Field

This application relates to assays useful to detect protein aggregates. More particularly, the application provides methods and kits useful to diagnose diseases marked by the presence in blood and other body fluids or tissues of aggregated forms of a normally non-aggregated protein.

Background

In its aggregated form, the normally non-aggregated, monomeric prion protein, PrP is a marker for brain wasting diseases such as bovine spongiform encephalopathy (BSE) or "mad cow" disease in cattle, scrapie in sheep, chronic wasting disease (CWD) in deer and elk, and Creutzfeldt- Jacob disease (CJD) including variant, classical and sporadic CJD in humans.

The high level of prion infectivity has prompted research into agents suitable for disinfecting medical instruments and equipment used in the food processing industry. Lemmer et al studied the effect of pH variation on prion aggregates extracted from surgical instruments (J. Gen. Virol, 2004, 85:3805). At relatively high pH (pH>12) established either with sodium hydroxide, sodium hypochlorite or an alkaline cleaner, detachment and destabilization of infective prion was noted, although degradation was observed only when the sample was either heated or treated with NaOH at a strength of at least 0.5M. There was very little effect on prions subjected to relatively low pH. Similarly, Bauman et al investigated factors that influence prion inactivation by NaOH (Vox Sanguinis, 2006, 91 :34). They found that incubation of hamster scrapie brain homogenate with 0.1M NaOH for 15 minutes at either 4°C or 18 ⁰ C had the effect of reducing detectable prion aggregates. A still further reduction in prion aggregates was noted when NaOH was combined with the detergent sarkosyl.

The detection of prion aggregates in blood is important for the surveillance of blood supplies for human transfusions, and for the monitoring of meat and other food products derived from livestock including sheep and cows as well as goats, elk and deer. Assays having the sensitivity and specificity sufficient to detect prion infectivity are accordingly essential to monitor the presence and spread of prion infectivity. The detection of prions is also important to ensure the safety of therapeutics and vaccines, which are often derived from animal or human blood or tissues.

Detection of PrP aggregates is complicated by many factors. To be useful, the detection method must discern between the presence in the sample of PrP in its pathogenic aggregated form, and PrP in its physiological non-aggregated form which, like the aggregated form, presents most of the same antigenic determinants and therefore displays substantially the same immunoreactivity. Moreover, monomeric PrP is present in samples at physiological levels that, while small, are many times greater than the levels of the pathogenic aggregated form. The detection method accordingly must be not only highly specific for the aggregated form of the prion, to avoid false positive detection; it must also be highly sensitive to avoid false negative results.

Various assay formats and protocols are now in development for detection of PrP aggregates in body fluids and tissues. In one approach referred to as the conformation dependent immunoassay, or CDI, a sample comprising both monomeric and aggregated PrP is denatured so that PrP monomers within the aggregate are liberated. Denatured and untreated aliquots of the same sample are then assayed using an antibody that binds the PrP monomer. A sample is positive for the aggregated form if the signal is greater in the denatured sample relative to an untreated sample. A pretreatment step, such as precipitation with phosphotungstic acid, is typically applied to enrich for aggregated PrP before denaturation. In an alternative format, the sample is digested with enzyme, such as proteinase K, to reduce background from monomeric PrP. In another related format, the entire sample is subjected to enzymatic digestion using conditions controlled so that monomeric PrP is eliminated, and detection of PrP epitopes in the subsequently denatured sample indicates the presence of PrP aggregates in the original sample.

Cashman et al (WO2005/019828 published March 3, 2005) have described a different approach, referred to as epitope protection, which also uses a pretreatment step but eliminates the need for such careful control over reaction conditions, and the need to run sample aliquots in duplicate. In this format, sample is pretreated with peroxynitrite to derivatize tyrosine and other residues that are abundant in PrP (and most proteins), effectively "masking" all immunoreactive sites on both monomeric and aggregated forms of PrP present in the sample when the reaction is run to completion. The PrP is then dissociated, and the sample is probed with PrP antibody that binds only when dissociation exposes PrP epitopes that were protected, by aggregation, from the peroxynitrite reaction. This assay reliably detects human brain prions in blood at a dilution of one in a million and sheep scrapie brain prions in blood at a dilution of one in two hundred thousand.

It is an object of the present application to provide an assay useful to detect protein aggregates. It is a particular object of the present application to provide a method useful for detecting the aggregated form of the prion protein, PrP.

Summary

It has now been discovered that detection of protein aggregates can be achieved using reagents that are relatively inexpensive and safe to handle and protocols that are relatively simple and easy to follow. It has more particularly been found that target proteins are modified in an epitope-protected manner when exposed to relatively high pH, such as by treatment of a sample with a suitable buffer or by addition of an alkali metal hydroxide such as potassium hydroxide and particularly sodium hydroxide (NaOH). In the present application, this phenomenon is exploited to detect protein aggregates, and preferably aggregates of proteins that are of diagnostic and clinical interest.

More particularly, and in the case of PrP for example, it has been determined that pH elevation yields a protein having an immunoreactivity that is altered relative to an untreated control. In aspects, the present application uses pH elevation to mask an epitope exposed on the surface of protein aggregate, and then probes the dissociated aggregate with an antibody to that epitope (e.g. to detect epitopes that were not modified but concealed in the aggregate form). Formation of an antibody:target protein complex indicates the presence of target protein aggregate in the sample. Elevated pH has the effect of masking only certain epitopes, and selection of an appropriate antibody in the probing step is therefore an important consideration in the present method.

According to one aspect of the present application, there is provided an assay useful to detect the presence of an aggregated form of a target protein in a sample that may also contain the target protein in non-aggregated form, the assay comprising the steps of:

(a) treating or contacting the sample, optionally with a pH elevating agent to elevate the pH thereof to a value of at least about 12, thereby to protect one or more epitopes accessible on the target protein aggregate without causing substantial hydrolysis of the target protein; (b) neutralizing the pH of the treated sample, optionally with a pH neutralizing agent, optionally wherein the pH is reduced to at least about 8, at least about 7.6, at least about 7.4 or at least about 7.0;

(c) exposing the pH-neutralized sample to dissociating conditions and/or dissociating aggregated protein in the neutralized sample;

(d) incubating and/or contacting the dissociated sample with a binding agent that binds selectively to one or more epitopes of the same type as that protected by the pH-elevating step; and

(e) determining the formation of a binding agenttarget protein complex, wherein the formation of complex indicates the presence in the sample, prior to the pH elevating step, of an aggregated form of the target protein.

The step of exposing the pH-neutralized sample to dissociating conditions and/or dissociating aggregated protein in the neutralized sample, converts aggregated target protein to non- aggregated target protein.

