Lateral flow device for diagnosis of alzheimer's disease using T14 peptide

Abstract

The present invention relates to neurodegenerative diseases, and to the diagnosis and / or prognosis of neurodegenerative diseases in test subjects using lateral flow testing or the like. The invention also relates to the detection of diagnostic and prognostic biomarkers in a variety of patient sample types for the diagnosis and / or prognosis of neurodegenerative diseases, such as Alzheimer's disease. The invention also provides biomarker detection methods and devices for diagnosis and prognosis of neurodegenerative diseases and methods of treating patients diagnosed or prognosed with neurodegenerative diseases. The invention also extends to detection of biomarkers and / or screening in subjects before symptoms for early diagnosis, so that diseases can be prevented or intervened.

Description

[0001] The present invention relates to neurodegenerative diseases and, in particular but not exclusively, to the diagnosis and / or prognosis of neurodegenerative diseases in a test subject using, for example, a lateral flow test. The present invention also relates to the detection of diagnostic and prognostic biomarkers in different patient sample types for the diagnosis and / or prognosis of neurodegenerative diseases such as Alzheimer's disease. The present invention also provides methods and devices for detecting biomarkers for the diagnosis and prognosis of neurodegenerative diseases, as well as methods for treating patients diagnosed or prognosed with a neurodegenerative disease. The present invention also extends to the detection and / or screening of biomarkers in pre-symptomatic subjects for early diagnosis, enabling the prevention or intervention of the disease.

[0002] Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease, Huntington's disease or motor neuron disease are one of the largest socio-economic burdens in the world, with the global incidence of dementia rising and nearly 10 million new dementia cases each year. As both the incidence and prevalence of AD increase with age, the number of patients is also increasing significantly with the aging of the population. In 2015, there were over 46 million dementia patients, with an estimated annual socio-economic cost of $800 billion, and it is predicted that the number of patients will increase to over 130 million by 2050, with a social cost of over $2 trillion per year. AD has recently been declared the leading cause of death in people over 65 in the UK, and in the US, AD has now become the sixth leading cause of death for all ages.

[0003] Currently, there is no single test available for the diagnosis of Alzheimer's disease or the prognosis of cognitive decline (e.g., Braak stage IV, V or VI). It is currently impossible to diagnose pre-symptomatic AD (e.g., Braak stage I, II or III). The clinical classification of AD relies on a combination of subjective reports, medical history assessment, cognitive function tests and expensive brain imaging scans, and true classification is not possible until a post-mortem examination of the brain is carried out. All of these tests require the involvement of a professional doctor, but the accuracy remains variable. They also take time and are therefore slow and expensive.

[0004] Accordingly, there is a need to provide improved methods, devices and / or kits for diagnosing the disease in a subject suspected of having a neurodegenerative disease (e.g., Braak stage I, II or III), in particular Alzheimer's disease, and for prognosticating the cause or progression of the disease in a subject diagnosed with the disease (e.g., Braak stage IV, V or VI). There is also a need to provide methods and devices for screening and detecting the risk of developing a neurodegenerative disorder in a subject before the onset of symptoms of a neurodegenerative disease or suspected of having such a condition (e.g., Braak stage I or II). Ideally, such improved methods and devices are rapid, quantitative (or at least semi-quantitative or qualitative), accurate, painless and inexpensive.

[0005] The inventors continued their previous research in the field, focusing on the toxic peptide "T14", which is derived from the C-terminus of acetylcholinesterase (AChE) and exists as a bioactive molecule naturally present in the brain tissue. WO

[0006] 2016 / 156803 describes antibodies generated against AChE-derived peptide (T14) peptides, and it has previously been shown that T14 found in cerebrospinal fluid (CSF) or venous blood samples can serve as a robust biomarker for the diagnosis of neurodegenerative diseases such as Alzheimer's disease.

[0007] However, as discussed in the examples and Figure 7 shown, the inventors have now surprisingly found that T14 peptide levels are elevated not only in patients with late-stage Alzheimer's disease (e.g., Braak stage V or VI), but also in the brains of those in the early stages of neurodegeneration, i.e., the pre-symptomatic stage of Alzheimer's disease (e.g., Braak stage I, II, or III), which was completely unexpected. Furthermore, as Figure 2 shown, the inventors have also unexpectedly found that the T14 biomarker is detectable not only in cerebrospinal fluid and venous blood draws, but also in various peripheral tissues at very low but still detectable concentrations, such as saliva, nasal secretions, and finger prick blood. This type of peripheral tissue can be easily analyzed using a lateral flow test (LFT), which, unlike cerebrospinal fluid and venous / arterial blood (as shown in the inventors' previous studies), is painless, socially acceptable, low-cost, can be used frequently (even daily or weekly), and provides very rapid and accurate results.

[0008] Accordingly, the inventors believe that, taken together, these data form the basis for a first-class reliable diagnosis or prognostic screening of neurodegenerative diseases, as well as for the diagnosis of pre-symptomatic neurodegenerative diseases (such as early-onset Alzheimer's disease).

[0009] Accordingly, in a first aspect of the present invention, there is provided a lateral flow method for diagnosing or prognosticating a neurodegenerative disorder in a subject, the method comprising using a lateral flow assay to detect a peptide comprising or consisting of SEQ ID No: 3 (T14) or a variant or fragment thereof in a sample obtained from the test subject, wherein detection of the presence of a peptide comprising or consisting of SEQ ID No: 3 (T14) or a variant or fragment thereof in the sample indicates that the test subject has a neurodegenerative disease, or is predisposed to having a neurodegenerative disease, or has a negative prognosis.

[0010] In a second aspect of the present invention, there is provided a lateral flow device for diagnosing or prognosing a neurodegenerative disease in a subject, the device comprising a lateral flow support for detecting a peptide in a sample obtained from a test subject, the peptide comprising or consisting of: SEQ ID NO:3 (T14) or a variant or fragment thereof, wherein the detection of a peptide comprising SEQ ID NO:3 (T14) or a variant or fragment thereof or consisting of SEQ ID NO:3 (T14) or a variant or fragment thereof corresponds to a subject having a neurodegenerative disease, or having a predisposition to a neurodegenerative disease, or having a negative prognosis for a neurodegenerative disease.

[0011] In a third aspect, there is provided a method of treating a subject having or suspected of having a neurodegenerative disease, pre-symptomatic neurodegenerative disease and / or having cognitive decline, the method comprising:

[0012] (a) using a lateral flow to detect a peptide in a sample obtained from a test subject that comprises SEQ ID No:3 (T14) or a variant or fragment thereof or consists of SEQ ID No:3 (T14) or a variant or fragment thereof, wherein the detection of the presence of a peptide in the sample that comprises SEQ IDNo:3 or a variant or consists of SEQ ID No:3 (T14) or a variant or fragment thereof indicates that the test subject has a neurodegenerative disease, or has a predisposition to a neurodegenerative disease or its negative prognosis; and

[0013] (b) administering or having administered to the subject a therapeutic agent that prevents, reduces or delays neurodegeneration and / or cognitive decline.

[0014] The inventors believe that they are the first to develop a method of using a lateral flow test to detect the T14 peptide in a patient sample.

[0015] Accordingly, in a fourth aspect, there is provided a method of detecting a peptide in a sample obtained from a test subject that comprises SEQ IDNo:3 (T14) or a variant or fragment thereof or consists of SEQ ID No:3 (T14) or a variant or fragment thereof, the method comprising using a lateral flow to detect a peptide in a sample obtained from a test subject that comprises SEQ ID No:3 (T14) or a variant or fragment thereof or consists of SEQID No:3 (T14) or a variant or fragment thereof.

[0016] Due to their much smaller volume, the concentration is significantly reduced, and quite unexpectedly, in any peripheral body sample (such as Figure 2Detectable levels of the T14 peptide (SEQ ID NO: 3) have been found in saliva, nasal secretions, or finger prick blood as shown, suitable for lateral flow detection. Advantageously, there are various situations in which the methods and devices of the present invention are of great value, as they enable the accurate and rapid screening, diagnosis, and prognosis of neurodegenerative diseases (preferably Alzheimer's disease) using lateral flow tests (LFTs).

[0017] LFTs are also known in the art as "rapid tests", "quick tests", or "lateral flow immunoassays (LFIA)", and are thus also covered by the present invention.

[0018] First, for people who have already been diagnosed with Alzheimer's disease, rapid and accurate detection is of great value and can be used to actively monitor the cause or progression of their condition. In addition, performing such a test can be used to influence the more accurate dosing of therapeutic drugs given to the patient and can also lead to better patient outcomes. Thus, preferably, the method and device serve as a prognostic method or prognostic device for prognosticating the progression of neurodegenerative diseases.

[0019] Second, accurate and rapid diagnostic tests are of considerable value for people who are suspected of having Alzheimer's disease but have not yet been diagnosed. Such tests would have significant advantages compared to the currently employed methods of cognitive testing in terms of cost, frequency, and efficacy, such as after a patient is referred to a memory clinic. Thus, preferably, the methods and devices of the present invention are a diagnostic method or diagnostic device for diagnosing neurodegenerative diseases.

[0020] Third, it is clearly very useful to provide a convenient test for people who are not currently suspected of having Alzheimer's disease (i.e., young and / or asymptomatic people). Thus, regardless of any predisposing factors or symptoms, the general population has a great opportunity to undergo pre-symptomatic testing, which can be performed in general practitioner clinics or even at home, i.e., as part of a general medical examination. For example, LFT tests can be routinely performed in a specific age group or in people above a certain age, similar to breast cancer or bowel cancer screening.

[0021] The prognostic pre-screening may be qualitative in nature. For example, for a predefined range (or window) of T14 values regarding the risk of having or being predisposed to a neurodegenerative disease, a binary result of "positive" or "negative" is given. Then, based on the results of the prognostic pre-screening, the subject may be referred to a doctor or other specialist who can then perform more accurate quantitative tests and provide expert medical intervention as needed. The prognostic pre-screening can be self-administered. Thus, it can filter out those subjects who are clearly outside the range of T14 values (i.e., have a "negative" result) and who are considered at risk of or predisposed to a neurodegenerative disease. So only those subjects with a "positive" result in the pre-screening for a neurodegenerative disease will undergo a qualitative test under supervision to then determine if they are indeed in the early stages of a neurodegenerative disease. Thus, preferably, the method and apparatus are used to diagnose the pre-symptomatic condition of patients who will or may develop into a neurodegenerative disease in the future. Preferably, the method is a diagnostic method, including diagnosing a neurodegenerative disease of a test subject, preferably a pre-symptomatic condition. The ability for early detection will enable early therapeutic intervention, thereby delaying the onset of the neurodegenerative disease and even preventing the disease.

[0022] Fourth, if the progression of the disease can be easily and accurately measured in terms of time scale, smaller sample groups, and other value metrics, pharmaceutical companies will be able to save a significant amount of time and cost in drug development because patients will effectively act as their own controls as it is possible to monitor how much they have deteriorated or not since an earlier test (e.g., a day ago, a week ago, or a month ago). Thus, preferably, the method and apparatus are used in clinical trials to monitor the activity or efficacy of a drug administered to a test subject and preferably to determine how much the subject has or has not deteriorated since an earlier test.

[0023] Preferably, the lateral flow device or method of the first to fourth aspects is performed in vitro. It should be understood that the lateral flow test is an immunoassay but not an ELISA. Thus, preferably, the peptide SEQ ID No:3 (T14) or its variants or fragments are not assayed using ELISA (i.e., non-ELISA methods and kits / devices).

[0024] Preferably, the lateral flow device or method of the first to fourth aspects is used to identify the presence of the peptide of SEQ ID No: 3 (T14) or its variants or fragments in a sample, and / or to determine its concentration in the sample, preferably the concentration of soluble T14. The T14 peptide (SEQ ID No: 3) can be assayed by a variety of lateral flow systems known to those skilled in the art. Lateral flow is a form of immunoassay used to detect the T14 peptide or determine T14 levels. Preferably, the method (or assay) or device is suitable for detecting 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, which can be compared with the assay.

