CN111323597A

CN111323597A - Methods, kits and methods of screening compounds for detecting MCI and/or AD in a subject

Info

Publication number: CN111323597A
Application number: CN201811534804.4A
Authority: CN
Inventors: 陈志成
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-12-14
Filing date: 2018-12-14
Publication date: 2020-06-23

Abstract

The invention discloses methods, kits and methods of screening compounds for detecting MCI and/or AD in a subject. A method of detecting mild cognitive impairment, MCI, and/or alzheimer's disease, AD, in a subject comprising: (1) a step of measuring the content of tau N-terminal fragment in a blood sample collected from a subject using a reagent to obtain a measurement value; (2) a step of comparing the measured value with a standard value; (3) when the measured value is higher than the standard value, the subject is detected as having, or predicted to be at risk for having, mild cognitive impairment MCI and/or alzheimer's disease AD.

Description

Methods, kits and methods of screening compounds for detecting MCI and/or AD in a subject

Technical Field

The present invention relates to the detection of mild-moderate cognitive impairment and/or alzheimer's disease, in particular to methods, kits and methods of screening compounds for the detection of MCI and/or AD in a subject.

Background

However, recent clinical drug studies show an urgent need for more effective clinical diagnostic tools and an urgent need to develop biomarkers responsive to treatment.measuring tau and β -amyloid in CSF, or quantifying amyloidosis or tangles by PET imaging enables identification of Mild Cognitive Impairment (MCI), which is generally considered a precursor of AD.

Numerous studies have shown an increase in tau protein in the CSF of AD. Typical assays are performed using monoclonal antibodies directed against the distinct regions of tau protein, but are often inaccurate as total tau protein assays. In fact, such assays (including the commonly used Innotest ELISA) are unable to detect tau protein fragments lacking the entire middle region or a portion thereof. In addition, more and more studies indicate that the primary structure of extracellular tau proteins is highly heterogeneous (see reports from Johnson et al 1997, Meredith et al 2013, Kanmert et al 2015, and Guix et al 2018).

Recent studies have revealed that small amounts of tau protein are detectable in human plasma (see, Yanamandra K et al, Anti-tau antibody administration in peptides proteins with tau opath. Sci Transl Med 2017; 9 (386)). Some studies have also revealed that certain types of Plasma tau proteins may increase in AD, but the results of these studies are contradictory due to the different populations at the time of the study (see Mattsson N et al, Plasma tau in Alzheimer disease. neurology 2016; 87(17): 1827-35).

Disclosure of Invention

The inventors have developed a sensitive and well-validated set of tau immunoassay systems that can be used to detect various tau fragments found in blood. The inventors, after analyzing different pre-classified diagnostic groups, found that blood NT1 in the sample pool used for the study and the sample pool used for the validation enabled good differentiation of AD and AD-MCI from normal controls. These results indicate that the NT1 assay can be used as a blood-based screening method for AD. The present invention has been accomplished, at least in part, based on this. Specifically, the present invention includes the following.

In a first aspect of the invention, there is provided a method of detecting mild cognitive impairment, MCI, and/or alzheimer's disease, AD, in a subject, comprising:

(1) a step of measuring the content of tau N-terminal fragment in a blood sample collected from the subject using a reagent to obtain a measurement value;

(2) a step of comparing the measured value with a standard value;

(3) when the measurement value is higher than the standard value, detecting the subject as suffering from mild cognitive impairment MCI and/or Alzheimer's disease AD, or predicting the subject as suffering from risk of mild cognitive impairment MCI and/or Alzheimer's disease AD.

Preferably, the N-terminal fragment of tau protein comprises the sequence between positions 6-198, preferably between positions 6-18, of the full-length sequence of tau protein.

Preferably, the standard value is a value obtained by the same method from a blood sample of a normal subject equivalent to the age of the subject.

Preferably, the standard value is measured as the amount in a blood sample T1 taken from the subject at a first point in time and the measured value is measured as the amount in a blood sample T2 taken from the same subject at a second point in time.

Preferably, the standard value is 3.00 ± 0.25 pg/ml.

Preferably, the measurement of the measured value and/or the standard value is performed using a single molecule array technique, optionally the reagent is an antibody.

