KR102029394B1 - Monoclonal antibody for diagnosis of chikungunya virus infection, hybribodma producing the monoclonal antibody, and diagnosis method using the same - Google Patents

Abstract

The present invention relates to a monoclonal antibody for diagnosing chikungunnia virus infection and its use, and more specifically, to a monoclonal antibody specifically binding to the chikungunnia virus epidermal protein, a hybridoma producing the monoclonal antibody, The present invention relates to a composition for diagnosing and diagnosing chikungunnia virus disease comprising the monoclonal antibody. The monoclonal antibody according to the present invention specifically binds to the epidermal protein of chikungunia virus, and thus can be usefully used for diagnosing diseases caused by chikungunia virus.

Description

Monoclonal antibody for diagnosing chikungunnia virus infection, hybridomas producing the same, and method for diagnosing chikungunya virus infection using the same {monoclonal antibody for diagnosis of chikungunya virus infection, hybribodma producing the monoclonal antibody, and diagnosis method using the same}

The present invention relates to a monoclonal antibody for diagnosing chikungunnia virus infection and its use, and more specifically, to a monoclonal antibody specifically binding to the chikungunnia virus epidermal protein, a hybridoma producing the monoclonal antibody, The present invention relates to a composition for diagnosing and diagnosing chikungunnia virus disease comprising the monoclonal antibody.

Chikkununya fever is a mosquito-borne recessive disease that is infected by the Chikkununya virus (CHIKV). Initial diagnosis is very important because there are no vaccines or treatments available to date. Chikungunia fever has a high probability that both diseases can occur simultaneously in a region because the distribution of the virus is largely overlapped with dengue fever and the mosquito species that carry it are the same. Moreover, in the diagnosis of patients, clinical symptoms (high fever, myalgia, rash) similar to dengue fever and Japanese encephalitis are shown, and early differential diagnosis is absolutely necessary.

White lined mosquitoes, which can mediate chikungunia fever in Korea, are in need throughout the country, so caution is required, and continuous monitoring and management measures such as differential diagnosis between Japanese encephalitis and dengue fever are needed. However, there is currently no research on chikungunnia virus and there is no diagnostic kit for early diagnosis.

In order to solve the above problems, the present inventors produce a hybridoma that produces a monoclonal antibody having the chikungunnia virus epidermal protein as an antigen, and monoclonal against the chikungunnia virus produced from the hybridoma. The present invention was completed by confirming that the antibody can be used for diagnosis of related diseases.

It is an object of the present invention to provide a monoclonal antibody that specifically binds to a chikungunian virus epidermal protein and a hybridoma producing the same.

It is another object of the present invention to provide a composition, kit and method for diagnosing chikungunia virus disease comprising the monoclonal antibody.

In order to solve the above problems, the present invention provides a monoclonal antibody produced by the hybridoma cell line 5H2 of Accession Number KCTC18590P.

In addition, the present invention provides a composition for diagnosing chikungunnia virus disease comprising the monoclonal antibody.

In addition, the present invention provides a kit for diagnosing chikungunia virus disease comprising the composition.

The present invention also provides a method for providing information for diagnosing chikungunnia virus disease, comprising contacting the monoclonal antibody with a sample.

The monoclonal antibody according to the present invention specifically binds to the epidermal protein of chikungunia virus, and thus can be usefully used for diagnosing diseases caused by chikungunia virus.

1 shows the genome structure of chikungunia virus.
Figure 2 is a schematic diagram of the baculovirus expression vector of chikungunia virus epidermal protein E2.
Figure 3 shows the results confirmed by Western blot amplification of recombinant virus.
Figure 4 shows the results confirmed by SDS-PAGE analysis of purified purified chikungunnia virus epidermal protein (E2) antigen expressed in insect cells.
5 is a result of analyzing the reactivity of the monoclonal antibody to the chikungunnia virus epidermal protein E2 according to the present invention by indirect ELISA method.
6 is a result of analyzing the reactivity of the monoclonal antibody against non-denatured antigens against chikungunnia virus according to the present invention by indirect ELISA method.
Figure 7 shows the results of analyzing the reactivity of the monoclonal antibody against the denatured E2 (1 ~ 216 aa) antigen against chikungunnia virus according to the present invention by immunoblot.

