WO2010093214A2 - Monoclonal antibody recognizing scye1 and use thereof - Google Patents

Monoclonal antibody recognizing scye1 and use thereof Download PDF

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WO2010093214A2
WO2010093214A2 PCT/KR2010/000934 KR2010000934W WO2010093214A2 WO 2010093214 A2 WO2010093214 A2 WO 2010093214A2 KR 2010000934 W KR2010000934 W KR 2010000934W WO 2010093214 A2 WO2010093214 A2 WO 2010093214A2
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scye1
monoclonal antibody
antibody
cells
quantifying
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PCT/KR2010/000934
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French (fr)
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WO2010093214A3 (en
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Kang Jun Yoon
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Stem Science
Cell & Bio
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines

Definitions

  • the present invention relates to a monoclonal antibody recognizing SCYE1 and a use thereof, more specifically to a monoclonal antibody recognizing SCYE1 protein, and a method and kit for quantifying SCYE1 by using the antibody.
  • SCYE1 small inducible cytokine subfamily E, member 1
  • SCYE1 acts as a cofactor of protein synthase in cells by binding to tRNA. It is secreted out of the cells by stimulation of cytokine.
  • the secreted SCYE1 inhibits angiogenesis by inhibiting proliferation and immigration of endothelial cells.
  • a conventional antibody produced in a body by stimulation of antigen is an antibody mixture which has various binding affinities to antigens (polyclonal antibody).
  • monoclonal antibodies are homogeneous antibodies that have the same structure formed by cells proliferated from a single antibody producing cells (monoclone) because the antibody producing cells produces one kind of antibody.
  • the method for preparing a monoclonal antibody producing cells was developed in 1975 by Koller and Milstein. This is the method by which B-lymphocyte derived from the spleen or lymph node of an animal which has been immunized by a specific antigen is fused with myeloma cells.
  • a B-lymphocyte is a normal cell having the ability to produce monoclonal antibodies.
  • a myeloma cell obtained from cancered B-lymphocyte is a cell that does not has an ability to produce immunoglobulin but has an abnormal proliferative ability that can consistently proliferate.
  • the fusion cell of these cells has both the ability to produce monoclonal antibodies of B-lymphocyte and be persistently proliferated without death.
  • the fusion cells need to be screened because fusion cells, non-fused spleen cells and myeloma cells are present in the culture media.
  • the cells are cultured in HAT media. Because purine (adenine and guanine) is biosynthesized through the two pathways of endogenous pathway and salvage pathway, it is not critical to a wild type strain to lose one of those pathways.
  • HAT media contain hypoxanthine, animopterin and thymidine.
  • non-fused myeloma cells on the salvage pathway are also blocked by a deficiency of hypoxanthine phosphoribosyl transferase (HGPRT) of the salvage pathway (thus having a tolerance to toxic base analog (thioguanine or azaguanine)), and the cells died.
  • HGPRT hypoxanthine phosphoribosyl transferase
  • the non-fused spleen cells naturally die within 1-2 weeks after separation. Accordingly, only the fusion cells grow in the media and they can be screened.
  • the monoclonal antibody which represents a specific reaction can be screened since it recognizes a single epitope.
  • There is a major antigen site influencing an antibody formation in a whole antigen and the use of a monoclonal antibody on this antigen site can improve the sensitivity compared with the antigen diagnostic method by using antiserum.
  • the present invention was in response to the above need.
  • the present inventors have worked persistently to obtain a monoclonal antibody recognizing SCYE1 protein.
  • the present invention was completed, in which the fusion cells are prepared by a cell fusion of a spleen cell obtained from a SCYE1 protein immunized mouse and a myeloma cell, and monoclonal antibodies are obtained by screening and culturing the fusion cells recognizing SCYE1.
  • the object of this invention is to provide monoclonal antibodies recognizing SCYE1 which causes an inflammation through the induction of secretion of TNF- ⁇ and IL-8 of immune cells including monocytes, macrophages, dendritic cells.
  • Another object of this invention is to provide the hybridoma which produces the monoclonal antibodies recognizing SCYE1.
  • Another object of this invention is to provide a kit for quantifying SCYE1 which includes the monoclonal antibodies recognizing SCYE1.
  • Another object of this invention is to provide s method for quantifying SCYE1 in the samples by using the monoclonal antibodies recognizing SCYE1.
  • the monoclonal antibodies recognizing SCYE1 protein can be used in diagnosis and treatment of inflammatory disease related to SCYE1 by measuring the concentration of SCYE1 in blood.
  • Figure 1 is a result of determining the isotype of a monoclonal antibody of the present invention.
  • FIG. 2 is a result that the monoclonal antibody cell line of the present invention which is specific to SCYE1.
  • Figure 3 is a result showing the epitope recognition site of the monoclonal antibody of the present invention.
  • Figure 4 represents the inhibition of TNF- ⁇ production by anti-SCYE1 IgG.
  • RAW264.7 cells were innoculated in 12-well plate, SCYE1 (0.4 ug/ml) was treated in a presence or an absence of anti-SCYE1 IgG (5ug/ml, 10ug/ml, 20ug/ml).
  • the supernatant of cell culture was obtained and the production of TNF- ⁇ was analyzed by using ELISA kit. The value is mean ⁇ SEM. *, p ⁇ 0.001.
  • Figure 5 represent the analysis of the total duration time or migration distance to reach the platform in Morris water maze test.
  • Anti-SCYE1 IgG (2mg/kg, 5mg/kg, 10mg/kg) or mock IgG (20mg/kg) was subcutaneously injected into Alzheimer's disease mouse caused by ⁇ -amyloid, once per week for a total of 4 times.
  • Aricept (2mg/kg) was injected daily for 4 weeks as a positive control.
  • the total duration time (A) or migration distance to reach the platform was evaluated and represented as mean ⁇ SEM. *, p ⁇ 0.05.
  • Figure 6 is the analysis result of target platform visiting rate and duration rate of Alzheimer mouse induced by ⁇ -amyloid in probe trial of Morris water maze test.
  • the target platform visiting rate and duration rate of the mouse were investigated and represented as a ratio of a number of platform visiting mice/ a number of total test mice. *, p ⁇ 0.05; **, p ⁇ 0.01.
  • antibody refers to a molecule or an active fragment of the molecule that binds to a known antigen.
  • the active fragment that binds to a known antigen include Fab and F(ab) 2 fragments.
  • the active fragment can be derived from the antibody of the present invention by numerous techniques. For example, a purified monoclonal antibody can be cleaved by an enzyme such as pepsin and subjected to an HPLC gel filtration. A proper fraction containing Fab fragments can be collected and concentrated by membrane filtration. Information on general techniques for separating an active fragment of an antibody can be referenced from the documents [for example, Khaw, B.A. et al., J. Nucl. Med. 23: 1011-1019 (1982)].
  • the term "antibody” also includes bispecific and chimeric antibodies.
  • the term "monoclonal antibody” is known in the art and is a highly specific antibody directed to a single antigenic moiety. In contrast to a polyclonal antibody that includes different antibodies directed to different determinants (epitopes), a monoclonal antibody is typically directed to a single determinant on the antigen.
  • the monoclonal antibody of the present invention can be prepared using conventional cloning and cell fusion techniques. For example, an immunogen (antigen) of interest is administered to a wild-type or bred mouse (for example, BALB/c) to produce a natural or human monoclonal antibody. The antigen can be administered alone or in admixture with an adjuvant, or can be expressed from a vector.
  • the antigen is DNA or a fusion protein that can induce an immune response.
  • the fusion protein includes a carrier protein, which is coupled to a peptide designed to induce an immune response - for example, ⁇ -galactosidase, glutathione S-transferase, keyhole limpet hemocyanin (KLH) and bovine serum albumin, but the carrier protein is not limited thereto.
  • the peptide functions as a hapten for the carrier protein.
  • the following is a simple explanation of a method for preparing a monoclonal antibody. After boosting an animal, the spleen is removed.
  • Spleen cells are extracted and fused with myeloma cells according to known methods 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 obtained hybrid cells are cloned using a conventional method - for example, a restricted dilution.
  • the obtained clone which produces a desired monoclonal antibody is cultured.
