CA2437999A1

CA2437999A1 - Epitope protection assay

Info

Publication number: CA2437999A1
Application number: CA002437999A
Authority: CA
Inventors: Neil Cashman; Marty Letho
Original assignee: Individual
Current assignee: Promis Neurosciences Inc
Priority date: 2003-08-20
Filing date: 2003-08-21
Publication date: 2005-02-20
Anticipated expiration: 2023-08-21
Also published as: ES2566565T3; DK1668369T3; CA2437999C

Abstract

The invention relates to an epitope protection assay for use in diagnosis, prognosis and therapeutic intervention in diseases involving polypeptide aggregation such as prion infections. The methods of the invention first block accessible polypeptide target epitope with a protecting agent. After denaturation of the polyeptide, a detecting agent is used to detect protein with target epitope that was inaccessible during contact with the protecting agent.

Description

TITLE: Epitope Protection Assay FIELD OF INVENTION
The invention relates to an epitope protection assay for use in diagnosis, prognosis and therapeutic intervention in diseases involving polypeptide aggregation such as prion infections.
BACKGROUND OF THE INVENTION
Prion diseases have become a major health concern since the outbreak of BSE or "Mad Cow Disease" (reviewed above, refs 1,2). 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.
Emeraina Prion Diseases.
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'~2.
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)3. The primary epidemic from consumption of contaminated cattle tissue has affected over 130 individuals as of this filing.

The spectre of vCJD "secondary epidemics" through blood, blood products, surgery, dentistry, vaccines, and cosmetics is of great concern ''2. Detection of blood priors infectivity in experimental BSE/vCJD infections of mice and sheep' suggests a special risk exists for the transmission of vCJD through blood and blood products. Canada and the United States have recently expanded vCJD
blood donor deferrals to all countries in Western Europe.
Although sheep scrapie has been known for centuries, the most important animal priors 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
priors strain. A recent report showed that prions can replicate in certain muscle groups, indicating a potential risk in tissues previously considered safe for human consumption.
Chronic wasting disease (CWD) of captive and wild cervids (deer and elk) represents another newly emergent animal priors disease in North America, whose impact on human health is yet unknown. It is apparent that newly-recognized priors diseases pose a threat to the safety of foods, blood products, and medical-surgical treatments.
Prions: Atyt~ical Pathogens.
Newly emergent priors diseases, and the polypeptide-only nature of prions, have created serious medical, veterinary, and economic challenges worldwide. To date, the only commercialised tests for priors 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 priors infection is a challenging task, in part due to the unusual nature of the priors 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 2.
According to the prion theory developed by Nobel Laureate Dr. Stanley Prusiner, infectivity resides in PrPS', a misfolded conformational isoform of the near-ubiquitous normal cellular prion polypeptide PrPc. PrPs° 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.
PrPS° is partially resistant to protease digestion, poorly soluble, and exists in an aggregated state, in contrast to the protease sensitive, soluble, monomeric isoform PrPc a.~o.
PrPS° is derived from its normal cellular isoform (PrPc), which is rich in a-helical structure, by a posttranslational process involving a conformational transition.
While the primary structure of PrPc is identical to that of PrPs~, secondary and tertiary structural changes are responsible for the distinct physicochemical properties of the two isoforms.
One of the difficulties in assessing the safety of food or blood products from potentially infected humans with prions is the Pack 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 6 months.
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.
Some tests that are in the process of being commercialized are based on surrogate markers of infection which are "once removed" from actual infectious pnons.
PrP protease resistance, 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, enzymes that can digest PrP~, but leave a protease-resistant core of PrPS°. The protease-resistant fragment of PrPs° 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 PrPS' which is infectious and aggregated, but which is not protease resistant. Protease-sensitive PrPs' also predominates early in infection and in cross-species transmission of disease 4.
Detection of protease-resistant PrP fragments is also the basis of a urine diagnostic test " 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 PrPs~ in late stages of the disease, not pre-symptomatically).
SUMMARY OF THE INVENTION
The inventors have recently discovered the epitope protection assay (EPA), a novel method that yields sensitive and specific antemortem detection of prions in blood and other accessible tissues and fluids. In prion diseases, the normal cellular monomeric prion polypeptide PrPc undergoes refolding to an abnormal, aggregated isoform, generically designated PrPs'.
According to the invention, the methods are useful where a target epitope is accessible in either one of a disease protein or a wild type protein and inaccessible in the other.
The invention relates to a detection method comprising:
reacting a sample of polypeptide with a chemical modifying agent, typically a protecting agent, which modifies exposed epitopes so that they cannot bind to detection agents;
disaggregating and/or denaturing the polypeptide in the sample; and probing with detection agents, such as antibodies against a target epitope to determine whether the polypeptide prior to disaggregation and/or denaturing, included inaccessible target epitopes.
The result indicates whether the polypeptide includes inaccessible epitope, which is indicative of the type of polypeptide that is present (i.e. wild type or disease protein).
In one embodiment, the prion detection method comprises:
reacting a sample of polypeptide (the sample typically contains PrPs°
and/or PrPc, and in many cases an abundance of one or the other) with a chemical modifying agent, typically a protecting agent such as peroxynitrite, which modifies exposed epitopes so that they cannot bind to a detection agent;
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.
PrPc 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 PrPS° are "protected" from chemical modification by virtue of being sequestered within aggregates.
In one embodiment, the Alzheimer's disease detection method comprises:
reacting a sample of polypeptide (the sample typically contains all or part of diseased amyloid precursor polypeptide or amyloid beta 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 protecting agent such as peroxynitrite, which modifies exposed epitopes so that they cannot bind to a detection agent;
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.
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 PrPS', the pathogenic molecule which constitutes the infectious particle.
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 s 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.
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:
contacting the candidate polypeptide with a protecting agent and next determining whether the target epitope is inacessible or accessible to chemical modification by the protecting agent. 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.
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:
contacting the polypeptide with a protecting 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 protecting agent, and wherein in the other conformation, the target epitope is inaccessible and does not react with the protecting agent;
removing unreacted protecting agent from contact with the polypeptide;
next modifying the candidate polypeptide to convert any inaccessible target epitope to accessible target epitope;

