WO2011064225A1 - Novel diagnostic method for the diagnosis of alzheimer's disease or mild cgnitive impairment - Google Patents

Novel diagnostic method for the diagnosis of alzheimer's disease or mild cgnitive impairment Download PDF

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WO2011064225A1
WO2011064225A1 PCT/EP2010/068069 EP2010068069W WO2011064225A1 WO 2011064225 A1 WO2011064225 A1 WO 2011064225A1 EP 2010068069 W EP2010068069 W EP 2010068069W WO 2011064225 A1 WO2011064225 A1 WO 2011064225A1
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epitope
peptide
antibody
seq
target
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PCT/EP2010/068069
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French (fr)
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WO2011064225A8 (en
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Hans-Ulrich Demuth
Claudia Goettlich
Martin Kleinschmidt
Jens-Ulrich Rahfeld
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Probiodrug Ag
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Priority to CN2010800531373A priority Critical patent/CN102666577A/en
Priority to JP2012540407A priority patent/JP2013511734A/en
Priority to CA2779565A priority patent/CA2779565A1/en
Priority to EP10785394A priority patent/EP2504356A1/en
Publication of WO2011064225A1 publication Critical patent/WO2011064225A1/en
Publication of WO2011064225A8 publication Critical patent/WO2011064225A8/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the invention relates to the detection and diagnosis of Alzheimer's disease with the use of the oligomeric state of fragments of amyloid ⁇ as a biomarker and further concerns a novel method to determine the oligomeric state of fragments of amyloid ⁇ in biological samples.
  • Alzheimer's disease is the most common form of dementia and has a prevalence of approximately 65-70% among all dementia disorders (Blennow et al., 2006). Resulting from increased life expectancy, this disease has become a particular issue in highly developed industrialised countries like Japan and China as well as in the US a nd Eu rope . The n u mber of Alzhei mer patients is esti mated to increase from 24 million in 2001 to 81 million in 2040 (Ferri et al. , 2005) . Currently, the costs for treatment and care of AD patients worldwide amount to approximately 250 billion US dollars per year.
  • Alzheimer's disease The progression of the sporadic form of the disease is relatively slow and Alzheimer's disease will usually last for about 10-12 years after the onset of first symptoms.
  • AD Alzheimer's disease
  • a good diagnosis with a reliability of more than 90% is only possible in the later stages of the disease.
  • diagnosis here relies on the use of certain criteria according to Knopman et al., 2001; Waldemar et al., 2007 or Dubois et al., 2007.
  • Neurodegeneration starts however 20 to 30 years before the first clinical symptoms are noticed (Blennow et al., 2006; Jellinger KA, 2007).
  • MCI mimild cognitive impairment
  • Biomarkers for Alzheimer's disease have already been described in the prior art. Alongside well known psychological tests such as e.g . ADAS-cog, MMSE, DemTect, SKT or the Clock Drawing test, biomarkers are supposed to improve diagnostic sensitivity and specificity for first diagnosis as well as for monitoring the progression of the disease. In relation to the current status of development of biomarkers for AD/MCI it was proposed to correlate the disease in the future with the other diagnostic criteria (Whitwell et al., 2007; Panza et al. , 2007; Hyman SE, 2007). Biomarkers are supposed to support the classical neuropsychological tests in the future.
  • Magnetic resonance imaging is an imaging process which allows detection of degenerative atrophies in the brain (Barnes J et a/., 2007; Vemuri et a/., 2008).
  • MTA medial temporal lobe
  • Mild MTA is not encountered more frequently in other dementias (Barkhof et a/., 2007) but it does correlate with MCI (Mevel et a/. , 2007) .
  • a further imaging method is Positron Emission Tomography (PET) which visualises the accumulation of a detector molecule (PIB) on amyloid deposits. It could be detected that the thioflavin T-analogue (n C)PIB will accumulate increasingly in certain regions of the brain of patients with MCI or mild Alzheimer's disease, respectively (Kemppainen et a/., 2007; Klunk et a/., 2004; Rowe et a/., 2007); unfortunately this can also be detected in subjects who do not have dementia (Pike et a/., 2007).
  • PTT Positron Emission Tomography
  • Senile plaques are one of the pathological characteristics of Alzheimer's disease. These plaques consist mostly of ⁇ (1-42) peptides (Attems J, 2005). In some studies it could be shown that a low level of ⁇ (1-42) in CSF of MCI patients correlates specifically with the further development of Alzheimer's disease in its progression (Blennow and Hampel, 2003; Hansson et a/., 2006 and 2007). The reduction in CSF is probably due to enhanced aggregation of ⁇ (1-42) in the brain (Fagan et a/., 2006; Prince et a/., 2004; Strozyk et a/. , 2003) .
  • CSF samples are usually analyzed via a comparative proteomic analysis which results in a diagnosis of AD with en h a n ced se nsitivity a n d a l so to en a b le the d iffere nti ati o n fro m other degenerative dementia disorders (Finehout et al., 2007; Castano et al., 2006; Zhang et al., 2005; Simonsen et al., 2007; Lescuyer et al., 2004; Abdi et al., 2006).
  • biomarker After a proteomic analysis, the potential new biomarker should be analyzed in detail for its suitability and correlation with pathological causes.
  • a typical example for a biomarker which was found by a proteomic analysis is truncated cystatin C as a biomarker for multiple sclerosis; this biomarker was later proven to be a storage artefact (Irani et al., 2006; Hansson et al., 2007(2)).
  • plasma ⁇ (1-42) level is not a reliable biomarker for MCI or AD (Blasko et al., 2008; Mehta et al., 2000; Brettschneider et al., 2005 )
  • a decrease of the ratio pl asma ⁇ (1-38) / ⁇ (1-40) is considered a biomarker for vascular dementia and comes close to the predictability of CSF markers (Bibl et a/., 2007).
  • ⁇ oligomers were disregarded as biomarkers for Alzheimer, however, they are supposed to play a decisive role in initiating the neurodegenerative process (Walsh & Selkoe, 2007).
  • the neurotoxic effect was shown for ⁇ dimers with 8 kDa to the point of protofibrils with over 100 kDa (Lambert et al., 1998; Walsh et al, 2002; Keayed et al., 2004; Cleary et al., 2005).
  • ⁇ oligomers were found in human liquor (Pitschke et al., 1998; Santos et al., 2007; Klyubin et al., 2008).
  • oligomers have also an influence on the determination of the ⁇ concentration in human samples.
  • the oligomerization leads to masking of the C-terminal epitopes of ⁇ peptides (Roher et a/., 2000) yielding to underestimated ⁇ levels detected by C-terminal specific ELISA (Stenh et al., 2005).
  • the existence of ⁇ oligomers in the sample results in lowering of the ELISA signal. This could be a problem for exact determination of the ⁇ concentration, however this fact offers also the chance to measure the amount of oligomers and the level of oligomerization in biological samples.
  • ELISA or ELISA-type systems are used for quantification of ⁇ , and recently also ⁇ oligomers, in plasma.
  • the specification of such detections systems is usually only unsatisfactorily analyzed or are completely disregarded .
  • a critical ite m l i ke the recove ry rate is n ot a n a lyzed o r is n ot me nti o n ed i n the publications.
  • the recovery rate is however decisive for giving a complete picture of those ⁇ peptides or oligomers which occur in plasma. Differences between the studies can also result from the differences in these rates.
  • a further important characteristic of an ELISA or multiplex system is its linearity.
  • the concentrations determined for the analytes in plasma should only depend on the dilution used in the measurement to a very low degree or not at all.
  • this is neither possible for ELISA nor for the multiplex systems for quantification of ⁇ in plasma.
  • the difference between the calculated plasma ⁇ (1-42) concentration for a dilution of 1-20 was three times as high as for the 1-2 dilution of the same sample (Hansson et al. , 2008) .
  • This example alone shows that the use of different dilutions of plasma samples in the several studies makes it impossible to compare the same.
  • the present invention uses also the indirect measurement of ⁇ oligomers, however, in contrast to the prior art, both values (under denaturing and non-denaturing conditions) were determined with ⁇ specific ELISA to ensure the comparability. Because of an initial immunoprecipitation step, which isolates ⁇ peptides in monomeric as well as oligomeric form followed by our novel disaggregation method, the subsequent ELISA is not constricted by recovery and/or linearity issues.
  • the present invention aims at providing diagnostic markers which can be determined with rel iable methods and can be used for rel iable and clear prediction of Alzheimer's disease.
  • a method of diagnosing or monitoring a neurodegenerative disorder such as Alzheimer's disease and Mild Cognitive Impairment, which comprises determining the oligomeric state of a target amyloid ⁇ peptide (Abeta or ⁇ ) in a biolog ical sample from a test subject, characterized in that said method comprises the following steps:
  • step (b) disaggregating the target ⁇ peptide from step (a);
  • a ratio of c d / c a which is lower than 1 .5 is ind icative of a positive diagnosis for a neurodegenerative disorder.
  • a method of determining the oligomeric state of a target amyloid ⁇ peptide (Abeta or ⁇ ) in a biological sample which comprises the following steps:
  • step (b) disaggregating the target ⁇ peptide from step (a);
  • Olemeric refers to a limited number of aggregated ⁇ peptide monomer u n its . Exa mples of su ch ol ig omers incl ude d i mers, tri mers and tetramers .
  • the term "d isagg reg ation” refers to the process of converti ng oligomeric forms of ⁇ peptide to monomeric forms of ⁇ peptide.
  • Capture antibody in the sense of the present application is intended to encompass those antibodies which bind to a target ⁇ peptide.
  • the capture antibodies bind to the target ⁇ peptide with a high affinity.
  • high affinity means an affinity with a K D value of 10 "7 M or better, such as a K D value of 10 "8 M or better or even more particularly, a K D value of 10 "9 M to 10 "12 M .
  • 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 as long as they exhibit the desired biological activity.
  • the antibody may be an IgM, IgG (e.g. IgGl, IgG2, IgG 3 or IgG4) , Ig D, IgA or Ig E, for example. Suitably however, the antibody is not an IgM antibody.
  • the "desired biological activity” is binding to a target ⁇ peptide.
  • 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.
  • 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 adva ntageous i n that they are synthesized by the hybridoma cu ltu re, 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 .
  • the monoclonal antibodies to be used in accord ance with the present i nvention may be made by the hybridoma method first described by Kohler et a/. , Nature, 256 : 495 (1975), or may be m ade by genera l ly wel l kn own recom bi na nt D NA meth ods .
  • 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 a/., J. Mol. Biol., 222 : 581-597 (1991), for example.
  • 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.
  • 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.
  • 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.
  • 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.
  • CDR complementarity-determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported
  • 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.
  • Fc immunoglobulin constant region
  • the humanized antibody includes a PrimatizedTM antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest or a "camelized" antibody.
  • Single-chain Fv or “sFv” antibody fragments comprise the V H and V L domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V
  • diabodies refers to small antibody fragments with two antigen- binding sites, which fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V D ) in the same polypeptide chain (V H - V D ).
  • V H heavy-chain variable domain
  • V D light-chain variable domain
  • 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.
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most particularly 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 non-reducing conditions using Coomassie blue or, suitably, 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.
  • the expressions "cell”, “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
  • 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.
  • polypeptide As used herein, are interchangeable and are defined to mean a biomolecule composed of amino acids linked by a peptide bond.
  • Amyloid ⁇ , ⁇ or ⁇ -amyloid is an in the art recognized term and refers to amyloid ⁇ proteins and peptides, amyloid ⁇ precursor protein (APP), as well as modifications, fragments and any functional equivalents thereof.
  • amyloid ⁇ as used herein is meant any fragment produced by proteolytic cleavage of APP but especia l ly those frag ments wh ich a re i nvolved i n or associated with the amyloid pathologies including, but not limited to, ⁇ (1-38) of SEQ ID NO. 3, ⁇ (1-40) of SEQ ID NO. 2, and ⁇ (1-42) of SEQ ID NO. 1.
  • fragments of amyloid ⁇ are all amyloid ⁇ peptides, which comprise a core amyloid ⁇ fragment ⁇ (3-38) of SEQ ID NO. 13, More suitably for the purpose of the present invention are all amyloid ⁇ peptides, which comprise the core amyloid ⁇ fragment ⁇ (11-38) of SEQ ID NO. 19. Such ⁇ fragments, which comprise the amino acid sequence of ⁇ (11-38) of SEQ ID NO.
  • ⁇ (x-y) fragments which have been shown to accumulate in a subject as a consequence of a neurodegenerative disorder, such as Alzheimer's disease and Mild Cognitive Impairment, wherein x is defined as an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11;
  • x is an integer selected from 1, 2,
  • x is 1.
  • x is 11.
  • y is defined as an integer selected from 38, 39, 40, 41, 42 and 43.
  • y is 38, 40 or 42, such as 40 or 42.
  • y is 40.
  • y is 38.
  • Functional equivalents encompass all those mutants or variants of ⁇ (x-y) which might naturally occur in the patient group which has been selected to undergo the method for detection or method for diagnosis as described according to the present invention. More particularly, “functional equivalent” in the present context means that the functional equivalent of ⁇ (x-y) are mutants or variants thereof and have been shown to accu m u late in Alzheimer's d isease . The functional equivalents have no more than 30, such as 20, e.g . 10, particularly 5 and most particularly 2, or only 1 mutation(s) compared to the respective ⁇ (x- y) peptide. Functional equivalents also encompass mutated variants, which comprise by way of example all ⁇ peptides starting with amino acids Asp-Ala- Glu and ending with Gly-Val-Val and Val-Ile Ala, respectively.
  • Particularly useful equivalents in the present context are those of ⁇ (1-40) (SEQ ID NO. 2) and ⁇ (1-42) (SEQ ID NO. 1), which are those described by Irie et al., 2005, namely the Tottori, Flemish, Dutch, Italian, Arctic and Iowa mutations of ⁇ .
  • Functional equivalents also encompass ⁇ peptides derived from amyloid precursor protein bearing mutations next to the ⁇ - or ⁇ -secretase cleavage site such as the Swedish, Austrian, French, German, Florida, London, Indiana and Australian variations (Irie et al., 2005).
  • Modified Amyloid ⁇ , ⁇ or ⁇ -amyloid encompasses all modifications at various amino acid positions in the amyloid ⁇ proteins and peptides, amyloid ⁇ precursor protein (APP), fragments and functional equivalents thereof.
  • Useful in the present context are modifications at the N- and/or C-terminal amino acids of said amyloid ⁇ proteins and peptides, amyloid ⁇ precursor protein (APP), fragments and functional equivalents.
  • Particularly useful are modifications at glutamine and glutamate residues, such as the cyclization of N-terminal glutamine or glutamate residues to pyroglutamate.
  • Suitable examples according to the present invention are the amyloid ⁇ peptides of SEQ ID Nos.
  • Suitable examples according to the present invention are the amyloid ⁇ peptides of SEQ ID Nos. 1 to 6, wherein the aspartate residues at amino acid positions 1 and/or 7 are converted to isoasparate.
  • Futher suitable examples are the amyloid ⁇ peptides of SEQ ID Nos. 7 to 12, wherein the aspartate residue at amino acid position 6 is converted to isoasparate.
  • suitable examples are the amyloid ⁇ peptides of SEQ ID Nos. 13 to 18, wherein the aspartate residue at amino acid position 5 is converted to isoasparate.
  • sandwich ELISAs usually involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected .
  • the test sample analyte is bound by a first antibody wh ich is i mmobil ized on a sol id 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).
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
  • Figure 2 Determination of the oligomeric state of ⁇ peptides derived from human plasma
  • the ratio of concentration determined after disaggregation to the concentration determined without disaggregation reflects the oligomeric state of ⁇ (1-40/42).
  • a method of diag nosing or monitoring a neurodegenerative disorder such as Alzheimer's disease and Mild Cognitive Impairment, which comprises determining the oligomeric state of a target amyloid ⁇ peptide (Abeta or ⁇ ) in a biological sample from a test subject, characterized in that said method comprises the following steps:
  • step (b) disaggregating the target ⁇ peptide from step (a);
  • a ratio of c d / c a which is lower than 1.5 is indicative of a positive diagnosis for a neurodegenerative disorder.
  • a rati o (c d / c a ) of 1.0 indicates that there are no oligomers in the sample.
  • Higher ratios of c d / c a i.e. ratios of > 1.0 reflect a greater amount of oligomers or more compactness of oligomers (less accessibility of epitopes) in the sample.
  • Ratios of c d / c a which are lower than 1.5 (i.e. a ratio between 1.0 and 1.5), such as lower than 1.4, lower than 1.3, lower than 1.2, lower than 1.1 or lower than 1.05 have been found to be indicative of a positive diagnosis for a neurodegenerative disorder, such as Alzheimer's disease.
  • a meth od of diagnosing or monitoring a neurodegenerative disorder such as Alzheimer's disease and Mild Cognitive Impairment, which comprises determining the oligomeric state of a target amyloid ⁇ peptide (Abeta or ⁇ ) in a biological sample from a test subject, characterized in that said method comprises the following steps:
  • step (b) disaggregating the target ⁇ peptide from step (a);
  • a sum of Cd and c a which is lower than 2.9, lower than 2.8, lower than 2.7, lower than 2.6, lower than 2.5, lower than 2.4 or lower than 2.3 have been found to be indicative of a positive d iagnosis for a neurodegenerative disorder, such as Alzheimer's disease.
  • the concentration of target ⁇ peptides in steps (a) and (c) was determined by sandwich ELISA systems consisting of a capture and a detection antibody. Com pared with the size of a n a nti body ( 150 kDa ) , ⁇ peptides (4.5 kDa) as monomers are very small . Because of the aggregation propensity of the peptides they tend to form oligomers, protofibrils to the point of fibrils. Within such an aggregate the ⁇ monomers are tightly packed with the consequence that not all monomers can be bound by the detection antibody due to sterical hindrance or epitope inaccessibility. The detected ⁇ concentration for oligomers is lower than for monomers.
  • the disaggregation step (b) comprises the use of an alkali.
  • the alkali used for disaggregation in step (b) is sodium hydroxide, such as 500mM sodium hydroxide.
  • the advantage of using an alkali and particularly a strong alkali such as sodium hydroxide is that more efficient disaggregation is achieved . For example, a higher proportion of monomers are obtained, with no observable quantities of dimers, trimers or tetramers.
  • the disaggregation step (b) additionally comprises the use of a suitable solvent, such as methanol, e.g. 50% (v/v) methanol.
  • the disaggregation step (b) comprises an incubation step.
  • the incubation step comprises incubation at room temperature for at least 2 minutes.
  • the incubation step comprises incubation at room temperature for at least 10 minutes.
  • ⁇ peptides are liberated from the amyloid precursor protein (APP) after a sequential cleavage by the enzymes ⁇ -and ⁇ -secretase.
  • the ⁇ -secretase cleavage results in the generation of primarily ⁇ (1-40) and ⁇ (1-42) peptides but also ending prominently at position 38 or 43, which differ in their C-termini and exhibit different potencies of aggregation, fibril formation and neurotoxicity.
  • ⁇ -secretase release can generate different N-termini and also subsequent modifications by peptidases and other enzymes resulting in prominent species such as ⁇ peptides starting at the positions e.g .
  • the present invention thus provides a method for the determination of the oligomeric states of the ⁇ (x-y) peptides, wherein x and y are as hereinbefore defined .
  • the ol igomeric state of a target ⁇ peptide to be determined is selected from the group consisting of:
  • the oiigomeric state of a target ⁇ peptide to be detected is ⁇ (1-40) (SEQ ID No : 2).
  • the oiigomeric state of a target ⁇ peptide to be detected is ⁇ (1-42) (SEQ ID No : 1).
  • the oiigomeric state of a target ⁇ peptide to be detected is ⁇ (1-40) (SEQ ID No : 2) and ⁇ (1-42) (SEQ ID No : 1).
