KR102201261B1 - Monoclonal antibody specific to Chikungunya virus and uses thereof - Google Patents

Abstract

The present invention relates to a monoclonal antibody specific for chikungunya virus and its use, and more particularly to a monoclonal antibody that specifically binds to chikungunya virus envelope protein 2 and its use.
The chikungunya virus-specific monoclonal antibody provided by the present invention recognizes the chikungunya virus envelope protein 2 with a very high binding affinity, and shows a very high specificity that other mosquito-borne viruses do not recognize. , Chikunguniya virus infection diagnosis, etc. can be very useful.

Description

Monoclonal antibody specific to Chikungunya virus and uses thereof {Monoclonal antibody specific to Chikungunya virus and uses thereof}

The present invention relates to a monoclonal antibody specific for chikungunya virus and its use, and more particularly to a monoclonal antibody that specifically binds to chikungunya virus envelope protein 2 and its use.

Chikungunya fever is a mosquito-borne disease infected with Chikungunya virus (CHIKV), a single-stranded positively susceptible RNA virus of the genus Alphavirus of the Togaviridae family. CHEKV is transmitted to people via Aedes mosquitoe and can cause a number of symptoms including fever, rash and multiple sclerosis. CHIKV infection has caused many outbreaks in Africa, Asia and Europe over the past decade, with serious consequences in the Indian Ocean region, including India in 2004. Since then, the risk of re-emergence of CHIKV has spread worldwide and has become a global health problem. Despite its importance, CHIKV infection in mosquito-borne diseases caused by various arboviruses, including DENV (Dengue virus), ZIKV (Zika virus), and JEV (Japanese Encephalitis virus), classified into the Flavivirus genus of Flaviviridae. Because the clinical features are similar, it is difficult to accurately identify an infected virus only by clinical symptoms.

Diagnosis of CHIKV infection is generally divided into two detection methods based on viral RNA and protein. The detection of viral RNA by reverse transcription-polymerase chain reaction (RT-PCR) is sensitive, but its use is limited because it can detect short-lived viruses only in the early stages of CHIKV infection. On the other hand, protein-based diagnosis can be used even after the acute phase of CHIKV infection and is a more accurate, complementary and reliable method. Protein-based methods have been applied to ELISA, immunofluorescence assay (IFA) and immunochromatographic assay (ICA), and a key component of the diagnosis is the robust and accurate response of viral antigens and antibodies. Several protein-based diagnostics are available to detect CHIKV infection, but low sensitivity remains a limitation of recent CHIKV diagnosis. Therefore, it is necessary to develop a specific antibody with more high sensitivity in order to ultimately improve the accuracy and sensitivity of CHIKV diagnosis.

CHIKV has two envelope proteins, E1 and E2, which cover the viral surface in the form of spikes and mediate viral entry into host cells. E2 is responsible for cell adhesion and E1 regulates membrane fusion during viral infection. The extracellular portion of the E2 protein contains domains for receptor binding and is immunogenic. Therefore, the most abundant antibody induced by CHIKV infection has been reported to target the CHIKV-E2 protein. That is, the CHIKV-E2 protein can be an excellent target to develop an anti-CHIKV-E2 monoclonal antibody (mAb) with high binding affinity and specificity to improve CHIKV diagnosis.

Accordingly, the present inventors have a chikungunya virus-specific monoclonal capable of accurately distinguishing and detecting various arboviruses including DENV, ZIKV and JEV that cause mosquito-borne diseases and chikungunya virus (CHIKV). As a result of extensive research in order to develop an antibody, it was found that an anti-CHIKV-E2 monoclonal antibody that binds to CHIKV envelope protein 2 with very high specificity and sensitivity through a hybridoma system can be produced. Finished.

Accordingly, it is an object of the present invention to provide a monoclonal antibody that specifically binds to Chikungunya virus produced by hybridoma cells with accession number KCTC13814BP.

Another object of the present invention is to provide a hybridoma cell with accession number KCTC13814BP producing the antibody.

Another object of the present invention is to provide a composition/kit for detecting chikungunya virus comprising the antibody.

Another object of the present invention is to provide a method for detecting chikungunya virus comprising the step of detecting the formation of an antigen-antibody complex by contacting the antibody with a sample sample.

