WO2016080591A1 - Antibody recognizing nucleocapsid of middle east respiratory syndrome coronavirus, and use thereof - Google Patents

Abstract

Provided are monoclonal antibodies, which specifically bind to the nucleocapsid of Middle East respiratory syndrome coronavirus (MERS-CoV), an antibody which is a combination thereof, a Middle East respiratory syndrome coronavirus diagnosis composition and kit comprising the antibody, and a Middle East respiratory syndrome coronavirus detection method using same.

Description

Antibodies Recognizing Middle East Respiratory Syndrome Coronavirus Nucleocapsid and Uses thereof

The present invention is an antibody recognizing a Middle East respiratory syndrome coronavirus (MERS-CoV) nucleocapsid and cells capable of producing the same, a composition, kit for diagnosing a Middle East respiratory syndrome coronavirus comprising the antibody, and the Middle East. It relates to a method for diagnosing a respiratory syndrome coronavirus infection.

The Middle East Respiratory Syndrome Corona Virus (MERS-CoV) is a virus that has been concentrated in the Middle East since it was first discovered in Saudi Arabia in 2012. It belongs to the Coroviridae family and is known to have a virus similar to SARS (Acute Respiratory Syndrome).

MERS-CoV has an incubation period of about one week and, like SARS, causes severe respiratory symptoms such as high fever, cough and difficulty breathing. Unlike SARS, however, it is accompanied by acute renal failure. More deadly than the SARS, the findings show that the mortality rate is about six times higher than the SARS. Depending on age, mortality rates are over 50%.

The source of infection is not yet known, but camels are pointed out. In some cases, the virus detected from an infected person coincided with the virus detected from a camel. However, the practice of eating camel meat or drinking milk is known to be difficult.

Therefore, the diagnosis of Middle East respiratory syndrome coronavirus (MERS-CoV) infection is required.

One aspect of the invention provides an antibody that recognizes Middle East respiratory syndrome coronavirus nucleocapsid.

Another aspect of the invention provides a hybridoma with accession number KCLRF-BP-00331 or KCLRF-BP-00332.

Another aspect of the present invention provides a composition for diagnosing Middle East respiratory syndrome coronavirus.

Another aspect of the present invention provides a kit for diagnosing Middle East respiratory syndrome coronavirus.

Another aspect of the present invention provides a method for diagnosing a Middle East respiratory syndrome coronavirus infection.

One aspect provides antibodies that recognize Middle East respiratory syndrome coronavirus nucleocapsid.

Middle East respiratory syndrome coronavirus (MERS-CoV, hereinafter referred to as 'MERS-CoV') has positive-sense single stranded RNA. The MERS-CoV is a species of the genus Betacoronavirus. The MERS-CoV is also known as HCoV-EMC or NcoV.

The antibody may specifically bind to nucleocapsid of MERS-CoV. The nucleocapsid of MERS-CoV may be one having an amino acid sequence of SEQ ID NO: 1. Nucleocapsids of MERS-CoV having amino acid sequences of 22-40, 126-146, 164-202, 234-259, 362-388, or combinations thereof in SEQ ID NO: 1 are hydrophilicity Can be. Nucleocapsids of MERS-CoV having amino acid sequences of 22-40, 126-146, 164-202, 234-259, 362-388, or a combination thereof in SEQ ID NO: 1 are respectively SEQ ID NO: 1, Amino acid sequences of 2, 3, 4, and 5. The nucleocapsid of MERS-CoV having the hydrophilicity may comprise an epitope. According to one embodiment of the present invention, the inventors investigated the hydrophilicity of the nucleocapsid protein of MERS-CoV, to prepare a polypeptide antigen of the high hydrophilic region of the protein, to prepare an antibody that specifically binds to it. .

According to one embodiment of the invention, a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid of MERS-CoV. In addition, according to one embodiment of the present invention, a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 4 in the nucleocapsid of MERS-CoV. In addition, a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid of MERS-CoV, and an epitope represented by the amino acid sequence of SEQ ID NO: 4 in the nucleocapsid of MERS-CoV An antibody is provided that is a combination of monoclonal antibodies that bind specifically.

The term "specifically binding" or "specifically recognizing" has the same meaning as is commonly known to those skilled in the art, wherein the antigen and antibody specifically interact to form an antigen-antibody complex. Can also mean an immunological response.

A complete antibody is a structure having two full length light chains and two full length heavy chains, each of which consists of a heavy chain constant region and a light chain constant region divided by a heavy chain and a disulfide bond (SS-bond) antibody. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, and subclasses gamma 1 (γ1), gamma 2 (γ2), and gamma. 3 (γ 3), gamma 4 (γ 4), alpha 1 (α 1), and alpha 2 (α 2). The constant region of the light chain may have kappa (κ) and lambda (λ) types.

The term “monoclonal antibody” means that the antibody molecule is made to consist of a single molecule. Monoclonal antibodies have specificity that responds only to antigens with the same epitope, and also show affinity only for specific epitopes.

