Novel Method for the Detection of pGlu-Abeta Peptides

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

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.

2. The method of claim 1, wherein said Aβ target peptide is a pGlu-Aβ peptide.

3. 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.

4. 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.

5. The method of claim 4, wherein the capture reagent is a capture antibody specific for a pGlu-Aβ peptide.

6. 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.

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. 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.

13. 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.

14. 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.

15. (canceled)

16. 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.

17. 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.

18. 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.

19. 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.

20. 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: TABLE-US-00012 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).

21. 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.

22. 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.

23. 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.

24. 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.

25. 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).

26. 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.

27. 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).

28. 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.

29. The method according to claim 2, wherein the detection of said pGlu-Aβ peptide is performed with an immuno-PCR reaction.

30. The method according to claim 2, wherein a mixture of different pGlu-Aβ peptides, is applied as a reference substance for quantification.

31. (canceled)

32. 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.

33. 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.

34. 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.

35. 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.

36. 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.

37. 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.

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.

39. 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.

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.

41. The detection complex of claim 40, further comprising at least one second linker molecule capable of specifically binding the first linker molecule.

42. The detection detectiondetection complex of claim 40, wherein the detection antibody specifically binds to the pyroglutamate carrying amino terminus of a pGlu-Aβ peptide.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0069] FIG. 1: shows the results of the investigation of 3 pan-specific β-amyloid antibodies (6E10, 13-28, 12F4) with different capture antibodies (clones 6-1-6, 17-4-3, 24-2-3 (all three Probiodrug AG) and clone 2-48 (Synaptic Systems). [0070] 1 Detection antibody 6E10, positive control of 300 pg/ml pGlu-Aβ(3-42) peptide (SEQ ID NO: 30), [0071] 2 Detection antibody 6E10, negative control [0072] 3 Detection antibody 13-28 (BAM 90.1, Sigma), positive control of 300 pg/ml pGlu-Aβ(3-42) peptide (SEQ ID NO: 30), [0073] 4 Detection antibody 13-28, negative control [0074] 5 Detection antibody 12F4 (Covance), positive control of 300 pg/ml pGlu-Aβ(3-42) peptide (SEQ ID NO: 30), [0075] 6 Detection antibody 12F4, negative control

[0076] FIG. 2: shows the recovery plot for high and low concentrations of standard pGlu-Aβ(3-42) peptide (SEQ ID NO: 30) mixtures used in the validation of the pGlu-Aβ(3-42) peptide (SEQ ID NO: 30).

[0077] FIG. 3: DemTect Test

[0078] Mean values (Mean±SD) of the results of classification differences in AD patients and healthy subjects (Group I: 18-30 years; Group II: 31-45 years; Group III: 46-65 years) by DemTect Scale.

[0079] FIG. 4: Mini-Mental-State Test

[0080] Mean values (Mean±SD) of the results of classification differences in AD patients and healthy subjects (Group I: 18-30 years; Group II: 31-45 years; Group III: 46-65 years) by Mini-Mental-State Test.

[0081] FIG. 5: Clock-Drawing Test

[0082] Mean values (Mean±SD) of the results of classification differences in AD patients and healthy subjects (Group I: 18-30 years; Group II: 31-45 years; Group III: 46-65 years) by Clock-Drawing Test.

DETAILED DESCRIPTION OF THE INVENTION

[0083] According to a first aspect of the invention there is provided 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: [0084] a) contacting a biological sample with said capture reagent and said detection complex; and [0085] b) detecting said Aβ target peptide;
wherein the detection complex comprises an Aβ target peptide specific antibody and a nucleic acid marker.

[0086] Preferably, said Aβ target peptide is a pGlu-Aβ peptide.

[0087] Suitably, said detection complex consists of, consists essentially of or comprises a detection antibody capable of binding a pGlu-Aβ peptide, one or more nucleic acid markers comprising a predetermined nucleotide sequence; and one or more first linker molecules adapted to bind said antibody and the nucleic acid marker.

[0088] In a preferred embodiment, the method according to the present invention comprises the steps of: [0089] a) contacting the biological sample with the detection complex under conditions allowing the binding of an Aβ target peptide to said detection complex; and [0090] 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 under conditions allowing the binding of said capture reagent to said Aβ target peptide.

[0091] Suitably, the capture reagent is a capture antibody specific for a pGlu-Aβ peptide.

[0092] According to a further preferred aspect of the invention there is provided a method for the detection of a pGlu-Aβ peptide in a biological sample, comprising the steps of: [0093] a) contacting a biological sample with at least one detection complex, wherein said detection complex consists of, consists essentially of or comprises a detection antibody capable of binding an Aβ target peptide, one or more nucleic acid markers comprising a predetermined nucleotide sequence and one or more first linker molecules adapted to bind said antibody and the nucleic acid marker; under conditions allowing the binding of said detection complex to said Aβ target peptide; [0094] 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 [0095] 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.

[0096] The data presented herein surprisingly demonstrate that the sensitivity of the detection of pGlu-Aβ peptides in biological samples was significantly increased by the method of invention, i.e. trace amounts down to 4.2 fg/ml of could be detected with high reliability. The limit of quantitation (LOQ) for the detection of pGlu- Aβ peptides could be improved at least 1000 fold compared to existing assay methods in the prior art.

[0097] The biological samples concerned by the present invention usually comprise a mixture of different Aβ peptides, fragments or functional derivatives thereof as well as different pGlu- Aβ peptides, fragments or functional derivatives thereof. For example, the biological samples may comprise a mixture of the peptides according to SEQ ID NOs: 1 to 37.

[0098] In one embodiment of the method invention, both of the detection antibody and the capture antibody specifically bind to the pyroglutamate carrying amino terminus of said pGlu-Aβ peptide. The advantage of this embodiment is that already in the step of (i) contacting a biological sample with at least one detection complex, wherein said detection complex comprises a detection antibody, only pGlu-Aβ peptides, e.g. the pGlu-Aβ peptides of at least one of SEQ ID NOs: 26-37 are bound by the detection antibody. As a result, there is already made a selective enrichment of said pGlu-Aβ peptides in the first step of the method of the invention. This embodiment of the method of the invention is particularly suitable for the detection of pGlu-Aβ peptides, which are comprised in Aβ oligomers.

[0099] In an alternative embodiment of the method of the invention, the capture antibody specifically binds to the pyroglutamate carrying amino terminus of said pGlu-Aβ peptide and the detection antibody binds to another epitope sequence of an Aβ peptide. The advantage of this embodiment is that in the step (i) of contacting a biological sample with at least one detection complex, wherein said detection complex comprises a detection antibody, all Aβ peptides, pGlu-Aβ peptides as well as fragments and functional variants thereof, which present in said biological sample, are bound by the detection antibody and are thus enriched in this method step. The highly selective discrimination between Aβ peptides and pGlu-Aβ peptides is then performed in method step ii) of further contacting the Aβ peptide, which is bound to the detection complex, with a capture antibody capable of binding a pGlu-Aβ peptide under conditions allowing the binding of said capture antibody to said pGlu-Aβ peptide. This alternative embodiment is especially advantageous when the pGlu-Aβ peptides are comprised not only in Aβ oligomers, but when the pGlu-Aβ peptides are comprised in the biological samples as free monomers or as monomers bound to proteins contained in the biological samples. This alternative embodiment is particularly advantageous when only one pGlu-Aβ monomer is bound to a protein contained in the biological samples. When both, the detection antibody and the capture antibody specifically bind to the same epitope such as to the pyroglutamate carrying amino terminus of said pGlu-Aβ peptide, monomeric pGlu-Aβ peptide bound to the could possibly not detected by the capture antibody, because the pyroglutamate carrying amino terminus of said pGlu-Aβ peptide is already masked or occupied by the detection antibody in the detection complex.

[0100] In a further alternative embodiment of the method of the invention, the detection antibody specifically binds to the pyroglutamate carrying amino terminus of said pGlu-Aβ peptide and the capture antibody binds to another epitope sequence of an Aβ peptide. This embodiment is as advantageous as the afore described embodiment for the detection of free or protein-bound monomeric pGlu-Aβ peptide and pGlu-Aβ containing oligomers.

[0101] The “other epitope sequence” of an Aβ peptide, to which the capture antibody and/or the detection antibody binds, when the capture antibody and/or the detection antibody do not bind to the pyroglutamate carrying amino terminus of a pGlu-Aβ peptide, may be a part of the amino acid sequence of a full-length Aβ peptide, such as Aβ (1-38) of SEQ ID NO: 1, Aβ (1-39) of SEQ ID NO: 2, Aβ (1-40) of SEQ ID NO: 3, Aβ (1-41) of SEQ ID NO: 4, Aβ (1-42) of SEQ ID NO: 5, and Aβ (1-43) of SEQ ID NO: 6. The capture antibody and/or the detection antibody, which do not bind to the pyroglutamate carrying amino terminus, may specifically detect the untruncated and/or unmodified N-terminus or C-terminus of an Aβ peptide. Further suitably, the other epitope sequence may be part of the amino acid sequence of one of SEQ ID NOs: 7 to 37.

