The present application claims priority of EP20168007.1 filed 3 April 2020, fully incorporated by reference herein.

Field of the invention

The present invention relates to rapid diagnostic assays for testing for disease in animals and humans, and more particularly to assays for detecting the pathogenic form of a target protein in a biological sample from fluids obtained from humans suspected of having a disease caused by and/or associated with the target protein and pathological conformer, respectively. In particular, the present invention relates to a method of increasing the level of a soluble target protein in a biological sample by subjecting the sample to a protein cleavage reaction which does not affect the target protein. The target protein may be Ab 42 and the biological sample may be a plasma sample from a subject, e.g. human subject. Together with appropriate detection means and methods such as cyclic amplification systems to increase the levels of the target protein, the method of the present invention is particularly suitable for quantifying the minimal detectable amount of a target protein in a biological sample, which could serve as a biomarker in the diagnosis of a disease associated with the target protein. In particular, such disease may be Alzheimer’s disease. Moreover, the simplicity of sample preparation makes the present invention suitable for use in all kinds of diagnostic applications and drug discovery.

Background of the invention

The term “Amyloid beta” (Ab or Abeta) denotes peptides of 36-43 amino acids that are the main component of the amyloid plaques found in the brains of people with Alzheimer's disease. AB peptides derive from the amyloid precursor protein (APP), which is cleaved by beta secretase and gamma secretase to yield Ab. Ab molecules can aggregate to form flexible soluble oligomers, which may exist in several forms. Certain misfolded oligomers can induce other Ab molecules to also take the misfolded oligomeric form, leading to a chain reaction akin to a prion infection. The oligomers are toxic to nerve cells. Another protein implicated in Alzheimer's disease, tau protein, also forms such prion-like misfolded oligomers.

A major challenge in measuring blood-based biomarkers is that their concentrations are much lower in plasma or serum than in the cerebrospinal fluid (CSF). For example, the concentrations of Ab42 and tau (Uniprot ID PI 0636) in plasma are approximately 30- and 100-fold lower, respectively, than those in the CSF. A second challenge is that plasma and serum have a higher total protein concentration and a more complex protein matrix than does the CSF. The binding of blood biomarkers such as Ab42 to many proteins in plasma or serum (e.g., albumin, lipoproteins, Ab autoantibodies, fibrinogen, immunoglobulin, apolipoprotein J, apolipoprotein E, transthyretin, a-2 -macroglobulin, serum amyloid p component, plasminogen, and amylin) can further reduce the concentration of the biomarker available for measurement. Though less pronounced the same problems may occur in CSF samples since most of the proteins present in the CSF are derived from plasma; see for review [13]

Over the past few years, technologies such as single molecule array (SiMoA), immunoprecipitation mass spectrometry (IP-MS), immunomagnetic reduction superconducting quantum interference (MagQu), and the interdigitated microelectrode sensor system have emerged for the development of assays for blood-based biomarkers of several diseases, in particular Alzheimer’s disease (AD) [13]

Nevertheless, despite those achievements in sensitivity there is still a need of improvement of assays for biomarkers in body fluids such as Ab42 for AD, which are one or more of easy to perform, cost-effective, highly precise, robust, fully automated, and amenable to high- throughput analysis.

The solution to this problem is provided by the embodiments as characterized in the claims and described further below.

Summary of the invention

The present invention is directed to a method for increasing the level of freely available soluble target protein in a biological sample, which is characterized in that the sample is subjected to a chemical protein cleavage reagent, wherein the cleavage reaction does not affect the target protein or portion thereof. In particular embodiments, the target protein is a biomarker and the biological sample is a body fluid, in particular plasma sample from a subject, e.g. human subject. The method of the present invention is particularly suitable in assays, where the target protein is a biomarker and intended for facilitating the determination of the presence, level or absence of the target protein. The method of the present invention can be advantageously implemented in a method for the diagnosis or detection of a conformational disease associated with a pathogenic conformer of a target protein by assaying for a biomarker of such disease within a sample, wherein the sample has been processed by protein cleavage before assaying the biomarker. In particular embodiments, the method comprises a cyclic amplification system to increase the levels of the pathogenic conformer. In particular, such conformational disease may be Alzheimer’s disease.

The present invention is based on experiments aiming at determining the level of Amyloid beta 42 peptide, hereinafter Abeta42 and Ab42, respectively, in blood from human volunteers and patients, respectively, in order to develop an Ab42 biomarker-based prodromal diagnosis of Alzheimer’s disease. The reliable detection of Ab42 in blood is hampered by several obstacles. For example, Ab42 is very well known to be sequestered by plasma proteins [17] and bound to them. Thus, the levels of freely available soluble Ab42 is very low and make it difficult for Ab detection in plasma through the currently available techniques. Hence, for the detection of Ab42 levels in blood, enrichment of Ab42 and depletion of contaminating proteins, respectively, would be desirable. This problem could surprisingly be solved by treating the sample, i.e. plasma sample prior to assaying Ab42 with 2-nitro-5-thiocyanobenzoic acid (NTCB), a chemical cleavage reagent that abundantly cleaves plasma proteins but not Ab42; see Fig. 1 and 2

As demonstrated in the Examples, the increase in the level of soluble Ab42 after treatment with the chemical cleavage reagent compared to a control has been verified by protein misfolding cyclic amplification (PMC A), a kind of PCR for protein detection that can be generally applied to all proteins that are prone to aggregation, in particular misfolded proteins such as Ab42 which rapidly aggregate in solution, and which was first used to amplify PrP ^Sc in vitro for biochemical detection of prions and adapted to misfolded Ab42; see [l]-[4] and Fig. 3 as well as [9] However, in principle any other detection method may be used as well; see the background section, supra and the description, infra. In case of Ab42 Ab oligomers (i.e. pathogenic conformer of Ab42 and thus target protein) in patient plasma sample serve as seeds for aggregation so that when adding a predetermined amount of soluble monomeric Ab42 (non- pathogenic) under conditions allowing a conformational transition between the non-pathogenic and the pathogenic conformer, and wherein the conformational transition can be propagated from {i.e. passed by) the pathogenic conformer to the non-pathogenic conformer the level of the pathogenic conformer is increased and can be easily detected by conventional means; see also the Examples.

As illustrated in the Examples and shown in Fig. 7, plasma concentration as low as lmg/ml can be used for the detection of the target protein, i.e. here Ab42, which translates into a plasma stock as low as 10m1 volume that is sufficient for performing a single experiment. Thus, due to the novel chemical protein cleavage-based sample processing, the detection limit could be reduced such that only a small sample volume is needed allowing for miniaturized assay formats and in particular the development of fully automated PMCA assay which a detection limit up to in the fmol range.

