WO2012137502A1 - Procédé de diagnostic ou procédé de prédiction de pronostic pour la démence ou la maladie d'alzheimer utilisant un produit de clivage de peptide alcadéine - Google Patents

Procédé de diagnostic ou procédé de prédiction de pronostic pour la démence ou la maladie d'alzheimer utilisant un produit de clivage de peptide alcadéine Download PDF

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WO2012137502A1
WO2012137502A1 PCT/JP2012/002370 JP2012002370W WO2012137502A1 WO 2012137502 A1 WO2012137502 A1 WO 2012137502A1 JP 2012002370 W JP2012002370 W JP 2012002370W WO 2012137502 A1 WO2012137502 A1 WO 2012137502A1
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dementia
alc
patient
treatment
level
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PCT/JP2012/002370
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English (en)
Japanese (ja)
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鈴木　利治
沙緒里 伴
勉 清藤
昌次 柏原
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株式会社免疫生物研究所
国立大学法人北海道大学
Ｄｓファーマバイオメディカル株式会社
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Publication of WO2012137502A1 publication Critical patent/WO2012137502A1/fr

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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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to the field of diagnosis or prognosis prediction of dementia or Alzheimer's disease. More specifically, the present invention relates to a method for diagnosing or predicting prognosis of dementia or Alzheimer's disease by detecting the amount or quantitative change of an alkadein peptide cleavage product in a body fluid. The present invention also relates to a kit for detecting the amount or quantitative change of an alkadein peptide cleavage product in a body fluid used for diagnosis or prognosis prediction of dementia or Alzheimer's disease.
  • AD Alzheimer's disease
  • radical treatment disease-modifying therapy
  • Alc Arcadeins
  • Alc Arcadeins
  • Alc ⁇ , Alc ⁇ , and Alc ⁇ are encoded by independent genes, while Alc ⁇ 1 and Alc ⁇ 2 are splice variants of the Alc ⁇ gene.
  • Alc is stabilized through an interaction with X11L and forms a complex with amyloid ⁇ protein precursor (hereinafter referred to as “APP”) indirectly.
  • APP amyloid ⁇ protein precursor
  • Alc forms a trimer with APP and X11L, they all show resistance to proteolytic metabolism (Non-patent Document 1).
  • Alc in the Alc-X11L-APP complex shows resistance to proteolysis, but Alc that does not form a complex has been reported to be cleaved by continuous proteolysis to produce a short peptide (non-patent document). Reference 3).
  • p3-Alc an Alc-derived short peptide that corresponds to the p3 domain of APP is secreted by cleaving Alc CTF by ⁇ -secretase.
  • PS mutation in familial AD changes the cleavage site of p3-Alc (see Patent Document 1 and Patent Document 2; Non-patent Document 4).
  • a change in the cleavage site of p3-Alc can be an indicator of familial AD, there has been a demand for a diagnostic method that enables more reliable diagnosis or progress prediction of AD including sporadic AD. Until now, no marker has been known for predicting the prognosis of AD progression.
  • CDR cognitive impairment
  • p3-Alc is useful for quantitative diagnosis of patients and prediction of prognosis, it has been found that it is also useful for determining the effects of drug administration and treatment stoppage.
  • the present invention provides an early diagnosis method (or a method for determining the degree of progression) or prognosis prediction method for dementia or AD by measuring p3-Alc level.
  • the present invention relates to the inventions described in the following (1) to (34).
  • the present invention also relates to a diagnostic agent for use in the following methods (1) to (23).
  • the present invention relates to a method for providing information for the following methods (1) to (23).
  • a method for predicting or diagnosing / determining the progress, onset, or progress of dementia or AD at an early stage comprising measuring p3-Alc levels in a specimen derived from a patient with dementia or a suspected dementia. (3) measuring p3-Alc levels in specimens from patients with or with suspected dementia; and The method according to (2), comprising diagnosing / determining or predicting the progress, onset, or progress of dementia or AD of the patient from the measured p3-Alc level in the specimen.
  • the method according to any one of (2) to (5) which is a method for predicting early onset of progression to AD.
  • Early prediction or determination of the treatment or prevention effect of dementia or AD of the treatment including measuring p3-Alc level in a sample from a dementia patient undergoing treatment or a suspected dementia patient how to.
  • a method for early prediction or determination of a change in dementia or AD symptoms after treatment cessation which comprises measuring p3-Alc level in a specimen derived from a dementia patient including an AD patient who has discontinued treatment .
  • the treatment is treatment with a ⁇ -secretase inhibitor, a ⁇ -secretase vaccine, a hyperlipidemia therapeutic agent, a lipid metabolism improving agent, or a drug having an effect of preventing cranial nerve injury (10) to (19) The method of any one of these.
  • a carrier selected from the group consisting of a solid phase, a hapten, a magnetic bead, and an insoluble carrier, on which a first antibody that specifically binds to human p3-Alc is immobilized, (1) A kit for immunochemical measurement for use in the method of (24).
  • the immunochemical measurement kit according to claim 26, comprising: (33) (i) an insoluble carrier on which a first antibody that specifically binds to human p3-Alc is immobilized; and (ii) an insoluble carrier on which a second antibody that specifically binds to human p3-Alc is immobilized. , 27.
  • the immunochemical measurement kit according to claim 26, comprising: (34) The immunochemical measurement kit according to any one of (25) to (33), further comprising a substrate that reacts with the label.
  • Alc ⁇ 1 SEQ ID NO: 1
  • Alc ⁇ SEQ ID NO: 2
  • Alc ⁇ SEQ ID NO: 3
  • P3-Alkadein or “p3-Alc” refers to a cell-bound C-terminal fragment of Alc (alcadein carboxylic terminal fragments, hereinafter referred to as “CTF”) produced by the primary cleavage of Alc by ⁇ -secretase, It is a peptide fragment on the N-terminal side of CTF produced by secondary cleavage with a transmembrane cleavage protease ( ⁇ -secretase).
  • p3-Alc encompasses all of these p3-Alc species (referring to each p3-Alc).
  • P3-Alc detected in the method of the present invention can be determined by appropriately selecting the antibody to be used.
  • the p3-Alc to be detected in the method of the present invention may be all p3-Alc species, two or more p3-Alc species, or one specific type of p3-Alc.
  • p3-Alc ⁇ includes p3-Alc ⁇ 34 (SEQ ID NO: 4), p3-Alc ⁇ 35 (SEQ ID NO: 5), p3-Alc ⁇ 36 (SEQ ID NO: 6), p3-Alc ⁇ 37 (SEQ ID NO: 7), p3-Alc ⁇ 38 (SEQ ID NO: 8), p3-Alc ⁇ 39 (SEQ ID NO: 9), p3-Alc ⁇ 2N + 35 (SEQ ID NO: 10), p3-Alc ⁇ 2N + 38 (SEQ ID NO: 11), p3-Alc ⁇ 2N + 39 (SEQ ID NO: 12), and p3-Alc ⁇ 2N + 40 (SEQ ID NO: 13) are known.
  • p3-Alc ⁇ X means a peptide consisting of X amino acids having an amino acid sequence from the 817th alanine (A) to the (816 + X) th amino acid of Alc ⁇ 1.
  • P3-Alc ⁇ 2N + Y means a peptide composed of (Y + 2) amino acids having an amino acid sequence from the 815th methionine (M) to the (816 + Y) th amino acid of Alc ⁇ 1.
  • p3-Alc ⁇ includes all these p3-Alc ⁇ species. P3-Alc ⁇ detected in the method of the present invention can be determined by appropriately selecting the antibody to be used.
  • the p3-Alc ⁇ to be detected in the method of the present invention may be all p3-Alc ⁇ species, two or more p3-Alc ⁇ species, or one specific type of p3-Alc ⁇ .