[0102] In a preferred embodiment of the method of the invention, the other epitope sequence of an Aβ peptide, to which the capture antibody and/or the detection antibody binds, when the capture antibody and/or the detection antibody do not bind to the pyroglutamate carrying amino terminus of a pGlu-Aβ peptide, is comprised in the core amyloid β sequence of Aβ (11-38) of

[0103] SEQ ID NO: 19 in the case that pGlu-Aβ peptides starting with the N-terminal pGlu residue at position 3, most preferably the pGlu-Aβ peptides of SEQ ID NOs: 26-31 shall be detected and/or quantified.

[0104] In a preferred embodiment of the method of the invention, the other epitope sequence of an Aβ peptide, to which the capture antibody and/or the detection antibody binds, when the capture antibody and/or the detection antibody do not bind to the pyroglutamate carrying amino terminus of a pGlu-Aβ peptide, is comprised in the core amyloid β sequence of Aβ(15-38) of SEQ ID NO: 25 in the case that pGlu-Aβ peptides starting with the N-terminal pGlu residue at position 11, most preferably the pGlu-Aβ peptides of SEQ ID NOs: 32-37 shall be detected and/or quantified.

[0105] Suitably, the other epitope sequence consists of the entire amino acid sequence of an Aβ peptide of one of SEQ ID NOs: 1 to 25. More suitably, other epitope sequence consists of 30, 25, 20 or 15 amino acids of an Aβ peptide of one of SEQ ID NOs: 1 to 25. Most preferably, the other epitope sequence consists of 10, 9, 8, 7, 6, 5, 4 or 3 amino acids of an Aβ peptide of one of SEQ ID NOs: 1 to 25.

[0106] Particularly good and reliable results are achieved with the method of the present invention, when the detection complex is provided in a matrix similar to the biological sample. Further suitably, the detection complex comprised in such a matrix similar to the biological sample is added directly to the biological sample. Surprisingly, best results can be obtained when the detection complex is provided in a matrix similar to the biological sample, is added directly to the biological in a ratio <1+1.

[0107] The method of the present invention is based on a new and surprising strategy in the assay protocol, which comprises a combined overnight incubation of the biological sample and the addition of the detection complex, which is contained in a matrix similar to the biological sample, directly to the biological sample in a ratio of 1+0.03. This assay protocol is quite unexpected and unconventional compared to methods used in the prior art, where a typical sample dilution is 1+1 to 1+9 and higher. Only this incubation strategy enabled the intended highly sensitive detection of the Aβ target peptide.

[0108] A further increase in the sensitivity and reliability of the method of the present invention is achieved, when a mixture of different pGlu- Aβ peptides is applied to human CSF or artificial human CSF as a reference substance for quantification. In a preferred embodiment, a 1+1 mixture of pGlu-Aβ(x-40) and pGlu-Aβ(x-42) is applied to human CSF or artificial human CSF as a reference substance for quantification, wherein x is an integer selected from 3 and 11. Most preferably, a 1+1 mixture of pGlu-Aβ(3-40) and pGlu-Aβ(3-42) is applied human CSF or artificial human CSF as a reference substance for quantification, when the Aβ target peptide is selected from SEQ ID NOs: 1 to 25. Yet most preferably, a 1+1 mixture of pGlu-Aβ(11-40) and pGlu-Aβ(11-42) is applied human CSF or artificial human CSF as a reference substance for quantification, when the Aβ target peptide is selected from SEQ ID NOs: 32-37. Such a use of a mixture of two Aβ target peptides as a reference standard is a new and innovative strategy.

[0109] Suitable examples for detection and/or capture antibodies, which do not bind to the pyroglutamate carrying amino terminus of pGlu-Aβ peptides are: [0110] 3D6, Epitope:1-5 (Elan Pharmaceuticals, Innogenetics), [0111] pAb-EL16, Epitope: 1-7, [0112] 2H4, Epitope: 1-8 (Covance), [0113] 1E11, Epitope: 1-8 (Covance), [0114] 20.1, Epitope: 1-10 (Covance, Santa Cruz Biotechnology), [0115] Rabbit Anti-Aβ Polyclonal Antibody, Epitope: 1-14 (Abcam), [0116] AB10, Epitope: 1-16 (Chemicon/Upstate—part of Millipore), [0117] 82E1, Epitope: 1-16 (IBL), [0118] pAb 1-42, Epitope: 1-11, [0119] NAB228, Epitope: 1-11 (Covance, Sigma-Aldrich, Cell Signaling, Santa Cruz Biotechnology, Zymed/Invitrogen), [0120] DE2, Epitope: 1-16 (Chemicon/Upstate—part of Millipore), [0121] DE2B4, Epitope: 1-17 (Novus Biologicals, Abcam, Accurate, AbD Serotec), [0122] 6E10, Epitope: 1-17 (Signet Covance, Sigma-Aldrich), [0123] 10D5, Epitope: 3-7 (Elan Pharmaceuticals), [0124] WO-2, Epitope: 4-10 (The Genetics Company), [0125] 1A3, Epitope 5-9 (Abbiotec), [0126] pAb-EL21, Epitope 5-11, [0127] 310-03, Epitope 5-16 (Abcam, Santa Cruz Biotechnology), [0128] Chicken Anti-Human Aβ Polyclonal Antibody, Epitope 12-28 (Abcam), [0129] Chicken Anti-Human Aβ Polyclonal Antibody, Epitope 25-35 (Abcam), [0130] Rabbit Anti-Human Aβ Polyclonal Antibody, Epitope: N-terminal (ABR), [0131] Rabbit Anti-Human Aβ Polyclonal Antibody (Anaspec), [0132] 12C3, Epitope 10-16 (Abbiotec, Santa Cruz Biotechnology), [0133] 16C9, Epitope 10-16 (Abbiotec, Santa Cruz Biotechnology), [0134] 19B8, Epitope 9-10 (Abbiotec, Santa Cruz Biotechnology), [0135] pAb-EL26, Epitope: 11-26, [0136] BAM90.1, Epitope: 13-28 (Sigma-Aldrich), [0137] Rabbit Anti-beta-Amyloid (pan) Polyclonal Antibody, Epitope: 15-30 (MBL), [0138] 22D12, Epitope: 18-21 (Santa Cruz Biotechnology), [0139] 266, Epitope: 16-24 (Elan Pharmaceuticals), [0140] pAb-EL17; Epitope: 15-24, [0141] 4G8, Epitope: 17-24 (Covance), [0142] Rabbit Anti-Aβ Polyclonal Antibody, Epitope: 22-35 (Abcam), [0143] G2-10; Epitope: 31-40 (The Genetics Company), [0144] Rabbit Anti-Aβ, aa 32-40 Polyclonal Antibody (GenScript Corporation), [0145] EP1876Y, Epitope: x-40 (Novus Biologicals), [0146] G2-11, Epitope: 33-42 (The Genetics Company), [0147] 16C11, Epitope: 33-42 (Santa Cruz Biotechnology), [0148] 21F12, Epitope: 34-42 (Elan Pharmaceuticals, Innogenetics), [0149] 1A10, Epitope: 35-40 (IBL), [0150] D-17 Goat anti-Aβ antibody, Epitope: C-terminal (Santa Cruz Biotechnology), [0151] 2C8, Epitope: 1-16 (Accurate), [0152] BAM-10, Epitope: 1-12 (Biotrend, Sigma-Aldrich), [0153] 12B2, Epitope: 11-28 (IBL, Biotrend), [0154] 6F/3D, Epitope: 8-17 (Accurate), [0155] 310-01, Epitope: 10-16, (Accurate), [0156] 11A5-10, Epitop: 34-40, (Millipore), [0157] 12F4, Epitope: 36-42, (Millipore, Covance), [0158] 9C4, Epitope: 37-43, (Milipore), [0159] 7N22, Epitope: 1-20, (Biosource), [0160] 11A50-B10, Epitope: 35-40) (Covance), [0161] G2-13, Epitope: C-terminus Aβ42 (Genetics Company), [0162] 2B9, Epitope: 1-17 (Santa Cruz), and [0163] 9C4, Epitope: 3-8 (Covance).

[0164] The pGlu-Aβ peptide, which is preferably detected by the method of the invention, is at least one pGlu-Aβ peptide selected from the group consisting of SEQ ID NOs: 26 to 37.

[0165] In a preferred embodiment of the invention, said detection antibody and/or said capture antibody is a monoclonal antibody, more preferably a humanized monoclonal antibody. Further preferred according to the invention is a detection antibody and/or a capture antibody, which is a diabody or a single chain antibody which retains the high affinity.

[0166] According to one embodiment of the invention, the capture and/or the detection antibody, which specifically binds to the pyroglutamate carrying amino terminus of said pGlu-Aβ peptides of SEQ ID NOs: 26-31, is selected from the group consisting of [0167] pGlu3-Aβ antibody clone 2-48 (monoclonal, mouse); Synaptic Systems, [0168] pGlu3-Aβ antibody (polyclonal, rabbit); Synaptic Systems, Biotrend, IBL, [0169] pGlu3-Aβ antibody clone 8E1 (monoclonal, mouse); Anawa, [0170] pGlu3-Aβ antibody clone 8E1 (monoclonal, mouse); Biotrend, [0171] Anti-Human Amyloidlβ (N3pE) Rabbit IgG (polyclonal, rabbit); IBL, [0172] Abeta-pE3 rabbit polyclonal, affinity purified, Synaptic systems, [0173] Anti-Human Aβ N3pE (8E1) Mouse IgG Fab (monoclonal, mouse); IBL, [0174] pGlu3-Aβ antibody clone 337.48 (monoclonal, mouse); Biolegend, [0175] pGlu3-Aβ antibody clone 1-57 (monoclonal, mouse); Synaptic Systems, [0176] pGlu3-Aβ antibody clone 70D7 (monoclonal, mouse); Synaptic Systems, and [0177] oligo pGlu3-Aβ antibody clone 9D5 (monoclonal, mouse); Synaptic Systems.