[0025] Particularly preferably, the method or device of the present invention includes the detection of soluble T14 (SEQ ID No: 3, or its variants or fragments). In one embodiment, the concentration of a soluble peptide comprising SEQ ID NO: 3 or its variants or fragments or consisting of SEQ ID NO: 3 or its variants or fragments is determined. Preferably, the method or device includes means for determining the concentration of SEQ ID No: 3 or its variants or fragments in a sample obtained from a test subject. The means for determining the concentration of SEQ ID NO: 3 or its variants or fragments in a sample obtained from a test subject may include an anti-T14 antibody or an antigen-binding fragment thereof, i.e., a T14 neutralizing antibody. The antibody or its antigen-binding fragment may be polyclonal or monoclonal. The antibody or its antigen-binding fragment may be produced in rabbits, mice or rats.

[0026] Embodiments of the lateral flow methods and devices of the first to fourth aspects are shown in Figure 1 and 3 -6. Preferably, the device method includes the use of a sample extraction device to obtain a sample from a test subject. For example, the sample extraction device may include a nasal swab, an oral swab or a needle prick blood extraction device. Figure 1 Each tool for lateral flow sampling is shown.

[0027] Once a sample is obtained from a subject, it can be inserted into a suitable sample collection container. The container preferably contains a buffer solution. In one embodiment, the buffer solution may contain 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. Preferably, the sample is mixed with the buffer solution until any T14 peptide therein is suspended.

[0028] Preferably, the device includes an LFT cassette having a sample well into which the buffered sample solution is delivered. Preferably, the cassette includes a well or window through which the test line and the control line can be observed.

[0029] Figure 4 and Figure 5 illustrate 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 includes an inhibition lateral flow test, as Figure 4 shown. In another embodiment, the device or method preferably includes a sandwich lateral flow test, as Figure 5 shown.

[0030] Preferably, the LFT cassette includes a sample pad on which a sample is placed. Preferably, the LFT cassette contains an anti - T14 antibody, which is preferably disposed on a conjugate pad. For example, the anti - T14 antibody can be as described in WO 2016 / 156803. Preferably, the conjugate pad is disposed substantially adjacent to the sample pad. Preferably, the anti - T14 antibody is labeled with a labeling moiety. For example, the labeling moiety can comprise nanoparticles, preferably gold nanoparticles. The average diameter of the nanoparticles can be between 10 nm and 65 nm, or between 20 nm and 60 nm, or between 30 nm and 5 nm.

[0031] Preferably, the LFT cassette includes a fixed T14 peptide test line that contains a T14 peptide linked thereto. Preferably, the LFT cassette contains a control line that contains an anti - species labeled antibody that is 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 IgY polyclonal antibody or a goat anti - rabbit IgY polyclonal antibody. Preferably, the control line is laterally spaced from the fixed T14 peptide test line and is preferably remote from the sample pad and the conjugate pad. Preferably, the LFT cassette includes a wick disposed at or towards the end of the cassette opposite the sample pad and configured to laterally draw a sample solution across the cassette.

[0032] As Figure 4 shown in the upper half of (showing a positive result for T14 detection using an inhibition lateral flow test), when the sample contains a sample T14 peptide and it flows laterally across a conjugate pad containing an anti - T14 antibody, it is preferably captured by the anti - T14 antibody, producing a T14 - antibody conjugate. This T14 - antibody conjugate preferably cannot bind to the fixed T14 peptide linked to the test line. However, preferably, the anti - T14 antibody that has not bound to the sample T14 peptide in the sample can flow and bind to the anti - species labeled antibody linked to the control line. It should be understood that the sample T14 blocks the anti - T14 antibody, which cannot bind to the fixed T14 peptide linked to the cassette. Thus, preferably, a single line is formed on the cassette as a positive result, as Figure 4 shown in the upper right of.

[0033] As Figure 4as shown in the lower half of Figure 4 as shown in the lower right corner of

[0034] Now referring to Figure 5 , due to the size of the T14 peptide, the inventors have also developed a sandwich (i.e., two antibody) assay format. Preferably, the method or device comprises a first antibody that binds to a first epitope located in one region of the T14 peptide (e.g., at the N-terminus or towards the N-terminus), and a second antibody that binds to a second epitope on the T14 peptide, which is spaced apart from the first antibody that binds to the first epitope (e.g., at the C-terminus or towards the C-terminus). Preferably, the first and second epitopes are different amino acids on the T14 peptide.

[0035] Preferably, the LFT cassette includes a sample pad on which the sample is placed. Preferably, the LFT cassette contains a first anti-T14 antibody, which is preferably disposed on the conjugate pad. For example, the anti-T14 antibody can be as described in WO 2016 / 156803. The first anti-T4 antibody preferably binds to the first epitope on the T14 peptide, such as the C-terminal residue of T14. Preferably, the conjugate pad is disposed substantially adjacent to the sample pad. Preferably, the anti-T14 antibody is labeled with a label moiety. For example, the label moiety can include nanoparticles, preferably gold nanoparticles.

[0036] Preferably, the LFT cassette includes a T14 peptide test line carrying a second anti-14 antibody. Preferably, the second anti-T14 antibody binds to the second epitope on the T14 peptide. For example, the second anti-T14 antibody can bind to the N-terminus of T14.

[0037] Preferably, the LFT cassette contains a control line that includes an anti-species labeled antibody that is 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 contain a goat anti-chicken or anti-rabbit IgY polyclonal antibody. Preferably, the control line is laterally spaced apart from the immobilized T14 peptide test line, and is preferably remote from the sample pad and the conjugate pad. Preferably, the LFT cassette includes a wicking core disposed at or towards the end of the cassette opposite the sample pad, and configured to laterally draw the sample solution across the cassette.

[0038] As Figure 5as shown in the upper half (showing a positive result for T14 detection using a sandwich lateral flow test), when the sample contains the T14 peptide and it flows laterally over a conjugate pad carrying the first anti-T14 antibody, it is preferably captured by the anti-T14 antibody, thereby generating a conjugate of the anti-T14 antibody. Preferably, the T14 antibody conjugate itself is captured by a second anti-T14 antibody bound to the cassette along the test line (16). This is possible because a second epitope on the T14 peptide remains exposed in the conjugate. Thus, the first line is preferably formed on the cassette, indicating binding of the antibody at the T14 test line. Preferably, any additional anti-T14 antibody not bound to the T14 peptide in the sample flows and preferably binds to an anti-species control immobilized to the control line. Advantageously, unlike the inhibition LFT assay described above, the sandwich assay, since the cassette does not include any LFT-immobilized T14, the sandwich assay using the first and second anti-T14 antibodies is a direct detection of the sample T14 present in the sample. Thus, the appearance of a second line on the test cassette is a positive result, as Figure 5 shown in the upper right corner.

[0039] as Figure 5 shown in the lower half (showing a negative result for T14 detection using a sandwich lateral flow test), when the sample does not contain the T14 peptide and it preferably flows laterally over a conjugate pad carrying the first anti-T14 antibody, there is no T14 captured by the anti-T14 antibody, and thus preferably no conjugate of T14-antibody is generated. Preferably, all unbound first anti-T14 antibodies flow through and are unable to bind to the second T14 antibody attached to the cassette. The T14 antibody preferably flows to and binds to the anti-species control immobilized on the control line, and preferably produces a single line on the cassette (10) as a negative result, as Figure 5 shown in the lower right corner of.

[0040] It should be understood from the foregoing that the lateral flow test described above provides a rapid and convenient means for detecting the presence of the T14 peptide or its fragments, variants in a sample taken from a subject.

[0041] 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 of the presence or absence of the T14 peptide or its variants or fragments in their sample. Visual assessment of the presence or absence of the test line provides a qualitative diagnostic or prognostic test, and visual determination of the relative thickness (or darkness of color) of the test line provides a useful semi-quantitative measurement. Thus, the use of the lateral flow test for screening T14 is highly valuable and can be performed by the subject at home (i.e., without the presence of a qualified medical personnel).

[0042] However, it is challenging to achieve an accurate and fully quantitative assessment solely based on the LFT results. Therefore, preferably, the method or device may include means for quantitatively measuring the results of the LFT cassette. In one embodiment, the lateral flow cassette may be connected to a reader that runs software for detecting the test line and the control line, and then accurately calculates the concentration of the T14 peptide in the sample based on the concentration / value of the test line and the control line.

[0043] In another embodiment, the cassette may be connected to a computer, tablet, or smartphone that runs software for detecting the test line and the control line, and then calculates the concentration of the T14 peptide in the sample based on the concentration / value of the test line and the control line. Alternatively, the user may take a photo of the LFT cassette, and the software determines the level of the T14 peptide in the sample based on the signal intensity in the test line and the control line.

[0044] Preferably, the sample includes a biological sample. The sample can be any biological material obtainable from a subject from which SEQ ID No:3 (T14) or its variant or fragment can be obtained.

[0045] The sample can be nasal secretion or fluid, saliva, blood, venous blood, arterial blood, plasma, serum, capillary blood, non-venous blood, non-arterial blood, finger prick blood, spinal fluid, urine, sweat, tears, breast aspirate, prostatic fluid, semen, vaginal fluid, feces, cervical smear, cells, amniotic fluid, intraocular fluid, mucus, respiratory moisture, animal tissue, cell lysate, tumor tissue, hair, skin, buccal swab, lymph, interstitial fluid, nail, bone marrow, cartilage, prion, bone meal, earwax, or a combination thereof.

[0046] Preferably, the sample does not contain blood, and most preferably does not contain venous blood or arterial blood. Preferably, the sample does not contain saliva.

[0047] However, preferably, the sample includes a peripheral tissue sample. Preferably, the sample includes nasal secretion or nasal fluid, saliva, capillary blood, or finger prick blood.

[0048] The device may include a sample collection container for receiving the extracted sample obtained from the subject. The T14 level of the blood sample can be detected immediately. Alternatively, the blood sample can be stored at a low temperature, such as in a refrigerator or even frozen before the T14 assay. The detection of the T14 peptide can be performed on whole blood taken from the blood sample. However, preferably, the blood sample contains the collected serum. Preferably, the blood sample contains plasma.

[0049] Prior to performing the T14 determination, the blood can be further processed. For example, an anticoagulant can be added, such as citrate (e.g., sodium citrate), hirudin, heparin, PPACK, or sodium fluoride. Thus, the sample collection container can contain an anticoagulant to prevent the blood sample from clotting. Alternatively, the blood sample can be centrifuged or filtered to prepare a plasma or serum fraction that can be used for analysis. Thus, it is preferred to analyze or determine T14 in a plasma or serum sample. Particularly preferably, the T14 concentration is measured in vitro from a serum sample or plasma sample taken from a subject.

[0050] Most preferably, the sample comprises a capillary blood sample, preferably a finger prick blood sample. Most preferably, the sample comprises a saliva sample. Most preferably, the sample comprises a nasal secretion or nasal fluid sample. Advantageously, any of the above samples can be used in the LFT method or device. However, these samples (especially nasal secretions / fluids and finger prick blood samples) can also be tested for the T14 peptide by non-LFT methods.

[0051] Thus, in a fifth aspect, there is provided a method of diagnosing or prognosticating a neurodegenerative disorder in a subject, the method comprising detecting a peptide comprising or consisting of SEQ ID NO:3 (T14), or a variant or fragment thereof, in a nasal secretion, nasal fluid, or finger prick blood sample obtained from a test subject, wherein the 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 disease, or is predisposed to having a neurodegenerative disease or has a negative prognosis thereof.

[0052] In a sixth aspect of the invention, there is provided a neurodegenerative disorder diagnostic or prognostic device for diagnosing or prognosticating a neurodegenerative disorder in a subject, the device comprising means for detecting a peptide comprising SEQ ID No:3 (T14) or a variant or fragment thereof, or consisting of SEQ ID No:3 (T14) or a variant or fragment thereof, in a nasal secretion, nasal fluid, or finger prick blood sample obtained from a test subject, wherein the detection of a peptide comprising SEQ ID No:3 (T14) or a variant or fragment thereof, or the detection of a peptide consisting of SEQ ID No:3 (T14) or a variant or fragment thereof, corresponds to a subject having a neurodegenerative disorder, or being predisposed to having a neurodegenerative disorder or having a negative prognosis for a neurodegenerative disorder.

