid support, and the endpoint method is used. Alternatively, transmitted light and scattered light are measured by a rate method.

When the measurement is carried out visually, the sample and the antibody sensitized to the solid phase carrier are reacted in a container such as a plate or a microtiter plate, and the state of aggregation is visually determined. Note that the measurement may be performed using a device such as a microplate reader instead of performing the measurement visually.

[0040] 4. Using the monoclonal antibody of the present invention
For detection and / or quantification of Ebola virus nucleoprotein
The kit of the present invention contains a monoclonal antibody or a fragment thereof that recognizes the nucleoprotein of Ebola virus provided by the present invention. The monoclonal antibody or a fragment thereof mentioned here may be an immobilized antibody or a labeled antibody described above in this specification. For example, when a monoclonal antibody that recognizes a nucleoprotein of Ebola virus provided by the present invention is used as a primary antibody, the kit of the present invention includes a secondary antibody for detecting a complex formed by an antigen-antibody binding reaction. Antibodies may be included. In order to use the kit efficiently and conveniently, the kit of the present invention may contain various auxiliary agents in addition to these antibodies. As an auxiliary agent, for example, a dissolving agent for dissolving a solid secondary antibody, a detergent used for washing an insolubilized carrier, and for measuring enzyme activity when an enzyme is used as a labeling substance for an antibody And those usually used as kits for immunological measurement reagents such as a substrate of the above and a reaction terminator. The present invention will be specifically described with reference to the following examples, but the present invention is not limited to the examples.

[0041]

EXAMPLES (A) Materials and Methods (1) Cell Culture Hybridoma and its parent cell line P3 / Ag5
68 is 10% fetal bovine serum (FBS), a non-essential amino acid (G
RPMI1640 (Gibco BRL) supplemented with ibco BRL) and antibiotics (streptomycin and penicillin, Gibco BRL)
And cultured. HAT supplements (Gibco BRL) were added to the medium during hybridoma selection. Tn5 cells are 10% F
The cells were cultured in TC100 (Gibco BRL) supplemented with BS, 2% tryptose phosphate broth (Difco) and kanamycin.

(2) Recombinant NP The recombinant nuclear protein of the Ebola virus Zaire strain (r
NP) was expressed by a baculovirus expression system with a histidine tag at the N-terminus. That is, the CD of Zaire strain
Complete open reading frame of NA fragment (Sanche
1989.The nucleoprotein gene of Ebola viru
s: cloning, sequencing, and in vitro expression.V
irology. 170: 81-91) was amplified by PCR and a histidine tag was added in-frame to the N-terminus of NP, pQE3.
1 plasmid (Qiagen). Then, cDNA
With the histidine tag, transfer vector (pA
cYM1) (Matsuura, Y. et al. 1987. Baculovirus expr
ession vectors: the requirements for high level ex
pression of proteins, including glycoproteins. J.
Gen. Virol. 68: 1233-1250). Recombinant baculovirus was produced by co-transfection of transfer plasmid and viral DNA. rNP was purified from Tn5 cell lysates infected with recombinant baculovirus by Ni agarose (Qiagen). Protein concentration was measured with a protein assay kit (Bio-Rad, Hercules, CA).
After PCR amplification, the polypeptides corresponding to the different parts of the NP were transferred to the pGEX2T vector (Amersham Pharmacia, Lis.
(G.ttle Chalfont, UK) using E. coli as a fusion protein with GST. Table 1 shows the primers used in this experiment. Sudan and Reston 2
To express 6 amino acids, primers SNP8EF and
Anneal SNP8ER or RNP8EF or RNP8ER, PC
Filled with R reaction and cloned into pGEX2T. To generate longer peptides, these fragments were sequentially extended by successive PCR reactions. Automatic nucleotide sequencer (Applied Biosystems. Fost)
er City, CA). Glutathione Sepharose 4B (Amersham Pharmac)
ia).

[0043]

[Table 1]

(3) ELISA microwell immunoplate (Falcon, Franklin Lak)
es, NJ) to 100 ng / phosphate phosphate buffer (PBS).
Wells were coated with purified rNP or partial NP at 4 ° C. overnight and blocked with 5% skim milk. Sample (10
0 μl) was added to each well, and the bound antibody was purified from horseradish peroxidase (HRPO) labeled anti-mouse IgG (Zyme
d, San Francisco, CA) and ABTS substrate solution (Boehri
nger Mannheim, Germany).