Hereinafter, the present invention will be described in detail.

The present invention provides a monoclonal antibody produced by hybridoma cell line 5H2 of accession number KCTC18590P.

As used herein, the term “antibody” refers to an antibody that specifically binds to a chikungunian virus epidermal protein antigen, and includes not only a complete antibody form but also an antigen binding fragment thereof.

The complete antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by heavy and disulfide bonds.

The antigen binding fragment of an antibody molecule means the fragment which has antigen binding function, and includes Fab, F (ab '), F (ab') 2, Fv, etc. The Fab has one antigen binding site in a structure having the variable region of the light and heavy chains, the constant region of the light chain and the first constant region of the heavy chain (CH1). The F (ab ') is different from Fab in that it has a hinge region including one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F (ab ') 2 is produced by disulfide bonds of cysteine residues in the hinge region of Fab'. The Fv refers to the minimum antibody fragment having only the heavy chain variable region and the light chain variable region. Such antibody fragments can be obtained using proteolytic enzymes according to methods known in the art. For example, restriction digestion of the entire antibody with papain yields a Fab, and digestion with pepsin yields F (ab ') 2 fragments, preferably through genetic recombination techniques.

As used herein, the term "variable region" refers to a portion of an antibody molecule that exhibits many variations in sequence while performing a function of specifically binding to an antigen, and in the variable region CDR1 which is a complementarity determining region (CDR). , CDR2 and CDR3 are present. A framework region (FR) portion exists between the CDRs to support the CDR rings.

As used herein, the term "complementarity determining region" is a ring-shaped region that is involved in the recognition of an antigen, and as the sequence of this region changes, the specificity of the antibody to the antigen is determined.

As used herein, the term “heavy chain” refers to a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and a full length heavy chain comprising three constant region domains CH1, CH2 and CH3 It means all fragments.

As used herein, the term "light chain" refers to both the full-length light chain and fragment thereof including the variable region domain VL and the constant region domain CL, including the amino acid sequence having sufficient variable region sequence to confer specificity to the antigen.

The antibody may be alone or conjugated with other substances. The other substance may be or capable of generating a detectable signal. The other substance may be, for example, a coloring substance such as a fluorescent substance or a protein such as an enzyme.

Antibodies of the invention were prepared by hybridoma methods with reference to techniques known in the art (see, eg, Kohler and Milstein (1976) European Jounral of Immunology 6: 511-519).

As used herein, the term “hybridoma (fusion cell line)” refers to a cell obtained by fusing B lymphocytes that produce antibodies to myeloma cancer cells (myeloma cells), and myeloma cells are transformed cells. Because of the long-term incubation, the myeloma hybridoma that produces only one antibody can be easily obtained in large quantities in monoclonal antibodies. Preferably, the hybridoma of the present invention is a hybridoma cell line of Accession No. KCTC18590P.

The inventors have identified hybridomas that secrete antibodies having the above characteristics, and according to the provisions of the Budapest Treaty on International Approval of Microbial Deposits in Patent Procedure, 2017. On July 31, it was deposited with Korea Research Institute of Bioscience and Biotechnology (Korean Collection for Type Cultures) as accession number KCTC18590P.

The chikungunnia virus epidermal protein antigen used to prepare the antibody in the present invention may be preferably composed of amino acids 1 to 360 of the amino acid sequence of SEQ ID NO: 1, and more preferably, fragments consisting of 1 to 216 amino acids. Can be.

