U.S. patent application number 13/821185 was filed with the patent office on 2014-01-09 for morphology and protein specific reagents as diagnostics for neurodegenerative diseases.
This patent application is currently assigned to Arizona Board of Regents, A Body Corporate of the State of Arizona, Acting for and on Behalf of Az.. The applicant listed for this patent is Sharareh Emadi, Srinath Kasturirangan, Shalini Prasad, Michael Sierks. Invention is credited to Sharareh Emadi, Srinath Kasturirangan, Shalini Prasad, Michael Sierks.
Application Number | 20140011691 13/821185 |
Document ID | / |
Family ID | 45994379 |
Filed Date | 2014-01-09 |
United States Patent
Application |
20140011691 |
Kind Code |
A1 |
Sierks; Michael ; et
al. |
January 9, 2014 |
MORPHOLOGY AND PROTEIN SPECIFIC REAGENTS AS DIAGNOSTICS FOR
NEURODEGENERATIVE DISEASES
Abstract
The invention relates to devices and diagnostic methods using
the devices for detecting the presence of neurodegenerative
diseases. The device (a "biosensor") distinguishes between
different neurodegenerative diseases and facilitates
pre-symptomatic diagnoses.
Inventors: |
Sierks; Michael; (Ft.
McDowell, AZ) ; Kasturirangan; Srinath; (Germantown,
MD) ; Emadi; Sharareh; (Scottsdale, AZ) ;
Prasad; Shalini; (Chandler, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sierks; Michael
Kasturirangan; Srinath
Emadi; Sharareh
Prasad; Shalini |
Ft. McDowell
Germantown
Scottsdale
Chandler |
AZ
MD
AZ
AZ |
US
US
US
US |
|
|
Assignee: |
Arizona Board of Regents, A Body
Corporate of the State of Arizona, Acting for and on Behalf of
Az.
|
Family ID: |
45994379 |
Appl. No.: |
13/821185 |
Filed: |
October 26, 2011 |
PCT Filed: |
October 26, 2011 |
PCT NO: |
PCT/US11/57925 |
371 Date: |
September 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61406721 |
Oct 26, 2010 |
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61406728 |
Oct 26, 2010 |
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61406734 |
Oct 26, 2010 |
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Current U.S.
Class: |
506/9 ; 506/18;
506/39 |
Current CPC
Class: |
A61K 38/1716 20130101;
G01N 33/6857 20130101; G01N 2333/4709 20130101; C07K 2317/569
20130101; G01N 2800/2821 20130101; G01N 33/5438 20130101; G01N
33/54386 20130101; A61K 39/3955 20130101; C07K 16/005 20130101;
G01N 33/6896 20130101; C07K 16/18 20130101; C07K 2317/76 20130101;
C07K 2317/622 20130101; C07K 2317/73 20130101 |
Class at
Publication: |
506/9 ; 506/18;
506/39 |
International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 33/68 20060101 G01N033/68 |
Goverment Interests
U.S. GOVERNMENT RIGHTS
[0002] This work was supported by the National Institutes of Health
Grant R01 AG017984. The United States Government has certain rights
to this invention.
Claims
1. A biosensor comprising: a. a microelectrode array base platform
containing an array of conductive-material sensing sites, wherein
each sensing site is comprised of a working electrode (WE) and a
counter electrode (CE) wherein the surface area ratio of CE: WE is
between about 20:1 and 300:1; b. a nanoporous membrane overlaid on
the platform wherein the membrane forms nanowells, wherein each
nanowell comprises immobilized therein an antibody agent that
specifically detects a marker of a neurodegenerative disease; c. a
microfluidic encapsulant to enable lateral flow of reagents over
and into the nanowells.
2. The biosensor of claim 1, wherein the conductive material is
platinum, gold, silver, or copper.
3-6. (canceled)
7. The biosensor of claim 1, wherein the nanoporous membrane is
aluminum.
8. The biosensor of claim 1, wherein the nanowell has an effective
diameter of about 10 nm to 500 nm.
9. (canceled)
10. The biosensor of claim 1, further comprising a readout
amplifier electrically connected to nano-pores in one or more
regions of the membrane.
11. The biosensor of claim 1, further comprising a fluid
chamber.
12. (canceled)
13. The biosensor of claim 1, wherein the base platform is an
insulator or includes an insulator portion.
14-15. (canceled)
16. The biosensor of claim 1, further comprising one or more
conductor strips coupling the biosensor to a sensor, wherein the
sensor is coupled to a multiplexer.
17. (canceled)
18. The biosensor of claim 1, wherein the marker of a
neurodegenerative disease is an A.beta., a-syn or tau
morphology.
19. The biosensor of claim 1, wherein the antibody agent is an
antibody, an Fab.sub.2 or a nanobody.
20. The biosensor of claim 19, wherein the nanobody is a C6, A4,
E1, D5, 10H, 6E, D10 or BSEC1 nanobody.
21. (canceled)
22. A method of detecting the presence of a neurodegenerative
disease comprising contacting a physiological sample from a subject
suspected of having a neurodegenerative disease with a biosensor of
claim 1 and measuring the output impedence of the biosensor in the
presence of the biological fluid wherein a change in the measured
output impedance across a sensing site is indicative of the
presence of a marker of a neurodegenerative disease.
23-29. (canceled)
30. A biosensor device comprising: (a) a printed circuit board
platform, (b) a nanoporous membrane, and (c) a micro fluidic
chamber.
31. The biosensor device of claim 30, wherein the printed circuit
board platform comprises inter-digitated working and counter
electrodes.
32. The biosensor device of claim 31, wherein the electrodes are
tin oxide, gold, platinum, silver, or copper, or a combination of
these materials.
33. (canceled)
34. The biosensor device of claim 30, wherein electrodes are about
600-1000 .mu.m in width, about 2-10 mm in length and about 600-1000
nm in thickness.
35. (canceled)
36. The biosensor device of claim 31, wherein the nanoporous
membrane is contacted onto the interdigitated electrodes.
37. (canceled)
38. The biosensor device of claim 30, wherein the nanoporous
membrane comprises alumina, polycarbonate, a metal oxide, or a
ceramic-based material.
39. (canceled)
40. The biosensor device of claim 30, wherein the nanoporous
membrane is 100-500 nm thick, has a lateral diameter of 5-20 mm
with pore diameters of 100-500 nm.
41. (canceled)
42. The biosensor device of claim 30, wherein the nanoporous
membrane has a porosity of about 25% to 50%.
43. The biosensor device of claim 30, wherein the micro fluidic
chamber is silicone, acrylic, plastic, or any other biocompatible
hydrophilic material.
44. (canceled)
45. The biosensor device of claim 30, wherein the micro fluidic
chamber forms an enclosure for the printed circuit board platform
and the nanoporous membrane to prevent evaporation.
46. The biosensor device of claim 45, wherein the micro fluidic
chamber has a volume of about 10 .mu.L to about 5 ml.
47. (canceled)
48. The biosensor device of claim 31, wherein the electrodes are
contacted with a negatively-charged substance to form a coated
electrode surface.
49. The biosensor device of claim 31, wherein the electrodes are
contacted with an amine or BST.
50. (canceled)
51. The biosensor device of claim 31, further comprising an
antibody agent immobilized onto the printed circuit board platform
or the coated electrode surface.
52. The biosensor device of claim 51, wherein the antibody agent is
an antibody, an Fab.sub.2 or a nanobody.
53. The biosensor device of claim 52, wherein the nanobody is a C6,
A4, E1, D5, 10H, 6E, D10 or BSEC1 nanobody.
54-57. (canceled)
Description
RELATED APPLICATIONS
[0001] This patent application claims the benefit of priority of
U.S. application Ser. No. 61/406,734, filed Oct. 26, 2010; U.S.
application Ser. No. 61/406,728, filed Oct. 26, 2010, and U.S.
application Ser. No. 61/406,721, filed Oct. 26, 2010, which
applications are incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0003] As our population ages, neurodegenerative diseases such as
Alzheimer's Disease (AD) and Parkinson's Disease (PD) will affect
an increasing number of people at rapidly increasing cost. For AD
alone, over 5 million Americans currently are living with the
disease, with nearly half a million new cases expected next year
with total yearly economic costs of nearly $150 billion. Despite
many impressive advances in brain research, current clinical
diagnostic methods cannot predict the development of AD in
cognitively unimpaired elderly individuals. Similar difficulties
exist in the diagnosis of other neurodegenerative diseases
including PD, Dementia with Lewy Bodies (DLB) and other
synucleinopathies and tauopathies, particularly for pre-symptomatic
diagnoses.
[0004] Therapies administered very early in the disease course,
before clinical signs and advanced brain destruction occur, are
more likely to be effective than later treatment, but this is not
possible at the present time due to the lack of an adequate
predictive test. Any such predictive test must be able to
distinguish the different neurodegenerative diseases from normal
decline in elderly individuals and from other less common diseases
that are clinically similar. Therefore, there is a crucial need for
new diagnostic tests that will detect these diseases before they
cause disability. While existing clinical diagnostic methods may
have a high sensitivity, they generally have much lower specificity
[1].
[0005] Certain neurodegenerative diseases correlate with various
misfolded and aggregated forms of different target proteins, such
as amyloid-beta (A.beta.) with AD, alpha-synuclein (a-syn) with PD,
DLB and other synucleinopathies, and tau with AD and various
tauopathies. Therefore, the concentration profile of various
aggregate forms of each of these respective proteins can provide a
pre-symptomatic indicator for each disease to facilitate diagnosis
and to monitor treatment.
[0006] A vast amount of literature implicates A.beta. accumulation
as being central to the progression of AD, leading to formation of
the A.beta. hypothesis [2]. The major weakness of the AP hypothesis
however, is that the presence of amyloid plaques does not correlate
well with the progression of AD [3, 4]. In addition to fibrillar
amyloid plaques, A.beta. can also form a number of soluble
intermediate or metastable structures which may contribute to
toxicity (reviewed in [5, 6]). Cortical levels of soluble A.beta.
correlated well with the cognitive impairment and loss of synaptic
function [7, 8]. Small, soluble aggregates of A.beta. termed
A.beta.-derived diffusible ligands (ADDLs) and spherical or annular
aggregates termed protofibrils are neurotoxic [9-11]. Oligomeric
forms of A.beta., created in vitro or derived from cell cultures,
inhibit long term potentiation (LTP) [10, 12, 13]. These small
oligomers are also called "low-n oligomers" (i.e., dimers, trimers,
or tetramers). The concentration of oligomeric forms of A.beta. are
also elevated in transgenic mouse models of AD [14] and in human AD
brain [15] and CSF samples [16]. Disruption of neural connections
near A.beta. plaques was also attributed to oligomeric A.beta.
species [17]. A halo of oligomeric A.beta. surrounds A.beta.
plaques causing synapse loss [18], and oligomeric AP was shown to
disrupt cognitive function in transgenic animal models of AD
[19-21]. Different size oligomers of A.beta. have been correlated
with AD, including a 56 kD aggregate [22] and smaller trimeric and
tetrameric species [5, 23].
[0007] The second major pathological feature of AD brains is the
presence of neurofibrillary tangles that contain aggregates of the
microtubule associated protein, tau. Tau is a natively unfolded
protein, and can aberrantly fold into various aggregate
morphologies including .beta.-sheet containing fibrillar forms
[24-27]. Tau has 21 different phosphorylation sites [28], and
excess phosphorylation can interfere with microtubule assembly.
Total tau concentration in CSF has been correlated with AD [29] as
has the presence of various phosphorylated tau forms or even the
ratio of phosphorylated tau to A.beta.42 (reviewed in [30]). Levels
of oligomeric tau have also been implicated as a potential early
diagnostic for AD [31]. Determination of total tau, phosphorylated
tau and oligomeric tau concentrations all have potential value as
diagnostics for AD.
[0008] Aggregation of alpha-synuclein (a-syn) plays a critical role
in PD and synucleinopathies. A-syn is a major component of Lewy
bodies and neurites [32, 33]. Wild-type a-syn along with the three
mutant forms, A30P, E46K and A53T can assemble into Lewy body like
fibrils in vitro [34-38]. Since all of the mutations increase the
total rate of oligomerization compared to the wild-type form of
a-syn [34, 36, 39], it has been postulated that the intermediate
oligomeric morphologies of a-syn are the toxic structures in PD
rather than fibrils. A partially folded intermediate of a-syn helps
to promote fibril formation in vitro [40] and a protofibrillar form
of a-syn is stabilized by formation of a dopamine adduct complex,
suggesting a possible connection between this morphology of a-syn
and dopaminergic cell death [41]. The different morphologies of
a-syn also have different affinities for various membranes, and
both the oligomeric forms [42-45] and fibrillar forms [45] have
been shown to disrupt membrane permeability and integrity.
[0009] Aggregated forms of a-syn were shown to induce toxicity in
dopaminergic neurons in vivo [46] and several different oligomeric
morphologies were shown to each have different toxic mechanisms and
effects on cells [47]. It has been shown that oligomeric but not
fibrillar forms of a-syn are toxic to neuronal cells [48]. Toxic
oligomeric a-syn forms were identified in living cells [49], in
human plasma from PD patients [50], and in human PD brain
tissue[48, 51].
[0010] Clearly protein misfolding and aggregation is a critically
important in many devastating neurodegenerative diseases.
Therefore, determining how concentration profiles of selected key
forms and morphologies of A.beta., tau and a-syn vary in healthy,
early, and late stage AD, PD and DLB patients will facilitate
development of an effective diagnostic assay for these diseases. In
order to define the role of these intermediates in the various
diseases, highly specific reagents to identify the different
protein forms are needed. Our lab has developed unique technology
that enables us to isolate reagents that bind specific morphologies
of a target protein. The inventors have combined the imaging
capabilities of AFM with the binding diversity of phage display
antibody technology to allow us to identify the presence of
specific protein morphologies and then isolate reagents that bind a
target morphology [52]. In order to determine how different protein
morphologies contribute to different diseases, it is important to
understand how the different diseases are currently diagnosed.
[0011] The diagnostic term "mild cognitive impairment" (MCI) was
originally introduced to define a progressive monosymptomatic
amnestic syndrome [53, 54], but more recently has evolved by
consensus into an entire classification scheme for early,
nondisabling cognitive disorders [55, 56] that include amnestic MCI
(single domain), amnestic MCI-multiple domains, nonamnestic,
MCI-single domain, and nonamnestic MCI-multiple domains. The
diagnostic criteria for probable AD [57] include dementia with
cognitive deficits in at least two cognitive domains including
progressive memory loss, normal level of consciousness, onset
between ages 40 and 90 years, and without another plausible medical
explanation. Diagnostic evaluation of symptomatic patients with MCI
and AD include MRI or CT of the brain, neuropsychological testing,
and a battery of blood tests all with the intention of excluding
potentially reversible (or other) causes of memory loss and
dementia. In patients with AD, CSF beta amyloid (A.beta.) levels
fall and tau levels rise with disease progression, but again are
diagnostically equivocal in early stage disease [58]. Blood levels
of amyloid have not been shown to be useful diagnostically, at any
stage. In asymptomatic people at genetic risk for AD, subtle
deficits can be disclosed suggesting that the disease process
begins much earlier than symptomatic onset of memory loss. For
example, otherwise healthy APOE e4 homozygotes perform less well on
memory tests when they are tired [59], less well on problem solving
tasks when they are anxious [60], and both cross sectional [61, 62]
and longitudinal neuropsychological studies [63, 64], show an
accelerated rate of memory decline relative to noncarriers that
predates symptomatic presentation of MCI or AD.
