Uses of antibodies directed against amyloid-β

Abstract

Diagnostic assays for the diagnosis of amyloidosis, in particular Alzheimer's disease, and related aspects. In particular, monoclonal antibodies and an antibody assay are provided.

Claims

1. A method for treatment or delay of an amyloid beta-associated amyloidosis comprising administering a therapeutically effective amount of an antibody or a humanized form of said antibody to a subject in need thereof, wherein the antibody comprises (i) a light chain comprising a variable part, the variable part of the light chain of said antibody comprising a polypeptide (a) encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, and SEQ ID NO: 61 or (b) having an amino acid sequence selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, and SEQ ID NO: 62; or (ii) a heavy chain comprising a variable part; the variable part of the heavy chain of said antibody comprising a polypeptide (a) encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, and SEQ ID NO: 63 or (b) having an amino acid sequence selected from the group consisting of SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, and SEQ ID NO: 64.

2. The method of claim 1, wherein said amyloidosis is a neurodegenerative disease selected from the group consisting of mild cognitive impairment, Alzheimer's disease and neurodegeneration in Down Syndrome.

3. The method of claim 1, wherein said amyloidosis is sporadic Alzheimer's disease or a Familial Alzheimer's dementia.

4. The method of claim 3, wherein said Familial Alzheimer's dementia is Familial British Dementia or Familial Danish Dementia.

5. The method according to claim 1, wherein the antibody binds an A peptide with a dissociation constant (KD) value of at least 10.sup.7 M.

6. The method according to claim 1, wherein said antibody is a monoclonal antibody.

7. The method according to claim 1, wherein: the antibody comprises the light chain; the light chain comprises the variable part; and the variable part of the light chain of said antibody comprises the polypeptide encoded by the nucleotide sequence selected from the group consisting of SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57 and SEQ ID NO: 61, or having the amino acid sequence selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, and SEQ ID NO: 62.

8. The method according to claim 1, wherein: the antibody comprises the heavy chain; the heavy chain comprises the variable part; and the variable part of the heavy chain of said antibody comprises the polypeptide encoded by the nucleotide sequence selected from SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59 and SEQ ID NO: 63, or having the amino acid sequence selected from SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60 and SEQ ID NO: 64.

9. The method according to claim 1, wherein: the isolated antibody comprises the light chain; the light chain comprises the variable part; the variable part of the light chain of said antibody comprises the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 49 or having the amino acid sequence of SEQ ID NO: 50; the isolated antibody comprises the heavy chain; the heavy chain comprises the variable part; and the variable part of the heavy chain of said antibody comprises the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 51, or having the amino acid sequence of SEQ ID NO: 52.

10. The method according to claim 1, wherein: the isolated antibody comprises the light chain; the light chain comprises the variable part; the variable part of the light chain of said antibody comprises the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 53 or having the amino acid sequence of SEQ ID NO: 54; the isolated antibody comprises the heavy chain; the heavy chain comprises the variable part; and the variable part of the heavy chain of said antibody comprises the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 55, or having the amino acid sequence of SEQ ID NO: 56.

11. The method according to claim 1, wherein: the isolated antibody comprises the light chain; the light chain comprises the variable part; the variable part of the light chain of said antibody comprises the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 57 or having the amino acid sequence of SEQ ID NO: 58; the isolated antibody comprises the heavy chain; the heavy chain comprises the variable part; and the variable part of the heavy chain of said antibody comprises the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 59, or having the amino acid sequence of SEQ ID NO: 60.

12. The method according to claim 1, wherein: the isolated antibody comprises the light chain; the light chain comprises the variable part; the variable part of the light chain of said antibody comprises the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 61 or having the amino acid sequence of SEQ ID NO: 62; the isolated antibody comprises the heavy chain; the heavy chain comprises the variable part; and a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 63, or having the amino acid sequence of SEQ ID NO: 64.

13. The method according to claim 1, wherein said antibody is selected from the group consisting of: A 5-5-6 (Deposit No. DSM ACC 2923); A 6-1-6 (Deposit No. DSM ACC 2924); A 17-4-3 (Deposit No. DSM ACC 2925); and A 24-2-3 (Deposit No. DSM ACC 2926).

14. The method according to claim 1, wherein said antibody is A 6-1-6 (Deposit No. DSM ACC 2924).

15. The method according to claim 1, wherein said antibody is A 24-2-3 (Deposit No. DSM ACC 2926).

16. The method according to claim 1, wherein said antibody is a humanized or chimeric antibody, or an antibody fragment.

17. The method according to claim 1, wherein the antibody is a human antibody other than the variable part of the light chain and the variable part of the heavy chain.

18. The method according to claim 1, wherein the antibody is a diabody or a single chain antibody.

19. The method according to claim 1, wherein the antibody binds to an epitope of an antigen selected from the group consisting of: pGlu-A3-38; pGlu-A3-40; pGlu-A3-42; and pGlu-A3-x variants, wherein x is an integer between 10 and 42.

20. The method according to claim 1, wherein the antibody comprises a complementarity determining region that binds to an antigen selected from the group consisting of: pGlu-A3-38; pGlu-A3-40; pGlu-A3-42; and pGlu-A3-x variants, wherein x is an integer between 10 and 42.

21. The method according to claim 1, wherein the antibody is labeled.

22. The method according to claim 1, wherein the antibody is obtained from any one of hybridoma cell lines DSM ACC 2923, DSM ACC 2924, DSM ACC 2925, or DSM ACC 2926.

23. The method according to claim 19, wherein x is an integer between 18 and 42.

24. The method according to claim 19, wherein x is an integer between 30 and 42.

25. The method according to claim 20, wherein x is an integer between 18 and 42.

26. The method according to claim 20, wherein x is an integer between 30 and 42.

Description

DESCRIPTION OF THE FIGURES

(1) 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.

(2) FIG. 1A

(3) Detection of 10 ng/ml amyloid N3pE-40 by increasing concentrations of pGlu-6166 antibody (clone 12-1).

(4) FIG. 1B

(5) Determination of the highest concentration of pGlu-6166 antibody (clone 12-1) required for detection of 10 ng/ml amyloid N3pE-40.

(6) FIG. 2

(7) Dot Blot analysis of hybridoma cell culture supernatants of individual IgG producing clones.

(8) FIG. 3

(9) PepSpot Analysis of pGlu-6166 Hybridoma Cell Clones and IBL-A N3pE antibody.

(10) FIG. 4

(11) 12% SDS-PAGE of 20 g pGlu-6166 antibody and hybridoma cell culture supernatants.

(12) FIG. 5

(13) Biacore analysis of hybridoma cell culture supernatants. An overlay of monitored binding courses is illustrated graphically.

(14) FIG. 6

(15) Sensograms of interaction of anti-AN3pE antibody clone 6-1-6 with ApE3-40.

(16) FIG. 7

(17) Sensograms of interaction of anti-AN3pE antibody clone 24-2-3 with ApE3-40.

(18) FIG. 8

(19) N3pE-ELISA for clone 6-1-6, standard curve of ApE3-40.

(20) FIG. 9:

(21) Sensograms of N3pE antibody clone 6-1-6.

(22) FIG. 10

(23) Quantification of ApE3-42 using the method of neutralization by 1:20 dilution in EIA buffer, pH titration with 860 l 3.5 M Tris.

(24) FIG. 11A-FIG. 11C

(25) Stained brain sections form Alzheimer's disease (AD) patients

(26) (FIG. 11A) Brain of a sporadic AD (SAD) patient stained with anti-A antibody 6E10, recognizing total A,

(27) (FIG. 11B) Brain of a sporadic AD (SAD) patient stained with N3pE antibody clone 24-2-3, recognizing ApE3-x,

(28) (FIG. 11C) Brain of a familial AD (FAD) patient stained with N3pE antibody clone 24-2-3, recognizing ApE3-x.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

Definitions

(29) The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

(30) The term antibody is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The antibody may be an IgM, IgG (e.g. IgG.sub.1, IgG2, IgG3 or IgG4), IgD, IgA or IgE, for example. Preferably however, the antibody is not an IgM antibody.

(31) Antibody fragments comprise a portion of an intact antibody, generally the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab, F(ab)2, and Fv fragments: diabodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

(32) The term monoclonal antibody as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies can frequently be advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Khler et al., Nature, 256:495 (1975), or may be made by generally well known recombinant DNA methods. The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.

(33) The monoclonal antibodies herein specifically include chimeric antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

(34) Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab, F(ab)2 or other antigen-binding subsequences of antibodies) which contain a minimal sequence derived from a non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.

(35) These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525 (1986), Reichmann et al, Nature. 332:323-329 (1988): and Presta, Curr. Op. Struct. Biel., 2:593-596 (1992). The humanized antibody includes a Primatized antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.

(36) Single-chain Fv or sFv antibody fragments comprise the V.sub.H and V.sub.L domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the V.sub.H and V.sub.L domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

(37) The term diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V.sub.H) connected to a light-chain variable domain (V.sub.D) in the same polypeptide chain (V.sub.H-V.sub.D). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in Hollinger et al., Proc. Natl. Acad. Sol. USA, 90:6444-6448 (1993).

(38) An isolated antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

(39) As used herein, the expressions cell, cell line, and cell culture are used interchangeably and all such designations include progeny. Thus, the words transformants and transformed cells include the primary subject cell and culture derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, this will be clear from the context.

(40) The terms polypeptide, peptide, and protein, as used herein, are interchangeable and are defined to mean a biomolecule composed of amino acids linked by a peptide bond.

(41) The terms a, an and the as used herein are defined to mean one or more and include the plural unless the context is inappropriate.

(42) The language diseases and disorders which are caused by or associated with amyloid or amyloid-like proteins includes, but is not limited to, diseases and disorders caused by the presence or activity of amyloid-like proteins in monomeric, fibril, or polymeric state, or any combination of the three. Such diseases and disorders include, but are not limited to, amyloidosis, endocrine tumors, and macular degeneration.

(43) The term amyloidosis refers to a group of diseases and disorders associated with amyloid plaque formation including, but not limited to, secondary amyloidosis and age-related amyloidosis such as diseases including, but not limited to, neurological disorders such as Alzheimer's Disease (AD), including diseases or conditions characterized by a loss of cognitive memory capacity such as, for example, mild cognitive impairment (MCI), sporadic Alzheimer's disease, Lewy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); the Guam Parkinson-Dementia complex, familial forms of Alzheimer's disease like Familial British Dementia (FBD) and Familial Danish Dementia (FDD); as well as other diseases which are based on or associated with amyloid-like proteins such as progressive supranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease, Parkinson's disease, HIV-related dementia, ALS (amyotropic lateral sclerosis), inclusion-body myositis (IBM), Adult Onset Diabetes, and senile cardiac amyloidosis; and various eye diseases including macular degeneration, drusen-related optic neuropathy, and cataract due to beta-amyloid deposition.

(44) Amyloid , A or/-amyloid is an art recognized term and refers to amyloid proteins and peptides, amyloid precursor protein (APP), as well as modifications, fragments and any functional equivalents thereof. In particular, by amyloid as used herein is meant any fragment produced by proteolytic cleavage of APP but especially those fragments which are involved in or associated with the amyloid pathologies including, but not limited to, A.sub.1-38, A.sub.1-40, A.sub.1-42. The amino acid sequences of these A peptides are as follows:

(45) TABLE-US-00001 A 1-42(SEQIDNO.1): Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val- His-His-Gln-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(SEQIDNO.2): Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val- His-His-Gln-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 A 1-38(SEQIDNO.3): Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val- His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val- Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val- Gly-Gly

(46) 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.sub.3-38, pGlu-A.sub.3-40, p-Glu-A.sub.3-42.

