A nasal fluid sample comprising aβ, ptau and / or ttau

Abstract

The present invention relates to a nasal fluid sample obtained from a subject, the nasal fluid sample comprising one or more marker protein amyloid beta (A beta), phosphorylated Tau (pTau) and / or total Tau (tTau). The invention further relates to a nasal fluid sample comprising one or more marker proteins A [beta], pTau and / or tTau for use in a method for aiding in the diagnosis of a degenerative disease of the nervous system, and to the use of a nasal fluid sample comprising one or more marker proteins A [beta], pTau and / or tTau for aiding in the diagnosis of a degenerative disease of the nervous system. The invention further relates to a method for aiding the diagnosis of a nervous system degenerative disease in a subject / individual.

Description

[0001] The present invention relates to a nasal fluid sample obtained from a subject, said nasal fluid sample comprising one or more marker proteins amyloid-beta (A-beta, (Abeta)), phosphorylated Tau (pTau) and / or total Tau (tTau). The present invention further relates to a nasal fluid sample comprising one or more marker proteins Abeta, pTau and / or tTau for use in a method of aiding in the diagnosis of a neurodegenerative disease, and the use of a nasal fluid sample comprising one or more marker proteins Abeta, pTau and / or tTau for aiding in the diagnosis of a neurodegenerative disease. The present invention further relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject / individual. BACKGROUND

[0002] Neurodegenerative diseases are a major threat to human health. These diseases are becoming more and more prevalent, partly because of the increase in the elderly population in recent years. Some examples of neurodegenerative diseases are Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), Creutzfeldt-Jakob disease (CJD), Lewy body dementia, vascular dementia (VD), Huntington's disease (HD), frontotemporal dementia (FTD), rapid eye movement sleep behavior disorder, multiple system atrophy (MSA), amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS).

[0003] As an example of a neurodegenerative disease (ND), Alzheimer’s disease (AD) is the most common form of dementia, accounting for 60-80% of all dementia syndromes (see “2020 Alzheimer’s disease facts and figures”, Alzheimers Dement (2020), 16(3), pp. 391-460, doi: 10.1002 / ALZ.12068). The “A / T / (N)” research framework introduced by the National Institute on Aging (NIA) and the Alzheimer’s Association (NIA-AA) in 2018 (see Jack et al., Alzheimer’s and Dementia, (2018), 14(4), pp. 535-562, doi: 10.1016 / j.jalz.2018.02.018) includes several biomarkers for the diagnosis of AD, including: low cerebrospinal fluid (CSF) levels or low CSF Abeta(1-42) / Abeta(1-40) ratio as a marker of Abeta pathology (labeled “A”); elevated CSF tau phosphorylated at threonine 181 (pTau181) as a marker of fibrillar tau (labeled “T”); and elevated CSF total tau (tTau) as a marker of neurodegeneration or neuronal injury (labeled “(N)”). The presence of different markers determines the individual disease state of each subject, i.e. whether the patient exhibits only pathological changes of AD (Abeta pathology: A+) or has AD (Abeta and fibrillar tau pathology: A+T+); see Jack et al., Alzheimer’s and Dementia, (2018), 14(4), pp. 535-562, doi: 10.1016 / j.jalz.2018.02.018.

[0004] Currently, marker proteins for diagnosis based on A, T and N are obtained by lumbar puncture and CSF measurement or by positron emission tomography (PET) (Hansson et al., Alzheimers Dement (2018), 14(11), pp. 1470-1481, doi: 10.1016 / J.JALZ.2018.01.010; Pannee et al., J Neurochem (2016), 139(4), pp. 651-658, doi: 10.1111 / JNC.13838; Bittner et al., Alzheimers Dement (2016), 12(5), pp. 517-526, doi: 10.1016 / J.JALZ.2015.09.009; Alcolea et al., Ann Clin Transl Neurol (2019), 6(9), pp. 1815-1824, doi: 10.1002 / ACN3.50873). Subjects with preclinical AD show changes in CSF amyloid and tau levels / biomarkers. Specifically, decreased levels of Ab42 and increased levels of total Tau (tTau) and phosphorylated Tau (pTau), in particular pTau181, in CSF can be used for early diagnosis of AD (Muller et al., PLoS One. (2019), 14(8):e0221365).

[0005] In relation to PET, CSF biomarkers can have several advantages. First, CSF testing is cheaper and there is no radiation exposure issue compared to PET. Second, previous studies have shown that changes in CSF Ab42 occur before PET uptake (Jack et al., Lancet Neurol. (2013); 12(2), pp. 207-216; Bateman et al., N Engl J Med. (2012), 367(9), pp. 795-804), suggesting that CSF biomarkers can be more sensitive in identifying subjects with preclinical AD. Third, the most notable benefit of CSF biomarker analysis is that CSF provides more information about AD-related pathology compared to PET. That is, CSF samples yield information not only about the marker protein Ab but also about the tau pathology biomarker phosphorylated Tau (p-Tau) and the neurodegeneration marker total tau (t-Tau) (Apostolova et al., Neurobiol Aging. (2010), 31(8), pp. 1284-1303).

[0006] In principle, after analyzing the marker proteins in CSF, the evaluation of these marker proteins in CSF is always based on a pooled evaluation of all individual markers to form a comprehensive overall result, so that a reliable and diagnostically valuable statement can be made. In one aspect, this diagnostic strategy allows the identification of typical patterns of marker proteins, and in another aspect, it enables plausibility checks to avoid analytical errors (see Guideline for Diagnosis and Therapy in Neurology: Tumani H., Petereit H.-F. et al., Lumbalpunktion und Liquordiagnostik, S1-Leitlinie, 2019, Deutsche Gesellschaft fur Neurologie (Hrsg.), Leitlinien fur Diagnostik und Therapie in der Neurologie).

[0007] The pooled evaluation of the marker proteins Ab42, Ab40, pTau181 and tTau reveals typical patterns of these proteins in CSF. It is well known from multiple examinations of CSF of patients with neurodegenerative disorders that these marker proteins do not change independently from each other, but show significant, very strong correlated changes. For example, it is known that pTau181 (a phosphorylated form of tTau) and tTau increase almost linearly together in the course of neurodegeneration. They are also negatively correlated with the Ab42 / Ab40 ratio, i.e. a lower Ab42 / Ab40 ratio in CSF is strongly correlated with elevated pTau181 and tTau levels. These correlations are all strong and highly significant, so they can be considered as patterns forming a comprehensive overall result, which can be diagnostically interpreted as a high likelihood of the presence of AD pathology and, in addition, can indirectly exclude analytical errors.

[0008] In the case of neurodegenerative diseases, the olfactory system is involved early on. Olfactory dysfunction is among the earliest preclinical prodromal symptoms of ND diseases. Several studies were able to detect single AD biomarkers in nasal samples (such as e.g. nasal biopsies and or nasal lavage fluid). The results of these studies are clearly inconsistent. Sometimes e.g. in clinically diagnosed AD cases an increase in the concentration of Abeta42 can be reported and in other studies a decrease in concentration was found. Other studies can only detect Abeta after a harsh pre-treatment of the nasal sample with a degenerative substance. Some studies report an increase in the concentration of pTau in clinically confirmed AD cases, which can be completely unrelated to any biomarker confirmed AD and / or ND disease. All previous working groups have to cope with the fact that the number and or concentration of marker proteins in nasal samples is significantly lower, in nearly 50% of the cases below the lower limit of quantification and detection, and they additionally report a high variability, thus severely limiting the interpretation of the data (see Kim et al., Sci Rep (2019), 9(1), p. 4966, doi: 10.1038 / s41598-019-41429-1). However, due to the detection of only single proteins of the brain, a consistent pattern of marker proteins for neurodegenerative diseases like AD cannot be determined and none of the previous studies reported CSF sample amounts or CSF like quaternary structures. The cribriform plate (CP) of the anterior skull base is a thin bony structure forming the roof of the nasal cavity. It surrounds the olfactory bulb (OB), an extension of the central nervous system (CNS). Millions of olfactory nerve fibers originating from the OB pass through the CP to spread over the roof of the nasal cavity. This large area (about 23 cm 2 )(where the CNS is in communication with the nose) is also defined as the brain-nose interface (BNI). Furthermore, the BNI can also be regarded as a physiological connection between the brain and the nasal lymphatic system, which constitutes a drainage pathway for CSF and its metabolites, which enter the nasal lymphatic system along the olfactory pathway and from there into the retropharyngeal and cervical lymph nodes.

[0009] The applicant reports an applicator for positioning an absorbent matrix (AM) element within the BNI to obtain a nasal fluid sample from a subject, in particular a healthy subject or a subject suffering from subjective or objective cognitive decline possibly due to a neurodegenerative disease like AD; EP 4 000 534 A1 and WO 2022 / 101311. This nasal secretion collection device is a tool for standardized collection of nasal secretions from the vicinity of the olfactory cleft and combines the following requirements: extraction of a nasal fluid sample of sufficient high quality volume from this location while also avoiding artifacts due to e.g. blood or cell debris or nasal fluid from other nasal regions in the subsequent analysis of the collected nasal fluid sample. Furthermore, the nasal mucosa in the vicinity of the olfactory cleft should be handled with care and not exposed to any damage.

[0010] In the present application, it has been proven for the first time that marker proteins associated with neurodegenerative diseases, in particular but not limited to AD, PD and CTE, can be detected in nasal fluid samples obtained from a subject, preferably as collected from the vicinity of the olfactory fissure, and more preferably collected with the application device reported by the applicant, without changing the CSF-like pattern of the marker proteins. This finding is surprising and unexpected, as the skilled person would not have expected that nasal fluid samples can be used like CSF samples obtained by lumbar puncture, i.e. that nasal fluid samples can be used as an alternative fluid sample allowing in vitro diagnosis of neurodegenerative diseases like AD, PD and CTE. Moreover, it was unexpected that nasal fluid samples can be equally evaluated on the pooled evaluation of all individual markers to form a comprehensive overall result allowing a reliable and diagnostically valuable statement. Thus, nasal fluid samples allow the same diagnostic strategy to interpret one or more marker proteins to identify typical features of one or more marker proteins and provide plausibility checks to avoid analytical errors. In particular, the claimed invention is unexpected and surprising, as there is only limited knowledge about the peripheral outflow system of CSF across the brain-nose interface. In several mammalian studies and post-mortem studies on human cadavers, it was reported that there is a direct pathway to drain CSF into a lymphatic network located in the submucosal layer of the nose and from there to continue to the retropharyngeal and cervical lymph nodes (see Spera et al., eBioMedicine (2023), 91 : 104558 (published online: https: / / doi.org / 10.1016 / j.ebiom.2023.104558)). However, the exact anatomy and physiological mechanisms of this drainage system are still unknown. Moreover, information such as the volume of CSF drained into the lymphatic system over time or the pattern of marker proteins of the drained CSF are also unknown. Furthermore, possible disturbances or changes of the marker proteins after drainage are still unclear. Given the fact that the previously described drainage pathway is described to drain CSF directly into the lymphatic vessels across the BNI, the skilled person would have expected that it would not be possible to obtain nasal sample fluids providing these CSF-like information (e.g. about the biomarker proteins Abeta, pTau and tTau) without entering the lymphatic system covered by mucosal tissue. Moreover, it would have been completely unexpected for the skilled person to find almost identical patterns or inter-protein correlations between one or more marker proteins, and the skilled person would also not have expected to find almost identical distributions of different oligomeric protein species of each marker protein or equal or even higher amounts of these marker proteins in nasal fluid samples.

[0011] Thus, the problem to be solved by the present application is to identify an alternative biological sample that can produce a comparable diagnosis of neurodegenerative diseases like AD, PD and CTE. SUMMARY

[0012] The present invention solves this technical problem by providing embodiments as defined in the claims. In particular, the present invention provides a nasal fluid sample obtained from a subject, said nasal fluid sample comprising the marker proteins amyloid-beta (A-beta / Aβ), phosphorylated Tau (p-Tau / pTau) and / or total Tau (t-Tau / tTau). The present invention further provides said nasal fluid sample for use in a method of diagnosing a neurodegenerative disease and the use of said nasal fluid sample for diagnosing a neurodegenerative disease. The present invention further relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject.

[0013] In the appended examples, by analyzing marker proteins associated with neurodegenerative diseases, the inventors surprisingly and surprisingly demonstrated that a nasal fluid sample obtained from a subject according to the present invention allows to make the same diagnosis as a CSF analysis of the same subject. It has been surprisingly and unexpectedly found that a nasal fluid sample according to the present invention comprises almost the same marker protein profile and / or protein correlations as also seen in a CSF sample. It is also completely unexpected that the distribution of different oligomeric protein species of one or more marker proteins is almost identical in a nasal fluid sample and a corresponding CSF sample from the same individual. Furthermore, it is unexpected that the concentration of one or more marker proteins in the nasal fluid is equal or even higher than in the corresponding CSF sample.

[0014] It has been shown for the first time that by analyzing a nasal fluid sample one can quantify the same one or more marker proteins as in a corresponding CSF sample. It has also been shown for the first time that the quantified one or more marker proteins in nasal fluid follow the same pattern as in CSF when evaluated in a pooled evaluation in the sense of a comprehensive overall result. This means that it has been shown for the first time that e.g. the Aβ42 / Aβ40 ratio is negatively correlated with the tau markers. Likewise, it has been shown that the two tau isoforms (pTau and tTau) show the same strong almost linear correlation with each other, which means that whenever pTau is elevated, tTau is also elevated. Thus, it has been shown for the first time that in a nasal fluid sample the same pattern can be used to provide a plausibility check to avoid analytical errors and that nasal fluid can be used to identify patterns or correlations of marker proteins which are typical for the presence of a neurodegenerative disease in a subject (e.g. low Aβ42 / Aβ40 ratio and high pTau and tTau levels are correlated with AD). In addition, by comparing two corresponding samples from the same subject (i.e. a nasal fluid sample and a CSF sample), it has also been shown that the distribution (different sizes and amounts) of monomers and oligomers also shows almost the same pattern. Thus, the oligomer pattern of a nasal sample represents the oligomer pattern in a CSF sample from the same subject. Thus, analyzing one or more nasal fluid samples from a subject with respect to the relative protein amounts and by quantifying different oligomer species can allow to identify subjects suffering from different neurodegenerative diseases. For example, when comparing nasal fluid samples of subjects with amyloid pathology (A+) or without amyloid pathology (A-), these individuals show different oligomeric structures and relative amounts of one or more marker proteins. This has never been shown before and since it was surprising to first find out that CSF is, it is surprising and completely unexpected that nasal fluid samples with equal or even higher amounts of one or more marker proteins can be measured or analyzed and that their almost identical pattern and oligomeric structures can be analyzed. BRIEF DESCRIPTION OF DRAWINGS

[0015] The accompanying drawings illustrate:

[0016] Figure 1: Color scale to assess blood content of nasal secretion eluate.

[0017] Figure 2: Aβ40: Different amounts of oligomers can be detected at 56 kDa and 100 kDa using Aβ40 specific antibodies from R and D by Simple Western™ Jess.

[0018] Figure 3: Aβ40: Examples of CSF-validated A- and A+ individuals classified by a 56 kDa band. Using an Aβ40-specific antibody from Rand D, A+ subjects had fewer 56 kDa Aβ40 oligomers compared to A- subjects.

[0019] Figure 4: Aβ40: Lower molecular weight oligomers and monomers can be detected by Simple Western™ Jess using Aβ40-specific antibodies from R and D. Subjects with A- had higher amounts of monomeric Aβ-40.

[0020] Figure 5: Aβ40: Compared to A+, A- subjects had more < 12 kDa monomers, as shown in this exemplary figure from two subjects using Aβ40-specific antibodies from R and D.

[0021] Figure 6: Aβ42: A+ with reduced total Aβ42 levels and altered ratio of oligomeric bands at 56 kDa to 32 kDa, as illustrated by an A+ subject and an A- subject using an Aβ42-specific antibody from Fujifilm Wako.

[0022] Figure 7: Using the 6E10 antibody, the pan-Aβ antibody recognizes multiple oligomeric Aβ structures in nasal secretions.

[0023] Figure 8: Increased tTau levels were detected in N+ subjects relative to N-, as illustrated in this example using a Tau-specific antibody from Biolegend in both N+ and N- subjects.

[0024] Figure 9: The level of pTau181 is reduced in T+ compared to T-, as shown in this example using a pTau-specific antibody from Cell Signaling in a T+ patient sample and a T- patient sample.

[0025] Figure 10: Aβ40: Direct comparison of Aβ40 structures identified in nasal fluid and CSF samples from the same individual / subject using an Aβ40-specific antibody from BioLegend (purified (azide-free) anti-β-amyloid, 1-40 (11A50-B10), BioLegend, catalog number 805409) revealed nearly identical structures at approximately 62 kDa and approximately 231 kDa. The applied CSF sample volume was 6 times larger, indicating that the amount of structures at approximately 62 kDa and approximately 231 kDa in the nasal secretion sample was at least equivalent to or higher than that in the CSF. Additional structures at approximately 20 kDa, 32 kDa, and 98 kDa were found in the nasal secretion sample.

[0026] Figure 11 : Αβ42: Direct comparison of Αβ42 structures recognized in nasal secretion samples and CSF samples from the same individuals using Αβ42 specific antibody from Fujifilm Wako (Anti-amyloid beta 42 (43), Monoclonal antibody (BC05), Fujifilm Wako, Cat. No. 010-26903) revealed structures at approximately 33 kDa, 56 kDa, 75 kDa and 99 kDa in nasal secretion samples. These structures were not observed in CSF even though the applied CSF sample volume was six times higher. This indicates that the amount of structures observed in nasal secretion samples is higher than in the corresponding CSF samples.