In order to achieve the above object of the present invention, the present invention provides a monoclonal antibody that specifically binds to Chikungunya virus produced by hybridoma cells with accession number KCTC13814BP.

In order to achieve another object of the present invention, the present invention provides a hybridoma cell with accession number KCTC13814BP producing the antibody.

In order to achieve another object of the present invention, the present invention provides a composition/kit for detecting chikungunya virus comprising the antibody.

In order to achieve another object of the present invention, the present invention provides a method for detecting chikungunya virus comprising the step of detecting the formation of an antigen-antibody complex by contacting the antibody with a sample sample.

Hereinafter, the present invention will be described in detail.

The present invention provides a monoclonal antibody that specifically binds to Chikungunya virus produced by hybridoma cells with accession number KCTC13814BP.

Chikungunya virus (CHIKV) is an enveloped positive-sense RNA virus of the genus Alphavirus of the Togaviridae family, and is transmitted by Aedes mosquitoes. The mature CHIKV virion contains two glycoproteins E1 and E2 generated from the precursor polyprotein p62-E1 by proteolytic cleavage. E2 functions in viral attachment, while E1 mediates membrane fusion to allow viral introduction. In humans, CHIKV infection is serious and in some cases causes fever and joint pain that can last for years. CHIKV has caused outbreaks in most of sub-Saharan Africa and parts of Asia, Europe and the Indian Ocean and Pacific. In December 2013, the first outbreak of CHIKV occurred in the Western Hemisphere, and indigenous cases were identified in St. Martin. These viruses have spread rapidly to almost all islands in the Caribbean, as well as Central America, South America and North America. Within a year, more than one million suspected cases of CHIKV were reported in the Western Hemisphere, and endemic transmission was recorded in more than 40 countries, including the United States.

According to an embodiment of the present invention, it was shown that the anti-CHIKV monoclonal antibody (mAb) produced in the present invention particularly effectively binds to the CHIKV E2 protein. In particular, the mAb of the present invention showed higher binding affinity than mAb and commercially available mAb produced by other hybridoma cells.

Several studies have shown that domain A of CHIKV E2 is located peripherally and is mainly exposed to the viral surface, while domains B and C are located in the center of E2 and close to the viral membrane. The surface structure of the CHIKV Virion is covered with 80 trimer spikes. A single spike consists of three E1/E2 heterodimers. Thus, while the mAb according to the present invention is capable of binding epitopes exposed on the viral surface, mAbs and commercially available mAbs produced by other hybridoma cells are viral surface or hidden by dimerization of E1 and E2. It was determined that the binding affinity for CHIKV E2 was lower than that of the mAb according to the present invention because the epitope inside the region was recognized.

According to another embodiment of the present invention, the mAb of the present invention hardly recognizes viruses transmitted by other mosquitoes, including ZIKV, DENV and JEV. In contrast, mAb produced by other hybridoma cells had significantly higher binding affinity for DENV 1-4 type as well as CHIKV than the PBS control. Currently, clinical diagnosis is difficult because of similar clinical symptoms between viruses transmitted by mosquitoes in the early stages of CHIKV infection (Chen and Wilson, 2010; Hoarau et al., 2010). Therefore, since CHIKV belongs to Alphavirus of Togaviridea family, and DENV, JEV and ZIKV belong to Flavivirus of Flavivirdea family, molecular diagnosis is very important to accurately diagnose infection. Therefore, the mAb according to the present invention can be very usefully used to identify CHIKV from other viruses transmitted by mosquitoes.

In the present invention, the term'binding affinity' generally refers to the strength of the total sum of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, as used herein,'binding affinity' refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of molecule X for partner Y can generally be expressed as the dissociation constant (Kd). Affinity can be determined by conventional methods known in the art, including those described herein. Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high-affinity antibodies generally bind antigens more quickly and tend to remain bound for a longer period of time. Various methods of measuring binding affinity are known in the art, and for the purposes of the present invention, any of these methods can be used.