The pH elevating and neutralization steps can be performed at one time point and the detection steps can be performed at a later time point. Accordingly another aspect provides an assay for detecting the presence of an aggregated form of a target protein in a sample that may also contain the target protein in non-aggregated form, the assay comprising the steps of:

a) contacting the sample with a binding agent, wherein the sample has been: i) contacted or treated with a pH elevating agent that increased the pH of the sample to at least about 12 and modified one or more epitopes accessible on the target protein, ii) contacted or treated with a pH neutralizing agent, that reduced the pH of the sample to at least about 8, at least about 7.5 or at least about 7.0; and iii) contacted or treated with a dissociating agent or dissociating conditions to dissociate aggregated proteins; and wherein the binding agent binds to the one or more epitopes in the target protein exposed by dissociating protein aggregates in the sample; and

b) detecting the presence of a binding agent: target protein complex; wherein the presence of complex indicates the target protein was aggregated in the sample prior to the pH elevating step. The present assays can be applied to assay samples for detecting the presence of an aggregated form of any target protein that presents in both non-aggregated and aggregated forms. In embodiments, the target protein is a protein that forms aggregates that have clinical significance, and is therefore a protein of diagnostic interest. Proteins that are members of the prion protein family are the target proteins in embodiments of the present application. In specific embodiments, the target protein is sheep PrP which in aggregated form is diagnostic for sheep scrapie. In other embodiments, the target protein is human PrP which in aggregated form is diagnostic for CJD.

The binding agent that binds selectively to the target protein epitope that is modifiable by pH elevation is preferably an antibody to that epitope, or an epitope binding fragment of that antibody.

In particular aspects, the assay is applied for the detection of an aggregated form of the normal, monomeric and cellular form of PrP, designated PrPc. The pathogenic PrPc aggregates are referred to herein as PrPSc. Thus, in aspects of the present application, there is provided an assay useful to detect PrPSc in a sample that may also contain PrPc, the method comprising the steps of:

(a) treating or contacting the sample, optionally with a pH elevating agent, to elevate the pH thereof to a value of at least about 12, thereby to protect one or more target epitopes common to PrPc and PrPSc without causing substantial hydrolysis of the PrPc and PrPSc;

(b) neutralizing the pH of the treated sample, optionally with a pH neutralizing agent, wherein the pH is reduced to at least about 8, at least about 7.6, at least about 7.4 or at least about 7;

(c) exposing the pH-neutralized sample to dissociating conditions and/or dissociating protein aggregates in the neutralized sample;

(d) incubating and/or contacting the dissociated sample with a binding agent that binds selectively to one or more epitopes of the same type as that protected by pH-elevating step (e.g. one or more unprotected epitopes); and

(e) determining the formation of a binding agent:target protein complex,

wherein the formation of complex indicates the presence in the sample of PrPSc prior to the pH elevating step. In embodiments, the pH elevating step is achieved using a pH elevating agent having for example a pH in the range of 12-14, such as an alkali metal hydroxide such as potassium hydroxide (KOH) and/or phosphate solutions (e.g. PO4 ^2" ). In a preferred embodiment, the pH elevating agent is sodium hydroxide (NaOH).

In embodiments, the pH-elevating step is performed under conditions that avoid causing substantial hydrolysis of the target protein in either aggregated or non-aggregated form. In an embodiment, treatment with NaOH is performed at a NaOH concentration of about O. IM for a period of about 5 minutes. In another embodiment, the sample is a sample of biological fluid comprising whole blood, plasma, serum and/or spinal fluid. In another embodiment, the sample comprises plasma. The sample is optionally pretreated. In an embodiment, the sample is pretreated prior to pH elevation to enrich for target protein.

In other embodiments, the binding agent that binds selectively to the target epitope modified by treatment with the pH-elevating agent is an antibody. In another embodiment, the target protein is a PrP species. In a further embodiment, the antibody binds for example an epitope that lies within the region C-terminal to and/or including amino acid residue 90, 93, 98 and/or 100 of PrP. In specific embodiments, the target protein is sheep PrP and detection of the aggregated form of the target protein is useful to diagnose sheep scrapie, including classical sheep scrapie and atypical sheep scrapie. In another embodiment, wherein the target protein is sheep PrP, the antibody binds at an epitope within amino acid residues 100-237, 100-230 or amino acid residues, 106-112, 144-152, 121-152, 214-230, 225-228 and/or 23-237. In yet a further embodiment, the antibody is selected from 3C8, 3F4, 6H4, POMl, 7D9, 1E4 and/or 6Dl 1. In other specific embodiments, the target protein is human PrP and detection of the aggregated form of the protein is useful in the diagnosis of human CJD including variant and sporadic CJD. In another embodiment, wherein the target protein is human PrP, the antibody binds at an epitope within amino acid residues 93-237, 100-230, 98-108, 93-109, 106-112, 144-152, 121-152, 214-230, 225-228 and/or 23-237. In yet a further embodiment, antibody is selected from 3C8, 3F4, 6H4, POMl, 7D9, 5G12, 1E4 and 6Dl 1.

In other embodiments, the step of determining the formation of a binding agent:target protein complex is performed using an antibody sandwich assay comprising a first antibody (or detection antibody) that binds said epitope and a second antibody (or capture antibody) that binds target protein, optionally simultaneously with said first antibody. In certain embodiments, at least one of said antibodies comprises a detectable label. In other embodiments, wherein the label is a fluorescent label. In further embodiments, the label is a fluorescent bead. For example, in certain embodiments, the first or detection antibody is coupled to a fluorescent bead. In other embodiments, the second or capture antibody is conjugated to a magnetic bead. In certain embodiments, the target protein is sheep PrP, and the first antibody and the second antibody are selected independently from 3C8, 3F4, 6H4, POMl, 7D9, 1E4 and 6Dl 1. In other embodiments, the target protein is human PrP, and the first antibody and the second antibody are selected independently from 3C8, 3F4, 6H4, POMl, 7D9, 5G12, 1E4 and 6Dl 1. In another embodiment, the step of determining the formation of the binding agent:target protein complex is performed using an antibody assay, such as a sandwich immunoassay comprising a first antibody or a detection antibody that binds said epitope and a second antibody or a capture antibody that binds target protein simultaneously with said first antibody, wherein the detection antibody is selected from 3C8, 3F4, 6H4, POMl , and 7D9 and the capture antibody is selected from 3C8, 3F4, 6H4, POMl, 7D9, 5G12, 1E4, 6Dl 1, CC2, 8B4, P0M2, and 5C4. In a further embodiment, wherein the step of determining the formation of a binding agent:target protein complex is performed using an antibody assay, such as a sandwich immunoassay comprising a first antibody or a detection antibody that binds said epitope and a second antibody or a capture antibody that binds target protein simultaneously with said first antibody, wherein the detection antibody is selected from 3C8, 3F4, 6H4, POMl, 7D9, 1E4 and 6Dl 1 and the capture antibody is selected from 3C8, 3F4, 6H4, POMl, 7D9, 5Gl 2, 1E4, 6Dl 1, CC2, 8B4, P0M2, and 5C4. In a more specific embodiment, the first antibody is POMl and the second antibody is 5C4. This antibody combination can for example be used where the target protein is sheep PrP. In other embodiments, the first antibody is P0M2 and the second antibody is 3F4; or the first antibody is 6H4 and the second antibody is 3F4. These antibody combinations are useful for example where the target protein is human PrP.