  • the data presented herein demonstrates the suitability of summation of the ⁇ (1-40) and ⁇ (1-42) peptides wherein it has been shown that summation of both oiigomeric states improves the significance of the diagnosis.
  • the oiigomeric state of a target ⁇ peptide to be detected is at least one ⁇ peptide selected from the SEQ ID NOs: 13 to 24, which start with a glutamate residue at the N-terminus.
  • the oiigomeric state of a target ⁇ peptide to be detected is ⁇ (3-38) (SEQ ID No : 13).
  • the oiigomeric state of a target ⁇ peptide to be detected is ⁇ (11-38) (SEQ ID No : 19).
  • the oiigomeric state of a target ⁇ peptide to be detected is at least one ⁇ peptide selected from SEQ ID NOs: 13 to 24, wherein the glutamate residue at the N-terminus of these peptides is cyclized to pyroglutamate.
  • the oiigomeric state of a target ⁇ peptide to be detected is at least one ⁇ peptide selected from SEQ ID Nos. 1 to 6, wherein the aspartate residues at amino acid positions 1 and/or 7 are converted to isoasparate.
  • the oligomeric state of a target ⁇ peptide to be detected is at least one ⁇ peptide selected from SEQ ID Nos. 7 to 12, wherein the aspartate residue at amino acid position 6 is converted to isoasparate.
  • the oligomeric state of a target ⁇ peptide to be detected is at least one ⁇ peptide selected from SEQ ID Nos. 13 to 18, wherein the aspartate residue at amino acid position 5 is converted to isoasparate.
  • the method of determining the oligomeric state of ⁇ constitutes a further aspect of the invention related to a novel and inventive assay which is not necessarily limited to the diagnosis of a neurodegenerative disorder such as Alzheimer's disease.
  • a method of determining the oligomeric state of a target amyloid ⁇ peptide (Abeta or ⁇ ) in a biological sample which comprises the following steps:
  • step (b) disaggregating the target ⁇ peptide from step (a);
  • the disaggregation step (b) comprises the use of an alkali as hereinbefore defined .
  • steps (a) and (c) comprise:
  • the quantifying step (iv) comprises analysis in an ⁇ specific ELISA.
  • the ⁇ specific ELISA is a sandwich-ELISA.
  • steps (a) and (c) both comprise analysis with an ⁇ specific ELISA.
  • Th is embod iment provides the advantage of allowing comparability between the first and second concentrations obtained in steps (a) and (c).
  • the biological sample is selected from the group consisting of blood, serum, urine, cerebrospinal fluid (CSF), plasma, lymph, saliva, sweat, pleural fluid, synovial fluid, tear fluid, bile and pancreas secretion.
  • the biological sample is plasma.
  • the biological sample can be obtained from a patient in a manner well-known to a person skilled in the art.
  • a blood sample can be obtained from a subject and the blood sample can be separated into serum and plasma by conventional methods.
  • the subject, from which the biological sample is obtained is suspected of being afflicted with Alzheimer's disease, at risk of developing Alzheimer's disease and/or being at risk of or having any other kind of dementia.
  • MCI Mild Cognitive Impairment
  • the present method has several advantages over the methods known in the art, i.e. the method of the present invention can be used to detect Alzheimer's disease at an early stage and to differentiate between Alzheimer's disease and other types of dementia in early stages of disease development and progression.
  • One possible early stage is Mild Cognitive Impairment (MCI). It is impossible with the methods currently known in the art to make a clear and reliable diagnosis of early stages of Alzheimer's disease and , i n particu l a r, it is i m possi ble to d ifferentiate between the onset of Alzheimer's d isease and other forms of dementia in said early stages. This especially applies for patients afflicted with MCI.
  • MCI Mild Cognitive Impairment
  • the methods provided by the present invention are suitable for a differential diagnosis of Alzheimer's disease.
  • the present invention provides a method, wherein the oligomeric state of target ⁇ peptides can be detected in biological samples obtained from any of the above described subjects in a highly reproducible manner.
  • the high reproducibility of the methods of the present invention is achieved by using at least two different capture antibodies in an initial immune-precipitation step (step (a)) which is identical to the process subsequently used in step (c).
  • these at least two different capture antibodies are directed to different epitopes of the ⁇ target peptide.
  • the biological sample is plasma.
  • ⁇ target peptide encompasses ⁇ (x-y) as hereinbefore defined .
  • the inventors of present invention have shown that it is possible to determine the oligomeric state of target ⁇ peptides, in a reliable manner, and, it also became clear for the first time that in fact the oligomeric state of ⁇ (x-y) is particularly suitable for the diagnosis of early onset Alzheimer's disease.
  • the method of determining the first and second concentration of a target ⁇ peptide in steps (a) and (c) specifically comprises the following steps: i) Contacting a biological sample with at least two different capture antibodies in an immunoprecipitation step. After contacting the biological sample with the aforementioned at least two different capture antibodies, an immune complex will form between the at least two different capture antibodies and the target ⁇ peptides. This step does not act for specific isolation of full length ⁇ (x-y) wherein x would be 1, rather than capturing and separating all ⁇ species, especially ending at position 38, 40 and/or 42. ii) This complex is then detected by secondary antibodies. Suitably, the secondary antibodies are immobilized on magnetic beads.
  • the immune complex can be easily separated from the body fluid (plasma/serum CSF etc.) using the magnetic separator.
  • the immune complex is eluted from the beads.
  • the elution step is performed by incubating the beads carrying the immune complex in a solution comprising 50 % Methanol / 0.5 % formic acid for lh at room temperature. Thereby, all intermolecular interactions are destructed and all ⁇ peptide molecules, which were isolated from the biological sample, are released from the beads in the solution.
  • the released, isolated ⁇ peptide will be quantified in a subsequent step, for example by a sandwich ELISA that specifically detects full length ⁇ (x-y), wherein full length ⁇ (x-y) in this step most suitably means ⁇ (1-40) and ⁇ (1-42).
  • Particular antibodies for the immunoprecipitation are: 3D6 (Elan), BAN50 (Takeda), 82E1 (IBL), 6E10 (Covance), WO-2 (The Genetics Company), 266(Elan), BAM90.1 (Sigma), 4G8 (Covance), G2-10 (The Genetics Company), 1A10 (IBL), BA27 (Takeda), 11A5-B10 (Millipore), 12F4 (Millipore), 21F12 (Elan).
  • ⁇ 3 ⁇ specific antibodies are:
  • Examples for ⁇ isoAsp 1 specific antibiodies are :
  • ⁇ 3 ⁇ specific antibodies are:
  • amyloid beta specific antibodies which are suitable for immunoprecipitation can be used in the concentration determining method (further suitable antibodies can e.g. be taken from www.alzforum.org).
  • Decisive for good capture efficiency is the use of two, three or more different antibodies with different epitopes.
  • the use of more than one antibody type for immunoprecipitation of ⁇ peptides offers cooperative and surprisingly synergistic binding effects (avidity), which finally allows to achieve a tremendously higher capture efficiency (see Figure 1).
  • the secondary antibodies in step ii) are specific against the host antibody type of the capture antibodies. Suitable secondary antibodies are anti-mouse antibodies and anti-rabbit antibodies.
  • the beads may be washed with washing buffer (see examples of the present invention). Washing buffers, which contain detergents or other additives preventing unspecific binding, can be used for this step. Non-limiting examples of washing buffers are:
  • dilution buffer Any dilution buffers, which can prevent unspecific interaction with surfaces and the immobilized first ELISA antibody can be used for this step.
  • dilution buffers are:
  • ELISA-Kits that a re a ble to q ua ntify fu l l len gth ⁇ ( 1-40) are commercially available. Suitable ELISA-Kits for the quantification of ⁇ ( 1-40) in the methods of the present invention are for example : Amyloid- ⁇ ( 1-40) ( N) ELISA (IBL, J P27714) ; ⁇ [ 1-40] H uman ELISA Kit (Invitrogen ) ; H uman Amyloid beta (Amyloid- ⁇ ), aa 1-40 ELISA Kit (Wako Chemicals USA, Inc.); Amyloid Beta 1-40 ELISA Kit (The Genetics Company).
  • ELISA-Kits that are able to quantify full length ⁇ ( 1-42) are also commercially available. Suitable ELISA-Kits for the quantification of ⁇ ( 1-42) in the methods of the present invention are for example : Amyloid- ⁇ ( 1-42) (N) ELISA (IBL, JP27712); ⁇ [1-42] Human ELISA Kit (Invitrogen), Human Amyloid beta (Amyloid- ⁇ ), aa 1-42 ELISA Kit (Wako Chemicals USA, Inc.), Amyloid Beta 1-40 ELISA Kit (The Genetics Company), INNOTEST® ⁇ - AMYLOID (1-42) (Innogenetics).
  • the concentration determining method is not limited to the exemplary aforementioned commercially available ELISA-Kits for ⁇ (1-40) or ⁇ (1-42). Numerous further sandwich ELISAs for full length ⁇ (1-40) or ⁇ (1-42) may be available in the prior art or may be developed by the skilled artisan. All these full length ⁇ 1-40 or ⁇ 1-42 sandwich ELISAs shall also be encompassed by the concentration determining method and should typically comprise a suitable pair of capture and detection antibodies, which are specific for the complete N- terminus of ⁇ (1-40) and/or ⁇ (l-42)and the C-terminus ending at amino acid 40 or 42, respectively.
  • Such a full length ⁇ (1-40) sandwich ELISA may comprise a first immobilized antibody recognizing specifically the C-terminus of ⁇ (1-40) and a second labeled detection antibody recognizing specifically the complete N-terminus of ⁇ (1-40).
  • a full length ⁇ (1-42) sandwich ELISA may comprise a first immobilized antibody recognizing specifically the C-terminus of ⁇ (1-42) and a second labeled detection antibody recognizing specifically the complete N-terminus of ⁇ (1-42).
  • a full length ⁇ (1-40) sandwich ELISA may also comprise a first immobilized antibody recognizing specifically the complete N-terminus of ⁇ (1-40) and a second labeled detection antibody recognizing specifically the C-terminus of ⁇ (1-40).
  • a full length ⁇ (1-42) sandwich ELISA may also comprise a first immobilized antibody recognizing specifically the complete N-terminus of ⁇ (1-42) and a second labeled detection antibody recognizing specifically the C-terminus of ⁇ (1-42).
  • Suitable ⁇ (1-40/42) N-terminal specific antibodies for use in the concentration determining method are for example 3D6 (Elan), WO-2 (The Genetics Company), 82E1 (IBL), BAN-50 (Ta ked a ) .
  • N u merous fu rther ⁇ ( 1-40/42 ) N-terminal specific antibodies may be available in the prior art or may be developed by the skilled artisan . All these ⁇ ( 1-40/42) N-terminal specific antibodies are also envisaged for the concentration determining method.
  • Suitable ⁇ ( 1-40) C-terminal specific antibodies are for example G2-10 (The Genetics Company); 11A5-B10 (Millipore); 1A10 (IBL); BA27 (Takeda); EP1876Y (Novus Biologicals). Numerous further ⁇ ( 1-40) C-terminal specific antibodies may be available in the prior art or may be developed by the skilled artisan . All these ⁇ ( 1-40 ) C-terminal specific antibodies are also envisaged for the concentration determining method .
  • Suitable ⁇ ( 1-42) C-terminal specific antibodies are for example G2-11 (The Genetics Company); 12F4 (Millipore); Anti- Human ⁇ (38-42) Rabbit IgG (IBL); 21 F12 (Elan); BC05 (Takeda); 16C11 (Santa Cruz Biotechnology) . Numerous further ⁇ (1-42) C-terminal specific antibodies may be available in the prior art or may be developed by the skil led artisan . Al l these ⁇ ( 1-42 ) C-terminal specific antibodies are envisaged for the concentration determining method . According to one embodiment, the detection antibodies are labeled .
  • the detection antibody will typically be labeled with a detectable moiety.
  • a detectable moiety Numerous labels are available which can be generally grouped into the following categories:
  • Radioisotopes such as 35 S, 14 C, 125 I, 3 H, and 131 I.
  • the antibody can be labeled with the rad ioisotope using the techniq ues described in Current Protocols in Immunology, Vol umes 1 and 2, Glois et al . , Ed . , Wiley-Interscience. New York, New York. Pubs., (1991) for example and radioactivity can be measured using scintillation counting .
  • 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.
  • 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 colour 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 electronical ly 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 incl ude l uciferases (e.
  • Ogalactosidase Ogalactosidase, 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.
  • saccharide oxidases e.g ., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase
  • heterocyclic oxidases such as uricase and xanthine oxidase
  • lactoperoxidase lactoperoxidase
  • microperoxidase and the like.
  • enzyme-substrate combinations include, for example: (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));
  • HRPO Horseradish peroxidase
  • OPD orthophenylene diamine
  • TMB 3,3',5,5'-tetramethyl benzidine hydrochloride
  • ⁇ -D-galactosidase ( ⁇ -D-Gal) with a chromogenic substrate (e.g. p- nitrophenyl-6-D-galactosidase) or the fluorogenic substrate 4- methylumbelliferyl-6-D-galactosidase.
  • a chromogenic substrate e.g. p- nitrophenyl-6-D-galactosidase
  • fluorogenic substrate 4- methylumbelliferyl-6-D-galactosidase.
  • Another possible label for a detection antibody is a short nucleotide sequence.
  • the concentration is then determined by a RT-PCR system (ImperacerTM, Chimera Biotech).
  • the label is indirectly conjugated with the antibody.
  • 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.
  • 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).
  • a small hapten e.g. digoxin
  • an anti-hapten antibody e.g. anti-digoxin antibody
  • the antibodies used in 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).
  • 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 ⁇ peptide in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies.
  • the antibodies generally are insolubilized before or after the competition, so that the standard and analyte that are bou nd to the anti bod ies may conven iently be separated from the standard and analyte which remain unbound .
  • body fluids can be used : blood, cerebrospinal fluid (CSF), urine, lymph, saliva, sweat, pleural fluid, synovial fluid, aqueous fluid, tear fluid, bile and pancreas secretion.
  • the novel method was established by the present inventors using blood samples (see the examples of the present invention).
  • the present method is however not to be construed to be limited to blood samples.
  • the method can also be employed using CSF, brain extract and urine samples, as well as all other human body fluids, e.g . the above mentioned in the same manner.
  • Particular samples include plasma samples.
  • the tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example.
  • a preservative such as formalin
  • Suitable alternative methods for determining the concentration of ⁇ are: 1. Amyloid ⁇ 1-40 HTRF ® Assay (CisBio Bioassavs) :
  • This assay principle is based on TR-FRET, which is a combination of Time- Resolved Fluorescence and Forster Resonance Energy Transfer. Similar to the usual sandwich ELISA the ⁇ (1-40) is bound by two antibodies; the antibodies are here, however, not bound on a surface, the interaction occurs in solution. Both antibodies are labeled with a fluorophor. When these two fluorophors are brought together by a biomolecular interaction a portion of energy captured by the donor fluorophor during excitation is transferred via FRET to an acceptor fluorophor, which will be excited as a result. The fluorescence of the acceptor fluorophor is measured . The measuring signal is correlated with the amount of FRET and thus, the amount of ⁇ (1-40) in solution.
  • the AlphascreenTM Assay from Lilly can be used .
  • a suitable example for use in the methods of the present invention is the INNO-BIA plasma ⁇ forms assay (Innogenetics).
  • This assay is a well standardized multiparameter bead-based immunoassay for the simultaneous quantification of human ⁇ -amyloid forms ⁇ (1-42) and ⁇ (1- 40) or ⁇ ( ⁇ -42) and ⁇ ( ⁇ -40) in plasma using xMAP® technology (xMAP is a registered trademark of Luminex Corp.).
  • This assay system is able to quantify up to 100 different analytes in parallel.
  • the basis of this method are small spherical polystyrol particles, called microspheres or beads.
  • microspheres or beads In analogy to ELISA and Western Blot these beads serve as a solid phase for the biochemical detection.
  • These beads are colour-coded, so that 100 different bead classes can be distinguished . Every bead class has one specific antibody (e.g . against ⁇ (1-40)) immobilized on the microsphere surface. If the ⁇ ( 1-40) concentration increases more peptide molecules will be bound by the beads of this class.
  • the detection of the binding of the analyte is carried out by a second anti- ⁇ ( 1-40) antibody, which is labeled with another fluorescence dye, emitting green light.
  • the sample is handled comparable to FACS analysis.
  • the microspheres are singularized by hydrodynamic focusing and analyzed by laser- based detection system, which can make a quantification on the basis of the green fluorescence and identify the bound analyte by the specific coloration of the bead . Thus, it is possible to determine the concentration of multiple analytes in one sample.
  • 2D-Gel electrophoresis cou pled with Western Blot analysis may be a suitable method to quantify ⁇ peptides (Sergeant et al., 2003; Casas et al., 2004).
  • the antibodies used in the method 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.
  • kits for diagnosing a neurodegenerative disorder such as Alzheimer's disease which comprises a suitable alkali and instructions to use said kit in accordance with the methods defined herein.
  • the kit additionally comprises at least two different capture antibodies as defined herein.
  • the kit will include substrates and cofactors required by the enzyme (e.g . a substrate precursor which provides the detectable chromophore or fluorophore).
  • substrates and cofactors required by the enzyme e.g . a substrate precursor which provides the detectable chromophore or fluorophore.
  • other additives may be included such as stabilizers, buffers (e.g . a block buffer or lysis buffer) and the l ike .
  • 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.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.
  • the diagnostic kit of the invention is especially useful for the detection and diagnosis of neurodegenerative disorders, such as amyloid-associated diseases and conditions, e.g. Alzheimer's disease.
  • the method of the present invention makes it possible for the first time to detect and quantify oligomeric target ⁇ peptides, in particular ⁇ (1-40), ⁇ (1-42), ⁇ (3-38) and/or ⁇ (11-38), or a functional equivalent thereof, in a reliable manner.
  • the present invention provides oligomeric ⁇ (1-40), ⁇ (1-42), ⁇ (3-38) and/or ⁇ (11-38) as a plasma biomarker, which is suitable for a differential diagnosis of Alzheimer's disease, in particular in the early stages of the disease.
  • the invention is directed to the use of the method of determining the oligomeric state of amyloid ⁇ peptide for the diagnosis of Alzheimer's disease, such as the differential diagnosis of Alzheimer's disease, in pa rticu l ar i n the early stages of the d isease .
  • the ea rly stage of Alzheimer's disease is Mild Cognitive impairment.
  • the invention is directed to the use of the oligomeric ⁇ target peptides for the diagnosis of Alzheimer's diseases, such as the differential diagnosis of Alzheimer's disease, in particular in the early stages of the disease.
  • the early stage of Alzheimer's disease is Mild Cognitive impairment.
  • the oligomeric ⁇ target peptide which shall be used for diagnosis of Alzheimer's disease, is detected and quantified with a method according to the present invention.
  • the ⁇ target peptide is ⁇ (x-y), as hereinbefore defined, or a functional equivalent thereof.
  • the method of the invention also has industrial applicability to monitoring the efficacy of a g iven treatment of a neu rod egenerative d isorder, su ch as Alzheimer's disease.
  • a method of monitoring efficacy of a therapy in a subject having , suspected of having, or of being predisposed to, a neurodegenerative disorder, such as Alzheimer's disease comprising determining the oligomeric state of a target amyloid ⁇ peptide (Abeta or ⁇ ) as defined herein in a biological sample from a test subject.
  • the biological sample will be taken on two or more occasions from a test subject.
  • the method additionally comprises comparing the level of the oligomeric state of a target amyloid ⁇ peptide (Abeta or ⁇ ) present in biological samples taken on two or more occasions from a test subject.
