HK1194468A - METHOD FOR DETECTING Aβ-SPECIFIC ANTIBODIES IN A BIOLOGICAL SAMPLE - Google Patents

Description

Method for detecting A beta-specific antibodies in biological samples

The present invention relates to a method for the detection of a β -specific antibodies in a biological sample, in particular in connection with Alzheimer's Disease (AD).

AD is a complex, progressive disorder involving interacting pathological cascades including amyloid- β aggregation and plaque formation in the brain, hyperphosphorylation of tau (hyperphosphorylation) and the formation of tangles within neurons. With the aggregation and hyperphosphorylation of these brain proteins, inflammatory processes contribute to loss of synaptic integrity and progressive neurodegeneration.

The conversion of amyloid beta peptide (A β or A-beta) from a soluble form with a predominantly alpha-helical or random coil secondary structure to an aggregated form with a beta-sheet secondary structure, which ultimately forms amyloid plaques in the brain, represents one of the first hallmarks of AD pathology. Several forms of a β, i.e., C-and N-terminally truncated or modified peptides, contribute to a β plaque formation in the brain. The three major C-terminal variants of A β include peptides A β 1-40 (consisting of 40 amino acids (aa), Val-40 being the last aa), A β 1-42, and A β 1-43. In addition to these major forms of C-terminal truncated peptides, there are other truncated forms that occur less frequently, namely A β 1-37, A β 1-38, and A β 1-39. The N-terminal variant of A β consists of A β 3-40/42/43 and A β 11-40/42/43. In all of these N-terminal truncations, glutamate occupies the first position. This aa is not stable but undergoes changes to establish pyroglutamic acid (pE), leading to the formation of A β p (E)3-40/42 and A β p (E) 11-40/42. The pE residues are formed either spontaneously or enzymatically by an enzyme called glutamyl cyclase.

Until recently, the diagnosis of AD was a complete clinical diagnosis based on the gradual onset of cognitive deficits in at least two areas (e.g., cognition, function) that negatively impacted the patient's daily life without another discernible cause (e.g., blood vessels). The limitations of clinical diagnosis of AD are the high rate of misdiagnosis (80% diagnostic specificity by experts) and the fact that a diagnosis can only be made at a late point in time when the disease has ceased to cause substantial neuronal loss of functional deficits.

The manner in which AD is diagnosed is rapidly changing based on knowledge gathered over the past 20 years. A group of researchers led by b.dubois (paris) integrated data for the first time, which had been derived from the investigation of the underlying pathology of AD, the emergence of diagnostic algorithms (Dubois et al, Lancet neurol.9(2010):1118 1127). According to the authors, the diagnosis of AD is based on specific cognitive deficits (changes in contextual memory) which must occur in combination with changes in disease-specific biomarkers (e.g. hippocampal atrophy assessed by structural MRI; cerebrospinal fluid signature (signature) typical of AD (low a42, high total Tau, high phosphate Tau); positive amyloid imaging; defined genetic risk). Recently, the NIH-NINCDS working group has largely adopted this pathophysiologically driven diagnostic algorithm (McKhann et al, Alzheimer's & Dementia7(2011): 263-269). For major practical purposes, the NIH NINCDS working group retained MCI (mild cognitive impairment) of the AD type as a diagnosis of early stages of AD.

The workgroup, designated by the National Institute of Aging (NIA, NIH, USA) and the Alzheimer's Association, has adopted the concept of enhancing the clinical diagnosis of AD by means of biomarkers reflecting the pathology of the disease. By doing so, AD is no longer diagnosed by exclusion, but begins to be a positive diagnosis. The fact that NIA and AA do not fully follow the biomarker-driven algorithm proposed by Dubois and coworkers reflects the limitations of currently available biomarkers. The AD cerebrospinal fluid (CSF) signature may be discussed as an example. The CSF of AD patients shows typical patterns, i.e. a decrease in Α β 1-42 and an increase in total tau (ttau) and tau phosphate (ptau).

The tag was present in AD patients, but no change was detected over time. Indeed, in a population of patients at risk for AD (i.e. MCI patients), it identifies subjects who continue to develop clinical symptoms. Already at this stage, CSF shows the same expression pattern and variation as in fully developed AD patients. Thus, while it is not possible to define the normal range of A β 1-42, tTau and pTau, no turning point is defined, i.e., the moment at which, for example, A β physiology is transformed from normal to pathological in a given patient. As is any biomarker currently being followed but not yet identified. The main reason for this is that there are only a few longitudinal studies evaluating this problem, since CSF, MRI, amyloid imaging examinations cannot be easily repeated due to their risks to the patient and/or the costs associated therewith.

As such, there remains a lack of reliable biomarkers that can be repeatedly applied at low risk and cost. This is particularly the case due to the failure of all the efforts hitherto expended to develop blood-based biomarkers (Hampel et al, nat. Rev. drug Discov.9(2010): 560-. The availability of such biomarkers would be of paramount importance for the development of disease modifying therapies. The earlier such therapies are administered, the greater the chance of success. Moreover, such efforts can be limited to true AD cases identified by means of specific biomarkers.

To date, a β (the various a β species and aggregate states tested) has been evaluated in AD and MCI (pre-dementing phase of AD). Recent findings have shown the presence of anti-amyloidogenic activity of IgG and IgM contained in plasma and cerebrospinal fluid of AD patients and healthy controls (O' Nuallain et al, J.Clin.Immunol.30(2010) Suppl.1: S37-S42; Marcello et al, J.neural.Transm.116 (2009): 913-920). Results obtained by ELISA or immunoprecipitation assays evaluating IgG and/or IgM specific for various a β forms/aggregation states show that AD and MCI patients exhibit lower levels of serum a β autoantibodies than healthy controls. Although these studies show differences in autoantibody concentrations, the methods used lack the sensitivity and specificity that would be necessary to use them as predictive diagnostic tools for identifying AD or MCI patients with high selectivity and specificity. Most of the methods used to date are based on ELISA techniques. To increase the sensitivity of these assays, some methods use radiolabeling of the A β 1-42 peptide. The difference between healthy controls and AD patients was measured using an immunoprecipitation assay with chloramine T labeled A β 1-42, and ROC (receiver operating characteristics) analysis of Brettschneider et al, (Brettschneider et al, biol. Psychiatyry 57(2005):813-817) achieved a specificity of 46.7% with the sensitivity set at 81.3%. In contrast, Bayer et al (WO2010/128139A1; Marcello et al, JNeural. Transm.116(2009):913-920) used an ELISA-based method in which pyroglutamic acid-A β fragments were coated on plates. In this case, detection of anti-a β -specific IgM-autoantibodies in healthy controls and AD patients with anti-IgM-HRP antibodies showed a specificity of 60% with the sensitivity set at 80%. To date, none of these approaches meet the criteria that would make them meet as predictive biomarkers (>80% specificity) for AD.