In other aspects of the present application, there is provided a kit comprising a binding agent such as an antibody that binds to an epitope on a target protein that is protectable by treatment thereof by pH elevation, preferably NaOH, and instructions for the use thereof in accordance with the method of the present application. In other embodiments, the kit further comprises a pH elevating agent and/or a neutralizing agent and/or target protein in quantity useful as a control or calibrator.

Other features and advantages of the present disclosure 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 disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

These and other aspects of the present application are now described in greater detail with reference to the accompanying drawings in which:

Brief Reference to the Figures

Figure 1 shows the results of a sandwich assay performed using antibodies 3F4 and POM2 to detect aggregated PrP in human vCJD brain spikes, using the protocol described in Example 2 herein;

Figure 2 shows the results of a sandwich assay performed using antibodies 3F4 and P0M2 to detect aggregated PrP in hamster scrapie brain spikes, using the protocol described in Example 2 herein;

Figure 3 shows results of an assay performed using antibodies POMl and 5C4 to detect aggregated PrP in human vCJD brain spikes at different dilutions (S/N is reported, where "N" is the background signal from an un-spiked sample of plasma); and

Figure 4 shows the effect of NaOH concentration on recombinant sheep PrPc, analyzed by SDS-PAGE using Coomassie staining.

Detailed Description and Preferred Embodiments

The application relates to assays useful to detect the presence of an aggregated form of a target protein that is normally in non-aggregated form. The normal, "non-aggregated" form of the target protein may be monomeric, such as the prion protein, or may be multimeric or oligomeric in normally comprising, in its wild type form, two or more different or identical subunits, such as the SODl homodimer, or a heterodimer that forms pathogenic aggregates. The application utilizes pH elevation in a sample the pH elevating step to protect one or more epitopes presented by the target protein while avoiding substantial hydrolysis, i.e., hydrolytic digestion, of the target protein into component amino acids or short peptides. It will be appreciated that some hydrolysis may be tolerated, for example less than about 1%, 2%, 3%, 5% or less than about 10%. Following pH elevation and subsequent neutralization, the treated sample is subjected to dissociating conditions so that non-aggregated protein is released from any aggregate in the treated sample, and the dissociated sample is then probed using an antibody or other binding agent that binds selectively to an epitope that is sensitive to pH elevation but was insulated therefrom in its aggregated form.

The binding agent binds the "same type" of epitope that was protected by the pH elevating step. The "same type" refers to the binding agent selectively binding one or more epitopes having for example the same amino acid sequence as the epitope modified or protected by the pH elevating step but which was insulated by the aggregate and therefore unavailable for modification.

An epitope is considered "protected" when, as a result of pH elevation such as by incubation with NaOH, an antibody or other binding agent to that epitope no longer binds with equal affinity to that epitope. Thus, epitopes amenable to such protection are those target protein epitopes that are exposed and accessible to the elevated pH agent or conditions. Antibodies or binding agents useful in the present assay are those which bind to an epitope (e.g. unmodified epitope) that are protectable or alterable by pH elevation, and is also presented in its native conformation when the pH elevated sample is neutralized and dissociated into component monomers exposing epitopes that were insulated by aggregation from the effects of pH elevation.

The mechanism by which epitope protection is achieved through pH elevation is not fully understood, but may occur by an effect of elevated pH on the conformation of target protein in the region to which a given antibody normally binds, by racemization of the amino acid residues, by elimination reactions involving the side chains of certain amino acids or by oxidation of amino acid residues. The effect of pH elevation is not mediated principally, however, by way of hydrolysis of the target protein, or by causing dissociation of a protein aggregate into its non-aggregated form. Conditions for pH elevation accordingly are selected on this basis.

In the pH elevating step of the present assay, the sample or a form thereof enriched for the aggregated form of a target protein is treated, for example with a pH elevating agent, to elevate the pH to a value that is at least about 12, i.e., at least 11.5, 1 1.6, 11.7, 11.8, or 11.9. A suitable pH lies in the range from 12 to 13.5, e.g., 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4 or 13.5. In an embodiment, the sample is treated or contacted with a pH elevating agent in an amount sufficient to increase the pH of the sample to at least about 12, or to a suitable pH in the range from 12 to 13.5, as mentioned above.

To elevate pH, a pH elevating agent is introduced to the sample. The pH elevating agent has for example a pH in the range of at least about 12-14 and has the effect of establishing within the sample a pH having a value recited above. Suitable pH elevating agents include for example the alkali metal hydroxides such as potassium hydroxide (KOH), lithium hydroxide (LiOH), rubidium hydroxide (RbOH), and cesium hydroxide (CsOH) and, in a preferred embodiment, NaOH. Others include for example phosphate solutions such as a solution

2- 3- 2- comprising PO4 , PO4 , and/or H PO4 , used optionally in combination with other pH elevating agents such as NaOH. In an embodiment, the concentration of the pH elevating agent is about l .OM, about 0.9M, about 0.8M, about 0.7M, about 0.6M, about 0.5M, about 0.4M, about 0.3M, about 0.2M, about 0.1M, about 0.09M, about 0.08 M, about 0.07M, about 0.06 M, about 0.05M, about 0.04M, about 0.03M, about 0.02M or about 0.0 IM. In an embodiment, the concentration of the pH elevating agent is less than about l.OM, and suitably lies in the range from 0.01-0.50M, more desirably 0.05-0.2OM and preferably in the range from 0.08-0.12M, e.g., about 0.1M.

Elevation of pH can affect a given protein in various ways depending on incubation conditions such as time and temperature of incubation, and the nature of the incubated protein. The pH elevation step used in the present assay is intended to cause an alteration in the reactivity of the protein to binding agents used in the assay. The conditions of pH elevation are chosen most desirably to avoid hydrolysis of the target protein, thereby to avoid reducing the signal generated by the assay. Also, because the assay will detect only protein monomer that was protected within the protein aggregate, there is no requirement to hydrolyze the non-aggregated protein present in the original sample to avoid contamination of the assay result. As well, it is desirable to avoid pH elevating conditions that result in dissociation of the target protein aggregate. Dissociation will result in blocking of epitopes presented by the dissociated monomers, and reduce signal in the assay result.

A variety of pH elevating agents and conditions can be tested to identify those which satisfy these desired end-points for a given target protein. More particularly, a given target protein can be treated with a selected set of pH elevating conditions and then examined such as by SDS-PAGE or the like to identify those conditions that allow the target protein to remain intact in the aggregated form (little or no dissociation) and in the non-aggregated form (little or no hydrolysis). Using those conditions, the effect of any pH elevating agent on the immunoreactivity of the target protein can be examined using a panel of antibodies raised against various epitopes on the target protein, to identify those antibodies that no longer bind as a result of pH elevation. It will be appreciated that the behavior of the protein can differ between experiments run with spiked "neat" purified recombinant protein samples and actual biological assay samples such as plasma, and that any results with a model system developed using protein standards can be adapted for use with extracted biological samples. This is the approach adopted in the work described in the examples herein, from which useful guidance can be taken.

