WO2021025412A1 - Méthode de diagnostic de la maladie d'alzheimer au moyen du composant c8 gamma du complément - Google Patents

Méthode de diagnostic de la maladie d'alzheimer au moyen du composant c8 gamma du complément Download PDF

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WO2021025412A1
WO2021025412A1 PCT/KR2020/010227 KR2020010227W WO2021025412A1 WO 2021025412 A1 WO2021025412 A1 WO 2021025412A1 KR 2020010227 W KR2020010227 W KR 2020010227W WO 2021025412 A1 WO2021025412 A1 WO 2021025412A1
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expression level
disease
alzheimer
gamma
complement component
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PCT/KR2020/010227
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Korean (ko)
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석경호
김종헌
이호원
고판우
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경북대학교 산학협력단
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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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to a method for diagnosing Alzheimer's disease using a complement component C8 gamma (C8G), and more particularly, a composition for diagnosis of Alzheimer's disease, including an agent for measuring the expression level of complement component C8 gamma, for diagnosis.
  • C8G complement component C8 gamma
  • the present invention relates to a kit and a method of qualitative or quantitative analysis of complement component C8 gamma in order to provide information necessary for diagnosis of Alzheimer's disease.
  • degenerative brain disease can be said to be important because it can be expected to overcome brain disease when its early diagnosis is made and timely treatment is performed.
  • Alzheimer's dementia is a disease characterized by progressive memory loss and cognitive impairment, mainly in the elderly over the age of 65.
  • the entanglement of beta-amyloid protein precipitate and nerve fiber tau protein is recognized as a representative etiology.
  • the prevalence of dementia in the world is expected to be the most fatal threat to public health, showing a very steep increase from 35.6 million in 2010 to 115.4 million in 2050 (Alzheimers Dement. (2007) 3:186-191).
  • Sexual dementia accounts for the largest share.
  • Biomarkers are recommended to be easily measured in order to facilitate accurate diagnosis at an early stage. Particularly, there are many difficulties in invasive collection of brain tissue or nerve tissue in brain diseases, such as causing significant risk and secondary complications. Should be considered Also, at present, quantitative analysis of beta-amyloid protein concentration in cerebrospinal fluid and brain imaging tests are used as tests to confirm dementia (Alzheimer's), but cerebrospinal fluid test is also an invasive test method, which is inappropriate for early diagnosis. Positron tomography (PET) tests for medical treatment are also expensive and have limitations in space and time constraints (Lancet Neurol. (2010) 9:119).
  • PET Positron tomography
  • US2010/0159486 reports a very wide range of protein expression patterns by performing proteomic analysis in a sample of a patient with neurological disease.
  • the transthyretin protein which is reported to be increased in samples of Alzheimer's patients in the above literature
  • other studies using ELISA technique reported that the expression of the protein was much lower in Alzheimer's patients compared to normal subjects (J Alzheimers Dis. 2015 ;47(3):761-71, J Alzheimers Dis. 2012;28(2):369-75, Curr Alzheimer Res. 2012 Oct;9(8):881-9).
  • the present inventors completed the present invention by confirming the effectiveness of C8G as a biomarker for the diagnosis of Alzheimer's disease through a marked increase in the level of C8G in body fluids (especially plasma) in Alzheimer's patients.
  • an object of the present invention is to provide a composition for diagnosis of Alzheimer's disease including an agent for measuring the expression level of complement component C8 gamma (C8G) and a kit for diagnosis of Alzheimer's disease comprising the same.
  • C8G complement component C8 gamma
  • a composition for diagnosis of Alzheimer's disease comprising a formulation for measuring the expression level of complement component C8 gamma (C8G), and a kit for diagnosis of Alzheimer's disease comprising the same.
  • C8G complement component C8 gamma
  • Another object of the present invention is to provide a method for qualitative or quantitative analysis of the expression level of complement component C8 gamma from a sample collected from a subject in order to provide information necessary for diagnosis of Alzheimer's disease.
  • Another object of the present invention is to provide a use of a formulation for measuring the expression level of complement component C8 gamma (C8G) for preparing a diagnostic formulation for Alzheimer's disease.
  • C8G complement component C8 gamma
  • Another object of the present invention is to provide a third object of the present invention.