In summary, experiments performed in accordance with the present invention demonstrate that sample processing according to the method of the present invention leads to enrichment of the target protein, while interfering proteins are depleted by cleavage in peptides and further smaller fragments or portions thereof. This procedure augments the detection of the protein in question, facilitates the further processing and improves the detection limit of a given assay. Accordingly, while the present invention may be predominantly illustrated for Ab42 in a plasma sample and PMCA assay as detection means, the person skilled in the art will immediately recognize the general applicability of the embodiments described herein.

Description of the figures

Fig. 1: Sample processing in accordance with the present invention illustrated with a plasma sample, NTCB and Ab42; see also Example 1.

Fig. 2: A: Analysis of cleavage sites of the chemical cleavage reagent NTCB in the most abundant plasma proteins. Amino acid sequences of plasma proteins as indicated have been analyzed using Expasy peptide cutter tool made available by the Swiss Institute of Bioinformatics https://web . expasv.org/pepti de_cutter/. B: Coomassie staining of plasma treatment with NTCB. Plasma stock of 80mg/ml was diluted down to 5mg/ml and lmg/ml. The samples were treated with lOmg/ml of NTCB. Protein cleavage was carried out under basic pH, 50°C. Proteins samples were neutralized to neutral pH with 0.1M Tris-HCl and loaded on the gel.

Fig.: 3 Schematic representation of protein misfolding cyclic amplification in prion proteins [3] PrPSc represents scrapie prion while recPrP represents the normal recombinant prions.

Fig. 4: Novel method of preparing monomeric solution of self-aggregating proteins illustrated with monomeric Ab42; see also Example 2. As indicated, initial experiments trying to obtain monomeric Ab42 via spin columns with cut-off filters described previously failed to give appropriate results.

Fig. 5: Characterization of monomeric Ab42 solution in NaOH solution using ThT assay. The ThT assay was carried out on Ab42 wildtype and scrambled peptide at 25°C, for 50 hours with peptide concentration at 5uM and ThT concentration at 20uM.and at 485nm wavelength. Relative fluorescence was measured for both supernatants S 1 (monomers) and S2 (aggregates). As shown, when wildtype Ab42 is dissolved in x PBS buffer and centrifuged, most of the peptide aggregates, since Ab42 has a high propensity for aggregation at biological pH. Thus, most of the peptide is lost in the supernatant fraction S2 and very low amounts of the peptide is available as monomer. This is reflected by the high Thioflavin T signal of the wildtype Ab42 S2 fraction vs Ab42 SI in PBS buffer, pH-7.4. In contrast, wildtype Ab42 when dissolved in 0.01M NaOH, the Thioflavin T signal of the S2 vs SI fraction is in similar fluorescence units range, with the S2 fraction having relatively higher signal. Thus, large amounts of peptide is still present in monomeric state. Hence, the peptide dissolved in NaOH with supernatant SI fraction was used in the overall assay development. As a negative control, scrambled Ab42 peptide dissolved in both PBS and NaOH has been used. The Thioflavin signal in both buffer conditions were similar, thus, no aggregation was seen as expected.

Fig. 6: Prototype of a PMCA assay a: Ab oligomers ranging between lOpmol to lOfmol were incubated with 5uM monomers. The fluorescence signal was measured every hour. The fluorescence signal was corrected for ThT baseline fluorescence b: At 24 hours fluorescence signal between oligomers and the monomer alone was compared.

Fig.7: PMCA assay on plasma spiked with oligomers. A and C depict the fluorescence kinetics of plasma containing oligomers ranging between 1.25uM-12.5pM. Plasma of different concentration, 1 mg/ml and 5mg/ml, were used for testing. B and D represent fluorescence after 10 hours i.e: 20 cycles of PMCA assay. Fluorescence measurements were taken at 485nm.

Fig. 8 is a graph of the real-time PMCA assay kinetics for upto 40 hours and each datapoint is the mean value of the ThT signal of the samples. As illustrated in Figure A, there is a stark difference in the Thioflavin T (ThT) signal beyond 24 hours for Plasma only vs Plasma with oligomers. Plasma samples containing Ab oligomers have a high ThT signal. This signal remains relatively constant. Fig 1 B. depicts the mean ThT values of the samples for 25-35 hours. T-test was performed to compare the signal difference between plasma with oligomers vs plasma alone samples and this difference is significant. The data demonstrate that the assay is sensitive to up to 20fM oligomers.

Fig. 9 shows that the ThT signal seems high for highest for MCI followed by AD and then by HC. This is furthered represented in Fig.9b, where the ThT data has been pooled for time points between 25-35 hours. Previous studies have shown that Ab oligomers are absent in healthy people, but are produced in significant amount in the brain primarily in the early disease stages of Alzheimer’s disease and are constantly entering the blood system. However, as the disease further proceeds, Ab oligomers aggregate a lot more in the brain, forming Ab plaque deposits. Thus, their levels are lowered in the blood. All this is reflected in the plasma. Thus, Fig 9b. illustrates that MCI patient level shows the highest Tht fluorescence and thus, maximum oligomeric level in plasma followed by AD patient. The HC has lowest ThT signal close to baseline. Detailed Description of the invention

For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of’ or “consisting of.”

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictate otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et ak, Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et ak, Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods. See also: Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19 850673 2; Second edition published 2006, ISBN 0-19-852917-1 978-0-19852917-0 and the references cited in brackets “[]” below.

The terms "target protein" and "biomarker", are used interchangeably herein. “AB” is used synonymously with “Abeta”.

The present invention generally relates to a method for enhancing the level of and/or determining the presence or absence of a soluble target protein or portion thereof in a sample of a body fluid from a subject. The method is characterized in that the sample is subjected to a chemical protein cleavage reagent, wherein the cleavage reaction does not affect the target protein or portion thereof.

Unless otherwise stated, a term as used herein is given the definition as provided in the "Biological sample" refers to fluid or tissue extracted from vertebrates, such as brain tissue, whole blood, serum, plasma, saliva, urine, and cerebral spinal fluid but other samples may be used as well, for example wherein the detection of a target protein is hampered by abundant other proteins such as matrix proteins and the target protein may be bound to proteins which prevent access of detections means such as antibodies to the target protein, or wherein the proteins otherwise interfere with assaying for the target protein.