[0178] In a further preferred embodiment of the invention, the capture and/or the detection antibody, specifically binds to the epitope sequence pGlu-FRHDSGC, SEQ ID NO: 38.

[0179] In a more preferred embodiment of the invention, the detection and/or the capture antibody is produced by a hybridoma cell line selected from the group consisting of:

TABLE-US-00003 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), and Aβ 24-2-3 (Deposit No. DSM ACC 2926),
which are disclosed in WO 2010/009987.

[0180] In a most preferred embodiment of the invention, 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 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.

[0181] In a further most preferred embodiment of the invention, the variable part of the light chain of said detection antibody and/or said capture antibody has the nucleotide sequence of SEQ ID

[0182] NO: 44 or the amino acid sequence of SEQ ID NO: 45, and 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.

[0183] In a further most preferred embodiment of the invention, 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 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.

[0184] In a further most preferred embodiment of the invention, 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 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.

[0185] In a preferred embodiment of the invention, the capture and/or the detection antibody, which specifically binds to the pyroglutamate carrying amino terminus of said pGlu-Aβ peptides of SEQ ID NOs: 32 to 37, is selected from the group consisting of [0186] pGlu11-Aβ antibody clone 173D8, (monoclonal, mouse); Synaptic Systems, and [0187] pGlu11-Aβ antibody (polyclonal rabbit); Synaptic Systems.

[0188] In a further preferred embodiment of the invention, the capture and/or the detection antibody, specifically binds to the epitope sequence pGlu-VHH, SEQ ID NO: 39.

[0189] More preferably, said detection antibody and/or said capture antibody is a monoclonal antibody produced by hybridoma cell line Aβ (Deposit No. DSM ACC 3100), which is disclosed in WO 2012/123562.

[0190] In a most preferred embodiment of the invention, 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 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.

[0191] It is further preferred according to invention, that the capture antibody is immobilized on a solid support, and thereafter the detection complex comprising the detection antibody and the analyte binds to the capture antibody, thus forming an insoluble complex.

[0192] General methods for preparing a detection complex, which is used in the method of the invention, has been described in DE 199 41 756 A1 and EP 2 189 539 A1, the disclosure of which is incorporated herein in their entirety. In particular, it has been found that by use of detection complexes that consist of, consist essentially of or comprise one or more non-nucleic acid receptors (e.g. an antibody such as a detection antibody), one or more nucleic acid markers, one or more first linker molecules adapted to bind the non-nucleic acid receptor and the nucleic acid marker, and one or more second linker molecules adapted to bind the first linker molecule the performance, in particular the assay sensitivity and the signal-to-background-ratio, of an Immuno-PCR (IPCR) reaction can be significantly improved.

[0193] According to a further aspect of the invention there is provided a method for the detection of a pGlu-Aβ peptide in a biological sample, comprising the steps of: [0194] i. contacting a biological sample with at least one detection complex, wherein said detection complex consist of, consist essentially of or comprises a detection antibody capable of binding an Aβ peptide, one or more nucleic acid markers comprising a predetermined nucleotide sequence and one or more first linker molecules adapted to bind said antibody and the nucleic acid marker; and one or more second linker molecules adapted to bind the first linker molecule under conditions allowing the binding of said detection complex to the Aβ peptide; [0195] ii. further contacting the Aβ peptide, which is bound to the detection complex, with a capture antibody capable of binding an Aβ peptide under conditions allowing the binding of said capture antibody to said Aβ peptide; and [0196] iii. detecting said pGlu-Aβ peptide;
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.

[0197] Thus, in one embodiment, the invention relates to detection complexes comprising one or more detection antibody molecules, one or more nucleic acid markers, one or more first linker molecules adapted to bind the non-nucleic acid receptor and the nucleic acid marker, and one or more second linker molecules adapted to bind the first linker molecule. In one specific embodiment of the present invention, the detection complexes comprise a plurality of detection antibody molecules, nucleic acid markers, first linker molecules and second linker molecules. It is desirable to include several detection antibodies with specific binding affinity for a certain Aβ peptide or pGlu-Aβ peptide in the detection complexes according to the invention in order to enhance the affinity for the analyte of choice by means of increased avidity. In turn, it is also desirable to include several nucleic acid markers in the detection complexes, because thus the positive signal, indicating the presence of the analyte in a sample, is enhanced and the signal-to-background ratio improved.

[0198] In the detection complexes according to the present invention, the first and second linker molecules serve the purpose to form supramolecular aggregates of the detection antibodies and the nucleic acid markers and thus increase the sensitivity of the complexes as detection reagents in IPCR assays. To achieve the self-assembly of supramolecular networks, the first linker molecules are adapted to bind the detection antibodies, the nucleic acid markers and the second linker molecules.

[0199] The supramolecular detection complexes according to the present invention may include 2-50, preferably 5-50 molecules of each the detection antibodies, the nucleic acid markers, the first linker molecules and the second linker molecules. In one embodiment of the invented detection complexes, the complexes include at least 2, preferably 3 or more detection antibody molecules and/or nucleic acid markers. In some embodiments of the invention, the invented detection complexes include about 10-40 nucleic acid marker and first linker molecules, about 5-15 detection antibody molecules and about 5-10 second linker molecules.

[0200] In accordance with a further embodiment of the present invention, the detection antibody may be an antibody fragment or functional variant of a detection antibody or detection antibody fragment that retains the ability to specifically bind an Aβ peptide or pGlu-Aβ peptide. The detection antibody may be a monoclonal or polyclonal antibody and the antibody fragment may be, for example, a Fab or F(ab′)2 fragment, a single chain variable fragment (scFv), an Fv diabody or a linear antibody. The detection antibodies, fragments or functional variants thereof may be biotinylated and thus include one or more biotin or biotin analog moieties.

[0201] The nucleic acid marker including a predetermined nucleotide sequence may be any nucleic acid, such as, for example, double- or single-stranded DNA, double- or single stranded RNA, or double-stranded hybrids of DNA and RNA. The nucleic acid marker may contain nucleotide analogs, such as those, in which the naturally occurring bases and sugars are replaced by base analogs or sugar analogs or in which the phosphate backbone is substituted by other suitable groups. Suitable modifications have been mentioned above. All afore-mentioned nucleic acid marker molecules may be biotinylated and thus include one or more biotin or biotin analog moieties. One particular example are mono- or bis-biotinylated DNA molecules.

[0202] In one embodiment of the invention, the detection complexes are formed by non-covalent interactions between the first linker molecules and the detection antibody and/or the nucleic acid marker. In such an embodiment, the binding of the first linker molecule to the second linker molecule may also be non-covalent.

[0203] According to one specific embodiment of the present invention, the binding of the first linker molecule to the detection antibody, the nucleic acid marker and/or the second linker molecule may be facilitated by coupling each the non-nucleic acid receptor, the nucleic acid marker and/or the second linker molecule to one or more, for example 2, 3, 4, 5 or more binding partners of the first linker molecule. These binding partners may be the same or different for the detection antibody, the nucleic acid marker and the second linker molecule. In one embodiment of the invention, these binding partners of the first linker molecule are covalently coupled to the detection antibody, the nucleic acid marker and/or the second linker molecule.

[0204] In accordance with one specific embodiment of the present invention, the binding partner of the first linker molecule may be a ligand of the first linker molecule. It is preferred that the first linker molecule is bivalent, trivalent, tetravalent or multivalent for the binding to the binding partner. In one embodiment, the first linker molecule specifically recognizes and binds its binding partner with a high affinity.

[0205] In one embodiment of the present invention, the first linker molecule may be avidin or streptavidin or a biotin-binding fragment or mutant thereof.

[0206] In a specific embodiment, the binding partner of the first linker molecule is biotin or a biotin analog. The biotin analogs of the present invention preferably retain the ability to specifically bind to avidin, streptavidin or a biotin-binding fragment or mutant thereof.

[0207] If the first linker molecule is avidin, streptavidin or a biotin-binding fragment or mutant thereof, the binding of the first linker molecule to the detection antibody, the nucleic acid marker and/or the second linker molecule may be facilitated by coupling the detection antibody, the nucleic acid marker and/or the second linker molecule to biotin or a biotin analog. This coupling may be covalent and either of the detection antibody, the nucleic acid marker and/or the second linker may be coupled to at least 2 biotin or biotin analog molecules.

[0208] In an alternative embodiment, the first linker molecule may be a fusion protein or an at least bivalent antibody or antibody-like molecule adapted to simultaneously bind at least two of the detection antibodies, the nucleic acid marker and the second linker molecule.

