KR20100093451A - Monoclonal antibody recognizing scye1 and use thereof - Google Patents

Abstract

PURPOSE: A monoclonal antibody which recognizes SCYE1(small inducible cytokine subfamily E, member 1) is provided to use in measuring SCYE1 concentration in blood and to treat and diagnose inflammatory diseases. CONSTITUTION: A monoclonal antibody which recognizes SCYE 1 protein recognizes 3-51th amino acid site from amino terminal of human SCYE 1 antigen comprising an amino acid sequence of sequence number1. The monoclonal antibody is bound with detection labeling agent which generates detectable signal. The detection labeling agent is enzyme, fluorescence, luminescence, or radioactive substance. A SCYE1 quantitative kit contains the monoclonal antibody. The SCYE1 quantitative kit contains a secondary antibody recognizing the monoclonal antibody.

Description

Monoclonal antibody recognizing SCVE1 and use thereof

The present invention relates to a monoclonal antibody that recognizes SCYE1 and its use, and more particularly to a monoclonal antibody that recognizes SCYE1 protein, SCYE1 quantification method and quantification kit using the same.

SCYE1 (small inducible cytokine subfamily E, member 1) causes inflammation by inducing the secretion of TNFa and IL-8 from immune cells, including monocytes, macrophages, and dendritic cells. Known. SCYE1 binds to tRNA and acts as a cofactor of protein synthase in the cell and is secreted out of the cell when cytokines are stimulated. Secreted SCYE1 is known to inhibit angiogenesis by inhibiting proliferation and migration of endothelial cells. Therefore, there is a need for the development of technology and materials that can be suppressed.

Antibodies usually produced in vivo by antigen stimulation are mixtures of antibodies (polyclonal antibodies) that have various binding forces to the antigen, but some antibody producing cells produce only one type of antibody. Monoclonal antibodies refer to homogeneous antibodies having the same structure formed by cells (monolone) that have grown from a single antibody producing cell. A method for preparing cells that produce monoclonal antibodies was developed in 1975 by Kohler and Milstein. This method involves fusion of B-lymphocytes obtained from the spleen or lymph nodes of an animal immunized with a particular antigen with myeloma cells, which are normal cells with the ability to produce monoclonal antibodies and myeloma cells become cancerous. As cells obtained from B-lymphocytes, which do not have the ability to produce immunoglobulins but have abnormal proliferative capacity to continue growing, consequently, the fusion cells of these cells can produce monoclonal antibodies of B-lymphocytes. The ability to grow and to continue to grow without killing myeloma cells.

When cell fusion is completed, the fusion cells, non-fusion splenocytes, and myeloma cells are mixed in the medium in which the fusion is performed. Therefore, only fusion cells need to be selected. Cells are grown in HAT medium for this purpose. Purine (adenine, guanine) is biosynthesized through two pathways, the endogenous pathway and the salvage pathway, so wild pathogens are not lethal if they lose either pathway. The HAT medium contains hypoxanthine (hypoxanthine), aminopterin (aminopterin) and thymidine (thymidine), which inhibits the action of the dihydrofolate reductase (DHFR) of the endogenes pathway. As it blocks the Genes pathway, it also lacks the HGPRT (hypoxanthine phosphoribosyl transferase) of the salvage pathway (and therefore also the salvage pathway, which is resistant to toxic base analogs (thioguanine or azaguuanine)). Unlike wild strains, non-fused myeloma cells die without growth in HAT medium, and non-fused splenocytes die naturally after 1 to 2 weeks after separation. It becomes possible.

However, in spite of the method for producing fusion cells producing such antibodies, no monoclonal antibody that recognizes human SCYE1 protein has been reported.

Since monoclonal antibodies recognize a single epitope, only monoclonal antibodies with specific reactions can be selected. In addition, there are major antigenic sites in the whole antigen that have a great effect on the formation of antibodies. The use of monoclonal antibodies against these antigenic sites can improve sensitivity compared to antiserum-based antigen diagnosis. Therefore, there has been a continuous need for the production of monoclonal antibodies directed against specific antigens and the development of diagnostic agents using the same.

The present invention has been made in accordance with the above requirements, and as a result, the present inventors have conducted continuous studies to obtain monoclonal antibodies recognizing the SCYE1 protein. As a result, splenocytes and myeloma cells of mice immunized with the SCYE1 protein have been produced. Fusion was prepared by fusion cells, and among them, SCYE1 was used to select and culture fusion cells that recognize this to successfully obtain monoclonal antibodies to complete the present invention.

In order to solve the above problems, an object of the present invention is SCYE1 to induce inflammation through the induction of secretion of TNFa, IL-8 of immune cells, including monocytes, macrophages, dendritic cells (dendritic cells) It is to provide a monoclonal antibody that recognizes.

