# US 2017/0363645 A1 · Novel Method for the Detection of pGlu-Abeta Peptides > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2017/0363645 A1 - Document ID: US20170363645A1 - Status: Published application - Kind: A1 - Application number: 15/536,014 - Filed: 2015-12-18 - Published: 2017-12-21 - Earliest claimed: 2014-12-19 - Assignees: Probiodrug AG - Applicants: Probiodrug AG - Inventors: Martin Kleinschmidt; Torsten Hoffmann; Jens-Ulrich Rahfeld; Stephan Schilling; Beena Punnamoottil; Michael Adler - Web page: https://patentgrep.com/patent/US20170363645A1 ## CPC - G01N 33/6896: {Neurological disorders, e.g. Alzheimer's disease} - C07K 16/18: against material from animals or humans - C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53) - C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature - C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues - C07K 2317/56: variable (Fv) region, i.e. VH and/or VL - G01N 2333/4709: Amyloid plaque core protein - G01N 2458/10: Oligonucleotides as tagging agents for labelling antibodies - G01N 2800/2821: Alzheimer - G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The invention relates to a highly sensitive method for the detection of pGlu-Abeta (pGlu-Aβ) peptides and the use of this method in the diagnosis of neurodegenerative diseases, such as Alzheimer's disease and Mild Cognitive Impairment. The invention further concerns a novel method for monitoring the effectiveness of a treatment of neurode-generative diseases by monitoring changes in the level of pGlu-Aβ peptides. ## Claims ### Claim 1 A highly sensitive method for the detection of an Aβ target peptide in a biological sample, comprising a capture reagent which is specific for said Aβ target peptide; and an Aβ target peptide detection complex, said method comprising the steps of: a) contacting a biological sample with said capture reagent and said detection complex; and b) detecting said Aβ target peptide; wherein the detection complex comprises an Aβ target peptide specific antibody and a nucleic acid marker. ### Claim 2 (depends on 1) The method of claim 1, wherein said Aβ target peptide is a pGlu-Aβ peptide. ### Claim 3 (depends on 1) The method of claim 1, wherein said detection complex comprises a detection antibody capable of binding a pGlu-Aβ peptide, at least one nucleic acid marker comprising a predetermined nucleotide sequence; and at least one first linker molecule adapted to bind said antibody and the nucleic acid marker. ### Claim 4 (depends on 1) The method according to claim 1, comprising the steps of: a) contacting the biological sample with the detection complex under conditions allowing the binding of an Aβ target peptide to said detection complex; and b) subsequently contacting said Aβ target peptide, which is bound to the detection complex, with a capture reagent capable of binding an Aβ target peptide peptide under conditions allowing the binding of said capture reagent to said Aβ target peptide. ### Claim 5 (depends on 4) The method of claim 4, wherein the capture reagent is a capture antibody specific for a pGlu-Aβ peptide. ### Claim 6 (depends on 1) The method according to claim 1, comprising the steps of: a) contacting a biological sample with at least one detection complex, wherein said detection complex comprises a detection antibody capable of binding an Aβ target peptide, at least one nucleic acid marker comprising a predetermined nucleotide sequence and at least one first linker molecule adapted to bind said antibody and the nucleic acid marker; under conditions allowing the binding of said detection complex to said Aβ target peptide; b) further contacting said Aβ target peptide, which is bound to the detection complex, with a capture antibody capable of binding said Aβ target peptide under conditions allowing the binding of said capture antibody to said Aβ target peptide; and c) detecting said Aβ target peptide; wherein said Aβ target peptide is a pGlu-Aβ peptide; and wherein at least one of the detection antibody and the capture antibody specifically binds to the pyroglutamate carrying amino terminus of said pGlu-Aβ peptide. ### Claim 7 (depends on 1) The method according to claim 1, wherein both of the detection antibody and the capture antibody specifically bind to the pyroglutamate carrying amino terminus of said pGlu-Aβ peptide. ### Claim 8 (depends on 1) The method according to claim 1, wherein the detection