U.S. patent application number 17/578728 was filed with the patent office on 2022-05-05 for detection of misfolded tau protein.
This patent application is currently assigned to Amprion, Inc.. The applicant listed for this patent is Amprion, Inc., Board of Regents of the University of Texas System. Invention is credited to Nicolas Mendez Dinamarca, Russell M. Lebovitz, Mohammad Shahnawaz, Claudio Soto-Jara, Benedikt K. Vollrath.
Application Number | 20220137073 17/578728 |
Document ID | / |
Family ID | |
Filed Date | 2022-05-05 |
United States Patent
Application |
20220137073 |
Kind Code |
A1 |
Soto-Jara; Claudio ; et
al. |
May 5, 2022 |
DETECTION OF MISFOLDED TAU PROTEIN
Abstract
Methods and kits are provided for amplifying and detecting
misfolded tau protein from samples, for example, from patients
having tauopathies such as Alzheimer's Disease, Progressive
Supranuclear Palsy, and the like.
Inventors: |
Soto-Jara; Claudio;
(Friendswood, TX) ; Lebovitz; Russell M.;
(Oakland, CA) ; Vollrath; Benedikt K.; (San Diego,
CA) ; Shahnawaz; Mohammad; (Houston, TX) ;
Dinamarca; Nicolas Mendez; (Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Amprion, Inc.
Board of Regents of the University of Texas System |
San Francisco
Austin |
CA
TX |
US
US |
|
|
Assignee: |
Amprion, Inc.
San Francisco
CA
Board of Regents of the University of Texas System
Austin
TX
|
Appl. No.: |
17/578728 |
Filed: |
January 19, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15981449 |
May 16, 2018 |
11249092 |
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17578728 |
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62507166 |
May 16, 2017 |
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International
Class: |
G01N 33/68 20060101
G01N033/68 |
Claims
1. A method for determining a presence or absence in a sample of a
first misfolded protein aggregate, the method comprising:
performing a first protein misfolding cyclic amplification (PMCA)
procedure, the first PMCA procedure comprising: forming a first
incubation mixture by contacting a first portion of the sample with
a first substrate protein, the first substrate protein comprising
4R tau; conducting an incubation cycle two or more times under
conditions effective to form a first amplified, misfolded protein
aggregate, each incubation cycle comprising: incubating the first
incubation mixture effective to cause misfolding and/or aggregation
of at least a portion of the first substrate protein in the
presence of the first misfolded protein aggregate; disrupting the
first incubation mixture effective to form the first amplified,
misfolded protein aggregate; and determining the presence or
absence in the sample of the first misfolded protein aggregate by
analyzing the first incubation mixture for the presence or absence
of the first amplified, misfolded protein aggregate, the first
misfolded protein aggregate comprising the first substrate protein
and the first amplified, misfolded protein aggregate comprising the
first substrate protein.
2. The method of claim 1, comprising determining the presence in
the sample of the first misfolded protein aggregate, the first
misfolded protein aggregate being misfolded 4R tau aggregate.
3. The method of claim 1, further comprising determining the
presence or absence in the sample of at least a second misfolded
protein aggregate.
4. The method of claim 3, further comprising performing at least a
second PMCA procedure to determine the presence or absence in the
sample of at least the second misfolded protein aggregate,
comprising: forming a second incubation mixture by contacting a
second portion of the sample with a second substrate protein, the
second substrate protein being subject to pathological misfolding
and/or aggregation in vivo to form the second misfolded protein
aggregate; conducting an incubation cycle two or more times under
conditions effective to form a second amplified, misfolded protein
aggregate, each incubation cycle comprising: incubating the second
incubation mixture effective to cause misfolding and/or aggregation
of at least a portion of the second substrate protein in the
presence of the second misfolded protein aggregate; disrupting the
second incubation mixture effective to form the second amplified,
misfolded protein aggregate; and determining the presence or
absence in the sample of the second misfolded protein aggregate by
analyzing the second incubation mixture for the presence or absence
of the second amplified, misfolded protein aggregate, the second
misfolded protein aggregate comprising the second substrate protein
and the second amplified, misfolded protein aggregate comprising
the second substrate protein, the second substrate protein
comprising one of: amyloid-beta (A.beta.), alpha synuclein, and 3R
tau.
5. The method of claim 4, the second substrate protein comprising
3R tau, the method further comprising determining a ratio of the 4R
tau and the 3R tau in the sample of between about 1:99 and about
99:1.
6. The method of claim 1, the sample being taken from a subject,
further comprising determining or diagnosing the presence or
absence of a tauopathy in the subject according to the presence or
absence of the first misfolded protein aggregate in the sample.
7. The method of claim 6, further comprising characterizing an
identity of the tauopathy by analyzing the first amplified,
misfolded protein aggregate or one or more corresponding PMCA
kinetic parameters thereof for a signature of at least one of:
Alzheimer's disease (AD), Parkinson's Disease (PD), Progressive
Supranuclear Palsy (PSP), FrontoTemporal Dementia (FTD),
Corticobasal degeneration (CBD), Mild cognitive impairment (MCI),
Argyrophilic grain disease (AgD) Traumatic Brain Injury (TBI),
Chronic Traumatic Encephalopathy (CTE), and Dementia Pugilistica
(DP).
8. The method of claim 7, characterizing the identity of the
tauopathy comprising using one or more of: an antibody selective
for a conformational epitope of a tauopathy-specific misfolded tau
protein aggregate; an indicator selective for the
tauopathy-specific misfolded tau protein aggregate; a spectrum
characteristic of the tauopathy-specific misfolded tau protein
aggregate; a proteolytic resistance of the tauopathy-specific
misfolded tau protein aggregate; and a stability to denaturation of
the tauopathy-specific misfolded tau protein aggregate.
9. The method of claim 1, the sample being taken from a subject
characterized by one of: exhibiting no clinical signs of dementia
according to cognitive testing; exhibiting no cortex plaques or
tangles according to contrast imaging; and exhibiting clinical
signs of dementia according to cognitive testing, further
comprising determining or diagnosing the presence or absence of a
tauopathy in the subject according to the presence or absence of
the first misfolded protein aggregate in the sample.
10. The method of claim 1, comprising preparing the first
incubation mixture characterized by at least one concentration of:
the first substrate protein of less than about 20 .mu.M; heparin of
less than about 75 .mu.M; NaCl of less than about 190 mM; and
Thioflavin T of less than about 9.5 .mu.M.
11. The method of claim 1, comprising preparing or maintaining the
first incubation mixture characterized by one or more of: the first
substrate protein at a concentration between about 0.001 .mu.M and
about 2000 .mu.M; heparin at a concentration between about 0.001
.mu.M and about 75 .mu.M; comprising a buffer composition of one or
more of: Tris-HCL, PBS, MES, PIPES, MOPS, BES, TES, and HEPES, the
buffer composition at a total concentration of between about 1
.mu.M and about 1 M; comprising a salt composition at a total
concentration of between about 1 .mu.M and about 1 M; a pH of
between about 5 and about 9; comprising an indicator at a total
concentration of between about 1 nM and about 1 mM; and a
temperature between about 5.degree. C. and about 60.degree. C.
12. The method of claim 1: further comprising contacting an
indicator of the first misfolded protein aggregate to the first
incubation mixture, the indicator of the first misfolded protein
aggregate being characterized by an indicating state in the
presence of the first misfolded protein aggregate and a
non-indicating state in the absence of the first misfolded protein
aggregate; and wherein the determining the presence of the first
misfolded protein aggregate in the sample comprises detecting the
indicating state of the indicator of the first misfolded protein
aggregate.
13. The method of claim 1, the detecting the first misfolded
protein aggregate comprising one or more of: a Western Blot assay;
a dot blot assay; an enzyme-linked immunosorbent assay (ELISA); a
fluorescent protein/peptide binding assay; a thioflavin binding
assay, a Congo Red binding assay; a sedimentation assay; electron
microscopy; atomic force microscopy; surface plasmon resonance;
spectroscopy; contacting the first incubation mixture with a
protease, and detecting the first misfolded protein aggregate using
anti-misfolded protein antibodies or antibodies specific for a
misfolded tau aggregate in one or more of: a Western Blot assay, a
dot blot assay, and an ELISA.
14. The method of claim 1, the sample characterized by one or more
of: comprising one or more of amniotic fluid; bile; blood;
cerebrospinal fluid; cerumen; skin; exudate; feces; gastric fluid;
lymph; milk; mucus; mucosal membrane; peritoneal fluid; plasma;
pleural fluid; pus; saliva; sebum; semen; sweat; synovial fluid;
tears; and urine; derived from cells or tissue of one or more of:
skin, brain, heart, liver, pancreas, lung, kidney,
gastro-intestine, nerve, mucous membrane, blood cell, gland, and
muscle; and each portion of the sample characterized by a volume
from about 1 .mu.L to about 1000 .mu.L.
15. The method of claim 1, further comprising obtaining the sample
from a subject, the subject being one or more of: at risk of a
tauopathy, having the tauopathy, and under treatment for the
tauopathy.
16. The method of claim 15, the subject being treated with a
tauopathy modulating therapy, further comprising: comparing the
amount of the first misfolded protein aggregate in the sample to an
amount of the first misfolded protein aggregate in a comparison
sample, the sample and the comparison sample being taken from the
subject at different times over a period of time under the
tauopathy modulating therapy; and determining the subject is one
of: responsive to the tauopathy modulating therapy according to a
change in the first misfolded protein aggregate over the period of
time, or non-responsive to the tauopathy modulating therapy
according to homeostasis of the first misfolded protein aggregate
over the period of time.
17. The method of claim 1, further comprising selectively
concentrating the first misfolded protein aggregate in one or more
of the sample and the first incubation mixture.
18. The method of claim 1, the disrupting the first incubation
mixture comprising one or more of: sonication, stirring, cyclic
agitation, shaking, freezing/thawing, laser irradiation, autoclave
incubation, high pressure, and homogenization.
19. The method of claim 1, comprising: contacting the sample with a
thioflavin and a molar excess of the first substrate protein to
form the first incubation mixture, the molar excess being greater
than an amount of the first substrate protein included in the first
misfolded protein aggregate in the sample; conducting the
incubation cycle two or more times effective to form the first
amplified, misfolded protein aggregate, each incubation cycle
comprising: incubating the first incubation mixture effective to
cause misfolding and/or aggregation of the first substrate protein
in the presence of the first misfolded protein aggregate; shaking
the first incubation mixture effective to form the first amplified,
misfolded protein aggregate; and determining the presence of the
first misfolded protein aggregate in the sample by detecting a
fluorescence of the thioflavin corresponding to the first misfolded
protein aggregate.
20. The method of claim 1, provided the sample excludes tau
fibrils.
Description
CROSS-REFERENCE TO RELATED A.beta. PLICATIONS
[0001] This application claims priority from U.S. Provisional
Patent Application No. 62/507,166, filed on May 16, 2017, the
entire contents of which are incorporated herein by reference.
BACKGROUND
[0002] Tauopathies may include, for example, Alzheimer's disease
(AD), Parkinson's Disease (PD), Progressive Supranuclear Palsy
(PSP), FrontoTemporal Dementia (FTD), Corticobasal degeneration
(CBD), Mild cognitive impairment (MCI), Argyrophilic grain disease
(AgD) Traumatic Brain Injury (TBI), Chronic Traumatic
Encephalopathy (CTE), and Dementia Pugilistica (DP), and the like.
Misfolded tau aggregates and fibrils may be formed and accumulate
via nucleation and growth. The misfolded tau aggregates may induce
cellular dysfunction and tissue damage, among other effects.
[0003] Real time quaking-induced conversion (RT-QuiC) has been
shown to cause replication of 3-repeat (3R) tau isoforms from brain
homogenate and cerebrospinal fluid samples drawn from Pick disease
subjects, allowing sensitive detection of this rare disease and
discrimination from other tauopathies. Surprisingly, however, for
more common tauopathies of clinical importance that include
misfolding of 4R tau, the efficacy of RT-QuiC was reduced by 3 to 5
orders of magnitude, rendering it ineffective and impractical for
clinical and laboratory use. Such adverse results were obtained by
seeding with brain samples containing predominant 4-repeat (4R) tau
aggregates from cases of CBD, AgD, and FTDP-17, and PSP, as well as
AD, a 4R+3R tauopathy. Some AD and PSP samples gave signals above
the detection limit, but the signals were outliers and much weaker
compared to Pick disease brain samples. Additionally, the AD and
PSP samples which generated weak responses were not analyzed for
contamination. The RT-QuiC analyses of 4R or 4R+3R tauopathies in
general do not appear to be significantly different from controls
using diseased subjects with no immunohistologically detected tau
pathology. Such controls included diagnoses of senile change (SC),
cerebrovascular disease (CVD), diffuse Lewy body disease (DLBD),
frontotemporal dementia with TDP-43 (FTD-TDP), and amyotrophic
lateral sclerosis (ALS). In sum, RT-QuiC analyses were shown to be
generally ineffective and impractical for 4R tauopathies including
4R predominant and 4R+3R mixed tauopathies.
[0004] The present application appreciates that detection of
misfolded tau protein, e.g., for diagnosis of related diseases, may
be a challenging endeavor.
SUMMARY
[0005] In one embodiment, a method is provided for determining a
presence or absence in a sample of a first misfolded protein
aggregate. The method may include performing a first protein
misfolding cyclic amplification (PMCA) procedure. The first PMCA
procedure may include forming a first incubation mixture by
contacting a first portion of the sample with a first substrate
protein. The first substrate protein may include 4R tau protein.
The first PMCA procedure may include conducting an incubation cycle
two or more times under conditions effective to form as first
amplified, misfolded protein aggregate. Each incubation cycle may
include incubating the first incubation mixture effective to cause
misfolding and/or aggregation of the first substrate protein in the
presence of the first misfolded protein aggregate. Each incubation
cycle may include disrupting the first incubation mixture effective
to form the first amplified, misfolded protein aggregate. The first
PMCA procedure may include determining the presence or absence in
the sample of the first misfolded protein aggregate by analyzing
the first incubation mixture for the presence or absence of the
first amplified, misfolded protein aggregate. The first misfolded
protein aggregate may include the first substrate protein. The
first amplified, misfolded protein aggregate may include the first
substrate protein.
[0006] In another embodiment, a method is provided for determining
a presence or absence in a subject of a tauopathy corresponding to
a first misfolded protein aggregate. The method may include
providing a sample from the subject. The method may include
performing at least a first PMCA procedure. The first PMCA
procedure may include forming a first incubation mixture by
contacting a first portion of the sample with a first substrate
protein. The first substrate protein may include a tau isoform. The
first substrate protein may be subject to pathological misfolding
and/or aggregation in vivo to form the first misfolded protein
aggregate. The first PMCA procedure may include conducting an
incubation cycle two or more times under conditions effective to
form a first amplified, misfolded protein aggregate. Each
incubation cycle may include incubating the first incubation
mixture effective to cause misfolding and/or aggregation of the
first substrate protein in the presence of the first misfolded
protein aggregate. Each incubation cycle may include disrupting the
first incubation mixture effective to form the first amplified,
misfolded protein aggregate. The first PMCA procedure may include
determining the presence or absence in the sample of the first
misfolded protein aggregate by analyzing the first incubation
mixture for the presence or absence of the first amplified,
misfolded protein aggregate. The first PMCA procedure may include
determining the presence or absence of the tauopathy in the subject
according the presence or absence of the first misfolded protein
aggregate in the sample. The first misfolded protein aggregate may
include the first substrate protein. The first amplified, misfolded
protein aggregate may include the first substrate protein. The
method may provide that the tauopathy excludes Pick's disease when
the first substrate protein consists of monomeric 3R tau.
[0007] In one embodiment, a method is provided using capturing for
determining a presence or absence in a sample of a first misfolded
protein aggregate. The method may include capturing the first
misfolded protein aggregate from the sample to form a captured
first misfolded protein aggregate. The method may include
performing at least a first PMCA procedure. The first PMCA
procedure may include forming a first incubation mixture by
contacting the captured first misfolded protein aggregate with a
molar excess of a first substrate protein. The first substrate
protein may be subject to pathological misfolding and/or
aggregation in vivo to form the first misfolded protein aggregate.
The molar excess may be greater than an amount of protein monomer
included in the captured first misfolded protein aggregate. The
method may include conducting an incubation cycle two or more times
effective to form a first amplified, misfolded protein aggregate.
Each incubation cycle may include incubating the first incubation
mixture effective to cause misfolding and/or aggregation of the
first substrate protein in the presence of the captured first
misfolded protein aggregate. Each incubation cycle may include
disrupting the first incubation mixture effective to form the first
amplified, misfolded protein aggregate. The first PMCA procedure
may include determining the presence or absence of the first
misfolded protein aggregate in the sample by detecting the first
amplified, misfolded protein aggregate. The first misfolded protein
aggregate may include the first substrate protein. The first
amplified, misfolded protein aggregate may include the first
substrate protein.
[0008] In another embodiment, a method is provided for determining
a presence or absence of a tauopathy in a subject, the tauopathy
including Alzheimer's disease (AD). The method may include
providing the subject. The method may include obtaining a sample
from the subject. The sample may include one or more of: a
bio-fluid, a biomaterial, a homogenized tissue, and a cell lysate.
The method may include performing at least a first PMCA procedure.
The first PMCA procedure may include forming a first incubation
mixture by contacting the sample with a first substrate protein.
The first substrate protein may include 4R tau. The first PMCA
procedure may include conducting an incubation cycle two or more
times effective to form a first amplified, misfolded protein
aggregate. Each incubation cycle may include incubating the first
incubation mixture effective to cause misfolding and/or aggregation
of the first substrate protein in the presence of the first
misfolded protein aggregate. Each incubation cycle may include
disrupting the incubation mixture effective to form the first
amplified, misfolded protein aggregate. The first PMCA procedure
may include determining the presence or absence in the sample of
the first misfolded protein aggregate by detecting in the first
incubation mixture the presence or absence of the first amplified,
misfolded protein aggregate. The method may include determining the
presence or absence in the subject of AD according to the presence
or absence of the first misfolded protein aggregate in the
sample.
[0009] In one embodiment, a method is provided for determining a
presence or absence of a tauopathy in a subject, the tauopathy
including Parkinson's disease (PD). The method may include
providing the subject. The method may include obtaining a sample
from the subject. The sample may include one or more of: a
bio-fluid, a biomaterial, a homogenized tissue, and a cell lysate.
The method may include performing at least a first PMCA procedure.
The first PMCA procedure may include forming a first incubation
mixture by contacting the sample with a first substrate protein.
The first substrate protein may include 4R tau. The first PMCA
procedure may include conducting an incubation cycle two or more
times effective to form a first amplified, misfolded protein
aggregate. Each incubation cycle may include incubating the first
incubation mixture effective to cause misfolding and/or aggregation
of the first substrate protein in the presence of the first
misfolded protein aggregate. Each incubation cycle may include
disrupting the incubation mixture effective to form the first
amplified, misfolded protein aggregate. The first PMCA procedure
may include determining the presence or absence in the sample of
the first misfolded protein aggregate by detecting in the first
incubation mixture the presence or absence of the first amplified,
misfolded protein aggregate. The method may include determining the
presence or absence in the subject of PD according to the presence
or absence of the first misfolded protein aggregate in the
sample.
[0010] In another embodiment, a method is provided for determining
a presence or absence of a tauopathy in a subject, the tauopathy
including Progressive Supranuclear Palsy (PSP). The method may
include providing the subject. The method may include obtaining a
sample from the subject. The sample may include one or more of: a
bio-fluid, a biomaterial, a homogenized tissue, and a cell lysate.
The method may include performing at least a first PMCA procedure.