In a second aspect of the invention, there is provided a method for determining the effectiveness of a treatment of mild cognitive impairment, MCI, and/or alzheimer's disease, AD, in a subject, comprising:

(1') a step of measuring the content of tau N-terminal fragments in a blood sample taken from the subject during or after treatment with an agent to obtain a measurement value, preferably a measurement of a measurement value and/or a standard value using a single molecule array technique;

(2') a step of comparing the measured value with a standard value, preferably, measuring the content of tau N-terminal fragment in a blood sample taken from the subject before the start of the treatment as a standard value; also preferably, the standard value is a value obtained from a blood sample of a subject comparable to the age of the subject, e.g. the standard value is 3.00 ± 0.25 pg/ml;

(3') when the measured value is higher than the standard value, the treatment is judged to be effective, and when the measured value is lower than the standard value, the treatment is judged not to be effective.

In a third aspect of the invention, there is provided a method for screening a compound useful for treating or alleviating mild intermediate cognitive impairment, MCI, and/or alzheimer's disease, AD, comprising:

a. a step of measuring the content of tau N-terminal fragments in a blood sample taken from a subject suffering from mild-moderate cognitive impairment MCI and/or alzheimer's disease AD to obtain a first measurement value;

b. a step of administering the compound to a subject suffering from mild-moderate cognitive impairment, MCI, and/or alzheimer's disease, AD;

c. a step of measuring the content of tau N-terminal fragments in a blood sample taken from a subject suffering from mild-moderate cognitive impairment MCI and/or alzheimer's disease AD after administration of said compound to obtain a second measurement value;

d. a step of comparing the first measurement value and the second measurement value;

e. screening the compound for a compound useful for treating or ameliorating mild intermediate cognitive impairment (MCI) and/or Alzheimer's Disease (AD) when the second measurement is less than the first measurement, and screening the compound for a compound not useful for treating or ameliorating mild intermediate cognitive impairment (MCI) and/or Alzheimer's Disease (AD) when the second measurement is greater than or equal to the first measurement.

In a fourth aspect of the invention, there is provided a kit for detecting mild cognitive impairment, MCI, and/or alzheimer's disease, AD, in a subject, comprising reagents for measuring the amount of an N-terminal fragment of tau in a blood sample taken from the subject.

Preferably, the N-terminal fragment of tau comprises the sequence between positions 6 and 198 of the full-length sequence of tau and the agent comprises an antibody directed against the N-terminal fragment of tau.

There are inaccuracies in the diagnosis of AD, with a large number of individuals diagnosed with increasing cognitive impairment by clinical criteria, but the results have not progressed to AD (Monsell et al, 2015; FDA, 2018). The present approach minimizes this problem and excludes cognitively intact controls that may have precursors to incipient AD.

Drawings

FIG. 1 is a standard curve for ELISA detection of tau in the middle region.

FIG. 2 Standard Curve for ELISA detection of NT1tau protein.

FIG. 3 Standard Curve for ELISA detection of NT2tau protein.

FIG. 4 Standard Curve for ELISA detection of full-length tau protein.

FIG. 5 Standard curves for each protein type obtained by the Simoa technique.

FIG. 6 results of FL Simoa analysis of tau protein in normal controls and subjects with AD-MCI and AD.

FIG. 7NT2Simoa results from analysis of tau protein in normal controls and subjects with AD-MCI and AD.

FIG. 8 tau protein levels measured in AD-MCI and AD were analyzed using NT 1.

FIG. 9 ROC analysis performed on plasma NT1 of AD and/or AD-MCI.

Fig. 10 repeat analysis of plasma NT1 collected from 87 subjects.

Fig. 11ROC analysis results of the study to test the effectiveness of NT1 in plasma.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

Novel biomarkers of AD can be identified by detecting the type and amount of tau protein in human body fluids, and the role of tau in AD can be better understood. For more than 20 years, tau protein in CSF has been known to be elevated when AD occurs (Tato et al, 1995; Andreasen et al, 1998), but little attention has been given to the specific type of elevated tau protein and why the elevated phenomenon detected in AD CSF was not observed in other neurodegenerative diseases.