[0012] Lewy bodies, intracytoplasmic fibrillar aggregates of a-syn
and ubiquitin, are a signature feature of Parkinson's disease (PD),
and a defining feature of Dementia with Lewy Bodies (DLB) [65]. The
synucleinopathies are a group of disorders that include PD, DLB,
and Multiple System Atrophy (MSA) [65]. Dementia commonly occurs
within the context of parkinsonism, but is an infrequent
accompaniment of MSA. Neuropathological studies have shown that
cortical Lewy bodies are a common feature in patients with PD (32%
with H&E stains, up to 76% with ubiquitin stains), and a
defining feature in the setting of concurrent parkinsonism and
dementia [66]. Distinctions currently made between "Parkinson's
Disease with Dementia" (PDD) and DLB are based on the duration of
parkinsonism preceding the onset of cognitive symptoms, but
neuropathological findings are similar whether dementia occurs as
an early or late feature. Dementia itself correlates with cortical
Lewy body burden whether or nor not there is concurrent AD
pathology [67]. Distinguishing the relative contributions to
dementia of lewy body and AD pathology in PDD and DLB patients is
difficult. More than half of patients with clinically diagnosed DLB
have AD pathology in addition to neocortical Lewy bodies at autopsy
[68], and among patients with clinically diagnosed AD, neocortical
Lewy bodies are found in 15% at autopsy [69]. Clearly additional
tools to facilitate diagnoses of neurodegenerative diseases,
particularly early in the course of the diseases would be very
beneficial. Thus, there is a need to develop such tools that can be
used for the diagnosis of neurodegenerative diseases and as
adjuncts to therapeutic regimens.
SUMMARY OF THE INVENTION
[0013] In the present invention, the inventors have developed an
electronic biosensor that can simultaneously determine not just
levels of these key protein biomarkers, but levels of specific key
morphologies of each of these critical proteins. Selected
biomarkers have already been identified to help diagnose some of
these diseases and these can be readily incorporated into the
biosensor as well, however because these diseases are connected
with aggregation of specific proteins, detecting levels of selected
forms of each protein will enable an earlier and more accurate
diagnosis of the specific neurological disease and provide a means
to follow therapeutic strategies to determine their efficacy. The
biosensor is designed so that it can be ultimately used in a
physician's office using serum or CSF samples.
[0014] More specifically, the invention describes a biosensensor
comprising: [0015] a. a microelectrode array base platform
containing an array of conductive-material sensing sites, wherein
each sensing site is comprised of a working electrode (WE) and a
counter electrode (CE) wherein the surface area ration of CE:WE is
between about 20:1 and 300:1; [0016] b. a nanoporous membrane
overlaid on the platform wherein the membrane forms nanowells
wherein each nanowell comprises immobilized therein an antibody
agent that specifically detects a marker of a neurodegenerative
disease; [0017] c. a microfluidic encapsulant to enable lateral
flow of reagents over and into the nanowells.
[0018] In certain embodiments, the conductive material is platinum,
gold, silver, or copper. In certain embodiments, the array
comprises circular conductive-material sensing sites. In certain
embodiments, the ratio of CE:WE is 50:1. In other embodiments, the
ratio of CE:WE is 200:1. In certain embodiments, the nanoporous
membrane is aluminum. In certain embodiments, the nanowell has an
effective diameter of about 10 nm to 500 nm. In certain
embodiments, the nanowells are rectangular, hexagonal, circular,
elliptical, or other shape.
[0019] In certain embodiments, the biosensor further comprises a
readout amplifier electrically connected to nano-pores in one or
more regions of the membrane. In certain embodiments, the biosensor
further comprises a fluid chamber. In certain embodiments, the
fluid chamber is formed of polydimethoxysilane (PDMS). In certain
embodiments, the base platform is an insulator or includes an
insulator portion. In certain embodiments, the base platform is
silicon, glass, fused silica, polycarbonate, polyamides, ceramics,
epoxy, or plastic, and may further comprise an oxide layer.
[0020] In certain embodiments, the biosensor further comprises one
or more conductor strips coupling the biosensor to a sensor,
wherein the sensor is coupled to a multiplexer, and the multiplexer
may be controlled by a computer.
[0021] In certain embodiments, the marker of a neurodegenerative
disease is an A.beta., a-syn or tau morphology. As used herein, the
term "antibody" includes scFv (also called a "nanobody"),
humanized, fully human or chimeric antibodies, single-chain
antibodies, diabodies, and antigen-binding fragments of antibodies
(e.g., Fab fragments). In certain embodiments, the antibody agent
is an antibody, an Fab.sub.2 or a nanobody. In certain embodiments,
the antibody agent is a C6, A4, E1, D5, 10H, 6E, D10 or BSEC1
nanobody.
[0022] In the biosensor of the invention the marker of a
neurodegenerative disease is a nanobody that detects to selected
A.beta., a-syn and tau morphologies. For example, the biosensor has
immobilized thereon a C6 nanobody. In certain embodiments, the C6
nanobody is less than 300 amino acids in length and comprises amino
acid residues 16-292 of SEQ ID NO:1. In specific embodiments, the
antibody fragment has an amino acid sequence of SEQ ID NO:1. The
antibody fragment is specific for a 12-16 kDa oligomeric species of
A.beta..
[0023] The present invention discloses an antibody or antibody
fragment that specifically recognizes oligomeric A.beta. that is at
least partially resistant to denaturation by SDS (i.e., is
SDS-stable) but does not bind monomeric or fibrillar A.beta. or in
vitro-generated oligomeric A.beta.. Also contemplated herein is a
binding molecule that binds to oligomeric A.beta. that is at least
partially resistant to denaturation by SDS but does not bind
monomeric A.beta., fibrillar A.beta. or oligomeric forms of A.beta.
that are generated in vitro, wherein the binding molecule comprises
the sequence of SEQ ID NO:1. As used herein, "SDS-stable" means
that the oligomeric aggregate does not dissociate into the monomer
units in SDS (such as in 1% SDS
[0024] The present invention also provides a biosensor device
comprising a solid substrate and immobilized thereon one or more of
a C6, A4, E1, D5, 10H, 6E, D10 or BSEC1 nanobody.
[0025] Also contemplated is a method of detecting the presence of a
neurodegenerative disease comprising contacting a biological fluid
from a subject suspected of having a neurodegenerative disease with
a biosensor of the invention and measuring the output impedence of
said biosensor in the presence of said biological fluid wherein a
change in the measured output impedance across a sensing site is
indicative of the presence of a marker of a neurodegenerative
disease.
[0026] The present invention further provides a biosensor device
comprising: (a) a printed circuit board platform, (b) a nanoporous
membrane, and (c) a micro fluidic chamber. In certain embodiments,
the printed circuit board platform comprises inter-digitated
working and counter electrodes. In certain embodiments, the
electrodes are tin oxide, gold, platinum, silver, or copper, or a
combination of these materials, such as tin oxide and gold. In
certain embodiments, the electrodes are about 600-1000 .mu.m in
width, about 2-10 mm in length and about 600-1000 nm in thickness.
In certain embodiments, the electrodes are about 800 .mu.m in
width, about 5 mm in length and about 800 nm in thickness. In
certain embodiments, the electrodes have rounded edges to minimize
fringe effects during the application of a sinusoidal voltage input
signal. In certain embodiments, the nanoporous membrane is soldered
onto the interdigitated electrodes generating a high density array
of nanowells. In certain embodiments the nanoporous membrane is
attached to the interdigitated electrodes by means of epoxy
bonding, pressure attachment. In certain embodiments, the
nanoporous membrane is flush on the substrate. In certain
embodiments, the nanoporous membrane is a nanoporous alumina
membrane. In certain embodiments, the nanoporous membrane is
polycarbonate, a metal oxide, or a ceramic-based material. In
certain embodiments, the nanoporous membrane is 100-500 nm thick,
has a lateral diameter of 5-20 mm with pore diameters of 100-500
nm. In certain embodiments, the nanoporous membrane is 250 nm
thick, has a lateral diameter of 13 mm with pore diameters of 200
nm. In certain embodiments, the nanoporous membrane has a porosity
of about 25% to 50%. In certain embodiments, the micro fluidic
chamber is silicone. In certain embodiments, the micro fluidic
chamber is acrylate, plastic, or any other biocompatible
hydrophilic material. In certain embodiments, the micro fluidic
chamber forms an enclosure for the printed circuit board platform
and the nanoporous membrane to prevent evaporation. In certain
embodiments, the micro fluidic chamber has a volume of about 10
.mu.L to about 5 ml. The volume needs to be sufficient to wet the
membrane and not dry out. In certain embodiments, the micro fluidic
chamber has a volume of about 1.6 ml. In certain embodiments, the
electrode is contacted with a negatively-charged substance to form
a coated electrode surface. In certain embodiments, the charged
substance is an amine (e.g., 3-aminopropyl triethoxysilane (APTES))
or BST. In certain embodiments, the biosensor device further
comprises an antibody agent immobilized onto the printed circuit
board platform or the coated electrode surface. In certain
embodiments, the antibody agent is an antibody, an Fab.sub.2 or a
nanobody. In certain embodiments, the nanobody is a C6, A4, E1, D5,
10H, 6E, D10 or BSEC1 nanobody. In certain embodiments, the
antibody agent is immobilized onto the printed circuit board
platform or the coated electrode surface by means of one or more
negatively- or positively-charged chemical linkers. In certain
embodiments, the chemical linker is a thiol linker, a carboxylic
linker, or a hydroxy linker. In certain embodiments, the thiol
linker is 3,3'-dithiobis succinimidyl propionate (DSP).
[0027] The present invention further provides a method of making a
biosensor device comprising: [0028] (a) functionalizing an
electrode surface with an amide to form a silanized electrode
surface, [0029] (b) soldering an alumina membrane to the silanized
electrode surface, [0030] (c) coating the silanized electrode
surface with a cross-linking agent to form a functionalized
surface, and [0031] (d) contacting an antibody agent to the
functionalized surface.
BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS
[0032] FIGS. 1A-1F. Nanodevice of the invention. (A) Nanomonitor
(NM) device, (B) Sensing Site--Working (WE) and Counter (CE)
electrodes, (C) Input/Output pads connected to sensing site through
interconnects, (D) Nanoporous membrane overlaying on NM, (E) SEM of
membrane, and (F) the microfluidic encapsulant fabricated out of
biocompatible acrylic to regulate the lateral flow of reagents onto
the sensing sites. The encapsulant is interfaced to the device
through a pressure sensitive adhesive. It consists of 8
microchannels, each encapsulating one sensing site and consisting
of an input and output port. Each microchannel supports the flow of
8 .mu.l onto the sensing site.
[0033] FIG. 2. Binding specificity of nanobodies toward different
oligomeric A.beta. forms by time course immunoreactivity assay.
A.beta.40 was incubated for 10 days and aliquots removed and probed
with E1 and A4 scFvs.
[0034] FIG. 3. E1 binds human AD but not healthy brain extract.
Brain extract from non-disease (ND) and AD patients were probed
with E1 nanobody.
[0035] FIG. 4. AFM images of 3 different anti-oligomeric A.beta.
phage displayed scFvs. Phage appear as long thin filaments. C6
reacts only with brain derived oligomers, E1 with 1 day synthetic
oligomers, and A4 with 3 day synthetic oligomers.
[0036] FIG. 5. Detection of oligomeric a-syn in human post-mortem
CSF samples on Nanomonitor devices. The D5 nanobody against
oligomeric a-syn was immobilized into the nanowell surfaces of the
sensing sites. CSF samples were injected onto the sensing sites.
Binding of the oligomeric a-syn was characterized by measuring
impedance changes from the baseline. A minimum of three replicates
were performed for each sample, and the data was extracted for the
100 Hz frequency.
[0037] FIGS. 6A-6B. Detection of oligomeric a-syn (A) and A.beta.
(B) in human post-mortem CSF samples using electronic biosensor.
The nanobodies D5 (oligomeric a-syn) and A4 (oligomeric A.beta.)
were separately immobilized onto nanowell surfaces. CSF samples
were injected and binding of specific oligomeric a-syn or A.beta.
species was determined by measuring impedance changes from
baseline. At least three replicates were performed for each sample.
Data was extracted for 100 Hz frequency. Three CSF samples of
Non-demented (ND1-3), AD (AD 1-3), and PD (PD1-3) cases were
analyzed. PD3 represents an MSA patient with Parkinsonism.
[0038] FIG. 7 illustrates conductive strips 403-406 with gaps 402
formed on a surface of a base substrate 400.
[0039] FIG. 8 provides nucleic acid and amino acid sequences for
several nanobodies. Underlining indicates CDR regions (or nucleic
acids that encode CDR regions).
[0040] FIGS. 9A-9D. (A) Physical layout of the sensor platform on
PCB surface (B) Integration of the alumina membrane (C)
Incorporation of silicone chamber and (D) Changes to the electrical
double layer within each nanowell due to the binding of the target
antigens onto the nanobodies.
[0041] FIG. 10. Impedance spectrum variation over the frequency
range from 50 Hz to 1 MHz. The inset shows a zoomed spectrum for
various doses at relevant frequencies. 100 Hz showed maximum
separation between the spectrums for individual doses, hence, 100
Hz was chosen as the sensing frequency.
[0042] FIG. 11. Calibration run of alpha-synuclein in pure samples.
The plot also establishes the specificity of the biosensor in
identifying the specific interaction between ScFv and
alpha-synuclein.
[0043] FIGS. 12A-12C. 4 (A) Normalized data with D5 antibody (B)
Normalized data with A4 antibody (C) Qualitative representation of
the data.
DETAILED DESCRIPTION OF THE INVENTION
[0044] Biosensors are very powerful tools for the detection of
biological markers or targets in samples obtained from media such
as blood and tissue. The successful coupling of electrochemistry
with naturally existing sophisticated biomolecules has enabled the
design of novel, real time molecular recognition technologies that
are both extremely sensitive and selective. The inventors have
developed a NanoMonitor biosensor that can detect low
concentrations (femtomolar) of multiple target antigens in
biological fluids such as serum and CSF [70-72]. The high
sensitivity the Nanomonitor combined with the high specificity of
morphology specific nanobodies provides a unique opportunity to
determine how serum and CSF levels of individual aggregate species
of A.beta., a-syn and tau correlate with various different
neurodegenerative diseases.
[0045] A biosensor that can accurately distinguish between
different neurodegenerative diseases and facilitate pre-symptomatic
diagnoses would be an extremely valuable clinical tool. It is shown
here that it is possible to construct a simple, robust, biosensor
that can be utilized in a physician's office to sensitively
differentiate between AD, PD, DLB and other neurodegenerative
diseases. Substantial efforts have been expended to identify
biomarkers that can distinguish between these diseases (see for
example: [73-75]). The most promising CSF biomarkers identified are
A.beta., tau and phosphorylated tau levels, however in general
studies employing these biomarkers have met with somewhat limited
success [76]. There are several unique aspects that distinguish
this approach from similar previous studies and strongly increase
our chances of succeeding. One major difference is that the
reagents to be utilized in the biosensor can selectively identify
specific toxic forms of individual proteins that are correlated
with disease progression. The presence of specific oligomeric
A.beta. aggregate species has been correlated with AD, the presence
of tau aggregates has been correlated with AD and other
tauopathies, and the presence of a-syn aggregates has been
correlated with PD, DLB and other synucleinopathies.
[0046] The present invention uses a set of reagents that can
selectively identify different A.beta. aggregate species, and
another set that selectively recognize different a-syn species. The
invention also can be used with reagents that will recognize
different tau species. The inventors have generated and
characterized recombinant antibody fragments or nanobodies against
specific protein morphologies that can be used in the biosensors.
In order to isolate such nanobodies to individual aggregate forms,
a novel AFM biopanning technology was used that enabled the
inventors to visualize the target protein morphology and monitor
the panning process [52]. Utilizing this technology nanobodies were
isolated that recognize different areas of monomeric A.beta. and
a-syn [77-80], fibrillar A.beta. and a-syn [52, 81], two different
oligomeric a-syn species [48, 51], two a-syn/dopamine adduct
species, and three different oligomeric A.beta. species ([82]). The
different oligomer specific nanobodies do not show
cross-reactivity, so the nanobodies binding oligomeric A.beta. do
not bind oligomeric a-syn and vice versa. Each of the different
aggregate species recognized by the different nanobodies naturally
occur in human AD or PD tissue, and that the nanobodies can be used
to distinguish between AD, PD and healthy brain tissue, and block
toxicity of different aggregate species [48, 51, 82]. Therefore the
inventors have a unique panel of well characterized and highly
selective reagents that can be used to correlate the levels of key
aggregated protein forms associated with different
neurodegenerative diseases.
