GLUTAMINYL CYCLASE AS A DIAGNOSTIC/PROGNOSTIC INDICATOR FOR NEURODEGENERATIVE DISEASES

Abstract

A method for predicting, diagnosing and prognosticating a neurodegenerative disease, such as Alzheimer's disease (AD), Mild Cognitive Impairment (MCI) and neurodegeneration in Down's syndrome (NDS) using glutaminyl cyclase (QC) as a diagnostic/prognostic indicator. The use of antibodies binding to QC and kits for performing said diagnostic method are also provided.

Claims

1. A method for diagnosing a neurodegenerative disease in a subject, the method comprising: (a) detecting an amount of glutaminyl cyclase (QC), or an isoform thereof, in a biological sample of said subject and comparing the detected amount of QC in the biological sample with an amount of QC characteristic of a normal control; wherein an elevated amount of QC in said biological sample relative to the normal control is a positive indicator of the neurodegenerative disease; and the neurodegenerative disease is selected from the group consisting of Alzheimer's Disease (AD), Neurodegeneration in Down's Syndrome (NDS) and Mild Cognitive Impairment (MCI); and optionally, (b) detecting an amount of A N3pE-X and comparing the detected amount A N3pE-X in the biological sample with an amount of A N3pE-X characteristic of a normal control; wherein an elevated amount of QC and A N3pE-X in said biological sample relative to the normal control is a positive indicator of the neurodegenerative disease; and X is an integer selected from the group consisting of 38, 40, and 42; and optionally, (c) detecting an amount of a chemokine and comparing the detected amount of the chemokine in the biological sample with an amount of chemokine characteristic of a normal control; wherein an elevated amount of QC and chemokine in said biological sample relative to the normal control is a positive indicator of the neurodegenerative disease; and optionally, (d) detecting an amount of A 1-42 and comparing the detected amount A 1-42 in the biological sample with an amount of A 1-42 characteristic of a normal control; wherein an elevated amount of QC and A 1-42 in said biological sample relative to the normal control is a positive indicator of the neurodegenerative disease.

2. The method of claim 1, comprising: b) detecting an amount of A N3pE-X and comparing the detected amount A N3pE-X in the biological sample with an amount of A N3pE-X characteristic of a normal control; wherein an elevated amount of QC and A N3pE-X in said biological sample relative to the normal control is a positive indicator of the neurodegenerative disease; and X is an integer selected from the group consisting of 38, 40 and 42.

3. The method of claim 1, comprising: c) detecting an amount of a chemokine and comparing the detected amount of the chemokine in the biological sample with an amount of chemokine characteristic of a normal control; wherein an elevated amount of QC and chemokine in said biological sample relative to the normal control is a positive indicator of the neurodegenerative disease.

4. The method according to claim 1, wherein said QC is human QC or an isoform thereof, having an amino acid sequence selected from the group consisting of SEQ ID NO 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID: NO: 4; and SEQ ID NO: 5.

5. The method according to claim 4, wherein said QC is human QC of SEQ ID NO: 1.

6. The method according to claim 1, wherein said biological sample is serum, plasma, urine or cerebrospinal fluid.

7. The method according to claim 6, wherein said biological sample is plasma.

8. The method according to claim 1, wherein the amount of QC is detected by immunoturbidimetric assay, immunofluorescence, immunodiffusion, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Western blot, protein activity assay, Northern Blot, PCR, high performance liquid chromatography (HPLC), mass spectrometry (MS), gas chromatography (GC), GC-MS, LC-MS, or LC-MS/MS.

9. The method according to claim 1, wherein the amount of QC, or an isoform thereof, is detected on the basis of the protein level of said QC or isoform thereof.

10. The method according to claim 1, wherein the amount of QC is detected using an antibody that specifically binds to QC, or an isoform thereof.

11. The method according to claim 1, wherein the amount of QC is detected by measuring the enzymatic activity of QC, or an isoform thereof.

12. The method according to claim 1, wherein the amount of QC, or an isoform thereof, is detected on the basis of the mRNA level of said QC or isoform thereof.

13. The method of claim 2, wherein X is 42.

14. The method of claim 2, wherein X is 40.

15. The method of claim 2, wherein X is 38.

16. The method according to claim 2, wherein detecting an amount of AR N pE-X comprises detecting (i) one or more of A N3pE-42, A N3pE-40, and A N3pE-38 and (ii) at least one of pGluABri or pGluADan.

17. The method according to claim 16, wherein detecting an amount of A N3pE-X comprises detecting (i) two or more of A N3pE-42, A N3pE-40, and A N3pE-38 and (ii) at least one of pGluABri or pGluADan.

18. The method according to claim 3, wherein said chemokine is selected from the group consisting of CCL2, CCL7, CCL8, CCL9/10, CCL13, CCL15, CCL16, CCL25 and Fractalkine.

19. The method according to claim 18, wherein said chemokine is CCL2.

20. The method of claim 1, further comprising: obtaining a biological sample from said subject; wherein detecting the amount of glutaminyl cyclase (QC), or an isoform thereof, in the biological sample of said subject comprises: i) contacting said biological sample with an antibody that binds to glutaminyl cyclase (QC), or its isoforms; ii) allowing the antibody and QC to form an immune complex; and iii) detecting the amount of immune complex formed as an indication of the amount of QC in said biological sample.