When NaOH is used as the pH elevating agent, for example, epitope protection without consequent hydrolysis or dissociation can be achieved generally by incubating the sample at an NaOH concentration less than about 1.0M, for a period of not more than about 60 minutes at about room temperature. It has been found more particularly, and in one embodiment of the application, that the pH elevating step of a sample of scrapie-infected sheep plasma can proceed at an NaOH concentration that is less than about 1.0M, and suitably lies in the range from 0.01-0.50M, more desirably 0.05-0.20M and preferably in the range from 0.08-0.12M, e.g., about 0.1 M. Incubation periods are generally less than 60 minutes, generally less than 20 minutes and usually about 2-10 minutes, e.g., 5 minutes. Incubation is suitably conducted at room temperature. Thus, in a specific embodiment, pH elevation of a sheep scrapie plasma sample is performed for about 5 minutes at 0. IM NaOH and at room temperature.

It has also been found, in another embodiment of the application in which the sample is a sample of a human vCJD brain homogenate diluted 1 in 50,000, that pH elevation can proceed at an NaOH concentration that is less than about 1.0M, and suitably lies in the range from 0.01-0.50M, more desirably 0.05-0.20M and preferably in the range from 0.08-0.12M, e.g., about 0.1M. Incubation periods are generally less than 60 minutes, generally less than 20 minutes and usually about 2-10 minutes, e.g., 5 minutes. Incubation is suitably conducted at room temperature. Thus, in a specific embodiment, pH elevation of a human vCJD brain homogenate sample is performed for about 5 minutes at 0.1 M NaOH and at room temperature.

Sample treatment using KOH as the pH elevating agent can proceed as described above for NaOH-based pH elevation. After subjecting the sample to pH elevation, the pH of the sample is desirably neutralized for example by contacting or adding a neutralization agent, before being dissociated and then probed for monomer reactive with the binding agent.

The term "neutralization agent" or "neutralizer" as used herein refers to an agent, such as an acid, that alters upon its addition to a sample the pH of the sample to a pH of at least about 8, at least about 7.6, at least about 7.4, at least about 7.2 or at least about 7.0, or at least about 6.8 or alters the pH of a sample to have a pH in the range from about 6.8 to 7.6, e.g., 7.0-7.4.

Neutralization is suitably achieved in a routine manner, to bring the pH down to about physiological pH, e.g. a pH having a value of at least about 8, at least about 7.6, at least about 7.4, at least about 7.2 or at least about 7.0, or having a pH in the range from about 6.8 to 7.6, e.g., 7.0-7.4, such as by addition of an acid such as an inorganic acid e.g., hydrochloric acid (HCl), hydrobromic acid (HBr), hydroiodic acid (HI), sulfuric acid (H ₂ SO ₄ ), nitric acid (HNO ₃ ) or a neutralizer such as a phosphate buffer, or any combination of the above. In an embodiment, the neutralizer comprises HCl and phosphate buffer in an amount sufficient to reduce the pH to at least about 8, at least about 7.6, at least about 7.4, at least about 7.2 or at least about 7.0, or having a pH in the range from about 6.8 to 7.6, e.g., 7.0-7.4.

In an embodiment, the pH neutralized sample is equilibrated, for example for NaCl, with for example an equilibration agent.

The pH neutralized sample, with or without equilibration, can then be subjected to dissociation, to release the monomers within the protein aggregate that were insulated from the effects of pH elevation. Dissociation can be achieved using any suitable agents such as disaggregating or denaturing agents such as guanidinium hydrochloride, guanidinium thiocyanate, urea, denaturing detergent (e.g. SDS), or the like. To ensure complete dissociation, the sample is desirably also denatured by heating, such as by incubation at or above 80°C, e.g., 83°C, as is well established. In a specific embodiment, the sample is treated with GdnHCl, e.g. 4M guanidine as GdnHCl and heated to and held at 83°C for 6 minutes. After dissociation, the sample is desirably treated such as by filtration/washing, to remove dissociating agent, thereby to reduce interference when probed subsequently with the binding agent.

The "binding agent" used in the present method to probe the dissociated sample for epitope(s) protected by pH elevation of the sample is suitably an antibody or a fragment thereof that binds selectively to that epitope. Binding agents that bind "selectively" have an affinity for a given epitope that is greater than, and preferably at least ten times greater than, the affinity with which they bind to a different epitope. The antibodies may be polyclonal, and may be monoclonal including chimeric and humanized antibodies or recombinant antibodies including human antibodies. Suitable antibody fragments include Fab fragments such as Fab, F(ab), F(ab)2, single chain Fvs and the like, and any other fragment that incorporates a complementarity determining region (CDR) having affinity for the target epitope. In the alternative, the agent can be an RNA, DNA or peptide aptamer. It will be appreciated that the binding agent is one that, generally, is unable to bind to the pH elevation- treated aggregated form of the target protein but does bind selectively to a non-aggregated form of the target protein that is released when the aggregated target protein is dissociated.

The particular format chosen to probe for unreacted non-aggregated target protein released by dissociation from the aggregate is a matter of design choice. It is however important to choose a binding agent that binds selectively to an epitope of the type sensitive to NaOH treatment. As exemplified herein, the antibodies suitable for this purpose can be determined empirically for any given target protein, simply by analyzing the binding pattern of an antibody panel toward the target protein before and after NaOH treatment.

The chosen antibody can be incorporated into an assay format of any useful type, including Western blotting, ELISA, RIA, and the like. In embodiments of the present application, an antibody sandwich assay is used, in which the chosen antibody is used in combination with a second antibody that binds the non-aggregated target protein simultaneously with the chosen antibody. In the sandwich format, either antibody can be used as the capture or detector (e.g. detection) antibody.

In one embodiment of the present application, the assay thus utilizes an immunoassay sandwich format in which at least one of the antibodies binds to the target protein at a site that is sensitive to but was insulated from pH elevation (e.g. the detection antibody). In another embodiment, another antibody, the capture antibody, can be any antibody that binds the target protein and can for example bind an epitope that is not sensitive to pH elevation

(e.g. in the N-terminus for PrP). In a preferred embodiment, both antibodies utilized in the immunoassay sandwich format are antibodies that bind at sites that are different sites but are both sensitive to pH elevation. As used herein, a "capture antibody" refers to an antibody that binds the target protein and is for example used to pull down or capture the target protein. For example any antibody that does not interfere with the detection antibody that specifically binds the target protein can be used as a capture antibody.

As used herein, a "detection antibody" or "detector antibody" refers to an antibody that binds an epitope in the target protein that is modifiable or protectable by a pH elevating step or agent, for example an antibody the binds an epitope present within amino acid C-terminus to and/or including residue 93 of PrP, e.g. 93-237.

In an embodiment, the target protein is PrP. As mentioned PrPc is the cellular form of PrP and PrPSc or Scrapie is the aggregated disease associated form of PrP, such as human aggregated PrP, sheep aggregated PrP and/or bovine aggregated PrP. Accordingly, in an embodiment, the assay determines whether a sample prior to treatment or contacting with a pH elevating agent comprises aggregated PrP e.g. comprises PrPSc.