  • the present invention provides a composition for diagnosis of Alzheimer's disease including an agent measuring the expression level of complement component C8 gamma (C8G), and a kit for diagnosis of Alzheimer's disease comprising the same.
  • C8G complement component C8 gamma
  • the present invention provides a composition for diagnosis of Alzheimer's disease comprising a formulation for measuring the expression level of complement component C8 gamma (C8G), and a kit for diagnosis of Alzheimer's disease comprising the same.
  • a composition for diagnosis of Alzheimer's disease comprising a formulation for measuring the expression level of complement component C8 gamma (C8G), and a kit for diagnosis of Alzheimer's disease comprising the same.
  • the present invention provides a composition for diagnosis of Alzheimer's disease and a kit for diagnosis of Alzheimer's disease comprising the same, which consists essentially of an agent for measuring the expression level of the complement component C8 gamma (C8G).
  • the present invention provides a method for qualitative or quantitative analysis of the expression level of complement component C8 gamma from a sample collected from a subject in order to provide information necessary for diagnosis of Alzheimer's disease.
  • the present invention provides a use of a formulation for measuring the expression level of complement component C8 gamma (C8G) for preparing a diagnostic formulation for Alzheimer's disease.
  • C8G complement component C8 gamma
  • diagnosis refers to determining the susceptibility of an object to a specific disease or disease, determining whether an object currently has a specific disease or disorder, as long as it has a specific disease or disorder. It is a concept that includes both determining the prognosis of an object, or therametrics (eg, monitoring the condition of an object to provide information about treatment efficacy).
  • the diagnosis in the present invention may be to confirm the presence or onset of Alzheimer's disease by measuring the expression level of the complement component C8 gamma polypeptide.
  • Alzheimer's disease is also called Alzheimer's dementia, as a brain disease accompanying aging. It is known that neurons are killed by extracellular beta-amyloid deposition and intracellular hyperphosphorylated tau protein throughout the brain. It is a disease that causes gradual memory impairment, cognitive deficit, and personal personality change. In the present invention, the term “Alzheimer's disease” is characterized in that it is particularly distinguished from mild cognitive impairment (MCI).
  • MCI mild cognitive impairment
  • the "Alzheimer's disease” means that it is determined as Alzheimer's disease according to the NINCDS-ADRDA diagnostic criteria (Neurology, 1984;34(7);939).
  • the term'expression' means that a protein (polypeptide) or nucleic acid is produced in a cell.
  • the term'protein' is used interchangeably with'polypeptide' or'peptide' and, for example, refers to a polymer of amino acid residues as commonly found in proteins in a natural state.
  • the term'polynucleotide' or'nucleic acid' refers to a deoxyribonucleotide (DNA) or ribonucleotide (RNA) in a single- or double-stranded form. Unless otherwise limited, also include known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to those of naturally occurring nucleotides.
  • 'mRNA' is an RNA that transfers genetic information (gene-specific nucleotide sequence) to a ribosome that specifies an amino acid sequence from a specific gene during protein synthesis.
  • C8G polypeptide (Complement component C8 gamma) in Alzheimer's disease patients is significantly elevated in body fluid (especially plasma), and the diagnostic use of C8G for Alzheimer's disease is the present invention. It is released for the first time in This is contrary to the previously known subunit polypeptides of complement protein C8, such as C8A (Complement component C8 alpha) and C8B (Complement component C8 beta), that have no significant change in Alzheimer's disease and thus have no diagnostic value ( Reichwald J et al., J Neuroinflammation. 2009 Nov 17; 6:35.).
  • C8A Complement component C8 alpha
  • C8B Complement component C8 beta
  • C8A and C8B genes are located in Chromosome 1 and have an expression pattern similar to other complement activities in general, but C8G has a locus adjacent to the lipocalin family gene in Chromosome 9, so the mechanism of inducing expression is different from that of other complements. Seeing it as something else. Accordingly, the present invention suggests that the C8G polypeptide can be utilized as a biological indicator independent of the in vivo behavior of other complements, including the C8 complement protein or subunit polypeptide thereof, (especially as an indicator of Alzheimer's disease).
  • C8 means a protein complex composed of C8A, C8B and C8G.
  • the structure of the C8 complex and the function of the subunits are atypical.