In one embodiment of the present invention, the monoclonal antibody that specifically binds to the epitope represented by the amino acid sequence of SEQ ID NO: 2 may be one produced in hybridoma cells having accession number KCLRFBP00331. In addition, the monoclonal antibody that specifically binds to the epitope represented by the amino acid sequence of SEQ ID NO: 4 may be one produced in hybridoma cells having accession number KCLRFBP00332.

The hybridoma cells can be prepared using methods known in the art. Specifically, the hybridoma cells are prepared by immunizing a polypeptide of an immunogen MERS-CoV nucleocapsid to an animal and fusing B cells, which are antibody-producing cells derived from the immunized animal, with myeloma cells. Next, it can be prepared by a method of selecting hybridomas that produce monoclonal antibodies that specifically bind to MERS-CoV nucleocapsid. The immunized animal can use an animal such as a goat, sheep, morph, rat or rabbit as well as the mouse used in the examples.

As a method for immunizing the immunized animal, a method already known in the art may be used. For example, when immunizing mice, 1 to 100 µg of an immunogen is emulsified with an antigen adjuvant such as physiological saline and / or Freund's adjuvant at one time to subcutaneously subcutaneously in the abdominal cavity of the immunized animal. Or 2-6 inoculations every 2-5 weeks intraperitoneally. After immunizing the immunized animals, spleens or lymph nodes are extracted after 3-5 days of final immunization, and according to cell fusion methods already known in the art, B cells contained in their tissues in the presence of a fusion promoter are myeloma. To fuse with the cell. The fusion promoter may use a material such as polyethylene glycol (PEG), for example. The myeloma cells are described, for example, in P3U1, NS-1, P3x63. Mouse derived cells such as Ag 8.653, Sp2 / 0-Ag14, and rat derived cells such as AG1, AG2 can be used. In addition, the cell fusion method known in the art, for example, B cells and myeloma cells are mixed at a ratio of 1: 1-10: 1, to which a PEG having a molecular weight of 1,000-6,000 is added at a concentration of 10-80%. For example, the method may be performed by incubating at 30-37 ° C. for 1-10 minutes. In addition, hybridomas that produce monoclonal antibodies that specifically bind to the nucleocapsid of MERS-CoV are cultured in a selective medium such as HAT medium, in which only hybridomas can survive, and hybridomas are produced. The antibody activity in the culture supernatant can be measured and selected using methods such as ELISA. Finally, hybridomas that produce monoclonal antibodies that specifically bind to nucleocapsids of MERS-CoV may, for example, produce high levels of monoclonal antibodies that specifically bind to nucleocapsids of MERS-CoV. For bridoma, it can be selected by repeating cloning by methods such as limiting dilution. Meanwhile, the monoclonal antibody may be of IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA1, IgA5, or IgD type.

Another aspect provides antigen binding fragments according to the monoclonal antibodies described above.

The term “antibody” is a specific antibody against MERS-CoV, which binds specifically to the nucleocapsid of MERS-CoV, and is a complete antibody, antigen binding fragment of an antibody molecule, synthetic antibody, recombinant antibody, or antibody. It may include a hybrid (antibody hybrid). The description of the complete antibody is as mentioned above.

The term “antigen binding fragment” refers to a portion of a polypeptide that includes a portion to which an antigen can bind, as a fragment thereof for the entire structure of an immunoglobulin. For example, it can be F (ab ') 2, Fab', Fab, Fv or scFv. Fab of the antigen-binding fragment has one antigen binding site in a structure having a variable region of the light and heavy chains, a constant region of the light chain and a first constant region of the heavy chain (C _H1 ). Fab 'differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain C _H1 domain. F (ab ') ₂ antibodies are produced when the cysteine residues of the hinge region of Fab' form disulfide bonds. Recombinant techniques for generating Fv fragments with minimal antibody fragments in which Fv has only heavy chain variable regions and light chain variable regions are well known in the art. Double-chain Fv is a non-covalent bond in which the heavy chain variable region and the light chain variable region are linked, and the single-chain Fv is generally shared by the variable region of the heavy chain and the short chain variable region through a peptide linker. It may be linked by bond or directly at the C-terminus to form a dimer-like structure such as a double chain Fv. The antigen-binding fragment can be obtained using proteolytic enzymes (for example, restriction digestion of the entire antibody with papain can yield Fab and cleavage with pepsin can yield F (ab ') ₂ fragment). In addition, the antigen-binding fragment can be produced through genetic recombination technology.

Such antibodies include monoclonal antibodies, bispecific antibodies, non-human antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fv (scFv), single chain antibodies, Fab fragments, F (ab ') fragments, disulfide-binding Fv (sdFv) and anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of the antibodies.

Another aspect provides hybridoma cells (Accession: KCLRFBP00331) that produce monoclonal antibodies that specifically bind to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid of MERS-CoV. Another aspect provides hybridoma cells (Accession: KCLRFBP00332) that produce monoclonal antibodies that specifically bind to an epitope represented by the amino acid sequence of SEQ ID NO: 4 in the nucleocapsid of MERS-CoV.

