WO2014011972A1 - Immunodosage de tau - Google Patents

Immunodosage de tau Download PDF

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Publication number
WO2014011972A1
WO2014011972A1 PCT/US2013/050242 US2013050242W WO2014011972A1 WO 2014011972 A1 WO2014011972 A1 WO 2014011972A1 US 2013050242 W US2013050242 W US 2013050242W WO 2014011972 A1 WO2014011972 A1 WO 2014011972A1
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tau
antibody
csf
level
protein
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PCT/US2013/050242
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English (en)
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Sethu SANKARANARAYANAN
Jere E. Meredith
Valerie Lee GUSS
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Bristol-Myers Squibb Company
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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
    • 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

  • AD Alzheimer's disease
  • ⁇ amyloid beta
  • NFTs intra-neuronal neurofibrillary tangles
  • NFT pathology is observed in tauopathies including frontotemporal dementia with Parkinson's disease with Tau mutations (FTDP-17), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Picks disease (Lee et al, 2001, Annu Rev Neuroscl, 24: 1 121-59).
  • Tau is a microtubule-associated protein expressed in the central nervous system with a primary function to stabilize microtubules.
  • insoluble Tau from the AD brains and brains from other tauopathies demonstrate varying levels of phosphorylation, including the existence of 3 -repeat and 4-repeat Tau isoforms and varying degrees of Tau cleavages.
  • the present invention fulfills a need in the art by providing alternative methods useful for identifying and quantitating a Tau protein.
  • the present invention provides a method of detecting the level of at least one Tau protein in a biological sample, comprising: (a) contacting a biological sample from a subject with a first antibody which binds to an N-terminal portion of Tau 441 (SEQ ID NO: 1), and a second antibody which binds to a middle portion of Tau 441 (SEQ ID NO: 1), wherein the first antibody and the second antibody both bind to the Tau protein to form a complex; and (b) detecting the level of the Tau protein in the complex.
  • the first antibody binds to an N- terminal portion of amino acid residues 9-158 of Tau 441 (SEQ ID NO: 1).
  • the first antibody binds to amino acid residues 9-18 of SEQ ID NO: 1.
  • the second antibody binds to a middle portion of amino acid residues 159-231 of Tau 441 (SEQ ID NO: 1).
  • the second antibody binds to amino acid residues 194-198, residues 159-163, the phosphorylated threonine residue 181 or the phosphorylated threonine residue 231 of SEQ ID NO: 1.
  • the first antibody is Taul2, and the second antibody is selected from BT2, HT7, Tau5, AT270, and PHF6.
  • the first antibody or the second antibody of the present methods is immobilized on a solid substrate.
  • the first antibody or the second antibody comprises a label.
  • the biological sample is a sample.
  • the subject has a Tau- related neurological disease, such as Alzheimer's disease.
  • the present invention provides a method of detecting the level of at least one Tau protein in a biological sample, comprising: (a) contacting a biological sample from a subject with a first antibody which binds to amino acid residues 159-163 of Tau 441 (SEQ ID NO: 1), and a second antibody which binds to amino acid residues 194-198, residues 218-225, or the phosphorylated threonine residue 231 of Tau 441 (SEQ ID NO: 1), wherein the first antibody and the second antibody both bind to the Tau protein to form a complex; and (b) detecting the level of the Tau protein in the complex.
  • a biological sample from a subject with a first antibody which binds to amino acid residues 159-163 of Tau 441 (SEQ ID NO: 1), and a second antibody which binds to amino acid residues 194-198, residues 218-225, or the phosphorylated threonine residue 231 of Tau 441 (SEQ ID NO: 1), wherein
  • the first antibody is HT7
  • the second antibody is selected from BT2, Tau5, and PHF6.
  • the first antibody or the second antibody of the present methods is immobilized on a solid substrate.
  • the first antibody or the second antibody comprises a label.
  • the biological sample is cerebrospinal fluid (CSF), blood, serum or plasma.
  • the subject has a Tau-related neurological disease, such as Alzheimer's disease.
  • the above-mentioned methods use a second antibody which binds to a phosphorylated threonine residue (e.g., at position 181 or 231). In such cases, the level of the phosphorylated Tau protein is detected by the present methods.
  • the present invention provides a method of identifying a subject having or at risk of having a Tau-related neurological disease, comprising: (a) isolating a biological sample from the subject; (b) detecting the level of the Tau protein in the biological sample according to any of the above-mentioned methods; and (c) comparing the level of the Tau protein to a reference; and (d) if the level of the Tau protein is increased compared to the reference, identifying the subject to have or at risk of having a Tau-related neurological disease.
  • the Tau- related neurological disease is Alzheimer's disease.
  • the present invention provides a method of identifying a subject having or at risk of having a Tau-related neurological disease, comprising: (a) isolating a biological sample from the subject; (b) detecting a first level of the Tau protein in the biological sample using HT7 as the first antibody and BT2 as the second antibody; (c) detecting a second level of the Tau protein in the biological sample using HT7 as the first antibody and Tau5 as the second antibody; (d) determining the ratio of the first level from (b) versus the second level from (c); (e) comparing the ratio to a reference; and (f) if the ratio is decreased compared to the reference, identifying the subject to have or at risk of having a Tau-related neurological disease.
  • the Tau-related neurological disease is
  • the present invention provides a method of identifying a subject having or at risk of having a Tau-related neurological disease, comprising: (a) isolating a biological sample from the subject; (b) detecting a first level of the Tau protein in the biological sample using HT7 as the first antibody and BT2 as the second antibody is BT2; (c) detecting a second level of the Tau protein in the biological sample using Taul2 as the first antibody is Taul2 and HT7 as the second antibody; (d) determining the ratio of the first level from (b) versus the second level from (c); (e) comparing the ratio to a reference; and (f) if the ratio is decreased compared to the reference, identifying the subject to have or at risk of having a Tau- related neurological disease.
  • the Tau-related neurological disease is Alzheimer's disease.
  • the present invention provides a kit comprising: (1) a first antibody which binds to an N-terminal portion of a Tau protein; (2) a second antibody which binds to a middle portion of a Tau protein; and (3) reagents necessary for facilitating an antibody-antigen complex formation.
  • the first antibody is Taul2
  • the second antibody is selected from BT2, HT7, Tau5, AT270, and PHF6.
  • the present invention provides a kit comprising: (1) a first antibody which binds to amino acid residues 159-163 of Tau 441 (SEQ ID NO: 1); (2) a second antibody which binds to amino acid residues 194-198, residues 218- 225, or the phosphorylated threonine residue 231 of Tau 441 (SEQ ID NO: 1); and (3) reagents necessary for facilitating an antibody-antigen complex formation.