  • the quantitative ratios of C8B and C8A or C8G exist differently compared to the same amount of the C8 complex. That is, it was confirmed that the amount of free C8A or C8G existed much higher than that of free C8B in the blood.
  • C8B was directly related to the activity and expression of the C8 complex, whereas C8A and C8G were decomposed from the C8 complex and some were derived. Rather, it can be seen as independently expressed.
  • C8A and C8G are highly sensitive to increase in gene levels after inflammation stimulation and decrease again after being secreted, whereas C8B reacts slowly and is synthesized. Reported that it was secreted.
  • C8G is not an essential element for C8 activity and is expressed independently of C8A. Therefore, when these prior literatures are summarized, the change of C8G is difficult to be an indicator for C8 complex and complement activity, and it is very likely to have independent expression and function. Therefore, it is difficult to see that the expression of C8G and the activity of the C8 complex have a direct causal relationship, and the present invention also provides C8G unique to C8 as a biomarker for diagnosis of Alzheimer's.
  • U.S. Patent Application Publication No. US2010/0159486 discloses a result of analyzing a polypeptide derived from a complement component C8 gamma in a sample derived from a patient with neurological disease, but the document is a protein analysis result based on LC-MS/MS. The reliability of the results cannot be guaranteed, and referring to the data described in the above literature, it is believed that the polypeptide derived from the complement component C8 gamma can be used as a biomarker to distinguish mild cognitive impairment (MCI) and Alzheimer's patients. There is a limitation in that it is difficult to know whether it can be a biomarker that can differentiate between normal people and Alzheimer's patients.
  • MCI mild cognitive impairment
  • the present invention Complement Complement component C8 gamma, C8G ) A composition for diagnosis of Alzheimer's disease containing an agent for measuring the expression level and for diagnosis of Alzheimer's disease containing the same Kit to provide.
  • The'C8G expression level measurement means measuring the expression level of a C8G polypeptide or a C8G coding gene.
  • the complement component C8 gamma (C8G) polypeptide is preferably known to be of human origin, its specific sequence is not particularly limited, but as an example GenBank (NCBI) accession number: NP_000597 (SEQ ID NO: 1) Known things such as can be used.
  • the complement component C8 gamma polypeptide of the present invention may be a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1, more preferably a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1.
  • the complement component C8 gamma polypeptide is known to be encoded by a polynucleotide sequence such as GenBank (NCBI) accession number: NM_000606 (mRNA, SEQ ID NO: 2), and gene sequences such as NG_029580 (RefSeqGene on chromosome 9). You can refer to it.
  • GenBank NCBI accession number: NM_000606 (mRNA, SEQ ID NO: 2)
  • gene sequences such as NG_029580 (RefSeqGene on chromosome 9). You can refer to it.
  • the type of the agent for measuring the expression level of the C8G polypeptide is not particularly limited as long as it is known to be usable in the art to measure the expression level of a protein, but is preferably an antibody or aptamer that specifically binds to the C8G polypeptide. I can.
  • the term'antibody' means an immunoglobulin that specifically binds to an antigenic site.
  • the C8G antibody in the present invention is an antibody that does not react with polypeptides other than C8G and specifically binds only to C8G polypeptides.
  • the antibody specifically binding to the C8G polypeptide may be an antibody that specifically binds to a polypeptide (C8G) comprising an amino acid sequence represented by SEQ ID NO: 1.
  • the antibody is a conventional method in the art, such as obtaining a protein (polypeptide) encoded by the gene by cloning a C8G coding gene into an expression vector, and injecting the obtained protein into an animal to obtain an antibody produced. It can be manufactured according to.
  • the C8G antibody may be prepared through a C8G full-length sequence polypeptide, or may be prepared using a fragment containing an antigenic site of a C8G polypeptide.
  • the form of the antibody of the present invention is not particularly limited, and includes a polyclonal antibody or a monoclonal antibody. In addition, as long as it has antigen-antibody binding (reaction), a part of the whole antibody is also included in the antibody of the present invention, and all kinds of immunoglobulin antibodies that specifically bind to C8G are included.
  • the antibody of the present invention includes special antibodies such as humanized antibodies and chimeric antibodies, and recombinant antibodies as long as it can specifically bind to the C8G polypeptide.