The average concentration of normal controls measured by conventional tau detection means is above 200 pg/ml. For example, the mean concentration of the normal control was 236. + -.23 pg/ml as determined by the innotest approach, 258. + -.19.5 pg/ml as determined by in-house median zone analysis, and higher for AD, 921. + -.96 pg/ml and 920. + -.124 pg/ml, respectively. The inventors have found that there are many different types of tau fragments in blood, and the content of these fragments is generally below 20 pg/ml. Thus, conventional tau detection means are not effective in detecting these tau fragments.

The inventors have exploited the technique of hypersensitivity immunoassay (Simoa technique, i.e. single molecule analysis) to explore low abundance types of tau protein in blood that could not be detected by traditional ELISA means. The hypersensitivity immunoassay technique of the present invention is capable of producing highly sensitive assays with LLoQs of less than 0.7 pg/ml. Using this technique, different types of tau protein fragments present in plasma can be detected.

The inventors found that in the control, tau protein levels measured by FL, NT1 and NT2 assays were comparable in range. This result is quite different from previous studies, suggesting that in the control equivalent amounts of plasma tau protein are full-length. Considering that neither the FL analysis nor the NT2 analysis gave more tau than the NT1 analysis, these measurements did not allow comparison of absolute amounts.

Nonetheless, when the results were compared to the diagnostic group, it was unexpectedly found that only the NT1 analysis showed a significant increase in disease association. Tau protein levels measured using NT2 analysis were elevated in AD/AD-MCI, but showed overlap with controls over a broad range. Tau protein levels measured by NT1 analysis were generally higher than NT2 or FL analysis at individual AD and AD-MCI levels. Indicating that an increase in a particular tau protein type was detected by NT1 analysis. In addition, existing studies have found that the levels of full-length tau protein do not change with the progression of AD.

The present invention has been completed based on the above-mentioned results. The present invention is described in detail below.

[ method for detecting Mild cognitive impairment MCI and/or Alzheimer's disease AD in a subject ]

In a first aspect of the invention, there is provided a method of detecting mild cognitive impairment, MCI, and/or alzheimer's disease, AD, in a subject, comprising at least:

(2) a step of comparing the measured value with a standard value;

Step (1)

Step (1) of the present invention is a step for obtaining a measured value of the content of an N-terminal fragment of tau protein in a blood sample. Wherein the agent is any agent that can be used to display the content or level of the N-terminal fragment of the tau protein to be tested. Preferably, the agent includes, but is not limited to, an antibody. Antibodies herein specifically encompass monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, such as variable domains and other portions of antibodies that exhibit the desired biological activity. The term "monoclonal antibody (mAb)" refers to an antibody that is highly specific and directed against a single antigenic determinant (epitope). Thus, the term "monoclonal" refers to an antibody to the same epitope and should not be construed as requiring the production of the antibody by any particular method. It is understood that monoclonal antibodies can be prepared by any technique or method known in the art; including, for example, the hybridoma method (Kohler et al, 1975, Nature256:495), or recombinant DNA methods known in the art (see, for example, U.S. Pat. No. 4,816,567), or methods using phage antibody libraries and isolating recombinantly produced monoclonal antibodies using techniques described in the following references: clackson et al, 1991, Nature 352: 624-; and Marks et al, 1991, J.mol.biol.222: 581-597.

In the present invention, an "antibody fragment" refers to a molecule that is different from an intact antibody, comprises a portion of an intact antibody, and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab') 2; a diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

The reagent of the present invention may comprise other components in addition to the above-mentioned antibody. Examples of the other components include ABC diluent, TMB color developing solution, TMB stop solution, washing solution, and the like. In certain embodiments, any one of the above substances may be separately present in a state separated from the other substances, and stored in different containers (e.g., vials), as long as they are capable of contacting each other at the time of use. In addition, preferably, any two or more of the above-mentioned substances may be mixed to exist as a mixture.

In certain embodiments, the other ingredients may be provided separately in the form of a dry powder, or in the form of a solution, e.g., an aqueous solution. The concentrations or contents of these substances, in the case of their presence in aqueous solution, are readily determinable by the person skilled in the art as a function of the various requirements. For example, for storage purposes, the concentration of the substance may be present in a higher form, and when in service or in use, the concentration may be reduced to a working concentration by, for example, diluting the higher concentration solution.