The first PMCA procedure may include forming a first incubation
mixture by contacting the sample with a first substrate protein.
The first substrate protein may include 4R tau. The first PMCA
procedure may include conducting an incubation cycle two or more
times effective to form a first amplified, misfolded protein
aggregate. Each incubation cycle may include incubating the first
incubation mixture effective to cause misfolding and/or aggregation
of the first substrate protein in the presence of the first
misfolded protein aggregate. Each incubation cycle may include
disrupting the incubation mixture effective to form the first
amplified, misfolded protein aggregate. The first PMCA procedure
may include determining the presence or absence in the sample of
the first misfolded protein aggregate by detecting in the first
incubation mixture the presence or absence of the first amplified,
misfolded protein aggregate. The method may include determining the
presence or absence in the subject of PSP according to the presence
or absence of the first misfolded protein aggregate in the
sample.
[0011] In one embodiment, a method is provided for determining a
presence or absence of a tauopathy in a subject, the tauopathy
including FrontoTemporal Dementia (FTD). The method may include
providing the subject. The method may include obtaining a sample
from the subject. The sample may include one or more of: a
bio-fluid, a biomaterial, a homogenized tissue, and a cell lysate.
The method may include performing at least a first PMCA procedure.
The first PMCA procedure may include forming a first incubation
mixture by contacting the sample with a first substrate protein.
The first substrate protein may include 4R tau. The first PMCA
procedure may include conducting an incubation cycle two or more
times effective to form a first amplified, misfolded protein
aggregate. Each incubation cycle may include incubating the first
incubation mixture effective to cause misfolding and/or aggregation
of the first substrate protein in the presence of the first
misfolded protein aggregate. Each incubation cycle may include
disrupting the incubation mixture effective to form the first
amplified, misfolded protein aggregate. The first PMCA procedure
may include determining the presence or absence in the sample of
the first misfolded protein aggregate by detecting in the first
incubation mixture the presence or absence of the first amplified,
misfolded protein aggregate. The method may include determining the
presence or absence in the subject of FTD according to the presence
or absence of the first misfolded protein aggregate in the
sample.
[0012] In another embodiment, a method is provided for determining
a presence or absence of a tauopathy in a subject, the tauopathy
including Corticobasal degeneration (CBD). The method may include
providing the subject. The method may include obtaining a sample
from the subject. The sample may include one or more of: a
bio-fluid, a biomaterial, a homogenized tissue, and a cell lysate.
The method may include performing at least a first PMCA procedure.
The first PMCA procedure may include forming a first incubation
mixture by contacting the sample with a first substrate protein.
The first substrate protein may include 4R tau. The first PMCA
procedure may include conducting an incubation cycle two or more
times effective to form a first amplified, misfolded protein
aggregate. Each incubation cycle may include incubating the first
incubation mixture effective to cause misfolding and/or aggregation
of the first substrate protein in the presence of the first
misfolded protein aggregate. Each incubation cycle may include
disrupting the incubation mixture effective to form the first
amplified, misfolded protein aggregate. The first PMCA procedure
may include determining the presence or absence in the sample of
the first misfolded protein aggregate by detecting in the first
incubation mixture the presence or absence of the first amplified,
misfolded protein aggregate. The method may include determining the
presence or absence in the subject of CBD according to the presence
or absence of the first misfolded protein aggregate in the
sample.
[0013] In one embodiment, a kit is provided for determining a
presence or absence in a sample of a first misfolded protein
aggregate. The kit may include a first substrate protein that may
include 4R tau. The kit may include an indicator of the first
misfolded protein aggregate. The first misfolded protein aggregate
may include the first substrate protein. The first misfolded
protein aggregate may correspond to a tauopathy. The kit may
include a buffer. The kit may include heparin. The kit may include
a salt. The kit may include instructions. The instructions may
direct a user to obtain the sample. The instructions may direct the
user to perform at least a first PMCA procedure. The first PMCA
procedure may include forming a first incubation mixture by
contacting a first portion of the sample with the first substrate
protein, the indicator of the first misfolded protein aggregate,
the buffer, the heparin, and the salt. The first incubation mixture
may be formed with a concentration of one or more of: the first
substrate protein of less than about 20 .mu.M; the heparin of less
than about 75 .mu.M; the salt as NaCl of less than about 190 mM;
and the indicator of the first misfolded protein aggregate as
Thioflavin T of less than about 9.5 .mu.M. The first PMCA procedure
may include conducting an incubation cycle two or more times
effective to form a first amplified, misfolded protein aggregate.
Each incubation cycle may include incubating the first incubation
mixture effective to cause misfolding and/or aggregation of the
first substrate protein in the presence of the first misfolded
protein aggregate. Each incubation cycle may include disrupting the
incubation mixture effective to form the first amplified, misfolded
protein aggregate. The instructions may direct the user to
determine the presence or absence in the sample of the first
misfolded protein aggregate by analyzing the first incubation
mixture for the presence or absence of the first amplified,
misfolded protein aggregate according to the indicator of the first
misfolded protein aggregate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The accompanying figures, which are incorporated in and
constitute a part of the specification, illustrate example methods
and results, and are used merely to illustrate example
embodiments.
[0015] FIG. 1A shows electron micrographs taken at 0 h, 5 h, 10 h,
and 24 h of incubation.
[0016] FIG. 1B is a western blot of soluble oligomeric A.beta.
protein aggregates.
[0017] FIG. 2A is a graph showing non-amplified amyloid formation
measured by ThT fluorescence as a function of time seeded by
various concentrations of synthetic soluble oligomeric A.beta.
protein of EXAMPLE 1.
[0018] FIG. 2B is a graph showing amplification cycle-accelerated
amyloid formation measured by ThT fluorescence as a function of
time seeded by various concentrations of synthetic soluble
oligomeric A.beta. protein of EXAMPLE 1.
[0019] FIG. 3A is a graph of amyloid formation versus time,
measured as a function of ThT fluorescence labeling, showing the
average kinetics of A.beta. aggregation seeded by CSF from 5
representative samples from the AD, NND, and NAND groups.
[0020] FIG. 3B is a graph of the lag phase time in h for A.beta.
aggregation in the presence of samples from the AD, NND, and NAND
groups.
[0021] FIG. 3C is a graph showing the extent of amyloid formation
obtained after 180 A.beta.-PMCA cycles, e.g. 90 h of incubation
(P90) in the presence of CSF samples from AD, NND and NAND
patients.
[0022] FIGS. 4A-D are plots of the true positive rate (sensitivity)
as a function of the false positive rate (specificity) for
different cut-off points using the lag phase values showed in FIG.
3B for AD vs NAND (FIG. 4A), AD vs NND (FIG. 4B) and AD vs All
control samples (FIG. 4C). FIG. 4D estimates the most reliable
cut-off point for the different set of group comparisons.
[0023] FIG. 5, Table 1 shows estimations of the sensitivity,
specificity and predictive value of the A.beta.-PMCA test,
calculated using the lag phase numbers.
[0024] FIG. 6 is a graph of the lag phase time in h for samples
obtained after 300 A.beta.-PMCA cycles, e.g. 150 h of incubation
(P90) in the presence of CSF samples from AD and control
patients.
[0025] FIG. 7A is a western blot showing results of immunodepletion
using synthetically prepared A.beta. oligomers spiked into human
CSF.
[0026] FIG. 7B is a graph showing the kinetics of A.beta.
aggregation seeded by control and immunodepleted CSF samples.
[0027] FIG. 7C is a graph showing the kinetics of A.beta.
aggregation seeded by control and immunodepleted CSF samples,
depleted only with the All conformational antibody.
[0028] FIG. 8A is a schematic representation of an ELISA solid
phase method employed to capture A.beta. oligomers from complex
biological samples.
[0029] FIG. 8B is a schematic representation of a magnetic bead
solid phase method employed to capture A.beta. oligomers from
complex biological samples.
[0030] FIG. 9, Table 2 shows the ability of specific antibodies to
capture the A.beta. oligomers.
[0031] FIG. 10 is a graph of amyloid formation versus time showing
the acceleration of A.beta. aggregation by the presence of
different quantities of synthetic oligomers spiked in human
plasma.
[0032] FIG. 11 is a graph showing time to reach 50% aggregation in
an A.beta.-PMCA assay in the presence of plasma samples from AD
patients and controls.
[0033] FIG. 12 is a western blot showing the results of
amplification of A.beta. aggregation by cycles of
incubation/sonication in the presence of distinct quantities of
synthetic A.beta. oligomers monitored by Western blot after
protease digestion.
[0034] FIG. 13A is a graph of Thioflavin T fluorescence versus time
showing the detection of .alpha.S seeds by PD-PMCA.
[0035] FIG. 13B is a graph of time to reach 50% aggregation plotted
as a function of the indicated amounts .alpha.S seeds.
[0036] FIG. 14 shows detection of .alpha.S seeds in CSF samples
from human PD patients by PD-PMCA, versus controls with Alzheimer's
disease (AD) or a non-neurodegenerative disease (NND).
[0037] FIG. 15, Table 3 demonstrates the ability of different
sequence or conformational antibodies to capture .alpha.S
oligomers.
[0038] FIG. 16A is a schematic representation of an ELISA solid
phase method employed to capture .alpha.S oligomers.
[0039] FIG. 16B is a schematic representation of a magnetic bead
solid phase method employed to capture .alpha.S oligomers.
[0040] FIGS. 17A, 17B, and 17C are a series of graphs that show the
results of immunoprecipitation/aggregation of .alpha.-Synuclein
oligomers from human blood plasma using three different
.alpha.-Synuclein antibodies. FIG. 17A shows results with antibody
N-19. FIG. 17B shows results with antibody 211. FIG. 17C shows
results with antibody C-20.
[0041] FIGS. 18A, 18B, and 18C are a series of graphs that show the
results of detection for .alpha.S seeds in CSF samples. FIG. 18A
shows results in control samples. FIG. 18B shows results in PD
patients. FIG. 18C shows results in patients with Multiple System
Atrophy (MSA).
[0042] FIG. 19 is a flow chart showing the preparation and
purification of recombinant full-length 4R tau protein.
[0043] FIG. 20A is a graph of aggregation in % according to ThT
fluorescence for various initial amounts of tau seeds and a
control. The values in FIG. 20A are the mean of two replicates,
with the error bars indicating standard deviation.
[0044] FIG. 20B is a graph of T.sub.50, the time to 50% aggregation
as measured by ThT fluorescence versus the log of the amount of
oligomeric tau seeds in fmol.
[0045] FIG. 20C is a graph of aggregation followed over time by ThT
fluorescence.
[0046] FIG. 20D is a graph of the relationship between the quantity
of tau oligomers and the Tau-PMCA signal (time to reach 50%
aggregation).
[0047] FIGS. 20E-20L are a series of graphs that display the
aggregation results based on ThT fluorescence of 8 of the
conditions tested, including 4 different time points (0, 7, 14 and
30 days) with samples subjected to freezing and thawing or not and
in the presence of buffer or CSF.
[0048] FIG. 20E is a graph of aggregation based on ThT fluorescence
of a first seed preparation at 0 days.
[0049] FIG. 20F is a graph of aggregation based on ThT fluorescence
of a first seed preparation at 7 days.
[0050] FIG. 20G is a graph of aggregation based on ThT fluorescence
of a first seed preparation at 14 days.
[0051] FIG. 20H is a graph of aggregation based on ThT fluorescence
of a first seed preparation at 30 days.
[0052] FIG. 20I is a graph of aggregation based on ThT fluorescence
of a second seed preparation at 0 days.
[0053] FIG. 20J is a graph of aggregation based on ThT fluorescence
of a second seed preparation at 7 days.
[0054] FIG. 20K is a graph of aggregation based on ThT fluorescence
of a second seed preparation at 14 days.
[0055] FIG. 20L is a graph of aggregation based on ThT fluorescence
of a second seed preparation at 30 days.
[0056] FIG. 20M is a table of Tso values showing reproducibility
across 16 different conditions.
[0057] FIG. 20N is a graph of ThT fluorescence vs time for the tau
assay seeded with 1 pm of tau, A.beta.40, AB42, His .alpha.Syn, Hu
.alpha.Syn, and a control with no seeds.
[0058] FIG. 21A is a graph showing ThT fluorescence at 447 h of
incubation for patients with AD, patients with MCI, patients with
other tauopathies, positive controls using samples of healthy CSF
spiked with synthetic Tau oligomers (12.5 fmol), negative controls
of samples of healthy CSF without Tau seeds; and control patients
with other neurological diseases.
[0059] FIG. 21B shows fluorescence signals for samples from
patients with AD or other tauopathies for tau-PMCA comparable to
that observed in samples containing recombinant tau oligomers.
[0060] FIG. 22 is a graph showing aggregation % based on ThT versus
time for patients affected by AD, FTD (frontotemporal dementia),
CBD (corticobasal degeneration), and PSP (progressive supranuclear
palsy), versus representative CSF samples from a control.
DETAILED DESCRIPTION
[0061] Methods and kits are provided for the detection or
characterization of misfolded tau protein in a sample, including
for the determination or diagnosis of tauopathies in a subject from
which the sample is taken. Misfolded aggregates of tau proteins may
be formed and accumulate. The misfolded aggregates may induce
cellular dysfunction and tissue damage among other effects. For
example, tauopathies may include those that predominantly regard
misfolding of 4R, or misfolding of mixtures of 4R and 3R:
Alzheimer's disease (AD), Parkinson's Disease (PD), Progressive
Supranuclear Palsy (PSP), FrontoTemporal Dementia (FTD),
Corticobasal degeneration (CBD), Mild cognitive impairment (MCI),
Argyrophilic grain disease (AgD) Traumatic Brain Injury (TBI),
Chronic Traumatic Encephalopathy (CTE), Dementia Pugilistica (DP),
and the like.
[0062] In some embodiments, tauopathies herein may exclude Pick's
disease. In some embodiments, the tauopathies described herein may
exclude those that predominantly regard 3R tau misfolding, e.g.,
Pick's disease.
[0063] The methods may include protein misfolding cyclic
amplification (PMCA), which may provide ultra-sensitive detection
of misfolded protein aggregates such as tau through artificial
acceleration and amplification of the misfolding and aggregation
process in vitro. The basic concept of PMCA has been previously
demonstrated experimentally for prions (Soto et al, WO 2002/04954;
Estrada, et al., U.S. Pat. App. Pub. No. 20080118938, each of which
is entirely incorporated herein by reference) and for other protein
misfolding, such as of "A.beta." or "beta amyloid" in Alzheimer's
disease and alpha synuclein in Parkinson's disease (Soto et al, WO
2016/040907, which is entirely incorporated herein by reference).
However, prior to the filing date of the present document, no
reference has described PCMA for the amplification and detection of
misfolded tau protein corresponding to any tauopathy that
predominantly regards misfolding of 4R, or that regards misfolding
of 3R tau in the presence of 4R tau, or for any tauopathy other
than Pick's disease. This document discloses specific examples and
details which enable PMCA technology for the detecting the presence
or absence of misfolded tau aggregates, and, in various
embodiments, one or more additional PMCA procedures for the
detection of other misfolded proteins such as misfolded A.beta. in
Alzheimer's disease and alpha synuclein in Parkinson's disease.
Such one or more additional PMCA procedures may provide
discrimination among the various tauopathies, for example, to
distinguish AD and PD from each other and from PSP, FTD, CBD, MCI,
AgD, TBI, CTE, DP, and the like.
[0064] As used herein, "A.beta." or "beta amyloid" refers to a
peptide formed via sequential cleavage of the amyloid precursor
protein (A.beta. P). Various A.beta. isoforms may include 38-43
amino acid residues. The A.beta. protein may be formed when A.beta.
P is processed by .beta.- and/or .gamma.-secretases in any
combination. The A.beta. may be a constituent of amyloid plaques in
brains of individuals suffering from or suspected of having AD.
Various A.beta. isoforms may include and are not limited to Abeta40
and Abeta42. Various A.beta. peptides may be associated with
neuronal damage associated with AD.
[0065] As used herein, ".alpha.S" or "alpha-synuclein" refers to
full-length, 140 amino acid .alpha.-synuclein protein, e.g.,
".alpha.S-140." Other isoforms or fragments may include
".alpha.S-126," alpha-synuclein-126, which lacks residues 41-54,
e.g., due to loss of exon 3; and ".alpha.S-112"
alpha-synuclein-112, which lacks residue 103-130, e.g., due to loss
of exon 5. The .alpha.S may be present in brains of individuals
suffering from PD or suspected of having PD. Various .alpha.S
isoforms may include and are not limited to .alpha.S-140,
.alpha.S-126, and .alpha.S-112. Various .alpha.S peptides may be
associated with neuronal damage associated with PD.
[0066] As used herein, "tau" refers to proteins are the product of
alternative splicing from a single gene, e.g., MA.beta. T
(microtubule-associated protein tau) in humans. Tau proteins
include up to full-length and truncated forms of any of tau's
isoforms. Various isoforms include, but are not limited to, the six
tau isoforms known to exist in human brain tissue, which correspond
to alternative splicing in exons 2, 3, and 10 of the tau gene.
Three isoforms have three binding domains and the other three have
four binding domains. Misfolded tau may be present in brains of
individuals suffering from AD or suspected of having AD, or other
tauopathies that, like AD, regard misfolding in the presence of
both 4R and 3R tau isoforms. Misfolded tau may also be present in
diseases that regard misfolding of primarily 4R tau isoforms, such
as progressive supranuclear palsy (PSP), tau-dependent
frontotemporal dementia (FTD), corticobasal degeneration (CBD),
mild cognitive impairment (MCI), argyrophilic grain disease (AgD),
and the like.
[0067] As used herein, a "misfolded protein aggregate" is a protein
that contains in part or in full a structural conformation of the
protein that differs from the structural conformation that exists
when involved in its typical, non-pathogenic normal function within
a biological system. A misfolded protein may aggregate. A misfolded
protein may localize in a protein aggregate. A misfolded protein
may be a non-functional protein. A misfolded protein may be a
pathogenic conformer of the protein. Monomeric protein compositions
may be provided in native, nonpathogenic conformations without the
catalytic activity for misfolding, oligomerization, and aggregation
associated with seeds (a misfolded protein oligomer capable of
catalyzing misfolding under PMCA conditions). Monomeric protein
compositions may be provided in seed-free form.
[0068] As used herein, "monomeric protein" refers to single protein
molecules. "Soluble, aggregated misfolded protein" refers to
oligomers or aggregations of monomeric protein that remain in
solution. Examples of soluble, misfolded protein may include any
number of protein monomers so long as the misfolded protein remains
soluble. For example, soluble, misfolded protein may include
monomers or aggregates of between 2 and about 50 units of monomeric
protein.
[0069] Monomeric and/or soluble, misfolded protein may aggregate to
form insoluble aggregates, higher oligomers, and/or tau fibrils.
For example, aggregation of A.beta. or tau protein may lead to
protofibrils, fibrils, and eventually misfolded plaques or tangles
that may be observed in AD or tauopathy subjects. "Seeds" or
"nuclei" refer to misfolded protein or short fragmented fibrils,
particularly soluble, misfolded protein with catalytic activity for
further misfolding, oligomerization, and aggregation. Such
nucleation-dependent aggregation may be characterized by a slow lag
phase wherein aggregate nuclei may form, which may then catalyze
rapid formation of further aggregates and larger oligomers and
polymers. The lag phase may be minimized or removed by addition of
pre-formed nuclei or seeds. Monomeric protein compositions may be
provided without the catalytic activity for misfolding and
aggregation associated with misfolded seeds. Monomeric protein
compositions may be provided in seed-free form.