  • the term "aptamer” refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure as a substance capable of specifically binding to an analyte to be detected in a sample. , Specifically, the presence of the target protein (polypeptide) in the sample can be confirmed.
  • the aptamer is synthesized by determining the sequence of an oligonucleotide having a high binding ability and selective to the target protein to be identified (C8G polypeptide in the present invention) according to a general aptamer production method.
  • The'end or 3'end may be modified by modifying -SH, -COOH, -OH or -NH2 so that the functional group of the aptamer chip can be bonded, but is not limited thereto.
  • the meaning of measuring the expression level of the C8G-encoding gene includes measuring the expression level of transcripts derived from the C8G-encoding gene, and for example, it includes measuring the expression level of C8G mRNA.
  • the agent for measuring the expression level of the C8G coding gene is not limited thereto, but may preferably mean an agent for detecting the mRNA expressed from the gene.
  • the type of the agent for detecting the C8G-encoding gene is not particularly limited as long as it is a ligand that specifically attaches or hybridizes to the mRNA expressed from the gene, but may be, for example, a primer (pair) or a probe.
  • the "primer” refers to a short nucleic acid sequence having a short free 3-terminal hydroxyl group, capable of forming a base pair with a complementary template and serving as a starting point for template strand copying. .
  • Primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature. PCR conditions, the length of the sense and antisense primers can be appropriately selected according to techniques known in the art.
  • the sequence of the primer does not need to have a sequence that is completely complementary to some of the base sequences of the template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with the template to function unique to the primer. Therefore, in the present invention, the primer for measuring the expression level of C8G mRNA does not need to have a sequence that is completely complementary to the C8G coding gene sequence, and the amount of C8G mRNA by amplifying a specific section of C8G mRNA or C8G cDNA through DNA synthesis It is sufficient to have a length and complementarity suitable for the purpose of measuring.
  • the primers for the amplification reaction consist of a set (pair) that complementarily bind to the template (or sense) at both ends of the specific section of the C8G mRNA to be amplified and the opposite side (antisense, antisense).
  • the primer can be easily designed by those skilled in the art by referring to the C8G mRNA or cDNA sequence.
  • 'Probe' refers to a fragment of polynucleotides such as RNA or DNA of several to several hundred base pairs in length that can specifically bind to mRNA or cDNA (complementary DNA) of a specific gene. It is labeled so that the presence or absence of the target mRNA or cDNA to be bound, and the amount of expression can be confirmed.
  • a probe complementary to C8G mRNA can be used for diagnosis of Alzheimer's disease by measuring the expression level of C8G mRNA by performing a hybridization reaction with a sample of a subject. Probe selection and hybridization conditions can be appropriately selected according to techniques known in the art.
  • primers or probes of the present invention can be chemically synthesized using a method of synthesizing a solid support of phosphoramidite or other well-known methods.
  • primers or probes can be variously modified according to methods known in the art within a range that does not interfere with hybridization with C8G mRNA. Examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkers (e.g. methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc. ) Or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.), and binding of a labeling material using fluorescence or enzymes.
  • uncharged linkers e.g. methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc.
  • charged linkers eg, phosphorothioate
  • antibodies that selectively recognize C8G polypeptides as markers, aptamers or primers that recognize C8G mRNA as markers, probes, as well as one or more suitable for analysis methods may be included.
  • the kit includes Western blot, ELISA (enzyme-linked immunospecific assay), radioimmunoassay, radioimmune diffusion method, octeroni immune diffusion method, rocket immunoelectrophoresis, immunostaining method, immunoprecipitation assay, complement fixation assay, FACS (Fluorescence activated cell sorter) or a diagnostic kit characterized by including known essential elements and ancillary elements necessary to perform the protein chip method, but is not limited thereto.
  • ELISA enzyme-linked immunospecific assay
  • radioimmunoassay radioimmune diffusion method
  • octeroni immune diffusion method octeroni immune diffusion method
  • rocket immunoelectrophoresis immunostaining method
  • immunoprecipitation assay complement fixation assay
  • FACS Fluorescence activated cell sorter
  • a diagnostic kit characterized by including known essential elements and ancillary elements necessary to perform the protein chip method, but is not limited thereto.
  • the kit includes an antibody specific for the C8G polypeptide.
