U.S. patent application number 10/568729 was filed with the patent office on 2007-01-04 for epitope protection assay and method for detecting protein conformations.
This patent application is currently assigned to Amorfix Life Sciences Ltd. Invention is credited to Neil Cashman, Marty Lehto.
Application Number | 20070003977 10/568729 |
Document ID | / |
Family ID | 34222433 |
Filed Date | 2007-01-04 |
United States Patent
Application |
20070003977 |
Kind Code |
A1 |
Cashman; Neil ; et
al. |
January 4, 2007 |
Epitope protection assay and method for detecting protein
conformations
Abstract
The invention relates to an epitope protection assay for use in
diagnosis, prognosis and therapeutic intervention in diseases, for
example, involving polypeptide aggregation, such as prion
infections. The methods of the invention first block accessible
polypeptide target epitope with a blocking agent. After
denaturation of the polypeptide, a detecting agent is used to
detect protein with target epitope that was inaccessible during
contact with the blocking agent.
Inventors: |
Cashman; Neil; (Vancouver,
CA) ; Lehto; Marty; (Burlington, CA) |
Correspondence
Address: |
SYNNESTVEDT & LECHNER, LLP
2600 ARAMARK TOWER
1101 MARKET STREET
PHILADELPHIA
PA
191072950
US
|
Assignee: |
Amorfix Life Sciences Ltd
3080 Yonge Street, Suite 6020
Toronto
CA
M4 N3N1
|
Family ID: |
34222433 |
Appl. No.: |
10/568729 |
Filed: |
August 20, 2004 |
PCT Filed: |
August 20, 2004 |
PCT NO: |
PCT/CA04/01503 |
371 Date: |
July 13, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60496381 |
Aug 20, 2003 |
|
|
|
60497362 |
Aug 21, 2003 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
435/7.23 |
Current CPC
Class: |
A61P 25/00 20180101;
C07K 2317/626 20130101; G01N 33/6896 20130101; G01N 2333/90283
20130101; C07K 2317/55 20130101; C07K 2317/54 20130101; G01N
2800/28 20130101; G01N 33/68 20130101; G01N 33/6845 20130101; C07K
2317/34 20130101; C07K 2317/624 20130101; C07K 16/18 20130101; G01N
33/58 20130101; A61K 38/00 20130101; C07K 2317/31 20130101; G01N
33/581 20130101; C07K 16/40 20130101; G01N 2800/2828 20130101; C07K
2317/622 20130101 |
Class at
Publication: |
435/007.1 ;
435/007.23 |
International
Class: |
G01N 33/53 20060101
G01N033/53; G01N 33/574 20060101 G01N033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 20, 2003 |
CA |
2 437 675 |
Aug 21, 2003 |
CA |
2 437 999 |
Claims
1. A method of detecting whether a candidate polypeptide including
a target epitope is in i) a wildtype conformation or ii) a
non-wildtype conformation, comprising: contacting the polypeptide
with a blocking agent that selectively blocks accessible target
epitope, wherein in the wildtype conformation, the target epitope
is accessible and reacts with the blocking agent, and wherein in
the non-wildtype conformation, the target epitope is inaccessible
and the target epitope cannot react with the blocking agent;
removing unreacted blocking agent from contact with the
polypeptide; modifying the candidate polypeptide to convert any
inaccessible target epitope to accessible target epitope; and
contacting the polypeptide with a detection agent that binds
selectively to the target epitope that was converted from
inaccessible target epitope to accessible target epitope, wherein
binding between detection agent and converted target epitope
indicates that the candidate polypeptide was in a non-wildtype
conformation and wherein lack of binding between the detection
agent and the target epitope indicates that the polypeptide was in
a wildtype conformation.
2. The method of claim 1, wherein the candidate polypeptide
comprises prion protein, the wild type conformation comprises the
conformation of wild type prion protein and the non-wildtype
conformation comprises the conformation of PrPSc.
3. The method of claim 1, wherein the candidate polypeptide
comprises beta-amyloid polypeptide, tau protein or APP protein.
4. The method of claim 1, wherein the candidate polypeptide
comprises SOD1.
5. The method of claim 1, wherein the candidate polypeptide
comprises alpha-synuclein.
6. The method of claim 1 wherein the candidate polypeptide
comprises huntingtin protein.
7. The method of claim 1, wherein the candidate polypeptide
comprises p53.
8. The method of claim 1, wherein the candidate polypeptide
comprises islet amyloid polypeptide or resistin.
9. The method of claim 1, wherein the blocking agent is selected
from the group consisting of peroxynitrite, hydrogen peroxide,
methylene compounds, succinic anhydride, epoxides, diethyl
pyrocarbonate, 4-hydroxynonenal (4HNE) and diazirine.
10. The method of claim 1, wherein the polypeptide is modified by
denaturing the polypeptide.
11. The method of claim 1, wherein the polypeptide is denatured by
heat and/or detergent and/or chaotropic agents.
12. The method of claim 1, wherein the polypeptide is modified by
treatment with a disaggregation agent to disaggregate the
polypeptide from the aggregated polypeptides.
13. The method of claim 12, wherein the disaggregation agent is
selected from at least one of the group consisting of chaotropic
agents, detergent and heat.
14. The method of claim 13, wherein the detergent comprises
SDS.
15. The method of claim 1, wherein the detection agent comprises an
aptamer or an antibody.
16. The method of claim 15, wherein the aptamer or antibody is
directed against a prion polypeptide epitope.
17. The method of claim 16, wherein the antibody comprises 6H4 or
3F4,
18. The method of claim 15, wherein the aptamer or antibody is
directed against an amyloid beta epitope.
19. The method of claim 16, wherein the antibody comprises 6E10 or
4G8.
20. The method of claim 1, wherein the non-wildtype conformation is
indicative of a disease caused by protein aggregation.
21. The method of claim 20, wherein the disease comprises prion
disease.
22. The method of claim 20, wherein the disease comprises BSE or
CJD.
23. The method of claim 20, wherein the disease comprises
Alzheimer's disease.
24. The method of claim 20, wherein the disease comprises
Parkinson's disease or Lewy body disease.
25. The method of claim 20, wherein the disease comprises
Huntington's disease.
26. The method of claim 20, wherein the disease comprises
amyotrophic lateral sclerosis.
27. The method of claim 20, wherein the disease comprises
cancer.
28. The method of claim 20, wherein the disease comprises
diabetes.
28. The method of claim 1, wherein prior to contacting the blocking
agent with the candidate polypeptide, the candidate polypeptide is
in a sample that is pretreated by one or more of the following
methods: adsorption, precipitation, or centrifugation.
29. The method of claim 1, wherein prior to contacting the blocking
agent with the candidate polypeptide, the target epitope is
mapped.
30. The method of claim 1, wherein the polypeptide is in a
postmortem or antemortem sample selected from the group consisting
of CSF, serum, blood, urine, biopsy sample and brain tissue.
31. A kit for detecting whether a candidate polypeptide including a
target epitope is in i) a wildtype conformation or ii) a
non-wildtype conformation, comprising a detecting agent that
recognizes the target epitope and instructions for at least one of
i) mapping a target epitope, ii) contacting a candidate polypeptide
with a blocking agent, and iii) contacting a candidate polypeptide
with a detecting agent.
32. The kit of claim 31 wherein the detecting agent comprises an
aptamer or an antibody.
33. The kit of claim 32 wherein the antibody comprises 6H4, 3F4,
6E10 or 4G8, optionally immobilized to a solid support.
34. The kit of claim 31, further comprising buffers and reagents
for ELISA, including sandwhich ELISA, fluorescent ELISA.
35. The kit of claim 31 further comprising a blocking agent.
36. The kit of claim 31 further comprising a denaturing agent
selected from at least one of the group of detergents and
chaotropic agents.
37. The kit of claim 31, further comprising a polypeptide
standard.
38. The kit of claim 34, wherein the polypeptide standard comprises
a recombinant disease protein or a recombinant protein that mimics
a disease protein.
39. A method of detecting whether a candidate polypeptide that has
been contacted with a blocking agent is i) a wildtype conformation
or ii) a non-wildtype conformation, wherein the candidate
polypeptide comprises at least one target epitope and, following
contact with the blocking agent and removal of the blocking agent,
the candidate polypeptide has been modified to convert any
inaccessible target epitope to accessible target epitope, the
method comprising: contacting the polypeptide with a detection
agent that binds selectively to the target epitope that was
converted from inaccessible target epitope to accessible target
epitope, wherein binding between detection agent and converted
target epitope indicates that the candidate polypeptide was in a
non-wildtype conformation and wherein lack of binding between the
detection agent and the target epitope indicates that the
polypeptide was in a wild type conformation.
40. The method of claim 1, wherein the target epitope is within the
superoxide dismutase I polypeptide and the target epitope comprises
all or part of the following amino acid sequences: Gln Lys Glu Ser
Asn Gly (SEQ ID NO:4); Glu Asp Asn Thr Ala Gly Cys Thr Ser Ala (SEQ
ID NO:5); Pro Lys Asp Glu Glu Arg His Val (SEQ ID NO:6); Ala Asp
Lys Asp Gly (SEQ ID NO:7); Gly Lys Gly Gly Asn Glu Gln Ser Thr Lys
(SEQ ID NO:8); Asp Leu Gly Lys Gly Gly Asn Glu Glu Ser Thr Lys Thr
Gly Asn Ala Gly Ser (SEQ ID NO:9); or Asn Pro Leu Ser Arg Lys His
Gly Gly Pro Lys Asp Glu Glu (SEQ ID NO:10).
41. The method of claim 1, wherein binding between the detection
agent and the converted target epitope is detected using
dissociation enhanced lanthanide fluoroimmunoassay and
time-resolved fluorescence.
42. An isolated polypeptide consisting of the amino acid sequence
Asp Leu Gly Lys Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly
Ser (SEQ ID NO:9).
43. An isolated polypeptide consisting of the amino acid sequence
Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu (SEQ ID
NO:10).
44. A method of making an antibody specific for the isolated
polypeptide according to claim 42.
45. A method of making an antibody specific for the isolated
polypeptide according to claim 43.
46. An antibody specific for an epitope comprising an amino acid
sequence selected from the group consisting of: Asp Leu Gly Lys Gly
Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser (SEQ ID NO:9);
and Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu (SEQ ID
NO:10).
47. The method of claim 1, wherein the target epitope is
inaccessible because the candidate polypeptide is aggregated.
48. The method of claim 1, wherein the target epitope is
inaccessible because the candidate polypeptide is in an different
conformation as compared to the wildtype conformation.
49. A method of detecting whether a candidate polypeptide including
a target epitope is in i) a wildtype conformation or ii) a
non-wildtype conformation, comprising: contacting the polypeptide
with a blocking agent that selectively blocks accessible target
epitope, wherein in the non-wildtype conformation, the target
epitope is accessible and reacts with the blocking agent, and
wherein in the wildtype conformation, the target epitope is
inaccessible and the target epitope cannot react with the blocking
agent; removing unreacted blocking agent from contact with the
polypeptide; modifying the candidate polypeptide to convert any
inaccessible target epitope to accessible target epitope; and
contacting the polypeptide with a detection agent that binds
selectively to the target epitope that was converted from
inaccessible target epitope to accessible target epitope, wherein
binding between detection agent and converted target epitope
indicates that the candidate polypeptide was in a wildtype
conformation and wherein lack of binding between the detection
agent and the target epitope indicates that the polypeptide was in
a non-wildtype conformation.
Description
FIELD OF THE INVENTION
[0001] The invention relates to an epitope protection assay for use
in diagnosis, prognosis and therapeutic intervention in diseases,
for example, diseases involving polypeptide aggregation such as
prion infections.
BACKGROUND OF THE INVENTION
Protein Misfolding and Aggregation
[0002] Proteins can fold into complex and close-packed structures.
Folding is not only crucial for biological activity but failure of
proteins to fold properly or remain folded can give rise to disease
(Dobson C M, Methods (2004) 34:4-14). Misfolding can in some cases
cause protein aggregation which can further give rise to discrete
deposits extracellularly (e.g., plaques) or intracellularly (e.g.,
inclusions in the cytosol or nucleus).
[0003] Neurogenerative diseases such as Alzheimer's disease (AD),
Parkinson's disease (PD), Huntington's disease (HD), amyotrophic
lateral sclerosis (ALS) and prion diseases are characterized by
neural deposits of misfolded aggregated protein. Type II diabetes
and cancer have also been linked to protein misfolding and it is
likely that there are yet to be identified diseases that result
from errors in protein folding and that in some cases lead to
consequences such as aggregation. The nature of the misfolding and
any aggregation in such diseases is typically not well
characterized.
Prion Diseases
[0004] Prion diseases have become a major health concern since the
outbreak of BSE or "Mad Cow Disease" (reviewed above, 40, 41). BSE
was first discovered in the United Kingdom but has now spread to
many other countries in Europe and Japan. In the UK alone there has
been close to 180,000 cases of BSE, which resulted in the
destruction of cattle and possible infection of an estimated 3-5
million head. The total cost estimated to the UK was in excess of
$2.5 billion. BSE is believed to be transmitted among cattle
through feed that contains prions rendered from infected cattle,
and it is thought to be transmitted to humans through eating beef
or other cattle products from infected animals.
Emerging Prion Diseases
[0005] The prion diseases are a group of rapidly progressive and
untreatable neurodegenerative syndromes, neuropathologically
characterized by spongiform change, neuronal cell loss, gliosis,
and brain accumulation of abnormal amyloid polypeptide. Human prion
diseases include classical Creutzfeldt-Jakob disease (CJD), which
has sporadic, iatrogenic, and familial forms. Since 1996, a "new
variant" of CJD (vCJD) has been identified in the United Kingdom,
France, the Republic of Ireland, Hong Kong, Italy, the United
States, and Canada (40,41). Variant CJD is capable of killing
individuals as young as age 14 with unknown incubation period.
There is little doubt that vCJD is a human form of bovine
spongiform encephalopathy (BSE)(42). The primary epidemic from
consumption of contaminated cattle tissue has affected over 130
individuals as of this filing.
[0006] The spectre of vCJD "secondary epidemics" through blood,
blood products, surgery, dentistry, vaccines, and cosmetics is of
great concern(40,41). Detection of blood prion infectivity in
experimental BSE/vCJD infections of mice and sheep (40) suggests a
special risk exists for the transmission of vCJD through blood and
blood products. The recent reports of vCJD in two recipients of a
donor who developed the disease is also troubling (52, 53). Canada
and the United States have recently expanded vCJD blood donor
deferrals to all countries in Western Europe.
[0007] Although sheep scrapie has been known for centuries, the
most important animal prion disease at present is BSE. More than
173,000 cattle, primarily from Britain, have developed symptomatic
BSE, and as many as 3 million have entered the food supply
undetected. BSE is now being increasingly reported in cattle which
were "born after the ban" in 1996 of food supplementation with meat
and bone meal, suggesting that alternate routes may exist to keep
the epidemic from being readily extinguished. Another troubling
issue is the possible transmission of BSE to sheep, which may
expose additional human populations to the BSE/vCJD prion strain.
Recent reports show that prions can replicate in certain muscle
groups of sheep, experimental animals and humans (54-57),
indicating a potential risk in tissues previously considered safe
for human consumption.
[0008] Chronic wasting disease (CWD) of captive and wild cervids
(deer and elk) represents another newly emergent animal prion
disease in North America, whose impact on human health is yet
unknown. It is apparent that newly-recognized prion diseases pose a
threat to the safety of foods, blood products, and medical-surgical
treatments.
Prions: Atypical Pathogens
[0009] Newly emergent prion diseases, and the polypeptide-only
nature of prions, have created serious medical, veterinary, and
economic challenges worldwide. To date, the only commercialised
tests for prion infection have been based on post-mortem brain
samples. No biochemical test exists to detect prions in the blood
of infected animals, despite detection by experimental transmission
studies. The development of sensitive and specific diagnostic tests
for prion infection is a challenging task, in part due to the
unusual nature of the prion infectious agent. The infectious agents
that transmit the prion diseases differ from other pathogens in
that no nucleic acid component has been detected in infectious
materials (41). According to the prion theory developed by Nobel
Laureate Dr. Stanley Prusiner, infectivity resides in PrP.sup.Sc, a
misfolded conformational isoform of the near-ubiquitous normal
cellular prion polypeptide PrP.sup.C. PrP.sup.Sc is indeed the most
prominent (or perhaps sole) macromolecule in preparations of prion
infectivity, and minimally appears to be a reliable surrogate for
prion infection. PrP.sup.Sc is partially resistant to protease
digestion, poorly soluble, and exists in an aggregated state, in
contrast to the protease sensitive, soluble, monomeric isoform
PrP.sup.C (29, 31, 4346).
[0010] PrP.sup.Sc is derived from its normal cellular isoform
(PrP.sup.C), which is rich in .alpha.-helical structure, by a
posftranslational process involving a conformational transition.
While the primary structure of PrP.sup.C is identical to that of
PrPSc, secondary and tertiary structural changes are responsible
for the distinct physicochemical properties of the two
isoforms.
[0011] One of the difficulties in assessing the safety of food or
blood products from potentially infected humans with prions is the
lack of an accurate diagnostic test for blood or other accessible
biosamples. Currently, there are no diagnostic tests that can be
applied for screening live animals, humans, blood or blood products
at an early stage. This also provides a further problem in organ
transplantation, adding unknown risk to organ recipients.
Therefore, as a preventative measure, countries such as the UK no
longer source plasma from its inhabitants. The risk of spreading
prion diseases has affected other countries as well. For example,
the United States and Canada do not accept blood donations from
individuals who have resided in the UK or France for more than 3-6
months.