As illustrated in the Examples, the sample to be analyzed may be derived from blood or, particularly, a plasma sample [4] However, other body fluids wherein proteins may interfere with the detection of a target protein or otherwise interfere with its detection may be applicable as well. For example, cerebrospinal fluid (CSF) is a proximal fluid which communicates closely with brain tissue, and contains numerous brain-derived proteins and thus represents a promising fluid for discovery of biomarkers of central nervous system (CNS) diseases. Approximately 80 % of the total CSF protein is derived from the plasma, upon crossing the blood-brain barrier, wherein the most abundant blood-derived proteins are albumin and immunoglobulins [12] Therefore, also CSF samples may be advantageously processed via cleavage treatment in according to the present invention. Typically, the plasma concentration is about 1 mg/ml. Particularly, the sample to be analyzed comprises 5 to 100 mΐ plasma, more particularly about 50, 40, 30, 20 or 10 mΐ plasma are used. The same applies to equivalent samples, for example CSF as mentioned.

In particular embodiments, the method of the present invention is applied to a target protein that features deposition of amyloids and is involved in common neurodegenerative diseases. Amyloids are ordered protein assemblies that can act as templates for their own replication through monomer addition. Evidence suggests that this characteristic may underlie the progression of pathology in neurodegenerative diseases. Many different amyloid proteins, including Ab, tau, and a-synuclein, exhibit properties similar to those of infectious prion protein in experimental systems: discrete and self-replicating amyloid structures, transcellular propagation of aggregation, and transmissible neuropathology [7] For example, prionoid proteins which propagate into pathological protein aggregates involved in the pathogenesis of neurodegenerative diseases are described in [5], [6] In a particular embodiment, the target protein is selected from Ab, tau, a-synuclein and PrP ^Sc .

In one specific embodiment, the target protein is Ab.

In one specific embodiment, the target protein is tau (Uniprot ID P10636).

In one specific embodiment, the target protein is a-synuclein.

In one specific embodiment, the target protein is PrP ^Sc .

However, in times of current pandemic infection with coronavirus COVID-19 (Coronavirus SARS-CoV-2) the means and methods of the present invention may also be helpful for detecting circulating virus and viral proteins at an early stage of infection and to confirm the healthy status of a subject, in particular after recovery of an infection and suspected immunity. In this context, it has been described that a 9 residue peptide (TK9) of the extra membrane C-terminal tail of the SARS Corona virus envelope is capable of self-assembly and are mimicking amyloid proteins in their aggregation propensity [20]

Regarding the selection of an appropriate chemical cleavage reagent for a given target protein, the target protein can be assessed for cleavage sites and the lack of them via computational analysis, for example using the Expasy peptide cutter tool made available by the Swiss Institute of Bioinformatics https://web . expasv.org/pepti de_cutter/. In the Examples, the method of the present invention has been illustrated with Ab42 and NTCB. As a further example, analysis with the Expasy peptide cutter tool reveals that the NTCB cleavage can be used for detecting alpha-synuclein protein which is involved in Parkinson’s disease. Of course, the person skilled in the art can easily identify other chemical cleavage reagents for any target protein.

In this context, the person skilled in the art will appreciate that the cleavage reagent for use in accordance with the present invention has recognition site residues in proteins, which may not be unique but low abundant so that a cleavage reagent can be selected which does cleave the target protein, at least under appropriate conditions but interfering, in particular large protein such as albumin, see also Fig. 2 for illustration. In contrast, for example Proteinase K, a serine protease often used in sample preparation cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids and results in general digestion of proteins in biological samples. Furthermore, compared to sequence-specific proteases chemical cleavage reagents such as NTCB could more easily access cleavage sites within proteins, in particular membrane proteins and protein complexes, aggregates and the like.

The chemical cleavage reagent is particularly selected from the group consisting of NTCB (2- nitro-5-thiocyanobenzoic acid), Cyanogen bromide (CNBr), BNPS-skatole [2-(2- nitrophenylsulfenyl)-3-methylindole], Formic Acid, Hydroxylamine (NH2OH) or Iodosobenzoic acid [8]

In certain embodiments, the cleavage reagent is NTCB, which cleaves Cys-X, leading to splits on the N-terminus. NTCB specifically cyanylates cysteine thiols, which subsequently cleave on the N-terminal side of the cyanylated residue under mildly alkaline conditions to form an amino-terminal peptide and a series of 2-iminothiazolidine-4-carboxylyl (ITC) peptides [19] In certain embodiments, the cleavage reagent is CNBr, which hydrolyzes peptide bonds at the C -terminus of methionine residues converting Met to Homoserine. An excess CNBr can cleave/degrade Trp and Tyr-side chains.

In certain embodiments, the cleavage reagent is BNPS-Skatole, a mild oxidant and brominating reagent that cleaves at the C-terminus of tryptophan. It is unstable under acidic conditions and must be prepared fresh prior to use to minimize side reactions. It shows an excellent specificity and yield. The cleavage of Met-Ser/Met-Thr bonds may give suboptimal yields.

In certain embodiments, the cleavage reagent is CHOOH. Formic Acid cleaves at the C- terminus of Asp- Pro. Yields of 40 % with high specificity can be achieved.

In certain embodiments, the cleavage reagent is hydroxylamine (NH2OH), which cleaves at the C-terminus of Asn and at the N-terminus of Gly. It also breaks imide links to Gly. Iodosobenzoic acid cleaves at the C-terminus of Trp.

The cleavage reaction is advantageously conducted with the cleavage reagent in an amount sufficient to substantially digest substantially all the proteins present in the sample except the target protein or a portion thereof, which itself my serve as the target protein to be detect, for example a self-aggregating peptide, domain of a receptor, antigenic determinant and the like. As mentioned, sample processing by protein cleavage according to the method of the present invention due to enrichment of the target protein versus abundant proteins in the original samples improves the detection limit of a given assay. Accordingly, once a sample has been processed according to the present invention the target protein may be detected with any conventional assay format including for example immunoassays which otherwise would not be sensitive enough [13], [14]; see also the background section, supra.

As discussed before and illustrated in the Examples, target proteins that are prone to aggregation, in particular misfolded proteins, can in certain embodiments be detected via misfolding cyclic amplification (PMCA), a kind of PCR for protein detection that can be generally applied to all proteins that are prone to aggregation, in particular misfolded proteins such Ab and corresponding protein aggregates.