The reason for the reduced serum concentration of a β -specific antibodies in these two groups of patients is unknown. There are two general and non-mutually exclusive explanations: reduced production (disease specificity versus general immunosenescence) and redistribution (entrapment of antibodies in amyloid deposits present in, for example, the brain). Support for the potential function of these a β -homologous (auto) antibodies comes from recent studies demonstrating that commercial blood products, pooled IgG fractions extracted from plasma derived from healthy donors, have been shown to contain antibodies specific for a β peptides. Two such products, i.e. intravenous immunoglobulin (IVIG) preparations, from two different companies are currently being clinically tested to assess their potential to interfere with or prevent AD pathology.

Another unanswered question is whether the level of a β -specific antibodies is reduced in disease entities having an a β component in their pathophysiology, i.e. parkinson's dementia (PDD), dementia-associated lewy bodies (DLB), Cerebral Amyloid Angiopathy (CAA), chronic head trauma (e.g. boxing). Investigation of the levels of Α β aggregate-specific antibodies in these diseases may add to the current understanding of the processes that lead to a reduction in serum concentrations of Α β aggregate-specific antibodies in AD. Investigation of sera of patients with these diseases may clarify the question as to whether AD originates from a specific immunodeficiency (in the case where a β -specific antibody titers are reduced only in AD and not in other diseases characterized by a β pathology) or whether the reduced a β -specific antibody levels originate from redistribution due to extensive a β pathology. In the former case, the test would be a highly disease-specific biomarker and should therefore help to distinguish AD from the aforementioned disease entities. Given the second case, the test may be suitable for identifying patients whose disease is driven by Α β pathology.

In WO2010/128139a1, biomarkers and methods for diagnosing AD, in particular antibodies against pGlu a β, are disclosed. Funke et al (curr. alz. res.,6(3) (2009): 285-. Maetzler et al (J.Alz.Dis.26(1) (2011): 171-. Funke et al (Rejuv. Res.13(2-3) (2010):206-209) disclose a single particle detection system for A β aggregates. Fukumoto et al (Faeb J.,1(24) (2010):2716-2726) disclose that high molecular weight beta-amyloid oligomers are elevated in the cerebrospinal fluid of AD patients.

Although AD is considered a degenerative brain disease, the immune system also plays an important role in the disease process. In recent years, immune-based therapies have been designed to remove amyloid-beta peptide from the brain. These therapy concepts give positive results in animal models of disease. Clinical trials to interrupt active amyloid-beta vaccination in alzheimer's patients after some patients develop Meningoencephalitis (Meningoencephalitis). New immunotherapies using humoral and cell-based approaches are currently being investigated for the treatment and prevention of alzheimer's disease and also other related diseases. In such immunotherapeutic approaches, there is an unmet need for reliable biomarkers that monitor or demonstrate the immune response of patients during disease treatment and development. There is also a need to identify relevant a β antibodies indicative of the vaccination target in a vaccinated patient.

It is therefore an object of the present invention to provide means for the detection of anti-a β antibodies in a biological sample, in particular in a sample of a human AD patient or a human individual suspected of having or at risk of developing AD. A further object is to provide reliable tools for observing the development of clinical trials for drugs, especially immunotherapeutic interventions for the treatment of AD, and for observing patients treated with such immunotherapeutic interventions.

Accordingly, the present invention provides a method for detecting an a β -specific antibody in a biological sample, comprising the steps of:

-contacting the sample with a β aggregates or with particles having an a β aggregate-like surface and allowing a β -specific antibodies to bind to a β aggregates, and

-detecting a β -specific antibodies bound to a β aggregates by a single particle detection technique, preferably by fluorescence activated cell sorting FACS.

By virtue of the present invention, a novel method of detecting a β -specific antibodies is disclosed which can be used as a diagnostic tool which is very useful for monitoring the development of clinical trials using immunotherapeutic methods for the treatment of AD. The present method is based on the invention of not only using a single a β peptide as a capture tool for a β -specific antibodies, but instead using a β aggregates, and thereby detecting the resulting antibody-a β aggregate complexes using a single particle detection technique. Briefly, a β aggregates (derived from different a β truncated and modified versions) are generated, for example, by overnight incubation. Subsequently, Α β aggregates were incubated with serum samples derived from Healthy Donors (HD) or AD patients to allow binding of the present antibodies (both IgG and IgM). Antibodies bound to a β aggregates can be detected by any suitable method available to those skilled in the art, for example by using a labeled secondary antibody that recognizes a β -specific antibodies bound to a β aggregates. For example, a secondary antibody labeled with Phycoerythrin (PE) can be used. Thereafter, immune complexes comprising Α β -specific antibodies (and optionally one or more detection agents, such as secondary antibodies) bound to Α β aggregates are measured using a single particle detection technique, such as FACS (fluorescence activated cell sorting) analysis (also known as flow cytometry). Furthermore, using the method according to the invention it can be shown that the reactivity of a β -specific immunoglobulins derived from AD patients (directed against a β aggregates provided according to the invention) can be improved by a method called unmasking (removal of potentially bound a β antigen from autoantibodies). On the other hand, reactivity of IgM antibodies after unmasking (= dissociation of already bound a β in serum) reveals elevated IgM levels in AD patients. Furthermore, by virtue of the present invention, data is provided showing that the present method has a much higher capacity to detect a β -specific antibodies than the methods disclosed so far, and thus a much higher capacity to monitor changes in a β -specific antibody concentration in AD serum samples. In view of these facts, the method according to the invention satisfies the theoretical premise that a suitable method tracks the clinical response of a given patient to treatment.

The present invention was developed for the analysis of a β -specific antibodies in human samples. Thus, a preferred embodiment is the detection of human a β -specific antibodies, preferably human IgG or IgM antibodies, especially human IgG antibodies. As already mentioned, the detection of a β -specific antibodies in humans is in principle known in the art; however, the role as a possible biomarker cannot be demonstrated. This is also due to the analytical deficiencies of the detection methods available in the art, as demonstrated by the present invention. Due to the superiority of the method according to the invention, the availability of these antibodies as biomarkers in human samples is achieved, in particular for monitoring AD immunotherapy. Thus, the present invention is particularly suitable for detecting antibodies in biological samples. Thus, in a preferred embodiment of the method, the antibody specific for a β to be detected and quantified is an antibody.