[0047] This panel of reagents is used in combination a label-free
biosensor technology that operates on the principle of
electrochemical impedance spectroscopy that is ideally suited to
identify protein biomarkers in clinical samples. Thus, the panel of
nanobodies is used in combination with the NanoMonitor assay (US
patent application number: 20070256941 incorporated by reference
herein) as a clinical diagnostic tool for simultaneous detection of
low nano- and picogram/ml levels of various target antigens in
patient serum samples [70-72]. The morphology specific reagents in
conjunction with the Nanomonitor can distinguish between PD, AD and
healthy human CSF samples. The biosensor has several features that
make it ideally suited for detecting low concentrations of specific
protein morphologies in human samples. First it uses a label-free
technology so only a single binding event and no modification of
target antigen are needed. Second, the nanoscale array includes a
porous filter to prevent cells and other large material from
blocking the antibody surface and to confine the target antigen in
the porous wells. Third, the Nanomonitor assay can determine
antigen concentrations over large concentrations ranges with
detection limits down to low femtomolar or even attomolar
levels.
[0048] Electrochemical devices are extremely useful for delivering
the diagnostic information in a fast, simple, and low cost fashion,
and are thus uniquely qualified for meeting the demands of
point-of-care screening for a variety of diseases. In particular,
Electronic Impedence
[0049] Spectroscopy (EIS) offers an elegant way for interfacing
biomolecular recognition events with electrochemical signal
transduction for simple, rapid, and label-free detection of
ultralow concentrations of biomarkers for disease. The remarkable
sensitivity of such electrochemical sensing protocols opens up the
possibility of detecting disease markers that cannot be measured by
conventional methods and has potential applications for early
detection of disease [83].
[0050] The Prasad lab has designed and developed a label-free
biosensor technology operating on the principle of electrochemical
impedance spectroscopy suitable for protein biomarker profiling
from clinical samples. This technology known as the NanoMonitor
assay (US patent application number: 20070256941) has been
demonstrated as a clinical diagnostics tool in detecting C-reactive
protein and Myeloperoxidase (protein biomarkers associated with
vulnerable coronary vascular plaque) from patient serum samples
collected through a pilot study conducted in collaboration with
Oregon Health Sciences University and the Portland Va. at
clinically relevant concentrations in the lower picogram/ml range
[70, 71]. The NanoMonitor assay was also tested for detecting
allergens such as Japanese tree pollen (cry j) from serum samples
at clinically relevant concentrations in the nanogram/ml regime
[72]. The notion of antibody confinement in nanoscale arrays for
enhancing the antibody density and enhancing antibody-antigen
interactions has been the genesis for designing the nano-template
based NanoMonitor. In comparison to other electrochemical
diagnostics assays currently under development, the NanoMonitors
have a significant advantage as they provide demonstrate
simultaneous detection of multiple protein biomarkers at clinically
relevant concentrations from complex samples such as purified human
serum with comparable sensitivity and specificity as that observed
for synthetic samples in isotonic buffers [70-72].
[0051] The NanoMonitor is comprised of three parts: the
microelectrode array base platform, a nanoporous alumina membrane
overlayer which forms the nanowells, and the microfluidic
encapsulant to enable the lateral flow of reagents over and into
the nanowells. The microelectrode base platform contains an array
of circular gold measurement/sensing sites where the binding of the
protein molecules occurs in a controlled manner (FIG. 1A). Each
sensing site is comprised of a working electrode (WE) --and a
counter electrode (CE) (FIG. 1B), wherein the surface area ratio of
the CE to WE is 225:1 (FIG. 1B). Additionally the distance of
separation between the circular portions of the WE and CE is very
small (2 .mu.m). The capacitance change due to biomolecule binding
is measured across the WE and CE. Based on fundamental principles
of electrical engineering, the capacitance change is maximized by
an increase in the differential surface area and a decrease in the
distance of separation between WE and CE. The metal electrodes
function as the base for multiple nanowells once the nanoporous
alumina membrane is overlaid. Protein binding and capacitance
changes occur at these electrodes (sensing sites). These electrodes
are connected, to input/output measurement leads. An input low
voltage alternating current (AC) signal is provided to the sensing
site through these leads and the output capacitance signal is
measured across the same leads (FIG. 1C). Both the electrodes are
designed to be circular in shape to attain maximum surface area of
interaction and to avoid any possible edge effects causing
impedance variations. The microelectrode is designed to get 8 such
sensing sites on a 20.times.20 mm.sup.2 silicon chip using the
standard process of photolithography used for fabricating
microelectronics The second part of the nanomonitor is the
nanoporous alumina membrane (FIGS. 1D and 1E). A commercially
available nanoporous alumina membrane with nanopore diameter of 200
nm and membrane thickness of 250 nm (Anodise, Watman, N.J., USA) is
overlaid and adhered using a non-toxic epoxy glue onto the base
microelectrode array [70]. The pore diameter is selected such that
the pore is large enough to function as a scaffold for one or more
antibody-antigen binding complexes. At 200 nm diameter there are
approximately one quarter million nanowells on a single sensing
site [119]. The third part is the microfluidic encapsulant
fabricated out of biocompatible acrylic (FIG. 1F) to regulate the
lateral flow of reagents onto the sensing sites. The encapsulant is
interfaced to the device through a pressure sensitive adhesive. It
consists of 8 microchannels, each encapsulating one sensing site
and consisting of an input and output port. Each microchannel
supports the flow of 8 .mu.l onto the sensing site.
[0052] Thus, in certain embodiments the invention uses nanoporous
membranes in which the aggregate detecting nanobodies are
mobilized. The skilled person will be aware of various materials
that can be used to form nanoporous membranes. The nanopores in the
membrane can all be of a uniform size, arranged in a series of rows
and columns, but other arrangements of pores of the same or
different sizes can be used. Such nano-pores can be arranged in
regular patterns, irregularly, or can be randomly distributed. The
nano-pores can be of similar sizes (length, cross-sectional area),
or different sizes or a selected distribution of sizes can be
provided in a single substrate. Nano-pores typically have
cross-sectional areas similar to the cross-sectional areas of
cylinders having diameters of between about 5 nm and 1000 nm.
Nano-pore aspect ratios (length/diameter) typically are in a range
from about 0.5 to 1000. Dimensions, shapes, and aspect ratios can
be selected based on a particular application. Nano-pore
cross-sectional areas can be rectangular, hexagonal, circular,
elliptical, or other shape. Nano-porous membranes can be used for
specimen analysis based on, for example, sample size and structure
(size based filtration) or based on nano-pore sensitization using
antibodies or other sensitizing agents.
[0053] Nano-pores in one or more regions of the membrane are
electrically connected to a readout amplifier, typically a
differential amplifier that can produce a signal based on a
difference in an electrical characteristic of the nano-pores in the
different regions. The electrical readout can be processed to
obtain, for example, a spectrum (using, for example, a fast Fourier
transform), a power spectral density, or to identify a particular
spectral component associated with an intended response. The
electrical readout can be configured to permit measurement of a
time evolution of response so that, for example, spectrum as a
function of exposure time is determined.
[0054] Substrates are generally selected for ease of nanopore
formation. Aluminum is convenient as it can be electrochemically
processed to produce alumina nano-pores of hexagonal
cross-sectional area, and having different aspect ratios. Aspect
ratios (length/diameter) of at least about 1000:1 can be achieved.
Aperture dimensions can be configured based on electrochemical bath
temperature and composition, applied voltage, current density,
and/or exposure duration. Different aperture dimensions can be
provided on a single substrate by selectively processing different
substrate regions. Different size pores can be particularly useful
in sized-based protein trapping in which the response of different
pore sizes can be associated with protein size or other analyte
property. For electrical measurements, the substrate is preferably
substantially non-conductive, although configurations in which the
nano-pores are electrically isolated from the substrate can be used
as well. Substrates such as silicon, silicon oxides and nitrides
can also be used, and apertures can be formed by wet or dry
etching, ion beam milling, or other process. Surface portions of
the substrate can be coated with a conductive material such as
platinum, gold, silver, copper, or other material by sputtering,
evaporation, or other processes so as to electrically couple a
pluralities of nanopores forming sets of nanopores. In typical
examples, antibodies or other sensitizing agents are immobilized on
surfaces of the nanopores, typically nanopore sidewalls.
[0055] In representative examples, sensors comprise a substrate
having defined therein at least one nanoporous membrane portion
that includes a plurality of nanopores. The nanopores are
electrically coupled with a sensing site. In preferred embodiments,
the sensing site is comprised of a working electrode and a counter
electrode wherein the surface area of the CE: WE is between about
20:1 to about 300:1. It has been found by the inventors that 20:1
and 50:1 ratios have a much better sensitivity than 10:1 ratio. In
specific embodiments, there are a plurality of nanopores and each
nanopore is electrically coupled to sensing site. Alternatively, a
plurality of nanopores can be electrically coupled to one sensing
site. In addition, it is contemplated that there are different
sensitizing agents (by sensitizing agents, the present invention
means morphology specific nanobodies that are use to determine the
presence of aggregate species of A.beta., a-syn and tau) coupled to
different nanopore sites such that there is a first sensor either
coupled to one nanopore or a plurality of nanopores each having
immobilized thereon the same sensitizing agent and a further
second, third, fourth etc, sensor that is either coupled to one
nanopore or a plurality of nanopores each having immobilized
thereon the same second, third, fourth etc. sensitizing agent.
[0056] In preferred embodiments, the sensing site in the present
invention is comprises of circular gold sensing sites. However, it
should be understood that the gold is simply a conductive material
for the sensing site and may be replaced with other materials such
as platinum, silver, copper, and the like.
[0057] In specific embodiments, a spectrum analyzer is in
communication with the sensing sites and is configured to produce
an estimate of a received signal portion associated with a
signature frequency or frequencies. In additional examples, the
spectrum analyzer is configured to produce an estimate of a
received signal portion associated with at least two frequencies
associated with a first signature and a second signature.
[0058] In operation, a test specimen of a fluid from a subject to
be diagnosed is contacted with a biosensor of the invention, and
evaluating an electrical signal associated with administration of
the test specimen to the nanopores. The test specimen can be
assessed based on the evaluation. In some examples, the electrical
signal is evaluated to identify a magnitude of at least one
electrical spectral peak associated with exposure of the sensitized
nanopores to the target compound, and the test specimen is assessed
based on the magnitude. In further examples, the electrical signal
is evaluated to identify an electrical signature associated with
the target compound, and the test specimen is assessed based on the
signature.
[0059] Thus, as noted above, the biosensor of the invention has
three components: a base substrate containing an array of
electrically conductive materials that are linked to nanopores in a
nanoporous membrane and a third component which is a fluid chamber
that allows channels to form to produce a lateral flow of reagents
over and into the nanowells. The fluid chamber includes an inlet
port and an exit port and is situated so that the nanowells in the
nano-membrane are exposed to reagents provided to the fluid chamber
through the inlet port. Fluid chamber volume can be selected based
on, for example, a convenient specimen volume, and is typically
between about 1 .mu.l and 1000 .mu.l. Conductor strips are provided
on the base substrate, and are electrically coupled to respective
portions of a second surface of the nano-membrane. The
nano-membrane includes a plurality of nanopores that couple the
first and second surfaces.
[0060] In a convenient example, the nano-membrane is an alumina
membrane formed from an aluminum foil, and gold conductor strips
are patterned and formed on the base substrate using contact
photolithography. Other membrane materials can be used, and
conductors of silver, gold, copper, or other conductor OP
semi-conductor materials can be used. The fluid chamber is formed
of polydimethoxysilane (PDMS), but other materials can be used.
Alternatively, the chamber can be omitted and test materials
dispensed directly onto the first surface of the nano-membrane.
[0061] The nanoporous membrane will have therein a plurality of
nanowells having effective diameters of about 10 nm to 500 nm. The
pores can have circular, elliptical, hexagonal, cross-sections, or
cross-sections of other shapes. In certain applications, pore
diameter is substantially uniform or variable within a
predetermined range. The nano-membrane is preferably an electrical
insulator so that the pores are not electrically coupled to each
other absent addition electrical connections such as the conductor
strips.
[0062] The base substrate is generally an insulator, or includes an
insulator portion. For example, silicon with an oxide layer can
serve as the base substrate, wherein the conductor strips are
defined on or in the oxide layer so as to be substantially
electrically isolated. Such a base substrate can be especially
convenient for inclusion of detection electronics in the base
substrate. However, other substrate materials such as glass, fused
silica, polycarbonate, polyamides, ceramics, epoxy, plastics, or
the like can be used.
[0063] For example, the base substrate is formed using a 2 cm by 2
cm section of silicon wafer cleaved from a larger wafer. This
substrate is cleaned in piranha solution, spin coated with a
positive photoresist, and a quartz photomask is used to define
features 1 .mu.m by 2 cm. A 10 nm thick gold film is sputter coated
onto the photoresist, and gold conductor strips 2 .mu.m by 2 cm can
be formed using a lift off process. FIG. 7 illustrates conductive
strips 403-406 with gaps 402 formed on a surface of a base
substrate 400.
[0064] Alumina membrane fabrication is known to those of skill in
the art. High purity aluminum foil substrates (99.99% pure) are
selected and sized, degreased in acetone, and cleaned in an aqueous
solution of HF, HNO.sub.3, and HCl in a volume ratio of about
1:1:2.5. After cleaning, the substrates are annealed in a nitrogen
ambient at 400.degree. C. for about 45-60 min. to remove mechanical
stresses and allow re-crystallization. Grain sizes can be measured
using electron microscopy, and grain sizes in the annealed
substrates are typically between about 100 nm and 200 nm. Surfaces
of the annealed substrates are electro-polished in a mixture of
HClO.sub.4 (perchloric acid) and C.sub.2H.sub.5OH (ethanol). The
substrates can be anodized at a constant cell potential in aqueous
H.sub.2SO.sub.4 (sulfuric acid) at concentrations of between about
1.8 M and 7.2 M. Sulfuric acid/oxalic acid mixtures can also be
used. Typical mixtures are combinations of 0.3 M oxalic acid with
0.18 M to 0.5 M sulfuric acid. Current densities typically range
from about 50-100 mA/cm.sup.2.
[0065] Multi-step anodizations can also be used. In a typical two
step anodization, a first step is used to form a concave texture,
and a second step is used to form nanostructures, typically at
locations at which texture changes were formed in the first step.
In a typical first anodization, the aluminum substrates are mounted
on a copper plate anode, and a graphite plate is used a cathode.
During anodization, the electrolyte is vigorously stirred and/or
recycled, and cell voltage, current, and temperature are monitored
and recorded. In this first anodization, cell potential is fixed at
about 40 V and the substrates are exposed to 0.3 M oxalic acid
(H.sub.2C.sub.2O.sub.4) electrolyte solution for about 3 hrs at
about 25.degree. C. In a second anodization, partially anodized
substrates are exposed to a mixture of 6% by weight of phosphoric
acid and 1.8% by weight chromic acid for about 10 hrs at a
temperature of about 60.degree. C. After this second anodization,
the first anodization is repeated for about 5 hrs. Pores are
generally about 20 nm wide and about 25 nm deep. Any remaining
aluminum in the substrates can be removed with a saturated mercuric
chloride solution.
[0066] For anodization, an aluminum substrate is secured to a
copper plate that serves as an anode. A graphite plate is used as a
cathode, and the aluminum substrate/copper plate and graphite plate
are exposed to an electrolyte solution at a selected applied
voltage. Electrolyte solution temperature, composition, and
concentration, and applied voltage are selected to provide an
intended pore size, aspect ratio, and/or pore density.
[0067] In typical examples, nanopores having diameters of about 25,
50, and 100 nm are produced using cell voltages of about 12 V, 25
V, and 40 V, respectively, at a cell temperature of about
60.degree. C. Current density varies from about 1.2 A/cm.sup.2 to 5
A/cm.sup.2. Pore densities can be varied from about
610.sup.8/cm.sup.2 to about 510.sup.10/cm.sup.2, and are typically
directly proportional to current density and inversely proportional
to cell temperature.