The method for preparing the hybridoma cells is as described above. In addition, the hybridoma cells prepared according to the following examples were deposited on October 14, 2014 to the Korean Collection for Type Cultures, an international depository institution under the Budapest Treaty (Accession No .: KCLRFBP00331 and KCLRFBP00332). On the other hand, the deposited hybridoma cells are stored according to the provisions of the Budapest Treaty for Microbial Deposits and can be distributed to the general public with reference to the accession number.

Another aspect provides a composition for diagnosing MERS CoV of an individual comprising said monoclonal antibody or antigen binding fragment and an anti-MERS CoV monoclonal antibody.

The composition may be a liquid composition comprising the antibody in a liquid. The liquid may be one capable of dissolving and maintaining the antibody. For example, it may be water or a buffer solution such as PBS. The composition may further include a substance for keeping the antibody stable.

Another aspect provides a MERS-CoV diagnostic kit comprising the above-described antibody or antigen-binding fragment thereof.

The kit may further comprise a detectable moiety. A detectable moiety can include a moiety in which the presence, relative amount and / or location (eg, location on the array) of the moiety can be determined directly or indirectly. Such detectable moieties are well known in the art. For example, a detectable moiety can be detected when exposed to certain conditions, such as a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, a radioactive moiety (eg, a radioactive atom), or an enzyme moiety. It may be selected from the group consisting of. For example, fluorescent moieties may need to be exposed to radiation at certain wavelengths and intensities that cause excitation of the fluorescent moiety, thereby detecting at a particular wavelength that can be detected. It can be made to emit as much fluorescence as possible.

The kit includes a sample pad (1) to which a liquid sample including an analyte is applied;

A storage pad 2 fluidly connected to the sample pad and movably supported by a conjugate of gold particles, wherein the conjugate of gold particles is a conjugate of a first antibody or fragment thereof and a gold particle Storage pad 2;

A chromatographic membrane material (3) in fluid communication with the storage pad and in which the liquid sample is moved by capillary movement, wherein the second antibody or fragment thereof is immobilized downstream of the storage pad. A chromatographic membrane material 3 comprising a detection region;

A hygroscopic pad 4 in fluid communication with the chromatography membrane material; And

It may be an analytical device for detecting the Middle East respiratory syndrome coronavirus, including the sample pad, the storage pad, the chromatographic membrane material, and the solid support 5 supporting the hygroscopic pad.

The first and second antibodies specifically bind to an epitope represented by the amino acid sequence of SEQ ID NO: 2, for example, in the nucleocapsid of Middle East respiratory syndrome coronavirus (MERS-CoV). One of a monoclonal antibody and a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 4. The first antibody and the second antibody may be different. In one embodiment, the first antibody is a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 4, and the second antibody is specifically directed to an epitope represented by the amino acid sequence of SEQ ID NO: 2 It may be a monoclonal antibody that binds. Further, in one embodiment, the first antibody is a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 2, and the second antibody is specific to an epitope represented by the amino acid sequence of SEQ ID NO: 4 It may be a monoclonal antibody that binds to.

1 is a side view showing an example of an analysis device for detecting and detecting the Middle East Respiratory Syndrome Coronavirus according to an embodiment of the present invention.

The kit is supported by a chromatography material membrane (3) having a detection region (T) and a control region (C) on which a second antibody is immobilized on a solid support (5), wherein the specimen pad (1) The storage pad 2 and the moisture absorption pad on which the first antibody and the gold conjugate are supported are overlapped and connected. When the sample is added to the sample pad 1 in the strip, it moves to the storage pad 2 by capillary action so that the antigen in the sample binds to the conjugate of the gold and the first antibody in the storage pad, Capillary movement is made downstream through the direction of the moisture absorption pad (4). When the conjugate of the antigen and the conjugate reaches the detection region (T), the conjugate and the second antibody specifically bind and develop color, and in the absence of the conjugate, no color development occurs. In addition, when the conjugate of the first antibody and the gold particles together with the sample passes through the control region (C), the conjugate of the first antibody and the gold particles is specific to the first antibody and the gold particle conjugate in the control region. In combination with the color development, it is possible to check whether the capillary movement is properly performed.

The kit may be an immunoassay kit using a porous material as a solid carrier of an immunochemical component such as an antigen or an antibody. Sample pads, storage pads, chromatographic membrane materials and hygroscopic pads used in one embodiment of the invention are materials having a porosity and volume sufficient to contain and contain a liquid sample to be analyzed, for example a microporous membrane. It may be a substance. Examples of such microporous membrane materials include nylon, cellulose materials, polysulfones, polyvinylidene difluorides, polyesters and glass fibers. Preferred examples of the microporous membrane material are nitrocellulose membranes. The analysis device may have the form of a strip.