In contrast to prior art methods, the present method uses a β aggregates as probes for binding a β -specific antibodies from a sample. Although such aggregates are in principle known in the art, it is not recognized that the use of such aggregates in the analysis of Α β -specific antibodies (especially in human samples) can significantly improve such methods also in combination with single particle detection techniques, such as FACS. Due to the use of such aggregates, detection with a single particle detection technique, which is a well established technique in a number of different fields and for different problems, is possible for analyzing a β -specific antibodies in human samples, such as blood, which are often very complex and difficult to handle.

Preferably, the size of the aggregates to be used according to the invention is standardized for analytical use. This can be done by establishing certain parameters during the generation of the aggregate. The size of the aggregates can be adjusted depending on the conditions applied during the generation of the aggregates. Preferred sizes of a β aggregates according to the invention are 50nm to 15 μm, preferably 100nm to 10 μm, especially 200nm to 5 μm (defined as the length (i.e. most often the extension) of the aggregates).

Preferred methods for providing aggregates suitable for the present invention comprise the step of incubating A β -1-42 peptide, A β -1-43 peptide, A β -3-42 or A β -p (E)3-42 peptide or less preferably C-terminally truncated A β peptide, such as A β -1-40 peptide, at a pH of 2 to 9 for at least 20 minutes, preferably at least 1 hour, especially at least 4 hours. The duration of incubation is one of the parameters that regulate the size of the aggregates: the longer the incubation, the larger the aggregates. Typical incubation times are 10 minutes to 24 hours. Shorter incubation times generally result in only very short aggregates and small amounts of aggregates; aggregates which are produced in the case of incubation times significantly longer than 48 hours are generally not preferred in the present method. Of course, aggregates can also be sorted and "screened" to achieve a desired size (if desired), such as by fractional centrifugation and similar techniques.

According to the method, a sample in which a β -specific antibodies are to be detected is contacted with a β aggregates to effect binding of a β -specific antibodies (and reactive versus a β aggregates) that may be present in the sample. Therefore, the concentration of a β aggregates must be adjusted to provide sufficient binding sites for the antibody. Thus, preferably, the concentration of a β aggregates for binding to the antibody in the sample is in the range of 0.001 to 1 μ M, preferably 0.01 to 0.1 μ M. The optimal concentration also depends on the nature of the antibody to be bound, the nature of the sample, the planned contact time and the size of the aggregates.

The method is mainly used for human sample application. Thus, it is preferred that the biological sample is human blood or a sample derived from human blood, preferably human serum or human plasma; human cerebrospinal fluid or human lymph. By virtue of such sample sources, continuous and routine testing (especially for blood-derived samples) can also be established.

Preferred contact times to allow for proper binding of the antibody in the sample to the aggregate are at least 10 minutes (e.g., 10 minutes to 48 hours), preferably 15 minutes to 24 hours, especially 30 minutes to 2 hours.

If the biological sample is not particularly pretreated, a β -specific antibodies having binding capacity for a β aggregates will be bound during contact with a β aggregates. The method according to the invention does not detect masked a β -specific antibodies (i.e. those that have bound to a binding partner (e.g. a β -containing structure, or endogenous a β peptide)) in the sample (in the absence of such specific sample pre-treatment). While identification and quantification of only reactive antibodies may be sufficient and desirable in many cases, there may be circumstances or diagnostic issues where the total amount of a β -specific antibodies (reactive and non-reactive) or all of the reactive a β -specific antibodies, the number of non-reactive ("masked") antibodies and the total number of a β -specific antibodies in a sample should be detected.

Thus, according to another preferred embodiment of the present invention, the sample is unmasked, i.e. the Α β -specific antibodies are "released" from any binding to the binding partners present in the sample before being contacted with the Α β aggregates according to the present invention. This allows the detection of all a β -specific antibodies in the sample and not only those antibodies that are not bound to the binding partner in the sample ("free" or "reactive" antibodies). As mentioned above, the method is also suitable for determining the total amount of a β -specific antibodies in a sample, i.e. free (or "reactive") antibodies as well as those antibodies that have bound (e.g. to a β structures) in the sample. This may help to establish the difference (Δ) of reactive versus non-reactive antibodies in the sample, a parameter that may also be of great significance for AD diagnosis. In order to use a β -specific IgM as a parameter for AD diagnosis, it is particularly preferred to perform the unmasking step prior to the method, such that the difference (Δ) of reactive versus non-reactive antibodies in the sample is defined and used as a relevant parameter.

The method according to the invention applies single particle detection techniques. Such techniques allow the identification and quantification ("counting") of the number and count of "positive" binding results of a β -specific antibodies to a β aggregates. A preferred embodiment of this technique is FACS, a technique established in the art. Other detection methods to be used for detecting antibodies bound to a β aggregates are, for example, Luminex or mass spectrometry.

Sample preparation can be performed as described in materials and methods, in accordance with the Luminex technology. After sample preparation, A β aggregates recognized by specific A β -specific antibodies can be detected by a secondary antibody coupled to fluorescently-stained microspheres that can be detected in a multiplex detection system, such as a Luminex reader (Binder et al, Lupus15(2005): 412-.

If mass spectrometry is used as the single particle detection technique, sample preparation can also be carried out as described in the materials and methods of the examples section below. Sample preparation was completed as described. After sample preparation, Α β aggregates recognized by the specific antibody can be detected by a secondary antibody coupled to a stable isotope of a transition element, which can be detected by atomic mass spectrometry. The sample can then be sprayed onto an induction coil filled with argon plasma heated to a temperature >5,500K. The sample was evaporated and ionized to its atomic composition and the number of isotope-labeled antibodies was quantified by time-of-flight mass spectrometry (Janes et al, nat. Biotechnol.29(2011): 602-604).