[0068] In the second anodization step, varying the electrolyte
temperature from 25.degree. C. to 50.degree. C. in increments of
1.degree. C. for every 10 minutes permits selection of pore widths
in a range of about 12 nm to 200 nm. Varying the applied voltage
from 40 V to 70 V at 5 V increments every 10 minutes permits
selection of pore surface density in a range of about 10.sup.5
pores/mm.sup.2 to 10.sup.15 pores/mm.sup.2, and pore depth can be
altered from about 10 nm to 250 nm by increasing the voltage. By
varying the concentrations of oxalic, phosphoric and chromic acids
from about (1:0.5:0.5) by volume to about (2:3:3) by volume, pore
width can be varied from about 12 nm to 750 nm.
[0069] Pores typically nucleate at surfaces of the substrates at
approximately random locations, and pores have random locations and
a broad distribution of sizes. Under certain specific conditions, a
hexagonal ordering of pores is produced. These pores are well
suited for trapping of nanometer sized particles. Pore sizes for a
particular application can be selected based on a protein size so
that the target protein "fits" the pores. Such a fit can reduce
non-specific binding events, increasing measurement sensitivity and
reliability.
[0070] Sensors can be interrogated by coupling one or more
conductor strips. A sensor includes a plurality of conductors that
are coupled to a multiplexer that selects one or more of the
conductors for coupling to a buffer amplifier. The multiplexer can
be controlled for such selection based on a user selection or under
control of a desktop, laptop, or palmtop computer. Alternatively,
each conductor can be coupled to a respective buffer amplifier, and
signals on all conductors made simultaneously available for signal
analysis. In other examples, a mechanical switch or probe can be
used to selectively couple to one or more conductors.
[0071] The conductors can be associated with different
sensitizations (for example, contacted to nano-pores on which
different types of antibodies are immobilized.). Electrical signals
from the conductors are based on, for example, effective
conductance variations associated with binding of antigen-antibody
complexes. These electrical signals exhibit complex time domain
behavior, but generally have characteristic features or
"signatures" when viewed in the frequency domain. Typically, a
specific bound complex is associated with one or more
characteristic frequencies, and signal magnitude at the
characteristic frequency (or frequencies) is a function of analyte
concentration.
[0072] Characteristic frequencies can be detected with a spectrum
analyzer that is coupled to the selected conductor (or conductors)
and that receives an electrical signal associated with the
sensitized conductors/nano-pores. The spectrum analyzer can be
implemented using a mixer and a swept oscillator with a detector
that is coupled to evaluate a magnitude and/or phase of a
difference or sum frequency from the mixer. Alternatively, a time
record of the coupled electrical signal can be stored, and a
spectrum obtained using, for example, a fast Fourier transform. In
some examples, a power spectrum is obtained in order to identify
presence of a targeted material, or a response to a compound under
investigation. A differential electrical signal is generally used
such that a difference signal associated with a reference conductor
and a conductor coupled to sensitized nano-pores is evaluated.
Signals are generally available within seconds after exposure of a
sensitized membrane to an analyte, and thus permit rapid analyte
assessment. A signature analysis processor is generally coupled to
receive the detected spectra and, based on signatures stored in a
signature database, determine presence and/or concentration of one
or more analytes.
[0073] Key to the biosensors of the present invention is that they
employ a antibodies that detect neuropathies such as A.beta., a-syn
and tau morphologies. Binding of the antigen aggregates to these
antibodies results in a detectable variation in signal. Detected
voltage variations are based on binding of the antibody-antigen
protein complex to a base substrate.
[0074] A.beta., tau and a-syn are proving to be critical components
in the etiology of several devastating neurodegenerative diseases
including AD, PD and DLB. Extensive studies show that accumulation
and aggregation of these different proteins are connected with
various AD, PD, DLB, and other synucleinopathies and tauopathies.
Accumulation of soluble A.beta., for example, has been correlated
with the severity of AD [8] and is thought to lead to diffuse
plaque formation, setting off a cascade of events including
activation of microglial cells, inflammation and misprocessing of
tau, which results in the other dominant pathological feature of
AD, neurofibrillary tangles (reviewed in [86]). Soluble oligomers
of A.beta. were shown to be cytotoxic, disrupt neuronal functions
including LTP and learning, and correlate better with progression
of AD and have been identified in AD brains (reviewed in [87]).
Since cellular stress can lead to an increase in protein
misfolding, it is likely that toxic aggregated species of A.beta.
can not only lead to misprocessing and aggregation of tau, but also
to aggregation of a-syn and other proteins. Similarly aggregation
of a-syn in PD can lead to misprocessing and aggregation of A.beta.
and tau. Therefore, well defined highly specific reagents that can
identify and quantify individual morphologies of A.beta., a-syn and
tau will be a valuable tool for use in biosensors to assist in
early diagnosis of various neurodegenerative diseases.
[0075] Thus the sensitizing agents used in the biosensing devices
of the invention are nanobody reagents specific for a variety of
different morphologies of key proteins involved in
neurodegenerative diseases including A.beta. and a-syn. A single
chain antibody variable domain fragments (scFvs or nanobodies) was
isolated that specifically recognize monomeric [77], fibrillar
[52], and two different oligomeric a-syn morphologies, one against
SDS stable trimers and hexamers [51], and one binding dimers and
tetramers [48]. The anti-oligomeric a-syn nanobodies do not cross
react with oligomeric A.beta., and specifically label PD brain
tissue but not AD or healthy tissue [51]. Dopamine interacts with
a-syn to form stable toxic adducts, and nanobodies that
specifically recognize monomeric and oligomeric a-syn/dopamine
adducts, but not monomeric or oligomeric a-syn alone (Table 1) were
isolated. Therefore, the devices of the invention may use
nanobodies that specifically recognize six conformationally
distinct a-syn aggregate morphologies.
TABLE-US-00001 TABLE 1 ELISA showing specificity of scFvs for
a-syn/dopamine adducts Oligomeric Clone Control Oligomeric a-syn
Dopamine alone a-syn/dop Olad A7 0.075 0.153 0.097 0.066 Olad C12
0.056 0.050 0.040 0.182
[0076] In addition, nanobodies to different regions of monomeric
[79, 80] and fibrillar A.beta. [81] also may be used. The inventors
also isolated a nanobody (A4) that specifically recognizes
oligomeric A.beta., does not cross react with oligomeric a-syn, and
specifically labels A.beta. aggregates in human AD brain samples,
but not PD or healthy brain tissue [82].
[0077] In addition, the biosensor may have attached a nanobody (C6)
that specifically recognizes A.beta. dimeric species derived from
human AD brain tissue [13, 87]. The different specificities of the
three different anti-oligomeric A.beta. nanobodies, A4, E1 and C6,
can be readily visualized by AFM when the nanobody fragments are
expressed on the surface of phage instead of as soluble proteins.
Using this technique, the C6 nanobody can be seen to bind brain
derived oligomeric A.beta., but not in vitro generated A.beta.
oligomeric species, while E1 binds the 1 day preaggregated in vitro
sample containing tetrameric A.beta., but not the brain derived
A.beta. particles or the 3 day preaggregated in vitro A.beta.
sample, and A4 binds the 3 day preaggregated in vitro A.beta.
species, but not the 1 day or brain derived samples (FIG. 4). Each
of these oligomeric forms occurs in human AD brain tissue as C6
binds to A.beta. oligomers recovered from brain tissue and A4 [82]
and E1 (FIG. 3) bind AD but not healthy brain tissue. There are
three different nanobodies (A4, C6, and E1) that recognize three
conformationally distinct small oligomeric A.beta. species, and do
not cross react with oligomeric a-synuclein aggregates. Thus there
is a pool of nanobodies to six different aggregate morphologies of
a-syn and five different morphologies of A.beta. that distinguish
between diseased and healthy brain tissue.
[0078] While A.beta., a-syn and tau are normally present in CSF at
low nanomolar concentrations [21, 22], the in vivo concentrations
of the various oligomeric forms of the proteins are likely
considerably lower. Based on preliminary results showing that it is
possible to obtain femtomolar sensitivity, it is contemplated that
the monomeric binding nanobodies will have high enough affinity to
detect the necessary concentrations in the serum and CSF samples.
However, if higher affinity is required to detect a signal without
background interference, the affinity can be readily improved by
affinity maturation protocols. In order to increase the stability
of the scFvs where necessary, the skilled person can convert the
scFvs into the corresponding Fab.sub.2 form. Conversion is readily
accomplished by splicing out the respective heavy and light chain
variable domain regions and splicing them into vectors containing a
constant heavy and light chain region, respectively. The resulting
Fab.sub.2 fragments can then be produced using a bicistronic
construct in E. coli. The protocols are standard and will be
performed essentially as described [100].
[0079] The skilled person can also increase the affinity of a given
nanobody for its target antigen or increase the specificity for
target A over target B by affinity maturation. To do this, it is
possible to sequentially randomize CDR regions from the antibody
heavy and light chain regions to generate a secondary library of
antibodies where the entire library is now based on antibody
sequences specific for the target protein morphology. The first
generation of this library will be constructed by randomizing the
CDR3 region of the light chain essentially as described and
subsequent generations will target other CDR regions as described
[101]. Affinity maturation using antibody libraries has routinely
improved the affinity of the parent antibody [10]-106]. For
increased specificity panning can be done for nanobodies that are
specific only for target A oligomers by first performing a negative
panning step to remove nanobodies that bind target B and C
oligomers, and then recover nanobodies that are specific for target
A oligomers. The inventors have successfully utilized this two step
panning protocol to isolate nanobodies to a specific protein among
a family of related proteins [107]. Such protein and morphology
specific nanobodies will be extremely valuable for diagnostic
applications to distinguish between different neurodegenerative
diseases.
[0080] In certain embodiments, the biosensor is a detection assay
such as an ELISA, Western blot, dot blot or the like.
Example 1
[0081] For the initial EIS experiments, nanobody fragments are
covalently attached to thiols through Dithiobis[succinimidyl
propionate]) cross linker into the nanowells. Serial dilutions of
purified nanobody are made in an isotonic buffer (15 mM phosphate
buffered saline) and EIS spectra taken. The data is processed
noting optimal frequency and/or equivalent circuit parameters for
the given target to determine reproducibility and linear range of
detection as well as limit of detection (LOD). The Gamry Ref 600
potentiostat is used to acquire the EIS spectra. This potentiostat
system has built in Randles and Warburg models, which are used to
determine the physical equivalent circuit element which is a
measure of the nanobody binding to the sensor surface: double layer
capacitance (C.sub.d1) which is a measure of the binding of the
nanobody fragments on to the sensor surface. Frequency analysis is
also performed to verify optimal frequency for time based studies.
Control samples, nonspecific antibodies or other targets are used
to verify specificity and operation of the array.
[0082] Purified antigen samples are then be spiked in 100%
commercially available human serum to replicate the complex media
of patient samples. Again, a linear range of concentrations of
target is run as before to determine LOD, linear range, and
reproducibility. Then the concentration of commercial human serum
is diluted with buffer from 25%-70% and the study repeated.
Comparisons of impedance at optimal frequency will be analyzed and
plotted. The highest concentration of commercial human serum that
still yields statistically significant results determines how much
(if any) purification of the patient samples is required. The
preliminary data shows that the antigens can be detected accurately
with a linear dose range from 1 pg/ml to 1 .mu.g/ml for 30% volume
of human serum, hence the lowest dilution used is 30%.
[0083] The protocol for detecting the proteins is as follows: The
nanowells on the sensing site are immobilized with nanobody through
the DSP linker chemistry. The nanobody saturated nanowell surfaces
are treated with a blocking agent to ensure that unbound linker
surfaces are blocked. Proteins are then added onto the nanobody
saturated substrate. Binding of proteins is monitored by changes to
the impedance through electrochemical impedance spectroscopy.
Protein dose response is performed by measuring impedance changes
from the NanoMonitor due to changing doses of the protein. All the
biomolecules (linker molecules, antibodies and proteins) have
surface charges. The binding of these molecules to the base of each
nano-well perturbs the charge distribution in the electrical double
layer that forms at the solid/liquid interface. This charge
perturbation produces a capacitance change in the electrical double
layer. The biomolecule binding induced capacitance change is
measured by the electrochemical impedance spectroscopy technique.
In this technique protein binding to the sensing site is achieved
through the immobilization linker specific antibodies to the
nano-wells. A low voltage (50 mV-1V) AC excitation signal at very
low frequency range (10 Hz to 1 kHz) is applied to a sensing site.
Binding of the biomolecules produces a change in the measured
output impedance across a sensing site. The measured impedance is
the sum total of two components: the resistance and capacitance of
the sensing site. It is only the capacitive component that is a
measure of the biomolecule binding event, as the capacitive
component indicates the surface charge differential in the
electrical double layer as a function of biomolecule binding. Hence
the input voltage parameters (frequency and voltage amplitude) need
to be optimized to measure this capacitive component. The
optimization range is focused to voltages below 1V as higher
voltages have demonstrated hydrolysis of biomolecules and
frequencies >1 kHz have been associated with impedances changes
in solution bulk and not to the electrical double layer (EDL).
[0084] The inventors have demonstrated that they can obtain
femtomolar sensitivity for the target antigen using the morphology
specific nanobodies in conjunction with the NanoMonitor assay even
in the presence of complex media. In addition, the inventors have
also shown that they can detect the presence of specific target
protein morphologies in complex media such as CSF.
[0085] The optimal signal frequency and amplitude (impedance), may
vary for each nanobody and target, however it is expected that the
linear signal range and limit of detection will be similar for most
of the systems to be studied. It may be necessary, with the use of
serum samples, to reduce background noise. One method to do this is
to use mixed alkanethiol monolayers [84] that allow for binding of
the capture element but block non-target molecules by use of
nonfunctional thiols groups or other cappers. Additionally due to
the viscosity of whole serum or whole blood clinical samples it is
possible that there will be fouling of the nanoporous alumina
preventing the transport of the antigens of interest into the
nanowells. By reducing the concentration of the whole serum, the
limit of detection can be ascertained in the sense of how pure of a
sample is needed in order to maintain detectibility.
[0086] Other noise reduction strategies including blocking and
washing steps may also be required to reduce nonspecific binding
effects particularly with serum samples. Different blocking agents,
such as, bovine serum albumin (BSA), casein (an isolate from,
bovine milk), polyethylene glycol (PEG), ethanolamine, commercial
combinations such as SuperBlock Blocking Buffer, etc. will be used
at various concentrations (low, e.g. 0.5% or 1 mM to high levels
10% or 100 mM depending upon blocking agent) and for various times
(ranging 1-120 min) An important component to the blocking step is
the washing step. Temperature of the wash, component(s), and time
and volume of rinses will all be investigated. Here, the expected
outcome is that a limit of % of human serum will be reached where
signal becomes either non-linear or altogether lost.
[0087] Protein Aggregation.
[0088] Soluble oligomers of A.beta., a-syn and tau are formed as
transient intermediates during the aggregation of each protein. The
inventors have extensive experience in studying aggregation of
proteins involved in neurodegenerative diseases [48, 51, 77-82,
89-92] and can readily generate a variety of different A.beta.
aggregates. Since the inventors already have nanobodies to several
oligomeric A.beta. forms generated either at neutral pH or isolated
from brain tissue, here the inventors will focus on generating
A.beta. aggregates at acidic pH. Since A.beta. may be generated at
least in part by proteolytic processing in endosomes, some A.beta.
aggregates may originate under these acidic conditions. To do this,
the inventors will aggregate A.beta. essentially as before, but use
a sodium acetate buffer (pH 4.5) instead of PBS buffer (pH 7.4).
The C-terminal truncated species of a-syn are abundant in Lewy body
deposits, aggregate more rapidly than the full length form [93, 94]
and are naturally produced by the proteosome [95]. The truncated
form of a-syn may serve as a seed to promote cytotoxic aggregation
of full length a-syn. Aggregates of truncated C-terminal truncated
a-syn will be produced using protocols essentially as described
[48, 51, 52]. To generate oligomeric and paired helical filamentous
forms of tau, aggregate proteins as described [96].
[0089] Biopanning and Nanobody Characterization.
[0090] Biopanning to isolate nanobodies to the different protein
aggregate forms will be performed essentially as described [48, 51,
52, 82, 91]. Nanobody specificity can be determined by a variety of
different assays depending on availability of the target
antigen.
[0091] a) Antibody Specificity Using ELISA, Western Blot or Dot
Blot.
