Biofluid-based methods for diagnosing alzheimer's disease-associated conditions

Abstract

To provide accurate biofluid-based methods for diagnosis, prediction of onset, and assessment of progression of Alzheimer's disease.SOLUTION: The method comprises the steps of; (a) admixing (i) a suitably diluted sample of a suitable fluid from the subject and (ii) a suitable amount of a labeled molecule, wherein the labeled molecule is subject to a matrix effect with respect to the suitable fluid in a subject afflicted with the condition; and (b) after a suitable duration under suitable conditions, determining an amount of matrix-unaffected labeled molecule present in a resulting admixture, wherein the subject is afflicted with the condition if the amount of matrix-unaffected labeled molecule determined in the step (b) correlates with a positive control for the condition, and wherein the subject is not afflicted with the condition if that amount correlates with a negative control for the condition. Related methods, kits, and compositions are also provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 161,524, filed March 16, 2021, the contents of which are incorporated herein by reference.

[0002] Throughout this application, various publications are cited, the disclosures of which are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0003] FIELD OF THE INVENTION The present invention relates to certain methods for determining whether a subject has a condition that is correlated with matrix effects, which conditions may be diagnostic for certain disorders (e.g., Alzheimer's disease), may be prognostic, and / or may be correlated with disease progression for that particular disorder. [Background technology]

[0004] BACKGROUND OF THE INVENTION Alzheimer's disease is characterized by a continuous change in the severity of various pathologies, such as β-amyloid (Aβ) accumulation in the brain. Other Alzheimer's disease-related pathologies include, for example, tauopathy and synapse loss. Diagnostic and prognostic methods for Alzheimer's disease based on measuring various plasma Aβ peptides or (phospho)tau have been developed. However, these methods have produced conflicting results, and the absolute distinction between healthy control subjects and diseased subjects is limited. Therefore, there remains a need for accurate biofluid-based methods for diagnosing Alzheimer's disease, predicting the onset of Alzheimer's disease, and assessing the progression of Alzheimer's disease. Summary of the Invention [Means for solving the problem]

[0005] (Summary of the Invention) The present invention provides a first method for determining whether a subject has a condition correlated with a matrix effect, the method comprising: (a) mixing (i) an appropriately diluted sample of an appropriate fluid from the subject with (ii) an appropriate amount of a labeled molecule, where the labeled molecule is affected by a matrix effect with respect to the appropriate fluid in a subject suffering from the condition; and (b) after an appropriate period of time under appropriate conditions, determining the amount of unaffected labeled molecule present in the resulting mixture; wherein if the amount of unaffected labeled molecule determined in step (b) correlates with a positive control for the condition, the subject has the condition; and if the amount correlates with a negative control for the condition, the subject does not have the condition.

[0006] The present invention also provides a second method for determining whether a subject has a condition correlated with a matrix effect, the second method comprising: (a) mixing (i) an appropriately diluted sample of an appropriate fluid from the subject with (ii) appropriate amounts of a first labeled molecule and a second labeled molecule, where the labeled molecules are affected by a matrix effect with respect to the appropriate fluid in a subject suffering from the condition; and (b) after an appropriate period of time under appropriate conditions, determining a quantitative relationship between the matrix-unaffected first labeled molecule and the matrix-unaffected second labeled molecule present in the resulting mixture; wherein if the relationship determined in step (b) correlates with a positive control for the condition, the subject has the condition; and if the relationship correlates with a negative control for the condition, the subject does not have the condition.

[0007] The present invention further provides a first kit for use in determining whether a subject (preferably a human subject) has a condition correlated with matrix effects, the first kit comprising, in separate compartments: (a) a labeled molecule that is subject to matrix effects with respect to an appropriate fluid in a subject suffering from the condition; and (b) an agent useful for processing the labeled molecule to facilitate measurement of the labeled molecule in a form that is not subject to matrix effects.

[0008] The present invention still further provides a second kit for use in determining whether a subject (preferably a human subject) has a condition correlated with a matrix effect, the second kit comprising, in separate compartments: (a) a first labeled molecule and a second labeled molecule, each of which is affected by a matrix effect with respect to an appropriate fluid in a subject suffering from the condition; and (b) an agent useful for treating each of the first labeled molecule and the second labeled molecule to facilitate measurement of the labeled molecule in a form that is not affected by the matrix.

[0009] The present invention provides a first method for determining whether a human subject (either symptomatic or asymptomatic) has a condition correlated with Alzheimer's disease (i.e., the subject may actually have Alzheimer's disease or may not yet have Alzheimer's disease), comprising: (a) mixing (i) an appropriately diluted sample of a suitable fluid from the subject with (ii) an appropriate amount of labeled Aβ peptide; and (b) after an appropriate period of time under appropriate conditions, determining the amount of matrix-free, labeled Aβ peptide present in the resulting mixture; wherein if the amount of matrix-free, labeled Aβ peptide determined in step (b) correlates with a positive control for the condition, then the subject has the condition; and if the amount correlates with a negative control for the condition, then the subject does not have the condition.

[0010] The present invention also provides a second method for determining whether a human subject (either symptomatic or asymptomatic) has a condition correlated with Alzheimer's disease (i.e., the subject may actually have Alzheimer's disease or may not yet have Alzheimer's disease), comprising: (a) mixing (i) an appropriately diluted sample of a suitable fluid from the subject with (ii) appropriate amounts of a first labeled Aβ peptide and a second labeled Aβ peptide; and (b) after an appropriate period of time under appropriate conditions, determining a quantitative relationship between the matrix-unaffected first labeled Aβ peptide and the matrix-unaffected second labeled Aβ peptide present in the resulting mixture; wherein if the relationship determined in step (b) correlates with a positive control for the condition, the subject has the condition; and if the relationship correlates with a negative control for the condition, the subject does not have the condition.

[0011] The present invention further provides a first kit for use in determining whether a human subject has a condition correlated with Alzheimer's disease, the kit comprising, in separate compartments: (a) a labeled Aβ peptide; and (b) an agent useful for processing the labeled Aβ peptide to facilitate measurement of the labeled Aβ peptide in its matrix-free form.

[0012] The present invention still further provides a second kit for use in determining whether a human subject has a condition correlated with Alzheimer's disease, the kit comprising, in separate compartments, (a) a first labeled Aβ peptide and a second labeled Aβ peptide, and (b) an agent useful for processing each of the first labeled Aβ peptide and the second labeled Aβ peptide to facilitate measurement of the labeled Aβ peptides in their matrix-free form.

[0013] The present invention provides a first method for determining whether a human subject (either symptomatic or asymptomatic) has a condition correlated with Parkinson's disease (i.e., the subject may actually have Parkinson's disease or may not yet have it), the method comprising: (a) mixing (i) a suitably diluted sample of a suitable fluid from the subject with (ii) a suitable amount of labeled alpha-synuclein; and (b) after a suitable period of time under suitable conditions, determining the amount of matrix-free labeled alpha-synuclein present in the resulting mixture; if the amount of matrix-free labeled alpha-synuclein determined in step (b) correlates with a positive control for the condition, then the subject has the condition; and if the amount correlates with a negative control for the condition, then the subject does not have the condition.

[0014] The present invention also provides a second method for determining whether a human subject (either symptomatic or asymptomatic) has a condition correlated with Parkinson's disease (i.e., the subject may actually have Parkinson's disease or may not yet have it), the method comprising: (a) mixing (i) a suitably diluted sample of a suitable fluid from the subject with (ii) suitable amounts of a first labeled alpha-synuclein and a second labeled alpha-synuclein; and (b) after a suitable period of time under suitable conditions, determining a quantitative relationship between the matrix-unaffected first labeled alpha-synuclein and the matrix-unaffected second labeled alpha-synuclein present in the resulting mixture; if the relationship determined in step (b) correlates with a positive control for the condition, then the subject has the condition; and if the relationship correlates with a negative control for the condition, then the subject does not have the condition.

[0015] The invention further provides a first kit for use in determining whether a human subject has a condition correlated with Parkinson's disease, the kit comprising, in separate compartments: (a) labeled alpha-synuclein; and (b) an agent useful for processing the labeled alpha-synuclein to facilitate measurement of the labeled alpha-synuclein in its matrix-free form.

[0016] Finally, the present invention provides a second kit for use in determining whether a human subject has a condition correlated with Parkinson's disease, the kit comprising, in separate compartments, (a) a first labeled α-synuclein and a second labeled α-synuclein, and (b) an agent useful for treating each of the first labeled α-synuclein and the second labeled α-synuclein to facilitate measurement of the labeled α-synuclein in its matrix-free form. [Brief explanation of the drawings]

[0017] [Figure 1] This figure shows the results from Assay 1, described in Example 2 below. Results are presented as box plots for each study. Details regarding outcome parameters are provided in Table 1.

[0018] [Figure 2] This figure shows the results from Assay 2, described below in Example 3. Results are expressed as mean ± standard error (n=8).

[0019] [Figure 3] This figure shows the results from Assay 3, described below in Example 4. Results are shown as parts per million (ppm) values ​​for one sample at various dilutions of that sample. DETAILED DESCRIPTION OF THE INVENTION

[0020] (Detailed Description of the Invention) The present invention provides certain methods for determining whether a subject has a condition that is correlated with matrix effects. These matrix-related correlations may be diagnostic and / or prognostic for certain disorders, particularly neurodegenerative disorders.

[0021] (definition) In this application, certain terms are used that have the meanings indicated below.

[0022] As used herein, "Aβ peptide" includes, but is not limited to, peptides having the amino acid sequence shown in Example 5. Aβ peptides also include, but are not limited to, any fragment of Aβ1-37 peptide, Aβ1-38 peptide, Aβ1-40 peptide, Aβ1-42 peptide, Aβ1-43 peptide, Aβ1-10 peptide, Aβ1-16 peptide, Aβ(-3)-37 peptide, Aβ(-3)-38 peptide, Aβ(-3)-40 peptide, Aβ(-3)-42 peptide, Aβ(-3)-43 peptide, Aβ(-3)-10 peptide, or Aβ(-3)-16 peptide. These fragments include, for example, (i) N-terminal truncated fragments of Aβ1-37 peptide, N-terminal truncated fragments of Aβ1-38 peptide, N-terminal truncated fragments of Aβ1-40 peptide, N-terminal truncated fragments of Aβ1-42 peptide, N-terminal truncated fragments of Aβ1-43 peptide, N-terminal truncated fragments of Aβ1-10 peptide, and N-terminal truncated fragments of Aβ1-16 peptide; (ii) C-terminal truncated fragments terminating at Aβ37 position, C-terminal truncated fragments terminating at Aβ38 position, C-terminal truncated fragments terminating at Aβ40 position, C-terminal truncated fragments terminating at Aβ42 position, C-terminal truncated fragments terminating at Aβ43 position, C-terminal truncated fragments terminating at Aβ10 position, and C-terminal truncated fragments terminating at Aβ11 position. and (iii) fragments cleaved or extended at both the N- and C-terminus of Aβ37 peptides, fragments cleaved or extended at both the N- and C-terminus of Aβ38 peptides, fragments cleaved or extended at both the N- and C-terminus of Aβ40 peptides, fragments cleaved or extended at both the N- and C-terminus of Aβ42 peptides, fragments cleaved or extended at both the N- and C-terminus of Aβ43 peptides, fragments cleaved or extended at both the N- and C-terminus of Aβ10 peptides, and fragments cleaved or extended at both the N- and C-terminus of Aβ16 peptides.