  • the method additionally comprises comparing the level of the oligomeric state of a target amyloid ⁇ peptide (Abeta or ⁇ ) present in a test sample with the amount present in one or more sample(s) taken from said subject prior to commencement of therapy, and/or one or more samples taken from said subject at an earlier stage of therapy.
  • the method additionally comprises comparing the level of the oligomeric state of a target amyloid ⁇ peptide (Abeta or ⁇ ) with one or more controls.
  • a target amyloid ⁇ peptide Abeta or ⁇
  • the present invention is further described by the fol lowing examples, which should however by no means be construed to limit the invention in any way; the invention is defined in its scope only by the claims as enclosed herewith.
  • the DemTect scale is a brief screening for dementia comprising five short subtests (10-word list repetition, number transcoding, semantic word fluency task, backward digit span, delayed word list recall) (Kessler et al. , 2000).
  • the raw scores are transformed to give age- and education-independent scores, classified as 'suspected dementia' (score ⁇ 8), 'mild cognitive impairment' (score 9 - 12), and 'appropriate for age' (score 13 - 18).
  • MMSE Mini-Mental State Examination
  • Folstein test is a brief 30-point questionnaire test that is used to assess cognition (see Table 1). It is commonly used in medicine to screen for dementia. In the time span of about 10 minutes it samples various functions including arithmetic, memory and orientation. It was introduced by Folstein et a/. , 1975, and is widely used with small modifications.
  • the MMSE includes simple questions and problems in a number of areas: the time and place of the test, repeating lists of words arithmetic, language use and comprehension, and basic motor skills. For example, one question asks to copy drawing of two pentagons (see next table). Any score over 27 (out of 30) is effectively normal .
  • Scoring of the clocks was based on a modification of the scale used by Shulmann et ai, 1986. All circles were pre-drawn and the instruction to subjects was to "set the time 10 after 11".
  • the scoring system (see Table 2) ranges in scores from 1 to 6 with higher scores reflecting a greater number of errors and more impairment. This scoring system is empirically derived and modified on the basis of clinical practice. Of necessity, it leaves considerable scope for individual judgment, but it is simple enough to have a high level of interrater reliability. Our study lends itself to the analysis of the three major components. These include cross-sectional comparisons of the clock-drawing test with other measures of cognitive function; a longitudinal description of the clock-drawing test over time, and the relationship between deterioration on the clock-drawing test and the decisions to institutionalize.
  • ⁇ 1-40 and /or ⁇ 1-42 concentration in humans all of the following body fluids can be used : blood, cerebrospinal fluid, urine, lymph, saliva, sweat, pleura fl uid, synovial fl uid, aq ueous fluid , tear fl uid , bile and pancreas secretion.
  • the novel method was established with blood samples and can be further used for CSF, brain extract and urine samples, followed by all other human body fluids.
  • Plasma or serum was pipetted off, filled in one 5 ml polypropylene cryo-tube (Carl-Roth, E295.1) and stored frozen at -80°C . Sa m ples were centrifu ged with i n one hou r after blood withdrawal. The appropriate labelling of the plasma or serum tubes according to the study protocol was duty of the CRO.
  • Beside wild type ⁇ 1-40 mutated variants can also be quantified by this method .
  • the mutated variants comprise all amyloid beta peptides starting with amino acids Asp-Ala-Glu and ending with Gly-Val-Val .
  • the ⁇ 1-40 assay can be also used for other familial Alzheimer's disease, which offer mutations outside the ⁇ 1-40 sequence producing the wild type ⁇ 1-40. Following familial Alzheimer's disease examples are also suitable for the assay: Swedish, Austrian, French, German, Florida, London, Indiana, Australian (Irie et a/., 2005)
  • Beside wild type ⁇ 1-42 also mutated variants can be quantified by this method .
  • the mutated variants comprise all amyloid beta peptides starting with amino acids Asp-Ala-Glu and ending with Val-Ile-Ala. Mutated ⁇ 1-42 examples:
  • the ⁇ 1-42 assay can be also used for other familial Alzheimer's disease, which offer mutations outside the ⁇ 1-42 sequence producing the wild type ⁇ 1-42. Following familial Alzheimer's disease examples are also suitable for the assay: Swedish, Austrian, French, German, Florida, London, Indiana, Australian (Irie et a/., 2005)
  • EDTA plasma samples (containing 4 ml plasma) (heparin plasma, serum also possible) were thawed and aliquoted at 1 ml in 2 ml polypropylene tubes (Eppendorf, 0030120.094).
  • protease inhibitor (Roche, Complete mini Protease inhibitor cocktail, 11836153001) was dissolved in 1 ml D-PBS (Invitrogen, 14190-094). 25 ⁇ of the protease inhibitor solution was added to 1 ml EDTA plasma. All aliquots were frozen and stored again at -80°C, except one tube of each sample. These plasma tubes were spiked with 10 ⁇ of 10 % Tween- 20. To ea ch tu be 2.
  • anti-mouse magnetic beads (Invitrogen, 112-02D) were used for a 1 ml plasma sample. Beside these special anti-mouse antibodies conjugated on mag netic beads al l other anti- mouse antibod ies or anti-host antibodies (host : origin of primary antibod ies listed above) can be used . These antibod ies can be immobilized on several matrices (col u m n matrices a nd bead matrices) via d ifferent conj ug atio n strategies, e.g.
  • Biotin-Streptavidin interaction tosyl-activated surface, epoxy- activated surface, amine-surface, carboxylic surface.
  • 100 ⁇ beads were pipetted off from the original bottle into a 2 ml tube and washed 3-times with 1 ml PBS. After washing the beads were resuspended in 200 ⁇ PBS.
  • the plasma tubes were centrifuged for 30sec at 2000 x g .
  • the supernatants were transferred into the tubes containing the anti-mouse magnetic beads.
  • the tubes were incubated overnight at 4°C in an overhead shaker. On the next day the tubes were placed into a magnetic separator to allow the bead to be drawn to the tube wall .
  • washing buffers which contain detergents or other additives preventing unspecific binding can be used for this step. Examples for washing buffers are:
  • the solution was drawn out, the tubes were taken from the magnetic separator and 100 ⁇ 50 % (v/v) Methanol / 0.5 % (v/v) formic acid were added to each tube and the beads were resuspended by slightly shaking . All tubes were incubated for 1 hour at room temperature. Afterwards the tubes were again placed in the mag netic separator and 40 ⁇ eluate from each tube were mixed with 440 ⁇ EIA buffer (dilution buffer of the IBL 1-40/42 (N) ELISA Kit). The pH of the diluted samples were adjusted with 16 ⁇ 400 mM Na 2 HP0 4 , 400 m M KH 2 P0 4 pH 8.0.
  • the determination of the peptide concentration was performed using the IBL 1-40(N) ELISA Kit (IBL, JP27714) and IBL 1-42(N) ELISA Kit (IBL, JP27712).
  • Amyloid beta Human Amyloid beta (Amyloid-b), Wako Chemicals USA, Inc. (aa 1-40 ELISA Kit)
  • Amyloid Beta 1-40 ELISA Kit The Genetics Company Self made ⁇ 1-40 ELISA comprise of a pair of capture and detection antibody, which are specific for the complete N-terminus of ⁇ 1-40 and the C-terminus ending at amino acid 40.
  • Such a self made fu l l len gth ⁇ 1-40 sandwich ELISA can comprise a first immobilized antibody recognizing specifically the C-terminus of ⁇ 1-40 and a second labeled detection antibody recog nizing specifical ly the complete N- terminus of ⁇ 1-40.
  • a full length ⁇ 1-40 sandwich ELISA can also comprise a first immobilized antibody recognizing specifically the complete N-terminus of ⁇ 1-40 and a second labeled detection antibody recog nizing specifically the C- term i n us of ⁇ 1-40 , th is type of ⁇ 1-40 sandwich ELISA is particularly envisaged .
  • Amyloid Beta 1-42 ELISA Kit The Genetics Company beta-Amyloid 1-42 ELISA Kit (SIGNET) Covance
  • Self made ⁇ 1-40 ELISA comprise of a pair of capture and detection antibody, which are specific for the complete N-terminus of ⁇ 1-42 and the C-terminus ending at amino acid 40.
  • Such a self made full length ⁇ 1-42 sandwich ELISA can comprise a first immobilized antibody recognizing specifically the C-terminus of ⁇ 1-42 and a second labeled detection antibody recog nizing specifical ly the complete N- terminus of ⁇ 1-42.
  • a full length ⁇ 1-42 sandwich ELISA can also comprise a first immobilized antibody recognizing specifically the complete N-terminus of ⁇ 1-42 and a second labeled detection antibody recog nizing specifically the C- term i n us of ⁇ 1-42 , th is type of ⁇ 1-42 sandwich ELISA is particularly envisaged .
  • the diluted samples (with and without disaggregation, respectively) were applied to the ELISA plate (100 ⁇ per well, repeat determination).
  • the ELISA standard were taken from the kit, dissolved and diluted according to the manufacture instruction protocol . After application of all samples and concentration standards the ELISA plate was incubated for 18 h at 4°C. On the following day, the ELISA was developed according to the manufacturers instruction protocol .
  • the calculated concentration was corrected by the EIA buffer dilution (including pH adjustment), factor 12.4, and the concentration effect ( 1 m l to 100 ⁇ ⁇ ) of the i mm u nopreci pitation by factor 0. 1 .
  • the calculated concentration was corrected by the EIA buffer dilution (including pH adjustment), factor 12.25, the dilution by adding 20 ⁇ 50 % (v/v) Methanol / 500 mM NaOH to the eluted sample, factor 1.4, and the concentration effect (1 ml to 100 ⁇ ) of the immunoprecipitation by factor 0.1.
  • the determined plasma ⁇ ( 1-40/42) concentrations (with and without disaggregation, respectively) were denoted in pg/ml.
  • Oiigomeric state ⁇ ( 1-40) ⁇ 1-40 (with disaggregation) / ⁇ 1-40 (without disaggregation)
  • Oiigomeric state ⁇ ( 1-42) ⁇ 1-42 (with disaggregation) / ⁇ 1-42 (without disaggregation)
  • the raw scores are transformed to give age- and education-independent scores, classified as 'suspected dementia' (score ⁇ 8), 'mild cognitive impairment' (score 9 - 12), and 'appropriate for age' (score 13 - 18).
  • the test results for all visits are shown in Figure 3.
  • the results from Figure 3 demonstrate that there are clear differences between the three groups of healthy subjects compared with the patients.
  • the scoring system ranges in scores from 1 to 6 with higher scores reflecting a greater number of errors and more impairment.
  • This scoring system is empirically derived and modified on the basis of clinical practice. Of necessity, it leaves considerable scope for individual judgment, but it is simple enough to have a high level of interrater reliability.
  • Plasma oligomeric state of ⁇ (1-40) and ⁇ (1-42) (TO+9 month series)
  • the mean values and the standard error of mean for all four groups were calculated .
  • the T-test has compared the AD group with each single control group.
  • the values for the oligomeric state in table 4 were calculated according the methods of the present invention and represent the ratio Cd / c a .
  • the oligomeric state of ⁇ (1-40) and ⁇ (1-42) of all control samples against all samples of the AD group was also evaluated .
  • the used method cannot determine the amount of ⁇ (1-40) or ⁇ (1-42) homo- oligomers in the sample, it displays the amount of ⁇ ( 1-40) and ⁇ ( 1-42) peptides within soluble aggregates compared with monomeric ⁇ (1-40) and ⁇ (1-42) in the sample. Because of this fact the summation of the values for ⁇ (1- 40) and ⁇ ( 1-42) can reflect the overall amount of ⁇ oligomers in plasma of AD patients and healthy controls ( Figure 7). The summation of oligomeric state values has further improved the p-value of the T-test, although the sample quantity was less compared with single evaluation for ⁇ (1-40) and ⁇ (1-42), respectively.
  • this assay which uses a monoclonal antibody (clone 13C3) generated by immunization of mice with a fibrillar form of ⁇ 42.
  • the characterization of the 13C3 antibody offers a good affinity to protofibrillar ⁇ 42, however also to monomeric ⁇ 42 (Schupf etal., 2008; supporting information), which can falsify the determined protofibrillar ⁇ 42 level. Therefore the usage of such assay systems based on oligomer or protofibrillar specific antibodies is hampered, if the detection antibody is not exclusively specific for higher molecular aggregates of Amyloid ⁇ .
  • the ⁇ 42 concentration is determined by a normal sandwich ELISA. As described above, such a simple sandwich ELISA could have problems with the recovery rate. For denatured conditions the ⁇ 42 concentration is determined by SDS-PAGE followed by Western Blot analysis. A critical issue of this method is the fact that ⁇ ( 1-42) assemblies cannot completely disaggregate to monomer by 2 % SDS. Our experiences show also trimer and tetramer species of ⁇ ( 1- 42) in SDS-PAGE. against this background a correct quantification of ⁇ (1-42) monomers is very doubtful . Furthermore this fact makes a comparison with ELISA determined concentration and subsequent the calculation of a ratio of both values very defective.
  • Dubois B Feldman HH, Jacova C, Dekosky ST, Barberger-Gateau P, Cummings J, Delacourte A, Galasko D, Gauthier S, Jicha G, Meguro K, O'brien J, Pasquier F, Robert P, Rossor M, Sa Noway S, Stern Y, Visser PJ, Scheltens P. Research criteria for the diagnosis of Alzheimer's disease : revising the NINCDS-ADRDA criteria. Lancet Neurol. 2007 Aug ;6(8):734-46.
  • Beta-amyloid imaging and memory in non- demented individuals evidence for preclinical Alzheimer's disease. Brain. 2007 Nov;130(Pt l l):2837-44
  • Fagan AM Mintun MA, Mach RH, Lee SY, Dence CS, Shah AR, LaRossa GN, Spinner ML, Klunk WE, Mathis CA, DeKosky ST, Morris JC, Holtzman DM.
  • Cerebrospinal fluid tau/beta-amyloid(42) ratio as a prediction of cognitive decline in nondemented older adults. Arch Neurol. 2007 Mar; 64(3): 343-9. Gustafson DR, Skoog I, Rosengren L, Zetterberg H, Blennow K.
  • Cerebrospinal fluid beta-amyloid 1-42 concentration may predict cognitive decline in older women .
  • Amyloid beta peptide ratio 42/40 but not A beta 42 correlates with phospho-Tau i n patie nts with l ow- and high-CSF A beta 40 load . J Neurochem. 2007 May; 101 (4) : 1053-9
  • the beta APP717 Alzheimer mutation increases the percentage of plasma amyloid-b eta p ro te i n e n d i n g a t A beta42(43) . Neurology. 1997 Mar;48(3):741 -5
  • Amyloid beta protein dimer-containing human CSF disrupts synaptic plasticity: prevention by systemic passive immunization. J Neurosci. 2008 Apr 16;28(16):4231 -7
  • Amyloid-beta oligomers are inefficiently measured by enzyme-linked immunosorbent assay. Ann Neurol. 2005 Jul;58(l): 147-50.
  • Kessler 3 Calabrese P, Kalbe E, Berger F. Ein 1468 Screening-Verfahren Kunststoff Unterstijtzung der Demenzdiagnostik. Psycho 26 343-347 (2000)

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Abstract

The invention relates to the detection and diagnosis of Alzheimer's disease with the use of the oligomeric state of fragments of amyloid β as a biomarker and further concerns a novel method to determine the oligomeric state of fragments of amyloid β in biological samples.

Description

NOVEL DIAGNOSTIC METHOD FOR THE DIAGNOSIS OF ALZHEIMER'S DISEASE OR MILD
COGNITIVE IMPAIRMENT
FIELD OF THE INVENTION
The invention relates to the detection and diagnosis of Alzheimer's disease with the use of the oligomeric state of fragments of amyloid β as a biomarker and further concerns a novel method to determine the oligomeric state of fragments of amyloid β in biological samples.
BACKGROUND OF THE INVENTION
Alzheimer's disease is the most common form of dementia and has a prevalence of approximately 65-70% among all dementia disorders (Blennow et al., 2006). Resulting from increased life expectancy, this disease has become a particular issue in highly developed industrialised countries like Japan and China as well as in the US a nd Eu rope . The n u mber of Alzhei mer patients is esti mated to increase from 24 million in 2001 to 81 million in 2040 (Ferri et al. , 2005) . Currently, the costs for treatment and care of AD patients worldwide amount to approximately 250 billion US dollars per year.
The progression of the sporadic form of the disease is relatively slow and Alzheimer's disease will usually last for about 10-12 years after the onset of first symptoms. Presently, it is extremely difficult to make a reliable and early diagnosis of AD and distinguish it from other forms of dementia. A good diagnosis with a reliability of more than 90% is only possible in the later stages of the disease. Prior to that, it is only possible to make a prediction that Alzheimer's is possible or probable; diagnosis here relies on the use of certain criteria according to Knopman et al., 2001; Waldemar et al., 2007 or Dubois et al., 2007. Neurodegeneration starts however 20 to 30 years before the first clinical symptoms are noticed (Blennow et al., 2006; Jellinger KA, 2007). The onset of the clinical phase is usually characterized by the so-called "mild cognitive impairment" ( MCI), where patients wil l show measurable cognitive deficits which are not sufficient to enable a diagnosis of a dementia disease in a clear fashion (Petersen et al., 1999; Chetkow et al., 2008). Many patients with MCI will have neuropathological changes which are typical for AD and which means that an earlier stage of AD is possible, but not certain (Scheff et al., 2006; Markesbery et al., 2006; Bouwman et al., 2007). There are however many MCI cases which will not progress to Alzheimer's; in these cases, other factors are responsible for the cognitive deficit (Saito et al., 2007; Jicha et al., 2006 and Petersen et al., 2006). While some MCI patients will not show any deterioration of their condition or even some kind of amelioration, for most MCI cases the cognitive deficit will continue to clinical dementia. The yearly rate of this conversion is approximately 10-19% (Gauthier et al., 2006; Fischer et al., 2007). At present there is a combination of clinical, neuropsychological and imaging processes which are capable of differentiating the various subtypes of Mild Cognitive Impairment (Devanand et al., 2007; Rossi et al., 2007; Whitwell et al., 2007; Panza et al., 2007). However, there is no significant difference between these subtypes in relation to the further progression of dementia (Fischer et al., 2007). Thus, it is of utmost importance to develop a method to enable a clear and reliable diagnosis of Alzheimer's disease in the early stages, suitably at its onset or during MCI.
Prior art biomarkers
Biomarkers for Alzheimer's disease have already been described in the prior art. Alongside well known psychological tests such as e.g . ADAS-cog, MMSE, DemTect, SKT or the Clock Drawing test, biomarkers are supposed to improve diagnostic sensitivity and specificity for first diagnosis as well as for monitoring the progression of the disease. In relation to the current status of development of biomarkers for AD/MCI it was proposed to correlate the disease in the future with the other diagnostic criteria (Whitwell et al., 2007; Panza et al. , 2007; Hyman SE, 2007). Biomarkers are supposed to support the classical neuropsychological tests in the future. There is a common belief that they will be of great importance as surrogate markers for the development of agents against Alzheimer's (Blennow K, 2004; Blennow K, 2005; Hampel et a/., 2006; Lewczuk et al., 2006; Irizarry MC, 2004).