[0027] Figure 12: tTau: Direct comparison of tTau structures recognized in nasal secretion samples and CSF samples from the same individuals using tTau specific antibody from BioLegend (Purified Anti-Tau, 404-441 antibody (Mouse), BioLegend, Cat. No. 806601) revealed almost identical structures at 63 kDa. The applied CSF sample volume was six times higher, indicating that the amount of structures at approximately 63 kDa in nasal secretion samples is at least comparable or higher than in CSF.

[0028] Figure 13: pTau: Direct comparison of pTau structures recognized in nasal secretion samples and CSF samples from the same individuals using pTau specific antibody from Cell Signaling Technology (Phospho-Tau (Thr181) (D9F4G) Rabbit mAb, Cell Signaling, Cat. No. 12885S) revealed almost identical structures at approximately 60 kDa, approximately 100 kDa, approximately 146 kDa and approximately 230 kDa. The applied CSF sample volume was six times higher than the nasal secretion volume, indicating that the amount of structures at approximately 60 kDa, approximately 100 kDa, approximately 146 kDa and approximately 230 kDa in nasal secretion samples is at least comparable or higher than in CSF. An additional structure can be found at approximately 20 kDa in nasal secretion samples.

[0029] Figure 14: Analysis within the two matrices (nasal fluid samples (Figure 14A) and CSF (Figure 14B)) of the respective individuals showed a similar significant inter-protein correlation between Αβ42 and Αβ40 (r = 0.63; p < 0.001, N = 79).

[0030] Figure 15: Analysis within both matrices (nasal fluid sample (Figure 15A) and CSF (Figure 15B) of respective individuals showed similar significant inter-protein correlation between pTau and tTau (r = 0.78, p < 0.001, N = 78).

[0031] Figure 16: Analysis within both matrices (nasal fluid sample (Figure 16A) and CSF (Figure 16B) of respective individuals showed similar significant inter-protein correlation between Αβ42 / Αβ40 and pTau (r = -0.345, p = 0.003, N = 73).

[0032] Figure 17: Comparison of marker protein correlations in respective nasal fluid samples and CSF. DETAILED DESCRIPTION

[0033] The application is described in more detail hereinafter.

[0034] In particular, the present application relates to the following items:

[0035] 1. A nasal fluid sample obtained from a subject, wherein the nasal fluid sample comprises the marker proteins amyloid-beta (Αβ), phosphorylated Tau (pTau) and / or total Tau (tTau).

[0036] 2. The nasal fluid sample according to item 1, wherein the nasal fluid sample is a purified nasal fluid sample.

[0037] 3. The nasal fluid sample according to item 1 or 2, wherein the nasal fluid sample of a patient suffering from cognitive impairment due to a neurodegenerative disease is characterized by a decrease of Αβ42 protein concentration relative to Αβ40 protein concentration.

[0038] 4. The nasal fluid sample according to any one of items 1 to 3, wherein the concentration of the one or more marker proteins is identical or increased by at least 1.1-fold compared to a cerebrospinal fluid (CSF) sample obtained from the same subject.

[0039] 5. The nasal fluid sample according to any one of items 1 to 4, wherein the nasal fluid sample is characterized by a pTau protein concentration that is less than the tTau protein concentration.

[0040] 6. The nasal fluid sample according to any one of items 1 to 5, wherein pTau and tTau are positively correlated and / or Αβ42 and Αβ40 are positively correlated.

[0041] 7. The nasal fluid sample according to any one of items 1 to 6, wherein the relative amount of Αβ42 / Αβ40 is greater than 0 and less than 1.

[0042] 8. The nasal fluid sample according to any one of items 1 to 7, wherein the relative amount of Αβ42 / Αβ40 is negatively correlated with pTau and / or tTau.

[0043] 9. The nasal fluid sample according to any one of items 1 to 8, wherein the Αβ marker is characterized by a molecular weight of about 4 kDa, about 8 kDa, about 12 kDa, about 16 kDa, about 19 kDa, about 24 kDa, about 32 kDa, about 40 kDa, about 44 kDa, about 48 kDa, about 52 kDa, about 56 kDa, about 60 kDa to 72 kDa, about 84 kDa to 120 kDa, and / or greater than about 140 kDa.

[0044] 10. The nasal fluid sample according to any one of items 1 to 8, wherein the pTau marker is characterized by a molecular weight of about 20 kDa, about 30 kDa, about 38 kDa, about 55 kDa to 62 kDa, about 96 kDa to 106 kDa, and about 140 kDa to 160 kDa, and / or greater than about 180 kDa.

[0045] 11. The nasal fluid sample according to any one of items 1 to 8, wherein the tTau marker is characterized by a molecular weight of about 30 kDa, about 38 kDa, about 48 kDa, about 55 kDa to 62 kDa, about 96 kDa to 106 kDa, about 140 kDa to 160 kDa, and greater than 160 kDa.

[0046] 12. Use of the nasal fluid sample according to any one of items 1 to 11 for aiding in the diagnosis of a neurodegenerative disease.

[0047] 13. A method for aiding in the diagnosis of a neurodegenerative disease in a subject in a nasal fluid sample, wherein the method comprises the following steps:

[0048] (a) determining the protein concentration of the marker proteins phospho-Tau (pTau), total Tau (tTau), and / or optionally amyloid-beta (Αβ) in a nasal fluid sample according to any one of items 1 to 11 ;

[0049] (b) comparing the protein concentration of the marker proteins as determined in the nasal fluid sample with the protein concentration in a control; and

[0050] (c) determining whether the values of the marker proteins are pathologically altered relative to the protein concentration of the control.

[0051] 14. The method according to item 13, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer’s disease (AD), Parkinson’s disease (PD), chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), Pick’s disease and Creutzfeldt-Jakob disease.

[0052] 15. The method according to item 13 or 14, wherein a decrease in the concentration of Ab42 protein relative to the concentration of Ab40 protein is evidence of AD in a subject with cognitive impairment.

[0053] 16. The method according to any one of items 13 to 15, wherein a negative correlation of the relative amount of Ab42 / Ab40 with pTau and / or tTau is evidence of a neurodegenerative disease in the subject.

[0054] 17. The method according to any one of items 13 to 15, wherein a positive correlation of the relative amount of Ab42 / Ab40 with pTau and / or tTau is evidence of a neurodegenerative disease in the subject.

[0055] 18. The method according to item 16, wherein any one of the following (i) or (ii) applies:

[0056] (i) pTau is positively correlated with tTau and Ab42 is positively correlated with Ab40; and

[0057] (ii) the relative amount of Ab42 / Ab40 is greater than 0 and less than 1.

[0058] 19. The method according to item 17, wherein any one of the following (i) or (ii) applies:

[0059] (i) pTau is positively correlated with Ab42 / Ab40 and Ab42 is negatively correlated with Ab40; and

[0060] (ii) the relative amount of Ab42 / Ab40 is greater than 0 and less than 1.

[0061] 20. The method according to any one of items 13, 14, 16, 17, 18 or 19, wherein the neurodegenerative disease is AD.

[0062] 21. The use of a nasal fluid sample according to item 12, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer’s disease (AD), Parkinson’s disease, chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), Pick’s disease and Creutzfeldt-Jakob disease (CJD).

[0063] As mentioned above, the present application provides a nasal fluid sample obtained from a subject, said nasal fluid sample comprising one or more marker proteins beta amyloid (A-beta, also described herein as Ab), phosphorylated Tau (pTau or also described as p-Tau) and / or total Tau (tTau or also described as t-Tau). The skilled person knows that beta amyloid (in particular Ab42 and / or Ab40), pTau (in particular pTau181, pTau231 and / or pTau217) and / or tTau are marker proteins reported to be relevant for the diagnosis of a neurodegenerative disease. As used herein, the term “comprising” is to be understood as indicating that the nasal fluid sample comprises Ab, pTau and / or tTau, but can also comprise one or more additional marker proteins relevant for a neurodegenerative disease, such as alpha synuclein, neurofilament light chain (NFL), glial fibrillary acidic protein (GFAP), MTBR-Tau243, myeloid cell trigger receptor 2 (sTREM2), ubiquitin (Ub), protein s100a, apolipoprotein E epsilon 4 (ApoE4), superoxide dismutase 1 (SOD1), RNA binding protein FUS / TLS (FUS), TAR DNA binding protein 43 (TDP-43), granulin (GRN), misfolded prion protein (PrPSc), mutant and wild-type huntingtin protein (Htt) and IgM and IgG against Epstein-Barr virus. Preferably, the one or more additional marker proteins is alpha synuclein, neurofilament light chain (NFL), glial fibrillary acidic protein (GFAP), protein s100a and / or apolipoprotein E epsilon 4 (ApoE4).

[0064] The terms “subject”, “patient” and “individual” can be used synonymously herein and refer to an organism (preferably a mammal, most preferably a human) which is diagnosed by using a nasal fluid sample according to the present application from said organism. Thus, the skilled person is well aware that a nasal fluid sample refers to an in vitro sample obtained from a subject.

[0065] The present application further relates to a nasal fluid sample comprising marker proteins specific for a neurodegenerative disease, and the use of such a nasal fluid sample for aiding the diagnosis of a neurodegenerative disease. The nasal fluid sample as described herein can be used in a method of diagnosing or aiding the diagnosis of one or more neurodegenerative diseases in a subject. Thus, it is considered that the present application encompasses the nasal fluid sample, the use of the nasal fluid sample and the method of aiding the diagnosis of a neurodegenerative disease by using a nasal fluid sample as described herein. Furthermore, the use of the nasal fluid sample relates to i) selecting a suitable therapy for a subject, ii) monitoring a subject’s response to a therapy, iii) evaluating the likelihood of a positive response to a therapy, and / or iv) evaluating the likelihood of adverse events in response to a therapy.

[0066] The present application further relates to the use of a nasal fluid sample as described herein. In particular, the present application relates to the use of a nasal fluid sample for aiding in the diagnosis of a neurodegenerative disease. According to the above definition, the use of a purified nasal fluid sample preferably relates to aiding in the diagnosis of a neurodegenerative disease.

[0067] It is envisaged that what is described herein in relation to a nasal fluid sample equally applies to the use of a nasal fluid sample as described herein, and to the method for aiding in the diagnosis as described herein.

[0068] The term "diagnosis" is used herein in the broadest sense, and refers to the determination or confirmation of a disease or condition in a subject, in particular based on the results of various diagnostic procedures, including the detection of protein levels or concentrations according to at least some embodiments of the present application in a biological sample obtained from the subject. The term "aiding in the diagnosis" is used herein as any method which can be used sequentially and / or simultaneously with diagnostic methods known in the art for pre-screening, complementing or verifying the diagnosis of a disease or condition in a subject.

[0069] In the context of the present application, the term "diagnosis" is to be understood as clinical diagnosis. In the context of the present application, the term "evidence for a neurodegenerative disease" is to be understood as aiding in the diagnosis of a neurodegenerative disease based on the results of the analysis of marker proteins in a nasal fluid sample obtained from a subject.

[0070] The skilled person is well aware of which diseases belong to the term "neurodegenerative disease". Examples of neurodegenerative diseases can be Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), Pick's disease, Creutzfeldt-Jakob disease (CJD), Lewy body dementia, vascular dementia (VD), Huntington's disease (HD), rapid eye movement sleep behavior disorder, multiple system atrophy (MSA), amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). Preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), Pick's disease and Creutzfeldt-Jakob disease (CJD). More preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD) and chronic traumatic encephalopathy (CTE). Even more preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease (AD) and Parkinson's disease (PD). Most preferably, the neurodegenerative disease is Alzheimer's disease (AD).

[0071] The present invention provides a nasal fluid sample of a subject comprising the marker proteins amyloid-beta (Αβ), phosphorylated Tau (pTau) and / or total Tau (tTau) for use in a method of diagnosing a neurodegenerative disease, and the use of said nasal fluid sample for diagnosing a neurodegenerative disease. Furthermore, the present invention relates to a nasal fluid sample of a subject comprising the marker proteins amyloid-beta (Αβ), phosphorylated Tau (pTau) and / or total Tau (tTau) for use in a method of diagnosing a neurodegenerative disease, and the use of said nasal fluid sample for diagnosing a neurodegenerative disease, wherein the neurodegenerative disease is preferably selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease, chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), Pick's disease and Creutzfeldt-Jakob disease (CJD).

[0072] The subject can be treated according to evidence of a neurodegenerative disease, and / or can be selected for a study, e.g. according to evidence of a neurodegenerative disease.

[0073] The research framework for AD diagnosis developed under the support of the National Institute on Aging and the Alzheimer’s Association (Jack (2018), Alzheimers. Dement., 14(4), 535-562) suggests the classification of individuals using the so-called A / T / N (amyloid, tau, neurodegeneration) classification system according to the evidence of pathological marker proteins. According to the ATN system, the presence of (i) Ab42 (CSF Ab42 or amyloid positron emission tomography-computed tomography (PET-CT): “A”), (ii) hyperphosphorylated Tau (CSF pTau181 or Tau PET-CT: “T”), and (iii) neurodegeneration (atrophy, fluorodeoxyglucose (FDG), PET-CT, or CSF total tau (tTau) based on structural magnetic resonance imaging (MRI): “N”) in each individual is assessed. This A / T / N classification is globally recognized as a system to diagnose AD and to initiate appropriate therapeutic measures (consequence) (Jack (2016), Neurology, 87(5), 539-547; Grontvedt (2020), J. Alzheimers Dis. 74, 829-837). According to the A / T / N classification, subjects are classified into three binary categories A, T, and N. That is, according to the A / T / N classification, subjects are classified as A+ or A-, T+ or T-, and N+ or N-. It is noted that for some applications, it can be sufficient to classify subjects in only one of the three categories. For example, when classifying subjects according to the A category only and classifying as A+, the subject can be diagnosed with AD. Typically, when classifying subjects as A+, the subject has AD.

[0074] As used herein, the term “marker protein” refers to a protein that can be associated with a neurodegenerative disease and is found in a nasal fluid sample and analyzed in the context of the methods described herein for aiding in the diagnosis of a neurodegenerative disease in a subject.

[0075] As used herein, the term “biomarker” refers to a protein that can be associated with a neurodegenerative disease and is found in a nasal fluid sample and analyzed in the context of the methods described herein for aiding in the diagnosis of a neurodegenerative disease in a subject.

[0076] As used herein, the term “analyte” refers to a protein that can be associated with a neurodegenerative disease and is found in a nasal fluid sample and analyzed in the context of the methods described herein for aiding in the diagnosis of a neurodegenerative disease in a subject.

[0077] The term "marker protein" can be used interchangeably with "analyte", "protein", "marker" or "biomarker". It will be apparent to the skilled person that depending on the context, "protein" can refer to a marker protein, an analyte or a biomarker which is analyzed in the methods described herein. However, "protein" can also refer to all proteins in a nasal fluid sample, i.e. all proteins in a nasal fluid sample, including proteins which are known to be not associated with a degenerative disease of the nervous system.

[0078] In the context of the present application, the term "marker protein", "analyte", "marker" or "biomarker" can refer to total protein, e.g. beta amyloid, for which one or more protein concentrations of one or several marker proteins are determined.

[0079] In other words, "marker protein", "analyte" or "biomarker" can refer to the sum of several "marker proteins", "analytes" or "biomarkers". Thus, when reference is made herein to analyzing / testing a "marker protein", "analyte" or "biomarker", this can mean that one or several marker proteins are determined. For example, when reference is made herein to analyzing / testing a "marker protein", "analyte" or "biomarker" beta amyloid, the protein concentrations of the marker proteins Ab40 and Ab42 can be determined. But it can also refer to only one of the isoforms of beta amyloid, e.g. Ab40 or Ab42, for which the protein concentration is determined.

[0080] Thus, it will also be apparent to the skilled person that, for example, the term "(total) protein level" can refer to the level of all proteins in a given sample. However, the term "(total) protein level of beta amyloid" refers to the protein level of beta amyloid. The term "(total) protein level of Ab40" refers to the protein level of the beta amyloid isoform Ab40 and the term "(total) protein level of Ab42" refers to the protein level of the beta amyloid isoform Ab42. It is envisaged that the explanations here for beta amyloid apply of course also to pTau and tTau.

[0081] The terms "protein concentration", "level", "amount", "protein level" and "protein amount" can be used synonymously herein. The terms "total protein concentration", "total level", "total amount", "total protein level", "total protein amount", "whole protein concentration", "whole protein level" and "whole protein amount" can be used synonymously herein. These terms can refer to a concentration or can be expressed as a concentration, such as mg / mL or µM. However, they can also be expressed as arbitrary units. For example, it is envisaged herein that the analyte / marker protein / biomarker / protein of interest is measured via immuno-detection. The analyte can first be contacted with a primary antibody specific for the analyte, and after the primary antibody has associated with the analyte, a secondary antibody specific for the primary antibody can be added. The secondary antibody can be labeled with a moiety that generates a detectable signal, such as a fluorophore, or a molecule that generates chemiluminescence. The fluorescence or chemiluminescence can be detected and can be proportional to the amount of analyte in the sample. The fluorescence or chemiluminescence can be expressed in arbitrary units.

[0082] It is envisaged that prior to association with the primary antibody, the analyte of interest in the sample can be separated, for example via molecular weight. In this case, the detectable signal can be proportional to different quaternary structures of the analyte in the sample, for example.