In the present invention, the term "monoclonal antibody" refers to a highly specific antibody directed against a single antigenic site as a term known in the art. Monoclonal antibodies are also referred to as single cell population antibodies or monoclonal antibodies. Typically, unlike polyclonal antibodies, which contain different antibodies directed against different epitopes (antigen determinants), monoclonal antibodies are directed against a single determinant on the antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and also have another advantage of not being contaminated by other immunoglobulins because they are synthesized by hybridoma culture.

The mAb provided herein is an antibody specific for chikungunya virus, and includes not only the entire antibody form, but also functional fragments of antibody molecules. The whole antibody is a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to a heavy chain by a disulfide bond. 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 (γ3). ), gamma4(γ4), alpha1(α1) and alpha2(α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

The functional fragment of an antibody molecule refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2, Fv, and the like. Among antibody fragments, Fab has a structure having a light chain and a heavy chain variable region, a light chain constant region, and a heavy chain first constant region (CH1), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. In the F(ab')2 antibody, a cysteine residue in the hinge region of Fab' forms a disulfide bond. Fv is the smallest antibody fragment having only a heavy chain variable region and a light chain variable region. Recombination techniques for generating Fv fragments are disclosed in International Patent Publications WO 88/10649, WO 88/106630, WO88/07085, WO 88/07086 and WO 88/ It is disclosed in 09344. dsFv (double-chain Fv) is a non-covalent bond between the heavy chain variable region and the light chain variable region, and scFv (single chain Fv) is generally a heavy chain variable region and a single chain variable region connected by a covalent bond through a peptide linker or C-terminal Since it is directly connected to dsFv, it can form a dimer-like structure.

Such antibody fragments can be obtained using proteolytic enzymes (e.g., restriction digestion of the entire antibody with papain yields Fab, and digestion with pepsin yields F(ab')2 fragments), preferably It can be produced through genetic recombination technology. In the present invention, the antibody is preferably in the form of Fab or in the form of a whole antibody.

According to an embodiment of the present invention, the mAb of the present invention was confirmed to be an IgG2 immunoglobulin isotype, more specifically, it was confirmed to be an IgG2b immunoglobulin isotype, and more specifically, an IgG2b kappa It was confirmed to be a chain isotype.

The advantage of using monoclonal antibodies to detect antigens is that they have specific interactions by recognizing a single epitope. As such, various approaches can be used to map epitopes involved in the interaction between antigen and antibody. For example, biopanning using a phage display peptide library, immunoblotting of fragments generated by digesting antigenic polypeptides with proteases and screening to determine fragments containing epitope sequences, activated membranes or solid phases such as polyethylene pins Screening the peptide arrays immobilized on, and a competitive ELISA assay using soluble peptides to determine the importance of each amino acid sequence residue in the epitope sequence.

The monoclonal antibody produced by the hybridoma may be used without purification, but in order to obtain the best result, it is used after purifying with high purity (eg, 95% or more) according to a method well known in the art. It is desirable. As such a purification technique, it can be separated from a culture medium or ascites fluid using a purification method such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.

In the present invention, the term'hybridoma' is known in the art and refers to a cell formed by fusion of an antibody-producing cell and an immortal cell. In general, hybridoma cells are made by fusing immune cells from an immunologically suitable host animal such as a mouse injected with an antigenic protein and myeloma cells, and can continuously supply monoclonal antibodies.

In the present invention, the term "specific binding" to "specific" means a non-specific interaction and a measurably different binding. Specific binding can be determined, for example, by determining the binding of a molecule by comparing the binding of a control molecule, which is a molecule of similar structure that generally does not retain binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, for example an excess of unlabeled target. In this case, it is specific binding if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. In one embodiment, such terms mean that the mAb according to the invention does not bind other similar viruses, but specifically binds only to the CHIKV viral antigen, more specifically CHIKV E2.

Hybridoma cell line 19-1 producing mAb according to the present invention was deposited with the Korea Research Institute of Bioscience and Biotechnology under the accession number KCTC13814BP on February 3, 2019.

The present invention also provides a composition for detecting chikungunya virus comprising the mAb according to the present invention.