  • a monoclonal antibody has the advantage of improving the selectivity and specificity of a diagnosis and an analytic assay that uses an antigen-antibody binding.
  • a monoclonal antibody is synthesized by culturing a hybridoma, it has another advantage of not being contaminated by other immunoglobulins.
  • epitope is known in the art and generally refers to a region of an antigen that interacts with an antibody.
  • An epitope of a peptide or protein antigen can be formed by continuous or non-continuous amino acid sequences of the antigen.
  • Many proteins can contain numerous epitopes.
  • An epitope recognized by the antibody of the present invention forms one embodiment of the present invention.
  • An antibody that can recognize a certain epitope can be used for the immunoaffinity column for purifying said certain epitope. Since it is reported that an antigenic epitope can be formed by a small number (such as five) of amino acid residues, a terminal cleavage form of a certain epitope also can be used.
  • hybridoma refers to a cell formed by fusion of an antibody-producing cell and an immortal cell - for example, a myeloma cell.
  • the hybridoma cell can continuously supply an antibody.
  • the term "forming an immunological complex” is meant to include confirming the existence or non-existence of the SCYE1 antigen in a sample.
  • the existence or non-existence of the SCYE1 antigen can be detected by use of an immunoassay.
  • Numerous immunoassays for detecting and/or quantifying an antigen would be known to a person having ordinary skill in the art. See the document [Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York 1988, 556-612].
  • detection marker refers to a marker for detecting an immunological complex, specific examples of which include a radioisotope marker; an enzyme; a chemoluminescent compound; a fluorescein such as phycobiliprotein, rare earth chelate and rhodamine; an enzyme cofactor; biotin and the like, but are not limited thereto.
  • biological sample is meant to include a biological substance including cells, tissues or a biological fluid.
  • the present invention provides a monoclonal antibody recognizing an amino acid region at positions 3 to 51 from the amino terminal of human SCYE1 antigen which consists of the amino acid sequence of SEQ ID NO. 1.
  • the monoclonal antibody can be combined with a detection marker that can produce a detectable signal.
  • the detection marker can be an enzyme, a fluorescein, a luminescent substance or a radio substance.
  • the present invention also provides a SCYE1 assay kit comprising the monoclonal antibody.
  • the kit can further comprise a second antibody that recognizes the monoclonal antibody, and the second antibody can be combined with a detection marker.
  • the detection marker is an enzyme
  • the kit can further comprise a substrate that can be used for measuring an enzyme activity, and a reaction-stopping reagent.
  • the present invention also provides a method for quantifying SCYE1 in a sample by using the SCYE1 monoclonal antibody comprising the steps of
  • step iv) quantifying the SCYE1 present in the biological sample by comparing the amount of protein detected to quantify in step ii) with the standard curve obtained in step iii).
  • the present invention provides an antibody which is immunologically reactive to the SCYE1 protein or epitopes thereof.
  • the antibody provided by this invention can be produced in an animal by inoculating cells expressing the SCYE1 protein or an epitope thereof according to a known method in the art.
  • the protein can be separated from the cells with various levels of homogeneity through a conventional biochemical method.
  • the synthetic peptide prepared by the synthetic method confirmed in vitro and optionally conjugated to a heterogeneous amino acid sequence also includes a method for preparing the antibody of this invention.
  • the animals used in the inoculation include cow, sheep, pig, mouse, rat, rabbit, hamster, goat and primate.
  • the preferable animals for the inoculation include rodent - for example, mouse, rat and hamster. The most preferable animal is mouse.
  • the monoclonal antibody provided in the present invention is also produced by a recombinant genetic engineering method known to a person having ordinary skill in the art, and the present invention includes an antibody according to the above method, which is immunologically reactive to the epitope of SCYE1 protein of this invention.
  • the present invention also includes fragments of the antibody - for example, F(ab) and F(ab) 2 , but is not limited thereto.
  • the fragments are prepared by any method which includes the preparation of the fragment according to a protein hydrolysis cleavage, chemical synthesis or genetic engineering method, but is not limited thereto.
  • the present invention may include a single-chain antibody immunologically reactive to SCYE1 protein and prepared by a method known to a person having ordinary skill in the art.
  • the present invention also includes chimeric antibody comprising light and heavy-chain peptide, which is immunologically reactive to the epitope induced by SCYE1 of this invention.
  • the chimeric antibody provided by this invention includes not only natural antibody but also chimeric antibody prepared according to genetic engineering techniques known to a person having ordinary skill in the art.
  • the monoclonal antibody of this invention should be understood to include any antibody recognizing SCYE1, which is prepared by using human SCYE1 or recombinant SCYE1 as an antigen.
  • the cells which can be used in this invention are any cells or cell lines that can express natural or genetically modified SCYE1 protein provided by this invention.
  • the cells which can be used in this invention include HEK-293 which is a mammalian cell, but is not limited thereto.
  • the monoclonal antibody provided in this invention is produced by a hybridoma cell line, which is prepared by a method known in the art [note: Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)].
  • the hybridoma cell line is prepared by fusing each cell of myeloma cell line with spleen cells induced from an animal immunized by an agent containing SCYE1 protein itself or heterogeneous or fusional protein construct of the protein, a membrane comprising the agent, cells encoding the protein or the protein.
  • the myeloma cell lines used in this invention include the cell lines derived from mouse, rat, hamster, primate and human myeloma.
  • the myeloma cells derived from a mouse can be used - for example, P3X63Ag8, p3-U1, NS-1, MPC-11, SP-2/0, F0, P3x63Ag8. V653 and S194.
  • the cell lines such as R-210, which are derived from rat, can be also used.
  • the preferable myeloma cell lines used in this invention include myeloma cell SP2/0-Ag14 (ATCC CRL-1581) and P3X63Ag8.653 (ATCC CRL-1580).
  • a preferable animal from which a spleen can be obtained after immunization includes rat, mouse and hamster, preferably mouse.
  • a spleen cell and a myeloma cell can be fused using many methods known in the art including an inoculation of Sendai virus and an inoculation of polyethylene glycol (PEG), but is not limited thereto.
  • the monoclonal antibody produced by hybridoma cell lines can be obtained from a supernatant of cell media by in vitro cell growth, or the hybridoma cells can be injected into an animal, preferably subcutaneously and/or intraperitoneally, and a monoclonal antibody is thus obtained from blood or ascite.
  • the hybridoma cell that produces the monoclonal antibody to SCYE1 according to this invention is the fusion cell of myeloma cell P3X63Ag8.653 cell (ATCC CRL-1580, USA) and spleen cell producing the antibody to SCYE1.
  • the present invention also provides a method for diagnosis and treatment of inflammatory disease by detecting the expression of SCYE1 protein in an animal, preferably in a human.
  • the diagnosing reagent to use in the method includes antibody, most preferably a monoclonal antibody of this invention.
  • the antibody can be used in conventional immunological techniques - for example, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), western blot analysis, immunological titer analysis, immunological diffusion analysis and a method known to a person having ordinary skill in the art, but is not limited thereto.
  • the reagents used in the immunological analysis include suitable carrier, detection marker which can produce a detectable signal, resolvent and detergent.
  • a marker substance is an enzyme
  • a substrate which can measure the enzyme activity and reaction stopping agent can be included.
  • the suitable carriers can be soluble carriers, e.g., physiologically acceptable buffer solution known in the art, or insoluble carriers, e.g., polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin, cross-linked dextran, polysaccharide, polymer such as magnetic particulate that is a latex plated with metal, paper, glass, metal agarose, and combination thereof, but is not limited thereto.
  • the antibody of the present invention can be easily detected by coupling with detectable substances, i.e., detection markers.
  • detection markers include various enzymes, prosthetic groups, fluorescein, luminescent substances, biological luminescent substances, radioactive substances and the like.
  • the detection marker can be directly coupled or linked to the SCYE1 monoclonal antibody of this invention by using techniques known in the art, indirectly coupled or linked through an intermediate (e.g., linkers known in the art), or coupled or linked to an another monoclonal antibody recognizing SCYE1 protein or a second antibody recognizing SCYE1 antibody.
