Lateral flow device for diagnosing Alzheimer's disease using T14 peptide

Abstract

The present invention relates to neurodegenerative disorders, and the diagnosis and / or prognosis of neurodegenerative disorders in test subjects using lateral flow assays. The present invention also relates to detecting diagnostic and prognostic biomarkers in a range of different patient sample types to diagnose and / or prognose neurodegenerative disorders, such as Alzheimer's disease. The present invention further provides biomarker detection methods and device(s) for diagnosing and prognosing neurodegenerative disorders, as well as methods of treating patients diagnosed or prognosed with a neurodegenerative disorder. The present invention also extends to the detection and / or screening of biomarkers in pre-symptomatic subjects for early diagnosis to enable disease prevention or intervention.

Description

[Technical Field]

[0001] The present invention relates to neurodegenerative disorders, and particularly, but not exclusively, to the diagnosis and / or prognosis of neurodegenerative disorders in test subjects using lateral flow assays. The present invention also relates to detecting diagnostic and prognostic biomarkers in a range of different patient sample types to diagnose and / or prognose neurodegenerative disorders, such as Alzheimer's disease. The present invention further provides biomarker detection methods and device(s) for diagnosing and prognosing neurodegenerative disorders, as well as methods of treating patients diagnosed or prognosed with a neurodegenerative disorder. The present invention also extends to the detection and / or screening of biomarkers in pre-symptomatic subjects for early diagnosis to enable disease prevention or intervention. [Background technology]

[0002] Neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, or motor neuron disease, are some of the world's greatest socioeconomic burdens, with an ever-increasing global incidence of nearly 10 million new cases of dementia each year. Because both the incidence and prevalence of AD increase with age, the number of patients continues to grow significantly with the aging population. In 2015, there were more than 46 million people living with dementia, with an estimated socioeconomic cost of $800 billion per year, and the number of patients is expected to rise to over 130 million by 2050, costing society more than $2 trillion per year. AD was recently declared the leading cause of death in people over 65 years of age in the UK, and is currently the sixth leading cause of death across all ages in the US. Summary of the Invention

[0003] Currently, there is no single test that can be performed to diagnose Alzheimer's disease or to prognose cognitive decline (e.g., Braak stages IV, V, or VI). It is also currently not possible to diagnose pre-symptomatic AD (e.g., Braak stages I, II, or III). Clinical classification of AD relies on a combination of subjective report, medical history assessment, cognitive function testing, and costly brain imaging scans, and true classification will not be possible until post-mortem brain examinations can be performed. All of these tests require the involvement of specialists, but accuracy remains variable. They are also time-consuming and therefore slow and expensive.

[0004] Thus, there is a need to provide improved methods, devices, and / or kits for diagnosing disease in subjects suspected of having a neurodegenerative disorder (e.g., Braak stage I, II, or III), particularly Alzheimer's disease, and for prognosing disease etiology or progression in subjects already diagnosed with the condition (e.g., Braak stage IV, V, or VI). There is also a need to provide methods and devices for screening for neurodegenerative disorders and detecting risk of developing a neurodegenerative disorder in subjects who are pre-symptomatic for, or suspected of having, such a condition (e.g., Braak stage I or II). Ideally, such improved methods and devices would be rapid, quantitative (or at least semi-quantitative or qualitative), accurate, painless, and inexpensive.

[0005] Continuing previous research in this field, the present inventors have focused on the toxic peptide "T14," which is derived from the C-terminus of acetylcholinesterase (AChE) and exists as a naturally occurring bioactive molecule in brain tissue. WO 2016 / 156803 describes antibodies against the AChE-derived peptide (T14), and it has previously been shown that T14 found in cerebrospinal fluid (CSF) or venous blood samples can act as a robust biomarker for diagnosing neurodegenerative disorders, such as Alzheimer's disease.

[0006] However, as discussed in the Examples and shown in Figure 7, the inventors have now surprisingly found that T14 peptide levels are elevated in the brain not only in late-stage Alzheimer's disease patients (e.g., Braak stage V or VI), but also at very early stages of neurodegeneration, i.e., pre-symptomatic stages of Alzheimer's disease (e.g., Braak stage I, II, or III). This was completely unexpected. Furthermore, as shown in Figure 2, the inventors have also unexpectedly found that the T14 biomarker is not only detectable in CSF and venous blood draws, but is also present at very small, but still detectable, concentrations in various peripheral tissues, such as saliva, nasal secretions, and pinprick blood. Such peripheral tissue types can be easily analyzed using lateral flow tests (LFTs), which, unlike CSF and venous / arterial blood (as shown in our previous work), are painless, socially acceptable by the general public, low cost, can be used frequently (daily or even weekly), and provide very fast and accurate results.

[0007] Therefore, we believe that, in combination, these data form the basis for a first-in-class reliable diagnostic or prognostic screen for neurodegenerative disorders, as well as for diagnosing pre-symptomatic neurodegenerative disorders, e.g., early-onset Alzheimer's disease.

[0008] Thus, in a first aspect of the present invention there is provided a lateral flow method for diagnosing or prognosing a neurodegenerative disorder in a subject, the method comprising the step of using lateral flow to detect a peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof (T14) in a sample obtained from the test subject, wherein detection of the presence of a peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in the sample indicates that the test subject has a neurodegenerative disorder or a predisposition to or poor prognosis thereof.

[0009] In a second aspect of the present invention, there is provided a lateral flow device for diagnosing or prognosing a neurodegenerative disorder in a subject, the device comprising a lateral flow support for detecting a peptide comprising or consisting of SEQ ID NO: 3 (T14) or a variant or fragment thereof in a sample obtained from a test subject, wherein detection of the peptide comprising or consisting of SEQ ID NO: 3 (T14) or a variant or fragment thereof corresponds to the subject having a neurodegenerative disorder or a predisposition to or poor prognosis thereof.

[0010] In a third aspect, there is provided a method of treating a subject having or suspected of having a neurodegenerative disorder, a pre-symptomatic neurodegenerative disorder and / or suffering from cognitive decline, comprising: (a) using lateral flow to detect a peptide (T14) comprising or consisting of SEQ ID NO: 3, or a variant or fragment thereof, in a sample obtained from a test subject, wherein detecting the presence of a peptide comprising or consisting of SEQ ID NO: 3, or a variant or fragment thereof, in the sample indicates that the test subject has a neurodegenerative disorder or a predisposition to or poor prognosis thereof; and (b) administering or administering to the subject a therapeutic agent that prevents, reduces, or delays neurodegeneration and / or cognitive decline. A method is provided, comprising:

[0011] The present inventors believe to be the first to develop a method for detecting T14 peptide in patient samples using a lateral flow test.

[0012] Thus, in a fourth aspect, there is provided a method for detecting a peptide (T14) comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in a sample obtained from a test subject, the method comprising using lateral flow to detect a peptide (T14) comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in a sample obtained from the test subject.

[0013] Due to its rather small volume and therefore significantly low concentration, it was totally unexpected that detectable levels of T14 peptide (SEQ ID NO: 3) would be found in any peripheral body sample suitable for lateral flow testing (e.g., saliva, nasal discharge, or pinch blood as shown in Figure 2). There are various scenarios in which the method and apparatus of the present invention would be of significant value, as it advantageously allows for accurate and rapid screening, diagnosis, and prognosis of neurodegenerative disorders (preferably Alzheimer's disease) using lateral flow testing (LFT).

[0014] LFTs are also known in the art as "rapid tests," "quick tests," or "lateral flow immunoassays (LFIA)," and are therefore also covered by the present invention.

[0015] First, for those who have already been diagnosed with Alzheimer's disease, a rapid and accurate test that can be used to actively monitor the etiology or progression of their condition would be of great value. Furthermore, conducting such a test could then influence more accurate dosage of therapeutic drugs administered to patients and also produce better patient outcomes. Therefore, preferably, the method and device are used as a prognostic method or device for prognosticating the progression of neurodegenerative disorders.

[0016] Second, there is considerable value in an accurate and fast diagnostic test for use in people who are suspected of having Alzheimer's disease but have not yet been diagnosed. Such a test would be significantly advantageous compared to the significantly higher cost, frequency, and effectiveness of currently deployed cognitive testing methods, for example, after patient referral to a memory clinic. Therefore, preferably, the method and device of the present invention are diagnostic methods or diagnostic devices for diagnosing neurodegenerative disorders.

[0017] Third, having a simple test available for people who are not currently suspected of having Alzheimer's, i.e., they are young and / or pre-symptomatic, would clearly be very useful. Thus, there is a great opportunity for the general population to have a pre-symptomatic test that can be performed at a GP surgery or even at home, i.e., as part of a general medical examination, regardless of any predisposing factors or symptoms. For example, LFT testing can be routinely performed in certain age groups or above a certain age, similar to breast cancer or bowel cancer screening. This pre-test screening can be qualitative in nature, for example, producing a binary result of either "positive" or "negative" for a predefined range (or window) of T14 values ​​regarding the risk of having or predisposing to a neurodegenerative disease. Depending on the results of the pre-test screening, the subject can then be referred to a physician or other specialized medical practitioner, who can then perform more accurate quantitative testing and, if necessary, provide specialist medical intervention. The pre-test screening can be self-administered. Therefore, subjects who are significantly outside the range of T14 values ​​considered to be at risk for or predisposed to neurodegenerative disease (i.e., have a "negative" result) can be filtered out, and only those subjects who have a "positive" result in the pre-test screening can be subjected to supervised qualitative testing to determine whether they are actually at an early stage of neurodegenerative disease. Therefore, preferably, the method and device are used to diagnose the pre-symptomatic state of patients who will or may develop a neurodegenerative disorder in the future. Preferably, the method is a diagnostic method that includes diagnosing a neurodegenerative disorder, preferably a pre-symptomatic state, in a test subject. The ability of early detection allows for early therapeutic intervention, thus delaying the onset of neurodegenerative disorders or even preventing the disease.

[0018] Fourth, because patients can effectively serve as their own controls and monitor how much they have worsened or not since their previous test (e.g., one day, one week, or one month ago), pharmaceutical companies can save a great deal of time and cost in drug development in terms of timescales, smaller sample groups, and other value metrics when accurate measures of disease progression are readily available. Thus, preferably, the methods and devices are used in clinical trials to monitor the activity or effectiveness of drugs administered to test subjects, preferably to determine how much a subject has worsened or not since their previous test.

[0019] Preferably, the lateral flow devices or methods of the first to fourth aspects are performed in vitro. It is understood that the lateral flow tests are immunoassays, but not ELISA. Thus, preferably, the activity of peptide SEQ ID NO:3 (T14) or variants or fragments thereof is not determined using ELISA, i.e., non-ELISA methods and kits / devices.

[0020] Preferably, the lateral flow device or method of the first to fourth aspects is used to identify the presence or absence of the peptide of SEQ ID NO: 3 (T14) or a variant or fragment thereof in a sample and / or to determine its concentration, preferably the concentration of soluble T14, in a sample. The T14 peptide (SEQ ID NO: 3) can be assayed by several lateral flow systems known to those skilled in the art. Lateral flow is a form of immunoassay used to detect T14 peptide or determine T14 levels. Preferably, the method (or assay) or device is adapted to detect the presence and / or absence of T14 in a sample. The lateral flow device or method may include the use of positive and / or negative controls against which the assay can be compared.

[0021] It is particularly preferred that the method or device of the present invention comprises the detection of soluble T14 (SEQ ID NO: 3 or a variant or fragment thereof). In one embodiment, the concentration of a soluble peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof is determined. Preferably, the method or device comprises a means for determining the concentration of SEQ ID NO: 3 or a variant or fragment thereof in a sample obtained from a test subject. The means for determining the concentration of SEQ ID NO: 3 or a variant or fragment thereof in a sample obtained from a test subject may comprise an anti-T14 antibody or an antigen-binding fragment thereof, i.e., a T14-neutralizing antibody. The antibody or antigen-binding fragment thereof may be polyclonal or monoclonal. The antibody or antigen-binding fragment thereof may be generated in rabbit, mouse, or rat.

[0022] Embodiments of the lateral flow method and device of the first through fourth aspects are shown in Figures 1 and 3-6. Preferably, the device method includes the use of a sample extraction means to obtain a sample from a test subject. For example, the sample extraction means may include a nasal swab, a mouth swab, or a needlestick blood extraction means. Each of these tools for obtaining a sample for lateral flow is shown in Figure 1.