The term "in flow communication with" means that a liquid sample is applied to one location (e.g. sample pad) and is available to another location (e.g. storage pad) by capillary movement when the liquid sample is applied. Say what it is. In addition, the term "movably supported" means that the conjugate of gold particles is immobilized to the storage pad movably with capillary movement of the liquid sample. In addition, the term "nondiffusively immobilized" means that the second antibody or fragment thereof is immobilized so that it does not diffuse together by capillary movement of the liquid sample in the detection region. The method for non-diffusion immobilizing the antigen and / or antibody can include any method as long as it is capable of non-diffusion immobilizing the chromatography membrane material and the antigen and / or antibody to the detection region of the chromatography membrane material. Examples include, but are not limited to, covalent bonding methods. The detection zone may take any shape, for example rectangular and circular in shape, the area of which is smaller than the area of the chromatography membrane material.

Antibodies and gold conjugates used in the kits of one embodiment of the invention can be prepared by methods well known in the art. For example, it can be prepared by making a gold colloidal solution and covalently binding the gold in the solution with the antibody. Antibodies and gold conjugates can be prepared, for example, by the methods disclosed in US Pat. Nos. 5,514,302 and 4,313,734, which are incorporated herein by reference in their entirety.

In addition, the kit can be used to diagnose MERS-CoV by detecting MERS-CoV according to an immunoassay method. Such immunoassays can be performed according to various immunoassays or immunostaining protocols developed in the prior art. The immunoassay or immunostaining format may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining. And immunoaffinity tablets.

For example, when the method of one embodiment of the invention is carried out according to a radioimmunoassay, the antibody labeled with the radioisotope (eg, C14, I125, P32 and S35) may be MERS-CoV or a nucleo thereof. It can be used to detect the capsid.

Further, for example, when the method according to one embodiment of the present invention is carried out in an ELISA method, certain embodiments of the present invention include the steps of coating an unknown cell sample lysate to be analyzed on the surface of a solid substrate; Reacting the cell lysate with a primary antibody against the nucleocapsid of MERS-CoV; Reacting the reaction product of the step of reacting the primary antibody against the nucleocapsid of MERS-CoV and the cell lysate with a secondary antibody to which an enzyme is bound; And measuring the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, and may be microtiter plates.

The enzyme bound to the secondary antibody may include an enzyme catalyzing a color reaction, a fluorescence reaction, a luminescence reaction or an infrared reaction. For example, alkaline phosphatase, β-galactosidase, and horse radish fur Oxidase, luciferase and cytochrome P450. When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT) naphthol-AS-B1-phosphate (naphthol-AS-) Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-phosphate) and chromogenic reaction substrates such as enhanced chemifluorescence (ECF) are used and hose radish peroxidase is used. N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), TMB ( tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS ( substrates such as phenzaine methosulfate can be used. All.

When the method according to one embodiment of the invention is carried out in a capture-ELISA mode, one embodiment of the invention comprises coating an anti-MERS-CoV antibody on the surface of a solid substrate as a capturing antibody; Reacting the capture antibody with the sample; The detection result of reacting the capture antibody with the sample is a detection antibody that binds to a label that generates a signal and specifically reacts to MERS-CoV or a nucleocaptide thereof (e.g., one embodiment of the present invention). Antibody or antigen-binding fragment); And measuring the signal generated from the label.

The detection antibody carries a label which generates a detectable signal. The label may include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P450), radioactive substances (eg C14, I125 , P32 and S35), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent, and fluorescence resonance energy transfer (FRET).

Measurement of the final enzyme activity or signal in the ELISA method and the capture-ELISA method can be carried out according to various methods known in the art. Detection of these signals allows for qualitative or quantitative analysis of MERS-CoV. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used.

Another aspect includes contacting a sample with an antibody or antigen-binding fragment thereof as described above to provide information necessary for diagnosing MERS-CoV; And detecting a complex of the antibody or an antigen-binding fragment thereof and MERS-CoV.

The method for detecting MERS-CoV may include contacting the sample with the above-described antibody or antigen-binding fragment thereof. The contacting step comprises contacting the sample with a plate coated with the above-described anti-MERS-CoV antibody or antigen-binding fragment thereof (referred to as 'primary antibody or antigen-binding fragment thereof'), followed by anti-MERS-CoV antibody or The antigen binding fragment thereof (referred to as 'secondary antibody or antigen binding fragment thereof') may further comprise the step of contacting the plate. The primary antibody or antigen-binding fragment thereof and the secondary antibody or antigen-binding fragment thereof may be the same or different. The primary antibody or antigen-binding fragment thereof and the secondary antibody or antigen-binding fragment thereof may be bound to a detectable moiety. For example, the secondary antibody or binding fragment thereof may be one that contains a detectable moiety such as gold. The primary and secondary antibodies or antigen-binding fragments thereof are monoclonal antibodies that specifically bind to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid of MERS-CoV, and the nucleo of MERS-CoV, respectively. It may be a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 4 in the capsid, or vice versa.

The sample may be a biological material derived from an individual. The subject may be a vertebrate. The vertebrate may be a mammal. The mammal may be a primate, including a human and a non-human primate, a camel, or a rodent, including a mouse and a rat. The sample may be frozen or left in its natural state.