In embodiments of the present application, where the target protein is sheep PrP, the binding agent is an antibody that binds to sheep PrP in the region C-terminal to and/or including residue 90, 95, 98, 100 or 105, for example in the region spanning residues 100-230, for example amino acids 106-1 12, 144-152, 121-152, 214-230, 225-228 and/or 23-237. In specific embodiments, the binding agent is an antibody that selected from 3C8, 3F4, 6H4, POMl, 7D9, 5Gl 2, 1E4, and 6Dl 1, or an antibody or binding fragment that competes therewith for binding to sheep PrP. In a more specific embodiment, the binding agent is an antibody or binding fragment thereof selected from 3C8, 3F4, 6H4, POMl, 7D9 and 5G12. In a preferred embodiment, the antibody is 3C8, or an antibody or binding fragment that competes therewith for binding to sheep PrP. In related embodiments, where the sheep scrapie assay is conducted in a sandwich format, the second antibody is an antibody that binds to any sheep PrP epitope, including for example antibodies 3C8, 3F4, 6H4, POMl, 7D9, 5G12, 1E4, 6Dl 1, CC2, 8B4, POM2, and 5C4. In a preferred embodiment, the second antibody is different from the first antibody, and is otherwise selected from the same list. In a particular embodiment, the antibodies used to detect sheep PrP are POMl (e.g. as the detection antibody and/or first antibody) and 5C4 (e.g. capture antibody and/or second antibody).

In one embodiment of the present application, the assay method is applied for purposes of diagnosing sheep scrapie. In a specific embodiment, the assay method is applied to diagnose classical sheep scrapie. In another specific embodiment, the assay method is applied to diagnose atypical sheep scrapie. About 45 % of currently diagnosed scrapie cases and 84 % of the affected flocks represent atypical scrapie. The atypical phenotype is characterized by the absence of or a faint and mostly granular deposition of PrPSc at the level of the obex. On the other hand, in the majority of cases, massive accumulations of PrPSc are detectable in the cerebellum and cerebrum. The PrPSc electrophoretic profile by Western blot is different from that of classical scrapie cases and includes an additional fragment with a molecular mass of approximately 12 kDa.

The abnormal PrPSc derived from atypical scrapie cases displays a lower resistance to proteinase K (PK) than does PrPSc from classical scrapie. Although atypical scrapie cases are frequently found only in single animals of an affected flock, atypical scrapie cases are indeed infectious when inoculated into transgenic mice. Notably, such atypical scrapie cases were often found in sheep carrying PrP genotypes that were believed to convey scrapie resistance.

Polymorphisms at codons 141 and 154 in the ovine prion protein gene may be correlated with atypical scrapie cases.

In other embodiments of the present application, where the target protein is human PrP, the binding agent is an antibody that binds to monomeric human PrP in the region C-terminal to and/or include residues 90, 93, 95, 98 or 100, for example within the region spanning residues 93-237, 100-230 or for example for example amino acids 98-108, 93-109, 106-1 12, 144-152, 121-152, 214-230, 225-228 and/or 23-237. In specific embodiments, the binding agent is an antibody selected from 3C8, 3F4, 6H4, POMl, 7D9, 5G12, 1E4, and 6Dl 1, or an antibody or binding fragment that competes therewith for binding to human PrP. In a more specific embodiment, the binding agent is an antibody selected from 3C8, 3F4, 6H4, POMl, 7D9 and 5G12. In a preferred embodiment, the antibody is 3C8, or an antibody or binding fragment that competes therewith for binding to human PrP. In related embodiments, where the human PrP assay is conducted in a sandwich format, the second antibody is an antibody that binds to any human PrP epitope including for example antibodies 3C8, 3F4, 6H4, POMl, 7D9, 5G12, 1E4, 6Dl 1, CC2, 8B4, POM2, and 5C4. In a preferred embodiment, the second antibody is different from the first antibody, and is otherwise selected from the same list. In specific embodiments, the antibodies used to detect human PrP are POMl (e.g. detection antibody) and 5C4 (e.g. capture antibody), or POM2 (e.g. capture antibody) and 3F4 (e.g. detection antibody). In an embodiment, the detection antibody is coupled or comprises a label. In one embodiment, the present method is applied to diagnose Creutzfeldt- Jakob Disease (CJD), which is a rare disease that affects the central nervous system. In a specific embodiment, the present method is applied to detect aggregated human PrP, thereby to diagnose either classical CJD, including sporadic, familial, and iatrogenic CJD, or to diagnose variant CJD. Sporadic CJD, which comprises 85-90% of all classical CJD cases, occurs spontaneously in the general population. Familial CJD is even rarer, accounting for 10 to 15 percent of all classical CJD cases. It occurs in individuals within specific families with a genetic predisposition. Iatrogenic CJD is the rarest of all, accounting for less than 1 percent of all classical CJD cases. It occurs accidentally as the result of certain medical procedures, where there is transmission of the causative agent from a patient with the disease to another patient. In classical CJD, the period between exposure to the infection in cases where this is known and the onset of symptoms can range from 1 to 30 years or more. Symptoms usually occur suddenly and the patient's condition rapidly declines. Early symptoms include lapses in memory, mood swings similar to depression, lack of interest and social withdrawal. The patient may become unsteady on his/her feet. Later symptoms may include blurred vision, sudden jerking movements and rigidity in the limbs. The patient may experience slurred speech and have difficulty swallowing. There is progressive mental deterioration and, eventually, movement and speech are lost. Detection of aggregated human PrP by the present method, particularly but not only in homogenates of tissues extracted post-mortem from subjects displaying these symptoms, is useful in the diagnosis of CJD.

In specific embodiments of the present method, a dual bead sandwich assay format is adopted, as exemplified herein, in which one antibody (e.g. the detection antibody), is coupled to a bead serving as or comprising a detectable label such as a fluorescent bead, and the other antibody (e.g. the capture antibody) is coupled to a magnetic bead to facilitate separation of bound material from unbound material during performance of the assay.

It will be appreciated that different labels can also be useful, including radioactive labels, i.e., radioisotopes, chemiluminescent labels, cytochromes, and enzymes including horseradish peroxidase and alkaline phosphatase, as well as colloidal gold. The coupling of such labels, and instruments useful for their detection, are all available and well established in the art.

The assay can be performed by a clinical lab, for example, samples can be obtained from a subject and sent to a clinical lab for processing. Alternatively, the assay can be performed by a research lab. Further the sample can be treated with a pH elevating agent, neutralizing agent and/or dissociated in one location, before being processed for epitope binding. The sample can for example be treated with a pH elevating agent, neutralized and dissociated with for example a sample buffer loading dye and/or frozen and sent for analysis e.g. sent for antibody detection of target protein.

The application further provides a kit useful for performing the assay of the application. The kit will comprise at least a quantity of the antibody that binds to the epitope alterable by a pH elevating agent such as NaOH, and instructions for the use thereof in accordance with the present methods. In addition, the kit may further comprise a known quantity of the non- aggregated form of the target protein, or other protein (e.g. molecular weight markers) for use as a standard, calibrator or control, in performance of the assay. Other components that can be included are for example, a quantity of a pH elevating agent and/or a quantity of a neutralizing agent, and/or a plate for conducting the assay.