  • suitable enzymes include horseradish peroxidase, acetylcholine esterase, peroxidase, alkaline phosphatase, ⁇ -D-galactosidase, glucose oxidase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, invertase and the like;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrine or phycobiliprotein;
  • the examples of luminescent substances include luminol, isolucinol and lucigenin;
  • biological luminous substances include luciferase, luciferin and aequorin;
  • suitable ratioactive substances include 125 I, 131 I, 111 In
  • the present invention also provides an analytical method or analytical kit which detects the formation of immunological complex of monoclonal antibody and SCYE1 by using monoclonal antibody to SCYE1, for detecting or quantifying SCYE1 in samples by applying an immunoassay or a kit for immunoassay.
  • an immunoassay and kit for immunoassay using a monoclonal antibody known in the art, e.g., ELISA analysis and radioimmunoassay, can be applied to this invention.
  • the kit used for SCYE1 quantification analysis of this invention may have a container including the SCYE1 monoclonal antibody of this invention, a suitable carrier solution, a detection marker which can produces a detectable signal, a resolvent and detergent, and a manual.
  • a marker substance is an enzyme
  • a substrate that can measure the enzyme activity and reaction stopping agent may be included.
  • a detection marker is linked directly or through a linker to the SCYE1 monoclonal antibody according to this invention, or may include other secondary antibody to SCYE1 linked to a detection marker.
  • One preferable embodiment of this invention provides the method for quantifying SCYE1, by which biological samples are added to a microtiter plate coated with the monoclonal antibody of this invention and reacted with the antibody. After the enzyme-antibody complex is reacted, the enzyme substance is treated and the absorbance is measured. SCYE1 presented in the biological samples is quantified by comparing the absorbance with a standard curve.
  • the antibody of the enzyme-antibody complex can be linked to biotin.
  • the enzyme also can be horseradish peroxidase (hereinafter referred to as HPR), but is not limited thereto, and any enzyme and substrate used in the ELISA analysis can be adapted in this invention.
  • ELISA analysis is an analysis method to determine the presence or absence of antigen and to quantify the amount of antibody present in the samples by using antibody and antigen reaction.
  • one ELISA kit about two to three antibodies are need.
  • the ELISA kit using the monoclonal antibody recognizing SCYE1 consists of the above-mentioned three antibodies. The constitution and principle are as follows.
  • a 96-well plate (in case of quantification of SCYE1 in human plasma) is coated with a monoclonal antibody (first antibody), the sample including SCYE1 is added to the well and reacted with the antibody, and an enzyme marked polyclonal antibody (secondary antibody) is reacted. After the substrate reactable with the enzyme marker is treated, the enzyme-substrate reaction is probed and the SCYE1 presented in the samples can be quantified.
  • biotin and streptavidine conjugate HRP can be used as an enzyme and a substrate, but is not limited thereto.
  • ORF(1-312aa) encoding human SCYE1 was amplified by using primer; 5'-GCGAATTCATGGCAAATAATGATGCTGTTC-3' (SEQ ID No. 2), R: 5'-CCGCTCGAGTTATTTGATTCCA CTGTTGCTCATG-3' (SEQ ID NO. 3) from HEK293 cDNA and performing 25 cycles of 95°C 1 min, 55°C 1 min and 72°C 1 min.
  • the PCR product was restricted by EcoRI/SalI, and ligased with pGEX4T-1 (Pharmacia) vector, formerly restricted with EcoRI/SalI, for 2 hours at room temperature. After the transformation of DH-5 ⁇ by heat shock for 90 sec at 42°C, the E. coli was cultured in LB media containing ampiciline (50 ⁇ g/ml), and colonies including SCYE1 were selected. The cloned SCYE1 gene was sequenced and there was no mutation.
  • E. coli BL21 (DE3) with SCYE1 plasmid
  • the E. coli was plated to LB media containing ampiciline (50 ⁇ g/ml), and cultured for 24 hours at 37°C.
  • the grown colony was cultured in 5 ml of LB media containing ampiciline (50 ⁇ g/ml) for 12 hours, and then 4ml of the cultured media was inoculated to 2L of LB media containing ampiciline (50 ⁇ g/ml). It is cultured at 37°C until O.D (600nm) reached 0.3.
  • IPTG was added in a final concentration of 0.1mM, and p43 protein was expressed by culturing for 6 hours at 30°C.
  • coli was centrifuged for 15 min at 7,000 rpm, the cells were resuspended by 1XPBS (8g NaCl, 0.2g KCl, 1.44g Na 2 HPO, 0.24g KH 2 PO 4 /L, pH 7.4), sonicated at 4°C and centrifuged for 40 min at 26,000 x g. The supernatant was obtained and loaded to a glutathione Sepharose 4B-column which become homogeneous by 1xPBS. The resin was washed with PBS, and GST-p43 was eluted by using 50mM of Tris-HCl (pH 8.0) containing 10mM of glutathione. After the purity was identified by using 10% SDS-PAGE, dialysis was performed by using PBS containing 20% glycerol, and the dialyzed product was stored at -70°C. The purity was over 90% on SDS-PAGE.
  • 1XPBS 8g NaCl, 0.2g KC
  • An emulsion was prepared by mixing 20 ⁇ g/mouse of the protein with the same volume of Complete Freund's Adjuvant (Sigma, USA). The emulsion was intraperitoneally injected into three 7-week-old female Balb/c mice (orient). 20 ⁇ g of antigen was injected into each mouse with the 400 ⁇ l of total volumn. After 2 weeks, the emulsion mixing Incomplete Freund's Adjuvant (Sigma, USA) and antigen was intraperitoneally injected into the mouse. After 2 weeks, the antigen (20 ⁇ g/mouse) dissolved in PBS was intraperitoneally injected to induce the production of antibody. After identifying the antibody by performing enzyme immunoassay and western blot analysis, antigen dissolved in PBS was further injected into a tail vein of the mouse 3 days before the cell fusion.
  • Blood was obtained from the eye ball of an immunized mouse according to the above method, placed into the 1.5ml microcentrifuge tube and centrifuged for 10 min at 13,000 rpm. The serum was separated and stored at -20°C until the experiment for identifying the production of antibody was performed. After identifying the production of antibody by performing enzyme immunoassay and western blot analysis using antigen protein, the fusion for spleen cells of the antibody-producing mouse was performed.
  • the mouse was sacrificed and the splenocytes were separated.
  • the splenocytes were fused with myeloma cells P3X63Ag8.653 (ATCC CRL-1580, USA). That is, P3X63Ag8.653 cells of the mouse were maintained in the culture plate by using RPMI1640 media supplemented with 10% fetal bovine serum.
  • P3X63Ag8.653 cells were washed two times with serum-free RPMI1640 media (Biowhittaker, USA) and adjusted to become the concentration of 1 x 10 7 cells.
  • the mouse was sacrificed by cervical dislocation, and its spleen was obtained.
  • 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. After the completely separated antibody-producing cells were centrifuged for 5 min at 400g, they were washed two times with serum-free media and resuspended into the 10ml media. The lymph cells were counted by using a haemocytometer, and 1x10 8 of lymphocytes were mixed with 1 x 10 7 of P3X63Ag8.653 cells (10:1) in the serum-free media. The centrifugation was performed for 5 min at 400g.
  • HAT selection media were replaced in 2 to 3 days intervals, and the cells were cultured for 14 days. After 14 days, subculture was performed by replacing RPMI1640 media supplemented with 20% fetal bovine serum and HT (media in which 0.4 ⁇ M aminopterine is removed from HAT media).
  • the supernatant of the culture fused in the above method 3) was obtained, and the enzyme immunoassay was performed to investigate the production of the provided antigen-specific antibody.
  • the culture solution of fusion cells which represents the titer more than 4 times compared with negative control was selected and transferred to a 24-well culture plate and a 25 cm 2 culture flask.
  • the isotype of monoclonal antibody was determined by using isotyping kit (Zymed Labomouseories Inc. USA) using enzyme immunoassay with culture supernatant ( Figure 1).
  • the antibody to SCYE1 was identified as IgG1.
  • the western blotting was performed by using whole-cell extract of HEK293 cells to determine whether the antibody derived from the prepared cell line specifically recognizes SCYE1 by using culture solution of the prepared SCYE1 monoclonal antibody cell line.