  • the p3-Alc ⁇ used in the method of the present invention is p3-Alc ⁇ 35.
  • p3-Alc ⁇ As p3-Alc ⁇ , p3-Alc ⁇ 37 (SEQ ID NO: 14) and p3-Alc ⁇ 40 (SEQ ID NO: 15) are known (see International Publication WO2009 / 077504).
  • p3-Alc ⁇ X means a peptide consisting of X amino acids having an amino acid sequence from the 813th valine (V) to the (812 + X) th amino acid of Alc ⁇ .
  • p3-Alc ⁇ includes all these p3-Alc ⁇ species.
  • P3-Alc ⁇ detected in the method of the present invention can be determined by appropriately selecting the antibody to be used.
  • the p3-Alc ⁇ detected in the method of the present invention may be all p3-Alc ⁇ species, two or more p3-Alc ⁇ species, or one specific type of p3-Alc ⁇ .
  • the p3-Alc ⁇ used in the method of the present invention is p3-Alc ⁇ 37 and p3-Alc ⁇ 40.
  • p3-Alc ⁇ 31 SEQ ID NO: 16
  • p3-Alc ⁇ 34 SEQ ID NO: 17
  • p3-Alc ⁇ X means a peptide consisting of X amino acids having an amino acid sequence from the 804th leucine (L) to the (803 + X) th amino acid of Alc ⁇ .
  • p3-Alc ⁇ includes all these p3-Alc ⁇ species.
  • the p3-Alc ⁇ detected in the method of the present invention can be determined by appropriately selecting the antibody to be used.
  • the p3-Alc ⁇ detected in the method of the present invention may be all p3-Alc ⁇ species, two or more p3-Alc ⁇ species, or one specific type of p3-Alc ⁇ .
  • the p3-Alc ⁇ used in the method of the present invention is p3-Alc ⁇ 31 and p3-Alc ⁇ 34.
  • the “specimen derived from a dementia patient or a suspected dementia patient” is not particularly limited as long as p3-Alc can be detected, but is preferably a sample reflecting the p3-Alc level in cerebrospinal fluid.
  • specimens that can be used include blood (plasma, serum), cerebrospinal fluid, lymph fluid, urine, serous fluid, joint fluid, aqueous humor, tear fluid, saliva, and other body fluids or fractions or processed products thereof, or Examples thereof include biological samples collected from human brain tissue or processed products thereof.
  • the detection specimen can be appropriately prepared according to the kind of specimen collected from the patient and the detection method.
  • the sample for detection when it can be used for detection as it is, such as cerebrospinal fluid, the sample for detection may be appropriately adjusted by centrifugation, dilution or the like.
  • the inhibitor when a sample that is collected from a patient such as blood contains a substance that inhibits detection, the inhibitor is removed / inactivated by pretreatment (for example, removal of immunoglobulin using a protein G column) for detection.
  • the specimen may be adjusted.
  • a low polarity organic solvent and a hydrophilic organic solvent an organic solvent having a dipole moment of 1.10 to 1.20 and an organic solvent having a dipole moment of 1.65 to 1.75, most preferably , Chloroform: methanol.
  • p3-Alc level may include the amount of p3-Alc as well as the ratio of p3-Alc species, unless otherwise specified.
  • the p3-Alc level includes the ratio of the p3-Alc ⁇ 35 level to the total amount of p3-Alc ⁇ , the ratio of the p3-Alc ⁇ 40 level to the p3-Alc ⁇ 37 level, and the like.
  • the present invention also includes an immunochemical measurement comprising a carrier selected from the group consisting of a solid phase, a hapten, and an insoluble carrier, to which a first antibody that specifically binds to human p3-Alc is immobilized.
  • a carrier selected from the group consisting of a solid phase, a hapten, and an insoluble carrier, to which a first antibody that specifically binds to human p3-Alc is immobilized.
  • the kit of the present invention may further contain a labeled second antibody that specifically binds to human p3-Alc, or a hapten or an insoluble carrier on which the second antibody is immobilized.
  • the measurement kit of the present invention can be based on a known method using an antibody molecule.
  • EIA method enzyme immunoassay
  • ELISA method enzyme-linked immunosorbent assay
  • RIA method radioimmunoassay
  • FIA method fluorescent immunoassay
  • Labeled immunoassay such as Western blotting method
  • immunochromatography method such as colloidal gold aggregation method
  • chromatography method such as ion exchange chromatography method and affinity chromatography method
  • TIA method turbidimetric method
  • TIA method ion exchange chromatography method and affinity chromatography method
  • TIA method turbidimetric method
  • TIA method turbidimetric method
  • TIA method ion exchange chromatography method
  • NIA method turbidimetric method
  • Colorimetric method Latex aggregation method
  • LIA method Particle counting method
  • CIA method Chemiluminescence measurement method
  • CLIA method, CLEIA method Precipitation reaction method
  • SPR method Surface plasmon resonance method
  • RMD method Resonant
  • the kit of the present invention can be used for qualitative, quantitative or semi-quantitative analysis, but preferably for quantitative analysis.
  • the antibody contained in the kit of the present invention is an antibody fragment (for example, F (ab ′) 2 , Fab ′, Fab, single chain Fv, disulfide bond Fv, or a polymer thereof, dimerized V region). There may be.
  • the kit of the present invention comprises (i) a solid phase or hapten on which a first antibody that specifically binds human p3-Alc is immobilized, and (ii) a labeled that specifically binds to human p3-Alc.
  • a kit for immunochemical measurement comprising a second antibody.
  • the kit of the present invention may further contain a solid phase on which a substance that specifically binds to the hapten is immobilized.
  • the kit of the present invention comprises: (i) a solid phase on which a first antibody that specifically binds to human p3-Alc is immobilized; and (ii) a second antibody that specifically binds to human p3-Alc. It can be set as the kit of the immunochemical measurement containing the fixed hapten.
  • the kit may further contain a labeled substance that specifically binds to the hapten.
  • the kit of the present invention comprises (i) an insoluble carrier on which a first antibody that specifically binds to human p3-Alc is immobilized; and (ii) a second antibody that specifically binds to human p3-Alc. It can be set as the kit of the immunochemical measurement containing the fixed insoluble support
  • the solid phase is not particularly limited as long as it can be used for immunochemical measurement.
  • the insoluble carrier means a suspendable insoluble solid
  • a detectable label such as a radioactive label, an enzyme, a fluorescent label, a bioluminescent label, or a chemiluminescent label metal
  • a detectable label such as a radioactive label, an enzyme, a fluorescent label, a bioluminescent label, or a chemiluminescent label metal
  • examples of such labels include, but are not limited to, radioactive labels such as 32 P, 3 H, 125 I, and 14 C; ⁇ -galactosidase, peroxidase, alkaline phosphatase, glucose oxidase, lactate oxidase, Enzymes such as alcohol oxidase, monoamine oxidase, horseradish peroxidase; coenzymes or prosthetic groups such as FAD, FMN, ATP, biotin, heme; fluorescein derivatives (fluorescein isothiocyanate (FITC), fluorescein ovalbamil, etc.), rhodamine derivatives (
  • the kit of the present invention may contain a coloring reagent, a reaction stopping reagent, a standard antigen reagent, a sample pretreatment reagent, a blocking reagent, and the like, if necessary.
  • a coloring reagent a reaction stopping reagent
  • a standard antigen reagent a sample pretreatment reagent
  • a blocking reagent a blocking reagent
  • the kit of the present invention may further contain a substrate that reacts with the label.
  • the kit of the present invention may include instructions for use, a container for storing reagents, a 96-well plate, and the like in the package.