Another object of the present invention is to provide hybridoma cells that produce monoclonal antibodies that recognize SCYE1.

Another object of the present invention is to provide a SCYE1 quantitative kit comprising a monoclonal antibody that recognizes SCYE1.

Another object of the present invention is to provide a method for quantifying SCYE1 in a sample using a monoclonal antibody that recognizes SCYE1.

According to the present invention, by measuring the concentration of SCYE1 in the blood using a monoclonal antibody that recognizes SCYE1 protein, it can be usefully used for the diagnosis and treatment of inflammatory diseases related thereto.

In the following description of the invention, many terms used in recombinant DNA and immunology are used extensively. In order to provide a clearer and more consistent understanding of the present specification and claims, the following definitions are provided.

The term "antibody" is a term known in the art and means a molecule or active fragment of a molecule that binds a known antigen. Examples of active fragments that bind known antigens include Fab and F (ab) ₂ fragments. Such active fragments can be derived from the antibodies of the invention by a number of techniques. For example, purified monoclonal antibodies can be cleaved by enzymes such as pepsin and filtered by HPLC gel. Suitable fractions containing Fab fragments can be collected and concentrated by membrane filtration and the like. A description of general techniques for active fragment separation of antibodies can be found in, for example, Khaw, BA et al., J. Nucl. Med. 23: 1011-1019 (1982). The term “antibody” also includes bispecific and chimeric antibodies.

The term “monoclonal antibody” is a term known in the art and is a highly specific antibody directed against a single antigenic site. Unlike polyclonal antibodies, which typically include different antibodies directed against different determinants (epitopes), monoclonal antibodies are directed against a single determinant on an antigen. Monoclonal antibodies of the invention can be prepared using conventional cloning and cell fusion techniques. For example, an immunogen of interest (antigen) can be administered to wild type or breeding mice (eg BALB / c) to produce natural or human monoclonal antibodies. Such antigens can be administered alone, in admixture with an adjuvant, can be expressed from a vector and can elicit an immune response as a DNA or fusion protein. Fusion proteins include carrier proteins coupled with peptides intended for an immune response, such as β-galactosidase, glutathione S-transferase, keyhole limpet hemocyanin (KLH), and bovine serum albumin, Carrier proteins are not limited thereto. In this case the peptide acts as a hapten for the carrier protein. The method for producing monoclonal antibody is briefly described as follows. After boosting the animal, the spleen is removed and the spleen cells are extracted by methods known in the art [Kohler and Milstein, Nature 256: 495-497 (1975); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988). The hybrid cells obtained are cloned by conventional methods, for example by limiting dilution, and the resulting clones are cultured to produce the desired monoclonal antibody. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and because they are synthesized by hybridoma culture, they also have another advantage that they are not contaminated by other immunoglobulins. Have

The term “epitope” is a term known in the art and generally refers to a region of an antigen that interacts with an antibody. Epitopes of peptide or protein antigens may be formed from consecutive or discontinuous amino acid sequences of the antigen. Many proteins can contain many epitopes. Epitopes recognized by the antibodies of the invention form an aspect of the invention. Antibodies that recognize specific epitopes can be used in immunoaffinity columns to purify specific epitopes. Since antigenic epitopes are reported to be exhibited by as few as five amino acid residues, terminal cleavage of certain epitopes may also be possible.

The term “hybridoma” is known in the art and refers to a cell formed by fusion of antibody producing cells with immortal cells, such as myeloma cells. The hybrid cell can continuously supply the antibody.

As used herein, the term "forming an immunological complex" is intended to include identifying the presence or absence of the SCYE1 antigen in a sample. The presence or absence of the SCYE1 antigen can be detected using immunoassays. Numerous immunoassays used to detect and / or quantify antigens are known to those of ordinary skill in the art and are described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York 1988, 556-612. ].

As used herein, the term "detection labeling agent" means a label for detection of an immunocomplex, and specific examples are radioisotope labels, enzymes, chemoluminescent compounds, fluorescein, picobili Fluorescent materials such as proteins, rare earth chelates, rhodamines, enzyme cofactors, biotin, and the like, but are not necessarily limited thereto.

The term "(biological) sample" is intended to include biological material, including cells, tissues or biological fluids.

The present invention relates to a monoclonal antibody that recognizes amino acid sites 3 to 51 from the amino terminus of the human SCYE1 antigen consisting of the amino acid sequence of SEQ ID NO: 1, wherein the monoclonal antibody is preferably a detection capable of generating a detectable signal The labeling agent may be bound, and the detection labeling agent may be an enzyme, a fluorescent material, a luminescent material, or a radioactive material.