The principle of protein misfolding cyclic amplification (PMCA)[1] is described in detail [9] and depicted in Fig. 3. This technique was first developed for the detection of prion proteins. PMCA involves incubating minute amounts of infectious prions (PrP ^Sc ) with normal prion proteins (recPrP). These infections prions serve as seeds and recruit more PrP, which get converted to infectious prions (p _r p ^PMCA ) on repeated quaking. This exponential amplification can be easily detected using western blotting. This assay can be exploited for detecting Ab42 oligomers since amyloid propagate like prion proteins and has been refined for the detection Ab42 [10] but also for synuclein and tau [11] Moreover, PMCA has already been shown to detect low levels of Ab oligomers in the CSF samples of MCI and AD patients [2] Accordingly, as illustrated in the Examples the method of processing a sample is particularly suitable for combining with PMCA, i.e. for incorporation in a method for the diagnosis or detection of a conformational disease which is characterized by a conformational transition of an underlying protein between a non-pathogenic and a pathogenic conformer and wherein the conformational transition can be propagated from {passed by) the pathogenic conformer to a non-pathogenic conformer, by assaying a marker of said disease within a sample from a subject, which method comprises:

(i) contacting said sample with an amount of the non-pathogenic conformer;

(ii) disaggregating any aggregates eventually formed during step (i); and

(iii) determining the presence and/or amount of said pathogenic conformer within the sample, the pathogenic conformer being a marker for the presence of said disease, insofar, that prior to step (i) the sample is subjected to the above-mentioned method wherein the sample is subjected to a chemical protein cleavage reagent, wherein the target protein comprises the pathogenic conformer and the cleavage reaction does not affect the pathogenic conformer. In particular embodiments, step (i) comprises step (ia) incubating said sample/non- pathogenic conformer and steps (ia) and (ii) form a cycle which is repeated at least twice before carrying out step (iii); see also Examples 1 and 3. The term "conformational disease" my be understood as defined in W02/04954A2 (incorporated by reference herein) at page 14, lines 3- 10 and includes any prion-like disease; see also [5]-[7] In this context, it is also envisaged to perform the PMCA assay the other way around, i.e. detecting and determining the level of the non-pathogenic conformer, e.g., monomeric Ab42 by adding a predetermined amount of a pathogenic conformer, e.g, oligomeric Ab42 as a seed as illustrated in Fig. 1. After processing the sample and optionally amplification, detection of the target protein can be performed by any conventional methods, including but not limited to immunoblotting after SDS-P AGE, ELISA assay, radioactivity assays, fluorescence assays, aggregation assays and structural assays such as NMR, Circular dichroism, Fourier transformed infrared spectroscopy, Raman spectroscopy, intrinsic fluorescence, UV absorption, etc.

In one embodiment of the present invention the pathogenic conformers are detected by fluorescence, in particular examples by a Thioflavin T (ThT) assay, which can be used as illustrated in the Examples. Thioflavin T is a fluorescent compound that binds only to aggregated forms of peptide, especially to the beta-sheet structures of amyloid fibrils and fluoresces at 485nm.

As mentioned in Example 2, when trying to analyze the plasma samples after processing with NTCB cleavage in accordance with Example 1 for the presence and level of pathogenic Ab oligomers by PMCA, problems were encountered when trying to provide a stable solution of soluble monomeric Ab42 as the non-pathogenic conformer for use in the assay and to avoid premature aggregation. Accordingly, a novel method to generate a monomeric solution of Ab42 has been developed as illustrated in Fig. 4 and 5. Therefore, in an exemplary particular embodiment of the PMCA assay as applied for Ab42 in a biological sample the non-pathogenic Abeta42 monomers are obtained by a method comprising

(a) dissolving monomeric Abeta in a solution of NaOH;

(b) sonication of the solution,

(c) ultracentrifugation of the solution and

(d) collection of the supernatant for use in step (i).

In particular embodiments, the NaOH-solution has a concentration of 0.01M. The sonication time is around 7-12 min, whereby 10 min is preferred. The ultrasonic bath contains icecold water, which prevents heating up of the peptide which may result in peptide aggregation. The ultracentrifugation of the solution should take place at 4°C for at least 1 hour at 100000 rpm.

A further object of the invention is a method for the prodromal diagnosis or detection of Alzheimer’s disease (AD) comprising a protein misfolding cyclic amplification (PMCA) of Abeta42 oligomers within a plasma sample of a subject, which is characterized in that prior to the amplification step the sample is subjected to NTCB. This method involves amplifying Ab oligomers, which serve as seeds, present in plasma samples of patients by incubating the plasma samples with externally added monomeric Ab42 solution as described hereinabove and explained in Example 3. In this embodiment, the conditions in step (i) in the above mentioned method comprise 5 mM Abeta42 monomers; and the incubation time should be about 1 hour and at 25°C; and shaking 510 rpm for 1 min. As illustrated in the Examples, this technique is relatively fast and simple to execute. Moreover, it would be inexpensive since it requires a functional fluorescence reader alone which is affordable and can be implemented in several clinics and diagnostic centers.

Before the treatment with NTCB the plasma sample can be subjected to a TCEP (Tris(2- carboxyethyljphosphinehydrochloride) treatment for breaking disulphide protein linkages. After the cleavage with NTCP a filtration step can optionally follow.

As mentioned before and illustrated in Example 2, another object of the present invention is a method of preparing Abeta42 monomers, which are useful as non-pathogenic conformers in the method described above, wherein the method comprises

(a) dissolving monomeric Abeta in a solution of NaOH;

(b) sonication of the solution;

(c) ultracentrifugation of the solution and

(d) collection of the supernatant for use in step (i).

In certain embodiments, the NaOH-solution has a concentration of 0.01M. The sonication time is around 7-12 min, whereby 10 min is preferred. The ultrasonic bath contains ice cold water, which prevents heating up of the peptide, which may result in peptide aggregation. The ultracentrifugation of the solution should take place at 4°C for at least 1 hour at 100000 rpm. As illustrated in Fig. 5, in NaOH solution wildtype Ab42 after sonification and collection upon subsequent ultracentrifugation and optionally neutralization with HC1 has a lesser propensity to aggregate than in physiological PBS buffer and thus the relative fluorescence is higher yet comparable to that of scrambled Ab42 monomers in either buffer, which are not capable of aggregating; see Example 2 and Fig. 5 and the legend thereto. Accordingly, in other aspect the present invention relates to an liquid solution of monomeric Ab42 obtainable by the above- described method, particularly wherein the solution of monomeric Ab42 at a concentration of 5uM when incubated with 20uM of ThT for at least 48, typically 50 hours at 25°C has a lesser propensity to aggregate compared to a corresponding liquid solution of monomeric Ab42 in lxPBS buffer, particularly by at least 20%, particularly 25% lower in terms of relative fluorescence as determined in a ThT assay as described in Example 2; see Example 2 and Fig. 5. The present invention relates to the "intermediate" solution of wildtype Ab42 in NaOH, particularly about 0.01 M NaOH as well as to the neutralized solution. In certain embodiments, the concentration of Ab42 in the solution is about 1 to 50uM, more particularly about 5 to 25 uM. The stable solution of monomeric wildtype Ab42 prepared in accordance with the present invention finds various applications, in particular in diagnostic assays such as the PMCA assay illustrated in the Examples but also in drug screening, generation and characterization of Ab and amyloid binding molecules, in particular antibodies.