[0070] As used herein, "soluble" species may form a solution in
biological fluids under physiological conditions, whereas
"insoluble" species may be present as precipitates, fibrils,
deposits, tangles, or other non-dissolved forms in such biological
fluids under physiological conditions. Such biological fluids may
include, for example, fluids, or fluids expressed from one or more
of: amniotic fluid; bile; blood; cerebrospinal fluid; cerumen;
skin; exudate; feces; gastric fluid; lymph; milk; mucus, e.g. nasal
secretions; mucosal membrane, e.g., nasal mucosal membrane;
peritoneal fluid; plasma; pleural fluid; pus; saliva; sebum; semen;
sweat; synovial fluid; tears; urine; and the like. Insoluble
species may include, for example, fibrils of A.beta., .alpha.S, 4R
tau, 3R tau, combinations thereof such as 3R tau+4R tau, and the
like. A species that dissolves in a non-biological fluid but not
one of the aforementioned biological fluids under physiological
conditions may be considered insoluble. For example, fibrils of
A.beta., .alpha.S, 4R tau, 3R tau, combinations thereof such as 3R
tau+4R tau, and the like may be dissolved in a solution of, e.g., a
surfactant such as sodium dodecyl sulfate (SDS) in water, but may
still be insoluble in one or more of the mentioned biological
fluids under physiological conditions.
[0071] In some embodiments, the sample may exclude insoluble
species of the misfolded proteins such as A.beta., .alpha.S, 4R
tau, 3R tau, combinations thereof such as 3R tau+4R tau and the
like as a precipitate, fibril, deposit, tangle, plaque, or other
form that may be insoluble in one or more of the described
biological fluids under physiological conditions.
[0072] For example, in some embodiments, the sample may exclude tau
in fibril form. The sample may exclude misfolded tau proteins in
insoluble form, e.g., the sample may exclude the misfolded tau
proteins as precipitates, fibrils, deposits, tangles, plaques, or
other insoluble forms, e.g., in fibril form. The methods described
herein may include preparing the sample by excluding the misfolded
protein in insoluble form, e.g., by excluding from the sample the
misfolded tau protein as precipitates, fibrils, deposits, tangles,
plaques, or other insoluble forms, e.g., in fibril form. The kits
described herein may include instructions directing a user to
prepare the sample by excluding from the sample the misfolded tau
protein as precipitates, fibrils, deposits, tangles, plaques, or
other insoluble forms, e.g., in fibril form. The exclusion of such
insoluble forms of the described misfolded proteins from the sample
may be substantial or complete.
[0073] As used herein, aggregates of misfolded protein refer to
non-covalent associations of protein including soluble, misfolded
protein. Aggregates of misfolded protein may be "de-aggregated", or
disrupted to break up or release soluble, misfolded protein. The
catalytic activity of a collection of soluble, misfolded protein
seeds may scale, at least in part with the number of such seeds in
a mixture. Accordingly, disruption of aggregates of misfolded
protein in a mixture to release misfolded protein seeds may lead to
an increase in catalytic activity for oligomerization or
aggregation of monomeric protein.
[0074] In various embodiments, a method is provided for determining
a presence or absence in a sample of a first misfolded protein
aggregate. The method may include performing a first protein
misfolding cyclic amplification (PMCA) procedure. The first PMCA
procedure may include forming a first incubation mixture by
contacting a first portion of the sample with a first substrate
protein. The first substrate protein may include 4R tau protein.
The first PMCA procedure may include conducting an incubation cycle
two or more times under conditions effective to form as first
amplified, misfolded protein aggregate. Each incubation cycle may
include incubating the first incubation mixture effective to cause
misfolding and/or aggregation of the first substrate protein in the
presence of the first misfolded protein aggregate. Each incubation
cycle may include disrupting the first incubation mixture effective
to form the first amplified, misfolded protein aggregate. The first
PMCA procedure may include determining the presence or absence in
the sample of the first misfolded protein aggregate by analyzing
the first incubation mixture for the presence or absence of the
first amplified, misfolded protein aggregate. The first misfolded
protein aggregate may include the first substrate protein. The
first amplified, misfolded protein aggregate may include the first
substrate protein.
[0075] In various embodiments, a method is provided for determining
a presence or absence in a subject of a tauopathy corresponding to
a first misfolded protein aggregate. The method may include
providing a sample from the subject. The method may include
performing at least a first PMCA procedure. The first PMCA
procedure may include forming a first incubation mixture by
contacting a first portion of the sample with a first substrate
protein. The first substrate protein may include a tau isoform. The
first substrate protein may be subject to pathological misfolding
and/or aggregation in vivo to form the first misfolded protein
aggregate. The first PMCA procedure may include conducting an
incubation cycle two or more times under conditions effective to
form a first amplified, misfolded protein aggregate. Each
incubation cycle may include incubating the first incubation
mixture effective to cause misfolding and/or aggregation of the
first substrate protein in the presence of the first misfolded
protein aggregate. Each incubation cycle may include disrupting the
first incubation mixture effective to form the first amplified,
misfolded protein aggregate. The first PMCA procedure may include
determining the presence or absence in the sample of the first
misfolded protein aggregate by analyzing the first incubation
mixture for the presence or absence of the first amplified,
misfolded protein aggregate. The first PMCA procedure may include
determining the presence or absence of the tauopathy in the subject
according the presence or absence of the first misfolded protein
aggregate in the sample. The first misfolded protein aggregate may
include the first substrate protein. The first amplified, misfolded
protein aggregate may include the first substrate protein. The
method may provide that the tauopathy excludes Pick's disease when
the first substrate protein consists of monomeric 3R tau.
[0076] In various embodiments, a method is provided using capturing
for determining a presence or absence in a sample of a first
misfolded protein aggregate. The method may include capturing the
first misfolded protein aggregate from the sample to form a
captured first misfolded protein aggregate. The method may include
performing at least a first PMCA procedure. The first PMCA
procedure may include forming a first incubation mixture by
contacting the captured first misfolded protein aggregate with a
molar excess of a first substrate protein. The first substrate
protein may be subject to pathological misfolding and/or
aggregation in vivo to form the first misfolded protein aggregate.
The molar excess may be greater than an amount of protein monomer
included in the captured first misfolded protein aggregate. The
method may include conducting an incubation cycle two or more times
effective to form a first amplified, misfolded protein aggregate.
Each incubation cycle may include incubating the first incubation
mixture effective to cause misfolding and/or aggregation of the
first substrate protein in the presence of the captured first
misfolded protein aggregate. Each incubation cycle may include
disrupting the first incubation mixture effective to form the first
amplified, misfolded protein aggregate. The first PMCA procedure
may include determining the presence or absence of the first
misfolded protein aggregate in the sample by detecting the first
amplified, misfolded protein aggregate. The first misfolded protein
aggregate may include the first substrate protein. The first
amplified, misfolded protein aggregate may include the first
substrate protein.
[0077] In various embodiments, a method is provided for determining
a presence or absence of a tauopathy in a subject, the tauopathy
including Alzheimer's disease (AD). The method may include
providing the subject. The method may include obtaining a sample
from the subject. The sample may include one or more of: a
bio-fluid, a biomaterial, a homogenized tissue, and a cell lysate.
The method may include performing at least a first PMCA procedure.
The first PMCA procedure may include forming a first incubation
mixture by contacting the sample with a first substrate protein.
The first substrate protein may include 4R tau. The first PMCA
procedure may include conducting an incubation cycle two or more
times effective to form a first amplified, misfolded protein
aggregate. Each incubation cycle may include incubating the first
incubation mixture effective to cause misfolding and/or aggregation
of the first substrate protein in the presence of the first
misfolded protein aggregate. Each incubation cycle may include
disrupting the incubation mixture effective to form the first
amplified, misfolded protein aggregate. The first PMCA procedure
may include determining the presence or absence in the sample of
the first misfolded protein aggregate by detecting in the first
incubation mixture the presence or absence of the first amplified,
misfolded protein aggregate. The method may include determining the
presence or absence in the subject of AD according to the presence
or absence of the first misfolded protein aggregate in the
sample.
[0078] In some embodiments, determining the presence or absence in
the subject of AD may include distinguishing AD from one or more
additional tauopathies by determining a signature of AD tau protein
aggregate. The signature AD tau protein aggregate may include one
or more of: one or more AD-specific corresponding PMCA kinetic
parameters of: lag phase, T.sub.50, amplification rate, and
amplification extent; an assay using an antibody selective for a
conformational epitope of AD tau protein aggregate; an indicator
selective for AD tau protein aggregate; and a spectrum
characteristic of AD tau protein aggregate.
[0079] In various embodiments, a method is provided for determining
a presence or absence of a tauopathy in a subject, the tauopathy
including Parkinson's disease (PD). The method may include
providing the subject. The method may include obtaining a sample
from the subject. The sample may include one or more of: a
bio-fluid, a biomaterial, a homogenized tissue, and a cell lysate.
The method may include performing at least a first PMCA procedure.
The first PMCA procedure may include forming a first incubation
mixture by contacting the sample with a first substrate protein.
The first substrate protein may include 4R tau. The first PMCA
procedure may include conducting an incubation cycle two or more
times effective to form a first amplified, misfolded protein
aggregate. Each incubation cycle may include incubating the first
incubation mixture effective to cause misfolding and/or aggregation
of the first substrate protein in the presence of the first
misfolded protein aggregate. Each incubation cycle may include
disrupting the incubation mixture effective to form the first
amplified, misfolded protein aggregate. The first PMCA procedure
may include determining the presence or absence in the sample of
the first misfolded protein aggregate by detecting in the first
incubation mixture the presence or absence of the first amplified,
misfolded protein aggregate. The method may include determining the
presence or absence in the subject of PD according to the presence
or absence of the first misfolded protein aggregate in the
sample.
[0080] In some embodiments, determining the presence or absence in
the subject of PD may include distinguishing PD from one or more
additional tauopathies by determining a signature of PD tau protein
aggregate. The signature PD tau protein aggregate may include one
or more of: one or more PD-specific corresponding PMCA kinetic
parameters of: lag phase, T.sub.50, amplification rate, and
amplification extent; an assay using an antibody selective for a
conformational epitope of PD tau protein aggregate; an indicator
selective for PD tau protein aggregate; and a spectrum
characteristic of PD tau protein aggregate.
[0081] In various embodiments, a method is provided for determining
a presence or absence of a tauopathy in a subject, the tauopathy
including Progressive Supranuclear Palsy (PSP). The method may
include providing the subject. The method may include obtaining a
sample from the subject. The sample may include one or more of: a
bio-fluid, a biomaterial, a homogenized tissue, and a cell lysate.
The method may include performing at least a first PMCA procedure.
The first PMCA procedure may include forming a first incubation
mixture by contacting the sample with a first substrate protein.
The first substrate protein may include 4R tau. The first PMCA
procedure may include conducting an incubation cycle two or more
times effective to form a first amplified, misfolded protein
aggregate. Each incubation cycle may include incubating the first
incubation mixture effective to cause misfolding and/or aggregation
of the first substrate protein in the presence of the first
misfolded protein aggregate. Each incubation cycle may include
disrupting the incubation mixture effective to form the first
amplified, misfolded protein aggregate. The first PMCA procedure
may include determining the presence or absence in the sample of
the first misfolded protein aggregate by detecting in the first
incubation mixture the presence or absence of the first amplified,
misfolded protein aggregate. The method may include determining the
presence or absence in the subject of PSP according to the presence
or absence of the first misfolded protein aggregate in the
sample.
[0082] In some embodiments, determining the presence or absence in
the subject of PSP may include distinguishing PSP from one or more
additional tauopathies by determining a signature of PSP tau
protein aggregate. The signature PSP tau protein aggregate may
include one or more of: one or more PSP-specific corresponding PMCA
kinetic parameters of: lag phase, T.sub.50, amplification rate, and
amplification extent; an assay using an antibody selective for a
conformational epitope of PSP tau protein aggregate; an indicator
selective for PSP tau protein aggregate; and a spectrum
characteristic of PSP tau protein aggregate.
[0083] In various embodiments, a method is provided for determining
a presence or absence of a tauopathy in a subject, the tauopathy
including FrontoTemporal Dementia (FTD). The method may include
providing the subject. The method may include obtaining a sample
from the subject. The sample may include one or more of: a
bio-fluid, a biomaterial, a homogenized tissue, and a cell lysate.
The method may include performing at least a first PMCA procedure.
The first PMCA procedure may include forming a first incubation
mixture by contacting the sample with a first substrate protein.
The first substrate protein may include 4R tau. The first PMCA
procedure may include conducting an incubation cycle two or more
times effective to form a first amplified, misfolded protein
aggregate. Each incubation cycle may include incubating the first
incubation mixture effective to cause misfolding and/or aggregation
of the first substrate protein in the presence of the first
misfolded protein aggregate. Each incubation cycle may include
disrupting the incubation mixture effective to form the first
amplified, misfolded protein aggregate. The first PMCA procedure
may include determining the presence or absence in the sample of
the first misfolded protein aggregate by detecting in the first
incubation mixture the presence or absence of the first amplified,
misfolded protein aggregate. The method may include determining the
presence or absence in the subject of FTD according to the presence
or absence of the first misfolded protein aggregate in the
sample.
[0084] In some embodiments, determining the presence or absence in
the subject of FTD may include distinguishing FTD from one or more
additional tauopathies by determining a signature of FTD tau
protein aggregate. The signature FTD tau protein aggregate may
include one or more of: one or more FTD-specific corresponding PMCA
kinetic parameters of: lag phase, T.sub.50, amplification rate, and
amplification extent; an assay using an antibody selective for a
conformational epitope of FTD tau protein aggregate; an indicator
selective for FTD tau protein aggregate; and a spectrum
characteristic of FTD tau protein aggregate.
[0085] In various embodiments, a method is provided for determining
a presence or absence of a tauopathy in a subject, the tauopathy
including Corticobasal degeneration (CBD). The method may include
providing the subject. The method may include obtaining a sample
from the subject. The sample may include one or more of: a
bio-fluid, a biomaterial, a homogenized tissue, and a cell lysate.
The method may include performing at least a first PMCA procedure.
The first PMCA procedure may include forming a first incubation
mixture by contacting the sample with a first substrate protein.
The first substrate protein may include 4R tau. The first PMCA
procedure may include conducting an incubation cycle two or more
times effective to form a first amplified, misfolded protein
aggregate. Each incubation cycle may include incubating the first
incubation mixture effective to cause misfolding and/or aggregation
of the first substrate protein in the presence of the first
misfolded protein aggregate. Each incubation cycle may include
disrupting the incubation mixture effective to form the first
amplified, misfolded protein aggregate. The first PMCA procedure
may include determining the presence or absence in the sample of
the first misfolded protein aggregate by detecting in the first
incubation mixture the presence or absence of the first amplified,
misfolded protein aggregate. The method may include determining the
presence or absence in the subject of CBD according to the presence
or absence of the first misfolded protein aggregate in the
sample.
[0086] In some embodiments, determining the presence or absence in
the subject of CBD may include distinguishing CBD from one or more
additional tauopathies by determining a signature of CBD tau
protein aggregate. The signature CBD tau protein aggregate may
include one or more of: one or more CBD-specific corresponding PMCA
kinetic parameters of: lag phase, T.sub.50, amplification rate, and
amplification extent; an assay using an antibody selective for a
conformational epitope of CBD tau protein aggregate; an indicator
selective for CBD tau protein aggregate; and a spectrum
characteristic of CBD tau protein aggregate.
[0087] In further embodiments, each of the methods described herein
above may incorporate one or more of the following features. In
particular, each feature described with reference to any protein
substrate, misfolded protein aggregate, amplified misfolded protein
aggregate, incubation mixture, PMCA procedure, portion of the
sample, and the like, should be understood to describe,
independently selected in various other embodiments, any other
protein substrate, misfolded protein aggregate, amplified misfolded
protein aggregate, incubation mixture, PMCA procedure, portion of
the sample, and the like. For example, features described for a
"first" protein substrate may, in some embodiments, also be
independently selected to describe a "second" protein substrate;
features described for a "first" misfolded protein aggregate may
also be independently selected to describe a "second" misfolded
protein aggregate; features described for a "first" incubation
mixture may also be independently selected to describe a "second"
incubation mixture; features described for a "first" PMCA procedure
may also be independently selected to describe a "second" PMCA
procedure; and the like. Further, for example, features described
with reference to "each" protein substrate, misfolded protein
aggregate, amplified misfolded protein aggregate, incubation
mixture, PMCA procedure, portion of the sample, and the like,
should be understood to describe, independently selected in various
other embodiments, any other enumerated element, e.g., "first,"
"second," "third," and the like, as applied to the protein
substrate, misfolded protein aggregate, amplified misfolded protein
aggregate, incubation mixture, PMCA procedure, portion of the
sample, and the like. For example, a description with reference to
"each substrate protein" may be independently selected to describe
and support recitations of a "first substrate protein," a "second
substrate protein," a "third substrate protein," and the like.
[0088] In several embodiments, features described generally for
enumerated or specified elements, e.g., "first," "second," "each,"
and the like, may be independently selected to be the same or
distinct. For example, in some embodiments, a first substrate
protein may include a 4R tau and a second substrate protein may
include A.beta.; a condition such as a temperature may be selected
independently for a first and second PMCA procedure, and the like.
In several embodiments, some features described generally for such
first and second elements may be selected to be the same, or to
overlap, while other features described generally for such first
and second elements may be independently selected to be distinct.
For example, in some embodiments, first and second portions of the
sample may be the same or combined, and first and second incubation
mixtures may be the same or combined, while corresponding first and
second PMCA procedures may be conducted in parallel or in series in
the combined incubation mixture using different first and second
substrate proteins, e.g., 4R tau and A.beta..
[0089] In several embodiments, one, two, or more instances may be
independently selected for each protein substrate, misfolded
protein aggregate, amplified misfolded protein aggregate,
incubation mixture, PMCA procedure, portion of the sample, and the
like. For example, various method embodiments may include a first
PMCA procedure using 4R tau as a first substrate protein, a second
PMCA procedure using A.beta. as a second substrate protein, a third
PMCA procedure using alpha synuclein as a third substrate protein,
a fourth PMCA procedure using 3R tau as a fourth substrate protein,
and the like. Such multiple PMCA procedures may be performed for a
sample, e.g., a laboratory sample, or a sample drawn from a
subject, such as a subject having a tauopathy. Such multiple PMCA
procedures may be performed in parallel for each protein substrate,
misfolded protein aggregate, amplified misfolded protein aggregate,
incubation mixture, PMCA procedure, portion of the sample, and the
like, for example as follows.
[0090] In various embodiments, the method may include determining
the presence in the sample of the first misfolded protein
aggregate. The method may include performing at least a second PMCA
procedure to determine the presence or absence in the sample of at
least a second misfolded protein aggregate. The second PMCA
procedure may include forming a second incubation mixture by
contacting a second portion of the sample with a second substrate
protein. The second substrate protein may be subject to
pathological misfolding and/or aggregation in vivo to form the
second misfolded protein aggregate. The second PMCA procedure may
include conducting an incubation cycle two or more times under
conditions effective to form a second amplified, misfolded protein
aggregate. Each incubation cycle may include incubating the second
incubation mixture effective to cause misfolding and/or aggregation
of the second substrate protein in the presence of the second
misfolded protein aggregate. Each incubation cycle may include
disrupting the second incubation mixture effective to form the
second amplified, misfolded protein aggregate. The second PMCA
procedure may include determining the presence or absence in the
sample of the second misfolded protein aggregate by analyzing the
second incubation mixture for the presence or absence of the second
amplified, misfolded protein aggregate. The second misfolded
protein aggregate may include the second substrate protein. The
second amplified, misfolded protein aggregate may include the
second substrate protein.