  • the method of the present invention it is possible to more accurately diagnose and predict the onset of AD or dementia, predict the prognosis of AD or dementia patients, and determine or predict the effects of drug administration and treatment stop.
  • the kit of the present invention in the method of the present invention, more accurate diagnosis of AD or dementia and onset prediction, prognosis prediction of AD or dementia patients, and determination of effects of drug administration or treatment stoppage or Prediction can be performed simply or accurately.
  • the vertical axis represents the subject's CDR
  • the horizontal axis represents the p3-Alc ⁇ level. It is the graph which showed the correlation with CDR and A (beta) 42 level with the box-and-whisker figure.
  • the vertical axis represents the subject's CDR
  • the horizontal axis represents the A ⁇ 42 level. It is a graph which shows the correlation with CDR and age.
  • the vertical axis represents the subject's CDR
  • the horizontal axis represents the age. It is the graph which showed the correlation with CDR and p3-Alc (beta) 37 level with a box-and-whisker plot.
  • the vertical axis represents the subject's CDR
  • the horizontal axis represents the p3-Alc ⁇ 37 level. It is the graph which showed the correlation with CDR and p3-Alc (beta) 40 level with the box-and-whisker plot.
  • the vertical axis represents the subject's CDR
  • the horizontal axis represents the p3-Alc ⁇ 40 level. It is the graph which showed the correlation with CDR and p3-Alc (alpha) level with the box-and-whisker plot.
  • the vertical axis represents the subject's CDR
  • the horizontal axis represents the p3-Alc ⁇ level.
  • the vertical axis represents the subject's CDR
  • the horizontal axis represents the A ⁇ 42 level.
  • the vertical axis represents the subject's CDR
  • the horizontal axis represents the age.
  • the vertical axis represents sensitivity and the horizontal axis represents 1-specificity.
  • the vertical axis represents the subject's CDR
  • the horizontal axis represents the p3-Alc ⁇ 37 level.
  • the vertical axis represents the subject's CDR
  • the horizontal axis represents the p3-Alc ⁇ 40 level.
  • the vertical axis represents the subject's CDR
  • the horizontal axis represents the p3-Alc ⁇ level.
  • the vertical axis represents the subject's CDR
  • the horizontal axis represents the A ⁇ 42 level.
  • CDR0.5 mild cognitive impairment
  • CDR1 mild dementia
  • CDR2 moderate dementia
  • CDR3 severe dementia
  • the vertical axis represents the subject's CDR
  • the horizontal axis represents the age.
  • the vertical axis represents sensitivity
  • the horizontal axis represents 1-specificity.
  • CDR0 control
  • CDR2 moderate dementia
  • CDR3 severe dementia
  • the vertical axis represents the subject's CDR
  • the horizontal axis represents the age.
  • the vertical axis represents sensitivity
  • the horizontal axis represents 1-specificity.
  • the vertical axis shows the concentration of each p3-Alc ⁇ (pg / mL), and the horizontal axis shows the elapsed time from the first measurement to the second measurement. It is a graph showing the change of the level of p3-Alc (beta) 37 and p3-Alc (beta) 40 of the patient whose dementia did not worsen.
  • the vertical axis shows the concentration of each p3-Alc ⁇ (pg / mL), and the horizontal axis shows the elapsed time from the first measurement to the second measurement.
  • the p3-Alc level is measured in specimens derived from patients with dementia or mild cognitive impairment (especially patients with suspected dementia that are difficult to distinguish from healthy individuals), which is higher than the p3-Alc level in healthy individuals Can be diagnosed as having dementia (particularly AD) or having an abnormality in ⁇ -secretase.
  • the conventional method it has been difficult to diagnose whether or not an early stage patient with dementia has developed dementia.
  • the method for measuring p3-Alc level according to the present invention it is possible to detect early. Diagnosis is possible.
  • the invention relates to a method for diagnosing mild dementia or mild cognitive impairment comprising measuring p3-Alc levels in a subject-derived specimen.
  • the subject is a patient who is unclear whether or not the patient has dementia or a patient suspected of having dementia.
  • the present method is effective for dementia (especially AD) when the p3-Alc level in the subject-derived specimen is higher than the p3-Alc level in the specimen from the healthy subject. It includes diagnosing the onset or abnormality of ⁇ -secretase.
  • CDR ⁇ 2 patients with severe dementia
  • CDR 0.5 patients with mild dementia
  • CDR1 patients with mild dementia
  • CDR0.5 patients with mild cognitive impairment
  • the p3-Alc level in a sample derived from a patient with dementia is measured multiple times or periodically over a period of time and the patient measures p3-Alc level after onset of dementia
  • dementia particularly AD
  • severe dementia CDR3
  • the p3-Alc level in the patient's body fluid shows a lower value than that of a healthy person. It was.
  • the present invention relates to a method for diagnosing (determining) severe dementia or severe dementia, comprising measuring p3-Alc level in a specimen derived from a subject.
  • the subject is a patient with mild or more dementia.
  • the present method is suitable for dementia (especially AD) when the p3-Alc level in the subject-derived specimen is lower than the p3-Alc level in the past subject specimen.
  • the method comprises determining severe dementia if the p3-Alc level in a subject-derived sample is low compared to the p3-Alc level in a healthy subject sample. .
  • a method for early detection of an abnormality in ⁇ -secretase comprising measuring p3-Alc level in a specimen from a patient with dementia or a suspected dementia, or a dementia (for example, , Mild dementia) or AD progression, onset, or method of early diagnosis / determination.
  • the present invention provides a method for determining the degree of progression of AD, comprising measuring p3-Alc levels in a specimen from a patient with dementia or a suspected dementia.
  • the method for early detection of abnormality of ⁇ -secretase of the present invention and the method for early determination of progression, onset, or progression of dementia (for example, mild dementia) or AD, (A) measuring p3-Alc levels in a specimen from a patient with dementia or a patient with suspected dementia (or a patient with unknown dementia); and (B) detecting an abnormality of ⁇ -secretase in the patient from the measured p3-Alc level in the specimen, or developing, developing, or progressing dementia (for example, mild dementia) or AD in the patient Including diagnosing / determining the degree.
  • dementia for example, mild dementia
  • AD Alzheimer'sive dementia
  • the present invention provides a change in p3-Alc level by continuously monitoring the p3-Alc level of a subject suspected of having dementia (or a subject whose dementia is unknown) or a patient with dementia.
  • a subject suspected of having dementia or a subject whose dementia is unknown
  • a patient with dementia can be diagnosed / determined at an early stage of dementia (for example, mild dementia) or AD progression, onset, or progression.
  • the method of diagnosing / determining the dementia (eg, mild dementia) or AD progression, onset, or progression of the present invention includes (A) measuring p3-Alc in a sample from a patient with dementia or a patient with suspected dementia (or a patient with unknown dementia) multiple times over a desired period, (B) diagnosing / determining the progression, onset, or progression of dementia (eg, mild dementia) or AD of the patient from the measured change in p3-Alc level in the specimen.
  • Diagnosis / determination of mild dementia or AD progression, onset, or progression depends on the degree of onset of dementia in the subject and past diagnosis results. For patients who are uncertain whether or not they have dementia or subjects with suspected dementia, etc., and who are diagnosed with the onset of dementia (including AD, the same applies in the following paragraph) or mild dementia And determining a subject having a high p3-Alc level in a specimen as a patient suffering from or having a high degree of mild dementia or AD. In the diagnosis of the onset of dementia or mild dementia, the ratio of p3-Alc species level may be used as an index instead of the p3-Alc level.
  • the ratio of the p3-Alc ⁇ 35 level to the total amount of p3-Alc ⁇ , the ratio of the p3-Alc ⁇ 40 level to the p3-Alc ⁇ 37 level, and the like can be used.