  • the first antibody is HT7
  • the second antibody is selected from BT2, Tau5, and PHF6.
  • Figure 1 shows detection of Tau fragments in human CSF.
  • Human control and AD CSF subjected to RP-HPLC, fractions collected and run on SDS-PAGE gels followed by western-blotting with different Tau antibodies.
  • human recombinant Tau441 (Tau) is included in lane 1 and molecular weight markers (mw) in lane 2 followed by the HPLC fractions from 1 to 6 or HPLC fractions 7 to 1 1.
  • Fractions 1 and 2 were pooled and run as a single sample, while fractions 3-10 were run as individual samples.
  • Control CSF (C) and AD CSF (D) samples for each fraction were run side by side for comparison.
  • FIG. 2 shows Tau and pTau ELISAs.
  • Antibody combinations used for the different Tau and pTau ELISAs are shown. For each assay, capture antibodies are highlighted in red, detection antibodies in black and the minimal Tau region required (aa numbering based on Tau 441) is indicated.
  • Antibodies used in the INNOTEST/INNO-BIA AlzBio3 total Tau and pl81 Tau assays are also shown for comparison.
  • FIG. 3 shows characterization of Tau ELISAs.
  • Tau 441 calibrators (Standards) and results for CSF sample dilutions (Samples) are shown.
  • the assay lower limit of quantitation (LLQ, vertical dashed line) is also indicated.
  • dilution- corrected Tau levels for a pooled control CSF sample and a pooled AD CSF sample relative to sample dilution are shown.
  • the vertical dashed lines indicate the dilution determined to be optimal for CSF analysis.
  • Figure 4 shows characterization of HT7+77G7 Tau ELISA. Representative Tau 441 standard curve (left panel) and CSF dilution linearity results (right panel) shown. On the standard curve graph, Tau 441 calibrators (Standards) and results for CSF sample dilutions (Samples) are shown. The assay lower limit of quantitation (LLQ, vertical dashed line) is also indicated. On the dilution linearity graph, Tau levels for a pooled control and pooled AD CSF samples tested with or without a 100 pg/ml Tau 441 spike are shown.
  • Figure 5 shows characterization of pTau assays. Representative pTau standard curves (left panels) and CSF dilution linearity results (right panels) shown for: A) HT7-AT270, B) HT7-PHF6, and C) Taul2-AT270 pTau ELISAs. On each standard curve graph, pTau calibrators (Standards) and results for CSF sample dilutions (Samples) are shown. The assay lower limit of quantitation (LLQ, vertical dashed line) is also indicated. On each dilution linearity graph, dilution-corrected pTau levels for a pooled control and a pooled AD CSF samples relative to sample dilution are shown. The vertical dashed lines indicate the dilution determined to be optimal for CSF analysis.
  • Figure 6 shows Tau and pTau levels in 20 AD and 20 control CSF samples.
  • a set of 20 AD and 20 age-matched normal control CSF samples were analyzed using the Tau ELISAs (HT7-BT2, HT7-Tau5, Taul2-BT2, Taul2-HT7 and HT7-77G7) and pTau ELISAs (HT7-AT270, HT7-PHF6 and Taul2-AT270). Dashed lines indicate the assay LLQ corrected for CSF dilution.
  • Figure 7 shows Tau peptides used for epitope mapping.
  • a set of 29 overlapping peptides spanning the length of human Tau 441 were generated, coupled to beads and used to map the epitope of Tau antibody 77G7 using a Luminex-based multiplex assay.
  • Figure 8 shows mapping the Tau epitope of antibody 77G7.
  • 77G7 exhibited binding to human Tau 441 and the C-terminal human Tau fragment aa 231- 441 but not to the Tau fragments aa 1-125 or aa 126-230;
  • 77G7 also bound to the other Tau isoforms (data not shown).
  • Mid-domain Tau antibody HT7 exhibited binding to Tau 441 and fragment aa 126-230 as expected.
  • FIG. 9 shows verification of signal specificity in Tau ELISAs by immunodepletion.
  • Pooled CSF was immunodepleted with Tau antibody HT7 (IP) or treated with protein A/G beads alone (control).
  • Samples were analyzed in Tau ELISAs A) HT7-BT2, B) HT7-Tau5, C) Taul2-BT2 and D) Taul2-HT7.
  • Data represents mean ⁇ SEM from 3-4 determinations. Dashed lines indicate the assay LLQ corrected for CSF dilution.
  • FIG. 10 shows spike recovery in Tau ELISAs. Pooled CSF samples were treated with Tau 441 spikes ranging from 10 - 800 pg/ml. Spiked samples and a matching untreated control were analyzed in Tau ELISAs. A) HT7-BT2, B) HT7- Tau5, C) Taul2-BT2 and D) Taul2-HT7 and spike recovery determined (%). Data represents mean ⁇ SEM from 3 determinations. Dashed lines indicate 100% spike recovery.
  • FIG 11 shows verification of signal specificity in pTau ELISAs by immunodepletion and peptide competition.
  • Pooled CSF samples from healthy control subjects (black bars) or AD patients (red bars) were immunodepleted with Tau antibody HT7 (IP) or protein A/G beads alone (control and AD).
  • CSF samples were also treated with pT 181 or pT231 peptides for competition analysis.
  • Samples were analyzed in pTau ELISAs A) HT7-AT270, B) HT7-PHF6, and C) Taul2-AT270. Data represents mean ⁇ SEM from 3 determinations. Dashed lines indicate the assay LLQ corrected for CSF dilution.
  • FIG. 12 shows spike recovery in pTau ELISAs.
  • Pooled CSF samples were treated with pT181 or pT231 spikes ranging from 12.5 - 200 pg/ml. Spiked samples and a matching untreated controls were analyzed in pTau ELISAs.
  • Figure 13 shows analysis of Tau and pTau levels in 20 AD and 20 control CSF samples.
  • a set of 20 AD and 20 age-matched normal control CSF samples were analyzed using ⁇ ⁇ - ⁇ AlzBio3.
  • the present invention relates to novel methods of identifying the level of at least a Tau protein in a biological sample (e.g., CSF) from a human subject.
  • a biological sample e.g., CSF
  • the present invention provides novel methods of identifying a subject having or at risk of having a Tau-related neurological disease (e.g., AD).
  • AD Tau-related neurological disease
  • Tau fragments have been detected in CSF primarily by using western blotting and ELISA assays.