  • the antibody is an antibody having high specificity and affinity for a target marker protein (C8G polypeptide in the present invention) and has little cross-reactivity with other proteins, and is a monoclonal antibody, polyclonal antibody, or recombinant antibody.
  • the kit may additionally include an antibody specific for a control protein.
  • the kit may contain reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (as conjugated with antibodies), and substrates or antibodies thereof capable of binding. Other materials may be included.
  • the kit of the present invention may include a washing solution or an eluent capable of removing excess chromogenic substrate and unbound protein, and retaining only protein markers bound to the antibody.
  • the kit is not particularly limited as long as it is known in the art as an assay kit that provides a primer (primer pair) or a probe as a component, but, for example, PCR (polymerase chain reaction, polymerase chain reaction), RNase protection assay , Northern blotting, Southern blotting, or DNA microarray chip kit.
  • the diagnostic kit may be a diagnostic kit characterized in that it includes essential elements necessary to perform the polymerase reaction.
  • the polymerase reaction kit contains each primer pair specific for a marker gene (mRNA).
  • the primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene (mRNA), and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length.
  • a primer specific to the nucleic acid sequence of the control gene may be included.
  • Other polymerase reaction kits include test tubes or other suitable containers, reaction buffers (varies in pH and magnesium concentration), deoxynucleotides (dNTPs), DNA polymerases (e.g. Taq-polymerase) and reverse transcriptase, DNAse, RNAse inhibitor DEPC-water, sterile water, and the like.
  • the invention Complement Provides a method for qualitative or quantitative analysis of the expression level of the component C8 gamma (C8G).
  • the method is (a) from a sample of a subject Complement Complement component C8 gamma, C8G ) Measuring the expression level;
  • the term "analysis” may preferably mean “measurement”, the qualitative analysis may mean measuring and confirming the presence or absence of a target substance, and the quantitative analysis It may mean measuring and confirming a change in the level of presence (level of expression) or quantity.
  • the analysis or measurement may be performed without limitation, including both a qualitative method and a quantitative method, and preferably, a quantitative measurement may be performed.
  • step (a) in the sample provided from the subject (potential patient) Complement Complement component C8 gamma, C8G ) This is the step of measuring the expression level.
  • the sample may be used without limitation as long as it is collected from a subject to be diagnosed with Alzheimer's disease.
  • cells or tissues obtained by biopsy, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, various secretions, It can be urine or feces.
  • body fluid sample for example blood, plasma, serum, saliva, nasal fluid, sputum, joint capsule fluid, amniotic fluid, ascites, cervical or vaginal discharge, urine and cerebrospinal fluid.
  • body fluid sample for example blood, plasma, serum, saliva, nasal fluid, sputum, joint capsule fluid, amniotic fluid, ascites, cervical or vaginal discharge, urine and cerebrospinal fluid.
  • the present invention has great technical significance in that diagnosis is possible through bodily fluid samples (for example, plasma) other than the direct brain lesion tissue in which Alzheimer's disease has occurred, and thus the effectiveness, rapidity, convenience and safety of diagnosis are increased.
  • the sample may most preferably be blood, plasma, or serum.
  • the sample can be pretreated prior to use for detection (measurement) or diagnosis.
  • it may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like.
  • Measuring the C8G expression level in step (a) means measuring the expression level of a C8G protein or a C8G coding gene.
  • the measurement of the protein expression level is not particularly limited as long as it is by a protein expression measurement method known in the art, but for example, Western blot, ELISA (enzyme-linked immunospecific assay), radioimmunoassay, radioimmunoassay, OY Cteroni immune diffusion method, rocket immunoelectrophoresis, immunostaining (including immunohistochemical staining and immunofluorescence staining, etc.), immunoprecipitation assay, complement fixation assay, FACS (Fluorescence activated cell sorter) or protein chip method Can be.
  • Western blot Western blot, ELISA (enzyme-linked immunospecific assay), radioimmunoassay, radioimmunoassay, OY Cteroni immune diffusion method, rocket immunoelectrophoresis, immunostaining (including immunohistochemical staining and immunofluorescence staining, etc.), immunoprecipitation assay, complement fixation assay, FACS (Fluor
  • the measurement of the gene expression level is not particularly limited as long as it is by a gene expression measurement method known in the art, but preferably PCR (polymerase chain reaction), RNase protection assay, northern blotting, and southern blotting It may be to use any one selected from the group consisting of (southern blotting) and DNA chips.