The present invention also relates to a SCYE1 quantitative kit comprising said monoclonal antibody, said kit further comprises a secondary antibody recognizing the monoclonal antibody, wherein the detection labeling agent can be bound to the secondary antibody. In addition, the kit may further include a substrate and a reaction terminator, wherein the detection labeling agent is an enzyme and can measure enzymatic activity.

The present invention also provides

i) contacting the biological sample with the SCYE1 monoclonal antibody of the invention in combination with a detection labeling agent,

ii) quantitatively detecting the immunological complex obtained in step i),

iii) quantifying the immunological complex obtained by contacting the purified SCYE1 antigen with the SCYE1 monoclonal antibody bound to the detection marker to obtain a standard curve, and

iv) quantifying SCYE1 in a sample using a SCYE1 monoclonal antibody, comprising quantifying SCYE1 present in a biological sample by comparing the amount of protein detected in step ii) to the standard curve obtained in step iii). It is about a method.

Hereinafter, the present invention is described in more detail.

The present invention provides antibodies that are immunologically reactive to the SCYE1 protein or epitope thereof. Antibodies provided herein can be generated in animals by inoculating cells expressing the SCYE1 protein or epitope thereof by methods known in the art. The protein can be isolated from the cells at various levels of homogeneity by conventional biochemical methods. Synthetic peptides prepared by synthetic methods established in vitro and optionally conjugated to heterologous amino acid sequences are also included in the antibody preparation methods of the invention. Animals used for the inoculation include cattle, sheep, pigs, mice, rats, rabbits, hamsters, goats and primates. Preferred animals for inoculation include rodents such as mice, rats, hamsters. The most preferred animal is a mouse.

Monoclonal antibodies provided by the present invention are also produced by recombinant genetic engineering methods known to those skilled in the art, and the present invention relates to antibodies by the above methods, which are immunologically reactive with epitopes of the SCYE1 protein of the present invention. Include. The invention also includes fragments of such antibodies, such as, but not limited to, F (ab) and F (ab) ₂ . The fragments are prepared by any method, including but not limited to the preparation of such fragments by proteolytic cleavage, chemical synthesis or genetic engineering methods. The present invention may also include single chain antibodies immunologically reactive with the SCYE1 protein, prepared by methods known to those skilled in the art. The invention also includes chimeric antibodies comprising light and heavy chain peptides that are immunologically reactive with the SCYE1 induced epitopes according to the invention. Chimeric antibodies provided herein include natural antibodies as well as chimeric antibodies prepared by methods of genetic engineering techniques known to those skilled in the art.

It is to be understood that the monoclonal antibody according to the present invention includes all antibodies that recognize SCYE1 as an antibody prepared from human SCYE1 or recombinant SCYE1 as antigen. Cells that can be used herein are any cells or cell lines capable of expressing the natural or genetically engineered SCYE1 protein provided by the present invention. Cells that can be used in the present invention include, but are not limited to, mammalian cells, HEK-293 cells.

Monoclonal antibodies provided in the present invention are produced by hybridoma cell lines, which are also provided by the present invention and prepared by methods known in the art. See Harlow and Lane, Antibodies: A Laboratory. Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988). Hybridoma cell lines immunize each cell of a myeloma cell line with a homogenizing agent of SCYE1 protein, including the SCYE1 protein itself or a heterologous or fusion protein construct, a membrane comprising the same, a cell encoding the protein, the protein Prepared by fusion with splenocytes derived from animals. Myeloma cell lines used in the present invention include cell lines derived from mice, rats, hamsters, primates, and human myeloma. As myeloma cells, for example, P3X63Ag8, p3-U1, NS-1, MPC-11, SP-2 / 0, F0, P3x63Ag8. Mice derived from mice such as V653 and S194 can be used. In addition, cell lines such as R-210 which are rat-derived cells can be used. Preferred myeloma cell lines for use in the present invention include myeloma cells SP2 / 0-Ag14 (ATCC CRL-1581) and P3X63Ag8.653 (ATCC CRL-1580).

Preferred animals from which spleen can be obtained after immunization include rats, mice and hamsters, preferably mice. Splenocytes and myeloma cells can be fused using a number of methods known in the art, including, but not limited to, inoculation with Sendai virus and inoculation with polyethylene glycol (PEG), and the like. . Monoclonal antibodies produced by hybridoma cell lines can be recovered from cell culture supernatants via in vitro cell growth, or hybridoma cells can be injected subcutaneously and / or intraperitoneally in animals, most preferably mice, Monoclonal antibodies are obtained from blood or ascite.

Hybridoma cells producing monoclonal antibodies against SCYE1 are fusions of myeloma cell P3X63Ag8.653 cells (ATCC CRL-1580, USA) and spleen cells producing antibodies to SCYE1.