The antibody produced by the hybridoma may be used without purification, but in order to obtain the best results, it is preferable to use the purified with high purity according to methods well known in the art. Antibodies prepared by the above method can be separated by methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

Screening of hybridomas in the present invention can be carried out by methods known in the art. For example, screening of hybridomas that produce antibodies of the present invention by binding the chikungonia virus epidermal protein antigen used as an immunogen to a solid support and performing known immunoassays such as ELISA on the hybridoma culture supernatant. can do.

In another aspect, the present invention provides a composition for diagnosing chikungunnia virus disease, comprising the monoclonal antibody, and a kit for diagnosing chikungunia virus disease comprising the composition.

In the present invention, the chikungunia virus disease refers to an infectious disease caused by chikungunia virus and includes chikungunia fever.

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.

The kit may comprise a second antibody that binds to the antibody. For example, it may include labeled or unlabeled anti-mouse antibodies. In addition, the kit may further include a reagent required for an assay reaction using an antibody such as ELISA or immunofluorescence assay.

In another aspect, the present invention provides a method for providing information for diagnosing chikungunnia virus disease, comprising performing an antigen-antibody reaction by contacting a monoclonal antibody with a sample.

The contact can be made static or dynamic in the liquid medium. The sample may be a biological sample including an organism or a substance derived therefrom. For example, it may be feces, saliva, blood, urine, cells or tissues.

The monoclonal antibody according to the present invention binds specifically to the chikungunian virus epidermal protein, induces an antigen-antibody reaction, and then detects the antigen-antibody complex by a known method, thereby detecting the chikungunnia virus in the biological sample. It can be determined whether there exists.

Detection of the complex can be made by methods known in the art. For example, the complex can be detected by using the antibody with a detectable label and measuring the signal coming from the detectable label from the complex. More specifically, the antigen-antibody reaction is tissue immunostaining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assay (Immunoprecipitation Assay), immunodiffusion assay (Immunodiffusion Assay) ), Complement fixation assay (Complement Fixation Assay), Fluorescence-activated cell sorter (FACS) or protein chip (protein chip) analysis, etc. can be identified, but is not limited thereto.

Hereinafter, the present invention will be described in detail through examples. The following examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples according to the gist of the present invention to those skilled in the art. Will be self-evident.

Example  One. Chikungunia  Isolation of viral epidermal protein (E2) and Cloning

In order to use the chikungunnia virus epidermal protein (E2) as an antigen, a chikungunnia virus epidermal protein (E2) consisting of the amino acid sequence of SEQ ID NO: 1 was obtained. A base sequence encoding the 1st to 216th amino acids of the viral epidermal protein (E2), which is known as an immunoreactive site, and the hydrophobic portion of the C terminus was removed to increase the expression rate. Base sequences encoding the 1 st to 360 th amino acids were separated, and then amplified by PCR. The amplified gene was inserted into a baculovirus expression vector (pAcGP67A) to prepare two recombinant expression vectors. The genomic structure of the chikungunnia virus is shown in FIG. 1 and the vector map of the recombinant expression vector is shown in FIG. 2.

Example  2. Recombination Baculovirus  And produced therefrom Chikungunia  Isolation of Viral Epidermal Protein (E2)

2-1. Recombination Baculovirus  Separation

Sf9 (S. frugiperda) insect cells (Invitrogen, # 11496-015) were maintained in Sf-900 II SFM medium. 1 × 10 ⁶ Sf9 insect cells were conventionally known as pAcGP67A-chik E2 DNA (0.5 ug) and 0.5 ug of linearized baculovirus genomic DNA ProEasy (0.5 ug, AB Vector # A10S) prepared in Example 1 above. It was transformed according to the method. After 3 days of transformation, the medium was centrifuged and a limiting dilution assay was performed using the supernatant. Recombinant baculovirus in the supernatant was amplified by infected Sf9 cells. The amplified recombinant baculovirus was confirmed by Western blot analysis. The results are shown in FIG.