  • step (b) the subject sample measured in the step (a) C8G Compare the expression level with the normal person, compared to the normal person C8G Manifestation Level of increased This is the step of determining that the subject has Alzheimer's disease.
  • the C8G expression level (including both gene and protein levels) of the subject sample measured by the method of step (a) described above is compared with the C8G expression level of a normal sample measured by the same method.
  • the sample is preferably a sample obtained by the same type or the same method between the subject and a normal person.
  • a subject whose expression level of C8G is up-regulated compared to that of a healthy normal person is determined to have Alzheimer's disease.
  • the present invention provides a use of a formulation for measuring the expression level of complement component C8 gamma (C8G) for preparing a diagnostic formulation for Alzheimer's disease.
  • C8G complement component C8 gamma
  • the present invention is a.
  • the present invention provides a method of diagnosing and treating Alzheimer's disease in a subject (subject) comprising the steps of:
  • the step iv) is a step of performing treatment of the disease through a means such as administration of a therapeutic drug such as Donepezil to an individual whose disease is diagnosed in step iii) or surgery.
  • The'treatment' of the present invention refers generically to improving the symptoms of Alzheimer's disease or Alzheimer's disease, which may include curing, substantially preventing, or improving the condition, and from Alzheimer's disease It includes, but is not limited to, alleviating, curing, or preventing one symptom or most symptoms resulting from it.
  • The'treatment drug' is not particularly limited as long as it is a kind of drug that is commonly used for the treatment of Alzheimer's disease.
  • the therapeutic drug is administered to an individual in a'therapeutically effective amount', and the therapeutically effective amount can be determined by those skilled in the art, as well as the age, weight, health condition, sex, and disease of the patient.
  • the effective dosage for a patient can be determined by considering various factors such as the severity of the patient, diet and excretion rate.
  • the route of administration of the therapeutic drug is not particularly limited, and may be administered orally or parenterally, and includes not only local administration but also systemic administration.
  • the parenteral administration may be, but is not limited to, intranasal drug application, subcutaneous injection, and the like, and as another example, intramuscular injection, intravenous injection, or the like may be used.
  • The'sample' of the present invention is obtained separately from an individual suspected of having a disease, but is not limited thereto, but is not limited to cells, tissues, blood, serum, plasma, saliva, and sputum. It may be selected from the group consisting of mucosal fluid and urine, and the'individual' may be an animal, preferably an animal including a mammal, particularly a human, and may be a cell, tissue, organ, etc. derived from an animal. The individual may be a patient in need of the therapeutic effect.
  • the term “comprising” is used with the same meaning as “including” or “characterized by”, and in the composition or method according to the present invention, specifically mentioned It does not exclude additional components or method steps that have not been made.
  • the term “consisting of” means excluding additional elements, steps, or ingredients that are not separately described.
  • the term “essentially consisting of” means that, in the scope of a composition or method, it is possible to include substances or steps that do not substantially affect their basic properties in addition to the substances or steps described.
  • Complement component C8 gamma (C8G) not only shows a significantly increased level compared to the normal group in Alzheimer's disease patients, but also can be measured through bodily fluid samples such as plasma, so it is easy to measure. It has excellent utility as a diagnostic biomarker.
  • C8G complement component C8 gamma
  • NOR normal control group
  • AD patient group AD patient group based on the unpaired two-tailed students t test (p ⁇ .0001).
  • the horizontal bar in each experimental group represents the average value of the C8G level.
  • Figure 2 shows the ROC (Receiver operating charateristic curve) for the plasma C8G level of the AD patient group as a biomarker (AUC: area under curve).
  • Example 1 In plasma for diagnosis of Alzheimer's disease C8G As a biomarker Check the effectiveness
  • Plasma samples from patients who fasted more than 8 hours the day before Put the blood collected from the vena cava into an EDTA tube, add a protease inhibitor cocktail (Roche Diagnostics) and mix gently. The EDTA-treated blood was centrifuged for 10 minutes at a speed of 3000 rpm to separate plasma. The collected plasma samples were immediately frozen on dry ice and then stored in a -80°C freezer.