[0023] Aβ peptide fragments contemplated herein include, for example, (i) Aβ37 peptide fragments having a length (in amino acid residues) of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36; (ii) Aβ37 peptide fragments having a length (in amino acid residues) of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 (iii) Aβ38 peptide fragments having a length (in amino acid residues) of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39. (iv) Aβ42 peptide fragments having a length (in amino acid residues) of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41; (v) Aβ42 peptide fragments having a length (in amino acid residues) of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 (vi) Aβ43 peptide fragments having a length (in amino acid residues) of 5, 6, 7, 8, or 9; and (vii) Aβ16 peptide fragments having a length (in amino acid residues) of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0024] Aβ peptide fragments contemplated herein also include, for example, the following, where the N-terminus of the fragment is defined as the corresponding amino acid residue number of its full-length counterpart Aβ peptide: (i) Aβ37 peptide having its N-terminus at residues 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 (ii) Aβ38 peptide fragments having their N-terminus at residues 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28; (iii) Aβ38 peptide fragments having their N-terminus at residues 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, (iv) an Aβ40 peptide fragment having its N-terminus at residues 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32; (v) an Aβ42 peptide fragment having its N-terminus at residues 2, 3, 4, 5, 6, 7, 8, 9, 10, (vi) an Aβ43 peptide fragment having its N-terminus at residue 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33; (vi) an Aβ10 peptide fragment having its N-terminus at residue 2, 3, 4, or 5; and (vii) an Aβ16 peptide fragment having its N-terminus at residue 2, 3, 4, 5, or 6. Aβ peptides and fragments thereof can optionally contain amino acid derivatives, such as pyroglutamic acid (e.g., pyroglutamic acid substitution at amino acid residues 3 and / or 11) (Gu and Viles (2016) and Rezaei-Ghaleh et al. (2016)).

[0025] As used herein, "conditions correlated with matrix effects" include amyloidopathies, tauopathies, neuronal damage, synucleinopathies, cerebral proteinopathies (e.g., These conditions include, but are not limited to, TDP-43 proteinopathy, progranulinopathy, and neuronal inflammation. The following conditions are contemplated in the present invention, along with the following proteins and peptides that, when labeled with matrix effects, are useful for carrying out the present method: (i) amyloidopathy (β-amyloid; BACE1 (amyloid precursor protein β-site cleaving enzyme); and APP (β-amyloid precursor protein)); (ii) tauopathy (tau and phosphorylated tau); and (iii) synucleopathy (α-synuclein; neurogranin; SNAP-25 (synaptosomal-associated protein 25); GAP-43 (growth-associated protein 43 or neuromodulin); synaptotagmin; VAMP2 (vesicle-associated membrane protein 2), and synaptobrevin). (iv) TDP-43 pathology (TDP-43 (transactivation response DNA-binding protein)); (v) neuroinflammation and glial activation (sTREM2 B36 (soluble triggering receptor expressed on myeloid cells 2); YKL-40 (chitinase 3-like protein 1); IP-10 (interferon-gamma-inducible protein 10); GFAP (glial fibrillary acidic protein); progranulin; osteopontin; MCP-1 (monocyte chemotactic protein 1); and IL-6 (interleukin 6)); (vi) vascular dysregulation (hFABP (heart-type fatty acid-binding protein)); and (vii) neuronal injury (VILIP-1 (visinin-like protein 1); and neurofilaments).

[0026] As used herein, the term "antibody" includes, but is not limited to, (a) immunoglobulin molecules comprising two heavy chains (i.e., H chains, e.g., μ, δ, γ, α, and ε) and two light chains (i.e., L chains, e.g., λ and κ) that recognize an antigen; (b) polyclonal and monoclonal immunoglobulin molecules; (c) monovalent and bivalent fragments thereof; and (d) bispecific forms thereof. Immunoglobulin molecules can be derived from any of the commonly known classes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art, and include, but are not limited to, human IgG1, human IgG2, human IgG3, and human IgG4. Antibodies can be both naturally occurring and non-naturally occurring. Furthermore, antibodies include chimeric antibodies, fully synthetic antibodies, single-chain antibodies (e.g., scFv), and fragments thereof. An antibody can, for example, include all or part of a constant region (e.g., Fc region) and a variable region, or can include only the variable region (responsible for antigen binding). The antibody can be human, humanized, or non-human (e.g., camelid) antibody.

[0027] As used herein, a "human subject" can be a subject of any age, sex, race, or comorbidity status. In one embodiment, the subject is male, and in another embodiment, the subject is female. In another embodiment, the subject is younger than 60 years old, younger than 55 years old, younger than 50 years old, younger than 45 years old, younger than 40 years old, younger than 35 years old, younger than 30 years old, younger than 25 years old, or younger than 20 years old. In another embodiment, the subject carries a genetic mutation (e.g., a mutation in APP) correlated with early onset of Alzheimer's disease. In yet another embodiment, the subject is at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, at least 80 years old, at least 85 years old, or at least 90 years old. In a further embodiment, the human subject has symptoms of a disorder (e.g., Alzheimer's disease or Parkinson's disease) associated with a condition detected using the present methods. In still further embodiments, the human subject is free of symptoms of a disorder associated with a condition detected using the present methods (eg, Alzheimer's disease or Parkinson's disease).

[0028] As used herein, "labeled" with respect to a molecule means covalently or non-covalently immobilized to a detectable moiety. In one embodiment, the detectable moiety is directly immobilized to the molecule (i.e., the detectable moiety is attached to the molecule without being attached through an intermediate moiety). In another embodiment, the detectable moiety is indirectly immobilized to the molecule (i.e., the detectable moiety is attached to the molecule through an intermediate moiety, such as a polypeptide or other type of linker). Detectable moieties include, for example, radioisotopes (e.g., 32 P, 35 S, and 125I), fluorophores, and other non-radioactive compounds (e.g., biotin). When the labeled molecule is a peptide or protein, for example, the label can be fixed to the N-terminus, C-terminus, central domain, or some or all of the amino acid residues of the peptide or protein. Labeled peptides and labeled proteins include, for example, labeled β-amyloid, labeled tau, labeled synuclein, and labeled TDP-43. For example, when the labeled peptide is biotin-labeled Aβ40 peptide, the biotin can be attached to the N-terminus, C-terminus, or central domain.Further examples of biotin-labeled peptides include: (i) N-terminally labeled Aβ37 peptide, C-terminally labeled Aβ37 peptide, and centrally labeled Aβ37 peptide; (ii) N-terminally labeled Aβ38 peptide, C-terminally labeled Aβ38 peptide, and centrally labeled Aβ38 peptide; (iii) N-terminally labeled Aβ42 peptide, C-terminally labeled Aβ42 peptide, and centrally labeled Aβ42 peptide; (iv) N-terminally labeled Aβ43 peptide, C-terminally labeled Aβ43 peptide, and centrally labeled Aβ43 peptide; (v) N-terminally labeled Aβ10 peptide, C-terminally labeled Aβ10 peptide, and centrally labeled Aβ10 peptide; (vi) N-terminally labeled α-synuclein, C-terminally labeled α-synuclein, and centrally labeled α-synuclein; (vii) N-terminally labeled Aβ42 peptide. (viii) N-terminally labeled TDP-43, C-terminally labeled TDP-43, and centrally labeled TDP-43; (ix) N-terminally labeled progranulin, C-terminally labeled progranulin, and centrally labeled progranulin; (x) N-terminally labeled GFAP, C-terminally labeled GFAP, and centrally labeled GFAP; (xi) N-terminally labeled neurogranin, C-terminally labeled neurogranin, and centrally labeled neurogranin; (xii) N-terminally labeled neurofilament, C-terminally labeled neurofilament, and centrally labeled neurofilament; and (xiii) N-terminally labeled phosphorylated tau protein, C-terminally labeled phosphorylated tau protein, and centrally labeled phosphorylated tau protein. N-terminally labeled fragments, C-terminally labeled fragments, and centrally labeled fragments of each of the labeled peptides and proteins set forth in items (i) through (xiii) above are also contemplated in the present invention.Preferably, for labeled molecules (e.g., biotin-labeled Aβ peptides), the label is attached to the molecule in a manner that does not interfere with (i) the measurement of the labeled molecule or (ii) binding to the capture antibody. The ideal manner for labeling a molecule to achieve its purpose depends on the molecule, label, and measurement method used. Thus, for example, if the molecule is Aβ40 peptide, the label is biotin, and the measurement method used uses an anti-biotin antibody, avidin, or streptavidin (e.g., radiolabeled streptavidin, enzyme-labeled streptavidin, or streptavidin bound to magnetic particles), the ideal manner for labeling Aβ40 peptide with biotin may be to attach biotin to the N-terminus of the peptide.

[0029] As used herein, "matrix effect" refers to the phenomenon that, for a particular condition (e.g., amyloidosis) in a subject, when a suitable diluted sample of a suitable fluid (e.g., a 32-fold diluted plasma sample) from the subject is mixed with a suitable amount of labeled molecule (e.g., 100 pg / ml biotin-labeled Aβ peptide) under suitable conditions for a suitable time (e.g., 3 hours at room temperature), the fluid from a subject with the condition will produce a greater amount of labeled molecule that is not affected by the matrix (i.e., a smaller amount of labeled molecule bound to a biomolecule (e.g., carrier protein) in the fluid) than a subject without the condition. For example, the matrix effect is demonstrated when a 32-fold diluted plasma sample from a subject with amyloidosis is mixed with 100 pg / ml biotin-labeled Aβ40 peptide at room temperature for 3 hours, producing a greater amount of biotin-labeled Aβ40 peptide that is not affected by the matrix than a plasma sample from a subject without amyloidosis. While not wishing to be bound by any particular scientific theory, it is believed that available biomolecules, such as carrier proteins (e.g., chaperone proteins), in the diluted fluid sample act as a "matrix" for the labeled molecule because the matrix binds to the labeled molecule to a degree that correlates with the presence or absence of the condition being detected. Specifically, when the condition is present, the matrix may have a different amount of available binding protein than it would have in the absence of the condition, binding protein with an altered three-dimensional structure, binding protein with secondary modifications (e.g., phosphorylation, nitration, and glycosylation), or binding protein that has a reduced capacity to carry the labeled molecule and therefore binds to a smaller amount of the labeled molecule. Thus, when the condition is present, a greater amount of labeled molecule is produced that is not affected by the matrix than when the condition is absent.

[0030] As used herein, a labeled molecule that is "matrix-free" means a labeled molecule that is not adsorbed or otherwise bound to any biomolecules (e.g., carrier proteins) from the fluid sample of interest that would interfere with the detection of the labeled molecule.

[0031] As used herein, the term "molecule" includes, but is not limited to, aptamers, polypeptides (e.g., peptides (e.g., synthetic peptides) or proteins (e.g., recombinant proteins)), nucleic acids (e.g., DNA or RNA molecules), lipoproteins, and carbohydrates.

[0032] As used herein, a "negative control" for the present method includes: (i) the amount of labeled molecules unaffected by the matrix (or, if applicable, labeled molecules unaffected by the first matrix and labeled molecules unaffected by the second matrix) determined in a parallel method performed in conjunction with a fluid from a subject known to not have the condition being tested; (ii) the amount of labeled molecules unaffected by the matrix (or, if applicable, labeled molecules unaffected by the first matrix and labeled molecules unaffected by the second matrix) determined in a parallel method previously performed with a fluid from a subject known to not have the condition being tested; These include, but are not limited to, (i) the mean or median amount (±15%) of labeled molecules unaffected by the matrix (or, if applicable, labeled molecules unaffected by the first matrix and labeled molecules unaffected by the second matrix) determined in multiple parallel methods previously performed using fluids from one or more subjects known to be free of the condition being tested; and (iv) the mean or median relationship (e.g., mean ratio) between labeled molecules unaffected by the first matrix and labeled molecules unaffected by the second matrix determined in multiple parallel methods previously performed using fluids from one or more subjects known to be free of the condition being tested. By way of example, in one embodiment, the method is for determining whether a human subject has an amyloidopathy correlated with Alzheimer's disease. The method includes (a) mixing (i) a 32-fold diluted sample of plasma from the subject with (ii) 100 pg / ml of biotin-labeled Aβ40 peptide, and (b) determining the amount of matrix-free biotin-labeled Aβ40 peptide present in the resulting mixture after 3 hours at room temperature, assuming the negative control is 100 ppm.It is the average amount of matrix-free biotin-labeled Aβ40 peptide determined in 20 previously performed parallel methods (i.e., performed similarly to the present method, except that these methods use 32-fold diluted plasma from 20 subjects known to be free of amyloidopathy correlated with Alzheimer's disease). Also, assume that the amount of matrix-free biotin-labeled Aβ40 peptide present in the resulting mixture is determined to be 100 ppm. The subject does not have amyloidopathy, because the amount of matrix-free labeled Aβ peptide determined in step (b) correlates with a negative control for amyloidopathy. In the present invention, perfect correlation with a negative control is not necessary to determine that the subject does not have the condition being tested. For example, in the above example, if the amount of matrix-free biotin-labeled Aβ40 peptide present in the mixture was determined to be 125 ppm, the negative control was 100 ppm, and the positive control (discussed herein) was 300 ppm, it could still be determined that the subject does not have amyloidopathy.