2-2. Chikungunia  Isolation of Viral E2 Protein

Cells in Sf-900 II SFM medium were infected with recombinant baculovirus of 0.01-10 m.o.i in a stirred flask. The supernatant was incubated for 3 days, and then centrifuged at 3000 g for 10 minutes to remove cell debris. The supernatant was injected into Ni-NTA resin and then equilibrated with 20 mM Tris buffer (Tris-HCl, pH7.5.0), 0.5 M calcium chloride. After washing with 5 mM imidazoloresin, the protein was separated with 500 mM imidazole. The isolated protein was dialyzed with PBS (pH7.5) and 5% glycerol and confirmed by SDS-PAGE. The results are shown in FIG.

Example  3. Chikungunia  virus To epidermal protein (E2)  About Monoclonal antibody production

In order to produce a monoclonal antibody, two mice (Balb / C mice) were intraperitoneally injected with 100 μg of each of the chikungunnia virus epidermal proteins (E2 1-216 aa) isolated in Example 2 to perform an immune response. . This was done a total of two times. After the immune response, the antigen-binding degree of the E2 polyclonal antibody of the mouse was confirmed by using an ELISA method.

After isolation of spleen B immune cells from mice, B immune cells producing antibodies that specifically bind only to chikungunia virus epidermal protein (E2) were isolated. This was fused with myeloma-derived cells to obtain hybridomas, which were deposited with the Korea Research Institute of Bioscience and Biotechnology on July 31, 2017 (Accession No. KCTC18590P).

The hybridomas were incubated at 37 ° C. in a 5% CO ₂ incubator, and monoclonal antibodies were purified from the cell supernatants obtained after incubation. Purification of the antibody was carried out using a protein G column, and purified monoclonal antibody at a concentration of about 1 mg / ml per ml of fusion cell culture was obtained. Isotyping of the produced monoclonal antibody, it was confirmed that the IgG1, kappa. Hereinafter, the monoclonal antibody against the chikungunnia virus epidermal protein (E2) was named 5H2.

Example  4. Chikungunia  virus To epidermal protein (E2)  About Monoclonal antibody  Affinity analysis

The affinity analysis of the monoclonal antibodies obtained in Example 3 was carried out by indirect ELISA using purified E2 antigen and step diluted monoclonal antibodies. First, the purified antigen was diluted with carbonate buffer (pH 9.4) to a concentration of 10 μg / ml, and then 100 μl was added to each well of the ELISA plate (Nunc), and reacted at 4 ° C. for 16 hours to coat the antigen. Each well coated with antigen was treated with a phosphate buffer solution containing 1% BSA to block nonspecific reaction at 37 ° C. for 1 hour. The monoclonal antibody obtained in Example 3 was diluted to 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, and 0.3 nM concentration using a phosphate buffer solution containing 1% BSA. Each well was added, reacted at 37 ° C. for 1 hour, and washed three times with phosphate buffer solution. After washing, 100 μl of HRP-condensed anti-mouse IgG antibody (bio-rad) diluted 1: 2000 times was added to each well, reacted at 37 ° C. for 1 hour, and washed three times with phosphate buffer solution. After washing, 100 μl of the TMB substrate solution was added to each well, followed by reaction for 30 minutes in the dark, followed by development of 1N H ₂ SO ₄ to stop the enzymatic reaction. After the reaction, the absorbance was measured at 450 nm using an ELISA reader.

As a result, a dose-response curve showing affinity according to the concentration of the monoclonal antibody was confirmed, and the concentration of the monoclonal antibody showing an absorbance (OD405) of 1.00 was calculated from the curve. It was found to be 10 ⁻⁷ M.

Example  5. Chikungunia  virus To epidermal protein (E2)  About Monoclonal antibodies  Preparation of indirect Enzyme-linked immunosorbent assay (ELISA) to confirm

An indirect ELISA was prepared to identify the antibody specific for the chikungunnia virus prepared in Example 3.