[0209] According to one embodiment of the invention, the second linker molecules may be selected from the group consisting of nucleic acids distinct from the nucleic acid marker, organic polymers, polypeptides and polysaccharides. In one embodiment of the present invention, the second linker molecules comprise at least two, three or four different molecules selected from the group consisting of nucleic acids distinct from the nucleic acid marker, organic polymers, proteins and polysaccharides.

[0210] If the second linker molecules consist of, consist essentially of or include organic polymer molecules, these may be selected from the group consisting of cationic polymers, such as linear, branched or dendritic polyethyleneimines, polyacrylamides, polyamines, and polyamidoamines according to one specific embodiment of the present invention.

[0211] In case the second linker molecules consist of, consist essentially of or include protein or polypeptide molecules, these may be selected from the group consisting of serum albumines and immunoglobulins or fragments thereof. In one embodiment, the second linker molecule may be BSA. Alternatively, the second linker molecules may be homo-polymers of cationic amino acids, such as poly-lysine, poly-histidine or poly-arginine.

[0212] In one alternative embodiment of the present invention, the second linker molecules consist of, consist essentially of or include polysaccharides selected from the group consisting of linear, cyclic or branched dextrans.

[0213] In still another embodiment of the present invention, the second linker molecules may also consist of, consist essentially of or include nucleic acid molecules distinct from the nucleic acid marker. The nucleic acid molecules may be nucleic acid oligomers, for example, oligonucleotides or nucleic acid polymers, such as polynucleotides. Exemplary nucleic acid oligomers that may be used as second linker molecules consist of two complementary nucleic acid strands, wherein each of these strands is independently adapted to bind to a first linker molecule. In one specific embodiment of the invention, this binding to a first linker molecule is facilitated by covalently coupling each single strand of the nucleic acid oligomer to one first linker molecule, with the result that each of these two strands is independently coupled to a first linker molecule by a covalent bond.

[0214] Another alternative embodiment may be a polynucleotide adapted to bind one or more first linker molecules.

[0215] In one embodiment of the present invention, the second linker molecules may also be a heterogeneous mixture of the above specified molecules. According to one embodiment of the present invention, the second linker molecules thus include two or more different molecules selected from the group consisting of linear, branched or dendritic polyethyleneimines, polyacrylamides, polyamines, polyamidoamines, homo-polymers of cationic amino acids, such as poly-lysine, serum albumines, immunoglobulins or fragments thereof, linear, cyclic or branched dextrans, poly- and oligonucleotides. In one specific embodiment of the present invention, the second linker molecules include nucleic acid oligomers consisting of two complementary nucleic acid strands, wherein each of these strands is independently adapted to bind to a first linker molecule, optionally be forming a covalent bond, and organic polymers, such as polyethyleneimines, polypeptides, such as albumines or immunoglobulins, polysaccharides and/or polynucleotides distinct from the nucleic acid oligomers and the nucleic acid marker.

[0216] All afore-mentioned second linker molecules may be coupled to one or more biotin or biotin analog molecules. Specific examples of second linker molecules according to the invention are polybiotinylated BSA, polybiotinylated polyethyleneimine, polybiotinylated poly(meth)acrylamide, polybiotinylated polyamine, or polybiotinylated polyamidoamine.

[0217] In one embodiment of the present invention, the detection complexes of the invention may further include one or more modulators adapted to bind to the first linker molecules. These modulators are used to saturate non-occupied binding sites of the first linker molecule for the detection antibody, the nucleic acid marker, the second linker molecule and/or a binding partner of the first linker molecule. In order to avoid that the modulators compete with the binding of the detection antibody, the nucleic acid marker, the second linker molecule and/or a binding partner of the first linker molecule coupled to the detection antibody, the nucleic acid marker and/or the second linker molecule, the modulator is preferably added after formation of a detection complex from the detection antibody, the nucleic acid marker, the first and the second linker molecule. The modulators may be positively charged and may be selected from the group consisting of amino-biotin, diamino-biotin and amino-substituted biotin analogs.

[0218] In a further aspect, the present invention relates to methods for the preparation of the above detection complexes. In one embodiment, such a method for the preparation of a detection complex according to the invention includes the steps of: [0219] (a) contacting one or more nucleic acid markers with one or more first linker molecules adapted to bind nucleic acid markers and detection antibodies to form a complex of one or more nucleic acid markers with one or more first linker molecules; [0220] (b) contacting the complex of step (a) with one or more detection antibodies to form a complex of one or more detection antibodies, one or more nucleic acid markers and one or more first linker molecules; and [0221] (c) contacting the complex of step (b) with one or more second linker molecules adapted to bind the first linker molecules to form a complex of one or more detection antibodies, one or more nucleic acid markers, one or more first linker molecules and one or more second linker molecules.

[0222] This method may optionally further include the step of: [0223] (d) contacting the complex of step (c) with one or more modulators adapted to bind to the first linker molecules to saturate non-occupied binding sites of the first linker molecule for the detection antibody, the nucleic acid marker and the second linker molecule to form a complex of one or more detection antibodies, one or more nucleic acid markers, one or more first linker molecules, one or more second linker molecules and one or more modulators.

[0224] In another embodiment, the invention encompasses a method for the preparation of a detection complex including: [0225] (i) one or more detection antibodies capable of specifically binding an Aβ peptide and/or a pGlu-Aβ peptide; [0226] (ii) one or more nucleic acid markers including a predetermined nucleotide sequence; [0227] (iii) one or more first linker molecules adapted to bind the detection antibody and the nucleic acid marker; [0228] (iv) one or more nucleic acid oligomers adapted to bind the first linker molecules, wherein the one or more nucleic acid oligomers comprise two complementary nucleic acid strands distinct from the nucleic acid marker; and [0229] (v) one or more organic polymers, polynucleotides distinct from the nucleic acid marker and the one or more nucleic acid oligomers, polypeptides or polysaccharides adapted to bind the first linker molecules;
wherein the method comprises the steps of: [0230] (a) contacting one nucleic acid strand of the one or more nucleic acid oligomers with one or more first linker molecules to form a first detection of one or more first linker molecules and one nucleic acid strand of the one or more nucleic acid oligomers; [0231] (b) contacting the nucleic acid strand of the one or more nucleic acid oligomers complementary to that used in step (a) with one or more first linker molecules to form a second detection of one or more first linker molecules and one nucleic acid strand of the one or more nucleic acid oligomers complementary to that used in step (a); [0232] (c) contacting the detection of step (a) with one or more nucleic acid markers to form a first complex of one or more first linker molecules detectiond to one nucleic acid strand of the one or more nucleic acid oligomers and one or more nucleic acid markers; [0233] (d) contacting the detection of step (b) with one or more detection antibodies to form a second complex of one or more first linker molecules detectiond to one nucleic acid strand of the one or more nucleic acid oligomers complementary to that used in step (a) and (c) and one or more detection antibodies; [0234] (e) contacting the first complex of step (c) with one or more organic polymers, polynucleotides, polypeptides or polysaccharides, to form a third complex of one or more first linker molecules detectiond to one nucleic acid strand of the one or more nucleic acid oligomers, one or more nucleic acid markers and one or more organic polymers, polynucleotides, polypeptides or polysaccharides; and [0235] (f) contacting the second complex of step (d) with the third complex of step (e) to form a complex detection of one or more first linker molecules detectiond to one nucleic acid strand of the one or more nucleic acid oligomers, one or more nucleic acid markers, one or more organic polymers, polynucleotides, polypeptides or polysaccharides, one or more first linker molecules detectiond to one nucleic acid strand of the one or more nucleic acid oligomers complementary to that used in step (a) and (c) and one or more detection antibodies.

[0236] In one embodiment of the invention, this method may further include the step of contacting the complexes of steps (d) and (e) with one or more modulators adapted to bind to the first linker molecule before step (f).

[0237] In the methods of the invention, the detection antibody, the nucleic acid marker, the first linker molecule, the second linker molecule and the modulator may be as defined above. In particular, the binding of the detection antibody, the nucleic acid marker and the second linker molecule to the first linker molecule may be facilitated by one or more binding partner(s) of the first linker molecule coupled to the detection antibody, the nucleic acid marker and the second linker molecule. In one embodiment, these binding partners are biotin and/or a biotin analog and the first linker molecule is streptavidin, avidin or a biotin-binding fragment thereof.

[0238] Also encompassed by the present invention are the detection complexes obtainable by the invented methods.

[0239] In another aspect, the invention is also directed to the use of the detection complex according to the invention in an immunoassay for the detection or the determination of the amount of a pGlu-Aβ peptide. Said pGlu-Aβ peptide may be as defined above and is specifically recognized and bound by the detection antibody. The immunoassay may include a nucleic acid amplification reaction to amplify the nucleic acid marker. The amplification reaction is preferably a polymerase chain reaction (PCR), more preferably a real-time PCR reaction.

[0240] In still another aspect, the invention features a method for detecting a pGlu-Aβ peptide in a sample, wherein the method includes the steps of: [0241] (a) contacting a detection complex according to the invention comprising one or more detection antibodies capable of specifically binding said pGlu-Aβ peptide with said sample to form a complex of said analyte and said detection complex; [0242] (b) specifically detecting the presence of the one or more nucleic acid markers in said complex; wherein the presence of the one or more nucleic acid markers indicates the presence of the pGlu-Aβ peptide in said sample.

