[go: up one dir, main page]

WO2018106889A1 - Diagnostic de la maladie d'alzheimer - Google Patents

Diagnostic de la maladie d'alzheimer Download PDF

Info

Publication number
WO2018106889A1
WO2018106889A1 PCT/US2017/065060 US2017065060W WO2018106889A1 WO 2018106889 A1 WO2018106889 A1 WO 2018106889A1 US 2017065060 W US2017065060 W US 2017065060W WO 2018106889 A1 WO2018106889 A1 WO 2018106889A1
Authority
WO
WIPO (PCT)
Prior art keywords
tau
acetylated
sample
csf
subject
Prior art date
Application number
PCT/US2017/065060
Other languages
English (en)
Inventor
Joel Steven ROSS
Original Assignee
Cogwellin L.L.C.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cogwellin L.L.C. filed Critical Cogwellin L.L.C.
Publication of WO2018106889A1 publication Critical patent/WO2018106889A1/fr

Links

Classifications

    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • 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 disclosure describes a biomarker for diagnosing Alzheimer's disease (AD).
  • the method involves detecting the biomarker using an immunoassay.
  • tauopathies which include frontotemporal dementia (FTD) and Alzheimer's disease (AD).
  • FTD frontotemporal dementia
  • AD Alzheimer's disease
  • AD is the sixth- leading cause of death in the United States.
  • AD is a progressive neurodegenerative disease affecting 60-70% of dementia patients.
  • AD is characterized by memory loss and disorientation. The hallmark of the disease is associated with the dysregulation of amyloid beta 42 (Ab-42), total tau (t-tau), and phosphorylated tau (p-tau) proteins, resulting in deposition of amyloid plaques, neuronal death, and accumulation of tangles respectively.
  • Ab-42 amyloid beta 42
  • t-tau total tau
  • p-tau phosphorylated tau
  • tau has been demonstrated to be modified by lysine acetylation, including lysine 280 (K280) within the microtubule-binding motif.
  • lysine acetylation including lysine 280 (K280) within the microtubule-binding motif.
  • Immunohistochemical and biochemical studies of brains from tau transgenic mice and patients with AD and related tauopathies showed that acetylated tau pathology is specifically associated with insoluble, Thioflavin-positive tau aggregates, and the acetylated form has been detected in diseased tissue, suggesting it may play a role in pathological tau transformation.
  • tau K280 acetylation is a potential target for drug discovery and biomarker development for AD and may represent a better predictive biomarker for monitoring the progression of Alzheimer's disease in patients.
  • This present disclosure describes a method of diagnosing AD using a biomarker.
  • the biomarker is acetylated tau.
  • the method includes detecting acetylated tau in the tissues of the central nervous system (CNS) and bodily fluids of subjects suspected of having AD. The method also quantitates acetylated tau in the bodily fluids the subjects.
  • the bodily fluid is cerebral spinal fluid (CSF)
  • acetylated tau is acetylated K280 tau.
  • FIG. 1A shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the white matter at the superior cerebellar peduncle of a control patient.
  • FIG. 1 B shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the white matter at the superior cerebellar peduncle of a Braaks Stage 1 AD patient.
  • FIG. 2A shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the cerebral white matter of a control patient.
  • FIG. 2B shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the cerebral white matter of a Braaks Stage 1 AD patient.
  • FIG. 3A shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the putamen of a Braaks Stage 1 AD patient.
  • FIG. 3B shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the neurons of the putamen of a Braaks Stage 1 AD patient.
  • FIG. 4A shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the caudate nucleus of a Braaks Stage I AD patient.
  • FIG. 4B shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the large cholinergic mterneurons in the caudate nucleus of a
  • FIG. 5A shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the reticular thalamic nucleus of a Braaks Stage 1 AD patient.
  • FIG. 5B shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the neurons of the reticular thalamic nucleus of a Braaks Stage 1 AD patient.
  • FIG. 6A shows the results of western blot analysis performed to detect acetylated 280 tau in the cerebrospinal fluid of a control patient (MEC-A-010, Lane 2) and a patient with AD (MEC-X-061 , Lane 3).
  • FIG. 6B shows the results of western blot analysis performed to detect acetylated 280 tau in the cerebrospinal fluid of a control patient (Lane 2) and a patient with AD (Lane 3) using a lighter stain than was used in the western blot analysis of FIG. 6A.
  • FIG. 7A shows Ponceau S staining of transferred proteins.
  • Lanes 1 - 8 are spinal fluid samples, showing dense protein staining in the 50-75kDa region, the expected location of acetylated tau.
  • Lane 9 shows the location of full-length tau peptide, having a molecular weight of 55-60kda.
  • Lane 10 is pre-stained molecular weight markers, with the position of the 50 kDa protein indicated.
  • FIGs. 7B-7D show a band for acetylated tau in the CSF of AD patients MECA 018 (FIG 7B), MEC-M 028 (FIG 7C), and MEC-B 038, MECA 004, and MEC-A-003 (FIG 7D).