First, a plate coated with purified protein and chikungunia virus was used to identify antibodies specific for chikungunia virus. Dilute the chikungunnia virus epidermal protein E2 and the control epidermal proteins E1 and BSA with carbonate buffer (pH 9.4) to a concentration of 10 μg / ml, and add 100 μl to each well of the ELISA plate (Nunc). Antigen was coated by reaction for 16 hours, and the other plate was coated by diluting the inactivated chikunkunia virus (1 × 10 ⁵ TCID / well) in carbonate buffer (pH 9.4) and incubating overnight at 4 ° C. Each plate was treated with a phosphate buffer solution containing 1% BSA to block nonspecific reactions at 37 ° C. for 1 hour. Dilute the monoclonal antibody (5H2) to a concentration of 10 µg / ml using a phosphate buffer solution containing 1% BSA, add 100 µl to each well, and react at 37 ° C for 1 hour, followed by phosphate buffer solution. Washed three times. After washing, 100 μl of HRP-condensed anti-mouse IgG antibody (bio-rad) diluted 1: 2000 times was added to each well, reacted at 37 ° C. for 1 hour, and washed three times with phosphate buffer solution. After washing, 100 µl of the TMB substrate solution was added to each well, followed by reaction for 30 minutes in the dark, followed by development of 1N H ₂ SO ₄ to stop the enzymatic reaction. After the reaction, the absorbance was measured at 450 nm using an ELISA reader. The results are shown in FIGS. 5 and 6.

As shown in FIG. 5, it was confirmed that the monoclonal antibody (5H2) according to the present invention specifically reacted to the chikungunian epidermal protein E2 without reacting to the chikungunian epidermal protein E1 or BSA.

In addition, as shown in FIG. 6, the monoclonal antibody (5H2) according to the present invention specifically reacted not only with the chikungunnia epidermal protein E2 antigen but also with the chikungunnia virus, and thus it was confirmed that it can be used for diagnostic purposes. .

Example  6. Monoclonal antibodies  Used Weston Blot  Perform

1 ug of purified Chikunkunia virus epidermal protein (E2) was suspended in sample buffer. Samples were loaded onto 12% polyacrylamide gels and subjected to electrophoresis at 120V. After SDS-PAGE, the protein was transferred to the membrane and monoclonal antibody (5H2) diluted 1: 1000 using primary antibody dilution buffer (1% skim milk powder, 10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20). ) Was incubated at room temperature for 1 hour. The membranes were washed six times with PBST, and HRP-condensed anti-mouse IgG antibodies diluted 1: 5000 in blocking buffer (1% skim milk powder, 10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20), respectively. Incubated at room temperature for 1 hour. After incubation the membrane was visualized using ECL. The results are shown in FIG.

As shown in FIG. 7, it was confirmed that the chikunkunia virus epidermal protein (E2 1 to 216 a.a.) could be detected using the monoclonal antibody according to the present invention.

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC18590P

Deposit Date: 20170731

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Claims (7)

Monoclonal antibody specific for chikungunnia virus epidermal protein produced by hybridoma cell line 5H2 of accession number KCTC18590P. The method of claim 1,
The chikungunnia virus epidermal protein is a monoclonal antibody, characterized in that represented by the amino acid sequence of SEQ ID NO: 1. The method of claim 2,
The chikungunnia virus epidermal protein is a monoclonal antibody, characterized in that the fragment represented by amino acids 1 to 216 of the amino acid sequence of SEQ ID NO: 1. Comprising a monoclonal antibody of claim 1, the composition for diagnosing chikungunnia virus disease. Chikungunia virus disease diagnostic kit comprising the composition of claim 4. A method for providing information for diagnosing chikungunnia virus disease, comprising contacting a monoclonal antibody of claim 1 with a sample to perform an antigen-antibody reaction. The method of claim 6,
The method is tissue immunostaining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assay (Immunoprecipitation Assay), immunodiffusion assay (Immunodiffusion Assay), complement fixation assay (Complement) Antigen-antibody complexes are detected using at least one method selected from the group consisting of fixation assay, Fluorescence-activated cell sorter (FACS), and protein chip analysis. How to provide information for diagnosis.

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