The biological material may be nasal swap, nasal aspirate, nasopharyngeal swab, nasopharyngeal aspirate, blood or blood constituent, body fluid fluid) or a combination thereof.

Another aspect includes contacting a sample with an antibody or antigen-binding fragment thereof described above; Detecting a complex of the antibody or antigen-binding fragment thereof with MERS-CoV; And it provides a MERS-CoV diagnostic method comprising the step of detecting whether the sample MERS-CoV infection from the detection result.

The MERS-CoV diagnostic method may include determining that an individual from which the sample is derived is infected with MERS-CoV when an antibody or an antigen-binding fragment thereof and a complex of MERS-CoV is detected. In addition, when the complex is not detected, it may include determining that the individual from which the sample is derived is not infected with MERS-CoV.

According to one embodiment, an antibody specifically binding to a nucleocapsid of MERS-CoV and a composition and kit for diagnosing Middle East respiratory syndrome coronavirus comprising the same, and a method for diagnosing Middle East respiratory syndrome coronavirus using the same, Middle East respiratory syndrome Coronavirus infection can be measured efficiently.

1 is a side view showing an example of an analysis device for detecting and detecting the Middle East Respiratory Syndrome Coronavirus according to an embodiment of the present invention.

2 is a diagram showing the hydrophilicity of the nucleocapsid protein of MERS-CoV of SEQ ID NO: 1.

3A is a diagram showing the results of SDS-PAGE of polypeptides of MERS-CoV NP P1 to P5.

Figure 3b is a view showing the Western blot results of the anti-MERS CoV P1 monoclonal antibody.

Figure 3c is a diagram showing the Western blot results of the anti-MERS CoV P3 monoclonal antibody.

Figure 4 is a schematic illustration of the operation of the kit used in one embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Preparation of Monoclonal Antibodies Against Nucleocapsids of MERS-CoV

1. Preparation of antigen for immunity

A peptide antigen was synthesized in a hydrophilic site in the nucleocapsid (NP) gene of MERS-CoV and used as an immunogen. The hydrophilicity of the gene encoding the nucleocapsid of MERS-CoV was examined to synthesize peptide antigens for high hydrophilic site portions. 2 is a diagram showing the hydrophilicity of the nucleocapsid protein of MERS-CoV of SEQ ID NO: 1. Parker Hydrophilicity prediction program of Immune epitope database was used. The synthesized peptide antigens are the 22-40 amino acids, 126-146 amino acids, 164-202 amino acids, 234-259 amino acids, and 362-388 amino acids in the nucleocapsid of MERS-CoV of SEQ ID NO: 1, respectively. It is referred to as MERS-CoV NP P1, MERS-CoV NP P2, MERS-CoV NP P3, MERS-CoV NP P4, and MERS-CoV NP P5.

2. Monoclonal Antibody Preparation

(1) Preparation of Immunogen

After synthesizing immunological peptides for producing antibodies of MERS-CoV NP P1, P2, P3, P4, and P5, immunogens were prepared as follows. Immunogens used in the first immunization were prepared by complete Freud's Adjuvant (Sigma), which was prepared by emulsifying the ratio of antigen to the adjuvant in a 1: 1 ratio, and used for the second to fourth immunizations. The immunogen was prepared by incomplete Freud's Adjuvant (Sigma Co., Ltd.) in a 1: 1 ratio of antigen to the adjuvant.

(2) Inoculation

The immunogen prepared by the above method was immunized to BALB / c mice at 6 weeks of age and females as follows. Four intraperitoneal injections at weekly intervals in an amount of 200 μl / horse and after one week the antigen was diluted three times at a daily interval by diluting the antigen to 20% concentration (v / v) in Phosphate buffered saline (PBS). Intravenously injected 20 μl / time. After the third immunization, a medium titer test was performed with serum obtained from the tail vein. As a result, mice obtained with sufficient amounts of antibodies were selected to perform a cell fusion process.

(3) Hybridoma Cell Line Preparation

After immunization of the peptides of MERS-CoV NP P1, P2, P3, P4, and P5, spleen cells of the antibody-producing mice were removed and filtered through a 70 μm pore cell strainer (BD Falcon). The precipitated splenocytes were centrifuged to obtain a precipitate, and 5 ml of red blood cell lysis buffer (Sigma) was mixed and left for 1 minute to remove red blood cells. Spleen cells from which red blood cells were removed were washed three times with DMEM (Dulbecco's Modified Eagle's Medium, Gibco) and subjected to cell counting. The cells to be fused with the splenocytes were SP2 / 0-Ag 14 (ATCC CRL-1581) cell line, a mouse-derived myeloma cell, and the mixing ratio was 10: 1 (splenocytes: SP2 / 0). Mixed cells were washed twice with DMEM and then fused using PEG1500 (Roche). The cell fusion was performed with 1.7 ml of PEG1500 for 1 minute, 1 second for 30 seconds, 1 ml of 1 minute for DMEM, 2 ml of 1 minute for DEME, 6 ml for 30 seconds of DMEM, and finally 30 minutes of DMEM after 30 seconds. 10 ml was dropped for a second. The cell solution after fusion is obtained by centrifugation as a precipitate, and well with DMEM (hereinafter referred to as 'HAT medium') containing 10% of HAT (Gibco), antibiotics (Gibco), and fetal bovine serum (Hyclone). After mixing, the cells were dispensed into 96 well plates at a dose of 200 μl / well and grown in the incubator for 3 days.