(47) The sequences of the N-terminally truncated forms of A, A.sub.3-38, A.sub.43-40, A.sub.43-42 are as follows:

(48) TABLE-US-00002 A 3-42(SEQIDNO.4): Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His- Gln-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 3-40(SEQIDNO.5): Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His- Gln-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 A 3-38(SEQIDNO.6): Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His- Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser- Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly

(49) In particular the present invention pertains to the following items: 1. Antibody, characterised in that it binds to A peptides or variants thereof, preferably with high affinity. 2. Antibody according to item 1, wherein said high affinity means a dissociation constant (K.sub.D) value of 10.sup.7 M, or better. 3. Antibody according to item 1 or 2, wherein said antibody is a monoclonal antibody. 4. Antibody according to any of the preceding items, wherein the variable part of the light chain of said antibody has a nucleotide sequence selected from SEQ ID NOs: 49, 53, 57 and 61, or having an amino acid sequence selected from SEQ ID NOs: 50, 54, 58, and 62. 5. Antibody according to any of the preceding items, wherein the variable part of the heavy chain of said antibody has a nucleotide sequence selected from SEQ ID NOs: 51, 55, 59 and 63, or having an amino acid sequence selected from SEQ ID NOs: 52, 56, 60 and 64. 6. Antibody according to any of the preceding items, wherein the variable part of the light chain of said antibody has the nucleotide sequence of SEQ ID NO: 49 or the amino acid sequence of SEQ ID NO: 50, and wherein the variable part of the heavy chain of said antibody has the nucleotide sequence of SEQ ID NO: 51, or the amino acid sequence of SEQ ID NO: 52. 7. Antibody according to any of the preceding items, wherein the variable part of the light chain of said antibody has the nucleotide sequence of SEQ ID NO: 53 or the amino acid sequence of SEQ ID NO: 54, and wherein the variable part of the heavy chain of said antibody has the nucleotide sequence of SEQ ID NO: 55, or the amino acid sequence of SEQ ID NO: 56. 8. Antibody according to any of the preceding items, wherein the variable part of the light chain of said antibody has the nucleotide sequence of SEQ ID NO: 57 or the amino acid sequence of SEQ ID NO: 58, and wherein the variable part of the heavy chain of said antibody has the nucleotide sequence of SEQ ID NO: 59, or the amino acid sequence of SEQ ID NO: 60. 9. Antibody according to any of the preceding items, wherein the variable part of the light chain of said antibody has the nucleotide sequence of SEQ ID NO: 61 or the amino acid sequence of SEQ ID NO: 62, and wherein the variable part of the heavy chain of said antibody has the nucleotide sequence of SEQ ID NO: 63, or the amino acid sequence of SEQ ID NO: 64. 10. Antibody according to any of the preceding items, wherein said antibody is selected from the following group: A 5-5-6 (Deposit No. DSM ACC 2923) A 6-1-6 (Deposit No. DSM ACC 2924) A 17-4-3 (Deposit No. DSM ACC 2925) A 24-2-3 (Deposit No. DSM ACC 2926) or functional variants thereof. 11. Antibody according to any of the preceding items, wherein said antibody is A 6-1-6 (Deposit No. DSM ACC 2924). 12. Antibody according to any of the preceding items, wherein said antibody is A 24-2-3 (Deposit No. DSM ACC 2926). 13. Antibody according to any of the preceding items, wherein said antibody is a humanized or chimeric antibody, or an antibody fragment which retains the high affinity. 14. Antibody according to any of the preceding items for use in the detection of A peptide or variants thereof. 15. Antibody according to item 14, wherein said variants are selected from the following group: pGlu-A.sub.3-38 pGlu-A.sub.3-40 pGlu-A.sub.3-42, and pGlu-A.sub.3-x variants, wherein x is an integer between 10 and 42; preferably 18 and 42, more preferably 30 and 42. 16. Antibody according to any of the preceding items, which is human. 17. Antibody according to any of the preceding items, which is a diabody or a single chain antibody which retains the high affinity. 18. Antibody according to any of the preceding items, which binds to the epitope bound by the antibodies defined in item 15. 19. Antibody according to any of the preceding items, which has the complementarity determining regions of the antibodies as defined in item 15. 20. Antibody according to any of the preceding items, which is labeled. 21. Antibody according to any of the preceding items, which is immobilised on a solid phase. 22. Antibody obtainable from any one of hybridoma cell lines DSM ACC 2923, DSM ACC 2924, DSM ACC 2925, DSM ACC 2926. 23. Composition comprising the antibody as defined in any of the preceding items. 24. Composition according to item 23 for the treatment, prevention or delay of amyloidosis. 25. Composition according to item 23 or 24, wherein said amyloidosis is a neurodegenerative disease selected from the group consisting of mild cognitive impairment, Alzheimer's disease and neurodegeneration in Down Syndrome. 26. Composition according to item 23 or 24, wherein said amyloidosis is sporadic Alzheimer's disease or a Familial Alzheimer's dementia. 27. Composition according to item 26, wherein said Familial Alzheimer's dementia is Familial British Dementia or Familial Danish Dementia. 28. Hybridoma cell line DSM ACC 2923. 29. Hybridoma cell line DSM ACC 2924 30. Hybridoma cell line DSM ACC 2925. 31. Hybridoma cell line DSM ACC 2926. 32. Use of the antibody as defined in any one of items 1 to 22 or the composition as defined in any one of items 23 to 27 in a diagnostic or therapeutic method. 33. The use according to item 32 for the diagnosis of an amyloid-associated disease or condition. 34. The use according to item 33, wherein said amyloidosis is a neurodegenerative disease selected from the group consisting of mild cognitive impairment, Alzheimer's disease and neurodegeneration in Down Syndrome. 35. The use according to item 33, wherein said amyloidosis is sporadic Alzheimer's disease or a Familial Alzheimer's dementia. 36. The use according to item 35, wherein said Familial Alzheimer's dementia is Familial British Dementia or Familial Danish Dementia. 37. In vitro diagnostic method for the diagnosis of an amyloid-associated disease or condition, in particular Alzheimer's disease, comprising the following steps: contacting an antibody according to any one of items 1 to 22 with a sample from a subject suspected to be afflicted with said disease or condition, and detecting binding of the antibody to a pGlu-amyloid protein, preferably pGlu-A peptide from the sample. 38. Diagnostic kit, comprising the antibody as defined in any one of items 1 to 22, and instructions for use, andoptionally(a) further biologically active substance(s). 39. The diagnostic kit of item 32, wherein said further biological substance is an inhibitor of glutaminy cyclase. 40. An oligonucleotide selected from the group consisting of SEQ ID Nos: 23 to 48.

(50) The antibodies of the invention may be useful for the diagnosis of amyloidosis.

(51) The antibodies of the invention may be used as affinity purification agents. In this process, the antibodies are immobilised on a solid phase such a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody is contacted with a sample containing the A-peptide (or fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the A-peptide, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0 that will release the A-peptide from the antibody.

(52) Anti-A-peptide antibodies may also be useful in diagnostic assays for A-peptide, e.g. detecting its occurrence in specific cells, tissues, or serum. Thus, the antibodies may be used in the diagnosis of amyloidosis, in particular a neurodegenerative disease selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), like for instance sporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias (FAD) such as Familial British Dementia (FBD) and Familial Danish Dementia (FDD) and neurodegeneration in Down Syndrome; preferably Alzheimer's disease.

(53) For diagnostic applications, the antibody typically will be labelled with a detectable moiety. Numerous labels are available which can be generally grouped into the following categories:

(54) (a) Radioisotopes, such as .sup.35S, .sup.14C, .sup.125I, .sup.3H, and .sup.131I. The antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Gtigen et al., Ed., Wiley-Interscience. New York, N.Y. Pubs., (1991) for example and radioactivity can be measured using scintillation counting.

(55) (b) Fluorescent labels such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are available. The fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra for example. Fluorescence can be quantified using a fluorimeter.

(56) (c) Various enzyme-substrate labels are available. The enzyme generally catalyses a chemical alteration of the chromogenic substrate which can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase. 0-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym (ed Langone & H. Van Vunakis), Academic Press, New York, 73: 147-166 (1981).

(57) Examples of enzyme-substrate combinations include, for example:

(58) (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g. orthophenylene diamine (OPD) or 3,3,5,5-tetramethyl benzidine hydrochloride (TMB));

(59) (ii) alkaline phosphatase (A) with para-Nitrophenyl phosphate as chromogenic substrate; and

(60) (iii) -D-galactosidase (-D-Gal) with a chromogenic substrate (e.g. p-nitrophenyl--D-galactosidase) or the fluorogenic substrate 4-methylumbelliferyl--D-galactosidase.

(61) Numerous other enzyme-substrate combinations are available to those skilled in the art.

(62) Sometimes, the label is indirectly conjugated with the antibody. The skilled artisan will be aware of various techniques for achieving this. For example, the antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten (e.g. digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g. anti-digoxin antibody). Thus, indirect conjugation of the label with the antibody can be achieved.

(63) The A-antibody needs not be labeled, and the presence thereof can be detected using a labeled antibody, which binds to the A-antibody.

(64) The antibodies of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies A Manual of Techniques, pp. 147-158 (CRC Press. Inc., 1987)

(65) Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody. The amount of A peptide in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies generally are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.

(66) Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one preferable type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.

(67) For immunohistochemistry, the tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example.

(68) Diagnostic Kits

(69) As a matter of convenience, the antibody of the present invention can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay. Where the antibody is labelled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e.g. a substrate precursor which provides the detectable chromophore or fluorophore). In addition, other additives may be included such as stabilizers, buffers (e.g. a block buffer or lysis buffer) and the like. 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.

(70) The diagnostic kit according to the invention may contain a further biologically active substance as described below. Especially preferred for the use in the diagnostic kit are inhibitors of glutaminyl cyclase.

(71) The diagnostic kit of the invention is especially useful for the detection and diagnosis of amyloid-associated diseases and conditions, in particular neurodegenerative diseases selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), like for instance sporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias (FAD) like Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in Down Syndrome; preferably Alzheimer's disease.

(72) The present invention pertains in particular to antibodies which are characterized in that they bind to A-peptide with a high affinity. The present invention also pertains to antibodies which are characterised in that they bind to A-peptides or variants thereof with a high affinity. Said high affinity means in the context of the present invention an affinity of a K.sub.D value of 10.sup.7 M or better, preferably a K.sub.D value of 10.sup.8 M or better, and even more preferably a K.sub.D value of 10.sup.9 M10.sup.12 M. Thereby, the inventive antibodies bind to A-peptide with a higher affinity than previously known antibodies.

(73) In particular the antibody is preferably a monoclonal antibody and is selected from the following group A 5-5-6 (DSM ACC 2923) A 6-1-6 (DSM ACC 2924) A 17-4-3 (DSM ACC 2925) A 24-2-3 (DSM ACC 2926)

(74) The antibody according to the present invention is especially useful in a diagnostic method to detect amyloidosis, in particular a neurodegenerative disease selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), like for instance sporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias (FAD) like Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in Down Syndrome; preferably Alzheimer's disease.