[0012] Currently, the diagnosis of vCJD can only be confirmed
following pathological examination of the brain at autopsy or
biopsy. Some complimentary strategies in early CJD detection
include electroencephalograms (EEG), magnetic resonance imaging
(MRI) scans, and cerebrospinal fluid (CSF) tests, which may be
useful "surrogate" or "proxy" markers. The absence of a "direct
test" for prion infection stands in stark contrast to conventional
infectious agents, such as viruses and bacteria.
[0013] Some tests that are in the process of being commercialized
are based on surrogate markers of infection which are "once
removed" from actual infectious prions.
[0014] PrP protease resistance is the basis of most commercially
available diagnostic tests for prion disease. In the current
methodologies, a sample of brain is removed and digested with
proteases that can eliminate PrP.sup.C, but leave a
protease-resistant core of PrP.sup.Sc. The protease-resistant
fragment of PrP.sup.Sc is then detected by immunoblotting (as in
the Prionics test) or by capture ELISA (as in the BioRad and Enfer
tests, and in a new test from Prionics). However, digestion with
proteases is cumbersome and variable, leading to false negatives
and positives. Moreover, there are some prion strains which are
reported to contain PrP.sup.Sc which is infectious and aggregated,
but which is not protease resistant. Protease-sensitive PrP.sup.Sc
also predominates early in infection and in cross-species
transmission of disease (31).
[0015] Detection of protease-resistant PrP fragments is also the
basis of a urine diagnostic test (47) which is being commercially
developed by Prionics. However, detection of protease-resistant PrP
in urine is subject to the same limitations as the post-mortem
brain test, and has the additional disadvantage of requiring
precipitation from large volumes of urine, and poor sensitivity
(for example, only detecting PrP.sup.Sc in late stages of the
disease, not pre-symptomatically).
Other Neurodegenerative Diseases
[0016] Neurodegenerative diseases, such as Alzheimer's disease
(AD), Huntington's disease, amyotrophic lateral sclerosis (ALS) and
Parkinson's disease/Lewy body dementia (PD, LBD) also pose major
challenges to our aging population and health care system (reviewed
in 1). An estimated 364,000 Canadians over 65 are currently
diagnosed with AD or a related dementia (http://www.alzheimer.ca/).
With increased life expectancy, the incidence of neurodegenerative
disease is expected to grow. By 2025, AD will affect as many as a
million Canadians, and by 2050, this number will double.
[0017] Sporadic AD, ALS, and PD/LBD are all associated with neural
accumulation of pathological multimers of misfolded polypeptides
(these could potentially be fibrils, protofilaments, and amorphous
aggregates), including the amyloid-beta (Abeta) fragment of the
amyloid precursor protein (APP) in AD; superoxide dismutase-1
(SOD1) in ALS, and alpha-synuclein in PD and LBD (1). Additionally
familial amyloidotic polyneuropathy (FAP) results from the
aggregation of transthyretin to form amyloid deposits. As with
prion diseases, mutations in genes encoding these polypeptides are
associated with autosomal dominant familial forms of AD, ALS, and
PD.
Alzheimer's Disease
[0018] AD is a common dementing (disordered memory and cognition)
neurodegenerative disease associated with brain accumulation of
extracellular plaques composed predominantly of the Abeta (1-40),
Abeta (1-42) and Abeta (1-43) peptides, all of which are
proteolytic products of APP (reviewed in 4). In addition,
neurofibrillary tangles, composed principally of abnormally
phosphorylated tau protein (a neuronal microtubule-associated
protein), accumulate intracellularly in dying neurons (4). Familial
forms of AD can be caused by mutations in the APP gene, or in the
presenilin 1 or 2 genes (www.websiteformutations.com), the protein
products of which are implicated in the processing of APP to Abeta.
Apolipoprotein E allelic variants also influence the age at onset
of both sporadic and familial forms of AD (reviewed in 5). Abeta
has been detected in the blood and CSF of AD patients and in normal
controls (6). Abeta is also present in vascular and plaque amyloid
filaments in trisomy 21 (Down's syndrome), hereditary cerebral
hemorrhage with amyloidosis (HCHWA)-Dutch type, and normal brain
aging (Mori, H et al. JBC (1992) 267: 17082-86). Tau and
phosphorylated tau have been detected in the cerebral spinal fluid
(CSF) of AD patients and patients with other neurological diseases
(7; reviewed in 8).
Amyotrophic Lateral Sclerosis
[0019] ALS is a fatal neuromuscular disease, with an incidence of 1
in 1000 adults, presenting as progressive weakness, muscle atrophy,
and spasticity, which is due to degeneration of .about.500,000
"lower motor neurons" in the spinal cord and brainstem, and
innumerable "upper motor neurons" in the brain cortex. An important
clue to the etiology of ALS came with the finding that about 20% of
familial ALS (fALS) cases are due to mutations in superoxide
dismutase-1 (SOD1) (10,11), a free radical defense enzyme. Over 100
fALS SOD1 missense, nonsense, and intronic splice-disrupting
mutations have been catalogued to date (12; www.alsod.org).
Transgenic mice expressing mutant human SOD1 (mtHuSOD1) develop a
motor neuron syndrome with clinical and pathological similarities
to human ALS (13, 14), whereas mice expressing wild-type human SOD1
(wtHuSOD1) do not develop disease (13). SOD1-containing cytoplasmic
inclusions can be detected in many diseased motor neurons from
familial and sporadic ALS patients (15), and in most transgenic
mouse (16, 17) and tissue culture (18) models of the disease.
Parkinson's and Lewy Body Disease
[0020] PD is a neurodegenerative movement disorder second only to
AD in prevalence (.about.350 per 100,000 population; 1). It is
clinically characterized by rigidity, slowness of movement, and
tremor (reviewed in 21). Most cases of Parkinson's disease are
sporadic, but both sporadic and familial forms of the disease are
characterized by intracellular Lewy bodies in dying neurons of the
substantia nigra, a population of midbrain neurons (.about.60,000)
that are selectively decimated in PD. Lewy bodies are predominantly
composed of alpha-synuclein (22). Mutations in the gene encoding
alpha-synuclein have been found in patients with familial
Parkinson's disease (reviewed in 23; www.parkinsonsmutation.com).
Another gene associated with autosomal recessive PD is parkin,
which is involved in alpha-synuclein degradation (22, 23). Diffuse
cortical Lewy bodies composed of alpha-synuclein are observed in
Lewy body disease (LBD), a dementing syndrome associated with
parkinsonian tone changes, hallucinations, and rapid symptom
fluctuation (24). LBD may be the second most common form of
neurodegenerative dementia after AD, accounting for 20 to 30
percent of cases among persons over the age of 60 years (1,
24).
Huntington's Disease and Related Diseases
[0021] HD is a progressive neurodegenerative disorder characterized
by expansion of polyglutamine encoding CAG repeats in the
N-terminus of the huntingtin protein (reviewed in 48).
Polyglutamine stretches of .gtoreq.36 cause disease and longer
repeats cause earlier onset (49, 50).
[0022] Other polyglutamine diseases such as dentate-rubral and
pallido-luysian atrophy (DRPLA) and some forms of sino-cerebellar
ataxia (SCA) also have intracellular inclusions that roughly
correlate to regions of neuronal death. Interruptions in the
expanded polyglutamine repeat in the SCA-1 gene product result in
the absence of disease (51),
[0023] Neurodegenerative diseases, such as Alzheimer's disease
(AD), amyotrophic lateral sclerosis (ALS) and Parkinson's
disease/Lewy body disease (PD, LBD) pose major challenges to our
aging population and health care system. No specific biochemical
test exists for neurodegenerative diseases as a group (1,2). Since
neurodegenerative diseases are regarded as "diagnoses of
exclusion," very broad investigation is required to achieve
"clinically probable" diagnosis for these progressive, incurable,
and usually fatal conditions. Expensive surrogate testing, such as
neuroimaging, is utilized to increase diagnostic probability (2).
The availability of specific, sensitive, and inexpensive
biochemical tests for this devastating group of diseases could
potentially conserve financial resources for over-burdened health
care systems. Moreover, secure diagnosis of these diseases at an
earlier symptomatic stage increases the window for enhanced
treatment efficacy at a time at which the disease pathophysiology
is generally more responsive to treatment (3).
[0024] Effective, efficient and inexpensive diagnostic and
screening strategies for antemortem diagnosis of human
neurodegenerative diseases are urgently needed, given the aging
population and continued financial pressure on the health care
system.
Diabetes
[0025] Protein aggregation is also observed in patients with type
II diabetes. Increased expression of the adipocyte-derived peptide,
resistin, has been observed in diabetes type II patients (Youn B S
et al. J Clin Endocrinol Metab. (2004); 89:150-6) and studies
suggest that elevated resistin levels may play a role in obesity
and insulin resistance. Additionally, islet amyloid polypeptide
(also known as amylin) deposition is pathogenically associated with
type 2 diabetes. These deposits contain islet amyloid polypeptide,
a unique amyloidogenic peptide and are associated with beta cell
death. Recent studies suggest that the species responsible for
islet amyloid-induced beta-cell death are formed early in islet
amyloid formation, when islet amyloid polypeptide accumulation
begins (Hull R L et al. J Clin Endocrinol Metab. (2004)
89:3629-43). A diagnostic test that can identify pathogenic islet
amyloid polypeptide would be very useful for detecting type 2
diabetes in its early stages, when dietary and therapeutic
interventions are most effective.
Cancer
[0026] Many forms of cancer are also considered to be protein
conformation diseases (Ishimaru D. et al. Biochemistry (2003)
42:9022-7). A subset of neuroblastomas, carcinomas and myelomas
show an abnormal accumulation of tumor suppressor p53 protein
aggregates (Butler J S et al. Biochemistry (2003) 42: 2396-403;
Ishimaru D. et al. Biochemistry (2003) 42:9022-7). This
accumulation could contribute to the loss of p53 function in some
cancerous cells (Ishimaru D. et al. Biochemistry (2003) 42:9022-7).
Assays able to detect accumulated p53 could provide a
diagnostically useful detection system and could enhance
therapeutic intervention by individualizing therapeutic
intervention.
SUMMARY OF THE INVENTION
[0027] The inventors have recently developed the epitope protection
assay (EPA), a novel method that yields sensitive and specific
antemortem detection of disease proteins in blood and other
accessible tissues and fluids. The invention shows the role of
aggregation in diseases, such as prion disease, and provides an
assay that overcomes problems in the prior art. In prion diseases,
the normal cellular monomeric prion polypeptide PrP.sup.C undergoes
refolding to an abnormal, aggregated isoform, generically
designated PrP.sup.Sc. Diseases such as AD, PD, LBD, ALS and HD are
also characterized by misfolded and/or aggregated conformations of
cellular proteins. This property is exploited by the methods of the
invention to provide sensitive and specific diagnostic tests for
these and other diseases.
[0028] According to the invention, the methods are useful where a
target epitope is accessible in either one of a non-wildtype
protein (i.e. disease protein) or a wild type protein and
inaccessible in the other. Inaccessibility is often due to
aggregation making the target epitope inaccessible.
[0029] The invention includes a method of detecting whether a
candidate polypeptide including a target epitope is a disease
(disorder) polypeptide or a wild type polypeptide, comprising:
[0030] contacting the candidate polypeptide with a blocking agent;
and [0031] determining whether the target epitope is inacessible or
accessible to chemical modification by the blocking agent.
[0032] The accessibility or inaccessibility of the target epitope
is indicative of whether the candidate polypeptide is a disease
(disorder) polypeptide or a wild type polypeptide because in one of
the disease (disorder) protein and the wild type protein, the
target epitope is accessible. In the other polypeptide the target
epitope is inaccessible.
[0033] In one embodiment, the invention provides a method of
detecting prion diseases, for example, determining whether a
candidate polypeptide including a target epitope is in a wildtype
conformation or in a non-wildtype conformation in which it is
aggregated, comprising: [0034] reacting a sample of polypeptide
(the sample typically contains PrP.sup.Sc and/or PrP.sup.C, and in
many cases an abundance of one or the other) with a chemical
modifying agent, typically an agent which chemically reacts with
proteins such as peroxynitrite, which modifies accessible epitopes
(target epitopes) so that they cannot bind to a detection agent;
[0035] disaggregating and/or denaturing the polypeptide in the
sample; and [0036] probing with detection agents, such as an
antibody against a target epitope, to determine whether the
polypeptide (such as prior to disaggregation and/or denaturing)
included inaccessible target epitopes.
[0037] PrP.sup.C is rendered "invisible" in the assay, because
epitopes on the monomeric molecules are blocked to antibody
recognition by the chemical modifying agent, whereas molecules of
PrP.sup.Sc are "protected" from chemical modification by virtue of
being sequestered within aggregates or otherwise unavailable for
reacting. Alternatively, epitopes on the multimeric molecules are
blocked to antibody recognition by the chemical modifying agent,
whereas molecules of PrP.sup.C are "protected" from chemical
modification by virtue of a difference in accessible epitopes.
[0038] In another embodiment, the Alzheimer's disease detection
method comprises: [0039] reacting a sample of polypeptide (the
sample typically contains all or part of diseased amyloid precursor
polypeptide or A beta or tau and/or the corresponding wild type
polypeptide, and in many cases an abundance of one or the other)
with a chemical modifying agent, typically an agent which
chemically reacts with proteins such as peroxynitrite, which
modifies exposed epitopes so that they cannot bind to a detection
agent; [0040] disaggregating and/or denaturing the polypeptide in
the sample; and [0041] probing with detection agents, such as an
antibody against a target epitope to determine whether the
polypeptide prior to disaggregation and/or denaturing, included
inaccessible target epitopes.
[0042] In further embodiments, the invention provides disease
detection methods for other diseases characterized by
differentially accessible target epitopes in disease and wildtype
conformations, for example, resulting from misfolded and/or
aggregated proteins such as Parkinson's disease (PD), Lewy Body
disease (LBD), Huntington's disease (HD), amyotrophic lateral
sclerosis (ALS), diabetes, and cancer. These methods similarly
include steps such as reacting a sample of polypeptide (eg. a
disease polypeptide described herein) with a chemical modifying
agent, which modifies exposed epitopes so that they cannot bind to
a detection agent; then disaggregating and/or denaturing the
polypeptide in the sample; and probing with detection agents, such
as an antibody against a target epitope to determine whether the
polypeptide prior to disaggregation and/or denaturing, included
inaccessible target epitopes. These steps are similarly adapted for
other purposes, such as screening blood and blood products, and
other uses described herein.
[0043] The method of the invention has many advantages over
existing technology. As noted above, the invention is optionally
referred to as "EPA", which in the case of prion protein disease
detection is a simple, efficient method for detecting aggregated
disease proteins such as PrP.sup.Sc, the pathogenic molecule which
is thought to constitute the infectious particle in prion diseases
and polypeptide, associated with AD.
[0044] The invention is useful in high-throughput robotic-capable
platforms. For example, EPA is not dependent on PrP protease
resistance, the basis of most commercially available diagnostic
tests for prion disease. Epitope protection technology does not
require a protease digestion step, which makes it more sensitive to
early infection. Certainly, the absence of a protease digestion
step permits EPA to be more amenable to high-throughput robotic
platforms.
[0045] In addition, the methods of the invention can be used to
detect any protein that exists in two or more conformations, where
one or more target epitopes are concealed in at least one
conformation.
[0046] Accordingly, the invention relates to a detection method
comprising: [0047] reacting a sample of polypeptide with a chemical
modifying agent, typically an agent which chemically reacts with
proteins, which is defined to modify exposed epitopes so that they
cannot bind to detection agents; [0048] disaggregating and/or
denaturing the polypeptide in the sample; and [0049] probing with
detection agents, such as antibodies against a target epitope to
determine whether the polypeptide prior to disaggregation and/or
denaturing, included target epitopes inaccessible to the chemical
modifying agent.
[0050] The result indicates whether the polypeptide includes
inaccessible epitopes, which is indicative of the type of
polypeptide that is present (i.e. wild type protein or non-wild
type protein).
[0051] In one embodiment, the invention includes a method of
detecting whether a candidate polypeptide including a target
epitope is in a wildtype conformation or a non-wildtype
conformation (in one embodiment, in the non-wildtype conformation,
the candidate polypeptide aggregates with aggregated polypeptide),
comprising: [0052] contacting the polypeptide with a blocking agent
that selectively blocks accessible target epitopes, wherein in one
of the non-wildtype conformation or the wildtype conformation, the
target epitope is accessible and reacts with the blocking agent,
and wherein in the other conformation, the target epitope is
inaccessible and does not react with the blocking agent. Unreacted
blocking agent is removed from contact with the polypeptide, for
example, by allowing time for blocking agent to be consumed or
degraded or by actively removing it by physical or chemical
processes as described below; [0053] modifying the candidate
polypeptide to convert any inaccessible target epitope to
accessible target epitope; and [0054] contacting the polypeptide
with a detection agent that binds selectively to target epitope
that was converted from inaccessible target epitope to accessible
target epitope, wherein binding between detection agent and
converted target epitope indicates that prior to conversion the
candidate polypeptide was in a conformation in which the target
epitope was inaccessible and wherein lack of binding between the
detection agent and the target epitope indicates that the
polypeptide was in a conformation in which the target epitope was
accessible, thereby indicating whether the polypeptide was in a
wildtype conformation or a non-wildtype conformation.