(4) cell culture

The cell line after the fusion was grown at 37 ° C., 5% CO ₂ , and in a cell incubator maintained at humid conditions, and the fused cell line was selected by replacing HAT (hypoxanthine-aminopterin-thymidine medium) for two days at a week. Was

(5) Positive clone cell line detection (selection of hybridoma cells producing monoclonal antibodies)

The cell lines selected by HAT medium were screened for positive clones by Enzyme-linked immunosorbent assay (ELISA). Specifically, 100 μl of the fused cell line culture grown in each well of the 96 well plate was placed in a 96 well adsorption plate (Costar) coated with a concentration of 2.5 ug / ml of MERS-CoV nucleocapsid (NP) gene antigen. Dispense at a dose of / well and react for 1 hour at 37 ° C. After 1 hour of reaction, the cells were washed five times, and the anti mouse IgG HRP conjugate and the anti mouse IgM HRP conjugate were dispensed at a dose of 100 μl / well, and reacted at 37 ° C. for 30 minutes. After the reaction for 30 minutes, it was washed five times, the substrate liquid was dispensed at a dose of 100 μl / well, and the color reaction was performed for 15 minutes, and the reaction stop solution was added with a dose of 100 μl / well. After stopping the reaction, the absorbance was measured at 450 nm in the reader to select the positive clones MERS-CoV NP P1 and MERS-CoV NP P3 which showed high values. Selected positive clones were transferred to 24 well tissue culture plates and incubated for 3 days, followed by secondary screening in the same manner as above, that is, by limiting dilution of hybridoma cell lines obtained through repeated selection, the final single positive clone candidate group. MERS-CoV NP P1, and MERS-CoV NP P3 were selected.

The final selected monoclonal antibody-producing hybridomas were named anti-MERS CoV P1 monoclonal antibodies, and anti-MERS CoV P3 monoclonal antibodies, which were deposited with the Bank of Korea Cell Line and assigned accession numbers.

(6) antibody purification

The final selected positive clones were transferred to a T75 (SPL) dish and incubated for 3 days, and injected into the abdominal cavity of mice at 1 × 10 6 cell / ml cell density, and collected one week later. The mouse ascites liquid obtained through the above method was precipitated by centrifugation (3000 rpm, 10 minutes, 4 ° C.), and the supernatant was mixed at a 1: 1 ratio with a binding buffer (Binding buffer, Thermo scientific) at room temperature. Reaction was carried out for 1 hour. After 1 hour, the suspension was precipitated once again by centrifugation (3000 rpm, 10 minutes, 4 ° C.), and the unprecipitated suspension was filtered again using a filter having a 1.2 μm pore size. Ascites fluid was completely removed from the suspension was placed in a column containing Protein G resin (Pierce) to induce binding between the antibody and Protein G. After passing through the column, the plural solution was washed three times with phosphate buffer solution and eluted using an elution buffer (Elution buffer, Thermo scientific). The eluted antibody was again applied to a PD-10 column (GE heathcare) and eluted with phosphate buffer solution for final purification.

Example 2: Identification of Epitopes in MERS-CoV Nucleocapsids to Which Specific Monoclonal Antibodies Binding

In order to determine whether the monoclonal antibody prepared in Example 1 specifically recognizes the amino acid sequence of which region of the MERS-CoV nucleocapsid (NP). The MERS-CoV NP gene was divided into 5 peptides, and the synthesized peptide was conjugated with Bovine serum albumine (BSA). The conjugate was prepared by Ab Frontier for the efficiency of the experiment. MERS-CoV NP P1, MERS-CoV NP P2, MERS-CoV NP P3, MERS-CoV NP P4, and MERS-CoV NP P5 conjugated to BSA were each expressed in the nucleocapsid of MERS-CoV of SEQ ID NO: 1. To 40th amino acid, 126-146 amino acids, 164-202 amino acids, 234-259 amino acids, and 362-388 amino acids.

For SDS-PAGE, a 10% polyacrylamide gel was prepared and then 5 MERS-CoV combined with bovine serum albumin (BSA) (EQUITECH-BIO, INC) and BSA prepared above as a control. Polypeptides of NP P1 to P5 were each loaded at 1.25 μg. Subsequently, SDS-PAGE was performed for 1 hour under conditions of 160V and 400 mA, and then, mini-Protean 3 Electrophoresis (BIO-RAD) was used on a nitrocellulose membrane (GE healthcare) under conditions of 160V and 400 mA. The polypeptides were transferred for time. 3A is a diagram showing the results of SDS-PAGE of polypeptides of MERS-CoV NP P1 to P5. In FIG. 3A, lane 1 represents BSA control, lane M represents a protein marker, and lanes 2 to 6 represent polypeptides of MERS-CoV NP P1 to P5, respectively.