(75) According to a preferred embodiment, the antibody can be humanised or is a chimeric antibody or is a human antibody.

(76) Further, the antibody as selected from the above-mentioned group can also be a functional variant of said group.

(77) In the context of the present invention, a variant of an p-Glu-A peptide is in particular pGlu-A.sub.3-38, pGlu-A.sub.3-40, pGlu-A.sub.3-42

(78) Further variants of A peptides are all pGlu-A.sub.3-x variants, which have been shown to accumulate in the brain as a consequence of Alzheimer's disease or preceding Alzheimer's disease. X is defined as an integer between 10 and 42, e.g. in the above pGlu-A.sub.3-42, 42 would be the integer for x.

(79) In the context of the present invention a functional variant of the inventive antibody is an antibody which retains the binding capacities, in particular binding capacities with high affinity to a pGlu-A.sub.3-x peptide or functional variant thereof. The provision of such functional variants is known in the art and encompasses the above-mentioned possibilities, which were indicated under the definition of antibodies and fragments thereof.

(80) In a preferred embodiment, the antibody is an antibody fragment, as defined above.

(81) In a further preferred embodiment, the inventive antibody is an antibody which binds to the epitope which is bound by the antibodies as defined above, in particular antibody 5-5-6, antibody 6-1-6, antibody 17-4-3 and antibody 24-2-3.

(82) In a further preferred embodiment, the antibody of the invention is an antibody which has the complementarity-determining regions (CDRs) of the above-defined antibodies. Preferably, the antibody can be labeled; possible labels are those as mentioned above and all those known to a person skilled in the art of diagnostic uses of antibodies in particular.

(83) Preferably, the antibody is immobilized on a solid phase.

(84) The present invention also concerns an antibody which is obtainable from hybridoma cell line 6-1-6 (DSM ACC 2924).

(85) The present invention also relates to a composition which comprises the antibody as defined above. In particular, said composition is a composition for a diagnostic use, especially for the diagnosis of a neurodegenerative disease selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), like for instance sporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias (FAD) like Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in Down Syndrome; preferably Alzheimer's disease; in particular by detection of A peptide or variants thereof in a biological sample.

(86) In another embodiment, the antibody according to the invention and as described herein before or a fragment thereof, exhibits an binding affinity to an A oligomer, fiber, fibril or filament which is at least 2 times, particularly at least 4 times, particularly at least 10 times, particularly at least 15 times, more particularly at least 20 times, but especially at least 25 times higher than the binding affinity to an A monomer.

(87) In still another embodiment, an antibody or a fragment thereof or a chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof is provided as described herein before, which antibody substantially binds to aggregated A, including A plaques, in the mammalian, particularly the human brain but, preferably, does not show any significant cross-reactivity with amyloid precursor protein (APP).

(88) In another aspect of the invention, the antibody or a fragment thereof or the chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof is provided as described herein before, which antibody substantially binds to soluble polymeric amyloid, particularly amyloid (A), including A monomers, in the mammalian, particularly the human brain but, preferably, does not show any significant cross-reactivity with amyloid precursor protein (APP).

(89) The present invention relates also to humanized forms of the antibodies as defined above, compositions comprising said humanized antibodies and the use of said compositions for the treatment of amyloidosis, especially for the treatment of neurodegenerative disease in a mammal, in particular in a human. Said neurodegenerative disease is in particular selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), like for instance sporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias (FAD) like Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in Down Syndrome. Preferably, said neurodegenerative disease is Alzheimer's disease.

(90) The present invention is also directed to the following hybridoma cell lines 5-5-6, 6-1-6, 17-4-3 and 24-2-3.

(91) The present invention also pertains to the use of the antibody or the composition comprising the antibody, both as defined above, for use in an in vitro diagnostic method. In particular, this diagnostic method is directed to diagnosis of a neurodegenerative disease selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), like for instance sporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias (FAD) like Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in Down Syndrome; preferably Alzheimer's disease; especially by detecting an A peptide or variants thereof in a biological sample.

(92) Preferably, said sample is a serum sample.

(93) According to another preferred embodiment, said sample is a liquor or cerebrospinal fluid (CSF) sample.

(94) In a particularly preferred embodiment, the present invention pertains to the following method:

(95) In vitro or in situ diagnostic method for the diagnosis of an amyloid-associated disease or condition, preferably Alzheimer's disease, comprising the following steps:

(96) contacting an antibody according to the invention with a sample, preferably selected from a serum, liquor or CSF sample, most preferably a serum sample; or a specific body part or body area of a subject suspected to be afflicted with said condition or disease, and

(97) detecting binding of the antibody to a pGlu-amyloid protein, preferably pGlu-A peptide, from the sample.

(98) More particularly, the invention relates to a method of diagnosis of an amyloid-associated disease or condition, preferably Alzheimer's disease, comprising detecting the immunospecific binding of an antibody or an active fragment thereof to a pGlu-amyloid protein, preferably pGlu-A peptide, in a sample or in situ which includes the steps of

(99) (a) bringing the sample or a specific body part or body area suspected to contain the amyloid protein into contact with an antibody, particularly a monoclonal antibody according to the invention, or a chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof according to the invention and as described herein before, and/or a functional part thereof, which antibody binds a pGlu-A peptide;

(100) (b) allowing the antibody and/or a functional part thereof, to bind to the pGlu-A peptide to form an immunological complex;

(101) (c) detecting the formation of the immunological complex; and

(102) (d) correlating the presence or absence of the immunological complex with the presence or absence of pGlu-A peptide in the sample or specific body part or area.

(103) Also comprised is a method of determining the extent of amyloidogenic plaque burden in a tissue and/or body fluids comprising

(104) (a) obtaining a sample representative of the tissue and/or body fluids under investigation;

(105) (b) testing said sample for the presence of amyloid protein with an antibody, particularly a monoclonal antibody according to the invention, or a chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof according to the invention and as described herein before, and/or a functional part thereof;

(106) (c) determining the amount of antibody bound to the protein; and

(107) (d) calculating the plaque burden in the tissue and/or body fluids.

(108) In particular, the invention relates to a method of determining the extent of amyloidogenic plaque burden in a tissue and/or body fluids, wherein the formation of the immunological complex in step c) is determined such that presence or absence of the immunological complex correlates with presence or absence of amyloid protein, in particular pGlu-A peptides.

(109) In still another embodiment, the invention relates to a composition comprising the antibody according to the invention, or a chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof according to the invention and as described herein before including any functionally equivalent antibody or any derivative or functional parts thereof, in a therapeutically effective amount, in particular a composition which is a pharmaceutical composition optionally further comprising a pharmaceutically acceptable carrier.

(110) In another embodiment of the invention, said composition comprises the antibody in a therapeutically effective amount. Further comprised by the invention is a mixture comprising an antibody, particularly a monoclonal antibody according to the invention, or a chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof according to the invention and as described herein before including any functionally equivalent antibody or any derivative or functional parts thereof, in a therapeutically effective amount and, optionally, a further biologically active substance and/or a pharmaceutically acceptable carrier and/or a diluent and/or an excipient.

(111) In particular, the invention relates to a mixture, wherein the further biologically active substance is a compound used in the medication of amyloidosis, a group of diseases and disorders associated with amyloid or amyloid-like protein such as the A protein involved in neurodegenerative diseases selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), like for instance sporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias (FAD) like Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in Down Syndrome; preferably Alzheimer's disease.

(112) In another embodiment of the invention, the other biologically active substance or compound may also be a therapeutic agent that may be used in the treatment of amyloidosis caused by amyloid or may be used in the medication of other neurological disorders.

(113) The other biologically active substance or compound may exert its biological effect by the same or a similar mechanism as the antibody according to the invention or by an unrelated mechanism of action or by a multiplicity of related and/or unrelated mechanisms of action.

(114) Generally, the other biologically active compound may include neutron-transmission enhancers, psychotherapeutic drugs, acetylcholine esterase inhibitors, calcium-channel blockers, biogenic amines, benzodiazepine tranquillizers, acetylcholine synthesis, storage or release enhancers, acetylcholine postsynaptic receptor agonists, monoamine oxidase-A or -B inhibitors, N-methyl-D-aspartate glutamate receptor antagonists, non-steroidal anti-inflammatory drugs, antioxidants, and serotonergic receptor antagonists. More particularly, the invention relates to a mixture comprising at least one compound selected from the group consisting of compounds effective against oxidative stress, anti-apoptotic compounds, metal chelators, inhibitors of DNA repair such as pirenzepin and metabolites, 3-amino-1-propanesulfonic acid (3 APS), 1,3-propanedisulfonate (1,3PDS), -secretase activators, - and -secretase inhibitors, tau proteins, neurotransmitter, /3-sheet breakers, attractants for amyloid beta clearing/depleting cellular components, inhibitors of N-terminal truncated amyloid beta including pyroglutamated amyloid beta 3-42, such as inhibitors of glutaminyl cyclase, anti-inflammatory molecules, or cholinesterase inhibitors (ChEIs) such as tacrine, rivastigmine, donepezil, and/or galantamine, M1 agonists and other drugs including any amyloid or tau modifying drug and nutritive supplements, and nutritive supplements, together with an antibody according to the present invention and, optionally, a pharmaceutically acceptable carrier and/or a diluent and/or an excipient.

(115) The invention further relates to a mixture, wherein the compound is a cholinesterase inhibitor (ChEIs), particularly a mixture, wherein the compound is one selected from the group consisting of tacrine, rivastigmine, donepezil, galantamine, niacin and memantine.

(116) In a further embodiment, the mixtures according to the invention may comprise niacin or memantine together with an antibody according to the present invention and, optionally, a pharmaceutically acceptable carrier and/or a diluent and/or an excipient.

(117) In a further embodiment, the mixtures according to the invention may comprise a glutaminyl cyclase inhibitor together with an antibody according to the present invention and, optionally, a pharmaceutically acceptable carrier and/or a diluent and/or an excipient.