  • the prepared monoclonal antibody cell line was specific to SCYE1 ( Figure 2).
  • the peptide of human SCYE1 was synthesized (3-54, 51-90, 103-139, 191-219aa of ORF) and a 96-well plate was coated with 100ng of the peptide per well. Blocking was performed by using 1X PBS containing 2% BSA (sigma) for 1 hour at room temperature. 100 ⁇ l of culture supernatant was loaded to each well and reacted for 1 hour at room temperature. After washing 3 times with PBS containing 0.1% Tween 20, HRP-conjugated anti-mouse IgG was loaded and reacted for 1 hour at room temperature. After rewashing 4 times with PBS containing 0.1% Tween 20, TMB (sigma) was added and reacted for 20 minutes. The result of the reading at 405nm represented that the epitope of the prepared antibody recognized 3-51 aa of SCYE1 ( Figure 3).
  • RWA264.7 cells mouse macrophage, were cultured in DMEM supplemented with 10% FBS and 1% streptomycin/penicillin.
  • RAW264.7 cells were seeded to a 12-well plate in 4X10 5 cells/well and cultured for 12 hours. In each well, RAW264.7 cells were treated for 30 min with 0.4 ⁇ g/ml of SCYE1 protein or with SCYE1 protein incubated with anti-SCYE1 IgG for 5 min. The culture solution was obtained and TNF- ⁇ was quantified by using mouse TNF- ⁇ ELISA kit (BD Biosciences) according to the method of the manual.
  • TNF- ⁇ was measured in the culture solution of the RAW264.7 cells. It was observed that the secretion of mouse TNF- ⁇ was increased about 14 times compared with the control after treating 400ng/ml of SCYE1 for 30 min (vehicle, TNF- ⁇ 0.11 ⁇ 0.01ng/ml; SCYE1 0.4 ⁇ g/ml, TNF- ⁇ 1.41 ⁇ 0.05ng/ml, p ⁇ 0.001).
  • Example 6 The effect of anti-SCYE1 antibody on the behavior of an Alzheimer's disease mouse
  • Alzheimer's disease was induced by injecting ⁇ -amyloid (2 ⁇ g/mouse) into the third ventricle of a male 12-week-old BALB/C mouse. After 24 hours, mice which showed normal reflex reactions in eye-blink tests were selected and used, with 10 mice per group.
  • the anti-SCYE1 IgG was subcutaneously injected into 3 groups of mice with the amount of 2mg/kg, 5mg/kg, 10mg/kg once per week. Aricept (2mg/kg, Eisai Korea Inc.) was orally administered as a positive control, once per day for 4 weeks, for a total of 28 times. 10mg/kg of normal IgG was subcutaneously injected once per week as a negative control.
  • the mean 2.1 ⁇ 0.42g of weight increase was observed in the mice used in the test for a total of 4 weeks in experiments in all groups, and there was no difference in the weight change among the groups.
  • the total duration time to find the platform was 49.37 ⁇ 3.45 sec in the group in which ⁇ -amyloid was not treated, whereas it was 56.19 ⁇ 2.19 sec in the group in which ⁇ -amyloid was treated ( p ⁇ 0.05). This is regarded as a result of inducing Alzheimer's disease caused by ⁇ -amyloid.
  • the total duration time was 51.84 ⁇ 2.67 sec (2mg/kg group), 51.2 ⁇ 2.14 sec (5mg/kg group), 49.07 ⁇ 2.24 sec (10mg/kg group) in the groups in which ⁇ -amyloid was treated and anti-SCYE1 IgG was subcutaneously injected.
  • the total duration time of 10mg/kg per group was similar to that of the group which was not treated with ⁇ -amyloid, whereas no time decrease was observed in the mock IgG (10mg/kg) group. It shows that anti-SCYE1 IgG specifically improved symptom of Alzheimer's disease.
  • Aricept was used as a positive control, there was no statistically significant change despite a tendency to decrease compared with the control ( Figure 5A).
  • the platform was divided and separated into 4 pieces and the ratio of the mouse that found the platform placed at first was measured.
  • the ratio finding platform placed at first in the group treated with anti-SCYE1 IgG was generally increased compared to that of the group treated only with ⁇ -amyloid.
  • a 300% increase was observed in the group treated with 10mg/kg of IgG ( p ⁇ 0.05) ( Figure 6A).
  • the ratio of the time that the mouse stayed on the platform placed at first among 4 platforms in the group treated with ⁇ -amyloid was decreased about 65% compared with the ratio in the non-treated group ( p ⁇ 0.05).
  • the present invention reidentified the fact that SCYE1 boosts the secretion of TNF- ⁇ by activating mouse macrophage and proved that anti-SCYE1 antibody can inhibit the secretion of TNF- ⁇ . It also identified that the symptom of Alzheimer's disease is improved by a subcutaneous injection of anti-SCYE1 antibody into a mouse with Alzheimer's disease induced by ⁇ -amyloid.

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Abstract

The present invention relates to a monoclonal antibody recognizing SCYE1 protein, and a method and kit for quantifying SCYE1. According to the present invention, the monoclonal antibody recognizing SCYE1 protein can be used in diagnosis and treatment of inflammatory disease related to SCYE1 by measuring the concentration of SCYE1 in blood.

Description

MONOCLONAL ANTIBODY RECOGNIZING SCYE1 AND USE THEREOF
The present invention relates to a monoclonal antibody recognizing SCYE1 and a use thereof, more specifically to a monoclonal antibody recognizing SCYE1 protein, and a method and kit for quantifying SCYE1 by using the antibody.
It is known that small inducible cytokine subfamily E, member 1 (SCYE1) causes inflammation by inducing the secretion of TNF-α and IL-8 from immune cells including monocytes, macrophages and dendritic cells. SCYE1 acts as a cofactor of protein synthase in cells by binding to tRNA. It is secreted out of the cells by stimulation of cytokine. The secreted SCYE1 inhibits angiogenesis by inhibiting proliferation and immigration of endothelial cells. Thus, there is a need to develop a technique and a material for inhibiting SCYE 1.
A conventional antibody produced in a body by stimulation of antigen is an antibody mixture which has various binding affinities to antigens (polyclonal antibody). However, monoclonal antibodies are homogeneous antibodies that have the same structure formed by cells proliferated from a single antibody producing cells (monoclone) because the antibody producing cells produces one kind of antibody. The method for preparing a monoclonal antibody producing cells was developed in 1975 by Koller and Milstein. This is the method by which B-lymphocyte derived from the spleen or lymph node of an animal which has been immunized by a specific antigen is fused with myeloma cells. A B-lymphocyte is a normal cell having the ability to produce monoclonal antibodies. A myeloma cell obtained from cancered B-lymphocyte is a cell that does not has an ability to produce immunoglobulin but has an abnormal proliferative ability that can consistently proliferate. As a result, the fusion cell of these cells has both the ability to produce monoclonal antibodies of B-lymphocyte and be persistently proliferated without death.
Completing the cell fusion, the fusion cells need to be screened because fusion cells, non-fused spleen cells and myeloma cells are present in the culture media. For this purpose, the cells are cultured in HAT media. Because purine (adenine and guanine) is biosynthesized through the two pathways of endogenous pathway and salvage pathway, it is not critical to a wild type strain to lose one of those pathways. HAT media contain hypoxanthine, animopterin and thymidine. Because the animopterin blocks the endogenesis pathway by inhibiting the action of dihydrofolate reductase (DHFR) of the pathway, non-fused myeloma cells on the salvage pathway are also blocked by a deficiency of hypoxanthine phosphoribosyl transferase (HGPRT) of the salvage pathway (thus having a tolerance to toxic base analog (thioguanine or azaguanine)), and the cells died. The non-fused spleen cells naturally die within 1-2 weeks after separation. Accordingly, only the fusion cells grow in the media and they can be screened.
Despite this method for preparing fusion cell producing antibodies, there is no report about monoclonal antibodies recognizing human SCYE 1 protein.