[0055] The invention also includes a method of detecting whether a
candidate polypeptide including a target epitope is in a wildtype
conformation or a non-wildtype conformation, comprising: [0056]
contacting the polypeptide with a blocking agent that selectively
blocks accessible target epitope, wherein in the wildtype
conformation, the target epitope is accessible and reacts with the
blocking agent, and wherein in the non-wildtype conformation, the
target epitope is inaccessible and does not react with the blocking
agent. Unreacted blocking agent is removed from contact with the
polypeptide, for example, by allowing time for blocking agent to be
consumed or degraded or by actively removing it by physical or
chemical processes as described below; [0057] modifying the
candidate polypeptide to convert any inaccessible target epitope to
accessible target epitope; and [0058] contacting the polypeptide
with a detection agent that binds selectively to the target epitope
that was converted from inaccessible target epitope to accessible
target epitope, wherein binding between detection agent and
converted target epitope indicates that the candidate polypeptide
was in a non-wildtype conformation and wherein lack of binding
between the detection agent and the target epitope indicates that
the polypeptide was in a wildtype conformation.
[0059] The invention also includes a method of detecting whether a
candidate polypeptide including a target epitope is in a wildtype
conformation or a non-wildtype conformation, comprising: [0060]
contacting the polypeptide with a blocking agent that selectively
blocks accessible target epitope, wherein in the non-wildtype
conformation, the target epitope is accessible and reacts with the
blocking agent, and wherein in the wildtype conformation, the
target epitope is inaccessible and does not react with the blocking
agent. Unreacted blocking agent is removed from contact with the
polypeptide, for example, by allowing time for blocking agent to be
consumed or degraded or by actively removing it by physical or
chemical processes as described below; [0061] modifying the
candidate polypeptide to convert any inaccessible target epitope to
accessible target epitope; and [0062] contacting the polypeptide
with a detection agent that binds selectively to target epitope
that was converted from inaccessible target epitope to accessible
target epitope, wherein binding between detection agent and
converted target epitope indicates that the candidate polypeptide
was in a wildtype conformation and wherein lack of binding between
the detection agent and the target epitope indicates that the
polypeptide was in a non-wildtype conformation.
[0063] The invention also includes a method of detecting whether a
candidate polypeptide including target epitope which has been
reacted with a blocking agent, is in a wildtype conformation or a
non-wildtype conformation, comprising: [0064] modifying the
candidate polypeptide to convert any inaccessible target epitope to
accessible target epitope; [0065] contacting the polypeptide with a
detection agent that binds selectively to the target epitope that
was converted from inaccessible target epitope to accessible target
epitope, wherein binding between detection agent and converted
target epitope indicates that the candidate polypeptide was in a
non-wildtype conformation and wherein lack of binding between the
detection agent and the target epitope indicates that the
polypeptide was in a wild type conformation.
[0066] In the methods of the invention, the epitope is in many
cases inaccessible in the misfolded or non-wild type conformation
because i) the differential misfolding of the polypeptide compared
to the wild type folded polypeptide prevents or reduces reaction
between the blocking agent and the target epitope, ii) the
polypeptide in the misfolded conformation aggregates with itself or
other polypeptides in the misfolded conformation to prevent or
reduce reaction between the protecting/blocking agent and the
target epitope, and/or iii) post translational modifications of the
polypeptide prevent or reduce reactions between the blocking agent
and the target epitope.
[0067] In one example, the candidate polypeptide comprises prion
protein, the wild type folded conformation comprises the
conformation of wild type folded prion protein and the misfolded
conformation comprises the conformation of PrP.sup.Sc.
Alternatively, the wild type folded protein comprises the
conformation of APP or its cleavage product amyloid beta, and the
misfolded conformation comprises the conformation of Alzheimer's
disease APP or its cleavage product amyloid beta.
[0068] In another example, the candidate polypeptide comprises,
SOD1, alpha-synuclein, islet amyloid polypeptide, resistin or p53
protein. The methods and kits of the invention described in the
application are useful, for example, for application to a
non-wildtype polypeptide having a conformation comprising multiple
copies of a polypeptide aggregated together through interactions of
beta-sheet-rich areas of the polypeptide. In one embodiment the
polypeptide is polypeptide that is aggregated in prion protein
aggregates. In another embodiment, the polypeptide is polypeptide
that is aggregated in amyloid plaques.
[0069] The invention also includes i) polypeptide of the invention
modified by reaction with a blocking agent listed herein and ii)
the polypeptide modified by reaction with a detecting agent. The
invention also includes compositions and kits of the invention
including these modified polypeptide.
[0070] The blocking agent is optionally peroxynitrite, hydrogen
peroxide, diethyl pyrocarbonate (DEPC), 4-hydroxynonenal (4HNE) an
epoxide such as conduritol-B-epoxide and
1,2-epoxy-3-(p-nitrophenoxy)propane, methylene or diazirine and
related compounds. In the methods, the polypeptide is optionally
modified by denaturing the polypeptide, for example with heat,
detergent and/or chaotropic agents. The polypeptide is optionally
modified by treatment with a disaggregation agent to disaggregate
the polypeptide from other polypeptides of the same type, and from
other molecules, wherein the disaggregation agent is optionally
selected from at least one of the group consisting of chaotropic
agent (including guanidine salts, urea or thiourea), detergent and
heat.
[0071] It is readily apparent to a skilled person that the method
steps of the invention recited here involving removing the blocking
agent typically involve physically, chemically or otherwise
removing the blocking agent away from the candidate polypeptide to
prevent further reaction. Removal optionally involves allowing a
sufficient time to pass so that the blocking agent is removed from
the candidate polypeptide by being consumed or degraded (for
example, such that the blocking agent becomes inert or oxidized).
Removal optionally involves adding a compound to react with any
excess blocking agent to inactivate it. Removal also optionally
involves physical filtering of the blocking agent by conventional
filtration techniques or centrifugation to separate the candidate
polypeptide and blocking agent, or physical binding to a substrate
useful for removing the blocking agent, such as by binding of
blocking agent or candidate polypeptide to an immobilized substrate
in a column.
[0072] Removing means preventing further reactions by the blocking
agent by, for example, physically or chemically inactivating the
blocking agent, taking the blocking agent out of contact with the
sample including the candidate polypeptide or allowing a sufficient
amount of time to pass for the blocking agent to be consumed or
degraded.
[0073] The detection agent optionally comprises an antibody
directed against a prion polypeptide epitope, an amyloid beta
epitope, an alpha-synuclein epitope or a SOD1 epitope. The antibody
optionally comprises all or part of the anti-prion antibodies 6H4
and 3F4, and the anti-amyloid beta antibodies 6E10 and 4G8.
[0074] The methods of the invention are preferably used with
mammals, such as humans. In addition, the methods of the invention
are preferably used with mammals, such as livestock. In addition
the methods of the invention are used with food items, cosmetic
items, dental and surgical instruments vacuums and pharmaceutical
products.
[0075] The invention also includes a method of testing a sample
from an animal, using methods of the invention described herein, to
determine if the animal has a disease characterized by the presence
of candidate polypeptide in a non-wildtype conformation in the
sample, wherein the candidate polypeptide includes a target
epitope. Such diseases are described herein. In one embodiment, the
method comprises; determining whether the candidate polypeptide is
in i) a wildtype conformation or ii) a non-wildtype conformation,
by a method comprising the steps of: [0076] contacting the sample
with a blocking agent that selectively blocks accessible target
epitope in the candidate polypeptide, wherein in the wildtype
conformation, the target epitope is accessible and reacts with the
blocking agent, and wherein in the non-wildtype conformation, the
target epitope is inaccessible because the candidate polypeptide is
aggregated with the aggregated polypeptide and the target epitope
cannot react with the blocking agent; [0077] contacting the sample
with a conversion agent to modify the candidate polypeptide to
convert any inaccessible target epitope in the sample to accessible
target epitope; [0078] contacting the polypeptide with a detection
agent that binds selectively to target epitope that was converted
from inaccessible target epitope to accessible target epitope,
wherein binding between detection agent and converted target
epitope indicates that the candidate polypeptide was in a
non-wildtype conformation and the animal has a disease and wherein
lack of binding between the detection agent and the target epitope
indicates that the polypeptide was in a wild type conformation.
[0079] The invention also includes a method of the invention
described herein for screening, for example, by testing a sample,
such as blood or blood products and other samples, to determine if
the sample comprises a candidate polypeptide in a non-wildtype
conformation wherein the candidate polypeptide includes a target
epitope. In one embodiment, the method comprises; determining
whether the candidate polypeptide is in i) a wildtype conformation
or ii) a non-wildtype conformation, by a method comprising the
steps of: [0080] contacting the sample with a blocking agent that
selectively blocks accessible target epitope in the candidate
polypeptide, wherein in the wildtype conformation, the target
epitope is accessible and reacts with the blocking agent, and
wherein in the non-wildtype conformation, the target epitope is
inaccessible and cannot react with the blocking agent; [0081]
contacting the sample with a conversion agent to modify the
candidate polypeptide to convert any inaccessible target epitope in
the sample to accessible target epitope; contacting the polypeptide
with a detection agent that binds selectively to target epitope
that was converted from inaccessible target epitope to accessible
target epitope, wherein binding between detection agent and
converted target epitope indicates that the candidate polypeptide
was in a non-wildtype conformation and the sample comprises a
candidate polypeptide in a non-wildtype conformation and wherein
lack of binding between the detection agent and the target epitope
indicates that the polypeptide was in a wild type conformation.
[0082] In another embodiment, the invention relates to a method of
detecting whether a candidate polypeptide including a target
epitope is in i) a wildtype conformation or ii) a non-wildtype
conformation (for example, wherein the polypeptide aggregates in
the non-wildtype conformation), comprising: [0083] contacting the
polypeptide with a blocking agent that selectively blocks
accessible target epitope, wherein in the wildtype conformation,
the target epitope is accessible and reacts with the blocking
agent, and wherein in the non-wildtype conformation, the target
epitope is inaccessible (for example, because the candidate
polypeptide is aggregated) and the target epitope cannot react with
the blocking agent; [0084] modifying the candidate polypeptide to
convert inaccessible target epitope to accessible target epitope;
and [0085] contacting the polypeptide with a detection agent that
binds selectively to the target epitope that was converted from
inaccessible target epitope to accessible target epitope, wherein
binding between detection agent and converted target epitope
indicates that the candidate polypeptide was in a non-wildtype
conformation and wherein lack of binding between the detection
agent and the target epitope indicates that the polypeptide was in
a wild type conformation. One also removes unreacted blocking agent
from contact with the polypeptide, for example, by allowing it to
be consumed or degraded or removing it from the reaction by
physical or chemical processes.
[0086] The candidate polypeptide optionally comprises prion
protein, the wild type conformation comprises the conformation of
wild type prion protein and the non-wildtype conformation comprises
the conformation of PrP.sup.Sc. The candidate polypeptide
optionally comprises beta-amyloid polypeptide, tau protein or APP
protein, SOD1, alpha-synuclein, huntingtin protein, p53 or islet
amyloid polypeptide or resistin. The blocking agent is optionally
selected from the group consisting of peroxynitrite, hydrogen
peroxide, methylene compounds, succinic anhydride, epoxides,
diethyl pyrocarbonate, 4-hydroxynonenal (4HNE) and diazirine. The
polypeptide is optionally modified by denaturing the polypeptide.
The polypeptide is also optionally denatured by heat and/or
detergent and/or chaotropic agents. The polypeptide is optionally
modified by treatment with a disaggregation agent to disaggregate
the polypeptide from the aggregated polypeptides. The
disaggregation agent is optionally selected from at least one of
the group consisting of chaotropic agents, detergent and heat. The
detergent optionally comprises SDS. The detection agent optionally
comprises an aptamer or an antibody, for example, directed against
a prion polypeptide epitope. The antibody optionally comprises 6H4
or 3F4. The aptamer or antibody is optionally directed against an
amyloid beta epitope. The antibody optionally comprises 6E10 or
4G8. The non-wildtype conformation is in certain embodiments
indicative of a disease caused by protein aggregation, such as
prion disease (eg. BSE or CJD), Alzheimer's disease, Parkinson's
disease or Lewy body disease, Huntington's disease, amyotrophic
lateral sclerosis, cancer or diabetes. Optionally, prior to
contacting the blocking agent with the candidate polypeptide, the
candidate polypeptide is in a sample that is pretreated by one or
more of the following methods: adsorption, precipitation, or
centrifugation. Optionally, prior to contacting the blocking agent
with the candidate polypeptide, the target epitope is mapped (ie.,
the epitope is identified, for example, as described below under
the "Target Epitopes" section). Optionally, the polypeptide is in a
postmortem or antemortem sample selected from the group of: CSF,
serum, blood, urine, biopsy sample or brain tissue. Another aspect
of the invention relates to a kit for detecting whether a candidate
polypeptide including a target epitope is in i) a wildtype
conformation or ii) a non-wildtype conformation, comprising a
detecting agent that recognizes the target epitope and instructions
for at least one of i) mapping a target epitope, ii) contacting a
candidate polypeptide with a blocking agent, and iii) contacting a
candidate polypeptide with a detecting agent. The kit is useful to
implement method of the invention described herein. The detecting
agent optionally comprises an aptamer or an antibody. The antibody
optionally comprises 6H4, 3F4, 6E10 or 4G8, optionally immobilized
to a solid support. The kit optionally further comprises buffers
and reagents, for example, for ELISA, such as sandwhich ELISA,
fluorescent ELISA. The kit optionally further comprises a blocking
agent. The kit optionally further comprises a denaturing agent
selected from at least one of the group of detergents and
chaotropic agents. The kit optionally further comprises a
polypeptide standard. The kit optionally comprises a recombinant
disease protein or a recombinant protein that mimics a disease
protein. In another embodiment, the invention relates to method of
detecting whether a candidate polypeptide that has been contacted
with a blocking agent is i) a wildtype conformation or ii) a
non-wildtype conformation, wherein the candidate polypeptide
comprises at least one target epitope and, following contact with
the blocking agent and removal of the blocking agent, the candidate
polypeptide has been modified to convert any inaccessible target
epitope to accessible target epitope, the method comprising:
contacting the polypeptide with a detection agent that binds
selectively to the target epitope that was converted from
inaccessible target epitope to accessible target epitope, wherein
binding between detection agent and converted target epitope
indicates that the candidate polypeptide was in a non-wildtype
conformation (for example, an aggregated conformation) and wherein
lack of binding between the detection agent and the target epitope
indicates that the polypeptide was in a wild type conformation.
Diseases, blocking agents, target epitopes, detecting agents and
other aspects described herein are also useful in this method. The
diseases, blocking agents, target epitopes, detecting agents and
other aspects described herein are also readily adapted for the
methods described in preceding paragraphs, such as methods for
testing a sample from an animal (such as a human, livestock etc.)
to determine if the animal has a disease or screening a sample.
[0087] The reverse situation to the methods described in some of
the aforementioned paragraphs is also usefully detected, for
example, where the wildtype conformation includes an inaccessible
epitope and the non-wild type conformation has an accessible
epitope. This situation is also readily adapted to methods
described herein, such as diagnosing disease or screening
samples.
[0088] Other features and advantages of the present invention will
become apparent from the following detailed description. It should
be understood, however, that the detailed description and the
specific examples while indicating preferred embodiments of the
invention are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0089] Embodiments of the invention are described in relation to
the drawings in which:
FIG. 1. Brain PrP Aggregated In Vitro by Acid Treatment is
Protected From Modification by Peroxynitrite
[0090] Mock or acid treated human brain homogenate was treated with
increasing concentrations of peroxynitrite (ONOO) and then
subjected to immunoblotting with 3F4 (panel A) or 6H4 (panel B).
Effect of peroxynitrite on the 3F4 (C) and 6H4 (D) epitope in mock
(.quadrature.) and acid treated (.circle-solid.) brain homogenate.
Immunoblot films were scanned and band intensities determined by
Unscanit software. The results are the combined relative
intensities of 3 separate experiments.
FIG. 2. PrP in Scrapie Infected Hamster Brain is Protected from
Modification by Peroxynitrite
[0091] (A) Effect of peroxynitrite treatment on the 6H4 epitope in
scrapie infected hamster brain. (B) The blot in (A) was scanned and
relative band intensities determined using Unscanit software.
(.circle-solid.) Scrapie infected hamster brain. (.quadrature.)
Normal hamster brain.
FIG. 3 Protection from Peroxynitrite Induced Modification is Due to
Aggregation in Acid Treated Brain
[0092] (A) Effect of peroxynitrite on the immunoprecipitation (IP)
of PrP in mock and acid treated brain homogenate. Brain homogenate
was treated with 10 mM peroxynitrite followed by incubation for 2 h
at RT with (+) or without (-) 2.5 M guanidine hydrochloride (Gu).
The resulting samples were immunoprecipitated with 6H4 or 3F4. More
PrP is precipitated in the acid treated sample following treatment
with peroxynitrite+Gu whereas in the mock sample, Gu has no effect.
This suggests that Gu is able to break up aggregated PrP in the
acid sample that is protected from destruction by peroxynitrite.
(B) Effect of peroxynitrite on PrP in mock and acid treated brain
homogenate as measured by ELISA. Brain homogenate was treated with
increasing concentrations of peroxynitrite followed by 2.5 M Gu.
Following a 10-fold dilution, the samples were analyzed by sandwich
ELISA with 6H4 as the capture Ab and 3F4 as the detection Ab.
Similar to the immunoblot and IP data, the results show that
misfolded PrP is protected from destruction by peroxynitrite
treatment, due to aggregation.
FIG. 4 Detection of Aggregated Amyloid Beta (Abeta) Using EPA
[0093] The 6E10 epitope in the Abeta region of APP is less
accessible to peroxynitrite modification in Alzheimer's disease
brain compared to normal brain (panel A), and in brain homogenates
that have been treated at low pH to induce protein aggregation
(panel B). Abeta 1-42 peptide aggregated in vitro shows prominent
epitope protection of the 6E10 epitope to peroxynitrite
modification, in comparison with soluble non-aggregated Abeta 1-42
(panel C). Normal and aggregated Abeta 1-42 treated with increasing
concentrations of peroxynitrite and them immunoblotted with 6E10
antibody also shows prominent epitope protection (panel D).