(118) Preferred inhibitors of glutaminyl cyclase are described in WO 2005/075436, in particular examples 1-141 as shown on pp. 31-40. The synthesis of examples 1-141 is shown on pp. 40-48 of WO 2005/075436. The disclosure of WO 2005/075436 regarding examples 1-141, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

(119) Further preferred inhibitors of glutaminyl cyclase are described in WO 2008/055945, in particular examples 1-473 as shown on pp. 46-155. The synthesis of examples 1-473 is shown on pp. 156-192 of WO 2008/055945. The disclosure of WO 2008/055945 regarding examples 1-473, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

(120) Further preferred inhibitors of glutaminyl cyclase are described in WO 2008/055947, in particular examples 1-345 as shown on pp. 53-118. The synthesis of examples 1-345 is shown on pp. 119-133 of WO 2008/055947. The disclosure of WO 2008/055947 regarding examples 1-345, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

(121) Further preferred inhibitors of glutaminyl cyclase are described in WO 2008/055950, in particular examples 1-212 as shown on pp. 57-120. The synthesis of examples 1-212 is shown on pp. 121-128 of WO 2008/055950. The disclosure of WO 2008/055950 regarding examples 1-212, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

(122) Further preferred inhibitors of glutaminyl cyclase are described in WO2008/065141, in particular examples 1-25 as shown on pp. 56-59. The synthesis of examples 1-25 is shown on pp. 60-67 of WO2008/065141. The disclosure of WO2008/065141 regarding examples 1-25, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

(123) Further preferred inhibitors of glutaminyl cyclase are described in WO 2008/110523, in particular examples 1-27 as shown on pp. 55-59. The synthesis of examples 1-27 is shown on pp. 59-71 of WO 2008/110523. The disclosure of WO 2008/110523 regarding examples 1-27, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

(124) Further preferred inhibitors of glutaminyl cyclase are described in WO 2008/128981, in particular examples 1-18 as shown on pp. 62-65. The synthesis of examples 1-18 is shown on pp. 65-74 of WO 2008/128981. The disclosure of WO 2008/128981 regarding examples 1-18, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

(125) Further preferred inhibitors of glutaminyl cyclase are described in WO 2008/128982, in particular examples 1-44 as shown on pp. 61-67. The synthesis of examples 1-44 is shown on pp. 68-83 of WO 2008/128982. The disclosure of WO 2008/128982 regarding examples 1-44, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

(126) Further preferred inhibitors of glutaminyl cyclase are described in WO 2008/128983, in particular examples 1-30 as shown on pp. 64-68. The synthesis of examples 1-30 is shown on pp. 68-80 of WO 2008/128983. The disclosure of WO 2008/128983 regarding examples 1-30, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

(127) Further preferred inhibitors of glutaminyl cyclase are described in WO 2008/128984, in particular examples 1-36 as shown on pp. 63-69. The synthesis of examples 1-36 is shown on pp. 69-81 of WO 2008/128984. The disclosure of WO 2008/128984 regarding examples 1-36, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

(128) Further preferred inhibitors of glutaminyl cyclase are described in WO 2008/128985, in particular examples 1-71 as shown on pp. 66-76. The synthesis of examples 1-71 is shown on pp. 76-98 of WO 2008/128985. The disclosure of WO 2008/128985 regarding examples 1-71, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

(129) Further preferred inhibitors of glutaminyl cyclase are described in WO 2008/128986, in particular examples 1-7 as shown on pp. 65-66. The synthesis of examples 1-7 is shown on pp. 66-73 of WO 2008/128986. The disclosure of WO 2008/128986 regarding examples 1-7, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

(130) In still another embodiment of the invention mixtures are provided that comprise atypical antipsychotics such as, for example clozapine, ziprasidone, risperidone, aripiprazole or olanzapine for the treatment of positive and negative psychotic symptoms including hallucinations, delusions, thought disorders (manifested by marked incoherence, derailment, tangentiality), and bizarre or disorganized behavior, as well as anhedonia, flattened affect, apathy, and social withdrawal, together with an antibody, particularly a monoclonal antibody according to the invention, but particularly a chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof according to the invention and as described herein and, optionally, a pharmaceutically acceptable carrier and/or a diluent and/or an excipient.

(131) In a specific embodiment of the invention, the compositions and mixtures according to the invention and as described herein before comprise the antibody and the biologically active substance, respectively, in a therapeutically effective amount.

(132) Other compounds that can be suitably used in mixtures in combination with the antibody according to the present invention are described in WO2008/065141 (see especially pages 37/38), including PEP-inhibitors (pp. 43/44), LiCl, inhibitors of dipeptidyl aminopeptidases, preferably inhibitors of DP IV or DP IV-like enzymes (see pp. 48/49); acetylcholinesterase (ACE) inhibitors (see p. 47), PIMT enhancers, inhibitors of beta secretases (see p. 41), inhibitors of gamma secretases (see pp. 41/42), inhibitors of neutral endopeptidase, inhibitors of phosphodiesterase-4 (PDE-4) (see pp. 42/43), TNFalpha inhibitors, muscarinic M1 receptor antagonists (see p. 46), NMDA receptor antagonists (see pp. 47/48), sigma-1 receptor inhibitors, histamine H3 antagonists (see p. 43), immunomodulatory agents, immunosuppressive agents or an agent selected from the group consisting of antegren (natalizumab), Neurelan (fampridine-SR), campath (alemtuzumab), IR 208, NBI 5788/MSP 771 (tiplimotide), paclitaxel, Anergix.MS (AG 284), SH636, Differin (CD 271, adapalene), BAY 361677 (interleukin-4), matrix-metalloproteinase-inhibitors (e.g. BB 76163), interferon-tau (trophoblastin) and SAIK-MS; beta-amyloid antibodies (see p. 44), cysteine protease inhibitors (see p. 44); MCP-1 antagonists (see pp. 44/45), amyloid protein deposition inhibitors (see 42) and beta amyloid synthesis inhibitors (see p. 42), which document is incorporated herein by reference.

(133) In another embodiment, the invention relates to a mixture comprising the antibody, particularly a monoclonal antibody according to the invention, or a chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof according to the invention and as described herein before and/or the biologically active substance in a therapeutically effective amount.

(134) The invention further relates to the use of an antibody, particularly a monoclonal antibody according to the invention, but particularly a chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof according to the invention and as described herein before and/or a functional part thereof and/or a pharmaceutical composition, or a mixture comprising said antibody, for the preparation of a medicament for treating or alleviating the effects of amyloidosis, a group of diseases and disorders associated with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis such as diseases including, but not limited to, neurological disorders such as Alzheimer's Disease (AD), Lewy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); the Guam Parkinson-Dementia complex; as well as other diseases which are based on or associated with amyloid-like proteins such as progressive supranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease, Parkinson's disease, HIV-related dementia, ALS (amyotropic lateral sclerosis), Adult Onset Diabetes; senile cardiac amyloidosis; endocrine tumors, and others, including macular degeneration.

(135) Also comprised by the present invention is a method for the preparation of an antibody, particularly a monoclonal antibody according to the invention, but particularly a chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof according to the invention and as described herein before and/or a functional part thereof and/or a pharmaceutical composition, or a mixture comprising said antibody and/or a functional part thereof, particularly in a therapeutically effective amount, for use in a method of preventing, treating or alleviating the effects of amyloidosis, a group of diseases and disorders associated with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis such as diseases including, but not limited toneurodegenerative diseases such as mild cognitive impairment (MCI), Alzheimer's disease (AD), like for instance sporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias (FAD) like Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in Down Syndrome; Lewy body dementia, hereditary cerebral hemorrhage with amyloidosis (Dutch type); the Guam Parkinson-Dementia complex; as well as other diseases which are based on or associated with amyloid-like proteins such as progressive supranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease, Parkinson's disease, HIV-related dementia, ALS (amyotropic lateral sclerosis), Adult Onset Diabetes; senile cardiac amyloidosis; endocrine tumors, and others, including macular degeneration comprising formulating an antibody, particularly a monoclonal antibody according to the invention, but particularly a chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof according to the invention in a pharmaceutically acceptable form.

(136) Further comprised by the present invention is a method for preventing, treating or alleviating the effects of amyloidosis, a group of diseases and disorders associated with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis such as diseases including, but not limited to, neurological disorders such as mild cognitive impairment (MCI), Alzheimer's disease (AD), like for instance sporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias (FAD) like Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in Down Syndrome; Lewy body dementia, hereditary cerebral hemorrhage with amyloidosis (Dutch type); the Guam Parkinson-Dementia complex; as well as other diseases which are based on or associated with amyloid-like proteins such as progressive supranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease, Parkinson's disease, HIV-related dementia, ALS (amyotropic lateral sclerosis), Adult Onset Diabetes; senile cardiac amyloidosis; endocrine tumors, and others, including macular degeneration by administering an antibody and/or a functional part thereof, but particularly a humanized antibody and/or a functional part thereof, or a composition or mixture comprising such an antibody and/or a functional part thereof, to an animal or a human affected by such a disorder comprising administering the antibody in a therapeutically effective amount.

(137) It is also an object of the invention to provide a method for the treatment of amyloidosis, a group of diseases and disorders associated with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis including, but not limited to, neurodegenerative diseases such as mild cognitive impairment (MCI), Alzheimer's disease (AD), like for instance sporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias (FAD) like Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in Down Syndrome; particularly a disease or condition characterized by a loss of cognitive memory capacity by administering to an animal, particularly a mammal or a human, an antibody, particularly a pharmaceutical composition according to the invention and as described herein.

(138) In a specific embodiment the invention provides a method for retaining or increasing cognitive memory capacity but, particularly, for restoring the cognitive memory capacity of an animal, particularly a mammal or a human, suffering from memory impairment by administering to an animal, particularly a mammal or a human, an antibody, particularly a pharmaceutical composition according to the invention and as described herein before.

(139) It is a further object of the invention to provide a therapeutic composition and a method of producing such a composition as well as a method for the treatment of amyloidosis, a group of diseases and disorders associated with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis including, but not limited to, neurodegenerative diseases such as mild cognitive impairment (MCI), Alzheimer's disease (AD), like for instance sporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias (FAD) like Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in Down Syndrome; particularly a disease or condition characterized by a loss of cognitive memory capacity, using an antibody according to the invention and as described herein before.

(140) In particular, the invention relates to the treatment of an animal, particularly a mammal or a human, suffering from an amyloid-associated condition characterized by a loss of cognitive memory capacity that leads to the retention of cognitive memory capacity.

(141) 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

(142) 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.

(143) 1. Material and Methods

(144) 1.1 Production of Antibodies

(145) Mice

(146) For the production of hybridomas, female BALB/C mice (Charles River) of 8 weeks age were used.

(147) Myeloma Cell Line

(148) For the generation of the hybridomas, the myeloma cell line SP2/0-Ag14 from Deutsche Stammsammlung von Mikoorganismen and Zellkulturen was used.

(149) Antigen

(150) The peptide pGlu-6166 (sequence pGlu-FRHDSGC, SEQ ID NO: 65) was used.

(151) Strategy

(152) As an immunogen, the peptide was coupled to bovine thyroglubulin (BTG, SIGMA) via maleimid groups from three different linkers. The three linkers of different length were used from N-[e-maleimidocaproyloxy]succinimide ester (EMCS), succiminidyl-4-(N-maleimidomethyl)-cyclohexan-1-carboxy-(6-amidocaproate) (LCSMCC) and N-[b-maleimidopropyloxy]succinimide ester (BMPS). For the detection of the generated antibodies, the same peptide was conjugated to bovine serum albumine (BSA, SIGMA) via maleimid groups from succinimidyl-6-[(b-maleimido-propionamido)hexanoate] (SMPH).

(153) Methods

(154) Conjugation of the Peptide for Immunization

(155) Conjugation was performed in two steps via SH-groups from the cystein residue of the peptide.

(156) 1. Maleoylation of the Carrier Protein

(157) 2 to 5 mg of the respective linker (50 mg/ml in N-methylpyrrolidone, NMP) was added to 2 ml of the carrier protein solution (10 mg/ml in 0.1 mM NaHCO3, pH 8.0). The reaction mixture was incubated for 1 h at room temperature (RT). The reaction mixture was thereafter desalted using a Sephadex G-50 column (1.514 cm), which was equilibrated with 50 mM sodium phosphate, 250 mM NaCl, pH 6.8.