For Western blot, the SDS-PAGE gel described above was transferred to NC-membrane (200V, 200mA, 1 hour). Placed in 5% skim milk and blocked for 30 minutes at room temperature, then 10 μg / mL of anti-MERS CoV P1 monoclonal antibody (in 5% Skim milk / TBS) and anti-MERS CoV P3 monoclonal antibody (in 5% Skim milk / TBS) was reacted at room temperature for 1 hour. Thereafter, washing was repeated three times for 5 minutes using a wash solution (1M Tris, 0.0025% Proclin 300, 0.1% Tween 20, and 0.15 mM NaCl) to remove monoclonal antibody that did not bind to the polypeptide on the membrane. Subsequently, 2 μg / mL Anti mouse IgG AP (SIGMA Cat. NoA4312) was added to the scheme milk and reacted at room temperature for 1 hour, and then the washing process was repeated. NoA4312) was removed and developed with BCIP / NBT solution (SIGMA Cat. NoB6404).

Results of Western blot, FIG. 3B is a diagram showing the Western blot results of the anti-MERS CoV P1 monoclonal antibody. As shown in FIG. 3B, the anti-MERS CoV P1 monoclonal antibody according to one embodiment of the present invention showed color development only in lane 2 and no color development in the remaining lanes 3-6. Therefore, it was confirmed that the anti-MERS CoV P1 monoclonal antibody according to an embodiment of the present invention specifically reacts with a polypeptide including amino acids 22 to 40 of lane 2. Therefore, the monoclonal antibody, that is, anti-MERS CoV P1 monoclonal antibody according to one embodiment of the present invention against MERS-CoV prepared in Example 1 from the above result is the nucleocapsid of MERS-CoV (SEQ ID NO: 1) It was confirmed that the 22-40 amino acid sequence part of (SEQ ID NO: 2) is an epitope.

In addition, Figure 3c is a diagram showing the Western blot results of the anti-MERS CoV P3 monoclonal antibody. As shown in FIG. 3C, the anti-MERS CoV P3 monoclonal antibody according to one embodiment of the present invention showed distinct color development only in lane 4, and did not show distinct color development in lanes 2, 3, 5, and 6. Therefore, it was confirmed that the anti-MERS CoV P3 monoclonal antibody according to an embodiment of the present invention specifically reacts with a polypeptide including amino acids 164 to 202 of lane 4. Therefore, the monoclonal antibody according to one embodiment of the present invention, the anti-MERS CoV P3 monoclonal antibody according to one embodiment of the present invention against MERS-CoV prepared in Example 1 from the above results It was confirmed that the 164 to 202th amino acid sequence portion (SEQ ID NO: 4) of cleopacapside (SEQ ID NO: 1) was an epitope.

Example 3: MERS-CoV Virus Diagnostic Experiment Using Antibodies Against MERS-CoV

To diagnose MERS-CoV, an antibody according to one embodiment of the present invention was used to sample 81 camels (Camel) nasal swabs, and the collected body fluid was used as a sample to diagnose MERS-CoV virus.

1. Use kit

A monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid of MERS-CoV, and an amino acid sequence of SEQ ID NO: 4 in the nucleocapsid of MERS-CoV Kits were prepared comprising monoclonal antibodies (called 'P3') that specifically bind to the indicated epitopes. Monoclonal antibodies that specifically bind to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid are those produced in hybridoma cells having accession number KCLRFBP00331. In addition, a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 4 in the nucleocapsid of MERS-CoV is one produced in hybridoma cells having accession number KCLRFBP00331. Figure 4 is a schematic illustration of the operation of the kit used in one embodiment of the present invention. (A), (b) and (c) of FIG. 4 indicate that the analyte 400 is moved by capillary movement in that order, and the analyte 400 operates with a kit according to one embodiment of the present invention. This is a schematic of the principle. In FIG. 4, there is a conjugate 100 of the first antibody and the gold particles in the storage pad of the kit, the second antibody 200 is immobilized in the detection region T of the chromatography membrane material, and the control region C ) Is an antibody 300 specific for the conjugate of the first antibody and the gold particles. In one embodiment of the present invention, a monoclonal antibody of P1, the second antibody 200, is immobilized in the detection region T, and is conjugated with a gold particle and a monoclonal antibody of P3, the first antibody 100. In the case of the analyte 400 complexing the MERS-CoV (referred to as 'MERS'), the presence of the MERS-CoV may be determined by detecting the presence of the complex of P1 and P3 in the detection region.

Specifically, a monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid of MERS-CoV was coated on a plate.