  • FIG. 8 shows the detection of acetylated K280 tau in the CSF of AD patient.
  • a strong signal in the CSF sample from AD patient (MEC-X-061 ) was detected as compared with the CSF sample from non-AD patient (MEC-A-010).
  • FIG. 9 shows detection and quantitation of acetylated tau in the CSF patient (MEC-X-061 ) using the Singulex Erenna Immunoassay System.
  • Tau proteins are soluble proteins that stabilize microtubules and are important in axonal maintenance and axonal transport. Tau proteins are found mostly in neurons. There are six tau isoforms which are formed from alternative splicing in exons 2, 3, and 10 of the tau gene. These six isoforms are distinguished by their number of binding domains. Tau is a phosphoprotein with serine and threonine phosphorylation sites. Accordingly, tau proteins control microtubule stability through the different isoforms and phosphorylation.
  • Hyperphosphorylation of tau proteins result in the self-assembly of insoluble neurofibrillary or gliofibrillary tangles in the brain.
  • An abnormal accumulation of neurofibrillary tangles composed of paired helical filaments (PHFs) and straight filaments are found in tauopathy patients including those diagnosed with AD.
  • tau acetylation has been associated with the progression of AD.
  • Min (2010) reported that acetylation of tau prevents its degradation.
  • Min (2010) also showed that tau acetylation was elevated in patients at early and moderate Braak stages of tauopathy.
  • Min (2015) reported the identification of tau acetylation at K174 at an early change in AD brains and a critical determinant in tau homeostasis and toxicity in mice.
  • Min (2015) showed that the acetyl-mimicking mutant K174Q slows tau turnover and induced cognitive deficits in vivo.
  • Salsalate and salicylate have been shown to enhance tau turnover and reduce tau levels.
  • acetylated tau may play a critical role in AD.
  • the conventional method for diagnosing AD involves brain imaging studies using magnetic resonance imaging (MRI) or positron emission tomography (PET).
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • U.S. Publication No. 2013/0251731 describes methods of diagnosing tauopathies using tau acetylation as a biomarker for these diseases.
  • acetylation of the tau at least one of the following lysine residues has been suggested as a biomarker for certain tauopathies: K150, K163, ⁇ 74, K234, K240, K259, K274, K280, K281 , K290, K31 1 , K369, and K395.
  • acetylated K280 tau is specifically associated with insoluble tau aggregates.
  • tau K280 acetylation was only detected in diseased tissues, suggesting that acetylated K280 tau may have a role in causing insoluble tau accumulation.
  • acetylated K280 tau can be considered a specific biomarker for AD.
  • the presence of a biomarker in a biological sample can be detected by immunohistochemistry, immunocytochemistry, immunofluorescence,
  • the biological sample to be tested can be obtained from a subject suspected of having AD.
  • the biological sample can be a tissue sample or a sample of bodily fluid obtained from the subject.
  • acetylated tau was shown to be present in the different types of brainstem nuclei from Braaks Stage 1 AD patient (see Table I). Moreover, acetylated K280 tau was also shown to be present in the superior cerebellar peduncle and cerebral white matter from the Braaks Stage 1 AD (see FIG. 1 B and FIG. 2B). Further, acetylated K280 tau was shown to be present in the putamen, caudate nucleus, and reticular thalamic nucleus from the Braaks Stage 1 AD patient (FIG. 3A to FIG. 5B).
  • the present disclosure describes the use of acetylated tau as a biomarker for diagnosing AD.
  • the detection of the presence of acetylated tau in various post mortem CNS tissues of subjects indicates the subject has AD.
  • CNS tissues that can be tested for diagnosing the subject include brainstem, superior cerebellar peduncle, cerebral white matter, cerebral grey matter, putamen, caudate nucleus, and reticular thalamic nucleus, as well as all subthalamic nuclei.
  • the acetylated tau is acetylated K280 tau.
  • the probe for detecting the biomarker can be an antibody, such as acetylated K280 tau specific antibody.
  • the antibody can be obtained commercially or by methods known in the art for preparing antibodies.
  • acetylated tau was shown to be present in the cerebral spinal fluid (CSF) of AD patients. It was also shown that the amount of acetylated tau in CSF of AD patients is very low, in the sub-nanogram to picogram range which makes detection using conventional methods, such as western blotting, very difficult. Therefore, there is a need to develop a new method for detecting acetylated tau.
  • CSF cerebral spinal fluid
  • acetylated tau When the amount of acetylated tau is low in a sample, as in a CSF sample, techniques including concentrating the sample, increasing the sample volume to run on a gel (for western blot or other immunoassay methods for detection), and collecting a larger volume of sample, could be used to enhance detection of acetylated tau.
  • the sample can be concentrated at least about four times, about five times, about six time, about seven times, about eight times, about nine times, about ten times, about 20 times, about 50 times, or about 100 times.