The reagent of the invention can be further prepared into a diagnostic agent for detecting Mild Cognitive Impairment (MCI) and/or Alzheimer's Disease (AD) in a subject. The diagnostic agent can be in the form of a diagnostic composition, a diagnostic kit, or any other form in which a plurality of separately present reagents are used in combination.

The sample of the invention is a blood sample in which the profile of tau protein is quite different from the profile in a CSF sample. Generally, tau protein content is lower and the type of tau protein is different in blood samples. In the present invention, the blood sample includes plasma, serum, whole blood and processed products thereof. Preferably plasma.

In the present invention, the "N-terminal fragment of tau protein" (sometimes referred to herein simply as N-terminal fragment, or NT) refers to a partial amino acid sequence located on the N-terminal side of full-length tau protein. The N-terminal fragment of tau protein refers to a fragment starting from amino acid N of the full-length sequence, wherein N is a natural number between 1 and 50. The length of the N-terminal fragment is not particularly limited, and may be generally 5 to 250 amino acids, preferably 10 to 200 amino acids, and more preferably 50 to 180 amino acids. In certain embodiments, the N-terminal fragment of the invention includes the sequence between positions 6-198 of the full-length sequence of tau protein (sometimes referred to herein simply as NT 1).

In the present invention, the measurement of the content of the "N-terminal fragment of tau" can be obtained by a known method. For example, by plotting a standard curve. In an exemplary embodiment, the content of the N-terminal fragment of tau protein is relative content, and the content of the N-terminal fragment of tau protein is detected using an antibody directed to the N-terminus and taken as the content of the N-terminal fragment of tau protein. In further exemplary embodiments, the content of the N-terminal fragment of tau is determined by using an antibody directed against the N-terminus, while the content of full-length tau is determined by using an antibody directed against full-length tau, and the content of the N-terminal fragment of tau as described herein is determined by subtracting the content of full-length tau from the content of the N-terminal fragment of tau.

In the present invention, tau proteins and fragments thereof are preferably detected by highly or ultrasensitive detection means. Highly sensitive or ultrasensitive detection means are known in the art, for example, measurement of measured values and/or standard values can be performed using single molecule array (Simoa) technology.

Step (2)

Step (2) of the present invention is a step of comparing the measured value with a standard value. The standard value may be a specific value or a range of values.

In certain embodiments, a standard value can be a blood sample test value from a normal subject. Preferably, the values of the blood sample from a normal subject of an age comparable to that of the subject to be tested, and also preferably, the standard values and the measured values are obtained by the same method.

In certain embodiments, the standard values are from different time periods of the same subject. For example, the amount in the blood sample T1 taken from the subject at a first time point is measured as the standard value and the amount in the blood sample T2 taken from the same subject at a second time point is measured as the measured value. Wherein the first time point T1 is preferably the time point before any sign of mild moderate cognitive impairment MCI and/or alzheimer's disease AD is not shown or present, the second time point T2 is the time point at which detection takes place.

In certain embodiments, a standard value of the invention is a specific value, for example, 3.00 ± 0.25 pg/ml. More preferably, the standard value may be set to 2.88pg/ml for AD-MCI and to 3.10pg/ml for AD.

Step (3)

Step (3) of the present invention is a result determination step. Specifically, when the measured value is higher than the standard value, the subject is detected as having, or predicted to be at risk of having, mild cognitive impairment MCI and/or alzheimer's disease AD. Also preferably, when the measured value is lower than or equal to the standard value, the subject is detected as not having mild cognitive impairment MCI and/or alzheimer's disease AD, or the subject is predicted as not having a risk of having mild cognitive impairment MCI and/or alzheimer's disease AD.