[0243] In one embodiment of the present invention, the detecting step (b) may comprise amplifying the one or more nucleic acid markers in a PCR reaction, preferably a real time PCR reaction.

[0244] In one embodiment, the detection of the pGlu-Aβ peptide includes the determination of the amount of the pGlu-Aβ peptide, that is a quantitative determination of the pGlu-Aβ peptide.

[0245] Detection and, in a specific embodiment, also quantitation of the pGlu-Aβ peptide may be achieved by detection and, optionally, quantitation of the number of amplicons generated in the PCR reaction using the nucleic acid marker as a template. Detection and, optionally quantitation may be achieved by using nucleic acid probes labeled with a detectable label or suitable dyes.

[0246] In one embodiment of the invention, the nucleic acid marker is detected by real time PCR, carried out in a commercially available instrument. Real-time PCR amplification is performed in the presence of a fluorescent-labelled probe which specifically binds to the amplified PCR product, for example a dual labelled primer including a fluorescent moiety quenched by another label which is in spatial proximity to the fluorescent label as long as the primer is not incorporated in an amplification product and separated from each other due to elongation of the primer during amplification.

[0247] In another embodiment, a non-primer detectable probe which specifically binds the PCR amplification product is used. The probe may include a covalently bonded reporter dye at the 5′-end and a downstream quencher dye at the 3′-end, which allows fluorescent resonance energy transfer (FRET).

[0248] Detection of the amplified PCR product may be carried out after each amplification cycle, as the amount of PCR product is at every stage of the amplification reaction proportional to the initial number of template copies. The number of template copies can be calculated by means of the detected fluorescence of the reporter dye. In an intact probe the fluorescence is quenched due to the close proximity of the reporter dye and quencher dye. During PCR, the nuclease activity of the DNA polymerase cleaves the probe in the 5′-3′ direction and thus separates the reporter dye from the quencher dye. Because reporter and quencher dye are then no longer in close proximity to each other, the fluorescence of the reporter dye is increased. The increase in fluorescence is measured and is directly proportional to the amplification during PCR. See Heid et al. (1996), “Real time quantitative PCR” Genome Research 6(10):986-994. This detection system is now commercially available as the TaqMan® PCR system from Perkin-Elmer, which allows real time PCR detection.

[0249] In an alternative embodiment, the reporter dye and quencher dye may be located on two separate probes which hybridize to the amplified PCR detector molecule in adjacent locations sufficiently close to allow the quencher dye to quench the fluorescence signal of the reporter dye (Rasmussen et al. (1998), “Quantitative PCR by continuous fluorescence monitoring of a double strand DNA specific binding dye” Biochemica 2:8-15). As with the detection system described above, the 5′-3′ nuclease activity of the polymerase cleaves the one dye from me probe containing it, separating the reporter dye from the quencher dye located on the adjacent probe preventing quenching of the reporter dye. As in the embodiment described above, detection of the PCR product is by measurement of the increase in fluorescence of the reporter dye.

[0250] In other embodiments of this invention, other real time PCR detection strategies may be used, including known techniques such as intercalating dyes (ethidium bromide) and other double stranded DNA binding dyes used for detection (e.g. SYBR green, FMC Bioproducts), dual fluorescent probes (Wittwer et al. (1977) BioTechniques 22:130-138 and Wittwer et al. (1997) BioTechniques 22:176-181) and panhandle fluorescent probes (i.e. molecular beacons; Tyagi and Kramer (1996) Nature Biotechnology 14:303-308). Although intercalating dyes and double stranded DNA binding dyes permit quantitation of PCR product accumulation in real time applications, they suffer from a lack of specificity, detecting primer dimer and any non-specific amplification product. Careful sample preparation and handling, as well as careful primer design, using known techniques are necessary to minimize the presence of matrix and contaminant DNA and to prevent primer dimer formation. Appropriate PCR instrument analysis software and melting temperature analysis permit a means to extract specificity (Ririe, K., et al. (1977) Anal. Biochem. 245:154-160) and may be used with these embodiments.

[0251] In still another embodiment of this invention, the Scorpions reaction is used as a real time PCR detection method. Scorpions are bi-functional molecules containing a PCR primer covalently linked to a probe. The fluorophore in the probe interacts with a quencher which reduces fluorescence. During the PCR reaction the primer binds to the template and is elongated by the polymerase. Once the elongation reaction is completed and primer and template are separated in the denaturation step, the elongated primer sequence can interact intramolecularly with the probe sequence in the next annealing step. The binding of the probe to the elongated primer sequence prevents interaction of the probe-bound fluorophore with the quencher, which leads to an increase in light output from the reaction tube. Currently, there are two formats for Scorpions, the bimolecular Scorpion format and the unimolecular format. In the bimolecular format the quencher is bound to a separate nucleic acid molecule which is complementary to the probe sequence, whereas in the unimolecular format both, fluorophore and quencher, are attached to the same molecule, and an integral stem loop sequence is used to bring the quencher close to the fluorophore.

[0252] The Scorpions technique is described more fully in Whitcombe et al. (1999), Detection of PCR products using self-probing amplicons and fluorescence, Nature Biotech 17, pages 804-807. This detection system is now commercially available as the scorpions system from DxS Ltd. (Manchester, UK).

[0253] The design of primers for the amplification reaction and nucleic acid probes is well-established in the art and thus routine practice for the skilled person. Suitable fluorescent reporter dyes are also known and commercially available, and include, without limitation 6-carboxy-fluorescein (FAM), tetrachloro-6-carboxy-fluorescein (TET), 2,7-dimethoxy-4,5-dichloro-6-carboxy-fluorescein (JOE) and hexachloro-6-carboxy-fluorescein (HEX). Another suitable reporter dye is 6-carboxy-tetramethylrhodamine (TAMRA).

[0254] In another aspect, the invention relates to a kit i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the detection method or the diagnostic method of the invention. Such a kit comprises one or more detection complexes according to the invention or manufactured according to the methods of the invention. Such a kit may additionally contain further components. Exemplary components that may be additionally comprised in the kits of the present invention include, but are not limited to stabilizers, buffers (e.g. a block buffer or lysis buffer), dyes, oligonucleotide primers or probes, which may be optionally labelled with a detectable label, etc. The components of the detection complexes according to the invention may be as defined above.

[0255] Furthermore, the antibodies used in the methods of the present invention can also be provided in the kit.

[0256] The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.

[0257] According to a further aspect of the invention, there is provided a kit for diagnosing a neurodegenerative disorder, such as Alzheimer's disease which comprises detection complexes according to the invention or manufactured according to the methods of the invention, at least one capture antibody and instructions for use. In one embodiment, the kit additionally comprises at least one capture antibody that specifically binds to the pyroglutamate carrying amino terminus of said pGlu-Aβ peptide.

[0258] In still another aspect, the invention is also directed to the use of one or more organic polymer, polypeptide, polysaccharide and/or oligo- or polynucleotide molecules, all of which may be optionally biotinylated, as additional linker molecules in a detection comprising one or more non-nucleic acid receptors, one or more nucleic acid markers and one or more first linker molecules to form a detection complex comprising one or more non-nucleic acid receptors, one or more nucleic acid markers, one or more first linker molecules and one or more organic polymer, polypeptide, polysaccharide and/or oligo- or polynucleotide molecules.

[0259] The biological sample may be any sample, for example from a human. In one specific example, the sample is a tissue sample, a body fluid sample or a cell sample. In one embodiment, the biological sample is selected from the group consisting of blood, serum, urine, cerebrospinal fluid (CSF), plasma, lymph, saliva, sweat, pleural fluid, synovial fluid, tear fluid, bile and pancreas secretion. In a further embodiment, the biological sample is plasma. In a preferred embodiment, the biological sample is CSF.

[0260] The biological sample can be obtained from a patient in a manner well-known to a person skilled in the art. In particular, a blood sample can be obtained from a subject and the blood sample can be separated into serum and plasma by conventional methods. The subject, from which the biological sample is obtained is preferably a subject suspected of being afflicted with Alzheimer's disease, at risk of developing Alzheimer's disease and/or being at risk of or having any other kind of dementia. In particular, the sample is obtained from a subject suspected of having Mild Cognitive Impairment (MCI) and/or being in the early stages of Alzheimer's disease.

[0261] The invention further relates to the use of the method for the detection of a pGlu-Aβ peptide according to the present invention in a method of diagnosing or monitoring a neurodegenerative disease, such as Alzheimer's disease and Mild Cognitive Impairment.

[0262] In particular, the invention provides 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 subject, comprising the following steps: [0263] 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 for the detection of a pGlu-Aβ peptide according to the present invention; [0264] ii. comparing the first level of the pGlu-A Aβ peptide with a second level of said pGlu-Aβ peptide in a healthy control subject; and [0265] 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.

[0266] In a further embodiment, the invention provides 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 the level of a pGlu-Aβ peptide in a biological sample from a subject with a method for the detection of a pGlu-Aβ peptide according to the present invention.

[0267] In a particular embodiment, said method of diagnosing or said 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, comprises the determination of the level of a pGlu-Aβ peptide in a biological sample taken on two or more occasions from a subject.