next 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.
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:
contacting the polypeptide with a protecting agent that selectively blocks accessible target epitope, wherein in the wildtype conformation, the target epitope is accessible and reacts with the protecting agent, and wherein in the non-wildtype conformation, the target epitope is inaccessible and does not react with the protecting agent;
removing unreacted protecting agent from contact with the polypeptide;
next modifying the candidate polypeptide to convert any inaccessible target epitope to accessible target epitope;
next 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 wherein lack of binding between the detection agent and the target epitope indicates that the polypeptide was in a wildtype conformation.

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:
contacting the polypeptide with a protecting agent that selectively blocks accessible target epitope, wherein in the non-wildtype conformation, the target epitope is accessible and reacts with the protecting agent, and wherein in the wildtype conformation, the target epitope is inaccessible and does not react with the protecting agent;
removing unreacted protecting agent from contact with the polypeptide;
next modifying the candidate polypeptide to convert any inaccessible target epitope to accessible target epitope;
next 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.
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 PrPs°.
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.
The blocking agent is optionally peroxynitrite, hydrogen peroxide, diethyl pyrocarbonate, 4-hydroxynonenal (4HNE) or diazerine. In the methods, the polypeptide is optionally modified by denaturing the polypeptide, for example with heat, detergent and/or 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 guanidine, detergent and heat. The detection agent optionally comprises an antibody directed against a prion polypeptide epitope or an amyloid beta 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 The misfolded conformation is typically indicative of a disease or disorder caused by protein misfolding, such as BSE, CJD or Alzheimer's disease. In the methods, the epitope is in many cases inaccessible in the misfolded 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, iii) 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 polyeptide prevent or reduce reactions between the blocking agent and the target epitope.
The methods of the invention are preferably used with mammals, such as humans.
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
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 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 (o) and acid treated ( ~ ) 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 (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. ( ~ ) Scrapie infected hamster brain. (o) Normal hamster brain.
Fig. 3 Protection from peroxynitrite induced modification is due to aggregation in acid treated brain (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 suggest that misfolded PrP
is protected from destruction by peroxynitrite treatment, possibly due to aggregation.
Fig. 4 Detection of aggregated amyloid beta (Abets) using EPA
The 6E10 epitope in the Abets 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). Abets 1-42 peptide aggregated in vitro shows prominent epitope protection of the 6E10 epitope to peroxynitrite modification, in comparison with soluble non-aggregated Abets 1-42 (panel C).
DETAILED DESCRIPTION OF THE INVENTION
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 polypeptide. In one embodiment, the method of the invention is applied to the detection of PrPS° in plasma, serum, urine or other biological sample.
"Epitope" refers to a portion of an antigen which is recognized by and bound by 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 an antibody. A conformational epitope includes non-contiguous amino acids. Sometimes confomational epitopes can re-establish themselves after denaturation by partial refolding on, e.g, an immunoblot membrane. The 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 antibody to recognize and bind to the amino acids. In an unfolded (denatured) protein only the linear epitope is recognized and bound by the antibody. Since the protein is unfolded prior to contact with the detection agent, the inaccessible epitope will typically be a linear epitope.
"Protecting agent" or "blocking agent" refers to an agent that covalently modifies and destroys 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 hydrogen peroxide, diethyl pyrocarbonate, 4-hydroxynonenal (4HNE) and diazerine,. Chemical modifying agents that completely saturate accessible amino acids critical for epitope recognition in native conditions are most useful in the applications of epitope protection technology.