Structural biomarkers
"Magnetic resonance imaging" (MRI) is an imaging process which allows detection of degenerative atrophies in the brain (Barnes J et a/., 2007; Vemuri et a/., 2008). Thus, atrophy of the medial temporal lobe (MTA) is sensitive to a degeneration of the hippocampal region in the brain of older patients; this can be made visible very clearly by MRI, but is not specific for Alzheimer's disease. Mild MTA is not encountered more frequently in other dementias (Barkhof et a/., 2007) but it does correlate with MCI (Mevel et a/. , 2007) . For this reason it is not possible to determine from MRI data alone whether the neurodegeneration is Alzheimer's disease or an early stage of Alzheimer's disease. A further imaging method is Positron Emission Tomography (PET) which visualises the accumulation of a detector molecule (PIB) on amyloid deposits. It could be detected that the thioflavin T-analogue (nC)PIB will accumulate increasingly in certain regions of the brain of patients with MCI or mild Alzheimer's disease, respectively (Kemppainen et a/., 2007; Klunk et a/., 2004; Rowe et a/., 2007); unfortunately this can also be detected in subjects who do not have dementia (Pike et a/., 2007). This would probably indicate that the detection of amyloid deposits via PET allows detection of pre-clinical stages of Alzheimer's; however, this has to be confirmed by further studies. Besides the most frequently used processes, MRI and PET, there are additional structural biomarkers for AD: CBF- SPECT, CMRg l-PET (glucose metabolism proton spectroscopy (H-l MRS), high field strength functional MRI, voxel-based morphometry, enhanced activation of the mediobasal temporal lobe (detected by fMRI, (R)-[(n)C]PK11195 PET for the detection of microglial cells (Huang et a/., 2007; Kantarci et a/., 2007; Petrella et a/., 2007; Hamalainen et a/., 2007; Kircher et a/., 2007; Kropholler et a/., 2007). CSF Biomarkers
Senile plaques are one of the pathological characteristics of Alzheimer's disease. These plaques consist mostly of Αβ (1-42) peptides (Attems J, 2005). In some studies it could be shown that a low level of Αβ (1-42) in CSF of MCI patients correlates specifically with the further development of Alzheimer's disease in its progression (Blennow and Hampel, 2003; Hansson et a/., 2006 and 2007). The reduction in CSF is probably due to enhanced aggregation of Αβ (1-42) in the brain (Fagan et a/., 2006; Prince et a/., 2004; Strozyk et a/. , 2003) . Another possibility is the occurrence of semi-soluble Αβ (1-42) oligomers (Walsh et a/., 2005) which would lead to a lower level of detection in CSF. In particular in the early stages of Alzheimer's, decreased concentrations of Αβ (1-42) would be detected, while increased amounts of Tau protein and phospho-tau proteins in CSF, respectively, could be detected (Ewers et a/., 2007; Lewczuk et a/., 2004). To provide a better predictability of biomarkers, it is usually attempted to use the Tau/ Αβ (1-42) ratio and correlate it with the prediction of cognitive deficiency in older persons who do not have dementia (Fagan et a/., 2007; Gustafson et a/., 2007; Hansson et a/., 2007; Li et a/., 2007; Stomrud et a/., 2007) as well as in MCI patients (Hampel et a/., 2004; Maccioni et a/., 2006; Schonknecht et a/., 2007). A further correlation between ante mortem CSF level of Αβ (1-42), Tau, phospho-Tau-Thr231 and post-mortem histopathological alterations of the brain could be detected in AD patients (Clark et a/., 2003; Buerger et a/., 2006). In other studies, however, no correlation between CSF biomarkers and Αβ (1-42), total Tau and phospho-Tau with APOE £4-allele, plaque and tangle load after autopsy could be detected (Engelborghs et a/., 2007; Buerger et a/., 2007) . An interesting aspect was detected in a multicenter study. It appears that increased level of total Tau and phospho-Tau (181) correlates with a decreased ratio of Αβ (1-42)/ Αβ (1-40), but not with the Αβ (1-42) alone (Wiltfang et a/., 2007). An increased level of CSF Tau was however also detected in other CNS diseases such as Creutzfeldt-Jakob d isease, brai n infarction , and cerebra l vascu la r dementia, which are all associated with a neuronal loss (Buerger et a/., 2006 (2); Bibl et a/., 2008). A further possible biomarker is the increase of BACE 1 activity in CSF as an indicator for MCI (Zhong et a/., 2007) . It is also discussed that the increased BACE 1 activity wil l result in increased Αβ production and therefore increased aggregation of the peptides. Alzheimer's disease is accompanied by neuroinflammatory processes. CSF anti-microglial cell antibodies are therefore possible biomarkers for these inflammatory processes in AD (McRea et a/., 2007). In spite of the multitude of biomarkers which are su pposed to enable early diagnosis of Alzheimer's disease, there is not a single biomarker that ensures reliable and clear diagnosis. This is usually because most studies use a com pa rison of the respective bioma rkers and cl i n ical d iag nosis . A better approach would be the correlation of biomarkers with the pathological causes of Alzheimer's disease.
A possible approach would be repeated analysis of immuno-precipitated CSF samples of clearly identified and defined neuropathological dementia diseases to clarify whether Αβ (1-40) a n d Αβ (1-42) are in fact suitable neurochemical dementia markers (Jellinger et a/., 2008). In order to discover novel, up to now unknown, biomarkers for Alzheimer's disease, CSF samples are usually analyzed via a comparative proteomic analysis which results in a diagnosis of AD with en h a n ced se nsitivity a n d a l so to en a b le the d iffere nti ati o n fro m other degenerative dementia disorders (Finehout et al., 2007; Castano et al., 2006; Zhang et al., 2005; Simonsen et al., 2007; Lescuyer et al., 2004; Abdi et al., 2006). After a proteomic analysis, the potential new biomarker should be analyzed in detail for its suitability and correlation with pathological causes. A typical example for a biomarker which was found by a proteomic analysis is truncated cystatin C as a biomarker for multiple sclerosis; this biomarker was later proven to be a storage artefact (Irani et al., 2006; Hansson et al., 2007(2)). Plasma Biomarkers
Besides the frequently used plasma biomarkers, i .e. the Αβ peptides, further inflammatory plasma markers are used for the early diagnosis of dementia (Ravaglia et al., 2007; Engelhart et al., 2004) in particular for Alzheimer's (Motta et al., 2007). All of these are still under discussion. Further possible biomarkers were also found via comparative proteomic analysis of plasma from AD patients and healthy controls (German et al., 2007; Ray et al., 2007). The future will show whether these biomolecules are indeed specific for Alzheimer's disease and are suitable as biomarkers. There is no convincing or suitable data which would show either specificity or suitability of any of the biomarkers discussed above.
Contrary to the analysis of amyloid β in CSF, the results until now with respect to suitable Αβ biomarkers in plasma are not reliable or clear. In some studies a correlation between a decreased ratio of Αβ (1-42)/ Αβ (1-40) in plasma and an enhanced conversion of cognitive normal persons to MCI or Alzheimer patients, respectively, was found ((Graff-Radford et al., 2007; van Oijen et al., 2006; Sundelof et al., 2008). Other studies however detected that a reduction of the Αβ (1-42) plasma level is more likely a marker for the conversion from MCI to AD (Song et al. , 2007) and is not suitable as a marker for neu rodegenerative purposes which are encountered with Alzheimer's (Pesaresi et al., 2006). Most of the studies however do not show a d ifference in Αβ plasma levels between healthy controls and patients with sporadic Alzheimer's (Fukumoto et al., 2003; Kosaka et al., 1997; Scheuner et a/., 1996; Sobow et a/., 2005; Tamaoka et a/., 1996; Vanderstichele et al., 2000). Some studies also showed that the level of Αβ in plasma does not correlate with the level as encountered in the brain (Fagan et al., 2006; Freeman et al., 2007) nor does it correlate with the level encountered in CSF (Mehta et al., 2001; Vanderstichele et al., 2000). In a recent study, a correlation was detected for Αβ (1-40) and Αβ (1-42) between CSF and plasma, but only in healthy controls. This correlation could not be detected in MCI and AD which is explained by destroying the balance between CSF and plasma Αβ d ue to Αβ deposits in the brain (Giedraitis et al., 2007). Generally, it is assumed that plasma Αβ (1-42) level is not a reliable biomarker for MCI or AD (Blasko et al., 2008; Mehta et al., 2000; Brettschneider et al., 2005 ) , whereas a decrease of the ratio pl asma Αβ (1-38) / Αβ (1-40) is considered a biomarker for vascular dementia and comes close to the predictability of CSF markers (Bibl et a/., 2007).
Until now, Αβ oligomers were disregarded as biomarkers for Alzheimer, however, they are supposed to play a decisive role in initiating the neurodegenerative process (Walsh & Selkoe, 2007). In several studies, the neurotoxic effect was shown for Αβ dimers with 8 kDa to the point of protofibrils with over 100 kDa (Lambert et al., 1998; Walsh et al, 2002; Keayed et al., 2004; Cleary et al., 2005). Furthermore, such Αβ oligomers were found in human liquor (Pitschke et al., 1998; Santos et al., 2007; Klyubin et al., 2008). Besides their neurotoxicity, oligomers have also an influence on the determination of the Αβ concentration in human samples. The oligomerization leads to masking of the C-terminal epitopes of Αβ peptides (Roher et a/., 2000) yielding to underestimated Αβ levels detected by C-terminal specific ELISA (Stenh et al., 2005). Thus, the existence of Αβ oligomers in the sample results in lowering of the ELISA signal. This could be a problem for exact determination of the Αβ concentration, however this fact offers also the chance to measure the amount of oligomers and the level of oligomerization in biological samples. The data presented herein surprisingly demonstrates that the content of Αβ oligomers can be determined indirectly by measuring the ELISA signal before and after disaggregation of the oligomers. The ratio of both values reflect the concentration of soluble Αβ oligomers and the oligomeric level, respectively, in human plasma. Independently from our present invention a similar approach was published very recently (Englund et a/., 2009). They determ i ned the Αβ 1-42 o l ig omer rati o i n h u m a n CS F sa m p les by measuring the Αβ 1-42 concentration under non-denaturing conditions via ELISA and under denaturing cond itions using SDS-PAG E fol lowed by Western Blot analysis. However, this approach of indirect determination of the oligomeric level has some critical issues:
1. SDS-PAGE is not able to fully disaggregate Αβ 1-42. Our experiences have shown also Αβ trimer and tetramer reflecting bands on the SDS gel.
2. The comparison of Αβ concentrations determined via ELISA and via Western Blot is defective.
Another more common approach is the direct measurement of Αβ oligomers. Such a method, especially with oligomeric plasma Αβ as a biomarker, is however extremely difficult to establish as the Αβ peptides are very hydrophobic. Currently described assay systems use Αβ oligomer specific antibodies in a ELISA system (Englund et a/., 2007; Schupf et a/, 2008). However, the usage of ELISAs based on such oligomer specific antibodies have the same problems as traditional Αβ ELISA systems. The methods only achieve very unsatisfactory analytical sensitivity and encounter great problems with the very complex interactions between analytes and matrix, i.e. plasma. Usually, ELISA or ELISA-type systems (Multiplex) are used for quantification of Αβ, and recently also Αβ oligomers, in plasma. The specification of such detections systems is usually only unsatisfactorily analyzed or are completely disregarded . For example a critical ite m l i ke the recove ry rate is n ot a n a lyzed o r is n ot me nti o n ed i n the publications. The recovery rate is however decisive for giving a complete picture of those Αβ peptides or oligomers which occur in plasma. Differences between the studies can also result from the differences in these rates. A further important characteristic of an ELISA or multiplex system is its linearity. Thus, the concentrations determined for the analytes in plasma should only depend on the dilution used in the measurement to a very low degree or not at all. However, this is neither possible for ELISA nor for the multiplex systems for quantification of Αβ in plasma. Thus, the difference between the calculated plasma Αβ (1-42) concentration for a dilution of 1-20 was three times as high as for the 1-2 dilution of the same sample (Hansson et al. , 2008) . This example alone shows that the use of different dilutions of plasma samples in the several studies makes it impossible to compare the same.
Thus, it is an objective of the present invention to provide a novel method which allows determination of oligomeric Αβ, i n pa rticu l a r in pl asma, with a h ig h reliability. The present invention uses also the indirect measurement of Αβ oligomers, however, in contrast to the prior art, both values (under denaturing and non-denaturing conditions) were determined with Αβ specific ELISA to ensure the comparability. Because of an initial immunoprecipitation step, which isolates Αβ peptides in monomeric as well as oligomeric form followed by our novel disaggregation method, the subsequent ELISA is not constricted by recovery and/or linearity issues.
Moreover, the present invention aims at providing diagnostic markers which can be determined with rel iable methods and can be used for rel iable and clear prediction of Alzheimer's disease.
SUMMARY OF THE INVENTION
According to a first aspect of the invention there is provided a method of diagnosing or monitoring a neurodegenerative disorder, such as Alzheimer's disease and Mild Cognitive Impairment, which comprises determining the oligomeric state of a target amyloid β peptide (Abeta or Αβ) in a biolog ical sample from a test subject, characterized in that said method comprises the following steps:
(a) determining a first concentration (ca) of a target Αβ peptide in a biological sample;
(b) disaggregating the target Αβ peptide from step (a);
(c) determining a second concentration (cd) of the d isaggregated Αβ peptide; and
(d) determining the ratio of cd / ca, wherein the value of the second concentration (cd) is divided by the value of the first concentration ca;
wherein a ratio of cd / ca, which is lower than 1 .5 is ind icative of a positive diagnosis for a neurodegenerative disorder.
According to a second aspect of the invention there is provided a method of determining the oligomeric state of a target amyloid β peptide (Abeta or Αβ) in a biological sample which comprises the following steps:
(a) determining a first concentration (ca) of a ta rget Αβ peptide in a biological sample;
(b) disaggregating the target Αβ peptide from step (a);
(c) determining a second concentration (cd) of the disaggregated Αβ peptide; and
(d) determining the ratio of cd / ca, wherein the value of the second concentration (cd) is divided by the value of the first concentration ca;
wherein a ratio of cd / ca, which is in excess of 1, is indicative of the presence of oligomeric Αβ.
DEFINITIONS
"Oligomeric" as used herein refers to a limited number of aggregated Αβ peptide monomer u n its . Exa mples of su ch ol ig omers incl ude d i mers, tri mers and tetramers . The term "d isagg reg ation" refers to the process of converti ng oligomeric forms of Αβ peptide to monomeric forms of Αβ peptide.
"Capture antibody" in the sense of the present application is intended to encompass those antibodies which bind to a target Αβ peptide. Suitably the capture antibodies bind to the target Αβ peptide with a high affinity. In the context of the present invention, high affinity means an affinity with a KD value of 10"7M or better, such as a KD value of 10"8M or better or even more particularly, a KD value of 10"9M to 10"12M .
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 as long as they exhibit the desired biological activity. The antibody may be an IgM, IgG (e.g. IgGl, IgG2, IgG 3 or IgG4) , Ig D, IgA or Ig E, for example. Suitably however, the antibody is not an IgM antibody. The "desired biological activity" is binding to a target Αβ peptide. "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.
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 adva ntageous i n that they are synthesized by the hybridoma cu ltu re, 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 accord ance with the present i nvention may be made by the hybridoma method first described by Kohler et a/. , Nature, 256 : 495 (1975), or may be m ade by genera l ly wel l kn own recom bi na nt D NA meth ods . 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 a/., J. Mol. Biol., 222 : 581-597 (1991), for example.
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.
"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.
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 or a "camelized" antibody.
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and V|_ 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).
The term "diabodies" refers to small antibody fragments with two antigen- binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VD) in the same polypeptide chain (VH - VD). 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 eta/., Proc. Natl. Acad. Sol. USA, 90:6444-6448 (1993). 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 suitable embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most particularly 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 non-reducing conditions using Coomassie blue or, suitably, 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.
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.
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.
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.
"Amyloid β, Αβ or β-amyloid" is an in the art recognized term and refers to amyloid β proteins and peptides, amyloid β precursor protein (APP), as well as modifications, fragments and any functional equivalents thereof. In particular, by amyloid β as used herein is meant any fragment produced by proteolytic cleavage of APP but especia l ly those frag ments wh ich a re i nvolved i n or associated with the amyloid pathologies including, but not limited to, Αβ (1-38) of SEQ ID NO. 3, Αβ (1-40) of SEQ ID NO. 2, and Αβ (1-42) of SEQ ID NO. 1.
In the context of the present invention, "fragments of amyloid β" are all amyloid β peptides, which comprise a core amyloid β fragment Αβ (3-38) of SEQ ID NO. 13, More suitably for the purpose of the present invention are all amyloid β peptides, which comprise the core amyloid β fragment Αβ (11-38) of SEQ ID NO. 19. Such Αβ fragments, which comprise the amino acid sequence of Αβ (11-38) of SEQ ID NO. 19, are in particular Αβ (x-y) fragments, which have been shown to accumulate in a subject as a consequence of a neurodegenerative disorder, such as Alzheimer's disease and Mild Cognitive Impairment, wherein x is defined as an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11;
Preferably, x is an integer selected from 1, 2,
More preferably, x is 1.
Even more preferably, x is 11. y is defined as an integer selected from 38, 39, 40, 41, 42 and 43.
Preferably, y is 38, 40 or 42, such as 40 or 42.
More preferably, y is 40.
Even more preferably, y is 38.
Suitable examples for Αβ (x-y) fragments are
Αβ (1-38) (SEQ ID NO. 3),
Αβ (1-39) (SEQ ID NO. 4),
Αβ (1-40) (SEQ ID NO. 2),
Αβ (1-41) (SEQ ID NO. 5)
Αβ (1-42) (SEQ ID NO. 1)
Αβ (1-43) (SEQ ID NO. 6)
Αβ (2-38) (SEQ ID NO. 7), Αβ (2-39) (SEQ ID NO. 8),
Αβ (2-40) (SEQ ID NO. 9),
Αβ (2-41) (SEQ ID NO. 10),
Αβ (2-42) (SEQ ID NO. 11),
Αβ (2-43) (SEQ ID NO. 12),
Αβ (3-38) (SEQ ID NO. 13),
Αβ (3-39) (SEQ ID NO. 14),
Αβ (3-40) (SEQ ID NO. 15),
Αβ (3-41) (SEQ ID NO. 16),
Αβ (3-42) (SEQ ID NO. 17),
Αβ (3-43) (SEQ ID NO. 18),
Αβ (11-38) (SEQ ID NO. 19),
Αβ (11-39) (SEQ ID NO. 20),
Αβ (11-40) (SEQ ID NO. 21),
Αβ (11-41) (SEQ ID NO. 22),
Αβ (11-42) (SEQ ID NO. 23),
Αβ (11-43) (SEQ ID NO. 24).
" Functional equivalents" encompass all those mutants or variants of Αβ (x-y) which might naturally occur in the patient group which has been selected to undergo the method for detection or method for diagnosis as described according to the present invention. More particularly, "functional equivalent" in the present context means that the functional equivalent of Αβ (x-y) are mutants or variants thereof and have been shown to accu m u late in Alzheimer's d isease . The functional equivalents have no more than 30, such as 20, e.g . 10, particularly 5 and most particularly 2, or only 1 mutation(s) compared to the respective Αβ (x- y) peptide. Functional equivalents also encompass mutated variants, which comprise by way of example all Αβ peptides starting with amino acids Asp-Ala- Glu and ending with Gly-Val-Val and Val-Ile Ala, respectively.
Particularly useful equivalents in the present context are those of Αβ (1-40) (SEQ ID NO. 2) and Αβ (1-42) (SEQ ID NO. 1), which are those described by Irie et al., 2005, namely the Tottori, Flemish, Dutch, Italian, Arctic and Iowa mutations of Αβ. Functional equivalents also encompass Αβ peptides derived from amyloid precursor protein bearing mutations next to the β- or γ-secretase cleavage site such as the Swedish, Austrian, French, German, Florida, London, Indiana and Australian variations (Irie et al., 2005).