[0023] Once the sample is obtained from the subject, it can then be inserted into an appropriate sample collection container. The container preferably contains a buffer solution. In one embodiment, the buffer solution can contain 0.5 M Tris HCl, pH 6.8, 10% glycerol, 2% (w / v) sodium dodecyl sulfate, 5% (v / v) 2-b-mercaptoethanol, and 0.05% bromophenol blue. Preferably, the sample is mixed with the buffer solution until all of the T14 peptide is suspended therein.

[0024] Preferably, the device includes an LFT cartridge having a sample opening into which a buffered sample solution is delivered. Preferably, the cartridge includes an opening or window through which the test line and control line can be viewed.

[0025] Figures 4 and 5 show two different preferred embodiments of the lateral flow device and method of the present invention. Advantageously, the LFT assays described herein are qualitative, semi-quantitative, or quantitative. In one embodiment, the device or method preferably comprises an inhibition lateral flow test, as shown in Figure 4. In another embodiment, the device or method preferably comprises a sandwich lateral flow test, as shown in Figure 5.

[0026] Preferably, the LFT cartridge includes a sample pad on which a sample is placed. Preferably, the LFT cartridge includes an anti-T14 antibody preferably placed on a conjugate pad. For example, the anti-T14 antibody may be as described in WO2016 / 156803. Preferably, the conjugate pad is placed substantially adjacent to the sample pad. Preferably, the anti-T14 antibody is labeled with a labeling moiety. For example, the labeling moiety may include nanoparticles, preferably gold nanoparticles. The average diameter of the nanoparticles may be between 10 nm and 65 nm, or between 20 nm and 60 nm, or between 30 nm and 50 nm.

[0027] Preferably, the LFT cartridge includes an immobilized T14 peptide test line containing a T14 peptide bound thereto. Preferably, the LFT cartridge includes a control line containing an anti-species labeled antibody not specific for T14. For example, the anti-species labeled antibody may include a goat anti-chicken antibody or a goat anti-rabbit antibody. For example, the anti-species labeled antibody may include a goat anti-chicken IgY polyclonal antibody or a goat anti-rabbit IgY polyclonal antibody. Preferably, the control line is laterally spaced from the immobilized T14 peptide test line and is preferably distal from the sample pad and conjugate pad. Preferably, the LFT cartridge includes a wick positioned at or toward the opposite end of the cartridge from the sample pad and configured to draw sample solution laterally across the cartridge.

[0028] As shown in the upper part of Figure 4 (showing a positive result for T14 detection using an inhibition lateral flow test), if the sample contains the sample T14 peptide, it flows laterally over the conjugate pad bearing the anti-T14 antibody and is preferably captured by the anti-T14 antibody to produce a T14-antibody conjugate. The T14-antibody conjugate preferably cannot bind to the immobilized T14 peptide attached to the test line. However, preferably, anti-T14 antibody not bound to the sample T14 peptide in the sample can flow through and bind to the anti-species-labeled antibody attached to the control line. It is understood that the sample T14 will bind the anti-T14 antibody that cannot bind to the immobilized T14 peptide attached to the cartridge. Therefore, preferably, a single line appears on the cartridge as a positive result, as shown in the upper right corner of Figure 4.

[0029] As shown in the lower part of Figure 4 (showing a negative result for T14 detection using an inhibition lateral flow test), if the sample does not contain sample T14 peptide, the sample preferably flows laterally over the conjugate pad carrying the anti-T14 antibody. Because there is no sample T14 to be captured by the anti-T14 antibody, preferably no T14-antibody conjugate is produced. Therefore, any unbound anti-T14 antibody preferably flows through and binds to the LFT-immobilized T14 peptide bound to the test line and, preferably, the anti-species antibody control bound to the control line. Thus, two spaced apart lines preferably appear on the cartridge as a negative result, as shown in the lower right of Figure 4.

[0030] Referring now to Figure 5, the inventors have also developed a sandwich (i.e., two-antibody) assay format due to the size of the T14 peptide. Preferably, the method or device includes a first antibody that binds to a first epitope located in one region of the T14 peptide (e.g., at or toward the N-terminus) and a second antibody that binds to a second epitope on the T14 peptide spaced from the first epitope (e.g., at or toward the C-terminus). Preferably, the first and second epitopes are different amino acids on the T14 peptide.

[0031] Preferably, the LFT cartridge comprises a sample pad on which a sample is placed. Preferably, the LFT cartridge comprises a first anti-T14 antibody, which is preferably placed on a conjugate pad. For example, the anti-T14 antibody may be as described in WO2016 / 156803. The first anti-T14 antibody preferably binds to a first epitope on the T14 peptide, for example, the C-terminal residue of T14. Preferably, the conjugate pad is placed substantially adjacent to the sample pad. Preferably, the anti-T14 antibody is labeled with a labeling moiety. For example, the labeling moiety may comprise nanoparticles, preferably gold nanoparticles.

[0032] Preferably, the LFT cartridge includes a T14 peptide test line containing a second anti-T14 antibody. Preferably, the second anti-T14 antibody binds to a second epitope on the T14 peptide. For example, the second anti-T14 antibody can bind to the N-terminus of T14.

[0033] Preferably, the LFT cartridge includes a control line containing an anti-species-labeled antibody not specific for T14. For example, the anti-species-labeled antibody can include a goat anti-chicken antibody or a goat anti-rabbit antibody. For example, the anti-species-labeled antibody can include a goat anti-chicken or anti-rabbit IgY polyclonal antibody. Preferably, the control line is laterally spaced from the immobilized T14 peptide test line and is preferably distal from the sample pad and conjugate pad. Preferably, the LFT cartridge includes a wick positioned at or toward the opposite end of the cartridge from the sample pad and configured to draw the sample solution laterally across the cartridge.

[0034] As shown in the top of Figure 5 (showing a positive result for T14 detection using a sandwich lateral flow test), if the sample contains the T14 peptide, it flows laterally over the conjugate pad bearing the first anti-T14 antibody and is preferably captured by the anti-T14 antibody, thereby producing a T14-antibody conjugate. Preferably, the T14-antibody conjugate itself is captured by a second anti-T14 antibody bound to the cartridge along the test line (16). This is possible because the second epitope on the T14 peptide is still exposed in the conjugate. Thus, preferably, a first line appears on the cartridge indicating antibody binding at the T14 test line. Preferably, any additional anti-T14 antibody in the sample that is not bound to the T14 peptide flows through and preferably binds to the anti-species control bound to the control line. Advantageously, unlike the inhibition LFT assay described above, the sandwich assay using the first and second anti-T14 antibodies is a direct detection of the sample T14 present in the sample because the cartridge does not contain any LFT-immobilized T14. Thus, a second line appears on the cartridge as a positive result, as shown in the top right of Figure 5.

[0035] As shown in the bottom of Figure 5 (showing a negative result for T14 detection using a sandwich lateral flow test), if the sample does not contain T14 peptide, it preferably flows laterally over the conjugate pad bearing the first anti-T14 antibody; no T14 is captured by the anti-T14 antibody, and therefore, preferably, no T14-antibody conjugate is produced. Preferably, all unbound first anti-T14 antibody flows away and cannot bind to the second T14 antibody bound to the cartridge. The T14 antibody preferably flows through and binds to the anti-species control bound to the control line, preferably producing a single line on the cartridge (10) as a negative result, as shown in the bottom right of Figure 5.

[0036] It will be appreciated from the above that the lateral flow test provides a fast and convenient means for detecting the presence or absence of a variant of the T14 peptide or a fragment thereof in a sample obtained from a subject.

[0037] Advantageously, the presence of the control line ensures that the method is robust and effective, and the presence or absence of the test line informs the subject about the presence or absence of T14 peptide or its variant or fragment in their sample.Visually assessing the presence or absence of the test line provides a qualitative diagnostic or prognostic test, and visually determining the relative density (or color darkness) of the test line provides a useful semi-quantitative measure.In this way, the use of lateral flow test to screen for T14 is highly valuable and can be performed by subjects at home by themselves (i.e., without the presence of qualified medical staff).

[0038] However, it is difficult to achieve accurate and completely quantitative evaluation from simply looking at the LFT results. Therefore, preferably, the method or device can include a means for quantitatively measuring the results of the LFT cartridge. In one embodiment, the lateral flow cartridge can be connected to a reader that runs software to detect the test line and the control line, and then accurately calculates the concentration of T14 peptide in the sample based on the concentration / value of the test line and the control line.

[0039] In another embodiment, the cartridge can be connected to a computer, tablet, or smartphone that runs software to detect the test and control lines and then calculates the concentration of T14 peptide in the sample based on the concentrations / values ​​of the test and control lines. Alternatively, the user can take a picture of the LFT cartridge, and the software will determine the level of T14 peptide in the sample based on the strength or intensity of the signal in the test and control lines.

[0040] Preferably, the sample comprises a biological sample. The sample may be any biological material obtainable from a subject from which SEQ ID NO: 3 (T14) or a variant or fragment thereof may be obtained.

[0041] The sample can be nasal secretion or mucus, saliva, blood, venous blood, arterial blood, plasma, serum, capillary blood, non-venous blood, non-arterial blood, pinprick blood, cerebrospinal fluid, urine, sweat, tears, breast aspirate, prostatic fluid, semen, vaginal fluid, feces, cervical scraping, cytes, amniotic fluid, ocular fluid, mucus, respiratory moisture, animal tissue, cell lysate, tumor tissue, hair, skin, cheek scraping, lymph, interstitial fluid, nail, bone marrow, cartilage, prion, bone powder, earwax, or a combination thereof.

[0042] Preferably, the sample does not contain blood, most preferably does not contain venous or arterial blood. Preferably, the sample does not contain saliva.

[0043] Preferably, however, the sample comprises a peripheral tissue sample. Preferably, the sample comprises nasal secretion or discharge, saliva, capillary blood or pinprick blood.

[0044] The device may include a sample collection container for receiving the extracted sample obtained from the subject. The blood sample may be immediately assayed for T14 levels. Alternatively, the blood sample may be stored at a low temperature, for example, in a refrigerator, or even frozen, before the T14 assay is performed. The detection of T14 peptide may be performed on whole blood obtained from the blood sample. However, preferably, the blood sample includes serum obtained. Preferably, the blood sample includes plasma.

[0045] The blood may be further processed before the T14 assay is performed. For example, an anticoagulant, such as citrate (e.g., sodium citrate), hirudin, heparin, PPACK, or sodium fluoride, may be added. Thus, the sample collection container may contain an anticoagulant to prevent the blood sample from clotting. Alternatively, the blood sample may be centrifuged or filtered to prepare a plasma or serum fraction that can be used for analysis. Therefore, it is preferable that T14 be analyzed or assayed in a plasma or serum sample. It is particularly preferable that the T14 concentration be measured in vitro from a serum or plasma sample obtained from a subject.

[0046] Most preferably, the sample comprises a capillary blood sample, preferably a pinprick blood sample. Most preferably, the sample comprises a saliva sample. Most preferably, the sample comprises a nasal secretion or nasal discharge sample. Advantageously, any of the above samples can be used in an LFT method or device. However, it is also possible to detect T14 peptide in these samples (especially nasal secretion / nasal discharge and pinprick blood samples) by non-LFT means.

[0047] Accordingly, in a fifth aspect, there is provided a method of diagnosing or prognosing a neurodegenerative disorder in a subject, the method comprising the step of detecting a peptide comprising or consisting of SEQ ID NO: 3 (T14) or a variant or fragment thereof in a nasal discharge, mucus or pinprick blood sample obtained from a test subject, wherein detection of the presence of a peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in the sample indicates that the test subject has a neurodegenerative disorder or a predisposition to or poor prognosis thereof.

[0048] In a sixth aspect of the present invention, there is provided a neurodegenerative disorder diagnostic or prognostic device for diagnosing or prognosing a neurodegenerative disorder in a subject, the device comprising means for detecting a peptide (T14) comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in a nasal discharge, mucus or pinprick blood sample obtained from a test subject, wherein detection of a peptide (T14) comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof corresponds to the subject having a neurodegenerative disorder or a predisposition to or poor prognosis thereof.

[0049] In a seventh aspect, there is provided a method of treating a subject having or suspected of having a neurodegenerative disorder, a pre-symptomatic neurodegenerative disorder and / or suffering from cognitive decline, comprising: (a) detecting a peptide (T14) comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in a nasal secretion, mucus, or pinprick blood sample obtained from the test subject, wherein detecting the presence of a peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in the sample indicates that the test subject has a neurodegenerative disorder or a predisposition to or poor prognosis thereof; and (b) administering or administering to the subject a therapeutic agent that prevents, reduces, or delays neurodegeneration and / or cognitive decline. A method is provided, comprising:

[0050] The methods or devices of the fifth to seventh aspects may be carried out in vivo, in vitro or ex vivo, however preferably the methods are carried out in vitro.