  • the sample also can be concentrated to dryness and reconstituted to the desired volume for detection by various methods.
  • the present disclosure describes a novel method for detecting acetylated tau in the bodily fluids of subjects which includes a combination of the above steps and others for diagnosing AD in such subjects.
  • the present disclosure describes a method of diagnosing AD using bodily fluids from subjects.
  • the method includes detecting the presence of acetylated tau in bodily fluids of subjects.
  • Bodily fluids include CSF, urine, semen, saliva, sweat, whole blood, plasma, serum, bile, lymph, tears, and pleural fluid.
  • the method includes detecting the presence of acetylated tau in the CSF of a subject for diagnosing AD in the subject.
  • the acetylated tau is acetylated K-280 tau.
  • Cerebrospinal fluid is a clear, colorless body fluid in the brain and spinal cord. It is produced by the choroid plexuses of the ventricles of the brain, absorbed in the arachnoid granulations, and circulates the subarachnoid space around the brain and spinal cord. It serves as a cushion for the brain within the skull and act as a shock absorber for the central nervous system. The CSF also circulates nutrients and chemicals filtered from the blood and removes waste products form the brain.
  • CSF can be obtained from a subject via a lumbar puncture (spinal tap) of a subject.
  • a lumbar puncture involves inserting a long thin hollow needle between two bones in the lower spine and into the space where the CSF circulates and withdrawing CSF using the syringe.
  • the bodily fluid includes exosomes.
  • Exosomes are cell-derived vesicles that can be isolated from many biological fluids. Exosomes have been shown to be involved in specialized functions, including intracellular signaling.
  • the method described herein includes isolating exosomes from bodily fluids for detecting the presence of acetylated tau.
  • the method includes isolating exosomes, for example from serum or CSF, and detecting the presence of acetylated tau.
  • the method detects the presence of acetylated K280 tau in serum exosomes and CSF exosomes.
  • the method for diagnosing AD includes an immunoassay.
  • an immunoassay the presence of a molecule (analyte) in a sample solution is detected and measured via a biochemical reaction using a probe that binds the analyte.
  • Immunoassays can be run in multiple steps with reagents being added and washed away or separated at different points in the assay. Immunoassays can be performed by mixing reagents and sample and making a measurement, such as a homogeneous immunoassay, and
  • immunoassays can involve multiple steps such as heterogeneous or separation immunoassay.
  • the analyte is the acetylated tau and the probe is the antibody that binds the acetylated tau.
  • the acetylated tau is acetylated K280 tau and the antibody is the acetylated K280 tau specific antibody.
  • Calibrators are often used in an immunoassay.
  • the calibrator contains the analyte in question in a known amount, so that the signal strength of the analyte in the sample and in the calibrator can be compared to for determining the presence and concentration of the analyte in the sample.
  • Immunoassays use various labels to allow for detection of antibody binding to analyte.
  • labels include enzymes (enzyme-linked immunosorbent assays (ELISA)), radioactive isotopes (radioimmunoassay (RIA)), fluorogenic reporters (used in protein microarrays), and electrochemiluminescent tags (light emits in response to electric current).
  • enzymes enzyme-linked immunosorbent assays (ELISA)
  • radioactive isotopes radioactive isotopes
  • RIA radioactive isotopes
  • fluorogenic reporters used in protein microarrays
  • electrochemiluminescent tags light emits in response to electric current.
  • Immunoassays include ELISA, RIA, fluorescence immunoassay (FIA) including time-resolved FIA, dot blot, slot blot, western blot, immunoprecipitation, enzyme immunoassay (EIA), immunohistostaining, immunochromatography, chemiluminescent immunoassay, surface plasmon resonance immunoassay, and other known
  • the method of diagnosing AD described herein also includes using
  • immunoassay system Some of these systems provide a kit for ease of testing. Many have enhanced sensitivity for detecting and quantitating very low amounts of analyte in a sample, for example in the picogram range. Examples of some companies that provide such immunoassay platforms include Siemens Healthcare Diagnostic, Randox Laboratories, Abbott Diagnostics, Quanterix Corporation, and EMD Millipore.
  • An example of an immunoassay system is the Singulex Erenna ⁇ immunoassay system owned by EMD Millipore.
  • the Erenna® is a bench-top analytical instrument that utilizes capillary flow, laser-induced fluorescence, and a highly sensitive detection optics module to achieve single molecule counting (SMC), Thus, it can detect and quantitate analyte in the lower analytical measurement range.
  • Immunoassay reagents are supplied by Singulex in a kit format.
  • Paramagnetic niicroparticles (MPs) are used as the solid phase for immune-capture and detection of analytes in a microplate format. Signal generated by fluorescently labeled detection molecules are counted as digital events, which corresponds to a single analyte molecule.