(4) Establishment of Monoclonal Antibody BALB / c mice were immunized three or four times with purified rNP. Three days after the last immunization, spleen cells were collected and PEG
(Gibco BRL) and fused to P3 / Ag568 cells. Purification using culture supernatant of hybridoma cells as antigen
Screening was performed by ELISA using NP. MAb Trap GII antibody purification kit (Amer
sham Pharmacia). The monoclonal antibody isotype was determined using a mouse monoclonal antibody isotyping kit (Gibco BRL).

(5) Polyclonal Antibody Rabbit polyclonal antibody was prepared by subcutaneous injection of purified rNP emulsified in aluminum adjuvant (Pierce, Rockford, Ill.). The titer of this antiserum was> 1: 10,000 in an immunofluorescence assay using rNP expressing cells as antigen.

(6) Antigen Capture ELISA Purified monoclonal antibody was added to 100 μl
After coating microwell immunoplates (Falcon) at a concentration of 100 ng / well in 1 PBS
Blocked for 1 hour at room temperature with 5% skim milk in PBS. Plates in PBS containing 0.5% Tween 20
After washing with (PBST), 100 μl of a sample containing rNP or a clinical sample was added and incubated at room temperature for 1 hour. Plates were washed with PBST and 100 μl of rabbit polyclonal antibody diluted 1: 1000 in 0.5% skim milk was added to each well. After 1 hour incubation at room temperature, the plate is washed with PBST,
HRPO-labeled anti-rabbit IgG (Zymed) was added. Plates were incubated for 1 hour at room temperature. After further washing with PBST, 100 μl of ABTS substrate solution (Boehringe
After addition of r) and incubation at room temperature for 30 minutes, the optical density (OD) was measured at 405 nm.

(7) Clinical samples Tissues and sera from cynomolgus monkeys ( Macaca fascicularis ) naturally infected with the Ebola virus reston strain in the Philippines were used as clinical samples. These samples are-
Stored at 80 ° C. or in liquid nitrogen. The state of Reston infection in these animals has already been established (Miranda,
ME et al. 1999. Epidemiology of Ebola (Subtype Resto
n) virus in the Philippines, 1996. J. Infect. Di
s. 179 Suppl. 1: S115-119). Liver and spleen tissues (about 10% w / vol) were added to 0.05% Tween20, 1% Triton X10
Homogenized in PBS containing 0 and 5% skim milk. After centrifugation, the supernatant was used as starting material. 1 serum
% Triton was added to inactivate and diluted in 5% skim milk.

(8) Western Blot SDS-PAGE and Western blot analysis were performed using 10% acrylamide gel and Immobilon nylon membrane (Mili
pore, Bedford, MA). The ascites fluid containing the monoclonal antibody was used at a dilution of 1: 1000. The bound antibody was HRPO-labeled anti-mouse IgG (Zyme
d) and POD substrate (Wako Pure Chemical Industries).
A polyclonal antibody against GST is available from Amersham Pharmac.
purchased from ia.

(B) Results (1) Establishment of Monoclonal Antibodies Twelve hybridoma strains secreting rNP-reactive IgG antibodies were established. After purifying these antibodies, the antigen capture EL
Tested by ISA. As shown in FIG. 1, two antibodies 3-
3D and 2-11G were reactive in this test. Other monoclonal antibodies did not show a specific reaction like antibodies 3-3D and 2-11G even when the concentration of rNP was increased. The isotype of antibodies 3-3D and 2-11G was IgG1. The hybridoma producing antibody 3-3D was deposited under the accession number FERM P-18279 on March 28, 2001 by the Ministry of Economy, Trade and Industry, National Institute of Advanced Industrial Science and Technology It has been deposited at 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture).