[0033] As used herein, "neurodegenerative disorders" include, but are not limited to, Alzheimer's disease and Parkinson's disease.

[0034] As used herein, "multiple serially diluted samples" (e.g., plasma samples) include, but are not limited to, two or more samples of various dilution factors. In one embodiment, the multiple serially diluted samples include two samples in which the fluid therein is diluted (i) 4-fold and 8-fold; (ii) 8-fold and 16-fold; (iii) 16-fold and 32-fold; (iv) 32-fold and 64-fold; (v) 64-fold and 128-fold; and (vi) 128-fold and 256-fold. In another embodiment, the multiple serially diluted samples include three samples in which the fluid therein is diluted (i) 4-fold, 8-fold, and 16-fold; (ii) 8-fold, 16-fold, and 32-fold; (iii) 16-fold, 32-fold, and 64-fold; (iv) 32-fold, 64-fold, and 128-fold; and (v) 64-fold, 128-fold, and 256-fold. In a further embodiment, the plurality of serially diluted samples comprises four samples in which the fluid therein is diluted (i) 4-fold, 8-fold, 16-fold, and 32-fold; (ii) 8-fold, 16-fold, 32-fold, and 64-fold; (iii) 16-fold, 32-fold, 64-fold, and 128-fold; and (iv) 32-fold, 64-fold, 128-fold, and 256-fold. In yet a further embodiment, the plurality of serially diluted samples comprises five samples in which the fluid therein is diluted (i) 4-fold, 8-fold, 16-fold, 32-fold, and 64-fold; (ii) 8-fold, 16-fold, 32-fold, 64-fold, and 128-fold; and (iii) 16-fold, 32-fold, 64-fold, 128-fold, and 256-fold. In yet a further embodiment, the plurality of serially diluted samples comprises six samples in which the fluid therein is diluted (i) 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, and 128-fold; and (ii) 8-fold, 16-fold, 32-fold, 64-fold, 128-fold, and 256-fold. In yet a further embodiment, the plurality of serially diluted samples comprises seven samples in which the fluid therein is diluted 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 128-fold, and 256-fold.

[0035] As used herein, a "positive control" for the present method includes: (i) the amount of labeled molecules unaffected by the matrix (or, if applicable, labeled molecules unaffected by the first matrix and labeled molecules unaffected by the second matrix) determined in a parallel method performed simultaneously using fluid from a subject known to have the condition being tested; (ii) the amount of labeled molecules unaffected by the matrix (or, if applicable, labeled molecules unaffected by the first matrix and labeled molecules unaffected by the second matrix) determined in a parallel method previously performed using fluid from a subject known to have the condition being tested; (iii) the mean or median amount of labeled molecules unaffected by the matrix (or, if applicable, labeled molecules unaffected by the first matrix and labeled molecules unaffected by the second matrix) determined in multiple parallel methods previously performed using fluids from one or more subjects known to have the condition being tested; and (iv) the mean or median relationship (e.g., mean ratio) between labeled molecules unaffected by the first matrix and labeled molecules unaffected by the second matrix determined in multiple parallel methods previously performed using fluids from one or more subjects known to have the condition being tested. By way of example, in one embodiment, the method is for determining whether a human subject has an amyloidopathy correlated with Alzheimer's disease. The method includes (a) mixing (i) a 32-fold diluted sample of plasma from the subject with (ii) 100 pg / ml of biotin-labeled Aβ40 peptide, and (b) determining the amount of matrix-free biotin-labeled Aβ40 peptide present in the resulting mixture after 3 hours at room temperature, assuming the positive control is 300 ppm.It is the average amount of matrix-free biotin-labeled Aβ40 peptide determined in 20 previously performed parallel methods (i.e., performed similarly to the present method, except that these methods used 32-fold diluted plasma from 20 subjects known to have an amyloidopathy correlated with Alzheimer's disease). Also, assume that the amount of matrix-free biotin-labeled Aβ40 peptide present in the resulting mixture is determined to be 300 ppm. The subject has amyloidopathy, because the amount of matrix-free labeled Aβ peptide determined in step (b) correlates with a positive control for amyloidopathy. In the present invention, perfect correlation with a positive control is not required to determine that the subject has the condition being tested. For example, in the above example, if the amount of matrix-free biotin-labeled Aβ40 peptide present in the mixture was determined to be 275 ppm, the positive control was 300 ppm, and the negative control (discussed herein) was 100 ppm, then the subject could still be determined to have amyloidopathy.

[0036] As used herein, the "quantitative relationship" between a first labeled molecule unaffected by the matrix and a second labeled molecule unaffected by the matrix present in the resulting mixture includes, but is not limited to, (i) the change in one of the molecules relative to the other; (ii) the ratio of the amount of one of the two molecules to the amount of the other (e.g., the ratio of the first labeled molecule unaffected by the matrix to the second labeled molecule unaffected by the matrix); (iii) an algorithmically defined relationship (e.g., x*A+b*B+z); (iv) the relationship between the slopes of the dilution curves of each molecule; (v) the relative final dilution potency of each molecule; and (vi) combinations thereof.

[0037] As used herein, an antibody "specifically binds" to its target (e.g., an epitope on an Aβ peptide) if the antibody does at least one of the following: (i) binds to the target with greater affinity than the antibody binds to any other target; or (ii) binds to the target with at least 5×10 -4 M (e.g., at least 1 × 10 -4 M, at least 5 × 10 -5 M, at least 1 × 10 -5 M, at least 5 × 10 -6 M, at least 1 × 10 -6 M, at least 5 × 10 -7 M, at least 1 × 10 -7 M, at least 5 × 10 -8 M, at least 1 × 10 -8 M, at least 5 × 10 -9 M, or at least 1 × 10 -9 M) Preferably, an antibody specifically binds to its target if it does both of items (i) and (ii) above.

[0038] As used herein, the term "subject" includes, but is not limited to, mammals, such as humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs, hamsters, rats, and mice. As the methods are directed to human subjects in the present invention, the methods are contemplated for these non-human embodiments, mutatis mutandis.

[0039] As used herein, an "appropriate amount" of a labeled molecule is an amount sufficient to allow experimental detection of the molecule in a matrix-free form in step (b) of the method. Suitable amounts of labeled molecule include (i) 1 pg / ml (i.e., the amount of target molecule that will achieve a concentration of 1 pg / ml), 5 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, 100 pg / ml, 110 pg / ml, 120 pg / ml, 130 pg / ml, 140 pg / ml, 150 pg / ml, 160 pg / ml, 170 pg / ml, 180 pg / ml, 190 pg / ml, 200 pg / ml, 300 pg / ml, 400 pg / ml, 500 pg / ml, 600 pg / ml, 700 pg / ml, 800 pg / ml, and 900 pg / ml. , 1 ng / ml, 5 ng / ml, 10 ng / ml, 50 ng / ml, and 100 ng / ml; (ii) 1 pg / ml to 20 pg / ml, 20 pg / ml to 50 pg / ml, 50 pg / ml to 100 pg / ml, 100 pg / ml to 150 pg / ml, 150 pg / ml to 200 pg / ml, 200 pg / ml to 500 pg / ml, 500 pg / ml to 1 ng / ml, 1 ng / ml to 10 ng / ml, and 10 ng / ml to 100 ng / ml; and (iii) 1 pg / ml to 100 pg / ml, 100 pg / ml to 1 ng / ml, 1 ng / ml to 10 ng / ml, and 10 ng / ml to 100 ng / ml.

[0040] As used herein, "suitable fluids" include, but are not limited to, whole blood, serum, plasma (e.g., untreated plasma, and EDTA-, heparin-, and / or citrate-treated plasma), cerebrospinal fluid (e.g., lumbar, ventricular, or cisternal cerebrospinal fluid, whether collected by a shunt or a drain), vitreous humor, saliva, tears, ocular fluid, nasal fluid, and interstitial fluid.

[0041] As used herein, "suitable conditions" for step (b) of the present method include, but are not limited to, (i) room temperature, 37°C, and / or 2°C to 8°C; (ii) rotation, orbital shaking, and / or static; (iii) fresh, frozen (various cycles / methods) (e.g., the temperature is reduced to freezing), heated (i.e., the temperature is increased to 56°C or higher), and / or static; (iv) sonication; (v) denaturation (e.g., with GuHCl, formic acid, high concentrations of detergent, and combinations thereof); and (vi) combinations thereof.

[0042] As used herein, a "suitable period of time" with respect to step (b) of the method includes, but is not limited to, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 48 ​​hours, 72 hours, or a longer period of time.

[0043] As used herein, an "appropriately diluted" sample of a suitable fluid (e.g., plasma) includes, but is not limited to, a sample in which the fluid is diluted 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 128-fold, and 256-fold.

[0044] As used herein, "treating" a labeled molecule (whether directly or indirectly) in a manner that facilitates its measurement includes, but is not limited to, (i) contacting the molecule with an antibody that specifically binds to the molecule (e.g., at the N-terminus, C-terminus, central domain, or any other domain); (ii) pretreating the molecule (e.g., by acidification, alkalinization, heating, freezing, and / or sonication); and (ii) chemically treating the molecule (e.g., by phosphorylation, dephosphorylation, oxidation, and / or oligomerization). Other agents, such as dyes or curcumin, can be used in place of antibodies for this purpose. Antibodies useful for treating labeled molecules in a manner that facilitates their measurement include, but are not limited to, 3D6 (Johnson-Wood et al. (1997) and Vanderstichele et al. (2005)); 1E8 (Wiltfang et al. (2001)); WO2 (Vanderstichele et al. (2005)); 82E1 (Horikoshia et al. (2004)); 6E10 (Baghallab et al. (2018) and Vanderstichele et al. (2005)); 2G3 (Vanderstichele et al. (2005)); and 21F12 (Johnson-Wood et al. (1997) and Vanderstichele et al. (2005)).

[0045] (Embodiments of the invention) The present invention provides certain methods for determining whether a subject has a condition correlated with matrix effects. These conditions (e.g., amyloidopathies) may be diagnostic and / or prognostic for certain disorders (e.g., Alzheimer's disease).

[0046] Specifically, the present invention provides a first method for determining whether a subject has a condition correlated with a matrix effect, the first method comprising: (a) mixing (i) an appropriately diluted sample of an appropriate fluid from the subject with (ii) an appropriate amount of a labeled molecule, where the labeled molecule is affected by a matrix effect with respect to the appropriate fluid in a subject suffering from the condition; and (b) after an appropriate period of time under appropriate conditions, determining the amount of unaffected labeled molecule present in the resulting mixture; wherein if the amount of unaffected labeled molecule determined in step (b) correlates with a positive control for the condition, then the subject has the condition; and if the amount correlates with a negative control for the condition, then the subject does not have the condition.