[0083] It is also envisaged that the methods described herein comprise electrophoretic techniques. Thus, it is envisaged that the protein level is determined via an electrophoretic technique. Thus, it is envisaged that the protein level is determined via an electrophoretic technique that comprises separation of quaternary structures and / or isoforms.

[0084] The term "fold increase" is herein to be understood synonymously with "fold", wherein the numerical value is a positive number greater than 1. Specifically, a value A which is increased 3-fold compared to a value B is to be understood as meaning that the value of A is equal to 3 times the value of B. Preferably, the concentration of the one or more marker proteins in the nasal fluid sample according to the application is equal or increased at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6-fold compared to a cerebrospinal fluid (CSF) sample obtained from the same subject. More preferably, the concentration of the one or more marker proteins is increased at least 2-fold compared to a cerebrospinal fluid (CSF) sample obtained from the same subject. Even more preferably, the concentration of the one or more marker proteins is increased at least 4-fold compared to a cerebrospinal fluid (CSF) sample obtained from the same subject. Most preferably, the concentration of the one or more marker proteins is increased at least 6-fold compared to a cerebrospinal fluid (CSF) sample obtained from the same subject.

[0085] It is therefore also apparent to the person skilled in the art that, for example, the term "(total) protein concentration" can refer to the concentration of all proteins in a given sample. However, the term "(total) protein concentration of amyloid-beta" refers to the protein concentration of amyloid-beta. The term "(total) protein concentration of Αβ40" refers to the protein concentration of amyloid-beta isoform Αβ40 and the term "(total) protein concentration of Αβ42" refers to the protein concentration of amyloid-beta isoform Αβ42. It is envisaged that the explanations here for amyloid-beta apply of course also to pTau and tTau.

[0086] It is also envisaged that the terms "relative concentration", "relative value", "relative amount", "relative level", "relative protein amount" and "relative protein level" are synonymous in the context of the present application.

[0087] The skilled person will understand that the concentration of one or more marker proteins (or fragments thereof, or precursors, or fragments thereof) in a nasal fluid sample of a subject can be further analyzed to identify subjects that can be diagnosed as having a neurodegenerative disorder. For example, a ratio of one marker protein to another protein, i.e. the relative amount of one marker protein relative to another marker protein, can be derived from the measured protein concentrations. This marker protein ratio from a subject can be compared to values (also referred to herein as reference values) of the respective marker proteins collected from a group of “control” subjects to determine whether the subject has any evidence of a neurodegenerative disorder.

[0088] It is envisaged that the protein concentration of one or more marker proteins of a key analyte is determined in the methods described herein. In the context of the present application, the term “protein concentration of a marker protein” means the protein concentration obtained from the measurement of the analyte / marker protein / biomarker / protein described herein and can include the total protein concentration of the analyte / marker protein / biomarker / protein.

[0089] In the context of the present application, the terms “marker protein for an analyte” and “marker protein of an analyte” can be used synonymously herein. Preferably, the key analyte is beta amyloid (e.g. beta amyloid 40 (Ab40) and beta amyloid 42 (Ab42), as well as pannamyloid beta), phosphorylated Tau (pTau) and total Tau (tTau). It is important to note that for each of beta amyloid, pTau and tTau, more than one marker protein concentration can be determined in the context of the methods described herein. In the context of the present application, further marker proteins can be determined in the nasal fluid sample. In particular, the skilled person is aware of one or more specific marker proteins that are associated with one or more of the neurodegenerative disorders described herein. For example, such one or more further marker proteins associated with a neurodegenerative disorder can include alpha synuclein, neurofilament light chain (NFL), glial fibrillary acidic protein (GFAP), MTBR-Tau243, triggering receptor expressed on myeloid cells 2 (sTREM2), ubiquitin (Ub), protein s100a, apolipoprotein E epsilon 4 (ApoE4), superoxide dismutase 1 (SOD1), RNA binding protein FUS / TLS (FUS), TAR DNA binding protein 43 (TDP-43), granulin (GRN), misfolded prion protein (PrPSc), mutant and wild-type huntingtin protein (Htt) and IgM and IgG against Epstein-Barr virus. Preferably, the one or more further marker proteins are alpha synuclein, neurofilament light chain (NFL), glial fibrillary acidic protein (GFAP), protein s100a and / or apolipoprotein E epsilon 4 (ApoE4).

[0090] As mentioned above, the nasal fluid sample described herein can comprise the marker protein Αβ (Αβ). The skilled person knows that amyloid-β is present in the organism in different isoforms which can form different quaternary structures (oligomers). The isoforms of amyloid-β can have any length of the amyloid-β peptide 1 to 38, 1 to 39, 1 to 40, 1 to 41, 1 to 42, 1 to 43, or any N-terminally truncated isoform x to 38, x to 39, x to 40, x to 41, x to 42, x to 43, wherein "x" stands for any N-terminally truncated position, e.g. any position between amino acid position 1 to 30, preferably 1 to 10, most preferably position 2, 3 or 4. In the present invention, preferably, Αβ is present as isoform amyloid-β 40 (Αβ40) and / or amyloid-β 42 (Αβ42).

[0091] The skilled person knows that Αβ40 and Αβ42 are derived from a common precursor called amyloid precursor protein (APP) and are produced in about the same amount in vivo. In the present invention, the nasal fluid sample as described herein can be characterized by an increased Αβ42 protein concentration relative to the Αβ40 protein concentration, but preferably by a decreased Αβ42 protein concentration relative to the Αβ40 protein concentration, preferably in case of a neurodegenerative condition. The skilled person knows that neurodegenerative diseases are usually associated with a molecular structural change of Αβ42 leading to plaque formation. The skilled person understands that in this preferred embodiment the Αβ42 / Αβ40 protein ratio is decreased which is indicative for the presence of a neurodegenerative disease. In particular, the Αβ42 / Αβ40 protein ratio is decreased when compared to a healthy subject, i.e. a subject, preferably a mammal, most preferably a human, who does not suffer from any cognitive impairment due to a neurodegenerative disease. Furthermore, the present invention relates to a purified nasal fluid sample obtained from a subject, wherein the nasal fluid sample of a patient suffering from a cognitive impairment due to a neurodegenerative disease is characterized by an increased Αβ42 protein concentration relative to the Αβ40 protein concentration or by a decreased Αβ42 protein concentration relative to the Αβ40 protein concentration. As shown in the accompanying examples, in particular in Figure 14A, the nasal fluid sample of a patient suffering from a cognitive impairment due to a neurodegenerative disease is characterized by a decreased Αβ42 protein concentration relative to the Αβ40 protein concentration.

[0092] As mentioned above, the nasal fluid sample described herein can comprise the marker protein pTau. The skilled person is aware that pTau exists in different isoforms in an organism. Isoforms of pTau are any phosphorylated form of Tau, including but not limited to pTau181, pTau202, pTau205, pTau217, pTau231, pTau199, pTau18, pTau396 and / or pTau422. Preferably, pTau is pTau181 and / or pTau231 and / or pTau217.

[0093] Isoforms of tTau include variants (fragments) produced by alternative splicing or proteolytic processing, including but not limited to the alternatively spliced forms 2N4R, 2N3R, 1N4R, 1N3R, ON4R, ON3R and / or fragments 1 to 314, 187 to 441, 1 to 255, 1 to 368, 151 to 421, 45 to 230, 243 to 441.

[0094] The present invention relates to a nasal fluid sample characterized by a pTau protein concentration that is less than a tTau protein concentration. The present invention further provides a nasal fluid sample for use in a method of aiding in the diagnosis of a neurodegenerative disease, said nasal fluid sample being characterized by a pTau protein concentration that is less than a tTau protein concentration; and the use of said nasal fluid sample for the diagnosis of a neurodegenerative disease. The present invention further relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject by using a nasal fluid sample, said nasal fluid sample being characterized by a pTau protein concentration that is less than a tTau protein concentration. The present invention also relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject by using a nasal fluid, wherein an increased or alternatively decreased Aβ42 protein concentration relative to Aβ40 protein concentration is evidence of AD in a subject with cognitive impairment. Preferably, a decreased Aβ42 protein concentration relative to Aβ40 protein concentration is evidence of AD in a subject with cognitive impairment.

[0095] The present invention relates to a nasal fluid sample characterized by a pTau protein concentration less than a tTau protein concentration, and wherein pTau is positively correlated with tTau. The present invention further provides a nasal fluid sample for use in a method of aiding in the diagnosis of a neurodegenerative disease, said nasal fluid sample characterized by a pTau protein concentration less than a tTau protein concentration, wherein pTau is positively correlated with tTau; and use of said nasal fluid sample for the diagnosis of a neurodegenerative disease. The present invention further relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject by using a nasal fluid sample, wherein a negative correlation of the relative amount of Aβ42 / Aβ40 with pTau and / or tTau is evidence of a neurodegenerative disease in the subject. Alternatively, a method for aiding in the diagnosis of a neurodegenerative disease in a subject by using a nasal fluid sample can be characterized in that a positive correlation of the relative amount of Aβ42 / Aβ40 with pTau and / or tTau is evidence of a neurodegenerative disease in the subject. The present invention also relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject by using a nasal fluid sample, wherein a negative correlation of the relative amount of Aβ42 / Aβ40 with pTau and / or tTau is evidence of a neurodegenerative disease in the subject, and wherein either of the following (i) or (ii) applies: (i) pTau is positively correlated with tTau and Aβ42 is positively correlated with Aβ40, alternatively pTau is positively correlated with tTau and Aβ42 is negatively correlated with Aβ40; and (ii) the relative amount of Aβ42 / Aβ40 is greater than 0 and less than 1. Alternatively, the present invention relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject by using a nasal fluid sample, wherein a positive correlation of the relative amount of Aβ42 / Aβ40 with pTau and / or tTau is evidence of a neurodegenerative disease in the subject, and wherein either of the following (i) or (ii) applies: (i) pTau is positively correlated with Aβ42 / Aβ40 and Aβ42 is negatively correlated with Aβ40; and (ii) the relative amount of Aβ42 / Aβ40 is greater than 0 and less than 1.

[0096] As shown in the appended examples, preferably, pTau is positively correlated with tTau and Aβ42 is positively correlated with Aβ40.

[0097] As explained herein, the amyloid-beta protein exists in different isoforms (e.g. A940 and A942). As also explained herein, these different isoforms in turn can form different quaternary structures (e.g. oligomers). Thus, there is a total concentration of a certain isoform (i.e. the sum of all quaternary structures of said isoform) and a concentration of a specific quaternary structure of said isoform. For example, for A940, there are quaternary structures with a molecular weight of 56 kDa and 97 kDa, for example. Said quaternary structures exist in different concentrations (as evident from the chemiluminescence signal in the examples).

[0098] As an example, when it is explained herein to determine one or more protein concentrations of one or more marker proteins of amyloid-beta, it is meant to determine one or more protein concentrations of one or several marker proteins. However, in addition, one or more protein concentrations of total amyloid-beta can be determined. Thus, it is envisaged to determine a protein concentration of a marker protein of total amyloid-beta, a protein concentration of a marker protein of A940 and a protein concentration of a marker protein of A942. All three protein concentrations of marker proteins can then be used to determine the ratio of interest.

[0099] It is envisaged that the explanations herein with respect to amyloid-beta apply of course also to pTau and tTau. In pTau, pTau-181 is preferred, but other isoforms can also be preferred, such as pTau231 and pTau217.

[0100] The term “quaternary structure” is used herein in the broadest sense. A protein / polypeptide can have a primary structure, a secondary structure, a tertiary structure and a quaternary structure. The quaternary structure of a protein is the association of several protein chains or polypeptide chains into an arrangement (closely packed). For example, two proteins / polypeptides / oligopeptides can form a dimer, and three proteins / polypeptides / oligopeptides can form a trimer. It is noted that quaternary structures as used herein refer to monomers and oligomers comprising two up to several thousand proteins / polypeptides. Quaternary structures can be distinguished by the number of protein chains or polypeptide chains comprised in the quaternary structure, or by the molecular weight (e.g. kDa) of the quaternary structure (e.g. as determined by the running behavior in e.g. electrophoretic techniques). It is envisaged that quaternary structures and / or isoforms between about 2 kDa and about 440 kDa can be isolated. In other words, the techniques comprising the isolation of quaternary structures and / or isoforms can isolate quaternary structures and / or isoforms with a molecular weight between about 2 kDa and about 440 kDa (such as about 2 kDa to about 40 kDa, about 12 kDa to about 230 kDa or about 66 kDa to about 440 kDa, preferably about 2 kDa to about 40 kDa, about 12 kDa to about 230 kDa).

[0101] One or more protein concentrations of amyloid-beta can include total amyloid-beta full protein levels and / or total amyloid-beta quaternary structure protein levels and / or amyloid-beta isoform full protein levels and / or amyloid-beta isoform quaternary structure protein levels. One or more protein concentrations of tTau can include total tTau full protein levels and / or total tTau quaternary structure protein levels and / or tTau isoform full protein levels and / or tTau isoform quaternary structure protein levels. One or more protein concentrations of pTau can include total pTau full protein levels and / or total pTau quaternary structure protein levels and / or pTau isoform full protein levels and / or pTau isoform quaternary structure protein levels.

[0102] As mentioned, the methods described herein can include determining protein levels via techniques that separate quaternary structures and / or isoforms including by size, molecular weight, or charge. For example, any form of gel electrophoresis, isoelectric focusing, size exclusion chromatography, or gel filtration chromatography.

[0103] Observed molecular weights in kDa can vary depending on the technique, device, buffer, individual / subject, etc. It is contemplated that observed molecular weights can vary by up to 10%. Thus, the term "about" as used herein can mean that the depicted value can vary + / - 10%.

[0104] Molecular weights of amyloid-beta can be about 4 kDa, about 19 kDa, about 24 kDa, about 32 kDa, about 40 kDa, about 44 kDa, about 48 kDa, about 52 kDa, about 56 kDa, about 60 kDa to 72 kDa, about 84 kDa to 120 kDa, and / or greater than about 140 kDa. Thus, it is contemplated that quaternary structures of amyloid-beta having the above molecular weights are included in the protein concentration of amyloid-beta. Thus, the present disclosure relates to a nasal fluid sample wherein the Abeta marker is characterized by a molecular weight of about 4 kDa, about 19 kDa, about 24 kDa, about 32 kDa, about 40 kDa, about 44 kDa, about 48 kDa, about 52 kDa, about 56 kDa, about 60 kDa to 72 kDa, about 84 kDa to 120 kDa, and / or greater than about 140 kDa.

[0105] The molecular weight of pTau can be about 20 kDa, about 30 kDa, about 38 kDa, about 55 kDa to 62 kDa, about 96 kDa to 106 kDa, about 140 kDa to 160 kDa and / or greater than about 180 kDa. Thus, it is envisaged that the quaternary structure of pTau having the above-mentioned molecular weights is comprised in the protein concentration of phosphorylated Tau. Accordingly, the present application relates to a nasal fluid sample wherein the pTau marker is characterized by a molecular weight of about 30 kDa, about 38 kDa, about 55 kDa to 62 kDa, about 96 kDa to 106 kDa and about 140 kDa to 160 kDa.

[0106] The molecular weight of tTau can be about 30 kDa, about 38 kDa, about 48 kDa, about 55 kDa to 62 kDa, about 96 kDa to 106 kDa, about 140 kDa to 160 kDa and greater than 160 kDa. Thus, it is envisaged that the quaternary structure of tTau having the above-mentioned molecular weights is comprised in the protein concentration of tTau. Accordingly, the present application relates to a nasal fluid sample wherein the tTau marker is characterized by a molecular weight of tTau can have a molecular weight of about 30 kDa, about 38 kDa, about 48 kDa, about 55 kDa to 62 kDa, about 96 kDa to 106 kDa, about 140 kDa to 160 kDa and greater than 160 kDa.

[0107] The term "ratio" refers to the ratio of the protein amount or concentration between the analytes and is well known in the art. For example, in the context of the present application, the term "ratio of Αβ42 / Αβ40" is to be understood as the relative value of the Αβ42 protein concentration relative to the Αβ40 protein concentration. The terms "ratio of Αβ42 / Αβ40", "Αβ42 / Αβ40 ratio" and "relative amount of Αβ42 / Αβ40" are used interchangeably herein.

[0108] The skilled person will understand that preferably, the nasal fluid sample of the present application is characterized by a relative amount of Αβ42 / Αβ40 greater than 0 and less than 1, 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1. More preferably, the nasal fluid sample of the present application is characterized by a relative amount of Αβ42 / Αβ40 greater than 0.001 and less than 1. Even more preferably, the nasal fluid sample of the present application is characterized by a relative amount of Αβ42 / Αβ40 greater than 0.003 and less than 0.75. Most preferably, the nasal fluid sample of the present application is characterized by a relative amount of Αβ42 / Αβ40 greater than 0.005 and less than 0.75.

[0109] The relative amount / ratio of Αβ42 / Αβ40 can be calculated by measuring the marker proteins Αβ40 and Αβ42 using an automated immunoassay platform such as the Simoa® Αβ42 Advantage kit, Simoa® Αβ40 Advantage kit (Quanterix, Billerica, MA, USA) and the Quanterix SIMOA SR-X analyzer. The skilled person will understand that the absolute value of the relative amount / ratio of Αβ42 / Αβ40 can increase due to measurement system variation when different automated immunoassay platforms are used.

[0110] In the context of the present application, the term "control" is to be understood as a predetermined level of the concentration of one or more marker proteins or a ratio derived from the concentration of two or more marker proteins. The "control subject" can be a healthy subject or a subject not suffering from any cognitive impairment due to a neurodegenerative disease. Methods for obtaining reference values from a selected group of "control subjects" are well known in the art. The term "control" is interchangeable with the term "reference value".