"Modified Amyloid β, Αβ or β-amyloid" encompasses all modifications at various amino acid positions in the amyloid β proteins and peptides, amyloid β precursor protein (APP), fragments and functional equivalents thereof. Useful in the present context are modifications at the N- and/or C-terminal amino acids of said amyloid β proteins and peptides, amyloid β precursor protein (APP), fragments and functional equivalents. Particularly useful are modifications at glutamine and glutamate residues, such as the cyclization of N-terminal glutamine or glutamate residues to pyroglutamate. Suitable examples according to the present invention are the amyloid β peptides of SEQ ID Nos. 13 to 24, which start with a glutamate residue at the N-terminus, wherein said the N-terminal glutamate residue is modified to pyroglutamate. Even useful are modifications at aspartate residues, such as the conversion of asparte to isoaspartate. Suitable examples according to the present invention are the amyloid β peptides of SEQ ID Nos. 1 to 6, wherein the aspartate residues at amino acid positions 1 and/or 7 are converted to isoasparate. Futher suitable examples are the amyloid β peptides of SEQ ID Nos. 7 to 12, wherein the aspartate residue at amino acid position 6 is converted to isoasparate. Moreover, suitable examples are the amyloid β peptides of SEQ ID Nos. 13 to 18, wherein the aspartate residue at amino acid position 5 is converted to isoasparate.
"Sandwich ELISAs" usually 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 wh ich is i mmobil ized on a sol id 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 suitable type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. BRIEF DESCRIPTION OF THE FIGURES
Figure 1: Bivalent Immunoprecipitation System improves capture efficiency
(A) Recovery of Αβ 1-40 from Cypl8 solution and human Plasma by usage of different antibody combinations
(B) Schematic of bivalent capture system (shaded : 4G8 antibody, grey: x-40 antibody, black: anti-mouse antibody conjugated to magnetic bead).
Figure 2: Determination of the oligomeric state of Αβ peptides derived from human plasma
The ratio of concentration determined after disaggregation to the concentration determined without disaggregation reflects the oligomeric state of Αβ (1-40/42).
Figure 3: DemTect Test
Mean values (Mean ± SD) of the results of classification differences in AD patients and healthy subjects (Group I : 18-30 years; Group II : 31-45 years; Group III: 46-65 years) by DemTect Scale.
Figure 4: Mini-Mental-State Test
Mean values (Mean ± SD) of the results of classification differences in AD patients and healthy subjects (Group I : 18-30 years; Group II : 31-45 years; Group III: 46-65 years) by Mini-Mental-State Test.
Figure 5: Clock-Drawing Test
Mean values (Mean ± SD) of the results of classification differences in AD patients and healthy subjects (Group I : 18-30 years; Group II : 31-45 years; Group III: 46-65 years) by Clock-Drawing Test. Figure 6: Mean values of oligomeric state, T-test of AD group vs. Control group
** T-test, p<0.01; *** T-test, p<0.001 Figure 7: Oligomeric state of Αβ (1-40) + Αβ (1-42) as reflection of overall oligomeric amount in plasma
DETAILED DESCRIPTION OF THE INVENTION
According to a first aspect of the invention there is provided a method of diag nosing or monitoring a neurodegenerative disorder, such as Alzheimer's disease and Mild Cognitive Impairment, which comprises determining the oligomeric state of a target amyloid β peptide (Abeta or Αβ) in a biological sample from a test subject, characterized in that said method comprises the following steps:
(a) determining a first concentration (ca) of a target Αβ peptide in a biological sample;
(b) disaggregating the target Αβ peptide from step (a);
(c) determining a second concentration (cd) of the disaggregated Αβ peptide; and
(d) determining the ratio of cd / ca, wherein the val ue of the second concentration (cd) is divided by the value of the first concentration ca;
wherein a ratio of cd / ca, which is lower than 1.5 is indicative of a positive diagnosis for a neurodegenerative disorder. The data presented herein surprisingly demonstrate that the oligomeric state of Αβ was significantly decreased in Alzheimer's disease patients when compared with control patients. Therefore, the oligomeric state of Αβ appears to be a reliable and clear prediction of Alzheimer's disease. A rati o (cd / ca) of 1.0 indicates that there are no oligomers in the sample. Higher ratios of cd / ca (i.e. ratios of > 1.0) reflect a greater amount of oligomers or more compactness of oligomers (less accessibility of epitopes) in the sample. Ratios of cd / ca, which are lower than 1.5 (i.e. a ratio between 1.0 and 1.5), such as lower than 1.4, lower than 1.3, lower than 1.2, lower than 1.1 or lower than 1.05 have been found to be indicative of a positive diagnosis for a neurodegenerative disorder, such as Alzheimer's disease.
I n a n other em bod i me nt of the i nventi on there is provi ded a meth od of diagnosing or monitoring a neurodegenerative disorder, such as Alzheimer's disease and Mild Cognitive Impairment, which comprises determining the oligomeric state of a target amyloid β peptide (Abeta or Αβ) in a biological sample from a test subject, characterized in that said method comprises the following steps:
(a) determining a first concentration (ca) of a target Αβ peptide in a biological sample;
(b) disaggregating the target Αβ peptide from step (a);
(c) determining a second concentration (cd) of the disaggregated Αβ peptide; and
(d) adding up the values of cd and ca, wherein the value of the sum of cd and Ca, which is lower than 3.0, is indicative of a positive diagnosis for a neurodegenerative disorder.
A sum of Cd and ca, which is lower than 2.9, lower than 2.8, lower than 2.7, lower than 2.6, lower than 2.5, lower than 2.4 or lower than 2.3 have been found to be indicative of a positive d iagnosis for a neurodegenerative disorder, such as Alzheimer's disease.
In nearly all studies, the concentration of target Αβ peptides in steps (a) and (c) was determined by sandwich ELISA systems consisting of a capture and a detection antibody. Com pared with the size of a n a nti body ( 150 kDa ) , Αβ peptides (4.5 kDa) as monomers are very small . Because of the aggregation propensity of the peptides they tend to form oligomers, protofibrils to the point of fibrils. Within such an aggregate the Αβ monomers are tightly packed with the consequence that not all monomers can be bound by the detection antibody due to sterical hindrance or epitope inaccessibility. The detected Αβ concentration for oligomers is lower than for monomers. This would lead to underestimated Αβ levels in plasma and CSF, respectively. The discrepancy between measured and actual concentration is dependent upon the amou nt of ol igomers and their compactness. Inversely, the amount of Αβ aggregates, or more precisely the burden epitopes, can be determined by comparison of the concentration detected in the presence of ol igomers and the concentration after d isagg regation of oligomers completely to monomers. The principle of the method is shown in Figure 2.
In one embodiment, the disaggregation step (b) comprises the use of an alkali. In a further embodiment, the alkali used for disaggregation in step (b) is sodium hydroxide, such as 500mM sodium hydroxide. The advantage of using an alkali and particularly a strong alkali such as sodium hydroxide is that more efficient disaggregation is achieved . For example, a higher proportion of monomers are obtained, with no observable quantities of dimers, trimers or tetramers. In one embodiment, the disaggregation step (b) additionally comprises the use of a suitable solvent, such as methanol, e.g. 50% (v/v) methanol.
In one embodiment, the disaggregation step (b) comprises an incubation step. In a further embodiment, the incubation step comprises incubation at room temperature for at least 2 minutes. In a yet further embodiment, the incubation step comprises incubation at room temperature for at least 10 minutes.
Αβ peptides are liberated from the amyloid precursor protein (APP) after a sequential cleavage by the enzymes β-and γ-secretase. The γ-secretase cleavage results in the generation of primarily Αβ (1-40) and Αβ (1-42) peptides but also ending prominently at position 38 or 43, which differ in their C-termini and exhibit different potencies of aggregation, fibril formation and neurotoxicity. Also, β-secretase release can generate different N-termini and also subsequent modifications by peptidases and other enzymes resulting in prominent species such as Αβ peptides starting at the positions e.g . 2, 3, 4 and also 11, while the species staring at positions glutamate 3 and 11 can be transformed into pyroglutamate, rendering these peptides especially hydrophobic and prone to fast aggregation (Schilling e a\, 2004; Piccini et a/., 2005; Schilling et al, 2006; Schlenzig et al, 2009). Such C- and N-terminal variants of Αβ can serve as functional equivalents of Αβ (1-40) and Αβ (1-42) peptides.
The present invention thus provides a method for the determination of the oligomeric states of the Αβ (x-y) peptides, wherein x and y are as hereinbefore defined .
Thus, according to one embodiment of the above-described method, the ol igomeric state of a target Αβ peptide to be determined is selected from the group consisting of:
Αβ (1-38) (SEQ ID NO. 3),
Αβ (1-39) (SEQ ID NO. 4),
Αβ (1-40) (SEQ ID NO. 2),
Αβ (1-41) (SEQ ID NO. 5)
Αβ (1-42) (SEQ ID NO. 1)
Αβ (1-43) (SEQ ID NO. 6)
Αβ (2-38) (SEQ ID NO. 7),
Αβ (2-39) (SEQ ID NO. 8),
Αβ (2-40) (SEQ ID NO. 9),
Αβ (2-41) (SEQ ID NO. 10)
Αβ (2-42) (SEQ ID NO. 11)
Αβ (2-43) (SEQ ID NO. 12)
Αβ (3-38) (SEQ ID NO. 13)
Αβ (3-39) (SEQ ID NO. 14)
Αβ (3-40) (SEQ ID NO. 15)
Αβ (3-41) (SEQ ID NO. 16)
Αβ (3-42) (SEQ ID NO. 17)
Αβ (3-43) (SEQ ID NO. 18)
Αβ (11-38) (SEQ ID NO. 19)
Αβ (11-39) (SEQ ID NO. 20)
Αβ (11-40) (SEQ ID NO. 21)
Αβ (11-41) (SEQ ID NO. 22)
Αβ (11-42) (SEQ ID NO. 23) and Αβ (11-43) (SEQ ID NO. 24).
In a particular embodiment, the oiigomeric state of a target Αβ peptide to be detected is Αβ (1-40) (SEQ ID No : 2).
In a particular embodiment, the oiigomeric state of a target Αβ peptide to be detected is Αβ (1-42) (SEQ ID No : 1).
In a particular embodiment, the oiigomeric state of a target Αβ peptide to be detected is Αβ (1-40) (SEQ ID No : 2) and Αβ (1-42) (SEQ ID No : 1). The data presented herein demonstrates the suitability of summation of the Αβ (1-40) and Αβ (1-42) peptides wherein it has been shown that summation of both oiigomeric states improves the significance of the diagnosis. In a particular embodiment, the oiigomeric state of a target Αβ peptide to be detected is at least one Αβ peptide selected from the SEQ ID NOs: 13 to 24, which start with a glutamate residue at the N-terminus.
In a particular embodiment, the oiigomeric state of a target Αβ peptide to be detected is Αβ (3-38) (SEQ ID No : 13).
In a particular embodiment, the oiigomeric state of a target Αβ peptide to be detected is Αβ (11-38) (SEQ ID No : 19). In a further particular embodiment, the oiigomeric state of a target Αβ peptide to be detected is at least one Αβ peptide selected from SEQ ID NOs: 13 to 24, wherein the glutamate residue at the N-terminus of these peptides is cyclized to pyroglutamate. In a further particular embodiment, the oiigomeric state of a target Αβ peptide to be detected is at least one Αβ peptide selected from SEQ ID Nos. 1 to 6, wherein the aspartate residues at amino acid positions 1 and/or 7 are converted to isoasparate. Even particularly, the oligomeric state of a target Αβ peptide to be detected is at least one Αβ peptide selected from SEQ ID Nos. 7 to 12, wherein the aspartate residue at amino acid position 6 is converted to isoasparate.
Even particularly, the oligomeric state of a target Αβ peptide to be detected is at least one Αβ peptide selected from SEQ ID Nos. 13 to 18, wherein the aspartate residue at amino acid position 5 is converted to isoasparate.
It will be appreciated that the method of determining the oligomeric state of Αβ constitutes a further aspect of the invention related to a novel and inventive assay which is not necessarily limited to the diagnosis of a neurodegenerative disorder such as Alzheimer's disease. Thus, according to a second aspect of the invention there is provided a method of determining the oligomeric state of a target amyloid β peptide (Abeta or Αβ) in a biological sample which comprises the following steps:
(a) determining a first concentration (ca) of a target Αβ peptide in a biological sample;
(b) disaggregating the target Αβ peptide from step (a);
(c) determining a second concentration (cd) of the disaggregated Αβ peptide; and
(d) determining the ratio of cd / ca, wherein the value of the second concentration (cd) is divided by the value of the first concentration ca;
wherein a ratio of cd / ca, which is in excess of 1, is indicative of the presence of oligomeric Αβ.
In one embodiment, the disaggregation step (b) comprises the use of an alkali as hereinbefore defined .
I n o n e e m bo d i m e nt, th e m eth o d of d ete rm i n i n g th e fi rst a n d se co n d concentration of a target Αβ peptide in steps (a) and (c) comprise:
i) contacting a biological sample with at least two different capture antibodies, ii) detection of the resulting immune complex,
iii) destruction of the immune complex, and,
iv) quantifying the captured Αβ peptides. In one embodiment, the quantifying step (iv) comprises analysis in an Αβ specific ELISA. In a further embodiment, the Αβ specific ELISA is a sandwich-ELISA. In one embodiment, steps (a) and (c) both comprise analysis with an Αβ specific ELISA. Th is embod iment provides the advantage of allowing comparability between the first and second concentrations obtained in steps (a) and (c).
In one embodiment, the biological sample is selected from the group consisting of blood, serum, urine, cerebrospinal fluid (CSF), plasma, lymph, saliva, sweat, pleural fluid, synovial fluid, tear fluid, bile and pancreas secretion. In a further embodiment, the biological sample is plasma.
The biological sample can be obtained from a patient in a manner well-known to a person skilled in the art. In particular, a blood sample can be obtained from a subject and the blood sample can be separated into serum and plasma by conventional methods. The subject, from which the biological sample is obtained is suspected of being afflicted with Alzheimer's disease, at risk of developing Alzheimer's disease and/or being at risk of or having any other kind of dementia.
In particular, it is a subject suspected of having Mild Cognitive Impairment (MCI) and/or being in the early stages of Alzheimer's disease.
The present method has several advantages over the methods known in the art, i.e. the method of the present invention can be used to detect Alzheimer's disease at an early stage and to differentiate between Alzheimer's disease and other types of dementia in early stages of disease development and progression. One possible early stage is Mild Cognitive Impairment (MCI). It is impossible with the methods currently known in the art to make a clear and reliable diagnosis of early stages of Alzheimer's disease and , i n particu l a r, it is i m possi ble to d ifferentiate between the onset of Alzheimer's d isease and other forms of dementia in said early stages. This especially applies for patients afflicted with MCI.
In contrast, the methods provided by the present invention are suitable for a differential diagnosis of Alzheimer's disease. In particular, the present invention provides a method, wherein the oligomeric state of target Αβ peptides can be detected in biological samples obtained from any of the above described subjects in a highly reproducible manner. The high reproducibility of the methods of the present invention is achieved by using at least two different capture antibodies in an initial immune-precipitation step (step (a)) which is identical to the process subsequently used in step (c). In one embodiment, these at least two different capture antibodies are directed to different epitopes of the Αβ target peptide.
In one embodiment, the biological sample is plasma.
The above-mentioned "Αβ target peptide" encompasses Αβ (x-y) as hereinbefore defined .
A specific problem, which had to be overcome by the present invention, is that the biomarker to be used is altered in early stages of Alzheimer's disease, e.g . during mild cognitive impairment. The inventors of present invention have shown that it is possible to determine the oligomeric state of target Αβ peptides, in a reliable manner, and, it also became clear for the first time that in fact the oligomeric state of Αβ (x-y) is particularly suitable for the diagnosis of early onset Alzheimer's disease.
The method of determining the first and second concentration of a target Αβ peptide in steps (a) and (c) specifically comprises the following steps: i) Contacting a biological sample with at least two different capture antibodies in an immunoprecipitation step. After contacting the biological sample with the aforementioned at least two different capture antibodies, an immune complex will form between the at least two different capture antibodies and the target Αβ peptides. This step does not act for specific isolation of full length Αβ (x-y) wherein x would be 1, rather than capturing and separating all Αβ species, especially ending at position 38, 40 and/or 42. ii) This complex is then detected by secondary antibodies. Suitably, the secondary antibodies are immobilized on magnetic beads. Together with the magnetic beads the immune complex can be easily separated from the body fluid (plasma/serum CSF etc.) using the magnetic separator. iii) The immune complex is eluted from the beads. Suitably, the elution step is performed by incubating the beads carrying the immune complex in a solution comprising 50 % Methanol / 0.5 % formic acid for lh at room temperature. Thereby, all intermolecular interactions are destructed and all Αβ peptide molecules, which were isolated from the biological sample, are released from the beads in the solution. iv) The released, isolated Αβ peptide will be quantified in a subsequent step, for example by a sandwich ELISA that specifically detects full length Αβ (x-y), wherein full length Αβ (x-y) in this step most suitably means Αβ (1-40) and Αβ (1-42).
Possible antibodies for immunoprecipitation, which would be suitable in the present context, are the following, although the present invention is not delimited to those specific working examples:
3D6, Epitope: 1-5 (Elan Pharmaceuticals, Innogenetics)
pAb-EL16, Epitope: 1-7
2H4, Epitope: 1-8 (Covance)
1E11, Epitope: 1-8 (Covance)
20.1, Epitope: 1-10 (Covance, Santa Cruz Biotechnology)
Rabbit Anti-Αβ Polyclonal Antibody, Epitope: 1-14 (Abeam) AB10, Epitope: 1-16 (Chemicon/Upstate - part of Millipore)
82E1, Epitope: 1-16 (IBL)
pAb 1-42, Epitope: 1-11
NAB228, Epitope: 1-11 (Covance, Sigma-Aldrich, Cell Signaling, Santa
Cruz
Biotechnology, Zymed/Invitrogen)
DE2, Epitope: 1-16 (Chemicon/Upstate - part of Millipore)
DE2B4, Epitope: 1-17 (Novus Biologicals, Abeam, Accurate, AbD Serotec)
6E10, Epitope: 1-17 (Signet Covance, Sigma-Aldrich)
10D5, Epitope: 3-7 (Elan Pharmaceuticals)
WO-2, Epitope: 4-10 (The Genetics Company)
1A3, Epitope 5-9 (Abbiotec)
pAb-EL21, Epitope 5-11
310-03, Epitope 5-16 (Abeam, Santa Cruz Biotechnology)
Chicken Anti-Human Αβ Polyclonal Antibody, Epitope 12-28 (Abeam)
Chicken Anti-Human Αβ Polyclonal Antibody, Epitope 25-35 (Abeam)
Rabbit Anti-Human Αβ Polyclonal Antibody, Epitope: N-terminal (ABR)
Rabbit Anti-Human Αβ Polyclonal Antibody (Anaspec)
12C3, Epitope 10-16 (Abbiotec, Santa Cruz Biotechnology)
16C9, Epitope 10-16 (Abbiotec, Santa Cruz Biotechnology)
19B8, Epitope 9-10 (Abbiotec, Santa Cruz Biotechnology)
pAb-EL26, Epitope: 11-26
BAM90.1, Epitope: 13-28 (Sigma-Aldrich)
Rabbit Anti-beta-Amyloid (pan) Polyclonal Antibody, Epitope: 15-30 (MBL) 22D12, Epitope: 18-21 (Santa Cruz Biotechnology)
266, Epitope: 16-24 (Elan Pharmaceuticals)
pAb-EL17; Epitope: 15-24
4G8, Epitope: 17-24 (Covance)
Rabbit Anti-Αβ Polyclonal Antibody, Epitope: 22-35 (Abeam)
G2-10; Epitope: 31-40 (The Genetics Company)
Rabbit Anti-Αβ, aa 32-40 Polyclonal Antibody (GenScript Corporation)
EP1876Y, Epitope: x-40 (Novus Biologicals)
G2-11, Epitope: 33-42 (The Genetics Company) 16C11, Epitope: 33-42 (Santa Cruz Biotechnology)
21F12, Epitope: 34-42 (Elan Pharmaceuticals, Innogenetics)
1A10, Epitope: 35-40 (IBL)
D-17 Goat anti-Α β a n t i b o d y , E p i to p e : C-te rm i n a l ( Sa nta Cruz Biotechnology)
Particular antibodies for the immunoprecipitation are: 3D6 (Elan), BAN50 (Takeda), 82E1 (IBL), 6E10 (Covance), WO-2 (The Genetics Company), 266(Elan), BAM90.1 (Sigma), 4G8 (Covance), G2-10 (The Genetics Company), 1A10 (IBL), BA27 (Takeda), 11A5-B10 (Millipore), 12F4 (Millipore), 21F12 (Elan).