[0047] In one embodiment of the first method, step (a) further comprises treating the labeled molecule in a manner that facilitates measurement of the labeled molecule in step (b). Preferably, treating the labeled molecule in a manner that facilitates measurement of the labeled molecule in step (b) comprises contacting the labeled molecule with an appropriate amount of an antibody that specifically binds to the labeled molecule.

[0048] In the first method, step (a) may be carried out by mixing the appropriate amount of the labeled molecule with either a single diluted sample of the fluid or multiple serially diluted samples of the fluid.

[0049] The present invention also provides a second method for determining whether a subject has a condition correlated with a matrix effect, comprising: (a) mixing (i) an appropriately diluted sample of an appropriate fluid from the subject and (ii) appropriate amounts of a first labeled molecule and a second labeled molecule (and optionally three or more labeled molecules), where the labeled molecules are affected by a matrix effect with respect to the appropriate fluid in a subject suffering from the condition; and (b) after an appropriate period of time under appropriate conditions, determining a quantitative relationship between the matrix-unaffected first labeled molecule and the matrix-unaffected second labeled molecule present in the resulting mixture; wherein if the relationship determined in step (b) correlates with a positive control for the condition, then the subject has the condition; and if the relationship correlates with a negative control for the condition, then the subject does not have the condition.

[0050] In one embodiment, the second method comprises: (a) mixing (i) an appropriately diluted sample of an appropriate fluid from the subject with (ii) appropriate amounts of three or more labeled molecules (e.g., labeled Aβ40 peptide, labeled Aβ42 peptide, and labeled Aβ10 peptide), wherein the labeled molecules are affected by a matrix effect with respect to the appropriate fluid in a subject suffering from the condition; and (b) after an appropriate period of time under appropriate conditions, determining a quantitative relationship between each of the matrix-unaffected molecules present in the resulting mixture; if the relationship determined in step (b) correlates with a positive control for the condition, then the subject is affected by the condition; and if the relationship correlates with a negative control for the condition, then the subject is not affected by the condition.

[0051] In another embodiment of the second method, step (a) further comprises treating the labeled molecules in a manner that facilitates measurement of the labeled molecules in step (b). Preferably, treating the labeled molecules in a manner that facilitates measurement of the labeled molecules in step (b) comprises contacting each labeled molecule with an appropriate amount of an antibody that specifically binds to each labeled molecule.

[0052] In the second method, step (a) may be performed by mixing the appropriate amount of the labeled molecule with either a single diluted sample of the fluid or multiple serially diluted samples of the fluid.

[0053] In the first and second methods, the subject is preferably a human.

[0054] In one embodiment of the first and second methods, the condition is a condition associated with a neurodegenerative disorder (e.g., Alzheimer's disease, Parkinson's disease, mild cognitive impairment, non-Alzheimer's disease dementia, frontotemporal dementia, age-related macular degeneration (e.g., wet age-related macular degeneration), and dementia with Lewy bodies). In this embodiment, the subject can either (i) have symptoms of Alzheimer's disease, Parkinson's disease, mild cognitive impairment, non-Alzheimer's disease dementia, frontotemporal dementia, age-related macular degeneration, or dementia with Lewy bodies, or (ii) do not have symptoms of Alzheimer's disease, Parkinson's disease, mild cognitive impairment, non-Alzheimer's disease dementia, frontotemporal dementia, age-related macular degeneration, or dementia with Lewy bodies. In another embodiment of the first and second methods, the condition is a condition associated with Down's syndrome. In further embodiments of the first and second methods, the condition is an amyloidopathy, a tauopathy, neuronal injury, a synucleinopathy, a cerebral proteinopathy (e.g., a TDP proteinopathy), a progranulinopathy, and neuronal inflammation.

[0055] In the first and second methods, the suitable fluid is preferably whole blood, serum, plasma, cerebrospinal fluid, saliva, tears, ocular fluid, vitreous humor, nasal fluid, and interstitial fluid.

[0056] In a preferred embodiment of the first method, the labeled molecule is a labeled Aβ peptide (e.g., a labeled Aβ40 peptide, a labeled Aβ42 peptide, a labeled Aβ37 peptide, a labeled Aβ38 peptide, a labeled Aβ10 peptide, a labeled Aβ16 peptide, or a labeled Aβ43 peptide) or a fragment thereof, a labeled tau protein or a fragment thereof, a labeled phosphorylated tau protein or a fragment thereof, a labeled progranulin or a fragment thereof, a labeled TDP-43 or a fragment thereof, a labeled GFAP or a fragment thereof, a labeled neurogranin or a fragment thereof, a labeled neurofilament or a fragment thereof, or a labeled synuclein (e.g., α-synuclein or β-synuclein) or a fragment thereof.

[0057] In a preferred embodiment of the second method, the first labeled molecule and the second labeled molecule are selected from the group consisting of a labeled Aβ peptide (e.g., a labeled Aβ40 peptide, a labeled Aβ42 peptide, a labeled Aβ37 peptide, a labeled Aβ38 peptide, a labeled Aβ10 peptide, a labeled Aβ16 peptide, or a labeled Aβ43 peptide) or a fragment thereof, a labeled tau protein or a fragment thereof, a labeled phosphorylated tau protein or a fragment thereof, a labeled progranulin or a fragment thereof, a labeled TDP-43 or a fragment thereof, a labeled GFAP or a fragment thereof, a labeled neurogranin or a fragment thereof, a labeled neurofilament or a fragment thereof, and a labeled synuclein (e.g., α-synuclein or β-synuclein) or a fragment thereof.

[0058] In the first and second methods, determining the amount of labeled molecule unaffected by matrix can be accomplished absolutely (e.g., in pg / ml or total pg) or comparatively (e.g., in ppm or fold difference compared to another molecule).

[0059] The present invention contemplates a method for measuring the progression of a condition correlated with matrix effects in a subject, the method comprising: (a) performing the first method described above at multiple time points (e.g., at one-month intervals, two-month intervals, or three-month intervals); and (b) comparing the amounts of labeled molecules not affected by matrix effects determined by the first method at each of the time points. In one embodiment of this method, the subject is undergoing treatment for the condition, and this method is used to monitor the success of the treatment. The present invention contemplates another method for measuring the progression of a condition correlated with matrix effects in a subject, the method comprising: (a) performing the second method described above at multiple time points (e.g., at one-month intervals, two-month intervals, or three-month intervals); and (b) comparing the amounts of labeled molecules not affected by matrix effects determined by the second method at each of the time points. In one embodiment of this method, the subject is undergoing treatment for the condition, and this method is used to monitor the success of the treatment. Various embodiments of methods for measuring the progression of these conditions are contemplated as they are for the first and second methods of the present invention, mutatis mutandis.

[0060] The present invention further provides a first kit for use in determining whether a subject (preferably a human subject) has a condition correlated with matrix effects, the first kit comprising, in separate compartments, (a) a labeled molecule that is subject to matrix effects with respect to an appropriate fluid in a subject suffering from the condition, and (b) an agent useful for processing the labeled molecule to facilitate measurement of the labeled molecule in its matrix-free form. In a preferred embodiment of the first kit, the kit further comprises, in a separate compartment, an agent useful for measuring the labeled molecule in its matrix-free form. In another preferred embodiment of the first kit, the labeled molecule is selected from the group consisting of a labeled Aβ peptide (e.g., a labeled Aβ40 peptide, a labeled Aβ42 peptide, a labeled Aβ37 peptide, a labeled Aβ38 peptide, a labeled Aβ10 peptide, a labeled Aβ16 peptide, or a labeled Aβ43 peptide) or a fragment thereof, a labeled tau protein or a fragment thereof, a labeled phosphorylated tau protein or a fragment thereof, a labeled progranulin or a fragment thereof, a labeled TDP-43 or a fragment thereof, a labeled GFAP or a fragment thereof, a labeled neurogranin or a fragment thereof, a labeled neurofilament or a fragment thereof, and a labeled synuclein (e.g., α-synuclein) or a fragment thereof.

[0061] The present invention still further provides a second kit for use in determining whether a subject (preferably a human subject) has a condition correlated with matrix effects, the second kit comprising, in separate compartments, (a) a first labeled molecule and a second labeled molecule, each of which is affected by matrix effects with respect to an appropriate fluid in a subject suffering from the condition, and (b) an agent useful for treating each of the first labeled molecule and the second labeled molecule to facilitate measurement of the labeled molecule in its matrix-free form. In a preferred embodiment of the second kit, the kit further comprises, in one or more separate compartments, an agent useful for measuring each of the first labeled molecule and the second labeled molecule in its matrix-free form. In another preferred embodiment of the second kit, the first labeled molecule and the second labeled molecule are selected from the group consisting of a labeled Aβ peptide (e.g., a labeled Aβ40 peptide, a labeled Aβ42 peptide, a labeled Aβ37 peptide, a labeled Aβ38 peptide, a labeled Aβ10 peptide, a labeled Aβ16 peptide, or a labeled Aβ43 peptide) or a fragment thereof, a labeled tau protein or a fragment thereof, a labeled phosphorylated tau protein or a fragment thereof, a labeled progranulin or a fragment thereof, a labeled TDP-43 or a fragment thereof, a labeled GFAP or a fragment thereof, a labeled neurogranin or a fragment thereof, a labeled neurofilament or a fragment thereof, and a labeled synuclein (e.g., α-synuclein) or a fragment thereof.

[0062] In one embodiment, the second kit comprises, in separate compartments: (a) three or more labeled molecules (e.g., a labeled Aβ40 peptide, a labeled Aβ42 peptide, and a labeled Aβ10 peptide), each of which is subject to a matrix effect with respect to an appropriate fluid in a subject suffering from the condition; and (b) one or more agents useful for processing the labeled molecules to facilitate their measurement in a matrix-free form.

[0063] The present invention provides a first method for determining whether a human subject (either symptomatic or asymptomatic) has a condition correlated with Alzheimer's disease (i.e., the subject may actually have Alzheimer's disease or may not yet have Alzheimer's disease), comprising: (a) mixing (i) an appropriately diluted sample of a suitable fluid from the subject with (ii) an appropriate amount of labeled Aβ peptide; and (b) after an appropriate period of time under appropriate conditions, determining the amount of matrix-free, labeled Aβ peptide present in the resulting mixture; wherein if the amount of matrix-free, labeled Aβ peptide determined in step (b) correlates with a positive control for the condition, then the subject has the condition; and if the amount correlates with a negative control for the condition, then the subject does not have the condition.

[0064] In one embodiment, the first method comprises: (a) mixing (i) a first, appropriately diluted sample of a suitable fluid from the subject with (ii) an appropriate amount of labeled Aβ peptide; and separately mixing (i) a second, appropriately diluted sample of a suitable fluid from the subject with (ii) an appropriate amount of labeled Aβ peptide; (b) after a suitable period of time and under suitable conditions, determining a quantitative relationship between the matrix-unaffected labeled Aβ peptide at the first dilution and the matrix-unaffected labeled Aβ peptide at the second dilution present in the resulting mixture; wherein if the relationship determined in step (b) correlates with a positive control for the condition, the subject is afflicted with the condition; and if the relationship correlates with a negative control for the condition, the subject is not afflicted with the condition. For example, in one embodiment, the relationship determined is the ratio of the matrix-free labeled Aβ peptides measured for a plasma sample diluted 1:8 (a first appropriately diluted sample) to the matrix-free labeled Aβ peptides measured for a plasma sample diluted 1:128 (a second appropriately diluted sample).