21. The method of claim 1, wherein detecting the amount of QC, or an isoform thereof, occurs in vitro.

22. The method of claim 1, wherein the neurodegenerative disease is AD.

23. The method of claim 1, wherein the neurodegenerative disease is NDS.

24. The method of claim 1, wherein the neurodegenerative disease is MCI.

25. A kit for diagnosing a neurodegenerative disease comprising an antibody that binds to QC and an established standard of an amount of QC characteristic of a normal control.

26. The kit of claim 25, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's Disease (AD), Neurodegeneration in Down's Syndrome (NDS) and Mild Cognitive Impairment (MCI).

Description

DESCRIPTION OF THE DRAWINGS

[0023] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0024] FIG. 1: FIG. 1(a) shows the analysis of QC transcript levels applying quantitative RT-PCR. Total RNA from human neocortical brain samples (Brodmann area 22) was isolated from normally aged and AD brains of different Braak stages as indicated. The QC transcript level was normalized to house-keeping transcript concentration. FIG. 1(b) shows the Western-Blot analysis for QC from the same cases and brain region as used for QC mRNA analysis. The extraction of soluble protein was normalized to the tissue weight. FIG. 1(c) shows the quantification of A N3(pE)-42 (indicated as A.sub.3(pE)-42) and of A 1-42 (A.sub.1-42) concentrations from the same cases and brain region applying ELISA analysis of SDS- and formic acid extracts of human neocortical brain samples. Note the robust increase in A N3(pE)-42 peptide concentrations at early AD stages compared to the much more moderate increase in A 1-42 peptides. FIG. 1(d) shows the immunohistochemical detection of total A peptides by the antibody 4G8 and of A N3(pE)-42 peptides in Brodmann area 22 from normally aged subjects and different AD stages. Sparse A plaques were detected in normal aging but these deposits lacked A N3(pE)-42 immunoreactivity. At all AD stages, however, the majority of A plaques contains A N3(pE)-42 peptides.

[0025] FIG. 2 shows the results of the determination of the gene expression rate of QC and CCL2 in stimulated THP-1 cells.

[0026] FIG. 3 shows the results of the determination of the specific QC activity in conditioned medium of THP-1 cells.

TABLE-US-00001 SEQUENCESOFAMYLOIDPEPTIDESANDCHEMOKINES A(1-42) Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln- (SEQIDNO:6) Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala- Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala A(1-40) Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln- (SEQIDNO:7) Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala- Ile-IIe-Gly-Leu-Met-Val-Gly-Gly-Val-Val A(3-42) Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu- (SEQIDNO:8) Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-lIe-Ile- Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala A3(3-40) Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu- (SEQIDNO:9) Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-lle-Ile- Gly-Leu-Met-Val-Gly-Gly-Val-Val A(1-38) Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln- (SEQIDNO:10) Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala- Ile-Ile-Gly-Leu-Met-Val-Gly-Gly A(3-38) Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu- (SEQIDNO:11) Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-lle-Ile- Gly-Leu-Met-Val-Gly-Gly ABri EASNCFAIRHFENKFAVETLICSRTVKKNIIEEN (SEQIDNO:12) ADan EASNCFAIRHFENKFAVETLICFNLFLNSQEKHY (SEQIDNO:13) CCL2(small QPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTI induciblecytokine VAKEICADPKQKWVQDSMDHLDKQTQTPKT A2)(SEQIDNO: 14) Swiss-Prot: P13500 CCL7(Small- QPVGINTSTTCCYRFINKKIPKQRLESYRRTTSSHCPREAVIFKT induciblecytokine KLDKEICADPTQKWVQDFMKHLDKKTQTPKL A7)(SEQIDNO: 15) Swiss-Prot: P80098 CCL8(small QPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKR induciblecytokine GKEVCADPKERWVRDSMKHLDQIFQNLKP A8)(SEQID NO: 16) Swiss-Prot: P80075 CCL9/10(Small- QITHATETKEVQSSLKAQQGLEIEMFHMGFQDSSDCCLSYNSRI induciblecytokine QCSRFIGYFPTSGGCTRPGIIFISKRGFQVCANPSDRRVQRCIE A9)(SEQIDNO: RLEQNSQPRTYKQ 17) Swiss-Prot: P51670 CCL13(Small- QPDALNVPSTCCFTFSSKKISLQRLKSYVITTSRCPQKAVIFRTK induciblecytokine LGKEICADPKEKWVQNYMKHLGRKAHTLKT A13)(SEQIDNO: 18) Swiss-Prot: Q99616 CCL15(Small- QFINDAETELMMSKLPLENPVVLNSFHFAADCCTSYISQSIPCSL induciblecytokine MKSYFETSSECSKPGVIFLTKKGRQVCAKPSGPGVQDCMKKLK A15)(SEQIDNO: PYSI 19) Swiss-Prot: Q16663 CCL16 QPKVPEWVNTPSTCCLKYYEKVLPRRLVVGYRKALNCHLPAIIF (Small-inducible VTKRNREVCTNPNDDWVQEYIKDPNLPLLPTRNLSTVKIITAKN cytokineA16) GQPQLLNSQ (SEQIDNO:20) Swiss-Prot: O15467 Fractalkine QHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQ (neurotactin)(SEQ HRLFCADPKEQWVKDAMQHLDRQAAALTRNGGTFEKQIGEVK IDNO:21) PRTTPAAGGMDESVVLEPEATGESSSLEPTPSSQEAQRALGTS Swiss-Prot: PELPTGVTGSSGTRLPPTPKAQDGGPVGTELFRVPPVSTAATW P78423 QSSAPHQPGPSLWAEAKTSEAPSTQDPSTQASTASSPAPEEN APSEGQRVWGQGQSPRPENSLEREEMGPVPAHTDAFQDWGP GSMAHVSVVPVSSEGTPSREPVASGSWTPKAEEPIHATMDPQ RLGVLITPVPDAQAATRRQAVGLLAFLGLLFCLGVAMFTYQSLQ GCPRKMAGEMAEGLRYIPRSCGSNSYVLVPV CCL25 QGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYL (Small-inducible PKRHRKVCGNPKSREVQRAMKLLDARNKVFAKLHHNTQTFQA cytokineA25) GPHAVKKLSSGNSKLSSSKFSNPISSSKRNVSLLISANSGL (SEQIDNO:22) Swiss-Prot: O15444