  • patients with a higher proportion of p3-Alc species (p3-Alc ⁇ 35, p3-Alc ⁇ 40) whose abundance increases in AD are affected by the onset of dementia or mild dementia compared to healthy individuals.
  • it may include determining as a patient having a high degree of morbidity.
  • mild cognition Including determining subjects with low p3-Alc levels in the specimen as compared to patients with disability (CDR 0.5) or mild dementia (CDR1) as patients with advanced or high degree of dementia be able to.
  • CDR 0.5 patients with disability
  • CDR1 mild dementia
  • the p3-Alc level in the specimen derived from the dementia patient is measured, and the p3-Alc level measured in the past is measured. When it shows a low value in comparison, it can include determining that dementia (particularly AD) is progressing.
  • diagnosis / determination may be performed using the ratio of p3-Alc species level as an index instead of the p3-Alc level.
  • the ratio of the p3-Alc ⁇ 35 level to the total amount of p3-Alc ⁇ , the ratio of the p3-Alc ⁇ 40 level to the p3-Alc ⁇ 37 level, and the like can be used.
  • patients with a low proportion of p3-Alc species p3-Alc ⁇ 35, p3-Alc ⁇ 40
  • p3-Alc ⁇ 35, p3-Alc ⁇ 40 patients with mild cognitive impairment (CDR0.5) or mild dementia (CDR1)
  • CDR0.5 mild cognitive impairment
  • CDR1 mild dementia
  • the method for early detection of an abnormality of ⁇ -secretase of the present invention and the method for early determination of progression, onset, or progression of dementia (for example, mild dementia) or AD, (A) measuring p3-Alc levels in specimens from patients with suspected dementia; and (B) A subject whose p3-Alc level in the specimen is higher than that of a healthy subject is determined as a patient having an abnormality in ⁇ -secretase, or a patient suffering from or likely to suffer from mild dementia or AD It may be a method to do.
  • the method for early detection of abnormality of ⁇ -secretase of the present invention and the method for early determination of progress, onset, or progress of dementia or AD include: (A) measuring p3-Alc levels in specimens from patients with dementia; and (B) A subject with a low p3-Alc level in a specimen compared to a patient with mild cognitive impairment (CDR 0.5) or mild dementia (CDR1), or a patient with abnormal ⁇ -secretase, or severe dementia (eg , CDR3).
  • CDR 0.5 mild cognitive impairment
  • CDR1 mild dementia
  • severe dementia eg , CDR3
  • the method for early detection of abnormality of ⁇ -secretase of the present invention and the method for early determination of progress, onset, or progress of dementia or AD include: (A) measuring p3-Alc levels in specimens from patients with dementia; and (B) A method in which a subject whose p3-Alc level in the specimen is lower than that of a healthy person is determined as a patient having an abnormality in ⁇ -secretase or severe dementia (eg, CDR3) may be used.
  • the method of determining the progress or progression of dementia or AD of the present invention (A) measuring p3-Alc in a specimen derived from a patient with dementia or suspected dementia multiple times over a desired period of time; (B) A method for determining that dementia (particularly AD) is progressing when the p3-Alc level in the measured specimen shows a low value compared to the p3-Alc level measured in the past. It may be.
  • the diagnosis / determination of dementia eg, mild dementia
  • AD progression, onset, or progression may be determined based on the measured p3-Alc level or change in p3-Alc level.
  • AD progression, onset, or progression may be quantified (numerical). Quantification is obtained from a subject by creating a standard curve for the degree of progression and p3-Alc level from information on p3-Alc level in patients with mild dementia or AD whose degree of progression has been confirmed in advance, for example. This can be done by quantifying the degree of progress based on the obtained p3-Alc level.
  • the method of the present invention further quantifies (numerizes) the progression, onset, or progression (particularly the progression) of mild dementia or AD from the measured change in p3-Alc level or p3-Alc level. ) May be included.
  • the method for early detection of ⁇ -secretase abnormality and the method for early determination of progression, onset, or progression of mild dementia or AD are different from p3-Alc together with p3-Alc in the specimen. It may include performing a test that is an indicator of the development, onset, or progression of mild dementia or AD or measuring a marker that is an indicator. Examples of such indexes include memory ability, judgment ability, orientation ability, problem solving ability, social adaptation ability, nursing care status, p-tau, A ⁇ (A ⁇ 40 and A ⁇ 42, etc.), and image diagnosis such as PET.
  • the method of the present invention comprises: (A) measuring p3-Alc levels in specimens from patients with or suspected of having dementia; (B) performing a test or measuring a marker that is an indicator of the progression, onset, or progression of mild dementia or AD that is different from p3-Alc in a patient with dementia or a suspected dementia; and (C) Mild dementia or AD progression of the patient from the degree or value of the indicator of progression, onset, or progression of mild dementia or AD different from p3-Alc and p3-Alc in the measured specimen, It may be a method including diagnosing / determining the onset or the degree of progression.
  • the method of the present invention also includes (A) measuring p3-Alc levels in specimens from patients with or suspected of having dementia; (B) performing a test that is an indicator of the progression, onset, or progression of mild dementia or AD that is different from p3-Alc of a dementia patient or a suspected dementia patient, or measuring a marker that is an indicator, (C) determining the degree of dementia in the patient from the degree or value of an indicator of the progression, onset, or progression of mild dementia or AD that is different from the measured p3-Alc; and (D-1) When the degree of dementia is low, a subject whose p3-Alc level in the sample is higher than that of a healthy subject is suffering from a patient with abnormal ⁇ -secretase or mild dementia or AD Diagnose / determine as a patient, (D-2) When the degree of dementia is high, a subject having a low p3-Alc level in a specimen compared to a healthy person or a patient with mild dementia suffers from
  • the early method of determining the progression, onset, or progression of AD (A) measuring p3-Alc and A ⁇ (preferably A ⁇ 42) in a specimen from a patient with or with suspected dementia; and (B) a method comprising diagnosing / determining the progression, onset, or progression of mild dementia or AD of the patient from the measured values of p3-Alc and A ⁇ (preferably A ⁇ 42) in the specimen. May be.
  • the method of the present invention also includes (A) measuring p3-Alc and A ⁇ (preferably A ⁇ 42) in a specimen from a patient with or with suspected dementia; and (B) A patient whose A ⁇ (preferably A ⁇ 42) value in the measured specimen is sufficiently low compared to a healthy person is determined to have a severe degree of dementia (or a high possibility of dementia), Determining patients with little or no difference compared to healthy individuals as having a low degree of dementia (or low possibility of dementia) (c-1) Diagnose / determine subjects with higher p3-Alc levels in the specimen compared to healthy individuals as patients with abnormal ⁇ -secretase, or patients with mild dementia or AD , (D-2) When the degree of dementia is high (or the possibility of dementia is high), subjects who have a low p3-Alc level in the specimen compared to healthy subjects or patients with mild dementia are referred to as ⁇ -secretase.
  • the method may include diagnosing / determining that the patient has an abnormality or suffers from AD (
  • the diagnosis / determination of dementia using the indicator needs to include the extent No. For example, if the indicator can determine whether or not the patient has dementia, a patient with a high p3-Alc level (compared to a healthy person) is mild, and a patient with a p3-Alc level equal to or low with a healthy person It can be determined that it is severe.
  • the method for early detection of abnormality of ⁇ -secretase of the present invention, and the method for early determination of progression, onset, or progression of dementia for example, mild dementia
  • B) a method comprising diagnosing / determining the progress, onset, or progression of dementia (for example, mild dementia) or AD of the patient from the measured values of p3-Alc and p-tau in the specimen It may be.