[0065] In another embodiment of the first method, step (a) further comprises treating the labeled Aβ peptide in a manner that facilitates measurement of the labeled Aβ peptide in step (b). Preferably, treating the labeled Aβ peptide in a manner that facilitates measurement of the labeled Aβ peptide in step (b) comprises contacting the labeled Aβ peptide with an appropriate amount of an agent (e.g., an antibody) that binds (preferably specifically) to the labeled Aβ peptide. Such agents include, but are not limited to, antibody 3D6 (Johnson-Wood et al. (1997) and Vanderstichele et al. (2005)); antibody 1E8 (Wiltfang et al. (2001)); antibody WO2 (Vanderstichele et al. (2005)); antibody 82E1 (Horikoshia et al. (2004)); antibody 6E10 (Baghallab et al. (2018) and Vanderstichele et al. (2005)); antibody 2G3 (Vanderstichele et al. (2005)); and antibody 21F12 (Johnson-Wood et al. (1997) and Vanderstichele et al. (2005)).

[0066] In the first method, step (a) may be carried out by mixing the appropriate amount of labeled Aβ peptide with either a single diluted sample of the fluid or multiple serially diluted samples of the fluid.

[0067] In a preferred embodiment of the first method, the labeled Aβ peptide is a labeled Aβ40 peptide or a fragment thereof, a labeled Aβ42 peptide or a fragment thereof, a labeled Aβ37 peptide or a fragment thereof, a labeled Aβ38 peptide or a fragment thereof, a labeled Aβ10 peptide or a fragment thereof, a labeled Aβ16 peptide or a fragment thereof, or a labeled Aβ43 peptide or a fragment thereof. In a further preferred embodiment of the first method, the labeled Aβ peptide is a biotinylated Aβ40 peptide, a biotinylated Aβ42 peptide, or a fragment thereof.

[0068] The present invention also provides a second method for determining whether a human subject (either symptomatic or asymptomatic) has a condition correlated with Alzheimer's disease (i.e., the subject may actually have Alzheimer's disease or may not yet have Alzheimer's disease), comprising: (a) mixing (i) an appropriately diluted sample of a suitable fluid from the subject with (ii) appropriate amounts of a first labeled Aβ peptide and a second labeled Aβ peptide; and (b) after an appropriate period of time under appropriate conditions, determining a quantitative relationship between the matrix-unaffected first labeled Aβ peptide and the matrix-unaffected second labeled Aβ peptide present in the resulting mixture; wherein if the relationship determined in step (b) correlates with a positive control for the condition, the subject has the condition; and if the relationship correlates with a negative control for the condition, the subject does not have the condition.

[0069] In one embodiment of the second method, step (a) further comprises treating the labeled Aβ peptide in a manner that facilitates measurement of the labeled Aβ peptide in step (b). Preferably, treating the labeled Aβ peptide in a manner that facilitates measurement of the labeled Aβ peptide in step (b) comprises contacting the labeled Aβ peptide with an appropriate amount of an agent (e.g., an antibody) that binds (preferably specifically) to the labeled Aβ peptide. Such agents include, but are not limited to, antibody 3D6 (Johnson-Wood et al. (1997) and Vanderstichele et al. (2005)); antibody 1E8 (Wiltfang et al. (2001)); antibody WO2 (Vanderstichele et al. (2005)); antibody 82E1 (Horikoshia et al. (2004)); antibody 6E10 (Baghallab et al. (2018) and Vanderstichele et al. (2005)); antibody 2G3 (Vanderstichele et al. (2005)); and antibody 21F12 (Johnson-Wood et al. (1997) and Vanderstichele et al. (2005)).

[0070] In the second method, step (a) may be carried out by mixing the appropriate amount of labeled Aβ peptide with either a single diluted sample of the fluid or multiple serially diluted samples of the fluid.

[0071] In a preferred embodiment of the second method, each of the labeled Aβ peptides is a labeled Aβ40 peptide or a fragment thereof, a labeled Aβ42 peptide or a fragment thereof, a labeled Aβ37 peptide or a fragment thereof, a labeled Aβ38 peptide or a fragment thereof, a labeled Aβ10 peptide or a fragment thereof, a labeled Aβ16 peptide or a fragment thereof, or a labeled Aβ43 peptide or a fragment thereof. In a further preferred embodiment of the second method, each of the labeled Aβ peptides is a biotinylated Aβ40 peptide, a biotinylated Aβ42 peptide, or a fragment thereof.

[0072] In the first and second methods, the condition correlated with Alzheimer's disease can be any condition that has this correlation and also correlates with matrix effect.Preferably, this condition is amyloidopathy, tauopathy, neuronal injury, synucleinopathy, cerebral proteinopathy (e.g., TDP-43), progranulinopathy, and neuronal inflammation.

[0073] In the first and second methods, the suitable fluid is preferably whole blood, serum, plasma, cerebrospinal fluid, saliva, tears, ocular fluid, vitreous fluid, nasal fluid, or interstitial fluid.

[0074] In the first and second methods, determining the amount of labeled molecule unaffected by matrix can be accomplished absolutely (e.g., in pg / ml or total pg) or comparatively (e.g., in ppm or fold difference compared to another molecule).

[0075] The present invention contemplates a method for measuring the progression of a subject with a condition correlated with Alzheimer's disease, comprising: (a) performing the first method described above at multiple time points (e.g., at monthly, 2-month, or 3-month intervals); and (b) comparing the amounts of matrix-free, labeled Aβ peptide determined by the first method at each of the time points. In one embodiment of this method, the subject is undergoing treatment for the condition, and this method is used to monitor the success of the treatment. The present invention contemplates another method for measuring the progression of a subject with a condition correlated with Alzheimer's disease, comprising: (a) performing the second method described above at multiple time points (e.g., at monthly, 2-month, or 3-month intervals); and (b) comparing the amounts of matrix-free, labeled Aβ peptide determined by the second method at each of the time points. In one embodiment of this method, the subject is undergoing treatment for the condition, and this method is used to monitor the success of the treatment. Various embodiments of methods for measuring the progression of these conditions are contemplated as are those for the first and second methods of the invention, mutatis mutandis.

[0076] The present invention further provides a first kit for use in determining whether a human subject has a condition correlated with Alzheimer's disease, the kit comprising, in separate compartments: (a) a labeled Aβ peptide; and (b) an agent useful for processing the labeled Aβ peptide to facilitate measurement of the labeled Aβ peptide in its matrix-free form. In a preferred embodiment of the first kit: In another preferred embodiment of the first kit, the kit further comprises, in a separate compartment, an agent useful for measuring the labeled Aβ peptide in a form unaffected by the matrix. In another preferred embodiment of the first kit, the labeled Aβ peptide is a labeled Aβ40 peptide or a fragment thereof, a labeled Aβ42 peptide or a fragment thereof, a labeled Aβ37 peptide or a fragment thereof, a labeled Aβ38 peptide or a fragment thereof, a labeled Aβ10 peptide or a fragment thereof, a labeled Aβ16 peptide or a fragment thereof, or a labeled Aβ43 peptide or a fragment thereof. In a further preferred embodiment of the first kit, the labeled Aβ peptide is a biotinylated Aβ40 peptide or a biotinylated Aβ42 peptide, or a fragment thereof.

[0077] The present invention still further provides a second kit for use in determining whether a human subject has a condition correlated with Alzheimer's disease, the kit comprising, in separate compartments, (a) a first labeled Aβ peptide and a second labeled Aβ peptide, and (b) an agent useful for processing each of the first labeled Aβ peptide and the second labeled Aβ peptide to facilitate measurement of the labeled Aβ peptides in their matrix-free forms. In a preferred embodiment of the second kit, the kit further comprises, in one or more separate compartments, an agent useful for measuring each of the first labeled Aβ peptide and the second labeled Aβ peptide in their matrix-free forms. In another preferred embodiment of the second kit, each of the labeled Aβ peptides is a labeled Aβ40 peptide or fragment thereof, a labeled Aβ42 peptide or fragment thereof, a labeled Aβ37 peptide or fragment thereof, a labeled Aβ38 peptide or fragment thereof, a labeled Aβ10 peptide or fragment thereof, a labeled Aβ16 peptide or fragment thereof, or a labeled Aβ43 peptide or fragment thereof. In a further preferred embodiment of the second kit, the first and second labeled Aβ peptides are biotinylated Aβ40 and biotinylated Aβ42 peptides, or fragments thereof.

[0078] The present invention provides a first method for determining whether a human subject (either symptomatic or asymptomatic) has a condition correlated with Parkinson's disease (i.e., the subject may actually have Parkinson's disease or may not yet have it), the method comprising: (a) mixing (i) a suitably diluted sample of a suitable fluid from the subject with (ii) a suitable amount of labeled alpha-synuclein; and (b) after a suitable period of time under suitable conditions, determining the amount of matrix-free labeled alpha-synuclein present in the resulting mixture; if the amount of matrix-free labeled alpha-synuclein determined in step (b) correlates with a positive control for the condition, then the subject has the condition; and if the amount correlates with a negative control for the condition, then the subject does not have the condition.

[0079] In one embodiment, the first method comprises the steps of: (a) mixing (i) a first, appropriately diluted sample of a suitable fluid from the subject with (ii) a suitable amount of labeled alpha-synuclein; and separately mixing (i) a second, appropriately diluted sample of a suitable fluid from the subject with (ii) a suitable amount of labeled alpha-synuclein; (b) after a suitable period of time under suitable conditions, determining a quantitative relationship between the matrix-free labeled alpha-synuclein at the first dilution and the matrix-free labeled alpha-synuclein at the second dilution present in the resulting mixture; wherein if the relationship determined in step (b) correlates with a positive control for the condition, then the subject is afflicted with the condition; and if the relationship correlates with a negative control for the condition, then the subject is not afflicted with the condition. For example, in one embodiment, the relationship determined is the ratio of matrix-free labeled alpha-synuclein measured in a plasma sample diluted 1:8 (a first appropriately diluted sample) to matrix-free labeled alpha-synuclein measured in a plasma sample diluted 1:128 (a second appropriately diluted sample).

[0080] In another embodiment of the first method, step (a) further comprises treating the labeled alpha-synuclein in a manner that facilitates measurement of the labeled alpha-synuclein in step (b). Preferably, treating the labeled alpha-synuclein in a manner that facilitates measurement of the labeled alpha-synuclein in step (b) comprises contacting the labeled alpha-synuclein with an appropriate amount of an antibody that specifically binds to the labeled alpha-synuclein.

[0081] In the first method, step (a) may be carried out by mixing the appropriate amount of labeled alpha-synuclein with either a single diluted sample of the fluid or multiple serially diluted samples of the fluid.

[0082] The present invention also provides a second method for determining whether a human subject (either symptomatic or asymptomatic) has a condition correlated with Parkinson's disease (i.e., the subject may actually have Parkinson's disease or may not yet have it), the method comprising: (a) mixing (i) a suitably diluted sample of a suitable fluid from the subject with (ii) suitable amounts of a first labeled alpha-synuclein and a second labeled alpha-synuclein; and (b) after a suitable period of time under suitable conditions, determining a quantitative relationship between the matrix-unaffected first labeled alpha-synuclein and the matrix-unaffected second labeled alpha-synuclein present in the resulting mixture; if the relationship determined in step (b) correlates with a positive control for the condition, then the subject has the condition; and if the relationship correlates with a negative control for the condition, then the subject does not have the condition.

[0083] In one embodiment of the second method, step (a) further comprises treating the labeled alpha-synuclein in a manner that facilitates measurement of the labeled alpha-synuclein in step (b). Preferably, treating the labeled alpha-synuclein in a manner that facilitates measurement of the labeled alpha-synuclein in step (b) comprises contacting the labeled alpha-synuclein with an appropriate amount of an antibody that specifically binds to the labeled alpha-synuclein.

[0084] In the second method, step (a) may be carried out by mixing the appropriate amount of labeled alpha-synuclein with either a single diluted sample of the fluid or multiple serially diluted samples of the fluid.

[0085] In the first and second methods, the suitable fluid is preferably whole blood, serum, plasma, cerebrospinal fluid, saliva, tears, nasal fluid, ocular fluid, vitreous humor, or interstitial fluid.