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

[0027] The present invention provides an efficient and rapid in vitro method for diagnosing a neurodegenerative disease by directly detecting an amount of QC in a biological sample obtained from a subject and comparing the detected amount of QC with an amount of QC characteristic of a normal control. An elevated amount of QC in the biological sample of the subject is a positive indication of AD or MCI or NDS. Thus, as described herein, it is demonstrated that QC is consistently and significantly elevated in a biological sample of AD, NDS or MCI patients compared to normal controls. As such, the methods for diagnosing AD, MCI or NDS of the present invention by detecting or quantifying the amount of QC in a patient sample will greatly improve current clinical diagnostic assessment for patients suffering from these neurodegenerative diseases.

[0028] Accordingly, there is provided a method for assessing whether a subject may be suffering from AD, MCI or NDS using QC as a biological marker.

[0029] Glutaminyl cyclase or glutaminyl-peptide cyclotransferase (QC, EC 2.3.2.5) catalyzes the intramolecular cyclization of N-terminal glutaminyl residues into pyroglutamic acid (5-oxo-proline, pGlu*) under liberation of ammonia and the intramolecular cyclization of N-terminal glutamyl residues into pyroglutamic acid under liberation of water.

[0030] A QC was first isolated by Messer from the Latex of the tropical plant Carica papaya in 1963 (Messer, M. 1963 Nature 4874, 1299). 24 years later, a corresponding enzymatic activity was discovered in animal pituitary (Busby, W. H. J. et al. 1987 J Biol Chem 262, 8532-8536; Fischer, W. H. and Spiess, J. 1987 Proc Natl Acad Sci U S A 84, 3628-3632). For the mammalian QCs, the conversion of Gln into pGlu by QC could be shown for the precursors of TRH and GnRH (Busby, W. H. J. et al. 1987 J Biol Chem 262, 8532-8536; Fischer, W. H. and Spiess, J. 1987 Proc Natl Acad Sci U S A 84, 3628-3632). In addition, initial localization experiments of QC revealed a co-localization with its putative products of catalysis in the bovine tractus hypothalamo-hypophysalisfurther improving the suggested function in peptide hormone maturation (Bockers, T. M. et al. 1995 J Neuroendocrinol 7, 445-453). In contrast, the physiological function of the plant QC is less clear. In case of the enzyme from C. papaya, a role in the plant defence against pathogenic microorganisms was suggested (El Moussaoui, A. et al. 2001 Cell Mol Life Sci 58, 556-570). Putative QCs from other plants were identified by sequence comparisons recently (Dahl, S. W. et al. 2000 Protein Expr Purif 20, 27-36). The physiological function of these enzymes, however, is still ambiguous.

[0031] The QCs known from plants and animals show a strict specificity for L-Glutamine in the N-terminal position of the substrates and their kinetic behaviour was found to obey the Michaelis-Menten equation (Pohl, T. et al. 1991 Proc Natl Acad Sci U S A 88, 10059-10063; Consalvo, A. P. et al. 1988 Anal Biochem 175, 131-138; Gololobov, M. Y. et al. 1996 Biol Chem Hoppe Seyler 377, 395-398). A comparison of the primary structures of the QCs from C. papaya and that of the highly conserved QC from mammals, however, did not reveal any sequence homology (Dahl, S. W. et al. (2000) Protein Expr Purif 20, 27-36). Whereas the plant QCs appear to belong to a new enzyme family (Dahl, S. W. et al. (2000) Protein Expr Purif 20, 27-36), the mammalian QCs were found to have a pronounced sequence homology to bacterial aminopeptidases (Bateman, R. C. et al. 2001 Biochemistry 40, 11246-11250), leading to the conclusion that the QCs from plants and animals have different evolutionary origins.

[0032] Gostranova et al. have found that glutaminyl cyclase activity is a characteristic feature of cerebrospinal fluid in multiple sclerosis patients and controls (Gostranova et al., Clin Chim Acta. 2008 389 (1-2), pp. 152-159).

[0033] Different isoforms of QC, the glutaminyl-peptide cyclotransferase-like proteins (QPCTLs) have been observed (WO 2008/034891). These novel proteins have significant sequence similarity to glutaminyl cyclase, e.g. the QPCTL from human (further named as isoQC) (GenBank accession no. NM_017659).