FIG. 5 Detection of Epitopes Modifiable by DEPC in SOD1
[0094] (A) Western blot showing soluble SOD1 treated with
increasing concentrations of DEPC and immunoblotted with sheep
anti-SOD1. (B) Graphical representation of decreasing antibody
binding to SOD1 with increasing DEPC-concentration.
FIG. 6 Detection of Aggregated Alpha-Synuclein Using EPA
[0095] (A) Western blot showing effect of increasing concentrations
of DEPC on antibody binding to soluble and insoluble
alpha-synuclein. (B) Graphical representation showing the extent of
antibody binding to normal (-) and insoluble (.pi.)
alpha-synuclein.
DETAILED DESCRIPTION OF THE INVENTION
[0096] The current invention provides a useful method for the
detection of a disease related polypeptide counterpart of a normal
cellular polypeptide which forms aggregates or otherwise leads to
the obscuration of one or more epitopes that are not obscured in
the normal or wild type polypeptide. The invention recognizes the
importance of aggregation in the pathology of diseases such as
prion disease. The invention also takes advantage of this
aggregation effect and provides an assay that overcomes problems
with prior art detection assays. In one embodiment, the method of
the invention is applied to the detection of PrP.sup.Sc in plasma,
serum, urine or other biological sample. The methods of the
invention are further useful for detecting any polypeptide that
exists in two or more conformations, where one or more target
epitopes are inaccessible in at least one conformation. In one
embodiment the invention includes a method of detecting whether a
candidate polypeptide including a target epitope is in a wild type
or non-wild type conformation.
[0097] "Epitope" refers to a portion of a sequence of contiguous or
non-contiguous amino acids (antigen) which is recognized by and
bound by a detection agent such as an antibody. Preferably, the
epitope is a linear epitope on a polypeptide which typically
includes 3 to 10 or 6 to 10 or more contiguous amino acids that are
recognized and bound by a detection agent. A conformational epitope
includes non-contiguous amino acids. Sometimes conformational
epitopes can re-establish themselves after denaturation by partial
refolding on, e.g, an immunoblot membrane. The detection agent such
as an antibody recognizes the 3-dimensional structure. When a
protein molecule is folded into a three dimensional structure the
amino acids forming the epitope are positioned in a manner that
permits the detection agent to recognize and bind to the amino
acids. In an unfolded (denatured) protein only the linear epitope
is recognized and bound by the detection agent. Since the protein
is unfolded prior to contact with the detection agent, the
inaccessible epitope will typically be a linear epitope.
[0098] "Blocking agent" refers to an agent that reduces epitope
reactivity, for example by binding to the epitope or by modifying
and destroying epitope reactivity, for example on an amino acid
side group within a linear epitope, so that the epitope is
prevented from binding to detection agent (usually but not always
an antibody). An example of a blocking agent is peroxynitrite.
Other examples would include methylene, hydrogen peroxide, diethyl
pyrocarbonate, 4-hydroxynonenal (4HNE) epoxides such as
conduritol-B-epoxide and 1,2-epoxy-3-(p-nitrophenoxy)propane and
diazirine. Chemical modifying agents that saturate accessible amino
acids critical for epitope recognition in native conditions are
most useful in the applications of epitope protection technology.
Additionally the blocking agent may phosphorylate, glycosylate or
otherwise modify a target-epitope. The blocking agent may also
include peptides, antibodies or antibody fragments that bind to the
epitope. The blocking agent should efficiently modify accessible
amino acids (e.g. modify at least: 50%, 75%, 90%, 95% or 99% of
accessible amino acids).
[0099] "Accessible epitope" is target epitope that is available to
react with blocking agent in methods of the invention. For example,
epitope that is available to react with blocking agent is
accessible epitope. After reacting with blocking agent, the
accessible epitope is prevented from binding to detection agent
(after this reacting step, the reacted epitope may be referred to
as the blocked epitope).
[0100] "Antibody" is intended to include whole antibodies and
fragments thereof which also specifically react with one or more
protein epitopes, such as disease protein epitopes. Antibodies can
be fragmented using conventional techniques and the fragments
screened for utility in the same manner as described below. For
example, F(ab')2 fragments can be generated by treating antibody
with pepsin. The resulting F(ab')2 fragment can be treated to
reduce disulfide bridges to produce Fab' fragments.
[0101] "Aptamer" means a macromolecule such as a peptide, RNA or
DNA molecule that is able to specifically interact with a protein
or peptide target.
[0102] "Inaccessible epitope" means that target epitope
modification by the chemical blocking agent is prevented or
significantly reduced (e.g. reduced by at least: 50%, 75%, 90%, or
95%), for example, by differential misfolding relative to the wild
type polypeptide, by aggregation of misfolded polypeptide or by
post-translational modifications of the polypeptide. In some cases,
inaccessible epitope is converted to accessible epitope by removing
the hindrance (e.g. misfolding or aggregation) that prevents or
significantly reduces target epitope modification by the blocking
agent. The inaccessible epitope that is converted to accessible
epitope may also be called "revealed epitope".
[0103] "Detection agent" refers to an agent that binds to epitope
and which may be detected, such as antibody specific for prion
polypeptide epitopes that can be used to probe the sample
containing the polypeptide. The detection agent is used after the
polypeptide is unfolded such that the detection preferentially
binds the unblocked, unmodified epitopes.
[0104] "Disease protein or disease polypeptide" refers to a
polypeptide associated with a disease or disorder state where the
modular or higher order conformation of the polypeptide differs
from the wild type or non-disease conformation and includes
mutants, variants and polymorphic versions thereof. A disease
protein or disease polypeptide can also be referred to as non-wild
type conformation protein or polypeptide. The modular conformation
refers to conformational changes in the three dimensional structure
of a single protein molecule. The higher order conformation refers
to conformational changes in the three dimensional structure of
many protein molecules aggregated together. The aggregation can
consist of one or more different proteins and can be associated
with non-protein molecules. The wildtype and non-wildtype candidate
polypeptides including disease proteins or polypeptides also
include recombinant proteins, such as cellularly expressed (i.e.
bacteria, using baculovirus sytems etc.) and in vitro translated
polypeptides
[0105] "Wildtype folded conformation" refers to the wild type,
folded conformation of protein in a non-disease or non-disorder
state.
[0106] "Misfolded conformation" refers to the folded conformation
of polypeptide in a disease or disorder state where the
conformation differs from the wild type conformation. The
difference in conformation is as a result of differential folding.
The differential folding may cause protein aggregation.
[0107] "Wildtype conformation" refers to the conformation of
polypeptide in its usual or normal state or in a reference or
desired state and can include polypeptide in a non-disease or
disorder state.
[0108] "Non-wildtype conformation" refers to a conformation of
polypeptide that differs from the conformation of the wild type
polypeptide and can include a conformation of polypeptide in a
disease or disorder, where the conformation differs from the wild
type conformation. The difference in conformation may be as a
result of differential folding, polypeptide aggregation or
differential post-translational modification compared to the wild
type polypeptide. In the case of polypeptide aggregation, the
aggregation may prevent accessibility of the epitope rather than
the changed conformation.
[0109] Neurodegenerative diseases, such as Alzheimer's disease
(AD), amyotrophic lateral sclerosis (ALS) and Parkinson's
disease/Lewy body dementia (PD, LBD) pose major challenges to the
aging population and health care system. No specific biochemical
test exists for neurodegenerative diseases as a group (1,2).
Sporadic AD, ALS, and PD/LBD are all associated with neural
accumulation of pathological multimers of misfolded polypeptides
(such as fibrils, protofilaments; and amorphous aggregates),
including the Abeta fragment of the amyloid precursor protein (APP)
in AD; superoxide dismutase-1 (SOD1) in ALS, and alpha-synuclein in
PD and LBD (1). As with prion diseases, mutations in genes encoding
these aggregation-prone polypeptides are associated with autosomal
dominant familial forms of AD, ALS, and PD. The detection of
disease-associated misfolded polypeptide aggregates enables
specific and sensitive antemortem diagnostic tests for
neurodegenerative diseases.
[0110] To this end the inventors have invented the "epitope
protection assay" (EPA), an innovative technology for detection of
aggregated polypeptides in tissues and accessible biological
fluids, such as blood and CSF, which serve as "sinks" for the
aggregates released from dying neurons. The method optionally
consists of: [0111] reacting a sample with a chemical modifying
agent; [0112] disaggregating and denaturing the treated
polypeptides; [0113] probing the sample with detection agents such
as antibodies against specific epitopes blocked by the chemical
modifier; and [0114] detection of agent-bound polypeptides (e.g.,
by ELISA).
[0115] Normal soluble polypeptides in the sample are rendered
"invisible" in the assay, because accessible epitopes are not
detected by a detecting agent (eg. blocked to antibody
recognition), whereas a proportion of polypeptides in aggregates
are "protected" from chemical modification by virtue of their
interior sequestration, and are still available to be detected by a
detecting agent (eg. bind antibody) after disaggregation.
[0116] The methods of the invention are useful to diagnose diseases
characterized by polypeptide misfolding and/or aggregation such as
in the diseases mentioned above or for diseases or disorders
characterized by polypeptides with otherwise differentially
accessible target epitopes in disease and wildtype protein
conformations.
[0117] The present inventors have also found that treatment of
recombinant mouse prion polypeptide (rmPrP) at low pH in the
presence of low concentrations of denaturants causes the
polypeptide to acquire increased beta-sheet content, reminiscent of
the misfolded disease-associated prion polypeptide isoform, PrPSc.
This conversion of rmPrP is associated with increased solvent
accessibility of tyrosine side chains.sup.4. The inventors have
found that treatment of normal brain homogenate with acid and
denaturants causes PrP to become detergent insoluble (29). In order
to probe the surface accessibility of tyrosines and other residues
in normal and misfolded PrP.sup.C, normal and acid-misfolded human
brain tissue was treated with the chemical nitrating compound
peroxynitrite. Peroxynitrite treatment of brain tissue caused a
reduction in the binding of the anti-PrP antibodies 3F4 and 6H4 as
measured by immunoblotting, immunoprecipitation and ELISA.
Peroxynitrite-induced epitope blocking was more pronounced on
normal brain PrP than on misfolded PrP, showing a protective effect
of aggregation. Similar findings were observed in normal and
scrapie-infected hamster brain, in which 3F4 and 6H4 epitopes of
scrapie brain PrP were partially protected from
peroxynitrite-induced modification. Immunoprecipitation of
peroxynitrite-treated brain with anti-nitrotyrosine antibodies
suggests that either PrP is nitrated on tyrosine residues or
another polypeptide in proximity to PrP is nitrated and
coimmunoprecipitates PrP.
[0118] Accordingly, the invention includes a method of determining
polypeptide aggregation, including but not limited to PrP.sup.Sc,
comprising: [0119] reacting a sample with a chemical modifying
agent where such agent could be, but is not limited to,
peroxynitrite [0120] disaggregating and/or denaturing the
chemically modified sample with heat, detergent, or chaotropic
agents; and [0121] probing with antibodies specific for prion
polypeptide epitopes.
[0122] The inventors have further shown that the methods of the
invention are useful for detecting Alzheimer disease proteins.
[0123] In one embodiment, the Alzheimer's disease detection method
comprises: [0124] reacting a sample of polypeptide (the sample
typically contains all or part of a disease protein or polypeptide
such as amyloid precursor polypeptide or amyloid beta or tau and/or
the corresponding wild type polypeptide, and in many cases an
abundance of one or the other) with a chemical modifying agent,
typically a blocking agent such as peroxynitrite, which modifies
exposed epitopes so that they cannot bind to a detection agent;
[0125] disaggregating and/or denaturing the polypeptide in the
sample; and [0126] probing with detection agents, such as an
antibody against a target epitope to determine whether the
polypeptide prior to disaggregation and/or denaturing, included
inaccessible target epitopes.
[0127] Abeta containing vascular or plaque filaments are also
associated with conditions such as trisomy 21 (Down's syndrome),
hereditary cerebral hemorrhage with amyloidosis (HCHWA)-Dutch type,
and normal brain aging (Mori, H et al. JBC (1992) 267: 17082-86).
Accordingly, in one embodiment detection of Abeta disease protein
is prognostic for diseases HCHWA-Dutch type or normal brain
aging.
[0128] In addition, the methods of the invention can be combined
with other diagnostic methods such as magnetic resonance imaging
(MRI) or computed tomography (CT) scans to confirm diagnosis.
[0129] The methods of the invention are useful to detect protein or
polypeptide including target that exists in two or more
conformations, where one or more target epitopes are concealed in
at least one conformation.
[0130] Accordingly, the invention relates to a detection method
comprising: [0131] reacting polypeptide with a chemical modifying
agent, typically a blocking agent, which is defined to modify
exposed epitopes so that they cannot bind to detection agents;
[0132] disaggregating and/or denaturing the polypeptide in the
sample; and
[0133] probing with detection agents, such as antibodies against
target epitope to determine whether the polypeptide prior to
disaggregation and/or denaturing, included target epitopes
inaccessible to the chemical modifying agent.
[0134] The result indicates whether the polypeptide includes
inaccessible epitopes, which is indicative of the type of
polypeptide that is present (i.e. wild type or non-wild type
protein).
[0135] The invention also includes a method of detecting whether a
candidate polypeptide including a target epitope that has been
reacted with a blocking agent, is in a wildtype conformation or a
non-wildtype conformation, comprising: [0136] modifying the
candidate polypeptide to convert any inaccessible target epitope to
accessible target epitope; and [0137] contacting the polypeptide
with a detection agent that binds selectively to the target epitope
that was converted from inaccessible target epitope to accessible
target epitope, wherein binding between detection agent and
converted target epitope indicates that the candidate polypeptide
was in a non-wildtype conformation and wherein lack of binding
between the detection agent and the target epitope indicates that
the polypeptide was in a wild type conformation.
[0138] In another application, the invention also includes a method
of detecting intrinsically modified polypeptide, wherein the
modification protects target epitope from reacting with the
detecting agent, comprising: [0139] contacting the polypeptide with
a blocking agent that selectively blocks accessible target epitope,
wherein in one of the non-wildtype conformation or the wildtype
conformation, the target epitope is accessible and reacts with the
blocking agent, and wherein in the other conformation, the target
epitope is inaccessible and does not react with the blocking agent;
[0140] reacting the sample with an agent that removes the intrinsic
modification from the intrinsically modified polypeptide target
epitope; [0141] disaggregating and/or denaturing the polypeptide in
the sample; and
[0142] probing with a detection agent, such as antibodies against
the target epitope, to determine whether the candidate polypeptide
is an intrinsically modified polypeptide.
Chemical Modifying Agents
[0143] The chemical modifying agent of the invention comprises any
chemical (including a biological agent) that modifies target
epitope residues such that the epitope is rendered invisible by the
methods of the invention (ie. not detected by the detecting agent
or detection is reduced). For example, peroxynitrite preferentially
modifies tyrosine, serine, methionine, histidine and tryptophan as
well as cysteine and other amino acids (25, 26). DEPC
preferentially modifies histidines (37), and succinic anhydride
preferentially modifies residues comprising amines. Epoxides,
including conduritol-B-epoxide and
1,2-epoxy-3-(p-nitrophenoxy)propane) are a reactive group used
widely for "suicide inhibition" of carboxyl group side chains, such
as the catalytic residues of aspartyl proteases (19, 20). Hydrogen
peroxide and methylene are also useful. The chemicals may modify
the target epitope by oxidizing, nitrating, reducing, or otherwise
modifying the epitope. In addition, the epitope may be modified by
a chemical modifying agent that is a phosphate group (by
phosphorylation), or a gylcosyl group (by gylcosylation), and/or
other chemical group that obscures the target epitope.
[0144] Accordingly, in one embodiment the chemical modifying agent
is chosen from the group peroxynitrite, DEPC, hydrogen peroxide,
succinic anhydride, methylene and epoxides (conduritol-B-epoxide
and 1,2-epoxy-3-)p-nitrophenoxy)propane and/or related variants
thereof.
[0145] After reacting with candidate polypeptides, the chemical
modifying agent is removed. It is readily apparent to a skilled
person that the method steps of the invention recited here
involving removing the blocking agent typically involve physically,
chemically or otherwise removing the blocking agent away from the
candidate polypeptide to prevent further reaction. Removal
optionally involves allowing a sufficient time to pass so that the
blocking agent is removed from the candidate polypeptide by being
consumed or degraded (for example, such that the blocking agent
becomes inert or oxidized). Removal optionally involves adding a
compound to react with any excess blocking agent to inactivate it.
Removal also optionally involves physical filtering of the blocking
agent by conventional filtration techniques or centrifugation to
separate the candidate polypeptide and blocking agent, or physical
binding to a substrate useful for removing the blocking agent, such
as by binding of blocking agent or candidate polypeptide to an
immobilized substrate in a column.
[0146] Removing means preventing further reactions by the blocking
agent by, for example, physically or chemically inactivating the
blocking agent, taking the blocking agent out of contact with the
sample including the candidate polypeptide or allowing a sufficient
amount of time to pass for the blocking agent to be consumed or
degraded.
[0147] Chemical modification of a target epitope leads to
obscuration of an epitope to antibody recognition. In one
embodiment treatment with a blocking agent such as peroxynitrite
leads to destruction of epitopes on monomeric proteins but not
epitopes on aggregated proteins such as non-wild type polypeptides
or disease proteins.