[0111] The skilled person will understand that a subject not suffering from any cognitive impairment due to a neurodegenerative disease has a ratio of Αβ42 / Αβ40 above a predetermined cut-off value in the sense that the ratio is not decreased.

[0112] The term "correlation" is to be understood herein as an interdependence or association between two variables. For example, the variables are considered to have a positive correlation when an increase in one variable is associated with a proportional increase in the other variable, or when a decrease in one variable is associated with a proportional decrease in the other variable. Thus, the variables are considered to have a negative correlation when an increase in one variable is associated with a proportional decrease in the other variable, or when a decrease in one variable is associated with a proportional increase in the other variable. For example, the skilled person will understand that when pTau is positively correlated with tTau, this means that when pTau increases or decreases, tTau also increases or decreases, respectively.

[0113] The present invention relates to a nasal fluid sample wherein the relative amount / ratio of Αβ42 / Αβ40 is negatively correlated with pTau and / or tTau. Thus, the skilled person will understand that when the relative amount / ratio of Αβ42 / Αβ40 is negatively correlated with pTau and / or tTau, this means that a decrease in the relative amount / ratio of Αβ42 / Αβ40 is associated with an increase in the concentration of pTau and / or that a decrease in the relative amount / ratio of Αβ42 / Αβ40 is associated with an increase in the concentration of tTau. The present invention further provides a nasal fluid sample for use in a method of aiding in the diagnosis of a neurodegenerative disease, wherein the relative amount / ratio of Αβ42 / Αβ40 is negatively correlated with pTau and / or tTau; and the use of said nasal fluid sample for the diagnosis of a neurodegenerative disease. The present invention further relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject by using a nasal fluid sample, wherein the relative amount / ratio of Αβ42 / Αβ40 is negatively correlated with pTau and / or tTau. Preferably, the relative amount / ratio of Αβ42 / Αβ40 is negatively correlated with pTau and / or tTau, as shown in the accompanying examples.

[0114] Alternatively, the present invention relates to a nasal fluid sample wherein the relative amount / ratio of Αβ42 / Αβ40 is positively correlated with pTau and / or tTau. Thus, the skilled person will understand that when the relative amount / ratio of Αβ42 / Αβ40 is positively correlated with pTau and / or tTau, this means that a decrease in the relative amount / ratio of Αβ42 / Αβ40 is associated with a decrease in the concentration of pTau and / or that an increase in the relative amount / ratio of Αβ42 / Αβ40 is associated with an increase in the concentration of tTau. The present invention further provides a nasal fluid sample for use in a method of aiding in the diagnosis of a neurodegenerative disease, wherein the relative amount / ratio of Αβ42 / Αβ40 is positively correlated with pTau and / or tTau; and the use of said nasal fluid sample for the diagnosis of a neurodegenerative disease. The present invention further relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject by using a nasal fluid sample, wherein the relative amount / ratio of Αβ42 / Αβ40 is positively correlated with pTau and / or tTau.

[0115] It will be understood that the variable can be a value, such as a protein concentration of a marker protein, and / or a ratio, i.e. a relative amount of one marker protein relative to another marker protein.

[0116] A statistical analysis tool can be used to calculate whether a correlation exists between different variables. One possible statistical analysis tool is the Pearson correlation coefficient. The Pearson correlation coefficient is a measure of association well known in the art. Specifically, the Pearson correlation coefficient is the most common method of measuring the linear relationship between two variables, where a change in one variable is associated with a proportional change in the other variable. The Pearson correlation coefficient is often denoted as "r" or the Greek letter p (rho). The statistical significance of the Pearson correlation coefficient is often denoted as the p-value, denoted by "p". The definition and calculation of the p-value is well known in the art. A positive correlation coefficient (> 0) describes a positive correlation between two variables. A negative correlation coefficient (< 0) describes a negative correlation between two variables. When the correlation coefficient is 0, it describes no correlation between two variables. The terms "Pearson correlation coefficient", "correlation coefficient", "coefficient" are used interchangeably herein.

[0117] As disclosed in the examples, the skilled person will understand that the correlation between the protein concentrations or ratios of marker proteins from nasal fluid samples according to the present application can be measured as a Pearson correlation coefficient. Furthermore, it is apparent to the skilled person that the Pearson correlation coefficient derived from nasal fluid is comparable to the Pearson correlation coefficient derived from CSF, allowing for equivalent diagnostics (Figures 14 to 17).

[0118] The term "pathologically altered" is used herein to mean that the value of a marker protein has increased or decreased as characterised in the CSF of a subject diagnosed with a neurodegenerative disease, compared to a subject not suffering from any cognitive impairment due to a neurodegenerative disease. For example, it is reported that marker proteins measured from the CSF of patients with AD show a lower Ab42 / Ab40 protein concentration ratio, a decrease in the level of Ab42 compared to the level of Ab40, an increase in the level of pTau and / or an increase in the level of tTau when compared to patients not suffering from AD.

[0119] The altered value of a marker protein can be a change in the protein concentration of the altered marker protein and / or the relative amount of one or more marker proteins relative to another marker protein.

[0120] The present application relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject in a nasal fluid sample, wherein the neurodegenerative disease is AD.

[0121] As described above, the present application relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject in a nasal fluid sample, wherein the method comprises the steps of:

[0122] (a) determining the protein concentration of the marker proteins phospho-Tau (pTau), total Tau (tTau) and / or optionally amyloid-beta (Aβ) in a nasal fluid sample as defined herein;

[0123] (b) comparing the protein concentration of the marker proteins as determined in the nasal fluid sample with the protein concentration of the control; and

[0124] (c) determining whether the value of the marker proteins is pathologically altered relative to the protein concentration of the control.

[0125] Preferably, the present application relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject in a nasal fluid sample, wherein the method comprises the following steps:

[0126] (a) determining the protein concentration of the marker proteins phospho-Tau (pTau), total Tau (tTau) and / or optionally amyloid-beta (Aβ) in a nasal fluid sample as defined herein;

[0127] (b) comparing the protein concentration of the marker proteins as determined in the nasal fluid sample with the protein concentration of the control; and

[0128] (c) determining whether the value of the marker proteins is pathologically altered relative to the protein concentration of the control,

[0129] wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), Pick's disease and Creutzfeldt-Jakob disease (CJD).

[0130] Further, the present application relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject in a nasal fluid sample, wherein the method comprises the following steps:

[0131] (a) determining the protein concentration of the marker proteins phospho-Tau (pTau), total Tau (tTau) and / or optionally amyloid-beta (Aβ) in a nasal fluid sample as defined herein;

[0132] (b) comparing the protein concentration of the marker proteins as determined in the nasal fluid sample with the protein concentration of the control; and

[0133] (c) determining whether the value of the marker proteins is pathologically altered relative to the protein concentration of the control,

[0134] wherein an increased or alternatively decreased Aβ42 protein concentration relative to the Aβ40 protein concentration is evidence for AD in a subject with cognitive impairment.

[0135] The present invention relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject in a nasal fluid sample, wherein the method comprises the following steps:

[0136] (a) determining the protein concentration of the marker proteins phospho-Tau (pTau), total Tau (tTau) and / or optionally beta amyloid (Aβ) in a nasal fluid sample as defined herein;

[0137] (b) comparing the protein concentration of the marker proteins as determined in the nasal fluid sample with the protein concentration in a control; and

[0138] (c) determining whether the values of the marker proteins are pathologically altered relative to the protein concentration of the control,

[0139] wherein the neurodegenerative disease is selected from the group consisting of Alzheimer’s disease (AD), Parkinson’s disease (PD), chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), Pick’s disease and Creutzfeldt-Jakob disease (CJD).

[0140] It is also envisaged that the present invention relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject in a nasal fluid sample, wherein the method comprises the following steps:

[0141] (a) determining the protein concentration of the marker proteins phospho-Tau (pTau), total Tau (tTau) and / or optionally beta amyloid (Aβ) in a nasal fluid sample as defined herein;

[0142] (b) comparing the protein concentration of the marker proteins as determined in the nasal fluid sample with the protein concentration in a control; and

[0143] (c) determining whether the values of the marker proteins are pathologically altered relative to the protein concentration of the control,

[0144] wherein either of the following (i) or (ii) applies:

[0145] (i) pTau is positively correlated with tTau and Aβ42 is positively correlated with Aβ40, alternatively pTau is positively correlated with tTau and Aβ42 is negatively correlated with Aβ40; and

[0146] (ii) the relative amount of Aβ42 / Aβ40 is greater than 0 and less than 1, and

[0147] wherein the neurodegenerative disease is selected from the group consisting of Alzheimer’s disease (AD), Parkinson’s disease (PD), chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), Pick’s disease and Creutzfeldt-Jakob disease (CJD).

[0148] Alternatively, it is also envisaged that the present application relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject in a nasal fluid sample, wherein the method comprises the following steps:

[0149] (a) determining the protein concentration of the marker proteins phospho-Tau (pTau), total Tau (tTau) and / or optionally beta amyloid (Aβ) in a nasal fluid sample as defined herein;

[0150] (b) comparing the protein concentration of the marker proteins as determined in the nasal fluid sample with the protein concentration in a control; and

[0151] (c) determining whether the values of the marker proteins are pathologically altered relative to the protein concentration of the control,

[0152] wherein any of the following (i) or (ii) apply:

[0153] (i) pTau positively correlates with Aβ42 / Aβ40 and Aβ42 negatively correlates with Aβ40; and

[0154] (ii) the relative amount of Aβ42 / Aβ40 is greater than 0 and less than 1, and

[0155] wherein the neurodegenerative disease is selected from the group consisting of Alzheimer’s disease (AD), Parkinson’s disease (PD), chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), Pick’s disease and Creutzfeldt-Jakob disease (CJD).

[0156] It is also envisaged that the present application relates to a method for aiding in the diagnosis of a neurodegenerative disease in a subject in a nasal fluid sample, wherein the method comprises the following steps:

[0157] (a) determining the protein concentration of the marker proteins phospho-Tau (pTau), total Tau (tTau) and / or optionally beta amyloid (Aβ) in a nasal fluid sample as defined herein;

[0158] (b) comparing the protein concentration of the marker proteins as determined in the nasal fluid sample with the protein concentration in a control;

[0159] (c) determining whether the values of the marker proteins are pathologically altered relative to the protein concentration of the control;

[0160] (d) the Aβ42 protein concentration is increased or alternatively decreased relative to the Aβ40 protein concentration; and

[0161] (e) the relative amount of Aβ42 / Aβ40 negatively correlates with pTau and / or tTau, alternatively wherein the relative amount of Aβ42 / Aβ40 positively correlates with pTau and / or tTau,

[0162] wherein either of (i) or (ii) below applies:

[0163] (i) pTau is positively correlated with tTau and Aβ42 is positively correlated with Aβ40, alternatively pTau is positively correlated with tTau and Aβ42 is negatively correlated with Aβ40; and

[0164] (ii) the relative amount of Aβ42 / Aβ40 is greater than 0 and less than 1,

[0165] is evidence of AD in a subject having cognitive impairment.

[0166] Marker proteins / analytes are analyzed by techniques described herein (e.g., using electrophoretic techniques utilizing different capillary sizes (low, medium, high molecular size)). Each measurement yields one or more features that are specific to the respective protein, and also specific to the individual from which the sample was first collected, and also specific to the individual disease state. The one or more features consist essentially of a number of values or parameters. These values or parameters include the total level of the analyte, separation of the analyte by molecular weight into different quaternary structures (e.g., monomers and oligomers), and the level of the different quaternary structures (e.g., the level of monomers, the level of oligomers). The sum of the information yields one or more biomarker-specific features for each analyte (referred to herein as protein-specific biomarker features) that include the protein concentration of the analyte. The protein concentration / level of, e.g., beta amyloid is also referred to herein as the beta amyloid concentration / level.

[0167] Measuring protein levels in analytes and determining protein concentrations can be determined by techniques known to those of skill in the art. Methods for measuring protein levels and determining protein concentrations include, but are not limited to, Western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, fluorescence-activated cell sorting (FACS), flow cytometry, and assays based on the properties of DJ-1 including, but not limited to, ligand binding or interaction with other protein partners.

[0168] In one example, the protein concentration of one or more marker proteins in a nasal fluid sample obtained as described herein can be measured by a BCA assay (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, 10678484). One sample aliquot can be removed from the -80ºC freezer and equilibrated to room temperature (RT) on a workbench for approximately 30 minutes. The protein concentration measurement assay should be performed according to the manufacturer’s protocol, and the concentration can be calculated in mg / ml. These results can be further used to normalize the total protein concentration in, for example, gel electrophoresis in the following measurements.

[0169] To determine the protein levels of the marker proteins, the respective samples can be run on an automated protein separation and immuno-detection system, such as Simple Western™ Jess, BioTechne, 004-650, and optionally on a traditional gel electrophoresis and Western blot. An aliquot of the nasal fluid sample eluate can be thawed at room temperature for 30 minutes. An appropriate volume can be removed from the sample such that the final total protein concentration in the assay can be about 0.25 mg / mL, about 0.5 mg / mL, about 0.75 mg / mL, about 1.0 mg / mL, about 1.25 mg / mL, about 1.5 mg / mL, about 1.75 mg / mL, or about 2.0 mg / mL.

[0170] Preferably, the final total protein concentration in the assay is about 1.0 mg / mL. The sample can be diluted in the sample buffer for the Simple Western™ Jess system provided by the manufacturer, such as BioTechne. The diluted sample can be loaded into the Simple Western™ Jess system, and can be initiated and run as a single plex assay according to the manufacturer’s instructions. Capillaries with different gel percentages suitable for low molecular weight (2 kDa to 40 kDa separation module, 8 x 25 capillary cartridge, BioTechne, SM-W012), medium molecular weight (12 kDa to 230 kDa separation module, 8 x 25 capillary cartridge, BioTechne, SM-W004), or high molecular weight (66 kDa to 440 kDa separation module, 8 x 25 capillary cartridge, BioTechne, SM-W008) separation can be used.

[0171] In the context of the present application, the skilled person is aware of means and methods for detecting marker proteins in a sample, such as Αβ, pTau and / or tTau. Exemplarily, for the detection of Αβ40, the following primary antibodies can be used: Human Amyloid Beta (aa 1-40) Antibody, BioTechne / R&D, MAB96181-100 and / or Purified (Azide-Free) Anti-beta-Amyloid, 1-40 (11A50-B10), BioLegend, 805409. Both antibodies are specific for the C-terminus of Αβ40. An enzyme-linked secondary antibody can then be added, such as Anti-Mouse Detection Module, BioTechne, DM-003, followed by a chemiluminescent substrate. The amount of Αβ40 protein can be measured, revealing information about the total level of analyte and the level of Αβ40 molecules of different molecular weight (quaternary structure) within the same sample. This allows for the simultaneous detection of different oligomeric Αβ40 molecules and the quantification of said molecules separately for each individual molecular weight, collectively and / or relative to each other. Fig. 2 and Fig. 4 show exemplary banding patterns of Αβ40 that can be measured in nasal fluid from patients with different clinical conditions. Each antibody-epitope recognition is represented by a chemiluminescent signal, so the higher the signal intensity, the more antibody-epitope recognition, and thus the higher the level of quaternary structure detected or the total level of analyte. By calculating the area under the curve (AUC), the amount of antibody-epitope recognition can be measured, and thus the level of a given quaternary structure or the total level of protein can be back-calculated. A graphical representation of the AUC calculated from the chemiluminescent signal of the bound antibodies of Αβ40 can be generated. Oligomers of approximately 19 kDa, 32 kDa, 56 kDa and 100 kDa, as well as monomers of approximately 4 kDa can be detected. In Fig. 5, the signal of the Αβ40 4 kDa monomer band can be higher in patients without AD (classified as A-) compared to patients with clear AD (classified as A+). Without necessarily being bound by scientific theory, it is believed that insoluble Αβ42 aggregates in Alzheimer’s disease patients sequester Αβ40 molecules, leading to a decrease in Αβ40 detected. The healthy monomer species detected can be higher in patients without AD (A-) relative to patients with AD (A+).

[0172] Figure 10 shows signals of A940 oligomers of approximately 20 kDa, 32 kDa, 62 kDa, 98 kDa and 231 kDa detected from a nasal fluid sample. Signals of A940 oligomers of approximately 62 kDa, 87 kDa and 233 kDa were detected from the corresponding CSF of the same individual. A940 oligomers of approximately 32 kDa and 20 kDa could not be detected from the corresponding CSF. Comparing the quaternary / oligomeric structures of the same individual in the corresponding samples nasal fluid and CSF one can observe a distribution of the same oligomeric species at approximately 62 kDa and 231 kDa (233 kDa + / -10% technical bias). The nasal fluid sample contains additional bands that could not be observed in the CSF.