[0034] Multiple isoforms of a protein, such as QC or human isoQC, can also be produced from a single gene by a variety of mechanisms, including alternative RNA splicing, post- translational proteolytic processing and cell type-specific glycosylation. Thus, the terms glutaminyl cyclase, QC and isoQC as used herein refer to QC in its native form, as well as any of its isoforms.

[0035] Preferred for the use of the present invention are human QC or its isoforms, having an amino acid sequence selected from the group of SEQ ID NO's: 1, 2, 3, 4 and 5.

[0036] More preferred for use in the methods of the present invention is the human QPCTL having an amino acid sequence of SEQ ID NO. 2, or even preferred of SEQ ID NO: 3.

[0037] Even preferred for use in the methods of the present invention are spliceforms of human QPCTL having an amino acid sequence of SEQ ID NO. 4 or of SEQ ID NO: 5.

[0038] Most preferred for use in the methods of the present invention is human QC having the amino acid sequence of SEQ ID NO: 1.

[0039] Thus, according to a first aspect of the present invention, there is provided a method for diagnosing probable Alzheimer's Disease (AD), Neurodegeneration in Down's Syndrome (NDS) or Mild Cognitive Impairment (MCI) in a subject, the method comprising:

[0040] (a) detecting the amount of glutaminyl cyclase (QC), or an isoform thereof, in a biological sample obtained from said subject; and

[0041] (b) comparing the detected amount of QC in the biological sample with an amount of QC characteristic of a normal control;

[0042] whereby an elevated amount of QC in said biological sample relative to the normal control is a positive indicator of AD, NDS or MCI.

[0043] It has been demonstrated by inventors of the present invention that an elevated amount of QC in a biological sample may correlate with an elevated amount of N-terminally truncated and pyroglutamated amyloid beta peptides, such as for example A6 N3pE-42 and/or A N3pE-40 and/or A6 N3pE-38.

[0044] Thus, according to a further aspect of the present invention, there is provided a method for diagnosing probable Alzheimer's Disease (AD), Neurodegeneration in Down's Syndrome (NDS) or Mild Cognitive Impairment (MCI) in a subject, the method comprising:

[0045] (a) detecting the amount of glutaminyl cyclase (QC), or an isoform thereof, in a biological sample obtained from said subject; and

[0046] (b) further detecting the amount of A N3pE-X,

[0047] (c) comparing the detected amount of QC and A N3pE-X in the biological sample with an amount of QC and A N3pE-X characteristic of a normal control;

[0048] whereby an elevated amount of QC and A N3pE-X in said biological sample relative to the normal control is a positive indicator of AD, NDS or MCI, and

[0049] wherein X is an integer selected from 38, 40 and 42.

[0050] In a preferred embodiment, X is 42.

[0051] In a further preferred embodiment, X is 40.

[0052] In a yet preferred embodiment, X is 38.

[0053] Further preferred are methods, wherein not only a single form of the N-terminally truncated and pyroglutamated amyloid beta peptides but a combination of A N3pE-42 and/or A N3pE-40 and/or A N3pE-38 is detected together with QC.

[0054] Further preferred are methods, wherein not only a single form of the N-terminally truncated and pyroglutamated amyloid beta peptides but a combination of A N3pE-42 and/or A N3pE-40 and/or A N3pE-38 and/or peptides occurring in familial Alzheimer's dementias, such as pGluABri or pGluADan, is detected together with QC.

[0055] pGlu-A or A N3pE refers to N-terminally truncated forms of A, that start at the glutamic acid residue at position 3 in the amino acid sequence of A, and wherein said glutamic acid residue is cyclized to form a pyroglutamic acid residue. In particular, by pGlu-A as used herein are meant those fragments which are involved in or associated with the amyloid pathologies including, but not limited to, pGlu-A 3-38, pGlu-A 3-40, p-Glu-A 3-42.

[0056] It has further been demonstrated by the inventors of the present invention that an elevated amount of QC in a biological sample may correlate with an elevated amount of a chemokine, such as for example CCL2, CCL7, CCL8, CCL9/10, CCL13, CCL15, CCL16, CCL25 and Fractalkine.

[0057] Thus, according to a further aspect of the present invention, there is provided a method for diagnosing Alzheimer's Disease (AD), Neurodegeneration in Down's Syndrome (NDS) or Mild Cognitive Impairment (MCI) in a subject, the method comprising:

[0058] (a) detecting the amount of glutaminyl cyclase (QC), or an isoform thereof, in a biological sample obtained from said subject; and

[0059] (b) further detecting the amount of a chemokine,

[0060] (c) comparing the detected amount of QC and the chemokine in the biological sample with an amount of QC and the chemokine characteristic of a normal control;

[0061] whereby an elevated amount of QC and chemokine in said biological sample relative to the normal control is a positive indicator of AD, NDS or MCI.

[0062] In a preferred embodiment, said chemokine is of mammalian origin. More preferably, said chemokine is a human chemokine. Most preferably, said chemokine is human CCL2.

[0063] In a further preferred embodiment, any of the aforementioned methods for diagnosing Alzheimer's Disease (AD), Neurodegeneration in Down's Syndrome (NDS) or Mild Cognitive Impairment (MCI) may also be performed in vitro in a biological sample of a subject.