In the present invention, in the detection composition, in addition to the mAb of the present invention, a carrier used for immunological analysis, a detection label capable of generating a detectable signal, and a reagent capable of measuring enzyme activity when the labeling substance is an enzyme are included. It may contain additionally. The carrier is not limited thereto, but may be a soluble carrier, for example, a physiologically acceptable buffer (eg PBS) known in the art, or an insoluble carrier, and a combination thereof, and the detection label and the detection label The reagent for measuring the activity of is to measure any one of color development, fluorescence, and luminescence characteristics by a reaction. Various detection markers used in general immunoassay methods and reagents for measuring activity according to each detection label may be used.

The present invention also provides a method for detecting chikungunya virus comprising the step of detecting antigen-antibody complex formation by contacting the mAb of the present invention with a sample sample.

In the present invention, the term "antigen-antibody complex" refers to a combination of a chikungunya virus E2 antigen in a sample and a monoclonal antibody or fragment thereof according to the present invention that recognizes it, and the antigen-antibody complex is a colormetric method (colormetric method), electrochemical method, fluorescence method, luminometry, particle counting method, visual assessment, and scintillation counting method It can be detected by any method. However, it is not necessarily limited to these, and various applications and applications are possible.

In the present invention, various markers can be used for detecting the antigen-antibody complex. Specific examples may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, and radioactive isotopes, but are not necessarily limited thereto.

Preferred enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, or β-lactamase. Preferred fluorescent substances include quantum dots, fluorine Lescein, Eu ³⁺ , Eu ³⁺ chelate or cryptate, preferred ligands include biotin derivatives, preferred luminescents include acridinium esters or isoluminol derivatives, and preferred microparticles include colloidal gold or There is a colored latex, and a preferred radioactive isotope is ⁵⁷ Co, ³ H, ¹²⁵ I or ¹²⁵ I-Bolton Hunter's reagent, but is not limited thereto.

The method of detecting the antigen-antibody complex may preferably be detected using an enzyme immunosorbent method (ELISA), but is not limited thereto. Enzyme immunosorbent is a direct ELISA using an antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled secondary antibody that recognizes the capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, and attaches to a solid support. Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the prepared antibody and antigen, and a label that recognizes this antibody after reacting with another antibody that recognizes the antigen in the complex of the antibody and the antigen attached to a solid support. It includes various ELISA methods, such as indirect sandwich ELISA using a secondary antibody.

According to the ELISA detection method, a biological sample such as urine, blood, serum or plasma is contacted with a monoclonal antibody of the present invention coated on a solid support such as a microtiter plate, a membrane, a test strip, or the like. As a specific example, after coating the wells of the microtiter plate with the monoclonal antibody of the present invention and blocking the nonoccupied binding sites with, for example, BSA, the wells of the coated plate are incubated with a sample sample, The presence of the antigen-antibody complex can then be determined. The presence of the antigen-antibody complex can be confirmed using an antibody specific for the antigen of the antigen-antibody complex, ie, the mAb according to the present invention.

The monoclonal antibody or fragment thereof may have a detection label, and if it does not have a detection label, these monoclonal antibodies or fragments thereof can be captured, and can be identified by treatment with another antibody having a detection label. .

The present invention also provides a kit for detecting chikungunya virus comprising the mAb according to the present invention.

The chikungunya virus detection kit may be prepared in the form of a protein chip to which the mAb of the present invention is attached. The kits of the present invention may include monoclonal antibodies or fragments thereof and tools/reagents used for immunological analysis.

Tools/reagents used in the immunological assay include a suitable carrier, a labeling substance capable of generating a detectable signal, a solubilizing agent, a detergent, and the like. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.

Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, Polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymers such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

The assay system for use in the detection method and diagnostic kit of the present invention includes, but is not limited to, an ELISA plate, a dip-stick device, an immunochromatography test strip and a radiation split immunoassay device, and a flow-through. Devices, etc.

The chikungunya virus-specific monoclonal antibody provided by the present invention recognizes the chikungunya virus envelope protein 2 with a very high binding affinity, and shows a very high specificity that other mosquito-borne viruses do not recognize. , Chikunguniya virus infection diagnosis, etc. can be very useful.