  • One study showed that there was no intact Tau and that the majority of the Tau fragments contained the amino (N)-terminus in human CSF samples (Johnson et al 1997).
  • Another report showed evidence of multiple bands in the 20-40 Kd range by western blotting using HT7, a mid-domain Tau antibody (Hanisch et al 2010).
  • Another report demonstrated that carboxy (C)-terminal Tau fragments are evident in CSF from traumatic brain injury patients but not in the normal controls (Zemlan et al 1999).
  • a proteomic analysis of Tau in CSF from an AD patient using immunoprecipitation with mass-spectrometry demonstrated that the majority of Tau sequence detected is in the N-terminal half of the molecule (Portelius et al 2008). Alterations in cleaved N-terminal Tau fragments were evident in brain extracts from PSP distinct from those in CBD samples. In this study, Applicants used reverse-phase high performance liquid chromatography to enrich and concentrate Tau prior to western-blot analysis. Multiple N-terminal and mid-domain fragments of Tau were detected in pooled CSF with apparent sizes ranging from ⁇ 20 kDa to ⁇ 40 kDa. The pattern of Tau fragments in AD and control samples were similar.
  • CSF Tau data uses the total Tau assays in commercially available kits such as the Luminex-based AlzBio3 triplex (Olsson et al 2005) and Innotest enzyme linked immunosorbant assays (ELISAs) (Hulstaert et al 1999, Vanderstichele et al 2006).
  • the antibodies used in these assays have epitopes localized to a mid-domain region of the Tau protein and, thus, are unable to detect the presence or alterations in the amino (N)- and carboxy (C)-terminal regions of the Tau protein.
  • CSF phospho-Tau Similar to the total Tau assays, the majority of the reported studies on CSF phospho-Tau utilize the pT181 assay in the Innotest ELISA kit and the Luminex- based AlzBio3 triplex kit. In addition to CSF pT181 Tau, levels of pT231 Tau show a robust increase in AD subjects compared to age matched controls (Kohnken et al 2000). CSF pT231 Tau is also significantly elevated in AD compared to other neurodegenerative diseases (Buerger et al 2002). The CSF pT231 assay uses a combination of CP27 and Taul, mid-domain Tau antibodies as capture antibodies, and CP9, a pT231 specific antibody for detection (Kohnken et al 2000).
  • the existing Tau assays utilize antibodies which bind to a mid-domain region of the Tau protein, these assays may not be able to detect certain Tau fragments (e.g., N-terminal or C-terminal fragments).
  • Applicants have developed novel assays to investigate CSF Tau and pTau as biomarkers for neurodegenerative diseases (e.g., AD). For example, five Tau ELISAs and three pTau ELISAs were developed to detect and quantitate different overlapping regions of the Tau protein. The discriminatory potential of each assay was determined using 20 AD and 20 age-matched control CSF samples.
  • ⁇ refers to amyloid beta
  • ⁇ 4 2 refers to Amyloid Beta 1-42.
  • Tau 441 refers to the native full-length Tau protein corresponding to the following amino acid sequence:
  • Tau protein and “Tau polypeptide” are used interchangeably, and refer to any of the six non-cleaved isoforms of the Tau protein that have a molecular weight in the range of 48 to 68 kDa.
  • Tau protein or “Tau polypeptide”, as used herein, includes a Tau fragment, a Tau variant, and a modified form of human Tau protein (such as a phosphorylated Tau protein).
  • Tau fragment refers to a Tau protein with a reduced molecular weight compared to the full-length Tau protein and can be comprised of any interior portion of the full-length Tau protein (e.g., the N-terminal portion, the C- terminal portion, and/or the middle portion).
  • Exemplary Tau fragments include a Tau fragment comprising at least residues 9-163 of SEQ ID NO: 1, a Tau fragment comprising at least residues 9-181 of SEQ ID NO: 1, a Tau fragment comprising at least residues 9-198 of SEQ ID NO: 1, a Tau fragment comprising at least residues 159-181 of SEQ ID NO: 1, a Tau fragment comprising at least residues 159-198 of SEQ ID NO: 1, a Tau fragment comprising at least residues 159-225 of SEQ ID NO: 1, and a Tau fragment comprising at least residues 159-231 of SEQ ID NO: 1.
  • pT181 refers to a Tau protein, polypeptide or fragment that is phosphorylated at the threonine residue at amino acid position 181.
  • pT231 refers to a Tau protein, polypeptide or fragment that is phosphorylated at the threonine residue at amino acid position 231.
  • an antibody is used in the broadest sense, and includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, antibody fragments (e.g., "antigen- binding portion"), and single chains antibodies.
  • An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3-
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • antibody portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a Tau protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VH, VL, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, Nature, 341 :544-546 (1989)), which consists of a V H domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VH, VL, CL and CHI domains
  • F(ab')2 fragment a bivalent fragment comprising two Fab fragments linked by a
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al, Science, 242:423-426 (1988); and Huston et al, Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988)).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • biological sample refers to any source of biological material, for example, body fluids, brain extract, peripheral blood or any other sample comprising a Tau protein.
  • body fluid refers to all fluids that are present in the human body including, but not limited to, whole blood, blood components (such as serum, plasma, blood cells, and platelets), urine, lymph, and cerebrospinal fluid (CSF).
  • Tau-related neurological disorder and “tauopathy” are used interchangeably, and refer to any form of dementia that is associated with a Tau pathology.
  • Alzheimer's disease and certain forms of Frontotemporal dementia are the most common forms of tauopathy.
  • Other tauopathies include, but are not limited to, Progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), and Subacute sclerosing panencephalitis.
  • the invention relates to methods for identifying the level of at least one Tau protein (e.g., a Tau fragment).
  • An immunoassay often requires biologically specific capture reagents, such as antibodies, to capture the analytes or a biomarker of interest (e.g., a Tau protein).
  • Antibodies can be produced by methods well known in the art, e.g., by immunizing animals with the biomarker (e.g., a Tau protein) as an antigen.
  • the methods can employ an immunoassay, e.g., an enzyme immunoassay
  • EIA enzyme-linked immunosorbant assay
  • RIA radioimmunoassay
  • indirect competitive immunoassay direct competitive immunoassay, non-competitive immunoassay, sandwich immunoassay, agglutination assay or other immunoassay describe herein and known in the art (see, e.g., Zola, Monoclonal Antibodies: A Manual of ' Techniques, pp. 147-158, CRC Press, Inc. (1987)).
  • the immunoassays may be fluorescence-based or enzyme-based immunoassays. Immunoassays may be constructed in heterogeneous or homogeneous formats.