[0064] The term subject refers to a mammal which is afflicted with, or suspected to be afflicted with a neurogenerative disease such as AD, MCI or NDS. Preferably, subject refers to a human.

[0065] The term biological sample refers to any source of biological material, including, but are not limited to, peripheral blood, plasma, lymphocytes, cerebrospinal fluid, urine, saliva, epithelia, fibroblasts, or any other sample comprising QC protein.

[0066] In a preferred embodiment, the amount of QC is detected in a body fluid sample obtained from a mammal, most preferably a human. The term body fluid refers to all fluids that are present in the human body including but not limited to blood, lymph, urine and cerebrospinal fluid (CSF) comprising QC. The blood sample may include a plasma sample or a serum sample, or fractions derived from these samples. The sample can be treated prior to use, such as preparing plasma from blood, diluting viscous fluids, and the like. Preferably, the plasma sample is treated with an anti-coagulant, such as EDTA.

[0067] According to a preferred embodiment of the present invention, the amount of QC is detected in a blood sample taken from the subject, more preferably a plasma sample. Thus, the present invention preferably relates to a method as described above, comprising the steps of: obtaining a plasma sample from said subject; detecting the amount of QC in the plasma sample; comparing the detected amount of QC in the plasma sample with the amount of QC in a plasma sample from a normal control, whereby an elevated amount of QC relative to the normal control is a positive indication of AD, NDS or MCI. Elevated amounts of QC have been shown to correlate with and are useful in aiding the diagnosis of AD, NDS and MCI.

[0068] An elevated amount of QC (or an isoform thereof) means that the amount of QC detected in the samples of the subjects is greater than the mean amount of QC characteristic of a normal control person beyond the range of experimental error, as known in the art. Preferably, the amount of QC detected in the samples of the subjects is 10% greater than said mean amount of QC characteristic of a normal control person. More preferably, the amount of QC (or an isoform thereof) detected in the samples of the subjects is 25% greater, or, even more preferred 50% or 75% greater than said mean amount of QC characteristic of a normal control person. Most preferably, the amount of QC (or an isoform thereof) detected in the samples of the subjects is several times greater than said mean amount of QC characteristic of a normal control person, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times greater.

[0069] A normal control is a biological sample of the same type obtained from the subject, for example that is obtained from at least one normal age-matched control person or from the patient at another time. In an embodiment, the normal control is taken from the patient at an earlier time. A normal control sample from a normal age-matched population should be isolated from an adequate population sample of healthy age matched controls with no history of AD, MCI or NDS in their family. By way of example, a plasma QC level higher than the control levels of QC, as determined by an adequate control population sample size, is indicative of AD, NDS or MCI. One of skill in the art will appreciate that the sample from the subject to be diagnosed is assessed against a normal age-matched control and that a significant elevation or reduction in the amount of QC in the subject's protein sample is determined based on comparison to the controls used in the given assay.

[0070] According to a further embodiment of the present invention, the amount of QC, or an isoform thereof, is detected either on the basis of the protein level or the mRNA level of said QC or isoform thereof.

[0071] The amount of QC detected or quantified in a subject's biological sample can be accomplished by any means known in the art. Such means may include, but are not limited to, for example by immunoturbidimetric assay, immunofluorescence, immunodiffusion, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Western Blot, protein activity assay, or, for the determination of the QC mRNA level, Northern Blot or polymerase chain reaction (PCR) analysis, for example real-time PCR. Also useful are high performance liquid chromatography (HPLC), mass spectrometry (MS) and gas chromatography (GC), as well as their various configurations, including gas chromatograph-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and liquid-chromatography-tandem mass spectrometry (LC-MS/MS) systems, to name a few.

[0072] While detection of QC can be accomplished by methods known in the art for detecting peptides, the use of immmunological detection techniques using antibodies, antibody fragments, recombinant antibodies, and the like, is preferred. Therefore, such detection of QC includes, but is not limited to, the use of antibodies, which specifically bind to QC, or its isoforms, to form an immune complex, as well as reagents for detecting the formation of the immune complex. Particularly suitable detection techniques employing one or more antibodies include immunoturbidimetric assay, immunofluorescence, immunodiffusion, ELISA, RIA and the like.

[0073] Such antibodies may be polyclonal or monoclonal. Methods to produce polyclonal or monoclonal antibodies are well known in the art. For a review, see Harlow and Lane (Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988) and Yelton et al. (Yelton D. E. and Scharff M. D. Monoclonal Antibodies: a powerful new tool in biology and medicine. Ann. Rev. Biochem. 50:657-680, 1981), both of which are herein incorporated by reference. For monoclonal antibodies, see Kohler and Milstein (Kohler G. and Milstein C, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256:495-497, 1975), herein incorporated by reference. The antibodies of the invention are of any isotype, e.g., IgG or IgA, and polyclonal antibodies are of a single isotype or a mixture of isotypes.

[0074] According to a preferred embodiment of the invention, the anti-QC antibody is a monoclonal antibody. Although anti-QC antibodies are widely commercially available, antibodies for use in the various immunoassays described herein, can be produced according to standard methods.

[0075] Further, the monoclonal anti-QC antibody is capable of recognizing QC in its native form, as well as any of its isoforms. Thus, any monoclonal antibody that specifically recognizes QC, including its isoforms, can be used in said method for the quantification of QC.