[0091] In some embodiments, the method may include determining the
presence in the sample of the first misfolded protein aggregate and
the second misfolded protein aggregate. The method may include
performing at least a third PMCA procedure to determine the
presence or absence in the sample of at least a third misfolded
protein aggregate. The third PMCA procedure may include forming a
third incubation mixture by contacting a third portion of the
sample with a third substrate protein. The third substrate protein
may be subject to pathological misfolding and/or aggregation in
vivo to form the third misfolded protein aggregate. The third PMCA
procedure may include conducting an incubation cycle two or more
times under conditions effective to form a third amplified,
misfolded protein aggregate. Each incubation cycle may include
incubating the third incubation mixture effective to cause
misfolding and/or aggregation of the third substrate protein in the
presence of the third misfolded protein aggregate. Each incubation
cycle may include disrupting the third incubation mixture effective
to form the third amplified, misfolded protein aggregate. The third
PMCA procedure may include determining the presence or absence in
the sample of the third misfolded protein aggregate by analyzing
the third incubation mixture for the presence or absence of the
third amplified, misfolded protein aggregate. The third misfolded
protein aggregate may include the third substrate protein. The
third amplified, misfolded protein aggregate may include the third
substrate protein.
[0092] In several embodiments, the method may include determining
the presence in the sample of the first misfolded protein
aggregate, the second misfolded protein aggregate, and the fourth
misfolded protein aggregate. The method may include performing at
least a fourth PMCA procedure to determine the presence or absence
in the sample of a fourth misfolded protein aggregate. The fourth
PMCA procedure may include forming a fourth incubation mixture by
contacting a fourth portion of the sample with a fourth substrate
protein. The fourth substrate protein may be subject to
pathological misfolding and/or aggregation in vivo to form the
fourth misfolded protein aggregate. The fourth PMCA procedure may
include conducting an incubation cycle two or more times under
conditions effective to form a fourth amplified, misfolded protein
aggregate. Each incubation cycle may include incubating the fourth
incubation mixture effective to cause misfolding and/or aggregation
of the fourth substrate protein in the presence of the fourth
misfolded protein aggregate. Each incubation cycle may include
disrupting the fourth incubation mixture effective to form the
fourth amplified, misfolded protein aggregate. The fourth PMCA
procedure may include determining the presence or absence in the
sample of the fourth misfolded protein aggregate by analyzing the
fourth incubation mixture for the presence or absence of the fourth
amplified, misfolded protein aggregate. The fourth misfolded
protein aggregate may include the fourth substrate protein. The
fourth amplified, misfolded protein aggregate may include the
fourth substrate protein.
[0093] In various embodiments, the first substrate protein may
independently include a tau isoform, e.g., 3R tau, 4R tau, and the
like. In several embodiments, the first substrate protein may
include 4R tau. The first substrate protein may include 3R tau. The
first substrate protein may exclude 3R tau, for example, when the
sample corresponds to Pick's disease or is drawn from a subject
having Pick's disease. The first substrate protein may be soluble.
The first substrate protein may be monomeric. The first substrate
protein may be in a native in vivo conformation, e.g., folded. The
first substrate protein may be distinct from each other substrate
protein.
[0094] In various embodiments, the second substrate protein may
independently include one of: a tau isoform, e.g., 3R tau, 4R tau,
and the like; A.beta., alpha synuclein, and the like. The second
substrate protein may include one of: 3R tau, A.beta., alpha
synuclein, and the like. The second substrate protein may include
3R tau. The second substrate protein may include A.beta.. The
second substrate protein may include alpha synuclein. The second
substrate protein may consist essentially of, or consist of, one
of: 3R tau, 4R tau, A.beta., or alpha synuclein. The second
substrate protein may be soluble. The second substrate protein may
be monomeric. The second substrate protein may be in a native in
vivo conformation, e.g., folded. The second substrate protein may
be distinct from each other substrate protein.
[0095] In various embodiments, the third substrate protein may
independently include one of: a tau isoform, e.g., 3R tau, 4R tau,
and the like; A.beta., alpha synuclein, and the like. The third
substrate protein may include one of: 3R tau, A.beta., alpha
synuclein, and the like. The third substrate protein may include 3R
tau. The third substrate protein may include A.beta.. The third
substrate protein may include alpha synuclein. The third substrate
protein may consist essentially of, or consist of, one of: 3R tau,
4R tau, A.beta., or alpha synuclein. The third substrate protein
may be soluble. The third substrate protein may be monomeric. The
third substrate protein may be in a native in vivo conformation,
e.g., folded. The third substrate protein may be distinct from each
other substrate protein.
[0096] In various embodiments, the fourth substrate protein may
independently include one of: a tau isoform, e.g., 3R tau, 4R tau,
and the like; A.beta., alpha synuclein, and the like. The fourth
substrate protein may include one of: 3R tau, A.beta., alpha
synuclein, and the like. The fourth substrate protein may include
3R tau. The fourth substrate protein may include A.beta.. The
fourth substrate protein may include alpha synuclein. The fourth
substrate protein may consist essentially of, or consist of, one
of: 3R tau, 4R tau, A.beta., or alpha synuclein. The fourth
substrate protein may be soluble. The fourth substrate protein may
be monomeric. The fourth substrate protein may be in a native in
vivo conformation, e.g., folded. The fourth substrate protein may
be distinct from each other substrate protein.
[0097] In some embodiments the sample may be taken from a subject.
The method may include determining or diagnosing the presence or
absence of a tauopathy in the subject according to the presence or
absence of the first misfolded protein aggregate in the sample.
[0098] In several embodiments, the method may include performing at
least a second PMCA procedure to determine the presence or absence
in the sample of second misfolded protein aggregate, e.g., a
misfolded protein aggregate that includes a second substrate
protein. The second PMCA procedure may include forming a second
incubation mixture by contacting a second portion of the sample
with a second substrate protein. The second substrate protein may
be subject to pathological misfolding and/or aggregation in vivo to
form the second misfolded protein aggregate. The methods may
include determining the presence or absence in the sample of the
second misfolded protein aggregate by analyzing the second
incubation mixture for the presence or absence of the second
amplified, misfolded protein aggregate. The second misfolded
protein aggregate may include the second substrate protein. The
second amplified, misfolded protein aggregate may include the
second substrate protein. The second substrate protein may include
one of: amyloid-beta (A.beta.), alpha synuclein, and 3R tau.
[0099] In some embodiments, the sample may be taken from a subject.
The methods may include determining or diagnosing the presence or
absence of a tauopathy in the subject according to the presence or
absence of the first misfolded protein aggregate in the sample. The
methods may include performing at least a second PMCA procedure to
determine the presence or absence in the sample of a second
misfolded protein aggregate. The second PMCA procedure may include
forming a second incubation mixture by contacting a second portion
of the sample with a second substrate protein. The second substrate
protein may be subject to pathological misfolding and/or
aggregation in vivo to form the second misfolded protein aggregate.
The second PMCA procedure may include conducting an incubation
cycle two or more times under conditions effective to form a second
amplified, misfolded protein aggregate. Each incubation cycle may
include incubating the second incubation mixture effective to cause
misfolding and/or aggregation of the second substrate protein in
the presence of the second misfolded protein aggregate. Each
incubation cycle may include disrupting the second incubation
mixture effective to form the second amplified, misfolded protein
aggregate. The second PMCA procedure may include determining the
presence or absence in the sample of the second misfolded protein
aggregate by analyzing the second incubation mixture for the
presence or absence of the second amplified, misfolded protein
aggregate. The second misfolded protein aggregate may include the
second substrate protein. The second amplified, misfolded protein
aggregate may include the second substrate protein.
[0100] In various embodiments, the subject may have the tauopathy.
The methods may include characterizing an identity of the tauopathy
in the subject according to: the presence in the sample of the
first misfolded protein aggregate; and the presence or absence in
the sample of the second misfolded protein aggregate. The second
substrate protein may include one of: amyloid-beta (A.beta.), alpha
synuclein, and 3R tau. For example, the presence of misfolded 4R
tau aggregate as the first misfolded protein aggregate and A.beta.
as the second misfolded protein aggregate may indicate the
tauopathy in the subject is AD; the presence of misfolded 4R tau
aggregate as the first misfolded protein aggregate and alpha
synuclein as the second misfolded protein aggregate may indicate
the tauopathy in the subject is PD; the presence of misfolded 4R
tau aggregate as the first misfolded protein aggregate and the
absence of the second misfolded protein aggregate including
A.beta., alpha synuclein, or 3R may indicate a 4R tauopathy, such
as PSP, CBD, AGD, and the like.
[0101] In various embodiments, the tauopathy may include a primary
tauopathy or a secondary tauopathy. The tauopathy may be
characterized at least in part by misfolding and/or aggregation of
4R tau protein. The tauopathy may be characterized at least in part
by misfolding and/or aggregation of 4R tau protein and 3R tau
protein. The tauopathy may be characterized at least in part by
misfolded and/or aggregated 4R tau protein, in a ratio to misfolded
and/or aggregated 3R tau protein, of one of about: 1:99, 5:95,
10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50,
55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, and
99:1, or a range between any two of the preceding ratios, for
example, between 1:99 and 99:1.
[0102] In several embodiments, the methods may include
characterizing an identity of the tauopathy by analyzing the first
amplified, misfolded protein aggregate or one or more corresponding
PMCA kinetic parameters thereof for a signature of at least one of:
Alzheimer's disease (AD), Parkinson's Disease (PD), Progressive
Supranuclear Palsy (PSP), FrontoTemporal Dementia (FTD),
Corticobasal degeneration (CBD), Mild cognitive impairment (MCI),
Argyrophilic grain disease (AgD) Traumatic Brain Injury (TBI),
Chronic Traumatic Encephalopathy (CTE), and Dementia Pugilistica
(DP). For example, characterizing the identity of the tauopathy may
include determining the one or more corresponding PMCA kinetic
parameters, including one or more of: lag phase, T.sub.50,
amplification rate, and amplification extent. Characterizing the
identity of the tauopathy may include comparing the one or more
corresponding PMCA kinetic parameters to one or more corresponding
predetermined corresponding PMCA kinetic parameters that are
characteristic of the identity of the tauopathy to determine a
similarity or difference effective to characterize the identity of
the tauopathy.
[0103] In some embodiments, the methods may include characterizing
the identity of the tauopathy using an antibody selective for a
conformational epitope of a tauopathy-specific misfolded tau
protein aggregate. The methods may include characterizing the
identity of the tauopathy using an indicator selective for each
tauopathy-specific misfolded tau protein aggregate. The indicator
selective for each tauopathy-specific misfolded tau protein
aggregate may include a small molecule, a peptide, or a DNA or RNA
aptamer; and the like. The methods may include characterizing the
identity of the tauopathy using a spectrum characteristic of each
tauopathy-specific misfolded tau protein aggregate.
[0104] In some embodiments, the methods may include, for example,
characterizing the identity of the tauopathy by analyzing the
proteolytic resistance of each tauopathy-specific misfolded tau
protein aggregate. For example, each tauopathy-specific misfolded
tau protein aggregate may be contacted with a proteinase, e.g.,
proteinase K, trypsin, chymotrypsin, and the like, at a proteinase
concentration of from 0.1 to 5000 .mu.g/mL, at various temperatures
from 20.degree. C. to 120.degree. C. and for various times, e.g.,
from 1 min to 4 h. The proteolytic resistance of each
tauopathy-specific misfolded tau protein aggregate may be
characterized and used to distinguish the various
tauopathy-specific misfolded tau protein aggregates.
[0105] In several embodiments, the methods may include
characterizing the identity of the tauopathy by analyzing the
stability to denaturation of each tauopathy-specific misfolded tau
protein aggregate. For example, each tauopathy-specific misfolded
tau protein aggregate may be treated with guanidinium or urea at a
sufficiently elevated temperature to induce protein denaturation of
each tauopathy-specific misfolded tau protein aggregate. The
concentration of guanidinium or urea may range from 0.1 M to 8 M.
The temperature may range between 20.degree. C. to 120.degree. C.
The stability of each tauopathy-specific misfolded tau protein
aggregate may be characterized and used to distinguish the various
tauopathy-specific misfolded tau protein aggregates.
[0106] The methods may include sedimentation of each
tauopathy-specific misfolded tau protein aggregate. The methods may
include gel chromatography to characterize the size of each
tauopathy-specific misfolded tau protein aggregate. The methods may
include circular dichroism spectroscopy of each tauopathy-specific
misfolded tau protein aggregate. The methods may include Fourier
transform infrared spectroscopy to analyze secondary structure of
each tauopathy-specific misfolded tau protein aggregate. The
methods may include nuclear magnetic resonance spectroscopy to
analyze structure of each tauopathy-specific misfolded tau protein
aggregate. The methods may include mass spectrometry, e.g.,
fragmentation and collision induced dissociation to analyze
secondary and tertiary structure of each tauopathy-specific
misfolded tau protein aggregate. The methods may include
microscopy, e.g., atomic force microscopy, cryo-electron
microscopy, and the like to analyze morphology of each
tauopathy-specific misfolded tau protein aggregate. Each of these
methods may be coupled with substitution using atomic isotopes of
different mass, magnetic properties, and/or isotopic stability to
complement the methods; for example, nuclear magnetic resonance
spectroscopy may be coupled with deuterium exchange in each
tauopathy-specific misfolded tau protein aggregate to obtain
structural information.
[0107] In various embodiments, the methods are provided such that
the tauopathy specifically excludes Pick's disease. In various
embodiments, the exclusion of Pick's disease does not encompass the
remainder of Pick's complex of diseases.
[0108] In several embodiments, the methods may include determining
or diagnosing the presence or absence of a tauopathy in the subject
including comparing the presence or absence of the first misfolded
protein aggregate in the sample to a control sample taken from a
control subject. The detecting may include detecting an amount of
the first misfolded protein aggregate in the sample. The sample may
be taken from a subject. The methods may include determining or
diagnosing the presence or absence of a tauopathy in the subject by
comparing the amount of the first misfolded protein aggregate in
the sample to a predetermined threshold amount. The sample may be
taken from a subject exhibiting no clinical signs of dementia
according to cognitive testing. The methods may include determining
or diagnosing the presence or absence of a tauopathy in the subject
according to the presence or absence of the first misfolded protein
aggregate in the sample. The sample may be taken from a subject
exhibiting no cortex plaques or tangles according to contrast
imaging. The methods may include determining or diagnosing the
presence or absence of a tauopathy in the subject according to the
presence or absence of the first misfolded protein aggregate in the
sample. The sample may be taken from a subject exhibiting clinical
signs of dementia according to cognitive testing. The methods may
include determining or diagnosing the presence or absence of a
tauopathy as a contributing factor to the clinical signs of
dementia in the subject according to the presence or absence of the
first misfolded protein aggregate in the sample. The sample may be
taken from a subject exhibiting no clinical signs of dementia
according to cognitive testing. The subject may exhibit a
predisposition to dementia according to genetic testing. The
genetic testing may indicate, for example, an increased risk of
tauopathy according to one or two copies of the ApoE4 allele,
variants of the brain derived neurotrophic factor (BDNF) gene, such
as the val66met allele, in which valine at AA position 66 is
replaced by methionine, and the like. The methods may include
determining or diagnosing the presence or absence of a tauopathy in
the subject according to the presence or absence of the first
misfolded protein aggregate in the sample.
[0109] In some embodiments, the methods may include preparing the
first incubation mixture characterized by at least one
concentration of: the first substrate protein of less than about 20
.mu.M; heparin of less than about 75 .mu.M; NaCl of less than about
190 mM; and Thioflavin T of less than about 9.5 .mu.M.
[0110] In various embodiments, the methods may include preparing
the first incubation mixture including the first substrate protein
at a concentration in .mu.M of one or more of about: 0.001, 0.01,
0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5,
5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12,
12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5,
19, 19.5, 20, 25, 50, 70, 100, 150, 200, 250, 500, 750, 1000, 1500,
or 2000, or a range between any two of the preceding values, for
example, between about 0.001 .mu.M and about 2000 .mu.M. The
methods may include preparing the first incubation mixture
characterized by heparin at a concentration in .mu.M of one or more
of about: 0.001, 0.01, 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2,
2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10 11, 12, 12.5, 15, 17.5, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, and 75, or a range between
any two of the preceding values, for example, between about 0.001
.mu.M and about 75 .mu.M. The methods may include preparing the
first incubation mixture including a buffer composition of one or
more of: Tris-HCL, PBS, MES, PIPES, MOPS, BES, TES, and HEPES. The
methods may include preparing the first incubation mixture
including the buffer composition at a total concentration of one or
more of about: 1 .mu.M, 10 .mu.M, 100 .mu.M, 250 .mu.M, 500 .mu.M,
750 .mu.M, 1 mM, 10 mM, 100 mM, 250 mM, 500 mM, 750 mM, and 1M, or
a range between any two of the preceding values, for example,
between about 1 .mu.M and about 1 M. The methods may include
preparing the first incubation mixture including a salt composition
at a total concentration of one or more of: 1 .mu.M, 10 .mu.M, 100
.mu.M, 250 .mu.M, 500 .mu.M, 750 .mu.M, 1 mM, 10 mM, 20 mM, 30 mM,
40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM,
130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 250
mM, 500 mM, 750 mM, and 1M, or a range between any two of the
preceding values, for example, between about 1 .mu.M and about 1 M.
The salt composition may include one or more of: NaCl and KCl.
[0111] In various embodiments, the methods may include preparing or
maintaining the first incubation mixture at a pH of one or more of
about: 5, 5.5, 6, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3,
7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, or 9, or
a range between any two of the preceding values, e.g., from about
pH 5 to about pH 9.
[0112] In some embodiments, the methods may include preparing the
first incubation mixture including an indicator at a total
concentration of one or more of: 1 nM, 10 nM, 100 nM, 250 nM, 500
nM, 750 nM, 1 .mu.M, 2 .mu.M, 3 .mu.M, 4 .mu.M, 5 .mu.M, 6 .mu.M, 7
.mu.M, 8 .mu.M, 9 .mu.M, 9.5 .mu.M, 10 .mu.M, 25 .mu.M, 50 .mu.M,
100 .mu.M, 250 .mu.M, 500 .mu.M, 750 .mu.M, 1 mM, or a range
between any two of the preceding values, for example, between about
1 nM and about 1 mM.
[0113] In some embodiments of the methods, the incubating may
include heating or maintaining the first incubation mixture at a
temperature in .degree. C. of one of: 5, 10, 15, 20, 22.5, 25,
27.5, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 50, 55, 60, or a range between any two of the preceding
values, for example, between about 5.degree. C. and about
60.degree. C.
[0114] In several embodiments, the methods may include contacting
an indicator of the first misfolded protein aggregate to the first
incubation mixture. The indicator of the first misfolded protein
aggregate may be characterized by an indicating state in the
presence of the first misfolded protein aggregate and a
non-indicating state in the absence of the first misfolded protein
aggregate. Determining the presence of the first misfolded protein
aggregate in the sample may include detecting the indicating state
of the indicator of the first misfolded protein aggregate. The
indicating state of the indicator and the non-indicating state of
the indicator may be characterized by a difference in fluorescence.
Determining the presence of the first misfolded protein aggregate
in the sample may include detecting the difference in fluorescence.
The methods may include contacting a molar excess of the indicator
of the first misfolded protein aggregate to the first incubation
mixture. The molar excess may be greater than a total molar amount
of protein monomer included in the first substrate protein and the
first misfolded protein aggregate in the first incubation mixture.
The indicator of the first misfolded protein aggregate may include
one or more of: a thioflavin, e.g., thioflavin T or thioflavin S;
Congo Red, m-I-Stilbene, Chrysamine G, PIB, BF-227, X-34, TZDM,
FDDNP, MeO-X-04, IMPY, NIAD-4, luminescent conjugated
polythiophenes, a fusion with a fluorescent protein such as green
fluorescent protein and yellow fluorescent protein, derivatives
thereof, and the like.