The monoclonal antibody which represents a specific reaction can be screened since it recognizes a single epitope. There is a major antigen site influencing an antibody formation in a whole antigen, and the use of a monoclonal antibody on this antigen site can improve the sensitivity compared with the antigen diagnostic method by using antiserum. Thus, there has been a need for the production of a monoclonal antibody to a specific antigen and the development of diagnostic agent using the antibody.
The present invention was in response to the above need. The present inventors have worked persistently to obtain a monoclonal antibody recognizing SCYE1 protein. As a result, the present invention was completed, in which the fusion cells are prepared by a cell fusion of a spleen cell obtained from a SCYE1 protein immunized mouse and a myeloma cell, and monoclonal antibodies are obtained by screening and culturing the fusion cells recognizing SCYE1.
In order to resolve the problem, the object of this invention is to provide monoclonal antibodies recognizing SCYE1 which causes an inflammation through the induction of secretion of TNF-α and IL-8 of immune cells including monocytes, macrophages, dendritic cells.
Another object of this invention is to provide the hybridoma which produces the monoclonal antibodies recognizing SCYE1.
Another object of this invention is to provide a kit for quantifying SCYE1 which includes the monoclonal antibodies recognizing SCYE1.
Another object of this invention is to provide s method for quantifying SCYE1 in the samples by using the monoclonal antibodies recognizing SCYE1.
According to the present invention, the monoclonal antibodies recognizing SCYE1 protein can be used in diagnosis and treatment of inflammatory disease related to SCYE1 by measuring the concentration of SCYE1 in blood.
Figure 1 is a result of determining the isotype of a monoclonal antibody of the present invention.
Figure 2 is a result that the monoclonal antibody cell line of the present invention which is specific to SCYE1.
Figure 3 is a result showing the epitope recognition site of the monoclonal antibody of the present invention.
Figure 4 represents the inhibition of TNF-α production by anti-SCYE1 IgG.　 RAW264.7 cells were innoculated in 12-well plate, SCYE1 (0.4 ug/ml) was treated in a presence or an absence of anti-SCYE1 IgG (5ug/ml, 10ug/ml, 20ug/ml).　 The supernatant of cell culture was obtained and the production of TNF-α was analyzed by using ELISA kit.　 The value is mean±SEM. *, p<0.001.
Figure 5 represent the analysis of the total duration time or migration distance to reach the platform in Morris water maze test. Anti-SCYE1 IgG (2mg/kg, 5mg/kg, 10mg/kg) or mock IgG (20mg/kg) was subcutaneously injected into Alzheimer's disease mouse caused by β-amyloid, once per week for a total of 4 times. Aricept (2mg/kg) was injected daily for 4 weeks as a positive control. The total duration time (A) or migration distance to reach the platform was evaluated and represented as mean±SEM. *, p<0.05.
Figure 6 is the analysis result of target platform visiting rate and duration rate of Alzheimer mouse induced by β-amyloid in probe trial of Morris water maze test. The target platform visiting rate and duration rate of the mouse were investigated and represented as a ratio of a number of platform visiting mice/ a number of total test mice. *, p<0.05; **, p<0.01.
In the following description of the present invention, many terms from a recombinant DNA field and immunology are extensively used. In order to provide a clear and consistent understanding of the present specification and claims, the following definitions are provided.
The term "antibody" is known in the art and refers to a molecule or an active fragment of the molecule that binds to a known antigen. Examples of the active fragment that binds to a known antigen include Fab and F(ab)2 fragments. The active fragment can be derived from the antibody of the present invention by numerous techniques. For example, a purified monoclonal antibody can be cleaved by an enzyme such as pepsin and subjected to an HPLC gel filtration. A proper fraction containing Fab fragments can be collected and concentrated by membrane filtration. Information on general techniques for separating an active fragment of an antibody can be referenced from the documents [for example, Khaw, B.A. et al., J. Nucl. Med. 23: 1011-1019 (1982)]. The term "antibody" also includes bispecific and chimeric antibodies.
The term "monoclonal antibody" is known in the art and is a highly specific antibody directed to a single antigenic moiety. In contrast to a polyclonal antibody that includes different antibodies directed to different determinants (epitopes), a monoclonal antibody is typically directed to a single determinant on the antigen. The monoclonal antibody of the present invention can be prepared using conventional cloning and cell fusion techniques. For example, an immunogen (antigen) of interest is administered to a wild-type or bred mouse (for example, BALB/c) to produce a natural or human monoclonal antibody. The antigen can be administered alone or in admixture with an adjuvant, or can be expressed from a vector. The antigen is DNA or a fusion protein that can induce an immune response. The fusion protein includes a carrier protein, which is coupled to a peptide designed to induce an immune response - for example, β-galactosidase, glutathione S-transferase, keyhole limpet hemocyanin (KLH) and bovine serum albumin, but the carrier protein is not limited thereto. In the above case, the peptide functions as a hapten for the carrier protein. The following is a simple explanation of a method for preparing a monoclonal antibody. After boosting an animal, the spleen is removed. Spleen cells are extracted and fused with myeloma cells according to known methods 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 obtained hybrid cells are cloned using a conventional method - for example, a restricted dilution. The obtained clone which produces a desired monoclonal antibody is cultured. A monoclonal antibody has the advantage of improving the selectivity and specificity of a diagnosis and an analytic assay that uses an antigen-antibody binding. In addition, since a monoclonal antibody is synthesized by culturing a hybridoma, it has another advantage of not being contaminated by other immunoglobulins.
The term "epitope" is known in the art and generally refers to a region of an antigen that interacts with an antibody. An epitope of a peptide or protein antigen can be formed by continuous or non-continuous amino acid sequences of the antigen. Many proteins can contain numerous epitopes. An epitope recognized by the antibody of the present invention forms one embodiment of the present invention. An antibody that can recognize a certain epitope can be used for the immunoaffinity column for purifying said certain epitope. Since it is reported that an antigenic epitope can be formed by a small number (such as five) of amino acid residues, a terminal cleavage form of a certain epitope also can be used.
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 - for example, a myeloma cell. The hybridoma cell can continuously supply an antibody.
As used herein, the term "forming an immunological complex" is meant to include confirming the existence or non-existence of the SCYE1 antigen in a sample. The existence or non-existence of the SCYE1 antigen can be detected by use of an immunoassay. Numerous immunoassays for detecting and/or quantifying an antigen would be known to a person having ordinary skill in the art. See the document [Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York 1988, 556-612].
As used herein, the term "detection marker" refers to a marker for detecting an immunological complex, specific examples of which include a radioisotope marker; an enzyme; a chemoluminescent compound; a fluorescein such as phycobiliprotein, rare earth chelate and rhodamine; an enzyme cofactor; biotin and the like, but are not limited thereto.
The term "(biological) sample" is meant to include a biological substance including cells, tissues or a biological fluid.
The present invention provides a monoclonal antibody recognizing an amino acid region at positions 3 to 51 from the amino terminal of human SCYE1 antigen which consists of the amino acid sequence of SEQ ID NO. 1. Preferably, the monoclonal antibody can be combined with a detection marker that can produce a detectable signal. The detection marker can be an enzyme, a fluorescein, a luminescent substance or a radio substance.
The present invention also provides a SCYE1 assay kit comprising the monoclonal antibody. The kit can further comprise a second antibody that recognizes the monoclonal antibody, and the second antibody can be combined with a detection marker. Furthermore, if the detection marker is an enzyme, the kit can further comprise a substrate that can be used for measuring an enzyme activity, and a reaction-stopping reagent.
The present invention also provides a method for quantifying SCYE1 in a sample by using the SCYE1 monoclonal antibody comprising the steps of
i) contacting SCYE1 monoclonal antibody of the present invention which is linked to a detection marker with a biological sample,
ii) detecting to quantify the immunological complex obtained from step i),
iii) obtaining the standard curve by quantifying the immunological complex obtained by contacting the monoclonal antibody of claim 1 which is linked to a detection marker and purified SCYE1 antigen, and
iv) quantifying the SCYE1 present in the biological sample by comparing the amount of protein detected to quantify in step ii) with the standard curve obtained in step iii).
In the following, the present invention is described in detail.