[0053] In a seventh aspect, there is provided a method of treating a subject having or suspected of having a neurodegenerative disease, pre-symptomatic neurodegenerative disease, and / or having cognitive decline, the method comprising:

[0054] (a) Detect a peptide comprising SEQ ID NO:3 (T14) or a variant or fragment thereof, or consisting of SEQ ID NO:3 (T14) or a variant or fragment thereof, in a nasal secretion, nasal fluid, or finger-prick blood sample obtained from a test subject, wherein the presence of a peptide comprising SEQ ID No:3 or a variant or fragment thereof, or consisting of SEQ ID No:3 or a variant or fragment thereof, in the test sample indicates that the test subject has a neurodegenerative disease, or is predisposed to a neurodegenerative disease, or has a negative prognosis;

[0055] (b) Administering or having administered to a subject a therapeutic agent that prevents, reduces, or delays neurodegenerative diseases and / or cognitive decline.

[0056] The methods or devices of the fifth to seventh aspects can be performed in vivo, in vitro, or ex vivo. However, preferably, the method is performed in vitro.

[0057] Most preferably, the sample comprises nasal secretion or nasal fluid. Most preferably, the sample comprises saliva.

[0058] Preferably, the device or method of the fifth to seventh aspects is used to identify the presence of T14 (SEQ ID No:3 or a variant or fragment thereof) in a sample, or to determine its concentration in the sample, preferably the concentration of soluble T14. The device for determining the T14 concentration can include an assay suitable for detecting the presence and / or absence of T14 in the sample. The device or method can include the use of a positive control and / or a negative control, which can be compared with the assay.

[0059] Although the methods or devices of the first to fourth aspects include the use of lateral flow, for the methods or devices of the fifth to sixth aspects, the T14 peptide (SEQ ID

[0060] No:3) can be determined by a variety of methods known to those skilled in the art, and it is not necessarily lateral flow. For example, immunoassays are preferably employed to detect the T14 peptide or to determine the T14 level. However, it should be understood that non-immuno-based assays can also be used, for example, labeling a compound having an affinity for the ligand of the T14 peptide and then measuring the label. The T14 peptide can also be determined by Western blot analysis, which can be used to determine the total protein level of the T14 peptide. Thus, the T14 peptide concentration can be detected by enzyme-linked immunosorbent assay (ELISA), fluorescence assay, chemiluminescence assay, or radioimmunoassay analysis.

[0061] Most preferably, immunoassays (such as ELISA for LFT) are used to detect the soluble T14 peptide. Most preferably, Western blot analysis is used to detect the aggregated T14 peptide. Particularly preferably, the methods, devices, and uses of the present invention include the detection of soluble T14 (SEQ ID No:3).

[0062] In one embodiment, the concentration of: (i) a soluble peptide comprising SEQ ID NO:3 or a variant or fragment thereof or consisting of SEQ ID NO:3 or a variant or fragment thereof, or (ii) an aggregated peptide comprising SEQ ID NO:3 or a variant or fragment thereof 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 SEQ ID No:3 or a variant or fragment thereof or consisting of SEQ ID No:3 or a variant or fragment thereof, and (ii) an aggregated peptide comprising SEQ ID No:3 or a variant or fragment thereof or consisting of SEQ ID No:3 or a variant or fragment thereof is determined. Preferably, soluble T14 and aggregated T14 can be detected in combination.

[0063] However, preferably, the apparatus or method of the fifth to seventh aspects includes using a lateral flow assay or test as in the apparatus or method of the first to fourth aspects.

[0064] Preferably, the method or apparatus of the fifth to seventh aspects includes 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 fluid or finger prick blood 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 an antigen-binding fragment thereof may be polyclonal or monoclonal. The antibody or an antigen-binding fragment thereof may be produced in rabbits, mice or rats.

[0065] Any method or apparatus described herein includes the use of an anti-T14 immunospecific antibody or an 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 at the C-terminus of SEQ ID No 13. Preferably, the antibody or antigen-binding fragment thereof specifically binds to one or more amino acids of SEQ ID No 11 (i.e., SYMVHWK, which is amino acids 7-14 at the C-terminus of SEQ ID No:3). Preferably, the antibody or antigen-binding fragment thereof specifically binds to the C-terminal lysine (K) residue in the epitope. The inventors surprisingly observed that the C-terminal amino acid sequence VHWK in SEQ IDNo:3, which is described herein as SEQ IDNo.12 (i.e., amino acids 11-14 at the C-terminus of SEQ ID N0.3), serves as an epitope for the antibody or an 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 N0.12. Thus, it should be understood that the epitope bound by the antibody comprises or consists of SEQ ID No:12. Thus, the antibody or antigen-binding fragment thereof specifically binds to SEQID No:3 or a fragment or variant thereof and can be used as a T14 peptide detection device or in a T14 peptide detection device.

[0066] Preferably, the antibody or antigen-binding fragment thereof does not bind to SEQ ID No:2 (i.e., T30).

[0067] Preferably, the antibody or antigen-binding fragment thereof does not bind to SEQ ID No:13 (i.e., T15), i.e., NQFDHYSKQDRCSDL.

[0068] Preferably, the antibody or antigen-binding fragment thereof does not bind to SEQ ID No:14 (i.e., β-amyloid (Aβ)), i.e., DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGWIA.

[0069] The device or method may further include the use of a label that can be detected in an assay. The term "label" may mean any moiety that can be attached to any part of the device for assaying the concentration of T14 peptide in a sample obtained from a test subject, whether soluble T14 and / or aggregated T14.

[0070] The moiety can be used for, for example, therapeutic or diagnostic procedures. Therapeutic labels include, for example, moieties that can be attached to the antibodies or fragments thereof described herein and used to monitor the binding of the antibody to the T14 peptide (i.e., SEQ ID No: 3 or its fragments or variants). Diagnostic labels include, for example, moieties that can be detected by an analytical method. 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 methods such as radioimmunoassay, ELISA, or FACS analysis methods. Analytical methods also include in vitro and in vivo imaging methods. Specific examples of diagnostic labels that can be detected by analytical means include enzymes, radioisotopes, fluorescent dyes, chemiluminescent labels, and biotin.

[0071] Preferably, the T14 peptide concentration can be measured by a sandwich ELISA or LFT. The ELISA can include using suitable antibodies, for example, for coating microtiter plates (for ELISA) or substrates (for LFT). For example, such suitable antibodies can include the anti - T14 peptide antibodies described herein (WO2016 / 156803). In addition, the ELISA can include using suitable antibodies for detection. For example, such suitable antibodies can include peroxidase - labeled monoclonal mouse anti - human T14 peptide antibodies. Human T14 peptide can be purified from plasma of nasal secretions and then can be quantified by amino acid analysis, and plasma or nasal secretion standards can be calibrated using standard techniques known to those skilled in the art. The label can be directly attached to the antibody or to a second binder that specifically binds T14. Such a second binder can be, for example, a second antibody. The second antibody can be polyclonal or monoclonal and can be of human, rodent, or chimeric origin.

[0072] The T14 level of the finger - prick blood sample used in the devices or methods of the fifth to seventh aspects can be measured immediately. Alternatively, the finger - prick blood sample can be stored at low temperature, for example, in a refrigerator or even frozen before the T14 measurement is performed. The detection of T14 can be performed on whole blood obtained from the finger - prick. However, preferably, the finger - prick blood sample contains serum. Preferably, the finger - prick blood sample contains plasma.

[0073] Before performing the T14 measurement, the blood can be further processed. For example, an anticoagulant can be added, such as citrate (e.g., sodium citrate), hirudin, heparin, PPACK, or sodium fluoride. Thus, the sample collection container can contain an anticoagulant to prevent the blood sample from clotting. Alternatively, the blood sample can be centrifuged or filtered to prepare plasma or serum fractions, which can be used for analysis. Thus, it is preferred to analyze or measure T14 in plasma or serum samples. Particularly preferably, the T14 concentration is measured in vitro from serum samples or plasma samples taken from a subject.

[0074] Preferably, the device or method is used to identify the presence of T14 (SEQ ID NO: 3 or its variants or fragments) in a sample, or to determine its concentration in the sample, preferably the concentration of T14. The device for determining the concentration of T14 may include an assay suitable for detecting the presence of T14 in a sample. The device or method may include using a positive control and / or a negative control that can be compared with the assay.

[0075] The concentration of the peptide contained in or consisting of SEQ ID No: 3 or its variants or fragments in the sample can be between 0.1 and 1000 ng / mg protein in the sample, or between 0.2 and 750 ng / mg protein, or between 0.5 and 500 ng / mg protein. Preferably, the concentration of the peptide contained in or consisting of SEQ ID No: 3 or its variants or fragments in the sample is between 1 and 400 ng / mg protein in the sample, or between 2 and 300 ng / mg protein, or between 3 and 200 ng / mg protein. Preferably, the concentration of the peptide contained in or consisting of SEQ ID No: 3 or its variants or fragments in the sample is between 4 and 100 ng / mg protein, or between 5 and 75 ng / mg protein, between 10 and 50 ng / mg protein, or between 20 and 40 ng / mg protein.

[0076] The present invention can be used in a method or device where the subject has or is suspected of having a neurodegenerative disease selected from: Alzheimer's disease, Parkinson's disease, Huntington's disease, motor neuron disease, spinocerebellar ataxia types 1, 2, and 3, amyotrophic lateral sclerosis (ALS), schizophrenia, dementia with Lewy bodies, and frontotemporal dementia.

[0077] However, preferably the present invention is used to study or prognose cognitive decline in any neurological disorder associated with the non-enzymatic function of AChE.

[0078] Therefore, preferably, the neurodegenerative disease is selected from: Alzheimer's disease, Parkinson's disease, and motor neuron disease, and preferably Alzheimer's disease or Parkinson's disease.

[0079] However, particularly preferably, when the subject has or is suspected of having Alzheimer's disease, the methods and devices of the present invention are used.

[0080] Therefore, it should be understood that in a preferred embodiment, the method or device of the present invention may include using lateral flow to detect SEQ ID NO: 3 or its variants or fragments in a sample, which is preferably nasal secretion, nasal fluid, or saliva, for diagnosing or prognosing Alzheimer's disease.

[0081] Examples of suitable therapeutic agents that can be administered to a subject for the prevention or treatment of neurodegenerative and / or cognitive decline include, but are not limited to, acetylcholinesterase inhibitors such as rivastigmine, galantamine, and donepezil, and / or N-methyl-D-aspartate (NMDA) antagonists such as memantine. Alternative therapies include the use of cyclic peptides as described in WO2015 /

[0082] 004430, linear peptides as described in WO2015 / 053601, or peptidomimetics as described in WO2018 / 033724, the entire contents of these three patent applications are incorporated herein by reference.

[0083] Preferably, the subject being tested is a living subject. The subject can be a vertebrate, a mammal, or a domestic animal. However, most preferably, the subject is a human, which can be male or female. The subject can be a child or an adult. The age of the subject can be at least 20, 30, 40, 50, 60, 65, or 70 years old. However, the subject can be less than 80, 70, 65, 60, or 50 years old.

[0084] In one embodiment, the method and apparatus are for a subject diagnosed with a neurodegenerative disease. Thus, the subject preferably has symptoms and exhibits signs of cognitive decline or dementia. Thus, the method and apparatus can predict and monitor disease progression.

[0085] In another embodiment, the method and apparatus are for a subject suspected of having a neurodegenerative disease. The subject may have symptoms of a neurodegenerative disease. Thus, the method and apparatus can be diagnostic.

[0086] In a preferred embodiment, the method and apparatus are for a subject not suspected of having a neurodegenerative disease. Preferably, the subject is tested before any symptoms of neurodegenerative, cognitive decline, or neurodegenerative disease appear. Preferably, the subject is pre-symptomatic. Preferably, the subject is in Braak stage I or II. Thus, the method and apparatus can be diagnostic.

[0087] Thus, it should be understood that in a preferred embodiment, the method or apparatus of the present invention can include using lateral flow to detect SEQ ID NO:3 or its variants or fragments in a sample, which is preferably nasal secretion, nasal fluid, or saliva, to diagnose or prognose Alzheimer's disease, most preferably pre-symptomatic AD.