antibody specifically binds to the pyroglutamate carrying amino terminus of said pGlu-Aβ peptide and the capture antibody binds to an epitope sequence of an Aβ peptide other than the pyroglutamate carrying amino terminus. ### Claim 9 (depends on 1) The method according to claim 1, wherein the capture antibody specifically binds to the pyroglutamate carrying amino terminus of said pGlu-Aβ peptide and the detection antibody binds to an epitope sequence of an Aβ peptide other than the pyroglutamate carrying amino terminus. ### Claim 10 (depends on 1) The method according to claim 1, wherein the detection complex is provided in a matrix similar to the biological sample and added directly to the biological sample in a ratio lower than 1+1. ### Claim 11 (depends on 2) The method according to claim 2, wherein pGlu-Aβ peptides are detected as monomers and/or in Aβ peptide oligomers and/or bound to proteins in the biological sample. ### Claim 12 (depends on 2) The method according to claim 2, wherein said pGlu-Aβ is at least one pGlu-Aβ peptide selected from SEQ ID NOs: 26 to 37. ### Claim 13 (depends on 6) The method according to claim 6, wherein the detection antibody and/or the capture antibody specifically binds to the pyroglutamate carrying amino terminus of said at least one pGlu-Aβ peptide selected from SEQ ID NOs: 26 to 31. ### Claim 14 (depends on 13) The method according to claim 13, wherein the detection antibody and/or the capture antibody that specifically binds to the pyroglutamate carrying amino terminus of said at least one pGlu-Aβ peptide of SEQ ID NOs: 26 to 31, is selected from the group consisting of: pGlu3-Aβ antibody clone 2-48 (monoclonal, mouse); Synaptic Systems, pGlu3-Aβ antibody (polyclonal, rabbit); Synaptic Systems, Biotrend, IBL, pGlu3-Aβ antibody clone 8E1 (monoclonal, mouse); Anawa, pGlu3-Aβ antibody clone 8E1 (monoclonal, mouse); Biotrend, Anti-Human Amyloidβ (N3pE) Rabbit IgG (polyclonal, rabbit); IBL, Abeta-pE3 rabbit polyclonal, affinity purified, Synaptic systems, Anti-Human Aβ N3pE (8E1) Mouse IgG Fab (monoclonal, mouse); IBL, pGlu3-Aβ antibody clone 337.48 (monoclonal, mouse); Biolegend, pGlu3-Aβ antibody clone 1-57 (monoclonal, mouse); Synaptic Systems, pGlu3-Aβ antibody clone 70D7 (monoclonal, mouse); Synaptic Systems, and oligo pGlu3-Aβ antibody clone 9D5 (monoclonal, mouse); Synaptic Systems. ### Claim 15 (canceled) ### Claim 16 (depends on 13) The method according to claim 13, wherein the variable part of the light chain of said detection antibody and/or said capture antibody has the nucleotide sequence of SEQ ID NO: 40 or the amino acid sequence of SEQ ID NO: 41, and wherein the variable part of the heavy chain of said detection antibody and/or said capture antibody has the nucleotide sequence of SEQ ID NO: 42, or the amino acid sequence of SEQ ID NO: 43. ### Claim 17 (depends on 13) The method according to claim 13, wherein the variable part of the light chain of said detection antibody and/or said capture antibody has the nucleotide sequence of SEQ ID NO: 44 or the amino acid sequence of SEQ ID NO: 45, and wherein the variable part of the heavy chain of said detection antibody and/or said capture antibody has the nucleotide sequence of SEQ ID NO: 46, or the amino acid sequence of SEQ ID NO: 47. ### Claim 18 (depends on 13) The method according to claim 13, wherein the variable part of the light chain of said detection antibody and/or said capture antibody has the nucleotide sequence of SEQ ID NO: 48 or the amino acid sequence of SEQ ID NO: 49, and wherein the variable part of the heavy chain of said detection antibody and/or said capture antibody has the nucleotide sequence of SEQ ID NO: 50, or the amino acid sequence of SEQ ID NO: 51. ### Claim 19 (depends on 13) The method according to claim 13, wherein the variable part of the light chain of said detection antibody and/or said capture antibody has the nucleotide sequence of SEQ ID NO: 52 or the amino acid sequence of SEQ ID NO: 53, and wherein the variable part of the heavy chain of said detection antibody and/or said capture antibody has the nucleotide sequence of SEQ ID NO: 54, or the amino acid sequence of SEQ ID NO: 55. ### Claim 20 (depends on 6) The method according to claim 6, wherein said detection antibody and/or said capture antibody is a monoclonal antibody produced by a hybridoma cell line selected from the group consisting of: Aβ 5-5-6 (Deposit No. DSM