The present invention provides an antibody which is immunologically reactive to the SCYE1 protein or epitopes thereof. The antibody provided by this invention can be produced in an animal by inoculating cells expressing the SCYE1 protein or an epitope thereof according to a known method in the art. The protein can be separated from the cells with various levels of homogeneity through a conventional biochemical method. The synthetic peptide prepared by the synthetic method confirmed in vitro and optionally conjugated to a heterogeneous amino acid sequence also includes a method for preparing the antibody of this invention. The animals used in the inoculation include cow, sheep, pig, mouse, rat, rabbit, hamster, goat and primate. The preferable animals for the inoculation include rodent - for example, mouse, rat and hamster. The most preferable animal is mouse.
The monoclonal antibody provided in the present invention is also produced by a recombinant genetic engineering method known to a person having ordinary skill in the art, and the present invention includes an antibody according to the above method, which is immunologically reactive to the epitope of SCYE1 protein of this invention. The present invention also includes fragments of the antibody - for example, F(ab) and F(ab)2, but is not limited thereto. The fragments are prepared by any method which includes the preparation of the fragment according to a protein hydrolysis cleavage, chemical synthesis or genetic engineering method, but is not limited thereto. The present invention may include a single-chain antibody immunologically reactive to SCYE1 protein and prepared by a method known to a person having ordinary skill in the art. The present invention also includes chimeric antibody comprising light and heavy-chain peptide, which is immunologically reactive to the epitope induced by SCYE1 of this invention. The chimeric antibody provided by this invention includes not only natural antibody but also chimeric antibody prepared according to genetic engineering techniques known to a person having ordinary skill in the art.
The monoclonal antibody of this invention should be understood to include any antibody recognizing SCYE1, which is prepared by using human SCYE1 or recombinant SCYE1 as an antigen. The cells which can be used in this invention are any cells or cell lines that can express natural or genetically modified SCYE1 protein provided by this invention. The cells which can be used in this invention include HEK-293 which is a mammalian cell, but is not limited thereto.
The monoclonal antibody provided in this invention is produced by a hybridoma cell line, which is prepared by a method known in the art [note: Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)]. The hybridoma cell line is prepared by fusing each cell of myeloma cell line with spleen cells induced from an animal immunized by an agent containing SCYE1 protein itself or heterogeneous or fusional protein construct of the protein, a membrane comprising the agent, cells encoding the protein or the protein. The myeloma cell lines used in this invention include the cell lines derived from mouse, rat, hamster, primate and human myeloma. The myeloma cells derived from a mouse can be used - for example, P3X63Ag8, p3-U1, NS-1, MPC-11, SP-2/0, F0, P3x63Ag8. V653 and S194. The cell lines such as R-210, which are derived from rat, can be also used. The preferable myeloma cell lines used in this invention include myeloma cell SP2/0-Ag14 (ATCC CRL-1581) and P3X63Ag8.653 (ATCC CRL-1580).
A preferable animal from which a spleen can be obtained after immunization includes rat, mouse and hamster, preferably mouse. A spleen cell and a myeloma cell can be fused using many methods known in the art including an inoculation of Sendai virus and an inoculation of polyethylene glycol (PEG), but is not limited thereto. The monoclonal antibody produced by hybridoma cell lines can be obtained from a supernatant of cell media by in vitro cell growth, or the hybridoma cells can be injected into an animal, preferably subcutaneously and/or intraperitoneally, and a monoclonal antibody is thus obtained from blood or ascite.
The hybridoma cell that produces the monoclonal antibody to SCYE1 according to this invention is the fusion cell of myeloma cell P3X63Ag8.653 cell (ATCC CRL-1580, USA) and spleen cell producing the antibody to SCYE1.
The present invention also provides a method for diagnosis and treatment of inflammatory disease by detecting the expression of SCYE1 protein in an animal, preferably in a human. The diagnosing reagent to use in the method includes antibody, most preferably a monoclonal antibody of this invention. The antibody can be used in conventional immunological techniques - for example, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), western blot analysis, immunological titer analysis, immunological diffusion analysis and a method known to a person having ordinary skill in the art, but is not limited thereto.
The reagents used in the immunological analysis include suitable carrier, detection marker which can produce a detectable signal, resolvent and detergent. When a marker substance is an enzyme, a substrate which can measure the enzyme activity and reaction stopping agent can be included. The suitable carriers can be soluble carriers, e.g., physiologically acceptable buffer solution known in the art, or insoluble carriers, e.g., polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin, cross-linked dextran, polysaccharide, polymer such as magnetic particulate that is a latex plated with metal, paper, glass, metal agarose, and combination thereof, but is not limited thereto.
The antibody of the present invention can be easily detected by coupling with detectable substances, i.e., detection markers. The examples of detection markers include various enzymes, prosthetic groups, fluorescein, luminescent substances, biological luminescent substances, radioactive substances and the like. The detection marker can be directly coupled or linked to the SCYE1 monoclonal antibody of this invention by using techniques known in the art, indirectly coupled or linked through an intermediate (e.g., linkers known in the art), or coupled or linked to an another monoclonal antibody recognizing SCYE1 protein or a second antibody recognizing 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 prosthetic group complexes include streptavidin/biotin and avidin/biotin; suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrine or phycobiliprotein; the examples of luminescent substances include luminol, isolucinol and lucigenin; biological luminous substances include luciferase, luciferin and aequorin; examples of suitable ratioactive substances include 125I, 131I, 111In, 99Tc 14C, 3H and the like.
The present invention also provides an analytical method or analytical kit which detects the formation of immunological complex of monoclonal antibody and SCYE1 by using monoclonal antibody to SCYE1, for detecting or quantifying SCYE1 in samples by applying an immunoassay or a kit for immunoassay. As mentioned above, any immunoassay and kit for immunoassay using a monoclonal antibody known in the art, e.g., ELISA analysis and radioimmunoassay, can be applied to this invention. For example, the kit used for SCYE1 quantification analysis of this invention may have a container including the SCYE1 monoclonal antibody of this invention, a suitable carrier solution, a detection marker which can produces a detectable signal, a resolvent and detergent, and a manual. When a marker substance is an enzyme, a substrate that can measure the enzyme activity and reaction stopping agent may be included. Furthermore, a detection marker is linked directly or through a linker to the SCYE1 monoclonal antibody according to this invention, or may include other secondary antibody to SCYE1 linked to a detection marker.
One preferable embodiment of this invention provides the method for quantifying SCYE1, by which biological samples are added to a microtiter plate coated with the monoclonal antibody of this invention and reacted with the antibody. After the enzyme-antibody complex is reacted, the enzyme substance is treated and the absorbance is measured. SCYE1 presented in the biological samples is quantified by comparing the absorbance with a standard curve. In this case, the antibody of the enzyme-antibody complex can be linked to biotin. The enzyme also can be horseradish peroxidase (hereinafter referred to as HPR), but is not limited thereto, and any enzyme and substrate used in the ELISA analysis can be adapted in this invention.
ELISA analysis is an analysis method to determine the presence or absence of antigen and to quantify the amount of antibody present in the samples by using antibody and antigen reaction. In one ELISA kit, about two to three antibodies are need. The ELISA kit using the monoclonal antibody recognizing SCYE1 consists of the above-mentioned three antibodies. The constitution and principle are as follows.
At first, a 96-well plate (in case of quantification of SCYE1 in human plasma) is coated with a monoclonal antibody (first antibody), the sample including SCYE1 is added to the well and reacted with the antibody, and an enzyme marked polyclonal antibody (secondary antibody) is reacted. After the substrate reactable with the enzyme marker is treated, the enzyme-substrate reaction is probed and the SCYE1 presented in the samples can be quantified. In one embodiment of this invention, biotin and streptavidine conjugate HRP can be used as an enzyme and a substrate, but is not limited thereto.
The present invention is set forth by the following examples. They only illustrate this invention, but the scope of this invention is not limited to the following examples.