[ method for determining the effectiveness of treatment of Mild cognitive impairment MCI and/or Alzheimer's disease AD in a subject ]

In a second aspect of the invention, there is provided a method for determining the effectiveness of a treatment of mild cognitive impairment, MCI, and/or alzheimer's disease, AD, in a subject, sometimes referred to simply as "method of determining the effectiveness of a treatment". As mentioned above, since changes in the amount of the N-terminal fragment of tau are associated with the progression of the disease, the effectiveness of treatment of mild cognitive impairment MCI and/or alzheimer's disease AD can be determined by measuring the amount of the N-terminal fragment of tau. Specifically, the following steps may be included:

(2') a step of comparing the measured value with a standard value, preferably, measuring the content of tau N-terminal fragment in a blood sample taken from the subject before the start of the treatment as a standard value; also preferably, the standard value is a value obtained from a blood sample comparable to the age of the subject, e.g. the standard value is 3.00 ± 0.25 pg/ml;

[ method for screening for Compounds useful for treating or alleviating Mild-Medium cognitive impairment MCI and/or Alzheimer's disease AD ]

In a third aspect of the invention, there is provided a method, sometimes referred to herein simply as a "screening method", for screening compounds useful for the treatment or alleviation of mild-moderate cognitive impairment, MCI, and/or alzheimer's disease, AD. Preferably, the method may comprise:

In the screening method of the present invention, the subject is preferably an animal model, for example, rat, mouse, dog, pig, monkey, chimpanzee, etc., having mild-moderate cognitive impairment MCI and/or alzheimer's disease AD. Such animals may be artificially induced to suffer from mild-moderate cognitive impairment and/or alzheimer's disease.

[ kit for detecting Mild cognitive impairment MCI and/or Alzheimer's disease AD in a subject ]

In a fourth aspect of the invention, there is provided a kit for detecting mild cognitive impairment, MCI, and/or alzheimer's disease, AD, in a subject, comprising reagents for measuring the amount of an N-terminal fragment of tau in a blood sample taken from the subject. The reagents of the kit of the invention may be those described in the first aspect. And will not be described in detail herein.

Examples

Reagent

The reagents used in the present invention are commercially available unless otherwise specified.

The antibodies and sources used in the present invention are shown in table 1 below.

TABLE 1 antibodies and information related thereto

Preparation of recombinant tau protein

Coli BL21(DE3) was transformed with pNG2/htau40, pNG2/N-terminal tau and pNG2/C-terminal tau. Reference is made to Barghorn S et al, Purification of Recombinant Tau Protein and preparation of Alzheimer-Pair viral fibers In vitro, Sigurdsson E, edition, Amyloid Proteins Humana Press; p.35-51 and O 'Down ST et al The ELISA-Measured incorporated in a mesoporous fluidized bed catalysts Alzheimer's diseases from other neurological compositions Recognition of Tau assembled molecular diagnostics J Alz Dis 2013; 33(4) 923-8 to express and purify the protein. Protein purity was determined by SDS-PAGE/silver staining and high performance liquid chromatography. Extinction coefficients predicted by measuring absorbance at 280nm and using various tau protein structures (ε for human tau441, NT and CT respectively₂₈₀7575, 4470 and 3105M^-1cm^-1) To determine the concentration.

Analysis of mid-region tau protein

Reference is made to Kanmert D, Cantlon A, Muratore CR, Jin M, O' Malley TT, Lee G, et al.C-Terminally Truncated Forms of Tau, But Not to Full-Length Tau or Its C-terminalFragments, and Are derived from nerves Independently of Cell Death.JNeurosci 2015; 35(30) 10851-65, the tau protein of the middle section region is analyzed. Specifically, a 96-well plate was coated with 25. mu.l of TBSpH7.4 per well, wherein 2.5. mu.g/ml of BT2 antibody was contained in TBS. Incubate at 37 ℃ for 1 hour. All steps were performed at room temperature unless otherwise indicated. The cells were washed 3 times with TBS (TBS-T) containing 0.05% Tween20 and then blocked with TBS containing 3% (w/v) BSA for 2 hours while shaking at 300rpm by an orbital shaker. After that, the reaction was washed 3 times with TBS-T. Each sample was analyzed twice (25. mu.l/well), while the blank and tau441/tau381 standards (7.8-8000pg/ml) were analyzed three times. Standards and samples were diluted with Simoa tau dilution reagent: 1 volume buffer plus 1 volume sample. All samples were incubated overnight at 4 ℃. The next day, the plates were shaken on an orbital shaker for 1 hour to bring the temperature back to room temperature. Subsequently, 25. mu.l of 1% (w/v) BSA in Tau-alkaline phosphatase TBS-T was added to each well and incubated for 1 hour while shaking on an orbital shaker. Each well was washed 5 times, 50. mu.l of a chemiluminescent substrate (Tropix CDP-Star Sapphire II; Applied Biosystems, Foster city, Calif.) was added, and incubated for 30 minutes with shaking. Chemiluminescence values were measured using a Synergy H1plate reader (Biotek, Winooski, VT). And a standard curve was prepared. The lower limit of quantitation (LLoQ) was defined as the minimum standard with a signal above the blank mean signal +9SDs, allowing percent recoveries equal to or greater than 100 ± 20% and Coefficient of Variation (CV) equal to or less than 20%. The LLoQ of each panel was calculated and ranged from 15.6-31.3 pg/ml. The standard curve is shown in figure 1.