[0092] The binding specificity can be determined by ELISA, western
or dot blot, depending on how readily we can purify the target
aggregate morphology. The protocols for each of these assays are
routinely used in our lab [48, 77-80, 90, 97].
[0093] b) Antibody Specificity Using AFM.
[0094] Nanobody target specificity can be determined using AFM. The
height distribution of aggregated particles will increase in size
after addition of a nanobody that binds the particles on the mica
surface. By monitoring the change in height distribution one can
determine target specificity using only minimal amounts of target
material [91]. In addition, the target material does not need to be
purified since we can monitor changes in selected target heights.
The inventors can qualitatively determine specificity by visually
identifying which morphologies the nanobody binds to,
quantitatively determine the specificity by plotting the change in
height distribution data as a function of nanobody concentration as
a Langmuir isotherm to calculate K.sub.D values. Alternatively,
nanobody specificity for various morphologies can be determined
using AFM recognition imaging as described [98].
[0095] Using affinity maturation protocols with a phage display
system, the inventors isolated antibodies with over 35-fold better
affinity than the original clone using a novel single step
selection protocol [108]. The inventors also utilized yeast display
libraries to improve the affinity of an scFv specific for A.beta.42
over 30-fold from 2.3.times.10.sup.-7M to 7.8.times.10.sup.-9M.
[0096] Methods.
[0097] Affinity maturation using antibody libraries has routinely
improved the affinity of the parent antibody [10]-106], even
evolving to obtain femtomolar affinity [109]. The protocols for
generating these second generation antibody libraries are described
in the various references [10]-106]. Basically a second generation
library will be constructed essentially as described [110].
Additional library generation can be constructed by varying
different CDR regions. While the primers will vary to flank the
target CDR sequence, the protocols are essentially the same.
[0098] Based on the results the inventors have obtained with the
nanobodies already isolated, the inventors do not anticipate that
there will be a need to increase the affinity or stability of the
majority of the nanobodies generated. For those clones that do not
have sufficient sensitivity in the biosensor assay, the inventors
expect that conversion to Fab.sub.2 format will provide sufficient
increase in stability and also in affinity due to the bivalent
structure. If the Fab.sub.2 format does not have the desired
specificity, the individual scFv will be subjected to affinity
maturation to increase specificity. If after affinity maturation
and conversion to Fab.sub.2 format, the antibody fragment does not
bind sufficiently well, the antibody will be dropped from further
studies.
[0099] Testing the Biosensor ability to detect A.beta., a-syn and
tau forms in frozen post-mortem samples of CSF taken from healthy,
early and late AD, PD and DLB patients: Once the biosensor
protocols are developed for sensitive detection of target antigen,
and have identified a panel of nanobodies to selected A.beta.,
a-syn and tau morphologies, for use in the initial test of the
biosensor using post-mortem CSF samples from AD, PD, DLB and
non-dementia individuals. The samples will be obtained from the
Banner/Sun Health Research Institute (BSHRI) Tissue Bank. The
inventors will test whether the capturing nanobodies and the EIS
arrays have sufficient sensitivity to detect the concentrations of
the target species at different stages of these neurodegenerative
diseases. For those ligands that cannot be detected, the affinity
of the nanobody for the target or the sensitivity of the sensor
will be increased as needed. If the target cannot be detected even
at femtomolar levels, then we will assume that it is not a suitable
biomarker for the selected neurodegenerative diseases. The goal of
this aim is to identify which forms of A.beta., a-syn and tau can
serve as biomarkers to distinguishing different stages of AD, PD,
DLB and other related neurodegenerative diseases.
[0100] To test whether the biosensor and nanobody reagents can
detect specific protein aggregate species in human CSF samples, the
inventors immobilized D5 nanobody, which recognizes an oligomeric
a-syn species, to biosensor chips. They then tested five different
CSF samples obtained from the BDP (two non-dementia (ND), two PD,
and one AD sample) for the presence of this a-syn aggregate
species. The two PD CSF samples had the highest concentration of
the a-syn aggregated species, the two ND samples had the lowest
concentration, and the AD sample was in the middle (FIG. 5). These
very promising preliminary results indicate our morphology specific
nanobodies can detect target protein species in human CSF samples
using the nanomonitor system. They also indicate that levels of
specific aggregate protein species have great potential as
biomarkers to distinguish between different neurodegenerative
diseases.
[0101] Methods:
[0102] The BDP currently has CSF available from 147 AD cases with
no additional major neuropathologic diagnosis, 46 cases of non-AD
dementia and no concurrent diagnosis of AD, 28 non-demented
individuals with moderate to severe AD histopathology (Braak III or
IV and moderate or frequent CERAD neuritic plaque density) and 29
non-demented individuals with minimal (Braak I or II, zero or
sparse CERAD neuritic plaque density) AD histopathology. The non-AD
dementia cases are free of significant AD histopathology (CERAD
neuritic plaque density zero or sparse; Braak stage I or II) and
consist of 18 cases of Parkinson's disease with dementia, 6 cases
of vascular dementia, 6 cases of dementia with Lewy bodies, 6 cases
of progressive supranuclear palsy, 5 cases of hippocampal sclerosis
dementia and 5 cases of dementia lacking distinctive
histopathology. In addition, the BDP has brain tissue and CSF from
131 patients with PD, 131 with AD with Lewy Bodies, and 82 with DLB
and over 200 elderly controls.
[0103] C6 Nanobody Recognizes A.beta. Aggregates in Brain
Tissue.
[0104] To determine whether C6 nanobody could also recognize small
oligomeric A.beta. aggregates in brain tissue, the inventors tested
different age mouse brain tissue from wild type and triple
transgenic (3.times.Tg) mice developed by LaFerla et al. (34).
Brain extracts from A.beta. over-expressing transgenic mice
(Tg2576) and control mice were homogenized and run on a 10%
Tris-Tricine gel, and transferred onto a nitro-cellulose membrane
for western blot analysis. The membrane was probed with C6 nanobody
using a 9E10-biotin primary and streptavidin-HRP as secondary
antibody and stained with DAB (Sigma). Staining intensity of bands
corresponding to 40 kDa was quantified using ImageJ software and
compared to the background. Samples with standard deviation a)
<2 times background are denoted as -; b) 2-3 times background as
+; and c) 3-4 times background as ++ and d) >4 times background
as +++. Thus, when the tissues were probed with C6 nanobody, strong
reactivity was observed with 22 and 27 weeks old 3.times.Tg
samples, while little or no binding was observed with similar aged
mice or 68.4 week 3.times.Tg mice (Table 2).
TABLE-US-00002 TABLE 2 C6 reactivity with mouse brain samples Mouse
Type Age Reactivity Wild Type .sup. 22 wks - Wild Type 28.7 wks -
Transgenic .sup. 10 wks ++ Transgenic .sup. 22 wks +++ Transgenic
27.3 wks +++ Transgenic 68.4 wks +
[0105] Next, the C6 antibody fragment was reacted with blotted
aliquots of soluble homogenized samples obtained from healthy (ND)
or AD human brain tissue. Brain extracts from the medial temporal
gyms of Non Diseased patients (ND) and Alzheimer's disease patients
(AD) were homogenized and deposited onto a nitrocellulose membrane
and probed with C6 nanobody. Staining intensity of the dot blot was
quantified using ImageJ software and compared to the background.
Samples with standard deviation a) <2 times background are
denoted as -; b) 2-3 times background as +; and c) 3-4 times
background as ++ and d) >4 times background as +++. Thus, C6
reacted strongly with brain tissue from AD patients who had
moderate plaque frequency, but showed little or no reaction with
brain tissue from AD patients with severe plaques or with the ND
patients (Table 3).
TABLE-US-00003 TABLE 3 C6 reactivity with human brain samples
Sample Sample Description Reactivity ND1 No plaque - ND2 No plaque
- ND3 No plaque + ND4 Moderate Frequency Plaque - ND5 Moderate
Frequency Plaque - ND6 Moderate Frequency Plaque + AD1 Moderate
Frequency Plaque ++ AD2 Moderate Frequency Plaque +++ AD3 Moderate
Frequency Plaque ++ AD4 Severe Plaques + AD5 Severe Plaques - AD6
Severe Plaques -
[0106] Testing Biosensor Ability to Detect A.beta., a-Syn and Tau
Forms in Frozen Post-Mortem Samples of CSF Taken from Healthy,
Early and Late AD, PD and DLB Patients.
[0107] Preliminary results were obtained with five post-mortem CSF
samples using our nanomonitor sensor with immobilized D5 nanobody
which recognizes an oligomeric a-syn species [2]. The inventors
have now completed testing on nine different CSF samples obtained
from the BSHRI BDP (three non-demented (ND), three PD, and three AD
samples) for reactivity with D5 and also have data with these same
nine samples using the A4.nanobody which recognizes an oligomeric
form of A.beta. [4]. The biosensor is sufficiently sensitive that
the CSF samples can be diluted 10 fold and the specific oligomeric
species are readily detected. Using the D5 anti a-syn nanobody, the
three ND CSF samples (ND1-3) all showed consistently low, non-zero
readings, the three AD samples (AD1-3) had substantially higher
values, and two of the three PD samples (PD1, PD2) had the highest
values, while the third PD sample (PD3) had a reading similar to
the ND samples (FIG. 6A). The pathology associated with the third
PD sample (PD3) indicates that the patient had Parkinsonism caused
by Multiple System Atrophy (MSA) and was not a true PD case. The
presence of aggregated a-syn in AD brain tissue has been documented
[5], and here it is shown that aggregated a-syn is also detected in
CSF from human AD patients, but at slightly lower levels than in PD
cases. Using the A4 anti-AP nanobody, the three ND samples (ND1-3)
and three PD samples (PD1-3) did not show any binding to A4, while
the three AD samples (AD1-3) all showed significant presence of
this oligomeric A.beta. species (FIG. 6B). Therefore using only a
positive or negative binding result based on a threshold value of
ND samples, one can readily distinguish between ND, AD and PD
samples using only two of the morphology specific reagents (Table
4). It is expected that evaluating concentrations of the different
oligomeric species will provide additional information about
disease progression and can be very valuable as a tool to assess
effectiveness of different therapeutic strategies.
TABLE-US-00004 TABLE 4 Summary of CSF analysis using two morphology
specific nanobodies Anti- body ND1 ND2 ND3 PD1 PD2 PD2 AD1 AD2 AD3
D5 - - - + + - + + + A4 - - - - - - + + +
[0108] The additional data presented herein provide further
evidence that different oligomeric protein forms, including A.beta.
species, exist in human tissue and in cell and animal models of AD,
and that our reagents can specifically recognize these species,
that they can be used to differentiate different stages of AD and
to distinguish between disease samples. It is also shown that one
can accurately and sensitively detect target protein species
directly from human CSF samples using the electronic biosensor
system. Using just two nanobodies from our proposed panel of
morphology specific reagents, we are able to very clearly
differentiate ND, PD and AD CSF samples. Our reagents not only
distinguish between different neurodegenerative diseases but
potentially between different causes of these diseases. These
results provide very encouraging evidence that levels of specific
aggregate protein species have great potential as biomarkers to
distinguish between different neurodegenerative diseases, to
characterize progression of these diseases, and to monitor the
effectiveness of different therapeutic strategies.
[0109] The following material provides additional data showing
morphology specific nanobodies specifically recognize different
naturally occurring A.beta. aggregates that are diagnostic of
different stages of Alzheimer's disease and data showing that the
nanomonitor biosensor in conjunction with the nanobodies can
readily and sensitively d3etect specific morphologies of target
antigens in CSF samples. The results indicate that detection of
specific toxic protein species in CSF has great potential as a
diagnostic for neurodegenerative diseases.
[0110] Table 5 summarizes additional data for tests using CSF
samples. In the table, (+) indicates reactivity of the sample with
the target antibody, (-) indicates no reactivity, (+?) indicates
that the testing is not completed yet, but the ones evaluated so
far are positive, (-/+) means some are positive and some are
negative, and (?) means that the experiments are not yet completed.
The data is very encouraging since with only four of the
nanobodies, the inventors are able distinguish between non-demented
(ND), Parkinson's (PD), Alzheimer's (AD) and Dementia with Lewy
Bodies (DLB).
TABLE-US-00005 TABLE 5 Summary of Results Showing Reactivity
Profiles of CSF with Different Nanobodies Disease D10 A4 D5 10H E6
ND + - - - ? PD + - + -/+ ? AD + + + + ? DLB + + +? - ?
[0111] In certain embodiments, the antibody fragments that can be
used in the present invention are those listed in Table 6 below
(See also FIG. 8):
TABLE-US-00006 TABLE 6 Antibody Library Assays to Applications
Fragment source Specificity validate demonstrated SEQ ID NO A4
Tomlinson Oligomeric Dot blot, Human AD brain amino acid (MRC)
Abeta 3-day time course, tissue, Human SEQ ID NO: 2; aggregates
ELISA, AFM CSF, Mouse AD nucleic acid (Note: 3-day brain tissue,
(Dot SEQ ID NO: 8 oligomeric blot assays). target is not
Immunohisto- stable for chemistry westerns) E1 Tomlinson Oligomeric
Dot blot, Human AD brain amino acid (MRC) Abeta 1-day time course,
tissue, Human SEQ ID NO: 3; aggregates ELISA, AFM CSF, Mouse AD
nucleic acid (Note: 1-day brain tissue, (Dot SEQ ID NO: 9
oligomeric blot assays) target is not stable for westerns) C6
Sheets Oligomeric AFM Human AD brain amino acid (UCSF) Abeta: brain
tissue, Human SEQ ID NO: 1; derived CSF, Mouse AD nucleic acid
brain tissue, (Dot SEQ ID NO: 10 blot assays) and amino acid SEQ ID
NO: 15 D5 Tomlinson Oligomeric Dot blot, Human PD brain amino acid
(MRC) a-synuclein time course, tissue, Human SEQ ID NO: 4; 3-day
ELISA, AFM, CSF, Mouse PD nucleic acid aggregates western blot
brain tissue, (Dot SEQ ID NO: 11 analysis blot assays, western
blot, Immunohisto- chemistry with tissue and cells 10H Tomlinson
Oligomeric Dot blot, Human PD brain amino acid (MRC) a-synuclein
time course, tissue, Mouse PD SEQ ID NO: 5; 7-day ELISA, AFM, brain
tissue, (Dot nucleic acid aggregates western blot blot assays, SEQ
ID NO: 12 analysis western blot, Immunohisto- chemistry with tissue
and cells 6E Tomlinson Fibrillar Dot blot, Human PD brain amino
acid (MRC) aggregates time course, tissue, Human SEQ ID NO: 6;
(likely not ELISA, AFM CSF, Mouse PD nucleic acid protein brain
tissue, (Dot SEQ ID NO: 13 specific) blot assasys, western blot,
Immunohisto- chemistry with tissue and cells D10 Tomlinson All
forms of Dot blot, Human PD brain amino acid (MRC) a-synuclein time
course, tissue, Human SEQ ID NO: 7; ELISA, AFM, CSF, Mouse PD
nucleic acid western brain tissue, (Dot SEQ ID NO: 14 blot assasys,
western blot, Immunohisto- chemistry with tissue and cells BSEC1
PNRL (Pacific BACE1 BACE-1 Cell toxicity, National cleavage
catalytic cell culture Research site on APP assay assays Labs?)
(Does not Yeast bind soluble library Abeta
[0112] In certain embodiments, the C6 nanobody has a sequence of
SEQ ID NO:1:
TABLE-US-00007 EXPIAYGSRWIVITRGPAGHGPGTAAGVGGGLVQPGGSLRLSCAASGFTF
SSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTL
YLQMNSLRAEDTAVYYCAKSYGSVKISCFDYWGQSTLVTVSSGGGGSGGG
GSGGGGSEIVLTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYLAWYQ
QKPGQSPELLIYWASTRESGVPDRFSGSGSGTEFTLTISSLQAEDVAVYY
CQQFYSTPPTFGQGTKLEIKRAAAHHHHHHGAAEQKLISEED
[0113] In certain embodiments, the C6 nanobody lacks the initial
1-15 amino acids of SEQ ID NO:1.