"Accessible epitope" is target epitope that is available to react with other compounds (eg. protecting agent) in methods of the invention. For example, epitope that is available to react with protecting agent is accessible epitope. After reacting with protecting agent, the accessible epitope is prevented from binding to detection agent (after this reacting step, the reacted epitope may be referred to as protected epitope or blocked epitope).
"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".
"Detection agent" refers to an agent that binds to epitope and which may be detected, such an 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 agent only has the opportunity to bind to unblocked, unmodified epitopes.
"Wildtype folded conformation" refers to the wild type, folded conformation of protein in a non-disease or non-disorder state.
"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.
"Wildtype conformation" refers to the wild type conformation of protein in a non-disease or non-disorder state.
"Non-wildtype conformation" refers to the conformation of polypeptide in a disease or disorder state 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.
The present inventors have 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, PrPs'.
This conversion of rmPrP is associated with increased solvent accessibility of tyrosine side chains 4. The inventors have found that treatment of normal brain homogenate with acid and denaturants causes PrP to become detergent insoluble 5. In order to probe the surface accessibility of tyrosines and other residues in normal and misfolded PrPc, 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, suggesting 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.
The invention includes a method of determining polypeptide aggregation, including but not limited to PrPS', comprising:
reacting said sample with a chemical modifying agent where such agent could be, but not limited to, peroxynitrite disaggregating and denaturing the chemically modified sample with heat, detergent, or chaotropic agents probing with antibodies specific for prion polypeptide epitopes.
Identifying prion conversion inhibitors 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 and APP in Alzheimer's disease. 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 PrPS° (or other disease or disorder polypeptides indicates that the candidate compound is useful for treating prion disease.
The assays of the invention may be used to screen candidate compounds to determine if they inhibit PrPs' 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 PrPs° or if PrPS° has been converted to wild type protein. Similar methods are used with respect to other disease or disorder polypeptides.
Therefore, the invention also provides methods for identifying substances that inhibit conversion to PrPS~ (e.g. prion protein conversion from wild type protein or intermediate to PrPs°) comprising the steps of:
(a) reacting a polypeptide and a candidate substance, and (b) determining whether the protein has been converted to PrPs° using the methods of the invention.
Similar methods may also be performed to identify compounds which stabilize the wild-type prion state, or bind to PrPs° and block conversion of recruitable PrP isoforms.
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 or APP protein in Alzheimer's disease) comprising the steps of:
(a) reacting a polypeptide and a candidate substance, and (b) determining whether the protein has been converted to the amyloid beta or APP protein in Alzheimer's disease using the methods of the invention.
Another aspect of the invention provides a method of identifying substances which reverse PrPs' formation comprising the steps of:
(a) reacting a polypeptide and a candidate substance, and (b) determining whether the PrPs° has been converted to wild type protein using the methods of the invention.
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:
(a) reacting a polypeptide and a candidate substance, and (b) 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.
The same methods are used with other polypeptides associated with diseases and disorders described in this application.
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 prion protein.
To achieve specificity, inhibitors should target the unique sequences and or conformational features of prion protein.
Prion protein conversion, Alzheimer's disease related polypeatide or other disease/disorder polypeptide may be periodically monitored in a subject over time (eg. 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 PrPc or increased or decreased levels of PrPs° in the subject. The methods of the invention are also useful to measure a subject's level of PrPc or PrPs°
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.