  • Data is analyzed with the Erenna ⁇ software or exported for analysis with standard quantitative ELISA curve fitting software.
  • the method of diagnosing AD described herein includes detecting and quantitating acetylated tau in a subject suspected of having AD.
  • the method includes comparing the results obtained from the subject with the results of a control or healthy subject, that is a subject that does not have AD, for determining whether acetylated tau is present and in what quantity in the subject suspected of having AD.
  • the presence of acetyl tau in a subject indicates that the subject has AD.
  • the amount of the acetyl tau can provide some information as to what stage of AD, mild (early), moderate (middle), or severe (advanced).
  • tau and “tau protein” are used interchangeably in this disclosure to refer to the “tau protein.”
  • acetylated tau and acetylated tau protein are used interchangeably to refer to the “acetylated tau protein,”
  • Methods disclosed herein include diagnosing subjects in need of being diagnosed for AD.
  • Subjects include mammals, for example human, mouse, dog, horse, pig, etc.
  • Subjects in need of diagnosing (in need thereof) are subjects suspected of having AD.
  • Such subjects include human patients diagnosed with mild cognitive impairment (MCI), dementia, cognitive disability in ages over 35, and/or
  • each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component.
  • the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.”
  • the transition term “comprise” or “comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts.
  • the transitional phrase “consisting of” excludes any element, step, ingredient or component not specified.
  • the transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment.
  • the term "about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ⁇ 20% of the stated value; ⁇ 19% of the stated value; ⁇ 18% of the stated value; ⁇ 17% of the stated value; ⁇ 16% of the stated value; ⁇ 15% of the stated value; ⁇ 14% of the stated value; ⁇ 13% of the stated value; ⁇ 12% of the stated value; ⁇ 1 1 % of the stated value; ⁇ 10% of the stated value; ⁇ 9% of the stated value; ⁇ 8% of the stated value; ⁇ 7% of the stated value; ⁇ 6% of the stated value; ⁇ 5% of the stated value; ⁇ 4% of the stated value; ⁇ 3% of the stated value; ⁇ 2% of the stated value; or ⁇ 1 % of the stated value.
  • a method of diagnosing Alzheimer's disease comprising obtaining a sample of bodily fluid from a subject suspected of having AD and detecting acetylated tau in the sample, thereby diagnosing the subject as having AD.
  • the bodily fluid is cerebral spinal fluid (CSF), whole blood, plasma, serum, bile, lymph, mucus, pleural fluid, semen, saliva, sweat, tears, or urine.
  • CSF cerebral spinal fluid
  • any one of embodiments 1 -9 wherein the method further comprises prior to detection of the acetylated tau in the sample, concentrating the sample at least four times, at least five times, at least six time, at least seven times, at least eight times, at least nine times, at least ten times, at least 20 times, at least 50 times, or at least 100 times.
  • a method of diagnosing AD comprising obtaining a CNS tissue from a subject suspected of having AD and detecting the presence of acetylated tau in the CNS tissue of the subject.
  • CNS tissue is brainstem, superior cerebellar peduncle white matter, cerebral white matter, putamen, caudate nucleus, or reticular thalamic nucleus.
  • acetylated tau is acetylated K280 tau. 19. The method of one of embodiments 16-18, wherein the method comprises performing immunohistochemistry, immunocytochemistry, or immunofluorescence.
  • Example 1 Immunohistochemical Staining of Acetylated K280 tau in the CNS of AD Patients.
  • the brainstem nuclei from a Braaks Stage 1 AD patient were tested for presence of acetylated K280 tau.
  • Table I shows semi-quantitative data (from +: mild to +++: severe staining) obtained from immunohistochemical staining using an anti- acetylated K280 tau antibody. The data indicate that acetylated K280 tau is present in all of the AD patient brainstem nuclei tested.
  • the subcortical nuclei putamen, caudate nucleus and reticular thalamic nucleus from a Braaks Stage 1 AD were tested for presence of acetylated K280 tau. As shown in FIG. 3A and FIG. 3B; FIG. 4A and FIG. 4B; and FIG. 5A and FIG. 5B, respectively, the putamen, caudate nucleus and reticular thalamic nucleus from the Braaks Stage 1 AD patient show distinct staining which indicates the presence of acetylated K280 tau.
  • Example 2 Acetylated Tau Screening of CSF Samples of Patients.
  • Exosome samples were denatured in SDS-PAGE sample buffer, heated 70°C 10 min and centrifuged 30s 10,000 xg.
  • the blots were incubated with chemiluminescent substrate for 5 min according to the manufacturer's protocol.
  • Proteins from the CSF samples were concentrated followed by western blotting for acetylated tau.
  • sample buffer heated 70°C 10 min and centrifuged 30s 10,000 xg.
  • Prestained molecular weight markers SeeBlue, Invitrogen were run in parallel to monitor the progress of electrophoresis as well as transfer.
  • Transfer was performed in 1x Transfer Buffer (Invitrogen) containing 10% methanol for 60 min at 35V.