[0076] Preferred are monoclonal antibodies, that specifically recognize QC but show low, or more preferably, no crossreactivity with isoforms of QC. Alternatively preferred are monoclonal antibodies that specifically recognize a particular isoform of QC but show low, or more preferably, no crossreactivity with QC.

[0077] Suitable anti-QC antibodies are, for example, those which are commercially available from Abnova (Taipei City, Taiwan), e.g. a mouse polyclonal antibody (Cat. # H00025797-B01 P) and a rabbit polyclonal antibody (Cat. # H00025797-DO1P).

[0078] A suitable anti-QPCTL antibody is, for example, the commercially available mouse polyclonal antibody from Abnova (Taipei City, Taiwan, Cat. # H00054814-B01P).

[0079] Also fragments derived from these monoclonal antibodies such as Fab, F(ab).sub.2/ ssFv (single chain variable fragment) and other antibody-like constructs that retain the variable region of the antibody, providing they have retained the original binding properties, can be used in a method of the present invention. Such fragments are commonly generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases. It is well known to the person skilled in the art that monoclonal antibodies, or fragments thereof, can be modified for various uses. Thus, antibodies of the invention, may be recombinant, e.g., chimeric (e.g., constituted by a variable region of murine origin associated with a human constant region), humanized (a human immunoglobulin constant backbone together with hypervariable region of animal, e.g., murine, origin), and/or single chain.

[0080] An antibody specific for QC, or its isoforms, used in a method of the present invention may be labelled by an appropriate label and identified in the biological sample based upon the presence of the label. The label allows for the detection of the antibody when it is bound to QC. Examples of labels include, but are not limited to, the following: radioisotopes (e.g., 3H, .sup.14C, .sup.35S, .sup.125I, .sup.131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), luminescent labels, enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, and biotinyl groups.

[0081] Methods for conjugating or labelling the antibodies discussed above may be readily accomplished by one of ordinary skill in the art (see for example Inman, Methods In Enzymology, Vol. 34, Affinity Techniques, Enzyme Purification: Part B, Jakoby and Wichek (eds.), Academic Press, New York, p. 30, 1974; and Wilchek and Bayer, The Avidin-Biotin Complex in Bioanalytical Applications, Anal. Biochem. 171:1-32, 1988).

[0082] For diagnostic applications, the anti-QC antibody is either in a free state or immobilized on a solid support, such as a tube, a bead, or any other conventional support used in the field. Immobilization is achieved using direct or indirect means. Direct means include passive adsorption (non-covalent binding) or covalent binding between the support and the reagent. By indirect means is meant that an anti-reagent compound that interacts with a reagent is first attached to the solid support. Indirect means may also employ a ligand-receptor system, for example, where a molecule such as a vitamin is grafted onto the reagent and the corresponding receptor immobilized on the solid phase. This is illustrated by the biotin-streptavidin system.

[0083] Those skilled in the art will readily understand that an immune complex is formed between QC in the biological sample and the antibody, and that any unbound material is removed prior to detecting the complex. It is understood that an antibody of the invention is used for quantifying an amount of QC in the biological sample, such as, for example, blood, plasma, lymphocytes, cerebrospinal fluid, urine, saliva, epithelia and fibroblasts.

[0084] As is known in the art, the determination of such antibody binding can be performed using a great variety of immunoassay formats including, but not limited to immunoturbidimetric assay (agglutination), enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) (see, for example, Principles and Practice of Immunoassay (1991) Christopher P. Price and David J. Neoman (eds), Stockton Press, New York, N.Y. and Ausubel et al, (eds) (1987) in Current Protocols in Molecular Biology John Wiley and Sons, New York, N.Y., both of which are incorporated herein by reference). Detection may be by colormetic or radioactive methods or any other conventional methods known to one skill in the art. Other standard techniques known in the art are described in Methods in Immunodiagnosis, 2nd Edition, Rose and Bigazzi, eds., John Wiley and Sons, New York 1980 and Campbell et al.; Methods of Immunology, W. A. Benjamin, Inc., 1964; U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168, the disclosures of which are incorporated herein by reference. For a review of the general immunoassays, see also Methods In Cell Biology, Vol. 37, Asai, ed. Academic Press, Inc. New York (1993); Basic And Clinical Immunology 7.sup.th Edition, Stites & Terr, eds. (1991).

[0085] Such assays for detecting QC may be a direct, indirect, competitive, or noncompetitive immunoassay as described in the art (see, for example, Principles and Practice of Immunoassay (1991) Christopher P. Price and David J. Neoman (eds), Stockton Press, New York, N.Y.; Ausubel et al. (eds) (1987) in Current Protocols in Molecular Biology John Wiley and Sons, New York, N.Y.; and Oellirich, M. 1984. J. Clin. Chem. Clin. Biochem. 22: 895-904, incorporated herein by reference).

[0086] Noncompetitive immunoassays are assays in which the amount of QC is directly detected. In the sandwich assay, for example, the anti-QC antibodies can be bound directly to a solid substrate where they are immobilized. These immobilized antibodies then capture the QC present in the biological sample. The QC thus immobilized is then bound by a labeling agent, such as a second human QC antibody bearing a label.