Examples for ΑβΝ3ρΕ specific antibodies are:
- the Pyro-Glu Abeta antibodies Αβ 5-5-6 (Deposit No. DSM ACC
2923), Αβ 6-1-6 (Deposit No. DSM ACC 2924)
Αβ 17-4-3 (Deposit No. DSM ACC 2925) and Αβ 24-2-3 (Deposit No. DSM
ACC 2926), which are described in PCT/EP2009/058803 (monoclonal, mouse), Probiodrug AG
Pyro-Glu Abeta antibody clone 2-48 (monoclonal , mouse) ; Synaptic
Systems
- Pyro-Glu Abeta antibody (polyclonal, rabbit); Synaptic Systems
Pyro-Glu Abeta antibody clone 8E1 (monoclonal, mouse); Anawa
Pyro-Glu Abeta antibody clone 8E1 (monoclonal, mouse); Biotrend
- Anti-Human Amyloidβ (N3pE) Rabbit IgG (polyclonal, rabbit); IBL
- Anti- Human Αβ N3pE (8E1) Mouse IgG Fab (monoclonal, mouse); IBL
Examples for Αβ isoAsp 1 specific antibiodies are :
- anti-human Αβ isoAsp 1 antibody (polyclonal, rabbit); disclosed in Saido TC, et al., Neurosci Lett. (1996) 13; 215(3) : 173-6.
Particular antibody pairs for the immunoprecipitation are:
4G8 and 11A5-B10, 3D6 and 4G8, 6E10 and 4G8, 82E1 and 4G8, 4G8 and 12F4, 4G8 and 21F12, 3D6 and 21F12, 6E10 and 21F12, BAN50 and 4G8, 3D6 and 11A5-B10, 3D6 and 1A10, 3D6 and BA27, 6E10 and 11A5-B10, 6E10 and 1A10, 6E10 and BA27, 4G8 and 11A5-B10, 4G8 and 1A10, 4G8 and BA27, 4G8 and 12F4, 4G8 and 21F12.
Examples for ΑβΝ3ρΕ specific antibodies are:
- the Pyro-Glu Abeta antibodies Αβ 5-5-6 (Deposit No. DSM ACC
2923), Αβ 6-1-6 (Deposit No. DSM ACC 2924)
Αβ 17-4-3 (Deposit No. DSM ACC 2925) and Αβ 24-2-3 (Deposit No. DSM
ACC 2926), which are described in PCT/EP2009/058803 (monoclonal, mouse), Probiodrug AG
- Pyro-Glu Abeta antibody clone 2-48 (monoclonal, mouse); Synaptic
Systems
- Pyro-Glu Abeta antibody (polyclonal, rabbit); Synaptic Systems
Pyro-Glu Abeta antibody clone 8E1 (monoclonal, mouse); Anawa
Pyro-Glu Abeta antibody clone 8E1 (monoclonal, mouse); Biotrend
- Anti-Human Amyloidβ (N3pE) Rabbit IgG (polyclonal, rabbit); IBL
- Anti- Human Αβ N3pE (8E1) Mouse IgG Fab (monoclonal, mouse); IBL
Examples for Αβ isoAsp 1 specific antibiodies:
- anti-human Αβ isoAsp 1 antibody (polyclonal, rabbit); Saido et al., 1996).
Apart from the above designated antibodies all other amyloid beta specific antibodies (monoclonal and polyclonal), which are suitable for immunoprecipitation can be used in the concentration determining method (further suitable antibodies can e.g. be taken from www.alzforum.org). Decisive for good capture efficiency is the use of two, three or more different antibodies with different epitopes. The use of more than one antibody type for immunoprecipitation of Αβ peptides offers cooperative and surprisingly synergistic binding effects (avidity), which finally allows to achieve a tremendously higher capture efficiency (see Figure 1).
The secondary antibodies in step ii) are specific against the host antibody type of the capture antibodies. Suitable secondary antibodies are anti-mouse antibodies and anti-rabbit antibodies. After incubation of the complex with the magnetic beads in step iii), the beads may be washed with washing buffer (see examples of the present invention). Washing buffers, which contain detergents or other additives preventing unspecific binding, can be used for this step. Non-limiting examples of washing buffers are:
- D-PBS containing 10 mg/ml Cyclophilin 18 (Cyp 18) and 0.05 % Tween- 20,
- PBS + 0.05 % Tween-20,
- TBS + 0.05 % Tween-20,
- PBS + 1 % (w/v) BSA + 0.05 % Tween-20,
- TBS + 1 % (w/v) BSA + 0.05 % Tween-20, and
- Pierce ELISA Blocker (with Tween-20). After elution of the immune complex from the beads in step iv), the solution is diluted in dilution buffer. Any dilution buffers, which can prevent unspecific interaction with surfaces and the immobilized first ELISA antibody can be used for this step. Non-limiting examples for dilution buffers are:
- EIA buffer (dilution buffer of the IBL 1-40 (N) ELISA Kit),
- PBS + 1 % (w/v) BSA + 0.05 % Tween-20,
- TBS + 1 % (w/v) BSA + 0.05 % Tween-20, and
- Pierce ELISA Blocker (with Tween-20).
ELISA-Kits that a re a ble to q ua ntify fu l l len gth Αβ ( 1-40) are commercially available. Suitable ELISA-Kits for the quantification of Αβ ( 1-40) in the methods of the present invention are for example : Amyloid-β ( 1-40) ( N) ELISA (IBL, J P27714) ; Αβ [ 1-40] H uman ELISA Kit (Invitrogen ) ; H uman Amyloid beta (Amyloid-β), aa 1-40 ELISA Kit (Wako Chemicals USA, Inc.); Amyloid Beta 1-40 ELISA Kit (The Genetics Company).
ELISA-Kits that are able to quantify full length Αβ ( 1-42) are also commercially available. Suitable ELISA-Kits for the quantification of Αβ ( 1-42) in the methods of the present invention are for example : Amyloid-β ( 1-42) (N) ELISA (IBL, JP27712); Αβ [1-42] Human ELISA Kit (Invitrogen), Human Amyloid beta (Amyloid-β), aa 1-42 ELISA Kit (Wako Chemicals USA, Inc.), Amyloid Beta 1-40 ELISA Kit (The Genetics Company), INNOTEST® β- AMYLOID (1-42) (Innogenetics).
The concentration determining method is not limited to the exemplary aforementioned commercially available ELISA-Kits for Αβ (1-40) or Αβ (1-42). Numerous further sandwich ELISAs for full length Αβ (1-40) or Αβ (1-42) may be available in the prior art or may be developed by the skilled artisan. All these full length Αβ 1-40 or Αβ 1-42 sandwich ELISAs shall also be encompassed by the concentration determining method and should typically comprise a suitable pair of capture and detection antibodies, which are specific for the complete N- terminus of Αβ (1-40) and/or Αβ (l-42)and the C-terminus ending at amino acid 40 or 42, respectively.
Such a full length Αβ (1-40) sandwich ELISA may comprise a first immobilized antibody recognizing specifically the C-terminus of Αβ (1-40) and a second labeled detection antibody recognizing specifically the complete N-terminus of Αβ (1-40).
A full length Αβ (1-42) sandwich ELISA may comprise a first immobilized antibody recognizing specifically the C-terminus of Αβ (1-42) and a second labeled detection antibody recognizing specifically the complete N-terminus of Αβ (1-42).
A full length Αβ (1-40) sandwich ELISA may also comprise a first immobilized antibody recognizing specifically the complete N-terminus of Αβ (1-40) and a second labeled detection antibody recognizing specifically the C-terminus of Αβ (1-40).
A full length Αβ (1-42) sandwich ELISA may also comprise a first immobilized antibody recognizing specifically the complete N-terminus of Αβ (1-42) and a second labeled detection antibody recognizing specifically the C-terminus of Αβ (1-42).
Suitable Αβ (1-40/42) N-terminal specific antibodies for use in the concentration determining method are for example 3D6 (Elan), WO-2 (The Genetics Company), 82E1 (IBL), BAN-50 (Ta ked a ) . N u merous fu rther Αβ ( 1-40/42 ) N-terminal specific antibodies may be available in the prior art or may be developed by the skilled artisan . All these Αβ ( 1-40/42) N-terminal specific antibodies are also envisaged for the concentration determining method.
Suitable Αβ ( 1-40) C-terminal specific antibodies are for example G2-10 (The Genetics Company); 11A5-B10 (Millipore); 1A10 (IBL); BA27 (Takeda); EP1876Y (Novus Biologicals). Numerous further Αβ ( 1-40) C-terminal specific antibodies may be available in the prior art or may be developed by the skilled artisan . All these Αβ ( 1-40 ) C-terminal specific antibodies are also envisaged for the concentration determining method .
Suitable Αβ ( 1-42) C-terminal specific antibodies are for example G2-11 (The Genetics Company); 12F4 (Millipore); Anti- Human Αβ(38-42) Rabbit IgG (IBL); 21 F12 (Elan); BC05 (Takeda); 16C11 (Santa Cruz Biotechnology) . Numerous further Αβ (1-42) C-terminal specific antibodies may be available in the prior art or may be developed by the skil led artisan . Al l these Αβ ( 1-42 ) C-terminal specific antibodies are envisaged for the concentration determining method . According to one embodiment, the detection antibodies are labeled .
For diagnostic applications, the detection antibody will typically be labeled with a detectable moiety. Numerous labels are available which can be generally grouped into the following categories:
(a) Radioisotopes, such as 35S, 14C, 125I, 3H, and 131I. The antibody can be labeled with the rad ioisotope using the techniq ues described in Current Protocols in Immunology, Vol umes 1 and 2, Gutigen et al . , Ed . , Wiley-Interscience. New York, New York. Pubs., (1991) for example and radioactivity can be measured using scintillation counting .
(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.
(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 colour 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 electronical ly 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 incl ude l uciferases (e. g , firefly luciferase and bacterial luciferase; U.S. Patent No, 4,737,456), luciferin, 2,3- dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase. Ogalactosidase, 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 a/., 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).
Examples of enzyme-substrate combinations include, for example: (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));
(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate; and
(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g. p- nitrophenyl-6-D-galactosidase) or the fluorogenic substrate 4- methylumbelliferyl-6-D-galactosidase.
Numerous other enzyme-substrate combinations are available to those skilled in the art.
(d) Another possible label for a detection antibody is a short nucleotide sequence. The concentration is then determined by a RT-PCR system (Imperacer™, Chimera Biotech).
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.
The antibodies used in 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). 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 Αβ 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 bou nd to the anti bod ies may conven iently be separated from the standard and analyte which remain unbound . For the analysis of the Αβ (1-40) concentration in human all of the following body fluids can be used : blood, cerebrospinal fluid (CSF), urine, lymph, saliva, sweat, pleural fluid, synovial fluid, aqueous fluid, tear fluid, bile and pancreas secretion.
The novel method was established by the present inventors using blood samples (see the examples of the present invention). The present method is however not to be construed to be limited to blood samples. The method can also be employed using CSF, brain extract and urine samples, as well as all other human body fluids, e.g . the above mentioned in the same manner. Particular samples include plasma samples.
For immunohistochemistry analyses, the tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example. It will be appreciated that although the sandwich ELISA system comprises one particular embodiment for determining Αβ concentration in steps (a) and (c) of the invention, other concentration determining methods may be used .
Suitable alternative methods for determining the concentration of Αβ are: 1. Amyloid β 1-40 HTRF® Assay (CisBio Bioassavs) :
This assay principle is based on TR-FRET, which is a combination of Time- Resolved Fluorescence and Forster Resonance Energy Transfer. Similar to the usual sandwich ELISA the Αβ (1-40) is bound by two antibodies; the antibodies are here, however, not bound on a surface, the interaction occurs in solution. Both antibodies are labeled with a fluorophor. When these two fluorophors are brought together by a biomolecular interaction a portion of energy captured by the donor fluorophor during excitation is transferred via FRET to an acceptor fluorophor, which will be excited as a result. The fluorescence of the acceptor fluorophor is measured . The measuring signal is correlated with the amount of FRET and thus, the amount of Αβ (1-40) in solution.
Similarly, based on a comparable principle, the Alphascreen™ Assay from Lilly can be used .
2. Multiplex Assay Systems
Multiplex Assay Systems are available from several manufacturers and are well known and broadly used in the field . A suitable example for use in the methods of the present invention is the INNO-BIA plasma Αβ forms assay (Innogenetics). This assay is a well standardized multiparameter bead-based immunoassay for the simultaneous quantification of human β-amyloid forms Αβ (1-42) and Αβ (1- 40) or Αβ(Χ-42) and Αβ(Χ-40) in plasma using xMAP® technology (xMAP is a registered trademark of Luminex Corp.).
This assay system is able to quantify up to 100 different analytes in parallel. The basis of this method are small spherical polystyrol particles, called microspheres or beads. In analogy to ELISA and Western Blot these beads serve as a solid phase for the biochemical detection. These beads are colour-coded, so that 100 different bead classes can be distinguished . Every bead class has one specific antibody (e.g . against Αβ (1-40)) immobilized on the microsphere surface. If the Αβ ( 1-40) concentration increases more peptide molecules will be bound by the beads of this class. The detection of the binding of the analyte is carried out by a second anti-Αβ ( 1-40) antibody, which is labeled with another fluorescence dye, emitting green light. The sample is handled comparable to FACS analysis. The microspheres are singularized by hydrodynamic focusing and analyzed by laser- based detection system, which can make a quantification on the basis of the green fluorescence and identify the bound analyte by the specific coloration of the bead . Thus, it is possible to determine the concentration of multiple analytes in one sample.
3. Quantification by mass spectrometry
- For quantification of Αβ (1-40) also the SELDI-TOF mass spectrometry was used (Simonsen et al. , 2007 (2)).
- Quantitative a na lysis of Αβ peptides using i mmu noprecipitation and MALDI-TOF mass spectrometry. 15N labeled standard Αβ peptides are used for calibration. (Gelfanova et a/., 2007).
4. Western Blot analysis
2D-Gel electrophoresis cou pled with Western Blot analysis may be a suitable method to quantify Αβ peptides (Sergeant et al., 2003; Casas et al., 2004).
Diagnostic Kits
As a matter of convenience, the antibodies used in the method 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.
Thus, according to a further aspect of the invention there is provided a kit for diagnosing a neurodegenerative disorder, such as Alzheimer's disease which comprises a suitable alkali and instructions to use said kit in accordance with the methods defined herein.
In one embodiment, the kit additionally comprises at least two different capture antibodies as defined herein.
Where the antibody is labeled 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 l ike . 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.
The diagnostic kit of the invention is especially useful for the detection and diagnosis of neurodegenerative disorders, such as amyloid-associated diseases and conditions, e.g. Alzheimer's disease. Uses
The method of the present invention makes it possible for the first time to detect and quantify oligomeric target Αβ peptides, in particular Αβ (1-40), Αβ (1-42), Αβ (3-38) and/or Αβ (11-38), or a functional equivalent thereof, in a reliable manner. In particular, the present invention provides oligomeric Αβ (1-40), Αβ (1-42), Αβ (3-38) and/or Αβ (11-38) as a plasma biomarker, which is suitable for a differential diagnosis of Alzheimer's disease, in particular in the early stages of the disease.
Therefore, in one embodiment, the invention is directed to the use of the method of determining the oligomeric state of amyloid β peptide for the diagnosis of Alzheimer's disease, such as the differential diagnosis of Alzheimer's disease, in pa rticu l ar i n the early stages of the d isease . Su ita bly, the ea rly stage of Alzheimer's disease is Mild Cognitive impairment. In a further embodiment, the invention is directed to the use of the oligomeric Αβ target peptides for the diagnosis of Alzheimer's diseases, such as the differential diagnosis of Alzheimer's disease, in particular in the early stages of the disease. Suitably, the early stage of Alzheimer's disease is Mild Cognitive impairment. In particular, the oligomeric Αβ target peptide, which shall be used for diagnosis of Alzheimer's disease, is detected and quantified with a method according to the present invention. In a further embodiment, the Αβ target peptide is Αβ (x-y), as hereinbefore defined, or a functional equivalent thereof.
The method of the invention also has industrial applicability to monitoring the efficacy of a g iven treatment of a neu rod egenerative d isorder, su ch as Alzheimer's disease. Thus, according to a further aspect of the invention there is provided a method of monitoring efficacy of a therapy in a subject having , suspected of having, or of being predisposed to, a neurodegenerative disorder, such as Alzheimer's disease, comprising determining the oligomeric state of a target amyloid β peptide (Abeta or Αβ) as defined herein in a biological sample from a test subject.
In one embodiment, the biological sample will be taken on two or more occasions from a test subject. In a further embodiment, the method additionally comprises comparing the level of the oligomeric state of a target amyloid β peptide (Abeta or Αβ) present in biological samples taken on two or more occasions from a test subject. In one embodiment, the method additionally comprises comparing the level of the oligomeric state of a target amyloid β peptide (Abeta or Αβ) present in a test sample with the amount present in one or more sample(s) taken from said subject prior to commencement of therapy, and/or one or more samples taken from said subject at an earlier stage of therapy. In one embodiment, the method additionally comprises comparing the level of the oligomeric state of a target amyloid β peptide (Abeta or Αβ) with one or more controls. The present invention is further described by the fol lowing examples, which should however by no means be construed to limit the invention in any way; the invention is defined in its scope only by the claims as enclosed herewith.
EXAMPLES OF THE INVENTION
1. Materials and Methods
1.1 Patients and healthy controls
Patients with a clinical diagnosis of AD and healthy controls were recruited through a CRO (GALMED GmbH). In a prestudy examination the neuropsychological functions of all participants of the study were tested by several psychometric tests (DemTect, Mini-Mental-State Test, Clock-drawing test). DemTect Test
The DemTect scale is a brief screening for dementia comprising five short subtests (10-word list repetition, number transcoding, semantic word fluency task, backward digit span, delayed word list recall) (Kessler et al. , 2000). The raw scores are transformed to give age- and education-independent scores, classified as 'suspected dementia' (score < 8), 'mild cognitive impairment' (score 9 - 12), and 'appropriate for age' (score 13 - 18).
MMSE
The Mini-Mental State Examination (MMSE) or Folstein test is a brief 30-point questionnaire test that is used to assess cognition (see Table 1). It is commonly used in medicine to screen for dementia. In the time span of about 10 minutes it samples various functions including arithmetic, memory and orientation. It was introduced by Folstein et a/. , 1975, and is widely used with small modifications. The MMSE includes simple questions and problems in a number of areas: the time and place of the test, repeating lists of words arithmetic, language use and comprehension, and basic motor skills. For example, one question asks to copy drawing of two pentagons (see next table). Any score over 27 (out of 30) is effectively normal . Below this, 20 -26 indicates mild dementia; 10 -19 moderate dementia, and below 10 severe dementia. The normal value is also corrected for degree of schooling and age. Low to very low scores correlate closely with the presence of dementia, although other mental disorders can also lead to abnormal findings on MMST testing . Table 1: Mini-Mental State Examination
Figure imgf000042_0001
Clock-Drawing Test
Scoring of the clocks was based on a modification of the scale used by Shulmann et ai, 1986. All circles were pre-drawn and the instruction to subjects was to "set the time 10 after 11". The scoring system (see Table 2) ranges in scores from 1 to 6 with higher scores reflecting a greater number of errors and more impairment. This scoring system is empirically derived and modified on the basis of clinical practice. Of necessity, it leaves considerable scope for individual judgment, but it is simple enough to have a high level of interrater reliability. Our study lends itself to the analysis of the three major components. These include cross-sectional comparisons of the clock-drawing test with other measures of cognitive function; a longitudinal description of the clock-drawing test over time, and the relationship between deterioration on the clock-drawing test and the decisions to institutionalize.