Pretreatment
[0148] The methods of the invention also contemplate pretreatment
of the sample to enhance EPA detection. For example if decreased
detection of aggregated proteins such as prions in blood or urine
is observed, pre-clearing strategies are readily employed to
enhance detection with detergents, precipitating agents, and
adsorbents such as those typically used in commercial ELISA assays
which are known to one skilled in the art. Polypeptide samples may
also be pretreated with agents such as detergents or quanidine or
heat. Finally samples may be concentrated or precleared by methods
such as centrifugation. Accordingly, in one embodiment the samples
are pretreated before employing a method of the invention.
Detecting Misfolded or Aggregated Proteins and Polypeptides
[0149] The inventors have found a method that detects polypeptides
that have target epitopes that are accessible to detection in one
conformation and inaccessible in another by modification of
inaccessible epitopes by a modifying agent. The inventors have
identified several epitopes that are useful as target epitopes in
the methods of the invention. Other target epitopes are identified
as described below.
Target Epitopes
[0150] Target epitopes are identified for polypeptides that exist
in two or more conformations wherein epitopes that can be detected
by detecting agents such as antibodies, aptamers or peptides, are
accessible in one conformation and inaccessible in the other
conformation. Where an epitope is found to be blocked from
detection by a blocking agent in one conformation of the
polypeptide, the epitope is a target epitope. To identify target
epitopes, a detection agent such as an antibody is chosen. If the
detection agent is an antibody it is preferably a monoclonal
antibody although polyclonal antibodies are also usable. The
epitope, which can be a linear or non-linear epitope, and which is
specifically recognized by the antibody, is optionally a known
epitope. A candidate chemical modifying agent such as peroxynitrate
is chosen. If the epitope recognized by the detection agent is
known, the candidate chemical modifying agent is preferably chosen
based on its ability to modify amino acid residues in the target
epitope. For example peroxynitrite preferentially modifies tyrosine
and histidine residues with some modification of cysteine and other
amino acids. Peroxynitrite is optionally chosen as chemical
modifying agent if tyrosines and/or histidines are present in the
target epitope. Aliquots of a sample comprising wild type
polypeptide and aliquots of a sample comprising non-wildtype
polypeptide are reacted with increasing concentrations of the
chosen chemical modifying agent. Each sample comprises one or more
of recombinant polypeptide, cell extracts or tissue samples known
to express the polypeptide in either the wild type or non-wild type
conformation. Preferably samples of polypeptide have similar
concentrations of polypeptide. The non-wildtype conformation
polypeptide sample is alternatively obtained by treating a
polypeptide in wild type conformation with an agent, such as acid,
that induces conversion to a non-wildtype conformation.
[0151] Each sample of polypeptide is denatured and/or disaggregated
to convert any inaccessible putative target epitope to accessible
target epitope. Each sample of polypeptide is then contacted with
the chosen detection agent. Detection is performed using techniques
known in the art such as ELISA, and Western blotting. The amount of
signal generated by the detection agent for sample comprising
polypeptide in a wildtype conformation treated with protection
agent and for sample comprising polypeptide in a non-wildtype
conformation treated with protection agent are compared. A
difference in detection at one or more concentrations of chemical
modifying agent indicates that the epitope is protected in one
conformation and further indicates that the epitope is a target
epitope. A difference over a range of chemical modifying agent
concentrations indicates that the target epitope is useful for EPA.
The process is repeated with different blocking agents and/or
detecting agents and target epitopes are identified. One typically
standardizes and titrates the blocking agent and to performs
experiments using a "universal" chemical modifier such as
methylene.sup.24,25, which optionally yields more uniform and
complete protection of the target epitope.
[0152] Accordingly in one example, a method of identifying a target
epitope in a polypeptide that has two or more conformations wherein
the target epitope is accessible to detection in one conformation
and inaccessible in another conformation comprises: [0153] reacting
a sample comprising polypeptide in a wild type conformation and a
sample comprising polypeptide in a non-wild type conformation
typically with one or more concentrations of a chemical modifying
agent; [0154] denaturing and/or disaggregating each sample to
convert any inaccessible target epitope to accessible target
epitope; [0155] contacting the samples with a detection agent; and
[0156] comparing the signal generated by the detection agent for
samples comprising polypeptide in a wildtype conformation treated
with chemical modifying agent and for samples comprising
polypeptide in a non-wildtype conformation treated with chemical
modifying agent wherein a difference in detection between sample
comprising wildtype polypeptide and sample comprising non-wild type
sample indicates that the epitope is protected in one conformation
and further indicates that the epitope is a target epitope.
[0157] Conditions for EPA
[0158] Titration experiments with peroxynitrite, hydrogen peroxide
and methylene (based on UV light photolysis of the precursor
diazirine) or other modifying agents, are useful to improve
conditions for epitope protection.
[0159] Samples known to contain polypeptides in two or more
conformations, including disease proteins, are optionally reacted
using immunoblotting and ELISA. In each case, samples are prepared
and optionally mixed with increasing concentrations of the
modifying agent and processed, for example, by immunoblotting,
ELISA and/or time resolved fluorescence. This defines the type and
concentration of chemical agent allowing the maximal distinction
between monomeric and aggregated proteins including disease
proteins. Additional informative experiments can involve using
recombinant disease (non-wild type) protein (such as acid treated
prion proteins or mutant p53 proteins and other protein listed in
this application) and normal (wild type) protein in place of
samples containing disease proteins.
[0160] In some cases, disease proteins may have different
properties for chemical modification than do recombinant disease
proteins, and disease proteins in one sample type (such as brain
tissue) may display different chemical modification properties than
disease proteins circulating in blood, or detectable in urine. One
of skill in the art shall readily identify the optimal conditions
for endogenous prions using known techniques.
[0161] The EPA achieves superior commercial utility by detecting
disease proteins in biological tissues and fluids for which no
present technology exists. Some disease proteins are in very low
abundance. For example prions are in very low abundance (10-100
prions/mL by bioassay), and protease-resistant PrP in urine is only
intermittently/sporadically detectable by precipitation of large
fluid volumes. Also, any prospective blood test must contend with
high concentrations of wildtype protein (i.e. for PrP.sup.C
(typically 10.sup.6-fold more than PrP.sup.Sc)) and "blocking" by
heterologous plasma proteins. Using the optimized chemical
modification regimen and the DELFIA-TRF system, the sensitivity
thresholds for EPA in blood and urine are optionally determined
using: [0162] 1. Animal and human plasma and urine "spiked" with a
titration of disease protein; [0163] 2. Plasma and urine from model
disease animals expressing a disease protein.
[0164] Biological fluids clinically accessible by non-invasive
routes provide a substrate for a practical antemortem test for
diagnosis and screening of diseases involving aggregated disease
proteins in humans and animals. The methods of this invention are
also useful in post-mortem testing. One of skill in the art readily
determines whether EPA with "disease protein spike" titration in
normal blood and urine reveals similar DELFIA-TRF signals to the
same disease protein titration in buffer, showing that the EPA is
not affected by "blocking factors" in these biological fluids.
Preferential "blocking" of disease protein by heterologous proteins
may actually enhance epitope protection to chemical modifying
agents. If decreased detection of disease protein in blood or urine
is observed, pre-clearing strategies are readily employed to
enhance disease protein detection, for example, with detergents,
precipitating agents, and adsorbents typically used in commercial
ELISA assays which are known to one skilled in the art.
[0165] In one embodiment, the methods of the invention involve the
detection of target epitopes in misfolded or aggregated
polypeptides. In another embodiment, the invention provides a
method for improving or optimizing the detection of polypeptides
that can exist in 2 or more conformations. In another embodiment
the detection of misfolded and/or aggregated polypeptides is
indicative of disease (disease proteins). In another embodiment,
polypeptides are detected using detection agents such as
antibodies, aptamers or peptides that specifically bind to epitopes
of polypeptides that can be chemically modified. In another
embodiment these polypeptides are disease proteins. Antibodies to
candidate protein epitopes are commercially available antibodies or
are readily prepared by a person skilled in the art and include
antibody fragments, and single chain antibodies. All the
aforementioned methods are readily implemented using steps
described in this application.
Antibodies
[0166] The invention contemplates the use of known antibodies as
the binding agent including biotin-3F4 and 3F4 and 6H4 which
recognize prion disease proteins. 3F4 reacts against the MKHV
epitope and 6H4 reacts against the DYEDRYYRE epitope. Additionally,
6E10 which recognizes Abeta, reacts against the EFRHDS epitope
(residues 3-8).
[0167] Other antibodies and the epitopes recognized (if known)
which are optionally used with the methods of the invention are
listed in the table below. TABLE-US-00001 TABLE Antibodies useful
to detect disease proteins Protein Antibody Mono/Poly Epitope
Company Abeta 4G8 Monoclonal Within aa18-22 of human Abeta Signet
6E10 Monoclonal Within aa3-8 of human Abeta Signet ab2539
Polyclonal NA Abcam Abeta-NT Polyclonal NA QED Bioscience DE2B4
Monoclonal Within aa1-17 of human Abeta Acris antibodies NBA-104E
Monoclonal Within aa1-16 of human Abeta Stressgen Alpha- 4D6
Monoclonal Unknown Acris antibodies synuclein ab6162 Polyclonal NA
Abcam LB509 Monoclonal Unknown Zymed Syn-1 Monoclonal Within
aa91-99 of human a-syn BD Biosciences Syn-204 Monoclonal Within
aa87-110 of human a- Lab Vision syn Syn-211 Monoclonal Within
aa121-125 of human a- Lab Vision syn Tau Mouse Anti-Tau-1
Monoclonal Within aa95-108 of human tau Biomeda Mouse Anti-Tau-2
Monoclonal Unknown Stressgen T14 Monoclonal Within aa141-178 of
human tau Zymed T46 Monoclonal Within aa404-441 of human tau Zymed
Tau-2 Monoclonal Unknown Acris antibodies Tau-5 (ab3931) Monoclonal
Unknown Abcam SOD1 Mouse SOD1 Monoclonal Unknown Sigma-aldrich
Rabbit SOD1 Polyclonal Unknown Stressgen Rat SOD1 Polyclonal
Unknown Stressgen Sheep SOD1 Polyclonal Unknown OxisResearch
[0168] Antibodies to disease protein epitopes are prepared using
techniques known in the art. For example, by using a peptide of a
disease protein including a putative target epitope, polyclonal
antisera or monoclonal antibodies are made using standard methods.
A mammal, (e.g., a mouse, hamster, or rabbit) can be immunized with
an immunogenic form of the peptide which elicits an antibody
response in the mammal. Techniques for conferring immunogenicity on
a peptide include conjugation to carriers or other techniques well
known in the art. For example, the protein or peptide is
administered in the presence of adjuvant. The progress of
immunization can be monitored by detection of antibody titers in
plasma or serum. Standard ELISA or other immunoassay procedures are
optionally used with the immunogen as antigen to assess the levels
of antibodies. Following immunization, antisera can be obtained
and, if desired, polyclonal antibodies isolated from the sera.
[0169] To produce monoclonal antibodies, antibody producing cells
(lymphocytes) are optionally harvested from an immunized animal and
fused with myeloma cells by standard somatic cell fusion procedures
thus immortalizing these cells and yielding hybridoma cells. Such
techniques are well known in the art, (e.g., the hybridoma
technique originally developed by Kohler and Milstein (Nature 256,
495-497 (1975)) as well as other techniques such as the human
B-cell hybridoma technique (Kozbor et al., Immunol. Today 4, 72
(1983)), the EBV-hybridoma technique to produce human monoclonal
antibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy
(1985) Allen R. Bliss, Inc., pages 77-96), and screening of
combinatorial antibody libraries (Huse et al., Science 246, 1275
(1989)). Hybridoma cells can be screened immunochemically for
production of antibodies specifically reactive with the peptide and
the monoclonal antibodies can be isolated.
[0170] Chimeric antibody derivatives, i.e., antibody molecules that
combine a non-human animal variable region and a human constant
region are also contemplated within the scope of the invention.
Chimeric antibody molecules include, for example, the antigen
binding domain from an antibody of a mouse, rat, or other species,
with human constant regions. Conventional methods used to make
chimeric antibodies containing the immunoglobulin variable region
which recognizes disease protein epitopes of the invention (See,
for example, Morrison et al., Proc. Natl Acad. Sci. U.S.A. 81,6851
(1985); Takeda et al., Nature 314, 452 (1985), Cabilly et al., U.S.
Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi
et al., European Patent Publication EP171496; European Patent
Publication 0173494, United Kingdom patent GB 2177096B).
[0171] Specific antibodies, or antibody fragments, such as, but not
limited to, single-chain Fv monoclonal antibodies reactive against
disease protein epitopes are readily generated by screening
expression libraries encoding immunoglobulin genes, or portions
thereof, expressed in bacteria with peptides produced from the
nucleic acid molecules of disease proteins. For example, complete
Fab fragments, VH regions and FV regions are expressed in bacteria
using phage expression libraries (See for example Ward et al.,
Nature 341, 544-546: (1989); Huse et al., Science 246, 1275-1281
(1989); and McCafferty et al. Nature 348, 552-554 (1990)).
Alternatively, a SCID-hu mouse, for example the model developed by
Genpharm, is used to produce antibodies or fragments thereof.
[0172] Antibodies specifically reactive with disease protein
epitopes, or derivatives, such as enzyme conjugates or labeled
derivatives, are useful to detect disease protein epitopes in
various samples (e.g. biological materials). They are useful as
diagnostic or prognostic reagents and are readily used to detect
abnormalities in the level of protein expression, or abnormalities
in the structure, and/or temporal, tissue, cellular, or subcellular
location of disease protein epitopes. In vitro immunoassays are
also useful to assess or monitor the efficacy of particular
therapies. The antibodies of the invention may also be used in
vitro to determine the level of expression of a gene of a
polypeptide that exists in two or more conformations such as a
disease protein in cells genetically engineered to produce the
disease protein.
[0173] The antibodies are useful in any known immunoassays which
rely on the binding interaction between an antigenic determinant of
the disease protein epitopes and the antibodies. Examples of such
assays are radioimmunoassays, enzyme immunoassays (e.g. ELISA
including Sandwich ELISA), immunofluorescence, immunoprecipitation,
latex agglutination, hemagglutination, and histochemical tests. The
antibodies are useful to detect and quantify the disease protein in
a sample in order to determine its role and to diagnose the disease
caused by the disease protein.
[0174] In particular, the antibodies of the invention are useful in
immunohistochemical analyses, for example, at the cellular and
subcellular level, to detect a disease protein, to localize it to
particular cells and tissues, and to specific subcellular
locations, and to quantitate the level of expression.
[0175] Cytochemical techniques known in the art for localizing
antigens using light and electron microscopy to detect polypeptides
such as disease proteins. Generally, an antibody of the invention
is optionally labeled with a detectable substance and the
recognized polypeptide is localised in tissues and cells based upon
the presence of the detectable substance. Examples of detectable
substances include, but are not limited to, the following:
radioisotopes (e.g., .sup.3H, .sup.14C, .sup.35S, .sup.125I,
.sup.131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide
phosphors), luminescent labels such as luminol; enzymatic labels
(e.g., horseradish peroxidase, beta-galactosidase, luciferase,
alkaline phosphatase, acetylcholinesterase), biotinyl groups (which
can be detected by marked avidin e.g., streptavidin containing a
fluorescent marker or enzymatic activity that can be detected by
optical or colorimetric methods), predetermined polypeptide
epitopes recognized by a secondary reporter (e.g., leucine zipper
pair sequences, binding sites for secondary antibodies, metal
binding domains, epitope tags). In some embodiments, labels are
attached via spacer arms of various lengths to reduce potential
steric hindrance. Antibodies may also be coupled to electron dense
substances, such as ferritin or colloidal gold, which are readily
visualized by electron microscopy.
[0176] The antibody or sample may be immobilized on a carrier or
solid support which is capable of immobilizing cells, antibodies
etc. For example, the carrier or support may be nitrocellulose, or
glass, polyacrylamides, gabbros, and magnetite. The support
material may have any possible configuration including spherical
(e.g. bead), cylindrical (e.g. inside surface of a test tube or
well, or the external surface of a rod), or flat (e.g. sheet, test
strip). Indirect methods may also be employed in which the primary
antigen-antibody reaction is amplified by the introduction of a
second antibody, having specificity for the antibody reactive
against disease protein epitopes. By way of example, if the
antibody having specificity against a polypeptide epitope such as a
disease protein epitope is a rabbit IgG antibody, the second
antibody may be goat anti-rabbit gamma-globulin labeled with a
detectable substance as described herein.
[0177] Where a radioactive label is used as a detectable substance,
disease proteins may be localized by autoradiography. The results
of autoradiography may be quantitated by determining the density of
particles in the autoradiographs by various optical methods, or by
counting the grains.
Aptamers
[0178] Aptamers are also useful in the methods of the invention to
detect polypeptides such as disease proteins. Aptamers are
macromolecules that can recognize targets such as proteins with
high specificity and sensitivity.
[0179] Nucleic acid aptamers are small molecules isolated from
combinatorial libraries by a procedure named systemic evolution of
ligands by exponential enrichment (SELEX) (reviewed in Cerchia L et
al., FEBS Letters 528 (2002) 12-12). Using this technology aptamers
that bind proteins with high target specificity and selectivity can
be identified. The affinities can be comparable to antibody antigen
interactions. Discrimination between native and denatured protein
has been shown (Bianchini et al. Immunol Methods (2001) 252:191-97)
making aptamers useful detection agents for the methods of the
invention.