Since many neurological diseases are associated with aggregated proteins, similar diagnostic 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), and others 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 with an anti-6E10 or anti-4G8 antibody (detection agent) known in the art.
All such assays could be adapted and optimised to a simple high-throughput platform.

EXAMPLES
Example 1 Peroxynitrite reacts differently with PrP in normal and acid treated or scrapie brain homogenate 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 PrPs' (5). This acid treated PrP is a 'model prion' which is partially misfolded and/or aggregated resembling characteristics of PrPS°. When mock (D) and acid treated ( ~ ) brain homogenate is incubated with increasing concentrations of peroxynitrate and then subjected to immunoblotting, there is less PrP recognized by both 3F4 (Figure 1A and C) and 6H4 (Figure 1 B
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 peroxynitrate.
Example 2 PrP in scrapie infected hamster brain is protected from modification by peroxynitrite 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 brain (Figure 2A and B). As with model prions, the 3F4 and 6H4 epitopes of PrP in HaS~ brain homogenate are protected from modification by peroxynitrite. It is clear that 'model prions' and haPrPS~ share characteristics that provide protection from chemical modification by peroxynitrite, such as differential misfolding or aggregation.
Example 3 Is aggregation responsible for the reduction in peroxynitrite-induced epitope modification of misfolded PrP~

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 (6-8) 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 epitopes as revealed by immunoprecipitation (Figure 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 (Figure 3A lanes 5-8). This shows that guanidine is able 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 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:
1. Normal hamster and human brain "model prions", using immunoblotting and conventional fluorescence ELISA, conducted at BSL1 (low level containment);
2. Infectious prions from hamster and human brain, using immunoblotting analysis and time-resolved fluorescence, conducted in BSL3 (high-level containment).
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 PrPc in buffer and in PrP -~~
knockout mouse brain, and by mouse normal and scrapie-infected brain (murine PrP is 6H4+ and 3F4-). Methylene is a useful modifying agent for epitope protection of prions, due to its more uniform and complete chemical modification of many epitopes.
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 PrPS° 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 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 (Figure 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 2.5-3x as much PrP detected in the acid treated sample as compared to the mock treated sample.
Example 6 Detection of a single brain prions Detection of single brain prions has been estimated to comprise 105-106 molecules of PrPs~~ Detection of 10$-109 molecules of recombinant PrP using conventional fluorescence ELISA has been accomplished. We use an assay 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.
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:
1. Characterize, optimize and quantify detection of recombinant prion protein by TRF;
2. Determine the sensitivity of the DELFIA-TRF for hamster and human brain prions.
Example 7 Detection of Prion Proteins in biological fluids The EPA achieves commercial utility by detecting PrPs° 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~
(106-fold more than PrPS°) 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:

1. Hamster and human plasma and urine "spiked" with a titration of 263K
hamster prions;
2. Plasma and urine from Syrian hamsters "endogenously" infected with 263K
prion disease 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 PrPS° 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 PrPs detection with detergents, precipitating agents, and adsorbents typically used in commercial ELISA assays which are known to one skilled in the art.
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 (Abets) using EPA
Amyloid beta peptide (Abets) is a normal cleavage product of the proteolytic processing of amyloid precursor protein (APP). Abets 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, we 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 (panel A).
Similarly, the 6E10 epitope is partially protected in brain homogenates that have been treated at low pH to induce protein aggregation (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 (panel C). 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.
Materials and Methods Materials 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).
Preparation of Acid-misfolded PrP and APP.
Acid misfolded PrP was used as "model prions" in this study and was prepared as in (5). Briefly, 100 wl of 10% brain homogenate was mixed with an equal volume of 3.0 M GdnHCI (final concentration 1.5 M) in PBS at pH 7.4 or pH 3.5 adjusted with 1 N HCI, 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 ~,I of lysis buffer. The samples treated at pH 7.4 were designated as mock-treated samples.