[0173] For the detection of A942 one can use the following primary antibodies: purified (without azide) anti-beta-amyloid, 1-42 (12F4), BioLegend, 805501 and / or anti-amyloid beta 42 (43), monoclonal antibody (BC05), Fujifilm Wako, 010-26903. Both antibodies are specific for A942 and can recognize one or several isoforms and oligomeric structures. One can then add a secondary enzyme-linked antibody (such as anti-mouse detection module, BioTechne, DM-003) followed by a chemiluminescent substrate. One can measure the amount of protein, revealing information about the total level of the analyte within the same sample and the level of different molecular weights of A942 molecules (quaternary structures). This allows for the simultaneous detection of different oligomeric A942 molecules and the quantification of said molecules separately for each individual molecular weight, globally and / or relative to each other. Comparing the different quaternary / oligomeric distribution and relative protein amounts of two different individuals / subjects, while one individual has an amyloid pathology (A+) and the other does not (A-) (see Figure 6), one can observe that there can be a globally decreased total level of A942 (calculated by the total area under the curve of the total protein concentration) detected in the AD patient (classified as A+) relative to the total level of A942 of the individual without AD (A-). The band ratio between the oligomers (53 kDa and 32 kDa, in Figure 6) can also change.

[0174] Comparing the quaternary / oligomeric structures of the same individual in the two corresponding samples (i.e. nasal fluid and CSF) from this individual (Figure 11), one can observe quaternary / oligomeric bands at approximately 99 kDa, 75 kDa, 56 kDa and 33 kDa in the nasal fluid sample that are not determinable in the corresponding CSF sample of the same individual. There is a much higher amount of the corresponding oligomeric bands present in the nasal secretion compared to in the corresponding CSF.

[0175] For the detection of total Abeta one can use the following primary antibodies: primary antibody 6E10 (purified anti-beta-amyloid, 1-16 antibody (6E10), BioLegend, 803001) and / or monoclonal antibody against amyloid beta A4 (N-terminal), Nanotools, 0315-100 / bA4N-1E8, which recognizes all forms of Abeta. One can then add an enzyme-linked secondary antibody (such as anti-mouse detection module, BioTechne, DM-003), followed by a chemiluminescent substrate. One can measure the amount of pan Abeta protein, revealing information about the total level of the analyte within the same sample as well as the level of pan Abeta molecules of different molecular weight (quaternary structure). This allows for the simultaneous detection of different oligomeric pan Abeta molecules and quantification of the molecules separately for each individual molecular weight, globally and / or relative to each other (Figure 7). In comparison to the species-specific antibodies described above, the pan Abeta antibody binds to an epitope closer to the N-terminus. This allows for the recognition of different quaternary structures in comparison to antibodies specific for Abeta40 or Abeta42. It is envisaged that the ratio of the 45 kDa and 32 kDa bands, the total level and / or the level of high order oligomeric structures (such as the 183 kDa band) change in A+ individuals versus A- individuals. It is further envisaged that these changes can be used for classification, e.g. A+ versus A-. The skilled person will understand that the terms “total Abeta”, “pan Abeta”, “total Abeta”, “pan Abeta”, “total beta amyloid”, “pan beta amyloid”, “total amyloid beta” and “pan amyloid beta” can be used synonymously in the context of the present invention.

[0176] For the detection of tTau, the following primary antibodies can be used: purified anti-Tau, 404-441 antibody (mouse), BioLegend, 806601 and / or Tau Monoclonal Antibody (HT7), Biotin (mouse), Thermo Fisher Scientific, MN1000B. Both are specific for tTau and recognize one or several isoforms and oligomeric structures. Then an enzyme-linked secondary antibody (anti-mouse detection module, BioTechne, DM-003) can be added and subsequently a chemiluminescent substrate. The amount of tTau protein can be measured, revealing information about the total level of analyte within the same sample as well as the level of the quaternary structure or isoform of the tTau molecule. This allows for the simultaneous detection of different oligomeric tTau molecules and the quantification of said molecules separately for each individual molecular weight, globally and / or relative to each other. Comparing the different oligomer distribution and relative protein amounts of two different individuals / subjects, one with a neurodegenerative process (N+) and the other without a neurodegenerative process (N-) (see Figure 8), one can observe that the level of the canonical isoform of tTau observed at approximately 60 kDa can be higher in patients with clear signs of neurodegeneration (classified as N+) compared to patients without signs of neurodegeneration (classified as N-). Different other quaternary structures can also be measured, such as total Tau dimers at 32 kDa. Comparing the quaternary / oligomeric structures of the same individual from two respective samples of this individual (i.e. nasal fluid and CSF) (Figure 12), one can observe almost identical oligomeric bands at approximately 63 kDa in the nasal fluid sample and the corresponding CSF sample. Given the fact that the volume of CSF loaded is 6 times the volume of the corresponding volume from nasal fluid, the level of this quaternary / oligomeric band is at least equivalent to CSF or higher.

[0177] For the detection of pTau, the following primary antibody can be used: Phospho-Tau (Thr181) (D9F4G) Rabbit mAb, Cell Signaling, 12885S specific for pTau-181 and recognizes one or several isoforms and oligomeric structures. An enzyme-linked secondary antibody (anti-rabbit detection module, BioTechne, DM-001) can be added followed by a chemiluminescent substrate. The amount of pTau protein can be measured, revealing information about the total level of the analyte within the same sample and the level of the quaternary structures with different molecular weights. This allows the simultaneous detection of different oligomeric pTau-181 molecules and the quantification of said molecules separately for each individual molecular weight, globally and / or relative to each other. Comparing the different oligomer distribution and relative protein amounts of two different individuals / subjects, while in this case one individual has Tau pathology (T+) and the other does not have Tau pathology (T-) (see Figure 9), one can observe that the total level of pTau detected can be lower in this patient with pronounced Tau pathology (T+) compared to the other patient without Tau pathology (T-).

[0178] Comparing the quaternary / oligomeric structures of the same individual in two respective samples from this individual, i.e. nasal fluid and CSF (Figure 13), one can observe almost identical oligomeric bands at approximately 60 kDa, 100 kDa, 146 kDa and 230 kDa in the nasal fluid sample and in the respective CSF sample. Given the fact that the loaded CSF volume is 6 times the respective volume from the nasal fluid sample, the level of these quaternary / oligomeric bands is at least equal to or higher than in CSF. An additional band at approximately 20 kDa can be observed in the nasal fluid sample that cannot be observed in the respective CSF sample, also indicating that the amount of this band is at least higher than in the respective CSF sample.

[0179] The present invention relates to a nasal fluid sample obtained from a subject, wherein the concentration of a marker protein is equal or increased by at least 1.1 fold compared to a cerebrospinal fluid (CSF) sample obtained from the same subject. In particular, the present invention relates to a purified nasal fluid sample obtained from a subject, wherein the concentration of a marker protein is equal or increased by at least 1.1 fold compared to a cerebrospinal fluid (CSF) sample obtained from the same subject. Preferably, the concentration of one or more marker proteins in a purified nasal fluid sample according to the present invention is equal or increased by at least 1.1 fold compared to a cerebrospinal fluid (CSF) sample obtained from the same subject. More preferably, the concentration of one or more marker proteins in a purified nasal fluid sample according to the present invention is increased by at least 2 fold compared to a cerebrospinal fluid (CSF) sample obtained from the same subject. Even more preferably, the concentration of one or more marker proteins in a purified nasal fluid sample according to the present invention is increased by at least 4 fold compared to a cerebrospinal fluid (CSF) sample obtained from the same subject. Most preferably, the concentration of one or more marker proteins in a purified nasal fluid sample according to the present invention is increased by at least 6 fold compared to a cerebrospinal fluid (CSF) sample obtained from the same subject.

[0180] The skilled person is aware that when analyzing a body fluid, often a matrix effect or matrix interference is observed. In particular, the matrix refers to components of the sample other than the analyte of interest. The matrix can have a considerable influence on the way the analysis is performed and on the quality of the results obtained. Such effects are referred to as matrix effects or matrix interferences.

[0181] A "purified nasal fluid sample" is herein understood as a nasal fluid sample wherein nasal fluid compounds causing the above-mentioned matrix effects and interferences (e.g. mucin related effects) with respect to e.g. antibody epitope recognition have been resolved prior to measuring the level of one or more marker proteins. Such nasal fluid compounds (proteins) causing matrix effects can be, but are not limited to, mucin, exosomes, lysozyme, lactotransferrin, immunoglobulins, albumin, cell debris, DNA fragments and bacteria. Removing nasal fluid compounds causing matrix effects and interferences to obtain a purified nasal fluid sample can be performed by techniques well known in the art. As an example, one step of purification can be performed by using an electrophoresis system (like the Simple Western™ Jess system). The nasal fluid sample can be denatured and reduced by boiling in an appropriate buffer. Another step can be the use of a gel filter, while applying an electric current to the gel and separating the molecules according to their size.

[0182] The skilled person is well aware that a nasal fluid sample does not mean nasal fluid in the body of a subject, but rather nasal fluid in vitro or a nasal fluid sample obtained from a subject by the methods described herein. The skilled person is also well aware of how a nasal fluid sample can be obtained from a subject. In the context of the present application, a nasal fluid sample is synonymous with a nasal secretion eluate or a nasal superficial lining fluid sample or a nasal secretion sample.

[0183] In the context of the present application, the skilled person understands that the terms "purified" and "refined" or the terms "purification" and "refinement" can be used interchangeably. Thus, "purification" and "refinement" can also be used synonymously herein.

[0184] A nasal fluid sample can be obtained by swabbing, brushing, preferably by swabbing or brushing anywhere near the olfactory cleft, more preferably the olfactory mucosa, nasal lavage / flushing or olfactory mucosa biopsy. However, these techniques have certain disadvantages. Aspiration can lead to loss of sample material, nasal lavage / flushing can lead to uncontrolled loss of sample material into the nasopharynx and uncontrolled dilution, and washing the whole nose leads to further dilution of the proteins of interest due to the fact that the area of actual interest, i.e. the vicinity of the olfactory cleft, only constitutes a small part of the nose. Brushing, swabbing and dabbing are invasive and the brush has to be guided. Since the vicinity of the olfactory cleft is in a sagittal alignment, the olfactory cleft cannot be reached manually without general or local anesthesia, blood mixtures can alter the measurement results since there are organs producing e.g. Αβ42 and plasma can for example contain Tau proteins, so the measurement results can be distorted. Biopsies are invasive and since the area of interest is at most 23 cm^2, small biopsies of e.g. 1 mm^2 do not show the complete pathology in its entirety. Therefore, it is preferred that the nasal fluid sample is collected by absorption. In the context of the present application, it is preferred that the nasal fluid sample is obtained from the vicinity of the olfactory cleft, in particular from the olfactory mucosa. One or more nasal fluid samples can be obtained from one or both olfactory clefts, preferably both olfactory clefts.

[0185] Nobody has a completely symmetrical nose. Thus, the olfactory cleft and the olfactory mucosa in both nostrils can have different sizes. Therefore, it is preferred that the nasal fluid samples obtained from the vicinity of both olfactory clefts of a subject are pooled at a certain point in time before analysis. Thus, the present application relates to a nasal fluid obtained from a subject, wherein the nasal fluid sample comprises one or more marker proteins beta amyloid (Αβ), phosphorylated Tau (pTau or p-Tau) and / or total Tau (tTau or t-Tau) obtained from the vicinity of the olfactory mucosa in the vicinity of both olfactory clefts of the subject.

[0186] Preferably, the absorbing material is placed in the vicinity of the olfactory cleft, preferably in the vicinity of the olfactory mucosa, and left in the vicinity of the olfactory cleft or in the vicinity of the olfactory mucosa for a certain amount of time. After the leaving, the absorbing material is recovered and the nasal fluid sample is isolated / obtained from the absorbing material.

[0187] Thus, it is envisaged that in the methods described herein, the nasal fluid sample is obtained by:

[0188] a) placing an absorbing material in the vicinity of one or more olfactory clefts;

[0189] b) incubating the absorbing material in the vicinity of the one or more olfactory clefts;

[0190] c) recovering the absorbing material; and

[0191] d) isolating the nasal fluid sample from the absorbing material.

[0192] It is also envisaged that the absorbing material is placed in the vicinity of the olfactory cleft.

[0193] The absorbing material used in the methods described herein is not particularly limited and the skilled person is able to readily select a suitable absorbing material. Non-limiting examples of absorbing materials that can be used in the context of the described methods can be synthetic materials such as polyvinyl alcohol (PVA) or organic materials such as cotton or mixtures thereof.

[0194] Preferably, the absorbing material is in the form of a sponge.

[0195] It is envisaged that the absorbent material is left in the vicinity of the olfactory cleft for about 1 to 60 min, such as about 1 min, about 2 min, about 3 min, about 4 min, about 5 min, about 6 min, about 7 min, about 8 min, about 9 min, about 10 min, about 11 min, about 12 min, about 13 min, about 14 min, about 15 min, about 16 min, about 17 min, about 18 min, about 19 min, about 20 min, about 21 min, about 22 min, about 23 min, about 24 min, about 25 min, about 26 min, about 27 min, about 28 min, about 29 min, about 30 min, about 31 min, about 32 min, about 33 min, about 34 min, about 35 min, about 36 min, about 37 min, about 38 min, about 39 min, about 40 min, about 41 min, about 42 min, about 43 min, about 44 min, about 45 min, about 46 min, about 47 min, about 48 min, about 49 min, about 50 min, about 51 min, about 52 min, about 53 min, about 54 min, about 55 min, about 56 min, about 57 min, about 58 min, about 59 min, or about 60 min, and all values therebetween, such as about 14.7 min. Preferably in the context of the methods described herein, the absorbent material is left in the vicinity of the olfactory cleft for 20 min.

[0196] Thus, it is envisaged in the methods described herein that a nasal fluid sample is obtained by:

[0197] a) placing an absorbent material in the vicinity of one or more olfactory clefts;

[0198] b) leaving the absorbent material in the vicinity of the one or more olfactory clefts for about 1 to 60 min, preferably 20 min;

[0199] c) recovering the absorbent material; and

[0200] d) isolating the nasal fluid sample from the absorbent material.

[0201] It is envisaged that the nasal fluid sample is obtained using a proprietary developed absorbent material (AM) and medical device to facilitate insertion of the AM in the correct position (nosecollect®), as described in WO 2022 / 101311.

[0202] Thus, it is envisaged in the methods described herein that a nasal fluid sample is obtained by:

[0203] a) placing an absorbent material in the vicinity of one or more olfactory clefts;

[0204] b) leaving the absorption material in the one or more olfactory clefts for about 1 to 60 min, preferably 20 min;

[0205] c) recovering the absorption material; and

[0206] d) isolating the nasal fluid sample from the absorption material

[0207] e) purifying the nasal fluid sample from the nasal secretion eluate.

[0208] The operator collecting the nasal secretion can typically be a psychiatrist or neurologist, but can also be a different physician or trained medical personnel. It is preferred that the sample is collected by covering the entire pathology in both nostrils by collecting the sample from the vicinity of the entire olfactory mucosa area of about 23 cmA2. It is preferred that the absorption material (e.g. in the form of a sponge) is inserted in a sagittal direction to cover the entire olfactory cleft in its entirety and that the absorption material expands in a caudal direction when inserted in the vicinity of the olfactory cleft and not in a coronal direction (laterally).

[0209] For the collection of the nasal fluid sample, the absorption material (e.g. polyvinyl alcohol (PVA)) can be applied to the olfactory clefts on both sides of the subject’s nose by nosecollect® (as described in e.g. WO 2022 / 101311) or manually by trained personnel. In case of manual insertion, a Hartmann nasal speculum (13 cm, Karl Storz SE & Co. KG, 400500) as well as a Jansen Bayonet nasal forceps (16.5 cm, Karl Storz SE & Co. KG, 426516) can be used. A thread attached to the absorption material and hanging outside the nostril can be carefully fixed to the subject’s cheek with adhesive to prevent accidental displacement. The absorption material can be left in place for 20 to 30 minutes (or any other time mentioned herein, preferably 20 min). In case the mucosa is dry, the subject can be subjected to physical activity (walking around, climbing stairs), eating or drinking water to stimulate nasal fluid secretion. Other methods can also be suitable to increase the production of nasal secretion, e.g. treatment of the nose with a saline spray prior to the sampling process.

[0210] How the nasal sample can be obtained and processed is exemplarily disclosed in the following:

[0211] After the retention in the nose, the thread fixed on the cheek of the subject can be loosened and the absorption material saturated with nasal fluid can be removed from both sides of the nose by pulling the thread. If the absorption material of one or both nostrils of the subject is bloodied, it can be collected by the collector in a separate tube (Eppendorf 50 ml Protein LoBind tube, Eppendorf, 0030122240). If the absorption material of both sides is clean or only small blood spots are visible, the saturated absorption material of the second side, including the thread, can be combined with the absorption material of the first side in the same pre-labeled tube (Eppendorf 50 ml Protein LoBind tube, Eppendorf, 0030122240). The tubes containing the removed absorption material can be collected upright in a suitable plastic box.

[0212] The cartridge with the sample tube can be kept at room temperature (RT) for up to 10 minutes. If several samples are collected and it cannot be guaranteed that no sample is at room temperature for more than 10 minutes, the cartridge with the first sample can be immediately stored at -80°C and new samples are put in it successively. A temperature logger (Testo 184 T4, Testo SE & Co. KGaA, 05721844) can be added to the samples to guarantee cold chain compliance. The cartridge containing the collected nasal fluid samples can be stored upside down at -80°C until transport to the analysis laboratory. Storage in a -80°C freezer is preferred, however other conditions like dry ice are possible. In this case, it should be ensured that the whole cartridge is surrounded by dry ice to guarantee that the whole cartridge is in isothermal conditions. The cartridge containing the collected samples and the temperature logger can be transferred upside down into a styrofoam box. The whole styrofoam box can be filled with dry ice. The sample cartridge should be completely surrounded by dry ice. The samples can then be transported from e.g. the clinical center to the analysis laboratory by e.g. a commercial logistic partner (overnight). The styrofoam box containing the nasal fluid samples collected as described herein can be opened at the analysis laboratory (preferably shortly after delivery). It can be checked if the collected samples are in good condition. This means that during the whole transport they were frozen at -80°C (checked by the state of the remaining dry ice and the report of the included temperature logger) and the integrity of the test tubes is good (not broken, sealed). The samples can then be stored in a specific shelf or box in the -80°C freezer of the analysis laboratory until pre-treatment. On the day of elution, the nasal secretion samples can be taken out of the -80°C freezer and thawed for 30 minutes at room temperature (RT). The eluted samples can be visually inspected under a class II safety cabinet for blood stains. Absorbent material with blood stains cannot be eluted together with blood-free absorbent material. Therefore, a sterile pair of scissors (microscope scissors, curved, sharp / sharp, VWR, 233-1454) and tweezers (straight, blunt, VWR, 232-2116) can be used to cut the blood stains out of the absorbent material and then the absorbent material from both sides of the nose of one subject can be eluted together. A sterile pair of scissors can be used to cut the threads from the PVA.