Alternatively, it is also possible to apply a single particle detection technique in which one binding partner (Α β aggregates or antibodies/serum) is immobilized, but binding is measured under flow conditions. Examples are the Hybcell technique and the surface plasmon resonance technique. Using the Hybcell technique in the present invention, a serum sample can be located on the Hybcell (rotating cylinder) surface and incubation can be performed with either directly fluorescently labeled pre-incubated Α β aggregates or alternatively with fluorescently labeled monoclonal Α β -specific secondary antibodies. Antibodies that bind to A β aggregates were detected with a laser (Ronacher, antibodies Technical Note ANA-005 (2010)). If surface plasmon resonance is used in the method according to the invention, the opposite arrangement can be applied: the pre-incubated a β aggregates may be immobilized on the chip surface. Binding of serum-derived A.beta. -specific antibodies to the A.beta.aggregates on the chip can be detected by mass gain on the chip surface, so label-free binding partners are necessary. In order to increase sensitivity or to determine the IgG subtype, continuous injection of anti-IgG-AB is possible (Cannon et al, anal. biochem.328(2004): 67-75). Instead of directly immobilizing the a β aggregates to the chip surface, a capture antibody may be used. For this setup, a β -specific antibodies were immobilized on the chip surface followed by injection of pre-incubated a β aggregates. After capturing the aggregates, the serum was injected and reactivity was measured by mass gain.

Detection of binding of A.beta. -specific antibodies to A.beta.aggregates according to the invention can be carried out by any suitable method known, for example, by fluorescence spectroscopy for detecting A.beta. -specific antibodies bound to A.beta.aggregates by means of a secondary antibody, for example a labeled anti-IgG or anti-IgM secondary antibody (Missailidis et al, Methods in molecular biology248(2003): 431-441).

Detection of autoantibodies bound to aggregates can also be carried out using substrates that specifically bind to the antibody, such as protein a or protein G. Another possibility is to precipitate a β aggregate-specific autoantibodies using a β aggregates, wash the complex, and biotinylate the antibody. Subsequently, streptavidin can then be used as a second step reagent.

According to a preferred embodiment of the present invention, the particles having an a β aggregate-like surface are beads, in particular magnetic beads, having a β aggregates immobilized on their surface.

By virtue of the present invention, a β -specific (auto) antibodies in human patients undergoing immunotherapy are provided as markers of AD status. AD patients were tested before the AD treatment was initiated. A β -specific antibodies are then tested during therapy (e.g., AD vaccination therapy with a specific a β vaccine, preferably a mimotope vaccine). Then, the amount of a β -specific antibodies should be increased in AD patients. This rise demonstrates successful priming of the immune response at the antibody level. Thus, if such levels are modified in an AD patient or a subject at risk of developing AD or suspected of having AD, such modified levels are associated with the success of AD immunotherapy. The "level of modification" may be an absolute number of modifications of a β -specific antibodies or a modification of the reactivity of the total number of a β -specific antibodies, e.g. a β -specific antibodies of a given class (IgG, IgM, etc.). For example, increased reactive Α β -specific IgG is associated with and is a marker of successful AD vaccination. In the case of IgM, on the other hand, the level of (non-reactive; i.e.bound or masked) A.beta.specific IgM changes to a level which otherwise has a marker function with respect to AD. With this method, a significant increase, e.g., to 125% and higher, to 150% or higher or to 200% and higher, in the "initial" level of reactive a β -specific IgG set at 100%, is indicative of successful immunotherapy. On the other hand, an increase of at least 30%, for example at least 50% or at least 100% in total IgM (reactive + non-reactive) in the blood sample when the "initial" level of reactive Α β -specific IgM is set to 100%, is indicative of AD formation.

Preferably, the detected amount of a β -specific antibodies is correlated with other AD status tests, in particular brief mental state examination (MMSE). Samples were taken from subjects with MMSE results of the same AD patient. For example, the MMSE results prior to the initiation of a vaccination strategy for this patient can be compared to subsequent MMSEs generated throughout the course of treatment (and thereafter) to observe no deterioration or even improvement (indicative of successful AD immunotherapy) or deterioration (indicative of unsuccessful immunotherapy). MMSE can be linked to the antibody detection method according to the invention: an increase in antibody amounts (indicative of successful AD immunotherapy) or a decrease in antibody amounts (indicative of unsuccessful immunotherapy).

The method according to the invention is therefore particularly suitable for monitoring a given AD patient with respect to the development of a disease treatment, in particular for linking the diagnostic results according to the invention with other diagnostic tools for determining the AD status and/or for monitoring the efficiency of a therapeutic intervention. Thus, in a preferred embodiment of the invention, the method is performed at least twice on samples of the same patient taken at different times. Preferably, the detected amount of a β -specific antibodies is correlated with MMSE results of the same patient at the same time the sample was taken from the patient. To monitor the development of AD in a given patient, it is preferred to carry out the method according to the invention at regular intervals, for example at least once every 6 months, preferably at least once every 3 months, in particular at least once every month.

It is also possible to define certain threshold values of a β -specific antibodies in a sample for defining a disease state (e.g. "healthy", mild AD, late AD, or according to the MMSE scale). The threshold value will of course depend on the sample and the precise detection system, but any standardized way of implementing the invention may be developed. The concentration of Α β -specific IgG in the serum of healthy subjects is typically between 1000 and 5000ng/ml, whereas the concentration of IgG in AD patients is typically much lower, e.g. ranging from 250 to 1500 ng/ml. Most AD patients have less than 1000 ng/ml. Thus, according to a preferred embodiment of the invention, detection of an amount of an A β -specific antibody in a sample below a threshold level is indicative for AD, wherein the threshold level is 1500ng/ml or less, preferably 1000ng/ml or less, especially 750ng/ml or less. On the other hand, this increase in IgG concentration during AD treatment (especially AD treatment by immunotherapy) is indicative of successful immunotherapy. For example, if the level rises from 750ng/ml to over 1000ng/ml or even over 1500ng/ml, this would indicate successful immunotherapy.

Thus, the present method is particularly suitable for use in conjunction with AD immunotherapy of a given patient in a monitored manner over time.

The invention is also useful in immunotherapy for all pathologies associated with or associated with a β ("a β pathology"), in particular pathologies in which a β deposits appear during the course of the disease. Examples of such a β pathologies are parkinson's dementia (PDD), dementia with lewy bodies (DLB), Cerebral Amyloid Angiopathy (CAA), including myositis (IBM; especially sporadic IBM (sbm)), or chronic head trauma (e.g. boxing) (see e.g. WO2004/062556A, WO2009/103105A, WO2009/149485A, WO2009/149486A, WO2009/149487a, WO2011/020133A, WO2006/005707a, WO2006/005706A, WO2005/025651A, etc.).

It is possible to use this method to observe the development of disease and the performance of possible therapeutic approaches, in particular whether the therapeutic approach enables the establishment of "healthy" or "elevated" levels of a β -specific antibodies, in particular IgG.