  • the method of the present invention also includes (A) measuring p3-Alc and p-tau in a specimen from a dementia patient or a suspected dementia patient; and (B) determining a patient with dementia whose measured p-tau value in the specimen is higher than that of a healthy person, and (C) For patients who have been determined to have dementia, subjects whose samples have a higher p3-Alc level compared to normal subjects are determined to be mild dementia, and samples which are compared to normal subjects (or patients with mild dementia) It may also be a method comprising determining a subject with moderate or low p3-Alc levels as severe dementia.
  • subjects whose p3-Alc level in the patient's body fluid is higher than that of healthy subjects are mild dementia (CDR1) patients and mild cognitive impairment patients ( It is thought that it tends to progress to CDR 0.5). Therefore, the level of p3-Alc in a sample from a patient with dementia or a patient with suspected dementia (especially a patient with suspected dementia that is difficult to distinguish from a healthy person) is measured, and a healthy person (especially with dementia or AD).
  • CDR 0.5 p3-Alc level in the body fluid of patients with mild dementia
  • CDR ⁇ 2 p3-Alc levels in specimens from patients with dementia were measured and patients with mild dementia (CDR1) and patients with mild cognitive impairment (CDR0.5) (especially did not progress to severe dementia or AD Patients or patients with severe dementia or those who are believed not to progress to AD) or dementia (especially AD if it shows a low value compared to the patient's previous p3-Alc level) ) Is likely to progress.
  • the present invention there is provided a method for predicting the onset or progress of dementia or AD at an early stage by measuring or monitoring the p3-Alc level in a specimen derived from a patient with dementia or a suspected dementia. .
  • the present invention provides a method for predicting the onset or progression of AD.
  • the method for predicting the progression or onset of dementia or AD of the present invention (A) measuring p3-Alc levels in specimens from patients with or suspected of having dementia; and (B) predicting the progression or onset of dementia or AD in the patient from the measured p3-Alc level in the specimen.
  • the present invention predicts the development or onset of mild dementia or AD by continuously monitoring the p3-Alc level of subjects with suspected dementia or patients with dementia from changes in p3-Alc levels. Can do.
  • the method of the present invention comprises: (A) measuring p3-Alc in a specimen derived from a patient with dementia or suspected dementia multiple times over a desired period of time; (B) predicting the progression or onset of mild dementia or AD in the patient from the measured change in p3-Alc level in the specimen.
  • the prediction method of the present invention is a method for predicting the onset of mild dementia at an early stage, a method for predicting the onset of AD at an early stage, or a method for predicting the onset of progression to AD at an early stage.
  • the method for early prediction of progression or onset of dementia or AD of the present invention is as follows: (A) measuring p3-Alc levels in a specimen from a healthy person or a suspected dementia; and (B) A subject whose p3-Alc level in the sample is higher than that of a healthy person (particularly, a healthy person who did not suffer from dementia or AD or a healthy person who is believed not to suffer from dementia or AD) May be a method of predicting a high possibility of suffering from mild dementia or AD.
  • the method for early prediction of progression or onset of dementia or AD of the present invention is as follows: (A) measuring p3-Alc levels in specimens from patients with dementia; and (B) Mild cognitive impairment (CDR 0.5) or mild dementia patient (CDR1) (especially patients who did not progress to severe dementia or AD or patients who are believed to not develop severe dementia or AD ) May be a method of predicting that a subject whose p3-Alc level in the specimen is low is likely to progress to severe dementia (eg, CDR3).
  • the method for early prediction of progression or onset of dementia or AD of the present invention comprises: (A) measuring p3-Alc levels in specimens from patients with dementia; and (B) A method of predicting that a subject whose p3-Alc level in a specimen is lower than that of a healthy person is likely to progress to severe dementia (eg, CDR3) may be used.
  • a method of predicting that a subject whose p3-Alc level in a specimen is lower than that of a healthy person is likely to progress to severe dementia eg, CDR3
  • the method of predicting the progress or onset of dementia or AD of the present invention at an early stage (A) measuring p3-Alc in a sample from a healthy person or a patient with suspected dementia multiple times over a desired period of time; (B) If the measured p3-Alc level in the specimen is higher than the previously measured p3-Alc level, it is predicted that dementia (especially AD) is likely to progress It may be a method.
  • the method of predicting early development or onset of dementia or AD of the present invention (A) measuring p3-Alc in a specimen derived from a patient with dementia multiple times over a desired period; (B) If the measured p3-Alc level in the specimen is low compared to the p3-Alc level measured in the past, it is predicted that dementia (especially AD) is likely to progress It may be a method to do.
  • the method for predicting the onset or progress of dementia or AD at an early stage of the present invention may be predicted using the ratio of p3-Alc species level as an index instead of the p3-Alc level.
  • the ratio of the p3-Alc ⁇ 35 level to the total amount of p3-Alc ⁇ , the ratio of the p3-Alc ⁇ 40 level to the p3-Alc ⁇ 37 level, and the like can be used.
  • the method of predicting the onset or progress of dementia or AD of the present invention at an early stage is an indicator of the progression, onset, or progress of mild dementia or AD that is different from p3-Alc together with p3-Alc in a sample. May be measured.
  • indexes include memory ability, judgment ability, orientation ability, problem solving ability, social adaptation ability, nursing care status, p-tau, A ⁇ (A ⁇ 40 and A ⁇ 42, etc.), and image diagnosis such as PET.
  • the method of predicting the progression or onset of dementia or AD from the p3-Alc level can be performed by statistical analysis. Statistical significance is determined by comparing two or more populations and determining confidence intervals and / or p-values (Dowdy and Wearden, Statistics for Research, John Wiery & Sons, New York, 1983).
  • the confidence interval of the present invention may be, for example, 90%, 95%, 98%, 99%, 99.5%, 99.9% or 99.99%.
  • the p value of the present invention is, for example, 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0002, or 0.0001. It may be.
  • the method for predicting the progress or onset of dementia or AD means a method for predicting the progress or turning point of a patient's condition, and can predict the progress or turning point of the state with 100% accuracy. It does not mean that.
  • Predicting the progression or onset of dementia or AD means whether or not there is an increased likelihood of a course or turning point, meaning that it is likely to occur on the basis of the case where the course or turning point does not occur Not what you want. That is, the predicted outcome of the development or onset of dementia or AD is greater in patients who are predicted to develop or develop dementia or AD by the methods of the invention compared to patients who are not so predicted. It means that the onset or progress of AD is more likely to occur.
  • the present invention relates to a method for early determination of the treatment or prevention effect of dementia or AD of the treatment, which comprises measuring p3-Alc level in a specimen from a dementia patient undergoing treatment.
  • the method of the present invention comprises measuring p3-Alc level in a specimen derived from mild dementia or AD patient before the start of treatment, Measure the p3-Alc level in the specimen from the patient after the start, and the therapeutic effect of mild dementia or AD on the patient from the measured change in the p3-Alc level before and after the start of the treatment It is related with the method of determining the treatment or prevention effect of the dementia or AD of the said treatment including determination early.
  • the therapeutic or preventive effect of the treatment on dementia or AD can be determined at an early stage.
  • the therapeutic effect continues to be effective if the value has decreased and the value has not changed compared to the previous p3-Alc level.
  • the therapeutic effect can be monitored by employing the method for measuring p3-Alc level of the present invention.
  • mild dementia after treatment cessation including measuring p3-Alc level in specimens from AD patients or dementia patients who discontinue treatment
  • changes in AD symptoms can be predicted or determined early. Specifically, measuring p3-Alc level in specimens from patients with mild dementia or AD who discontinued treatment, and mild dementia after treatment cessation in the patient from the measured p3-Alc level or By predicting changes in AD symptoms, it is possible to predict or determine mild dementia after treatment cessation or changes in AD symptoms early.