As mentioned, sample processing in accordance with the present invention is particularly suitable and therefore intended for implementation in assaying biomarkers in context with a disease or physical condition. The "biomarker concept" includes a variety of possible research strategies: (a) predictive biomarkers for estimating disease probability at the pre-clinical stage, (b) diagnostic biomarkers, e.g. for precise differential diagnosis, (c) prognostic biomarkers for prognosis/chance of healing, (d) treatment response biomarkers ("theramarkers") for estimating the response to therapy, (e) surrogate biomarkers for getting evidence, how intervention influences the endpoint of interest, (f) trait markers as invariable characteristics of a disease e.g. gene mutations, and (g) state markers to follow disease progression, e.g. enzymes, ions, etc. [15] The present invention specifically envisages the use of any one of the afore-described methods, in particular sample processing in assaying and the detection of cerebrospinal fluid and blood biomarkers for neurodegenerative dementias.

As discussed before, a particular object of the invention is an assay for a marker of a conformational disease which is characterized by a conformational transition of an underlying protein between a non-pathogenic and a pathogenic conformer and wherein the conformational transition can be propagated from {passed by) the pathogenic conformer to a non-pathogenic conformer, within a sample, which assay comprises the steps of the above-mentioned method. As discussed hereinbefore, one goal of the present invention was to develop an Ab42 based biomarker assay using the principle of protein misfolding cyclic amplification (PMC A), i.e. wherein the target protein is Ab42, the non-pathogenic conformers are Ab42 monomers, the pathogenic conformers are Ab42 oligomers and/or the chemical cleavage reagent is NTCB. As demonstrated in the Examples, this goal has successfully been accomplished. Accordingly, in a particular preferred embodiment, the present invention relates to a method for detecting the presence of Abeta42 oligomers within a plasma sample from a subject, comprising:

(i) contacting the sample with an amount of Abeta42 monomers;

(a) incubating the sample/Abeta42 monomers;

(ii) disaggregating any aggregates formed during step (ia); repeating steps (ia)-(ii) two or more times; and then

(iii) determining the presence and/or amount of Ab42 oligomers within the sample; characterized in that prior to step (i) the sample is subjected to a protein cleavage reagent, wherein the cleavage reaction does not affect b42Abeta42, particularly wherein the protein cleavage reagent is a chemical cleavage reagent such as NTCB and/or the AB42 monomers are obtained by the method comprising

(a) dissolving monomeric Ap42beta in a solution of NaOH;

(b) sonication of the solution;

(c) ultracentrifugation of the solution and

(d) collection of the supernatant for use in step (i).

The conditions and step of any one of (i) to (iii) and (a) to (d) are particularly chosen and performed as described hereinbefore and most particularly as illustrated in the Examples.

And another object of the present invention is a method for obtaining, identifying and/or validating an inhibitor and a drug, respectively, or toxicity of a compound which modulates the conformational transition of an underlying protein between a non-pathogenic and a pathogenic conformer, the method comprising the steps of the method, mentioned above, wherein

(a) step (i) comprises contacting an amount of the non-pathogenic conformer with an amount of the pathogenic conformer (1) in the presence of said compound, and

(b) step (iii) comprises determining the amount of the pathogenic conformer (1) in the presence of said compound; wherein a decrease of the amount of pathogenic conformer (1) in the presence of said compound compared to a control (2) is indicative for a putative inhibitor and drug, respectively, and an increase of the amount of pathogenic conformer (1) compared to a control (2) is indicative for the putative toxicity of the compound, particularly wherein the control (2) is provided by contacting in step (i) an amount of the non-pathogenic conformer with an amount of the pathogenic conformer (2) in the absence of said compound and step (iii) comprises determining the amount of the pathogenic conformer (2) in the absence of said compound. According to the above method, "identifying" should also be interpreted to mean "screening" of a series of compounds.

A further object of the present invention is a diagnostic kit for use in the above-mentioned method or assay, which comprises a known amount of the non-pathogenic conformer, particularly monomeric Ab42 and a chemical protein cleavage reagent, particularly NTCB; and optionally, a multi-well microtiter plate, a multi-well sonicator, and or a known amount of pathogenic conformer as a positive control, particularly oligomeric Ab42.

Another further object of the present invention is the use of a chemical protein cleavage reagent, particularly selected from the group consisting of NTCB (2-nitro-5-thiocyanobenzoic acid), Cyanogen bromide (CNBr), BNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methylindole], Formic Acid, Hydroxylamine (NFEOH) or Iodosobenzoic acid for processing a biological sample, particularly of a bodily fluid from a subject, particularly plasma sample, optionally in an above mentioned method.

Still another object of the present invention is an apparatus for use in the above-described method or assay, comprising a comprising a microtiter plate reader, multi-well sonicator; and optionally an amount of a non-pathogenic conformer and/or reservoir for chemical protein cleavage reagent.

Still a further object of the present invention is a therapeutic agent for use in treating or ameliorating the symptoms of a patient which has been diagnosed to suffer from or being at risk to develop a conformational disease by a method described above, particularly wherein the disease is AD and the therapeutic agent is selected from an anti-beta amyloid antibody, particularly Aducanumab, cholinesterase inhibitor and a N-methyl D-aspartate (NMDA) antagonist.

The invention further encompasses the following embodiments:

Embodiment 1: A method of determining the presence of an analyte, particularly a target protein, in a biological sample, wherein the analyte is present in low abundance relative to a matrix protein present in the biological sample, particularly wherein the molar ratio of analyte to matrix protein is smaller than (<) 1:1000, even more particularly wherein the molar ratio of analyte to matrix protein is smaller than (<) 1:10.000 or even < 1:100.000, wherein the matrix protein interacts with the analyte, thereby reducing analyte detection, characterized in that in a sample pretreatment step, the sample is subjected to a selective protein cleavage reaction leading to a sequence-specific cleavage of the matrix protein, thereby reducing analyte binding to said matrix protein; optionally, in a pretreatment workup step, a reagent effecting said selective protein cleavage reaction is removed, and in a detection step, said analyte is detected.

Embodiment 2: The method according to embodiment 1, wherein the biological sample is a mammalian -particularly human- blood or plasma sample.

Embodiment 3: The method according to embodiment 1, wherein the biological sample is a mammalian -particularly human- sample obtained from a nervous system compartment, particularly wherein the sample is a cerebrospinal fluid sample.