Therefore, it is preferred to use the present method for monitoring AD patients, especially AD patients treated with drugs that cure or ameliorate AD. The present method can be successfully applied to observe patients in clinical trials of AD vaccines (e.g. with AD mimotopes according to WO2004/062556A, WO2006/005707a, WO2009/149486A and WO2009/149485 a; or with a β -derived vaccines according to WO 99/27944A) or a β -targeted disease modifying drugs.

By virtue of the present invention, it is in principle possible to detect changes in the immunological settings of a patient with respect to a β -specific antibodies. This makes patients eligible for early stage treatment regimens and/or prophylactic (or delay) strategies for AD, especially vaccination.

According to another aspect, the present invention relates to a kit for carrying out the method according to the invention, comprising:

-a β aggregates, and

sample containers, in particular for human samples (e.g. blood, serum, plasma).

Preferably, the kit according to the invention may further comprise means for detecting a β aggregates bound to an a β -specific antibody, preferably a secondary antibody, in particular a labeled secondary antibody, such as an anti-IgG or anti-IgM antibody). Other components may be standard samples, positive and/or negative controls, instructions for use, and appropriate packaging means (e.g., a robust cartridge, a colored bottle, etc.).

Brief Description of Drawings

The present invention is further illustrated by the following examples and figures, but is not limited thereto.

Figure 1 shows a β aggregate size determination using FACS analysis. Thioflavin (Thioflavin) T positive A β 1-42 aggregates can be detected using flow cytometry and are depicted as homogeneous populations in FL1-FITC A (log-scale) and FSC-A (log-scale) channels in dot blots. The size distribution of Α β aggregates (defined as FCS-A signal) was determined using commercial calibrated size beads (1, 2, 4, 6, 10, and 15 μm), as shown in the FSC-A histogram (B).

Figure 2 shows the detection of monoclonal antibody reactivity to a β aggregates using FACS-based assays. The A beta 1-42 monoclonal antibody 3A5 specifically binds to A beta 1-42 aggregates (A), but does not interact with A beta p (E)3-42 aggregates (C). In comparison, the A β p (E)3-42 specific antibody D129 binds to A β p (E)3-42 but not to A β 1-42 aggregates (B + D). Reactivity was determined in FL2-PE channel using an anti-immunoglobulin-PE labeled secondary antibody. The fluorescence intensity as shown in B and C represents background staining as seen when aggregates were incubated with PE-labeled secondary antibody alone.

FIG. 3 shows a comparison of the assay sensitivity of three different methods. (A) The A β 1-42 aggregates were incubated with the IVIG dilution series and the reactivity was determined in the FL-2 channel using flow cytometry. (B) IVIG titration on Maxisorp ELISA plates coated overnight with A β 1-42 at pH 9.2. (C) Label-free detection of different concentrations of IVIG interacting with the immobilized predominantly biotinylated a β 1-42 on the SA-chip was performed using surface plasmon resonance (BiaCore). Note that for comparison, all results are given as fold background signals.

Figure 4 shows the correlation of IgG reactivity with a brief mental state examination (MMSE) score. 24 AD patients were examined for MMSE status and the sera of those patients were subjected to the described FACS assay and the median FI was determined. The association of the accepted data is shown in the results and previous figures.

FIG. 5A shows the determination of A.beta.specific IgM autoantibody reactivity in a given serum sample. Control sera from healthy donors, or sera from AD patients were subjected to the FACS assay described. Fluorescence intensity of a β aggregates was evaluated in FL2-PE channel and expressed as Median Fluorescence Intensity (MFI). FIG. 5B shows ROC curve analysis. Sera from AD patients were compared to sera from healthy donors for IgM-specific anti- Α β 1-42 reactivity.

FIG. 6 shows the determination of the reactivity of A.beta.specific IgM autoantibodies in a given serum sample after antibody demasking. Control sera from healthy donors (HI), or sera derived from AD patients, were subjected to the unmasking method described and the subsequent FACS assay. Fluorescence intensity of a β aggregates was evaluated in FL2-PE channel and expressed as Median Fluorescence Intensity (MFI).

FIG. 7 shows ROC curve analysis. Sera from AD patients were compared for IgM-specific anti-a β 1-42 reactivity with sera from healthy donors after autoantibody unmasking.

Fig. 8 shows absolute MFI values representing Α β aggregate-specific IgG antibodies before, during and after vaccination in patients treated with AFFITOPE vaccine. The a β 1-42 reactivity of sera from patients immunized with (patient a) and without alum (patient B) is shown. Fluorescence intensity of a β aggregates was evaluated in FL2-PE channel and expressed as MFI.

Figure 9 shows a statistical analysis of immunoreactivity against Α β 1-42 aggregates expressed as median MFI values for patients immunized with AFFITOPE vaccine with and without alum. Due to one extreme in the adjuvant group, Mann-Whitney test (non-normal distribution) (A) was performed to give normal distribution after removing the extreme, and Student t test (B) was performed. Both tests were statistically significant (p <0, 05).

Examples

Materials and methods

Detection of Abeta-specific antibodies using surface plasmon resonance (SPR-BIAcore)

The Biacore system provides a variety of chips that can be used for immobilization. For this experiment, Streptavidin (SA) -coated chips were used. To avoid non-specific binding of the ligand to the chip surface via the side chain, C-terminal biotinylated a β peptide (purchased from Anaspec) was used. Since the biotin-streptavidin complex is very stable, it is most suitable for prolonged continuous experiments. To optimizeThe chip robustness and hence reproducibility, the largest response unit (about 1500RU) was immobilized onto the flow cell, while flow cell 1 was kept empty and used as a reference. To avoid non-specific binding on the chip surface, free biotin was used to saturate free SA binding sites on all 4 flow cells. Human samples (already diluted 1:10 to 1:100 in HBS ph 7.4) and IVIG dilution series ranging from 1mg/ml to 10 μ g/ml (also diluted in HBS ph 7.4) were measured. The chip surface was regenerated with 10mM glycine-HCl (pH1.8) after each sample injection. All experiments were performed at 25 ℃. Injections were performed using a standardized protocol, starting with an association phase of 200 seconds followed by a dissociation phase of 600 seconds. R_maxValues are defined as the level of response (measured in RU) at the end of sample injection. For signal evaluation, R was determined using integrated BIA evaluation software_maxThe value is obtained.