The invention also provides methods for diagnosing and treating inflammatory diseases by detecting expression of the SCYE1 protein in animals, preferably humans. Diagnostic reagents for use in the methods include antibodies, most preferably monoclonal antibodies of the invention. The antibody can be, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Westton blot assay, immunological titer assay, immunological diffusion assay, and others known to those skilled in the art. It can be used in conventional immunological techniques, including but not limited to methods.

Reagents used in immunological assays include suitable carriers, detection labels capable of producing detectable signals, solubilizers, cleaning agents. 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 (eg PBS) or insoluble carriers known in the art such as polystyrene, polyethylene, polypropylene, polyesters, polyacrylonitrile , Fluorine resins, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated with latex metal, other papers, glass, metals, agarose and combinations thereof.

Antibodies of the invention may be coupled with a detectable substance, ie a detection labeling agent, to facilitate detection. Examples of detection markers include various enzymes, prosthesis molecules, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, and the like. Detection markers can be directly coupled or coupled to SCYE1 monoclonal antibodies of the invention using techniques known in the art or indirectly coupled or bound through intermediates (eg, linkers known in the art). Or may be coupled or coupled to another polyclonal antibody that recognizes the SCYE1 protein or to a secondary antibody that is an antibody that recognizes the SCYE1 antibody. Examples of suitable enzymes include horseradish peroxidase, acetylcholine esterase, peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, malate dehydrogenase, glucose-6-phosphate Dehydrogenase, invertase, and the like; Examples of suitable submolecular complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin or picobiliprotein, and the like; Examples of luminescent materials include luminol, isoleucinol, lucigenin; Examples of bioluminescent materials include luciferase, luciferin and aequorin; Examples of suitable radioactive materials include 125I, 131I, 111In, 99Tc 14C, 3H, and the like.

In addition, the present invention is applied to an immunoassay or immunoassay kit for detecting the formation of an immunological complex with a monoclonal antibody using the monoclonal antibody against SCYE1, an assay method or assay kit for detection or quantification of SCYE1 in a sample To provide. As mentioned above, immunoassays and kits for immunoassays using monoclonal antibodies known in the art, such as ELISA assays and radioimmunoassays, are all applicable to the present invention. For example, a kit for use in the SCYE1 quantitative assay of the present invention may contain a SCYE1 monoclonal antibody according to the present invention, a suitable carrier solution, a container containing a detection label capable of generating a detectable signal, a dissolving agent, a cleaning agent, and instructions for use. It may include. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. In addition, the detection labeling agent may further comprise another secondary antibody against SCYE1 bound directly or via a linker to the SCYE1 monoclonal antibody according to the invention.

In a preferred embodiment of the present invention, by adding a biological sample to a microtiter plate coated with a monoclonal antibody according to the present invention, reacting the enzyme-antibody conjugate and then treating the substrate of the enzyme to measure the absorbance, A method of quantifying SCYE1 is provided that quantifies SCYE1 present in a biological sample by comparing absorbance with a standard curve. Herein, biotin may be combined with an antibody in the enzyme-antibody conjugate. In addition, the enzyme may be horse radish peroxidase (hereinafter referred to as HRP), but is not limited thereto, and all enzymes and substrates used in the ELISA assay may be applied to the present invention.

ELISA analysis is an analysis method that can quantify the presence or absence of antigen present in a sample by using antibody and antigen reaction. There are about two or three antibodies required for one ELISA kit. The ELISA kit using monoclonal antibodies recognizing SCYE1 consists of the three antibodies mentioned above. The composition and principle are as follows.

First, a monoclonal antibody (primary antibody) is coated on a 96-well plate (when quantifying SCYE1 in human plasma), a sample containing SCYE1 is added to the well to react, and an enzyme-labeled polyclonal antibody (secondary antibody). ), The enzyme label and the substrate capable of reacting, the enzyme-substrate reaction can be detected, and the SCYE1 present in the sample can be quantified by comparison with the standard curve. In a specific example of the present invention, enzymes and substrates may use, but are not limited to, biotin and streptavidin conjugate HRP.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the present invention, the content of the present invention is not limited to the following examples.

Example 1: SCYE1 Cloning and Protein Isolation

ORF (1-312aa) encoding human SCYE1 was obtained from primer F from HEK293 cDNA; 5'-GCGAATTCATGGCAAATAATGATGCTGTTC-3 '(SEQ ID NO: 2), R: 5'-CCGCTCGAGTTATTTGATTCCA CTGTTGCTCATG-3' (SEQ ID NO: 3) using 25 cycles of 95 ° C 1 minute, 55 ° C 1 minute, 72 ° C 1 minute Amplified. PCR products were cut with EcoRI / SalI and then ligated to pGEX4T-1 (Pharmacia) vector previously cut with EcoRI / SalI at room temperature for 2 hours. After transforming DH5a with heat shock at 42 ° C. for 90 seconds, E. coli was grown in LB medium containing ampicillin (50 ug / ml) to select colonies containing SCYE1. Sequencing confirmed that the cloned SCYE1 gene was free of mutations.