[0088] Following their previous research, the inventors continued their research on acetylcholinesterase, as well as their antibodies that exhibit immunological specificity for a specific region of the C-terminus of the enzyme, as described in WO 2016 / 156803, the content of which is incorporated herein by reference.

[0089] 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 called 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, as follows:

[0090] 1mrppqcllht pslaspllll llwllgggvg aegredaell vtvrggrlrg irlktpggpv

[0091] 61saflgipfae ppmgprrflp pepkqpwsgv vdattfqsvc yqyvdtlypg fegtemwnpn

[0092] 121relsedclyl nvwtpyprpt sptpvlvwiy gggfysgass ldvydgrflv qaertvlvsm

[0093] 181nyrvgafgfl alpgsreapg nvglldqrla lqwvqenvaa fggdptsvtl fgesagaasv

[0094] 241gmhllsppsr glfhravlqs gapngpwatv gmgearrrat qlahlvgcpp ggtggndtel

[0095] 301vaclrtrpaq vlvnhewhvl pqesvfrfsf vpvvdgdfls dtpealinag dfhglqvlvg

[0096] 361vvkdegsyfl vygapgfskd neslisraef lagvrvgvpq vsdlaaeavv lhytdwlhpe

[0097] 421dparlreals dvvgdhnvvc pvaqlagrla aqgarvyayv fehrastlsw plwmgvphgy

[0098] 481eiefifgipl dpsrnytaee kifaqrlmry wanfartgdp neprdpkapq wppytagaqq

[0099] 541yvsldlrple vrrglraqac afwnrflpkl lsatdtldea erqwkaefhr wssymvhwkn

[0100] 601qfdhyskqdr csdl

[0101] [SEQ ID No:1]

[0102] 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, as follows:

[0103] KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL

[0104] [SEQ ID No:2]

[0105] The amino acid sequence of T14 (which corresponds to the 14 amino acid residues located at the end of SEQ ID No:1 and lacks the last 15 amino acids found in T30) is provided herein as SEQ ID No:3, as follows:

[0106] AEFHRWSSYMVHWK

[0107] [SEQ ID No:3]

[0108] Thus, preferably, the peptide of SEQ ID No:3 or its variant or fragment is T14. Most preferably, the methods and devices of the present invention relate to detecting a soluble peptide comprising or consisting of SEQ ID No:3.

[0109] However, fragments of T14 (SEQ ID No:3) are also detectable in the methods and devices of the present invention and can serve as diagnostic or prognostic markers for use according to the present invention.

[0110] Thus, in one embodiment, a fragment of SEQ ID No:3 preferably comprises the amino acid sequence of SEQ ID No:4 (i.e., T7), i.e., SYMVHWK.

[0111] In another embodiment, a fragment of SEQ ID No:3 preferably comprises the amino acid sequence of SEQ ID No:5 (i.e., T8), i.e., SSYMVHWK.

[0112] In another embodiment, the fragment of SEQ ID No:3 preferably comprises the amino acid sequence of SEQ ID No:6 (i.e., T9), i.e., WSSYMVHWK.

[0113] In another embodiment, the fragment of SEQ ID No:3 preferably comprises the amino acid sequence of SEQ ID No:7 (i.e., T10), i.e., RWSSYMVHWK.

[0114] In another embodiment, the fragment of SEQ ID No:3 preferably comprises the amino acid sequence of SEQ ID No:8 (i.e., T11), i.e., HRWSSYMVHWK.

[0115] In another embodiment, the fragment of SEQ ID No:3 preferably comprises the amino acid sequence of SEQ ID No:9 (i.e., T12), i.e., FHRWSSYMVHWK.

[0116] In another embodiment, the fragment of SEQ ID No:3 preferably comprises the amino acid sequence of SEQ ID No:10 (i.e., T13), i.e., EFHRWSSYMVHWK.

[0117] In other words, although it is preferred to detect T14 (i.e., SEQ ID No:3), the present invention can also rely on the detection of any one or more of T7 - T13 (i.e., SEQ ID No:4 - 10).

[0118] Preferably, the method or apparatus of the present invention includes determining the Braak stage of a subject. The subject can be any one of Braak stage I, II, or III. Preferably, the subject is in Braak stage I. Preferably, the subject is in Braak stage II. Preferably, the subject is in Braak stage III.

[0119] Preferably, the subject is a living subject. So far, Braak staging can only be performed on deceased subjects, so the invention described herein represents a significant advancement over currently available methods.

[0120] Thus, preferably, the method comprises:

[0121] (a) Analyzing the concentration of a peptide comprising SEQ ID No:3 (T14) or a variant or fragment thereof or consisting of SEQ ID No:3 (T14) or a variant or fragment thereof in a sample obtained from a test subject; and

[0122] (b) Compare this concentration with a reference value of a control group from deceased subjects with known Braak stages, which contains the concentration of SEQ ID No:3 or its variants or fragments or a soluble peptide consisting of SEQ ID No:3 or its variants or fragments;

[0123] Wherein the Braak stage of a living body is determined for a test subject by comparing the concentration of a peptide containing SEQ ID No:3 or its variants or fragments or consisting of SEQ ID No:3 or its variants or fragments with the corresponding reference value related to the Braak stage.

[0124] It should be understood that the methods and devices of the present invention can be used to determine and monitor disease progression in a method closely related to the Braak stage. Advantageously, the results described in the examples support the inventors' hypothesis that the 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, so the methods and devices of the present invention represent a significant advancement over these existing methods. The use of the T14 biomarker (i.e., the peptide of SEQ ID No:3 or its variants or fragments) allows the Braak staging of a patient to be determined with very high specificity and sensitivity by non-invasive, easily repeatable, and cost-effective methods, such as blood, urine, or cerebrospinal fluid collection, and thus routine screening, Braak staging diagnosis, and appropriate therapeutic interventions can be carried out.

[0125] It should be understood that the Braak staging has six stages based on the location of neurofibrillary tangles, where Braak stage 0 corresponds to healthy subjects. The first and second stages involve the early stages of the disease, when neurofibrillary tangles are limited to the entorhinal area of the brain. Braak stages I and II are pre-symptomatic stages, and as Figure 7 shown, the inventors surprisingly demonstrated that T14 can be detected in samples at each of these stages. Stages III and IV define the involvement of neurofibrillary tangles in the limbic region (including the hippocampus), and stages V and VI are the widespread presence of neurofibrillary tangles in the neocortical region of the brain. Therefore, the methods and devices of the present invention can be used to determine the Braak stage 0, I, II, III, IV, V, or VI of a living body. Braak staging is a good method for recording the progression of Parkinson's disease and Alzheimer's disease in postmortem brains and is currently much more reliable than any pre-mortem method.

[0126] It should also be understood that the methods of the present invention can be used to enable a clinician to accurately diagnose the stage of neurodegenerative and / or cognitive decline and thus make an informed decision on the optimal course of treatment for a patient based on the T14 concentration detected in a sample or based on the Braak stage. In addition, these methods can be used to monitor the efficacy of a putative treatment for neurodegenerative and cognitive decline. Accordingly, the devices of the present invention can be used to provide a prognosis of the condition of a subject such that a clinician can administer treatment according to the third or seventh aspect. The device can also be used to monitor the efficacy of a putative treatment for neurodegenerative and cognitive decline. Thus, the methods and devices are very useful for guiding a clinician's treatment regimen and monitoring the efficacy of such a treatment regimen.

[0127] Preferably, the concentration of the following is analyzed: (i) the concentration of a soluble peptide comprising SEQ ID No: 3 (T14) or a variant or fragment thereof or consisting of SEQ ID No: 3 (T14) or a variant or fragment thereof, and / or (ii) an aggregated peptide comprising SEQ ID No: 3 or a variant or fragment thereof or consisting of SEQ ID No: 3 (T14) or a variant or fragment thereof, and the Braak stage of a living test subject is determined by comparing the concentration of the soluble or aggregated peptide comprising SEQ ID NO: 3 or a variant or fragment thereof or consisting of SEQ ID NO: 3 or a variant or fragment thereof with the corresponding reference value associated with the Braak stage.

[0128] Preferably, the soluble peptide is analyzed. In some embodiments, a higher concentration of the soluble peptide comprising SEQ ID No: 3 or a variant or fragment thereof or consisting of SEQ ID No: 3 or a variant or fragment thereof indicates a later Braak stage compared to the reference value. In other words, the higher the concentration of soluble T14, the greater the correlation with the late Braak stage (e.g., stage IV, V or VI) of a living body. It is preferred to use ELISA to assay the soluble peptide SEQ ID NO: 3 (T14) or a variant or fragment thereof, most preferably on a saliva, nasal fluid / nasal secretion or finger prick plasma sample taken from a subject.

[0129] However, preferably, a lower concentration of the soluble peptide comprising SEQ ID NO: 3 or a variant or fragment thereof or consisting of SEQ ID NO: 3 or a variant or fragment thereof indicates a later Braak stage compared to the reference value. In other words, the lower the concentration of soluble T14, the greater the correlation with the late Braak stage (e.g., stage IV, V or VI) of a living body. The soluble peptide SEQ ID No: 3 (T14) or a variant or fragment thereof is preferably determined using a lateral flow or ELISA, most preferably on a saliva, nasal fluid / nasal secretion or finger prick plasma sample taken from a subject.

[0130] Thus, preferably, compared to a reference value, a lower concentration of a soluble peptide comprising or consisting of SEQ ID No:3 or a variant or fragment thereof indicates that the patient is β-amyloid positive; and / or compared to a reference value, a higher concentration of a soluble peptide comprising or consisting of SEQ ID No:3 or a variant or fragment thereof indicates that the patient is β-amyloid negative. Preferably, compared to a reference value, a lower concentration of a soluble peptide comprising or consisting of SEQ ID No:3 or a variant or fragment thereof indicates that the subject has cognitive impairment; and / or compared to a reference value, a higher concentration of a soluble peptide comprising or consisting of SEQ ID No:3 or a variant or fragment thereof indicates that the patient has normal cognition.

[0131] Thus, in a preferred embodiment, compared to a reference value, a lower concentration of a soluble peptide comprising SEQ ID No:3 or a variant or fragment thereof or consisting of SEQ ID No:3 or a variant or fragment thereof indicates Braak stage IV. Alternatively, in another preferred embodiment, compared to a reference value, a lower concentration of a soluble peptide comprising SEQ ID NO:3 or a variant or fragment thereof or consisting of SEQ ID No:3 or a variant or fragment thereof indicates Braak stage V. Alternatively, in another preferred embodiment, compared to a reference value, a lower concentration of a soluble peptide comprising SEQ ID No:3 or a variant or fragment thereof or consisting of SEQ ID No:3 or a variant or fragment thereof indicates Braak stage VI.

[0132] The inventors have also surprisingly found that the concentration of T14 varies between different early Braak stages (see Figure 7 ), thus demonstrating that T14 levels can be used to determine the early pre-symptomatic Braak stages (e.g., I, II, and III) in living patients. Thus, in one embodiment, compared to a reference value, a lower concentration of a soluble peptide comprising SEQ ID No:3 or a variant or fragment thereof or consisting of SEQ ID No:3 or a variant or fragment thereof indicates Braak stage I. Alternatively, in another embodiment, compared to a reference value, a lower concentration of a soluble peptide comprising or consisting of SEQ ID NO:3 or a variant or fragment thereof indicates Braak stage II. Alternatively, in another embodiment, compared to a reference value, a lower concentration of a soluble peptide comprising SEQ ID No:3 or a variant or fragment thereof or consisting of SEQ ID No:3 or a variant or fragment thereof indicates Braak stage III.

[0133] In another embodiment, a higher concentration of a soluble peptide comprising or consisting of SEQ ID No:3 or a variant or fragment thereof as compared to a reference value indicates that the patient is β-amyloid positive; and / or a lower concentration of a soluble peptide comprising or consisting of SEQ ID No:3 or a variant or fragment thereof as compared to a reference value indicates that the patient is β-amyloid negative. Preferably, a higher concentration of a soluble peptide comprising SEQ ID No:3 or a variant or fragment thereof or consisting of SEQ ID No:3 or a variant or fragment thereof as compared to a reference value indicates that the subject has a cognitive impairment; and / or a lower concentration of a soluble peptide comprising or consisting of SEQ ID No:3 or a variant or fragment thereof as compared to a reference value indicates that the patient has normal cognition.