[0268] In one embodiment, the biological sample will be taken on two or more occasions from a test subject. In a further embodiment, the method additionally comprises comparing the level of the pGlu-Aβ peptides present in biological samples taken on two or more occasions from a test subject. In one embodiment, the method additionally comprises comparing the level of the pGlu-Aβ peptides present in a test sample with the amount present in one or more sample(s) taken from said subject prior to commencement of therapy, and/or one or more samples taken from said subject at an earlier stage of therapy. In one embodiment, the method additionally comprises comparing the level of the pGlu-Aβ peptides with one or more controls.

[0269] In a further embodiment, said method of diagnosing or said method of monitoring the efficacy of a therapy in a subject further comprises a step, wherein the state of the neurodegenerative disease of the subjects that are donors of the biological samples is characterized in one or more psychometric tests. Suitable psychometric tests for characterization of the state of the neurodegenerative disease of a subject are 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 7 Minute Screen.

[0270] Suitable treatments of neurodegenerative diseases, such as Alzheimer's diseases and/or Mild Cognitive Impairment, the efficacy of which can be monitored with the methods of the present invention, are treatments that inhibit the formation of the pGlu-residue at the N-terminus of N-terminally truncated Aβ peptides.

[0271] Particularly suitable treatments in this regard are inhibitors of the enzyme glutaminyl cyclase. Glutaminyl cyclase has been shown to catalyse the formation of pGlu at the N-terminus of peptides not only from a glutamine residue, but also from a glutamate residue. Accordingly, glutminyl cyclase is responsible for the posttranslational formation of glutamate residues at position 3 or 11 of Aβ peptide to pGlu.

[0272] Suitable glutaminyl cyclase inhibitors for the treatment of neurodegenerative diseases, such as Alzheimer's diseases and/or Mild Cognitive Impairment, are for example disclosed in WO 2005/075436, WO 2008/055945, WO 2008/055947, WO 2008/055950, W02008/065141, WO 2008/110523, WO 2008/128981, WO 2008/128982, WO 2008/128983, WO 2008/128984, WO 2008/128985, WO 2008/128986, WO 2008/128987, WO 2010/026212, WO 2010/012828, WO 2011/107530, WO 2011/110613, WO 2011/131748, WO 2012/123563 and WO 2014/140279.

[0273] Further suitable treatments of neurodegenerative diseases, such as Alzheimer's diseases and/or Mild Cognitive Impairment, are antibodies, preferably beta-amyloid antibodies, more preferably antibodies that specifically recognize pGlu-Aβ peptides. Suitable pGlu-Aβ antibodies are for example disclosed in WO 2010/009987, WO 2012/123562, U.S. Pat. No. 7,122,374 81, WO 2011/151076, WO 2012/021469; WO 2012/136552 and WO 2010/129276.

[0274] In a preferred embodiment, the invention provides a method for monitoring the efficacy of inhibitors of glutaminyl cyclase and/or beta-amyloid antibodies, most preferably antibodies that specifically recognize pGlu-Aβ peptides, in the treatment of neurodegenerative diseases, such as Alzheimer's diseases and/or Mild Cognitive Impairment.

[0275] The present method of diagnosis has several advantages over the methods known in the art, i.e. the method of the present invention can be used to detect Alzheimer's disease at an early stage and to differentiate between Alzheimer's disease and other types of dementia in early stages of disease development and progression. One possible early stage is Mild Cognitive Impairment (MCI). It is impossible with the methods currently known in the art to make a clear and reliable diagnosis of early stages of Alzheimer's disease and, in particular, it is impossible to differentiate between the onset of Alzheimer's disease and other forms of dementia in said early stages. This especially applies for patients afflicted with MCI.

[0276] In contrast, the methods provided by the present invention are suitable for a differential diagnosis of Alzheimer's disease. In particular, the present invention provides a diagnostic method, wherein the level of pGlu-Aβ peptides can be detected in biological samples obtained from any of the above described subjects in a highly sensitive and reproducible manner. The high sensitivity of the methods of the present invention is achieved by using the detection complex of the invention, the antibodies that are highly specific for the detection of pGlu-Aβ peptides; and the immune-PCR method for the detection and/or quantification of pGlu-Aβ peptides. With the method of the present invention, it is for the first time possible to detect trace amounts or very low amounts of pGlu-Aβ peptides, i.e. down to 4.2 fg/ml, in biological samples such as plasma or CSF. The invention provides a method for the detection of pGlu-Aβ peptides, which is highly sensitive, independently from whether the pGlu-Aβ peptides are present as monomers, in oligomers or bound to proteins in the sample. It is especially possible to detect the occurrence of pGlu-Aβ peptides in a biological sample already closely to or even prior to the onset of Alzheimer's diseases.

[0277] The method of the present invention makes it possible for the first time to detect and quantify pGlu-Aβ peptides, in particular those of SEQ ID NOs: 26-37, preferably of SEQ ID NOs: 26-31 and even preferably of SEQ ID NOs: 32-37; or fragments or functional variants thereof, in a highly sensitive manner. In particular, the present invention provides pGlu-Aβ peptides as a biomarker biological fluids, such as plasma or CSF, which is suitable for a differential diagnosis of Alzheimer's disease, in particular in the early stages of the disease.

[0278] Therefore, in one embodiment, the invention is directed to the use of the method of determining pGlu-Aβ peptides for the diagnosis of Alzheimer's disease, such as the differential diagnosis of Alzheimer's disease, in particular in the early stages of the disease. Suitably, the early stage of Alzheimer's disease is Mild Cognitive impairment.

[0279] In a further embodiment, the invention is directed to the use of the pGlu-Aβ peptides for the diagnosis of Alzheimer's diseases, such as the differential diagnosis of Alzheimer's disease, in particular in the early stages of the disease. Suitably, the early stage of Alzheimer's disease is Mild Cognitive impairment.

[0280] In particular, the pGlu-Aβ peptides, which shall be used for diagnosis of Alzheimer's disease, are detected and quantified with a method according to the present invention.

Deposits of Biological Material

[0281] The monoclonal antibodies expressing hybridoma cell lines 5-5-6, 6-1-6, 17-4-3, and 24-2-3 have been deposited in accordance with the Budapest Treaty and are available at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures) GmbH, DSMZ, Inhoffenstrasse 7B, 38124 Braunschweig,

[0282] Germany, with a deposit date of Jun. 17, 2008, and with the respective deposit numbers: [0283] (clone 5-5-6): DSM ACC2923 [0284] (clone 6-1-6): DSM ACC2924 [0285] (clone 17-4-3): DSM ACC2925 [0286] (clone 24-2-3): DSM ACC2926.

[0287] The monoclonal antibody expressing hybridoma cell line 13-11-6 has been deposited in accordance with the Budapest Treaty and is available at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures) GmbH, DSMZ, Inhoffenstrasse 7B, 38124 Braunschweig, Germany, with a deposit date of Dec. 14, 2010, and with the deposit number: [0288] (clone 13-11-6): DSM ACC 3100.

[0289] The present invention is further described by the following examples, which should however by no means be construed to limit the invention in any way; the invention is defined in its scope only by the claims as enclosed herewith.

EXAMPLES OF THE INVENTION

Example 1

Highly Sensitive Detection of pGlu-Aβ Peptide

[0290] Microplate modules (Chimera biotec C-001) were coated with 30 μl/well capture antibody (clone 6, 17 or 24, probiodrug) at a concentration of 5 μg/m1 in coating buffer (Chimera biotec C-010). Coating was carried out overnight at 4° C. Subsequently, coated modules were washed with wash buffer A (Chimera Biotec, C-011). All washing steps were carried out according to wash buffer manufacturer's instructions. The washed modules were incubated with 30 μl/well sample material, consisting of artificial CSF (Chimera biotec,) spiked with pGlu-Aβ (3-40) or (3-42) (Probiodrug), at different concentration levels and diluted 1+9 in sample dilution buffer (SDB-9100, Chimera Biotec). Incubation was carried out for 45 min at room temperature, followed by a washing step with wash buffer B (Chimera Biotec, C-012). Subsequently, wells were incubated with 30 μl/well biotinylated detection antibody (clone 17 or clone 24, Probiodrug) in a concentration of 0.2 μg/m1 in antibody dilution buffer (SDB-6000, Chimera Biotec). Incubation was carried out for 45 min at room temperature, followed by a washing step with wash buffer B. Subsequently, wells were incubated with 30 μl/well CHI—BIO biotin-binding detection conjugate containing DNA-marker (Chimera biotec,), applied in 1:200 dilution in conjugate dilution buffer (CDB-b, Chimera biotec) for 30 min at room temperature. Following a final washing step with buffer B and buffer A, 30 μl/well PCR-mastermix (Chimera Biotec, C-022) corresponding to the DNA-marker in CHI—BIO are added to each well. The microplate is sealed with PCR-foil (Chimera biotec, C-069) and DNA-amplification & data read-out is carried out according to manufacturer's instructions by application of an lmperacer® workstation (Chimera Biotec 25-002).