[0213] For elution, Pierce spin columns (10 ml, Thermo Fisher Scientific, PIER89898) can be prepared by removing the silica gel membrane. The absorbent material can be placed into these prepared columns, which can be put back into the original sample tube of the respective subject. If the blood stains cannot be removed, the absorbent material of both nostrils can be placed in separate spin columns and tubes, respectively.

[0214] The tube with the centrifuge column containing the absorption material can be centrifuged at room temperature (RT) at 4566 ref for 5 min. Afterwards, the centrifuge column and the absorption material can be discarded. The entire volume of eluted nasal fluid can be transferred from the original Eppendorf 50 ml Protein LoBind tube used for centrifugation into a pre-cooled Eppendorf 1.5 ml Protein LoBind tube (Eppendorf, 0030108116) and kept on ice.

[0215] The transferred eluate can be centrifuged at room temperature at 17000 ref for 10 min in a suitable benchtop centrifuge to precipitate the solid components. After centrifugation, the tube can be placed on ice under a class II safety cabinet and the supernatant can be transferred into a new pre-cooled Eppendorf 1.5 ml Protein LoBind tube without pipetting the solid or viscous parts. The volume of the transferred eluate can be estimated. If blood stains cannot be removed from the absorption material before centrifugation, the absorption material from both sides of the nose of one subject can be eluted separately. In this case, the two eluates from one subject are continued to be handled separately. The eluate with the lowest value on the color scale can be analyzed separately or the eluates from both can be mixed for analysis.

[0216] The eluate can be evaluated based on the available color scale (1-5) (Figure 1). If a sample is rated color scale 5, the sample can not be further processed. Several working aliquots can be prepared in a pre-cooled Eppendorf 1.5 ml Protein LoBind tube under a class II safety cabinet. During preparation, the sample and aliquots can be kept on ice. They can be stored at -80 °C for long-term storage.

[0217] As mentioned above, the present application also provides a method for aiding in the diagnosis of a neurodegenerative disease in a subject in a nasal fluid sample, wherein the method comprises the following steps:

[0218] (a) determining the protein concentration of the marker proteins phospho-Tau (pTau), total Tau (tTau) and / or optionally amyloid-beta (Ab) in the nasal fluid sample;

[0219] (b) comparing the protein concentration of the marker proteins as determined in the nasal fluid sample with the protein concentration in a control; and

[0220] (c) determining whether the value of the marker proteins is pathologically altered relative to the protein concentration of the control.

[0221] It is envisaged that in the methods described herein, the protein concentration of the marker protein amyloid-β Aβ40 and / or Aβ42 is analyzed. In other words, it is envisaged that the isoform of amyloid-β is Aβ40 and / or Aβ42. It is also envisaged that in the methods described herein, the protein concentration of the marker protein amyloid-β Aβ40, Aβ42 and / or total amyloid-β is analyzed. Total amyloid-β is also referred to as pannamyloid-β or pannAβ. Total amyloid-β can mean that all isoforms are analyzed and the respective information is provided as protein concentration of total amyloid-β. This can be done with an antibody that does not distinguish between different isoforms of amyloid-β (e.g. as described in Example section 3.2.3) but detects all isoforms (e.g. by binding to all isoforms). However, it is noted that for total amyloid-β as used herein, it can also simply mean that several isoforms are detected that are not single isoforms, but do not necessarily represent all amyloid-β molecules in the sample. For example, it is envisaged that e.g. amyloid-β peptides 1 to 41, 1 to 42 (Aβ42) and 1 to 43 are detected, but amyloid-β peptides 1 to 38, 1 to 39 and 1 to 40 (Aβ40) are not. Thus, the resulting protein concentration will include values for amyloid-β peptides 1 to 41, 1 to 42 and 1 to 43. This can also be referred to as total amyloid-β protein concentration, although it does not necessarily represent all amyloid-β molecules in the sample.

[0222] The nose fluid based measurements of the core markers can be compared to the CSF based measurements of the same core markers and analyzed for any evidence of a neurodegenerative disease such as AD. The nose fluid sample and the CSF sample can be collected from the same individual, thereby generating the respective samples.

[0223] In the case of AD, the respective samples can,

[0224] i) show the amount of one or more core AD marker proteins and the inter- and intra-protein correlations (pattern (signature) of one or more proteins);

[0225] ii) analyze the respective samples with respect to the distribution and amount of one or more protein specific monomeric and oligomeric structures of one or more AD marker proteins in the nose fluid sample and in the respective CSF sample obtained from the same subject. Surprisingly, the signature of one or more AD marker proteins Aβ40, tTau and pTau181 was found to be almost identical by direct comparison. Notably, the signature of the Aβ42 marker protein was found to be different in the nose fluid when compared to the respective CSF sample obtained from the same subject;

[0226] iii) the distribution pattern of protein specific monomeric and oligomeric structures and their respective amounts within the two respective body fluids; and

[0227] iv) show a neurodegenerative disease specific difference in the relative protein amounts of one or more marker proteins and in the distribution of monomeric and oligomeric structures of one or more marker proteins within a different subject suffering from e.g. an underlying amyloid pathology compared to another subject not suffering from an underlying amyloid pathology.

[0228] A representative neurodegenerative disease suitable to be assessed by the analysis of brain-derived markers in the context of the present application can be AD for the following reasons:

[0229] i) it is the most common neurodegenerative disease and therefore there is a large amount of data and knowledge;

[0230] ii) there are a variety of markers whose amounts can be reliably distinguished in CSF by in vitro diagnostic methods so far; and

[0231] iii) the AD specific inter- and intra-protein correlations in CSF (protein patterns) have been evaluated in extensive studies worldwide and the level of confidence in the authenticity is high on a global scale.

[0232] Pathological changes in the marker proteins pTau and or tTau as well as pathological changes in the marker protein amyloid beta can also be found in other neurodegenerative diseases. Tauopathies include a variety of different neurodegenerative disorders which can be detected by different distinguished patterns, e.g. pathological increase of tau markers. As a further example, patients suffering from PD can show changes in the marker proteins tau, amyloid beta and alpha synuclein. Since the range of said proteins which can be found in nasal fluid samples ranges from 4 kDa up to 400 kDa, all other CSF marker proteins in this range can also be measured in the same nasal fluid sample. Therefore, the claimed nasal fluid sample as well as its use in a method for aiding the diagnosis of a neurodegenerative disease will not be limited to AD but also include other known neurodegenerative diseases as disclosed herein.

[0233] For measuring the protein concentration of the marker proteins in the CSF of the respective subject, the CSF can be collected at the clinical site by lumbar puncture, preferably in the morning, and can be performed between the 3rdand 4thor 4thand 5thlumbar vertebra. It can be collected in a polypropylene collection tube following international guidelines, discarding the first 20 drops. Too much empty space in the tube should be avoided. Within the next 4 hours after collection, the CSF can be centrifuged at room temperature (RT) for 10 min at approximately 2000 rcf and transferred into a new polypropylene collection tube. 1500 mΐ of the transferred CSF can be aliquoted into separate polypropylene collection tubes. Within 4 hours after collection, the CSF samples and aliquots can be stored at -80ºC in suitable boxes. The boxes containing the CSF aliquots and a temperature logger can be transferred cap-side-up into a styrofoam box. The whole styrofoam box can be filled with dry ice. The sample boxes can be completely surrounded by dry ice. The samples can then be shipped (overnight) to the analysis laboratory, e.g. by a commercial logistics partner. The styrofoam box can be opened at the analysis laboratory shortly after delivery. The collected samples can be checked for being in good condition. This means that they should be frozen at -80ºC throughout the shipping period (checked by the state of the remaining dry ice and the report of the included temperature logger) and the integrity of the test tubes should be good (not broken, sealed). The samples can then be stored in a specific shelf or box in the -80ºC freezer of the analysis laboratory until pre-processing. The CSF aliquots can be thawed and measured for all core analytes according to the manufacturer’s protocol using immunoassay-based measurements (e.g. on the Fuji Rebio Lumipulse platform): amyloid-beta 40, amyloid-beta 42, pTau181 and tTau.

[0234] As described herein, the nasal fluid sample can be subjected to a technique comprising separating the quaternary structure and / or isoforms of the relevant marker proteins / analytes / biomarkers according to size, molecular weight or charge. It is envisaged that the technique generates raw data which is further processed to measure the protein concentration of the marker proteins / analytes / biomarkers.

[0235] Methods for separating quaternary structures and / or isoforms according to size, molecular weight or charge are well known to the person skilled in the art. As an example, the technique can be an electrophoretic technique, such as ELISA, Simoa® kit and / or Quanterix SIMOA SR-X analyzer (as exemplarily performed in the examples).

[0236] The subsequent data analysis is performed using the CSF- and nasal fluid-based measurements of all four analytes.

[0237] The respective datasets are stored in Excel files and processed with Python scripts using standard libraries like pandas and NumPy. Linear and logistic regressions are calculated using the sklearn package. Plots are generated using the matplotlib and seaborn packages. For quantitative analysis, these methods are used to draw conclusions from the data. If the underlying distributions are indeed identical, the P-value measures the probability of observing the same or a greater difference between the samples observed; a smaller value indicates a significant difference. Values of p < 0.05 are described as significant. It has also been verified that non-parametric alternatives to the Student's t-test (Mann-Whitney test, Welch's test) lead to similar values and conclusions. When comparing two variables (e.g., different markers / analytes) from the same cohort, linear regression is used. The result (in addition to the fitted line that gives the best linear relationship between the quantities) is Pearson's "r", which is a parameter of the goodness of fit, and Pearson's p, which is a parameter of statistical significance. The parameter "r" indicates the degree to which one quantity depends on the other, with a range of 0.1 to 0.3 counted as weakly correlated, a range of 0.3 to 0.5 counted as moderately correlated, and a range of 0.5 and greater counted as strongly correlated. The significance p still indicates the probability of measuring this or a greater degree of correlation by chance if the quantities were independent.

[0238] The following describes measuring the protein quantity or concentration of a marker protein when using techniques that include separating the quaternary structure and / or isoforms of the relevant marker protein / analyte according to molecular weight and immunodetection.

[0239] Corresponding nasal fluid and CSF samples are run on an automated protein separation and immuno-detection system (Simple Western™ Jess, BioTechne, 004-650) and optionally on traditional gel electrophoresis and Western Blot to show the almost identical distribution of protein specific monomeric and oligomeric structures and to show the respective amounts within these different quaternary structures and their relationship. The described protocol refers to the automated system. Nasal fluid aliquots are first thawed for 30 minutes at room temperature. Appropriate volume amounts of each nasal fluid sample are taken so that the final total protein concentration in the assay is 1.25 mg / ml. The samples are diluted in the manufacturer provided sample buffer. The diluted samples are loaded into the Simple Western™ Jess system and the system is started and run as a single assay according to the manufacturer’s instructions. Different capillaries with different gel percentages suitable for low molecular weight (2 kDa to 40 kDa separation module, 8 x 25 capillary cartridge, BioTechne, SM-W012), medium molecular weight (12 kDa to 230 kDa separation module, 8 x 25 capillary cartridge, BioTechne, SM-W004) or high molecular weight (66 kDa to 440 kDa separation module, 8 x 25 capillary cartridge, BioTechne, SM-W008) separation are used. Primary antibodies specific for core AD marker proteins are used which recognize one or several marker protein specific isoforms and oligomeric structures. Then enzyme linked secondary antibodies are added followed by a chemiluminescent substrate. Marker protein specific biomarker signatures are measured revealing information about i) the total level of the analyte and ii) the position of the maximum and the amount of the quaternary structure of different molecular weight within the same sample. This allows for the simultaneous detection of monomeric and oligomeric molecules and the quantification of the molecules separately for each individual molecular weight, globally and / or relative to each other as shown for example in Figures 10, 11, 12 and 13. Corresponding CSF samples from the same individual are applied according to the above described protocol on the same measurement platform, the only difference being that the applied CSF volume is 6 times the applied nasal fluid sample volume. The CSF is collected as previously described in the lumbar region. In the CSF the core protein markers of AD are diluted in a large volume, therefore the fluid itself is not highly protein rich.

[0240] A data set for each marker is generated for each measurement of a corresponding set of body fluid samples on an automated protein separation and immunoassay system. As shown in Figures 10, 11, 12, and 13, the measurement results are displayed in a spectrum that gives the intensity (corresponding to the concentration of protein-antibody complexes, displayed on the y-axis) as a function of the molecular weight (monomeric or oligomeric structure displayed on the x-axis). The software can provide a method to apply a baseline correction algorithm to the spectrum, and can use the corrected spectrum, as shown in Figures 3, 5, 6, 7, 8, 9, 10, 11, 12, and 13. The total intensity in the resulting spectrum can also be compared by numerically integrating the intensity for molecular weights in the quantifiable range (i.e., calculating the area under the curve (AUC) with the correct weighting), and by applying a fitting algorithm that fits a set of peaks to calculate the contribution to the signal from individual bands or groups of bands.

[0241] The result for each sample and run is a set of measurements for one analyte, which is referred to as a protein-specific biomarker signature, including but not limited to the total intensity and the intensity from each of the bands shown.

[0242] So in addition to the intensity of each band (which is equal to the amount of each molecular size), the distribution of these bands over a range of molecular sizes can also be observed. In addition, this allows the evaluation of the ratio or relationship of different band sizes and amounts to each other.

[0243] Since the similarities and or differences between the two body fluids are visible by eye in Figures 10, 11, 12, and 13, no additional data is provided here to compare the corresponding nasal fluid and CSF-based measurements.

[0244] It is also contemplated herein to calibrate the marker protein / analyte measurements to improve the quantification of the protein-specific biomarker signature.

[0245] The "run processing SW" and "sample processing SW" can be used as command line tools to process the run data and generate sample reports as needed. It can also be incorporated into an automated setup, where new data from an automated measurement device run is processed as soon as it is available, and a sample report including the measurements of the protein levels / concentrations as well as the position and total intensity of each of the different bands or features is generated as soon as the necessary measurements for this sample are complete.

[0246] As described herein, certain measurements or calculated values of the protein-specific biomarker signature can be used to determine one or more protein concentrations of one or more marker proteins and / or a relative amount / ratio of one marker protein to another marker protein. It is apparent to the skilled person that the corresponding protein-specific biomarker signature includes the values or the corresponding information that allows the calculation of the values.

[0247] It is apparent to the skilled person that further physiological parameters and / or further markers of neurodegenerative diseases are measured in the methods described herein.

[0248] Therefore, it is envisaged that the methods described herein can further comprise the measurement of further physiological parameters and / or further markers of neurodegenerative diseases.

[0249] Further physiological parameters and / or further markers of neurodegenerative diseases can be included to further simplify the diagnosis and support the physician’s decision. Further parameters can include, but are not limited to, age, cognitive performance measured by psychometric means (e.g. Mini Mental State Test (MMST)); risk factors like genetic predisposition, sleep disorders, neurofilament light chain (NFL), glial fibrillary acidic protein (GFAP), alpha synuclein, MTBR-Tau243, apolipoprotein E epsilon 4 (ApoE4), protein S100b, protein S100a, neurogranin, myeloid cell trigger receptor 2 (sTREM2), interleukins, ubiquitin (Ub), superoxide dismutase 1 (SOD1), RNA binding protein FUS / TLS (FUS), TAR DNA binding protein 43 (TDP-43), granulin (GRN), misfolded prion protein (PrPSc), mutant and wild-type huntingtin protein (Htt), and IgM and IgG against Epstein-Barr virus, immunoglobulins; imaging parameters like brain volume reduction, vascular pathology, enlargement of the cerebrospinal fluid space; or general consumption of drugs like smoking and alcohol.

[0250] It is envisaged that evidence identified based on the methods described herein can aid in the diagnosis of neurodegenerative diseases. Therefore, the methods described herein can further comprise pre-screening a subject for the diagnosis and / or prognosis of neurodegenerative diseases. Furthermore, evidence of neurodegenerative diseases identified based on a nasal fluid sample and the methods described herein can be combined with a respective diagnosis to select a treatment suitable for a neurodegenerative disease.

[0251] Reliable and approved identification of pathological alteration values of marker proteins for aiding in the diagnosis of neurodegenerative diseases based on nasal fluids can be fully validated in the near future. The robustness of neurodegenerative marker proteins detected in the nasal fluids described herein allows for an efficient identification of subjects for subsequent diagnosis or treatment, as well as monitoring over time to evaluate treatment effects or side effects, which can also be suitable for evaluating therapy efficacy or therapy failure.