Detection of A beta-specific antibodies using ELISA

Different a β peptides (purchased from rPeptide) were diluted in 100mM NaHCO3(ph9.2) at a concentration of 5 μ g/ml and coated overnight on Maxisorp96 well plates. To prevent non-specific binding, plates were blocked using 1% BSA/PBS for 1 hour at 37 ℃. The ELISA was performed for 1 hour at 37 ℃ with serial dilutions of human serum samples (starting with 1:10 dilutions) or IVIG diluted in a range of 1mg/ml down to a concentration of 10. mu.g/ml by addition of binding buffer (PBS/0.1% BSA/0.1% Tween 20). After repeating the washing step (3 times) with PBS/0.1% Tween20, secondary antibodies were detected by adding anti-human Ig HRP (0.5. mu.g/ml) at 37 ℃ for 1 hour. The samples were washed 3 more times and ABTS (0.68 mM in 0.1M citric acid pH 4.3) was added for 30 minutes to advance the assay, after which OD was measured on a plate reader (Biotek-Gen5Program) at a wavelength of 405 nm.

Detection of A beta-specific antibodies using FACS analysis

Prior to analysis, the lyophilized β -amyloid peptides were subjected to a disaggregation method. For this purpose, lyophilized A β species were in 1% NH₄OH (pH 11). Subsequently, the solubilized A β peptide was sampled in aliquots and stored at-20 ℃. To induce aggregate formation, willDifferent A.beta.species were incubated in aqueous solution (pH5) at a concentration of 20. mu.M overnight at 37 ℃ on a shaker (350rpm) in Eppendorf tubes. Formation of a β aggregates can be confirmed by thioflavin t (tht) staining in FACS (FSC/FL 1). Aggregated a β species were diluted to 0.15 μ M in sterile filtered 0.5% BSA and preincubated in 96-well plates for 30 min in a final volume of 95 μ l. Mu.l of pre-diluted human plasma or antibody (in 0.5% BSA/PBS) was added to 95. mu. l A. beta. aggregate solution. The final dilution of plasma ranged from 1:1000 up to 1: 10.000. For monoclonal antibodies, concentrations below 0.5. mu.g/ml were used. After 45 or 60 min incubation at Room Temperature (RT) on a shaker (350rpm), 200. mu.l of 0.5% BSA/PBS was added to each well and centrifuged at 3000rpm (96 well plate centrifuge) for 5 min. The supernatant was removed and the washing step repeated again with 200. mu.l of 0.5% BSA/PBS. After the second wash, the SN was discarded and the pellet resuspended in 100. mu.l of 0.5% BSA/PBS containing 1:1000 dilution of labeled anti-IgG (H + L) -PE or 1:500 dilution of anti-IgM, Fc 5. mu.secondary antibody (both Jackson immuno Research). Samples were incubated at RT on a shaker (350rpm) for an additional 45 or 60 minutes and measured on a FACS Canto equipped with a high-throughput sampler (HTS). Aggregates were gated in FSC/SSC and mean Median Fluorescence Intensity (MFI) was measured and evaluated in the FL2-PE channel and reactivity of ThT to A β aggregates was measured and evaluated in the FL1-FITC channel, respectively, using FACS Diva software.

Shielding releasing

To disrupt the binding of a β -specific autoantibodies to a β that may be present in the patient's serum and thus prevent detection of these a β bound to autoantibodies by antigen-based methods (such as ELISA or FACS), the serum was pre-diluted in 10mM glycine ph2.6 at a dilution of 1:16.7 for 5 minutes.

To disrupt the potential binding of A.beta.antigen to autoantibodies, serum was pre-diluted 5 minutes at 1:16.7 in 10mM glycine pH 2.6. Then, 5. mu.l of acidified serum was incubated with 3. mu. l A. beta.1-42 (100. mu.g/ml) for an additional 5 minutes. The mixture was then neutralized by adding 92. mu.l of 0.5% BSA/PBS and incubated for 20 to 60 minutes. The washing step and incubation with secondary antibody was performed as described above for the non-unmasked serum.

Results

A beta aggregate: oligomerization and fibril (fibril) formation

The formation of Α β aggregates (including Α β oligomers, fibrils and fibril aggregates) from monomeric Α β has been extensively investigated in recent years under a variety of conditions. It has been found that aggregation of a β peptides is very much dependent on different conditions, including pH, temperature, buffer composition and protein concentration. Aggregation begins with the formation of a β -hairpin from monomer, producing a soluble oligomer. Conformational transitions to parallel β -sheets result in the formation of fibrils and fibril aggregates that can be precipitated by centrifugation. Recent findings have shown that the Α β 40 and Α β 42 aggregates generated at ph7.4 comprise fibrils having 5nm wide filaments with a slight tendency to bind laterally into bundles (15 to 25nm wide filaments). The length of such single fibrils lies in the range from sub-micron (50-100nm) up to 10-15 μm as determined by transmission electron microscopy. One feature of these Α β fibrils is that they are specifically intercalated with the fluorescent dye thioflavin t (tht).

In accordance with the present invention, A β aggregates of different A β peptide variants (A β 1-42, A β 3-40, and A β p (E)3-40) are generated, which are inserted with ThT. FACS analysis can be used to detect these Α β aggregates. For this purpose, the non-nuclear (seedless) soluble A.beta.peptide was incubated at a concentration of 20. mu.M for 20 hours at 37 ℃ as described in MM. As shown in figure 1A (upper panel), a clear ThT positive homogenous population (measured as FL1(FITC) positive signal), FACS assays developed according to the present invention allow analysis of in vitro generated Α β aggregates. Size distribution of Α β aggregates (defined by forward scattering FSC-a) was analyzed using calibration large beads (flow cytometry size calibration kit from Molecular probes (catalog No. F-13838)) (bottom panel of fig. 1) ranging from 1 to 15 μm. Using this analysis, it was shown that the expected size range of the resulting a β aggregates was up to 10 μm in the submicron range, with most of the resulting aggregates ranging from about 200nm up to 3 μm.

Reactivity of monoclonal antibodies with Abeta aggregates

To define whether a β aggregates allow a β -specific antibody binding and determine whether such interactions can be monitored using the FACS-based assays described herein, another set of experiments was performed. For this purpose, A β 1-42 and A β p (E)3-42 aggregates are generated and incubated with monoclonal antibodies specific for A β 1-42(3A5) or specific for A β p (E)3-42 (D129). As shown in the FACS histogram in fig. 2, monoclonal antibody 3a5 only bound to a β 1-42 aggregates, whereas mab D129 only interacted with N-terminally truncated pyroglutamated a β species. This shows that the FACS-based assay described allows detection of a β -specific antibodies in a specific manner.