[0051] Most preferably, the sample comprises nasal secretions or mucus. Most preferably, the sample comprises saliva.

[0052] Preferably, the devices or methods of the fifth to seventh aspects are used to identify the presence or absence of T14 (SEQ ID NO: 3 or a variant or fragment thereof) in a sample or to determine its concentration in a sample, preferably the concentration of soluble T14. The means for determining the T14 concentration may include an assay adapted to detect the presence and / or absence of T14 in a sample. The device or method may include the use of positive and / or negative controls against which the assay can be compared.

[0053] While the methods or devices of the first through fourth aspects involve the use of lateral flow for the methods or devices of the fifth and sixth aspects, T14 peptide (SEQ ID NO: 3) can be assayed by several methods known to those skilled in the art, but not necessarily via lateral flow. For example, immunoassays are preferably used to detect T14 peptide or determine T14 levels. However, it is understood that non-immune-based assays can also be used, for example, by labeling a compound with affinity for a ligand of T14 peptide and then assaying for the label. T14 peptide can also be determined using Western blot analysis, which can be used to determine total protein levels of T14 peptide. Thus, T14 peptide concentrations can be detected by enzyme-linked immunosorbent assay (ELISA), fluorescence assay, chemiluminescence assay, or radioimmunoassay analysis.

[0054] Immunoassays (e.g., LFT ELISA) are most preferably used to detect soluble T14 peptide. Western blot analysis is most preferably used to detect aggregated T14 peptide. It is particularly preferred that the methods, devices, and uses of the present invention include the detection of soluble T14 (SEQ ID NO: 3).

[0055] In one embodiment, the concentration of (i) a soluble peptide comprising or consisting of SEQ ID NO: 3, or a variant or fragment thereof, or (ii) an aggregated peptide comprising or consisting of SEQ ID NO: 3, or a variant or fragment thereof, is determined. However, in a preferred embodiment, the concentration of (i) a soluble peptide comprising or consisting of SEQ ID NO: 3, or a variant or fragment thereof, and (ii) an aggregated peptide comprising or consisting of SEQ ID NO: 3, or a variant or fragment thereof, is determined. Preferably, both soluble and aggregated T14 can be detected in combination.

[0056] Preferably, however, the devices or methods of the fifth to seventh aspects involve the use of a lateral flow assay or test, similar to the devices or methods of the first to fourth aspects.

[0057] Preferably, the method or device of the fifth to seventh aspects includes a means for determining the concentration of SEQ ID NO: 3 or a variant or fragment thereof in a sample obtained from a test subject. The means for determining the concentration of (i) soluble T14 and / or (ii) aggregated T14 in a nasal secretion, nasal mucus, or pinprick blood sample obtained from the test subject may include an anti-T14 antibody or antigen-binding fragment thereof, i.e., a T14-neutralizing antibody. The antibody or antigen-binding fragment thereof may be polyclonal or monoclonal. The antibody or antigen-binding fragment thereof may be generated in rabbits, mice, or rats.

[0058] Any of the methods or devices described herein involve the use of an anti-T14 immunospecific antibody or antigen-binding fragment thereof. Preferably, the antibody or antigen-binding fragment thereof specifically binds to SEQ ID NO: 3. Preferably, the antibody or antigen-binding fragment thereof specifically binds to one or more amino acids in the C-terminus of SEQ ID NO: 3. Preferably, the antibody or antigen-binding fragment thereof specifically binds to one or more amino acids in SEQ ID NO: 11 (i.e., SYMVHWK, which are the C-terminal amino acids 7-14 of SEQ ID NO: 3). Preferably, the antibody or antigen-binding fragment thereof specifically binds to a C-terminal lysine (K) residue in the epitope. The present inventors have surprisingly observed that the C-terminal amino acid sequence VHWK in SEQ ID NO: 3, set forth herein as SEQ ID NO: 12 (i.e., the C-terminal amino acids 11-14 of SEQ ID NO: 3), functions as an epitope for the antibody or antigen-binding fragment thereof. Thus, more preferably, the antibody or antigen-binding fragment thereof specifically binds to one or more amino acids in SEQ ID NO: 12. Most preferably, the antibody or antigen-binding fragment thereof specifically binds to SEQ ID NO: 12. It is therefore understood that the epitope to which the antibody binds comprises or consists of SEQ ID NO: 12. Thus, the antibody or antigen-binding fragment thereof specifically binds to SEQ ID NO: 3 or a fragment or variant thereof and can be used as or in a means of detecting T14 peptide.

[0059] Preferably, the antibody or antigen-binding fragment thereof does not bind to SEQ ID NO: 2 (ie, T30).

[0060] Preferably, the antibody or antigen-binding fragment thereof does not bind to SEQ ID NO: 13 (ie, T15), ie, NQFDHYSKQDRCSDL.

[0061] Preferably, the antibody or antigen-binding fragment thereof does not bind to SEQ ID NO: 14 (i.e., β-amyloid (Aβ), i.e., DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA).

[0062] The device or method may further include the use of a label that can be detected in the assay. The term "label" may refer to any moiety that can be attached to a means for determining the concentration of T14 peptide, be it soluble T14 and / or aggregated T14, in a sample obtained from a test subject. The moieties can be used, for example, for therapeutic or diagnostic procedures. Therapeutic labels include, for example, moieties that can be attached to the antibodies or fragments thereof described herein and moieties that can be used to monitor antibody binding to the T14 peptide (i.e., SEQ ID NO: 3 or a fragment or variant thereof). Diagnostic labels include, for example, moieties that can be detected by analytical methods. Analytical methods include, for example, qualitative, semi-quantitative, and quantitative procedures. Qualitative analytical methods include, for example, immunohistochemistry and indirect immunofluorescence. Quantitative analytical methods include, for example, immunoaffinity procedures, such as radioimmunoassays, ELISA, or FACS analysis. Analytical methods also include both in vitro and in vivo imaging procedures. Specific examples of diagnostic labels that can be detected by analytical means include enzymes, radioisotopes, fluorescent dyes, chemiluminescent markers, and biotin.

[0063] Preferably, T14 peptide concentration can be measured by double-antibody sandwich ELISA or LFT. ELISA can involve, for example, using an appropriate antibody to coat a microtiter plate (for ELISA) or a substrate (for LFT). For example, such an appropriate antibody can include the anti-T14 peptide antibody described herein (WO 2016 / 156803). Furthermore, ELISA can involve using an appropriate antibody for detection. For example, such an antibody can include a peroxidase-labeled monoclonal mouse anti-human T14 peptide antibody. Human T14 peptide, which can be purified from nasal plasma and then quantified by amino acid analysis, can be used to calibrate plasma or nasal secretion standards using standard techniques known to those skilled in the art. The label can be directly attached to the antibody or to a secondary binding agent that specifically binds to T14. Such a secondary binding agent can be, for example, a secondary antibody. The secondary antibody can be either polyclonal or monoclonal and can be of human, rodent, or chimeric origin.

[0064] The pinprick blood sample used in the device or method of the fifth to seventh aspects can be assayed immediately for T14 levels. Alternatively, the pinprick blood sample can be stored at a low temperature, for example, in a refrigerator, or even frozen, before the T14 assay is performed. Detection of T14 can be performed on whole blood obtained from a pinprick. However, preferably, the pinprick blood sample contains serum. Preferably, the pinprick blood sample contains plasma.

[0065] The blood may be further processed before the T14 assay is performed. For example, an anticoagulant, such as citrate (e.g., sodium citrate), hirudin, heparin, PPACK, or sodium fluoride, may be added. Thus, the sample collection container may contain an anticoagulant to prevent the blood sample from clotting. Alternatively, the blood sample may be centrifuged or filtered to prepare a plasma or serum fraction that can be used for analysis. Therefore, it is preferable that T14 be analyzed or assayed in a plasma or serum sample. It is particularly preferable that the T14 concentration be measured in vitro from a serum or plasma sample collected from a subject.

[0066] Preferably, the device or method is used to identify the presence or absence of T14 (SEQ ID NO: 3 or a variant or fragment thereof) in a sample or to determine its concentration in a sample, preferably the concentration of T14. The means for determining the T14 concentration may include an assay adapted to detect the presence and / or absence of T14 in a sample. The device or method may include the use of positive and / or negative controls against which the assay can be compared.

[0067] The concentration of a peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in a sample can be between 0.1 ng and 1000 ng, or between 0.2 ng and 750 ng, or between 0.5 ng and 500 ng per mg of protein in the sample. Preferably, the concentration of a peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in a sample is between 1 ng and 400 ng, or between 2 ng and 300 ng, or between 3 ng and 200 ng per mg of protein in the sample. Preferably, the concentration of a peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in a sample is between 4 ng and 100 ng, or between 5 ng and 75 ng, or between 10 ng and 50 ng, or between 20 ng and 40 ng per mg of protein in the sample.

[0068] The present invention may be used in a method or device where the subject has or is suspected of having a neurodegenerative disease selected from the group consisting of Alzheimer's disease; Parkinson's disease; Huntington's disease; motor neuron disease; spinocerebellar types 1, 2 and 3; amyotrophic lateral sclerosis (ALS); schizophrenia; dementia with Lewy bodies; and frontotemporal dementia.

[0069] However, the present invention is preferably used to study or predict cognitive decline in any neurological disorder associated with the non-enzymatic function of AChE.Therefore, preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease and motor neuron disease, preferably Alzheimer's disease or Parkinson's disease.

[0070] However, it is particularly preferred that the methods and devices of the present invention be used when a subject has or is suspected of having Alzheimer's disease.

[0071] It is therefore understood that in a preferred embodiment, the method or device of the present invention may involve the use of lateral flow to detect SEQ ID NO:3 or a variant or fragment thereof in a sample, preferably nasal secretion, mucus or saliva, to diagnose or prognose Alzheimer's disease.

[0072] Examples of suitable therapeutic agents that can be administered to subjects to prevent or treat neurodegeneration and / or cognitive decline include, but are not limited to, acetylcholinesterase inhibitors, such as rivastigmine, galantamine and donepezil, and / or N-methyl-D-aspartic acid (NMDA) antagonists, such as memantine.Alternative treatments include the use of cyclic peptides as described in WO2015 / 004430, linear peptides as described in WO2015 / 053601, or peptidomimetics as described in WO2018 / 033724, the contents of all three patent applications of which are incorporated herein by reference.

[0073] Preferably, the subject to be tested is a living subject. The subject may be a vertebrate, a mammal, or a livestock animal. However, most preferably, the subject is a human, which may be male or female. The subject may be a child or an adult. The subject's age may be at least 20, 30, 40, 50, 60, 65, or 70 years old. However, the subject may be less than 80 years old, less than 70 years old, less than 65 years old, less than 60 years old, or less than 50 years old.

[0074] In one embodiment, the method and device are used for subjects who have already been diagnosed with neurodegenerative disorders.Therefore, the subject is preferably symptomatic and shows signs of cognitive decline or dementia.Therefore, the method and device can be prognostic and can monitor disease progression.

[0075] In another embodiment, the methods and devices are used on a subject suspected of having a neurodegenerative disorder. The subject may be symptomatic for the neurodegenerative disorder. Thus, the methods and devices may be diagnostic.

[0076] In a preferred embodiment, the method and device are used for subjects who are not suspected of having neurodegenerative disorders.Preferably, the subject is examined before any symptoms of neurodegeneration, cognitive decline or neurodegenerative disorders appear.Preferably, the subject is before symptoms appear.Preferably, the subject is in Braak stage I or II.Therefore, the method and device can be diagnostic.

[0077] It will therefore be appreciated that in a preferred embodiment the method or device of the present invention may involve the use of lateral flow to detect SEQ ID NO:3 or a variant or fragment thereof in a sample, preferably nasal secretions, mucus or saliva, to diagnose or prognose Alzheimer's disease, most preferably pre-symptomatic AD.

[0078] Continuing from previous work, the inventors continued their work using acetylcholinesterase and their antibodies that demonstrate immunospecificity for a particular region in the C-terminus of this enzyme, as described in WO2016 / 156803, the contents of which are incorporated herein by reference.