Results

[0291]

TABLE-US-00004 TABLE 1 Quantification of pGlu-Aβ(3-40) with capture antibody clone 17 or clone 24 Calculated conc [pg/ml] nominal Capture: % RE [pg/ml] Clone 17 Clone 24 Clone 17 Clone 24 100000 112278.5 102939.9 12 3 10000 9516.6 9619.4 5 4 1000 1075.8 1169.0 8 17 100 33.3 77.7 67 22 10 51.1 10.5 411 5

TABLE-US-00005 TABLE 2 Quantification of pGlu-Aβ(3-42) with capture antibody clone 6 nominal calc. Conc. [pg/ml] [pg/ml] % RE 1 1.1 8.9 10 8.3 16.7 100 114.2 14.2 1000 958.4 4.2 10000 9542.0 4.6 100000 115430.7 15.4 1000000 872503.9 12.7

Example 2

Application of an Antibody—DNA Detection Complex for the Detection of pGlu-Aβ Peptides in a Biological Sample

[0292] Microplate modules (Chimera biotec C-001) were coated with 30 μl/well capture antibody (clone 24, Probiodrug) at a concentration of 5 μg/m1 in coating buffer (Chimera biotec C-010). Coating was carried out overnight at 4° C. Subsequently, coated modules were washed with wash buffer A (Chimera Biotec, C-011). All washing steps were carried out according to wash buffer manufacturer's instructions. The washed modules were incubated with 30 μl/well sample material, consisting of artificial CSF (Chimera biotec) spiked with pGlu-Aβ (1+1 mixture of 40 & 42, Probiodrug) at different concentration levels as reference standards and individual CSF for analysis. The sample material was additionally mixed 1+0.03 (one part sample+0.03 part reagent) with an antibody-DNA detection complex (CHI-pGlu, Chimera Biotec, synthesized from clone 17-24, Probiodrug) at sub μg/m1 in artificial CSF (Chimera biotec). Pre-incubation of samples and detection complex was carried out overnight at 4° C. in vials previous to incubation on capture-coated modules; subsequent incubation on capture-coated wells was carried out for 60 min at room temperature, respectively. Following a final washing step with buffer B and buffer A, 30 μl/well PCR-mastermix (Chimera Biotec, C-022) corresponding to the

[0293] DNA-marker in CHI—BIO are added to each well. The microplate is sealed with PCR-foil (Chimera biotec, C-069) and DNA-amplification & data read-out is carried out according to manufacturer's instructions by application of an Imperacer® workstation (Chimera Biotec 25-002).

Results:

[0294]

TABLE-US-00006 TABLE 3 Standards nominal Measured calc. Conc. Accuracy Precision [pg/ml] delta Ct [pg/ml] % RE % CV 0.046 11.79 0.036 22.11 2.4 0.137 12.85 0.168 22.39 13.6 0.412 13.75 0.487 18.28 62.8 1.235 14.51 1.065 13.79 10.3 3.703 15.91 3.755 1.40 13.8 11.11 17.28 10.96 1.33 5.3 33.33 18.81 32.23 3.31 12.1 100 20.60 103.2 3.19 21.2

TABLE-US-00007 TABLE 4 Individuals Calculated Concentration # [pg/ml] CV % I 0.2 31.2 II 1.0 9.3 III 1.9 1.4 IV 2.9 10.3

Example 4

Detection of pGlu3-Aβ Peptides with Pan-Specific anti-β-amyloid Antibodies

[0295] The assay protocol of example 3 was repeated with the following modifications: [0296] a) pan-specific antibodies 6E10, BAM90.1 (epitope aa 13-28) or 12F4(specific for Aβ42

[0297] C-terminus) were used as detection antibodies in the detection antibody-DNA-conjugate (ADC); and [0298] b) pGlu3-Aβ peptide specific monoclonal antibody clone 6-1-6, clone 17-4-3 or clone 24-2-3 were used as capture antibody for coating the microplate modules.

[0299] The preparation of the antibody-DNA-conjugate (ADC) and the detection of pGlu3-Aβ40 and pGlu3-Aβ42 was performed as described in examples 1, 2 and 3.

[0300] 300 pg/ml of pGlu3-Aβ40 and pGlu3-Aβ42 peptides were detected in the analyzed CSF samples. Antibody clone 6E10 as detection antibody revealed best performance with pGlu3-Aβ peptide specific monoclonal antibody clone 6-1-6 or clone 24-2-3 as capture antibody (see FIG. 1).

Example 4

Preparation of Antibody—DNA Detection Complexes for Detection of pGlu-Aβ Peptides

[0301] 30 μI of a 2.11 pmol/ml solution of 169 bp bis-biotinylated DNA (DNA-marker “1” (SEQ ID NO: 60); 63.3. pmol) were incubated for 30 min at RT with 3.24 μI of a 19.5 pmol/μl solution of recombinant STV (streptavidin, IBA) to form a STV-DNA conjugate (“SDC”). 30 μI of this SDC were mixed with 30 μI of a 500 μg/mI solution of a biotinylated anti-pGlu3-Aβ detection antibody selected from clones 6, 17 and 24 and incubated for 60 min at RT/orbital shaking. The antibody-DNA-STV conjugate was purified by FPLC (Superdex 200) and the 1 ml product fraction was mixed with 2 ml NaCl solution (300 mM) for a final solution of 10.5 pmol/ml detection antibody-DNA-conjugate (“ADC”) (cf. Niemeyer et al., (1999). Nucleic Acids Res 27(23): 4553-61).

Example 5

Validation of the pGlu-Aβ Assay

[0302] Standard curve and quality controls (QCs) samples with concentrations of pGlu3-Aβ(40/42) of SEQ ID NOs: 28 and 30 were prepared and evaluated according to Table 5. The “low series” contained pGlu3-Aβ(40/42) SEQ ID NOs: 28 and 30 in the range from 4.2 fg/ml up to 9 pg/ml in artificial human CFS. The “high series” contained pGlu3-Aβ(40/42) SEQ ID NOs: 28 and 30 in the range from 78 fg/ml up to 27 pg/ml in artificial human CSF.

[0303] The artificial human CSF consists of: [0304] NaCl: 125 mM [0305] KCI: 2.5 mM [0306] MgCI×6 H.sub.2O: 1 mM [0307] NaH.sub.2PO.sub.4: 1.25 mM [0308] CaCl.sub.2×2 H.sub.2O: 2.0 mM [0309] NaHCO.sub.3: 25 mM [0310] Glucose: 25 mM [0311] pH adjusted with NaOH to 7.3 [0312] Human serum albumine: 0.3 g/I

[0313] Detection of pGlu3-Aβ(40/42) of SEQ ID NOs: 28 and 30 was performed as described in Example 3.

[0314] Acceptance criteria for precision (% CV) and accuracy (% RE) for different series (“high” and “low”) of standards and QCs were <20% for the lower limit of quantitation (LLOQ) and <25% for the upper limit of quantitation (ULOQ).

[0315] FIG. 2 shows the recovery plot for high and low concentration of standards. It can be seen from FIG. 2 that the developed method allows the detection and quantitation of pGlu-Aβ peptides over a broad linear range of the calibration curve with high precision and accuracy. The method is very sensitive and allows the quantitation of pGlu-Aβ peptides down to 4.2 fg/ml.

TABLE-US-00008 TABLE 5 Inter-Assay Average: Precision (% CV) and Accuracy (% RE) for “high” and “low” standards and QCs pg/ml “high series” pg/ml “low series” [nominal] % CV % RE [nominal] % CV % RE Standards Standards 27 1.18 0.8 9 3.10 1.5 13.5 0.94 1.6 3 0.08 2.3 6.75 1.29 0.7 1 5.24 2.6 3.375 7.06 5.8 0.34 11.10 1.8 1.6875 2.76 12.0 0.12 5.92 11.2 0.84375 7.34 9.0 0.042 21.55 7.8 0.421875 5.14 1.5 0.0124 0.66 8.3 0.210938 16.14 6.5 0.0042 17.71 8.3 0.078125 6.80 2.3 QCs QCs 10 12.20 8.6 3.34 0.64 11.7 5 30.03 1.5 1.12 11.21 8.3 2.5 18.95 6.5 0.38 26.29 16.10.2014 1.25 1.52 3.0 0.13 12.35 0.7 0.625 18.71 3.2 0.0138 4.64 26.2 0.3125 17.59 5.7 0.0046 5.52 22.0 0.15625 2.44 4.7

Example 5

Determination of pGlu3-Aβ40 (SEQ ID NO: 28) and pGlu3-Aβ42 (SEQ ID NO: 30) in CSF Samples

[0316] CSF samples were obtained from patients with a clinical diagnosis of AD and healthy controls according to standard procedures.

[0317] A standard curve and quality control samples (QCs) were generated and used as described in Example 5. Acceptance criteria for precision (% CV) and accuracy (% RE) for the CSF samples and QCs were <20% for the lower limit of quantitation (LLOQ) and <25% for the upper limit of quantitation (ULOQ). Individual CSF samples (see sample # in Table 6) were measured as described in Example 3. The pGlu3-Aβ concentration was calculated based on the standard curve.

[0318] Table 6 shows the results of the quantitation of pGlu3-Aβ40 (SEQ ID NO: 28) and pGlu3-Aβ42) (SEQ ID NO: 30). The values for the pGlu-Aβ concentration represent the concentration of pGlu3-Aβ40 (SEQ ID NO: 28) and pGlu3-Aβ42 (SEQ ID NO: 30) as a sum parameter.