Sensitivity of different detection methods defining A.beta.binding of human autoantibodies using IVIG

IVIG (intravenous immunoglobulin) is a commercial blood product. It contains pooled IgG fractions extracted from plasma from healthy donors (human plasma from at least 1000 donors). IVIG preparations have been shown to contain naturally occurring antibodies (autoantibodies) specific for a β peptides.

The purpose of this experiment was to define and compare the detection limits of three independent detection methods (Biacore, ELISA and FACS-based assays) for the a β reactivity of IVIG (IVIG-Subcuvia, available from Baxter, Austria). Thus, a β 1-42 was immobilized onto the chip surface or on a Maxisorp microtiter plate for SPR or ELISA assays, respectively. Alternatively, A β 1-42 aggregates were generated for FACS analysis. Subsequently, different IVIG dilutions were applied to each system and R was defined_maxValues (in the case of SPR), OD values (in the case of ELISA) or fluorescence intensity (MFI value) (in the case of FACS assay). For comparison reasons, the signal-to-noise ratio was evaluated for all IVIG concentrations, and the signals were expressed as fold background signals (xBG) (fig. 3). In the case of SPR measurements, none of the IVIG concentrations gave a signal above background (fig. 3C). In contrast, the control antibody 3A5 is givenA strong signal, indicating successful immobilization of a β 1-42 to the chip surface, and a β peptides can in principle be recognized by antibodies on the chip surface. Using ELISA as a detection method, 1000. mu.g/ml IVIG gave a clear signal (7 times BG) above background, whereas the signal induced by 100. mu.g/ml IVIG was only 2 times background. IVIG at 10. mu.g/ml produced no signal above background (FIG. 3B). As depicted in fig. 3A, FACS-based assays provide a much higher signal than delivered by the other two detection methods. Not only 1000. mu.g/ml (24 times BG), or 100. mu.g/ml (11 times BG), but also 10. mu.g/ml (5 times BG), and 1. mu.g/ml (2 times BG geometry) IVIG dilutions produced a signal significantly above background. This indicates that the newly developed FACS-based assay that detects Α β -specific autoantibodies is at least 100-fold more sensitive than conventional assays such as ELISA or Biacore.

Defining anti-beta amyloid antibodies in human blood from healthy donors and AD patients

IgG reactivity to A.beta.1-42

In an attempt to define a correlation between cognitive and immunological data, the reactivity of measured IgG to Α β aggregates in AD patient sera (n =24) was correlated with the score of the patient's brief mental state examination (MMSE) at the time of serum sampling. This correlation reveals a trend for patients with weak test performance to show reduced IgG reactivity, as depicted in figure 4. Thus, this reduction in Α β -specific IgG reactivity reflects AD progression.

In an attempt to define a correlation between cognitive and immunological data, measured IgG reactivity of AD patient sera (n =24) was correlated with results from a brief mental state examination (MMSE). This test is commonly used to screen for cognitive impairment and dementia, and to assess the severity of cognitive impairment in an individual at a given time point. The correlation of MFI with MMSE score reveals a trend for patients with weak test performance to show reduced IgG reactivity, as depicted in fig. 4.

IgG reactivity to A.beta.3-42 and A.beta.p (E)3-42

In addition to defining the reactivity of immunoglobulins naturally present in the sera of AD patients and healthy subjects to A β 1-42 aggregates, the reactivity of these sera is also defined against A β 3-42 and against A β p (E) 3-42.

Reactivity of IgM to different Abeta aggregates

In the next set of experiments, a β aggregate-specific IgM reactivity was defined in serum samples derived from healthy individuals or AD patients using the same sera as described above. As can be seen in fig. 5A, naturally occurring antibodies of IgM isotype specific for a β aggregates were detected in all samples tested. In contrast to IgG reactivity, however, serum samples derived from healthy controls did not show a higher reactivity of IgM for Α β aggregates than serum samples derived from AD patients. Therefore, ROC curve analysis did not produce a curve that plots sensitivity or specificity (fig. 5B).

d. Reactivity of unmasked IgG and IgM to A β aggregates

In previous studies, it has been shown that a β -specific autoantibodies (mainly of the IgM isotype) can be harboured with a β antigen to establish immune complexes that are stable and circulating in human blood (WO2010/128139a1; Marcello et al, 2009; lindagen-Persson et al, PLoS ONE5(2010): e 13928). To define whether the a β antigens potentially binding to autoantibodies can block the reactivity of these antibodies to a β aggregates generated in vitro, individual sera were subjected to unmasking methods, as described in materials and methods. With low pH, the potential immune complex is destroyed, resulting in removal of a β antigen from the antibody binding domain (antigen dissociation). Thus, if immune complexes are present in the serum sample, the unmasking method can produce higher autoantibody signals in FACS-based assays. Interestingly, as depicted in figure 6, IgM reactivity of AD patient sera was significantly elevated after unmasking compared to untreated sera, while the reactivity of healthy control sera was unchanged (compare figure 9A).

In fig. 7, the results depicted in fig. 6 have been summarized in ROC curves. This ROC curve analysis showed that at a sensitivity of 78%, the specificity was 84% and the area under the curve (AUC) was 0.822.

Conclusion

Based on these results, it can be shown that the higher sensitivity of the FACS-aggregation assay according to the invention is directly linked to the different aggregation states of A β 1-42. For BIAcore analysis, freshly dissolved and biotinylated A.beta.1-40 was used for immobilization on a streptavidin chip. This method facilitates the binding of the monomers a β 1-42 on the chip surface, since the peptide is immobilized immediately without pre-incubation, and the biotin-tag also slows down the formation of aggregates. Coating of a β 1-42 on Maxisorp plates at pH9 also appears to favor monomeric forms of a β, although coating of some aggregates cannot be excluded. In these assays, the affinity between the antibody and the A β 1-42 peptide poses a limit of detection.

In contrast to these two methods for FACS-based assays according to the present invention, Α β -1-42 aggregates are specifically induced and used to detect antibodies specific for Α β present in IVIG. These larger molecules provide multiple antibody binding sites. The resulting avidity effect (sum of multiple synergistic low affinity antibody-antigen interactions) may result in higher sensitivity of the assay and is performed by resulting in detection of low affinity antibodies within the IVIG fraction. The reactivity of IVIG towards a β aggregates can also be explained by the presence of epitopes present only on the a β aggregated form.

The examples clearly show the superiority of the present invention over the methods currently available in the art, particularly with respect to analytical properties such as specificity and selectivity.