[0180] Peptide aptamers, also known as paptamers,
thioredoxin-insert proteins or pertubagens are artificial proteins
where an inserted peptide is expressed on a solvent exposed surface
of a structurally stable protein which functions as a scaffold
(Crawford M. et al. Brief Funct Genomic Proteomic. 2003 April;
2:72-9). Peptide aptamers can function similarly to antibodies and
have dissociation constants that are comparable to, and sometimes
better than, antibodies. They can be used to probe immobilized
proteins on nitrocellulose (Crawford M. et al. Brief Funct Genomic
Proteomic. 2003 April; 2:72-9). Peptide aptamers have been shown to
exhibit different affinities for small changes such as single amino
acid differences making them useful for the detection of
polypeptides that exist in two or more conformations such as
disease proteins that exhibit different folding or aggregation
conformations.
[0181] Accordingly, in one embodiment of the invention, nucleic
acid and/or peptide aptamers are used with the methods of the
invention to distinguish between wild-type and disease conformation
proteins. In one embodiment the disease protein is a prion protein.
In another embodiment the disease protein is amyloid-beta. In
another embodiment the disease protein is tau protein. In another
embodiment the disease protein is alpha-synuclein. In another
embodiment the disease protein is SOD-1.
Denaturing and Disaggregation
[0182] In the methods, the polypeptide is optionally modified by
denaturing the polypeptide, for example with heat, detergent and/or
chaotropic agents. The polypeptide is optionally modified by
treatment with a disaggregation agent to disaggregate the
polypeptide from other polypeptides of the same type, and from
other molecules, wherein the disaggregation agent is optionally
selected from at least one of the group consisting of chaotropic
agents, detergent and heat. Chaotropic agents can include but are
not limited to such as guanidine salts, urea, and thiourea.
[0183] The inventors have shown that treating proteins with
guanidine hydrochloride increases the amount of protected protein
detectable.
[0184] Combining disaggregation methods can result in optimised
disaggregation. For example boiling samples in sodium dodecyl
sulfate (SDS; also known as sodium lauryl sulfate) loading buffer
can increase solubilization of polypeptides such as disease
proteins, increasing the epitopes available for interacting with
the detecting agent. For example, boiling samples in SDS loading
buffer results in enhanced solubilization, and allows detection of
protected epitopes by sandwich ELISA. The sandwich ELISA assay
system is able to identify aggregated disease protein in tissue
homogenate samples if the samples are boiled in SDS loading buffer
after peroxynitrite treatment. At peroxynitrite concentrations
greater than 8 mM, there is 2.5-3.times. as much PrP detected in
the acid treated sample as compared to the mock treated sample.
Accordingly, in one embodiment the sample is boiled in SDS loading
after treatment with modifying agent and before detection with a
detecting agent such as an antibody.
Time Resolved Fluorescence (TRF) Two Point ELISA and
Dissociation
Enhanced Lanthanide FluoroImmunoassay (DELFIA)
[0185] As previously mentioned ELISA techniques can be employed by
the methods of the invention. Time resolved flouresence two-point
ELISA employing Dissociation Enhanced Lanthanide FluoroImmunassay
(DELFIA) technology is 1000 fold more sensitive than conventional
ELISA techniques and can be used with the methods of the invention
to detect polypeptides aggregated in vitro, in neural tissue of
transgenic mouse models of neurodegeneration, and in human AD, ALS,
PD and LBD patient brain samples.
[0186] The DELFIA assay uses a chelated lanthanide-labeled tracer,
such as europium (Eu) and time-resolved fluorescence (TRF) to
measure output signal (33). The benefit of lanthanide chelates is
that their fluorescence is intense and lasts up to 200,000 times
longer than conventional fluorophores, allowing signal capture
after non-specific interfering fluorescence has faded (particularly
critical for biological samples, which may possess considerable
intrinsic fluorescence, the emission of which is comparatively
short-lived). DELFIA-based systems can measure as little as 100
fmol/well of Eu (33).
[0187] In one embodiment of the invention, a chemical modifying
agent and antibody are employed in a sensitive capture-detection
"sandwich" 96-well plate DELFIA TRF system in the detection of
aggregated disease specific proteins described herein, such as
Abeta, tau, SOD1, huntingtin alpha-synuclein, islet amyloid
polypeptide, resistin and p53.
[0188] A two-point EPA increases the specificity for detection of
proteins sequestered in aggregates of a clinical sample. In one
embodiment, two or more chemically modifiable epitopes are present
in each test polypeptide, which would increase the specificity of
diagnostic tests employing this technology (e.g., use in two-point
ELISA). In one embodiment, the chemically modifiable epitopes are
modified by the same chemical. In another embodiment, the epitopes
are modified by one of two or more different chemicals. The
modified epitopes may be recognized by the same antibody or they
may be recognized by two or more different antibodies. For clinical
and commercial use, EPA must be sensitive and specific for
polypeptides aggregated in vitro and in vivo. With optimal
antibodies and chemical modifying regimens, and the DELFIA-TRF
system EPA can detect 10.sup.5-10.sup.6 molecules of soluble
polypeptides This may correspond to a single polypeptide aggregate,
if these aggregates are of similar size to prion protein aggregates
in disease (35, 36).
[0189] Accordingly in one embodiment, the DELFIA-TRF system EPA can
be used to identify disease proteins that are in very low
abundance, as low as a single polypeptide aggregate.
Diagnostic and Screening Applications
[0190] Effective, efficient and inexpensive diagnostic and
screening strategies for antemortem diagnosis of human
neurodegenerative diseases are urgently needed, given the aging
population and continued financial pressure on the health care
system. EPA will achieve clinical utility by detecting polypeptide
aggregates in relevant and accessible biological tissues and
fluids, for which no present technology exists. In one embodiment
the methods of the invention are used to diagnose individuals who
have a disease protein related disease. In one embodiment, the
invention is used to diagnose individuals who have a
neurodegenerative disease. In another embodiment, the invention is
used to diagnose individuals who have a neurodegenerative disease
selected from the group comprising priori related diseases, AD, HD,
ALS and PD. In a further embodiment, the methods of the invention
are used post-mortem to determine if the individual had a disease
protein related disease.
[0191] The methods of the invention are used to detect whether a
human has a disease protein related disease. In another embodiment,
the methods are used to detect if a non-human animal has a disease
protein related disease. In a further embodiment the non-human
animal is one of the group comprising cattle, sheep and cervids. In
another embodiment, the methods of the invention are used to detect
if livestock has a disease protein related disease.
[0192] In one embodiment the methods of the invention are used to
detect disease proteins in biological specimens. The biological
specimens may comprise biological fluids, such as CSF, serum,
blood, tears, peritoneal exudates, or urine, or tissue samples such
as biopsies or brain tissue. The samples in one embodiment are
antemortem samples. In another embodiment they are postmortem
samples.
[0193] The methods of the invention are useful to quantify
detection of soluble form of disease related proteins such as
Abeta, tau, SOD1 huntingtin, alpha-synuclein, islet amyloid
polypeptide, resistin and p53 protein.
[0194] In another embodiment, EPA is used to determine the
sensitivity and specificity of aggregate detection in homogenates
from CRND8 (human mutant APP) mouse brain and CSF (34) and G93A
human mutant SOD1 transgenic mice (13).
[0195] In another embodiment the invention is used to determine the
sensitivity and specificity of aggregate detection in homogenates
from normal (treated and untreated at low pH) and diseased frozen
human brain (AD, ALS, PD, LBD).
[0196] The methods of the invention are used in one embodiment to
ensure preparations derived from mammalian blood or tissues or
involving processes where mammalian blood or tissues come into
contact with preparations, are free of disease proteins. In one
embodiment the preparation is a pharmaceutical product. In another
embodiment the preparation is a vaccine. In a further embodiment,
the preparation is a cosmetic. In one embodiment, the preparations
are tested for prion proteins. In another embodiment, the
preparations are tested for amyloid-beta. In another embodiment,
the preparations are tested for tau protein. In another embodiment,
the preparations are tested for alpha-synuclein. In a further
embodiment, the preparations are tested for SOD-1.
[0197] In another embodiment, the methods of the invention are used
to screen blood, and blood products (eg. blood fractions such as
blood plasma or compounds isolated or manufactured from blood) used
for transfusions or other medical procedures for disease proteins.
In another embodiment, the invention is used to screen organ
transplants for disease proteins. In one embodiment, the
preparations are screened for prion proteins. In another
embodiment, the preparations are screened for amyloid-beta. In one
embodiment, the preparations are screened for tau protein. In
another one embodiment, the preparations are screened for
alpha-synuclein. In a further embodiment, the preparations are
screened for SOD-1.
[0198] The invention is also useful for ensuring that food sources
are free of disease proteins. In another embodiment the methods of
the invention are used to test edible products derived from mammals
such as meats and meat products; and dairy products. Foods
potentially contaminated with neural tissue (such as "mechanically
separated meat," and meat cuts containing dorsal root ganglia or
other neural tissue) are particularly important to screen for prion
contamination.
[0199] Instruments that are used for invasive procedures may also
be a source of transmitting disease. In one embodiment instruments
used for medical and surgical procedures are tested for the
presence of disease proteins using methods of the invention. In
another embodiment instruments used for dental hygiene are tested
for the presence of disease proteins.
[0200] In a further embodiment, the invention provides methods to
ensure that decontamination methods for removing disease proteins
and disease protein containing tissues, have been successful. In
one embodiment the methods of the invention are used to assess
decontamination procedures in a meat processing plant. In another
embodiment the methods of the invention are used to assess
decontamination in a food processing plant. In another embodiment
instruments used for surgery or dentistry are tested for the
presence of disease proteins.
Prognostic Applications
[0201] Prion protein conversion, Alzheimer's disease related
polypeptide or other disease/disorder polypeptide may be
periodically monitored in a subject over time (e.g. at a first time
and a second time at least a week or at least a month after the
first time) to identify, for example, increased or decreased levels
of PrP.sup.C or increased or decreased levels of PrP.sup.Sc in the
subject. The methods of the invention are also useful to measure a
subject's level of PrP.sup.C or PrP.sup.Sc to determine the
subject's response to drug therapy. Decreasing levels of prion
protein in the subject over time indicate a positive response to
drug therapy. The same methods are used with other disease or
disorder protein.
[0202] Since many neurological diseases are associated with
aggregated proteins, similar methods are useful for these diseases
and their aggregated proteins, including, but not limited to:
amyotrophic lateral sclerosis (superoxide dismutase 1), Alzheimer's
disease (amyloid beta), Parkinson's disease (alpha synuclein),
Huntington's disease (huntingtin), cancer (p53), diabetes (eg.
islet amyloid polypeptide and resistin) and other diseases
involving abnormal protein folding, aggregation or
post-translational modification. Such a test is useful in the
spinal fluid and other bodily fluids in addition to peripheral
blood. In Alzheimer's disease, the aggregation status of the
amyloid beta peptide is optionally monitored by determining the
accessibility of two epitopes detected by the monoclonal antibodies
6E10 and 4G8, in addition to other amyloid beta epitopes, using the
methods described in this application, for example, with an
anti-6E10 or anti-4G8 antibody (detection agent) known in the
art.
Identifying Prion Conversion Inhibitors
[0203] Since the invention is useful for detecting differences
between polypeptides, the invention further includes an assay for
evaluating whether a candidate compound is capable of inhibiting or
stabilizing prion conversion or formation of other disease or
disorder polypeptides, such as amyloid beta, tau and APP in
Alzheimer's disease, SOD1 in amyotrophic lateral sclerosis,
alpha-synuclein in Parkinson's and Lewy body disease, huntingtin in
Huntington's disease islet amyloid polypeptide and resistin in
diabetes and p53 in cancer. The invention also includes compounds
for inhibiting or stabilizing prion conversion (or conversion of
other disease or disorder polypeptides) identified by the methods
described in the application. Decreased protein conversion to an
intermediate prion protein substrate or PrP.sup.Sc (or other
disease or disorder polypeptides shows that the candidate compound
is useful for treating prion disease.
[0204] The assays of the invention are useful to screen candidate
compounds to determine if they inhibit PrP.sup.Sc formation (or
formation of other disease or disorder polypeptides from wild type
protein). Protein may be contacted with a candidate compound in
vivo or in vitro and then used in the methods of the invention to
determine if wild type protein has been converted to PrP.sup.Sc or
if PrP.sup.Sc has been converted to wild type protein. Similar
methods are used with respect to other disease or disorder
polypeptides. Recombinant proteins are useful for identifying
aggregation inhibitors.
[0205] Therefore, the invention also provides methods for
identifying substances that inhibit conversion to PrP.sup.Sc (e.g.
prion protein conversion from wild type protein or intermediate to
PrP.sup.Sc) comprising the steps of: [0206] reacting a polypeptide
and a candidate substance, and
[0207] determining whether the protein has been converted to
PrP.sup.Sc using the methods of the invention.
[0208] Similar methods are optionally performed to identify
compounds which stabilize the wild-type prion state, or bind to
PrP.sup.Sc and block conversion of recruitable PrP isoforms.
[0209] The invention also provides methods for identifying
substances that inhibit conversion to disease or disorder
polypeptides (e.g. conversion from wild type protein to the amyloid
beta, tau or APP protein in Alzheimer's disease and other proteins
and diseases described in this application) comprising the steps
of: [0210] reacting a polypeptide and a candidate substance, and
[0211] determining whether the protein has been converted to the
amyloid betaor APP protein in Alzheimer's disease using the methods
of the invention.
[0212] Another aspect of the invention provides a method of
identifying substances which reverse PrP.sup.Sc formation
comprising the steps of: [0213] reacting a polypeptide and a
candidate substance, and
[0214] determining whether the PrP.sup.Sc has been converted to
wild type protein using the methods of the invention.
[0215] Another aspect of the invention provides a method of
identifying substances which reverse amyloid beta or APP protein in
Alzheimer's disease formation comprising the steps of: [0216]
reacting a polypeptide and a candidate substance; and [0217]
determining whether the amyloid beta or APP protein in Alzheimer's
disease has been converted to wild type protein using the methods
of the invention.
[0218] The same methods are used with other polypeptides associated
with diseases and disorders described in this application.
[0219] Biological samples and commercially available libraries may
be tested for substances such as proteins or small organic
molecules that bind to a protein. Inhibitors are preferably
directed towards specific domains of disease proteins such as prion
protein. To achieve specificity, inhibitors should target the
unique sequences and or conformational features of the disease
protein.
Protein Conformation Detection
[0220] The invention includes a method of detecting whether a
candidate polypeptide including a target epitope is a non-wild type
conformation polypeptide or a wild type conformation polypeptide,
comprising: [0221] contacting the candidate polypeptide with a
blocking agent; and [0222] determining whether the target epitope
is inaccessible or accessible to chemical modification by the
blocking agent.
[0223] The accessibility or inaccessibility of the target epitope
is indicative of whether the candidate polypeptide is non-wild type
conformation polypeptide or a wild type conformation polypeptide
because in one of the non-wild type protein and the wild type
protein, the target epitope is accessible. In the other
polypeptide, the target epitope is inaccessible.
[0224] In one embodiment, the invention includes a method of
detecting whether a candidate polypeptide including a target
epitope is in a wildtype conformation or a non-wildtype
conformation, comprising: [0225] contacting the polypeptide with a
blocking agent that selectively blocks accessible target epitope,
wherein in one of the non-wildtype conformation or the wildtype
conformation, the target epitope is accessible and reacts with the
blocking agent, and wherein in the other conformation, the target
epitope is inaccessible and does not react with the blocking agent;
[0226] removing unreacted blocking agent from contact with the
polypeptide (eg. by allowing blocking agent to be consumed or
degraded in the sample comprising the candidate polypeptide or by
physical or chemical removal processes); [0227] modifying the
candidate polypeptide to convert any inaccessible target epitope to
accessible target epitope; and [0228] contacting the polypeptide
with a detection agent that binds selectively to target epitope
that was converted from inaccessible target epitope to accessible
target epitope, wherein binding between detection agent and
converted target epitope indicates that prior to conversion the
candidate polypeptide was in a conformation in which the target
epitope was inaccessible and wherein lack of binding between the
detection agent and the target epitope indicates that the
polypeptide was in a conformation in which the target epitope was
inaccessible, thereby indicating whether the polypeptide was in a
wildtype conformation or a non-wildtype conformation.
[0229] A polypeptide may have more than two conformations. For
example a polypeptide may exist in a wild-type conformation, in a
benign misfolded, aggregated or otherwise non-wildtype conformation
not associated with disease, and a disease associated conformation
(i.e. aggregated in higher order structures). The methods of the
invention can be applied to distinguish each of these states
through the use of one or more chemical modifying agents and/or one
or more detecting agents such as antibodies.
Detection of Intrinsically Modified Polypeptides
[0230] The invention also provides a method of detecting
polypeptides that exist in two or more conformations wherein the
target epitopes in one of the conformations is modified by an
intrinsic mechanism. The intrinsic mechanism can include
intracellular and/or post-translational modification of a
polypeptide such as phosphorylation and/or glycosylation or a
modification resulting from an additive used in a process. The
intrinsic modification blocks a target epitope obscuring it from
detection with a detection agent. The sample of polypeptide is
reacted with a blocking agent that reacts with available target
epitope in polypeptide that is not intrinsically modified. The
intrinsic modification is then removed. For example if the
intrinsic modification is phosphorylation, the polypeptide is
treated with a phosphatase which removes the phosphorylation and
converts the inaccessible target epitope in the previously
intrinsically modified polypeptide, to accessible epitope. The
polypeptide is then detected with a detecting agent such as an
antibody.