[0079] Acetylcholinesterase is a serine protease that hydrolyzes acetylcholine and is well known to those skilled in the art. The major form of acetylcholinesterase found in the brain is known as tailed acetylcholinesterase (T-AChE). The protein sequence of one embodiment of human tailed acetylcholinesterase (GenBank: AAA68151.1) is 614 amino acids in length and is provided herein as SEQ ID NO: 1: 1 mrppqcllht pslaspllll llwllgggvg aegredaell vtvrggrlrg irlktpggpv 61 saflgipfae ppmgprrflp pepkqpwsgv vdattfqsvc yqyvdtlypg fegtemwnpn 121 relsedclyl nvwtpyprpt sptpvlvwiy gggfysgass ldvydgrflv qaertvlvsm 181 nyrvgafgfl alpgsreapg nvglldqrla lqwvqenvaa fggdptsvtl fgesagaasv 241 gmhllsppsr glfhravlqs gapngpwatv gmgearrrat qlahlvgcpp ggtggndtel 301 vaclrtrpaq vlvnhewhvl pqesvfrfsf vpvvdgdfls dtpealinag dfhglqvlvg 361 vvkdegsyfl vygapgfskd neslisraef lagvrvgvpq vsdlaaeavv lhytdwlhpe 421 dparlreals dvvgdhnvvc pvaqlagrla aqgarvyayv fehrastlsw plwmgvphgy 481 eiefifgipl dpsrnytaee kifaqrlmry wanfartgdp neprdpkapq wppytagaqq 541 yvsldlrple vrrglraqac afwnrflpkl lsatdtldea erqwkaefhr wssymvhwkn 601 qfdhyskqdr csdl[SEQ ID NO:1] The amino acid sequence of T30 (which corresponds to the last 30 amino acid residues of SEQ ID NO:1) is provided herein as SEQ ID NO:2 below: KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL [SEQ ID NO: 2] The amino acid sequence of T14 (which corresponds to the 14 amino acid residues located towards the end of SEQ ID NO: 1 and lacks the final 15 amino acids found in T30) is provided herein as SEQ ID NO: 3 below: AEFHRWSSYMVHWK [SEQ ID NO: 3] Thus, preferably the peptide of SEQ ID NO: 3 or a variant or fragment thereof is T14. Most preferably, the methods and devices of the present invention involve the detection of a soluble peptide comprising or consisting of SEQ ID NO: 3.

[0080] However, fragments of T14 (SEQ ID NO: 3) are also detectable in the methods and devices of the present invention and may act as diagnostic or prognostic markers for use in accordance with the present invention.

[0081] Thus, in one embodiment, a fragment of SEQ ID NO: 3 preferably comprises the amino acid sequence of SEQ ID NO: 4 (ie, T7), ie, SYMVHWK.

[0082] In another embodiment, the fragment of SEQ ID NO:3 preferably comprises the amino acid sequence of SEQ ID NO:5 (ie, T8), ie, SSYMVHWK.

[0083] In another embodiment, the fragment of SEQ ID NO:3 preferably comprises the amino acid sequence of SEQ ID NO:6 (ie, T9), ie, WSSYMVHWK.

[0084] In another embodiment, the fragment of SEQ ID NO: 3 preferably comprises the amino acid sequence of SEQ ID NO: 7 (ie, T10), ie, RWSSYMVHWK.

[0085] In another embodiment, the fragment of SEQ ID NO: 3 preferably comprises the amino acid sequence of SEQ ID NO: 8 (ie, T11), ie, HRWSSYMVHWK.

[0086] In another embodiment, the fragment of SEQ ID NO: 3 preferably comprises the amino acid sequence of SEQ ID NO: 9 (ie, T12), ie, FHRWSSYMVHWK.

[0087] In another embodiment, the fragment of SEQ ID NO: 3 preferably comprises the amino acid sequence of SEQ ID NO: 10 (ie, T13), ie, EFHRWSSYMVHWK.

[0088] In other words, although detection of T14 (ie, SEQ ID NO: 3) is preferred, the invention may also rely on detection of one or more of any of T7 to T13 (ie, SEQ ID NOs: 4 to 10).

[0089] Preferably, the method or apparatus of the present invention includes determining a subject's Braak stage. The subject may be at any of Braak stages I, II, or III. Preferably, the subject is at Braak stage I. Preferably, the subject is at Braak stage II. Preferably, the subject is at Braak stage III.

[0090] Preferably, the subject is a living subject. To date, it has only been possible to Braak stage deceased subjects, and therefore the invention described herein represents a significant advance over currently available approaches.

[0091] Thus, preferably, the method comprises: (a) analyzing the concentration of a peptide (T14) comprising or consisting of SEQ ID NO: 3, or a variant or fragment thereof, in a sample obtained from a test subject; and (b) comparing this concentration with a reference value from a control population of deceased subjects with known Braak stage for the concentration of a soluble peptide comprising or consisting of SEQ ID NO: 3, or a variant or fragment thereof. wherein the Braak stage of a live test subject is determined by comparing the concentration of a peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof with a respective reference value associated with the Braak stage.

[0092] It is understood that the methods and devices of the present invention can be used to determine and monitor disease progression in a manner that correlates well with the Braak stage. Advantageously, the results described in the Examples support the inventors' hypothesis that detection of the peptide of SEQ ID NO: 3 in an individual can be used to determine the Braak stage of a living subject. Currently, Braak staging can only be performed on postmortem brains, and therefore the methods and devices of the present invention provide a significant advance over these current methods. The use of the T14 biomarker (i.e., the peptide of SEQ ID NO: 3 or a variant or fragment thereof) allows for the determination of a patient's Braak stage with a very high degree of specificity and sensitivity through a non-invasive, easily repeatable, and cost-effective procedure, such as blood, urine, or CSF collection, thereby enabling routine screening, diagnosis of the Braak stage, and appropriate intervention through therapeutic treatment.

[0093] Braak staging has six stages based on the location of neurofibrillary tangles, with Braak stage 0 corresponding to a healthy subject. Stages I and II relate to early-stage disease, at which point neurofibrillary tangles are limited to the transitional entorhinal region of the brain. Braak stages I and II are pre-symptomatic, and as shown in Figure 7, the inventors surprisingly demonstrated that it is possible to detect T14 in samples at each of these stages. Stages III and IV define the involvement of neurofibrillary tangles in limbic regions, including the hippocampus, and stages V and VI are the time points at which neurofibrillary tangles become widespread in the neocortical region of the brain. Thus, the methods and devices of the present invention can be used to determine Braak stages 0, I, II, III, IV, V, or VI in living subjects. Braak staging is a good method for recording the progression of Parkinson's disease and Alzheimer's disease in postmortem brains and is currently far more reliable than any premortem method.

[0094] It will also be appreciated that the methods of the present invention are useful for allowing clinicians to accurately diagnose the stage of neurodegeneration and / or cognitive decline and, therefore, make informed decisions regarding the best course of treatment for a patient based on the detected T14 concentration in the sample or based on their Braak stage. Furthermore, the methods are useful for monitoring the effectiveness of putative treatments for neurodegeneration and cognitive decline. Thus, the devices of the present invention are useful for providing a prognosis of a subject's condition, so that clinicians can implement treatment according to the third or seventh aspect. The devices can also be used to monitor the effectiveness of putative treatments for neurodegeneration and cognitive decline. Thus, the methods and devices are highly useful for guiding clinicians in treatment regimes and for monitoring the effectiveness of such treatment regimes.

[0095] Preferably, the concentrations of (i) a soluble peptide (T14) comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof and / or (ii) an aggregated peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof are analyzed, and the Braak stage of the live test subject is determined by comparing the concentration of either the soluble or aggregated peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof with respective reference values ​​related to the Braak stage.

[0096] Preferably, soluble peptides are analyzed. In some embodiments, a higher concentration of soluble peptides comprising or consisting of SEQ ID NO: 3 or its variants or fragments compared to a reference value indicates a later Braak stage. In other words, the higher the concentration of soluble T14, the stronger the correlation with the later Braak stage of a living subject, for example, stage IV, V or VI. Soluble peptide SEQ ID NO: 3 (T14) or its variants or fragments is preferably determined using ELISA, which is most preferably performed on saliva, nasal discharge / nasal secretion or needle-prick plasma samples obtained from subjects.

[0097] Preferably, however, a lower concentration of a soluble peptide comprising or consisting of SEQ ID NO:3 or a variant or fragment thereof compared to the reference value indicates a later Braak stage. In other words, the lower the concentration of soluble T14, the stronger the correlation with a later Braak stage, e.g., stage IV, V, or VI, in a living subject. The soluble peptide SEQ ID NO:3 (T14) or a variant or fragment thereof is preferably determined using lateral flow or ELISA, which is most preferably performed on saliva, nasal discharge / nasal secretion, or needle stick plasma samples obtained from the subject.

[0098] Therefore, preferably, a lower concentration of the soluble peptide or its variant or fragment comprising SEQ ID NO:3 compared to the reference value indicates that the patient is beta-amyloid positive, and / or a higher concentration of the soluble peptide or its variant or fragment comprising SEQ ID NO:3 compared to the reference value indicates that the patient is beta-amyloid negative. Preferably, a lower concentration of the soluble peptide or its variant or fragment comprising SEQ ID NO:3 compared to the reference value indicates that the subject is cognitively impaired, and / or a higher concentration of the soluble peptide or its variant or fragment comprising SEQ ID NO:3 compared to the reference value indicates that the patient is cognitively normal.

[0099] Thus, in a preferred embodiment, a soluble peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof at a lower concentration compared to the reference value indicates Braak stage IV. Alternatively, in another preferred embodiment, a soluble peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof at a lower concentration compared to the reference value indicates Braak stage V. Alternatively, in another preferred embodiment, a soluble peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof at a lower concentration compared to the reference value indicates Braak stage VI.

[0100] The inventors have also surprisingly discovered that the concentration of T14 differs among early Braak stages (see FIG. 7), thus demonstrating that T14 levels can be used to determine early pre-symptomatic Braak stages (e.g., I, II, and III) in living patients. Thus, in one embodiment, a lower concentration of a soluble peptide comprising or consisting of SEQ ID NO:3, or a variant or fragment thereof, compared to a reference value, indicates Braak stage I. Alternatively, in another embodiment, a lower concentration of a soluble peptide comprising or consisting of SEQ ID NO:3, or a variant or fragment thereof, compared to a reference value, indicates Braak stage II. Alternatively, in another embodiment, a lower concentration of a soluble peptide comprising or consisting of SEQ ID NO:3, or a variant or fragment thereof, compared to a reference value, indicates Braak stage III.

[0101] In another embodiment, a higher concentration of the soluble peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof compared to the reference value indicates that the patient is beta-amyloid positive, and / or a lower concentration of the soluble peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof compared to the reference value indicates that the patient is beta-amyloid negative. Preferably, a higher concentration of the soluble peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof compared to the reference value indicates that the subject is cognitively impaired, and / or a lower concentration of the soluble peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof compared to the reference value indicates that the patient is cognitively normal.

[0102] Those skilled in the art will understand how to measure the concentration of T14 peptide (either soluble or aggregated) in a statistically significant number of control individuals and the concentration of T14 in a test subject, and then use these respective numbers to determine the test subject's Braak stage. Comparing the levels of SEQ ID NO: 3 peptide (i.e., T14) in samples (preferably plasma) collected as close as possible to the subject's death from a large group of well-characterized Braak stage 0 individuals (i.e., disease-free or "normal" health) may be a preferred method of defining a control population (i.e., cohort) of reference values.

[0103] In another embodiment, after collection of postmortem CSF samples from a sufficient number of control and Alzheimer's disease subjects (e.g., n>50 for each group), their soluble T14 and aggregated T14 levels can be measured by ELISA and Western blot, respectively. These levels are preferably calibrated to the subject's Braak stage (=0 for controls) to determine the relationship between changes in T14 and progressive increases in Braak staging. The resulting standard curve can be used with future ex-vivo CSF ​​samples from living patients to extrapolate their Braak stage from their T14 levels in asymptomatic stages of the patient's disease.

[0104] In another embodiment, a sufficient number of control and AD subject samples (e.g., n>200 for each group) may be required. Their soluble T14 and / or aggregated T14 levels can be measured by ELISA and Western blot, respectively. These levels can be normalized to the levels from healthy subjects. This normalization step may not be necessary. Then, the ranges of control and AD values ​​can be plotted with confidence intervals. By detecting whether T14 values ​​fall within or outside the control T14 range or within or outside the AD T14 range, a single sample from a patient without disease symptoms can be diagnosed or disease progression can be predicted.