  • the blots were incubated with chemiluminescent substrate for 5 min according to the manufacturer's protocol.
  • the AD patient sample (Lane 3) shows a faint but discernable band at the molecular weight expected for acetvlated K280 Tau when probed using the anti-tau antibody (Acetyl K280), Rabbit Polyclonal available from AnaSpec (Catalog #AS-56077).
  • the molecular weight of this protein is between 55,000- 60,000 Daltons, which is the expected molecular weight range for tau family.
  • the control experiment does not show such a band (FIG. 6A and FIG. 6B, Lane 2).
  • putative acetylated K280 tau has been identified in a concentrated cerebrospinal fluid sample from an Alzheimer's patient by western blot analysis.
  • Concentration of the CSF samples enabled the detection of acetylated tau by western blot analysis, using acetyl-K280-specific polyclonal antibody.
  • Example 3 Analysis of Concentrated Spinal Fluid Samples for Acetylated Tau.
  • CSF samples were collected from several AD patients.
  • the CSF samples were concentrated.
  • Western blotting was performed for acetylated K280 tau.
  • Prestained molecular weight markers SeeBlue, Invitrogen were run in parallel to monitor the progress of electrophoresis as well as transfer.
  • Transfer was performed in 1x Transfer Buffer (Invitrogen) containing 10% methanol for 60 min at 35V.
  • the blots were incubated with chemiluminescent substrate for 5 min according to the manufacturer's protocol.
  • FIG. 7A shows Ponceau S staining of transferred proteins.
  • Lanes 1 - 8 are spinal fluid samples, showing dense protein staining in the 50-75kDa region, the expected location of acetylated tau.
  • Lane 9 shows the location of full-length tau peptide, having a molecular weight of 55-60kda.
  • Lane 10 is pre-stained molecular weight markers,, with the position of the 50 kDa protein indicated.
  • acetylated tau has been identified in concentrated patient spinal fluid samples by western blot analysis.
  • Evidence in support of this statement includes an acetyl-tau immunoreactive band migrating parallel to full-length tau peptide, in the approximate molecular weight range of 50-60kDa.
  • the predicted molecular weight of tau isoforms reported in the literature is 45-65kda. It needs to be pointed out that the abundance of tau/acetyl-tau in spinal fluid is very, very low, in the nanogram to picogram amounts in the different samples which makes detection by western blotting challenging.
  • the limit for detection of this and other immunoreactive methods is generally in the microgram to high nanogram levels of protein.
  • the results of this Example support the idea that acetylated tau can be detected immunoreactively in the CSF of AD patients.
  • 1 Further concentrate the spinal fluid samples. In this study, the capability was limited to concentrating the sample 4-fold (from 1000uL to 250uL). Additional technologies exist to concentrate the sample to dryness, then reconstitute to a desired volume for gel electrophoresis; 2. Increase sample volume run on the gel. In this Example, capability was limited to 60uL of sample.
  • AD patients' spinal fluid samples were concentrated.
  • Western blot analysis was performed to detect acetylated tau, using acetyl-K280-specific polyclonal antibody.
  • a protein band was detected in two of these samples tested, and a possible band detected in an additional 3 samples (FIGs. 7B, 7C, and 7D).
  • This band had an approximate molecular weight of 55,000-60,000 Daltons, which is the expected molecular weight range for tau family members.
  • Full-length tau peptide was run as a molecular weight control, and the immunoreactive band was found to run at the same position.
  • This Example confirms that acetylated K280 tau can be detected in the CSF of AD patients.
  • Dot Blot 1 . Load acetylated tau (acTau) standards onto nitrocellulose membrane at 100, 50, 10, 5, and 1 ng tau/dot in duplicate (reaction products diluted in TBS) in 200 ⁇ _ volume using dot blot concentrator
  • FIG 8 shows a strong signal in the CSF sample from AD patient (MEC-X-061 ) as compared with the CSF sample from non-AD patient (MEC- A-010).
  • Example 5 Detecting Acetylated Tau in the CSF of AD Patient using the Singulex Erenna® Platform.
  • FIG. 9 shows acetylated K280 tau detected in the CSF of advanced AD patient.
  • the dot blot sensitivity with the Erenna® Immunoassay system is about 1 ng.
  • the predicated concentration of acetylated tau in the CSF of the AD Patient is about 10 pg/mL.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Neurology (AREA)
  • Food Science & Technology (AREA)
  • Neurosurgery (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La protéine tau acétylée est détectée dans les échantillons de tissu et l'échantillon de liquides corporels de patients atteints de la maladie d'Alzheimer (MA). Des méthodes de diagnostic de la maladie d'Alzheimer chez des sujets ont été mises au point à l'aide de la protéine tau acétylée en tant que biomarqueur.
PCT/US2017/065060 2016-12-09 2017-12-07 Diagnostic de la maladie d'alzheimer WO2018106889A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201662432295P 2016-12-09 2016-12-09
US62/432,295 2016-12-09
US201762447167P 2017-01-17 2017-01-17
US62/447,167 2017-01-17