[0115] In various embodiments, the method may include determining
an amount of the first misfolded protein aggregate in the sample.
For example, known amounts of in vitro, synthetic misfolded protein
aggregate seeds may be added to various portions of a biological
fluid of a healthy patient, e.g., CSF. Subsequently, PMCA may be
performed on the various portions. In each of the various portions,
a fluorescent indicator of the misfolded protein aggregate may be
added, and fluorescence may be measured as a function of, e.g.,
number of PMCA cycles, to determine various PMCA kinetics
parameters, e.g., number of PMCA cycles to maximum fluorescence
signal, number of PMCA cycles to 50% of maximum fluorescence
signal, lag phase in increase of fluorescence signal, rate of
increase in fluorescence signal versus PMCA cycles, and the like. A
calibration curve showing the relationship between the
concentration of synthetic seeds added and the PMCA kinetic
parameters. The kinetic parameters may be measured for unknown
samples and compared to the calibration curve to determine the
expected amount of seeds present in a particular sample.
Alternatively, the amount of the first misfolded protein aggregate
in the sample may be determined by a series of known dilutions of
the sample, and PMCA of each serial dilution to determine whether
the first misfolded protein aggregate can be detected or not in a
particular dilution. The amount of the first misfolded protein
aggregate in the undiluted sample can be estimated based on the
known dilution that results in no detection of the first misfolded
protein aggregate by PMCA. In another example, the amount of the
first misfolded protein aggregate in the sample may be determined
by a series of known dilutions of the sample, and PMCA to determine
a detection signal in each serial dilution. The collected detection
signals in the serial dilutions can be fit, e.g., via least squares
analysis, to determine whether the first misfolded protein
aggregate can be detected or not in a particular dilution. In
another example, the amount of the first misfolded protein
aggregate in the sample may be determined by known amounts of
antibodies to the first misfolded protein aggregate.
[0116] In some embodiments, the methods may include detecting the
amount of the first misfolded protein aggregate in the sample at a
sensitivity of at least about one or more of: 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
and 100%, e.g., at least about 70%. The methods may include
detecting the amount of the first misfolded protein aggregate in
the sample at less than about one or more of: 625, 62.5, 6.25, 630
.mu.g, 63 .mu.g, 6.3 .mu.g, 630 ng, 63 ng, 6.3 ng, 630 pg, 200 pg,
63 pg, 6.3 pg, 630 fg, 300 fg, 200 fg, 125 fg, 63 fg, 50 fg, 30 fg,
15 fg, 12.5 fg, 10 fg, 5 fg, or 2.5 fg, The methods may include
detecting the amount of the first misfolded protein aggregate in
the sample at less than about one or more of: 100 nmol, 10 nmol, 1
nmol, 100 pmol, 10 pmol, 1 .mu.mol, 100 fmol, 10 fmol, 3 fmol, 1
fmol, 100 attomol, 10 attomol, 5 attomol, 2 attomol, 1 attomol,
0.75 attomol, 0.5 attomol, 0.25 attomol, 0.2 attomol, 0.15 attomol,
0.1 attomol, and 0.05 attomol, e.g., less than about 100 nmol. The
methods may include detecting the amount of the first misfolded
protein aggregate in the sample in a molar ratio to the first
substrate protein included by the sample. The molar ratio may be
less than about one or more of: 1:100, 1:10,000, 1:100,000, and
1:1,000,000, e.g., less than about 1:100. The methods may include
determining the amount of the first misfolded protein aggregate in
the sample compared to a control sample.
[0117] In several embodiments, the methods may include detecting
the first misfolded protein aggregate in the sample with a
specificity of at least about one or more of: 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
and 100%, e.g. at least about 70%. The methods may include
detecting the first misfolded protein aggregate including one or
more of: a Western Blot assay, a dot blot assay, an enzyme-linked
immunosorbent assay (ELISA), a fluorescent protein/peptide binding
assay, a thioflavin binding assay, a Congo Red binding assay, a
sedimentation assay, electron microscopy, atomic force microscopy,
surface plasmon resonance, and spectroscopy. The ELISA may include
a two-sided sandwich ELISA. The spectroscopy may include one or
more of: quasi-light scattering spectroscopy, multispectral
ultraviolet spectroscopy, confocal dual-color fluorescence
correlation spectroscopy, Fourier-transform infrared spectroscopy,
capillary electrophoresis with spectroscopic detection, electron
spin resonance spectroscopy, nuclear magnetic resonance
spectroscopy, and Fluorescence Resonance Energy Transfer (FRET)
spectroscopy. Detecting the first misfolded protein aggregate may
include contacting the first incubation mixture with a protease;
and detecting the first misfolded protein aggregate using
anti-misfolded protein antibodies or antibodies specific for a
misfolded tau aggregate in one or more of: a Western Blot assay, a
dot blot assay, and an ELISA.
[0118] In various embodiments, the methods may include providing
the first substrate protein in labeled form. The first substrate
protein in labeled form may include one or more of: a covalently
incorporated radioactive amino acid, a covalently incorporated,
isotopically labeled amino acid, and a covalently incorporated
fluorophore. The methods may include detecting the first substrate
protein in labeled form as incorporated into the first amplified,
misfolded protein aggregate.
[0119] In some embodiments, the sample may include one or more of a
bio-fluid, e.g., blood, a biomaterial, e.g., cerumen, a homogenized
tissue, and a cell lysate. The sample may include one or more of:
amniotic fluid; bile; blood; cerebrospinal fluid; cerumen; skin;
exudate; feces; gastric fluid; lymph; milk; mucus; mucosal
membrane; peritoneal fluid; plasma; pleural fluid; pus; saliva;
sebum; semen; sweat; synovial fluid; tears; and urine. The sample
may be derived from cells or tissue of one or more of: skin, brain,
heart, liver, pancreas, lung, kidney, gastro-intestine, nerve,
mucous membrane, blood cell, gland, and muscle. The methods may
include obtaining the sample from a subject, such as by drawing a
bio-fluid or biomaterial, performing a tissue biopsy, and the like.
The volume of each portion of the sample added to a particular PMCA
reaction, e,g., in fluid or homogenized form, may be a volume in
.mu.L of one of about 5,000, 4,000, 3,000, 2,000, 1000, 900, 800,
750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150,
125, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 5, or 1, or a
range between any two of the preceding values, e.g., from about 1
.mu.L to about 1000 .mu.L. In some embodiments, when the sample is
CSF, the amount of each portion added to a particular PMCA reaction
may be a volume in .mu.L of any of the preceding, for example, one
of about 80, 70, 60, 50, 40, 30, 25, 20, 15, or 10, or a range
between any two of the preceding values, e.g., e.g., from about 10
.mu.L to about 80 .mu.L, e.g., about 40 .mu.L. In some embodiments,
when the sample is plasma, the amount of each portion added to a
particular PMCA reaction may be a volume in .mu.L of any of the
preceding, for example, one of about 750, 700, 650, 600, 550, 500,
450, 400, 350, 300, 250, or a range between any two of the
preceding values, e.g., e.g., from about 250 .mu.L to about 750
.mu.L, e.g., about 500 .mu.L. In some embodiments, when the sample
is blood, the amount of each portion added to a particular PMCA
reaction may be a volume in .mu.L of any of the preceding, for
example, one of about 5,000, 4,000, 3,000, 2,000, 1000, 900, 800,
750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, or 200, or a
range between any two of the preceding values, e.g., from about 200
.mu.L to about 1000 .mu.L.
[0120] In several embodiments, the subject may be one of a: human,
mouse, rat, dog, cat, cattle, horse, deer, elk, sheep, goat, pig,
and non-human primate. The subject may be one or more of: at risk
of a tauopathy, having the tauopathy, and under treatment for the
tauopathy. The methods may include determining a progression or
homeostasis of a tauopathy in the subject by comparing the amount
of the first misfolded protein aggregate in the sample to an amount
of the first misfolded protein aggregate in a comparison sample
taken from the subject at a different time compared to the sample.
The subject may be treated with a tauopathy modulating therapy. The
methods may include comparing the amount of the first misfolded
protein aggregate in the sample to an amount of the first misfolded
protein aggregate in a comparison sample. The sample and the
comparison sample may be taken from the subject at different times
over a period of time under the tauopathy modulating therapy. The
methods may include determining the subject is one of: responsive
to the tauopathy modulating therapy according to a change in the
first misfolded protein aggregate over the period of time, or
non-responsive to the tauopathy modulating therapy according to
homeostasis of the first misfolded protein aggregate over the
period of time. The methods may include treating the subject
determined to be responsive to the tauopathy modulating therapy
with the tauopathy modulating therapy. The methods may include
treating the subject with a tauopathy modulating therapy to inhibit
production of the first substrate protein or to inhibit aggregation
of the first misfolded protein aggregate.
[0121] In some embodiments, the subject may be treated with a
protein misfolding disorder (PMD) modulating therapy. The method
may include comparing the amount of the each misfolded protein
aggregate in the sample to an amount of the each misfolded protein
aggregate in a comparison sample. The sample and the comparison
sample may be taken from the subject at different times over a
period of time under the each misfolded protein aggregate
modulating therapy. The method may include determining or
diagnosing the subject is one of: responsive to the each misfolded
protein aggregate modulating therapy according to a change in the
each misfolded protein aggregate over the period of time, or
non-responsive to the each misfolded protein aggregate modulating
therapy according to homeostasis of the each misfolded protein
aggregate over the period of time. The method may include treating
the subject determined to be responsive to the each misfolded
protein aggregate modulating therapy with the each misfolded
protein aggregate modulating therapy. For AD, for example, the PMD
modulating therapy may include administration of one or more of: an
inhibitor of BACE1 (beta-secretase 1); an inhibitor of
.gamma.-secretase; and a modulator of A.beta. homeostasis, e.g., an
immunotherapeutic modulator of A.beta. homeostasis. The A.beta.
modulating therapy may include administration of one or more of:
E2609; MK-8931; LY2886721; AZD3293; semagacestat (LY-450139);
avagacestat (BMS-708163); solanezumab; crenezumab; bapineuzumab;
BIIB037; CAD106; 8F5 or 5598 or other antibodies raised against
A.beta. globulomers, e.g., as described by Barghorn et al,
"Globular amyloid .beta.-peptide.sub.1-42 oligomer--a homogenous
and stable neuropathological protein in Alzheimer's disease" J.
Neurochem., 2005, 95, 834-847, the entire teachings of which are
incorporated herein by reference; ACC-001; V950; Affitrope AD02;
and the like.
[0122] For PD, for example, the PMD modulating therapy may include
active immunization, such as PDO1A+ or PDO3A+, passive immunization
such as PRX002, and the like. The PMD modulating therapy may also
include treatment with GDNF (Glia cell-line derived neurotrophic
factor), inosine, Calcium-channel blockers, specifically Cav1.3
channel blockers such as isradipine, nicotine and nicotine-receptor
agonists, GM-CSF, glutathione, PPAR-gamma agonists such as
pioglitazone, and dopamine receptor agonists, including D2/D3
dopamine receptor agonists and LRRK2 (leucine-rich repeat kinase 2)
inhibitors.
[0123] In several embodiments, the amount of misfolded protein may
be measured in samples from patients using PMCA. Patients with
elevated misfolded protein measurements may be treated with disease
modifying therapies for a PMD. Patients with normal misfolded
protein measurements may not be treated. A response of a patient to
disease-modifying therapies may be followed. For example, misfolded
protein levels may be measured in a patient sample at the beginning
of a therapeutic intervention. Following treatment of the patient
for a clinical meaningful period of time, another patient sample
may be obtained and misfolded protein levels may be measured.
Patients who show a change in misfolded protein levels following
therapeutic intervention may be considered to respond to the
treatment. Patients who show unchanged misfolded protein levels may
be considered non-responding. The methods may include detection of
misfolded protein aggregates in patient samples containing
components that may interfere with the PMCA reaction.
[0124] In various embodiments, the methods may include selectively
concentrating the first misfolded protein aggregate in one or more
of the sample and the first incubation mixture. The selectively
concentrating the first misfolded protein aggregate may include
pre-treating the sample prior to forming the first incubation
mixture. The selectively concentrating the first misfolded protein
aggregate may include pre-treating the first incubation mixture
prior to incubating the first incubation mixture. The selectively
concentrating the first misfolded protein aggregate may include
contacting one or more antibodies capable of binding the first
misfolded protein aggregate to form a captured first misfolded
protein aggregate. The one or more antibodies capable of binding
the first misfolded protein aggregate may include one or more of:
an antibody specific for an amino acid epitope sequence of the
first misfolded protein aggregate, and an antibody specific for a
conformation of the first misfolded protein aggregate. The antibody
specific for a conformation of the first misfolded protein
aggregate may be selective for a conformational epitope of a
tauopathy-specific misfolded tau aggregate. The one or more one or
more antibodies capable of binding the first misfolded protein
aggregate may be coupled to a solid phase. The solid phase may
include one or more of a magnetic bead and a multiwell plate.
[0125] For example, ELISA plates may be coated with the antibodies
used to capture first misfolded protein aggregate from the patient
sample. The antibody-coated ELISA plates may be incubated with a
patient sample, unbound materials may be washed off, and the PMCA
reaction may be performed. Antibodies may also be coupled to beads.
The beads may be incubated with the patient sample and used to
separate first misfolded protein aggregate-antibody complexes from
the remainder of the patient sample.
[0126] In some embodiments, contacting the sample with the first
substrate protein to form the first incubation mixture may include
contacting a molar excess of the first substrate protein to the
sample including the captured first misfolded protein aggregate.
The molar excess of the first substrate protein may be greater than
a total molar amount of protein monomer included in the captured
first misfolded protein aggregate. Incubating the first incubation
mixture may be effective to cause misfolding and/or aggregation of
the first substrate protein in the presence of the captured first
misfolded protein aggregate to form the first amplified, misfolded
protein aggregate.
[0127] In several embodiments, disrupting the first incubation
mixture may include physically disrupting and/or thermally
disrupting. For example, the disrupting may include one or more of:
sonication, stirring, shaking, freezing/thawing, laser irradiation,
autoclave incubation, high pressure, and homogenization. Disrupting
the first incubation mixture may include cyclic agitation. The
cyclic agitation may be conducted for one or more of: between about
50 rotations per minute (RPM) and 10,000 RPM, between about 200 RPM
and about 2000 RPM, and at about 500 RPM. Disrupting the first
incubation mixture may be conducted in each incubation cycle for
one or more of: between about 5 seconds and about 10 minutes,
between about 30 sec and about 1 minute, between about 45 sec and
about 1 minute, and about 1 minute. Incubating the first incubation
mixture may be independently conducted, in each incubation cycle
for one or more of: between about 1 minute and about 5 hours,
between about 5 minutes and about 5 hours, between about 10 minutes
and about 2 hours, between about 15 minutes and about 1 hour, and
between about 25 minutes and about 45 minutes. Each incubation
cycle may include independently incubating and disrupting the first
incubation mixture for one or more of: incubating between about 1
minute and about 5 hours and disrupting between about 5 seconds and
about 10 minutes; incubating between about 5 minutes and about 5
hours and disrupting between about 5 seconds and about 10 minutes;
incubating between about 10 minutes and about 2 hours and
disrupting between about 30 sec and about 1 minute; incubating
between about 15 minutes and about 1 hour and disrupting between
about 45 sec and about 1 minute; incubating between about 25
minutes and about 45 minutes and disrupting between about 45 sec
and about 90 seconds; incubating for about 29 minutes and for about
1 minute; and incubating about 1 minute and disrupting about 1
minute. Conducting the incubation cycle may be repeated for one or
more of: between about 2 times and about 1000 times, between about
5 times and about 500 times, between about 50 times and about 500
times, and between about 150 times and about 250 times.
[0128] In various embodiments, contacting the sample with the first
substrate protein to form the first incubation mixture may be
conducted under physiological conditions. the methods may include
contacting the sample with a molar excess of the first substrate
protein to form the first incubation mixture. The molar excess may
be greater than a total molar amount of protein monomer included in
the first misfolded protein aggregate in the sample.
[0129] In some embodiments, the methods may include contacting the
sample with a thioflavin, e.g., thioflavin T or thioflavin S, and a
molar excess of the first substrate protein to form the first
incubation mixture. The molar excess may be greater than an amount
of the first substrate protein included in the first misfolded
protein aggregate in the sample. The method may include conducting
the incubation cycle two or more times effective to form the first
amplified, misfolded protein aggregate. Each incubation cycle may
include incubating the first incubation mixture effective to cause
misfolding and/or aggregation of at least the portion of the first
substrate protein in the presence of the first misfolded protein
aggregate. Each incubation cycle may include shaking the first
incubation mixture effective to form the first amplified, misfolded
protein aggregate. The methods may include determining the presence
of the first misfolded protein aggregate in the sample by detecting
a fluorescence of the thioflavin corresponding to the first
misfolded protein aggregate.
[0130] In several embodiments, the first substrate protein may be
produced by one of: chemical synthesis, recombinant production, and
extraction from non-recombinant biological samples. The first
misfolded protein aggregate may include one or more of a soluble
first misfolded protein aggregate and an insoluble first misfolded
protein aggregate. The first amplified, misfolded protein aggregate
may include one or more of: a soluble portion and an insoluble
portion. The first misfolded protein aggregate may be substantially
be a soluble first misfolded protein aggregate. In some
embodiments, the methods may provide that the sample excludes tau
fibrils. For example, the sample may be filtered or centrifuged to
remove tau fibrils.
[0131] In various embodiments, the second substrate protein may be
distinct from the first substrate protein. The second substrate
protein may include one of: amyloid-beta (A.beta.), alpha
synuclein, 3R tau, and 4R tau. The first substrate protein may
include 4R tau.
[0132] In some embodiments, the methods may include performing at
least a second PMCA procedure to determine the presence or absence
in the sample of a second misfolded protein aggregate. The second
PMCA procedure may include forming a second incubation mixture by
contacting a second portion of the sample with a second substrate
protein. The second substrate protein may be subject to
pathological misfolding and/or aggregation in vivo to form the
second misfolded protein aggregate. The second PMCA procedure may
include conducting an incubation cycle two or more times under
conditions effective to form a second amplified, misfolded protein
aggregate. Each incubation cycle may include incubating the second
incubation mixture effective to cause misfolding and/or aggregation
of the second substrate protein in the presence of the second
misfolded protein aggregate. Each incubation cycle may include
disrupting the second incubation mixture effective to form the
second amplified, misfolded protein aggregate. The second PMCA
procedure may include determining the presence or absence in the
sample of the second misfolded protein aggregate by analyzing the
second incubation mixture for the presence or absence of the second
amplified, misfolded protein aggregate. The second misfolded
protein aggregate may include the second substrate protein. The
second amplified, misfolded protein aggregate may include the
second substrate protein.
[0133] In several embodiments, the tauopathy may be present in the
subject. The methods may include characterizing an identity of the
tauopathy in the subject according to the presence in the sample of
the first misfolded protein aggregate. The methods may include
characterizing an identity of the tauopathy in the subject
according to the presence or absence in the sample of the second
misfolded protein aggregate.
[0134] In some embodiments, the methods may provide that that the
tauopathy is not primarily characterized by misfolding and/or
aggregation of 3R tau protein. For example, the tauopathy may be
characterized at least in part by misfolded and/or aggregated 4R
tau protein, in a ratio to misfolded and/or aggregated 3R tau
protein, of one of about: 1:99, 5:95, 10:90, 15:85, 20:80, 25:75,
30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30,
75:25, 80:20, 85:15, 90:10, 95:5, and 99:1, or a range between any
two of the preceding ratios, for example, between 1:99 and
99:1.