The protein targets of the assay can be any target protein that forms aggregates. In embodiments, the target protein is a protein of diagnostic interest, i.e., a target protein for which aggregation is indicative of a human or livestock/animal disease, condition or disorder (collectively, a medical "condition"). Included among the target proteins are the prion protein, PrP, which is associated with a variety of diseases including bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD) particularly in cervids such as deer and elk, and Creutzfeldt- Jakob disease (CJD) in humans including the classic and variant forms; the beta sheet forms of the Abeta protein associated with Alzheimer's disease (AD); the SODl protein associated with amyotrophic lateral sclerosis (ALS) and associated with AD; the alpha-synuclein protein associated with Parkinson's disease (PD) and Lewy body disease (LBD); the huntingtin protein associated with Huntington's disease (HD); transthyretin associated with familial amyloidotic polyneuropathy (FAP); islet amyloid polypeptide and resistin associated with diabetes; the tumour suppressor p53 protein associated with certain forms of cancer including neuroblastomas, carcinomas, and myelomas; the tau protein implicated in AD, and the like.

The samples useful in the present assay can be any body fluid or tissue, a food sample such as meat or milk, or any environmental sample such as water, soil or the like, in which an aggregated form of the target protein is suspected to reside. Body fluids include whole blood, as well as serum and plasma, spinal fluid, urine, peritoneal exudates, tears, and the like. Tissue samples can include for example tissue block or slide samples or homogenates of brain, spleen, eyelid, rectal tissue, or other organs, for post-mortem and anti-mortem analysis.

The sample can be treated before the assay and before the introduction of NaOH to prepare it for testing. For example, procedures have been developed to eliminate contaminants such as clots, cells, cell debris, or other substances that may interfere with the assay. The sample can also be pre-treated to concentrate the target protein aggregates. A variety of enrichment procedures have been developed particularly for enrichment of PrP aggregates.

The sample can, for instance, be treated first with an affinity matrix that binds PrPSc. Suitable affinity matrices include phosphotungstic acid (PTA), streptomycin, plasminogen or fibrinogen, certain PrP-binding peptide ligands such as GWGQPHGG (see WOO 1077687), polyionic agents such as glycosaminoglycans and poly lysine (see WO03073106), as well as antibodies having a binding preference for the aggregated form of the target protein. In the alternative, the sample can be treated to remove PrPc monomer from the sample, such as by treating the sample using a chelator-based immobilized metal affinity chromatography bead (IMAC) available, for instance, from Invitrogen. Following incubation of the sample with the affinity matrix, contaminants can then be washed away and the bound PrPSc can then be collected following elution using conditions appropriate for the chosen matrix.

In the alternative, or in addition, the sample can be subjected to filtration. Suitable filtering means for collecting PrPSc include the use of a perlite bed having a pore size less than about 6um; cellulose acetate which is said to have a greater affinity for PrPSc than for PrPc; and certain positively charged filter membranes available from Pall Corporation which are described in WO2008/005960, which also describes their use to concentrate and collect PrPSc from samples including whole blood and plasma.

It will be appreciated that the affinity matrices and/or the filtration steps can be used alone or in combination with other separation methods including centrifugation and sedimentation and the like in order to concentrate and thus enrich for the target protein aggregates to be detected by the assay.

As noted herein above, the sample, e.g., in its concentrated form, is subjected to a pH elevation step which alters the immunoreactivity of resident proteins. The pH of the sample is then neutralized, for example with a neutralizing agent, and the neutralized sample is then dissociated, for example by contacting the sample with a dissociation agent and/or heating. As an optional step, the dissociated sample may then be treated to remove the dissociating agent, to avoid contamination of the subsequent immunoassay. That sample is then subjected to an assay protocol that indicates the presence of protein target aggregates by detecting protein target monomers that were insulated, by aggregation, from epitope protection performed on the sample. In the present assay, such epitope protection is performed using pH elevation, which, in the case of PrPSc, has the effect of altering immunogenicity of the aggregated PrPSc but not the monomeric PrPc that is released for detection when the treated PrPSc is dissociated.

While the present disclosure has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure 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 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. Sequences associated with accession numbers described herein including for example the Tables, are herein specifically incorporated by reference.

The following non-limiting examples are illustrative of the present application:

Examples

Example 1 - Detection of PrPSc in scrapie-infected sheep

The detection of PrPSc in plasma taken from scrapie-infected sheep was performed as follows. Plasma can be obtained from sheep using any one of a number of standard blood collection and processing protocols. For example, an animal is restrained and a needle is inserted into its jugular vein. When blood flow is established, the other end of the needle is placed into the blood collection container containing the anticoagulant (usually Citrate, EDTA, ACD, or CPD). The blood is set at room temperature for an hour and centrifuged at 2750rpm (1500-1800rcf) for lOmin.The centrifuge is allowed to slow without using a brake and the plasma component is suctioned off and frozen. Generally, the plasma is first enriched for PrPSc, for example by as described in the literature and/or herein for example in Example 3, the enriched sample is then treated with NaOH under defined conditions, the treated sample is then dissociated and probed with antibody reactive to a site altered by NaOH treatment in the aggregate but not in the dissociated monomer. Binding is detected and reveals the presence of monomeric PrP that was protected from NaOH while in the aggregated state, and thus indicates the presence of PrPSc aggregate in the original plasma sample and in the blood of the animal from which the plasma was extracted.

900ul of each plasma sample was loaded onto a Prion extraction plate (PEP) plate (Pall Corp) while positioned on a 1.1ml collection plate, and then spun at 2,00Og for 2 minutes. Flow through was then discarded from the collection plate. The plate was then washed by adding 900ul PBS, spinning for 2 minutes in a centrifuge and then discarding the flow through. In a second wash, 500ul PBS was added, spun for 4 minutes at 2,00Og and then flow through was discarded.

Material bound to the PEP plate was then eluted using lOOul of 10OmM NaOH, and then spinning at 2,00Og for 2 minutes directly into a 450ul collection plate. At 4 + 0.5 minutes after spinning, 20ul of 0.5M NaH ₂ PO ₄ ZlOOmM HCl was added rapidly. The solution was then pipetted up and down 3 times. The mixture was then shaken at 900rpm for 1 minute.

120ul of 8M guanidine hydrochloride was added to all wells and the plate was shaken at 450rpm for 2 minutes to ensure mixing.

The collection plate was then heated for 6 minutes at 83°C, and the plate was then cooled to room temperature, over a period often minutes to three hours as required.

The samples so prepared were then transferred to pretreated 1OK plates (Pall Corp). To pretreat these plates, 200ul PBSTB was added to wells, incubated for 1 hour and then washed with 20OuL of water three times. The water was pipetted off, but lOOul of water was left on the plates until use. Immediately before use, the plates were spun at 3,000g for 10 minutes.

The 1OK plate containing the samples was placed on top of a 1.1ml collection plate, spun at 3,000g for 35 minutes or until dry. Plates were then washed with 200ul PBST, centrifugation at 3,000g for 40 minutes or until dry and the flow through was discarded from the collection plate. Material bound to the 1OK plate were then eluted by adding 55ul of PBSTB to all wells that were then covered and shaken at 900rpm for 10 minutes.