NT1tau ELISA

The same procedure as for the mid-region Tau ELISA was used for NT1Tau ELISA, except that anti-Tau mAb, Tau12 was used for capture and BT-2 alkaline phosphatase was used for detection. The LLoQ of each panel was calculated to be in the range of 15.6-62.5 pg/ml. The standard curve is shown in figure 2.

NT2tau ELISA

The same procedure as for the NT1ELISA was used for the second N-terminal Tau protein (NT2) except that anti-Tau mAb, Tau12 was used for capture and ADx202 alkaline phosphatase was used for detection. The LLoQ of each panel was calculated to be in the range of 15.6-31.3 pg/ml. The standard curve is shown in fig. 3.

full-Length (FL) tau ELISA

The ELISA referred to herein is Guix FX, Corbett GT, Cha DJ, Mustapic M, Liu W, Menel D, et. detection of Aggregation-competition Tau in Neuron-Derived excellen vehicles. int J Mol Sci 2018; 19(3) and the same procedure as the other ELISA described above was used except that Tau12 was used for capture and Tau ab alkaline phosphatase was used for detection. The LLoQ of each panel was calculated to be in the range of 7.8-31 pg/ml. The values obtained for a particular sample set are noted in the results and the standard curve is shown in figure 4.

II, analyzing:

the capture antibodies TauAB (435-. ADx202 recognizes an epitope similar to Tau5, and ADx202 performed better than Tau5 in the preliminary Simoa assay, thus using ADx202 instead of Tau5 in the NT2Simoa analysis. The detection antibodies (Tau12 and Tau5) were biotinylated using 40 parts of the biochemical reagent and 1 part of the antibody according to the manufacturer's recommendations.

Plasma samples were centrifuged for 4 minutes at 14000 and then diluted 1:4(1 volume sample +3 volumes diluent) with Tau 2.0 sample dilution reagent. The Tau381 standard was linearly diluted with Tau 2.0 sample dilution reagent to give a concentration range interval of 540-0.02 pg/ml. The sample, standard and blank were placed in a 1.5ml Eppendorf tube. In all experiments, samples, standards and blanks were analyzed in at least three replicates.

FL, NT1 and NT2 analyses were performed using a three-step method, the analyses were performed on a room temperature Simoa HD-1 analyzer (Quanterix corp., Lexington, MA)

step

1, 100 μ l of standard, blank or sample was added to the magnetic beads coated with capture antibody and mixed for 45 minutes, the magnetic beads were harvested and washed with buffer step 2, biotin detection antibody (0.6 μ g/ml) was added and incubated for 35 minutes, then the magnetic beads were washed three times step 3, 150pM streptavidin- β -galactosidase was added, followed by a further washing step, enzyme substrate, resorufin β -D-galactopyranoside was added and the complexes loaded onto the Simoa analyzer, which contained 216000 femto liter sized wells reference rin et, average enzyme units per magnetic bead (aev) wt. 2010, the average enzyme concentration per magnetic bead was determined as a function of the curve for each standard curve calculated for 0.5, 25, 5, 25, 2, g, of the standard curve for each.

Third, verification analysis

Test immunoassay response to sample dilution and recovery of "spiked" recombinant tau protein. Plasma samples were diluted from 1:2 to 1:8 in Tau sample dilution buffer. In a spiking assay, known amounts of recombinant NT tau and/or FLtau were added to samples diluted from 1:2 to 1: 8. The concentration of endogenous tau protein and the concentration of exogenous tau protein spiked to buffer in samples diluted before and after spiking were measured and% recovery was calculated using the formula:

recovery% (concentration of sample spiked with tau/(concentration of sample alone + concentration spiked with tau)) × 100.