Peroxynitrite treatment of Brain Homogenates An aliquot (18p1) of normal or misfolded/diseased brain homogenate was vortexed while 2 pl of peroxynitrite in 100mM NaOH/60 mM H202 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.
Western Blotting 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 Samples were incubated with 50 NI of Ab-conjugated (100Ng/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) x3 and boiled in 30 NI SDS loading buffer without beta-mercaptoethanol for 5 min. Supernatants were analyzed by Western blotting as described above.
Sandwich ELISA
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 °C. After blocking with 1 % BSA in 0.05% TBST for 2 h, plates were washed 3x in TBST and incubated overnight at 4°C with standard concentrations of rhuPrP or rHaPrP along with unknown brain homogenates. Plates were washed 3x and incubated with the detecting antibody biotin-3F4 (1:5000) at RT for 1 h.
After washing 3x, avidin-HRP (1:5000) was added and incubated for 30 min. at RT. Following a final wash step (x3) the plate was developed with Quantablu fluorescent substrate for 10-90 min at RT and fluorescent intensities determined with an excitation of 325nm and emission of 420 nm.
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.
All publications, patents and patent applications, including Canadian application no. (to be assigned) and US application no. (to be assigned) entitled Methods of Detecting Prion Protein (Cashman & Lehto), filed on August 20, 2003 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.

Claims

1. A method of detecting whether a candidate polypeptide including a target epitope is in a wildtype folded conformation or a misfolded conformation, comprising:
contacting the polypeptide with a protecting agent that selectively blocks accessible target epitope, wherein in the wildtype folded conformation, the target epitope is accessible and reacts with the protecting agent, and~
wherein in the misfolded conformation, the target epitope is inaccessible and does not react with the protecting agent;
removing unreacted protecting agent from contact with the polypeptide;
next modifying the candidate polypeptide to convert any inaccessible target epitope to accessible target epitope;
next 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 misfolded conformation and wherein lack of binding between the detection agent and the target epitope indicates that the polypeptide was in a wild type folded conformation.

2. The method of claim 1, wherein 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 Sc.

3. The method of claim 1 or 2, wherein the blocking agent is selected from the group consisting of peroxynitrite, hydrogen peroxide, diethyl pyrocarbonate, 4-hydroxynonenal (4HNE) and diazirine.

4. The method of claims 1-3, wherein the polypeptide is modified by denaturing the polypeptide.

5. The method of claims 1-4, wherein the polypeptide is denatured by heat and/or detergent.

6. The method of claims 1-5, wherein the polypeptide is modified by treatment with a disaggregation agent to disaggregate the polypeptide from other polyeptides.

7. The method of claims 1-6, wherein the disaggregation agent is selected from at least one of the group consisting of guanidine, detergent and heat.

8. The method of claims 1-7, wherein the detection agent comprises an antibody directed against a prion polypeptide epitope or an amyloid beta epitope.

9. The method of claims 1-8, wherein the antibody comprises 6H4 or 3F4.

10. The method of claims 1-9, wherein the misfolded conformation is indicative of a disease caused by protein misfolding.

11. The method of claims 1-10, wherein the disease comprises BSE, CJD or Alzheimer's disease.

12. The method of any one of claims 1 to 11, wherein the epitope is inaccessible in the misfolded 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 epitope, iii) the polypeptide in the misfolded conformation aggregates with other polypeptides in the misfolded conformation to prevent or reduce reaction between the blocking agent and the epitope, and/or iii) post translational modifications of the polypeptide prevent or reduce reactions between the blocking agent and the epitope.