[0231] Accordingly in one embodiment, the invention provides a
method of detecting intrinsically modified target epitopes in a
polypeptide having two or more conformations comprising; [0232]
contacting the polypeptide with a blocking agent that selectively
blocks accessible target epitope, wherein in one of the
non-wildtype conformation or the wildtype conformation, the target
epitope is accessible and reacts with the blocking agent, and
wherein in the other conformation, the target epitope is
inaccessible and does not react with the blocking agent; [0233]
reacting the sample with an agent that removes the intrinsic
modification from the intrinsically modified polypeptide target
epitope; [0234] disaggregating and/or denaturing the polypeptide in
the sample; and [0235] probing with a detection agent, such as
antibodies against the target epitope, to determine whether the
candidate polypeptide is an intrinsically modified polypeptide.
[0236] In one application, the methods of the invention can be used
to detect whether polypeptides present in food items have been
chemically modified by manufacturing processes. For example dairy
products can be tested for the presence of formaldehyde, which is
used as a bacteriostatic agent. Formaldehyde formylates gamma(2)
casein (Pizzano R. et al J. Agric Food Chem (2004) 52:649-54)
obscuring modified epitopes from subsequent detection by the
detecting agent.
Kits
[0237] The methods described herein are optionally performed by
utilizing pre-packaged diagnostic kits comprising the necessary
reagents to perform any of the methods of the invention. For
example, the kits typically include at least one specific nucleic
acid, peptide orantibody described herein, which are conveniently
used, e.g., in clinical settings, to screen and diagnose patients
and to screen and identify those individuals expressing a disease
conformation protein. Kit antibodies can comprise whole antibody,
antibody fragments, single chain antibody, monoclonal antibody
and/or polyclonal antibody. The kits optionally also include at
least one chemical agent for modifying epitopes recognized by an
antibody or aptamer. The kit is optionally based on ELISA
technology such as sandwich ELISA and DELFIA and may employ
detergents, precipitation agents (such as phosphotungstic acid) and
adsorbents typically used in ELISA technology and known to one
skilled in the art. The kit will also include detailed instructions
for carrying out the methods of the invention. Recombinant protein
are useful for standards in kits.
[0238] All such assays could be adapted and optimised to a simple
high-throughput platform.
[0239] The following non-limiting examples are illustrative of the
present invention.
EXAMPLES
Example 1
Peroxynitrite Reacts Differently with PrP in Normal and Acid
Treated or Scrapie Brain Homogenate
[0240] When brain homogenate is incubated at pH 3.5 in the presence
of guanidine, PrP becomes detergent insoluble and is more
susceptible to misfolding to a PK-resistant isoform in the presence
of PrP.sup.Sc (29). This acid treated PrP is a `model prion` which
is partially misfolded and/or aggregated resembling characteristics
of PrP.sup.Sc. When mock (.quadrature.) and acid treated
(.circle-solid.) brain homogenate is incubated with increasing
concentrations of peroxynitrite and then subjected to
immunoblotting, there is less PrP recognized by both 3F4 (FIGS. 1A
and C) and 6H4 (FIGS. 1B and D) in mock treated brain homogenate
than in acid treated brain homogenate. The PrP in the acid treated
brain homogenate is protected from modification by
peroxynitrite.
Example 2
PrP in Scrapie Infected Hamster Brain is Protected from
Modification by Peroxynitrite
[0241] The epitope protection phenomenon for `model prions` as
observed in example 1 was also observed for authentic
disease-misfolded prion protein in scrapie infected hamster (Ha)
brain (FIGS. 2A and B). As with model prions, the 3F4 and 6H4
epitopes of PrP in Ha.sup.Sc brain homogenate are protected from
modification by peroxynitrite. It is clear that `model prions` and
HaPrp.sup.sc share characteristics that provide protection from
chemical modification by peroxynitrite, such as differential
misfolding or aggregation.
Example 3
Aggregation is Responsible for the Reduction in
Peroxynitrite-Induced Epitope Modification of Misfolded PrP
[0242] To show that epitope protection of acid treated and scrapie
brain was due to aggregation, samples were treated with
peroxynitrite and then incubated with or without guanidine before
immunoprecipitation. Treatment of the samples with guanidine
dissociates aggregates of PrP (43-45) that protect the polypeptide
from modification by peroxynitrite. Incubation of mock treated
brain with 2.5 M guanidine after peroxynitrite treatment did not
show an increase in 3F4 and 6H4 epitopes as revealed by
immunoprecipitation (FIG. 3A lanes 1-4). However, when
peroxynitrite-treated acid brain homogenate was incubated with
guanidine, there was an increase in PrP that could be detected by
immunoprecipitation with 3F4 and 6H4 immunobeads (FIG. 3A lanes
5-8). This shows that guanidine is able to dissociate aggregates of
acid treated brain homogenate and release PrP that is protected
from modification by peroxynitrite. Other means of solubilizing PrP
aggregates were used and boiling samples in SDS loading buffer
resulted in the greatest observed solubilization to date.
Example 4
Optimization of EPA Parameters
[0243] Titration experiments with peroxynitrite, hydrogen peroxide
and methylene (based on UV light photolysis of the precursor
diazirine) or other modifying agents, identify the optimal
conditions for epitope protection in:
[0244] 1. Normal hamster and human brain "model prions", using
immunoblotting and conventional fluorescence ELISA.
[0245] 2. Infectious prions from hamster and human brain, using
immunoblotting analysis and time-resolved fluorescence In each
case, brain homogenates are prepared and mixed with increasing
concentrations of the modifying agent and processed as described
(immunoblotting, and time resolved fluorescence). This defines the
type and concentration of chemical agent allowing the maximal
distinction between monomeric and aggregated prion proteins.
Additional informative control experiments include using
recombinant hamster PrP.sup.C in buffer and in PrP.sup.-/- knockout
mouse brain, and by mouse normal and scrapie-infected brain (murine
PrP is 6H4+ and 3F4-).
[0246] In some cases, infectious prions may have different
properties for chemical modification than do "model prions," and
brain prions may display different chemical modification properties
than do endogenous prions circulating in blood, or PrP.sup.Sc
detectable in urine of infected animals. One of skill in the art
shall readily identify the optimal conditions for authentic
endogenous prions using known techniques.
Example 5
EPA Adapted to a Fluorescent ELISA System
[0247] The epitope protection assay for aggregated PrP was adapted
to a fluorescent sandwich ELISA system using 6H4 as the capture
antibody and 3F4 as the detection antibody (FIG. 3B). The sandwich
ELISA assay system is able to identify aggregated PrP in acid
treated brain homogenate but only if the samples are boiled in SDS
loading buffer after peroxynitrite treatment. At peroxynitrite
concentrations greater than 8 mM, there is 2.5-3.times. as much PrP
detected in the acid treated sample as compared to the mock treated
sample.
Example 6
Detection of a Single Brain Prion
[0248] A single brain prion has been estimated to comprise
10.sup.5-10.sup.6 molecules of PrP.sup.Sc. Detection of
10.sup.8-10.sup.9 molecules of recombinant PrP using conventional
fluorescence ELISA has been accomplished. The assay used is about
1000-fold more sensitive for single-prion detection--the necessary
sensitivity is provided by the Dissociation enhanced lanthanide
fluoroimmunoassay (DELFIA). DELFIA uses a chelated
lanthanide-labeled tracer, such as europium (Eu) and time-resolved
fluorescence (TRF) to measure output signal. The benefit of
lanthanide chelates is that their fluorescence duration is 200,000
times longer than conventional fluorophors, allowing signal capture
after non-specific interfering fluorescence has faded (particularly
critical for biological samples, which may possess considerable
non-specific fluorescence). DELFIA-based systems can measure as
little as 100 fmol/well of Eu which is >1000 times more
sensitive than conventional ELISA assays, which detects single
prions by EPA. The optimal TRF 96-well plate reader for the DELFIA
system is manufactured by Wallac-Victor (Perkin-Elmer), and is used
to automate sample analysis.
[0249] Using an optimal chemical modifier and optimal conditions a
sensitive capture 96-well plate assay for detection of hamster and
human prions, using the DELFIA TRF system is provided. This is used
to: [0250] 1. Characterize, optimize and quantify detection of
recombinant prion protein by TRF. [0251] 2. Determine the
sensitivity of the DELFIA-TRF for hamster and human brain
prions.
Example 7
[0251] Detection of Prion Proteins in Biological Fluids
[0252] The EPA achieves commercial utility by detecting PrPSc in
biological tissues and fluids for which no present technology
exists. Blood prions are in very low abundance (10-100 prions/mL by
bioassay, and protease-resistant PrP in urine is only
intermittently/sporadically detectable by precipitation of large
fluid volumes. Also, any prospective blood test must contend with
high concentrations of PrP.sup.C (10.sup.6-fold more than
PrP.sup.Sc) and "blocking" by heterologous plasma proteins. Using
the optimized chemical modification regimen and the DELFIA-TRF
system, the sensitivity thresholds for EPA in blood and urine are
determined using: [0253] 1. Hamster and human plasma and urine
"spiked" with a titration of 263K hamster prions; [0254] 2. Plasma
and urine from Syrian hamsters "endogenously" infected with 263K
prion disease
[0255] Biological fluids clinically accessible by non-invasive
routes provide a substrate for a practical antemortem test for
diagnosis and screening of prion infection in humans and animals.
The methods of this invention may also be used in post-mortem
testing. One of skill in the art readily determines whether EPA
with "prion spike" titration in normal blood and urine reveals
similar DELFIA-TRF signals to the same prion titration in buffer,
showing that the EPA is not affected by "blocking factors" in these
biological fluids. Interestingly, preferential "blocking" of
PrP.sup.Sc by heterologous proteins may actually enhance epitope
protection to chemical modifying agents. If decreased detection of
prions in blood or urine is observed, pre-clearing strategies are
readily employed to enhance PrP.sup.Sc detection with detergents,
precipitating agents, and adsorbents typically used in commercial
ELISA assays which are known to one skilled in the art.
[0256] Human and bovine plasma and urine and other bodily fluids
are tested using optimized EPA conditions and compared to samples
from human variant CJD and BSE, respectively. Although the
monoclonal antibody 6H4 recognizes PrP from all relevant species,
other antibodies (commercially available) are used for the DELFIA
TRF system for cattle, sheep, and cervids, which lack the 3F4
epitope. Other antibodies and epitopes useful in methods described
in this application will be readily apparent to those of skill in
the art.
Example 8
Detection of Aggregated Amyloid Beta (Abeta) Using EPA
[0257] Amyloid beta peptide (Abeta) is a normal cleavage product of
the proteolytic processing of amyloid precursor protein (APP).
Abeta accumulates in discrete plaques in affected regions of
Alzheimer's disease brain, and triggers neuronal death and gliosis
observed in this disease. Plaque Abeta is aggregated and rich in
beta-sheet structure, in contrast to the Abeta region of APP
expressed by normal cells. Using epitope protection technology, it
was demonstrated that the 6E10 epitope in the Abeta region of APP
is less accessible to peroxynitrite modification in Alzheimer's
disease brain compared to normal brain (FIG. 4 panel A). Similarly,
the 6E10 epitope is partially protected in brain homogenates that
have been treated at low pH to induce protein aggregation (FIG. 4
panel B). Abeta 1-42 peptide aggregated by overnight incubation at
1 mg/ml in water shows prominent epitope protection of the 6E10
epitope to peroxynitrite modification, in comparison with soluble
non-aggregated Abeta 1-42 (FIG. 4 panel C). A further example of
this phenomenon is presented in FIG. 4, panel D which shows normal
(.largecircle.) and aggregated (.circle-solid.) Abeta 1-42 treated
with increasing concentrations of peroxynitrite, subjected to
immunoblotting with 6E10 antibody.
[0258] The 6E10 epitope of APP is also unavailable to peroxynitrite
modification in AD brain homogenates and in normal brain
homogenates aggregated by low pH, but normal untreated brain does
not show this protection (FIG. 4 panel A and B), showing molecular
interaction in vivo of APP with an Abeta-domain blocking molecule
(perhaps Abeta itself; ref. 9).
[0259] The sensitive and specific EPA detection of aggregated Abeta
in biological fluids (such as blood and spinal fluid), or
protection of Abeta epitopes in APP in cells and tissues, provides
an antemortem diagnostic test for Alzheimer's disease. The methods
of the invention described in this application are used for this
diagnostic test.
Detection of Aggregated Tau Protein by EPA
[0260] Dying neurons release intracellular proteins such as tau
into the CSF (39) and likely ultimately blood. Tau studies are
directly performed on brain specimens including Alzheimer patient
samples and control brain.
Example 9
Detection of Aggregated Superoxide Dismutase 1 (SOD1) by EPA
[0261] SOD1-containing cytoplasmic inclusions are detected in many
diseased motor neurons from familial and sporadic ALS patients
(15), and in most transgenic mouse (16, 17) and tissue culture
models (18) of the disease. Human SOD1 can be aggregated in vitro.
Further SOD1 is modifiable by succinic anhydride and DEPC. This
property can be exploited by EPA technology to discriminate between
aggregated and unaggregated SOD1 protein.
[0262] Purified SOD1 from human erythrocytes (Sigma) was aggregated
in a metal-catalyzed oxidation reaction. Soluble SOD1 was treated
with varying concentrations of DEPC, denatured with heat, and
immunoblotted with anti-SOD1 antibodies. Western blotting shows
that increasing concentrations of DEPC are associated with
decreases in antibody binding in soluble SOD1 (FIG. 5) showing that
sites are available for modification by a blocking agent.
[0263] Antibodies against SOD1 selected a priori for utility in EPA
are used to distinguish disease specific aggregated SOD1 from
wildtype SOD1. Relevant factors include: [0264] 1) selecting an
epitope on the molecular surface of the native dimer to be
accessible to chemical modification in the native soluble state;
[0265] 2) identifying a linear epitope to optimize detection in the
denatured state on immunoblots and ELISA; [0266] 3) immunogenicity;
[0267] 4) uniqueness to SOD1; and [0268] 5) presence of acidic
amino acids (Glu and Asp) that are readily modified by
epoxides.
[0269] The five SOD1 sequences that meet these criteria are:
22QKESNG27; 51E D N T A G C T S A 60; 74PK D E E R H V 81; 89A D K
D G93; and 127GKGGNEQSTK136 (in bold: solvated side chains).
[0270] Additionally the electrostatic loop sequence and zinc
binding loop of human SOD1 are surface-accessible sequences and are
involved in aggregate formation (Elan, J. et al. Nature Structural
Biology (2003) 10:461-67).
[0271] These sequences are: TABLE-US-00002 Electrostatic loop of
human SOD1: Asp Leu Gly Lys Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly
Asn Ala Gly Ser Zinc-binding loop of human SOD1: Asn Pro Leu Ser
Arg Lys His Gly Gly Pro Lys Asp Glu Glu
Example 10
Detection of Aggregated Alpha-Synuclein by EPA
[0272] Most cases of Parkinson's disease are sporadic, but both
sporadic and familial forms of the disease are characterized by
intracellular Lewy bodies in dying neurons of the substantia nigra,
a population of midbrain neurons (.about.60,000) that are
selectively decimated in PD. Lewy bodies are predominantly composed
of alpha-synuclein (22). Mutations in the gene encoding
alpha-synuclein have been found in patients with familial
Parkinson's disease (reviewed in 23). Another gene associated with
autosomal recessive PD is parkin, which is involved in
alpha-synuclein degradation (22, 23). Diffuse cortical Lewy bodies
composed of alpha-synuclein are observed in Lewy body disease
(LBD), a dementing syndrome associated with Parkinsonian tone
changes, hallucinations, and rapid symptom fluctuation (24).
[0273] The Syn-1 epitope is optionally blocked by chemical
modification of recombinant alpha-synuclein with DEPC (histidine
reactive), and alpha-synuclein aggregated in vitro is partially
protected from DEPC epitope blocking (FIG. 6).
[0274] Aggregated alpha-synuclein in vitro is protected from
modification by DEPC whereas normal protein is not. Three mg/mL
mutant A53T alpha-synuclein was incubated at 37.degree. C. for
three days for aggregation. The aggregation reaction was applied to
ultracentrifugation. Normal protein prior to aggregation
(containing soluble alpha-synuclein) and the pellet resuspension
from the ultracentrifugation (containing insoluble alpha-synuclein)
were treated with varying concentrations of DEPC, denatured with
heat, and blotted with Syn-1 antibody from BD Biosciences. FIG. 6A
shows that increasing concentrations of DEPC are associated with a
gradual decrease in antibody binding in normal alpha-synuclein.
Insoluble alpha-synuclein shows little change in antibody binding
with increasing concentrations of DEPC until the DEPC
concentrations reach 1 mM. A graphical representation of these
findings is presented in FIG. 6B. The extent of antibody binding to
DEPC-treated normal alpha-synuclein (-) decreases gradually overall
but more rapidly at higher concentrations of DEPC. Insoluble
alpha-synuclein (.pi.), on the other hand, shows little change in
the extent of antibody binding. The last data point at 0.01 M DEPC
for insoluble alpha-synuclein increases due to the darkening of the
film.
Example 11
Detection of Aggregated Proteins in CSF
[0275] Extracellularly deposited Abeta has been quantified in CSF
and blood of patients with AD and normal controls (6-8).
Intracellular neuronal proteins, such as alpha-synuclein, have been
detected in CSF and blood (37, 38). Dying neurons release
intracellular proteins 14-3-3, neuron-specific enolase, tau, and
alpha-synuclein into the CSF (39), and likely ultimately blood. A
proportion of released protein in disease is in an aggregated form.
EPA technology is applied to determine the proportion of
polypeptide aggregates in CSF samples from patients with AD, ALS,
PD, and LBD. Signal is measured for polypeptides disaggregated
before and after chemical treatment, representing "total" and
"protected" epitopes, respectively, to determine the proportion of
polypeptide in the aggregated state. Using the optimized mAbs and
chemical modification regimens, and the DELFIA-TRF system, EPA
sensitivity is determined in: [0276] 1. Normal CSF "spiked" with
polypeptides aggregated in vitro. [0277] 2. CSF from patients with
AD, ALS, PD, and LBD.