Example 2
[0114] Protein misfolding and aggregation is a critically important
feature in many devastating neurodegenerative diseases, therefore
characterization of the CSF concentration profiles of selected key
forms and morphologies of proteins involved in these diseases,
including beta-amyloid (A.beta.) and a-synuclein (a-syn), can be an
effective diagnostic assay for these diseases. CSF levels of tau
and A.beta. have been shown to have great promise as biomarkers for
Alzheimer's disease. However since the onset and progression of
many neurodegenerative diseases have been strongly correlated with
the presence of soluble oligomeric aggregates of proteins including
various A.beta. and a-syn aggregate species, specific detection and
quantification of levels of each of these different toxic protein
species in CSF may provide a simple and accurate means to
presymptomatically diagnose and distinguish between these diseases.
Here we show that the presence of different protein morphologies in
human CSF samples can be readily detected using highly selective
morphology specific reagents in conjunction with a sensitive
electronic biosensor. We further show that these morphology
specific reagents can readily distinguish between post-mortem CSF
samples from AD, PD and cognitively normal sources. These studies
suggest that detection of specific oligomeric aggregate species
holds great promise as sensitive biomarkers for neurodegenerative
disease.
[0115] Neurodegenerative diseases such as Alzheimer's disease (AD)
and Parkinson's disease (PD) will affect an increasing number of
people as our population ages. For AD alone, over 5 million
Americans currently are living with the disease, with nearly half a
million new cases expected each year with total yearly economic
costs of over $170 billion. Diagnosis of these diseases is
challenging as other neurodegenerative diseases such as Lewy are
often classified as having Mild-Cognitive Impairment (MCI), a term
describes early, nondisabling cognitive disorders.
[0116] Although MCI describes a transitional state between normal
Body Dementia (LBD), frontotemporal dementia and vascular dementia
may share similar symptoms, but have different mechanisms and
pathology. Patients in early stages of dementia aging and dementia,
not all MCI cases progress to dementia. Pathological changes
associated with these different dementias have been shown to occur
long before symptoms are evident, suggesting that an appropriate
set of biomarkers would have great promise to study toxic
mechanisms and pathways in these different diseases and to
facilitate early diagnoses.
[0117] Numerous studies have looked at key biomarkers for
diagnosing neurodegenerative diseases, especially AD where levels
of tau, phosphorylated tau and amyloid-beta 42 (A.beta.42) have
shown promise for predicting AD. A recent study using these
biomarkers correctly identified 94% of autopsy verified AD cases
and also accurately predicted (100% sensitivity) which MCI cases
would progress to AD. These very promising studies indicate that
CSF biomarkers can be a valuable tool to both facilitate diagnosis
of neurodegenerative disease and to assess effectiveness of
different therapeutic strategies.
[0118] While CSF protein biomarkers such as tau and A.beta. hold
promise as diagnostics for neurodegenerative diseases, a better
more selective diagnostic biomarker set can potentially be obtained
by detecting specific toxic protein species that are associated
with each disease. Since many neurodegenerative diseases are
correlated with misfolding and aggregation of different target
proteins; amyloid-beta (A.beta.) with AD, alpha-synuclein (a-syn)
with PD, LBD and other synucleinopathies, and tau with AD and
various tauopathies, specific detection and quantification of
levels of each of these different toxic protein species may provide
a means study mechanisms of toxicity and progression in these
diseases and to presymptomatically diagnose and distinguish between
these diseases. For example, a vast amount of literature implicates
A.beta. accumulation and plaque formation as being central to the
progression of AD, however, while the presence of amyloid plaques
does not correlate well with the progression of AD, the presence of
various different soluble A.beta. species does. Similarly, soluble
aggregate forms of tau have been correlated with AD and soluble
forms of a-syn have also been correlated with PD.
[0119] Since protein misfolding and aggregation are closely
associated with many neurodegenerative diseases, determining the
CSF concentration profiles of selected key forms and morphologies
of proteins involved in these diseases, including A.beta. and
a-syn, can facilitate development of an effective diagnostic assay
to help study these diseases. Since specific soluble aggregate
morphologies of these proteins are likely present at only very low
concentrations in CSF, in order to detect and quantify levels of
these proteins, highly selective reagents that specifically
recognize each of the target species are needed along with a
sensitive biosensor system. We have used novel protocols to isolate
reagents that bind specific morphologies of target proteins by
combining the imaging capabilities of AFM with the binding
diversity of phage display antibody technology (Barkhordarian et
al., Isolating recombinant antibodies against specific protein
morphologies using atomic force microscopy and phage display
technologies. Protein Eng Des Sel 2006, 19, (11), 497-502; Zameeret
al., Anti-oligomeric Abeta single-chain variable domain antibody
blocks Abeta-induced toxicity against human neuroblastoma cells. J
Mol Biol 2008, 384, (4), 917-28; et al., Isolation of a human
single chain antibody fragment against oligomeric alpha-synuclein
that inhibits aggregation and prevents alpha-synuclein-induced
toxicity. J Mol Biol 2007, 368, (4), 1132-44; Emadi et al.,
Detecting morphologically distinct oligomeric forms of
alpha-synuclein. J Biol Chem 2009, 284, (17), 11048-58.). These
morphology specific reagents are ideal candidates to determine
whether specific aggregate species of A.beta. and a-syn can be
detected in CSF and whether they have potential as markers to help
study and diagnose neurodegenerative diseases. We have also
designed and developed a sensitive label-free biosensor technology
operating on the principle of electrochemical impedance
spectroscopy that is well suited to identify protein biomarkers in
clinical samples (Botharawt al., Nanomonitors: electrical
immunoassays for protein biomarker profiling. Nanomed 2008, 3, (4),
423-36; Reddy et al., Nanomonitors: Protein biosensors for rapid
analyte analysis. Ieee Sensors Journal 2008, 8, (5-6), 720-723;
Venkatraman et al., Iridium oxide nanomonitors: Clinical diagnostic
devices for health monitoring systems. Biosensors &
Bioelectronics 2009, 24, (10), 3078-3083). The biosensor has
several features that are ideally suited for detecting low
concentrations of specific protein morphologies in human samples.
First it uses a label-free technology so only a single binding
event and no modification of target antigen are needed. Second, the
nanoscale array includes a porous filter to prevent cells and other
large material from blocking the antibody surface and to confine
the target antigen in the porous wells. Third, the sensor can
determine antigen concentrations over large ranges with detection
limits down to low femtomolar or even attomolar levels. We used the
NanoMonitor assay (US patent application number: 20070256941) as a
clinical diagnostic tool for simultaneous detection of low nano-
and picogram/ml levels of various target antigens in patient serum
samples. The high sensitivity of the Nanomonitor combined with the
high selectivity of the morphology specific nanobodies provides a
uniquely powerful tool to determine whether CSF levels of various
different aggregate morphologies of A.beta. and a-syn have
potential as biomarkers to study and diagnose neurodegenerative
diseases. Here we show that morphology specific nanobodies in
conjunction with an electronic impedence biosensor can readily
distinguish post-mortem CSF samples taken from human AD, PD and age
matched non-diseased sources.
[0120] Materials and Methods
[0121] Fabrication of the Biosensor Device
[0122] The biosensor device is comprised of three integrated parts;
(a) a printed circuit board platform, (b) a nanoporous alumina
membrane and (c) a silicone micro fluidic chamber. The measurement
surface for the biosensor device is a printed circuit board
platform comprised of inter-digitated working and counter
electrodes. The tin oxide electrodes are 800 .mu.m in width, 5 mm
in length and 800 nm in thickness with rounded edges to minimize
fringe effects during the application of a sinusoidal voltage input
signal (FIG. 9A).
[0123] A nanoporous alumina membrane is soldered onto the
interdigitated electrodes generating a high density array of
nanowells. The membrane is 250 nm thick, has a lateral diameter of
13 mm with pore diameters of 200 nm. The porosity of the membranes
varies between 25% and 50%. An alumina membrane was utilized since
it offers electrical isolation between each individual nanowell, as
well as good biocompatibility (FIG. 9B).
[0124] Finally, a circular silicone chamber encloses the
nanotextured electrode surface to confine the fluid onto the device
surface and prevent evaporation which could lead to electrical
signal instability. The chamber has a maximum working volume of 1.6
ml. Thus, the combination of the alumina membrane on a PCB
substrate enclosed by a silicone chamber forms an inexpensive
bio-sensing device capable of detecting various bio-molecules (FIG.
9C).
[0125] Detection Methodology--Electrochemical Impedance
Spectroscopy (EIS)
[0126] The electronic biosensor measures impedance changes to the
electrical double layer at the solid-liquid interface within the
nanowells induced when target proteins contained in the sample bind
to reagents such as antibodies immobilized on the sensor surface.
Impedance measurements provide very detailed information about the
electrical changes occurring at the conductive or semi-conductive
interfaces.
[0127] When target antigens bind immobilized antibody inside the
nanowells, the double layer capacitance changes due to the change
in the surface charge concentrations. Thus, the double layer
capacitance directly correlates to the amount of binding taking
place at the solid--liquid interface, and the amount of binding is
directly proportional to the concentration of the target species.
Thus, by characterizing the double layer capacitance, we can get an
accurate estimate of the concentration of the target species. The
changes to the double layer capacitance can be represented as the
measured impedance changes especially at low frequencies (i.e.
below 1 kHz). In the sensor configuration used here, redox probes
are not used, and it can be assumed that all the conduction
occurring at the interface is non-faradaic in nature, so the charge
distribution dynamics at the metal--solution interface
characterizing the bio-molecular interactions at the surface can be
modeled using the Helmholtz-Gouy-Chapman model with Stems
correction (Chang, B. Y., Park, S. M., Electrochemical impedance
spectroscopy. Annu Rev Anal Chem (Palo Alto Calif.) 3, 207-29;
Lisdat, F., Schafer, D., The use of electrochemical impedance
spectroscopy for biosensing. Anal Bioanal Chem 2008, 391, (5),
1555-67). Since binding of antigens to the nanobodies is free of
any bio-chemical mediators, the impedance changes within the
electrical double layer (FIG. 9D) is non-Faradaic, and the
electrical circuit model of the sensor can be represented as a
simple resistive-capacitive (RC) series circuit whose values are
extracted by a frequency response analyzer potentiostat (Bothara,
Met al., Nanomonitors: electrical immunoassays for protein
biomarker profiling. Nanomed 2008, 3, (4), 423-36). For probing the
impedance changes to the electrical double layer of the nanowell
electrodes, a very small amplitude sinusoidal voltage is applied to
the electrochemical system, and the output current response is
sensed, the ratio of the applied voltage phasor to the output
current phasor is the resulting impedance, which is characterized
using a frequency response analyzer (Gamry Industries Reference 600
potentiostat).
[0128] Another commonly used electrochemical immuno-sensing
technique is the pulsed amperometry. This method involves the
immobilization of an immune-reagent component on the electrode
transducer and the use of an electrochemical active substance
produced by enzymatic reaction for signal generation (Lei, C. X.,
F. C. Gong, et al., Amperometric immunosensor for Schistosoma
japonicum antigen using antibodies loaded on a nano-Au monolayer
modified chitosan-entrapped carbon paste electrode. Sensors and
Actuators B: Chemical 2003, 96(3): 582-588). As simple as this
appears, there can be numerous problems associated with an
inadequate supply of enzyme inhibitors in the sample, instability
of the enzyme over time, irreproducibility of the electrode
kinetics for reoxidizing reagent or reducing oxidising agent, redox
active interferences which either react at the electrode and/or
couple with the reagent couple, and inadequate temperature control
(Kissinger, P. T., Introduction to Amperometric Biosensor
Configuration. CURRENTSEPARATIONS.COM and Drug Development 1997,
16(3)). The EIS technique eliminates most of these problems since
it doesn't rely on the redox properties of the analyte and does not
need an enzyme inhibitor. Another fundamental difference between
the two techniques is the sensing mechanism. Amperometry involves
detection of ions in the solution by applying a fixed voltage
through electrodes, and measuring the current/change in current;
whereas EIS involves characterizing the electrical double layer at
the electrode by sweeping a range of frequencies, and measuring the
current.
[0129] Nanobody Immobilization to Sensor Surface
[0130] Nanobodies are immobilized onto the electrode sensor surface
using a chemical linker. The electrode surface is first amine
functionalized using 3-Aminopropyl Triethoxysilane (APTES, 2% in
acetone buffer, Thermo Scientific Inc.). A 100 .mu.l aliquot of 2%
APTES is applied on the electrode surface and incubated at room
temperature for 30 seconds. Excess APTES was then removed by
flowing acetone over the surface. The alumina membrane is then
soldered to the silanized electrode surface. Then 3,3'-dithiobis
succinimidyl propionate (DSP, Thermo Scientific Inc.) dissolved in
DMSO solvent (4 mg/ml) is used to cross link the nanobodies to the
electrode surface. The DSP (thiol linker) is added for 30 minutes
to allow conjugation to the silanized electrode surfaces which form
the base of the nanowells. After conjugation of the linker to the
nanowell surfaces (primarily to the base of the nanowells), a 40
.mu.l aliquot of the nanobody (1 mg/ml) is added for 15 minutes,
followed by addition of 40 .mu.l of Bovine Serum Albumin (BSA) (2
mg/ml) to block any unbound amine sites on the sensor surface.
Phosphate buffered saline (1.times.PBS) is used to prepare antibody
aliquots and for the wash steps due to its isotonic properties.
[0131] Preparation of CSF Samples.
[0132] Post mortem CSF Samples from AD, PD, MSA and non-diseased
autopsy confirmed sources were generously provided by Dr. Thomas
Beach (Civin Laboratory for Neuropathology, Banner/Sun Health
Research Institute, Sun City, Ariz.). The post-mortem interval for
collection of samples is less than three hours. Samples were
diluted to one-tenth of their original concentration in 1.times.PBS
buffer and frozen.
[0133] Expression and Purification of Nanobodies.
[0134] D10, D5 and A4 nanobodies were produced and purified
essentially as previously described (Zameer et al., Anti-oligomeric
Abeta single-chain variable domain antibody blocks Abeta-induced
toxicity against human neuroblastoma cells. J Mol Biol 2008, 384,
(4), 917-28; Emadi et al., Detecting morphologically distinct
oligomeric forms of alpha-synuclein. J Biol Chem 2009, 284, (17),
11048-58). D10 was used to calibrate the sensor chip since it binds
to all forms of a-syn including monomeric facilitating preparation
of a-syn standards of known concentration. The D5 nanobody
specifically recognizes a small oligomeric a-syn species (Emadi et
al., Isolation of a human single chain antibody fragment against
oligomeric alpha-synuclein that inhibits aggregation and prevents
alpha-synuclein-induced toxicity. J Mol Biol 2007, 368, (4),
1132-44), while A4 specifically recognizes a small oligomeric
A.beta. species (Zameer et al., Anti-oligomeric Abeta single-chain
variable domain antibody blocks Abeta-induced toxicity against
human neuroblastoma cells. J Mol Biol 2008, 384, (4), 917-28).
Nanobody specificity was demonstrated by several different assays
including time course dot blot, ELISA, western blot and Atomic
force microscopy based assays (Zameer et al., Anti-oligomeric Abeta
single-chain variable domain antibody blocks Abeta-induced toxicity
against human neuroblastoma cells. J Mol Biol 2008, 384, (4),
917-28; Emadi et al., Isolation of a human single chain antibody
fragment against oligomeric alpha-synuclein that inhibits
aggregation and prevents alpha-synuclein-induced toxicity. J Mol
Biol 2007, 368, (4), 1132-44; Wang et al., Characterizing Antibody
Specificity to Different Protein Morphologies by AFM. Langmuir
2008. The supernatant and cell lysate from a 1 L culture were
combined and concentrated in a tangential flow filter (Millipore)
using a 10 kDa filter membrane (Millipore). Concentrated samples
were purified using a protein A--Sepharose column (GE healthcare,
NJ) as previously described (Zameer et al., Anti-oligomeric Abeta
single-chain variable domain antibody blocks Abeta-induced toxicity
against human neuroblastoma cells. J Mol Biol 2008, 384, (4),
917-28). Fractions containing nanobody were pooled, dialyzed
against PBS, lyophilized and stored at -20.degree. C. The purity of
the nanobody sample was determined by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) on 15% polyacrylamide
gels (Bio-Rad, Hercules, Calif.) and western blotting, and the
concentration was determined using bicinchonic acid (BCA) protein
assay (Pierce, Ill.).