(2) Antigen capture ELISA Antigen capture ELISA using antibodies 3-3D and 2-11G
The sensitivity of A was examined. RNP detected by this ELISA
Was approximately 33 ng / well for all antibodies (FIG. 1). Natural NP and antibodies 3-3D and 2-11
Clinical samples from monkeys infected with the Ebola virus reston strain were used to confirm reactivity with G. As shown in FIG. 2, Ebola NP could be detected in Reston-positive tissue (A) and serum (B) samples by antigen capture ELISA using antibody 3-3D. In two liver samples from two animals (liver 1 and liver 2):
Positive reactions were shown up to a dilution factor of 160 or 1: 320. The spleen sample showed a positive reaction up to a dilution of 1: 160, and the serum sample was positive up to a dilution of 1: 160. In similarly treated tissues from uninfected monkeys, results were negative even at dilutions as low as 1:10 for tissue samples and 1:20 for serum samples. These results indicate an antigen capture ELISA using antibody 3-3D.
The method is sensitive and shows that it can be applied to clinical samples from monkeys infected with Ebola virus reston.

(3) Epitope mapping of monoclonal antibody 3-3D ELISA using monoclonal antibody 3-3D
NP of Ebola virus other than Reston strain and Saile strain
In order to determine whether the method is applicable to the above case, mapping of the epitope recognized by the monoclonal antibody 3-3D was performed. Eight overlapping peptides each consisting of about 100 amino acids were made to encompass the entire amino acid sequence of Zaire strain NP. Monoclonal antibody 3-3D showed reactivity with the peptide corresponding to amino acid residues 631 to 739 in Western blot and ELISA. To identify the smallest epitope,
The C-terminal amino acid residues required for antibody recognition were first determined. As shown in FIG. 3A, the amino acid at position 673 was required to show sufficient reactivity in Western blot. In order to show sufficient reactivity, 26 amino acids upstream from the 673th amino acid at the C-terminus are required,
The reaction was weak at 23 amino acids (FIG. 3B). These results indicate that monoclonal antibody 3-3D recognizes a steric epitope formed by 26 amino acids consisting of amino acids 648 to 673 of NP.

(4) Cross-reactivity of monoclonal antibody 3-3D with other strains of Ebola virus C-terminal 109 amino acids including 26 amino acids required for antibody recognition in the Zaire strain (Zaire strain and Reston strain) Or 10
When tested with a long peptide consisting of 6 amino acids (Sudan strain), monoclonal antibody 3-3D reacted with these three peptides by Western blot and ELISA (FIG. 4). These results indicate that monoclonal antibody 3-
ELISA using 3D shows that NP of Sudan, Zaire and Reston strains can be detected.

[0054]

The monoclonal antibody of the present invention can specifically recognize Ebola virus. Also provided is a method for specifically detecting Ebola virus in serum and organs by ELISA using the monoclonal antibody of the present invention. In addition, since the ELISA system of the present invention does not contain live virus except for clinical samples, a high degree of security containment is not required, and the system can be reproduced in a normal laboratory, and non-infectious It is also possible to obtain secondary polyclonal antibodies that can be prepared with recombinant proteins.

[0055]

[Sequence List] SEQUENCE LISTING <110> Kokuritsu Kansensyo Kenkyusyocho <120> A monoclonal antibody recognizing Ebola virus <130> A11166A <160> 26

<210> 1 <211> 24 <212> DNA <213> Synthetic DNA <400> 1 caaggatccg agtatggatt ctcg 24

<210> 2 <211> 24 <212> DNA <213> Synthetic DNA <400> 2 atggatccat gctcattcac tgat 24

<210> 3 <211> 24 <212> DNA <213> Synthetic DNA <400> 3 ccgggatcca ctcaagaggc ca 22

<210> 4 <211> 23 <212> DNA <213> Synthetic DNA <400> 4 tgcgaattca ctgatgatgt tgc 23

<210> 5 <211> 25 <212> DNA <213> Synthetic DNA <400> 5 aaaagaattc actcatcctt catca 25

<210> 6 <211> 30 <212> DNA <213> Synthetic DNA <400> 6 ctagaattca atccttcatc atatgataat 30

<210> 7 <211> 29 <212> DNA <213> Synthetic DNA <400> 7 caggaattca cttcatcata tgataatac 29