[0086] In the first and second methods, determining the amount of labeled molecule unaffected by matrix can be accomplished absolutely (e.g., in pg / ml or total pg) or comparatively (e.g., in ppm or fold difference compared to another molecule).

[0087] The present invention contemplates a method for measuring the progression of a subject with a condition correlated with Parkinson's disease, comprising: (a) performing the first method described above at multiple time points (e.g., at monthly, bimonthly, or trimonthly intervals); and (b) comparing the amounts of matrix-free, labeled α-synuclein determined by the first method at each of the time points. In one embodiment of this method, the subject is undergoing treatment for the condition, and this method is used to monitor the success of the treatment. The present invention contemplates another method for measuring the progression of a subject with a condition correlated with Parkinson's disease, comprising: (a) performing the second method described above at multiple time points (e.g., at monthly, bimonthly, or trimonthly intervals); and (b) comparing the amounts of matrix-free, labeled α-synuclein determined by the second method at each of the time points. In one embodiment of this method, the subject is undergoing treatment for the condition, and this method is used to monitor the success of the treatment. Various embodiments of methods for measuring the progression of these conditions are contemplated as they are for the first and second methods of the present invention, mutatis mutandis.

[0088] The present invention further provides a first kit for use in determining whether a human subject has a condition correlated with Parkinson's disease, the kit comprising, in separate compartments, (a) labeled α-synuclein and (b) an agent useful for processing the labeled α-synuclein to facilitate measurement of the labeled α-synuclein in its matrix-free form. In a preferred embodiment of the first kit, the kit further comprises, in a separate compartment, an agent useful for measuring the labeled α-synuclein in its matrix-free form. In a further preferred embodiment of the first kit, the labeled α-synuclein is biotinylated α-synuclein or a fragment thereof.

[0089] The present invention still further provides a second kit for use in determining whether a human subject has a condition correlated with Parkinson's disease, the kit comprising (a) a first labeled α-synuclein and a second labeled α-synuclein, and (b) an agent useful for treating each of the first labeled α-synuclein and the second labeled α-synuclein in separate compartments to facilitate measurement of the labeled α-synuclein in its matrix-free form. In a preferred embodiment of the second kit, the kit further comprises, in one or more separate compartments, an agent useful for measuring each of the first labeled α-synuclein and the second labeled α-synuclein in its matrix-free form. In a further preferred embodiment of the second kit, the first labeled α-synuclein and the second labeled α-synuclein are biotinylated α-synuclein, or fragments thereof.

[0090] In the above first and second methods and first and second kits for use in relation to Parkinson's disease, alpha-synuclein may be replaced with one or more phosphorylated proteins, neurogranin, or one or more Rab proteins.

[0091] Finally, the present invention provides specific compositions. The first composition comprises a mixture of labeled Aβ peptide (e.g., biotinylated Aβ peptide) and a diluted fluid sample derived from a human subject. In one embodiment, the composition comprises: (i) a mixture of biotinylated Aβ37 peptide (or a fragment thereof) and a diluted human plasma sample; (ii) a mixture of biotinylated Aβ38 peptide (or a fragment thereof) and a diluted human plasma sample; (iii) a mixture of biotinylated Aβ40 peptide (or a fragment thereof) and a diluted human plasma sample; (iv) a mixture of biotinylated Aβ42 peptide (or a fragment thereof) and a diluted human plasma sample; (v) a mixture of biotinylated Aβ43 peptide (or a fragment thereof) and a diluted human plasma sample; (vi) a mixture of biotinylated Aβ10 peptide (or a fragment thereof) and a diluted human plasma sample; or (vii) a mixture of biotinylated Aβ16 peptide (or a fragment thereof) and a diluted human plasma sample. The second composition comprises a mixture of a labeled Aβ peptide (or a fragment thereof), an antibody that specifically binds to the labeled Aβ peptide (or a fragment thereof), and a diluted fluid sample derived from a human subject.In one embodiment, the composition comprises: (i) a mixture of biotinylated Aβ37 peptide (or a fragment thereof), an antibody that specifically binds to the labeled Aβ peptide (or a fragment thereof), and a diluted human plasma sample; (ii) a mixture of biotinylated Aβ38 peptide (or a fragment thereof), an antibody that specifically binds to the labeled Aβ peptide (or a fragment thereof), and a diluted human plasma sample; (iii) a mixture of biotinylated Aβ40 peptide (or a fragment thereof), an antibody that specifically binds to the labeled Aβ peptide (or a fragment thereof), and a diluted human plasma sample; (iv) a mixture of biotinylated Aβ42 peptide (or a fragment thereof), an antibody that specifically binds to the labeled Aβ peptide (or a fragment thereof), and a diluted human plasma sample. (v) a mixture of a biotinylated Aβ43 peptide (or a fragment thereof), an antibody that specifically binds to the biotinylated Aβ peptide (or a fragment thereof), and a diluted human plasma sample; (vi) a mixture of a biotinylated Aβ10 peptide (or a fragment thereof), an antibody that specifically binds to the biotinylated Aβ peptide (or a fragment thereof), and a diluted human plasma sample; or (vii) a mixture of a biotinylated Aβ16 peptide (or a fragment thereof), an antibody that specifically binds to the biotinylated Aβ peptide (or a fragment thereof), and a diluted human plasma sample.

[0092] The third composition comprises a mixture of labeled tau protein (e.g., biotinylated tau protein) (or a fragment thereof) and a diluted fluid sample derived from a human subject. In one embodiment, the composition comprises a mixture of biotinylated tau protein (or a fragment thereof) and a diluted human plasma sample. The fourth composition comprises a mixture of labeled tau protein (or a fragment thereof), an antibody that specifically binds to the labeled tau protein (or a fragment thereof), and a diluted fluid sample derived from a human subject. In one embodiment, the composition comprises a mixture of biotinylated tau protein (or a fragment thereof), an antibody that specifically binds to the biotinylated tau protein (or a fragment thereof), and a diluted human plasma sample.

[0093] The fifth composition comprises a mixture of labeled phosphorylated tau protein (e.g., biotinylated phosphorylated tau protein) (or a fragment thereof) and a diluted fluid sample derived from a human subject. In one embodiment, the composition comprises a mixture of biotinylated phosphorylated tau protein (or a fragment thereof) and a diluted human plasma sample. The sixth composition comprises a mixture of labeled phosphorylated tau protein (or a fragment thereof), an antibody that specifically binds to the labeled phosphorylated tau protein (or a fragment thereof), and a diluted fluid sample derived from a human subject. In one embodiment, the composition comprises a mixture of biotinylated phosphorylated tau protein (or a fragment thereof), an antibody that specifically binds to the biotinylated phosphorylated tau protein (or a fragment thereof), and a diluted human plasma sample.

[0094] The seventh composition comprises a mixture of a diluted fluid sample from a human subject and labeled synuclein (e.g., biotinylated alpha-synuclein) (or a fragment thereof). In one embodiment, the composition comprises a mixture of biotinylated alpha-synuclein (or a fragment thereof) and a diluted human plasma sample. The eighth composition comprises a mixture of labeled synuclein (or a fragment thereof), an antibody that specifically binds to the labeled synuclein (or a fragment thereof), and a diluted fluid sample from a human subject. In one embodiment, the composition comprises a mixture of biotinylated alpha-synuclein (or a fragment thereof), an antibody that specifically binds to the biotinylated alpha-synuclein (or a fragment thereof), and a diluted human plasma sample.

[0095] The ninth composition comprises a mixture of a diluted fluid sample from a human subject and labeled progranulin (e.g., biotinylated progranulin) (or a fragment thereof). The tenth composition comprises a mixture of a diluted fluid sample from a human subject and labeled TDP-43 (e.g., biotinylated TDP-43) (or a fragment thereof). The eleventh composition comprises a mixture of a diluted fluid sample from a human subject and labeled GFAP (e.g., biotinylated GFAP) (or a fragment thereof). The twelfth composition comprises a mixture of a diluted fluid sample from a human subject and labeled neurogranin (e.g., biotinylated neurogranin) (or a fragment thereof). The thirteenth composition comprises a mixture of a diluted fluid sample from a human subject and labeled neurofilament (e.g., biotinylated neurofilament) (or a fragment thereof).

[0096] As the present methods and kits are for Aβ peptide-based methods and kits in the present invention, the present methods and kits are contemplated, mutatis mutandis, for tau protein-based methods and kits, phosphorylated tau protein-based methods and kits, and synuclein-based methods and kits.

[0097] Because the methods and kits are for labeled molecules in the present invention, the methods and kits are contemplated, mutatis mutandis, for unlabeled molecules (e.g., alpha-synuclein). Because the methods and kits are for determining whether a human subject (whether symptomatic or asymptomatic) has a condition that is correlated with Alzheimer's disease in the present invention, the methods and kits are also contemplated, mutatis mutandis, for determining whether a human subject (whether symptomatic or asymptomatic) has a condition that is correlated with cognitive decline.

[0098] The present invention will be better understood by reference to the following examples, although those skilled in the art will readily recognize that the specific examples detailed are merely illustrative of the invention as more fully described in the appended claims. [Example]

[0099] Example 1 - Technology All data in Figure 1 were generated with MagArray technology (Milpitas, California). The assays were performed on a MagArray MR-813 instrument system (i.e., Hanno incubation unit and reader unit). MagArray Giant Magneto-resistant (GMR) sensor technology and associated instrumentation enable ultrasensitive detection of low-abundance biomolecules in a multiplexed format without the need for optics or microfluidics, while providing simultaneous real-time readouts (JR Lee et al. (2017) and R S Gaster et al. (2009)).

[0100] The assay format used in this study consists of one or more monoclonal antibodies (mAbs) spotted onto sensors on a printed circuit board (PCB). Each PCB contains eight chips. Each chip contains 80 sensors that can be spotted with the same or different antibodies. Several sensors were spotted with the same antibody to obtain a more robust and accurate signal per analyte or target antigen. Bound analytes (biotinylated peptides) were detected using custom magnetic nanotechnology (anti-biotin antibodies coupled to magnetic nanoparticles (MNPs)) obtained from Miltenyi Biotec (California, USA). All PCBs were surface-treated and prepared by MagArray using a proprietary process. After PCB surface treatment, antibodies were spotted, and these pre-functionalized PCBs were either freshly prepared, freshly frozen, or baked and frozen. If stored frozen, the PCBs were allowed to warm to room temperature (RT) before use in this assay. Bovine serum albumin was spotted on some sensors as a reference protein. Reference protein sensor signals were used to normalize for chip-specific variability in sensor rows and columns, while blank sensors allowed assessment of non-specific signals in clinical samples.

[0101] Example 2 - Assay 1 This assay was performed on a MagArray MR-813 instrument system (i.e., Hanno incubation unit and reader unit). Each plasma sample was serially diluted in phosphate-buffered saline (PBS) containing albumin as a stabilizing buffer. Each component was prepared in the same PBS buffer. Briefly, the process involves a pre-incubation step on an orbital shaker with pre-diluted EDTA-plasma samples (dilution factors: 1 / 8 to 1 / 1024) containing specific concentrations of N-terminally biotinylated Aβ1-40 peptide or N-terminally biotinylated Aβ1-42 peptide (rPeptide, Watkinsville, Georgia, US), followed by a 2-hour incubation at room temperature in the Hanno unit, where the plasma sample is exposed to the capture antibody on the chip. The PCB is then transferred to the reader unit and immersed in the above-mentioned MNP reagent for 20 to 30 minutes in the presence of a magnetic field, during which time signals from each GMR sensor are acquired.

[0102] (antibody) Monoclonal antibodies were obtained from various sources. The characteristics of antibodies 21F12, 2G3, and 3D6 have been previously described (Johnson-Wood et al. (1997) and Bard et al. (2003)). Further epitope mapping experiments were described in Vanderstichele et al. (2005).