[0134] Those skilled in the art will understand how to measure the concentration of T14 peptide (soluble or aggregated) in a statistically significant number of control individuals and the T14 concentration in a test subject, and then use these respective numbers to determine the Braak stage of the test subject. Comparing the peptide levels of SEQ ID No:3 (i.e., T14) in samples (preferably plasma) collected from a large group of well-characterized individuals at different Braak stages (i.e., without disease or "normal" health), and preferably, death can be the preferred method for defining the reference value of the control population (i.e., cohort).

[0135] In another embodiment, after collecting postmortem CSF samples from a sufficient number of control and Alzheimer's disease subjects (e.g., n > 50 per group), their soluble and aggregated T14 levels can be measured by ELISA and Western blot, respectively. These levels are preferably calibrated according to the Braak stage of the subject (for controls = 0) to determine the relationship between the change in T14 and the incremental increase in the Braak stage. The resulting standard curve can be used for future ex vivo cerebrospinal fluid samples of living patients to infer their Braak stage based on the T14 level in the asymptomatic disease stage of the patient.

[0136] In another embodiment, samples from a sufficient number of control and AD subjects may be required (e.g., n > 200 per group). Their soluble and / or aggregated T14 levels can be measured by ELISA and Western Blot, respectively. These levels may be normalized to the data of healthy subjects. This normalization step may not be required. Next, the ranges of control and AD values can be plotted with confidence intervals. By detecting whether the T14 value falls 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 the disease progression can be prognosed.

[0137] Accordingly, the inventors have recognized that for aggregated or soluble T14, the difference in T14 concentration between normal levels and elevated / decreased levels can be used as a physiological marker to determine the Braak stage of a living subject. It should be understood that if a subject has a lower soluble T14 concentration 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 will indicate a higher Braak stage. The inventors have also found that the concentration of T14 varies between different early Braak stages, thus demonstrating that T14 levels can be used to determine the early pre-symptomatic Braak stages (I, II, and III) of a living patient.

[0138] For example, the decrease in soluble T14 concentration relative to the reference concentration can be at least 10%, preferably at least 20% decrease, more preferably at least 30% decrease, even more preferably at least 40% decrease, and most preferably a decrease of at least 50% of the reference value concentration. Such a decrease in soluble T14 concentration indicates that the test subject will have a higher Braak stage. Alternatively, the increase in aggregated T14 concentration relative to the reference concentration can be about at least 10%, preferably about at least 20% increase, more preferably at least 30% increase, even more preferably an increase of 40% compared to the reference value concentration, and most preferably an increase of at least 50%. Such an increase in aggregated T14 concentration indicates that the test subject will have a higher Braak stage. Accordingly, the clinician will be able to make an informed decision regarding the preferred treatment course required, such as the type and dosage of the therapeutic agent according to the third or seventh aspect to be administered.

[0139] In another embodiment, it should be understood that 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 will indicate a lower Braak stage.

[0140] For example, the increase in soluble T14 concentration relative to the reference concentration can be at least 10%, preferably at least 20% increase, more preferably at least 30% increase, even more preferably at least 40% increase, and most preferably an increase of at least 50% of the reference value concentration. Such an increase in soluble T14 concentration indicates that the test subject will have a higher Braak stage. Alternatively, the decrease in aggregated T14 concentration relative to the reference concentration can be about at least 10%, preferably about at least 20% decrease, more preferably at least 30% decrease, even more preferably at least 40% decrease, and most preferably a decrease of at least 50% of the reference value concentration. Such a decrease in aggregated T14 concentration indicates that the test subject will have a higher Braak stage. Accordingly, the clinician will be able to make an informed decision regarding the preferred treatment course required, such as the type and dosage of the therapeutic agent according to the third or seventh aspect to be administered.

[0141] The method or apparatus of the present invention may further include measuring the rate of cognitive decline by Mini - Mental State Examination (MMSE) score and / or Preclinical Alzheimer's Cognitive Composite (PACC) score.

[0142] MMSE is a questionnaire that is almost universally applicable to patients suspected of having AD and a wider research cohort as a measure of cognitive impairment. It is widely regarded as the gold standard for AD diagnosis because it is easy to apply, requires little training, and is reproducible, valid, and reliable. It is also particularly useful when considering longitudinal assessments of AD and its progression. Repeated measurements of regularly collected MMSE scores can be used to calculate the rate of cognitive decline of a subject. Then, preferably, linear regression is performed to calculate the slope of the MMSE score over time, and it is this slope that is interpreted as the rate of cognitive change (decline / rise). Thus, preferably, the decline in cognitive ability is measured according to the MMSE score. The slope can be calculated as the number of points by which the MMSE score decreases per month.

[0143] On the other hand, the PACC test combines tests that evaluate episodic memory, timed executive function, and global cognition. This is the primary outcome measure for the first pre - clinical AD clinical trial.

[0144] The method of the present invention may further include the step of age - adjusting the T14 concentration from a test subject, whether soluble T14 or aggregated T14, against a corresponding reference value.

[0145] It should be understood that the present invention extends to any nucleic acid or peptide or its variants, derivatives, or analogs that substantially comprise the amino acid or nucleic acid sequence of any sequence mentioned herein, including its variants or fragments. The terms "substantially amino acid / nucleotide / peptide sequence", "variant", and "fragment" may be sequences having at least 40% sequence identity with the amino acid / nucleotide / peptide sequence of one of the sequences mentioned. In the present context, for example, having 40% identity with any sequence described herein.

[0146] Amino acid / polynucleotide / polypeptide sequences having greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, still more preferably greater than 80% sequence identity with any of the sequences mentioned are also contemplated. 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 having at least 97% identity with any of the sequences mentioned herein, even more preferably at least 98% identity, and most preferably at least 99% identity.

[0147] Those skilled in the art will understand how to calculate the percent identity between two amino acid / polynucleotide / polypeptide sequences. To calculate the percent identity between two amino acid / polynucleotide / polypeptide sequences, an alignment of the two sequences must first be prepared, and then the sequence identity value is calculated. The percent identity between two sequences can take different values, depending on: (i) the method used to align the sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or a structural alignment from 3D comparisons; and (ii) the parameters used by the alignment method, such as local alignment global alignment, the pairwise scoring matrix used (e.g., BLOSUM 62, PAM250, Gonnet, etc.), and the gap penalties, such as the functional form and constants.

[0148] After performing the alignment, there are many different ways to calculate the percent identity between two sequences. For example, the number of identities can be divided by: (i) the length of the shortest sequence; (ii) the alignment length; (iii) the average length of the sequences; (iv) the number of positions without gaps; (v) the number of equivalent positions excluding overhangs. In addition, it should be understood that the percent identity also strongly depends on the length. Thus, the shorter a pair of sequences, the higher the sequence identity that one would expect to occur by chance.

[0149] Therefore, it should be understood that the precise 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 way to generate the multiple alignments of the proteins or DNAs of the present invention. Suitable parameters for ClustalW can 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, matrix = Gonnet. For DNA and protein alignment: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will realize that these and other parameters may need to be changed to obtain the best sequence alignment.

[0150] Preferably, the calculation of the percent identity between two amino acid / polynucleotide / polypeptide sequences can then be calculated from an alignment such as (N / T)*100, where N is the number of positions where the sequences share the same residue, and T is the total number of positions being compared, including gaps and including or excluding overhangs. Preferably, overhangs are included in the calculation. Thus, the most preferred method for calculating the percent identity between two sequences includes (i) preparing a sequence alignment using the ClustalW program with a suitable set of parameters, e.g., as described above; (ii) substituting the values of N and T into the following formula: - sequence identity = (N / T)*100.

[0151] Alternative methods for identifying similar sequences are known to those skilled in the art. For example, substantially similar nucleotide sequences will be encoded by sequences that hybridize to the DNA sequence or its complementary sequence under stringent conditions. By stringent conditions, the inventors mean that nucleotides hybridize to DNA or RNA bound to a filter in 3x sodium chloride / sodium citrate (SSC) at approximately 45°C, and then are washed at approximately 20 - 65°C in 0.2x SSC / 0.1% SDS at least once. Alternatively, substantially similar polypeptides can differ from the sequence shown in, for example, SEQ ID NO:3 by at least 1, 2, 3, 4, but less than 5, 10, 20, 50, or 100 amino acids.

[0152] Due to the degeneracy of the genetic code, it is apparent that any nucleic acid sequence described herein can be altered or varied substantially without affecting the sequence of the protein encoded thereby, to provide functional variants thereof. Suitable nucleotide variants are those having sequences that are altered by substituting different codons within the sequence that encode the same amino acid, thereby producing silent (synonymous) changes. Other suitable variants are those having homologous nucleotide sequences but containing variants of all or part of the sequence that are altered by the substitution of different codons that encode amino acids having side chains with similar biophysical properties to the amino acid being substituted, to produce conservative changes. 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 should be understood which amino acids can be substituted by amino acids having similar biophysical properties, and those skilled in the art will know the nucleotide sequences encoding these amino acids.

[0153] All features described in this document (including any appended claims, abstract, and drawings) and / or all steps of any method or process thus disclosed can be combined in any combination with any one of the above aspects, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0154] For a better understanding of the present invention and to show how embodiments of the present invention may be implemented, reference will now be made, by way of example, to the accompanying drawings, in which:

[0155] Figure 1 (From left to right) shows a nasal swab for obtaining nasal secretions or fluids, an oral swab for obtaining oral fluids (such as saliva), and a device for performing a needle prick to obtain a blood sample by needle prick. Each of these sample types is referred to as peripheral tissue.

[0156] Figure 2 Shows the use of Figure 1 The T14 immunoassay data obtained from saliva samples of 6 individuals (labeled 1 - 6) using the oral swab shown in.

[0157] Figure 3 Shows a perspective view of a lateral flow test kit having a sample dropper for delivering a sample (such as Figure 2 The blood by needle prick, nasal secretions, or saliva shown in) to a sample well (S).

[0158] Figure 4 Shows a side view of a first embodiment of the lateral flow device of the present invention, which is referred to as an inhibition LFT (wet type) for detecting the T14 peptide biomarker.

[0159] Figure 5 Shows a side view of a second embodiment of the lateral flow device of the present invention, which is referred to as a sandwich LFT (wet type) for detecting the T14 peptide biomarker.

[0160] Figure 6 Illustrates a perspective view of an embodiment of a desktop reader (left side) or a smartphone running an application app (right side) for reading the LFT cassette shown in Figure 4 After the inhibition LFT of Figure 4 Or the sandwich LFT of Figure 3 These can be used to quantify the amount of T14 in the sample. Figure 5 These can be used to quantify the amount of T14 in the sample.

[0161] Figure 7 Shows three illustrations of the brain with clinical symptoms and corresponding Braak stages (from left to right), and the corresponding western blots of hippocampal T14 peptide for Braak stages I, II, and VI, showing an increase in the later stages.

[0162] Figure 8 Shows the therapeutic diagnostic interaction between the detection of T14 levels using the method of the present invention (e.g., LFT) and the therapeutic intervention by administration of an acetylcholinesterase inhibitor, or an N-methyl-D-aspartic acid (NMDA) antagonist, or a cyclic peptide (referred to as the NBP-14 cyclic peptide as described in WO2015 / 004430) to achieve permanent prevention of symptom onset.

[0163] Figure 9 Shows a device for implementing a wet reagent or "semi-strip" test method.

[0164] Figure 10 Shows the results of inhibiting a lateral flow assay.

[0165] Figure 11 Shows the results of a sandwich lateral flow assay. Examples

[0166] The inventors have observed that the T14 biomarker peptide surprisingly exists in saliva, nasal secretions, and finger prick blood at very low but detectable concentrations. Such sample types can generally be analyzed using a lateral flow test (LFT), which is painless, socially acceptable, low-cost, can be used frequently, and provides very rapid and accurate results, unlike cerebrospinal fluid and venous / arterial blood.

[0167] Materials and Methods

[0168] Generating Braak stage data, CSF data, and saliva data

[0169] Western blotting - brain tissue, CSF, and saliva

[0170] Human hippocampal samples and postmortem cerebrospinal fluid analysis were provided by the Oxford Brain Bank and classified as Braak stage 0, I, II, III, V, or VI. Saliva samples were collected from 6 donors and placed in separate tubes.