1, including FIGS. 1A to 1C, shows the results of preparing a hybridoma generating an anti-CHIKV-E2 antibody.
(a) CHIKV-E2 protein was separated by 12% SDS-PAGE and visualized by Coomassie blue staining. For immunoblotting, proteins were transferred from gel to PVDF membrane and probed with commercial monoclonal antibodies (Chk265 or 16A12) followed by HRP-binding anti-mouse IgG. Lane M represents the protein marker (kDa).
(b) and (c) BALB/c mice were immunized three times with CHIKV-E2 protein by footpad injection at intervals of 2 weeks. Two weeks after each immunization, serum was obtained to evaluate the amount of antibody produced (b), and the amount of antibody produced in the culture supernatant of hybridomas prepared using lymphocytes obtained from immunized mice was evaluated (c). The binding affinity for the CHIKV-E2 protein was measured by ELISA.
Figure 2, including Figures 2a to 2c, is a result of comparing the binding affinity of anti-CHIKV-E2 mAbs to CHIKV-E2 protein.
(a) CHIKV-E2 protein was coated on an ELISA plate in a dose dependent manner. Plates were blocked with 5% skim milk in PBS, treated with anti-CHIKV-E2 mAb followed by HRP-binding anti-mouse IgG and absorbance measured. Commercially available anti-CHIKV-E2 mAb, Chk265 and 16A12, were used for comparison.
(b) ELISA plates were coated with CHIKV-E2 protein, blocked, treated with serially diluted anti-CHIKV-E2 mAb and HRP-binding anti-mouse IgG, and absorbance was measured. Commercially available anti-CHIKV-E2 mAb, Chk265 and 16A12, were used for comparison.
(c) CHIKV-E2 protein was separated by 12% SDS-PAGE and transferred to a PVDF membrane. The membrane was probed with each anti-CHIKV-E2 mAb followed by HRP-binding anti-mouse IgG to confirm that each mAb detected CHIKV-E2. Lane M represents the protein marker (kDa). Commercially available anti-CHIKV-E2 mAb, Chk265 and 16A12, were used for comparison.
Figure 3, including Figures 3a to 3c, is a result of evaluating the binding affinity of anti-CHIKV-E2 mAb for inactivated CHIKV.
(a) Serially diluted CHIKV was coated on an ELISA plate. Plates were blocked with 5% skim milk in PBS, treated with anti-CHIKV-E2 mAb followed by HRP-binding anti-mouse IgG and absorbance was measured. Commercially available anti-CHIKV-E2 mAb, Chk265 and 16A12, were used for comparison.
(b) ELISA plates were coated with inactivated CHIKV, blocked, treated with serially diluted anti-CHIKV-E2 mAb, followed by HRP-binding anti-mouse IgG and absorbance was measured. Commercially available anti-CHIKV-E2 mAb, Chk265 and 16A12, were used for comparison.
(c) Inactivated CHIKV was separated by 12% SDS-PAGE and transferred to a PVDF membrane. The membrane was probed with anti-CHIKV-E2 mAb followed by HRP-binding anti-mouse IgG. Commercially available anti-CHIKV-E2 mAb, Chk265 and 16A12, were used for comparison. Lane M represents the protein marker (kDa).
Figure 4, including Figures 4a to 4c, is a result of evaluating the specificity of anti-CHIKV E2 mAb.
Inactivated arboviruses (CHIKV, ZIKV, JEV and DENV 1-4) were coated on ELISA plates. After blocking with 5% skim milk in PBS, the plate was treated with (a) 19-1 mAb, (b) 21-1 mAb or (c) Chk265 mAb and HRP-binding anti-mouse IgG and absorbance was measured. Statistically significant differences were analyzed by one-way ANOVA. * P <0.05, ** P <0.01, *** P <0.001.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are for illustrative purposes only, and the present invention is not limited thereto.

Experiment method

1. Mouse immunization and production of hybridomas

Six week old female BALB/c mice were purchased from Orient Bio (Gyeonggi-do, Republic of Korea) and reared in sterile facilities. CHIKV-E2 was purchased from Sino biological (Sino Biological, Beijing, China). Mice were immunized three times with 10 μg of CHIKV-E2 mixed with TiterMax Gold adjuvant (Sigma-Aldrich, St. Louis, MO, USA) by foodpad injection at 2 weeks intervals. Two weeks after each immunization, serum was collected from immunized mice. Two weeks after the final immunization, cells were isolated from the popliteal lymph nodes and used to prepare B cell hybridomas producing anti-CHIKV-E2 antibodies. Fusion with myeloma FO cells was performed as previously described (Heo et al., 2010). The binding affinity of the antibodies produced by these hybridoma cells was measured by ELISA. Monoclonal antibodies were purified using a protein G column (GE Healthcare, Uppsals, Sweden). The isotype of each mAb was determined using an immunoglobulin isotyping kit (Roche Diagnostics, Mannheim, Germany).