Embodiment 4: The method according to any one of the preceding embodiments, wherein the analyte is an analyte polypeptide, wherein the analyte polypeptide is not amenable to cleavage by said sequence specific cleavage reaction, and where the analyte polypeptide is capable of conformational transition between a non-pathogenic conformer and a pathogenic conformer and wherein said conformational transition can be propagated from ( passed by) the pathogenic conformer to a non-pathogenic conformer.

Embodiment 5: The method according to embodiment 4, wherein the detection step employs protein misfolding cyclic amplification (PMCA).

Embodiment 6: The method according to any one of the preceding embodiments, wherein said sequence specific cleavage reaction is effected by a reagent specifically reactive to cysteine residues, leading to cleavage of said matrix protein, particularly wherein said reagent is 2-nitro-5-thiocyanobenzoic acid (NTCB).

Embodiment 7 : The method according to embodiment 6, wherein prior to reaction of the sample with NTCB, the sample is exposed to a reducing agent capable of breaking treatment for breaking cystine disulphide bonds, particularly wherein the reducing agent is Tris(2- carboxyethyl)phosphinehydrochloride (TCEP).

Embodiment 8: The method according to any one of the preceding embodiments, wherein said sequence specific cleavage reaction is effected by a reagent specifically reactive to methionine residues, leading to cleavage of said matrix protein, particularly wherein said reagent is cyanogen bromide.

Embodiment 9: The method according to any one of the preceding embodiments, wherein the analyte is selected from AB protein and tau protein.

Embodiment 10: The method according to embodiment 9, wherein the analyte is AB42. Embodiment 11: A method of diagnosis related to Alzheimer’s disease (AD), particularly a method of a) diagnosing AD, particularly diagnosing prodromal AD, b) following progress of AD, c) assessing response to medical intervention, particularly pharmaceutical intervention targeting AD, d) assessing efficacy of an experimental intervention, particularly in context of a drug trial, targeting AD, in a patient, comprising pretreatment of a sample obtained from the patient with a method according to any one of the preceding claims 9 or 10, and detecting the presence of an AD related analyte selected from tau protein and AB protein, particularly AB42.

The invention further encompasses the following items: Item 1: A method of increasing the level of and/or determining the presence or absence of a soluble target protein in a biological sample, characterized in that the sample is subjected to a chemical protein cleavage reagent, wherein the cleavage reaction does not affect the target protein.

Item 2. The method of item 1, wherein the sample is derived from of a body fluid such as blood or cerebrospinal fluid (CSF), particularly wherein the sample is a plasma sample.

Item 3. The method of item 1 or 2, wherein the chemical cleavage reagent is selected from the group consisting of NTCB (2-nitro-5-thiocyanobenzoic acid), Cyanogen bromide (CNBr), BNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methylindole], Formic Acid, Hydroxylamine (NFFOH) or Iodosobenzoic acid.

Item 4. A method for the diagnosis or detection of a conformational disease which is characterized by a conformational transition of an underlying protein between a non- pathogenic and a pathogenic conformer and wherein the conformational transition can be propagated from {passed by) the pathogenic conformer to a non-pathogenic conformer, by assaying a marker of said disease within a sample from a subject, which method comprises:

(i) contacting said sample with an amount of the non-pathogenic conformer;

(ii) disaggregating any aggregates eventually formed during step (i); and

(iii) determining the presence and/or amount of said pathogenic conformer within the sample, the pathogenic conformer being a marker for the presence of said disease, characterized in that prior to step (i) the sample is subjected to a method of any one of items 1 to 3, wherein the target protein comprises the pathogenic conformer and the cleavage reaction does not affect the pathogenic conformer.

Item 5. The method of clam 4, wherein the pathogenic conform ers are detected by fluorescence, particularly by a Thioflavin T (ThT) assay.

Item 6. The method of any one of items 1 to 5, wherein the target protein is Abeta42, the non- pathogenic conformers are Abeta42 monomers, the pathogenic conformers are Abeta42 oligomers and/or the chemical cleavage reagent is NTCB.

Item 7. The method of item 6, wherein the non-pathogenic Abeta42 monomers are obtained by a method comprising

(a) dissolving monomeric Abeta in a solution of NaOH;

(b) sonication of the solution;

(c) ultracentrifugation of the solution; and

(d) collection of the supernatant for use in step (i). Item 8. A method for the prodromal diagnosis or detection of Alzheimer’s disease comprising a protein misfolding cyclic amplification (PMCA) of Abeta42 oligomers within a plasma sample of a subject, characterized in that prior to the amplification step the sample is subjected to NTCB.

Item 9. A method of preparing a liquid solution of Abeta42 monomers useful as non-pathogenic conformers in a method of any one of items 4 to 8, wherein the method comprises

(a) dissolving monomeric Abeta in a solution of NaOH;

(b) sonication of the solution;

(c) ultracentrifugation of the solution; and

(d) collection of the supernatant for use in step (i).

Item 10. An assay for a marker of a conformational disease which is characterized by a conformational transition of an underlying protein between a non-pathogenic and a pathogenic conformer and wherein the conformational transition can be propagated from (passed by) the pathogenic conformer to a non-pathogenic conformer, within a sample, which assay comprises the steps of the method of any one of items 1 to 7 or 9.

Item 11. A method for obtaining, identifying and/or validating an inhibitor and a drug, respectively, or toxicity of a compound which modulates the conformational transition of an underlying protein between a non-pathogenic and a pathogenic conformer, the method comprising the steps of the method of any one of items 1 to 7 or 9, wherein

(a) step (i) comprises contacting an amount of the non-pathogenic conformer with an amount of the pathogenic conformer (1) in the presence of said compound, and

(b) step (iii) comprises determining the amount of the pathogenic conformer (1) in the presence of said compound; wherein a decrease of the amount of pathogenic conformer (1) in the presence of said compound compared to a control (2) is indicative for a putative inhibitor and drug, respectively, and an increase of the amount of pathogenic conformer (1) compared to a control (2) is indicative for the putative toxicity of the compound, particularly wherein the control (2) is provided by contacting in step (i) an amount of the non-pathogenic conformer with an amount of the pathogenic conformer (2) in the absence of said compound and step (iii) comprises determining the amount of the pathogenic conformer (2) in the absence of said compound.

Item 12. A diagnostic kit for use in the method of any one of items 1 to 8, or 11 or in the assay of item 10, which comprises a known amount of the non-pathogenic conformer, particularly monomeric Abeta42 and a chemical protein cleavage reagent, particularly NTCB; and optionally, a multi-well microtiter plate, a multi-well sonicator, and or a known amount of pathogenic conformer as a positive control, particularly oligomeric Abeta42.