[0135] Sensor Calibration.
[0136] A-syn was prepared and purified in our lab as previously
described (Emadi et al., Isolation of a human single chain antibody
fragment against oligomeric alpha-synuclein that inhibits
aggregation and prevents alpha-synuclein-induced toxicity. J Mol
Biol 2007, 368, (4), 1132-44; Volles et al., Relationships between
the sequence of alpha-synuclein and its membrane affinity,
fibrillization propensity, and yeast toxicity. J Mol Biol 2007,
366, (5), 1510-22). Purified stocks of a-syn were lyophilized and
stored at -80.degree. C. Stocks were first dissolved in DI water
and subsequent dilutions were made in Tris buffer (25 mM Tris, 150
mM NaCl, pH 7.4). A 40 .mu.l aliquot of test sample is added,
incubated for 10 minutes, and then impedance measurements are
taken.
[0137] Results
[0138] Operating Parameters and Detectability Limits of Biosensor
with Nanobodies as Capturing Agents.
[0139] Specific aggregate morphologies of a-syn and A.beta. are
likely to be present in CSF samples only at very low concentrations
(nanomolar or less), therefore successful detection of these
targets requires a biosensor with very low detection limits. In
order to determine the sensitivity of the biosensor, we utilized
the D10 nanobody as a capture agent. D10 recognizes all forms of
a-syn (Zhou, C.; Emadi, S.; Sierks, M. R.; Messer, A., A human
single-chain Fv intrabody blocks aberrant cellular effects of
overexpressed alpha-synuclein. Mol Ther 2004, 10, (6), 1023-31),
and therefore we can accurately control target a-syn
concentrations. The first step to determine biosensor sensitivity
is to determine suitable electrical parameters which will enable
detection of bound target using electrochemical impedance
spectroscopy.
[0140] Since binding of antigen to target takes place over a high
density array of nano-pores, the impedance signal we obtain
correlates to the average signal obtained over all the pores,
ensuring that even if some pores do not contain immobilized capture
agent the measured impedance will be reproducible within an
acceptable margin of error. The dimensions of the electrical double
layer within the nanopores is approximately 50 nm. Based on
previous studies (Bothara et al., Nanomonitors: electrical
immunoassays for protein biomarker profiling. Nanomed 2008, 3, (4),
423-36; Reddy, et al., Nanomonitors: Protein biosensors for rapid
analyte analysis. Ieee Sensors Journal 2008, 8, (5-6), 720-723), a
100 mv peak to peak pulse should be utilized to characterize
changes to the capacitance of the electrical double layer induced
by biomolecule binding. The second parameter that needs to be
defined is the sensing frequency. Since double layer capacitance
dominates the impedance spectrum for frequencies less than 1 KHz in
order to determine the optimum frequency for these studies, we
tested the frequency response by adding a range of monomeric a-syn
concentrations (1 ng/ml to 1 ug/ml) to immobilized D10 using a
frequency range from 50 Hz to 1 MHz. A frequency of 100 Hz gave
maximum visible shifts in the impedance induced by biomolecule
binding (FIG. 10).
[0141] Using the defined electrical parameters, we generated a
calibration curve using different concentrations of a-syn using two
controls; BSA was added to immobilized D10 to test for nonspecific
binding to nanobody, and a-syn without immobilized D10 was used to
measure background a-syn binding to the sensor surface.
[0142] From the calibration curve, we can accurately detect antigen
down to a limit of detection (LOD) of 1 picogram/ml (FIG. 11)
indicating that it should be possible to detect low femtomolar
concentrations of target antigen in clinical patient samples. The
results also indicate that the target antigen, a-syn, binds to the
immobilized nanobody and not the conjugation linker since the
signal obtained with antigen binding to immobilized nanobody, even
at antigen concentrations six orders of magnitude lower, is higher
than the signal observed without nanobody (FIG. 11). The nanobody
also specifically reacts with the a-syn target since no signal is
observed with the control protein antigen, BSA (FIG. 11).
[0143] After demonstrating that we can detect femtomolar
concentrations of a-syn target using immobilized nanobodies, we
next determined whether we could detect specific morphologies of
a-syn in post-mortem CSF samples. While we utilized the D10
nanobody, which recognizes all forms of a-syn, for the calibration
studies since we could accurately measure the concentration of
monomeric a-syn present, in order to detect specific oligomeric
forms of a-syn and A.beta. in CSF samples, we next utilized two
other nanobodies which selectively recognize either a specific
oligomeric form of a-syn (D5) or of A.beta. (A4). We immobilized
either D5 or A4 to sensor chips and then analyzed nine different
CSF samples, three taken from 3 AD patients, 3 PD-like (2 PD and
one Multiple System Atrophy (MSA) which shares some similar
symptoms to PD), and 3 aged matched non-demented control samples
(ND). Since all nine samples could not be sequentially analyzed on
a single chip, we divided the samples into groups of three, where
each group contained one ND, one AD and one PD-like sample.
[0144] The experiments were repeated several times and performed
simultaneously utilizing multiple sensor chips. In order to control
for inter-sensor variability associated with the sensor
manufacturing process where there is heterogeneous integration of
multiple materials, we utilized a common sample in each data set
(AD1) performed on each chip, and normalized data from different
chips to this common reference point. An illustrative example of
the comparison of percentage change in impedance values obtained in
the raw data compared to normalized data is shown in Table 7. The
percentage change in impedance values after normalization to a
common reference point are shown for all nine CSF samples obtained
with immobilized D5 (FIG. 12A) and A4 (FIG. 12B).
TABLE-US-00008 TABLE 7 Percentage change in impedance from baseline
for the D5 antibody. The normalized data set represents the raw
data set normalized to group 1 AD reference point. Raw Data Set
Normalized Data Set ND AD PD ND AD PD Group 1 4.787248 6.108411
7.383063 4.787248 6.108411 7.383063 Group 2 9.754458 15.3544
16.84676 3.815685 6.006235 6.590008 Group 3 5.5921 7.9426 6.1691
2.871514 4.078518 3.16783
[0145] Oligomeric species of both a-syn and A.beta. are readily
detected in post-mortem CSF samples even when assayed at 10-fold
dilution (FIG. 12A, B). The results indicate that the levels of
oligomeric a-syn and A.beta. species in CSF varies depending on the
disease, and that these oligomeric species have great promise as
biomarkers for distinguishing between different neurodegenerative
diseases.
[0146] Discussion
[0147] CSF levels of A.beta., tau and phosphorylated tau were shown
to have promise as biomarkers for diagnosing AD. However increasing
evidence indicate that various soluble aggregated oligomeric forms
of A.beta., a-syn and tau are the relevant toxic species in
different neurodegenerative diseases, and specific detection of
different aggregate species in CSF may provide a more refined and
powerful tool to facilitate early and accurate diagnosis of a
variety of neurodegenerative diseases and to study the mechanisms
involved in the onset and progression of these diseases. Protein
aggregation is a common thread behind numerous neurodegenerative
diseases including AD, PD, LBD, tauopathies and synucleinopathies.
Aggregation of A.beta. has been correlated with AD, aggregation of
a-syn with PD, LBD and other synucleinopathies, and aggregation of
tau with AD and various tauopathies. While the presence of
fibrillar aggregates of these different proteins has been a classic
diagnostic feature of the respective diseases, increasing evidence
suggests that soluble oligomeric forms of these proteins are the
relevant toxic species. During the polymerization process from
monomeric to fibrillar form, each of the protein species must pass
through different oligomeric states, suggesting that various
oligomeric species may represent earlier biomarkers for these
diseases compared to the presence of fibrillar forms.
[0148] A rapidly growing body of evidence indicates that oligomeric
forms of A.beta. are key factors in the onset and progression of
AD. A.beta. forms a number of soluble intermediate or metastable
structures which may contribute to toxicity. Cortical levels of
soluble A.beta. correlate well with the cognitive impairment and
loss of synaptic function. Small, soluble spherical or annular
aggregates of A.beta. were shown to be neurotoxic, and oligomeric
forms of A.beta., created in vitro or derived from cell cultures
inhibit long term potentiation (LTP). The concentration of
oligomeric forms of A.beta. are elevated in transgenic mouse models
of AD and human AD brain and CSF samples and the presence of an SDS
stable dimeric form of A.beta. associates well with dementia in AD
patients. Disruption of neural connections near A.beta. plaques was
also attributed to oligomeric A.beta. species, a halo of oligomeric
A.beta. surrounds A.beta. plaques causing synapse loss, and
oligomeric AP was shown to disrupt cognitive function in transgenic
animal models of AD. Different size oligomers of A.beta. have been
correlated with AD, including a 56 kD aggregate and smaller
dimeric, trimeric and tetrameric species. Therefore specific
detection of soluble oligomeric A.beta. species holds great promise
as a biomarker for studying the progression of AD.
[0149] Similarly, formation of oligomeric aggregates of a-syn has
also been correlated with PD and other synucleinopathies. A-syn is
a major component of Lewy bodies and neurites. While wild-type and
mutant forms of a-syn associated with familial cases of PD, A30P,
E46K and A53T, can assemble into Lewy body like fibrils in vitro,
the mutations increase the total rate of oligomerization compared
to the wild-type form of a-syn. Different morphologies of a-syn
have different affinities for various membranes, and both
oligomeric and fibrillar forms have been shown to disrupt membrane
permeability and integrity. Aggregated forms of a-syn were shown to
induce toxicity in dopaminergic neurons in vivo and several
different oligomeric morphologies were shown to each have different
toxic mechanisms and effects on cells. Oligomeric forms of a-syn
were shown to be toxic to neuronal cells, and toxic oligomeric
a-syn forms were identified in living cells, in human plasma from
PD patients, and in human PD brain tissue. Elevated levels of
soluble oligomeric a-syn species were also detected in post-mortem
brain extracts from patients with LBD, even though monomeric a-syn
levels in CSF were not able to discriminate between LBD and AD.
Therefore the presence of various oligomeric a-syn species in CSF
is also a very promising biomarker for studying the progression of
various neurodegenerative diseases.
[0150] We developed a novel biosensor for sensitive detection of
biomolecules from clinical samples.sup.6 and show here that the
Nanomonitor has sufficient sensitivity to detect low femtomolar
concentrations of target directly from human CSF samples using
single chain antibody fragments or nanobodies as the capture agent.
We also developed technology which enables us to isolate nanobodies
that selectively recognize specific protein morphologies, and have
isolated nanobodies that bind two different oligomeric a-syn
species and two different oligomeric A.beta. species. The different
oligomer specific nanobodies do not show cross-reactivity, so the
nanobodies binding oligomeric A.beta. do not bind oligomeric a-syn
and vice versa. The different aggregate species recognized by each
of the morphology specific nanobodies can be detected in
post-mortem human AD or PD tissue, and can distinguish between AD,
PD and healthy brain tissue. Therefore these nanobodies represent
excellent tools to detect specific oligomeric aggregates of A.beta.
and a-syn in both clinical and animal model samples.
[0151] Here we show that detection of an oligomeric A.beta. species
in CSF samples using the A4 nanobody very sensitively distinguishes
the AD CSF samples from the PD and ND samples (FIG. 12B). The
oligomeric a-syn species recognized by the D5 nanobody, is present
in highest concentrations in the PD samples, lower concentrations
in the AD samples and lowest concentrations in the ND and MSA
samples (FIG. 12A). Interestingly, the a-syn species recognized by
D5 readily distinguishes between the PD and MSA CSF samples
indicating the value of this technology in studying disease
mechanisms. Using the results obtained with both the A4 and D5
nanobodies, we can clearly distinguish between AD, PD, and ND or
MSA samples, where ND cases do not show above threshold levels of
target with either nanobody, PD cases are distinguished by positive
binding signal with D5, but not A4, and AD cases show a positive
signal with A4 and also D5 in some cases (FIG. 12C). In order to
determine whether detection of specific oligomeric forms of
proteins are useful as biomarkers for studying and diagnosing
neurodegenerative disease, a much larger CSF sample size will need
to be analyzed using the various different morphology specific
nanobodies against a-syn and A.beta..
CONCLUSION
[0152] Diagnosis of neurodegenerative diseases including
Alzheimer's, Dementia with Lewy Bodies, frontotemporal dementia and
Parkinson's diseases is a challenging prospect. Since misfolding
and aggregation of proteins including A.beta., a-syn and tau have
been correlated with these neurodegenerative diseases, the presence
of different forms of these target proteins in clinical samples has
great promise as potential biomarkers to both study and diagnose
these diseases. CSF levels of different A.beta. and tau species can
correctly predict progression to AD validating this concept. Since
increasing evidence indicates that small soluble oligomeric forms
of proteins including A.beta. and a-syn are the relevant toxic
species associated with neurodegeneration, oligomeric forms of
these proteins may be sensitive biomarkers to study the onset and
progression of these different neurodegenerative diseases. Here we
show that the presence of different protein morphologies in human
CSF samples can be readily detected using highly selective
morphology specific reagents in conjunction with a sensitive
electronic biosensor. We further show that using morphology
specific reagents, we can readily distinguish between post-mortem
CSF samples from AD, PD and cognitively normal sources. These
studies suggest that detection of specific oligomeric aggregate
species holds great promise for use as sensitive biomarkers for
neurodegenerative disease.
[0153] All publications, patents and patent applications are
incorporated herein by reference. While in the foregoing
specification this invention has been described in relation to
certain embodiments thereof, and many details have been set forth
for purposes of illustration, it will be apparent to those skilled
in the art that the invention is susceptible to additional
embodiments and that certain of the details described herein may be
varied considerably without departing from the basic principles of
the invention.
[0154] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms e., meaning "including, but not
limited to") unless otherwise noted. Recitation of ranges of values
herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0155] Embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Variations of those embodiments may become apparent to
those of ordinary skill in the art upon reading the foregoing
description. The inventors expect skilled artisans to employ such
variations as appropriate, and the inventors intend for the
invention to be practiced otherwise than as specifically described
herein. Accordingly, this invention includes all modifications and
equivalents of the subject matter recited in the claims appended
hereto as permitted by applicable law. Moreover, any combination of
the above-described elements in all possible variations thereof is
encompassed by the invention unless otherwise indicated herein or
otherwise clearly contradicted by context.