<210> 8 <211> 30 <212> DNA <213> Synthetic DNA <400> 8 ctgaattcac atcatatgat aatacaaaac 30

<210> 9 <211> 22 <212> DNA <213> Synthetic DNA <400> 9 ggatccatgt atcgccacat tc

<210> 10 <211> 21 <212> DNA <213> Synthetic DNA <400> 10 ggatccgaga tgtatcgcca c 21

<210> 11 <211> 21 <212> DNA <213> Synthetic DNA <400> 11 ggatccgagg agatgtatcg c 21

<210> 12 <211> 18 <212> DNA <213> Synthetic DNA <400> 12 ggatcctttg aggagatg 18

<210> 13 <211> 55 <212> DNA <213> Synthetic DNA <400> 18 cgcggatccc tagaagaaac atattatcat ctcctaaaaa cacagggtcc atttg 55

<210> 14 <211> 57 <212> DNA <213> Synthetic DNA <400> 14 gctgaattca atcactcatt aggtgataat aattgattgc ctcaaatgga ccctgtg 57

<210> 15 <211> 55 <212> DNA <213> Synthetic DNA <400> 15 cgcggatcct taggagagac atatcaccat ctgctgagaa ctcaaggtcc atttg 55

<210> 16 <211> 57 <212> DNA <213> Synthetic DNA <400> 16 gctgaattca atccttcatc atgtgataat aattgatagc ttcaaatgga ccttgag 57

<210> 17 <211> 58 <212> DNA <213> Synthetic DNA <400> 17 ggatccctcc caatcaactc taaaaagagt tccgcactag aagaaacata ttatcatc 58

<210> 18 <211> 62 <212> DNA <213> Synthetic DNA <400> 18 gaattcagcc actttcagtg ctaaaagcaa tgggttcatc actcattagg tgataataat 60 tg

<210> 19 <211> 37 <212> DNA <213> Synthetic DNA <400> 19 ggatcctctg aaagtgaagc tctcccaatc aactcta 37

<210> 20 <211> 40 <212> DNA <213> Synthetic DNA <400> 20 gaattctcaa agatatattc cttgccactt tcagtgctaa 40

<210> 21 <211> 40 <212> DNA <213> Synthetic DNA <400> 21 gaattcattc aagggagtct ggaaagatat attccttgcc 40

<210> 22 <211> 73 <212> DNA <213> Synthetic DNA <400> 22 gaattcactt ctctaaggcc tccttctcac tcaaccacgg cgggtaggct tcttcaaggg 60 agtctggaaa gat 73

<210> 23 <211> 73 <212> DNA <213> Synthetic DNA <400> 23 gaattcatac cggccagagg aattgctggc catcaatgac cagataacga ttttccttct 60 ctaaggcctc ctt 73

<210> 24 <211> 67 <212> DNA <213> Synthetic DNA <400> 24 gaattcagtc atgttgaaga acggcaagga acttgtcccg taggctcatt accggccaga 60 ggaattg 67

<210> 25 <211> 28 <212> DNA <213> Synthetic DNA <400> 25 gctggatcct cacaattgaa tgaagacc 28

<210> 26 <211> 27 <212> DNA <213> Synthetic DNA <400> 26 gtggaattct tactgatggt gctgcaa 27

[Brief description of the drawings]

FIG. 1 is a diagram showing the reactivity of each monoclonal antibody in an antigen capture ELISA. Purified monoclonal antibodies (●; 3-3D, Δ; 2-11G, Δ; 2-1G,
×; 3-3D) was coated on a microplate at a concentration of 100 ng / well, and the ability to capture rNP was measured using an antigen capture EL.
Varying amounts of rNP were examined by ISA.

FIG. 2 shows the reactivity of clinical samples infected with the Reston strain in an antigen capture ELISA. (A) Two livers (◆ and ●) and spleen (▲) from two infected monkeys were examined by capture ELISA. Liver (×) and spleen (□) from uninfected monkeys are used to characterize the reaction. (B) Sera from infected (■) and two uninfected (○ and △) monkeys were examined by capture ELISA.