Item 13. Use of a chemical protein cleavage reagent, particularly selected from the group consisting of NTCB (2-nitro-5-thiocyanobenzoic acid), Cyanogen bromide (CNBr),

BNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methylindole], Formic Acid, Hydroxylamine (NH2OH) or Iodosobenzoic acid for processing a biological sample, particularly a bodily fluid from a subject, particularly plasma sample, optionally in a method of any one of items 1 to 8, or 11 or in the assay of item 10. Item 14. An apparatus for use in the method of any one of items 1 to 8, or 11 or in the assay of item 10, comprising a microtiter plate reader, multi -well sonicator; and optionally an amount of a non-pathogenic conformer and/or reservoir for chemical protein cleavage reagent.

Item 15. A therapeutic agent for use in treating or ameliorating the symptoms of a patient which has been diagnosed to suffer from or being at risk to develop a conformational disease by a method of any one of items 4 to 8 or 10, particularly wherein the disease is AD and the therapeutic agent is selected from an anti-beta amyloid antibody, particularly Aducanumab, cholinesterase inhibitor and aN-methyl D-aspartate (NMD A) antagonist. Several documents are cited throughout the text of this specification and in the list of references preceding the claims. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application including the background section and manufacturer's specifications, instructions, etc.) are hereby expressly incorporated by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.

A more complete understanding can be obtained by reference to the following specific Examples which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention. EXAMPLES

Example 1: Plasma Processing by Chemical Protein Cleavage Reagent

As pointed out above Ab42 peptide in blood is very well known to be sequestered by plasma proteins [1] and is bound to them. Thus, the levels of freely available soluble Ab42 is very low. Low protein levels make it difficult for Ab detection in plasma through the currently available techniques. Hence, for the detection of Ab42 levels in blood, protein enrichment becomes necessary.

In order to enhance the levels of freely soluble Ab42 in plasma, a novel way of plasma processing and enrichment of Ab oligomers by chemical cleavage using 2-nitro 5-cyanobenzoic acid (NTCB) compound has been investigated; see Fig. 1. NTCB cleaves proteins containing cysteine residues in basic condition (between pH-9 and 10) [18] The major advantage in using this method for plasma processing is that cysteine residues are abundant in plasma proteins but absent in Ab42 peptide sequence; see Fig. 2A. Thus, unspecific cleavage of Ab oligomers can be avoided and the level of freely soluble oligomers can be increased.

Briefly, as illustrated in Fig. 1 plasma samples are diluted to the working concentration ( e.g 5mg/ml, lmg/ml). Disulfide protein linkages are broken by TCEP ((Tris[2-carboxyethyl] phosphine hydrochloride), Thermofischer scientific, Pierce Immobilized TCEP Disulfide Reducing Gel, Catalogue no.77712) treatment for 1 hour. Then, samples are treated with NTCB (8.75 mg/ml in 1ml of 0.1M Tris-HCl pH 8.0) at 37°C for 1 hour. The sample is then subjected to column filtration for removal of NTCB using Sephadex G25 (Mol.wt >5000 Da; Sephadex G25 desalting columns: GE Healthcare Life Science, Catalogue no. 28918007). Sample cleavage is induced at 50°C for 1 hour at pH 9.5 with 3M Tris Base. After the cleavage reaction the sample is equilibrated to biological pH values (between 7-7.5) with Tris-HCl.

In order to test the theory underlying the present invention, human plasma samples of concentration 5mg/ml and lmg/ml were treated with NTCB as indicated above. In order to determine the cleavage of the plasma sample Coomassie staining was carried out as shown in Fig. 2B. Albumin, being the most abundant plasma protein (marked with arrow) was used as a reference to study the cleavage. Major amount of albumin gets cleaved into smaller fragments. Thus, the object of enhancing the level of soluble target protein in a sample of a body fluid from a subject by way of processing the sample with a chemical protein cleavage reagent, wherein the cleavage reaction does not affect the target protein had been achieved.

Example 2: Generation of monomeric solution of non-pathogenic Ab42 conformers As mentioned hereinbefore in the description, when trying to move forward with analyzing the plasma samples after processing with NTCB cleavage in accordance with Example 1 for the presence and level of Ab by PMCA, problems were encountered when trying to provide a stable solution of soluble monomeric Ab42 as the non-pathogenic conformer for use in the assay and to avoid premature aggregation. Accordingly, a novel method to generate monomeric solution of Ab42 has been developed as illustrated in Fig. 4.

Materials

Recombinant Ab42 peptide: Purchased from Rpeptide: (i) Beta-Amyloid (1-42), Ultra Pure, HFIP, Catalogue no. A-l 163-2; (ii) Beta-Amyloid (1-42), Ultra Pure, TFA, Catalogue no. A- 1002-2; (iii) Scrambled Beta-Amyloid (1-42), Catalogue no. A-1004-2.

Ultrapure water: Invitrogen, Catalogue no. 12060346

Ultracentrifuge: Beckmann Coulter, Model: Optima Max-XP and Ultracentrifugation tubes: Thick-walled polypropelene tubes, lmL, Beckmann Coulter, Catalogue no.41121703 Thioflavin T: Sigma Aldrich, Catalogue no. 2390-54-7 Tecan Spark 10 M plate reader lx PBS (phosphate buffer saline): Life Technologies, Catalogue no. 10010-015 Briefly, 25uM of Ab42 was dissolved in 0.01M of NaOH solution made using Ultrapure water. The solution was sonicated for 10 mins in an ultrasonic bath containing ice cold water. The ice- cold water prevents heating up of the peptide which may result in peptide aggregation. The samples were transferred to thick walled-polypropylene tubes and spun for 1 hour at 100,000rpm at 4°C.

Note: The ultracentrifugation process was tested for 1, 2 and 3 hours and 1 hour seems to be sufficient for generating monomers in NaOH solution.

Once the centrifugation step is completed, the supernatant is collected, leaving some drops of supernatant left since it may contain Ab42 aggregates. The supernatant is kept on ice until further for the assay. Right before the assay, peptide is neutralized with 0.01M HC1.

For verifying the presence of monomeric Ab42 with the above-mentioned process, Ab42 wildtype peptide (occurring in natural form) was tested with a scrambled Ab42 peptide as a negative control. A scrambled peptide has the same amino acids as a naturally occurring wildtype peptide, but the amino acids are arranged in a fashion such that the peptide does not aggregate. For positive control, the peptides were dissolved in lxPBS. Wildtype peptide aggregates in physiological buffer like PBS while scramble peptide does not.