  • Heterogeneous immunoassays are distinguished by incorporating a solid phase separation of bound analyte from free analyte or bound label from free label.
  • Solid phases can take a variety of forms well known in the art, including but not limited to tubes, plates, beads, and strips. One particular form is the microtiter plate.
  • the solid phase material may be comprised of a variety of glasses, polymers, plastics, papers, or membranes. Particularly desirable are plastics such as polystyrene.
  • Heterogeneous immunoassays may be competitive or non-competitive ⁇ i.e., sandwich formats) (see, e.g., U.S. Patent No. 7, 195,882).
  • a biospecific capture reagent for the biomarker is attached to the surface of a mass spectrometry (MS) probe, such as a pre-activated ProteinChip array.
  • MS mass spectrometry
  • the biomarker is then specifically captured on the biochip through this reagent, and the captured biomarker is detected by mass spectrometry.
  • the immunoassay may be a sandwich immunoassay, a single antibody immunoassay (often run in a competitive or "competition” mode for immunoreactive binding sites), or a double sandwich immunoassay or ELISA.
  • reverse-phase HPLC coupled with western blot is used to detect multiple CSF fragments of Tau using mid-domain, C- terminal and N-terminal region antibodies by Western blotting.
  • RP-HPLC can eliminate matrix interference and enrich analytes for western blot, ELISA, and other measurements.
  • CSF Tau can be fractionated by RP-HPLC, appropriate fractions concentrated and then run by electron-spray ionization mass- spectrometry (ESI-MS) or Tau peptide fragmentation and quantitative mass- identification.
  • ESI-MS electron-spray ionization mass- spectrometry
  • the methods of the present invention are used to screen for clinically significant biomarkers, for example novel Tau fragments, which are associated with a neurological disease.
  • the disease is a tauopathy.
  • the tauopathy is associated with a disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, including frontotemporal dementia with Parkinson's disease with Tau mutations (FTDP-17), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Picks disease.
  • the methods of the present invention may be used for clinical diagnosis of a tauopathy.
  • the present invention provides a method of detecting the level of at least one Tau protein in a biological sample, comprising: (a) contacting a biological sample from a subject with a first antibody which binds to an N-terminal portion of Tau 441 (SEQ ID NO: 1), and a second antibody which binds to a middle portion of Tau 441 (SEQ ID NO: 1), wherein the first antibody and the second antibody both bind to the Tau protein to form a complex; and (b) detecting the level of the Tau protein in the complex.
  • the first antibody binds to an N-terminal portion of amino acid residues 9-158 of Tau 441 (SEQ ID NO: 1).
  • the first antibody binds to amino acid residues 9-18 of SEQ ID NO: 1.
  • the second antibody binds to a middle portion of amino acid residues
  • the second antibody binds to amino acid residues 194-198, residues 159-163, the phosphorylated threonine residue 181 or the phosphorylated threonine residue 231 of SEQ ID NO: 1.
  • the first antibody is Taul2
  • the second antibody is selected from BT2, HT7, Tau5, AT270, and PHF6.
  • the methods use a second antibody which binds to a phosphorylated threonine residue (e.g., at position 181 or 231), the level of the phosphorylated Tau protein is detected by the present methods.
  • the first antibody or the second antibody of the present methods is immobilized on a solid substrate.
  • the first antibody or the second antibody comprises a label.
  • the biological sample is cerebrospinal fluid (CSF), blood, serum or plasma.
  • the subject has a Tau-related neurological disease, such as Alzheimer's disease.
  • the present invention provides a method of detecting the level of at least one Tau protein in a biological sample, comprising: (a) contacting a biological sample from a subject with a first antibody which binds to amino acid residues 159-163 of Tau 441 (SEQ ID NO: 1), and a second antibody which binds to amino acid residues 194-198, residues 218-225, or the phosphorylated threonine residue 231 of Tau 441 (SEQ ID NO: 1), wherein the first antibody and the second antibody both bind to the Tau protein to form a complex; and (b) detecting the level of the Tau protein in the complex.
  • the first antibody is HT7
  • the second antibody is selected from BT2, Tau5, and PHF6.
  • the methods use a second antibody which binds to a phosphorylated threonine residue (e.g., at position 181 or 231)
  • the level of the phosphorylated Tau protein is detected by the present methods.
  • the first antibody or the second antibody of the present methods is immobilized on a solid substrate.
  • the first antibody or the second antibody comprises a label.
  • the biological sample is cerebrospinal fluid (CSF), blood, serum or plasma.
  • the subject has a Tau-related neurological disease, such as Alzheimer's disease.
  • the present methods utilize enzyme amplification for detecting the presence or the level of the antibody:antigen complex.
  • Enzyme amplification can occur when the second antibody is linked to an enzyme.
  • the enzyme converts the substrate to a detectable product.
  • suitable substrates include fluorescent substrates, chemiluminescent substrates and chromogenic substrates.
  • suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, and acetylcholinesterase.
  • the present methods utilize fluorescence readout for detecting the presence or the level of the antibody:antigen complex.
  • readout by fluorescence can occur when a non-enzyme light emitting label is bound to the second antibody.
  • the non-enzyme light emitting label can be any fluorescent label known within the art. Illustrative examples of a non-enzyme light emitting label include fluorescein, and rhodamine.
  • a fluorescently labeled second antibody can be detected when the fluorescent label is exposed to a light of the proper wavelength and can be quantified using known methods in the art including a fluorometer. Readout by fluorsecence can also include enzyme amplification of a fluorogenic substrate.
  • the relative fluorescence units (emitted photons of light) that are detected are typically proportional to the amount of analyte being measured.
  • Suitable enzymes for enzyme amplification of a fluorogenic substrate include, but are not limited to, alkaline phosphatase, ⁇ -galactosidase or peroxidase.
  • a fluorogenic substrate may be chosen for its quantitative emission of light following excitation. Examples of suitable substrates include, but are not limited to, 4-methylumbelliferyl phosphate, 4- methylumbelliferyl galactoside, hydroxyphenylacetic acid, 3-p- hydroxyphenylproprionic acid.
  • the present methods utilize luminescence readout for detecting the presence or the level of the antibody:antigen complex.
  • Readout by luminescence may include the presence of a luminescent-tagged second antibody that can be detected by the presence of luminescence that occurs during the course of a chemical reaction.
  • luminescent labeling compounds include, but are not limited to, luminol, isoluminol, acridinium ester, imidazole, acridinium salt and oxalate ester.
  • Luminescence detection can also include enzyme amplification wherein an enzyme converts a substrate to a reaction product that emits photons of light instead of developing a visible color.