  • the treatment or prevention effect of the treatment dementia or AD can be determined at an early stage it can.
  • p3-Alc level is measured in samples from patients with mild dementia or AD during and after treatment discontinuation, and p3-Alc level after treatment discontinuation is different from p3-Alc level before treatment discontinuation. If the value is low compared, it is determined that the disease progresses due to treatment cessation, and if the value does not change compared to the p3-Alc level before the treatment is discontinued, it can be determined that the disease is in a lull state. it can. In particular, according to the conventional method, it was difficult to determine the effect of cessation of treatment for dementia, but by adopting the method for measuring p3-Alc level of the present invention, the effect of cessation of treatment can be determined.
  • the present invention measures p3-Alc levels in specimens from patients with mild dementia or AD who have been treated, and treatment for dementia or AD for the patient from the measured p3-Alc levels.
  • the present invention relates to a method for early predicting the treatment or prevention effect of dementia or AD of the treatment, including predicting the effect.
  • the p3-Alc level in a specimen from a dementia patient undergoing treatment is measured and shows a low value compared to the p3-Alc level before treatment, there is a possibility that there is no therapeutic effect
  • the numerical value does not change compared with the p3-Alc level before treatment, it can be predicted that there is a high possibility of having a therapeutic effect.
  • the therapeutic effect can be predicted by adopting the method for measuring the p3-Alc level of the present invention.
  • the method for determining or predicting the effect of treatment or cessation of treatment for dementia or AD at an early stage of the present invention predicts the ratio of p3-Alc species level as an index instead of the level of p3-Alc.
  • the ratio of the p3-Alc ⁇ 35 level to the total amount of p3-Alc ⁇ , the ratio of the p3-Alc ⁇ 40 level to the p3-Alc ⁇ 37 level, and the like can be used.
  • a patient with a low proportion of p3-Alc species p3-Alc ⁇ 35, p3-Alc ⁇ 40
  • the therapeutic effect may be low
  • the method for early determination or prediction of the therapeutic effect or therapeutic cessation effect on dementia or AD includes dementia (for example, mild dementia) different from p3-Alc together with p3-Alc in a sample.
  • dementia for example, mild dementia
  • it may include measuring an indicator of AD progression, onset, or progression.
  • indexes include memory ability, judgment ability, orientation ability, problem solving ability, social adaptation ability, nursing care status, p-tau, A ⁇ (A ⁇ 40 and A ⁇ 42, etc.), and image diagnosis such as PET.
  • the method for determining or predicting the effect of treatment or cessation of treatment on dementia or AD of the present invention at an early stage quantifies the treatment effect from the measured change in p3-Alc level or p3-Alc level (quantification). ).
  • the quantification can be obtained from a subject by creating a standard curve for the therapeutic effect and p3-Alc level from information on the p3-Alc level in patients with mild dementia or AD for which a previously digitized therapeutic effect has been confirmed, for example. This can be done by quantifying the therapeutic effect based on the determined p3-Alc level.
  • the method of the present invention may further comprise quantifying (quantifying) the therapeutic effect from the measured p3-Alc level or a change in p3-Alc level.
  • the treatment employed in the method for early determination or prediction of the therapeutic effect or treatment cessation effect on dementia or AD of the present invention is not particularly limited as long as it is a treatment for dementia or AD.
  • therapeutic agents include ⁇ -secretase inhibitors, ⁇ -secretase vaccines, hyperlipidemia therapeutic agents (eg, statins), lipid metabolism improving agents, or agents that have an effect of preventing cranial nerve cell injury. it can.
  • the term “dementia” includes AD unless otherwise contradicted.
  • the above-described disease diagnosis / determination method, disease prediction method, and therapeutic effect determination method can all be read as a method of providing information for prediction of a disease unless there is a particular contradiction.
  • the biomarker of the present invention is an indicator of the onset and progress of dementia or AD, it can be used for screening inhibitors of ⁇ -secretase or activity modifiers or therapeutic agents for dementia or AD. it can. For example, by administering a test drug to an AD patient or an AD model animal and measuring the p3-Alc level after a certain time, it is determined whether or not the therapeutic drug has a dementia or AD therapeutic effect can do.
  • the screening method of the present invention includes a step of detecting p3-Alc in a specimen, and can make a determination using a change in p3-Alc level as an index.
  • the screening method for a ⁇ -secretase inhibitor or activity modifier or a therapeutic agent for dementia or AD of the present invention comprises the following steps: a) administering a test drug to a model animal or human (or dementia or AD patient or dementia or AD model animal) having ⁇ -secretase abnormality; b) adjusting the body fluid of the administered subject as a sample; c) measuring the level of p3-Alc, and d) It can be carried out by a step of associating the level of p3-Alc with the presence or absence or extent of a therapeutic or preventive effect on a disease (or dementia or AD) based on ⁇ -secretase abnormality of the test drug.
  • the screening method for the inhibitor or activity modifier of ⁇ -secretase or the therapeutic agent for dementia or AD of the present invention comprises the following steps: a) adjusting a body fluid of a model animal or human (or dementia or AD patient or dementia or AD model animal) having a ⁇ -secretase abnormality as a specimen; b) administering a test drug to a model animal or human (or dementia or AD patient or dementia or AD model animal) having a ⁇ -secretase abnormality; c) adjusting the subject's body fluid after administration as a specimen; d) measuring the level of p3-Alc in the specimen before and after administration, and e) If the p3-Alc level in the specimen after treatment shows a low value compared to the p3-Alc level before treatment, the test drug has no therapeutic effect, and the p3-Alc level before treatment When the numerical value does not change in comparison, it can be determined by determining that the test drug has a therapeutic effect.
  • the measurement of p3-Alc is a method capable of measuring the p3-Alc level, and can be performed by methods well known to those skilled in the art.
  • the measurement of p3-Alc can include the identification and quantification of p3-Alc.
  • p3-Alc can also be measured by using ELISA, radioimmunoassay, immunohistochemical method, Western blot, or the like by using an antibody specific for p3-Alc.
  • P3-Alc can also be performed by separating the desired p3-Alc from the specimen and then detecting the separated p3-Alc.
  • Separation of p3-Alc from a sample is a method used for separation of peptides, and can be performed by methods well known to those skilled in the art, for example, by immunoprecipitation, Western blot, affinity chromatography, or the like. it can.
  • the method for detecting the separated p3-Alc is limited as long as it is information on the amino acid sequence, molecular weight, physical property, or chemical property of the peptide, and can obtain information that can identify p3-Alc. However, it can be carried out by methods well known to those skilled in the art, and examples thereof include mass spectrum, protein sequencer, western blotting, ELISA and the like.
  • an sELISA system can be appropriately constructed and measured according to the method described in International Publication No. WO2009 / 077504 and this specification.
  • Kit also includes a carrier selected from the group consisting of a solid phase, a hapten, a magnetic bead, and an insoluble carrier, to which a first antibody that specifically binds to human p3-Alc is immobilized.
  • the present invention relates to a kit for immunochemical measurement.
  • An antibody is prepared by using a peptide consisting of an amino acid sequence having a desired p3-Alc amino acid sequence as an antigen and, if necessary, an immunostimulant (for example, mineral oil or aluminum precipitate and heat-killed bacteria or lipopolysaccharide) Body, Freund's complete adjuvant, Freund's incomplete adjuvant, etc.) and non-human immunized animals.
  • an immunostimulant for example, mineral oil or aluminum precipitate and heat-killed bacteria or lipopolysaccharide
  • Body Freund's complete adjuvant, Freund's incomplete adjuvant, etc.
  • non-human immunized animals is not particularly limited as long as it can produce hybridomas such as mice, rats, hamsters, rabbits, chickens and ducks, but mice, rats or rabbits are preferred.