ACC 2923) Aβ 6-1-6 (Deposit No. DSM ACC 2924) Aβ 17-4-3 (Deposit No. DSM ACC 2925) Aβ 24-2-3 (Deposit No. DSM ACC 2926). ### Claim 21 (depends on 2) The method according to claim 2, wherein said pGlu-Aβ peptide is at least one pGlu-Aβ peptide of SEQ ID NOs: 32 to 37. ### Claim 22 (depends on 6) The method according to claim 6, wherein the detection antibody and/or the capture antibody that specifically binds to the pyroglutamate carrying amino terminus of said pGlu-Aβ(11-x) peptide pGlu-Aβ peptide of SEQ ID NOs: 32 to 37, is selected from the group consisting of pGlu11-Aβ antibody clone 173D8, (monoclonal, mouse); Synaptic Systems, and pGlu11-Aβ antibody (polyclonal rabbit); Synaptic Systems. ### Claim 23 (depends on 6) The method according to claim 6, wherein the detection antibody and/or the capture antibody specifically binds to epitope sequence pGlu-VHH of SEQ ID NO: 39. ### Claim 24 (depends on 6) The method according to claim 6, wherein the variable part of the light chain of said detection antibody and/or said capture antibody has the nucleotide sequence of SEQ ID NO: 56 or the amino acid sequence of SEQ ID NO: 57, and wherein the variable part of the heavy chain of said detection antibody and/or said capture antibody has the nucleotide sequence of SEQ ID NO: 58, or the amino acid sequence of SEQ ID NO: 59. ### Claim 25 (depends on 6) The method according to claim 6, wherein said detection antibody and/or said capture antibody is a monoclonal antibody produced by hybridoma cell line Aβ 13-11-6 (Deposit No. DSM ACC 3100). ### Claim 26 (depends on 6) The method according to claim 6, wherein said detection antibody and/or said capture antibody binds to an epitope of the Aβ peptide other than the pyroglutamate carrying amino terminus of a said pGlu-Aβ peptide. ### Claim 27 (depends on 26) The method according to claim 26, wherein said detection antibody and/or said capture antibody is selected from the group consisting of: 3D6, Epitope:1-5 (Elan Pharmaceuticals, Innogenetics), pAb-EL16, Epitope: 1-7, 2H4, Epitope: 1-8 (Covance), 1E11, Epitope: 1-8 (Covance), 20.1, Epitope: 1-10 (Covance, Santa Cruz Biotechnology), Rabbit Anti-Aβ (3 Polyclonal Antibody, Epitope: 1-14 (Abcam), AB10, Epitope: 1-16 (Chemicon/Upstate—part of Millipore), 82E1, Epitope: 1-16 (IBL), pAb 1-42, Epitope: 1-11, NAB228, Epitope: 1-11 (Covance, Sigma-Aldrich, Cell Signaling, Santa Cruz Biotechnology, Zymed/Invitrogen), DE2, Epitope: 1-16 (Chemicon/Upstate—part of Millipore), DE2B4, Epitope: 1-17 (Novus Biologicals, Abcam, Accurate, AbD Serotec), 6E10, Epitope: 1-17 (Signet Covance, Sigma-Aldrich), 10D5, Epitope: 3-7 (Elan Pharmaceuticals), WO-2, Epitope: 4-10 (The Genetics Company), 1A3, Epitope 5-9 (Abbiotec), pAb-EL21, Epitope 5-11, 310-03, Epitope 5-16 (Abeam, Santa Cruz Biotechnology), Chicken Anti-Human Aβ Polyclonal Antibody, Epitope 12-28 (Abeam), Chicken Anti-Human Aβ Polyclonal Antibody, Epitope 25-35 (Abeam), Rabbit Anti-Human Aβ Polyclonal Antibody, Epitope: N-terminal (ABR), Rabbit Anti-Human Aβ Polyclonal Antibody (Anaspec), 12C3, Epitope 10-16 (Abbiotec, Santa Cruz Biotechnology), 16C9, Epitope 10-16 (Abbiotec, Santa Cruz Biotechnology), 19B8, Epitope 9-10 (Abbiotec, Santa Cruz Biotechnology), pAb-EL26, Epitope: 11-26, BAM90.1, Epitope: 13-28 (Sigma-Aldrich), Rabbit Anti-beta-Amyloid (pan) Polyclonal Antibody, Epitope: 15-30 (MBL), 22D12, Epitope: 18-21 (Santa Cruz Biotechnology), 266, Epitope: 16-24 (Elan Pharmaceuticals), pAb-EL17; Epitope: 15-24, 4G8, Epitope: 17-24 (Covance), Rabbit Anti-Aβ Polyclonal Antibody, Epitope: 22-35 (Abeam), G2-10; Epitope: 31-40 (The Genetics Company), Rabbit Anti-Aβ aa 32-40 Polyclonal Antibody (GenScript Corporation), EP1876Y, Epitope: x-40 (Novus Biologicals), G2-11, Epitope: 33-42 (The Genetics Company), 16C11, Epitope: 33-42 (Santa Cruz Biotechnology), 21F12, Epitope: 34-42 (Elan Pharmaceuticals, Innogenetics), 1A10, Epitope: 35-40 (IBL), D-17 Goat anti-Aβ antibody, Epitope: C-terminal (Santa Cruz Biotechnology), 2C8, Epitope: 1-16 (Accurate), BAM-10, Epitope: 1-12 (Biotrend, Sigma-Aldrich), 12B2, Epitope: 11-28 (IBL, Biotrend), 6F/3D, Epitope: 8-17 (Accurate), 310-01, Epitope: 10-16, (Accurate), 11A5-B10, Epitop: 34-40, (Millipore), 12F4, Epitope: 36-42, (Millipore, Covance), 9C4, Epitope: 37-43, (Milipore), 7N22, Epitope: 1-20, (Biosource), 11A50-B10, Epitope: 35-40) (Covance), G2-13, Epitope: C-terminus Aβ42 (Genetics Company), 