  • Luminescence can be bioluminescence
  • a suitable enzyme includes alkaline phosphatase, ⁇ -galactosidase or peroxidase.
  • suitable substrates include luminol, polyphenols and acridine esters, luciferin.
  • the present methods utilize colorimetric readout for detecting the presence or the level of the antibody :antigen complex. Colorimetric detection results in a colored reaction product that absorbs light in the visible range.
  • the rate of color development is proportional, over a certain range, to the amount of enzyme conjugate present.
  • suitable enzymes for colorimetric detection include, but are not limited to, alkaline phosphatase, B-galactosidase or peroxidase.
  • Suitable enzymes include, but are not limited to, 5-bromo-4- chloro-3-indolyl-phosphate/nitroblue tetrazolium, p nitrophenylphosphate, 3,3',5,5' tetramethylbenzidine, 3,3',4,4' diaminobenzidine, 4-chloro- 1 -naphthol, TMB (dual function substrate), 2,2'-azino-di [3-ethylbenzthiazoline and o-phenylenediamine.
  • Antibodies include, but are not limited to, 5-bromo-4- chloro-3-indolyl-phosphate/nitroblue tetrazolium, p nitrophenylphosphate, 3,3',5,5' tetramethylbenzidine, 3,3',4,4' diaminobenzidine, 4-chloro- 1 -naphthol, TMB (dual function substrate), 2,2'-azino-di [3-eth
  • the methods of the instant invention were developed through a systematic evaluation of Tau-specific antibodies to detect Tau fragments containing the N- or C-terminal regions of Tau in disease samples compared to control samples.
  • the instant invention discloses multiple Tau fragment ELISA assays which have been developed using antibody pairs that are specific to the mid-domain and the N-terminal region (N-terminus to the beginning of microtubule repeat domain of Tau), respectively.
  • the instant invention discloses a novel pT231 Tau assay (e.g., using the HT7 and the PHF6 antibodies) and an N-terminal version of the pT181 Tau assay (e.g., using the Taul2 and AT270 antibodies) to complement the well-established mid-domain pT181 Tau assay (Vanderstichele et al 2006).
  • an N-terminal version of the pT181 Tau assay e.g., using the Taul2 and AT270 antibodies
  • the existence of N-terminal and mid-domain Tau fragments in human CSF is demonstrated.
  • a significant increase in the Tau fragment and pTau fragments in AD compared to age-matched controls is shown.
  • Antibody supports and test membranes are also known to the art.
  • the antibody support is a cuvette or a nitrocellulose membrane.
  • membranes to which the antibodies are removably, or fixedly attached may be employed.
  • the assays provided by this invention may utilize polyclonal or monoclonal antibodies to the selected antigens. They may use the same or different polyclonal or monoclonal antibodies for capture and/or detection.
  • the antibodies may be labeled using labels known in the art. Preferably, the label is readily detectable.
  • Antibodies that bind either phosphorylated or unphosphorylated Tau are examples of antibodies that bind either phosphorylated or unphosphorylated Tau.
  • Antibodies that bind both phosphorylated and unphosphorylated Tau are also contemplated. Examples of antibodies that bind Tau are listed in Table 1. Kits
  • kits that can be used in the assays described above, which comprise at least two antibodies (monoclonal or polyclonal) against a Tau protein, as well as reagents necessary for facilitating an antibody-antigen complex formation and/or detection.
  • a kit of the present invention is a packaged combination including the basic elements of: (a) capture reagents comprising at least one anti-Tau antibody (herein referred to as a "capture antibody"); and (b) at least one detectable (labeled or unlabeled) anti-Tau antibody that binds to a different epitope on Tau.
  • the kit may further comprise reagents necessary for facilitating an antibody-antigen complex formation.
  • the kit may further comprise instructions on how to perform the assay using these reagents.
  • the kit further comprises a solid support for the capture antibodies, which may be provided as a separate element or on which the capture antibodies are already immobilized.
  • the capture antibodies in the kit may be immobilized on a solid support, or they may be immobilized on such support that is included with the kit or provided separately from the kit.
  • the capture antibodies are coated on a microtiter plate.
  • the detectable antibodies may be labeled antibodies detected directly or unlabeled antibodies that are detected by labeled antibodies directed against the unlabeled antibodies raised in a different species.
  • the kit will ordinarily include substrates and cofactors required by the enzyme, where the label is a fluorophore, a dye precursor that provides the detectable chromophore, and where the label is biotin, an avidin such as avidin, streptavidin, or streptavidin conjugated to HRP or ⁇ -galactosidase with MUG.
  • the present invention provides a kit comprising: (1) a first antibody which binds to an N-terminal portion of a Tau protein; (2) a second antibody which binds to a middle portion of a Tau protein; and (3) reagents necessary for facilitating an antibody-antigen complex formation.
  • the first antibody is Taul2
  • the second antibody is selected from BT2, HT7, Tau5, AT270, and PHF6.
  • the present invention provides a kit comprising: (1) a first antibody which binds to amino acid residues 159-163 of Tau 441 (SEQ ID NO: 1); (2) a second antibody which binds to amino acid residues 194- 198, residues 218-225, or the phosphorylated threonine residue 231 of Tau 441 (SEQ ID NO: 1); and (3) reagents necessary for facilitating an antibody-antigen complex formation.
  • the first antibody is HT7
  • the second antibody is selected from BT2, Tau5, and PHF6.
  • kits may further comprise, as a positive control, a Tau protein
  • kits may further comprise, as a negative control, a Tau fragment which does not bind to a capture antibody or a detection antibody.
  • the kit may further comprise other additives such as stabilizers, washing and incubation buffers, and the like.
  • the components of the kit will be provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to provide for concentrations in solution of the reagents that substantially maximize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentration for combining with the sample to be tested.
  • the present invention provides methods of identifying a subject having or at risk of having a Tau-related neurological disease (e.g., tauopathy).
  • a Tau-related neurological disease e.g., tauopathy
  • Such diagnostic methods utilize the above-mentioned immunoassays for detecting the level of at least one Tau protein (e.g., a Tau fragment) in a biological sample from a subject.
  • the level of the Tau protein is then compared to a reference (e.g., a level of the Tau protein in a sample from a healthy control subject). If the level of the Tau protein is increased relative to the reference, then identifying the subject to have or at risk of having a Tau-related neurological disease.
  • such diagnostic methods utilize the above-mentioned immunoassays for detecting the level of at least two Tau proteins (e.g., two Tau fragments) in a biological sample from a subject.