  • Administration of the immunogen to the animal can be performed by, for example, subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection, or footpad injection, preferably by subcutaneous injection or intraperitoneal injection. is there.
  • the amount of the immunogen used is not particularly limited as long as it is an amount capable of producing an antibody, but is preferably 0.1 to 1000 ⁇ g, more preferably 1 to 500 ⁇ g, and still more preferably 10 to 100 ⁇ g.
  • Immunization can be performed once or several times at appropriate intervals. Preferably, immunization is performed 2 to 5 times in total for 1 to 5 weeks, and more preferably, immunization is performed 3 times in total for 3 weeks.
  • Antibody titer can be measured by methods well known to those skilled in the art. For example, a radioisotope immunoassay (RIA method), a solid-phase enzyme immunoassay (ELISA method), a fluorescent antibody method, and a passive hemagglutination method can be exemplified, and the ELISA method is preferable.
  • the antibody of the present invention can be obtained by purification from the serum of an animal exhibiting a sufficient antibody titer.
  • a monoclonal antibody When a monoclonal antibody is used as an antibody, it can be obtained by culturing a hybridoma obtained by fusing an antibody-producing cell obtained from an immunized animal immunized by the above-described method and a myeloma cell (myeloma cell). It can.
  • Examples of the fusion method include the method of Milstein et al. (Galfre, G. & Milstein, C., Methods Enzymol. 73: 3-46, 1981).
  • the antibody-producing cells to be used can be collected from the spleen, pancreas, lymph nodes, and peripheral blood of immunized animals that have been immunized by the above-described method and have a sufficient antibody titer, and are preferably collected from the spleen.
  • the myeloma cell to be used is not particularly limited as long as it is a cell derived from a mammal such as mouse, rat, guinea pig, hamster, rabbit or human and can be proliferated in vitro. Examples of such cells include P3-X63Ag8 (X63) (Nature, 256, 495, 1975), P3 / NS1 / 1-Ag4-1 (NS1) (Eur. J.
  • the antibody-producing cells and myeloma cells obtained according to the above method are washed with a medium, phosphate buffered saline (PBS), etc., and then added with a cell-aggregating medium such as polyethylene glycol (hereinafter referred to as “PEG”). To fuse the cells.
  • PBS phosphate buffered saline
  • PEG polyethylene glycol
  • the ratio of antibody-producing cells and myeloma cells at the time of fusion is, for example, 2: 1 to 1: 2.
  • the hybridoma is selectively grown by culturing in a medium such as HAT (hypoxanthine-aminopterin-thymidine) medium.
  • HAT hyperxanthine-aminopterin-thymidine
  • the culture supernatant is collected, and a sample that binds to the antigen protein and does not bind to the non-antigen protein is selected by ELISA or the like.
  • the sample is made into a single cell by the limiting dilution method, and cells that stably show a high antibody titer are selected.
  • Monoclonal antibodies can be obtained by culturing the hybridoma obtained by the above method in vitro and purifying the culture solution. Monoclonal antibodies can also be obtained by transplanting hybridomas into syngeneic animals or immunodeficient animals in which pristane has been administered intraperitoneally in advance, then ascites, and purifying the collected ascites. Purification can be obtained by collecting the IgG fraction using a protein A column, protein G column or the like after centrifugation.
  • the labeling of the antibody can be performed by a general method in this field. For example, when fluorescently labeling a protein or peptide, after washing the protein or peptide with a phosphate buffer, a dye adjusted with DMSO, buffer, etc. is added, mixed, and then allowed to bind at room temperature for 10 minutes. be able to.
  • a biotin labeling kit Biotin Labeling Kit-NH2, Biotin Labeling Kit-SH: Dojin Chemical Laboratory Co., Ltd.
  • an alkaline phosphatase labeling kit Alkaline Phosphatase Labeling Kit-NH2, Alkaline Phosphatase Kit-NH2
  • Kit The kit of the present invention is an enzyme immunoassay method (EIA method), a simplified EIA method, an enzyme-linked immunosorbent assay method (ELISA method) using the antibody (or labeled antibody) prepared according to the method described above. ), Radioimmunoassay method (RIA method), labeled immunoassay method such as fluorescent immunoassay method (FIA method); immunoblotting method such as Western blotting method; immunochromatography method such as colloidal gold aggregation method; ion exchange chromatography method, affinity Chromatographic methods such as chromatographic methods; turbidimetric method (TIA method); specific wax method (NIA method); colorimetric method; latex agglutination method (LIA method); particle counting method (CIA method); chemiluminescence measurement method (CLIA method) , CLEIA method); sedimentation reaction method; surface plasmon resonance method (SPR method); resonant mirror detector -Method (RMD method): As a kit for comparative inter
  • the kit of the present invention includes, for example, an anti-p3-Alc monoclonal antibody immobilized plate, a biotin-labeled anti-p3-Alc polyclonal antibody solution, a streptavidin POD solution, a washing solution, a TMB reagent, 2M HCl, a standard substance (p3-Alc Peptide).
  • the kit of the present invention can be produced as a kit comprising an anti-p3-Alc monoclonal antibody, an anti-p3-Alc monoclonal antibody-conjugated gold colloid, a rabbit immunoglobulin-conjugated gold colloid, and a test plate. .
  • the test plate comprises a specimen collecting part for inserting a specimen, a sensitized gold colloid application part containing an anti-p3-Alc monoclonal antibody-conjugated gold colloid, a judgment part (test line) containing an anti-p3-Alc monoclonal antibody, The determination part (reference line) containing a rabbit immunoglobulin polyclonal antibody, an absorber, and a membrane filter may be provided.
  • Example 1 Construction of p3-Alc ELISA Measurement System A sELISA kit for measuring p3-Alc ⁇ was prepared according to the description in International Publication WO2009 / 077504. Specifically, a polyclonal rabbit antibody 817 was prepared using a peptide containing a sequence between positions 817 to 822 of human Alc ⁇ 1 as an antigen, and affinity purified with an antigen-binding resin. Polyclonal rabbit antibody 839 was affinity-purified with an antigen-binding resin using a peptide containing a sequence between positions 839 and 852 of human Alc ⁇ 1 as an antigen.
  • the 839 antibody was used as an antibody to capture p3-Alc ⁇ , and horseradish peroxidase-conjugated pan p3-Alc ⁇ antibody 817 and tetramethylbenzidine were used to detect the supplemented p3-Alc ⁇ by colorimetric analysis at OD 450 .
  • Synthetic p3-Alc ⁇ 35 peptide (Hata S. et al., J. Bio. Chem. (2009) 284: 36024-36033) was used as a standard protein. It was confirmed by using p3-Alc ⁇ 35 peptide and p3-Alc ⁇ 37 peptide that the prepared sELISA system was specific to p3-Alc ⁇ (not shown).
  • pan p3-Alc ⁇ mouse monoclonal antibody 3B5 and the polyclonal rabbit antibody UT135 were prepared by the methods described (Hata S. et al., J. Bio. Chem. (2009) 284: 36024-36033).
  • the constructed sELISA system was able to detect the standard substance as a standard curve in the range of 39 to 625 pg / mL.
  • the anti-p3-Alc ⁇ 40 antibody (2A1 antibody) is a peptide having the amino acid sequence from the 32nd to the 40th amino acid sequence of p3-Alc ⁇ (CIPSAATLLII; SEQ ID NO: 18) as an antigen according to the description of International Publication WO2009 / 077504. Made. It was confirmed that the p3-Alc ⁇ 40 antibody reacts with p3-Alc ⁇ 40 in a dose-dependent manner (not shown). Further, the cross-reactivity of the p3-Alc ⁇ 40 antibody to p3-Alc ⁇ 37 was measured, and it was confirmed that no cross-reactivity occurred (not shown).