Four different recombinant tau constructs, FL tau (381 and 441), NT tau and CT tau were used and tested for analyte specificity of Simoa analysis by immunodepleting the samples with anti-tau mAb. For immunoprecipitation assays, Tau5 or control antibody mAb 46-4 was conjugated to paramagnetic tosyl activated M280 magnetic beads (Thermo Scientific). mAb18 hours in 0.1M sodium phosphate (pH7.4) was incubated in the presence of 1M ammonium sulfate. The beads were then blocked with 0.5% BSA in 0.01M sodium phosphate buffer (pH7.4, containing 0.15M sodium chloride) PBS at 37 ℃ for 1 hour and stored in 0.1% BSA at 4 ℃. After conjugation, the concentration of mAb in the supernatant was determined and used to calculate the amount of antibody conjugated to the magnetic beads. Plasma samples (0.5ml) were incubated with Tau or 46-4 conjugated to magnetic beads at 4 ℃ for 24 hours at a final antibody concentration of 10. mu.g/ml. The magnetic beads were collected and the supernatant was subjected to a second round of antibody treatment. The supernatants were then harvested and analyzed.

Fourthly, the result

1. Elevated plasma NT1 was found in AD-MCI and AD subjects in the study group

A reliable and hypersensitive Simoa assay was established for N-terminal and full-length tau proteins. Plasma from the study group was used in the study. FL and NT2Simoa analysis of normal controls and subjects with AD-MCI and AD revealed no significant difference in tau between normal controls and diseased subjects (fig. 6 and 7), in contrast, tau protein levels measured using NT1 analysis were significantly elevated in AD-MCI and AD compared to both normal controls (fig. 8).

To further investigate the potential use of NT1 in plasma for the AD and/or AD-MCI screening assays, ROC analysis was performed (FIGS. 9A and 9B). The results showed that the plasma NT1 assay was able to distinguish the control from AD-MCI with an AUC of 0.88 (95% CI: 0.77-0.99).

To investigate the specificity and sensitivity of this discrimination, a Youden index was used for ranking. It was found that setting the threshold (cut-off value) to 3.4pg/ml gave excellent sensitivity (0.95; 95% CI:0.74-1.00) and specificity (0.76; 95% CI: 0.53-0.92). It was found that when AUC was 0.96 (95% CI:0.90-1.02) and the threshold was 3.07pg/ml, NT1 levels distinguished well between controls and AD patients and gave excellent sensitivity (0.95; 95% CI:0.74-1.00) and specificity (0.92; 95% CI: 0.74-0.99).

2. Elevated plasma NT1 was confirmed in AD-MCI and AD subjects in the validation group

Many potential markers of AD show promise in a single test group, but do not perform well when performed again in another group (see, e.g., Olsson et al, 2016.) to further validate the use of NT1 for the detection of AD or AD-MCI, the inventors performed a detection experiment on a completely different validation group using methods similar to section 1.

Plasma was collected from a total of 87 subjects and repeated experiments of samples yielded highly similar results with an R2 value of 0.84 (fig. 10A). The 86 of the 87 samples had replicate values within 20%, with only one sample varying by more than 20% (fig. 10A). These results indicate that the results of the NT1 analysis are very stable. Thus, the NT1 results were further analyzed based on the diagnostic panel. Including (i) day 1 results for all 87 samples, (ii) day 2 results for all 87 samples, (iii) mean of day 1 and day 2 results for 86 samples, thereby yielding a day-to-day variation of less than 20% (fig. 10B). In all cases, the level of NT1 was similar in the AD-MCI and AD groups, with these results being significantly elevated compared to the normal control group (fig. 10B). As with the study groups, we further investigated the effectiveness of NT1 in plasma by ROC analysis, with the results shown in fig. 11C and 11D. These results indicate that in the validation group, the plasma NT1 assay distinguished control from AD-MCI by an AUC of 0.794 (95% CI:0.62-0.88) and control from AD by an AUC of 0.750 (95% CI: 0.68-0.91).