2B9, Epitope: 1-17 (Santa Cruz), and 9C4, Epitope: 3-8 (Covance). ### Claim 28 (depends on 3) The method according to claim 3, wherein the detection complex further comprises at least one second linker molecule capable of specifically binding the first linker molecule. ### Claim 29 (depends on 2) The method according to claim 2, wherein the detection of said pGlu-Aβ peptide is performed with an immuno-PCR reaction. ### Claim 30 (depends on 2) The method according to claim 2, wherein a mixture of different pGlu-Aβ peptides, is applied as a reference substance for quantification. ### Claim 31 (canceled) ### Claim 32 (depends on 2) A method of diagnosing or monitoring a neurodegenerative disease, such as Alzheimer's disease and Mild Cognitive Impairment, which comprises determining the level of a pGlu-Aβ peptide in a biological sample from a test subject, comprising the following steps: i. determining a first level of a pGlu-Aβ peptide in a biological sample from a subject suspected to be afflicted with said neurodegenerative disease with a method according to claim 2; ii. comparing the first level of the pGlu-Aβ peptide with a second level of said pGlu-Aβ peptide in a healthy control subject; and iii. diagnosing the subject with a neurodegenerative disease where the level of said pGlu-Aβ peptide in said biological sample is increased compared to the level of said pGlu-Aβ peptide in the healthy control subject. ### Claim 33 (depends on 2) A method of monitoring the efficacy of a therapy in a subject having, suspected of having, or being predisposed to a neurodegenerative disease, such as Alzheimer's disease or Mild Cognitive Impairment, comprising determining determining the level of a pGlu-Aβ peptide in a biological sample from a test subject with a method according to claim 2. ### Claim 34 (depends on 32) The method of diagnosing or monitoring as defined in claim 32, which comprises determining the level of a pGlu-Aβ peptide in a biological sample taken on at least two or more occasions from a test subject. ### Claim 35 (depends on 32) The method according to claim 32, wherein the state of the neurodegenerative disease of the subjects that are donors of the biological samples is characterized in at least one or more psychometric test. ### Claim 36 (depends on 35) The method according to claim 35, wherein said psychometric test is selected from the DemTect Test, Mini-Mental-State Test, Clock-Drawing Test, ADAS-Cog, Blessed Test, CANTAB, Cognistat, NPI, BEHAVE-AD, CERAD, CSDD, GDS and the The 7 Minute Screen. ### Claim 37 (depends on 1) The method or use according to claim 1, wherein the biological sample is selected from the group consisting of tissue, blood, serum, urine, cerebrospinal fluid (CSF), plasma, lymph, saliva, sweat, pleural fluid, synovial fluid, tear fluid, bile and pancreas secretion. ### Claim 38 A kit for diagnosing a neurodegenerative disease, such as Alzheimer's disease or Mild Cognitive Impairment, which comprises at least one detection complex, at least one capture antibody and instructions for using the kit, wherein said detection complex comprises, essentially consists of or consists of a detection antibody capable of binding an Aβ target peptide, one or more nucleic acid markers comprising a predetermined nucleotide sequence and at least one first linker molecule adapted to bind said antibody and the nucleic acid marker, and wherein at least one of the detection antibody and the capture antibody specifically binds to the pyroglutamate carrying amino terminus of the Aβ target peptide. ### Claim 39 (depends on 38) The kit of claim 38, wherein the detection complex further comprises at least one second linker molecule capable of specifically binding the first linker molecule. ### Claim 40 A detection complex comprising, a detection antibody capable of binding an Aβ target peptide, at least one or more nucleic acid marker markers comprising a predetermined nucleotide sequence and at least one first linker molecule adapted to bind said antibody and the nucleic acid marker. ### Claim 41 (depends on 40) The detection complex of claim 40, further comprising at least one second linker molecule capable of specifically binding the first linker molecule. ### Claim 42 (depends on 40) The detection detectiondetection complex of claim 40, wherein the detection antibody specifically binds to the pyroglutamate carrying amino terminus of a pGlu-Aβ peptide.