  • the ratio of two Tau protein levels is then compared to a reference (e.g., the ratio of the two Tau protein levels in a sample from a healthy control subject). If the ratio of the two Tau protein levels is decreased relative to the reference, then identifying the subject to have or at risk of having a Tau-related neurological disease.
  • Tau-related neurological disorder and “tauopathy” are used interchangeably herein, including, for example, Alzheimer's disease, Pick's disease, sporadic Frontotemporal dementia and Frontotemporal dementia with Parkinsonism linked to chromosome 17.
  • the present invention provides a method of identifying a subject having or at risk of having a Tau-related neurological disease, comprising: (a) isolating a biological sample from the subject; (b) detecting the level of the Tau protein in the biological sample according to any of the above-mentioned methods; and (c) comparing the level of the Tau protein to a reference; and (d) if the level of the Tau protein is increased compared to the reference, then identifying the subject to have or at risk of having a Tau-related neurological disease.
  • the Tau-related neurological disease is Alzheimer's disease.
  • the present invention provides a method of identifying a subject having or at risk of having a Tau-related neurological disease, comprising: (a) isolating a biological sample from the subject; (b) detecting a first level of the Tau protein in the biological sample using HT7 as the first antibody and BT2 as the second antibody; (c) detecting a second level of the Tau protein in the biological sample using HT7 as the first antibody and Tau5 as the second antibody;
  • the Tau-related neurological disease is Alzheimer's disease.
  • the present invention provides a method of identifying a subject having or at risk of having a Tau-related neurological disease, comprising: (a) isolating a biological sample from the subject; (b) detecting a first level of the Tau protein in the biological sample using HT7 as the first antibody and BT2 as the second antibody is BT2; (c) detecting a second level of the Tau protein in the biological sample using Taul2 as the first antibody is Taul2 and HT7 as the second antibody; (d) determining the ratio of the first level from (b) versus the second level from (c); (e) comparing the ratio to a reference ratio; and (f) if the ratio is decreased compared to the reference ratio, then identifying the subject to have or at risk of having a Tau-related neurological disease.
  • the Tau-related neurological disease is Alzheimer's disease.
  • Diagnosis of a Tau-related neurological disease can include any diagnostic tool that can be used to separate data points of healthy control individuals from data points of diseased patients. Such detection tools can be based on optimizing either specificity or sensitivity or both.
  • detection of a Tau-related neurological disease includes use of Receiver Operating Characteristic (ROC) curve analysis.
  • ROC curve analysis is a graphical plot of sensitivity vs. (1 -specificity). The cut-off value for the assay can be derived from the ROC curve analysis at a point where the sum of specificity and sensitivity is maximized.
  • the ratio of two Tau protein levels can be plotted in a scattergram to
  • AD Alzheimer's disease
  • Human CSF was denatured in guanidine-HCl (VWR, West Chester, PA) to a final concentration of 6 M guanidine-HCl.
  • 24 ml injections of the denatured CSF (6 ml CSF + 18 ml guanidine-HCl) were fractionated with an Agilent 1 100 series HPLC running at 1.5 ml/min over a Poros Rl/10 protein column (4.6 mm X 100 mm, Applied Biosystems, Foster City, CA) heated to 65° C.
  • 30 x 2 ml fractions were collected for each sample using a water/acetonitrile gradient (0-60% acetonitrile over 35 min) in the presence of 0.1% (volume/volume) trifluoroacetic acid.
  • Membranes were probed with HT7 (Pierce), Taul2 (Covance, Dedham, MA), KJ9A (Dako, Carpinteria, CA), and mouse IgGl monoclonal isotype control (Abeam, Cambridge, MA) conjugated to HRP (LYNX Rapid HRP Antibody Conjugation kit, AbD Serotec, Oxford, UK) in TBST with 1% BSA for 16 hrs at room temperature. Probed membranes were developed using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL).
  • suspension array bead sets were incubated with reference standards, QC samples, human CSF along with biotinylated reporter overnight.
  • Tau ELISAs were developed using the following mouse monoclonal antibodies for capture: Taul2 (aa 9-18, SIG-39416, Covance, Princeton, NJ), HT7 (aa 159-163, MN1000, Thermo Scientific, Rockford, IL) or BT2 (aa 194-198, MN1010, Thermo Scientific, Rockford, IL).
  • the antibody information is listed in Table 1 and "Supplemental Methods".
  • the respective analytes were detected using the following alkaline phosphatase (AP) conjugated mouse monoclonal antibodies: BT2, HT7, Tau 5 (aa 218-225, SIG-39413, Covance, Princeton, NJ) or 77G7 (aa 316-335, SIG-34905, Covance, Princeton, NJ).
  • Human Tau441 (Tau441) recombinant protein (rPeptide, Bogart, GA) was used to generate standard curves for each of the assays. Standards were run in two-fold serial dilutions in assay buffer containing 1% BSA (w/v) and 0.05% tween-20 (v/v) in Tris buffered saline (TBS), pH 8.
  • the Tau441 standard curve range for each of the ELISAs was 400-2 pg/ml (Taul2-BT2 and HT7-Tau5), 1000-16 pg/ml (Taul2-HT7), 1000-4 pg/ml (HT7-BT2), 1000-8 pg/ml (HT7-77G7).
  • Human CSF dilution linearity curves were run for each of the Tau ELISAs with CSF at 2-fold serial dilutions from 2- to 64-fold to determine the optimal sample dilution for each of the assays.
  • Tau ELISAs were run as follows. High binding black 96 well plates (Costar 3925, Corning, NY) were coated by the addition of 2.5 ⁇ g/ml (BT2, HT7) or 5 ⁇ g/ml (Tau 12) capture antibodies which were diluted in Tris buffered saline (TBS), pH 8. Plate sealers were attached then the plates were incubated at 37° C for 1 hr. Plates were washed with TBST (TBS containing 0.05% Tween-20) before blocking nonspecific binding sites with 3% bovine serum albumin (BSA; protease free, fraction V; Roche Biochemicals, Indianapolis, IN) (w/v) in TBS.
  • BSA bovine serum albumin
  • Plate sealers were attached and the plates were incubated at room temperature for 2-4 hrs while shaking. Plates were washed with TBST before the addition of 50 ⁇ per well diluted human CSF and human Tau441 standard curves which were each prepared in a final assay buffer concentration of 1% BSA (w/v) and 0.05% Tween-20 (v/v) in Tris buffered saline (TBS), pH 8. Plate sealers were attached; then assay plates containing human CSF and standard curves were incubated overnight at 4° C while shaking.