[0087] In a competitive immunoassay, the amount of antigen present in the biological sample is determined indirectly following addition of a known amount of labeled antigen to the sample and detecting the amount of labeled antigen bound with antibodies. For example, a known amount of, in this case, labeled QC is added to the biological sample and the sample is then contacted with anti-QC antibodies. The amount of labeled QC bound to the anti-QC antibody is inversely proportional to the concentration of QC in the biological sample. This is because the greater the amount of labeled QC detected, the less the amount of QC was available in the biological sample to compete with the labeled QC.

[0088] Diagnostic kits for carrying out the assays for diagnosing AD, MCI or NDS in a subject are also provided. Thus, the present invention can be practiced using a diagnostic kit that includes at least one antibody specific for QC, and its isoforms, as described herein as well as any reagents necessary for the detection of antibody-QC binding immune complexes. Generally, the kit may include a single antibody that specifically recognizes QC, and its isoforms. On the other hand, the kit may include a primary antibody that specifically recognizes QC, and its isoforms, as well as a secondary antibody that is conjugated with a signal-producing label and is capable of binding to the primary antibody, or at a site different from the site where the primary antibody binds. The signal-producing label linked to the secondary antibody may be, but is not limited to, an enzyme, such as horseradish peroxidase or alkaline phosphatase. The kits may further comprise other reagents for carrying out the assay such as buffers, a solid support, solutions and the like. The kit may also contain instructions for carrying out the method of the invention using one or more antibodies in diagnostic assays.

[0089] In some embodiments, the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term about. Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0090] In some embodiments, the terms a and an and the and similar references used in the context of describing a particular embodiment of the invention (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. such as) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0091] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0092] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.

[0093] Having described the invention in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the invention defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

[0094] The following non-limiting examples are provided to further illustrate the present invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the invention, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Formation of A N3PE-42 and QC Expression in vivo

[0095] A widespread QC distribution has been detected in mammalian brain with considerable expression in hippocampus and cortex. In order to assess whether QC expression in AD can be correlated with generation of A N3pE-42, QC mRNA and protein concentrations were analyzed in human neocortical brain samples postmortem (see e.g., FIG. 1a,b). Intriguingly, the inventors found an upregulation of QC mRNA and protein in AD brain samples, compared to normal aging. Moreover, significant concentrations of A N3pE-42 were detected in samples from AD patients in contrast to non-demented individuals supporting a role of QC in generation of A N3pE-42 (see e.g., FIG. 1c). On the other hand, ELISA analysis revealed high A x-42 concentrations in normally aged control subjects and a much smaller increase at early AD stages (see e.g., FIG. 1c). This observation was corroborated by immunohistochemistry applying antibodies detecting total A (4G8) or specifically A N3pE-42 (see e.g., FIG. 1d). Conspicuous immunoreactivity for A was detected in brain sections from all groups. In contrast, A N3pE-42 staining was absent in normal aging but specific for AD brain tissue, where A N3pE-42-immunoreactive plaque load was almost as high as the total of A plaque density.

[0096] Human Brain Tissue

[0097] The definite diagnosis of AD for all cases used in this study was based on the presence of neurofibrillary tangles and neuritic plaques in the hippocampal formation and neocortical areas and met the criteria of the National Institute of Neurologic and Communicative Disorders and Stroke (NINDS) and the Alzheimer's Disease and Related Disorders Association (ADRDA). Cortical tissue (Brodmann area 22) from the same cases was used for the quantification of QC mRNA concentrations, QC protein and A N3pE-42. In total, 10 control cases and 10 AD cases each of Braak staging I-II and V-VI were analyzed. The groups were matched for gender and age (control: mean 72 years6.6 years; AD I-II: mean 73 years3.1 years; AD V-VI: mean 77 years6.6 years). The mean post morten interval (PMI) was similar among the groups and ranged from 26 to 96 hours. The duration of PMI was neither related to the detection of QC by Western blot analysis nor to quantification of A by ELISA. For QC mRNA detection by qRT-PCR, only tissue samples with a PMI below 48 hours were included.

[0098] QC mRNA quantification and QC Western blot analysis

[0099] Tissue samples were homogenized by means of the homogenizer Precellys with 1.4 mm ceramic beads (5000 rpm, 30 sec, peqlab). RNA was isolated using the NucleoSpin RNA II kit (Macherey Nagel) according to the manufacturer's instructions. Constant 100 ng of RNA were reverse transcribed to cDNA using random primers (Roche) and Superscript II (Invitrogen). Quantitative real-time PCR was performed in a Rotorgene3000 (Corbett Research) using the QuantiTect Primer Assay for QPCT (QT00013881, Qiagen) as well as the QuantiTect SYBR Green RT-PCR kit (Qiagen). Absolute amounts of QC were determined using six dilutions of the external QC standard DNA (full length QC cloned in the pcDNA vector) in duplicate. For verification of the PCR, product melting curves were generated and single amplicons were confirmed by agarose gel electrophoresis. Absolute amounts were determined with the Rotorgene software version 4.6 in quantitation mode. Normalization was done against the two most stably expressed housekeeping genes HPRT and GAPDH (geNorm). For Western-Blot analysis, the brain samples (50 mg) were homogenized in buffer (1 ml) containing 10 mM Tris pH 7.5, 100 mM NaCl, 5 mM EDTA and 0.5% Triton X-100 and 10% glycerol. The tissue was homogenized by several strokes in Downs-homogenizer and subjected to 310s of ultrasonic shock. The resulting homogenate was cleared by centrifugation at 20000g for 25 min. A total of 12 pg protein of each sample was separated in Tris-Glycine SDS-PAGE. QC was detected using purified rabbit polyclonal antibodies raised against recombinant human QC. For visualization, blot membranes were incubated with secondary antibody conjugated with horseradish peroxidase (Cell Signaling) in TBS-T containing 5% (w/v) dry milk and subsequently developed using the SuperSignal West Pico System (Pierce) according to the manufacturer's protocol.