[0278] The proportion of aggregated polypeptides in a CSF sample,
is determined even if it constitutes only 10.sup.5-10.sup.6
molecules. Detergents, precipitating agents (such as
phosphotungstic acid), and adsorbents typically used in commercial
ELISA assays to enrich for relevant species are optionally
employed. Biological fluids clinically accessible by non-invasive
routes provides an ideal substrate for a practical antemortem test
for diagnosis and screening of neurodegenerative diseases.
Materials and Methods
Materials
[0279] Recombinant hamster PrP (rhaPrP) and 6H4 was from Prionics.
Recombinant human PrP (rhuPrP) was from Roboscreen. Biotin-3F4 and
3F4 were from Signet. 3F4 reacts against MKHV and 6H4 reacts
against DYEDRYYRE. 6E10 anti-Abeta (from Signet) reacts against
EFRHDS (residues 3-8).
[0280] Other antibodies and the epitopes recognized if known, are
provided in table 1 above.
Preparation of Acid-Misfolded PrP and APP.
[0281] Acid misfolded PrP was used as "model prions" in this study
and was prepared as in (29). Briefly, 100 .mu.l of 10% brain
homogenate was mixed with an equal volume of 3.0 M GdnHCl (final
concentration 1.5 M) in PBS at pH 7.4 or pH 3.5 adjusted with 1 N
HCl, followed by rotation at room temperature. After 5 h
incubation, samples were methanol precipitated with 5 volumes of
ice-cold methanol and pellets were resuspended in 100 .mu.l of
lysis buffer. The samples treated at pH 7.4 were designated as
mock-treated samples. Peroxynitrite treatment of Brain
Homogenates
[0282] An aliquot (18 .mu.l) of normal or misfolded/diseased brain
homogenate was vortexed while 2 .mu.l of peroxynitrite in 100 mM
NaOH/60 mM H.sub.2O.sub.2 was added to give a final peroxynitrite
concentration of 0-15 mM. After vortexing for a further 15 s, the
samples were subjected to Western blotting, immunoprecipitation or
sandwich ELISA.
DEPC Treatment of Erythrocyte SOD1
[0283] Purified SOD1 from human erythrocytes (Sigma) is aggregated
in a metal-catalyzed oxidation reaction consisting of 40 _M SOD1, 4
mM ascorbic acid, and 0.2 mM CuCl.sub.2 in 10 mM Tris-acetate
buffer (pH7) at 37.degree. C. for three days. Ultracentrifuged
supernatant (containing soluble SOD1) and the pellet resuspension
(containing insoluble SOD1) are treated with varying concentrations
of DEPC (100 pM to 0.1 M), denatured with heat, and immunoblotted
with anti-SOD1.
Western Blotting
[0284] Samples were boiled in SDS loading buffer (62 mM Tris (pH
6.8), 10% glycerol, 2% SDS, 5% beta-mercaptoethanol and 0.01%
bromphenol blue) for 5 min. and separated on 12% Tris-Glycine
polyacrylamide gels followed by transfer to Hybond-P. PrP was
detected using 3F4 (1:50000) 6H4 (1:10000) or 6E10 (1:1000) as the
primary antibodies and HRP-conjugated goat anti-mouse (1:10000) as
the secondary antibody followed by exposure to ECL-Plus and
visualization by exposure to Kodak X-OMAT film. Band intensities
were quantitated using UnScan-IT software.
Immunoprecipitation
[0285] Samples were incubated with 50 .mu.l of Ab-conjugated (100
.mu.g/ml) Dynal M-280 magnetic beads in a final volume of 1 ml
binding buffer (3% NP-40; 3% Tween-20) for 3 h at room temperature
with rotation. Beads were washed in wash buffer (2% NP-40; 2%
Tween-20) .times.3 and boiled in 30 .mu.l SDS loading buffer
without beta-mercaptoethanol for 5 min. Supernatants were analyzed
by Western blotting as described above.
Sandwich ELISA
[0286] The capture antibody (6H4; 1:5000 in 50 mM bicarbonate
binding buffer, pH 9.6) was bound to an opaque 96-well plate (Nunc
Maxisorp) by overnight incubation at 4.degree. C. After blocking
with 1% BSA in 0.05% TBST for 2 h, plates were washed 3.times. in
TBST and incubated overnight at 4.degree. C. with standard
concentrations of rhuPrP or rHaPrP along with unknown brain
homogenates. Plates were washed 3.times. and incubated with the
detecting antibody biotin-3F4 (1:5000) at RT for 1 h. After washing
3.times., avidin-HRP (1:5000) was added and incubated for 30 min.
at RT. Following a final wash step (.times.3) the plate was
developed with Quantablu fluorescent substrate for 10-90 min at RT
and fluorescent intensities determined with an excitation of 325 nm
and emission of 420 nm.
[0287] While the present invention has been described with
reference to what are presently considered to be the preferred
examples, it is to be understood that the invention is not limited
to the disclosed examples. To the contrary, the invention is
intended to cover various modifications and equivalent arrangements
included within the spirit and scope of the appended claims.
[0288] All publications, patents and patent applications, including
U.S. application No. 60/496,381 (entitled Methods of Detecting
Prion Protein (Cashman & Lehto), filed on Aug. 20, 2003 and
U.S. application No. 60/497,362 (entitled Epitope Protection Assay
(Cashman & Lehto), filed on Aug. 21, 2003 and the Corresponding
Canadian applications nos. 2,437,675 and 2,437,999 are herein
incorporated by reference in their entirety to the same extent as
if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety.
REFERENCES
[0289] 1. Prusiner S B. Shattuck lecture--neurodegenerative
diseases and prions. N Engl J Med. 344:1516-26, 2001. [0290] 2.
Caselli R J. Current issues in the diagnosis and management of
dementia. Semin Neurol. 23:231-40, 2003. [0291] 3. Cashman N R. Do
the benefits of currently available treatments justify early
diagnosis and announcement? Arguments for. Neurology. 53(Suppl
5):S50-2, 1999. [0292] 4. Selkoe D J. Alzheimer's disease: genes,
proteins, and therapy. Physiol Rev. 81:741-66, 2001. [0293] 5.
Puglielli L, Tanzi R E, Kovacs D M. Alzheimer's disease: the
cholesterol connection. Nat Neurosci. 6:345-51, 2003. [0294] 6.
Mehta P D, Pirttila T, Mehta S P. Plasma and cerebrospinal fluid
levels of amyloid beta proteins 1-40 and 1-42 in Alzheimer disease.
Arch Neurol. 57:100-5, 2000. [0295] 7. Clark C M, Xie S, Chittams J
et al. Cerebrospinal fluid tau and beta-amyloid: how well do these
biomarkers reflect autopsy-confirmed dementia diagnoses? Arch
Neurol. 60:1696-702, 2003. [0296] 8. Green A J. Cerebrospinal fluid
brain-derived proteins in the diagnosis of Alzheimer's disease and
Creutzfeldt-Jakob disease. Neuropathol Appl Neurobiol. 28:427-40,
2002. [0297] 9. Lorenzo A, Yuan M, Zhang Z, et al. Amyloid beta
interacts with the amyloid precursor protein: a potential toxic
mechanism in Alzheimer's disease. Nat Neurosci. 3:460-4, 2000.
[0298] 10. Rosen D R, Siddique T, Patterson D. et al. Mutations in
Cu/Zn superoxide dismutase gene are associated with familial
amyotrophic lateral sclerosis. Nature. 362:59-62, 1993. [0299] 11.
Deng H X, Hentati A, Tainer J A et al. Amyotrophic lateral
sclerosis and structural defects in Cu,Zn superoxide dismutase.
Science. 20; 261:1047-51, 1993. [0300] 12. Anderson, P M in Brown R
H, Meininger V, Swash eds. Amyotrophic Lateral Sclerosis. London:
Martin Dunitz. 2000. [0301] 13. Gurney, M. E., Pu, H., Chiu, A. Y.,
et al. Motor neuron degeneration in mice that express a human Cu,Zn
superoxide dismutase mutation. Science 264:1772-1775, 1994. [0302]
14. Ripps, M. E., Huntley, G. W., Hof, P. R., et al. Transgenic
mice expressing an altered murine superoxide dismutase gene provide
an animal model of amyotrophic lateral sclerosis. Proc. Natl. Acad.
Sci. U.S.A 92: 689-693, 1995. [0303] 15. Kato, S., Takikawa, M.,
Nakashima, K., et al. New consensus research on neuropathological
aspects of familial amyotrophic lateral sclerosis with superoxide
dismutase 1 (SOD1) gene mutations: inclusions containing SOD1 in
neurons and astrocytes. Amyotroph. Lateral. Scler. Other Motor
Neuron Disord. 1: 163-184, 2000. [0304] 16. Bruijn, L. I., Becher,
M. W., Lee, M. K. ALS-linked SOD1 mutant G85R mediates damage to
astrocytes and promotes rapidly progressive disease with
SOD1-containing inclusions. Neuron 18: 327-338, 1997. [0305] 17.
Bruijn, L. I., Houseweart, M. K., Kato, S., Aggregation and motor
neuron toxicity of an ALS-linked SOD1 mutant independent from
wild-type SOD1. Science 281: 1851-1854, 1998. [0306] 18. Durham, H.
D., Roy, J., Dong, L., and Figlewicz, D. A. Aggregation of mutant
Cu/Zn superoxide dismutase proteins in a culture model of ALS. J.
Neuropathol. Exp. Neurol. 56:523-530, 1997. [0307] 19 Li Y K, Chir
J. Chen F Y. Catalytic mechanism of a family 3 beta-glucosidase and
mutagenesis study on residue Asp-247. Biochem J 355(Pt 3):835-40,
2001 [0308] 20 Rose R B, Rose J R, Salto R, Craik C S, Stroud R M.
Structure of the protease from simian immunodeficiency virus:
complex with an irreversible nonpeptide inhibitor. Biochemistry
32:12498-507, 1993. [0309] 21. Olanow C W. The scientific basis for
the current treatment of Parkinson's disease. Annu Rev Med
55:41-60, 2004. [0310] 22. Iwatsubo T. Aggregation of
alpha-synuclein in the pathogenesis of Parkinson's disease. J
Neurol 250 Suppl 3:III 11-4, 2003. [0311] 23. Eriksen J L, Dawson T
M, Dickson D W, Petrucelli L Caught in the ac: alpha-synuclein is
the culprit in Parkinson's disease. Neuron 40:453-6, 2003. [0312]
24. McKeith I, Mintzer J, Aarsland D et al. Dementia with Lewy
bodies. Lancet Neurol 3:19-28, 2004. [0313] 25 Alvarez B,
Ferrer-Sueta G, Freeman B A, Radi R. Kinetics of peroxynitrite
reaction with amino acids and human serum albumin. J Biol Chem
274:842-8, 1999. [0314] 26. Alvarez B, Radi R Peroxynitrite
reactivity with amino acids and proteins. Amino Acids 25:295-311,
2003. [0315] 27. Sokol P P, Holohan P D, Ross C R. Arginyl and
histidyl groups are essential for organic anion exchange in renal
brush-border membrane vesicles. J Biol Chem 263:7118-23, 1988.
[0316] 28. Zou W-Q, Yang D-S, Fraser P E, Cashman N R, Chakrabartty
A. All-or-none fibrillogenesis of a prion peptide. Europ J Biochem
268:4885-4891, 2001. [0317] 29. Zou W-Q, Cashman N R. Acidic pH and
detergents enhance in vitro conversion of human brain PrPC to a
PrPSc-like form. J Biol Chem 277:43942-43947 2002. [0318] 30.
Rakhit R, Cunningham P, Furtos-Matei A, Dahan S, Qi X-F, Crow J,
Cashman N R, Kondejewski L H, Chakrabartty A. Oxidation-induced
misfolding and aggregation of superoxide dismutase and its
implications for amyotrophic lateral sclerosis. J Biol Chem
277:47551-62002, 2002. [0319] 31. Paramithiotis E, Pinard M, Lawton
T, LaBoissiere S, Leathers V L, Zou W-Q, Estey L A., Kondejewski L
H, Francoeur G P, Papadopoulos M, Haghighat A, Spatz S J, Tonelli
Q, Ledebur H C, Chakrabartty A, Cashman N R. A PrPSc-specific
immunological epitope. Nature Medicine 9:893-9, 2003. [0320] 32.
Rakhit R, Crow J P, Lepock J R, Kondejewski L H, Cashman N R,
Chakrabartty A. Monomeric Cu/Zn superoxide dismutase is a common
misfolding intermediate in the oxidation models of sporadic and
familial ALS. J Biol Chem e-pub January 2004. [0321] 33. MacGregor,
I., Hope, J., Barnard, G. Application of a time-resolved
fluoroimmunoassay for the analysis of normal prion protein in human
blood and its components. Vox Sang 77:88-96, 1999. [0322] 34.
Chishti M A, Yang D S, Janus C et al. Early-onset amyloid
deposition and cognitive deficits in transgenic mice expressing a
double mutant form of amyloid precursor protein 695. J Biol Chem
276:21562-70, 2001. [0323] 35. Bolton D. C., McKinley M. P., and
Prusiner S. B. Identification of a protein that purifies with the
scrapie prion. Science 218:1309-1311, 1982. [0324] 36. Beekes M.,
Baldauf E., and Diringer H. Sequential appearance and accumulation
of pathognomic markers in the central nervous system of hamsters
orally infected with scrapie J. Gen. Virol 77:1925-1934, 1996.
[0325] 37. Borghi R, Marchese R, Negro A, et al. Full length
alpha-synuclein is present in cerebrospinal fluid from Parkinson's
disease and normal subjects. Neurosci Left. 287:65-7, 2000. [0326]
38. El-Agnaf O M, Salem S A, Paleologou K E, et al. Alpha-synuclein
implicated in Parkinson's disease is present in extracellular
biological fluids, including human plasma. FASEB J 17:1945-7, 2003.
[0327] 39. Verbeek M M, De Jong D, Kremer H P. Brain-specific
proteins in cerebrospinal fluid for the diagnosis of
neurodegenerative diseases. Ann Clin Biochem 40(Pt 1):25-40, 2003.
[0328] 40. Coulthart, M. B. and Cashman, N. R. (2001) CMAJ. 165,
51-58 [0329] 41. Prusiner, S. B. (1998) Proc. Natl. Acad. Sci.
U.S.A 95, 13363-13383 [0330] 42 Will, R. G., Ironside, J. W.,
Zeidler, M., Cousens, S. N., Estibeiro, K., Alperovitch, A., Poser,
S., Pocchiari, M., Hofman, A., and Smith, P. G. (1996) Lancet 347,
921-925. [0331] 43. Kocisko, D. A., Lansbury, P. T., Jr., and
Caughey, B. (1996) Biochemistry 35, 13434-13442 [0332] 44, Barnard,
G., Helmick, B., Madden, S., Gilbourne, C., and Patel, R. (2000)
Luminescence. 15, 357-362 [0333] 45. Meyer, R. K., Oesch, B.,
Fatzer, R., Zurbriggen, A., and Vandevelde, M. (1999) J. Virol. 73,
9386-9392 [0334] 46. Kang, S. C., Li, R., Wang, C., Pan, T., Liu,
T., Rubenstein, R., Barnard, G., Wong, B. S., and Sy, M. S. (2003)
J. Pathol. 199, 534-541. [0335] 47. Shaked G M, Shaked Y,
Kariv-lnbal Z, Halimi M, Avraham I, Gabizon R. (2001) J Biol Chem.
276, 31479-82. [0336] 48. Ross C A et al. Nature Medicine, (2004)
S10-17.) [0337] 49. Davies S W et al Cell 90, 537-548 (1997).
[0338] 50. Scherzinger E et al. Proc. Natl. Acad. Sci. USA 96,
4604-9, (1999). [0339] 51. Sen S et al Protein Sci. (2003)
12:953-962. [0340] 52. Llewelyn C A et al. Lancet (2004)
363:417-421 [0341] 53. Peden A H et al. Lancet (2004) 364:527-529
[0342] 54. Andreoletti O et al. Nat. Med. (2004) 6:591-593 [0343]
55. Thomzig A et al. J Clin Invest. (2004) 10:1465-72. [0344] 56.
Glatzel M et al. N Engl J Med. (2003) 349:1812-20 [0345] 57. Bosque
P J et al. Proc Natl Acad Sci USA. (2002) 99:3812-7
Sequence CWU 1
1
10 1 4 PRT Artificial sequence epitope 1 Met Lys His Val 1 2 9 PRT
Artificial sequence epitope 2 Asp Tyr Glu Asp Arg Tyr Tyr Arg Glu 1
5 3 6 PRT Artificial sequence epitope 3 Glu Phe Arg His Asp Ser 1 5
4 6 PRT Homo sapiens 4 Gln Lys Glu Ser Asn Gly 1 5 5 10 PRT Homo
sapiens 5 Glu Asp Asn Thr Ala Gly Cys Thr Ser Ala 1 5 10 6 8 PRT
Homo sapiens 6 Pro Lys Asp Glu Glu Arg His Val 1 5 7 5 PRT Homo
sapiens 7 Ala Asp Lys Asp Gly 1 5 8 10 PRT Homo sapiens 8 Gly Lys
Gly Gly Asn Glu Gln Ser Thr Lys 1 5 10 9 18 PRT Homo sapiens 9 Asp
Leu Gly Lys Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala 1 5 10
15 Gly Ser 10 14 PRT Homo sapiens 10 Asn Pro Leu Ser Arg Lys His
Gly Gly Pro Lys Asp Glu Glu 1 5 10
* * * * *
References