[0171] Brain tissue:

[0172] Western blotting was 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 (16000 x g, 30 minutes, 4 °C) and the supernatant was quantified for protein.

[0173] Protein was measured using the Thermo Scientific Pierce 660 nm Protein Assay. The assay is a ready-to-use assay compatible with detergents and reducing agents and allows for the rapid measurement of total protein concentration compared to a protein standard of bovine serum albumin. For the assay, 10 ml of each human brain homogenate sample was added to a microtiter 96-well plate, followed by the addition of 150 ml of Pierce assay. After incubation for 5 minutes, absorbance was measured at 660 nm in a Vmax microplate reader (Molecular Devices, Wokingham, UK), and the optical density results were extrapolated to a BSA standard curve to obtain mg / ml.

[0174] 100 μg 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 minutes), and loaded onto a 4%-20% Mini-PROTEAN TGX precast protein gel, 10 wells, 50 μL. Proteins were separated by electrophoresis, transferred to a PVDF (0.45 μm) membrane, and blocked with 5% blotting-grade blocker non-fat milk (dissolved in Tris-buffered saline supplemented with 0.05% Tween20 (TBS-T 0.05%)) (1 hour, RT). The membrane was incubated overnight with the T14 antibody (stock solution 1 mg / mL, dilution 1:1000) as previously described in WO 2016 / 156803. The membrane was washed and incubated with a secondary antibody (1:10000). After washing, an enhanced chemiluminescence detection kit was used to visualize immunoreactive proteins according to the manufacturer's protocol (Thermo Scientific Pierce ECL Plus Western Blotting Substrate) and a CCD camera (G-Box, Syngene, Cambridge, UK) gel system. The scanned blots were analyzed using GensSnap software (Syngene, Cambridge, UK), and the spot density was expressed as a percentage of the spot density obtained from the control. Bands were quantified using ImageJ, and unpaired tests were performed using GraphPad Prism 9.0.

[0175] Detection of T14 in cerebrospinal fluid:

[0176] The cerebrospinal fluid samples were used directly for electrophoresis. Proteins were assayed 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-b-mercaptoethanol, 0.05% bromophenol blue, final concentration), boiled for 10 minutes, and then loaded onto a 10% acrylamide gel. Proteins were separated by electrophoresis until the leading edge of the elution migrated to allow proper separation of high molecular weight fragments. The proteins were then transferred from the gel to a polyvinylidene difluoride sheet (ThermoFisher). These sheets were blocked for 1 hour at room temperature with Tris-buffered saline buffer with 5% non-fat milk added with 0.05% Tween 20 (TBS-T buffer). They were then incubated overnight with the T14 antibody (1:1000, Genosphere) diluted with TBS-T buffer with 5% non-fat milk added. Thereafter, the membranes were washed with TBS-T buffer and incubated with a secondary antibody conjugated to anti-rabbit IgG horseradish peroxidase (HRP) (ab6 21Abeam, Cambridge, UK, 1:5000 dilution) for 45 minutes. After washing, the immunoreactive proteins were visualized using an enhanced chemiluminescence detection kit, according to the manufacturer's protocol (ThermoScientific Pierce ECL Plus Western Blotting Substrate) and a CCD camera (G-Box, Syngene, Cambridge, UK) gel system. The scanned blots were analyzed using GensSnap software (Syngene, Cambridge, UK), and the spot density was expressed as a percentage of the spot density obtained from the control. The bands were quantified using ImageJ, and unpaired tests were performed using GraphPad Prismg9.0.

[0177] Detection of T14 in saliva:

[0178] Saliva samples were obtained from 6 donors by using oral swabs or by directly spitting into tubes. For western blotting, 10 μl of total protein (unknown concentration) was loaded onto 5 μl of sample buffer (0.5 M Tris HCl, pH

[0179] 6.8, 10% glycerol, 2% (w / v) sodium dodecyl sulfate, 5% (v / v) 2-b-mercaptoethanol, 0.05% bromophenol blue, final concentration). Then the samples were boiled for 10 minutes and loaded onto a 4%-20% Mini-PROTEAN TGX precast protein gel, 10-well, 50 μL. Proteins were separated by electrophoresis until the leading edge of the elution migrated to allow proper separation of high molecular weight fragments. Then the proteins were transferred from the gel to polyvinylidene difluoride sheets (ThermoFisher). These sheets were blocked with Tris-buffered saline buffer containing 5% non-fat dry milk (added with 0.05% Tween 20 (TBS-T buffer)) at room temperature for 1 hour. Then they were incubated overnight with the T14 antibody (1:1000, Genosphere), and the T14 antibody was diluted with TBS-T buffer containing 5% non-fat dry milk. Thereafter, the membrane was washed with TBS-T buffer and incubated with a secondary antibody conjugated to anti-rabbit IgG horseradish peroxidase (HRP) (ab6721 Abeam, Cambridge, UK, 1:10,000 dilution) for 45 minutes. After washing, an enhanced chemiluminescence detection kit was used to visualize the immunoreactive proteins according to the manufacturer's protocol (Thermo Scientific Pierce ECL Plus Western Blotting Substrate) and a CCD camera (G-Box, Syngene, Cambridge, UK) gel system. The scanned blots were analyzed using GensSnap software (Syngene, Cambridge, UK), and the spot density was expressed as a percentage of the spot density obtained from the control. The bands were quantified using ImageJ and unpaired tests were performed using GraphPad Prism 9.0. For the immunoneutralization experiment, the T14 primary antibody (1:1000) was incubated with 1 mg / ml T14 peptide on a rocker at room temperature for 3 h and then added to the membrane.

[0180] Detection of T14 in nasal secretions:

[0181] The inventors believe that the concentration of T14 in nasal secretions (or saliva) is approximately 20 - 40 ng T14 / mg protein. For the LFT embodiment, there are multiple embodiments of devices based on antibody detection systems. This technology was developed using polyclonal and monoclonal IgG antibodies and three different types of immunoassays (i.e., indirect, competitive, and sandwich assays). These antibodies have been shown to perform well in Western blotting, ELISA, AlphaLISA, immunohistochemical staining for detecting synthetic and endogenous T14, and their binding specificities and epitopes have been determined to be good candidates for indirect and competitive immunoassays.

[0182] Lateral flow test (LFT) reagent

[0183] -(Ab16) affinity-purified polyclonal anti-peptide T14 antibody;

[0184] - Monoclonal liquid THK-1-102 purified rabbit IgG (approx. 15 μl);

[0185] - Monoclonal liquid THK-1-104 purified rabbit IgG (~600 μl);

[0186] - Monoclonal liquid THK-1-117 purified rabbit IgG (1 ml);

[0187] - T14 peptide (powder form / lyophilized trifluoroacetate (1 mg); and

[0188] - T30 peptide (powder form / lyophilized trifluoroacetate (1 mg).

[0189] LFT antibody binding

[0190] The detection reagent is 40 nm gold particles passively bound to the antibody. Conjugates were prepared: AB16 (rabbit polyclonal), AB117 (rabbit monoclonal), and AB104 (rabbit monoclonal).

[0191] pH titration and antibody loading onto 40 nm gold colloids were completed.

[0192] Preferred conditions for the conjugate are:

[0193] antibody antibody-loaded binding buffer concentration (OD) AB16 (polyclonal) 18 μg PBS 2.08 AB117 (monoclonal) 14 μg carbonate 1.28 AB104 (monoclonal) 18 μg carbonate 1.66

[0194] Each antibody was bound to 40 nm gold colloids.

[0195] LFT method

[0196] Using a wet reagent or “semi-strip” test method, as Figure 9 shown. In this form, the gold conjugate is in liquid form (in the wells) rather than in dry form, enabling high-throughput testing conditions. The test line was “spotted” onto the membrane with a pipette to facilitate the evaluation of a range of conditions.

[0197] The nitrocellulose membrane is CN180.

[0198] Test line: A) Inhibition assay: T14 peptide

[0199] T30 peptide

[0200] B) Sandwich assay: rabbit polyclonal, AB16

[0201] rabbit monoclonal antibody, AB117

[0202] rabbit monoclonal antibody, AB104

[0203] Rabbit monoclonal antibody, AB102

[0204] Control line: Goat anti-rabbit antibody

[0205] Inhibition of LFT method

[0206] Membrane preparation:

[0207] Dilute the peptide (T14 or T30) in deionized water to 1, 0.5 and 0.25 mg / ml. "Spot" the peptide onto a CN180 nitrocellulose membrane (1 μl for each concentration), and dry the membrane at 37 °C for 15 minutes. Apply goat anti-rabbit antibody to the membrane as the control line.

[0208] Testing method:

[0209] (i) Add 20 μl of gold conjugate (pure) and 20 μl of TBST (Tris-buffered saline 1% Tween 20) to well 1;

[0210] (ii) Add the test strip to well 1;

[0211] (iii) The test strip runs completely (no liquid residue); and

[0212] (iv) Add the test strip to well 2 containing 20 μl of TBST, only for washing the test strip.

[0213] Sandwich LFT method

[0214] Membrane preparation: "Spot" each antibody onto a CN180 membrane (1 μl pure, i.e., 1 mg / ml concentration), and dry the membrane at 37 °C for 15 minutes. Apply goat anti-rabbit antibody to the membrane as the control line. Then evaluate the conjugate and test line antibody combinations in a matrix study in lateral flow format.

[0215] Testing method (continuous sample addition):

[0216] (i) Add 10 μl of peptide (T14 or T30, 0.1 mg / ml) and 10 μl of TBST (Tris-buffered saline 1% Tween 20) to well 1;

[0217] (ii) Add the test strip to well 1;

[0218] (iii) The test strip runs completely (no liquid residue);

[0219] (iv) Add the test strip to well 2 containing 20 μl of conjugate;

[0220] (v) The test strip runs completely (no liquid residue); and

[0221] (vi) Add the test strip to well 3 containing 20 μl of TBST.

[0222] AlphaLISA Detection of the T14-α-7 Complex

[0223] Samples were extracted from homogenized human brain tissue using PerkinElmer lysis buffer (AL003C), and protein concentration was determined using the BCA method. For 100 mg of tissue homogenate, 1 mL of lysis buffer was used. Each sample was subjected to 5 cycles of 40-second pulses and 10-second rests on a benchtop homogenizer. Samples were centrifuged at 4 °C, 15,000 rpm (15 minutes) to obtain the supernatant; these were diluted in PerkinElmer detection buffer (AL000F) and used to measure the T14-α-7 nicotinic receptor complex using AlphaLISA according to the manufacturer's protocol in the presence of NBP14 (concentration 0.065 μM - 900 μM) (Genosphere). The antibody on the SA donor bead was biotinylated BTX (B1196; Invitrogen Life Technologies, Waltham, MA, USA), and anti-rabbit T14 (Genosphere) on the acceptor bead; results were read in an AlphaLISA Reader (model #EnSpire 2300 Multilabel Reader; PerkinElmer).

[0224] Example 1 - Performing a Lateral Flow Test (LFT) to Detect the T14 Peptide

[0225] See Figure 1 , which shows three different types of peripheral tissue samples that can be easily used for subsequent lateral flow tests, including nasal swabs (2) to obtain nasal secretions or nasal fluid from the nasal passage, oral swabs (4) for obtaining oral fluid (e.g., saliva in the buccal cavity or mouth), or a lancet (6) for pricking the skin to obtain blood (capillary blood or whole blood), typically obtained from the finger (but can also be from other parts of the body).

[0226] For example, oral fluid was obtained from six test subjects using either direct spitting or oral swabs (2) and then analyzed. As Figure 2As shown, the inventors surprisingly demonstrated that the T14 biomarker peptide (SEQ ID No: 3) surprisingly exists in saliva at a very low but detectable concentration. Western blot results showed that the T14 band was detected in all samples. To verify that the band was T14 and that the antibody did not cross-contaminate with other molecules, the samples were immunoneutralized (as described in the above method) to show that the antibody specifically detected T14. The concentration of the T14 peptide in saliva was approximately 20 - 40 ng of T14 peptide per milligram of protein in the sample. Therefore, the inventors set out to analyze T14 in the samples using a lateral flow test (LFT), and believed that finger prick blood and nasal secretions could also be used as samples instead of saliva.