Example 1: Cloning SCYE1 and separation of protein
ORF(1-312aa) encoding human SCYE1 was amplified by using primer; 5'-GCGAATTCATGGCAAATAATGATGCTGTTC-3' (SEQ ID No. 2), R: 5'-CCGCTCGAGTTATTTGATTCCA CTGTTGCTCATG-3' (SEQ ID NO. 3) from HEK293 cDNA and performing 25 cycles of 95℃ 1 min, 55℃ 1 min and 72℃ 1 min. The PCR product was restricted by EcoRI/SalI, and ligased with pGEX4T-1 (Pharmacia) vector, formerly restricted with EcoRI/SalI, for 2 hours at room temperature. After the transformation of DH-5α by heat shock for 90 sec at 42℃, the E. coli was cultured in LB media containing ampiciline (50㎍/ml), and colonies including SCYE1 were selected. The cloned SCYE1 gene was sequenced and there was no mutation.
Example 2: SCYE1 purification
After the transformation of E. coli BL21 (DE3) with SCYE1 plasmid, the E. coli was plated to LB media containing ampiciline (50㎍/ml), and cultured for 24 hours at 37℃. The grown colony was cultured in 5 ml of LB media containing ampiciline (50 ㎍/ml) for 12 hours, and then 4ml of the cultured media was inoculated to 2L of LB media containing ampiciline (50㎍/ml). It is cultured at 37℃ until O.D (600nm) reached 0.3. IPTG was added in a final concentration of 0.1mM, and p43 protein was expressed by culturing for 6 hours at 30℃. After E. coli was centrifuged for 15 min at 7,000 rpm, the cells were resuspended by 1XPBS (8g NaCl, 0.2g KCl, 1.44g Na2HPO, 0.24g KH2PO4/L, pH 7.4), sonicated at 4℃ and centrifuged for 40 min at 26,000 x g. The supernatant was obtained and loaded to a glutathione Sepharose 4B-column which become homogeneous by 1xPBS. The resin was washed with PBS, and GST-p43 was eluted by using 50mM of Tris-HCl (pH 8.0) containing 10mM of glutathione. After the purity was identified by using 10% SDS-PAGE, dialysis was performed by using PBS containing 20% glycerol, and the dialyzed product was stored at -70℃. The purity was over 90% on SDS-PAGE.
Example 3: Preparation of the SCYE1 antibody monoclone cell line
1) Antigen immune
An emulsion was prepared by mixing 20㎍/mouse of the protein with the same volume of Complete Freund's Adjuvant (Sigma, USA). The emulsion was intraperitoneally injected into three 7-week-old female Balb/c mice (orient). 20㎍ of antigen was injected into each mouse with the 400㎕ of total volumn. After 2 weeks, the emulsion mixing Incomplete Freund's Adjuvant (Sigma, USA) and antigen was intraperitoneally injected into the mouse. After 2 weeks, the antigen (20㎍/mouse) dissolved in PBS was intraperitoneally injected to induce the production of antibody. After identifying the antibody by performing enzyme immunoassay and western blot analysis, antigen dissolved in PBS was further injected into a tail vein of the mouse 3 days before the cell fusion.
2) Identifying and screening the cells producing antibody
Blood was obtained from the eye ball of an immunized mouse according to the above method, placed into the 1.5㎖ microcentrifuge tube and centrifuged for 10 min at 13,000 rpm. The serum was separated and stored at -20℃ until the experiment for identifying the production of antibody was performed. After identifying the production of antibody by performing enzyme immunoassay and western blot analysis using antigen protein, the fusion for spleen cells of the antibody-producing mouse was performed.
3) Preparation of a fusion cell
After the production of the antibody was identified, the mouse was sacrificed and the splenocytes were separated. The splenocytes were fused with myeloma cells P3X63Ag8.653 (ATCC CRL-1580, USA). That is, P3X63Ag8.653 cells of the mouse were maintained in the culture plate by using RPMI1640 media supplemented with 10% fetal bovine serum. To perform the cell fusion, P3X63Ag8.653 cells were washed two times with serum-free RPMI1640 media (Biowhittaker, USA) and adjusted to become the concentration of 1 x 107 cells. The mouse was sacrificed by cervical dislocation, and its spleen was obtained. It was placed into the mesh (Sigma, USA) container, and the cells were separated. After making suspension of the spleen cells, the suspension was washed by centrifugation. The spleen cell solution was exposed to Tris-NH4Cl solution (Tris 20.6g/L, NH4Cl 8.3 g/L) to lyse red blood cells. After the completely separated antibody-producing cells were centrifuged for 5 min at 400g, they were washed two times with serum-free media and resuspended into the 10㎖ media. The lymph cells were counted by using a haemocytometer, and 1x108 of lymphocytes were mixed with 1 x 107 of P3X63Ag8.653 cells (10:1) in the serum-free media. The centrifugation was performed for 5 min at 400g.
By using 50% (M/V) polyethylene glycol 1500 (Sigma, USA) pretreated at 37℃, 1㎖ solution was dropped to mix for 1 min. The fusion mixture solution produced in the above was diluted with serum-free RPMI1640 and centrifuged for 3 min at 400g. The cells were suspended in the 35㎖ of RPMI1640 selection media supplemented with 20% fetal bovine serum and HAT (100μM hypoxanthine, 0.4μM aminopterin, 16μM thymidine). 100㎕ of the suspension solution was loaded to 96 well plates coated with feeder cells (macrophages separated from abdominal cavity using RPMI1640) one day before and cultured at 37℃, 5% CO2. After 5 days, HAT selection media were replaced in 2 to 3 days intervals, and the cells were cultured for 14 days. After 14 days, subculture was performed by replacing RPMI1640 media supplemented with 20% fetal bovine serum and HT (media in which 0.4μM aminopterine is removed from HAT media).
4) Selection and separation of fusion cells producing each antibody
The supernatant of the culture fused in the above method 3) was obtained, and the enzyme immunoassay was performed to investigate the production of the provided antigen-specific antibody. The culture solution of fusion cells which represents the titer more than 4 times compared with negative control was selected and transferred to a 24-well culture plate and a 25 cm2 culture flask.
5) Separation of immunoglobulin and determination of isotype
The isotype of monoclonal antibody was determined by using isotyping kit (Zymed Labomouseories Inc. USA) using enzyme immunoassay with culture supernatant (Figure 1). The antibody to SCYE1 was identified as IgG1. The western blotting was performed by using whole-cell extract of HEK293 cells to determine whether the antibody derived from the prepared cell line specifically recognizes SCYE1 by using culture solution of the prepared SCYE1 monoclonal antibody cell line. The prepared monoclonal antibody cell line was specific to SCYE1 (Figure 2).
Example 4: Domain mapping
The peptide of human SCYE1 was synthesized (3-54, 51-90, 103-139, 191-219aa of ORF) and a 96-well plate was coated with 100ng of the peptide per well. Blocking was performed by using 1X PBS containing 2% BSA (sigma) for 1 hour at room temperature. 100㎕ of culture supernatant was loaded to each well and reacted for 1 hour at room temperature. After washing 3 times with PBS containing 0.1% Tween 20, HRP-conjugated anti-mouse IgG was loaded and reacted for 1 hour at room temperature. After rewashing 4 times with PBS containing 0.1% Tween 20, TMB (sigma) was added and reacted for 20 minutes. The result of the reading at 405nm represented that the epitope of the prepared antibody recognized 3-51 aa of SCYE1 (Figure 3).
Example 5: In vitro effect of anti-SCYE1 IgG on inflammation
RWA264.7 cells, mouse macrophage, were cultured in DMEM supplemented with 10% FBS and 1% streptomycin/penicillin. RAW264.7 cells were seeded to a 12-well plate in 4X105 cells/well and cultured for 12 hours. In each well, RAW264.7 cells were treated for 30 min with 0.4㎍/ml of SCYE1 protein or with SCYE1 protein incubated with anti-SCYE1 IgG for 5 min. The culture solution was obtained and TNF-α was quantified by using mouse TNF-α ELISA kit (BD Biosciences) according to the method of the manual.