Example 2: SCYE1 Tablet

The SCYE1 plasmid was transformed into E. coli BL21 (DE3), plated in LB medium containing ampicillin (50ug / ml), and incubated at 37 ° C for 24 hours. The grown colonies were grown in LB medium containing 5 ml of ampicillin (50 ug / ml) for 12 hours, and then again inoculated with 4 ml of LB medium containing 2 L of ampicillin (50 ug / ml). Incubate until IPTG was added to a final 0.1 mM and incubated at 30 ° C. for 6 hours to express p43 protein. After E. coli was centrifuged at 7,000 rpm for 15 minutes, the cells were resuspended in 1X PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na ₂ HPO ₄ , 0.24 g KH ₂ PO ₄ / L, pH 7.4) at 4 ° C. After sonication at 4 ℃, centrifuged for 40 minutes at 26,000xg. Supernatants were taken and loaded into glutathione Sepharose 4B-columns homogenized with 1XPBS. After washing the resin with PBS, GST-p43 was eluted using 50 mM Tris-HCl (pH 8.0) containing 10 mM glutathione. Purity was confirmed using 10% SDS-PAGE, and dialyzed with PBS containing 20% glycerol and stored at -70 ° C. Purity was found to be greater than 90% on SDS-PAGE.

Example 3: Construction of SCYE1 Antibody Monoclonal Cell Line

1) antigen immunity

An emulsion was prepared in which the protein was mixed with the same volume of Complete Freund's Adjuvant (Sigma, USA) at a concentration of 20 μg / mouse. The emulsion was intraperitoneally injected into three 7-week-old female Balb / c mice (Orients). 20 μg of antigen was injected per animal, but the total volume was 400 μl. Two weeks later, an emulsion containing Incomplete Freund's Adjuvant (Sigma, USA) and an antigen was injected into the abdominal cavity of the mouse, and then, two weeks later, the antigen was dissolved in PBS (20 μg / mouse) intraperitoneally to induce antibody production. . Antibodies were generated by enzyme-immunoassay and Western blot analysis to confirm the antibodies, and then further inoculated with antigen dissolved in PBS in the tail vein of rats 3 days before cell fusion.

2) Identification and Screening of Antibody Producing Cells

Blood was obtained from the orbital vein gun (eye ball) of immunized rats in a 1.5 ml centrifuge tube, serum was separated by 13,000 rpm, 10 minutes centrifugation, and tested for antibody production. Stored at. After the production of the antibody was confirmed by enzyme-immunoassay and Western blot analysis with the antigenic protein, fusion to spleen cells of antibody-producing mice was started.

3) fusion cell manufacturing

After antibody production was confirmed, mice were sacrificed to isolate antibody-producing cells (splenocytes), and myeloma cells P3X63Ag8.653 (ATCC CRL-1580, USA) were fused. That is, the mouse P3X63Ag8.653 cells were maintained in an optimal growth phase using RPMI1640 medium containing 10% fetal bovine serum (Biowhittaker, USA) in a culture dish. For cell fusion, P3X63Ag8.653 cells were washed twice with serum free RPMI1640 medium (Biowhittaker, USA) and then adjusted to 1 × 10 ⁷ cells. Mice were sacrificed by cervical dislocation to obtain a spleen and placed in a mesh (Sigma, USA) container to separate cells one by one. After making the splenocyte suspension and centrifuging, the spleen cell solution was exposed to Tris-NH ₄ Cl solution (Tris 20.6g / L, NH ₄ Cl 8.3 g / L) to lyse red blood cells, and then completely isolated antibody-producing cells were obtained. After centrifugation at 400 g for 5 minutes, the cells were washed twice with serum-free medium and suspended in 10 ml of medium. Lymphocytes were counted with a haemocytometer and 1 × 10 ⁸ lymphocytes were mixed with 1 × 10 ⁷ P3X63Ag8.653 cells (10: 1) in serum-free medium and centrifuged at 400 g for 5 minutes. Mix 1 ml solution slowly dropping over 1 minute using 50% (M / V) polyethylene glycol 1500 (Sigma, USA) pretreated at 37 ° C. The fusion mixture produced above was diluted with serum-free RPMI1640 medium and centrifuged at 400 g for 3 minutes followed by 20% fetal bovine serum and HAT (100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine). The cells were suspended in 35 ml of RPMI1640 selective medium. 100 μl of the suspension was dispensed into 96-well plates coated with feeder cells (a macrophages isolated from RPMI1640 medium from the abdominal cavity of the mouse) a day before, and incubated at 37 ° C. and 5% CO ₂ . Incubated for 14 days with alternating HAT selective medium. After 14 days, passage was performed with RPMI1640 medium containing 20% fetal bovine serum and HT (medium 0.4 μM aminopterin removed from HAT selective medium).