2. Virus

ZIKV (week MR766) was proliferated and maintained in Vero E6 cells. JEV (SA14-14-2 weeks) proliferated and maintained in BHK-21 cells. Vero E6 (ATCC CRL1586) and BHK-21 (ATCC CCL10) cells were maintained in Eagle's minimum essential medium (HyClone Laboratories, Inc., UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, Inc.). I did. All cell lines were cultured at 37° C. in 5% CO ₂ conditions. Cells were seeded in T75 flasks to 60% confluence and incubated for 16 hours. Cells were infected with 5 ml of suspended virus stock diluted 1:25 with fresh culture medium. The infectious suspension was replaced for virus propagation and the culture supernatant was harvested on days 3-5 to obtain the virus. The culture supernatant was filtered and precipitated for 12 hours in PBS for 4 hours. The virus precipitate was pelleted by centrifugation (14,000 xg, 1 hour, 4° C.), resuspended in 300 ul sterile PBS, and stored at -80° C. Infectious factor titers were estimated according to the number of PFUs by plaque assay. After incubation at 65° C. for 30 minutes to inactivate the virus, it was used in the experiment. Inactivated viral lysates of CHIKV and DENV type 1-4 were obtained from ZeptoMetrix corperation (ZeptoMetrix Corp., Franklin, MA, USA).

3. ELISA assay

MaxiSorp 96-well plate (Thermo Scientific, Roskilde, Denmark) was coated with CHIKV-E2 (5 μg / ml) or inactivated CHIKV lysate (90 μg / ml) in PBS at 4° C. for 16 hours, and 5% skim milk Treated with PBS at 37° C. for 1 hour and washed with 0.05% Tween-20 in PBS (PBST). Plates were incubated with 1:50,000 diluted serum of CHIKV-E2 immunized mice at 37° C. for 2 hours, culture supernatant of primary clone or purified mAb, and then horse-radish peroxidase (HRP)-conjugated anti-mouse It was incubated with IgG (1:5000) (Cell Signaling Technologies, Danvers, MA, USA) at 37°C for 1 hour. After washing the plate with PBST, the reaction was colored using a colored tetramethylbenzidine substrate (BD Biosciences, San Diego, CA, USA) and terminated with 2N H ₂ SO ₄ . Optical density (OD) was measured at 450 nm using a Versamax microplate reader (Molecular Devices, San Francisco, CA, USA). Commercially available anti-CHIKV-E2 mAb, 16A12 and Chk265 were purchased from native antigen (Oxford, UK) and absolute antibody (Oxford, UK), respectively.

In the cross-reaction experiment, MaxiSorp 96-well plates were used for CHIKV (10 μg / ml), ZIKV (1.7 x 10 ⁵ PFU / ml), JEV (1.7 x 10 ⁵ PFU / ml) or DENV type 1-4 (10 μg / ml). ) Containing carbonate-bicarbonate buffer (pH 9.6) and treated with an inactivated virus lysate at 4° C. for 16 hours. The plate was incubated with 5% skim milk in PBS for 2 hours at constant temperature, washed with 0.025% Tween-20 in PBS, and incubated with the resulting anti-CHIKV-E2 mAb at 37° C. for 1 hour 30 minutes. . It was treated with HRP-conjugated anti-mouse IgG (1:5000) (Thermo Fisher, Rockford, IL, USA) at 37° C. for 1 hour. The plate was washed and colored with chromogenic tetramethylbenzidine substrate (Thermo Fisher). The reaction was terminated with 2N H ₂ SO ₄ and absorbance was measured at 450 nm with a Versamax micro plate reader (Molecular Devices).