[0105] Therefore, the inventors have found that the difference in T14 concentration between normal and elevated / decreased levels of aggregated T14 or soluble T14, respectively, can be used as a physiological marker to determine the Braak stage of a living subject.If a subject has a decreased concentration of soluble T14 that is significantly lower than the reference soluble T14 concentration, or an elevated concentration of aggregated T14 that is significantly higher than the reference aggregated T14 concentration, this is understood to indicate a higher Braak stage.The inventors have also discovered that T14 concentration differs between early Braak stages, thus demonstrating that T14 levels can be used to determine the early pre-symptomatic Braak stage (I, II, and III) in living patients.

[0106] For example, the decrease in soluble T14 concentration from the reference concentration can be at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, and most preferably at least 50% from the reference concentration. Such a decrease in soluble T14 concentration indicates that the test subject has a higher Braak stage. Alternatively, the increase in aggregated T14 concentration from the reference concentration can be approximately at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, and most preferably at least 50% from the reference concentration. Such an increase in aggregated T14 concentration indicates that the test subject has a higher Braak stage. Thus, the clinician can make an informed decision regarding the required preferred course of treatment, for example, the type and dosage of the therapeutic agent according to the third or seventh embodiment to be administered.

[0107] In another embodiment, if a subject has an elevated concentration of soluble T14 that is significantly higher than the reference soluble T14 concentration, or a lower concentration of aggregated T14 that is significantly lower than the reference aggregated T14 concentration, this is understood to indicate a lower Braak stage.

[0108] For example, the increase in soluble T14 concentration from the reference concentration can be at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, and most preferably at least 50% from the reference concentration. Such an increase in soluble T14 concentration indicates that the test subject has a higher Braak stage. Alternatively, the decrease in aggregated T14 concentration from the reference concentration can be approximately at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, and most preferably at least 50% from the reference concentration. Such a decrease in aggregated T14 concentration indicates that the test subject has a higher Braak stage. Thus, the clinician can make an informed decision regarding the required preferred course of treatment, for example, the type and dosage of the therapeutic agent according to the third or seventh embodiment to be administered.

[0109] The methods or devices of the invention may further comprise measuring the rate of cognitive decline by Mini Mental State Examination (MMSE) score and / or Preclinical Alzheimer Cognitive Composite (PACC) score.

[0110] MMSE is a questionnaire that is practically universally administered to people with suspected AD and in wider research groups as a measure of cognitive impairment.It is widely regarded as the gold standard for diagnosis of AD due to its ease of application, repeatability, validity and reliability, which requires little training.It is also particularly useful when considering the long-term evaluation of AD and its progression.The repeated measurement of MMSE score that is collected at regular intervals can be used to calculate the speed of cognitive decline for subjects.Then, linear regression is preferably carried out to calculate the slope of MMSE score change over time, and this slope is interpreted as the speed of cognitive change (decline / slope).Therefore, cognitive decline is preferably measured in terms of MMSE score.Slope can be calculated in terms of the decline in MMSE score per month.

[0111] The PACC trial, on the other hand, combines tests assessing episodic memory, timed executive function, and global cognition, which is the primary outcome measure for the first clinical trial in preclinical AD.

[0112] The methods of the present invention may further comprise the step of age-adjusting the T14 concentration from the test subject to a corresponding reference value, whether soluble or aggregated T14.

[0113] It is understood that the present invention extends to any nucleic acid or peptide or variant, derivative or analog thereof that substantially comprises the amino acid or nucleic acid sequence of any of the sequences referred to herein, including variants or fragments thereof. The terms "substantially an amino acid / nucleotide / peptide sequence," "variant" and "fragment" may refer to a sequence that has at least 40% sequence identity with the amino acid / nucleotide / peptide sequence of any one of the sequences referred to herein, for example, 40% identity with any of the sequences described herein.

[0114] Also contemplated are amino acid / polynucleotide / polypeptide sequences that have greater than 65% sequence identity with any of the sequences mentioned, more preferably greater than 70%, even more preferably greater than 75%, and even more preferably greater than 80% sequence identity. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity with any of the sequences mentioned, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, and most preferably at least 99% identity with any of the sequences mentioned herein.

[0115] Those skilled in the art will understand how to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences.To calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences, the alignment of two sequences must first be prepared, and then sequence identity value is calculated.The percentage identity of two sequences can take different values ​​depending on (i) the method used to align sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by alignment method, such as local versus global alignment, the pair score matrix used (for example, BLOSUM62, PAM250, Gonnet, etc.) and gap penalty, such as function type and constant.

[0116] Once aligned, there are many different ways to calculate the percentage identity between two sequences.For example, it can divide the number of identities by (i) the length of the shortest sequence; (ii) the length of alignment; (iii) the average length of sequence; (iv) the number of non-gap positions; or (v) the number of equivalent positions excluding overhang.It is also understood that percentage identity is also strongly length-dependent.Therefore, the shorter the pair of sequences, the higher the sequence identity that can be expected to exist by chance.

[0117] Therefore, it is understood that accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred method for generating multiple alignments of proteins or DNA according to the present invention. Suitable parameters for ClustalW may be as follows: for DNA alignment: gap opening penalty = 15.0, gap extension penalty = 6.66, and matrix = Identity; for protein alignment: gap opening penalty = 10.0, gap extension penalty = 0.2, and matrix = Gonnet; for DNA and protein alignment: ENDGAP = -1 and GAPDIST = 4. Those skilled in the art will recognize that these and other parameters may need to be varied for optimal sequence alignment.

[0118] The calculation of the percentage identity between two amino acid / polynucleotide / polypeptide sequences is then preferably performed from such an alignment by calculating (N / T) *The percentage identity between two sequences can be calculated as 100, where N is the number of positions where the sequences share the same residue, and T is the total number of positions compared, including gaps, and either including or excluding overhangs.Preferably, overhangs are included in the calculation.Therefore, the most preferred method for calculating the percentage identity between two sequences is to (i) prepare a sequence alignment using the ClustalW program with an appropriate set of parameters, such as those shown above; and (ii) calculate the values ​​of N and T using the following formula: sequence identity=(N / T) * 100.

[0119] Alternative methods for identifying similar sequences are known to those skilled in the art. For example, a substantially similar nucleotide sequence is encoded by a sequence that hybridizes to a DNA sequence or its complement under stringent conditions. By stringent conditions, we mean that the nucleotides hybridize to filter-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by at least one wash in 0.2x SSC / 0.1% SDS at approximately 20-65°C. Alternatively, a substantially similar polypeptide may differ from the sequence set forth in, for example, SEQ ID NO: 3 by at least 1, 2, 3, or 4 amino acids, but less than 5, 10, 20, 50, or 100 amino acids.

[0120] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be varied or altered to provide functional variants thereof without substantially affecting the sequence of the protein encoded thereby. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons encoding the same amino acid within the sequence, thereby producing a silent (synonymous) change. Other suitable variants include all sequences or portions of sequences that have a homologous nucleotide sequence but have been altered by the substitution of different codons encoding amino acids with side chains with similar biophysical properties to the amino acid they replace, resulting in a conservative change. For example, small nonpolar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar, hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Thus, it is understood which amino acids can be replaced by amino acids with similar biophysical properties, and one of skill in the art knows the nucleotide sequences that encode these amino acids.

[0121] Every feature described in this specification (including any accompanying claims, abstract and drawings), and / or every step of any method or process so disclosed, may be combined in any combination with any of the above aspects, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0122] For a better understanding of the present invention, and to show how embodiments thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: [Brief explanation of the drawings]

[0123] [Figure 1] Shown (from left to right) are a nasal swab for obtaining nasal secretions or mucus, a mouth swab for obtaining oral fluids such as saliva, and a device for performing a needle prick to obtain a needle prick blood sample. Each of these sample types is known as a peripheral tissue. [Figure 2] FIG. 2 shows T14 immunoassay data obtained from saliva samples from six individuals (labeled 1-6) using the mouth swabs shown in FIG. 1. [Figure 3] FIG. 2 shows a perspective view of a lateral flow test cartridge with a sample dropper for delivering a sample (e.g., needle stick blood, nasal mucus, or saliva as shown in FIG. 2) to the sample opening (S). [Figure 4] FIG. 1 shows a side view of a first embodiment of a lateral flow device of the present invention, called Inhibition LFT (wet format), for detecting the T14 peptide biomarker. [Figure 5] FIG. 1 shows a side view of a second embodiment of a lateral flow device of the present invention, called sandwich LFT (wet format), for detecting the T14 peptide biomarker. [Figure 6] FIG. 1 shows a perspective view of an embodiment of a desktop reader (left) or a smartphone (right) running an app for reading the LFT cartridge shown in FIG. 3 according to either the inhibition LFT of FIG. 4 or the sandwich LFT of FIG. 5, which can be used to quantify the amount of T14 in a sample. [Figure 7] FIG. 1 shows three illustrations (from left to right) of brains with clinical symptoms and corresponding Braak stages showing an increase in the later stages, and corresponding Western blots for hippocampal T14 peptide at Braak stages I, II, and VI. [Figure 8]FIG. 1 shows the theranostic interaction between detecting T14 levels using the methods of the invention (e.g., LFT) and therapeutic intervention with an acetylcholinesterase inhibitor or an N-methyl-D-aspartate (NMDA) antagonist or a cyclic peptide (described in WO 2015 / 004430 and known as NBP-14 cyclic peptide) administered to achieve durable prevention of symptom onset. [Figure 9] FIG. 1 shows the apparatus used to perform the wet reagent or "half-dipstick" test method. [Figure 10] FIG. 1 shows the results of an inhibition lateral flow assay. [Figure 11] FIG. 1 shows the results of a sandwich lateral flow assay. DETAILED DESCRIPTION OF THE INVENTION

[0124] [Example] The inventors have surprisingly observed that the T14 biomarker peptide is present in very small but detectable concentrations in saliva, nasal secretions, and pinprick blood. Such sample types can generally be analyzed using lateral flow tests (LFTs), which, unlike CSF and venous / arterial blood, are painless, socially acceptable by the general public, low-cost, can be used frequently, and provide very fast and accurate results.

[0125] material and method Generation of Braak stage data, CSF data, and saliva data Western Blot – Brain tissue, CSF and saliva The analyzed human hippocampal samples and postmortem CSF were provided by the Oxford Brain Bank and classified as Braak stage 0, I, II, III, V, or VI. Saliva samples were collected from six donors and placed in separate tubes.

[0126] Brain tissue: Western blots were performed as described herein: Briefly, approximately 0.2 g of frozen human brain tissue was thawed and homogenized in ice-cold Neuronal Protein Extraction Reagent supplemented with phosphatase and protease inhibitors. 1 mL of buffer was added per 0.2 g of brain tissue. The homogenate was centrifuged (16,000 × g, 30 min, 4 °C), and the supernatant was quantified for protein.

[0127] Protein was determined using the Thermo Scientific Pierce 660nm Protein Assay, a ready-to-use, detergent- and reducing-agent-compatible assay for rapid measurement of total protein concentration relative to a bovine serum albumin protein standard. For this assay, 10 ml of each human brain homogenate sample was added to a microtiter 96-well plate, followed by 150 ml of Pierce assay. After a 5-minute incubation, absorbance was measured at 660 nm in a Vmax plate reader (Molecular Devices, Wokingham, UK), and optical density results were extrapolated to a BSA standard curve to obtain mg per ml.

[0128] One hundred micrograms of protein was mixed with 4x Laemmli sample buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 1% LDS, 0.005% bromophenol blue, 50 mM dithiothreitol [DTT]), heated to 50°C (10 min), and loaded onto 4%-20% Mini-PROTEANTGX Precast Protein Gels, 10 wells, 50 μL. Proteins were separated by electrophoresis, transferred to PVDF (0.45 μm) membranes, and blocked with 5% Blotting Grade Blocker Non-Fat Milk in Tris-buffered saline + 0.05% Tween 20 (TBS-T0.05%) (1 h; RT). The membranes were incubated overnight with T14 antibody (stock 1 mg / mL, diluted 1:1000) as previously described in WO2016 / 156803. The membrane was washed and incubated with secondary antibody (1:10,000). After washing, immunoreactive proteins were visualized using an enhanced chemiluminescence-based detection kit (Thermo Scientific Pierce ECL Plus Western Blotting Substrate) according to the manufacturer's protocol, using a CCD camera (G-Box, Syngene, Cambridge, UK) gel system. Scanned blots were analyzed using GensSnap software (Syngene, Cambridge, UK), and dot densities were expressed as a percentage of those obtained from the control. Bands were quantified using ImageJ, and unpaired analyses were performed using GraphPad Prism 9.0.