4) Screening and Isolation of Fusion Cells Producing Each Antibody

In the method of 3), the culture supernatant obtained by the fusion was obtained and searched by enzyme-immunoassay to investigate whether the specific antibody to the provided antigen was generated, and the culture solution of the fusion cell was four times more titer than the negative control group. Only cells were selected and transferred to 24-well culture plates and 25 cm ² culture flasks.

5) Immune Globulin Isolation and Individual Determination

Individual types of monoclonal antibodies were determined using an Isotyping kit (Zymed Labomouseories Inc. USA) using enzyme immunoassay using culture supernatants (FIG. 1). As a result, the antibody against SCYE1 was found to be IgG1. In addition, the monoclonal antibody cell line prepared by Western blot using whole cell extract of HEK 293 cells was used to confirm whether the antibody from the cell line prepared using the cultured SCYE1 monoclonal antibody cell line specifically recognized SCYE1. It was confirmed that it was SCYE1 specific (FIG. 2).

Example 4: Mapping the domain (Domain mapping)

Peptides of human SCYE1 were synthesized (ORF 3-54, 51-90, 103-139, 191-219aa) and coated in 100 wells per well in 96 well plates and then at 1 ambient temperature with 2% BSA (sigma) in 1X PBS. Blocking was performed for a time. 100 μl of the culture supernatant was loaded into each well and reacted at room temperature for 1 hour. After washing three times with PBS containing 0.1% Tween 20, HRP-conjugated anti-mouse IgG was loaded and reacted at room temperature for 1 hour, followed by washing four times with PBS containing 0.1% Tween 20, followed by TMB (sigma). ) Was added and reacted at room temperature for 20 minutes and then read at 405 nm (FIG. 3). As a result, the epitope of the produced antibody was found to recognize 3-51 aa of SCYE1.

Example  5: In in vitro Anti- SCYE1 IgG Has inflammation ( inflammation ) Impact

RAW264.7 cells, which are mouse macrophages, were cultured in DMEM containing 1% streptomycin / penicillin, 10% FBS. RAW264.7 cells were seeded in 12 well plates with ⁴ × 10 ⁵ cells and incubated for 12 hours. Each well was treated with 0.4 ug / ml of SCYE1 protein or incubated with anti-SCYE1 IgG for 5 minutes and then treated with RAW264.7 for 30 minutes. The culture was recovered and quantified TNFα according to the method supplied by the manufacturer using a mouse TNFα ELISA kit (BD Biosciences).

To determine whether anti-SCYE1 IgG could inhibit the cytokine function of SCYE1, the amount of TNFα was measured in culture of RAW264.7 cells. After 30 min treatment of 400ng / ml SCYE1, the secretion of mouse TNFα was increased about 14 times compared to the control (vehicle, TNFα 0.11 ± 0.01ng / ml; SCYE1 0.4ug / ml, TNFα 1.41 ± 0.05ng) / ml, p <0.001). On the other hand, when SCYE1 and anti-SCYE1 IgG were incubated for 5 minutes in advance and then treated with RAW264.7 cells, the concentration of TNFα secretion in RAW264.7 cells by SCYE1 stimulation was inhibited in a concentration dependent manner (SCYE1 0.4ug / ml, TNFα 1.41 ± 0.05 ng / ml; SCYE1 0.4 ug / ml + anti-SCYE1 IgG 5 ug / ml, 1.11 ± 0.09 ng / ml; SCYE1 0.4 ug / ml + anti-SCYE1 IgG 10 ug / ml, 0.48 ± 0.06 ng / ml; SCYE1 0.4 ug / ml + anti-SCYE1 IgG 20 ug / ml, 0.25 ± 0.03; SCYE1 0.4ug / ml + mock IgG 20ug / ml, 1.28 ± 0.07 ng / ml, p <0.001). Treatment with mock IgG did not inhibit the secretion of TNFα by SCYE1 (FIG. 4).