  • Alkaline phosphatase (AP) conjugated BT2, HT7, Tau5 or 77G7 antibodies were diluted into assay buffer before being added to the assay plate (50 ⁇ per well) to co-incubate with human CSF and hTau441 standard curves for 1 hr at room temperature while shaking. Plates were washed with TBST before being developed using alkaline phosphatase substrate (T2214; Applied Biosystems, Foster City, CA). Luminescence counts were measured using a Packard TopCount (PerkinElmer, MA). Log-transformed luminescence counts from individual samples were interpolated to concentration using a second-order polynomial fit to the respective standards (GraphPad Prism 5.00, GraphPad Software, San Diego, CA). CSF Tau levels were plotted after correction for dilution factor in the respective assays. Assay lower limit of quantitation (LLQ) was set based on the lowest calibrator demonstrating acceptable total error (bias + precision of ⁇ 30%).
  • a pooled CSF sample from AD patients and an age-matched pooled control CSF sample were 2-fold serially diluted from 2- to 256-fold in a final assay buffer concentration of 1% BSA (w/v) and 0.05% tween-20 (v/v) in Tris buffered saline (TBS), pH 8 before an aliquot of each was spiked with recombinant human Tau441 protein (rPeptide, Bogart, GA) at a final concentration of 100 pg/ml. Diluted CSF samples with and without Tau441 spike were assayed in the HT7-77G7 ELISA as described above.
  • HT7-AT270 pl81
  • HT7-PHF6 p231
  • Taul2-AT270 pl81
  • the HT7-AT270 and HT7-PHF6 ELISAs utilized HT7 (amino acids 159-163)
  • the Taul2-AT270 assay used Taul2 (amino acids 9-18), as the capture antibody
  • the respective analytes were detected using alkaline phosphatase (AP) conjugated AT270 pT181 Tau antibody (MN1050, Thermo, Rockford, IL) or PHF6 pT231 specific monoclonal antibody (SIG-39430, Covance, Dedham, MA); antibody information listed in Table 1.
  • the HT7-AT270 and HT7-PHF6 assay standards used native human Tau sequence of aa 155-207 and aa 155-236, respectively, with the Thrl81 and Thr 231 residues being phosphorylated;
  • the Taul2-AT270 assay standard consisted of aa 5-28 linked with a polyethylene glycol (PEG 12) linker to aa 174-187, with a
  • pTau ELISAs were run as follows. Black high-binding plates (Costar,
  • a multiplexed immunoassay was developed on the Luminex platform for mapping Tau antibody epitopes.
  • the assay consisted of 41 different antigens, each covalently coupled to a unique Luminex bead set using standard amine coupling protocols. These antigens included all six human Tau isoforms (rPeptide, Bogart, GA), a set of 29 synthetic overlapping Tau sequence peptides (GenScript, Piscataway NJ) spanning the length of human Tau 441 (Fig. 7), and 3 Tau fragments (aa 1-125, 126-230, 231-441) generated using standard in vitro transcription/ translation system. Beads were conjugated using a two-step carbodiimide procedure.
  • Beads were washed 2x times with activation buffer, resuspended in 200 ⁇ ⁇ of freshly prepared 5 mg/mL of EDC (l-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, inactivation buffer), and incubated in a rotator for 20 min at RT protected from light. Beads were then washed and resuspended in 500 ⁇ , of antigen in PBS (100 ⁇ g for the Tau isoforms and 20 ⁇ g for the peptides) and incubated for 2 hrs at RT in a rotator protected from light.
  • EDC l-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, inactivation buffer
  • Beads were then washed and incubated with 0.5 mL of blocking buffer (PBS, 1% (w/v) BSA, 0.02% (w/v) Tween-20) in a rotator for 1-hour at RT protected from light. Finally, the beads were counted with a hemacytometer and resuspended in blocking buffer at 2 x 10 6 beads/mL. Beads were stored protected from light at 4° C. Prior to testing, the bead sets were mixed together to form a suspension array. Antibodies for epitope mapping were incubated with the bead mix, washed, and subsequently incubated with PE-labeled anti-mouse IgG (H+L) reporter antibodies. The beads were then analyzed on a Bioplex Luminex 100 instrument (Bio-Rad Laboratories, Hercules, CA)
  • Human control CSF was diluted 2-fold into assay buffer (final concentration of 1% BSA in TBS with 0.05% Tween-20) before an aliquot was removed and HT7 was added to a final concentration of 1 ⁇ g/ml.
  • CSF and HT7 were co-incubated for 1 hour at 4° C before being added to protein A/G agarose (Thermo Fisher Scientific, Rockford, IL) at a 9: 1 ratio.
  • the protein A/G beads were blocked with 10 volumes of 2% BSA (w/v) in TBS for 1 hour before use.
  • the remaining diluted CSF sample that was not immunodepleted with HT7 was incubated with protein A/G beads to serve as a control.
  • CSF and protein A/G beads were incubated overnight at 4° C. Beads were spun out before the supernatant was collected, diluted and assayed in CSF Tau ELISAs. CSF was assayed at a 3-fold (HT7+Tau5), 5-fold (Taul2+BT2 and
  • Taul2+HT7 or 10-fold dilution (BT2+HT7).
  • Sepharose 4B beads or unconjugated inactive sepharose 4B beads (GE Healthcare, Pittsburgh, PA) following overnight incubation at 4° C in a rotating holder.
  • CSF samples were spun at 1000 rpm for 5 min and supernatants (depleted CSF) were run in respective pTau assays.
  • CSF samples were spiked with 100 ng/ml of pT181 (PPAPK-T(P04)-PP) or pT231 (KVAVVR- T(P04)-PPK) peptides and run in respective pTau assays (HT7-AT270, HT7-PHF6, and Taul2-AT270).
  • control CSF samples diluted in 0.3% BSA/PBS were spiked with the respective pTau standards over a range of concentrations and run in ELISA's along with non-spiked samples.
  • Spike recovery was estimated as a percent recovery of signal above basal signal levels in non-spiked CSF samples.
  • RP-HPLC reverse-phase high performance liquid chromatography column
  • Fig. 2 To more accurately quantify CSF Tau levels, a set of novel Tau and pTau ELISAs were developed (Fig. 2). Assays were designed to measure overlapping regions of Tau using different combinations of Tau and pTau antibodies (Table 1). Each assay is specific for different minimal regions of the Tau protein, as defined by the epitopes of the antibodies used, and thus may measure different subsets of fragments in CSF.