To identify whether anti-SCYE1 IgG can suppress the cytokine function of SCYE1, the amount of TNF-α was measured in the culture solution of the RAW264.7 cells. It was observed that the secretion of mouse TNF-α was increased about 14 times compared with the control after treating 400ng/ml of SCYE1 for 30 min (vehicle, TNF-α 0.11±0.01ng/ml; SCYE1 0.4㎍/ml, TNF-α 1.41±0.05ng/ml, p<0.001). On the other hand, when RAW264.7 cells were treated after SCYE1 was incubated with anti-SCYE1 IgG for 5 min, TNF-α secretion of RAW 264.7 cells caused by SCYE1 stimulation was inhibited in a concentration-dependent manner (SCYE1 0.4㎍/ml, TNF-α 1.41±0.05ng/ml; SCYE1 0.4㎍/ml + anti-SCYE1 IgG 5㎍/ml, 1.11±0.09ng/ml; SCYE1 0.4㎍/ml + anti-SCYE1 IgG 10㎍/ml, 0.48±0.06ng/ml; SCYE1 0.4㎍/ml + anti-SCYE1 IgG 20㎍/ml, 0.25±0.03; SCYE1 0.4㎍/ml + mock IgG 20㎍/ml, 1.28±0.07ng/ml, p<0.001). In case mock IgG was treated, the secretion of TNF-α caused by SCYE1 was not inhibited (Figure 4).
Example 6: The effect of anti-SCYE1 antibody on the behavior of an Alzheimer's disease mouse
Alzheimer's disease was induced by injecting β-amyloid (2㎍/mouse) into the third ventricle of a male 12-week-old BALB/C mouse. After 24 hours, mice which showed normal reflex reactions in eye-blink tests were selected and used, with 10 mice per group. The anti-SCYE1 IgG was subcutaneously injected into 3 groups of mice with the amount of 2mg/kg, 5mg/kg, 10mg/kg once per week. Aricept (2mg/kg, Eisai Korea Inc.) was orally administered as a positive control, once per day for 4 weeks, for a total of 28 times. 10mg/kg of normal IgG was subcutaneously injected once per week as a negative control. After 4 weeks, a Morris water maze test was performed to identify the effect on memory. The test was performed for 7 days. The total duration time and total migration distance to find the platform, and probe trial (visiting duration and distance) were investigated. All results were expressed as mean±SEM, and statistical significance was accepted at p<0.05 in a t-test.
The mean 2.1±0.42g of weight increase was observed in the mice used in the test for a total of 4 weeks in experiments in all groups, and there was no difference in the weight change among the groups. The total duration time to find the platform was 49.37±3.45 sec in the group in which β-amyloid was not treated, whereas it was 56.19±2.19 sec in the group in which β-amyloid was treated (p<0.05). This is regarded as a result of inducing Alzheimer's disease caused by β-amyloid.
On the other hand, the total duration time was 51.84±2.67 sec (2mg/kg group), 51.2±2.14 sec (5mg/kg group), 49.07±2.24 sec (10mg/kg group) in the groups in which β-amyloid was treated and anti-SCYE1 IgG was subcutaneously injected. This represented an improved effect of about 4.3 sec, 4.99 sec and 7 sec compared with negative control treated with β-amyloid , and it was statistically significant (p<0.05). In particular, the total duration time of 10mg/kg per group was similar to that of the group which was not treated with β-amyloid, whereas no time decrease was observed in the mock IgG (10mg/kg) group. It shows that anti-SCYE1 IgG specifically improved symptom of Alzheimer's disease. However, when Aricept was used as a positive control, there was no statistically significant change despite a tendency to decrease compared with the control (Figure 5A).
Next, the effect of administration of anti-SCYE1 antibody on the total distance which Alzheimer's disease mouse migrated until the mouse found the platform was investigated. The total migration distance was increased significantly in the group treated with β-amyloid compared with the group not treated with β-amyloid (normal, 797.85±50.43mm; β-amyloid control, 925±35mm, p<0.05). However, there was no significant difference among control, the anti-SCYE1 IgG treated group, and the Aricept and mock IgG treated groups in the groups treated with β-amyloid (Figure 5B).
To test the memory of the mouse, the platform was divided and separated into 4 pieces and the ratio of the mouse that found the platform placed at first was measured. As a result, the ratio finding platform placed at first in the group treated with anti-SCYE1 IgG was generally increased compared to that of the group treated only with β-amyloid. In particular, a 300% increase was observed in the group treated with 10mg/kg of IgG (p<0.05) (Figure 6A). Moreover, the ratio of the time that the mouse stayed on the platform placed at first among 4 platforms in the group treated with β-amyloid was decreased about 65% compared with the ratio in the non-treated group (p<0.05). When anti-SCYE1 IgG was reinjected to the group treated with β-amyloid, the staying time in the platform placed at first was increased in a concentration-dependent manner (Figure 6B). In particular, the staying time in the platform of the group treated with 10mg/kg was increased about 400% compared with the group not treated with β-amyloid (p<0.01). On the other hand, there was no improvement in the mock IgG group and no statistically significant increase in the Aricept group despite the improved effect.
As a result, the present invention reidentified the fact that SCYE1 boosts the secretion of TNF-α by activating mouse macrophage and proved that anti-SCYE1 antibody can inhibit the secretion of TNF-α. It also identified that the symptom of Alzheimer's disease is improved by a subcutaneous injection of anti-SCYE1 antibody into a mouse with Alzheimer's disease induced by β-amyloid.
<110> YOON, Kang Jun
<120> Monoclonal antibody recognizing SCYE1 and use thereof
<130> PC09023-CNB
<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
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Gln Ile Gln Pro Asp Leu His Thr Asn Asp Glu Cys Val Ala Thr Tyr
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gcgaattcat ggcaaataat gatgctgttc 30
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ccgctcgagt tatttgattc cactgttgct catg 34

Claims (7)

  1. A monoclonal antibody recognizing amino acids at positions 3 to 51 from the amino-terminal of human SCYE1 antigen which consists of the amino acid sequence of SEQ ID No. 1.
  2. The monoclonal antibody of claim 1, which is linked to detection marker that can produce a detectable signal.
  3. The monoclonal antibody of claim 2, wherein the detection maker is enzyme, fluorescein, luminescent substances or radioactive substances.
  4. A kit for quantifying SCYE1 containing the monoclonal antibody of claim 1.
  5. The kit for quantifying SCYE1 of claim 4 further containing a secondary antibody that recognizes the monoclonal antibody, wherein a detection marker is linked to the secondary antibody.
  6. The kit for quantifying SCYE1 of claim 5, wherein the detection marker is an enzyme, and the kit further contains a substrate that can measure the enzyme activity and a reaction stopping agent.
  7. A method for quantifying SCYE1 in a sample by using the SCYE1 monoclonal antibody of claim 1 comprising the steps of
    i) contacting the SCYE1 monoclonal antibody of claim 1 which is linked to a detection marker with a biological sample,
    ii) detecting to quantify the immunological complex obtained from step i),
    iii) obtaining the standard curve by quantifying the immunological complex obtained by contacting the monoclonal antibody of claim 1 which is linked to a detection marker with purified SCYE1 antigen, and
    iv) quantifying the SCYE1 present in the biological sample by comparing the amount of protein detected to quantify in step ii) with the standard curve obtained in step iii).
PCT/KR2010/000934 2009-02-16 2010-02-16 Monoclonal antibody recognizing scye1 and use thereof WO2010093214A2 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2717913A2 (en) * 2011-06-08 2014-04-16 Indiana University Research and Technology Corporation Monoclonal antibody and antigens for diagnosing and treating lung disease and injury
US20140154245A1 (en) * 2011-04-20 2014-06-05 Hee Koung Kim Humanized anti-emap ii antibody and use thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140154245A1 (en) * 2011-04-20 2014-06-05 Hee Koung Kim Humanized anti-emap ii antibody and use thereof
US9382316B2 (en) * 2011-04-20 2016-07-05 Cell & Bio Co., Ltd. Humanized anti-EMAP II antibody and use thereof
EP2717913A2 (en) * 2011-06-08 2014-04-16 Indiana University Research and Technology Corporation Monoclonal antibody and antigens for diagnosing and treating lung disease and injury
EP2717913A4 (en) * 2011-06-08 2014-11-26 Univ Indiana Res & Tech Corp Monoclonal antibody and antigens for diagnosing and treating lung disease and injury
US10450371B2 (en) 2011-06-08 2019-10-22 Indiana University Research And Technology Corporation Monoclonal antibody and antigens for diagnosing and treating lung disease and injury

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