(158) 2. Coupling of the Maleoylated BTG with the Peptide

(159) 250 l of the peptide solution (10 mg/ml in Aqua bidest) were mixed with 2 ml of a solution containing the maleoylated carrier proteins (2.5 mg/ml) in 50 mM sodium phosphate, 250 ml NaCl, pH 6.8 and incubated for 2 h at 4 C. and further 4 h at RT. Unreacted maleimid groups were blocked by addition of 2-mercaptoethanole up to a concentration of 10 mM and over night incubation at 4 C. The resulting conjugate was dialysed against 10 mM sodium phosphate, 150 mM NaCl, pH 7.5 at 4 C. (3 times buffer exchange, MW cut-off 10.000).

(160) Conjugation of the Peptide for ELISA

(161) Conjugation was performed in two steps via SH-groups from the cystein residue of the peptide.

(162) 1. Maleoylation of the Carrier Protein

(163) 2 mg SMPH (50 mg/ml in N-methylpyrrolidone, NMP) was added to 2 ml of the carrier protein solution (BSA, 10 mg/ml in 0.1 mM NaHCO3, pH 8.0). The reaction mixture was incubated for 1 h at room temperature (RT). The reaction mixture was thereafter desalted using a Sephadex G-50 column (1.514 cm), which was equilibrated with 50 mM sodium phosphate, 250 mM NaCl, pH 6.8.

(164) 2. Coupling of the Maleoylated BTG with the Peptide

(165) 100 l of the peptide solution (10 mg/ml in 50 mM sodium phosphate, 250 mM NaCl, pH 6.8) were mixed with 1 ml of a solution containing the maleoylated carrier proteins (2.5 mg/ml) in 50 mM sodium phosphate, 250 ml NaCl, pH 6.8 and incubated for 2 h at 4 C. and further 4 h at RT. Unreacted maleimid groups were blocked by addition of 2-mercaptoethanole up to a concentration of 10 mM and over night incubation at 4 C. The resulting conjugate was dialysed against 10 mM sodium phosphate, 150 mM NaCl, pH 7.5 at 4 C. (3 times buffer exchange, MW cut-off 10.000).

(166) Immunization

(167) Five mice were immunized intraperitoneally for 39 days. For immunization, a water-in-oil emulsion consisting of equal parts of the antigen solution (consisting of equal parts of the three different peptide-BTG-conjugates) and complete or incomplete Freundt's adjuvants was used.

(168) Fusion

(169) Three of the immunized mice were sacrificed by CO2 incubation. Spleens were taken and homogenized under sterile conditions. Spleen cells and myeloma SP2/0 cells were washed several times in Dulbecco's Modified Eagle Medium (DMEM, SIGMA) and fused in the ratio of 2,3 spleen cells: 1 SP2/0 cell using polyethylenglycole 3350 (1 ml 50% (w/v)). Further handling of the fused hybridomas was performed according to standard methodologies.

(170) ELISA

(171) An IgG directed ELISA was used to screen the cell culture supernatant. The test was performed in 96-well polystyrol microtiter plates (Greiner, Cat. No. 655061). The plates were coated with the BSA-pGlu-6166 peptide. 100 l undiluted cell culture supernatant was added to each well and incubated for 1 h at RT. Supernatant from SP2/0 cells was used as negative control. Supernatant from the spleen cells was used as positive control.

(172) Positive wells were detected using goat-anti-mouse IgG, which was conjugated with alkaline phosphatase. The optical density (OD) was measured in a Dynex Opsys MR Microplate Reader at 405 nm.

(173) Selection of stable antibody producing hybridoma cells Cells from positive wells were transferred to 24-well plates and cultivated for several days. Cells were again transferred and tested for BSA-pGlu-6199 binding and cross-reactivity in the ELISA. Positive wells were used for cryo-conservation of the hybridoma cell lines.

(174) Cloning Via Limited Dilution

(175) Two consecutive cloning steps were performed in order to separate antibody producing cells from non-producing cells and to assure that the selected cells are monoclonal. Both cloning steps were performed according to the method of limited dilution.

(176) Cryoconservation

(177) Selected hybridomas were cryo-conserved using DMSO and standard methods.

(178) 1.2. ELISA Assays

(179) Capturing of A N3pE-40 was performed using hA (x-40) ELISA (HS) from TGC (The GENETICS Company; Switzerland), basically according to the manufacturer's instructions.

(180) Biotinylated detection antibodies for A N3pE (pGlu-6166) were generated. Where indicated, IBL HRP-conjugated A N3pE antibody was used as positive control (available only in combination with the IBL ELISA Human Amyloid (N3pE) Assay Kit). Corresponding A N3pE-40 peptides (50 g aliquots in Hexafluoroisopropanol (HFIP) stored at 80 C.) were synthesized. Shortly before use, HFIP was evaporated and the peptide was diluted with 100 mM Tris/HCl pH 10, 4 to 1 g/l. This stock solution was diluted further with TGC antibody diluent. Subsequent capturing and detection was carried out according to manufacturer's instructions.

(181) 1.3. PepSpot Analysis

(182) Specificity and biological integrity of A N3pE antibodies and cell culture supernatants was determined by using the PepSpot technology of JPT Peptide Technologies GmbH, Volmerstrasse 5 (UTZ), 12489 Berlin, Germany.

(183) Corresponding PepSpot membranes were prepared at JPT. The principle of this method was introduced and described before by Kramer et al. 1997 Cell 91, 799-809.

(184) For analysis, membranes were blocked for two hours with TBST-M (10 mM Tris-Hcl, pH 7.5, 150 mM NaCl, 0.005% Tween20+5% skimmed milk) at room temperature with gentle shaking. Membranes were incubated over night at 4 C. on a rocking platform with the individual cell culture supernatants diluted in equal volumes of TBST-M. Secondary anti-mouse antibody conjugated with alkaline phosphatase was used for signal detection, following standard procedures.

(185) 1.4. DotBlot Analysis

(186) A simple DotBlot protocol was accomplished to obtain information about the sensitivity of A N3pE antibody and cell culture supernatants towards the respective native peptide. To that end, A N3pE-40 peptide in descending concentrations (1000 ng-8 ng) was spotted onto small pieces of nitrocellulose membrane, and subsequent experimental procedure was performed as for the PepSpot membranes.

(187) 1.5. SDS PAGE

(188) 12% SDS Polyacrylamide gels were cast following standard protocols. 15 l of cell culture supernatants and 10 g of biotinylated antibody were separated on a 12% SDS polyacrylamide gel. Electrophoresis was carried out for 2 hours at 100 V constant.

(189) 1.6. BIACORE Analysis

(190) A N3pE-40 peptide (positive control) and A N3E-40 peptide (negative control) were coupled on a Biacore CM5 Chip. Unmodified chips are used to determine blank values. Association and dissociation of biotinylated antibody diluted from 20 g/ml to 1 g/ml in TGC diluent was monitored to allow for subsequent determination of the respective KD value. In this way also binding characteristics of the individual cell culture supernatants were determined.

(191) 1.6.1 Affinity of AN3pE Specific Antibody Clone 6-1-6 and 24-2-3

(192) The purified antibody clone 6-1-6 was diluted in HBS-EP buffer (Biacore) down to 100, 50, 30, 20, 15, 10, 7, 4, 2, 1 nM. The affinity was determined using a Biacore 3000 with CM5-Chip, on which ApE3-40 was immobilized. The system was run with 30 l/min. Measured bulk effects and unspecific binding to the chip surface were corrected by subtraction of the signal of flow cell 4, at which ApE3-40 was immobilized, and the empty flow cell 3. The association (10 min) was obtained by injection of 300 l of each concentration. The dissociation was observed over 10 min. Remaining antibody molecules were removed by injection of 5 l 0.1 M HCL. For every antibody concentration the association and dissociation was recorded. The determination of the association and dissociation rate and the dissociation constant was performed by a global simultaneously fit of association and dissociation phase for all recorded antibody concentrations using the Bivalent analyte model.

(193) 1.7. Sequencing Antibody Variable Regions

(194) Cultivation of Hybridoma Cells:

(195) Hybridoma cells were grown in D-MEM (+L-Glutamin, +Na-Pyruvat, 4, 5 g/l Glucose, Gibco) with the addition of 15% FBS, 1% MEM-NEA (non essential amino acids, Gibco), 50 g/ml Gentamycin (Gibco) and 50 M -mercaptoethanol at 37 C. and 5% CO.sub.2. Subcultivation occurred after 3-4 days depending on cell density. Cells were seeded in a concentration of 0.510.sup.6 cells/ml, splitting occurred at a cell density of 2-510.sup.6 cells/ml.

(196) cDNA Synthesis and Reverse Transcription:

(197) Total RNA was isolated from 210.sup.6 cells according to the manual of the NucleospinRNA Isolation Kit (Macherey-Nagel). 100 ng RNA were applied for cDNA synthesis by using Oligo (dT).sub.15 primer (Promega) and SuperScript III Reverse Transcriptase (Invitrogen).

(198) PCR-Amplification of Heavy and Light Chain Variable Regions:

(199) Heavy chain variable regions were amplified from the template cDNA by using Phusion High-Fidelity DNA Polymerase (NEW ENGLAND BioLabs) with the primer MHCG1 (in case of clone 5-5-6 and 6-1-6) and MHCG2b (clone 17-4-3 and 24-2-3) in combination with primers MHV1-12. For amplification of light chain variable regions the primer MKC in combination with the primers MKV1-MKV11 were used. Primer sequences are shown in table 1.

(200) Cloning of PCR Products in pJET1.2:

(201) Heavy and light chain variable regions, amplified by PCR, were cloned into pJET1.2/blunt vector according to the protocol of CloneJET PCR Cloning Kit (Fermentas). Sequencing occurred with pJET1.2 sequencing primers.