Publications (1)

Publication Number Publication Date
WO2018106889A1 true WO2018106889A1 (fr) 2018-06-14

Family

ID=62491314

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2017/065060 WO2018106889A1 (fr) 2016-12-09 2017-12-07 Diagnostic de la maladie d'alzheimer

Country Status (1)

Country Link
WO (1) WO2018106889A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112946300A (zh) * 2021-03-19 2021-06-11 苏冬梅 基于“Western Blot”法的阿尔茨海默病早期检测试剂盒及其制备方法
WO2021142128A1 (fr) * 2020-01-07 2021-07-15 University Hospitals Cleveland Medical Center Biomarqueur et cible de neurodégénérescence pouvant être traitée par médicament
EP4001305A4 (fr) * 2019-07-15 2022-10-12 Adel Inc. Anticorps anti-tau et son utilisation
WO2023039456A1 (fr) * 2021-09-09 2023-03-16 The University Of North Carolina At Chapel Hill Anticorps monoclonaux ciblant la proteine tau acétylée et leurs procédés d'utilisation

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5492812A (en) * 1991-08-01 1996-02-20 Provost, Fellows And Scholars Of Trinity College Diagnostic method for Alzheimer's disease by screening for tau-peptides in the blood of a patient
US20100316564A1 (en) * 2009-06-10 2010-12-16 New York University Immunological targeting of pathological tau proteins
WO2011056300A1 (fr) * 2009-11-06 2011-05-12 The J. David Gladstone Institutes Procédés et compositions pour moduler les taux de tau
US20130251731A1 (en) * 2012-03-22 2013-09-26 The Trustees Of The University Of Pennsylvania Tau Acetylation in the Pathogenesis of Alzheimers Disease and Other Related Tauopathies