After Prestudy examination the study started 2 weeks later with blood withdrawal from all participants. Over one year with an interval of 3 months all participants had visited the center for the psychometric tests and blood samples withdrawal. The study was approved by the Ethics Committee of the "Arztekammer Sachsen- Anhalt". All patients (or their nearest relatives) and controls gave informed consent to participate in the study.
Table 2: Clock-drawing test
Figure imgf000044_0001
1.2 Blood samples
For the analysis of the Αβ 1-40 and /or Αβ 1-42 concentration in humans all of the following body fluids can be used : blood, cerebrospinal fluid, urine, lymph, saliva, sweat, pleura fl uid, synovial fl uid, aq ueous fluid , tear fl uid , bile and pancreas secretion.
The novel method was established with blood samples and can be further used for CSF, brain extract and urine samples, followed by all other human body fluids.
Blood samples for the determination of AD biomarkers were collected into three polypropylene tubes:
1. containing potassium-EDTA (Sarstedt Monovette, 02.1066.001) for EDTA plasma
2. containing Li-heparine (Sartstedt Monovette, 02.1065.001) for heparine plasma
3. blank (Sarstedt Monovette, 02.1063.001) for serum
All samples were collected by venous puncture or by repeated withdrawal out of an inserted forearm vein indwelling cannula. Blood was collected according to the time schedule (as described in section 1.1 above) . It was centrifuged at 1550 g (3000 rpm) for 10 min at 4°C to provide plasma. Plasma or serum was pipetted off, filled in one 5 ml polypropylene cryo-tube (Carl-Roth, E295.1) and stored frozen at -80°C . Sa m ples were centrifu ged with i n one hou r after blood withdrawal. The appropriate labelling of the plasma or serum tubes according to the study protocol was duty of the CRO.
1.3 Laboratory methods
Beside wild type Αβ 1-40 mutated variants can also be quantified by this method . The mutated variants comprise all amyloid beta peptides starting with amino acids Asp-Ala-Glu and ending with Gly-Val-Val . Mutated Αβ 1-40 examples:
Tottori, Flemish, Dutch, Italian, Arctic, Iowa (Irie et a/. , 2005) The Αβ 1-40 assay can be also used for other familial Alzheimer's disease, which offer mutations outside the Αβ 1-40 sequence producing the wild type Αβ 1-40. Following familial Alzheimer's disease examples are also suitable for the assay: Swedish, Austrian, French, German, Florida, London, Indiana, Australian (Irie et a/., 2005)
Beside wild type Αβ 1-42 also mutated variants can be quantified by this method . The mutated variants comprise all amyloid beta peptides starting with amino acids Asp-Ala-Glu and ending with Val-Ile-Ala. Mutated Αβ 1-42 examples:
Tottori, Flemish, Dutch, Italian, Arctic, Iowa (Irie et a/. , 2005)
The Αβ 1-42 assay can be also used for other familial Alzheimer's disease, which offer mutations outside the Αβ 1-42 sequence producing the wild type Αβ 1-42. Following familial Alzheimer's disease examples are also suitable for the assay: Swedish, Austrian, French, German, Florida, London, Indiana, Australian (Irie et a/., 2005)
Immunoprecipitation
EDTA plasma samples (containing 4 ml plasma) (heparin plasma, serum also possible) were thawed and aliquoted at 1 ml in 2 ml polypropylene tubes (Eppendorf, 0030120.094). One pill of protease inhibitor (Roche, Complete mini Protease inhibitor cocktail, 11836153001) was dissolved in 1 ml D-PBS (Invitrogen, 14190-094). 25 μΙ of the protease inhibitor solution was added to 1 ml EDTA plasma. All aliquots were frozen and stored again at -80°C, except one tube of each sample. These plasma tubes were spiked with 10 μΙ of 10 % Tween- 20. To ea ch tu be 2. 5 p g a nti-amyloid β (17-24) antibody 4G8 (Millipore, MAB1561), 2.5 pg anti-amyloid β (x-42) antibody 12F4 (Millipore, 05-831) and 2.5 pg anti-amyloid β (x-40) antibody 11A5-B10 (Millipore, 05-799) were added .
Other possible antibodies for immunoprecipitation are as defined hereinbefore. Beside these listed antibodies all other amyloid beta specific antibodies (monoclonal and polyclonal), which are suitable for immunoprecipitation can be used for this method (see also www.alzforum.org). Decisive for good capture efficiency is usage of two, three or more different antibodies with different epitopes. The usage of more than one antibody type for immunoprecipitation of Αβ peptides offer cooperative binding effects (avidity), which yield tremendously higher capture efficiency (see Figure 1). All plasma tubes were incubated overnight at 4°C in an overhead shaker. For immobilization of the amyloid β-antibody complex, 100 μΙ anti-mouse magnetic beads (Invitrogen, 112-02D) were used for a 1 ml plasma sample. Beside these special anti-mouse antibodies conjugated on mag netic beads al l other anti- mouse antibod ies or anti-host antibodies (host : origin of primary antibod ies listed above) can be used . These antibod ies can be immobilized on several matrices (col u m n matrices a nd bead matrices) via d ifferent conj ug atio n strategies, e.g. Biotin-Streptavidin interaction, tosyl-activated surface, epoxy- activated surface, amine-surface, carboxylic surface. Before usage, 100 μΙ beads were pipetted off from the original bottle into a 2 ml tube and washed 3-times with 1 ml PBS. After washing the beads were resuspended in 200 μΙ PBS. The plasma tubes were centrifuged for 30sec at 2000 x g . The supernatants were transferred into the tubes containing the anti-mouse magnetic beads. The tubes were incubated overnight at 4°C in an overhead shaker. On the next day the tubes were placed into a magnetic separator to allow the bead to be drawn to the tube wall . After about one minute the supernatant was ca refu l ly rem oved a n d the bea ds were washed twi ce with 500 μ Ι D-PBS containing 10 mg/ml Cyclophilin 18 and 0.05 % Tween-20. Other washing buffers, which contain detergents or other additives preventing unspecific binding can be used for this step. Examples for washing buffers are:
- PBS + 0.05 % Tween-20
- TBS + 0.05 % Tween-20
- Pierce ELISA Blocker (with Tween-20)
Elution and disaggregation of captured Amyloid β
After the last wash step, the solution was drawn out, the tubes were taken from the magnetic separator and 100 μΙ 50 % (v/v) Methanol / 0.5 % (v/v) formic acid were added to each tube and the beads were resuspended by slightly shaking . All tubes were incubated for 1 hour at room temperature. Afterwards the tubes were again placed in the mag netic separator and 40 μΙ eluate from each tube were mixed with 440 μΙ EIA buffer (dilution buffer of the IBL 1-40/42 (N) ELISA Kit). The pH of the diluted samples were adjusted with 16 μΙ 400 mM Na2HP04, 400 m M KH2P04 pH 8.0. From these samples the concentrations without disaggregation were determined . For disaggregation 50 μΙ eluate from each tube were transferred in new tubes and m ixed with 20 μ Ι 50 % (v/v) Methanol / 500 mM NaOH for every tube. The disaggregation was performed for 10 min at room temperature. Afterwards 40 μΙ from each disaggregation tube were mixed with 440 μΙ EIA buffer (dilution buffer of the IBL 1-40/42 (N) ELISA Kit). The pH of the diluted samples were adjusted with 10 μΙ 0.85 % (v/v) H3P04. From these samples the concentration after disaggregation were determined . Beside the special ELISA dilution buffer from manufacturer IBL all other dilution buffer, wh ich can prevent unspecific interaction with surfaces and capture antibodies, can be used for this step. Examples for dilution buffers are:
- PBS + 1 % (w/v) BSA + 0.05 % Tween-20
- TBS + 1 % (w/v) BSA + 0.05 % Tween-20
- Pierce ELISA Blocker (with Tween-20)
Quantification of the eluted amyloid β peptides
The determination of the peptide concentration (with and without disaggregation, respectively) was performed using the IBL 1-40(N) ELISA Kit (IBL, JP27714) and IBL 1-42(N) ELISA Kit (IBL, JP27712).
Beside this special Αβ 1-40 ELISA all other commercially available, which are able to detect full length Αβ 1-40 can be used.
Examples for commercially ELISA-Kits:
Human Abeta, aa 1-40 ELISA Kit Invitrogen
Human Amyloid beta (Amyloid-b), Wako Chemicals USA, Inc. (aa 1-40 ELISA Kit)
Amyloid Beta 1-40 ELISA Kit The Genetics Company Self made Αβ 1-40 ELISA comprise of a pair of capture and detection antibody, which are specific for the complete N-terminus of Αβ 1-40 and the C-terminus ending at amino acid 40.
Possible N-terminal specific antibodies are:
3D6 (Elan Pharmaceuticals)
WO-2 (The Genetics Company)
1-40(N) detection antibody (IBL)
BAN50 (Takeda Chemicals Industries)
Possible C-terminal specific antibodies:
G2-10 (The Genetics Company)
11A5-B10 (Millipore)
1A10 (IBL)
Rabbit Anti-beta-Amyloid, aa 32-40 Polyclonal Antibody (GenScript Corporation)
EP1876Y, Epitope: x-40 (Novus Biologicals) Such a self made fu l l len gth Αβ 1-40 sandwich ELISA can comprise a first immobilized antibody recognizing specifically the C-terminus of Αβ 1-40 and a second labeled detection antibody recog nizing specifical ly the complete N- terminus of Αβ 1-40. A full length Αβ 1-40 sandwich ELISA can also comprise a first immobilized antibody recognizing specifically the complete N-terminus of Αβ 1-40 and a second labeled detection antibody recog nizing specifically the C- term i n us of Αβ 1-40 , th is type of Αβ 1-40 sandwich ELISA is particularly envisaged .
Beside this special Αβ 1-42 ELISA all other commercially available, which are able to detect full length Αβ 1-42 can be used.
Examples for commercially ELISA-Kits:
Human Abeta, aa 1-40 ELISA Kit Invitrogen Human Amyloid beta (Amyloid-b), Wako Chemicals USA, Inc. (aa 1-42 ELISA Kit)
Amyloid Beta 1-42 ELISA Kit The Genetics Company beta-Amyloid 1-42 ELISA Kit (SIGNET) Covance
INNOTEST® 6- AMYLOID (1-42) Innogenetics
Self made Αβ 1-40 ELISA comprise of a pair of capture and detection antibody, which are specific for the complete N-terminus of Αβ 1-42 and the C-terminus ending at amino acid 40.
Possible N-terminal specific antibodies are:
3D6 (Elan Pharmaceuticals)
WO-2 (The Genetics Company)
1-40(N) detection antibody (IBL)
BAN50 (Takeda Chemicals Industries)
Possible C-terminal specific antibodies:
G2-11 (The Genetics Company)
16C11 (Santa Cruz Biotechnology)
21F12 (Elan Pharmaceuticals, Innogeneti
BC05 (Takeda Chemicals Industries)
Such a self made full length Αβ 1-42 sandwich ELISA can comprise a first immobilized antibody recognizing specifically the C-terminus of Αβ 1-42 and a second labeled detection antibody recog nizing specifical ly the complete N- terminus of Αβ 1-42. A full length Αβ 1-42 sandwich ELISA can also comprise a first immobilized antibody recognizing specifically the complete N-terminus of Αβ 1-42 and a second labeled detection antibody recog nizing specifically the C- term i n us of Αβ 1-42 , th is type of Αβ 1-42 sandwich ELISA is particularly envisaged .
The diluted samples (with and without disaggregation, respectively) were applied to the ELISA plate (100 μΙ per well, repeat determination). The ELISA standard were taken from the kit, dissolved and diluted according to the manufacture instruction protocol . After application of all samples and concentration standards the ELISA plate was incubated for 18 h at 4°C. On the following day, the ELISA was developed according to the manufacturers instruction protocol .
After stopping the colorimetric reaction the absorbance in each well was determined at 450 nm corrected by absorbance at 550 nm using a plate reader (TECAN Sunrise).
The determination of the standard cu rve was completed by plotting of the corrected absorbance at 450 nm versus the corresponding standard peptide concentration. The curve was fitted with the four-parameter equation (Equ. 1) using Origin 7.0 (Microcal).
Figure imgf000051_0001
wherein y represents the measured absorbance and x represents the corresponding concentration
The calculation of the Αβ (1-40) and Αβ ( 1-42) concentrations on ELISA of each sample was completed based on the according absorbance value using Equ. 2.
Figure imgf000051_0002
To determine the concentration in the plasma sample, determined without disaggregation, the calculated concentration was corrected by the EIA buffer dilution (including pH adjustment), factor 12.4, and the concentration effect ( 1 m l to 100 μ Ι ) of the i mm u nopreci pitation by factor 0. 1 . To determ ine the concentration in the plasma sample, determined with disaggregation, the calculated concentration was corrected by the EIA buffer dilution (including pH adjustment), factor 12.25, the dilution by adding 20 μΙ 50 % (v/v) Methanol / 500 mM NaOH to the eluted sample, factor 1.4, and the concentration effect (1 ml to 100 μΙ) of the immunoprecipitation by factor 0.1. The determined plasma Αβ ( 1-40/42) concentrations (with and without disaggregation, respectively) were denoted in pg/ml.
Calculation and Statistical analysis
For every plasma sample four parameters were determined :
1. Αβ (1-40) concentration (with disaggregation)
2. Αβ (1-40) concentration (without disaggregation)
3. Αβ (1-42) concentration (with disaggregation)
4. Αβ (1-42) concentration (without disaggregation)
From these data the ratio values for:
Oiigomeric state Αβ ( 1-40) = Αβ 1-40 (with disaggregation) / Αβ 1-40 (without disaggregation)
Oiigomeric state Αβ ( 1-42) = Αβ 1-42 (with disaggregation) / Αβ 1-42 (without disaggregation)
were calculated .
The association of plasma oiigomeric state of Αβ ( 1-40 ) an d Αβ ( 1-42) was examined with the existence of a positive clinical diagnosis of Alzheimer's disease using the Student 's t-Test.
2. Results
2.1 Demographic Characteristics
Overall 45 persons have participated in the study, 30 healthy controls and 15 AD patients. To observe possible influences of age on plasma Αβ, control persons were selected over a wide range of age and subclassified into three groups, Group I contains age of 18 to 30, Group II from 31 to 45 and Group III from 46 to 65. The demographic characteristics are shown in Table 3. Table 3 Demographic Characteristics
Figure imgf000053_0001
2.2 Psychometric tests
For eva l u ation of the neu ropsycho l og ica l fu nctions a l l pa rtici pa nts h ave performed the DemTect, Mini-Mental-State Test and Clock-Drawing test. These tests have been made in prestudy, 3 month, 6 month, 9 month and 12 month after the start of the study. DemTect Test
The raw scores are transformed to give age- and education-independent scores, classified as 'suspected dementia' (score < 8), 'mild cognitive impairment' (score 9 - 12), and 'appropriate for age' (score 13 - 18). The test results for all visits are shown in Figure 3. The results from Figure 3 demonstrate that there are clear differences between the three groups of healthy subjects compared with the patients. Mini-Mental-State Test
Any score over 27 (out of 30) is effectively normal . Below this, 20 -26 indicates mild dementia; 10 -19 moderate dementia, and below 10 severe dementia. The normal value is also corrected for degree of schooling and age. Low to very low scores correlate closely with the presence of dementia, although other mental disorders can also lead to abnormal findings on MMST testing . The test results are shown in Figure 4. The results from Figure 4 demonstrate that there are clear differences between the three g roups of healthy subjects compared with the patients.
Clock-Drawing Test
The scoring system ranges in scores from 1 to 6 with higher scores reflecting a greater number of errors and more impairment. This scoring system is empirically derived and modified on the basis of clinical practice. Of necessity, it leaves considerable scope for individual judgment, but it is simple enough to have a high level of interrater reliability.
Our study lends itself to the analysis of the three major components. These include cross-sectional comparisons of the clock-drawing test with other measures of cognitive function; a longitudinal description of the clock-drawing test over time, and the relationship between deterioration on the clock-drawing test and the decisions to institutionalize. The test results are shown in Figure 5. The results from Figure 5 demonstrate that there are clear differences between the three groups of healthy subjects compared with the patients.
2.3 Plasma oliqomeric state of Αβ (1-40) and Αβ (1-42)
The Αβ (1-40/42) concentrations (with and without disaggregation, respectively) were determined in EDTA plasma of the TO + 9 month series. Because of two serious adverse events, AD patient Nr. 34 and 35 were late, only 13 AD samples were unhanded by the CRO for investigations. Further samples of TO+9 series were used to optimize and establish the new immunoprecipitation method. Overall, the final optimized method was tested with 11 AD samples and 26 control samples. The determined concentrations are shown in Table 4. Table 4
Plasma oligomeric state of Αβ (1-40) and Αβ (1-42) (TO+9 month series)
The mean values and the standard error of mean for all four groups were calculated . The T-test has compared the AD group with each single control group. The values for the oligomeric state in table 4 were calculated according the methods of the present invention and represent the ratio Cd / ca.
Figure imgf000055_0001
Concerning the oligomeric state of Αβ (1-42) for all control groups, a significant increased value was obtained compared with the AD group. The same result was obtained by comparison of oligomeric state of Αβ ( 1-40) . Only group 18-30 has curtly missed the significance.
The oligomeric state of Αβ (1-40) and Αβ (1-42) of all control samples against all samples of the AD group (Figure 6) was also evaluated . The oligomeric state of Αβ ( 1-40 ) a n d Αβ ( 1-42), respectively, were sig nificantly decreased in AD patients compared with healthy controls. For the oligomeric state of Αβ ( 1-40) and Αβ (1-42) p-values of 0.0074 and 0.00067, respectively, were obtained . The used method cannot determine the amount of Αβ (1-40) or Αβ (1-42) homo- oligomers in the sample, it displays the amount of Αβ ( 1-40) and Αβ ( 1-42) peptides within soluble aggregates compared with monomeric Αβ (1-40) and Αβ (1-42) in the sample. Because of this fact the summation of the values for Αβ (1- 40) and Αβ ( 1-42) can reflect the overall amount of Αβ oligomers in plasma of AD patients and healthy controls (Figure 7). The summation of oligomeric state values has further improved the p-value of the T-test, although the sample quantity was less compared with single evaluation for Αβ (1-40) and Αβ (1-42), respectively.