[0103] (sample) EDTA-plasma samples were obtained from PrecisionMed (Solana Beach, California, USA), GoldonWest Biosolutions (Temecula, California, USA), or University Centers. Detailed information on gender, age, mini-mental status examinations (MMSE), and CSF biomarker profile was available at the time the samples were ordered. Samples were shipped on dry ice, and upon arrival at the laboratory, they were stored at <-20°C. Care was taken to always use polypropylene recipients with low protein adsorption capacity during sample preparation or aliquoting.

[0104] Three different sample sets were used (Study 1, Study 2, Study 3). Each sample set was divided into EDTA-plasma obtained from healthy controls or subjects diagnosed with Alzheimer's disease. The number of subjects varied as a function of the study protocol, as well as the study design or outcome of this assay (see Table 1).

[0105] Each sample was serially diluted with PBS containing albumin as a stabilizing buffer solution.

[0106] [Table 1]

[0107] Example 3 - Assay 2 This assay protocol was performed on a MagArray MR-813 instrument system. Each sample was serially diluted with PBS containing albumin as a stabilizing buffer. Each component was prepared in the same PBS buffer. Briefly, the process involves a pre-incubation step on an orbital shaker with pre-diluted EDTA-plasma samples (dilution factors: 1 / 8 to 1 / 1024) containing specific concentrations of Aβ1-10-bio (AnaSpec, Fremont, California, USA), followed by a 2-hour incubation at room temperature in a Hanno unit, where the plasma sample is exposed to the capture antibody on the chip. The PCB is then transferred to the Reader unit and immersed in the above-mentioned MNP reagent for 20 to 30 minutes in the presence of a magnetic field, during which time signals from each GMR sensor are acquired.

[0108] (antibody) The characteristics of antibodies 21F12, 2G3, and 3D6 have been previously described (Johnson-Wood et al. (1997) and Bard et al. (2003)). Further epitope mapping experiments were described in Vanderstichele et al. (2005).

[0109] (sample) EDTA-plasma samples were obtained from PrecisionMed (Solana Beach, California, USA), GoldonWest Biosolutions (Temecula, California, USA), or University Centers. Detailed information regarding gender, age, MMSE, and CSF biomarker profile was available at the time of ordering the materials. Materials were shipped on dry ice and stored at <-20°C upon arrival at the laboratory. Care was taken to always use polypropylene receiving containers with low protein adsorption capacity during sample preparation or aliquoting. Four EDTA-plasma samples obtained from healthy controls and four subjects diagnosed with Alzheimer's disease were used in this study. Each sample was serially diluted in PBS solution containing albumin as a stabilizer. Each sample was individually tested on four different 3D6-coated substrates along with other mAbs used as negative, internal, and performance validation controls.

[0110] Example 4 - Assay 3 Substrates were prepared using a mixture of tau-specific or phospho-tau-specific capture antibodies. Monoclonal antibodies were obtained from Thermo Fisher Scientific (Waltham, MA, USA). In this example, AT270 (a mAb specific for tau phosphorylated at position 181) was used as the capture antibody, while biotinylated HT7 (epitope 159–163 of the tau protein) was used as the detection antibody. The characteristics of these mAbs have been previously published (E. Vanmechelen et al. (2000) and H. Vanderstichele et al. (2006)). EDTA-plasma samples were obtained from GoldonWest Biosolutions (Temecula, California, USA). Samples were shipped on dry ice and stored at <−20°C upon arrival at the laboratory. Care was taken to always use polypropylene receiving containers with low protein adsorption capacity during the preparation of various dilutions of the plasma.

[0111] This assay was performed on a MagArray MR-813 instrument system. All components in the assay (peptide, diluted plasma, and biotinylated antibody) were prepared using the same PBS buffer. Plasma samples were serially diluted (1:2 to 1:32) with PBS containing albumin as a stabilizing protein. 240 μL of this prediluted plasma was mixed with 10 μL of buffer containing 2,500 pg / mL of a synthetic peptide (obtained from Proteogenix, Schiltigheim, France) containing the HT7 epitope and phosphorylated at position 181. This added peptide was not biotinylated. An equal volume of buffer containing biotinylated HT7 (200 ng / mL) was then added. This mixture was preincubated in a Hanno unit at room temperature for 1 hour, followed by a 4-hour incubation with the capture antibody in PCB. At the end of this incubation period, the PCB was immersed in the above MNP reagent for 20-30 minutes in the presence of a magnetic field, during which time signals from each GMR sensor were acquired.

[0112] Example 5 - Amino acid sequences of specific human Aβ peptides This example shows the amino acid sequences of certain human Aβ peptides. The amino acid sequence of Aβ1-37 is as follows: 1-DAEFRHDSGYEVHHQKL-VFFAEDVGSNKGAIIGLMVG-37. The amino acid sequence of Aβ1-38 is as follows: 1-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG-38. The amino acid sequence of Aβ1-40 is as follows: 1-DAEFRHDSGYEVHHQKL-VFFAEDVGSNKGAIIGLMVGGVV-40. The amino acid sequence of Aβ1-42 is as follows: 1-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA-42. The amino acid sequence of Aβ43 is as follows: 1-DAEFRHDSGYEVHHQKLV-FFAEDVGSNKGAIIGLMVGGVVIAT-43. The amino acid sequence of Aβ-3-40 is as follows: (-3)-VKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMV-GGVV-40.