[0129] Detect T14 in CSF: CSF samples were used directly for electrophoresis. Protein was determined using the Thermo Scientific Pierce 660nm Protein Assay as described above. For each sample, 20 μg of CSF sample was mixed with sample buffer (0.5 M Tris HCl, pH 6.8, 10% glycerol, 2% (w / v) sodium dodecyl sulfate, 5% (v / v) 2-β-mercaptoethanol, 0.05% bromophenol blue, final concentrations), boiled for 10 minutes, and loaded onto a 10% acrylamide gel. Proteins were separated by electrophoresis until elution of the migration front, allowing for proper separation of high molecular weight fragments. Proteins were then transferred from the gel to polyvinylidene fluoride sheets (ThermoFisher). These sheets were blocked with 5% nonfat milk in Tris-buffered saline buffer + 0.05% Tween 20 (TBS-T buffer) at room temperature for 1 hour. They were then incubated overnight with T14 antibody (1:1000, Genosphere) diluted in TBS-T buffer + 5% nonfat milk. The membranes were then washed with TBS-T buffer and incubated for 45 minutes with anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody (ab6721, Abcam, Cambridge, UK, 1:5000 dilution). After washing, immunoreactive proteins were visualized using an enhanced chemiluminescence-based detection kit (Thermo Scientific Pierce ECL Plus Western Blotting Substrate) according to the manufacturer's protocol, using a CCD camera (G-Box, Syngene, Cambridge, UK) gel system. Scanned blots were analyzed using GensSnap software (Syngene, Cambridge, UK), and dot density was expressed as a percentage of that obtained from the control. Bands were quantified using ImageJ, and unpaired analyses were performed using GraphPad Prism 9.0.

[0130] Detect T14 in saliva: Saliva samples were obtained from six donors using oral swabs or by spitting directly into tubes. For Western blot analysis, 10 μl of total protein (unknown concentration) was loaded with 5 μl of sample buffer (0.5 M Tris HCl, pH 6.8, 10% glycerol, 2% (w / v) sodium dodecyl sulfate, 5% (v / v) 2-b-mercaptoethanol, 0.05% bromophenol blue, final concentrations). Samples were then boiled for 10 minutes and loaded onto 4%-20% Mini-PROTEANTGX Precast Protein Gels, 10 wells, 50 μL each. Proteins were separated by electrophoresis until elution of the migration front allowed for proper separation of high molecular weight fragments. Proteins were then transferred from the gel to polyvinylidene fluoride sheets (ThermoFisher). These sheets were blocked with 5% nonfat milk in Tris-buffered saline buffer + 0.05% Tween 20 (TBS-T buffer) for 1 hour at room temperature. They were then incubated overnight with T14 antibody (1:1000, Genosphere) diluted in TBS-T buffer + 5% nonfat milk. The membranes were then washed with TBS-T buffer and incubated for 45 minutes with anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody (ab6721, Abcam, Cambridge, UK, 1:10,000 dilution). After washing, immunoreactive proteins were visualized using an enhanced chemiluminescence-based detection kit (Thermo Scientific Pierce ECL Plus Western Blotting Substrate) according to the manufacturer's protocol, using a CCD camera (G-Box, Syngene, Cambridge, UK) gel system. Scanned blots were analyzed using GensSnap software (Syngene, Cambridge, UK), and dot densities were expressed as a percentage of those obtained from the control. Bands were quantified using ImageJ, and unpaired analyses were performed using GraphPad Prism 9.0.For immunoneutralization experiments, T14 primary antibody (1:1000) was incubated with 1 mg / ml T14 peptide for 3 h at room temperature on a rocker and then added to the membrane.

[0131] Detect T14 in nasal secretions: We believe that the concentration of T14 in nasal secretions (or saliva) is approximately 20-40 ng of T14 / mg of protein. For LFT applications, several device embodiments exist that use antibody detection systems. This technology has been developed using polyclonal and monoclonal IgG antibodies, and three different types of immunoassays exist: indirect, competitive, and sandwich. These antibodies have been shown to work well in Western blots, ELISAs, AlphaLISAs, and immunohistochemical staining to detect both synthetic and endogenous T14, and their binding specificity and epitopes have been determined to be good candidates for indirect and competitive immunoassays.

[0132] Lateral Flow Test (LFT) Reagents - (Ab16) affinity-purified polyclonal anti-peptide T14 antibody; - Monoclonal liquid THK-1-102 purified rabbit IgG (approximately 15 μl); - Monoclonal liquid THK-1-104 purified rabbit IgG (approximately 600 μl); - Monoclonal liquid THK-1-117 purified rabbit IgG (1 ml); - T14 peptide (powder form / lyophilized trifluoroacetate salt (1 mg); and - T30 peptide (powder form / lyophilized trifluoroacetate salt (1 mg).

[0133] Antibody conjugation for LFT The detection reagent was 40 nm gold particles passively conjugated to antibodies. Conjugates were prepared for the following: AB16 (rabbit polyclonal), AB117 (rabbit monoclonal), and AB104 (rabbit monoclonal).

[0134] pH titration and antibody loading onto 40 nm gold colloids was completed.

[0135] The preferred conditions for conjugation were:

[0136] [Table 1]

[0137] Each of the antibodies was conjugated to 40 nm colloidal gold.

[0138] LFT method A wet reagent or "semi-dipstick" test method was used, as shown in Figure 9. In this format, the gold conjugate is in liquid form (in the well) rather than dry, allowing for high-throughput test conditions. Test lines are "dotted" onto the membrane by pipette to facilitate evaluation of a range of conditions.

[0139] The nitrocellulose membrane was CN180.

[0140] Testing Line: A) Inhibition assay: T14 peptide T30 peptide B) Sandwich assay: rabbit polyclonal, AB16 Rabbit monoclonal, AB117 Rabbit monoclonal, AB104 Rabbit monoclonal, AB102 Control line: goat anti-rabbit antibody Inhibitory LFT method Membrane preparation: Peptides (T14 or T30) were diluted to 1, 0.5, and 0.25 mg / ml in deionized water. Peptides were "dotted" (1 μl of each concentration) onto a CN180 nitrocellulose membrane, and the membrane was allowed to dry at 37°C for 15 minutes. Goat anti-rabbit antibody was applied to the membrane as a control line.

[0141] Test Method: (i) 20 μl of gold conjugate (neat) and 20 μl of TBST (Tris-buffered saline 1% Tween 20) were added to well 1; (ii) dipstick was added to well 1; (iii) the dipstick was run to completion (no residual liquid); and (iv) The dipstick was added to well 2, which contained 20 μl of TBST only, to wash the test strip.

[0142] Sandwich LFT method Membrane preparation: Each antibody was "dotted" (1 μl neat, i.e., at 1 mg / ml concentration) onto a CN180 membrane, and the membrane was allowed to dry at 37° C. for 15 minutes. A goat anti-rabbit antibody was applied to the membrane as a control line. The conjugate and test line antibody combinations were then evaluated in a matrix study in a lateral flow format.

[0143] Test method (sequential sample addition): (i) 10 μl of peptide (either T14 or T30 at 0.1 mg / ml) and 10 μl of TBST (Tris-buffered saline 1% Tween 20) were added to well 1; (ii) dipstick was added to well 1; (iii) the dipstick was run to completion (no residual liquid); (iv) The dipstick was added to well 2 containing 20 μl of conjugate; (v) the dipstick was run to completion (no residual liquid); and (vi) The dipstick was added to well 3 containing 20 μl of TBST.

[0144] AlphaLISA detection of T14-alpha-7 complex Samples were extracted from homogenized human brain tissue using PerkinElmer lysis buffer (AL003C), and protein concentrations were determined using the BCA method. 1 mL of lysis buffer was used for 100 mg of tissue homogenization. Five cycles of 40-second pulses and 10-second pauses were used for each sample on a shielded homogenizer. The samples were centrifuged at 40°C and 15,000 rpm (15 min) for the supernatants; these were diluted in PerkinElmer assay buffer (AL000F) and used to measure T14-alpha-7 nicotinic receptor complexes in the presence of NBP14 (concentrations 0.065 μM to 900 μM) (Genosphere) using AlphaLISA according to the manufacturer's protocol. The antibodies were biotinylated BTX (B1196; Invitrogen Life Technologies, Waltham, MA, USA) on SA-donor beads and anti-rabbit T14 (Genosphere) on acceptor beads; results were read in an AlphaLISA reader (model number EnSpire 2300 Multilabel Reader; PerkinElmer).

[0145] Example 1 Implementing a Lateral Flow Test (LFT) to detect T14 peptide Referring to Figure 1, three different peripheral tissue sample types that can be readily used for subsequent lateral flow testing are shown, including a nasal swab (2) to obtain nasal secretions or mucus from the nostrils, an oral swab (4) to obtain oral fluids such as saliva from the buccal cavity or mouth, or a finger prick (6) to create a small prick in the skin to obtain blood (capillary or whole blood), usually from the finger (but can also be elsewhere on the body).

[0146] As an example, oral fluid was obtained from six test subjects using direct spit or mouth swabs (2), which were then analyzed. As shown in Figure 2, we surprisingly demonstrated that the T14 biomarker peptide (SEQ ID NO: 3) is present in saliva at very small but detectable concentrations. Western blot results show the detection of a T14 band in all samples. To verify that the band was T14 and not due to cross-contamination of the antibody with another molecule, the samples were immunoneutralized (as described in the methods above) to demonstrate that the antibody specifically detected T14. The concentration of T14 peptide in saliva was approximately 20-40 ng of T14 peptide per mg of protein in the sample. Therefore, we attempted to analyze T14 in samples using a lateral flow test (LFT), and we believe that pinprick blood and nasal secretions could also be used as samples instead of saliva.

[0147] Thus, once a sample (nasal secretion, blood, or saliva) is obtained from a subject, it is then inserted into a sample tube 8, to which a buffer solution (sample buffer: 0.5 M Tris HCl, pH 6.8, 10% glycerol, 2% (w / v) sodium dodecyl sulfate, 5% (v / v) 2-b-mercaptoethanol, 0.05% bromophenol blue, final concentrations) is then added and mixed for a sufficient time to ensure that the sample, and all of the T14 peptide therein, is properly suspended and dispersed. Referring to Figure 3, an LFT cartridge (10) is shown with a sample opening (12, S) toward one end, into which a few drops of buffered sample solution are dispensed from a tube (8). The cartridge (10) has a window (14) through which the test line (16, T) and control line (18, C) can be viewed. The mechanism of lateral flow detection of the T14 peptide is now described below.

[0148] Figures 4 and 5 show two different embodiments of lateral flow devices: an inhibition lateral flow test (20) shown in Figure 4 and a sandwich lateral flow test (22) shown in Figure 5. The LFT techniques described below, using either polyclonal or monoclonal antibodies, can be qualitative, semi-quantitative, or quantitative.

[0149] Inhibitory Lateral Flow Test (LFT) Referring to Figure 4, in inhibitory LFT (20), the LFT cartridge (10) has a sample nitrocellulose membrane CN180 pad (24) onto which a sample (26) is placed through the opening (12). The sample (26) may or may not contain a sample T14 peptide (15), thus producing different results, as discussed below. A conjugate pad (28) is provided adjacent to the sample pad (24), onto which an anti-T14 antibody (30) (labeled with 40 nm gold nanoparticle colloids passively conjugated to a T14 immunospecific antibody) is placed. For example, the anti-T14 antibody (30) may be as described in WO2016 / 156803.

[0150] The cartridge (10) has an LFT immobilized T14 peptide test line (16) consisting of a pre-immobilized T14 peptide (17) bound thereto. Spaced laterally from the immobilized T14 peptide test line (16) is provided a control line (18) consisting of a secondary anti-species labeled antibody (31) (i.e., a goat anti-chicken IgY polyclonal antibody) that is not specific for T14. An upper wick (32) is provided toward the opposite end of the cartridge (10) and functions to draw the sample (26) solution laterally across the cartridge (10).