[0135] In various embodiments, the methods may include contacting
the sample with a thioflavin, e.g., thioflavin S or thioflavin T,
and a molar excess of the first substrate protein to form the first
incubation mixture. The molar excess may be greater than an amount
of protein monomer included in the first misfolded protein
aggregate in the sample. The methods may include conducting the
incubation cycle two or more times effective to form the first
amplified, misfolded protein aggregate. Each incubation cycle may
include incubating the first incubation mixture effective to cause
misfolding and/or aggregation of at least the portion of the first
substrate protein in the presence of the first misfolded protein
aggregate. Each incubation cycle may include shaking the first
incubation mixture effective to form the first amplified, misfolded
protein aggregate. The methods may include determining the presence
or absence of the first misfolded protein aggregate in the sample
by detecting a fluorescence of the thioflavin corresponding to the
first misfolded protein aggregate.
[0136] In some embodiments of the methods, the capturing the first
misfolded protein aggregate from the sample to form a captured
first misfolded protein aggregate may be conducted using one or
more antibodies specific for the first misfolded protein aggregate.
The one or more antibodies specific for the first misfolded protein
aggregate may include one or more of: an antibody specific for an
amino acid epitope sequence of the first misfolded protein
aggregate and an antibody specific for a conformation of the first
misfolded protein aggregate. The antibody specific for a
conformation of the first misfolded protein aggregate may be
selective for a conformational epitope of a tauopathy-specific
first misfolded protein aggregate. The antibody specific for the
conformation of the first misfolded protein aggregate may
correspond to one of: Alzheimer's disease (AD), Parkinson's Disease
(PD), Progressive Supranuclear Palsy (PSP), FrontoTemporal Dementia
(FTD), Corticobasal degeneration (CBD), Mild cognitive impairment
(MCI), Argyrophilic grain disease (AgD) Traumatic Brain Injury
(TBI), Chronic Traumatic Encephalopathy (CTE), and Dementia
Pugilistica (DP). The one or more antibodies specific for the first
misfolded protein aggregate may be coupled to a solid phase. The
solid phase may include one or more of a magnetic bead and a
multiwell plate. Contacting the sample with the first substrate
protein to form the first incubation mixture may include contacting
a molar excess of the first substrate protein to the sample. The
molar excess of the first substrate protein may be greater than a
total molar amount of protein monomer included in the captured
first misfolded protein aggregate. Incubating the first incubation
mixture may be effective to cause misfolding and/or aggregation of
the first substrate protein in the presence of the captured first
misfolded protein aggregate to form the first amplified, misfolded
protein aggregate. The first substrate protein may include 4R tau
protein.
[0137] In various embodiments, the methods may include performing
at least a second PMCA procedure to determine the presence or
absence in the sample of a second misfolded protein aggregate. The
second PMCA procedure may include forming a second incubation
mixture by contacting a second portion of the sample with a second
substrate protein, the second substrate protein may be subject to
pathological misfolding and/or aggregation. The second PMCA
procedure may include conducting an incubation cycle two or more
times under conditions effective to form a second amplified,
misfolded protein aggregate. Each incubation cycle may include
incubating the second incubation mixture effective to cause
misfolding and/or aggregation of the second substrate protein in
the presence of the second misfolded protein aggregate. Each
incubation cycle may include disrupting the second incubation
mixture effective to form the second amplified, misfolded protein
aggregate The second PMCA procedure may include determining the
presence or absence in the sample of the second misfolded protein
aggregate by analyzing the second incubation mixture for the
presence or absence of the second amplified, misfolded protein
aggregate. The tauopathy may be present in the subject. The methods
may include characterizing an identity of the tauopathy in the
subject according to: the presence in the sample of the first
misfolded protein aggregate; and the presence or absence in the
sample of the second misfolded protein aggregate. The second
misfolded protein aggregate may include the second substrate
protein. The second amplified, misfolded protein aggregate may
include the second substrate protein. The second substrate protein
may be distinct from the first substrate protein. The second
substrate protein may include one of: amyloid-beta (A.beta.), alpha
synuclein, and 3R tau.
[0138] In some embodiments, the methods may include distinguishing
each tauopathy from one or more additional tauopathies by analyzing
for at least one signature of one or more misfolded aggregates each
corresponding to one of A.beta., alpha synuclein, and 3R tau. Each
signature may correspond to one or more of: an assay using an
antibody selective for a conformational epitope of any of the one
or more misfolded aggregates; an assay using an antibody selective
for a conformational epitope of any of the one or more misfolded
aggregates; one or more PMCA kinetic parameters of the one or more
misfolded aggregates, including one or more of: lag phase,
T.sub.50, amplification rate, and amplification extent; an
indicator selective for any of the one or more misfolded
aggregates; and a spectrum characteristic of any of the one or more
misfolded aggregates.
[0139] Further, for example, specific antibodies may be employed
for second misfolded protein aggregates. For example, for AD,
amyloid antibodies may include one or more of: 6E10, 4G8, 82E1,
A11, X-40/42, 16ADV; and the like. Such antibodies may be obtained
as follows: 6E10 and 4G8 (Covance, Princeton, N.J.); 82E1 (IBL
America, Minneapolis, Minn.); All (Invitrogen, Carlsbad, Calif.);
X-40/42 (MyBioSource, Inc., San Diego, Calif.); and 16ADV (Acumen
Pharmaceuticals, Livermore, Calif.).
[0140] Further, for PD, for example, the one or more synuclein
specific antibodies may include PD specific antibodies including
one or more of: .alpha./.beta.-synuclein N-19; .alpha.-synuclein
C-20-R; .alpha.-synuclein 211; .alpha.-synuclein Syn 204;
.alpha.-synuclein 2B2D1; .alpha.-synuclein LB 509;
.alpha.-synuclein SPM451; .alpha.-synuclein 3G282;
.alpha.-synuclein 3H2897; a/.beta.-synuclein Syn 202;
a/.beta.-synuclein 3B6; .alpha./.beta./.gamma.-synuclein FL-140;
and the like. In some examples, the one or more specific antibodies
may include one or more of: a/.beta.-synuclein N-19;
.alpha.-synuclein C-20-R; .alpha.-synuclein 211; .alpha.-synuclein
Syn 204; and the like. Such antibodies may be obtained as follows:
a/.beta.-synuclein N-19 (cat. No. SC-7012, Santa Cruz Biotech,
Dallas, Tex.); .alpha.-synuclein C-20-R (SC-7011-R);
.alpha.-synuclein 211 (SC-12767); .alpha.-synuclein Syn 204
(SC-32280); .alpha.-synuclein 2B2D1 (SC-53955); .alpha.-synuclein
LB 509 (SC-58480); .alpha.-synuclein SPM451 (SC-52979);
.alpha.-synuclein 3G282 (SC-69978); .alpha.-synuclein 3H2897
(SC-69977); a/.beta.-synuclein Syn 202 (SC-32281);
a/.beta.-synuclein 3B6 (SC-69699); or
.alpha./.beta./.gamma.-synuclein FL-140 (SC-10717).
[0141] In various embodiments, a kit is provided for determining a
presence or absence in a sample of a first misfolded protein
aggregate. The kit may include a first substrate protein that may
include 4R tau. The kit may include an indicator of the first
misfolded protein aggregate. The first misfolded protein aggregate
may include the first substrate protein. The first misfolded
protein aggregate may correspond to a tauopathy. The kit may
include a buffer. The kit may include heparin. The kit may include
a salt. The kit may include instructions. The instructions may
direct a user to obtain the sample. The instructions may direct the
user to perform at least a first PMCA procedure. The first PMCA
procedure may include forming a first incubation mixture by
contacting a first portion of the sample with the first substrate
protein, the indicator of the first misfolded protein aggregate,
the buffer, the heparin, and the salt. The first incubation mixture
may be formed with a concentration of one or more of: the first
substrate protein of less than about 20 .mu.M; the heparin of less
than about 75 .mu.M; the salt as NaCl of less than about 190 mM;
and the indicator of the first misfolded protein aggregate as
Thioflavin T of less than about 9.5 .mu.M. The first PMCA procedure
may include conducting an incubation cycle two or more times
effective to form a first amplified, misfolded protein aggregate.
Each incubation cycle may include incubating the first incubation
mixture effective to cause misfolding and/or aggregation of the
first substrate protein in the presence of the first misfolded
protein aggregate. Each incubation cycle may include disrupting the
incubation mixture effective to form the first amplified, misfolded
protein aggregate. The instructions may direct the user to
determine the presence or absence in the sample of the first
misfolded protein aggregate by analyzing the first incubation
mixture for the presence or absence of the first amplified,
misfolded protein aggregate according to the indicator of the first
misfolded protein aggregate.
[0142] In several embodiments, the kit may include any element of
the methods described herein. Moreover, the kit may include
instructions directing the user to conduct any of the steps of the
methods described herein.
[0143] In some embodiments, for example, the instructions may
include directing the user to obtain the sample from a subject. The
sample may include one or more of: a bio-fluid, a biomaterial, a
homogenized tissue, and a cell lysate. The instructions directing
the user to determine or diagnose a tauopathy in the subject
according to the presence or absence in the sample of the first
misfolded protein aggregate.
[0144] In various embodiments, the kit may include a second
substrate protein and an indicator of a second misfolded protein
aggregate. The second misfolded protein aggregate may include the
second substrate protein. The second substrate protein may be
distinct from the first substrate protein. The second substrate
protein may include one of: amyloid-beta (A.beta.), alpha
synuclein, 3R tau, and 4R tau. The instructions may direct the user
to perform at least a second PMCA procedure. The second PMCA
procedure may include forming a second incubation mixture by
contacting a second portion of the sample with the second substrate
protein and the indicator of the second misfolded protein
aggregate. The second PMCA procedure may include conducting an
incubation cycle two or more times effective to form a second
amplified, misfolded protein aggregate. Each incubation cycle may
include incubating the second incubation mixture effective to cause
misfolding and/or aggregation of the second substrate protein in
the presence of the second misfolded protein aggregate. Each
incubation cycle may include disrupting the incubation mixture
effective to form the second amplified, misfolded protein
aggregate. The second PMCA procedure may include determining the
presence or absence in the sample of the second misfolded protein
aggregate by analyzing the second incubation mixture for the
presence or absence of the second amplified, misfolded protein
aggregate. The instructions may also direct the user to
characterize the sample for an identity of a tauopathy according
to: the presence in the sample of the first misfolded protein
aggregate; and the presence or absence in the sample of the second
misfolded protein aggregate.
[0145] In some embodiments, the kit may include a PMCA apparatus.
The PMCA apparatus may include one or more of: a multiwall
microtitre plate; a microfluidic plate; a shaking apparatus; a
spectrometer; and an incubator. The apparatus may be included
either as one or more of the individual plates or apparatuses, as a
combination device, and the like. For example, a shaking microplate
reader may be used to perform cycles of incubation and shaking and
automatically measure the ThT fluorescence emission during the
course of an experiment (e.g., FLUOstar OPTIMA, BMG LABTECH Inc.,
Cary, N.C.).
[0146] The antibody specific for the conformation of the first
misfolded protein aggregate may correspond to one of: Alzheimer's
disease (AD), Parkinson's Disease (PD), Progressive Supranuclear
Palsy (PSP), FrontoTemporal Dementia (FTD), Corticobasal
degeneration (CBD), Mild cognitive impairment (MCI), Argyrophilic
grain disease (AgD) Traumatic Brain Injury (TBI), Chronic Traumatic
Encephalopathy (CTE), and Dementia Pugilistica (DP). The
instructions may include determining, according to a binding assay
using the antibody specific for the conformation of the first
misfolded protein aggregate, the presence or absence in the subject
of one of AD, PD, PSP, FTD, CBD, MCI, AgD, TBI, CTE, and DP.
EXAMPLES
Example 1: Preparation of Synthetic A.beta. Oligomers
[0147] A.beta.1-42 was synthesized using solid-phase
N-tert-butyloxycarbonyl chemistry at the W. Keck Facility at Yale
University and purified by reverse-phase HPLC. The final product
was lyophilized and characterized by amino acid analysis and mass
spectrometry. To prepare stock solutions free of aggregated,
misfolded A.beta. protein, aggregates were dissolved high pH and
filtration through 30 kDa cut-off filters to remove remaining
aggregates. To prepare different types of aggregates, solutions of
seed-free A.beta.1-42 (10 .mu.M) were incubated for different times
at 25.degree. C. in 0.1 M Tris-HCl, pH 7.4 with agitation. This
preparation contained a mixture of A.beta. monomers as well as
fibrils, protofibrils and soluble, misfolded A.beta. protein in
distinct proportions depending on the incubation time. The degree
of aggregation was characterized by ThT fluorescence emission,
electron microscopy after negative staining, dot blot studies with
the A11 conformational antibody and western blot after gel
electrophoresis using the 4G8 anti-A.beta. antibody.
[0148] A mixture of A.beta. oligomers of different sizes were
generated during the process of fibril formation. Specifically,
soluble, misfolded A.beta. protein was prepared by incubation of
monomeric synthetic A.beta.1-42 (10 .mu.M) at 25.degree. C. with
stirring. After 5 h of incubation, an abundance of soluble,
misfolded A.beta. protein, globular in appearance, was observed by
electron microscopy after negative staining, with only a small
amount of protofibrils and fibrils observed. At 10 h there are
mostly protofibrils and at 24 h, a large amount of long fibrils are
observed. FIG. 1A shows electron micrographs taken at 0 h, 5 h, 10
h, and 24 h of incubation.
[0149] The soluble, misfolded A.beta. protein aggregates tested
positive using A11 anti-oligomer specific antibody according to the
method of Kayed, et al. "Common structure of soluble amyloid
oligomers implies common mechanism of pathogenesis," Science 2003,
300, 486-489. After further incubation at 10 h and 24 h,
protofibrillar and fibrillar structures were observed. The size of
the aggregates was determined by filtration through filters of
defined pore size and western blotting after SDS-PAGE separation.
Soluble, misfolded A.beta. protein formed by incubation for 5 h was
retained in filters of 30 kDa cut-off and passed through 1000 kDa
cutoff filters. FIG. 1B is a western blot of soluble, misfolded
A.beta. protein aggregates. Electrophoretic separation of this
soluble, misfolded A.beta. protein showed that the majority of the
material migrated as .about.170 kDa SDS-resistant aggregates, with
a minor band at 17 kDa.
Example 2: A.beta.-PMCA Detects Synthetic A.beta. Oligomers
[0150] EXAMPLE 2A. Seeding of A.beta. aggregation was studied by
incubating a solution of seed-free A.beta.1-42 in the presence of
Thioflavin T with or without different quantities of synthetic
soluble, misfolded A.beta. protein (Control (no oligomer); or 3,
80, 300, and 8400 femtomolar in synthetic soluble, misfolded
A.beta. protein). A.beta.-PMCA general procedure: Solutions of 2
.mu.M aggregate-free A.beta.1-42 in 0.1 M Tris-HCl pH 7.4 (200
.mu.L total volume) were placed in opaque 96-wells plates and
incubated alone or in the presence of synthetic A.beta. aggregates
(prepared by incubation over 5 h as described in EXAMPLE 1) or 40
.mu.L of CSF aliquots. Samples were incubated in the presence of 5
.mu.M Thioflavin T (ThT) and subjected to cyclic agitation (1 min
at 500 rpm followed by 29 min without shaking) using an Eppendorf
thermomixer, at a constant temperature of 22.degree. C. At various
time points, ThT fluorescence was measured in the plates at 485 nm
after excitation at 435 nm using a plate spectrofluorometer. FIG.
2A is a graph of amyloid formation (without cyclic amplification)
versus time as measured by Thioflavin T fluorescence, using the
indicated femtomolar concentration of synthetic soluble, misfolded
A.beta. protein seeds. The peptide concentration, temperature and
pH of the buffer were monitored to control the extent of the lag
phase and reproducibility among experiments. Under these
conditions, no spontaneous A.beta. aggregation was detected during
the time in which the experiment was performed (125 h). Aggregation
of monomeric A.beta.1-42 protein was observed in the presence of
0.3 to 8.4 fmol of the synthetic soluble, misfolded A.beta. protein
of EXAMPLE 1.
[0151] EXAMPLE 2B: Amplification cycles, combining phases of
incubation and physical disruption were employed. The same samples
as in FIG. 2A were incubated with cyclic agitation (1 min stirring
at 500 rpm followed by 29 min without shaking). Aggregation was
measured over time by the thioflavin T (ThT) binding to amyloid
fibrils using a plate spectrofluorometer (excitation: 435;
emission: 485 nm). Graphs show the mean and standard error of 3
replicates. The concentration of A.beta. oligomers was estimated
assuming an average molecular weight of 170 kDa. FIG. 2B is a graph
showing amplification cycle-accelerated amyloid formation measured
by ThT fluorescence as a function of time for various
concentrations of the synthetic soluble, misfolded A.beta. protein
of EXAMPLE 1. Under these conditions, the aggregation of monomeric
A.beta.1-42 protein induced by 8400, 300, 80 and 3 fmol of the
synthetic soluble, misfolded A.beta. protein was clearly faster and
more easily distinguished from that observed in the absence of the
synthetic soluble, misfolded A.beta. protein. This result indicates
the detection limit, under these conditions, is 3 fmol of soluble,
misfolded A.beta. protein or less in a given sample.
Example 3: A.beta.-PMCA Detects Misfolded A.beta. in the
Cerebrospinal Fluid of AD Patients
[0152] Aliquots of CSF were obtained from 50 AD patients, 39
cognitively normal individuals affected by non-degenerative
neurological diseases (NND), and 37 patients affected by non-AD
neurodegenerative diseases including other forms of dementia
(NAND). Test CSF samples were obtained from 50 patients with the
diagnosis of probable AD as defined by the DSM-IV and the
NINCDS-ADRA guidelines (McKhann et al., 1984) and determined using
a variety of tests, including routine medical examination,
neurological evaluation, neuropsychological assessment, magnetic
resonance imaging and measurements of CSF levels of A.beta.1-42,
total Tau and phospho-Tau. The mean age of AD patients at the time
of sample collection was 71.0.+-.8.1 years (range 49-84). Control
CSF samples were obtained from 39 patients affected by
non-degenerative neurological diseases (NND), including 12 cases of
normal pressure hydrocephalus, 7 patients with peripheral
neuropathy, 7 with diverse forms of brain tumor, 2 with ICTUS, 1
with severe cephalgia, 3 with encephalitis, 1 with hypertension and
6 with unclear diagnosis. The mean age at which CSF samples were
taken from this group of patients was 64.6.+-.14.7 years (range
31-83). Control CSF samples were also taken from 37 individuals
affected by non-AD neurodegenerative diseases (NAND), including 10
cases of fronto-temporal dementia (5 behavioral and 5 language
variants), 6 patients with Parkinson's disease (including 4
associated with dementia and 1 with motor neuron disease), 6 with
progressive supranuclear palsy, 6 with spinocerebellar ataxia (1
associated with dementia), 4 with amyotrophic lateral sclerosis, 2
with Huntington's disease, 1 with MELAS, 1 with Lewy body dementia,
and 1 with vascular dementia. The mean age at sample collection for
this group was 63.8.+-.11.1 years (range 41-80). CSF samples were
collected in polypropylene tubes following lumbar puncture at the
L4/LS or L3/L4 interspace with atraumatic needles after one night
fasting. The samples were centrifuged at 3,000 g for 3 min at
4.degree. C., aliquoted and stored at -80.degree. C. until
analysis. CSF cell counts, glucose and protein concentration were
determined. Albumin was measured by rate nephelometry. To evaluate
the integrity of the blood brain barrier and the intrathecal IgG
production, the albumin quotient (CSF albumin/serum
albumin).times.10.sup.3 and the IgG index (CSF albumin/serum
albumin)/(CSF IgG/serum IgG) were calculated. The study was
conducted according to the provisions of the Helsinki Declaration
and was approved by the Ethics Committee.