[0113] (References) I. Baghallab, et al., Epitomic Characterization of the Specificity of the Anti-Amyloid A Monoclonal Antibodies 6E10 and 4G8, Journal of Alzheimer's Disease 66 (2018) 1235-1244 DOI 10.3233 / JAD-180582. F. Bard, et al., Epitope and isotype specificities of antibodies to beta-amyloid peptide for protection against Alzheimer's disease-like neuropathology. Proc Natl Acad Sci USA 2003; 100:2023-2028. S. Barrio, Methods and reagents for improved detection of amyloid beta peptides, U.S. Publication No. 2012 / 0264642 (October 18, 2012). M. Bjerke, et al., Confounding Factors Influencing Amyloid Beta Concentration in Cerebrospinal Fluid, Int’l J. of Alzheimer’s Disease (2010), https: / / doi.org / 10.4061 / 2010 / 986310. C. Carroll, et al., Heart-type fatty acid binding protein as an early marker for myocardial infarction: systematic review and meta-analysis. J. Emerg Med 2013 (30):280-286. V.C. Cullen, et al., Development and Advanced Validation of an Optimized Method for the Quantitation of Aβ 42 in Human Cerebrospinal Fluid, The AAPS Journal (2012), 14, 510-518. R.S. Gaster, et al., Matrix-insensitive protein assays push the limits of biosensors in medicine. Nat Med. 2009 Nov;15(11):1327-32. doi: 10.1038 / nm.2032). N.P. Groome, et al., Region-specific immunoassays for human myelin basic protein, 1986, J. Neuroimmunol 12: 253-264. M. Gu and J. Viles, Methionine oxidation reduces lag-times for amyloid-β(1-40) fiber formation but generates highly fragmented fibers, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2016; 1864: 1260-1269. M.K. Herbert, et al., Optimisation of the quantification of glutamine synthetase and myelin basic protein in cerebrospinal fluid by a combined acidification and neutralisation protocol, J. Immunol Methods 381:1-8 (2012). Y. Horikoshia, et al., Development of Aβ terminal end-specific antibodies and sensitive ELISA for Aβ variant, Biochemical and Biophysical Research Communications, Volume 319, Issue 3, July 2, 2004, 733-737. J. Ilzecka, Decreased cerebrospinal fluid cGMP levels in patients with amyotrophic lateral sclerosis, 2004 J. Neural Transm 111: 167-72. K. Johnson-Wood, et al., Amyloid precursor protein processing and A beta42 deposition in a transgenic mouse model of Alzheimer disease, Proc Natl Acad Sci USA. 1997 Feb 18;94(4):1550-1555. doi: 10.1073 / pnas.94.4.1550. J.R. Lee, et al., Longitudinal monitoring of antibody responses against tumor cells using magneto-nanosensors with a nanoliter of blood. Nano Lett 17: 6644-6652 (2017). Q. Li, et al., Improvement of a low pH antigen-antibody dissociation procedure for ELISA measurement of circulating anti-Aβ antibodies. BMC Neurosci 8, 22 (2007). https: / / doi.org / 10.1186 / 1471-2202-8-22. R.M. Lyons, et al., Proteolytic activation of latent transforming growth factor-beta from fibroblast-conditioned medium, 1988 J. Cell Biol 106: 1659-1665. E. Mandelkow, et al., Structural principles of tau and the paired helical filaments of Alzheimer's disease, Brain Pathol 2007; 17:83-90. C.M. Mastroianni, et al., Detection of cerebrospinal fluid antibodies against myelin basic protein in patients with AIDS dementia complex, 1991 Mol Cell Neuropathol 14: 227-236. D.A. McGrowder, et al., Cerebrospinal Fluid Biomarkers of Alzheimer's Disease: Current Evidence and Future Perspectives. Brain Sci 2021; 11:215. doi: 10.3390 / brainsci11020215. N. Rezaei-Ghaleh, et al., Phosphorylation modifies the molecular stability of β-amyloid deposits, Nat Commun 2016; Apr 13;7:11359. J.R. Slemmon, et al., Measurement of Aβ1-42 in cerebrospinal fluid is influenced by matrix effects, Journal of Neurochemistry, 2012, 120:325-333. H. Shahpasand-Kroner, et al., A two-step immunoassay for the simultaneous assessment of Aβ38, Aβ40 and Aβ42 in human blood plasma supports the Aβ42 / Aβ40 ratio as a promising biomarker candidate of Alzheimer’s disease, Alzheimer’s Research & Therapy (2018), 10:121. H. Vanderstichele, et al., Amino-truncated beta-amyloid42 peptides in cerebrospinal fluid and prediction of progression of mild cognitive impairment, Clin Chem. 2005 Sep;51(9):1650-60. doi: 10.1373 / clinchem.2005.051201. H. Vanderstichele, et al., Analytical performance and clinical utility of the INNOTEST PHOSPHO-TAU181P assay for discrimination between Alzheimer's disease and dementia with Lewy bodies. Clin Chem Lab Med 2006; 44:1472-80. E. Vanmechelen, et al., Quantification of tau phosphorylated at threonine 181 in human cerebrospinal fluid: a sandwich ELISA with a synthetic phosphopeptide for standardization. Neurosci Lett 2000; 285:49-52. K.G. Warren and I. Catz, A correlation between cerebrospinal fluid mylein basic protein and anti-myelin basic protein in multiple sclerosis patients, 1987 Annal Neurol 21: 183-189. N.C. Wildburger, et al., Diversity of Amyloid-beta Proteoforms in the Alzheimer's Disease Brain, Sci Rep 2017; 7:9520. doi: 10.1038. J. Wiltfang, et al. (2001) Elevation of β-Amyloid Peptide 2-42 in Sporadic and Familial Alzheimer’s Disease and its Generation in PS1 Knockout Cells. J. Biol Chem 276:42645-42657. C. Zettler, et al., Detection of increased tissue concentrations of nerve growth factor with an improved extraction procedure, 1996 J. Neurosci Res 46: 581-594. The present invention provides, for example, the following items. (Item 1) 1. A method for determining whether a subject has a condition correlated with a matrix effect, comprising: (a) mixing (i) an appropriately diluted sample of a suitable fluid from the subject and (ii) an appropriate amount of a labeled molecule, wherein the labeled molecule is subject to a matrix effect with respect to the suitable fluid in a subject suffering from the condition; and (b) determining the amount of matrix-free labeled molecule present in the resulting mixture after a suitable period of time under suitable conditions. Including, if the amount of matrix-unaffected labeled molecule determined in step (b) correlates with a positive control for the condition, then the subject is afflicted with the condition; If the amount correlates with a negative control for the condition, then the subject is not afflicted with the condition. (Item 2) 2. The method of claim 1, wherein step (a) further comprises treating the labeled molecule in a manner that facilitates measurement of the labeled molecule in step (b). (Item 3) 3. The method of claim 2, wherein treating the labeled molecule in a manner that facilitates measurement of the labeled molecule in step (b) comprises contacting the labeled molecule with an appropriate amount of an antibody that specifically binds to the labeled molecule. (Item 4) 4. The method according to any one of items 1 to 3, wherein step (a) comprises mixing the appropriate amount of the labeled molecule with a plurality of serially diluted samples of the fluid. (Item 5) 1. A method for determining whether a subject has a condition correlated with a matrix effect, comprising: (a) mixing (i) an appropriately diluted sample of a suitable fluid from the subject and (ii) an appropriate amount of a first labeled molecule and a second labeled molecule, wherein the labeled molecules are subject to a matrix effect with respect to the suitable fluid in a subject suffering from the condition; and (b) determining a quantitative relationship between the matrix-unaffected first labeled molecules and the matrix-unaffected second labeled molecules present in the resulting mixture after a suitable period of time under suitable conditions; Including, if the relationship determined in step (b) correlates with a positive control for the condition, then the subject is afflicted with the condition; If the correlation correlates with a negative control for the condition, then the subject does not have the condition. method. (Item 6) 6. The method of claim 5, wherein step (a) further comprises treating the labeled molecule in a manner that facilitates measurement of the labeled molecule in step (b). (Item 7) 7. The method of claim 6, wherein treating the labeled molecules in a manner that facilitates measurement of the labeled molecules in step (b) comprises contacting each labeled molecule with an appropriate amount of an antibody that specifically binds to each labeled molecule. (Item 8) 8. The method according to any one of items 5 to 7, wherein step (a) comprises mixing the appropriate amount of the labeled molecule with a plurality of serially diluted samples of the fluid. (Item 9) 9. The method according to any one of items 1 to 8, wherein the subject is a human. (Item 10) 10. The method according to any of items 1 to 9, wherein the condition is a condition associated with a neurodegenerative disorder selected from the group consisting of Alzheimer's disease, Parkinson's disease, mild cognitive impairment, and non-Alzheimer's disease dementia. (Item 11) 10. The method according to any of items 1 to 9, wherein the condition is selected from the group consisting of amyloidopathy, tauopathy, neuronal injury, synucleinopathy, cerebral proteinopathy, progranulinopathy, and neuronal inflammation. (Item 12) 12. The method according to any one of items 1 to 11, wherein the suitable fluid is selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, saliva, tears, ocular fluid, nasal fluid, vitreous humor, and interstitial fluid. (Item 13) 13. The method according to any one of items 1 to 4 and 9 to 12, wherein the labeled molecule is selected from the group consisting of a labeled Aβ peptide or a fragment thereof, a labeled tau protein or a fragment thereof, a labeled phosphorylated tau protein or a fragment thereof, a labeled progranulin or a fragment thereof, a labeled TDP-43 or a fragment thereof, a labeled GFAP or a fragment thereof, a labeled neurogranin or a fragment thereof, a labeled neurofilament or a fragment thereof, and a labeled synuclein or a fragment thereof. (Item 14) 13. The method according to any one of items 5 to 12, wherein the first labeled molecule and the second labeled molecule are selected from the group consisting of a labeled Aβ peptide or a fragment thereof, a labeled tau protein or a fragment thereof, a labeled phosphorylated tau protein or a fragment thereof, a labeled progranulin or a fragment thereof, a labeled TDP-43 or a fragment thereof, a labeled GFAP or a fragment thereof, a labeled neurogranin or a fragment thereof, a labeled neurofilament or a fragment thereof, and a labeled synuclein or a fragment thereof. (Item 15) 1. A kit for use in determining whether a subject has a condition correlated with a matrix effect, comprising: (a) a labeled molecule that is subject to matrix effects with respect to an appropriate fluid in a subject suffering from the condition; and (b) an agent useful for processing the labeled molecule to facilitate measurement of the labeled molecule in a matrix-free form; in separate compartments. (Item 16) 16. The kit of item 15, further comprising, in a separate compartment, an agent useful for measuring the labeled molecule in its matrix-free form. (Item 17) 17. The kit of item 15 or 16, wherein the labeled molecule is selected from the group consisting of a labeled Aβ peptide or a fragment thereof, a labeled tau protein or a fragment thereof, a labeled phosphorylated tau protein or a fragment thereof, a labeled progranulin or a fragment thereof, a labeled TDP-43 or a fragment thereof, a labeled GFAP or a fragment thereof, a labeled neurogranin or a fragment thereof, a labeled neurofilament or a fragment thereof, and a labeled synuclein or a fragment thereof. (Item 18) 1. A kit for use in determining whether a subject has a condition correlated with a matrix effect, comprising: (a) a first labeled molecule and a second labeled molecule, each of which is subject to a matrix effect with respect to an appropriate fluid in a subject suffering from the condition; and (b) an agent useful for treating each of the first labeled molecule and the second labeled molecule to facilitate measurement of the labeled molecule in a matrix-free form; in separate compartments. (Item 19) 19. The kit of item 18, further comprising, in one or more separate compartments, an agent useful for measuring each of the first labeled molecule and the second labeled molecule in its matrix-free form. (Item 20) 20. The kit of item 18 or 19, wherein the first labeled molecule and the second labeled molecule are selected from the group consisting of a labeled Aβ peptide or a fragment thereof, a labeled tau protein or a fragment thereof, a labeled phosphorylated tau protein or a fragment thereof, a labeled progranulin or a fragment thereof, a labeled TDP-43 or a fragment thereof, a labeled GFAP or a fragment thereof, a labeled neurogranin or a fragment thereof, a labeled neurofilament or a fragment thereof, and a labeled synuclein or a fragment thereof. (Item 21) 1. A method for determining whether a human subject has a condition correlated with Alzheimer's disease, comprising: (a) mixing (i) an appropriately diluted sample of a suitable fluid from the subject and (ii) an appropriate amount of labeled Aβ peptide; and (b) determining the amount of matrix-free labeled Aβ peptide present in the resulting mixture after a suitable period of time under suitable conditions. wherein if the amount of matrix-unaffected labeled Aβ peptide determined in step (b) correlates with a positive control for the condition, the subject is affected with the condition, and if the amount correlates with a negative control for the condition, the subject is not affected with the condition. (Item 22) 22. The method according to item 21, wherein step (a) further comprises treating the labeled Aβ peptide in a manner that facilitates measurement of the labeled Aβ peptide in step (b). (Item 23) 23. The method according to item 21 or 22, wherein step (a) comprises mixing the appropriate amount of labeled Aβ peptide with a plurality of serially diluted samples of the fluid. (Item 24) 24. The method according to any one of items 21 to 23, wherein the labeled Aβ peptide is selected from the group consisting of a labeled Aβ40 peptide or a fragment thereof, a labeled Aβ42 peptide or a fragment thereof, a labeled Aβ37 peptide or a fragment thereof, a labeled Aβ38 peptide or a fragment thereof, a labeled Aβ10 peptide or a fragment thereof, a labeled Aβ16 peptide or a fragment thereof, and a labeled Aβ43 peptide or a fragment thereof. (Item 25) 25. The method according to any one of items 21 to 24, wherein the labeled Aβ peptide is a biotinylated Aβ40 peptide or a biotinylated Aβ42 peptide. (Item 26) 1. A method for determining whether a human subject has a condition correlated with Alzheimer's disease, comprising: (a) mixing (i) an appropriately diluted sample of a suitable fluid from the subject and (ii) an appropriate amount of a first labeled Aβ peptide and a second labeled Aβ peptide; and (b) determining a quantitative relationship between the matrix-free first labeled Aβ peptide and the matrix-free second labeled Aβ peptide present in the resulting mixture after a suitable period of time under suitable conditions; Including, if the relationship determined in step (b) correlates with a positive control for the condition, then the subject is afflicted with the condition; If the correlation correlates with a negative control for the condition, then the subject does not have the condition. method. (Item 27) 27. The method according to item 26, wherein step (a) further comprises treating the labeled Aβ peptide in a manner that facilitates measurement of the labeled Aβ peptide in step (b). (Item 28) 28. The method according to item 26 or 27, wherein step (a) comprises mixing the appropriate amount of labeled Aβ peptide with a plurality of serially diluted samples of the fluid. (Item 29) 29. The method according to any one of Items 26 to 28, wherein each of the labeled Aβ peptides is selected from the group consisting of a labeled Aβ40 peptide or a fragment thereof, a labeled Aβ42 peptide or a fragment thereof, a labeled Aβ37 peptide or a fragment thereof, a labeled Aβ38 peptide or a fragment thereof, a labeled Aβ10 peptide or a fragment thereof, a labeled Aβ16 peptide or a fragment thereof, and a labeled Aβ43 peptide or a fragment thereof. (Item 30) 30. The method according to any one of items 26 to 29, wherein the first labeled Aβ peptide and the second labeled Aβ peptide are biotinylated Aβ40 peptide and biotinylated Aβ42 peptide. (Item 31) 31. The method according to any of items 21 to 30, wherein the condition is selected from the group consisting of amyloidopathy, tauopathy, neuronal injury, synucleinopathy, cerebral proteinopathy, progranulinopathy, and neuronal inflammation. (Item 32) 32. The method according to any one of items 21 to 31, wherein the suitable fluid is selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, saliva, tears, ocular fluid, nasal fluid, vitreous humor, and interstitial fluid. (Item 33) (a) labeled Aβ peptide, and (b) an agent useful for processing the labeled Aβ peptide to facilitate measurement of the labeled Aβ peptide in its matrix-free form; in separate compartments. (Item 34) Agents useful for measuring the labeled Aβ peptide in its matrix-free form 34. The kit of item 33, further comprising in a separate compartment: (Item 35) 35. The kit of item 33 or 34, wherein the labeled Aβ peptide is selected from the group consisting of a labeled Aβ40 peptide or a fragment thereof, a labeled Aβ42 peptide or a fragment thereof, a labeled Aβ37 peptide or a fragment thereof, a labeled Aβ38 peptide or a fragment thereof, a labeled Aβ10 peptide or a fragment thereof, a labeled Aβ16 peptide or a fragment thereof, and a labeled Aβ43 peptide or a fragment thereof. (Item 36) 36. The kit according to any one of items 33 to 35, wherein the labeled Aβ peptide is a biotinylated Aβ40 peptide or a biotinylated Aβ42 peptide. (Item 37) (a) a first labeled Aβ peptide and a second labeled Aβ peptide, and (b) an agent useful for treating each of the first labeled Aβ peptide and the second labeled Aβ peptide to facilitate measurement of the labeled Aβ peptide in its matrix-free form; in separate compartments. (Item 38) Agents useful for measuring each of the first labeled Aβ peptide and the second labeled Aβ peptide in a form unaffected by the matrix. 38. The kit of item 37, further comprising in one or more separate compartments: (Item 39) 39. The method of claim 37 or 38, wherein each of the labeled Aβ peptides is selected from the group consisting of a labeled Aβ40 peptide or a fragment thereof, a labeled Aβ42 peptide or a fragment thereof, a labeled Aβ37 peptide or a fragment thereof, a labeled Aβ38 peptide or a fragment thereof, a labeled Aβ10 peptide or a fragment thereof, a labeled Aβ16 peptide or a fragment thereof, and a labeled Aβ43 peptide or a fragment thereof. (Item 40) 40. The kit according to any one of items 37 to 39, wherein the first labeled Aβ peptide and the second labeled Aβ peptide are biotinylated Aβ40 peptide and biotinylated Aβ42 peptide.

Claims

[Claim 1] The invention described in the present specification.

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