[0252] The treatment can include, but is not limited to, an anti-amyloid based antibody drug therapy like lecanemab. Lecanemab (Leqembi®) is for example approved in the US for the treatment of early AD (mild cognitive impairment [MCI] or mild AD dementia due to AD) with confirmed brain amyloid pathology. The subject to be treated should have amyloid pathology as evidenced by amyloid positive positron emission tomography (PET) results or cerebrospinal fluid (CSF) tests indicative of AD. Furthermore, it can be feasible to confirm efficacy of anti-amyloid drugs like lecanemab in a clinical setting, as the change in rate of decline is relatively small, but the change in values of marker proteins can be reliably detected and thus read out of efficacy of the selected treatment, also supporting dose adjustment and thus reduction of side effects.

[0253] It is also envisaged that the nasal fluid samples and methods described herein can be used to monitor the treatment response. Thus, the values of the marker proteins of the methods described herein can be used to monitor the treatment response. For example, if a patient is identified as having pathologically altered values of the marker proteins from their nasal fluid sample, the patient can be selected based on their characterization, e.g. for a given anti-amyloid treatment like lecanemab. In a successful treatment, the patient is expected to e.g. show one or more of the following changes: a change in one or more oligomeric bands and a change in the relative protein amount of one or more of the marker proteins Aβ42, Aβ40, pTau and tTau (e.g. an increase in the level of the relative protein amount of Aβ42 leading to a change in the Aβ42 / Aβ40 ratio), and possibly also a decrease in the level of the relative amount of pTau and / or tTau, and a change in the oligomerization pattern of these marker proteins.

[0254] Another possibility can be that the patient’s profile does not change, but rather, remains unchanged over time, which can also be seen as a successful treatment and can indicate a stable state, i.e. the progression of the disease can be significantly delayed or even stopped.

[0255] Unless defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure is related. References set forth herein to techniques generally understood by those having ordinary skill in the art are intended to refer to those techniques as commonly understood by those having ordinary skill in the art, including those that are obvious substitutions or equivalents to those techniques.

[0256] The present disclosure is further described by reference to the following non-limiting figures and examples.

[0257] Examples

[0258] 1. Collection, storage, transport, elution and preparation of nasal fluid samples

[0259] Samples were collected at four different clinical centers. Therefore, the absorbent material (AM) polyvinyl alcohol (PVA) was applied by trained personnel with nosecollect® or manually to the olfactory clefts on both sides of the nose of the subjects (N = 2). In case of manual insertion, a Hartmann nasal speculum (13 cm, Karl Storz SE & Co. KG, 400500) and a Jansen Bayonet nasal forceps (16.5 cm, Karl Storz SE & Co. KG, 426516) were used. The thread attached to the AM and hanging outside the nostril was carefully fixed to the subject's cheek with adhesive to prevent accidental displacement. The AM was left in place for 20 to 30 minutes. In case of dry mucosa, the subjects performed physical activity (walk around, climb stairs), ate or drank water to stimulate nasal secretion. After the nasal stay, the thread fixed to the subject's cheek was loosened and the AM saturated with nasal secretion was removed from both sides of the nose by pulling the thread. If one or both AMs of the subject were bloodied, they could be collected by the collector in a separate tube (Eppendorf 50 ml Protein LoBind tubes, Eppendorf, 0030122240).

[0260] The tubes containing the removed AMs were collected upright in a suitable plastic box. The box with the sample tubes was kept at room temperature (RT) for a maximum of 10 minutes and immediately stored at -80°C. A temperature logger (Testo 184 T4, Testo SE & Co. KGaA, 05721844) was added to the samples to guarantee cold chain compliance. The box containing the collected nasal fluid samples was stored upside down at -80°C until shipment to the analysis laboratory. Storage in a -80°C freezer is preferred, however other conditions like dry ice are possible. In this case, make sure that the whole box is surrounded by dry ice to guarantee that the whole box is in isothermal conditions.

[0261] The box containing the collected samples and the temperature logger was transferred upside down into a styrofoam box. The whole styrofoam box was filled with dry ice. The sample box was completely surrounded by dry ice. The samples were then shipped overnight to the analysis laboratory by a commercial logistics partner.

[0262] Shortly after delivery, the styrofoam box was opened at the analysis laboratory. The collected samples were checked for good condition. This means that they were frozen at -80°C during the whole shipment (checked by the state of the remaining dry ice and the report of the included temperature logger) and the integrity of the test tubes was good (not broken, sealed). The samples were then stored in a specific rack or box in the -80°C freezer of the analysis laboratory until pre-treatment.

[0263] On the day of elution, the nasal secretion samples were taken out of the -80°C freezer and thawed for 30 minutes at room temperature (RT). The thawed samples were visually inspected for blood traces under a class II safety cabinet. AM with blood spots were never eluted with blood-free AM. Therefore, blood spots were cut off the AM using a sterile pair of scissors (micro scissors, curved, sharp / sharp, VWR, 233-1454) and forceps (straight, blunt, VWR, 232-2116) and then two AM from one subject were eluted together. The threads were cut off the PVA using a sterile pair of scissors. For elution, Pierce spin columns (10 ml, Thermo Fisher Scientific, PIER89898) were prepared by removing the silica gel membrane. The two AM were placed into these prepared columns and the columns were put back into the original sample tubes of the respective subjects. If blood spots could not be removed, the two AM were placed into separate spin columns and tubes, respectively. The tubes with the spin columns containing the AM were centrifuged at 4566 ref for 5 minutes at room temperature (RT). Afterwards, the spin columns and the AM were discarded. The entire volume of the eluted nasal secretion was transferred from the original Eppendorf 50 ml Protein LoBind tubes used for centrifugation into pre-cooled Eppendorf 1.5 ml Protein LoBind tubes (Eppendorf, 0030108116) and kept on ice. The transferred eluate was centrifuged at 17000 ref for 10 minutes at room temperature in a suitable benchtop centrifuge to pellet the solid components. After centrifugation, the tubes were placed on ice under a class II safety cabinet and the supernatant was transferred into new pre-cooled Eppendorf 1.5 ml Protein LoBind tubes without pipetting the solid or viscous parts. The volume of the transferred eluate was estimated. If blood traces could not be removed from the AM before centrifugation, two AM from one subject can be eluted separately. In this case, the two eluates from one subject were continued to be handled separately. The eluates were evaluated based on the available color scale (1-5) (Figure 1). If a sample was rated color scale 5, the sample was not further processed.

[0264] Several working aliquots were prepared in pre-cooled Eppendorf 1.5 ml Protein LoBind tubes under a class II safety cabinet. During preparation, the samples and aliquots were kept on ice. They were stored at -80°C for long-term storage.

[0265] 2. Analysis of nasal fluid samples

[0266] 2.1 Measurement of total protein content

[0267] The total protein content of the nasal fluid samples (nasal secretion eluates) was measured by BCA assay (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, 10678484). One sample aliquot was taken from the -80°C freezer and equilibrated to room temperature on the workbench for about 30 minutes. The total protein assay (BCA) was performed according to the manufacturer's protocol and the concentration was calculated in mg / mL. In later measurements these results were further used for normalization of the total protein concentration in gel electrophoresis.

[0268] 2.2 Determination of protein-specific biomarker signatures of amyloid-β and Tau

[0269] The following steps describe the determination of protein-specific biomarker signatures of the following key analytes: amyloid-β 40 (Αβ40), amyloid-β 42 (Αβ42), total amyloid-β, phosphorylated Tau (p-Tau), and total Tau (t-Tau). The analysis was performed using one or two antibodies for each analyte and using different capillary sizes (low, medium, high molecular size). Each measurement generates a signature that is specific to the respective marker protein and also specific to the individual disease state. The signature is essentially composed of a multitude of parameters. These parameters include the total level of the analyte, the separation of the analyte by molecular weight based on the quaternary structure of the analyte (e.g. monomers and different oligomers), and the level of the different quaternary structures (e.g. level of monomers, level of oligomers).

[0270] 2.3 Determination of Αβ40

[0271] An aliquot of the nasal fluid sample is used to determine the Ab40-specific biomarker signature. The sample is run on an automated protein separation and immuno-detection system (Simple Western™ Jess, BioTechne, 004-650) and optionally on a traditional gel electrophoresis and Western blot (see protocols below). The aliquot is thawed at room temperature (RT) for 30 minutes. An appropriate volume of the sample is taken so that the final total protein concentration in the assay is 1.25 mg / ml. The sample is diluted in the manufacturer-provided sample buffer. The diluted sample is loaded into the Simple Western™ Jess system and initiated and run as a singleplex assay according to the manufacturer’s instructions. Capillaries with a gel percentage suitable for medium (12 kDa to 230 kDa separation module, 8 x 25 capillary column, BioTechne, SM-W004) are used. A primary antibody #1 (human amyloid-beta (aai-40) antibody, BioTechne / R&D, MAB96181-100) is used and the primary antibody has specificity for the C-terminal end of Ab40. An enzyme-linked secondary antibody (anti-mouse detection module, BioTechne, DM-003) is then added, followed by a chemiluminescent substrate. The Ab40 biomarker signature is measured, showing information about the total level of the analyte within the same sample and the level of Ab40 molecules of different molecular weight size (quaternary structure). As shown in Figures 2 and 4, this allows for the simultaneous detection of monomeric and oligomeric Ab40 molecules and the quantification of the molecules separately, overall and / or relative to each other for each individual molecular weight. Figures 2 and 4 show exemplary banding patterns of Ab40 that can be measured in nasal secretions from patients with different clinical conditions. Each lane represents an individual patient. The molecular weight is shown along the x-axis. Larger Ab40 quaternary structures (e.g. oligomers formed from aggregated monomers in the brain) do not separate into monomers by SDS, thus have a higher molecular weight. Each antibody-epitope recognition is represented by a chemiluminescent signal, so the higher the signal intensity indicates more antibody-epitope recognition, and thus a higher amount of quaternary structure detected or total level of analyte. By calculating the area under the curve (AUC), the amount of antibody-epitope recognition can be measured, and thus the level of a given quaternary structure or the total level of protein can be back-calculated. A graphical representation of the AUC (calculated from the chemiluminescent signal of the bound antibody of Ab40) can be found in Figures 3 and 6 (for more information, see Protein Simple - Jess User Guide Rev G, December 2022; https: / / resources.bio-techne.com / bio-techne-assets / docs / software / Simple%20Western / Jess / Jess%20User%20Guide_Rev%20G.pdf).Oligomers of approximately 100 kDa, 56 kDa and 32 kDa, and 19 kDa, as well as monomers of approximately 4 kDa can be detected. As can be seen for example in Figure 3, the 56 kDa oligomer band shows a higher signal in patients not suffering from AD (corresponding to the A- classification) compared to patients suffering from overt AD (classified as A+). Each curve represents a single patient. The relative decrease of the 56 kDa band in patients suffering from AD (A+) can be explained by the accumulation of Aβ40 in larger Aβ42 aggregates that are insoluble in the brain. Insoluble Aβ42 aggregates in AD patients are known to sequester Aβ40 molecules as well, resulting in a decrease of detected Aβ-40. Figure 4 shows an increase of the monomer species detected in patients not suffering from AD (A-) compared to patients suffering from AD (A+) (see *monomer Figure 4). Figure 5 shows the biomarker signature of Aβ40 from patients suffering from AD (A+) compared to patients not suffering from AD (A-) that also show a higher level of Aβ40 monomers of 4 kDa that can be detected.

[0272] 2.4 Determination of the Aβ42 specific biomarker signature

[0273] An aliquot of the nasal secretion is used to determine the Ab42-specific biomarker signature. The sample is run on an automated protein separation and immuno-detection system (Simple Western™ Jess, BioTechne, 004-650) and optionally on a traditional gel electrophoresis and Western blot. The protocol described below refers to the automated system. The aliquot is thawed for 30 minutes at room temperature. An appropriate volume of the sample is taken so that the final total protein concentration in the assay is 1.25 mg / ml. The sample is diluted in the sample buffer provided by the manufacturer. The diluted sample is loaded into the Simple Western™ Jess system and the system is started and run as a single assay according to the manufacturer’s instructions. Capillaries with a gel percentage suitable for medium (12 to 230 kDa separation module, 8 x 25 capillary column, BioTechne, SM-W004) molecular weight separation are used. A primary antibody #6 (anti-amyloid beta 42 (43), monoclonal antibody (BC05), Fujifilm Wako, 010-26903) is used and the primary antibody is specific for Ab42 and can recognize one or several isoforms and oligomeric structures. Then a secondary enzyme-linked antibody (anti-mouse detection module, BioTechne, DM-003) is added followed by a chemiluminescent substrate. The Ab42-specific biomarker signature is generated showing information about the total amount of analyte within the same sample and the amount of Ab42 molecules of different molecular weight size (quaternary structures). As shown in Figure 6, this allows for the simultaneous detection of different oligomeric Ab42 molecules and the quantification of the molecules separately for each individual size, globally and / or relative to each other. Figure 6 shows two Ab42-specific biomarker signatures, one from a patient with AD (A+) compared to another one from a patient without AD (A-). As evident from Figure 6, oligomers of approximately 32 kDa, 56 kDa, 70 kDa and 92 kDa can be detected. The two samples run on the medium molecular weight gel show that i) the total amount / level of Ab42 can be measured as well as ii) different (oligomeric) quaternary structures and their respective levels. As shown here, there is a global decrease in the total level of Ab42 detected (calculated by the total area under the curve of the whole biomarker signature) in the AD patient (classified as A+) relative to the total level of the A- individual. Both patients also show an altered band ratio between the 56 kDa oligomer and the 32 kDa oligomer. Furthermore, as evident from Figure 11, comparing the relative amount of the same marker protein and the amount of structures in nasal fluid samples and CSF samples from the same individual, oligomeric bands of approximately 32 kDa, 56 kDa as well as additional bands of approximately 75 kDa and 100 kDa can be detected in the nasal fluid samples which are not determinable in the respective CSF samples.

[0274] 2.5 Determination of the total Abeta-specific biomarker signature

[0275] An aliquot of the nasal secretion is used to determine the total Abeta-specific biomarker signature. The sample is run on an automated protein separation and immuno-detection system (Simple Western™ Jess, BioTechne, 004-650) and optionally on a traditional gel electrophoresis and Western blot. The protocol described below refers to the automated system. The aliquot is thawed for 30 minutes at room temperature. An appropriate volume of the sample is taken so that the final total protein concentration in the assay is 1.25 mg / ml. The sample is diluted in the sample buffer provided by the manufacturer. The diluted sample is loaded into the Simple Western™ Jess system and the system is started and run as a single assay according to the manufacturer's instructions. Capillaries with different gel percentages suitable for medium (12 to 230 kDa separation module, 8 x 25 capillary column, BioTechne, SM-W004) molecular weight separation are used. The primary antibody 6E10 (purified anti-beta-amyloid, 1-16 antibody (6E10), BioLegend, 803001) recognizes all forms of Abeta. Then the enzyme-linked secondary antibody (anti-mouse detection module, BioTechne, DM-003) is added followed by the chemiluminescent substrate. The pan Abeta-specific biomarker signature is generated showing information about the total level of the analyte within the same sample and the level of pan Abeta molecules (quaternary structures) of different molecular weight sizes. As shown in Figure 7, this allows the simultaneous detection of monomeric and oligomeric Abeta molecules and the quantification of the molecules separately for each individual size, globally and / or relative to each other. In comparison to the species-specific antibodies described above, the pan Abeta antibody binds to an epitope closer to the N-terminus. This allows the recognition of different quaternary structures in comparison to the antibodies used in 3.2.1 and 3.2.2 above. Figure 7 shows the pan Abeta-specific biomarker signature of a patient not suffering from Alzheimer's disease (AD). It is envisaged that the ratio of the 45 kDa band and the 32 kDa band, the total level and / or the level of high order oligomeric structures (like the 183 kDa band) change in A+ individuals versus A- individuals.

[0276] 2.6 Determination of the total Tau (t-Tau) specific biomarker signature

[0277] An aliquot of the nasal secretion is used to determine the t-Tau specific biomarker signature. The sample is run on an automated protein separation and immuno-detection system (Simple Western™ Jess, BioTechne, 004-650) and optionally on a traditional gel electrophoresis and Western blot. The protocol described below refers to the automated system. The aliquot is thawed for 30 minutes at room temperature. An appropriate volume of the sample is taken so that the final total protein concentration in the assay is 1.25 mg / ml. The sample is diluted in the sample buffer provided by the manufacturer. The diluted sample is loaded into the Simple Western™ Jess system and the system is started and run as a single assay according to the manufacturer’s instructions. Capillaries with different gel percentages suitable for low molecular weight (2 kDa to 40 kDa separation module, 8 x 25 capillary cartridge, BioTechne, SM-W012), medium molecular weight (12 kDa to 230 kDa separation module, 8 x 25 capillary cartridge, BioTechne, SM-W004) or high molecular weight (66 kDa to 440 kDa separation module, 8 x 25 capillary cartridge, BioTechne, SM-W008) separation are used. A primary antibody #7 (purified anti-Tau, 404-441 antibody (mouse), BioLegend, 806601) is used and the primary antibody is specific for t-Tau and recognizes one or several isoforms and oligomeric structures. An enzyme-linked secondary antibody (anti-mouse detection module, BioTechne, DM-003) is then added followed by a chemiluminescent substrate. The t-Tau specific biomarker signature is generated showing information about the total level of the analyte within the same sample and the level of the quaternary structures with different molecular weights. As shown in Figure 8, this allows for the simultaneous detection of monomeric and oligomeric t-Tau molecules and the quantification of the molecules separately for each individual molecular weight, overall and / or relative to each other. Higher levels of the canonical isoform of t-Tau observed at approximately 55 kDa are detected in patients with pronounced signs of neurodegeneration (classified as N+) compared to patients without signs of neurodegeneration (classified as N-) (Figure 8). Different other quaternary structures can also be measured, for example total Tau dimers. The ratio of t-Tau dimers to monomers can also be used to calculate a classification specific score for the N classification (Figure 8). Furthermore, as evident from Figure 12, when comparing the relative amount of the same marker protein tTau and the amount of structures in nasal fluid samples and CSF samples from the same individual (note: the experiment was performed with samples from another subject than reported in Figure 8), the oligomeric band at approximately 63 kDa can be detected with almost the same intensity in both samples from the same individual.Since the applied sample volume of CSF was 6 times the corresponding nasal fluid, this indirectly indicates that the amount of structure observed at 63 kDa in the nasal secretion sample was at least comparable to or higher than in CSF.