Example  6: anti- SCYE1  Effect of Antibodies on the Behavior of Alzheimer's Disease Mice

Dementia was induced by injecting β-amyloid (2ug / mouse) into the third ventricle of male 12-week-old BLAB / C mice. After 24 hours, eyes blink test was performed to select only animals that showed normal reflexes. Used. Anti-SCYE1 IgG was divided into 3 groups and injected subcutaneously once a week at 2mg / kg, 5mg / kg and 10mg / kg. As a positive control, Aricept (2mg / kg, Korea Ezai) was administered orally once a total of 28 times for 4 weeks. As a negative control, normal IgG was injected subcutaneously once a week at 10 mg / kg. Four weeks later, mice were subjected to a Morris water maze test to evaluate the effect on memory. The test was conducted for 7 days and the total duration time, total distance, and probe trial (visiting duration and distance) were investigated. All results were expressed as mean ± SEM and statistical significance was judged to be significant only when p <0.05 or less through t- test.

The weight change of the rats used in the test during the 4 week trial period was 2.1 ± 0.42g in all groups, and no difference in body weight was observed between the groups. The time required to find the platform in Morris's maze test was 49.37 ± 3.45 seconds in the group not treated with β-amyloid, while 56.19 ± 2.19 seconds was taken in the group treated with β-amyloid ( p <0.05). . This is believed to be due to Alzheimer's induction by β-amyloid.

On the other hand, in the group treated with β-amyloid and subcutaneously injected with anti-SCYE1 IgG for 4 weeks, 51.84 ± 2.67 sec (2 mg / kg group), 51.2 ± 2.14 sec (5 mg / kg group), and 49.07 ± 2.24 sec (10 mg / kg) kg group). This was about 4.3 seconds, 4.99 seconds, 7 seconds improvement compared to the β-amyloid-treated negative control ( p <0.05). In particular, the 10 mg / kg group showed similar time to the untreated group without β-amyloid, while the mock IgG (10 mg / kg) group showed no time-reducing effect. The symptoms seem to have improved. On the other hand, Aricept used as a positive control tended to decrease slightly compared to the control, but no statistically significant change was observed (FIG. 5A).

Next, the effect of anti-SCYE1 antibody administration on the total distance traveled to visit the platform of Alzheimer's mice was investigated. The total travel distance to the platform was significantly increased in the β-amyloid treated group compared to the non-beta-treated group (normal, 797.85 ± 50.43mm; β-amyloid control, 925 ± 35mm, p <0.05). In the β-amyloid treated group, no significant change was observed in the control group, the anti-SCYE1 IgG group, the Aricept, or the mock IgG group (FIG. 5B).

To test the memory of the mouse, the platform was divided into 4 pieces in the probe trial, and the ratio of the mice to the platform where they were first placed was measured.The group treated with anti-SCYE1 IgG compared to the group treated with β-amyloid only. The rate of visits to the platform from which it was first located increased overall. In particular, a 300% increase was observed in the 10 mg / kg IgG treated group ( p <0.05) (FIG. 6A). In addition, as a result of measuring the percentage of time mice stayed on the platform of the first position among the four platforms, it was observed that the group treated with β-amyloid decreased about 65% compared to the untreated group (p <0.05). When the anti-SCYE1 IgG was administered to the group treated with β-amyloid, it was observed that the time to stay on the first platform was increased in a concentration-dependent manner (FIG. 6B). In particular, in the 10 mg / kg treatment group, the time to stay on the platform was increased about 400% compared to the group not treated with β-amyloid (p <0.01). On the other hand, the mock IgG group showed no improvement at all and the aricept showed an improvement but no statistically significant increase.

In conclusion, the present invention confirmed once again that SCYE1 promotes the secretion of TNFα by activating mouse macrophages, and demonstrated that anti-SCYE1 antibody can inhibit the secretion of TNFα. In addition, it was confirmed that the anti-SCYE1 antibody improves dementia symptoms when subcutaneously injected into β-amyloid-induced Alzheimer's mice.

1 shows the results of determining the individual types of the monoclonal antibodies of the present invention.

Figure 2 shows the results confirming that the monoclonal antibody cell line of the present invention is SCYE1 specific.

Figure 3 is a result showing the epitope recognition site of the monoclonal antibody of the present invention.

4 shows inhibition of TNFα production by anti-SCYE1 IgG. RAW264.7 cells were seeded in 12 well plates and treated with SCYE1 (0.4 ug / ml) with or without anti-SCYE1 IgG (5 ug / ml, 10 ug / ml, 20 ug / ml). Cell culture supernatants were harvested and analyzed for TNFα production using an ELISA kit. Values are mean ± SEM. *, p <0.001.

FIG. 5 shows a total duration time or migration distance analysis reaching the platform in a Morris water maze test. Alzheimer's mice induced by β-amyloid were injected into the subcutaneous area four times a week with anti-SCYE1 IgG (2 mg / kg, 5 mg / kg, 10 mg / kg) or mock IgG (20 mg / kg). Aricept (2 mg / kg) was administered daily for 4 weeks as a positive control. The total duration (A) or distance traveled (B) to reach the platform was evaluated and expressed as mean ± SEM. *, p <0.05.