[0153] The experiments as well as the initial part of the analysis
were conducted blind. FIG. 3A is a graph of amyloid formation
versus time, measured as a function of ThT fluorescence labeling,
showing the average kinetics of A.beta. aggregation of 5
representative samples from the AD, NND, and NAND groups.
[0154] The results indicate that CSF from AD patients significantly
accelerates A.beta. aggregation as compared to control CSF
(P<0.001). The significance of the differences in A.beta.
aggregation kinetics in the presence of human CSF samples was
analyzed by one-way ANOVA, followed by the Tukey's multiple
comparison post-test. The level of significance was set at
P<0.05. The differences between AD and samples from the other
two groups were highly significant with P<0.001 (***).
[0155] FIG. 3B is a graph of the lag phase time in h for samples
from the AD, NND, and NAND groups. To determine the effect of
individual samples on A.beta. aggregation, the lag phase was
estimated, defined as the time to ThT fluorescence larger than 40
arbitrary units after subtraction of a control buffer sample. This
value was selected considering that it corresponds to .about.5
times the reading of the control buffer sample.
[0156] FIG. 3C is a graph showing the extent of amyloid formation
obtained after 180 A.beta.-PMCA cycles, e.g. 90 h of incubation
(P90). Comparison of the lag phase and P90 among the experimental
groups reveals a significant difference between AD and control
samples from individuals with non-degenerative neurological
diseases or with non-AD neurodegenerative diseases. Further, no
correlation was detected between the aggregation parameters and the
age of the AD patients, which indicates that the levels of the
marker corresponds to aggregated A.beta. protein in patient CSF,
and not patient age.
[0157] FIG. 5, Table 1 shows estimations of the sensitivity,
specificity and predictive value of the A.beta.-PMCA test,
calculated using the lag phase numbers.
[0158] To study reproducibility, an experiment similar to the one
shown in FIGS. 3A-C was independently done with different samples,
reagents and a new batch of A.beta. peptide as substrate for
A.beta.-PMCA. The extent of amyloid formation obtained after 300
A.beta.-PMCA cycles, e.g. 150 h of incubation (P150), was measured
in each patient. The control group includes both people affected by
other neurodegenerative diseases and non-neurologically sick
patients. Data for each sample represent the average of duplicate
tubes. Statistical differences were analyzed by student-t test.
FIG. 6 is a graph of the lag phase time in h for samples obtained
after 300 A.beta.-PMCA cycles, e.g. 150 h of incubation (P90).
[0159] During the course of the study an entire set of CSF samples
coming from a fourth location did not aggregate at all, even after
spiking with large concentrations of synthetic oligomers. It is
expected that reagent contamination during sample collection
interfered with the assay.
[0160] The differences in aggregation kinetics between different
samples were evaluated by the estimation of various different
kinetic parameters, including the lag phase, A50, and P90. Lag
phase is defined as the time required to reach a ThT fluorescence
higher than 5 times the background value of the buffer alone. The
A50 corresponds to the time to reach 50% of maximum aggregation.
P90 corresponds to the extent of aggregation (measured as ThT
fluorescence) at 90 h. Sensitivity, specificity and predictive
value were determined using this data, with cutoff thresholds
determined by Receiver Operating Characteristics (ROC) curve
analysis, using MedCalc software (MedCalc Software, Ostend,
Belgium).
Example 4: Determination of Threshold Values of Misfolded for
A.beta.-PMCA Detection of Ad in CSF
[0161] In support of FIG. 5, TABLE 1, sensitivity, specificity and
predictive value were determined using the lag phase data, with
cutoff thresholds determined by Receiver Operating Characteristics
(ROC) curve analysis, using the MedCalc software (version 12.2.1.0,
MedCalc, Belgium). As shown in FIG. 5, TABLE 1, a 90.0% sensitivity
and 84.2% specificity was estimated for the control group
consisting of age-matched individuals with non-degenerative
neurological diseases. By contrast, for the clinically more
relevant differentiation of AD from other neurodegenerative
diseases including other forms of dementia, 100% sensitivity and
94.6% specificity was estimated. This ability of A.beta.-PMCA to
distinguish AD from other forms of neurodegenerative diseases is
clinically significant. The overall sensitivity and specificity
considering all control individuals was 90% and 92%,
respectively.
[0162] To evaluate the performance of the A.beta.-PMCA test to
distinguish AD patients from controls, the true positive rate
(sensitivity) was plotted as a function of the false positive rate
(specificity) for different cut-off points. For this analysis the
lag phase values for AD vs NAND (FIG. 4A), AD vs NND (FIG. 4B) and
AD vs All control samples (FIG. 4C) was used. The performance of
the test, estimated as the area under the curve was
0.996.+-.0.0033, 0.95.+-.0.020 and 0.97.+-.0.011 for the comparison
of AD with NAND, NND and all controls, respectively. Statistical
analysis was done using the MedCalc ROC curve analysis software
(version 12.2.1.0) and the result indicated that the test can
distinguish AD from the controls with a P<0.0001. To estimate
the most reliable cut-off point for the different set of group
comparisons, sensitivity (blue line) and specificity (red line)
were plotted for each cut-off value (FIG. 4D). The graph shows the
curve and the 95% confidence intervals for the AD vs all control
samples (including NAND and NND groups). These cut-off values were
used to estimate sensitivity, specificity and predictive value in
FIG. 5, Table 1.
Example 5: A.beta.-Oligomer Immunodepletion Removes A.beta. Seeds
in Human Cerebrospinal Fluid and Confirms A.beta.-PMCA Detects
Soluble Misfolded A.beta. Protein in Ad CSF
[0163] Immunodepletion experiments were performed to confirm that
A.beta.-PMCA detects a seeding activity associated to soluble,
misfolded A.beta. protein present in CSF. The methodology for
efficient immunodepletion of soluble, misfolded A.beta. protein was
first optimized by using synthetically prepared soluble, misfolded
A.beta. protein. Immunodepletion was performed by incubation with
dynabeads conjugated with a mixture of antibodies recognizing
specifically the sequence of A.beta. (4G8) and conformational (A11)
antibodies. FIG. 7A is a western blot showing results of
immunodepletion using synthetically prepared A.beta. oligomers
spiked into human CSF. Soluble, misfolded A.beta. protein was
efficiently removed by this immunodepletion.
[0164] FIGS. 7A and 7B are graphs of amyloid formation versus time
as measured by Thioflavin T fluorescence, demonstrating that
seeding activity in AD CSF is removed by soluble, misfolded A.beta.
protein immuno-depletion. Samples of AD CSF before or after
immunodepletion with 4G8 and All antibodies were used to seed
A.beta. aggregation in the A.beta.-PMCA assay. Immunodepletion was
applied to 3 AD CSF. FIG. 7B is a graph showing the kinetics of
control and immunodepleted CSF samples. FIG. 7B shows that for
immunodepleted AD CSF, the kinetics of A.beta. aggregation in the
A.beta.-PMCA reaction was comparable to that observed in control
CSF samples, and both were significantly different from the
aggregation observed with AD CSF prior to immunodepletion. FIG. 7C
is a graph showing the kinetics of control and immunodepleted CSF
samples, depleted only with the A11 conformational antibody and
aggregation monitored by A.beta.-PMCA assay. FIG. 7C shows similar
results, obtained using AD CSF immunodepleted using the A11
conformational antibody, which specifically recognizes, misfolded
A.beta.. These results confirm that A.beta.-PMCA detects soluble,
misfolded .beta. protein in AD CSF.
Example 6: Solid Phase Immuno Capturing
[0165] FIGS. 8A and 8B are schematic representations of two solid
phase methods used to capture soluble, misfolded A.beta. protein
from complex samples such as blood plasma. Strategy 1 employed
ELISA plates pre-coated with specific antibodies bound to a solid
phase on the ELISA plate. After washing the plates, the
A.beta.-PMCA reaction was carried out in the same plates. Strategy
2 used magnetic beads as the solid phase coated with specific
antibodies. This approach provided concentration of the
samples.
Example 7: Specificity of Immuno Capturing
[0166] FIG. 9 shows Table 2, demonstrating the ability of specific
antibodies to capture the A.beta. oligomers. The top panel shows a
schematic representation of the epitope recognition site on the
A.beta. protein of the diverse sequence antibodies used in this
study. Table 2 in FIG. 9 demonstrates the efficiency of different
sequence or conformational antibodies to capture A.beta. oligomers.
The capacity to capture oligomers was measured by spiking synthetic
A.beta. oligomers in healthy human blood plasma and detection by
A.beta.-PMCA. The symbols indicate that the detection limits using
the different antibodies were: <12 fmol (+++); between 10-100
fmol (++); >1 pmol (+) and not significantly higher than without
capturing reagent (-).
Example 8: Detection of A.beta. Oligomers Spiked in Human
Plasma
[0167] FIG. 10 is a graph of amyloid formation versus time as
measured by Thioflavin T fluorescence showing detection of soluble,
misfolded A.beta. protein spiked in human plasma. ELISA plates
pre-coated with protein G were coated with an anti-conformational
antibody (16ADV from Acumen). Thereafter, plates were incubated
with human blood plasma (100 .mu.l) as such (control) or spiked
with different concentrations of synthetic soluble, misfolded
A.beta. protein. After incubation, plates were subjected to
A.beta.-PMCA (29 min incubation and 30 s shaking) in the presence
of A.beta.40 monomer (2 .mu.M) and ThT (5 .mu.M). Amyloid formation
was measured by Thioflavin fluorescence. FIG. 10 is representative
of several experiments done with 3 different antibodies which
worked similarly.
Example 9: Capturing of Soluble Misfolded A.beta. from Ad Patient
Samples Vs Controls
[0168] FIG. 11 is a graph showing time to reach 50% aggregation in
an A.beta.-PMCA assay in plasma samples from AD patients and
controls. Blood plasma samples from patients affected by AD, non-AD
neurodegenerative diseases (NAD), and healthy controls were
incubated with anti-A.beta. antibody (82E1) coated beads.
A.beta.-PMCA was carried out as described in EXAMPLE 2. The time
needed to reach 50% aggregation was recorded in individual patients
in each group. Differences were analyzed by one-way ANOVA followed
by the Tukey's post-hoc test. ROC analysis of this data showed a
82% sensitivity and 100% specificity for correctly identifying AD
patients from controls.
Example 10: Sonication and Shaking are Effective with Various
Detection Methods
[0169] FIG. 12 is a western blot showing the results of
amplification of A.beta. aggregation by using sonication instead of
shaking as a mean to fragment aggregates. The experiment was done
in the presence of distinct quantities of synthetic A.beta.
oligomers. Samples of 10 .mu.g/ml of seed-free monomeric
A.beta.1-42 were incubated alone (lane 1) or with 300 (lane 2), 30
(lane 3) and 3 (lane 4) fmols of, misfolded A.beta.. Samples were
either frozen without amplification (non-amplified) or subjected to
96 PMCA cycles (amplified), each including 30 min incubation
followed by 20 sec sonication. Aggregated A.beta. was detected by
western blot using anti-A.beta. antibody after treatment of the
samples with proteinase K (PK). In our experiments, it was observed
that detection using thioflavin T fluorescence was not compatible
with sonication, but works very well with shaking as a physical
disruption method. FIG. 12 shows that using a different detection
method for the A.beta. aggregates, in this case Western Blotting,
sonication works as well as shaking.
Example 11: Production of Monomeric A.beta. as PMCA Substrate
[0170] Seed-free monomeric A.beta. was obtained by size exclusion
chromatography. Briefly, an aliquot of a 1 mg/mL peptide solution
prepared in dimethylsulfoxide was fractionated using a Superdex 75
column eluted at 0.5 mL/min with 10 mM sodium phosphate at pH 7.5.
Peaks will be detected by UV absorbance at 220 nm. The peak
corresponding to 4-10 kDa molecular weight containing
monomer/dimmers of A.beta. was collected and concentration
determined by amino acid analysis. Samples were stored lyophilized
at -80.degree. C.
Example 12: Production and Purification of A3
[0171] E. coli cells harboring pET28 GroES-Ub-A1342 plasmid were
grown in Luria broth (LB) at 37.degree. C., and expression was
induced with 0.4 mM IPTG. After 4 h, cells were harvested and lysed
in a lysis buffer (20 mM Tris-Cl, pH 8.0, 10 mM NaCl, 0.1 mM PMSF,
0.1 mM EDTA and 1 mM .beta.-mercaptoethanol) and centrifuged at
18,000 rpm for 30 min. Inclusion bodies were re-suspended in a
resuspension buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, and 1 mM
DTT) containing 6 M urea. Insoluble protein was removed by
centrifugation at 18,000 rpm for 30 min. The supernatant containing
GroES-Ub-A.beta.42 fusion protein will be collected. To cleave off
A.beta.42 from fusion protein, the fusion protein was diluted
2-fold with resuspension buffer and treated with recombinant
de-ubiquinating enzyme (Usp2cc) 1:100 enzyme to substrate molar
ratio at 37.degree. C. for 2 h. After that, samples was loaded on a
C18 column (25 mm.times.250 mm, Grace Vydac, USA). A.beta.42 was
purified with a solvent system buffer 1 (30 mM ammonium acetate, pH
10, 2% acetonitrile) and buffer 2 (70% acetonitrile) at a flow rate
10 ml/min using a 20-40% linear gradient of buffer 2 over 35 min.
Purified A.beta.42 was lyophilized and stored at -80.degree. C.,
until use.
Example 13: Detection of .alpha.S Seeds by PD-PMCA
[0172] EXAMPLE 13A: Seeding of .alpha.S aggregation was studied by
incubating a solution of seed-free .alpha.S in the presence of
Thioflavin T with or without different quantities of synthetic
soluble oligomeric .alpha.S protein: Control (no .alpha.S
oligomer); or 1 ng/mL, 10 ng/mL, 100 ng/mL, and 1 .mu.g/mL of the
synthetic soluble oligomeric .alpha.S protein seed. .alpha.S-PMCA
general procedure: Solutions of 100 .mu.g/mL .alpha.S seed-free
.alpha.S in PBS, pH 7.4 (200 .mu.L total volume) were placed in
opaque 96-wells plates and incubated alone or in the presence of
the indicated concentrations of synthetic .alpha.S aggregates or 40
.mu.L of CSF aliquots. Samples were incubated in the presence of 5
.mu.M Thioflavin T (ThT) and subjected to cyclic agitation (1 min
at 500 rpm followed by 29 min without shaking) using an Eppendorf
thermomixer, at a constant temperature of 22.degree. C. At various
time points, ThT fluorescence was measured in the plates at 485 nm
after excitation at 435 nm using a plate spectrofluorometer. FIG.
13A is a graph of Thioflavin T fluorescence as a function of time,
showing the detection of .alpha.S seeds by PD-PMCA, using the
indicated concentration of synthetic soluble oligomeric .alpha.S
protein seeds. The peptide concentration, temperature and pH of the
buffer were monitored to control the extent of the lag phase and
reproducibility among experiments. Aggregation of monomeric
.alpha.S protein was observed in the presence of 1 ng/mL, 10 ng/mL,
100 ng/mL, and 1 .mu.g/mL .alpha.S of the synthetic soluble
oligomeric .alpha.S protein seed.
[0173] EXAMPLE 13B: The time to reach 50% aggregation as a function
of amounts of .alpha.S seeds added was determined using the samples
in EXAMPLE 1A. FIG. 13B is a graph showing time to reach 50%
aggregation plotted as a function of amounts of .alpha.S seeds
added.
Example 14: .alpha.S-PMCA Detects Oligomeric .alpha.S in the
Cerebrospinal Fluid of Pd Patients
[0174] Detection of seeding activity in human CSF samples from
controls and PD patients was performed by PD-PMCA. Purified seed
free alpha-synuclein (100 .mu.g/mL) in PBS, pH 7.4 was allowed to
aggregate at 37.degree. C. with shaking at 500 rpm in the presence
of CSF from human patients with confirmed PD, AD or
non-neurodegenerative neurological diseases (NND). The extend of
aggregation was monitored by Thioflavin fluorescence at 485 nm
after excitation at 435 nm using a plate spectrofluorometer.
[0175] Aliquots of CSF were obtained from PD patients, cognitively
normal individuals affected by non-degenerative neurological
diseases (NND), and patients affected by Alzheimer's disease (AD).
Test CSF samples were obtained from patients with the diagnosis of
probable PD as defined by the DSM-IV and determined using a variety
of tests, including routine medical examination, neurological
evaluation, neuropsychological assessment, and magnetic resonance
imaging. CSF samples were collected in polypropylene tubes
following lumbar puncture at the L4/L5 or L3/L4 interspace with
atraumatic needles after one night fasting. The samples were
centrifuged at 3,000 g for 3 min at 4.degree. C., aliquoted and
stored at -80.degree. C. until analysis. CSF cell counts, glucose
and protein concentration were determined. Albumin was measured by
rate nephelometry. To evaluate the integrity of the blood brain
barrier and the intrathecal IgG production, the albumin quotient
(CSF albumin/serum albumin).times.10.sup.3 and the IgG index (CSF
albumin/serum albumin)/(CSF IgG/serum IgG) were calculated. The
study was conducted according to the provisions of the Helsinki
Declaration and was approved by the Ethics Committee.
[0176] The experiments as well as the initial part of the analysis
were conducted blind. FIG. 14 is a graph of .alpha.S
oligomerization versus time, measured as a function of ThT
fluorescence labeling, showing the average kinetics of .alpha.S
aggregation of representative samples from the PD, AD, and NND
groups.
[0177] The results indicate that CSF from PD patients significantly
accelerates .alpha.S aggregation as compared to control CSF
(P<0.001). The significance of the differences in .alpha.S
aggregation kinetics in the presence of human CSF samples was
analyzed by one-way ANOVA, followed by the Tukey's multiple
comparison post-test. The level of significance was set at
P<0.05. The differences between PD and samples from the other
two groups were highly significant with P<0.001 (***).
Example 15: Specificity of Immuno Capturing
[0178] FIG. 15 shows Table 3, demonstrating the ability of
different sequence or conformational antibodies to capture .alpha.S
oligomers. The capacity to capture oligomers was measured by
spiking synthetic .alpha.S oligomers in healthy human blood plasma
and detection by .alpha.S-PMCA. The first column shows various
antibodies tested and corresponding commercial sources. The second
column lists the epitope recognition site on the .alpha.S protein
of the diverse sequence antibodies used in this study. The third
column indicates the observed ability of specific antibodies to
capture the .alpha.S oligomers. The symbols indicate that the
detection limits using the different antibodies were: <12 fmol
(+++); between 10-100 fmol (++); >1 pmol (+) and not
significantly higher than without capturing reagent (-).
Alpha/beta-synuclein antibody N-19 (N-terminal epitope) and
alpha-synuclein antibody C-20-R (C-terminal epitope) showed the
best results; and alpha-synuclein antibody 211 (epitope: amino
acids 121-125) showed very good results; alpha-synuclein antibody
204 (epitope: fragment 1-130) showed good results; and 16 ADV Mouse
IgG1 (conformational epitope) showed no result.
Example 16: Solid Phase Immuno Capturing
[0179] FIGS. 16A and 16B are schematic representations of two solid
phase methods used to capture soluble, misfolded .alpha.S protein
from complex samples such as blood plasma. Strategy 1 employed
ELISA plates pre-coated with specific antibodies bound to a solid
phase on the ELISA plate. After washing the plates, the
.alpha.S-PMCA reaction was carried out in the same plates. Strategy
2 used magnetic beads as the solid phase coated with specific
antibodies. This approach provided concentration of the
samples.