(202) TABLE-US-00003 TABLE1 PrimersequencesforPCR-amplificationof heavyandlightchainvariableregions SEQ ID Name Sequence NO. MKV1 ATGAAGTTGCCTGTTAGGCTGTTGGTGCTG 23 MKV2 ATGGAGWCAGACACACTCCTGYTATGGGTG 24 MKV3 ATGAGTGTGCTCACTCAGGTCCTGGSGTTG 25 MKV4 ATGAGGRCCCCTGCTCAGWTTYTTGGMWTCTTG 26 MKV5 ATGGATTTWCAGGTGCAGATTWTCAGCTTC 27 MKV6 ATGAGGTKCYYTGYTSAGYTYCTGRGG 28 MKV7 ATGGGCWTCAAGATGGAGTCACAKWYYCWGG 29 MKV8 ATGTGGGGAYCTKTTTYCMMTTTTTCAATTG 30 MKV9 ATGGTRTCCWCASCTCAGTTCCTTG 31 MKV10 ATGTATATATGTTTGTTGTCTATTTCT 32 MKV11 ATGGAAGCCCCAGCTCAGCTTCTCTTCC 33 MKC ACTGGATGGTGGGAAGATGG 34 MHV1 ATGAAATGCAGCTGGGGCATSTTCTTC 35 MHV2 ATGGGATGGAGCTRTATCATSYTCTT 36 MHV3 ATGAAGWTGTGGTTAAACTGGGTTTTT 37 MHV4 ATGRACTTTGGGYTCAGCTTGRTTT 38 MHV5 ATGGACTCCAGGCTCAATTTAGTTTTCCTT 39 MHV6 ATGGCTTGTCYTRGSGCTRCTCTTCTGC 40 MHV7 ATGGRATGGAGCKGGRTCTTTMTCTT 41 MHV8 ATGAGAGTGCTGATTCTTTTGTG 42 MHV9 ATGGMTTGGGTGTGGAMCTTGCTATTCCTG 43 MHV10 ATGGGCAGACTTACATTCTCATTCCTG 44 MHV11 ATGGATTTTGGGCTGATTTTTTTTATTG 45 MHV12 ATGATGGTGTTAAGTCTTCTGTACCTG 46 MHCG1 CAGTGGATAGACAGATGGGGG 47 MHCG2b CAGTGGATAGACTGATGGGGG 48

(203) 1.8 Application of Antibody Clone 6-1-6 for N3pE ELISA

(204) A 96-well maxisorb plate (Nunc) was coated with capture antibody by incubation of 100 l per well of 2 g/ml anti-A antibody 4G8, diluted in D-PBS, overnight at 4 C. The plated was sealed. The coating solution was removed and the surface of the plate was blocked with 200 l per well PIERCE Protein-free ELISA-Blocker (without Tween-20) for 2 hours at room temperature. Afterwards the plate was washed with 6-times with TBS+0.05% (v/v) Tween-20. Remaining washing solution was removed by tapping of the plate. The ApE3-40 standard peptide was diluted in PIERCE Protein-free ELISA-Blocker (with Tween-20) down to 200, 100, 50, 25, 12.5, 6.25, 3.125 pg/ml. 100 l of every concentration and 100 l of dilution buffer (Blank) were pipetted on the plate. The plate was sealed and incubated at 4 C. for 2 hours. Afterwards the plate was washed 6-times with TBS+0.05% (v/v) Tween-20. Remaining washing solution was removed by tapping of the plate. 100 l of the detection antibody-enzyme conjugate solution, which contains 1 g/ml AN3pE specific antibody clone 6-1-6 and 2 pg/ml Streptavidin-HRP conjugate (Sigma) solved in PIERCE Protein-free ELISA-Blocker (with Tween-20), was pipetted in each well. The plate was sealed and incubated at 4 C. for 1 hour. Afterwards the plate was washed 6-times with TBS+0.05% (v/v) Tween-20. Remaining washing solution is removed by tapping of the plate. In every well 100 l SureBlue substrate solution (KPL) are pipetted and the plate was incubated in the dark at room temperature for 30 min. The reaction was stopped by addition of 100 l per well of 1 M H.sub.2SO.sub.4. The absorbance was measured with TECAN Sunrise at 450 nm corrected by the absorbance at 540 nm.

(205) 1.9 Investigation of Cross Reactivity, Analyzed Via ELISA and Surface-Plasmon-Resonance (SPR)

(206) ELISA:

(207) A 96-well maxisorb plate (Nunc) was coated with capture antibody by incubation of 100 l per well of 2 g/ml anti-A antibody 4G8, diluted in D-PBS, overnight at 4 C. The plated was sealed. The coating solution was removed and the surface of the plate was blocked with 200 l per well PIERCE Protein-free ELISA-Blocker (without Tween-20) for 2 hours at room temperature. Afterwards the plate was washed with 6-times with TBS+0.05% (v/v) Tween-20. Remaining washing solution was removed by tapping of the plate. The ApE3-40 standard peptide and other A-Peptides (2-40, 3-40, 4-40, 1-42, 3-42 and pE11-40) were diluted in PIERCE Protein-free ELISA-Blocker (with Tween-20) down to 800, 400, 200, 100, 50, 25, 12.5. 100 l of every concentration and 100 l of dilution buffer (Blank) were pipetted on the plate. The plate was sealed and incubated at 4 C. for 2 hours. Afterwards the plate was washed 6-times with TBS+0.05% (v/v) Tween-20. Remaining washing solution was removed by tapping of the plate. 100 l of the detection antibody-enzyme conjugate solution, which contains 1 g/ml AN3pE specific antibody clone 6-1-6 and 2 g/ml Streptavidin-HRP conjugate (Sigma) diluted in PIERCE Protein-free ELISA-Blocker (with Tween-20), was pipetted in each well. The plate was sealed and incubated at 4 C. for 1 hour. Afterwards the plate was washed 6-times with TBS+0.05% (v/v) Tween-20. Remaining washing solution was removed by tapping of the plate. In every well 100 l SureBlue substrate solution (KPL) were pipetted and the plate was incubated in the dark at room temperature for 30 min. The reaction was stopped by addition of 100 l per well of 1M H.sub.2SO.sub.4. The absorbance was measured with TECAN Sunrise at 450 nm corrected by the absorbance at 540 nm.

(208) SPR:

(209) Beside different A species also the cross reactivity to other pGlu-Peptide, which occur in the human body, was determined. This was made by surface plasmon resonance. Following peptides or there N-terminal region of them were immobilized on the surface of CM5-Chips:

(210) MCP1, MCP2, big gastrin, gonadoliberin, neurotensin, orexin A, fibronectin, collagen 1 and TRH. As positive control also the binding to ApE3-40 was analyzed. The N3pE antibody clones 6-1-6 and 24-2-3 were diluted in HBS-EP (Biacore) down to 25 g/ml. The binding was observed using a Biacore 3000 with several CM5-Chips, on which the respective peptides (on flow cell 2, 3 and 4 was immobilized. The system was run with 20 l/min. Measured bulk effects and unspecific binding to the chip surface were corrected by subtraction of the signal of flow cell 2, 3 and 4, at which the tested peptides was immobilized, and the empty flow cell 1. The association (9 min) was obtained by injection of 180 l of antibody clones 6-1-6 and 24-2-3, respectively. The dissociation was observed over 9 min. Remaining antibody molecules were removed by injection of 5 l 0.1 M HCL. For every interaction of the antibody with the different peptides the association and dissociation was recorded. The cross reactivity was determined by evaluation of the association phase concerning rate and signal at the end. The values for all pGlu-Peptides compared with the signal for ApE3-40.

(211) 1.10 Optimization and Validation of N3pE ELISA for Brain Analysis

(212) Our developed N3pE ELISA should be used for analysis of ApE3-42 concentration in brain of transgenic mice. Generally, hemisphere and brainstem were separately analyzed concerning ApE3-42 content. Mouse brain was homogenized in 500 l 2-SDS solution with protease inhibitor in Precelly (Peqlab) homogenizer using ceramic beads. The suspension was pipetted off from the beads and transferred into centrifuge tube. Beads were washed again with 250 l 2-SDS solution with protease inhibitor and solution transferred into the centrifuge tube. The 750 l SDS brain suspension was sonificated on crashed ice for 20 sec. The sample was centrifuged for 1 hour at 4 C. with 75000g. Afterwards the supernatant was removed, aliquoted and stored until ELISA analysis at 80 C. The remaining SDS insoluble pellet was mixed with 150 l 70% formic acid and sonificated on crashed ice for 20 sec. Immediately after sonification the solution was neutralized with 2850 l 1 M Tris, which was the old method, or 2850 l EIA buffer (PBS+10 mg/ml BSA+0.05% Tween-20)+860 l 3.5 M Tris for neutralization, representing the new method. The formic acid fraction samples were stored until ELISA at 80 C.

(213) The N3pE ELISA was performed by following protocol:

(214) A 96-well maxisorb plate (Nunc) was coated with capture antibody by incubation of 100 l per well of 2 g/ml anti-A antibody 4G8, diluted in D-PBS, overnight at 4 C. The plated was sealed. The coating solution was removed and the surface of the plate was blocked with 200 l per well PIERCE Protein-free ELISA-Blocker (without Tween-20) for 2 hours at room temperature. Afterwards the plate was washed with 6-times with TBS+0.05% (v/v) Tween-20. Remaining washing solution was removed by tapping of the plate. The ApE3-42 standard peptide was diluted in PIERCE Protein-free ELISA-Blocker (with Tween-20) (old method) or EIA buffer (new method) down to 1029.2, 514.6, 257.3, 128.65, 64.32, 31.16, 16.08 g/ml. 100 l of every concentration and 100 l of dilution buffer (Blank) were pipetted on the plate. The SDS samples were thawed, diluted 1:25 and 1:100, respectively, in PIERCE Protein-free ELISA-Blocker (with Tween-20) (old method) or EIA buffer (new method) and pipetted on ELISA plate. The formic acid samples (old method: formic acid/Tris; new method: formic acid/EIA buffer/Tris) were thawed and undiluted pipetted on ELISA plate. The plate was sealed and incubated at 4 C. for 2 hours. Afterwards the plate was washed 6-times with TBS+0.05% (v/v) Tween-20. Remaining washing solution was removed by tapping of the plate. 100 l of the detection antibody-enzyme conjugate solution, which contains 1 g/ml AN3pE specific antibody clone 6-1-6 and 2 g/ml Streptavidin-HRP conjugate (Sigma) solved in PIERCE Protein-free ELISA-Blocker (with Tween-20), was pipetted in each well. The plate was sealed and incubated at 4 C. for 1 hour. Afterwards the plate was washed 6-times with TBS+0.05% (v/v) Tween-20. Remaining washing solution was removed by tapping of the plate. In every well 100 l SureBlue substrate solution (KPL) are pipetted and the plate was incubated in the dark at room temperature for 30 min. The reaction was stopped by addition of 100 l per well of 1 M H.sub.2SO.sub.4. The absorbance was measured with TECAN Sunrise at 450 nm corrected by the absorbance at 540 nm.

(215) 1.11 Application of N3pE Antibody Clones for Immunohistochemistry

(216) Formalin-fixed and paraffin-embedded sections from human brain (cortex) were treated as follows:

(217) 1. Deparaffinizing and rehydrating sections (immobilized on slides): a. Incubation of slides in Histoclear or xylene for 3 minutes b. Remove cleaning solution c. Incubation of slides again in Histoclear or xylene for 3 minutes d. Incubation of slides in Histoclear or xylene 1:1 with 100% ethanol for 3 minutes e. Incubation of slides in 100% ethanol for 3 minutes, remove solution f. Incubation of slides again in 100% ethanol for 3 min g. Incubation of slides in 95% ethanol for 3 minutes h. Incubation of slides in 70% ethanol for 3 minutes i. Incubation of slides in 50% ethanol for 3 minutes j. Incubation of slides in distilled water for 3 minutes

(218) 2. Quenching endogenous peroxidase activity:

(219) Incubation of slides with 99 ml methanol+1 ml 30% hydrogen peroxide for 10 minutes at room temperature.

(220) 3. Washing the slides with water: 25 minutes

(221) 4. Removing water in individual slides and place slides on slide rack in a humidity chamber to prevent sections from drying. Cover section with 88% formic acid at room temperature for 10 minutes under fume hood. Rinse in water several times and allow to shake in a water-filled staining dish for 10 minutes.

(222) 5. Blocking in 10% horse serum for 20 minutes at room temperature.

(223) 6. Shaking off (or aspirate) blocking solution and apply primary antibody (N3pE antibody clone 6 or 24) for overnight at 4 C.

(224) 7. Washing slides separately with TBS for 10 minutes to avoid dragging from one slide to another.

(225) 8. Addition of biotinylated secondary antibody (goat anti-mouse from Vector Laboratories): 9 ml TBS, 1 ml goat serum, 45 l 2.sup.nd antibody). Incubate 30 minutes at room temperature.