4. Immunoblotting

CHIKV-E2 (1 μg) or inactivated CHIKV lysate (3 μg) was incubated at 37° C. for 5 minutes with reducing sample buffer and heated at 95° C. for 5 minutes. Samples were separated by 12% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to a polyvinylidene fluoride membrane (Bio-Rad). After blocking with 5% skim milk in TBS containing 0.05% Tween-20, the membranes were incubated with anti-CHIKV E2 mAb (2 μg/ml) or commercially available mAb for 12 hours at 4°C. Subsequently, the membrane was washed and further incubated with HRP-conjugated anti-mouse IgG (1:5000) (Cell Signaling Technologies). Immunoreactive bands were reacted with Amersham ECL Western Blotting Detection Reagents (GE healthcare, Buckinghamshire, UK) and visualized using an Azure C300 Western blot imager (Azure Biosystems, D2ublin, CA, USA).

Experiment result

1. Preparation of anti-CHIKV-E2 monoclonal antibody (mAb)

To generate anti-CHIKV-E2 mAb secreting hybridomas, the present inventors used a recombinant CHIKV-E2 protein as an antigen. As shown in Figure 1a, the size of the recombinant CHIKV-E2 protein is almost 40 kDa. It was confirmed through immunoblotting that the recombinant CHIKV-E2 protein was detected by commercial anti-CHIKV-E2 mAb. Therefore, the present inventor immunized the sole of the foot three times with a recombinant CHIKV-E2 protein mixed with an adjuvant at intervals of two weeks. Two weeks after each immunization, serum was obtained from the immunized mice and evaluated by ELISA to determine the production of anti-CHIKV-E2 Ab. The levels of anti-CHIKV-E2 Abs appeared to increase fourfold in serum after the first immunization (Fig. 1b) and showed that immunization of the recombinant CHIKV-E2 protein repeatedly induced anti-CHIKV-E2 Abs production. . Two weeks after the last immunization, lymphocytes were obtained from the popliteal lymph nodes of mice and fused with FO myeloma cells to generate hybridomas secreting anti-CHIKV-E2 mAb. ELISA used the culture supernatant of hybridomas to determine the production of Ab-secreting hybridomas. Among the 55 primary clones, 10 clones had higher optical density (OD) values for CHIKV-E2 protein than BSA as a negative control (FIG. 1C). In particular, the four primary clones called 19, 21, 22 and 26 exhibit OD values of 2.5 or higher, suggesting that these hybridomas can effectively produce anti-CHIKV-E2 Ab. Accordingly, the inventors performed subcloning and selected four monoclonals designated 19-1, 21-1, 22-2 and 26-4 for further experiments.

2. Comparison of binding affinity and evaluation of properties of anti-CHIKV-E2 mAb

To compare the binding affinity of anti-CHIKV-E2 mAb, we performed ELISA with varying amounts of mAb or CHIKV-E2. Commercially available anti-CHIKV-E2 mAb, 16A12 and Chk265 were used for comparison. When the resulting mAb was applied to a plate coated with serially diluted CHIKV-E2 protein, all mAbs showed higher OD values compared to commercially available mAbs (FIG. 2A ). In particular, 19-1 mAb showed the highest OD value from 1.2 ng/ml to 312.5 ng/ml in the plate coated with CHIKV-E2 protein. Next, ELISA was performed using various amounts of mAb on the CHIKV-E2 protein coated plate. The 21-1 mAb showed a higher OD value than the commercially available mAb, and the 19-1 mAb showed almost the same OD value as Chk265. The other mAbs (22-2 and 26-4) showed lower OD values than commercial mAbs. The isotype of the monoclonal antibody was determined using an antibody isotyping kit (Table 1). The 19-1 and 22-2 mAb were of the IgG2b kappa-chain isotype, and the 21-1 and 26-4 mAb were the IgG1 kappa-chain isotype.

In addition, the present inventors investigated whether the mAb recognizes a linear or conformal epitope of the CHIKV-E2 protein. As shown in Figure 2c, immunoblotting suggests that all anti-CHIKV-E2 mAbs detect the CHIKV-E2 protein, so that the mAb can recognize the linear epitope of the CHIKV-E2 protein (Figure 2c). These results indicate that 19-1 and 21-1 mAb have a higher binding affinity to the CHIKV-E2 protein than commercially available monoclonal antibodies and recognize the linear epitope of the CHIKV-E2 protein.