Example 17: .alpha.S-PMCA for the Detection of .alpha.-Synuclein
Oligomers Spiked in Human Blood Plasma
[0180] Immunoprecipitation of .alpha.-Synuclein oligomers from
human blood plasma was performed by anti-.alpha.-Synuclein
antibody-coated beads (Dynabeads) and a seeding aggregation assay
using .alpha.-Synuclein monomers as seeding substrate along with
thioflavin-T for detection. The anti-.alpha.-Synuclein coated beads
(1.times.10.sup.7 beads) were incubated with human blood plasma
(500 .mu.L) with .alpha.-Synuclein seeds (+0.2 .mu.g Seed) and
without .alpha.-Synuclein seeds (-Seed). After immunoprecipitation,
the beads were re-suspended in 20 .mu.L, of reaction buffer
(1.times.PBS), and 10 .mu.L of beads were added to each well of a
96-well plate. The aggregation assay was performed by adding
.alpha.-Synuclein monomers (200 .mu.g/mL) and thioflavin-T (5
.mu.M). The increase in florescence was monitored by a fluorimeter
using an excitation of 435 nm and emission of 485 nm. FIG. 17A
illustrates immunoprecipitation/aggregation results with N-19
antibody in blood plasmas with and without seed. FIG. 17B
illustrates immunoprecipitation/aggregation results with 211
antibody in blood plasmas with and without seed. FIG. 17C
illustrates immunoprecipitation/aggregation results with C-20
antibody in blood plasmas with and without seed.
Example 18: .alpha.S-PMCA Detects Oligomeric .alpha.S in the
Cerebrospinal Fluid of Patients Affected by Pd and Multiple System
Atrophy with High Sensitivity and Specificity
[0181] To study the efficiency of .alpha.S-PMCA for biochemical
diagnosis of PD and related .alpha.-synucleinopathies, such as
multiple system atrophy (MSA), tests were performed on CSF from
many patients affected by these diseases as well as controls
affected by other diseases. FIGS. 18A, 18B, and 18C show detection
of seeding activity in human CSF samples from controls and patients
affected by PD and MSA, respectively, using .alpha.S-PMCA. Purified
seed free alpha-synuclein (100 .mu.g/mL) in buffer MES, pH 6.0 was
allowed to aggregate at 37.degree. C. with shaking at 500 rpm in
the presence of CSF from human patients and controls. The extent of
aggregation was monitored by Thioflavin T fluorescence at 485 nm
after excitation at 435 nm using a plate spectrofluorometer.
[0182] Test CSF samples were obtained from patients with the
diagnosis of probable PD and MSA as defined by the DSM-IV and
determined using a variety of tests, including routine medical
examination, neurological evaluation, neuropsychological
assessment, and magnetic resonance imaging. CSF samples were
collected in polypropylene tubes following lumbar puncture at the
L4/LS or L3/L4 interspace with atraumatic needles after one night
fasting. The samples were centrifuged at 3,000 g for 3 min at
4.degree. C., aliquoted and stored at -80.degree. C. until
analysis. CSF cell counts, glucose and protein concentration were
determined. Albumin was measured by rate nephelometry. The study
was conducted according to the provisions of the Helsinki
Declaration and was approved by the Ethics Committee.
[0183] The experiments as well as the initial part of the analysis
were conducted blind. FIGS. 18A, 18B, and 18C are graphs of
.alpha.S aggregation versus time, measured as a function of ThT
fluorescence labeling, showing the average kinetics of .alpha.S
aggregation, respectively, for controls and two representative
samples from the PD and MSA groups.
[0184] The results indicate that CSF from PD patients significantly
accelerates .alpha.S aggregation as compared to control CSF
(P<0.001). The significance of the differences in .alpha.S
aggregation kinetics in the presence of human CSF samples was
analyzed by one-way ANOVA, followed by the Tukey's multiple
comparison post-test. The level of significance was set at
P<0.05. The differences between PD and samples from the other
two groups were highly significant with P<0.001 (***).
[0185] The outcome of the overall set of 29 PD or MSA samples and
41 controls was that 26 of the 29 PD or MSA samples were positive,
whereas 3 of the 41 control samples were positive, which
corresponded to a 90% sensitivity and 93% specificity.
Example 19: Synthesis of Full-Length 4R Tau Protein and Seeds
[0186] FIG. 19 is a flow chart showing the preparation and
purification of recombinant full-length 4R tau protein. A gene for
hTau40 was transfected into E. coli and incubated under standard
conditions to express the hTau40. After a period of growth, the E.
coli cells were pelted, lysed, heat treated, and precipitated with
ammonium sulfate to produce a crude product. The crude product was
subjected to cation exchange chromatography, dialysis, then
concentrated and the buffer exchanged. Cut-off filtration at a mass
of 100 kDa was employed to further purify the hTau40. The yield of
the purified hTau 40 was 20 mg/L of bacterial culture. Full-length
4R Tau seeds were then prepared by incubating hTau 40 monomer with
12.5 .mu.M heparin in 10 mM HEPES pH 7.4, 100 mM NaCl for 3 days at
37.degree. C. using cyclic agitation (1 min shaking at 500 rpm
followed by 29 min without shaking).
Example 20a: Tau PMCA
[0187] A Tau-PMCA assay was performed on 96 well plates using 12.5
.mu.M Tau monomer, 1.25 .mu.M heparin, 5 .mu.M Thioflavin T, using
cyclic agitation (1 min shaking at 500 rpm followed by 29 min
without shaking). Seeds were added to the wells in amounts of 12.5
pmol, 1.25 pmol, 125 fmol, 12.5 fmol, and 1.25 fmol. Controls were
performed without seeds. Aggregation was followed over time by ThT
fluorescence using a plate spectrofluorometer (excitation: 435;
emission: 485). FIG. 20A is a graph of aggregation in % for the
various initial amounts of seeds and the control. The values in
FIG. 20A are the mean of two replicates, with the error bars
indicating standard deviation. FIG. 20B is a graph of T.sub.50, the
time to 50% aggregation as measured by ThT fluorescence versus the
log of the amount of oligomeric tau seeds in fmol.
Example 20B: Tau PMCA
[0188] For optimization of the tau-PMCA assay, full-length human
Tau40 that includes four imperfect tandem microtubule binding
repeats (4R). Tau oligomers were generated by incubation of
full-length Tau (50 .mu.M) in the presence of heparin (12.5 .mu.M)
for 3 days at 37.degree. C. Seeds were characterized by ability to
seed tau aggregation, binding to thioflavin T, western blot and
electron microscopy. The preformed aggregates were used to nucleate
and induce the aggregation of Tau. For the assay, seed-free
monomeric tau (15 .mu.M) in 10 mM HEPES pH 7.4, 100 mM NaCl
containing 3 .mu.M of heparin in the absence or the presence of
different quantities of synthetic seeds was subjected to cycles of
tau-PMCA by incubating at 20.degree. C. for 29.5 min followed by
shaking for 30 sec at 500 rpm. Under these conditions, Tau was only
observed to aggregate in the presence of preformed seeds and the
kinetic of aggregation was dependent on the amount of seeds added.
Importantly, the PMCA signal was observed to be directly
proportional to the amount of seeds added to the reaction. This
assay corresponded to a detection threshold of 0.125 pg of
oligomeric tau. This detection threshold corresponds to -2
atto-mol, based on the molecular weight of the tau monomer, or 0.15
atto-mol, based on the molecular weight of a 12-mer oligomer as a
proxy for average oligomer size.
Example 20C: Tau PMCA
[0189] A further Tau-PMCA assay was performed using full-length Tau
seeds prepared by incubating Tau monomer with 12.5 .mu.M heparin in
10 mM HEPES pH 7.4, 100 mM NaCl for 3 days at 37.degree. C. with
shaking. The assay was performed on 96 well plates using 12.5 .mu.M
Tau monomer, 1.25 .mu.M heparin, and 5 .mu.M Thioflavin T, using
cyclic agitation (1 min shaking at 500 rpm followed by 29 min
without shaking). FIG. 20C is a graph of aggregation followed over
time by ThT fluorescence using a plate spectrofluorometer
(excitation: 435; emission: 485). FIG. 20C shows the mean and SD of
two replicates. FIG. 20D is a graph of the relationship between the
quantity of tau oligomers and the Tau-PMCA signal (time to reach
50% aggregation).
Example 20D: Tau PMCA is Reproducible
[0190] A large scale experiment was conducted to evaluate the
robustness and reproducibility of the tau-PMCA assay to analyze the
performance at four different times (0, 14, 28 and 30 days) with or
without freezing/thawing. Two different set of synthetic seeds and
five different concentrations of synthetic seeds (1250, 125, 12.5,
1.25 and 0.125 pg of seeds) were employed, spiked either in buffer
or control CSF. Each sample was run in triplicate. The experiment
encompassed several steps, including the large-scale expression and
purification of tau in quantities needed to perform all
experiments, quality control of the material produced, generation
and characterization of synthetic tau oligomeric seeds and the
tau-PMCA experiments to investigate assay precision and
reproducibility in buffer and in the biological matrix (CSF). In
total the experiment employed 32 different conditions (2 different
seeds.times.4 time points x: 2 manners of dilution (freezing or not
freezing).times.2 different matrices (buffer or CSF)). Since all
conditions were tested with five different concentrations of
oligomeric seeds and each was done in triplicate, the entire
experiment involved 480 wells. The protein concentration, buffer
and PMCA conditions were the same as EXAMPLE 20B. From the 32
conditions tested only one gave results that were slightly
significantly different from the others, indicating high precision
and reproducibility. FIGS. 20E-20L are a series of graphs that
display the aggregation results based on ThT fluorescence of 8 of
the conditions tested, including 4 different time points (0, 7, 14
and 30 days) with samples subjected to freezing and thawing or not
and in the presence of buffer or CSF, and two different seed
preparations (FIGS. 20E-20H and FIGS. 20I-20L). FIGS. 20E-20L
demonstrate that the results obtained are very similar between the
triplicates, different time points, and distinct seeds. Data
correspond to average.+-.standard error of triplicate samples. Tau
substrate in the absence of seeds was not observed to aggregate
under any condition within the time in which experiments were
done.
[0191] To analyze the reproducibility of the assay, T.sub.50 values
(time to reach 50% aggregation) for the experiments in the presence
of 1250 pg of Tau seeds. The Tso values for the experiments done at
different days, with one of two seed preparations A or B and using
fresh or frozen seeds did not show any statistically significant
difference and an average Tso of 71.5.+-.1.8 h was obtained.
Similar non-significant differences were observed for the studies
done in the presence of all the other seed concentrations or for
the experiments done in buffer. FIG. 20M is a table of T.sub.50
values showing reproducibility across 16 different conditions. All
values were analyzed by one way ANOVA, followed by Tukey multiple
comparison test.
Example 20E: Tau PMCA is Specific
[0192] The tau PMCA assay was investigated for specificity,
particularly for the ability to detect aggregates composed of other
amyloidogenic proteins. A.beta. and .alpha.Syn oligomeric species
were prepared and used to cross-seed monomeric tau. FIG. 20N is a
graph of ThT fluorescence vs time for the tau assay seeded with 1
pm of tau, A.beta.40, AB42, His .alpha.Syn, Hu .alpha.Syn, and no
seeds. FIG. 20N shows that no significant signal was detectable in
the presence of A.beta. or .alpha.Syn seeds and no signal was
detected before about 100 h, even when the concentration of these
particles was relatively high (equivalent to 2 ng of tau seeds).
These A.beta. are .alpha.Syn seeds are very efficient in inducing
aggregation in the respective A.beta.- or .alpha.Syn-PMCA assays
described in preceding EXAMPLES. These results indicate that under
the conditions and concentrations used there is no cross-seeding
between other protein aggregates and that tau-PMCA is specific for
detecting tau oligomers.
Example 21a: Tau PMCA Detects Tau in Human CSF
[0193] Human CSF samples from AD patients (7 cases), 5 other
Tauopathies (1FTD, 2PSP, 2CBD), people affected by mild cognitive
impairment (MCI) and controls affected by other neurological
diseases (7 samples) were analyzed by Tau-PMCA. FIG. 21A is a graph
showing ThT fluorescence at 447 h of incubation, in which most
samples have reached the maximum fluorescence. Positive controls
used samples of healthy CSF spiked with synthetic Tau oligomers
(12.5 fmol). Negative controls correspond to samples of healthy CSF
without Tau seeds. FIG. 21A shows that patients with AD, other
tauopathies, and MCI showed Tau aggregation significantly above the
negative control and consistent with the positive control. 6 of the
7 control patients with other neurological diseases were consistent
with the negative control. One control patients in the other
neurological disease group showed maximum fluorescence consistent
with a tauopathy. This may indicate an undiagnosed tauopathy in
that patient, or alternatively, inadvertent contamination.
Example 21B: Tau PMCA Detects Tau in Human CSF
[0194] Human CSF samples from 11 patients affected by AD, 11 from
other tauopathies (4 PSP, 1 FTD, 5 CBD and 1 CTE) and 7 controls
affected from unrelated neurological disorders were examined.
Positive controls were prepared by spiking CSF with 20 ng of
recombinant tau oligomers. Negative controls were healthy CSF
without tau seeds. FIG. 21B is a graph of fluorescence signals for
samples from patients with AD or other tauopathies for tau-PMCA
comparable to that observed in samples containing recombinant tau
oligomers. Consequently, samples from patients with AD or other
tauopathies were able to accelerate tau aggregation. Conversely, 6
out of 7 of the controls produced a low signal in tau-PMCA, with
values equivalent to those observed in CSF without seeds. Despite
the small sample size, the differences between controls and
patients were statistically significant. These positive results
indicate that tau-PMCA is capable of detecting tau aggregates in
CSF of patients. FIG. 21B shows the maximum ThT fluorescence,
expressed as arbitrary units. Differences were analyzed by one-way
ANOVA followed by the Tukey's multiple comparison post-test. **
P<0.01.
Example 22: Tau PMCA Detects Tau in Human CSF
[0195] The performance of Tau-PMCA assay was examined in the
presence of representative CSF samples from a control, and patients
affected by AD, FTD (frontotemporal dementia), CBD (corticobasal
degeneration), and PSP (progressive supranuclear palsy). The
Tau-PMCA assay was performed on 96 well plates using 12.5 .mu.M Tau
monomer, 1.25 .mu.M heparin, 5 .mu.M Thioflavin T, using cyclic
agitation (1 min shaking at 500 rpm followed by 29 min without
shaking). Aggregation was followed over time by ThT fluorescence
using a plate spectrofluorometer (excitation: 435; emission: 485).
FIG. 22 is a graph showing aggregation % based on ThT versus time.
The various tauopathies differed in T.sub.50, amplification rate,
and amplification extent. For example, the T.sub.50 of PSP and AD
samples was about the same at 150 h, while the PSP sample appeared
to have a shorter lag phase, a lower amplification rate, and a
lower extent of amplification compared to AD. CBD appeared to have
a T.sub.50 of about 175 h, with a lower amplification rate, and a
lower extent of amplification compared to both AD and PSP. FTD
appeared to have a T.sub.50 of about 210 h, with a higher
amplification rate, and a lower extent of amplification compared to
each of AD, PSP, and CBD. The control CSF sample had a similar Tso
and amplification rate compared to FTD, with a far lower extent of
amplification. The amplification in the control CSF sample may be
due to spontaneous (seed-free) amplification, inadvertent
contamination, or an undiagnosed tauopathy with somewhat similar
kinetics to FTD.
Discussion
[0196] The present tau-PMCA may provide various advantages. For
example, embodiments of the present invention may detect the tau
misfolded protein directly, by contrast with known indirect
measures such as non-pathogenic biomarkers, measurement of the
total pool of tau of which only a small fraction forms the
synapto-toxic oligomeric aggregates, or measurement of variously
phosphorylated species of tau. In various embodiments, the present
invention detects the misfolded tau oligomers that seed tau
misfolding and are believed to contribute to spreading the damage
in the brain during the disease.
[0197] To the extent that the term "includes" or "including" is
used in the specification or the claims, it is intended to be
inclusive in a manner similar to the term "comprising" as that term
is interpreted when employed as a transitional word in a claim.
Furthermore, to the extent that the term "or" is employed (e.g., A
or B) it is intended to mean "A or B or both." When the applicants
intend to indicate "only A or B but not both" then the term "only A
or B but not both" will be employed. Thus, use of the term "or"
herein is the inclusive, and not the exclusive use. See Bryan A.
Garner, A Dictionary of Modern Legal Usage 624 (2d. Ed. 1995).
Also, to the extent that the terms "in" or "into" are used in the
specification or the claims, it is intended to additionally mean
"on" or "onto." To the extent that the term "selectively" is used
in the specification or the claims, it is intended to refer to a
condition of a component wherein a user of the apparatus may
activate or deactivate the feature or function of the component as
is necessary or desired in use of the apparatus. To the extent that
the term "operatively connected" is used in the specification or
the claims, it is intended to mean that the identified components
are connected in a way to perform a designated function. To the
extent that the term "substantially" is used in the specification
or the claims, it is intended to mean that the identified
components have the relation or qualities indicated with degree of
error as would be acceptable in the subject industry.
[0198] As used in the specification and the claims, the singular
forms "a," "an," and "the" include the plural unless the singular
is expressly specified. For example, reference to "a compound" may
include a mixture of two or more compounds, as well as a single
compound.
[0199] As used herein, the term "about" in conjunction with a
number is intended to include .+-.10% of the number. In other
words, "about 10" may mean from 9 to 11.
[0200] As used herein, the terms "optional" and "optionally" mean
that the subsequently described circumstance may or may not occur,
so that the description includes instances where the circumstance
occurs and instances where it does not.
[0201] In addition, where features or aspects of the disclosure are
described in terms of Markush groups, those skilled in the art will
recognize that the disclosure is also thereby described in terms of
any individual member or subgroup of members of the Markush group.
As will be understood by one skilled in the art, for any and all
purposes, such as in terms of providing a written description, all
ranges disclosed herein also encompass any and all possible
sub-ranges and combinations of sub-ranges thereof .DELTA.ny listed
range can be easily recognized as sufficiently describing and
enabling the same range being broken down into at least equal
halves, thirds, quarters, fifths, tenths, and the like. As a
non-limiting example, each range discussed herein can be readily
broken down into a lower third, middle third and upper third, and
the like. As will also be understood by one skilled in the art all
language such as "up to," "at least," "greater than," "less than,"
include the number recited and refer to ranges which can be
subsequently broken down into sub-ranges as discussed above.
Finally, as will be understood by one skilled in the art, a range
includes each individual member. For example, a group having 1-3
cells refers to groups having 1, 2, or 3 cells. Similarly, a group
having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells,
and so forth. While various aspects and embodiments have been
disclosed herein, other aspects and embodiments will be apparent to
those skilled in the art.
[0202] As stated above, while the present application has been
illustrated by the description of embodiments thereof, and while
the embodiments have been described in considerable detail, it is
not the intention of the applicants to restrict or in any way limit
the scope of the appended claims to such detail. Additional
advantages and modifications will readily appear to those skilled
in the art, having the benefit of the present application.
Therefore, the application, in its broader aspects, is not limited
to the specific details, illustrative examples shown, or any
apparatus referred to. Departures may be made from such details,
examples, and apparatuses without departing from the spirit or
scope of the general inventive concept.
[0203] The various aspects and embodiments disclosed herein are for
purposes of illustration and are not intended to be limiting, with
the true scope and spirit being indicated by the following
claims.
* * * * *