3. Discussion
The results presented herein show that a decrease of the oligomeric state of Αβ 1-40 and Αβ 1-42 were associated with a positive clinical diagnosis of Alzheimer's Disease. The summation of both oligomeric states (Αβ 1-40 + Αβ 1-42) improves the significance (p = 1.41e-4). Until now, there exist only a few comparable studies in the literature. In one study they could show that the plasma level of proto-fibrillar Αβ42 declined over the follow-up in those who had developed mild AD by the second assessment (Schupf et al., 2008), what supports our data. However, the plasma levels of protofibrillar Αβ42 were only detectable in 34 % of all participants (1125 elderly persons). This fact constrains the usability of this assay, which uses a monoclonal antibody (clone 13C3) generated by immunization of mice with a fibrillar form of Αβ42. The characterization of the 13C3 antibody offers a good affinity to protofibrillar Αβ42, however also to monomeric Αβ42 (Schupf etal., 2008; supporting information), which can falsify the determined protofibrillar Αβ42 level. Therefore the usage of such assay systems based on oligomer or protofibrillar specific antibodies is hampered, if the detection antibody is not exclusively specific for higher molecular aggregates of Amyloid β. In another study (Xia et al., 2009) a sandwich ELISA used the same antibody for capture and detection for detection of oligomeric Αβ. Thus a detection is only possible if the Αβ assembly contains at least two exposed copies of the same epitope that is accessible by the identical capturing and detection antibody (El-Agnaf et al., 2000; Howlett et al., 1999). Xia and co-workers found an increased plasma level of oligomeric Αβ with a p-value < 0.05, which is contradictory to the findings presented herein. However also in this study only in 30% of healthy controls and in 52 % of AD patients oligomeric Αβ were detectable, which constrains the usability also of this assay. Both studies show the same problem, the aggregated amyloid β is not detectable in all samples. It is possible that the issue is caused by an inefficient and not rel ia ble recovery rate of amyloid beta by a si mple sandwich ELISA. In the invention the bivalent capture system ensures a complete recovery of al l Αβ molecules from the sample, which makes the present assay more reliable.
A very recent publication offers a method that uses also a indirect quantification of oligomeric amyloid β (Englund et al., 2009). This study analyzed CSF samples a nd q u a ntified the Αβ ( 1-42) l evel u n der denatu ri ng a nd n on-denaturing conditions and calculated the Αβ42 oligomeric ratio CSF samples. They found an increased ratio in samples of AD and MCI com pared with hea lthy controls . However, this assay is constrained by the usage of different methods for quantification of denatured and non-d en atu red Αβ42 . Fo r n o n-denatured condition the Αβ42 concentration is determined by a normal sandwich ELISA. As described above, such a simple sandwich ELISA could have problems with the recovery rate. For denatured conditions the Αβ42 concentration is determined by SDS-PAGE followed by Western Blot analysis. A critical issue of this method is the fact that Αβ ( 1-42) assemblies cannot completely disaggregate to monomer by 2 % SDS. Our experiences show also trimer and tetramer species of Αβ ( 1- 42) in SDS-PAGE. Against this background a correct quantification of Αβ (1-42) monomers is very doubtful . Furthermore this fact makes a comparison with ELISA determined concentration and subsequent the calculation of a ratio of both values very defective.
Until now, all published methods for quantification of amyloid β oligomers or protofibrils exh ibit critical issues, they a re on ly co nstricted appl ica ble for analyzing human plasma and CSF, respectively. The invention overcomes these issues and show reliable detection of Αβ aggregates in human plasma. REFERENCES
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Claims

1. A method of diagnosing or monitoring a neurodegenerative disorder, such as Alzheimer's d isease a nd Mi ld Cognitive Im pairment, which com prises determining the oligomeric state of a target amyloid β peptide (Abeta or Αβ) in a biological sample from a test subject, characterized in that said method comprises the following steps:
(a) determining a first concentration (ca) of a target Αβ peptide in a biological sample;
(b) disaggregating the target Αβ peptide from step (a);
(c) determining a second concentration (Cd) of the disaggregated Αβ peptide; and
(d) determining the ratio of cd / ca, wherein the value of the second concentration (cd) is divided by the value of the first concentration ca;
wherein a ratio of Cd / ca, which is lower than 1.5, is indicative of a positive diagnosis for a neurodegenerative disorder.
2. The method according to claim 1, wherein the disaggregation step (b) comprises the use of an alkali.
3. The method according to claim 2, wherein the alkali used for disaggregation in step (b) is sodium hydroxide, such as 500mM sodium hydroxide.
4. The method according to claim 2 or claim 3, wherein the disaggregation step (b) additionally comprises the use of a suitable solvent, such as methanol, particularly 50% (v/v) methanol.
5. The method according to any of the preceding claims, wherein the disaggregation step (b) comprises an incubation step.
6. The method according to claim 5, wherein the disaggregation step (b) comprises an incubation step at room temperature for at least 2 minutes.
7. The method according to claim 6, wherein the disaggregation step (b) comprises an incubation step at room temperature for at least 10 minutes.
8. The method according to any of the preceding claims, wherein a ratio of cd / Ca lower than 1.4, such as lower than 1.3 is indicative of a positive diagnosis for a neurodegenerative disorder.
9. The method according to claim 8, wherein a ratio of cd / ca lower than 1.2, such as lower than 1.1 is indicative of a positive diagnosis for a neurodegenerative disorder.
10. The method according to any of the preceding claims, wherein the target Αβ peptide comprises the amino acid sequence of Αβ (3-38) of SEQ ID NO. 13.
11. The method according to any of the preceding claims, wherein the target Αβ peptide comprises the amino acid sequence of Αβ (11-38) of SEQ ID NO. 19.
12. The method according to any one of the preceding claims, wherein the target Αβ peptide is Αβ (x-y) including functional equivalents thereof, wherein x is defined as an integer selected from x is defined as an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 and y is defined as an integer selected from 38, 39, 40, 41, 42 and 43.
13. The method according to claim 10, wherein x is an integer selected from 1, 2, 3 and 11, such as 1, in particular 11.
14. The method according to claim 10, wherein y is an integer selected from 38, 40 or 42, such as 40, in particular 38.
15. The method according to any one of the preceding claims, wherein the target Αβ peptide is selected from the group consisting of SEQ ID NOs. 1 to 24 including functional equivalents thereof.
16. The method according to any one of the preceding claims, wherein the target Αβ peptide is Αβ (1-40) of SEQ ID NO : 2 including functional equivalents thereof.
17. The method according to any one of claims 1 to 15, wherein the target Αβ peptide is Αβ (1-42) of SEQ ID NO : 1 including functional equivalents thereof.
18. The method according to any one of claims 1 to 15, wherein the target Αβ peptide comprises Αβ (1-40) of SEQ ID NO. 2 and Αβ (1-42) of SEQ ID NO. 1 including functional equivalents thereof.
19. The method according to any one of claims 1 to 15, wherein the target Αβ peptide is selected from the group consisting of SEQ ID NOs. 13 to 24 including functional equivalents thereof.
20. The method according to any one of claims 1 to 15 and 19, wherein the target Αβ peptide is Αβ (3-38) of SEQ ID NO: 13 including functional equivalents thereof.
21. The method according to any one of claims 1 to 15 and 19, wherein the target Αβ peptide is Αβ ( 11-38) of SEQ ID NO: 19 including functional equivalents thereof.
22. The method according to any one of claims 19 to 21, wherein the glutamate residue at the N-terminus of said target Αβ peptide is cyclized to pyroglutamate.
23. The method according to any one of claims 1 to 15, wherein the target Αβ peptide is selected from the group consisting of SEQ ID Nos. 1 to 6 including functional equivalents thereof, and wherein the aspartate residues at amino acid positions 1 and/or 7 are converted to isoasparate.
24. The method according to any one of claims 1 to 15, wherein the target Αβ peptide is selected from the group consisting of SEQ ID Nos. 7 to 12 including functional equivalents thereof, and wherein the aspartate residue at amino acid position 6 is converted to isoasparate,
25. The method according to any one of claims 1 to 15, wherein the target Αβ peptide is selected from the group consisting of SEQ ID Nos. 13 to 18 including functional equivalents thereof, and wherein the aspartate residue at amino acid position 5 is converted to isoasparate.
26. The method according to any one of the preceding claims, wherein the biological sample is selected from the group consisting of blood, serum, urine, cerebrospinal fluid (CSF), plasma, lymph, saliva, sweat, pleural fluid, synovial fluid, tear fluid, bile and pancreas secretion.
27. The method according to claim 26, wherein the biological sample is plasma.
28. The method according to any of the preceding claims, wherein the steps of determining the concentration of a target Αβ peptide comprise:
i) contacting a biological sample with at least two different capture antibodies,
ii) detection of the resulting immune complex,
iii) destruction of the immune complex, and,
iv) quantifying the captured Αβ peptides.
29. The method according to claim 28, wherein the at least two different capture antibodies are each specific for a different epitope on the Αβ peptide.
30. The method according to claim 28 or claim 29, wherein the capture antibodies are selected from the group consisting of
3D6, Epitope: 1-5,
pAb-EL16, Epitope: 1-7,
2H4, Epitope: 1-8,
1E11, Epitope: 1-8,
20.1, Epitope: 1-10, Rabbit Anti-Αβ Polyclonal Antibody, Epitope: 1-14 (Abeam),
AB10, Epitope: 1-16,
82E1, Epitope: 1-16,
pAbl-42, Epitope: 1-11,
NAB228, Epitope: 1-11,
DE2, Epitope: 1-16,
DE2B4, Epitope: 1-17,
6E10, Epitope: 1-17,
10D5, Epitope: 3-7,
WO-2, Epitope: 4-10,
1A3, Epitope 5-9,
pAb-EL21, Epitope 5-11,
310-03, Epitope 5-16,
Chicken Anti-Human Αβ Polyclonal Antibody, Epitope 12-28 (Abeam), Chicken Anti-Human Αβ Polyclonal Antibody, Epitope 25-35 (Abeam), Rabbit Anti-H uman Αβ Polyclonal Antibody, Epitope : N-terminal
(ABR),
Rabbit Anti-Human Αβ Polyclonal Antibody (Anaspec),
12C3, Epitope 10-16,
16C9, Epitope 10-16,
19B8, Epitope 9-10,
pAb-EL26, Epitope: 11-26,
BAM90.1, Epitope: 13-28,
Rabbit Anti-beta-Amyloid (pan) Polyclonal Antibody, Epitope: 15-30
(MBL),
22D12, Epitope: 18-21,
266, Epitope: 16-24,
pAb-EI_17; Epitope: 15-24,
4G8, Epitope: 17-24,
Rabbit Anti-Αβ Polyclonal Antibody, Epitope: 22-35 (Abeam)
G2-10; Epitope: 31-40,
Rabbit Anti-Α β , a a 3 2-40 Polycl ona l Anti body (Gen Scri pt Corporation),
EP1876Y, Epitope: x-40, G2-11, Epitope: 33-42,
16C11, Epitope: 33-42,
21F12, Epitope: 34-42,
1A10, Epitope: 35-40, and
D-17 Goat anti-Αβ antibody, Epitope: C-terminal.
pyro-Glu Abeta antibodies, Probiodrug AG
Αβ 5-5-6,
Αβ 6-1-6,
Αβ 17-4-3,
Αβ 24-2-3,
Pyro-Glu Abeta antibody clone 2-48 (monoclonal, mouse); Synaptic Systems,
Pyro-Glu Abeta antibody (polyclonal, rabbit); Synaptic Systems, Pyro-Glu Abeta antibody clone 8E1 (monoclonal, mouse); Anawa, Pyro-Glu Abeta antibody clone 8E1 (monoclonal, mouse); Biotrend, Anti-Human Amyloid (N3pE) Rabbit IgG (polyclonal, rabbit); IBL, Anti- Human Αβ N3pE (8E1) Mouse IgG Fab (monoclonal, mouse); IBL
isoAsp antibody (group of T. Saido)
31. The method according to any one of claims 28 to 30, wherein the capture antibodies are selected from the group consisting of 3D6, BAN50, 82E1, 6E10, WO-2, 266, BAM90.1, 4G8, G2-10, 1A10, BA27, 11A5-B10, 12F4, and 21F12.
32. The method according to any one of claims 28 to 31, wherein the following antibody pairs are used as capture antibodies:
4G8 and 11A5-B10,
3D6 and 4G8,
6E10 and 4G8,
82E1 and 4G8,
4G8 and 12F4,
4G8 and 21F12,
3D6 and 21F12,
6E10 and 21F12, BAN50 and 4G8,
3D6 and 11A5-B10,
3D6 and 1A10,
3D6 and BA27,
6E10 and 11A5-B10,
6E10 and 1A10,
6E10 and BA27,
4G8 and 11A5-B10,
4G8 and 1A10,
4G8 and BA27,
4G8 and 12F4, and
4G8 and 21F12.
33. The method according to any one of claims 30 to 32, wherein the detection of the complex is carried out by using secondary antibodies, specifically reacting with each capture antibody.
34. The method according to claim 33, wherein the secondary antibodies are anti-mouse antibodies or anti-rabbit antibodies.
35. The method according to claim 33 or 34, wherein the secondary antibodies are labeled.
36. The method according to any one of claims 33 to 34, wherein the secondary antibodies are immobilized on magnetic beads.
37. The method according to claim 36, wherein the magnetic beads carrying the immune complex are separated from the biological sample using a magnetic separator.
38. The method according to any one of claims 30 to 37, wherein the destruction of the immune complex is performed in the presence of 50 % (v/v) Methanol / 0.5 % (v/v) formic acid.
39. The method according to any one of claims 30 to 38, wherein the detected immune complex is quantified.
40. The method according to any one of claims 30 to 39, wherein the captured Αβ peptides are quantified by a quantification means selected from the group consisting of sandwich ELISA, Amyloid β 1-40 HTRF® Assay, Alphascreen™ Assay, Multiplex Assay Systems, mass spectrometry and Western Blot analysis.
41. The method according to claim 40, wherein the captured Αβ peptides are quantified by a sandwich ELISA as quantification means.
42. The method according to claim 41, wherein the sandwich ELISA comprises a first antibody, which is specific for the complete N-terminus of Αβ (x-y); and a detection antibody, which is specific for the C-terminus ending at amino acid y of Αβ (x-y).
43. The method according to claim 42, wherein the sandwich ELISA comprises a first antibody, which is specific for the complete N-terminus of Αβ (1-40) of SEQ ID NO. 2; and a detection antibody, which is specific for the C-terminus ending at amino acid 40 of Αβ (1-40) of SEQ ID NO. 2.
44. The method according to claim 42, wherein the sandwich ELISA comprises a first antibody, which is specific for the complete N-terminus of Αβ (1-42) of SEQ ID NO. 1; and a detection antibody, which is specific for the C-terminus ending at amino acid 42 of Αβ (1-42) of SEQ ID NO. 1.
45. The method according to claim 42, wherein the sandwich ELISA comprises a first antibody, which is specific for the C-terminus of Αβ ( 1-40) of SEQ ID NO. 2; and a detection a ntibody, which is specific for the complete N-terminus starting with Asp-Ala-Glu of Αβ (1-40) of SEQ ID NO. 2.
46. The method according to claim 42, wherein the sandwich ELISA comprises a first antibody, which is specific for the C-terminus of Αβ (1-42) of SEQ ID NO. 1; and a detection antibody, which is specific for the complete N-terminus starting with Asp-Ala-Glu of Αβ (1-42) of SEQ ID NO. 1.
47. The method according to claim 42, wherein the sandwich ELISA comprises a first antibody, which is specific for the complete N-terminus of an Αβ target peptide selected from the group consisting of SEQ ID NOs. 13 to 24; and a detection antibody, which is specific for the C-terminus of said Αβ target peptide selected from the group consisting of SEQ ID NOs. 13 to 24.
48. The method according to claim 42, wherein the sandwich ELISA comprises a first antibody, which is specific for the complete N-terminus of Αβ (3-38) of SEQ ID NO. 13; and a detection antibody, which is specific for the C-terminus ending at amino acid 38 of Αβ (3-38) of SEQ ID NO. 13.
49. The method according to claim 42, wherein the sandwich ELISA comprises a first antibody, which is specific for the complete N-terminus of Αβ ( 11-38) of SEQ ID NO. 19; and a detection antibody, which is specific for the C-terminus ending at amino acid 38 of Αβ ( 11-38) of SEQ ID NO. 19.
50. Method according to any of claims 47 to 49, wherein the N-terminus of the Αβ target peptide selected from the group consisting of SEQ ID NOs. 13 to 24 is cyclized to pyroglutamate and wherein the first antibody is specifically detecting the pyroglutamated form of said Αβ target peptide selected from the group consisting of SEQ ID NOs. 13 to 24
51. The method according to any one of claims 42 to 50, wherein the first antibody is immobilized.
52. The method according to any one of claims 42 to 51, wherein the detection antibody is labeled.
53. The method according to claim 42, wherein an ELISA-K i t fo r th e quantification of Αβ (x-y) is used.
54. The method according to claim 53, wherein the ELISA-Kit is an ELISA-Kit for the quantification of Αβ ( 1-40) of SEQ I D NO . 2selected from the group consisting of: Amyloid-β (1-40) (N) ELISA (IBL, J P27714) ; Αβ [ 1-40] Human ELISA Kit (Invitrogen) ; Human Amyloid beta (Amyloid-b) , a a 1-40 ELISA Kit (Wako Chemicals USA, Inc.); and Amyloid Beta 1-40 ELISA Kit (The Genetics Company).
55. The method according to claim 53, wherein the ELISA-Kit is an ELISA-Kit for the quantification of Αβ ( 1-42) of SEQ ID N . 1 selected from the grou p consisting of: Amyloid-β ( 1-42) (N ) ELISA (IBL, JP27712) ; Αβ [ 1-42] Human ELISA Kit (Invitrogen), Human Amyloid beta (Amyloid-β) , a a 1-42 ELISA Kit (Wako Chemicals USA, Inc.), Amyloid Beta 1-40 E LISA Kit (The Genetics Company), INNOTEST® β-AMYLOID (1-42) (Innogenetics).
56. The method according to any of the preceding claims, for the differential diagnosis of Alzheimer's disease.
57. The method according to any of claims 1 to 55 for the diagnosis of early stages of Alzheimer' disease.
58. The method according to claim 57 for the diagnosis of Mild Cognitive Impairment.
59. Use of an oligomeric Αβ peptide, such as the target Αβ peptide as defined in any of claims 10 to 25, for the diagnosis of Alzheimer's disease.
60. The use according to claim 59 for the differential diagnosis of Alzheimer's disease.
61. The use according to claim 59 or 60 for the diagnosis of early stages of Alzheimer' disease.
62. The use according to claim 61 for the diagnosis of Mild Cognitive Impairment.
63. A method of determining the oligomeric state of a target amyloid β peptide (Abeta or Αβ) in a biological sample which comprises the following steps:
(a) determining a first concentration (ca) of a target Αβ peptide in a biological sample;
(b) disaggregating the target Αβ peptide from step (a);
(c) determining a second concentration (cd) of the disaggregated Αβ peptide; and
(d) determining the ratio of cd / ca, wherein the value of the second concentration (cd) is divided by the value of the first concentration ca;
wherein a ratio of cd / ca, which is in excess of 1, is indicative of the presence of oligomeric Αβ.
64. The method according to claim 65, wherein the disaggregation step (b) comprises the use of an alkali.
65. An in vitro method for the diagnosis of Alzheimer's disease, wherein the method of determining the oligomeric state of amyloid β peptide according to claim 63 or claim 64 is used.
66. A kit for diagnosing a neurodegenerative disorder, such as Alzheimer's disease which comprises a suitable alkali and instructions to use said kit in accordance with the methods according to any one of claims 1 to 55.
67. A method of monitoring efficacy of a thera py in a subject having, suspected of having, or of being predisposed to, a neurodegenerative disorder, such as Alzheimer's disease, comprising determining the oligomeric state of a target amyloid β peptide (Abeta or Αβ) according to any one of claims 1 to 55 in a biological sample from a test subject.
68. A method of diagnosing or monitoring as defined in any one of claims 1 to 55 or 67, which comprises determining the oligomeric state of a target amyloid β peptide in a biological sample taken on two or more occasions from a test subject.
69. A method of diagnosing or monitoring as defined in claim 68, which comprises comparing the level of the oligomeric state of a target amyloid β peptide in the biological samples taken on two or more occasions.
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