To verify whether the threshold derived from the study group can be applied to the validation group, we again obtained the Youden index for the validation group and tested the threshold identified using the study group. A threshold of 3.07pg/ml, which resulted in excellent AD discrimination from the study group controls, was applied to the validation group, resulting in a specificity of 0.83 (95% CI:0.68-0.93) and a sensitivity of 0.65 (95% CI: 0.43-0.84). Similarly, a threshold of 3.40pg/ml, which yields excellent AD-MCI discrimination from the study group controls, was applied, yielding a specificity of 0.88(0.74-0.96), and a sensitivity of 0.45 (95% CI: 0.24-0.68).

A possible reason for the poor performance of the threshold when moving from study to validation was that the NT1 values for AD-MCI and AD were higher in the study compared to the validation (4.85vs.3.42pg/ml, and 5.12vs.3.40pg/ml), thus making the threshold of versatility difficult. But the Youden index analysis within the validation set produced very encouraging results. Setting a threshold of 2.88pg/ml resulted in very good sensitivity (0.73; 95% CI:0.50-0.89) and specificity (0.80; 95% CI:0.63-0.91), distinguishing the control from AD-MCI. Similarly, a threshold of 3.10pg/ml distinguishes controls from AD with good sensitivity (0.6; 95% CI:0.43-0.84) and specificity (0.85; 95% CI: 0.71-0.94). All these results indicate that the detection of tau protein in plasma using the NT1 assay is a potential marker for screening for AD-MCI and AD.

It is noted that sample collection and processing was performed in the same manner in both the study and validation groups, and that samples were subjected to the same number of freeze-thaw cycles in a given group.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

Claims

1. A method of detecting mild cognitive impairment, MCI, and/or alzheimer's disease, AD, in a subject, comprising:

(2) a step of comparing the measured value with a standard value;

2. A method of detecting Mild Cognitive Impairment (MCI) and/or Alzheimer's Disease (AD) in a subject according to claim 1, wherein the N-terminal fragment of tau comprises a sequence between positions 6-198 of the full-length sequence of tau.

3. A method of detecting Mild Cognitive Impairment (MCI) and/or Alzheimer's Disease (AD) in a subject according to claim 1, wherein the standard value is a value obtained from a blood sample of a normal subject of comparable age to the subject.

4. A method of detecting Mild Cognitive Impairment (MCI) and/or Alzheimer's Disease (AD) in a subject according to claim 1, wherein the amount in a blood sample T1 taken from the subject at a first point in time is measured as the standard value and the amount in a blood sample T2 taken from the same subject at a second point in time is measured as the measured value.

5. A method of detecting mild cognitive impairment, MCI, and/or alzheimer's disease, AD, in a subject according to claim 1, wherein the standard value is 3.00 ± 0.25 pg/ml.

6. A method of detecting Mild Cognitive Impairment (MCI) and/or Alzheimer's Disease (AD) in a subject according to any one of claims 1 to 5, wherein the measurement of the measured value and/or the standard value is performed using single molecule array technology, optionally the agent is an antibody.

7. A method for determining the effectiveness of a treatment of mild cognitive impairment, MCI, and/or alzheimer's disease, AD, in a subject, comprising:

(2') a step of comparing the measured value with a standard value, preferably, measuring the content of tau N-terminal fragment in a blood sample taken from the subject before the start of the treatment as a standard value; also preferably, the standard value is a value obtained from a blood sample of a normal subject comparable to the age of the subject, e.g. the standard value is 3.00 ± 0.25 pg/ml;

8. A method for screening a compound useful for treating or ameliorating mild intermediate cognitive impairment, MCI, and/or alzheimer's disease, AD, comprising:

9. A kit for detecting mild cognitive impairment, MCI, and/or alzheimer's disease, AD, in a subject comprising reagents for measuring the amount of tau N-terminal fragment in a blood sample taken from the subject to obtain a measurement.

10. A kit for detecting mild cognitive impairment MCI and/or alzheimer's disease AD in a subject according to claim 9, wherein the N-terminal fragment of tau comprises a sequence between positions 6-198 of the full-length sequence of tau and the agent comprises an antibody against the N-terminal fragment of tau.