[0227] Thus, once a sample (nasal secretion, blood, or saliva) has been obtained from a subject, it is inserted into sample tube 8, where 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 concentration) is added and mixed for a sufficient time such that the sample and any T14 peptide therein are properly suspended and dispersed. Refer Figure 3 , a LFT cassette (10) is shown which has a sample well (12, S) towards one end, and a few drops of the buffered sample solution are applied from tube (8) into the sample well. The cassette (10) has a window (14) through which the test line (16, T) and the control line (18, C) can be viewed. The lateral flow detection mechanism for the T14 peptide will be described below.

[0228] Figure 4 and Figure 5 show two different embodiments of the lateral flow device, namely Figure 4 the inhibition lateral flow test (20) shown in Figure 5 and the sandwich lateral flow test (22) shown in

[0229] Inhibition Lateral Flow Test (LFT)

[0230] See Figure 4, in the inhibition of LFT (20), the LFT cassette (10) has a sample nitrocellulose membrane CN180 pad (24) on which the sample (26) is placed through the hole (12). The sample (26) may or may not contain the sample T14 peptide (15), thus producing different results as described below. Adjacent to the sample pad (24) is arranged a conjugate pad (28) on which is arranged an anti-T14 antibody (30) (labeled with 40 nm colloidal gold nanoparticles passively conjugated to a T14 immunospecific antibody). For example, the anti-T14 antibody (30) may be as described in WO 2016 / 156803.

[0231] The cassette (10) has attached thereto an LFT immobilized T14 peptide line (16) test consisting of a pre-immobilized T14 peptide (17). Lateral to the immobilized T14 peptide test line (16), a control line (18) consisting of a second anti-species labeled antibody (31) that is not specific for T14 (i.e., goat anti-chicken Ig polyclonal antibody) is provided. The upper wick (32) is provided towards the opposite end of the cassette (10) and is used to laterally draw the sample (26) solution across the cassette (10).

[0232] As Figure 4 shown in the upper part (showing a positive result for T14 detection), when the sample (26) contains the sample T14 peptide (15), it flows laterally across the conjugate pad containing the anti-T14 antibody (30), and it is captured by the anti-T14 antibody (30) to form a conjugate of the T14 antibody (34). This T14-antibody conjugate (34) cannot bind to the immobilized T14 peptide (17) immobilized on the test line (16), while the anti-T14 antibody (30) in the sample (26) that does not bind to the sample T14 peptide (15) can flow and bind to the anti-species control (31) immobilized on the control line (18). Thus, the sample T14 (15) captures the antibody (30), which cannot bind to the immobilized T14 (17) attached to the cassette (10). Therefore, a single line appears on the test cassette (10) as a positive result, as Figure 4 shown in the upper right.

[0233] As Figure 4as shown in the lower part of Figure 4 and showing a negative result for the T14 test, when the sample (26) does not contain the sample T14 peptide (15), the sample (26) flows laterally over a conjugate pad containing anti-T14 antibody (30). Since there is no sample T14 (15) available for the anti-T14 antibody (30) to capture, no conjugate of Ti4-antibody (34) is produced as in the positive result discussed above. Thus, all unbound anti-T14 antibodies (30) can flow and bind to the LFT immobilized T14 peptide (17) immobilized on the test line (16) and the anti-species control (31) immobilized on the control line (18). Thus, two spaced lines are formed on the cassette (10) as a negative result, as

[0234] Now referring to Figure 10 which shows the results of the "half-strip" LFT method as shown in Figure 9 . As can be seen in Figure 10 , the antigen can bind to the membrane in an inhibitory form. It can be seen that the LFT can detect T14 and T30 peptides. When the amount of peptide on the membrane is reduced from 1 mg / ml to 0.25 mg / ml, a decrease in the signal intensity in the test line region can be seen, which demonstrates specific binding. However, some non-specific binding is sometimes observed.

[0235] Sandwich lateral flow test (LFT)

[0236] Now referring to Figure 5 , due to the size of the T14 peptide, the inventors 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 (e.g., at the N-terminus) of the T14 peptide, and a second antibody (38) binds to a second epitope (40) spaced from the first epitope (e.g., at the C-terminus).

[0237] In the sandwich LFT (22), the LFT cassette (10) has a sample pad (24) on which the sample (26) is placed through a hole (12). The sample (26) may or may not contain the sample T14 peptide (15). Adjacent to the sample pad (24) is a conjugate pad (28) on which is disposed a first anti-T14 antibody (30) (labeled with 40 nm gold nanoparticle colloids passively bound to the antibody). For example, the first T14 antibody (30) may be as described in WO 2016 / 156803. These anti-T14 antibodies (30) bind to the first epitope (36) on the T14 peptide, such as the C-terminal residue of T14.

[0238] The cassette (10) has a T14 peptide test line (16) which consists of a pre-fixed 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.

[0239] Laterally spaced from the test line (16), the cassette (10) has a control line (18) consisting of an anti-species labeled antibody (31) which is not specific for T14 (e.g., goat anti-chicken IgY polyclonal antibody), and an upper wick (32) disposed towards the opposite end of the cassette (10) which is for laterally drawing in a sample (26) solution across the cassette (10).

[0240] As Figure 5 shown in the upper portion (showing a positive result for T14 detection), when the sample (26) contains the T14 peptide (15), it flows laterally across the conjugate pad (28) carrying the first anti-T14 antibody (30), it is captured by the anti-T14 antibody (30), producing a conjugate of T14-antibody (34). This T14-antibody conjugate (34) itself is captured by the second anti-T14 antibody (38) fixed along the test line (16) to the cassette (10) because the second epitope (40) remains exposed. Thus, a first line appears on the cassette (10), indicating binding of the antibody at the T14 test line (16). Additionally, the additional anti-T14 antibody (30) not bound to the T14 peptide in the sample (26) flows and binds to the anti-species control (31) fixed to the control line (18). As Figure 4 described in the inhibition LFT assay (20) different, in Figure 5 the sandwich assay (22) shown in Figure 5 the upper right, since the cassette (10) does not include any LFT-fixed T14 (17), the assay using two antibodies (30, 38) is a direct detection of the presence of sample T14 (15) in the sample. Thus, a second line appears on the cassette (10) as a positive result, as

[0241] As Figure 5 shown in the lower portion (showing a negative result for T14 detection), when the sample (26) does not contain the T14 peptide (15), it flows laterally across the conjugate pad carrying the anti-T14 antibody (30), there is no T14 (15) that can be captured by the anti-T14 antibody (30), and thus no conjugate of T14-antibody (34) is produced, as in the positive result discussed above. Thus, all unbound anti-T14 antibody (30) flows and cannot bind to the second T14 antibody (38) fixed to the cassette (10). The T14 antibody (30) flows and binds to the anti-species control (31) fixed to the control line (18), and creates a line on the kit (10) as a negative result, as shown in the lower right of Figure 5.

[0242] Now referring to Figure 11 , which shows the results of the "half-strip" LFT method as shown in Figure 9 . It can be seen that the LFT is able to detect T14 and T30 peptides. Some non-specific binding was observed in the strip, indicating that the test line and the binding antibody are interacting. However, these interactions can occur for a variety of reasons, including suboptimal binding conditions.

[0243] The inventors noted that when T14 or T30 peptides were added at 0.1 mg / ml, there was no significant increase in the test line signal. The only exception was the AB16 capture conjugate pair, which showed a slight increase in the concentration of the positive sample (highlighted in the gold box).

[0244] Qualitative and quantitative analysis of LFT results. From the foregoing, it can be understood that the above-mentioned lateral flow test provides a rapid and convenient means for detecting the presence or absence of T14 peptide (15) in a peripheral body sample taken from a subject. The presence of a control line (18) is required to ensure that the test is robust and effective, and the presence or absence of the test line (16) informs the subject whether T14 peptide (15) is present in their sample. Visual assessment of the presence or absence of the test line (16) provides a useful qualitative diagnostic or prognostic test, and visually determining the relative thickness (or color intensity) of the test line (16) provides a useful semi-quantitative measurement. However, it is challenging to achieve an accurate and fully quantitative assessment based solely on LFT results.

[0245] Referring to Figure 6 , an embodiment of a bench-top reader (42, on the left) and an embodiment of a smart phone running an app (44, on the right) for quantitatively measuring the results of an LFT cassette (10) are shown, after using Figure 4 to inhibit the LFT or Figure 5 in a sandwich LFT. The cassette can be connected to the reader (42), which runs software for detecting the test line (16) and the control line (18), and then, using these concentrations / values, accurately calculates the concentration of T14 peptide (15) in the sample (26). Similarly, the cassette (10) can be connected to a computer, tablet, or smart phone (44) running software for detecting the test line (16) and the control line (18), and then calculating the concentration of T14 peptide (15) in the sample (26). Alternatively, the user can take a photo of the LFT cassette (10), and the software determines the level of T14 peptide (15) in the sample (26) based on the signal intensity in the test line (16) and the control.

[0246] Example 2 - Braak staging using LFT

[0247] As Figure 7As shown, the inventors surprisingly showed that there is a distinct and surprising correlation between the concentration of the T14 peptide and the Braak stage of the subject, which in turn corresponds to the clinical symptoms of the subject. For example, the inventors have now detected T14 in samples obtained from living patients in Braak stages I and II (brain pathology) and asymptomatic (clinically). Braak stages III and IV are patients showing mild symptoms, while Braak stages V and VI correspond to dementia and cognitive decline.

[0248] As Figure 7 shown, the western blot of the Alzheimer's hippocampus shows a single T14-reactive band, which increases by approximately 2-fold from early (Braak 0-II) to late (Braak V-VI). It can be seen that there is a very clear signal in the western blot of the Braak VI stage sample. Surprisingly, the data also show that changes in T14 levels can be used to determine early Braak stages I and II. Therefore, importantly, this shows that the concentration of T14 can be used to determine the Braak stage of living asymptomatic patients (i.e., patients in Braak stages I and II).

[0249] Therefore, the present inventors believe that the lateral flow assay described in Example 1 can be conveniently used to detect the T14 peptide and, based on the concentration of T14, determine the Braak stage of the subject from Braak stage 0 to Braak stage VI. Given that Braak staging is currently performed post-mortem, the ability to provide a Braak staging score for living patients by LFT testing would be highly beneficial, as discussed in more detail below.

[0250] Example 3 - Diagnostic, Prognostic, and Therapeutic Uses of the LFT Test

[0251] The inventors were surprised that detectable levels of the T14 peptide (SEQ ID No: 3) will be present in any peripheral tissue suitable for lateral flow testing, such as saliva, nasal secretions, or finger prick blood. Thus, there are at least four scenarios in which these methods and devices can be used to test the general public: -

[0252] 1) Prognostic testing - Subjects diagnosed with Alzheimer's disease

[0253] For those who have been confirmed to have a neurodegenerative disease and / or cognitive decline, the rapid and accurate test of the present invention has great value, which can be used to actively monitor the cause or progression of their condition (i.e., prognosis), and is expected to improve accordingly, slowing down or even reversing cognitive decline. Such individuals may be in Braak stages III, IV, V, or VI.

[0254] Reference Figure 8, which shows the correlation between detecting T14 levels using the diagnostic or prognostic methods of the present invention (such as LFT tests or saliva / finger prick blood tests) and the therapeutic intervention of treating or preventing the onset of neurodegenerative diseases or diseases of cognitive decline with the administered drugs. For example, an acetylcholinesterase inhibitor, or an N-methyl-D-aspartic acid (NMDA) antagonist, or a cyclic peptide (referred to as the NBP-14 cyclic peptide, as described in WO2015 / 004430) can be administered to achieve a permanent prevention of symptom appearance or delay or even reverse the symptoms. Then, such tests can be used to influence the more accurate dosage of the therapeutic drugs given to the patient and can also lead to better patient outcomes. This is essentially a companion diagnosis for clinical trials and patient treatment, serving as a very sensitive monitoring system for treatment efficacy.