TABLE-US-00009 TABLE 6 Analyzed sample concentration (pGlu3-Aβ40 (SEQ ID NO: 28) and pGlu3-Aβ42 (SEQ ID NO: 30) in pg/ml) and precision (% CV) pGlu-Aβ concentration Sample # [pg/ml] % CV 1 0.3 26.5 2 0.7 21.1 3 0.9 20.4 4 1.5 1.4 5 1.5 22.0 6 1.6 29.5 7 5.0 1.1 8 5.1 8.6 9 6.8 22.4

[0319] The precision (% CV) is used as an acceptance criterion for biomarkers. The precision threshold for a biomarker to accepted is % CV <30%. The results in Table 6 show that the precision (% CV) in all measurements was <30% and therefore meet the precision acceptance criterion for biomarkers.

Example 7

Psychometric Tests for Identification of Subjects Suffering from a Neurodegenerative Disease

7.1 Materials and Methods

[0320] 7.1.1 Patients and healthy controls

[0321] Patients with a clinical diagnosis of AD and healthy controls were recruited through a CRO. In a prestudy examination the neuropsychological functions of all participants of the study were tested by several psychometric tests (DemTect, Mini-Mental-State Test, Clock-drawing test).

DemTect Test

[0322] The DemTect scale is a brief screening for dementia comprising five short subtests (10-word list repetition, number transcoding, semantic word fluency task, backward digit span, delayed word list recall) (Kessler et al., 2000). The raw scores are transformed to give age- and education-independent scores, classified as ‘suspected dementia’ (score ≦8), ‘mild cognitive impairment’ (score 9-12), and ‘appropriate for age’ (score 13-18).

MMSE

[0323] The Mini-Mental State Examination (MMSE) or Folstein test is a brief 30-point questionnaire test that is used to assess cognition (see Table 4). It is commonly used in medicine to screen for dementia. In the time span of about 10 minutes it samples various functions including arithmetic, memory and orientation. It was introduced by Folstein et al., 1975, and is widely used with small modifications.

[0324] The MMSE includes simple questions and problems in a number of areas: the time and place of the test, repeating lists of words arithmetic, language use and comprehension, and basic motor skills. For example, one question asks to copy drawing of two pentagons (see next table). Any score over 27 (out of 30) is effectively normal. Below this, 20-26 indicates mild dementia; 10-19 moderate dementia, and below 10 severe dementia. The normal value is also corrected for degree of schooling and age. Low to very low scores correlate closely with the presence of dementia, although other mental disorders can also lead to abnormal findings on MMST testing.

Clock-Drawing Test

[0325] Scoring of the clocks was based on a modification of the scale used by Shulmann et al., 1986. All circles were pre-drawn and the instruction to subjects was to “set the time 10 after 11”. The scoring system (see Table 5) ranges in scores from 1 to 6 with higher scores reflecting a greater number of errors and more impairment. This scoring system is empirically derived and modified on the basis of clinical practice. Of necessity, it leaves considerable scope for individual judgment, but it is simple enough to have a high level of interrater reliability. Our study lends itself to the analysis of the three major components. These include cross-sectional comparisons of the clock-drawing test with other measures of cognitive function; a longitudinal description of the clock-drawing test over time, and the relationship between deterioration on the clock-drawing test and the decisions to institutionalize.

TABLE-US-00010 TABLE 7 Mini-Mental State Examination Max Section Questions Points Score 1) Orienta- a) Can you tell me today's 5 tion (date)/(month)/(year)? Which day is it today? Can you tell me which (season) it is? b) What town/city are we in? 5 What is the (county)! (country)? What (building) are we in and on what (floor) 2) Registra- I should like to test your memory. 3 tion (name three common objects: “ball, car, man”) Can you repeat the words I said? (1 point per word) (repeat up to 6 trials until all three are remembered) 3) Attention a) From 100 keep subtracting 7 and given 5 and each answer. Stop after 5 answers. Calculation (93-86-79-72-65) Alternatively: b) Spell the word “World” backwards. (D_L_R_O_W) 4) Recall What were the three words I asked you to 3 say earlier? (skip this test if all of these objects were not remembered during the registration test) 5) Language Name the following objects (show a watch) 2 Naming and (show a pencil) Repeating Repeat the following: “no if, ands or buts” 1 6) Reading (show card or write: “Close your Eyes”) 1 Read this sentence and do what it says. Writing Now can you write a short sentence for me? 1 7) Three (present paper) 3 stage Take this paper in your left (or right) hand, command fold it in half, and place it on the floor. 8) Con- Will you copy this drawing please? 1 struction [00001] Total score 30 

[0326] After prestudy examination the study started 2 weeks later with blood withdrawal from all participants. Over one year with an interval of 3 months all participants had visited the center for the psychometric tests and blood samples withdrawal. The study was approved by the Ethics Committee of the “Ärztekammer Sachsen-Anhalt”. All patients (or their nearest relatives) and controls gave informed consent to participate in the study.

6.1.2 Biological Samples

[0327] For the analysis of the pGlu-Aβ concentration in humans all of the following body fluids can be used: blood, cerebrospinal fluid, urine, lymph, saliva, sweat, pleura fluid, synovial fluid, aqueous fluid, tear fluid, bile and pancreas secretion.

[0328] The novel method was established with CSF samples and can be further used for blood, brain extract and urine samples, followed by all other human body fluids.

[0329] CSF samples for the determination of AD biomarkers were collected into three polypropylene tubes: [0330] 1. containing potassium-EDTA (Sarstedt Monovette, 02.1066.001) for EDTA plasma [0331] 2. containing Li-heparine (Sartstedt Monovette, 02.1065.001) for heparine plasma [0332] 3. blank (Sarstedt Monovette, 02.1063.001) for serum

[0333] All samples were collected by venous puncture or by repeated withdrawal out of an inserted forearm vein indwelling cannula. Blood was collected according to the time schedule (as described in section 1.1 above). It was centrifuged at 1550 g (3000 rpm) for 10 min at 4° C. to provide plasma. Plasma or serum was pipetted off, filled in one 5 ml polypropylene cryo-tube (Carl-Roth, E295.1) and stored frozen at −80° C. Samples were centrifuged within one hour after blood withdrawal. The appropriate labelling of the plasma or serum tubes according to the study protocol was duty of the CRO.

7.2 Results

7.2.1 Demographic Characteristics

[0334] Overall 45 persons have participated in the study, 30 healthy controls and 15 AD patients. To observe possible influences of age on plasma Aβ, control persons were selected over a wide range of age and subclassified into three groups, Group I contains age of 18 to 30, Group II from 31 to 45 and Group III from 46 to 65. The demographic characteristics are shown in Table 9.

TABLE-US-00011 TABLE 9 Demographic Characteristics Healthy controls Group I Group II Group III (18-30) (31-45) (46-65) AD patients No. 10 10 10 15 Age at baseline 25.8 ± 2.9 38.4 ± 4.7 .sup. 54 ± 6.9 79.13 ± 7.09  (mean ± SDEV), Height, cm 175.5 ± 11.6 175.1 ± 7.2  167.5 ± 10.9 168.4 ± 10.34 (mean ± SDEV) Weight, kg 71.33 ± 11.8 71.36 ± 13.5 75.81 ± 13.3 72.00 ± 12.31 (mean ± SDEV) Sex (% women) 50 50 50 40

2.2 Psychometric Tests

[0335] For evaluation of the neuropsychological functions all participants have performed the DemTect, Mini-Mental-State Test and Clock-Drawing test. These tests have been made in prestudy, 3 month, 6 month, 9 month and 12 month after the start of the study.

DemTect Test The raw scores are transformed to give age- and education-independent scores, classified as ‘suspected dementia’ (score ≦8), ‘mild cognitive impairment’ (score 9-12), and ‘appropriate for age’ (score 13-18). The test results for all visits are shown in FIG. 3. The results from FIG. 3 demonstrate that there are clear differences between the three groups of healthy subjects compared with the patients.

Mini-Mental-State Test

[0336] Any score over 27 (out of 30) is effectively normal. Below this, 20-26 indicates mild dementia; 10-19 moderate dementia, and below 10 severe dementia. The normal value is also corrected for degree of schooling and age. Low to very low scores correlate closely with the presence of dementia, although other mental disorders can also lead to abnormal findings on MMST testing. The test results are shown in FIG. 4. The results from FIG. 4 demonstrate that there are clear differences between the three groups of healthy subjects compared with the patients.

Clock-Drawing Test

[0337] The scoring system ranges in scores from 1 to 6 with higher scores reflecting a greater number of errors and more impairment. This scoring system is empirically derived and modified on the basis of clinical practice. Of necessity, it leaves considerable scope for individual judgment, but it is simple enough to have a high level of interrater reliability.

[0338] Our study lends itself to the analysis of the three major components. These include cross-sectional comparisons of the clock-drawing test with other measures of cognitive function; a longitudinal description of the clock-drawing test over time, and the relationship between deterioration on the clock-drawing test and the decisions to institutionalize. The test results are shown in FIG. 5. The results from FIG. 5 demonstrate that there are clear differences between the three groups of healthy subjects compared with the patients.

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