  • the anti-p3-Alc ⁇ 37 antibody was prepared in accordance with the description of International Publication WO2009 / 077504 using a peptide having the amino acid sequence from the 29th to the 37th amino acid sequence (CNSMIPSAAT) of p3-Alc ⁇ as the antigen.
  • the reactivity of the obtained antibodies with p3-Alc ⁇ 37, p3-Alc ⁇ 40, and BSA was measured, and 14A1 antibody and 15A1 antibody were obtained as antibodies that specifically recognize p3-Alc ⁇ 37.
  • a sandwich ELISA system was constructed using the obtained antibodies, and the detection sensitivity was confirmed (not shown). It was confirmed that the 14A1 antibody with higher detection sensitivity did not cross-react with p3-Alc ⁇ 40 (not shown).
  • p3-Alc ⁇ 37 was measured using a sandwich ELISA system prepared using 14A1.
  • the sELISA for measuring A ⁇ 40 and A ⁇ 42 is a commercially available kit (Human Amyloid ⁇ (1-40) (N) Assay Kit-IBL (# 27714), Human Amyloid ⁇ (1-42) (N) Assay Kit-IBL (# 27712), Institute for Immunobiology, Japan).
  • Example 2 Measurement of each marker level of a CSF sample derived from a subject (slight cognitive impairment, AD patient and control, with CDR determination)
  • Subjects of measurement and analysis Measurement and analysis were performed on 65 patients who developed mild cognitive impairment and AD, and 19 elderly people (control) who were not dementia appropriate for the patient.
  • CDR0 health / non-disease
  • CDR0.5 mimild cognitive impairment
  • CDR1 mimild dementia
  • CDR2 the progression of dementia symptoms
  • CDR3 severe dementia
  • ⁇ Yotsusen ⁇ obtained cerebrospinal fluid (CSF) (25 ⁇ L) diluted 10 times with PBS containing 1% (w / v) BSA and 0.5% (v / v) Tween-20 250 ⁇ L of the sample was centrifuged at 15,000 g for 10 minutes to remove debris, and 100 ⁇ L of the supernatant was used as an assay sample and measured in duplicate by sELISA.
  • p3-Alc ⁇ and p3-Alc ⁇ were measured using the sELISA prepared in Example 1.
  • Example 3 Measurement of each marker level of a CSF sample derived from a subject (control, mild cognitive impairment (CDR0.5), mild dementia (CDR1)) (1) Analysis of marker measurement result In the measurement result of Example 2, the marker measurement result was compared with the control because there was a difference in the marker concentration between control and mild cognitive impairment.
  • CDR0.5 patients with mild cognitive impairment
  • CDR1 mild dementia
  • ROC Receiveiver
  • AUC Area under the curve of each marker was confirmed using an Operating Characteristic curve (FIG. 11).
  • the area under the curve (AUC) was 0.68772 for p3-Alc ⁇ 37, 0.77193 for p3-Alc ⁇ 40, 0.57632 for A ⁇ 42, 0.77333 for p3-Alc ⁇ , and p3-Alc ⁇ 40 was the largest.
  • Example 4 Measurement of each marker level of a CSF sample derived from a subject (control, mild cognitive impairment (CDR0.5), mild dementia (CDR1), moderate dementia (CDR2), severe dementia (CDR3)) (1) Analysis of marker measurement results In the measurement results of Example 2, moderate dementia and mild dementia peaked in moderate dementia (CDR2) and severe dementia (CDR3), indicating a low value. We compared the measurement results of dementia of higher degree with mild cognitive impairment / mild dementia or control.
  • CDR0.5 mild cognitive impairment
  • CDR1 mild dementia
  • CDR2 moderate dementia
  • CDR3 severe dementia
  • P3-Alc ⁇ , p3-Alc ⁇ 37, p3-Alc ⁇ 40, and A ⁇ 42 all showed a significantly low value as dementia progressed from CDR 0.5 and 1 (p ⁇ 0.0001).
  • ROC Receiveiver
  • AUC area under the curve of each marker was confirmed (FIG. 17).
  • the area under the curve (AUC) showed high values in p3-Alc ⁇ 37 (0.948), p3-Alc40 (0.952), and p3-Alc ⁇ (0.952).
  • a ⁇ 42 was 0.99.
  • the area under the curve (AUC) of each marker is confirmed using ROC (Receiver Operating Characteristic) curves to diagnose moderate and severe dementia compared to controls. (FIG. 23).
  • the area under the curve (AUC) was high in p3-Alc ⁇ 37 (0.883), p3-Alc40 (0.880), and p3-Alc ⁇ (0.891).
  • CDR 0.5 mild cognitive impairment
  • CDR1 mild dementia
  • CDR2 moderate dementia
  • CDR3 patients with severe dementia
  • a ⁇ 42 The same tendency was not confirmed with A ⁇ 42. No statistical difference was observed for A ⁇ 42. This is considered due to the aggregation property of A ⁇ 42.
  • CDR2 The decrease in p3-Alc ⁇ and A ⁇ 40 in AD patients (CDR2) is thought to be due to the progression of neurodegeneration in which the expression of Alc and APP weakens with a decrease in the number of neurons and / or a decrease in neural activity.
  • the CDR determined for the second time for patients A to C was CDR2, and all had dementia worsening.
  • the CDR determined for the second time for patients D and E was CDR0.5
  • the CDR determined for the second time for patient F was CDR1, and no change was observed in the degree of dementia.
  • the results of measuring p3-Alc ⁇ 37 and p3-Alc ⁇ 40 for patients A to C in which dementia worsened are shown in FIG. 24, and the results of measurements in patients D to F in which dementia did not worsen are shown in 25.
  • the vertical axis indicates the concentration (pg / mL) of each p3-Alc ⁇
  • the horizontal axis indicates the elapsed period from the first measurement.
  • Diagnosis and prediction of onset of AD or dementia of the present invention prognosis prediction of AD or dementia patients, and the determination or prediction method of the effects of drug administration and treatment stop early on the progress, onset of dementia or Alzheimer's disease, or Since the degree of progression can be diagnosed / determined or predicted, it can be used in the field of diagnosis of AD and dementia.

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Abstract

La présente invention porte sur un procédé qui permet le diagnostic plus précis de la maladie d'Alzheimer et la prédiction plus précise de l'apparition de la maladie d'Alzheimer. De manière spécifique, la présente invention porte sur : un procédé pour détecter l'anomalie de γ-secrétase à un stade précoce, ce qui consiste à mesurer le niveau de p3-Alc dans un échantillon provenant d'un patient qui est affecté de démence ou est suspecté d'être affecté de démence et qui est obtenu sur la base d'une découverte que le changement dans des niveaux numériques de p3-Alc (en particulier p3-Alcβ37 et p3-Alc40) est mis en corrélation avec l'évolution de CDR ; et autres. Il a également été découvert que p3-Alc est utile pour le diagnostic quantitatif du patient ou la prédiction de pronostic du patient et est donc également utile pour la détermination de l'effet de l'administration d'un médicament ou de l'effet de l'abandon d'une thérapie.
PCT/JP2012/002370 2011-04-04 2012-04-04 Procédé de diagnostic ou procédé de prédiction de pronostic pour la démence ou la maladie d'alzheimer utilisant un produit de clivage de peptide alcadéine WO2012137502A1 (fr)

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WO2016060190A1 (fr) * 2014-10-16 2016-04-21 国立大学法人北海道大学 AGENT THÉRAPEUTIQUE DESTINÉ AU TROUBLE COGNITIF INDUIT PAR LA β-PROTÉINE AMYLOÏDE

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