  • a protease inhibitor cocktail Roche Diagnostics
  • Plasma C8G measurement Plasma was diluted 10,000 times using PBS, and then ELISA assay was performed according to the manufacturer's protocol using Sandwich enzyme-linked immunosorbent assay kit (LSBio) for C8G. 100 ⁇ l of diluted plasma per well was added to the 96 well plate of the kit, and the C8G concentration of each patient was measured. The standard concentration of the used human recombinant C8G protein is 31.25 ⁇ 2000 pg/ml. All measurements were performed in duplicate.
  • LSBio Sandwich enzyme-linked immunosorbent assay kit
  • the predictive significance of plasma C8G levels in AD patients was evaluated using the ROC curve. As shown in FIG. 2, the area under the ROC curve (AUC, that is, accuracy) in AD patients was 0.9048.
  • the present invention relates to a method for diagnosing Alzheimer's disease using complement component C8 gamma, and more particularly, a formulation for measuring the expression level of complement component C8 gamma.
  • the present invention relates to a composition for diagnosis of Alzheimer's disease, a kit for diagnosis, and a method of qualitative or quantitative analysis of complement component C8 gamma in order to provide information necessary for diagnosis of Alzheimer's disease.
  • the complement component C8 gamma not only shows a significantly increased level compared to the normal group in Alzheimer's disease patients, but also can be measured through bodily fluid samples such as plasma, so it is easy to measure, so it is useful as a biomarker for diagnosis of Alzheimer's disease. As it is excellent, it has great industrial applicability in fields such as in vitro diagnostics industry.

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Abstract

La présente invention concerne une méthode de diagnostic de la maladie d'Alzheimer au moyen du composant C8 gamma du complément et, plus particulièrement, une composition pour diagnostiquer la maladie d'Alzheimer comprenant un agent pour mesurer le niveau d'expression du composant C8 gamma du complément, un kit pour le diagnostic et un procédé d'analyse qualitative ou quantitative du composant C8 gamma du complément afin de fournir des informations nécessaires au diagnostic de la maladie d'Alzheimer.
PCT/KR2020/010227 2019-08-02 2020-08-03 Méthode de diagnostic de la maladie d'alzheimer au moyen du composant c8 gamma du complément WO2021025412A1 (fr)

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KR102089032B1 (ko) * 2019-08-02 2020-05-29 경북대학교 산학협력단 보체 성분 c8 감마를 이용한 알츠하이머병의 진단방법

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000050639A2 (fr) * 1999-02-22 2000-08-31 Variagenics, Inc. Variations de sequences geniques presentant une utilite pour la selection du traitement d'une maladie
US20100159486A1 (en) * 2006-11-01 2010-06-24 George Mason Intellectual Properties, Inc. Biomarkers for neurological conditions
KR20100101465A (ko) * 2009-03-09 2010-09-17 경북대학교 산학협력단 퇴행성신경질환 진단용 마커
KR102089032B1 (ko) * 2019-08-02 2020-05-29 경북대학교 산학협력단 보체 성분 c8 감마를 이용한 알츠하이머병의 진단방법

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000050639A2 (fr) * 1999-02-22 2000-08-31 Variagenics, Inc. Variations de sequences geniques presentant une utilite pour la selection du traitement d'une maladie
US20100159486A1 (en) * 2006-11-01 2010-06-24 George Mason Intellectual Properties, Inc. Biomarkers for neurological conditions
KR20100101465A (ko) * 2009-03-09 2010-09-17 경북대학교 산학협력단 퇴행성신경질환 진단용 마커
KR102089032B1 (ko) * 2019-08-02 2020-05-29 경북대학교 산학협력단 보체 성분 c8 감마를 이용한 알츠하이머병의 진단방법

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
EMMA L. VAN DER ENDE, LIEKE H. MEETER, CHRISTOPH STINGL, JEROEN G. J. VAN ROOIJ, MARCEL P. STOOP, DIANA A. T. NIJHOLT, RAQUEL: "Novel CSF biomarkers in genetic frontotemporal dementia identified by proteomics", ANNALS OF CLINICAL AND TRANSLATIONAL NEUROLOGY, vol. 6, no. 4, 7 March 2019 (2019-03-07), pages 698 - 707, XP055778251, ISSN: 2328-9503, DOI: 10.1002/acn3.745 *

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