Clinical studies with a mimetic vaccine according to EP1583774B1 and EP1679319B 1.

AFFITOPE-vaccine as claimed in EP1583774B1 and EP1679319B1, which is capable of inducing antibodies specifically targeting a β peptide, was tested in a clinical study containing two arms. Patients in cohort 1 were immunized with AFFITOPE vaccine containing adjuvant and patients in cohort 2 were immunized with AFFITOPE vaccine lacking adjuvant as immune response enhancer. In contrast to non-adjuvanted vaccines, where an increase in Α β -specific IgG antibodies cannot be expected, adjuvanted vaccines should have the ability to induce antibodies targeting Α β.

To assess the ability of FACS-based assays according to the invention to monitor induced IgG antibodies during clinical studies, sera from vaccine recipients were subjected to this assay. 1 pre-immune serum (collected before starting the immunization protocol) and 3 post-immune sera from each single patient were analyzed. Two separate measurements for each time point and patient were performed on different aliquots of serum samples. An increase in Α β aggregate-specific IgG antibodies in each patient over time would indicate that FACS assays may be suitable for monitoring the efficacy of immunotherapeutic interventions over time, and in addition, such results would also indicate successful vaccination.

Using the FACS-based assay according to the present invention, it was found that pre-immune sera of all study participants already contained Α β aggregate-specific IgG, as expected. This is consistent with recent publications showing that body fluids such as blood (plasma/serum) and CSF contain IgM and IgG antibodies specific for Α β aggregates.

The values shown in fig. 8 depict the mean values of two measurements over time. In this figure, illustratively, the development of Α β -specific IgG antibody responses in patients derived from cohort 1 (patient a) and patients immunized with the vaccine lacking adjuvant (cohort 2-patient B) is depicted. As can be seen in fig. 8, the MFI signal from patient a immune sera 2 and 3 was significantly higher than the pre-serum derived fluorescence signal, indicating increased Α β -specific IgG. In contrast, immune serum derived from patient B did not produce an elevated fluorescence signal. These results are representative for the respective groups.

To define the increase in a β 1-42 reactivity in individual patients, the difference in mean MFI values of two independent measurements of the last immune serum and the corresponding pre-serum has been calculated. In fig. 9, it can be seen that analysis of immune sera using FACS assay resulted in a clear difference between the two groups. Immune sera from patients immunized with the adjuvanted vaccine consistently showed an increase in Α β -specific IgG.

To assess the statistical difference between median FI values for patients receiving adjuvanted or unadjuvanted vaccination, two different tests were performed. When all patient data were included, no normal distribution was given between the two groups due to one extreme in the adjuvanted group. Under this precondition, the Mann-Whitney test was performed, which resulted in a p-value of 0.0473 (fig. 9). To perform statistical analysis also under normal distribution conditions, the outer layer (out-layer) was removed from the data set and the student t-test was calculated. This analysis also yielded a statistically significant p-value of 0.0089 (figure 9).

Claims (17)

1. A method for detecting a β -specific antibodies in a biological sample, comprising the steps of:

-contacting the sample with a β aggregates or with particles having an a β aggregate-like surface and allowing the a β -specific antibodies to bind to the a β aggregates, and

-detecting said a β -specific antibodies bound to said a β aggregates by single particle detection technology (single particle detection technology), preferably by Fluorescence Activated Cell Sorting (FACS).

2. Method according to claim 1, characterized in that the antibody specific for a β is a human antibody, preferably a human IgG or IgM antibody, in particular a human IgG antibody.

3. Method according to claim 1 or 2, characterized in that the a β -specific antibody is an autoantibody.

4. The method according to any one of claims 1 to 3, characterized in that the A β aggregates have a size of 50nm to 15 μm, preferably 100nm to 10 μm, especially 200nm to 5 μm.

5. Method according to any one of claims 1 to 4, characterized in that the A β aggregates have been prepared by incubating the A β -1-42 peptide, the A β -1-43 peptide, the A β -3-42, or the A β -p (E)3-42 peptide at a pH of between 2 and 9 for at least 20 minutes, preferably for at least 1 hour, in particular for at least 4 hours.

6. Method according to any one of claims 1 to 5, characterized in that the A β aggregates are present in an amount of 0.001 to 1 μ M, preferably 0.01 to 0.1 μ M, for contacting the sample with A β aggregates.

7. The method according to any one of claims 1 to 6, characterized in that the biological sample is human blood or a sample derived from human blood, preferably human serum or human plasma; human cerebrospinal fluid or human lymph.

8. Method according to any one of claims 1 to 7, characterized in that the A β aggregates are brought into contact with the sample for at least 10 minutes, preferably from 10 minutes to 24 hours, in particular from 20 minutes to 2 hours.

9. Method according to any one of claims 1 to 8, characterized in that a demasking step is performed on said A β -specific antibodies in said sample before contacting said sample with A β aggregates, in particular when IgM antibodies are detected.

10. Method according to any one of claims 1 to 9, characterized in that the particles having an a β aggregate-like surface are beads, in particular magnetic beads, having a β aggregates immobilized on their surface.

11. The method according to any one of claims 1 to 10, wherein the biological sample is a sample of an AD patient undergoing or supposed to undergo AD immunotherapy, in particular AD vaccination.

12. The method according to any one of claims 1 to 11, wherein the biological sample is a sample of an AD patient undergoing or supposed to undergo AD immunotherapy, and wherein the detected amount of Α β -specific antibodies is associated with a Mini-Mental state examination (MMSE) result of the same patient at the same time the sample was taken from the patient.

13. Method according to any one of claims 1 to 12, wherein the method is performed at least twice on samples of the same patient taken at different times and wherein preferably the detected amount of Α β -specific antibodies is correlated to the MMSE outcome of the same patient at the same time the samples were taken from the patient.

14. The method according to claim 13, wherein the method is performed at least once every 6 months, preferably at least once every 3 months, in particular at least once every month.

15. Use of a method according to any one of claims 1 to 14 for monitoring AD patients, in particular AD patients treated with a medicament for curing or ameliorating AD.

16. Use of a method according to any one of claims 1 to 14 for the diagnosis of Parkinson's Dementia (PDD), Dementia with Lewy Bodies (DLB), Cerebral Amyloid Angiopathy (CAA), Inclusion Body Myositis (IBM), or chronic head trauma (chronic head trauma).

17. A kit for carrying out the method according to any one of claims 1 to 10, comprising:

-a β aggregates, and

-a sample container.