In addition, monoclonal antibodies that specifically bind to an epitope represented by the amino acid sequence of SEQ ID NO: 4 in the nucleocapsid of MERS-CoV are conjugated with gold particles. The conjugation with the gold particles was carried out as follows: Gold chloride was dissolved in distilled water at 0.01% and then heated with addition of 0.1% sodium citrate solution. After heating for about 10 minutes, the mixture was cooled and refrigerated to prepare a colloidal gold solution. Thereafter, anti-MERS-CoV P3 monoclonal antibody was reacted with the colloidal gold solution for 10 minutes to the final concentration of 20 ㎍ / ㎖ to induce conjugation reaction. After stabilizing the gold particles, centrifuge at 10,000 g for 30 minutes to form a precipitate. The precipitate was again dissolved in physiological saline, filtered through a filter of 0.45 μm, and then absorbed at 540 nm, diluted to a final 20, and conjugated with gold to anti-MERS-CoV P3.

A kit comprising a P1 monoclonal antibody immobilized on a plate and a P3 monoclonal antibody conjugated with gold particles was used as a MERS-CoV diagnostic kit.

2 sample used

The fluids collected from the nasal swabs of 81 camels were used.

3 results

The results of the presence or absence of MERS-CoV for the sample used using the MERS-CoV diagnostic kit and RT-PCR are shown in Tables 3 and 4.

TABLE 3 Sample number Diagnostic Results of the Kit of the Invention RT-PCR Results Sample number Diagnostic Results of the Kit of the Invention RT-PCR Results One P P 42 N N 2 N N 43 N N 3 N N 44 N N 4 P P 45 N N 5 N N 46 N N 6 N N 47 N N 7 N N 48 N N 8 N N 49 N N 9 P P 50 P P 10 P P 51 N N 11 N N 52 N N 12 P N 53 N N 13 N N 54 P P 14 N N 55 P P 15 N N 56 N N 16 P N 57 N N 17 N N 58 P P 18 N N 59 N N 19 N N 60 N N 20 N N 61 N N 21 P P 62 N N 22 N N 63 N N 23 N N 64 N N 24 N N 65 N N 25 N N 66 P P 26 N N 67 N N 27 N N 68 N N 28 N N 69 N N 29 N N 70 N N 30 P P 71 N N 31 N N 72 N N 32 N N 73 P N 33 P P 74 N N 34 P N 75 N N 35 N N 76 N N 36 P N 77 N N 37 N N 78 N N 38 N N 79 N N 39 N N 80 P P 40 P P 81 N N 41 N N

Table 4 RT-PCR Results Sum positivity voice Kit of the invention positivity 14 5 19 voice 5 57 62 Sum 19 62 81

In Table 3, P means positive, N means negative, and Table 4 summarizes the contents of Table 3. Thus, the sensitivity of the kit of the present invention is 73.68% and specificity is 91.94%.

The positive likelihood ratio is 9.14 and the negative likelihood ratio is 0.29. The positive likelihood ratio was calculated by {(14/19) / (5/62)}. Since the positive likelihood ratio was 9.14, it was confirmed that the diagnostic kit including the antigen specifically binding to the epitope of the nucleocapsid of MERS-CoV of the present invention has significance as a diagnostic kit. The negative likelihood ratio was calculated by {(5/19) / (57/62)}. In addition, the prevalence by MERS-CoV is 23.46%.

Depositary Name: Korea Cell Line Research Foundation

Accession number: KCLRFBP00331

Trust Date: 20141014

Depositary Name: Korea Cell Line Research Foundation

Accession number: KCLRFBP00332

Trust Date: 20141014

[Revision according to Rule 26 09.03.2015]

[Revision according to Rule 26 09.03.2015]

Claims (9)

A monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid of Middle East respiratory syndrome coronavirus (MERS-CoV), represented by the amino acid sequence of SEQ ID NO: 4 An antibody that is a monoclonal antibody that specifically binds to an epitope, or a combination thereof. The antibody of claim 1, wherein the monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 2 is produced in a hybridoma cell having accession number KCLRFBP00331. The antibody of claim 1, wherein the monoclonal antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 4 is produced in a hybridoma cell having accession number KCLRFBP00332. An antigen binding fragment of the antibody according to claim 1. Hybridoma cells producing monoclonal antibodies that specifically bind to an epitope represented by the amino acid sequence of SEQ ID NO: 2 in the nucleocapsid of MERS-CoV (Accession No .: KCLRFBP00331). Hybridoma cells producing monoclonal antibodies that specifically bind to an epitope represented by the amino acid sequence of SEQ ID NO: 4 in the nucleocapsid of MERS-CoV (Accession No .: KCLRFBP00332). MERS-CoV diagnostic composition of the individual comprising the antibody of claim 1, or an antigen-binding fragment thereof. MERS-CoV diagnostic kit of an individual comprising the antibody of claim 1, or an antigen-binding fragment thereof. Contacting the sample with the antibody of claim 1 or an antigen-binding fragment thereof; And Detecting the complex of the antibody or antigen-binding fragment thereof and MERS-CoV; method for detecting MERS-CoV of a subject.

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