(226) 9. Washing slides separately with TBS for 10 minutes to avoid dragging from one slide to another.

(227) 10. Addition of ABC-solution (10 ml TBS, 100 l horse serum, 90 l component A, 90 l component B). Incubate 30 minutes at room temperature.

(228) 11. Washing slides with 50 mM Tris: 210 minutes

(229) 12. Color reaction: Incubation of sections with DAB solution (20 mg DAB from Sigma in 100 ml 50 mM Tris, filtered, and add 33 l 33% hydrogen peroxide). Using microscope to observe color reaction. The reaction product is brown colored. Stop the reaction by putting slides into staining dishes containing water.

(230) 13. Washing slides with water for 10 minutes

(231) 14. Counterstaining with hematoxylin, washing with water.

(232) 15. Dehydrating and clearing: Follow step 1 in reverse order (e.g. water, ethanols to 100% histoclean)

(233) 16. Coverslip with permount (Fisher Scientific). Drying slides on air. Clean slides with razor blade and ethanol.

(234) 2. Results

(235) 2.1 Production of Antibodies

(236) Six clones were isolated that stably produce antibodies against the pGlu-6166-BSA peptide: clones 1-8-12, 5-5-6, 6-1-6, 12-1-8, 17-4-3 and 24-2-3. These clones were subject to further characterization.

(237) 2.2. Determination of Required Antibody Concentration:

(238) The intensity of signals in the ELISA assays correlates not only with the concentration of analyte/A variant but is also strongly dependent on the concentration of deployed antibody. Since A variants are only present in low concentration in serum samples it is necessary to determine antibody concentrations that are able to detect low concentrations of the corresponding A variants. Commercially available A ELISA kits have a specified detection limit towards A in the low pg range. In standard curves the highest concentration is usually 500 pg/ml. General information about deployed antibody concentration is typically lacking in data sheets/instruction manuals, however, due to further information as derivable from the general literature 1 g/ml of antibody is used as default.

(239) In a first series of experiments it was not possible to detect 500 pg/ml A N3pE-40 with the corresponding pGlu-6166 12-1 biotin-conjugated antibody. In fact, relatively high A N3pE concentrations (10 ng/ml) were required to obtain signals with 1 g/ml antibody (see FIG. 1A: middle bar). The intensity of this signal was tremendously enhanced by increasing the antibody concentration to 10 g/ml (see FIG. 1A: left-hand bar). Until 20 g/ml antibody the intensity of signal can be further elevated (see FIG. 1B). Beyond this concentration no further increase in signal intensity can be achieved. Therefore, 20 g/ml antibody was used to determine the detection limit for the pGlu-6166 antibody.

(240) 2.3. DotBlot Analysis

(241) The A N3pE-x antibody pGlu-6166 was chosen in the screening process because the original cell clone (designated 12-1-8) exhibited strong binding towards the peptide taken for immunization and very low cross reactivity (see table 2).

(242) TABLE-US-00004 TABLE 2 Screening results demonstrating signals in ELISA assays obtained with several hybridoma cell clone supernatants. N3pE- isoDAE- N3E- N11E- N11pE- Clone BSA BSA BSA BSA BSA 1-8-12 1.787 0.012 0.142 0.011 0.005 5-5-6 1.649 0.015 0.126 0.004 0.006 6-1-6 1.377 0.013 0.125 0.007 0.014 12-1-8 2.123 0.005 0.009 0.001 0.005 17-4-3 1.915 0.007 0.320 0.003 0.004 24-2-3 1.768 0.014 0.218 0.003 0.002 positive 1.824 1.227 1.596 1.243 1.346 control negative 0.045 0.005 0.008 0.001 0.003 control

(243) No screening step, dealing with the full length, native A N3pE-40 peptide had been included so far. Therefore the pool of available pGlu6166 hybridoma cell clones was screened for clones expressing antibodies which might exhibit a higher affinity for the native A N3pE-40 peptide.

(244) As seen in FIG. 2, cell clones could be identified which indeed exhibit higher sensitivity towards the native full length A N3pE-40 peptide. Whereas the pGlu-6166 antibody clone 12-1 could only detect 1 g peptide, clones 6-1-6 and 24-2-3 also gave signals with as little as 8 ng peptide. Hence, clones 6-1-6 and 24-2-3 are 125 times more sensitive. With these clones, a detection limit of 8 g/ml A N3pE-40 peptide in a corresponding ELISA could be attainable.

(245) 2.4. PepSpot Analysis

(246) Specificity was checked next by PepSpot analysis to compare biotinylated A N3pE-x antibody pGlu-6166 with hybridoma cell clones. In table 3, all peptides are listed which correspond to spots on the PepSpot membrane. As seen in FIG. 3 pGlu-6166 clones 6-1-6 and 24-2-3 do not produce more cross signals than pGlu-6166 antibody clone 12-1. All clones investigated recognized primarily spot number 6the specific A N3pE-x spot (pEFRHD . . . , i.e. SEQ ID No: 12), followed by spots number 5 (EFRHD . . . SEQ ID No: 11) and 7 (FRHD . . . SEQ ID No: 13). A faint signal was also attained with spot number 4 (AEFRHD . . . SEQ ID No: 10).

(247) TABLE-US-00005 TABLE3 SequencesofA peptidesspottedontoPepSpot Membranes(JPTPeptideTechnologiesGmbH)and detectionbypGlu-6166hybridomacellclones SEQ PeptideSequenceA 1-40(SEQIDNo:2) IBL- 5- 6- 17- 24- 12- ID No. --DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV AK 5-6 1-6 4-3 2-3 4 No. 1 KMDAEFRHDSGYE +/ 7 2 DAEFRHDSGYEVH +/ 8 3 iDAEFRHDSGYEVH +/ 9 4 AEFRHDSGYEVHH +/ +/ +/ +/ 10 5 EFRHDSGYEVHHQ + + + + + 11 6 pEFRHDSGYEVHHQ +++ +++ +++ +++ +++ 12 7 FRHDSGYEVHHQK + + + + + + 13 8 GYEVHHQKLVFFA 14 9 EVHHQKLVFFAED 15 10 pEVHHQKLVFFAED 16 11 VHHQKLVFFAEDV 17 12 DAEFRHiDSGYEVH 18 13 iDSGYEVHHQKLVF 19 14 LVFFAEDVGSNKG 20 15 GSNKGAIIGLMVG 21 16 AIIGLMVGGVV 22 pE in table 3 means pGlu, pyroglutamate. iD in table 3 means isoAsp, isoaspartate.

(248) 2.5. SDS-PAGE Analysis

(249) Biological integrity of the A-N3pE antibody and hybridoma cell culture supernatants was determined roughly by SDS-PAGE analysis (for details see Material & Methods supra).

(250) As seen in FIG. 4, all samples loaded onto the gel revealed sharp bands without smears, indicating the integrity of the pGlu-6166 12-1 antibody and the hybridoma cell clone supernatants.

(251) 2.6. BIACORE Analysis

(252) With DotBlot analysis significant differences in sensitivity toward the A N3pE-40 peptide of hybridoma cell clone supernatants compared to biotinylated pGlu-6166 antibody were diagnosed. However, with this method only an end point result is monitored. Biacore analysis on the other hand allows timewise resolution of the binding course of a given antibody. In order to check whether the poor binding of the pGlu-6166 12-1 antibody was a result of a low association to the A N3pE-40 peptide, a Biacore analysis was performed as described in Materials and Methods, supra.

(253) Monitoring binding courses of increasing concentrations of pGlu-6166 antibody allowed for calculation of a KD value of 30 nM. A comparison of the hybridoma cell clone supernatant 12-1 with cell clone supernatant 6-1-6 revealed striking differences in binding characteristics. The association of clone 6-1-6 was approximately 5 times higher than that observed with clone 12-1. Most markedly, however, is the difference in dissociation behavior. Whereas clone 6-1-6 hardly dissociates from the A N3pE-40 peptide, 12-1 is readily washed off within a few minutes. Hence, the poor binding of clone 12-1 is very likely to be the consequence of the observed off-rate. This assumption is further supported by the finding that clone 24-3-2, which gives particularly advantageous results in the DotBlot analysis, exhibits a very slow association to the A N3pE-40 peptide butin contrast to clone 12-1has no observable off-rate (see also FIG. 5).

(254) 2.6.1 Affinity of AN3pE Specific Antibody Clone 6-1-6 and 24-2-3

(255) For N3pE antibody clone 6-1-6 the association rate, dissociation rate and dissociation constant was calculated by a global fit of all sensograms shown in FIG. 6.

(256) The association rate was calculated with 1.67e5 M.sup.1s.sup.1, the dissociation rate with 2.63e4 s.sup.1 and the dissociation constant with 1.57 nM.

(257) For N3pE antibody clone 24-2-3 the association rate, dissociation rate and dissociation constant was calculated by a global fit of all sensograms shown in FIG. 7.

(258) The association rate was calculated with 3.25e3 M.sup.1s.sup.1, the dissociation rate with 3.29e4 s.sup.1 and the dissociation constant with 101 nM.

(259) 2.7 Sequencing Antibody Variable Regions

(260) The following sequences were identified:

(261) TABLE-US-00006 2.7.1Clone5-5-6 Variablepartlightchain,nucleotidesequence (SEQIDNO:49) ATGGTGTCCTCAGCTCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCAGGAAACCAACGGT GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCT ATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTGATGGAAAAACCTATTTGAATTGG TTATTACAGAGGCCAGGCCAGTCTCCAATGCGCCTAATCTATCTGGTGTCTAAACTGGAC TCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATC AGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCGTGCAAGGTACACATTTTCCA TTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTA TCCATCTTCCCACCAT Variablepartlightchain,proteinsequence (SEQIDNO:50) MVSSAQFLFLLVLWIQETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLYSDGKTYLNW LLQRPGQSPMRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFP FTFGSGTKLEIKRADAAPTVSIFPP Variablepartheavychain,nucleotidesequence (SEQIDNO.51) ATGGGATGGAGCGGGGTCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCTGAG GTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCC TGCAAGGCTTCTGGTTACTCATTCACTGGCTATACCATGAACTGGGTGAAGCAGAGCCAT GGAAAGAACCTTGAGTGGATTGGACTTATTAATCCTTACAGTGGTGTTACTAGGTACAAC CAGAAATTCAAGGGCAAGGCCACATTAATTGTAGACAAGTCATCCAGCACAGCCTACATG GAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTATTGTACAAGAGAGGCTAAA CGGGAGTGGGACGAGACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAA ACGACACCCCCATCTGTCTA Variablepartheavychain,proteinsequence (SEQIDNO:52) MGWSGVFLFLLSGTAGVHSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSH GKNLEWIGLINPYSGVTRYNQKFKGKATLIVDKSSSTAYMELLSLTSEDSAVYYCTREAK REWDETYWGQGTLVTVSAAKTTPPSV 2.7.2Clone6-1-6 Variablepartlightchainnucleotidesequence (SEQIDNO:53) ATGGTGTCCACAGCTCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCAGGAAACCAACGGT GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCT ATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTGACGGAAAAACCTATTTGAATTGG TTATTACAGAGGCCAGGCCAGTCTCCAATGCGCCTAATCTATCTGGTGTCTAAACTGGAC TCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATC AGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCGTGCAAGGTACACATTTTCCA TTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTA TCCATCTTCCCACCATCCAG Variablepartlightchain,proteinsequence (SEQIDNO:54) MVSTAQFLFLLVLWIQETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLYSDGKTYLNW LLQRPGQSPMRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFP FTFGSGTKLEIKRADAAPTVSIFPPS Variablepartheavychain,nucleotidesequence (SEQIDNO:55) ATGGGATGGAGCGGGGTCTTTATCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCTGAGGTCC AGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGC TTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTT GAGTGGATTGGACTTATTAATCCTTACAATGGTGTTACTAGGTACAACCAGAAGTTCAAGGGCA AGGCCACATTAATTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCCTCAGTCTGACATC TGAGGACTCTGCAGTCTATTACTGTACAAGAGAGGCTAAACGGGAGTGGGACGAGACTTACTGG GGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTG Variablepartheavychainproteinsequence (SEQIDNO:56) MGWSGVFIFLLSGTAGVHSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSH GKNLEWIGLINPYNGVTRYNQKFKGKATLIVDKSSSTAYMELLSLTSEDSAVYYCTREAK REWDETYWGQGTLVTVSAAKTTPPSVYPL 2.7.3Clone17-4-3 Variablepartlightchain,nucleotidesequence (SEQIDNO:57) ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGGTGTTCTGGATTCCTGTTTCCAGCAGTGATGTTG TGATGACCCAGACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAG ATCTAGTCAGAGCCTTGTACACAGTGATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCA GGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGT TCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCT GGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTG GAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGT Variablepartlightchainproteinsequence (SEQIDNO:58) MKLPVRLLVLVFWIPVSSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWY LQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPP TFGGGTKLEIKRADAAPTVSIFPPSS Variablepartheavychainnucleotidesequence (SEQIDNO:59) ATGGACTTTGGGCTCAGCTTACTTATTTTTGTCCTTATTTTAAAAGGTGTCCAGTGTGAG GTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCC TGTGCAGCCTCTGGATTCACTTTCAGTGACTACGGAATGGCGTGGGTTCGACAGGCTCCA GGGAAGGGGCCTGAGTGGGTAGCATTCATTAGTAATTTGGCATATAGTATCTACTATGCA GACACTGTGACGGGCCGATTCACCATCTCTAGAGAGAATGCCAAGAACACCCTGTACCTG GAAATGAGCAGTCTGAGGTCTGAGGACACAGCCATGTACTACTGTGCAAGGTATGACTAC GATAATATCTTGGACTATGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCC TCAGCCAAAACAACACCCCCATCAGTCTATCCACTG Variablepartheavychainproteinsequence (SEQIDNO:60) MDFGLSLLIFVLILKGVQCEVKLVESGGGLVQPGGSRKLSCAASGFTFSDYGMAWVRQAP GKGPEWVAFISNLAYSIYYADTVTGRFTISRENAKNTLYLEMSSLRSEDTAMYYCARYDY DNILDYVMDYWGQGTSVTVSSAKTTPPSVYPL 2.7.4Clone24-2-3 Variablepartlightchainnucleotidesequence (SEQIDNO:61) ATGAAGTTGCCTGTTAGGCTGTTGGTGCTCTGGATTCAGGAAACCAAGGGTGATGTTGTGCTGA CCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTATCTCTTGCAAGTCAAG TCAGAGCCTCTTATATAGTAATGGAAAAACCTATTTGAATTGGTTATTACAGAGGCCAGGCCAG TCTCCAAAGCGCCTAATCTATGTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTG GCAGTGGATCAGGAACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGT TTATTATTGCGTGCAAGGTACACATTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATA AAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGT Variablepartlightchainproteinsequence (SEQIDNO:62) MKLPVRLLVLWIQETKGDVVLTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQ RPGQSPKRLIYVVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPFTF GSGTKLEIKRADAAPTVSIFPPSS Variablepartheavychainnucleotidesequence (SEQIDNO:63) ATGGGATGGAGCGGGGTCTTTCTCTTCCTCCTGTCAGTAACTGAAGGTGTCCACTCCCAGGTTC AGCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGC TTCTGGCTATATATTCAATAACTACTGGATAAACTGGGTGAAGCAGAGGCCTGGTCAGGGTCTT GAGTGGATTGGACAGATTTATCCTGGAGATGGTGATACTAACTACAATGGGAAGTTCAAGGGTA AAGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTAACATC TGAGGACTCTGCGGTCTATTTCTGTGCAAGAGAGGGATATATTGTTTATTGGGGCCAAGGGACT CTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTG Variablepartheavychainproteinsequence (SEQIDNO:64) MGWSGVFLFLLSVTEGVHSQVQLQQSGAELVRPGSSVKISCKASGYIFNNYWINWVKQRP GQGLEWIGQIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAREGY IVYWGQGTLVTVSAAKTTPPSVYPL

(262) 2.8 Application of Antibody Clone 6-1-6 for N3pE ELISA

(263) The final N3pE ELISA protocol was tested concerning limit of quantification (LOQ) and signal-to-noise ratio (S/N). The standard curve of the ELISA is shown in FIG. 8.

(264) The shape of the standard curve looks very good especially for the low concentration range, which shows a nearly linear dependency of the absorbance. Based on this standard curve the LOQ is determined with 3.125 g/ml with a S/N=1.3.

(265) 2.9 Investigation of Cross Reactivity, Analyzed Via ELISA and SPR

(266) ELISA:

(267) The cross reactivity to other A variants was determined using our N3pE-ELISA. The raw date are shown in table 4.

(268) TABLE-US-00007 TABLE 4 Raw date of N3pE-ELISA with clone 6-1-6: Test of cross reactivity Con- centration pE3-40 pE3-40 pE11- (pg/ml) (28.04.) (21.04.) 2-40 3-40 4-40 1-42 3-42 40 800 1.8280 1.806 0.048 0.090 0.055 0.053 0.052 0.047 400 0.8750 0.912 0.045 0.065 0.044 0.048 0.052 0.044 200 0.4350 0.484 0.044 0.057 0.046 0.048 0.049 0.048 100 0.2290 0.248 0.045 0.052 0.045 0.047 0.050 0.048 50 0.1330 0.143 0.044 0.048 0.044 0.050 0.045 0.045 25 0.0820 0.086 0.044 0.046 0.044 0.048 0.047 0.048 12.5 0.0570 0.063 0.039 0.042 0.040 0.043 0.042 0.046 0 0.0410 0.040 0.038 0.065 0.061 0.059 0.063 0.066

(269) Only for ApE3-40 a dependency of the absorbance from the concentration was observed. All tested A variants have shown cross reactivity below 1%, except of 43-40. The signal (corrected by the blank) for 800 pg/ml was about 2.7% of the signal for ApE3-40. This is a very good value, considering that the N-terminus of both peptides have the same amino acids, except the first one, this is cyclized in the case of ApE3-40. Overall, the A N3pE antibody clone 6, which is generally used for ELISA, is very high specific for the N-terminus of A-peptides starting with pGlu at position 3.

(270) SPR:

(271) The cross reactivity of clones 6-1-6 and 24-2-3 to other non-A pGlu peptides was analyzed by surface plasmon resonance. Instead of ApE3-40, which shows a typical binding sensogram, all other tested pGlu peptides have shown nearly no interaction with clones 6-1-6 and 24-2-3, respectively, see also FIG. 9 (data only shown for clone 6-1-6). The sensograms of the pGlu peptides were compared with the sensogram for ApE3-40. The estimated cross reactivities were all below 1%. A summary of all analyzed peptides are shown in table 5.

(272) TABLE-US-00008 TABLE 5 Estimated Cross reactivity of clones 6- 1-6 and 24-2-3 to other pGlu-peptides pGlu Peptides % cross reactivity MCP-1 <1 MCP-2 <1 Big Gastrin <1 Gonadoliberin <1 Neurotensin <1 Orexin A <1 Fibronectin <1 Collagen 1 <1 TRH <1

(273) All experiments have confirmed the fact that N3pE antibody clone 6-1-6 and 24-2-3 are specific for the N-terminal epitope of ApE3-x. Neither other pGlu N-termini were recognized nor other A peptide variants, which do not bear an N-terminal pE residue.

(274) 2.10 Optimization and Validation of N3pE ELISA for Brain Analysis

(275) ApE3-42 concentration in mouse brainstem was analyzed dependent on the used method. The samples were derived from transgenic mice (tg) overexpressing human AQ3-42 in the brain, which is cyclized by QC to ApE3-42. Compared were samples from heterozygous transgenic mice (tg het) and homozygous transgenic mice (tg hom) and from wildtype, non-transgenic mice (wt). The mice used for sample generation were produced as described in WO2009034158.

(276) For all further experiments samples and standards were diluted in the EIA buffer. In a next step, the neutralization method for analyzing formic acid fraction samples was optimized, i.e. the neutralization was N3pE ELISA. The resulting and herein developed N3pE ELISA works well, it detected significant levels of human ApE3-42 in brains of the tg hom mice, significantly lower levels of human ApE3-42 in brains of the tg het mice and no human ApE3-42 in brains of the wt mice (see FIG. 10). The ELISA according to the present invention delivers high signals and consequently a very acceptable LOQ and is thus suitable for analysis of formic acid samples, in particular of formic acid brain samples.

(277) 2.11 Application of N3pE Antibody Clones for Immunohistochemistry

(278) With the N3pE antibodies of the present invention, ApE3-x was stained in brain sections of patients in the late stage of sporadic Alzheimer's disease (SAD) and familial forms of Alzheimer's disease (FAD), i.e. patients which bear a mutation in the presenilin 1 (PS1) gene. The stained brain sections are shown in FIG. 11. FIG. 11 shows that the N3pE antibodies of the present invention are suitable for immunohistochemistry. The antibodies specifically detect pGlu-A in brain of SAD and FAD patients. The N3pE antibodies show no background signals on the images, which proves the specific binding shown by ELISA and SPR analysis.

(279) 3. Deposits

(280) Monoclonal antibodies specifically recognizing A N3pE-x, were generated. Currently all corresponding 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 fr Mikroorganismen and Zellkulturen (DSMZ) in Braunschweig, DE, with a deposit date of Jun. 17, 2008, and with the respective deposit numbers (clone 5-5-6) DSM ACC2923 (clone 6-1-6) DSM ACC2924 (clone 17-4-3) DSM ACC2925 (clone 24-2-3) DSM ACC2926.

(281) Specificity of those antibodies for their respective target sequences could be confirmed. For A N3pE-x, high affinity antibody clones could be identified that should give strong signals in an ELISA set up with an expected detection limit in the low pg range.

(282) 4. Summary

(283) One objective of the present invention was the establishment of a highly sensitive and robust detection technique that allows quantitative determination of A variants in biological samples.

(284) Preferably, an ELISA based technique can be pursued. The task was started with A N3pE ELISA, because for this A variant an appropriate ELISA system was already commercially available (IBL). This system was used as reference and internal quality control.

(285) Applicability of the pGlu-6166 antibody in the chosen ELISA assay set up was investigated. To obtain clearly measurable signals, high antibody concentrations needed to be deployed (20 g/ml). High affinity A N3pE-x antibody clones could be identified. A detection limit in the low pg range (3-8 g/ml) can be achieved with these clones.