6 is a result of analysis of target platform visiting rate and duration rate of Alzheimer's-induced mice induced by β-amyloid in a probe trial of Morris's underwater maze test. The target platform visiting rate and duration rate of the four pieces of the platform were examined and expressed as the ratio of platform visiting mice / total test mice. *, p <0.05; **, p <0.01.

<110> YOON, Kang Jun <120> Monoclonal antibody recognizing SCYE1 and use <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 312 <212> PRT <213> Homo sapiens <400> 1 Met Ala Asn Asn Asp Ala Val Leu Lys Arg Leu Glu Gln Lys Gly Ala   1 5 10 15 Glu Ala Asp Gln Ile Ile Glu Tyr Leu Lys Gln Gln Val Ser Leu Leu              20 25 30 Lys Glu Lys Ala Ile Leu Gln Ala Thr Leu Arg Glu Glu Lys Lys Leu          35 40 45 Arg Val Glu Asn Ala Lys Leu Lys Lys Glu Ile Glu Glu Leu Lys Gln      50 55 60 Glu Leu Ile Gln Ala Glu Ile Gln Asn Gly Val Lys Gln Ile Ala Phe  65 70 75 80 Pro Ser Gly Thr Pro Leu His Ala Asn Ser Met Val Ser Glu Asn Val                  85 90 95 Ile Gln Ser Thr Ala Val Thr Thr Val Ser Ser Gly Thr Lys Glu Gln             100 105 110 Ile Lys Gly Gly Thr Gly Asp Glu Lys Lys Ala Lys Glu Lys Ile Glu         115 120 125 Lys Lys Gly Glu Lys Lys Glu Lys Lys Gln Gln Ser Ile Ala Gly Ser     130 135 140 Ala Asp Ser Lys Pro Ile Asp Val Ser Arg Leu Asp Leu Arg Ile Gly 145 150 155 160 Cys Ile Ile Thr Ala Arg Lys His Pro Asp Ala Asp Ser Leu Tyr Val                 165 170 175 Glu Glu Val Asp Val Gly Glu Ile Ala Pro Arg Thr Val Val Ser Gly             180 185 190 Leu Val Asn His Val Pro Leu Glu Gln Met Gln Asn Arg Met Val Ile         195 200 205 Leu Leu Cys Asn Leu Lys Pro Ala Lys Met Arg Gly Val Leu Ser Gln     210 215 220 Ala Met Val Met Cys Ala Ser Ser Pro Glu Lys Ile Glu Ile Leu Ala 225 230 235 240 Pro Pro Asn Gly Ser Val Pro Gly Asp Arg Ile Thr Phe Asp Ala Phe                 245 250 255 Pro Gly Glu Pro Asp Lys Glu Leu Asn Pro Lys Lys Lys Ile Trp Glu             260 265 270 Gln Ile Gln Pro Asp Leu His Thr Asn Asp Glu Cys Val Ala Thr Tyr         275 280 285 Lys Gly Val Pro Phe Glu Val Lys Gly Lys Gly Val Cys Arg Ala Gln     290 295 300 Thr Met Ser Asn Ser Gly Ile Lys 305 310 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 2 gcgaattcat ggcaaataat gatgctgttc 30 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 3 ccgctcgagt tatttgattc cactgttgct catg 34  

Claims (7)

A monoclonal antibody that recognizes amino acid positions 3 to 51 from the amino terminus of the human SCYE1 antigen consisting of the amino acid sequence of SEQ ID NO: 1. The monoclonal antibody according to claim 1, wherein a detection label is bound to a detectable signal. The monoclonal antibody according to claim 2, wherein the detection labeling agent is an enzyme, a fluorescent substance, a luminescent substance or a radioactive substance. SCYE1 quantification kit comprising the monoclonal antibody of claim 1. 5. The SCYE1 quantitative kit of claim 4, further comprising a secondary antibody recognizing a monoclonal antibody, wherein the detection marker binds to the secondary antibody. 6. The SCYE1 quantification kit according to claim 5, wherein the detection labeling agent is an enzyme, and further comprises a substrate capable of measuring enzymatic activity and a reaction terminator. i) contacting the biological sample with the SCYE1 monoclonal antibody of claim 1 in combination with a detection labeling agent, ii) quantitatively detecting the immunological complex obtained in step i), iii) quantifying the immunological complex obtained by contacting the purified SCYE1 antigen with the SCYE1 monoclonal antibody of claim 1 coupled with a detection label to obtain a standard curve, and iv) quantifying SCYE1 present in the biological sample by comparing the amount of protein quantified in step ii) with the standard curve obtained in step iii) A method for quantifying SCYE1 in a sample using a SCYE1 monoclonal antibody comprising a.

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