In order to verify if the peptide aggregates, a Thioflavin T (ThT)-test was carried out. As already mentioned, Thioflavin T is a fluorescent compound that binds only to aggregated forms of peptide, especially to the beta-sheet structures of amyloid fibrils and fluoresces at 485nm. Wildtype and scrambled peptide of concentration 5uM were incubated with 20uM of ThT. The fluorescence was measured for 48 hours as shown in Fig. 5. Both Supernatant 1 and 2 (SI and S2) were used to measure fluorescence. Supernatant SI has monomer alone. Supernatant S2 being close to the bottom of the tube has aggregates primarily.

As shown in Fig. 5 wildtype Ab42 aggregates very quickly in PBS buffer and thus, S2 of this peptide has high fluorescence in comparison to the other three samples, while in NaOH solution, the wildtype peptide has lesser propensity to aggregate and thus the relative fluorescence is higher yet comparable to the other 3 samples. Hence, the peptide dissolved in NaOH with supernatant SI fraction was used in the overall assay development.

Example 3: Detection of a pathogenic Ab42 conformers post plasma processing

For proof of principle, in that the processing of a plasma sample suspected to contain the target protein does not interfere with a corresponding detection assay, the PMCA assay illustrated in Example 1 was performed on human plasma samples spike with pathogenic conformers/oligomeric forms of Ab42, i.e. a biomarker for AD and typically found in blood of AD patients, though as mentioned in low amounts and thus difficult to detect.

Generation of Ab oligomers

Ab42 oligomers were freshly generated using the protocol by W.B Stine et.al [16] lOOug of Ab42 peptide was taken and to this 30ul DMSO was added. The mixture was then sonicated for lOmins in an ultrasonic water bath for lOmins. 196.2 ul of lxPBS buffer was then added to this to generate lOOuM stock of peptide. The samples were vortexed for about 30 sec and were incubated at 4°C overnight to generate oligomers.

PMCA assay in vitro

In order to test the working prototype of the PMCA assay, 5uM of Ab42 monomers prepared according to Example 2 were incubated with oligomers of concentration ranging from 10 pmol to 10 fmol. The assay was carried out with following conditions: Temperature: 25°C, shaking: for 1 min at 510 rpm and incubation time of 1 hour. The PMCA assay kinetics was studied using ThT fluorescence. At the end of this, second cycle began. The assay was executed for 4 days as depicted in Fig. 6a. Thus, fluorescence signal was measured every hour, i.e. at the end of every cycle. As a control, Ab42 monomer alone was incubated. Fig. 6b depicts ThT signal at 24 hours. There is a clear difference between the monomeric fluorescence signal alone and when oligomers are incubated with monomers. Up to 10 fmol sensitivity in terms of oligomer detection was achieved with p<0.001 significance.

Note: The assay has also been tested and works well for the following condition: shaking time: lmin at 216rpm and incubation time: 30mins and repeat the cycle for 48 hours. PMCA assay post plasma processing

In principle, plasma processing has been performed as described in Example 1 and shown in Fig. 1. Briefly, i. Plasma stock of 70mg/ml was spiked with oligomers of the following concentration: 1.25uM, 0.125uM, 1.25nM, 12.5pM. Equal volumes of plasma and oligomers were incubated together at room temp for 1 hour, 350 rpm to mimic the natural binding of Ab42 to plasma proteins. ii. Samples were then diluted to 5mg/ml and lmg/ml. iii. Equal volumes of the samples were then mixed with TCEP, for lhour at room temperature at 30pm in a rotamixer. iv. 20ul of NTCB solution was added to the 200ul samples and the mixtures was incubated for 2 hours at 37°C at 400rpm. v. The samples were then purified with a Sephadex G-25 column which was equilibrated prior use with 0.1M Tris-HCl, pH-8 buffer. vi. Samples were then cleaved at 50°C with 3M Tris-Base buffer. vii. Samples were brought to biological pH value. viii. For PMCA assay, Ab42 monomers as generated in Example 2, of final concentration 5uM was used with 20uM ThT. ix. PMCA assay condition: Temperature: 25°C, Shaking time: lmin at 360 rpm, incubation time: 30mins.

Results

From Fig. 7A, for lmg/ml concentration of plasma, oligomers up to 1.25nM concentration can be detected easily and has a higher fluorescence signal in comparison to the plasma alone. Whereas for 5mg/ml concentration, as seen in Fig. 7C, it is difficult to separate oligomers of different concentrations below 1.25uM. Therefore, it can be said that the plasma concentration of lmg/ml is better for the assay as the plasma matrix effect seen at higher concentrations can be avoided. This is further apparent from Fig. 7B and D, which represent the fluorescence measurements at 10 hours.

Example 4: PMCA assay post plasma processing

The following example illustrates a prototype PMCA assay in plasma samples spiked with oligomers.

Materials:

Plasma dilution buffer recipe (PDB): 9.3ml lxPBS, 400ul PI stock (used from 1 ^st thaw). 300ul of 0.5MEDTA, pH-8 2-nitro 5-thiocynatobenzoic acid (NTCB) : 9 mg/ml in 0.1M Tris, pH-8 Prototype Method:

1. Plasma samples were spiked with different concentration of oligomers ranging from 2pM- 20fM. Equal volumes of plasma and oligomers were incubated together at room temp for 1 hour, 300 rpm to mimic the natural binding of Ab42 to plasma proteins

2. Plasma samples were diluted by 1 :20 in PDB

3. 250ul of the diluted plasma was treated with 500ul TCEP beads and incubated together for 1 hr at room temperature, with gently mixing constantly in a shaker. The TCEP beads were at the end by centrifugation

4. 250 ul of the centrifuged samples were each added to a Eppendorf tube containing 400ul of NTCB. The tubes were quickly seal with Argon gas to avoid oxidation of NTCB. Samples were incubated for 2 hrs, at 37°C with constant mixing

5. The samples were then passed through a G25-Sephadex column to remove NTCB

6. The samples were then cleaved at 50°C with 3M Tris-Base buffer

7. The samples were brought to biological pH value and were used for the PMCA assay

8. Next set up the PMCA assay with one 4mm bead/well the following conditions:

• Temperature set to 25°C

• lmin shaking at 216rpm with 60mins incubation (for the elongation phase). [Also worked for lmin shaking at 360rpm with 30mins incubation]

Method for actual patient sample:

The above method was repeated for patient plasma samples. Plasma was obtained from a healthy control (HC), a mild cognitively impaired (MCI) and one Alzheimer’s disease (AD) patient. These patients were diagnosed clinically based on neuropsychological test and PET imaging. Mild cognitive impairment means that the patients have some mild cognition problems but yet to get Alzheimer’s disease.

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