The recovered samples were then transferred to a bead assay plate, and subjected to a bead- based sandwich immunoassay using an antibody bound to magnetic bead (M-beads, which are M280-tosylactivated, 2.8um magnetic beads available from Dynal, product number 142.03, wherein the beads and antibody were conjugated according to the supplier's instructions) (e.g. capture antibody), and another antibody bound to fluorescent beads (F- beads which are 0.2um polystyrene beads that are dyed with Europium chelate, and called Fluoro-Max fluorescent carboxylate modified particles, having catalog number 8347-0550 from Seradyn, and prepared according to the supplier's instructions) (e.g. detection antibody). For this assay, 50ul of sample was transferred to each well of the bead assay plate. Samples included test samples, as well as spiked reference samples. Reference samples included 50ul of recombinant sheep PrP at various concentrations in PBSTB and 0.05% azide (Alicon, cat# P0033)

To each well was added 5ul of Ab-F-beads. The plate was then sealed with film. The plate was then incubated at 37°C with shaking at 1200rpm on a Heidolph shaker for one hour. The film was removed and 5ul of Ab-M-beads were added, followed by plate sealing and shaking at 1200rpm on the Heidolph shaker for 2 hours. The plates were washed 3 times with 200ul PBST by first applying a 96- well plate magnet for 2 minutes to pellet the beads, aspirating the supernatant and adding 200ul PBST without mixing. The PBST wash was then aspirated, and the washing step was repeated twice more.

A further 150ul of PBST was then added, the plate sealed, removed from the magnet and then shaken once more in the Heidolph shaker for 5 minutes at 37°C. The lid was removed under fume hood to release any aerosols that may have been generated.

The plate was then transferred to a Perkin Elmer 1420 multilabel counter Victor ³ V to read fluorescence generated by the F-beads. The factory default settings for reading europium were used (excitation filter: 340nm, emission filter: 615nm, flash energy area: low, emission aperture and beam size: both "normal", counting delay and counting window: both at 400μs, counting cycle: lOOOμs)

Using this assay format and protocol, and using POMl antibodies bound to M-beads and 5C4 antibodies bound to F-beads, samples of scrapie-infected and normal control sheep blood were analyzed in a blind fashion. The results are tabulated below in Table 1. From these results, it will be appreciated that the NaOH-based PrP assay format is suitable for detecting aggregated prion protein in the blood of infected subjects as it allowed for the correct detection of 1 1 of 12 samples derived from scrapie-infected sheep and a low false-positive rate.

Example 2 - Detection of PrPSc in human vCJD brain and Hamster Scrapie (HaSc) brain homogenates using the NaOH Epitope Protection assay.

A modified protocol can detect the presence of infectious prions in human vCJD brain and HaSc brain homogenates diluted in plasma up to 400,000-fold. Generally, the protocol is similar to the detection of PrPSc in sheep plasma except that a different affinity matrix is used to remove the disaggregant (guanidine) and a variety of antibodies can be used in the detection assay. The vCJD or HaSc brain homogenates are first diluted into human plasma up to 400,000-fold, and the samples are enriched for PrPSc. In one protocol, the enriched PrPSc is treated with NaOH under defined conditions, dissociated and probed with any one of a number of defined antibodies reactive to sites altered by NaOH treatment of the aggregate. Binding is detected using a fluorescent assay and reveals the presence of monomeric PrP that was protected from NaOH while in the aggregated state, and thus indicates the presence of PrPSc aggregates in the original diluted brain homogenate.

In the experiments using plasma-diluted brain homogenates, the procedure is exactly the same as described in Example 1 for the sheep scrapie plasma up to the point just before loading the samples onto 10k plates. Briefly: 900ul of sample containing human vCJD or HaSc brain homogenate was loaded onto PEP plate on a 1. ImI collection plate, and then spun at 2,00Og for 2 minutes. Flow through was then discarded from the collection plate. The plate was then washed by adding 900ul PBS, spun for 2 minutes in a centrifuge and the flow- through was discarded. In a second wash, 500ul PBS was added, spun for 4 minutes at 2,00Og and the flow through was discarded.

Material bound to the PEP plate was then eluted using 1 OOul of 10OmM NaOH and spun at 2,00Og for 2 minutes directly into a 450ul collection plate. At 4 + 0.5 minutes after spinning, 20ul of 0.5M NaH ₂ PO ₄ ZlOOmM HCl was added rapidly. The solution was pipetted up and down 3 times and shaken at 900rpm for 1 minute. The mixture was treated with 120ul of 8M guanidine hydrochloride and shaken at 450rpm for 2 minutes to ensure mixing. The collection plate was then heated for 6 minutes at 83°C, and the plate was cooled to room temperature over a period of ten minutes to three hours as required.

The samples were allowed to cool to room temperature and then incubated with 5ul of copper-bound Immobilized Metal Affinity Chromatography (Cu-IMAC, Invitrogeή) beads and lOul of 5OmM NaOH. The PrPSc was allowed to bind to the IMAC beads for 15min while shaking at 600rpm at 37C. The PrPSc-bound beads were washed 3 times with 200ul of PBSTB. PrPSc was eluted from the IMAC beads by incubating the beads in 50ul of PBSTB containing EDTA. Beads were pipetted until resuspended and shaken at 1200rpm for 15min at 37°C. The PrPSc-containing eluate was then transferred to another 96- well plate and subjected to a bead-based sandwich immunoassay using an antibody bound to magnetic bead (M-beads), and another antibody bound to fluorescent beads (F-beads). For the detection assay, 50ul of sample was transferred to each well of the bead assay plate. Samples included test samples, as well as spiked reference samples. Reference samples included 50ul of recombinant human or hamster PrP at various concentrations in PBSTB and 0.05% azide (Alicon, cat# P0033)

To each well was added 5ul of Ab-F-beads and the plate was sealed with film. The plate was then incubated at 37°C with shaking at 1200rpm on a Heidolph shaker for 0.5-1 hour. The film was removed and 5ul of Ab-M-beads was added, followed by plate sealing and shaking at 1200rpm on the Heidolph shaker for 1-2 hours. The plates were washed 3 times with 200ul PBST by first applying a 96- well plate magnet for 2 minutes to pellet the beads, aspirating the supernatant and adding 200ul PBST without mixing. The PBST wash was then aspirated, and the washing step was repeated twice.

A further 150ul of PBST was then added, the plate sealed, removed from the magnet and then shaken once more in the Heidolph shaker for 5 minutes at 37 ⁰ C. The lid was removed under fume hood to release any aerosols that may have been generated.

The plate was then transferred to a Perkin Elmer 1420 multilabel counter Victor ³ V to read fluorescence generated by the F-beads. The factory default settings for reading europium were used (excitation filter: 340nm, emission filter: 615nm, flash energy area: low, emission aperture and beam size: both "normal", counting delay and counting window: both at 400μs, counting cycle: lOOOμs)

A number of F-bead/M-bead combinations can be used in the detection assay following the sample prep method described above. POMl antibodies bound to M-beads and 5C4 antibodies bound to F-beads were used to detect 1 :200,000-fold dilutions of human vCJD brain homogenates in plasma (Figure 3), but this combination of beads was not as sensitive in detecting HaSc brain homogenates.