[0151] As shown in the top portion of Figure 4 (showing a positive result for T14 detection), if the sample (26) contains the sample T14 peptide (15), it flows laterally over the conjugate pad bearing the anti-T14 antibody (30) and is captured by the anti-T14 antibody (30), producing a T14-antibody conjugate (34). This T14-antibody conjugate (34) cannot bind to the immobilized T14 peptide (17) attached to the test line (16), but the anti-T14 antibody (30) in the sample (26) that is not bound to the sample T14 peptide (15) can flow through and bind to the anti-species control (31) attached to the control line (18). Thus, the sample T14 (15) binds the antibody (30) that cannot bind to the immobilized T14 (17) attached to the cartridge (10). Thus, a single line appears on the cartridge (10) as a positive result, as shown in the top right of Figure 4.

[0152] As shown in the bottom of Figure 4 (showing a negative result for T14 detection), if the sample (26) does not contain the sample T14 peptide (15), the sample (26) flows laterally over the conjugate pad bearing the anti-T14 antibody (30). Because no sample T14 (15) is captured by the anti-T14 antibody (30), no T14-antibody conjugate (34) is produced, similar to the positive result discussed above. Therefore, any unbound anti-T14 antibody (30) can flow through and bind to the LFT-immobilized T14 peptide (17) bound to the test line (16) as well as the anti-species control (31) bound to the control line (18). Thus, two spaced apart lines appear on the cartridge (10) indicating a negative result, as shown in the bottom right of Figure 4.

[0153] Referring now to Figure 10, the results of the "half-dipstick" LFT method shown in Figure 9 are shown. As can be seen from Figure 10, the antigen was able to bind to the membrane in an inhibitory manner. As can be seen from the figure, LFT was able to detect both the T14 and T30 peptides. When the amount of peptide on the membrane was reduced from 1 mg / ml to 0.25 mg / ml, indicating specific binding, a decrease in signal intensity was observed in the test line zone. However, some non-specific binding was occasionally observed.

[0154] Sandwich Lateral Flow Test (LFT) Referring now to FIG. 5, due to the size of the T14 peptide, the inventors have also developed a sandwich (i.e., two-antibody) assay format (22) in which a first antibody (30) binds to a first epitope (36) located in one region of the T14 peptide (e.g., at the N-terminus) and a second antibody (38) binds to a second epitope (40) spaced from the first epitope (e.g., at the C-terminus).

[0155] In sandwich LFT (22), the LFT cartridge (10) has a sample pad (24) onto which a sample (26) is placed through the opening (12). The sample (26) may or may not contain a sample T14 peptide (15). A conjugate pad (28) is provided adjacent to the sample pad (24), onto which a first anti-T14 antibody (30) (labeled with a 40 nm gold nanoparticle colloid passively conjugated to the antibody) is placed. For example, the first T14 antibody (30) can be as described in WO2016 / 156803. These anti-T14 antibodies (30) bind to a first epitope (36) on the T14 peptide, e.g., the C-terminal residue of T14.

[0156] The cartridge (10) has a T14 peptide test line (16) that comprises a pre-immobilized second anti-T14 antibody (38) attached thereto. The second T14 antibody (38) binds to a second epitope (40) on the T14 peptide. For example, the second T14 antibody can bind to the N-terminus of T14.

[0157] Spaced laterally from the test line (16), the cartridge (10) has a control line (18) consisting of an anti-species labeled antibody (31) not specific for T14 (e.g., a goat anti-chicken IgY polyclonal antibody), and an upper wick (32) is provided toward the opposite end of the cartridge (10) and functions to draw the sample (26) solution laterally across the cartridge (10).

[0158] As shown in the top portion of Figure 5 (showing a positive result for T14 detection), if sample 26 contains T14 peptide 15, it flows laterally over conjugate pad 28 bearing first anti-T14 antibody 30 and is captured by anti-T14 antibody 30 to produce T14-antibody conjugate 34. Because the second epitope 40 is still exposed, this T14-antibody conjugate 34 is itself captured by second anti-T14 antibody 38 immobilized on cartridge 10 along test line 16. Thus, a first line appears on cartridge 10, indicating antibody binding at T14 test line 16. Additionally, additional anti-T14 antibody 30 not bound to the T14 peptide in sample 26 flows through and binds to anti-species control 31 bound to control line 18. Unlike the inhibition LFT assay (20) described in Figure 4, in the sandwich assay (22) shown in Figure 5, the cartridge (10) does not contain any LFT-immobilized T14 (17), so the assay using two antibodies (30, 38) is a direct detection of the sample T14 (15) present in the sample. Therefore, a second line appears on the cartridge (10) as a positive result, as shown in the upper right corner of Figure 5.

[0159] As shown in the bottom of Figure 5 (showing a negative result for T14 detection), if the sample (26) does not contain the T14 peptide (15), it will flow laterally over the conjugate pad bearing the anti-T14 antibody (30). There will be no T14 (15) captured by the anti-T14 antibody (30), and thus, similar to the positive result discussed above, no T14-antibody conjugate (34) will be produced. Therefore, any unbound anti-T14 antibody (30) will flow away and will not be able to bind to the second T14 antibody (38) bound to the cartridge (10). The T14 antibody (30) will flow through and bind to the anti-species control (31) bound to the control line (18), producing a single line on the cartridge (10) as a negative result, as shown in the bottom right of Figure 5.

[0160] Referring now to Figure 11, the results of the "half-dipstick" LFT method shown in Figure 9 are shown. As can be seen, LFT was able to detect both the T14 and T30 peptides. Some nonspecific binding was observed in the dipstick, indicating interactions between the test line and the conjugated antibody. However, these interactions may be occurring for several reasons, including suboptimal conjugation conditions.

[0161] We note that there is no significant increase in test line signal when either the T14 or T30 peptide is added at 0.1 mg / ml, with the only exception being the AB16 capture conjugate pair (highlighted in gold box) which showed a slight increase with positive samples.

[0162] Qualitative and quantitative analysis of LFT results It will be appreciated from the foregoing that the lateral flow test provides a fast and convenient means for detecting the presence or absence of T14 peptide 15 in peripheral body samples obtained from a subject. The presence of a control line 18 is required to ensure the test is robust and valid, and the presence or absence of a test line 16 informs the subject about the presence or absence of T14 peptide 15 in their sample. Visual assessment of the presence or absence of a test line 16 provides a useful qualitative diagnostic or prognostic test, and visual determination of the relative intensity (or darkness) of the test line 16 provides a useful semi-quantitative measure. However, achieving an accurate and fully quantitative assessment from simply viewing LFT results can be challenging.

[0163] Referring to FIG. 6, a desktop reader embodiment (42, left) and a smartphone embodiment (44, right) running an app are shown for quantitatively measuring the results of an LFT cartridge (10) following the use of either the inhibition LFT of FIG. 4 or the sandwich LFT of FIG. 5. The cartridge can be connected to the reader (42) running software to detect the test line (16) and the control line (18) and then use these concentrations / values ​​to accurately calculate the concentration of T14 peptide (15) in the sample (26). Similarly, the cartridge (10) can be connected to a computer, tablet, or smartphone (44) running software to detect the test line (16) and the control line (18) and then calculate the concentration of T14 peptide (15) in the sample (26). Alternatively, a user can take a photograph of the LFT cartridge (10), and the software determines the level of T14 peptide (15) in the sample (26) based on the strength or intensity of the signal in the test line (16) and the control line (18).

[0164] Example 2 Braak staging using LFT As shown in Figure 7, the inventors have surprisingly shown that there is a clear and surprising correlation between the concentration of T14 peptide and a subject's Braak stage, which in turn corresponds to the subject's clinical symptoms. For example, the inventors have now detected T14 in samples obtained from living patients who are asymptomatic (clinical) and in Braak stages I and II (brain pathology). Braak stages III and IV are patients who exhibit mild symptoms, while Braak stages V and VI correspond to dementia and cognitive decline.

[0165] As can be seen in Figure 7, Western blots of Alzheimer's disease hippocampus showed a single T14-reactive band that increased approximately two-fold from early (Braak 0-II) to late stages (Braak V-VI). As can be seen, there is a very clear signal in the Western blot of the Braak stage VI sample. Surprisingly, the data also show that changes in T14 levels can be used to determine early Braak stages I and II. Importantly, this therefore demonstrates that T14 concentrations can be used to determine Braak stage in living, asymptomatic patients (i.e., patients in Braak stages I and II).

[0166] Thus, the inventors believe that the lateral flow test described in Example 1 can be conveniently used to detect T14 peptide and, based on the concentration of T14, determine a subject's Braak stage from Braak stage 0 to Braak stage VI. Given that Braak staging is currently performed postmortem, the ability to provide a Braak stage score for living patients from an LFT test would be highly beneficial, as discussed in more detail below.

[0167] Example 3 Diagnostic, Prognostic, and Therapeutic Uses of LFT Tests The inventors were surprised to find that detectable levels of T14 peptide (SEQ ID NO: 3) are present in any peripheral tissue suitable for lateral flow testing, such as saliva, nasal secretions, or pinprick blood. Thus, there are at least four scenarios in which the method and device can be used to test the public: 1) Prognostic test - subjects already diagnosed with Alzheimer's disease For individuals already identified as suffering from a neurodegenerative disorder and / or cognitive decline, the rapid and accurate test of the present invention will be of great value as it can be used to actively monitor (i.e., prognose) the etiology or progression of their condition, hopefully leading to an improvement, slowing, or even reversal of cognitive decline. Such individuals may be at Braak stage III, IV, V, or VI.

[0168] Referring to Figure 8, the correlation between detecting T14 levels using the diagnostic or prognostic methods of the present invention (e.g., LFT tests or saliva / prick blood tests) and therapeutic intervention with drugs administered to treat or prevent the onset of neurodegenerative disorders or cognitive decline is shown. For example, acetylcholinesterase inhibitors or N-methyl-D-aspartate (NMDA) antagonists or cyclic peptides (such as those described in WO 2015 / 004430 and known as NBP-14 cyclic peptides) can be administered to achieve permanent prevention of the onset of symptoms or to delay or even reverse symptoms. Such tests can then be used to influence more precise dosing of therapeutic drugs administered to patients and also to produce better patient outcomes. This is essentially a combined diagnostic method for clinical testing and treatment of patients as a highly sensitive monitoring system for the effectiveness of treatment.

[0169] Because patients can be tested daily, weekly, or monthly, at a frequency consistent with the therapeutic drug regimen, and the effects can be monitored constantly and regularly, the inventors envision that a quantitative readout of T14 levels will inform the amount of treatment that needs to be received. It is expected that subjects will see a reduction in T14 levels as a direct result of the therapeutic drug being administered, in such a way that a successful outcome is achieved by either further delaying the onset of symptoms, preventing the onset of symptoms, or even reversing symptoms such as cognitive decline.

[0170] 2) Diagnostic Testing - Subjects suspected of having Alzheimer's disease This relates to an accurate and fast diagnostic test for use in people suspected (but not yet diagnosed) of having a neurodegenerative disorder, i.e., they may be pre-symptomatic (Braak stage I or II), which compares in cost, frequency, and effectiveness to currently deployed cognitive testing methods, for example, after patient referral to a memory clinic.

[0171] 3) Asymptomatic Study - Subjects not suspected of having Alzheimer's disease It would be useful to have a simple test available for people who are not currently suspected of having a neurodegenerative disorder, i.e., they are young and / or pre-symptomatic (Braak stage I or II). The opportunity to have a pre-symptomatic test to diagnose a neurodegenerative disorder, which can be performed at a GP surgery, or by a nurse, or even at home. For example, LFT tests, like breast cancer or bowel cancer screening, can be routinely performed (or suggested or required by employers, the NHS, etc.) in certain age groups or above a certain age. Depending on the results, the person can then be referred to a doctor or a specialized medical practitioner.

[0172] 4) Drug development and clinical trials Because patients can effectively serve as their own controls and monitor how much they have or have not worsened since the previous test (e.g., one day, one week, or one month ago), pharmaceutical companies can save time and cost in drug development in terms of timescales, smaller sample groups, and other value metrics when accurate measures of disease progression are readily available.

[0173] conclusion The inventors have shown that T14 levels are elevated in the brain in the pre-symptomatic stages of Alzheimer's disease, and have therefore translated this profile for detection in peripheral tissues, such as saliva / nasal discharge, as the basis for a reliable diagnostic method for early stage Alzheimer's disease. Thus, lateral flow can be used as a rapid and convenient means to detect T14 for diagnostic purposes.