[0278] 2.7 Determination of the p-Tau-181 -specific biomarker signature

[0279] An aliquot of the nasal secretion is used to determine the p-Tau-181 specific biomarker signature. The sample is run on an automated protein separation and immuno-detection system (Simple Western™ Jess, BioTechne, 004-650) and optionally on a traditional gel electrophoresis and Western blot. The protocol described below refers to the automated system. The aliquot is thawed for 30 minutes at room temperature. An appropriate volume of the sample is taken so that the final total protein concentration in the assay is 1.25 mg / ml. The sample is diluted in the sample buffer provided by the manufacturer. The diluted sample is loaded into the Simple Western™ Jess system and the system is started and run as a single assay according to the manufacturer’s instructions. Capillaries with different gel percentages suitable for low molecular weight (2 kDa to 40 kDa separation module, 8 x 25 capillary cartridge, BioTechne, SM-W012), medium molecular weight (12 kDa to 230 kDa separation module, 8 x 25 capillary cartridge, BioTechne, SM-W004) or high molecular weight (66 kDa to 440 kDa separation module, 8 x 25 capillary cartridge, BioTechne, SM-W008) separation are used. The primary antibody #8 (Phospho-Tau (Thr181) (D9F4G) Rabbit mAb, Cell Signaling, 12885S) is specific for pTau-181 and recognizes one or several isoforms and oligomeric structures. An enzyme-linked secondary antibody (Anti-Rabbit Detection Module, BioTechne, DM-001) is then added followed by a chemiluminescent substrate. The pTau-181 specific biomarker signature is measured, showing information about the total level of the analyte within the same sample and the level of the quaternary structures with different molecular weights. As shown in Figure 9, this allows us to detect monomeric and oligomeric pTau-181 molecules simultaneously and to quantify the molecules separately for each individual molecular weight, globally and / or relative to each other. As shown in Figure 9, the total level of p-Tau-181 can be calculated by the area under the curve (AUC). As shown here, the total level of p-Tau detected in this individual is reduced in patients with pronounced Tau pathology (T+) compared to patients without Tau pathology (T-). It is also shown here that a reduced level of monomers presenting at around 56 kDa and dimers and trimers with apparent molecular sizes of 96 kDa and 139 kDa, respectively, can be observed in patients with pronounced tau pathology (T+).Furthermore, analyzing another individual's respective samples (note: the experiment was performed with samples from another subject than reported in Fig. 9), as apparent from Fig. 13, and when comparing the relative amounts and amounts of structures of the same marker protein pTau in the nasal fluid sample and the CSF sample from the same individual, the oligomeric bands of approximately 60 kDa, 100 kDa, 146 kDa and 230 kDa can be detected in both samples from the same individual with almost the same intensity. Since the applied CSF sample volume is 6 times the respective nasal fluid volume, this indirectly indicates that the amounts of structures observed in the nasal secretion sample at 60 kDa, 100 kDa, 146 kDa and 230 kDa are at least comparable or higher than the respective CSF from the same individual.

[0280] 3. Generation of CSF reference data

[0281] 3.1 Comparison of respective CSF and nasal fluid core marker patterns

[0282] In order to compare the nasal fluid based measurements of core markers with the CSF based measurements of the same core markers, nasal fluid samples and CSF samples were collected from the same individual, resulting in respective samples.

[0283] Within these respective samples, it was intended to

[0284] i) show the amounts of one or more core AD marker proteins as well as the inter- and intra-protein correlations (pattern (signature) of one or more proteins);

[0285] ii) analyze the respective samples with respect to the distribution and amounts of one or more protein specific monomeric and oligomeric structures of one or more AD marker proteins in the nasal fluid sample as well as in the respective CSF sample obtained from the same subject. Surprisingly, by direct comparison it was found that the signature of one or more AD marker proteins Aβ40, tTau and pTau181 was almost identical. Notably, it was found that the signature of the Aβ42 marker protein in the nasal fluid was different when compared to the respective CSF sample obtained from the same subject;

[0286] iii) the distribution pattern of protein specific monomeric and oligomeric structures within both respective body fluids as well as their respective amounts; and

[0287] iv) show the neurodegenerative disease specific differences with respect to the relative protein amounts of one or more marker proteins as well as the distribution of monomeric and oligomeric structures of one or more marker proteins within different subjects suffering from e.g. underlying amyloid pathology compared to subjects not suffering from amyloid pathology.

[0288] side note AD was chosen as a representative of all neurodegenerative diseases that can be assessed by analysis of brain-derived markers because

[0289] i) it is the most common neurodegenerative disease, and therefore there is a large amount of data and knowledge;

[0290] ii) there are a variety of markers whose amounts can be reliably distinguished in CSF by in vitro diagnostic methods to date; and

[0291] iii) the AD-specific inter-protein and intra-protein correlations in CSF (protein patterns) have been evaluated in extensive studies worldwide, and the level of confidence in authenticity is high on a global scale.

[0292] 3.2 CSF collection

[0293] CSF samples were also obtained from subjects from whom nasal fluid samples were obtained (see sections 1 to 3 above). CSF samples were collected at the clinical center by lumbar puncture, preferably in the morning, and between the 3rdand 4thor 4thand 5thlumbar vertebrae. Following international guidelines, they were collected in polypropylene collection tubes, discarding the first 20 drops. Excessive empty space in the tubes was avoided. Within 4 hours after collection, the CSF was centrifuged at approximately 2000 rcfor 10 minutes at room temperature (RT) and transferred to new polypropylene collection tubes. 1500 mΐ of the transferred CSF was aliquoted into separate polypropylene collection tubes. Within 4 hours after collection, the CSF samples and aliquots were stored at -80ºC in suitable boxes.

[0294] 3.3 CSF sample transport

[0295] The boxes containing the CSF aliquots and temperature loggers were transferred cap-side-up into styrofoam boxes. The entire styrofoam box was filled with dry ice. The sample boxes were completely surrounded by dry ice. The samples were then shipped overnight to the analysis laboratory by a commercial logistics partner.

[0296] 3.4 Sample handling and pre-analysis

[0297] 3.4.1 Sample reception, inspection and storage

[0298] Shortly after delivery, the styrofoam boxes were opened at the analysis laboratory. It was checked whether the collected samples were in good condition. This means that they were frozen at -80ºC throughout the transport (checked by the state of the remaining dry ice and the report of the included temperature loggers), and the integrity of the test tubes was good (not broken, sealed). The samples were then stored in a specific shelf or box in the -80ºC freezer of the analysis laboratory until pre-treatment.

[0299] 3.4.2 Sample Measurement of CSF

[0300] The CSF aliquots were thawed and measured using immunoassay-based measurements (e.g. on the Fuji RebioLumipulse platform) according to the manufacturer’s protocol for all the following core analytes: amyloid-β40, amyloid-β42, pTau181, and tTau.

[0301] 3.4.3 Sample Measurement of Nasal Fluid Samples

[0302] To evaluate and compare biomarker proteins in a pooled assessment in the sense of overall results, and to compare biomarker protein patterns between two body fluids, comparable measurement techniques were used to analyze the correlation of biomarker proteins for these assessments.

[0303] As mentioned above, since the in vitro CSF ​​analysis for analyzing AD biomarker proteins is performed using an immunoassay, the analysis of nasal fluid samples for AD biomarker proteins is also performed using a semi-automated immunoassay (see Figures 14 to 17).

[0304] Analyze nasal fluid samples using the following SIMOA kit for all core AD analytes on an SR-X instrument (Quanterix) and following the manufacturer's instructions.

[0305] • SIMOA pTau-181 Advantage V2 kit (Quanterix, 103714), 1:5 dilution

[0306] • SIMOA Tau Advantage kit (Quanterix, 101552), 1:20 dilution

[0307] • SIMOA Aß-40 Advantage Kit (Quanterix, 101672), 1:5 dilution

[0308] • SIMOA Aß-42 Advantage kit (Quanterix, 101664), 1:2.5 dilution.

[0309] 4. Data processing for measurement results based on immunoassays

[0310] Subsequent data analysis was performed using CSF-based measurements and nasal fluid-based measurements of all four analytes.

[0311] The respective datasets were stored in Excel files and processed with Python scripts using standard libraries like pandas and NumPy. Linear and logistic regressions were calculated using the sklearn package. Plots were generated using the matplotlib and seaborn packages. For quantitative analysis, mainly these methods were used to draw conclusions from the data. If the underlying distributions are indeed identical, the P-value measures the probability of observing the same or a greater difference between the samples observed; a smaller value indicates a significant difference. Values of p < 0.05 were described as significant. We also verified that the non-parametric alternatives of the Student's t-test (Mann-Whitney test, Wilcoxon test) lead to similar values and conclusions.

[0312] When comparing two variables (e.g., different markers / analytes) from the same cohort, we used linear regression. The result (in addition to the fitted line that gives the best linear relationship between the quantities) is Pearson's "r", which is a parameter of the goodness of fit, and Pearson's p, which is a parameter of statistical significance. The parameter "r" indicates the degree to which one quantity depends on the other, with a range of 0.1 to 0.3 counted as weak correlation, a range of 0.3 to 0.5 as moderate correlation, and a range of 0.5 and greater as strong correlation. The significance p still indicates the probability of measuring this degree or greater of correlation by chance if the quantities were independent.

[0313] 4.1 Data processing of protein-specific biomarker signatures

[0314] Corresponding nasal fluid and CSF samples are additionally run on an automated protein separation and immuno-detection system (Simple Western™ Jess, BioTechne, 004-650) and optionally on traditional gel electrophoresis and Western Blot to show the almost identical distribution of protein specific monomeric and oligomeric structures and to show the respective amounts within these different quaternary structures and their relationship. The described protocol refers to the automated system. Nasal fluid aliquots are first thawed for 30 minutes at room temperature. Appropriate volume amounts of each nasal fluid sample are taken so that the final total protein concentration in the assay is 1.25 mg / ml. The samples are diluted in the manufacturer provided sample buffer. The diluted samples are loaded into the Simple Western™ Jess system and the system is started and run as a single assay according to the manufacturer’s instructions. Different capillaries with different gel percentages suitable for low molecular weight (2 kDa to 40 kDa separation module, 8 x 25 capillary cartridge, BioTechne, SM-W012), medium molecular weight (12 kDa to 230 kDa separation module, 8 x 25 capillary cartridge, BioTechne, SM-W004) or high molecular weight (66 kDa to 440 kDa separation module, 8 x 25 capillary cartridge, BioTechne, SM-W008) separation are used. Primary antibodies specific for core AD marker proteins are used which recognize one or several marker protein specific isoforms and oligomeric structures. Then enzyme linked secondary antibodies are added followed by the addition of chemiluminescent substrate. Marker protein specific biomarker signatures are measured revealing information about i) the total level of the analyte and ii) the maximum position and amount of the quaternary structures of different molecular weight within the same sample. This allows for the simultaneous detection of monomeric and oligomeric molecules and the quantification of the molecules separately for each individual molecular weight, globally and / or relative to each other as shown in Figures 10, 11, 12 and 13.

[0315] On the same measurement platform, corresponding CSF samples from the same individual / subject are applied according to the above described protocol, the only difference being that 6 times the volume of CSF is applied and compared to the applied volume of the nasal fluid samples. CSF is collected as before in the lumbar region. In CSF, the core protein markers of AD are diluted in a large volume, therefore the fluid itself is not highly protein rich.

[0316] A data set for each marker protein is generated for each measurement of a corresponding set of body fluid samples on an automated protein separation and immuno-detection system. As shown in Figures 10, 11, 12, and 13, the measurements are displayed in a spectrum that gives the intensity (corresponding to the concentration of protein-antibody complex, displayed on the y-axis) as a function of molecular weight (monomeric or oligomeric structure displayed on the x-axis). The software provides a method to apply a baseline correction algorithm to the spectrum, and uses the corrected spectrum, as shown in Figures 3, 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0317] The total intensity in the generated spectrum can also be compared by numerically integrating the intensity for molecular weights in the quantifiable range (i.e., calculating the area under the curve (AUC) with the correct weighting), and the contribution to the signal from individual bands or groups of bands can also be calculated by applying a fitting algorithm that fits a set of peaks.

[0318] The result for each sample and run is a set of measurements for one analyte, which is referred to as a protein-specific biomarker signature, and which includes, but is not limited to, the total intensity and the intensity from each of the displayed bands.

[0319] So in addition to the intensity of each band (which is equal to the amount of each molecular size), the distribution of these bands over a range of molecular sizes can also be observed. In addition, this allows the evaluation of the ratio or relationship of different band sizes and amounts to each other.

[0320] Since similarities and / or differences between the two body fluids are visible to the naked eye here, no additional data is provided to compare the corresponding nasal fluid- and CSF-based measurements here.

Claims

1. A nasal fluid sample obtained from a subject, wherein the nasal fluid sample comprises biomarker proteins β-amyloid (Aβ), phosphorylated Tau (pTau), and / or total Tau (tTau).

2. The nasal fluid sample according to claim 1, wherein the nasal fluid sample is a purified nasal fluid sample.

3. The nasal fluid sample according to claim 1 or 2, wherein the concentration of the one or more marker proteins is equivalent to or at least 1.1 times higher than that of a cerebrospinal fluid (CSF) sample obtained from the same subject.

4. The nasal fluid sample according to any one of claims 1 to 3, wherein the nasal fluid sample is characterized in that the pTau protein concentration is less than the tTau protein concentration.

5. The nasal fluid sample according to any one of claims 1 to 4, wherein the relative amount of Aβ42 / Aβ40 is greater than 0 and less than 1.

6. The nasal fluid sample according to any one of claims 1 to 5, wherein the Aβ marker is characterized by a molecular weight of about 4 kDa, about 8 kDa, about 12 kDa, about 16 kDa, about 19 kDa, about 24 kDa, about 32 kDa, about 40 kDa, about 44 kDa, about 48 kDa, about 52 kDa, about 56 kDa, about 60 kDa to 72 kDa, about 84 kDa to 120 kDa and / or greater than about 140 kDa.

7. The nasal fluid sample according to any one of claims 1 to 5, wherein the pTau marker is characterized by a molecular weight of about 20 kDa, about 30 kDa, about 38 kDa, about 55 kDa to 62 kDa, about 96 kDa to 106 kDa, and about 140 kDa to 160 kDa and / or greater than about 180 kDa.

8. The nasal fluid sample according to any one of claims 1 to 5, wherein the tTau marker is characterized by a molecular weight of about 30 kDa, about 38 kDa, about 48 kDa, about 55 kDa to 62 kDa, about 96 kDa to 106 kDa, about 140 kDa to 160 kDa and greater than 160 kDa.

9. Use of a nasal fluid sample according to any one of claims 1 to 8 for the adjunctive diagnosis of neurodegenerative diseases.

10. A method for assisting in the diagnosis of a neurodegenerative disease in a subject using a nasal fluid sample, wherein the method comprises the following steps: (a) Determine the protein concentrations of the biomarker proteins phosphorylated Tau (pTau), total Tau (tTau), and / or optionally β-amyloid (Aβ) in a nasal fluid sample according to any one of claims 1 to 8; (b) Compare the protein concentration of the marker protein as determined in the nasal fluid sample with the protein concentration in the control; as well as (c) Determine whether the value of the marker protein has changed pathologically relative to the concentration of the protein in the control.

11. The method of claim 10, wherein the neurodegenerative disease is selected from Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), Pick's disease, and Kreutzfeldt-Jacob disease (CJD).

12. The method according to claim 10 or 11, wherein the relative amount of Aβ42 / Aβ40 negatively correlated with pTau and / or tTau is evidence of neurodegenerative disease in the subject.

13. The method according to claim 10 or 11, wherein the relative amount of Aβ42 / Aβ40 is positively correlated with pTau and / or tTau as evidence of neurodegenerative disease in the subject.

14. The method of claim 12, wherein any one of (i) or (ii) below applies: (i) pTau is positively correlated with tTau, and Aβ42 is positively correlated with Aβ40; and (ii) The relative amount of Aβ42 / Aβ40 is greater than 0 and less than 1.

15. The method according to item 13, wherein either (i) or (ii) below applies: (i) pTau is positively correlated with Aβ42 / Aβ40, and Aβ42 is negatively correlated with Aβ40; and (ii) The relative amount of Aβ42 / Aβ40 is greater than 0 and less than 1.

16. The method according to any one of claims 10 to 15, wherein the neurodegenerative disease is AD.

17. Use of the nasal fluid sample according to claim 9, wherein the neurodegenerative disease is selected from Alzheimer's disease (AD), Parkinson's disease, chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), Pick's disease, and Kreutzfeldt-Jacob disease (CJD).

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