3. Sensitivity and specificity of anti-CHIKV-E2 mAb to virus

ELISA was performed using 19-1 and 21-1 mAb capable of strongly binding to the CHIKV-E2 protein to investigate whether the resulting monoclonal antibody recognizes CHIKV or other mosquito transfer viruses. First, inactivated CHIKV was serially diluted and coated on an ELISA plate. When a certain amount of anti-CHIKV-E2 mAb was applied to the plate, ELISA showed that 19-1 mAb had the highest OD value compared to the OD value of 21-1 mAb and commercially available mAb (FIG. 3A ). The 21-1 mAb had almost the same OD value as the commercial Chk265 mAb. In contrast, the commercial 16A12 mAb showed the lowest OD value. Next, we tested ELISA using varying amounts of mAb on inactivated CHIKV coated plates. Consistently, the 19-1 mAb had the highest OD value than that of the other mAb, whereas the 21-1 mAb showed an OD value similar to that of the commercial Chk265 mAb (Figure 3b). Then, we quantitatively analyzed the binding affinity of mAh by calculating half of the maximum effective concentration (EC50). As shown in Table 1, the EC50 of the 19-1 mAb (13.86 ng/ml) is the other mAb (20.45 ng/ml for the 21-1 mAb, 22.75 ng/ml for the Chk265 mAb and 24.58 ng/ml for the 16A12). ) Was found to be the lowest. In addition, it was confirmed by immunoblotting that the 19-1 and 21-1 mAb detect inactivated CHIKV at 50 kDa, which almost matches the predicted molecular weight of CHIKV-E2. In the commercial monoclonal antibody, Chk265 recognized the viral CHIKV-E2 protein, but 16A12 weakly detected the protein (Fig. 3C). This result indicates that 19-1 mAb effectively binds to CHIKV.

Since CHIKV is known to be one of the arboviruses that cause mosquito-induced disease, we are able to differentiate CHIKV from other mosquito-mediated arboviruses such as ZIKV, JEV and DENV type 1-4 by the monoclonal antibodies produced. We finally checked if there is any. The ELISA plate was coated with an inactivated arbovirus, and the binding affinity was compared using an anti-CHIKV-E2 monoclonal antibody, and then HRP-conjugated anti-mouse IgG was incubated at 37° C. for one hour. For comparison, Chk265 was used, and PBS was used as a negative control. In the ELISA results, it was shown that 19-1 mAb showed a significantly higher OD value only in CHIKV (FIG. 4A). In contrast, 21-1 mAb showed a significant increase in OD values in DENV 1-4 type and CHIKV compared to the PBS control (Fig. 4b). Commercial Chk265 mAb exhibited significantly higher OD values in CHIKV and DENV types 2-4 (Figure 4c). These results indicate that 19-1 mAb significantly binds only to CHIKV but not to other arboviruses.

The chikungunya virus-specific monoclonal antibody provided by the present invention recognizes the chikungunya virus envelope protein 2 with a very high binding affinity, and shows a very high specificity that other mosquito-borne viruses do not recognize. , Chikunguniya virus infection diagnosis, etc. can be used very usefully, industrial applicability is very excellent.

Korea Research Institute of Bioscience and Biotechnology KCTC13814BP 20190213

Claims (7)

A monoclonal antibody specifically binding to Chikungunya virus produced by hybridoma cells with accession number KCTC13814BP.
The monoclonal antibody of claim 1, wherein the antibody specifically binds to chikunguniya virus envelope protein 2.
The monoclonal antibody of claim 1, wherein the antibody is an IgG2 immunoglobulin isotype.
Hybridoma cells having accession number KCTC13814BP producing the antibody of claim 1.
A composition for detecting chikungunya virus comprising the antibody of any one of claims 1 to 3.
A method for detecting chikungunya virus, comprising the step of contacting the antibody of any one of claims 1 to 3 with a sample sample to detect formation of an antigen-antibody complex.
A kit for detecting chikungunya virus comprising the antibody of any one of claims 1 to 3.

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