[0255] The inventors envision a quantitative reading of T14 levels, which will inform the amount of treatment required, as patients can be tested daily, weekly, or monthly at a frequency consistent with the dosage regimen of the therapeutic agent and continuously monitor its effects regularly. It is hoped that the subjects will see a decrease in T14 levels as a direct result of the treatment administered, thereby achieving a successful outcome by delaying further symptom onset, preventing symptom onset, or even reversing the symptoms (e.g., cognitive decline).

[0256] 2) Diagnostic test - Subjects suspected of having Alzheimer's disease

[0257] This involves an accurate and rapid diagnostic test for people suspected (but not yet diagnosed) of having a neurodegenerative disease, i.e., those who may be in the pre-symptomatic stage (Braak I or II). Such tests will be compared with the cost, frequency, and efficacy of currently deployed cognitive test methods (e.g., after a patient is referred to a memory clinic).

[0258] 3) Asymptomatic detection - Subjects not suspected of having Alzheimer's disease

[0259] For people who are currently not suspected of having a neurodegenerative disease, i.e., those who are young and / or pre-symptomatic (Braak I or II), it would be useful to provide a convenient test. The opportunity for pre-symptomatic testing can be carried out at a general practitioner's clinic or by a nurse or even at home to diagnose neurodegenerative diseases. For example, LFT tests can be routinely performed in a specific age group or in people above a certain age (or recommended or required by an employer or the NHS, etc.), similar to breast cancer or bowel cancer screening. Then, based on the results, they may be referred to a doctor or a specialist.

[0260] 4) Drug development and clinical trials

[0261] If the disease progression can be accurately measured, pharmaceutical companies will be able to save time and costs in drug development.

[0262] In terms of time scale, smaller sample groups, and other value metrics, since patients will effectively serve as their own controls, as the degree to which their condition has deteriorated or not deteriorated since early testing (e.g., the previous day, week, or month) can be monitored.

[0263] Conclusion

[0264] The inventors have demonstrated that in the pre-symptomatic stage of Alzheimer's disease, the level of T14 in the brain is elevated, and thus this characteristic is translated into detection in peripheral tissues (such as saliva / nasal fluid) as a reliable basis for early diagnosis of Alzheimer's disease. Therefore, lateral flow can be used as a rapid and convenient means for detecting T14 for diagnosis.

[0265] The LFT format can be inhibitory LFT or sandwich LFT, as Figure 4 , 5 , shown in 9, 10, and 11. The antibody successfully binds to 40nm gold colloids. The antigen can bind to the membrane in both inhibitory and sandwich forms. When the amount of peptide on the membrane decreases, a decrease in the signal intensity in the test line region can be seen, indicating specific binding.

[0266] Therefore, this state-of-the-art pre-symptomatic test can not only be used as a diagnostic monitor but also as a prognostic test. In addition, the inventors believe that the methods and devices described herein can be used to diagnose a subject before any dementia symptoms occur (i.e., in the pre-symptomatic stage) and to determine when symptoms may occur in the future. After detecting T14 in the pre-symptomatic stage, the subject can immediately implement therapeutic interventions (such as medications or lifestyle changes), and then use the methods and devices as prognostic measurements to monitor and evaluate the efficacy of the medications.

Claims

1. A lateral flow method for diagnosing or prognosticating a neurodegenerative disease in a subject, the method comprising using lateral flow to detect a peptide comprising SEQ ID No:3 (T14) or a variant or fragment thereof or a peptide consisting of SEQ ID No:3 (T14) or a variant or fragment thereof in a sample obtained from a test subject, wherein, The detection of a peptide in a sample that comprises SEQ ID No:3 (T14) or a variant or fragment thereof, or a peptide consisting of SEQ ID No:3 (T14) or a variant or fragment thereof, indicates that the tested subject has a neurodegenerative disease, or is predisposed to a neurodegenerative disease or has a negative prognosis thereof.

2. A lateral flow device for diagnosing or prognosticating a neurodegenerative disease in a subject, the device comprising a lateral flow support for detecting a peptide comprising SEQ ID No:3 (T14) or a variant or fragment thereof or a peptide consisting of SEQ ID No:3 (T14) or a variant or fragment thereof in a sample obtained from a test subject, wherein the detection of the presence of a peptide comprising SEQ ID No:3 (T14) or a variant or fragment thereof or a peptide consisting of SEQ ID No:3 (T14) or a variant or fragment thereof corresponds to a subject suffering from a neurodegenerative disorder, or being predisposed to a neurodegenerative disease or having a negative prognosis.

3. The method or device according to claim 1 or claim 2, comprising prognosticating the progression of a neurodegenerative disease.

4. The method or device according to any one of the preceding claims, comprising diagnosing a neurodegenerative disease.

5. The method or device according to any one of the preceding claims, comprising diagnosing a pre-symptomatic condition of a test subject.

6. The method or device according to any one of the preceding claims, comprising conducting a clinical trial to monitor the activity or efficacy of a drug administered to a test subject, and preferably determining the extent to which the subject has deteriorated or not deteriorated since a previous test.

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

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

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

10. The method or device according to any one of the preceding claims, wherein, The sample is nasal secretion or nasal fluid, saliva, blood, venous blood, arterial blood, plasma, serum, capillary blood, non-venous blood, non-arterial blood, finger prick blood, spinal fluid, urine, sweat, tear, breast aspirate, prostatic fluid, semen, vaginal fluid, feces, cervical smear, cell, amniotic fluid, intraocular fluid, mucus, respiratory moisture, animal tissue, cell lysate, tumor tissue, hair, skin, buccal smear, lymph interstitial fluid, nail, bone marrow, cartilage, prion, bone meal, earwax, or a combination thereof.

11. The method or apparatus according to claim 10, wherein, The sample comprises nasal secretion or nasal fluid.

12. The method or apparatus according to claim 10, wherein, The sample comprises saliva.

13. The method or apparatus according to claim 10, wherein, The sample includes capillary blood or finger prick blood.

14. The method or apparatus according to any one of the preceding claims, comprising inhibiting a lateral flow assay.

15. The method or apparatus according to any one of the preceding claims, comprising a sandwich lateral flow test.

16. The method or apparatus according to any one of the preceding claims, wherein, The device comprises an LFT cassette that includes a sample pad on which the sample is placed, and wherein the LFT cassette includes an anti-T14 antibody, which is optionally labeled with a labeling moiety (such as gold nanoparticles).

17. The method or apparatus according to claim 16, wherein, The LFT cassette comprises a fixed T14 peptide test line that comprises a T14 peptide linked thereto, and / or wherein the LFT cassette comprises a control line that comprises an anti-species labeled antibody that is not specific for T14.

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

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

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

21. The method or apparatus according to claim 20, wherein the LFT cassette contains a control line that includes an anti-species labeled antibody that is not specific for T14.

22. The method or apparatus according to claim 20 or 21, wherein, When the sample contains the T14 peptide, it is captured by the first anti-T14 antibody, thereby producing a T14-antibody conjugate, which conjugate itself is captured by a second anti-T14 antibody linked to the cassette along the test line, and any additional T14 antibody not bound to the T14 peptide in the sample flows and binds to the anti-species control fixed on the control line.

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

24. The method or apparatus according to any one of the preceding claims, wherein, The method or device is qualitative, semi-quantitative or quantitative.

25. The method or device according to any one of claims 16 - 24 includes a device for quantitatively measuring the result of an LFT cassette. Optionally, the lateral flow cassette is suitable for connection to a reader, computer, tablet, or smartphone, which operates to detect test and control lines and calculates the concentration of the peptide of SEQ ID No:3 or its variant or fragment in the sample based on the values of the concentration / test and control lines.

26. A method for diagnosing or prognosticating a neurodegenerative disorder in a subject, the method comprising detecting a peptide comprising SEQ ID No:3 (T14) or its variant or fragment or consisting of SEQ ID No:3 (T14) or its variant or fragment in a nasal secretion, nasal fluid, or finger prick blood sample obtained from a test subject, wherein detecting the presence of a peptide comprising SEQ ID No:3 or its variant or fragment or detecting a peptide consisting of SEQ ID No:3 or its variant or fragment in the sample indicates that the test subject has or is predisposed to a neurodegenerative disease or has a negative prognosis for it.

27. A device for diagnosing or prognosticating a neurodegenerative disorder, for diagnosing or prognosticating a neurodegenerative disorder in a subject, the device comprising means for detecting a peptide comprising SEQ ID No:3 (T14) or its variant or fragment or consisting of SEQ ID No:3 (T14) or its variant or fragment in a nasal secretion, nasal fluid, or finger prick blood sample obtained from a test subject, wherein detecting a peptide comprising SEQ ID No:3 (T14) or its variant or fragment or consisting of SEQ ID No:3 (T14) or its variant or fragment corresponds to a subject having or being predisposed to a neurodegenerative disorder or having a negative prognosis for it.

28. The method or device according to claim 26 or claim 27 includes an assay suitable for detecting the presence of a peptide of SEQ ID No:3 or its variant or fragment in a sample.

29. The method or device according to claim 28, wherein, The assay comprises a lateral flow, immunoassay, non-immunoassay, western blot analysis, enzyme-linked immunosorbent assay (ELISA), fluorescence assay, chemiluminescence assay or radioimmunoassay analysis.

30. The method or device according to claim 29, wherein, The assay comprises a lateral flow.

31. The method or device according to any one of claims 26 - 30, wherein the assay (i) measures the concentration of a soluble peptide comprising SEQ ID No:3 or its variant or fragment or consisting of SEQ ID No:3 or its variant or fragment, and / or (ii) measures the concentration of an aggregated peptide comprising SEQ ID No:3 or its variant or fragment or consisting of SEQ ID No:3 or its variant or fragment.

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

33. A method or apparatus according to any one of claims 9 - 25 or claim 32, wherein the antibody or antigen - binding fragment thereof specifically binds to SEQ ID NO:3, optionally binds to one or more amino acids in SEQ ID No:11, and preferably wherein 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. A method or apparatus according to any one of the preceding claims, wherein, The concentration of SEQ ID No:3 or a variant or fragment thereof in the sample is: (i) between 0.1 and 1000 ng, or between 0.2 and 750 ng, or between 0.5 and 500 ng per milligram of protein in the sample; (ii) between 1 and 400 ng, or between 2 and 300 ng, or between 3 and 200 ng per milligram of protein in the sample; and / or (iii) between 4 and 100 ng, or between 5 and 75 ng, between 10 and 50 ng, or between 20 and 40 ng per milligram of protein in the sample.

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

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

37. A method or apparatus according to any one of the preceding claims, wherein, These methods and devices are for the following subjects: (i) Persons who have been diagnosed with a neurodegenerative disease; (ii) Those with symptoms and showing signs of cognitive decline or dementia; and / or (iii) Persons who are not suspected of having a neurodegenerative disease and are pre-symptomatic.

38. A method or apparatus according to any one of the preceding claims, comprising detecting a soluble and / or aggregated peptide comprising any one of or consisting of one or more of T7 - T13 (i.e., SEQ ID No:4 - 10).

39. A method or apparatus according to any one of the preceding claims, comprising determining the Braak stage of the subject.

40. A method or apparatus according to claim 39, wherein, The subject is in any of Braak I, II or III stages, preferably Braak I or II stage.

41. A method or apparatus according to claim 39 or 40, comprising: (a) analyzing the concentration of a peptide comprising SEQ ID No:3 (T14) or a variant or fragment thereof or consisting of SEQ ID No:3 (T14) or a variant or fragment thereof in a sample obtained from a test subject; and (b) Compare this concentration with a reference value of the concentration of SEQ ID No: 3 or its variant or fragment or a soluble peptide consisting of SEQ ID No: 3 or its variant or fragment from a control group of deceased subjects with a known Braak stage; wherein the Braak stage of a living test subject is determined by comparing the concentration of a peptide comprising SEQ ID No: 3 or its variant or fragment or consisting of SEQ ID No: 3 or its variant or fragment with the corresponding reference value associated with the Braak stage.

42. The method or device according to claim 41, wherein, Soluble SEQ ID No:3 (T14) or its variant or fragment is analyzed.

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

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

45. The method or device according to any one of the preceding claims, comprising using a lateral flow assay to detect SEQ ID No: 3 or its variant or fragment in nasal secretions, nasal fluid or saliva for the diagnosis or prognosis of pre - symptomatic Alzheimer's disease.

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