FIG. 3 shows mapping of the epitope of monoclonal antibody 3-3D by Western blot. (A) Amino acid position 631- of each NP (Zaire strain)
Bacterial lysates containing the GST fusion peptide corresponding to 671, 672, 673 or 674 were tested for reactivity with monoclonal antibody 3-3D. (B) amino acid position 648 of each NP (Zaire strain);
GST corresponding to 649, 650 and 651 to 673
Bacterial lysates containing the fusion peptide were examined for reactivity with monoclonal antibody 3-3D. At the same time, similarly prepared membranes were stained with anti-GST antibody to show similar expression levels of each fusion peptide.

FIG. 4. NPs from three strains of Ebola virus.
Monoclonal antibody 3-3 against the above epitope region
It is a figure which shows the reactivity of D. A GST fusion peptide (100 ng / well) corresponding to amino acid positions 631-739 of the Zaire strain (■) and Reston strain (●) or amino acid position 633-738 of the Sudan strain (▲) was coated on a microplate and subjected to ELISA. The reactivity with the monoclonal antibody 3-3D was examined. Irrelevant G
The ST fusion peptide (◇) was used as a negative control.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/569 C12N 5/00 B (72) Inventor Tetsuro Ikegami 4-7-1 Gakuen, Musashimurayama-shi, Tokyo National Institute of Infectious Diseases Virus Part 1 Exogenous Virus Room (72) Inventor Masayuki Saijo 4-7-1 Musashimurayama City Gakuen, Tokyo Metropolitan Infectious Diseases Research Institute Virus Section 1 Exogenous Virus Room (72) Invention Person Shigeru Morikawa 4-7-1, Musashimurayama City Gakuen, Tokyo Metropolitan Institute for Infectious Diseases Virus Section 1, Exogenous Virus Room (72) Inventor Kurane, Ichiro Kurane 1-2-1 Toyama, Shinjuku-ku, Tokyo National Infectious Disease Research Virus part 1 F term (reference) 4B024 AA14 BA32 GA03 HA15 4B064 AG27 CA10 CA20 CC24 DA15 4B065 AA91X AB05 BA08 CA25 CA46 4H045 AA11 AA30 CA01 DA76 EA52 FA72

Claims (13)

[Claims] 1. A monoclonal antibody or a fragment thereof that recognizes a nucleoprotein of Ebola virus. 2. The monoclonal antibody or a fragment thereof according to claim 1, which recognizes a nuclear protein of a Zaire strain, a Sudan strain and a Reston strain of Ebola virus. 3. The monoclonal antibody or a fragment thereof according to claim 1 or 2, which recognizes an epitope consisting of amino acids 631 to 739 of the nucleoprotein of Ebola virus Zaire strain. 4. The monoclonal antibody or a fragment thereof according to claim 1, which recognizes an epitope consisting of 26 amino acids consisting of amino acids 648 to 673 of the nucleoprotein of Ebola virus Zaire strain. 5. A monoclonal antibody or a fragment thereof produced by a hybridoma having accession number FERM P-18279. 6. A gene encoding the monoclonal antibody according to claim 1 or a fragment thereof. 7. A hybridoma that produces the monoclonal antibody according to any one of claims 1 to 5. 8. The hybridoma according to claim 7, obtained by fusing mammalian immune cells immunized with an immunogen containing a nucleoprotein of Ebola virus and mammalian myeloma cells. 9. A hybridoma having accession number FERM P-18279. 10. The monoclonal antibody according to any one of claims 1 to 5, comprising a step of culturing the hybridoma according to any one of claims 7 to 9, and a step of collecting a monoclonal antibody produced by the hybridoma. Manufacturing method. 11. A method for detecting and / or quantifying a nucleoprotein of Ebola virus, comprising performing an immunoassay using the monoclonal antibody or a fragment thereof according to any one of claims 1 to 5. 12. The method for detecting and / or quantifying a nucleoprotein of Ebola virus according to claim 10, comprising at least the following steps (a) and (b). (A) reacting the monoclonal antibody or fragment thereof according to any one of claims 1 to 5 with a sample; and (b) the antigen-antibody complex formed in step (1) and a labeled antibody for detection. Reacting with A kit for detecting and / or quantifying a nucleoprotein of Ebola virus, comprising the monoclonal antibody according to any one of claims 1 to 5.