A detection assay using POM2 antibodies bound to M-beads and 3F4 antibodies bound to F- beads was used to detect 1 :200,000 -fold dilutions of human vCJD brain homogenates in plasma (Figure 1), and provided sufficient sensitivity to detect HaSc brain homogenate dilutions of up to 1 :400,000 (Figure 2). Furthermore, preliminary experiments show that 6H4 antibodies bound to M-beads and 3F4 antibodies bound to F-beads can also be used in combination with the NaOH epitope protection assay to detect HaSc and human vCJD brain homogenate dilutions with similar sensitivity. The examples which follow describe experiments to determine the effect of NaOH at different concentrations and incubation periods on sheep PrP and human PrP provided as monomeric, recombinant protein.

Example 3 - Examination of NaOH-affected PrP epitopes

The effect of NaOH incubation on immunoreactivity of monomeric PrP was investigated by Western blot using recombinant, monomeric sheep or human PrP, at varying incubation conditions, and with a panel of antibodies reactive with different PrP epitopes. The primary antibodies and the PrP epitopes to which they bind are set out below. The effect of NaOH on the immunoreactivity of each antibody is also noted in Table 2, as being either sensitive (S) to NaOH incubation as revealed by decreasing immunoreactivity with increasing NaOH concentration, or resistant (R) to NaOH incubation, as indicated by substantially no change in immunoreactivity with increasing NaOH concentration. Immunoreactivity is noted for both recombinant sheep PrP (Sh-PrP) and recombinant human PrP (Hu-PrP).

TABLE 2

Hu-Prp Sensitive/Resistant

Antibodv Region Epitope Source Supplied As Sh-Prp/Hu-PrP

CC2 N-terminal 23-96 Priontype 1.0mg/ml R/R

8B4 N-terminal 37-44 Alicon 1.0mg/ml R/R

P0M2 N-terminal 51-91 Amorfix 2.0mg/ml R/R

5C4 N-terminal 51-91 Abeam 2.5mg/ml R/R

1E4 Mid 98-108 Fitzgerald 0.5mg/ml R/S

6Dl 1 Mid 93-109 Covance 2.0mg/ml R/S

3F4 Mid 106-1 12 Signet Labs 2.0mg/ml s/s

6H4 Mid 144-152 Prionics* 0.88mg/ml s/s

POMl Mid 121-152 Aguzzi 2.5mg/ml s/s

3C8 C-terminal 214-230 Alicon 1.0mg/ml s/s

5G12 C-terminal 225-228 Jena BioSci** 1.0mg/ml s/s

7D9 unknown 23-237 Abeam 1.0mg/ml s/s

* US 6765088

**Jena Bioscience in Germany catalog #ABD-022 Particularly, recombinant sheep PrP (500ng/ul, supplied by Alicon) was diluted 1 :4 (lOul PrP solution added to 30ul water) and used in aliquots of 6ul (750ng) per reaction. Aliquots were exposed for 60 minutes to increasing concentration of NaOH at room temperature. Reactions were then neutralized using HCl, sodium phosphate and then equilibrated for NaCl, and sample loading dye was added, followed by transfer to an eppendorf tube, 83 ⁰ C for 10 min, and cooling.

The cooled samples were then run by SDS-PAGE using the following conditions: 4-12% Bis- Tris gradient gel, lmm, 10 well NuPAGE; MES SDS running buffer, sample were prepared in sample buffer LDS (Ix final); and sample reducing agent (Ix final) with SeeBlue Plus2 (Ix) as the prestained MW marker (all from Invitrogen). Samples resolved on the gel were then transferred to nitrocellulose by blotting for analysis.

Transferred blots were placed in 25ml antibody buffer (24.2gTris base, 35.05g NaCl, 7.45g EDTA as disodium salt, 2ml NP40 and 1Og BSA, QS to 4 litres with USP water). Then, 12.5ug of primary anti-prion antibody was added. Blots were shaken at 60rpm at RT for 2 hours then washed four times with IX PBST. To each blot was then added lOug of secondary antibody (goat anti-murine IgG-HRP supplied by Pierce), and blots were shaken at 60rpm at RT for one hour then washed four times with IX PBST. TMB reagent (7ml) was then added to the washed blots and colour was developed until background became visible.

As shown by results with Coomassie staining in Figure 4, NaOH incubation for 60 min had the effect at higher concentrations (1.0M and above) of destroying the sheep PrP. However, at concentrations up to about 0.5M NaOH, the protein remained intact and was not hydrolyzed by NaOH. When the NaOH-treated and intact PrP was then examined for immunoreactivity, a pattern emerged which indicated that NaOH had the effect of altering immunoreactivity in the mid region and particularly the C-terminal region of PrP. The N- terminal region was not altered significantly.

More particularly, the results of Western blotting reveal that NaOH-treated PrP has altered immunoreactivity to antibodies 3C8, 3F4, 6H4, POMl, 7D9, 5G12, 1E4 and 6Dl 1. The immunoreactivity to antibodies POM2, 8B4, 5C4 and CC2 was substantially unaffected. This pattern reveals that pH elevation has the effect of altering immunoreactivity of PrP particularly in the C-terminal region of PrP (for example residues 100-230), and motivates the use of antibodies to this region to detect PrP after dissociation of aggregates that have been treated with NaOH or other pH elevating agents under conditions that leave the PrP protein intact.

It will be appreciated that the present method can also be applied to detect the aggregated form of other target proteins, including the Abeta protein that, in aggregated form, is associated with Alzheimer's disease. By following the format generally described above, but applied to a sample that is either spinal fluid or blood taken ante-mortem or a homogenate of brain tissue taken post-mortem, from a subject suspected of affliction with Alzheimer's disease as determined by established medical diagnosis, the presence of an aggregated form of Abeta can be detected for instance using antibodies such as 4G8 and 6E10, available from

Signet, or any other antibody(ies) directed to one or more epitopes on Abeta that are altered when Abeta aggregates are exposed to elevated pH.

TABLE 3 - Examples of Prion Sequences

Human Protein

In humans, the major prion protein (PrPc) consists of residues 23-230 of a precursor having residues 1-253 and UniProtKB accession #P04156, as follows:

MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGW GQP HGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCV NITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV ILLISFLIFLIVG (SEQ ID NO: 1)

The first 22 residues are a cleaved secretion signal.

Sheep Protein

In sheep, the major prion protein consists of residues 25-233 of a precursor having residues 1-256 and UniProtKB #P23907, as follows:

MVKSHIGSWILVLFVAMWSDVGLCKKRPKPGGGWNTGGSRYPGQGSPGGNRYPPQGG GGW GQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGGWGQGGSHSQWNKPSKPKTNMKHVAGAAA AGAVVGGLGGYMLGSAMSRPLIHFGNDYEDRYYRENMYRYPNQVYYRPVDRYSNQNNFVH DCVNITVKQHTVTTTTKGENFTETDIKIMERVVEQMCITQYQRESQAYYQRGASVILFSS PPVILLISFLIFLIVG (SEQ ID NO: 2)