Example 2

Determination of Gene Expression Rate of QC and CCL2 in Stimulated THP-1 Cells

[0100] Human monocytic leukaemia cell line THP-1 cells were cultivated in suspension (510.sup.5 cells per ml medium) in RPMI-1640 (Rosewell Park Memorial Institute Medium 1640 (Invitrogen)) containing 10% FCS (=FBS, Fetal Bovine Serum (Invitrogen)) and 60 g/ml gentamycin (Invitrogen) at 37 C. in 5% CO.sub.2 and 95% air humidified atmosphere.

[0101] To investigate stimulation effects of QC and CCL2 210.sup.6 cells were seeded in 24 well plates (Greiner) into 1 ml culture medium without FCS containing different concentrations of lipopolysaccharides (LPS; Sigma). After 24 h incubation the medium was removed from the cells by centrifugation (5 min 300g).

[0102] RNA isolation was carried out with the Nucleo-Spin RNA II Kit (Macherey & Nagel) followed by the determination of the RNA concentration. Using the SuperScript II Reverse Transcriptase Kit from Invitrogen 1 g RNA was transcribed into cDNA.

[0103] The gene expression rate of QC and CCL2 was determined via quantitative PCR with the real time cycler Rotor-Gene.sup.TM 3000. Using the comparative method of the operating software the change of the gene expression rate of the stimulated probes compared to the unstimulated control could be shown. The normalisation was performed against the reference gene YWHAZ (Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein). The results are shown in FIG. 2.

Example 3

Determination of the Specific QC Activity in Conditioned Medium of THP-1 Cells

[0104] 510.sup.6 THP-1 cells were seeded into 5 ml RPMI-1640 (Invitrogen) without phenol red and without FCS into 25 cm.sup.2 suspension flasks (Greiner) and stimulated with different concentrations of LPS (Sigma). After 24 h incubation at 37 C. and 5% CO.sub.2 cells were separated from the medium, which was reduced by centrifugation (4000g) using U-Tube Concentrators 6-10 (Merck, Novagen) with a MWCO (Moleculare Weight Cut Off) 10 kDa to a final volume of 250 l. The analysis of the protein concentration via Bradford method followed. The determination of the specific QC activity was realised by using a in-house established HPLC method. The results are shown in FIG. 3.

Example 4

Determination of QC Activity

[0105] Fluorometric assays

[0106] All measurements were performed with a BioAssay Reader HTS-7000Plus for microplates (Perkin Elmer) at 30 C. QC activity was evaluated fluorometrically using H-Gln-bNA. The samples consisted of 0.2 mM fluorogenic substrate, 0.25 U pyroglutamyl aminopeptidase (Unizyme, Hsholm, Denmark) in 0.2 M Tris/HCl, pH 8.0 containing 20 mM EDTA and an appropriately diluted aliquot of QC in a final volume of 250 l. Excitation/emission wavelengths were 320/410 nm. The assay reactions were initiated by addition of glutaminyl cyclase. QC activity was determined from a standard curve of b-naphthylamine under assay conditions. One unit is defined as the amount of QC catalyzing the formation of 1 mol pGlu-bNA from H-Gln-bNA per minute under the described conditions.

[0107] In a second fluorometric assay, QC activity was determined using H-Gln-AMC as substrate. Reactions were carried out at 30 C. utilizing the NOVOStar reader for microplates (BMG labtechnologies). The samples consisted of varying concentrations of the fluorogenic substrate, 0.1 U pyroglutamyl aminopeptidase (Qiagen) in 0.05 M Tris/HCl, pH 8.0 containing 5 mM EDTA and an appropriately diluted aliquot of QC in a final volume of 250 l. Excitation/emission wavelengths were 380/460 nm. The assay reactions were initiated by addition of glutaminyl cyclase. QC activity was determined from a standard curve of 7-amino-4-methylcoumarin under assay conditions. The kinetic data were evaluated using GraFit software.

[0108] Spectrophotometric assay of QC

[0109] In this assay, QC activity was analyzed spectrophotometrically using a continuous method, that was derived by adapting a previous discontinuous assay (Bateman, R. C. J. 1989 J Neurosci Methods 30, 23-28) utilizing glutamate dehydrogenase as auxiliary enzyme. Samples consisted of the respective QC substrate, 0.3 mM NADH, 14 mM a-Ketoglutaric acid and 30 U/ml glutamate dehydrogenase in a final volume of 250 l. Reactions were started by addition of QC and persued by monitoring of the decrease in absorbance at 340 nm for 8-15 min.

[0110] The initial velocities were evaluated and the enzymatic activity was determined from a standard curve of ammonia under assay conditions. All samples were measured at 30 C., using either the SPECTRAFluor Plus or the Sunrise (both from TECAN) reader for microplates. Kinetic data was evaluated using GraFit software.