  • the minimal regions of the Tau assays are aa 9-163 (Taul2-HT7), aa 9-198 (Taul2-BT2), aa 159-198 (HT7-BT2), aa 159-225 (HT7-Tau5), aa 159-335 (HT7-77G7); the minimal regions of the pTau assays are aa 9-pl81 (Taul2-AT270), aa 159-pl 81 (HT7-AT270) and aa 159-p231 (HT7-PHF6) (Fig 2).
  • the HT7-BT2, HT7-Tau5, Taul2-BT2 and Taul2-HT7 Tau assays demonstrated ⁇ 100-fold dynamic range of quantitation using the Tau 441 standard, with the LLQ ranging from 1.6 pg/ml to 7.8 pg/ml (Fig. 3).
  • Signal from pooled AD and control CSF samples were within the dynamic range of the HT7-BT2 assay when diluted 2- to 64-fold (left panel, Fig. 3A).
  • Consistent dilution-corrected Tau levels were observed with CSF dilutions ranging from 16- to 64-fold; similar results were observed for both AD and control samples (right panel, Fig. 3A).
  • the HT7-77G7 assay is specific for Tau species containing more C-terminal sequences (aa 159-335).
  • the dynamic range and LLQ observed were similar to the other Tau ELISAs (Fig. 4); however, a HT7-77G7 signal was not detected in either the pooled control or pooled AD samples, regardless of sample dilution (Fig. 4).
  • the lack of signal was not an artifact of matrix interference as robust recovery of a 100 pg/ml Tau 441 spike was observed in both control and AD CSF over a range of dilutions (Fig. 4).
  • Taul2-BT2 (aa 9-198) 591 (194) 1162 (639) 2.747 (0.155) 3.011 (0.223) 1.8 0.0001 0.86 0.0001
  • Taul2-AT270 (aa 9 -pl81) 21 (7) 29 (10) 1.306 (0.162) 1.439 (0.148) 1.4 0.0100 0.71 0.0266
  • iValues represent mean (SD) from 20 control and 20 AD samples
  • CSF Tau levels were found to be significantly higher in AD samples compared to controls in all but the HT7-77G7 assay (Fig. 6; Table 3).
  • ROC AUC values were also significant, ranging from 0.77 to 0.86 (Table 3).
  • the highest ROC AUCs (0.85, 0.86) were observed using assays specific for Tau containing aa 9-163 (Taul2-HT7) and aa 9-198 (Taul2-BT2), respectively, while a significantly lower AUC (0.77) was detected using the HT7-BT2 assay, specific for Tau species containing aa 159-198.
  • the 20x20 samples were also analyzed using the pTau ELISAs. All three pTau ELISAs exhibited significantly higher levels in AD compared to control samples, with increases ranging from 1.4-fold to 2.4-fold (Fig 6; Table 3). Of these, HT7-AT270, specific for Tau species containing aa 159-pl81, exhibited the highest ROC AUC (0.81), similar to the AUC generated using the comparable ⁇ ⁇ - ⁇ AlzBio3 pTau assay (0.80, Table 3). Interestingly, pl81 failed to exhibit a significant
  • r 2 0.67-0.72
  • HT7-PHF6 aul2-AT270 1.18 (1.96) 1.50 (1.02) ; 0.5282 0.70 i 0.0285 ⁇
  • HT7-PHF6 Taul2-BT2 0.0401 (0.0530) : 0.0434 (0.0448) ⁇ 0.8340 0.65 ; 0.1167 :
  • HT7-PHF6 aul2-HT7 0.0711 (0.0902) : 0.0762 (0.0886) ⁇ 0.8578 0.62 : 0.1851 :
  • HT7-PHF6 HT7-Tau5 0.0469 (0.0513) ; 0.0521 (0.0667) ⁇ 0.7839 0.59 ; 0.3235 ;
  • Val ues represent mean (SD) from 20 control and 20 AD samples
  • RP-HPLC was utilized to enrich and concentrate Tau from a large volume of pooled, denatured CSF thereby enabling analysis of the relatively low levels of Tau by western-blotting.
  • N-terminal and mid-domain Tau fragments were detected in both AD and control CSF, ranging in size from ⁇ 20 kDa to ⁇ 40 kDa.
  • C-terminal-containing fragments were not detected using the K9JA polyclonal antibody.
  • full-length Tau was not detected with any of the antibodies tested. The lack of detectable C-terminal fragments was surprising given reports indicating that MTBR-containing fragments are present in CSF
  • Tau is a putative substrate for various proteases such as calpain, caspases, cathepsins and thrombin (reviewed in [46,47]). Tau fragments observed in CSF could be a direct result of processing by these proteases. Indeed, cleavages at many of the known sites may partly explain differences in absolute levels detected in the different Tau ELISAs [46,47,48]. However, technical issues related to the range of Tau species present and the relative affinity of antibodies for those species could also contribute to assay differences. Thus, comparisons of absolute levels between ELISAs must be interpreted with caution.
  • each ELISA was evaluated for its ability to differentiate between 20 AD and 20 matched control CSF samples.
  • all of the Tau ELISAs with the exception of HT7-77G7, behaved in a similar manner exhibiting significant differences in levels and significant discrimination between AD and control.
  • These findings are consistent with the high degree of correlation between assays.
  • subtle differences in Tau assay performance were also noted.
  • Most interesting was the fact that the two assays specific for Tau species containing N-terminal sequences, aa 9-163 (Taul2- HT7) and aa 9-198 (Taul2-BT2), exhibited the highest ROC AUCs of any measured.
  • the HT7-AT270 (aa 159-pl 81) assay exhibited the highest level of discrimination, though only slightly better than HT7- PHF6 (aa 159-p231). This finding is consistent with data reporting that pl81, p231 and pi 99 were equivalent in their ability to discriminate AD from controls [1 11. Interestingly, significant discrimination of AD from control was lost when pi 81 was measured in the context of Tau species containing additional N-terminal sequence aa 9-pl81 (Taul2-AT270). This finding is surprising given that the Tau ELISAs dependent on the same N-terminal regions exhibited the highest ROC AUCs measured. These results suggest that there may be distinct pathways leading to increased CSF levels of these Tau and pTau species.
  • Phosphorylated tau in human cerebrospinal fluid is a diagnostic marker for
  • Alzheimer disease a comparative cerebrospinal fluid study. Arch Gen Psychiatry 61 : 95-102.
  • Alzheimer's disease identification of phosphorylation sites in tau protein. Biochem J 301 (Pt 3): 871-877.

Abstract

Dans certains modes de réalisation, cette invention concerne des procédés et des trousses pour la détection du taux d'au moins une protéine Tau dans un échantillon biologique.
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