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5492812A (en) * 1991-08-01 1996-02-20 Provost, Fellows And Scholars Of Trinity College Diagnostic method for Alzheimer's disease by screening for tau-peptides in the blood of a patient
US20100316564A1 (en) * 2009-06-10 2010-12-16 New York University Immunological targeting of pathological tau proteins
WO2011056300A1 (fr) * 2009-11-06 2011-05-12 The J. David Gladstone Institutes Procédés et compositions pour moduler les taux de tau
US20130251731A1 (en) * 2012-03-22 2013-09-26 The Trustees Of The University Of Pennsylvania Tau Acetylation in the Pathogenesis of Alzheimers Disease and Other Related Tauopathies

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SAMAN ET AL.: "Exosome-associated Tau Is Secreted in Tauopathy Models and Is Selectively Phosphorylated in Cerebrospinal Fluid in Early Alzheimer Disease", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 287, 3 February 2012 (2012-02-03), pages 3842 - 3849, XP055089563 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4001305A4 (fr) * 2019-07-15 2022-10-12 Adel Inc. Anticorps anti-tau et son utilisation
GB2600599B (en) * 2019-07-15 2025-05-21 Adel Inc Anti-Tau antibody and use thereof
WO2021142128A1 (fr) * 2020-01-07 2021-07-15 University Hospitals Cleveland Medical Center Biomarqueur et cible de neurodégénérescence pouvant être traitée par médicament
EP4087610A4 (fr) * 2020-01-07 2024-06-19 University Hospitals Cleveland Medical Center Biomarqueur et cible de neurodégénérescence pouvant être traitée par médicament
CN112946300A (zh) * 2021-03-19 2021-06-11 苏冬梅 基于“Western Blot”法的阿尔茨海默病早期检测试剂盒及其制备方法
WO2023039456A1 (fr) * 2021-09-09 2023-03-16 The University Of North Carolina At Chapel Hill Anticorps monoclonaux ciblant la proteine tau acétylée et leurs procédés d'utilisation

Similar Documents

Publication Publication Date Title
Kasai et al. Increased TDP-43 protein in cerebrospinal fluid of patients with amyotrophic lateral sclerosis
US20160161508A1 (en) Method for diagnosing neuro-degenerative disease
Roher et al. Proteomics-derived cerebrospinal fluid markers of autopsy-confirmed Alzheimer’s disease
WO2018106889A1 (fr) Diagnostic de la maladie d'alzheimer
JP7492711B2 (ja) アルツハイマー病バイオマーカー
Montero-Calle et al. The molecular misreading of APP and UBB induces a humoral immune response in Alzheimer’s disease patients with diagnostic ability
Grant et al. Human cerebrospinal fluid 6E10-immunoreactive protein species contain amyloid precursor protein fragments
WO2018030252A1 (fr) Procédé d'aide au diagnostic de la maladie d'alzheimer mettant en œuvre un biomarqueur de l'urine
Nakajima et al. Protein tyrosine phosphatase receptor type Q in cerebrospinal fluid reflects ependymal cell dysfunction and is a potential biomarker for adult chronic hydrocephalus
CN104487847B (zh) 与阿尔茨海默氏病和轻度认知障碍相关的原肌球蛋白同种型
US20150111777A1 (en) Biomarker for early neurodegeneration detection
WO2023068173A1 (fr) BIOMARQUEUR POUR ÉVALUER L'ACCUMULATION D'AMYLOÏDE-β INTRACÉRÉBRALE
US20030119074A1 (en) Diagnosis and treatment of dementia utilizing thrombospondin
Gatto et al. Microscopy assessment of a fluorescence [18F] flortaucipir analog (T726) shows neuropathological overlap with 3R and 4R tau lesions
US20220308073A1 (en) Biomarker for alzheimer's disease
JP2025073338A (ja) アルツハイマー病罹患者の認知機能障害の進行リスク又は発症リスクの検査方法、及びアルツハイマー病の検査方法
Khan et al. Fluid Markers of Tau Pathology
Liu et al. A cytometric bead array for the measurement of plasma biomarker levels in patients with Alzheimer’s

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17877514

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17877514

Country of ref document: EP

Kind code of ref document: A1