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Sequence CWU 1
1
271292PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Glu Xaa Pro Ile Ala Tyr Gly Ser Arg Trp Ile
Val Ile Thr Arg Gly 1 5 10 15 Pro Ala Gly His Gly Pro Gly Thr Ala
Ala Gly Val Gly Gly Gly Leu 20 25 30 Val Gln Pro Gly Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Ser Ser Tyr
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu
Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr 65 70 75 80 Tyr
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90
95 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
100 105 110 Ala Val Tyr Tyr Cys Ala Lys Ser Tyr Gly Ser Val Lys Ile
Ser Cys 115 120 125 Phe Asp Tyr Trp Gly Gln Ser Thr Leu Val Thr Val
Ser Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Ile Val 145 150 155 160 Leu Thr Gln Ser Pro Asp Ser
Leu Ala Val Ser Leu Gly Glu Arg Ala 165 170 175 Thr Ile Asn Cys Lys
Ser Ser Gln Ser Val Leu Tyr Asn Ser Asn Asn 180 185 190 Lys Asn Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Glu 195 200 205 Leu
Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg 210 215
220 Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
225 230 235 240 Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
Phe Tyr Ser 245 250 255 Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg Ala 260 265 270 Ala Ala His His His His His His Gly
Ala Ala Glu Gln Lys Leu Ile 275 280 285 Ser Glu Glu Asp 290
2268PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 2Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Pro Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Ala Ile
Gln His Thr Gly Ala Pro Thr Thr Tyr Ala Asp 50 55 60 Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Ala Lys Ala Phe Pro Pro Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 115 120 125 Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Ile Ser Ser Tyr Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Leu Leu
Ile Tyr Ser Ala Ser Ser Leu Gln Ser Gly Val 180 185 190 Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210 215
220 Arg Glu Thr Gly Pro Lys Ala Phe Gly Gln Gly Thr Lys Val Glu Ile
225 230 235 240 Lys Arg Ala Ala Ala His His His His His His Gly Ala
Ala Glu Gln 245 250 255 Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala
Ala 260 265 3268PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 3Met Ala Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser
Ser Ile Gln Pro Glu Gly Arg Arg Thr Ala Tyr Val Asp 50 55 60 Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr 85 90 95 Tyr Cys Ala Lys Pro Pro Glu Arg Phe Asp Tyr Trp Gly
Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Thr Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Ile Ser
Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro
Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val 180 185 190
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195
200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln 210 215 220 Ser Tyr Ser Thr Pro Asn Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile 225 230 235 240 Lys Arg Ala Ala Ala His His His His His
His Gly Ala Ala Glu Gln 245 250 255 Lys Leu Ile Ser Glu Glu Asp Leu
Asn Gly Ala Ala 260 265 4268PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 4Met Ala Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45
Trp Val Ser Ser Ile Gly Gln Lys Gly Gly Gly Thr Gln Tyr Ala Asp 50
55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr 85 90 95 Tyr Cys Ala Lys His Phe Glu Asn Phe Asp Tyr
Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Thr Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser
Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175
Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser His Leu Gln Ser Gly Val 180
185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln 210 215 220 Thr Arg Arg Pro Pro Ser Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile 225 230 235 240 Lys Arg Ala Ala Ala His His His
His His His Gly Ala Ala Glu Gln 245 250 255 Lys Leu Ile Ser Glu Glu
Asp Leu Asn Gly Ala Ala 260 265 5281PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
5Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 1
5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser 20 25 30 Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu 35 40 45 Trp Val Ser Asn Ile Ser Ser Ala Gly Lys Gly
Leu Glu Trp Val Ser 50 55 60 Ser Ile Asp Asp Ser Gly Ala Ser Thr
Tyr Tyr Ala Asp Ser Val Lys 65 70 75 80 Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr Leu 85 90 95 Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 100 105 110 Lys Asp Ser
Ala Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 115 120 125 Val
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135
140 Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
145 150 155 160 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Ile 165 170 175 Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys 180 185 190 Leu Leu Ile Tyr Thr Ala Ser Ser Leu
Gln Ser Gly Val Pro Ser Arg 195 200 205 Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser 210 215 220 Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ala Ala 225 230 235 240 Ser Pro
Ser Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ala 245 250 255
Ala Ala His His His His His His Gly Ala Ala Glu Gln Lys Leu Ile 260
265 270 Ser Glu Glu Asp Leu Asn Gly Ala Ala 275 280
6268PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 6Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Ala Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Tyr Ile
Ala Ser Gly Gly Asp Thr Thr Asn Tyr Ala Asp 50 55 60 Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Ala Lys Gly Ala Ser Ala Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 115 120 125 Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Ile Ser Ser Tyr Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Leu Leu
Ile Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val 180 185 190 Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210 215
220 Ser Ser Asn Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
225 230 235 240 Lys Arg Ala Ala Ala His His His His His His Gly Ala
Ala Glu Gln 245 250 255 Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala
Ala 260 265 7263PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 7Met Ala Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala
Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp 50 55 60 Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Val Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr 85 90 95 Tyr Cys Ala Arg Ile Asn Ala Lys Trp Gly Gln Gly Thr
Leu Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Ser 115 120 125 Ala Leu Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser 130 135 140 Val Gly Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser 145 150 155 160 Ser Tyr Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 165 170 175 Leu Ile
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe 180 185 190
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 195
200 205 Gln Pro Gly Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser
Thr 210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Ala Ala Ala 225 230 235 240 His His His His His His Gly Ala Ala Glu
Gln Lys Leu Ile Ser Glu 245 250 255 Glu Asp Leu Asn Gly Ala Ala 260
8877DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 8ttgttattac tcgcggccca gccggccatg
gccgaggtgc agctgttgga gtctggggga 60ggcttggtac agcctggggg gtccctgaga
ctctcctgtg cagcctctgg attcaccttt 120agcagctatc ccatgagctg
ggtccgccag gctccaggga aggggctgga gtgggtctca 180gcgattcagc
atactggtgc gccgacaact tacgcagact ccgtgaaggg ccggttcacc
240atctccagag acaattccaa gaacacgctg tatctgcaaa tgaacagcct
gagagccgag 300gacacggccg tatattactg tgcgaaagcg tttccgccgt
ttgactactg gggccaggga 360accctggtca ccgtctcgag cggtggaggc
ggttcaggcg gaggtggcag cggcggtggc 420gggtcgacgg acatccagat
gacccagtct ccatcctccc tgtctgcatc tgtaggagac 480agagtcacca
tcacttgccg ggcaagtcag agcattagca gctatttaaa ttggtatcag
540cagaaaccag ggaaagcccc taagctcctg atctattctg catcctcttt
gcaaagtggg 600gtcccatcaa ggttcagtgg cagtggatct gggacagatt
tcactctcac catcagcagt 660ctgcaacctg aagattttgc aacttactac
tgtcaacagc gggagactgg gcctnnnngt 720tcggncaang gancaangtg
gaaatcaaac gggcggccgc acatcatcat caccatcacg 780gggccgcana
acaaaaactc atctcanaan aggatctgaa tggggccgca tanactgttg
840aaanttgttt ancaaacnnc atacnnnaaa ttcattt 8779877DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
9ttgttattac tcgcggccca gccggcctgg ccgaggtgca gctgttggag tctgggggag
60gcttggtaca gcctgggggg tccctgagac tctcctgtgc agcctctgga ttcaccttta
120gcagctatgc catgagctgg gtccgccagg ctccagggaa ggggctggag
tgggtctcat 180ctattcagcc tgagggtagg cggacagcgt acgtagactc
cgtgaagggc cggttcacca 240tctccagaga caattccaag aacacgctgt
atctacaaat gaacagcctg agagccgagg 300acacggccgt atattactgt
gcgaaaccgc cggagaggtt tgactactgg ggccagggaa 360ccctggtcac
cgtctcgagc ggtggaggcg gttcaggcgg aggtggcagc ggcggtggcg
420ggtcgacgga catccagatg acccagtctc catcctccct gtctgcatct
gtaggagaca 480gagtcaccat cacttgccgg gcaagtcaga gcattagcag
ctatttaaat tggtatcagc 540agaaaccagg gaaagcccct aagctcctga
tctatgctgc atccagtttg caaagtgggg 600tcccatcaag gttcagtggc
agtggatctg ggacagattt cactctcacc atcagcagtc 660tgcaacctga
agattttgca acttactact gtcaacagag ttacagtacc cctaatacgt
720tcggccaagg gaccaaggtg gaaatcaaac gggcggccgc acatcatcat
caccatcacg 780gggccgcaga acaaaaactc atctcanaan aggatctgaa
tggggccgca tagactgttg 840aaagttgttt ancaaacctc atacagaaaa ttcattt
87710821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 10ccatggccca ggtacagctg
caggagtcgg gggaggcttg gtacagcctg gggggtccct 60gagactctcc tgtgcagcct
ctggattcac ctttagcagc tatgccatga gctgggtccg 120ccaggctcca
gggaaggggc tggagtgggt ctcagctatt agtggtagtg gtggtagcac
180atactacgca gactccgtga agggccgatt caccatctcc agagacaatt
ccaagaacac 240gctgtatctg caaatgaaca gcctgagagc tgaggacacg
gctgtgtatt actgtgcgaa 300gagctatggt tcagttaaaa taagctgctt
tgactactgg ggccagagca ccctggtcac 360cgtctcctca ggtggaggcg
gttcaggcgg aggtggctct ggcggtggcg gatcggaaat 420tgtgctgacg
cagtctccag actccctggc tgtgtctctg ggcgagaggg ccaccatcaa
480ctgcaagtcc agccagagtg ttctttacaa ctccaacaat aagaactact
tagcttggta 540ccagcagaaa ccaggacagt ctcctgagtt gctcatttac
tgggcatcaa cccgggaatc 600cggggtccct gaccgattca gtggcagcgg
gtctgggaca gaattcactc ttaccatcag 660cagcctgcag gctgaggatg
tggcagttta ttactgtcag caattttata gtactcctcc 720gacttttggc
caggggacca agctggagat caaacgtgcg gccgcacatc atcatcacca
780tcacggggcc gcagaacaaa aactcatctc agaagaggat c
82111822DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 11ccatggccga ggtgcagctg ttggagtctg
ggggaggctt ggtacagcct ggggggtccc 60tgagactctc ctgtgcagcc tctggattca
cctttagcag ctatgccatg agctgggtcc 120gccaggctcc agggaagggg
ctggagtggg tctcatcgat tggtcagaag ggtggtggta 180cacagtacgc
agactccgtg aagggccggt tcaccatctc cagagacaat tccaagaaca
240cgctgtatct gcaaatgaac agcctgagag ccgaggacac ggccgtatat
tactgtgcga 300aacattttga gaattttgac tactggggcc agggaaccct
ggtcaccgtc tcgagcggtg 360gaggcggttc aggcggaggt ggcagcggcg
gtggcgggtc gacggacatc cagatgaccc 420agtctccatc ctccctgtct
gcatctgtag gagacagagt caccatcact tgccgggcaa 480gtcagagcat
tagcagctat ttaaattggt atcagcagaa accagggaaa gcccctaagc
540tcctgatcta tgctgcatcc catttgcaaa gtggggtccc atcaaggttc
agtggcagtg 600gatctgggac agatttcact ctcaccatca gcagtctgca
acctgaagat tttgcaactt 660actactgtca acagacgcgt aggccgcctt
ctacgttcgg ccaagggacc aaggtggaaa 720tcaaacgggc ggccgcacat
catcatcacc atcacggggc cgcagaacaa aaactcatct 780cagaagagaa
tcactagtgc ggccgcctgc aggtcgacca ta 82212843DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
12ccatggccga ggtgcagctg ttggagtctg ggggaggctt ggtacagcct ggggggtccc
60tgagactctc ctgtgcagcc tctggattca cctttagcag ctatgccatg agctgggtcc
120gccaggctcc agggaagggg ctggagtggg tctcaaatat tagtagtgca
gggaaggggc 180tggagtgggt ctcaagtatt gatgattctg gtgcttctac
atattacgca gactccgtga 240agggccggtt caccatctcc agagacaatt
ccaagaacac gctgtatctg caaatgaaca 300gcctgagagc cgaggacacg
gccgtatatt actgtgcgaa agattctgct tcttttgact 360actggggcca
gggaaccctg gtcaccgtct cgagcggtgg aggcggttca ggcggaggtg
420gcagcggcgg tggcgggtcg acggacatcc agatgaccca gtctccatcc
tccctgtctg 480catctgtagg agacagagtc accatcactt gccgggcaag
tcagagcatt agcagctatt 540taaattggta tcagcagaaa ccagggaaag
cccctaagct cctgatctat actgcatcca 600gtttgcaaag tggggtccca
tcaaggttca gtggcagtgg atctgggaca gatttcactc 660tcaccatcag
cagtctgcaa cctgaagatt ttgcaactta ctactgtcaa cagtctgctg
720ctagtccttc tacgttcggc caagggacca aggtggaaat caaacgggcg
gccgcacatc 780accatcacca tcacggggcc gcagaacaaa aactcntctc
agaagnggat cnnaangggn 840ccg 84313879DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
13ttgttattac tcgcggccca gccggccatg gccgaggtgc agctgttgga gtctggggga
60ggcttggtac agcctggggg gtccctgaga ctctcctgtg cagcctctgg attcaccttt
120agcagctatg ccatgagctg ggtccgccag gctccaggga aggggctgga
gtgggtctca 180tatattgcta gtggtggtga tactacaaat tacgcagact
ccgtgaaggg ccggttcacc 240atctccagag acaattccaa gaacacgctg
tatctgcaaa tgaacagcct gagagccgag 300gacacggccg tatattactg
tgcgaaaggt gcttctgctt ttgactactg gggccaggga 360accctggtca
ccgtctcgag cggtggaggc ggttcaggcg gaggtggcag cggcggtggc
420gggtcgacgg acatccagat gacccagtct ccatcctccc tgtctgcatc
tgtaggagac 480agagtcacca tcacttgccg ggcaagtcag agcattagca
gctatttaaa ttggtatcag 540cagaaaccag ggaaagcccc taagctcctg
atctatgctg catcctattt gcaaagtggg 600gtcccatcaa ggttcagtgg
cagtggatct gggacagatt tcactctcac catcagcagt 660ctgcaacctg
aagattttgc aacttactac tgtcaacaga gttctaatga tccttatacg
720ttcggccaag ggaccaaggt ggaaatcaaa cgggcggccg cacatcatca
tcaccatcac 780gggngccnna naacaaaaac tcatctcaaa nnnnntctga
atgggggccn catanactgt 840tgaaagttgt ttnnaaacct cntacanaaa antcnnttt
87914776DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 14atggccgagg tgcagctggt ggagtctggg
ggaggcgtgg tccagcctgg gaggtccctg 60agactctcct gtgcagcctc tggattcacc
ttcagtagct atggcatgca ctgggtccgc 120caggccccag gcaaggggct
ggagtgggtg gcagttatat catatgatgg aagtaataaa 180tactatgcag
actccgtgaa gggccgattc accatctcca gagacaattc caagaacacg
240ctgtatctgc aagtgaacag cctgagagct gaggacacgg ccgtgtatta
ctgtgcaaga 300attaatgcga agtggggcca aggtaccctg gtcaccgtct
cgagtggtgg aggcggttca 360ggcggaggtg gctctggcgg tagtgcactt
gacatccaga tgacccagtc tccatcctcc 420ctgtctgcat ctgtaggaga
cagagtcacc atcacttgcc gggcaagtca gagcattagc 480agctatttaa
attggtatca gcagaaacca gggaaagccc ctaagctcct gatctatgct
540gcatccagtt tgcaaagtgg ggtcccatca aggttcagtg gcagtggatc
tgggacagat 600ttcactctca ccatcagcag tctgcaacct ggagattttg
caacttacta ctgtcaacag 660agttacagta ccccgacgtt cgggcaaggg
accaaggtgg aaatcaaacg tgcggccgca 720catcatcatc accatcacgg
ggccgcagaa caaaaactca tctcagaaga ggatct 77615278PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
15Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro 1
5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser 20 25 30 Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu 35 40 45 Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser
Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Ser
Tyr Gly Ser Val Lys Ile Ser Cys Phe Asp Tyr 100 105 110 Trp Gly Gln
Ser Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln 130 135
140 Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn
145 150 155 160 Cys Lys Ser Ser Gln Ser Val Leu Tyr Asn Ser Asn Asn
Lys Asn Tyr 165 170 175 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser
Pro Glu Leu Leu Ile 180 185 190 Tyr Trp Ala Ser Thr Arg Glu Ser Gly
Val Pro Asp Arg Phe Ser Gly 195 200 205 Ser Gly Ser Gly Thr Glu Phe
Thr Leu Thr Ile Ser Ser Leu Gln Ala 210 215 220 Glu Asp Val Ala Val
Tyr Tyr Cys Gln Gln Phe Tyr Ser Thr Pro Pro 225 230 235 240 Thr Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala His 245 250 255
His His His His His Gly Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu 260
265 270 Asp Leu Asn Gly Ala Ala 275 165PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Ser
Tyr Ala Met Ser 1 5 1717PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 17Ala Ile Ser Gly Ser Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 1812PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Ser
Tyr Gly Ser Val Lys Ile Ser Cys Phe Asp Tyr 1 5 10
1917PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19Lys Ser Ser Gln Ser Val Leu Tyr Asn Ser Asn Asn
Lys Asn Tyr Leu 1 5 10 15 Ala 207PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 20Trp Ala Ser Thr Arg Glu
Ser 1 5 219PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Gln Gln Phe Tyr Ser Thr Pro Pro Thr 1 5
2215DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 22agctatgcca tgagc 152351DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 23gctattagtg gtagtggtgg tagcacatac tacgcagact
ccgtgaaggg c 512436DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 24agctatggtt cagttaaaat
aagctgcttt gactac 362551DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 25aagtccagcc
agagtgttct ttacaactcc aacaataaga actacttagc t 512621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 26tgggcatcaa cccgggaatc c 212727DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 27cagcaatttt atagtactcc tccgact 27
* * * * *