[0174] The LFT modality can be either an inhibition LFT or a sandwich LFT, as shown in Figures 4, 5, 9, 10, and 11. Antibodies were successfully conjugated to 40 nm gold colloids. Antigens could be bound to the membrane in both the inhibition and sandwich formats. A decrease in signal intensity in the test line zone was observed when the amount of peptide on the membrane was reduced, indicating specific binding.

[0175] Thus, this first-in-class pre-symptomatic test can function not only as a diagnostic monitor but also as a prognostic test. Furthermore, the inventors believe that the methods and devices described herein can be used to diagnose subjects before the onset of any symptoms of dementia (i.e., in the pre-symptomatic stage) or to determine whether symptoms may be present in the future. Upon detection of T14 at the pre-symptomatic stage, subjects can be immediately administered a therapeutic intervention (e.g., a drug or lifestyle change), and the methods and devices can then be used as a prognostic measure to monitor and evaluate drug efficacy.

Claims

1. A lateral flow method for diagnosing or prognosing a neurodegenerative disorder in a subject, the method comprising using lateral flow to detect a peptide (T14) comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in a sample obtained from a test subject, wherein detection of the presence of said peptide (T14) comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in said sample indicates that the test subject has a neurodegenerative disorder or a predisposition to or poor prognosis thereof.

2. A lateral flow device for diagnosing or prognosing a neurodegenerative disorder in a subject, the device comprising a lateral flow support for detecting a peptide (T14) comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in a sample obtained from a test subject, wherein detection of said peptide (T14) comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof corresponds to said subject having a neurodegenerative disorder or a predisposition to developing such a disorder or a poor prognosis thereof.

3. 3. The method or apparatus of claim 1 or claim 2, comprising prognosing the progression of said neurodegenerative disorder.

4. 4. The method or apparatus of any one of claims 1 to 3, comprising diagnosing a neurodegenerative disorder.

5. 5. The method or apparatus of any one of claims 1 to 4, comprising diagnosing a pre-symptomatic condition in said test subject.

6. 6. The method or apparatus of any one of claims 1 to 5, comprising conducting a clinical trial to monitor the activity or effectiveness of a drug administered to said test subject, preferably further comprising determining how much said subject has or has not worsened since a previous test.

7. The method or device according to any one of claims 1 to 6, wherein the peptide (T14) comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof is not determined using ELISA.

8. 8. The method or apparatus of any one of claims 1 to 7, comprising determining the concentration of a soluble peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof.

9. 9. The method or apparatus of any one of claims 1 to 8, comprising means for determining the concentration of SEQ ID NO:3 or a variant or fragment thereof in the sample obtained from the test subject, wherein the means for determining the concentration of SEQ ID NO:3 or a variant or fragment thereof in the sample obtained from the test subject optionally comprises an anti-T14 antibody or antigen-binding fragment thereof.

10. 10. The method or apparatus of any one of claims 1 to 9, wherein the sample is nasal secretion or mucus, saliva, blood, venous blood, arterial blood, plasma, serum, capillary blood, non-venous blood, non-arterial blood, pinprick blood, cerebrospinal fluid, urine, sweat, tears, breast aspirate, prostatic fluid, semen, vaginal fluid, feces, cervical scraping, cells, amniotic fluid, ocular fluid, mucus, respiratory moisture, animal tissue, cell lysate, tumor tissue, hair, skin, cheek scraping, lymph, interstitial fluid, nail, bone marrow, cartilage, prion, bone powder, earwax, or a combination thereof.

11. The method or apparatus of claim 10 , wherein the sample comprises nasal secretions or mucus.

12. The method or device of claim 10 , wherein the sample comprises saliva.

13. 11. The method or apparatus of claim 10, wherein the sample comprises capillary blood or needle stick blood.

14. The method or device of any one of claims 1 to 13, comprising an inhibition lateral flow test.

15. 15. The method or apparatus of any one of claims 1 to 14, comprising a sandwich lateral flow test.

16. 16. The method or apparatus of any one of claims 1 to 15, wherein the apparatus comprises an LFT cartridge including a sample pad on which the sample is placed, the LFT cartridge containing T14 antibody, optionally labeled with a labeling moiety such as a gold nanoparticle.

17. 17. The method or apparatus of claim 16, wherein the LFT cartridge comprises an immobilized T14 peptide test line having a T14 peptide bound thereto, and / or the LFT cartridge comprises a control line comprising an anti-species labeled antibody that is not specific for T14.

18. 18. The method or apparatus of claim 17, wherein if the sample contains T14 peptide, the peptide is captured by the anti-T14 antibody to produce a T14-antibody conjugate, the T14-antibody conjugate cannot bind to the immobilized T14 peptide bound to the test line, and anti-T14 antibody in the sample that is not bound to the sample T14 peptide flows through and binds to the anti-species labeled antibody bound to the control line.

19. 19. The method or apparatus of any one of claims 14 to 18, wherein if the sample does not contain T14 peptide, no T14-antibody conjugate is produced and unbound anti-T14 antibody flows through and binds to the LFT-immobilized T14 peptide bound to the test line and the anti-species antibody control bound to the control line.

20. 20. The method or apparatus of any one of claims 15 to 19, wherein the LFT cartridge comprises a first anti-T14 antibody that binds to a first epitope on the T14 peptide, and the LFT cartridge comprises a T14 peptide test line that comprises a second anti-T14 antibody that binds to a second epitope on the T14 peptide.

21. 21. The method or apparatus of claim 20, wherein the LFT cartridge comprises a control line containing an anti-species labeled antibody that is not specific for T14.

22. 22. The method or apparatus of claim 20 or 21, wherein if the sample contains a T14 peptide, the T14 peptide is captured by the first anti-T14 antibody to produce a T14-antibody conjugate, the conjugate itself being captured by the second anti-T14 antibody bound to the cartridge along the test line, and any further anti-T14 antibody in the sample that is not bound to the T14 peptide flows through and binds to the anti-species control bound to the control line.

23. 23. The method or apparatus of any one of claims 20 to 22, wherein if the sample does not contain T14 peptide, no T14-antibody conjugate is produced, unbound first anti-T14 antibody cannot bind to the second T14 antibody, and the first anti-T14 antibody flows through and binds to the anti-species control bound to the control line.

24. 24. The method or apparatus of any one of claims 1 to 23, which is qualitative, semi-quantitative or quantitative.

25. 25. The method or apparatus of any one of claims 16 to 24, comprising means for quantitatively measuring the results of the LFT cartridge, wherein the lateral flow cartridge is optionally adapted to be connected to a reader, computer, tablet or smartphone that runs software for detecting the test and control lines and calculates the concentration of the peptide of SEQ ID NO: 3 or a variant or fragment thereof in the sample based on the concentrations / values ​​of the test and control lines.

26. 1. A method for diagnosing or prognosing a neurodegenerative disorder in a subject, the method comprising detecting a peptide (T14) comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in a nasal discharge, mucus or pinprick blood sample obtained from a test subject, wherein detecting the presence of said peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in said sample indicates that the test subject has a neurodegenerative disorder or a predisposition to or poor prognosis for said disorder.

27. A neurodegenerative disorder diagnostic or prognostic device for diagnosing or prognosing a neurodegenerative disorder in a subject, the device comprising means for detecting a peptide (T14) comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof in a nasal discharge, nasal mucus or pinprick blood sample obtained from a test subject, wherein detection of said peptide (T14) comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof corresponds to said subject having a neurodegenerative disorder or a predisposition to developing such a disorder or a poor prognosis thereof.

28. 28. The method or apparatus of claim 26 or claim 27, comprising an assay adapted to detect the presence and / or absence of a peptide of SEQ ID NO: 3 or a variant or fragment thereof in said sample.

29. 29. The method or apparatus of claim 28, wherein the assay comprises lateral flow, an immunoassay, a non-immune-based assay, a Western blot analysis, an enzyme-linked immunosorbent assay (ELISA), a fluorescent assay, a chemiluminescent assay, or a radioimmunoassay analysis.

30. 30. The method or apparatus of claim 29, wherein the assay comprises lateral flow.

31. 31. The method or apparatus of any one of claims 26 to 30, wherein the concentration of (i) a soluble peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof and / or (ii) an aggregated peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof is determined.

32. 32. The method or apparatus of claim 31 , wherein the device comprises means for determining the concentration of SEQ ID NO:3 or a variant or fragment thereof in the sample obtained from the test subject, and wherein the means for determining the concentration of SEQ ID NO:3 or a variant or fragment thereof in the sample obtained from the test subject optionally comprises an anti-T14 antibody or antigen-binding fragment thereof.

33. 33. The method or apparatus of any one of claims 9 to 25 and 32, wherein the antibody or antigen-binding fragment thereof specifically binds to SEQ ID NO:3, which may be one or more amino acids in SEQ ID NO:11, and preferably the antibody or antigen-binding fragment thereof does not bind to SEQ ID NO:2 (i.e., T30), SEQ ID NO:13 (i.e., T15), and / or SEQ ID NO:14 (i.e., Aβ).

34. the concentration of SEQ ID NO: 3 or a variant or fragment thereof in the sample is (i) between 0.1 ng and 1000 ng, or between 0.2 ng and 750 ng, or between 0.5 ng and 500 ng per mg of protein in the sample; (ii) between 1 ng and 400 ng, or between 2 ng and 300 ng, or between 3 ng and 200 ng per mg of protein in the sample; and / or (iii) between 4 ng and 100 ng, or between 5 ng and 75 ng, or between 10 ng and 50 ng, or between 20 ng and 40 ng per mg of protein in the sample; 34. The method or apparatus according to any one of claims 1 to 33, wherein

35. 35. The method or apparatus of any one of claims 1 to 34, wherein the subject has or is suspected of having a neurodegenerative disease selected from the group consisting of Alzheimer's disease; Parkinson's disease; Huntington's disease; motor neuron disease; spinocerebellar dementia types 1, 2 and 3; amyotrophic lateral sclerosis (ALS); schizophrenia; dementia with Lewy bodies; and frontotemporal dementia.

36. 36. The method or apparatus of any one of claims 1 to 35, wherein the subject has or is suspected of having Alzheimer's disease.

37. (i) a subject already diagnosed with a neurodegenerative disorder; (ii) a subject who is symptomatic and exhibits signs of cognitive decline or dementia; and / or (iii) a pre-symptomatic subject not suspected of having a neurodegenerative disorder; The method or device according to any one of claims 1 to 36, for use in

38. 38. A method or apparatus according to any one of claims 1 to 37, comprising the detection of soluble and / or aggregated peptides comprising or consisting of any one or more of T7 to T13 (i.e. SEQ ID NOs: 4 to 10).

39. 39. The method or apparatus of any preceding claim, comprising determining the subject's Braak stage.

40. 40. The method or apparatus of claim 39, wherein the subject is in Braak stage I, II or III, preferably either Braak stage I or II.

41. (a) analyzing the concentration of the peptide (T14) comprising or consisting of SEQ ID NO: 3, or a variant or fragment thereof, in the sample obtained from the test subject; and (b) comparing this concentration with a reference value from a control population of deceased subjects with known Braak stage for the concentration of a soluble peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof; 41. The method or apparatus of claim 39 or 40, comprising: wherein the Braak stage of a living test subject is determined by comparing the concentration of said peptide comprising or consisting of SEQ ID NO: 3 or a variant or fragment thereof with a respective reference value associated with the Braak stage.

42. 42. The method or apparatus of claim 41, wherein soluble SEQ ID NO: 3 (T14) or a variant or fragment thereof is analyzed.

43. (i) a lower concentration of a soluble peptide comprising or consisting of SEQ ID NO: 3, or a variant or fragment thereof, compared to the reference value indicates Braak stage I; (ii) a lower concentration of a soluble peptide comprising or consisting of SEQ ID NO: 3, or a variant or fragment thereof, compared to the reference value indicates Braak stage II; and / or (iii) a lower concentration of a soluble peptide comprising or consisting of SEQ ID NO: 3, or a variant or fragment thereof, compared to the reference value indicates Braak stage III; 43. The method or apparatus of claim 42.

44. 44. The method or apparatus of any one of claims 1 to 43, comprising measuring the rate of cognitive decline by Mini-Mental State Examination (MMSE) score and / or Preclinical Alzheimer's Cognitive Composite (PACC) score.

45. 45. The method or apparatus of any one of claims 1 to 44, comprising the use of lateral flow to detect SEQ ID NO:3 or a variant or fragment thereof in nasal secretions, mucus or saliva to diagnose or prognose pre-symptomatic Alzheimer's disease.

Images